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Sample records for capsid binding avidity

  1. Dengue Virus Capsid Protein Binds Core Histones and Inhibits Nucleosome Formation in Human Liver Cells

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    Colpitts, Tonya M.; Barthel, Sebastian; Wang, Penghua; Fikrig, Erol

    2011-01-01

    Dengue virus (DENV) is a member of the Flaviviridae and a globally (re)emerging pathogen that causes serious human disease. There is no specific antiviral or vaccine for dengue virus infection. Flavivirus capsid (C) is a structural protein responsible for gathering the viral RNA into a nucleocapsid that forms the core of a mature virus particle. Flaviviral replication is known to occur in the cytoplasm yet a large portion of capsid protein localizes to the nucleus during infection. The reasons for the nuclear presences of capsid are not completely understood. Here, we expressed mature DENV C in a tandem affinity purification assay to identify potential binding partners in human liver cells. DENV C targeted the four core histones, H2A, H2B, H3 and H4. DENV C bound recombinant histones in solution and colocalized with histones in the nucleus and cytoplasm of liver cells during DENV infection. We show that DENV C acts as a histone mimic, forming heterodimers with core histones, binding DNA and disrupting nucleosome formation. We also demonstrate that DENV infection increases the amounts of core histones in livers cells, which may be a cellular response to C binding away the histone proteins. Infection with DENV additionally alters levels of H2A phosphorylation in a time-dependent manner. The interactions of C and histones add an interesting new role for the presence of C in the nucleus during DENV infection. PMID:21909430

  2. Virulent variants emerging in mice infected with the apathogenic prototype strain of the parvovirus minute virus of mice exhibit a capsid with low avidity for a primary receptor.

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    Rubio, Mari-Paz; López-Bueno, Alberto; Almendral, José M

    2005-09-01

    The mechanisms involved in the emergence of virulent mammalian viruses were investigated in the adult immunodeficient SCID mouse infected by the attenuated prototype strain of the parvovirus Minute Virus of Mice (MVMp). Cloned MVMp intravenously inoculated in mice consistently evolved during weeks of subclinical infection to variants showing altered plaque phenotypes. All the isolated large-plaque variants spread systemically from the oronasal cavity and replicated in major organs (brain, kidney, liver), in sharp contrast to the absolute inability of the MVMp and small-plaque variants to productively invade SCID organs by this natural route of infection. The virulent variants retained the MVMp capacity to infect mouse fibroblasts, consistent with the lack of genetic changes across the 220-to-335 amino acid sequence of VP2, a capsid domain containing main determinants of MVM tropism. However, the capsid of the virulent variants shared a lower affinity than the wild type for a primary receptor used in the cytotoxic infection. The capsid gene of a virulent variant engineered in the MVMp background endowed the recombinant virus with a large-plaque phenotype, lower affinity for the receptor, and productive invasiveness by the oronasal route in SCID mice, eventually leading to 100% mortality. In the analysis of virulence in mice, both MVMp and the recombinant virus similarly gained the bloodstream 1 to 2 days postoronasal inoculation and remained infectious when adsorbed to blood cells in vitro. However, the wild-type MVMp was cleared from circulation a few days afterwards, in contrast to the viremia of the recombinant virus, which was sustained for life. Significantly, attachment to an abundant receptor of primary mouse kidney epithelial cells by both viruses could be quantitatively competed by wild-type MVMp capsids, indicating that virulence is not due to an extended receptor usage in target tissues. We conclude that the selection of capsid-receptor interactions of

  3. DC8 and DC13 var genes associated with severe malaria bind avidly to diverse endothelial cells.

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    Marion Avril

    Full Text Available During blood stage infection, Plasmodium falciparum infected erythrocytes (IE bind to host blood vessels. This virulence determinant enables parasites to evade spleen-dependent killing mechanisms, but paradoxically in some cases may reduce parasite fitness by killing the host. Adhesion of infected erythrocytes is mediated by P. falciparum erythrocyte membrane protein 1 (PfEMP1, a family of polymorphic adhesion proteins encoded by var genes. Whereas cerebral binding and severe malaria are associated with parasites expressing DC8 and DC13 var genes, relatively little is known about the non-brain endothelial selection on severe malaria adhesive types. In this study, we selected P. falciparum-IEs on diverse endothelial cell types and demonstrate that DC8 and DC13 var genes were consistently among the major var transcripts selected on non-brain endothelial cells (lung, heart, bone marrow. To investigate the molecular basis for this avid endothelial binding activity, recombinant proteins were expressed from the predominant upregulated DC8 transcript, IT4var19. In-depth binding comparisons revealed that multiple extracellular domains from this protein bound brain and non-brain endothelial cells, and individual domains largely did not discriminate between different endothelial cell types. Additionally, we found that recombinant DC8 and DC13 CIDR1 domains exhibited a widespread endothelial binding activity and could compete for DC8-IE binding to brain endothelial cells, suggesting they may bind the same host receptor. Our findings provide new insights into the interaction of severe malaria adhesive types and host blood vessels and support the hypothesis that parasites causing severe malaria express PfEMP1 variants with a superior ability to adhere to diverse endothelial cell types, and may therefore endow these parasites with a growth and transmission advantage.

  4. BI-2 destabilizes HIV-1 cores during infection and Prevents Binding of CPSF6 to the HIV-1 Capsid.

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    Fricke, Thomas; Buffone, Cindy; Opp, Silvana; Valle-Casuso, Jose; Diaz-Griffero, Felipe

    2014-12-11

    The recently discovered small-molecule BI-2 potently blocks HIV-1 infection. BI-2 binds to the N-terminal domain of HIV-1 capsid. BI-2 utilizes the same capsid pocket used by the small molecule PF74. Although both drugs bind to the same pocket, it has been proposed that BI-2 uses a different mechanism to block HIV-1 infection when compared to PF74. This work demonstrates that BI-2 destabilizes the HIV-1 core during infection, and prevents the binding of the cellular factor CPSF6 to the HIV-1 core. Overall this short-form paper suggests that BI-2 is using a similar mechanism to the one used by PF74 to block HIV-1 infection.

  5. Requirements for capsid-binding and an effector function in TRIMCyp-mediated restriction of HIV-1

    International Nuclear Information System (INIS)

    Diaz-Griffero, Felipe; Vandegraaff, Nick; Li Yuan; McGee-Estrada, Kathleen; Stremlau, Matthew; Welikala, Sohanya; Si Zhihai; Engelman, Alan; Sodroski, Joseph

    2006-01-01

    In owl monkeys, a retrotransposition event replaced the gene encoding the retroviral restriction factor TRIM5α with one encoding TRIMCyp, a fusion between the RING, B-box 2 and coiled-coil domains of TRIM5 and cyclophilin A. TRIMCyp restricts human immunodeficiency virus (HIV-1) infection by a mechanism dependent on the interaction of the cyclophilin A moiety and the HIV-1 capsid protein. Here, we show that infection by retroviruses other than HIV-1 can be restricted by TRIMCyp, providing an explanation for the evolutionary retention of the TRIMCyp gene in owl monkey lineages. The TRIMCyp-mediated block to HIV-1 infection occurs before the earliest step of reverse transcription. TRIMCyp-mediated restriction involves at least two functions: (1) capsid binding, which occurs most efficiently for trimeric TRIMCyp proteins that retain the coiled-coil and cyclophilin A domains, and (2) an effector function that depends upon the B-box 2 domain

  6. Use of Cre/loxP recombination to swap cell binding motifs on the adenoviral capsid protein IX

    International Nuclear Information System (INIS)

    Poulin, Kathy L.; Tong, Grace; Vorobyova, Olga; Pool, Madeline; Kothary, Rashmi; Parks, Robin J.

    2011-01-01

    We used Cre/loxP recombination to swap targeting ligands present on the adenoviral capsid protein IX (pIX). A loxP-flanked sequence encoding poly-lysine (pK-binds heparan sulfate proteoglycans) was engineered onto the 3'-terminus of pIX, and the resulting fusion protein allowed for routine virus propagation. Growth of this virus on Cre-expressing cells removed the pK coding sequence, generating virus that could only infect through alternative ligands, such as a tyrosine kinase receptor A (TrkA)-binding motif engineered into the capsid fibre protein for enhanced infection of neuronal cells. We used a similar approach to swap the pK motif on pIX for a sequence encoding a single-domain antibody directed towards CD66c for targeted infection of cancer cells; Cre-mediated removal of the pK-coding sequence simultaneously placed the single-domain antibody coding sequence in frame with pIX. Thus, we have developed a simple method to propagate virus lacking native viral tropism but containing cell-specific binding ligands. - Highlights: → We describe a method to grow virus lacking native tropism but containing novel cell-binding ligands. → Cre/loxP recombination was used to modify the adenovirus genome. → A targeting ligand present on capsid protein IX was removed or replaced using recombination. → Cre-loxP was also used to 'swap' the identity of the targeting ligand present on pIX.

  7. Essential C-Terminal region of the baculovirus minor capsid protein VP80 binds DNA

    NARCIS (Netherlands)

    Marek, M.; Merten, O.W.; Francis-Devaraj, F.; Oers, van M.M.

    2012-01-01

    The essential Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV) minor capsid protein VP80 has been recently shown to interact with the virus-triggered, nuclear F-actin cytoskeleton. A role for VP80 in virus morphogenesis has been proposed in the maturation of progeny nucleocapsids and

  8. Thermodynamic characterization of the peptide assembly inhibitor binding to HIV-1 capsid protein

    Czech Academy of Sciences Publication Activity Database

    Kožíšek, Milan; Durčák, Jindřich; Konvalinka, Jan

    2013-01-01

    Roč. 10, Suppl. 1 (2013), S37-S37 ISSN 1742-4690. [Frontiers of Retrovirology: Complex retorviruses, retroelements and their hosts. 16.09.2013-18.09.2013, Cambridge] R&D Projects: GA ČR GA13-19561S Institutional support: RVO:61388963 Keywords : HIV -1 capsid protein * CAI Subject RIV: EE - Microbiology, Virology http://www.retrovirology.com/content/10/S1/P108

  9. Multivalent Soluble Antigen Arrays Exhibit High Avidity Binding and Modulation of B Cell Receptor-Mediated Signaling to Drive Efficacy against Experimental Autoimmune Encephalomyelitis.

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    Hartwell, Brittany L; Pickens, Chad J; Leon, Martin; Berkland, Cory

    2017-06-12

    A pressing need exists for antigen-specific immunotherapies (ASIT) that induce selective tolerance in autoimmune disease while avoiding deleterious global immunosuppression. Multivalent soluble antigen arrays (SAgA PLP:LABL ), consisting of a hyaluronic acid (HA) linear polymer backbone cografted with multiple copies of autoantigen (PLP) and cell adhesion inhibitor (LABL) peptides, are designed to induce tolerance to a specific multiple sclerosis (MS) autoantigen. Previous studies established that hydrolyzable SAgA PLP:LABL , employing a degradable linker to codeliver PLP and LABL, was therapeutic in experimental autoimmune encephalomyelitis (EAE) in vivo and exhibited antigen-specific binding with B cells, targeted the B cell receptor (BCR), and dampened BCR-mediated signaling in vitro. Our results pointed to sustained BCR engagement as the SAgA PLP:LABL therapeutic mechanism, so we developed a new version of the SAgA molecule using nonhydrolyzable conjugation chemistry, hypothesizing it would enhance and maintain the molecule's action at the cell surface to improve efficacy. "Click SAgA" (cSAgA PLP:LABL ) uses hydrolytically stable covalent conjugation chemistry (Copper-catalyzed Azide-Alkyne Cycloaddition (CuAAC)) rather than a hydrolyzable oxime bond to attach PLP and LABL to HA. We explored cSAgA PLP:LABL B cell engagement and modulation of BCR-mediated signaling in vitro through flow cytometry binding and calcium flux signaling assays. Indeed, cSAgA PLP:LABL exhibited higher avidity B cell binding and greater dampening of BCR-mediated signaling than hydrolyzable SAgA PLP:LABL . Furthermore, cSAgA PLP:LABL exhibited significantly enhanced in vivo efficacy compared to hydrolyzable SAgA PLP:LABL , achieving equivalent efficacy at one-quarter of the dose. These results indicate that nonhydrolyzable conjugation increased the avidity of cSAgA PLP:LABL to drive in vivo efficacy through modulated BCR-mediated signaling.

  10. Identification of binding domains in the herpes simplex virus type 1 small capsid protein pUL35 (VP26).

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    Apcarian, Arin; Cunningham, Anthony L; Diefenbach, Russell J

    2010-11-01

    In this study, fragments of the small capsid protein pUL35 (VP26) from herpes simplex virus type 1 (HSV-1) were generated to identify binding domains for a number of known ligands. Analysis of the binding of dynein light chain subunits, DYNLT1 and DYNLT3, as well the HSV-1 structural proteins pUL19 (VP5) and pUL37 was then undertaken using the LexA yeast two-hybrid assay. The N-terminal half of pUL35, in particular residues 30-43, was identified as a common region for the binding of DYNLT1 and DYNLT3. Additional distinct regions in the C terminus of pUL35 also contribute to the binding of DYNLT1 and DYNLT3. In contrast, only the C-terminal half of pUL35 was found to mediate the binding of pUL19 and pUL37 through distinct regions. The relevance of this information to the role of pUL35 in viral transport and assembly is discussed.

  11. The lectin from Musa paradisiaca binds with the capsid protein of tobacco mosaic virus and prevents viral infection.

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    Liu, Xiao-Yu; Li, Huan; Zhang, Wei

    2014-05-04

    It has been demonstrated that the lectin from Musa paradisiaca (BanLec-1) could inhibit the cellular entry of human immunodeficiency virus (HIV). In order to evaluate its effects on tobacco mosaic virus (TMV), the banlec-1 gene was cloned and transformed into Escherichia coli and tobacco, respectively. Recombinant BanLec-1 showed metal ions dependence, and higher thermal and pH stability. Overexpression of banlec-1 in tobacco resulted in decreased leaf size, and higher resistance to TMV infection, which includes reduced TMV cellular entry, more stable chlorophyll contents, and enhanced antioxidant enzymes. BanLec-1 was found to bind directly to the TMV capsid protein in vitro , and to inhibit TMV infection in a dose-dependent manner. In contrast to limited prevention in vivo , purified rBanLec-1 exhibited more significant effects on TMV infection in vitro . Taken together, our study indicated that BanLec-1 could prevent TMV infection in tobacco, probably through the interaction between BanLec-1 and TMV capsid protein.

  12. Direct binding of retromer to human papillomavirus type 16 minor capsid protein L2 mediates endosome exit during viral infection.

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    Andreea Popa

    2015-02-01

    Full Text Available Trafficking of human papillomaviruses to the Golgi apparatus during virus entry requires retromer, an endosomal coat protein complex that mediates the vesicular transport of cellular transmembrane proteins from the endosome to the Golgi apparatus or the plasma membrane. Here we show that the HPV16 L2 minor capsid protein is a retromer cargo, even though L2 is not a transmembrane protein. We show that direct binding of retromer to a conserved sequence in the carboxy-terminus of L2 is required for exit of L2 from the early endosome and delivery to the trans-Golgi network during virus entry. This binding site is different from known retromer binding motifs and can be replaced by a sorting signal from a cellular retromer cargo. Thus, HPV16 is an unconventional particulate retromer cargo, and retromer binding initiates retrograde transport of viral components from the endosome to the trans-Golgi network during virus entry. We propose that the carboxy-terminal segment of L2 protein protrudes through the endosomal membrane and is accessed by retromer in the cytoplasm.

  13. Specific Inhibitors of HIV Capsid Assembly Binding to the C-Terminal Domain of the Capsid Protein: Evaluation of 2-Arylquinazolines as Potential Antiviral Compounds

    Czech Academy of Sciences Publication Activity Database

    Machara, A.; Lux, V.; Kožíšek, Milan; Grantz Šašková, Klára; Štěpánek, O.; Kotora, M.; Parkan, Kamil; Pávová, Marcela; Glass, B.; Sehr, P.; Lewis, J.; Müller, B.; Kräusslich, H. G.; Konvalinka, Jan

    2016-01-01

    Roč. 59, č. 2 (2016), s. 545-558 ISSN 0022-2623 R&D Projects: GA ČR GA13-19561S EU Projects: European Commission(XE) 201095 - HIV ACE Institutional support: RVO:61388963 Keywords : HIV -1 assembly * capsid * high-throughput screening * AlphaScreen assay Subject RIV: CE - Biochemistry Impact factor: 6.259, year: 2016

  14. General Model for Retroviral Capsid Pattern Recognition by TRIM5 Proteins.

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    Wagner, Jonathan M; Christensen, Devin E; Bhattacharya, Akash; Dawidziak, Daria M; Roganowicz, Marcin D; Wan, Yueping; Pumroy, Ruth A; Demeler, Borries; Ivanov, Dmitri N; Ganser-Pornillos, Barbie K; Sundquist, Wesley I; Pornillos, Owen

    2018-02-15

    Restriction factors are intrinsic cellular defense proteins that have evolved to block microbial infections. Retroviruses such as HIV-1 are restricted by TRIM5 proteins, which recognize the viral capsid shell that surrounds, organizes, and protects the viral genome. TRIM5α uses a SPRY domain to bind capsids with low intrinsic affinity ( K D of >1 mM) and therefore requires higher-order assembly into a hexagonal lattice to generate sufficient avidity for productive capsid recognition. TRIMCyp, on the other hand, binds HIV-1 capsids through a cyclophilin A domain, which has a well-defined binding site and higher affinity ( K D of ∼10 μM) for isolated capsid subunits. Therefore, it has been argued that TRIMCyp proteins have dispensed with the need for higher-order assembly to function as antiviral factors. Here, we show that, consistent with its high degree of sequence similarity with TRIM5α, the TRIMCyp B-box 2 domain shares the same ability to self-associate and facilitate assembly of a TRIMCyp hexagonal lattice that can wrap about the HIV-1 capsid. We also show that under stringent experimental conditions, TRIMCyp-mediated restriction of HIV-1 is indeed dependent on higher-order assembly. Both forms of TRIM5 therefore use the same mechanism of avidity-driven capsid pattern recognition. IMPORTANCE Rhesus macaques and owl monkeys are highly resistant to HIV-1 infection due to the activity of TRIM5 restriction factors. The rhesus macaque TRIM5α protein blocks HIV-1 through a mechanism that requires self-assembly of a hexagonal TRIM5α lattice around the invading viral core. Lattice assembly amplifies very weak interactions between the TRIM5α SPRY domain and the HIV-1 capsid. Assembly also promotes dimerization of the TRIM5α RING E3 ligase domain, resulting in synthesis of polyubiquitin chains that mediate downstream steps of restriction. In contrast to rhesus TRIM5α, the owl monkey TRIM5 homolog, TRIMCyp, binds isolated HIV-1 CA subunits much more tightly

  15. Novel antibody binding determinants on the capsid surface of serotype O foot-and-mouth disease virus

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    Asfor, Amin S.; Upadhyaya, Sasmita; Knowles, Nick J.; King, Donald P.; Paton, David J.

    2014-01-01

    Five neutralizing antigenic sites have been described for serotype O foot-and-mouth disease viruses (FMDV) based on monoclonal antibody (mAb) escape mutant studies. However, a mutant virus selected to escape neutralization of mAb binding at all five sites was previously shown to confer complete cross-protection with the parental virus in guinea pig challenge studies, suggesting that amino acid residues outside the mAb binding sites contribute to antibody-mediated in vivo neutralization of FMDV. Comparison of the ability of bovine antisera to neutralize a panel of serotype O FMDV identified three novel putative sites at VP2-74, VP2-191 and VP3-85, where amino acid substitutions correlated with changes in sero-reactivity. The impact of these positions was tested using site-directed mutagenesis to effect substitutions at critical amino acid residues within an infectious copy of FMDV O1 Kaufbeuren (O1K). Recovered viruses containing additional mutations at VP2-74 and VP2-191 exhibited greater resistance to neutralization with both O1K guinea pig and O BFS bovine antisera than a virus that was engineered to include only mutations at the five known antigenic sites. The changes at VP2-74 and VP3-85 are adjacent to critical amino acids that define antigenic sites 2 and 4, respectively. However VP2-191 (17 Å away from VP2-72), located at the threefold axis and more distant from previously identified antigenic sites, exhibited the most profound effect. These findings extend our knowledge of the surface features of the FMDV capsid known to elicit neutralizing antibodies, and will improve our strategies for vaccine strain selection and rational vaccine design. PMID:24584474

  16. Binding of glutathione to enterovirus capsids is essential for virion morphogenesis.

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    Hendrik Jan Thibaut

    2014-04-01

    Full Text Available Enteroviruses (family of the Picornaviridae cover a large group of medically important human pathogens for which no antiviral treatment is approved. Although these viruses have been extensively studied, some aspects of the viral life cycle, in particular morphogenesis, are yet poorly understood. We report the discovery of TP219 as a novel inhibitor of the replication of several enteroviruses, including coxsackievirus and poliovirus. We show that TP219 binds directly glutathione (GSH, thereby rapidly depleting intracellular GSH levels and that this interferes with virus morphogenesis without affecting viral RNA replication. The inhibitory effect on assembly was shown not to depend on an altered reducing environment. Using TP219, we show that GSH is an essential stabilizing cofactor during the transition of protomeric particles into pentameric particles. Sequential passaging of coxsackievirus B3 in the presence of low GSH-levels selected for GSH-independent mutants that harbored a surface-exposed methionine in VP1 at the interface between two protomers. In line with this observation, enteroviruses that already contained this surface-exposed methionine, such as EV71, did not rely on GSH for virus morphogenesis. Biochemical and microscopical analysis provided strong evidence for a direct interaction between GSH and wildtype VP1 and a role for this interaction in localizing assembly intermediates to replication sites. Consistently, the interaction between GSH and mutant VP1 was abolished resulting in a relocalization of the assembly intermediates to replication sites independent from GSH. This study thus reveals GSH as a novel stabilizing host factor essential for the production of infectious enterovirus progeny and provides new insights into the poorly understood process of morphogenesis.

  17. Binding of Glutathione to Enterovirus Capsids Is Essential for Virion Morphogenesis

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    Thibaut, Hendrik Jan; Thys, Bert; Canela, María-Dolores; Aguado, Leire; Wimmer, Eckard; Paul, Aniko; Pérez-Pérez, María-Jesús; van Kuppeveld, Frank J. M.; Neyts, Johan

    2014-01-01

    Enteroviruses (family of the Picornaviridae) cover a large group of medically important human pathogens for which no antiviral treatment is approved. Although these viruses have been extensively studied, some aspects of the viral life cycle, in particular morphogenesis, are yet poorly understood. We report the discovery of TP219 as a novel inhibitor of the replication of several enteroviruses, including coxsackievirus and poliovirus. We show that TP219 binds directly glutathione (GSH), thereby rapidly depleting intracellular GSH levels and that this interferes with virus morphogenesis without affecting viral RNA replication. The inhibitory effect on assembly was shown not to depend on an altered reducing environment. Using TP219, we show that GSH is an essential stabilizing cofactor during the transition of protomeric particles into pentameric particles. Sequential passaging of coxsackievirus B3 in the presence of low GSH-levels selected for GSH-independent mutants that harbored a surface-exposed methionine in VP1 at the interface between two protomers. In line with this observation, enteroviruses that already contained this surface-exposed methionine, such as EV71, did not rely on GSH for virus morphogenesis. Biochemical and microscopical analysis provided strong evidence for a direct interaction between GSH and wildtype VP1 and a role for this interaction in localizing assembly intermediates to replication sites. Consistently, the interaction between GSH and mutant VP1 was abolished resulting in a relocalization of the assembly intermediates to replication sites independent from GSH. This study thus reveals GSH as a novel stabilizing host factor essential for the production of infectious enterovirus progeny and provides new insights into the poorly understood process of morphogenesis. PMID:24722756

  18. Cyclophilin A potentiates TRIM5α inhibition of HIV-1 nuclear import without promoting TRIM5α binding to the viral capsid.

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    Mallori Burse

    Full Text Available The host immunophilin cyclophilin A (CypA binds to the capsid protein (CA of HIV-1 and regulates its infectivity. Depending on the target cell type, CypA can either promote or inhibit HIV-1 infection. The ability of CypA to promote HIV-1 infection has been extensively studied and linked to several steps in early replication including uncoating, reverse transcription and nuclear import. By contrast, the mechanism by which CypA inhibits infection is less well understood. We investigated the mechanism by which CypA potentiates restriction of HIV-1 by the tripartite motif-containing protein 5 (TRIM5α. Depletion of TRIM5α in the African green monkey cell line Vero, resulted in a loss of inhibition of infection by CypA, demonstrating that inhibition by CypA is mediated by TRIM5α. Complementary genetic and biochemical assays failed to demonstrate an ability of CypA to promote binding of TRIM5α to the viral capsid. TRIM5α inhibits HIV-1 reverse transcription in a proteasome-dependent manner; however, we observed that inhibition of proteasome activity did not reduce the ability of CypA to inhibit infection, suggesting that CypA acts at a step after reverse transcription. Accordingly, we observed a CypA-dependent reduction in the accumulation of nuclear HIV-1 DNA, indicating that CypA specifically promotes TRIM5α inhibition of HIV-1 nuclear import. We also observed that the ability of CypA to inhibit HIV-1 infection is abolished by amino acid substitutions within the conserved CPSF6-binding surface in CA. Our results indicate that CypA inhibits HIV-1 infection in Vero cells not by promoting TRIM5α binding to the capsid but by blocking nuclear import of the HIV-1 preintegration complex.

  19. Plus- and minus-end directed microtubule motors bind simultaneously to herpes simplex virus capsids using different inner tegument structures.

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    Kerstin Radtke

    2010-07-01

    Full Text Available Many viruses depend on host microtubule motors to reach their destined intracellular location. Viral particles of neurotropic alphaherpesviruses such as herpes simplex virus 1 (HSV1 show bidirectional transport towards the cell center as well as the periphery, indicating that they utilize microtubule motors of opposing directionality. To understand the mechanisms of specific motor recruitment, it is necessary to characterize the molecular composition of such motile viral structures. We have generated HSV1 capsids with different surface features without impairing their overall architecture, and show that in a mammalian cell-free system the microtubule motors dynein and kinesin-1 and the dynein cofactor dynactin could interact directly with capsids independent of other host factors. The capsid composition and surface was analyzed with respect to 23 structural proteins that are potentially exposed to the cytosol during virus assembly or cell entry. Many of these proteins belong to the tegument, the hallmark of all herpesviruses located between the capsid and the viral envelope. Using immunoblots, quantitative mass spectrometry and quantitative immunoelectron microscopy, we show that capsids exposing inner tegument proteins such as pUS3, pUL36, pUL37, ICP0, pUL14, pUL16, and pUL21 recruited dynein, dynactin, kinesin-1 and kinesin-2. In contrast, neither untegumented capsids exposing VP5, VP26, pUL17 and pUL25 nor capsids covered by outer tegument proteins such as vhs, pUL11, ICP4, ICP34.5, VP11/12, VP13/14, VP16, VP22 or pUS11 bound microtubule motors. Our data suggest that HSV1 uses different structural features of the inner tegument to recruit dynein or kinesin-1. Individual capsids simultaneously accommodated motors of opposing directionality as well as several copies of the same motor. Thus, these associated motors either engage in a tug-of-war or their activities are coordinately regulated to achieve net transport either to the nucleus during

  20. The differences in heparin binding for the C-terminal basic-sequence-rich peptides of HPV-16 and HPV-18 capsid protein L1

    International Nuclear Information System (INIS)

    Sun Jian; Yu Jisheng; Yu Zhiwu; Zha Xiao; Wu Yuqing

    2012-01-01

    Graphial abstract: The differences in heparin binding for the C-terminal basic-sequence-rich peptides of HPV-16 and HPV-18 capsid protein L1. Highlights: ► Several driving forces contribute to the interaction between heparin and peptides. ► C-terminal of HPV L1 is a potential candidate for the attachment to host cells. ► The C-terminal peptides of HPV-16 and -18 L1 have different heparin-binding. ► The different heparin-binding provides an explanation for the distinct prevalences. - Abstract: The high-risk types of human papillomaviruses (HPV) HPV-16 and -18 are the predominant types associated with cervical cancer. HPV-16 and -18 account for about 50% and 20%, respectively, of cervical cancers worldwide. While the reason and molecular mechanism of the distinct prevalence and distributions between them remain poorly understood, the binding affinity of cell surface receptor with capsid proteins, especially L1, may be involved. We examined heparin binding with two synthetic peptides corresponding to the 14 amino acid C-terminal peptides of HPV-16 and -18 L1 with the goal of comparing the equivalent residues in different HPV types. Using isothermal titration calorimetry (ITC) and static right-angle light scattering (SLS), we determined the binding constant K, reaction enthalpy ΔH, and other thermodynamic parameters in the interaction. Especially, we assessed the role of specific residues in binding with heparin by comparing the NMR spectra of free and heparin-bound peptides.

  1. Delineation of pulmonary airway fluid protein fractions with HRPO binding-avidity by far-Western ligand blot and mass spectrometry analyses: a model methodology for detecting mannose-binding protein expression profiles.

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    Coyne, Cody P; Rashmir-Raven, Ann; Jones, Toni; Mochal, Cathleen; Linford, Robert L; Brashier, Michael; Eddy, Alison

    2009-01-01

    Limited research to date has characterized the potential for HRPO to function as a primary molecular probe. Pulmonary airway fluid was developed by non-reducing far-Western (ligand) blot analyses utilizing conjugated HRPO-strepavidin or non-conjugated HRPO without the presence of primary immunoglobulin. Endogenous esterase-like biochemical activity of fractions within pulmonary airway fluid was inactivated to determine if they were capable of biochemically converting HRPO chemiluminescent substrate. Complementary analyses modified pulmonary fluid and HRPO with beta-galactosidase and alpha-mannosidase respectively, in addition to determining the influence of mannose and maltose competitive binding on HRPO far-Western (ligand) blot analyses. Identification of pulmonary fluid fractions detected by HRPO far-Western blot analyses was determined by mass spectrometry. Modification of pulmonary fluid with beta-galactosidase, and HRPO with alpha-mannosidase in concert with maltose and mannose competitive binding analyses altered the intensity and spectrum of pulmonary fluid fractions detected by HRPO far-Western blot analysis. Identity of pulmonary airway fluid fractions detected by HRPO far-Western (ligand) blot analysis were transferrin, dynein, albumin precursor, and two 156 kDa equine peptide fragments. HRPO can function as a partially-selective primary molecular probe when applied in either a conjugated or non-conjugated form. Some protein fractions can form complexes with HRPO through molecular mechanisms that involve physical interactions at the terminal alpha-mannose-rich regions of HRPO glycan side-chains. Based on its known molecular composition and structure, HRPO provides an opportunity for the development of diagnostics methodologies relevant to disease biomarkers that possess mannose-binding avidity.

  2. Stabilising the Herpes Simplex Virus capsid by DNA packaging

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    Wuite, Gijs; Radtke, Kerstin; Sodeik, Beate; Roos, Wouter

    2009-03-01

    Three different types of Herpes Simplex Virus type 1 (HSV-1) nuclear capsids can be distinguished, A, B and C capsids. These capsids types are, respectively, empty, contain scaffold proteins, or hold DNA. We investigate the physical properties of these three capsids by combining biochemical and nanoindentation techniques. Atomic Force Microscopy (AFM) experiments show that A and C capsids are mechanically indistinguishable whereas B capsids already break at much lower forces. By extracting the pentamers with 2.0 M GuHCl or 6.0 M Urea we demonstrate an increased flexibility of all three capsid types. Remarkably, the breaking force of the B capsids without pentamers does not change, while the modified A and C capsids show a large drop in their breaking force to approximately the value of the B capsids. This result indicates that upon DNA packaging a structural change at or near the pentamers occurs which mechanically reinforces the capsids structure. The reported binding of proteins UL17/UL25 to the pentamers of the A and C capsids seems the most likely candidate for such capsids strengthening. Finally, the data supports the view that initiation of DNA packaging triggers the maturation of HSV-1 capsids.

  3. Yellow fever virus capsid protein is a potent suppressor of RNA silencing that binds double-stranded RNA.

    Science.gov (United States)

    Samuel, Glady Hazitha; Wiley, Michael R; Badawi, Atif; Adelman, Zach N; Myles, Kevin M

    2016-11-29

    Mosquito-borne flaviviruses, including yellow fever virus (YFV), Zika virus (ZIKV), and West Nile virus (WNV), profoundly affect human health. The successful transmission of these viruses to a human host depends on the pathogen's ability to overcome a potentially sterilizing immune response in the vector mosquito. Similar to other invertebrate animals and plants, the mosquito's RNA silencing pathway comprises its primary antiviral defense. Although a diverse range of plant and insect viruses has been found to encode suppressors of RNA silencing, the mechanisms by which flaviviruses antagonize antiviral small RNA pathways in disease vectors are unknown. Here we describe a viral suppressor of RNA silencing (VSR) encoded by the prototype flavivirus, YFV. We show that the YFV capsid (YFC) protein inhibits RNA silencing in the mosquito Aedes aegypti by interfering with Dicer. This VSR activity appears to be broadly conserved in the C proteins of other medically important flaviviruses, including that of ZIKV. These results suggest that a molecular "arms race" between vector and pathogen underlies the continued existence of flaviviruses in nature.

  4. Herpesvirus capsid assembly and DNA packaging

    Science.gov (United States)

    Heming, Jason D.; Conway, James F.; Homa, Fred L.

    2017-01-01

    Herpes simplex virus type I (HSV-1) is the causative agent of several pathologies ranging in severity from the common cold sore to life-threatening encephalitic infection. During productive lytic infection, over 80 viral proteins are expressed in a highly regulated manner, resulting in the replication of viral genomes and assembly of progeny virions. The virion of all herpesviruses consists of an external membrane envelope, a proteinaceous layer called the tegument, and an icosahedral capsid containing the double-stranded linear DNA genome. The capsid shell of HSV-1 is built from four structural proteins: a major capsid protein, VP5, which forms the capsomers (hexons and pentons), the triplex consisting of VP19C and VP23 found between the capsomers, and VP26 which binds to VP5 on hexons but not pentons. In addition, the dodecameric pUL6 portal complex occupies one of the 12 capsid vertices, and the capsid vertex specific component (CVSC), a heterotrimer complex of pUL17, pUL25 and pUL36 binds specifically to the triplexes adjacent to each penton. The capsid is assembled in the nucleus where the viral genome is packaged into newly assembled closed capsid shells. Cleavage and packaging of replicated, concatemeric viral DNA requires the seven viral proteins encoded by the UL6, UL15, UL17, UL25, UL28, UL32, and UL33 genes. Considerable advances have been made in understanding the structure of the herpesvirus capsid and the function of several of the DNA packaging proteins by applying biochemical, genetic, and structural techniques. This review is a summary of recent advances with respect to the structure of the HSV-1 virion capsid and what is known about the function of the seven packaging proteins and their interactions with each other and with the capsid shell. PMID:28528442

  5. In vitro measurement of avidity of radioiodinated antibodies

    International Nuclear Information System (INIS)

    Badger, C.C.; Krohn, K.A.; Bernstein, I.D.

    1987-01-01

    A determination of the ability of radiolabeled antibodies to bind to their target antigen is an essential step in the initial selection of antibodies for clinical use as well as a quality control measure. In our studies of the 131 I-labeled anti-Thy 1.1 antibody treatment of murine lymphoma, we have used cell binding assays with a combination of Lineweaver-Burk analysis to determine immunoreactivity and Scatchard analysis to determine antibody avidity. Both assays were systematically influenced by target cell fixation and measurement of avidity was dependent on immunoreactivity. For 131 I-labeled anti-Thy 1.1 antibody, avidity was a much more sensitive indicator of iodination damage and predictor of in vivo behavior than was immunoreactivity, while for other antibodies immunoreactivity has been a better indicator of labeling damage. Thus, immunoreactivity and avidity assays are complementary and knowledge of both factors is required for the design of sensitive quality control procedures for radiolabeled antibodies. (author)

  6. Large-scale functional purification of recombinant HIV-1 capsid.

    Directory of Open Access Journals (Sweden)

    Magdeleine Hung

    Full Text Available During human immunodeficiency virus type-1 (HIV-1 virion maturation, capsid proteins undergo a major rearrangement to form a conical core that protects the viral nucleoprotein complexes. Mutations in the capsid sequence that alter the stability of the capsid core are deleterious to viral infectivity and replication. Recently, capsid assembly has become an attractive target for the development of a new generation of anti-retroviral agents. Drug screening efforts and subsequent structural and mechanistic studies require gram quantities of active, homogeneous and pure protein. Conventional means of laboratory purification of Escherichia coli expressed recombinant capsid protein rely on column chromatography steps that are not amenable to large-scale production. Here we present a function-based purification of wild-type and quadruple mutant capsid proteins, which relies on the inherent propensity of capsid protein to polymerize and depolymerize. This method does not require the packing of sizable chromatography columns and can generate double-digit gram quantities of functionally and biochemically well-behaved proteins with greater than 98% purity. We have used the purified capsid protein to characterize two known assembly inhibitors in our in-house developed polymerization assay and to measure their binding affinities. Our capsid purification procedure provides a robust method for purifying large quantities of a key protein in the HIV-1 life cycle, facilitating identification of the next generation anti-HIV agents.

  7. Quantitative relationship between antibody affinity and antibody avidity

    International Nuclear Information System (INIS)

    Griswold, W.R.

    1987-01-01

    The relationship between antibody avidity, measured by the dissociation of the antigen-antibody bond in antigen excess, and antibody affinity was studied. Complexes of radiolabelled antigen and antibody of known affinity were prepared in vitro and allowed to stand for seven days to reach equilibrium. Then nonlabelled antigen in one hundred fold excess was added to dissociate the complexes. After an appropriate incubation the fraction of antigen bound to antibody was measured by the ammonium sulfate precipitation method. The dissociation index was the fraction bound in the experimental sample divided by the fraction bound in the control. The correlation coefficient between the dissociation index and the antibody binding constant was 0.92 for early dissociation and 0.98 for late dissociation. The regression equation relating the binding constant to the dissociation index was K = 6.4(DI) + 6.25, where DI is the late dissociation index and K is the logarithm to the base 10 of the binding constant. There is a high correlation between avidity and affinity of antibody. Antibody affinity can be estimated from avidity data. The stability of antigen-antibody complexes can be predicted from antibody affinity

  8. Partnering with AVID to create transportation scholars

    Science.gov (United States)

    2010-10-01

    The goal of this project was to team with the Advancement Via Individual Determination (AVID) : program in local schools to stimulate student awareness of transportation and engineering careers and to : encourage interest in the science, technology, ...

  9. Residues of the UL25 Protein of Herpes Simplex Virus That Are Required for Its Stable Interaction with Capsids

    Science.gov (United States)

    Cockrell, Shelley K.; Huffman, Jamie B.; Toropova, Katerina; Conway, James F.; Homa, Fred L.

    2011-01-01

    The herpes simplex virus 1 (HSV-1) UL25 gene product is a minor capsid component that is required for encapsidation, but not cleavage, of replicated viral DNA. UL25 is located on the capsid surface in a proposed heterodimer with UL17, where five copies of the heterodimer are found at each of the capsid vertices. Previously, we demonstrated that amino acids 1 to 50 of UL25 are essential for its stable interaction with capsids. To further define the UL25 capsid binding domain, we generated recombinant viruses with either small truncations or amino acid substitutions in the UL25 N terminus. Studies of these mutants demonstrated that there are two important regions within the capsid binding domain. The first 27 amino acids are essential for capsid binding of UL25, while residues 26 to 39, which are highly conserved in the UL25 homologues of other alphaherpesviruses, were found to be critical for stable capsid binding. Cryo-electron microscopy reconstructions of capsids containing either a small tag on the N terminus of UL25 or the green fluorescent protein (GFP) fused between amino acids 50 and 51 of UL25 demonstrate that residues 1 to 27 of UL25 contact the hexon adjacent to the penton. A second region, most likely centered on amino acids 26 to 39, contacts the triplex that is one removed from the penton. Importantly, both of these UL25 capsid binding regions are essential for the stable packaging of full-length viral genomes. PMID:21411517

  10. Parvovirus Capsid Structures Required for Infection: Mutations Controlling Receptor Recognition and Protease Cleavages.

    Science.gov (United States)

    Callaway, Heather M; Feng, Kurtis H; Lee, Donald W; Allison, Andrew B; Pinard, Melissa; McKenna, Robert; Agbandje-McKenna, Mavis; Hafenstein, Susan; Parrish, Colin R

    2017-01-15

    Parvovirus capsids are small but complex molecular machines responsible for undertaking many of the steps of cell infection, genome packing, and cell-to-cell as well as host-to-host transfer. The details of parvovirus infection of cells are still not fully understood, but the processes must involve small changes in the capsid structure that allow the endocytosed virus to escape from the endosome, pass through the cell cytoplasm, and deliver the single-stranded DNA (ssDNA) genome to the nucleus, where viral replication occurs. Here, we examine capsid substitutions that eliminate canine parvovirus (CPV) infectivity and identify how those mutations changed the capsid structure or altered interactions with the infectious pathway. Amino acid substitutions on the exterior surface of the capsid (Gly299Lys/Ala300Lys) altered the binding of the capsid to transferrin receptor type 1 (TfR), particularly during virus dissociation from the receptor, but still allowed efficient entry into both feline and canine cells without successful infection. These substitutions likely control specific capsid structural changes resulting from TfR binding required for infection. A second set of changes on the interior surface of the capsid reduced viral infectivity by >100-fold and included two cysteine residues and neighboring residues. One of these substitutions, Cys270Ser, modulates a VP2 cleavage event found in ∼10% of the capsid proteins that also was shown to alter capsid stability. A neighboring substitution, Pro272Lys, significantly reduced capsid assembly, while a Cys273Ser change appeared to alter capsid transport from the nucleus. These mutants reveal additional structural details that explain cell infection processes of parvovirus capsids. Parvoviruses are commonly found in both vertebrate and invertebrate animals and cause widespread disease. They are also being developed as oncolytic therapeutics and as gene therapy vectors. Most functions involved in infection or transduction

  11. Parvovirus Capsid Structures Required for Infection: Mutations Controlling Receptor Recognition and Protease Cleavages

    Science.gov (United States)

    Callaway, Heather M.; Feng, Kurtis H.; Lee, Donald W.; Pinard, Melissa; McKenna, Robert; Agbandje-McKenna, Mavis; Hafenstein, Susan

    2016-01-01

    ABSTRACT Parvovirus capsids are small but complex molecular machines responsible for undertaking many of the steps of cell infection, genome packing, and cell-to-cell as well as host-to-host transfer. The details of parvovirus infection of cells are still not fully understood, but the processes must involve small changes in the capsid structure that allow the endocytosed virus to escape from the endosome, pass through the cell cytoplasm, and deliver the single-stranded DNA (ssDNA) genome to the nucleus, where viral replication occurs. Here, we examine capsid substitutions that eliminate canine parvovirus (CPV) infectivity and identify how those mutations changed the capsid structure or altered interactions with the infectious pathway. Amino acid substitutions on the exterior surface of the capsid (Gly299Lys/Ala300Lys) altered the binding of the capsid to transferrin receptor type 1 (TfR), particularly during virus dissociation from the receptor, but still allowed efficient entry into both feline and canine cells without successful infection. These substitutions likely control specific capsid structural changes resulting from TfR binding required for infection. A second set of changes on the interior surface of the capsid reduced viral infectivity by >100-fold and included two cysteine residues and neighboring residues. One of these substitutions, Cys270Ser, modulates a VP2 cleavage event found in ∼10% of the capsid proteins that also was shown to alter capsid stability. A neighboring substitution, Pro272Lys, significantly reduced capsid assembly, while a Cys273Ser change appeared to alter capsid transport from the nucleus. These mutants reveal additional structural details that explain cell infection processes of parvovirus capsids. IMPORTANCE Parvoviruses are commonly found in both vertebrate and invertebrate animals and cause widespread disease. They are also being developed as oncolytic therapeutics and as gene therapy vectors. Most functions involved in

  12. Overcoming preexisting humoral immunity to AAV using capsid decoys.

    Science.gov (United States)

    Mingozzi, Federico; Anguela, Xavier M; Pavani, Giulia; Chen, Yifeng; Davidson, Robert J; Hui, Daniel J; Yazicioglu, Mustafa; Elkouby, Liron; Hinderer, Christian J; Faella, Armida; Howard, Carolann; Tai, Alex; Podsakoff, Gregory M; Zhou, Shangzhen; Basner-Tschakarjan, Etiena; Wright, John Fraser; High, Katherine A

    2013-07-17

    Adeno-associated virus (AAV) vectors delivered through the systemic circulation successfully transduce various target tissues in animal models. However, similar attempts in humans have been hampered by the high prevalence of neutralizing antibodies to AAV, which completely block vector transduction. We show in both mouse and nonhuman primate models that addition of empty capsid to the final vector formulation can, in a dose-dependent manner, adsorb these antibodies, even at high titers, thus overcoming their inhibitory effect. To further enhance the safety of the approach, we mutated the receptor binding site of AAV2 to generate an empty capsid mutant that can adsorb antibodies but cannot enter a target cell. Our work suggests that optimizing the ratio of full/empty capsids in the final formulation of vector, based on a patient's anti-AAV titers, will maximize the efficacy of gene transfer after systemic vector delivery.

  13. Human rhinovirus capsid dynamics is controlled by canyon flexibility

    International Nuclear Information System (INIS)

    Reisdorph, Nichole; Thomas, John J.; Katpally, Umesh; Chase, Elaine; Harris, Ken; Siuzdak, Gary; Smith, Thomas J.

    2003-01-01

    Quantitative enzyme accessibility experiments using nano liquid chromatography electrospray mass spectrometry combined with limited proteolysis and isotope-labeling was used to examine the dynamic nature of the human rhinovirus (HRV) capsid in the presence of three antiviral compounds, a neutralizing Fab, and drug binding cavity mutations. Using these methods, it was found that the antivirals WIN 52084 and picovir (pleconaril) stabilized the capsid, while dansylaziridine caused destabilization. Site-directed mutations in the drug-binding cavity were found to stabilize the HRV14 capsid against proteolytic digestion in a manner similar to WIN 52084 and pleconaril. Antibodies that bind to the NIm-IA antigenic site and penetrate the canyon were also observed to protect the virion against proteolytic cleavage. These results demonstrate that quantifying the effects of antiviral ligands on protein 'breathing' can be used to compare their mode of action and efficacy. In this case, it is apparent that hydrophobic antiviral agents, antibodies, or mutations in the canyon region block viral breathing. Therefore, these studies demonstrate that mobility in the canyon region is a major determinant in capsid breathing

  14. Direct block scheduling technology: Analysis of Avidity

    Directory of Open Access Journals (Sweden)

    Felipe Ribeiro Souza

    Full Text Available Abstract This study is focused on Direct Block Scheduling testing (Direct Multi-Period Scheduling methodology which schedules mine production considering the correct discount factor of each mining block, resulting in the final pit. Each block is analyzed individually in order to define the best target period. This methodology presents an improvement of the classical methodology derived from Lerchs-Grossmann's initial proposition improved by Whittle. This paper presents the differences between these methodologies, specially focused on the algorithms' avidity. Avidity is classically defined by the voracious search algorithms, whereupon some of the most famous greedy algorithms are Branch and Bound, Brutal Force and Randomized. Strategies based on heuristics can accentuate the voracity of the optimizer system. The applied algorithm use simulated annealing combined with Tabu Search. The most avid algorithm can select the most profitable blocks in early periods, leading to higher present value in the first periods of mine operation. The application of discount factors to blocks on the Lerchs-Grossmann's final pit has an accentuated effect with time, and this effect may make blocks scheduled for the end of the mine life unfeasible, representing a trend to a decrease in reported reserves.

  15. Cyclophilin A interacts with diverse lentiviral capsids

    Directory of Open Access Journals (Sweden)

    Emerman Michael

    2006-10-01

    Full Text Available Abstract Background The capsid (CA protein of HIV-1 binds with high affinity to the host protein cyclophilin A (CypA. This binding positively affects some early stage of the viral life-cycle because prevention of binding either by drugs that occupy that active site of cyclophilin A, by mutation in HIV-1 CA, or RNAi that knocks down intracellular CypA level diminishes viral infectivity. The closely related lentivirus, SIVcpz also binds CypA, but it was thought that this interaction was limited to the HIV-1/SIVcpz lineage because other retroviruses failed to interact with CypA in a yeast two-hybrid assay. Results We find that diverse lentiviruses, FIV and SIVagmTAN also bind to CypA. Mutagenesis of FIV CA showed that an amino acid that is in a homologous position to the proline at amino acid 90 of HIV-1 CA is essential for FIV interactions with CypA. Conclusion These results demonstrate that CypA binding to lentiviruses is more widespread than previously thought and suggest that this interaction is evolutionarily important for lentiviral infection.

  16. Nanobodies targeting norovirus capsid reveal functional epitopes and potential mechanisms of neutralization.

    Directory of Open Access Journals (Sweden)

    Anna D Koromyslova

    2017-11-01

    Full Text Available Norovirus is the leading cause of gastroenteritis worldwide. Despite recent developments in norovirus propagation in cell culture, these viruses are still challenging to grow routinely. Moreover, little is known on how norovirus infects the host cells, except that histo-blood group antigens (HBGAs are important binding factors for infection and cell entry. Antibodies that bind at the HBGA pocket and block attachment to HBGAs are believed to neutralize the virus. However, additional neutralization epitopes elsewhere on the capsid likely exist and impeding the intrinsic structural dynamics of the capsid could be equally important. In the current study, we investigated a panel of Nanobodies in order to probe functional epitopes that could trigger capsid rearrangement and/ or interfere with HBGA binding interactions. The precise binding sites of six Nanobodies (Nano-4, Nano-14, Nano-26, Nano-27, Nano-32, and Nano-42 were identified using X-ray crystallography. We showed that these Nanobodies bound on the top, side, and bottom of the norovirus protruding domain. The impact of Nanobody binding on norovirus capsid morphology was analyzed using electron microscopy and dynamic light scattering. We discovered that distinct Nanobody epitopes were associated with varied changes in particle structural integrity and assembly. Interestingly, certain Nanobody-induced capsid morphological changes lead to the capsid protein degradation and viral RNA exposure. Moreover, Nanobodies employed multiple inhibition mechanisms to prevent norovirus attachment to HBGAs, which included steric obstruction (Nano-14, allosteric interference (Nano-32, and violation of normal capsid morphology (Nano-26 and Nano-85. Finally, we showed that two Nanobodies (Nano-26 and Nano-85 not only compromised capsid integrity and inhibited VLPs attachment to HBGAs, but also recognized a broad panel of norovirus genotypes with high affinities. Consequently, Nano-26 and Nano-85 have a great

  17. Nanobodies targeting norovirus capsid reveal functional epitopes and potential mechanisms of neutralization

    Science.gov (United States)

    2017-01-01

    Norovirus is the leading cause of gastroenteritis worldwide. Despite recent developments in norovirus propagation in cell culture, these viruses are still challenging to grow routinely. Moreover, little is known on how norovirus infects the host cells, except that histo-blood group antigens (HBGAs) are important binding factors for infection and cell entry. Antibodies that bind at the HBGA pocket and block attachment to HBGAs are believed to neutralize the virus. However, additional neutralization epitopes elsewhere on the capsid likely exist and impeding the intrinsic structural dynamics of the capsid could be equally important. In the current study, we investigated a panel of Nanobodies in order to probe functional epitopes that could trigger capsid rearrangement and/ or interfere with HBGA binding interactions. The precise binding sites of six Nanobodies (Nano-4, Nano-14, Nano-26, Nano-27, Nano-32, and Nano-42) were identified using X-ray crystallography. We showed that these Nanobodies bound on the top, side, and bottom of the norovirus protruding domain. The impact of Nanobody binding on norovirus capsid morphology was analyzed using electron microscopy and dynamic light scattering. We discovered that distinct Nanobody epitopes were associated with varied changes in particle structural integrity and assembly. Interestingly, certain Nanobody-induced capsid morphological changes lead to the capsid protein degradation and viral RNA exposure. Moreover, Nanobodies employed multiple inhibition mechanisms to prevent norovirus attachment to HBGAs, which included steric obstruction (Nano-14), allosteric interference (Nano-32), and violation of normal capsid morphology (Nano-26 and Nano-85). Finally, we showed that two Nanobodies (Nano-26 and Nano-85) not only compromised capsid integrity and inhibited VLPs attachment to HBGAs, but also recognized a broad panel of norovirus genotypes with high affinities. Consequently, Nano-26 and Nano-85 have a great potential to

  18. L2, the minor capsid protein of papillomavirus

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Joshua W. [Department of Pathology, The Johns Hopkins University, Baltimore, MD 21287 (United States); Roden, Richard B.S., E-mail: roden@jhmi.edu [Department of Pathology, The Johns Hopkins University, Baltimore, MD 21287 (United States); Department of Oncology, The Johns Hopkins University, Baltimore, MD 21287 (United States); Department of Gynecology and Obstetrics, The Johns Hopkins University, Baltimore, MD 21287 (United States)

    2013-10-15

    The capsid protein L2 plays major roles in both papillomavirus assembly and the infectious process. While L1 forms the majority of the capsid and can self-assemble into empty virus-like particles (VLPs), L2 is a minor capsid component and lacks the capacity to form VLPs. However, L2 co-assembles with L1 into VLPs, enhancing their assembly. L2 also facilitates encapsidation of the ∼8 kbp circular and nucleosome-bound viral genome during assembly of the non-enveloped T=7d virions in the nucleus of terminally differentiated epithelial cells, although, like L1, L2 is not detectably expressed in infected basal cells. With respect to infection, L2 is not required for particles to bind to and enter cells. However L2 must be cleaved by furin for endosome escape. L2 then travels with the viral genome to the nucleus, wherein it accumulates at ND-10 domains. Here, we provide an overview of the biology of L2. - Highlights: • L2 is the minor antigen of the non-enveloped T=7d icosahedral Papillomavirus capsid. • L2 is a nuclear protein that can traffic to ND-10 and facilitate genome encapsidation. • L2 is critical for infection and must be cleaved by furin. • L2 is a broadly protective vaccine antigen recognized by neutralizing antibodies.

  19. L2, the minor capsid protein of papillomavirus

    International Nuclear Information System (INIS)

    Wang, Joshua W.; Roden, Richard B.S.

    2013-01-01

    The capsid protein L2 plays major roles in both papillomavirus assembly and the infectious process. While L1 forms the majority of the capsid and can self-assemble into empty virus-like particles (VLPs), L2 is a minor capsid component and lacks the capacity to form VLPs. However, L2 co-assembles with L1 into VLPs, enhancing their assembly. L2 also facilitates encapsidation of the ∼8 kbp circular and nucleosome-bound viral genome during assembly of the non-enveloped T=7d virions in the nucleus of terminally differentiated epithelial cells, although, like L1, L2 is not detectably expressed in infected basal cells. With respect to infection, L2 is not required for particles to bind to and enter cells. However L2 must be cleaved by furin for endosome escape. L2 then travels with the viral genome to the nucleus, wherein it accumulates at ND-10 domains. Here, we provide an overview of the biology of L2. - Highlights: • L2 is the minor antigen of the non-enveloped T=7d icosahedral Papillomavirus capsid. • L2 is a nuclear protein that can traffic to ND-10 and facilitate genome encapsidation. • L2 is critical for infection and must be cleaved by furin. • L2 is a broadly protective vaccine antigen recognized by neutralizing antibodies

  20. Structure of the capsid of Kilham rat virus from small-angle neutron scattering

    International Nuclear Information System (INIS)

    Wobbe, C.R.; Mitra, S.; Ramakrishnan, V.

    1984-01-01

    The structure of empty capsids of Kilham rat virus, an autonomous parvovirus with icosahedral symmetry, was investigated by small-angle neutron scattering. From the forward scatter, the molecular weight was determined to be 4.0 x 10(6), and from the Guinier region, the radius of gyration was found to be 105 A in D2O and 104 A in H 2 O. On the basis of the capsid molecular weight and the molecular weights and relative abundances of the capsid proteins, the authors propose that the capsid has a triangulation number of 1. Extended scattering curves and mathematical modeling revealed that the capsid consists of two shells of protein, the inner shell extending from 58 to 91 A in D2O and from 50 to 91 A in H 2 O and containing 11% of the capsid scattering mass, and the outer shell extending to 121 A in H 2 O and D2O. The inner shell appears to have a higher content of basic amino acids than the outer shell, based on its lower scattering density in D2O than in H 2 O. The authors propose that all three capsid proteins contribute to the inner shell and that this basic region serves DNA binding and partial charge neutralization functions

  1. Hepatitis B virus core protein allosteric modulators can distort and disrupt intact capsids.

    Science.gov (United States)

    Schlicksup, Christopher John; Wang, Joseph Che-Yen; Francis, Samson; Venkatakrishnan, Balasubramanian; Turner, William W; VanNieuwenhze, Michael; Zlotnick, Adam

    2018-01-29

    Defining mechanisms of direct-acting antivirals facilitates drug development and our understanding of virus function. Heteroaryldihydropyrimidines (HAPs) inappropriately activate assembly of hepatitis B virus (HBV) core protein (Cp), suppressing formation of virions. We examined a fluorophore-labeled HAP, HAP-TAMRA. HAP-TAMRA induced Cp assembly and also bound pre-assembled capsids. Kinetic and spectroscopic studies imply that HAP-binding sites are usually not available but are bound cooperatively. Using cryo-EM, we observed that HAP-TAMRA asymmetrically deformed capsids, creating a heterogeneous array of sharp angles, flat regions, and outright breaks. To achieve high resolution reconstruction (HAP-TAMRA caused quasi-sixfold vertices to become flatter and fivefold more angular. This transition led to asymmetric faceting. That a disordered crosslink could rescue symmetry implies that capsids have tensegrity properties. Capsid distortion and disruption is a new mechanism by which molecules like the HAPs can block HBV infection. © 2017, Schlicksup et al.

  2. Hepatitis B virus core protein allosteric modulators can distort and disrupt intact capsids

    Science.gov (United States)

    Schlicksup, Christopher John; Wang, Joseph Che-Yen; Francis, Samson; Venkatakrishnan, Balasubramanian; Turner, William W; VanNieuwenhze, Michael

    2018-01-01

    Defining mechanisms of direct-acting antivirals facilitates drug development and our understanding of virus function. Heteroaryldihydropyrimidines (HAPs) inappropriately activate assembly of hepatitis B virus (HBV) core protein (Cp), suppressing formation of virions. We examined a fluorophore-labeled HAP, HAP-TAMRA. HAP-TAMRA induced Cp assembly and also bound pre-assembled capsids. Kinetic and spectroscopic studies imply that HAP-binding sites are usually not available but are bound cooperatively. Using cryo-EM, we observed that HAP-TAMRA asymmetrically deformed capsids, creating a heterogeneous array of sharp angles, flat regions, and outright breaks. To achieve high resolution reconstruction (particle symmetry. We deduced that HAP-TAMRA caused quasi-sixfold vertices to become flatter and fivefold more angular. This transition led to asymmetric faceting. That a disordered crosslink could rescue symmetry implies that capsids have tensegrity properties. Capsid distortion and disruption is a new mechanism by which molecules like the HAPs can block HBV infection. PMID:29377794

  3. Ebselen, a Small-Molecule Capsid Inhibitor of HIV-1 Replication.

    Science.gov (United States)

    Thenin-Houssier, Suzie; de Vera, Ian Mitchelle S; Pedro-Rosa, Laura; Brady, Angela; Richard, Audrey; Konnick, Briana; Opp, Silvana; Buffone, Cindy; Fuhrmann, Jakob; Kota, Smitha; Billack, Blase; Pietka-Ottlik, Magdalena; Tellinghuisen, Timothy; Choe, Hyeryun; Spicer, Timothy; Scampavia, Louis; Diaz-Griffero, Felipe; Kojetin, Douglas J; Valente, Susana T

    2016-04-01

    The human immunodeficiency virus type 1 (HIV-1) capsid plays crucial roles in HIV-1 replication and thus represents an excellent drug target. We developed a high-throughput screening method based on a time-resolved fluorescence resonance energy transfer (HTS-TR-FRET) assay, using the C-terminal domain (CTD) of HIV-1 capsid to identify inhibitors of capsid dimerization. This assay was used to screen a library of pharmacologically active compounds, composed of 1,280in vivo-active drugs, and identified ebselen [2-phenyl-1,2-benzisoselenazol-3(2H)-one], an organoselenium compound, as an inhibitor of HIV-1 capsid CTD dimerization. Nuclear magnetic resonance (NMR) spectroscopic analysis confirmed the direct interaction of ebselen with the HIV-1 capsid CTD and dimer dissociation when ebselen is in 2-fold molar excess. Electrospray ionization mass spectrometry revealed that ebselen covalently binds the HIV-1 capsid CTD, likely via a selenylsulfide linkage with Cys198 and Cys218. This compound presents anti-HIV activity in single and multiple rounds of infection in permissive cell lines as well as in primary peripheral blood mononuclear cells. Ebselen inhibits early viral postentry events of the HIV-1 life cycle by impairing the incoming capsid uncoating process. This compound also blocks infection of other retroviruses, such as Moloney murine leukemia virus and simian immunodeficiency virus, but displays no inhibitory activity against hepatitis C and influenza viruses. This study reports the use of TR-FRET screening to successfully identify a novel capsid inhibitor, ebselen, validating HIV-1 capsid as a promising target for drug development. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

  4. Determining the Epitope Dominance on the Capsid of a Serotype SAT2 Foot-and-Mouth Disease Virus by Mutational Analyses

    Science.gov (United States)

    Opperman, Pamela A.; Rotherham, Lia S.; Esterhuysen, Jan; Charleston, Bryan; Juleff, Nicholas; Capozzo, Alejandra V.; Theron, Jacques

    2014-01-01

    ABSTRACT Monoclonal-antibody (MAb)-resistant mutants were used to map antigenic sites on foot-and-mouth disease virus (FMDV), which resulted in the identification of neutralizing epitopes in the flexible βG-βH loop in VP1. For FMDV SAT2 viruses, studies have shown that at least two antigenic sites exist. By use of an infectious SAT2 cDNA clone, 10 structurally exposed and highly variable loops were identified as putative antigenic sites on the VP1, VP2, and VP3 capsid proteins of SAT2/Zimbabwe (ZIM)/7/83 (topotype II) and replaced with the corresponding regions of SAT2/Kruger National Park (KNP)/19/89 (topotype I). Virus neutralization assays using convalescent-phase antisera raised against the parental virus, SAT2/ZIM/7/83, indicated that the mutant virus containing the TQQS-to-ETPV mutation in the N-terminal part of the βG-βH loop of VP1 showed not only a significant increase in the neutralization titer but also an increase in the index of avidity to the convalescent-phase antisera. Furthermore, antigenic profiling of the epitope-replaced and parental viruses with nonneutralizing SAT2-specific MAbs led to the identification of two nonneutralizing antigenic regions. Both regions were mapped to incorporate residues 71 to 72 of VP2 as the major contact point. The binding footprint of one of the antigenic regions encompasses residues 71 to 72 and 133 to 134 of VP2 and residues 48 to 50 of VP1, and the second antigenic region encompasses residues 71 to 72 and 133 to 134 of VP2 and residues 84 to 86 and 109 to 11 of VP1. This is the first time that antigenic regions encompassing residues 71 to 72 of VP2 have been identified on the capsid of a SAT2 FMDV. IMPORTANCE Monoclonal-antibody-resistant mutants have traditionally been used to map antigenic sites on foot-and-mouth disease virus (FMDV). However, for SAT2-type viruses, which are responsible for most of the FMD outbreaks in Africa and are the most varied of all seven serotypes, only two antigenic sites have been

  5. Structure of a Human Astrovirus Capsid-Antibody Complex and Mechanistic Insights into Virus Neutralization

    Energy Technology Data Exchange (ETDEWEB)

    Bogdanoff, Walter A.; Campos, Jocelyn; Perez, Edmundo I.; Yin, Lu; Alexander, David L.; DuBois, Rebecca M. (UCSC)

    2016-11-02

    ABSTRACT

    Human astroviruses (HAstVs) are a leading cause of viral diarrhea in young children, the immunocompromised, and the elderly. There are no vaccines or antiviral therapies against HAstV disease. Several lines of evidence point to the presence of protective antibodies in healthy adults as a mechanism governing protection against reinfection by HAstV. However, development of anti-HAstV therapies is hampered by the gap in knowledge of protective antibody epitopes on the HAstV capsid surface. Here, we report the structure of the HAstV capsid spike domain bound to the neutralizing monoclonal antibody PL-2. The antibody uses all six complementarity-determining regions to bind to a quaternary epitope on each side of the dimeric capsid spike. We provide evidence that the HAstV capsid spike is a receptor-binding domain and that the antibody neutralizes HAstV by blocking virus attachment to cells. We identify patches of conserved amino acids that overlap the antibody epitope and may comprise a receptor-binding site. Our studies provide a foundation for the development of therapies to prevent and treat HAstV diarrheal disease.

    IMPORTANCEHuman astroviruses (HAstVs) infect nearly every person in the world during childhood and cause diarrhea, vomiting, and fever. Despite the prevalence of this virus, little is known about how antibodies in healthy adults protect them against reinfection. Here, we determined the crystal structure of a complex of the HAstV capsid protein and a virus-neutralizing antibody. We show that the antibody binds to the outermost spike domain of the capsid, and we provide evidence that the antibody blocks virus attachment to human cells. Importantly, our findings suggest that a subunit-based vaccine focusing the immune system on the HAstV capsid spike domain could be effective in protecting children against HAstV disease.

  6. High avidity antibodies to full-length VAR2CSA correlate with absence of placental malaria.

    Directory of Open Access Journals (Sweden)

    Yeung Lo Tutterrow

    Full Text Available VAR2CSA mediates sequestration of Plasmodium falciparum-infected erythrocytes in the placenta, increasing the risk of poor pregnancy outcomes. Naturally acquired antibodies (Ab to placental parasites at delivery have been associated with improved pregnancy outcomes, but Ab levels and how early in pregnancy Ab must be present in order to eliminate placental parasites before delivery remains unknown. Antibodies to individual Duffy-binding like domains of VAR2CSA have been studied, but the domains lack many of the conformational epitopes present in full-length VAR2CSA (FV2. Thus, the purpose of this study was to describe the acquisition of Ab to FV2 in women residing in high and low transmission areas and determine how Ab levels during pregnancy correlate with clearance of placental parasites. Plasma samples collected monthly throughout pregnancy from pregnant women living in high and low transmission areas in Cameroon were evaluated for Ab to FV2 and the proportion of high avidity Ab (i.e., Ab that remain bound in the presence of 3M NH(4SCN was assessed. Ab levels and proportion of high avidity Ab were compared between women with placental malaria (PM(+ and those without (PM(- at delivery. Results showed that PM(- women had significantly higher Ab levels (p = 0.0047 and proportion of high avidity Ab (p = 0.0009 than PM(+ women throughout pregnancy. Specifically, women with moderate to high Ab levels (>5,000 MFI and those with ≥ 35% high avidity Ab at 5-6 months were found to have 2.3 (95% CI, 1.0-4.9 and 7.6-fold (p = 0.0013, 95% CI: 1.2-50.0 reduced risk of placental malaria, respectively. These data show that high levels of Ab to FV2, particularly those with high avidity for FV2, produced by mid-pregnancy are important in clearing parasites from the placenta. Both high Ab levels and proportion of high avidity Ab to FV2 may serve as correlates of protection for assessing immunity against placental malaria.

  7. Structural Characterization of H-1 Parvovirus: Comparison of Infectious Virions to Empty Capsids

    Science.gov (United States)

    Halder, Sujata; Nam, Hyun-Joo; Govindasamy, Lakshmanan; Vogel, Michèle; Dinsart, Christiane; Salomé, Nathalie; McKenna, Robert

    2013-01-01

    The structure of single-stranded DNA (ssDNA) packaging H-1 parvovirus (H-1PV), which is being developed as an antitumor gene delivery vector, has been determined for wild-type (wt) virions and noninfectious (empty) capsids to 2.7- and 3.2-Å resolution, respectively, using X-ray crystallography. The capsid viral protein (VP) structure consists of an α-helix and an eight-stranded anti-parallel β-barrel with large loop regions between the strands. The β-barrel and loops form the capsid core and surface, respectively. In the wt structure, 600 nucleotides are ordered in an interior DNA binding pocket of the capsid. This accounts for ∼12% of the H-1PV genome. The wt structure is identical to the empty capsid structure, except for side chain conformation variations at the nucleotide binding pocket. Comparison of the H-1PV nucleotides to those observed in canine parvovirus and minute virus of mice, two members of the genus Parvovirus, showed both similarity in structure and analogous interactions. This observation suggests a functional role, such as in capsid stability and/or ssDNA genome recognition for encapsulation. The VP structure differs from those of other parvoviruses in surface loop regions that control receptor binding, tissue tropism, pathogenicity, and antibody recognition, including VP sequences reported to determine tumor cell tropism for oncotropic rodent parvoviruses. These structures of H-1PV provide insight into structural features that dictate capsid stabilization following genome packaging and three-dimensional information applicable for rational design of tumor-targeted recombinant gene delivery vectors. PMID:23449783

  8. Inflammatory pseudotumor: A gallium-avid mobile mesenteric mass

    International Nuclear Information System (INIS)

    Auringer, S.T.; Scott, M.D.; Sumner, T.E.

    1991-01-01

    An 8-yr-old boy with a 1-mo history of culture-negative fever and anemia underwent gallium, ultrasound, and computed tomography studies as part of the evaluation of a fever of unknown origin. These studies revealed a mobile gallium-avid solid abdominal mass subsequently proven to be an inflammatory pseudotumor of the mesentery, a rare benign mass. This report documents the gallium-avid nature of this rare lesion and discusses associated characteristic clinical, pathologic, and radiographic features

  9. Ultrasound stratification of the FDG-avid thyroid nodule

    International Nuclear Information System (INIS)

    Beech, P.; Lavender, I.; Jong, I.; Soo, G.; Ramdave, S.; Chong, A.; Nandurkar, D.

    2016-01-01

    Aim: To determine whether the malignancy risk in an 2-["1"8F]-fluoro-2-deoxy-D-glucose (FDG)-avid thyroid nodule can be stratified according to the presence or absence of suspicious ultrasound features and thereby identify which nodules require further cytological assessment. Materials and methods: A retrospective review of FDG-positron-emission tomography (PET) combined with computed tomography (CT) studies with FDG-avid thyroid nodules (defined as FDG uptake greater than blood pool) that were further assessed with ultrasound and fine-needle aspiration cytology or surgery was performed. FDG-avid thyroid nodules were classified as having either suspicious ultrasound features (marked hypo-echogenicity, irregular margins, microcalcifications, marked hypervascularity, or nodules that were taller than they were wide) or no suspicious ultrasound features and these findings were correlated with the subsequent cytological results. Results: Forty-eight FDG-avid thyroid nodules were assessed. On cytological assessment five nodules were malignant (10.4%), nine were indeterminate (18.75%), and 34 were benign (70.8%). On ultrasound, 24 (50%) had no suspicious features and 24 (50%) had one or more suspicious features. Of the nodules with no suspicious features, 22 (91.6%) were benign, two (8.3%) were indeterminate, and none were malignant. Of the nodules with suspicious features, five (20.8%) were malignant, seven (29.1%) were indeterminate, and 12 (50%) were benign. The absence of suspicious ultrasound features demonstrated a strong association with benign cytology (p=0.009). Out of the suspicious sonographic features, marked hypoechoic appearance (p=0.02), irregular margins (p=0.009), and taller than wide morphology (p=0.04) were statistically most significantly associated with malignancy. Conclusion: The rate of malignancy in FDG-avid thyroid nodules is low in the absence of specific suspicious ultrasound features. The SUV values are non-discriminatory to differentiate

  10. AAV8 capsid variable regions at the two-fold symmetry axis contribute to high liver transduction by mediating nuclear entry and capsid uncoating

    International Nuclear Information System (INIS)

    Tenney, Rebeca M.; Bell, Christie L.; Wilson, James M.

    2014-01-01

    Adeno-associated virus serotype 8 (AAV8) is a promising vector for liver-directed gene therapy. Although efficient uncoating of viral capsids has been implicated in AAV8's robust liver transduction, much about the biology of AAV8 hepatotropism remains unclear. Our study investigated the structural basis of AAV8 liver transduction efficiency by constructing chimeric vector capsids containing sequences derived from AAV8 and AAV2 – a highly homologous yet poorly hepatotropic serotype. Engineered vectors containing capsid variable regions (VR) VII and IX from AAV8 in an AAV2 backbone mediated near AAV8-like transduction in mouse liver, with higher numbers of chimeric genomes detected in whole liver cells and isolated nuclei. Interestingly, chimeric capsids within liver nuclei also uncoated similarly to AAV8 by 6 weeks after administration, in contrast with AAV2, of which a significantly smaller proportion were uncoated. This study links specific AAV capsid regions to the transduction ability of a clinically relevant AAV serotype. - Highlights: • We construct chimeric vectors to identify determinants of AAV8 liver transduction. • An AAV2-based vector with 17 AAV8 residues exhibited high liver transduction in mice. • This vector also surpassed AAV2 in cell entry, nuclear entry and onset of expression. • Most chimeric vector particles were uncoated at 6 weeks, like AAV8 and unlike AAV2. • Chimera retained heparin binding and was antigenically distinct from AAV2 and AAV8

  11. AAV8 capsid variable regions at the two-fold symmetry axis contribute to high liver transduction by mediating nuclear entry and capsid uncoating

    Energy Technology Data Exchange (ETDEWEB)

    Tenney, Rebeca M.; Bell, Christie L.; Wilson, James M., E-mail: wilsonjm@mail.med.upenn.edu

    2014-04-15

    Adeno-associated virus serotype 8 (AAV8) is a promising vector for liver-directed gene therapy. Although efficient uncoating of viral capsids has been implicated in AAV8's robust liver transduction, much about the biology of AAV8 hepatotropism remains unclear. Our study investigated the structural basis of AAV8 liver transduction efficiency by constructing chimeric vector capsids containing sequences derived from AAV8 and AAV2 – a highly homologous yet poorly hepatotropic serotype. Engineered vectors containing capsid variable regions (VR) VII and IX from AAV8 in an AAV2 backbone mediated near AAV8-like transduction in mouse liver, with higher numbers of chimeric genomes detected in whole liver cells and isolated nuclei. Interestingly, chimeric capsids within liver nuclei also uncoated similarly to AAV8 by 6 weeks after administration, in contrast with AAV2, of which a significantly smaller proportion were uncoated. This study links specific AAV capsid regions to the transduction ability of a clinically relevant AAV serotype. - Highlights: • We construct chimeric vectors to identify determinants of AAV8 liver transduction. • An AAV2-based vector with 17 AAV8 residues exhibited high liver transduction in mice. • This vector also surpassed AAV2 in cell entry, nuclear entry and onset of expression. • Most chimeric vector particles were uncoated at 6 weeks, like AAV8 and unlike AAV2. • Chimera retained heparin binding and was antigenically distinct from AAV2 and AAV8.

  12. Labeling and localization of the herpes simplex virus capsid protein UL25 and its interaction with the two triplexes closest to the penton

    Science.gov (United States)

    Conway, James F.; Cockrell, Shelley K.; Copeland, Anna Maria; Newcomb, William W.; Brown, Jay C.; Homa, Fred L.

    2010-01-01

    The herpes simplex virus type 1 (HSV-1) UL25 protein is one of seven viral proteins that are required for DNA cleavage and packaging. Together with UL17, UL25 forms part of an elongated molecule referred to as the C-capsid-specific component or CCSC. Five copies of the CCSC are located at each of the capsid vertices on DNA-containing capsids. To study the conformation of UL25 as it is folded on the capsid surface, we identified the sequence recognized by a UL25-specific monoclonal antibody and localized the epitope on the capsid surface by immunogold electron microscopy. The epitope mapped to amino acids 99-111 adjacent to the region of the protein (amino acids 1-50) that is required for capsid binding. In addition, cryo-EM reconstructions of C-capsids in which the green fluorescent protein (GFP) was fused within the N-terminus of UL25 localized the point of contact between UL25 and GFP. The result confirmed the modeled location of the UL25 protein in the CCSC density as the region that is distal to the penton with the N-terminus of UL25 making contact with the triplex one removed from the penton. Immunofluorescence experiments at early times during infection demonstrated that UL25-GFP was present on capsids located within the cytoplasm and adjacent to the nucleus. These results support the view that UL25 is present on incoming capsids with the capsid binding domain of UL25 located on the surface of the mature DNA-containing capsid. PMID:20109467

  13. Polarized DNA Ejection from the Herpesvirus Capsid

    Science.gov (United States)

    Newcomb, William W.; Cockrell, Shelley K.; Homa, Fred L.; Brown, Jay C.

    2009-01-01

    Ejection of DNA from the capsid is an early step in infection by all herpesviruses. Ejection or DNA uncoating occurs after a parental capsid has entered the host cell cytoplasm, migrated to the nucleus and bound to a nuclear pore. DNA exits the capsid through the portal vertex and proceeds by way of the nuclear pore complex into the nucleoplasm where it is transcribed and replicated. Here we describe use of an in vitro uncoating system to determine which genome end exits first from the herpes simplex virus (HSV-1) capsid. Purified DNA-containing capsids were bound to a solid surface and warmed under conditions in which some, but not all, of the DNA was ejected. Restriction endonuclease digestion was then used to identify the genomic origin of the ejected DNA. The results support the view that the S segment end exits the capsid first. Preferential release at the S end demonstrates that herpesvirus DNA uncoating conforms to the paradigm in dsDNA bacteriophage where the last end packaged is the first to be ejected. Release of HSV-1 DNA beginning at the S end causes the first gene to enter the host cell nucleus to be α4, a transcription factor required for expression of early genes. PMID:19631662

  14. Importin α1 is required for nuclear import of herpes simplex virus proteins and capsid assembly in fibroblasts and neurons

    Science.gov (United States)

    Anderson, Fenja; Rother, Franziska; Rudolph, Kathrin; Prank, Ute; Binz, Anne; Hügel, Stefanie; Hartmann, Enno; Bader, Michael; Bauerfeind, Rudolf; Sodeik, Beate

    2018-01-01

    Herpesviruses are large DNA viruses which depend on many nuclear functions, and therefore on host transport factors to ensure specific nuclear import of viral and host components. While some import cargoes bind directly to certain transport factors, most recruit importin β1 via importin α. We identified importin α1 in a small targeted siRNA screen to be important for herpes simplex virus (HSV-1) gene expression. Production of infectious virions was delayed in the absence of importin α1, but not in cells lacking importin α3 or importin α4. While nuclear targeting of the incoming capsids, of the HSV-1 transcription activator VP16, and of the viral genomes were not affected, the nuclear import of the HSV-1 proteins ICP4 and ICP0, required for efficient viral transcription, and of ICP8 and pUL42, necessary for DNA replication, were reduced. Furthermore, quantitative electron microscopy showed that fibroblasts lacking importin α1 contained overall fewer nuclear capsids, but an increased proportion of mature nuclear capsids indicating that capsid formation and capsid egress into the cytoplasm were impaired. In neurons, importin α1 was also not required for nuclear targeting of incoming capsids, but for nuclear import of ICP4 and for the formation of nuclear capsid assembly compartments. Our data suggest that importin α1 is specifically required for the nuclear localization of several important HSV1 proteins, capsid assembly, and capsid egress into the cytoplasm, and may become rate limiting in situ upon infection at low multiplicity or in terminally differentiated cells such as neurons. PMID:29304174

  15. The Role of Leaders in AVID Schools and the Impact on Student Achievement

    OpenAIRE

    Noone, Colleen Callahan

    2010-01-01

    The Advancement Via Individual Determination (AVID) program is designed to provide resources and strategies to enable underrepresented minority students to attend four-year colleges. Research indicates that the success of the students enrolled in AVID depends on the strict adherence to the AVID components. The purpose of this study was to determine whether the principal and administratorâ s understanding of, commitment to and involvement in AVID accounted for the difference in student achi...

  16. The tyrosine B10 hydroxyl is crucial for oxygen avidity of Ascaris hemoglobin.

    Science.gov (United States)

    Kloek, A P; Yang, J; Mathews, F S; Frieden, C; Goldberg, D E

    1994-01-28

    The parasitic nematode Ascaris suum has a gene encoding a two-domain hemoglobin with remarkable oxygen avidity. The strong interaction with oxygen is a consequence of a particularly slow oxygen off-rate. The single polypeptide chain consists of two domains, each of which can be expressed separately in Escherichia coli as a globin-like protein exhibiting oxygen binding characteristics comparable with the native molecule. Site-directed mutagenesis was performed on the gene segment encoding domain one. The E7 position, involved in forming a hydrogen bond with the liganded oxygen in vertebrate globins, is a glutamine in both Ascaris domains. Conversion of this residue to leucine or alanine produced a hemoglobin variant with an oxygen off-rate 5- or 60-fold faster than that of unaltered domain one. Replacement of the tyrosine B10 with either phenylalanine or leucine (as found in vertebrate globins) yielded hemoglobin mutants with oxygen off-rates 280- or 570-fold faster, approaching rates found with vertebrate myoglobins. The data suggest that the distal glutamine hydrogen bonds with the liganded oxygen and that the tyrosine B10 hydroxyl contributes an additional hydrogen bond that appears substantially responsible for the extreme oxygen avidity of Ascaris hemoglobin.

  17. Characterization of Tumor-Avid Antibody Fragments Genetically Engineered for Mono-Specific Radionuclide Chelation

    International Nuclear Information System (INIS)

    Quinn, T.P.

    2003-01-01

    The successful clinical application of targeted-radiopharmaceuticals depends on the development of molecules that optimize tumor specific radionuclide deposition and minimize non-specific organ irradiation. To this end, this proposal outlines a research effort to identify and evaluate novel antibodies and antibody fragments that bind breast tumors. The tumor-avid antibodies will be investigated for as imaging and therapeutic agents and to gain a better understanding of the pharmacokinetics and metabolism of radiolabeled tumor-avid antibody fragments through the use of site-specifically labeled molecules. Antibodies or antibody fragments, that bind breast carcinoma carbohydrate antigens, will be obtained from hybridoma or bacteriophage library screening. More specifically, antibody fragments that bind the carcinoma-associated Thomsen-Friedenreich (T) antigen will be radiolabeled with 99m Tc and 188 Re at a natural amino acid chelation site and will be investigated in vivo for their abilities to target human breast tumors. In addition, site-specific radiolabeled antibody fragments will be biosynthesized using misacylated suppressor tRNAs. Homogeneously radiolabeled populations of antibody fragments will be used to investigate the effects of radionuclide location and chelation chemistries on their biodistribution and metabolism. It is hypothesized that site-specifically radiolabeled antibody fragments will possess enhanced tumor imaging and therapeutic properties due to optimal label location and conjugation chemistries. New insights into the factors that govern antibody metabolism in vivo are also expected from this work. Results from these studies should enhance our ability to design and synthesize radiolabeled antibody fragments that have improved pharmacokinetic properties. The studies in this proposal involve basic research into the development of antibody-based radiopharmaceuticals, with the ultimate goal of application in humans. This type of basic nuclear

  18. Stochastic modeling of virus capsid assembly pathways

    Science.gov (United States)

    Schwartz, Russell

    2009-03-01

    Virus capsids have become a key model system for understanding self-assembly due to their high complexity, robust and efficient assembly processes, and experimental tractability. Our ability to directly examine and manipulate capsid assembly kinetics in detail nonetheless remains limited, creating a need for computer models that can infer experimentally inaccessible features of the assembly process and explore the effects of hypothetical manipulations on assembly trajectories. We have developed novel algorithms for stochastic simulation of capsid assembly [1,2] that allow us to model capsid assembly over broad parameter spaces [3]. We apply these methods to study the nature of assembly pathway control in virus capsids as well as their sensitivity to assembly conditions and possible experimental interventions. [4pt] [1] F. Jamalyaria, R. Rohlfs, and R. Schwartz. J Comp Phys 204, 100 (2005). [0pt] [2] N. Misra and R. Schwartz. J Chem Phys 129, in press (2008). [0pt] [3] B. Sweeney, T. Zhang, and R. Schwartz. Biophys J 94, 772 (2008).

  19. AVID: An integrative framework for discovering functional relationships among proteins

    Directory of Open Access Journals (Sweden)

    Keating Amy E

    2005-06-01

    Full Text Available Abstract Background Determining the functions of uncharacterized proteins is one of the most pressing problems in the post-genomic era. Large scale protein-protein interaction assays, global mRNA expression analyses and systematic protein localization studies provide experimental information that can be used for this purpose. The data from such experiments contain many false positives and false negatives, but can be processed using computational methods to provide reliable information about protein-protein relationships and protein function. An outstanding and important goal is to predict detailed functional annotation for all uncharacterized proteins that is reliable enough to effectively guide experiments. Results We present AVID, a computational method that uses a multi-stage learning framework to integrate experimental results with sequence information, generating networks reflecting functional similarities among proteins. We illustrate use of the networks by making predictions of detailed Gene Ontology (GO annotations in three categories: molecular function, biological process, and cellular component. Applied to the yeast Saccharomyces cerevisiae, AVID provides 37,451 pair-wise functional linkages between 4,191 proteins. These relationships are ~65–78% accurate, as assessed by cross-validation testing. Assignments of highly detailed functional descriptors to proteins, based on the networks, are estimated to be ~67% accurate for GO categories describing molecular function and cellular component and ~52% accurate for terms describing biological process. The predictions cover 1,490 proteins with no previous annotation in GO and also assign more detailed functions to many proteins annotated only with less descriptive terms. Predictions made by AVID are largely distinct from those made by other methods. Out of 37,451 predicted pair-wise relationships, the greatest number shared in common with another method is 3,413. Conclusion AVID provides

  20. Antimicrobial peptide capsids of de novo design.

    Science.gov (United States)

    De Santis, Emiliana; Alkassem, Hasan; Lamarre, Baptiste; Faruqui, Nilofar; Bella, Angelo; Noble, James E; Micale, Nicola; Ray, Santanu; Burns, Jonathan R; Yon, Alexander R; Hoogenboom, Bart W; Ryadnov, Maxim G

    2017-12-22

    The spread of bacterial resistance to antibiotics poses the need for antimicrobial discovery. With traditional search paradigms being exhausted, approaches that are altogether different from antibiotics may offer promising and creative solutions. Here, we introduce a de novo peptide topology that-by emulating the virus architecture-assembles into discrete antimicrobial capsids. Using the combination of high-resolution and real-time imaging, we demonstrate that these artificial capsids assemble as 20-nm hollow shells that attack bacterial membranes and upon landing on phospholipid bilayers instantaneously (seconds) convert into rapidly expanding pores causing membrane lysis (minutes). The designed capsids show broad antimicrobial activities, thus executing one primary function-they destroy bacteria on contact.

  1. Serological IgG avidity test for ocular toxoplasmosis

    Directory of Open Access Journals (Sweden)

    Suresh S

    2012-01-01

    Full Text Available Subramaniam Suresh1, Saidin Nor-Masniwati1, Muhd Nor Nor-Idahriani1, Wan-Hitam Wan-Hazabbah1, Mohamed Zeehaida2, Embong Zunaina11Department of Ophthalmology, 2Department of Medical Microbiology and Parasitology, School of Medical Sciences, Universiti Sains Malaysia, Kelantan, MalaysiaBackground: The purpose of this study was to evaluate the immunoglobulin (Ig G avidity of serological toxoplasmosis testing in patients with ocular inflammation and to determine the clinical manifestations of ocular toxoplasmosis.Methods: A retrospective review of all patients presenting with ocular inflammation to the Hospital Universiti Sains Malaysia, Kelantan, Malaysia between 2005 and 2009 was undertaken. Visual acuity, clinical manifestations at presentation, toxoplasmosis antibody testing, and treatment records were analyzed.Results: A total of 130 patients with ocular inflammation were reviewed retrospectively. The patients had a mean age of 38.41 (standard deviation 19.24, range 6–83 years. Seventy-one patients (54.6% were found to be seropositive, of whom five (3.8% were both IgG and IgM positive (suggestive of recently acquired ocular toxoplasmosis while one (0.8% showed IgG avidity ≤40% (suggestive of recently acquired ocular toxoplasmosis and 65 patients (50.0% showed IgG avidity >40% (suggestive of reactivation of toxoplasmosis infection. Chorioretinal scarring as an ocular manifestation was significantly more common in patients with seropositive toxoplasmosis (P = 0.036. Eighteen patients (13.8% were diagnosed as having recent and/or active ocular toxoplasmosis based on clinical manifestations and serological testing.Conclusion: Ocular toxoplasmosis is a clinical diagnosis, but specific toxoplasmosis antibody testing helps to support the diagnosis and to differentiate between reactivation of infection and recently acquired ocular toxoplasmosis.Keywords: ocular toxoplasmosis, chorioretinal scar, toxoplasmosis antibody, IgG avidity test

  2. Radiolabeled Rhein as Small-Molecule Necrosis Avid Agents for Imaging of Necrotic Myocardium.

    Science.gov (United States)

    Luo, Qi; Jin, Qiaomei; Su, Chang; Zhang, Dongjian; Jiang, Cuihua; Fish, Anne Folta; Feng, Yuanbo; Ni, Yicheng; Zhang, Jian; Yin, Zhiqi

    2017-01-17

    A rapid and accurate identification of necrotic myocardium is of great importance for diagnosis, risk stratification, clinical decision-making, and prognosis evaluation of myocardial infarction. Here, we explored technetium-99m labeled rhein derivatives for rapid imaging of the necrotic myocardium. Three hydrazinonicotinic acid-linker-rhein (HYNIC-linker-rhein) derivatives were synthesized, and then, these synthetic compounds were labeled with technetium-99m using ethylenediaminediacetic acid (EDDA) and tricine as coligands [ 99m Tc(EDDA)-HYNIC-linker-rhein]. The necrosis avidity of the three 99m Tc-labeled rhein derivatives was tested in a mouse model of ethanol-induced muscular necrosis by gamma counting, histochemical staining, and autoradiography. A lead tracer for visualization of necrotic myocardium was assessed by single photon emission computed tomography/computed tomography (SPECT/CT) imaging in a rat model with reperfused myocardial infarction. The necrosis avidity mechanism of the tracer was explored by DNA binding studies in vitro and blocking experiments in vivo. Results showed that the uptake in necrotic muscles of the three 99m Tc-compounds was higher than that in viable muscles (P EDDA)-HYNIC-ethylenediamine-rhein [ 99m Tc(EDDA)-HYNIC-2C-rhein] displayed the best distribution profiles for imaging. The necrotic myocardium lesions were clearly visualized by SPECT/CT using 99m Tc(EDDA)-HYNIC-2C-rhein at 1 h after injection. The necrotic-to-viable myocardium and necrotic myocardium-to-blood uptake ratios of 99m Tc(EDDA)-HYNIC-2C-rhein were 4.79 and 3.02 at 1 h after injection. DNA binding studies suggested HYNIC-linker-rhein bound to DNA through intercalation. The uptake of 99m Tc(EDDA)-HYNIC-2C-rhein in necrotic muscle was significantly blocked by excessive unlabeled rhein, with 77.61% decline at 1 h after coinjection. These findings suggested 99m Tc(EDDA)-HYNIC-2C-rhein emerged as a "hot spot" imaging probe that has a potential for rapid imaging of

  3. Characterization of the mode of action of a potent dengue virus capsid inhibitor.

    Science.gov (United States)

    Scaturro, Pietro; Trist, Iuni Margaret Laura; Paul, David; Kumar, Anil; Acosta, Eliana G; Byrd, Chelsea M; Jordan, Robert; Brancale, Andrea; Bartenschlager, Ralf

    2014-10-01

    Dengue viruses (DV) represent a significant global health burden, with up to 400 million infections every year and around 500,000 infected individuals developing life-threatening disease. In spite of attempts to develop vaccine candidates and antiviral drugs, there is a lack of approved therapeutics for the treatment of DV infection. We have previously reported the identification of ST-148, a small-molecule inhibitor exhibiting broad and potent antiviral activity against DV in vitro and in vivo (C. M. Byrd et al., Antimicrob. Agents Chemother. 57:15-25, 2013, doi:10 .1128/AAC.01429-12). In the present study, we investigated the mode of action of this promising compound by using a combination of biochemical, virological, and imaging-based techniques. We confirmed that ST-148 targets the capsid protein and obtained evidence of bimodal antiviral activity affecting both assembly/release and entry of infectious DV particles. Importantly, by using a robust bioluminescence resonance energy transfer-based assay, we observed an ST-148-dependent increase of capsid self-interaction. These results were corroborated by molecular modeling studies that also revealed a plausible model for compound binding to capsid protein and inhibition by a distinct resistance mutation. These results suggest that ST-148-enhanced capsid protein self-interaction perturbs assembly and disassembly of DV nucleocapsids, probably by inducing structural rigidity. Thus, as previously reported for other enveloped viruses, stabilization of capsid protein structure is an attractive therapeutic concept that also is applicable to flaviviruses. Dengue viruses are arthropod-borne viruses representing a significant global health burden. They infect up to 400 million people and are endemic to subtropical and tropical areas of the world. Currently, there are neither vaccines nor approved therapeutics for the prophylaxis or treatment of DV infections, respectively. This study reports the characterization of the

  4. Virus Capsids as Targeted Nanoscale Delivery Vessels of Photoactive Compounds for Site-Specific Photodynamic Therapy

    Science.gov (United States)

    Cohen, Brian A.

    The research presented in this work details the use of a viral capsid as an addressable delivery vessel of photoactive compounds for use in photodynamic therapy. Photodynamic therapy is a treatment that involves the interaction of light with a photosensitizing molecule to create singlet oxygen, a reactive oxygen species. Overproduction of singlet oxygen in cells can cause oxidative damage leading to cytotoxicity and eventually cell death. Challenges with the current generation of FDA-approved photosensitizers for photodynamic therapy primarily stem from their lack of tissue specificity. This work describes the packaging of photoactive cationic porphyrins inside the MS2 bacteriophage capsid, followed by external modification of the capsid with cancer cell-targeting G-quadruplex DNA aptamers to generate a tumor-specific photosensitizing agent. First, a cationic porphyrin is loaded into the capsids via nucleotide-driven packaging, a process that involves charge interaction between the porphyrin and the RNA inside the capsid. Results show that over 250 porphyrin molecules associate with the RNA within each MS2 capsid. Removal of RNA from the capsid severely inhibits the packaging of the cationic porphyrins. Porphyrin-virus constructs were then shown to photogenerate singlet oxygen, and cytotoxicity in non-targeted photodynamic treatment experiments. Next, each porphyrin-loaded capsid is externally modified with approximately 60 targeting DNA aptamers by employing a heterobifunctional crosslinking agent. The targeting aptamer is known to bind the protein nucleolin, a ubiquitous protein that is overexpressed on the cell surface by many cancer cell types. MCF-7 human breast carcinoma cells and MCF-10A human mammary epithelial cells were selected as an in vitro model for breast cancer and normal tissue, respectively. Fluorescently tagged virus-aptamer constructs are shown to selectively target MCF-7 cells versus MCF-10A cells. Finally, results are shown in which porphyrin

  5. Electrostatic potential of human immunodeficiency virus type 2 and rhesus macaque simian immunodeficiency virus capsid proteins

    Directory of Open Access Journals (Sweden)

    Katarzyna eBozek

    2012-06-01

    Full Text Available Human immunodeficiency virus type 2 (HIV-2 and simian immunodeficiency virus isolated from a macaque monkey (SIVmac are assumed to have originated from simian immunodeficiency virus isolated from sooty mangabey (SIVsm. Despite their close similarity in genome structure, HIV-2 and SIVmac show different sensitivities to TRIM5α, a host restriction factor against retroviruses. The replication of HIV-2 strains is potently restricted by rhesus (Rh monkey TRIM5α, while that of SIVmac strain 239 (SIVmac239 is not. Viral capsid protein is the determinant of this differential sensitivity to TRIM5α, as the HIV-2 mutant carrying SIVmac239 capsid protein evaded Rh TRIM5α-mediated restriction. However, the molecular determinants of this restriction mechanism are unknown. Electrostatic potential on the protein-binding site is one of the properties regulating protein-protein interactions. In this study, we investigated the electrostatic potential on the interaction surface of capsid protein of HIV-2 strain GH123 and SIVmac239. Although HIV-2 GH123 and SIVmac239 capsid proteins share more than 87% amino acid identity, we observed a large difference between the two molecules with the HIV-2 GH123 molecule having predominantly positive and SIVmac239 predominantly negative electrostatic potential on the surface of the loop between α-helices 4 and 5 (L4/5. As L4/5 is one of the major determinants of Rh TRIM5α sensitivity of these viruses, the present results suggest that the binding site of the Rh TRIM5α may show complementarity to the HIV-2 GH123 capsid surface charge distribution.

  6. Determination of prestress and elastic properties of virus capsids

    Science.gov (United States)

    Aggarwal, Ankush

    2018-03-01

    Virus capsids are protein shells that protect the virus genome, and determination of their mechanical properties has been a topic of interest because of their potential use in nanotechnology and therapeutics. It has been demonstrated that stresses exist in virus capsids, even in their equilibrium state, due to their construction. These stresses, termed "prestresses" in this study, closely affect the capsid's mechanical behavior. Three methods—shape-based metric, atomic force microscope indentation, and molecular dynamics—have been proposed to determine the capsid elastic properties without fully accounting for prestresses. In this paper, we theoretically analyze the three methods used for mechanical characterization of virus capsids and numerically investigate how prestresses affect the capsid's mechanical properties. We consolidate all the results and propose that by using these techniques collectively, it is possible to accurately determine both the mechanical properties and prestresses in capsids.

  7. Generation of polyclonal antibody with high avidity to rosuvastatin and its use in development of highly sensitive ELISA for determination of rosuvastatin in plasma

    Directory of Open Access Journals (Sweden)

    Al-Malaq Hamoud A

    2011-07-01

    Full Text Available Abstract In this study, a polyclonal antibody with high avidity and specificity to the potent hypocholesterolaemic agent rosuvastatin (ROS has been prepared and used in the development of highly sensitive enzyme-linked immunosorbent assay (ELISA for determination of ROS in plasma. ROS was coupled to keyhole limpt hemocyanin (KLH and bovine serum albumin (BSA using carbodiimide reagent. ROS-KLH conjugate was used for immunization of female 8-weeks old New Zealand white rabbits. The immune response of the rabbits was monitored by direct ELISA using ROS-BSA immobilized onto microwell plates as a solid phase. The rabbit that showed the highest antibody titer and avidity to ROS was scarified and its sera were collected. The IgG fraction was isolated and purified by avidity chromatography on protein A column. The purified antibody showed high avidity to ROS; IC50 = 0.4 ng/ml. The specificity of the antibody for ROS was evaluated by indirect ELISA using various competitors from the ROS-structural analogues and the therapeutic agents used with ROS in a combination therapy. The proposed ELISA involved a competitive binding reaction between ROS, in plasma sample, and the immobilized ROS-BSA for the binding sites on a limited amount of the anti-ROS antibody. The bound anti-ROS antibody was quantified with horseradish peroxidase-labeled second anti-rabbit IgG antibody (HRP-IgG and 3,3',5,5'-tetramethylbenzidine (TMB as a substrate for the peroxidase enzyme. The concentration of ROS in the sample was quantified by its ability to inhibit the binding of the anti-ROS antibody to the immobilized ROS-BSA and subsequently the color intensity in the assay wells. The assay enabled the determination of ROS in plasma at concentrations as low as 40 pg/ml.

  8. Properties and Functions of the Dengue Virus Capsid Protein.

    Science.gov (United States)

    Byk, Laura A; Gamarnik, Andrea V

    2016-09-29

    Dengue virus affects hundreds of millions of people each year around the world, causing a tremendous social and economic impact on affected countries. The aim of this review is to summarize our current knowledge of the functions, structure, and interactions of the viral capsid protein. The primary role of capsid is to package the viral genome. There are two processes linked to this function: the recruitment of the viral RNA during assembly and the release of the genome during infection. Although particle assembly takes place on endoplasmic reticulum membranes, capsid localizes in nucleoli and lipid droplets. Why capsid accumulates in these locations during infection remains unknown. In this review, we describe available data and discuss new ideas on dengue virus capsid functions and interactions. We believe that a deeper understanding of how the capsid protein works during infection will create opportunities for novel antiviral strategies, which are urgently needed to control dengue virus infections.

  9. EPSTEIN-BARR VIRUS INFECTIONS – AVIDITY TEST FOR IgG ANTIBODIES

    Directory of Open Access Journals (Sweden)

    Katja Strašek

    2001-06-01

    Full Text Available Background. We wish to introduce specific IgG avidity test as a supplementary assay in serological screening for Epstein-Barr virus infection if the status of patient cannot be resolved from a single serum sample with routine testing.Methods. Avidity of IgG antibodies was determined in sera of 57 patients with different stage of Epstein-Barr virus infection. Enzyme-immuno assay was used with a short incubation of 6-molar urea included in the procedure. Urea should remove low avidity antibodies. Avidity was expressed as the avidity index. Avidity testing with commercial kit was done as well.Results. Low avidity index was found for IgG antibodies of acute phase sera and high for those of past infection, recent infection and reactivation of endogenic virus.Conclusions. Avidity test for IgG antibodies might be supplementary assay to prove acute infection but also to resolve some other clinical states related to Epstein-Barr virus.

  10. Studies towards the sex pheromone of the green capsid bug

    NARCIS (Netherlands)

    Drijfhout, F.P.

    2001-01-01

    The green capsid bug, Lygocoris pabulinus (L.) (Heteroptera: Miridae) is a serious pest in fruit orchards, which is difficult to control. Because it is difficult to determine the actual population density, fruit growers apply insecticides against the green capsid bug on

  11. Structure of the Triatoma virus capsid.

    Science.gov (United States)

    Squires, Gaëlle; Pous, Joan; Agirre, Jon; Rozas-Dennis, Gabriela S; Costabel, Marcelo D; Marti, Gerardo A; Navaza, Jorge; Bressanelli, Stéphane; Guérin, Diego M A; Rey, Felix A

    2013-06-01

    The members of the Dicistroviridae family are non-enveloped positive-sense single-stranded RNA (+ssRNA) viruses pathogenic to beneficial arthropods as well as insect pests of medical importance. Triatoma virus (TrV), a member of this family, infects several species of triatomine insects (popularly named kissing bugs), which are vectors for human trypanosomiasis, more commonly known as Chagas disease. The potential use of dicistroviruses as biological control agents has drawn considerable attention in the past decade, and several viruses of this family have been identified, with their targets covering honey bees, aphids and field crickets, among others. Here, the crystal structure of the TrV capsid at 2.5 Å resolution is reported, showing that as expected it is very similar to that of Cricket paralysis virus (CrPV). Nevertheless, a number of distinguishing structural features support the introduction of a new genus (Triatovirus; type species TrV) under the Dicistroviridae family. The most striking differences are the absence of icosahedrally ordered VP4 within the infectious particle and the presence of prominent projections that surround the fivefold axis. Furthermore, the structure identifies a second putative autoproteolytic DDF motif in protein VP3, in addition to the conserved one in VP1 which is believed to be responsible for VP0 cleavage during capsid maturation. The potential meaning of these new findings is discussed.

  12. Structural determination of importin alpha in complex with beak and feather disease virus capsid nuclear localization signal

    International Nuclear Information System (INIS)

    Patterson, Edward I.; Dombrovski, Andrew K.; Swarbrick, Crystall M.D.; Raidal, Shane R.; Forwood, Jade K.

    2013-01-01

    Highlights: •Circovirus capsid proteins contain large nuclear localization signals (NLS). •A method of nuclear import has not been elucidated. •Beak and feather disease virus (BFDV) capsid NLS was crystallized with importin α. •The structure showed BFDV NLS binding to the major site of importin α. •Result shows implications for mechanism of nuclear transport for all circoviruses. -- Abstract: Circoviruses represent a rapidly increasing genus of viruses that infect a variety of vertebrates. Replication requires shuttling viral molecules into the host cell nucleus, a process facilitated by capsid-associated protein (Cap). Whilst a nuclear localization signal (NLS) has been shown to mediate nuclear translocation, the mode of nuclear transport remains to be elucidated. To better understand this process, beak and feather disease virus (BFDV) Cap NLS was crystallized with nuclear import receptor importin-α (Impα). Diffraction yielded structural data to 2.9 Å resolution, and the binding site on both Impα and BFDV Cap NLS were well resolved. The binding mechanism for the major site is likely conserved across circoviruses as supported by the similarity of NLSs in circovirus Caps. This finding illuminates a crucial step for infection of host cells by this viral family, and provides a platform for rational drug design against the binding interface

  13. Processing of the VP1/2A junction is not necessary for production of foot-and-mouth disease virus empty capsids and infectious viruses: characterization of "self-tagged" particles.

    Science.gov (United States)

    Gullberg, Maria; Polacek, Charlotta; Bøtner, Anette; Belsham, Graham J

    2013-11-01

    The foot-and-mouth disease virus (FMDV) capsid protein precursor, P1-2A, is cleaved by 3C(pro) to generate VP0, VP3, VP1, and the peptide 2A. The capsid proteins self-assemble into empty capsid particles or viruses which do not contain 2A. In a cell culture-adapted strain of FMDV (O1 Manisa [Lindholm]), three different amino acid substitutions (E83K, S134C, and K210E) were identified within the VP1 region of the P1-2A precursor compared to the field strain (wild type [wt]). Expression of the O1 Manisa P1-2A (wt or with the S134C substitution in VP1) plus 3C(pro), using a transient expression system, resulted in efficient capsid protein production and self-assembly of empty capsid particles. Removal of the 2A peptide from the capsid protein precursor had no effect on capsid protein processing or particle assembly. However, modification of E83K alone abrogated particle assembly with no apparent effect on protein processing. Interestingly, the K210E substitution, close to the VP1/2A junction, completely blocked processing by 3C(pro) at this cleavage site, but efficient assembly of "self-tagged" empty capsid particles, containing the uncleaved VP1-2A, was observed. These self-tagged particles behaved like the unmodified empty capsids in antigen enzyme-linked immunosorbent assays and integrin receptor binding assays. Furthermore, mutant viruses with uncleaved VP1-2A could be rescued in cells from full-length FMDV RNA transcripts encoding the K210E substitution in VP1. Thus, cleavage of the VP1/2A junction is not essential for virus viability. The production of such engineered self-tagged empty capsid particles may facilitate their purification for use as diagnostic reagents and vaccines.

  14. Retargeting of rat parvovirus H-1PV to cancer cells through genetic engineering of the viral capsid.

    Science.gov (United States)

    Allaume, Xavier; El-Andaloussi, Nazim; Leuchs, Barbara; Bonifati, Serena; Kulkarni, Amit; Marttila, Tiina; Kaufmann, Johanna K; Nettelbeck, Dirk M; Kleinschmidt, Jürgen; Rommelaere, Jean; Marchini, Antonio

    2012-04-01

    The rat parvovirus H-1PV is a promising anticancer agent given its oncosuppressive properties and the absence of known side effects in humans. H-1PV replicates preferentially in transformed cells, but the virus can enter both normal and cancer cells. Uptake by normal cells sequesters a significant portion of the administered viral dose away from the tumor target. Hence, targeting H-1PV entry specifically to tumor cells is important to increase the efficacy of parvovirus-based treatments. In this study, we first found that sialic acid plays a key role in H-1PV entry. We then genetically engineered the H-1PV capsid to improve its affinity for human tumor cells. By analogy with the resolved crystal structure of the closely related parvovirus minute virus of mice, we developed an in silico three-dimensional (3D) model of the H-1PV wild-type capsid. Based on this model, we identified putative amino acids involved in cell membrane recognition and virus entry at the level of the 2-fold axis of symmetry of the capsid, within the so-called dimple region. In situ mutagenesis of these residues significantly reduced the binding and entry of H-1PV into permissive cells. We then engineered an entry-deficient viral capsid and inserted a cyclic RGD-4C peptide at the level of its 3-fold axis spike. This peptide binds α(v)β(3) and α(v)β(5) integrins, which are overexpressed in cancer cells and growing blood vessels. The insertion of the peptide rescued viral infectivity toward cells overexpressing α(v)β(5) integrins, resulting in the efficient killing of these cells by the reengineered virus. This work demonstrates that H-1PV can be genetically retargeted through the modification of its capsid, showing great promise for a more efficient use of this virus in cancer therapy.

  15. Characterization of Three Novel Linear Neutralizing B-Cell Epitopes in the Capsid Protein of Swine Hepatitis E Virus.

    Science.gov (United States)

    Chen, Yiyang; Liu, Baoyuan; Sun, Yani; Li, Huixia; Du, Taofeng; Nan, Yuchen; Hiscox, Julian A; Zhou, En-Min; Zhao, Qin

    2018-04-18

    Hepatitis E virus (HEV) causes liver disease in humans and is thought to be a zoonotic infection with domestic animals being a reservoir including swine and rabbits. One of the proteins encoded by the virus is the capsid protein. This is likely the major immune-dominant protein and a target for vaccination. Four monoclonal antibodies (MAbs); three novel; 1E4, 2C7, 2G9, and one previously characterized (1B5), were evaluated for binding to the capsid protein from genotype 4 (swine) hepatitis E virus (HEV). The results indicated that 625 DFCP 628 , 458 PSRPF 462 , and 407 EPTV 410 peptides on the capsid protein comprised minimal amino acid sequence motifs recognized by 1E4, 2C7, and 2G9, respectively. The data suggested that 2C7 and 2G9 epitopes were partially exposed on the surface of the capsid protein. Truncated genotype 4 swine HEV capsid protein (sp239, amino acids 368-606), can exist in multimeric forms. Pre-incubation of swine HEV with 2C7, 2G9, or 1B5 before addition to HepG2 cells partially blocked sp239 cell binding and inhibited swine HEV infection. The study indicated that 2C7, 2G9, and 1B5 partially blocked swine HEV infection of rabbits better than 1E4 or normal mouse IgG. The cross reactivity of antibodies suggested that capsid epitopes recognized by 2C7 and 2G9 are common to HEV strains infecting most host species. Collectively, MAbs 2C7, 2G9, and 1B5 were shown to recognize three novel linear neutralizing B-cell epitopes of genotype 4 HEV capsid protein. These results enhance understanding of HEV capsid protein structure to guide vaccine and anti-viral design. IMPORTANCE Genotype 3 and 4 HEVs are zoonotic viruses. Here, genotype 4 HEV was studied due to its prevalence in human populations and pig herds in China. To improve HEV disease diagnosis and prevention, a better understanding of antigenic structure and neutralizing epitopes of HEV capsid protein are needed. In this study, the locations of three novel linear B-cell recognition epitopes within

  16. Synergy of two low-affinity NLSs determines the high avidity of influenza A virus nucleoprotein NP for human importin α isoforms.

    Science.gov (United States)

    Wu, Wei; Sankhala, Rajeshwer S; Florio, Tyler J; Zhou, Lixin; Nguyen, Nhan L T; Lokareddy, Ravi K; Cingolani, Gino; Panté, Nelly

    2017-09-12

    The influenza A virus nucleoprotein (NP) is an essential multifunctional protein that encapsidates the viral genome and functions as an adapter between the virus and the host cell machinery. NPs from all strains of influenza A viruses contain two nuclear localization signals (NLSs): a well-studied monopartite NLS1 and a less-characterized NLS2, thought to be bipartite. Through site-directed mutagenesis and functional analysis, we found that NLS2 is also monopartite and is indispensable for viral infection. Atomic structures of importin α bound to two variants of NLS2 revealed NLS2 primarily binds the major-NLS binding site of importin α, unlike NLS1 that associates with the minor NLS-pocket. Though peptides corresponding to NLS1 and NLS2 bind weakly to importin α, the two NLSs synergize in the context of the full length NP to confer high avidity for importin α7, explaining why the virus efficiently replicates in the respiratory tract that exhibits high levels of this isoform. This study, the first to functionally characterize NLS2, demonstrates NLS2 plays an important and unexpected role in influenza A virus infection. We propose NLS1 and NLS2 form a bipartite NLS in trans, which ensures high avidity for importin α7 while preventing non-specific binding to viral RNA.

  17. Assembly and characterization of foot-and-mouth disease virus empty capsid particles expressed within mammalian cells

    DEFF Research Database (Denmark)

    Gullberg, Maria; Muszynski, Bartosz; Organtini, Lindsey J.

    2013-01-01

    The foot-and-mouth disease virus (FMDV) structural protein precursor, P1-2A, is cleaved by the virus-encoded 3C protease (3Cpro) into the capsid proteins VP0, VP1 and VP3 (and 2A). In some systems, it is difficult to produce large amounts of these processed capsid proteins since 3Cpro can be toxic...... (from serotypes O and A) and 3Cpro were expressed from monocistronic cDNA cassettes as P1-2A-3C, or from dicistronic cassettes with the 3Cpro expression dependent on a mutant FMDV internal ribosome entry site (IRES) (designated P1-2A-mIRES-3C). The effects of using a mutant 3Cpro with reduced catalytic....... These products self-assembled to form FMDV empty capsid particles, which have a related, but distinct, morphology (as determined by electron microscopy and reconstruction) from that determined previously by X-ray crystallography. The assembled empty capsids bind, in a divalent cation-dependent manner, to the RGD...

  18. An elastic network model of HK97 capsid maturation.

    Science.gov (United States)

    Kim, Moon K; Jernigan, Robert L; Chirikjian, Gregory S

    2003-08-01

    The structure of the capsid of bacteriophage HK97 has been solved at various stages of maturity by crystallography and cryo-electron microscopy, and has been reported previously in the literature. Typically the capsid assembles through polymerization and maturation processes. Maturation is composed of proteolytic cleavages to the precursor capsid (called Prohead II), expansion triggered by DNA packaging (in which the largest conformational changes of the capsid appear), and covalent cross-links of neighboring subunits to create the mature capsid called Head II. We apply a coarse-grained elastic network interpolation (ENI) to generate a feasible pathway for conformational change from Prohead II to Head II. The icosahedral symmetry of the capsid structure offers a significant computational advantage because it is not necessary to consider the whole capsid structure but only an asymmetric unit consisting of one hexamer plus an additional subunit from an adjacent pentamer. We also analyze normal modes of the capsid structure using an elastic network model which is also subject to symmetry constraints. Using our model, we can visualize the smooth evolution of capsid expansion and revisit in more detail several interesting geometric changes recognized in early experimental works such as rigid body motion of two compact domains (A and P) with two refolding extensions (N-arm and E-loop) and track the approach of the two particular residues associated with isopeptide bonds that make hexagonal cross-links in Head II. The feasibility of the predicted pathway is also supported by the results of our normal mode analysis.

  19. AVIDITY EVALUATION OF LOCAL IgA ANTIBODIES IN PERSONS IMMUNIZED WITH LIVE INFLUENZA VACCINE

    Directory of Open Access Journals (Sweden)

    S. A. Donina

    2008-01-01

    Full Text Available Abstract. At present, immunogenicity evaluation of influenza vaccines is performed by quantitative assessment of increased serum antibodies. It was, however, shown that the degree of human defense against influenza is mostly related to their qualitative characteristics, i.e., avidity (functional activity. Leading role of local immunity is demonstrated in protection against influenza. Such immunity is mediated by IgA antibodies from mucosal airways. Meanwhile, the avidity issues for local antibodies still remain open.In present study, an attempt was undertaken to evaluate post-vaccination local immunological memory for influenza A virus, according to IgA antibodies from upper respiratory secretions. Two techniques were used to evaluate antibody avidity, that were previously applied for studying this phenomenon with serum imunoglobulins, i.e., a dynamic test (measurement of antigen-antibody reaction rates, and a test with urea, a chaotropic agent (avidity is determined as a strength of antigen-antibody complex. A total of 202 persons (18 to 20 years old were enrolled into the study.With both tests, a broad range of individual avidity values was observed for the antibodies. A significant cohort (up to 30 per cent of persons immunized with live influenza vaccine, showed sharply increased avidity of secretory IgA antibodies by both methods, along with accumulation of these immunoglobulins after vaccination. A reverse relationship is revealed between avidity levels of these antibodies before vaccination, and increase of this parameter post-immunization. The data present convincing arguments for specific renewal of local humoral immunological memory, as induced by live influenza vaccine. The study substantiates a necessity for application of the both tests in parallel, when determining avidity of secretory IgA antibodies. (Med. Immunol., vol. 10, N 4-5, pp 423-430.

  20. Structure of the Triatoma virus capsid

    International Nuclear Information System (INIS)

    Squires, Gaëlle; Pous, Joan; Agirre, Jon; Rozas-Dennis, Gabriela S.; Costabel, Marcelo D.; Marti, Gerardo A.; Navaza, Jorge; Bressanelli, Stéphane; Guérin, Diego M. A.; Rey, Felix A.

    2013-01-01

    The crystallographic structure of TrV shows specific morphological and functional features that clearly distinguish it from the type species of the Cripavirus genus, CrPV. The members of the Dicistroviridae family are non-enveloped positive-sense single-stranded RNA (+ssRNA) viruses pathogenic to beneficial arthropods as well as insect pests of medical importance. Triatoma virus (TrV), a member of this family, infects several species of triatomine insects (popularly named kissing bugs), which are vectors for human trypanosomiasis, more commonly known as Chagas disease. The potential use of dicistroviruses as biological control agents has drawn considerable attention in the past decade, and several viruses of this family have been identified, with their targets covering honey bees, aphids and field crickets, among others. Here, the crystal structure of the TrV capsid at 2.5 Å resolution is reported, showing that as expected it is very similar to that of Cricket paralysis virus (CrPV). Nevertheless, a number of distinguishing structural features support the introduction of a new genus (Triatovirus; type species TrV) under the Dicistroviridae family. The most striking differences are the absence of icosahedrally ordered VP4 within the infectious particle and the presence of prominent projections that surround the fivefold axis. Furthermore, the structure identifies a second putative autoproteolytic DDF motif in protein VP3, in addition to the conserved one in VP1 which is believed to be responsible for VP0 cleavage during capsid maturation. The potential meaning of these new findings is discussed

  1. Structure of the Triatoma virus capsid

    Energy Technology Data Exchange (ETDEWEB)

    Squires, Gaëlle; Pous, Joan [Laboratoire de Virologie Moléculaire et Structurale, CNRS, 1 Avenue de la Terrasse, 91198 Gif-sur-Yvette CEDEX (France); Agirre, Jon [Fundación Biofísica Bizkaia, Barrio Sarriena S/N, 48940 Leioa, Bizkaia (FBB) (Spain); Unidad de Biofísica (UBF, CSIC, UPV/EHU), PO Box 644, 48080 Bilbao (Spain); Rozas-Dennis, Gabriela S. [U.N.S., San Juan 670 (8000) Bahía Blanca (Argentina); U.N.S., Avenida Alem 1253 (8000) Bahía Blanca (Argentina); Costabel, Marcelo D. [U.N.S., Avenida Alem 1253 (8000) Bahía Blanca (Argentina); Marti, Gerardo A. [Centro de Estudios Parasitológicos y de Vectores (CEPAVE-CCT, La Plata, CONICET-UNLP), Calle 2 No. 584 (1900) La Plata (Argentina); Navaza, Jorge; Bressanelli, Stéphane [Laboratoire de Virologie Moléculaire et Structurale, CNRS, 1 Avenue de la Terrasse, 91198 Gif-sur-Yvette CEDEX (France); Guérin, Diego M. A., E-mail: diego.guerin@ehu.es [Fundación Biofísica Bizkaia, Barrio Sarriena S/N, 48940 Leioa, Bizkaia (FBB) (Spain); Unidad de Biofísica (UBF, CSIC, UPV/EHU), PO Box 644, 48080 Bilbao (Spain); Rey, Felix A., E-mail: diego.guerin@ehu.es [Laboratoire de Virologie Moléculaire et Structurale, CNRS, 1 Avenue de la Terrasse, 91198 Gif-sur-Yvette CEDEX (France)

    2013-06-01

    The crystallographic structure of TrV shows specific morphological and functional features that clearly distinguish it from the type species of the Cripavirus genus, CrPV. The members of the Dicistroviridae family are non-enveloped positive-sense single-stranded RNA (+ssRNA) viruses pathogenic to beneficial arthropods as well as insect pests of medical importance. Triatoma virus (TrV), a member of this family, infects several species of triatomine insects (popularly named kissing bugs), which are vectors for human trypanosomiasis, more commonly known as Chagas disease. The potential use of dicistroviruses as biological control agents has drawn considerable attention in the past decade, and several viruses of this family have been identified, with their targets covering honey bees, aphids and field crickets, among others. Here, the crystal structure of the TrV capsid at 2.5 Å resolution is reported, showing that as expected it is very similar to that of Cricket paralysis virus (CrPV). Nevertheless, a number of distinguishing structural features support the introduction of a new genus (Triatovirus; type species TrV) under the Dicistroviridae family. The most striking differences are the absence of icosahedrally ordered VP4 within the infectious particle and the presence of prominent projections that surround the fivefold axis. Furthermore, the structure identifies a second putative autoproteolytic DDF motif in protein VP3, in addition to the conserved one in VP1 which is believed to be responsible for VP0 cleavage during capsid maturation. The potential meaning of these new findings is discussed.

  2. Identification of two functional nuclear localization signals in the capsid protein of duck circovirus

    Energy Technology Data Exchange (ETDEWEB)

    Xiang, Qi-Wang; Zou, Jin-Feng; Wang, Xin [Department of Preventive Veterinary Medicine, College of Veterinary Medicine, Shandong Agricultural University, Shandong, Taian 271018 (China); Shandong Provincial Key Laboratory of Animal Biotechnology and Disease Control and Prevention, Shandong, Taian 271018 (China); Sun, Ya-Ni [College of Veterinary Medicine, Northwest A and F University, Shanxi, Yangling 712100 (China); Gao, Ji-Ming; Xie, Zhi-Jing [Department of Preventive Veterinary Medicine, College of Veterinary Medicine, Shandong Agricultural University, Shandong, Taian 271018 (China); Shandong Provincial Key Laboratory of Animal Biotechnology and Disease Control and Prevention, Shandong, Taian 271018 (China); Wang, Yu [Department of Basic Medical Sciences, Taishan Medical College, Shandong, Taian 271000 (China); Zhu, Yan-Li [Department of Preventive Veterinary Medicine, College of Veterinary Medicine, Shandong Agricultural University, Shandong, Taian 271018 (China); Shandong Provincial Key Laboratory of Animal Biotechnology and Disease Control and Prevention, Shandong, Taian 271018 (China); Jiang, Shi-Jin, E-mail: sjjiang@sdau.edu.cn [Department of Preventive Veterinary Medicine, College of Veterinary Medicine, Shandong Agricultural University, Shandong, Taian 271018 (China); Shandong Provincial Key Laboratory of Animal Biotechnology and Disease Control and Prevention, Shandong, Taian 271018 (China)

    2013-02-05

    The capsid protein (CP) of duck circovirus (DuCV) is the major immunogenic protein and has a high proportion of arginine residues concentrated at the N terminus of the protein, which inhibits efficient mRNA translation in prokaryotic expression systems. In this study, we investigated the subcellular distribution of DuCV CP expressed via recombinant baculoviruses in Sf9 cells and the DNA binding activities of the truncated recombinant DuCV CPs. The results showed that two independent bipartite nuclear localization signals (NLSs) situated at N-terminal 1-17 and 18-36 amino acid residue of the CP. Moreover, two expression level regulatory signals (ELRSs) and two DNA binding signals (DBSs) were also mapped to the N terminus of the protein and overlapped with the two NLSs. The ability of CP to bind DNA, coupled with the karyophilic nature of this protein, strongly suggests that it may be responsible for nuclear targeting of the viral genome.

  3. Identification of two functional nuclear localization signals in the capsid protein of duck circovirus

    International Nuclear Information System (INIS)

    Xiang, Qi-Wang; Zou, Jin-Feng; Wang, Xin; Sun, Ya-Ni; Gao, Ji-Ming; Xie, Zhi-Jing; Wang, Yu; Zhu, Yan-Li; Jiang, Shi-Jin

    2013-01-01

    The capsid protein (CP) of duck circovirus (DuCV) is the major immunogenic protein and has a high proportion of arginine residues concentrated at the N terminus of the protein, which inhibits efficient mRNA translation in prokaryotic expression systems. In this study, we investigated the subcellular distribution of DuCV CP expressed via recombinant baculoviruses in Sf9 cells and the DNA binding activities of the truncated recombinant DuCV CPs. The results showed that two independent bipartite nuclear localization signals (NLSs) situated at N-terminal 1–17 and 18–36 amino acid residue of the CP. Moreover, two expression level regulatory signals (ELRSs) and two DNA binding signals (DBSs) were also mapped to the N terminus of the protein and overlapped with the two NLSs. The ability of CP to bind DNA, coupled with the karyophilic nature of this protein, strongly suggests that it may be responsible for nuclear targeting of the viral genome.

  4. Specific interaction between hnRNP H and HPV16 L1 proteins: Implications for late gene auto-regulation enabling rapid viral capsid protein production

    Energy Technology Data Exchange (ETDEWEB)

    Zheng, Zi-Zheng; Sun, Yuan-Yuan; Zhao, Min; Huang, Hui [National Institute of Diagnostics and Vaccine Development in Infectious Diseases, Xiamen University, Xiamen, Fujian 361005 (China); School of Life Sciences, Xiamen University, Xiamen, Fujian 361005 (China); Zhang, Jun; Xia, Ning-Shao [National Institute of Diagnostics and Vaccine Development in Infectious Diseases, Xiamen University, Xiamen, Fujian 361005 (China); School of Life Sciences, Xiamen University, Xiamen, Fujian 361005 (China); School of Public Health, Xiamen University, Xiamen, Fujian 361005 (China); Miao, Ji, E-mail: jmiao@xmu.edu.cn [National Institute of Diagnostics and Vaccine Development in Infectious Diseases, Xiamen University, Xiamen, Fujian 361005 (China); School of Life Sciences, Xiamen University, Xiamen, Fujian 361005 (China); Zhao, Qinjian, E-mail: qinjian_zhao@xmu.edu.cn [National Institute of Diagnostics and Vaccine Development in Infectious Diseases, Xiamen University, Xiamen, Fujian 361005 (China); School of Public Health, Xiamen University, Xiamen, Fujian 361005 (China)

    2013-01-18

    Highlights: ► The RNA-binding hnRNP H regulates late viral gene expression. ► hnRNP H activity was inhibited by a late viral protein. ► Specific interaction between HPV L1 and hnRNP H was demonstrated. ► Co-localization of HPV L1 and hnRNP H inside cells was observed. ► Viral capsid protein production, enabling rapid capsid assembly, was implicated. -- Abstract: Heterogeneous nuclear ribonucleoproteins (hnRNPs), including hnRNP H, are RNA-binding proteins that function as splicing factors and are involved in downstream gene regulation. hnRNP H, which binds to G triplet regions in RNA, has been shown to play an important role in regulating the staged expression of late proteins in viral systems. Here, we report that the specific association between hnRNP H and a late viral capsid protein, human papillomavirus (HPV) L1 protein, leads to the suppressed function of hnRNP H in the presence of the L1 protein. The direct interaction between the L1 protein and hnRNP H was demonstrated by complex formation in solution and intracellularly using a variety of biochemical and immunochemical methods, including peptide mapping, specific co-immunoprecipitation and confocal fluorescence microscopy. These results support a working hypothesis that a late viral protein HPV16 L1, which is down regulated by hnRNP H early in the viral life cycle may provide an auto-regulatory positive feedback loop that allows the rapid production of HPV capsid proteins through suppression of the function of hnRNP H at the late stage of the viral life cycle. In this positive feedback loop, the late viral gene products that were down regulated earlier themselves disable their suppressors, and this feedback mechanism could facilitate the rapid production of capsid proteins, allowing staged and efficient viral capsid assembly.

  5. Avidity of Antibodies against HSV-2 and Risk to Neonatal Transmission among Mexican Pregnant Women

    Directory of Open Access Journals (Sweden)

    Antonia Herrera-Ortiz

    2013-01-01

    Full Text Available Objective. To determine HSV-2 seroprevalence, risk factors, and antibody avidity among a sample of Mexican pregnant women. Material and Methods. The avidity test was standardized with different urea concentrations and incubation times; the cut-off point was calculated to determine the low avidity (early infection. IgG antibodies against HSV-2 were detected from pregnant and postpartum women from Morelos, Mexico, and the avidity test was performed to positive samples. Multivariate regression logistic analysis was employed to evaluate demographic and sexual behavior characteristics associated with HSV-2 infection. Results. HSV-2 seroprevalence among Mexican women analyzed was 14.5% (333/2300, demographic factors (location of General Hospital, age, education level, and civil status, and risky sexual behaviors (STI self-report and number of sexual partners during last year were associated with HSV-2 infection. Seventeen women were detected with low avidity antibodies (early infection with a cut-off point of 66.1%. Conclusions. HSV-2 infection was common among this group of women from Mexico; the avidity test detected women with recent infections, and these women were more likely to transmit HSV-2 to their neonates. Neonatal herpes has no epidemiological surveillance, the disease could be overlooked, and so more studies are needed to estimate the magnitude of neonatal infection.

  6. Primary cytomegalovirus infection in pregnant Egyptian women confirmed by cytomegalovirus IgG avidity testing.

    Science.gov (United States)

    Kamel, N; Metwally, L; Gomaa, N; Sayed Ahmed, W A; Lotfi, M; Younis, S

    2014-01-01

    To determine the frequency of primary cytomegalovirus (CMV) infection in pregnant Egyptian women using CMV IgG avidity testing. A cross-sectional study was conducted at Suez Canal University Hospital, Ismailia, Egypt. A total of 546 pregnant women, presenting for routine antenatal screening, were tested for CMV IgG and IgM using a commercially available enzyme-linked immunosorbent assay (ELISA). Sera from CMV IgM-positive women were tested by CMV IgG avidity assay. All the 546 pregnant women were seropositive for anti-CMV IgG. Of the 546 women, 40 (7.3%) were positive or equivocal for IgM antibodies. All sera from the 40 women (IgG+/IgM+) showed a high or intermediate CMV IgG avidity index. Of the 40 women, 23 (57.5%) were in the second or third trimesters of pregnancy and had their first-trimester blood retrieved, and the tested CMV IgG avidity assay showed a high avidity index. Women who were IgM positive had no primary CMV infection in the index pregnancy as evidenced by the high CMV IgG avidity testing. © 2013 S. Karger AG, Basel.

  7. Investigating the thermal dissociation of viral capsid by lattice model

    Science.gov (United States)

    Chen, Jingzhi; Chevreuil, Maelenn; Combet, Sophie; Lansac, Yves; Tresset, Guillaume

    2017-11-01

    The dissociation of icosahedral viral capsids was investigated by a homogeneous and a heterogeneous lattice model. In thermal dissociation experiments with cowpea chlorotic mottle virus and probed by small-angle neutron scattering, we observed a slight shrinkage of viral capsids, which can be related to the strengthening of the hydrophobic interaction between subunits at increasing temperature. By considering the temperature dependence of hydrophobic interaction in the homogeneous lattice model, we were able to give a better estimate of the effective charge. In the heterogeneous lattice model, two sets of lattice sites represented different capsid subunits with asymmetric interaction strengths. In that case, the dissociation of capsids was found to shift from a sharp one-step transition to a gradual two-step transition by weakening the hydrophobic interaction between AB and CC subunits. We anticipate that such lattice models will shed further light on the statistical mechanics underlying virus assembly and disassembly.

  8. Bovine adenovirus type 3 containing heterologous protein in the C-terminus of minor capsid protein IX

    International Nuclear Information System (INIS)

    Zakhartchouk, Alexander; Connors, Wayne; Van Kessel, Andrew; Tikoo, Suresh Kumar

    2004-01-01

    Earlier, we detected pIX of BAdV-3 as a 14-kDa protein in purified virions. Analysis of BAdV-3 pIX using different region antibodies revealed that the N-terminus and central domain of the pIX contain immunogenic sites and are not exposed on the surface of BAdV-3 virion. This suggested that the C-terminus of BAdV-3 pIX (125 amino acid) may be exposed on the virion and may be used as a site for incorporation of heterologous peptides or proteins. We constructed recombinant BAV950 containing a small peptide (21 amino acid), including the RGD motif or recombinant BAV951 containing enhanced yellow-green fluorescent protein (EYFP) fused to the C-terminus of pIX. Western blot analysis demonstrated that the chimeric pIX-RGD was incorporated into virion capsids. Incorporation of the RGD motif into the pIX resulted in significant augmentation of BAdV-3 fiber knob-independent infection of the integrin-positive cells, suggesting that RGD motifs are displayed on the surface of virion capsids and are accessible for binding to integrins. Analysis of BAV951 revealed that the chimeric pIX is incorporated into virion capsids and EYFP containing the C-terminus of pIX is exposed on the surface of the virion. Moreover, insertion of chimeric pIXs was maintained without change through successive rounds of viral replication. These results suggested that in contrast to major capsid proteins (hexon, penton, fiber), the minor capsid protein IX can be use for the incorporation of targeting ligands based on either small peptides or longer polypeptides

  9. A theory for viral capsid assembly around electrostatic cores

    Science.gov (United States)

    Hagan, Michael F.

    2009-03-01

    We develop equilibrium and kinetic theories that describe the assembly of viral capsid proteins on a charged central core, as seen in recent experiments in which brome mosaic virus capsids assemble around nanoparticles functionalized with polyelectrolyte. We model interactions between capsid proteins and nanoparticle surfaces as the interaction of polyelectrolyte brushes with opposite charge using the nonlinear Poisson Boltzmann equation. The models predict that there is a threshold density of functionalized charge, above which capsids efficiently assemble around nanoparticles, and that light scatter intensity increases rapidly at early times without the lag phase characteristic of empty capsid assembly. These predictions are consistent with and enable interpretation of preliminary experimental data. However, the models predict a stronger dependence of nanoparticle incorporation efficiency on functionalized charge density than measured in experiments and do not completely capture a logarithmic growth phase seen in experimental light scatter. These discrepancies may suggest the presence of metastable disordered states in the experimental system. In addition to discussing future experiments for nanoparticle-capsid systems, we discuss broader implications for understanding assembly around charged cores such as nucleic acids.

  10. A Prime-Boost Vaccination Strategy in Cattle to Prevent Foot-and-Mouth Disease Using a "Single-Cycle" Alphavirus Vector and Empty Capsid Particles

    DEFF Research Database (Denmark)

    Gullberg, Maria; Lohse, Louise; Bøtner, Anette

    2016-01-01

    Foot-and-mouth disease (FMD) remains one of the most economically important infectious diseases of production animals globally. Vaccination can successfully control this disease, however, current vaccines are imperfect. They are made using chemically inactivated FMD virus (FMDV) that is produced...... in large-scale mammalian cell culture under high containment conditions. Here, we have expressed the FMDV capsid protein precursor (P1-2A) of strain O1 Manisa alone or with the FMDV 3C protease (3Cpro) using a "single cycle" packaged alphavirus self-replicating RNA based on Semliki Forest virus (SFV). When...... the FMDV P1-2A was expressed with 3Cpro then processing of the FMDV capsid precursor protein is observed within cells and the proteins assemble into empty capsid particles. The products interact with anti-FMDV antibodies in an ELISA and bind to the integrin αvβ6 (a cellular receptor for FMDV). In cattle...

  11. The pattern-recognition molecule mannan-binding lectin (MBL) in the pathophysiology of diabetic nephropathy

    DEFF Research Database (Denmark)

    Axelgaard, Esben; Thiel, Steffen; Hansen, Troels Krarup

    to carbohydrates of both specific type and density, which thus provides sufficient binding avidity. The character of MBL binding-sites on host cells remain unknown, but it is speculated that altered protein glycation in diabetes permits MBL binding. Based on new studies using MBL/double knockout C57bl/6j mice, we...

  12. Processing of the VP1/2A Junction Is Not Necessary for Production of Foot-and-Mouth Disease Virus Empty Capsids and Infectious Viruses: Characterization of “Self-Tagged” Particles

    DEFF Research Database (Denmark)

    Gullberg, Maria; Polacek, Charlotta; Bøtner, Anette

    2013-01-01

    The foot-and-mouth disease virus (FMDV) capsid protein precursor, P1-2A, is cleaved by 3Cpro to generate VP0, VP3, VP1, and the peptide 2A. The capsid proteins self-assemble into empty capsid particles or viruses which do not contain 2A. In a cell culture-adapted strain of FMDV (O1 Manisa [Lindholm...... the unmodified empty capsids in antigen enzyme-linked immunosorbent assays and integrin receptor binding assays. Furthermore, mutant viruses with uncleaved VP1-2A could be rescued in cells from full-length FMDV RNA transcripts encoding the K210E substitution in VP1. Thus, cleavage of the VP1/2A junction...

  13. Quantitative modeling assesses the contribution of bond strengthening, rebinding and force sharing to the avidity of biomolecule interactions.

    Directory of Open Access Journals (Sweden)

    Valentina Lo Schiavo

    Full Text Available Cell adhesion is mediated by numerous membrane receptors. It is desirable to derive the outcome of a cell-surface encounter from the molecular properties of interacting receptors and ligands. However, conventional parameters such as affinity or kinetic constants are often insufficient to account for receptor efficiency. Avidity is a qualitative concept frequently used to describe biomolecule interactions: this includes incompletely defined properties such as the capacity to form multivalent attachments. The aim of this study is to produce a working description of monovalent attachments formed by a model system, then to measure and interpret the behavior of divalent attachments under force. We investigated attachments between antibody-coated microspheres and surfaces coated with sparse monomeric or dimeric ligands. When bonds were subjected to a pulling force, they exhibited both a force-dependent dissociation consistent with Bell's empirical formula and a force- and time-dependent strengthening well described by a single parameter. Divalent attachments were stronger and less dependent on forces than monovalent ones. The proportion of divalent attachments resisting a force of 30 piconewtons for at least 5 s was 3.7 fold higher than that of monovalent attachments. Quantitative modeling showed that this required rebinding, i.e. additional bond formation between surfaces linked by divalent receptors forming only one bond. Further, experimental data were compatible with but did not require stress sharing between bonds within divalent attachments. Thus many ligand-receptor interactions do not behave as single-step reactions in the millisecond to second timescale. Rather, they exhibit progressive stabilization. This explains the high efficiency of multimerized or clustered receptors even when bonds are only subjected to moderate forces. Our approach provides a quantitative way of relating binding avidity to measurable parameters including bond

  14. Quantitative Modeling Assesses the Contribution of Bond Strengthening, Rebinding and Force Sharing to the Avidity of Biomolecule Interactions

    Science.gov (United States)

    Lo Schiavo, Valentina; Robert, Philippe; Limozin, Laurent; Bongrand, Pierre

    2012-01-01

    Cell adhesion is mediated by numerous membrane receptors. It is desirable to derive the outcome of a cell-surface encounter from the molecular properties of interacting receptors and ligands. However, conventional parameters such as affinity or kinetic constants are often insufficient to account for receptor efficiency. Avidity is a qualitative concept frequently used to describe biomolecule interactions: this includes incompletely defined properties such as the capacity to form multivalent attachments. The aim of this study is to produce a working description of monovalent attachments formed by a model system, then to measure and interpret the behavior of divalent attachments under force. We investigated attachments between antibody-coated microspheres and surfaces coated with sparse monomeric or dimeric ligands. When bonds were subjected to a pulling force, they exhibited both a force-dependent dissociation consistent with Bell’s empirical formula and a force- and time-dependent strengthening well described by a single parameter. Divalent attachments were stronger and less dependent on forces than monovalent ones. The proportion of divalent attachments resisting a force of 30 piconewtons for at least 5 s was 3.7 fold higher than that of monovalent attachments. Quantitative modeling showed that this required rebinding, i.e. additional bond formation between surfaces linked by divalent receptors forming only one bond. Further, experimental data were compatible with but did not require stress sharing between bonds within divalent attachments. Thus many ligand-receptor interactions do not behave as single-step reactions in the millisecond to second timescale. Rather, they exhibit progressive stabilization. This explains the high efficiency of multimerized or clustered receptors even when bonds are only subjected to moderate forces. Our approach provides a quantitative way of relating binding avidity to measurable parameters including bond maturation, rebinding and

  15. Engineering bacterial surface displayed human norovirus capsid proteins: A novel system to explore interaction between norovirus and ligands

    Directory of Open Access Journals (Sweden)

    Mengya eNiu

    2015-12-01

    Full Text Available Human noroviruses (HuNoVs are major contributors to acute nonbacterial gastroenteritis outbreaks. Many aspects of HuNoVs are poorly understood due to both the current inability to culture HuNoVs, and the lack of efficient small animal models. Surrogates for HuNoVs, such as recombinant viral like particles (VLPs expressed in eukaryotic system or P particles expressed in prokaryotic system, have been used for studies in immunology and interaction between the virus and its receptors. However, it is difficult to use VLPs or P particles to collect or isolate potential ligands binding to these recombinant capsid proteins. In this study, a new strategy was used to collect HuNoVs binding ligands through the use of ice nucleation protein (INP to display recombinant capsid proteins of HuNoVs on bacterial surfaces. The viral protein-ligand complex could be easily separated by a low speed centrifugation step. This system was also used to explore interaction between recombinant capsid proteins of HuNoVs and their receptors. In this system, the VP1 capsid encoding gene (ORF2 and the protruding domain (P domain encoding gene (3’ terminal fragment of ORF2 of HuNoVs GI.1 and GII.4 were fused with 5’ terminal fragment of ice nucleation protein encoding gene (inaQn. The results demonstrated that the recombinant VP1 and P domains of HuNoVs were expressed and anchored on the surface of Escherichia coli BL21 cells after the bacteria were transformed with the corresponding plasmids. Both cell surface displayed VP1 and P domains could be recognized by HuNoVs specific antibodies and interact with the viral histo-blood group antigens receptors. In both cases, displayed P domains had better binding abilities than VP1. This new strategy of using displayed HuNoVs capsid proteins on the bacterial surface could be utilized to separate HuNoVs binding components from complex samples, to investigate interaction between the virus and its receptors, as well as to develop an

  16. CD8 T Cell Sensory Adaptation Dependent on TCR Avidity for Self-Antigens

    DEFF Research Database (Denmark)

    Marquez, M.-E.; Ellmeier, W.; Sanchez-Guajardo, Vanesa Maria

    2005-01-01

    dephosphorylation of linker for activation of T cells and ERK upon activation. Normal TCR levels and cytokine production were restored by culturing cells in the absence of TCR/spMHC interaction, demonstrating dynamic tuning of peripheral T cell responses. The effect of avidity for self-ligand(s) on this sensory...... ZAP-YEEI cells were enhanced. Our data provide support for central and peripheral sensory T cell adaptation induced as a function of TCR avidity for self-ligands and signaling level. This may contribute to buffer excessive autoreactivity while optimizing TCR repertoire usage....

  17. Biophysical characterization of the feline immunodeficiency virus p24 capsid protein conformation and in vitro capsid assembly.

    Directory of Open Access Journals (Sweden)

    Jennifer Serrière

    Full Text Available The Feline Immunodeficiency Virus (FIV capsid protein p24 oligomerizes to form a closed capsid that protects the viral genome. Because of its crucial role in the virion, FIV p24 is an interesting target for the development of therapeutic strategies, although little is known about its structure and assembly. We defined and optimized a protocol to overexpress recombinant FIV capsid protein in a bacterial system. Circular dichroism and isothermal titration calorimetry experiments showed that the structure of the purified FIV p24 protein was comprised mainly of α-helices. Dynamic light scattering (DLS and cross-linking experiments demonstrated that p24 was monomeric at low concentration and dimeric at high concentration. We developed a protocol for the in vitro assembly of the FIV capsid. As with HIV, an increased ionic strength resulted in FIV p24 assembly in vitro. Assembly appeared to be dependent on temperature, salt concentration, and protein concentration. The FIV p24 assembly kinetics was monitored by DLS. A limit end-point diameter suggested assembly into objects of definite shapes. This was confirmed by electron microscopy, where FIV p24 assembled into spherical particles. Comparison of FIV p24 with other retroviral capsid proteins showed that FIV assembly is particular and requires further specific study.

  18. Quantum dot-induced viral capsid assembling in dissociation buffer

    Directory of Open Access Journals (Sweden)

    Gao D

    2013-06-01

    Full Text Available Ding Gao,1,2 Zhi-Ping Zhang,1 Feng Li,3 Dong Men,1 Jiao-Yu Deng,1 Hong-Ping Wei,1 Xian-En Zhang,1 Zong-Qiang Cui1 1State Key Laboratory of Virology, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan, 2Graduate University of Chinese Academy of Sciences, Beijing, 3Division of Nanobiomedicine and i-Lab, Suzhou Institute of Nano-Tech and Nano-Bionics, Chinese Academy of Sciences, Suzhou, People's Republic of China Abstract: Viruses encapsulating inorganic nanoparticles are a novel type of nanostructure with applications in biomedicine and biosensors. However, the encapsulation and assembly mechanisms of these hybridized virus-based nanoparticles (VNPs are still unknown. In this article, it was found that quantum dots (QDs can induce simian virus 40 (SV40 capsid assembly in dissociation buffer, where viral capsids should be disassembled. The analysis of the transmission electron microscope, dynamic light scattering, sucrose density gradient centrifugation, and cryo-electron microscopy single particle reconstruction experimental results showed that the SV40 major capsid protein 1 (VP1 can be assembled into ≈25 nm capsids in the dissociation buffer when QDs are present and that the QDs are encapsulated in the SV40 capsids. Moreover, it was determined that there is a strong affinity between QDs and the SV40 VP1 proteins (KD = 2.19E-10 M, which should play an important role in QD encapsulation in the SV40 viral capsids. This study provides a new understanding of the assembly mechanism of SV40 virus-based nanoparticles with QDs, which may help in the design and construction of other similar virus-based nanoparticles. Keywords: quantum dots, simian virus 40, self-assembly, encapsulation, virus-based nanoparticles

  19. The Mammalian Cell Cycle Regulates Parvovirus Nuclear Capsid Assembly

    Science.gov (United States)

    Riolobos, Laura; Domínguez, Carlos; Kann, Michael; Almendral, José M.

    2015-01-01

    It is unknown whether the mammalian cell cycle could impact the assembly of viruses maturing in the nucleus. We addressed this question using MVM, a reference member of the icosahedral ssDNA nuclear parvoviruses, which requires cell proliferation to infect by mechanisms partly understood. Constitutively expressed MVM capsid subunits (VPs) accumulated in the cytoplasm of mouse and human fibroblasts synchronized at G0, G1, and G1/S transition. Upon arrest release, VPs translocated to the nucleus as cells entered S phase, at efficiencies relying on cell origin and arrest method, and immediately assembled into capsids. In synchronously infected cells, the consecutive virus life cycle steps (gene expression, proteins nuclear translocation, capsid assembly, genome replication and encapsidation) proceeded tightly coupled to cell cycle progression from G0/G1 through S into G2 phase. However, a DNA synthesis stress caused by thymidine irreversibly disrupted virus life cycle, as VPs became increasingly retained in the cytoplasm hours post-stress, forming empty capsids in mouse fibroblasts, thereby impairing encapsidation of the nuclear viral DNA replicative intermediates. Synchronously infected cells subjected to density-arrest signals while traversing early S phase also blocked VPs transport, resulting in a similar misplaced cytoplasmic capsid assembly in mouse fibroblasts. In contrast, thymidine and density arrest signals deregulating virus assembly neither perturbed nuclear translocation of the NS1 protein nor viral genome replication occurring under S/G2 cycle arrest. An underlying mechanism of cell cycle control was identified in the nuclear translocation of phosphorylated VPs trimeric assembly intermediates, which accessed a non-conserved route distinct from the importin α2/β1 and transportin pathways. The exquisite cell cycle-dependence of parvovirus nuclear capsid assembly conforms a novel paradigm of time and functional coupling between cellular and virus life

  20. Correlation of Naturally Occurring HIV-1 Resistance to DEB025 with Capsid Amino Acid Polymorphisms

    Directory of Open Access Journals (Sweden)

    Brigitte Rosenwirth

    2013-03-01

    Full Text Available DEB025 (alisporivir is a synthetic cyclosporine with inhibitory activity against human immunodeficiency virus type-1 (HIV-1 and hepatitis C virus (HCV. It binds to cyclophilin A (CypA and blocks essential functions of CypA in the viral replication cycles of both viruses. DEB025 inhibits clinical HIV-1 isolates in vitro and decreases HIV-1 virus load in the majority of patients. HIV-1 isolates being naturally resistant to DEB025 have been detected in vitro and in nonresponder patients. By sequence analysis of their capsid protein (CA region, two amino acid polymorphisms that correlated with DEB025 resistance were identified: H87Q and I91N, both located in the CypA-binding loop of the CA protein of HIV-1. The H87Q change was by far more abundant than I91N. Additional polymorphisms in the CypA-binding loop (positions 86, 91 and 96, as well as in the N-terminal loop of CA were detected in resistant isolates and are assumed to contribute to the degree of resistance. These amino acid changes may modulate the conformation of the CypA-binding loop of CA in such a way that binding and/or isomerase function of CypA are no longer necessary for virus replication. The resistant HIV-1 isolates thus are CypA-independent.

  1. All-atom molecular dynamics calculation study of entire poliovirus empty capsids in solution

    Energy Technology Data Exchange (ETDEWEB)

    Andoh, Y.; Yoshii, N.; Yamada, A.; Kojima, H.; Mizutani, K.; Okazaki, S., E-mail: okazaki@apchem.nagoya-u.ac.jp [Department of Applied Chemistry, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8603 (Japan); Fujimoto, K. [Department of Pharmacy, College of Pharmaceutical Sciences, Ritsumeikan University, Nojihigashi, Kusatsu, Shiga 525-8577 (Japan); Nakagawa, A. [Institute for Protein Research, Osaka University, Yamadaoka, Suita, Osaka 565-0871 (Japan); Nomoto, A. [Institute of Microbial Chemistry, Kamiosaki, Shinagawa-ku, Tokyo 141-0021 (Japan)

    2014-10-28

    Small viruses that belong, for example, to the Picornaviridae, such as poliovirus and foot-and-mouth disease virus, consist simply of capsid proteins and a single-stranded RNA (ssRNA) genome. The capsids are quite stable in solution to protect the genome from the environment. Here, based on long-time and large-scale 6.5 × 10{sup 6} all-atom molecular dynamics calculations for the Mahoney strain of poliovirus, we show microscopic properties of the viral capsids at a molecular level. First, we found equilibrium rapid exchange of water molecules across the capsid. The exchange rate is so high that all water molecules inside the capsid (about 200 000) can leave the capsid and be replaced by water molecules from the outside in about 25 μs. This explains the capsid's tolerance to high pressures and deactivation by exsiccation. In contrast, the capsid did not exchange ions, at least within the present simulation time of 200 ns. This implies that the capsid can function, in principle, as a semipermeable membrane. We also found that, similar to the xylem of trees, the pressure of the solution inside the capsid without the genome was negative. This is caused by coulombic interaction of the solution inside the capsid with the capsid excess charges. The negative pressure may be compensated by positive osmotic pressure by the solution-soluble ssRNA and the counter ions introduced into it.

  2. All-atom molecular dynamics calculation study of entire poliovirus empty capsids in solution

    Science.gov (United States)

    Andoh, Y.; Yoshii, N.; Yamada, A.; Fujimoto, K.; Kojima, H.; Mizutani, K.; Nakagawa, A.; Nomoto, A.; Okazaki, S.

    2014-10-01

    Small viruses that belong, for example, to the Picornaviridae, such as poliovirus and foot-and-mouth disease virus, consist simply of capsid proteins and a single-stranded RNA (ssRNA) genome. The capsids are quite stable in solution to protect the genome from the environment. Here, based on long-time and large-scale 6.5 × 106 all-atom molecular dynamics calculations for the Mahoney strain of poliovirus, we show microscopic properties of the viral capsids at a molecular level. First, we found equilibrium rapid exchange of water molecules across the capsid. The exchange rate is so high that all water molecules inside the capsid (about 200 000) can leave the capsid and be replaced by water molecules from the outside in about 25 μs. This explains the capsid's tolerance to high pressures and deactivation by exsiccation. In contrast, the capsid did not exchange ions, at least within the present simulation time of 200 ns. This implies that the capsid can function, in principle, as a semipermeable membrane. We also found that, similar to the xylem of trees, the pressure of the solution inside the capsid without the genome was negative. This is caused by coulombic interaction of the solution inside the capsid with the capsid excess charges. The negative pressure may be compensated by positive osmotic pressure by the solution-soluble ssRNA and the counter ions introduced into it.

  3. All-atom molecular dynamics calculation study of entire poliovirus empty capsids in solution

    International Nuclear Information System (INIS)

    Andoh, Y.; Yoshii, N.; Yamada, A.; Kojima, H.; Mizutani, K.; Okazaki, S.; Fujimoto, K.; Nakagawa, A.; Nomoto, A.

    2014-01-01

    Small viruses that belong, for example, to the Picornaviridae, such as poliovirus and foot-and-mouth disease virus, consist simply of capsid proteins and a single-stranded RNA (ssRNA) genome. The capsids are quite stable in solution to protect the genome from the environment. Here, based on long-time and large-scale 6.5 × 10 6 all-atom molecular dynamics calculations for the Mahoney strain of poliovirus, we show microscopic properties of the viral capsids at a molecular level. First, we found equilibrium rapid exchange of water molecules across the capsid. The exchange rate is so high that all water molecules inside the capsid (about 200 000) can leave the capsid and be replaced by water molecules from the outside in about 25 μs. This explains the capsid's tolerance to high pressures and deactivation by exsiccation. In contrast, the capsid did not exchange ions, at least within the present simulation time of 200 ns. This implies that the capsid can function, in principle, as a semipermeable membrane. We also found that, similar to the xylem of trees, the pressure of the solution inside the capsid without the genome was negative. This is caused by coulombic interaction of the solution inside the capsid with the capsid excess charges. The negative pressure may be compensated by positive osmotic pressure by the solution-soluble ssRNA and the counter ions introduced into it

  4. Networks for Success: Preparing Mexican American AVID College Students for Credentials, Completion, and the Workforce

    Science.gov (United States)

    Kirk, Richard; Watt, Karen M.

    2018-01-01

    This qualitative study examines how Mexican American students participating in an AVID for Higher Education course perceived their preparation for the workforce and efficacy of completing a college credential. A focus group approach was used to explore how social and cultural networks (networks for success) contribute to college completion. The…

  5. Experimental evaluation of radioiodinated sennoside B as a necrosis-avid tracer agent.

    Science.gov (United States)

    Zhang, Dongjian; Huang, Dejian; Ji, Yun; Jiang, Cuihua; Li, Yue; Gao, Meng; Yao, Nan; Liu, Xuejiao; Shao, Haibo; Jing, Su; Ni, Yicheng; Yin, Zhiqi; Zhang, Jian

    2015-02-01

    Necrosis-avid agents are a class of compounds that selectively accumulate in the necrotic tissues after systemic administration, which can be used for in vivo necrosis imaging and targeted therapies. In order to search for a necrosis-avid tracer agent with improved drugability, we labelled iodine-131 on sennoside B (SB) as a naturally occurring median dianthrone compound. The necrosis targetability and clearance properties of (131)I-SB were evaluated in model rats with liver and muscle necrosis. On SPECT/CT images, a "hot spot" in the infarcted liver lobe and necrotic muscle was persistently observed at 24 h and 72 h post-injection (p.i.). Gamma counting of the tissues of interest revealed a radioactivity ratio of necrotic to viable liver at 4.6 and 3.4 and of necrotic to viable muscle at 7.0 and 8.8 at 24 h and 72 h p.i., respectively. The good match of autoradiographs and fluoromicroscopic images with corresponding histochemical staining suggested preferential uptake of (131)I-SB in necrotic tissue. Pharmacokinetic study revealed that (131)I-SB has an elimination half-life of 8.6 h. This study indicates that (131)I-SB shows not only prominent necrosis avidity but also favourable pharmacokinetics, which may serve as a potential necrosis-avid diagnostic agent for assessment of tissue viability.

  6. Benign Bone Conditions That May Be FDG-avid and Mimic Malignancy

    NARCIS (Netherlands)

    Kwee, Thomas C.; de Klerk, John M. H.; Nix, Maarten; Heggelman, Ben G. F.; Dubois, Stefan V.; Adams, Hugo J. A.

    Positron emission tomography with the radiotracer F-18-fluoro-2-deoxy-D-glucose (FDG) plays an important role in the evaluation of bone pathology. However, FDG is not a cancer-specific agent, and knowledge of the differential diagnosis of benign FDG-avid bone alterations that may resemble malignancy

  7. Porin A-specific antibody avidity in patients who are convalescing from meningococcal B disease.

    NARCIS (Netherlands)

    Vermont, C.L.; Dijken, H.H. van; Groot, R. de; Dobbelsteen, G.P. van den

    2005-01-01

    Porin A (PorA), which determines the serosubtype of Neisseria meningitidis, is the main antigen of a candidate vaccine against serogroup B meningococci, which has been shown to induce high-avidity antibodies in children. We characterized the immune response of children after convalescing from

  8. Ta(l)king sides: ethical and methodological challenges in comparative fieldwork on avid football rivalries

    NARCIS (Netherlands)

    Spaaij, R.; Geilenkirchen, M.

    2011-01-01

    Previous research on football (soccer) fan cultures has documented the lived experiences of devoted football supporters. Few studies, however, have used participant observation and intensive interviewing to examine the deep-rooted inter-group oppositions that characterize avid football rivalries.

  9. Changes in avidity and level of immunoglobulin G antibodies to Mycobacterium tuberculosis in sera of patients undergoing treatment for pulmonary tuberculosis

    NARCIS (Netherlands)

    Arias-Bouda, Lenka M. Pereira; Kuijper, Sjoukje; van der Werf, Anouk; Nguyen, Lan N.; Jansen, Henk M.; Kolk, Arend H. J.

    2003-01-01

    Much is known about specific antibodies and their titers in patients with tuberculosis. However, little is known about the avidity of these antibodies or whether changes in avidity occur during the progression of the disease or during treatment. The aims of this study were to determine the avidity

  10. Molecular characterization of capsid protein gene of potato virus X ...

    African Journals Online (AJOL)

    Molecular characterization of capsid protein gene of potato virus X from Pakistan. Arshad Jamal, Idrees Ahmad Nasir, Bushra Tabassum, Muhammad Tariq, Abdul Munim Farooq, Zahida Qamar, Mohsin Ahmad Khan, Nadeem Ahmad, Muhammad Shafiq, Muhammad Saleem Haider, M. Arshad Javed, Tayyab Husnain ...

  11. Specific interaction of capsid protein and importin-α/β influences West Nile virus production

    International Nuclear Information System (INIS)

    Bhuvanakantham, Raghavan; Chong, Mun-Keat; Ng, Mah-Lee

    2009-01-01

    West Nile virus (WNV) capsid (C) protein has been shown to enter the nucleus of infected cells. However, the mechanism by which C protein enters the nucleus is unknown. In this study, we have unveiled for the first time that nuclear transport of WNV and Dengue virus C protein is mediated by their direct association with importin-α. This interplay is mediated by the consensus sequences of bipartite nuclear localization signal located between amino acid residues 85-101 together with amino acid residues 42 and 43 of C protein. Elucidation of biological significance of importin-α/C protein interaction demonstrated that the binding efficiency of this association influenced the nuclear entry of C protein and virus production. Collectively, this study illustrated the molecular mechanism by which the C protein of arthropod-borne flavivirus enters the nucleus and showed the importance of importin-α/C protein interaction in the context of flavivirus life-cycle.

  12. Specific interaction of capsid protein and importin-{alpha}/{beta} influences West Nile virus production

    Energy Technology Data Exchange (ETDEWEB)

    Bhuvanakantham, Raghavan; Chong, Mun-Keat [Flavivirology Laboratory, Department of Microbiology, 5 Science Drive 2, National University of Singapore, Singapore 117597 (Singapore); Ng, Mah-Lee, E-mail: micngml@nus.edu.sg [Flavivirology Laboratory, Department of Microbiology, 5 Science Drive 2, National University of Singapore, Singapore 117597 (Singapore)

    2009-11-06

    West Nile virus (WNV) capsid (C) protein has been shown to enter the nucleus of infected cells. However, the mechanism by which C protein enters the nucleus is unknown. In this study, we have unveiled for the first time that nuclear transport of WNV and Dengue virus C protein is mediated by their direct association with importin-{alpha}. This interplay is mediated by the consensus sequences of bipartite nuclear localization signal located between amino acid residues 85-101 together with amino acid residues 42 and 43 of C protein. Elucidation of biological significance of importin-{alpha}/C protein interaction demonstrated that the binding efficiency of this association influenced the nuclear entry of C protein and virus production. Collectively, this study illustrated the molecular mechanism by which the C protein of arthropod-borne flavivirus enters the nucleus and showed the importance of importin-{alpha}/C protein interaction in the context of flavivirus life-cycle.

  13. Flexible Connectors between Capsomer Subunits that Regulate Capsid Assembly.

    Science.gov (United States)

    Hasek, Mary L; Maurer, Joshua B; Hendrix, Roger W; Duda, Robert L

    2017-08-04

    Viruses build icosahedral capsids of specific size and shape by regulating the spatial arrangement of the hexameric and pentameric protein capsomers in the growing shell during assembly. In the T=7 capsids of Escherichia coli bacteriophage HK97 and other phages, 60 capsomers are hexons, while the rest are pentons that are correctly positioned during assembly. Assembly of the HK97 capsid to the correct size and shape has been shown to depend on specific ionic contacts between capsomers. We now describe additional ionic interactions within capsomers that also regulate assembly. Each is between the long hairpin, the "E-loop," that extends from one subunit to the adjacent subunit within the same capsomer. Glutamate E153 on the E-loop and arginine R210 on the adjacent subunit's backbone alpha-helix form salt bridges in hexamers and pentamers. Mutations that disrupt these salt bridges were lethal for virus production, because the mutant proteins assembled into tubes or sheets instead of capsids. X-ray structures show that the E153-R210 links are flexible and maintained during maturation despite radical changes in capsomer shape. The E153-R210 links appear to form early in assembly to enable capsomers to make programmed changes in their shape during assembly. The links also prevent flattening of capsomers and premature maturation. Mutant phenotypes and modeling support an assembly model in which flexible E153-R210 links mediate capsomer shape changes that control where pentons are placed to create normal-sized capsids. The E-loop may be conserved in other systems in order to play similar roles in regulating assembly. Copyright © 2017 Elsevier Ltd. All rights reserved.

  14. Scaffold expulsion and genome packaging trigger stabilization of herpes simplex virus capsids

    Science.gov (United States)

    Roos, Wouter H.; Radtke, Kerstin; Kniesmeijer, Edward; Geertsema, Hylkje; Sodeik, Beate; Wuite, Gijs J. L.

    2009-01-01

    Herpes simplex virus type 1 (HSV1) capsids undergo extensive structural changes during maturation and DNA packaging. As a result, they become more stable and competent for nuclear egress. To further elucidate this stabilization process, we used biochemical and nanoindentation approaches to analyze the structural and mechanical properties of scaffold-containing (B), empty (A), and DNA-containing (C) nuclear capsids. Atomic force microscopy experiments revealed that A and C capsids were mechanically indistinguishable, indicating that the presence of DNA does not account for changes in mechanical properties during capsid maturation. Despite having the same rigidity, the scaffold-containing B capsids broke at significantly lower forces than A and C capsids. An extraction of pentons with guanidine hydrochloride (GuHCl) increased the flexibility of all capsids. Surprisingly, the breaking forces of the modified A and C capsids dropped to similar values as those of the GuHCl-treated B capsids, indicating that mechanical reinforcement occurs at the vertices. Nonetheless, it also showed that HSV1 capsids possess a remarkable structural integrity that was preserved after removal of pentons. We suggest that HSV1 capsids are stabilized after removal of the scaffold proteins, and that this stabilization is triggered by the packaging of DNA, but independent of the actual presence of DNA. PMID:19487681

  15. Periodic table of virus capsids: implications for natural selection and design.

    Science.gov (United States)

    Mannige, Ranjan V; Brooks, Charles L

    2010-03-04

    For survival, most natural viruses depend upon the existence of spherical capsids: protective shells of various sizes composed of protein subunits. So far, general evolutionary pressures shaping capsid design have remained elusive, even though an understanding of such properties may help in rationally impeding the virus life cycle and designing efficient nano-assemblies. This report uncovers an unprecedented and species-independent evolutionary pressure on virus capsids, based on the the notion that the simplest capsid designs (or those capsids with the lowest "hexamer complexity", C(h)) are the fittest, which was shown to be true for all available virus capsids. The theories result in a physically meaningful periodic table of virus capsids that uncovers strong and overarching evolutionary pressures, while also offering geometric explanations to other capsid properties (rigidity, pleomorphy, auxiliary requirements, etc.) that were previously considered to be unrelatable properties of the individual virus. Apart from describing a universal rule for virus capsid evolution, our work (especially the periodic table) provides a language with which highly diverse virus capsids, unified only by geometry, may be described and related to each other. Finally, the available virus structure databases and other published data reiterate the predicted geometry-derived rules, reinforcing the role of geometry in the natural selection and design of virus capsids.

  16. Persistent Low Toxoplasma IgG Avidity Is Common in Pregnancy: Experience from Antenatal Testing in Norway.

    Directory of Open Access Journals (Sweden)

    Gry Findal

    Full Text Available The parasite Toxoplasma gondii might harm the fetus if a woman is infected during pregnancy. IgG seroconversion and significant increase in IgG antibody amount in pregnancy indicates maternal infection. Presence of toxoplasma immunoglobulin M (IgM, immunoglobulin G (IgG and low IgG avidity in a single serum sample indicates possible maternal infection, but positive toxoplasma IgM and low IgG avidity may persist for months and even years. We aimed to evaluate avidity development during pregnancy in a retrospective study. Serial blood samples from 176 pregnant women admitted to Oslo University Hospital 1993-2013 for amniocentesis because of suspected toxoplasma infection were included. Data were obtained from journals and laboratory records. The avidity method used was based on Platelia Toxo IgG assay. Mean maternal age at first serology was 29.9 years (SD 5.2, range 18-42. In 37 (21% women only the avidity increased from low to high in < 3 months. In 139 (79% the IgG avidity remained below the high threshold ≥ 3 months and within this group 74 (42% women had stable low IgG avidity during the observation period. Median gestational age at first test was 10.6 weeks (range 4.6-28.7. Fetal infection was detected in four children, but none among children whose mother had stable low IgG avidity. The first antenatal toxoplasma serology should ideally be collected in early pregnancy and if stable values of toxoplasma IgM and low IgG-avidity are detected in a second sample after three to four weeks, the need for amniocentesis can be questioned.

  17. Five of Five VHHs Neutralizing Poliovirus Bind the Receptor-Binding Site.

    Science.gov (United States)

    Strauss, Mike; Schotte, Lise; Thys, Bert; Filman, David J; Hogle, James M

    2016-01-13

    Nanobodies, or VHHs, that recognize poliovirus type 1 have previously been selected and characterized as candidates for antiviral agents or reagents for standardization of vaccine quality control. In this study, we present high-resolution cryo-electron microscopy reconstructions of poliovirus with five neutralizing VHHs. All VHHs bind the capsid in the canyon at sites that extensively overlap the poliovirus receptor-binding site. In contrast, the interaction involves a unique (and surprisingly extensive) surface for each of the five VHHs. Five regions of the capsid were found to participate in binding with all five VHHs. Four of these five regions are known to alter during the expansion of the capsid associated with viral entry. Interestingly, binding of one of the VHHs, PVSS21E, resulted in significant changes of the capsid structure and thus seems to trap the virus in an early stage of expansion. We describe the cryo-electron microscopy structures of complexes of five neutralizing VHHs with the Mahoney strain of type 1 poliovirus at resolutions ranging from 3.8 to 6.3Å. All five VHHs bind deep in the virus canyon at similar sites that overlap extensively with the binding site for the receptor (CD155). The binding surfaces on the VHHs are surprisingly extensive, but despite the use of similar binding surfaces on the virus, the binding surface on the VHHs is unique for each VHH. In four of the five complexes, the virus remains essentially unchanged, but for the fifth there are significant changes reminiscent of but smaller in magnitude than the changes associated with cell entry, suggesting that this VHH traps the virus in a previously undescribed early intermediate state. The neutralizing mechanisms of the VHHs and their potential use as quality control agents for the end game of poliovirus eradication are discussed. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

  18. Efficient Capsid Antigen Presentation From Adeno-Associated Virus Empty Virions In Vivo.

    Science.gov (United States)

    Pei, Xiaolei; Earley, Lauriel Freya; He, Yi; Chen, Xiaojing; Hall, Nikita Elexa; Samulski, Richard Jude; Li, Chengwen

    2018-01-01

    Adeno-associated virus (AAV) vectors have been successfully applied in clinical trials for hemophilic patients. Although promising, the clinical results suggest that the capsid-specific CD8+T cell response has a negative effect on therapeutic success. In an in vitro analysis using an engineered AAV virus carrying immune-dominant SIINFEKL peptide in the capsid backbone, we have previously demonstrated that capsid antigen presentation from full (genome containing) AAV capsids requires endosome escape and is proteasome dependent and that no capsid antigen presentation is induced from empty virions. In the present study, we examined capsid antigen presentation from administration of empty virions in animal models. In wild-type mice, similar to AAV full particles, capsid antigen presentation from AAV empty virion infection was dose dependent, and the kinetics studies showed that antigen presentation was detected from 2 to 40 days after AAV empty virion administration. In the transporter associated with antigen processing 1 deficient (TAP-/-) mice, capsid antigen presentation was inhibited from both AAV full and empty virions, but higher inhibition was achieved from AAV full particle administration than that from empty virions. This indicates that the pathway of capsid antigen presentation from AAV transduction is dependent on proteasome-mediated degradation of AAV capsids (mainly for full particles) and that the endosomal pathway may also play a role in antigen presentation from empty particles but not full virions. The capsid antigen presentation efficiency from AAV preparations was positively correlated with the amount of empty virions contaminated with full particles. Collectively, the results indicate that contamination of AAV empty virions induces efficient antigen presentation in vivo and the mechanism of capsid antigen presentation from empty virions involves both endosomal and proteasomal pathways. The elucidation of capsid antigen presentation from AAV empty

  19. FDG avidity and PET/CT patterns in primary gastric lymphoma

    International Nuclear Information System (INIS)

    Radan, Lea; Fischer, Doron; Bar-Shalom, Rachel; Israel, Ora; Dann, Eldad J.; Epelbaum, Ron; Haim, Nissim; Gaitini, Diana

    2008-01-01

    The use of 18F-fluoro-deoxyglucose positron emission tomography/computed tomography (FDG-PET/CT) in primary gastric lymphoma (PGL) is challenging due to physiologic FDG activity in the stomach and variability in the degree of uptake in various histologic subtypes. This study assesses FDG avidity and PET/CT patterns in newly diagnosed PGL. Sixty-two PET/CT studies of newly diagnosed PGL were reviewed (24 low-grade mucosa-associated lymphoid tissue [MALT], 38 aggressive non-Hodgkin's lymphoma [AGNHL]). FDG avidity, patterns (focal/diffuse), and intensity (visually vs. the liver and SUVmax) were assessed and compared to 27 controls. Gastric CT abnormalities and extragastric sites were recorded. Gastric FDG uptake was found in 55/62 (89%) PGL (71% MALT vs. 100% AGNHL, p < 0.001) and 63% controls. A diffuse pattern was found in 60% PGL (76% MALT vs. 53% AGNHL, p = NS) and 47% controls. FDG uptake higher than liver was found in 82% PGL (58% MALT vs. 97% AGNHL, p < 0.05) and 63% controls. SUVmax in FDG-avid PGLs was 15.3 ± 11.7 (5.4 ± 2.9 MALT vs. 19.7 ± 11.5 AGNHL, p < 0.001) and 4.6 ± 1.4 in controls. CT abnormalities were found in 79% PGL (thickening, n = 49; ulcerations, n = 22). Extra-gastric FDG-avid sites were seen in none of MALT, but 61% of AGNHL (nodal, n = 18; nodal and extranodal, n 5). FDG avidity was present in 89% of PGLs, including all patients with AGNHL but only 71% of MALT. FDG uptake can be differentiated, in particular in AGNHL-PGL, from physiologic tracer activity by intensity but not by pattern. Extragastric foci on PET and structural CT abnormalities are additional parameters that can improve PET/CT assessment of PGL. Defining FDG avidity and PET/CT patterns in AGNHL and a subgroup of MALT-PGL before treatment may be important for further monitoring therapy response. (orig.)

  20. Avidity of anti-malarial antibodies inversely related to transmission intensity at three sites in Uganda.

    Science.gov (United States)

    Ssewanyana, Isaac; Arinaitwe, Emmanuel; Nankabirwa, Joaniter I; Yeka, Adoke; Sullivan, Richard; Kamya, Moses R; Rosenthal, Philip J; Dorsey, Grant; Mayanja-Kizza, Harriet; Drakeley, Chris; Greenhouse, Bryan; Tetteh, Kevin K A

    2017-02-10

    People living in malaria endemic areas acquire protection from severe malaria quickly, but protection from clinical disease and control of parasitaemia is acquired only after many years of repeated infections. Antibodies play a central role in protection from clinical disease; however, protective antibodies are slow to develop. This study sought to investigate the influence of Plasmodium falciparum exposure on the acquisition of high-avidity antibodies to P. falciparum antigens, which may be associated with protection. Cross-sectional surveys were performed in children and adults at three sites in Uganda with varied P. falciparum transmission intensity (entomological inoculation rates; 3.8, 26.6, and 125 infectious bites per person per year). Sandwich ELISA was used to measure antibody responses to two P. falciparum merozoite surface antigens: merozoite surface protein 1-19 (MSP1-19) and apical membrane antigen 1 (AMA1). In individuals with detectable antibody levels, guanidine hydrochloride (GuHCl) was added to measure the relative avidity of antibody responses by ELISA. Within a site, there were no significant differences in median antibody levels between the three age groups. Between sites, median antibody levels were generally higher in the higher transmission sites, with differences more apparent for AMA-1 and in ≥5 year group. Similarly, median avidity index (proportion of high avidity antibodies) showed no significant increase with increasing age but was significantly lower at sites of higher transmission amongst participants ≥5 years of age. Using 5 M GuHCl, the median avidity indices in the ≥5 year group at the highest and lowest transmission sites were 19.9 and 26.8, respectively (p = 0.0002) for MSP1-19 and 12.2 and 17.2 (p = 0.0007) for AMA1. Avidity to two different P. falciparum antigens was lower in areas of high transmission intensity compared to areas with lower transmission. Appreciation of the mechanisms behind these findings as

  1. FDG avidity and PET/CT patterns in primary gastric lymphoma

    Energy Technology Data Exchange (ETDEWEB)

    Radan, Lea [Rambam Health Care Campus, Department of Nuclear Medicine, Haifa (Israel); Fischer, Doron [Rambam Health Care Campus, Department of Diagnostic Imaging, Haifa (Israel); Bar-Shalom, Rachel; Israel, Ora [Technion - Israel Institute of Technology, Department of Nuclear Medicine, Rambam Health Care Campus and R. and B. Rappaport School of Medicine, Haifa (Israel); Dann, Eldad J. [Technion - Israel Institute of Technology, Department of Hematology, Rambam Health Care Campus, and R. and B. Rappaport School of Medicine, Haifa (Israel); Epelbaum, Ron; Haim, Nissim [Technion - Israel Institute of Technology, Department of Oncology, Rambam Health Care Campus, and R. and B. Rappaport School of Medicine, Haifa (Israel); Gaitini, Diana [Rambam Health Care Campus, and R. and B. Rappaport School of Medicine, Technion - Israel Institute of Technology, Department of Diagnostic Imaging, Haifa (Israel)

    2008-08-15

    The use of 18F-fluoro-deoxyglucose positron emission tomography/computed tomography (FDG-PET/CT) in primary gastric lymphoma (PGL) is challenging due to physiologic FDG activity in the stomach and variability in the degree of uptake in various histologic subtypes. This study assesses FDG avidity and PET/CT patterns in newly diagnosed PGL. Sixty-two PET/CT studies of newly diagnosed PGL were reviewed (24 low-grade mucosa-associated lymphoid tissue [MALT], 38 aggressive non-Hodgkin's lymphoma [AGNHL]). FDG avidity, patterns (focal/diffuse), and intensity (visually vs. the liver and SUVmax) were assessed and compared to 27 controls. Gastric CT abnormalities and extragastric sites were recorded. Gastric FDG uptake was found in 55/62 (89%) PGL (71% MALT vs. 100% AGNHL, p < 0.001) and 63% controls. A diffuse pattern was found in 60% PGL (76% MALT vs. 53% AGNHL, p = NS) and 47% controls. FDG uptake higher than liver was found in 82% PGL (58% MALT vs. 97% AGNHL, p < 0.05) and 63% controls. SUVmax in FDG-avid PGLs was 15.3 {+-} 11.7 (5.4 {+-} 2.9 MALT vs. 19.7 {+-} 11.5 AGNHL, p < 0.001) and 4.6 {+-} 1.4 in controls. CT abnormalities were found in 79% PGL (thickening, n = 49; ulcerations, n = 22). Extra-gastric FDG-avid sites were seen in none of MALT, but 61% of AGNHL (nodal, n = 18; nodal and extranodal, n = 5). FDG avidity was present in 89% of PGLs, including all patients with AGNHL but only 71% of MALT. FDG uptake can be differentiated, in particular in AGNHL-PGL, from physiologic tracer activity by intensity but not by pattern. Extragastric foci on PET and structural CT abnormalities are additional parameters that can improve PET/CT assessment of PGL. Defining FDG avidity and PET/CT patterns in AGNHL and a subgroup of MALT-PGL before treatment may be important for further monitoring therapy response. (orig.)

  2. Lipid-binding proteins modulate ligand-dependent trans-activation by peroxisome proliferator-activated receptors and localize to the nucleus as well as the cytoplasm

    DEFF Research Database (Denmark)

    Helledie, T; Antonius, M; Sorensen, R V

    2000-01-01

    Peroxisome proliferator-activated receptors (PPARs) are activated by a variety of fatty acids, eicosanoids, and hypolipidemic and insulin-sensitizing drugs. Many of these compounds bind avidly to members of a family of small lipid-binding proteins, the fatty acid-binding proteins (FABPs). Fatty...

  3. The structure of avian polyomavirus reveals variably sized capsids, non-conserved inter-capsomere interactions, and a possible location of the minor capsid protein VP4

    International Nuclear Information System (INIS)

    Shen, Peter S.; Enderlein, Dirk; Nelson, Christian D.S.; Carter, Weston S.; Kawano, Masaaki; Xing Li; Swenson, Robert D.; Olson, Norman H.; Baker, Timothy S.; Cheng, R. Holland; Atwood, Walter J.; Johne, Reimar; Belnap, David M.

    2011-01-01

    Avian polyomavirus (APV) causes a fatal, multi-organ disease among several bird species. Using cryogenic electron microscopy and other biochemical techniques, we investigated the structure of APV and compared it to that of mammalian polyomaviruses, particularly JC polyomavirus and simian virus 40. The structure of the pentameric major capsid protein (VP1) is mostly conserved; however, APV VP1 has a unique, truncated C-terminus that eliminates an intercapsomere-connecting β-hairpin observed in other polyomaviruses. We postulate that the terminal β-hairpin locks other polyomavirus capsids in a stable conformation and that absence of the hairpin leads to the observed capsid size variation in APV. Plug-like density features were observed at the base of the VP1 pentamers, consistent with the known location of minor capsid proteins VP2 and VP3. However, the plug density is more prominent in APV and may include VP4, a minor capsid protein unique to bird polyomaviruses.

  4. Functional requirements of the yellow fever virus capsid protein.

    Science.gov (United States)

    Patkar, Chinmay G; Jones, Christopher T; Chang, Yu-hsuan; Warrier, Ranjit; Kuhn, Richard J

    2007-06-01

    Although it is known that the flavivirus capsid protein is essential for genome packaging and formation of infectious particles, the minimal requirements of the dimeric capsid protein for virus assembly/disassembly have not been characterized. By use of a trans-packaging system that involved packaging a yellow fever virus (YFV) replicon into pseudo-infectious particles by supplying the YFV structural proteins using a Sindbis virus helper construct, the functional elements within the YFV capsid protein (YFC) were characterized. Various N- and C-terminal truncations, internal deletions, and point mutations of YFC were analyzed for their ability to package the YFV replicon. Consistent with previous reports on the tick-borne encephalitis virus capsid protein, YFC demonstrates remarkable functional flexibility. Nearly 40 residues of YFC could be removed from the N terminus while the ability to package replicon RNA was retained. Additionally, YFC containing a deletion of approximately 27 residues of the C terminus, including a complete deletion of C-terminal helix 4, was functional. Internal deletions encompassing the internal hydrophobic sequence in YFC were, in general, tolerated to a lesser extent. Site-directed mutagenesis of helix 4 residues predicted to be involved in intermonomeric interactions were also analyzed, and although single mutations did not affect packaging, a YFC with the double mutation of leucine 81 and valine 88 was nonfunctional. The effects of mutations in YFC on the viability of YFV infection were also analyzed, and these results were similar to those obtained using the replicon packaging system, thus underscoring the flexibility of YFC with respect to the requirements for its functioning.

  5. The Cellular Chaperone Heat Shock Protein 90 Is Required for Foot-and-Mouth Disease Virus Capsid Precursor Processing and Assembly of Capsid Pentamers.

    Science.gov (United States)

    Newman, Joseph; Asfor, Amin S; Berryman, Stephen; Jackson, Terry; Curry, Stephen; Tuthill, Tobias J

    2018-03-01

    Productive picornavirus infection requires the hijacking of host cell pathways to aid with the different stages of virus entry, synthesis of the viral polyprotein, and viral genome replication. Many picornaviruses, including foot-and-mouth disease virus (FMDV), assemble capsids via the multimerization of several copies of a single capsid precursor protein into a pentameric subunit which further encapsidates the RNA. Pentamer formation is preceded by co- and posttranslational modification of the capsid precursor (P1-2A) by viral and cellular enzymes and the subsequent rearrangement of P1-2A into a structure amenable to pentamer formation. We have developed a cell-free system to study FMDV pentamer assembly using recombinantly expressed FMDV capsid precursor and 3C protease. Using this assay, we have shown that two structurally different inhibitors of the cellular chaperone heat shock protein 90 (hsp90) impeded FMDV capsid precursor processing and subsequent pentamer formation. Treatment of FMDV permissive cells with the hsp90 inhibitor prior to infection reduced the endpoint titer by more than 10-fold while not affecting the activity of a subgenomic replicon, indicating that translation and replication of viral RNA were unaffected by the drug. IMPORTANCE FMDV of the Picornaviridae family is a pathogen of huge economic importance to the livestock industry due to its effect on the restriction of livestock movement and necessary control measures required following an outbreak. The study of FMDV capsid assembly, and picornavirus capsid assembly more generally, has tended to be focused upon the formation of capsids from pentameric intermediates or the immediate cotranslational modification of the capsid precursor protein. Here, we describe a system to analyze the early stages of FMDV pentameric capsid intermediate assembly and demonstrate a novel requirement for the cellular chaperone hsp90 in the formation of these pentameric intermediates. We show the added complexity

  6. Cleavage sites within the poliovirus capsid protein precursors

    International Nuclear Information System (INIS)

    Larsen, G.R.; Anderson, C.W.; Dorner, A.J.; Semler, B.L.; Wimmer, E.

    1982-01-01

    Partial amino-terminal sequence analysis was performed on radiolabeled poliovirus capsid proteins VP1, VP2, and VP3. A computer-assisted comparison of the amino acid sequences obtained with that predicted by the nucleotide sequence of the poliovirus genome allows assignment of the amino terminus of each capsid protein to a unique position within the virus polyprotein. Sequence analysis of trypsin-digested VP4, which has a blocked amino terminus, demonstrates that VP4 is encoded at or very near to the amino terminus of the polyprotein. The gene order of the capsid proteins is VP4-VP2-VP3-VP1. Cleavage of VP0 to VP4 and VP2 is shown to occur between asparagine and serine, whereas the cleavages that separate VP2/VP3 and VP3/VP1 occur between glutamine and glycine residues. This finding supports the hypothesis that the cleavage of VP0, which occurs during virion morphogenesis, is distinct from the cleavages that separate functional regions of the polyprotein

  7. Assembly of recombinant Israeli Acute Paralysis Virus capsids.

    Directory of Open Access Journals (Sweden)

    Junyuan Ren

    Full Text Available The dicistrovirus Israeli Acute Paralysis Virus (IAPV has been implicated in the worldwide decline of honey bees. Studies of IAPV and many other bee viruses in pure culture are restricted by available isolates and permissive cell culture. Here we show that coupling the IAPV major structural precursor protein ORF2 to its cognate 3C-like processing enzyme results in processing of the precursor to the individual structural proteins in a number of insect cell lines following expression by a recombinant baculovirus. The efficiency of expression is influenced by the level of IAPV 3C protein and moderation of its activity is required for optimal expression. The mature IAPV structural proteins assembled into empty capsids that migrated as particles on sucrose velocity gradients and showed typical dicistrovirus like morphology when examined by electron microscopy. Monoclonal antibodies raised to recombinant capsids were configured into a diagnostic test specific for the presence of IAPV. Recombinant capsids for each of the many bee viruses within the picornavirus family may provide virus specific reagents for the on-going investigation of the causes of honeybee loss.

  8. Improved testing of recent HIV-1 infections with the BioRad avidity assay compared to the limiting antigen avidity assay and BED Capture enzyme immunoassay: evaluation using reference sample panels from the German Seroconverter Cohort.

    Directory of Open Access Journals (Sweden)

    Andrea Hauser

    Full Text Available BACKGROUND: The variety and limitations of current laboratory methods for estimating HIV-incidence has driven attempts to improve and standardize the performance of serological 'Tests for Recent HIV-Infections' (TRI. Primary and follow-up HIV-1 positive plasma samples from individuals with well-defined dates of infection collected as part of the German Seroconverter Cohort provided specimens highly suitable for use in comparing the performance of three TRIs: the AWARE™ BED™ EIA HIV-1 Incidence test (BED-CEIA, Genetic systems HIV-1/HIV-2 Plus O EIA antibody avidity-based assay (BioRad Avidity and Sedia™ HIV-1 LAg Avidity EIA (LAg Avidity. METHODS: The evaluation panel included 180 specimens: 44 from antiretroviral (ARV-naïve individuals with recently acquired HIV-infection (≤ 130 days; 25 B and 19 non-B subtypes and 136 from long-term (>12 months infected individuals [101 ARV-naïve subtype B, 16 non-B subtypes, 14 ARV-treated individuals, 5 slow progressors (SLP]. RESULTS: For long-term infected, ARV-naïve individuals the false recent rates (FRR of both the BioRad and LAg Avidity assays were 2% (2/101 for subtype B and 6% (1/16 for subtype 'non-B', while the FRR of the BED-CEIA was 7% (7/101 for subtype B and 25% (4/16 for subtype 'non-B' (all p>0.05. Misclassification of ARV-treated individuals and SLP was rare by LAg (1/14, 0/5 and BioRad Avidity assays (2/14, 1/5 but more frequent by BED-CEIA (5/14, 3/5. Among recently-infected individuals (subtype B, 60% (15/25 were correctly classified by BED-CEIA, 88% (22/25 by BioRad Avidity and significantly fewer by LAg (48%, 12/25 compared to BioRad Avidity (p = 0.005 with a higher true-recency rate among non-B infections for all assays. CONCLUSIONS: This study using well-characterized specimens demonstrated lower FRRs for both avidity methods than with the BED-CEIA. For recently infected individuals the BioRad Avidity assay was shown to give the most accurate results.

  9. Purification of recombinant budgerigar fledgling disease virus VP1 capsid protein and its ability for in vitro capsid assembly

    Science.gov (United States)

    Rodgers, R. E.; Chang, D.; Cai, X.; Consigli, R. A.; Spooner, B. S. (Principal Investigator)

    1994-01-01

    A recombinant system for the major capsid VP1 protein of budgerigar fledgling disease virus has been established. The VP1 gene was inserted into a truncated form of the pFlag-1 vector and expressed in Escherichia coli. The budgerigar fledgling disease virus VP1 protein was purified to near homogeneity by immunoaffinity chromatography. Fractions containing highly purified VP1 were pooled and found to constitute 3.3% of the original E. coli-expressed VP1 protein. Electron microscopy revealed that the VP1 protein was isolated as pentameric capsomeres. Electron microscopy also revealed that capsid-like particles were formed in vitro from purified VP1 capsomeres with the addition of Ca2+ ions and the removal of chelating and reducing agents.

  10. Facilitating the use of alternative capsid control methods towards sustainable production of organic cocoa in Ghana

    OpenAIRE

    Ayenor, G.K.; Huis, van, A.; Obeng-Ofori, D.; Padi, B.; Röling, N.G.

    2007-01-01

    Cocoa (Theobroma cacao L.) is an important foreign exchange earner for Ghana. However, production is constrained by a high incidence of pests and diseases. Based on farmers' needs, this study focused on the control of capsids, mainly Sahlbergella singularis Haglund and Distantiella theobroma (Distant) (both Hemiptera: Miridae). Annual crop loss caused by capsids is estimated at 25¿30%. To control capsids, formal research recommends application of synthetic insecticides four times between Augu...

  11. Highly specific salt bridges govern bacteriophage P22 icosahedral capsid assembly: identification of the site in coat protein responsible for interaction with scaffolding protein.

    Science.gov (United States)

    Cortines, Juliana R; Motwani, Tina; Vyas, Aashay A; Teschke, Carolyn M

    2014-05-01

    Icosahedral virus assembly requires a series of concerted and highly specific protein-protein interactions to produce a proper capsid. In bacteriophage P22, only coat protein (gp5) and scaffolding protein (gp8) are needed to assemble a procapsid-like particle, both in vivo and in vitro. In scaffolding protein's coat binding domain, residue R293 is required for procapsid assembly, while residue K296 is important but not essential. Here, we investigate the interaction of scaffolding protein with acidic residues in the N-arm of coat protein, since this interaction has been shown to be electrostatic. Through site-directed mutagenesis of genes 5 and 8, we show that changing coat protein N-arm residue 14 from aspartic acid to alanine causes a lethal phenotype. Coat protein residue D14 is shown by cross-linking to interact with scaffolding protein residue R293 and, thus, is intimately involved in proper procapsid assembly. To a lesser extent, coat protein N-arm residue E18 is also implicated in the interaction with scaffolding protein and is involved in capsid size determination, since a cysteine mutation at this site generated petite capsids. The final acidic residue in the N-arm that was tested, E15, is shown to only weakly interact with scaffolding protein's coat binding domain. This work supports growing evidence that surface charge density may be the driving force of virus capsid protein interactions. Bacteriophage P22 infects Salmonella enterica serovar Typhimurium and is a model for icosahedral viral capsid assembly. In this system, coat protein interacts with an internal scaffolding protein, triggering the assembly of an intermediate called a procapsid. Previously, we determined that there is a single amino acid in scaffolding protein required for P22 procapsid assembly, although others modulate affinity. Here, we identify partners in coat protein. We show experimentally that relatively weak interactions between coat and scaffolding proteins are capable of driving

  12. Specificity and sensitivity of binding proteins in the radioimmunoassay of cortisol

    International Nuclear Information System (INIS)

    Gijzen, A.H.J.

    1977-01-01

    A comparison concerning avidity towards cortisol and 10 other steroids was made between several binding proteins either in solution or bound to cellulose as so called ''solid phase'' reagent. Human blood cortisol binding protein (CBP, transcortin), and two distinctly different cortisol-binding rabbit antisera and the isolated immunoglobulins thereof were compared in their avidity to bind cortisol and several other steroids. The antisera were harvested from rabbits immunized with either cortisol-21-succinyl-albumin (CSA) or cortisol-3-oxim-albumin (COA). The latter antiserum, having the highest titre in cortisol titration, showed the greatest specificity and was most useful as a binding reagent in cortisol radioimmunoassay when used as a solid phase reagent. The determination of cortisol in micro samples of blood serum is possible without steroid extraction or serum protein denaturation and with only minor influence of steroid impurities in the sample to be analyzed. Affinity constants for all compared binding reagents and steroids are given

  13. Low levels of foot-and-mouth disease virus 3C protease expression are required to achieve optimal capsid protein expression and processing in mammalian cells

    DEFF Research Database (Denmark)

    Polacek, Charlotta; Gullberg, Maria; Li, Jiong

    2013-01-01

    transient-expression assays, within mammalian cells, it is possible to modify the relative amounts of the substrate and protease. It has now been shown that optimal production of the processed capsid proteins from P1-2A is achieved with reduced levels of 3Cpro expression, relative to the P1-2A, compared...... detected by FMDV antigen detection assays. Furthermore, the P1-2A and the processed forms each bind to the integrin αvβ6, the major FMDV receptor. These results contribute to the development of systems which efficiently express the components of empty capsid particles and may represent the basis for safer...... production of diagnostic reagents and improved vaccines against foot-and-mouth disease....

  14. The tripartite capsid gene of Salmonella phage Gifsy-2 yields a capsid assembly pathway engaging features from HK97 and λ

    International Nuclear Information System (INIS)

    Effantin, Gregory; Figueroa-Bossi, Nara; Schoehn, Guy; Bossi, Lionello; Conway, James F.

    2010-01-01

    Phage Gifsy-2, a lambdoid phage infecting Salmonella, has an unusually large composite gene coding for its major capsid protein (mcp) at the C-terminal end, a ClpP-like protease at the N-terminus, and a ∼ 200 residue central domain of unknown function but which may have a scaffolding role. This combination of functions on a single coding region is more extensive than those observed in other phages such as HK97 (scaffold-capsid fusion) and λ (protease-scaffold fusion). To study the structural phenotype of the unique Gifsy-2 capsid gene, we have purified Gifsy-2 particles and visualized capsids and procapsids by cryoelectron microscopy, determining structures to resolutions up to 12 A. The capsids have lambdoid T = 7 geometry and are well modeled with the atomic structures of HK97 mcp and phage λ gpD decoration protein. Thus, the unique Gifsy-2 capsid protein gene yields a capsid maturation pathway engaging features from both phages HK97 and λ.

  15. Serodiagnosis of Toxoplasmosis: The effect of measurement of IgG avidity in pregnant women in Rabat in Morocco.

    Science.gov (United States)

    Laboudi, Majda; Sadak, Abderrahim

    2017-08-01

    The diagnosis of Toxoplasmosis in pregnant women during the early first trimester of pregnancy is very important for preventing congenital infection of the fetus; it will not only prevent the risk of transmitting the infection to the fetus but it will also enable to give these women a preventive treatment. In this study, the avidity test was performed on pregnant women during their first prenatal visit at the National Institute of Hygiene in Rabat, Morocco. One hundred and twenty-eight sera samples were collected from 128 pregnant women between August 2015 and June 2016; these women were chosen retrospectively and were in their first four months of pregnancy. The samples were screened using the specific anti-Toxoplasma IgG and IgM antibodies and were subjected to an IgG avidity test. After the serological screening, only 54 women (42.4%) were tested positive for IgG antibodies and five women (3.9%) were tested positive for both anti-Toxoplasma IgG and IgM antibodies. Four IgM-negative women had low-avidity antibodies. However, none of the IgG-avidity test had detected low-avidity antibodies in the five IgM-positive women; three women (60%) had high-avidity antibodies, indicating that the infection was acquired in the distant past. The avidity test is a helpful tool to exclude a recently acquired toxoplasmosis infection within IgM-positive serum samples in pregnant women during their first trimester of pregnancy. Thus, allowing to perform an appropriate therapeutic intervention. Copyright © 2017 Elsevier B.V. All rights reserved.

  16. Excretion and toxicity evaluation of 131I-Sennoside A as a necrosis-avid agent.

    Science.gov (United States)

    Yin, Zhiqi; Sun, Lidan; Jin, Qiaomei; Song, Shaoli; Feng, Yuanbo; Liao, Hong; Ni, Yicheng; Zhang, Jian; Liu, Wei

    2017-11-01

    1. Sennoside A (SA) is a newly identified necrosis-avid agent that shows capability for imaging diagnosis and tumor necrosis targeted radiotherapy. As a water-soluble compound, 131 I-Sennoside A ( 131 I-SA) might be excreted predominately through the kidneys with the possibility of nephrotoxicity. 2. To further verify excretion pathway and examine nephrotoxicity of 131 I-SA, excretion and nephrotoxicity were appraised. The pharmacokinetics, hepatotoxicity and hematotoxicity of 131 I-SA were also evaluated to accelerate its possible clinical translation. All these studies were conducted in mice with ethanol-induced muscular necrosis following a single intravenous administration of 131I-SA at 18.5 MBq/kg or 370 MBq/kg. 3. Excretion data revealed that 131 I-SA was predominately (73.5% of the injected dose (% ID)) excreted via the kidneys with 69.5% ID detected in urine within 72 h post injection. Biodistribution study indicated that 131 I-SA exhibited initial high distribution in the kidneys but subsequently a fast renal clearance, which was further confirmed by the results of autoradiography and single-photon emission computed tomography-computed tomography (SPECT-CT) imaging. The maximum necrotic to normal muscle ratio reached to 7.9-fold at 48 h post injection, which further verified the necrosis avidity of 131 I-SA. Pharmacokinetic parameters showed that 131 I-SA had fast blood clearance with an elimination half-life of 6.7 h. Various functional indexes were no significant difference (p > 0.05) between before administration and 1 d, 8 d, 16 d after administration. Histopathology showed no signs of tissue damage. 4. These data suggest 131 I-SA is a safe and promising necrosis-avid agent applicable in imaging diagnosis and tumor necrosis targeted radiotherapy.

  17. Motion-specific internal target volumes for FDG-avid mediastinal and hilar lymph nodes

    International Nuclear Information System (INIS)

    Lamb, James M.; Robinson, Clifford G.; Bradley, Jeffrey D.; Low, Daniel A.

    2013-01-01

    Background and purpose: To quantify the benefit of motion-specific internal target volumes for FDG-avid mediastinal and hilar lymph nodes generated using 4D-PET, vs. conventional internal target volumes generated using non-respiratory gated PET and 4D-CT scans. Materials and methods: Five patients with FDG-avid tumors metastatic to 11 hilar or mediastinal lymph nodes were imaged with respiratory-correlated FDG-PET (4D-PET) and 4D-CT. FDG-avid nodes were contoured by a radiation oncologist in two ways. Standard-of-care volumes were contoured using conventional un-gated PET, 4D-CT, and breath-hold CT. A second, motion-specific, set of volumes were contoured using 4D-PET.Contours based on 4D-PET corresponded directly to an internal target volume (ITV 4D ), whereas contours based on un-gated PET were expanded by a series of exploratory isotropic margins (from 5 to 13 mm) based on literature recommendations on lymph node motion to form internal target volumes (ITV 3D ). Results: A 13 mm expansion of the un-gated PET nodal volume was needed to cover the ITV 4D for 10 of 11 nodes studied. The ITV 3D based on a 13 mm expansion included on average 45 cm 3 of tissue that was not included in the ITV 4D . Conclusions: Motion-specific lymph-node internal target volumes generated from 4D-PET imaging could be used to improve accuracy and/or reduce normal-tissue irradiation compared to the standard-of-care un-gated PET based internal target volumes

  18. Clinical significance of pretreatment FDG PET/CT IN MOBG-avid pediatric neuroblastoma

    Energy Technology Data Exchange (ETDEWEB)

    Kang, Seo Young; Kim, Yong Il; Cheon, Gi Jeong; Kang, Keon Wook; Chung, June Key; Lee, Dong Soo; Kang, Hyoung Jin; Shin, Hee Young [Seoul National University Hospital, Seoul (Korea, Republic of); Kim, E. Edmund [Seoul National University, Seoul (Korea, Republic of); Rahim, Muhammad Kashif [Nishtar Medical College and Hospital, Multan (Pakistan)

    2017-06-15

    {sup 18}F-fluorodeoxyglucose-positron emission tomography (FDG-PET) imaging is well known to have clinical significance in the initial staging and response evaluation of the many kinds of neoplasms. However, its role in the pediatric neuroblastoma is not clearly defined. In the present study, the clinical significance of FDG-PET/computed tomography (CT) in 123I- or 131I-metaiodobenzylguanidine (MIBG)-avid pediatric neuroblastoma was investigated. Twenty patients with neuroblastoma who undertook pretreatment FDG PET/CT at our institute between 2008 and 2015 and showed MIBG avidity were retrospectively enrolled in the present study. Clinical information—including histopathology, and serum markers—and several PET parameters—including SUVmax of the primary lesion (Psuv), target-to-background ratio (TBR), metabolic tumor volume (MTV), and coefficient of variation (CV)—were analyzed. The prognostic effect of PET parameters was evaluated in terms of progression-free survival (PFS). Total 20 patients (4.5 ± 3.5 years) were divided as two groups by disease progression. Six patients (30.0 %) experienced disease progression and one patient (5.0 %) died during follow-up period. There were not statistically significant in age, stage, MYCN status, primary tumor size, serum lactate dehydrogenase (LDH), neuron-specific enolase (NSE), and ferritin level between two groups with progression or no progression. However, Psuv (p = 0.017), TBR (p = 0.09), MTV (p = 0.02), and CV (p = 0.036) showed significant differences between two groups. In univariate analysis, PFS was significantly associated with Psuv (p = 0.021) and TBR (p = 0.023). FDG-PET parameters were significantly related with progression of neuroblastoma. FDG-PET/CT may have the potential as a valuable modality for evaluating prognosis in the patients with MIBG-avid pediatric neuroblastoma.

  19. MIBG avidity correlates with clinical features, tumor biology, and outcomes in neuroblastoma: A report from the Children's Oncology Group.

    Science.gov (United States)

    DuBois, Steven G; Mody, Rajen; Naranjo, Arlene; Van Ryn, Collin; Russ, Douglas; Oldridge, Derek; Kreissman, Susan; Baker, David L; Parisi, Marguerite; Shulkin, Barry L; Bai, Harrison; Diskin, Sharon J; Batra, Vandana; Maris, John M; Park, Julie R; Matthay, Katherine K; Yanik, Gregory

    2017-11-01

    Prior studies suggest that neuroblastomas that do not accumulate metaiodobenzylguanidine (MIBG) on diagnostic imaging (MIBG non-avid) may have more favorable features compared with MIBG avid tumors. We compared clinical features, biologic features, and clinical outcomes between patients with MIBG nonavid and MIBG avid neuroblastoma. Patients had metastatic high- or intermediate-risk neuroblastoma and were treated on Children's Oncology Group protocols A3973 or A3961. Comparisons of clinical and biologic features according to MIBG avidity were made with chi-squared or Fisher exact tests. Event-free (EFS) and overall (OS) survival compared using log-rank tests and modeled using Cox models. Thirty of 343 patients (8.7%) had MIBG nonavid disease. Patients with nonavid tumors were less likely to have adrenal primary tumors (34.5 vs. 57.2%; P = 0.019), bone metastases (36.7 vs. 61.7%; P = 0.008), or positive urine catecholamines (66.7 vs. 91.0%; P neuroblastoma have lower rates of adrenal primary tumors, bone metastasis, and catecholamine secretion. Despite being more likely to have MYCN-amplified tumors, these patients have superior outcomes compared with patients with MIBG avid disease. © 2017 Wiley Periodicals, Inc.

  20. Antigenic structure of the capsid protein of rabbit haemorrhagic disease virus

    DEFF Research Database (Denmark)

    Martinez-Torrecuadrada, Jorge L.; Cortes, Elena; Vela, Carmen

    1998-01-01

    Rabbit haemorrhagic disease virus (RHDV) causes an important disease in rabbits. The virus capsid is composed of a single 60 kDa protein. The capsid protein gene was cloned in Escherichia coli using the pET3 system, and the antigenic structure of RHDV VP60 was dissected using 11 monoclonal...

  1. Identification of Factors Promoting HBV Capsid Self-Assembly by Assembly-Promoting Antivirals.

    Science.gov (United States)

    Rath, Soumya Lipsa; Liu, Huihui; Okazaki, Susumu; Shinoda, Wataru

    2018-02-26

    Around 270 million individuals currently live with hepatitis B virus (HBV) infection. Heteroaryldihydropyrimidines (HAPs) are a family of antivirals that target the HBV capsid protein and induce aberrant self-assembly. The capsids formed resemble the native capsid structure but are unable to propagate the virus progeny because of a lack of RNA/DNA. Under normal conditions, self-assembly is initiated by the viral genome. The mode of action of HAPs, however, remains largely unknown. In this work, using molecular dynamics simulations, we attempted to understand the action of HAP by comparing the dynamics of capsid proteins with and without HAPs. We found that the inhibitor is more stable in higher oligomers. It retains its stability in the hexamer throughout 1 μs of simulation. Our results also show that the inhibitor might help in stabilizing the C-terminus, the HBc 149-183 arginine-rich domain of the capsid protein. The C-termini of dimers interact with each other, assisted by the HAP inhibitor. During capsid assembly, the termini are supposed to directly interact with the viral genome, thereby suggesting that the viral genome might work in a similar way to stabilize the capsid protein. Our results may help in understanding the underlying molecular mechanism of HBV capsid self-assembly, which should be crucial for exploring new drug targets and structure-based drug design.

  2. Varicella-zoster virus induces the formation of dynamic nuclear capsid aggregates

    Energy Technology Data Exchange (ETDEWEB)

    Lebrun, Marielle [University of Liege (ULg), GIGA-Infection Immunity and Inflammation, Laboratory of Virology and Immunology, Liege (Belgium); Thelen, Nicolas; Thiry, Marc [University of Liege (ULg), GIGA-Neurosciences, Laboratory of Cellular and Tissular Biology, Liege (Belgium); Riva, Laura; Ote, Isabelle; Condé, Claude; Vandevenne, Patricia [University of Liege (ULg), GIGA-Infection Immunity and Inflammation, Laboratory of Virology and Immunology, Liege (Belgium); Di Valentin, Emmanuel [University of Liege (ULg), GIGA-Viral Vectors Platform, Liege (Belgium); Bontems, Sébastien [University of Liege (ULg), GIGA-Infection Immunity and Inflammation, Laboratory of Virology and Immunology, Liege (Belgium); Sadzot-Delvaux, Catherine, E-mail: csadzot@ulg.ac.be [University of Liege (ULg), GIGA-Infection Immunity and Inflammation, Laboratory of Virology and Immunology, Liege (Belgium)

    2014-04-15

    The first step of herpesviruses virion assembly occurs in the nucleus. However, the exact site where nucleocapsids are assembled, where the genome and the inner tegument are acquired, remains controversial. We created a recombinant VZV expressing ORF23 (homologous to HSV-1 VP26) fused to the eGFP and dually fluorescent viruses with a tegument protein additionally fused to a red tag (ORF9, ORF21 and ORF22 corresponding to HSV-1 UL49, UL37 and UL36). We identified nuclear dense structures containing the major capsid protein, the scaffold protein and maturing protease, as well as ORF21 and ORF22. Correlative microscopy demonstrated that the structures correspond to capsid aggregates and time-lapse video imaging showed that they appear prior to the accumulation of cytoplasmic capsids, presumably undergoing the secondary egress, and are highly dynamic. Our observations suggest that these structures might represent a nuclear area important for capsid assembly and/or maturation before the budding at the inner nuclear membrane. - Highlights: • We created a recombinant VZV expressing the small capsid protein fused to the eGFP. • We identified nuclear dense structures containing capsid and procapsid proteins. • Correlative microscopy showed that the structures correspond to capsid aggregates. • Procapsids and partial capsids are found within the aggregates of WT and eGFP-23 VZV. • FRAP and FLIP experiments demonstrated that they are dynamic structures.

  3. Nucleolin Interacts with the Dengue Virus Capsid Protein and Plays a Role in Formation of Infectious Virus Particles

    Science.gov (United States)

    Balinsky, Corey A.; Schmeisser, Hana; Ganesan, Sundar; Singh, Kavita; Pierson, Theodore C.

    2013-01-01

    Dengue virus (DENV) is a mosquito-transmitted flavivirus that can cause severe disease in humans and is considered a reemerging pathogen of significant importance to public health. The DENV capsid (C) protein functions as a structural component of the infectious virion; however, it may have additional functions in the virus replicative cycle. Here, we show that the DENV C protein interacts and colocalizes with the multifunctional host protein nucleolin (NCL). Furthermore, we demonstrate that this interaction can be disrupted by the addition of an NCL binding aptamer (AS1411). Knockdown of NCL with small interfering RNA (siRNA) or treatment of cells with AS1411 results in a significant reduction of viral titers after DENV infection. Western blotting and quantitative RT-PCR (qRT-PCR) analysis revealed no differences in viral RNA or protein levels at early time points postinfection, suggesting a role for NCL in viral morphogenesis. We support this hypothesis by showing that treatment with AS1411 alters the migration characteristics of the viral capsid, as visualized by native electrophoresis. Here, we identify a critical interaction between DENV C protein and NCL that represents a potential new target for the development of antiviral therapeutics. PMID:24027323

  4. Evolutionary changes in the capsid P2 region of Australian strains of the norovirus GII.Pe_GII.4.

    Science.gov (United States)

    Bruggink, Leesa D; Moselen, Jean M; Roberts, Jason A; Marshall, John A

    2017-07-01

    The protruding (P) 2 region of the norovirus capsid is thought to include hypervariable sites involved in receptor binding. This study examines the changes that occurred in the P2 region of GII.Pe_GII.4 norovirus in the course of its evolution from a precursor phase (2008-2009), to an intermediate phase (2010) and finally to an epidemic phase (2012-2015). Twenty-two P2 region amino acid (aa) sequences (166 aa long) from all phases of the evolution of the virus were compared and the changes analysed.Results/key findings. Twenty sites in the P2 region underwent aa change and of these, 10 corresponded to previously proposed hypervariable sites and 10 to novel hypervariable sites. It was notable that aa changes at two sites, X and Y, only emerged as the epidemic phase progressed. 3D computer modelling of the P2 region indicated that neither X nor Y were in the uppermost 'crown', but further down in the 'neck' portion. The location of X and Y and the nature of aa change at Y suggest these sites were important in enhancing the structural integrity of the capsid, which in turn may have facilitated the longer term viability of the virus. The current study helps establish the validity of previously proposed hypervariable sites in the P2 region as well as indicating new ones. It also provides quantitative and qualitative data on how these sites changed over the evolutionary history of a particular norovirus strain.

  5. Inner tegument proteins of Herpes Simplex Virus are sufficient for intracellular capsid motility in neurons but not for axonal targeting

    Science.gov (United States)

    Müller, Oliver; Ivanova, Lyudmila; Bialy, Dagmara; Pohlmann, Anja; Binz, Anne; Hegemann, Maike; Viejo-Borbolla, Abel; Rosenhahn, Bodo; Bauerfeind, Rudolf; Sodeik, Beate

    2017-01-01

    Upon reactivation from latency and during lytic infections in neurons, alphaherpesviruses assemble cytosolic capsids, capsids associated with enveloping membranes, and transport vesicles harboring fully enveloped capsids. It is debated whether capsid envelopment of herpes simplex virus (HSV) is completed in the soma prior to axonal targeting or later, and whether the mechanisms are the same in neurons derived from embryos or from adult hosts. We used HSV mutants impaired in capsid envelopment to test whether the inner tegument proteins pUL36 or pUL37 necessary for microtubule-mediated capsid transport were sufficient for axonal capsid targeting in neurons derived from the dorsal root ganglia of adult mice. Such neurons were infected with HSV1-ΔUL20 whose capsids recruited pUL36 and pUL37, with HSV1-ΔUL37 whose capsids associate only with pUL36, or with HSV1-ΔUL36 that assembles capsids lacking both proteins. While capsids of HSV1-ΔUL20 were actively transported along microtubules in epithelial cells and in the somata of neurons, those of HSV1-ΔUL36 and -ΔUL37 could only diffuse in the cytoplasm. Employing a novel image analysis algorithm to quantify capsid targeting to axons, we show that only a few capsids of HSV1-ΔUL20 entered axons, while vesicles transporting gD utilized axonal transport efficiently and independently of pUL36, pUL37, or pUL20. Our data indicate that capsid motility in the somata of neurons mediated by pUL36 and pUL37 does not suffice for targeting capsids to axons, and suggest that capsid envelopment needs to be completed in the soma prior to targeting of herpes simplex virus to the axons, and to spreading from neurons to neighboring cells. PMID:29284065

  6. Expression of Aleutian mink disease parvovirus capsid proteins in defined segments: localization of immunoreactive sites and neutralizing epitopes to specific regions.

    Science.gov (United States)

    Bloom, M E; Martin, D A; Oie, K L; Huhtanen, M E; Costello, F; Wolfinbarger, J B; Hayes, S F; Agbandje-McKenna, M

    1997-01-01

    The capsid proteins of the ADV-G isolate of Aleutian mink disease parvovirus (ADV) were expressed in 10 nonoverlapping segments as fusions with maltose-binding protein in pMAL-C2 (pVP1, pVP2a through pVP2i). The constructs were designed to capture the VP1 unique sequence and the portions analogous to the four variable surface loops of canine parvovirus (CPV) in individual fragments (pVP2b, pVP2d, pVP2e, and pVP2g, respectively). The panel of fusion proteins was immunoblotted with sera from mink infected with ADV. Seropositive mink infected with either ADV-TR, ADV-Utah, or ADV-Pullman reacted preferentially against certain segments, regardless of mink genotype or virus inoculum. The most consistently immunoreactive regions were pVP2g, pVP2e, and pVP2f, the segments that encompassed the analogs of CPV surface loops 3 and 4. The VP1 unique region was also consistently immunoreactive. These findings indicated that infected mink recognize linear epitopes that localized to certain regions of the capsid protein sequence. The segment containing the hypervariable region (pVP2d), corresponding to CPV loop 2, was also expressed from ADV-Utah. An anti-ADV-G monoclonal antibody and a rabbit anti-ADV-G capsid antibody reacted exclusively with the ADV-G pVP2d segment but not with the corresponding segment from ADV-Utah. Mink infected with ADV-TR or ADV-Utah also preferentially reacted with the pVP2d sequence characteristic of that virus. These results suggested that the loop 2 region may contain a type-specific linear epitope and that the epitope may also be specifically recognized by infected mink. Heterologous antisera were prepared against the VP1 unique region and the four segments capturing the variable surface loops of CPV. The antisera against the proteins containing loop 3 or loop 4, as well as the anticapsid antibody, neutralized ADV-G infectivity in vitro and bound to capsids in immune electron microscopy. These results suggested that regions of the ADV capsid proteins

  7. Eclipse Phase of Herpes Simplex Virus Type 1 Infection: Efficient Dynein-Mediated Capsid Transport without the Small Capsid Protein VP26

    Science.gov (United States)

    Döhner, Katinka; Radtke, Kerstin; Schmidt, Simone; Sodeik, Beate

    2006-01-01

    Cytoplasmic dynein,together with its cofactor dynactin, transports incoming herpes simplex virus type 1 (HSV-1) capsids along microtubules (MT) to the MT-organizing center (MTOC). From the MTOC, capsids move further to the nuclear pore, where the viral genome is released into the nucleoplasm. The small capsid protein VP26 can interact with the dynein light chains Tctex1 (DYNLT1) and rp3 (DYNLT3) and may recruit dynein to the capsid. Therefore, we analyzed nuclear targeting of incoming HSV1-ΔVP26 capsids devoid of VP26 and of HSV1-GFPVP26 capsids expressing a GFPVP26 fusion instead of VP26. To compare the cell entry of different strains, we characterized the inocula with respect to infectivity, viral genome content, protein composition, and particle composition. Preparations with a low particle-to-PFU ratio showed efficient nuclear targeting and were considered to be of higher quality than those containing many defective particles, which were unable to induce plaque formation. When cells were infected with HSV-1 wild type, HSV1-ΔVP26, or HSV1-GFPVP26, viral capsids were transported along MT to the nucleus. Moreover, when dynein function was inhibited by overexpression of the dynactin subunit dynamitin, fewer capsids of HSV-1 wild type, HSV1-ΔVP26, and HSV1-GFPVP26 arrived at the nucleus. Thus, even in the absence of the potential viral dynein receptor VP26, HSV-1 used MT and dynein for efficient nuclear targeting. These data suggest that besides VP26, HSV-1 encodes other receptors for dynein or dynactin. PMID:16873277

  8. First preclinical evaluation of mono-[123I]iodohypericin as a necrosis-avid tracer agent

    International Nuclear Information System (INIS)

    Ni, Yicheng; Chen, Feng; Marchal, Guy; Huyghe, Dieter; Verbeke, Kristin; Verbruggen, Alfons M.; Bormans, Guy M.; Witte, Peter A. de; Nuyts, Johan; Mortelmans, Luc

    2006-01-01

    We have labelled hypericin, a polyphenolic polycyclic quinone found in St. John's wort (Hypericum perforatum), with 123 I and evaluated mono-[ 123 I]iodohypericin (MIH) as a potential necrosis-avid diagnostic tracer agent. MIH was prepared by an electrophilic radioiodination method. The new tracer agent was evaluated in animal models of liver infarction in the rat and heart infarction in the rabbit using single-photon emission computed tomography (SPECT), triphenyltetrazolium chloride (TTC) histochemical staining, serial sectional autoradiography and microscopy, and radioactivity counting techniques. Using in vivo SPECT imaging, hepatic and cardiac infarctions were persistently visualised as well-defined hot spots over 48 h. Preferential uptake of the tracer agent in necrotic tissue was confirmed by perfect match of images from post-mortem TTC staining, autoradiography (ARX) and histology. Radioactivity concentration in infarcted tissues was over 10 times (liver; 3.51% ID/g in necrotic tissue vs 0.38% ID/g in normal tissue at 60 h p.i.) and over 6 times (myocardium; 0.36% ID/g in necrotic tissue vs 0.054% ID/g in normal tissue; ratios up to 18 for selected parts on ARX images) higher than in normal tissues. The results suggest that hypericin derivatives may serve as powerful necrosis-avid diagnostic agents for assessment of tissue viability. (orig.)

  9. Breaking Symmetry in Viral Icosahedral Capsids as Seen through the Lenses of X-ray Crystallography and Cryo-Electron Microscopy

    Directory of Open Access Journals (Sweden)

    Kristin N. Parent

    2018-02-01

    Full Text Available The majority of viruses on Earth form capsids built by multiple copies of one or more types of a coat protein arranged with 532 symmetry, generating an icosahedral shell. This highly repetitive structure is ideal to closely pack identical protein subunits and to enclose the nucleic acid genomes. However, the icosahedral capsid is not merely a passive cage but undergoes dynamic events to promote packaging, maturation and the transfer of the viral genome into the host. These essential processes are often mediated by proteinaceous complexes that interrupt the shell’s icosahedral symmetry, providing a gateway through the capsid. In this review, we take an inventory of molecular structures observed either internally, or at the 5-fold vertices of icosahedral DNA viruses that infect bacteria, archea and eukaryotes. Taking advantage of the recent revolution in cryo-electron microscopy (cryo-EM and building upon a wealth of crystallographic structures of individual components, we review the design principles of non-icosahedral structural components that interrupt icosahedral symmetry and discuss how these macromolecules play vital roles in genome packaging, ejection and host receptor-binding.

  10. Reactive oxygen species promote heat shock protein 90-mediated HBV capsid assembly

    International Nuclear Information System (INIS)

    Kim, Yoon Sik; Seo, Hyun Wook; Jung, Guhung

    2015-01-01

    Hepatitis B virus (HBV) infection induces reactive oxygen species (ROS) production and has been associated with the development of hepatocellular carcinoma (HCC). ROS are also an important factor in HCC because the accumulated ROS leads to abnormal cell proliferation and chromosome mutation. In oxidative stress, heat shock protein 90 (Hsp90) and glutathione (GSH) function as part of the defense mechanism. Hsp90 prevents cellular component from oxidative stress, and GSH acts as antioxidants scavenging ROS in the cell. However, it is not known whether molecules regulated by oxidative stress are involved in HBV capsid assembly. Based on the previous study that Hsp90 facilitates HBV capsid assembly, which is an important step for the packing of viral particles, here, we show that ROS enrich Hsp90-driven HBV capsid formation. In cell-free system, HBV capsid assembly was facilitated by ROS with Hsp90, whereas it was decreased without Hsp90. In addition, GSH inhibited the function of Hsp90 to decrease HBV capsid assembly. Consistent with the result of cell-free system, ROS and buthionine sulfoximine (BS), an inhibitor of GSH synthesis, increased HBV capsid formation in HepG2.2.15 cells. Thus, our study uncovers the interplay between ROS and Hsp90 during HBV capsid assembly. - Highlights: • We examined H 2 O 2 and GSH modulate HBV capsid assembly. • H 2 O 2 facilitates HBV capsid assembly in the presence of Hsp90. • GSH inhibits function of Hsp90 in facilitating HBV capsid assembly. • H 2 O 2 and GSH induce conformation change of Hsp90

  11. Revised Mimivirus major capsid protein sequence reveals intron-containing gene structure and extra domain

    Directory of Open Access Journals (Sweden)

    Suzan-Monti Marie

    2009-05-01

    Full Text Available Abstract Background Acanthamoebae polyphaga Mimivirus (APM is the largest known dsDNA virus. The viral particle has a nearly icosahedral structure with an internal capsid shell surrounded with a dense layer of fibrils. A Capsid protein sequence, D13L, was deduced from the APM L425 coding gene and was shown to be the most abundant protein found within the viral particle. However this protein remained poorly characterised until now. A revised protein sequence deposited in a database suggested an additional N-terminal stretch of 142 amino acids missing from the original deduced sequence. This result led us to investigate the L425 gene structure and the biochemical properties of the complete APM major Capsid protein. Results This study describes the full length 3430 bp Capsid coding gene and characterises the 593 amino acids long corresponding Capsid protein 1. The recombinant full length protein allowed the production of a specific monoclonal antibody able to detect the Capsid protein 1 within the viral particle. This protein appeared to be post-translationnally modified by glycosylation and phosphorylation. We proposed a secondary structure prediction of APM Capsid protein 1 compared to the Capsid protein structure of Paramecium Bursaria Chlorella Virus 1, another member of the Nucleo-Cytoplasmic Large DNA virus family. Conclusion The characterisation of the full length L425 Capsid coding gene of Acanthamoebae polyphaga Mimivirus provides new insights into the structure of the main Capsid protein. The production of a full length recombinant protein will be useful for further structural studies.

  12. Reactive oxygen species promote heat shock protein 90-mediated HBV capsid assembly

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Yoon Sik, E-mail: yumshak@naver.com; Seo, Hyun Wook, E-mail: suruk@naver.com; Jung, Guhung, E-mail: drjung@snu.ac.kr

    2015-02-13

    Hepatitis B virus (HBV) infection induces reactive oxygen species (ROS) production and has been associated with the development of hepatocellular carcinoma (HCC). ROS are also an important factor in HCC because the accumulated ROS leads to abnormal cell proliferation and chromosome mutation. In oxidative stress, heat shock protein 90 (Hsp90) and glutathione (GSH) function as part of the defense mechanism. Hsp90 prevents cellular component from oxidative stress, and GSH acts as antioxidants scavenging ROS in the cell. However, it is not known whether molecules regulated by oxidative stress are involved in HBV capsid assembly. Based on the previous study that Hsp90 facilitates HBV capsid assembly, which is an important step for the packing of viral particles, here, we show that ROS enrich Hsp90-driven HBV capsid formation. In cell-free system, HBV capsid assembly was facilitated by ROS with Hsp90, whereas it was decreased without Hsp90. In addition, GSH inhibited the function of Hsp90 to decrease HBV capsid assembly. Consistent with the result of cell-free system, ROS and buthionine sulfoximine (BS), an inhibitor of GSH synthesis, increased HBV capsid formation in HepG2.2.15 cells. Thus, our study uncovers the interplay between ROS and Hsp90 during HBV capsid assembly. - Highlights: • We examined H{sub 2}O{sub 2} and GSH modulate HBV capsid assembly. • H{sub 2}O{sub 2} facilitates HBV capsid assembly in the presence of Hsp90. • GSH inhibits function of Hsp90 in facilitating HBV capsid assembly. • H{sub 2}O{sub 2} and GSH induce conformation change of Hsp90.

  13. Analysis of mumps vaccine failure by means of avidity testing for mumps virus-specific immunoglobulin G.

    Science.gov (United States)

    Narita, M; Matsuzono, Y; Takekoshi, Y; Yamada, S; Itakura, O; Kubota, M; Kikuta, H; Togashi, T

    1998-11-01

    To characterize patients with mumps vaccine failure, avidity testing was performed with the Enzygnost Anti-Parotitis Virus/IgG kit using a single-dilution-6 M urea denaturation method. Five groups of patients were tested. Group 1 consisted of 29 patients with primary mumps infections; group 2 was 20 children and adults with a definite history of natural infection; group 3 was 7 patients with a recent mumps vaccination, 1 of whom developed parotid gland swelling and aseptic meningitis; group 4 was 14 patients with mumps vaccine failure; and group 5 was 6 patients with recurrent episodes of parotitis in addition to a history of vaccination. On the basis of the results of groups 1 and 2, an avidity of /=32% was determined to be high. Avidity maturation from low to high appears to occur around 180 days after the acute illness. The results of group 3 showed that the vaccine-induced immunoglobulin G (IgG) had very low avidity. Among the 14 patients in group 4, 12 patients, including 7 with a positive IgM response, were diagnosed as having secondary vaccine failures. The results of group 5 suggested the possibility that the avidity of the mumps vaccine-induced IgG remains low or borderline. These results showed that secondary mumps vaccine failure occurs not infrequently, even among school age children under condition in which the vaccine coverage is low (i.e., 33% in our study population), and therefore, vaccinees are prone to be exposed to wild-type viruses. Avidity testing should provide information useful for the analysis of mumps virus infections.

  14. Pregnancy does not affect HIV incidence test results obtained using the BED capture enzyme immunoassay or an antibody avidity assay.

    Directory of Open Access Journals (Sweden)

    Oliver Laeyendecker

    2010-10-01

    Full Text Available Accurate incidence estimates are needed for surveillance of the HIV epidemic. HIV surveillance occurs at maternal-child health clinics, but it is not known if pregnancy affects HIV incidence testing.We used the BED capture immunoassay (BED and an antibody avidity assay to test longitudinal samples from 51 HIV-infected Ugandan women infected with subtype A, C, D and intersubtype recombinant HIV who were enrolled in the HIVNET 012 trial (37 baseline samples collected near the time of delivery and 135 follow-up samples collected 3, 4 or 5 years later. Nineteen of 51 women were also pregnant at the time of one or more of the follow-up visits. The BED assay was performed according to the manufacturer's instructions. The avidity assay was performed using a Genetic Systems HIV-1/HIV-2 + O EIA using 0.1M diethylamine as the chaotropic agent.During the HIVNET 012 follow-up study, there was no difference in normalized optical density values (OD-n obtained with the BED assay or in the avidity test results (% when women were pregnant (n = 20 results compared to those obtained when women were not pregnant (n = 115; for BED: p = 0.9, generalized estimating equations model; for avidity: p = 0.7, Wilcoxon rank sum. In addition, BED and avidity results were almost exactly the same in longitudinal samples from the 18 women who were pregnant at only one study visit during the follow-up study (p = 0.6, paired t-test.These results from 51 Ugandan women suggest that any changes in the antibody response to HIV infection that occur during pregnancy are not sufficient to alter results obtained with the BED and avidity assays. Confirmation with larger studies and with other HIV subtypes is needed.

  15. Kinetic and HPV infection effects on cross-type neutralizing antibody and avidity responses induced by Cervarix®

    Science.gov (United States)

    Kemp, Troy J.; Safaeian, Mahboobeh; Hildesheim, Allan; Pan, Yuanji; Penrose, Kerri J.; Porras, Carolina; Schiller, John T.; Lowy, Douglas R.; Herrero, Rolando; Pinto, Ligia A.

    2012-01-01

    Background We previously demonstrated that Cervarix® elicits antibody responses against vaccine-related types for which clinical efficacy was demonstrated (HPV-31 and -45). Here, we evaluated the kinetics of neutralization titers and avidity of Cervarix®-induced antibodies up to 36 months of follow-up in unexposed and HPV infected women. Methods A subset of women who participated in the Cost Rica HPV-16/18 Vaccine Trial had pre- and post-vaccination sera tested for antibody responses to HPV-16, -18, -31, -45, and -58 using a pseudovirion-based neutralization assay, and HPV-16 antibody avidity using an HPV-16 L1 VLP (virus-like particle)-based ELISA developed in our laboratory. Results In uninfected women, neutralizing antibody titers did not reach significance until after the 3rd dose for HPV-31 (month 12, p=0.009) and HPV-45 (month 12, p=0.003), but then persisted up to month 36 (HPV-31, p=0.01; HPV-45, p=0.002). Individuals infected with HPV-16 or HPV-31 at enrollment developed a significantly higher median antibody response to the corresponding HPV type after one dose, but there was not a difference between median titers after three doses compared to the HPV negative group. Median HPV-16 antibody avidity and titer increased over time up to month 12; however, the HPV-16 avidity did not correlate well with HPV-16 neutralizing antibody titers at each time point examined, except for month 6. The median avidity levels were higher in HPV-16 infected women at month 1 (p=0.04) and lower in HPV-16 infected women at month 12 (p=0.006) compared to the HPV negative women. Conclusions The persistence of cross-neutralization titers at month 36 suggests cross-reactive antibody responses are likely to persist long-term and are not influenced by infection status at enrollment. However, the weak correlation between avidity and neutralization titers emphasizes the need for examining avidity in efficacy studies to determine if high avidity antibodies play a critical role in

  16. Packaging and structural phenotype of brome mosaic virus capsid protein with altered N-terminal β-hexamer structure

    International Nuclear Information System (INIS)

    Wispelaere, Melissanne de; Chaturvedi, Sonali; Wilkens, Stephan; Rao, A.L.N.

    2011-01-01

    The first 45 amino acid region of brome mosaic virus (BMV) capsid protein (CP) contains RNA binding and structural domains that are implicated in the assembly of infectious virions. One such important structural domain encompassing amino acids 28 QPVIV 32 , highly conserved between BMV and cowpea chlorotic mottle virus (CCMV), exhibits a β-hexamer structure. In this study we report that alteration of the β-hexamer structure by mutating 28 QPVIV 32 to 28 AAAAA 32 had no effect either on symptom phenotype, local and systemic movement in Chenopodium quinoa and RNA profile of in vivo assembled virions. However, sensitivity to RNase and assembly phenotypes distinguished virions assembled with CP subunits having β-hexamer from those of wild type. A comparison of 3-D models obtained by cryo electron microscopy revealed overall similar structural features for wild type and mutant virions, with small but significant differences near the 3-fold axes of symmetry.

  17. Structural rigidity in the capsid assembly of cowpea chlorotic mottle virus

    International Nuclear Information System (INIS)

    Hespenheide, B M; Jacobs, D J; Thorpe, M F

    2004-01-01

    The cowpea chlorotic mottle virus (CCMV) has a protein cage, or capsid, which encloses its genetic material. The structure of the capsid consists of 180 copies of a single protein that self-assemble inside a cell to form a complete capsid with icosahedral symmetry. The icosahedral surface can be naturally divided into pentagonal and hexagonal faces, and the formation of either of these faces has been proposed to be the first step in the capsid assembly process. We have used the software FIRST to analyse the rigidity of pentameric and hexameric substructures of the complete capsid to explore the viability of certain capsid assembly pathways. FIRST uses the 3D pebble game to determine structural rigidity, and a brief description of this algorithm, as applied to body-bar networks, is given here. We find that the pentameric substructure, which corresponds to a pentagonal face on the icosahedral surface, provides the best structural properties for nucleating the capsid assembly process, consistent with experimental observations

  18. A molecular breadboard: Removal and replacement of subunits in a hepatitis B virus capsid.

    Science.gov (United States)

    Lee, Lye Siang; Brunk, Nicholas; Haywood, Daniel G; Keifer, David; Pierson, Elizabeth; Kondylis, Panagiotis; Wang, Joseph Che-Yen; Jacobson, Stephen C; Jarrold, Martin F; Zlotnick, Adam

    2017-11-01

    Hepatitis B virus (HBV) core protein is a model system for studying assembly and disassembly of icosahedral structures. Controlling disassembly will allow re-engineering the 120 subunit HBV capsid, making it a molecular breadboard. We examined removal of subunits from partially crosslinked capsids to form stable incomplete particles. To characterize incomplete capsids, we used two single molecule techniques, resistive-pulse sensing and charge detection mass spectrometry. We expected to find a binomial distribution of capsid fragments. Instead, we found a preponderance of 3 MDa complexes (90 subunits) and no fragments smaller than 3 MDa. We also found 90-mers in the disassembly of uncrosslinked HBV capsids. 90-mers seem to be a common pause point in disassembly reactions. Partly explaining this result, graph theory simulations have showed a threshold for capsid stability between 80 and 90 subunits. To test a molecular breadboard concept, we showed that missing subunits could be refilled resulting in chimeric, 120 subunit particles. This result may be a means of assembling unique capsids with functional decorations. © 2017 The Protein Society.

  19. Structural rigidity in the capsid assembly of cowpea chlorotic mottle virus

    Science.gov (United States)

    Hespenheide, B. M.; Jacobs, D. J.; Thorpe, M. F.

    2004-11-01

    The cowpea chlorotic mottle virus (CCMV) has a protein cage, or capsid, which encloses its genetic material. The structure of the capsid consists of 180 copies of a single protein that self-assemble inside a cell to form a complete capsid with icosahedral symmetry. The icosahedral surface can be naturally divided into pentagonal and hexagonal faces, and the formation of either of these faces has been proposed to be the first step in the capsid assembly process. We have used the software FIRST to analyse the rigidity of pentameric and hexameric substructures of the complete capsid to explore the viability of certain capsid assembly pathways. FIRST uses the 3D pebble game to determine structural rigidity, and a brief description of this algorithm, as applied to body-bar networks, is given here. We find that the pentameric substructure, which corresponds to a pentagonal face on the icosahedral surface, provides the best structural properties for nucleating the capsid assembly process, consistent with experimental observations.

  20. Structural rigidity in the capsid assembly of cowpea chlorotic mottle virus

    Energy Technology Data Exchange (ETDEWEB)

    Hespenheide, B M [Department of Physics and Astronomy, Arizona State University, PO Box 871504, Tempe, AZ 85287-1504 (United States); Jacobs, D J [Department of Physics and Astronomy, California State University, 18111 Nordhoff Street, Northridge, CA 91330-8268 (United States); Thorpe, M F [Department of Physics and Astronomy, Arizona State University, PO Box 871504, Tempe, AZ 85287-1504 (United States)

    2004-11-10

    The cowpea chlorotic mottle virus (CCMV) has a protein cage, or capsid, which encloses its genetic material. The structure of the capsid consists of 180 copies of a single protein that self-assemble inside a cell to form a complete capsid with icosahedral symmetry. The icosahedral surface can be naturally divided into pentagonal and hexagonal faces, and the formation of either of these faces has been proposed to be the first step in the capsid assembly process. We have used the software FIRST to analyse the rigidity of pentameric and hexameric substructures of the complete capsid to explore the viability of certain capsid assembly pathways. FIRST uses the 3D pebble game to determine structural rigidity, and a brief description of this algorithm, as applied to body-bar networks, is given here. We find that the pentameric substructure, which corresponds to a pentagonal face on the icosahedral surface, provides the best structural properties for nucleating the capsid assembly process, consistent with experimental observations.

  1. Intracellular cargo delivery by virus capsid protein-based vehicles: From nano to micro.

    Science.gov (United States)

    Gao, Ding; Lin, Xiu-Ping; Zhang, Zhi-Ping; Li, Wei; Men, Dong; Zhang, Xian-En; Cui, Zong-Qiang

    2016-02-01

    Cellular delivery is an important concern for the efficiency of medicines and sensors for disease diagnoses and therapy. However, this task is quite challenging. Self-assembly virus capsid proteins might be developed as building blocks for multifunctional cellular delivery vehicles. In this work, we found that SV40 VP1 (Simian virus 40 major capsid protein) could function as a new cell-penetrating protein. The VP1 protein could carry foreign proteins into cells in a pentameric structure. A double color structure, with red QDs (Quantum dots) encapsulated by viral capsids fused with EGFP, was created for imaging cargo delivery and release from viral capsids. The viral capsids encapsulating QDs were further used for cellular delivery of micron-sized iron oxide particles (MPIOs). MPIOs were efficiently delivered into live cells and controlled by a magnetic field. Therefore, our study built virus-based cellular delivery systems for different sizes of cargos: protein molecules, nanoparticles, and micron-sized particles. Much research is being done to investigate methods for efficient and specific cellular delivery of drugs, proteins or genetic material. In this article, the authors describe their approach in using self-assembly virus capsid proteins SV40 VP1 (Simian virus 40 major capsid protein). The cell-penetrating behavior provided excellent cellular delivery and should give a new method for biomedical applications. Copyright © 2015 Elsevier Inc. All rights reserved.

  2. Effects of Point Mutations in the Major Capsid Protein of Beet Western Yellows Virus on Capsid Formation, Virus Accumulation, and Aphid Transmission

    Science.gov (United States)

    Brault, V.; Bergdoll, M.; Mutterer, J.; Prasad, V.; Pfeffer, S.; Erdinger, M.; Richards, K. E.; Ziegler-Graff, V.

    2003-01-01

    Point mutations were introduced into the major capsid protein (P3) of cloned infectious cDNA of the polerovirus beet western yellows virus (BWYV) by manipulation of cloned infectious cDNA. Seven mutations targeted sites on the S domain predicted to lie on the capsid surface. An eighth mutation eliminated two arginine residues in the R domain, which is thought to extend into the capsid interior. The effects of the mutations on virus capsid formation, virus accumulation in protoplasts and plants, and aphid transmission were tested. All of the mutants replicated in protoplasts. The S-domain mutant W166R failed to protect viral RNA from RNase attack, suggesting that this particular mutation interfered with stable capsid formation. The R-domain mutant R7A/R8A protected ∼90% of the viral RNA strand from RNase, suggesting that lower positive-charge density in the mutant capsid interior interfered with stable packaging of the complete strand into virions. Neither of these mutants systemically infected plants. The six remaining mutants properly packaged viral RNA and could invade Nicotiana clevelandii systemically following agroinfection. Mutant Q121E/N122D was poorly transmitted by aphids, implicating one or both targeted residues in virus-vector interactions. Successful transmission of mutant D172N was accompanied either by reversion to the wild type or by appearance of a second-site mutation, N137D. This finding indicates that D172 is also important for transmission but that the D172N transmission defect can be compensated for by a “reverse” substitution at another site. The results have been used to evaluate possible structural models for the BWYV capsid. PMID:12584348

  3. ATP-Driven Contraction of Phage T3 Capsids with DNA Incompletely Packaged In Vivo

    Directory of Open Access Journals (Sweden)

    Philip Serwer

    2017-05-01

    Full Text Available Adenosine triphosphate (ATP cleavage powers packaging of a double-stranded DNA (dsDNA molecule in a pre-assembled capsid of phages that include T3. Several observations constitute a challenge to the conventional view that the shell of the capsid is energetically inert during packaging. Here, we test this challenge by analyzing the in vitro effects of ATP on the shells of capsids generated by DNA packaging in vivo. These capsids retain incompletely packaged DNA (ipDNA and are called ipDNA-capsids; the ipDNA-capsids are assumed to be products of premature genome maturation-cleavage. They were isolated via preparative Nycodenz buoyant density centrifugation. For some ipDNA-capsids, Nycodenz impermeability increases hydration and generates density so low that shell hyper-expansion must exist to accommodate associated water. Electron microscopy (EM confirmed hyper-expansion and low permeability and revealed that 3.0 mM magnesium ATP (physiological concentration causes contraction of hyper-expanded, lowpermeability ipDNA-capsids to less than mature size; 5.0 mM magnesium ATP (border of supraphysiological concentration or more disrupts them. Additionally, excess sodium ADP reverses 3.0 mM magnesium ATP-induced contraction and re-generates hyper-expansion. The Nycodenz impermeability implies assembly perfection that suggests selection for function in DNA packaging. These findings support the above challenge and can be explained via the assumption that T3 DNA packaging includes a back-up cycle of ATP-driven capsid contraction and hyper-expansion.

  4. Use of IgG avidity ELISA to differentiate acute from persistent infection with Salmonella Dublin in cattle

    DEFF Research Database (Denmark)

    Hansen, K.R.; Nielsen, L.R.; Lind, Peter

    2006-01-01

    Aims: To investigate whether an immunoglobulin (Ig)G avidity ELISA can be used to differentiate between acute and persistent infection with Salmonella (S.) Dublin in cattle. To determine whether the IgG isotype, IgG(1) and IgG(2) responses in acute and persistent infections differ. Methods...

  5. IgG avidity to Pseudomonas aeruginosa over the course of chronic lung biofilm infection in cystic fibrosis

    DEFF Research Database (Denmark)

    Mauch, Renan Marrichi; Nørregaard, Lena Lingren; Ciofu, Oana

    2018-01-01

    Background and objectives: The mechanisms leading to low effectiveness of the humoral immune response against P. aeruginosa in cystic fibrosis (CF) are poorly understood. The aim of the present study was to assess the avidity maturation of specific antipseudomonal IgG before and during the develo...

  6. Enterovirus 71 viral capsid protein linear epitopes: Identification and characterization

    Directory of Open Access Journals (Sweden)

    Gao Fan

    2012-01-01

    Full Text Available Abstract Background To characterize the human humoral immune response against enterovirus 71 (EV71 infection and map human epitopes on the viral capsid proteins. Methods A series of 256 peptides spanning the capsid proteins (VP1, VP2, VP3 of BJ08 strain (genomic C4 were synthesized. An indirect enzyme-linked immunosorbent assay (ELISA was carried out to detect anti-EV71 IgM and IgG in sera of infected children in acute or recovery phase. The partially overlapped peptides contained 12 amino acids and were coated in the plate as antigen (0.1 μg/μl. Sera from rabbits immunized with inactivated BJ08 virus were also used to screen the peptide panel. Results A total of 10 human anti-EV71 IgM epitopes (vp1-14 in VP1; vp2-6, 21, 40 and 50 in VP2 and vp3-10, 12, 15, 24 and 75 in VP3 were identified in acute phase sera. In contrast, only one anti-EV71 IgG epitope in VP1 (vp1-15 was identified in sera of recovery stage. Four rabbit anti-EV71 IgG epitopes (vp1-14, 31, 54 and 71 were identified and mapped to VP1. Conclusion These data suggested that human IgM epitopes were mainly mapped to VP2 and VP3 with multi-epitope responses occurred at acute infection, while the only IgG epitope located on protein VP1 was activated in recovery phase sera. The dynamic changes of humoral immune response at different stages of infection may have public health significance in evaluation of EV71 vaccine immunogenicity and the clinical application of diagnostic reagents.

  7. HIV avidity index performance using a modified fourth-generation immunoassay to detect recent HIV infections.

    Science.gov (United States)

    Suligoi, Barbara; Regine, Vincenza; Raimondo, Mariangela; Rodella, Anna; Terlenghi, Luigina; Caruso, Arnaldo; Bagnarelli, Patrizia; Capobianchi, Maria Rosaria; Zanchetta, Nadia; Ghisetti, Valeria; Galli, Claudio

    2017-10-26

    Detecting recent HIV infections is important to evaluate incidence and monitor epidemic trends. We aimed to evaluate the diagnostic performance and accuracy of the avidity index (AI) for discriminating for recent HIV infections. We collected serum samples from HIV-1 positive individuals: A) with known date of infection (midpoint in time between last HIV-negative and first HIV-positive test); B) infected for >1 year. Samples were divided into two aliquots: one diluted with phosphate buffered saline (PBS) and the other with 1 M guanidine. Both aliquots were assayed by the Architect HIV Ag/Ab Combo 4th generation assay (Abbott). We compared AI found in recent (RI=HIV subtype had no impact on AI misclassifications. All individuals in group A reached the AI threshold of 0.80 within 24 months after seroconversion. The AI is an accurate serological marker for discriminating recent from established HIV infections and meets WHO requirements for HIV incidence assays.

  8. Accuracy of different thresholds for the anti-HIV avidity index

    Directory of Open Access Journals (Sweden)

    Claudio Galli

    2008-03-01

    Full Text Available Surveillance programs for human immunodeficiency virus (HIV infection are based on the reporting of newly diagnosed cases. In order to guarantee a more accurate estimate of the trends and behaviours of infected people, simple and reliable methods aimed at identifying recent (< 6 months HIV infections are needed. We evaluated the accuracy of the avidity index (AI of anti-HIV antibodies on 357 serum samples obtained from 127 subjects for whom an estimated date of seroconversion was calculated on the basis of the interval between the last negative and first positive anti-HIV test result.The ROC curve analysis performed at different thresholds of the AI showed that a cutoff of 0.80 (93.0% sensitivity and 98.5% specificity yields the best overall accuracy (95.8% and should be employed for surveillance purposes, whereas the application of the anti-HIV AI on individual cases is not recommended.

  9. The influence of dengue virus serotype-2 infection on Aedes aegypti (Diptera: Culicidae motivation and avidity to blood feed.

    Directory of Open Access Journals (Sweden)

    Rafael Maciel-de-Freitas

    Full Text Available BACKGROUND: Dengue virus (DENV is transmitted by Aedes aegypti, a species that lives in close association with human dwellings. The behavior of DENV-infected mosquitoes needs further investigation, especially regarding the potential influence of DENV on mosquito biting motivation and avidity. METHODOLOGY/PRINCIPAL FINDINGS: We orally challenged 4-5 day-old Ae. aegypti females with a low passage DENV serotype -2 (DENV-2 to test whether the virus influences motivation to feed (the likelihood that a mosquito obtains a blood-meal and the size of its blood meal and avidity (the likelihood to re-feed after an interrupted first blood-meal. To assay motivation, we offered mosquitoes an anesthetized mouse for 2, 3, 4 or 5 minutes 7 or 14 days after the initial blood meals and measured the time they started feeding. 60.5% of the unexposed mosquitoes fed on the mouse, but only 40.5% of the positive ones did. Exposed but negative mosquitoes behaved similarly to unexposed ones (55.0% feeding. Thus DENV-2 infection decreased the mosquitoes' motivation to feed. To assay avidity, we offered the same mosquitoes a mouse two hours after the first round of feeding, and we measured the time at which they started probing. The exposed (positive or negative mosquitoes were more likely to re-feed than the unexposed ones and, in particular, the size of the previous blood-meal that kept mosquitoes from re-feeding was larger in the exposed than in the unexposed mosquitoes. Thus, DENV-2 infection increased mosquito avidity. CONCLUSIONS/SIGNIFICANCE: DENV-2 significantly decreased the mosquitoes' motivation to feed, but increased their avidity (even after taking account the amount of blood previously imbibed. As these are important components of transmission, we expect that the changes of the blood-feeding behaviour impact the vectorial capacity Ae. aegypti for dengue.

  10. The influence of dengue virus serotype-2 infection on Aedes aegypti (Diptera: Culicidae) motivation and avidity to blood feed.

    Science.gov (United States)

    Maciel-de-Freitas, Rafael; Sylvestre, Gabriel; Gandini, Mariana; Koella, Jacob C

    2013-01-01

    Dengue virus (DENV) is transmitted by Aedes aegypti, a species that lives in close association with human dwellings. The behavior of DENV-infected mosquitoes needs further investigation, especially regarding the potential influence of DENV on mosquito biting motivation and avidity. We orally challenged 4-5 day-old Ae. aegypti females with a low passage DENV serotype -2 (DENV-2) to test whether the virus influences motivation to feed (the likelihood that a mosquito obtains a blood-meal and the size of its blood meal) and avidity (the likelihood to re-feed after an interrupted first blood-meal). To assay motivation, we offered mosquitoes an anesthetized mouse for 2, 3, 4 or 5 minutes 7 or 14 days after the initial blood meals and measured the time they started feeding. 60.5% of the unexposed mosquitoes fed on the mouse, but only 40.5% of the positive ones did. Exposed but negative mosquitoes behaved similarly to unexposed ones (55.0% feeding). Thus DENV-2 infection decreased the mosquitoes' motivation to feed. To assay avidity, we offered the same mosquitoes a mouse two hours after the first round of feeding, and we measured the time at which they started probing. The exposed (positive or negative) mosquitoes were more likely to re-feed than the unexposed ones and, in particular, the size of the previous blood-meal that kept mosquitoes from re-feeding was larger in the exposed than in the unexposed mosquitoes. Thus, DENV-2 infection increased mosquito avidity. DENV-2 significantly decreased the mosquitoes' motivation to feed, but increased their avidity (even after taking account the amount of blood previously imbibed). As these are important components of transmission, we expect that the changes of the blood-feeding behaviour impact the vectorial capacity Ae. aegypti for dengue.

  11. Pt, Co–Pt and Fe–Pt alloy nanoclusters encapsulated in virus capsids

    International Nuclear Information System (INIS)

    Okuda, M; Eloi, J-C; Jones, S E Ward; Schwarzacher, W; Verwegen, M; Cornelissen, J J L M

    2016-01-01

    Nanostructured Pt-based alloys show great promise, not only for catalysis but also in medical and magnetic applications. To extend the properties of this class of materials, we have developed a means of synthesizing Pt and Pt-based alloy nanoclusters in the capsid of a virus. Pure Pt and Pt-alloy nanoclusters are formed through the chemical reduction of [PtCl 4 ] − by NaBH 4 with/without additional metal ions (Co or Fe). The opening and closing of the ion channels in the virus capsid were controlled by changing the pH and ionic strength of the solution. The size of the nanoclusters is limited to 18 nm by the internal diameter of the capsid. Their magnetic properties suggest potential applications in hyperthermia for the Co–Pt and Fe–Pt magnetic alloy nanoclusters. This study introduces a new way to fabricate size-restricted nanoclusters using virus capsid. (paper)

  12. Specific cross-linking of capsid proteins to virus RNA by ultraviolet irradiation of polio virus

    Energy Technology Data Exchange (ETDEWEB)

    Wetz, K.; Habermehl, K.O. (Freie Univ. Berlin (Germany, F.R.))

    1982-04-01

    Poliovirus was irradiated with u.v. light under conditions causing approx. 5% cross-linking of capsid protein to virus RNA. Cross-linked RNA-protein complexes, freed from unbound protein, were treated with nuclease, and then analysed on SDS-polyacrylamide gels. The smallest capsid polypeptide VP4 was found to be associated with the RNA to the greatest degree, followed by VP2 and VP1, while VP3 was attached only in trace amounts. Low radiation doses, which produced cross-linking of RNA to protein, did not cause breakdown of the virus particles or conformational changes of the capsid as examined physically and serologically. However, higher doses caused structural alterations of the virus capsid.

  13. Specific cross-linking of capsid proteins to virus RNA by ultraviolet irradiation of polio virus

    International Nuclear Information System (INIS)

    Wetz, K.; Habermehl, K.-O.

    1982-01-01

    Poliovirus was irradiated with u.v. light under conditions causing approx. 5% cross-linking of capsid protein to virus RNA. Cross-linked RNA-protein complexes, freed from unbound protein, were treated with nuclease, and then analysed on SDS-polyacrylamide gels. The smallest capsid polypeptide VP4 was found to be associated with the RNA to the greatest degree, followed by VP2 and VP1, while VP3 was attached only in trace amounts. Low radiation doses, which produced cross-linking of RNA to protein, did not cause breakdown of the virus particles or conformational changes of the capsid as examined physically and serologically. However, higher doses caused structural alterations of the virus capsid. (author)

  14. The Assembly-Activating Protein Promotes Stability and Interactions between AAV’s Viral Proteins to Nucleate Capsid Assembly

    Directory of Open Access Journals (Sweden)

    Anna C. Maurer

    2018-05-01

    Full Text Available Summary: The adeno-associated virus (AAV vector is a preferred delivery platform for in vivo gene therapy. Natural and engineered variations of the AAV capsid affect a plurality of phenotypes relevant to gene therapy, including vector production and host tropism. Fundamental to these aspects is the mechanism of AAV capsid assembly. Here, the role of the viral co-factor assembly-activating protein (AAP was evaluated in 12 naturally occurring AAVs and 9 putative ancestral capsid intermediates. The results demonstrate increased capsid protein stability and VP-VP interactions in the presence of AAP. The capsid’s dependence on AAP can be partly overcome by strengthening interactions between monomers within the assembly, as illustrated by the transfer of a minimal motif defined by a phenotype-to-phylogeny mapping method. These findings suggest that the emergence of AAP within the Dependovirus genus relaxes structural constraints on AAV assembly in favor of increasing the degrees of freedom for the capsid to evolve. : Maurer et al. describe a phenotype-to-phylogeny mapping strategy correlating phenotypic variation in AAVs to a reconstructed phylogeny, revealing capsid structure-function relationships relevant to that phenotype. Dependence on the viral co-factor AAP for capsid assembly is examined, and capsid functional motifs, in addition to mechanistic roles of AAP, are elucidated. Keywords: AAV, AAP, adeno-associated virus, capsid assembly, manufacturing, capsid, vector engineering, structure-function, gene therapy

  15. SCHEMA computational design of virus capsid chimeras: calibrating how genome packaging, protection, and transduction correlate with calculated structural disruption.

    Science.gov (United States)

    Ho, Michelle L; Adler, Benjamin A; Torre, Michael L; Silberg, Jonathan J; Suh, Junghae

    2013-12-20

    Adeno-associated virus (AAV) recombination can result in chimeric capsid protein subunits whose ability to assemble into an oligomeric capsid, package a genome, and transduce cells depends on the inheritance of sequence from different AAV parents. To develop quantitative design principles for guiding site-directed recombination of AAV capsids, we have examined how capsid structural perturbations predicted by the SCHEMA algorithm correlate with experimental measurements of disruption in seventeen chimeric capsid proteins. In our small chimera population, created by recombining AAV serotypes 2 and 4, we found that protection of viral genomes and cellular transduction were inversely related to calculated disruption of the capsid structure. Interestingly, however, we did not observe a correlation between genome packaging and calculated structural disruption; a majority of the chimeric capsid proteins formed at least partially assembled capsids and more than half packaged genomes, including those with the highest SCHEMA disruption. These results suggest that the sequence space accessed by recombination of divergent AAV serotypes is rich in capsid chimeras that assemble into 60-mer capsids and package viral genomes. Overall, the SCHEMA algorithm may be useful for delineating quantitative design principles to guide the creation of libraries enriched in genome-protecting virus nanoparticles that can effectively transduce cells. Such improvements to the virus design process may help advance not only gene therapy applications but also other bionanotechnologies dependent upon the development of viruses with new sequences and functions.

  16. Binding of glutathione to enterovirus capsids is essential for virion morphogenesis.

    NARCIS (Netherlands)

    Thibaut, H.J.; Linden, L. van der; Jiang, P.; Thys, B.; Canela, M.D.; Aguado, L.; Rombaut, B.; Wimmer, E.; Paul, A.; Perez-Perez, M.J.; Kuppeveld, F.J.M. van; Neyts, J.

    2014-01-01

    Enteroviruses (family of the Picornaviridae) cover a large group of medically important human pathogens for which no antiviral treatment is approved. Although these viruses have been extensively studied, some aspects of the viral life cycle, in particular morphogenesis, are yet poorly understood. We

  17. Binding of glutathione to enterovirus capsids is essential for virion morphogenesis

    NARCIS (Netherlands)

    Thibaut, Hendrik Jan; van der Linden, Lonneke; Jiang, Ping; Thys, Bert; Canela, María-Dolores; Aguado, Leire; Rombaut, Bart; Wimmer, Eckard; Paul, Aniko; Pérez-Pérez, María-Jesús; van Kuppeveld, Frank J M; Neyts, Johan

    Enteroviruses (family of the Picornaviridae) cover a large group of medically important human pathogens for which no antiviral treatment is approved. Although these viruses have been extensively studied, some aspects of the viral life cycle, in particular morphogenesis, are yet poorly understood. We

  18. Oral Administration of Astrovirus Capsid Protein Is Sufficient To Induce Acute Diarrhea In Vivo

    Directory of Open Access Journals (Sweden)

    Victoria A. Meliopoulos

    2016-11-01

    Full Text Available The disease mechanisms associated with the onset of astrovirus diarrhea are unknown. Unlike other enteric virus infections, astrovirus infection is not associated with an inflammatory response or cellular damage. In vitro studies in differentiated Caco-2 cells demonstrated that human astrovirus serotype 1 (HAstV-1 capsid protein alone disrupts the actin cytoskeleton and tight junction complex, leading to increased epithelial barrier permeability. In this study, we show that oral administration of purified recombinant turkey astrovirus 2 (TAstV-2 capsid protein results in acute diarrhea in a dose- and time-dependent manner in turkey poults. Similarly to that induced by infectious virus, TAstV-2 capsid-induced diarrhea was independent of inflammation or histological changes but was associated with increased intestinal barrier permeability, as well as redistribution of sodium hydrogen exchanger 3 (NHE3 from the membrane to the cytoplasm of the intestinal epithelium. Unlike other viral enterotoxins that have been identified, astrovirus capsid induces diarrhea after oral administration, reproducing the natural route of infection and demonstrating that ingestion of intact noninfectious capsid protein may be sufficient to provoke acute diarrhea. Based on these data, we hypothesize that the astrovirus capsid acts like an enterotoxin and induces intestinal epithelial barrier dysfunction.

  19. How viral capsids adapt to mismatched cargoes—identifying mechanisms of morphology control with simulations

    Science.gov (United States)

    Elrad, Oren

    2009-03-01

    During the replication of many viruses, hundreds to thousands of protein subunits assemble around the viral nucleic acid to form a protein shell called a capsid. Most viruses form one particular structure with astonishing fidelity; yet, recent experiments demonstrate that capsids can assemble with different sizes and morphologies to accommodate nucleic acids or other cargoes such as functionalized nanoparticles. In this talk, we will explore the mechanisms of simultaneous assembly and cargo encapsidation with a computational model that describes the assembly of icosahedral capsids around functionalized nanoparticles. With this model, we find parameter values for which subunits faithfully form empty capsids with a single morphology, but adaptively assemble into different icosahedral morphologies around nanoparticles with different diameters. Analyzing trajectories in which adaptation is or is not successful sheds light on the mechanisms by which capsid morphology may be controlled in vitro and in vivo, and suggests experiments to test these mechanisms. We compare the simulation results to recent experiments in which Brome Mosaic Virus capsid proteins assemble around functionalized nanoparticles, and describe how future experiments can test the model predictions.

  20. High Relaxivity Gadolinium Hydroxypyridonate-Viral Capsid Conjugates: Nano-sized MRI Contrast Agents

    Energy Technology Data Exchange (ETDEWEB)

    Meux, Susan C.; Datta, Ankona; Hooker, Jacob M.; Botta, Mauro; Francis, Matthew B.; Aime, Silvio; Raymond, Kenneth N.

    2007-08-29

    High relaxivity macromolecular contrast agents based on the conjugation of gadolinium chelates to the interior and exterior surfaces of MS2 viral capsids are assessed. The proton nuclear magnetic relaxation dispersion (NMRD) profiles of the conjugates show up to a five-fold increase in relaxivity, leading to a peak relaxivity (per Gd{sup 3+} ion) of 41.6 mM{sup -1}s{sup -1} at 30 MHz for the internally modified capsids. Modification of the exterior was achieved through conjugation to flexible lysines, while internal modification was accomplished by conjugation to relatively rigid tyrosines. Higher relaxivities were obtained for the internally modified capsids, showing that (1) there is facile diffusion of water to the interior of capsids and (2) the rigidity of the linker attaching the complex to the macromolecule is important for obtaining high relaxivity enhancements. The viral capsid conjugated gadolinium hydroxypyridonate complexes appear to possess two inner-sphere water molecules (q = 2) and the NMRD fittings highlight the differences in the local motion for the internal ({tau}{sub RI} = 440 ps) and external ({tau}{sub RI} = 310 ps) conjugates. These results indicate that there are significant advantages of using the internal surface of the capsids for contrast agent attachment, leaving the exterior surface available for the installation of tissue targeting groups.

  1. Label-free Fab and Fc affinity/avidity profiling of the antibody complex half-life for polyclonal and monoclonal efficacy screening.

    Science.gov (United States)

    Read, Thomas; Olkhov, Rouslan V; Williamson, E Diane; Shaw, Andrew M

    2015-09-01

    A unified approach to affinity screening for Fab and Fc interactions of an antibody for its antigen and FcγR receptor has been developed. An antigen array is used for the Fab affinity and cross-reactivity screening and protein A/G proxy is the FcγR receptor. The affinities are derived using a simple 1:1 binding model with a consistent error analysis. The association and dissociation kinetics are measured over optimised times for accurate determination. The Fab/Fc affinities are derived for ten antibodies: mAb-actin (mouse), pAb-BSA (sheep), pAb-collagen V (rabbit), pAb-CRP (goat), mAb-F1 (mouse), mAbs (mouse) 7.3, 12.3, 29.3, 36.3 and 46.3 raised against LcrV in Yersinia pestis. The rate of the dissociation of antigen-antibody complexes relates directly to their immunological function as does the Fc-FcγR complex and a new half-life plot has been defined with a Fab/Fc half-life range of 17-470 min. The upper half-life value points to surface avidity. Two antibodies that are protective as an immunotherapy define a Fab half-life >250 min and an Fc half-life >50 min as characteristics of ideal interactions which can form the basis of an antibody screen for immunotherapy.

  2. Adenoviruses using the cancer marker EphA2 as a receptor in vitro and in vivo by genetic ligand insertion into different capsid scaffolds.

    Directory of Open Access Journals (Sweden)

    Michael Behr

    Full Text Available Adenoviral gene therapy and oncolysis would critically benefit from targeted cell entry by genetically modified capsids. This requires both the ablation of native adenovirus tropism and the identification of ligands that remain functional in virus context. Here, we establish cell type-specific entry of HAdV-5-based vectors by genetic ligand insertion into a chimeric fiber with shaft and knob domains of the short HAdV-41 fiber (Ad5T/41sSK. This fiber format was reported to ablate transduction in vitro and biodistribution to the liver in vivo. We show that the YSA peptide, binding to the pan-cancer marker EphA2, can be inserted into three positions of the chimeric fiber, resulting in strong transduction of EphA2-positive but not EphA2-negative cells of human melanoma biopsies and of tumor xenografts after intratumoral injection. Transduction was blocked by soluble YSA peptide and restored for EphA2-negative cells after recombinant EphA2 expression. The YSA peptide could also be inserted into three positions of a CAR binding-ablated HAdV-5 fiber enabling specific transduction; however, the Ad5T/41sSK format was superior in vivo. In conclusion, we establish an adenovirus capsid facilitating functional insertion of targeting peptides and a novel adenovirus using the tumor marker EphA2 as receptor with high potential for cancer gene therapy and viral oncolysis.

  3. Interaction of the Mouse Polyomavirus Capsid Proteins with Importins Is Required for Efficient Import of Viral DNA into the Cell Nucleus.

    Science.gov (United States)

    Soldatova, Irina; Prilepskaja, Terezie; Abrahamyan, Levon; Forstová, Jitka; Huérfano, Sandra

    2018-03-31

    The mechanism used by mouse polyomavirus (MPyV) overcomes the crowded cytosol to reach the nucleus has not been fully elucidated. Here, we investigated the involvement of importin α/β1 mediated transport in the delivery of MPyV genomes into the nucleus. Interactions of the virus with importin β1 were studied by co-immunoprecipitation and proximity ligation assay. For infectivity and nucleus delivery assays, the virus and its capsid proteins mutated in the nuclear localization signals (NLSs) were prepared and produced. We found that at early times post infection, virions bound importin β1 in a time dependent manner with a peak of interactions at 6 h post infection. Mutation analysis revealed that only when the NLSs of both VP1 and VP2/3 were disrupted, virus did not bind efficiently to importin β1 and its infectivity remarkably decreased (by 80%). Nuclear targeting of capsid proteins was improved when VP1 and VP2 were co-expressed. VP1 and VP2 were effectively delivered into the nucleus, even when one of the NLS, either VP1 or VP2, was disrupted. Altogether, our results showed that MPyV virions can use VP1 and/or VP2/VP3 NLSs in concert or individually to bind importins to deliver their genomes into the cell nucleus.

  4. Adenoviruses Using the Cancer Marker EphA2 as a Receptor In Vitro and In Vivo by Genetic Ligand Insertion into Different Capsid Scaffolds

    Science.gov (United States)

    Behr, Michael; Kaufmann, Johanna K.; Ketzer, Patrick; Engelhardt, Sarah; Mück-Häusl, Martin; Okun, Pamela M.; Petersen, Gabriele; Neipel, Frank; Hassel, Jessica C.; Ehrhardt, Anja; Enk, Alexander H.; Nettelbeck, Dirk M.

    2014-01-01

    Adenoviral gene therapy and oncolysis would critically benefit from targeted cell entry by genetically modified capsids. This requires both the ablation of native adenovirus tropism and the identification of ligands that remain functional in virus context. Here, we establish cell type-specific entry of HAdV-5-based vectors by genetic ligand insertion into a chimeric fiber with shaft and knob domains of the short HAdV-41 fiber (Ad5T/41sSK). This fiber format was reported to ablate transduction in vitro and biodistribution to the liver in vivo. We show that the YSA peptide, binding to the pan-cancer marker EphA2, can be inserted into three positions of the chimeric fiber, resulting in strong transduction of EphA2-positive but not EphA2-negative cells of human melanoma biopsies and of tumor xenografts after intratumoral injection. Transduction was blocked by soluble YSA peptide and restored for EphA2-negative cells after recombinant EphA2 expression. The YSA peptide could also be inserted into three positions of a CAR binding-ablated HAdV-5 fiber enabling specific transduction; however, the Ad5T/41sSK format was superior in vivo. In conclusion, we establish an adenovirus capsid facilitating functional insertion of targeting peptides and a novel adenovirus using the tumor marker EphA2 as receptor with high potential for cancer gene therapy and viral oncolysis. PMID:24760010

  5. Antiviral activity of α-helical stapled peptides designed from the HIV-1 capsid dimerization domain

    Directory of Open Access Journals (Sweden)

    Cowburn David

    2011-05-01

    Full Text Available Abstract Background The C-terminal domain (CTD of HIV-1 capsid (CA, like full-length CA, forms dimers in solution and CTD dimerization is a major driving force in Gag assembly and maturation. Mutations of the residues at the CTD dimer interface impair virus assembly and render the virus non-infectious. Therefore, the CTD represents a potential target for designing anti-HIV-1 drugs. Results Due to the pivotal role of the dimer interface, we reasoned that peptides from the α-helical region of the dimer interface might be effective as decoys to prevent CTD dimer formation. However, these small peptides do not have any structure in solution and they do not penetrate cells. Therefore, we used the hydrocarbon stapling technique to stabilize the α-helical structure and confirmed by confocal microscopy that this modification also made these peptides cell-penetrating. We also confirmed by using isothermal titration calorimetry (ITC, sedimentation equilibrium and NMR that these peptides indeed disrupt dimer formation. In in vitro assembly assays, the peptides inhibited mature-like virus particle formation and specifically inhibited HIV-1 production in cell-based assays. These peptides also showed potent antiviral activity against a large panel of laboratory-adapted and primary isolates, including viral strains resistant to inhibitors of reverse transcriptase and protease. Conclusions These preliminary data serve as the foundation for designing small, stable, α-helical peptides and small-molecule inhibitors targeted against the CTD dimer interface. The observation that relatively weak CA binders, such as NYAD-201 and NYAD-202, showed specificity and are able to disrupt the CTD dimer is encouraging for further exploration of a much broader class of antiviral compounds targeting CA. We cannot exclude the possibility that the CA-based peptides described here could elicit additional effects on virus replication not directly linked to their ability to bind

  6. Epitope specificity is critical for high and moderate avidity cytotoxic T lymphocytes associated with control of viral load and clinical disease in horses with equine infectious anemia virus

    International Nuclear Information System (INIS)

    Mealey, Robert H.; Zhang Baoshan; Leib, Steven R.; Littke, Matt H.; McGuire, Travis C.

    2003-01-01

    Equine infectious anemia virus (EIAV) is a lentivirus that causes persistent infections in horses. We hypothesized that high-avidity CTL specific for nonvariable epitopes might be associated with low viral load and minimal disease in EIAV-infected horses. To test this hypothesis, memory CTL (CTLm) responses were analyzed in two infected horses with high plasma viral loads and recurrent disease (progressors), and in two infected horses with low-to-undetectable viral loads and mild disease (nonprogressors). High-avidity CTLm in one progressor recognized an envelope gp90 epitope, and the data documented for the first time in EIAV that viral variation led to CTL escape. Each of the nonprogressors had high-to-moderate avidity CTLm directed against epitopes within Rev, including the nuclear export and nuclear localization domains. These results suggested that the epitope specificity of high- and moderate-avidity CTLm was an important determinant for disease outcome in the EIAV-infected horses examined

  7. High Concentrations of Measles Neutralizing Antibodies and High-Avidity Measles IgG Accurately Identify Measles Reinfection Cases

    Science.gov (United States)

    Rota, Jennifer S.; Hickman, Carole J.; Mercader, Sara; Redd, Susan; McNall, Rebecca J.; Williams, Nobia; McGrew, Marcia; Walls, M. Laura; Rota, Paul A.; Bellini, William J.

    2016-01-01

    In the United States, approximately 9% of the measles cases reported from 2012 to 2014 occurred in vaccinated individuals. Laboratory confirmation of measles in vaccinated individuals is challenging since IgM assays can give inconclusive results. Although a positive reverse transcription (RT)-PCR assay result from an appropriately timed specimen can provide confirmation, negative results may not rule out a highly suspicious case. Detection of high-avidity measles IgG in serum samples provides laboratory evidence of a past immunologic response to measles from natural infection or immunization. High concentrations of measles neutralizing antibody have been observed by plaque reduction neutralization (PRN) assays among confirmed measles cases with high-avidity IgG, referred to here as reinfection cases (RICs). In this study, we evaluated the utility of measuring levels of measles neutralizing antibody to distinguish RICs from noncases by receiver operating characteristic curve analysis. Single and paired serum samples with high-avidity measles IgG from suspected measles cases submitted to the CDC for routine surveillance were used for the analysis. The RICs were confirmed by a 4-fold rise in PRN titer or by RT-quantitative PCR (RT-qPCR) assay, while the noncases were negative by both assays. Discrimination accuracy was high with serum samples collected ≥3 days after rash onset (area under the curve, 0.953; 95% confidence interval [CI], 0.854 to 0.993). Measles neutralizing antibody concentrations of ≥40,000 mIU/ml identified RICs with 90% sensitivity (95% CI, 74 to 98%) and 100% specificity (95% CI, 82 to 100%). Therefore, when serological or RT-qPCR results are unavailable or inconclusive, suspected measles cases with high-avidity measles IgG can be confirmed as RICs by measles neutralizing antibody concentrations of ≥40,000 mIU/ml. PMID:27335386

  8. The Influence of Dengue Virus Serotype-2 Infection on Aedes aegypti (Diptera: Culicidae) Motivation and Avidity to Blood Feed

    OpenAIRE

    Maciel-de-Freitas, Rafael; Sylvestre, Gabriel; Gandini, Mariana; Koella, Jacob C.

    2013-01-01

    BACKGROUND: Dengue virus (DENV) is transmitted by Aedes aegypti, a species that lives in close association with human dwellings. The behavior of DENV-infected mosquitoes needs further investigation, especially regarding the potential influence of DENV on mosquito biting motivation and avidity. METHODOLOGY/PRINCIPAL FINDINGS: We orally challenged 4-5 day-old Ae. aegypti females with a low passage DENV serotype -2 (DENV-2) to test whether the virus influences motivation to feed (the likelihood ...

  9. Mutation of a Conserved Nuclear Export Sequence in Chikungunya Virus Capsid Protein Disrupts Host Cell Nuclear Import.

    Science.gov (United States)

    Jacobs, Susan C; Taylor, Adam; Herrero, Lara J; Mahalingam, Suresh; Fazakerley, John K

    2017-10-20

    Transmitted by mosquitoes; chikungunya virus (CHIKV) is responsible for frequent outbreaks of arthritic disease in humans. CHIKV is an arthritogenic alphavirus of the Togaviridae family. Capsid protein, a structural protein encoded by the CHIKV RNA genome, is able to translocate to the host cell nucleus. In encephalitic alphaviruses nuclear translocation induces host cell shut off; however, the role of capsid protein nuclear localisation in arthritogenic alphaviruses remains unclear. Using replicon systems, we investigated a nuclear export sequence (NES) in the N-terminal region of capsid protein; analogous to that found in encephalitic alphavirus capsid but uncharacterised in CHIKV. The chromosomal maintenance 1 (CRM1) export adaptor protein mediated CHIKV capsid protein export from the nucleus and a region within the N-terminal part of CHIKV capsid protein was required for active nuclear targeting. In contrast to encephalitic alphaviruses, CHIKV capsid protein did not inhibit host nuclear import; however, mutating the NES of capsid protein (∆NES) blocked host protein access to the nucleus. Interactions between capsid protein and the nucleus warrant further investigation.

  10. Structural Studies of Adeno-Associated Virus Serotype 8 Capsid Transitions Associated with Endosomal Trafficking

    Energy Technology Data Exchange (ETDEWEB)

    Nam, Hyun-Joo; Gurda, Brittney L.; McKenna, Robert; Potter, Mark; Byrne, Barry; Salganik, Maxim; Muzyczka, Nicholas; Agbandje-McKenna, Mavis (Florida)

    2012-09-17

    The single-stranded DNA (ssDNA) parvoviruses enter host cells through receptor-mediated endocytosis, and infection depends on processing in the early to late endosome as well as in the lysosome prior to nuclear entry for replication. However, the mechanisms of capsid endosomal processing, including the effects of low pH, are poorly understood. To gain insight into the structural transitions required for this essential step in infection, the crystal structures of empty and green fluorescent protein (GFP) gene-packaged adeno-associated virus serotype 8 (AAV8) have been determined at pH values of 6.0, 5.5, and 4.0 and then at pH 7.5 after incubation at pH 4.0, mimicking the conditions encountered during endocytic trafficking. While the capsid viral protein (VP) topologies of all the structures were similar, significant amino acid side chain conformational rearrangements were observed on (i) the interior surface of the capsid under the icosahedral 3-fold axis near ordered nucleic acid density that was lost concomitant with the conformational change as pH was reduced and (ii) the exterior capsid surface close to the icosahedral 2-fold depression. The 3-fold change is consistent with DNA release from an ordering interaction on the inside surface of the capsid at low pH values and suggests transitions that likely trigger the capsid for genome uncoating. The surface change results in disruption of VP-VP interface interactions and a decrease in buried surface area between VP monomers. This disruption points to capsid destabilization which may (i) release VP1 amino acids for its phospholipase A2 function for endosomal escape and nuclear localization signals for nuclear targeting and (ii) trigger genome uncoating.

  11. Interference of daratumumab with pretransfusion testing, mimicking a high-titer, low avidity like antibody

    Directory of Open Access Journals (Sweden)

    Mei-Hwa Lin

    2017-01-01

    Full Text Available Daratumumab is a monoclonal immunoglobulin against CD38 and has been approved for treating patients with refractory multiple myeloma. The presence of daratumumab in the sera can interfere with pretransfusion testing due to the weakly expression of CD38 on red cells. The reactivity could be mistaken as autoantibody (if autocontrol is positive or alloantibody (if autocontrol is negative. We present a case that demonstrates daratumumab could mimic a high titer low avidity (HTLA alloantibody. A 34-year-old male patient of refractory myeloma was recruited in phase three clinical trial involving daratumumab. Samples were sent to the blood bank for pretransfusion testing. Without knowledge of patient having used daratumumab, we mistook the reactivity in the patient's sera as an HTLA antibody due to the results of negative autocontrol and high titers of antibody activity. Antibody screen showed a panreactive pattern and the reactivity against screening cells was up to a titer of 1: 1240. The reactivity was weaker against cord cells than adult cells, became weaker against ZZAP-treated cells and became negative against DDT-treated cells. A discussion with attending physician finally revealed the reactivity was due to the interference caused by daratumumab. The case demonstrates good communication is essential in performing pretransfusion testing for patients receiving daratumumab and other new biological regimens that can interfere with compatibility test.

  12. Enhanced change detection performance reveals improved strategy use in avid action video game players.

    Science.gov (United States)

    Clark, Kait; Fleck, Mathias S; Mitroff, Stephen R

    2011-01-01

    Recent research has shown that avid action video game players (VGPs) outperform non-video game players (NVGPs) on a variety of attentional and perceptual tasks. However, it remains unknown exactly why and how such differences arise; while some prior research has demonstrated that VGPs' improvements stem from enhanced basic perceptual processes, other work indicates that they can stem from enhanced attentional control. The current experiment used a change-detection task to explore whether top-down strategies can contribute to VGPs' improved abilities. Participants viewed alternating presentations of an image and a modified version of the image and were tasked with detecting and localizing the changed element. Consistent with prior claims of enhanced perceptual abilities, VGPs were able to detect the changes while requiring less exposure to the change than NVGPs. Further analyses revealed this improved change detection performance may result from altered strategy use; VGPs employed broader search patterns when scanning scenes for potential changes. These results complement prior demonstrations of VGPs' enhanced bottom-up perceptual benefits by providing new evidence of VGPs' potentially enhanced top-down strategic benefits. Copyright © 2010 Elsevier B.V. All rights reserved.

  13. A case report of hyperfunctioning metastatic thyroid cancer and rare I-131 avid liver metastasis

    International Nuclear Information System (INIS)

    Kunawudhi, Anchisa; Promteangtrong, Chetsadaporn; Chotipanich, Chanisa

    2016-01-01

    Thyroid cancer is usually, relatively hypofunctional; most patients with thyroid cancer are clinically euthyroid. The combination of thyroid cancer and thyrotoxicosis is not common. We herein, report a case of follicular thyroid cancer with hyperfunctioning metastasis in a 43-year-old woman who presented with thyrotoxicosis, a cold right thyroid nodule, and low I-131 uptake at the thyroid bed. An additional total body scan with I-131 revealed a large radioiodine avid osteolytic bone metastasis with soft tissue masses and liver metastasis. The patient received treatment with total thyroidectomy, methimazole, and I-131 at a cumulative dose of 600 mCi along with recombinant human thyroid-stimulating hormone before the first I-131 treatment and palliative radiation. The patient had normal liver function test and experienced a mild degree of bone marrow suppression after I-131. At the 2-year follow-up, the patient was still alive with the progression of bone metastases but was doing well with less severe thyrotoxicosis, good ambulation, and an Eastern Cooperative Oncology Group performance status of 2. Clinicians should be aware of the unusual concurrent presentation of thyrotoxicosis and thyroid cancer, a differential diagnosis in patients with thyrotoxicosis and low or normal radioiodine uptake over the neck and also potential pitfalls during radionuclide treatment

  14. High Avidity dsDNA Autoantibodies in Brazilian Women with Systemic Lupus Erythematosus: Correlation with Active Disease and Renal Dysfunction

    Directory of Open Access Journals (Sweden)

    Rodrigo C. Oliveira

    2015-01-01

    Full Text Available We investigated in Brazilian women with SLE the prevalence and levels of high avidity (HA dsDNA antibodies and tested their correlation with lupus activity and biomarkers of renal disease. We also compared these correlations to those observed with total dsDNA antibodies and antibodies against nucleosome (ANuA. Autoantibodies were detected by ELISA, while C3 and C4 levels were determined by nephelometry. Urine protein/creatinine ratio was determined, and lupus activity was measured by SLEDAI-2K. The prevalence of total and HA dsDNA antibodies was similar to but lower than that verified for ANuA. The levels of the three types of antibodies were correlated, but the correlation was more significant between HA dsDNA antibodies and ANuA. High avidity dsDNA antibodies correlated positively with ESR and SLEDAI and inversely with C3 and C4. Similar correlations were observed for ANuA levels, whereas total dsDNA antibodies only correlated with SLEDAI and C3. The levels of HA dsDNA antibodies were higher in patients with proteinuria, but their levels of total dsDNA antibodies and ANuA were unaltered. High avidity dsDNA antibodies can be found in high prevalence in Brazilian women with SLE and are important biomarkers of active disease and kidney dysfunction.

  15. Influence of maternal vaccination against diphtheria, tetanus, and pertussis on the avidity of infant antibody responses to a pertussis containing vaccine in Belgium.

    Science.gov (United States)

    Caboré, Raïssa Nadège; Maertens, Kirsten; Dobly, Alexandre; Leuridan, Elke; Van Damme, Pierre; Huygen, Kris

    2017-10-03

    Maternal antibodies induced by vaccination during pregnancy cross the placental barrier and can close the susceptibility gap to pertussis in young infants up to the start of primary immunization. As not only the quantity but also the quality of circulating antibodies is important for protection, we assessed whether maternal immunization affects the avidity of infant vaccine-induced IgG antibodies, in the frame of a prospective clinical trial on pregnancy vaccination in Belgium. Infants born from Tdap (Boostrix®) vaccinated (N = 55) and unvaccinated (N = 26) mothers were immunized with a hexavalent pertussis containing vaccine (Infanrix Hexa®) at 8, 12 and 16 weeks, followed by a fourth dose at 15 months of age. Right before and one month after this fourth vaccine dose, the avidity of IgG antibodies against diphtheria toxin (DT), tetanus toxin (TT), pertussis toxin (PT), filamentous hemagglutinin (FHA) and pertactin (Prn) was determined using 1.5 M ammonium thiocyanate as dissociating agent. In both groups, antibody avidity was moderate for TT, PT, FHA and Prn and low for DT after priming. After a fourth dose, antibody avidity increased significantly to high avidity for TT and PT, whereas it remained moderate for FHA and Prn and low for DT. The avidity correlated positively with antibody level in both study groups, yet not significantly for PT. When comparing both study groups, only PT-specific antibodies showed significantly lower avidity in infants born from vaccinated than from unvaccinated mothers after the fourth vaccine dose. The clinical significance of lower avidity of vaccine induced infant antibodies after maternal vaccination, if any, needs further investigation.

  16. Synthesis of dispersive iron or iron–silver nanoparticles on engineered capsid pVIII of M13 virus with electronegative terminal peptides

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, Shuai; Nakano, Kazuhiko; Zhang, Shu-liang; Yu, Hui-min, E-mail: yuhm@tsinghua.edu.cn [Tsinghua University, Key Laboratory of Industrial Biocatalysis of the Ministry of Education, Department of Chemical Engineering (China)

    2015-10-15

    M13 is a filamentous Escherichia coli virus covered with five types of capsid proteins, in which pVIII with ∼2700 copies was around the cylindered surface and pIII with five copies located at one end of the phage particle. The pIII-engineered M13 phages with enhanced binding specificity toward Fe were screened after five rounds of biopanning, and the one containing ATPTVAMSLSPL peptide at pIII-terminus was selected for mediated synthesis of zero valent (ZV) Fe nanoparticles (NPs) with the wild M13 as control. Under a reducing environment, uniformly dispersed ZVFeNPs with diameter of 5–10 nm were both synthesized and the morphologies after annealing were confirmed to be face-centered cubic type. The synthesized FeNPs mediated by the two phages showed no significant difference, revealing that the pVIII capsid did dominant contribution to metal binding in comparison with the pIII. A novel pVIII-engineered M13 containing AAEEEDPAK at terminus, named as 4ED-pVIII-M13, was constructed and it carried one more negatively charged residue than the wild one (AEGDDPAK). Metal adsorption quantification showed that the binding affinity of the 4ED-pVIII-M13 toward Ag and Ni ions improved to 62 and 18 % from original 21 and 6 %, respectively. The binding affinity toward Fe remained constant (∼85 %). ZVFe–Ag bi-NPs were successfully synthesized through mediation of 4ED-pVIII-M13. Particularly, the Fe:Ag ratio in the bi-NPs was conveniently controlled through changing the molar concentration of FeCl{sub 2} and AgNO{sub 3} solution before reduction.

  17. Diagnosis of acute toxoplasmosis in pregnant women referred to therapeutic centers of Alborz Province (Iran using immunoglobulin G avidity ELISA technique

    Directory of Open Access Journals (Sweden)

    Lame Akhlaghi

    2016-11-01

    Full Text Available Objective: To evaluate immunoglobulin G (IgG avidity as a useful and reliable technique in diagnosing toxoplasmosis in pregnant women referring to therapeutic centers of Alborz Province (Iran in 2014, against two other tests, IgG and immunoglobulin G (IgM antiToxoplasma. Methods: Serum samples (468 in total were obtained from different therapeutic centers in Karaj City. ELISA method was used to test the anti-Toxoplasma avidity of IgG, IgM and IgG. The data were analyzed by descriptive statistical methods and Chi-square test (P < 0.05 using SPSS 17.0. Results: Anti-Toxoplasma tests of IgM and IgG were positive in 9 and 86 samples respectively. Also, a borderline IgM was detected in 2 suspected samples. In addition, among all positive and suspected samples, 79 cases indicated high titers of IgG avidity, 7 cases were of low titers and 1 case was of a borderline titer. The prevalence of anti-Toxoplasma antibodies was 20%. The sera which showed high avidity index was obtained from patients at chronic phase of infection (92% while those which showed low avidity levels were from patients at acute toxoplasmosis (77.7%. Conclusions: This study clearly showed that acute and chronic phases of toxoplasmosis could be differentiated with the aid of IgG avidity test. This test may also assist in recognizing old and newly acquired infections.

  18. Fragment-derived inhibitors of human N-myristoyltransferase block capsid assembly and replication of the common cold virus

    Science.gov (United States)

    Mousnier, Aurélie; Bell, Andrew S.; Swieboda, Dawid P.; Morales-Sanfrutos, Julia; Pérez-Dorado, Inmaculada; Brannigan, James A.; Newman, Joseph; Ritzefeld, Markus; Hutton, Jennie A.; Guedán, Anabel; Asfor, Amin S.; Robinson, Sean W.; Hopkins-Navratilova, Iva; Wilkinson, Anthony J.; Johnston, Sebastian L.; Leatherbarrow, Robin J.; Tuthill, Tobias J.; Solari, Roberto; Tate, Edward W.

    2018-06-01

    Rhinoviruses (RVs) are the pathogens most often responsible for the common cold, and are a frequent cause of exacerbations in asthma, chronic obstructive pulmonary disease and cystic fibrosis. Here we report the discovery of IMP-1088, a picomolar dual inhibitor of the human N-myristoyltransferases NMT1 and NMT2, and use it to demonstrate that pharmacological inhibition of host-cell N-myristoylation rapidly and completely prevents rhinoviral replication without inducing cytotoxicity. The identification of cooperative binding between weak-binding fragments led to rapid inhibitor optimization through fragment reconstruction, structure-guided fragment linking and conformational control over linker geometry. We show that inhibition of the co-translational myristoylation of a specific virus-encoded protein (VP0) by IMP-1088 potently blocks a key step in viral capsid assembly, to deliver a low nanomolar antiviral activity against multiple RV strains, poliovirus and foot and-mouth disease virus, and protection of cells against virus-induced killing, highlighting the potential of host myristoylation as a drug target in picornaviral infections.

  19. Relevance of capsid structure in the buckling and maturation of spherical viruses

    International Nuclear Information System (INIS)

    Aznar, María; Luque, Antoni; Reguera, David

    2012-01-01

    The shape and mechanical properties of viral capsids play an important role in several biological processes during the virus life cycle. In particular, to become infective, many viruses require a maturation stage where the capsid undergoes a buckling transition, from an initial spherical procapsid into a final icosahedral faceted shell. Here we study, using a minimal physical model, how the capsid shape and the buckling transition depend on the triangulation number T and the icosahedral class P of the virus structure. We find that, for small shells, capsids with P = 1 are most likely to produce polyhedral shapes that minimize their energy and accumulated stress, whereas viruses with P = 3 prefer to remain spherical. For big capsids, all shells are more stable adopting an icosahedral shape, in agreement with continuum elastic theory. Moreover, spherical viruses show a buckling transition to polyhedral shells under expansion, in consonance with virus maturation. The resulting icosahedral shell is mechanically stiffer, tolerates larger expansions and withstands higher internal pressures before failing, which could explain why some dsDNA viruses, which rely on the pressurization of their genetic material to facilitate the infection, undergo a buckling transition. We emphasize that the results are general and could also be applied to non-biological systems. (paper)

  20. A molecular thermodynamic model for the stability of hepatitis B capsids

    Science.gov (United States)

    Kim, Jehoon; Wu, Jianzhong

    2014-06-01

    Self-assembly of capsid proteins and genome encapsidation are two critical steps in the life cycle of most plant and animal viruses. A theoretical description of such processes from a physiochemical perspective may help better understand viral replication and morphogenesis thus provide fresh insights into the experimental studies of antiviral strategies. In this work, we propose a molecular thermodynamic model for predicting the stability of Hepatitis B virus (HBV) capsids either with or without loading nucleic materials. With the key components represented by coarse-grained thermodynamic models, the theoretical predictions are in excellent agreement with experimental data for the formation free energies of empty T4 capsids over a broad range of temperature and ion concentrations. The theoretical model predicts T3/T4 dimorphism also in good agreement with the capsid formation at in vivo and in vitro conditions. In addition, we have studied the stability of the viral particles in response to physiological cellular conditions with the explicit consideration of the hydrophobic association of capsid subunits, electrostatic interactions, molecular excluded volume effects, entropy of mixing, and conformational changes of the biomolecular species. The course-grained model captures the essential features of the HBV nucleocapsid stability revealed by recent experiments.

  1. A molecular thermodynamic model for the stability of hepatitis B capsids

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Jehoon; Wu, Jianzhong, E-mail: jwu@engr.ucr.edu [Department of Chemical and Environmental Engineering, University of California, Riverside, California 92521 (United States)

    2014-06-21

    Self-assembly of capsid proteins and genome encapsidation are two critical steps in the life cycle of most plant and animal viruses. A theoretical description of such processes from a physiochemical perspective may help better understand viral replication and morphogenesis thus provide fresh insights into the experimental studies of antiviral strategies. In this work, we propose a molecular thermodynamic model for predicting the stability of Hepatitis B virus (HBV) capsids either with or without loading nucleic materials. With the key components represented by coarse-grained thermodynamic models, the theoretical predictions are in excellent agreement with experimental data for the formation free energies of empty T4 capsids over a broad range of temperature and ion concentrations. The theoretical model predicts T3/T4 dimorphism also in good agreement with the capsid formation at in vivo and in vitro conditions. In addition, we have studied the stability of the viral particles in response to physiological cellular conditions with the explicit consideration of the hydrophobic association of capsid subunits, electrostatic interactions, molecular excluded volume effects, entropy of mixing, and conformational changes of the biomolecular species. The course-grained model captures the essential features of the HBV nucleocapsid stability revealed by recent experiments.

  2. The mechanism of DNA ejection in the Bacillus anthracis spore-binding phage 8a revealed by cryo-electron tomography

    Energy Technology Data Exchange (ETDEWEB)

    Fu, Xiaofeng [Department of Pathology and Laboratory Medicine, University of Texas Medical School at Houston, Houston, TX 77030 (United States); Walter, Michael H. [Department of Biology, University of Northern Iowa, Cedar Falls, IA 50614 (United States); Paredes, Angel [Department of Pathology and Laboratory Medicine, University of Texas Medical School at Houston, Houston, TX 77030 (United States); Morais, Marc C., E-mail: mcmorais@utmb.edu [Sealy Center for Structural Biology and Molecular Biophysics, University of Texas Medical Branch, Galveston, TX 77555 (United States); Department of Biochemistry and Molecular Biology, University of Texas Medical Branch, Galveston, TX 77555 (United States); Liu, Jun, E-mail: Jun.Liu.1@uth.tmc.edu [Department of Pathology and Laboratory Medicine, University of Texas Medical School at Houston, Houston, TX 77030 (United States)

    2011-12-20

    The structure of the Bacillus anthracis spore-binding phage 8a was determined by cryo-electron tomography. The phage capsid forms a T = 16 icosahedron attached to a contractile tail via a head-tail connector protein. The tail consists of a six-start helical sheath surrounding a central tail tube, and a structurally novel baseplate at the distal end of the tail that recognizes and attaches to host cells. The parameters of the icosahedral capsid lattice and the helical tail sheath suggest protein folds for the capsid and tail-sheath proteins, respectively, and indicate evolutionary relationships to other dsDNA viruses. Analysis of 2518 intact phage particles show four distinct conformations that likely correspond to four sequential states of the DNA ejection process during infection. Comparison of the four observed conformations suggests a mechanism for DNA ejection, including the molecular basis underlying coordination of tail sheath contraction and genome release from the capsid.

  3. Electrical storm presages nonsudden death: the antiarrhythmics versus implantable defibrillators (AVID) trial.

    Science.gov (United States)

    Exner, D V; Pinski, S L; Wyse, D G; Renfroe, E G; Follmann, D; Gold, M; Beckman, K J; Coromilas, J; Lancaster, S; Hallstrom, A P

    2001-04-24

    Electrical storm, multiple temporally related episodes of ventricular tachycardia (VT) or ventricular fibrillation (VF), is a frequent problem among recipients of implantable cardioverter defibrillators (ICDs). However, insufficient data exist regarding its prognostic significance. This analysis includes 457 patients who received an ICD in the Antiarrhythmics Versus Implantable Defibrillators (AVID) trial and who were followed for 31 +/- 13 months. Electrical storm was defined as > or = 3 separate episodes of VT/VF within 24 hours. Characteristics and survival of patients surviving electrical storm (n = 90), those with VT/VF unrelated to electrical storm (n = 184), and the remaining patients (n = 183) were compared. The 3 groups differed in terms of ejection fraction, index arrhythmia, revascularization status, and baseline medication use. Survival was evaluated using time-dependent Cox modeling. Electrical storm occurred 9.2 +/- 11.5 months after ICD implantation, and most episodes (86%) were due to VT. Electrical storm was a significant risk factor for subsequent death, independent of ejection fraction and other prognostic variables (relative risk [RR], 2.4; 95% confidence interval [CI], 1.3 to 4.2; P = 0.003), but VT/VF unrelated to electrical storm was not (RR, 1.0; 95% CI, 0.6 to 1.7; P = 0.9). The risk of death was greatest 3 months after electrical storm (RR, 5.4; 95% Cl, 2.4 to 12.3; P = 0.0001) and diminished beyond this time (RR, 1.9; 95% CI, 1.0 to 3.6; P=0.04). Electrical storm is an important, independent marker for subsequent death among ICD recipients, particularly in the first 3 months after its occurrence. However, the development of VT/VF unrelated to electrical storm does not seem to be associated with an increased risk of subsequent death.

  4. Dual time point FDG PET imaging in evaluating pulmonary nodules with low FDG avidity

    International Nuclear Information System (INIS)

    Chen Xiang; Zhao Jinhua; Song Jianhua; Xing Yan; Wang Taisong; Qiao Wenli

    2010-01-01

    A standardized uptake value (SUV) of 2.5 is frequently used as criteria to evaluate pulmonary lesions. However, false results may occur. Some studies have shown the usefulness of delayed PET for improving accuracy, while others recently have shown fewer promising results. This study was designed to investigate the accuracy of dual time point (DTP) FDG PET imaging in the evaluation of pulmonary lesions with an initial SUV less than 2.5. DTP FDG PET studies were conducted about 1 and 2 hours after FDG injection, and pulmonary lesions with an initial SUV less than 2.5 were identified. Nodules with pathologic results or imaging follow up were included. The differences in SUV and retention index (RI) between benign and malignant pulmonary lesions were analyzed. Receiver operating characteristics (ROC) analysis was performed to evaluate the discriminating validity of SUV and RI. 51 lesions were finally included. A RI greater than 0% was observed in 64% of the benign lesions; 56% had a RI greater than 10%. Among the malignancies, 80.8% had a RI greater than 0%, and 61.5% had a RI greater than 10%. We found no significant differences in SUV and RI between benign and malignant lesions. The area under the ROC curve did not differ from 0.5 whether using SUV or the retention index. Utilizing a SUV increase of 10%, the sensitivity was 61.5%, specificity 44% and accuracy was 52.9%. Dual time point FDG PET may not be of benefit in the evaluation of pulmonary nodules with low FDG avidity. (authors)

  5. Parvovirus B19 does not bind to membrane-associated globoside in vitro

    International Nuclear Information System (INIS)

    Kaufmann, Baerbel; Baxa, Ulrich; Chipman, Paul R.; Rossmann, Michael G.; Modrow, Susanne; Seckler, Robert

    2005-01-01

    The glycosphingolipid globoside (globotetraosylceramide, Gb4Cer) has been proposed to be the cellular receptor of human parvovirus B19. Quantitative measurements of the binding of parvovirus B19 to Gb4Cer were performed to explore the molecular basis of the virus tropism. Solid-phase assays with fluorescence-labeled liposomes or 125 iodine-labeled empty capsids were used to characterize the specificity of binding. In addition, surface plasmon resonance on lipid layers, as well as isothermal titration microcalorimetry, was utilized for real-time analysis of the virus-receptor interaction. These studies did not confirm binding of Gb4Cer to recombinant B19 VP2 capsids, suggesting that Gb4Cer does not function on its own as the cellular receptor of human parvovirus B19, but might be involved in a more complex recognition event. The biochemical results were further confirmed by cryo-electron microscopy image reconstructions at 10 A resolution, in which the structures of empty capsids were compared with empty capsids incubated with Gb4Cer

  6. Poliovirus-associated protein kinase: Destabilization of the virus capsid and stimulation of the phosphorylation reaction by Zn2+

    International Nuclear Information System (INIS)

    Ratka, M.; Lackmann, M.; Ueckermann, C.; Karlins, U.; Koch, G.

    1989-01-01

    The previously described poliovirus-associated protein kinase activity phosphorylates viral proteins VP0 and VP2 as well as exogenous proteins in the presence of Mg 2+ . In this paper, the effect of Zn 2+ on the phosphorylation reaction and the stability of the poliovirus capsid has been studied in detail and compared to that of Mg 2+ . In the presence of Zn 2+ , phosphorylation of capsid proteins VP2 and VP4 is significantly higher while phosphorylation of VP0 and exogenous phosphate acceptor proteins is not detected. The results indicate the activation of more than one virus-associated protein kinase by Zn 2+ . The ion-dependent behavior of the enzyme activities is observed independently of whether the virus was obtained from HeLa or green monkey kidney cells. The poliovirus capsid is destabilized by Zn 2+ . This alteration of the poliovirus capsid structure is a prerequisite for effective phosphorylation of viral capsid proteins. The increased level of phosphorylation of viral capsid proteins results in further destabilization of the viral capsid. As a result of the conformational changes, poliovirus-associated protein kinase activities dissociate from the virus particle. The authors suggest that the destabilizing effect of phosphorylation on the viral capsid plays a role in uncoating of poliovirus

  7. Structural Transitions and Energy Landscape for Cowpea Chlorotic Mottle Virus Capsid Mechanics from Nanomanipulation in Vitro and in Silico

    Science.gov (United States)

    Kononova, Olga; Snijder, Joost; Brasch, Melanie; Cornelissen, Jeroen; Dima, Ruxandra I.; Marx, Kenneth A.; Wuite, Gijs J. L.; Roos, Wouter H.; Barsegov, Valeri

    2013-10-01

    Physical properties of capsids of plant and animal viruses are important factors in capsid self-assembly, survival of viruses in the extracellular environment, and their cell infectivity. Virus shells can have applications as nanocontainers and delivery vehicles in biotechnology and medicine. Combined AFM experiments and computational modeling on sub-second timescales of the indentation nanomechanics of Cowpea Chlorotic Mottle Virus (CCMV) capsid show that the capsid's physical properties are dynamic and local characteristics of the structure, which depend on the magnitude and geometry of mechanical input. Surprisingly, under large deformations the CCMV capsid transitions to the collapsed state without substantial local structural alterations. The enthalpy change in this deformation state dH = 11.5 - 12.8 MJ/mol is mostly due to large-amplitude out-of-plane excitations, which contribute to the capsid bending, and the entropy change TdS = 5.1 - 5.8 MJ/mol is mostly due to coherent in-plane rearrangements of protein chains, which result in the capsid stiffening. Dynamic coupling of these modes defines the extent of elasticity and reversibility of capsid mechanical deformation. This emerging picture illuminates how unique physico-chemical properties of protein nanoshells help define their structure and morphology, and determine their viruses' biological function.

  8. Human Cytomegalovirus Nuclear Capsids Associate with the Core Nuclear Egress Complex and the Viral Protein Kinase pUL97.

    Science.gov (United States)

    Milbradt, Jens; Sonntag, Eric; Wagner, Sabrina; Strojan, Hanife; Wangen, Christina; Lenac Rovis, Tihana; Lisnic, Berislav; Jonjic, Stipan; Sticht, Heinrich; Britt, William J; Schlötzer-Schrehardt, Ursula; Marschall, Manfred

    2018-01-13

    The nuclear phase of herpesvirus replication is regulated through the formation of regulatory multi-component protein complexes. Viral genomic replication is followed by nuclear capsid assembly, DNA encapsidation and nuclear egress. The latter has been studied intensely pointing to the formation of a viral core nuclear egress complex (NEC) that recruits a multimeric assembly of viral and cellular factors for the reorganization of the nuclear envelope. To date, the mechanism of the association of human cytomegalovirus (HCMV) capsids with the NEC, which in turn initiates the specific steps of nuclear capsid budding, remains undefined. Here, we provide electron microscopy-based data demonstrating the association of both nuclear capsids and NEC proteins at nuclear lamina budding sites. Specifically, immunogold labelling of the core NEC constituent pUL53 and NEC-associated viral kinase pUL97 suggested an intranuclear NEC-capsid interaction. Staining patterns with phospho-specific lamin A/C antibodies are compatible with earlier postulates of targeted capsid egress at lamina-depleted areas. Important data were provided by co-immunoprecipitation and in vitro kinase analyses using lysates from HCMV-infected cells, nuclear fractions, or infectious virions. Data strongly suggest that nuclear capsids interact with pUL53 and pUL97. Combined, the findings support a refined concept of HCMV nuclear trafficking and NEC-capsid interaction.

  9. Human Cytomegalovirus Nuclear Capsids Associate with the Core Nuclear Egress Complex and the Viral Protein Kinase pUL97

    Directory of Open Access Journals (Sweden)

    Jens Milbradt

    2018-01-01

    Full Text Available The nuclear phase of herpesvirus replication is regulated through the formation of regulatory multi-component protein complexes. Viral genomic replication is followed by nuclear capsid assembly, DNA encapsidation and nuclear egress. The latter has been studied intensely pointing to the formation of a viral core nuclear egress complex (NEC that recruits a multimeric assembly of viral and cellular factors for the reorganization of the nuclear envelope. To date, the mechanism of the association of human cytomegalovirus (HCMV capsids with the NEC, which in turn initiates the specific steps of nuclear capsid budding, remains undefined. Here, we provide electron microscopy-based data demonstrating the association of both nuclear capsids and NEC proteins at nuclear lamina budding sites. Specifically, immunogold labelling of the core NEC constituent pUL53 and NEC-associated viral kinase pUL97 suggested an intranuclear NEC-capsid interaction. Staining patterns with phospho-specific lamin A/C antibodies are compatible with earlier postulates of targeted capsid egress at lamina-depleted areas. Important data were provided by co-immunoprecipitation and in vitro kinase analyses using lysates from HCMV-infected cells, nuclear fractions, or infectious virions. Data strongly suggest that nuclear capsids interact with pUL53 and pUL97. Combined, the findings support a refined concept of HCMV nuclear trafficking and NEC-capsid interaction.

  10. Intra- and inter-subunit disulfide bond formation is nonessential in adeno-associated viral capsids.

    Directory of Open Access Journals (Sweden)

    Nagesh Pulicherla

    Full Text Available The capsid proteins of adeno-associated viruses (AAV have five conserved cysteine residues. Structural analysis of AAV serotype 2 reveals that Cys289 and Cys361 are located adjacent to each other within each monomer, while Cys230 and Cys394 are located on opposite edges of each subunit and juxtaposed at the pentamer interface. The Cys482 residue is located at the base of a surface loop within the trimer region. Although plausible based on molecular dynamics simulations, intra- or inter-subunit disulfides have not been observed in structural studies. In the current study, we generated a panel of Cys-to-Ser mutants to interrogate the potential for disulfide bond formation in AAV capsids. The C289S, C361S and C482S mutants were similar to wild type AAV with regard to titer and transduction efficiency. However, AAV capsid protein subunits with C230S or C394S mutations were prone to proteasomal degradation within the host cells. Proteasomal inhibition partially blocked degradation of mutant capsid proteins, but failed to rescue infectious virions. While these results suggest that the Cys230/394 pair is critical, a C394V mutant was found viable, but not the corresponding C230V mutant. Although the exact nature of the structural contribution(s of Cys230 and Cys394 residues to AAV capsid formation remains to be determined, these results support the notion that disulfide bond formation within the Cys289/361 or Cys230/394 pair appears to be nonessential. These studies represent an important step towards understanding the role of inter-subunit interactions that drive AAV capsid assembly.

  11. Evolution to pathogenicity of the parvovirus minute virus of mice in immunodeficient mice involves genetic heterogeneity at the capsid domain that determines tropism.

    Science.gov (United States)

    López-Bueno, Alberto; Segovia, José C; Bueren, Juan A; O'Sullivan, M Gerard; Wang, Feng; Tattersall, Peter; Almendral, José M

    2008-02-01

    mice. The parental consensus genotype prevailed during leukopenia development, but plaque-forming viruses with the reversion of the 575A residue to valine emerged in affected organs. The disease caused by the DNA virus in mice, therefore, involves the generation of heterogeneous viral populations that may cooperatively interact for the hemopoietic syndrome. The evolutionary changes delineate a sector of the surface of the capsid that determines tropism and that surrounds the sialic acid receptor binding domain.

  12. Single Mutations in the VP2 300 Loop Region of the Three-Fold Spike of the Carnivore Parvovirus Capsid Can Determine Host Range

    Science.gov (United States)

    Organtini, Lindsey J.; Zhang, Sheng; Hafenstein, Susan L.; Holmes, Edward C.

    2015-01-01

    ABSTRACT Sylvatic carnivores, such as raccoons, have recently been recognized as important hosts in the evolution of canine parvovirus (CPV), a pandemic pathogen of domestic dogs. Although viruses from raccoons do not efficiently bind the dog transferrin receptor (TfR) or infect dog cells, a single mutation changing an aspartic acid to a glycine at capsid (VP2) position 300 in the prototype raccoon CPV allows dog cell infection. Because VP2 position 300 exhibits extensive amino acid variation among the carnivore parvoviruses, we further investigated its role in determining host range by analyzing its diversity and evolution in nature and by creating a comprehensive set of VP2 position 300 mutants in infectious clones. Notably, some position 300 residues rendered CPV noninfectious for dog, but not cat or fox, cells. Changes of adjacent residues (residues 299 and 301) were also observed often after cell culture passage in different hosts, and some of the mutations mimicked changes seen in viruses recovered from natural infections of alternative hosts, suggesting that compensatory mutations were selected to accommodate the new residue at position 300. Analysis of the TfRs of carnivore hosts used in the experimental evolution studies demonstrated that their glycosylation patterns varied, including a glycan present only on the domestic dog TfR that dictates susceptibility to parvoviruses. Overall, there were significant differences in the abilities of viruses with alternative position 300 residues to bind TfRs and infect different carnivore hosts, demonstrating that the process of infection is highly host dependent and that VP2 position 300 is a key determinant of host range. IMPORTANCE Although the emergence and pandemic spread of canine parvovirus (CPV) are well documented, the carnivore hosts and evolutionary pathways involved in its emergence remain enigmatic. We recently demonstrated that a region in the capsid structure of CPV, centered around VP2 position 300

  13. Identification of a novel cell culture adaptation site on the capsid of foot-and-mouth disease virus.

    Science.gov (United States)

    Chamberlain, Kyle; Fowler, Veronica L; Barnett, Paul V; Gold, Sarah; Wadsworth, Jemma; Knowles, Nick J; Jackson, Terry

    2015-09-01

    Vaccination remains the most effective tool for control of foot-and-mouth disease both in endemic countries and as an emergency preparedness for new outbreaks. Foot-and-mouth disease vaccines are chemically inactivated virus preparations and the production of new vaccines is critically dependent upon cell culture adaptation of field viruses, which can prove problematic. A major driver of cell culture adaptation is receptor availability. Field isolates of foot-and-mouth disease virus (FMDV) use RGD-dependent integrins as receptors, whereas cell culture adaptation often selects for variants with altered receptor preferences. Previously, two independent sites on the capsid have been identified where mutations are associated with improved cell culture growth. One is a shallow depression formed by the three major structural proteins (VP1-VP3) where mutations create a heparan sulphate (HS)-binding site (the canonical HS-binding site). The other involves residues of VP1 and is located at the fivefold symmetry axis. For some viruses, changes at this site result in HS binding; for others, the receptors are unknown. Here, we report the identification of a novel site on VP2 where mutations resulted in an expanded cell tropism of a vaccine variant of A/IRN/87 (called A - ). Furthermore, we show that introducing the same mutations into a different type A field virus (A/TUR/2/2006) resulted in the same expanded cell culture tropism as the A/IRN/87 A -  vaccine variant. These observations add to the evidence for multiple cell attachment mechanisms for FMDV and may be useful for vaccine manufacture when cell culture adaptation proves difficult.

  14. Effects of egg-adaptation on receptor-binding and antigenic properties of recent influenza A (H3N2) vaccine viruses.

    Science.gov (United States)

    Parker, Lauren; Wharton, Stephen A; Martin, Stephen R; Cross, Karen; Lin, Yipu; Liu, Yan; Feizi, Ten; Daniels, Rodney S; McCauley, John W

    2016-06-01

    Influenza A virus (subtype H3N2) causes seasonal human influenza and is included as a component of influenza vaccines. The majority of vaccine viruses are isolated and propagated in eggs, which commonly results in amino acid substitutions in the haemagglutinin (HA) glycoprotein. These substitutions can affect virus receptor-binding and alter virus antigenicity, thereby, obfuscating the choice of egg-propagated viruses for development into candidate vaccine viruses. To evaluate the effects of egg-adaptive substitutions seen in H3N2 vaccine viruses on sialic acid receptor-binding, we carried out quantitative measurement of virus receptor-binding using surface biolayer interferometry with haemagglutination inhibition (HI) assays to correlate changes in receptor avidity with antigenic properties. Included in these studies was a panel of H3N2 viruses generated by reverse genetics containing substitutions seen in recent egg-propagated vaccine viruses and corresponding cell culture-propagated wild-type viruses. These assays provide a quantitative approach to investigating the importance of individual amino acid substitutions in influenza receptor-binding. Results show that viruses with egg-adaptive HA substitutions R156Q, S219Y, and I226N, have increased binding avidity to α2,3-linked receptor-analogues and decreased binding avidity to α2,6-linked receptor-analogues. No measurable binding was detected for the viruses with amino acid substitution combination 156Q+219Y and receptor-binding increased in viruses where egg-adaptation mutations were introduced into cell culture-propagated virus. Substitutions at positions 156 and 190 appeared to be primarily responsible for low reactivity in HI assays with post-infection ferret antisera raised against 2012-2013 season H3N2 viruses. Egg-adaptive substitutions at position 186 caused substantial differences in binding avidity with an insignificant effect on antigenicity.

  15. Radiation Therapy to the Primary and Postinduction Chemotherapy MIBG-Avid Sites in High-Risk Neuroblastoma

    Energy Technology Data Exchange (ETDEWEB)

    Mazloom, Ali; Louis, Chrystal U.; Nuchtern, Jed; Kim, Eugene; Russell, Heidi; Allen-Rhoades, Wendy; Krance, Robert; Paulino, Arnold C., E-mail: apaulino@mdanderson.org

    2014-11-15

    Purpose: Although it is generally accepted that consolidation therapy for neuroblastoma includes irradiation of the primary site and any remaining metaiodobenzylguanidine (MIBG)-avid metastatic sites, limited information has been published regarding the efficacy of this approach. Methods and Materials: Thirty patients with high-risk neuroblastoma were treated at 1 radiation therapy (RT) department after receiving 5 cycles of induction chemotherapy and resection. All patients had at least a partial response after induction therapy, based upon international neuroblastoma response criteria. The primary sites were treated with 24 to 30 Gy whereas the MIBG-avid metastatic sites were treated with 24 Gy. RT was followed by high-dose chemotherapy with autologous stem cell rescue and 6 months of cis-retinoic acid. Results: The 5-year progression-free survival (PFS) and overall survival (OS) rates were 48% and 59%, respectively. The 5-year locoregional control at the primary site was 84%. There were no differences in locoregional control according to degree of primary surgical resection. The 5-year local control rate for metastatic sites was 74%. The 5-year PFS rates for patients with 0, 1, 2, and >3 postinduction MIBG sites were 66%, 57%, 20%, and 0% (P<.0001), respectively, whereas 5-year OS rates were 80%, 57%, 50%, and 0%, respectively (P<.0001). Conclusions: RT to the primary site and postinduction MIBG-positive metastatic sites was associated with 84% and 74% local control, respectively. The number of MIBG-avid sites present after induction chemotherapy and surgery was predictive of progression-free and overall survival.

  16. Work in progress. Gastrointestinal ulcerations: detection using a technetium-99m-labeled ulcer-avid agent

    International Nuclear Information System (INIS)

    Vasquez, T.E.; Bridges, R.L.; Braunstein, P.; Jansholt, A.L.; Meshkinpour, H.

    1983-01-01

    Technetium-99m-labeled sucralfate, an ulcer-avid material, was shown in preliminary animal and human studies to be stable in vivo with good sensitivity. Eight experimentally produced discrete gastric ulcers in three rabbits were visualized using this material. Of seven human studies, four studies were true-positive and three were true-negative. It is suggested that Tc-99m-labeled sucralfate may prove to have significant clinical advantages for the evaluation of gastrointestinal ulcer disease and other diseases that are associated with loss of mucosal integrity. The method for labeling sucralfate with Tc-99m was developed by the authors

  17. FDG-avid portal vein tumor thrombosis from hepatocellular carcinoma in contrast-enhanced FDG PET/CT

    Directory of Open Access Journals (Sweden)

    Canh Nguyen

    2015-01-01

    Full Text Available Objective(s: In this study, we aimed to describe the characteristics of portal vein tumor thrombosis (PVTT, complicating hepatocellular carcinoma (HCC in contrast-enhanced FDG PET/CT scan. Methods: In this retrospective study, 9 HCC patients with FDG-avid PVTT were diagnosed by contrast-enhanced fluorodeoxyglucose positron emission tomography/computed tomography (FDG PET/CT, which is a combination of dynamic liver CT scan, multiphase imaging, and whole-body PET scan. PET and CT DICOM images of patients were imported into the PET/CT imaging system for the re-analysis of contrast enhancement and FDG uptake in thrombus, the diameter of the involved portal vein, and characteristics of liver tumors and metastasis. Results: Two patients with previously untreated HCC and 7 cases with previously treated HCC had FDG-avid PVTT in contrast-enhanced FDG PET/CT scan. During the arterial phase of CT scan, portal vein thrombus showed contrast enhancement in 8 out of 9 patients (88.9%. PET scan showed an increased linear FDG uptake along the thrombosed portal vein in all patients. The mean greatest diameter of thrombosed portal veins was 1.8 ± 0.2 cm, which was significantly greater than that observed in normal portal veins (P<0.001. FDG uptake level in portal vein thrombus was significantly higher than that of blood pool in the reference normal portal vein (P=0.001. PVTT was caused by the direct extension of liver tumors. All patients had visible FDG-avid liver tumors in contrast-enhanced images. Five out of 9 patients (55.6% had no extrahepatic metastasis, 3 cases (33.3% had metastasis of regional lymph nodes, and 1 case (11.1% presented with distant metastasis. The median estimated survival time of patients was 5 months. Conclusion: The intraluminal filling defect consistent with thrombous within the portal vein, expansion of the involved portal vein, contrast enhancement, and linear increased FDG uptake of the thrombus extended from liver tumor are

  18. The Importance of IgG Avidity and the Polymerase Chain Reaction in Treating Toxoplasmosis during Pregnancy: Current Knowledge

    Directory of Open Access Journals (Sweden)

    João Bortoletti Filho

    2013-01-01

    Full Text Available A brief report on the nature and epidemiology of T. gondii infection is firstly presented. The importance of the specific IgG avidity test and polymerase chain reaction (PCR for toxoplasmosis is discussed, along with their significance and importance as auxiliary methods for determining the most likely time for the initial infection by this coccidian and for defining the therapeutic strategy. Lastly, practical comments are made in relation to the classical therapeutic regimens, with special attention to the indications for fetal treatment, when this is necessary.

  19. Comparison of Toxoplasma gondii IgG avidity Architect and Vidas assays with the estimated date of infection in pregnant women

    Directory of Open Access Journals (Sweden)

    Smets Aurélie

    2016-01-01

    Full Text Available A maternal Toxoplasma gondii infection during pregnancy is a risk for congenital infection through maternal-fetal transplacental transmission. Estimation of the date of infection is of the utmost importance for management and treatment recommendations. In this setting, IgG avidity has been shown to be useful as high avidity rules out an infection dating less than 4 months. The estimated date of infection can also be obtained by the ratio of T. gondii IgG titers measured by the Vidas (bioMérieux assay versus T. gondii IgG titers measured by the Architect (Abbott Laboratories test, together with T. gondii IgM and IgA antibody responses. In this study, using 117 serum samples from pregnant women, we compared the IgG avidity values obtained by Architect and Vidas with the presumed date of T. gondii infection established by the T. gondii IgG ratio of IgG Vidas and IgG Architect plus the IgM and IgA results. To date, IgG avidity Vidas seems to exhibit better performance than Architect. For both assays, gray zone results were most likely obtained from patients infected more than 4 months before sampling. These data should be taken into account for a possible reconsideration of the interpretation of avidity results in the gray zone.

  20. A new series of HAPs as anti-HBV agents targeting at capsid assembly.

    Science.gov (United States)

    Yang, Xiu-yan; Xu, Xiao-qian; Guan, Hua; Wang, Li-li; Wu, Qin; Zhao, Guo-ming; Li, Song

    2014-09-01

    A series of novel Heteroaryldihydropyrimidines (HAPs) derivatives were designed and synthesized as potent inhibitors of HBV capsid assembly. These compounds were prepared from efforts to optimize an earlier series of HAPs, and compounds Mo1, Mo7, Mo8, Mo10, Mo12, and Mo13 demonstrated potent inhibition of HBV DNA replication at submicromolar range. Copyright © 2014. Published by Elsevier Ltd.

  1. Formation of newly synthesized adeno-associated virus capsids in the cell nucleus.

    Science.gov (United States)

    Bell, Peter; Vandenberghe, Luk H; Wilson, James M

    2014-06-01

    Adeno-associated virus (AAV) particles inside the nucleus of a HEK 293 cell are shown by electron microscopy. Cells have been triple-transfected for vector production and were analyzed for capsid formation three days later. Newly assembled particle are visible as seemingly unstructured conglomerates or crystal-like arrays.

  2. The VP7 Outer Capsid Protein of Rotavirus Induces Polyclonal B-Cell Activation

    Science.gov (United States)

    Blutt, Sarah E.; Crawford, Sue E.; Warfield, Kelly L.; Lewis, Dorothy E.; Estes, Mary K.; Conner, Margaret E.

    2004-01-01

    The early response to a homologous rotavirus infection in mice includes a T-cell-independent increase in the number of activated B lymphocytes in the Peyer's patches. The mechanism of this activation has not been previously determined. Since rotavirus has a repetitively arranged triple-layered capsid and repetitively arranged antigens can induce activation of B cells, one or more of the capsid proteins could be responsible for the initial activation of B cells during infection. To address this question, we assessed the ability of rotavirus and virus-like particles to induce B-cell activation in vivo and in vitro. Using infectious rotavirus, inactivated rotavirus, noninfectious but replication-competent virus, and virus-like particles, we determined that neither infectivity nor RNA was necessary for B-cell activation but the presence of the rotavirus outer capsid protein, VP7, was sufficient for murine B-cell activation. Preincubation of the virus with neutralizing VP7 antibodies inhibited B-cell activation. Polymyxin B treatment and boiling of the virus preparation were performed, which ruled out possible lipopolysaccharide contamination as the source of activation and confirmed that the structural conformation of VP7 is important for B-cell activation. These findings indicate that the structure and conformation of the outer capsid protein, VP7, initiate intestinal B-cell activation during rotavirus infection. PMID:15194774

  3. Functional dissection of the alphavirus capsid protease: sequence requirements for activity.

    Science.gov (United States)

    Thomas, Saijo; Rai, Jagdish; John, Lijo; Günther, Stephan; Drosten, Christian; Pützer, Brigitte M; Schaefer, Stephan

    2010-11-18

    The alphavirus capsid is multifunctional and plays a key role in the viral life cycle. The nucleocapsid domain is released by the self-cleavage activity of the serine protease domain within the capsid. All alphaviruses analyzed to date show this autocatalytic cleavage. Here we have analyzed the sequence requirements for the cleavage activity of Chikungunya virus capsid protease of genus alphavirus. Amongst alphaviruses, the C-terminal amino acid tryptophan (W261) is conserved and found to be important for the cleavage. Mutating tryptophan to alanine (W261A) completely inactivated the protease. Other amino acids near W261 were not having any effect on the activity of this protease. However, serine protease inhibitor AEBSF did not inhibit the activity. Through error-prone PCR we found that isoleucine 227 is important for the effective activity. The loss of activity was analyzed further by molecular modelling and comparison of WT and mutant structures. It was found that lysine introduced at position 227 is spatially very close to the catalytic triad and may disrupt electrostatic interactions in the catalytic site and thus inactivate the enzyme. We are also examining other sequence requirements for this protease activity. We analyzed various amino acid sequence requirements for the activity of ChikV capsid protease and found that amino acids outside the catalytic triads are important for the activity.

  4. The two capsid proteins of maize rayado fino virus contain common peptide sequences.

    Science.gov (United States)

    Falk, B W; Tsai, J H

    1986-01-01

    Virions of maize rayado fino virus (MRFV) were purified and two major capsid proteins (ca. Mr 29,000 and 22,000) were resolved by SDS-PAGE. When the two major capsid proteins were isolated from gels and compared by one-dimensional peptide mapping after digestion with Staphylococcus aureus V-8 protease, indistinguishable peptide maps were obtained, suggesting that these two proteins contain common peptide sequences. Some preparations also showed minor protein components that were intermediate between the Mr 22,000 and Mr 29,000 capsid proteins. One of the minor proteins, ca. Mr 27,000, gave a peptide map indistinguishable from the major capsid proteins. In vitro ageing of partially purified preparations or virion treatment with proteolytic enzymes failed to show conversion of the Mr 29,000 protein to a Mr 22,000. Protease inhibitors added to the buffers used for virion purification did not affect the apparent 1:3 ratio of 29,000 to 22,000 proteins in the purified preparations.

  5. Promoter analysis of the Chilo iridescent virus DNA polymerase and major capsid protein genes

    NARCIS (Netherlands)

    Nalcacioglu, R.; Marks, H.; Vlak, J.M.; Demirbag, Z.; Oers, van M.M.

    2003-01-01

    The DNA polymerase (DNApol) and major capsid protein (MCP) genes were used as models to study promoter activity in Chilo iridescent virus (CIV). Infection of Bombyx mori SPC-BM-36 cells in the presence of inhibitors of DNA or protein synthesis showed that DNApol, as well as helicase, is an

  6. Porcine circovirus-2 capsid protein induces cell death in PK15 cells

    Energy Technology Data Exchange (ETDEWEB)

    Walia, Rupali; Dardari, Rkia, E-mail: rdardari@ucalgary.ca; Chaiyakul, Mark; Czub, Markus

    2014-11-15

    Studies have shown that Porcine circovirus (PCV)-2 induces apoptosis in PK15 cells. Here we report that cell death is induced in PCV2b-infected PK15 cells that express Capsid (Cap) protein and this effect is enhanced in interferon gamma (IFN-γ)-treated cells. We further show that transient PCV2a and 2b-Cap protein expression induces cell death in PK15 cells at rate similar to PCV2 infection, regardless of Cap protein localization. These data suggest that Cap protein may have the capacity to trigger different signaling pathways involved in cell death. Although further investigation is needed to gain deeper insights into the nature of the pathways involved in Cap-induced cell death, this study provides evidence that PCV2-induced cell death in kidney epithelial PK15 cells can be mapped to the Cap protein and establishes the need for future research regarding the role of Cap-induced cell death in PCV2 pathogenesis. - Highlights: • IFN-γ enhances PCV2 replication that leads to cell death in PK15 cells. • IFN-γ enhances nuclear localization of the PCV2 Capsid protein. • Transient PCV2a and 2b-Capsid protein expression induces cell death. • Cell death is not dictated by specific Capsid protein sub-localization.

  7. Facilitating the use of alternative capsid control methods towards sustainable production of organic cocoa in Ghana

    NARCIS (Netherlands)

    Ayenor, G.K.; Huis, van A.; Obeng-Ofori, D.; Padi, B.; Röling, N.G.

    2007-01-01

    Cocoa (Theobroma cacao L.) is an important foreign exchange earner for Ghana. However, production is constrained by a high incidence of pests and diseases. Based on farmers' needs, this study focused on the control of capsids, mainly Sahlbergella singularis Haglund and Distantiella theobroma

  8. Four levels of hierarchical organization, including noncovalent chainmail, brace the mature tumor herpesvirus capsid against pressurization.

    Science.gov (United States)

    Zhou, Z Hong; Hui, Wong Hoi; Shah, Sanket; Jih, Jonathan; O'Connor, Christine M; Sherman, Michael B; Kedes, Dean H; Schein, Stan

    2014-10-07

    Like many double-stranded DNA viruses, tumor gammaherpesviruses Epstein-Barr virus and Kaposi's sarcoma-associated herpesvirus withstand high internal pressure. Bacteriophage HK97 uses covalent chainmail for this purpose, but how this is achieved noncovalently in the much larger gammaherpesvirus capsid is unknown. Our cryoelectron microscopy structure of a gammaherpesvirus capsid reveals a hierarchy of four levels of organization: (1) Within a hexon capsomer, each monomer of the major capsid protein (MCP), 1,378 amino acids and six domains, interacts with its neighboring MCPs at four sites. (2) Neighboring capsomers are linked in pairs by MCP dimerization domains and in groups of three by heterotrimeric triplex proteins. (3) Small (∼280 amino acids) HK97-like domains in MCP monomers alternate with triplex heterotrimers to form a belt that encircles each capsomer. (4) One hundred sixty-two belts concatenate to form noncovalent chainmail. The triplex heterotrimer orchestrates all four levels and likely drives maturation to an angular capsid that can withstand pressurization. Copyright © 2014 Elsevier Ltd. All rights reserved.

  9. Scaffold expulsion and genome packaging trigger stabilization of herpes simplex virus capsids

    NARCIS (Netherlands)

    Roos, W.H.; Radtke, K.; Kniesmeijer, E.G.R.; Geertsema, H.J.; Sodeik, B.; Wuite, G.J.L.

    2009-01-01

    Herpes simplex virus type 1 (HSV1) capsids undergo extensive structural changes during maturation and DNA packaging. As a result, they become more stable and competent for nuclear egress. To further elucidate this stabilization process, we used biochemical and nanoindentation approaches to analyze

  10. Scaffold expulsion and genome packaging trigger stabilization of Herpes Simplex Virus capsids

    NARCIS (Netherlands)

    Roos, W.H.; Radtke, K.; Kniesmeijer, E.; Geertsema, H.J.; Sodeik, B.; Wuite, G.J.L.

    2009-01-01

    Herpes simplex virus type 1 (HSV1) capsids undergo extensive structural changes during maturation and DNA packaging. As a result, they become more stable and competent for nuclear egress. To further elucidate this stabilization process, we used biochemical and nanoindentation approaches to analyze

  11. Exploring the role of genome and structural ions in preventing viral capsid collapse during dehydration

    Science.gov (United States)

    Martín-González, Natalia; Guérin Darvas, Sofía M.; Durana, Aritz; Marti, Gerardo A.; Guérin, Diego M. A.; de Pablo, Pedro J.

    2018-03-01

    Even though viruses evolve mainly in liquid milieu, their horizontal transmission routes often include episodes of dry environment. Along their life cycle, some insect viruses, such as viruses from the Dicistroviridae family, withstand dehydrated conditions with presently unknown consequences to their structural stability. Here, we use atomic force microscopy to monitor the structural changes of viral particles of Triatoma virus (TrV) after desiccation. Our results demonstrate that TrV capsids preserve their genome inside, conserving their height after exposure to dehydrating conditions, which is in stark contrast with other viruses that expel their genome when desiccated. Moreover, empty capsids (without genome) resulted in collapsed particles after desiccation. We also explored the role of structural ions in the dehydration process of the virions (capsid containing genome) by chelating the accessible cations from the external solvent milieu. We observed that ion suppression helps to keep the virus height upon desiccation. Our results show that under drying conditions, the genome of TrV prevents the capsid from collapsing during dehydration, while the structural ions are responsible for promoting solvent exchange through the virion wall.

  12. Effects of immunosuppression on circulating adeno-associated virus capsid-specific T cells in humans.

    Science.gov (United States)

    Parzych, Elizabeth M; Li, Hua; Yin, Xiangfan; Liu, Qin; Wu, Te-Lang; Podsakoff, Gregory M; High, Katherine A; Levine, Matthew H; Ertl, Hildegund C J

    2013-04-01

    In humans adeno-associated virus (AAV)-mediated gene transfer is followed by expansion of AAV capsid-specific T cells, evidence of cell damage, and loss of transgene product expression, implicating immunological rejection of vector-transduced cells, which may be prevented by immunosuppressive drugs. We undertook this study to assess the effect of immunosuppression (IS) used for organ transplantation on immune responses to AAV capsid antigens. Recipients of liver or kidney transplants were tested before and 4 weeks after induction of IS in comparison with matched samples from healthy human adults and an additional cohort with comorbid conditions similar to those of the transplant patients. Our data show that transplant patients and comorbid control subjects have markedly higher frequencies of circulating AAV capsid-specific T cells compared with healthy adults. On average, IS resulted in a reduction of AAV-specific CD4⁺ T cells, whereas numbers of circulating CD8⁺ effector and central memory T cells tended to increase. Independent of the type of transplant or the IS regimens, the trend of AAV capsid-specific T cell responses after drug treatment varied; in some patients responses were unaffected whereas others showed decreases or even pronounced increases, casting doubt on the usefulness of prophylactic IS for AAV vector recipients.

  13. Expression and purification of capsid proteins of Aichi virus and in vitro reassembly of empty virion

    Czech Academy of Sciences Publication Activity Database

    Smola, Miroslav; Dubánková, Anna; Šilhán, Jan; Bouřa, Evžen

    2017-01-01

    Roč. 284, Suppl 1 (2017), s. 107 ISSN 1742-464X. [FEBS Congress /42./ From Molecules to Cells and Back. 10.09.2017-14.09.2017, Jerusalem] R&D Projects: GA ČR GJ15-21030Y; GA MŠk LO1302 Institutional support: RVO:61388963 Keywords : Aichi virus * capsid proteins Subject RIV: CE - Biochemistry

  14. Portal protein functions akin to a DNA-sensor that couples genome-packaging to icosahedral capsid maturation

    Science.gov (United States)

    Lokareddy, Ravi K.; Sankhala, Rajeshwer S.; Roy, Ankoor; Afonine, Pavel V.; Motwani, Tina; Teschke, Carolyn M.; Parent, Kristin N.; Cingolani, Gino

    2017-01-01

    Tailed bacteriophages and herpesviruses assemble infectious particles via an empty precursor capsid (or ‘procapsid') built by multiple copies of coat and scaffolding protein and by one dodecameric portal protein. Genome packaging triggers rearrangement of the coat protein and release of scaffolding protein, resulting in dramatic procapsid lattice expansion. Here, we provide structural evidence that the portal protein of the bacteriophage P22 exists in two distinct dodecameric conformations: an asymmetric assembly in the procapsid (PC-portal) that is competent for high affinity binding to the large terminase packaging protein, and a symmetric ring in the mature virion (MV-portal) that has negligible affinity for the packaging motor. Modelling studies indicate the structure of PC-portal is incompatible with DNA coaxially spooled around the portal vertex, suggesting that newly packaged DNA triggers the switch from PC- to MV-conformation. Thus, we propose the signal for termination of ‘Headful Packaging' is a DNA-dependent symmetrization of portal protein. PMID:28134243

  15. The complex subcellular distribution of satellite panicum mosaic virus capsid protein reflects its multifunctional role during infection

    International Nuclear Information System (INIS)

    Qi Dong; Omarov, Rustem T.; Scholthof, Karen-Beth G.

    2008-01-01

    Satellite panicum mosaic virus (SPMV) depends on its helper Panicum mosaic virus for replication and movement in host plants. The positive-sense single-stranded genomic RNA of SPMV encodes a 17-kDa capsid protein (CP) to form 16-nm virions. We determined that SPMV CP accumulates in both cytosolic and non-cytosolic fractions, but cytosolic accumulation of SPMV CP is exclusively associated with virions. An N-terminal arginine-rich motif (N-ARM) on SPMV CP is used to bind its cognate RNA and to form virus particles. Intriguingly, virion formation is dispensable for successful systemic SPMV RNA accumulation, yet this process still depends on an intact N-ARM. In addition, a C-terminal domain on the SPMV CP is necessary for self-interaction. Biochemical fractionation and fluorescent microscopy of green fluorescent protein-tagged SPMV CP demonstrated that the non-cytosolic SPMV CP is associated with the cell wall, the nucleus and other membranous organelles. To our knowledge, this is the first report that a satellite virus CP not only accumulates exclusively as virions in the cytosol but also is directed to the nucleolus and membranes. That SPMV CP is found both in the nucleus and the cell wall suggests its involvement in viral nuclear import and cell-to-cell transport

  16. Portal protein functions akin to a DNA-sensor that couples genome-packaging to icosahedral capsid maturation

    Energy Technology Data Exchange (ETDEWEB)

    Lokareddy, Ravi K.; Sankhala, Rajeshwer S.; Roy, Ankoor; Afonine, Pavel V.; Motwani, Tina; Teschke, Carolyn M.; Parent, Kristin N.; Cingolani, Gino (Rutgers); (LBNL); (Connecticut); (TJU); (MSU)

    2017-01-30

    Tailed bacteriophages and herpesviruses assemble infectious particles via an empty precursor capsid (or ‘procapsid’) built by multiple copies of coat and scaffolding protein and by one dodecameric portal protein. Genome packaging triggers rearrangement of the coat protein and release of scaffolding protein, resulting in dramatic procapsid lattice expansion. Here, we provide structural evidence that the portal protein of the bacteriophage P22 exists in two distinct dodecameric conformations: an asymmetric assembly in the procapsid (PC-portal) that is competent for high affinity binding to the large terminase packaging protein, and a symmetric ring in the mature virion (MV-portal) that has negligible affinity for the packaging motor. Modelling studies indicate the structure of PC-portal is incompatible with DNA coaxially spooled around the portal vertex, suggesting that newly packaged DNA triggers the switch from PC- to MV-conformation. Thus, we propose the signal for termination of ‘Headful Packaging’ is a DNA-dependent symmetrization of portal protein.

  17. The porcine circovirus type 1 capsid gene promoter improves antigen expression and immunogenicity in a HIV-1 plasmid vaccine

    Directory of Open Access Journals (Sweden)

    Burger Marieta

    2011-02-01

    Full Text Available Abstract Background One of the promising avenues for development of vaccines against Human immunodeficiency virus type 1 (HIV-1 and other human pathogens is the use of plasmid-based DNA vaccines. However, relatively large doses of plasmid must be injected for a relatively weak response. We investigated whether genome elements from Porcine circovirus type 1 (PCV-1, an apathogenic small ssDNA-containing virus, had useful expression-enhancing properties that could allow dose-sparing in a plasmid vaccine. Results The linearised PCV-1 genome inserted 5' of the CMV promoter in the well-characterised HIV-1 plasmid vaccine pTHgrttnC increased expression of the polyantigen up to 2-fold, and elicited 3-fold higher CTL responses in mice at 10-fold lower doses than unmodified pTHgrttnC. The PCV-1 capsid gene promoter (Pcap alone was equally effective. Enhancing activity was traced to a putative composite host transcription factor binding site and a "Conserved Late Element" transcription-enhancing sequence previously unidentified in circoviruses. Conclusions We identified a novel PCV-1 genome-derived enhancer sequence that significantly increased antigen expression from plasmids in in vitro assays, and improved immunogenicity in mice of the HIV-1 subtype C vaccine plasmid, pTHgrttnC. This should allow significant dose sparing of, or increased responses to, this and other plasmid-based vaccines. We also report investigations of the potential of other circovirus-derived sequences to be similarly used.

  18. Protruding Features of Viral Capsids Are Clustered on Icosahedral Great Circles.

    Directory of Open Access Journals (Sweden)

    David P Wilson

    Full Text Available Spherical viruses are remarkably well characterized by the Triangulation (T number developed by Casper and Klug. The T-number specifies how many viral capsid proteins are required to cover the virus, as well as how they are further subdivided into pentamer and hexamer subunits. The T-number however does not constrain the orientations of these proteins within the subunits or dictate where the proteins should place their protruding features. These protrusions often take the form of loops, spires and helices, and are significant because they aid in stability of the capsid as well as recognition by the host organism. Until now there has be no overall understanding of the placement of protrusions for spherical viruses, other than they have icosahedral symmetry. We constructed a set of gauge points based upon the work affine extensions of Keef and Twarock, which have fixed relative angular locations with which to measure the locations of these features. This work adds a new element to our understanding of the geometric arrangement of spherical viral capsid proteins; chiefly that the locations of protruding features are not found stochastically distributed in an icosahedral manner across the viral surface, but instead these features are found only in specific locations along the 15 icosahedral great circles. We have found that this result holds true as the T number and viral capsids size increases, suggesting an underlying geometric constraint on their locations. This is in spite of the fact that the constraints on the pentamers and hexamer orientations change as a function of T-number, as you need to accommodate more hexamers in the same solid angle between pentamers. The existence of this angular constraint of viral capsids suggests that there is a fitness or energetic benefit to the virus placing its protrusions in this manner. This discovery may have profound impacts on identifying and eliminating viral pathogens, understanding evolutionary

  19. Functional characterization of Kaposi's sarcoma-associated herpesvirus small capsid protein by bacterial artificial chromosome-based mutagenesis

    International Nuclear Information System (INIS)

    Sathish, Narayanan; Yuan Yan

    2010-01-01

    A systematic investigation of interactions amongst KSHV capsid proteins was undertaken in this study to comprehend lesser known KSHV capsid assembly mechanisms. Interestingly the interaction patterns of the KSHV small capsid protein, ORF65 suggested its plausible role in viral capsid assembly pathways. Towards further understanding this, ORF65-null recombinant mutants (BAC-Δ65 and BAC-stop65) employing a bacterial artificial chromosome (BAC) system were generated. No significant difference was found in both overall viral gene expression and lytic DNA replication between stable monolayers of 293T-BAC36 (wild-type) and 293T-BAC-ORF65-null upon induction with 12-O-tetradecanoylphorbol-13-acetate, though the latter released 30-fold fewer virions to the medium than 293T-BAC36 cells. Sedimentation profiles of capsid proteins of ORF65-null recombinant mutants were non-reflective of their organization into the KSHV capsids and were also undetectable in cytoplasmic extracts compared to noticeable levels in nuclear extracts. These observations collectively suggested the pivotal role of ORF65 in the KSHV capsid assembly processes.

  20. The Dual Role of an ESCRT-0 Component HGS in HBV Transcription and Naked Capsid Secretion

    Science.gov (United States)

    Chou, Shu-Fan; Tsai, Ming-Lin; Huang, Jyun-Yuan; Chang, Ya-Shu; Shih, Chiaho

    2015-01-01

    The Endosomal Sorting Complex Required for Transport (ESCRT) is an important cellular machinery for the sorting and trafficking of ubiquitinated cargos. It is also known that ESCRT is required for the egress of a number of viruses. To investigate the relationship between ESCRT and hepatitis B virus (HBV), we conducted an siRNA screening of ESCRT components for their potential effect on HBV replication and virion release. We identified a number of ESCRT factors required for HBV replication, and focused our study here on HGS (HRS, hepatocyte growth factor-regulated tyrosine kinase substrate) in the ESCRT-0 complex. Aberrant levels of HGS suppressed HBV transcription, replication and virion secretion. Hydrodynamic delivery of HGS in a mouse model significantly suppressed viral replication in the liver and virion secretion in the serum. Surprisingly, overexpression of HGS stimulated the release of HBV naked capsids, irrespective of their viral RNA, DNA, or empty contents. Mutant core protein (HBc 1–147) containing no arginine-rich domain (ARD) failed to secrete empty virions with or without HGS. In contrast, empty naked capsids of HBc 1–147 could still be promoted for secretion by HGS. HGS exerted a strong positive effect on the secretion of naked capsids, at the expense of a reduced level of virions. The association between HGS and HBc appears to be ubiquitin-independent. Furthermore, HBc is preferentially co-localized with HGS near the cell periphery, instead of near the punctate endosomes in the cytoplasm. In summary, our work demonstrated the importance of an optimum level of HGS in HBV propagation. In addition to an effect on HBV transcription, HGS can diminish the pool size of intracellular nucleocapsids with ongoing genome maturation, probably in part by promoting the secretion of naked capsids. The secretion routes of HBV virions and naked capsids can be clearly distinguished based on the pleiotropic effect of HGS involved in the ESCRT-0 complex. PMID

  1. Drosophila Nora virus capsid proteins differ from those of other picorna-like viruses.

    Science.gov (United States)

    Ekström, Jens-Ola; Habayeb, Mazen S; Srivastava, Vaibhav; Kieselbach, Thomas; Wingsle, Gunnar; Hultmark, Dan

    2011-09-01

    The recently discovered Nora virus from Drosophila melanogaster is a single-stranded RNA virus. Its published genomic sequence encodes a typical picorna-like cassette of replicative enzymes, but no capsid proteins similar to those in other picorna-like viruses. We have now done additional sequencing at the termini of the viral genome, extending it by 455 nucleotides at the 5' end, but no more coding sequence was found. The completeness of the final 12,333-nucleotide sequence was verified by the production of infectious virus from the cloned genome. To identify the capsid proteins, we purified Nora virus particles and analyzed their proteins by mass spectrometry. Our results show that the capsid is built from three major proteins, VP4A, B and C, encoded in the fourth open reading frame of the viral genome. The viral particles also contain traces of a protein from the third open reading frame, VP3. VP4A and B are not closely related to other picorna-like virus capsid proteins in sequence, but may form similar jelly roll folds. VP4C differs from the others and is predicted to have an essentially α-helical conformation. In a related virus, identified from EST database sequences from Nasonia parasitoid wasps, VP4C is encoded in a separate open reading frame, separated from VP4A and B by a frame-shift. This opens a possibility that VP4C is produced in non-equimolar quantities. Altogether, our results suggest that the Nora virus capsid has a different protein organization compared to the order Picornavirales. Copyright © 2011 Elsevier B.V. All rights reserved.

  2. The Dual Role of an ESCRT-0 Component HGS in HBV Transcription and Naked Capsid Secretion.

    Directory of Open Access Journals (Sweden)

    Shu-Fan Chou

    2015-10-01

    Full Text Available The Endosomal Sorting Complex Required for Transport (ESCRT is an important cellular machinery for the sorting and trafficking of ubiquitinated cargos. It is also known that ESCRT is required for the egress of a number of viruses. To investigate the relationship between ESCRT and hepatitis B virus (HBV, we conducted an siRNA screening of ESCRT components for their potential effect on HBV replication and virion release. We identified a number of ESCRT factors required for HBV replication, and focused our study here on HGS (HRS, hepatocyte growth factor-regulated tyrosine kinase substrate in the ESCRT-0 complex. Aberrant levels of HGS suppressed HBV transcription, replication and virion secretion. Hydrodynamic delivery of HGS in a mouse model significantly suppressed viral replication in the liver and virion secretion in the serum. Surprisingly, overexpression of HGS stimulated the release of HBV naked capsids, irrespective of their viral RNA, DNA, or empty contents. Mutant core protein (HBc 1-147 containing no arginine-rich domain (ARD failed to secrete empty virions with or without HGS. In contrast, empty naked capsids of HBc 1-147 could still be promoted for secretion by HGS. HGS exerted a strong positive effect on the secretion of naked capsids, at the expense of a reduced level of virions. The association between HGS and HBc appears to be ubiquitin-independent. Furthermore, HBc is preferentially co-localized with HGS near the cell periphery, instead of near the punctate endosomes in the cytoplasm. In summary, our work demonstrated the importance of an optimum level of HGS in HBV propagation. In addition to an effect on HBV transcription, HGS can diminish the pool size of intracellular nucleocapsids with ongoing genome maturation, probably in part by promoting the secretion of naked capsids. The secretion routes of HBV virions and naked capsids can be clearly distinguished based on the pleiotropic effect of HGS involved in the ESCRT-0 complex.

  3. Changes in the stability and biomechanics of P22 bacteriophage capsid during maturation.

    Science.gov (United States)

    Kant, Ravi; Llauró, Aida; Rayaprolu, Vamseedhar; Qazi, Shefah; de Pablo, Pedro J; Douglas, Trevor; Bothner, Brian

    2018-03-15

    The capsid of P22 bacteriophage undergoes a series of structural transitions during maturation that guide it from spherical to icosahedral morphology. The transitions include the release of scaffold proteins and capsid expansion. Although P22 maturation has been investigated for decades, a unified model that incorporates thermodynamic and biophysical analyses is not available. A general and specific model of icosahedral capsid maturation is of significant interest to theoreticians searching for fundamental principles as well as virologists and material scientists seeking to alter maturation to their advantage. To address this challenge, we have combined the results from orthogonal biophysical techniques including differential scanning fluorimetry, atomic force microscopy, circular dichroism, and hydrogen-deuterium exchange mass spectrometry. By integrating these results from single particle and population measurements, an energy landscape of P22 maturation from procapsid through expanded shell to wiffle ball emerged, highlighting the role of metastable structures and the thermodynamics guiding maturation. The propagation of weak quaternary interactions across symmetric elements of the capsid is a key component for stability in P22. A surprising finding is that the progression to wiffle ball, which lacks pentamers, shows that chemical and thermal stability can be uncoupled from mechanical rigidity, elegantly demonstrating the complexity inherent in capsid protein interactions and the emergent properties that can arise from icosahedral symmetry. On a broader scale, this work demonstrates the power of applying orthogonal biophysical techniques to elucidate assembly mechanisms for supramolecular complexes and provides a framework within which other viral systems can be compared. Copyright © 2018 Elsevier B.V. All rights reserved.

  4. Evaluation of an antibody avidity index method for detecting recent human immunodeficiency virus type 1 infection using an automated chemiluminescence immunoassay.

    Science.gov (United States)

    Fernández, Gema; Manzardo, Christian; Montoliu, Alexandra; Campbell, Colin; Fernández, Gregorio; Casabona, Jordi; Miró, José Maria; Matas, Lurdes; Rivaya, Belén; González, Victoria

    2015-04-01

    Recent infection testing algorithms (RITAs) are used in public health surveillance to estimate the incidence of recently acquired HIV-1 infection. Our aims were (i) to evaluate the precision of the VITROS® Anti-HIV 1+2 automated antibody avidity assay for qualitative detection of antibodies to HIV 1+2 virus; (ii) to validate the accuracy of an automated guanidine-based antibody avidity assay to discriminate between recent and long standing infections using the VITROS 3600 platform; (iii) to compare this method with BED-CEIA assay; and (iv) to evaluate the occurrence of false recent misclassifications by the VITROS antibody avidity assay in patients with a CD4 count de Enfermedades Infecciosas y Microbiología Clínica. All rights reserved.

  5. First preclinical evaluation of mono-[{sup 123}I]iodohypericin as a necrosis-avid tracer agent

    Energy Technology Data Exchange (ETDEWEB)

    Ni, Yicheng; Chen, Feng; Marchal, Guy [University Hospital Gasthuisberg, Department of Radiology, Leuven (Belgium); Huyghe, Dieter; Verbeke, Kristin; Verbruggen, Alfons M.; Bormans, Guy M. [University of Leuven, Laboratory of Radiopharmaceutical Chemistry, Faculty of Pharmaceutical Sciences, Leuven (Belgium); Witte, Peter A. de [University of Leuven, Laboratory of Pharmaceutical Biology and Phytopharmacology, Faculty of Pharmaceutical Sciences, Leuven (Belgium); Nuyts, Johan; Mortelmans, Luc [University Hospital Gasthuisberg, Department of Nuclear Medicine, Leuven (Belgium)

    2006-05-15

    We have labelled hypericin, a polyphenolic polycyclic quinone found in St. John's wort (Hypericum perforatum), with{sup 123}I and evaluated mono-[{sup 123}I]iodohypericin (MIH) as a potential necrosis-avid diagnostic tracer agent. MIH was prepared by an electrophilic radioiodination method. The new tracer agent was evaluated in animal models of liver infarction in the rat and heart infarction in the rabbit using single-photon emission computed tomography (SPECT), triphenyltetrazolium chloride (TTC) histochemical staining, serial sectional autoradiography and microscopy, and radioactivity counting techniques. Using in vivo SPECT imaging, hepatic and cardiac infarctions were persistently visualised as well-defined hot spots over 48 h. Preferential uptake of the tracer agent in necrotic tissue was confirmed by perfect match of images from post-mortem TTC staining, autoradiography (ARX) and histology. Radioactivity concentration in infarcted tissues was over 10 times (liver; 3.51% ID/g in necrotic tissue vs 0.38% ID/g in normal tissue at 60 h p.i.) and over 6 times (myocardium; 0.36% ID/g in necrotic tissue vs 0.054% ID/g in normal tissue; ratios up to 18 for selected parts on ARX images) higher than in normal tissues. The results suggest that hypericin derivatives may serve as powerful necrosis-avid diagnostic agents for assessment of tissue viability. (orig.)

  6. Differential expression profiling of circulation microRNAs in PTC patients with non-131I and 131I-avid lungs metastases: a pilot study

    International Nuclear Information System (INIS)

    Qiu, Zhong-Ling; Shen, Chen-Tian; Song, Hong-Jun; Wei, Wei-Jun; Luo, Quan-Yong

    2015-01-01

    Introduction: Loss of the ability to concentrate 131 I is one of the important causes of radioiodine-refractory disease in papillary thyroid carcinoma (PTC). Recent advantages of serum microRNAs (miRNAs) open a new realm of possibilities for noninvasive diagnosis and prognosis of many cancers. The aim of the current study was to identify differential expression profiling of circulation miRNAs in PTC patients with non- 131 I and 131 I-avid lungs metastases. Methods: The expressions of miRNAs were examined using miRNA microarray chip. The most significantly changed miRNAs from microarray were verified by using qRT-PCR. The potential miRNAs regulating target genes and their preliminary biological functions were forecasted by Bioinformatic analysis. Results: Compared to 131 I-avid lung metastases, 13 kinds of significantly differential serum miRNAs including 5 upregulated miRNAs (miR-1249, miR-106a, miR-503, miR-34c-5p, miR-1281) and 8 downregulated miRNAs (miR-1915, miR-2861, miR-3196, miR-500, miR-572, miR-33b, miR-554, miR-18a) in PTC patients with non- 131 I-avid lung metastases were identified. Bioinformatic analysis demonstrated that miR-106a was the core miRNA regulating 193 genes in the network. The results of validation confirmed the up-regulation of miR-106a in non- 131 I-avid lungs metastatic PTC patients. Conclusion: Differentially expressed serum miRNA profiles between PTC patients with non- 131 I and 131 I-avid lungs metastases were analyzed. These findings in our present study could represent new clues for the diagnostic and therapeutic strategy in PTC patients with non- 131 I-avid metastatic disease

  7. Immunoglobulin G Avidities in Infants in Mexico after Primary Immunization with Three Doses of Polyribosylribitol Phosphate-Tetanus Toxoid Haemophilus influenzae Type b Vaccine▿ †

    Science.gov (United States)

    Gómez-de-León, Patricia; Díaz-García, F. Javier; Villaseñor-Sierra, Alberto; Segura, Jorge; Carranza, Martha I.; Arredondo-Garcia, José Luis; Santos, José Ignacio

    2008-01-01

    Serum immunoglobulin G concentrations and avidities specific to Haemophilus influenzae type b (Hib) were measured in 208 children living in Guadalajara and Mexico City. Protective concentrations were found in 98.9% and 100.0% of participants, respectively. Geometric mean concentrations differed between both populations and/or among age groups. Mean avidities differed only among the 7- to 12-month-old children. Diphtheria-tetanus-whole-cell pertussis-hepatitis B-Hib primary vaccination seems to induce protection in Mexican children. PMID:18417667

  8. Immunoglobulin G avidities in infants in Mexico after primary immunization with three doses of polyribosylribitol phosphate-tetanus toxoid Haemophilus influenzae type b vaccine.

    Science.gov (United States)

    Gómez-de-León, Patricia; Díaz-García, F Javier; Villaseñor-Sierra, Alberto; Segura, Jorge; Carranza, Martha I; Arredondo-Garcia, José Luis; Santos, José Ignacio

    2008-06-01

    Serum immunoglobulin G concentrations and avidities specific to Haemophilus influenzae type b (Hib) were measured in 208 children living in Guadalajara and Mexico City. Protective concentrations were found in 98.9% and 100.0% of participants, respectively. Geometric mean concentrations differed between both populations and/or among age groups. Mean avidities differed only among the 7- to 12-month-old children. Diphtheria-tetanus-whole-cell pertussis-hepatitis B-Hib primary vaccination seems to induce protection in Mexican children.

  9. Human Cytomegalovirus pUL47 Modulates Tegumentation and Capsid Accumulation at the Viral Assembly Complex

    Science.gov (United States)

    Cappadona, Ilaria; Villinger, Clarissa; Schutzius, Gabi; Mertens, Thomas

    2015-01-01

    ABSTRACT Human cytomegalovirus (HCMV) tegument protein pUL47 is an interaction partner of pUL48 and highly conserved among herpesviruses. It is closely associated with the capsid and has an important function early in infection. Here, we report a specific role of pUL47 in the tegumentation of capsids in the cytoplasm. A newly generated mutant virus (TB-47stop), in which expression of pUL47 is blocked, exhibited a severe impairment in cell-to-cell spread and release of infectivity from infected cells. Ultrastructural analysis of TB-47stop-infected cells clearly showed cytoplasmic accumulations of nonenveloped capsids that were only partially tegumented, indicating that these capsids failed to complete tegumentation. Nevertheless, these accumulations were positive for HCMV inner tegument proteins pp150 and pUL48, suggesting that their attachment to capsids occurs independently of pUL47. Despite these morphological alterations, fully enveloped virus particles were found in the extracellular space and at the viral assembly complex (vAC) of TB-47stop-infected cells, indicating that pUL47 is not essential for the generation of virions. We confirmed findings that incorporation of pUL48 into virions is impaired in the absence of pUL47. Interestingly, pUL47 exhibited a strong nuclear localization in transfected cells, whereas it was found exclusively at the vAC in the context of virus infection. Colocalization of pUL47 and pUL48 at the vAC is consistent with their interaction. We also found a shift to a more nuclear localization of pUL47 when the expression of pUL48 was reduced. Summarizing our results, we hypothesize that pUL48 directs pUL47 to the vAC to promote tegumentation and secondary envelopment of capsids. IMPORTANCE Generation of infectious HCMV particles requires an organized and multistep process involving the action of several viral and cellular proteins as well as protein-protein interactions. A better understanding of these processes is important for

  10. Recalibration of the limiting antigen avidity EIA to determine mean duration of recent infection in divergent HIV-1 subtypes.

    Directory of Open Access Journals (Sweden)

    Yen T Duong

    Full Text Available Mean duration of recent infection (MDRI and misclassification of long-term HIV-1 infections, as proportion false recent (PFR, are critical parameters for laboratory-based assays for estimating HIV-1 incidence. Recent review of the data by us and others indicated that MDRI of LAg-Avidity EIA estimated previously required recalibration. We present here results of recalibration efforts using >250 seroconversion panels and multiple statistical methods to ensure accuracy and consensus.A total of 2737 longitudinal specimens collected from 259 seroconverting individuals infected with diverse HIV-1 subtypes were tested with the LAg-Avidity EIA as previously described. Data were analyzed for determination of MDRI at ODn cutoffs of 1.0 to 2.0 using 7 statistical approaches and sub-analyzed by HIV-1 subtypes. In addition, 3740 specimens from individuals with infection >1 year, including 488 from patients with AIDS, were tested for PFR at varying cutoffs.Using different statistical methods, MDRI values ranged from 88-94 days at cutoff ODn = 1.0 to 177-183 days at ODn = 2.0. The MDRI values were similar by different methods suggesting coherence of different approaches. Testing for misclassification among long-term infections indicated that overall PFRs were 0.6% to 2.5% at increasing cutoffs of 1.0 to 2.0, respectively. Balancing the need for a longer MDRI and smaller PFR (<2.0% suggests that a cutoff ODn = 1.5, corresponding to an MDRI of 130 days should be used for cross-sectional application. The MDRI varied among subtypes from 109 days (subtype A&D to 152 days (subtype C.Based on the new data and revised analysis, we recommend an ODn cutoff = 1.5 to classify recent and long-term infections, corresponding to an MDRI of 130 days (118-142. Determination of revised parameters for estimation of HIV-1 incidence should facilitate application of the LAg-Avidity EIA for worldwide use.

  11. Recalibration of the limiting antigen avidity EIA to determine mean duration of recent infection in divergent HIV-1 subtypes.

    Science.gov (United States)

    Duong, Yen T; Kassanjee, Reshma; Welte, Alex; Morgan, Meade; De, Anindya; Dobbs, Trudy; Rottinghaus, Erin; Nkengasong, John; Curlin, Marcel E; Kittinunvorakoon, Chonticha; Raengsakulrach, Boonyos; Martin, Michael; Choopanya, Kachit; Vanichseni, Suphak; Jiang, Yan; Qiu, Maofeng; Yu, Haiying; Hao, Yan; Shah, Neha; Le, Linh-Vi; Kim, Andrea A; Nguyen, Tuan Anh; Ampofo, William; Parekh, Bharat S

    2015-01-01

    Mean duration of recent infection (MDRI) and misclassification of long-term HIV-1 infections, as proportion false recent (PFR), are critical parameters for laboratory-based assays for estimating HIV-1 incidence. Recent review of the data by us and others indicated that MDRI of LAg-Avidity EIA estimated previously required recalibration. We present here results of recalibration efforts using >250 seroconversion panels and multiple statistical methods to ensure accuracy and consensus. A total of 2737 longitudinal specimens collected from 259 seroconverting individuals infected with diverse HIV-1 subtypes were tested with the LAg-Avidity EIA as previously described. Data were analyzed for determination of MDRI at ODn cutoffs of 1.0 to 2.0 using 7 statistical approaches and sub-analyzed by HIV-1 subtypes. In addition, 3740 specimens from individuals with infection >1 year, including 488 from patients with AIDS, were tested for PFR at varying cutoffs. Using different statistical methods, MDRI values ranged from 88-94 days at cutoff ODn = 1.0 to 177-183 days at ODn = 2.0. The MDRI values were similar by different methods suggesting coherence of different approaches. Testing for misclassification among long-term infections indicated that overall PFRs were 0.6% to 2.5% at increasing cutoffs of 1.0 to 2.0, respectively. Balancing the need for a longer MDRI and smaller PFR (<2.0%) suggests that a cutoff ODn = 1.5, corresponding to an MDRI of 130 days should be used for cross-sectional application. The MDRI varied among subtypes from 109 days (subtype A&D) to 152 days (subtype C). Based on the new data and revised analysis, we recommend an ODn cutoff = 1.5 to classify recent and long-term infections, corresponding to an MDRI of 130 days (118-142). Determination of revised parameters for estimation of HIV-1 incidence should facilitate application of the LAg-Avidity EIA for worldwide use.

  12. Computational design of trimeric influenza-neutralizing proteins targeting the hemagglutinin receptor binding site

    Energy Technology Data Exchange (ETDEWEB)

    Strauch, Eva-Maria; Bernard, Steffen M.; La, David; Bohn, Alan J.; Lee, Peter S.; Anderson, Caitlin E.; Nieusma, Travis; Holstein, Carly A.; Garcia, Natalie K.; Hooper, Kathryn A.; Ravichandran, Rashmi; Nelson, Jorgen W.; Sheffler, William; Bloom, Jesse D.; Lee, Kelly K.; Ward, Andrew B.; Yager, Paul; Fuller, Deborah H.; Wilson, Ian A.; Baker , David (UWASH); (Scripps); (FHCRC)

    2017-06-12

    Many viral surface glycoproteins and cell surface receptors are homo-oligomers1, 2, 3, 4, and thus can potentially be targeted by geometrically matched homo-oligomers that engage all subunits simultaneously to attain high avidity and/or lock subunits together. The adaptive immune system cannot generally employ this strategy since the individual antibody binding sites are not arranged with appropriate geometry to simultaneously engage multiple sites in a single target homo-oligomer. We describe a general strategy for the computational design of homo-oligomeric protein assemblies with binding functionality precisely matched to homo-oligomeric target sites5, 6, 7, 8. In the first step, a small protein is designed that binds a single site on the target. In the second step, the designed protein is assembled into a homo-oligomer such that the designed binding sites are aligned with the target sites. We use this approach to design high-avidity trimeric proteins that bind influenza A hemagglutinin (HA) at its conserved receptor binding site. The designed trimers can both capture and detect HA in a paper-based diagnostic format, neutralizes influenza in cell culture, and completely protects mice when given as a single dose 24 h before or after challenge with influenza.

  13. Historically aggressive types of follicular cell-derived thyroid cancer often have radioactive avid distant metastases: a study of 314 patients with distant metastases at a single institution

    Energy Technology Data Exchange (ETDEWEB)

    Tala, H.P.; Rondeau, G.; Fagin, J.A.; Tuttle, R.M. [Endocrinology Division, Department of Medicine, Nuclear Medicine Division, Memorial Sloan Kettering Cancer Center, New-York (United States); Ghossein, R.A. [Pathology Department, Nuclear Medecine Division, Memorial Sloan Kettering Cancer Center, New-York (United States); Grewal, R.K.; Larson, S.M. [Radiology Department, Nuclear Medicine Division, Memorial Sloan Kettering Cancer Center, New-York (United States)

    2012-07-01

    Radioactive iodine (RAI) remains one of the primary treatment options for metastatic, follicular cell derived thyroid cancers. The aim of this study was to determine the likelihood that metastatic lesions arising from one of the aggressive thyroid cancer histologies [tall cell variant of papillary thyroid carcinoma (TCV-PTC), poorly differentiated thyroid carcinoma (PDTC) and Hurthle cell carcinoma (HCC)] would demonstrate sufficient RAI avidity for visualization on RAI scanning and therefore could potentially benefit from RAI therapy. The study shows that in patients selected for RAI scanning or therapy at our center, RAI avid lesions can be identified in more than two thirds of the patients with distant metastases arising in the setting of C-PTC, WD-FTC, FV-PTC, TCV-PTC, or PDTC primary tumors. While RAI avidity on a post-therapy scan does not always correlate with clinically significant tumor killing activity, it is likely that some of these patients with RAI avid metastatic disease did obtain a clinical benefit

  14. Duration of (18)F-FDG avidity in lymph nodes after pandemic H1N1v and seasonal influenza vaccination

    DEFF Research Database (Denmark)

    Thomassen, Anders; Lerberg Nielsen, Anne; Gerke, Oke

    2011-01-01

    PURPOSE: The aim of our study was to investigate the occurrence of fluorodeoxyglucose (FDG) avidity in draining axillary lymph nodes after vaccination against influenza (H1N1v pandemic and seasonal) and to determine the period of increased FDG uptake. METHODS: During December 2009, patients...

  15. Usefulness of Toxoplasma gondii recombinant antigens (GRA1, GRA7 and SAG1) in an immunoglobulin G avidity test for the serodiagnosis of toxoplasmosis

    DEFF Research Database (Denmark)

    Pietkiewicz, H; Hiszczyńska-Sawicka, E; Kur, J

    2007-01-01

    The precise diagnosis of an acute and recent Toxoplasma infection in pregnant women and the newborn child is important before treatment. This study describes a new Toxoplasma gondii IgG avidity test based on a combination of recombinant GRA1, GRA7 and SAG1 antigens and shows that this test is use...

  16. Dual Testing Algorithm of BED-CEIA and AxSYM Avidity Index Assays Performs Best in Identifying Recent HIV Infection in a Sample of Rwandan Sex Workers

    NARCIS (Netherlands)

    Braunstein, Sarah L.; Nash, Denis; Kim, Andrea A.; Ford, Ken; Mwambarangwe, Lambert; Ingabire, Chantal M.; Vyankandondera, Joseph; van de Wijgert, Janneke H. H. M.

    2011-01-01

    To assess the performance of BED-CEIA (BED) and AxSYM Avidity Index (Ax-AI) assays in estimating HIV incidence among female sex workers (FSW) in Kigali, Rwanda. Eight hundred FSW of unknown HIV status were HIV tested; HIV-positive women had BED and Ax-AI testing at baseline and ≥12 months later to

  17. Promoter analysis of the Chilo iridescent virus DNA polymerase and major capsid protein genes

    International Nuclear Information System (INIS)

    Nalcacioglu, Remziye; Marks, Hendrik; Vlak, Just M.; Demirbag, Zihni; Oers, Monique M. van

    2003-01-01

    The DNA polymerase (DNApol) and major capsid protein (MCP) genes were used as models to study promoter activity in Chilo iridescent virus (CIV). Infection of Bombyx mori SPC-BM-36 cells in the presence of inhibitors of DNA or protein synthesis showed that DNApol, as well as helicase, is an immediate-early gene and confirmed that the major capsid protein (MCP) is a late gene. Transcription of DNApol initiated 35 nt upstream and that of MCP 14 nt upstream of the translational start site. In a luciferase reporter gene assay both promoters were active only when cells were infected with CIV. For DNApol sequences between position -27 and -6, relative to the transcriptional start site, were essential for promoter activity. Furthermore, mutation of a G within the sequence TTGTTTT located just upstream of the DNApol transcription initiation site reduced the promoter activity by 25%. Sequences crucial for MCP promoter activity are located between positions -53 and -29

  18. Chronic hepatitis B infection and HBV DNA-containing capsids: Modeling and analysis

    Science.gov (United States)

    Manna, Kalyan; Chakrabarty, Siddhartha P.

    2015-05-01

    We analyze the dynamics of chronic HBV infection taking into account both uninfected and infected hepatocytes along with the intracellular HBV DNA-containing capsids and the virions. While previous HBV models have included either the uninfected hepatocytes or the intracellular HBV DNA-containing capsids, our model accounts for both these two populations. We prove the conditions for local and global stability of both the uninfected and infected steady states in terms of the basic reproduction number. Further, we incorporate a time lag in the model to encompass the intracellular delay in the production of the infected hepatocytes and find that this delay does not affect the overall dynamics of the system. The results for the model and the delay model are finally numerically illustrated.

  19. Structures of foot and mouth disease virus pentamers: Insight into capsid dissociation and unexpected pentamer reassociation.

    Directory of Open Access Journals (Sweden)

    Nayab Malik

    2017-09-01

    Full Text Available Foot-and-mouth disease virus (FMDV belongs to the Aphthovirus genus of the Picornaviridae, a family of small, icosahedral, non-enveloped, single-stranded RNA viruses. It is a highly infectious pathogen and is one of the biggest hindrances to the international trade of animals and animal products. FMDV capsids (which are unstable below pH6.5 release their genome into the host cell from an acidic compartment, such as that of an endosome, and in the process dissociate into pentamers. Whilst other members of the family (enteroviruses have been visualized to form an expanded intermediate capsid with holes from which inner capsid proteins (VP4, N-termini (VP1 and RNA can be released, there has been no visualization of any such state for an aphthovirus, instead the capsid appears to simply dissociate into pentamers. Here we present the 8-Å resolution structure of isolated dissociated pentamers of FMDV, lacking VP4. We also found these pentamers to re-associate into a rigid, icosahedrally symmetric assembly, which enabled their structure to be solved at higher resolution (5.2 Å. In this assembly, the pentamers unexpectedly associate 'inside out', but still with their exposed hydrophobic edges buried. Stabilizing interactions occur between the HI loop of VP2 and its symmetry related partners at the icosahedral 3-fold axes, and between the BC and EF loops of VP3 with the VP2 βB-strand and the CD loop at the 2-fold axes. A relatively extensive but subtle structural rearrangement towards the periphery of the dissociated pentamer compared to that in the mature virus provides insight into the mechanism of dissociation of FMDV and the marked difference in antigenicity.

  20. HIV Incidence Estimates Using the Limiting Antigen Avidity EIA Assay at Testing Sites in Kiev City, Ukraine: 2013-2014

    Science.gov (United States)

    Kruglov, Yuri; Yurchenko, Alexander

    2016-01-01

    Objective To estimate HIV incidence and highlight the characteristics of persons at greatest risk of HIV in the Ukraine capital, Kiev. Method Residual samples from newly-diagnosed persons attending the Kiev City AIDS Centre were tested for evidence of recent HIV infection using an avidity assay. Questions on possible risk factors for HIV acquisition and testing history were introduced. All persons (≥16yrs) presenting for an HIV test April’13–March’14 were included. Rates per 100,000 population were calculated using region-specific denominators. Results During the study period 6370 individuals tested for HIV. Of the 467 individuals newly-diagnosed with HIV, 21 had insufficient samples for LAg testing. Of the remaining 446, 39 (8.7%) were classified as recent with an avidity index <1.5ODn, 10 were reclassified as long-standing as their viral load was <1000 copies/mL, resulting in 29 (6.5%) recent HIV infections. The only independent predictor for a recent infection was probable route of exposure, with MSM more likely to present with a recent infection compared with heterosexual contact [Odds Ratio 8.86; 95%CI 2.65–29.60]. We estimated HIV incidence at 21.5 per 100,000 population, corresponding to 466 new infections. Using population estimates for MSM and PWID, incidence was estimated to be between 2289.6 and 6868.7/100,000 MSM, and 350.4 for PWID. Conclusion A high proportion of persons newly-infected remain undiagnosed, with MSM disproportionally affected with one in four newly-HIV-diagnosed and one in three recently-HIV-infected. Our findings should be used for targeted public health interventions and health promotion. PMID:27276170

  1. HIV Incidence Estimates Using the Limiting Antigen Avidity EIA Assay at Testing Sites in Kiev City, Ukraine: 2013-2014.

    Directory of Open Access Journals (Sweden)

    Ruth Simmons

    Full Text Available To estimate HIV incidence and highlight the characteristics of persons at greatest risk of HIV in the Ukraine capital, Kiev.Residual samples from newly-diagnosed persons attending the Kiev City AIDS Centre were tested for evidence of recent HIV infection using an avidity assay. Questions on possible risk factors for HIV acquisition and testing history were introduced. All persons (≥16yrs presenting for an HIV test April'13-March'14 were included. Rates per 100,000 population were calculated using region-specific denominators.During the study period 6370 individuals tested for HIV. Of the 467 individuals newly-diagnosed with HIV, 21 had insufficient samples for LAg testing. Of the remaining 446, 39 (8.7% were classified as recent with an avidity index <1.5ODn, 10 were reclassified as long-standing as their viral load was <1000 copies/mL, resulting in 29 (6.5% recent HIV infections. The only independent predictor for a recent infection was probable route of exposure, with MSM more likely to present with a recent infection compared with heterosexual contact [Odds Ratio 8.86; 95%CI 2.65-29.60]. We estimated HIV incidence at 21.5 per 100,000 population, corresponding to 466 new infections. Using population estimates for MSM and PWID, incidence was estimated to be between 2289.6 and 6868.7/100,000 MSM, and 350.4 for PWID.A high proportion of persons newly-infected remain undiagnosed, with MSM disproportionally affected with one in four newly-HIV-diagnosed and one in three recently-HIV-infected. Our findings should be used for targeted public health interventions and health promotion.

  2. HIV Incidence Estimates Using the Limiting Antigen Avidity EIA Assay at Testing Sites in Kiev City, Ukraine: 2013-2014.

    Science.gov (United States)

    Simmons, Ruth; Malyuta, Ruslan; Chentsova, Nelli; Karnets, Iryna; Murphy, Gary; Medoeva, Antonia; Kruglov, Yuri; Yurchenko, Alexander; Copas, Andrew; Porter, Kholoud

    2016-01-01

    To estimate HIV incidence and highlight the characteristics of persons at greatest risk of HIV in the Ukraine capital, Kiev. Residual samples from newly-diagnosed persons attending the Kiev City AIDS Centre were tested for evidence of recent HIV infection using an avidity assay. Questions on possible risk factors for HIV acquisition and testing history were introduced. All persons (≥16yrs) presenting for an HIV test April'13-March'14 were included. Rates per 100,000 population were calculated using region-specific denominators. During the study period 6370 individuals tested for HIV. Of the 467 individuals newly-diagnosed with HIV, 21 had insufficient samples for LAg testing. Of the remaining 446, 39 (8.7%) were classified as recent with an avidity index <1.5ODn, 10 were reclassified as long-standing as their viral load was <1000 copies/mL, resulting in 29 (6.5%) recent HIV infections. The only independent predictor for a recent infection was probable route of exposure, with MSM more likely to present with a recent infection compared with heterosexual contact [Odds Ratio 8.86; 95%CI 2.65-29.60]. We estimated HIV incidence at 21.5 per 100,000 population, corresponding to 466 new infections. Using population estimates for MSM and PWID, incidence was estimated to be between 2289.6 and 6868.7/100,000 MSM, and 350.4 for PWID. A high proportion of persons newly-infected remain undiagnosed, with MSM disproportionally affected with one in four newly-HIV-diagnosed and one in three recently-HIV-infected. Our findings should be used for targeted public health interventions and health promotion.

  3. Endocytosis of Integrin-Binding Human Picornaviruses

    Directory of Open Access Journals (Sweden)

    Pirjo Merilahti

    2012-01-01

    Full Text Available Picornaviruses that infect humans form one of the largest virus groups with almost three hundred virus types. They include significant enteroviral pathogens such as rhino-, polio-, echo-, and coxsackieviruses and human parechoviruses that cause wide range of disease symptoms. Despite the economic importance of picornaviruses, there are no antivirals. More than ten cellular receptors are known to participate in picornavirus infection, but experimental evidence of their role in cellular infection has been shown for only about twenty picornavirus types. Three enterovirus types and one parechovirus have experimentally been shown to bind and use integrin receptors in cellular infection. These include coxsackievirus A9 (CV-A9, echovirus 9, and human parechovirus 1 that are among the most common and epidemic human picornaviruses and bind to αV-integrins via RGD motif that resides on virus capsid. In contrast, echovirus 1 (E-1 has no RGD and uses integrin α2β1 as cellular receptor. Endocytosis of CV-A9 has recently been shown to occur via a novel Arf6- and dynamin-dependent pathways, while, contrary to collagen binding, E-1 binds inactive β1 integrin and enters via macropinocytosis. In this paper, we review what is known about receptors and endocytosis of integrin-binding human picornaviruses.

  4. Solid-to-fluid DNA transition inside HSV-1 capsid close to the temperature of infection

    Energy Technology Data Exchange (ETDEWEB)

    Sae-Ueng, Udom; Li, Dong; Zuo, Xiaobing; Huffman, Jamie B.; Homa, Fred L.; Rau, Donald; Evilevitch, Alex

    2014-10-01

    DNA in the human Herpes simplex virus type 1 (HSV-1) capsid is packaged to a tight density. This leads to tens of atmospheres of internal pressure responsible for the delivery of the herpes genome into the cell nucleus. In this study we show that, despite its liquid crystalline state inside the capsid, the DNA is fluid-like, which facilitates its ejection into the cell nucleus during infection. We found that the sliding friction between closely packaged DNA strands, caused by interstrand repulsive interactions, is reduced by the ionic environment of epithelial cells and neurons susceptible to herpes infection. However, variations in the ionic conditions corresponding to neuronal activity can restrict DNA mobility in the capsid, making it more solid-like. This can inhibit intranuclear DNA release and interfere with viral replication. In addition, the temperature of the human host (37 °C) induces a disordering transition of the encapsidated herpes genome, which reduces interstrand interactions and provides genome mobility required for infection.

  5. Structure of a Spumaretrovirus Gag Central Domain Reveals an Ancient Retroviral Capsid.

    Directory of Open Access Journals (Sweden)

    Neil J Ball

    2016-11-01

    Full Text Available The Spumaretrovirinae, or foamy viruses (FVs are complex retroviruses that infect many species of monkey and ape. Despite little sequence homology, FV and orthoretroviral Gag proteins perform equivalent functions, including genome packaging, virion assembly, trafficking and membrane targeting. However, there is a paucity of structural information for FVs and it is unclear how disparate FV and orthoretroviral Gag molecules share the same function. To probe the functional overlap of FV and orthoretroviral Gag we have determined the structure of a central region of Gag from the Prototype FV (PFV. The structure comprises two all α-helical domains NtDCEN and CtDCEN that although they have no sequence similarity, we show they share the same core fold as the N- (NtDCA and C-terminal domains (CtDCA of archetypal orthoretroviral capsid protein (CA. Moreover, structural comparisons with orthoretroviral CA align PFV NtDCEN and CtDCEN with NtDCA and CtDCA respectively. Further in vitro and functional virological assays reveal that residues making inter-domain NtDCEN-CtDCEN interactions are required for PFV capsid assembly and that intact capsid is required for PFV reverse transcription. These data provide the first information that relates the Gag proteins of Spuma and Orthoretrovirinae and suggests a common ancestor for both lineages containing an ancient CA fold.

  6. In vitro protease cleavage and computer simulations reveal the HIV-1 capsid maturation pathway

    Science.gov (United States)

    Ning, Jiying; Erdemci-Tandogan, Gonca; Yufenyuy, Ernest L.; Wagner, Jef; Himes, Benjamin A.; Zhao, Gongpu; Aiken, Christopher; Zandi, Roya; Zhang, Peijun

    2016-12-01

    HIV-1 virions assemble as immature particles containing Gag polyproteins that are processed by the viral protease into individual components, resulting in the formation of mature infectious particles. There are two competing models for the process of forming the mature HIV-1 core: the disassembly and de novo reassembly model and the non-diffusional displacive model. To study the maturation pathway, we simulate HIV-1 maturation in vitro by digesting immature particles and assembled virus-like particles with recombinant HIV-1 protease and monitor the process with biochemical assays and cryoEM structural analysis in parallel. Processing of Gag in vitro is accurate and efficient and results in both soluble capsid protein and conical or tubular capsid assemblies, seemingly converted from immature Gag particles. Computer simulations further reveal probable assembly pathways of HIV-1 capsid formation. Combining the experimental data and computer simulations, our results suggest a sequential combination of both displacive and disassembly/reassembly processes for HIV-1 maturation.

  7. Transient Bluetongue virus serotype 8 capsid protein expression in Nicotiana benthamiana

    Directory of Open Access Journals (Sweden)

    Albertha R. van Zyl

    2016-03-01

    Full Text Available Bluetongue virus (BTV causes severe disease in domestic and wild ruminants, and has recently caused several outbreaks in Europe. Current vaccines include live-attenuated and inactivated viruses; while these are effective, there is risk of reversion to virulence by mutation or reassortment with wild type viruses. Subunit or virus-like particle (VLP vaccines are safer options: VLP vaccines produced in insect cells by expression of the four BTV capsid proteins are protective against challenge; however, this is a costly production method. We investigated production of BTV VLPs in plants via Agrobacterium-mediated transient expression, an inexpensive production system very well suited to developing country use. Leaves infiltrated with recombinant pEAQ-HT vectors separately encoding the four BTV-8 capsid proteins produced more proteins than recombinant pTRA vectors. Plant expression using the pEAQ-HT vector resulted in both BTV-8 core-like particles (CLPs and VLPs; differentially controlling the concentration of infiltrated bacteria significantly influenced yield of the VLPs. In situ localisation of assembled particles was investigated by using transmission electron microscopy (TEM and it was shown that a mixed population of core-like particles (CLPs, consisting of VP3 and VP7 and VLPs were present as paracrystalline arrays in the cytoplasm of plant cells co-expressing all four capsid proteins.

  8. Antibody Competition Reveals Surface Location of HPV L2 Minor Capsid Protein Residues 17–36

    Directory of Open Access Journals (Sweden)

    Stephanie M. Bywaters

    2017-11-01

    Full Text Available The currently available nonavalent human papillomavirus (HPV vaccine exploits the highly antigenic L1 major capsid protein to promote high-titer neutralizing antibodies, but is limited to the HPV types included in the vaccine since the responses are highly type-specific. The limited cross-protection offered by the L1 virus-like particle (VLP vaccine warrants further investigation into cross-protective L2 epitopes. The L2 proteins are yet to be fully characterized as to their precise placement in the virion. Adding to the difficulties in localizing L2, studies have suggested that L2 epitopes are not well exposed on the surface of the mature capsid prior to cellular engagement. Using a series of competition assays between previously mapped anti-L1 monoclonal antibodies (mAbs (H16.V5, H16.U4 and H16.7E and novel anti-L2 mAbs, we probed the capsid surface for the location of an L2 epitope (aa17–36. The previously characterized L1 epitopes together with our competition data is consistent with a proposed L2 epitope within the canyons of pentavalent capsomers.

  9. Antibody Competition Reveals Surface Location of HPV L2 Minor Capsid Protein Residues 17-36.

    Science.gov (United States)

    Bywaters, Stephanie M; Brendle, Sarah A; Tossi, Kerstin P; Biryukov, Jennifer; Meyers, Craig; Christensen, Neil D

    2017-11-10

    The currently available nonavalent human papillomavirus (HPV) vaccine exploits the highly antigenic L1 major capsid protein to promote high-titer neutralizing antibodies, but is limited to the HPV types included in the vaccine since the responses are highly type-specific. The limited cross-protection offered by the L1 virus-like particle (VLP) vaccine warrants further investigation into cross-protective L2 epitopes. The L2 proteins are yet to be fully characterized as to their precise placement in the virion. Adding to the difficulties in localizing L2, studies have suggested that L2 epitopes are not well exposed on the surface of the mature capsid prior to cellular engagement. Using a series of competition assays between previously mapped anti-L1 monoclonal antibodies (mAbs) (H16.V5, H16.U4 and H16.7E) and novel anti-L2 mAbs, we probed the capsid surface for the location of an L2 epitope (aa17-36). The previously characterized L1 epitopes together with our competition data is consistent with a proposed L2 epitope within the canyons of pentavalent capsomers.

  10. Structure of a Spumaretrovirus Gag Central Domain Reveals an Ancient Retroviral Capsid

    Science.gov (United States)

    Dutta, Moumita; Pollard, Dominic J.; Goldstone, David C.; Ramos, Andres; Müllers, Erik; Stirnnagel, Kristin; Stanke, Nicole; Lindemann, Dirk; Taylor, William R.; Rosenthal, Peter B.

    2016-01-01

    The Spumaretrovirinae, or foamy viruses (FVs) are complex retroviruses that infect many species of monkey and ape. Despite little sequence homology, FV and orthoretroviral Gag proteins perform equivalent functions, including genome packaging, virion assembly, trafficking and membrane targeting. However, there is a paucity of structural information for FVs and it is unclear how disparate FV and orthoretroviral Gag molecules share the same function. To probe the functional overlap of FV and orthoretroviral Gag we have determined the structure of a central region of Gag from the Prototype FV (PFV). The structure comprises two all α-helical domains NtDCEN and CtDCEN that although they have no sequence similarity, we show they share the same core fold as the N- (NtDCA) and C-terminal domains (CtDCA) of archetypal orthoretroviral capsid protein (CA). Moreover, structural comparisons with orthoretroviral CA align PFV NtDCEN and CtDCEN with NtDCA and CtDCA respectively. Further in vitro and functional virological assays reveal that residues making inter-domain NtDCEN—CtDCEN interactions are required for PFV capsid assembly and that intact capsid is required for PFV reverse transcription. These data provide the first information that relates the Gag proteins of Spuma and Orthoretrovirinae and suggests a common ancestor for both lineages containing an ancient CA fold. PMID:27829070

  11. Perspective on Adeno-Associated Virus Capsid Modification for Duchenne Muscular Dystrophy Gene Therapy.

    Science.gov (United States)

    Nance, Michael E; Duan, Dongsheng

    2015-12-01

    Duchenne muscular dystrophy (DMD) is a X-linked, progressive childhood myopathy caused by mutations in the dystrophin gene, one of the largest genes in the genome. It is characterized by skeletal and cardiac muscle degeneration and dysfunction leading to cardiac and/or respiratory failure. Adeno-associated virus (AAV) is a highly promising gene therapy vector. AAV gene therapy has resulted in unprecedented clinical success for treating several inherited diseases. However, AAV gene therapy for DMD remains a significant challenge. Hurdles for AAV-mediated DMD gene therapy include the difficulty to package the full-length dystrophin coding sequence in an AAV vector, the necessity for whole-body gene delivery, the immune response to dystrophin and AAV capsid, and the species-specific barriers to translate from animal models to human patients. Capsid engineering aims at improving viral vector properties by rational design and/or forced evolution. In this review, we discuss how to use the state-of-the-art AAV capsid engineering technologies to overcome hurdles in AAV-based DMD gene therapy.

  12. Viral capsid is a pathogen-associated molecular pattern in adenovirus keratitis.

    Directory of Open Access Journals (Sweden)

    Ashish V Chintakuntlawar

    2010-04-01

    Full Text Available Human adenovirus (HAdV infection of the human eye, in particular serotypes 8, 19 and 37, induces the formation of corneal subepithelial leukocytic infiltrates. Using a unique mouse model of adenovirus keratitis, we studied the role of various virus-associated molecular patterns in subsequent innate immune responses of resident corneal cells to HAdV-37 infection. We found that neither viral DNA, viral gene expression, or viral replication was necessary for the development of keratitis. In contrast, empty viral capsid induced keratitis and a chemokine profile similar to intact virus. Transfected viral DNA did not induce leukocyte infiltration despite CCL2 expression similar to levels in virus infected corneas. Mice without toll-like receptor 9 (Tlr9 signaling developed clinical keratitis upon HAdV-37 infection similar to wild type mice, although the absolute numbers of activated monocytes in the cornea were less in Tlr9(-/- mice. Virus induced leukocytic infiltrates and chemokine expression in mouse cornea could be blocked by treatment with a peptide containing arginine glycine aspartic acid (RGD. These results demonstrate that adenovirus infection of the cornea induces chemokine expression and subsequent infiltration by leukocytes principally through RGD contact between viral capsid and the host cell, possibly through direct interaction between the viral capsid penton base and host cell integrins.

  13. AAV capsid CD8+ T-cell epitopes are highly conserved across AAV serotypes

    Directory of Open Access Journals (Sweden)

    Daniel J Hui

    Full Text Available Adeno-associated virus (AAV has become one of the most promising vectors in gene transfer in the last 10 years with successful translation to clinical trials in humans and even market approval for a first gene therapy product in Europe. Administration to humans, however, revealed that adaptive immune responses against the vector capsid can present an obstacle to sustained transgene expression due to the activation and expansion of capsid-specific T cells. The limited number of peripheral blood mononuclear cells (PBMCs obtained from samples within clinical trials allows for little more than monitoring of T-cell responses. We were able to identify immunodominant major histocompatibility complex (MHC class I epitopes for common human leukocyte antigen (HLA types by using spleens isolated from subjects undergoing splenectomy for non-malignant indications as a source of large numbers of lymphocytes and restimulating them with single AAV capsid peptides in vitro. Further experiments confirmed that these epitopes are naturally processed and functionally relevant. The design of more effective and less immunogenic AAV vectors, and precise immune monitoring of vector-infused subjects, are facilitated by these findings.

  14. AAV capsid CD8+ T-cell epitopes are highly conserved across AAV serotypes.

    Science.gov (United States)

    Hui, Daniel J; Edmonson, Shyrie C; Podsakoff, Gregory M; Pien, Gary C; Ivanciu, Lacramioara; Camire, Rodney M; Ertl, Hildegund; Mingozzi, Federico; High, Katherine A; Basner-Tschakarjan, Etiena

    2015-01-01

    Adeno-associated virus (AAV) has become one of the most promising vectors in gene transfer in the last 10 years with successful translation to clinical trials in humans and even market approval for a first gene therapy product in Europe. Administration to humans, however, revealed that adaptive immune responses against the vector capsid can present an obstacle to sustained transgene expression due to the activation and expansion of capsid-specific T cells. The limited number of peripheral blood mononuclear cells (PBMCs) obtained from samples within clinical trials allows for little more than monitoring of T-cell responses. We were able to identify immunodominant major histocompatibility complex (MHC) class I epitopes for common human leukocyte antigen (HLA) types by using spleens isolated from subjects undergoing splenectomy for non-malignant indications as a source of large numbers of lymphocytes and restimulating them with single AAV capsid peptides in vitro. Further experiments confirmed that these epitopes are naturally processed and functionally relevant. The design of more effective and less immunogenic AAV vectors, and precise immune monitoring of vector-infused subjects, are facilitated by these findings.

  15. Performance Evaluation of the VIDAS® Measles IgG Assay and Its Diagnostic Value for Measuring IgG Antibody Avidity in Measles Virus Infection

    Science.gov (United States)

    Dina, Julia; Creveuil, Christian; Gouarin, Stephanie; Viron, Florent; Hebert, Amelie; Freymuth, Francois; Vabret, Astrid

    2016-01-01

    The objective of this study is primarily to compare the performance of the VIDAS® Measles immunoglobulin (Ig)G assay to that of two other serological assays using an immunoassay technique, Enzygnost® Anti-measles Virus/IgG (Siemens) and Measles IgG CAPTURE EIA® (Microimmune). The sensitivity and the agreement of the VIDAS® Measles IgG assay compared to the Enzygnost® Anti-measles Virus/IgG assay and the Measles IgG CAPTURE EIA® assay are 100%, 97.2% and 99.0%, 98.4%, respectively. The very low number of negative sera for IgG antibodies does not allow calculation of specificity. As a secondary objective, we have evaluated the ability of the VIDAS® Measles IgG assay to measure anti-measles virus IgG antibody avidity with the help of the VIDAS® CMV IgG Avidity reagent, using 76 sera from subjects with measles and 238 other sera. Different groups of populations were analyzed. In the primary infection measles group, the mean IgG avidity index was 0.16 (range of 0.07 to 0.93) compared to 0.79 (range of 0.25 to 1) in the serum group positive for IgG antibodies and negative for IgM. These data allow to define a weak anti-measles virus IgG antibody avidity as an avidity index (AI) 0.6. The VIDAS® Measles IgG assay has a performance equivalent to that of other available products. Its use, individual and quick, is well adapted to testing for anti-measles immunity in exposed subjects. PMID:27556477

  16. Chimeric PD-1:28 Receptor Upgrades Low-Avidity T cells and Restores Effector Function of Tumor-Infiltrating Lymphocytes for Adoptive Cell Therapy.

    Science.gov (United States)

    Schlenker, Ramona; Olguín-Contreras, Luis Felipe; Leisegang, Matthias; Schnappinger, Julia; Disovic, Anja; Rühland, Svenja; Nelson, Peter J; Leonhardt, Heinrich; Harz, Hartmann; Wilde, Susanne; Schendel, Dolores J; Uckert, Wolfgang; Willimsky, Gerald; Noessner, Elfriede

    2017-07-01

    Inherent intermediate- to low-affinity T-cell receptors (TCR) that develop during the natural course of immune responses may not allow sufficient activation for tumor elimination, making the majority of T cells suboptimal for adoptive T-cell therapy (ATT). TCR affinity enhancement has been implemented to provide stronger T-cell activity but carries the risk of creating undesired cross-reactivity leading to potential serious adverse effects in clinical application. We demonstrate here that engineering of low-avidity T cells recognizing a naturally processed and presented tumor-associated antigen with a chimeric PD-1:28 receptor increases effector function to levels seen with high-avidity T cells of identical specificity. Upgrading the function of low-avidity T cells without changing the TCR affinity will allow a large arsenal of low-avidity T cells previously thought to be therapeutically inefficient to be considered for ATT. PD-1:28 engineering reinstated Th1 function in tumor-infiltrating lymphocytes that had been functionally disabled in the human renal cell carcinoma environment without unleashing undesired Th2 cytokines or IL10. Involved mechanisms may be correlated to restoration of ERK and AKT signaling pathways. In mouse tumor models of ATT, PD-1:28 engineering enabled low-avidity T cells to proliferate stronger and prevented PD-L1 upregulation and Th2 polarization in the tumor milieu. Engineered T cells combined with checkpoint blockade secreted significantly more IFNγ compared with T cells without PD-1:28, suggesting a beneficial combination with checkpoint blockade therapy or other therapeutic strategies. Altogether, the supportive effects of PD-1:28 engineering on T-cell function make it an attractive tool for ATT. Cancer Res; 77(13); 3577-90. ©2017 AACR . ©2017 American Association for Cancer Research.

  17. A central region in the minor capsid protein of papillomaviruses facilitates viral genome tethering and membrane penetration for mitotic nuclear entry.

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    Inci Aydin

    2017-05-01

    Full Text Available Incoming papillomaviruses (PVs depend on mitotic nuclear envelope breakdown to gain initial access to the nucleus for viral transcription and replication. In our previous work, we hypothesized that the minor capsid protein L2 of PVs tethers the incoming vDNA to mitotic chromosomes to direct them into the nascent nuclei. To re-evaluate how dynamic L2 recruitment to cellular chromosomes occurs specifically during prometaphase, we developed a quantitative, microscopy-based assay for measuring the degree of chromosome recruitment of L2-EGFP. Analyzing various HPV16 L2 truncation-mutants revealed a central chromosome-binding region (CBR of 147 amino acids that confers binding to mitotic chromosomes. Specific mutations of conserved motifs (IVAL286AAAA, RR302/5AA, and RTR313EEE within the CBR interfered with chromosomal binding. Moreover, assembly-competent HPV16 containing the chromosome-binding deficient L2(RTR313EEE or L2(IVAL286AAAA were inhibited for infection despite their ability to be transported to intracellular compartments. Since vDNA and L2 were not associated with mitotic chromosomes either, the infectivity was likely impaired by a defect in tethering of the vDNA to mitotic chromosomes. However, L2 mutations that abrogated chromatin association also compromised translocation of L2 across membranes of intracellular organelles. Thus, chromatin recruitment of L2 may in itself be a requirement for successful penetration of the limiting membrane thereby linking both processes mechanistically. Furthermore, we demonstrate that the association of L2 with mitotic chromosomes is conserved among the alpha, beta, gamma, and iota genera of Papillomaviridae. However, different binding patterns point to a certain variance amongst the different genera. Overall, our data suggest a common strategy among various PVs, in which a central region of L2 mediates tethering of vDNA to mitotic chromosomes during cell division thereby coordinating membrane

  18. Generation of neutralizing monoclonal antibodies against a conformational epitope of human adenovirus type 7 (HAdv-7 incorporated in capsid encoded in a HAdv-3-based vector.

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    Minglong Liu

    Full Text Available The generation of monoclonal antibodies (MAbs by epitope-based immunization is difficult because the immunogenicity of simple peptides is poor and T cells must be potently stimulated and immunological memory elicited. A strategy in which antigen is incorporated into the adenoviral capsid protein has been used previously to develop antibody responses against several vaccine targets and may offer a solution to this problem. In this study, we used a similar strategy to develop HAdv-7-neutralizing MAbs using rAdMHE3 virions into which hexon hypervariable region 5 (HVR5 of adenovirus type 7 (HAdv-7 was incorporated. The epitope mutant rAdMHE3 was generated by replacing HVR5 of Ad3EGFP, a recombinant HAdv-3-based vector expressing enhanced green fluorescence protein, with HVR5 of HAdv-7. We immunized BALB/c mice with rAdMHE3 virions and produced 22 different MAbs against them, four of which showed neutralizing activity against HAdv-7 in vitro. Using an indirect enzyme-linked immunosorbent assay (ELISA analysis and an antibody-binding-competition ELISA with Ad3EGFP, HAdv-7, and a series of chimeric adenoviral particles containing epitope mutants, we demonstrated that the four MAbs recognize the neutralization site within HVR5 of the HAdv-7 virion. Using an immunoblotting analysis and ELISA with HAdv-7, recombinant peptides, and a synthetic peptide, we also showed that the neutralizing epitope within HVR5 of the HAdv-7 virion is a conformational epitope. These findings suggest that it is feasible to use a strategy in which antigen is incorporated into the adenoviral capsid protein to generate neutralizing MAbs. This strategy may also be useful for developing therapeutic neutralizing MAbs and designing recombinant vector vaccines against HAdv-7, and in structural analysis of adenoviruses.

  19. Genetic modification of adeno-associated viral vector type 2 capsid enhances gene transfer efficiency in polarized human airway epithelial cells.

    Science.gov (United States)

    White, April F; Mazur, Marina; Sorscher, Eric J; Zinn, Kurt R; Ponnazhagan, Selvarangan

    2008-12-01

    Cystic fibrosis (CF) is a common genetic disease characterized by defects in the expression of the CF transmembrane conductance regulator (CFTR) gene. Gene therapy offers better hope for the treatment of CF. Adeno-associated viral (AAV) vectors are capable of stable expression with low immunogenicity. Despite their potential in CF gene therapy, gene transfer efficiency by AAV is limited because of pathophysiological barriers in these patients. Although a few AAV serotypes have shown better transduction compared with the AAV2-based vectors, gene transfer efficiency in human airway epithelium has still not reached therapeutic levels. To engineer better AAV vectors for enhanced gene delivery in human airway epithelium, we developed and characterized mutant AAV vectors by genetic capsid modification, modeling the well-characterized AAV2 serotype. We genetically incorporated putative high-affinity peptide ligands to human airway epithelium on the GH loop region of AAV2 capsid protein. Six independent mutant AAV were constructed, containing peptide ligands previously reported to bind with high affinity for known and unknown receptors on human airway epithelial cells. The vectors were tested on nonairway cells and nonpolarized and polarized human airway epithelial cells for enhanced infectivity. One of the mutant vectors, with the peptide sequence THALWHT, not only showed the highest transduction in undifferentiated human airway epithelial cells but also indicated significant transduction in polarized cells. Interestingly, this modified vector was also able to infect cells independently of the heparan sulfate proteoglycan receptor. Incorporation of this ligand on other AAV serotypes, which have shown improved gene transfer efficiency in the human airway epithelium, may enhance the application of AAV vectors in CF gene therapy.

  20. Mutation of the N-Terminal Region of Chikungunya Virus Capsid Protein: Implications for Vaccine Design.

    Science.gov (United States)

    Taylor, Adam; Liu, Xiang; Zaid, Ali; Goh, Lucas Y H; Hobson-Peters, Jody; Hall, Roy A; Merits, Andres; Mahalingam, Suresh

    2017-02-21

    Mosquito-transmitted chikungunya virus (CHIKV) is an arthritogenic alphavirus of the Togaviridae family responsible for frequent outbreaks of arthritic disease in humans. Capsid protein, a structural protein encoded by the CHIKV RNA genome, is able to translocate to the host cell nucleolus. In encephalitic alphaviruses, nuclear translocation induces host cell transcriptional shutoff; however, the role of capsid protein nucleolar localization in arthritogenic alphaviruses remains unclear. Using recombinant enhanced green fluorescent protein (EGFP)-tagged expression constructs and CHIKV infectious clones, we describe a nucleolar localization sequence (NoLS) in the N-terminal region of capsid protein, previously uncharacterized in CHIKV. Mutation of the NoLS by site-directed mutagenesis reduced efficiency of nuclear import of CHIKV capsid protein. In the virus, mutation of the capsid protein NoLS (CHIKV-NoLS) attenuated replication in mammalian and mosquito cells, producing a small-plaque phenotype. Attenuation of CHIKV-NoLS is likely due to disruption of the viral replication cycle downstream of viral RNA synthesis. In mice, CHIKV-NoLS infection caused no disease signs compared to wild-type CHIKV (CHIKV-WT)-infected mice; lack of disease signs correlated with significantly reduced viremia and decreased expression of proinflammatory factors. Mice immunized with CHIKV-NoLS, challenged with CHIKV-WT at 30 days postimmunization, develop no disease signs and no detectable viremia. Serum from CHIKV-NoLS-immunized mice is able to efficiently neutralize CHIKV infection in vitro Additionally, CHIKV-NoLS-immunized mice challenged with the related alphavirus Ross River virus showed reduced early and peak viremia postchallenge, indicating a cross-protective effect. The high degree of CHIKV-NoLS attenuation may improve CHIKV antiviral and rational vaccine design. IMPORTANCE CHIKV is a mosquito-borne pathogen capable of causing explosive epidemics of incapacitating joint pain

  1. High avidity antibodies to full-length VAR2CSA correlate with absence of placental malaria

    DEFF Research Database (Denmark)

    Tutterrow, Yeung Lo; Salanti, Ali; Avril, Marion

    2012-01-01

    VAR2CSA mediates sequestration of Plasmodium falciparum-infected erythrocytes in the placenta, increasing the risk of poor pregnancy outcomes. Naturally acquired antibodies (Ab) to placental parasites at delivery have been associated with improved pregnancy outcomes, but Ab levels and how early...... in pregnancy Ab must be present in order to eliminate placental parasites before delivery remains unknown. Antibodies to individual Duffy-binding like domains of VAR2CSA have been studied, but the domains lack many of the conformational epitopes present in full-length VAR2CSA (FV2). Thus, the purpose...... of this study was to describe the acquisition of Ab to FV2 in women residing in high and low transmission areas and determine how Ab levels during pregnancy correlate with clearance of placental parasites. Plasma samples collected monthly throughout pregnancy from pregnant women living in high and low...

  2. Antibodies with High Avidity to the gp120 Envelope Protein in Protection from Simian Immunodeficiency Virus SIVmac251 Acquisition in an Immunization Regimen That Mimics the RV-144 Thai Trial

    Science.gov (United States)

    Pegu, Poonam; Vaccari, Monica; Gordon, Shari; Keele, Brandon F.; Doster, Melvin; Guan, Yongjun; Ferrari, Guido; Pal, Ranajit; Ferrari, Maria Grazia; Whitney, Stephen; Hudacik, Lauren; Billings, Erik; Rao, Mangala; Montefiori, David; Tomaras, Georgia; Alam, S. Munir; Fenizia, Claudio; Lifson, Jeffrey D.; Stablein, Donald; Tartaglia, Jim; Michael, Nelson; Kim, Jerome; Venzon, David

    2013-01-01

    The recombinant canarypox vector, ALVAC-HIV, together with human immunodeficiency virus (HIV) gp120 envelope glycoprotein, has protected 31.2% of Thai individuals from HIV acquisition in the RV144 HIV vaccine trial. This outcome was unexpected, given the limited ability of the vaccine components to induce CD8+ T-cell responses or broadly neutralizing antibodies. We vaccinated macaques with an immunization regimen intended to mimic the RV144 trial and exposed them intrarectally to a dose of the simian immunodeficiency virus SIVmac251 that transmits few virus variants, similar to HIV transmission to humans. Vaccination induced anti-envelope antibodies in all vaccinees and CD4+ and CD8+ T-cell responses. Three of the 11 macaques vaccinated with ALVAC-SIV/gp120 were protected from SIVmac251 acquisition, but the result was not significant. The remaining vaccinees were infected and progressed to disease. The magnitudes of vaccine-induced SIVmac251-specific T-cell responses and binding antibodies were not significantly different between protected and infected animals. However, sera from protected animals had higher avidity antibodies to gp120, recognized the variable envelope regions V1/V2, and reduced SIVmac251 infectivity in cells that express high levels of α4β7 integrins, suggesting a functional role of antibodies to V2. The current results emphasize the utility of determining the titer of repeated mucosal challenge in the preclinical evaluation of HIV vaccines. PMID:23175374

  3. Antibodies with high avidity to the gp120 envelope protein in protection from simian immunodeficiency virus SIV(mac251) acquisition in an immunization regimen that mimics the RV-144 Thai trial.

    Science.gov (United States)

    Pegu, Poonam; Vaccari, Monica; Gordon, Shari; Keele, Brandon F; Doster, Melvin; Guan, Yongjun; Ferrari, Guido; Pal, Ranajit; Ferrari, Maria Grazia; Whitney, Stephen; Hudacik, Lauren; Billings, Erik; Rao, Mangala; Montefiori, David; Tomaras, Georgia; Alam, S Munir; Fenizia, Claudio; Lifson, Jeffrey D; Stablein, Donald; Tartaglia, Jim; Michael, Nelson; Kim, Jerome; Venzon, David; Franchini, Genoveffa

    2013-02-01

    The recombinant canarypox vector, ALVAC-HIV, together with human immunodeficiency virus (HIV) gp120 envelope glycoprotein, has protected 31.2% of Thai individuals from HIV acquisition in the RV144 HIV vaccine trial. This outcome was unexpected, given the limited ability of the vaccine components to induce CD8(+) T-cell responses or broadly neutralizing antibodies. We vaccinated macaques with an immunization regimen intended to mimic the RV144 trial and exposed them intrarectally to a dose of the simian immunodeficiency virus SIV(mac251) that transmits few virus variants, similar to HIV transmission to humans. Vaccination induced anti-envelope antibodies in all vaccinees and CD4(+) and CD8(+) T-cell responses. Three of the 11 macaques vaccinated with ALVAC-SIV/gp120 were protected from SIV(mac251) acquisition, but the result was not significant. The remaining vaccinees were infected and progressed to disease. The magnitudes of vaccine-induced SIV(mac251)-specific T-cell responses and binding antibodies were not significantly different between protected and infected animals. However, sera from protected animals had higher avidity antibodies to gp120, recognized the variable envelope regions V1/V2, and reduced SIV(mac251) infectivity in cells that express high levels of α(4)β(7) integrins, suggesting a functional role of antibodies to V2. The current results emphasize the utility of determining the titer of repeated mucosal challenge in the preclinical evaluation of HIV vaccines.

  4. Development and validation of novel AAV2 random libraries displaying peptides of diverse lengths and at diverse capsid positions.

    Science.gov (United States)

    Naumer, Matthias; Ying, Ying; Michelfelder, Stefan; Reuter, Antje; Trepel, Martin; Müller, Oliver J; Kleinschmidt, Jürgen A

    2012-05-01

    Libraries based on the insertion of random peptide ligands into the capsid of adeno-associated virus type 2 (AAV2) have been widely used to improve the efficiency and selectivity of the AAV vector system. However, so far only libraries of 7-mer peptide ligands have been inserted at one well-characterized capsid position. Here, we expanded the combinatorial AAV2 display system to a panel of novel AAV libraries, displaying peptides of 5, 7, 12, 19, or 26 amino acids in length at capsid position 588 or displaying 7-mer peptides at position 453, the most prominently exposed region of the viral capsid. Library selections on two unrelated cell types-human coronary artery endothelial cells and rat cardiomyoblasts-revealed the isolation of cell type-characteristic peptides of different lengths mediating strongly improved target-cell transduction, except for the 26-mer peptide ligands. Characterization of vector selectivity by transduction of nontarget cells and comparative gene-transduction analysis using a panel of 44 human tumor cell lines revealed that insertion of different-length peptides allows targeting of distinct cellular receptors for cell entry with similar efficiency, but with different selectivity. The application of such novel AAV2 libraries broadens the spectrum of targetable receptors by capsid-modified AAV vectors and provides the opportunity to choose the best suited targeting ligand for a certain application from a number of different candidates.

  5. Single Amino Acid Modification of Adeno-Associated Virus Capsid Changes Transduction and Humoral Immune Profiles

    Science.gov (United States)

    Diprimio, Nina; Bowles, Dawn E.; Hirsch, Matthew L.; Monahan, Paul E.; Asokan, Aravind; Rabinowitz, Joseph; Agbandje-McKenna, Mavis

    2012-01-01

    Adeno-associated virus (AAV) vectors have the potential to promote long-term gene expression. Unfortunately, humoral immunity restricts patient treatment and in addition provides an obstacle to the potential option of vector readministration. In this study, we describe a comprehensive characterization of the neutralizing antibody (NAb) response to AAV type 1 (AAV1) through AAV5 both in vitro and in vivo. These results demonstrated that NAbs generated from one AAV type are unable to neutralize the transduction of other types. We extended this observation by demonstrating that a rationally engineered, muscle-tropic AAV2 mutant containing 5 amino acid substitutions from AAV1 displayed a NAb profile different from those of parental AAV2 and AAV1. Here we found that a single insertion of Thr from AAV1 into AAV2 capsid at residue 265 preserved high muscle transduction, while also changing the immune profile. To better understand the role of Thr insertion at position 265, we replaced all 20 amino acids and evaluated both muscle transduction and the NAb response. Of these variants, 8 mutants induced higher muscle transduction than AAV2. Additionally, three classes of capsid NAb immune profile were defined based on the ability to inhibit transduction from AAV2 or mutants. While no relationship was found between transduction, amino acid properties, and NAb titer or its cross-reactivity, these studies map a critical capsid motif involved in all steps of AAV infectivity. Our results suggest that AAV types can be utilized not only as templates to generate mutants with enhanced transduction efficiency but also as substrates for repeat administration. PMID:22593151

  6. Capsid coding sequences of foot-and-mouth disease viruses are determinants of pathogenicity in pigs.

    Science.gov (United States)

    Lohse, Louise; Jackson, Terry; Bøtner, Anette; Belsham, Graham J

    2012-05-24

    The surface exposed capsid proteins, VP1, VP2 and VP3, of foot-and-mouth disease virus (FMDV) determine its antigenicity and the ability of the virus to interact with host-cell receptors. Hence, modification of these structural proteins may alter the properties of the virus.In the present study we compared the pathogenicity of different FMDVs in young pigs. In total 32 pigs, 7-weeks-old, were exposed to virus, either by direct inoculation or through contact with inoculated pigs, using cell culture adapted (O1K B64), chimeric (O1K/A-TUR and O1K/O-UKG) or field strain (O-UKG/34/2001) viruses. The O1K B64 virus and the two chimeric viruses are identical to each other except for the capsid coding region.Animals exposed to O1K B64 did not exhibit signs of disease, while pigs exposed to each of the other viruses showed typical clinical signs of foot-and-mouth disease (FMD). All pigs infected with the O1K/O-UKG chimera or the field strain (O-UKG/34/2001) developed fulminant disease. Furthermore, 3 of 4 in-contact pigs exposed to the O1K/O-UKG virus died in the acute phase of infection, likely from myocardial infection. However, in the group exposed to the O1K/A-TUR chimeric virus, only 1 pig showed symptoms of disease within the time frame of the experiment (10 days). All pigs that developed clinical disease showed a high level of viral RNA in serum and infected pigs that survived the acute phase of infection developed a serotype specific antibody response. It is concluded that the capsid coding sequences are determinants of FMDV pathogenicity in pigs.

  7. Outer capsid proteins induce the formation of pores in epithelial cells

    International Nuclear Information System (INIS)

    Ruiz, M; Abad M; Michelangely, F; Charpilienne, A; Cohen, J

    1995-01-01

    Two mechanisms of entrance in cell of the rotavirus, during the infection, were proposed: a direct entrance through the plasmatic membrane or by means of endocytosis. In the two cases, a permeabilization mechanism of the membrane (cellular or of the endocytic vesicle, respectively) should occur. It has been shown that the rotavirus induces permeabilization of liposomes and of membrane vesicles. In this work, are studied the changes of intact cells permeability, measuring the entrance of e tide bromides. Viral particles of double capsid of the RF stump produce an increase of the cells membrane MA104 permeability, while the simple capsid ones don't induce effect. This phenomenon requires the particles trypsinization, and occurs in a means where the concentration of free Ca is lower to 1 micromolar. The temporary course of the fluorescence increase is sigmoid. The latency, the speed and the width depend on the relationship of virus / cell, and it can be observed up to 100% of permeabilization in relation to the effect of digitonin. The pores induced in the membrane by the rotavirus are irreversible. The permeabilizer effect of the rotavirus on the membrane was observed in other cellular lines as Hela and HT29, but not in the L929 ones. These results suggest that one or more proteins of the external capsid are responsible s of the effect. These could be involved in the penetration process of the virus towards the cytoplasm and could be one of the restrictive factor of the cell infection by means of the virus [es

  8. Ultrastructural visualization of the transmembranous and cytomatrix-related part of nicotinic acetylcholine receptor of frog motor endplate by means of an immunochemical avidity of IgG for d-tubocurarine.

    Directory of Open Access Journals (Sweden)

    Shigeru Tsuji

    2008-02-01

    Full Text Available In the present study, a fine ultrastructural localization of nicotinic acetylcholine receptor (nAChR was attempted, using d-tubocurarine (d-TC, a quaternary ammonium compound binding to nAChR. The localization was based on the binding avidity of immunoglobulin G (IgG for acetylcholine (ACh and other quaternary ammonium compounds, such as d-TC. d-TC was applied to the frog neuromuscular preparation and caused a blockade of neuromuscular transmission. Then, d-TC was rendered insoluble in situ by silicotungstic acid (STA, a precipitating agent of soluble proteins and quaternary ammonium compounds. After tissue fixation, a normal rabbit serum was applied to the fine precipitate of the insoluble salt of d-TC silicotungstate (quaternary ammonium radical of d-TC to form the immunochemical complex d-TC- rabbit IgG at ACh binding sites. The IgG of the complex was revealed by means of the conventional immunoperoxidase procedure used for ultrastructural localization. Under the electron microscope, fine diaminobenzidine (DAB precipitates appeared as regular rod-like structures oriented to cytoplasmic side of the horizontal part (crest of the postsynaptic membrane (between the junctional folds which is known to be endowed with nAChR. The rod-like precipitates were not observed in the postsynaptic junctional folds which are devoid of nAChR. The distance separating the rods each other was rather constant (12 - 15 nm, while the length of the rods was variable and exceeded the usual length of nAChR. The present work indicates that the rod-like structures, already observed in association with sarcoplasmic side of the postsynaptic membrane, did correspond to the intramembranous and intracytoplasmic part of nAChR and related proteins. These cytochemical results confirm that d-TC binds to ACh binding sites in the pore of nAChR, and raise the question of DAB staining of cytoskeletal proteins related to the nAChR complex.

  9. Modeling capsid kinetics assembly from the steady state distribution of multi-sizes aggregates

    Energy Technology Data Exchange (ETDEWEB)

    Hozé, Nathanaël; Holcman, David

    2014-01-24

    The kinetics of aggregation for particles of various sizes depends on their diffusive arrival and fusion at a specific nucleation site. We present here a mean-field approximation and a stochastic jump model for aggregates at equilibrium. This approach is an alternative to the classical Smoluchowski equations that do not have a close form and are not solvable in general. We analyze these mean-field equations and obtain the kinetics of a cluster formation. Our approach provides a simplified theoretical framework to study the kinetics of viral capsid formation, such as HIV from the self-assembly of the structural proteins Gag.

  10. Nanofluidic Devices with Two Pores in Series for Resistive-Pulse Sensing of Single Virus Capsids

    DEFF Research Database (Denmark)

    Harms, Zachary D.; Mogensen, Klaus Bo; Rodrigues de Sousa Nunes, Pedro André

    2011-01-01

    We report fabrication and characterization of nanochannel devices with two nanopores in series for resistive-pulse sensing of hepatitis B virus (HBV) capsids. The nanochannel and two pores are patterned by electron beam lithography between two microchannels and etched by reactive ion etching....... The two nanopores are 50-nm wide, 50-nm deep, and 40-nm long and are spaced 2.0-μm apart. The nanochannel that brackets the two pores is 20 wider (1 μm) to reduce the electrical resistance adjacent to the two pores and to ensure the current returns to its baseline value between resistive-pulse events...

  11. Nasal Immunization Confers High Avidity Neutralizing Antibody Response and Immunity to Primary and Recurrent Genital Herpes in Guinea Pigs

    Science.gov (United States)

    Persson, Josefine; Zhang, Yuan; Olafsdottir, Thorunn A.; Thörn, Karolina; Cairns, Tina M.; Wegmann, Frank; Sattentau, Quentin J.; Eisenberg, Roselyn J.; Cohen, Gary H.; Harandi, Ali M.

    2016-01-01

    Genital herpes is one of the most prevalent sexually transmitted infections in both the developing and developed world. Following infection, individuals experience life-long latency associated with sporadic ulcerative outbreaks. Despite many efforts, no vaccine has yet been licensed for human use. Herein, we demonstrated that nasal immunization with an adjuvanted HSV-2 gD envelope protein mounts significant protection to primary infection as well as the establishment of latency and recurrent genital herpes in guinea pigs. Nasal immunization was shown to elicit specific T cell proliferative and IFN-γ responses as well as systemic and vaginal gD-specific IgG antibody (Ab) responses. Furthermore, systemic IgG Abs displayed potent HSV-2 neutralizing properties and high avidity. By employing a competitive surface plasmon resonance (SPR) analysis combined with a battery of known gD-specific neutralizing monoclonal Abs (MAbs), we showed that nasal immunization generated IgG Abs directed to two major discontinuous neutralizing epitopes of gD. These results highlight the potential of nasal immunization with an adjuvanted HSV-2 envelope protein for induction of protective immunity to primary and recurrent genital herpes. PMID:28082979

  12. Primary Nocardia Infection Causing a Fluorodeoxyglucose-Avid Right Renal Mass in a Redo Lung Transplant Recipient

    Directory of Open Access Journals (Sweden)

    Sreeja Biswas Roy

    2018-01-01

    Full Text Available Immunosuppression after lung transplantation may increase susceptibility to opportunistic infection and is associated with early and delayed deaths in lung transplant recipients. Factors that may predispose lung transplant recipients to opportunistic bacterial and fungal infections include prolonged corticosteroid use, renal impairment, treatment of acute rejection, and post-transplant diabetes mellitus. We present a unique case of a 63-year-old woman with diabetes mellitus who underwent redo lung transplantation. Three years after her right-sided single redo lung transplant, she presented with right-sided abdominal pain, nausea, and vomiting. Upon examination, computed tomography showed a 4.5 × 3.3 cm heterogeneous, enhancing right renal mass with a patent renal vein. Magnetic resonance imaging confirmed a T1/T2 hypointense, diffusion-restricting, right mid-renal mass that was fluorodeoxyglucose-avid on positron emission tomography. We initially suspected primary renal cell carcinoma. However, after a right nephrectomy, no evidence of neoplasia was observed; instead, a renal abscess containing filamentous bacteria was noted, raising suspicion for infection of the Nocardia species. Special stains confirmed a diagnosis of Nocardia renal abscess. Computed tomography of the chest and brain revealed no lesions consistent with infection. We initiated a long-term therapeutic regimen of anti-Nocardia therapy with imipenem and trimethoprim-sulfamethoxazole.

  13. Metastatic Follicular Thyroid Carcinoma Secreting Thyroid Hormone and Radioiodine Avid without Stimulation: A Case Report and Literature Review

    Directory of Open Access Journals (Sweden)

    Syed A. Abid

    2014-01-01

    Full Text Available Introduction. This is an extremely rare case of a patient with metastatic follicular thyroid cancer who continued to produce thyroid hormone and was iodine scan positive without stimulation after thyroidectomy and radioiodine (I-131 therapy. Patient Findings. A 76-year-old Caucasian male was diagnosed with metastatic follicular thyroid carcinoma on lung nodule biopsy. Total thyroidectomy was performed and he was ablated with 160 mCi of I-131 after recombinant human thyrotropin (rhTSH stimulation. Whole body scan (WBS after treatment showed uptake in bilateral lungs, right sacrum, and pelvis. The thyroglobulin decreased from 2,063 to 965 four months after treatment but rapidly increased to 2,506 eleven months after I-131. Thyroid stimulating hormone (TSH remained suppressed and free T4 remained elevated after I-131 therapy without thyroid hormone supplementation. He was treated with an additional 209 mCi with WBS findings positive in lung and pelvis. Despite I-131, new metastatic lesions were noted in the left thyroid bed and large destructive lesion to the first cervical vertebrae four months after the second I-131 dose. Conclusions. This case is exceptional because of its rarity and also due to the dissociation between tumor differentiation and aggressiveness. The metastatic lesions continued to secrete thyroid hormone and remained radioiodine avid with rapid progression after I-131 therapy.

  14. Nasal Immunization Confers High Avidity Neutralizing Antibody Response and Immunity to Primary and Recurrent Genital Herpes in Guinea Pigs.

    Science.gov (United States)

    Persson, Josefine; Zhang, Yuan; Olafsdottir, Thorunn A; Thörn, Karolina; Cairns, Tina M; Wegmann, Frank; Sattentau, Quentin J; Eisenberg, Roselyn J; Cohen, Gary H; Harandi, Ali M

    2016-01-01

    Genital herpes is one of the most prevalent sexually transmitted infections in both the developing and developed world. Following infection, individuals experience life-long latency associated with sporadic ulcerative outbreaks. Despite many efforts, no vaccine has yet been licensed for human use. Herein, we demonstrated that nasal immunization with an adjuvanted HSV-2 gD envelope protein mounts significant protection to primary infection as well as the establishment of latency and recurrent genital herpes in guinea pigs. Nasal immunization was shown to elicit specific T cell proliferative and IFN-γ responses as well as systemic and vaginal gD-specific IgG antibody (Ab) responses. Furthermore, systemic IgG Abs displayed potent HSV-2 neutralizing properties and high avidity. By employing a competitive surface plasmon resonance (SPR) analysis combined with a battery of known gD-specific neutralizing monoclonal Abs (MAbs), we showed that nasal immunization generated IgG Abs directed to two major discontinuous neutralizing epitopes of gD. These results highlight the potential of nasal immunization with an adjuvanted HSV-2 envelope protein for induction of protective immunity to primary and recurrent genital herpes.

  15. Discovery of Radioiodinated Monomeric Anthraquinones as a Novel Class of Necrosis Avid Agents for Early Imaging of Necrotic Myocardium.

    Science.gov (United States)

    Wang, Qin; Yang, Shengwei; Jiang, Cuihua; Li, Jindian; Wang, Cong; Chen, Linwei; Jin, Qiaomei; Song, Shaoli; Feng, Yuanbo; Ni, Yicheng; Zhang, Jian; Yin, Zhiqi

    2016-02-16

    Assessment of myocardial viability is deemed necessary to aid in clinical decision making whether to recommend revascularization therapy for patients with myocardial infarction (MI). Dianthraquinones such as hypericin (Hyp) selectively accumulate in necrotic myocardium, but were unsuitable for early imaging after administration to assess myocardial viability. Since dianthraquinones can be composed by coupling two molecules of monomeric anthraquinone and the active center can be found by splitting chemical structure, we propose that monomeric anthraquinones may be effective functional groups for necrosis targetability. In this study, eight radioiodinated monomeric anthraquinones were evaluated as novel necrosis avid agents (NAAs) for imaging of necrotic myocardium. All (131)I-anthraquinones showed high affinity to necrotic tissues and (131)I-rhein emerged as the most promising compound. Infarcts were visualized on SPECT/CT images at 6 h after injection of (131)I-rhein, which was earlier than that with (131)I-Hyp. Moreover, (131)I-rhein showed satisfactory heart-to-blood, heart-to-liver and heart-to-lung ratios for obtaining images of good diagnostic quality. (131)I-rhein was a more promising "hot spot imaging" tracer for earlier visualization of necrotic myocardium than (131)I-Hyp, which supported further development of radiopharmaceuticals based on rhein for SPECT/CT ((123)I and (99m)Tc) or PET/CT imaging ((18)F and (124)I) of myocardial necrosis.

  16. IL-4 and IL-13 mediated down-regulation of CD8 expression levels can dampen anti-viral CD8⁺ T cell avidity following HIV-1 recombinant pox viral vaccination.

    Science.gov (United States)

    Wijesundara, Danushka K; Jackson, Ronald J; Tscharke, David C; Ranasinghe, Charani

    2013-09-23

    We have shown that mucosal HIV-1 recombinant pox viral vaccination can induce high, avidity HIV-specific CD8(+) T cells with reduced interleukin (IL)-4 and IL-13 expression compared to, systemic vaccine delivery. In the current study how these cytokines act to regulate anti-viral CD8(+) T, cell avidity following HIV-1 recombinant pox viral prime-boost vaccination was investigated. Out of a panel of T cell avidity markers tested, only CD8 expression levels were found to be enhanced on, KdGag197-205 (HIV)-specific CD8(+) T cells obtained from IL-13(-/-), IL-4(-/-) and signal transducer and, activator of transcription of 6 (STAT6)(-/-) mice compared to wild-type (WT) controls following, vaccination. Elevated CD8 expression levels in this instance also correlated with polyfunctionality, (interferon (IFN)-γ, tumour necorsis factor (TNF)-α and IL-2 production) and the avidity of HIVspecific CD8(+) T cells. Furthermore, mucosal vaccination and vaccination with the novel adjuvanted IL-13 inhibitor (i.e. IL-13Rα2) vaccines significantly enhanced CD8 expression levels on HIV-specific CD8(+), T cells, which correlated with avidity. Using anti-CD8 antibodies that blocked CD8 availability on CD8(+), T cells, it was established that CD8 played an important role in increasing HIV-specific CD8(+) T cell avidity and polyfunctionality in IL-4(-/-), IL-13(-/-) and STAT6(-/-) mice compared to WT controls, following vaccination. Collectively, our data demonstrate that IL-4 and IL-13 dampen CD8 expression levels on anti-viral CD8(+) T cells, which can down-regulate anti-viral CD8(+) T cell avidity and, polyfunctionality following HIV-1 recombinant pox viral vaccination. These findings can be exploited to, design more efficacious vaccines not only against HIV-1, but many chronic infections where high, avidity CD8(+) T cells help protection. Copyright © 2013 Elsevier Ltd. All rights reserved.

  17. Essential role of the unordered VP2 n-terminal domain of the parvovirus MVM capsid in nuclear assembly and endosomal enlargement of the virion fivefold channel for cell entry

    Energy Technology Data Exchange (ETDEWEB)

    Sanchez-Martinez, Cristina; Grueso, Esther [Centro de Biologia Molecular Severo Ochoa (CSIC-UAM), Universidad Autonoma de Madrid, 28049 Cantoblanco, Madrid (Spain); Carroll, Miles [Health Protection Agency, Centre for Emergency Preparedness and Response, Porton Down, Salisbury SP4 OJG, Wilts (United Kingdom); Rommelaere, Jean [Deutsches Krebsforschungszentrum Division F010, Im Neuenheimer Feld 242, D-69120 Heidelberg (Germany); Almendral, Jose M., E-mail: jmalmendral@cbm.uam.es [Centro de Biologia Molecular Severo Ochoa (CSIC-UAM), Universidad Autonoma de Madrid, 28049 Cantoblanco, Madrid (Spain)

    2012-10-10

    The unordered N-termini of parvovirus capsid proteins (Nt) are translocated through a channel at the icosahedral five-fold axis to serve for virus traffick. Heterologous peptides were genetically inserted at the Nt of MVM to study their functional tolerance to manipulations. Insertion of a 5T4-single-chain antibody at VP2-Nt (2Nt) yielded chimeric capsid subunits failing to enter the nucleus. The VEGFR2-binding peptide (V1) inserted at both 2Nt and VP1-Nt efficiently assembled in virions, but V1 disrupted VP1 and VP2 entry functions. The VP2 defect correlated with restricted externalization of V1-2Nt out of the coat. The specific infectivity of MVM and wtVP-pseudotyped mosaic MVM-V1 virions, upon heating and/or partial 2Nt cleavage, demonstrated that some 2Nt domains become intracellularly translocated out of the virus shell and cleaved to initiate entry. The V1 insertion defines a VP2-driven endosomal enlargement of the channel as an essential structural rearrangement performed by the MVM virion to infect.

  18. Essential role of the unordered VP2 n-terminal domain of the parvovirus MVM capsid in nuclear assembly and endosomal enlargement of the virion fivefold channel for cell entry

    International Nuclear Information System (INIS)

    Sánchez-Martínez, Cristina; Grueso, Esther; Carroll, Miles; Rommelaere, Jean; Almendral, José M.

    2012-01-01

    The unordered N-termini of parvovirus capsid proteins (Nt) are translocated through a channel at the icosahedral five-fold axis to serve for virus traffick. Heterologous peptides were genetically inserted at the Nt of MVM to study their functional tolerance to manipulations. Insertion of a 5T4-single-chain antibody at VP2-Nt (2Nt) yielded chimeric capsid subunits failing to enter the nucleus. The VEGFR2-binding peptide (V1) inserted at both 2Nt and VP1-Nt efficiently assembled in virions, but V1 disrupted VP1 and VP2 entry functions. The VP2 defect correlated with restricted externalization of V1-2Nt out of the coat. The specific infectivity of MVM and wtVP-pseudotyped mosaic MVM-V1 virions, upon heating and/or partial 2Nt cleavage, demonstrated that some 2Nt domains become intracellularly translocated out of the virus shell and cleaved to initiate entry. The V1 insertion defines a VP2-driven endosomal enlargement of the channel as an essential structural rearrangement performed by the MVM virion to infect.

  19. Structures of the major capsid proteins of the human Karolinska Institutet and Washington University polyomaviruses.

    Science.gov (United States)

    Neu, Ursula; Wang, Jianbo; Macejak, Dennis; Garcea, Robert L; Stehle, Thilo

    2011-07-01

    The Karolinska Institutet and Washington University polyomaviruses (KIPyV and WUPyV, respectively) are recently discovered human viruses that infect the respiratory tract. Although they have not yet been linked to disease, they are prevalent in populations worldwide, with initial infection occurring in early childhood. Polyomavirus capsids consist of 72 pentamers of the major capsid protein viral protein 1 (VP1), which determines antigenicity and receptor specificity. The WUPyV and KIPyV VP1 proteins are distant in evolution from VP1 proteins of known structure such as simian virus 40 or murine polyomavirus. We present here the crystal structures of unassembled recombinant WUPyV and KIPyV VP1 pentamers at resolutions of 2.9 and 2.55 Å, respectively. The WUPyV and KIPyV VP1 core structures fold into the same β-sandwich that is a hallmark of all polyomavirus VP1 proteins crystallized to date. However, differences in sequence translate into profoundly different surface loop structures in KIPyV and WUPyV VP1 proteins. Such loop structures have not been observed for other polyomaviruses, and they provide initial clues about the possible interactions of these viruses with cell surface receptors.

  20. Rapid increase of near atomic resolution virus capsid structures determined by cryo-electron microscopy.

    Science.gov (United States)

    Ho, Phuong T; Reddy, Vijay S

    2018-01-01

    The recent technological advances in electron microscopes, detectors, as well as image processing and reconstruction software have brought single particle cryo-electron microscopy (cryo-EM) into prominence for determining structures of bio-molecules at near atomic resolution. This has been particularly true for virus capsids, ribosomes, and other large assemblies, which have been the ideal specimens for structural studies by cryo-EM approaches. An analysis of time series metadata of virus structures on the methods of structure determination, resolution of the structures, and size of the virus particles revealed a rapid increase in the virus structures determined by cryo-EM at near atomic resolution since 2010. In addition, the data highlight the median resolution (∼3.0 Å) and size (∼310.0 Å in diameter) of the virus particles determined by X-ray crystallography while no such limits exist for cryo-EM structures, which have a median diameter of 508 Å. Notably, cryo-EM virus structures in the last four years have a median resolution of 3.9 Å. Taken together with minimal sample requirements, not needing diffraction quality crystals, and being able to achieve similar resolutions of the crystal structures makes cryo-EM the method of choice for current and future virus capsid structure determinations. Copyright © 2017 Elsevier Inc. All rights reserved.

  1. Genetically Thermo-Stabilised, Immunogenic Poliovirus Empty Capsids; a Strategy for Non-replicating Vaccines.

    Directory of Open Access Journals (Sweden)

    Helen Fox

    2017-01-01

    Full Text Available While wild type polio has been nearly eradicated there will be a need to continue immunisation programmes for some time because of the possibility of re-emergence and the existence of long term excreters of poliovirus. All vaccines in current use depend on growth of virus and most of the non-replicating (inactivated vaccines involve wild type viruses known to cause poliomyelitis. The attenuated vaccine strains involved in the eradication programme have been used to develop new inactivated vaccines as production is thought safer. However it is known that the Sabin vaccine strains are genetically unstable and can revert to a virulent transmissible form. A possible solution to the need for virus growth would be to generate empty viral capsids by recombinant technology, but hitherto such particles are so unstable as to be unusable. We report here the genetic manipulation of the virus to generate stable empty capsids for all three serotypes. The particles are shown to be extremely stable and to generate high levels of protective antibodies in animal models.

  2. Production of highly knotted DNA by means of cosmid circularization inside phage capsids

    Directory of Open Access Journals (Sweden)

    Trigueros Sonia

    2007-12-01

    Full Text Available Abstract Background The formation of DNA knots is common during biological transactions. Yet, functional implications of knotted DNA are not fully understood. Moreover, potential applications of DNA molecules condensed by means of knotting remain to be explored. A convenient method to produce abundant highly knotted DNA would be highly valuable for these studies. Results We had previously shown that circularization of the 11.2 kb linear DNA of phage P4 inside its viral capsid generates complex knots by the effect of confinement. We demonstrate here that this mechanism is not restricted to the viral genome. We constructed DNA cosmids as small as 5 kb and introduced them inside P4 capsids. Such cosmids were then recovered as a complex mixture of highly knotted DNA circles. Over 250 μg of knotted cosmid were typically obtained from 1 liter of bacterial culture. Conclusion With this biological system, DNA molecules of varying length and sequence can be shaped into very complex and heterogeneous knotted forms. These molecules can be produced in preparative amounts suitable for systematic studies and applications.

  3. Gaussian fluctuation of the diffusion exponent of virus capsid in a living cell nucleus

    Science.gov (United States)

    Itto, Yuichi

    2018-05-01

    In their work [4], Bosse et al. experimentally showed that virus capsid exhibits not only normal diffusion but also anomalous diffusion in nucleus of a living cell. There, it was found that the distribution of fluctuations of the diffusion exponent characterizing them takes the Gaussian form, which is, quite remarkably, the same form for two different types of the virus. This suggests high robustness of such fluctuations. Here, the statistical property of local fluctuations of the diffusion exponent of the virus capsid in the nucleus is studied. A maximum-entropy-principle approach (originally proposed for a different virus in a different cell) is applied for obtaining the fluctuation distribution of the exponent. Largeness of the number of blocks identified with local areas of interchromatin corrals is also examined based on the experimental data. It is shown that the Gaussian distribution of the local fluctuations can be derived, in accordance with the above form. In addition, it is quantified how the fluctuation distribution on a long time scale is different from the Gaussian distribution.

  4. TensorCalculator: exploring the evolution of mechanical stress in the CCMV capsid

    Science.gov (United States)

    Kononova, Olga; Maksudov, Farkhad; Marx, Kenneth A.; Barsegov, Valeri

    2018-01-01

    A new computational methodology for the accurate numerical calculation of the Cauchy stress tensor, stress invariants, principal stress components, von Mises and Tresca tensors is developed. The methodology is based on the atomic stress approach which permits the calculation of stress tensors, widely used in continuum mechanics modeling of materials properties, using the output from the MD simulations of discrete atomic and C_α -based coarse-grained structural models of biological particles. The methodology mapped into the software package TensorCalculator was successfully applied to the empty cowpea chlorotic mottle virus (CCMV) shell to explore the evolution of mechanical stress in this mechanically-tested specific example of a soft virus capsid. We found an inhomogeneous stress distribution in various portions of the CCMV structure and stress transfer from one portion of the virus structure to another, which also points to the importance of entropic effects, often ignored in finite element analysis and elastic network modeling. We formulate a criterion for elastic deformation using the first principal stress components. Furthermore, we show that von Mises and Tresca stress tensors can be used to predict the onset of a viral capsid’s mechanical failure, which leads to total structural collapse. TensorCalculator can be used to study stress evolution and dynamics of defects in viral capsids and other large-size protein assemblies.

  5. Controlled immobilisation of active enzymes on the cowpea mosaic virus capsid

    Science.gov (United States)

    Aljabali, Alaa A. A.; Barclay, J. Elaine; Steinmetz, Nicole F.; Lomonossoff, George P.; Evans, David J.

    2012-08-01

    Immobilisation of horseradish peroxidase (HRP) and glucose oxidase (GOX) via covalent attachment of modified enzyme carbohydrate to the exterior of the cowpea mosaic virus (CPMV) capsid gave high retention of enzymatic activity. The number of enzymes bound per virus was determined to be about eleven for HRP and 2-3 for GOX. This illustrates that relatively large biomacromolecules can be readily coupled to the virus surface using simple conjugation strategies. Virus-biomacromolecule hybrids have great potential for uses in catalysis, diagnostic assays or biosensors.Immobilisation of horseradish peroxidase (HRP) and glucose oxidase (GOX) via covalent attachment of modified enzyme carbohydrate to the exterior of the cowpea mosaic virus (CPMV) capsid gave high retention of enzymatic activity. The number of enzymes bound per virus was determined to be about eleven for HRP and 2-3 for GOX. This illustrates that relatively large biomacromolecules can be readily coupled to the virus surface using simple conjugation strategies. Virus-biomacromolecule hybrids have great potential for uses in catalysis, diagnostic assays or biosensors. Electronic supplementary information (ESI) available: Alternative conjugation strategies, agarose gel electrophoresis of CPMV and CPMV-HRP conjugates, UV-vis spectrum of HRP-ADHCPMV, agarose gel electrophoresis of GOX-ADHCPMV particles and corresponding TEM image, calibration curves for HRP-ADHCPMV and GOX-ADHCPMV, DLS data for GOX-ADHCPMV are made available. See DOI: 10.1039/c2nr31485a

  6. Cross-serotype immunity induced by immunization with a conserved rhinovirus capsid protein.

    Directory of Open Access Journals (Sweden)

    Nicholas Glanville

    Full Text Available Human rhinovirus (RV infections are the principle cause of common colds and precipitate asthma and COPD exacerbations. There is currently no RV vaccine, largely due to the existence of ∼150 strains. We aimed to define highly conserved areas of the RV proteome and test their usefulness as candidate antigens for a broadly cross-reactive vaccine, using a mouse infection model. Regions of the VP0 (VP4+VP2 capsid protein were identified as having high homology across RVs. Immunization with a recombinant VP0 combined with a Th1 promoting adjuvant induced systemic, antigen specific, cross-serotype, cellular and humoral immune responses. Similar cross-reactive responses were observed in the lungs of immunized mice after infection with heterologous RV strains. Immunization enhanced the generation of heterosubtypic neutralizing antibodies and lung memory T cells, and caused more rapid virus clearance. Conserved domains of the RV capsid therefore induce cross-reactive immune responses and represent candidates for a subunit RV vaccine.

  7. Immune response to pneumococcal polysaccharides 4 and 14 in elderly and young adults. I Antibody concentrations, avidity and functional activity

    Directory of Open Access Journals (Sweden)

    Carlone George M

    2005-06-01

    Full Text Available Abstract Streptococcus pneumoniae is a serious worldwide pathogen and the focus of numerous vaccine development projects. Currently the most widely accepted surrogate marker for evaluating the efficacy of a given vaccine is to utilize ELISA. Measurement of antibody concentration by ELISA without reduction in cross-reactive antibodies causes an overestimation of antibody concentration and therefore protection, this is most notable in the aged, an at risk group for this infection. We compared the immune response to the pneumococcal polysaccharides (PPS 4 and 14 of 20 young to 20 elderly adults. Pre-and post-vaccination IgG antibody concentrations and antibody avidity against PPS4 and PPS14 were measured using two different enzyme-linked immunosorbant assay (ELISA absorption protocols. All sera were pre-absorbed with either cell-wall polysaccharide (CPS, or CPS and serotype 22F polysaccharide. Pre- and post-vaccination IgG antibody concentrations for serotype 4, but not 14, were significantly lowered with the additional absorption with serotype 22F polysaccharide in both age groups. Young and elderly demonstrated a significant increase from pre- to post-immunization antibody concentration, using either absorption method; and opsonophagocytic antibody titers in response to both PPS4 and PPS14. The correlation coefficients between ELISA and opsonophagocytic assays were improved by additional absorption with serotype 22F in response to serotype 4, but not serotype 14 in all age groups. Opsonophagocytic antibody titers in a sub-group of elderly (>77 years of age were significantly lower than the opsonophagocytic antibody concentrations in young adults. These results suggest the importance of eliminating cross-reactive antibodies from ELISA measurements by absorption of serum and an age-related impairment in the antibody response to pneumococcal polysaccharides.

  8. Normal physiologic and Benign foci with F-18 FDG avidity on PET/CT in patients with breast cancer

    Energy Technology Data Exchange (ETDEWEB)

    Park, Soon Ah; Lee, Kwang Man; Choi, Un Jong; Kim, Hun Soo; Kim, Hye Won; Song, Jeong Hoon [College of Medicine, Wonkwnag University, Iksan (Korea, Republic of)

    2010-12-15

    The aim of this study was to evaluate the physiologic and benign F-18 fluorodeoxyglucose (FDG) avid foci in patients with breast cancer. On 309 F-18 FDG PET/CT scans of 241 women with breast cancer, the hypermetabolic lesions compared with the surrounding normal region were evaluated retrospectively. Available reports of other relevant radiological imaging medical records, and follow-up PET/CT were reviewed for explanations of the abnormal uptake. Among the 70 physiologic foci, muscular uptake of the lower neck following the surgical and/or radiation therapy of ipsilateral breast (29%), hypermetabolic ovaries (16%) and uterine (10%) uptake during the ovulatory and menstrual phases during the normal menstrual cycle were identified, and also hypermetabolic brown fat in cold-induced thermogenesis (7%), non-specific bowel uptake (35%) were observed. Among the 147 benign lesions, sequelae of the chest wall and breasts following surgical and/or radiation therapy, were often observed (27%). Hypermetabolic thyroid glands were noted as adenomas and chronic thyroiditis (18%). Reactive hyperplasia of cervical or mediastinal lymph nodes (32%), degenerative osteoarthritis and healed fractures (15%), hypermetabolic benign lung lesions (6%) were observed. Altered physiologic and benign F-18 FDG uptake in the cervical muscle and chest wall following ipsilateral breast surgery or radiotherapy were common, and also normal physiologic uptake in ovary and uterus, brown fat, thyroid were considered as predominant findings in women patients with breast cancer. Knowledge of these findings might aid in the interpretation of FDG PET/CT in patients with breast cancer

  9. Understanding the in vivo uptake kinetics of a phosphatidylethanolamine-binding agent 99mTc-Duramycin

    International Nuclear Information System (INIS)

    Audi, Said; Li Zhixin; Capacete, Joseph; Liu Yu; Fang, Wei; Shu, Laura G.; Zhao Ming

    2012-01-01

    Introduction: 99m Tc-Duramycin is a peptide-based molecular probe that binds specifically to phosphatidylethanolamine (PE). The goal was to characterize the kinetics of molecular interactions between 99m Tc-Duramycin and the target tissue. Methods: High level of accessible PE is induced in cardiac tissues by myocardial ischemia (30 min) and reperfusion (120 min) in Sprague–Dawley rats. Target binding and biodistribution of 99m Tc-duramycin were captured using SPECT/CT. To quantify the binding kinetics, the presence of radioactivity in ischemic versus normal cardiac tissues was measured by gamma counting at 3, 10, 20, 60 and 180 min after injection. A partially inactivated form of 99m Tc-Duramycin was analyzed in the same fashion. A compartment model was developed to quantify the uptake kinetics of 99m Tc-Duramycin in normal and ischemic myocardial tissue. Results: 99m Tc-duramycin binds avidly to the damaged tissue with a high target-to-background radio. Compartment modeling shows that accessibility of binding sites in myocardial tissue to 99m Tc-Duramycin is not a limiting factor and the rate constant of target binding in the target tissue is at 2.2 ml/nmol/min/g. The number of available binding sites for 99m Tc-Duramycin in ischemic myocardium was estimated at 0.14 nmol/g. Covalent modification of D15 resulted in a 9-fold reduction in binding affinity. Conclusion: 99m Tc-Duramycin accumulates avidly in target tissues in a PE-dependent fashion. Model results reflect an efficient uptake mechanism, consistent with the low molecular weight of the radiopharmaceutical and the relatively high density of available binding sites. These data help better define the imaging utilities of 99m Tc-Duramycin as a novel PE-binding agent.

  10. Highly conserved serine residue 40 in HIV-1 p6 regulates capsid processing and virus core assembly

    Directory of Open Access Journals (Sweden)

    Solbak Sara MØ

    2011-02-01

    Full Text Available Abstract Background The HIV-1 p6 Gag protein regulates the final abscission step of nascent virions from the cell membrane by the action of two late assembly (L- domains. Although p6 is located within one of the most polymorphic regions of the HIV-1 gag gene, the 52 amino acid peptide binds at least to two cellular budding factors (Tsg101 and ALIX, is a substrate for phosphorylation, ubiquitination, and sumoylation, and mediates the incorporation of the HIV-1 accessory protein Vpr into viral particles. As expected, known functional domains mostly overlap with several conserved residues in p6. In this study, we investigated the importance of the highly conserved serine residue at position 40, which until now has not been assigned to any known function of p6. Results Consistently with previous data, we found that mutation of Ser-40 has no effect on ALIX mediated rescue of HIV-1 L-domain mutants. However, the only feasible S40F mutation that preserves the overlapping pol open reading frame (ORF reduces virus replication in T-cell lines and in human lymphocyte tissue cultivated ex vivo. Most intriguingly, L-domain mediated virus release is not dependent on the integrity of Ser-40. However, the S40F mutation significantly reduces the specific infectivity of released virions. Further, it was observed that mutation of Ser-40 selectively interferes with the cleavage between capsid (CA and the spacer peptide SP1 in Gag, without affecting cleavage of other Gag products. This deficiency in processing of CA, in consequence, led to an irregular morphology of the virus core and the formation of an electron dense extra core structure. Moreover, the defects induced by the S40F mutation in p6 can be rescued by the A1V mutation in SP1 that generally enhances processing of the CA-SP1 cleavage site. Conclusions Overall, these data support a so far unrecognized function of p6 mediated by Ser-40 that occurs independently of the L-domain function, but selectively

  11. α-Defensin HD5 Inhibits Human Papillomavirus 16 Infection via Capsid Stabilization and Redirection to the Lysosome

    Directory of Open Access Journals (Sweden)

    Mayim E. Wiens

    2017-01-01

    Full Text Available α-Defensins are an important class of abundant innate immune effectors that are potently antiviral against a number of nonenveloped viral pathogens; however, a common mechanism to explain their ability to block infection by these unrelated viruses is lacking. We previously found that human defensin 5 (HD5 blocks a critical host-mediated proteolytic processing step required for human papillomavirus (HPV infection. Here, we show that bypassing the requirement for this cleavage failed to abrogate HD5 inhibition. Instead, HD5 altered HPV trafficking in the cell. In the presence of an inhibitory concentration of HD5, HPV was internalized and reached the early endosome. The internalized capsid became permeable to antibodies and proteases; however, HD5 prevented dissociation of the viral capsid from the genome, reduced viral trafficking to the trans-Golgi network, redirected the incoming viral particle to the lysosome, and accelerated the degradation of internalized capsid proteins. This mechanism is equivalent to the mechanism by which HD5 inhibits human adenovirus. Thus, our data support capsid stabilization and redirection to the lysosome during infection as a general antiviral mechanism of α-defensins against nonenveloped viruses.

  12. RNA packaging of MRFV virus-like particles: The interplay between RNA pools and capsid coat protein

    Science.gov (United States)

    Virus-like particles (VLPs) can be produced through self-assembly of capsid protein (CP) into particles with discrete shapes and sizes and containing different types of RNA molecules. The general principle that governs particle assembly and RNA packaging is determined by unique interactions between ...

  13. Structure of the immature HIV-1 capsid in intact virus particles at 8.8 angstrom resolution

    Czech Academy of Sciences Publication Activity Database

    Schur, F. K. M.; Hagen, W. J. H.; Rumlová, Michaela; Ruml, T.; Müller, B.; Kräusslich, H. G.; Briggs, J. A. G.

    2015-01-01

    Roč. 517, č. 7535 (2015), s. 505-508 ISSN 0028-0836 R&D Projects: GA ČR(CZ) GA14-15326S Institutional support: RVO:61388963 Keywords : retrovirus * HIV * M-PMV * capsid protein * CA * assembly * immature particles Subject RIV: CE - Biochemistry Impact factor: 38.138, year: 2015

  14. Herpes Simplex Virus 1 Us3 Deletion Mutant is Infective Despite Impaired Capsid Translocation to the Cytoplasm

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    Peter Wild

    2015-01-01

    Full Text Available Herpes simplex virus 1 (HSV-1 capsids are assembled in the nucleus bud at the inner nuclear membrane into the perinuclear space, acquiring envelope and tegument. In theory, these virions are de-enveloped by fusion of the envelope with the outer nuclear membrane and re-enveloped by Golgi membranes to become infective. Us3 enables the nucleus to cytoplasm capsid translocation. Nevertheless, Us3 is not essential for the production of infective progeny viruses. Determination of phenotype distribution by quantitative electron microscopy, and calculation per mean nuclear or cell volume revealed the following: (i The number of R7041(∆US3 capsids budding at the inner nuclear membrane was significantly higher than that of wild type HSV-1; (ii The mean number of R7041(∆US3 virions per mean cell volume was 2726, that of HSV-1 virions 1460 by 24 h post inoculation; (iii 98% of R7041(∆US3 virions were in the perinuclear space; (iv The number of R7041(∆US3 capsids in the cytoplasm, including those budding at Golgi membranes, was significantly reduced. Cell associated R7041(∆US3 yields were 2.37 × 108 and HSV-1 yields 1.57 × 108 PFU/mL by 24 h post inoculation. We thus conclude that R7041(∆US3 virions, which acquire envelope and tegument by budding at the inner nuclear membrane into the perinuclear space, are infective.

  15. Sequence Analysis of the Capsid Gene during a Genotype II.4 Dominated Norovirus Season in One University Hospital

    DEFF Research Database (Denmark)

    Holzknecht, Barbara Juliane; Franck, Kristina Træholt; Nielsen, Rikke Thoft

    2015-01-01

    Norovirus (NoV) is a leading cause of gastroenteritis and genotype II.4 (GII.4) is responsible for the majority of nosocomial NoV infections. Our objective was to examine whether sequencing of the capsid gene might be a useful tool for the hospital outbreak investigation to define possible...

  16. Cell culture adaptation mutations in foot-and-mouth disease virus serotype A capsid proteins: implications for receptor interactions

    Science.gov (United States)

    In this study we describe the adaptive changes fixed on the capsid of several foot-and-mouth disease virus serotype A strains during propagation in cell monolayers. Viruses passaged extensively in three cell lines (BHK-21, LFBK and IB-RS-2), consistently gained several positively charged amino acids...

  17. Crystal Structure of the Full-Length Feline Immunodeficiency Virus Capsid Protein Shows an N-Terminal β-Hairpin in the Absence of N-Terminal Proline

    Directory of Open Access Journals (Sweden)

    Christelle Folio

    2017-11-01

    Full Text Available Feline immunodeficiency virus (FIV is a member of the Retroviridae family. It is the causative agent of an acquired immunodeficiency syndrome (AIDS in cats and wild felines. Its capsid protein (CA drives the assembly of the viral particle, which is a critical step in the viral replication cycle. Here, the first atomic structure of full-length FIV CA to 1.67 Å resolution is determined. The crystallized protein exhibits an original tetrameric assembly, composed of dimers which are stabilized by an intermolecular disulfide bridge induced by the crystallogenesis conditions. The FIV CA displays a standard α-helical CA topology with two domains, separated by a linker shorter than other retroviral CAs. The β-hairpin motif at its amino terminal end, which interacts with nucleotides in HIV-1, is unusually long in FIV CA. Interestingly, this functional β-motif is formed in this construct in the absence of the conserved N-terminal proline. The FIV CA exhibits a cis Arg–Pro bond in the CypA-binding loop, which is absent in known structures of lentiviral CAs. This structure represents the first tri-dimensional structure of a functional, full-length FIV CA.

  18. Venture from the Interior-Herpesvirus pUL31 Escorts Capsids from Nucleoplasmic Replication Compartments to Sites of Primary Envelopment at the Inner Nuclear Membrane.

    Science.gov (United States)

    Bailer, Susanne M.

    2017-11-25

    Herpesviral capsid assembly is initiated in the nucleoplasm of the infected cell. Size constraints require that newly formed viral nucleocapsids leave the nucleus by an evolutionarily conserved vescular transport mechanism called nuclear egress. Mature capsids released from the nucleoplasm are engaged in a membrane-mediated budding process, composed of primary envelopment at the inner nuclear membrane and de-envelopment at the outer nuclear membrane. Once in the cytoplasm, the capsids receive their secondary envelope for maturation into infectious virions. Two viral proteins conserved throughout the herpesvirus family, the integral membrane protein pUL34 and the phosphoprotein pUL31, form the nuclear egress complex required for capsid transport from the infected nucleus to the cytoplasm. Formation of the nuclear egress complex results in budding of membrane vesicles revealing its function as minimal virus-encoded membrane budding and scission machinery. The recent structural analysis unraveled details of the heterodimeric nuclear egress complex and the hexagonal coat it forms at the inside of budding vesicles to drive primary envelopment. With this review, I would like to present the capsid-escort-model where pUL31 associates with capsids in nucleoplasmic replication compartments for escort to sites of primary envelopment thereby coupling capsid maturation and nuclear egress.

  19. Atomic force microscopy investigation of Turnip Yellow Mosaic Virus capsid disruption and RNA extrusion

    International Nuclear Information System (INIS)

    Kuznetsov, Yu. G.; McPherson, Alexander

    2006-01-01

    Turnip Yellow Mosaic Virus (TYMV) was subjected to a variety of procedures which disrupted the protein capsids and produced exposure of the ssRNA genome. The results of the treatments were visualized by atomic force microscopy (AFM). Both in situ and ex situ freeze-thawing produced RNA emission, though at low efficiency. The RNA lost from such particles was evident, in some cases in the process of exiting the virions. More severe disruption of TYMV and extrusion of intact RNA onto the substrate were produced by drying the virus and rehydrating with neutral buffer. Similar products were also obtained by heating TYMV to 70-75 deg. C and by exposure to alkaline pH. Experiments showed the nucleic acid to have an elaborate secondary structure distributed linearly along its length

  20. Identification of immunogenic hot spots within plum pox potyvirus capsid protein for efficient antigen presentation.

    Science.gov (United States)

    Fernández-Fernández, M Rosario; Martínez-Torrecuadrada, Jorge L; Roncal, Fernando; Domínguez, Elvira; García, Juan Antonio

    2002-12-01

    PEPSCAN analysis has been used to characterize the immunogenic regions of the capsid protein (CP) in virions of plum pox potyvirus (PPV). In addition to the well-known highly immunogenic N- and C-terminal domains of CP, regions within the core domain of the protein have also shown high immunogenicity. Moreover, the N terminus of CP is not homogeneously immunogenic, alternatively showing regions frequently recognized by antibodies and others that are not recognized at all. These results have helped us to design efficient antigen presentation vectors based on PPV. As predicted by PEPSCAN analysis, a small displacement of the insertion site in a previously constructed vector, PPV-gamma, turned the derived chimeras into efficient immunogens. Vectors expressing foreign peptides at different positions within a highly immunogenic region (amino acids 43 to 52) in the N-terminal domain of CP were the most effective at inducing specific antibody responses against the foreign sequence.

  1. Kinetics of the association of dengue virus capsid protein with the granular component of nucleolus.

    Science.gov (United States)

    Tiwary, Ashish Kumar; Cecilia, D

    2017-02-01

    Dengue virus (DENV) replicates in the cytoplasm but translocation of the capsid protein (C) to the nucleoli of infected cells has been shown to facilitate virus multiplication for DENV-2. This study demonstrates that the nucleolar localization of C occurs with all four serotypes of DENV. The interaction of C with the nucleolus was found to be dynamic with a mobile fraction of 66% by FRAP. That the C shuttled between the nucleus and cytoplasm was suggested by FLIP and translation inhibition experiments. Colocalization with B23 indicated that DENV C targeted the granular component (GC) of the nucleolus. Presence of DENV C in the nucleolus affected the recovery kinetics of B23 in infected and transfected cells. Sub-nucleolar localization of DENV C of all serotypes to the GC, its mobility in and out of the nucleolus and its affect on the dynamics of B23 is being shown for the first time. Copyright © 2016 Elsevier Inc. All rights reserved.

  2. Capsid coding sequences of foot-and-mouth disease viruses are determinants of pathogenicity in pigs

    DEFF Research Database (Denmark)

    Lohse, Louise; Jackson, Terry; Bøtner, Anette

    2012-01-01

    The surface exposed capsid proteins, VP1, VP2 and VP3, of foot-and-mouth disease virus (FMDV) determine its antigenicity and the ability of the virus to interact with host-cell receptors. Hence, modification of these structural proteins may alter the properties of the virus. In the present study we...... compared the pathogenicity of different FMDVs in young pigs. In total 32 pigs, 7-weeks-old, were exposed to virus, either by direct inoculation or through contact with inoculated pigs, using cell culture adapted (O1K B64), chimeric (O1K/A-TUR and O1K/O-UKG) or field strain (O-UKG/34/2001) viruses. The O1K...... coding sequences are determinants of FMDV pathogenicity in pigs....

  3. The Polerovirus Minor Capsid Protein Determines Vector Specificity and Intestinal Tropism in the Aphid

    Science.gov (United States)

    Brault, Véronique; Périgon, Sophie; Reinbold, Catherine; Erdinger, Monique; Scheidecker, Danièle; Herrbach, Etienne; Richards, Ken; Ziegler-Graff, Véronique

    2005-01-01

    Aphid transmission of poleroviruses is highly specific, but the viral determinants governing this specificity are unknown. We used a gene exchange strategy between two poleroviruses with different vectors, Beet western yellows virus (BWYV) and Cucurbit aphid-borne yellows virus (CABYV), to analyze the role of the major and minor capsid proteins in vector specificity. Virus recombinants obtained by exchanging the sequence of the readthrough domain (RTD) between the two viruses replicated in plant protoplasts and in whole plants. The hybrid readthrough protein of chimeric viruses was incorporated into virions. Aphid transmission experiments using infected plants or purified virions revealed that vector specificity is driven by the nature of the RTD. BWYV and CABYV have specific intestinal sites in the vectors for endocytosis: the midgut for BWYV and both midgut and hindgut for CABYV. Localization of hybrid virions in aphids by transmission electron microscopy revealed that gut tropism is also determined by the viral origin of the RTD. PMID:16014930

  4. Tyrosine Mutation in AAV9 Capsid Improves Gene Transfer to the Mouse Lung.

    Science.gov (United States)

    Martini, Sabrina V; Silva, Adriana L; Ferreira, Debora; Rabelo, Rafael; Ornellas, Felipe M; Gomes, Karina; Rocco, Patricia R M; Petrs-Silva, Hilda; Morales, Marcelo M

    2016-01-01

    Adeno-associated virus (AAV) vectors are being increasingly used as the vector of choice for in vivo gene delivery and gene therapy for many pulmonary diseases. Recently, it was shown that phosphorylation of surface-exposed tyrosine residues from AAV capsid targets the viral particles for ubiquitination and proteasome-mediated degradation, and mutations of these tyrosine residues lead to highly efficient vector transduction in vitro and in vivo in different organs. In this study, we evaluated the pulmonary transgene expression efficacy of AAV9 vectors containing point mutations in surface-exposed capsid tyrosine residues. Eighteen C57BL/6 mice were randomly assigned into three groups: (1) a control group (CTRL) animals underwent intratracheal (i.t.) instillation of saline, (2) the wild-type AAV9 group (WT-AAV9, 1010 vg), and (3) the tyrosine-mutant Y731F AAV9 group (M-AAV9, 1010 vg), which received (i.t.) self-complementary AAV9 vectors containing the DNA sequence of enhanced green fluorescence protein (eGFP). Four weeks after instillation, lung mechanics, morphometry, tissue cellularity, gene expression, inflammatory cytokines, and growth factor expression were analyzed. No significant differences were observed in lung mechanics and morphometry among the experimental groups. However, the number of polymorphonuclear cells was higher in the WT-AAV9 group than in the CTRL and M-AAV9 groups, suggesting that the administration of tyrosine-mutant AAV9 vectors was better tolerated. Tyrosine-mutant AAV9 vectors significantly improved transgene delivery to the lung (30%) compared with their wild-type counterparts, without eliciting an inflammatory response. Our results provide the impetus for further studies to exploit the use of AAV9 vectors as a tool for pulmonary gene therapy. © 2016 The Author(s) Published by S. Karger AG, Basel.

  5. SEROPREVALANCE OF TOXOPLASMA IgG AMONG PREGNANT WOMEN IN THE PROVINCE OF HATAY AND CONTRIBUTION OF AVIDITY TEST TO THE DIAGNOSE

    Directory of Open Access Journals (Sweden)

    Ayşe Güler Okyay

    2013-09-01

    Full Text Available Objective: Toxoplasma gondii, an obligate intracellular parasite, is a common zoonosis in the world and causes toxoplasmosis. The infection is usually asymptomatic and if it occurs during pregnancy it may cause congenital anomalies, miscarriage, stillbirth and premature delivery. In this study, determination of seroprevalance of Toxoplazma IgG and IgM among pregnant women in the province of Hatay was aimed. In addition, IgG avidity test was performed in subjects who had positive test results for both of the two antibodies. Design: In this study, 3340 pregnant women who admitted to the outpatient clinic of gynecelogy and obstetrics department of Mustafa Kemal University/Hatay between the years of 2007 and 2012were included. Toxoplazma IgM and IgG antibody test results of the subjects were examined retrospectively by screening the records of microbiology laboratory of the hospital. Additionally, IgG avidity test was performed in 114 cases whoose test results for both Toxoplazma IgM and IgG were positive. Main outcome measures: IgM and IgG seropositivity rates were found as 3.6% (n = 120 and 57% (n = 1910, respectively. The rate of new infection was detected with avidity test as 31%. Conclusions: Remarkably high seropositivity rate was observed in the Mediterranean region especially in the province of Hatay. Antibody testing for Toxoplasma gondii may be useful during routine examinations of women who are in the first trimester of pregnancy and who plan gestation. In addition, measures should be taken to raise awareness of hygiene in our region in terms of water and sanitation.

  6. Vesicular monoamine transporter protein expression correlates with clinical features, tumor biology, and MIBG avidity in neuroblastoma: a report from the Children's Oncology Group

    International Nuclear Information System (INIS)

    Temple, William; Mendelsohn, Lori; Nekritz, Erin; Gustafson, W.C.; Matthay, Katherine K.; Kim, Grace E.; Lin, Lawrence; Giacomini, Kathy; Naranjo, Arlene; Van Ryn, Collin; Yanik, Gregory A.; Kreissman, Susan G.; Hogarty, Michael; DuBois, Steven G.

    2016-01-01

    Vesicular monoamine transporters 1 and 2 (VMAT1 and VMAT2) are thought to mediate MIBG uptake in adult neuroendocrine tumors. In neuroblastoma, the norepinephrine transporter (NET) has been investigated as the principal MIBG uptake protein, though some tumors without NET expression concentrate MIBG. We investigated VMAT expression in neuroblastoma and correlated expression with MIBG uptake and clinical features. We evaluated VMAT1 and VMAT2 expression by immunohistochemistry (IHC) in neuroblastoma tumors from 76 patients with high-risk metastatic disease treated in a uniform cooperative group trial (COG A3973). All patients had baseline MIBG diagnostic scans centrally reviewed. IHC results were scored as the product of intensity grading (0 - 3+) and percent of tumor cells expressing the protein of interest. The association between VMAT1 and VMAT2 scores and clinical and biological features was tested using Wilcoxon rank-sum tests. Patient characteristics were typical of high-risk neuroblastoma, though the cohort was intentionally enriched in patients with MIBG-nonavid tumors (n = 20). VMAT1 and VMAT2 were expressed in 62 % and 75 % of neuroblastoma tumors, respectively. VMAT1 and VMAT2 scores were both significantly lower in MYCN amplified tumors and in tumors with high mitotic karyorrhectic index. MIBG-avid tumors had significantly higher VMAT2 scores than MIBG-nonavid tumors (median 216 vs. 45; p = 0.04). VMAT1 expression did not correlate with MIBG avidity. VMAT1 and VMAT2 are expressed in the majority of neuroblastomas. Expression correlates with other biological features. The expression level of VMAT2 but not that of VMAT1 correlates with avidity for MIBG. (orig.)

  7. Vesicular monoamine transporter protein expression correlates with clinical features, tumor biology, and MIBG avidity in neuroblastoma: a report from the Children's Oncology Group

    Energy Technology Data Exchange (ETDEWEB)

    Temple, William; Mendelsohn, Lori; Nekritz, Erin; Gustafson, W.C.; Matthay, Katherine K. [UCSF School of Medicine, Department of Pediatrics, San Francisco, CA (United States); UCSF Benioff Children' s Hospital, San Francisco, CA (United States); Kim, Grace E. [UCSF School of Medicine, Department of Pathology, San Francisco, CA (United States); Lin, Lawrence; Giacomini, Kathy [UCSF School of Pharmacy, Department of Bioengineering and Therapeutic Sciences, San Francisco, CA (United States); Naranjo, Arlene; Van Ryn, Collin [University of Florida, Children' s Oncology Group Statistics and Data Center, Gainesville, FL (United States); Yanik, Gregory A. [University of Michigan, CS Mott Children' s Hospital, Ann Arbor, MI (United States); Kreissman, Susan G. [Duke University Medical Center, Durham, NC (United States); Hogarty, Michael [University of Pennsylvania, Children' s Hospital of Philadelphia and Perelman School of Medicine, Philadelphia, PA (United States); DuBois, Steven G. [UCSF School of Medicine, Department of Pediatrics, San Francisco, CA (United States); UCSF Benioff Children' s Hospital, San Francisco, CA (United States); UCSF School of Medicine, San Francisco, CA (United States)

    2016-03-15

    Vesicular monoamine transporters 1 and 2 (VMAT1 and VMAT2) are thought to mediate MIBG uptake in adult neuroendocrine tumors. In neuroblastoma, the norepinephrine transporter (NET) has been investigated as the principal MIBG uptake protein, though some tumors without NET expression concentrate MIBG. We investigated VMAT expression in neuroblastoma and correlated expression with MIBG uptake and clinical features. We evaluated VMAT1 and VMAT2 expression by immunohistochemistry (IHC) in neuroblastoma tumors from 76 patients with high-risk metastatic disease treated in a uniform cooperative group trial (COG A3973). All patients had baseline MIBG diagnostic scans centrally reviewed. IHC results were scored as the product of intensity grading (0 - 3+) and percent of tumor cells expressing the protein of interest. The association between VMAT1 and VMAT2 scores and clinical and biological features was tested using Wilcoxon rank-sum tests. Patient characteristics were typical of high-risk neuroblastoma, though the cohort was intentionally enriched in patients with MIBG-nonavid tumors (n = 20). VMAT1 and VMAT2 were expressed in 62 % and 75 % of neuroblastoma tumors, respectively. VMAT1 and VMAT2 scores were both significantly lower in MYCN amplified tumors and in tumors with high mitotic karyorrhectic index. MIBG-avid tumors had significantly higher VMAT2 scores than MIBG-nonavid tumors (median 216 vs. 45; p = 0.04). VMAT1 expression did not correlate with MIBG avidity. VMAT1 and VMAT2 are expressed in the majority of neuroblastomas. Expression correlates with other biological features. The expression level of VMAT2 but not that of VMAT1 correlates with avidity for MIBG. (orig.)

  8. Allelic Variation in KIR2DL3 Generates a KIR2DL2-like Receptor with Increased Binding to Its HLA-C Ligand12

    OpenAIRE

    Frazier, William R.; Steiner, Noriko; Hou, Lihua; Dakshanamurthy, Sivanesan; Hurley, Carolyn Katovich

    2013-01-01

    Although extensive homology exists between their extracellular domains, natural killer cell inhibitory receptors KIR2DL2*001 and KIR2DL3*001 have previously been shown to differ substantially in their HLA-C binding avidity. To explore the largely uncharacterized impact of allelic diversity, the most common KIR2DL2/3 allelic products in European American and African American populations were evaluated for surface expression and binding affinity to their HLA-C group 1 and 2 ligands. Although no...

  9. Molecular characterization of genome segments 1 and 3 encoding two capsid proteins of Antheraea mylitta cytoplasmic polyhedrosis virus

    Directory of Open Access Journals (Sweden)

    Chakrabarti Mrinmay

    2010-08-01

    Full Text Available Abstract Background Antheraea mylitta cytoplasmic polyhedrosis virus (AmCPV, a cypovirus of Reoviridae family, infects Indian non-mulberry silkworm, Antheraea mylitta, and contains 11 segmented double stranded RNA (S1-S11 in its genome. Some of its genome segments (S2 and S6-S11 have been previously characterized but genome segments encoding viral capsid have not been characterized. Results In this study genome segments 1 (S1 and 3 (S3 of AmCPV were converted to cDNA, cloned and sequenced. S1 consisted of 3852 nucleotides, with one long ORF of 3735 nucleotides and could encode a protein of 1245 amino acids with molecular mass of ~141 kDa. Similarly, S3 consisted of 3784 nucleotides having a long ORF of 3630 nucleotides and could encode a protein of 1210 amino acids with molecular mass of ~137 kDa. BLAST analysis showed 20-22% homology of S1 and S3 sequence with spike and capsid proteins, respectively, of other closely related cypoviruses like Bombyx mori CPV (BmCPV, Lymantria dispar CPV (LdCPV, and Dendrolimus punctatus CPV (DpCPV. The ORFs of S1 and S3 were expressed as 141 kDa and 137 kDa insoluble His-tagged fusion proteins, respectively, in Escherichia coli M15 cells via pQE-30 vector, purified through Ni-NTA chromatography and polyclonal antibodies were raised. Immunoblot analysis of purified polyhedra, virion particles and virus infected mid-gut cells with the raised anti-p137 and anti-p141 antibodies showed specific immunoreactive bands and suggest that S1 and S3 may code for viral structural proteins. Expression of S1 and S3 ORFs in insect cells via baculovirus recombinants showed to produce viral like particles (VLPs by transmission electron microscopy. Immunogold staining showed that S3 encoded proteins self assembled to form viral outer capsid and VLPs maintained their stability at different pH in presence of S1 encoded protein. Conclusion Our results of cloning, sequencing and functional analysis of AmCPV S1 and S3 indicate that S3

  10. The Chikungunya Virus Capsid Protein Contains Linear B Cell Epitopes in the N- and C-Terminal Regions that are Dependent on an Intact C-Terminus for Antibody Recognition

    Directory of Open Access Journals (Sweden)

    Lucas Y. H. Goh

    2015-06-01

    Full Text Available Chikungunya virus (CHIKV is an arthropod-borne agent that causes severe arthritic disease in humans and is considered a serious health threat in areas where competent mosquito vectors are prevalent. CHIKV has recently been responsible for several millions of cases of disease, involving over 40 countries. The recent re-emergence of CHIKV and its potential threat to human health has stimulated interest in better understanding of the biology and pathogenesis of the virus, and requirement for improved treatment, prevention and control measures. In this study, we mapped the binding sites of a panel of eleven monoclonal antibodies (mAbs previously generated towards the capsid protein (CP of CHIKV. Using N- and C-terminally truncated recombinant forms of the CHIKV CP, two putative binding regions, between residues 1–35 and 140–210, were identified. Competitive binding also revealed that five of the CP-specific mAbs recognized a series of overlapping epitopes in the latter domain. We also identified a smaller, N-terminally truncated product of native CP that may represent an alternative translation product of the CHIKV 26S RNA and have potential functional significance during CHIKV replication. Our data also provides evidence that the C-terminus of CP is required for authentic antigenic structure of CP. This study shows that these anti-CP mAbs will be valuable research tools for further investigating the structure and function of the CHIKV CP.

  11. Assessment of the diagnostic performance of the IDS-iSYS tests for toxo IgG, toxo IgM and avidity.

    Science.gov (United States)

    Levigne, Pauline; Peyron, François; Wallon, Martine

    2016-10-01

    When acquired during pregnancy toxoplasmosis can have devastating consequences on the fetus. As maternal infection is in the majority of cases subclinical, the diagnosis of toxoplasmosis relies on serological tests for the detection of IgG, IGM and the mesure of IgG avidity. We evaluated the performance of IDS-iSYS a new automatized instrument based on chemiluminescence for the diagnosis of the disease. Our study was based on non-selected samples received in our laboratory either for the determination of serological status or for distinguishing acute from chronic infection. Seven hundred eighty three samples were enrolled in the study. Compared with Architect IgG and IgM assays, the sensitivity and specificity were respectively 99% and 99% for IgG, and 75% and 97% for IgM. We observed higher remaining titers for IDS iSYS IgG, which could reduce the proportions of patients who have to be retested because of doubtful titers. IgM detection and avidity scored equivalent performance with both methods. This automate appears to be a reliable and easy-to-use tool for diagnosing toxoplasmosis in different clinical settings. Copyright © 2016 Elsevier Inc. All rights reserved.

  12. The quantum chemical causality of pMHC-TCR biological avidity: Peptide atomic coordination data and the electronic state of agonist N termini

    Directory of Open Access Journals (Sweden)

    Georgios S.E. Antipas

    2015-06-01

    Full Text Available The quantum state of functional avidity of the synapse formed between a peptide-Major Histocompatibility Complex (pMHC and a T cell receptor (TCR is a subject not previously touched upon. Here we present atomic pair correlation meta-data based on crystalized tertiary structures of the Tax (HTLV-1 peptide along with three artificially altered variants, all of which were presented by the (Class I HLA-A201 protein in complexation with the human (CD8+ A6TCR. The meta-data reveal the existence of a direct relationship between pMHC-TCR functional avidity (agonist/antagonist and peptide pair distribution function (PDF. In this context, antagonist peptides are consistently under-coordinated in respect to Tax. Moreover, Density Functional Theory (DFT datasets in the BLYP/TZ2P level of theory resulting from relaxation of the H species on peptide tertiary structures reveal that the coordination requirement of agonist peptides is also expressed as a physical observable of the protonation state of their N termini: agonistic peptides are always found to retain a stable ammonium (NH3+ terminal group while antagonist peptides are not.

  13. Magnetic resonance imaging after radiofrequency ablation in a rodent model of liver tumor: tissue characterization using a novel necrosis-avid contrast agent

    International Nuclear Information System (INIS)

    Ni, Yicheng; Yu, Jie; Marchal, Guy; Chen, Feng; Mulier, Stefaan; Sun, Xihe; Landuyt, Willy; Verbruggen, Alfons

    2006-01-01

    We exploited a necrosis-avid contrast agent ECIV-7 for magnetic resonance imaging (MRI) in rodent liver tumors after radiofrequency ablation (RFA). Rats bearing liver rhabdomyosarcoma (R1) were randomly allocated to three groups: group I, complete RFA, group II, incomplete RFA, and group III, sham ablation. Within 24 h after RFA, T1-weighted (T1-w) MRI was performed before and after injection of ECIV-7 at 0.05 mmol/kg and followed up from 6-24 h. Signal intensities (SIs) were measured with relative enhancement (RE) and contrast ratio (CR) calculated. The MRI findings were verified histomorphologically. On plain T1-w MRI the contrasts between normal liver, RFA lesion, residual and/or intact tumor were vague. Early after administration of ECIV-7, the liver SI was strongly enhanced (RE=40-50%), leaving the RFA lesion as a hypointense region in groups I and II. At delayed phase, two striking peri-ablational enhancement patterns appeared (RE=90% and CR=1.89%), i.e., ''O'' type of hyperintense rim in group I and ''C'' type of incomplete rim in group II. These MRI manifestations could be proven histologically. In this study, tissue components after RFA could be characterized with discernable contrasts by necrosis-avid contrast agent (NACA)-enhanced MRI, especially at delayed phase. This approach may prove useful for defining the ablated area and identifying residual tumor after RFA. (orig.)

  14. Sugar-binding sites of the HA1 subcomponent of Clostridium botulinum type C progenitor toxin.

    Science.gov (United States)

    Nakamura, Toshio; Tonozuka, Takashi; Ide, Azusa; Yuzawa, Takayuki; Oguma, Keiji; Nishikawa, Atsushi

    2008-02-22

    Clostridium botulinum type C 16S progenitor toxin contains a hemagglutinin (HA) subcomponent, designated HA1, which appears to play an important role in the effective internalization of the toxin in gastrointestinal epithelial cells and in creating a broad specificity for the oligosaccharide structure that corresponds to various targets. In this study, using the recombinant protein fused to glutathione S-transferase, we investigated the binding specificity of the HA1 subcomponent to sugars and estimated the binding sites of HA1 based on X-ray crystallography and soaking experiments using various sugars. N-Acetylneuraminic acid, N-acetylgalactosamine, and galactose effectively inhibited the binding that occurs between glutathione S-transferase-HA1 and mucins, whereas N-acetylglucosamine and glucose did not inhibit it. The crystal structures of HA1 complex with N-acetylneuraminic acid, N-acetylgalactosamine, and galactose were also determined. There are two sugar-binding sites, sites I and II. Site I corresponds to the electron densities noted for all sugars and is located at the C-terminal beta-trefoil domain, while site II corresponds to the electron densities noted only for galactose. An aromatic amino acid residue, Trp176, at site I has a stacking interaction with the hexose ring of the sugars. On the other hand, there is no aromatic residue at site II; thus, the interaction with galactose seems to be poor. The double mutant W176A at site I and D271F at site II has no avidity for N-acetylneuraminic acid but has avidity for galactose. In this report, the binding specificity of botulinum C16S toxin HA1 to various sugars is demonstrated based on its structural features.

  15. Random Insertion of mCherry Into VP3 Domain of Adeno-associated Virus Yields Fluorescent Capsids With no Loss of Infectivity

    Directory of Open Access Journals (Sweden)

    Justin Judd

    2012-01-01

    Full Text Available Adeno-associated virus (AAV-derived vectors are promising gene delivery systems, and a number of design strategies have been pursued to improve their performance. For example, genetic insertion of proteins into the capsid may be used to achieve vector retargeting, reduced immunogenicity, or to track vector transport. Unfortunately, rational approaches to genetic insertion have experienced limited success due to the unpredictable context-dependent nature of protein folding and the complexity of the capsid's macroassembly. We report the construction and use of a frame-enriched DNase-based random insertion library based on AAV2 cap, called pAAV2_RaPID (Random Peptide Insertion by DNase. The fluorescent mCherry protein was inserted randomly throughout the AAV2 capsid and the library was selected for fluorescent and infectious variants. A capsid site was identified in VP3 that can tolerate the large protein insertion. In contrast to previous efforts to incorporate fluorescent proteins into the AAV2 capsid, the isolated mCherry mutant maintains native infectivity while displaying robust fluorescence. Collectively, these results demonstrate that the pAAV2_RaPID platform library can be used to create fully infectious AAV vectors carrying large functional protein domains on the capsid.

  16. Dengue Virus Uses a Non-Canonical Function of the Host GBF1-Arf-COPI System for Capsid Protein Accumulation on Lipid Droplets.

    Science.gov (United States)

    Iglesias, Nestor G; Mondotte, Juan A; Byk, Laura A; De Maio, Federico A; Samsa, Marcelo M; Alvarez, Cecilia; Gamarnik, Andrea V

    2015-09-01

    Dengue viruses cause the most important human viral disease transmitted by mosquitoes. In recent years, a great deal has been learned about molecular details of dengue virus genome replication; however, little is known about genome encapsidation and the functions of the viral capsid protein. During infection, dengue virus capsid progressively accumulates around lipid droplets (LDs) by an unknown mechanism. Here, we examined the process by which the viral capsid is transported from the endoplasmic reticulum (ER) membrane, where the protein is synthesized, to LDs. Using different methods of intervention, we found that the GBF1-Arf1/Arf4-COPI pathway is necessary for capsid transport to LDs, while the process is independent of both COPII components and Golgi integrity. The transport was sensitive to Brefeldin A, while a drug resistant form of GBF1 was sufficient to restore capsid subcellular distribution in infected cells. The mechanism by which LDs gain or lose proteins is still an open question. Our results support a model in which the virus uses a non-canonical function of the COPI system for capsid accumulation on LDs, providing new ideas for antiviral strategies. © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  17. Structural basis for the development of avian virus capsids that display influenza virus proteins and induce protective immunity.

    Science.gov (United States)

    Pascual, Elena; Mata, Carlos P; Gómez-Blanco, Josué; Moreno, Noelia; Bárcena, Juan; Blanco, Esther; Rodríguez-Frandsen, Ariel; Nieto, Amelia; Carrascosa, José L; Castón, José R

    2015-03-01

    Bioengineering of viruses and virus-like particles (VLPs) is a well-established approach in the development of new and improved vaccines against viral and bacterial pathogens. We report here that the capsid of a major avian pathogen, infectious bursal disease virus (IBDV), can accommodate heterologous proteins to induce protective immunity. The structural units of the ~70-nm-diameter T=13 IBDV capsid are trimers of VP2, which is made as a precursor (pVP2). The pVP2 C-terminal domain has an amphipathic α helix that controls VP2 polymorphism. In the absence of the VP3 scaffolding protein, 466-residue pVP2 intermediates bearing this α helix assemble into genuine VLPs only when expressed with an N-terminal His6 tag (the HT-VP2-466 protein). HT-VP2-466 capsids are optimal for protein insertion, as they are large enough (cargo space, ~78,000 nm(3)) and are assembled from a single protein. We explored HT-VP2-466-based chimeric capsids initially using enhanced green fluorescent protein (EGFP). The VLP assembly yield was efficient when we coexpressed EGFP-HT-VP2-466 and HT-VP2-466 from two recombinant baculoviruses. The native EGFP structure (~240 copies/virion) was successfully inserted in a functional form, as VLPs were fluorescent, and three-dimensional cryo-electron microscopy showed that the EGFP molecules incorporated at the inner capsid surface. Immunization of mice with purified EGFP-VLPs elicited anti-EGFP antibodies. We also inserted hemagglutinin (HA) and matrix (M2) protein epitopes derived from the mouse-adapted A/PR/8/34 influenza virus and engineered several HA- and M2-derived chimeric capsids. Mice immunized with VLPs containing the HA stalk, an M2 fragment, or both antigens developed full protection against viral challenge. Virus-like particles (VLPs) are multimeric protein cages that mimic the infectious virus capsid and are potential candidates as nonliving vaccines that induce long-lasting protection. Chimeric VLPs can display or include foreign

  18. Viable adenovirus vaccine prototypes: High-level production of a papillomavirus capsid antigen from the major late transcriptional unit

    OpenAIRE

    Berg, Michael; DiFatta, Julie; Hoiczyk, Egbert; Schlegel, Richard; Ketner, Gary

    2005-01-01

    Safe, effective, orally delivered, live adenovirus vaccines have been in use for three decades. Recombinant derivatives of the live adenovirus vaccines may prove an economical alternative to current vaccines for a variety of diseases. To explore that possibility, we constructed a series of recombinants that express the major capsid protein (L1) of canine oral papillomavirus (COPV), a model for mucosal human papillomavirus (HPV) infection. Vaccination with virus-like particles (VLPs) composed ...

  19. Portal protein functions akin to a DNA-sensor that couples genome-packaging to icosahedral capsid maturation

    OpenAIRE

    Lokareddy, Ravi K.; Sankhala, Rajeshwer S.; Roy, Ankoor; Afonine, Pavel V.; Motwani, Tina; Teschke, Carolyn M.; Parent, Kristin N.; Cingolani, Gino

    2017-01-01

    Tailed bacteriophages and herpesviruses assemble infectious particles via an empty precursor capsid (or ?procapsid') built by multiple copies of coat and scaffolding protein and by one dodecameric portal protein. Genome packaging triggers rearrangement of the coat protein and release of scaffolding protein, resulting in dramatic procapsid lattice expansion. Here, we provide structural evidence that the portal protein of the bacteriophage P22 exists in two distinct dodecameric conformations: a...

  20. Protection against myxomatosis and rabbit viral hemorrhagic disease with recombinant myxoma viruses expressing rabbit hemorrhagic disease virus capsid protein

    OpenAIRE

    Bertagnoli, Stéphane; Gelfi, Jacqueline; Le Gall, Ghislaine; Boilletot, Eric; Vautherot, Jean-François; Rasschaert, Denis; Laurent, Sylvie; Petit, Frédérique; Boucraut-Baralon, Corine; Milon, Alain

    1996-01-01

    Two myxoma virus-rabbit hemorrhagic disease virus (RHDV) recombinant viruses were constructed with the SG33 strain of myxoma virus to protect rabbits against myxomatosis and rabbit viral hemorrhagic disease. These recombinant viruses expressed the RHDV capsid protein (VP60). The recombinant protein, which is 60 kDa in size, was antigenic, as revealed by its reaction in immunoprecipitation with antibodies raised against RHDV. Both recombinant viruses induced high levels of RHDV- and myxoma vir...

  1. Detention of HPV L1 Capsid Protein and hTERC Gene in Screening of Cervical Cancer

    Directory of Open Access Journals (Sweden)

    Huang Bin

    2013-06-01

    Full Text Available   Objective(s: To investigate the expression of human papilloma virus (HPV L1 capsid protein, and human telomerase RNA component (hTERC in cervical cancer and the role of detection of both genes in screening of cervical cancer.   Materials and Methods: A total of 309 patients were recruited and cervical exfoliated cells were collected. Immunocytochemistry was employed to detect HPV L1 capsid protein, and fluorescent in situ hybridization (FISH was performed to detect the hTERC. Results: The expression of HPV L1 capsid protein reduced with the increase of the histological grade of cervical cells and was negatively related to the grade of cervical lesions. However, the expression of hTERC increased with the increase of the histological grade and positively associated with the grade of cervical lesions. The proportion of patients with L1(-/hTERC(+ was higher in patients with histological grade of CIN2 or higher than that in those with histological grade of CIN1. The L1(+/hTERC(- and L1(-/hTERC(- were negatively related to the grade of cervical lesions. L1(-/hTERC(+ was positively associated with the grade of cervical lesions. The L1/hTERC ratio increased. The negative predictive value of both HPV L1 and hTERC was higher than that of HPV L1 or hTERC, but there was no marked difference in the screening efficacy of cervical cancer among HPV L1, hTERC and HPV L1+hTERC. Conclusion: HPV L1 capsid protein and hTERC gene may serve as markers for the early diagnosis and prediction of cervical lesions. The increase in L1/hTERC ratio reflects the progression of cervical lesions to a certain extent.

  2. Minor Capsid Protein L2 Polytope Induces Broad Protection against Oncogenic and Mucosal Human Papillomaviruses.

    Science.gov (United States)

    Pouyanfard, Somayeh; Spagnoli, Gloria; Bulli, Lorenzo; Balz, Kathrin; Yang, Fan; Odenwald, Caroline; Seitz, Hanna; Mariz, Filipe C; Bolchi, Angelo; Ottonello, Simone; Müller, Martin

    2018-02-15

    The amino terminus of the human papillomavirus (HPV) minor capsid protein L2 contains a major cross-neutralization epitope which provides the basis for the development of a broadly protecting HPV vaccine. A wide range of protection against different HPV types would eliminate one of the major drawbacks of the commercial, L1-based prophylactic vaccines. Previously, we have reported that insertion of the L2 epitope into a scaffold composed of bacterial thioredoxin protein generates a potent antigen inducing comprehensive protection against different animal and human papillomaviruses. We also reported, however, that although protection is broad, some oncogenic HPV types escape the neutralizing antibody response, if L2 epitopes from single HPV types are used as immunogen. We were able to compensate for this by applying a mix of thioredoxin proteins carrying L2 epitopes from HPV16, -31, and -51. As the development of a cost-efficient HPV prophylactic vaccines is one of our objectives, this approach is not feasible as it requires the development of multiple good manufacturing production processes in combination with a complex vaccine formulation. Here, we report the development of a thermostable thioredoxin-based single-peptide vaccine carrying an L2 polytope of up to 11 different HPV types. The L2 polytope antigens have excellent abilities in respect to broadness of protection and robustness of induced immune responses. To further increase immunogenicity, we fused the thioredoxin L2 polytope antigen with a heptamerization domain. In the final vaccine design, we achieve protective responses against all 14 oncogenic HPV types that we have analyzed plus the low-risk HPVs 6 and 11 and a number of cutaneous HPVs. IMPORTANCE Infections by a large number of human papillomaviruses lead to malignant and nonmalignant disease. Current commercial vaccines based on virus-like particles (VLPs) effectively protect against some HPV types but fail to do so for most others. Further, only

  3. Foot-and-mouth disease virus capsid proteins; analysis of protein processing, assembly and utility as vaccines

    DEFF Research Database (Denmark)

    Belsham, Graham

    Foot-and-mouth disease (FMD) remains one of the most economically important infectious diseases of production animals globally. The infection is caused by foot-and-mouth disease virus (FMDV), a member of the picornavirus family. The positive sense RNA genome of the virus includes a single, large......, open reading frame that encodes a polyprotein. The intact polyprotein is never observed as it is processed, both during and after translation, to 15 different mature proteins plus a variety of precursors. The FMDV capsid protein precursor, P1-2A, is cleaved by the virus encoded 3C protease (3Cpro......) to generate VP0, VP3, VP1 and the peptide 2A. Sixty copies of each of the capsid proteins “self-assemble” into empty capsid particles or with the RNA genome into infectious viruses. These particles normally lack 2A but it is possible to construct and isolate mutant FMDVs in which the cleavage of the VP1/2A...

  4. Plum pox virus capsid protein suppresses plant pathogen-associated molecular pattern (PAMP)-triggered immunity.

    Science.gov (United States)

    Nicaise, Valerie; Candresse, Thierry

    2017-08-01

    The perception of pathogen-associated molecular patterns (PAMPs) by immune receptors launches defence mechanisms referred to as PAMP-triggered immunity (PTI). Successful pathogens must suppress PTI pathways via the action of effectors to efficiently colonize their hosts. So far, plant PTI has been reported to be active against most classes of pathogens, except viruses, although this defence layer has been hypothesized recently as an active part of antiviral immunity which needs to be suppressed by viruses for infection success. Here, we report that Arabidopsis PTI genes are regulated upon infection by viruses and contribute to plant resistance to Plum pox virus (PPV). Our experiments further show that PPV suppresses two early PTI responses, the oxidative burst and marker gene expression, during Arabidopsis infection. In planta expression of PPV capsid protein (CP) was found to strongly impair these responses in Nicotiana benthamiana and Arabidopsis, revealing its PTI suppressor activity. In summary, we provide the first clear evidence that plant viruses acquired the ability to suppress PTI mechanisms via the action of effectors, highlighting a novel strategy employed by viruses to escape plant defences. © 2016 BSPP AND JOHN WILEY & SONS LTD.

  5. A novel inhibitor of dengue virus replication that targets the capsid protein.

    Science.gov (United States)

    Byrd, Chelsea M; Dai, Dongcheng; Grosenbach, Douglas W; Berhanu, Aklile; Jones, Kevin F; Cardwell, Kara B; Schneider, Christine; Wineinger, Kristin A; Page, Jessica M; Harver, Chris; Stavale, Eric; Tyavanagimatt, Shanthakumar; Stone, Melialani A; Bartenschlager, Ralf; Scaturro, Pietro; Hruby, Dennis E; Jordan, Robert

    2013-01-01

    Dengue viruses (DENV) infect 50 to 100 million people worldwide per year, of which 500,000 develop severe life-threatening disease. This mosquito-borne illness is endemic in most tropical and subtropical countries and has spread significantly over the last decade. While there are several promising vaccine candidates in clinical trials, there are currently no approved vaccines or therapeutics available for treatment of dengue infection. Here, we describe a novel small-molecule compound, ST-148, that is a potent inhibitor of all four serotypes of DENV in vitro. ST-148 significantly reduced viremia and viral load in vital organs and tended to lower cytokine levels in the plasma in a nonlethal model of DENV infection in AG129 mice. Compound resistance mapped to the DENV capsid (C) gene, and a direct interaction of ST-148 with C protein is suggested by alterations of the intrinsic fluorescence of the protein in the presence of compound. Thus, ST-148 appears to interact with the DENV C protein and inhibits a distinct step(s) of the viral replication cycle.

  6. Roles of three amino acids of capsid proteins in mink enteritis parvovirus replication.

    Science.gov (United States)

    Mao, Yaping; Su, Jun; Wang, Jigui; Zhang, Xiaomei; Hou, Qiang; Bian, Dawei; Liu, Weiquan

    2016-08-15

    Virulent mink enteritis parvovirus (MEV) strain MEV-LHV replicated to higher titers in feline F81 cells than attenuated strain MEV-L. Phylogenetic and sequence analyses of the VP2 gene of MEV-LHV, MEV-L and other strains in GenBank revealed two evolutionary branches separating virulent and attenuated strains. Three residues, 101, 232 and 411, differed between virulent and attenuated strains but were conserved within the two branches. Site-directed mutagenesis of the VP2 gene of infectious plasmids of attenuated strain MEV-L respectively replacing residues 101 Ile and 411 Ala with Thr and Glu of virulent strains (MEV-L I101T and MEV-L A411E) increased replication efficiency but still to lower levels than MEV-LHV. However, viruses with mutation of residue 232 (MEV-L I232V and MEV-L I101T/I232V/A411E) decreased viral transcription and replication levels. The three VP2 residues 101, 232 and 411, located on or near the capsid surface, played different roles in the infection processes of MEV. Copyright © 2016 Elsevier B.V. All rights reserved.

  7. Mechanical and assembly units of viral capsids identified via quasi-rigid domain decomposition.

    Directory of Open Access Journals (Sweden)

    Guido Polles

    Full Text Available Key steps in a viral life-cycle, such as self-assembly of a protective protein container or in some cases also subsequent maturation events, are governed by the interplay of physico-chemical mechanisms involving various spatial and temporal scales. These salient aspects of a viral life cycle are hence well described and rationalised from a mesoscopic perspective. Accordingly, various experimental and computational efforts have been directed towards identifying the fundamental building blocks that are instrumental for the mechanical response, or constitute the assembly units, of a few specific viral shells. Motivated by these earlier studies we introduce and apply a general and efficient computational scheme for identifying the stable domains of a given viral capsid. The method is based on elastic network models and quasi-rigid domain decomposition. It is first applied to a heterogeneous set of well-characterized viruses (CCMV, MS2, STNV, STMV for which the known mechanical or assembly domains are correctly identified. The validated method is next applied to other viral particles such as L-A, Pariacoto and polyoma viruses, whose fundamental functional domains are still unknown or debated and for which we formulate verifiable predictions. The numerical code implementing the domain decomposition strategy is made freely available.

  8. Nuclear localization signal regulates porcine circovirus type 2 capsid protein nuclear export through phosphorylation.

    Science.gov (United States)

    Hou, Qiang; Hou, Shaohua; Chen, Qing; Jia, Hong; Xin, Ting; Jiang, Yitong; Guo, Xiaoyu; Zhu, Hongfei

    2018-02-15

    The open reading frame 2 (ORF2) of Porcine circovirus type 2 (PCV2) encodes the major Capsid (Cap) protein, which self-assembles into virus-like particle (VLP) of similar morphology to the PCV2 virion and accumulates in the nucleus through the N-terminal arginine-rich nuclear localization signal (NLS). In this study, PCV2 Cap protein and its derivates were expressed via the baculovirus expression system, and the cellular localization of the recombinant proteins were investigated using anti-Cap mAb by imaging flow cytometry. Analysis of subcellular localization of Cap protein and its variants demonstrated that NLS mediated Cap protein nuclear export as well as nuclear import, and a phosphorylation site (S17) was identified by liquid chromatography-tandem mass spectrometry (LC-MS/MS) in the NLS domain to regulate Cap protein nuclear export. Phosphorylation of NLS regulating the PCV2 Cap protein nuclear export was also demonstrated in PK15 cells by fluorescence microscopy. Moreover, the influence of Rep and Rep' protein on Cap protein subcellular localization was investigated in PK15 cells. Phosphorylation of NLS regulating Cap protein nuclear export provides more detailed knowledge of the PCV2 viral life cycle. Copyright © 2018 Elsevier B.V. All rights reserved.

  9. Whole-Chain Tick Saliva Proteins Presented on Hepatitis B Virus Capsid-Like Particles Induce High-Titered Antibodies with Neutralizing Potential.

    Directory of Open Access Journals (Sweden)

    Philipp Kolb

    Full Text Available Ticks are vectors for various, including pathogenic, microbes. Tick saliva contains multiple anti-host defense factors that enable ticks their bloodmeals yet also facilitate microbe transmission. Lyme disease-causing borreliae profit specifically from the broadly conserved tick histamine release factor (tHRF, and from cysteine-rich glycoproteins represented by Salp15 from Ixodes scapularis and Iric-1 from Ixodes ricinus ticks which they recruit to their outer surface protein C (OspC. Hence these tick proteins are attractive targets for anti-tick vaccines that simultaneously impair borrelia transmission. Main obstacles are the tick proteins´ immunosuppressive activities, and for Salp15 orthologs, the lack of efficient recombinant expression systems. Here, we exploited the immune-enhancing properties of hepatitis B virus core protein (HBc derived capsid-like particles (CLPs to generate, in E. coli, nanoparticulate vaccines presenting tHRF and, as surrogates for the barely soluble wild-type proteins, cysteine-free Salp15 and Iric-1 variants. The latter CLPs were exclusively accessible in the less sterically constrained SplitCore system. Mice immunized with tHRF CLPs mounted a strong anti-tHRF antibody response. CLPs presenting cysteine-free Salp15 and Iric-1 induced antibodies to wild-type, including glycosylated, Salp15 and Iric-1. The broadly distributed epitopes included the OspC interaction sites. In vitro, the anti-Salp15 antibodies interfered with OspC binding and enhanced human complement-mediated killing of Salp15 decorated borreliae. A mixture of all three CLPs induced high titered antibodies against all three targets, suggesting the feasibility of combination vaccines. These data warrant in vivo validation of the new candidate vaccines´ protective potential against tick infestation and Borrelia transmission.

  10. Whole-Chain Tick Saliva Proteins Presented on Hepatitis B Virus Capsid-Like Particles Induce High-Titered Antibodies with Neutralizing Potential

    Science.gov (United States)

    Kolb, Philipp; Wallich, Reinhard; Nassal, Michael

    2015-01-01

    Ticks are vectors for various, including pathogenic, microbes. Tick saliva contains multiple anti-host defense factors that enable ticks their bloodmeals yet also facilitate microbe transmission. Lyme disease-causing borreliae profit specifically from the broadly conserved tick histamine release factor (tHRF), and from cysteine-rich glycoproteins represented by Salp15 from Ixodes scapularis and Iric-1 from Ixodes ricinus ticks which they recruit to their outer surface protein C (OspC). Hence these tick proteins are attractive targets for anti-tick vaccines that simultaneously impair borrelia transmission. Main obstacles are the tick proteins´ immunosuppressive activities, and for Salp15 orthologs, the lack of efficient recombinant expression systems. Here, we exploited the immune-enhancing properties of hepatitis B virus core protein (HBc) derived capsid-like particles (CLPs) to generate, in E. coli, nanoparticulate vaccines presenting tHRF and, as surrogates for the barely soluble wild-type proteins, cysteine-free Salp15 and Iric-1 variants. The latter CLPs were exclusively accessible in the less sterically constrained SplitCore system. Mice immunized with tHRF CLPs mounted a strong anti-tHRF antibody response. CLPs presenting cysteine-free Salp15 and Iric-1 induced antibodies to wild-type, including glycosylated, Salp15 and Iric-1. The broadly distributed epitopes included the OspC interaction sites. In vitro, the anti-Salp15 antibodies interfered with OspC binding and enhanced human complement-mediated killing of Salp15 decorated borreliae. A mixture of all three CLPs induced high titered antibodies against all three targets, suggesting the feasibility of combination vaccines. These data warrant in vivo validation of the new candidate vaccines´ protective potential against tick infestation and Borrelia transmission. PMID:26352137

  11. Complement-mediated bactericidal activity of anti-factor H binding protein monoclonal antibodies against the meningococcus relies upon blocking factor H binding.

    Science.gov (United States)

    Giuntini, Serena; Reason, Donald C; Granoff, Dan M

    2011-09-01

    Binding of the complement-downregulating protein factor H (fH) to the surface of the meningococcus is important for survival of the organism in human serum. The meningococcal vaccine candidate factor H binding protein (fHbp) is an important ligand for human fH. While some fHbp-specific monoclonal antibodies (MAbs) block binding of fH to fHbp, the stoichiometry of blocking in the presence of high serum concentrations of fH and its effect on complement-mediated bactericidal activity are unknown. To investigate this question, we constructed chimeric antibodies in which the human IgG1 constant region was paired with three murine fHbp-specific binding domains designated JAR 3, JAR 5, and MAb502. By surface plasmon resonance, the association rates for binding of all three MAbs to immobilized fHbp were >50-fold higher than that for binding of fH to fHbp, and the MAb dissociation rates were >500-fold lower than that for fH. While all three MAbs elicited similar C1q-dependent C4b deposition on live bacteria (classical complement pathway), only those antibodies that inhibited binding of fH to fHbp (JAR 3 and JAR 5) had bactericidal activity with human complement. MAb502, which did not inhibit fH binding, had complement-mediated bactericidal activity only when tested with fH-depleted human complement. When an IgG1 anti-fHbp MAb binds to sparsely exposed fHbp on the bacterial surface, there appears to be insufficient complement activation for bacteriolysis unless fH binding also is inhibited. The ability of fHbp vaccines to elicit protective antibodies, therefore, is likely to be enhanced if the antibody repertoire is of high avidity and includes fH-blocking activity.

  12. Imaging and Quantitation of a Succession of Transient Intermediates Reveal the Reversible Self-Assembly Pathway of a Simple Icosahedral Virus Capsid.

    Science.gov (United States)

    Medrano, María; Fuertes, Miguel Ángel; Valbuena, Alejandro; Carrillo, Pablo J P; Rodríguez-Huete, Alicia; Mateu, Mauricio G

    2016-11-30

    Understanding the fundamental principles underlying supramolecular self-assembly may facilitate many developments, from novel antivirals to self-organized nanodevices. Icosahedral virus particles constitute paradigms to study self-assembly using a combination of theory and experiment. Unfortunately, assembly pathways of the structurally simplest virus capsids, those more accessible to detailed theoretical studies, have been difficult to study experimentally. We have enabled the in vitro self-assembly under close to physiological conditions of one of the simplest virus particles known, the minute virus of mice (MVM) capsid, and experimentally analyzed its pathways of assembly and disassembly. A combination of electron microscopy and high-resolution atomic force microscopy was used to structurally characterize and quantify a succession of transient assembly and disassembly intermediates. The results provided an experiment-based model for the reversible self-assembly pathway of a most simple (T = 1) icosahedral protein shell. During assembly, trimeric capsid building blocks are sequentially added to the growing capsid, with pentamers of building blocks and incomplete capsids missing one building block as conspicuous intermediates. This study provided experimental verification of many features of self-assembly of a simple T = 1 capsid predicted by molecular dynamics simulations. It also demonstrated atomic force microscopy imaging and automated analysis, in combination with electron microscopy, as a powerful single-particle approach to characterize at high resolution and quantify transient intermediates during supramolecular self-assembly/disassembly reactions. Finally, the efficient in vitro self-assembly achieved for the oncotropic, cell nucleus-targeted MVM capsid may facilitate its development as a drug-encapsidating nanoparticle for anticancer targeted drug delivery.

  13. {sup 18}F-FDG-Avid Adenocarcinoma of the Rectum Presenting as a Subtle Filling Defect on Maximum Intensity Projection Image: Report of a Case

    Energy Technology Data Exchange (ETDEWEB)

    Bahk, Yong Whee; Lee, Kwang Chan [SungAe General Hospital, Seoul (Korea, Republic of)

    2009-04-15

    {sup 18}F-FDG avid polypoid or tumefacient carcinomas of the gastrointestinal (GI) tract including the rectum are characteristically featured on PET-CT as an intraluminal tumor surrounded by completely or partially cleared background. However, the carcinomas of intramural or sessile variant may not so easily be detected especially when tumors are obliterated by feces or mucus retained in the host bowel loop. Recently, we observed a case of cauliflower-like adenocarcinoma of the rectum that was diagnosed by noting a subtle, flat filling defect created against the background of 'black' feces-mucus filled rectum. To our knowledge such a 'filling defect' produced by sessile tumor has not previously been reported as a useful diagnostic sign of GI tract carcinoma on PET-CT.

  14. Compensating for cross-reactions using avidity and computation in a suspension multiplex immunoassay for serotyping of Zika versus other flavivirus infections.

    Science.gov (United States)

    Rönnberg, Bengt; Gustafsson, Åke; Vapalahti, Olli; Emmerich, Petra; Lundkvist, Åke; Schmidt-Chanasit, Jonas; Blomberg, Jonas

    2017-10-01

    The recent spread of Zika virus (ZIKV) in the Americas and Asia necessitates an increased preparedness for improved maternal and perinatal health and blood safety. However, serological cross-reactions, especially to Dengue virus (DENV), complicate ZIKV antibody serodiagnosis. A novel "pan-Flavi" suspension multiplex immunoassay (PFSMIA) using 25 antigens, whole virus (WV), non-structural protein 1 (NS1), and envelope (E) proteins, from 7 zoonotic flaviviruses for specific detection of ZIKV and DENV IgM and IgG was developed. Patterns of antibody cross-reactivity, avidity, and kinetics were established in 104 sera from returning travelers with known ZIKV and DENV infections. PFSMIA gave IgM- and IgG-sensitivities for both viruses of 96-100%, compared to an immunofluorescence assay. Main IgM cross-reactions were to NS1, for IgG to the E and WV antigens. Infecting virus yielded reactivity to several antigens of the homologous virus, while cross-reactions tended to occur only to a single antigen from heterologous virus(es). A specificity-enhancing computer procedure took into account antibody isotype, number of antibody-reactive antigens per virus, avidity, average degree of cross-reactivity to heterologous flavivirus antigens, and reactivity changes in serial sera. It classified all 50 cases correctly. Applied to sera from 200 pregnant women and 173 blood donors from Sweden, one blood donor was found ZIKV NS1 IgM positive, and another as ZIKV NS1 IgG positive. These samples did not react with other ZIKV antigens and were thereby judged as false-positives. PFSMIA provided sensitive and specific ZIKV and DENV serology, warranting high-throughput serological surveillance and a minimized need for laborious and expensive virus neutralization assays.

  15. Duration of 18F-FDG avidity in lymph nodes after pandemic H1N1v and seasonal influenza vaccination

    International Nuclear Information System (INIS)

    Thomassen, Anders; Lerberg Nielsen, Anne; Gerke, Oke; Johansen, Allan; Petersen, Henrik

    2011-01-01

    The aim of our study was to investigate the occurrence of fluorodeoxyglucose (FDG) avidity in draining axillary lymph nodes after vaccination against influenza (H1N1v pandemic and seasonal) and to determine the period of increased FDG uptake. During December 2009, patients referred for 18 F-FDG positron emission tomography (PET)/CT scans (n = 293) filled in a questionnaire concerning vaccination type (seasonal and/or H1N1v), time and anatomical localization of vaccination. Only injections in deltoid regions were evaluated, thus ensuring that draining lymph nodes were axillary. If more vaccinations had been given, only the latest vaccination was evaluated in each deltoid region. Of all patients who underwent PET/CT scans during December 2009, 26% had been vaccinated with at least one influenza vaccination in the deltoid region. A total of 92 'draining' and 60 'reference' (i.e. contralateral, non-vaccinated) axillary lymph nodes were evaluated in 61 patients (19 of 61 patients were scanned twice). The maximal intensity in FDG uptake (SUV max ) in draining lymph nodes was 5 g/ml body weight (BW), whereas the maximal intensity in reference lymph nodes was 1.9 g/ml BW. The SUV max was normalized approximately 40 days after vaccination. No significant enlargement of metabolically active draining lymph nodes could be demonstrated on CT scan. Chemotherapy or immunosuppressive drugs given within 2 weeks from vaccination did not affect SUV max in the axillary lymph nodes. Influenza vaccination may lead to FDG-avid draining lymph nodes beyond 1 month. (orig.)

  16. Duration of {sup 18}F-FDG avidity in lymph nodes after pandemic H1N1v and seasonal influenza vaccination

    Energy Technology Data Exchange (ETDEWEB)

    Thomassen, Anders; Lerberg Nielsen, Anne; Gerke, Oke; Johansen, Allan; Petersen, Henrik [OUH, Odense University Hospital, Department of Nuclear Medicine, Odense C (Denmark)

    2011-05-15

    The aim of our study was to investigate the occurrence of fluorodeoxyglucose (FDG) avidity in draining axillary lymph nodes after vaccination against influenza (H1N1v pandemic and seasonal) and to determine the period of increased FDG uptake. During December 2009, patients referred for {sup 18}F-FDG positron emission tomography (PET)/CT scans (n = 293) filled in a questionnaire concerning vaccination type (seasonal and/or H1N1v), time and anatomical localization of vaccination. Only injections in deltoid regions were evaluated, thus ensuring that draining lymph nodes were axillary. If more vaccinations had been given, only the latest vaccination was evaluated in each deltoid region. Of all patients who underwent PET/CT scans during December 2009, 26% had been vaccinated with at least one influenza vaccination in the deltoid region. A total of 92 'draining' and 60 'reference' (i.e. contralateral, non-vaccinated) axillary lymph nodes were evaluated in 61 patients (19 of 61 patients were scanned twice). The maximal intensity in FDG uptake (SUV{sub max}) in draining lymph nodes was 5 g/ml body weight (BW), whereas the maximal intensity in reference lymph nodes was 1.9 g/ml BW. The SUV{sub max} was normalized approximately 40 days after vaccination. No significant enlargement of metabolically active draining lymph nodes could be demonstrated on CT scan. Chemotherapy or immunosuppressive drugs given within 2 weeks from vaccination did not affect SUV{sub max} in the axillary lymph nodes. Influenza vaccination may lead to FDG-avid draining lymph nodes beyond 1 month. (orig.)

  17. Interaction of the host protein NbDnaJ with Potato virus X minus-strand stem-loop 1 RNA and capsid protein affects viral replication and movement.

    Science.gov (United States)

    Cho, Sang-Yun; Cho, Won Kyong; Sohn, Seong-Han; Kim, Kook-Hyung

    2012-01-06

    Plant viruses must interact with host cellular components to replicate and move from cell to cell. In the case of Potato virus X (PVX), it carries stem-loop 1 (SL1) RNA essential for viral replication and movement. Using two-dimensional electrophoresis northwestern blot analysis, we previously identified several host proteins that bind to SL1 RNA. Of those, we further characterized a DnaJ-like protein from Nicotiana benthamiana named NbDnaJ. An electrophoretic mobility shift assay confirmed that NbDnaJ binds only to SL1 minus-strand RNA, and bimolecular fluorescence complementation (BiFC) indicated that NbDnaJ interacts with PVX capsid protein (CP). Using a series of deletion mutants, the C-terminal region of NbDnaJ was found to be essential for the interaction with PVX CP. The expression of NbDnaJ significantly changed upon infection with different plant viruses such as PVX, Tobacco mosaic virus, and Cucumber mosaic virus, but varied depending on the viral species. In transient experiments, both PVX replication and movement were inhibited in plants that over-expressed NbDnaJ but accelerated in plants in which NbDnaJ was silenced. In summary, we suggest that the newly identified NbDnaJ plays a role in PVX replication and movement by interacting with SL1(-) RNA and PVX CP. Copyright © 2011 Elsevier Inc. All rights reserved.

  18. Relevance of Assembly-Activating Protein for Adeno-associated Virus Vector Production and Capsid Protein Stability in Mammalian and Insect Cells.

    Science.gov (United States)

    Grosse, Stefanie; Penaud-Budloo, Magalie; Herrmann, Anne-Kathrin; Börner, Kathleen; Fakhiri, Julia; Laketa, Vibor; Krämer, Chiara; Wiedtke, Ellen; Gunkel, Manuel; Ménard, Lucie; Ayuso, Eduard; Grimm, Dirk

    2017-10-15

    The discovery that adeno-associated virus 2 (AAV2) encodes an eighth protein, called assembly-activating protein (AAP), transformed our understanding of wild-type AAV biology. Concurrently, it raised questions about the role of AAP during production of recombinant vectors based on natural or molecularly engineered AAV capsids. Here, we show that AAP is indeed essential for generation of functional recombinant AAV2 vectors in both mammalian and insect cell-based vector production systems. Surprisingly, we observed that AAV2 capsid proteins VP1 to -3 are unstable in the absence of AAP2, likely due to rapid proteasomal degradation. Inhibition of the proteasome led to an increase of intracellular VP1 to -3 but neither triggered assembly of functional capsids nor promoted nuclear localization of the capsid proteins. Together, this underscores the crucial and unique role of AAP in the AAV life cycle, where it rapidly chaperones capsid assembly, thus preventing degradation of free capsid proteins. An expanded analysis comprising nine alternative AAV serotypes (1, 3 to 9, and rh10) showed that vector production always depends on the presence of AAP, with the exceptions of AAV4 and AAV5, which exhibited AAP-independent, albeit low-level, particle assembly. Interestingly, AAPs from all 10 serotypes could cross-complement AAP-depleted helper plasmids during vector production, despite there being distinct intracellular AAP localization patterns. These were most pronounced for AAP4 and AAP5, congruent with their inability to rescue an AAV2/AAP2 knockout. We conclude that AAP is key for assembly of genuine capsids from at least 10 different AAV serotypes, which has implications for vectors derived from wild-type or synthetic AAV capsids. IMPORTANCE Assembly of adeno-associated virus 2 (AAV2) is regulated by the assembly-activating protein (AAP), whose open reading frame overlaps with that of the viral capsid proteins. As the majority of evidence was obtained using virus

  19. Ultrastructural Localization and Molecular Associations of HCV Capsid Protein in Jurkat T Cells

    Directory of Open Access Journals (Sweden)

    Cecilia Fernández-Ponce

    2018-01-01

    Full Text Available Hepatitis C virus core protein is a highly basic viral protein that multimerizes with itself to form the viral capsid. When expressed in CD4+ T lymphocytes, it can induce modifications in several essential cellular and biological networks. To shed light on the mechanisms underlying the alterations caused by the viral protein, we have analyzed HCV-core subcellular localization and its associations with host proteins in Jurkat T cells. In order to investigate the intracellular localization of Hepatitis C virus core protein, we have used a lentiviral system to transduce Jurkat T cells and subsequently localize the protein using immunoelectron microscopy techniques. We found that in Jurkat T cells, Hepatitis C virus core protein mostly localizes in the nucleus and specifically in the nucleolus. In addition, we performed pull-down assays combined with Mass Spectrometry Analysis, to identify proteins that associate with Hepatitis C virus core in Jurkat T cells. We found proteins such as NOLC1, PP1γ, ILF3, and C1QBP implicated in localization and/or traffic to the nucleolus. HCV-core associated proteins are implicated in RNA processing and RNA virus infection as well as in functions previously shown to be altered in Hepatitis C virus core expressing CD4+ T cells, such as cell cycle delay, decreased proliferation, and induction of a regulatory phenotype. Thus, in the current work, we show the ultrastructural localization of Hepatitis C virus core and the first profile of HCV core associated proteins in T cells, and we discuss the functions and interconnections of these proteins in molecular networks where relevant biological modifications have been described upon the expression of Hepatitis C virus core protein. Thereby, the current work constitutes a necessary step toward understanding the mechanisms underlying HCV core mediated alterations that had been described in relevant biological processes in CD4+ T cells.

  20. Rubella virus capsid protein modulation of viral genomic and subgenomic RNA synthesis

    International Nuclear Information System (INIS)

    Tzeng, W.-P.; Frey, Teryl K.

    2005-01-01

    The ratio of the subgenomic (SG) to genome RNA synthesized by rubella virus (RUB) replicons expressing the green fluorescent protein reporter gene (RUBrep/GFP) is substantially higher than the ratio of these species synthesized by RUB (4.3 for RUBrep/GFP vs. 1.3-1.4 for RUB). It was hypothesized that this modulation of the viral RNA synthesis was by one of the virus structural protein genes and it was found that introduction of the capsid (C) protein gene into the replicons as an in-frame fusion with GFP resulted in an increase of genomic RNA production (reducing the SG/genome RNA ratio), confirming the hypothesis and showing that the C gene was the moiety responsible for the modulation effect. The N-terminal one-third of the C gene was required for the effect of be exhibited. A similar phenomenon was not observed with the replicons of Sindbis virus, a related Alphavirus. Interestingly, modulation was not observed when RUBrep/GFP was co-transfected with either other RUBrep or plasmid constructs expressing the C gene, demonstrating that modulation could occur only when the C gene was provided in cis. Mutations that prevented translation of the C protein failed to modulate RNA synthesis, indicating that the C protein was the moiety responsible for modulation; consistent with this conclusion, modulation of RNA synthesis was maintained when synonymous codon mutations were introduced at the 5' end of the C gene that changed the C gene sequence without altering the amino acid sequence of the C protein. These results indicate that C protein translated in proximity of viral replication complexes, possibly from newly synthesized SG RNA, participate in regulating the replication of viral RNA

  1. Identification and classification of human cytomegalovirus capsids in textured electron micrographs using deformed template matching

    Directory of Open Access Journals (Sweden)

    Söderberg-Nauclér Cecilia

    2006-08-01

    Full Text Available Abstract Background Characterization of the structural morphology of virus particles in electron micrographs is a complex task, but desirable in connection with investigation of the maturation process and detection of changes in viral particle morphology in response to the effect of a mutation or antiviral drugs being applied. Therefore, we have here developed a procedure for describing and classifying virus particle forms in electron micrographs, based on determination of the invariant characteristics of the projection of a given virus structure. The template for the virus particle is created on the basis of information obtained from a small training set of electron micrographs and is then employed to classify and quantify similar structures of interest in an unlimited number of electron micrographs by a process of correlation. Results Practical application of the method is demonstrated by the ability to locate three diverse classes of virus particles in transmission electron micrographs of fibroblasts infected with human cytomegalovirus. These results show that fast screening of the total number of viral structures at different stages of maturation in a large set of electron micrographs, a task that is otherwise both time-consuming and tedious for the expert, can be accomplished rapidly and reliably with our automated procedure. Using linear deformation analysis, this novel algorithm described here can handle capsid variations such as ellipticity and furthermore allows evaluation of properties such as the size and orientation of a virus particle. Conclusion Our methodological procedure represents a promising objective tool for comparative studies of the intracellular assembly processes of virus particles using electron microscopy in combination with our digitized image analysis tool. An automated method for sorting and classifying virus particles at different stages of maturation will enable us to quantify virus production in all stages of the

  2. Maize rayado fino virus capsid proteins assemble into virus-like particles in Escherichia coli.

    Science.gov (United States)

    Hammond, Rosemarie W; Hammond, John

    2010-02-01

    Maize rayado fino virus (MRFV; genus Marafivirus; family Tymoviridae) is an isometric plant virus of 30 nm containing two components: empty shells and complete virus particles (encapsidating the 6.3 kb genomic RNA). Both particles are composed of two serologically related, carboxy co-terminal, coat proteins (CP) of apparent molecular mass 21-22 kDa (CP2) and 24-28 kDa (CP1) in a molar ratio of 3:1, respectively; CP1 contains a 37 amino acid amino terminal extension of CP2. In our study, expression of CP1 or CP2 in Escherichia coli resulted in assembly of each capsid protein into virus-like particles (VLPs), appearing in electron microscopy as stain-permeable (CP2) or stain-impermeable particles (CP1). CP1 VLPs encapsidated bacterial 16S ribosomal RNA, but not CP mRNA, while CP2 VLPs encapsidated neither CP mRNA nor 16S ribosomal RNA. Expression of CP1 and CP2 in E. coli using a co-expression vector resulted in the assembly of VLPs which were stain-impermeable and encapsidated CP mRNA. These results suggest that the N-terminal 37 amino acid residues of CP1, although not required for particle formation, may be involved in the assembly of complete virions and that the presence of both CP1 and CP2 in the particle is required for specific encapsidation of MRFV CP mRNA. (c) 2009 Elsevier B.V. All rights reserved.

  3. Observations on the expression of human papillomavirus major capsid protein in HeLa cells.

    Science.gov (United States)

    Xiao, Chang-Yi; Fu, Bing-Bing; Li, Zhi-Ying; Mushtaq, Gohar; Kamal, Mohammad Amjad; Li, Jia-Hua; Tang, Gui-Cheng; Xiao, Shuo-Shuang

    2015-01-01

    The goal of this study was to identify the nature of the inclusion bodies that have been found in HeLa cells (cervical cancer immortal cell line) by electron microscope and to determine whether the major capsid protein (L1) of human papillomavirus (HPV) can be expressed in HPV-positive uterine cervix cancer cells. HPV L1 protein expression in HeLa cells was detected with anti-HPV L1 multivalent mice monoclonal antibody and rabbit polyclonal anti-HPV L1 antibody by ELISA, light microscope immunohistochemistry, electron microscope immunocytochemistry and Western blotting assays. Reverse transcriptional PCR (RT-PCR) was performed to detect the transcription of L1 mRNA in HeLa cells. The immortalized human keratinocyte HeCat was used as the negative control. HPV L1 proteins reacted positively in the lysate of HeLa cells by ELISA assays. HRP labeled light microscope immunohistochemistry assay showed that there was a strong HPV L1 positive reaction in HeLa cells. Under the electron microscope, irregular shaped inclusion bodies, assembled by many small and uniform granules, had been observed in the cytoplasm of some HeLa cells. These granules could be labeled by the colloidal gold carried by HPV L1 antibody. The Western blotting assay showed that there was a L1 reaction strap at 80-85 kDa in the HeLa cell lysates, hence demonstrating the existence of HPV18 L1 in HeLa cells. RT-PCR assay showed that the L1 mRNA was transcribed in HeLa cells. The inclusion bodies found in the cytoplasm of HeLa cells are composed of HPV18 L1 protein. Since HeLa cell line is a type of cervical cancer cells, this implies that HeLa cells have the ability to express HPV L1 proteins.

  4. [Expression and activity determination of recombinant capsid protein VP2 gene of enterovirus type 71].

    Science.gov (United States)

    Huang, Xueyong; Liu, Guohua; Hu, Xiaoning; Du, Yanhua; Li, Xingle; Xu, Yuling; Chen, Haomin; Xu, Bianli

    2014-04-01

    To clone and express the recombinant capsid protein VP2 of enterovirus type 71 (EV71) and to identify the immune activity of expressed protein in order to build a basis for the investigation work of vaccine and diagnostic antigen. VP2 gene of EV71 was amplified by PCR, and then was cut by restriction enzyme and inserted into expression vector pMAL-c2X. The positive recombinants were transferred into E.coli TB1, the genetically engineered bacteria including pMAL-c2X-VP2 plasmids were induced by isopropyl thiogalactoside ( IPTG) , and the expression products were analyzed by SDS-PAGE and western blotting method. EV71 IgM antibody detection method by ELISA was set up, and the sensitivity and specificity of this method was assessed; 60 neutralizing antibody positive serum samples from hand foot and mouth disease (HFMD) patients were determined, of which 52 samples were positive and 8 samples were negative; a total of 88 acute phase serum samples of HFMD patients diagnosed in clinical were also detected. VP2 gene of 762 bp was obtained by PCR, the gene segment inserted into the recombinant vector was identified using restriction enzyme digestion. The recombinant vector could express a specific about 71 500 fusion protein in E.coli by SDS-PAGE. The purified recombinant protein of EV71-VP2 can react with the serum of HFMD patients to produce a specific band by western blotting. The sensitivity and specificity of ELISA was 87% and 83%, respectively. Of the 88 acute phase serum samples from children with HFMD, 48 samples (55%) were positive by the ELISA assay. VP2 gene of EV71 has been cloned and a prokaryotic high expression system for VP2 gene was successfully constructed in the present study. The recombination EV71-VP2 has well antigenicity, which could be useful for developing diagnose reagent or vaccine of EV71.

  5. Biochemical and biophysical characterization of cell-free synthesized Rift Valley fever virus nucleoprotein capsids enables in vitro screening to identify novel antivirals.

    Science.gov (United States)

    Broce, Sean; Hensley, Lisa; Sato, Tomoharu; Lehrer-Graiwer, Joshua; Essrich, Christian; Edwards, Katie J; Pajda, Jacqueline; Davis, Christopher J; Bhadresh, Rami; Hurt, Clarence R; Freeman, Beverly; Lingappa, Vishwanath R; Kelleher, Colm A; Karpuj, Marcela V

    2016-05-14

    Viral capsid assembly involves the oligomerization of the capsid nucleoprotein (NP), which is an essential step in viral replication and may represent a potential antiviral target. An in vitro transcription-translation reaction using a wheat germ (WG) extract in combination with a sandwich ELISA assay has recently been used to identify small molecules with antiviral activity against the rabies virus. Here, we examined the application of this system to viruses with capsids with a different structure, such as the Rift Valley fever virus (RVFV), the etiological agent of a severe emerging infectious disease. The biochemical and immunological characterization of the in vitro-generated RVFV NP assembly products enabled the distinction between intermediately and highly ordered capsid structures. This distinction was used to establish a screening method for the identification of potential antiviral drugs for RVFV countermeasures. These results indicated that this unique analytical system, which combines nucleoprotein oligomerization with the specific immune recognition of a highly ordered capsid structure, can be extended to various viral families and used both to study the early stages of NP assembly and to assist in the identification of potential antiviral drugs in a cost-efficient manner. Reviewed by Jeffry Skolnick and Noah Isakov. For the full reviews please go to the Reviewers' comments section.

  6. Structures of the Streptococcus sanguinis SrpA Binding Region with Human Sialoglycans Suggest Features of the Physiological Ligand.

    Science.gov (United States)

    Loukachevitch, Lioudmila V; Bensing, Barbara A; Yu, Hai; Zeng, Jie; Chen, Xi; Sullam, Paul M; Iverson, T M

    2016-10-11

    Streptococcus sanguinis is a leading cause of bacterial infective endocarditis, a life-threatening infection of heart valves. S. sanguinis binds to human platelets with high avidity, and this adherence is likely to enhance virulence. Previous studies suggest that a serine-rich repeat adhesin termed SrpA mediates the binding of S. sanguinis to human platelets via its interaction with sialoglycans on the receptor GPIbα. However, in vitro binding assays with SrpA and defined sialoglycans failed to identify specific high-affinity ligands. To improve our understanding of the interaction between SrpA and human platelets, we determined cocrystal structures of the SrpA sialoglycan binding region (SrpA BR ) with five low-affinity ligands: three sialylated trisaccharides (sialyl-T antigen, 3'-sialyllactose, and 3'-sialyl-N-acetyllactosamine), a sialylated tetrasaccharide (sialyl-Lewis X ), and a sialyl galactose disaccharide component common to these sialoglyans. We then combined structural analysis with mutagenesis to further determine whether our observed interactions between SrpA BR and glycans are important for binding to platelets and to better map the binding site for the physiological receptor. We found that the sialoglycan binding site of SrpA BR is significantly larger than the sialoglycans cocrystallized in this study, which suggests that binding of SrpA to platelets either is multivalent or occurs via a larger, disialylated glycan.

  7. Protection against myxomatosis and rabbit viral hemorrhagic disease with recombinant myxoma viruses expressing rabbit hemorrhagic disease virus capsid protein.

    Science.gov (United States)

    Bertagnoli, S; Gelfi, J; Le Gall, G; Boilletot, E; Vautherot, J F; Rasschaert, D; Laurent, S; Petit, F; Boucraut-Baralon, C; Milon, A

    1996-08-01

    Two myxoma virus-rabbit hemorrhagic disease virus (RHDV) recombinant viruses were constructed with the SG33 strain of myxoma virus to protect rabbits against myxomatosis and rabbit viral hemorrhagic disease. These recombinant viruses expressed the RHDV capsid protein (VP60). The recombinant protein, which is 60 kDa in size, was antigenic, as revealed by its reaction in immunoprecipitation with antibodies raised against RHDV. Both recombinant viruses induced high levels of RHDV- and myxoma virus-specific antibodies in rabbits after immunization. Inoculations by the intradermal route protected animals against virulent RHDV and myxoma virus challenges.

  8. Structure, Immunogenicity, and Protective Mechanism of an Engineered Enterovirus 71-Like Particle Vaccine Mimicking 80S Empty Capsid.

    Science.gov (United States)

    Wang, Xiaoli; Ku, Zhiqiang; Zhang, Xiang; Ye, Xiaohua; Chen, Jinhuan; Liu, Qingwei; Zhang, Wei; Zhang, Chao; Fu, Zhenglin; Jin, Xia; Cong, Yao; Huang, Zhong

    2018-01-01

    Enterovirus 71 (EV71) is the major causative agent of severe hand, foot, and mouth disease, which affects millions of young children in the Asia-Pacific region annually. In this study, we engineered a novel EV71 virus-like particle (VLP) that lacks VP4 (therefore designated VLP ΔVP4 ) and investigated its structure, antigenicity, and vaccine potential. The cryo-electron microscopy (cryo-EM) structure of VLP ΔVP4 was reconstructed to 3.71-Å resolution. Results from structural and biochemical analyses revealed that VLP ΔVP4 resembles the end product of the viral uncoating process, the 80S empty capsid. VLP ΔVP4 is able to elicit high-titer neutralizing antibodies and to fully protect mice against lethal viral challenge. Mechanistic studies showed that, at the cellular level, the anti-VLP ΔVP4 sera exert neutralization effects at both pre- and postattachment stages by inhibiting both virus attachment and internalization, and at the molecular level, the antisera can block multiple interactions between EV71 and its key receptors. Our study gives a better understanding of EV71 capsid assembly and provides important information for the design and development of new-generation vaccines for EV71, and perhaps for other enteroviruses, as well. IMPORTANCE Enterovirus 71 (EV71) infection may lead to severe hand, foot, and mouth disease, with significant morbidity and mortality. Knowledge regarding EV71 particle assembly remains limited. Here, we report the generation and characterization of a novel EV71 virus-like particle that lacks the VP4 capsid subunit protein. This particle, termed VLP ΔVP4 , structurally mimics the 80S empty capsid, which is the end stage of EV71 uncoating. We further show that VLP ΔVP4 exhibits desirable immunogenicity and protective efficacy in proof-of-concept studies. In addition, the inhibitory mechanisms of the VLP ΔVP4 -induced antibodies are unraveled at both the cellular and molecular levels. Our work provides the first evidence of

  9. NMR structure of the N-terminal domain of capsid protein from the Mason-Pfizer monkey virus

    Czech Academy of Sciences Publication Activity Database

    Macek, Pavel; Chmelík, Josef; Křížová, Ivana; Kadeřávek, P.; Padrta, P.; Žídek, L.; Wildová, Marcela; Hadravová, Romana; Chaloupková, R.; Pichová, Iva; Ruml, T.; Rumlová, Michaela; Sklenář, V.

    2009-01-01

    Roč. 392, č. 1 (2009), s. 100-114 ISSN 0022-2836 R&D Projects: GA MŠk LC545; GA MŠk 1M0508; GA ČR GA204/09/1388; GA ČR GESCO/06/E001 Grant - others:GA MŠk(CZ) 1M0520; MŠk(CZ) LC06030 Program:1M; LC Institutional research plan: CEZ:AV0Z40550506; CEZ:AV0Z50200510 Keywords : M-PMV * betaretroviruses * capsid protein * NMR structure * internal dynamics Subject RIV: CE - Biochemistry Impact factor: 3.871, year: 2009

  10. Stabilization of the beta-hairpin in Mason-Pfizer monkey virus capsid protein- a critical step for infectivity

    Czech Academy of Sciences Publication Activity Database

    Obr, M.; Hadravová, Romana; Doležal, Michal; Křížová, Ivana; Papoušková, V.; Žídek, L.; Hrabal, R.; Ruml, T.; Rumlová, Michaela

    2014-01-01

    Roč. 11, Oct 30 (2014), 94/1-94/14 ISSN 1742-4690 R&D Projects: GA ČR(CZ) GA14-15326S; GA MŠk LO1302 Grant - others:GA MŠk(CZ) ED1.1.00/02.0068; Seventh Framework Programme of the European Union(XE) FP7-261863 Program:ED Institutional support: RVO:61388963 Keywords : retrovirus * assembly * M-PMV * capsid protein * maturation * beta-hairpin Subject RIV: EE - Microbiology, Virology Impact factor: 4.185, year: 2014 http://www.retrovirology.com/content/11/1/94

  11. Characterization of the invariable residue 51 mutations of human immunodeficiency virus type 1 capsid protein on in vitro CA assembly and infectivity

    Directory of Open Access Journals (Sweden)

    Höglund Stefan

    2007-09-01

    Full Text Available Abstract Background The mature HIV-1 conical core formation proceeds through highly regulated protease cleavage of the Gag precursor, which ultimately leads to substantial rearrangements of the capsid (CAp24 molecule involving both inter- and intra-molecular contacts of the CAp24 molecules. In this aspect, Asp51 which is located in the N-terminal domain of HIV-1 CAp24 plays an important role by forming a salt-bridge with the free imino terminus Pro1 following proteolytic cleavage and liberation of the CAp24 protein from the Pr55Gag precursor. Thus, previous substitution mutation of Asp51 to alanine (D51A has shown to be lethal and that this invariable residue was found essential for tube formation in vitro, virus replication and virus capsid formation. Results We extended the above investigation by introducing three different D51 substitution mutations (D51N, D51E, and D51Q into both prokaryotic and eukaryotic expression systems and studied their effects on in vitro capsid assembly and virus infectivity. Two substitution mutations (D51E and D51N had no substantial effect on in vitro capsid assembly, yet they impaired viral infectivity and particle production. In contrast, the D51Q mutant was defective both for in vitro capsid assembly and for virus replication in cell culture. Conclusion These results show that substitutions of D51 with glutamate, glutamine, or asparagine, three amino acid residues that are structurally related to aspartate, could partially rescue both in vitro capsid assembly and intra-cellular CAp24 production but not replication of the virus in cultured cells.

  12. Intraoperative detection of 18F-FDG-avid tissue sites using the increased probe counting efficiency of the K-alpha probe design and variance-based statistical analysis with the three-sigma criteria

    International Nuclear Information System (INIS)

    Povoski, Stephen P; Chapman, Gregg J; Murrey, Douglas A; Lee, Robert; Martin, Edward W; Hall, Nathan C

    2013-01-01

    Intraoperative detection of 18 F-FDG-avid tissue sites during 18 F-FDG-directed surgery can be very challenging when utilizing gamma detection probes that rely on a fixed target-to-background (T/B) ratio (ratiometric threshold) for determination of probe positivity. The purpose of our study was to evaluate the counting efficiency and the success rate of in situ intraoperative detection of 18 F-FDG-avid tissue sites (using the three-sigma statistical threshold criteria method and the ratiometric threshold criteria method) for three different gamma detection probe systems. Of 58 patients undergoing 18 F-FDG-directed surgery for known or suspected malignancy using gamma detection probes, we identified nine 18 F-FDG-avid tissue sites (from amongst seven patients) that were seen on same-day preoperative diagnostic PET/CT imaging, and for which each 18 F-FDG-avid tissue site underwent attempted in situ intraoperative detection concurrently using three gamma detection probe systems (K-alpha probe, and two commercially-available PET-probe systems), and then were subsequently surgical excised. The mean relative probe counting efficiency ratio was 6.9 (± 4.4, range 2.2–15.4) for the K-alpha probe, as compared to 1.5 (± 0.3, range 1.0–2.1) and 1.0 (± 0, range 1.0–1.0), respectively, for two commercially-available PET-probe systems (P < 0.001). Successful in situ intraoperative detection of 18 F-FDG-avid tissue sites was more frequently accomplished with each of the three gamma detection probes tested by using the three-sigma statistical threshold criteria method than by using the ratiometric threshold criteria method, specifically with the three-sigma statistical threshold criteria method being significantly better than the ratiometric threshold criteria method for determining probe positivity for the K-alpha probe (P = 0.05). Our results suggest that the improved probe counting efficiency of the K-alpha probe design used in conjunction with the three

  13. Intraoperative detection of ¹⁸F-FDG-avid tissue sites using the increased probe counting efficiency of the K-alpha probe design and variance-based statistical analysis with the three-sigma criteria.

    Science.gov (United States)

    Povoski, Stephen P; Chapman, Gregg J; Murrey, Douglas A; Lee, Robert; Martin, Edward W; Hall, Nathan C

    2013-03-04

    Intraoperative detection of (18)F-FDG-avid tissue sites during 18F-FDG-directed surgery can be very challenging when utilizing gamma detection probes that rely on a fixed target-to-background (T/B) ratio (ratiometric threshold) for determination of probe positivity. The purpose of our study was to evaluate the counting efficiency and the success rate of in situ intraoperative detection of (18)F-FDG-avid tissue sites (using the three-sigma statistical threshold criteria method and the ratiometric threshold criteria method) for three different gamma detection probe systems. Of 58 patients undergoing (18)F-FDG-directed surgery for known or suspected malignancy using gamma detection probes, we identified nine (18)F-FDG-avid tissue sites (from amongst seven patients) that were seen on same-day preoperative diagnostic PET/CT imaging, and for which each (18)F-FDG-avid tissue site underwent attempted in situ intraoperative detection concurrently using three gamma detection probe systems (K-alpha probe, and two commercially-available PET-probe systems), and then were subsequently surgical excised. The mean relative probe counting efficiency ratio was 6.9 (± 4.4, range 2.2-15.4) for the K-alpha probe, as compared to 1.5 (± 0.3, range 1.0-2.1) and 1.0 (± 0, range 1.0-1.0), respectively, for two commercially-available PET-probe systems (P < 0.001). Successful in situ intraoperative detection of 18F-FDG-avid tissue sites was more frequently accomplished with each of the three gamma detection probes tested by using the three-sigma statistical threshold criteria method than by using the ratiometric threshold criteria method, specifically with the three-sigma statistical threshold criteria method being significantly better than the ratiometric threshold criteria method for determining probe positivity for the K-alpha probe (P = 0.05). Our results suggest that the improved probe counting efficiency of the K-alpha probe design used in conjunction with the three-sigma statistical

  14. Synthesis and characterization of different immunogenic viral nanoconstructs from rotavirus VP6 inner capsid protein

    Directory of Open Access Journals (Sweden)

    Bugli F

    2014-05-01

    Full Text Available Francesca Bugli,1 Valeria Caprettini,2 Margherita Cacaci,1 Cecilia Martini,1 Francesco Paroni Sterbini,1 Riccardo Torelli,1 Stefano Della Longa,3 Massimiliano Papi,4 Valentina Palmieri,4 Bruno Giardina,5 Brunella Posteraro,1 Maurizio Sanguinetti,1 Alessandro Arcovito5 1Istituto di Microbiologia, Università Cattolica del Sacro Cuore, 2Dipartimento di Fisica, Sapienza Università di Roma, Rome, 3Dipartimento di Medicina Clinica, Sanità Pubblica, Scienze della Vita e dell’Ambiente, Università dell’Aquila, L’Aquila, 4Istituto di Fisica, 5Istituto di Biochimica e Biochimica Clinica, Università Cattolica del Sacro Cuore, Rome, Italy Abstract: In order to deliver low-cost viral capsomeres from a large amount of soluble viral VP6 protein from human rotavirus, we developed and optimized a biotechnological platform in Escherichia coli. Specifically, three different expression protocols were compared, differing in their genetic constructs, ie, a simple native histidine-tagged VP6 sequence, VP6 fused to thioredoxin, and VP6 obtained with the newly described small ubiquitin-like modifier (SUMO fusion system. Our results demonstrate that the histidine-tagged protein does not escape the accumulation in the inclusion bodies, and that SUMO is largely superior to the thioredoxin-fusion tag in enhancing the expression and solubility of VP6 protein. Moreover, the VP6 protein produced according to the SUMO fusion tag displays well-known assembly properties, as observed in both transmission electron microscopy and atomic force microscopy images, giving rise to either VP6 trimers, 60 nm spherical virus-like particles, or nanotubes a few micron long. This different quaternary organization of VP6 shows a higher level of immunogenicity for the elongated structures with respect to the spheres or the protein trimers. Therefore, the expression and purification strategy presented here – providing a large amount of the viral capsid protein in the native

  15. Characterization of a protein kinase activity associated with purified capsids of the granulosis virus infecting Plodia interpunctella.

    Science.gov (United States)

    Wilson, M E; Consigli, R A

    1985-06-01

    A cyclic-nucleotide independent protein kinase activity has been demonstrated in highly purified preparations of the granulosis virus infecting the Indian meal moth, Plodia interpunctella. A divalent cation was required for activity. Manganese was the preferred cation and a pH of 8.0 resulted in optimal incorporation of 32P radiolabel into acid-precipitable protein. Although both ATP and GTP could serve as phosphate donors, ATP was utilized more efficiently by the enzyme. The kinase activity was localized to purified capsids; and the basic, internal core protein, VP12, was found to be the predominant viral acceptor. Histones and protamine sulfate could also serve as acceptors for the capsid-associated kinase activity. Using acid hydrolysis and phosphoamino acid analysis of phosphorylated nucleocapsid protein and nuclear magnetic resonance of phosphorylated VP12, it was determined that the enzyme catalyzes the transfer of phosphate to both serine and arginine residues of acceptor proteins. We believe this kinase activity may play a significant role in the viral replication cycle.

  16. Impact of reducing and oxidizing agents on the infectivity of Qβ phage and the overall structure of its capsid.

    Science.gov (United States)

    Loison, Pauline; Majou, Didier; Gelhaye, Eric; Boudaud, Nicolas; Gantzer, Christophe

    2016-11-01

    Qβ phages infect Escherichia coli in the human gut by recognizing F-pili as receptors. Infection therefore occurs under reducing conditions induced by physiological agents (e.g. glutathione) or the intestinal bacterial flora. After excretion in the environment, phage particles are exposed to oxidizing conditions and sometimes disinfection. If inactivation does not occur, the phage may infect new hosts in the human gut through the oral route. During such a life cycle, we demonstrated that, outside the human gut, cysteines of the major protein capsid of Qβ phage form disulfide bonds. Disinfection with NaClO does not allow overoxidation to occur. Such oxidation induces inactivation rather by irreversible damage to the minor proteins. In the presence of glutathione, most disulfide bonds are reduced, which slightly increases the capacity of the phage to infect E. coli in vitro Such reduction is reversible and barely alters infectivity of the phage. Reduction of all disulfide bonds by dithiothreitol leads to complete capsid destabilization. These data provide new insights into how the phages are impacted by oxidizing-reducing conditions outside their host cell and raises the possibility of the intervention of the redox during life cycle of the phage. © FEMS 2016. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  17. African Swine Fever Virus Undergoes Outer Envelope Disruption, Capsid Disassembly and Inner Envelope Fusion before Core Release from Multivesicular Endosomes.

    Directory of Open Access Journals (Sweden)

    Bruno Hernáez

    2016-04-01

    Full Text Available African swine fever virus (ASFV is a nucleocytoplasmic large DNA virus (NCLDV that causes a highly lethal disease in domestic pigs. As other NCLDVs, the extracellular form of ASFV possesses a multilayered structure consisting of a genome-containing nucleoid successively wrapped by a thick protein core shell, an inner lipid membrane, an icosahedral protein capsid and an outer lipid envelope. This structural complexity suggests an intricate mechanism of internalization in order to deliver the virus genome into the cytoplasm. By using flow cytometry in combination with pharmacological entry inhibitors, as well as fluorescence and electron microscopy approaches, we have dissected the entry and uncoating pathway used by ASFV to infect the macrophage, its natural host cell. We found that purified extracellular ASFV is internalized by both constitutive macropinocytosis and clathrin-mediated endocytosis. Once inside the cell, ASFV particles move from early endosomes or macropinosomes to late, multivesicular endosomes where they become uncoated. Virus uncoating requires acidic pH and involves the disruption of the outer membrane as well as of the protein capsid. As a consequence, the inner viral membrane becomes exposed and fuses with the limiting endosomal membrane to release the viral core into the cytosol. Interestingly, virus fusion is dependent on virus protein pE248R, a transmembrane polypeptide of the inner envelope that shares sequence similarity with some members of the poxviral entry/fusion complex. Collective evidence supports an entry model for ASFV that might also explain the uncoating of other multienveloped icosahedral NCLDVs.

  18. EPIDERMAL GROWTH FACTOR RECEPTOR (EGFR AND HUMAN PAPILLOMAVIRUS (HPV L1 CAPSID PROTEIN IN CERVICAL SQUAMOUS INTRAEPITHELIAL LESIONS

    Directory of Open Access Journals (Sweden)

    Balan Raluca

    2010-09-01

    Full Text Available We analyzed the immunohistochemical pattern of epidermal growth factor receptor (EGFR in cervical squamous intraepithelial lesions (SILs in correlation with L1 HPV capsid protein, in order to determine the relationship between EGFR expression and the infection status of human papillomavirus (HPV. The study included 40 cases, 24 LSIL (low grade SIL (CIN1, cervical intraepithelial neoplasia and 16 HSIL (high grade SIL (6 cases of CIN2 and 10 cases of CIN3. The immunoexpression of L1 HPV protein was assessed on conventional cervico-vaginal smears and EGFR was immunohistochemically evaluated on the corresponding cervical biopsies. The HPV L1 capsid protein was expressed in 45.83% of LSIL and 25% of HSIL. EGFR was overexpressed in 62,4% of HSIL (58,4% CIN2 and 41,6% CIN3 and 37,6% LSIL. The immunoexpression of L1 HPV has clinical application in the progression assessment of the cervical precancerous lesions without a correlation to the grade of the cervical SIL. EGFR is expressed by all proliferating squamous epithelial cells, thus corresponding with the grade of SIL. The evaluation of EGFR status, correlated with L1 HPV protein expression, can provide useful data of progression risk of cervical squamous intraepithelial lesions

  19. Enhanced sensitivity in detection of antiviral antibody responses using biotinylation of foot-and-mouth disease virus (FMDV) capsids.

    Science.gov (United States)

    Kenney, Mary; Waters, Ryan A; Rieder, Elizabeth; Pega, Juan; Perez-Filguera, Mariano; Golde, William T

    2017-11-01

    Analysis of the immune response to infection of livestock by foot-and-mouth disease virus (FMDV) is most often reported as the serum antibody response to the virus. While measurement of neutralizing antibody has been sensitive and specific, measurements of the quality of the antibody response are less robust. Determining the immunoglobulin (Ig) isotype of the serum antibody response provides a deeper understanding of the biology of the response and more sensitive methods for these assays will facilitate analyses of B cell mediated immunity. We tested the hypothesis that using the virus as the molecular probe could be achieved by adding tags to the surface of the FMDV capsid, and that would enhance sensitivity in assays for anti-FMDV antibody responses. The use of a FLAG-tagged virus in these assays failed to yield improvement whereas chemically biotinylating the virus capsid resulted in significant enhancement of the signal. Here we describe methods using biotinylated virus for measuring anti-viral antibody in serum and antibody secreting cells (ASCs) in blood that are sensitive and specific. Finally, we describe using the biotinylated virus in flow cytometry where such assays should greatly enhance the analysis of anti-virus antibody producing B cells, allowing the investigator to focus on only the FMDV specific B cells when analyzing the development of the B cell response to either infection or vaccination. Published by Elsevier B.V.

  20. Synthesis and Preclinical Evaluation of Radioiodinated Hypericin Dicarboxylic Acid as a Necrosis Avid Agent in Rat Models of Induced Hepatic, Muscular, and Myocardial Necroses.

    Science.gov (United States)

    Li, Jindian; Zhang, Jian; Yang, Shengwei; Jiang, Cuihua; Zhang, DongJian; Jin, Qiaomei; Wang, Qin; Wang, Cong; Ni, Yicheng; Yin, Zhiqi; Song, Shaoli

    2016-01-04

    Myocardial infarction (MI) leads to substantial morbidity and mortality around the world. Accurate assessment of myocardial viability is essential to assist therapies and improve patient outcomes. (131)I-hypericin dicarboxylic acid ((131)I-HDA) was synthesized and evaluated as a potential diagnostic agent for earlier assessment of myocardium viability compared to its preceding counterpart (131)I-hypericin ((131)I-Hyp) with strong hydrophobic property, long plasma half-life, and high uptake in mononuclear phagocyte system (MPS). Herein, HDA was synthesized and characterized, and self-aggregation constant Kα was analyzed by spectrophotometry. Plasma half-life was determined in healthy rats by γ-counting. (131)I-HDA and (131)I-Hyp were prepared with iodogen as oxidant. In vitro necrosis avidity of (131)I-HDA and (131)I-Hyp was evaluated in necrotic cells induced by hyperthermia. Biodistribution was determined in rat models of induced necrosis using γ-counting, autoradiography, and histopathology. Earlier imaging of necrotic myocardium to assess myocardial viability was performed in rat models of reperfused myocardium infarction using single photon emission computed tomography/computed tomography (SPECT/CT). As a result, the self-aggregation constant Kα of HDA was lower than that of Hyp (105602 vs 194644, p HDA displayed a shorter blood half-life compared with (131)I-Hyp (9.21 vs 31.20 h, p HDA relative to that with (131)I-Hyp (5.48 vs 4.63, p HDA showed a higher necrotic-viable myocardium ratio (7.32 vs 3.20, p HDA achieved imaging of necrotic myocardium at 6 h postinjection (p.i.) with SPECT/CT, earlier than what (131)I-Hyp did. Therefore, (131)I-HDA may serve as a promising necrosis-avid diagnostic agent for earlier imaging of necrotic myocardium compared with (131)I-Hyp. This may support further development of radiopharmaceuticals ((123)I and (99m)Tc) based on HDA for SPECT/CT of necrotic myocardium.

  1. PET/CT-guided percutaneous biopsy of FDG-avid metastatic bone lesions in patients with advanced lung cancer: a safe and effective technique

    Energy Technology Data Exchange (ETDEWEB)

    Guo, Wei; Hao, Bing; Chen, Hao-jun; Zhao, Long; Luo, Zuo-ming; Wu, Hua; Sun, Long [The First Affiliated Hospital of Xiamen University, Department of Nuclear Medicine and Minnan PET Center, Xiamen Cancer Hospital, Xiamen (China)

    2017-01-15

    {sup 18}F-FDG PET/CT should be performed before a diagnostic biopsy site is chosen in patients with a high clinical suspicion of aggressive, advanced tumour. The aim of this study was to evaluate the safety and efficacy of {sup 18}F-FDG PET/CT in guiding biopsy of bone metastases in patients with advanced lung cancer. PET/CT-guided percutaneous core biopsies were performed in 51 consecutive patients with suspected lung cancer and {sup 18}F-FDG-avid bone lesions after whole-body {sup 18}F-FDG PET/CT scans. Generally, one tissue sample was obtained from each patient. The final diagnoses were established on the basis of the histology results. The histopathological and molecular testing results were systematically evaluated. A total of 53 samples were obtained for histological examination or molecular testing as a second biopsy was required in two patients in whom the pathological diagnosis was unclear following the first biopsy. The pathological diagnosis and lung cancer classification were confirmed in 48 patients. The epidermal growth factor receptor mutation status was determined in 23 biopsies, and the mutation rate was 30.4 % (7/23). The anaplastic lymphoma kinase mutation status was determined in 19 biopsies, and the mutation rate was 31.6 % (6/19). Two of the 51 biopsies were positive for non-Hodgkin's lymphoma and one was positive for metastatic renal cell carcinoma. The first-time diagnostic success rate of biopsy was 96.1 % (49/51) and the overall diagnostic success rate and sensitivity were 100 %. All 51 patients were eventually confirmed as having stage IV disease. No serious complications were encountered and the average biopsy time was 30 min. PET/CT-guided percutaneous biopsy of {sup 18}F-FDG-avid bone metastases is an effective and safe method that yields a high diagnostic success rate in the evaluation of hypermetabolic bone lesions in patients with suspected advanced lung cancer. (orig.)

  2. Cryo-electron Microscopy Reconstruction and Stability Studies of the Wild Type and the R432A Variant of Adeno-associated Virus Type 2 Reveal that Capsid Structural Stability Is a Major Factor in Genome Packaging.

    Science.gov (United States)

    Drouin, Lauren M; Lins, Bridget; Janssen, Maria; Bennett, Antonette; Chipman, Paul; McKenna, Robert; Chen, Weijun; Muzyczka, Nicholas; Cardone, Giovanni; Baker, Timothy S; Agbandje-McKenna, Mavis

    2016-10-01

    The adeno-associated viruses (AAV) are promising therapeutic gene delivery vectors and better understanding of their capsid assembly and genome packaging mechanism is needed for improved vector production. Empty AAV capsids assemble in the nucleus prior to genome packaging by virally encoded Rep proteins. To elucidate the capsid determinants of this process, structural differences between wild-type (wt) AAV2 and a packaging deficient variant, AAV2-R432A, were examined using cryo-electron microscopy and three-dimensional image reconstruction both at an ∼5.0-Å resolution (medium) and also at 3.8- and 3.7-Å resolutions (high), respectively. The high resolution structures showed that removal of the arginine side chain in AAV2-R432A eliminated hydrogen bonding interactions, resulting in altered intramolecular and intermolecular interactions propagated from under the 3-fold axis toward the 5-fold channel. Consistent with these observations, differential scanning calorimetry showed an ∼10°C decrease in thermal stability for AAV2-R432A compared to wt-AAV2. In addition, the medium resolution structures revealed differences in the juxtaposition of the less ordered, N-terminal region of their capsid proteins, VP1/2/3. A structural rearrangement in AAV2-R432A repositioned the βA strand region under the icosahedral 2-fold axis rather than antiparallel to the βB strand, eliminating many intramolecular interactions. Thus, a single amino acid substitution can significantly alter the AAV capsid integrity to the extent of reducing its stability and possibly rendering it unable to tolerate the stress of genome packaging. Furthermore, the data show that the 2-, 3-, and 5-fold regions of the capsid contributed to producing the packaging defect and highlight a tight connection between the entire capsid in maintaining packaging efficiency. The mechanism of AAV genome packaging is still poorly understood, particularly with respect to the capsid determinants of the required capsid

  3. Comparison of two threshold detection criteria methodologies for determination of probe positivity for intraoperative in situ identification of presumed abnormal 18F-FDG-avid tissue sites during radioguided oncologic surgery.

    Science.gov (United States)

    Chapman, Gregg J; Povoski, Stephen P; Hall, Nathan C; Murrey, Douglas A; Lee, Robert; Martin, Edward W

    2014-09-13

    Intraoperative in situ identification of (18)F-FDG-avid tissue sites during radioguided oncologic surgery remains a significant challenge for surgeons. The purpose of our study was to evaluate the 1.5-to-1 ratiometric threshold criteria method versus the three-sigma statistical threshold criteria method for determination of gamma detection probe positivity for intraoperative in situ identification of presumed abnormal (18)F-FDG-avid tissue sites in a manner that was independent of the specific type of gamma detection probe used. From among 52 patients undergoing appropriate in situ evaluation of presumed abnormal (18)F-FDG-avid tissue sites during (18)F-FDG-directed surgery using 6 available gamma detection probe systems, a total of 401 intraoperative gamma detection probe measurement sets of in situ counts per second measurements were cumulatively taken. For the 401 intraoperative gamma detection probe measurement sets, probe positivity was successfully met by the 1.5-to-1 ratiometric threshold criteria method in 150/401 instances (37.4%) and by the three-sigma statistical threshold criteria method in 259/401 instances (64.6%) (P < 0.001). Likewise, the three-sigma statistical threshold criteria method detected true positive results at target-to-background ratios much lower than the 1.5-to-1 target-to-background ratio of the 1.5-to-1 ratiometric threshold criteria method. The three-sigma statistical threshold criteria method was significantly better than the 1.5-to-1 ratiometric threshold criteria method for determination of gamma detection probe positivity for intraoperative in situ detection of presumed abnormal (18)F-FDG-avid tissue sites during radioguided oncologic surgery. This finding may be extremely important for reshaping the ongoing and future research and development of gamma detection probe systems that are necessary for optimizing the in situ detection of radioisotopes of higher-energy gamma photon emissions used during radioguided oncologic surgery.

  4. Potential role of combined FDG PET/CT & contrast enhancement MRI in a rectal carcinoma model with nodal metastases characterized by a poor FDG-avidity.

    Science.gov (United States)

    Farace, Paolo; Conti, Giamaica; Merigo, Flavia; Tambalo, Stefano; Marzola, Pasquina; Sbarbati, Andrea; Quarta, Carmelo; D'Ambrosio, Daniela; Chondrogiannis, Sotirios; Nanni, Cristina; Rubello, Domenico

    2012-04-01

    To investigate the additional role of MRI contrast enhancement (CE) in the primary tumor and the FDG uptake at PET in the lymph-node metastases. A model of colorectal cancer induced by orthotopic HT-29 cells microinjection, producing pelvic lymph node metastases, was assessed using CE-MRI and FDG-PET. Histology and GLUT-1 immunohistochemistry were performed on primary tumors and iliac lymph nodes. Primary tumors were characterized by low FDG-uptake but high CE-MRI, particularly at tumor periphery. Undetectable FDG-uptake characterized the metastatic lymph-nodes. Histology revealed large stromal bundles at tumor periphery and a dense network of stromal fibers and neoplastic cells in the inner portion of the tumors. Both primary tumors and positive lymph nodes showed poor GLUT-1 staining. Our data support the complementary role of MRI-CE and FDG PET in some types of carcinomas characterized by abundant cancer-associated stroma and poor FDG avidity consequent to poor GLUT-1 transported. In these tumors FDG-PET alone may be not completely adequate to obtain an adequate tumor radiotherapy planning, and a combination with dual CE-MRI is strongly recommended. Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.

  5. Potential role of combined FDG PET/CT and contrast enhancement MRI in a rectal carcinoma model with nodal metastases characterized by a poor FDG-avidity

    International Nuclear Information System (INIS)

    Farace, Paolo; Conti, Giamaica; Merigo, Flavia; Tambalo, Stefano; Marzola, Pasquina; Sbarbati, Andrea; Quarta, Carmelo; D’Ambrosio, Daniela; Chondrogiannis, Sotirios; Nanni, Cristina; Rubello, Domenico

    2012-01-01

    Purpose: To investigate the additional role of MRI contrast enhancement (CE) in the primary tumor and the FDG uptake at PET in the lymph-node metastases. Materials and methods: A model of colorectal cancer induced by orthotopic HT-29 cells microinjection, producing pelvic lymph node metastases, was assessed using CE-MRI and FDG-PET. Histology and GLUT-1 immunohistochemistry were performed on primary tumors and iliac lymph nodes. Results: Primary tumors were characterized by low FDG-uptake but high CE-MRI, particularly at tumor periphery. Undetectable FDG-uptake characterized the metastatic lymph-nodes. Histology revealed large stromal bundles at tumor periphery and a dense network of stromal fibers and neoplastic cells in the inner portion of the tumors. Both primary tumors and positive lymph nodes showed poor GLUT-1 staining. Conclusion: Our data support the complementary role of MRI-CE and FDG PET in some types of carcinomas characterized by abundant cancer-associated stroma and poor FDG avidity consequent to poor GLUT-1 transported. In these tumors FDG-PET alone may be not completely adequate to obtain an adequate tumor radiotherapy planning, and a combination with dual CE-MRI is strongly recommended.

  6. Total iron binding capacity

    Science.gov (United States)

    ... page: //medlineplus.gov/ency/article/003489.htm Total iron binding capacity To use the sharing features on this page, please enable JavaScript. Total iron binding capacity (TIBC) is a blood test to ...

  7. Evidence that a synthetic amyloid-ß oligomer-binding peptide (ABP) targets amyloid-ß deposits in transgenic mouse brain and human Alzheimer's disease brain.

    Science.gov (United States)

    Chakravarthy, Balu; Ito, Shingo; Atkinson, Trevor; Gaudet, Chantal; Ménard, Michel; Brown, Leslie; Whitfield, James

    2014-03-14

    The synthetic ~5 kDa ABP (amyloid-ß binding peptide) consists of a region of the 228 kDa human pericentrioloar material-1 (PCM-1) protein that selectively and avidly binds in vitro Aβ1-42 oligomers, believed to be key co-drivers of Alzheimer's disease (AD), but not monomers (Chakravarthy et al., (2013) [3]). ABP also prevents Aß1-42 from triggering the apoptotic death of cultured human SHSY5Y neuroblasts, likely by sequestering Aß oligomers, suggesting that it might be a potential AD therapeutic. Here we support this possibility by showing that ABP also recognizes and binds Aβ1-42 aggregates in sections of cortices and hippocampi from brains of AD transgenic mice and human AD patients. More importantly, ABP targets Aβ1-42 aggregates when microinjected into the hippocampi of the brains of live AD transgenic mice. Crown Copyright © 2014. Published by Elsevier Inc. All rights reserved.

  8. A new human IgG avidity test, using mixtures of recombinant antigens (rROP1, rSAG2, rGRA6), for the diagnosis of difficult-to-identify phases of toxoplasmosis.

    Science.gov (United States)

    Drapała, Dorota; Holec-Gąsior, Lucyna; Kur, Józef; Ferra, Bartłomiej; Hiszczyńska-Sawicka, Elżbieta; Lautenbach, Dariusz

    2014-07-01

    The preliminary diagnostic utility of two mixtures of Toxoplasma gondii recombinant antigens (rROP1+rSAG2 and rROP1+rGRA6) in IgG ELISA and IgG avidity test has been evaluated. A total of 173 serum samples from patients with toxoplasmosis and seronegative people were examined. The sensitivity of IgG ELISA for rROP1+rSAG2 and rROP1+rGRA6 was 91.1% and 76.7%, respectively, while the reactivity for sera from patients where acute toxoplasmosis was suspected was higher, at 100% and 95.4%, respectively, than for people with chronic infection, at 88.2% and 70.6%. In this study a different trend in avidity maturation of IgG antibodies for two mixtures of proteins in comparison with native antigen was observed. The results suggest that a new IgG avidity test using the mixtures of recombinant antigens may be useful for the diagnosis of difficult-to-identify phases of toxoplasmosis. For this reason, selected mixtures after the additional tests on groups of sera with well-defined dates of infection could be used as a better alternative to the native antigens of the parasite in the serodiagnosis of human T. gondii infection. Copyright © 2014 Elsevier Inc. All rights reserved.

  9. Use of an in vivo FTA assay to assess the magnitude, functional avidity and epitope variant cross-reactivity of T cell responses following HIV-1 recombinant poxvirus vaccination.

    Directory of Open Access Journals (Sweden)

    Danushka K Wijesundara

    Full Text Available Qualitative characteristics of cytotoxic CD8+ T cells (CTLs are important in measuring the effectiveness of CTLs in controlling HIV-1 infections. Indeed, in recent studies patients who are naturally resistant to HIV-1 infections have been shown to possess CTLs that are of high functional avidity and have a high capacity to recognize HIV epitope variants, when compared to HIV-1 infection progressors. When developing efficacious vaccines, assays that can effectively measure CTL quality specifically in vivo are becoming increasingly important. Here we report the use of a recently developed high-throughput multi-parameter technique, known as the fluorescent target array (FTA assay, to simultaneously measure CTL killing magnitude, functional avidity and epitope variant cross-reactivity in real time in vivo. In the current study we have applied the FTA assay as a screening tool to assess a large cohort of over 20 different HIV-1 poxvirus vaccination strategies in mice. This screen revealed that heterologous poxvirus prime-boost vaccination regimes (i.e., recombinant fowlpox (FPV-HIV prime followed by a recombinant vaccinia virus (VV-HIV booster were the most effective in generating high quality CTL responses in vivo. In conclusion, we have demonstrated how the FTA assay can be utilized as a cost effective screening tool (by reducing the required number of animals by >100 fold, to evaluate a large range of HIV-1 vaccination strategies in terms of CTL avidity and variant cross-reactivity in an in vivo setting.

  10. Use of an in vivo FTA assay to assess the magnitude, functional avidity and epitope variant cross-reactivity of T cell responses following HIV-1 recombinant poxvirus vaccination.

    Science.gov (United States)

    Wijesundara, Danushka K; Ranasinghe, Charani; Jackson, Ronald J; Lidbury, Brett A; Parish, Christopher R; Quah, Benjamin J C

    2014-01-01

    Qualitative characteristics of cytotoxic CD8+ T cells (CTLs) are important in measuring the effectiveness of CTLs in controlling HIV-1 infections. Indeed, in recent studies patients who are naturally resistant to HIV-1 infections have been shown to possess CTLs that are of high functional avidity and have a high capacity to recognize HIV epitope variants, when compared to HIV-1 infection progressors. When developing efficacious vaccines, assays that can effectively measure CTL quality specifically in vivo are becoming increasingly important. Here we report the use of a recently developed high-throughput multi-parameter technique, known as the fluorescent target array (FTA) assay, to simultaneously measure CTL killing magnitude, functional avidity and epitope variant cross-reactivity in real time in vivo. In the current study we have applied the FTA assay as a screening tool to assess a large cohort of over 20 different HIV-1 poxvirus vaccination strategies in mice. This screen revealed that heterologous poxvirus prime-boost vaccination regimes (i.e., recombinant fowlpox (FPV)-HIV prime followed by a recombinant vaccinia virus (VV)-HIV booster) were the most effective in generating high quality CTL responses in vivo. In conclusion, we have demonstrated how the FTA assay can be utilized as a cost effective screening tool (by reducing the required number of animals by >100 fold), to evaluate a large range of HIV-1 vaccination strategies in terms of CTL avidity and variant cross-reactivity in an in vivo setting.

  11. Role of a nuclear localization signal on the minor capsid Proteins VP2 and VP3 in BKPyV nuclear entry

    Energy Technology Data Exchange (ETDEWEB)

    Bennett, Shauna M. [Cellular and Molecular Biology Program University of Michigan 1150W Medical Center Dr 5724 Medical Science Bldg II Ann Arbor, MI 48109 (United States); Zhao, Linbo [Doctoral Program in Cancer Biology Program University of Michigan 1150W Medical Center Dr 5724 Medical Science Bldg II Ann Arbor, MI 48109 (United States); Bosard, Catherine [Department of Microbiology and Immunology University of Michigan 1150W Medical Center Dr 5724 Medical Science Bldg II Ann Arbor, MI 48109 (United States); Imperiale, Michael J., E-mail: imperial@umich.edu [Cellular and Molecular Biology Program University of Michigan 1150W Medical Center Dr 5724 Medical Science Bldg II Ann Arbor, MI 48109 (United States); Doctoral Program in Cancer Biology Program University of Michigan 1150W Medical Center Dr 5724 Medical Science Bldg II Ann Arbor, MI 48109 (United States); Department of Microbiology and Immunology University of Michigan 1150W Medical Center Dr 5724 Medical Science Bldg II Ann Arbor, MI 48109 (United States)

    2015-01-01

    BK Polyomavirus (BKPyV) is a ubiquitous nonenveloped human virus that can cause severe disease in immunocompromised populations. After internalization into renal proximal tubule epithelial cells, BKPyV traffics through the ER and enters the cytosol. However, it is unclear how the virus enters the nucleus. In this study, we elucidate a role for the nuclear localization signal located on the minor capsid proteins VP2 and VP3 during infection. Site-directed mutagenesis of a single lysine in the basic region of the C-terminus of the minor capsid proteins abrogated their nuclear localization, and the analogous genomic mutation reduced infectivity. Additionally, through use of the inhibitor ivermectin and knockdown of importin β1, we found that the importin α/β pathway is involved during infection. Overall these data are the first to show the significance of the NLS of the BKPyV minor capsid proteins during infection in a natural host cell. - Highlights: • Polyomaviruses must deliver their genome to the nucleus to replicate. • The minor capsid proteins have a well-conserved nuclear localization signal. • Mutation of this NLS diminishes, but does not completely inhibit, infection.

  12. Novel Infectivity-Enhanced Oncolytic Adenovirus with a Capsid-Incorporated Dual-Imaging Moiety for Monitoring Virotherapy in Ovarian Cancer

    Directory of Open Access Journals (Sweden)

    Kristopher J. Kimball

    2009-09-01

    Full Text Available We sought to develop a cancer-targeted, infectivity-enhanced oncolytic adenovirus that embodies a capsid-labeling fusion for non-invasive dual-modality imaging of ovarian cancer virotherapy. A functional fusion protein composed of fluorescent and nuclear imaging tags was genetically incorporated into the capsid of an infectivity-enhanced conditionally replicative adenovirus. Incorporation of herpes simplex virus thymidine kinase (HSV-tk and monomeric red fluorescent protein 1 (mRFP1 into the viral capsid and its genomic stability were verified by molecular analyses. Replication and oncolysis were evaluated in ovarian cancer cells. Fusion functionality was confirmed by in vitro gamma camera and fluorescent microscopy imaging. Comparison of tk-mRFP virus to single-modality controls revealed similar replication efficiency and oncolytic potency. Molecular fusion did not abolish enzymatic activity of HSV-tk as the virus effectively phosphorylated thymidine both ex vivo and in vitro. In vitro fluorescence imaging demonstrated a strong correlation between the intensity of fluorescent signal and cytopathic effect in infected ovarian cancer cells, suggesting that fluorescence can be used to monitor viral replication. We have in vitro validated a new infectivity-enhanced oncolytic adenovirus with a dual-imaging modality-labeled capsid, optimized for ovarian cancer virotherapy. The new agent could provide incremental gains toward climbing the barriers for achieving conditionally replicated adenovirus efficacy in human trials.

  13. Recombinant human adenovirus-5 expressing capsid proteins of Indian vaccine strains of foot-and-mouth disease virus elicits effective antibody response in cattle

    Science.gov (United States)

    Recombinant adenovirus-5 vectored foot-and-mouth disease constructs (Ad5- FMD) were made for three Indian vaccine virus serotypes O,A and Asia 1. Constructs co-expressing foot-and- mouth disease virus (FMDV) capsid and viral 3C protease sequences, were evaluated for their ability to induce a neutral...

  14. Bacterial surface-displayed GII.4 human norovirus capsid proteins bound to surface of Romaine lettuce through HBGA-like molecules

    Science.gov (United States)

    Human Noroviruses (HuNoVs) are the main cause of nonbacterial gastroenteritis. Contaminated produce is a main vehicle for dissemination of HuNoVs. In this study, we used an ice nucleation protein (INP) mediated surface display system to present the protruding domain of GII.4 HuNoV capsid protein (G...

  15. Vaccination of mice with plasmids expressing processed capsid protein of foot-and-mouth disease virus - Importance of dominant and subdominant epitopes for antigenicity and protection

    DEFF Research Database (Denmark)

    Frimann, Tine; Barfoed, Annette Malene; Aasted, Bent

    2007-01-01

    The capsid of foot-and-mouth disease virus (FMDV) displays several independent B cell epitopes, which stimulate the production of neutralising antibodies. Some of these epitopes are highly variable between virus strains, but dominate the immune response. The site A on VP1 is the most prominent...

  16. Role of a nuclear localization signal on the minor capsid Proteins VP2 and VP3 in BKPyV nuclear entry

    International Nuclear Information System (INIS)

    Bennett, Shauna M.; Zhao, Linbo; Bosard, Catherine; Imperiale, Michael J.

    2015-01-01

    BK Polyomavirus (BKPyV) is a ubiquitous nonenveloped human virus that can cause severe disease in immunocompromised populations. After internalization into renal proximal tubule epithelial cells, BKPyV traffics through the ER and enters the cytosol. However, it is unclear how the virus enters the nucleus. In this study, we elucidate a role for the nuclear localization signal located on the minor capsid proteins VP2 and VP3 during infection. Site-directed mutagenesis of a single lysine in the basic region of the C-terminus of the minor capsid proteins abrogated their nuclear localization, and the analogous genomic mutation reduced infectivity. Additionally, through use of the inhibitor ivermectin and knockdown of importin β1, we found that the importin α/β pathway is involved during infection. Overall these data are the first to show the significance of the NLS of the BKPyV minor capsid proteins during infection in a natural host cell. - Highlights: • Polyomaviruses must deliver their genome to the nucleus to replicate. • The minor capsid proteins have a well-conserved nuclear localization signal. • Mutation of this NLS diminishes, but does not completely inhibit, infection

  17. Comparison of classical and affinity purification techniques of Mason-Pfizer monkey virus capsid protein: The Alteration of the product by an affinity tag

    Czech Academy of Sciences Publication Activity Database

    Rumlová, Michaela; Benedíková, Jitka; Cubínková, Romana; Pichová, Iva; Ruml, Tomáš

    2001-01-01

    Roč. 23, - (2001), s. 75-83 ISSN 1046-5928 R&D Projects: GA ČR GA203/00/1005 Institutional research plan: CEZ:AV0Z4055905 Keywords : Mason-Pfizer monkey virus * capsid protein Subject RIV: CE - Biochemistry Impact factor: 1.497, year: 2001

  18. Prognostic relevance of human papillomavirus L1 capsid protein detection within mild and moderate dysplastic lesions of the cervix uteri in combination with p16 biomarker

    DEFF Research Database (Denmark)

    Hilfrich, Ralf; Hariri, Jalil

    2008-01-01

    OBJECTIVE: To proof the prognostic relevance of HPV L1 capsid protein detection on colposcopically-guided punch biopsies in combination with p16. STUDY DESIGN: Sections of colposcopically-guided punch biopsies from 191 consecutive cases with at least 5 years of follow-up were stained with HPV L1 ...

  19. Structures of Adenovirus Incomplete Particles Clarify Capsid Architecture and Show Maturation Changes of Packaging Protein L1 52/55k.

    Science.gov (United States)

    Condezo, Gabriela N; Marabini, Roberto; Ayora, Silvia; Carazo, José M; Alba, Raúl; Chillón, Miguel; San Martín, Carmen

    2015-09-01

    Adenovirus is one of the most complex icosahedral, nonenveloped viruses. Even after its structure was solved at near-atomic resolution by both cryo-electron microscopy and X-ray crystallography, the location of minor coat proteins is still a subject of debate. The elaborated capsid architecture is the product of a correspondingly complex assembly process, about which many aspects remain unknown. Genome encapsidation involves the concerted action of five virus proteins, and proteolytic processing by the virus protease is needed to prime the virion for sequential uncoating. Protein L1 52/55k is required for packaging, and multiple cleavages by the maturation protease facilitate its release from the nascent virion. Light-density particles are routinely produced in adenovirus infections and are thought to represent assembly intermediates. Here, we present the molecular and structural characterization of two different types of human adenovirus light particles produced by a mutant with delayed packaging. We show that these particles lack core polypeptide V but do not lack the density corresponding to this protein in the X-ray structure, thereby adding support to the adenovirus cryo-electron microscopy model. The two types of light particles present different degrees of proteolytic processing. Their structures provide the first glimpse of the organization of L1 52/55k protein inside the capsid shell and of how this organization changes upon partial maturation. Immature, full-length L1 52/55k is poised beneath the vertices to engage the virus genome. Upon proteolytic processing, L1 52/55k disengages from the capsid shell, facilitating genome release during uncoating. Adenoviruses have been extensively characterized as experimental systems in molecular biology, as human pathogens, and as therapeutic vectors. However, a clear picture of many aspects of their basic biology is still lacking. Two of these aspects are the location of minor coat proteins in the capsid and the

  20. Mapping the Structural Determinants Responsible for Enhanced T Cell Activation to the Immunogenic Adeno-Associated Virus Capsid from Isolate Rhesus 32.33

    Science.gov (United States)

    Mays, Lauren E.; Wang, Lili; Tenney, Rebeca; Bell, Peter; Nam, Hyun-Joo; Lin, Jianping; Gurda, Brittney; Van Vliet, Kim; Mikals, Kyle; Agbandje-McKenna, Mavis

    2013-01-01

    Avoiding activation of immunity to vector-encoded proteins is critical to the safe and effective use of adeno-associated viral (AAV) vectors for gene therapy. While commonly used serotypes, such as AAV serotypes 1, 2, 7, 8, and 9, are often associated with minimal and/or dysfunctional CD8+ T cell responses in mice, the threshold for immune activation appears to be lower in higher-order species. We have modeled this discrepancy within the mouse by identifying two capsid variants with differential immune activation profiles: AAV serotype 8 (AAV8) and a hybrid between natural rhesus isolates AAVrh32 and AAVrh33 (AAVrh32.33). Here, we aimed to characterize the structural determinants of the AAVrh32.33 capsid that augment cellular immunity to vector-encoded proteins or those of AAV8 that may induce tolerance. We hypothesized that the structural domain responsible for differential immune activation could be mapped to surface-exposed regions of the capsid, such as hypervariable regions (HVRs) I to IX of VP3. To test this, a series of hybrid AAV capsids was constructed by swapping domains between AAV8 and AAVrh32.33. By comparing their ability to generate transgene-specific T cells in vivo versus the stability of transgene expression in the muscle, we confirmed that the functional domain lies within the VP3 portion of the capsid. Our studies were able to exclude the regions of VP3 which are not sufficient for augmenting the cellular immune response, notably, HVRs I, II, and V. We have also identified HVR IV as a region of interest in conferring the efficiency and stability of muscle transduction to AAVrh32.33. PMID:23720715

  1. Uncoating-like modification of poliovirus capsid resulting from the cooperative effects of subfreezing temperature and submolar concentrations of urea

    International Nuclear Information System (INIS)

    Mandel, B.

    1982-01-01

    Inactivation of poliovirus at subfreezing temperature in the presence of unusually low concentrations of urea (<=0.5 M) was investigated. Whereas serotypes 1 and 2 are very sensitive, type 3 is resistant. Inactivation cannot be attributed to concentration of solutes since temperature must be reduced below -13degC for loss of infectivity. Characteristics of the inactivated virion are similar to those of virions in the early stages of uncoating in HeLa cells, viz., loss of infectivity, sensitivity to proteases and detergents, change in isoelectric point, retention of intact genome, and in some instances, loss of VP4. The molecular basis for inactivation is considered to be dissociation of water bound to capsid proteins thereby causing irreversible denaturation of native tertiary structure. The results of this study are discussed in terms of their relevance to the early stages of uncoating in vivo. (Author)

  2. Effect of capsid proteins to ICG mass ratio on fluorescent quantum yield of virus-resembling optical nano-materials

    Science.gov (United States)

    Gupta, Sharad; Ico, Gerardo; Matsumura, Paul; Rao, A. L. N.; Vullev, Valentine; Anvari, Bahman

    2012-03-01

    We recently reported construction of a new type of optical nano-construct composed of genome-depleted plant infecting brome mosaic virus (BMV) doped with Indocyanine green (ICG), an FDA-approved chromophore. We refer to these constructs as optical viral ghosts (OVGs) since only the capsid protein (CP) subunits of BMV remain to encapsulate ICG. To utilize OVGs as effective nano-probes in fluorescence imaging applications, their fluorescence quantum yield needs to be maximized. In this study, we investigate the effect of altering the CP to ICG mass ratio on the fluorescent quantum yield of OVGs. Results of this study provide the basis for construction of OVGs with optimal amounts of CP and ICG to yield maximal fluorescence quantum yield.

  3. Sequence analysis of malacoherpesvirus proteins: Pan-herpesvirus capsid module and replication enzymes with an ancient connection to "Megavirales".

    Science.gov (United States)

    Mushegian, Arcady; Karin, Eli Levy; Pupko, Tal

    2018-01-01

    The order Herpesvirales includes animal viruses with large double-strand DNA genomes replicating in the nucleus. The main capsid protein in the best-studied family Herpesviridae contains a domain with HK97-like fold related to bacteriophage head proteins, and several virion maturation factors are also homologous between phages and herpesviruses. The origin of herpesvirus DNA replication proteins is less well understood. While analyzing the genomes of herpesviruses in the family Malacohepresviridae, we identified nearly 30 families of proteins conserved in other herpesviruses, including several phage-related domains in morphogenetic proteins. Herpesvirus DNA replication factors have complex evolutionary history: some are related to cellular proteins, but others are closer to homologs from large nucleocytoplasmic DNA viruses. Phylogenetic analyses suggest that the core replication machinery of herpesviruses may have been recruited from the same pool as in the case of other large DNA viruses of eukaryotes. Published by Elsevier Inc.

  4. Down-Regulation of Na+/K+ ATPase Activity by Human Parvovirus B19 Capsid Protein VP1

    Directory of Open Access Journals (Sweden)

    Ahmad Almilaji

    2013-05-01

    Full Text Available Background/Aims: Human parvovirus B19 (B19V may cause inflammatory cardiomyopathy (iCMP which is accompanied by endothelial dysfunction. The B19V capsid protein VP1 contains a lysophosphatidylcholine producing phospholipase A2 (PLA sequence. Lysophosphatidylcholine has in turn been shown to inhibit Na+/K+ ATPase. The present study explored whether VP1 modifies Na+/K+ ATPase activity. Methods: Xenopus oocytes were injected with cRNA encoding VP1 isolated from a patient suffering from fatal B19V-iCMP or cRNA encoding PLA2-negative VP1 mutant (H153A and K+ induced pump current (Ipump as well as ouabain-inhibited current (Iouabain both reflecting Na+/K+-ATPase activity were determined by dual electrode voltage clamp. Results: Injection of cRNA encoding VP1, but not of VP1(H153A or water, was followed by a significant decrease of both, Ipump and Iouabain in Xenopus oocytes. The effect was not modified by inhibition of transcription with actinomycin (10 µM for 36 hours but was abrogated in the presence of PLA2 specific blocker 4-bromophenacylbromide (50 µM and was mimicked by lysophosphatidylcholine (0.5 - 1 µg/ml. According to whole cell patch clamp, lysophosphatidylcholine (1 µg /ml similarly decreased Ipump in human microvascular endothelial cells (HMEC. Conclusion: The B19V capsid protein VP1 is a powerful inhibitor of host cell Na+/K+ ATPase, an effect at least partially due to phospholipase A2 (PLA2 dependent formation of lysophosphatidylcholine.

  5. B-cell depletion is protective against anti-AAV capsid immune response: a human subject case study

    Directory of Open Access Journals (Sweden)

    M Corti

    2014-01-01

    Full Text Available Gene therapy strategies for congenital myopathies may require repeat administration of adeno-associated viral (AAV vectors due to aspects of the clinical application, such as: (i administration of doses below therapeutic efficacy in patients enrolled in early phase clinical trials; (ii progressive reduction of the therapeutic gene expression over time as a result of increasing muscle mass in patients treated at a young age; and (iii a possibly faster depletion of pathogenic myofibers in this patient population. Immune response triggered by the first vector administration, and to subsequent doses, represents a major obstacle for successful gene transfer in young patients. Anti-capsid and anti-transgene product related humoral and cell-mediated responses have been previously observed in all preclinical models and human subjects who received gene therapy or enzyme replacement therapy (ERT for congenital myopathies. Immune responses may result in reduced efficacy of the gene transfer over time and/or may preclude for the possibility of re-administration of the same vector. In this study, we evaluated the immune response of a Pompe patient dosed with an AAV1-GAA vector after receiving Rituximab and Sirolimus to modulate reactions against ERT. A key finding of this single subject case report is the observation that B-cell ablation with rituximab prior to AAV vector exposure results in non-responsiveness to both capsid and transgene, therefore allowing the possibility of repeat administration in the future. This observation is significant for future gene therapy studies and establishes a clinically relevant approach to blocking immune responses to AAV vectors.

  6. Gu-4 suppresses affinity and avidity modulation of CD11b and improves the outcome of mice with endotoxemia and sepsis.

    Directory of Open Access Journals (Sweden)

    TingTing Yan

    Full Text Available BACKGROUND: Systemic leukocyte activation and disseminated leukocyte adhesion will impair the microcirculation and cause severe decrements in tissue perfusion and organ function in the process of severe sepsis. Gu-4, a lactosyl derivative, could selectively target CD11b to exert therapeutic effect in a rat model of severe burn shock. Here, we addressed whether Gu-4 could render protective effects on septic animals. METHODOLOGY/PRINCIPAL FINDINGS: On a murine model of endotoxemia induced by lipopolysaccharide (LPS, we found that the median effective dose (ED50 of Gu-4 was 0.929 mg/kg. In vivo treatment of Gu-4 after LPS challenge prominently attenuated LPS-induced lung injury and decreased lactic acid level in lung tissue. Using the ED50 of Gu-4, we also demonstrated that Gu-4 treatment significantly improved the survival rate of animals underwent sepsis induced by cecal ligation and puncture. By adhesion and transwell migration assays, we found that Gu-4 treatment inhibited the adhesion and transendothelial migration of LPS-stimulated THP-1 cells. By flow cytometry and microscopy, we demonstrated that Gu-4 treatment inhibited the exposure of active I-domain and the cluster formation of CD11b on the LPS-stimulated polymorphonuclear leukocytes. Western blot analyses further revealed that Gu-4 treatment markedly inhibited the activation of spleen tyrosine kinase in LPS-stimulated THP-1 cells. CONCLUSIONS/SIGNIFICANCE: Gu-4 improves the survival of mice underwent endotoxemia and sepsis, our in vitro investigations indicate that the possible underlying mechanism might involve the modulations of the affinity and avidity of CD11b on the leukocyte. Our findings shed light on the potential use of Gu-4, an interacting compound to CD11b, in the treatment of sepsis and septic shock.

  7. Diverse Epitope Specificity, Immunodominance Hierarchy, and Functional Avidity of Effector CD4 T Cells Established During Priming Is Maintained in Lung After Influenza A Virus Infection.

    Science.gov (United States)

    Richards, Katherine A; DiPiazza, Anthony T; Rattan, Ajitanuj; Knowlden, Zackery A G; Yang, Hongmei; Sant, Andrea J

    2018-01-01

    One of the major contributions to protective immunity to influenza viruses that is provided by virus-specific CD4 T cells is delivery of effector function to the infected lung. However, there is little known about the selection and breadth of viral epitope-specific CD4 T cells that home to the lung after their initial priming. In this study, using a mouse model of influenza A infection and an unbiased method of epitope identification, the viral epitope-specific CD4 T cells elicited after infection were identified and quantified. We found that a very diverse specificity of CD4 T cells is primed by infection, including epitopes from hemagglutinin, neuraminidase, matrix protein, nucleoprotein, and non-structural protein-1. Using peptide-specific cytokine EliSpots, the diversity and immunodominance hierarchies established in the lung-draining lymph node were compared with specificities of CD4 T cells that home to the lung. Our studies revealed that CD4 T cells of all epitope specificities identified in peripheral lymphoid tissue home back to the lung and that most of these lung-homing cells are localized within the tissue rather than the pulmonary vasculature. There is a striking shift of CD4 T cell functionality that enriches for IFN-γ production as cells are primed in the lymph node, enter the lung vasculature, and finally establish residency in the tissue, but with no apparent shifts in their functional avidity. We conclude that CD4 T cells of broad viral epitope specificity are recruited into the lung after influenza infection, where they then have the opportunity to encounter infected or antigen-bearing antigen-presenting cells.

  8. A Heterogeneous Nuclear Ribonucleoprotein A/B-Related Protein Binds to Single-Stranded DNA near the 5′ End or within the Genome of Feline Parvovirus and Can Modify Virus Replication

    Science.gov (United States)

    Wang, Dai; Parrish, Colin R.

    1999-01-01

    Phage display of cDNA clones prepared from feline cells was used to identify host cell proteins that bound to DNA-containing feline panleukopenia virus (FPV) capsids but not to empty capsids. One gene found in several clones encoded a heterogeneous nuclear ribonucleoprotein (hnRNP)-related protein (DBP40) that was very similar in sequence to the A/B-type hnRNP proteins. DBP40 bound specifically to oligonucleotides representing a sequence near the 5′ end of the genome which is exposed on the outside of the full capsid but did not bind most other terminal sequences. Adding purified DBP40 to an in vitro fill-in reaction using viral DNA as a template inhibited the production of the second strand after nucleotide (nt) 289 but prior to nt 469. DBP40 bound to various regions of the viral genome, including a region between nt 295 and 330 of the viral genome which has been associated with transcriptional attenuation of the parvovirus minute virus of mice, which is mediated by a stem-loop structure of the DNA and cellular proteins. Overexpression of the protein in feline cells from a plasmid vector made them largely resistant to FPV infection. Mutagenesis of the protein binding site within the 5′ end viral genome did not affect replication of the virus. PMID:10438866

  9. Feature Binding in Zebrafish

    Directory of Open Access Journals (Sweden)

    P Neri

    2012-07-01

    Full Text Available Binding operations are primarily ascribed to cortex or similarly complex avian structures. My experiments show that the zebrafish, a lower vertebrate lacking cortex, supports visual feature binding of form and motion for the purpose of social behavior. These results challenge the notion that feature binding may require highly evolved neural structures and demonstrate that the nervous system of lower vertebrates can afford unexpectedly complex computations.

  10. VP1, the major capsid protein of the mouse polyomavirus, binds microtubules, promotes their acetylation and blocks the host cell cycle

    Czech Academy of Sciences Publication Activity Database

    Horníková, L.; Fraiberk, M.; Man, Petr; Janovec, V.; Forstová, J.

    2017-01-01

    Roč. 284, č. 2 (2017), s. 301-323 E-ISSN 1742-4658 R&D Projects: GA MŠk(CZ) ED1.1.00/02.0109; GA MŠk(CZ) LO1509 Grant - others:Ministerstvo pro místní rozvoj(CZ) CZ.2.16./3.1.00/24023 Institutional support: RVO:61388971 Keywords : cell cycle arrest * chaperone Hsp90 * microtubules Subject RIV: EE - Microbiology, Virology OBOR OECD: Microbiology

  11. Dextran as a Generally Applicable Multivalent Scaffold for Improving Immunoglobulin-Binding Affinities of Peptide and Peptidomimetic Ligands

    Science.gov (United States)

    2015-01-01

    Molecules able to bind the antigen-binding sites of antibodies are of interest in medicine and immunology. Since most antibodies are bivalent, higher affinity recognition can be achieved through avidity effects in which a construct containing two or more copies of the ligand engages both arms of the immunoglobulin simultaneously. This can be achieved routinely by immobilizing antibody ligands at high density on solid surfaces, such as ELISA plates, but there is surprisingly little literature on scaffolds that routinely support bivalent binding of antibody ligands in solution, particularly for the important case of human IgG antibodies. Here we show that the simple strategy of linking two antigens with a polyethylene glycol (PEG) spacer long enough to span the two arms of an antibody results in higher affinity binding in some, but not all, cases. However, we found that the creation of multimeric constructs in which several antibody ligands are displayed on a dextran polymer reliably provides much higher affinity binding than is observed with the monomer in all cases tested. Since these dextran conjugates are simple to construct, they provide a general and convenient strategy to transform modest affinity antibody ligands into high affinity probes. An additional advantage is that the antibody ligands occupy only a small number of the reactive sites on the dextran, so that molecular cargo can be attached easily, creating molecules capable of delivering this cargo to cells displaying antigen-specific receptors. PMID:25073654

  12. Melanin-binding radiopharmaceuticals

    International Nuclear Information System (INIS)

    Packer, S.; Fairchild, R.G.; Watts, K.P.; Greenberg, D.; Hannon, S.J.

    1980-01-01

    The scope of this paper is limited to an analysis of the factors that are important to the relationship of radiopharmaceuticals to melanin. While the authors do not attempt to deal with differences between melanin-binding vs. melanoma-binding, a notable variance is assumed

  13. Competitive protein binding assay

    International Nuclear Information System (INIS)

    Kaneko, Toshio; Oka, Hiroshi

    1975-01-01

    The measurement of cyclic GMP (cGMP) by competitive protein binding assay was described and discussed. The principle of binding assay was represented briefly. Procedures of our method by binding protein consisted of preparation of cGMP binding protein, selection of 3 H-cyclic GMP on market, and measurement procedures. In our method, binding protein was isolated from the chrysalis of silk worm. This method was discussed from the points of incubation medium, specificity of binding protein, the separation of bound cGMP from free cGMP, and treatment of tissue from which cGMP was extracted. cGMP existing in the tissue was only one tenth or one scores of cGMP, and in addition, cGMP competed with cGMP in binding with binding protein. Therefore, Murad's technique was applied to the isolation of cGMP. This method provided the measurement with sufficient accuracy; the contamination by cAMP was within several per cent. (Kanao, N.)

  14. Specific in vitro cleavage of Mason-Pfizer monkey virus capsid protein: evidence for a potential role of retroviral protease in early stages of infection

    International Nuclear Information System (INIS)

    Rumlova, Michaela; Ruml, Tomas; Pohl, Jan; Pichova, Iva

    2003-01-01

    Processing of Gag polyproteins by viral protease (PR) leads to reorganization of immature retroviral particles and formation of a ribonucleoprotein core. In some retroviruses, such as HIV and RSV, cleavage of a spacer peptide separating capsid and nucleocapsid proteins is essential for the core formation. We show here that no similar spacer peptide is present in the capsid-nucleocapsid (CA-NC) region of Mason-Pfizer monkey virus (M-PMV) and that the CA protein is cleaved in vitro by the PR within the major homology region (MHR) and the NC protein in several sites at the N-terminus. The CA cleavage product was also identified shortly after penetration of M-PMV into COS cells, suggesting that the protease-catalyzed cleavage is involved in core disintegration

  15. Cyclophilin A Levels Dictate Infection Efficiency of Human Immunodeficiency Virus Type 1 Capsid Escape Mutants A92E and G94D ▿

    Science.gov (United States)

    Ylinen, Laura M. J.; Schaller, Torsten; Price, Amanda; Fletcher, Adam J.; Noursadeghi, Mahdad; James, Leo C.; Towers, Greg J.

    2009-01-01

    Cyclophilin A (CypA) is an important human immunodeficiency virus type 1 (HIV-1) cofactor in human cells. HIV-1 A92E and G94D capsid escape mutants arise during CypA inhibition and in certain cell lines are dependent on CypA inhibition. Here we show that dependence on CypA inhibition is due to high CypA levels. Restricted HIV-1 is stable, and remarkably, restriction is augmented by arresting cell division. Nuclear entry is not inhibited. We propose that high CypA levels and capsid mutations combine to disturb uncoating, leading to poor infectivity, particularly in arrested cells. Our data suggest a role for CypA in uncoating the core of HIV-1 to facilitate integration. PMID:19073742

  16. Formation of RNA Granule-Derived Capsid Assembly Intermediates Appears To Be Conserved between Human Immunodeficiency Virus Type 1 and the Nonprimate Lentivirus Feline Immunodeficiency Virus.

    Science.gov (United States)

    Reed, Jonathan C; Westergreen, Nick; Barajas, Brook C; Ressler, Dylan T B; Phuong, Daryl J; Swain, John V; Lingappa, Vishwanath R; Lingappa, Jaisri R

    2018-05-01

    During immature capsid assembly in cells, human immunodeficiency virus type 1 (HIV-1) Gag co-opts a host RNA granule, forming a pathway of intracellular assembly intermediates containing host components, including two cellular facilitators of assembly, ABCE1 and DDX6. A similar assembly pathway has been observed for other primate lentiviruses. Here we asked whether feline immunodeficiency virus (FIV), a nonprimate lentivirus, also forms RNA granule-derived capsid assembly intermediates. First, we showed that the released FIV immature capsid and a large FIV Gag-containing intracellular complex are unstable during analysis, unlike for HIV-1. We identified harvest conditions, including in situ cross-linking, that overcame this problem, revealing a series of FIV Gag-containing complexes corresponding in size to HIV-1 assembly intermediates. Previously, we showed that assembly-defective HIV-1 Gag mutants are arrested at specific assembly intermediates; here we identified four assembly-defective FIV Gag mutants, including three not previously studied, and demonstrated that they appear to be arrested at the same intermediate as the cognate HIV-1 mutants. Further evidence that these FIV Gag-containing complexes correspond to assembly intermediates came from coimmunoprecipitations demonstrating that endogenous ABCE1 and the RNA granule protein DDX6 are associated with FIV Gag, as shown previously for HIV-1 Gag, but are not associated with a ribosomal protein, at steady state. Additionally, we showed that FIV Gag associates with another RNA granule protein, DCP2. Finally, we validated the FIV Gag-ABCE1 and FIV Gag-DCP2 interactions with proximity ligation assays demonstrating colocalization in situ Together, these data support a model in which primate and nonprimate lentiviruses form intracellular capsid assembly intermediates derived from nontranslating host RNA granules. IMPORTANCE Like HIV-1 Gag, FIV Gag assembles into immature capsids; however, it is not known whether

  17. QA prime-boost vaccination strategy in prevent serotype O FMDV infection using a "single-cycle" alphavirus vector and empty capsid particles

    DEFF Research Database (Denmark)

    Gullberg, Maria; Lohse, Louise; Bøtner, Anette

    Introduction Foot-and-mouth disease (FMD) remains one of the most economically important infectious diseases of production animals globally. Vaccination can help to control this disease, however, current vaccines based on chemically inactivated FMDV, are imperfect and there is a need for new, safe...... and effective vaccines to control FMD. There is no cross protection between the 7 serotypes but serotype O is the most abundant globally. Material and methods The FMDV capsid protein precursor (P1-2A) of strain O1 Manisa has been expressed with the FMDV 3C protease (3Cpro) using a “single cycle” packaged...... alphavirus self-replicating RNA based on Semliki Forest virus (SFV). Purified O1 Manisa empty capsid particles (ECs) have been prepared using a recombinant vaccinia virus expression system. Cattle have been vaccinated with the SFV-FMDV vectors and boosted subsequently with the ECs and then challenged...

  18. Structure, dynamics and RNA binding of the multi-domain splicing factor TIA-1

    Science.gov (United States)

    Wang, Iren; Hennig, Janosch; Jagtap, Pravin Kumar Ankush; Sonntag, Miriam; Valcárcel, Juan; Sattler, Michael

    2014-01-01

    Alternative pre-messenger ribonucleic acid (pre-mRNA) splicing is an essential process in eukaryotic gene regulation. The T-cell intracellular antigen-1 (TIA-1) is an apoptosis-promoting factor that modulates alternative splicing of transcripts, including the pre-mRNA encoding the membrane receptor Fas. TIA-1 is a multi-domain ribonucleic acid (RNA) binding protein that recognizes poly-uridine tract RNA sequences to facilitate 5′ splice site recognition by the U1 small nuclear ribonucleoprotein (snRNP). Here, we characterize the RNA interaction and conformational dynamics of TIA-1 by nuclear magnetic resonance (NMR), isothermal titration calorimetry (ITC) and small angle X-ray scattering (SAXS). Our NMR-derived solution structure of TIA-1 RRM2–RRM3 (RRM2,3) reveals that RRM2 adopts a canonical RNA recognition motif (RRM) fold, while RRM3 is preceded by an non-canonical helix α0. NMR and SAXS data show that all three RRMs are largely independent structural modules in the absence of RNA, while RNA binding induces a compact arrangement. RRM2,3 binds to pyrimidine-rich FAS pre-mRNA or poly-uridine (U9) RNA with nanomolar affinities. RRM1 has little intrinsic RNA binding affinity and does not strongly contribute to RNA binding in the context of RRM1,2,3. Our data unravel the role of binding avidity and the contributions of the TIA-1 RRMs for recognition of pyrimidine-rich RNAs. PMID:24682828

  19. Heterologous expression of full-length capsid protein of porcine circovirus 2 in Escherichia coli and its potential use for detection of antibodies

    Czech Academy of Sciences Publication Activity Database

    Marčeková, Zuzana; Psikal, P.; Kosinová, E.; Benada, Oldřich; Šebo, Peter; Bumba, Ladislav

    2009-01-01

    Roč. 162, 1-2 (2009), s. 133-141 ISSN 0166-0934 R&D Projects: GA ČR GP310/07/P115; GA MŠk 2B06161 Institutional research plan: CEZ:AV0Z50200510 Keywords : PCV 2 * Porcine circovirus * Capsid protein Subject RIV: EE - Microbiology, Virology Impact factor: 2.133, year: 2009

  20. 1H, 13C, and 15N resonance assignment of the N-terminal domainof Mason-Pfizer monkey virus capsid protein, CA 1-140

    Czech Academy of Sciences Publication Activity Database

    Macek, Pavel; Žídek, L.; Rumlová, Michaela; Pichová, Iva; Sklenář, V.

    2008-01-01

    Roč. 2, č. 1 (2008), s. 43-45 ISSN 1874-2718 R&D Projects: GA MŠk LC545; GA MŠk(CZ) LC06030; GA MŠk 1M0508 Institutional research plan: CEZ:AV0Z50200510; CEZ:AV0Z40550506 Keywords : nmr * assignment * capsid protein Subject RIV: EE - Microbiology, Virology Impact factor: 0.015, year: 2008

  1. Specific in vitro cleavage of Mason-Pfizer monkey virus capsid protein: evidence for a potential role of retroviral protease in early stages of infection

    Czech Academy of Sciences Publication Activity Database

    Rumlová, Michaela; Ruml, T.; Pohl, J.; Pichová, Iva

    2003-01-01

    Roč. 310, - (2003), s. 310-318 ISSN 0042-6822 R&D Projects: GA ČR GA203/00/1241; GA AV ČR IAB4055202 Institutional research plan: CEZ:AV0Z4055905 Keywords : M-PMV protease * HIV-1 capsid protein * HIV-1 protease Subject RIV: CE - Biochemistry Impact factor: 3.391, year: 2003

  2. A sialoreceptor binding motif in the Mycoplasma synoviae adhesin VlhA.

    Directory of Open Access Journals (Sweden)

    Meghan May

    Full Text Available Mycoplasma synoviae depends on its adhesin VlhA to mediate cytadherence to sialylated host cell receptors. Allelic variants of VlhA arise through recombination between an assemblage of promoterless vlhA pseudogenes and a single transcription promoter site, creating lineages of M. synoviae that each express a different vlhA allele. The predicted full-length VlhA sequences adjacent to the promoter of nine lineages of M. synoviae varying in avidity of cytadherence were aligned with that of the reference strain MS53 and with a 60-a.a. hemagglutinating VlhA C-terminal fragment from a Tunisian lineage of strain WVU1853(T. Seven different sequence variants of an imperfectly conserved, single-copy, 12-a.a. candidate cytadherence motif were evident amid the flanking variable residues of the 11 total sequences examined. The motif was predicted to adopt a short hairpin structure in a low-complexity region near the C-terminus of VlhA. Biotinylated synthetic oligopeptides representing four selected variants of the 12-a.a. motif, with the whole synthesized 60-a.a. fragment as a positive control, differed (P<0.01 in the extent they bound to chicken erythrocyte membranes. All bound to a greater extent (P<0.01 than scrambled or irrelevant VlhA domain negative control peptides did. Experimentally introduced branched-chain amino acid (BCAA substitutions Val3Ile and Leu7Ile did not significantly alter binding, whereas fold-destabilizing substitutions Thr4Gly and Ala9Gly tended to reduce it (P<0.05. Binding was also reduced to background levels (P<0.01 when the peptides were exposed to desialylated membranes, or were pre-saturated with free sialic acid before exposure to untreated membranes. From this evidence we conclude that the motif P-X-(BCAA-X-F-X-(BCAA-X-A-K-X-G binds sialic acid and likely mediates VlhA-dependent M. synoviae attachment to host cells. This conserved mechanism retains the potential for fine-scale rheostasis in binding avidity, which could be a

  3. A triclinic crystal structure of the carboxy-terminal domain of HIV-1 capsid protein with four molecules in the asymmetric unit reveals a novel packing interface

    International Nuclear Information System (INIS)

    Lampel, Ayala; Yaniv, Oren; Berger, Or; Bacharach, Eran; Gazit, Ehud; Frolow, Felix

    2013-01-01

    The triclinic structure of the HIV-1 capsid protein contains four molecules in the asymmetric unit that form a novel packing interface that could conceivably resemble an intermediate structure that is involved in the early steps of HIV-1 assembly. The Gag precursor is the major structural protein of the virion of human immunodeficiency virus-1 (HIV-1). Capsid protein (CA), a cleavage product of Gag, plays an essential role in virus assembly both in Gag-precursor multimerization and in capsid core formation. The carboxy-terminal domain (CTD) of CA contains 20 residues that are highly conserved across retroviruses and constitute the major homology region (MHR). Genetic evidence implies a role for the MHR in interactions between Gag precursors during the assembly of the virus, but the structural basis for this role remains elusive. This paper describes a novel triclinic structure of the HIV-1 CA CTD at 1.6 Å resolution with two canonical dimers of CA CTD in the asymmetric unit. The canonical dimers form a newly identified packing interface where interactions of four conserved MHR residues take place. This is the first structural indication that these MHR residues participate in the putative CTD–CTD interactions. These findings suggest that the molecules forming this novel interface resemble an intermediate structure that participates in the early steps of HIV-1 assembly. This interface may therefore provide a novel target for antiviral drugs

  4. Transient gene expression in serum-free suspension-growing mammalian cells for the production of foot-and-mouth disease virus empty capsids.

    Directory of Open Access Journals (Sweden)

    Ana Clara Mignaqui

    Full Text Available Foot-and-mouth disease (FMD is a highly contagious disease of cloven-hoofed animals. It produces severe economic losses in the livestock industry. Currently available vaccines are based on inactivated FMD virus (FMDV. The use of empty capsids as a subunit vaccine has been reported to be a promising candidate because it avoids the use of virus in the vaccine production and conserves the conformational epitopes of the virus. In this report, we explored transient gene expression (TGE in serum-free suspension-growing mammalian cells for the production of FMDV recombinant empty capsids as a subunit vaccine. The recombinant proteins produced, assembled into empty capsids and induced protective immune response against viral challenge in mice. Furthermore, they were recognized by anti-FMDV bovine sera. By using this technology, we were able to achieve expression levels that are compatible with the development of a vaccine. Thus, TGE of mammalian cells is an easy to perform, scalable and cost-effective technology for the production of a recombinant subunit vaccine against FMDV.

  5. Structures of minute virus of mice replication initiator protein N-terminal domain: Insights into DNA nicking and origin binding

    International Nuclear Information System (INIS)

    Tewary, Sunil K.; Liang, Lingfei; Lin, Zihan; Lynn, Annie; Cotmore, Susan F.; Tattersall, Peter; Zhao, Haiyan; Tang, Liang

    2015-01-01

    Members of the Parvoviridae family all encode a non-structural protein 1 (NS1) that directs replication of single-stranded viral DNA, packages viral DNA into capsid, and serves as a potent transcriptional activator. Here we report the X-ray structure of the minute virus of mice (MVM) NS1 N-terminal domain at 1.45 Å resolution, showing that sites for dsDNA binding, ssDNA binding and cleavage, nuclear localization, and other functions are integrated on a canonical fold of the histidine-hydrophobic-histidine superfamily of nucleases, including elements specific for this Protoparvovirus but distinct from its Bocaparvovirus or Dependoparvovirus orthologs. High resolution structural analysis reveals a nickase active site with an architecture that allows highly versatile metal ligand binding. The structures support a unified mechanism of replication origin recognition for homotelomeric and heterotelomeric parvoviruses, mediated by a basic-residue-rich hairpin and an adjacent helix in the initiator proteins and by tandem tetranucleotide motifs in the replication origins. - Highlights: • The structure of a parvovirus replication initiator protein has been determined; • The structure sheds light on mechanisms of ssDNA binding and cleavage; • The nickase active site is preconfigured for versatile metal ligand binding; • The binding site for the double-stranded replication origin DNA is identified; • A single domain integrates multiple functions in virus replication

  6. Structures of minute virus of mice replication initiator protein N-terminal domain: Insights into DNA nicking and origin binding

    Energy Technology Data Exchange (ETDEWEB)

    Tewary, Sunil K.; Liang, Lingfei; Lin, Zihan; Lynn, Annie [Department of Molecular Biosciences, University of Kansas, Lawrence, KS 66045 (United States); Cotmore, Susan F. [Departments of Laboratory Medicine, Yale University Medical School, New Haven, CT 06510 (United States); Tattersall, Peter [Departments of Laboratory Medicine, Yale University Medical School, New Haven, CT 06510 (United States); Departments of Genetics, Yale University Medical School, New Haven, CT 06510 (United States); Zhao, Haiyan, E-mail: zhaohy@ku.edu [Department of Molecular Biosciences, University of Kansas, Lawrence, KS 66045 (United States); Tang, Liang, E-mail: tangl@ku.edu [Department of Molecular Biosciences, University of Kansas, Lawrence, KS 66045 (United States)

    2015-02-15

    Members of the Parvoviridae family all encode a non-structural protein 1 (NS1) that directs replication of single-stranded viral DNA, packages viral DNA into capsid, and serves as a potent transcriptional activator. Here we report the X-ray structure of the minute virus of mice (MVM) NS1 N-terminal domain at 1.45 Å resolution, showing that sites for dsDNA binding, ssDNA binding and cleavage, nuclear localization, and other functions are integrated on a canonical fold of the histidine-hydrophobic-histidine superfamily of nucleases, including elements specific for this Protoparvovirus but distinct from its Bocaparvovirus or Dependoparvovirus orthologs. High resolution structural analysis reveals a nickase active site with an architecture that allows highly versatile metal ligand binding. The structures support a unified mechanism of replication origin recognition for homotelomeric and heterotelomeric parvoviruses, mediated by a basic-residue-rich hairpin and an adjacent helix in the initiator proteins and by tandem tetranucleotide motifs in the replication origins. - Highlights: • The structure of a parvovirus replication initiator protein has been determined; • The structure sheds light on mechanisms of ssDNA binding and cleavage; • The nickase active site is preconfigured for versatile metal ligand binding; • The binding site for the double-stranded replication origin DNA is identified; • A single domain integrates multiple functions in virus replication.

  7. Allelic Variation in KIR2DL3 Generates a KIR2DL2-like Receptor with Increased Binding to Its HLA-C Ligand12

    Science.gov (United States)

    Frazier, William R.; Steiner, Noriko; Hou, Lihua; Dakshanamurthy, Sivanesan; Hurley, Carolyn Katovich

    2013-01-01

    Although extensive homology exists between their extracellular domains, natural killer cell inhibitory receptors KIR2DL2*001 and KIR2DL3*001 have previously been shown to differ substantially in their HLA-C binding avidity. To explore the largely uncharacterized impact of allelic diversity, the most common KIR2DL2/3 allelic products in European American and African American populations were evaluated for surface expression and binding affinity to their HLA-C group 1 and 2 ligands. Although no significant differences in the degree of cell membrane localization were detected in a transfected human NKL cell line by flow cytometry, surface plasmon resonance and KIR binding to a panel of HLA allotypes demonstrated that KIR2DL3*005 differed significantly from other KIR2DL3 allelic products in its ability to bind HLA-C. The increased affinity and avidity of KIR2DL3*005 for its ligand was also demonstrated to have a larger impact on the inhibition of IFN-γ production by the human KHYG-1 NK cell line compared to KIR2DL3*001, a low affinity allelic product. Site-directed mutagenesis established that the combination of arginine at residue 11 and glutamic acid at residue 35 in KIR2DL3*005 were critical to the observed phenotype. Although these residues are distal to the KIR/HLA-C interface, molecular modeling suggests that alteration in the interdomain hinge angle of KIR2DL3*005 towards that found in KIR2DL2*001, another strong receptor of the KIR2DL2/3 family, may be the cause of this increased affinity. The regain of inhibitory capacity by KIR2DL3*005 suggests that the rapidly evolving KIR locus may be responding to relatively recent selective pressures placed upon certain human populations. PMID:23686481

  8. Allelic variation in KIR2DL3 generates a KIR2DL2-like receptor with increased binding to its HLA-C ligand.

    Science.gov (United States)

    Frazier, William R; Steiner, Noriko; Hou, Lihua; Dakshanamurthy, Sivanesan; Hurley, Carolyn Katovich

    2013-06-15

    Although extensive homology exists between their extracellular domains, NK cell inhibitory receptors killer Ig-like receptor (KIR) 2DL2*001 and KIR2DL3*001 have previously been shown to differ substantially in their HLA-C binding avidity. To explore the largely uncharacterized impact of allelic diversity, the most common KIR2DL2/3 allelic products in European American and African American populations were evaluated for surface expression and binding affinity to their HLA-C group 1 and 2 ligands. Although no significant differences in the degree of cell membrane localization were detected in a transfected human NKL cell line by flow cytometry, surface plasmon resonance and KIR binding to a panel of HLA allotypes demonstrated that KIR2DL3*005 differed significantly from other KIR2DL3 allelic products in its ability to bind HLA-C. The increased affinity and avidity of KIR2DL3*005 for its ligand was also demonstrated to have a larger impact on the inhibition of IFN-γ production by the human KHYG-1 NK cell line compared with KIR2DL3*001, a low-affinity allelic product. Site-directed mutagenesis established that the combination of arginine at residue 11 and glutamic acid at residue 35 in KIR2DL3*005 were critical to the observed phenotype. Although these residues are distal to the KIR/HLA-C interface, molecular modeling suggests that alteration in the interdomain hinge angle of KIR2DL3*005 toward that found in KIR2DL2*001, another strong receptor of the KIR2DL2/3 family, may be the cause of this increased affinity. The regain of inhibitory capacity by KIR2DL3*005 suggests that the rapidly evolving KIR locus may be responding to relatively recent selective pressures placed upon certain human populations.

  9. Analysis of the functional compatibility of SIV capsid sequences in the context of the FIV gag precursor.

    Directory of Open Access Journals (Sweden)

    César A Ovejero

    Full Text Available The formation of immature lentiviral particles is dependent on the multimerization of the Gag polyprotein at the plasma membrane of the infected cells. One key player in the virus assembly process is the capsid (CA domain of Gag, which establishes the protein-protein interactions that give rise to the hexagonal lattice of Gag molecules in the immature virion. To gain a better understanding of the functional equivalence between the CA proteins of simian and feline immunodeficiency viruses (SIV and FIV, respectively, we generated a series of chimeric FIV Gag proteins in which the CA-coding region was partially or totally replaced by its SIV counterpart. All the FIV Gag chimeras were found to be assembly-defective; however, all of them are able to interact with wild-type SIV Gag and be recruited into extracellular virus-like particles, regardless of the SIV CA sequences present in the chimeric FIV Gag. The results presented here markedly contrast with our previous findings showing that chimeric SIVs carrying FIV CA-derived sequences are assembly-competent. Overall, our data support the notion that although the SIV and FIV CA proteins share 51% amino acid sequence similarity and exhibit a similar organization, i.e., an N-terminal domain joined by a flexible linker to a C-terminal domain, their functional exchange between these different lentiviruses is strictly dependent on the context of the recipient Gag precursor.

  10. Application of the major capsid protein as a marker of the phylogenetic diversity of Emiliania huxleyi viruses.

    Science.gov (United States)

    Rowe, Janet M; Fabre, Marie-Françoise; Gobena, Daniel; Wilson, William H; Wilhelm, Steven W

    2011-05-01

    Studies of the Phycodnaviridae have traditionally relied on the DNA polymerase (pol) gene as a biomarker. However, recent investigations have suggested that the major capsid protein (MCP) gene may be a reliable phylogenetic biomarker. We used MCP gene amplicons gathered across the North Atlantic to assess the diversity of Emiliania huxleyi-infecting Phycodnaviridae. Nucleotide sequences were examined across >6000 km of open ocean, with comparisons between concentrates of the virus-size fraction of seawater and of lysates generated by exposing host strains to these same virus concentrates. Analyses revealed that many sequences were only sampled once, while several were over-represented. Analyses also revealed nucleotide sequences distinct from previous coastal isolates. Examination of lysed cultures revealed a new richness in phylogeny, as MCP sequences previously unrepresented within the existing collection of E. huxleyi viruses (EhV) were associated with viruses lysing cultures. Sequences were compared with previously described EhV MCP sequences from the North Sea and a Norwegian Fjord, as well as from the Gulf of Maine. Principal component analysis indicates that location-specific distinctions exist despite the presence of sequences common across these environments. Overall, this investigation provides new sequence data and an assessment on the use of the MCP gene. © 2011 Federation of European Microbiological Societies Published by Blackwell Publishing Ltd. All rights reserved.

  11. Capsid protein VP4 of human rhinovirus induces membrane permeability by the formation of a size-selective multimeric pore.

    Directory of Open Access Journals (Sweden)

    Anusha Panjwani

    2014-08-01

    Full Text Available Non-enveloped viruses must deliver their viral genome across a cell membrane without the advantage of membrane fusion. The mechanisms used to achieve this remain poorly understood. Human rhinovirus, a frequent cause of the common cold, is a non-enveloped virus of the picornavirus family, which includes other significant pathogens such as poliovirus and foot-and-mouth disease virus. During picornavirus cell entry, the small myristoylated capsid protein VP4 is released from the virus, interacts with the cell membrane and is implicated in the delivery of the viral RNA genome into the cytoplasm to initiate replication. In this study, we have produced recombinant C-terminal histidine-tagged human rhinovirus VP4 and shown it can induce membrane permeability in liposome model membranes. Dextran size-exclusion studies, chemical crosslinking and electron microscopy demonstrated that VP4 forms a multimeric membrane pore, with a channel size consistent with transfer of the single-stranded RNA genome. The membrane permeability induced by recombinant VP4 was influenced by pH and was comparable to permeability induced by infectious virions. These findings present a molecular mechanism for the involvement of VP4 in cell entry and provide a model system which will facilitate exploration of VP4 as a novel antiviral target for the picornavirus family.

  12. A physical interaction between viral replicase and capsid protein is required for genome-packaging specificity in an RNA virus.

    Science.gov (United States)

    Seo, Jang-Kyun; Kwon, Sun-Jung; Rao, A L N

    2012-06-01

    Genome packaging is functionally coupled to replication in RNA viruses pathogenic to humans (Poliovirus), insects (Flock house virus [FHV]), and plants (Brome mosaic virus [BMV]). However, the underlying mechanism is not fully understood. We have observed previously that in FHV and BMV, unlike ectopically expressed capsid protein (CP), packaging specificity results from RNA encapsidation by CP that has been translated from mRNA produced from replicating genomic RNA. Consequently, we hypothesize that a physical interaction with replicase increases the CP specificity for packaging viral RNAs. We tested this hypothesis by evaluating the molecular interaction between replicase protein and CP using a FHV-Nicotiana benthamiana system. Bimolecular fluorescence complementation in conjunction with fluorescent cellular protein markers and coimmunoprecipitation assays demonstrated that FHV replicase (protein A) and CP physically interact at the mitochondrial site of replication and that this interaction requires the N-proximal region from either amino acids 1 to 31 or amino acids 32 to 50 of the CP. In contrast to the mitochondrial localization of CP derived from FHV replication, ectopic expression displayed a characteristic punctate pattern on the endoplasmic reticulum (ER). This pattern was altered to relocalize the CP throughout the cytoplasm when the C-proximal hydrophobic domain was deleted. Analysis of the packaging phenotypes of the CP mutants defective either in protein A-CP interactions or ER localization suggested that synchronization between protein A-CP interaction and its subcellular localization is imperative to confer packaging specificity.

  13. Antigenic heterogeneity of capsid protein VP1 in foot-and-mouth disease virus (FMDV serotype Asia1

    Directory of Open Access Journals (Sweden)

    Alam SM

    2013-08-01

    Full Text Available SM Sabbir Alam,1 Ruhul Amin,1 Mohammed Ziaur Rahman,2 M Anwar Hossain,1 Munawar Sultana11Department of Microbiology, University of Dhaka, Dhaka, Bangladesh; 2International Centre for Diarrhoeal Disease Research, Dhaka, BangladeshAbstract: Foot and mouth disease virus (FMDV, with its seven serotypes, is a highly contagious virus infecting mainly cloven-hoofed animals. The serotype Asia1 occurs mainly in Asian regions. An in-silico approach was taken to reveal the antigenic heterogeneities within the capsid protein VP1 of Asia1. A total of 47 VP1 sequences of Asia1 isolates from different countries of South Asian regions were selected, retrieved from database, and were aligned. The structure of VP1 protein was modeled using a homology modeling approach. Several antigenic sites were identified and mapped onto the three-dimensional protein structure. Variations at these antigenic sites were analyzed by calculating the protein variability index and finding mutation combinations. The data suggested that vaccine escape mutants have derived from only few mutations at several antigenic sites. Five antigenic peptides have been identified as the least variable epitopes, with just fewer amino acid substitutions. Only a limited number of serotype Asia1 antigenic variants were found to be circulated within the South Asian region. This emphasizes a possibility of formulating synthetic vaccines for controlling foot-and-mouth disease by Asia1 serotypes.Keywords: protein modeling, antigenic sites, sequence variation

  14. Fine-tuning translation kinetics selection as the driving force of codon usage bias in the hepatitis A virus capsid.

    Science.gov (United States)

    Aragonès, Lluís; Guix, Susana; Ribes, Enric; Bosch, Albert; Pintó, Rosa M

    2010-03-05

    Hepatitis A virus (HAV), the prototype of genus Hepatovirus, has several unique biological characteristics that distinguish it from other members of the Picornaviridae family. Among these, the need for an intact eIF4G factor for the initiation of translation results in an inability to shut down host protein synthesis by a mechanism similar to that of other picornaviruses. Consequently, HAV must inefficiently compete for the cellular translational machinery and this may explain its poor growth in cell culture. In this context of virus/cell competition, HAV has strategically adopted a naturally highly deoptimized codon usage with respect to that of its cellular host. With the aim to optimize its codon usage the virus was adapted to propagate in cells with impaired protein synthesis, in order to make tRNA pools more available for the virus. A significant loss of fitness was the immediate response to the adaptation process that was, however, later on recovered and more associated to a re-deoptimization rather than to an optimization of the codon usage specifically in the capsid coding region. These results exclude translation selection and instead suggest fine-tuning translation kinetics selection as the underlying mechanism of the codon usage bias in this specific genome region. Additionally, the results provide clear evidence of the Red Queen dynamics of evolution since the virus has very much evolved to re-adapt its codon usage to the environmental cellular changing conditions in order to recover the original fitness.

  15. Fine-tuning translation kinetics selection as the driving force of codon usage bias in the hepatitis A virus capsid.

    Directory of Open Access Journals (Sweden)

    Lluís Aragonès

    2010-03-01

    Full Text Available Hepatitis A virus (HAV, the prototype of genus Hepatovirus, has several unique biological characteristics that distinguish it from other members of the Picornaviridae family. Among these, the need for an intact eIF4G factor for the initiation of translation results in an inability to shut down host protein synthesis by a mechanism similar to that of other picornaviruses. Consequently, HAV must inefficiently compete for the cellular translational machinery and this may explain its poor growth in cell culture. In this context of virus/cell competition, HAV has strategically adopted a naturally highly deoptimized codon usage with respect to that of its cellular host. With the aim to optimize its codon usage the virus was adapted to propagate in cells with impaired protein synthesis, in order to make tRNA pools more available for the virus. A significant loss of fitness was the immediate response to the adaptation process that was, however, later on recovered and more associated to a re-deoptimization rather than to an optimization of the codon usage specifically in the capsid coding region. These results exclude translation selection and instead suggest fine-tuning translation kinetics selection as the underlying mechanism of the codon usage bias in this specific genome region. Additionally, the results provide clear evidence of the Red Queen dynamics of evolution since the virus has very much evolved to re-adapt its codon usage to the environmental cellular changing conditions in order to recover the original fitness.

  16. Quantitative characterization of all single amino acid variants of a viral capsid-based drug delivery vehicle.

    Science.gov (United States)

    Hartman, Emily C; Jakobson, Christopher M; Favor, Andrew H; Lobba, Marco J; Álvarez-Benedicto, Ester; Francis, Matthew B; Tullman-Ercek, Danielle

    2018-04-11

    Self-assembling proteins are critical to biological systems and industrial technologies, but predicting how mutations affect self-assembly remains a significant challenge. Here, we report a technique, termed SyMAPS (Systematic Mutation and Assembled Particle Selection), that can be used to characterize the assembly competency of all single amino acid variants of a self-assembling viral structural protein. SyMAPS studies on the MS2 bacteriophage coat protein revealed a high-resolution fitness landscape that challenges some conventional assumptions of protein engineering. An additional round of selection identified a previously unknown variant (CP[T71H]) that is stable at neutral pH but less tolerant to acidic conditions than the wild-type coat protein. The capsids formed by this variant could be more amenable to disassembly in late endosomes or early lysosomes-a feature that is advantageous for delivery applications. In addition to providing a mutability blueprint for virus-like particles, SyMAPS can be readily applied to other self-assembling proteins.

  17. Phylogenetic Distribution of the Capsid Assembly Protein Gene (g20) of Cyanophages in Paddy Floodwaters in Northeast China

    Science.gov (United States)

    Jing, Ruiyong; Liu, Junjie; Yu, Zhenhua; Liu, Xiaobing; Wang, Guanghua

    2014-01-01

    Numerous studies have revealed the high diversity of cyanophages in marine and freshwater environments, but little is currently known about the diversity of cyanophages in paddy fields, particularly in Northeast (NE) China. To elucidate the genetic diversity of cyanophages in paddy floodwaters in NE China, viral capsid assembly protein gene (g20) sequences from five floodwater samples were amplified with the primers CPS1 and CPS8. Denaturing gradient gel electrophoresis (DGGE) was applied to distinguish different g20 clones. In total, 54 clones differing in g20 nucleotide sequences were obtained in this study. Phylogenetic analysis showed that the distribution of g20 sequences in this study was different from that in Japanese paddy fields, and all the sequences were grouped into Clusters α, β, γ and ε. Within Clusters α and β, three new small clusters (PFW-VII∼-IX) were identified. UniFrac analysis of g20 clone assemblages demonstrated that the community compositions of cyanophage varied among marine, lake and paddy field environments. In paddy floodwater, community compositions of cyanophage were also different between NE China and Japan. PMID:24533125

  18. Intracellular Distribution of Capsid-Associated pUL77 of Human Cytomegalovirus and Interactions with Packaging Proteins and pUL93.

    Science.gov (United States)

    Köppen-Rung, Pánja; Dittmer, Alexandra; Bogner, Elke

    2016-07-01

    DNA packaging into procapsids is a common multistep process during viral maturation in herpesviruses. In human cytomegalovirus (HCMV), the proteins involved in this process are terminase subunits pUL56 and pUL89, which are responsible for site-specific cleavage and insertion of the DNA into the procapsid via portal protein pUL104. However, additional viral proteins are required for the DNA packaging process. We have shown previously that the plasmid that encodes capsid-associated pUL77 encodes another potential player during capsid maturation. Pulse-chase experiments revealed that pUL77 is stably expressed during HCMV infection. Time course analysis demonstrated that pUL77 is expressed in the early late part of the infectious cycle. The sequence of pUL77 was analyzed to find nuclear localization sequences (NLSs), revealing monopartite NLSm at the N terminus and bipartite NLSb in the middle of pUL77. The potential NLSs were inserted into plasmid pHM829, which encodes a chimeric protein with β-galactosidase and green fluorescent protein. In contrast to pUL56, neither NLSm nor NLSb was sufficient for nuclear import. Furthermore, we investigated by coimmunoprecipitation whether packaging proteins, as well as pUL93, the homologue protein of herpes simplex virus 1 pUL17, are interaction partners of pUL77. The interactions between pUL77 and packaging proteins, as well as pUL93, were verified. We showed that the capsid-associated pUL77 is another potential player during capsid maturation of HCMV. Protein UL77 (pUL77) is a conserved core protein of HCMV. This study demonstrates for the first time that pUL77 has early-late expression kinetics during the infectious cycle and an intrinsic potential for nuclear translocation. According to its proposed functions in stabilization of the capsid and anchoring of the encapsidated DNA during packaging, interaction with further DNA packaging proteins is required. We identified physical interactions with terminase subunits pUL56 and p

  19. Distinct binding interactions of HIV-1 Gag to Psi and non-Psi RNAs: implications for viral genomic RNA packaging.

    Science.gov (United States)

    Webb, Joseph A; Jones, Christopher P; Parent, Leslie J; Rouzina, Ioulia; Musier-Forsyth, Karin

    2013-08-01

    Despite the vast excess of cellular RNAs, precisely two copies of viral genomic RNA (gRNA) are selectively packaged into new human immunodeficiency type 1 (HIV-1) particles via specific interactions between the HIV-1 Gag and the gRNA psi (ψ) packaging signal. Gag consists of the matrix (MA), capsid, nucleocapsid (NC), and p6 domains. Binding of the Gag NC domain to ψ is necessary for gRNA packaging, but the mechanism by which Gag selectively interacts with ψ is unclear. Here, we investigate the binding of NC and Gag variants to an RNA derived from ψ (Psi RNA), as well as to a non-ψ region (TARPolyA). Binding was measured as a function of salt to obtain the effective charge (Zeff) and nonelectrostatic (i.e., specific) component of binding, Kd(1M). Gag binds to Psi RNA with a dramatically reduced Kd(1M) and lower Zeff relative to TARPolyA. NC, GagΔMA, and a dimerization mutant of Gag bind TARPolyA with reduced Zeff relative to WT Gag. Mutations involving the NC zinc finger motifs of Gag or changes to the G-rich NC-binding regions of Psi RNA significantly reduce the nonelectrostatic component of binding, leading to an increase in Zeff. These results show that Gag interacts with gRNA using different binding modes; both the NC and MA domains are bound to RNA in the case of TARPolyA, whereas binding to Psi RNA involves only the NC domain. Taken together, these results suggest a novel mechanism for selective gRNA encapsidation.

  20. SHBG (Sex Hormone Binding Globulin)

    Science.gov (United States)

    ... Links Patient Resources For Health Professionals Subscribe Search Sex Hormone Binding Globulin (SHBG) Send Us Your Feedback ... As Testosterone-estrogen Binding Globulin TeBG Formal Name Sex Hormone Binding Globulin This article was last reviewed ...

  1. Pharmacological Induction of Heme Oxygenase-1 Impairs Nuclear Accumulation of Herpes Simplex Virus Capsids upon Infection

    Directory of Open Access Journals (Sweden)

    Francisco J. Ibáñez

    2017-10-01

    Full Text Available Heme oxygenase-1 (HO-1 is an inducible enzyme that is expressed in response to physical and chemical stresses, such as ultraviolet radiation, hyperthermia, hypoxia, reactive oxygen species (ROS, as well as cytokines, among others. Its activity can be positively modulated by cobalt protoporphyrin (CoPP and negatively by tin protoporphirin (SnPP. Once induced, HO-1 degrades iron-containing heme into ferrous iron (Fe2+, carbon monoxide (CO and biliverdin. Importantly, numerous products of HO-1 are cytoprotective with anti-apoptotic, anti-oxidant, anti-inflammatory, and anti-cancer effects. The products of HO-1 also display antiviral properties against several viruses, such as the human immunodeficiency virus (HIV, influenza, hepatitis B, hepatitis C, and Ebola virus. Here, we sought to assess the effect of modulating HO-1 activity over herpes simplex virus type 2 (HSV-2 infection in epithelial cells and neurons. There are no vaccines against HSV-2 and treatment options are scarce in the immunosuppressed, in which drug-resistant variants emerge. By using HSV strains that encode structural and non-structural forms of the green fluorescent protein (GFP, we found that pharmacological induction of HO-1 activity with CoPP significantly decreases virus plaque formation and the expression of virus-encoded genes in epithelial cells as determined by flow cytometry and western blot assays. CoPP treatment did not affect virus binding to the cell surface or entry into the cytoplasm, but rather downstream events in the virus infection cycle. Furthermore, we observed that treating cells with a CO-releasing molecule (CORM-2 recapitulated some of the anti-HSV effects elicited by CoPP. Taken together, these findings indicate that HO-1 activity interferes with the replication cycle of HSV and that its antiviral effects can be recapitulated by CO.

  2. Comparison of immunofluorescence and enzyme-linked immunosorbent assay and immunoglobulin G avidity techniques for screening of anti: Toxoplasma antibodies among single serum sample pregnant women in Tabriz, Iran

    Directory of Open Access Journals (Sweden)

    Mehrangiz Rajaii

    2015-01-01

    Full Text Available Background: Congenital toxoplasmosis is that pregnant women acquire the infection during gestation; diagnosis of the acute infection during pregnancy is a complex subject of maternal toxoplasmosis. Thus, the presence of immunoglobulin G (IgG and/or IgM Toxoplasma antibodies in a single serum sample drawn during gestation cannot be used to define whether the infection was recently acquired or chronic. Materials and Methods: At this cross-sectional descriptive study, sera of 391 pregnant women examined and compared. They were in an age range of 21-35 years, referred by gynecologists and infectious disease specialists, during March 2012-April 2013. They have referred, 215 (54.98%, 102 (26%, 74 (18.92% in the first, second and third trimesters of gestation, respectively. For each of them, a questionnaire was completed and serum samples were prepared in an equal condition, examined according to the procedures of indirect immunofluorescence (IIF, enzyme-linked immunosorbent assay (ELISA and IgG Avidity techniques. Results: We have found 111 (28.38% seronegative and 280 (71.61% seropositive cases by IIF and 124 (31.70% seronegative, 267 (68.28% seropositive cases by ELISA. The IgG avidity test confirmed 45 (69.23% and 7 (10.76% doubtful cases of IgM test in IIF and ELISA techniques. Conclusions: This study highlights how to manage pregnant women with toxoplasmosis, especially in a single serum sample condition.

  3. Avidity of IgG for rubella: an evaluation of the need for implementation at the Materno-Infantil Presidente Vargas Hospital in Porto Alegre, Rio Grande do Sul, Brazil

    Directory of Open Access Journals (Sweden)

    M.M. Reis

    Full Text Available Rubella serum assays performed in the laboratory of the Materno-Infantil Presidente Vargas Hospital (HMIPV from 1998 to 2002 were reviewed to determine if IgG avidity assays should be implemented. IgG was determined using the Enzyme Linked Fluorescent Assay, ELFA, VIDAS® system, bioMérieux or the Microparticle Enzyme Immunoassay, MEIA, Axsym® system, Abbott, and IgM was determined using the ELFA, VIDAS® system, bioMérieux, a capture format assay. Specific IgG was assayed in 2,863 samples, with positive results for 84% of the patients, for the most part with high levels of antibodies. IgM was assayed in 2,851 samples, being positive in 14 (0.49% and inconclusive in 25 (0.88%. Serology for toxoplasmosis was also positive or inconclusive in 5 patients. After a cost-effectiveness analysis, it was decided not to implement avidity assays, considering that the HMIPV is a public institution, with limited funding. Difficulties concerning the integration of the Clinical Pathology Service with the Clinical Staff of the institution were also considered.

  4. The best and the brightest: exploiting tryptophan-sensitized Tb(3+) luminescence to engineer lanthanide-binding tags.

    Science.gov (United States)

    Martin, Langdon J; Imperiali, Barbara

    2015-01-01

    Consider the lanthanide metals, comprising lanthanum through lutetium. Lanthanides form stable cations with a +3 charge, and these ions exhibit a variety of useful physical properties (long-lifetime luminescence, paramagnetism, anomalous X-ray scattering) that are amenable to studies of biomolecules. The absence of lanthanide ions in living systems means that background signals are generally a nonissue; however, to exploit the advantageous properties it is necessary to engineer a robust lanthanide-binding sequence that can be appended to any macromolecules of interest. To this end, the luminescence produced by tryptophan-sensitized Tb(3+) has been used as a selection marker for peptide sequences that avidly chelate these ions. A combinatorial split-and-pool library that uses two orthogonal linkers-one that is cleaved for selection and one that is cleaved for sequencing and characterization-has been used to develop lanthanide-binding tags (LBTs): peptides of 15-20 amino acids with low-nM affinity for Tb(3+). Further validating the success of this screen, knowledge about LBTs has enabled the introduction of a lanthanide-binding loop in place of one of the four native calcium-binding loops within the protein calcineurin B.

  5. Structural Basis for a Switch in Receptor Binding Specificity of Two H5N1 Hemagglutinin Mutants

    Directory of Open Access Journals (Sweden)

    Xueyong Zhu

    2015-11-01

    Full Text Available Avian H5N1 influenza viruses continue to spread in wild birds and domestic poultry with sporadic infection in humans. Receptor binding specificity changes are a prerequisite for H5N1 viruses and other zoonotic viruses to be transmitted among humans. Previous reported hemagglutinin (HA mutants from ferret-transmissible H5N1 viruses of A/Vietnam/1203/2004 and A/Indonesia/5/2005 showed slightly increased, but still very weak, binding to human receptors. From mutagenesis and glycan array studies, we previously identified two H5N1 HA mutants that could more effectively switch receptor specificity to human-like α2-6-linked sialosides with avidity comparable to wild-type H5 HA binding to avian-like α2-3-linked sialosides. Here, crystal structures of these two H5 HA mutants free and in complex with human and avian glycan receptor analogs reveal the structural basis for their preferential binding to human receptors. These findings suggest continuous surveillance should be maintained to monitor and assess human-to-human transmission potential of H5N1 viruses.

  6. Synthesis and characterization of time-resolved fluorescence probes for evaluation of competitive binding to melanocortin receptors.

    Science.gov (United States)

    Alleti, Ramesh; Vagner, Josef; Dehigaspitiya, Dilani Chathurika; Moberg, Valerie E; Elshan, N G R D; Tafreshi, Narges K; Brabez, Nabila; Weber, Craig S; Lynch, Ronald M; Hruby, Victor J; Gillies, Robert J; Morse, David L; Mash, Eugene A

    2013-09-01

    Probes for use in time-resolved fluorescence competitive binding assays at melanocortin receptors based on the parental ligands MSH(4), MSH(7), and NDP-α-MSH were prepared by solid phase synthesis methods, purified, and characterized. The saturation binding of these probes was studied using HEK-293 cells engineered to overexpress the human melanocortin 4 receptor (hMC4R) as well as the human cholecystokinin 2 receptor (hCCK2R). The ratios of non-specific binding to total binding approached unity at high concentrations for each probe. At low probe concentrations, receptor-mediated binding and uptake was discernable, and so probe concentrations were kept as low as possible in determining Kd values. The Eu-DTPA-PEGO-MSH(4) probe exhibited low specific binding relative to non-specific binding, even at low nanomolar concentrations, and was deemed unsuitable for use in competition binding assays. The Eu-DTPA-PEGO probes based on MSH(7) and NDP-α-MSH exhibited Kd values of 27±3.9nM and 4.2±0.48nM, respectively, for binding with hMC4R. These probes were employed in competitive binding assays to characterize the interactions of hMC4R with monovalent and divalent MSH(4), MSH(7), and NDP-α-MSH constructs derived from squalene. Results from assays with both probes reflected only statistical enhancements, suggesting improper ligand spacing on the squalene scaffold for the divalent constructs. The Ki values from competitive binding assays that employed the MSH(7)-based probe were generally lower than the Ki values obtained when the probe based on NDP-α-MSH was employed, which is consistent with the greater potency of the latter probe. The probe based on MSH(7) was also competed with monovalent, divalent, and trivalent MSH(4) constructs that previously demonstrated multivalent binding in competitive binding assays against a variant of the probe based on NDP-α-MSH. Results from these assays confirm multivalent binding, but suggest a more modest increase in avidity for these

  7. CARBOHYDRATE-CONTAINING COMPOUNDS WHICH BIND TO CARBOHYDRATE BINDING RECEPTORS

    DEFF Research Database (Denmark)

    1995-01-01

    Carbohydrate-containing compounds which contain saccharides or derivatives thereof and which bind to carbohydrate binding receptors are useful in pharmaceutical products for treatment of inflammatory diseases and other diseases.......Carbohydrate-containing compounds which contain saccharides or derivatives thereof and which bind to carbohydrate binding receptors are useful in pharmaceutical products for treatment of inflammatory diseases and other diseases....

  8. Bat Caliciviruses and Human Noroviruses Are Antigenically Similar and Have Overlapping Histo-Blood Group Antigen Binding Profiles.

    Science.gov (United States)

    Kocher, Jacob F; Lindesmith, Lisa C; Debbink, Kari; Beall, Anne; Mallory, Michael L; Yount, Boyd L; Graham, Rachel L; Huynh, Jeremy; Gates, J Edward; Donaldson, Eric F; Baric, Ralph S

    2018-05-22

    Emerging zoonotic viral diseases remain a challenge to global public health. Recent surveillance studies have implicated bats as potential reservoirs for a number of viral pathogens, including coronaviruses and Ebola viruses. Caliciviridae represent a major viral family contributing to emerging diseases in both human and animal populations and have been recently identified in bats. In this study, we blended metagenomics, phylogenetics, homology modeling, and in vitro assays to characterize two novel bat calicivirus (BtCalV) capsid sequences, corresponding to strain BtCalV/A10/USA/2009, identified in Perimyotis subflavus near Little Orleans, MD, and bat norovirus. We observed that bat norovirus formed virus-like particles and had epitopes and receptor-binding patterns similar to those of human noroviruses. To determine whether these observations stretch across multiple bat caliciviruses, we characterized a novel bat calicivirus, BtCalV/A10/USA/2009. Phylogenetic analysis revealed that BtCalV/A10/USA/2009 likely represents a novel Caliciviridae genus and is most closely related to "recoviruses." Homology modeling revealed that the capsid sequences of BtCalV/A10/USA/2009 and bat norovirus resembled human norovirus capsid sequences and retained host ligand binding within the receptor-binding domains similar to that seen with human noroviruses. Both caliciviruses bound histo-blood group antigens in patterns that overlapped those seen with human and animal noroviruses. Taken together, our results indicate the potential for bat caliciviruses to bind histo-blood group antigens and overcome a significant barrier to cross-species transmission. Additionally, we have shown that bat norovirus maintains antigenic epitopes similar to those seen with human noroviruses, providing further evidence of evolutionary descent. Our results reiterate the importance of surveillance of wild-animal populations, especially of bats, for novel viral pathogens. IMPORTANCE Caliciviruses are

  9. Surface plasmon resonance imaging reveals multiple binding modes of Agrobacterium transformation mediator VirE2 to ssDNA.

    Science.gov (United States)

    Kim, Sanghyun; Zbaida, David; Elbaum, Michael; Leh, Hervé; Nogues, Claude; Buckle, Malcolm

    2015-07-27

    VirE2 is the major secreted protein of Agrobacterium tumefaciens in its genetic transformation of plant hosts. It is co-expressed with a small acidic chaperone VirE1, which prevents VirE2 oligomerization. After secretion into the host cell, VirE2 serves functions similar to a viral capsid in protecting the single-stranded transferred DNA en route to the nucleus. Binding of VirE2 to ssDNA is strongly cooperative and depends moreover on protein-protein interactions. In order to isolate the protein-DNA interactions, imaging surface plasmon resonance (SPRi) studies were conducted using surface-immobilized DNA substrates of length comparable to the protein-binding footprint. Binding curves revealed an important influence of substrate rigidity with a notable preference for poly-T sequences and absence of binding to both poly-A and double-stranded DNA fragments. Dissociation at high salt concentration confirmed the electrostatic nature of the interaction. VirE1-VirE2 heterodimers also bound to ssDNA, though by a different mechanism that was insensitive to high salt. Neither VirE2 nor VirE1-VirE2 followed the Langmuir isotherm expected for reversible monomeric binding. The differences reflect the cooperative self-interactions of VirE2 that are suppressed by VirE1. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

  10. Inhibition of enterovirus 71 (EV-71 infections by a novel antiviral peptide derived from EV-71 capsid protein VP1.

    Directory of Open Access Journals (Sweden)

    Chee Wah Tan

    Full Text Available Enterovirus 71 (EV-71 is the main causative agent of hand, foot and mouth disease (HFMD. In recent years, EV-71 infections were reported to cause high fatalities and severe neurological complications in Asia. Currently, no effective antiviral or vaccine is available to treat or prevent EV-71 infection. In this study, we have discovered a synthetic peptide which could be developed as a potential antiviral for inhibition of EV-71. Ninety five synthetic peptides (15-mers overlapping the entire EV-71 capsid protein, VP1, were chemically synthesized and tested for antiviral properties against EV-71 in human Rhabdomyosarcoma (RD cells. One peptide, SP40, was found to significantly reduce cytopathic effects of all representative EV-71 strains from genotypes A, B and C tested, with IC(50 values ranging from 6-9.3 µM in RD cells. The in vitro inhibitory effect of SP40 exhibited a dose dependent concentration corresponding to a decrease in infectious viral particles, total viral RNA and the levels of VP1 protein. The antiviral activity of SP40 peptide was not restricted to a specific cell line as inhibition of EV-71 was observed in RD, HeLa, HT-29 and Vero cells. Besides inhibition of EV-71, it also had antiviral activities against CV-A16 and poliovirus type 1 in cell culture. Mechanism of action studies suggested that the SP40 peptide was not virucidal but was able to block viral attachment to the RD cells. Substitutions of arginine and lysine residues with alanine in the SP40 peptide at positions R3A, R4A, K5A and R13A were found to significantly decrease antiviral activities, implying the importance of positively charged amino acids for the antiviral activities. The data demonstrated the potential and feasibility of SP40 as a broad spectrum antiviral agent against EV-71.

  11. Self-assembly of virus-like particles of porcine circovirus type 2 capsid protein expressed from Escherichia coli

    Directory of Open Access Journals (Sweden)

    Cai Xuepeng

    2010-07-01

    Full Text Available Abstract Background Porcine circovirus 2 (PCV2 is a serious problem to the swine industry and can lead to significant negative impacts on profitability of pork production. Syndrome associated with PCV2 is known as porcine circovirus closely associated with post-weaning multisystemic wasting syndrome (PMWS. The capsid (Cap protein of PCV2 is a major candidate antigen for development of recombinant vaccine and serological diagnostic method. The recombinant Cap protein has the ability to self-assemble into virus-like particles (VLPs in vitro, it is particularly opportunity to develop the PV2 VLPs vaccine in Escherichia coli,(E.coli , because where the cost of the vaccine must be weighed against the value of the vaccinated pig, when it was to extend use the VLPs vaccine of PCV2. Results In this report, a highly soluble Cap-tag protein expressed in E.coli was constructed with a p-SMK expression vector with a fusion tag of small ubiquitin-like modifiers (SUMO. The recombinant Cap was purified using Ni2+ affinity resins, whereas the tag was used to remove the SUMO protease. Simultaneously, the whole native Cap protein was able to self-assemble into VLPs in vitro when viewed under an electron microscope. The Cap-like particles had a size and shape that resembled the authentic Cap. The result could also be applied in the large-scale production of VLPs of PCV2 and could be used as a diagnostic antigen or a potential VLP vaccine against PCV2 infection in pigs. Conclusion we have, for the first time, utilized the SUMO fusion motif to successfully express the entire authentic Cap protein of PCV2 in E. coli. After the cleavage of the fusion motif, the nCap protein has the ability to self-assemble into VLPs, which can be used as as a potential vaccine to protect pigs from PCV2-infection.

  12. Self-assembly of virus-like particles of porcine circovirus type 2 capsid protein expressed from Escherichia coli

    Science.gov (United States)

    2010-01-01

    Background Porcine circovirus 2 (PCV2) is a serious problem to the swine industry and can lead to significant negative impacts on profitability of pork production. Syndrome associated with PCV2 is known as porcine circovirus closely associated with post-weaning multisystemic wasting syndrome (PMWS). The capsid (Cap) protein of PCV2 is a major candidate antigen for development of recombinant vaccine and serological diagnostic method. The recombinant Cap protein has the ability to self-assemble into virus-like particles (VLPs) in vitro, it is particularly opportunity to develop the PV2 VLPs vaccine in Escherichia coli,(E.coli ), because where the cost of the vaccine must be weighed against the value of the vaccinated pig, when it was to extend use the VLPs vaccine of PCV2. Results In this report, a highly soluble Cap-tag protein expressed in E.coli was constructed with a p-SMK expression vector with a fusion tag of small ubiquitin-like modifiers (SUMO). The recombinant Cap was purified using Ni2+ affinity resins, whereas the tag was used to remove the SUMO protease. Simultaneously, the whole native Cap protein was able to self-assemble into VLPs in vitro when viewed under an electron microscope. The Cap-like particles had a size and shape that resembled the authentic Cap. The result could also be applied in the large-scale production of VLPs of PCV2 and could be used as a diagnostic antigen or a potential VLP vaccine against PCV2 infection in pigs. Conclusion we have, for the first time, utilized the SUMO fusion motif to successfully express the entire authentic Cap protein of PCV2 in E. coli. After the cleavage of the fusion motif, the nCap protein has the ability to self-assemble into VLPs, which can be used as as a potential vaccine to protect pigs from PCV2-infection. PMID:20646322

  13. Vaccination with Recombinant Baculovirus Expressing Ranavirus Major Capsid Protein Induces Protective Immunity in Chinese Giant Salamander, Andrias davidianus

    Directory of Open Access Journals (Sweden)

    Xiaoyuan Zhou

    2017-07-01

    Full Text Available The Chinese giant salamander iridovirus (CGSIV, belonging to the genus Ranavirus in the family Iridoviridae, is the causative agent of an emerging infectious disease causing high mortality of more than 90% and economic losses in Chinese giant salamanders in China. In this study, a recombinant baculovirus-based vaccine expressing the CGSIV major capsid protein (MCP was developed and its protective immunity in Chinese giant salamanders was evaluated. The recombinant Autographa californica nucleopolyhedrosis virus (AcNPV, expressing CGSIV MCP, designated as AcNPV-MCP, was generated with the highest titers of 1 × 108 plaque forming units/mL (PFU/mL and confirmed by Western blot and indirect immunofluorescence (IIF assays. Western blot analysis revealed that the expressed MCP reacted with mouse anti-MCP monoclonal antibodies at the band of about 53 kDa. The results of IIF indicated that the MCP was expressed in the infected Spodoptera frugiperda 9 (Sf9 cells with the recombinant baculovirus, and the Chinese giant salamander muscle cells also transduced with the AcNPV-MCP. Immunization with the recombinant baculovirus of AcNPV-MCP elicited robust specific humoral immune responses detected by ELISA and neutralization assays and potent cellular immune responses in Chinese giant salamanders. Importantly, the effective immunization conferred highly protective immunity for Chinese giant salamanders against CGSIV challenge and produced a relative percent of survival rate of 84%. Thus, the recombinant baculovirus expressing CGSIV MCP can induce significant immune responses involving both humoral and cell-mediated immunity in Chinese giant salamanders and might represent a potential baculovirus based vaccine candidate for Chinese giant salamanders against CGSIV.

  14. Absolute quantification of norovirus capsid protein in food, water, and soil using synthetic peptides with electrospray and MALDI mass spectrometry

    International Nuclear Information System (INIS)

    Hartmann, Erica M.; Colquhoun, David R.; Schwab, Kellogg J.; Halden, Rolf U.

    2015-01-01

    Highlights: • Mass spectrometry-based methods for norovirus quantification are developed. • Absolute quantification is achieved using internal heavy isotope-labeled standards. • A single labeled peptide serves in two distinct detection strategies. • These methods are validated for food, water, and soil analysis. • MS-based detection limits are lowered by two orders of magnitude. - Abstract: Norovirus infections are one of the most prominent public health problems of microbial origin in the U.S. and other industrialized countries. Surveillance is necessary to prevent secondary infection, confirm successful cleanup after outbreaks, and track the causative agent. Quantitative mass spectrometry, based on absolute quantitation with stable-isotope labeled peptides, is a promising tool for norovirus monitoring because of its speed, sensitivity, and robustness in the face of environmental inhibitors. In the current study, we present two new methods for the detection of the norovirus genogroup I capsid protein using electrospray and matrix-assisted laser desorption/ionization (MALDI) mass spectrometry. The peptide TLDPIEVPLEDVR was used to quantify norovirus-like particles down to 500 attomoles with electrospray and 100 attomoles with MALDI. With MALDI, we also demonstrate a detection limit of 1 femtomole and a quantitative dynamic range of 5 orders of magnitude in the presence of an environmental matrix effect. Due to the rapid processing time and applicability to a wide range of environmental sample types (bacterial lysate, produce, milk, soil, and groundwater), mass spectrometry-based absolute quantitation has a strong potential for use in public health and environmental sciences

  15. Absolute quantification of norovirus capsid protein in food, water, and soil using synthetic peptides with electrospray and MALDI mass spectrometry

    Energy Technology Data Exchange (ETDEWEB)

    Hartmann, Erica M. [Center for Environmental Security and Security Defense Systems Initiative, The Biodesign Institute, Arizona State University, 781 E. Terrace Mall, Tempe, AZ 85287-5904 (United States); Colquhoun, David R.; Schwab, Kellogg J. [Department of Environmental Health Sciences, The Johns Hopkins University, Bloomberg School of Public Health, 615 N. Wolfe St., Baltimore, MD 21205 (United States); Halden, Rolf U., E-mail: halden@asu.edu [Center for Environmental Security and Security Defense Systems Initiative, The Biodesign Institute, Arizona State University, 781 E. Terrace Mall, Tempe, AZ 85287-5904 (United States); Department of Environmental Health Sciences, The Johns Hopkins University, Bloomberg School of Public Health, 615 N. Wolfe St., Baltimore, MD 21205 (United States)

    2015-04-09

    Highlights: • Mass spectrometry-based methods for norovirus quantification are developed. • Absolute quantification is achieved using internal heavy isotope-labeled standards. • A single labeled peptide serves in two distinct detection strategies. • These methods are validated for food, water, and soil analysis. • MS-based detection limits are lowered by two orders of magnitude. - Abstract: Norovirus infections are one of the most prominent public health problems of microbial origin in the U.S. and other industrialized countries. Surveillance is necessary to prevent secondary infection, confirm successful cleanup after outbreaks, and track the causative agent. Quantitative mass spectrometry, based on absolute quantitation with stable-isotope labeled peptides, is a promising tool for norovirus monitoring because of its speed, sensitivity, and robustness in the face of environmental inhibitors. In the current study, we present two new methods for the detection of the norovirus genogroup I capsid protein using electrospray and matrix-assisted laser desorption/ionization (MALDI) mass spectrometry. The peptide TLDPIEVPLEDVR was used to quantify norovirus-like particles down to 500 attomoles with electrospray and 100 attomoles with MALDI. With MALDI, we also demonstrate a detection limit of 1 femtomole and a quantitative dynamic range of 5 orders of magnitude in the presence of an environmental matrix effect. Due to the rapid processing time and applicability to a wide range of environmental sample types (bacterial lysate, produce, milk, soil, and groundwater), mass spectrometry-based absolute quantitation has a strong potential for use in public health and environmental sciences.

  16. Influence of minor displacements in loops of the porcine parvovirus VP2 capsid on virus-like particles assembly and the induction of antibody responses.

    Science.gov (United States)

    Pan, Qunxing; He, Kongwang; Wang, Yongshan; Wang, Xiaoli; Ouyang, Wei

    2013-06-01

    An antigen-delivery system based on hybrid virus-like particles (VLPs) formed by the self-assembly of the capsid VP2 protein of porcine parvovirus (PPV) and expressing foreign peptides offers an alternative method for vaccination. In this study, the three-dimensional structure of the PPV capsid protein and surface loops deletion mutants were analyzed to define essential domains in PPV VP2 for the assembly of VLPs. Electron microscopic analysis and SDS-PAGE analysis confirmed the presence of abundant VLPs in a loop2 deletion mutant of expected size and appropriate morphology. Loop4 and loop2-loop4 deletion mutants, however, resulted in a lower number of particles and the morphology of the particles was not well preserved. Furthermore, the green fluorescent protein (gfp) gene was used as a model. GFP was observed at the same level in displacements mutants. However, GFP displacement mutants in loop2 construct allowed better adaptation for the fusion GFP to be further displayed on the surface of the capsid-like structure. Immunogenicity study showed that there is no obvious difference in mice inoculated with rAd-VP2(Δloop2), rAd-VP2(Δloop4), rAd-VP2(Δloop2-Δloop4), and PPV inactivated vaccine. The results suggested the possibility of inserting simultaneously B and T cell epitopes in the surface loop2 and the N-terminus. The combination of different types of epitopes (B, CD4+, and CD8+) in different positions of the PPV particles opens the way to the development of highly efficient vaccines, able to stimulate at the same time the different branches of the immune system.

  17. Effects of two amino acid substitutions in the capsid proteins on the interaction of two cell-adapted PanAsia-1 strains of foot-and-mouth disease virus serotype O with heparan sulfate receptor.

    Science.gov (United States)

    Bai, Xingwen; Bao, Huifang; Li, Pinghua; Wei, Wei; Zhang, Meng; Sun, Pu; Cao, Yimei; Lu, Zengjun; Fu, Yuanfang; Xie, Baoxia; Chen, Yingli; Li, Dong; Luo, Jianxun; Liu, Zaixin

    2014-07-24

    encoding Gln-2080 → Leu in VP2 of O/Tibet/CHA/6/99tc could bind to HS, but there was no expression of the 3A protein of these two viruses in WT-CHO cells. The results suggest that the cooperation of certain specific amino acid residues in the capsid proteins of these two cell-adapted PanAsia-1 strains is essential for viral infectivity, the heparin affinity and the capability on FMDV-HS interaction.

  18. Direct interaction between two viral proteins, the nonstructural protein 2C and the capsid protein VP3, is required for enterovirus morphogenesis.

    Directory of Open Access Journals (Sweden)

    Ying Liu

    2010-08-01

    Full Text Available In spite of decades-long studies, the mechanism of morphogenesis of plus-stranded RNA viruses belonging to the genus Enterovirus of Picornaviridae, including poliovirus (PV, is not understood. Numerous attempts to identify an RNA encapsidation signal have failed. Genetic studies, however, have implicated a role of the non-structural protein 2C(ATPase in the formation of poliovirus particles. Here we report a novel mechanism in which protein-protein interaction is sufficient to explain the specificity in PV encapsidation. Making use of a novel "reporter virus", we show that a quasi-infectious chimera consisting of the capsid precursor of C-cluster coxsackie virus 20 (C-CAV20 and the nonstructural proteins of the closely related PV translated and replicated its genome with wild type kinetics, whereas encapsidation was blocked. On blind passages, encapsidation of the chimera was rescued by a single mutation either in capsid protein VP3 of CAV20 or in 2C(ATPase of PV. Whereas each of the single-mutation variants expressed severe proliferation phenotypes, engineering both mutations into the chimera yielded a virus encapsidating with wild type kinetics. Biochemical analyses provided strong evidence for a direct interaction between 2C(ATPase and VP3 of PV and CAV20. Chimeras of other C-CAVs (CAV20/CAV21 or CAV18/CAV20 were blocked in encapsidation (no virus after blind passages but could be rescued if the capsid and 2C(ATPase coding regions originated from the same virus. Our novel mechanism explains the specificity of encapsidation without apparent involvement of an RNA signal by considering that (i genome replication is known to be stringently linked to translation, (ii morphogenesis is known to be stringently linked to genome replication, (iii newly synthesized 2C(ATPase is an essential component of the replication complex, and (iv 2C(ATPase has specific affinity to capsid protein(s. These conditions lead to morphogenesis at the site where newly

  19. Production of a recombinant capsid protein VP1 from a newly described polyomavirus (RacPyV for downstream use in virus characterization

    Directory of Open Access Journals (Sweden)

    Molly E. Church

    2016-06-01

    Full Text Available Here we describe the methods for production of a recombinant viral capsid protein and subsequent use in an indirect enzyme linked immunosorbent assay (ELISA, and for use in production of a rabbit polyclonal antibody. These reagents were utilized in development and optimization of an ELISA, which established the extent of exposure of free ranging raccoons to a newly described polyomavirus (RacPyV [1]. Production of a polyclonal antibody has allowed for further characterization of RacPyV, including immunohistochemistry and immunocytochemistry techniques, in order to answer questions about pathogenesis of this virus.

  20. Sequential memory: Binding dynamics

    Science.gov (United States)

    Afraimovich, Valentin; Gong, Xue; Rabinovich, Mikhail

    2015-10-01

    Temporal order memories are critical for everyday animal and human functioning. Experiments and our own experience show that the binding or association of various features of an event together and the maintaining of multimodality events in sequential order are the key components of any sequential memories—episodic, semantic, working, etc. We study a robustness of binding sequential dynamics based on our previously introduced model in the form of generalized Lotka-Volterra equations. In the phase space of the model, there exists a multi-dimensional binding heteroclinic network consisting of saddle equilibrium points and heteroclinic trajectories joining them. We prove here the robustness of the binding sequential dynamics, i.e., the feasibility phenomenon for coupled heteroclinic networks: for each collection of successive heteroclinic trajectories inside the unified networks, there is an open set of initial points such that the trajectory going through each of them follows the prescribed collection staying in a small neighborhood of it. We show also that the symbolic complexity function of the system restricted to this neighborhood is a polynomial of degree L - 1, where L is the number of modalities.