WorldWideScience

Sample records for virus membrane fusion

  1. Cryomicroscopy provides structural snapshots of influenza virus membrane fusion.

    Science.gov (United States)

    Calder, Lesley J; Rosenthal, Peter B

    2016-09-01

    The lipid-enveloped influenza virus enters host cells during infection by binding cell-surface receptors and, after receptor-mediated endocytosis, fusing with the membrane of the endosome and delivering the viral genome and transcription machinery into the host cell. These events are mediated by the hemagglutinin (HA) surface glycoprotein. At the low pH of the endosome, an irreversible conformational change in the HA, including the exposure of the hydrophobic fusion peptide, activates membrane fusion. Here we used electron cryomicroscopy and cryotomography to image the fusion of influenza virus with target membranes at low pH. We visualized structural intermediates of HA and their interactions with membranes during the course of membrane fusion as well as ultrastructural changes in the virus that accompany membrane fusion. Our observations are relevant to a wide range of protein-mediated membrane-fusion processes and demonstrate how dynamic membrane events may be studied by cryomicroscopy.

  2. Characterization of the functional requirements of West Nile virus membrane fusion

    NARCIS (Netherlands)

    Moesker, Bastiaan; Rodenhuis-Zybert, Izabela A.; Meijerhof, Tjarko; Wilschut, Jan; Smit, Jolanda M.

    Flaviviruses infect their host cells by a membrane fusion reaction. In this study, we performed a functional analysis of the membrane fusion properties of West Nile virus (WNV) with liposomal target membranes. Membrane fusion was monitored continuously using a lipid mixing assay involving the

  3. Herpes Simplex Virus 1 Envelope Cholesterol Facilitates Membrane Fusion

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    George A. Wudiri

    2017-12-01

    Full Text Available Methyl beta-cyclodextrin (MβCD treatment of herpes simplex virus 1 (HSV-1 reduced envelope cholesterol levels and inhibited viral entry and infectivity in several cell types, regardless of the dependence of entry on endocytosis or low pH. Viral protein composition was similar in MβCD-treated and untreated virions, and ultrastructural analysis by electron microscopy revealed that cholesterol removal did not grossly affect virion structure or integrity. Removal of envelope cholesterol greatly reduced virion fusion activity as measured by fusion-from-without, suggesting that virion cholesterol is critical for the step of membrane fusion. MβCD-treatment of HSV-1 did not reduce viral attachment to the cells nor endocytic uptake of HSV-1 from the cell surface. The pre-fusion form of gB present in the HSV-1 envelope undergoes conformational changes in response to mildly acidic pH. These gB changes occurred independently of envelope cholesterol. Removal of cholesterol compromised virion stability as measured by recovery of infectivity following cycles of freeze-thaw. Taken together, the data suggest that HSV-1 envelope cholesterol is important for viral entry and infectivity due to a critical role in membrane fusion.

  4. Henipavirus Mediated Membrane Fusion, Virus Entry and Targeted Therapeutics

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    Dimitar B. Nikolov

    2012-02-01

    Full Text Available The Paramyxoviridae genus Henipavirus is presently represented by the type species Hendra and Nipah viruses which are both recently emerged zoonotic viral pathogens responsible for repeated outbreaks associated with high morbidity and mortality in Australia, Southeast Asia, India and Bangladesh. These enveloped viruses bind and enter host target cells through the coordinated activities of their attachment (G and class I fusion (F envelope glycoproteins. The henipavirus G glycoprotein interacts with host cellular B class ephrins, triggering conformational alterations in G that lead to the activation of the F glycoprotein, which facilitates the membrane fusion process. Using the recently published structures of HeV-G and NiV-G and other paramyxovirus glycoproteins, we review the features of the henipavirus envelope glycoproteins that appear essential for mediating the viral fusion process, including receptor binding, G-F interaction, F activation, with an emphasis on G and the mutations that disrupt viral infectivity. Finally, recent candidate therapeutics for henipavirus-mediated disease are summarized in light of their ability to inhibit HeV and NiV entry by targeting their G and F glycoproteins.

  5. Membrane fusion

    DEFF Research Database (Denmark)

    Bendix, Pól Martin

    2015-01-01

    At Stanford University, Boxer lab, I worked on membrane fusion of small unilamellar lipid vesicles to flat membranes tethered to glass surfaces. This geometry closely resembles biological systems in which liposomes fuse to plasma membranes. The fusion mechanism was studied using DNA zippering...... between complementary strands linked to the two apposing membranes closely mimicking the zippering mechanism of SNARE fusion complexes....

  6. Influenza-virus membrane fusion by cooperative fold-back of stochastically induced hemagglutinin intermediates

    NARCIS (Netherlands)

    Ivanovic, Tijana; Choi, Jason L.; Whelan, Sean P.; Oijen, Antoine M. van; Harrison, Stephen C.

    2013-01-01

    Influenza virus penetrates cells by fusion of viral and endosomal membranes catalyzed by the viral hemagglutinin (HA). Structures of the initial and final states of the HA trimer define the fusion endpoints, but do not specify intermediates. We have characterized these transitions by analyzing

  7. Early Events in Chikungunya Virus Infection—From Virus CellBinding to Membrane Fusion

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    Mareike K. S. van Duijl-Richter

    2015-07-01

    Full Text Available Chikungunya virus (CHIKV is a rapidly emerging mosquito-borne alphavirus causing millions of infections in the tropical and subtropical regions of the world. CHIKV infection often leads to an acute self-limited febrile illness with debilitating myalgia and arthralgia. A potential long-term complication of CHIKV infection is severe joint pain, which can last for months to years. There are no vaccines or specific therapeutics available to prevent or treat infection. This review describes the critical steps in CHIKV cell entry. We summarize the latest studies on the virus-cell tropism, virus-receptor binding, internalization, membrane fusion and review the molecules and compounds that have been described to interfere with virus cell entry. The aim of the review is to give the reader a state-of-the-art overview on CHIKV cell entry and to provide an outlook on potential new avenues in CHIKV research.

  8. Membrane fusion-competent virus-like proteoliposomes and proteinaceous supported bilayers made directly from cell plasma membranes.

    Science.gov (United States)

    Costello, Deirdre A; Hsia, Chih-Yun; Millet, Jean K; Porri, Teresa; Daniel, Susan

    2013-05-28

    Virus-like particles are useful materials for studying virus-host interactions in a safe manner. However, the standard production of pseudovirus based on the vesicular stomatitis virus (VSV) backbone is an intricate procedure that requires trained laboratory personnel. In this work, a new strategy for creating virus-like proteoliposomes (VLPLs) and virus-like supported bilayers (VLSBs) is presented. This strategy uses a cell blebbing technique to induce the formation of nanoscale vesicles from the plasma membrane of BHK cells expressing the hemagglutinin (HA) fusion protein of influenza X-31. These vesicles and supported bilayers contain HA and are used to carry out single particle membrane fusion events, monitored using total internal reflection fluorescence microscopy. The results of these studies show that the VLPLs and VLSBs contain HA proteins that are fully competent to carry out membrane fusion, including the formation of a fusion pore and the release of fluorophores loaded into vesicles. This new strategy for creating spherical and planar geometry virus-like membranes has many potential applications. VLPLs could be used to study fusion proteins of virulent viruses in a safe manner, or they could be used as therapeutic delivery particles to transport beneficial proteins coexpressed in the cells to a target cell. VLSBs could facilitate high throughput screening of antiviral drugs or pathogen-host cell interactions.

  9. Influenza-virus membrane fusion by cooperative fold-back of stochastically induced hemagglutinin intermediates.

    Science.gov (United States)

    Ivanovic, Tijana; Choi, Jason L; Whelan, Sean P; van Oijen, Antoine M; Harrison, Stephen C

    2013-02-19

    Influenza virus penetrates cells by fusion of viral and endosomal membranes catalyzed by the viral hemagglutinin (HA). Structures of the initial and final states of the HA trimer define the fusion endpoints, but do not specify intermediates. We have characterized these transitions by analyzing low-pH-induced fusion kinetics of individual virions and validated the analysis by computer simulation. We detect initial engagement with the target membrane of fusion peptides from independently triggered HAs within the larger virus-target contact patch; fusion then requires engagement of three or four neighboring HA trimers. Effects of mutations in HA indicate that withdrawal of the fusion peptide from a pocket in the pre-fusion trimer is rate-limiting for both events, but the requirement for cooperative action of several HAs to bring the fusing membranes together leads to a long-lived intermediate state for single, extended HA trimers. This intermediate is thus a fundamental aspect of the fusion mechanism. DOI:http://dx.doi.org/10.7554/eLife.00333.001.

  10. Cell-based analysis of Chikungunya virus membrane fusion using baculovirus-expression vectors.

    Science.gov (United States)

    Kuo, Szu-Cheng; Chen, Ying-Ju; Wang, Yu-Ming; Kuo, Ming-Der; Jinn, Tzyy-Rong; Chen, Wen-Shuo; Chang, Yen-Chung; Tung, Kuo-Lun; Wu, Tzong-Yuan; Lo, Szecheng J

    2011-08-01

    Chikungunya virus infection has emerged in many countries over the past decade. There are no effective drugs for controlling the disease. To develop cell-based system for screening anti-virus drugs, a bi-cistronic baculovirus expression system was utilized to co-express viral structural proteins C (capsid), E2 and E1 and the enhanced green fluorescence protein (EGFP) in Spodoptera frugiperda insect cells (Sf21). The EGFP-positive Sf21 cells fused with each other and with uninfected cells to form a syncytium, allowing characterization of cholesterol and low pH requirements for syncytium formation. Western blot analysis showed three structural proteins were expressed in baculovirus infected cells. The structural proteins of Chikungunya virus that is required for cell fusion was determined with various recombinant baculoviruses bearing different lengths of the viral structural protein genes. Protein E1 was required for cell fusion and indicating that Chikungunya viral membrane fusion was a class II membrane fusion. It was also demonstrated that the heterologous expression of alphavirus monomeric E1 can induce insect cell fusions. Furthermore, this cell-based system provides a model for studying class II viral membrane fusion. Copyright © 2011 Elsevier B.V. All rights reserved.

  11. Measuring the Strength of Interaction between the Ebola Fusion Peptide and Lipid Rafts: Implications for Membrane Fusion and Virus Infection

    Science.gov (United States)

    Freitas, Mônica S.; Follmer, Cristian; Costa, Lilian T.; Vilani, Cecília; Bianconi, M. Lucia; Achete, Carlos Alberto; Silva, Jerson L.

    2011-01-01

    The Ebola fusion peptide (EBO16) is a hydrophobic domain that belongs to the GP2 membrane fusion protein of the Ebola virus. It adopts a helical structure in the presence of mimetic membranes that is stabilized by the presence of an aromatic-aromatic interaction established by Trp8 and Phe12. In spite of its infectious cycle becoming better understood recently, several steps still remain unclear, a lacuna that makes it difficult to develop strategies to block infection. In order to gain insight into the mechanism of membrane fusion, we probed the structure, function and energetics of EBO16 and its mutant W8A, in the absence or presence of different lipid membranes, including isolated domain-resistant membranes (DRM), a good experimental model for lipid rafts. The depletion of cholesterol from living mammalian cells reduced the ability of EBO16 to induce lipid mixing. On the other hand, EBO16 was structurally sensitive to interaction with lipid rafts (DRMs), but the same was not observed for W8A mutant. In agreement with these data, W8A showed a poor ability to promote membrane aggregation in comparison to EBO16. Single molecule AFM experiments showed a high affinity force pattern for the interaction of EBO16 and DRM, which seems to be a complex energetic event as observed by the calorimetric profile. Our study is the first to show a strong correlation between the initial step of Ebola virus infection and cholesterol, thus providing a rationale for Ebola virus proteins being co-localized with lipid-raft domains. In all, the results show how small fusion peptide sequences have evolved to adopt highly specific and strong interactions with membrane domains. Such features suggest these processes are excellent targets for therapeutic and vaccine approaches to viral diseases. PMID:21249196

  12. Outer nuclear membrane fusion of adjacent nuclei in varicella-zoster virus-induced syncytia.

    Science.gov (United States)

    Wang, Wei; Yang, Lianwei; Huang, Xiumin; Fu, Wenkun; Pan, Dequan; Cai, Linli; Ye, Jianghui; Liu, Jian; Xia, Ningshao; Cheng, Tong; Zhu, Hua

    2017-12-01

    Syncytia formation has been considered important for cell-to-cell spread and pathogenesis of many viruses. As a syncytium forms, individual nuclei often congregate together, allowing close contact of nuclear membranes and possibly fusion to occur. However, there is currently no reported evidence of nuclear membrane fusion between adjacent nuclei in wild-type virus-induced syncytia. Varicella-zoster virus (VZV) is one typical syncytia-inducing virus that causes chickenpox and shingles in humans. Here, we report, for the first time, an interesting observation of apparent fusion of the outer nuclear membranes from juxtaposed nuclei that comprise VZV syncytia both in ARPE-19 human epithelial cells in vitro and in human skin xenografts in the SCID-hu mouse model in vivo. This work reveals a novel aspect of VZV-related cytopathic effect in the context of multinucleated syncytia. Additionally, the information provided by this study could be helpful for future studies on interactions of viruses with host cell nuclei. Copyright © 2017 Elsevier Inc. All rights reserved.

  13. The Fusion Loops of the Initial Prefusion Conformation of Herpes Simplex Virus 1 Fusion Protein Point Toward the Membrane.

    Science.gov (United States)

    Fontana, Juan; Atanasiu, Doina; Saw, Wan Ting; Gallagher, John R; Cox, Reagan G; Whitbeck, J Charles; Brown, Lauren M; Eisenberg, Roselyn J; Cohen, Gary H

    2017-08-22

    All enveloped viruses, including herpesviruses, must fuse their envelope with the host membrane to deliver their genomes into target cells, making this essential step subject to interference by antibodies and drugs. Viral fusion is mediated by a viral surface protein that transits from an initial prefusion conformation to a final postfusion conformation. Strikingly, the prefusion conformation of the herpesvirus fusion protein, gB, is poorly understood. Herpes simplex virus (HSV), a model system for herpesviruses, causes diseases ranging from mild skin lesions to serious encephalitis and neonatal infections. Using cryo-electron tomography and subtomogram averaging, we have characterized the structure of the prefusion conformation and fusion intermediates of HSV-1 gB. To this end, we have set up a system that generates microvesicles displaying full-length gB on their envelope. We confirmed proper folding of gB by nondenaturing electrophoresis-Western blotting with a panel of monoclonal antibodies (MAbs) covering all gB domains. To elucidate the arrangement of gB domains, we labeled them by using (i) mutagenesis to insert fluorescent proteins at specific positions, (ii) coexpression of gB with Fabs for a neutralizing MAb with known binding sites, and (iii) incubation of gB with an antibody directed against the fusion loops. Our results show that gB starts in a compact prefusion conformation with the fusion loops pointing toward the viral membrane and suggest, for the first time, a model for gB's conformational rearrangements during fusion. These experiments further illustrate how neutralizing antibodies can interfere with the essential gB structural transitions that mediate viral entry and therefore infectivity.IMPORTANCE The herpesvirus family includes herpes simplex virus (HSV) and other human viruses that cause lifelong infections and a variety of diseases, like skin lesions, encephalitis, and cancers. As enveloped viruses, herpesviruses must fuse their envelope

  14. Structural insights into the membrane fusion mechanism mediated by influenza virus hemagglutinin.

    Science.gov (United States)

    Ni, Fengyun; Chen, Xiaorui; Shen, Jun; Wang, Qinghua

    2014-02-11

    Membrane fusion is involved in many fundamental cellular processes and entry of enveloped viruses into host cells. Influenza type A virus HA has long served as a paradigm for mechanistic studies of protein-mediated membrane fusion via large-scale structural rearrangements induced by acidic pH. Here we report the newly determined crystal structure of influenza B virus HA2 in the postfusion state. Together with a large number of previously determined prefusion structures of influenza A and B virus HA and a postfusion structure of influenza A/H3N2 HA2, we identified conserved features that are shared between influenza A and B virus HA in the conformational transition and documented substantial differences that likely influence the detailed mechanisms of this process. Further studies are needed to dissect the effects of these and other structural differences in HA conformational changes and influenza pathogenicity and transmission, which may ultimately expedite the discovery of novel anti-influenza fusion inhibitors.

  15. Membrane tension and membrane fusion

    OpenAIRE

    Kozlov, Michael M.; Chernomordik, Leonid V.

    2015-01-01

    Diverse cell biological processes that involve shaping and remodeling of cell membranes are regulated by membrane lateral tension. Here we focus on the role of tension in driving membrane fusion. We discuss the physics of membrane tension, forces that can generate the tension in plasma membrane of a cell, and the hypothesis that tension powers expansion of membrane fusion pores in late stages of cell-to-cell and exocytotic fusion. We propose that fusion pore expansion can require unusually la...

  16. The Fusion Loops of the Initial Prefusion Conformation of Herpes Simplex Virus 1 Fusion Protein Point Toward the Membrane

    Directory of Open Access Journals (Sweden)

    Juan Fontana

    2017-08-01

    Full Text Available All enveloped viruses, including herpesviruses, must fuse their envelope with the host membrane to deliver their genomes into target cells, making this essential step subject to interference by antibodies and drugs. Viral fusion is mediated by a viral surface protein that transits from an initial prefusion conformation to a final postfusion conformation. Strikingly, the prefusion conformation of the herpesvirus fusion protein, gB, is poorly understood. Herpes simplex virus (HSV, a model system for herpesviruses, causes diseases ranging from mild skin lesions to serious encephalitis and neonatal infections. Using cryo-electron tomography and subtomogram averaging, we have characterized the structure of the prefusion conformation and fusion intermediates of HSV-1 gB. To this end, we have set up a system that generates microvesicles displaying full-length gB on their envelope. We confirmed proper folding of gB by nondenaturing electrophoresis-Western blotting with a panel of monoclonal antibodies (MAbs covering all gB domains. To elucidate the arrangement of gB domains, we labeled them by using (i mutagenesis to insert fluorescent proteins at specific positions, (ii coexpression of gB with Fabs for a neutralizing MAb with known binding sites, and (iii incubation of gB with an antibody directed against the fusion loops. Our results show that gB starts in a compact prefusion conformation with the fusion loops pointing toward the viral membrane and suggest, for the first time, a model for gB’s conformational rearrangements during fusion. These experiments further illustrate how neutralizing antibodies can interfere with the essential gB structural transitions that mediate viral entry and therefore infectivity.

  17. Ebola virus glycoprotein needs an additional trigger, beyond proteolytic priming for membrane fusion.

    Directory of Open Access Journals (Sweden)

    Shridhar Bale

    2011-11-01

    Full Text Available Ebolavirus belongs to the family filoviridae and causes severe hemorrhagic fever in humans with 50-90% lethality. Detailed understanding of how the viruses attach to and enter new host cells is critical to development of medical interventions. The virus displays a trimeric glycoprotein (GP(1,2 on its surface that is solely responsible for membrane attachment, virus internalization and fusion. GP(1,2 is expressed as a single peptide and is cleaved by furin in the host cells to yield two disulphide-linked fragments termed GP1 and GP2 that remain associated in a GP(1,2 trimeric, viral surface spike. After entry into host endosomes, GP(1,2 is enzymatically cleaved by endosomal cathepsins B and L, a necessary step in infection. However, the functional effects of the cleavage on the glycoprotein are unknown.We demonstrate by antibody binding and Hydrogen-Deuterium Exchange Mass Spectrometry (DXMS of glycoproteins from two different ebolaviruses that although enzymatic priming of GP(1,2 is required for fusion, the priming itself does not initiate the required conformational changes in the ectodomain of GP(1,2. Further, ELISA binding data of primed GP(1,2 to conformational antibody KZ52 suggests that the low pH inside the endosomes also does not trigger dissociation of GP1 from GP2 to effect membrane fusion.The results reveal that the ebolavirus GP(1,2 ectodomain remains in the prefusion conformation upon enzymatic cleavage in low pH and removal of the glycan cap. The results also suggest that an additional endosomal trigger is necessary to induce the conformational changes in GP(1,2 and effect fusion. Identification of this trigger will provide further mechanistic insights into ebolavirus infection.

  18. Role of a Transbilayer pH Gradient in the Membrane Fusion Activity of the Influenza Virus Hemagglutinin: Use of the R18 Assay to Monitor Membrane Merging

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    Pedroso de Lima Maria C.

    1999-01-01

    Full Text Available It had been suggested that influenza virus-mediated membrane fusion might be dependent on a pH gradient across a target membrane. We have designed experiments in which this issue could be addressed. Two populations of liposomes were prepared, both simulating the plasma membrane of target cells, but with the pH of the internal aqueous medium buffered either at pH 7.4 (physiological cytosol pH or at pH 5.0 (endosomal pH at which influenza virus displays maximal fusion activity. By monitoring fusion using the R18 assay, we found that the internal pH of the target liposomes did not influence membrane merging as mediated by the influenza virus hemagglutinin, thus demonstrating that a transmembrane pH gradient is not required in this fusion process.

  19. Reciprocal complementation of bovine parainfluenza virus type 3 lacking either the membrane or fusion gene.

    Science.gov (United States)

    Takada, Marina; Matsuura, Ryosuke; Kokuho, Takehiro; Tsuboi, Takamitsu; Kameyama, Ken-Ichiro; Takeuchi, Kaoru

    2017-11-01

    Two defective bovine parainfluenza virus type 3 (BPIV3) strains were generated, one lacking the membrane (M) protein gene and expressing EGFP (ΔM-EGFP) and the other lacking the fusion (F) protein gene and expressing mStrawberry (ΔF-mSB), by supplying deficient proteins in trans. When Madin-Darby bovine kidney (MDBK) cells were co-infected with ΔM-EGFP and ΔF-mSB at a multiplicity of infection (MOI) of 0.1, complemented viruses were easily obtained. Complemented viruses grew as efficiently as wild-type BPIV3 and could be passaged in MDBK cell cultures even at an MOI of 0.01, possibly due to multiploid virus particles containing genomes of both ΔM-EGFP and ΔF-mSB. This reciprocal complementation method using two defective viruses would be useful to express large or multiple proteins in cell cultures using paramyxovirus vectors. Copyright © 2017 Elsevier B.V. All rights reserved.

  20. The membrane fusion step of vaccinia virus entry is cooperatively mediated by multiple viral proteins and host cell components.

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    Jason P Laliberte

    2011-12-01

    Full Text Available For many viruses, one or two proteins allow cell attachment and entry, which occurs through the plasma membrane or following endocytosis at low pH. In contrast, vaccinia virus (VACV enters cells by both neutral and low pH routes; four proteins mediate cell attachment and twelve that are associated in a membrane complex and conserved in all poxviruses are dedicated to entry. The aim of the present study was to determine the roles of cellular and viral proteins in initial stages of entry, specifically fusion of the membranes of the mature virion and cell. For analysis of the role of cellular components, we used well characterized inhibitors and measured binding of a recombinant VACV virion containing Gaussia luciferase fused to a core protein; viral and cellular membrane lipid mixing with a self-quenching fluorescent probe in the virion membrane; and core entry with a recombinant VACV expressing firefly luciferase and electron microscopy. We determined that inhibitors of tyrosine protein kinases, dynamin GTPase and actin dynamics had little effect on binding of virions to cells but impaired membrane fusion, whereas partial cholesterol depletion and inhibitors of endosomal acidification and membrane blebbing had a severe effect at the later stage of core entry. To determine the role of viral proteins, virions lacking individual membrane components were purified from cells infected with members of a panel of ten conditional-lethal inducible mutants. Each of the entry protein-deficient virions had severely reduced infectivity and except for A28, L1 and L5 greatly impaired membrane fusion. In addition, a potent neutralizing L1 monoclonal antibody blocked entry at a post-membrane lipid-mixing step. Taken together, these results suggested a 2-step entry model and implicated an unprecedented number of viral proteins and cellular components involved in signaling and actin rearrangement for initiation of virus-cell membrane fusion during poxvirus entry.

  1. Association of the pr Peptides with Dengue Virus at Acidic pH Blocks Membrane Fusion

    Energy Technology Data Exchange (ETDEWEB)

    Yu, I.-M.; Holdaway, H.A.; Chipman, P.R.; Kuhn, R.J.; Rossmann, M.G.; Chen, J.; Purdue

    2010-07-27

    Flavivirus assembles into an inert particle that requires proteolytic activation by furin to enable transmission to other hosts. We previously showed that immature virus undergoes a conformational change at low pH that renders it accessible to furin (I. M. Yu, W. Zhang, H. A. Holdaway, L. Li, V. A. Kostyuchenko, P. R. Chipman, R. J. Kuhn, M. G. Rossmann, and J. Chen, Science 319:1834-1837, 2008). Here we show, using cryoelectron microscopy, that the structure of immature dengue virus at pH 6.0 is essentially the same before and after the cleavage of prM. The structure shows that after cleavage, the proteolytic product pr remains associated with the virion at acidic pH, and that furin cleavage by itself does not induce any major conformational changes. We also show by liposome cofloatation experiments that pr retention prevents membrane insertion, suggesting that pr is present on the virion in the trans-Golgi network to protect the progeny virus from fusion within the host cell.

  2. Fusion between perinuclear virions and the outer nuclear membrane requires the fusogenic activity of herpes simplex virus gB.

    Science.gov (United States)

    Wright, Catherine C; Wisner, Todd W; Hannah, Brian P; Eisenberg, Roselyn J; Cohen, Gary H; Johnson, David C

    2009-11-01

    Herpesviruses cross nuclear membranes (NMs) in two steps, as follows: (i) capsids assemble and bud through the inner NM into the perinuclear space, producing enveloped virus particles, and (ii) the envelopes of these virus particles fuse with the outer NM. Two herpes simplex virus (HSV) glycoproteins, gB and gH (the latter, likely complexed as a heterodimer with gL), are necessary for the second step of this process. Mutants lacking both gB and gH accumulate in the perinuclear space or in herniations (membrane vesicles derived from the inner NM). Both gB and gH/gL are also known to act directly in fusing the virion envelope with host cell membranes during HSV entry into cells, i.e., both glycoproteins appear to function directly in different aspects of the membrane fusion process. We hypothesized that HSV gB and gH/gL also act directly in the membrane fusion that occurs during virus egress from the nucleus. Previous studies of the role of gB and gH/gL in nuclear egress involved HSV gB and gH null mutants that could potentially also possess gross defects in the virion envelope. Here, we produced recombinant HSV-expressing mutant forms of gB with single amino acid substitutions in the hydrophobic "fusion loops." These fusion loops are thought to play a direct role in membrane fusion by insertion into cellular membranes. HSV recombinants expressing gB with any one of four fusion loop mutations (W174R, W174Y, Y179K, and A261D) were unable to enter cells. Moreover, two of the mutants, W174Y and Y179K, displayed reduced abilities to mediate HSV cell-to-cell spread, and W174R and A261D exhibited no spread. All mutant viruses exhibited defects in nuclear egress, enveloped virions accumulated in herniations and in the perinuclear space, and fewer enveloped virions were detected on cell surfaces. These results support the hypothesis that gB functions directly to mediate the fusion between perinuclear virus particles and the outer NM.

  3. Fusion of biological membranes

    Indian Academy of Sciences (India)

    The process of membrane fusion has been examined by Monte Carlo simulation, and is found to be very different than the conventional picture. The differences in mechanism lead to several predictions, in particular that fusion is accompanied by transient leakage. This prediction has recently been verified. Self-consistent ...

  4. Development of a membrane fusible drug carrier from erythrocytes by the spontaneous transfer of viral fusion protein from influenza virus-infected cells.

    Science.gov (United States)

    Kogure, K; Okuda, O; Itoh, T; Hayashi, K; Ueno, M

    1997-05-01

    In order to develop a membrane fusible drug carrier from human erythrocytes, we attempted the reconstitution of influenza virus fusion protein hemagglutinin (HA) to an erythrocyte membrane. In this study, we succeeded in the preparation of HA-reconstituted erythrocytes (HA-erythrocytes) by the incubation of erythrocytes with influenza virus-infected CV-1 cells, and confirmed the ability of HA-erythrocytes to fuse with the cell membrane. Furthermore, by using an HA-reconstituted ghost (HA-ghost), which entrapped fluorescent-labeled ovalbumin, 25% of the protein was incorporated into cells through the fusion of the HA-ghost with the cell membrane.

  5. The soluble ectodomain of herpes simplex virus gD contains a membrane-proximal pro-fusion domain and suffices to mediate virus entry.

    Science.gov (United States)

    Cocchi, Francesca; Fusco, Daniela; Menotti, Laura; Gianni, Tatiana; Eisenberg, Roselyn J; Cohen, Gary H; Campadelli-Fiume, Gabriella

    2004-05-11

    Entry of herpes simplex virus (HSV) 1 into cells requires the interaction of HSV gD with herpesvirus entry mediator or nectin1 receptors, and fusion with cell membrane mediated by the fusion glycoproteins gB, gH, and gL. We report that the gD ectodomain in soluble form (amino acids 1-305) was sufficient to rescue the infectivity of a gD-null HSV mutant, indicating that gD does not need to be anchored to the virion envelope to mediate entry. Entry mediated by soluble gD required, in addition to the receptor-binding sites contained within residues 1-250, a discrete downstream portion (amino acids 261-305), located proximal to the transmembrane segment in full-length gD. We named it as profusion domain. The pro-fusion domain was required for entry mediated by virion-bound gD, because its substitution with the corresponding region of CD8 failed to complement the infectivity of gD(-/+) HSV. Furthermore, a receptor-negative gD (gD(Delta6-259)) inhibited virus infectivity when coexpressed with wild-type gD; i.e., it acted as a dominant-negative gD mutant. The pro-fusion domain is proline-rich, which is characteristic of regions involved in protein-protein interactions. P291L-P292A substitutions diminished the gD capacity to complement gD(-/+) HSV infectivity. We propose that gD forms a tripartite complex with its receptor and, by way of the proline-rich pro-fusion domain, with the fusion glycoproteins, or with one of them. The tripartite complex would serve to recruit/activate the fusion glycoproteins and bring them from a fusion-inactive to a fusion-active state, such that they execute fusion of the virion envelope with cell membrane.

  6. Early Events in Chikungunya Virus Infection-From Virus Cell Binding to Membrane Fusion

    NARCIS (Netherlands)

    van Duijl-Richter, Mareike K. S.; Hoornweg, Tabitha E.; Rodenhuis-Zybert, Izabela A.; Smit, Jolanda M.

    Chikungunya virus (CHIKV) is a rapidly emerging mosquito-borne alphavirus causing millions of infections in the tropical and subtropical regions of the world. CHIKV infection often leads to an acute self-limited febrile illness with debilitating myalgia and arthralgia. A potential long-term

  7. pH-Dependent Formation and Disintegration of the Influenza A Virus Protein Scaffold To Provide Tension for Membrane Fusion.

    Science.gov (United States)

    Batishchev, O V; Shilova, L A; Kachala, M V; Tashkin, V Y; Sokolov, V S; Fedorova, N V; Baratova, L A; Knyazev, D G; Zimmerberg, J; Chizmadzhev, Y A

    2015-10-14

    Influenza virus is taken up from a pH-neutral extracellular milieu into an endosome, whose contents then acidify, causing changes in the viral matrix protein (M1) that coats the inner monolayer of the viral lipid envelope. At a pH of ~6, M1 interacts with the viral ribonucleoprotein (RNP) in a putative priming stage; at this stage, the interactions of the M1 scaffold coating the lipid envelope are intact. The M1 coat disintegrates as acidification continues to a pH of ~5 to clear a physical path for the viral genome to transit from the viral interior to the cytoplasm. Here we investigated the physicochemical mechanism of M1's pH-dependent disintegration. In neutral media, the adsorption of M1 protein on the lipid bilayer was electrostatic in nature and reversible. The energy of the interaction of M1 molecules with each other in M1 dimers was about 10 times as weak as that of the interaction of M1 molecules with the lipid bilayer. Acidification drives conformational changes in M1 molecules due to changes in the M1 charge, leading to alterations in their electrostatic interactions. Dropping the pH from 7.1 to 6.0 did not disturb the M1 layer; dropping it lower partially desorbed M1 because of increased repulsion between M1 monomers still stuck to the membrane. Lipid vesicles coated with M1 demonstrated pH-dependent rupture of the vesicle membrane, presumably because of the tension generated by this repulsive force. Thus, the disruption of the vesicles coincident with M1 protein scaffold disintegration at pH 5 likely stretches the lipid membrane to the point of rupture, promoting fusion pore widening for RNP release. Influenza remains a top killer of human beings throughout the world, in part because of the influenza virus's rapid binding to cells and its uptake into compartments hidden from the immune system. To attack the influenza virus during this time of hiding, we need to understand the physical forces that allow the internalized virus to infect the cell. In

  8. Peptides and membrane fusion : Towards an understanding of the molecular mechanism of protein-induced fusion

    NARCIS (Netherlands)

    Pecheur, EI; Sainte-Marie, J; Bienvenue, A; Hoekstra, D

    1999-01-01

    Processes such as endo- or exocytosis, membrane recycling, fertilization and enveloped viruses infection require one or more critical membrane fusion reactions. A key feature in viral and cellular fusion phenomena is the involvement of specific fusion proteins. Among the few well-characterized

  9. Induction of heterosubtypic cross-protection against influenza by a whole inactivated virus vaccine: the role of viral membrane fusion activity.

    Directory of Open Access Journals (Sweden)

    Natalija Budimir

    Full Text Available BACKGROUND: The inability of seasonal influenza vaccines to effectively protect against infection with antigenically drifted viruses or newly emerging pandemic viruses underlines the need for development of cross-reactive influenza vaccines that induce immunity against a variety of virus subtypes. Therefore, potential cross-protective vaccines, e.g., whole inactivated virus (WIV vaccine, that can target conserved internal antigens such as the nucleoprotein (NP and/or matrix protein (M1 need to be explored. METHODOLOGY/PRINCIPAL FINDINGS: In the current study we show that a WIV vaccine, through induction of cross-protective cytotoxic T lymphocytes (CTLs, protects mice from heterosubtypic infection. This protection was abrogated after depletion of CD8+ cells in vaccinated mice, indicating that CTLs were the primary mediators of protection. Previously, we have shown that different procedures used for virus inactivation influence optimal activation of CTLs by WIV, most likely by affecting the membrane fusion properties of the virus. Specifically, inactivation with formalin (FA severely compromises fusion activity of the virus, while inactivation with β-propiolactone (BPL preserves fusion activity. Here, we demonstrate that vaccination of mice with BPL-inactivated H5N1 WIV vaccine induces solid protection from lethal heterosubtypic H1N1 challenge. By contrast, vaccination with FA-inactivated WIV, while preventing death after lethal challenge, failed to protect against development of disease and severe body weight loss. Vaccination with BPL-inactivated WIV, compared to FA-inactivated WIV, induced higher levels of specific CD8+ T cells in blood, spleen and lungs, and a higher production of granzyme B in the lungs upon H1N1 virus challenge. CONCLUSION/SIGNIFICANCE: The results underline the potential use of WIV as a cross-protective influenza vaccine candidate. However, careful choice of the virus inactivation procedure is important to retain membrane

  10. Tin oxide nanowires suppress herpes simplex virus-1 entry and cell-to-cell membrane fusion.

    Directory of Open Access Journals (Sweden)

    James Trigilio

    Full Text Available The advent of nanotechnology has ushered in the use of modified nanoparticles as potential antiviral agents against diseases such as herpes simplex virus 1 and 2 (HSV-1 (HSV-2, human immunodeficiency virus (HIV, monkeypox virus, and hepatitis B virus. Here we describe the application of tin oxide (SnO(2 nanowires as an effective treatment against HSV-1 infection. SnO(2 nanowires work as a carrier of negatively charged structures that compete with HSV-1 attachment to cell bound heparan sulfate (HS, therefore inhibiting entry and subsequent cell-to-cell spread. This promising new approach can be developed into a novel form of broad-spectrum antiviral therapy especially since HS has been shown to serve as a cellular co-receptor for a number of other viruses as well, including the respiratory syncytial virus, adeno-associated virus type 2, and human papilloma virus.

  11. Sphingolipids activate membrane fusion of Semliki Forest virus in a stereospecific manner

    DEFF Research Database (Denmark)

    Moesby, Lise; Corver, J; Erukulla, R K

    1995-01-01

    process. Pyrene-labeled SFV fused rapidly and extensively with liposomes consisting of a mixture of phosphatidylcholine, phosphatidylethanolamine, and cholesterol, supplemented with low concentrations of D-erythro-ceramide, representing the naturally occurring sphingolipid stereoisomer. Fusion...

  12. Refining the Mechanisms of Heniparvirus-Mediated Membrane Fusion Through Mutagenesis of Hendra virus Envelope Glycoproteins

    Science.gov (United States)

    2007-09-06

    Nipah viruses ..........................................................2 Pathogenesis ...many emerging viral diseases. Bats have long been known to serve as reservoirs for Rabies virus, and are now known to be reservoirs of the closely...purposes. Pathogenesis HeV and NiV are unusual paramyxoviruses in that they are zoonotic and highly pathogenic (reviewed (27)). Henipaviruses have

  13. Enhanced Growth of Influenza Vaccine Seed Viruses in Vero Cells Mediated by Broadening the Optimal pH Range for Virus Membrane Fusion

    Science.gov (United States)

    Murakami, Shin; Ito, Mutsumi; Takano, Ryo; Katsura, Hiroaki; Shimojima, Masayuki

    2012-01-01

    Vaccination is one of the most effective preventive measures to combat influenza. Prospectively, cell culture-based influenza vaccines play an important role for robust vaccine production in both normal settings and urgent situations, such as during the 2009 pandemic. African green monkey Vero cells are recommended by the World Health Organization as a safe substrate for influenza vaccine production for human use. However, the growth of influenza vaccine seed viruses is occasionally suboptimal in Vero cells, which places limitations on their usefulness for enhanced vaccine production. Here, we present a strategy for the development of vaccine seed viruses with enhanced growth in Vero cells by changing an amino acid residue in the stem region of the HA2 subunit of the hemagglutinin (HA) molecule. This mutation optimized the pH for HA-mediated membrane fusion in Vero cells and enhanced virus growth 100 to 1,000 times in the cell line, providing a promising strategy for cell culture-based influenza vaccines. PMID:22090129

  14. pH Optimum of Hemagglutinin-Mediated Membrane Fusion Determines Sensitivity of Influenza A Viruses to the Interferon-Induced Antiviral State and IFITMs.

    Science.gov (United States)

    Gerlach, Thomas; Hensen, Luca; Matrosovich, Tatyana; Bergmann, Janina; Winkler, Michael; Peteranderl, Christin; Klenk, Hans-Dieter; Weber, Friedemann; Herold, Susanne; Pöhlmann, Stefan; Matrosovich, Mikhail

    2017-06-01

    The replication and pathogenicity of influenza A viruses (IAVs) critically depend on their ability to tolerate the antiviral interferon (IFN) response. To determine a potential role for the IAV hemagglutinin (HA) in viral sensitivity to IFN, we studied the restriction of IAV infection in IFN-β-treated human epithelial cells by using 2:6 recombinant IAVs that shared six gene segments of A/Puerto Rico/8/1934 virus (PR8) and contained HAs and neuraminidases of representative avian, human, and zoonotic H5N1 and H7N9 viruses. In A549 and Calu-3 cells, viruses displaying a higher pH optimum of HA-mediated membrane fusion, H5N1-PR8 and H7N9-PR8, were less sensitive to the IFN-induced antiviral state than their counterparts with HAs from duck and human viruses, which fused at a lower pH. The association between a high pH optimum of fusion and reduced IFN sensitivity was confirmed by using HA point mutants of A/Hong Kong/1/1968-PR8 that differed solely by their fusion properties. Furthermore, similar effects of the viral fusion pH on IFN sensitivity were observed in experiments with (i) primary human type II alveolar epithelial cells and differentiated cultures of human airway epithelial cells, (ii) nonrecombinant zoonotic and pandemic IAVs, and (iii) preparations of IFN-α and IFN-λ1. A higher pH of membrane fusion and reduced sensitivity to IFN correlated with lower restriction of the viruses in MDCK cells stably expressing the IFN-inducible transmembrane proteins IFITM2 and IFITM3, which are known to inhibit viral fusion. Our results reveal that the pH optimum of HA-driven membrane fusion of IAVs is a determinant of their sensitivity to IFN and IFITM proteins. IMPORTANCE The IFN system constitutes an important innate defense against viral infection. Substantial information is available on how IAVs avoid detection by sensors of the IFN system and disable IFN signaling pathways. Much less is known about the ability of IAVs to tolerate the antiviral activity of IFN

  15. Mutational analyses of Epstein-Barr virus glycoprotein 42 reveal functional domains not involved in receptor binding but required for membrane fusion.

    Science.gov (United States)

    Silva, Amanda L; Omerovic, Jasmina; Jardetzky, Theodore S; Longnecker, Richard

    2004-06-01

    Epstein-Barr virus (EBV) is a human gammaherpesvirus associated with malignancies of both epithelial and lymphoid origin. Efficient infection of the latent host reservoir B lymphocytes involves the binding of glycoproteins gp350/220 for initial attachment, followed by the concerted action of gH, gL, gB, and gp42 for membrane fusion. The type II membrane protein gp42 is required for infection of B cells and assembles into a complex with gH and gL. The cellular host receptor for gp42, class II human leukocyte antigen (HLA), has been structurally verified by crystallization analyses of gp42 bound to HLA-DR1. Interestingly, the crystal structure revealed a hydrophobic pocket consisting of many aromatic and aliphatic residues from the predicted C-type lectin domain of gp42 that in other members of the C-type lectin family binds major histocompatibility complex class I or other diverse ligands. Although the hydrophobic pocket does not bind HLA class II, mutational analyses presented here indicate that this domain is essential for EBV-induced membrane fusion. In addition, mutational analysis of the region of gp42 contacting HLA class II in the gp42-HLA-DR1 cocrystal confirms that this region interacts with HLA class II and that this interaction is also important for EBV-induced membrane fusion.

  16. Herpes simplex virus glycoproteins gB and gH function in fusion between the virion envelope and the outer nuclear membrane.

    Science.gov (United States)

    Farnsworth, Aaron; Wisner, Todd W; Webb, Michael; Roller, Richard; Cohen, Gary; Eisenberg, Roselyn; Johnson, David C

    2007-06-12

    Herpesviruses must traverse the nuclear envelope to gain access to the cytoplasm and, ultimately, to exit cells. It is believed that herpesvirus nucleocapsids enter the perinuclear space by budding through the inner nuclear membrane (NM). To reach the cytoplasm these enveloped particles must fuse with the outer NM and the unenveloped capsids then acquire a second envelope in the trans-Golgi network. Little is known about the process by which herpesviruses virions fuse with the outer NM. Here we show that a herpes simplex virus (HSV) mutant lacking both the two putative fusion glycoproteins gB and gH failed to cross the nuclear envelope. Enveloped virions accumulated in the perinuclear space or in membrane vesicles that bulged into the nucleoplasm (herniations). By contrast, mutants lacking just gB or gH showed only minor or no defects in nuclear egress. We concluded that either HSV gB or gH can promote fusion between the virion envelope and the outer NM. It is noteworthy that fusion associated with HSV entry requires the cooperative action of both gB and gH, suggesting that the two types of fusion (egress versus entry) are dissimilar processes.

  17. A Model for Membrane Fusion

    Science.gov (United States)

    Ngatchou, Annita

    2010-01-01

    Pheochromocytoma is a tumor of the adrenal gland which originates from chromaffin cells and is characterized by the secretion of excessive amounts of neurotransmitter which lead to high blood pressure and palpitations. Pheochromocytoma contain membrane bound granules that store neurotransmitter. The release of these stored molecules into the extracellular space occurs by fusion of the granule membrane with the cell plasma membrane, a process called exocytosis. The molecular mechanism of this membrane fusion is not well understood. It is proposed that the so called SNARE proteins [1] are the pillar of vesicle fusion as their cleavage by clostridial toxin notably, Botulinum neurotoxin and Tetanus toxin abrogate the secretion of neurotransmitter [2]. Here, I describe how physical principles are applied to a biological cell to explore the role of the vesicle SNARE protein synaptobrevin-2 in easing granule fusion. The data presented here suggest a paradigm according to which the movement of the C-terminal of synaptobrevin-2 disrupts the lipid bilayer to form a fusion pore through which molecules can exit.

  18. Mutation of the dengue virus type 2 envelope protein heparan sulfate binding sites or the domain III lateral ridge blocks replication in Vero cells prior to membrane fusion.

    Science.gov (United States)

    Roehrig, John T; Butrapet, Siritorn; Liss, Nathan M; Bennett, Susan L; Luy, Betty E; Childers, Thomas; Boroughs, Karen L; Stovall, Janae L; Calvert, Amanda E; Blair, Carol D; Huang, Claire Y-H

    2013-07-05

    Using an infectious cDNA clone we engineered seven mutations in the putative heparan sulfate- and receptor-binding motifs of the envelope protein of dengue virus serotype 2, strain 16681. Four mutant viruses, KK122/123EE, E202K, G304K, and KKK305/307/310EEE, were recovered following transfection of C6/36 cells. A fifth mutant, KK291/295EE, was recovered from C6/36 cells with a compensatory E295V mutation. All mutants grew in and mediated fusion of virus-infected C6/36 cells, but three of the mutants, KK122/123EE, E202K, G304K, did not grow in Vero cells without further modification. Two Vero cell lethal mutants, KK291/295EV and KKK307/307/310EEE, failed to replicate in DC-SIGN-transformed Raji cells and did not react with monoclonal antibodies known to block DENV attachment to Vero cells. Additionally, both mutants were unable to initiate negative-strand vRNA synthesis in Vero cells by 72h post-infection, suggesting that the replication block occurred prior to virus-mediated membrane fusion. Published by Elsevier Inc.

  19. Membrane Trafficking and Vesicle Fusion

    Indian Academy of Sciences (India)

    Home; Journals; Resonance – Journal of Science Education; Volume 19; Issue 5. Membrane Trafficking and Vesicle Fusion: Post-Palade Era Researchers Win the Nobel Prize. Riddhi Atul Jani Subba Rao Gangi Setty. General Article Volume 19 Issue 5 May 2014 pp 421-445 ...

  20. Fusion of biological membranes

    Indian Academy of Sciences (India)

    This may be one of the reasons that lipid composition of membranes is tightly regulated. The free energy of stalk-like structures for a given architecture, f = 0.35, for different tensions is shown in figure 4b. Tensions there, are in units of that of the bare hydrophilic, hydrophobic interface, γint. One sees that the low free energy.

  1. A fusion-inhibiting peptide against Rift Valley fever virus inhibits multiple, diverse viruses.

    Directory of Open Access Journals (Sweden)

    Jeffrey W Koehler

    Full Text Available For enveloped viruses, fusion of the viral envelope with a cellular membrane is critical for a productive infection to occur. This fusion process is mediated by at least three classes of fusion proteins (Class I, II, and III based on the protein sequence and structure. For Rift Valley fever virus (RVFV, the glycoprotein Gc (Class II fusion protein mediates this fusion event following entry into the endocytic pathway, allowing the viral genome access to the cell cytoplasm. Here, we show that peptides analogous to the RVFV Gc stem region inhibited RVFV infectivity in cell culture by inhibiting the fusion process. Further, we show that infectivity can be inhibited for diverse, unrelated RNA viruses that have Class I (Ebola virus, Class II (Andes virus, or Class III (vesicular stomatitis virus fusion proteins using this single peptide. Our findings are consistent with an inhibition mechanism similar to that proposed for stem peptide fusion inhibitors of dengue virus in which the RVFV inhibitory peptide first binds to both the virion and cell membranes, allowing it to traffic with the virus into the endocytic pathway. Upon acidification and rearrangement of Gc, the peptide is then able to specifically bind to Gc and prevent fusion of the viral and endocytic membranes, thus inhibiting viral infection. These results could provide novel insights into conserved features among the three classes of viral fusion proteins and offer direction for the future development of broadly active fusion inhibitors.

  2. Characterization of a structural intermediate of flavivirus membrane fusion.

    Directory of Open Access Journals (Sweden)

    Karin Stiasny

    2007-02-01

    Full Text Available Viral membrane fusion proceeds through a sequence of steps that are driven by triggered conformational changes of viral envelope glycoproteins, so-called fusion proteins. Although high-resolution structural snapshots of viral fusion proteins in their prefusion and postfusion conformations are available, it has been difficult to define intermediate structures of the fusion pathway because of their transient nature. Flaviviruses possess a class II viral fusion protein (E mediating fusion at acidic pH that is converted from a dimer to a trimer with a hairpin-like structure during the fusion process. Here we show for tick-borne encephalitis virus that exposure of virions to alkaline instead of acidic pH traps the particles in an intermediate conformation in which the E dimers dissociate and interact with target membranes via the fusion peptide without proceeding to the merger of the membranes. Further treatment to low pH, however, leads to fusion, suggesting that these monomers correspond to an as-yet-elusive intermediate required to convert the prefusion dimer into the postfusion trimer. Thus, the use of nonphysiological conditions allows a dissection of the flavivirus fusion process and the identification of two separate steps, in which membrane insertion of multiple copies of E monomers precedes the formation of hairpin-like trimers. This sequence of events provides important new insights for understanding the dynamic process of viral membrane fusion.

  3. Mutation of the dengue virus type 2 envelope protein heparan sulfate binding sites or the domain III lateral ridge blocks replication in Vero cells prior to membrane fusion

    Energy Technology Data Exchange (ETDEWEB)

    Roehrig, John T., E-mail: jtr1@cdc.gov [Division of Vector-Borne Diseases, Centers for Disease Control and Prevention, Fort Collins, CO 80521 (United States); Butrapet, Siritorn; Liss, Nathan M. [Division of Vector-Borne Diseases, Centers for Disease Control and Prevention, Fort Collins, CO 80521 (United States); Bennett, Susan L. [Arthropod-borne and Infectious Diseases Laboratory, Department of Microbiology, Immunology, and Pathology, Colorado State University, Fort Collins, CO 80523 (United States); Luy, Betty E.; Childers, Thomas; Boroughs, Karen L.; Stovall, Janae L.; Calvert, Amanda E. [Division of Vector-Borne Diseases, Centers for Disease Control and Prevention, Fort Collins, CO 80521 (United States); Blair, Carol D. [Arthropod-borne and Infectious Diseases Laboratory, Department of Microbiology, Immunology, and Pathology, Colorado State University, Fort Collins, CO 80523 (United States); Huang, Claire Y.-H. [Division of Vector-Borne Diseases, Centers for Disease Control and Prevention, Fort Collins, CO 80521 (United States)

    2013-07-05

    Using an infectious cDNA clone we engineered seven mutations in the putative heparan sulfate- and receptor-binding motifs of the envelope protein of dengue virus serotype 2, strain 16681. Four mutant viruses, KK122/123EE, E202K, G304K, and KKK305/307/310EEE, were recovered following transfection of C6/36 cells. A fifth mutant, KK291/295EE, was recovered from C6/36 cells with a compensatory E295V mutation. All mutants grew in and mediated fusion of virus-infected C6/36 cells, but three of the mutants, KK122/123EE, E202K, G304K, did not grow in Vero cells without further modification. Two Vero cell lethal mutants, KK291/295EV and KKK307/307/310EEE, failed to replicate in DC-SIGN-transformed Raji cells and did not react with monoclonal antibodies known to block DENV attachment to Vero cells. Additionally, both mutants were unable to initiate negative-strand vRNA synthesis in Vero cells by 72 h post-infection, suggesting that the replication block occurred prior to virus-mediated membrane fusion. - Highlights: • Heparan sulfate- and receptor-binding motifs of DENV2 envelope protein were mutated. • Four mutant viruses were isolated—all could fuse C6/36 cells. • Two of these mutants were lethal in Vero cells without further modification. • Lethal mutations were KK291/295EV and KKK305/307/310EEE. • Cell attachment was implicated as the replication block for both mutants.

  4. Mitochondrial fusion through membrane automata.

    Science.gov (United States)

    Giannakis, Konstantinos; Andronikos, Theodore

    2015-01-01

    Studies have shown that malfunctions in mitochondrial processes can be blamed for diseases. However, the mechanism behind these operations is yet not sufficiently clear. In this work we present a novel approach to describe a biomolecular model for mitochondrial fusion using notions from the membrane computing. We use a case study defined in BioAmbient calculus and we show how to translate it in terms of a P automata variant. We combine brane calculi with (mem)brane automata to produce a new scheme capable of describing simple, realistic models. We propose the further use of similar methods and the test of other biomolecular models with the same behaviour.

  5. Mechanisms of influenza viral membrane fusion

    NARCIS (Netherlands)

    Blijleven, Jelle S; Boonstra, Sander; Onck, Patrick R; van der Giessen, Erik; van Oijen, Antoine M

    2016-01-01

    Influenza viral particles are enveloped by a lipid bilayer. A major step in infection is fusion of the viral and host cellular membranes, a process with large kinetic barriers. Influenza membrane fusion is catalyzed by hemagglutinin (HA), a class I viral fusion protein activated by low pH. The exact

  6. Herpesvirus gB-induced fusion between the virion envelope and outer nuclear membrane during virus egress is regulated by the viral US3 kinase.

    Science.gov (United States)

    Wisner, Todd W; Wright, Catherine C; Kato, Akihisa; Kawaguchi, Yasushi; Mou, Fan; Baines, Joel D; Roller, Richard J; Johnson, David C

    2009-04-01

    Herpesvirus capsids collect along the inner surface of the nuclear envelope and bud into the perinuclear space. Enveloped virions then fuse with the outer nuclear membrane (NM). We previously showed that herpes simplex virus (HSV) glycoproteins gB and gH act in a redundant fashion to promote fusion between the virion envelope and the outer NM. HSV mutants lacking both gB and gH accumulate enveloped virions in herniations, vesicles that bulge into the nucleoplasm. Earlier studies had shown that HSV mutants lacking the viral serine/threonine kinase US3 also accumulate herniations. Here, we demonstrate that HSV gB is phosphorylated in a US3-dependent manner in HSV-infected cells, especially in a crude nuclear fraction. Moreover, US3 directly phosphorylated the gB cytoplasmic (CT) domain in in vitro assays. Deletion of gB in the context of a US3-null virus did not add substantially to defects in nuclear egress. The majority of the US3-dependent phosphorylation of gB involved the CT domain and amino acid T887, a residue present in a motif similar to that recognized by US3 in other proteins. HSV recombinants lacking gH and expressing either gB substitution mutation T887A or a gB truncated at residue 886 displayed substantial defects in nuclear egress. We concluded that phosphorylation of the gB CT domain is important for gB-mediated fusion with the outer NM. This suggested a model in which the US3 kinase is incorporated into the tegument layer (between the capsid and envelope) in HSV virions present in the perinuclear space. By this packaging, US3 might be brought close to the gB CT tail, leading to phosphorylation and triggering fusion between the virion envelope and the outer NM.

  7. Virus separation using membranes.

    Science.gov (United States)

    Grein, Tanja A; Michalsky, Ronald; Czermak, Peter

    2014-01-01

    Industrial manufacturing of cell culture-derived viruses or virus-like particles for gene therapy or vaccine production are complex multistep processes. In addition to the bioreactor, such processes require a multitude of downstream unit operations for product separation, concentration, or purification. Similarly, before a biopharmaceutical product can enter the market, removal or inactivation of potential viral contamination has to be demonstrated. Given the complexity of biological solutions and the high standards on composition and purity of biopharmaceuticals, downstream processing is the bottleneck in many biotechnological production trains. Membrane-based filtration can be an economically attractive and efficient technology for virus separation. Viral clearance, for instance, of up to seven orders of magnitude has been reported for state of the art polymeric membranes under best conditions.This chapter summarizes the fundamentals of virus ultrafiltration, diafiltration, or purification with adsorptive membranes. In lieu of an impractical universally applicable protocol for virus filtration, application of these principles is demonstrated with two examples. The chapter provides detailed methods for production, concentration, purification, and removal of a rod-shaped baculovirus (Autographa californica M nucleopolyhedrovirus, about 40 × 300 nm in size, a potential vector for gene therapy, and an industrially important protein expression system) or a spherical parvovirus (minute virus of mice, 22-26 nm in size, a model virus for virus clearance validation studies).

  8. Hendra virus fusion protein transmembrane domain contributes to pre-fusion protein stability.

    Science.gov (United States)

    Webb, Stacy; Nagy, Tamas; Moseley, Hunter; Fried, Michael; Dutch, Rebecca

    2017-04-07

    Enveloped viruses utilize fusion (F) proteins studding the surface of the virus to facilitate membrane fusion with a target cell membrane. Fusion of the viral envelope with a cellular membrane is required for release of viral genomic material, so the virus can ultimately reproduce and spread. To drive fusion, the F protein undergoes an irreversible conformational change, transitioning from a metastable pre-fusion conformation to a more thermodynamically stable post-fusion structure. Understanding the elements that control stability of the pre-fusion state and triggering to the post-fusion conformation is important for understanding F protein function. Mutations in F protein transmembrane (TM) domains implicated the TM domain in the fusion process, but the structural and molecular details in fusion remain unclear. Previously, analytical ultracentrifugation was utilized to demonstrate that isolated TM domains of Hendra virus F protein associate in a monomer-trimer equilibrium (Smith, E. C., Smith, S. E., Carter, J. R., Webb, S. R., Gibson, K. M., Hellman, L. M., Fried, M. G., and Dutch, R. E. (2013) J. Biol. Chem. 288, 35726-35735). To determine factors driving this association, 140 paramyxovirus F protein TM domain sequences were analyzed. A heptad repeat of β-branched residues was found, and analysis of the Hendra virus F TM domain revealed a heptad repeat leucine-isoleucine zipper motif (LIZ). Replacement of the LIZ with alanine resulted in dramatically reduced TM-TM association. Mutation of the LIZ in the whole protein resulted in decreased protein stability, including pre-fusion conformation stability. Together, our data suggest that the heptad repeat LIZ contributed to TM-TM association and is important for F protein function and pre-fusion stability. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  9. Potent peptidic fusion inhibitors of influenza virus

    Energy Technology Data Exchange (ETDEWEB)

    Kadam, Rameshwar U.; Juraszek, Jarek; Brandenburg, Boerries; Buyck, Christophe; Schepens, Wim B. G.; Kesteleyn, Bart; Stoops, Bart; Vreeken, Rob J.; Vermond, Jan; Goutier, Wouter; Tang, Chan; Vogels, Ronald; Friesen, Robert H. E.; Goudsmit, Jaap; van Dongen, Maria J. P.; Wilson, Ian A.

    2017-09-28

    Influenza therapeutics with new targets and mechanisms of action are urgently needed to combat potential pandemics, emerging viruses, and constantly mutating strains in circulation. We report here on the design and structural characterization of potent peptidic inhibitors of influenza hemagglutinin. The peptide design was based on complementarity-determining region loops of human broadly neutralizing antibodies against the hemagglutinin (FI6v3 and CR9114). The optimized peptides exhibit nanomolar affinity and neutralization against influenza A group 1 viruses, including the 2009 H1N1 pandemic and avian H5N1 strains. The peptide inhibitors bind to the highly conserved stem epitope and block the low pH–induced conformational rearrangements associated with membrane fusion. These peptidic compounds and their advantageous biological properties should accelerate the development of new small molecule– and peptide-based therapeutics against influenza virus.

  10. Inhibition of HIV-1 endocytosis allows lipid mixing at the plasma membrane, but not complete fusion

    Directory of Open Access Journals (Sweden)

    de la Vega Michelle

    2011-12-01

    Full Text Available Abstract Background We recently provided evidence that HIV-1 enters HeLa-derived TZM-bl and lymphoid CEMss cells by fusing with endosomes, whereas its fusion with the plasma membrane does not proceed beyond the lipid mixing step. The mechanism of restriction of HIV-1 fusion at the cell surface and/or the factors that aid the virus entry from endosomes remain unclear. Results We examined HIV-1 fusion with a panel of target cells lines and with primary CD4+ T cells. Kinetic measurements of fusion combined with time-resolved imaging of single viruses further reinforced the notion that HIV-1 enters the cells via endocytosis and fusion with endosomes. Furthermore, we attempted to deliberately redirect virus fusion to the plasma membrane, using two experimental strategies. First, the fusion reaction was synchronized by pre-incubating the viruses with cells at reduced temperature to allow CD4 and coreceptors engagement, but not the virus uptake or fusion. Subsequent shift to a physiological temperature triggered accelerated virus uptake followed by entry from endosomes, but did not permit fusion at the cell surface. Second, blocking HIV-1 endocytosis by a small-molecule dynamin inhibitor, dynasore, resulted in transfer of viral lipids to the plasma membrane without any detectable release of the viral content into the cytosol. We also found that a higher concentration of dynasore is required to block the HIV-endosome fusion compared to virus internalization. Conclusions Our results further support the notion that HIV-1 enters disparate cell types through fusion with endosomes. The block of HIV-1 fusion with the plasma membrane at a post-lipid mixing stage shows that this membrane is not conducive to fusion pore formation and/or enlargement. The ability of dynasore to interfere with the virus-endosome fusion suggests that dynamin could be involved in two distinct steps of HIV-1 entry - endocytosis and fusion within intracellular compartments.

  11. Induction of cell-cell fusion by ectromelia virus is not inhibited by its fusion inhibitory complex

    Directory of Open Access Journals (Sweden)

    Fuchs Pinhas

    2009-09-01

    Full Text Available Abstract Background Ectromelia virus, a member of the Orthopox genus, is the causative agent of the highly infectious mousepox disease. Previous studies have shown that different poxviruses induce cell-cell fusion which is manifested by the formation of multinucleated-giant cells (polykaryocytes. This phenomenon has been widely studied with vaccinia virus in conditions which require artificial acidification of the medium. Results We show that Ectromelia virus induces cell-cell fusion under neutral pH conditions and requires the presence of a sufficient amount of viral particles on the plasma membrane of infected cells. This could be achieved by infection with a replicating virus and its propagation in infected cells (fusion "from within" or by infection with a high amount of virus particles per cell (fusion "from without". Inhibition of virus maturation or inhibition of virus transport on microtubules towards the plasma membrane resulted in a complete inhibition of syncytia formation. We show that in contrast to vaccinia virus, Ectromelia virus induces cell-cell fusion irrespectively of its hemagglutination properties and cell-surface expression of the orthologs of the fusion inhibitory complex, A56 and K2. Additionally, cell-cell fusion was also detected in mice lungs following lethal respiratory infection. Conclusion Ectromelia virus induces spontaneous cell-cell fusion in-vitro and in-vivo although expressing an A56/K2 fusion inhibitory complex. This syncytia formation property cannot be attributed to the 37 amino acid deletion in ECTV A56.

  12. A soluble form of Epstein-Barr virus gH/gL inhibits EBV-induced membrane fusion and does not function in fusion

    Energy Technology Data Exchange (ETDEWEB)

    Rowe, Cynthia L. [Department of Microbiology and Immunology, Feinberg School of Medicine, Northwestern University, Chicago, IL 60611 (United States); Connolly, Sarah A. [Department of Health Sciences, DePaul University, Chicago, IL 60614 (United States); Chen, Jia [Department of Microbiology and Immunology, Feinberg School of Medicine, Northwestern University, Chicago, IL 60611 (United States); Jardetzky, Theodore S. [Department of Structural Biology, Stanford University School of Medicine, 371 Serra Mall, Stanford, CA 94305 (United States); Longnecker, Richard, E-mail: r-longnecker@northwestern.edu [Department of Microbiology and Immunology, Feinberg School of Medicine, Northwestern University, Chicago, IL 60611 (United States)

    2013-02-05

    We investigated whether soluble EBV gH/gL (sgH/gL) functions in fusion and made a series of truncations of gH/gL domains based on the gH/gL crystal structure. We found sgH/gL failed to mediate cell-cell fusion both when co-expressed with the other entry glycoproteins and when added exogenously to fusion assays. Interestingly, sgH/gL inhibited cell-cell fusion in a dose dependent manner when co-expressed. sgH/gL from HSV was unable to inhibit EBV fusion, suggesting the inhibition was specific to EBV gH/gL. sgH/gL stably binds gp42, but not gB nor gH/gL. The domain mutants, DI/gL, DI-II/gL and DI-II-III/gL were unable to bind gp42. Instead, DI-II/gL, DI-II-III/gL and sgH/gL but not DI/gL decreased the expression of gp42, resulting in decreased overall fusion. Overall, our results suggest that domain IV may be required for proper folding and the transmembrane domain and cytoplasmic tail of EBV gH/gL are required for the most efficient fusion.

  13. Membrane interaction and structure of the transmembrane domain of influenza hemagglutinin and its fusion peptide complex

    Directory of Open Access Journals (Sweden)

    Lin Chi-Hui

    2008-01-01

    Full Text Available Abstract Background To study the organization and interaction with the fusion domain (or fusion peptide, FP of the transmembrane domain (TMD of influenza virus envelope glycoprotein for its role in membrane fusion which is also essential in the cellular trafficking of biomolecules and sperm-egg fusion. Results The fluorescence and gel electrophoresis experiments revealed a tight self-assembly of TMD in the model membrane. A weak but non-random interaction between TMD and FP in the membrane was found. In the complex, the central TMD oligomer was packed by FP in an antiparallel fashion. FP insertion into the membrane was altered by binding to TMD. An infrared study exhibited an enhanced membrane perturbation by the complex formation. A model was built to illustrate the role of TMD in the late stages of influenza virus-mediated membrane fusion reaction. Conclusion The TMD oligomer anchors the fusion protein in the membrane with minimal destabilization to the membrane. Upon associating with FP, the complex exerts a synergistic effect on the membrane perturbation. This effect is likely to contribute to the complete membrane fusion during the late phase of fusion protein-induced fusion cascade. The results presented in the work characterize the nature of the interaction of TMD with the membrane and TMD in a complex with FP in the steps leading to pore initiation and dilation during virus-induced fusion. Our data and proposed fusion model highlight the key role of TMD-FP interaction and have implications on the fusion reaction mediated by other type I viral fusion proteins. Understanding the molecular mechanism of membrane fusion may assist in the design of anti-viral drugs.

  14. Flavivirus cell entry and membrane fusion

    NARCIS (Netherlands)

    Smit, Jolanda M.; Moesker, Bastiaan; Rodenhuis-Zybert, Izabela; Wilschut, Jan

    Flaviviruses, such as dengue virus and West Nile virus, are enveloped viruses that infect cells through receptor-mediated endocytosis and fusion from within acidic endosomes. The cell entry process of flaviviruses is mediated by the viral E glycoprotein. This short review will address recent

  15. Flavivirus cell entry and membrane fusion

    NARCIS (Netherlands)

    Smit, Jolanda M.; Moesker, Bastiaan; Rodenhuis-Zybert, Izabela; Wilschut, Jan

    2011-01-01

    Flaviviruses, such as dengue virus and West Nile virus, are enveloped viruses that infect cells through receptor-mediated endocytosis and fusion from within acidic endosomes. The cell entry process of flaviviruses is mediated by the viral E glycoprotein. This short review will address recent

  16. C-terminal tyrosine residues modulate the fusion activity of the Hendra virus fusion protein.

    Science.gov (United States)

    Popa, Andreea; Pager, Cara Teresia; Dutch, Rebecca Ellis

    2011-02-15

    The paramyxovirus family includes important human pathogens such as measles, mumps, respiratory syncytial virus, and the recently emerged, highly pathogenic Hendra and Nipah viruses. The viral fusion (F) protein plays critical roles in infection, promoting both the virus-cell membrane fusion events needed for viral entry as well as cell-cell fusion events leading to syncytia formation. We describe the surprising finding that addition of the short epitope HA tag to the cytoplasmic tail (CT) of the Hendra virus F protein leads to a significant increase in the extent of cell-cell membrane fusion. This increase was not due to alterations in surface expression, cleavage state, or association with lipid microdomains. Addition of a Myc tag of similar length did not alter Hendra F protein fusion activity, indicating that the observed stimulation was not solely a result of lengthening the CT. Three tyrosine residues within the HA tag were critical for the increase in the extent of fusion, suggesting C-terminal tyrosines may modulate Hendra fusion activity. The effects of addition of the HA tag varied with other fusion proteins, as parainfluenza virus 5 F-HA showed a decreased level of surface expression and no stimulation of fusion. These results indicate that additions to the C-terminal end of the F protein CT can modulate protein function in a sequence specific manner, reinforcing the need for careful analysis of epitope-tagged glycoproteins. In addition, our results implicate C-terminal tyrosine residues in the modulation of the membrane fusion reaction promoted by these viral glycoproteins.

  17. Full inactivation of human influenza virus by high hydrostatic pressure preserves virus structure and membrane fusion while conferring protection to mice against infection.

    Directory of Open Access Journals (Sweden)

    Carlos H Dumard

    Full Text Available Whole inactivated vaccines (WIVs possess greater immunogenicity than split or subunit vaccines, and recent studies have demonstrated that WIVs with preserved fusogenic activity are more protective than non-fusogenic WIVs. In this work, we describe the inactivation of human influenza virus X-31 by high hydrostatic pressure (HHP and analyze the effects on the structure by spectroscopic measurements, light scattering, and electron microscopy. We also investigated the effects of HHP on the glycoprotein activity and fusogenic activity of the viral particles. The electron microscopy data showed pore formation on the viral envelope, but the general morphology was preserved, and small variations were seen in the particle structure. The activity of hemagglutinin (HA during the process of binding and fusion was affected in a time-dependent manner, but neuraminidase (NA activity was not affected. Infectious activity ceased after 3 hours of pressurization, and mice were protected from infection after being vaccinated. Our results revealed full viral inactivation with overall preservation of viral structure and maintenance of fusogenic activity, thereby conferring protection against infection. A strong response consisting of serum immunoglobulin IgG1, IgG2a, and serum and mucosal IgA was also detected after vaccination. Thus, our data strongly suggest that applying hydrostatic pressure may be an effective method for developing new vaccines against influenza A as well as other viruses.

  18. Functional fluorescent protein insertions in herpes simplex virus gB report on gB conformation before and after execution of membrane fusion.

    Directory of Open Access Journals (Sweden)

    John R Gallagher

    2014-09-01

    Full Text Available Entry of herpes simplex virus (HSV into a target cell requires complex interactions and conformational changes by viral glycoproteins gD, gH/gL, and gB. During viral entry, gB transitions from a prefusion to a postfusion conformation, driving fusion of the viral envelope with the host cell membrane. While the structure of postfusion gB is known, the prefusion conformation of gB remains elusive. As the prefusion conformation of gB is a critical target for neutralizing antibodies, we set out to describe its structure by making genetic insertions of fluorescent proteins (FP throughout the gB ectodomain. We created gB constructs with FP insertions in each of the three globular domains of gB. Among 21 FP insertion constructs, we found 8 that allowed gB to remain membrane fusion competent. Due to the size of an FP, regions in gB that tolerate FP insertion must be solvent exposed. Two FP insertion mutants were cell-surface expressed but non-functional, while FP insertions located in the crown were not surface expressed. This is the first report of placing a fluorescent protein insertion within a structural domain of a functional viral fusion protein, and our results are consistent with a model of prefusion HSV gB constructed from the prefusion VSV G crystal structure. Additionally, we found that functional FP insertions from two different structural domains could be combined to create a functional form of gB labeled with both CFP and YFP. FRET was measured with this construct, and we found that when co-expressed with gH/gL, the FRET signal from gB was significantly different from the construct containing CFP alone, as well as gB found in syncytia, indicating that this construct and others of similar design are likely to be powerful tools to monitor the conformation of gB in any model system accessible to light microscopy.

  19. Effect of viral membrane fusion activity on antibody induction by influenza H5N1 whole inactivated virus vaccine

    NARCIS (Netherlands)

    Geeraedts, Felix; ter Veer, Wouter; Wilschut, Jan; Huckriede, Anke; de Haan, Aalzen

    2012-01-01

    Whole inactivated virus (WIV) influenza vaccines are more immunogenic in unprimed individuals than split-virus or subunit vaccines. In mice, this superior immunogenicity has been linked to the recognition of the viral ssRNA by endosomal TLR7 receptors in immune cells, leading to IFN alpha production

  20. Membrane Fusion and Infection of the Influenza Hemagglutinin.

    Science.gov (United States)

    Smrt, Sean T; Lorieau, Justin L

    2017-01-01

    The influenza virus is a major health concern associated with an estimated 5000 to 30,000 deaths every year (Reed et al. 2015) and a significant economic impact with the development of treatments, vaccinations and research (Molinari et al. 2007). The entirety of the influenza genome is comprised of only eleven coding genes. An enormous degree of variation in non-conserved regions leads to significant challenges in the development of inclusive inhibitors for treatment. The fusion peptide domain of the influenza A hemagglutinin (HA) is a promising candidate for treatment since it is one of the most highly conserved sequences in the influenza genome (Heiny et al. 2007), and it is vital to the viral life cycle. Hemagglutinin is a class I viral fusion protein that catalyzes the membrane fusion process during cellular entry and infection. Impediment of the hemagglutinin's function, either through incomplete post-translational processing (Klenk et al. 1975; Lazarowitz and Choppin 1975) or through mutations (Cross et al. 2001), leads to non-infective virus particles. This review will investigate current research on the role of hemagglutinin in the virus life cycle, its structural biology and mechanism as well as the central role of the hemagglutinin fusion peptide (HAfp) to influenza membrane fusion and infection.

  1. Hemagglutinin-esterase-fusion (HEF protein of influenza C virus

    Directory of Open Access Journals (Sweden)

    Mingyang Wang

    2015-07-01

    Full Text Available ABSTRACT Influenza C virus, a member of the Orthomyxoviridae family, causes flu-like disease but typically only with mild symptoms. Humans are the main reservoir of the virus, but it also infects pigs and dogs. Very recently, influenza C-like viruses were isolated from pigs and cattle that differ from classical influenza C virus and might constitute a new influenza virus genus. Influenza C virus is unique since it contains only one spike protein, the hemagglutinin-esterase-fusion glycoprotein HEF that possesses receptor binding, receptor destroying and membrane fusion activities, thus combining the functions of Hemagglutinin (HA and Neuraminidase (NA of influenza A and B viruses. Here we briefly review the epidemiology and pathology of the virus and the morphology of virus particles and their genome. The main focus is on the structure of the HEF protein as well as on its co- and post-translational modification, such as N-glycosylation, disulfide bond formation, S-acylation and proteolytic cleavage into HEF1 and HEF2 subunits. Finally, we describe the functions of HEF: receptor binding, esterase activity and membrane fusion.

  2. An anti-human immunodeficiency virus multiple antigen peptide encompassing the cleavage region of the env precursor interferes with membrane fusion at a post-CD4 binding step.

    Science.gov (United States)

    Barbouche, R; Decroly, E; Kieny, M P; Fenouillet, E

    2000-07-20

    CLIV is a multiple antigen peptide ([PTKAKRRVVQREKR](4)-K(2)-K-betaA) that encompasses the cleavage region of the human immunodeficiency virus type 1 (HIV-1) envelope precursor. It displays an antiviral activity against HIV-1 and HIV-2 and inhibits HIV-1 Env-mediated cell-to-cell fusion. This effect has previously been attributed to interference with Env processing, resulting in the expression of a nonfusogenic envelope [Virology (1998) 247, 137]. However, we show here that CLIV does not alter the status of Env cleavage at steady state. Using various aggregation/syncytium assays that allow us to discriminate between gp120/CD4 binding and binding followed by gp41-mediated fusion, we demonstrate that CLIV inhibits a step of the cell-to-cell fusion process after CD4 binding. We demonstrate also that CLIV binds at 37 degrees C to a single class of protein present at the CD4(+) cell surface (Scatchard analysis: K(d) = 8 nM; B(max) = 10(4) sites/cell) and that the fusion inhibition activity seems to correlate with binding to this proteic component. In contrast, CLIV interacts with neither membrane-inserted nor CD4-associated Env. We therefore propose that CLIV interferes after Env/CD4 binding with a step of the membrane fusion process that may involve the C-terminal domain of gp120. Copyright 2000 Academic Press.

  3. Measles Virus Fusion Protein: Structure, Function and Inhibition

    Directory of Open Access Journals (Sweden)

    Philippe Plattet

    2016-04-01

    Full Text Available Measles virus (MeV, a highly contagious member of the Paramyxoviridae family, causes measles in humans. The Paramyxoviridae family of negative single-stranded enveloped viruses includes several important human and animal pathogens, with MeV causing approximately 120,000 deaths annually. MeV and canine distemper virus (CDV-mediated diseases can be prevented by vaccination. However, sub-optimal vaccine delivery continues to foster MeV outbreaks. Post-exposure prophylaxis with antivirals has been proposed as a novel strategy to complement vaccination programs by filling herd immunity gaps. Recent research has shown that membrane fusion induced by the morbillivirus glycoproteins is the first critical step for viral entry and infection, and determines cell pathology and disease outcome. Our molecular understanding of morbillivirus-associated membrane fusion has greatly progressed towards the feasibility to control this process by treating the fusion glycoprotein with inhibitory molecules. Current approaches to develop anti-membrane fusion drugs and our knowledge on drug resistance mechanisms strongly suggest that combined therapies will be a prerequisite. Thus, discovery of additional anti-fusion and/or anti-attachment protein small-molecule compounds may eventually translate into realistic therapeutic options.

  4. Measles Virus Fusion Protein: Structure, Function and Inhibition

    Science.gov (United States)

    Plattet, Philippe; Alves, Lisa; Herren, Michael; Aguilar, Hector C.

    2016-01-01

    Measles virus (MeV), a highly contagious member of the Paramyxoviridae family, causes measles in humans. The Paramyxoviridae family of negative single-stranded enveloped viruses includes several important human and animal pathogens, with MeV causing approximately 120,000 deaths annually. MeV and canine distemper virus (CDV)-mediated diseases can be prevented by vaccination. However, sub-optimal vaccine delivery continues to foster MeV outbreaks. Post-exposure prophylaxis with antivirals has been proposed as a novel strategy to complement vaccination programs by filling herd immunity gaps. Recent research has shown that membrane fusion induced by the morbillivirus glycoproteins is the first critical step for viral entry and infection, and determines cell pathology and disease outcome. Our molecular understanding of morbillivirus-associated membrane fusion has greatly progressed towards the feasibility to control this process by treating the fusion glycoprotein with inhibitory molecules. Current approaches to develop anti-membrane fusion drugs and our knowledge on drug resistance mechanisms strongly suggest that combined therapies will be a prerequisite. Thus, discovery of additional anti-fusion and/or anti-attachment protein small-molecule compounds may eventually translate into realistic therapeutic options. PMID:27110811

  5. The dynamic envelope of a fusion class II virus. Prefusion stages of semliki forest virus revealed by electron cryomicroscopy.

    Science.gov (United States)

    Wu, Shang-Rung; Haag, Lars; Hammar, Lena; Wu, Bomu; Garoff, Henrik; Xing, Li; Murata, Kazuyoshi; Cheng, R Holland

    2007-03-02

    Semliki Forest virus is among the prototypes for Class II virus fusion and targets the endosomal membrane. Fusion protein E1 and its envelope companion E2 are both anchored in the viral membrane and form an external shell with protruding spikes. In acid environments, mimicking the early endosomal milieu, surface epitopes in the virus rearrange along with exposure of the fusion loop. To visualize this transformation into a fusogenic stage, we determined the structure of the virus at gradually lower pH values. The results show that while the fusion loop is available for external interaction and the shell and stalk domains of the spike begin to deteriorate, the E1 and E2 remain in close contact in the spike head. This unexpected observation points to E1 and E2 cooperation beyond the fusion loop exposure stage and implies a more prominent role for E2 in guiding membrane close encounter than has been earlier anticipated.

  6. Side chain packing below the fusion peptide strongly modulates triggering of the Hendra virus F protein.

    Science.gov (United States)

    Smith, Everett Clinton; Dutch, Rebecca Ellis

    2010-10-01

    Triggering of the Hendra virus fusion (F) protein is required to initiate the conformational changes which drive membrane fusion, but the factors which control triggering remain poorly understood. Mutation of a histidine predicted to lie near the fusion peptide to alanine greatly reduced fusion despite wild-type cell surface expression levels, while asparagine substitution resulted in a moderate restoration in fusion levels. Slowed kinetics of six-helix bundle formation, as judged by sensitivity to heptad repeat B-derived peptides, was observed for all H372 mutants. These data suggest that side chain packing beneath the fusion peptide is an important regulator of Hendra virus F triggering.

  7. Cholesterol mediates membrane curvature during fusion events.

    Science.gov (United States)

    Ivankin, Andrey; Kuzmenko, Ivan; Gidalevitz, David

    2012-06-08

    Biomembranes undergo extensive shape changes as they perform vital cellular functions. The mechanisms by which lipids and proteins control membrane curvature remain unclear. We use x-ray reflectivity, grazing incidence x-ray diffraction, and epifluorescence microscopy to study binding of HIV-1 glycoprotein gp41's membrane-bending domain to DPPC/cholesterol monolayers of various compositions at the air-liquid interface. The results offer a new insight into how membrane curvature could be regulated by cholesterol during fusion of the viral lipid envelope and the host cell membranes.

  8. Membrane Trafficking and Vesicle Fusion

    Indian Academy of Sciences (India)

    IAS Admin

    hemophagocytic syndrome) and metabolic (diabe- tes) disorders [2, 23, 33]. Mutations in the genes of the basic secretory protein machinery lead to a number of membrane trafficking diseases such as Charcot–Marie–Tooth disease, Cohen.

  9. Role of Conserved Histidine Residues in the Low-pH Dependence of the Semliki Forest Virus Fusion Protein▿

    OpenAIRE

    Qin, Zhao-ling; Zheng, Yan; Kielian, Margaret

    2009-01-01

    A wide variety of enveloped viruses infects cells by taking advantage of the low pH in the endocytic pathway to trigger virus-membrane fusion. For alphaviruses such as Semliki Forest virus (SFV), acidic pH initiates a series of conformational changes in the heterodimeric virus envelope proteins E1 and E2. Low pH dissociates the E2/E1 dimer, releasing the membrane fusion protein E1. E1 inserts into the target membrane and refolds to a trimeric hairpin conformation, thus driving the fusion reac...

  10. Imaging single retrovirus entry through alternative receptor isoforms and intermediates of virus-endosome fusion.

    Science.gov (United States)

    Jha, Naveen K; Latinovic, Olga; Martin, Erik; Novitskiy, Gennadiy; Marin, Mariana; Miyauchi, Kosuke; Naughton, John; Young, John A T; Melikyan, Gregory B

    2011-01-20

    A large group of viruses rely on low pH to activate their fusion proteins that merge the viral envelope with an endosomal membrane, releasing the viral nucleocapsid. A critical barrier to understanding these events has been the lack of approaches to study virus-cell membrane fusion within acidic endosomes, the natural sites of virus nucleocapsid capsid entry into the cytosol. Here we have investigated these events using the highly tractable subgroup A avian sarcoma and leukosis virus envelope glycoprotein (EnvA)-TVA receptor system. Through labeling EnvA pseudotyped viruses with a pH-sensitive fluorescent marker, we imaged their entry into mildly acidic compartments. We found that cells expressing the transmembrane receptor (TVA950) internalized the virus much faster than those expressing the GPI-anchored receptor isoform (TVA800). Surprisingly, TVA800 did not accelerate virus uptake compared to cells lacking the receptor. Subsequent steps of virus entry were visualized by incorporating a small viral content marker that was released into the cytosol as a result of fusion. EnvA-dependent fusion with TVA800-expressing cells occurred shortly after endocytosis and delivery into acidic endosomes, whereas fusion of viruses internalized through TVA950 was delayed. In the latter case, a relatively stable hemifusion-like intermediate preceded the fusion pore opening. The apparent size and stability of nascent fusion pores depended on the TVA isoforms and their expression levels, with TVA950 supporting more robust pores and a higher efficiency of infection compared to TVA800. These results demonstrate that surface receptor density and the intracellular trafficking pathway used are important determinants of efficient EnvA-mediated membrane fusion, and suggest that early fusion intermediates play a critical role in establishing low pH-dependent virus entry from within acidic endosomes.

  11. Imaging single retrovirus entry through alternative receptor isoforms and intermediates of virus-endosome fusion.

    Directory of Open Access Journals (Sweden)

    Naveen K Jha

    2011-01-01

    Full Text Available A large group of viruses rely on low pH to activate their fusion proteins that merge the viral envelope with an endosomal membrane, releasing the viral nucleocapsid. A critical barrier to understanding these events has been the lack of approaches to study virus-cell membrane fusion within acidic endosomes, the natural sites of virus nucleocapsid capsid entry into the cytosol. Here we have investigated these events using the highly tractable subgroup A avian sarcoma and leukosis virus envelope glycoprotein (EnvA-TVA receptor system. Through labeling EnvA pseudotyped viruses with a pH-sensitive fluorescent marker, we imaged their entry into mildly acidic compartments. We found that cells expressing the transmembrane receptor (TVA950 internalized the virus much faster than those expressing the GPI-anchored receptor isoform (TVA800. Surprisingly, TVA800 did not accelerate virus uptake compared to cells lacking the receptor. Subsequent steps of virus entry were visualized by incorporating a small viral content marker that was released into the cytosol as a result of fusion. EnvA-dependent fusion with TVA800-expressing cells occurred shortly after endocytosis and delivery into acidic endosomes, whereas fusion of viruses internalized through TVA950 was delayed. In the latter case, a relatively stable hemifusion-like intermediate preceded the fusion pore opening. The apparent size and stability of nascent fusion pores depended on the TVA isoforms and their expression levels, with TVA950 supporting more robust pores and a higher efficiency of infection compared to TVA800. These results demonstrate that surface receptor density and the intracellular trafficking pathway used are important determinants of efficient EnvA-mediated membrane fusion, and suggest that early fusion intermediates play a critical role in establishing low pH-dependent virus entry from within acidic endosomes.

  12. IM30 triggers membrane fusion in cyanobacteria and chloroplasts

    OpenAIRE

    Hennig, R.; Heidrich, J.; Saur, M.; Schmüser, L.; Roeters, S.J.; Hellmann, N.; Woutersen, S.; Bonn, M.; Weidner, T.; Markl, J.; Schneider, D.

    2015-01-01

    The thylakoid membrane of chloroplasts and cyanobacteria is a unique internal membrane system harbouring the complexes of the photosynthetic electron transfer chain. Despite their apparent importance, little is known about the biogenesis and maintenance of thylakoid membranes. Although membrane fusion events are essential for the formation of thylakoid membranes, proteins involved in membrane fusion have yet to be identified in photosynthetic cells or organelles. Here we show that IM30, a con...

  13. Physico-chemical requirements and kinetics of membrane fusion of flavivirus-like particles.

    Science.gov (United States)

    Espósito, Danillo L A; Nguyen, Jennifer B; DeWitt, David C; Rhoades, Elizabeth; Modis, Yorgo

    2015-07-01

    Flaviviruses deliver their RNA genome into the host-cell cytoplasm by fusing their lipid envelope with a cellular membrane. Expression of the flavivirus pre-membrane and envelope glycoprotein genes in the absence of other viral genes results in the spontaneous assembly and secretion of virus-like particles (VLPs) with membrane fusion activity. Here, we examined the physico-chemical requirements for membrane fusion of VLPs from West Nile and Japanese encephalitis viruses. In a bulk fusion assay, optimal hemifusion (or lipid mixing) efficiencies were observed at 37 °C. Fusion efficiency increased with decreasing pH; half-maximal hemifusion was attained at pH 5.6. The anionic lipids bis(monoacylglycero)phosphate and phosphatidylinositol-3-phosphate, when present in the target membrane, significantly enhanced fusion efficiency, consistent with the emerging model that flaviviruses fuse with intermediate-to-late endosomal compartments, where these lipids are most abundant. In a single-particle fusion assay, VLPs catalysed membrane hemifusion, tracked as lipid mixing with the cellular membrane, on a timescale of 7-20 s after acidification. Lipid mixing kinetics suggest that hemifusion is a kinetically complex, multistep process.

  14. Dynamic Viral Glycoprotein Machines: Approaches for Probing Transient States That Drive Membrane Fusion

    Directory of Open Access Journals (Sweden)

    Natalie K. Garcia

    2016-01-01

    Full Text Available The fusion glycoproteins that decorate the surface of enveloped viruses undergo dramatic conformational changes in the course of engaging with target cells through receptor interactions and during cell entry. These refolding events ultimately drive the fusion of viral and cellular membranes leading to delivery of the genetic cargo. While well-established methods for structure determination such as X-ray crystallography have provided detailed structures of fusion proteins in the pre- and post-fusion fusion states, to understand mechanistically how these fusion glycoproteins perform their structural calisthenics and drive membrane fusion requires new analytical approaches that enable dynamic intermediate states to be probed. Methods including structural mass spectrometry, small-angle X-ray scattering, and electron microscopy have begun to provide new insight into pathways of conformational change and fusion protein function. In combination, the approaches provide a significantly richer portrait of viral fusion glycoprotein structural variation and fusion activation as well as inhibition by neutralizing agents. Here recent studies that highlight the utility of these complementary approaches will be reviewed with a focus on the well-characterized influenza virus hemagglutinin fusion glycoprotein system.

  15. Pulling force and surface tension drive membrane fusion.

    Science.gov (United States)

    Liu, Xuejuan; Tian, Falin; Yue, Tongtao; Zhang, Xianren; Zhong, Chongli

    2017-11-21

    Despite catalyzed by fusion proteins of quite different molecular architectures, intracellular, viral, and cell-to-cell fusions are found to have the essential common features and the nearly same nature of transition states. The similarity inspires us to find a more general catalysis mechanism for membrane fusion that minimally depends on the specific structures of fusion proteins. In this work, we built a minimal model for membrane fusion, and by using dissipative particle dynamics simulations, we propose a mechanism that the pulling force generated by fusion proteins initiates the fusion process and the membrane tension regulates the subsequent fusion stages. The model shows different features compared to previous computer simulation studies: the pulling force catalyzes membrane fusion through lipid head overcrowding in the contacting region, leading to an increase in the head-head repulsion and/or the unfavorable head-tail contacts from opposing membranes, both of which destabilize the contacting leaflets and thus promote membrane fusion or vesicle rupture. Our simulations produce a variety of shapes and intermediates, closely resembling cases seen experimentally. Our work strongly supports the view that the tight pulling mechanism is a conserved feature of fusion protein-mediated fusion and that the membrane tension plays an essential role in fusion.

  16. Pulling force and surface tension drive membrane fusion

    Science.gov (United States)

    Liu, Xuejuan; Tian, Falin; Yue, Tongtao; Zhang, Xianren; Zhong, Chongli

    2017-11-01

    Despite catalyzed by fusion proteins of quite different molecular architectures, intracellular, viral, and cell-to-cell fusions are found to have the essential common features and the nearly same nature of transition states. The similarity inspires us to find a more general catalysis mechanism for membrane fusion that minimally depends on the specific structures of fusion proteins. In this work, we built a minimal model for membrane fusion, and by using dissipative particle dynamics simulations, we propose a mechanism that the pulling force generated by fusion proteins initiates the fusion process and the membrane tension regulates the subsequent fusion stages. The model shows different features compared to previous computer simulation studies: the pulling force catalyzes membrane fusion through lipid head overcrowding in the contacting region, leading to an increase in the head-head repulsion and/or the unfavorable head-tail contacts from opposing membranes, both of which destabilize the contacting leaflets and thus promote membrane fusion or vesicle rupture. Our simulations produce a variety of shapes and intermediates, closely resembling cases seen experimentally. Our work strongly supports the view that the tight pulling mechanism is a conserved feature of fusion protein-mediated fusion and that the membrane tension plays an essential role in fusion.

  17. Dissipative Particle Dynamics of tension-induced membrane fusion

    DEFF Research Database (Denmark)

    Shillcock, Julian C.

    2009-01-01

    Recent studies of tension-induced membrane fusion using dissipative particle dynamics (DPD) simulations are briefly reviewed. The stochastic nature of the fusion process makes it necessary to simulate a large number of fusion attempts in order to obtain reliable fusion statistics and to extract...

  18. The Dynamic Envelope of a Fusion Class II Virus

    Science.gov (United States)

    Wu, Shang-Rung; Haag, Lars; Sjöberg, Mathilda; Garoff, Henrik; Hammar, Lena

    2008-01-01

    In alphaviruses, here represented by Semliki Forest virus, infection requires an acid-responsive spike configuration to facilitate membrane fusion. The creation of this relies on the chaperon function of glycoprotein E2 precursor (p62) and its maturation cleavage into the small external E3 and the membrane-anchored E2 glycoproteins. To reveal how the E3 domain of p62 exerts its control of spike functions, we determine the structure of a p62 cleavage-impaired mutant virus particle (SQL) by electron cryomicroscopy. A comparison with the earlier solved wild type virus structure reveals that the E3 domain of p62SQL forms a bulky side protrusion in the spike head region. This establishes a gripper over part of domain II of the fusion protein, with a cotter-like connection downward to a hydrophobic cluster in its central β-sheet. This finding reevaluates the role of the precursor from being only a provider of a shield over the fusion loop to a structural playmate in formation of the fusogenic architecture. PMID:18596032

  19. IM30 triggers membrane fusion in cyanobacteria and chloroplasts.

    Science.gov (United States)

    Hennig, Raoul; Heidrich, Jennifer; Saur, Michael; Schmüser, Lars; Roeters, Steven J; Hellmann, Nadja; Woutersen, Sander; Bonn, Mischa; Weidner, Tobias; Markl, Jürgen; Schneider, Dirk

    2015-05-08

    The thylakoid membrane of chloroplasts and cyanobacteria is a unique internal membrane system harbouring the complexes of the photosynthetic electron transfer chain. Despite their apparent importance, little is known about the biogenesis and maintenance of thylakoid membranes. Although membrane fusion events are essential for the formation of thylakoid membranes, proteins involved in membrane fusion have yet to be identified in photosynthetic cells or organelles. Here we show that IM30, a conserved chloroplast and cyanobacterial protein of approximately 30 kDa binds as an oligomeric ring in a well-defined geometry specifically to membranes containing anionic lipids. Triggered by Mg(2+), membrane binding causes destabilization and eventually results in membrane fusion. We propose that IM30 establishes contacts between internal membrane sites and promotes fusion to enable regulated exchange of proteins and/or lipids in cyanobacteria and chloroplasts.

  20. A compensatory mutation provides resistance to disparate HIV fusion inhibitor peptides and enhances membrane fusion.

    Directory of Open Access Journals (Sweden)

    Matthew P Wood

    Full Text Available Fusion inhibitors are a class of antiretroviral drugs used to prevent entry of HIV into host cells. Many of the fusion inhibitors being developed, including the drug enfuvirtide, are peptides designed to competitively inhibit the viral fusion protein gp41. With the emergence of drug resistance, there is an increased need for effective and unique alternatives within this class of antivirals. One such alternative is a class of cyclic, cationic, antimicrobial peptides known as θ-defensins, which are produced by many non-human primates and exhibit broad-spectrum antiviral and antibacterial activity. Currently, the θ-defensin analog RC-101 is being developed as a microbicide due to its specific antiviral activity, lack of toxicity to cells and tissues, and safety in animals. Understanding potential RC-101 resistance, and how resistance to other fusion inhibitors affects RC-101 susceptibility, is critical for future development. In previous studies, we identified a mutant, R5-tropic virus that had evolved partial resistance to RC-101 during in vitro selection. Here, we report that a secondary mutation in gp41 was found to restore replicative fitness, membrane fusion, and the rate of viral entry, which were compromised by an initial mutation providing partial RC-101 resistance. Interestingly, we show that RC-101 is effective against two enfuvirtide-resistant mutants, demonstrating the clinical importance of RC-101 as a unique fusion inhibitor. These findings both expand our understanding of HIV drug-resistance to diverse peptide fusion inhibitors and emphasize the significance of compensatory gp41 mutations.

  1. Fusion of Semliki Forest virus with cholesterol-containing liposomes at low pH

    DEFF Research Database (Denmark)

    Wilschut, J; Corver, J; Nieva, J L

    1995-01-01

    Semliki Forest virus (SFV) utilizes a membrane fusion strategy to introduce its genome into the host cell. After binding to cell-surface receptors, virus particles are internalized through receptor-mediated endocytosis and directed to the endosomal cell compartment. Subsequently, triggered by the...

  2. Spatial regulation of membrane fusion controlled by modification of phosphoinositides.

    Directory of Open Access Journals (Sweden)

    Fabrice Dumas

    Full Text Available Membrane fusion plays a central role in many cell processes from vesicular transport to nuclear envelope reconstitution at mitosis but the mechanisms that underlie fusion of natural membranes are not well understood. Studies with synthetic membranes and theoretical considerations indicate that accumulation of lipids characterised by negative curvature such as diacylglycerol (DAG facilitate fusion. However, the specific role of lipids in membrane fusion of natural membranes is not well established. Nuclear envelope (NE assembly was used as a model for membrane fusion. A natural membrane population highly enriched in the enzyme and substrate needed to produce DAG has been isolated and is required for fusions leading to nuclear envelope formation, although it contributes only a small amount of the membrane eventually incorporated into the NE. It was postulated to initiate and regulate membrane fusion. Here we use a multidisciplinary approach including subcellular membrane purification, fluorescence spectroscopy and Förster resonance energy transfer (FRET/two-photon fluorescence lifetime imaging microscopy (FLIM to demonstrate that initiation of vesicle fusion arises from two unique sites where these vesicles bind to chromatin. Fusion is subsequently propagated to the endoplasmic reticulum-derived membranes that make up the bulk of the NE to ultimately enclose the chromatin. We show how initiation of multiple vesicle fusions can be controlled by localised production of DAG and propagated bidirectionally. Phospholipase C (PLCgamma, GTP hydrolysis and (phosphatidylinsositol-(4,5-bisphosphate (PtdIns(4,5P(2 are required for the latter process. We discuss the general implications of membrane fusion regulation and spatial control utilising such a mechanism.

  3. Deduced sequences of the membrane fusion and attachment proteins of canine distemper viruses isolated from dogs and wild animals in Korea.

    Science.gov (United States)

    Bae, Chae-Wun; Lee, Joong-Bok; Park, Seung-Yong; Song, Chang-Seon; Lee, Nak-Hyung; Seo, Kun-Ho; Kang, Young-Sun; Park, Choi-Kyu; Choi, In-Soo

    2013-08-01

    Canine distemper virus (CDV) causes highly contagious respiratory, gastrointestinal, and neurological diseases in wild and domestic animal species. Despite a broad vaccination campaign, the disease is still a serious problem worldwide. In this study, six field CDV strains were isolated from three dogs, two raccoon dogs, and one badger in Korea. The full sequence of the genes encoding fusion (F) and hemagglutinin (H) proteins were compared with those of other CDVs including field and vaccine strains. The phylogenetic analysis for the F and H genes indicated that the two CDV strains isolated from dogs were most closely related to Chinese strains in the Asia-1 genotype. Another four strains were closely related to Japanese strains in the Asia-2 genotype. The six currently isolated strains shared 90.2-92.1% and 88.2-91.8% identities with eight commercial vaccine strains in their nucleotide and amino acid sequences of the F protein, respectively. They also showed 90.1-91.4% and 87.8-90.7% identities with the same vaccine strains in their nucleotide and deduced amino acid sequences of the H protein, respectively. Different N-linked glycosylation sites were identified in the F and H genes of the six isolates from the prototype vaccine strain Onderstepoort. Collectively, these results demonstrate that at least two different CDV genotypes currently exist in Korea. The considerable genetic differences between the vaccine strains and wild-type isolates would be a major factor of the incomplete protection of dogs from CDV infections.

  4. Fusion Pore Diameter Regulation by Cations Modulating Local Membrane Anisotropy

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    Doron Kabaso

    2012-01-01

    Full Text Available The fusion pore is an aqueous channel that is formed upon the fusion of the vesicle membrane with the plasma membrane. Once the pore is open, it may close again (transient fusion or widen completely (full fusion to permit vesicle cargo discharge. While repetitive transient fusion pore openings of the vesicle with the plasma membrane have been observed in the absence of stimulation, their frequency can be further increased using a cAMP-increasing agent that drives the opening of nonspecific cation channels. Our model hypothesis is that the openings and closings of the fusion pore are driven by changes in the local concentration of cations in the connected vesicle. The proposed mechanism of fusion pore dynamics is considered as follows: when the fusion pore is closed or is extremely narrow, the accumulation of cations in the vesicle (increased cation concentration likely leads to lipid demixing at the fusion pore. This process may affect local membrane anisotropy, which reduces the spontaneous curvature and thus leads to the opening of the fusion pore. Based on the theory of membrane elasticity, we used a continuum model to explain the rhythmic opening and closing of the fusion pore.

  5. Two single mutations in the fusion protein of Newcastle disease virus confer hemagglutinin-neuraminidase independent fusion promotion and attenuate the pathogenicity in chickens

    Science.gov (United States)

    The fusion (F) protein of Newcastle disease virus (NDV) plays an important role in viral infection and pathogenicity through mediating membrane fusion between the virion and host cells in the presence of the hemagglutinin-neuraminidase (HN). Previously, we obtained a velogenic NDV genotype VII muta...

  6. Solid-state nuclear magnetic resonance (NMR) spectroscopy of human immunodeficiency virus gp41 protein that includes the fusion peptide: NMR detection of recombinant Fgp41 in inclusion bodies in whole bacterial cells and structural characterization of purified and membrane-associated Fgp41.

    Science.gov (United States)

    Vogel, Erica P; Curtis-Fisk, Jaime; Young, Kaitlin M; Weliky, David P

    2011-11-22

    Human immunodeficiency virus (HIV) infection of a host cell begins with fusion of the HIV and host cell membranes and is mediated by the gp41 protein, a single-pass integral membrane protein of HIV. The 175 N-terminal residues make up the ectodomain that lies outside the virus. This work describes the production and characterization of an ectodomain construct containing the 154 N-terminal gp41 residues, including the fusion peptide (FP) that binds to target cell membranes. The Fgp41 sequence was derived from one of the African clade A strains of HIV-1 that have been less studied than European/North American clade B strains. Fgp41 expression at a level of ~100 mg/L of culture was evidenced by an approach that included amino acid type (13)CO and (15)N labeling of recombinant protein and solid-state NMR (SSNMR) spectroscopy of lyophilized whole cells. The approach did not require any protein solubilization or purification and may be a general approach for detection of recombinant protein. The purified Fgp41 yield was ~5 mg/L of culture. SSNMR spectra of membrane-associated Fgp41 showed high helicity for the residues C-terminal of the FP. This was consistent with a "six-helix bundle" (SHB) structure that is the final gp41 state during membrane fusion. This observation and negligible Fgp41-induced vesicle fusion supported a function for SHB gp41 of membrane stabilization and fusion arrest. SSNMR spectra of residues in the membrane-associated FP provided evidence of a mixture of molecular populations with either helical or β-sheet FP conformation. These and earlier SSNMR data strongly support the existence of these populations in the SHB state of membrane-associated gp41. © 2011 American Chemical Society

  7. Obatoclax Inhibits Alphavirus Membrane Fusion by Neutralizing the Acidic Environment of Endocytic Compartments.

    Science.gov (United States)

    Varghese, Finny S; Rausalu, Kai; Hakanen, Marika; Saul, Sirle; Kümmerer, Beate M; Susi, Petri; Merits, Andres; Ahola, Tero

    2017-03-01

    As new pathogenic viruses continue to emerge, it is paramount to have intervention strategies that target a common denominator in these pathogens. The fusion of viral and cellular membranes during viral entry is one such process that is used by many pathogenic viruses, including chikungunya virus, West Nile virus, and influenza virus. Obatoclax, a small-molecule antagonist of the Bcl-2 family of proteins, was previously determined to have activity against influenza A virus and also Sindbis virus. Here, we report it to be active against alphaviruses, like chikungunya virus (50% effective concentration [EC 50 ] = 0.03 μM) and Semliki Forest virus (SFV; EC 50 = 0.11 μM). Obatoclax inhibited viral entry processes in an SFV temperature-sensitive mutant entry assay. A neutral red retention assay revealed that obatoclax induces the rapid neutralization of the acidic environment of endolysosomal vesicles and thereby most likely inhibits viral fusion. Characterization of escape mutants revealed that the L369I mutation in the SFV E1 fusion protein was sufficient to confer partial resistance against obatoclax. Other inhibitors that target the Bcl-2 family of antiapoptotic proteins inhibited neither viral entry nor endolysosomal acidification, suggesting that the antiviral mechanism of obatoclax does not depend on its anticancer targets. Obatoclax inhibited the growth of flaviviruses, like Zika virus, West Nile virus, and yellow fever virus, which require low pH for fusion, but not that of pH-independent picornaviruses, like coxsackievirus A9, echovirus 6, and echovirus 7. In conclusion, obatoclax is a novel inhibitor of endosomal acidification that prevents viral fusion and that could be pursued as a potential broad-spectrum antiviral candidate. Copyright © 2017 American Society for Microbiology.

  8. Dengue virus ensures its fusion in late endosomes using compartment-specific lipids.

    Directory of Open Access Journals (Sweden)

    Elena Zaitseva

    2010-10-01

    Full Text Available Many enveloped viruses invade cells via endocytosis and use different environmental factors as triggers for virus-endosome fusion that delivers viral genome into cytosol. Intriguingly, dengue virus (DEN, the most prevalent mosquito-borne virus that infects up to 100 million people each year, fuses only in late endosomes, while activation of DEN protein fusogen glycoprotein E is triggered already at pH characteristic for early endosomes. Are there any cofactors that time DEN fusion to virion entry into late endosomes? Here we show that DEN utilizes bis(monoacylglycerophosphate, a lipid specific to late endosomes, as a co-factor for its endosomal acidification-dependent fusion machinery. Effective virus fusion to plasma- and intracellular- membranes, as well as to protein-free liposomes, requires the target membrane to contain anionic lipids such as bis(monoacylglycerophosphate and phosphatidylserine. Anionic lipids act downstream of low-pH-dependent fusion stages and promote the advance from the earliest hemifusion intermediates to the fusion pore opening. To reach anionic lipid-enriched late endosomes, DEN travels through acidified early endosomes, but we found that low pH-dependent loss of fusogenic properties of DEN is relatively slow in the presence of anionic lipid-free target membranes. We propose that anionic lipid-dependence of DEN fusion machinery protects it against premature irreversible restructuring and inactivation and ensures viral fusion in late endosomes, where the virus encounters anionic lipids for the first time during entry. Currently there are neither vaccines nor effective therapies for DEN, and the essential role of the newly identified DEN-bis(monoacylglycerophosphate interactions in viral genome escape from the endosome suggests a novel target for drug design.

  9. The structural dynamics of the flavivirus fusion peptide-membrane interaction.

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    Ygara S Mendes

    Full Text Available Membrane fusion is a crucial step in flavivirus infections and a potential target for antiviral strategies. Lipids and proteins play cooperative roles in the fusion process, which is triggered by the acidic pH inside the endosome. This acidic environment induces many changes in glycoprotein conformation and allows the action of a highly conserved hydrophobic sequence, the fusion peptide (FP. Despite the large volume of information available on the virus-triggered fusion process, little is known regarding the mechanisms behind flavivirus-cell membrane fusion. Here, we evaluated the contribution of a natural single amino acid difference on two flavivirus FPs, FLA(G ((98DRGWGNGCGLFGK(110 and FLA(H ((98DRGWGNHCGLFGK(110, and investigated the role of the charge of the target membrane on the fusion process. We used an in silico approach to simulate the interaction of the FPs with a lipid bilayer in a complementary way and used spectroscopic approaches to collect conformation information. We found that both peptides interact with neutral and anionic micelles, and molecular dynamics (MD simulations showed the interaction of the FPs with the lipid bilayer. The participation of the indole ring of Trp appeared to be important for the anchoring of both peptides in the membrane model, as indicated by MD simulations and spectroscopic analyses. Mild differences between FLA(G and FLA(H were observed according to the pH and the charge of the target membrane model. The MD simulations of the membrane showed that both peptides adopted a bend structure, and an interaction between the aromatic residues was strongly suggested, which was also observed by circular dichroism in the presence of micelles. As the FPs of viral fusion proteins play a key role in the mechanism of viral fusion, understanding the interactions between peptides and membranes is crucial for medical science and biology and may contribute to the design of new antiviral drugs.

  10. Tension-induced fusion of bilayer membranes and vesicles

    Science.gov (United States)

    Shillcock, Julian C.; Lipowsky, Reinhard

    2005-03-01

    Maintaining the integrity of their protective plasma membrane is a primary requirement of cells. Accordingly, cellular events that breach the membrane are tightly regulated. Artificial vesicles used in drug delivery must also stay intact until they have reached the desired target. In both cases, the intrinsic resistance of the membrane to rupture must be overcome to allow the efflux of the vesicle's contents. Here, we use mesoscopic simulations to study the fusion of 28-nm-diameter vesicles to 50 × 50 nm2 planar membrane patches over 2 μs. We monitor the time evolution of 93 different fusion attempts. This allows us to construct a global morphology diagram, using the initial tensions of the vesicle and the planar membrane patch as control parameters, and to determine the corresponding fusion statistics. All successful fusion events are observed to occur within 350 ns, which reflects the presence of alternative pathways for the tension relaxation.

  11. Universal antibodies against the highly conserved influenza fusion peptide cross-neutralize several subtypes of influenza A virus

    Energy Technology Data Exchange (ETDEWEB)

    Hashem, Anwar M. [Centre for Vaccine Evaluation, Biologics and Genetic Therapies Directorate, HPFB, Health Canada, Ottawa, ON (Canada); Department of Microbiology, Faculty of Medicine, King Abdulaziz University, Jeddah (Saudi Arabia); Department of Biochemistry, Microbiology and Immunology, University of Ottawa, Ottawa, ON (Canada); Van Domselaar, Gary [National Microbiology Laboratory, Public Health Agency of Canada, Winnipeg, MB (Canada); Li, Changgui; Wang, Junzhi [National Institute for the Control of Pharmaceutical and Biological Products, Beijing (China); She, Yi-Min; Cyr, Terry D. [Centre for Vaccine Evaluation, Biologics and Genetic Therapies Directorate, HPFB, Health Canada, Ottawa, ON (Canada); Sui, Jianhua [Department of Cancer Immunology and AIDS, Dana-Farber Cancer Institute, Department of Medicine, Harvard Medical School, 44 Binney Street, Boston, MA 02115 (United States); He, Runtao [National Microbiology Laboratory, Public Health Agency of Canada, Winnipeg, MB (Canada); Marasco, Wayne A. [Department of Cancer Immunology and AIDS, Dana-Farber Cancer Institute, Department of Medicine, Harvard Medical School, 44 Binney Street, Boston, MA 02115 (United States); Li, Xuguang, E-mail: Sean.Li@hc-sc.gc.ca [Centre for Vaccine Evaluation, Biologics and Genetic Therapies Directorate, HPFB, Health Canada, Ottawa, ON (Canada); Department of Biochemistry, Microbiology and Immunology, University of Ottawa, Ottawa, ON (Canada)

    2010-12-10

    Research highlights: {yields} The fusion peptide is the only universally conserved epitope in all influenza viral hemagglutinins. {yields} Anti-fusion peptide antibodies are universal antibodies that cross-react with all influenza HA subtypes. {yields} The universal antibodies cross-neutralize different influenza A subtypes. {yields} The universal antibodies inhibit the fusion process between the viruses and the target cells. -- Abstract: The fusion peptide of influenza viral hemagglutinin plays a critical role in virus entry by facilitating membrane fusion between the virus and target cells. As the fusion peptide is the only universally conserved epitope in all influenza A and B viruses, it could be an attractive target for vaccine-induced immune responses. We previously reported that antibodies targeting the first 14 amino acids of the N-terminus of the fusion peptide could bind to virtually all influenza virus strains and quantify hemagglutinins in vaccines produced in embryonated eggs. Here we demonstrate that these universal antibodies bind to the viral hemagglutinins in native conformation presented in infected mammalian cell cultures and neutralize multiple subtypes of virus by inhibiting the pH-dependant fusion of viral and cellular membranes. These results suggest that this unique, highly-conserved linear sequence in viral hemagglutinin is exposed sufficiently to be attacked by the antibodies during the course of infection and merits further investigation because of potential importance in the protection against diverse strains of influenza viruses.

  12. The SI Strain of Measles Virus Derived from a Patient with Subacute Sclerosing Panencephalitis Possesses Typical Genome Alterations and Unique Amino Acid Changes That Modulate Receptor Specificity and Reduce Membrane Fusion Activity ▿ ‡

    Science.gov (United States)

    Seki, Fumio; Yamada, Kentaro; Nakatsu, Yuichiro; Okamura, Koji; Yanagi, Yusuke; Nakayama, Tetsuo; Komase, Katsuhiro; Takeda, Makoto

    2011-01-01

    Subacute sclerosing panencephalitis (SSPE) is a fatal sequela associated with measles and is caused by persistent infection of the brain with measles virus (MV). The SI strain was isolated in 1976 from a patient with SSPE and shows neurovirulence in animals. Genome nucleotide sequence analyses showed that the SI strain genome possesses typical genome alterations for SSPE-derived strains, namely, accumulated amino acid substitutions in the M protein and cytoplasmic tail truncation of the F protein. Through the establishment of an efficient reverse genetics system, a recombinant SI strain expressing a green fluorescent protein (rSI-AcGFP) was generated. The infection of various cell types with rSI-AcGFP was evaluated by fluorescence microscopy. rSI-AcGFP exhibited limited syncytium-forming activity and spread poorly in cells. Analyses using a recombinant MV possessing a chimeric genome between those of the SI strain and a wild-type MV strain indicated that the membrane-associated protein genes (M, F, and H) were responsible for the altered growth phenotype of the SI strain. Functional analyses of viral glycoproteins showed that the F protein of the SI strain exhibited reduced fusion activity because of an E300G substitution and that the H protein of the SI strain used CD46 efficiently but used the original MV receptors on immune and epithelial cells poorly because of L482F, S546G, and F555L substitutions. The data obtained in the present study provide a new platform for analyses of SSPE-derived strains as well as a clear example of an SSPE-derived strain that exhibits altered receptor specificity and limited fusion activity. PMID:21917959

  13. An Alphavirus E2 Membrane-Proximal Domain Promotes Envelope Protein Lateral Interactions and Virus Budding

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    Emily A. Byrd

    2017-11-01

    Full Text Available Alphaviruses are members of a group of small enveloped RNA viruses that includes important human pathogens such as Chikungunya virus and the equine encephalitis viruses. The virus membrane is covered by a lattice composed of 80 spikes, each a trimer of heterodimers of the E2 and E1 transmembrane proteins. During virus endocytic entry, the E1 glycoprotein mediates the low-pH-dependent fusion of the virus membrane with the endosome membrane, thus initiating virus infection. While much is known about E1 structural rearrangements during membrane fusion, it is unclear how the E1/E2 dimer dissociates, a step required for the fusion reaction. A recent Alphavirus cryo-electron microscopy reconstruction revealed a previously unidentified D subdomain in the E2 ectodomain, close to the virus membrane. A loop within this region, here referred to as the D-loop, contains two highly conserved histidines, H348 and H352, which were hypothesized to play a role in dimer dissociation. We generated Semliki Forest virus mutants containing the single and double alanine substitutions H348A, H352A, and H348/352A. The three D-loop mutations caused a reduction in virus growth ranging from 1.6 to 2 log but did not significantly affect structural protein biosynthesis or transport, dimer stability, virus fusion, or specific infectivity. Instead, growth reduction was due to inhibition of a late stage of virus assembly at the plasma membrane. The virus particles that are produced show reduced thermostability compared to the wild type. We propose the E2 D-loop as a key region in establishing the E1-E2 contacts that drive glycoprotein lattice formation and promote Alphavirus budding from the plasma membrane.

  14. Inhibition of Henipavirus fusion and infection by heptad-derived peptides of the Nipah virus fusion glycoprotein

    Science.gov (United States)

    Bossart, Katharine N; Mungall, Bruce A; Crameri, Gary; Wang, Lin-Fa; Eaton, Bryan T; Broder, Christopher C

    2005-01-01

    Background The recent emergence of four new members of the paramyxovirus family has heightened the awareness of and re-energized research on new and emerging diseases. In particular, the high mortality and person to person transmission associated with the most recent Nipah virus outbreaks, as well as the very recent re-emergence of Hendra virus, has confirmed the importance of developing effective therapeutic interventions. We have previously shown that peptides corresponding to the C-terminal heptad repeat (HR-2) of the fusion envelope glycoprotein of Hendra virus and Nipah virus were potent inhibitors of both Hendra virus and Nipah virus-mediated membrane fusion using recombinant expression systems. In the current study, we have developed shorter, second generation HR-2 peptides which include a capped peptide via amidation and acetylation and two poly(ethylene glycol)-linked (PEGylated) peptides, one with the PEG moity at the C-terminus and the other at the N-terminus. Here, we have evaluated these peptides as well as the corresponding scrambled peptide controls in Nipah virus and Hendra virus-mediated membrane fusion and against infection by live virus in vitro. Results Unlike their predecessors, the second generation HR-2 peptides exhibited high solubility and improved synthesis yields. Importantly, both Nipah virus and Hendra virus-mediated fusion as well as live virus infection were potently inhibited by both capped and PEGylated peptides with IC50 concentrations similar to the original HR-2 peptides, whereas the scrambled modified peptides had no inhibitory effect. These data also indicate that these chemical modifications did not alter the functional properties of the peptides as inhibitors. Conclusion Nipah virus and Hendra virus infection in vitro can be potently blocked by specific HR-2 peptides. The improved synthesis and solubility characteristics of the second generation HR-2 peptides will facilitate peptide synthesis for pre-clinical trial

  15. Inhibition of Henipavirus fusion and infection by heptad-derived peptides of the Nipah virus fusion glycoprotein

    Directory of Open Access Journals (Sweden)

    Eaton Bryan T

    2005-07-01

    Full Text Available Abstract Background The recent emergence of four new members of the paramyxovirus family has heightened the awareness of and re-energized research on new and emerging diseases. In particular, the high mortality and person to person transmission associated with the most recent Nipah virus outbreaks, as well as the very recent re-emergence of Hendra virus, has confirmed the importance of developing effective therapeutic interventions. We have previously shown that peptides corresponding to the C-terminal heptad repeat (HR-2 of the fusion envelope glycoprotein of Hendra virus and Nipah virus were potent inhibitors of both Hendra virus and Nipah virus-mediated membrane fusion using recombinant expression systems. In the current study, we have developed shorter, second generation HR-2 peptides which include a capped peptide via amidation and acetylation and two poly(ethylene glycol-linked (PEGylated peptides, one with the PEG moity at the C-terminus and the other at the N-terminus. Here, we have evaluated these peptides as well as the corresponding scrambled peptide controls in Nipah virus and Hendra virus-mediated membrane fusion and against infection by live virus in vitro. Results Unlike their predecessors, the second generation HR-2 peptides exhibited high solubility and improved synthesis yields. Importantly, both Nipah virus and Hendra virus-mediated fusion as well as live virus infection were potently inhibited by both capped and PEGylated peptides with IC50 concentrations similar to the original HR-2 peptides, whereas the scrambled modified peptides had no inhibitory effect. These data also indicate that these chemical modifications did not alter the functional properties of the peptides as inhibitors. Conclusion Nipah virus and Hendra virus infection in vitro can be potently blocked by specific HR-2 peptides. The improved synthesis and solubility characteristics of the second generation HR-2 peptides will facilitate peptide synthesis for pre

  16. The influenza hemagglutinin fusion domain is an amphipathic helical hairpin that functions by inducing membrane curvature.

    Science.gov (United States)

    Smrt, Sean T; Draney, Adrian W; Lorieau, Justin L

    2015-01-02

    The highly conserved N-terminal 23 residues of the hemagglutinin glycoprotein, known as the fusion peptide domain (HAfp23), is vital to the membrane fusion and infection mechanism of the influenza virus. HAfp23 has a helical hairpin structure consisting of two tightly packed amphiphilic helices that rest on the membrane surface. We demonstrate that HAfp23 is a new class of amphipathic helix that functions by leveraging the negative curvature induced by two tightly packed helices on membranes. The helical hairpin structure has an inverted wedge shape characteristic of negative curvature lipids, with a bulky hydrophobic region and a relatively small hydrophilic head region. The F3G mutation reduces this inverted wedge shape by reducing the volume of its hydrophobic base. We show that despite maintaining identical backbone structures and dynamics as the wild type HAfp23, the F3G mutant has an attenuated fusion activity that is correlated to its reduced ability to induce negative membrane curvature. The inverted wedge shape of HAfp23 is likely to play a crucial role in the initial stages of membrane fusion by stabilizing negative curvature in the fusion stalk. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  17. Crystal structure of Venezuelan hemorrhagic fever virus fusion glycoprotein reveals a class 1 postfusion architecture with extensive glycosylation.

    Science.gov (United States)

    Parsy, Marie-Laure; Harlos, Karl; Huiskonen, Juha T; Bowden, Thomas A

    2013-12-01

    Guanarito virus (GTOV) is an emergent and deadly pathogen. We present the crystal structure of the glycosylated GTOV fusion glycoprotein to 4.1-Å resolution in the postfusion conformation. Our structure reveals a classical six-helix bundle and presents direct verification that New World arenaviruses exhibit class I viral membrane fusion machinery. The structure provides visualization of an N-linked glycocalyx coat, and consideration of glycan dynamics reveals extensive coverage of the underlying protein surface, following virus-host membrane fusion.

  18. Homotypic fusion of endoplasmic reticulum membranes in plant cells

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    Junjie eHu

    2013-12-01

    Full Text Available The endoplasmic reticulum (ER is a membrane-bounded organelle whose membrane comprises a network of tubules and sheets. The formation of these characteristic shapes and maintenance of their continuity through homotypic membrane fusion appears to be critical for the proper functioning of the ER. The atlastins (ATLs, a family of ER-localized dynamin-like GTPases, have been identified as fusogens of the ER membranes in metazoans. Mutations of the ATL proteins in mammalian cells cause morphological defects in the ER, and purified Drosophila ATL mediates membrane fusion in vitro. Plant cells do not possess ATL, but a family of similar GTPases, named root hair defective 3 (RHD3, are likely the functional orthologs of ATLs. In this review, we summarize recent advances in our understanding of how RHD3 proteins play a role in homotypic ER fusion. We also discuss the possible physiological significance of forming a tubular ER network in plant cells.

  19. Structure-function dissection of the Pseudorabies virus glycoprotein B fusion loops.

    Science.gov (United States)

    Vallbracht, Melina; Brun, Delphine; Tassinari, Matteo; Vaney, Marie-Christine; Pehau-Arnaudet, Gérard; Guardado-Calvo, Pablo; Haouz, Ahmed; Klupp, Barbara G; Mettenleiter, Thomas C; Rey, Felix A; Backovic, Marija

    2017-10-18

    Conserved across the Herpesviridae family, glycoprotein B (gB) is responsible for driving fusion of the viral envelope with the host cell membrane for entry upon receptor binding and activation by the viral gH/gL complex. Although crystal structures of the gB ectodomain of several herpesviruses have been reported, the membrane fusion mechanism has remained elusive. Here, we report the X-ray structure of the Pseudorabies virus (PrV) gB ectodomain, revealing a typical class III post-fusion trimer that binds membranes via its fusion loops (FLs) in a cholesterol-dependent manner. Mutagenesis of FL residues allowed us to dissect those interacting with distinct sub-regions of the lipid bilayer and their role for membrane interactions. We tested 15 gB variants for their ability to bind to liposomes, and further investigated a subset of them in functional assays. We found that PrV gB FL residues Trp187, Tyr192, Phe275 and Tyr276, which were essential for liposome binding and for fusion in a cellular and viral context, form a continuous hydrophobic patch at the gB trimer surface. Together with reported results from other alpha-herpesvirus gBs, our data suggest a model in which Phe275 from the tip of FL2 protrudes deeper into the hydrocarbon core of the lipid bilayer, while the side chains of Trp187, Tyr192 and Tyr276 form a rim that inserts into the more superficial, interfacial region of the membrane to catalyze the fusion process. Comparative analysis with gB from beta- and gamma-herpesviruses suggest that this membrane-interaction mode is valid for gB from all herpesviruses.IMPORTANCE Herpesviruses are common human and animal pathogens, which infect cells by entering via fusion of viral and cellular membranes and which cause life-long and incurable infections. Central to the membrane fusion event for entry is glycoprotein B (gB), which is the most conserved envelope protein across the herpesvirus family. Like other viral fusion proteins, gB anchors itself into the target

  20. Role of Conserved Histidine Residues in the Low-pH Dependence of the Semliki Forest Virus Fusion Protein▿

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    Qin, Zhao-ling; Zheng, Yan; Kielian, Margaret

    2009-01-01

    A wide variety of enveloped viruses infects cells by taking advantage of the low pH in the endocytic pathway to trigger virus-membrane fusion. For alphaviruses such as Semliki Forest virus (SFV), acidic pH initiates a series of conformational changes in the heterodimeric virus envelope proteins E1 and E2. Low pH dissociates the E2/E1 dimer, releasing the membrane fusion protein E1. E1 inserts into the target membrane and refolds to a trimeric hairpin conformation, thus driving the fusion reaction. The means by which E1 senses and responds to low pH is unclear, and protonation of conserved E1 histidine residues has been proposed as a possible mechanism. We tested the role of four conserved histidines by mutagenesis of the wild-type (wt) SFV infectious clone to create virus mutants with E1 H3A, H125A, H331A, and H331A/H333A mutations. The H125A, H331A, and H331A/H333A mutants had growth properties similar to those of wt SFV and showed modest change or no change in the pH dependence of virus-membrane fusion. By contrast, the E1 H3A mutation produced impaired virus growth and a markedly more acidic pH requirement for virus-membrane fusion. The dissociation of the H3A heterodimer and the membrane insertion of the mutant E1 protein were comparable to those of the wt in efficiency and pH dependence. However, the formation of the H3A homotrimer required a much lower pH and showed reduced efficiency. Together, these results and the location of H3 suggest that this residue acts to regulate the low-pH-dependent refolding of E1 during membrane fusion. PMID:19244325

  1. Role of conserved histidine residues in the low-pH dependence of the Semliki Forest virus fusion protein.

    Science.gov (United States)

    Qin, Zhao-Ling; Zheng, Yan; Kielian, Margaret

    2009-05-01

    A wide variety of enveloped viruses infects cells by taking advantage of the low pH in the endocytic pathway to trigger virus-membrane fusion. For alphaviruses such as Semliki Forest virus (SFV), acidic pH initiates a series of conformational changes in the heterodimeric virus envelope proteins E1 and E2. Low pH dissociates the E2/E1 dimer, releasing the membrane fusion protein E1. E1 inserts into the target membrane and refolds to a trimeric hairpin conformation, thus driving the fusion reaction. The means by which E1 senses and responds to low pH is unclear, and protonation of conserved E1 histidine residues has been proposed as a possible mechanism. We tested the role of four conserved histidines by mutagenesis of the wild-type (wt) SFV infectious clone to create virus mutants with E1 H3A, H125A, H331A, and H331A/H333A mutations. The H125A, H331A, and H331A/H333A mutants had growth properties similar to those of wt SFV and showed modest change or no change in the pH dependence of virus-membrane fusion. By contrast, the E1 H3A mutation produced impaired virus growth and a markedly more acidic pH requirement for virus-membrane fusion. The dissociation of the H3A heterodimer and the membrane insertion of the mutant E1 protein were comparable to those of the wt in efficiency and pH dependence. However, the formation of the H3A homotrimer required a much lower pH and showed reduced efficiency. Together, these results and the location of H3 suggest that this residue acts to regulate the low-pH-dependent refolding of E1 during membrane fusion.

  2. Conflicting views on the membrane fusion machinery and the fusion pore

    DEFF Research Database (Denmark)

    Sørensen, Jakob B

    2009-01-01

    Fast exocytosis of synaptic vesicles differs from other membrane fusion reactions by being under tight temporal control by the intracellular calcium concentration. This is achieved by subjecting the SNARE-dependent fusion pathway to additional layers of control, both upstream and downstream...... of the water-soluble vesicle content. The fusion pore remains the most elusive part of the exocytosis pathway, owing to its short lifetime....

  3. Herpesvirus glycoproteins undergo multiple antigenic changes before membrane fusion.

    Directory of Open Access Journals (Sweden)

    Daniel L Glauser

    Full Text Available Herpesvirus entry is a complicated process involving multiple virion glycoproteins and culminating in membrane fusion. Glycoprotein conformation changes are likely to play key roles. Studies of recombinant glycoproteins have revealed some structural features of the virion fusion machinery. However, how the virion glycoproteins change during infection remains unclear. Here using conformation-specific monoclonal antibodies we show in situ that each component of the Murid Herpesvirus-4 (MuHV-4 entry machinery--gB, gH/gL and gp150--changes in antigenicity before tegument protein release begins. Further changes then occurred upon actual membrane fusion. Thus virions revealed their final fusogenic form only in late endosomes. The substantial antigenic differences between this form and that of extracellular virions suggested that antibodies have only a limited opportunity to block virion membrane fusion.

  4. Efficient Fusion at Neutral pH by Human Immunodeficiency Virus gp41 Trimers containing the Fusion Peptide and Transmembrane Domain.

    Science.gov (United States)

    Liang, Shuang; Ratnayake, Punsisi; Keinath, Craig; Jia, Lihui; Wolfe, Robert; Ranaweera, Ahinsa; Weliky, David P

    2018-01-18

    Human immunodeficiency virus (HIV) is membrane-enveloped and an initial infection step is joining/fusion of viral and cell membranes. This step is catalyzed by gp41 which is a single-pass integral viral membrane protein. The protein contains a ~170-residue ectodomain located outside the virus that is important for fusion, and includes the fusion peptide (FP), N-helix, loop, C-helix, and viral membrane-proximal external region (MPER). The virion initially has non-covalent complexes between three gp41 ectodomains and three gp120 proteins. A gp120 contains ~500-residues and functions to identify target T-cells and macrophages via binding to specific protein receptors of the target cell membrane. Gp120 moves away from the gp41 ectodomain, and the ectodomain is thought to bind to the target cell membrane and mediate membrane fusion. The secondary and tertiary structures of the ectodomain are different in the initial complex with gp120 and the final state without gp120. There isn't yet imaging of gp41 during fusion, so the temporal relationship between the gp41 and membrane structures isn't known. The present study describes biophysical and functional characterization of large gp41 constructs that include the ectodomain and transmembrane domain (TM). Significant fusion is observed of both neutral and anionic vesicles at neutral pH which reflects the expected conditions of HIV/cell fusion. Fusion is enhanced by the FP, which in HIV/cell fusion likely contacts the host membrane, and the MPER and TM, which respectively interfacially contact and traverse the HIV membrane. Initial contact with vesicles is made by protein trimers which are in a native oligomeric state that reflects the initial complex with gp120, and also is commonly observed for the ectodomain without gp120. Circular dichroism data support helical structure for the N-helix, C-helix, and MPER, and non-helical structure for the FP and loop. Distributions of monomer, trimer, and hexamer states are observed by

  5. Optimization of membrane protein overexpression and purification using GFP fusions

    NARCIS (Netherlands)

    Drew, David; Lerch, Mirjam; Kunji, Edmund; Slotboom, Dirk-Jan; de Gier, Jan-Willem

    Optimizing conditions for the overexpression and purification of membrane proteins for functional and structural studies is usually a Laborious and time-consuming process. This process can be accelerated using membrane protein-GFP fusions(1-3), which allows direct monitoring and visualization of

  6. Development of Lead Compounds as Fusion Inhibitors for Dengue Virus

    Science.gov (United States)

    2009-08-01

    Identification of antiviral agents is critical to the development of therapeutic treatments directed against dengue virus (DENV), for which there is no...infection and exposure of US military personnel to the disease might be offered by antiviral agents as a treatment strategy. The dengue virus...0285 TITLE: DEVELOPMENT OF LEAD COMPOUNDS AS FUSION INHIBITORS FOR DENGUE VIRUS PRINCIPAL INVESTIGATOR: TOMAS PEREZ-ACLE

  7. Identification of bioflavonoid as fusion inhibitor of dengue virus using molecular docking approach

    Directory of Open Access Journals (Sweden)

    Asif Mir

    Full Text Available Dengue virus with four distinct serotypes belongs to Flavivirus, poses a significant threat to human health and becomes an emerging global problem. Membrane fusion is a central molecular event during viral entry into host cell. To prevent viral infection it is necessary to interrupt the virus replication at an early stage of attachment. Dengue Virus (DENV envelope protein experiences conformational changes and it causes the virus to fuse with host cell. Hinge region movement of domain I and II in envelope protein facilitates the fusion process. Small molecules that bind in this pocket may have the ability to interrupt the conformational changes that trigger fusion process. We chose different flavonoids (baicalein, fisetin, hesperetin, naringenin/ naringin, quercetin and rutin that possess anti dengue activity. Molecular docking analysis was done to examine the inhibitory effect of flavonoids against envelope protein of DENV-2. Results manifest quercetin (flavonoid found in Carica papaya, apple and even in lemon as the only flavone that can interrupt the fusion process of virus by inhibiting the hinge region movement and by blocking the conformational rearrangement in envelope protein. These novel findings using computational approach are worthwhile and will be a bridge to check the efficacy of compounds using appropriate animal model under In vivo studies. This information can be used by new techniques and provides a way to control dengue virus infection. Keywords: Dengue virus, Inhibitor identification, Molecular docking, Interaction analysis

  8. Vesicle fusion with bilayer lipid membrane controlled by electrostatic interaction

    Directory of Open Access Journals (Sweden)

    Azusa Oshima

    2017-09-01

    Full Text Available The fusion of proteoliposomes is a promising approach for incorporating membrane proteins in artificial lipid membranes. In this study, we employed an electrostatic interaction between vesicles and supported bilayer lipid membranes (s-BLMs to control the fusion process. We combined large unilamellar vesicles (LUVs containing anionic lipids, which we used instead of proteoliposomes, and s-BLMs containing cationic lipids to control electrostatic interaction. Anionic LUVs were never adsorbed or ruptured on the SiO2 substrate with a slight negative charge, and selectively fused with cationic s-BLMs. The LUVs can be fused effectively to the target position. Furthermore, as the vesicle fusion proceeds and some of the positive charges are neutralized, the attractive interaction weakens and finally the vesicle fusion saturates. In other words, we can control the number of LUVs fused with s-BLMs by controlling the concentration of the cationic lipids in the s-BLMs. The fluidity of the s-BLMs after vesicle fusion was confirmed to be sufficiently high. This indicates that the LUVs attached to the s-BLMs were almost completely fused, and there were few intermediate state vesicles in the fusion process. We could control the position and amount of vesicle fusion with the s-BLMs by employing an electrostatic interaction.

  9. Inner membrane fusion mediates spatial distribution of axonal mitochondria

    Science.gov (United States)

    Yu, Yiyi; Lee, Hao-Chih; Chen, Kuan-Chieh; Suhan, Joseph; Qiu, Minhua; Ba, Qinle; Yang, Ge

    2016-01-01

    In eukaryotic cells, mitochondria form a dynamic interconnected network to respond to changing needs at different subcellular locations. A fundamental yet unanswered question regarding this network is whether, and if so how, local fusion and fission of individual mitochondria affect their global distribution. To address this question, we developed high-resolution computational image analysis techniques to examine the relations between mitochondrial fusion/fission and spatial distribution within the axon of Drosophila larval neurons. We found that stationary and moving mitochondria underwent fusion and fission regularly but followed different spatial distribution patterns and exhibited different morphology. Disruption of inner membrane fusion by knockdown of dOpa1, Drosophila Optic Atrophy 1, not only increased the spatial density of stationary and moving mitochondria but also changed their spatial distributions and morphology differentially. Knockdown of dOpa1 also impaired axonal transport of mitochondria. But the changed spatial distributions of mitochondria resulted primarily from disruption of inner membrane fusion because knockdown of Milton, a mitochondrial kinesin-1 adapter, caused similar transport velocity impairment but different spatial distributions. Together, our data reveals that stationary mitochondria within the axon interconnect with moving mitochondria through fusion and fission and that local inner membrane fusion between individual mitochondria mediates their global distribution. PMID:26742817

  10. Molecular View of the Role of Fusion Peptides in Promoting Positive Membrane Curvature

    NARCIS (Netherlands)

    Fuhrmans, Marc; Marrink, Siewert J.

    2012-01-01

    Fusion peptides are moderately hydrophobic segments of viral and nonviral membrane fusion proteins that enable these proteins to fuse two closely apposed biological membranes. In vitro assays furthermore show that even isolated fusion peptides alone can support membrane fusion in model systems. In

  11. Structural basis of influenza virus fusion inhibition by the antiviral drug Arbidol

    Energy Technology Data Exchange (ETDEWEB)

    Kadam, Rameshwar U.; Wilson, Ian A.

    2016-12-21

    The broad-spectrum antiviral drug Arbidol shows efficacy against influenza viruses by targeting the hemagglutinin (HA) fusion machinery. However, the structural basis of the mechanism underlying fusion inhibition by Arbidol has remained obscure, thereby hindering its further development as a specific and optimized influenza therapeutic. We determined crystal structures of Arbidol in complex with influenza virus HA from pandemic 1968 H3N2 and recent 2013 H7N9 viruses. Arbidol binds in a hydrophobic cavity in the HA trimer stem at the interface between two protomers. This cavity is distal to the conserved epitope targeted by broadly neutralizing stem antibodies and is ~16 Å from the fusion peptide. Arbidol primarily makes hydrophobic interactions with the binding site but also induces some conformational rearrangements to form a network of inter- and intraprotomer salt bridges. By functioning as molecular glue, Arbidol stabilizes the prefusion conformation of HA that inhibits the large conformational rearrangements associated with membrane fusion in the low pH of the endosome. This unique binding mode compared with the small-molecule inhibitors of other class I fusion proteins enhances our understanding of how small molecules can function as fusion inhibitors and guides the development of broad-spectrum therapeutics against influenza virus.

  12. Organelle acidification negatively regulates vacuole membrane fusion in vivo

    Science.gov (United States)

    Desfougères, Yann; Vavassori, Stefano; Rompf, Maria; Gerasimaite, Ruta; Mayer, Andreas

    2016-01-01

    The V-ATPase is a proton pump consisting of a membrane-integral V0 sector and a peripheral V1 sector, which carries the ATPase activity. In vitro studies of yeast vacuole fusion and evidence from worms, flies, zebrafish and mice suggested that V0 interacts with the SNARE machinery for membrane fusion, that it promotes the induction of hemifusion and that this activity requires physical presence of V0 rather than its proton pump activity. A recent in vivo study in yeast has challenged these interpretations, concluding that fusion required solely lumenal acidification but not the V0 sector itself. Here, we identify the reasons for this discrepancy and reconcile it. We find that acute pharmacological or physiological inhibition of V-ATPase pump activity de-acidifies the vacuole lumen in living yeast cells within minutes. Time-lapse microscopy revealed that de-acidification induces vacuole fusion rather than inhibiting it. Cells expressing mutated V0 subunits that maintain vacuolar acidity were blocked in this fusion. Thus, proton pump activity of the V-ATPase negatively regulates vacuole fusion in vivo. Vacuole fusion in vivo does, however, require physical presence of a fusion-competent V0 sector. PMID:27363625

  13. The Cytoplasmic Tail Domain of Epstein-Barr Virus gH Regulates Membrane Fusion Activity through Altering gH Binding to gp42 and Epithelial Cell Attachment

    Directory of Open Access Journals (Sweden)

    Jia Chen

    2016-11-01

    Full Text Available Epstein-Barr virus (EBV is associated with infectious mononucleosis and a variety of cancers as well as lymphoproliferative disorders in immunocompromised patients. EBV mediates viral entry into epithelial and B cells using fusion machinery composed of four glycoproteins: gB, the gH/gL complex, and gp42. gB and gH/gL are required for both epithelial and B cell fusion. The specific role of gH/gL in fusion has been the most elusive among the required herpesvirus entry glycoproteins. Previous mutational studies have focused on the ectodomain of EBV gH and not on the gH cytoplasmic tail domain (CTD. In this study, we chose to examine the function of the gH CTD by making serial gH truncation mutants as well as amino acid substitution mutants to determine the importance of the gH CTD in epithelial and B cell fusion. Truncation of 8 amino acids (aa 698 to 706 of the gH CTD resulted in diminished fusion activity using a virus-free syncytium formation assay and fusion assay. The importance of the amino acid composition of the gH CTD was also investigated by amino acid substitutions that altered the hydrophobicity or hydrophilicity of the CTD. These mutations also resulted in diminished fusion activity. Interestingly, some of the gH CTD truncation mutants and hydrophilic tail substitution mutants lost the ability to bind to gp42 and epithelial cells. In summary, our studies indicate that the gH CTD is an important functional domain.

  14. 5-(Perylen-3-yl)ethynyl-arabino-uridine (aUY11), an arabino-based rigid amphipathic fusion inhibitor, targets virion envelope lipids to inhibit fusion of influenza virus, hepatitis C virus, and other enveloped viruses.

    Science.gov (United States)

    Colpitts, Che C; Ustinov, Alexey V; Epand, Raquel F; Epand, Richard M; Korshun, Vladimir A; Schang, Luis M

    2013-04-01

    Entry of enveloped viruses requires fusion of viral and cellular membranes. Fusion requires the formation of an intermediate stalk structure, in which only the outer leaflets are fused. The stalk structure, in turn, requires the lipid bilayer of the envelope to bend into negative curvature. This process is inhibited by enrichment in the outer leaflet of lipids with larger polar headgroups, which favor positive curvature. Accordingly, phospholipids with such shape inhibit viral fusion. We previously identified a compound, 5-(perylen-3-yl)ethynyl-2'-deoxy-uridine (dUY11), with overall shape and amphipathicity similar to those of these phospholipids. dUY11 inhibited the formation of the negative curvature necessary for stalk formation and the fusion of a model enveloped virus, vesicular stomatitis virus (VSV). We proposed that dUY11 acted by biophysical mechanisms as a result of its shape and amphipathicity. To test this model, we have now characterized the mechanisms against influenza virus and HCV of 5-(perylen-3-yl)ethynyl-arabino-uridine (aUY11), which has shape and amphipathicity similar to those of dUY11 but contains an arabino-nucleoside. aUY11 interacted with envelope lipids to inhibit the infectivity of influenza virus, hepatitis C virus (HCV), herpes simplex virus 1 and 2 (HSV-1/2), and other enveloped viruses. It specifically inhibited the fusion of influenza virus, HCV, VSV, and even protein-free liposomes to cells. Furthermore, aUY11 inhibited the formation of negative curvature in model lipid bilayers. In summary, the arabino-derived aUY11 and the deoxy-derived dUY11 act by the same antiviral mechanisms against several enveloped but otherwise unrelated viruses. Therefore, chemically unrelated compounds of appropriate shape and amphipathicity target virion envelope lipids to inhibit formation of the negative curvature required for fusion, inhibiting infectivity by biophysical, not biochemical, mechanisms.

  15. Inner/Outer Nuclear Membrane Fusion in Nuclear Pore Assembly

    Science.gov (United States)

    Fichtman, Boris; Ramos, Corinne; Rasala, Beth; Harel, Amnon

    2010-01-01

    Nuclear pore complexes (NPCs) are large proteinaceous channels embedded in double nuclear membranes, which carry out nucleocytoplasmic exchange. The mechanism of nuclear pore assembly involves a unique challenge, as it requires creation of a long-lived membrane-lined channel connecting the inner and outer nuclear membranes. This stabilized membrane channel has little evolutionary precedent. Here we mapped inner/outer nuclear membrane fusion in NPC assembly biochemically by using novel assembly intermediates and membrane fusion inhibitors. Incubation of a Xenopus in vitro nuclear assembly system at 14°C revealed an early pore intermediate where nucleoporin subunits POM121 and the Nup107-160 complex were organized in a punctate pattern on the inner nuclear membrane. With time, this intermediate progressed to diffusion channel formation and finally to complete nuclear pore assembly. Correct channel formation was blocked by the hemifusion inhibitor lysophosphatidylcholine (LPC), but not if a complementary-shaped lipid, oleic acid (OA), was simultaneously added, as determined with a novel fluorescent dextran-quenching assay. Importantly, recruitment of the bulk of FG nucleoporins, characteristic of mature nuclear pores, was not observed before diffusion channel formation and was prevented by LPC or OA, but not by LPC+OA. These results map the crucial inner/outer nuclear membrane fusion event of NPC assembly downstream of POM121/Nup107-160 complex interaction and upstream or at the time of FG nucleoporin recruitment. PMID:20926687

  16. Inhibition of influenza A virus (H1N1 fusion by benzenesulfonamide derivatives targeting viral hemagglutinin.

    Directory of Open Access Journals (Sweden)

    Lei Zhu

    Full Text Available Hemagglutinin (HA of the influenza virus plays a crucial role in the early stage of the viral life cycle by binding to sialic acid on the surface of host epithelial cells and mediating fusion between virus envelope and endosome membrane for the release of viral genomes into the cytoplasm. To initiate virus fusion, endosome pH is lowered by acidification causing an irreversible conformational change of HA, which in turn results in a fusogenic HA. In this study, we describe characterization of an HA inhibitor of influenza H1N1 viruses, RO5464466. One-cycle time course study in MDCK cells showed that this compound acted at an early step of influenza virus replication. Results from HA-mediated hemolysis of chicken red blood cells and trypsin sensitivity assay of isolated HA clearly showed that RO5464466 targeted HA. In cell-based assays involving multiple rounds of virus infection and replication, RO5464466 inhibited an established influenza infection. The overall production of progeny viruses, as a result of the compound's inhibitory effect on fusion, was dramatically reduced by 8 log units when compared with a negative control. Furthermore, RO5487624, a close analogue of RO5464466, with pharmacokinetic properties suitable for in vivo efficacy studies displayed a protective effect on mice that were lethally challenged with influenza H1N1 virus. These results might benefit further characterization and development of novel anti-influenza agents by targeting viral hemagglutinin.

  17. Ecotropic murine leukemia virus-induced fusion of murine cells

    Energy Technology Data Exchange (ETDEWEB)

    Pinter, A.; Chen, T.; Lowy, A.; Cortez, N.G.; Silagi, S.

    1986-03-01

    Extensive fusion occurs upon cocultivation of murine fibroblasts producing ecotropic murine leukemia viruses (MuLVs) with a large variety of murine cell lines in the presence of the polyene antibiotic amphotericin B, the active component of the antifungal agent Fungizone. The resulting polykaryocytes contain nuclei from both infected and uninfected cells, as evidenced by autoradiographic labeling experiments in which one or the other parent cell type was separately labeled with (/sup 3/H)thymidine and fused with an unlabeled parent. This cell fusion specifically requires the presence of an ecotropic MuLV-producing parent and is not observed for cells producing xenotropic, amphotropic, or dualtropic viruses. Mouse cells infected with nonecotropic viruses retain their sensitivity toward fusion, whereas infection with ecotropic viruses abrogates the fusion of these cells upon cocultivation with other ecotropic MuLV-producing cells. Nonmurine cells lacking the ecotropic gp70 receptor are not fused under similar conditions. Fusion is effectively inhibited by monospecific antisera to gp70, but not by antisera to p15(E), and studies with monoclonal antibodies identify distinct amino- and carboxy-terminal gp70 regions which play a role in the fusion reaction. The enhanced fusion which occurs in the presence of amphotericin B provides a rapid and sensitive assay for the expression of ecotropic MuLVs and should facilitate further mechanistic studies of MuLV-induced fusion of murine cells.

  18. Mouse C2 myoblast cells resist HVJ (Sendai virus)-mediated cell fusion in the proliferating stage but become capable of fusion after differentiation.

    Science.gov (United States)

    Hirayama, E; Nakanishi, M; Honda, N; Kim, J

    1999-05-01

    To investigate the mechanism of myoblast fusion, we attempted to prepare artificial myotubes of mouse C2 myoblast cells using the hemagglutinating virus of Japan (HVJ, Sendai virus). Proliferating C2 cells showed strong resistance to HVJ-mediated cell fusion and remained morphologically unchanged even though massive numbers of virions adsorbed onto their surface. They showed no membrane disruption, which occurs in the early stage of cell fusion induced by HVJ. These observations suggest that proliferating C2 cells are resistant to HVJ-mediated cell fusion. However, upon induction of differentiation, C2 cells gradually became capable of fusion induced by HVJ and then even generated heterokaryons with Ehrlich ascites tumor cells. When differentiated C2 cells that had become fusion-sensitive were treated with HVJ in the presence of EDTA, they did not fuse but degenerated, suggesting that their cell membranes were transiently disrupted by interaction with HVJ. These results suggest that the cell membranes of myoblasts change to a fusion-capable state during the process of differentiation.

  19. Dependence of herpes simplex virus type 1-induced cell fusion on cell type

    Energy Technology Data Exchange (ETDEWEB)

    Bzik, D.J.; Person, S.

    1981-04-15

    Syncytial mutants of herpes simplex virus type 1 (HSV-1), such as syn20, cause extensive fusion of human embryonic lung (HEL) cells but only a small amount of fusion of human epidermoid carcinoma No. 2 (HEp-2) cells. In order to determine the cellular basis of this difference in fusion, sparse cultures of syn20-infected HEL or HEp-2 cells, previously labeled with (/sup 3/H)thymidine, were surrounded with uninfected, unlabeled HEL or HEp-2 cells. The fusion of radioactive with nonradioactive cells was determined at different times after infection using radioautography. The major difference in the fusion capacity of HEL and HEp-2 cells was not due to a difference in cell-surface receptors for a fusion factor in the two cell types. The process of infection of HEp-2 cells did not cause the plasma membranes of the cells to become refractory to fusion, because syn20-infected HEL cells fused equally well with either uninfected or infected HEp-2 cells. In a mixed infection with equal numbers of MP and its nonsyncytial parent, mP, extensive fusion was observed for infected HEL cells and significantly less fusion was observed for infected African green monkey (CV-1), baby hamster kidney (BHK-21), and HEp-2 cells.

  20. Intra- and interspecies virus transfer in Aspergilli via protoplast fusion

    NARCIS (Netherlands)

    van Diepeningen, A D; Debets, A J; Hoekstra, R F

    1998-01-01

    Intra- and interspecies transfer of dsRNA viruses between black Aspergilli and Aspergillus nidulans strains has been investigated using protoplast fusion. We found interspecies transfer of virus in all combinations of black Aspergillus and A. nidulans strains and vice versa. Using the same

  1. Intra- and interspecies virus transfer in Aspergilli via protoplast fusion.

    NARCIS (Netherlands)

    Diepeningen, van A.D.; Debets, A.J.M.; Hoekstra, R.F.

    1998-01-01

    Intra- and interspecies transfer of dsRNA viruses between black Aspergilli and Aspergillus nidulans strains has been investigated using protoplast fusion. We found interspecies transfer of virus in all combinations of black Aspergillus and A. nidulans strains and vice versa. Using the same

  2. Slow fusion of liposomes composed of membrane-spanning lipids

    NARCIS (Netherlands)

    Elferink, MGL; vanBreemen, J; Konings, WN; Driessen, AJM; Wilschut, J; Elferink, Marieke G.L.

    1997-01-01

    The fusion characteristics of large unilamellar liposomes composed of bipolar tetraether lipids extracted from the thermophilic archaeon Sulfolobus acidocaldarius, was investigated. These lipids span the entire membrane and form single monolayer liposomes in aqueous media [Elferink, M.G.L., de Wit,

  3. Membrane remodelling during vaccinia virus morphogenesis.

    Science.gov (United States)

    Chichón, Francisco Javier; Rodríguez, María Josefa; Risco, Cristina; Fraile-Ramos, Alberto; Fernández, José Jesús; Esteban, Mariano; Carrascosa, José L

    2009-07-01

    VACV (vaccinia virus) is one of the most complex viruses, with a size exceeding 300 nm and more than 100 structural proteins. Its assembly involves sequential interactions and important rearrangements of its structural components. We have used electron tomography of sections of VACV-infected cells to follow, in three dimensions, the remodelling of the membrane components of the virus during envelope maturation. The tomograms obtained suggest that a number of independent 'crescents' interact with each other to enclose the volume of an incomplete ellipsoid in the viral factory area, attaining the overall shape and size characteristic of the first immature form of the virus [IV (immature virus)]. The incorporation of the DNA into these forms leads to particles with a nucleoid [IVN (IV with nucleoid)] that results in local disorganization of the envelope in regions near the condensed DNA. These particles suffer the progressive disappearance of the membrane outer spikes with a change in the shape of the membrane, becoming locally curled. The transformation of the IVN into the mature virus involves an extreme rearrangement of the particle envelope, which becomes fragmented and undulated. During this process, we also observed connections between the outer membranes with internal ones, suggesting that the latter originate from internalization of the IV envelope. The main features observed for VACV membrane maturation during morphogenesis resemble the breakdown and reassembly of cellular endomembranes.

  4. Polyclonal and monoclonal antibodies specific for the six-helix bundle of the human respiratory syncytial virus fusion glycoprotein as probes of the protein post-fusion conformation

    Energy Technology Data Exchange (ETDEWEB)

    Palomo, Concepción; Mas, Vicente; Vázquez, Mónica; Cano, Olga [Unidad de Biología Viral, Centro Nacional de Microbiología, Madrid (Spain); CIBER de Enfermedades Respiratorias, Instituto de Salud Carlos III, Majadahonda, 28220 Madrid (Spain); Luque, Daniel; Terrón, María C. [Unidad de Microscopía Electrónica y Confocal, Centro Nacional de Microbiología, Instituto de Salud Carlos III, Majadahonda, 28220 Madrid (Spain); Calder, Lesley J. [National Institute for Medical Research, MRC, Mill Hill, London NW7 1AA (United Kingdom); Melero, José A., E-mail: jmelero@isciii.es [Unidad de Biología Viral, Centro Nacional de Microbiología, Madrid (Spain); CIBER de Enfermedades Respiratorias, Instituto de Salud Carlos III, Majadahonda, 28220 Madrid (Spain)

    2014-07-15

    Human respiratory syncytial virus (hRSV) has two major surface glycoproteins (G and F) anchored in the lipid envelope. Membrane fusion promoted by hRSV{sub F} occurs via refolding from a pre-fusion form to a highly stable post-fusion state involving large conformational changes of the F trimer. One of these changes results in assembly of two heptad repeat sequences (HRA and HRB) into a six-helix bundle (6HB) motif. To assist in distinguishing pre- and post-fusion conformations of hRSV{sub F}, we have prepared polyclonal (α-6HB) and monoclonal (R145) rabbit antibodies specific for the 6HB. Among other applications, these antibodies were used to explore the requirements of 6HB formation by isolated protein segments or peptides and by truncated mutants of the F protein. Site-directed mutagenesis and electron microscopy located the R145 epitope in the post-fusion hRSV{sub F} at a site distantly located from previously mapped epitopes, extending the repertoire of antibodies that can decorate the F molecule. - Highlights: • Antibodies specific for post-fusion respiratory syncytial virus fusion protein are described. • Polyclonal antibodies were obtained in rabbit inoculated with chimeric heptad repeats. • Antibody binding required assembly of a six-helix bundle in the post-fusion protein. • A monoclonal antibody with similar structural requirements is also described. • Binding of this antibody to the post-fusion protein was visualized by electron microscopy.

  5. Shear-Induced Membrane Fusion in Viscous Solutions

    KAUST Repository

    Kogan, Maxim

    2014-05-06

    Large unilamellar lipid vesicles do not normally fuse under fluid shear stress. They might deform and open pores to relax the tension to which they are exposed, but membrane fusion occurring solely due to shear stress has not yet been reported. We present evidence that shear forces in a viscous solution can induce lipid bilayer fusion. The fusion of 1,2-dioleoyl-sn-glycero-3- phosphocholine (DOPC) liposomes is observed in Couette flow with shear rates above 3000 s-1 provided that the medium is viscous enough. Liposome samples, prepared at different viscosities using a 0-50 wt % range of sucrose concentration, were studied by dynamic light scattering, lipid fusion assays using Förster resonance energy transfer (FRET), and linear dichroism (LD) spectroscopy. Liposomes in solutions with 40 wt % (or more) sucrose showed lipid fusion under shear forces. These results support the hypothesis that under suitable conditions lipid membranes may fuse in response to mechanical-force- induced stress. © 2014 American Chemical Society.

  6. Membrane-on-a-chip: microstructured silicon/silicon-dioxide chips for high-throughput screening of membrane transport and viral membrane fusion.

    Science.gov (United States)

    Kusters, Ilja; van Oijen, Antoine M; Driessen, Arnold J M

    2014-04-22

    Screening of transport processes across biological membranes is hindered by the challenge to establish fragile supported lipid bilayers and the difficulty to determine at which side of the membrane reactants reside. Here, we present a method for the generation of suspended lipid bilayers with physiological relevant lipid compositions on microstructured Si/SiO2 chips that allow for high-throughput screening of both membrane transport and viral membrane fusion. Simultaneous observation of hundreds of single-membrane channels yields statistical information revealing population heterogeneities of the pore assembly and conductance of the bacterial toxin α-hemolysin (αHL). The influence of lipid composition and ionic strength on αHL pore formation was investigated at the single-channel level, resolving features of the pore-assembly pathway. Pore formation is inhibited by a specific antibody, demonstrating the applicability of the platform for drug screening of bacterial toxins and cell-penetrating agents. Furthermore, fusion of H3N2 influenza viruses with suspended lipid bilayers can be observed directly using a specialized chip architecture. The presented micropore arrays are compatible with fluorescence readout from below using an air objective, thus allowing high-throughput screening of membrane transport in multiwell formats in analogy to plate readers.

  7. A mechanistic paradigm for broad-spectrum antivirals that target virus-cell fusion.

    Directory of Open Access Journals (Sweden)

    Frederic Vigant

    Full Text Available LJ001 is a lipophilic thiazolidine derivative that inhibits the entry of numerous enveloped viruses at non-cytotoxic concentrations (IC50 ≤ 0.5 µM, and was posited to exploit the physiological difference between static viral membranes and biogenic cellular membranes. We now report on the molecular mechanism that results in LJ001's specific inhibition of virus-cell fusion. The antiviral activity of LJ001 was light-dependent, required the presence of molecular oxygen, and was reversed by singlet oxygen ((1O2 quenchers, qualifying LJ001 as a type II photosensitizer. Unsaturated phospholipids were the main target modified by LJ001-generated (1O2. Hydroxylated fatty acid species were detected in model and viral membranes treated with LJ001, but not its inactive molecular analog, LJ025. (1O2-mediated allylic hydroxylation of unsaturated phospholipids leads to a trans-isomerization of the double bond and concurrent formation of a hydroxyl group in the middle of the hydrophobic lipid bilayer. LJ001-induced (1O2-mediated lipid oxidation negatively impacts on the biophysical properties of viral membranes (membrane curvature and fluidity critical for productive virus-cell membrane fusion. LJ001 did not mediate any apparent damage on biogenic cellular membranes, likely due to multiple endogenous cytoprotection mechanisms against phospholipid hydroperoxides. Based on our understanding of LJ001's mechanism of action, we designed a new class of membrane-intercalating photosensitizers to overcome LJ001's limitations for use as an in vivo antiviral agent. Structure activity relationship (SAR studies led to a novel class of compounds (oxazolidine-2,4-dithiones with (1 100-fold improved in vitro potency (IC50<10 nM, (2 red-shifted absorption spectra (for better tissue penetration, (3 increased quantum yield (efficiency of (1O2 generation, and (4 10-100-fold improved bioavailability. Candidate compounds in our new series moderately but significantly (p≤0

  8. Towards understanding of Nipah virus attachment protein assembly and the role of protein affinity and crowding for membrane curvature events.

    Energy Technology Data Exchange (ETDEWEB)

    Stachowiak, Jeanne C.; Hayden, Carl C.; Negrete, Oscar.; Davis, Ryan Wesley; Sasaki, Darryl Y

    2013-10-01

    Pathogenic viruses are a primary threat to our national security and to the health and economy of our world. Effective defense strategies to combat viral infection and spread require the development of understanding of the mechanisms that these pathogens use to invade the host cell. We present in this report results of our research into viral particle recognition and fusion to cell membranes and the role that protein affinity and confinement in lipid domains plays in membrane curvature in cellular fusion and fission events. Herein, we describe 1) the assembly of the G attachment protein of Nipah virus using point mutation studies to define its role in viral particle fusion to the cell membrane, 2) how lateral pressure of membrane bound proteins induce curvature in model membrane systems, and 3) the role of membrane curvature in the selective partitioning of molecular receptors and specific affinity of associated proteins.

  9. The SARS-CoV Fusion Peptide Forms an Extended Bipartite Fusion Platform that Perturbs Membrane Order in a Calcium-Dependent Manner.

    Science.gov (United States)

    Lai, Alex L; Millet, Jean K; Daniel, Susan; Freed, Jack H; Whittaker, Gary R

    2017-12-08

    Coronaviruses (CoVs) are a major infectious disease threat and include the pathogenic human pathogens of zoonotic origin: severe acute respiratory syndrome CoV (SARS-CoV) and Middle East respiratory syndrome CoV (MERS-CoV). Entry of CoVs into host cells is mediated by the viral spike (S) protein, which is structurally categorized as a class I viral fusion protein, within the same group as influenza virus and HIV. However, S proteins have two distinct cleavage sites that can be activated by a much wider range of proteases. The exact location of the CoV fusion peptide (FP) has been disputed. However, most evidence suggests that the domain immediately downstream of the S2' cleavage site is the FP (amino acids 798-818 SFIEDLLFNKVTLADAGFMKQY for SARS-CoV, FP1). In our previous electron spin resonance spectroscopic studies, the membrane-ordering effect of influenza virus, HIV, and Dengue virus FPs has been consistently observed. In this study, we used this effect as a criterion to identify and characterize the bona fide SARS-CoV FP. Our results indicate that both FP1 and the region immediately downstream (amino acids 816-835 KQYGECLGDINARDLICAQKF, FP2) induce significant membrane ordering. Furthermore, their effects are calcium dependent, which is consistent with in vivo data showing that calcium is required for SARS-CoV S-mediated fusion. Isothermal titration calorimetry showed a direct interaction between calcium cations and both FPs. This Ca2+-dependency membrane ordering was not observed with influenza FP, indicating that the CoV FP exhibits a mechanistically different behavior. Membrane-ordering effects are greater and penetrate deeper into membranes when FP1 and FP2 act in a concerted manner, suggesting that they form an extended fusion "platform." Copyright © 2017 Elsevier Ltd. All rights reserved.

  10. Therapeutic efficacy of a respiratory syncytial virus fusion inhibitor.

    Science.gov (United States)

    Roymans, Dirk; Alnajjar, Sarhad S; Battles, Michael B; Sitthicharoenchai, Panchan; Furmanova-Hollenstein, Polina; Rigaux, Peter; Berg, Joke Van den; Kwanten, Leen; Ginderen, Marcia Van; Verheyen, Nick; Vranckx, Luc; Jaensch, Steffen; Arnoult, Eric; Voorzaat, Richard; Gallup, Jack M; Larios-Mora, Alejandro; Crabbe, Marjolein; Huntjens, Dymphy; Raboisson, Pierre; Langedijk, Johannes P; Ackermann, Mark R; McLellan, Jason S; Vendeville, Sandrine; Koul, Anil

    2017-08-01

    Respiratory syncytial virus is a major cause of acute lower respiratory tract infection in young children, immunocompromised adults, and the elderly. Intervention with small-molecule antivirals specific for respiratory syncytial virus presents an important therapeutic opportunity, but no such compounds are approved today. Here we report the structure of JNJ-53718678 bound to respiratory syncytial virus fusion (F) protein in its prefusion conformation, and we show that the potent nanomolar activity of JNJ-53718678, as well as the preliminary structure-activity relationship and the pharmaceutical optimization strategy of the series, are consistent with the binding mode of JNJ-53718678 and other respiratory syncytial virus fusion inhibitors. Oral treatment of neonatal lambs with JNJ-53718678, or with an equally active close analog, efficiently inhibits established acute lower respiratory tract infection in the animals, even when treatment is delayed until external signs of respiratory syncytial virus illness have become visible. Together, these data suggest that JNJ-53718678 is a promising candidate for further development as a potential therapeutic in patients at risk to develop respiratory syncytial virus acute lower respiratory tract infection.Respiratory syncytial virus causes lung infections in children, immunocompromised adults, and in the elderly. Here the authors show that a chemical inhibitor to a viral fusion protein is effective in reducing viral titre and ameliorating infection in rodents and neonatal lambs.

  11. Sialic Acids on Varicella-Zoster Virus Glycoprotein B Are Required for Cell-Cell Fusion.

    Science.gov (United States)

    Suenaga, Tadahiro; Matsumoto, Maki; Arisawa, Fuminori; Kohyama, Masako; Hirayasu, Kouyuki; Mori, Yasuko; Arase, Hisashi

    2015-08-07

    Varicella-zoster virus (VZV) is a member of the human Herpesvirus family that causes varicella (chicken pox) and zoster (shingles). VZV latently infects sensory ganglia and is also responsible for encephalomyelitis. Myelin-associated glycoprotein (MAG), a member of the sialic acid (SA)-binding immunoglobulin-like lectin family, is mainly expressed in neural tissues. VZV glycoprotein B (gB) associates with MAG and mediates membrane fusion during VZV entry into host cells. The SA requirements of MAG when associating with its ligands vary depending on the specific ligand, but it is unclear whether the SAs on gB are involved in the association with MAG. In this study, we found that SAs on gB are essential for the association with MAG as well as for membrane fusion during VZV infection. MAG with a point mutation in the SA-binding site did not bind to gB and did not mediate cell-cell fusion or VZV entry. Cell-cell fusion and VZV entry mediated by the gB-MAG interaction were blocked by sialidase treatment. N-glycosylation or O-glycosylation inhibitors also inhibited the fusion and entry mediated by gB-MAG interaction. Furthermore, gB with mutations in N-glycosylation sites, i.e. asparagine residues 557 and 686, did not associate with MAG, and the cell-cell fusion efficiency was low. Fusion between the viral envelope and cellular membrane is essential for host cell entry by herpesviruses. Therefore, these results suggest that SAs on gB play important roles in MAG-mediated VZV infection. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  12. Mechanisms of virus assembly on membranes

    Science.gov (United States)

    Lazaro, Guillermo; Hagan, Michael

    We present a computational model motivated by icosahedral enveloped viruses, which consist of nucleocapsid (a protein shell encasing the genome) and an outer envelope composed of a lipid membrane and transmembrane glycoproteins. Viruses acquire their envelope by budding through a host cell membrane. Despite extensive experimental efforts, it remains an open question whether the nucleocapsid is necessary for budding (nucleocapsid-driven assembly), or whether interactions between glycoproteins are sufficient to simultaneously drive membrane deformation and assembly of an icosahedral structure (glycoprotein-driven assembly). To study this question, we use a coarse-grained computational model for the nucleocapsid, glycoproteins, and the membrane. Our simulations demonstrate that glycoproteins alone are sufficient to drive budding; however, barriers due to membrane elasticity can lead to malformed capsids lacking icosahedral symmetry. In contrast, with a nucleocapsid present, icosahedral structures form over a broad range of parameter values. Our simulations also identify a key role for glycoprotein geometry in reshaping the membrane and avoiding membrane deformations that frustrate assembly. Supported by the NIH (R01GM108021) and NSF Brandeis MRSEC (DMR-1420382).

  13. Redistribution of Endosomal Membranes to the African Swine Fever Virus Replication Site

    Directory of Open Access Journals (Sweden)

    Miguel Ángel Cuesta-Geijo

    2017-06-01

    Full Text Available African swine fever virus (ASFV infection causes endosomal reorganization. Here, we show that the virus causes endosomal congregation close to the nucleus as the infection progresses, which is necessary to build a compact viral replication organelle. ASFV enters the cell by the endosomal pathway and reaches multivesicular late endosomes. Upon uncoating and fusion, the virus should exit to the cytosol to start replication. ASFV remodels endosomal traffic and redistributes endosomal membranes to the viral replication site. Virus replication also depends on endosomal membrane phosphoinositides (PtdIns synthesized by PIKfyve. Endosomes could act as platforms providing membranes and PtdIns, necessary for ASFV replication. Our study has revealed that ASFV reorganizes endosome dynamics, in order to ensure a productive infection.

  14. Relative contributions of measles virus hemagglutinin- and fusion protein- specific serum antibodies to virus neutralization.

    NARCIS (Netherlands)

    R.L. de Swart (Rik); S. Yüksel (Selma); A.D.M.E. Osterhaus (Albert)

    2005-01-01

    textabstractThe relative contribution of measles virus hemagglutinin (H)- or fusion protein (F)-specific antibodies to virus neutralization (VN) has not been demonstrated. We have depleted these specific antibodies from sera collected from young adults, who had been vaccinated during childhood, by

  15. 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.

  16. In vitro assay using engineered yeast vacuoles for neuronal SNARE-mediated membrane fusion

    Science.gov (United States)

    Ko, Young-Joon; Lee, Miriam; Kang, KyeongJin; Song, Woo Keun; Jun, Youngsoo

    2014-01-01

    Intracellular membrane fusion requires not only SNARE proteins but also other regulatory proteins such as the Rab and Sec1/Munc18 (SM) family proteins. Although neuronal SNARE proteins alone can drive the fusion between synthetic liposomes, it remains unclear whether they are also sufficient to induce the fusion of biological membranes. Here, through the use of engineered yeast vacuoles bearing neuronal SNARE proteins, we show that neuronal SNAREs can induce membrane fusion between yeast vacuoles and that this fusion does not require the function of the Rab protein Ypt7p or the SM family protein Vps33p, both of which are essential for normal yeast vacuole fusion. Although excess vacuolar SNARE proteins were also shown to mediate Rab-bypass fusion, this fusion required homotypic fusion and vacuole protein sorting complex, which bears Vps33p and was accompanied by extensive membrane lysis. We also show that this neuronal SNARE-driven vacuole fusion can be stimulated by the neuronal SM protein Munc18 and blocked by botulinum neurotoxin serotype E, a well-known inhibitor of synaptic vesicle fusion. Taken together, our results suggest that neuronal SNARE proteins are sufficient to induce biological membrane fusion, and that this new assay can be used as a simple and complementary method for investigating synaptic vesicle fusion mechanisms. PMID:24821814

  17. Point-like protrusion as a prestalk intermediate in membrane fusion pathway.

    Science.gov (United States)

    Efrat, Avishay; Chernomordik, Leonid V; Kozlov, Michael M

    2007-04-15

    The widely accepted pathway of membrane fusion begins with the fusion stalk representing the initial intermediate of hemifusion. The lipid structures preceding hemifusion and their possible influence on fusion kinetics were not addressed. Here, we suggest the point-like protrusion as a prestalk fusion intermediate, which has energy lower than that of stalk and, therefore, does not limit the fusion rate. We demonstrate that by calculating the energy of the point-like protrusion, which depends on the lipid monolayer elastic parameters and the strength of the intermembrane hydration repulsion. The point-like protrusion completes the fusion-through-hemifusion model of membrane merger.

  18. Point-Like Protrusion as a Prestalk Intermediate in Membrane Fusion Pathway

    OpenAIRE

    Efrat, Avishay; Chernomordik, Leonid V.; Kozlov, Michael M.

    2007-01-01

    The widely accepted pathway of membrane fusion begins with the fusion stalk representing the initial intermediate of hemifusion. The lipid structures preceding hemifusion and their possible influence on fusion kinetics were not addressed. Here, we suggest the point-like protrusion as a prestalk fusion intermediate, which has energy lower than that of stalk and, therefore, does not limit the fusion rate. We demonstrate that by calculating the energy of the point-like protrusion, which depends ...

  19. New influenza A Virus Entry Inhibitors Derived from the Viral Fusion Peptides.

    Directory of Open Access Journals (Sweden)

    Wenjiao Wu

    Full Text Available Influenza A viral (IAV fusion peptides are known for their important role in viral-cell fusion process and membrane destabilization potential which are compatible with those of antimicrobial peptides. Thus, by replacing the negatively or neutrally charged residues of FPs with positively charged lysines, we synthesized several potent antimicrobial peptides derived from the fusogenic peptides (FPs of hemagglutinin glycoproteins (HAs of IAV. The biological screening identified that in addition to the potent antibacterial activities, these positively charged fusion peptides (pFPs effectively inhibited the replication of influenza A viruses including oseltamivir-resistant strain. By employing pseudovirus-based entry inhibition assays including H5N1 influenza A virus (IAV, and VSV-G, the mechanism study indicated that the antiviral activity may be associated with the interactions between the HA2 subunit and pFP, of which, the nascent pFP exerted a strong effect to interrupt the conformational changes of HA2, thereby blocking the entry of viruses into host cells. In addition to providing new peptide "entry blockers", these data also demonstrate a useful strategy in designing potent antibacterial agents, as well as effective viral entry inhibitors. It would be meaningful in treatment of bacterial co-infection during influenza pandemic periods, as well as in our current war against those emerging pathogenic microorganisms such as IAV and HIV.

  20. Human keratinocytes restrict chikungunya virus replication at a post-fusion step

    Energy Technology Data Exchange (ETDEWEB)

    Bernard, Eric [Centre d' étude d’agents Pathogènes et Biotechnologies pour la Santé, CPBS CNRS- UMR5236/UM1/UM2, Montpellier (France); Hamel, Rodolphe [Laboratoire Maladies Infectieuses et Vecteurs: Ecologie, Génétique, Evolution, Contrôle, UMR 5290 CNRS/IRD/UM1, Montpellier (France); Neyret, Aymeric [Centre d' étude d’agents Pathogènes et Biotechnologies pour la Santé, CPBS CNRS- UMR5236/UM1/UM2, Montpellier (France); Ekchariyawat, Peeraya [Laboratoire Maladies Infectieuses et Vecteurs: Ecologie, Génétique, Evolution, Contrôle, UMR 5290 CNRS/IRD/UM1, Montpellier (France); Molès, Jean-Pierre [INSERM U1058, UM1, CHU Montpellier (France); Simmons, Graham [Blood Systems Research Institute, San Francisco, CA 94118 (United States); Chazal, Nathalie [Centre d' étude d’agents Pathogènes et Biotechnologies pour la Santé, CPBS CNRS- UMR5236/UM1/UM2, Montpellier (France); Desprès, Philippe [Unité Interactions Moléculaires Flavivirus-Hôtes, Institut Pasteur, Paris (France); and others

    2015-02-15

    Transmission of chikungunya virus (CHIKV) to humans is initiated by puncture of the skin by a blood-feeding Aedes mosquito. Despite the growing knowledge accumulated on CHIKV, the interplay between skin cells and CHIKV following inoculation still remains unclear. In this study we questioned the behavior of human keratinocytes, the predominant cell population in the skin, following viral challenge. We report that CHIKV rapidly elicits an innate immune response in these cells leading to the enhanced transcription of type I/II and type III interferon genes. Concomitantly, we show that despite viral particles internalization into Rab5-positive endosomes and efficient fusion of virus and cell membranes, keratinocytes poorly replicate CHIKV as attested by absence of nonstructural proteins and genomic RNA synthesis. Accordingly, human keratinocytes behave as an antiviral defense against CHIKV infection rather than as a primary targets for initial replication. This picture significantly differs from that reported for Dengue and West Nile mosquito-borne viruses. - Highlights: • Human keratinocytes support endocytosis of CHIKV and fusion of viral membranes. • CHIKV replication is blocked at a post entry step in these cells. • Infection upregulates type-I, –II and –III IFN genes expression. • Keratinocytes behave as immune sentinels against CHIKV.

  1. Specific Movement of Cell Membranes Fused with HVJ (Sendai Virus)

    Science.gov (United States)

    Okada, Yoshio; Kim, Jeman; Maeda, Yumiko; Koseki, Ignez

    1974-01-01

    When Ehrlich ascites tumor cells fused with UV-inactivated HVJ (Sendai virus) were cultured, the viral envelope antigens, which had been integrated at random into the cell membranes, accumulated at one site on the fused cell. The site showed a specific structure, namely, a projection of the cytoplasm from the spherical fused cell surface with clusters of many long microvillilike protrusions from it. When the fused cells were conjugated with antibody against Ehrlich ascites tumor cells and cultured, the antigen-antibody complex also accumulated on the projection. Associated with the development of the projection and accumulation of viral antigens, vacuoles developed at the base of the projection in the cytoplasm of the fused cells in culture. The development of the projection and vacuoles and the accumulation of viral antigens were all inhibited by glucose, mannose, galactose, sodium azide, and cycloheximide, and these inhibitions were reversible. All these structures were observed in multinucleated cells, but few in mononucleated cells after reaction with HVJ (Sendai virus). We consider that these three phenomena correlate with each other and may result from changes in cell-membrane systems caused by fusion of cells. Images PMID:4365583

  2. Respiratory Syncytial Virus Attachment Glycoprotein Contribution to Infection Depends on the Specific Fusion Protein.

    Science.gov (United States)

    Meng, Jia; Hotard, Anne L; Currier, Michael G; Lee, Sujin; Stobart, Christopher C; Moore, Martin L

    2015-10-14

    Human respiratory syncytial virus (RSV) is an important pathogen causing acute lower respiratory tract disease in children. The RSV attachment glycoprotein (G) is not required for infection, as G-null RSV replicates efficiently in several cell lines. Our laboratory previously reported that the viral fusion (F) protein is a determinant of strain-dependent pathogenesis. Here, we hypothesized that virus dependence on G is determined by the strain specificity of F. We generated recombinant viruses expressing G and F, or null for G, from the laboratory A2 strain (Katushka RSV-A2GA2F [kRSV-A2GA2F] and kRSV-GstopA2F) or the clinical isolate A2001/2-20 (kRSV-2-20G2-20F and kRSV-Gstop2-20F). We quantified the virus cell binding, entry kinetics, infectivity, and growth kinetics of these four recombinant viruses in vitro. RSV expressing the 2-20 G protein exhibited the greatest binding activity. Compared to the parental viruses expressing G and F, removal of 2-20 G had more deleterious effects on binding, entry, infectivity, and growth than removal of A2 G. Overall, RSV expressing 2-20 F had a high dependence on G for binding, entry, and infection. RSV is the leading cause of childhood acute respiratory disease requiring hospitalization. As with other paramyxoviruses, two major RSV surface viral glycoproteins, the G attachment protein and the F fusion protein, mediate virus binding and subsequent membrane fusion, respectively. Previous work on the RSV A2 prototypical strain demonstrated that the G protein is functionally dispensable for in vitro replication. This is in contrast to other paramyxoviruses that require attachment protein function as a prerequisite for fusion. We reevaluated this requirement for RSV using G and F proteins from clinical isolate 2-20. Compared to the laboratory A2 strain, the G protein from 2-20 had greater contributions to virus binding, entry, infectivity, and in vitro growth kinetics. Thus, the clinical isolate 2-20 F protein function depended

  3. Inhibition of EBV-mediated membrane fusion by anti-gHgL antibodies

    Energy Technology Data Exchange (ETDEWEB)

    Sathiyamoorthy, Karthik; Jiang, Jiansen; Möhl, Britta S.; Chen, Jia; Zhou, Z. Hong; Longnecker, Richard; Jardetzky, Theodore S. (UCLA); (Stanford-MED); (NWU)

    2017-09-22

    Herpesvirus entry into cells requires the coordinated action of multiple virus envelope glycoproteins, including gH, gL, and gB. For EBV, the gp42 protein assembles into complexes with gHgL heterodimers and binds HLA class II to activate gB-mediated membrane fusion with B cells. EBV tropism is dictated by gp42 levels in the virion, as it inhibits entry into epithelial cells while promoting entry into B cells. The gHgL and gB proteins are targets of neutralizing antibodies and potential candidates for subunit vaccine development, but our understanding of their neutralizing epitopes and the mechanisms of inhibition remain relatively unexplored. Here we studied the structures and mechanisms of two anti-gHgL antibodies, CL40 and CL59, that block membrane fusion with both B cells and epithelial cells. We determined the structures of the CL40 and CL59 complexes with gHgL using X-ray crystallography and EM to identify their epitope locations. CL59 binds to the C-terminal domain IV of gH, while CL40 binds to a site occupied by the gp42 receptor binding domain. CL40 binding to gHgL/gp42 complexes is not blocked by gp42 and does not interfere with gp42 binding to HLA class II, indicating that its ability to block membrane fusion with B cells represents a defect in gB activation. These data indicate that anti-gHgL neutralizing antibodies can block gHgL-mediated activation of gB through different surface epitopes and mechanisms.

  4. Role for the αV Integrin Subunit in Varicella-Zoster Virus-Mediated Fusion and Infection.

    Science.gov (United States)

    Yang, Edward; Arvin, Ann M; Oliver, Stefan L

    2016-08-15

    Varicella-zoster virus (VZV) is an alphaherpesvirus that causes varicella and herpes zoster. Membrane fusion is essential for VZV entry and the distinctive syncytium formation in VZV-infected skin and neuronal tissue. Herpesvirus fusion is mediated by a complex of glycoproteins gB and gH-gL, which are necessary and sufficient for VZV to induce membrane fusion. However, the cellular requirements of fusion are poorly understood. Integrins have been implicated to facilitate entry of several human herpesviruses, but their role in VZV entry has not yet been explored. To determine the involvement of integrins in VZV fusion, a quantitative cell-cell fusion assay was developed using a VZV-permissive melanoma cell line. The cells constitutively expressed a reporter protein and short hairpin RNAs (shRNAs) to knock down the expression of integrin subunits shown to be expressed in these cells by RNA sequencing. The αV integrin subunit was identified as mediating VZV gB/gH-gL fusion, as its knockdown by shRNAs reduced fusion levels to 60% of that of control cells. A comparable reduction in fusion levels was observed when an anti-αV antibody specific to its extracellular domain was tested in the fusion assay, confirming that the domain was important for VZV fusion. In addition, reduced spread was observed in αV knockdown cells infected with the VZV pOka strain relative to that of the control cells. This was demonstrated by reductions in plaque size, replication kinetics, and virion entry in the αV subunit knockdown cells. Thus, the αV integrin subunit is important for VZV gB/gH-gL fusion and infection. Varicella-zoster virus (VZV) is a highly infectious pathogen that causes chickenpox and shingles. A common complication of shingles is the excruciating condition called postherpetic neuralgia, which has proven difficult to treat. While a vaccine is now available, it is not recommended for immunocompromised individuals and its efficacy decreases with the recipient's age. These

  5. FUSION OF ARTIFICIAL MEMBRANES WITH MAMMALIAN SPERMATOZOA - SPECIFIC INVOLVEMENT OF THE EQUATORIAL SEGMENT AFTER ACROSOME REACTION

    NARCIS (Netherlands)

    ARTS, EGJM; KUIKEN, J; JAGER, S; HOEKSTRA, D

    1993-01-01

    The fusogenic properties of bovine and human spermatozoa membranes were investigated, using phospholipid bilayers (liposomes) as target membranes. Fusion was monitored by following lipid mixing, as revealed by an assay based on resonance-energy transfer. In addition, fusion was visualized by

  6. Enhanced Membrane Fusion in Sterol-enriched Vacuoles Bypasses the Vrp1p RequirementD⃞

    OpenAIRE

    Tedrick, Kelly; Trischuk, Tim; Lehner, Richard; Eitzen, Gary

    2004-01-01

    Organization of lipids into membrane microdomains is a vital mechanism of protein processing. Here we show that overexpression of ERG6, a gene involved in ergosterol synthesis, elevates sterol levels 1.5-fold on the vacuole membrane and enhances their homotypic fusion. The mechanism of sterol-enhanced fusion is not via more efficient sorting, but instead promotes increased kinetics of fusion subreactions. We initially isolated ERG6 as a suppressor of a vrp1Δ growth defect selective for vacuol...

  7. Progress in the Identification of Dengue Virus Entry/Fusion Inhibitors

    Directory of Open Access Journals (Sweden)

    Carolina De La Guardia

    2014-01-01

    Full Text Available Dengue fever, a reemerging disease, is putting nearly 2.5 billion people at risk worldwide. The number of infections and the geographic extension of dengue fever infection have increased in the past decade. The disease is caused by the dengue virus, a flavivirus that uses mosquitos Aedes sp. as vectors. The disease has several clinical manifestations, from the mild cold-like illness to the more serious hemorrhagic dengue fever and dengue shock syndrome. Currently, there is no approved drug for the treatment of dengue disease or an effective vaccine to fight the virus. Therefore, the search for antivirals against dengue virus is an active field of research. As new possible receptors and biological pathways of the virus biology are discovered, new strategies are being undertaken to identify possible antiviral molecules. Several groups of researchers have targeted the initial step in the infection as a potential approach to interfere with the virus. The viral entry process is mediated by viral proteins and cellular receptor molecules that end up in the endocytosis of the virion, the fusion of both membranes, and the release of viral RNA in the cytoplasm. This review provides an overview of the targets and progress that has been made in the quest for dengue virus entry inhibitors.

  8. Kinetics of interaction of HIV fusion protein (gp41) with lipid membranes studied by real-time AFM imaging

    Energy Technology Data Exchange (ETDEWEB)

    Bitler, Arkady, E-mail: arkady.bitler@weizmann.ac.il [Department of Chemical Research Support (Israel); Lev, Naama; Fridmann-Sirkis, Yael; Blank, Lior [Department of Biological Chemistry, Weizmann Institute of Science, Rehovot 76100 (Israel); Cohen, Sidney R. [Department of Chemical Research Support (Israel); Shai, Yechiel [Department of Biological Chemistry, Weizmann Institute of Science, Rehovot 76100 (Israel)

    2010-05-15

    One of the most important steps in the process of viral infection is a fusion between cell membrane and virus, which is mediated by the viral envelope glycoprotein. The study of activity of the glycoprotein in the post-fusion state is important for understanding the progression of infection. Here we present a first real-time kinetic study of the activity of gp41 (the viral envelope glycoprotein of human immunodeficiency virus-HIV) and its two mutants in the post-fusion state with nanometer resolution by atomic force microscopy (AFM). Tracking the changes in the phosphatidylcholine (PC) and phosphatidylcholine-phosphatidylserine (PC:PS) membrane integrity over one hour by a set of AFM images revealed differences in the interaction of the three types of protein with zwitterionic and negatively charged membranes. A quantitative analysis of the slow kinetics of hole formation in the negatively charged lipid bilayer is presented. Specifically, analysis of the rate of roughness change for the three types of proteins suggests that they exhibit different types of kinetic behavior.

  9. Crystal Structure of the Conserved Herpes Virus Fusion Regulator Complex gH–gL

    Energy Technology Data Exchange (ETDEWEB)

    Chowdary, T.; Cairns, T; Atanasiu, D; Cohen, G; Eisenberg, R; Heldwein, E

    2010-01-01

    Herpesviruses, which cause many incurable diseases, infect cells by fusing viral and cellular membranes. Whereas most other enveloped viruses use a single viral catalyst called a fusogen, herpesviruses, inexplicably, require two conserved fusion-machinery components, gB and the heterodimer gH-gL, plus other nonconserved components. gB is a class III viral fusogen, but unlike other members of its class, it does not function alone. We determined the crystal structure of the gH ectodomain bound to gL from herpes simplex virus 2. gH-gL is an unusually tight complex with a unique architecture that, unexpectedly, does not resemble any known viral fusogen. Instead, we propose that gH-gL activates gB for fusion, possibly through direct binding. Formation of a gB-gH-gL complex is critical for fusion and is inhibited by a neutralizing antibody, making the gB-gH-gL interface a promising antiviral target.

  10. Crystal structure of the conserved herpes virus fusion regulator complex gH-gL

    Energy Technology Data Exchange (ETDEWEB)

    Chowdary, Tirumala K; Cairns, Tina M; Atanasiu, Doina; Cohen, Gary H; Eisenberg, Roselyn J; Heldwein, Ekaterina E [UPENN; (Tufts-MED)

    2010-09-13

    Herpesviruses, which cause many incurable diseases, infect cells by fusing viral and cellular membranes. Whereas most other enveloped viruses use a single viral catalyst called a fusogen, herpesviruses, inexplicably, require two conserved fusion-machinery components, gB and the heterodimer gH-gL, plus other nonconserved components. gB is a class III viral fusogen, but unlike other members of its class, it does not function alone. We determined the crystal structure of the gH ectodomain bound to gL from herpes simplex virus 2. gH-gL is an unusually tight complex with a unique architecture that, unexpectedly, does not resemble any known viral fusogen. Instead, we propose that gH-gL activates gB for fusion, possibly through direct binding. Formation of a gB-gH-gL complex is critical for fusion and is inhibited by a neutralizing antibody, making the gB-gH-gL interface a promising antiviral target.

  11. Residues in the Hendra Virus Fusion Protein Transmembrane Domain Are Critical for Endocytic Recycling

    OpenAIRE

    Popa, Andreea; Carter, James R.; Smith, Stacy E.; Hellman, Lance; Fried, Michael G.; Dutch, Rebecca Ellis

    2012-01-01

    Hendra virus is a highly pathogenic paramyxovirus classified as a biosafety level four agent. The fusion (F) protein of Hendra virus is critical for promoting viral entry and cell-to-cell fusion. To be fusogenically active, Hendra virus F must undergo endocytic recycling and cleavage by the endosomal/lysosomal protease cathepsin L, but the route of Hendra virus F following internalization and the recycling signals involved are poorly understood. We examined the intracellular distribution of H...

  12. The TIP30 protein complex, arachidonic acid and coenzyme A are required for vesicle membrane fusion.

    Directory of Open Access Journals (Sweden)

    Chengliang Zhang

    Full Text Available Efficient membrane fusion has been successfully mimicked in vitro using artificial membranes and a number of cellular proteins that are currently known to participate in membrane fusion. However, these proteins are not sufficient to promote efficient fusion between biological membranes, indicating that critical fusogenic factors remain unidentified. We have recently identified a TIP30 protein complex containing TIP30, acyl-CoA synthetase long-chain family member 4 (ACSL4 and Endophilin B1 (Endo B1 that promotes the fusion of endocytic vesicles with Rab5a vesicles, which transport endosomal acidification enzymes vacuolar (H⁺-ATPases (V-ATPases to the early endosomes in vivo. Here, we demonstrate that the TIP30 protein complex facilitates the fusion of endocytic vesicles with Rab5a vesicles in vitro. Fusion of the two vesicles also depends on arachidonic acid, coenzyme A and the synthesis of arachidonyl-CoA by ACSL4. Moreover, the TIP30 complex is able to transfer arachidonyl groups onto phosphatidic acid (PA, producing a new lipid species that is capable of inducing close contact between membranes. Together, our data suggest that the TIP30 complex facilitates biological membrane fusion through modification of PA on membranes.

  13. Oncogenic virus-mediated cell fusion: new insights into initiation and progression of oncogenic viruses--related cancers.

    Science.gov (United States)

    Gao, Peng; Zheng, Jie

    2011-04-01

    Cell fusion is fundamental to the development and physiology of multicellular organisms, such as fertilization, placentation, development of skeletal muscle and bone. Oncogenic virus-mediated cell fusion, however, may lead to chromosomal instability (CIN) by various mechanisms when tumor suppressor p53 is deregulated and produce oncogenic aneuploid cells. It is worth noting that all human oncogenic viruses, including human papillomavirus (HPV), hepatitis B virus (HBV), hepatitis C virus (HCV), Epstein-Barr virus (EBV), human herpesviruses-8/Kaposi sarcoma herpesvirus (HHV-8/KSHV) and human T-cell lymphotropic virus type 1 (HTLV-1), are capable of both inducing cell fusion and inhibiting the functions of p53 as well as pRb. Although it is now not clear whether a link between virus-mediated cell fusion and cancer established in experimental systems also exists in humans, the fact that the observation of tetraploid cells is more frequent in virus-positive than virus-negative premalignant lesions supports this link. Additionally, there are now no available vaccines against most oncogenic viruses except for HBV and HPV. Given these, developing fusion inhibitors is beneficial to cancer prevention and therapy of virus-associated cancers via inhibiting virus entry, spread and oncogenic role. Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.

  14. Binding and Fusion of Extracellular Vesicles to the Plasma Membrane of Their Cell Targets.

    Science.gov (United States)

    Prada, Ilaria; Meldolesi, Jacopo

    2016-08-09

    Exosomes and ectosomes, extracellular vesicles of two types generated by all cells at multivesicular bodies and the plasma membrane, respectively, play critical roles in physiology and pathology. A key mechanism of their function, analogous for both types of vesicles, is the fusion of their membrane to the plasma membrane of specific target cells, followed by discharge to the cytoplasm of their luminal cargo containing proteins, RNAs, and DNA. Here we summarize the present knowledge about the interactions, binding and fusions of vesicles with the cell plasma membrane. The sequence initiates with dynamic interactions, during which vesicles roll over the plasma membrane, followed by the binding of specific membrane proteins to their cell receptors. Membrane binding is then converted rapidly into fusion by mechanisms analogous to those of retroviruses. Specifically, proteins of the extracellular vesicle membranes are structurally rearranged, and their hydrophobic sequences insert into the target cell plasma membrane which undergoes lipid reorganization, protein restructuring and membrane dimpling. Single fusions are not the only process of vesicle/cell interactions. Upon intracellular reassembly of their luminal cargoes, vesicles can be regenerated, released and fused horizontally to other target cells. Fusions of extracellular vesicles are relevant also for specific therapy processes, now intensely investigated.

  15. Ca(2+)-independent fusion of secretory granules with phospholipase A2-treated plasma membranes in vitro.

    OpenAIRE

    Nagao, T.; Kubo, T.; Fujimoto, R.; Nishio, H; Takeuchi, T.; Hata, F.

    1995-01-01

    The fusion of secretory granules with plasma membranes prepared from rat parotid gland was studied in vitro to clarify the mechanism of exocytosis. Fusion of the granules with plasma membranes was measured by a fluorescence-dequenching assay with octadecyl rhodamine B, and release of amylase was also measured to confirm the fusion as a final step of the secretory process. Plasma membranes that had been pretreated with porcine phospholipase A2 (PLA2) in the presence of 20 microM Ca2+ fused wit...

  16. Lamp1 Increases the Efficiency of Lassa Virus Infection by Promoting Fusion in Less Acidic Endosomal Compartments.

    Science.gov (United States)

    Hulseberg, Christine E; Fénéant, Lucie; Szymańska, Katarzyna M; White, Judith M

    2018-01-02

    Lassa virus (LASV) is an arenavirus whose entry into host cells is mediated by a glycoprotein complex (GPC) comprised of a receptor binding subunit, GP1, a fusogenic transmembrane subunit, GP2, and a stable signal peptide. After receptor-mediated internalization, arenaviruses converge in the endocytic pathway, where they are thought to undergo low-pH-triggered, GPC-mediated fusion with a late endosome membrane. A unique feature of LASV entry is a pH-dependent switch from a primary cell surface receptor (α-dystroglycan) to an endosomal receptor, lysosomal-associated membrane protein (Lamp1). Despite evidence that the interaction between LASV GP1 and Lamp1 is critical, the function of Lamp1 in promoting LASV infection remains poorly characterized. Here we used wild-type (WT) and Lamp1 knockout (KO) cells to show that Lamp1 increases the efficiency of, but is not absolutely required for, LASV entry and infection. We then used cell-cell and pseudovirus-cell surface fusion assays to demonstrate that LASV GPC-mediated fusion occurs at a significantly higher pH when Lamp1 is present compared to when Lamp1 is missing. Correspondingly, we found that LASV entry occurs through less acidic endosomes in WT (Lamp1-positive) versus Lamp1 KO cells. We propose that, by elevating the pH threshold for fusion, Lamp1 allows LASV particles to exit the endocytic pathway before they encounter an increasingly acidic and harsh proteolytic environment, which could inactivate a significant percentage of incoming viruses. In this manner Lamp1 increases the overall efficiency of LASV entry and infection. IMPORTANCE Lassa virus is the most clinically important member of the Arenaviridae , a family that includes six additional biosafety level 4 (BSL4) hemorrhagic fever viruses. The lack of specific antiviral therapies for Lassa fever drives an urgent need to identify druggable targets, and interventions that block infection at the entry stage are particularly attractive. Lassa virus is only the

  17. IM30 triggers membrane fusion in cyanobacteria and chloroplasts

    NARCIS (Netherlands)

    Hennig, R.; Heidrich, J.; Saur, M.; Schmüser, L.; Roeters, S.J.; Hellmann, N.; Woutersen, S.; Bonn, M.; Weidner, T.; Markl, J.; Schneider, D.

    2015-01-01

    The thylakoid membrane of chloroplasts and cyanobacteria is a unique internal membrane system harbouring the complexes of the photosynthetic electron transfer chain. Despite their apparent importance, little is known about the biogenesis and maintenance of thylakoid membranes. Although membrane

  18. Structural rearrangements in the membrane penetration protein of a non-enveloped virus

    Science.gov (United States)

    Dormitzer, Philip R.; Nason, Emma B.; Venkataram Prasad, B. V.; Harrison, Stephen C.

    2007-01-01

    Non-enveloped virus particles (those that lack a lipid-bilayer membrane) must breach the membrane of a target host cell to gain access to its cytoplasm. So far, the molecular mechanism of this membrane penetration step has resisted structural analysis. The spike protein VP4 is a principal component in the entry apparatus of rotavirus, a non-enveloped virus that causes gastro-enteritis and kills 440,000 children each year1. Trypsin cleavage of VP4 primes the virus for entry by triggering a rearrangement that rigidifies the VP4 spikes2. We have determined the crystal structure, at 3.2 Å resolution, of the main part of VP4 that projects from the virion. The crystal structure reveals a coiled-coil stabilized trimer. Comparison of this structure with the two-fold clustered VP4 spikes in a ~12 Å resolution image reconstruction from electron cryomicroscopy of trypsin-primed virions shows that VP4 also undergoes a second rearrangement, in which the oligomer reorganizes and each subunit folds back on itself, translocating a potential membrane-interaction peptide from one end of the spike to the other. This rearrangement resembles the conformational transitions of membrane fusion proteins of enveloped viruses3–6. PMID:15329727

  19. Traficking of Intracellular Membranes: From Molecular Sorting to Membrane Fusion : Proceedings of the NATO Advanced Study Institute

    NARCIS (Netherlands)

    Pedroso de Lima, Maria C; Duzgunes, Nejat; Hoekstra, Dick

    1995-01-01

    This volume contains the lectures presented at the NATO Advanced Study Institute (ASI) on "Trafficking of Intracellular Membranes: From Molecular Sorting to Membrane Fusion", held in Espinho, Portugal, from June 19 to June 30,1994. The objective of this Institute was to survey recent developments

  20. Ca(2+)-independent fusion of secretory granules with phospholipase A2-treated plasma membranes in vitro.

    Science.gov (United States)

    Nagao, T; Kubo, T; Fujimoto, R; Nishio, H; Takeuchi, T; Hata, F

    1995-04-15

    The fusion of secretory granules with plasma membranes prepared from rat parotid gland was studied in vitro to clarify the mechanism of exocytosis. Fusion of the granules with plasma membranes was measured by a fluorescence-dequenching assay with octadecyl rhodamine B, and release of amylase was also measured to confirm the fusion as a final step of the secretory process. Plasma membranes that had been pretreated with porcine phospholipase A2 (PLA2) in the presence of 20 microM Ca2+ fused with the granules within 30 s, and induced amylase release by reacting with the membranes of granules, whereas without this pretreatment they had no significant effect. The fusion process accompanied by amylase release was induced in the presence of 10 mM EGTA, and therefore was apparently Ca(2+)-independent. On the other hand, the presence of EGTA or 100 microM quinacrine, an inhibitor of PLA2, during treatment of plasma membranes with PLA2 inhibited their fusogenic activity, suggesting the importance of activation of PLA2. Arachidonic acid and linoleic acid were released from the plasma membranes during the PLA2 treatment. The presence of albumin, an adsorbent of fatty acids, during the treatment also inhibited the activity. Pretreatment of the membranes with arachidonic acid or linoleic acid did not have any effect, but the presence of exogenously added arachidonic acid during PLA2 treatment enhanced the membrane-fusion-inducing effect of PLA2. Pretreatment of the membranes with lysophosphatidylcholine induced fusogenic activity. These findings suggest that the conformational change in the plasma-membrane phospholipids induced by PLA2 and the presence of arachidonic acid or linoleic acid produced by PLA2 are important in the process of fusion of secretory granules with the plasma membranes of rat parotid acinar cells and that the fusion process itself is independent of Ca2+.

  1. Mitochondrial DNA mutations provoke dominant inhibition of mitochondrial inner membrane fusion.

    Directory of Open Access Journals (Sweden)

    Cécile Sauvanet

    Full Text Available Mitochondria are highly dynamic organelles that continuously move, fuse and divide. Mitochondrial dynamics modulate overall mitochondrial morphology and are essential for the proper function, maintenance and transmission of mitochondria and mitochondrial DNA (mtDNA. We have investigated mitochondrial fusion in yeast cells with severe defects in oxidative phosphorylation (OXPHOS due to removal or various specific mutations of mtDNA. We find that, under fermentative conditions, OXPHOS deficient cells maintain normal levels of cellular ATP and ADP but display a reduced mitochondrial inner membrane potential. We demonstrate that, despite metabolic compensation by glycolysis, OXPHOS defects are associated to a selective inhibition of inner but not outer membrane fusion. Fusion inhibition was dominant and hampered the fusion of mutant mitochondria with wild-type mitochondria. Inhibition of inner membrane fusion was not systematically associated to changes of mitochondrial distribution and morphology, nor to changes in the isoform pattern of Mgm1, the major fusion factor of the inner membrane. However, inhibition of inner membrane fusion correlated with specific alterations of mitochondrial ultrastructure, notably with the presence of aligned and unfused inner membranes that are connected to two mitochondrial boundaries. The fusion inhibition observed upon deletion of OXPHOS related genes or upon removal of the entire mtDNA was similar to that observed upon introduction of point mutations in the mitochondrial ATP6 gene that are associated to neurogenic ataxia and retinitis pigmentosa (NARP or to maternally inherited Leigh Syndrome (MILS in humans. Our findings indicate that the consequences of mtDNA mutations may not be limited to OXPHOS defects but may also include alterations in mitochondrial fusion. Our results further imply that, in healthy cells, the dominant inhibition of fusion could mediate the exclusion of OXPHOS-deficient mitochondria from

  2. Beyond anchoring: the expanding role of the hendra virus fusion protein transmembrane domain in protein folding, stability, and function.

    Science.gov (United States)

    Smith, Everett Clinton; Culler, Megan R; Hellman, Lance M; Fried, Michael G; Creamer, Trevor P; Dutch, Rebecca Ellis

    2012-03-01

    While work with viral fusion proteins has demonstrated that the transmembrane domain (TMD) can affect protein folding, stability, and membrane fusion promotion, the mechanism(s) remains poorly understood. TMDs could play a role in fusion promotion through direct TMD-TMD interactions, and we have recently shown that isolated TMDs from three paramyxovirus fusion (F) proteins interact as trimers using sedimentation equilibrium (SE) analysis (E. C. Smith, et al., submitted for publication). Immediately N-terminal to the TMD is heptad repeat B (HRB), which plays critical roles in fusion. Interestingly, addition of HRB decreased the stability of the trimeric TMD-TMD interactions. This result, combined with previous findings that HRB forms a trimeric coiled coil in the prefusion form of the whole protein though HRB peptides fail to stably associate in isolation, suggests that the trimeric TMD-TMD interactions work in concert with elements in the F ectodomain head to stabilize a weak HRB interaction. Thus, changes in TMD-TMD interactions could be important in regulating F triggering and refolding. Alanine insertions between the TMD and HRB demonstrated that spacing between these two regions is important for protein stability while not affecting TMD-TMD interactions. Additional mutagenesis of the C-terminal end of the TMD suggests that β-branched residues within the TMD play a role in membrane fusion, potentially through modulation of TMD-TMD interactions. Our results support a model whereby the C-terminal end of the Hendra virus F TMD is an important regulator of TMD-TMD interactions and show that these interactions help hold HRB in place prior to the triggering of membrane fusion.

  3. [Proteolytic mechanism of deproteinization of influenza virus by plasmatic membranes].

    Science.gov (United States)

    Degtiarenko, V I; Fedchuk, A S

    1977-01-01

    A mechanism responsible for proteolytic deproteinization of influenza virus A2 Hong-Kong (I)68 by plasmatic membranes of sensitive cells was studied. Presence of trypsinlike protease in plasmatic membranes of white mice lungs was demonstrated. A considerable inhibition of the membrane proteolitic activity was obtained in the presence of epsilon-aminocaproic acid. Disintegration of the virus labeled by [3H]uridine by plasmatic membranes was investigated and it was found that this process required ATP. Inhibition of the protease activity by epsilon-aminocaproic acid led to the suppression of deproteinization of influenza virus. The experimental data obtained indicate that the proteolytic enzymes of plasmatic membranes participate in the complex process of virus "uncoating".

  4. An induced pocket for the binding of potent fusion inhibitor CL-385319 with H5N1 influenza virus hemagglutinin.

    Directory of Open Access Journals (Sweden)

    Runming Li

    Full Text Available The influenza glycoprotein hemagglutinin (HA plays crucial roles in the early stage of virus infection, including receptor binding and membrane fusion. Therefore, HA is a potential target for developing anti-influenza drugs. Recently, we characterized a novel inhibitor of highly pathogenic H5N1 influenza virus, CL-385319, which specifically inhibits HA-mediated viral entry. Studies presented here identified the critical binding residues for CL-385319, which clustered in the stem region of the HA trimer by site-directed mutagenesis. Extensive computational simulations, including molecular docking, molecular dynamics simulations, molecular mechanics generalized Born surface area (MM_GBSA calculations, charge density and Laplacian calculations, have been carried out to uncover the detailed molecular mechanism that underlies the binding of CL-385319 to H5N1 influenza virus HA. It was found that the recognition and binding of CL-385319 to HA proceeds by a process of "induced fit" whereby the binding pocket is formed during their interaction. Occupation of this pocket by CL-385319 stabilizes the neutral pH structure of hemagglutinin, thus inhibiting the conformational rearrangements required for membrane fusion. This "induced fit" pocket may be a target for structure-based design of more potent influenza fusion inhibitors.

  5. Multi-layered nanoparticles for penetrating the endosome and nuclear membrane via a step-wise membrane fusion process.

    Science.gov (United States)

    Akita, Hidetaka; Kudo, Asako; Minoura, Arisa; Yamaguti, Masaya; Khalil, Ikramy A; Moriguchi, Rumiko; Masuda, Tomoya; Danev, Radostin; Nagayama, Kuniaki; Kogure, Kentaro; Harashima, Hideyoshi

    2009-05-01

    Efficient targeting of DNA to the nucleus is a prerequisite for effective gene therapy. The gene-delivery vehicle must penetrate through the plasma membrane, and the DNA-impermeable double-membraned nuclear envelope, and deposit its DNA cargo in a form ready for transcription. Here we introduce a concept for overcoming intracellular membrane barriers that involves step-wise membrane fusion. To achieve this, a nanotechnology was developed that creates a multi-layered nanoparticle, which we refer to as a Tetra-lamellar Multi-functional Envelope-type Nano Device (T-MEND). The critical structural elements of the T-MEND are a DNA-polycation condensed core coated with two nuclear membrane-fusogenic inner envelopes and two endosome-fusogenic outer envelopes, which are shed in stepwise fashion. A double-lamellar membrane structure is required for nuclear delivery via the stepwise fusion of double layered nuclear membrane structure. Intracellular membrane fusions to endosomes and nuclear membranes were verified by spectral imaging of fluorescence resonance energy transfer (FRET) between donor and acceptor fluorophores that had been dually labeled on the liposome surface. Coating the core with the minimum number of nucleus-fusogenic lipid envelopes (i.e., 2) is essential to facilitate transcription. As a result, the T-MEND achieves dramatic levels of transgene expression in non-dividing cells.

  6. Mitotic phosphorylation of VCIP135 blocks p97ATPase-mediated Golgi membrane fusion

    Energy Technology Data Exchange (ETDEWEB)

    Totsukawa, Go; Matsuo, Ayaka; Kubota, Ayano; Taguchi, Yuya; Kondo, Hisao, E-mail: hk228@med.kyushu-u.ac.jp

    2013-04-05

    Highlights: •VCIP135 is mitotically phosphorylated on Threonine-760 and Serine-767 by Cdc2. •Phosphorylated VCIP135 does not bind to p97ATPase. •The phosphorylation of VCIP135 inhibits p97ATPase-mediated Golgi membrane fusion. -- Abstract: In mammals, the Golgi apparatus is disassembled early mitosis and reassembled at the end of mitosis. For Golgi disassembly, membrane fusion needs to be blocked. Golgi biogenesis requires two distinct p97ATPase-mediated membrane fusion, the p97/p47 and p97/p37 pathways. We previously reported that p47 phosphorylation on Serine-140 and p37 phosphorylation on Serine-56 and Threonine-59 result in mitotic inhibition of the p97/p47 and the p97/p37 pathways, respectively [11,14]. In this study, we show another mechanism of mitotic inhibition of p97-mediated Golgi membrane fusion. We clarified that VCIP135, an essential factor in both p97 membrane fusion pathways, is phosphorylated on Threonine-760 and Serine-767 by Cdc2 at mitosis and that this phosphorylated VCIP135 does not bind to p97. An in vitro Golgi reassembly assay revealed that VCIP135(T760E, S767E), which mimics mitotic phosphorylation, caused no cisternal regrowth. Our results indicate that the phosphorylation of VCIP135 on Threonine-760 and Serine-767 inhibits p97-mediated Golgi membrane fusion at mitosis.

  7. A high throughput Cre–lox activated viral membrane fusion assay identifies pharmacological inhibitors of HIV entry

    Energy Technology Data Exchange (ETDEWEB)

    Esposito, Anthony M. [Division of Infectious Diseases, Department of Medicine, Icahn School of Medicine at Mount Sinai, Immunology Institute, New York, NY (United States); Cheung, Pamela [Integrated Screening Core, Icahn School of Medicine at Mount Sinai, New York, NY (United States); Swartz, Talia H.; Li, Hongru [Division of Infectious Diseases, Department of Medicine, Icahn School of Medicine at Mount Sinai, Immunology Institute, New York, NY (United States); Tsibane, Tshidi [Department of Microbiology, Icahn School of Medicine at Mount Sinai, New York, NY (United States); Durham, Natasha D. [Division of Infectious Diseases, Department of Medicine, Icahn School of Medicine at Mount Sinai, Immunology Institute, New York, NY (United States); Basler, Christopher F. [Department of Microbiology, Icahn School of Medicine at Mount Sinai, New York, NY (United States); Felsenfeld, Dan P. [Integrated Screening Core, Icahn School of Medicine at Mount Sinai, New York, NY (United States); Chen, Benjamin K., E-mail: benjamin.chen@mssm.edu [Division of Infectious Diseases, Department of Medicine, Icahn School of Medicine at Mount Sinai, Immunology Institute, New York, NY (United States)

    2016-03-15

    Enveloped virus entry occurs when viral and cellular membranes fuse releasing particle contents into the target cell. Human immunodeficiency virus (HIV) entry occurs by cell-free virus or virus transferred between infected and uninfected cells through structures called virological synapses. We developed a high-throughput cell-based assay to identify small molecule inhibitors of cell-free or virological synapse-mediated entry. An HIV clone carrying Cre recombinase as a Gag-internal gene fusion releases active Cre into cells upon viral entry activating a recombinatorial gene switch changing dsRed to GFP-expression. A screen of a 1998 known-biological profile small molecule library identified pharmacological HIV entry inhibitors that block both cell-free and cell-to-cell infection. Many top hits were noted as HIV inhibitors in prior studies, but not previously recognized as entry antagonists. Modest therapeutic indices for simvastatin and nigericin were observed in confirmatory HIV infection assays. This robust assay is adaptable to study HIV and heterologous viral pseudotypes. - Highlights: • Cre recombinase viral fusion assay screens cell-free or cell–cell entry inhibitors. • This Gag-iCre based assay is specific for the entry step of HIV replication. • Screened a library of known pharmacologic compounds for HIV fusion antagonists. • Many top hits were previously noted as HIV inhibitors, but here are classified as entry antagonists. Many top hits were previously noted as HIV inhibitors, but not as entry antagonists. • The assay is compatible with pseudotyping with HIV and heterologous viruses.

  8. Nuclear envelope breakdown induced by herpes simplex virus type 1 involves the activity of viral fusion proteins

    Energy Technology Data Exchange (ETDEWEB)

    Maric, Martina; Haugo, Alison C. [Department of Microbiology, University of Iowa, Iowa City, IA 52242 (United States); Dauer, William [Department of Neurology, University of Michigan, Ann Arbor, MI 48109 (United States); Johnson, David [Department of Microbiology and Immunology, Oregon Health Sciences University, Portland, OR 97201 (United States); Roller, Richard J., E-mail: richard-roller@uiowa.edu [Department of Microbiology, University of Iowa, Iowa City, IA 52242 (United States)

    2014-07-15

    Herpesvirus infection reorganizes components of the nuclear lamina usually without loss of integrity of the nuclear membranes. We report that wild-type HSV infection can cause dissolution of the nuclear envelope in transformed mouse embryonic fibroblasts that do not express torsinA. Nuclear envelope breakdown is accompanied by an eight-fold inhibition of virus replication. Breakdown of the membrane is much more limited during infection with viruses that lack the gB and gH genes, suggesting that breakdown involves factors that promote fusion at the nuclear membrane. Nuclear envelope breakdown is also inhibited during infection with virus that does not express UL34, but is enhanced when the US3 gene is deleted, suggesting that envelope breakdown may be enhanced by nuclear lamina disruption. Nuclear envelope breakdown cannot compensate for deletion of the UL34 gene suggesting that mixing of nuclear and cytoplasmic contents is insufficient to bypass loss of the normal nuclear egress pathway. - Highlights: • We show that wild-type HSV can induce breakdown of the nuclear envelope in a specific cell system. • The viral fusion proteins gB and gH are required for induction of nuclear envelope breakdown. • Nuclear envelope breakdown cannot compensate for deletion of the HSV UL34 gene.

  9. Entropic forces drive self-organization and membrane fusion by SNARE proteins.

    Science.gov (United States)

    Mostafavi, Hakhamanesh; Thiyagarajan, Sathish; Stratton, Benjamin S; Karatekin, Erdem; Warner, Jason M; Rothman, James E; O'Shaughnessy, Ben

    2017-05-23

    SNARE proteins are the core of the cell's fusion machinery and mediate virtually all known intracellular membrane fusion reactions on which exocytosis and trafficking depend. Fusion is catalyzed when vesicle-associated v-SNAREs form trans-SNARE complexes ("SNAREpins") with target membrane-associated t-SNAREs, a zippering-like process releasing ∼65 kT per SNAREpin. Fusion requires several SNAREpins, but how they cooperate is unknown and reports of the number required vary widely. To capture the collective behavior on the long timescales of fusion, we developed a highly coarse-grained model that retains key biophysical SNARE properties such as the zippering energy landscape and the surface charge distribution. In simulations the ∼65-kT zippering energy was almost entirely dissipated, with fully assembled SNARE motifs but uncomplexed linker domains. The SNAREpins self-organized into a circular cluster at the fusion site, driven by entropic forces that originate in steric-electrostatic interactions among SNAREpins and membranes. Cooperative entropic forces expanded the cluster and pulled the membranes together at the center point with high force. We find that there is no critical number of SNAREs required for fusion, but instead the fusion rate increases rapidly with the number of SNAREpins due to increasing entropic forces. We hypothesize that this principle finds physiological use to boost fusion rates to meet the demanding timescales of neurotransmission, exploiting the large number of v-SNAREs available in synaptic vesicles. Once in an unfettered cluster, we estimate ≥15 SNAREpins are required for fusion within the ∼1-ms timescale of neurotransmitter release.

  10. Influenza virus M2 protein ion channel activity helps to maintain pandemic 2009 H1N1 virus hemagglutinin fusion competence during transport to the cell surface.

    Science.gov (United States)

    Alvarado-Facundo, Esmeralda; Gao, Yamei; Ribas-Aparicio, Rosa María; Jiménez-Alberto, Alicia; Weiss, Carol D; Wang, Wei

    2015-02-01

    The influenza virus hemagglutinin (HA) envelope protein mediates virus entry by first binding to cell surface receptors and then fusing viral and endosomal membranes during endocytosis. Cleavage of the HA precursor (HA0) into a surface receptor-binding subunit (HA1) and a fusion-inducing transmembrane subunit (HA2) by host cell enzymes primes HA for fusion competence by repositioning the fusion peptide to the newly created N terminus of HA2. We previously reported that the influenza virus M2 protein enhances pandemic 2009 influenza A virus [(H1N1)pdm09] HA-pseudovirus infectivity, but the mechanism was unclear. In this study, using cell-cell fusion and HA-pseudovirus infectivity assays, we found that the ion channel function of M2 was required for enhancement of HA fusion and HA-pseudovirus infectivity. The M2 activity was needed only during HA biosynthesis, and proteolysis experiments indicated that M2 proton channel activity helped to protect (H1N1)pdm09 HA from premature conformational changes as it traversed low-pH compartments during transport to the cell surface. While M2 has previously been shown to protect avian influenza virus HA proteins of the H5 and H7 subtypes that have polybasic cleavage motifs, this study demonstrates that M2 can protect HA proteins from human H1N1 strains that lack a polybasic cleavage motif. This finding suggests that M2 proton channel activity may play a wider role in preserving HA fusion competence among a variety of HA subtypes, including HA proteins from emerging strains that may have reduced HA stability. Influenza virus infects cells when the hemagglutinin (HA) surface protein undergoes irreversible pH-induced conformational changes after the virus is taken into the cell by endocytosis. HA fusion competence is primed when host cell enzymes cleave the HA precursor. The proton channel function of influenza virus M2 protein has previously been shown to protect avian influenza virus HA proteins that contain a polybasic cleavage

  11. Epstein Barr virus latent membrane protein-1 in Hodgkin's ...

    African Journals Online (AJOL)

    Epstein-Barr virus latent membrane protein-1 (LMP-1), CD15 and CD30 immunohistochemistry was also performed. The clinical characteristics of each patient were documented. Objectives: To document the frequency of involvement of Epstein-Barr virus in cases of HL seen in a university hospital in Nigeria. Results: Out of ...

  12. Mutations in the Transmembrane Domain and Cytoplasmic Tail of Hendra Virus Fusion Protein Disrupt Virus-Like-Particle Assembly.

    Science.gov (United States)

    Cifuentes-Muñoz, Nicolás; Sun, Weina; Ray, Greeshma; Schmitt, Phuong Tieu; Webb, Stacy; Gibson, Kathleen; Dutch, Rebecca Ellis; Schmitt, Anthony P

    2017-07-15

    Hendra virus (HeV) is a zoonotic paramyxovirus that causes deadly illness in horses and humans. An intriguing feature of HeV is the utilization of endosomal protease for activation of the viral fusion protein (F). Here we investigated how endosomal F trafficking affects HeV assembly. We found that the HeV matrix (M) and F proteins each induced particle release when they were expressed alone but that their coexpression led to coordinated assembly of virus-like particles (VLPs) that were morphologically and physically distinct from M-only or F-only VLPs. Mutations to the F protein transmembrane domain or cytoplasmic tail that disrupted endocytic trafficking led to failure of F to function with M for VLP assembly. Wild-type F functioned normally for VLP assembly even when its cleavage was prevented with a cathepsin inhibitor, indicating that it is endocytic F trafficking that is important for VLP assembly, not proteolytic F cleavage. Under specific conditions of reduced M expression, we found that M could no longer induce significant VLP release but retained the ability to be incorporated as a passenger into F-driven VLPs, provided that the F protein was competent for endocytic trafficking. The F and M proteins were both found to traffic through Rab11-positive recycling endosomes (REs), suggesting a model in which F and M trafficking pathways converge at REs, enabling these proteins to preassemble before arriving at plasma membrane budding sites. IMPORTANCE Hendra virus and Nipah virus are zoonotic paramyxoviruses that cause lethal infections in humans. Unlike that for most paramyxoviruses, activation of the henipavirus fusion protein occurs in recycling endosomal compartments. In this study, we demonstrate that the unique endocytic trafficking pathway of Hendra virus F protein is required for proper viral assembly and particle release. These results advance our basic understanding of the henipavirus assembly process and provide a novel model for the interplay between

  13. Genetic studies of cell fusion induced by herpes simplex virus type 1

    Energy Technology Data Exchange (ETDEWEB)

    Read, G.S.; Person, S.; Keller, P.M.

    1980-07-01

    Eight cell fusion-causing syn mutants were isolated from the KOS strain of herpes simplex virus type 1. Unlike the wild-type virus, the mutants produced plaques containing multinucleated cells, or syncytia. Fusion kinetics curves were established with a Coulter Counter assay for the mutants and wild-type virus in single infections of human embryonic lung (HEL) cells, for the mutants and wild-type virus in mixed infections (dominance test), and for pairs of mutants in mixed infection and proceeded with an exponential decrease in the number of small single cells. At some later time that was characteristic of the mutant, there was a significant reduction in the rate of fusion for all but possibly one of the mutants. Although the wild-type virus did not produce syncytial plaques, it did induce a small amount of fusion that stopped abruptly about 2 h after it started. These data are consistent with the hypothesis that both mutants and wild type induce an active fusion inducer and that the activity of this inducer is subsequently inhibited. The extent of fusion is apparently determined by the length of the interval during which the fusion inducer is active. That fusion is actively inhibited in wild-type infections is indicated by the observation that syn mutant-infected cells fused more readily with uninfected cells than with wild type-infected cells.

  14. Exploring the membrane fusion mechanism through force-induced disassembly of HIV-1 six-helix bundle

    Energy Technology Data Exchange (ETDEWEB)

    Gao, Kai [Key Laboratory of RNA Biology, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101 (China); Beijing Key Laboratory of Noncoding RNA, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101 (China); University of Chinese Academy of Sciences, Beijing 100049 (China); Zhang, Yong [Key Laboratory of RNA Biology, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101 (China); Beijing Key Laboratory of Noncoding RNA, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101 (China); Lou, Jizhong, E-mail: jlou@ibp.ac.cn [Key Laboratory of RNA Biology, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101 (China); Beijing Key Laboratory of Noncoding RNA, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101 (China)

    2016-05-13

    Enveloped virus, such as HIV-1, employs membrane fusion mechanism to invade into host cell. HIV-1 gp41 ectodomain uses six-helix bundle configuration to accomplish this process. Using molecular dynamic simulations, we confirmed the stability of this six-helix bundle by showing high occupancy of hydrogen bonds and hydrophobic interactions. Key residues and interactions important for the bundle integration were characterized by force-induced unfolding simulations of six-helix bundle, exhibiting the collapse order of these groups of interactions. Moreover, our results in some way concerted with a previous theory that the formation of coiled-coil choose a route which involved cooperative interactions between the N-terminal and C-terminal helix. -- Highlights: •Unfolding of HIV-1 gp41 six-helix bundle is studied by molecular dynamics simulations. •Specific interactions responsible for the stability of HIV-1 envelope post-fusion conformation were identified. •The gp41 six-helix bundle transition inducing membrane fusion might be a cooperative process of the three subunits.

  15. Cytosol-dependent membrane fusion in ER, nuclear envelope and nuclear pore assembly: biological implications.

    Science.gov (United States)

    Rafikova, Elvira R; Melikov, Kamran; Chernomordik, Leonid V

    2010-01-01

    Endoplasmic reticulum and nuclear envelope rearrangements after mitosis are often studied in the reconstitution system based on Xenopus egg extract. In our recent work we partially replaced the membrane vesicles in the reconstitution mix with protein-free liposomes to explore the relative contributions of cytosolic and transmembrane proteins. Here we discuss our finding that cytosolic proteins mediate fusion between membranes lacking functional transmembrane proteins and the role of membrane fusion in endoplasmic reticulum and nuclear envelope reorganization. Cytosol-dependent liposome fusion has allowed us to restore, without adding transmembrane nucleoporins, functionality of nuclear pores, their spatial distribution and chromatin decondensation in nuclei formed at insufficient amounts of membrane material and characterized by only partial decondensation of chromatin and lack of nuclear transport. Both the mechanisms and the biological implications of the discovered coupling between spatial distribution of nuclear pores, chromatin decondensation and nuclear transport are discussed.

  16. Assessing the efficacy of vesicle fusion with planar membrane arrays using a mitochondrial porin as reporter

    DEFF Research Database (Denmark)

    Pszon-Bartosz, Kamila Justyna; Hansen, Jesper S.; Stibius, Karin B.

    2011-01-01

    establish a protein incorporation efficacy assay using the major non-specific porin of Fusobacterium nucleatum (FomA) as reporter. We use electrical conductance measurements and fluorescence microscopy to characterize proteoliposome fusion with an array of planar membranes. We show that protein...... in a bilayer array with a total membrane area of 2mm2 within 20min. This novel assay for quantifying protein delivery into lipid bilayers may be a useful tool in developing biomimetic membrane applications....

  17. Low energy cost for optimal speed and control of membrane fusion.

    Science.gov (United States)

    François-Martin, Claire; Rothman, James E; Pincet, Frederic

    2017-02-07

    Membrane fusion is the cell's delivery process, enabling its many compartments to receive cargo and machinery for cell growth and intercellular communication. The overall activation energy of the process must be large enough to prevent frequent and nonspecific spontaneous fusion events, yet must be low enough to allow it to be overcome upon demand by specific fusion proteins [such as soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs)]. Remarkably, to the best of our knowledge, the activation energy for spontaneous bilayer fusion has never been measured. Multiple models have been developed and refined to estimate the overall activation energy and its component parts, and they span a very broad range from 20 kBT to 150 kBT, depending on the assumptions. In this study, using a bulk lipid-mixing assay at various temperatures, we report that the activation energy of complete membrane fusion is at the lowest range of these theoretical values. Typical lipid vesicles were found to slowly and spontaneously fully fuse with activation energies of ∼30 kBT Our data demonstrate that the merging of membranes is not nearly as energy consuming as anticipated by many models and is ideally positioned to minimize spontaneous fusion while enabling rapid, SNARE-dependent fusion upon demand.

  18. Functional Carboxy-Terminal Fluorescent Protein Fusion to Pseudorabies Virus Small Capsid Protein VP26.

    Science.gov (United States)

    Hogue, Ian B; Jean, Jolie; Esteves, Andrew D; Tanneti, Nikhila S; Scherer, Julian; Enquist, Lynn W

    2018-01-01

    Fluorescent protein fusions to herpesvirus capsids have proven to be a valuable method to study virus particle transport in living cells. Fluorescent protein fusions to the amino terminus of small capsid protein VP26 are the most widely used method to visualize pseudorabies virus (PRV) and herpes simplex virus (HSV) particles in living cells. However, these fusion proteins do not incorporate to full occupancy and have modest effects on virus replication and pathogenesis. Recent cryoelectron microscopy studies have revealed that herpesvirus small capsid proteins bind to capsids via their amino terminus, whereas the carboxy terminus is unstructured and therefore may better tolerate fluorescent protein fusions. Here, we describe a new recombinant PRV expressing a carboxy-terminal VP26-mCherry fusion. Compared to previously characterized viruses expressing amino-terminal fusions, this virus expresses more VP26 fusion protein in infected cells and incorporates more VP26 fusion protein into virus particles, and individual virus particles exhibit brighter red fluorescence. We performed single-particle tracking of fluorescent virus particles in primary neurons to measure anterograde and retrograde axonal transport, demonstrating the usefulness of this novel VP26-mCherry fusion for the study of viral intracellular transport.IMPORTANCE Alphaherpesviruses are among the very few viruses that are adapted to invade the mammalian nervous system. Intracellular transport of virus particles in neurons is important, as this process underlies both mild peripheral nervous system infection and severe spread to the central nervous system. VP26, the small capsid protein of HSV and PRV, was one of the first herpesvirus proteins to be fused to a fluorescent protein. Since then, these capsid-tagged virus mutants have become a powerful tool to visualize and track individual virus particles. Improved capsid tags will facilitate fluorescence microscopy studies of virus particle intracellular

  19. Specific interaction of IM30/Vipp1 with cyanobacterial and chloroplast membranes results in membrane remodeling and eventually in membrane fusion.

    Science.gov (United States)

    Heidrich, Jennifer; Thurotte, Adrien; Schneider, Dirk

    2017-04-01

    The photosynthetic light reaction takes place within the thylakoid membrane system in cyanobacteria and chloroplasts. Besides its global importance, the biogenesis, maintenance and dynamics of this membrane system are still a mystery. In the last two decades, strong evidence supported the idea that these processes involve IM30, the inner membrane-associated protein of 30kDa, a protein also known as the vesicle-inducing protein in plastids 1 (Vipp1). Even though we just only begin to understand the precise physiological function of this protein, it is clear that interaction of IM30 with membranes is crucial for biogenesis of thylakoid membranes. Here we summarize and discuss forces guiding IM30-membrane interactions, as the membrane properties as well as the oligomeric state of IM30 appear to affect proper interaction of IM30 with membrane surfaces. Interaction of IM30 with membranes results in an altered membrane structure and can finally trigger fusion of adjacent membranes, when Mg(2+) is present. Based on recent results, we finally present a model summarizing individual steps involved in IM30-mediated membrane fusion. This article is part of a Special Issue entitled: Lipid order/lipid defects and lipid-control of protein activity edited by Dirk Schneider. Copyright © 2016 Elsevier B.V. All rights reserved.

  20. Cytoplasmic Motifs in the Nipah Virus Fusion Protein Modulate Virus Particle Assembly and Egress.

    Science.gov (United States)

    Johnston, Gunner P; Contreras, Erik M; Dabundo, Jeffrey; Henderson, Bryce A; Matz, Keesha M; Ortega, Victoria; Ramirez, Alfredo; Park, Arnold; Aguilar, Hector C

    2017-05-15

    Nipah virus (NiV), a paramyxovirus in the genus Henipavirus, has a mortality rate in humans of approximately 75%. While several studies have begun our understanding of NiV particle formation, the mechanism of this process remains to be fully elucidated. For many paramyxoviruses, M proteins drive viral assembly and egress; however, some paramyxoviral glycoproteins have been reported as important or essential in budding. For NiV the matrix protein (M), the fusion glycoprotein (F) and, to a much lesser extent, the attachment glycoprotein (G) autonomously induce the formation of virus-like particles (VLPs). However, functional interactions between these proteins during assembly and egress remain to be fully understood. Moreover, if the F-driven formation of VLPs occurs through interactions with host cell machinery, the cytoplasmic tail (CT) of F is a likely interactive domain. Therefore, we analyzed NiV F CT deletion and alanine mutants and report that several but not all regions of the F CT are necessary for efficient VLP formation. Two of these regions contain YXXØ or dityrosine motifs previously shown to interact with cellular machinery involved in F endocytosis and transport. Importantly, our results showed that F-driven, M-driven, and M/F-driven viral particle formation enhanced the recruitment of G into VLPs. By identifying key motifs, specific residues, and functional viral protein interactions important for VLP formation, we improve our understanding of the viral assembly/egress process and point to potential interactions with host cell machinery.IMPORTANCE Henipaviruses can cause deadly infections of medical, veterinary, and agricultural importance. With recent discoveries of new henipa-like viruses, understanding the mechanisms by which these viruses reproduce is paramount. We have focused this study on identifying the functional interactions of three Nipah virus proteins during viral assembly and particularly on the role of one of these proteins, the fusion

  1. Fusion activity of influenza virus PR8/34 correlates with a temperature-induced conformational change within the hemagglutinin ectodomain detected by photochemical labeling

    Energy Technology Data Exchange (ETDEWEB)

    Brunner, J.; Zugliani, C. (Swiss Federal Inst. of Tech., Zuerich (Switzerland)); Mischler, R. (Swiss Serum and Vaccine Inst., Bern (Switzerland))

    1991-03-05

    Fusion of influenza viruses with membranes is catalyzed by the viral spike protein hemagglutinin (HA). Under mildly acidic conditions ({approximately}pH 5) this protein undergoes a conformational change that triggers the exposure of the fusion peptide, the hydrophobic N-terminal segment of the HA2 polypeptide chain. Insertion of this segment into the target membrane (or viral membrane ) is likely to represent a key step along the fusion pathway, but the details are far from being clear. The photoreactive phospholipid 1-palmitoyl-2-(11-(4-(3-(trifluoromethyl)diazirinyl)phenyl)(2-{sup 3}H)undecanoyl)-sn-glycero-3-phosphocholine (({sup 3}H)PTPC/11), inserted into the bilayer of large unilamellar vesicles (LUVs), allowed the authors to investigate both the interaction of viruses with the vesicles under perfusion conditions and the fusion process itself occurring at elevated temperatures only. Despite the observed binding of viruses to LUVs at pH 5 and 0C, labeling of HA2 was very weak. They have studied also the effect of temperature on the acid-induced (pH 5) interaction of bromelain-solubilized HA (BHA) with vesicles.

  2. Inner/Outer nuclear membrane fusion in nuclear pore assembly: biochemical demonstration and molecular analysis.

    Science.gov (United States)

    Fichtman, Boris; Ramos, Corinne; Rasala, Beth; Harel, Amnon; Forbes, Douglass J

    2010-12-01

    Nuclear pore complexes (NPCs) are large proteinaceous channels embedded in double nuclear membranes, which carry out nucleocytoplasmic exchange. The mechanism of nuclear pore assembly involves a unique challenge, as it requires creation of a long-lived membrane-lined channel connecting the inner and outer nuclear membranes. This stabilized membrane channel has little evolutionary precedent. Here we mapped inner/outer nuclear membrane fusion in NPC assembly biochemically by using novel assembly intermediates and membrane fusion inhibitors. Incubation of a Xenopus in vitro nuclear assembly system at 14°C revealed an early pore intermediate where nucleoporin subunits POM121 and the Nup107-160 complex were organized in a punctate pattern on the inner nuclear membrane. With time, this intermediate progressed to diffusion channel formation and finally to complete nuclear pore assembly. Correct channel formation was blocked by the hemifusion inhibitor lysophosphatidylcholine (LPC), but not if a complementary-shaped lipid, oleic acid (OA), was simultaneously added, as determined with a novel fluorescent dextran-quenching assay. Importantly, recruitment of the bulk of FG nucleoporins, characteristic of mature nuclear pores, was not observed before diffusion channel formation and was prevented by LPC or OA, but not by LPC+OA. These results map the crucial inner/outer nuclear membrane fusion event of NPC assembly downstream of POM121/Nup107-160 complex interaction and upstream or at the time of FG nucleoporin recruitment.

  3. The destructive effect of botulinum neurotoxins on the SNARE protein: SNAP-25 and synaptic membrane fusion

    Directory of Open Access Journals (Sweden)

    Bin Lu

    2015-06-01

    Full Text Available Synaptic exocytosis requires the assembly of syntaxin 1A and SNAP-25 on the plasma membrane and synaptobrevin 2 (VAMP2 on the vesicular membrane to bridge the two opposite membranes. It is believed that the three SNARE proteins assemble in steps along the dynamic assembly pathway. The C-terminus of SNAP-25 is known to be the target of botulinum neurotoxins (BoNT/A and BoNT/E that block neurotransmitters release in vivo. In this study, we employed electron paramagnetic resonance (EPR spectroscopy to investigate the conformation of the SNAP-25 C-terminus in binary and ternary SNARE complexes. The fluorescence lipid mixing assay shows that the C-terminal of SNAP-25 is essential for membrane fusion, and that the truncated SNAP-25 mutants cleaved by BoNT/A and BoNT/E display different inhibition effects on membrane fusion: SNAP-25E (Δ26 abolishes the fusion activity of the SNARE complex, while SNAP-25A (Δ9 loses most of its function, although it can still form a SDS-resistant SNARE complex as the wild-type SNAP-25. CW-EPR spectra validate the unstable structures of the SNARE complex formed by SNAP-25 mutants. We propose that the truncated SNAP-25 mutants will disrupt the assembly of the SNARE core complex, and then inhibit the synaptic membrane fusion accordingly.

  4. The destructive effect of botulinum neurotoxins on the SNARE protein: SNAP-25 and synaptic membrane fusion.

    Science.gov (United States)

    Lu, Bin

    2015-01-01

    Synaptic exocytosis requires the assembly of syntaxin 1A and SNAP-25 on the plasma membrane and synaptobrevin 2 (VAMP2) on the vesicular membrane to bridge the two opposite membranes. It is believed that the three SNARE proteins assemble in steps along the dynamic assembly pathway. The C-terminus of SNAP-25 is known to be the target of botulinum neurotoxins (BoNT/A and BoNT/E) that block neurotransmitters release in vivo. In this study, we employed electron paramagnetic resonance (EPR) spectroscopy to investigate the conformation of the SNAP-25 C-terminus in binary and ternary SNARE complexes. The fluorescence lipid mixing assay shows that the C-terminal of SNAP-25 is essential for membrane fusion, and that the truncated SNAP-25 mutants cleaved by BoNT/A and BoNT/E display different inhibition effects on membrane fusion: SNAP-25E (Δ26) abolishes the fusion activity of the SNARE complex, while SNAP-25A (Δ9) loses most of its function, although it can still form a SDS-resistant SNARE complex as the wild-type SNAP-25. CW-EPR spectra validate the unstable structures of the SNARE complex formed by SNAP-25 mutants. We propose that the truncated SNAP-25 mutants will disrupt the assembly of the SNARE core complex, and then inhibit the synaptic membrane fusion accordingly.

  5. α-SNAP Interferes with the Zippering of the SNARE Protein Membrane Fusion Machinery

    Science.gov (United States)

    Park, Yongsoo; Vennekate, Wensi; Yavuz, Halenur; Preobraschenski, Julia; Hernandez, Javier M.; Riedel, Dietmar; Walla, Peter Jomo; Jahn, Reinhard

    2014-01-01

    Neuronal exocytosis is mediated by soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins. Before fusion, SNARE proteins form complexes bridging the membrane followed by assembly toward the C-terminal membrane anchors, thus initiating membrane fusion. After fusion, the SNARE complex is disassembled by the AAA-ATPase N-ethylmaleimide-sensitive factor that requires the cofactor α-SNAP to first bind to the assembled SNARE complex. Using chromaffin granules and liposomes we now show that α-SNAP on its own interferes with the zippering of membrane-anchored SNARE complexes midway through the zippering reaction, arresting SNAREs in a partially assembled trans-complex and preventing fusion. Intriguingly, the interference does not result in an inhibitory effect on synaptic vesicles, suggesting that membrane properties also influence the final outcome of α-SNAP interference with SNARE zippering. We suggest that binding of α-SNAP to the SNARE complex affects the ability of the SNARE complex to harness energy or transmit force to the membrane. PMID:24778182

  6. Yeast Lipin 1 Orthologue Pah1p Regulates Vacuole Homeostasis and Membrane Fusion*

    Science.gov (United States)

    Sasser, Terry; Qiu, Quan-Sheng; Karunakaran, Surya; Padolina, Mark; Reyes, Anna; Flood, Blake; Smith, Sheena; Gonzales, Chad; Fratti, Rutilio A.

    2012-01-01

    Vacuole homotypic fusion requires a group of regulatory lipids that includes diacylglycerol, a fusogenic lipid that is produced through multiple metabolic pathways including the dephosphorylation of phosphatidic acid (PA). Here we examined the relationship between membrane fusion and PA phosphatase activity. Pah1p is the single yeast homologue of the Lipin family of PA phosphatases. Deletion of PAH1 was sufficient to cause marked vacuole fragmentation and abolish vacuole fusion. The function of Pah1p solely depended on its phosphatase activity as complementation studies showed that wild type Pah1p restored fusion, whereas the phosphatase dead mutant Pah1pD398E had no effect. We discovered that the lack of PA phosphatase activity blocked fusion by inhibiting the binding of SNAREs to Sec18p, an N-ethylmaleimide-sensitive factor homologue responsible for priming inactive cis-SNARE complexes. In addition, pah1Δ vacuoles were devoid of the late endosome/vacuolar Rab Ypt7p, the phosphatidylinositol 3-kinase Vps34p, and Vps39p, a subunit of the HOPS (homotypic fusion and vacuole protein sorting) tethering complex, all of which are required for vacuole fusion. The lack of Vps34p resulted in the absence of phosphatidylinositol 3-phosphate, a lipid required for SNARE activity and vacuole fusion. These findings demonstrate that Pah1p and PA phosphatase activity are critical for vacuole homeostasis and fusion. PMID:22121197

  7. Yeast lipin 1 orthologue pah1p regulates vacuole homeostasis and membrane fusion.

    Science.gov (United States)

    Sasser, Terry; Qiu, Quan-Sheng; Karunakaran, Surya; Padolina, Mark; Reyes, Anna; Flood, Blake; Smith, Sheena; Gonzales, Chad; Fratti, Rutilio A

    2012-01-13

    Vacuole homotypic fusion requires a group of regulatory lipids that includes diacylglycerol, a fusogenic lipid that is produced through multiple metabolic pathways including the dephosphorylation of phosphatidic acid (PA). Here we examined the relationship between membrane fusion and PA phosphatase activity. Pah1p is the single yeast homologue of the Lipin family of PA phosphatases. Deletion of PAH1 was sufficient to cause marked vacuole fragmentation and abolish vacuole fusion. The function of Pah1p solely depended on its phosphatase activity as complementation studies showed that wild type Pah1p restored fusion, whereas the phosphatase dead mutant Pah1p(D398E) had no effect. We discovered that the lack of PA phosphatase activity blocked fusion by inhibiting the binding of SNAREs to Sec18p, an N-ethylmaleimide-sensitive factor homologue responsible for priming inactive cis-SNARE complexes. In addition, pah1Δ vacuoles were devoid of the late endosome/vacuolar Rab Ypt7p, the phosphatidylinositol 3-kinase Vps34p, and Vps39p, a subunit of the HOPS (homotypic fusion and vacuole protein sorting) tethering complex, all of which are required for vacuole fusion. The lack of Vps34p resulted in the absence of phosphatidylinositol 3-phosphate, a lipid required for SNARE activity and vacuole fusion. These findings demonstrate that Pah1p and PA phosphatase activity are critical for vacuole homeostasis and fusion.

  8. Convenient synthesis and application of versatile nucleic acid lipid membrane anchors in the assembly and fusion of liposomes

    DEFF Research Database (Denmark)

    Ries, Oliver; Löffler, Philipp M. G.; Vogel, Stefan

    2015-01-01

    Hydrophobic moieties like lipid membrane anchors are highly demanded modifications for nucleic acid oligomers. Membrane-anchor modified oligonucleotides are applicable in biomedicine leading to new delivery strategies as well as in biophysical investigations towards assembly and fusion of liposom...

  9. Trans-complex formation by proteolipid channels in the terminal phase of membrane fusion

    DEFF Research Database (Denmark)

    Peters, C; Bayer, M J; Bühler, S

    2001-01-01

    -complex formation occurs downstream from trans-SNARE pairing, and depends on both the Rab-GTPase Ypt7 and calmodulin. The maintenance of existing complexes and completion of fusion are independent of trans-SNARE pairs. Reconstituted proteolipids form sealed channels, which can expand to form aqueous pores in a Ca2......+/calmodulin-dependent fashion. V0 trans-complexes may therefore form a continuous, proteolipid-lined channel at the fusion site. We propose that radial expansion of such a protein pore may be a mechanism for intracellular membrane fusion....

  10. Homotypic vacuole fusion in yeast requires organelle acidification and not the V-ATPase membrane domain.

    Science.gov (United States)

    Coonrod, Emily M; Graham, Laurie A; Carpp, Lindsay N; Carr, Tom M; Stirrat, Laura; Bowers, Katherine; Bryant, Nia J; Stevens, Tom H

    2013-11-25

    Studies of homotypic vacuole-vacuole fusion in the yeast Saccharomyces cerevisiae have been instrumental in determining the cellular machinery required for eukaryotic membrane fusion and have implicated the vacuolar H(+)-ATPase (V-ATPase). The V-ATPase is a multisubunit, rotary proton pump whose precise role in homotypic fusion is controversial. Models formulated from in vitro studies suggest that it is the proteolipid proton-translocating pore of the V-ATPase that functions in fusion, with further studies in worms, flies, zebrafish, and mice appearing to support this model. We present two in vivo assays and use a mutant V-ATPase subunit to establish that it is the H(+)-translocation/vacuole acidification function, rather than the physical presence of the V-ATPase, that promotes homotypic vacuole fusion in yeast. Furthermore, we show that acidification of the yeast vacuole in the absence of the V-ATPase rescues vacuole-fusion defects. Our results clarify the in vivo requirements of acidification for membrane fusion. Copyright © 2013 Elsevier Inc. All rights reserved.

  11. Studies on virus-induced cell fusion. Progress report, July 1, 1978-July 31, 1979

    Energy Technology Data Exchange (ETDEWEB)

    Person, S.

    1979-08-01

    We have determined the extent of fusion in mixed Herpes Simplex Virus type 1 (HSV-1) cell populations. Labeled, sparse, infected cells were surrounded by unlabeled, excess, uninfected (or infected) cells to determine if fusion inhibition is active when present in the same cell as fusion factor, when present in cells that do not contain fusion factor, or in both situations. We found that wild type infected cells fuse to the same extent with each other as with uninfected cells. Therefore fusion inhibitor is active when present in the same cell as fusion factor. Other experiments indicate that the inhibition activity can also cause a small decrease of fusion when fusion factor and fusion inhibitor are present in different, but neighboring, cells. Marked variations are found in the capacity of different cell types, such as human embryonic lung (HEDL) and HEp-2 to support fusion by particular syn mutants. The difference in fusion capacity of the two cell types may be due to a difference in a cellular factor affecting the activity of viral, fusion-associated molecules, and not to cell surface differences of the uninfected cells. (PCS)

  12. Radiation inactivation analysis of fusion and hemolysis by vesicular stomatitis virus

    Energy Technology Data Exchange (ETDEWEB)

    Bundo-Morita, K.; Gibson, S.; Lenard, J.

    1988-04-01

    Radiation inactivation analysis was used to determine the size of the functional unit responsible for fusion of vesicular stomatitis virus (VSV) with cardiolipin or phosphatidylcholine-phosphatidylethanolamine (1:1) liposomes, and for VSV-induced hemolysis. When radiation-insensitive background values were subtracted, the calculated functional units for all three activities were similar, ranging from 866 to 957 kDa, equivalent to about 15 G protein molecules. This is in striking contrast to results of similar studies with influenza and Sendai viruses, in which the functional unit corresponded in size to a single fusion protein monomer, and suggests that VSV fusion may occur by a different mechanism.

  13. Two coiled-coil domains of Chlamydia trachomatis IncA affect membrane fusion events during infection.

    Directory of Open Access Journals (Sweden)

    Erik Ronzone

    Full Text Available Chlamydia trachomatis replicates in a parasitophorous membrane-bound compartment called an inclusion. The inclusions corrupt host vesicle trafficking networks to avoid the degradative endolysosomal pathway but promote fusion with each other in order to sustain higher bacterial loads in a process known as homotypic fusion. The Chlamydia protein IncA (Inclusion protein A appears to play central roles in both these processes as it participates to homotypic fusion and inhibits endocytic SNARE-mediated membrane fusion. How IncA selectively inhibits or activates membrane fusion remains poorly understood. In this study, we analyzed the spatial and molecular determinants of IncA's fusogenic and inhibitory functions. Using a cell-free membrane fusion assay, we found that inhibition of SNARE-mediated fusion requires IncA to be on the same membrane as the endocytic SNARE proteins. IncA displays two coiled-coil domains showing high homology with SNARE proteins. Domain swap and deletion experiments revealed that although both these domains are capable of independently inhibiting SNARE-mediated fusion, these two coiled-coil domains cooperate in mediating IncA multimerization and homotypic membrane interaction. Our results support the hypothesis that Chlamydia employs SNARE-like virulence factors that positively and negatively affect membrane fusion and promote infection.

  14. Nuclear envelope breakdown induced by herpes simplex virus type 1 involves the activity of viral fusion proteins.

    Science.gov (United States)

    Maric, Martina; Haugo, Alison C; Dauer, William; Johnson, David; Roller, Richard J

    2014-07-01

    Herpesvirus infection reorganizes components of the nuclear lamina usually without loss of integrity of the nuclear membranes. We report that wild-type HSV infection can cause dissolution of the nuclear envelope in transformed mouse embryonic fibroblasts that do not express torsinA. Nuclear envelope breakdown is accompanied by an eight-fold inhibition of virus replication. Breakdown of the membrane is much more limited during infection with viruses that lack the gB and gH genes, suggesting that breakdown involves factors that promote fusion at the nuclear membrane. Nuclear envelope breakdown is also inhibited during infection with virus that does not express UL34, but is enhanced when the US3 gene is deleted, suggesting that envelope breakdown may be enhanced by nuclear lamina disruption. Nuclear envelope breakdown cannot compensate for deletion of the UL34 gene suggesting that mixing of nuclear and cytoplasmic contents is insufficient to bypass loss of the normal nuclear egress pathway. Copyright © 2014 Elsevier Inc. All rights reserved.

  15. Membrane fusion is induced by a distinct peptide sequence of the sea urchin fertilization protein bindin

    NARCIS (Netherlands)

    Ulrich, AS; Glabe, CG; Hoekstra, D

    1998-01-01

    Fertilization in the sea urchin is mediated by the membrane-associated acrosomal protein bindin, which plays a key role in the adhesion and fusion between sperm and egg. We have investigated the structure/function relationship of an 18-amino acid peptide fragment "B18," which represents the minimal

  16. Amino Acid substitutions in matrix, fusion and hemagglutinin proteins of wild measles virus for adaptation to vero cells.

    Science.gov (United States)

    Xin, Ji Yi; Ihara, Toshiaki; Komase, Katsuhiro; Nakayama, Tetsuo

    2011-01-01

    Wild-type measles virus (MV) is isolated in B95a but not in Vero cells. Through an adaptation process of wild-type MV to Vero cells, several amino acid substitutions were reported. Six strains were adapted to Vero cells and membrane (M), fusion (F) and hemagglutinin (H) genes were sequenced. Cell fusion was assessed and recombinant MVs were constructed, having wild-type H or M gene with or without mutations. No F gene substitution was noted. Amino-acid substitutions at positions 481 from Asn to Tyr (N481Y) and 546 from Ser to Gly (S546G) were observed in the H protein. Glu at position 89 of the M protein was substituted for Gly (E89G) and two mutations were noted at positions 62 (S62R) and 83 (S83P) in M protein. Recombinant viruses with mutation(s) detected in Vero-adapted strains induced a cytopathic effect and grew well in Vero cells, but those with the wild type did not. Recombinant viruses with mutation(s) demonstrated lower viral growth in B95a cells. Substitutions of E89G, S62R and S83P of the M protein were newly observed through adaptation to Vero cells, besides the mutations described in previous reports, with varying adaptation for each strain. Copyright © 2011 S. Karger AG, Basel.

  17. Respiratory Syncytial Virus Associated Myocarditis Requiring Venoarterial Extracorporeal Membrane Oxygenation

    Directory of Open Access Journals (Sweden)

    Anamaria Milas

    2017-01-01

    Full Text Available Severe fulminant myocarditis causing cardiogenic shock can be a rapidly progressing, life threatening condition. Respiratory syncytial virus (RSV is a very rare infectious culprit infrequently described in medical literature as a cause of myocarditis, particularly in adults. We present a case of acute fulminant myocarditis in a patient with PCR positive RSV infection requiring venoarterial extracorporeal membrane oxygenation (VA-ECMO.

  18. Virus assembly and plasma membrane domains: which came first?

    Science.gov (United States)

    Kerviel, A; Thomas, A; Chaloin, L; Favard, C; Muriaux, D

    2013-02-01

    Viral assembly is a key step in the virus life cycle. In this review, we focus mainly on the ability of retroviruses, especially HIV-1, to assemble at the plasma membrane of their host cells. The assembly process of RNA enveloped viruses necessitates a fine orchestration between the different viral components and specific interactions between viral proteins and lipids of the host cell membrane. Searching for a comparison with another RNA enveloped virus, we refer to influenza virus to show how it could share (or not) some common features with HIV-1 assembly since both viruses are believed to assemble mainly in raft microdomains. We also discuss the role of RNA and the cellular actin cytoskeleton in enhancing these viral assembly processes. Finally, based on the literature and on new results we have obtained by molecular docking, we propose another mechanism for HIV-1 assembly in membrane domains. This mechanism involves the trapping of acidic lipids by the viral Gag protein by means of ionic protein-lipid interactions, inducing thereby formation of acidic lipid-enriched microdomains (ALEM). Copyright © 2012 Elsevier B.V. All rights reserved.

  19. Coarse-grained molecular dynamics study of membrane fusion: Curvature effects on free energy barriers along the stalk mechanism

    Energy Technology Data Exchange (ETDEWEB)

    Kawamoto, Shuhei; Shinoda, Wataru, E-mail: w.shinoda@apchem.nagoya-u.ac.jp [Department of Applied Chemistry, Nagoya University, Furo-cho, Chikusa-ku, Nagoya, Aichi 464-8603 (Japan); Klein, Michael L. [Institute for Computational Molecular Science, Temple University, SERC Building 1925 North 12th Street, Philadelphia, Pennsylvania 19122 (United States)

    2015-12-28

    The effects of membrane curvature on the free energy barrier for membrane fusion have been investigated using coarse-grained molecular dynamics (CG-MD) simulations, assuming that fusion takes place through a stalk intermediate. Free energy barriers were estimated for stalk formation as well as for fusion pore formation using the guiding potential method. Specifically, the three different geometries of two apposed membranes were considered: vesicle–vesicle, vesicle–planar, and planar–planar membranes. The free energy barriers for the resulting fusion were found to depend importantly on the fusing membrane geometries; the lowest barrier was obtained for vesicular membranes. Further, lipid sorting was observed in fusion of the mixed membranes of dimyristoyl phosphatidylcholine and dioleoyl phosphatidylethanolamine (DOPE). Specifically, DOPE molecules were found to assemble around the stalk to support the highly negative curved membrane surface. A consistent result for lipid sorting was observed when a simple continuum model (CM) was used, where the Helfrich energy and mixing entropy of the lipids were taken into account. However, the CM predicts a much higher free energy barrier than found using CG-MD. This discrepancy originates from the conformational changes of lipids, which were not considered in the CM. The results of the CG-MD simulations reveal that a large conformational change in the lipid takes place around the stalk region, which results in a reduction of free energy barriers along the stalk mechanism of membrane fusion.

  20. Coarse-grained molecular dynamics study of membrane fusion: Curvature effects on free energy barriers along the stalk mechanism.

    Science.gov (United States)

    Kawamoto, Shuhei; Klein, Michael L; Shinoda, Wataru

    2015-12-28

    The effects of membrane curvature on the free energy barrier for membrane fusion have been investigated using coarse-grained molecular dynamics (CG-MD) simulations, assuming that fusion takes place through a stalk intermediate. Free energy barriers were estimated for stalk formation as well as for fusion pore formation using the guiding potential method. Specifically, the three different geometries of two apposed membranes were considered: vesicle-vesicle, vesicle-planar, and planar-planar membranes. The free energy barriers for the resulting fusion were found to depend importantly on the fusing membrane geometries; the lowest barrier was obtained for vesicular membranes. Further, lipid sorting was observed in fusion of the mixed membranes of dimyristoyl phosphatidylcholine and dioleoyl phosphatidylethanolamine (DOPE). Specifically, DOPE molecules were found to assemble around the stalk to support the highly negative curved membrane surface. A consistent result for lipid sorting was observed when a simple continuum model (CM) was used, where the Helfrich energy and mixing entropy of the lipids were taken into account. However, the CM predicts a much higher free energy barrier than found using CG-MD. This discrepancy originates from the conformational changes of lipids, which were not considered in the CM. The results of the CG-MD simulations reveal that a large conformational change in the lipid takes place around the stalk region, which results in a reduction of free energy barriers along the stalk mechanism of membrane fusion.

  1. Coarse-grained molecular dynamics study of membrane fusion: Curvature effects on free energy barriers along the stalk mechanism

    Science.gov (United States)

    Kawamoto, Shuhei; Klein, Michael L.; Shinoda, Wataru

    2015-12-01

    The effects of membrane curvature on the free energy barrier for membrane fusion have been investigated using coarse-grained molecular dynamics (CG-MD) simulations, assuming that fusion takes place through a stalk intermediate. Free energy barriers were estimated for stalk formation as well as for fusion pore formation using the guiding potential method. Specifically, the three different geometries of two apposed membranes were considered: vesicle-vesicle, vesicle-planar, and planar-planar membranes. The free energy barriers for the resulting fusion were found to depend importantly on the fusing membrane geometries; the lowest barrier was obtained for vesicular membranes. Further, lipid sorting was observed in fusion of the mixed membranes of dimyristoyl phosphatidylcholine and dioleoyl phosphatidylethanolamine (DOPE). Specifically, DOPE molecules were found to assemble around the stalk to support the highly negative curved membrane surface. A consistent result for lipid sorting was observed when a simple continuum model (CM) was used, where the Helfrich energy and mixing entropy of the lipids were taken into account. However, the CM predicts a much higher free energy barrier than found using CG-MD. This discrepancy originates from the conformational changes of lipids, which were not considered in the CM. The results of the CG-MD simulations reveal that a large conformational change in the lipid takes place around the stalk region, which results in a reduction of free energy barriers along the stalk mechanism of membrane fusion.

  2. Autographa californica multiple nucleopolyhedrovirus GP64 protein: Analysis of domain I and V amino acid interactions and membrane fusion activity

    Energy Technology Data Exchange (ETDEWEB)

    Yu, Qianlong [State Key Laboratory of Crop Stress Biology for Arid Areas, Key Laboratory of Northwest Loess Plateau Crop Pest Management of Ministry of Agriculture, College of Plant Protection, Northwest A& F University, Yangling, Shaanxi 712100 (China); Blissard, Gary W. [Boyce Thompson Institute, Cornell University, Ithaca, NY 14853, United State (United States); Liu, Tong-Xian [State Key Laboratory of Crop Stress Biology for Arid Areas, Key Laboratory of Northwest Loess Plateau Crop Pest Management of Ministry of Agriculture, College of Plant Protection, Northwest A& F University, Yangling, Shaanxi 712100 (China); Li, Zhaofei, E-mail: zhaofeili73@outlook.com [State Key Laboratory of Crop Stress Biology for Arid Areas, Key Laboratory of Northwest Loess Plateau Crop Pest Management of Ministry of Agriculture, College of Plant Protection, Northwest A& F University, Yangling, Shaanxi 712100 (China)

    2016-01-15

    The Autographa californica multiple nucleopolyhedrovirus GP64 is a class III viral fusion protein. Although the post-fusion structure of GP64 has been solved, its pre-fusion structure and the detailed mechanism of conformational change are unknown. In GP64, domain V is predicted to interact with two domain I segments that flank fusion loop 2. To evaluate the significance of the amino acids involved in these interactions, we examined 24 amino acid positions that represent interacting and conserved residues within domains I and V. In several cases, substitution of a single amino acid involved in a predicted interaction disrupted membrane fusion activity, but no single amino acid pair appears to be absolutely required. We identified 4 critical residues in domain V (G438, W439, T452, and T456) that are important for membrane fusion, and two residues (G438 and W439) that appear to be important for formation or stability of the pre-fusion conformation of GP64. - Highlights: • The baculovirus envelope glycoprotein GP64 is a class III viral fusion protein. • The detailed mechanism of conformational change of GP64 is unknown. • We analyzed 24 positions that might stabilize the post-fusion structure of GP64. • We identified 4 residues in domain V that were critical for membrane fusion. • Two residues are critical for formation of the pre-fusion conformation of GP64.

  3. β2 adrenergic receptor fluorescent protein fusions traffic to the plasma membrane and retain functionality.

    Directory of Open Access Journals (Sweden)

    Jaclyn Bubnell

    Full Text Available Green fluorescent protein (GFP has proven useful for the study of protein interactions and dynamics for the last twenty years. A variety of new fluorescent proteins have been developed that expand the use of available excitation spectra. We have undertaken an analysis of seven of the most useful fluorescent proteins (XFPs, Cerulean (and mCerulean3, Teal, GFP, Venus, mCherry and TagRFP657, as fusions to the archetypal G-protein coupled receptor, the β2 adrenergic receptor (β2AR. We have characterized these β2AR::XFP fusions in respect to membrane trafficking and G-protein activation. We noticed that in the mouse neural cell line, OP 6, that membrane bound β2AR::XFP fusions robustly localized in the filopodia identical to gap::XFP fusions. All β2AR::XFP fusions show responses indistinguishable from each other and the non-fused form after isoprenaline exposure. Our results provide a platform by which G-protein coupled receptors can be dissected for their functionality.

  4. Sequential analysis of trans-SNARE formation in intracellular membrane fusion.

    Directory of Open Access Journals (Sweden)

    Kannan Alpadi

    2012-01-01

    Full Text Available SNARE complexes are required for membrane fusion in the endomembrane system. They contain coiled-coil bundles of four helices, three (Q(a, Q(b, and Q(c from target (t-SNAREs and one (R from the vesicular (v-SNARE. NSF/Sec18 disrupts these cis-SNARE complexes, allowing reassembly of their subunits into trans-SNARE complexes and subsequent fusion. Studying these reactions in native yeast vacuoles, we found that NSF/Sec18 activates the vacuolar cis-SNARE complex by selectively displacing the vacuolar Q(a SNARE, leaving behind a Q(bcR subcomplex. This subcomplex serves as an acceptor for a Q(a SNARE from the opposite membrane, leading to Q(a-Q(bcR trans-complexes. Activity tests of vacuoles with diagnostic distributions of inactivating mutations over the two fusion partners confirm that this distribution accounts for a major share of the fusion activity. The persistence of the Q(bcR cis-complex and the formation of the Q(a-Q(bcR trans-complex are both sensitive to the Rab-GTPase inhibitor, GDI, and to mutations in the vacuolar tether complex, HOPS (HOmotypic fusion and vacuolar Protein Sorting complex. This suggests that the vacuolar Rab-GTPase, Ypt7, and HOPS restrict cis-SNARE disassembly and thereby bias trans-SNARE assembly into a preferred topology.

  5. Sequential Analysis of Trans-SNARE Formation in Intracellular Membrane Fusion

    Science.gov (United States)

    Alpadi, Kannan; Kulkarni, Aditya; Comte, Veronique; Reinhardt, Monique; Schmidt, Andrea; Namjoshi, Sarita; Mayer, Andreas; Peters, Christopher

    2012-01-01

    SNARE complexes are required for membrane fusion in the endomembrane system. They contain coiled-coil bundles of four helices, three (Qa, Qb, and Qc) from target (t)-SNAREs and one (R) from the vesicular (v)-SNARE. NSF/Sec18 disrupts these cis-SNARE complexes, allowing reassembly of their subunits into trans-SNARE complexes and subsequent fusion. Studying these reactions in native yeast vacuoles, we found that NSF/Sec18 activates the vacuolar cis-SNARE complex by selectively displacing the vacuolar Qa SNARE, leaving behind a QbcR subcomplex. This subcomplex serves as an acceptor for a Qa SNARE from the opposite membrane, leading to Qa-QbcR trans-complexes. Activity tests of vacuoles with diagnostic distributions of inactivating mutations over the two fusion partners confirm that this distribution accounts for a major share of the fusion activity. The persistence of the QbcR cis-complex and the formation of the Qa-QbcR trans-complex are both sensitive to the Rab-GTPase inhibitor, GDI, and to mutations in the vacuolar tether complex, HOPS (HOmotypic fusion and vacuolar Protein Sorting complex). This suggests that the vacuolar Rab-GTPase, Ypt7, and HOPS restrict cis-SNARE disassembly and thereby bias trans-SNARE assembly into a preferred topology. PMID:22272185

  6. On the entry of an emerging arbovirus into host cells: Mayaro virus takes the highway to the cytoplasm through fusion with early endosomes and caveolae-derived vesicles

    Directory of Open Access Journals (Sweden)

    Carlos A.M. Carvalho

    2017-04-01

    Full Text Available Mayaro virus (MAYV is an emergent sylvatic alphavirus in South America, related to sporadic outbreaks of a chikungunya-like human febrile illness accompanied by severe arthralgia. Despite its high potential for urban emergence, MAYV is still an obscure virus with scarce information about its infection cycle, including the corresponding early events. Even for prototypical alphaviruses, the cell entry mechanism still has some rough edges to trim: although clathrin-mediated endocytosis is quoted as the putative route, alternative paths as distinct as direct virus genome injection through the cell plasma membrane seems to be possible. Our aim was to clarify crucial details on the entry route exploited by MAYV to gain access into the host cell. Tracking the virus since its first contact with the surface of Vero cells by fluorescence microscopy, we show that its entry occurs by a fast endocytic process and relies on fusion with acidic endosomal compartments. Moreover, blocking clathrin-mediated endocytosis or depleting cholesterol from the cell membrane leads to a strong inhibition of viral infection, as assessed by plaque assays. Following this clue, we found that early endosomes and caveolae-derived vesicles are both implicated as target membranes for MAYV fusion. Our findings unravel the very first events that culminate in a productive infection by MAYV and shed light on potential targets for a rational antiviral therapy, besides providing a better comprehension of the entry routes exploited by alphaviruses to get into the cell.

  7. The tail domain of tomosyn controls membrane fusion through tomosyn displacement by VAMP2

    Energy Technology Data Exchange (ETDEWEB)

    Yamamoto, Yasunori; Fujikura, Kohei; Sakaue, Mio; Okimura, Kenjiro; Kobayashi, Yuta; Nakamura, Toshihiro [Division of Membrane Dynamics, Department of Physiology and Cell Biology, Kobe University Graduate School of Medicine, Kobe 650-0017 (Japan); Sakisaka, Toshiaki, E-mail: sakisaka@med.kobe-u.ac.jp [Division of Membrane Dynamics, Department of Physiology and Cell Biology, Kobe University Graduate School of Medicine, Kobe 650-0017 (Japan)

    2010-08-13

    Research highlights: {yields} The tail domain of tomosyn has no effect on the tomosyn-SNARE complex formation. {yields} The tail domain binding to the VAMP-like domain allows VAMP2 to displace tomosyn. {yields} Tomosyn displacement by VAMP2 leads to SNARE complex formation. {yields} The SNARE complex formation drives membrane fusion. -- Abstract: Neurotransmitter release is regulated by SNARE complex-mediated synaptic vesicle fusion. Tomosyn sequesters target SNAREs (t-SNAREs) through its C-terminal VAMP-like domain (VLD). Cumulative biochemical results suggest that the tomosyn-SNARE complex is so tight that VAMP2 cannot displace tomosyn. Based on these results, the tomosyn-SNARE complex has been believed to be a dead-end complex to inhibit neurotransmitter release. On the other hand, some studies using siRNA depletion of tomosyn suggest that tomosyn positively regulates exocytosis. Therefore, it is still controversial whether tomosyn is a simple inhibitor for neurotransmitter release. We recently reported that the inhibitory activity of tomosyn is regulated by the tail domain binding to the VLD. In this study, we employed the liposome fusion assay in order to further understand modes of action of tomosyn in detail. The tail domain unexpectedly had no effect on binding of the VLD to t-SNARE-bearing liposomes. Nonetheless, the tail domain decreased the inhibitory activity of the VLD on the SNARE complex-mediated liposome fusion. These results indicate that the tail domain controls membrane fusion through tomosyn displacement by VAMP2. Deletion of the tail domain-binding region in the VLD retained the binding to t-SNAREs and promoted the liposome fusion. Together, we propose here a novel mechanism of tomosyn that controls synaptic vesicle fusion positively by serving as a placeholder for VAMP2.

  8. The lipidomes of vesicular stomatitis virus, semliki forest virus, and the host plasma membrane analyzed by quantitative shotgun mass spectrometry

    DEFF Research Database (Denmark)

    Kalvodova, Lucie; Sampaio, Julio L; Cordo, Sandra

    2009-01-01

    Although enveloped virus assembly in the host cell is a crucial step in the virus life cycle, it remains poorly understood. One issue is how viruses include lipids in their membranes during budding from infected host cells. To analyze this issue, we took advantage of the fact that baby hamster...... kidney cells can be infected by two different viruses, namely, vesicular stomatitis virus and Semliki Forest virus, from the Rhabdoviridae and Togaviridae families, respectively. We purified the host plasma membrane and the two different viruses after exit from the host cells and analyzed the lipid...

  9. Residues in the hendra virus fusion protein transmembrane domain are critical for endocytic recycling.

    Science.gov (United States)

    Popa, Andreea; Carter, James R; Smith, Stacy E; Hellman, Lance; Fried, Michael G; Dutch, Rebecca Ellis

    2012-03-01

    Hendra virus is a highly pathogenic paramyxovirus classified as a biosafety level four agent. The fusion (F) protein of Hendra virus is critical for promoting viral entry and cell-to-cell fusion. To be fusogenically active, Hendra virus F must undergo endocytic recycling and cleavage by the endosomal/lysosomal protease cathepsin L, but the route of Hendra virus F following internalization and the recycling signals involved are poorly understood. We examined the intracellular distribution of Hendra virus F following endocytosis and showed that it is primarily present in Rab5- and Rab4-positive endosomal compartments, suggesting that cathepsin L cleavage occurs in early endosomes. Hendra virus F transmembrane domain (TMD) residues S490 and Y498 were found to be important for correct Hendra virus F recycling, with the hydroxyl group of S490 and the aromatic ring of Y498 important for this process. In addition, changes in association of isolated Hendra virus F TMDs correlated with alterations to Hendra virus F recycling, suggesting that appropriate TMD interactions play an important role in endocytic trafficking.

  10. Production of FMDV virus-like particles by a SUMO fusion protein approach in Escherichia coli

    Directory of Open Access Journals (Sweden)

    Liang Shu-Mei

    2009-08-01

    Full Text Available Abstract Virus-like particles (VLPs are formed by the self-assembly of envelope and/or capsid proteins from many viruses. Some VLPs have been proven successful as vaccines, and others have recently found applications as carriers for foreign antigens or as scaffolds in nanoparticle biotechnology. However, production of VLP was usually impeded due to low water-solubility of recombinant virus capsid proteins. Previous studies revealed that virus capsid and envelope proteins were often posttranslationally modified by SUMO in vivo, leading into a hypothesis that SUMO modification might be a common mechanism for virus proteins to retain water-solubility or prevent improper self-aggregation before virus assembly. We then propose a simple approach to produce VLPs of viruses, e.g., foot-and-mouth disease virus (FMDV. An improved SUMO fusion protein system we developed recently was applied to the simultaneous expression of three capsid proteins of FMDV in E. coli. The three SUMO fusion proteins formed a stable heterotrimeric complex. Proteolytic removal of SUMO moieties from the ternary complexes resulted in VLPs with size and shape resembling the authentic FMDV. The method described here can also apply to produce capsid/envelope protein complexes or VLPs of other disease-causing viruses.

  11. Analysis of the fusion protein gene of Newcastle disease viruses isolated in Japan.

    Science.gov (United States)

    Mase, Masaji; Murayama, Kazunori; Karino, Ayako; Inoue, Toshikazu

    2011-01-01

    The complete nucleotide sequences of the fusion (F) protein gene of Newcastle disease viruses (NDV) isolated in Japan from 1930 to 2007 (45 strains total) were determined and genetically analyzed. In the deduced amino acid sequences of fusion protein, the 5 potential asparagine-linked glycosylation sites and 10 cysteine residues were all conserved in the NDV examined in this study. The major epitopes involved in virus neutralization are conserved in most of the NDV strains isolated in Japan except a few strains. By virus neutralization test, no major antigenic differences were observed among representative strains of each genotype in Japan. All chickens vaccinated with the B1 strain survived without clinical signs after challenge with 2 NDV strains isolated in Japan (velogenic strains, JP/Ibaraki/2000 and JP/Kagoshima/91), which possess amino acids substitutions involved in virus neutralization in the F protein gene.

  12. Tri-membrane nanoparticles produced by combining liposome fusion and a novel patchwork of bicelles to overcome endosomal and nuclear membrane barriers to cargo delivery.

    Science.gov (United States)

    Yamada, Asako; Mitsueda, Asako; Hasan, Mahadi; Ueda, Miho; Hama, Susumu; Warashina, Shota; Nakamura, Takashi; Harashima, Hideyoshi; Kogure, Kentaro

    2016-03-01

    Membrane fusion is a rational strategy for crossing intracellular membranes that present barriers to liposomal nanocarrier-mediated delivery of plasmid DNA into the nucleus of non-dividing cells, such as dendritic cells. Based on this strategy, we previously developed nanocarriers consisting of a nucleic acid core particle coated with four lipid membranes [Akita, et al., Biomaterials, 2009, 30, 2940-2949]. However, including the endosomal membrane and two nuclear membranes, cells possess three intracellular membranous barriers. Thus, after entering the nucleus, nanoparticles coated with four membranes would still have one lipid membrane remaining, and could impede cargo delivery. Until now, coating a core particle with an odd number of lipid membranes was challenging. To produce nanocarriers with an odd number of lipid membranes, we developed a novel coating method involving lipid nano-discs, also known as bicelles, as a material for packaging DNA in a carrier with an odd number of lipid membranes. In this procedure, bicelles fuse to form an outer coating that resembles a patchwork quilt, which allows the preparation of nanoparticles coated with only three lipid membranes. Moreover, the transfection activity of dendritic cells with these three-membrane nanoparticles was higher than that for nanoparticles coated with four lipid membranes. In summary, we developed novel nanoparticles coated with an odd number of lipid membranes using the novel "patchwork-packaging method" to deliver plasmid DNA into the nucleus via membrane fusion.

  13. Nuclear inner membrane fusion facilitated by yeast Jem1p is required for spindle pole body fusion but not for the first mitotic nuclear division during yeast mating.

    Science.gov (United States)

    Nishikawa, Shuh-ichi; Hirata, Aiko; Endo, Toshiya

    2008-11-01

    During mating of budding yeast, Saccharomyces cerevisiae, two haploid nuclei fuse to produce a diploid nucleus. The process of nuclear fusion requires two J proteins, Jem1p in the endoplasmic reticulum (ER) lumen and Sec63p, which forms a complex with Sec71p and Sec72p, in the ER membrane. Zygotes of mutants defective in the functions of Jem1p or Sec63p contain two haploid nuclei that were closely apposed but failed to fuse. Here we analyzed the ultrastructure of nuclei in jem1 Delta and sec71 Delta mutant zygotes using electron microscope with the freeze-substituted fixation method. Three-dimensional reconstitution of nuclear structures from electron microscope serial sections revealed that Jem1p facilitates nuclear inner-membrane fusion and spindle pole body (SPB) fusion while Sec71p facilitates nuclear outer-membrane fusion. Two haploid SPBs that failed to fuse could duplicate, and mitotic nuclear division of the unfused haploid nuclei started in jem1 Delta and sec71 Delta mutant zygotes. This observation suggests that nuclear inner-membrane fusion is required for SPB fusion, but not for SPB duplication in the first mitotic cell division.

  14. Membrane fusion induced by the membrane mobility agent, A2C. Differentiation between fusible and non-fusible cells. Transfer of fusibility.

    Science.gov (United States)

    Tavassoli, M; Kosower, N S; Halverson, C; Aoki, M; Kosower, E M

    1980-10-02

    Red cells of different species respond differently to the treatment with the membrane mobility agent, A2C, with respect to both the A2C interaction and the subsequent cell-cell interaction. Depending on whether both, one or neither of the processes are effective, some red cells (e.g., nucleated Leghorn hen red cells, rat red cells) fuse easily, some (human red cells) show morphological changes but do not fuse, and others (nucleated Rock hen red cells) show little or no response. Mixed fusion (i.e., between fusible cells of different species) is readily obtained, indicating that no species-specific recognition sites are required for A2C-induced fusion. the potential for fusion is a transferable characteristic. In the presence of fusible cells, A2C induces both heterologous and homologous fusion of otherwise 'non-fusible' cells. Electron micrographs of fusing cells after treatment with A2C reveal 'onion-ring' structures ('whorls'), free of intramembranous protein particles but different from the smooth appearance of A(2)C particles. Whorls are considered to arise from fusion-potent membrane areas. Fusion is apparent at multiple sites along the contact line between apposed membranes. The postulated appearance of vesicle-like structures along the fusion line (Kosower, E.M., Kosower, N.S. and Wegman, P. (1977) Biochim. Biophys. Acta 471, 311-329) is confirmed by micrographs. The mechanism of this fusion process is duscussed and compared to other types of fusion process.

  15. Destabilization of a model membrane by a predicted fusion peptide of fertilin α

    Science.gov (United States)

    Schanck, A.; Brasseur, R.; Peuvot, J.

    1998-02-01

    The subunit of the guinea pig fertilin (previously known as PH-30, an integral membrane protein involved in sperm-egg binding and fusion) is predicted to be a potential fusion protein. The structure of this putative fusion protein was analysed by molecular modeling and we have found a peptidic sequence of 17 residues (D83{-P99}) organized in helix that inserts obliquely in lipid phases. The effect of this synthesized peptide was studied on a model membrane by 31P NMR and light scattering. It appears to increase the size of lipid vesicles and induces structural modifications. We interpret these observations as a destabilization of the lipid organization by this peptide because of its tilted insertion in phospholipid layers. This destabilization could favor membrane fusion. La sous-unité α de la fertiline du cochon d'inde (précédemment appelée PH-30, une protéine membranaire impliquée dans la liaison et la fusion ovule-spermatozoïde) est prédite comme étant une protéine de fusion potentielle. Nous avons analysé la structure de cette protéine par modélisation moléculaire et nous avons trouvé une séquence peptidique de 17 résidus (D83 P99) organisée en hélice qui s'insère de façon oblique dans une phase lipidique. L'effet de ce peptide synthétique a été étudié sur membrane modèle par RMN du 31P et par diffusion de la lumière. Il provoque une augmentation de taille de vésicules lipidiques et induit des modifications structurales. Nous interprétons ces observations en termes de déstabilisation de l'organisation lipidique par ce peptide à cause de son insertion oblique dans la couche lipidique. Cette déstabilisation pourrait favoriser la fusion membranaire.

  16. Interaction of a synthetic peptide corresponding to the N-terminus of canine distemper virus fusion protein with phospholipid vesicles: a biophysical study.

    Science.gov (United States)

    Aranda, Francisco J; Teruel, José A; Ortiz, Antonio

    2003-12-03

    The F protein of canine distemper virus (CDV) is a classic type I glycoprotein formed by two polypeptides, F1 and F2. The N-terminal regions of the F1 polypeptides of CDV, measles virus and other paramyxoviruses present moderate to high homology, supporting the existence of a high conservation within these structures, which emphasises its role in viral-host cell membrane fusion. This N-terminal polypeptide is usually termed the fusion peptide. The fusion peptides of most viral fusion-mediating glycoproteins contain a high proportion of hydrophobic amino acids, which facilitates its insertion into target membranes during fusion. In this work we report on the interaction of a 31-residue synthetic peptide (FP31) corresponding to the N terminus of CDV F1 protein with phospholipid membranes composed of various phospholipids, as studied by means of various biophysical techniques. FTIR investigation of FP31 secondary structure in aqueous medium and in membranes resulted in a major proportion of alpha-helical structure which increased upon membrane insertion. Differential scanning calorimetry (DSC) showed that the presence of concentrations of FP31 as low as 0.1 mol%, in mixtures with L-alpha-dimyristoylphosphatidylcholine (DMPC), L-alpha-dipalmitoylphosphatidylcholine (DPPC) and L-alpha-distearoylphosphatidylcholine (DSPC), already affected the thermotropic properties of the gel to liquid-crystalline phase transition. In mixtures with the three lipids, increasing the concentration of peptide made the pretransition to disappear, and lowered and broadened the main transition. This effect was slightly stronger as the acyl chain length of the phospholipid grew larger. In the corresponding partial phase diagrams, no immiscibilities or critical points were observed. FTIR showed that incorporation of 1 mol% of peptide in DPPC shifted the antisymmetric and symmetric CH2 stretching bands to higher values, indicating the existence of an additional disordering of the acyl chain

  17. Matrix protein 2 of influenza A virus blocks autophagosome fusion with lysosomes

    DEFF Research Database (Denmark)

    Gannagé, Monique; Dormann, Dorothee; Albrecht, Randy

    2009-01-01

    Influenza A virus is an important human pathogen causing significant morbidity and mortality every year and threatening the human population with epidemics and pandemics. Therefore, it is important to understand the biology of this virus to develop strategies to control its pathogenicity. Here, we...... demonstrate that influenza A virus inhibits macroautophagy, a cellular process known to be manipulated by diverse pathogens. Influenza A virus infection causes accumulation of autophagosomes by blocking their fusion with lysosomes, and one viral protein, matrix protein 2, is necessary and sufficient...... for this inhibition of autophagosome degradation. Macroautophagy inhibition by matrix protein 2 compromises survival of influenza virus-infected cells but does not influence viral replication. We propose that influenza A virus, which also encodes proapoptotic proteins, is able to determine the death of its host cell...

  18. Palmitoylation of SARS-CoV S protein is necessary for partitioning into detergent-resistant membranes and cell-cell fusion but not interaction with M protein

    Science.gov (United States)

    McBride, Corrin E.; Machamer, Carolyn E.

    2010-01-01

    Coronaviruses are enveloped RNA viruses that generally cause mild disease in humans. However, the recently emerged coronavirus that caused severe acute respiratory syndrome (SARS-CoV) is the most pathogenic human coronavirus discovered to date. The SARS-CoV spike (S) protein mediates virus entry by binding cellular receptors and inducing fusion between the viral envelope and the host cell membrane. Coronavirus S proteins are palmitoylated, which may affect function. Here, we created a non-palmitoylated SARS-CoV S protein by mutating all nine cytoplasmic cysteine residues. Palmitoylation of SARS-CoV S was required for partitioning into detergent-resistant membranes and for cell-cell fusion. Surprisingly, however, palmitoylation of S was not required for interaction with SARS-CoV M protein. This contrasts with the requirement for palmitoylation of mouse hepatitis virus S protein for interaction with M protein, and may point to important differences in assembly and infectivity of these two coronaviruses. PMID:20580052

  19. Sites of glucose transporter-4 vesicle fusion with the plasma membrane correlate spatially with microtubules.

    Directory of Open Access Journals (Sweden)

    Jennine M Dawicki-McKenna

    Full Text Available In adipocytes, vesicles containing glucose transporter-4 (GLUT4 redistribute from intracellular stores to the cell periphery in response to insulin stimulation. Vesicles then fuse with the plasma membrane, facilitating glucose transport into the cell. To gain insight into the details of microtubule involvement, we examined the spatial organization and dynamics of microtubules in relation to GLUT4 vesicle trafficking in living 3T3-L1 adipocytes using total internal reflection fluorescence (TIRF microscopy. Insulin stimulated an increase in microtubule density and curvature within the TIRF-illuminated region of the cell. The high degree of curvature and abrupt displacements of microtubules indicate that substantial forces act on microtubules. The time course of the microtubule density increase precedes that of the increase in intensity of fluorescently-tagged GLUT4 in this same region of the cell. In addition, portions of the microtubules are highly curved and are pulled closer to the cell cortex, as confirmed by Parallax microscopy. Microtubule disruption delayed and modestly reduced GLUT4 accumulation at the plasma membrane. Quantitative analysis revealed that fusions of GLUT4-containing vesicles with the plasma membrane, detected using insulin-regulated aminopeptidase with a pH-sensitive GFP tag (pHluorin, preferentially occur near microtubules. Interestingly, long-distance vesicle movement along microtubules visible at the cell surface prior to fusion does not appear to account for this proximity. We conclude that microtubules may be important in providing spatial information for GLUT4 vesicle fusion.

  20. Sites of Glucose Transporter-4 Vesicle Fusion with the Plasma Membrane Correlate Spatially with Microtubules

    Science.gov (United States)

    Dawicki-McKenna, Jennine M.; Goldman, Yale E.; Ostap, E. Michael

    2012-01-01

    In adipocytes, vesicles containing glucose transporter-4 (GLUT4) redistribute from intracellular stores to the cell periphery in response to insulin stimulation. Vesicles then fuse with the plasma membrane, facilitating glucose transport into the cell. To gain insight into the details of microtubule involvement, we examined the spatial organization and dynamics of microtubules in relation to GLUT4 vesicle trafficking in living 3T3-L1 adipocytes using total internal reflection fluorescence (TIRF) microscopy. Insulin stimulated an increase in microtubule density and curvature within the TIRF-illuminated region of the cell. The high degree of curvature and abrupt displacements of microtubules indicate that substantial forces act on microtubules. The time course of the microtubule density increase precedes that of the increase in intensity of fluorescently-tagged GLUT4 in this same region of the cell. In addition, portions of the microtubules are highly curved and are pulled closer to the cell cortex, as confirmed by Parallax microscopy. Microtubule disruption delayed and modestly reduced GLUT4 accumulation at the plasma membrane. Quantitative analysis revealed that fusions of GLUT4-containing vesicles with the plasma membrane, detected using insulin-regulated aminopeptidase with a pH-sensitive GFP tag (pHluorin), preferentially occur near microtubules. Interestingly, long-distance vesicle movement along microtubules visible at the cell surface prior to fusion does not appear to account for this proximity. We conclude that microtubules may be important in providing spatial information for GLUT4 vesicle fusion. PMID:22916292

  1. Gene transfer mediated by fusion protein hemagglutinin reconstituted in cationic lipid vesicles

    NARCIS (Netherlands)

    Schoen, P; Chonn, A; Cullis, PR; Wilschut, J; Scherrer, P

    Hemagglutinin, the membrane fusion protein of influenza virus,is known to mediate a low-pH-dependent fusion reaction between the viral envelope and the limiting membrane of the endosomal cell compartment following cellular uptake of the virus particles by receptor-mediated endocytosis. Here we

  2. Porcine reproductive and respiratory syndrome virus nonstructural protein 2 (nsp2) topology and selective isoform integration in artificial membranes

    Science.gov (United States)

    Membrane modification of host subcellular compartments is critical to the replication of many RNA viruses. Enveloped viruses additionally require the ability to requisition cellular membranes during egress for the development of infectious progeny. Porcine reproductive and respiratory syndrome virus...

  3. Triggering of the Newcastle Disease Virus Fusion Protein by a Chimeric Attachment Protein That Binds to Nipah Virus Receptors*

    Science.gov (United States)

    Mirza, Anne M.; Aguilar, Hector C.; Zhu, Qiyun; Mahon, Paul J.; Rota, Paul A.; Lee, Benhur; Iorio, Ronald M.

    2011-01-01

    The fusion (F) proteins of Newcastle disease virus (NDV) and Nipah virus (NiV) are both triggered by binding to receptors, mediated in both viruses by a second protein, the attachment protein. However, the hemagglutinin-neuraminidase (HN) attachment protein of NDV recognizes sialic acid receptors, whereas the NiV G attachment protein recognizes ephrinB2/B3 as receptors. Chimeric proteins composed of domains from the two attachment proteins have been evaluated for fusion-promoting activity with each F protein. Chimeras having NiV G-derived globular domains and NDV HN-derived stalks, transmembranes, and cytoplasmic tails are efficiently expressed, bind ephrinB2, and trigger NDV F to promote fusion in Vero cells. Thus, the NDV F protein can be triggered by binding to the NiV receptor, indicating that an aspect of the triggering cascade induced by the binding of HN to sialic acid is conserved in the binding of NiV G to ephrinB2. However, the fusion cascade for triggering NiV F by the G protein and that of triggering NDV F by the chimeras can be distinguished by differential exposure of a receptor-induced conformational epitope. The enhanced exposure of this epitope marks the triggering of NiV F by NiV G but not the triggering of NDV F by the chimeras. Thus, the triggering cascade for NiV G-F fusion may be more complex than that of NDV HN and F. This is consistent with the finding that reciprocal chimeras having NDV HN-derived heads and NiV G-derived stalks, transmembranes, and tails do not trigger either F protein for fusion, despite efficient cell surface expression and receptor binding. PMID:21460213

  4. Triggering of the newcastle disease virus fusion protein by a chimeric attachment protein that binds to Nipah virus receptors.

    Science.gov (United States)

    Mirza, Anne M; Aguilar, Hector C; Zhu, Qiyun; Mahon, Paul J; Rota, Paul A; Lee, Benhur; Iorio, Ronald M

    2011-05-20

    The fusion (F) proteins of Newcastle disease virus (NDV) and Nipah virus (NiV) are both triggered by binding to receptors, mediated in both viruses by a second protein, the attachment protein. However, the hemagglutinin-neuraminidase (HN) attachment protein of NDV recognizes sialic acid receptors, whereas the NiV G attachment protein recognizes ephrinB2/B3 as receptors. Chimeric proteins composed of domains from the two attachment proteins have been evaluated for fusion-promoting activity with each F protein. Chimeras having NiV G-derived globular domains and NDV HN-derived stalks, transmembranes, and cytoplasmic tails are efficiently expressed, bind ephrinB2, and trigger NDV F to promote fusion in Vero cells. Thus, the NDV F protein can be triggered by binding to the NiV receptor, indicating that an aspect of the triggering cascade induced by the binding of HN to sialic acid is conserved in the binding of NiV G to ephrinB2. However, the fusion cascade for triggering NiV F by the G protein and that of triggering NDV F by the chimeras can be distinguished by differential exposure of a receptor-induced conformational epitope. The enhanced exposure of this epitope marks the triggering of NiV F by NiV G but not the triggering of NDV F by the chimeras. Thus, the triggering cascade for NiV G-F fusion may be more complex than that of NDV HN and F. This is consistent with the finding that reciprocal chimeras having NDV HN-derived heads and NiV G-derived stalks, transmembranes, and tails do not trigger either F protein for fusion, despite efficient cell surface expression and receptor binding. © 2011 by The American Society for Biochemistry and Molecular Biology, Inc.

  5. A GALA lipopeptide mediates pH- and membrane charge dependent fusion with stable giant unilamellar vesicles

    DEFF Research Database (Denmark)

    Etzerodt, Thomas P.; Trier, Sofie; Henriksen, Jonas R.

    2012-01-01

    Peptides capable of mediating fusion between lipid membranes are widely observed in nature, and have attracted considerable attention in the liposome drug delivery field. However, studies that are proving the benefit of small synthetic fusion peptides as components in drug delivery systems remain...

  6. Low-pH-induced membrane fusion mediated by human metapneumovirus F protein is a rare, strain-dependent phenomenon

    NARCIS (Netherlands)

    S. Herfst (Sander); V. Mas (Vicente); L.S. Ver (Lorena); R.J. Wierda (Rutger); A.D.M.E. Osterhaus (Albert); R.A.M. Fouchier (Ron); J.A. Melero (José)

    2008-01-01

    textabstractMembrane fusion promoted by human metapneumovirus (HMPV) fusion (F) protein was suggested to require low pH (R. M. Schowalter, S. E. Smith, and R. E. Dutch, J. Virol. 80:10931-10941, 2006). Using prototype F proteins representing the four HMPV genetic lineages, we detected

  7. Molecular evolution of respiratory syncytial virus fusion gene, Canada, 2006-2010.

    Science.gov (United States)

    Papenburg, Jesse; Carbonneau, Julie; Hamelin, Marie-Ève; Isabel, Sandra; Bouhy, Xavier; Ohoumanne, Najwa; Déry, Pierre; Paes, Bosco A; Corbeil, Jacques; Bergeron, Michel G; De Serres, Gaston; Boivin, Guy

    2012-01-01

    To assess molecular evolution of the respiratory syncytial virus (RSV) fusion gene, we analyzed RSV-positive specimens from 123 children in Canada who did or did not receive RSV immunoprophylaxis (palivizumab) during 2006-2010. Resistance-conferring mutations within the palivizumab binding site occurred in 8.7% of palivizumab recipients and none of the nonrecipients.

  8. Deltabaculoviruses encode a functional type I budded virus envelope fusion protein

    Science.gov (United States)

    Envelope fusion proteins (F proteins) are major constituents of budded viruses (BVs) of alpha- and betabaculoviruses (Baculoviridae) and are essential for the systemic infection of insect larvae and insect cells in culture. An F protein homolog gene was absent in gammabaculoviruses. Here we show tha...

  9. Fusion of Legionella pneumophila outer membrane vesicles with eukaryotic membrane systems is a mechanism to deliver pathogen factors to host cell membranes.

    Science.gov (United States)

    Jäger, Jens; Keese, Susanne; Roessle, Manfred; Steinert, Michael; Schromm, Andra B

    2015-05-01

    The formation and release of outer membrane vesicles (OMVs) is a phenomenon observed in many bacteria, including Legionella pneumophila. During infection, this human pathogen primarily invades alveolar macrophages and replicates within a unique membrane-bound compartment termed Legionella-containing vacuole. In the current study, we analysed the membrane architecture of L. pneumophila OMVs by small-angle X-ray scattering and biophysically characterized OMV membranes. We investigated the interaction of L. pneumophila OMVs with model membranes by Förster resonance energy transfer and Fourier transform infrared spectroscopy. These experiments demonstrated the incorporation of OMV membrane material into liposomes composed of different eukaryotic phospholipids, revealing an endogenous property of OMVs to fuse with eukaryotic membranes. Cellular co-incubation experiments showed a dose- and time-dependent binding of fluorophore-labelled OMVs to macrophages. Trypan blue quenching experiments disclosed a rapid internalization of OMVs into macrophages at 37 and 4 °C. Purified OMVs induced tumour necrosis factor-α production in human macrophages at concentrations starting at 300 ng ml(-1). Experiments on HEK293-TLR2 and TLR4/MD-2 cell lines demonstrated a dominance of TLR2-dependent signalling pathways. In summary, we demonstrate binding, internalization and biological activity of L. pneumophila OMVs on human macrophages. Our data support OMV membrane fusion as a mechanism for the remote delivery of virulence factors to host cells. © 2014 John Wiley & Sons Ltd.

  10. Fusion

    CERN Document Server

    Mahaffey, James A

    2012-01-01

    As energy problems of the world grow, work toward fusion power continues at a greater pace than ever before. The topic of fusion is one that is often met with the most recognition and interest in the nuclear power arena. Written in clear and jargon-free prose, Fusion explores the big bang of creation to the blackout death of worn-out stars. A brief history of fusion research, beginning with the first tentative theories in the early 20th century, is also discussed, as well as the race for fusion power. This brand-new, full-color resource examines the various programs currently being funded or p

  11. Conformations and membrane-driven self-organization of rodlike fd virus particles on freestanding lipid membranes.

    Science.gov (United States)

    Petrova, Anastasiia B; Herold, Christoph; Petrov, Eugene P

    2017-10-11

    Membrane-mediated interactions and aggregation of colloidal particles adsorbed to responsive elastic membranes are challenging problems relevant for understanding the microscopic organization and dynamics of biological membranes. We experimentally study the behavior of rodlike semiflexible fd virus particles electrostatically adsorbed to freestanding cationic lipid membranes and find that their behavior can be controlled by tuning the membrane charge and ionic strength of the surrounding medium. Three distinct interaction regimes of rodlike virus particles with responsive elastic membranes can be observed. (i) A weakly charged freestanding cationic lipid bilayer in a low ionic strength medium represents a gentle quasi-2D substrate preserving the integrity, structure, and mechanical properties of the membrane-bound semiflexible fd virus, which under these conditions is characterized by a monomer length of 884 ± 4 nm and a persistence length of 2.5 ± 0.2 μm, in perfect agreement with its properties in bulk media. (ii) An increase in the membrane charge leads to the membrane-driven collapse of fd virus particles on freestanding lipid bilayers and lipid nanotubes into compact globules. (iii) When the membrane charge is low, and the mutual electrostatic repulsion of membrane-bound virus particles is screened to a considerable degree, membrane-driven self-organization of membrane-bound fd virus particles into long linear tip-to-tip aggregates showing dynamic self-assembly/disassembly and quasi-semiflexible behavior takes place. These observations are in perfect agreement with the results of recent theoretical and simulation studies predicting that membrane-mediated interactions can control the behavior of colloidal particles adsorbed on responsive elastic membranes.

  12. Dysregulated Glycoprotein B-Mediated Cell-Cell Fusion Disrupts Varicella-Zoster Virus and Host Gene Transcription during Infection

    Science.gov (United States)

    Yang, Edward; Arvin, Ann M.

    2016-01-01

    ABSTRACT The highly conserved herpesvirus glycoprotein complex gB/gH-gL mediates membrane fusion during virion entry and cell-cell fusion. Varicella-zoster virus (VZV) characteristically forms multinucleated cells, or syncytia, during the infection of human tissues, but little is known about this process. The cytoplasmic domain of VZV gB (gBcyt) has been implicated in cell-cell fusion regulation because a gB[Y881F] substitution causes hyperfusion. gBcyt regulation is necessary for VZV pathogenesis, as the hyperfusogenic mutant gB[Y881F] is severely attenuated in human skin xenografts. In this study, gBcyt-regulated fusion was investigated by comparing melanoma cells infected with wild-type-like VZV or hyperfusogenic mutants. The gB[Y881F] mutant exhibited dramatically accelerated syncytium formation in melanoma cells caused by fusion of infected cells with many uninfected cells, increased cytoskeleton reorganization, and rapid displacement of nuclei to dense central structures compared to pOka using live-cell confocal microscopy. VZV and human transcriptomes were concurrently investigated using whole transcriptome sequencing (RNA-seq) to identify viral and cellular responses induced when gBcyt regulation was disrupted by the gB[Y881F] substitution. The expression of four vital VZV genes, ORF61 and the genes for glycoproteins gC, gE, and gI, was significantly reduced at 36 h postinfection for the hyperfusogenic mutants. Importantly, hierarchical clustering demonstrated an association of differential gene expression with dysregulated gBcyt-mediated fusion. A subset of Ras GTPase genes linked to membrane remodeling were upregulated in cells infected with the hyperfusogenic mutants. These data implicate gBcyt in the regulation of gB fusion function that, if unmodulated, triggers cellular processes leading to hyperfusion that attenuates VZV infection. IMPORTANCE The highly infectious, human-restricted pathogen varicella-zoster virus (VZV) causes chickenpox and shingles

  13. Dysregulated Glycoprotein B-Mediated Cell-Cell Fusion Disrupts Varicella-Zoster Virus and Host Gene Transcription during Infection.

    Science.gov (United States)

    Oliver, Stefan L; Yang, Edward; Arvin, Ann M

    2017-01-01

    The highly conserved herpesvirus glycoprotein complex gB/gH-gL mediates membrane fusion during virion entry and cell-cell fusion. Varicella-zoster virus (VZV) characteristically forms multinucleated cells, or syncytia, during the infection of human tissues, but little is known about this process. The cytoplasmic domain of VZV gB (gBcyt) has been implicated in cell-cell fusion regulation because a gB[Y881F] substitution causes hyperfusion. gBcyt regulation is necessary for VZV pathogenesis, as the hyperfusogenic mutant gB[Y881F] is severely attenuated in human skin xenografts. In this study, gBcyt-regulated fusion was investigated by comparing melanoma cells infected with wild-type-like VZV or hyperfusogenic mutants. The gB[Y881F] mutant exhibited dramatically accelerated syncytium formation in melanoma cells caused by fusion of infected cells with many uninfected cells, increased cytoskeleton reorganization, and rapid displacement of nuclei to dense central structures compared to pOka using live-cell confocal microscopy. VZV and human transcriptomes were concurrently investigated using whole transcriptome sequencing (RNA-seq) to identify viral and cellular responses induced when gBcyt regulation was disrupted by the gB[Y881F] substitution. The expression of four vital VZV genes, ORF61 and the genes for glycoproteins gC, gE, and gI, was significantly reduced at 36 h postinfection for the hyperfusogenic mutants. Importantly, hierarchical clustering demonstrated an association of differential gene expression with dysregulated gBcyt-mediated fusion. A subset of Ras GTPase genes linked to membrane remodeling were upregulated in cells infected with the hyperfusogenic mutants. These data implicate gBcyt in the regulation of gB fusion function that, if unmodulated, triggers cellular processes leading to hyperfusion that attenuates VZV infection. The highly infectious, human-restricted pathogen varicella-zoster virus (VZV) causes chickenpox and shingles. Postherpetic

  14. Epstein-Barr Virus Fusion with Epithelial Cells Triggered by gB Is Restricted by a gL Glycosylation Site.

    Science.gov (United States)

    Möhl, Britta S; Chen, Jia; Park, Seo Jin; Jardetzky, Theodore S; Longnecker, Richard

    2017-12-01

    Epstein-Barr virus (EBV) entry into epithelial cells is mediated by the conserved core fusion machinery, composed of the fusogen gB and the receptor-binding complex gH/gL. The heterodimeric gH/gL complex binds to the EBV epithelial cell receptor or gp42, which binds to the B-cell receptor, triggering gB-mediated fusion of the virion envelope with cellular membranes. Our previous study found that the gL glycosylation mutant N69L/S71V had an epithelial cell-specific hyperfusogenic phenotype. To study the influence of this gL mutant on the initiation and kinetics of gB-driven epithelial cell fusion, we established a virus-free split-green fluorescent protein cell-cell fusion assay that enables real-time measurements of membrane fusion using live cells. The gL_N69L/S71V mutant had a large increase in epithelial cell fusion activity of up to 300% greater than that of wild-type gL starting at early time points. The hyperfusogenicity of the gL mutant was not a result of alterations in complex formation with gH or alterations in cellular localization. Moreover, the hyperfusogenic phenotype of the gL mutant correlated with the formation of enlarged syncytia. In summary, our present findings highlight an important role of gL in the kinetics of gB-mediated epithelial cell fusion, adding to previous findings indicating a direct interaction between gL and gB in EBV membrane fusion.IMPORTANCE EBV predominantly infects epithelial cells and B lymphocytes, which are the cells of origin for the EBV-associated malignancies Hodgkin and Burkitt lymphoma as well as nasopharyngeal carcinoma. Contrary to the other key players of the core fusion machinery, gL has the most elusive role during EBV-induced membrane fusion. We found that the glycosylation site N69/S71 of gL is involved in restricting epithelial cell fusion activity, strongly correlating with syncytium size. Interestingly, our data showed that the gL glycosylation mutant increases the fusion activity of the hyperfusogenic g

  15. Chimeric Bovine Respiratory Syncytial Virus with Attachment and Fusion Glycoproteins Replaced by Bovine Parainfluenza Virus Type 3 Hemagglutinin-Neuraminidase and Fusion Proteins

    Science.gov (United States)

    Stope, Matthias B.; Karger, Axel; Schmidt, Ulrike; Buchholz, Ursula J.

    2001-01-01

    Chimeric bovine respiratory syncytial viruses (BRSV) expressing glycoproteins of bovine parainfluenza virus type 3 (BPIV-3) instead of BRSV glycoproteins were generated from cDNA. In the BRSV antigenome cDNA, the open reading frames of the major BRSV glycoproteins, attachment protein G and fusion protein F, were replaced individually or together by those of the BPIV-3 hemagglutinin-neuraminidase (HN) and/or fusion (F) glycoproteins. Recombinant virus could not be recovered from cDNA when the BRSV F open reading frame was replaced by the BPIV-3 F open reading frame. However, cDNA recovery of the chimeric virus rBRSV-HNF, with both glycoproteins replaced simultaneously, and of the chimeric virus rBRSV-HN, with the BRSV G protein replaced by BPIV-3 HN, was successful. The replication rates of both chimeras were similar to that of standard rBRSV. Moreover, rBRSV-HNF was neutralized by antibodies specific for BPIV-3, but not by antibodies specific to BRSV, demonstrating that the BRSV glycoproteins can be functionally replaced by BPIV-3 glycoproteins. In contrast, rBRSV-HN was neutralized by BRSV-specific antisera, but not by BPIV-3 specific sera, showing that infection of rBRSV-HN is mediated by BRSV F. Hemadsorption of cells infected with rBRSV-HNF and rBRSV-HN proved that BPIV-3 HN protein expressed by rBRSV is functional. Colocalization of the BPIV-3 glycoproteins with BRSV M protein was demonstrated by confocal laser scan microscopy. Moreover, protein analysis revealed that the BPIV-3 glycoproteins were present in chimeric virions. Taken together, these data indicate that the heterologous glycoproteins were not only expressed but were incorporated into the envelope of recombinant BRSV. Thus, the envelope glycoproteins derived from a member of the Respirovirus genus can together functionally replace their homologs in a Pneumovirus background. PMID:11533200

  16. Recombinant mumps viruses expressing the batMuV fusion glycoprotein are highly fusion active and neurovirulent.

    Science.gov (United States)

    Krüger, Nadine; Sauder, Christian; Hoffmann, Markus; Örvell, Claes; Drexler, Jan Felix; Rubin, Steven; Herrler, Georg

    2016-11-01

    A recent study reported the detection of a bat-derived virus (BatPV/Epo_spe/AR1/DCR/2009, batMuV) with phylogenetic relatedness to human mumps virus (hMuV). Since all efforts to isolate infectious batMuV have reportedly failed, we generated recombinant mumps viruses (rMuVs) in which the open reading frames (ORFs) of the fusion (F) and haemagglutinin-neuraminidase (HN) glycoproteins of an hMuV strain were replaced by the corresponding ORFs of batMuV. The batMuV F and HN proteins were successfully incorporated into viral particles and the resultant chimeric virus was able to mediate infection of Vero cells. Distinct differences were observed between the fusogenicity of rMuVs expressing one or both batMuV glycoproteins: viruses expressing batMuV F were highly fusogenic, regardless of the origin of HN. In contrast, rMuVs expressing human F and bat-derived HN proteins were less fusogenic compared to hMuV. The growth kinetics of chimeric MuVs expressing batMuV HN in combination with either hMuV or batMuV F were similar to that of the backbone virus, whereas a delay in virus replication was obtained for rMuVs harbouring batMuV F and hMuV HN. Replacement of the hMuV F and HN genes or the HN gene alone by the corresponding batMuV genes led to a slight reduction in neurovirulence of the highly neurovirulent backbone strain. Neutralizing antibodies inhibited infection mediated by all recombinant viruses generated. Furthermore, group IV anti-MuV antibodies inhibited the neuraminidase activity of bat-derived HN. Our study reports the successful generation of chimeric MuVs expressing the F and HN proteins of batMuV, providing a means for further examination of this novel batMuV.

  17. Use of a novel cell-based fusion reporter assay to explore the host range of human respiratory syncytial virus F protein

    Directory of Open Access Journals (Sweden)

    Sarisky Robert T

    2005-07-01

    Full Text Available Abstract Human respiratory syncytial virus (HRSV is an important respiratory pathogen primarily affecting infants, young children, transplant recipients and the elderly. The F protein is the only virion envelope protein necessary and sufficient for virus replication and fusion of the viral envelope membrane with the target host cell. During natural infection, HRSV replication is limited to respiratory epithelial cells with disseminated infection rarely, if ever, occurring even in immunocompromised patients. However, in vitro infection of multiple human and non-human cell types other than those of pulmonary tract origin has been reported. To better define host cell surface molecules that mediate viral entry and dissect the factors controlling permissivity for HRSV, we explored the host range of HRSV F protein mediated fusion. Using a novel recombinant reporter gene based fusion assay, HRSV F protein was shown to mediate fusion with cells derived from a wide range of vertebrate species including human, feline, equine, canine, bat, rodent, avian, porcine and even amphibian (Xenopus. That finding was extended using a recombinant HRSV engineered to express green fluorescent protein (GFP, to confirm that viral mRNA expression is limited in several cell types. These findings suggest that HRSV F protein interacts with either highly conserved host cell surface molecules or can use multiple mechanisms to enter cells, and that the primary determinants of HRSV host range are at steps post-entry.

  18. Membrane cholesterol regulates lysosome-plasma membrane fusion events and modulates Trypanosoma cruzi invasion of host cells.

    Directory of Open Access Journals (Sweden)

    Bárbara Hissa

    Full Text Available BACKGROUND: Trypomastigotes of Trypanosoma cruzi are able to invade several types of non-phagocytic cells through a lysosomal dependent mechanism. It has been shown that, during invasion, parasites trigger host cell lysosome exocytosis, which initially occurs at the parasite-host contact site. Acid sphingomyelinase released from lysosomes then induces endocytosis and parasite internalization. Lysosomes continue to fuse with the newly formed parasitophorous vacuole until the parasite is completely enclosed by lysosomal membrane, a process indispensable for a stable infection. Previous work has shown that host membrane cholesterol is also important for the T. cruzi invasion process in both professional (macrophages and non-professional (epithelial phagocytic cells. However, the mechanism by which cholesterol-enriched microdomains participate in this process has remained unclear. METHODOLOGY/PRINCIPAL FINDING: In the present work we show that cardiomyocytes treated with MβCD, a drug able to sequester cholesterol from cell membranes, leads to a 50% reduction in invasion by T. cruzi trypomastigotes, as well as a decrease in the number of recently internalized parasites co-localizing with lysosomal markers. Cholesterol depletion from host membranes was accompanied by a decrease in the labeling of host membrane lipid rafts, as well as excessive lysosome exocytic events during the earlier stages of treatment. Precocious lysosomal exocytosis in MβCD treated cells led to a change in lysosomal distribution, with a reduction in the number of these organelles at the cell periphery, and probably compromises the intracellular pool of lysosomes necessary for T. cruzi invasion. CONCLUSION/SIGNIFICANCE: Based on these results, we propose that cholesterol depletion leads to unregulated exocytic events, reducing lysosome availability at the cell cortex and consequently compromise T. cruzi entry into host cells. The results also suggest that two different pools of

  19. Expression and Immunogenicity of Two Recombinant Fusion Proteins Comprising Foot-and-Mouth Disease Virus Structural Protein VP1 and DC-SIGN-Binding Glycoproteins.

    Science.gov (United States)

    Liu, Xinsheng; Lv, Jianliang; Fang, Yuzhen; Zhou, Peng; Lu, Yanzhen; Pan, Li; Zhang, Zhongwang; Ma, Junwu; Zhang, Yongguang; Wang, Yonglu

    2017-01-01

    Improving vaccine immunogenicity by targeting antigens to dendritic cells has recently emerged as a new design strategy in vaccine development. In this study, the VP1 gene of foot-and-mouth disease virus (FMDV) serotype A was fused with the gene encoding human immunodeficiency virus (HIV) membrane glycoprotein gp120 or C2-V3 domain of hepatitis C virus (HCV) envelope glycoprotein E2, both of which are DC-SIGN-binding glycoproteins. After codon optimization, the VP1 protein and the two recombinant VP1-gp120 and VP1-E2 fusion proteins were expressed in Sf9 insect cells using the insect cell-baculovirus expression system. Western blotting showed that the VP1 protein and two recombinant VP1-gp120 and VP1-E2 fusion proteins were correctly expressed in the Sf9 insect cells and had good reactogenicity. Guinea pigs were then immunized with the purified proteins, and the resulting humoral and cellular immune responses were analyzed. The VP1-gp120 and VP1-E2 fusion proteins induced significantly higher specific anti-FMDV antibody levels than the VP1 protein and stronger cell-mediated immune responses. This study provides a new perspective for the development of novel FMDV subunit vaccines.

  20. Expression and Immunogenicity of Two Recombinant Fusion Proteins Comprising Foot-and-Mouth Disease Virus Structural Protein VP1 and DC-SIGN-Binding Glycoproteins

    Directory of Open Access Journals (Sweden)

    Xinsheng Liu

    2017-01-01

    Full Text Available Improving vaccine immunogenicity by targeting antigens to dendritic cells has recently emerged as a new design strategy in vaccine development. In this study, the VP1 gene of foot-and-mouth disease virus (FMDV serotype A was fused with the gene encoding human immunodeficiency virus (HIV membrane glycoprotein gp120 or C2-V3 domain of hepatitis C virus (HCV envelope glycoprotein E2, both of which are DC-SIGN-binding glycoproteins. After codon optimization, the VP1 protein and the two recombinant VP1-gp120 and VP1-E2 fusion proteins were expressed in Sf9 insect cells using the insect cell-baculovirus expression system. Western blotting showed that the VP1 protein and two recombinant VP1-gp120 and VP1-E2 fusion proteins were correctly expressed in the Sf9 insect cells and had good reactogenicity. Guinea pigs were then immunized with the purified proteins, and the resulting humoral and cellular immune responses were analyzed. The VP1-gp120 and VP1-E2 fusion proteins induced significantly higher specific anti-FMDV antibody levels than the VP1 protein and stronger cell-mediated immune responses. This study provides a new perspective for the development of novel FMDV subunit vaccines.

  1. Principles of membrane tethering and fusion in endosome and lysosome biogenesis.

    Science.gov (United States)

    Kümmel, Daniel; Ungermann, Christian

    2014-08-01

    Endosomes and lysosomes receive cargo via vesicular carriers that arrive along multiple trafficking routes. On both organelles, tethering proteins have been identified that interact specifically with Rab5 on endosomes and Rab7 on late endosomes/lysosomes and that facilitate the SNARE-driven membrane fusion. Even though the structure and stoichiometry of the involved proteins and protein complexes differ strongly, they may operate by similar principles. Within this review, we will provide insights into their common functions and discuss the open questions in the field. Copyright © 2014 Elsevier Ltd. All rights reserved.

  2. Negative regulation of syntaxin4/SNAP-23/VAMP2-mediated membrane fusion by Munc18c in vitro.

    Directory of Open Access Journals (Sweden)

    Fiona M Brandie

    Full Text Available Translocation of the facilitative glucose transporter GLUT4 from an intracellular store to the plasma membrane is responsible for the increased rate of glucose transport into fat and muscle cells in response to insulin. This represents a specialised form of regulated membrane trafficking. Intracellular membrane traffic is subject to multiple levels of regulation by conserved families of proteins in all eukaryotic cells. Notably, all intracellular fusion events require SNARE proteins and Sec1p/Munc18 family members. Fusion of GLUT4-containing vesicles with the plasma membrane of insulin-sensitive cells involves the SM protein Munc18c, and is regulated by the formation of syntaxin 4/SNAP23/VAMP2 SNARE complexes.Here we have used biochemical approaches to characterise the interaction(s of Munc18c with its cognate SNARE proteins and to examine the role of Munc18c in regulating liposome fusion catalysed by syntaxin 4/SNAP23/VAMP2 SNARE complex formation. We demonstrate that Munc18c makes contacts with both t- and v-SNARE proteins of this complex, and directly inhibits bilayer fusion mediated by the syntaxin 4/SNAP23/VAMP2 SNARE complex.Our reductionist approach has enabled us to ascertain a direct inhibitory role for Munc18c in regulating membrane fusion mediated by syntaxin 4/SNAP23/VAMP2 SNARE complex formation. It is important to note that two different SM proteins have recently been shown to stimulate liposome fusion mediated by their cognate SNARE complexes. Given the structural similarities between SM proteins, it seems unlikely that different members of this family perform opposing regulatory functions. Hence, our findings indicate that Munc18c requires a further level of regulation in order to stimulate SNARE-mediated membrane fusion.

  3. Automatically Identifying Fusion Events between GLUT4 Storage Vesicles and the Plasma Membrane in TIRF Microscopy Image Sequences.

    Science.gov (United States)

    Wu, Jian; Xu, Yingke; Feng, Zhouyan; Zheng, Xiaoxiang

    2015-01-01

    Quantitative analysis of the dynamic behavior about membrane-bound secretory vesicles has proven to be important in biological research. This paper proposes a novel approach to automatically identify the elusive fusion events between VAMP2-pHluorin labeled GLUT4 storage vesicles (GSVs) and the plasma membrane. The differentiation is implemented to detect the initiation of fusion events by modified forward subtraction of consecutive frames in the TIRFM image sequence. Spatially connected pixels in difference images brighter than a specified adaptive threshold are grouped into a distinct fusion spot. The vesicles are located at the intensity-weighted centroid of their fusion spots. To reveal the true in vivo nature of a fusion event, 2D Gaussian fitting for the fusion spot is used to derive the intensity-weighted centroid and the spot size during the fusion process. The fusion event and its termination can be determined according to the change of spot size. The method is evaluated on real experiment data with ground truth annotated by expert cell biologists. The evaluation results show that it can achieve relatively high accuracy comparing favorably to the manual analysis, yet at a small fraction of time.

  4. Automatically Identifying Fusion Events between GLUT4 Storage Vesicles and the Plasma Membrane in TIRF Microscopy Image Sequences

    Directory of Open Access Journals (Sweden)

    Jian Wu

    2015-01-01

    Full Text Available Quantitative analysis of the dynamic behavior about membrane-bound secretory vesicles has proven to be important in biological research. This paper proposes a novel approach to automatically identify the elusive fusion events between VAMP2-pHluorin labeled GLUT4 storage vesicles (GSVs and the plasma membrane. The differentiation is implemented to detect the initiation of fusion events by modified forward subtraction of consecutive frames in the TIRFM image sequence. Spatially connected pixels in difference images brighter than a specified adaptive threshold are grouped into a distinct fusion spot. The vesicles are located at the intensity-weighted centroid of their fusion spots. To reveal the true in vivo nature of a fusion event, 2D Gaussian fitting for the fusion spot is used to derive the intensity-weighted centroid and the spot size during the fusion process. The fusion event and its termination can be determined according to the change of spot size. The method is evaluated on real experiment data with ground truth annotated by expert cell biologists. The evaluation results show that it can achieve relatively high accuracy comparing favorably to the manual analysis, yet at a small fraction of time.

  5. Second Sialic Acid Binding Site in Newcastle Disease Virus Hemagglutinin-Neuraminidase: Implications for Fusion

    OpenAIRE

    Zaitsev, Viatcheslav; von Itzstein, Mark; Groves, Darrin; Kiefel, Milton; Takimoto, Toru; Portner, Allen; Taylor, Garry

    2004-01-01

    Paramyxoviruses are the leading cause of respiratory disease in children. Several paramyxoviruses possess a surface glycoprotein, the hemagglutinin-neuraminidase (HN), that is involved in attachment to sialic acid receptors, promotion of fusion, and removal of sialic acid from infected cells and progeny virions. Previously we showed that Newcastle disease virus (NDV) HN contained a pliable sialic acid recognition site that could take two states, a binding state and a catalytic state. Here we ...

  6. Computational analysis of perturbations in the post-fusion Dengue virus envelope protein highlights known epitopes and conserved residues in the Zika virus [version 2; referees: 3 approved

    Directory of Open Access Journals (Sweden)

    Sandeep Chakraborty

    2016-09-01

    Full Text Available The dramatic transformation of the Zika virus (ZIKV from a relatively unknown virus to a pathogen generating global-wide panic has exposed the dearth of detailed knowledge about this virus. Decades of research in the related Dengue virus (DENV, finally culminating in a vaccine registered for use in endemic regions (CYD-TDV in three countries, provides key insights in developing strategies for tackling ZIKV, which has caused global panic to microcephaly and Guillain-Barre Syndrome. Dengue virus (DENV, a member of the family Flaviviridae, the causal agent of the self-limiting Dengue fever and the potentially fatal hemorrhagic fever/dengue shock syndrome, has been a scourge in tropical countries for many centuries. The recently solved structure of mature ZIKV (PDB ID:5IRE has provided key insights into the structure of the envelope (E and membrane (M proteins, the primary target of neutralizing antibodies. The previously established MEPP methodology compares two conformations of the same protein and identifies residues with significant spatial and electrostatic perturbations. In the current work, MEPP analyzed the pre-and post-fusion DENV type 2 envelope (E protein, and identified several known epitopes (His317, Tyr299, Glu26, Arg188, etc. (MEPPitope. These residues are overwhelmingly conserved in ZIKV and all DENV serotypes, and also enumerates residue pairs that undergo significant polarity reversal. Characterization of α-helices in E-proteins show that α1 is not conserved in the sequence space of ZIKV and DENV. Furthermore, perturbation of α1 in the post-fusion DENV structure includes a known epitope Asp215, a residue absent in the pre-fusion α1. A cationic β-sheet in the GAG-binding domain that is stereochemically equivalent in ZIKV and all DENV serotypes is also highlighted due to a residue pair (Arg286-Arg288 that has a significant electrostatic polarity reversal upon fusion. Finally, two highly conserved residues (Thr32 and Thr40, with

  7. Computational analysis of perturbations in the post-fusion Dengue virus envelope protein highlights known epitopes and conserved residues in the Zika virus.

    Science.gov (United States)

    Chakraborty, Sandeep

    2016-01-01

    The dramatic transformation of the Zika virus (ZIKV) from a relatively unknown virus to a pathogen generating global-wide panic has exposed the dearth of detailed knowledge about this virus. Decades of research in the related Dengue virus (DENV), finally culminating in a vaccine registered for use in endemic regions (CYD-TDV) in three countries, provides key insights in developing strategies for tackling ZIKV, which has caused global panic to microcephaly and Guillain-Barre Syndrome. Dengue virus (DENV), a member of the family Flaviviridae, the causal agent of the self-limiting Dengue fever and the potentially fatal hemorrhagic fever/dengue shock syndrome, has been a scourge in tropical countries for many centuries. The recently solved structure of mature ZIKV (PDB ID:5IRE) has provided key insights into the structure of the envelope (E) and membrane (M) proteins, the primary target of neutralizing antibodies. The previously established MEPP methodology compares two conformations of the same protein and identifies residues with significant spatial and electrostatic perturbations. In the current work, MEPP analyzed the pre-and post-fusion DENV type 2 envelope (E) protein, and identified several known epitopes (His317, Tyr299, Glu26, Arg188, etc.) (MEPPitope). These residues are overwhelmingly conserved in ZIKV and all DENV serotypes, and also enumerates residue pairs that undergo significant polarity reversal. Characterization of α-helices in E-proteins show that α1 is not conserved in the sequence space of ZIKV and DENV. Furthermore, perturbation of α1 in the post-fusion DENV structure includes a known epitope Asp215, a residue absent in the pre-fusion α1. A cationic β-sheet in the GAG-binding domain that is stereochemically equivalent in ZIKV and all DENV serotypes is also highlighted due to a residue pair (Arg286-Arg288) that has a significant electrostatic polarity reversal upon fusion. Finally, two highly conserved residues (Thr32 and Thr40), with little

  8. Specific and efficient cleavage of fusion proteins by recombinant plum pox virus NIa protease.

    Science.gov (United States)

    Zheng, Nuoyan; Pérez, José de Jesús; Zhang, Zhonghui; Domínguez, Elvira; Garcia, Juan Antonio; Xie, Qi

    2008-02-01

    Site-specific proteases are the most popular kind of enzymes for removing the fusion tags from fused target proteins. Nuclear inclusion protein a (NIa) proteases obtained from the family Potyviridae have become promising due to their high activities and stringencies of sequences recognition. NIa proteases from tobacco etch virus (TEV) and tomato vein mottling virus (TVMV) have been shown to process recombinant proteins successfully in vitro. In this report, recombinant PPV (plum pox virus) NIa protease was employed to process fusion proteins with artificial cleavage site in vitro. Characteristics such as catalytic ability and affecting factors (salt, temperature, protease inhibitors, detergents, and denaturing reagents) were investigated. Recombinant PPV NIa protease expressed and purified from Escherichia coli demonstrated efficient and specific processing of recombinant GFP and SARS-CoV nucleocapsid protein, with site F (N V V V H Q black triangle down A) for PPV NIa protease artificially inserted between the fusion tags and the target proteins. Its catalytic capability is similar to those of TVMV and TEV NIa protease. Recombinant PPV NIa protease reached its maximal proteolytic activity at approximately 30 degrees C. Salt concentration and only one of the tested protease inhibitors had minor influences on the proteolytic activity of PPV NIa protease. Recombinant PPV NIa protease was resistant to self-lysis for at least five days.

  9. Glycosphingolipides et fusion virus-cellule : données actuelles montrant le rôle des micro-domaines membranaires dans le cycle d’infection du VIH-1

    Directory of Open Access Journals (Sweden)

    Hammache Djilali

    2000-09-01

    Full Text Available Depuis plusieurs années, nous étudions les mécanismes moléculaires responsables de la fusion du virus de l’immunodéficience humaine (VIH avec la membrane plasmique des cellules cibles. Ces travaux ont permis de préciser le rôle essentiel joué par les micro-domaines de glycosphingolipides au cours de la fusion virus-cellule. En particulier, nous avons pu reconstituer un complexe de fusion fonctionnel faisant intervenir les différents partenaires moléculaires de la fusion : un micro-domaine de glycosphingolipide se présentant sous la forme d’un film monomoléculaire à l’interface eau-air, le récepteur CD4 et la glycoprotéine externe de l’enveloppe du virus, la gp120. La dynamique des interactions moléculaires dans ce complexe de fusion a pu être mesurée à l’aide d’un micro-tensiomètre. Ce système expérimental pourrait permettre d’évaluer l’activité d’inhibiteurs de fusion tels que des analogues synthétiques de glycosphingolipides.

  10. Identification and Characterization of LFD-2, a Predicted Fringe Protein Required for Membrane Integrity during Cell Fusion in Neurospora crassa

    OpenAIRE

    Palma-Guerrero, J; Zhao, J; Pedro Gonçalves, A.; Starr, TL; N Louise Glass

    2015-01-01

    © 2015, American Society for Microbiology. All Rights Reserved. The molecular mechanisms of membrane merger during somatic cell fusion in eukaryotic species are poorly understood. In the filamentous fungus Neurospora crassa, somatic cell fusion occurs between genetically identical germinated asexual spores (germlings) and between hyphae to form the interconnected network characteristic of a filamentous fungal colony. In N. crassa, two proteins have been identified to function at the step of m...

  11. Identification and characterization of LFD-2, a predicted fringe protein required for membrane integrity during cell fusion in neurospora crassa.

    Science.gov (United States)

    Palma-Guerrero, Javier; Zhao, Jiuhai; Gonçalves, A Pedro; Starr, Trevor L; Glass, N Louise

    2015-03-01

    The molecular mechanisms of membrane merger during somatic cell fusion in eukaryotic species are poorly understood. In the filamentous fungus Neurospora crassa, somatic cell fusion occurs between genetically identical germinated asexual spores (germlings) and between hyphae to form the interconnected network characteristic of a filamentous fungal colony. In N. crassa, two proteins have been identified to function at the step of membrane fusion during somatic cell fusion: PRM1 and LFD-1. The absence of either one of these two proteins results in an increase of germling pairs arrested during cell fusion with tightly appressed plasma membranes and an increase in the frequency of cell lysis of adhered germlings. The level of cell lysis in ΔPrm1 or Δlfd-1 germlings is dependent on the extracellular calcium concentration. An available transcriptional profile data set was used to identify genes encoding predicted transmembrane proteins that showed reduced expression levels in germlings cultured in the absence of extracellular calcium. From these analyses, we identified a mutant (lfd-2, for late fusion defect-2) that showed a calcium-dependent cell lysis phenotype. lfd-2 encodes a protein with a Fringe domain and showed endoplasmic reticulum and Golgi membrane localization. The deletion of an additional gene predicted to encode a low-affinity calcium transporter, fig1, also resulted in a strain that showed a calcium-dependent cell lysis phenotype. Genetic analyses showed that LFD-2 and FIG1 likely function in separate pathways to regulate aspects of membrane merger and repair during cell fusion. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

  12. New and conventional pore size tests in virus-removing membranes

    NARCIS (Netherlands)

    Duek, A.; Arkhangelsky, E.; Krush, R.; Brenner, A.; Gitis, V.

    2012-01-01

    Microorganisms are retained by ultrafiltration (UF) membranes mainly due to size exclusion. The sizes of viruses and membrane pores are close to each other and retention of viruses can be guaranteed only if the precise pore diameter is known. Unfortunately and rather surprisingly, there is no direct

  13. A computational approach identifies two regions of Hepatitis C Virus E1 protein as interacting domains involved in viral fusion process

    Directory of Open Access Journals (Sweden)

    El Sawaf Gamal

    2009-07-01

    Full Text Available Abstract Background The E1 protein of Hepatitis C Virus (HCV can be dissected into two distinct hydrophobic regions: a central domain containing an hypothetical fusion peptide (FP, and a C-terminal domain (CT comprising two segments, a pre-anchor and a trans-membrane (TM region. In the currently accepted model of the viral fusion process, the FP and the TM regions are considered to be closely juxtaposed in the post-fusion structure and their physical interaction cannot be excluded. In the present study, we took advantage of the natural sequence variability present among HCV strains to test, by purely sequence-based computational tools, the hypothesis that in this virus the fusion process involves the physical interaction of the FP and CT regions of E1. Results Two computational approaches were applied. The first one is based on the co-evolution paradigm of interacting peptides and consequently on the correlation between the distance matrices generated by the sequence alignment method applied to FP and CT primary structures, respectively. In spite of the relatively low random genetic drift between genotypes, co-evolution analysis of sequences from five HCV genotypes revealed a greater correlation between the FP and CT domains than respect to a control HCV sequence from Core protein, so giving a clear, albeit still inconclusive, support to the physical interaction hypothesis. The second approach relies upon a non-linear signal analysis method widely used in protein science called Recurrence Quantification Analysis (RQA. This method allows for a direct comparison of domains for the presence of common hydrophobicity patterns, on which the physical interaction is based upon. RQA greatly strengthened the reliability of the hypothesis by the scoring of a lot of cross-recurrences between FP and CT peptides hydrophobicity patterning largely outnumbering chance expectations and pointing to putative interaction sites. Intriguingly, mutations in the CT

  14. Respiratory syncytial virus fusion glycoprotein expressed in insect cells form protein nanoparticles that induce protective immunity in cotton rats.

    Directory of Open Access Journals (Sweden)

    Gale Smith

    Full Text Available Respiratory Syncytial Virus (RSV is an important viral agent causing severe respiratory tract disease in infants and children as well as in the elderly and immunocompromised individuals. The lack of a safe and effective RSV vaccine represents a major unmet medical need. RSV fusion (F surface glycoprotein was modified and cloned into a baculovirus vector for efficient expression in Sf9 insect cells. Recombinant RSV F was glycosylated and cleaved into covalently linked F2 and F1 polypeptides that formed homotrimers. RSV F extracted and purified from insect cell membranes assembled into 40 nm protein nanoparticles composed of multiple RSV F oligomers arranged in the form of rosettes. The immunogenicity and protective efficacy of purified RSV F nanoparticles was compared to live and formalin inactivated RSV in cotton rats. Immunized animals induced neutralizing serum antibodies, inhibited virus replication in the lungs, and had no signs of disease enhancement in the respiratory track of challenged animals. RSV F nanoparticles also induced IgG competitive for binding of palivizumab neutralizing monoclonal antibody to RSV F antigenic site II. Antibodies to this epitope are known to protect against RSV when passively administered in high risk infants. Together these data provide a rational for continued development a recombinant RSV F nanoparticle vaccine candidate.

  15. 'a'-Position-mutated and G4-mutated hemagglutinin-neuraminidase proteins of Newcastle disease virus impair fusion and hemagglutinin-neuraminidase-fusion interaction by different mechanisms.

    Science.gov (United States)

    Chu, Fu-lu; Wen, Hong-ling; Zhang, Wen-qiang; Lin, Bin; Zhang, Yan; Sun, Cheng-xi; Ren, Gui-jie; Song, Yan-yan; Wang, Zhiyu

    2013-01-01

    To determine the effects of heptad repeat regions (HRs) and N-linked carbohydrate sites of the Newcastle disease virus hemagglutinin-neuraminidase (HN) protein on fusion of HN and fusion (F) proteins and HN-F interaction. We mutated six 'a' residues in the HRs and four asparagines in N-linked carbohydrate sites to alanine in the HN protein. A vaccinia-T7 RNA polymerase expression system was used to express HN cDNAs in BHK-21 cells to determine the HN functions. Deglycosylation was treated with PGNase F digestion. The formation of HN-F protein complexes was determined by the coimmunoprecipitation assay. Each HR-mutated protein interfered with fusion and the HN-F interaction. The G4-mutated protein not only impaired fusion and HN-F interaction but also decreased activities in cell fusion promotion, hemadsorption and neuraminidase. It is assumed that two different mechanisms for mutations in these two regions are responsible for the decreased fusion promotion activity and the reduced ability of interaction with F protein. Mutations in the HRs impair fusion and HN-F interaction by altering the transmission of a signal from the globular domain to the F-specific region in the stalk, but the G4 mutation modulates fusion and HN-F interaction by the misfolding of some important structures. Copyright © 2012 S. Karger AG, Basel.

  16. Genetic characteristic of protein membran of avian influenza viruses H5N1 subtype

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    N.L.P Indi Dharmayanti

    2013-10-01

    Full Text Available In 2006-2008 there were findings about the antigenic drift on AI virus due to vaccination and the AI H5N1 subtype viruses which was similar to H5N1 viruses in human. The findings indicated that the AI viruses continue and undergoing to mutate and try to adapt with their environment. The objective of this study was to characterize the mutation of recent AI viruses (2009 on the membran protein namely Hemagglutinin (HA, Neuraminidase (NA and Matrix 2 (M2. In this study RT-PCR – sequencing methods and genetic analysis for the protein membran of AI viruses were used. Result revealed that there were specific mutation belong to AI 2009 viruses on HA and NA protein such as AI virus mutation in 2008 which was isolated from backyard chicken. The mutations were non synonimous and not caused by immunological pressure. Furthermore, M2 analysis indicated that the viruses were resistant to amantadine.

  17. [Shifts in the antioxidant activity in the membrane structures of virus-infected cells].

    Science.gov (United States)

    Petrenko, Iu M; Anan'ev, V A; Konstantinova, L A; Vladimirov, Iu A

    1985-01-01

    It is assumed that the process of virus infection of cells causes significant changes in the conditions of lipid peroxidation in membrane structures of such cells. Experiments with virus-infected HeLa cells demonstrated noticeable decrease in the capacity of membrane lipids for peroxidation induced by ultraviolet irradiation and iron monoxide ions. Similar changes were also observed in membranes of chick embryo liver cells infected with viruses. The established reduced capacity of lipids in membranes of virus-infected cells to induced peroxidation (interpreted as the increase of their antioxidant activity) is characteristic of whole membranes only, because the lipids extracted from infected and control cells showed no differences in their capacity to peroxidation in liposomal membranes when oxidation was induced by iron monoxide.

  18. Cdc42p and Rho1p are sequentially activated and mechanistically linked to vacuole membrane fusion

    Energy Technology Data Exchange (ETDEWEB)

    Logan, Michael R.; Jones, Lynden [Department of Cell Biology, University of Alberta, Edmonton, Alta., Canada T6G 2H7 (Canada); Eitzen, Gary, E-mail: gary.eitzen@ualberta.ca [Department of Cell Biology, University of Alberta, Edmonton, Alta., Canada T6G 2H7 (Canada)

    2010-03-26

    Small monomeric GTPases act as molecular switches, regulating many biological functions via activation of membrane localized signaling cascades. Activation of their switch function is controlled by GTP binding and hydrolysis. Two Rho GTPases, Cdc42p and Rho1p, are localized to the yeast vacuole where they regulate membrane fusion. Here, we define a method to directly examine vacuole membrane Cdc42p and Rho1p activation based on their affinity to probes derived from effectors. Cdc42p and Rho1p showed unique temporal activation which aligned with distinct subreactions of in vitro vacuole fusion. Cdc42p was rapidly activated in an ATP-independent manner while Rho1p activation was kinetically slower and required ATP. Inhibitors that are known to block vacuole membrane fusion were examined for their effect on Cdc42p and Rho1p activation. Rdi1p, which inhibits the dissociation of GDP from Rho proteins, blocked both Cdc42p and Rho1p activation. Ligands of PI(4,5)P{sub 2} specifically inhibited Rho1p activation while pre-incubation with U73122, which targets Plc1p function, increased Rho1p activation. These results define unique activation mechanisms for Cdc42p and Rho1p, which may be linked to the vacuole membrane fusion mechanism.

  19. Biophysical Characterization of a Vaccine Candidate against HIV-1: The Transmembrane and Membrane Proximal Domains of HIV-1 gp41 as a Maltose Binding Protein Fusion

    Science.gov (United States)

    Gong, Zhen; Martin-Garcia, Jose M.; Daskalova, Sasha M.; Craciunescu, Felicia M.; Song, Lusheng; Dörner, Katerina; Hansen, Debra T.; Yang, Jay-How; LaBaer, Joshua; Hogue, Brenda G.; Mor, Tsafrir S.; Fromme, Petra

    2015-01-01

    The membrane proximal region (MPR, residues 649–683) and transmembrane domain (TMD, residues 684–705) of the gp41 subunit of HIV-1’s envelope protein are highly conserved and are important in viral mucosal transmission, virus attachment and membrane fusion with target cells. Several structures of the trimeric membrane proximal external region (residues 662–683) of MPR have been reported at the atomic level; however, the atomic structure of the TMD still remains unknown. To elucidate the structure of both MPR and TMD, we expressed the region spanning both domains, MPR-TM (residues 649–705), in Escherichia coli as a fusion protein with maltose binding protein (MBP). MPR-TM was initially fused to the C-terminus of MBP via a 42 aa-long linker containing a TEV protease recognition site (MBP-linker-MPR-TM). Biophysical characterization indicated that the purified MBP-linker-MPR-TM protein was a monodisperse and stable candidate for crystallization. However, crystals of the MBP-linker-MPR-TM protein could not be obtained in extensive crystallization screens. It is possible that the 42 residue-long linker between MBP and MPR-TM was interfering with crystal formation. To test this hypothesis, the 42 residue-long linker was replaced with three alanine residues. The fusion protein, MBP-AAA-MPR-TM, was similarly purified and characterized. Significantly, both the MBP-linker-MPR-TM and MBP-AAA-MPR-TM proteins strongly interacted with broadly neutralizing monoclonal antibodies 2F5 and 4E10. With epitopes accessible to the broadly neutralizing antibodies, these MBP/MPR-TM recombinant proteins may be in immunologically relevant conformations that mimic a pre-hairpin intermediate of gp41. PMID:26295457

  20. Autoprocessing of human immunodeficiency virus type 1 protease miniprecursor fusions in mammalian cells

    Directory of Open Access Journals (Sweden)

    Chen Chaoping

    2010-07-01

    Full Text Available Abstract Background HIV protease (PR is a virus-encoded aspartic protease that is essential for viral replication and infectivity. The fully active and mature dimeric protease is released from the Gag-Pol polyprotein as a result of precursor autoprocessing. Results We here describe a simple model system to directly examine HIV protease autoprocessing in transfected mammalian cells. A fusion precursor was engineered encoding GST fused to a well-characterized miniprecursor, consisting of the mature protease along with its upstream transframe region (TFR, and small peptide epitopes to facilitate detection of the precursor substrate and autoprocessing products. In HEK 293T cells, the resulting chimeric precursor undergoes effective autoprocessing, producing mature protease that is rapidly degraded likely via autoproteolysis. The known protease inhibitors Darunavir and Indinavir suppressed both precursor autoprocessing and autoproteolysis in a dose-dependent manner. Protease mutations that inhibit Gag processing as characterized using proviruses also reduced autoprocessing efficiency when they were introduced to the fusion precursor. Interestingly, autoprocessing of the fusion precursor requires neither the full proteolytic activity nor the majority of the N-terminal TFR region. Conclusions We suggest that the fusion precursors provide a useful system to study protease autoprocessing in mammalian cells, and may be further developed for screening of new drugs targeting HIV protease autoprocessing.

  1. HCIV-1 and Other Tailless Icosahedral Internal Membrane-Containing Viruses of the Family Sphaerolipoviridae

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    Tatiana A. Demina

    2017-02-01

    Full Text Available Members of the virus family Sphaerolipoviridae include both archaeal viruses and bacteriophages that possess a tailless icosahedral capsid with an internal membrane. The genera Alpha- and Betasphaerolipovirus comprise viruses that infect halophilic euryarchaea, whereas viruses of thermophilic Thermus bacteria belong to the genus Gammasphaerolipovirus. Both sequence-based and structural clustering of the major capsid proteins and ATPases of sphaerolipoviruses yield three distinct clades corresponding to these three genera. Conserved virion architectural principles observed in sphaerolipoviruses suggest that these viruses belong to the PRD1-adenovirus structural lineage. Here we focus on archaeal alphasphaerolipoviruses and their related putative proviruses. The highest sequence similarities among alphasphaerolipoviruses are observed in the core structural elements of their virions: the two major capsid proteins, the major membrane protein, and a putative packaging ATPase. A recently described tailless icosahedral haloarchaeal virus, Haloarcula californiae icosahedral virus 1 (HCIV-1, has a double-stranded DNA genome and an internal membrane lining the capsid. HCIV-1 shares significant similarities with the other tailless icosahedral internal membrane-containing haloarchaeal viruses of the family Sphaerolipoviridae. The proposal to include a new virus species, Haloarcula virus HCIV1, into the genus Alphasphaerolipovirus was submitted to the International Committee on Taxonomy of Viruses (ICTV in 2016.

  2. Membrane fusion inducers, chloroquine and spermidine increase lipoplex-mediated gene transfection

    Energy Technology Data Exchange (ETDEWEB)

    Wong-Baeza, Carlos; Bustos, Israel; Serna, Manuel; Tescucano, Alonso; Alcantara-Farfan, Veronica; Ibanez, Miguel [Biochemistry Department, National Polytechnic Institute (IPN), Mexico City 11340 (Mexico); Montanez, Cecilia [Department of Genetics and Molecular Biology, Centre for Research and Advanced Studies (CINVESTAV), IPN, Mexico City 07360 (Mexico); Wong, Carlos [Biochemistry Department, National Polytechnic Institute (IPN), Mexico City 11340 (Mexico); Baeza, Isabel, E-mail: ibaeza@encb.ipn.mx [Biochemistry Department, National Polytechnic Institute (IPN), Mexico City 11340 (Mexico)

    2010-05-28

    Gene transfection into mammalian cells can be achieved with viral and non-viral vectors. Non-viral vectors, such as cationic lipids that form lipoplexes with DNA, are safer and more stable than viral vectors, but their transfection efficiencies are lower. Here we describe that the simultaneous treatment with a membrane fusion inducer (chlorpromazine or procainamide) plus the lysosomotropic agent chloroquine increases lipoplex-mediated gene transfection in human (HEK293 and C-33 A) and rat (PC12) cell lines (up to 9.2-fold), as well as in situ in BALB/c mice spleens and livers (up to 6-fold); and that the polyamine spermidine increases lipoplex-mediated gene transfection and expression in cell cultures. The use of these four drugs provides a novel, safe and relatively inexpensive way to considerably increase lipoplex-mediated gene transfection efficiency.

  3. Probing plasma membrane microdomains in cowpea protoplasts using lipidated GFP-fusion proteins and multimode FRET microscopy

    NARCIS (Netherlands)

    Vermeer, J.E.M.; van Munster, E.B.; Vischer, N.O.; Gadella, T.

    2004-01-01

    Multimode fluorescence resonance energy transfer (FRET) microscopy was applied to study the plasma membrane organization using different lipidated green fluorescent protein (GFP)-fusion proteins co-expressed in cowpea protoplasts. Cyan fluorescent protein (CFP) was fused to the hyper variable region

  4. Different sets of ER-resident J-proteins regulate distinct polar nuclear-membrane fusion events in Arabidopsis thaliana.

    Science.gov (United States)

    Maruyama, Daisuke; Yamamoto, Masaya; Endo, Toshiya; Nishikawa, Shuh-ichi

    2014-11-01

    Angiosperm female gametophytes contain a central cell with two polar nuclei. In many species, including Arabidopsis thaliana, the polar nuclei fuse during female gametogenesis. We previously showed that BiP, an Hsp70 in the endoplasmic reticulum (ER), was essential for membrane fusion during female gametogenesis. Hsp70 function requires partner proteins for full activity. J-domain containing proteins (J-proteins) are the major Hsp70 functional partners. A. thaliana ER contains three soluble J-proteins, AtERdj3A, AtERdj3B, and AtP58(IPK). Here, we analyzed mutants of these proteins and determined that double-mutant ovules lacking AtP58(IPK) and AtERdj3A or AtERdj3B were defective in polar nuclear fusion. Electron microscopy analysis identified that polar nuclei were in close contact, but no membrane fusion occurred in mutant ovules lacking AtP58(IPK) and AtERdj3A. The polar nuclear outer membrane appeared to be connected via the ER remaining at the inner unfused membrane in mutant ovules lacking AtP58(IPK) and AtERdj3B. These results indicate that ER-resident J-proteins, AtP58(IPK)/AtERdj3A and AtP58(IPK)/AtERdj3B, function at distinct steps of polar nuclear-membrane fusion. Similar to the bip1 bip2 double mutant female gametophytes, the aterdj3a atp58(ipk) double mutant female gametophytes defective in fusion of the outer polar nuclear membrane displayed aberrant endosperm proliferation after fertilization with wild-type pollen. However, endosperm proliferated normally after fertilization of the aterdj3b atp58(ipk) double mutant female gametophytes defective in fusion of the inner membrane. Our results indicate that the polar nuclear fusion defect itself does not cause an endosperm proliferation defect. © The Author 2014. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com.

  5. Endocytosis Plays a Critical Role in Proteolytic Processing of the Hendra Virus Fusion Protein

    OpenAIRE

    Meulendyke, Kelly Ann; Wurth, Mark Allen; McCann, Richard O.; Dutch, Rebecca Ellis

    2005-01-01

    The Hendra virus fusion (F) protein is synthesized as a precursor protein, F0, which is proteolytically processed to the mature form, F1+F2. Unlike the case for the majority of paramyxovirus F proteins, the processing event is furin independent, does not require the addition of exogenous proteases, is not affected by reductions in intracellular Ca2+, and is strongly affected by conditions that raise the intracellular pH (C. T. Pager, M. A. Wurth, and R. E. Dutch, J. Virol. 78:9154-9163, 2004)...

  6. Evaluation of Measles Vaccine Virus as a Vector to Deliver Respiratory Syncytial Virus Fusion Protein or Epstein-Barr Virus Glycoprotein gp350.

    Science.gov (United States)

    Mok, Hoyin; Cheng, Xing; Xu, Qi; Zengel, James R; Parhy, Bandita; Zhao, Jackie; Wang, C Kathy; Jin, Hong

    2012-01-01

    Live attenuated recombinant measles vaccine virus (MV) Edmonston-Zagreb (EZ) strain was evaluated as a viral vector to express the ectodomains of fusion protein of respiratory syncytial virus (RSV F) or glycoprotein 350 of Epstein-Barr virus (EBV gp350) as candidate vaccines for prophylaxis of RSV and EBV. The glycoprotein gene was inserted at the 1(st) or the 3(rd) position of the measles virus genome and the recombinant viruses were generated. Insertion of the foreign gene at the 3(rd) position had a minimal impact on viral replication in vitro. RSV F or EBV gp350 protein was secreted from infected cells. In cotton rats, EZ-RSV F and EZ-EBV gp350 induced MV- and insert-specific antibody responses. In addition, both vaccines also induced insert specific interferon gamma (IFN-γ) secreting T cell response. EZ-RSV F protected cotton rats from pulmonary replication of RSV A2 challenge infection. In rhesus macaques, although both EZ-RSV F and EZ-EBV gp350 induced MV specific neutralizing antibody responses, only RSV F specific antibody response was detected. Thus, the immunogenicity of the foreign antigens delivered by measles vaccine virus is dependent on the nature of the insert and the animal models used for vaccine evaluation.

  7. Reassessment of the lineage fusion hypothesis for the origin of double membrane bacteria.

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    Kristen S Swithers

    Full Text Available In 2009, James Lake introduced a new hypothesis in which reticulate phylogeny reconstruction is used to elucidate the origin of gram-negative bacteria (Nature 460: 967-971. The presented data supported the gram-negative bacteria originating from an ancient endosymbiosis between the Actinobacteria and Clostridia. His conclusion was based on a presence-absence analysis of protein families that divided all prokaryotes into five groups: Actinobacteria, Double Membrane bacteria (DM, Clostridia, Archaea and Bacilli. Of these five groups, the DM are by far the largest and most diverse group compared to the other groupings. While the fusion hypothesis for the origin of double membrane bacteria is enticing, we show that the signal supporting an ancient symbiosis is lost when the DM group is broken down into smaller subgroups. We conclude that the signal detected in James Lake's analysis in part results from a systematic artifact due to group size and diversity combined with low levels of horizontal gene transfer.

  8. An extended model of vesicle fusion at the plasma membrane to estimate protein lateral diffusion from TIRF microscopy images.

    Science.gov (United States)

    Basset, Antoine; Bouthemy, Patrick; Boulanger, Jérôme; Waharte, François; Salamero, Jean; Kervrann, Charles

    2017-07-24

    Characterizing membrane dynamics is a key issue to understand cell exchanges with the extra-cellular medium. Total internal reflection fluorescence microscopy (TIRFM) is well suited to focus on the late steps of exocytosis at the plasma membrane. However, it is still a challenging task to quantify (lateral) diffusion and estimate local dynamics of proteins. A new model was introduced to represent the behavior of cargo transmembrane proteins during the vesicle fusion to the plasma membrane at the end of the exocytosis process. Two biophysical parameters, the diffusion coefficient and the release rate parameter, are automatically estimated from TIRFM image sequences, to account for both the lateral diffusion of molecules at the membrane and the continuous release of the proteins from the vesicle to the plasma membrane. Quantitative evaluation on 300 realistic computer-generated image sequences demonstrated the efficiency and accuracy of the method. The application of our method on 16 real TIRFM image sequences additionally revealed differences in the dynamic behavior of Transferrin Receptor (TfR) and Langerin proteins. An automated method has been designed to simultaneously estimate the diffusion coefficient and the release rate for each individual vesicle fusion event at the plasma membrane in TIRFM image sequences. It can be exploited for further deciphering cell membrane dynamics.

  9. Pandemic H1N1 influenza A directly induces a robust and acute inflammatory gene signature in primary human bronchial epithelial cells downstream of membrane fusion

    Energy Technology Data Exchange (ETDEWEB)

    Paquette, Stéphane G. [Division of Experimental Therapeutics, Toronto General Hospital Research Institute, University Health Network, Toronto, Ontario (Canada); Institute of Medical Science, Faculty of Medicine, University of Toronto, Toronto, Ontario (Canada); Banner, David [Division of Experimental Therapeutics, Toronto General Hospital Research Institute, University Health Network, Toronto, Ontario (Canada); Chi, Le Thi Bao [Department of Microbiology, Hue University of Medicine and Pharmacy, Thua Thien Hue (Viet Nam); Carlo Urbani Centre, Hue University of Medicine and Pharmacy, Thua Thien Hue (Viet Nam); Leon, Alberto J. [Division of Experimental Therapeutics, Toronto General Hospital Research Institute, University Health Network, Toronto, Ontario (Canada); International Institute of Infection and Immunity, Shantou University Medical College, Shantou, Guangdong (China); Xu, Luoling; Ran, Longsi [Division of Experimental Therapeutics, Toronto General Hospital Research Institute, University Health Network, Toronto, Ontario (Canada); Huang, Stephen S.H. [Division of Experimental Therapeutics, Toronto General Hospital Research Institute, University Health Network, Toronto, Ontario (Canada); Department of Immunology, Faculty of Medicine, University of Toronto, Toronto, Ontario (Canada); Farooqui, Amber [Division of Experimental Therapeutics, Toronto General Hospital Research Institute, University Health Network, Toronto, Ontario (Canada); International Institute of Infection and Immunity, Shantou University Medical College, Shantou, Guangdong (China); and others

    2014-01-05

    Pandemic H1N1 influenza A (H1N1pdm) elicits stronger pulmonary inflammation than previously circulating seasonal H1N1 influenza A (sH1N1), yet mechanisms of inflammatory activation in respiratory epithelial cells during H1N1pdm infection are unclear. We investigated host responses to H1N1pdm/sH1N1 infection and virus entry mechanisms in primary human bronchial epithelial cells in vitro. H1N1pdm infection rapidly initiated a robust inflammatory gene signature (3 h post-infection) not elicited by sH1N1 infection. Protein secretion inhibition had no effect on gene induction. Infection with membrane fusion deficient H1N1pdm failed to induce robust inflammatory gene expression which was rescued with restoration of fusion ability, suggesting H1N1pdm directly triggered the inflammatory signature downstream of membrane fusion. Investigation of intra-virion components revealed H1N1pdm viral RNA (vRNA) triggered a stronger inflammatory phenotype than sH1N1 vRNA. Thus, our study is first to report H1N1pdm induces greater inflammatory gene expression than sH1N1 in vitro due to direct virus–epithelial cell interaction. - Highlights: • We investigated H1N1pdm/sH1N1 infection in primary epithelial cells. • H1N1pdm directly initiated a robust inflammatory gene signature, sH1N1 did not. • H1N1pdm viral RNA triggered a stronger response than sH1N1. • H1N1pdm induces greater response due to direct virus–cell interaction. • These results have potential to impact vaccine and therapeutic development.

  10. Improvement of virus removal using ultrafiltration membranes modified with grafted zwitterionic polymer hydrogels.

    Science.gov (United States)

    Lu, Ruiqing; Zhang, Chang; Piatkovsky, Maria; Ulbricht, Mathias; Herzberg, Moshe; Nguyen, Thanh H

    2017-06-01

    Potable water reuse has been adopted by cities suffering water scarcity in recent years. The microbial safety in water reuse, especially with respect to pathogenic viruses, is still a concern for water consumers. Membrane filtration can achieve sufficient removal of pathogenic viruses without disinfection byproducts, but the required energy is intensive. In this study, we graft-polymerized zwitterionic SPP ([3-(methacryloylamino) propyl] dimethyl (3-sulfopropyl) ammonium hydroxide) on a 150 kDa ultrafiltration polyethersulfone membrane to achieve a significantly higher virus removal. The redox-initiated graft-polymerization was performed in an aqueous solution during filtration of the monomer and initiators, allowing for functionalizing the membrane pores with hydrophilic polySPP. Bacteriophage MS2 and human adenovirus type 2 (HAdV-2) were used as surrogates for pathogenic human norovirus and human adenovirus. The grafting resulted in ∼18% loss of the membrane permeability but an increase of 4 log10 in HAdV-2 removal and 3 log10 in MS2 removal. The pristine and the grafted membranes were both conditioned with soluble microbial products (SMP) extracted from a full-scale membrane bioreactor (MBR) in order to test the virus removal after fouling the membranes. After fouling, the HAdV-2 removal by the grafted membrane was 1 log10 higher than that of the pristine membrane. For MS2, the grafted membrane after fouling with SMP achieved an additional 5 log10 removal compared to the unmodified membrane. The simple graft-polymerization functionalization of commercialized membrane achieving enhanced virus removal efficiency highlights the promise of membrane filtration for pathogen control in potable water reuse. Copyright © 2017 Elsevier Ltd. All rights reserved.

  11. Crystal structure of the membrane fusion protein CusB from Escherichia coli.

    Science.gov (United States)

    Su, Chih-Chia; Yang, Feng; Long, Feng; Reyon, Deepak; Routh, Mathew D; Kuo, Dennis W; Mokhtari, Adam K; Van Ornam, Jonathan D; Rabe, Katherine L; Hoy, Julie A; Lee, Young Jin; Rajashankar, Kanagalaghatta R; Yu, Edward W

    2009-10-23

    Gram-negative bacteria, such as Escherichia coli, frequently utilize tripartite efflux complexes belonging to the resistance-nodulation-division family to expel diverse toxic compounds from the cell. These systems contain a periplasmic membrane fusion protein (MFP) that is critical for substrate transport. We here present the x-ray structures of the CusB MFP from the copper/silver efflux system of E. coli. This is the first structure of any MFPs associated with heavy-metal efflux transporters. CusB bridges the inner-membrane efflux pump CusA and outer-membrane channel CusC to mediate resistance to Cu(+) and Ag(+) ions. Two distinct structures of the elongated molecules of CusB were found in the asymmetric unit of a single crystal, which suggests the flexible nature of this protein. Each protomer of CusB can be divided into four different domains, whereby the first three domains are mostly beta-strands and the last domain adopts an entirely helical architecture. Unlike other known structures of MFPs, the alpha-helical domain of CusB is folded into a three-helix bundle. This three-helix bundle presumably interacts with the periplasmic domain of CusC. The N- and C-termini of CusB form the first beta-strand domain, which is found to interact with the periplasmic domain of the CusA efflux pump. Atomic details of how this efflux protein binds Cu(+) and Ag(+) were revealed by the crystals of the CusB-Cu(I) and CusB-Ag(I) complexes. The structures indicate that CusB consists of multiple binding sites for these metal ions. These findings reveal novel structural features of an MFP in the resistance-nodulation-division efflux system and provide direct evidence that this protein specifically interacts with transported substrates.

  12. The membrane fusion enigma: SNAREs, Sec1/Munc18 proteins, and their accomplices--guilty as charged?

    Science.gov (United States)

    Rizo, Josep; Südhof, Thomas C

    2012-01-01

    Neurotransmitter release is governed by proteins that have homo-logs in most types of intracellular membrane fusion, including the Sec1/Munc18 protein Munc18-1 and the SNARE proteins syntaxin-1, synaptobrevin/VAMP, and SNAP-25. The SNAREs initiate fusion by forming tight SNARE complexes that bring the vesicle and plasma membranes together. SNARE maintenance in a functional state depends on two chaperone systems (Hsc70/αCSP/SGT and synuclein); defects in these systems lead to neurodegeneration. Munc18-1 binds to an autoinhibitory closed conformation of syntaxin-1, gating formation of SNARE complexes, and also binds to SNARE complexes, which likely underlies the crucial function of Munc18-1 in membrane fusion by an as-yet unclear mechanism. Syntaxin-1 opening is mediated by Munc13s through their MUN domain, which is homologous to diverse tethering factors and may also have a general role in fusion. MUN domain activity is likely modulated in diverse presynaptic plasticity processes that depend on Ca(2+) and RIM proteins, among others.

  13. Subcellular Localization and Calcium and pH Requirements for Proteolytic Processing of the Hendra Virus Fusion Protein

    OpenAIRE

    Pager, Cara Theresia; Wurth, Mark Allen; Dutch, Rebecca Ellis

    2004-01-01

    Proteolytic cleavage of the Hendra virus fusion (F) protein results in the formation of disulfide-linked F1 and F2 subunits, with cleavage occurring after residue K109 in the sequence GDVK↓L. This unusual cleavage site and efficient propagation of Hendra virus in a furin-deficient cell line indicate that the Hendra F protein is not cleaved by furin, the protease responsible for proteolytic activation of many viral fusion proteins. To identify the subcellular site of Hendra F processing, Vero ...

  14. The Matrix protein M1 from influenza C virus induces tubular membrane invaginations in an in vitro cell membrane model.

    Science.gov (United States)

    Saletti, David; Radzimanowski, Jens; Effantin, Gregory; Midtvedt, Daniel; Mangenot, Stéphanie; Weissenhorn, Winfried; Bassereau, Patricia; Bally, Marta

    2017-01-25

    Matrix proteins from enveloped viruses play an important role in budding and stabilizing virus particles. In order to assess the role of the matrix protein M1 from influenza C virus (M1-C) in plasma membrane deformation, we have combined structural and in vitro reconstitution experiments with model membranes. We present the crystal structure of the N-terminal domain of M1-C and show by Small Angle X-Ray Scattering analysis that full-length M1-C folds into an elongated structure that associates laterally into ring-like or filamentous polymers. Using negatively charged giant unilamellar vesicles (GUVs), we demonstrate that M1-C full-length binds to and induces inward budding of membrane tubules with diameters that resemble the diameter of viruses. Membrane tubule formation requires the C-terminal domain of M1-C, corroborating its essential role for M1-C polymerization. Our results indicate that M1-C assembly on membranes constitutes the driving force for budding and suggest that M1-C plays a key role in facilitating viral egress.

  15. Click labeling of unnatural sugars metabolically incorporated into viral envelope glycoproteins enables visualization of single particle fusion

    OpenAIRE

    Oum, Yoon Hyeun; Desai, Tanay M.; Marin, Mariana; Melikyan, Gregory B

    2016-01-01

    Enveloped viruses infect target cells by fusing their membrane with cellular membrane through a process that is mediated by specialized viral glycoproteins. The inefficient and highly asynchronous nature of viral fusion complicates studies of virus entry on a population level. Single virus imaging in living cells has become an important tool for delineating the entry pathways and for mechanistic studies of viral fusion. We have previously demonstrated that incorporation of fluorescent labels ...

  16. Structure of Respiratory Syncytial Virus Fusion Glycoprotein in the Postfusion Conformation Reveals Preservation of Neutralizing Epitopes

    Energy Technology Data Exchange (ETDEWEB)

    McLellan, Jason S.; Yang, Yongping; Graham, Barney S.; Kwong, Peter D. (NIAID)

    2011-09-16

    Respiratory syncytial virus (RSV) invades host cells via a type I fusion (F) glycoprotein that undergoes dramatic structural rearrangements during the fusion process. Neutralizing monoclonal antibodies, such as 101F, palivizumab, and motavizumab, target two major antigenic sites on the RSV F glycoprotein. The structures of these sites as peptide complexes with motavizumab and 101F have been previously determined, but a structure for the trimeric RSV F glycoprotein ectodomain has remained elusive. To address this issue, we undertook structural and biophysical studies on stable ectodomain constructs. Here, we present the 2.8-{angstrom} crystal structure of the trimeric RSV F ectodomain in its postfusion conformation. The structure revealed that the 101F and motavizumab epitopes are present in the postfusion state and that their conformations are similar to those observed in the antibody-bound peptide structures. Both antibodies bound the postfusion F glycoprotein with high affinity in surface plasmon resonance experiments. Modeling of the antibodies bound to the F glycoprotein predicts that the 101F epitope is larger than the linear peptide and restricted to a single protomer in the trimer, whereas motavizumab likely contacts residues on two protomers, indicating a quaternary epitope. Mechanistically, these results suggest that 101F and motavizumab can bind to multiple conformations of the fusion glycoprotein and can neutralize late in the entry process. The structural preservation of neutralizing epitopes in the postfusion state suggests that this conformation can elicit neutralizing antibodies and serve as a useful vaccine antigen.

  17. Fusion Peptide Improves Stability and Bioactivity of Single Chain Antibody against Rabies Virus.

    Science.gov (United States)

    Xi, Hualong; Zhang, Kaixin; Yin, Yanchun; Gu, Tiejun; Sun, Qing; Shi, Linqing; Zhang, Renxia; Jiang, Chunlai; Kong, Wei; Wu, Yongge

    2017-04-28

    The combination of rabies immunoglobulin (RIG) with a vaccine is currently effective against rabies infections, but improvements are needed. Genetic engineering antibody technology is an attractive approach for developing novel antibodies to replace RIG. In our previous study, a single-chain variable fragment, scFv57R, against rabies virus glycoprotein was constructed. However, its inherent weak stability and short half-life compared with the parent RIG may limit its diagnostic and therapeutic application. Therefore, an acidic tail of synuclein (ATS) derived from the C-terminal acidic tail of human alpha-synuclein protein was fused to the C-terminus of scFv57R in order to help it resist adverse stress and improve the stability and halflife. The tail showed no apparent effect on the preparation procedure and affinity of the protein, nor did it change the neutralizing potency in vitro. In the ELISA test of molecular stability, the ATS fusion form of the protein, scFv57R-ATS, showed an increase in thermal stability and longer half-life in serum than scFv57R. The protection against fatal rabies virus challenge improved after fusing the tail to the scFv, which may be attributed to the improved stability. Thus, the ATS fusion approach presented here is easily implemented and can be used as a new strategy to improve the stability and half-life of engineered antibody proteins for practical applications.

  18. Tamiflu-resistant but HA-mediated cell-to-cell transmission through apical membranes of cell-associated influenza viruses.

    Directory of Open Access Journals (Sweden)

    Kotaro Mori

    Full Text Available The infection of viruses to a neighboring cell is considered to be beneficial in terms of evasion from host anti-virus defense systems. There are two pathways for viral infection to "right next door": one is the virus transmission through cell-cell fusion by forming syncytium without production of progeny virions, and the other is mediated by virions without virus diffusion, generally designated cell-to-cell transmission. Influenza viruses are believed to be transmitted as cell-free virus from infected cells to uninfected cells. Here, we demonstrated that influenza virus can utilize cell-to-cell transmission pathway through apical membranes, by handover of virions on the surface of an infected cell to adjacent host cells. Live cell imaging techniques showed that a recombinant influenza virus, in which the neuraminidase gene was replaced with the green fluorescence protein gene, spreads from an infected cell to adjacent cells forming infected cell clusters. This type of virus spreading requires HA activation by protease treatment. The cell-to-cell transmission was also blocked by amantadine, which inhibits the acidification of endosomes required for uncoating of influenza virus particles in endosomes, indicating that functional hemagglutinin and endosome acidification by M2 ion channel were essential for the cell-to-cell influenza virus transmission. Furthermore, in the cell-to-cell transmission of influenza virus, progeny virions could remain associated with the surface of infected cell even after budding, for the progeny virions to be passed on to adjacent uninfected cells. The evidence that cell-to-cell transmission occurs in influenza virus lead to the caution that local infection proceeds even when treated with neuraminidase inhibitors.

  19. Palmitoylation of the feline immunodeficiency virus envelope glycoprotein and its effect on fusion activity and envelope incorporation into virions

    Energy Technology Data Exchange (ETDEWEB)

    Gonzalez, Silvia A.; Paladino, Monica G. [Laboratorio de Virologia, CONICET-Universidad de Belgrano (UB), Villanueva 1324 (C1426BMJ), Buenos Aires (Argentina); Affranchino, Jose L., E-mail: jose.affranchino@comunidad.ub.edu.ar [Laboratorio de Virologia, CONICET-Universidad de Belgrano (UB), Villanueva 1324 (C1426BMJ), Buenos Aires (Argentina)

    2012-06-20

    The feline immunodeficiency virus (FIV) envelope glycoprotein (Env) possesses a short cytoplasmic domain of 53 amino acids containing four highly conserved cysteines at Env positions 804, 811, 815 and 848. Since palmitoylation of transmembrane proteins occurs at or near the membrane anchor, we investigated whether cysteines 804, 811 and 815 are acylated and analyzed the relevance of these residues for Env functions. Replacement of cysteines 804, 811 and 815 individually or in combination by serine residues resulted in Env glycoproteins that were efficiently expressed and processed. However, mutations C804S and C811S reduced Env fusogenicity by 93% and 84%, respectively, compared with wild-type Env. By contrast, mutant C815S exhibited a fusogenic capacity representing 50% of the wild-type value. Remarkably, the double mutation C804S/C811S abrogated both Env fusion activity and Env incorporation into virions. Finally, by means of Click chemistry assays we demonstrated that the four FIV Env cytoplasmic cysteines are palmitoylated.

  20. An alpha-helical domain within the carboxyl terminus of herpes simplex virus type 1 (HSV-1) glycoprotein B (gB) is associated with cell fusion and resistance to heparin inhibition of cell fusion.

    Science.gov (United States)

    Foster, T P; Melancon, J M; Kousoulas, K G

    2001-08-15

    Previous studies from our laboratory indicated that a 28-amino-acid carboxyl-terminal truncation of gB caused extensive virus-induced cell fusion (Baghian et al., 1993, J Virol 67, 2396-2401). We tested the ability of additional truncations and mutations within gB to cause cell fusion in the recently established virus-free cell fusion assay (Turner et al., 1998, J. Virol. 72, 873-875). Deletion of the carboxyl-terminal 28 amino acids of gB (gBDelta28), which removed part of the predicted alpha-helical structure H17b, caused extensive cell fusion. A gB truncation specified by gBDelta36, which removed the entire H17b domain, caused as much cell fusion as the gBDelta28 truncation. Similarly, gB(A874P) containing a substitution of an Ala with Pro within H17b caused cell fusion. Heparin, a gB-specific inhibitor of virus-induced cell fusion, inhibited both wild-type gB and gB(syn3)-mediated cell fusion. In contrast, fusion of cells transfected with gB(Delta28), gB(Delta36), or gB(A874P) was resistant to heparin inhibition of cell fusion. We concluded the following: (1) The predicted alpha-helical structure of H17b within the carboxyl terminus of gB is involved in both virus-induced and virus-free cell fusion. (2) Heparin is a specific inhibitor of gB-mediated fusion in both systems. (3) Resistance to heparin inhibition of gB-mediated cell fusion is associated with the predicted alpha-helical structure H17b within the carboxyl terminus of gB. Copyright 2001 Academic Press.

  1. Membrane dynamics and interactions in measles virus dendritic cell infections.

    Science.gov (United States)

    Avota, Elita; Koethe, Susanne; Schneider-Schaulies, Sibylle

    2013-02-01

    Viral entry, compartmentalization and transmission depend on the formation of membrane lipid/protein microdomains concentrating receptors and signalosomes. Dendritic cells (DCs) are prime targets for measles virus (MV) infection, and this interaction promotes immune activation and generalized immunosuppression, yet also MV transport to secondary lymphatics where transmission to T cells occurs. In addition to MV trapping, DC-SIGN interaction can enhance MV uptake by activating cellular sphingomyelinases and, thereby, vertical surface transport of its entry receptor CD150. To exploit DCs as Trojan horses for transport, MV promotes DC maturation accompanied by mobilization, and restrictions of viral replication in these cells may support this process. MV-infected DCs are unable to support formation of functional immune synapses with conjugating T cells and signalling via viral glycoproteins or repulsive ligands (such as semaphorins) plays a key role in the induction of T-cell paralysis. In the absence of antigen recognition, MV transmission from infected DCs to T cells most likely involves formation of polyconjugates which concentrate viral structural proteins, viral receptors and with components enhancing either viral uptake or conjugate stability. Because DCs barely support production of infectious MV particles, these organized interfaces are likely to represent virological synapses essential for MV transmission. © 2012 Blackwell Publishing Ltd.

  2. The role of blood cell membrane lipids on the mode of action of HIV-1 fusion inhibitor sifuvirtide

    Energy Technology Data Exchange (ETDEWEB)

    Matos, Pedro M.; Freitas, Teresa; Castanho, Miguel A.R.B. [Instituto de Medicina Molecular, Faculdade de Medicina da Universidade de Lisbon, Av. Prof. Egas Moniz, 1649-028 Lisboa (Portugal); Santos, Nuno C., E-mail: nsantos@fm.ul.pt [Instituto de Medicina Molecular, Faculdade de Medicina da Universidade de Lisbon, Av. Prof. Egas Moniz, 1649-028 Lisboa (Portugal)

    2010-12-17

    Research highlights: {yields} Sifuvirtide interacts with erythrocyte and lymphocyte membrane in a concentration dependent manner by decreasing its dipole potential. {yields} Dipole potential variations in lipid vesicles show sifuvirtide's lipid selectivity towards saturated phosphatidylcholines. {yields} This peptide-membrane interaction may direct the drug towards raft-like membrane domains where the receptors used by HIV are located, facilitating its inhibitory action. -- Abstract: Sifuvirtide is a gp41 based peptide that inhibits HIV-1 fusion with the host cells and is currently under clinical trials. Previous studies showed that sifuvirtide partitions preferably to saturated phosphatidylcholine lipid membranes, instead of fluid-phase lipid vesicles. We extended the study to the interaction of the peptide with circulating blood cells, by using the dipole potential sensitive probe di-8-ANEPPS. Sifuvirtide decreased the dipole potential of erythrocyte and lymphocyte membranes in a concentration dependent manner, demonstrating its interaction. Also, the lipid selectivity of the peptide towards more rigid phosphatidylcholines was confirmed based on the dipole potential variations. Overall, the interaction of the peptide with the cell membranes is a contribution of different lipid preferences that presumably directs the peptide towards raft-like domains where the receptors are located, facilitating the reach of the peptide to its molecular target, the gp41 in its pre-fusion conformation.

  3. Binding of white spot syndrome virus to Artemia sp. cell membranes.

    Science.gov (United States)

    Feng, Shuying; Li, Guangda; Feng, Wenpo; Huang, Jie

    2013-10-01

    Using differential velocity centrifugation, cell membranes of Artemia sp. were prepared, and their binding to white spot syndrome virus (WSSV) was analyzed in vitro. The results indicated that WSSV can specifically bind to Artemia cell membranes, and that WSSV receptor very likely existed in this membrane, which suggested that Artemia sp. may be a reservoir of WSSV. This study investigated the specific WSSV binding site by performing competitive inhibition experiments using shrimp gill cell membranes to bind WSSV to Artemia cell membranes. The results showed that shrimp gill cell membranes had a distinct inhibition effect on the specific binding of Artemia cell membranes to WSSV. Thus, potentially similar WSSV receptors or binding sites existed on Artemia sp. cell membranes and shrimp gill cell membranes. Taken together, these findings may provide experimental basis for the development of an effective approach to controlling WSSV, and theoretical basis for the study of WSSV receptors. Copyright © 2013 Elsevier B.V. All rights reserved.

  4. The role of myristoylation in the membrane association of the Lassa virus matrix protein Z

    Directory of Open Access Journals (Sweden)

    Eichler Robert

    2006-11-01

    Full Text Available Abstract The Z protein is the matrix protein of arenaviruses and has been identified as the main driving force for budding. Both LCMV and Lassa virus Z proteins bud from cells in the absence of other viral proteins as enveloped virus-like particles. Z accumulates near the inner surface of the plasma membrane where budding takes place. Furthermore, biochemical data have shown that Z is strongly membrane associated. The primary sequence of Z lacks a typical transmembrane domain and until now it is not understood by which mechanism Z is able to interact with cellular membranes. In this report, we analyzed the role of N-terminal myristoylation for the membrane binding of Lassa virus Z. We show that disruption of the N-terminal myristoylation signal by substituting the N-terminal glycine with alanine (Z-G2A mutant resulted in a significant reduction of Z protein association with cellular membranes. Furthermore, removal of the myristoylation site resulted in a relocalization of Z from a punctuate distribution to a more diffuse cellular distribution pattern. Finally, treatment of Lassa virus-infected cells with various myristoylation inhibitors drastically reduced efficient Lassa virus replication. Our data indicate that myristoylation of Z is critical for its binding ability to lipid membranes and thus, for effective virus budding.

  5. Host Cell Plasma Membrane Phosphatidylserine Regulates the Assembly and Budding of Ebola Virus.

    Science.gov (United States)

    Adu-Gyamfi, Emmanuel; Johnson, Kristen A; Fraser, Mark E; Scott, Jordan L; Soni, Smita P; Jones, Keaton R; Digman, Michelle A; Gratton, Enrico; Tessier, Charles R; Stahelin, Robert V

    2015-09-01

    Lipid-enveloped viruses replicate and bud from the host cell where they acquire their lipid coat. Ebola virus, which buds from the plasma membrane of the host cell, causes viral hemorrhagic fever and has a high fatality rate. To date, little has been known about how budding and egress of Ebola virus are mediated at the plasma membrane. We have found that the lipid phosphatidylserine (PS) regulates the assembly of Ebola virus matrix protein VP40. VP40 binds PS-containing membranes with nanomolar affinity, and binding of PS regulates VP40 localization and oligomerization on the plasma membrane inner leaflet. Further, alteration of PS levels in mammalian cells inhibits assembly and egress of VP40. Notably, interactions of VP40 with the plasma membrane induced exposure of PS on the outer leaflet of the plasma membrane at sites of egress, whereas PS is typically found only on the inner leaflet. Taking the data together, we present a model accounting for the role of plasma membrane PS in assembly of Ebola virus-like particles. The lipid-enveloped Ebola virus causes severe infection with a high mortality rate and currently lacks FDA-approved therapeutics or vaccines. Ebola virus harbors just seven genes in its genome, and there is a critical requirement for acquisition of its lipid envelope from the plasma membrane of the human cell that it infects during the replication process. There is, however, a dearth of information available on the required contents of this envelope for egress and subsequent attachment and entry. Here we demonstrate that plasma membrane phosphatidylserine is critical for Ebola virus budding from the host cell plasma membrane. This report, to our knowledge, is the first to highlight the role of lipids in human cell membranes in the Ebola virus replication cycle and draws a clear link between selective binding and transport of a lipid across the membrane of the human cell and use of that lipid for subsequent viral entry. Copyright © 2015, American

  6. Reprogramming of somatic cells induced by fusion of embryonic stem cells using hemagglutinating virus of Japan envelope (HVJ-E)

    Energy Technology Data Exchange (ETDEWEB)

    Yue, Xiao-shan [Nano Medical Engineering Laboratory, RIKEN Advanced Science Institute, 2-1 Hirosawa, Wako-shi, Saitama 351-0198 (Japan); Department of Biomolecular Engineering, Graduate School of Bioscience and Technology, Tokyo Institute of Technology, Nagatsuta-cho, Midori-ku, Yokohama-shi, Kanagawa 226-8501 (Japan); Fujishiro, Masako [Nano Medical Engineering Laboratory, RIKEN Advanced Science Institute, 2-1 Hirosawa, Wako-shi, Saitama 351-0198 (Japan); Toyoda, Masashi [Department of Reproductive Biology, National Institute for Child Health and Development, 2-10-1, Okura, Setagaya-ku, Tokyo 157-8535 (Japan); Akaike, Toshihiro [Department of Biomolecular Engineering, Graduate School of Bioscience and Technology, Tokyo Institute of Technology, Nagatsuta-cho, Midori-ku, Yokohama-shi, Kanagawa 226-8501 (Japan); Ito, Yoshihiro, E-mail: y-ito@riken.jp [Nano Medical Engineering Laboratory, RIKEN Advanced Science Institute, 2-1 Hirosawa, Wako-shi, Saitama 351-0198 (Japan); Department of Biomolecular Engineering, Graduate School of Bioscience and Technology, Tokyo Institute of Technology, Nagatsuta-cho, Midori-ku, Yokohama-shi, Kanagawa 226-8501 (Japan)

    2010-04-16

    In this research, hemagglutinating virus of Japan envelope (HVJ-E) was used to reprogram somatic cells by fusion with mouse embryonic stem (ES) cells. Neomycin-resistant mouse embryonic fibroblasts (MEFs) were used as somatic cells. Nanog-overexpressing puromycin-resistant EB3 cells were used as mouse ES cells. These two cells were fused by exposing to HVJ-E and the generated fusion cells were selected by puromycin and G418 to get the stable fusion cell line. The fusion cells form colonies in feeder-free culture system. Microsatellite analysis of the fusion cells showed that they possessed genes from both ES cells and fibroblasts. The fusion cells were tetraploid, had alkali phosphatase activity, and expressed stem cell marker genes such as Pou5f1, Nanog, and Sox2, but not the fibroblast cell marker genes such as Col1a1 and Col1a2. The pluripotency of fusion cells was confirmed by their expression of marker genes for all the three germ layers after differentiation induction, and by their ability to form teratoma which contained all the three primary layers. Our results show that HVJ-E can be used as a fusion reagent for reprogramming of somatic cells.

  7. Reprogramming of somatic cells induced by fusion of embryonic stem cells using hemagglutinating virus of Japan envelope (HVJ-E).

    Science.gov (United States)

    Yue, Xiao-shan; Fujishiro, Masako; Toyoda, Masashi; Akaike, Toshihiro; Ito, Yoshihiro

    2010-04-16

    In this research, hemagglutinating virus of Japan envelope (HVJ-E) was used to reprogram somatic cells by fusion with mouse embryonic stem (ES) cells. Neomycin-resistant mouse embryonic fibroblasts (MEFs) were used as somatic cells. Nanog-overexpressing puromycin-resistant EB3 cells were used as mouse ES cells. These two cells were fused by exposing to HVJ-E and the generated fusion cells were selected by puromycin and G418 to get the stable fusion cell line. The fusion cells form colonies in feeder-free culture system. Microsatellite analysis of the fusion cells showed that they possessed genes from both ES cells and fibroblasts. The fusion cells were tetraploid, had alkali phosphatase activity, and expressed stem cell marker genes such as Pou5f1, Nanog, and Sox2, but not the fibroblast cell marker genes such as Col1a1 and Col1a2. The pluripotency of fusion cells was confirmed by their expression of marker genes for all the three germ layers after differentiation induction, and by their ability to form teratoma which contained all the three primary layers. Our results show that HVJ-E can be used as a fusion reagent for reprogramming of somatic cells. Crown Copyright 2010. Published by Elsevier Inc. All rights reserved.

  8. Mildly Acidic pH Triggers an Irreversible Conformational Change in the Fusion Domain of Herpes Simplex Virus 1 Glycoprotein B and Inactivation of Viral Entry.

    Science.gov (United States)

    Weed, Darin J; Pritchard, Suzanne M; Gonzalez, Floricel; Aguilar, Hector C; Nicola, Anthony V

    2017-03-01

    Herpes simplex virus (HSV) entry into a subset of cells requires endocytosis and endosomal low pH. Preexposure of isolated virions to mildly acidic pH of 5 to 6 partially inactivates HSV infectivity in an irreversible manner. Acid inactivation is a hallmark of viruses that enter via low-pH pathways; this occurs by pretriggering conformational changes essential for fusion. The target and mechanism(s) of low-pH inactivation of HSV are unclear. Here, low-pH-treated HSV-1 was defective in fusion activity and yet retained normal levels of attachment to cell surface heparan sulfate and binding to nectin-1 receptor. Low-pH-triggered conformational changes in gB reported to date are reversible, despite irreversible low-pH inactivation. gB conformational changes and their reversibility were measured by antigenic analysis with a panel of monoclonal antibodies and by detecting changes in oligomeric conformation. Three-hour treatment of HSV-1 virions with pH 5 or multiple sequential treatments at pH 5 followed by neutral pH caused an irreversible >2.5 log infectivity reduction. While changes in several gB antigenic sites were reversible, alteration of the H126 epitope was irreversible. gB oligomeric conformational change remained reversible under all conditions tested. Altogether, our results reveal that oligomeric alterations and fusion domain changes represent distinct conformational changes in gB, and the latter correlates with irreversible low-pH inactivation of HSV. We propose that conformational change in the gB fusion domain is important for activation of membrane fusion during viral entry and that in the absence of a host target membrane, this change results in irreversible inactivation of virions. IMPORTANCE HSV-1 is an important pathogen with a high seroprevalence throughout the human population. HSV infects cells via multiple pathways, including a low-pH route into epithelial cells, the primary portal into the host. HSV is inactivated by low-pH preexposure, and g

  9. Endothelial galectin-1 binds to specific glycans on nipah virus fusion protein and inhibits maturation, mobility, and function to block syncytia formation.

    Directory of Open Access Journals (Sweden)

    Omai B Garner

    2010-07-01

    Full Text Available Nipah virus targets human endothelial cells via NiV-F and NiV-G envelope glycoproteins, resulting in endothelial syncytia formation and vascular compromise. Endothelial cells respond to viral infection by releasing innate immune effectors, including galectins, which are secreted proteins that bind to specific glycan ligands on cell surface glycoproteins. We demonstrate that galectin-1 reduces NiV-F mediated fusion of endothelial cells, and that endogenous galectin-1 in endothelial cells is sufficient to inhibit syncytia formation. Galectin-1 regulates NiV-F mediated cell fusion at three distinct points, including retarding maturation of nascent NiV-F, reducing NiV-F lateral mobility on the plasma membrane, and directly inhibiting the conformational change in NiV-F required for triggering fusion. Characterization of the NiV-F N-glycome showed that the critical site for galectin-1 inhibition is rich in glycan structures known to bind galectin-1. These studies identify a unique set of mechanisms for regulating pathophysiology of NiV infection at the level of the target cell.

  10. The UL24 protein of herpes simplex virus 1 affects the sub-cellular distribution of viral glycoproteins involved in fusion

    Energy Technology Data Exchange (ETDEWEB)

    Ben Abdeljelil, Nawel; Rochette, Pierre-Alexandre; Pearson, Angela, E-mail: angela.pearson@iaf.inrs.ca

    2013-09-15

    Mutations in UL24 of herpes simplex virus type 1 can lead to a syncytial phenotype. We hypothesized that UL24 affects the sub-cellular distribution of viral glycoproteins involved in fusion. In non-immortalized human foreskin fibroblasts (HFFs) we detected viral glycoproteins B (gB), gD, gH and gL present in extended blotches throughout the cytoplasm with limited nuclear membrane staining; however, in HFFs infected with a UL24-deficient virus (UL24X), staining for the viral glycoproteins appeared as long, thin streaks running across the cell. Interestingly, there was a decrease in co-localized staining of gB and gD with F-actin at late times in UL24X-infected HFFs. Treatment with chemical agents that perturbed the actin cytoskeleton hindered the formation of UL24X-induced syncytia in these cells. These data support a model whereby the UL24 syncytial phenotype results from a mislocalization of viral glycoproteins late in infection. - Highlights: • UL24 affects the sub-cellular distribution of viral glycoproteins required for fusion. • Sub-cellular distribution of viral glycoproteins varies in cell-type dependent manner. • Drugs targeting actin microfilaments affect formation of UL24-related syncytia in HFFs.

  11. Surface Density of the Hendra G Protein Modulates Hendra F Protein-Promoted Membrane Fusion: Role for Hendra G Protein Trafficking and Degradation

    OpenAIRE

    Whitman, Shannon D.; Dutch, Rebecca Ellis

    2007-01-01

    Hendra virus, like most paramyxoviruses, requires both a fusion (F) and attachment (G) protein for promotion of cell-cell fusion. Recent studies determined that Hendra F is proteolytically processed by the cellular protease cathepsin L after endocytosis. This unique cathepsin L processing results in a small percentage of Hendra F on the cell surface. To determine how the surface densities of the two Hendra glycoproteins affect fusion promotion, we performed experiments that varied the levels ...

  12. Solid-state nuclear magnetic resonance measurements of HIV fusion peptide 13CO to lipid 31P proximities support similar partially inserted membrane locations of the α helical and β sheet peptide structures.

    Science.gov (United States)

    Gabrys, Charles M; Qiang, Wei; Sun, Yan; Xie, Li; Schmick, Scott D; Weliky, David P

    2013-10-03

    Fusion of the human immunodeficiency virus (HIV) membrane and the host cell membrane is an initial step of infection of the host cell. Fusion is catalyzed by gp41, which is an integral membrane protein of HIV. The fusion peptide (FP) is the ∼25 N-terminal residues of gp41 and is a domain of gp41 that plays a key role in fusion catalysis likely through interaction with the host cell membrane. Much of our understanding of the FP domain has been accomplished with studies of "HFP", i.e., a ∼25-residue peptide composed of the FP sequence but lacking the rest of gp41. HFP catalyzes fusion between membrane vesicles and serves as a model system to understand fusion catalysis. HFP binds to membranes and the membrane location of HFP is likely a significant determinant of fusion catalysis perhaps because the consequent membrane perturbation reduces the fusion activation energy. In the present study, many HFPs were synthesized and differed in the residue position that was (13)CO backbone labeled. Samples were then prepared that each contained a singly (13)CO labeled HFP incorporated into membranes that lacked cholesterol. HFP had distinct molecular populations with either α helical or oligomeric β sheet structure. Proximity between the HFP (13)CO nuclei and (31)P nuclei in the membrane headgroups was probed by solid-state NMR (SSNMR) rotational-echo double-resonance (REDOR) measurements. For many samples, there were distinct (13)CO shifts for the α helical and β sheet structures so that the proximities to (31)P nuclei could be determined for each structure. Data from several differently labeled HFPs were then incorporated into a membrane location model for the particular structure. In addition to the (13)CO labeled residue position, the HFPs also differed in sequence and/or chemical structure. "HFPmn" was a linear peptide that contained the 23 N-terminal residues of gp41. "HFPmn_V2E" contained the V2E mutation that for HIV leads to greatly reduced extent of fusion and

  13. Solid-State Nuclear Magnetic Resonance Measurements of HIV Fusion Peptide 13CO to Lipid 31P Proximities Support Similar Partially Inserted Membrane Locations of the α Helical and β Sheet Peptide Structures

    Science.gov (United States)

    Gabrys, Charles M.; Qiang, Wei; Sun, Yan; Xie, Li; Schmick, Scott D.; Weliky, David P.

    2013-10-01

    Fusion of the human immunodeficiency virus (HIV) membrane and the host cell membrane is an initial step of infection of the host cell. Fusion is catalyzed by gp41, which is an integral membrane protein of HIV. The fusion peptide (FP) is the -25 N-terminal residues of gp41 and is a domain of gp41 that plays a key role in fusion catalysis likely through interaction with the host cell membrane. Much of our understanding of the FP domain has been accomplished with studies of -HFP-, i.e., a -25-residue peptide composed of the FP sequence but lacking the rest of gp41. HFP catalyzes fusion between membrane vesicles and serves as a model system to understand fusion catalysis. HFP binds to membranes and the membrane location of HFP is likely a significant determinant of fusion catalysis perhaps because the consequent membrane perturbation reduces the fusion activation energy. In the present study, many HFPs were synthesized and differed in the residue position that was 13CO backbone labeled. Samples were then prepared that each contained a singly 13CO labeled HFP incorporated into membranes that lacked cholesterol. HFP had distinct molecular populations with either α helical or oligomeric - sheet structure. Proximity between the HFP 13CO nuclei and 31P nuclei in the membrane headgroups was probed by solid-state NMR (SSNMR) rotational-echo double-resonance (REDOR) measurements. For many samples, there were distinct 13CO shifts for the α helical and - sheet structures so that the proximities to 31P nuclei could be determined for each structure. Data from several differently labeled HFPs were then incorporated into a membrane location model for the particular structure. In addition to the 13CO labeled residue position, the HFPs also differed in sequence and/or chemical structure. -HFPmn- was a linear peptide that contained the 23 N-terminal residues of gp41. -HFPmn_V2E- contained the V2E mutation that for HIV leads to greatly reduced extent of fusion and infection. The

  14. Additive protection induced by mixed virus-like particles presenting respiratory syncytial virus fusion or attachment glycoproteins.

    Science.gov (United States)

    Lee, Sujin; Quan, Fu-Shi; Kwon, Youngman; Sakamoto, Kaori; Kang, Sang-Moo; Compans, Richard W; Moore, Martin L

    2014-11-01

    Respiratory syncytial virus (RSV) is the most important pathogen for lower respiratory tract illness in infants and a high priority for vaccine development. We previously reported that RSV virus-like particles (VLPs) expressing either the fusion (F) or attachment (G) glycoprotein could confer protection against RSV challenge in BALB/c mice. Here, we tested the hypothesis that RSV VLP vaccine efficacy can be enhanced by mixing RSV VLP F and RSV VLP G, and we analyzed host responses to these RSV VLPs. Mice were immunized with VLP F, VLP G, or VLP F+VLP G. Lung viral loads in BALB/c mice following RSV strain A2-line19F challenge were lower in mice vaccinated with RSV VLP F+VLP G compared to VLP F- or VLP G-vaccinated mice. Vaccination with VLP F or VLP F+VLP G induced similar levels of neutralizing antibodies. The enhanced protection against RSV challenge induced by vaccination with RSV VLP F+VLP G correlated with CD8 T cells producing T helper type 1 cytokines. VLP G vaccination alone followed by challenge resulted in immunopathology similar to formalin-inactivated RSV vaccination and RSV challenge. Taken together, mixed VLP F+VLP G provided a high level of protection against RSV without vaccine-induced immunopathology, but VLP G vaccination enhanced disease when used alone. Copyright © 2014 Elsevier B.V. All rights reserved.

  15. Naja naja atra and Naja nigricollis cardiotoxins induce fusion of Escherichia coli and Staphylococcus aureus membrane-mimicking liposomes.

    Science.gov (United States)

    Kao, Pei-Hsiu; Lin, Shinne-Ren; Hu, Wan-Ping; Chang, Long-Sen

    2012-09-01

    Our previous studies showed that the bactericidal effect of Naja naja atra cardiotoxin 3 (CTX3) and Naja nigricollis toxin γ was associated with their membrane-damaging activity. To elucidate the mechanism responsible for CTX3- and toxin γ-induced membrane permeability, we investigated the interacted mode of CTX3 and toxin γ with model membrane of Escherichia coli (phosphatidylethanolamine (PE)/phosphatidylglycerol (PG), mol/mol, 75/25) and Staphylococcus aureus (PG/cardiolipin, mol/mol, 60/40) in this study. Membrane-damaging activity of toxin γ on PE/PG and PG/cardiolipin vesicles were similar, while CTX3-induced leakage of PG/cardiolipin vesicles was notably higher than that of PE/PG vesicles. Noticeably, fusogenic activity of CTX3 and toxin γ on the phospholipid vesicles correlated positively with their membrane-damaging activity. Unlike toxin γ, CTX3 induced increasingly leakage and fusion of phospholipid vesicles with increased cardiolipin content. Changes in membrane fluidity and lipid packing occurred with the binding of CTX3 and toxin γ with vesicles, reflecting the penetration of toxin molecules into membrane bilayers. Consistent with the finding that PE/PG and PG/cardiolipin vesicles induced differently conformational changes of CTX3 and toxin γ, CTX3 and toxin γ adopted different membrane bound-mode upon absorption onto either PE/PG or PG/cardiolipin vesicles. Taken together, our data indicate that membrane-bound mode and membrane-perturbing effect of CTX3 and toxin γ in concert with targeted membrane compositions determine their fusogenicity and membrane-damaging activity, and suggest a causal relationship between bactericidal activity and fusogenicity of CTX3 and toxin γ. Copyright © 2012 Elsevier Ltd. All rights reserved.

  16. Structure of a Major Antigenic Site on the Respiratory Syncytial Virus Fusion Glycoprotein in Complex with Neutralizing Antibody 101F

    Energy Technology Data Exchange (ETDEWEB)

    McLellan, Jason S.; Chen, Man; Chang, Jung-San; Yang, Yongping; Kim, Albert; Graham, Barney S.; Kwong, Peter D. (NIH)

    2010-11-19

    Respiratory syncytial virus (RSV) is a major cause of pneumonia and bronchiolitis in infants and elderly people. Currently there is no effective vaccine against RSV, but passive prophylaxis with neutralizing antibodies reduces hospitalizations. To investigate the mechanism of antibody-mediated RSV neutralization, we undertook structure-function studies of monoclonal antibody 101F, which binds a linear epitope in the RSV fusion glycoprotein. Crystal structures of the 101F antigen-binding fragment in complex with peptides from the fusion glycoprotein defined both the extent of the linear epitope and the interactions of residues that are mutated in antibody escape variants. The structure allowed for modeling of 101F in complex with trimers of the fusion glycoprotein, and the resulting models suggested that 101F may contact additional surfaces located outside the linear epitope. This hypothesis was supported by surface plasmon resonance experiments that demonstrated 101F bound the peptide epitope {approx}16,000-fold more weakly than the fusion glycoprotein. The modeling also showed no substantial clashes between 101F and the fusion glycoprotein in either the pre- or postfusion state, and cell-based assays indicated that 101F neutralization was not associated with blocking virus attachment. Collectively, these results provide a structural basis for RSV neutralization by antibodies that target a major antigenic site on the fusion glycoprotein.

  17. Point mutations in the paramyxovirus F protein that enhance fusion activity shift the mechanism of complement-mediated virus neutralization.

    Science.gov (United States)

    Johnson, John B; Schmitt, Anthony P; Parks, Griffith D

    2013-08-01

    Parainfluenza virus 5 (PIV5) activates and is neutralized by the alternative pathway (AP) in normal human serum (NHS) but not by heat-inactivated (HI) serum. We have tested the relationship between the fusion activity within the PIV5 F protein, the activation of complement pathways, and subsequent complement-mediated virus neutralization. Recombinant PIV5 viruses with enhanced fusion activity were generated by introducing point mutations in the F fusogenic peptide (G3A) or at a distal site near the F transmembrane domain (S443P). In contrast to wild-type (WT) PIV5, the mutant G3A and S443P viruses were neutralized by both NHS and HI serum. Unlike WT PIV5, hyperfusogenic G3A and S443P viruses were potent C4 activators, C4 was deposited on NHS-treated mutant virions, and the mutants were neutralized by factor B-depleted serum but not by C4-depleted serum. Antibodies purified from HI human serum were sufficient to neutralize both G3A and S443P viruses in vitro but were ineffective against WT PIV5. Electron microscopy data showed greater deposition of purified human antibodies on G3A and S443P virions than on WT PIV5 particles. These data indicate that single amino acid changes that enhance the fusion activity of the PIV5 F protein shift the mechanism of complement activation in the context of viral particles or on the surface of virus-infected cells, due to enhanced binding of antibodies. We present general models for the relationship between enhanced fusion activity in the paramyxovirus F protein and increased susceptibility to antibody-mediated neutralization.

  18. Selective Fusion of Heterogeneous Classifiers for Predicting Substrates of Membrane Transporters.

    Science.gov (United States)

    Shaikh, Naeem; Sharma, Mahesh; Garg, Prabha

    2017-03-27

    Membrane transporters play a crucial role in determining fate of administered drugs in a biological system. Early identification of plausible transporters for a drug molecule can provide insights into its therapeutic, pharmacokinetic, and toxicological profiles. In the present study, predictive models for classifying small molecules into substrates and nonsubstrates of various pharmaceutically important membrane transporters were developed using quantitative structure-activity relationship (QSAR) and proteochemometric (PCM) approaches. For this purpose, 4575 substrate interactions for these transporters were collected from the Metabolism and Transport Database (Metrabase) and the literature. The transporters selected for this study include (i) six efflux transporters, viz., breast cancer resistance protein (BCRP/ABCG2), P-glycoprotein (P-gp/MDR1), and multidrug resistance proteins (MRP1, MRP2, MRP3, and MRP4), and (ii) seven influx transporters, viz., organic cation transporter (OCT1/SO22A1), peptide transporter (PEPT1/SO15A1), apical sodium-bile acid transporter (ASBT/NTCP2), and organic anion transporting peptides (OATP1A2/SO1A2, OATP1B/SO1B1, OATP1B3/SO1B3, and OATP2B1/SO2B1). Various types of descriptors and machine learning methods (classifiers) were evaluated for the development of robust predictive models. Additionally, ensemble models were developed by bagging of homogeneous classifiers and selective fusion of heterogeneous classifiers. It was observed that the latter approach improves the accuracy of substrate/nonsubstrate prediction for transporters (average correct classification rate of more than 0.80 for external validation). Moreover, structural fragments important in determining the substrate specificity across the various transporters were identified. To demonstrate these fragments on the query molecule, contour maps were generated. The prediction efficacy of the developed models was illustrated by a good correlation between the reported logBB value

  19. Critical role of the fusion protein cytoplasmic tail sequence in parainfluenza virus assembly.

    Directory of Open Access Journals (Sweden)

    Raychel Stone

    Full Text Available Interactions between viral glycoproteins, matrix protein and nucleocapsid sustain assembly of parainfluenza viruses at the plasma membrane. Although the protein interactions required for virion formation are considered to be highly specific, virions lacking envelope glycoprotein(s can be produced, thus the molecular interactions driving viral assembly and production are still unclear. Sendai virus (SeV and human parainfluenza virus type 1 (hPIV1 are highly similar in structure, however, the cytoplasmic tail sequences of the envelope glycoproteins (HN and F are relatively less conserved. To unveil the specific role of the envelope glycoproteins in viral assembly, we created chimeric SeVs whose HN (rSeVhHN or HN and F (rSeVh(HN+F were replaced with those of hPIV1. rSeVhHN grew as efficiently as wt SeV or hPIV1, suggesting that the sequence difference in HN does not have a significant impact on SeV replication and virion production. In sharp contrast, the growth of rSeVh(HN+F was significantly impaired compared to rSeVhHN. rSeVh(HN+Fstail which expresses a chimeric hPIV1 F with the SeV cytoplasmic tail sequence grew similar to wt SeV or rSeVhHN. Further analysis indicated that the F cytoplasmic tail plays a critical role in cell surface expression/accumulation of HN and F, as well as NP and M association at the plasma membrane. Trafficking of nucelocapsids in infected cells was not significantly affected by the origin of F, suggesting that F cytoplasmic tail is not involved in intracellular movement. These results demonstrate the role of the F cytoplasmic tail in accumulation of structural components at the plasma membrane assembly sites.

  20. Addition of a Magnetite Layer onto a Polysulfone Water Treatment Membrane to Enhance Virus Removal

    Science.gov (United States)

    Raciny, Isabel

    The applicability of low-pressure membranes systems in distributed (point of use) water treatment is hindered by, among other things, their inability to remove potentially harmful viruses and ions via size exclusion. According to the USEPA and the Safe Drinking Water Act, drinking water treatment processes must be designed for 4-log virus removal. Batch experiments using magnetite nanoparticle (nano-Fe3O4) suspensions and water filtration experiments with Polysulfone (PSf) membranes coated with nano-Fe3O 4 were conducted to assess the removal of a model virus (bacteriophage MS2). The membranes were coated via a simple filtration protocol. Unmodified membranes were a poor adsorbent for MS2 bacteriophage with less than 0.5-log removal, whereas membranes coated with magnetite nanoparticles exhibited a removal efficiency exceeding 99.99% (4-log). Thus, a cartridge of PSf membranes coated with nano-Fe3O4 particles could be used to remove viruses from water. Such membranes showed negligible iron leaching into the filtrate, thus obviating concern about colored water. Further research is needed to reduce the loss of water flux caused by coating.

  1. Probing a self-assembled fd virus membrane with a microtubule.

    Science.gov (United States)

    Xie, Sheng; Pelcovits, Robert A; Hagan, Michael F

    2016-06-01

    The self-assembly of highly anisotropic colloidal particles leads to a rich variety of morphologies whose properties are just beginning to be understood. This article uses computer simulations to probe a particle-scale perturbation of a commonly studied colloidal assembly, a monolayer membrane composed of rodlike fd viruses in the presence of a polymer depletant. Motivated by experiments currently in progress, we simulate the interaction between a microtubule and a monolayer membrane as the microtubule "pokes" and penetrates the membrane face-on. Both the viruses and the microtubule are modeled as hard spherocylinders of the same diameter, while the depletant is modeled using ghost spheres. We find that the force exerted on the microtubule by the membrane is zero either when the microtubule is completely outside the membrane or when it has fully penetrated the membrane. The microtubule is initially repelled by the membrane as it begins to penetrate but experiences an attractive force as it penetrates further. We assess the roles played by translational and rotational fluctuations of the viruses and the osmotic pressure of the polymer depletant. We find that rotational fluctuations play a more important role than the translational ones. The dependence on the osmotic pressure of the depletant of the width and height of the repulsive barrier and the depth of the attractive potential well is consistent with the assumed depletion-induced attractive interaction between the microtubule and viruses. We discuss the relevance of these studies to the experimental investigations.

  2. Viruses budding from either the apical or the basolateral plasma membrane domain of MDCK cells have unique phospholipid compositions

    NARCIS (Netherlands)

    van Meer, G.; Simons, K.

    1982-01-01

    Influenza virus and vesicular stomatitis virus (VSV) obtain their lipid envelope by budding through the plasma membrane of infected cells. When monolayers of Madin-Darby canine kidney (MDCK) cells, a polarized epithelial cell line, are infected with fowl plague virus (FPV), an avian influenza virus,

  3. Three-Dimensional Architecture and Biogenesis of Membrane Structures Associated with Plant Virus Replication.

    Science.gov (United States)

    Jin, Xuejiao; Cao, Xiuling; Wang, Xueting; Jiang, Jun; Wan, Juan; Laliberté, Jean-François; Zhang, Yongliang

    2018-01-01

    Positive-sense (+) RNA viruses represent the most abundant group of viruses and are dependent on the host cell machinery to replicate. One remarkable feature that occurs after (+) RNA virus entry into cells is the remodeling of host endomembranes, leading to the formation of viral replication factories. Recently, rapid progress in three-dimensional (3D) imaging technologies, such as electron tomography (ET) and focused ion beam-scanning electron microscopy (FIB-SEM), has enabled researchers to visualize the novel membrane structures induced by viruses at high resolution. These 3D imaging technologies provide new mechanistic insights into the viral infection cycle. In this review, we summarize the latest reports on the cellular remodeling that occurs during plant virus infection; in particular, we focus on studies that provide 3D architectural information on viral replication factories. We also outline the mechanisms underlying the formation of these membranous structures and discuss possible future research directions.

  4. Assessment of the efficacy of membrane filtration processes to remove human enteric viruses and the suitability of bacteriophages and a plant virus as surrogates for those viruses.

    Science.gov (United States)

    Shirasaki, N; Matsushita, T; Matsui, Y; Murai, K

    2017-05-15

    Here, we evaluated the efficacy of direct microfiltration (MF) and ultrafiltration (UF) to remove three representative human enteric viruses (i.e., adenovirus [AdV] type 40, coxsackievirus [CV] B5, and hepatitis A virus [HAV] IB), and one surrogate of human caliciviruses (i.e., murine norovirus [MNV] type 1). Eight different MF membranes and three different UF membranes were used. We also examined the ability of coagulation pretreatment with high-basicity polyaluminum chloride (PACl) to enhance virus removal by MF. The removal ratios of two bacteriophages (MS2 and φX174) and a plant virus (pepper mild mottle virus; PMMoV) were compared with the removal ratios of the human enteric viruses to assess the suitability of these viruses to be used as surrogates for human enteric viruses. The virus removal ratios obtained with direct MF with membranes with nominal pore sizes of 0.1-0.22 μm differed, depending on the membrane used; adsorptive interactions, particularly hydrophobic interactions between virus particles and the membrane surface, were dominant factors for virus removal. In contrast, direct UF with membranes with nominal molecular weight cutoffs of 1-100 kDa effectively removed viruses through size exclusion, and >4-log10 removal was achieved when a membrane with a nominal molecular weight cutoff of 1 kDa was used. At pH 7 and 8, in-line coagulation-MF with nonsulfated high-basicity PACls containing Al30 species had generally a better virus removal (i.e., >4-log10 virus removal) than the other aluminum-based coagulants, except for φX174. For all of the filtration processes, the removal ratios of AdV, CV, HAV, and MNV were comparable and strongly correlated with each other. The removal ratios of MS2 and PMMoV were comparable or smaller than those of the three human enteric viruses and MNV, and were strongly correlated with those of the three human enteric viruses and MNV. The removal ratios obtained with coagulation-MF for φX174 were markedly smaller than

  5. Adhesion and fusion efficiencies of human immunodeficiency virus type 1 (HIV-1) surface proteins

    Science.gov (United States)

    Dobrowsky, Terrence M.; Rabi, S. Alireza; Nedellec, Rebecca; Daniels, Brian R.; Mullins, James I.; Mosier, Donald E.; Siliciano, Robert F.; Wirtz, Denis

    2013-10-01

    In about half of patients infected with HIV-1 subtype B, viral populations shift from utilizing the transmembrane protein CCR5 to CXCR4, as well as or instead of CCR5, during late stage progression of the disease. How the relative adhesion efficiency and fusion competency of the viral Env proteins relate to infection during this transition is not well understood. Using a virus-cell fusion assay and live-cell single-molecule force spectroscopy, we compare the entry competency of viral clones to tensile strengths of the individual Env-receptor bonds of Env proteins obtained from a HIV-1 infected patient prior to and during coreceptor switching. The results suggest that the genetic determinants of viral entry were predominantly enriched in the C3, HR1 and CD regions rather than V3. Env proteins can better mediate entry into cells after coreceptor switch; this effective entry capacity does not correlate with the bond strengths between viral Env and cellular receptors.

  6. Glycosyl-phosphatidylinositol (GPI)-anchored membrane association of the porcine reproductive and respiratory syndrome virus GP4 glycoprotein and its co-localization with CD163 in lipid rafts

    Energy Technology Data Exchange (ETDEWEB)

    Du, Yijun [Department of Pathobiology, University of Illinois at Urbana-Champaign, 2001 South Lincoln Ave, Urbana, IL 61802 (United States); Shandong Key Laboratory of Animal Disease Control and Breeding, Institute of Animal Science and Veterinary Medicine, Shandong Academy of Agricultural Sciences, Jinan (China); Pattnaik, Asit K. [School of Veterinary Medicine and Biomedical Sciences and the Nebraska Center for Virology, University of Nebraska-Lincoln, Lincoln, NE 68583-0900 (United States); Song, Cheng [Department of Pathobiology, University of Illinois at Urbana-Champaign, 2001 South Lincoln Ave, Urbana, IL 61802 (United States); Yoo, Dongwan, E-mail: dyoo@illinois.edu [Department of Pathobiology, University of Illinois at Urbana-Champaign, 2001 South Lincoln Ave, Urbana, IL 61802 (United States); Li, Gang, E-mail: dyoo@illinois.edu [Department of Pathobiology, University of Illinois at Urbana-Champaign, 2001 South Lincoln Ave, Urbana, IL 61802 (United States); Institute of Animal Science and Veterinary Medicine, Chinese Academy of Agricultural Sciences, Beijing (China)

    2012-03-01

    The porcine reproductive and respiratory syndrome virus (PRRSV) glycoprotein 4 (GP4) resembles a typical type I membrane protein in its structure but lacks a hydrophilic tail at the C-terminus, suggesting that GP4 may be a lipid-anchored membrane protein. Using the human decay-accelerating factor (DAF; CD55), a known glycosyl-phosphatidylinositol (GPI) lipid-anchored protein, chimeric constructs were made to substitute the GPI-anchor domain of DAF with the putative lipid-anchor domain of GP4, and their membrane association and lipase cleavage were determined in cells. The DAF-GP4 fusion protein was transported to the plasma membrane and was cleaved by phosphatidylinositol-specific phospholipase C (PI-PLC), indicating that the C-terminal domain of GP4 functions as a GPI anchor. Mutational studies for residues adjacent to the GPI modification site and characterization of respective mutant viruses generated from infectious cDNA clones show that the ability of GP4 for membrane association corresponded to virus viability and growth characteristics. The residues T158 ({omega} - 2, where {omega} is the GPI moiety at E160), P159 ({omega} - 1), and M162 ({omega} + 2) of GP4 were determined to be important for virus replication, with M162 being of particular importance for virus infectivity. The complete removal of the peptide-anchor domain in GP4 resulted in a complete loss of virus infectivity. The depletion of cholesterol from the plasma membrane of cells reduced the virus production, suggesting a role of lipid rafts in PRRSV infection. Remarkably, GP4 was found to co-localize with CD163 in the lipid rafts on the plasma membrane. Since CD163 has been reported as a cellular receptor for PRRSV and GP4 has been shown to interact with this receptor, our data implicates an important role of lipid rafts during entry of the virus.

  7. Vesicular PtdIns(3,4,5)P3 and Rab7 are key effectors of sea urchin zygote nuclear membrane fusion.

    Science.gov (United States)

    Lete, Marta G; Byrne, Richard D; Alonso, Alicia; Poccia, Dominic; Larijani, Banafshé

    2017-01-15

    Regulation of nuclear envelope dynamics is an important example of the universal phenomena of membrane fusion. The signalling molecules involved in nuclear membrane fusion might also be conserved during the formation of both pronuclear and zygote nuclear envelopes in the fertilised egg. Here, we determine that class-I phosphoinositide 3-kinases (PI3Ks) are needed for in vitro nuclear envelope formation. We show that, in vivo, PtdIns(3,4,5)P 3 is transiently located in vesicles around the male pronucleus at the time of nuclear envelope formation, and around male and female pronuclei before membrane fusion. We illustrate that class-I PI3K activity is also necessary for fusion of the female and male pronuclear membranes. We demonstrate, using coincidence amplified Förster resonance energy transfer (FRET) monitored using fluorescence lifetime imaging microscopy (FLIM), a protein-lipid interaction of Rab7 GTPase and PtdIns(3,4,5)P 3 that occurs during pronuclear membrane fusion to create the zygote nuclear envelope. We present a working model, which includes several molecular steps in the pathways controlling fusion of nuclear envelope membranes. © 2017. Published by The Company of Biologists Ltd.

  8. Decoding distinct membrane interactions of HIV-1 fusion inhibitors using a combined atomic force and fluorescence microscopy approach.

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    Franquelim, Henri G; Gaspar, Diana; Veiga, A Salomé; Santos, Nuno C; Castanho, Miguel A R B

    2013-08-01

    Enfuvirtide and T-1249 are two potent HIV-1 fusion inhibitor peptides. Recent studies indicate that lipids play an important role in the mode of action of those bioactive molecules. Using a combined tandem atomic force microscopy (AFM)-epifluorescence microscopy approach, we studied the interaction of both enfuvirtide and T-1249 with supported lipid bilayers. Fluid (ld)-gel (so) and ld-liquid ordered (lo) phase-separated membrane systems were tested. Results, especially for T-1249, show significant lipid membrane activity at a 15μM peptide concentration. T-1249, in opposition to enfuvirtide, induces an increase in membrane surface roughness, decrease in membrane fluidity, bilayer thinning at ld domains and disruption of the so domain borders. In terms of structural properties, both enfuvirtide and T-1249 possess distinct functional hydrophobic and amphipathic domains of HIV gp41. While enfuvirtide only yields the tryptophan-rich domain (TRD), T-1249 possesses both TRD and pocket-binding domain (PBD). TRD increases the hydrophobicity of the peptide while PBD enhances the amphipathic characteristics. As such, the enhanced membrane activity of T-1249 may be explained by a synergism between its amphipathic N-terminal segment and its hydrophophic C-terminal. Our findings provide valuable insights on the molecular-level mode of action of HIV-1 fusion inhibitors, unraveling the correlation between their structural properties and membrane interactions as a factor influencing their antiviral activity. Ultimately, this work validates the applicability of a combined AFM and fluorescence approach to evaluate the mechanic and structural properties of supported lipid bilayers upon interaction with membrane-active peptides. Copyright © 2013 Elsevier B.V. All rights reserved.

  9. Lateral Organization of Influenza Virus Proteins in the Budozone Region of the Plasma Membrane.

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    Leser, George P; Lamb, Robert A

    2017-05-01

    Influenza virus assembles and buds at the plasma membrane of virus-infected cells. The viral proteins assemble at the same site on the plasma membrane for budding to occur. This involves a complex web of interactions among viral proteins. Some proteins, like hemagglutinin (HA), NA, and M2, are integral membrane proteins. M1 is peripherally membrane associated, whereas NP associates with viral RNA to form an RNP complex that associates with the cytoplasmic face of the plasma membrane. Furthermore, HA and NP have been shown to be concentrated in cholesterol-rich membrane raft domains, whereas M2, although containing a cholesterol binding motif, is not raft associated. Here we identify viral proteins in planar sheets of plasma membrane using immunogold staining. The distribution of these proteins was examined individually and pairwise by using the Ripley K function, a type of nearest-neighbor analysis. Individually, HA, NA, M1, M2, and NP were shown to self-associate in or on the plasma membrane. HA and M2 are strongly coclustered in the plasma membrane; however, in the case of NA and M2, clustering depends upon the expression system used. Despite both proteins being raft resident, HA and NA occupy distinct but adjacent membrane domains. M2 and M1 strongly cocluster, but the association of M1 with HA or NA is dependent upon the means of expression. The presence of HA and NP at the site of budding depends upon the coexpression of other viral proteins. Similarly, M2 and NP occupy separate compartments, but an association can be bridged by the coexpression of M1.IMPORTANCE The complement of influenza virus proteins necessary for the budding of progeny virions needs to accumulate at budozones. This is complicated by HA and NA residing in lipid raft-like domains, whereas M2, although an integral membrane protein, is not raft associated. Other necessary protein components such as M1 and NP are peripherally associated with the membrane. Our data define spatial relationships

  10. Chloroquine Increases Glucose Uptake via Enhancing GLUT4 Translocation and Fusion with the Plasma Membrane in L6 Cells

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    Qi Zhou

    2016-05-01

    Full Text Available Background/Aims: Chloroquine can induce an increase in the cellular uptake of glucose; however, the underlying mechanism is unclear. Methods: In this study, translocation of GLUT4 and intracellular Ca2+ changes were simultaneously observed by confocal microscope in L6 cells stably over-expressing IRAP-mOrange. The GLUT4 fusion with the plasma membrane (PM was traced using HA-GLUT4-GFP. Glucose uptake was measured using a cell-based glucose uptake assay. GLUT4 protein was detected by Western blotting and mRNA level was detected by RT-PCR. Results: We found that chloroquine induced significant increases in glucose uptake, glucose transporter GLUT4 translocation to the plasma membrane (GTPM, GLUT4 fusion with the PM, and intracellular Ca2+ in L6 muscle cells. Chloroquine-induced increases of GTPM and intracellular Ca2+ were inhibited by Gallein (Gβγ inhibitor and U73122 (PLC inhibitor. However, 2-APB (IP3R blocker only blocked the increase in intracellular Ca2+ but did not inhibit GTPM increase. These results indicate that chloroquine, via the Gβγ-PLC-IP3-IP3R pathway, induces elevation of Ca2+, and this Ca2+ increase does not play a role in chloroqui-ne-evoked GTPM increase. However, GLUT4 fusion with the PM and glucose uptake were significantly inhibited with BAPTA-AM. This suggests that Ca2+ enhances GLUT4 fusion with the PM resulting in glucose uptake increase. Conclusion: Our data indicate that chloroquine via Gβγ-PLC-IP3-IP3R induces Ca2+ elevation, which in turn promotes GLUT4 fusion with the PM. Moreover, chloroquine can enhance GLUT4 trafficking to the PM. These mechanisms eventually result in glucose uptake increase in control and insulin-resistant L6 cells. These findings suggest that chloroquine might be a potential drug for improving insulin tolerance in diabetic patients.

  11. Three-dimensional architecture and biogenesis of membrane structures associated with hepatitis C virus replication.

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    Romero-Brey, Inés; Merz, Andreas; Chiramel, Abhilash; Lee, Ji-Young; Chlanda, Petr; Haselman, Uta; Santarella-Mellwig, Rachel; Habermann, Anja; Hoppe, Simone; Kallis, Stephanie; Walther, Paul; Antony, Claude; Krijnse-Locker, Jacomine; Bartenschlager, Ralf

    2012-01-01

    All positive strand RNA viruses are known to replicate their genomes in close association with intracellular membranes. In case of the hepatitis C virus (HCV), a member of the family Flaviviridae, infected cells contain accumulations of vesicles forming a membranous web (MW) that is thought to be the site of viral RNA replication. However, little is known about the biogenesis and three-dimensional structure of the MW. In this study we used a combination of immunofluorescence- and electron microscopy (EM)-based methods to analyze the membranous structures induced by HCV in infected cells. We found that the MW is derived primarily from the endoplasmic reticulum (ER) and contains markers of rough ER as well as markers of early and late endosomes, COP vesicles, mitochondria and lipid droplets (LDs). The main constituents of the MW are single and double membrane vesicles (DMVs). The latter predominate and the kinetic of their appearance correlates with kinetics of viral RNA replication. DMVs are induced primarily by NS5A whereas NS4B induces single membrane vesicles arguing that MW formation requires the concerted action of several HCV replicase proteins. Three-dimensional reconstructions identify DMVs as protrusions from the ER membrane into the cytosol, frequently connected to the ER membrane via a neck-like structure. In addition, late in infection multi-membrane vesicles become evident, presumably as a result of a stress-induced reaction. Thus, the morphology of the membranous rearrangements induced in HCV-infected cells resemble those of the unrelated picorna-, corona- and arteriviruses, but are clearly distinct from those of the closely related flaviviruses. These results reveal unexpected similarities between HCV and distantly related positive-strand RNA viruses presumably reflecting similarities in cellular pathways exploited by these viruses to establish their membranous replication factories.

  12. Mimivirus reveals Mre11/Rad50 fusion proteins with a sporadic distribution in eukaryotes, bacteria, viruses and plasmids

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    Ogata Hiroyuki

    2011-09-01

    Full Text Available Abstract Background The Mre11/Rad50 complex and the homologous SbcD/SbcC complex in bacteria play crucial roles in the metabolism of DNA double-strand breaks, including DNA repair, genome replication, homologous recombination and non-homologous end-joining in cellular life forms and viruses. Here we investigated the amino acid sequence of the Mimivirus R555 gene product, originally annotated as a Rad50 homolog, and later shown to have close homologs in marine microbial metagenomes. Results Our bioinformatics analysis revealed that R555 protein sequence is constituted from the fusion of an N-terminal Mre11-like domain with a C-terminal Rad50-like domain. A systematic database search revealed twelve additional cases of Mre11/Rad50 (or SbcD/SbcC fusions in a wide variety of unrelated organisms including unicellular and multicellular eukaryotes, the megaplasmid of a bacterium associated to deep-sea hydrothermal vents (Deferribacter desulfuricans and the plasmid of Clostridium kluyveri. We also showed that R555 homologs are abundant in the metagenomes from different aquatic environments and that they most likely belong to aquatic viruses. The observed phyletic distribution of these fusion proteins suggests their recurrent creation and lateral gene transfers across organisms. Conclusions The existence of the fused version of protein sequences is consistent with known functional interactions between Mre11 and Rad50, and the gene fusion probably enhanced the opportunity for lateral transfer. The abundance of the Mre11/Rad50 fusion genes in viral metagenomes and their sporadic phyletic distribution in cellular organisms suggest that viruses, plasmids and transposons played a crucial role in the formation of the fusion proteins and their propagation into cellular genomes.

  13. IFITM proteins restrict viral membrane hemifusion.

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    Kun Li

    2013-01-01

    Full Text Available The interferon-inducible transmembrane (IFITM protein family represents a new class of cellular restriction factors that block early stages of viral replication; the underlying mechanism is currently not known. Here we provide evidence that IFITM proteins restrict membrane fusion induced by representatives of all three classes of viral membrane fusion proteins. IFITM1 profoundly suppressed syncytia formation and cell-cell fusion induced by almost all viral fusion proteins examined; IFITM2 and IFITM3 also strongly inhibited their fusion, with efficiency somewhat dependent on cell types. Furthermore, treatment of cells with IFN also markedly inhibited viral membrane fusion and entry. By using the Jaagsiekte sheep retrovirus envelope and influenza A virus hemagglutinin as models for study, we showed that IFITM-mediated restriction on membrane fusion is not at the steps of receptor- and/or low pH-mediated triggering; instead, the creation of hemifusion was essentially blocked by IFITMs. Chlorpromazine (CPZ, a chemical known to promote the transition from hemifusion to full fusion, was unable to rescue the IFITM-mediated restriction on fusion. In contrast, oleic acid (OA, a lipid analog that generates negative spontaneous curvature and thereby promotes hemifusion, virtually overcame the restriction. To explore the possible effect of IFITM proteins on membrane molecular order and fluidity, we performed fluorescence labeling with Laurdan, in conjunction with two-photon laser scanning and fluorescence-lifetime imaging microscopy (FLIM. We observed that the generalized polarizations (GPs and fluorescence lifetimes of cell membranes expressing IFITM proteins were greatly enhanced, indicating higher molecularly ordered and less fluidized membranes. Collectively, our data demonstrated that IFITM proteins suppress viral membrane fusion before the creation of hemifusion, and suggested that they may do so by reducing membrane fluidity and conferring a positive

  14. Determination of the topology of endoplasmic reticulum membrane proteins using redox-sensitive green-fluorescence protein fusions.

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    Tsachaki, Maria; Birk, Julia; Egert, Aurélie; Odermatt, Alex

    2015-07-01

    Membrane proteins of the endoplasmic reticulum (ER) are involved in a wide array of essential cellular functions. Identification of the topology of membrane proteins can provide significant insight into their mechanisms of action and biological roles. This is particularly important for membrane enzymes, since their topology determines the subcellular site where a biochemical reaction takes place and the dependence on luminal or cytosolic co-factor pools and substrates. The methods currently available for the determination of topology of proteins are rather laborious and require post-lysis or post-fixation manipulation of cells. In this work, we have developed a simple method for defining intracellular localization and topology of ER membrane proteins in living cells, based on the fusion of the respective protein with redox-sensitive green-fluorescent protein (roGFP). We validated the method and demonstrated that roGFP fusion proteins constitute a reliable tool for the study of ER membrane protein topology, using as control microsomal 11β-hydroxysteroid dehydrogenase (11β-HSD) proteins whose topology has been resolved, and comparing with an independent approach. We then implemented this method to determine the membrane topology of six microsomal members of the 17β-hydroxysteroid dehydrogenase (17β-HSD) family. The results revealed a luminal orientation of the catalytic site for three enzymes, i.e. 17β-HSD6, 7 and 12. Knowledge of the intracellular location of the catalytic site of these enzymes will enable future studies on their biological functions and on the role of the luminal co-factor pool. Copyright © 2015 Elsevier B.V. All rights reserved.

  15. Archaeal Haloarcula californiae Icosahedral Virus 1 Highlights Conserved Elements in Icosahedral Membrane-Containing DNA Viruses from Extreme Environments

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    Tatiana A. Demina

    2016-07-01

    Full Text Available Despite their high genomic diversity, all known viruses are structurally constrained to a limited number of virion morphotypes. One morphotype of viruses infecting bacteria, archaea, and eukaryotes is the tailless icosahedral morphotype with an internal membrane. Although it is considered an abundant morphotype in extreme environments, only seven such archaeal viruses are known. Here, we introduce Haloarcula californiae icosahedral virus 1 (HCIV-1, a halophilic euryarchaeal virus originating from salt crystals. HCIV-1 also retains its infectivity under low-salinity conditions, showing that it is able to adapt to environmental changes. The release of progeny virions resulting from cell lysis was evidenced by reduced cellular oxygen consumption, leakage of intracellular ATP, and binding of an indicator ion to ruptured cell membranes. The virion contains at least 12 different protein species, lipids selectively acquired from the host cell membrane, and a 31,314-bp-long linear double-stranded DNA (dsDNA. The overall genome organization and sequence show high similarity to the genomes of archaeal viruses in the Sphaerolipoviridae family. Phylogenetic analysis based on the major conserved components needed for virion assembly—the major capsid proteins and the packaging ATPase—placed HCIV-1 along with the alphasphaerolipoviruses in a distinct, well-supported clade. On the basis of its virion morphology and sequence similarities, most notably, those of its core virion components, we propose that HCIV-1 is a member of the PRD1-adenovirus structure-based lineage together with other sphaerolipoviruses. This addition to the lineage reinforces the notion of the ancient evolutionary links observed between the viruses and further highlights the limits of the choices found in nature for formation of a virion.

  16. Conservation of proteo-lipid nuclear membrane fusion machinery during early embryogenesis.

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    Byrne, Richard D; Veeriah, Selvaraju; Applebee, Christopher J; Larijani, Banafshé

    2014-01-01

    The fusogenic lipid diacylglycerol is essential for remodeling gamete and zygote nuclear envelopes (NE) during early embryogenesis. It is unclear whether upstream signaling molecules are likewise conserved. Here we demonstrate PLCγ and its activator SFK1, which co-operate during male pronuclear envelope formation, also promote the subsequent male and female pronuclear fusion. PLCγ and SFK1 interact directly at the fusion site leading to PLCγ activation. This is accompanied by a spatially restricted reduction of PtdIns(4,5)P2. Consequently, pronuclear fusion is blocked by PLCγ or SFK1 inhibition. These findings identify new regulators of events in the early embryo and suggest a conserved "toolkit" of fusion machinery drives successive NE fusion events during embryogenesis.

  17. Hepatitis C virus NS4B carboxy terminal domain is a membrane binding domain

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    Spaan Willy JM

    2009-05-01

    Full Text Available Abstract Background Hepatitis C virus (HCV induces membrane rearrangements during replication. All HCV proteins are associated to membranes, pointing out the importance of membranes for HCV. Non structural protein 4B (NS4B has been reported to induce cellular membrane alterations like the membranous web. Four transmembrane segments in the middle of the protein anchor NS4B to membranes. An amphipatic helix at the amino-terminus attaches to membranes as well. The carboxy-terminal domain (CTD of NS4B is highly conserved in Hepaciviruses, though its function remains unknown. Results A cytosolic localization is predicted for the NS4B-CTD. However, using membrane floatation assays and immunofluorescence, we now show targeting of the NS4B-CTD to membranes. Furthermore, a profile-profile search, with an HCV NS4B-CTD multiple sequence alignment, indicates sequence similarity to the membrane binding domain of prokaryotic D-lactate dehydrogenase (d-LDH. The crystal structure of E. coli d-LDH suggests that the region similar to NS4B-CTD is located in the membrane binding domain (MBD of d-LDH, implying analogy in membrane association. Targeting of d-LDH to membranes occurs via electrostatic interactions of positive residues on the outside of the protein with negative head groups of lipids. To verify that anchorage of d-LDH MBD and NS4B-CTD is analogous, NS4B-CTD mutants were designed to disrupt these electrostatic interactions. Membrane association was confirmed by swopping the membrane contacting helix of d-LDH with the corresponding domain of the 4B-CTD. Furthermore, the functionality of these residues was tested in the HCV replicon system. Conclusion Together these data show that NS4B-CTD is associated to membranes, similar to the prokaryotic d-LDH MBD, and is important for replication.

  18. Membrane vesiculation induced by proteins of the dengue virus envelope studied by molecular dynamics simulations

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    de Oliveira dos Santos Soares, Ricardo; Oliveira Bortot, Leandro; van der Spoel, David; Caliri, Antonio

    2017-12-01

    Biological membranes are continuously remodeled in the cell by specific membrane-shaping machineries to form, for example, tubes and vesicles. We examine fundamental mechanisms involved in the vesiculation processes induced by a cluster of envelope (E) and membrane (M) proteins of the dengue virus (DENV) using molecular dynamics simulations and a coarse-grained model. We show that an arrangement of three E-M heterotetramers (EM3) works as a bending unit and an ordered cluster of five such units generates a closed vesicle, reminiscent of the virus budding process. In silico mutagenesis of two charged residues of the anchor helices of the envelope proteins of DENV shows that Arg-471 and Arg-60 are fundamental to produce bending stress on the membrane. The fine-tuning between the size of the EM3 unit and its specific bending action suggests this protein unit is an important factor in determining the viral particle size.

  19. Evaluation of membrane filtration system using The “Pore Diffusion” for eliminating viruses

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    HASHIMOTO-GOTOH, Akira; MATSUKI, Takahiro; MIYAZAWA, Takayuki

    2015-01-01

    Here, we report a first study of virus removal by a novel membrane filtration system, named the “Pore Diffusion”. The “Pore Diffusion” manipulated the direction of circulating flow from vertical to parallel to the membrane, thereby achieved to alter the trans-membrane pressure as low as possible. We compared the viral activity between before and after filtration by both infectivity assay and real-time reverse transcription-PCR. Among 4 “Pore Diffusion” modules tested, the big module with average pore size of 80 nm showed the highest log reduction value of viral activity. Our study shows the possibility of “The Pore Diffusion” to filtrate viruses from bioproducts without increasing the trans-membrane pressure, so that the filtration process can be carried out effectively and economically. PMID:25715959

  20. Respiratory syncytial virus fusion protein promotes TLR-4-dependent neutrophil extracellular trap formation by human neutrophils.

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    Giselle A Funchal

    Full Text Available Acute viral bronchiolitis by Respiratory Syncytial Virus (RSV is the most common respiratory illness in children in the first year of life. RSV bronchiolitis generates large numbers of hospitalizations and an important burden to health systems. Neutrophils and their products are present in the airways of RSV-infected patients who developed increased lung disease. Neutrophil Extracellular Traps (NETs are formed by the release of granular and nuclear contents of neutrophils in the extracellular space in response to different stimuli and recent studies have proposed a role for NETs in viral infections. In this study, we show that RSV particles and RSV Fusion protein were both capable of inducing NET formation by human neutrophils. Moreover, we analyzed the mechanisms involved in RSV Fusion protein-induced NET formation. RSV F protein was able to induce NET release in a concentration-dependent fashion with both neutrophil elastase and myeloperoxidase expressed on DNA fibers and F protein-induced NETs was dismantled by DNase treatment, confirming that their backbone is chromatin. This viral protein caused the release of extracellular DNA dependent on TLR-4 activation, NADPH Oxidase-derived ROS production and ERK and p38 MAPK phosphorylation. Together, these results demonstrate a coordinated signaling pathway activated by F protein that led to NET production. The massive production of NETs in RSV infection could aggravate the inflammatory symptoms of the infection in young children and babies. We propose that targeting the binding of TLR-4 by F protein could potentially lead to novel therapeutic approaches to help control RSV-induced inflammatory consequences and pathology of viral bronchiolitis.

  1. Sulfated polysaccharide, curdlan sulfate, efficiently prevents entry/fusion and restricts antibody-dependent enhancement of dengue virus infection in vitro: a possible candidate for clinical application.

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    Koji Ichiyama

    Full Text Available Curdlan sulfate (CRDS, a sulfated 1→3-β-D glucan, previously shown to be a potent HIV entry inhibitor, is characterized in this study as a potent inhibitor of the Dengue virus (DENV. CRDS was identified by in silico blind docking studies to exhibit binding potential to the envelope (E protein of the DENV. CRDS was shown to inhibit the DENV replication very efficiently in different cells in vitro. Minimal effective concentration of CRDS was as low as 0.1 µg/mL in LLC-MK2 cells, and toxicity was observed only at concentrations over 10 mg/mL. CRDS can also inhibit DENV-1, 3, and 4 efficiently. CRDS did not inhibit the replication of DENV subgenomic replicon. Time of addition experiments demonstrated that the compound not only inhibited viral infection at the host cell binding step, but also at an early post-attachment step of entry (membrane fusion. The direct binding of CRDS to DENV was suggested by an evident reduction in the viral titers after interaction of the virus with CRDS following an ultrafiltration device separation, as well as after virus adsorption to an alkyl CRDS-coated membrane filter. The electron microscopic features also showed that CRDS interacted directly with the viral envelope, and caused changes to the viral surface. CRDS also potently inhibited DENV infection in DC-SIGN expressing cells as well as the antibody-dependent enhancement of DENV-2 infection. Based on these data, a probable binding model of CRDS to DENV E protein was constructed by a flexible receptor and ligand docking study. The binding site of CRDS was predicted to be at the interface between domains II and III of E protein dimer, which is unique to this compound, and is apparently different from the β-OG binding site. Since CRDS has already been tested in humans without serious side effects, its clinical application can be considered.

  2. Sulfated polysaccharide, curdlan sulfate, efficiently prevents entry/fusion and restricts antibody-dependent enhancement of dengue virus infection in vitro: a possible candidate for clinical application.

    Science.gov (United States)

    Ichiyama, Koji; Gopala Reddy, Sindhoora Bhargavi; Zhang, Li Feng; Chin, Wei Xin; Muschin, Tegshi; Heinig, Lars; Suzuki, Youichi; Nanjundappa, Haraprasad; Yoshinaka, Yoshiyuki; Ryo, Akihide; Nomura, Nobuo; Ooi, Eng Eong; Vasudevan, Subhash G; Yoshida, Takashi; Yamamoto, Naoki

    2013-01-01

    Curdlan sulfate (CRDS), a sulfated 1→3-β-D glucan, previously shown to be a potent HIV entry inhibitor, is characterized in this study as a potent inhibitor of the Dengue virus (DENV). CRDS was identified by in silico blind docking studies to exhibit binding potential to the envelope (E) protein of the DENV. CRDS was shown to inhibit the DENV replication very efficiently in different cells in vitro. Minimal effective concentration of CRDS was as low as 0.1 µg/mL in LLC-MK2 cells, and toxicity was observed only at concentrations over 10 mg/mL. CRDS can also inhibit DENV-1, 3, and 4 efficiently. CRDS did not inhibit the replication of DENV subgenomic replicon. Time of addition experiments demonstrated that the compound not only inhibited viral infection at the host cell binding step, but also at an early post-attachment step of entry (membrane fusion). The direct binding of CRDS to DENV was suggested by an evident reduction in the viral titers after interaction of the virus with CRDS following an ultrafiltration device separation, as well as after virus adsorption to an alkyl CRDS-coated membrane filter. The electron microscopic features also showed that CRDS interacted directly with the viral envelope, and caused changes to the viral surface. CRDS also potently inhibited DENV infection in DC-SIGN expressing cells as well as the antibody-dependent enhancement of DENV-2 infection. Based on these data, a probable binding model of CRDS to DENV E protein was constructed by a flexible receptor and ligand docking study. The binding site of CRDS was predicted to be at the interface between domains II and III of E protein dimer, which is unique to this compound, and is apparently different from the β-OG binding site. Since CRDS has already been tested in humans without serious side effects, its clinical application can be considered.

  3. An immunoreceptor tyrosine-based inhibition motif in varicella-zoster virus glycoprotein B regulates cell fusion and skin pathogenesis.

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    Oliver, Stefan L; Brady, Jennifer J; Sommer, Marvin H; Reichelt, Mike; Sung, Phillip; Blau, Helen M; Arvin, Ann M

    2013-01-29

    Herpesvirus entry functions of the conserved glycoproteins gB and gH-gL have been delineated, but their role in regulating cell-cell fusion is poorly understood. Varicella-zoster virus (VZV) infection provides a valuable model for investigating cell-cell fusion because of the importance of this process for pathogenesis in human skin and sensory ganglia. The present study identifies a canonical immunoreceptor tyrosine-based inhibition motif (ITIM) in the gB cytoplasmic domain (gBcyt) and demonstrates that the gBcyt is a tyrosine kinase substrate. Orbitrap mass spectrometry confirmed that Y881, central to the ITIM, is phosphorylated. To determine whether the gBcyt ITIM regulates gB/gH-gL-induced cell-cell fusion in vitro, tyrosine residues Y881 and Y920 in the gBcyt were substituted with phenylalanine separately or together. Recombinant viruses with these substitutions were generated to establish their effects on syncytia formation in replication in vitro and in the human skin xenograft model of VZV pathogenesis. The Y881F substitution caused significantly increased cell-cell fusion despite reduced cell-surface gB. Importantly, the Y881F or Y881/920F substitutions in VZV caused aggressive syncytia formation, reducing cell-cell spread. These in vitro effects of aggressive syncytia formation translated to severely impaired skin infection in vivo. In contrast, the Y920F substitution did not affect virus replication in vitro or in vivo. These observations suggest that gB modulates cell-cell fusion via an ITIM-mediated Y881 phosphorylation-dependent mechanism, supporting a unique concept that intracellular signaling through this gBcyt motif regulates VZV syncytia formation and is essential for skin pathogenesis.

  4. CNS myelin sheath is stochastically built by homotypic fusion of myelin membranes within the bounds of an oligodendrocyte process.

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    Szuchet, Sara; Nielsen, Lauren L; Domowicz, Miriam S; Austin, Jotham R; Arvanitis, Dimitrios L

    2015-04-01

    Myelin - the multilayer membrane that envelops axons - is a facilitator of rapid nerve conduction. Oligodendrocytes form CNS myelin; the prevailing hypothesis being that they do it by extending a process that circumnavigates the axon. It is pertinent to ask how myelin is built because oligodendrocyte plasma membrane and myelin are compositionally different. To this end, we examined oligodendrocyte cultures and embryonic avian optic nerves by electron microscopy, immuno-electron microscopy and three-dimensional electron tomography. The results support three novel concepts. Myelin membranes are synthesized as tubules and packaged into "myelinophore organelles" in the oligodendrocyte perikaryon. Myelin membranes are matured in and transported by myelinophore organelles within an oligodendrocyte process. The myelin sheath is generated by myelin membrane fusion inside an oligodendrocyte process. These findings abrogate the dogma of myelin resulting from a wrapping motion of an oligodendrocyte process and open up new avenues in the quest for understanding myelination in health and disease. Copyright © 2015 Elsevier Inc. All rights reserved.

  5. The pro-fusion domain of herpes simplex virus glycoprotein D (gD) interacts with the gD N terminus and is displaced by soluble forms of viral receptors.

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    Fusco, Daniela; Forghieri, Cristina; Campadelli-Fiume, Gabriella

    2005-06-28

    Entry of herpes simplex virus into the cell requires the interaction of gD with one of its receptors, herpesvirus entry mediator or nectin 1, and the intervention of gB, gH, or gL, required to execute fusion of the virion envelope with cell membranes. The gD ectodomain is organized in two structurally and functionally differentiated regions. The N terminus (residues 1-260) carries the receptor binding sites, and the C terminus (residues 260-310) functions as the pro-fusion domain (PFD), which is required for viral infectivity and fusion but not for receptor binding. The objective of our studies is to elucidate how gD links receptor recognition to the triggering of fusion. Here, we show that PFD is made of subdomains 1 and 2 (amino acids 260-285 and 285-310). Each one partially contributed to herpes simplex virus infectivity. By means of glutathione S-transferase (GST) fusion proteins, we show that PFD bound soluble forms of gD, truncated at residue 260 (gD260t) or downstream. Both PFD subdomains bound gD260t, highlighting multiple contact sites between the N and C termini of gD. When gD260t was in complex with either receptor, it failed to bind GST-PFD. In turn, the receptors did not bind GST-PFD, irrespective of whether they were in complex with gD. Thus, gD260t interacted with the C terminus only if unbound to the receptor. We propose that (i) before receptor binding, gD adopts a "closed" conformation in which the N and C termini interact; and (ii) on encounter with a receptor, gD modifies its conformation and the N and C termini are released from reciprocal interactions ("opened" conformation) and enabled to trigger fusion.

  6. Interaction of Mason-Pfizer monkey virus matrix protein with plasma membrane.

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    Jan ePrchal

    2014-01-01

    Full Text Available Budding is the final step of the late phase of retroviral life cycle. It begins with the interaction of Gag precursor with plasma membrane through its N-terminal domain, the matrix protein. However, single generas of Retroviridae family differ in the way how they interact with plasma membrane. While in case of lentiviruses (e.g. human immunodeficiency virus (HIV the structural polyprotein precursor Gag interacts with cellular membrane prior to the assembly, betaretroviruses (Mason-Pfizer monkey virus (M-PMV first assemble their virus-like particles in the pericentriolar region of the infected cell and therefore, already assembled particles interact with the membrane. Although both these types of retroviruses use similar mechanism of the interaction of Gag with the membrane, the difference in the site of assembly leads to some differences in the mechanism of the interaction. Here we describe the interaction of M-PMV matrix protein with plasma membrane with emphasis on the structural aspects of the interaction with single phospholipids.

  7. Characterization of heterokaryons between skeletal myoblasts and somatic cells formed by fusion with HVJ (Sendai virus); effects on myogenic differentiation.

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    Hirayama, E; Udaka, Y; Kawai, T; Kim, J

    2001-02-01

    In skeletal myogenic differentiation, myoblasts fuse with myogenic cells spontaneously, but do not fuse with non-myogenic cells either in vivo or in vitro, suggesting that the fusion of myoblasts with non-myogenic cells is unsuitable for differentiation. To understand the inevitability of the fusion among myoblasts, we prepared heterokaryons in crosses between quail myoblasts transformed with a temperature-sensitive mutant of Rous sarcoma virus (QM-RSV cells) and rodent non-myogenic cells, such as tumor cells, fibroblasts, or neurogenic cells by HVJ (Sendai virus) and examined how myogenic differentiation was influenced in the prepared heterokaryons, focusing on myogenin expression and myofibril formation as markers of differentiation. When presumptive QM-RSV cells were fused with non-myogenic cells by HVJ and induced to differentiate, both myogenin expression and myofibril formation were suppressed. When myotubes of QM-RSV cells that had already expressed myogenin and formed myofibrils were fused with non-myogenic cells, both myogenin and myofibrils disappeared. Especially, fibrous structures of myofibrils were significantly lost and dots or aggregations of F-actin were formed within 24 hr after formation of heterokaryons. However, the fusion of presumptive or differentiated QM-RSV cells with rodent myoblasts did not disturb myogenin expression or myofibril formation. These results suggest that mutual fusion of myoblasts is indispensable for normal myogenic differentiation irrespective of the species, and that some factors inhibiting myogenic differentiation exist in the cytoplasm of non-myogenic cells, but not in myoblasts.

  8. Role of lipid phase separations and membrane hydration in phospholipid vesicle fusion.

    NARCIS (Netherlands)

    Hoekstra, D.

    1982-01-01

    The relationship between lipid phase separation and fusion of small unilamellar phosphatidylserine-containing vesicles was investigated. The kinetics of phase separation were monitored by following the increase of self-quenching of the fluorescent phospholipid analogue

  9. Requirement of sperm-oocyte plasma membrane fusion for establishment of the plasma membrane block to polyspermy in human pronuclear oocytes.

    Science.gov (United States)

    Sengoku, K; Tamate, K; Takaoka, Y; Horikawa, M; Goishi, K; Okada, R; Tsuchiya, K; Ishikawa, M

    1999-02-01

    We investigated whether the incorporation of the sperm membrane into the oolemma contributes to the human plasma membrane block to polyspermy. We used zona pellucida-free oocytes fertilized by intracytoplasmic sperm injection (ICSI) or activated by parthenogenetic activation. Only two of the 35 pronuclear oocytes fertilized by spermatozoa (control) demonstrated one single penetrating spermatozoa. In contrast, the majority of ICSI and parthenogenetically activated pronuclear oocytes were penetrated with an average of three spermatozoa per oocyte. The number of fused and binding spermatozoa of ICSI and parthenogenetically activated oocytes were significantly higher than in control oocytes (3.5+/-0.6 and 4.3+/-0.6 for ICSI; 3.0+/-0.3 and 3.8+/-0.4 for activated and 0.2+/-0.1 and 0.6+/-0.2 for controls, respectively, P < 0.01). Furthermore, the cortical granules were released from the cortex of ICSI and calcium ionophore-puromycin-activated pronuclear oocytes to the same extent as that of pronuclear oocytes fertilized by spermatozoa. These results suggest that the establishment of the plasma membrane block to sperm penetration in the human oocyte may require a fusion process between sperm and oocyte plasma membranes.

  10. Molecular evolution of the fusion protein (F) gene in human respiratory syncytial virus subgroup B.

    Science.gov (United States)

    Kimura, Hirokazu; Nagasawa, Koo; Kimura, Ryusuke; Tsukagoshi, Hiroyuki; Matsushima, Yuki; Fujita, Kiyotaka; Hirano, Eiko; Ishiwada, Naruhiko; Misaki, Takako; Oishi, Kazunori; Kuroda, Makoto; Ryo, Akihide

    2017-08-01

    In this study, we examined the molecular evolution of the fusion protein (F) gene in human respiratory syncytial virus subgroup B (HRSV-B). First, we performed time-scale evolution analyses using the Bayesian Markov chain Monte Carlo (MCMC) method. Next, we performed genetic distance, linear B-cell epitope prediction, N-glycosylation, positive/negative selection site, and Bayesian skyline plot analyses. We also constructed a structural model of the F protein and mapped the amino acid substitutions and the predicted B-cell epitopes. The MCMC-constructed phylogenetic tree indicated that the HRSV F gene diverged from the bovine respiratory syncytial virus gene approximately 580years ago and had a relatively low evolutionary rate (7.14×10(-4)substitutions/site/year). Furthermore, a common ancestor of HRSV-A and -B diverged approximately 290years ago, while HRSV-B diverged into three clusters for approximately 60years. The genetic similarity of the present strains was very high. Although a maximum of 11 amino acid substitutions were observed in the structural model of the F protein, only one strain possessed an amino acid substitution located within the palivizumab epitope. Four epitopes were predicted, although these did not correspond to the neutralization sites of the F protein including the palivizumab epitope. In addition, five N-glycosylation sites of the present HRSV-B strains were inferred. No positive selection sites were identified; however, many sites were found to be under negative selection. The effective population size of the gene has remained almost constant. On the basis of these results, it can be concluded that the HRSV-B F gene is highly conserved, as is the F protein of HRSV-A. Moreover, our prediction of B-cell epitopes does not show that the palivizumab reaction site may be recognized as an epitope during naturally occurring infections. Copyright © 2017 Elsevier B.V. All rights reserved.

  11. Molecular epidemiology and phylodynamics of the human respiratory syncytial virus fusion protein in northern Taiwan.

    Directory of Open Access Journals (Sweden)

    Hsin Chi

    Full Text Available BACKGROUND AND AIMS: The glycoprotein (G protein and fusion protein (F protein of respiratory syncytial virus (RSV both show genetic variability, but few studies have examined the F protein gene. This study aimed to characterize the molecular epidemiology and phylodynamics of the F protein gene in clinical RSV strains isolated in northern Taiwan from 2000-2011. METHODS: RSV isolates from children presenting with acute respiratory symptoms between July 2000 and June 2011 were typed based on F protein gene sequences. Phylogeny construction and evaluation were performed using the neighbor-joining (NJ and maximum likelihood (ML methods. Phylodynamic patterns in RSV F protein genes were analyzed using the Bayesian Markov Chain Monte Carlo framework. Selection pressure on the F protein gene was detected using the Datamonkey website interface. RESULTS: From a total of 325 clinical RSV strains studied, phylogenetic analysis showed that 83 subgroup A strains (RSV-A could be further divided into three clusters, whereas 58 subgroup B strains (RSV-B had no significant clustering. Three amino acids were observed to differ between RSV-A and -B (positions 111, 113, and 114 in CTL HLA-B*57- and HLA-A*01-restricted epitopes. One positive selection site was observed in RSV-B, while none was observed in RSV-A. The evolution rate of the virus had very little change before 2000, then slowed down between 2000 and 2005, and evolved significantly faster after 2005. The dominant subtypes of RSV-A in each epidemic were replaced by different subtypes in the subsequent epidemic. CONCLUSIONS: Before 2004, RSV-A infections were involved in several small epidemics and only very limited numbers of strains evolved and re-emerged in subsequent years. After 2005, the circulating RSV-A strains were different from those of the previous years and continued evolving through 2010. Phylodynamic pattern showed the evolutionary divergence of RSV increased significantly in the recent 5

  12. Bovine parainfluenza virus type 3 (PIV3) expressing the respiratory syncytial virus (RSV) attachment and fusion proteins protects hamsters from challenge with human PIV3 and RSV.

    Science.gov (United States)

    Haller, Aurelia A; Mitiku, Misrach; MacPhail, Mia

    2003-08-01

    Parainfluenza virus type 3 (PIV3) and respiratory syncytial virus (RSV) are the main causes of ubiquitous acute respiratory diseases of infancy and early childhood, causing 20-25 % of pneumonia and 45-50 % of bronchiolitis in hospitalized children. The primary goal of this study was to create an effective and safe RSV vaccine based on utilizing attenuated bovine PIV3 (bPIV3) as a virus vector backbone. bPIV3 had been evaluated in human clinical trials and was shown to be attenuated and immunogenic in children as young as 2 months of age. The ability of bPIV3 to function as a virus vaccine vector was explored further by introducing the RSV attachment (G) and fusion (F) genes into the bPIV3 RNA genome. The resulting virus, bPIV3/RSV(I), contained an insert of 2900 nt, comprising two translationally competent transcription units. Despite this increase in genetic material, the virus replicated to high titres in Vero cells. This recombinant virus expressed the RSV G and F proteins sufficiently to evoke a protective immune response in hamsters upon challenge with RSV or human PIV3 and to elicit RSV neutralizing and PIV3 haemagglutinin inhibition serum antibodies. In effect, a bivalent vaccine was produced that could protect vaccinees from RSV as well as PIV3. Such a vaccine would vastly reduce the respiratory disease burden, the associated hospitalization costs and, most importantly, decrease morbidity and mortality of infants, immunocompromised individuals and the elderly.

  13. Relationship between the loss of neutralizing antibody binding and fusion activity of the F protein of human respiratory syncytial virus

    Directory of Open Access Journals (Sweden)

    Sarisky Robert T

    2007-07-01

    Full Text Available Abstract To elucidate the relationship between resistance to HRSV neutralizing antibodies directed against the F protein and the fusion activity of the F protein, a recombinant approach was used to generate a panel of mutations in the major antigenic sites of the F protein. These mutant proteins were assayed for neutralizing mAb binding (ch101F, palivizumab, and MAb19, level of expression, post-translational processing, cell surface expression, and fusion activity. Functional analysis of the fusion activity of the panel of mutations revealed that the fusion activity of the F protein is tolerant to multiple changes in the site II and IV/V/VI region in contrast with the somewhat limited spectrum of changes in the F protein identified from the isolation of HRSV neutralizing antibody virus escape mutants. This finding suggests that aspects other than fusion activity may limit the spectrum of changes tolerated within the F protein that are selected for by neutralizing antibodies.

  14. Cowpea mosaic virus 32- and 60-kilodalton replication proteins target and change the morphology of endoplasmic reticulum membranes

    NARCIS (Netherlands)

    Carette, J.E.; Lent, van J.; MacFarlance, S.A.; Wellink, J.E.; Kammen, van A.

    2002-01-01

    Cowpea mosaic virus (CPMV) replicates in close association with small membranous vesicles that are formed by rearrangements of intracellular membranes. To determine which of the viral proteins are responsible for the rearrangements of membranes and the attachment of the replication complex, we have

  15. Different host cell proteases activate the SARS-coronavirus spike-protein for cell-cell and virus-cell fusion

    Science.gov (United States)

    Simmons, Graham; Bertram, Stephanie; Glowacka, Ilona; Steffen, Imke; Chaipan, Chawaree; Agudelo, Juliet; Lu, Kai; Rennekamp, Andrew J.; Hofmann, Heike; Bates, Paul; Pöhlmann, Stefan

    2011-01-01

    Severe acute respiratory syndrome coronavirus (SARS-CoV) poses a considerable threat to human health. Activation of the viral spike (S)-protein by host cell proteases is essential for viral infectivity. However, the cleavage sites in SARS-S and the protease(s) activating SARS-S are incompletely defined. We found that R667 was dispensable for SARS-S-driven virus-cell fusion and for SARS-S-activation by trypsin and cathepsin L in a virus-virus fusion assay. Mutation T760R, which optimizes the minimal furin consensus motif 758-RXXR-762, and furin overexpression augmented SARS-S-activity, but did not result in detectable SARS-S cleavage. Finally, SARS-S-driven cell-cell fusion was independent of cathepsin L, a protease essential for virus-cell fusion. Instead, a so far unknown leupeptin-sensitive host cell protease activated cellular SARS-S for fusion with target cells expressing high levels of ACE2. Thus, different host cell proteases activate SARS-S for virus-cell and cell-cell fusion and SARS-S cleavage at R667 and 758-RXXR-762 can be dispensable for SARS-S activation. PMID:21435673

  16. HIV-1 variants with a single-point mutation in the gp41 pocket region exhibiting different susceptibility to HIV fusion inhibitors with pocket- or membrane-binding domain.

    Science.gov (United States)

    Lu, Lu; Tong, Pei; Yu, Xiaowen; Pan, Chungen; Zou, Peng; Chen, Ying-Hua; Jiang, Shibo

    2012-12-01

    Enfuvirtide (T20), the first FDA-approved peptide HIV fusion/entry inhibitor derived from the HIV-1 gp41 C-terminal heptad-repeat (CHR) domain, is believed to share a target with C34, another well-characterized CHR-peptide, by interacting with the gp41 N-terminal heptad-repeat (NHR) to form six-helix bundle core. However, our previous studies showed that T20 mainly interacts with the N-terminal region of the NHR (N-NHR) and lipid membranes, while C34 mainly binds to the NHR C-terminal pocket region. But so far, no one has shown that C34 can induce drug-resistance mutation in the gp41 pocket region. In this study, we constructed pseudoviruses in which the Ala at the position of 67 in the gp41 pocket region was substituted with Asp, Gly or Ser, respectively, and found that these mutations rendered the viruses highly resistant to C34, but sensitive to T20. The NHR-peptide N36 with mutations of A67 exhibited reduced anti-HIV-1 activity and decreased α-helicity. The stability of six-helix bundle formed by C34 and N36 with A67 mutations was significantly lower than that formed by C34 and N36 with wild-type sequence. The combination of C34 and T20 resulted in potent synergistic anti-HIV-1 effect against the viruses with mutations in either N- or C-terminal region in NHR. These results suggest that C34 with a pocket-binding domain and T20 containing the N-NHR- and membrane-binding domains inhibit HIV-1 fusion by interacting with different target sites and the combinatorial use of C34 and T20 is expected to be effective against HIV-1 variants resistant to HIV fusion inhibitors. Copyright © 2012 Elsevier B.V. All rights reserved.

  17. Structural and Functional Studies on the Fusion and Attachment Envelope Glycoproteins of Nipah Virus and Hendra Virus

    Science.gov (United States)

    2003-01-01

    Embo J, 1990. 9(6): p. 2017-22. 37. Pelet, T., J. Curran, and D. Kolakofsky, The P gene of bovine parainfluenza virus 3 expresses all three reading... parainfluenza viruses (hPIV) 1-4, respiratory syncytial virus (RSV), and simian virus 5 (SV5) (reviewed in (33)). The genomes of paramyxoviruses, as a...virus (NDV) (58), human parainfluenza virus (hPIV) (59), and most recently with MeV (60), but these observations have often been with the aid of

  18. New model for cardiomyocyte sheet transplantation using a virus-cell fusion technique.

    Science.gov (United States)

    Takahashi, Yuto; Tomotsune, Daihachiro; Takizawa, Sakiko; Yue, Fengming; Nagai, Mika; Yokoyama, Tadayuki; Hirashima, Kanji; Sasaki, Katsunori

    2015-06-26

    To facilitate close contacts between transplanted cardiomyocytes and host skeletal muscle using cell fusion mediated by hemagglutinating virus of Japan envelope (HVJ-E) and tissue maceration. Cardiomyocytes (1.5 × 10(6)) from fetal rats were first cultured. After proliferation, some cells were used for fusion with adult muscle fibers using HVJ-E. Other cells were used to create cardiomyocyte sheets (area: about 3.5 cm(2) including 2.1 × 10(6) cells), which were then treated with Nile blue, separated, and transplanted between the latissimus dorsi and intercostal muscles of adult rats with four combinations of HVJ-E and/or NaOH maceration: G1: HVJ-E(+), NaOH(+), Cardiomyocytes(+); G2: HVJ-E(-), NaOH(+), Cardiomyocytes(+); G3: HVJ-E(+), NaOH(-), Cardiomyocytes(+); G4: HVJ-E(-), NaOH(-), Cardiomyocytes(-). At 1 and 2 wk after transplantation, the four groups were compared by detection of beating domains, motion images using moving target analysis software, action potentials, gene expression of MLC-2v and Mesp1 by reverse transcription-polymerase chain reaction, hematoxylin-eosin staining, and immunostaining for cardiac troponin and skeletal myosin. In vitro cardiomyocytes were fused with skeletal muscle fibers using HVJ-E. Cardiomyocyte sheets remained in the primary transplanted sites for 2 wk. Although beating domains were detected in G1, G2, and G3 rats, G1 rats prevailed in the number, size, motion image amplitudes, and action potential compared with G2 and G3 rats. Close contacts were only found in G1 rats. At 1 wk after transplantation, the cardiomyocyte sheets showed adhesion at various points to the myoblast layer in the latissimus dorsi muscle. At 2 wk after transplantation, close contacts were seen over a broad area. Part of the skeletal muscle sarcoplasma seemed to project into the myocardiocyte plasma and some nuclei appeared to share both sarcoplasmas. The present results show that close contacts were acquired and facilitated the beating function

  19. Improving the yeast two-hybrid system with permutated fusions proteins: the Varicella Zoster Virus interactome

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    Haas Jürgen

    2010-02-01

    Full Text Available Abstract Background Yeast two-hybrid (Y2H screens have been among the most powerful methods to detect and analyze protein-protein interactions. However, they suffer from a significant degree of false negatives, i.e. true interactions that are not detected, and to a certain degree from false positives, i.e. interactions that appear to take place only in the context of the Y2H assay. While the fraction of false positives remains difficult to estimate, the fraction of false negatives in typical Y2H screens is on the order of 70-90%. Here we present novel Y2H vectors that significantly decrease the number of false negatives and help to mitigate the false positive problem. Results We have constructed two new vectors (pGBKCg and pGADCg that allow us to make both C-terminal fusion proteins of DNA-binding and activation domains. Both vectors can be combined with existing vectors for N-terminal fusions and thus allow four different bait-prey combinations: NN, CC, NC, and CN. We have tested all ~4,900 pairwise combinations of the 70 Varicella-Zoster-Virus (VZV proteins for interactions, using all possible combinations. About ~20,000 individual Y2H tests resulted in 182 NN, 89 NC, 149 CN, and 144 CC interactions. Overlap between screens ranged from 17% (NC-CN to 43% (CN-CC. Performing four screens (i.e. permutations instead of one resulted in about twice as many interactions and thus much fewer false negatives. In addition, interactions that are found in multiple combinations confirm each other and thus provide a quality score. This study is the first systematic analysis of such N- and C-terminal Y2H vectors. Conclusions Permutations of C- and N-terminal Y2H vectors dramatically increase the coverage of interactome studies and thus significantly reduce the number of false negatives. We suggest that future interaction screens should use such vector combinations on a routine basis, not the least because they provide a built-in quality score for Y2H

  20. In Vitro Evidence Supports Membrane Alanyl Aminopeptidase N as a Receptor for a Plant Virus in the Pea Aphid Vector

    OpenAIRE

    Linz, Lucas B.; LIU, Sijun; Chougule, Nanasaheb P.; Bonning, Bryony C.

    2015-01-01

    Insect-borne plant viruses cause significant agricultural losses and jeopardize sustainable global food production. Although blocking plant virus transmission would allow for crop protection, virus receptors in insect vectors are unknown. Here we identify membrane alanyl aminopeptidase N (APN) as a receptor for pea enation mosaic virus (PEMV) coat protein (CP) in the gut of the pea aphid, Acyrthosiphon pisum, using a far-Western blot method. Pulldown and immunofluorescence binding assays and ...

  1. NSF- and SNARE-mediated membrane fusion is required for nuclear envelope formation and completion of nuclear pore complex assembly in Xenopus laevis egg extracts.

    Science.gov (United States)

    Baur, Tina; Ramadan, Kristijan; Schlundt, Andreas; Kartenbeck, Jürgen; Meyer, Hemmo H

    2007-08-15

    Despite the progress in understanding nuclear envelope (NE) reformation after mitosis, it has remained unclear what drives the required membrane fusion and how exactly this is coordinated with nuclear pore complex (NPC) assembly. Here, we show that, like other intracellular fusion reactions, NE fusion in Xenopus laevis egg extracts is mediated by SNARE proteins that require activation by NSF. Antibodies against Xenopus NSF, depletion of NSF or the dominant-negative NSF(E329Q) variant specifically inhibited NE formation. Staging experiments further revealed that NSF was required until sealing of the envelope was completed. Moreover, excess exogenous alpha-SNAP that blocks SNARE function prevented membrane fusion and caused accumulation of non-flattened vesicles on the chromatin surface. Under these conditions, the nucleoporins Nup107 and gp210 were fully recruited, whereas assembly of FxFG-repeat-containing nucleoporins was blocked. Together, we define NSF- and SNARE-mediated membrane fusion events as essential steps during NE formation downstream of Nup107 recruitment, and upstream of membrane flattening and completion of NPC assembly.

  2. Characterization of the sites of proteolytic activation of Newcastle disease virus membrane glycoprotein precursors.

    Science.gov (United States)

    Gorman, J J; Nestorowicz, A; Mitchell, S J; Corino, G L; Selleck, P W

    1988-09-05

    The F1- and F2-polypeptide components of the fusion proteins and the hemagglutinin/neuraminidase proteins of the avirulent Queensland (V4) and virulent Australia-Victoria (AuV) strains of Newcastle disease virus have been isolated and subjected to extensive primary structural analysis including amino-terminal sequence analysis and fast atom bombardment-mass spectrometry mapping. Nucleotide sequence analysis was performed on the gene which encodes the V4 hemagglutinin/neuraminidase protein. Signal peptidase cleavage was found to have occurred at the Ser31-Leu32 peptide bond of the primary translation products of the fusion protein genes. Activation cleavage of the V4 fusion protein precursor generated a sequence of -Gly-Lys-Gln-Gly84 at the carboxyl terminus of the F2-polypeptide and an amino-terminal sequence of the F1-polypeptide commencing with 86Leu-Ile-Gly-. The V4 hemagglutinin/neuraminidase protein gene was found to encode a primary translation product 45 amino acids longer at the carboxyl terminus than obtainable from the corresponding gene of the AuV strain (McGinnes, L. W., and Morrison, T. G. (1986) Virus Res. 5, 343-356). However, post-translational proteolytic processing, exclusive to the primary translation product of the V4 hemagglutinin/neuraminidase protein gene, was found to have removed the last 42 residues of this carboxyl-terminal appendage.

  3. The Role of the Hendra Virus and Nipah Virus Attachment Glycoproteins in Receptor Binding and Antibody Neutralization

    Science.gov (United States)

    2014-01-31

    The Role of the Hendra Virus and Nipah Virus Attachment Glycoproteins in Receptor Binding and Antibody Neutralization by...Henipavirus mediated membrane fusion, virus entry and targeted therapeutics. Viruses 4:280-308. and Bossart KN, Fusco DL, Broder CC. 2013. Paramyxovirus...of the hendra virus attachment G glycoprotein bound to a potent cross-reactive neutralizing human monoclonal antibody. PLoS pathogens 9:e1003684

  4. Proteomic analysis of membrane proteins of vero cells: exploration of potential proteins responsible for virus entry.

    Science.gov (United States)

    Guo, Donghua; Zhu, Qinghe; Zhang, Hong; Sun, Dongbo

    2014-01-01

    Vero cells are highly susceptible to many viruses in humans and animals, and its membrane proteins (MPs) are responsible for virus entry. In our study, the MP proteome of the Vero cells was investigated using a shotgun LC-MS/MS approach. Six hundred twenty-seven proteins, including a total of 1839 peptides, were identified in MP samples of the Vero cells. In 627 proteins, 307 proteins (48.96%) were annotated in terms of biological process of gene ontology (GO) categories; 356 proteins (56.78%) were annotated in terms of molecular function of GO categories; 414 proteins (66.03%) were annotated in terms of cellular components of GO categories. Of 627 identified proteins, seventeen proteins had been revealed to be virus receptor proteins. The resulting protein lists and highlighted proteins may provide valuable information to increase understanding of virus infection of Vero cells.

  5. Ion-Exchange Membrane Chromatography as an Alternative Method of Separation of Potato y Virus

    Directory of Open Access Journals (Sweden)

    Treder Krzysztof

    2015-12-01

    Full Text Available Procedures of separation of virus particles from a plant material are multistage. Furthermore often they are difficult in terms of methodology and require use of expensive, highly specialist equipment and yield of separation is often low. The antigen obtained is often degraded and contains admixtures of other proteins. Therefore, generation of high quality and specificity antibodies based on such antigen is very difficult and quality of the antibodies has impact on reliability, sensitivity and unambiguity of results of immunodiagnostic tests (e.g. ELISA that are currently conventionally used to detect vegetable viruses. In this study three conventionally-performed methods of separation of potato virus Y (PVY were compared and a method of separation based on membrane chromatography, as an alternative separation technique, has been presented. It has been demonstrated that in proper process conditions good quality virus preparation can be obtained.

  6. Structural and Functional Studies on the Fusion and Attachment Envelope Glycoproteins of Nipah Virus and Hendra Virus

    National Research Council Canada - National Science Library

    Bossart, Katharine

    2003-01-01

    Nipah virus (NiV) and Hendra (HeV) virus are emerging, biosafety level 4 paramyxoviruses responsible for fatal zoonotic infections of humans from pigs and horses, respectively, and are the prototypic members of a new Paramyxovirinae...

  7. Topographical distribution of a membrane-inserted fluorescent phospholipid analogue during cell fusion

    NARCIS (Netherlands)

    Hoekstra, D

    1983-01-01

    Potential alterations in the transbilayer distribution of lipid molecules during cell-cell fusion were studied, using the fluorescent phospholipid analogue 1-acyl, 2-(N-4-nitrobenzo-2-oxa-1,3-diazole)-aminocaproyl phosphatidylcholine (C6-NBD-PC). The fluophore was inserted into the outer leaflet of

  8. PSITE vectors for stable integration or transient expression of autofluorescent protein fusions in plants: probing Nicotiana benthamiana-virus interactions.

    Science.gov (United States)

    Chakrabarty, Romit; Banerjee, Rituparna; Chung, Sang-Min; Farman, Mark; Citovsky, Vitaly; Hogenhout, Saskia A; Tzfira, Tzvi; Goodin, Michael

    2007-07-01

    Plant functional proteomics research is increasingly dependent upon vectors that facilitate high-throughput gene cloning and expression of fusions to autofluorescent proteins. Here, we describe the pSITE family of plasmids, a new set of Agrobacterium binary vectors, suitable for the stable integration or transient expression of various autofluorescent protein fusions in plant cells. The pSITE vectors permit single-step Gateway-mediated recombination cloning for construction of binary vectors that can be used directly in transient expression studies or for the selection of transgenic plants on media containing kanamycin. These vectors can be used to express native proteins or fusions to monmeric red fluorescent protein or the enhanced green fluorescent protein and its cyan and yellow-shifted spectral variants. We have validated the vectors for use in transient expression assays and for the generation of transgenic plants. Additionally, we have generated markers for fluorescent highlighting of actin filaments, chromatin, endoplasmic reticulum, and nucleoli. Finally, we show that pSITE vectors can be used for targeted gene expression in virus-infected cells, which should facilitate high-throughput characterization of protein dynamics in host-virus interactions.

  9. Fusion of protegrin-1 and plectasin to MAP30 shows significant inhibition activity against dengue virus replication.

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    Hussin A Rothan

    Full Text Available Dengue virus (DENV broadly disseminates in tropical and sub-tropical countries and there are no vaccine or anti-dengue drugs available. DENV outbreaks cause serious economic burden due to infection complications that requires special medical care and hospitalization. This study presents a new strategy for inexpensive production of anti-DENV peptide-fusion protein to prevent and/or treat DENV infection. Antiviral cationic peptides protegrin-1 (PG1 and plectasin (PLSN were fused with MAP30 protein to produce recombinant antiviral peptide-fusion protein (PG1-MAP30-PLSN as inclusion bodies in E. coli. High yield production of PG1-MAP30-PLSN protein was achieved by solubilization of inclusion bodies in alkaline buffer followed by the application of appropriate refolding techniques. Antiviral PG1-MAP30-PLSN protein considerably inhibited DENV protease (NS2B-NS3pro with half-maximal inhibitory concentration (IC50 0.5±0.1 μM. The real-time proliferation assay (RTCA and the end-point proliferation assay (MTT assay showed that the maximal-nontoxic dose of the peptide-fusion protein against Vero cells is approximately 0.67±0.2 μM. The cell-based assays showed considerable inhibition of the peptide-fusion protein against binding and proliferating stages of DENV2 into the target cells. The peptide-fusion protein protected DENV2-challeged mice with 100% of survival at the dose of 50 mg/kg. In conclusion, producing recombinant antiviral peptide-fusion protein by combining short antiviral peptide with a central protein owning similar activity could be useful to minimize the overall cost of short peptide production and take advantage of its synergistic antiviral activities.

  10. Inhibition of neuraminidase inhibitor-resistant influenza virus by DAS181, a novel sialidase fusion protein.

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    Gallen B Triana-Baltzer

    2009-11-01

    Full Text Available Antiviral drug resistance for influenza therapies remains a concern due to the high prevalence of H1N1 2009 seasonal influenza isolates which display H274Y associated oseltamivir-resistance. Furthermore, the emergence of novel H1N1 raises the potential that additional reassortments can occur, resulting in drug resistant virus. Thus, additional antiviral approaches are urgently needed. DAS181 (Fludase, a sialidase fusion protein, has been shown to have inhibitory activity against a large number of seasonal influenza strains and a highly pathogenic avian influenza (HPAI strain (H5N1. Here, we examine the in vitro activity of DAS181 against a panel of 2009 oseltamivir-resistant seasonal H1N1 clinical isolates. The activity of DAS181 against nine 2009, two 2007, and two 2004 clinical isolates of seasonal IFV H1N1 was examined using plaque number reduction assay on MDCK cells. DAS181 strongly inhibited all tested isolates. EC50 values remained constant against isolates from 2004, 2007, and 2009, suggesting that there was no change in DAS181 sensitivity over time. As expected, all 2007 and 2009 isolates were resistant to oseltamivir, consistent with the identification of the H274Y mutation in the NA gene of all these isolates. Interestingly, several of the 2007 and 2009 isolates also exhibited reduced sensitivity to zanamivir, and accompanying HA mutations near the sialic acid binding site were observed. DAS181 inhibits IFV that is resistant to NAIs. Thus, DAS181 may offer an alternative therapeutic option for seasonal or pandemic IFVs that become resistant to currently available antiviral drugs.

  11. Virus movements on the plasma membrane support infection and transmission between cells.

    Directory of Open Access Journals (Sweden)

    Christoph J Burckhardt

    2009-11-01

    Full Text Available How viruses are transmitted across the mucosal epithelia of the respiratory, digestive, or excretory tracts, and how they spread from cell to cell and cause systemic infections, is incompletely understood. Recent advances from single virus tracking experiments have revealed conserved patterns of virus movements on the plasma membrane, including diffusive motions, drifting motions depending on retrograde flow of actin filaments or actin tail formation by polymerization, and confinement to submicrometer areas. Here, we discuss how viruses take advantage of cellular mechanisms that normally drive the movements of proteins and lipids on the cell surface. A concept emerges where short periods of fast diffusive motions allow viruses to rapidly move over several micrometers. Coupling to actin flow supports directional transport of virus particles during entry and cell-cell transmission, and local confinement coincides with either nonproductive stalling or infectious endocytic uptake. These conserved features of virus-host interactions upstream of infectious entry offer new perspectives for anti-viral interference.

  12. Amino Acid Changes in the HIV-1 gp41 Membrane Proximal Region Control Virus Neutralization Sensitivity

    Directory of Open Access Journals (Sweden)

    Todd Bradley

    2016-10-01

    Full Text Available Most HIV-1 vaccines elicit neutralizing antibodies that are active against highly sensitive (tier-1 viruses or rare cases of vaccine-matched neutralization-resistant (tier-2 viruses, but no vaccine has induced antibodies that can broadly neutralize heterologous tier-2 viruses. In this study, we isolated antibodies from an HIV-1-infected individual that targeted the gp41 membrane-proximal external region (MPER that may have selected single-residue changes in viral variants in the MPER that resulted in neutralization sensitivity to antibodies targeting distal epitopes on the HIV-1 Env. Similarly, a single change in the MPER in a second virus from another infected-individual also conferred enhanced neutralization sensitivity. These gp41 single-residue changes thus transformed tier-2 viruses into tier-1 viruses that were sensitive to vaccine-elicited tier-1 neutralizing antibodies. These data demonstrate that Env amino acid changes within the MPER bnAb epitope of naturally-selected escape viruses can increase neutralization sensitivity to multiple types of neutralizing antibodies, and underscore the critical importance of the MPER for maintaining the integrity of the tier-2 HIV-1 trimer.

  13. Membrane-on-a-Chip : Microstructured Silicon/Silicon-Dioxide Chips for High-Throughput Screening of Membrane Transport and Viral Membrane Fusion

    NARCIS (Netherlands)

    Kusters, Ilja; van Oijen, Antoine M.; Driessen, Arnold J. M.

    Screening of transport processes across biological membranes is hindered by the challenge to establish fragile supported lipid bilayers and the difficulty to determine at which side of the membrane reactants reside. Here, we present a method for the generation of suspended lipid bilayers with

  14. Application of virus-like particles (VLP) to NMR characterization of viral membrane protein interactions

    Energy Technology Data Exchange (ETDEWEB)

    Antanasijevic, Aleksandar; Kingsley, Carolyn [University of Illinois at Chicago, Department of Biochemistry and Molecular Genetics (United States); Basu, Arnab; Bowlin, Terry L. [Microbiotix Inc. (United States); Rong, Lijun [University of Illinois at Chicago, Department of Microbiology and Immunology (United States); Caffrey, Michael, E-mail: caffrey@uic.edu [University of Illinois at Chicago, Department of Biochemistry and Molecular Genetics (United States)

    2016-03-15

    The membrane proteins of viruses play critical roles in the virus life cycle and are attractive targets for therapeutic intervention. Virus-like particles (VLP) present the possibility to study the biochemical and biophysical properties of viral membrane proteins in their native environment. Specifically, the VLP constructs contain the entire protein sequence and are comprised of native membrane components including lipids, cholesterol, carbohydrates and cellular proteins. In this study we prepare VLP containing full-length hemagglutinin (HA) or neuraminidase (NA) from influenza and characterize their interactions with small molecule inhibitors. Using HA-VLP, we first show that VLP samples prepared using the standard sucrose gradient purification scheme contain significant amounts of serum proteins, which exhibit high potential for non-specific interactions, thereby complicating NMR studies of ligand-target interactions. We then show that the serum contaminants may be largely removed with the addition of a gel filtration chromatography step. Next, using HA-VLP we demonstrate that WaterLOGSY NMR is significantly more sensitive than Saturation Transfer Difference (STD) NMR for the study of ligand interactions with membrane bound targets. In addition, we compare the ligand orientation to HA embedded in VLP with that of recombinant HA by STD NMR. In a subsequent step, using NA-VLP we characterize the kinetic and binding properties of substrate analogs and inhibitors of NA, including study of the H274Y-NA mutant, which leads to wide spread resistance to current influenza antivirals. In summary, our work suggests that VLP have high potential to become standard tools in biochemical and biophysical studies of viral membrane proteins, particularly when VLP are highly purified and combined with control VLP containing native membrane proteins.

  15. Enhancing membrane protein subcellular localization prediction by parallel fusion of multi-view features.

    Science.gov (United States)

    Yu, Dongjun; Wu, Xiaowei; Shen, Hongbin; Yang, Jian; Tang, Zhenmin; Qi, Yong; Yang, Jingyu

    2012-12-01

    Membrane proteins are encoded by ~ 30% in the genome and function importantly in the living organisms. Previous studies have revealed that membrane proteins' structures and functions show obvious cell organelle-specific properties. Hence, it is highly desired to predict membrane protein's subcellular location from the primary sequence considering the extreme difficulties of membrane protein wet-lab studies. Although many models have been developed for predicting protein subcellular locations, only a few are specific to membrane proteins. Existing prediction approaches were constructed based on statistical machine learning algorithms with serial combination of multi-view features, i.e., different feature vectors are simply serially combined to form a super feature vector. However, such simple combination of features will simultaneously increase the information redundancy that could, in turn, deteriorate the final prediction accuracy. That's why it was often found that prediction success rates in the serial super space were even lower than those in a single-view space. The purpose of this paper is investigation of a proper method for fusing multiple multi-view protein sequential features for subcellular location predictions. Instead of serial strategy, we propose a novel parallel framework for fusing multiple membrane protein multi-view attributes that will represent protein samples in complex spaces. We also proposed generalized principle component analysis (GPCA) for feature reduction purpose in the complex geometry. All the experimental results through different machine learning algorithms on benchmark membrane protein subcellular localization datasets demonstrate that the newly proposed parallel strategy outperforms the traditional serial approach. We also demonstrate the efficacy of the parallel strategy on a soluble protein subcellular localization dataset indicating the parallel technique is flexible to suite for other computational biology problems. The

  16. Selective adhesion, lipid exchange and membrane-fusion processes between vesicles of various sizes bearing complementary molecular recognition groups.

    Science.gov (United States)

    Marchi-Artzner, V; Gulik-Krzywicki, T; Guedeau-Boudeville, M A; Gosse, C; Sanderson, J M; Dedieu, J C; Lehn, J M

    2001-06-18

    Equimolar mixtures of large unilamellar vesicles (LUVs) obtained from mixtures of egg lecithin and lipids containing complementary hydrogen bonding head groups (barbituric acid (BAR) and 2,4,6-triaminopyrimidine (TAP)) were shown to aggregate and fuse. These events have been studied in detail using electron microscopy and dynamic light scattering, and by fluorimetry using membrane or water-soluble fluorescence probes. It was shown that aggregation was followed by two competitive processes: a) lipid mixing leading to redispersion of the vesicles; b) fusion events generating much larger vesicles. In order to better understand the nature of the interaction, the effects of ionic strength and surface concentration of recognition lipids on the aggregation process were investigated by dynamic light scattering. Additionally, it was possible to inhibit the aggregation kinetics through addition of a soluble barbituric acid competitor. The study was extended to giant unilamellar vesicles (GUVs) to investigate the size effect and visualise the phenomena in situ. The interactions between complementary LUVs and GUVs or GUVs and GUVs were studied by optical microscopy using dual fluorescent labelling of both vesicle populations. A selective adhesion of LUVs onto GUVs was observed by electron and optical microscopies, whereas no aggregation took place in case of a GUV/GUV mixture. Furthermore, a fusion assay of GUV and LUV using the difference of size between GUV and LUV and calceine self-quenching showed that no mixing between the aqueous pools occured. © 2001 WILEY-VCH Verlag GmbH, Weinheim, Fed. Rep. of Germany.

  17. Virus Disinfection in Water by Biogenic Silver Immobilized in Polyvinylidene Fluoride Membranes

    Energy Technology Data Exchange (ETDEWEB)

    B De Gusseme; T Hennebel; E Christiaens; H Saveyn; K Verbeken; J Fitts; N Boon; W Vertraete

    2011-12-31

    The development of innovative water disinfection strategies is of utmost importance to prevent outbreaks of waterborne diseases related to poor treatment of (drinking) water. Recently, the association of silver nanoparticles with the bacterial cell surface of Lactobacillus fermentum (referred to as biogenic silver or bio-Ag{sup 0}) has been reported to exhibit antiviral properties. The microscale bacterial carrier matrix serves as a scaffold for Ag{sup 0} particles, preventing aggregation during encapsulation. In this study, bio-Ag{sup 0} was immobilized in different microporous PVDF membranes using two different pre-treatments of bio-Ag{sup 0} and the immersion-precipitation method. Inactivation of UZ1 bacteriophages using these membranes was successfully demonstrated and was most probably related to the slow release of Ag{sup +} from the membranes. At least a 3.4 log decrease of viruses was achieved by application of a membrane containing 2500 mg bio-Ag{sup 0}{sub powder} m{sup -2} in a submerged plate membrane reactor operated at a flux of 3.1 L m{sup -2} h{sup -1}. Upon startup, the silver concentration in the effluent initially increased to 271 {mu}g L{sub -1} but after filtration of 31 L m{sup -2}, the concentration approached the drinking water limit (= 100 {mu}g L{sup -1}). A virus decline of more than 3 log was achieved at a membrane flux of 75 L m{sup -2} h{sup -1}, showing the potential of this membrane technology for water disinfection on small scale.

  18. A Novel Peptide Derived from the Fusion Protein Heptad Repeat Inhibits Replication of Subacute Sclerosing Panencephalitis Virus In Vitro and In Vivo.

    Science.gov (United States)

    Watanabe, Masahiro; Hashimoto, Koichi; Abe, Yusaku; Kodama, Eiichi N; Nabika, Ryota; Oishi, Shinya; Ohara, Shinichiro; Sato, Masatoki; Kawasaki, Yukihiko; Fujii, Nobutaka; Hosoya, Mitsuaki

    2016-01-01

    Subacute sclerosing panencephalitis (SSPE) is a persistent, progressive, and fatal degenerative disease resulting from persistent measles virus (MV) infection of the central nervous system. Most drugs used to treat SSPE have been reported to have limited effects. Therefore, novel therapeutic strategies are urgently required. The SSPE virus, a variant MV strain, differs virologically from wild-type MV strain. One characteristic of the SSPE virus is its defective production of cell-free virus, which leaves cell-to-cell infection as the major mechanism of viral dissemination. The fusion protein plays an essential role in this cell-to-cell spread. It contains two critical heptad repeat regions that form a six-helix bundle in the trimer similar to most viral fusion proteins. In the case of human immunodeficiency virus type-1 (HIV-1), a synthetic peptide derived from the heptad repeat region of the fusion protein enfuvirtide inhibits viral replication and is clinically approved as an anti-HIV-1 agent. The heptad repeat regions of HIV-1 are structurally and functionally similar to those of the MV fusion protein. We therefore designed novel peptides derived from the fusion protein heptad repeat region of the MV and examined their effects on the measles and SSPE virus replication in vitro and in vivo. Some of these synthetic novel peptides demonstrated high antiviral activity against both the measles (Edmonston strain) and SSPE (Yamagata-1 strain) viruses at nanomolar concentrations with no cytotoxicity in vitro. In particular, intracranial administration of one of the synthetic peptides increased the survival rate from 0% to 67% in an SSPE virus-infected nude mouse model.

  19. A Novel Peptide Derived from the Fusion Protein Heptad Repeat Inhibits Replication of Subacute Sclerosing Panencephalitis Virus In Vitro and In Vivo.

    Directory of Open Access Journals (Sweden)

    Masahiro Watanabe

    Full Text Available Subacute sclerosing panencephalitis (SSPE is a persistent, progressive, and fatal degenerative disease resulting from persistent measles virus (MV infection of the central nervous system. Most drugs used to treat SSPE have been reported to have limited effects. Therefore, novel therapeutic strategies are urgently required. The SSPE virus, a variant MV strain, differs virologically from wild-type MV strain. One characteristic of the SSPE virus is its defective production of cell-free virus, which leaves cell-to-cell infection as the major mechanism of viral dissemination. The fusion protein plays an essential role in this cell-to-cell spread. It contains two critical heptad repeat regions that form a six-helix bundle in the trimer similar to most viral fusion proteins. In the case of human immunodeficiency virus type-1 (HIV-1, a synthetic peptide derived from the heptad repeat region of the fusion protein enfuvirtide inhibits viral replication and is clinically approved as an anti-HIV-1 agent. The heptad repeat regions of HIV-1 are structurally and functionally similar to those of the MV fusion protein. We therefore designed novel peptides derived from the fusion protein heptad repeat region of the MV and examined their effects on the measles and SSPE virus replication in vitro and in vivo. Some of these synthetic novel peptides demonstrated high antiviral activity against both the measles (Edmonston strain and SSPE (Yamagata-1 strain viruses at nanomolar concentrations with no cytotoxicity in vitro. In particular, intracranial administration of one of the synthetic peptides increased the survival rate from 0% to 67% in an SSPE virus-infected nude mouse model.

  20. Solid-State Nuclear Magnetic Resonance Investigation of the Structural Topology and Lipid Interactions of a Viral Fusion Protein Chimera Containing the Fusion Peptide and Transmembrane Domain.

    Science.gov (United States)

    Yao, Hongwei; Lee, Myungwoon; Liao, Shu-Yu; Hong, Mei

    2016-12-13

    The fusion peptide (FP) and transmembrane domain (TMD) of viral fusion proteins play important roles during virus-cell membrane fusion, by inducing membrane curvature and transient dehydration. The structure of the water-soluble ectodomain of viral fusion proteins has been extensively studied crystallographically, but the structures of the FP and TMD bound to phospholipid membranes are not well understood. We recently investigated the conformations and lipid interactions of the separate FP and TMD peptides of parainfluenza virus 5 (PIV5) fusion protein F using solid-state nuclear magnetic resonance. These studies provide structural information about the two domains when they are spatially well separated in the fusion process. To investigate how these two domains are structured relative to each other in the postfusion state, when the ectodomain forms a six-helix bundle that is thought to force the FP and TMD together in the membrane, we have now expressed and purified a chimera of the FP and TMD, connected by a Gly-Lys linker, and measured the chemical shifts and interdomain contacts of the protein in several lipid membranes. The FP-TMD chimera exhibits α-helical chemical shifts in all the membranes examined and does not cause strong curvature of lamellar membranes or membranes with negative spontaneous curvature. These properties differ qualitatively from those of the separate peptides, indicating that the FP and TMD interact with each other in the lipid membrane. However, no 13 C- 13 C cross peaks are observed in two-dimensional correlation spectra, suggesting that the two helices are not tightly associated. These results suggest that the ectodomain six-helix bundle does not propagate into the membrane to the two hydrophobic termini. However, the loosely associated FP and TMD helices are found to generate significant negative Gaussian curvature to membranes that possess spontaneous positive curvature, consistent with the notion that the FP-TMD assembly may

  1. H3N2 Virus as Causative Agent of ARDS Requiring Extracorporeal Membrane Oxygenation Support

    Directory of Open Access Journals (Sweden)

    Adriano Peris

    2014-01-01

    Full Text Available Pandemic influenza virus A(H1N1 2009 was associated with a higher risk of viral pneumonia in comparison with seasonal influenza viruses. The influenza season 2011-2012 was characterized by the prevalent circulation of influenza A(H3N2 viruses. Whereas most H3N2 patients experienced mild, self-limited influenza-like illness, some patients were at increased risk for influenza complications because of age or underlying medical conditions. Cases presented were patients admitted to the Intensive Care Unit (ICU of ECMO referral center (Careggi Teaching Hospital, Florence, Italy. Despite extracorporeal membrane oxygenation treatment (ECMO, one patient with H3N2-induced ARDS did not survive. Our experience suggests that viral aetiology is becoming more important and hospitals should be able to perform a fast differential diagnosis between bacterial and viral aetiology.

  2. Paramyxovirus Fusion and Entry: Multiple Paths to a Common End

    Science.gov (United States)

    Chang, Andres; Dutch, Rebecca E.

    2012-01-01

    The paramyxovirus family contains many common human pathogenic viruses, including measles, mumps, the parainfluenza viruses, respiratory syncytial virus, human metapneumovirus, and the zoonotic henipaviruses, Hendra and Nipah. While the expression of a type 1 fusion protein and a type 2 attachment protein is common to all paramyxoviruses, there is considerable variation in viral attachment, the activation and triggering of the fusion protein, and the process of viral entry. In this review, we discuss recent advances in the understanding of paramyxovirus F protein-mediated membrane fusion, an essential process in viral infectivity. We also review the role of the other surface glycoproteins in receptor binding and viral entry, and the implications for viral infection. Throughout, we concentrate on the commonalities and differences in fusion triggering and viral entry among the members of the family. Finally, we highlight key unanswered questions and how further studies can identify novel targets for the development of therapeutic treatments against these human pathogens. PMID:22590688

  3. Paramyxovirus Fusion and Entry: Multiple Paths to a Common End

    Directory of Open Access Journals (Sweden)

    Rebecca E. Dutch

    2012-04-01

    Full Text Available The paramyxovirus family contains many common human pathogenic viruses, including measles, mumps, the parainfluenza viruses, respiratory syncytial virus, human metapneumovirus, and the zoonotic henipaviruses, Hendra and Nipah. While the expression of a type 1 fusion protein and a type 2 attachment protein is common to all paramyxoviruses, there is considerable variation in viral attachment, the activation and triggering of the fusion protein, and the process of viral entry. In this review, we discuss recent advances in the understanding of paramyxovirus F protein-mediated membrane fusion, an essential process in viral infectivity. We also review the role of the other surface glycoproteins in receptor binding and viral entry, and the implications for viral infection. Throughout, we concentrate on the commonalities and differences in fusion triggering and viral entry among the members of the family. Finally, we highlight key unanswered questions and how further studies can identify novel targets for the development of therapeutic treatments against these human pathogens.

  4. The herpes simplex virus UL20 protein functions in glycoprotein K (gK intracellular transport and virus-induced cell fusion are independent of UL20 functions in cytoplasmic virion envelopment

    Directory of Open Access Journals (Sweden)

    Kousoulas Konstantin G

    2007-11-01

    Full Text Available Abstract The HSV-1 UL20 protein (UL20p and glycoprotein K (gK are both important determinants of cytoplasmic virion morphogenesis and virus-induced cell fusion. In this manuscript, we examined the effect of UL20 mutations on the coordinate transport and Trans Golgi Network (TGN localization of UL20p and gK, virus-induced cell fusion and infectious virus production. Deletion of 18 amino acids from the UL20p carboxyl terminus (UL20 mutant 204t inhibited intracellular transport and cell-surface expression of both gK and UL20, resulting in accumulation of UL20p and gK in the endoplasmic reticulum (ER in agreement with the inability of 204t to complement UL20-null virus replication and virus-induced cell fusion. In contrast, less severe carboxyl terminal deletions of either 11 or six amino acids (UL20 mutants 211t and 216t, respectively allowed efficient UL20p and gK intracellular transport, cell-surface expression and TGN colocalization. However, while both 211t and 216t failed to complement for infectious virus production, 216t complemented for virus-induced cell fusion, but 211t did not. These results indicated that the carboxyl terminal six amino acids of UL20p were crucial for infectious virus production, but not involved in intracellular localization of UL20p/gK and concomitant virus-induced cell fusion. In the amino terminus of UL20, UL20p mutants were produced changing one or both of the Y38 and Y49 residues found within putative phosphorylation sites. UL20p tyrosine-modified mutants with both tyrosine residues changed enabled efficient intracellular transport and TGN localization of UL20p and gK, but failed to complement for either infectious virus production, or virus-induced cell fusion. These results show that UL20p functions in cytoplasmic envelopment are separable from UL20 functions in UL20p intracellular transport, cell surface expression and virus-induced cell fusion.

  5. Groundnut bud necrosis virus encoded NSm associates with membranes via its C-terminal domain.

    Directory of Open Access Journals (Sweden)

    Pratibha Singh

    Full Text Available Groundnut Bud Necrosis Virus (GBNV is a tripartite ambisense RNA plant virus that belongs to serogroup IV of Tospovirus genus. Non-Structural protein-m (NSm, which functions as movement protein in tospoviruses, is encoded by the M RNA. In this communication, we demonstrate that despite the absence of any putative transmembrane domain, GBNV NSm associates with membranes when expressed in E. coli as well as in N. benthamiana. Incubation of refolded NSm with liposomes ranging in size from 200-250 nm resulted in changes in the secondary and tertiary structure of NSm. A similar behaviour was observed in the presence of anionic and zwitterionic detergents. Furthermore, the morphology of the liposomes was found to be modified in the presence of NSm. Deletion of coiled coil domain resulted in the inability of in planta expressed NSm to interact with membranes. Further, when the C-terminal coiled coil domain alone was expressed, it was found to be associated with membrane. These results demonstrate that NSm associates with membranes via the C-terminal coiled coil domain and such an association may be important for movement of viral RNA from cell to cell.

  6. Virus disinfection in water by biogenic silver immobilized in polyvinylidene fluoride membranes

    Energy Technology Data Exchange (ETDEWEB)

    Gusseme, B.D.; Fitts, J.; Hennebel, T.; Christiaens, E.; Saveyn, H.; Verbeken, K.; Boon, N.; Verstraete, W.

    2011-03-01

    The development of innovative water disinfection strategies is of utmost importance to prevent outbreaks of waterborne diseases related to poor treatment of (drinking) water. Recently, the association of silver nanoparticles with the bacterial cell surface of Lactobacillus fermentum (referred to as biogenic silver or bio-Ag{sup 0}) has been reported to exhibit antiviral properties. The microscale bacterial carrier matrix serves as a scaffold for Ag{sup 0} particles, preventing aggregation during encapsulation. In this study, bio-Ag{sup 0} was immobilized in different microporous PVDF membranes using two different pre-treatments of bio-Ag{sup 0} and the immersion-precipitation method. Inactivation of UZ1 bacteriophages using these membranes was successfully demonstrated and was most probably related to the slow release of Ag{sup +} from the membranes. At least a 3.4 log decrease of viruses was achieved by application of a membrane containing 2500 mg bio-Ag{sub powder}{sup 0} m{sup -2} in a submerged plate membrane reactor operated at a flux of 3.1 L m{sup -2} h{sup -1}. Upon startup, the silver concentration in the effluent initially increased to 271 {micro}g L{sup -1} but after filtration of 31 L m{sup -2}, the concentration approached the drinking water limit (= 100 {micro}g L{sup -1}). A virus decline of more than 3 log was achieved at a membrane flux of 75 L m{sup -2} h{sup -1}, showing the potential of this membrane technology for water disinfection on small scale. In biogenic silver, silver nanoparticles are attached to a bacterial carrier matrix. Bio-Ag{sup 0} was successfully immobilized in PVDF membranes using immersion-precipitation. The antiviral activity of this material was demonstrated in a plate membrane reactor. The antimicrobial mechanism was most probably related to the slow release of Ag{sup +} ions. The membranes can be applied for treatment of limited volumes of contaminated water.

  7. Dynamics of HIV-1 RNA Near the Plasma Membrane during Virus Assembly.

    Science.gov (United States)

    Sardo, Luca; Hatch, Steven C; Chen, Jianbo; Nikolaitchik, Olga; Burdick, Ryan C; Chen, De; Westlake, Christopher J; Lockett, Stephen; Pathak, Vinay K; Hu, Wei-Shau

    2015-11-01

    To increase our understanding of the events that lead to HIV-1 genome packaging, we examined the dynamics of viral RNA and Gag-RNA interactions near the plasma membrane by using total internal reflection fluorescence microscopy. We labeled HIV-1 RNA with a photoconvertible Eos protein via an RNA-binding protein that recognizes stem-loop sequences engineered into the viral genome. Near-UV light exposure causes an irreversible structural change in Eos and alters its emitted fluorescence from green to red. We studied the dynamics of HIV-1 RNA by photoconverting Eos near the plasma membrane, and we monitored the population of photoconverted red-Eos-labeled RNA signals over time. We found that in the absence of Gag, most of the HIV-1 RNAs stayed near the plasma membrane transiently, for a few minutes. The presence of Gag significantly increased the time that RNAs stayed near the plasma membrane: most of the RNAs were still detected after 30 min. We then quantified the proportion of HIV-1 RNAs near the plasma membrane that were packaged into assembling viral complexes. By tagging Gag with blue fluorescent protein, we observed that only a portion, ∼13 to 34%, of the HIV-1 RNAs that reached the membrane were recruited into assembling particles in an hour, and the frequency of HIV-1 RNA packaging varied with the Gag expression level. Our studies reveal the HIV-1 RNA dynamics on the plasma membrane and the efficiency of RNA recruitment and provide insights into the events leading to the generation of infectious HIV-1 virions. Nascent HIV-1 particles assemble on plasma membranes. During the assembly process, HIV-1 RNA genomes must be encapsidated into viral complexes to generate infectious particles. To gain insights into the RNA packaging and virus assembly mechanisms, we labeled and monitored the HIV-1 RNA signals near the plasma membrane. Our results showed that most of the HIV-1 RNAs stayed near the plasma membrane for only a few minutes in the absence of Gag, whereas

  8. Phosphatidylserine Lateral Organization Influences the Interaction of Influenza Virus Matrix Protein 1 with Lipid Membranes.

    Science.gov (United States)

    Bobone, Sara; Hilsch, Malte; Storm, Julian; Dunsing, Valentin; Herrmann, Andreas; Chiantia, Salvatore

    2017-06-15

    Influenza A virus matrix protein 1 (M1) is an essential component involved in the structural stability of the virus and in the budding of new virions from infected cells. A deeper understanding of the molecular basis of virion formation and the budding process is required in order to devise new therapeutic approaches. We performed a detailed investigation of the interaction between M1 and phosphatidylserine (PS) (i.e., its main binding target at the plasma membrane [PM]), as well as the distribution of PS itself, both in model membranes and in living cells. To this end, we used a combination of techniques, including Förster resonance energy transfer (FRET), confocal microscopy imaging, raster image correlation spectroscopy, and number and brightness (N&B) analysis. Our results show that PS can cluster in segregated regions in the plane of the lipid bilayer, both in model bilayers constituted of PS and phosphatidylcholine and in living cells. The viral protein M1 interacts specifically with PS-enriched domains, and such interaction in turn affects its oligomerization process. Furthermore, M1 can stabilize PS domains, as observed in model membranes. For living cells, the presence of PS clusters is suggested by N&B experiments monitoring the clustering of the PS sensor lactadherin. Also, colocalization between M1 and a fluorescent PS probe suggest that, in infected cells, the matrix protein can specifically bind to the regions of PM in which PS is clustered. Taken together, our observations provide novel evidence regarding the role of PS-rich domains in tuning M1-lipid and M1-M1 interactions at the PM of infected cells.IMPORTANCE Influenza virus particles assemble at the plasma membranes (PM) of infected cells. This process is orchestrated by the matrix protein M1, which interacts with membrane lipids while binding to the other proteins and genetic material of the virus. Despite its importance, the initial step in virus assembly (i.e., M1-lipid interaction) is still

  9. Preterm premature rupture of membranes in pregnancies complicated by human immunodeficiency virus infection: a single center's five-year experience.

    Science.gov (United States)

    Alvarez, Jesus R; Bardeguez, Arlene; Iffy, Leslie; Apuzzio, Joseph J

    2007-12-01

    The objective of this study was to describe one center's five-year experience of the management of human immunodeficiency virus (HIV) positive gravidas with preterm premature rupture of the membranes (PPROM) not in labor at review of all HIV positive gravidas with PPROM at rupture of membranes prior to delivery.

  10. Ultrastructural Characterization of Turnip Mosaic Virus-Induced Cellular Rearrangements Reveals Membrane-Bound Viral Particles Accumulating in Vacuoles.

    Science.gov (United States)

    Wan, Juan; Basu, Kaustuv; Mui, Jeannie; Vali, Hojatollah; Zheng, Huanquan; Laliberté, Jean-François

    2015-12-01

    Positive-strand RNA [(+) RNA] viruses remodel cellular membranes to facilitate virus replication and assembly. In the case of turnip mosaic virus (TuMV), the viral membrane protein 6K2 plays an essential role in endomembrane alterations. Although 6K2-induced membrane dynamics have been widely studied by confocal microscopy, the ultrastructure of this remodeling has not been extensively examined. In this study, we investigated the formation of TuMV-induced membrane changes by chemical fixation and high-pressure freezing/freeze substitution (HPF/FS) for transmission electron microscopy at different times of infection. We observed the formation of convoluted membranes connected to rough endoplasmic reticulum (rER) early in the infection process, followed by the production of single-membrane vesicle-like (SMVL) structures at the midstage of infection. Both SMVL and double-membrane vesicle-like structures with electron-dense cores, as well as electron-dense bodies, were found late in the infection process. Immunogold labeling results showed that the vesicle-like structures were 6K2 tagged and suggested that only the SMVL structures were viral RNA replication sites. Electron tomography (ET) was used to regenerate a three-dimensional model of these vesicle-like structures, which showed that they were, in fact, tubules. Late in infection, we observed filamentous particle bundles associated with electron-dense bodies, which suggests that these are sites for viral particle assembly. In addition, TuMV particles were observed to accumulate in the central vacuole as membrane-associated linear arrays. Our work thus unravels the sequential appearance of distinct TuMV-induced membrane structures for viral RNA replication, viral particle assembly, and accumulation. Positive-strand RNA viruses remodel cellular membranes for different stages of the infection process, such as protein translation and processing, viral RNA synthesis, particle assembly, and virus transmission. The

  11. NS2 Proteins of GB Virus B and Hepatitis C Virus Share Common Protease Activities and Membrane Topologies

    Science.gov (United States)

    Boukadida, Célia; Marnata, Caroline; Montserret, Roland; Cohen, Lisette; Blumen, Brigitte; Gouttenoire, Jérôme; Moradpour, Darius; Penin, François

    2014-01-01

    ABSTRACT GB virus B (GBV-B), which is hepatotropic in experimentally infected small New World primates, is a member of the Hepacivirus genus but phylogenetically relatively distant from hepatitis C virus (HCV). To gain insights into the role and specificity of hepaciviral nonstructural protein 2 (NS2), which is required for HCV polyprotein processing and particle morphogenesis, we investigated whether NS2 structural and functional features are conserved between HCV and GBV-B. We found that GBV-B NS2, like HCV NS2, has cysteine protease activity responsible for cleavage at the NS2/NS3 junction, and we experimentally confirmed the location of this junction within the viral polyprotein. A model for GBV-B NS2 membrane topology was experimentally established by determining the membrane association properties of NS2 segments fused to green fluorescent protein (GFP) and their nuclear magnetic resonance structures using synthetic peptides as well as by applying an N-glycosylation scanning approach. Similar glycosylation studies confirmed the HCV NS2 organization. Together, our data show that despite limited amino acid sequence similarity, GBV-B and HCV NS2 proteins share a membrane topology with 3 N-terminal transmembrane segments, which is also predicted to apply to other recently discovered hepaciviruses. Based on these data and using trans-complementation systems, we found that intragenotypic hybrid NS2 proteins with heterologous N-terminal membrane segments were able to efficiently trans-complement an assembly-deficient HCV mutant with a point mutation in the NS2 C-terminal domain, while GBV-B/HCV or intergenotypic NS2 chimeras were not. These studies indicate that virus- and genotype-specific intramolecular interactions between N- and C-terminal domains of NS2 are critically involved in HCV morphogenesis. IMPORTANCE Nonstructural protein 2 (NS2) of hepatitis C virus (HCV) is a multifunctional protein critically involved in polyprotein processing and virion

  12. Development of a virus concentration method using lanthanum-based chemical flocculation coupled with modified membrane filtration procedures.

    Science.gov (United States)

    Zhang, Yanyan; Riley, Lela K; Lin, Mengshi; Purdy, Gregory A; Hu, Zhiqiang

    2013-06-01

    Direct membrane filtration is often used to concentrate viruses in water but it may suffer from severe membrane fouling and clogging. Here, a lanthanum-based flocculation method coupled with modified membrane filtration procedures was developed and evaluated to detect viruses in large volume (40 L) water samples. The lanthanum-based flocculation method could easily reduce the water sample volume by a factor of 40. Additional volume reduction was achieved by a two-step membrane filtration approach. First, selected membrane filters (including 1MDS electropositive filters and nitrocellulose electronegative filters-Millipore HATF filters) were used to reduce water sample volume further and compare their efficiencies in virus recovery. The Mg²⁺-modified HATF membrane performed better on MS2 retention with an average virus recovery of 83.4% (±4.5% [standard deviation]). After HATF membrane filtration and elution, centrifugal ultrafiltration through a 30 kDa cut-off membrane resulted in an overall concentration factor of 20,000. Results from the infectivity assay showed that the MS2 recovery efficiencies from the NanoCeram- and 1MDS-based direct filtration and the lanthanum-based concentration coupled with the modified filtration procedure were 10.1% (±1.0%), 3.3% (±0.1%), and 17.5% (±1.1%), respectively. Results from the PCR analysis showed that the virus recoveries of the lanthanum-based method were 20.6% (±2.9%) and 19.5% (±3.4%) for MS2 and adenovirus, respectively, while no adenovirus could be detected through the NanoCeram- and 1MDS-based direct filtration. The lanthanum-based concentration method coupled with modified membrane filtration procedures is therefore a promising method for detecting waterborne viruses. Copyright © 2013 Elsevier B.V. All rights reserved.

  13. Chimeric rabies virus-like particles containing membrane-anchored GM-CSF enhances the immune response against rabies virus.

    Science.gov (United States)

    Kang, Hongtao; Qi, Yinglin; Wang, Hualei; Zheng, Xuexing; Gao, Yuwei; Li, Nan; Yang, Songtao; Xia, Xianzhu

    2015-03-11

    Rabies remains an important public health threat in most developing countries. To develop a more effective and safe vaccine against rabies, we have constructed a chimeric rabies virus-like particle (VLP), which containing glycoprotein (G) and matrix protein (M) of rabies virus (RABV) Evelyn-Rokitnicki-Abelseth (ERA) strain, and membrane-anchored granulocyte-macrophage colony-stimulating factor (GM-CSF), and it was named of EVLP-G. The immunogenicity and protective efficacy of EVLP-G against RABV were evaluated by intramuscular administration in a mouse model. The EVLP-G was successfully produced in insect cells by coinfection with three recombinant baculoviruses expressing G, M, and GM-CSF, respectively. The membrane-anchored GM-CSF possesses a strong adjuvant activity. More B cells and dendritic cells (DCs) were recruited and/or activated in inguinal lymph nodes in mice immunized with EVLP-G. EVLP-G was found to induce a significantly increased RABV-specific virus-neutralizing antibody and elicit a larger and broader antibody subclass responses compared with the standard rabies VLP (sRVLP, consisting of G and M). The EVLP-G also elicited significantly more IFN-γ- or IL-4-secreting CD4+ and CD8+ T cells than the sRVLP. Moreover, the immune responses induced by EVLP-G protect all vaccinated mice from lethal challenge with RABV. These results suggest that EVLP-G has the potential to be developed as a novel vaccine candidate for the prevention and control of animal rabies.

  14. Chimeric Rabies Virus-Like Particles Containing Membrane-Anchored GM-CSF Enhances the Immune Response against Rabies Virus

    Directory of Open Access Journals (Sweden)

    Hongtao Kang

    2015-03-01

    Full Text Available Rabies remains an important public health threat in most developing countries. To develop a more effective and safe vaccine against rabies, we have constructed a chimeric rabies virus-like particle (VLP, which containing glycoprotein (G and matrix protein (M of rabies virus (RABV Evelyn-Rokitnicki-Abelseth (ERA strain, and membrane-anchored granulocyte-macrophage colony-stimulating factor (GM-CSF, and it was named of EVLP-G. The immunogenicity and protective efficacy of EVLP-G against RABV were evaluated by intramuscular administration in a mouse model. The EVLP-G was successfully produced in insect cells by coinfection with three recombinant baculoviruses expressing G, M, and GM-CSF, respectively. The membrane-anchored GM-CSF possesses a strong adjuvant activity. More B cells and dendritic cells (DCs were recruited and/or activated in inguinal lymph nodes in mice immunized with EVLP-G. EVLP-G was found to induce a significantly increased RABV-specific virus-neutralizing antibody and elicit a larger and broader antibody subclass responses compared with the standard rabies VLP (sRVLP, consisting of G and M. The EVLP-G also elicited significantly more IFN-γ- or IL-4-secreting CD4+ and CD8+ T cells than the sRVLP. Moreover, the immune responses induced by EVLP-G protect all vaccinated mice from lethal challenge with RABV. These results suggest that EVLP-G has the potential to be developed as a novel vaccine candidate for the prevention and control of animal rabies.

  15. Myxoma virus expressing a fusion protein of interleukin-15 (IL15 and IL15 receptor alpha has enhanced antitumor activity.

    Directory of Open Access Journals (Sweden)

    Vesna Tosic

    Full Text Available Myxoma virus, a rabbit poxvirus, can efficiently infect various types of mouse and human cancer cells. It is a strict rabbit-specific pathogen, and is thought to be safe as a therapeutic agent in all non-rabbit hosts tested including mice and humans. Interleukin-15 (IL15 is an immuno-modulatory cytokine with significant potential for stimulating anti-tumor T lymphocytes and NK cells. Co-expression of IL15 with the α subunit of IL15 receptor (IL15Rα greatly enhances IL15 stability and bioavailability. Therefore, we engineered a new recombinant myxoma virus (vMyx-IL15Rα-tdTr, which expresses an IL15Rα-IL15 fusion protein plus tdTomato red fluorescent reporter protein. Permissive rabbit kidney epithelial (RK-13 cells infected with vMyx-IL15Rα-tdTr expressed and secreted the IL15Rα-IL15 fusion protein. Functional activity was confirmed by demonstrating that the secreted fusion protein stimulated proliferation of cytokine-dependent CTLL-2 cells. Multi-step growth curves showed that murine melanoma (B16-F10 and B16.SIY cell lines were permissive to vMyx-IL15Rα-tdTr infection. In vivo experiments in RAG1-/- mice showed that subcutaneous B16-F10 tumors treated with vMyx-IL15Rα-tdTr exhibited attenuated tumor growth and a significant survival benefit for the treated group compared to the PBS control and the control viruses (vMyx-IL15-tdTr and vMyx-tdTr. Immunohistological analysis of the subcutaneous tumors showed dramatically increased infiltration of NK cells in vMyx-IL15Rα-tdTr treated tumors compared to the controls. In vivo experiments with immunocompetent C57BL/6 mice revealed a strong infiltrate of both NK cells and CD8+ T cells in response to vMyx-IL15Rα-tdTr, and prolonged survival. We conclude that delivery of IL15Rα-IL15 in a myxoma virus vector stimulates both innate and adaptive components of the immune system.

  16. Myxoma virus expressing a fusion protein of interleukin-15 (IL15) and IL15 receptor alpha has enhanced antitumor activity.

    Science.gov (United States)

    Tosic, Vesna; Thomas, Diana L; Kranz, David M; Liu, Jia; McFadden, Grant; Shisler, Joanna L; MacNeill, Amy L; Roy, Edward J

    2014-01-01

    Myxoma virus, a rabbit poxvirus, can efficiently infect various types of mouse and human cancer cells. It is a strict rabbit-specific pathogen, and is thought to be safe as a therapeutic agent in all non-rabbit hosts tested including mice and humans. Interleukin-15 (IL15) is an immuno-modulatory cytokine with significant potential for stimulating anti-tumor T lymphocytes and NK cells. Co-expression of IL15 with the α subunit of IL15 receptor (IL15Rα) greatly enhances IL15 stability and bioavailability. Therefore, we engineered a new recombinant myxoma virus (vMyx-IL15Rα-tdTr), which expresses an IL15Rα-IL15 fusion protein plus tdTomato red fluorescent reporter protein. Permissive rabbit kidney epithelial (RK-13) cells infected with vMyx-IL15Rα-tdTr expressed and secreted the IL15Rα-IL15 fusion protein. Functional activity was confirmed by demonstrating that the secreted fusion protein stimulated proliferation of cytokine-dependent CTLL-2 cells. Multi-step growth curves showed that murine melanoma (B16-F10 and B16.SIY) cell lines were permissive to vMyx-IL15Rα-tdTr infection. In vivo experiments in RAG1-/- mice showed that subcutaneous B16-F10 tumors treated with vMyx-IL15Rα-tdTr exhibited attenuated tumor growth and a significant survival benefit for the treated group compared to the PBS control and the control viruses (vMyx-IL15-tdTr and vMyx-tdTr). Immunohistological analysis of the subcutaneous tumors showed dramatically increased infiltration of NK cells in vMyx-IL15Rα-tdTr treated tumors compared to the controls. In vivo experiments with immunocompetent C57BL/6 mice revealed a strong infiltrate of both NK cells and CD8+ T cells in response to vMyx-IL15Rα-tdTr, and prolonged survival. We conclude that delivery of IL15Rα-IL15 in a myxoma virus vector stimulates both innate and adaptive components of the immune system.

  17. Hydrodynamic and Membrane Binding Properties of Purified Rous Sarcoma Virus Gag Protein

    Energy Technology Data Exchange (ETDEWEB)

    Dick, Robert A.; Datta, Siddhartha A.K.; Nanda, Hirsh; Fang, Xianyang; Wen, Yi; Barros, Marilia; Wang, Yun-Xing; Rein, Alan; Vogt, Volker M. (NCI); (Cornell); (CM); (NIST)

    2016-05-06

    Previously, no retroviral Gag protein has been highly purified in milligram quantities and in a biologically relevant and active form. We have purified Rous sarcoma virus (RSV) Gag protein and in parallel several truncation mutants of Gag and have studied their biophysical properties and membrane interactionsin vitro. RSV Gag is unusual in that it is not naturally myristoylated. From its ability to assemble into virus-like particlesin vitro, we infer that RSV Gag is biologically active. By size exclusion chromatography and small-angle X-ray scattering, Gag in solution appears extended and flexible, in contrast to previous reports on unmyristoylated HIV-1 Gag, which is compact. However, by neutron reflectometry measurements of RSV Gag bound to a supported bilayer, the protein appears to adopt a more compact, folded-over conformation. At physiological ionic strength, purified Gag binds strongly to liposomes containing acidic lipids. This interaction is stimulated by physiological levels of phosphatidylinositol-(4,5)-bisphosphate [PI(4,5)P2] and by cholesterol. However, unlike HIV-1 Gag, RSV Gag shows no sensitivity to acyl chain saturation. In contrast with full-length RSV Gag, the purified MA domain of Gag binds to liposomes only weakly. Similarly, both an N-terminally truncated version of Gag that is missing the MA domain and a C-terminally truncated version that is missing the NC domain bind only weakly. These results imply that NC contributes to membrane interactionin vitro, either by directly contacting acidic lipids or by promoting Gag multimerization.

    Retroviruses like HIV assemble at and bud from the plasma membrane of cells. Assembly requires the interaction between thousands of Gag molecules to form a lattice. Previous work indicated that lattice formation at the plasma membrane is influenced by the conformation of monomeric HIV. We have extended this work to the more tractable RSV Gag. Our

  18. A minimal phycobilisome: fusion and chromophorylation of the truncated core-membrane linker and phycocyanin.

    Science.gov (United States)

    Tang, Kun; Zeng, Xiao-Li; Yang, Yi; Wang, Zhi-Bin; Wu, Xian-Jun; Zhou, Ming; Noy, Dror; Scheer, Hugo; Zhao, Kai-Hong

    2012-07-01

    Phycobilisomes, the light-harvesting antennas in cyanobacteria and red algae, consist of an allophycocyanin core that is attached to the membrane via a core-membrane linker, and rods comprised of phycocyanin and often also phycoerythrin or phycoerythrocyanin. Phycobiliproteins show excellent energy transfer among the chromophores that renders them biomarkers with large Stokes-shifts absorbing over most of the visible spectrum and into the near infrared. Their application is limited, however, due to covalent binding of the chromophores and by solubility problems. We report construction of a water-soluble minimal chromophore-binding unit of the red-absorbing and fluorescing core-membrane linker. This was fused to minimal chromophore-binding units of phycocyanin. After double chromophorylation with phycocyanobilin, in E. coli, the fused phycobiliproteins absorbed light in the range of 610-660nm, and fluoresced at ~670nm, similar to phycobilisomes devoid of phycoerythr(ocyan)in. The fused phycobiliprotein could also be doubly chromophorylated with phycoerythrobilin, resulting in a chromoprotein absorbing around 540-575nm, and fluorescing at ~585nm. The broad absorptions and the large Stokes shifts render these chromoproteins candidates for imaging; they may also be helpful in studying phycobilisome assembly. Copyright © 2012 Elsevier B.V. All rights reserved.

  19. Factors that can interfere with virus concentration from wastewater when using zeta plus 60S filter membranes

    Directory of Open Access Journals (Sweden)

    Queiroz APS

    2000-01-01

    Full Text Available Zeta plus filter membranes (ZP60S have been shown to be efficient for rotavirus concentration from wastewater and for the reduction of cytotoxicity for cell cultures. Recently a variability in both properties was observed. In view of the low costs and the high virus recovery rates obtained in the past, we re-evaluated the application of ZP60S filter membranes for virus concentration from environmental samples. Some factors that could interfere with the concentration strategy using ZP60S were also considered and assessed including the type of water to be filtered and the possible release of toxic substances from the membrane matrix during filtration.

  20. Protection against respiratory syncytial virus by inactivated influenza virus carrying a fusion protein neutralizing epitope in a chimeric hemagglutinin.

    Science.gov (United States)

    Lee, Yu-Na; Hwang, Hye Suk; Kim, Min-Chul; Lee, Young-Tae; Kim, Yu-Jin; Lee, F Eun-Hyung; Kang, Sang-Moo

    2016-04-01

    A desirable vaccine against respiratory syncytial virus (RSV) should induce neutralizing antibodies without eliciting abnormal T cell responses to avoid vaccine-enhanced pathology. In an approach to deliver RSV neutralizing epitopes without RSV-specific T cell antigens, we genetically engineered chimeric influenza virus expressing RSV F262-276 neutralizing epitopes in the globular head domain as a chimeric hemagglutinin (HA) protein. Immunization of mice with formalin-inactivated recombinant chimeric influenza/RSV F262-276 was able to induce RSV protective neutralizing antibodies and lower lung viral loads after challenge. Formalin-inactivated RSV immune mice showed high levels of pulmonary inflammatory cytokines, macrophages, IL-4-producing T cells, and extensive histopathology. However, RSV-specific T cell responses and enhancement of pulmonary histopathology were not observed after RSV infection of inactivated chimeric influenza/RSV F262-276. This study provides evidence that an inactivated vaccine platform of chimeric influenza/RSV virus can be developed into a safe RSV vaccine candidate without priming RSV-specific T cells and immunopathology. Respiratory syncytial virus (RSV) is a major cause of respiratory tract illness and morbidity in children. Hence, there is a need to develop an effective vaccine against this virus. In this article, the authors engineered chimeric influenza virus to express RSV neutralizing epitopes. The positive findings in in-vivo experiments provide a beginning for future clinical trials and perhaps eventual product realization. Copyright © 2015 Elsevier Inc. All rights reserved.

  1. Ready, Set, Fuse! The Coronavirus Spike Protein and Acquisition of Fusion Competence

    Science.gov (United States)

    Heald-Sargent, Taylor; Gallagher, Tom

    2012-01-01

    Coronavirus-cell entry programs involve virus-cell membrane fusions mediated by viral spike (S) proteins. Coronavirus S proteins acquire membrane fusion competence by receptor interactions, proteolysis, and acidification in endosomes. This review describes our current understanding of the S proteins, their interactions with and their responses to these entry triggers. We focus on receptors and proteases in prompting entry and highlight the type II transmembrane serine proteases (TTSPs) known to activate several virus fusion proteins. These and other proteases are essential cofactors permitting coronavirus infection, conceivably being in proximity to cell-surface receptors and thus poised to split entering spike proteins into the fragments that refold to mediate membrane fusion. The review concludes by noting how understanding of coronavirus entry informs antiviral therapies. PMID:22590686

  2. The yeast ATP-binding cassette (ABC) transporter Ycf1p enhances the recruitment of the soluble SNARE Vam7p to vacuoles for efficient membrane fusion.

    Science.gov (United States)

    Sasser, Terry L; Lawrence, Gus; Karunakaran, Surya; Brown, Christopher; Fratti, Rutilio A

    2013-06-21

    The Saccharomyces cerevisiae vacuole contains five ATP-binding cassette class C (ABCC) transporters, including Ycf1p, a family member that was originally characterized as a Cd(2+) transporter. Ycf1p has also been found to physically interact with a wide array of proteins, including factors that regulate vacuole homeostasis. In this study, we examined the role of Ycf1p and other ABCC transporters in the regulation of vacuole homotypic fusion. We found that deletion of YCF1 attenuated in vitro vacuole fusion by up to 40% relative to wild-type vacuoles. Plasmid-expressed wild-type Ycf1p rescued the deletion phenotype; however, Ycf1p containing a mutation of the conserved Lys-669 to Met in the Walker A box of the first nucleotide-binding domain (Ycf1p(K669M)) was unable to complement the fusion defect of ycf1Δ vacuoles. This indicates that the ATPase activity of Ycf1p is required for its function in regulating fusion. In addition, we found that deleting YCF1 caused a striking decrease in vacuolar levels of the soluble SNARE Vam7p, whereas total cellular levels were not altered. The attenuated fusion of ycf1Δ vacuoles was rescued by the addition of recombinant Vam7p to in vitro experiments. Thus, Ycf1p contributes in the recruitment of Vam7p to the vacuole for efficient membrane fusion.

  3. The Yeast ATP-binding Cassette (ABC) Transporter Ycf1p Enhances the Recruitment of the Soluble SNARE Vam7p to Vacuoles for Efficient Membrane Fusion*

    Science.gov (United States)

    Sasser, Terry L.; Lawrence, Gus; Karunakaran, Surya; Brown, Christopher; Fratti, Rutilio A.

    2013-01-01

    The Saccharomyces cerevisiae vacuole contains five ATP-binding cassette class C (ABCC) transporters, including Ycf1p, a family member that was originally characterized as a Cd2+ transporter. Ycf1p has also been found to physically interact with a wide array of proteins, including factors that regulate vacuole homeostasis. In this study, we examined the role of Ycf1p and other ABCC transporters in the regulation of vacuole homotypic fusion. We found that deletion of YCF1 attenuated in vitro vacuole fusion by up to 40% relative to wild-type vacuoles. Plasmid-expressed wild-type Ycf1p rescued the deletion phenotype; however, Ycf1p containing a mutation of the conserved Lys-669 to Met in the Walker A box of the first nucleotide-binding domain (Ycf1pK669M) was unable to complement the fusion defect of ycf1Δ vacuoles. This indicates that the ATPase activity of Ycf1p is required for its function in regulating fusion. In addition, we found that deleting YCF1 caused a striking decrease in vacuolar levels of the soluble SNARE Vam7p, whereas total cellular levels were not altered. The attenuated fusion of ycf1Δ vacuoles was rescued by the addition of recombinant Vam7p to in vitro experiments. Thus, Ycf1p contributes in the recruitment of Vam7p to the vacuole for efficient membrane fusion. PMID:23658021

  4. West Nile virus infection causes endocytosis of a specific subset of tight junction membrane proteins.

    Directory of Open Access Journals (Sweden)

    Zaikun Xu

    Full Text Available West Nile virus (WNV is a blood-borne pathogen that causes systemic infections and serious neurological disease in human and animals. The most common route of infection is mosquito bites and therefore, the virus must cross a number of polarized cell layers to gain access to organ tissue and the central nervous system. Resistance to trans-cellular movement of macromolecules between epithelial and endothelial cells is mediated by tight junction complexes. While a number of recent studies have documented that WNV infection negatively impacts the barrier function of tight junctions, the intracellular mechanism by which this occurs is poorly understood. In the present study, we report that endocytosis of a subset of tight junction membrane proteins including claudin-1 and JAM-1 occurs in WNV infected epithelial and endothelial cells. This process, which ultimately results in lysosomal degradation of the proteins, is dependent on the GTPase dynamin and microtubule-based transport. Finally, infection of polarized cells with the related flavivirus, Dengue virus-2, did not result in significant loss of tight junction membrane proteins. These results suggest that neurotropic flaviviruses such as WNV modulate the host cell environment differently than hemorrhagic flaviviruses and thus may have implications for understanding the molecular basis for neuroinvasion.

  5. Retrograde Transport from Early Endosomes to the trans-Golgi Network Enables Membrane Wrapping and Egress of Vaccinia Virus Virions.

    Science.gov (United States)

    Sivan, Gilad; Weisberg, Andrea S; Americo, Jeffrey L; Moss, Bernard

    2016-10-01

    The anterograde pathway, from the endoplasmic reticulum through the trans-Golgi network to the cell surface, is utilized by trans-membrane and secretory proteins. The retrograde pathway, which directs traffic in the opposite direction, is used following endocytosis of exogenous molecules and recycling of membrane proteins. Microbes exploit both routes: viruses typically use the anterograde pathway for envelope formation prior to exiting the cell, whereas ricin and Shiga-like toxins and some nonenveloped viruses use the retrograde pathway for cell entry. Mining a human genome-wide RNA interference (RNAi) screen revealed a need for multiple retrograde pathway components for cell-to-cell spread of vaccinia virus. We confirmed and extended these results while discovering that retrograde trafficking was required for virus egress rather than entry. Retro-2, a specific retrograde trafficking inhibitor of protein toxins, potently prevented spread of vaccinia virus as well as monkeypox virus, a human pathogen. Electron and confocal microscopy studies revealed that Retro-2 prevented wrapping of virions with an additional double-membrane envelope that enables microtubular transport, exocytosis, and actin polymerization. The viral B5 and F13 protein components of this membrane, which are required for wrapping, normally colocalize in the trans-Golgi network. However, only B5 traffics through the secretory pathway, suggesting that F13 uses another route to the trans-Golgi network. The retrograde route was demonstrated by finding that F13 was largely confined to early endosomes and failed to colocalize with B5 in the presence of Retro-2. Thus, vaccinia virus makes novel use of the retrograde transport system for formation of the viral wrapping membrane. Efficient cell-to-cell spread of vaccinia virus and other orthopoxviruses depends on the wrapping of infectious particles with a double membrane that enables microtubular transport, exocytosis, and actin polymerization

  6. Characterization of Pre-F-GCN4t, a Modified Human Respiratory Syncytial Virus Fusion Protein Stabilized in a Noncleaved Prefusion Conformation.

    Science.gov (United States)

    Blais, Normand; Gagné, Martin; Hamuro, Yoshitomo; Rheault, Patrick; Boyer, Martine; Steff, Ann-Muriel; Baudoux, Guy; Dewar, Vincent; Demers, Josée; Ruelle, Jean-Louis; Martin, Denis

    2017-07-01

    The human respiratory syncytial virus (hRSV) fusion (F) protein is considered a major target of the neutralizing antibody response to hRSV. This glycoprotein undergoes a major structural shift from the prefusion (pre-F) to the postfusion (post-F) state at the time of virus-host cell membrane fusion. Recent evidences suggest that the pre-F state is a superior target for neutralizing antibodies compared to the post-F state. Therefore, for vaccine purposes, we have designed and characterized a recombinant hRSV F protein, called Pre-F-GCN4t, stabilized in a pre-F conformation. To show that Pre-F-GCN4t does not switch to a post-F conformation, it was compared with a recombinant post-F molecule, called Post-F-XC. Pre-F-GCN4t was glycosylated and trimeric and displayed a conformational stability different from that of Post-F-XC, as shown by chemical denaturation. Electron microscopy analysis suggested that Pre-F-GCN4t adopts a lollipop-like structure. In contrast, Post-F-XC had a typical elongated conical shape. Hydrogen/deuterium exchange mass spectrometry demonstrated that the two molecules had common rigid folding core and dynamic regions and provided structural insight for their biophysical and biochemical properties and reactivity. Pre-F-GCN4t was shown to deplete hRSV-neutralizing antibodies from human serum more efficiently than Post-F-XC. Importantly, Pre-F-GCN4t was also shown to bind D25, a highly potent monoclonal antibody specific for the pre-F conformation. In conclusion, this construct presents several pre-F characteristics, does not switch to the post-F conformation, and presents antigenic features required for a protective neutralizing antibody response. Therefore, Pre-F-GCN4t can be considered a promising candidate vaccine antigen.IMPORTANCE Human respiratory syncytial virus (RSV) is a global leading cause of infant mortality and adult morbidity. The development of a safe and efficacious RSV vaccine remains an important goal. The RSV class I fusion (F

  7. Fusion Machinery

    DEFF Research Database (Denmark)

    Sørensen, Jakob Balslev; Milosevic, Ira

    2015-01-01

    the vesicular SNARE VAMP2/synaptobrevin-2 and the target (plasma membrane) SNAREs SNAP25 and syntaxin-1 results in fusion and release of neurotransmitter, synchronized to the electrical activity of the cell by calcium influx and binding to synaptotagmin. Formation of the SNARE complex is tightly regulated...... and appears to start with syntaxin-1 bound to an SM (Sec1/Munc18-like) protein. Proteins of the Munc13-family are responsible for opening up syntaxin and allowing sequential binding of SNAP-25 and VAMP2/synaptobrevin-2. N- to C-terminal “zippering” of the SNARE domains leads to membrane fusion...

  8. Deletion of the Vaccinia Virus I2 Protein Interrupts Virion Morphogenesis, Leading to Retention of the Scaffold Protein and Mislocalization of Membrane-Associated Entry Proteins.

    Science.gov (United States)

    Hyun, Seong-In; Weisberg, Andrea; Moss, Bernard

    2017-08-01

    The I2L open reading frame of vaccinia virus (VACV) encodes a conserved 72-amino-acid protein with a putative C-terminal transmembrane domain. Previous studies with a tetracycline-inducible mutant demonstrated that I2-deficient virions are defective in cell entry. The purpose of the present study was to determine the step of replication or entry that is affected by loss of the I2 protein. Fluorescence microscopy experiments showed that I2 colocalized with a major membrane protein of immature and mature virions. We generated a cell line that constitutively expressed I2 and allowed construction of the VACV I2L deletion mutant vΔI2. As anticipated, vΔI2 was unable to replicate in cells that did not express I2. Unexpectedly, morphogenesis was interrupted at a stage after immature virion formation, resulting in the accumulation of dense spherical particles instead of brick-shaped mature virions with well-defined core structures. The abnormal particles retained the D13 scaffold protein of immature virions, were severely deficient in the transmembrane proteins that comprise the entry fusion complex (EFC), and had increased amounts of unprocessed membrane and core proteins. Total lysates of cells infected with vΔI2 also had diminished EFC proteins due to instability attributed to their hydrophobicity and failure to be inserted into viral membranes. A similar instability of EFC proteins had previously been found with unrelated mutants blocked earlier in morphogenesis that also accumulated viral membranes retaining the D13 scaffold. We concluded that I2 is required for virion morphogenesis, release of the D13 scaffold, and the association of EFC proteins with viral membranes.IMPORTANCE Poxviruses comprise a large family that infect vertebrates and invertebrates, cause disease in both in humans and in wild and domesticated animals, and are being engineered as vectors for vaccines and cancer therapy. In addition, investigations of poxviruses have provided insights into many

  9. The cellular receptors for infectious bursal disease virus | Zhu ...

    African Journals Online (AJOL)

    Virus receptors are simplistically defined as cell surface molecules that mediate binding (attachment, adsorption) and/or trigger membrane fusion or entry through other processes. Infectious bursal disease virus (IBDV) entry into host cells occurs by recognition of specific cellular receptor(s) with viral envelope glycoprotein, ...

  10. The current status and challenges in the development of fusion inhibitors as therapeutics for HIV-1 infection.

    Science.gov (United States)

    Tan, Jian Jun; Ma, Xue Ting; Liu, Chang; Zhang, Xiao Yi; Wang, Cun Xin

    2013-01-01

    HIV-1 membrane fusion as a part of the process of viral entry in the target cells is facilitated by gp41 and gp120, which are encoded by Env gene of HIV-1. Based on the structure and the mechanism researches, new treatment options targeting HIV-1 entry process have been proposed. Enfuvirtide, which mimics amino acid sequences of viral envelope glycoprotein gp41, is the first HIV-1 fusion inhibitor approved by FDA. Although it fulfills vital functions by binding to gp41 and abolishing the membrane fusion reaction when used in combination, it could induce drug resistant virus variants. Currently, a number of design and modification schemes have been presented, a large number of prospective fusion peptides have emerged. For these fusion inhibitors, multiple mutations in gp41 have been associated with the loss of susceptibility to agents. This review reported the current developments and innovative designs of HIV-1 membrane fusion inhibitors.

  11. Virus-cell fusion as a trigger of innate immunity dependent on the adaptor STING

    NARCIS (Netherlands)

    Holm, C.K.; Jensen, S.B.; Jakobsen, M.R.; Cheshenko, N.; Horan, K.A.; Moeller, H.B.; Gonzalez-Dosal, R.; Rasmussen, S.B.; Christensen, M.H.; Yarovinsky, T.O.; Rixon, F.J.; Herold, B.C.; Fitzgerald, K.A.; Paludan, S.R.

    2012-01-01

    The innate immune system senses infection by detecting either evolutionarily conserved molecules essential for the survival of microbes or the abnormal location of molecules. Here we demonstrate the existence of a previously unknown innate detection mechanism induced by fusion between viral

  12. A single L288I substitution in the fusion protein of bovine parainfluenza virus type 3 enhances virus growth in semi-suitable cell lines.

    Science.gov (United States)

    Matsuura, Ryosuke; Takada, Marina; Kokuho, Takehiro; Tsuboi, Takamitsu; Kameyama, Ken-Ichiro; Takeuchi, Kaoru

    2017-08-01

    The bovine parainfluenza virus type 3 BN-CE vaccine strain was obtained by serial passage of the BN-1 strain in chicken embryonic fibroblasts (CEF). We previously identified a substitution (L288I) in the fusion (F) protein between the two strains. To examine the effect of the substitution on CEF adaptation and attenuation, we generated a recombinant BN-1 strain with the L288I substitution in the F protein (F L288I -EGFP). F L288I -EGFP replicated more efficiently than a recombinant BN-1 strain (wt-EGFP) in semi-suitable cell lines, suggesting that the L288I substitution was established in the BN-1 strain during the process of adaptation in CEF.

  13. Improved immunogenicity of Newcastle disease virus inactivated vaccine following DNA vaccination using Newcastle disease virus hemagglutinin-neuraminidase and fusion protein genes.

    Science.gov (United States)

    Firouzamandi, Masoumeh; Moeini, Hassan; Hosseini, Davood; Bejo, Mohd Hair; Omar, Abdul Rahman; Mehrbod, Parvaneh; Ideris, Aini

    2016-03-01

    The present study describes the development of DNA vaccines using the hemagglutinin-neuraminidase (HN) and fusion (F) genes from AF2240 Newcastle disease virus strain, namely pIRES/HN, pIRES/F and pIRES-F/HN. Transient expression analysis of the constructs in Vero cells revealed the successful expression of gene inserts in vitro. Moreover, in vivo experiments showed that single vaccination with the constructed plasmid DNA (pDNA) followed by a boost with inactivated vaccine induced a significant difference in enzyme-linked immunosorbent assay antibody levels (p inactivated vaccine alone. Taken together, these results indicated that recombinant pDNA could be used to increase the efficacy of the inactivated vaccine immunization procedure.

  14. Generation of a dual-functional split-reporter protein for monitoring membrane fusion using self-associating split GFP.

    Science.gov (United States)

    Ishikawa, Hirohito; Meng, Fanxia; Kondo, Naoyuki; Iwamoto, Aikichi; Matsuda, Zene

    2012-12-01

    Split reporter proteins capable of self-association and reactivation have applications in biomedical research, but designing these proteins, especially the selection of appropriate split points, has been somewhat arbitrary. We describe a new methodology to facilitate generating split proteins using split GFP as a self-association module. We first inserted the entire GFP module at one of several candidate split points in the protein of interest, and chose clones that retained the GFP signal and high activity relative to the original protein. Once such chimeric clones were identified, a final pair of split proteins was generated by splitting the GFP-inserted chimera within the GFP domain. Applying this strategy to Renilla reniformis luciferase, we identified a new split point that gave 10 times more activity than the previous split point. The process of membrane fusion was monitored with high sensitivity using a new pair of split reporter proteins. We also successfully identified new split points for HaloTag protein and firefly luciferase, generating pairs of self-associating split proteins that recovered the functions of both GFP and the original protein. This simple method of screening will facilitate the designing of split proteins that are capable of self-association through the split GFP domains.

  15. Glutamic Acid Residues in HIV-1 p6 Regulate Virus Budding and Membrane Association of Gag.

    Science.gov (United States)

    Friedrich, Melanie; Setz, Christian; Hahn, Friedrich; Matthaei, Alina; Fraedrich, Kirsten; Rauch, Pia; Henklein, Petra; Traxdorf, Maximilian; Fossen, Torgils; Schubert, Ulrich

    2016-04-25

    The HIV-1 Gag p6 protein regulates the final abscission step of nascent virions from the cell membrane by the action of its two late (L-) domains, which recruit Tsg101 and ALIX, components of the ESCRT system. Even though p6 consists of only 52 amino acids, it is encoded by one of the most polymorphic regions of the HIV-1 gag gene and undergoes various posttranslational modifications including sumoylation, ubiquitination, and phosphorylation. In addition, it mediates the incorporation of the HIV-1 accessory protein Vpr into budding virions. Despite its small size, p6 exhibits an unusually high charge density. In this study, we show that mutation of the conserved glutamic acids within p6 increases the membrane association of Pr55 Gag followed by enhanced polyubiquitination and MHC-I antigen presentation of Gag-derived epitopes, possibly due to prolonged exposure to membrane bound E3 ligases. The replication capacity of the total glutamic acid mutant E0A was almost completely impaired, which was accompanied by defective virus release that could not be rescued by ALIX overexpression. Altogether, our data indicate that the glutamic acids within p6 contribute to the late steps of viral replication and may contribute to the interaction of Gag with the plasma membrane.

  16. Fusion of reconstituted influenza virus envelopes with liposomes mediated by streptavidin/biotin interactions

    NARCIS (Netherlands)

    Schoen, P; Leserman, L; Wilschut, J

    1996-01-01

    Reconstituted influenza virus envelopes (virosomes) containing the viral hemagglutinin (HA) represent an efficient fusogenic cellular delivery system, By interaction of HA with its natural receptors, sialylated lipids (gangliosides) or proteins, virosomes bind to cells and, following endocytic

  17. Inhibition of human immunodeficiency virus type-1 (HIV-1 glycoprotein-mediated cell-cell fusion by immunor (IM28

    Directory of Open Access Journals (Sweden)

    Akoume Marie-Yvonne

    2005-02-01

    Full Text Available Abstract Background Immunor (IM28, an analog of dehydroepiandrosterone (DHEA, inhibits human immunodeficiency virus type-1 (HIV-1 by inhibiting reverse transcriptase. We assessed the ability of IM28 to inhibit the cell-cell fusion mediated by HIV envelope glycoprotein in an in vitro system. For this purpose, we co-cultured TF228.1.16, a T-cell line expressing stably HIV-1 glycoprotein envelopes, with an equal number of 293/CD4+, another T cell line expressing CD4, and with the SupT1 cell line with or without IM28. Results In the absence of IM28, TF228.1.16 fused with 293/CD4+, inducing numerous large syncytia. Syncytia appeared more rapidly when TF228.1.16 was co-cultured with SupT1 cells than when it was co-cultured with the 293/CD4+ cell line. IM28 (1.6 – 45 μg/ml completely inhibits cell-cell fusion. IM28 also prevented the development of new syncytia in infected cells and protected naive SupT1 cells from HIV-1 infection. Evaluation of 50% inhibitory dose (IC50 of IM28 revealed a decrease in HIV-1 replication with an IC50 of 22 mM and 50% cytotoxicity dose (CC50 as determined on MT2 cells was 75 mM giving a selectivity index of 3.4 Conclusions These findings suggest that IM28 exerts an inhibitory action on the env proteins that mediate cell-cell fusion between infected and healthy cells. They also suggest that IM28 interferes with biochemical processes to stop the progression of existing syncytia. This property may lead to the development of a new class of therapeutic drug.

  18. Investigation of the curvature induction and membrane localization of the influenza virus M2 protein using static and off-magic-angle spinning solid-state nuclear magnetic resonance of oriented bicelles.

    Science.gov (United States)

    Wang, Tuo; Hong, Mei

    2015-04-07

    A wide variety of membrane proteins induce membrane curvature for function; thus, it is important to develop new methods to simultaneously determine membrane curvature and protein binding sites in membranes with multiple curvatures. We introduce solid-state nuclear magnetic resonance (NMR) methods based on magnetically oriented bicelles and off-magic-angle spinning (OMAS) to measure membrane curvature and the binding site of proteins in mixed-curvature membranes. We demonstrate these methods on the influenza virus M2 protein, which not only acts as a proton channel but also mediates virus assembly and membrane scission. An M2 peptide encompassing the transmembrane (TM) domain and an amphipathic helix, M2(21-61), was studied and compared with the TM peptide (M2TM). Static (31)P NMR spectra of magnetically oriented 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC)/1,2-dihexanoyl-sn-glycero-3-phosphocholine (DHPC) bicelles exhibit a temperature-independent isotropic chemical shift in the presence of M2(21-61) but not M2TM, indicating that the amphipathic helix confers the ability to generate a high-curvature phase. Two-dimensional (2D) (31)P spectra indicate that this high-curvature phase is associated with the DHPC bicelle edges, suggestive of the structure of budding viruses from the host cell. (31)P- and (13)C-detected (1)H relaxation times of the lipids indicate that the majority of M2(21-61) is bound to the high-curvature phase. Using OMAS experiments, we resolved the (31)P signals of lipids with identical headgroups based on their distinct chemical shift anisotropies. On the basis of this resolution, 2D (1)H-(31)P correlation spectra show that the amide protons in M2(21-61) correlate with the DMPC but not DHPC (31)P signal of the bicelle, indicating that a small percentage of M2(21-61) partitions into the planar region of the bicelles. These results show that the amphipathic helix induces high membrane curvature and localizes the protein to this phase, in good

  19. Investigation of the Curvature Induction and Membrane Localization of the Influenza Virus M2 Protein Using Static and Off-Magic-Angle Spinning Solid-State NMR of Oriented Bicelles

    Science.gov (United States)

    Wang, Tuo; Hong, Mei

    2015-01-01

    A wide variety of membrane proteins induce membrane curvature for function, thus it is important to develop new methods to simultaneously determine membrane curvature and protein binding sites in membranes with multiple curvatures. We introduce solid-state NMR methods based on magnetically oriented bicelles and off-magic-angle spinning (OMAS) to measure membrane curvature and the binding site of proteins in mixed-curvature membranes. We demonstrate these methods on the influenza virus M2 protein, which not only acts as a proton channel but also mediates virus assembly and membrane scission. An M2 peptide encompassing the transmembrane (TM) domain and an amphipathic helix, M2(21-61), was studied and compared with the TM peptide (M2TM). Static 31P NMR spectra of magnetically oriented DMPC/DHPC bicelles exhibit a temperature-independent isotropic chemical shift in the presence of M2(21-61) but not M2TM, indicating that the amphipathic helix confers the peptide with the ability to generate a high-curvature phase. 2D 31P spectra indicate that this high-curvature phase is associated with the DHPC bicelle edges, suggestive of the structure of budding viruses from the host cell. 31P- and 13C-detected 1H relaxation times of the lipids indicate that the majority of M2(21-61) is bound to the high-curvature phase. Using OMAS experiments, we resolved the 31P signals of lipids with identical headgroups based on their distinct chemical shift anisotropies. Based on this resolution, 2D 1H-31P correlation spectra show that the amide protons in M2(21-61) correlate with the DMPC but not the DHPC 31P signal of the bicelle, indicating that a small percentage of M2(21-61) partitions into the planar region of the bicelles. These results show that the M2 amphipathic helix induces high membrane curvature and localizes the protein to this phase, in excellent agreement with the membrane-scission function of the protein. These bicelle-based relaxation and OMAS solid-state NMR techniques are

  20. An Open Receptor-Binding Cavity of Hemagglutinin-Esterase-Fusion Glycoprotein from Newly-Identified Influenza D Virus: Basis for Its Broad Cell Tropism.

    Directory of Open Access Journals (Sweden)

    Hao Song

    2016-01-01

    Full Text Available Influenza viruses cause seasonal flu each year and pandemics or epidemic sporadically, posing a major threat to public health. Recently, a new influenza D virus (IDV was isolated from pigs and cattle. Here, we reveal that the IDV utilizes 9-O-acetylated sialic acids as its receptor for virus entry. Then, we determined the crystal structures of hemagglutinin-esterase-fusion glycoprotein (HEF of IDV both in its free form and in complex with the receptor and enzymatic substrate analogs. The IDV HEF shows an extremely similar structural fold as the human-infecting influenza C virus (ICV HEF. However, IDV HEF has an open receptor-binding cavity to accommodate diverse extended glycan moieties. This structural difference provides an explanation for the phenomenon that the IDV has a broad cell tropism. As IDV HEF is structurally and functionally similar to ICV HEF, our findings highlight the potential threat of the virus to public health.

  1. Fabrication of Photofunctional Nanoporous Membrane and Its Photoinactivation Effect of Vesicular Stomatitis Virus

    Directory of Open Access Journals (Sweden)

    Kang-Kyun Wang

    2012-01-01

    Full Text Available Fabrication and photophysical study of photofunctional nanoporous alumina membrane (PNAM were performed, and its application of photodynamic antimicrobial chemotherapy (PACT was investigated. Nanoporous alumina membrane (NAM was fabricated by two-step aluminium anodic oxidation process. Surface of the fabricated NAM was modified with organo-silane agent to induce covalent bonding between NAM and a photosensitizer (PtCP: [5,10,15-triphenyl-20-(4-methoxycarbonylphenyl-porphyrin] platinum. PtCP was covalently bonded to the surface of the modified NAM by nucleophilic acyl substitution reaction process. The morphology and the photophysical properties of the fabricated PNAM were confirmed with field emission scanning electron microscope (FE-SEM, steady-state spectroscopies, and nanosecond laser-induced time-resolved spectroscopy. For the efficacy study of PNAM in PACT, an enveloped animal virus, vesicular stomatitis virus (VSV, was utilized as a target organism. Antiviral effect of the PNAM-PACT was measured by the extent of suppression of plaque-forming units (PFU after the light irradiation. In the cultures inoculated with PACT-treated VSV, the suppression of PFU was prominent, which demonstrates that PNAM is a potential bio clean-up tool.

  2. Combined virus-like particle and fusion protein-encoding DNA vaccination of cotton rats induces protection against respiratory syncytial virus without causing vaccine-enhanced disease.

    Science.gov (United States)

    Hwang, Hye Suk; Lee, Young-Tae; Kim, Ki-Hye; Park, Soojin; Kwon, Young-Man; Lee, Youri; Ko, Eun-Ju; Jung, Yu-Jin; Lee, Jong Seok; Kim, Yu-Jin; Lee, Yu-Na; Kim, Min-Chul; Cho, Minkyoung; Kang, Sang-Moo

    2016-07-01

    A safe and effective vaccine against respiratory syncytial virus (RSV) should confer protection without causing vaccine-enhanced disease. Here, using a cotton rat model, we investigated the protective efficacy and safety of an RSV combination vaccine composed of F-encoding plasmid DNA and virus-like particles containing RSV fusion (F) and attachment (G) glycoproteins (FFG-VLP). Cotton rats with FFG-VLP vaccination controlled lung viral replication below the detection limit, and effectively induced neutralizing activity and antibody-secreting cell responses. In comparison with formalin inactivated RSV (FI-RSV) causing severe RSV disease after challenge, FFG-VLP vaccination did not cause weight loss, airway hyper-responsiveness, IL-4 cytokines, histopathology, and infiltrates of proinflammatory cells such as eosinophils. FFG-VLP was even more effective in preventing RSV-induced pulmonary inflammation than live RSV infections. This study provides evidence that FFG-VLP can be developed into a safe and effective RSV vaccine candidate. Copyright © 2016. Published by Elsevier Inc.

  3. Combined virus-like particle and fusion protein-encoding DNA vaccination of cotton rats induces protection against respiratory syncytial virus without causing vaccine-enhanced disease

    Energy Technology Data Exchange (ETDEWEB)

    Hwang, Hye Suk; Lee, Young-Tae; Kim, Ki-Hye; Park, Soojin; Kwon, Young-Man; Lee, Youri; Ko, Eun-Ju; Jung, Yu-Jin [Center for Inflammation, Immunity & Infection, Institute for Biomedical Sciences and Department of Biology, Georgia State University, Atlanta, GA (United States); Lee, Jong Seok [Center for Inflammation, Immunity & Infection, Institute for Biomedical Sciences and Department of Biology, Georgia State University, Atlanta, GA (United States); National Institute of Biological Resources, Incheon (Korea, Republic of); Kim, Yu-Jin [Center for Inflammation, Immunity & Infection, Institute for Biomedical Sciences and Department of Biology, Georgia State University, Atlanta, GA (United States); Lee, Yu-Na; Kim, Min-Chul [Center for Inflammation, Immunity & Infection, Institute for Biomedical Sciences and Department of Biology, Georgia State University, Atlanta, GA (United States); Animal and Plant Quarantine Agency, Gyeonggi-do, Gimcheon, Gyeongsangbukdo (Korea, Republic of); Cho, Minkyoung [Center for Inflammation, Immunity & Infection, Institute for Biomedical Sciences and Department of Biology, Georgia State University, Atlanta, GA (United States); Kang, Sang-Moo, E-mail: skang24@gsu.edu [Center for Inflammation, Immunity & Infection, Institute for Biomedical Sciences and Department of Biology, Georgia State University, Atlanta, GA (United States)

    2016-07-15

    A safe and effective vaccine against respiratory syncytial virus (RSV) should confer protection without causing vaccine-enhanced disease. Here, using a cotton rat model, we investigated the protective efficacy and safety of an RSV combination vaccine composed of F-encoding plasmid DNA and virus-like particles containing RSV fusion (F) and attachment (G) glycoproteins (FFG-VLP). Cotton rats with FFG-VLP vaccination controlled lung viral replication below the detection limit, and effectively induced neutralizing activity and antibody-secreting cell responses. In comparison with formalin inactivated RSV (FI-RSV) causing severe RSV disease after challenge, FFG-VLP vaccination did not cause weight loss, airway hyper-responsiveness, IL-4 cytokines, histopathology, and infiltrates of proinflammatory cells such as eosinophils. FFG-VLP was even more effective in preventing RSV-induced pulmonary inflammation than live RSV infections. This study provides evidence that FFG-VLP can be developed into a safe and effective RSV vaccine candidate. - Highlights: • Combined RSV FFG VLP vaccine is effective in inducing F specific responses. • FFG VLP vaccine confers RSV neutralizing activity and viral control in cotton rats. • Cotton rats with RSV FFG VLP vaccination do not show vaccine-enhanced disease. • Cotton rats with FFG VLP vaccine induce F specific antibody secreting cell responses. • Cotton rats with FFG VLP do not induce lung cellular infiltrates and Th2 cytokine.

  4. INFLUENZA VIRUS AND ITS MUCOPROTEIN SUBSTRATE IN THE CHORIOALLANTOIC MEMBRANE OF THE CHICK EMBRYO

    Science.gov (United States)

    Schlesinger, R. Walter; Karr, H. Valerie

    1956-01-01

    As a preliminary to a study of the fate of mucoprotein substrate in tissues infected with influenza virus, some characteristics of soluble hemagglutination inhibitors (HI) extracted from chorioallantoic membranes (CAM) have been investigated. The inhibitory material was found to be heat-stable, precipitable with cold ethanol, and subject to progressive inactivation by active viruses or by receptor-destroying enzyme (RDE). Certain changes in the slope of titration curves obtained with precipitated and non-precipitated fractions upon alcohol fractionation suggested that the HI in heated CAM extracts was heterogeneous. Alcohol in low concentration precipitated preferentially the more efficient (longer?) HI leaving the "weak" component in the non-precipitated fraction. With higher concentrations of alcobol, "strong" HI were converted to "weak" ones, either by denaturation or as result of reduced solubility. These changes in slope of titration curves were reflected in significant discrepancies when densitometric titers (HI50) of fractions were compared with their titers in pattern tests. The action of active viruses on HI, on the other hand, did not induce qualitative changes in the composition of the inhibitory principle: Titration curves retained parallel slopes even when their position was markedly displaced from that of control curves. In its essential properties, the HI from the CAM appeared to be similar to inhibitors isolated from various other biological sources which have been identified as mucoprotein in nature. Standards for reproducibility of inhibitory titers obtained by the densitometric method of Hirst and Pickels have been presented. The relation of HI to the allantoic epithelium has been analyzed. It has been concluded that the HI is a normal constituent and secretion product of these cells. Under physiological conditions, i.e. in the intact egg, the HI contained in the mucoid outer layer of allantoic cells appears to be protected from enzymatic action

  5. MacB ABC transporter is a dimer whose ATPase activity and macrolide-binding capacity are regulated by the membrane fusion protein MacA.

    Science.gov (United States)

    Lin, Hong Ting; Bavro, Vassiliy N; Barrera, Nelson P; Frankish, Helen M; Velamakanni, Saroj; van Veen, Hendrik W; Robinson, Carol V; Borges-Walmsley, M Inês; Walmsley, Adrian R

    2009-01-09

    Gram-negative bacteria utilize specialized machinery to translocate drugs and protein toxins across the inner and outer membranes, consisting of a tripartite complex composed of an inner membrane secondary or primary active transporter (IMP), a periplasmic membrane fusion protein, and an outer membrane channel. We have investigated the assembly and function of the MacAB/TolC system that confers resistance to macrolides in Escherichia coli. The membrane fusion protein MacA not only stabilizes the tripartite assembly by interacting with both the inner membrane protein MacB and the outer membrane protein TolC, but also has a role in regulating the function of MacB, apparently increasing its affinity for both erythromycin and ATP. Analysis of the kinetic behavior of ATP hydrolysis indicated that MacA promotes and stabilizes the ATP-binding form of the MacB transporter. For the first time, we have established unambiguously the dimeric nature of a noncanonic ABC transporter, MacB that has an N-terminal nucleotide binding domain, by means of nondissociating mass spectrometry, analytical ultracentrifugation, and atomic force microscopy. Structural studies of ABC transporters indicate that ATP is bound between a pair of nucleotide binding domains to stabilize a conformation in which the substrate-binding site is outward-facing. Consequently, our data suggest that in the presence of ATP the same conformation of MacB is promoted and stabilized by MacA. Thus, MacA would facilitate the delivery of drugs by MacB to TolC by enhancing the binding of drugs to it and inducing a conformation of MacB that is primed and competent for binding TolC. Our structural studies are an important first step in understanding how the tripartite complex is assembled.

  6. Data fusion and machine learning to identify threat vectors for the Zika virus and classify vulnerability

    Science.gov (United States)

    Gentle, J. N., Jr.; Kahn, A.; Pierce, S. A.; Wang, S.; Wade, C.; Moran, S.

    2016-12-01

    With the continued spread of the zika virus in the United States in both Florida and Virginia, increased public awareness, prevention and targeted prediction is necessary to effectively mitigate further infection and propagation of the virus throughout the human population. The goal of this project is to utilize publicly accessible data and HPC resources coupled with machine learning algorithms to identify potential threat vectors for the spread of the zika virus in Texas, the United States and globally by correlating available zika case data collected from incident reports in medical databases (e.g., CDC, Florida Department of Health) with known bodies of water in various earth science databases (e.g., USGS NAQWA Data, NASA ASTER Data, TWDB Data) and by using known mosquito population centers as a proxy for trends in population distribution (e.g., WHO, European CDC, Texas Data) while correlating historical trends in the spread of other mosquito borne diseases (e.g., chikungunya, malaria, dengue, yellow fever, west nile, etc.). The resulting analysis should refine the identification of the specific threat vectors for the spread of the virus which will correspondingly increase the effectiveness of the limited resources allocated towards combating the disease through better strategic implementation of defense measures. The minimal outcome of this research is a better understanding of the factors involved in the spread of the zika virus, with the greater potential to save additional lives through more effective resource utilization and public outreach.

  7. Proteins specified by herpes simplex virus. IX. Contiguity of host and viral proteins in the plasma membrane of infected cells.

    Science.gov (United States)

    Heine, J W; Roizman, B

    1973-05-01

    Artificial mixtures of plasma membrane vesicles produced by microcavitation from infected and uninfected cells band at the same density on isopycnic centrifugation in sucrose density gradient. However, after reaction with antiviral antibody, the density of the infected cell plasma membrane vesicles increases, and the infected and uninfected cell membranes are quantitatively separable on isopycnic centrifugation. Plasma membrane vesicles prepared from cells doubly labeled before and after infection with radioactive amino acids and reacted with antibody banded at a high density. Polyacrylamide gel electropherograms show that the vesicles reacted with antibody consist of both host- and virus-specific membrane proteins. Microcavitation does not disrupt viral envelopes since infectivity is not affected by this procedure. We conclude that viral and cellular proteins in the plasma membrane preparations are contiguous.

  8. Lysosome-associated membrane glycoprotein 3 is involved in influenza A virus replication in human lung epithelial (A549 cells

    Directory of Open Access Journals (Sweden)

    Wang Jianwei

    2011-08-01

    Full Text Available Abstract Background Influenza A virus mutates rapidly, rendering antiviral therapies and vaccines directed against virus-encoded targets ineffective. Knowledge of the host factors and molecular pathways exploited by influenza virus will provide further targets for novel antiviral strategies. However, the critical host factors involved in influenza virus infection have not been fully defined. Results We demonstrated that LAMP3, a member of lysosome-associated membrane glycoprotein (LAMP family, was significantly induced in human lung epithelial (A549 cells upon influenza A virus infection. Knockdown of LAMP3 expression by RNA interference attenuated production of viral nucleoprotein (NP as well as virus titers. Confocal microscopy results demonstrated that viral NP is colocalized within LAMP3 positive vesicles at early stages of virus infection. Furthermore, knockdown of LAMP3 expression led to a reduction in nuclear accumulation of viral NP and impeded virus replication. Conclusions LAMP3 is an influenza A virus inducible gene, and plays an important role in viral post-entry steps. Our observations may provide insights into the mechanism of influenza virus replication and potential targets for novel anti-influenza therapeutics.

  9. Synthetic peptides of hepatitis G virus (GBV-C/HGV) in the selection of putative peptide inhibitors of the HIV-1 fusion peptide.

    Science.gov (United States)

    Herrera, Elena; Gomara, Maria J; Mazzini, Stefania; Ragg, Enzio; Haro, Isabel

    2009-05-21

    The GB virus C or hepatitis G virus (GBV-C/HGV) is a single-stranded positive sense RNA virus that belongs to the Flaviviridae family. Recent years have seen the publication of numerous works in which coinfection with GBV-C/HGV and HIV has been associated with slower progression of the illness and a higher survival rate of patients once AIDS has developed. The mechanism by which the GBV-C/HGV virus has a "protective effect" in patients with HIV has still not been defined. Study of the interaction of the GBV-C/HGV and HIV viruses could lead to the development of new therapeutic agents for the treatment of AIDS. Given that the mechanism responsible for the beneficial effect exercised by the GBV-C/HGV virus in the course of HIV infection has not been defined, the present work is intended as a study of the structure and interactions between the fusion peptide of HIV-1, gp41(1-23), and synthetic peptide sequences of the E2 envelope protein of GBV-C/HGV using biophysical techniques. Our results highlight that the E2(269-286) sequence interacts with the target fusion peptide of HIV-1 and modifies its conformation.

  10. African swine fever virus is enveloped by a two-membraned collapsed cisterna derived from the endoplasmic reticulum.

    Science.gov (United States)

    Andrés, G; García-Escudero, R; Simón-Mateo, C; Viñuela, E

    1998-11-01

    During the cytoplasmic maturation of African swine fever virus (ASFV) within the viral factories, the DNA-containing core becomes wrapped by two shells, an inner lipid envelope and an outer icosahedral capsid. We have previously shown that the inner envelope is derived from precursor membrane-like structures on which the capsid layer is progressively assembled. In the present work, we analyzed the origin of these viral membranes and the mechanism of envelopment of ASFV. Electron microscopy studies on permeabilized infected cells revealed the presence of two tightly apposed membranes within the precursor membranous structures as well as polyhedral assembling particles. Both membranes could be detached after digestion of intracellular virions with proteinase K. Importantly, membrane loop structures were observed at the ends of open intermediates, which suggests that the inner envelope is derived from a membrane cisterna. Ultraestructural and immunocytochemical analyses showed a close association and even direct continuities between the endoplasmic reticulum (ER) and assembling virus particles at the bordering areas of the viral factories. Such interactions become evident with an ASFV recombinant that inducibly expresses the major capsid protein p72. In the absence of the inducer, viral morphogenesis was arrested at a stage at which partially and fully collapsed ER cisternae enwrapped the core material. Together, these results indicate that ASFV, like the poxviruses, becomes engulfed by a two-membraned collapsed cisterna derived from the ER.

  11. Recombinant Wild-Type and Edmonston Strain Measles Viruses Bearing Heterologous H Proteins: Role of H Protein in Cell Fusion and Host Cell Specificity

    OpenAIRE

    Takeuchi, Kaoru; Takeda, Makoto; Miyajima, Naoko; Kobune, Fumio; Tanabayashi, Kiyoshi; Tashiro, Masato

    2002-01-01

    Wild-type measles virus (MV) isolated from B95a cells has a restricted host cell specificity and hardly replicates in Vero cells, whereas the laboratory strain Edmonston (Ed) replicates in a variety of cell types including Vero cells. To investigate the role of H protein in the differential MV host cell specificity and cell fusion activity, H proteins of wild-type MV (IC-B) and Ed were coexpressed with the F protein in Vero cells. Cell-cell fusion occurred in Vero cells when Ed H protein, but...

  12. Flagellar membrane fusion and protein exchange in trypanosomes; a new form of cell-cell communication? [version 1; referees: 2 approved

    Directory of Open Access Journals (Sweden)

    Simon Imhof

    2016-04-01

    Full Text Available Diverse structures facilitate direct exchange of proteins between cells, including plasmadesmata in plants and tunnelling nanotubes in bacteria and higher eukaryotes.  Here we describe a new mechanism of protein transfer, flagellar membrane fusion, in the unicellular parasite Trypanosoma brucei. When fluorescently tagged trypanosomes were co-cultured, a small proportion of double-positive cells were observed. The formation of double-positive cells was dependent on the presence of extracellular calcium and was enhanced by placing cells in medium supplemented with fresh bovine serum. Time-lapse microscopy revealed that double-positive cells arose by bidirectional protein exchange in the absence of nuclear transfer.  Furthermore, super-resolution microscopy showed that this process occurred in ≤1 minute, the limit of temporal resolution in these experiments. Both cytoplasmic and membrane proteins could be transferred provided they gained access to the flagellum. Intriguingly, a component of the RNAi machinery (Argonaute was able to move between cells, raising the possibility that small interfering RNAs are transported as cargo. Transmission electron microscopy showed that shared flagella contained two axonemes and two paraflagellar rods bounded by a single membrane. In some cases flagellar fusion was partial and interactions between cells were transient. In other cases fusion occurred along the entire length of the flagellum, was stable for several hours and might be irreversible. Fusion did not appear to be deleterious for cell function: paired cells were motile and could give rise to progeny while fused. The motile flagella of unicellular organisms are related to the sensory cilia of higher eukaryotes, raising the possibility that protein transfer between cells via cilia or flagella occurs more widely in nature.

  13. The African Swine Fever Virus Virion Membrane Protein pE248R Is Required for Virus Infectivity and an Early Postentry Event ▿

    Science.gov (United States)

    Rodríguez, Irene; Nogal, María L.; Redrejo-Rodríguez, Modesto; Bustos, María J.; Salas, María L.

    2009-01-01

    The African swine fever virus (ASFV) protein pE248R, encoded by the gene E248R, is a late structural component of the virus particle. The protein contains intramolecular disulfide bonds and has been previously identified as a substrate of the ASFV-encoded redox system. Its amino acid sequence contains a putative myristoylation site and a hydrophobic transmembrane region near its carboxy terminus. We show here that the protein pE248R is myristoylated during infection and associates with the membrane fraction in infected cells, behaving as an integral membrane protein. Furthermore, the protein localizes at the inner envelope of the virus particles in the cytoplasmic factories. The function of the protein pE248R in ASFV replication was investigated by using a recombinant virus that inducibly expresses the gene E248R. Under repressive conditions, the ASFV polyproteins pp220 and pp62 are normally processed and virus particles with morphology indistinguishable from that of those produced in a wild-type infection or under permissive conditions are generated. Moreover, the mutant virus particles can exit the cell as does the parental virus. However, the infectivity of the pE248R-deficient virions was reduced at least 100-fold. An investigation of the defect of the mutant virus indicated that neither virus binding nor internalization was affected by the absence of the protein pE248R, but a cytopathic effect was not induced and early and late gene expression was impaired, indicating that the protein is required for some early postentry event. PMID:19793823

  14. The African swine fever virus virion membrane protein pE248R is required for virus infectivity and an early postentry event.

    Science.gov (United States)

    Rodríguez, Irene; Nogal, María L; Redrejo-Rodríguez, Modesto; Bustos, María J; Salas, María L

    2009-12-01

    The African swine fever virus (ASFV) protein pE248R, encoded by the gene E248R, is a late structural component of the virus particle. The protein contains intramolecular disulfide bonds and has been previously identified as a substrate of the ASFV-encoded redox system. Its amino acid sequence contains a putative myristoylation site and a hydrophobic transmembrane region near its carboxy terminus. We show here that the protein pE248R is myristoylated during infection and associates with the membrane fraction in infected cells, behaving as an integral membrane protein. Furthermore, the protein localizes at the inner envelope of the virus particles in the cytoplasmic factories. The function of the protein pE248R in ASFV replication was investigated by using a recombinant virus that inducibly expresses the gene E248R. Under repressive conditions, the ASFV polyproteins pp220 and pp62 are normally processed and virus particles with morphology indistinguishable from that of those produced in a wild-type infection or under permissive conditions are generated. Moreover, the mutant virus particles can exit the cell as does the parental virus. However, the infectivity of the pE248R-deficient virions was reduced at least 100-fold. An investigation of the defect of the mutant virus indicated that neither virus binding nor internalization was affected by the absence of the protein pE248R, but a cytopathic effect was not induced and early and late gene expression was impaired, indicating that the protein is required for some early postentry event.

  15. Rab3A is a new interacting partner of synaptotagmin I and may modulate synaptic membrane fusion through a competitive mechanism

    Energy Technology Data Exchange (ETDEWEB)

    Xie, Chunliang [Key Laboratory of Protein Chemistry and Developmental Biology of Ministry of Education, College of Life Sciences, Hunan Normal University, Changsha 410081 (China); Institute of Bast Fiber Crops, Chinese Academy of Agricultural Sciences, Changsha 410205 (China); Li, Jianglin; Guo, Tianyao; Yan, Yizhong; Tang, Cheng; Wang, Ying; Chen, Ping [Key Laboratory of Protein Chemistry and Developmental Biology of Ministry of Education, College of Life Sciences, Hunan Normal University, Changsha 410081 (China); Wang, Xianchun, E-mail: wang_xianchun@263.net [Key Laboratory of Protein Chemistry and Developmental Biology of Ministry of Education, College of Life Sciences, Hunan Normal University, Changsha 410081 (China); Liang, Songping, E-mail: liangsp@hunnu.edu.cn [Key Laboratory of Protein Chemistry and Developmental Biology of Ministry of Education, College of Life Sciences, Hunan Normal University, Changsha 410081 (China)

    2014-02-21

    Highlights: • Rab3A has been found to be a novel interacting protein of synaptotagmin I. • Rab3A binds to synaptotagmin I in a Ca{sup 2+}-independent manner. • KKKK motif in C2B domain of synaptotagmin I is a key site for Rab3A binding. • Rab3A competitively inhibits the binding of C2B in synaptotagmin I to syntaxin 1B. • Rab3A may regulate synaptic membrane fusion and exocytosis in a competitive manner. - Abstract: Rab3 and synaptotagmin have been reported to be the key proteins that have opposite actions but cooperatively play critical regulatory roles in selecting and limiting the number of vesicles released at central synapses. However, the exact mechanism has not been fully understood. In this study, Rab3A and synaptotagmin I, the most abundant isoforms of Rab3 and synaptotagmin, respectively, in brain were for the first time demonstrated to directly interact with each other in a Ca{sup 2+}-independent manner, and the KKKK motif in the C2B domain of synaptotagmin I was a key site for the Rab3A binding, which was further confirmed by the competitive inhibition of inositol hexakisphosphate. Further studies demonstrated that Rab3A competitively affected the synaptotagmin I interaction with syntaxin 1B that was involved in membrane fusion during the synaptic vesicle exocytosis. These data indicate that Rab3A is a new synaptotagmin I interacting partner and may participate in the regulation of synaptic membrane fusion and thus the vesicle exocytosis by competitively modulating the interaction of synaptotagmin with syntaxin of the t-SNARE complex in presynaptic membranes.

  16. Sequence analysis of the Epstein-Barr virus (EBV) latent membrane protein-1 gene and promoter region

    DEFF Research Database (Denmark)

    Sandvej, K; Gratama, J W; Munch, M

    1997-01-01

    Sequence variations in the Epstein-Barr virus (EBV) encoded latent membrane protein-1 (LMP-1) gene have been described in a Chinese nasopharyngeal carcinoma-derived isolate (CAO), and in viral isolates from various EBV-associated tumors. It has been suggested that these genetic changes, which...

  17. Cell fusion and nuclear fusion in plants.

    Science.gov (United States)

    Maruyama, Daisuke; Ohtsu, Mina; Higashiyama, Tetsuya

    2016-12-01

    Eukaryotic cells are surrounded by a plasma membrane and have a large nucleus containing the genomic DNA, which is enclosed by a nuclear envelope consisting of the outer and inner nuclear membranes. Although these membranes maintain the identity of cells, they sometimes fuse to each other, such as to produce a zygote during sexual reproduction or to give rise to other characteristically polyploid tissues. Recent studies have demonstrated that the mechanisms of plasma membrane or nuclear membrane fusion in plants are shared to some extent with those of yeasts and animals, despite the unique features of plant cells including thick cell walls and intercellular connections. Here, we summarize the key factors in the fusion of these membranes during plant reproduction, and also focus on "non-gametic cell fusion," which was thought to be rare in plant tissue, in which each cell is separated by a cell wall. Copyright © 2016 Elsevier Ltd. All rights reserved.

  18. Inferior immunogenicity and efficacy of respiratory syncytial virus fusion protein-based subunit vaccine candidates in aged versus young mice.

    Directory of Open Access Journals (Sweden)

    Corinne Cayatte

    Full Text Available Respiratory syncytial virus (RSV is recognized as an important cause of lower and upper respiratory tract infections in older adults, and a successful vaccine would substantially lower morbidity and mortality in this age group. Recently, two vaccine candidates based on soluble purified glycoprotein F (RSV F, either alone or adjuvanted with glucopyranosyl lipid A formulated in a stable emulsion (GLA-SE, failed to reach their primary endpoints in clinical efficacy studies, despite demonstrating the desired immunogenicity profile and efficacy in young rodent models. Here, one of the RSV F vaccine candidates (post-fusion conformation, RSV post-F, and a stabilized pre-fusion form of RSV F (RSV pre-F, DS-Cav1 were evaluated in aged BALB/c mice. Humoral and cellular immunogenicity elicited after immunization of naïve, aged mice was generally lower compared to young animals. In aged mice, RSV post-F vaccination without adjuvant poorly protected the respiratory tract from virus replication, and addition of GLA-SE only improved protection in the lungs, but not in nasal turbinates. RSV pre-F induced higher neutralizing antibody titers compared to RSV post-F (as previously reported but interestingly, RSV F-specific CD8 T cell responses were lower compared to RSV post-F responses regardless of age. The vaccines were also tested in RSV seropositive aged mice, in which both antigen forms similarly boosted neutralizing antibody titers, although GLA-SE addition boosted neutralizing activity only in RSV pre-F immunized animals. Cell-mediated immune responses in the aged mice were only slightly boosted and well below levels induced in seronegative young mice. Taken together, the findings suggest that the vaccine candidates were not able to induce a strong anti-RSV immune response in recipient mice with an aged immune system, in agreement with recent human clinical trial results. Therefore, the aged mouse model could be a useful tool to evaluate improved vaccine

  19. Membraner

    DEFF Research Database (Denmark)

    Bach, Finn

    2009-01-01

    Notatet giver en kort introduktion til den statiske virkemåde af membraner og membrankonstruktioner......Notatet giver en kort introduktion til den statiske virkemåde af membraner og membrankonstruktioner...

  20. Virus removal retention challenge tests performed at lab scale and pilot scale during operation of membrane units.

    Science.gov (United States)

    Humbert, H; Machinal, C; Labaye, Ivan; Schrotter, J C

    2011-01-01

    The determination of the virus retention capabilities of UF units during operation is essential for the operators of drinking water treatment facilities in order to guarantee an efficient and stable removal of viruses through time. In previous studies, an effective method (MS2-phage challenge tests) was developed by the Water Research Center of Veolia Environnement for the measurement of the virus retention rates (Log Removal Rate, LRV) of commercially available hollow fiber membranes at lab scale. In the present work, the protocol for monitoring membrane performance was transferred from lab scale to pilot scale. Membrane performances were evaluated during pilot trial and compared to the results obtained at lab scale with fibers taken from the pilot plant modules. PFU culture method was compared to RT-PCR method for the calculation of LRV in both cases. Preliminary tests at lab scale showed that both methods can be used interchangeably. For tests conducted on virgin membrane, a good consistency was observed between lab and pilot scale results with the two analytical methods used. This work intends to show that a reliable determination of the membranes performances based on RT-PCR analytical method can be achieved during the operation of the UF units.

  1. Studies on factors affecting herpesvirus-induced cell fusion. Progress report, July 1, 1980-June 30, 1981

    Energy Technology Data Exchange (ETDEWEB)

    Person, S.

    1981-06-01

    The difference in the fusion sensitivity of HEL and HEp-2 cells was shown not to be due to receptors for fusion factor on the surfaces of the infected cells. Fusion inhibition of syn mutant-infected cells by nonsyncytial-infected cells was shown to require the synthesis of virus glycoproteins up to about 6 h after infection. The DNA sequences required for virus entry have been identified. Cell fractionation studies have revealed a slower accumulation of fully glycosylated glycoproteins in the smooth membranes of syncytial-infected cells relative to those of nonsyncytial-infected cells.

  2. Remorin, a Solanaceae Protein Resident in Membrane Rafts and Plasmodesmata, Impairs Potato virus X Movement[W

    Science.gov (United States)

    Raffaele, Sylvain; Bayer, Emmanuelle; Lafarge, David; Cluzet, Stéphanie; German Retana, Sylvie; Boubekeur, Tamy; Leborgne-Castel, Nathalie; Carde, Jean-Pierre; Lherminier, Jeannine; Noirot, Elodie; Satiat-Jeunemaître, Béatrice; Laroche-Traineau, Jeanny; Moreau, Patrick; Ott, Thomas; Maule, Andrew J.; Reymond, Philippe; Simon-Plas, Françoise; Farmer, Edward E.; Bessoule, Jean-Jacques; Mongrand, Sébastien

    2009-01-01

    Remorins (REMs) are proteins of unknown function specific to vascular plants. We have used imaging and biochemical approaches and in situ labeling to demonstrate that REM clusters at plasmodesmata and in ∼70-nm membrane domains, similar to lipid rafts, in the cytosolic leaflet of the plasma membrane. From a manipulation of REM levels in transgenic tomato (Solanum lycopersicum) plants, we show that Potato virus X (PVX) movement is inversely related to REM accumulation. We show that REM can interact physically with the movement protein TRIPLE GENE BLOCK PROTEIN1 from PVX. Based on the localization of REM and its impact on virus macromolecular trafficking, we discuss the potential for lipid rafts to act as functional components in plasmodesmata and the plasma membrane. PMID:19470590

  3. Targeting Membrane-Bound Viral RNA Synthesis Reveals Potent Inhibition of Diverse Coronaviruses Including the Middle East Respiratory Syndrome Virus

    Science.gov (United States)

    Bergström, Tomas; Kann, Nina; Adamiak, Beata; Hannoun, Charles; Kindler, Eveline; Jónsdóttir, Hulda R.; Muth, Doreen; Kint, Joeri; Forlenza, Maria; Müller, Marcel A.; Drosten, Christian; Thiel, Volker; Trybala, Edward

    2014-01-01

    Coronaviruses raise serious concerns as emerging zoonotic viruses without specific antiviral drugs available. Here we screened a collection of 16671 diverse compounds for anti-human coronavirus 229E activity and identified an inhibitor, designated K22, that specifically targets membrane-bound coronaviral RNA synthesis. K22 exerts most potent antiviral activity after virus entry during an early step of the viral life cycle. Specifically, the formation of double membrane vesicles (DMVs), a hallmark of coronavirus replication, was greatly impaired upon K22 treatment accompanied by near-complete inhibition of viral RNA synthesis. K22-resistant viruses contained substitutions in non-structural protein 6 (nsp6), a membrane-spanning integral component of the viral replication complex implicated in DMV formation, corroborating that K22 targets membrane bound viral RNA synthesis. Besides K22 resistance, the nsp6 mutants induced a reduced number of DMVs, displayed decreased specific infectivity, while RNA synthesis was not affected. Importantly, K22 inhibits a broad range of coronaviruses, including Middle East respiratory syndrome coronavirus (MERS–CoV), and efficient inhibition was achieved in primary human epithelia cultures representing the entry port of human coronavirus infection. Collectively, this study proposes an evolutionary conserved step in the life cycle of positive-stranded RNA viruses, the recruitment of cellular membranes for viral replication, as vulnerable and, most importantly, druggable target for antiviral intervention. We expect this mode of action to serve as a paradigm for the development of potent antiviral drugs to combat many animal and human virus infections. PMID:24874215

  4. Reconstituted Fusion Pore

    OpenAIRE

    Jeremic, Aleksandar; Kelly, Marie; Cho, Sang-Joon; Stromer, Marvin H.; Jena, Bhanu P.

    2003-01-01

    Fusion pores or porosomes are basket-like structures at the cell plasma membrane, at the base of which, membrane-bound secretory vesicles dock and fuse to release vesicular contents. Earlier studies using atomic force microscopy (AFM) demonstrated the presence of fusion pores at the cell plasma membrane in a number of live secretory cells, revealing their morphology and dynamics at nm resolution and in real time. ImmunoAFM studies demonstrated the release of vesicular contents through the por...

  5. Poor immune responses of newborn rhesus macaques to measles virus DNA vaccines expressing the hemagglutinin and fusion glycoproteins.

    Science.gov (United States)

    Polack, Fernando P; Lydy, Shari L; Lee, Sok-Hyong; Rota, Paul A; Bellini, William J; Adams, Robert J; Robinson, Harriet L; Griffin, Diane E

    2013-02-01

    A vaccine that would protect young infants against measles could facilitate elimination efforts and decrease morbidity and mortality in developing countries. However, immaturity of the immune system is an important obstacle to the development of such a vaccine. In this study, DNA vaccines expressing the measles virus (MeV) hemagglutinin (H) protein or H and fusion (F) proteins, previously shown to protect juvenile macaques, were used to immunize groups of 4 newborn rhesus macaques. Monkeys were inoculated intradermally with 200 μg of each DNA at birth and at 10 months of age. As controls, 2 newborn macaques were similarly vaccinated with DNA encoding the influenza virus H5, and 4 received one dose of the current live attenuated MeV vaccine (LAV) intramuscularly. All monkeys were monitored for development of MeV-specific neutralizing and binding IgG antibody and cytotoxic T lymphocyte (CTL) responses. These responses were poor compared to the responses induced by LAV. At 18 months of age, all monkeys were challenged intratracheally with a wild-type strain of MeV. Monkeys that received the DNA vaccine encoding H and F, but not H alone, were primed for an MeV-specific CD8(+) CTL response but not for production of antibody. LAV-vaccinated monkeys were protected from rash and viremia, while DNA-vaccinated monkeys developed rashes, similar to control monkeys, but had 10-fold lower levels of viremia. We conclude that vaccination of infant macaques with DNA encoding MeV H and F provided only partial protection from MeV infection.

  6. Specific detection of dengue and Zika virus antibodies using envelope proteins with mutations in the conserved fusion loop.

    Science.gov (United States)

    Rockstroh, Alexandra; Moges, Beyene; Barzon, Luisa; Sinigaglia, Alessandro; Palù, Giorgio; Kumbukgolla, Widuranga; Schmidt-Chanasit, Jonas; Sarno, Manoel; Brites, Carlos; Moreira-Soto, Andres; Drexler, Jan Felix; Ferreira, Orlando C; Ulbert, Sebastian

    2017-11-08

    Detection of antibodies is widely used for the diagnosis of infections with arthropod-borne flaviviruses including dengue (DENV) and Zika virus (ZIKV). Due to the emergence of ZIKV in areas endemic for DENV, massive co-circulation is observed and methods to specifically diagnose these infections and differentiate them from each other are mandatory. However, serological assays for flaviviruses in general, and for DENV and ZIKV in particular, are compromised by the high degree of similarities in their proteins which can lead to cross-reacting antibodies and false-positive test results. Cross-reacting flavivirus antibodies mainly target the highly conserved fusion loop (FL) domain in the viral envelope (E-) protein, and we and others have shown previously that recombinant E-proteins bearing FL-mutations strongly reduce cross-reactivity. Here we investigate whether such mutant E-proteins can be used to specifically detect antibodies against DENV and ZIKV in an ELISA-format. IgM antibodies against DENV and ZIKV virus were detected with 100% and 94.2% specificity and 90.7% and 87.5% sensitivity, respectively. For IgG the mutant E-proteins showed cross-reactivity, which was overcome by pre-incubation of the sera with the heterologous antigen. This resulted in specificities of 97.1% and 97.9% and in sensitivities of 100% and 100% for the DENV and ZIKV antigens, respectively. Our results suggest that E-proteins bearing mutations in the FL-domain have a high potential for the development of serological DENV and ZIKV tests with high specificity.

  7. Expression and Immunogenicity of Two Recombinant Fusion Proteins Comprising Foot-and-Mouth Disease Virus Structural Protein VP1 and DC-SIGN-Binding Glycoproteins

    OpenAIRE

    Xinsheng Liu; Jianliang Lv; Yuzhen Fang; Peng Zhou; Yanzhen Lu; Li Pan; Zhongwang Zhang; Junwu Ma; Yongguang Zhang; Yonglu Wang

    2017-01-01

    Improving vaccine immunogenicity by targeting antigens to dendritic cells has recently emerged as a new design strategy in vaccine development. In this study, the VP1 gene of foot-and-mouth disease virus (FMDV) serotype A was fused with the gene encoding human immunodeficiency virus (HIV) membrane glycoprotein gp120 or C2-V3 domain of hepatitis C virus (HCV) envelope glycoprotein E2, both of which are DC-SIGN-binding glycoproteins. After codon optimization, the VP1 protein and the two recombi...

  8. Intercellular adhesion molecule 1 promotes HIV-1 attachment but not fusion to target cells.

    Directory of Open Access Journals (Sweden)

    Naoyuki Kondo

    Full Text Available Incorporation of intercellular adhesion molecule 1 (ICAM-1 into HIV-1 particles is known to markedly enhance the virus binding and infection of cells expressing lymphocyte function-associated antigen-1 (LFA-1. At the same time, ICAM-1 has been reported to exert a less pronounced effect on HIV-1 fusion with lymphoid cells. Here we examined the role of ICAM-1/LFA-1 interactions in productive HIV-1 entry into lymphoid cells using a direct virus-cell fusion assay. ICAM-1 promoted HIV-1 attachment to cells in a temperature-dependent manner. It exerted a marginal effect on virus binding in the cold, but enhanced binding up to 4-fold at physiological temperature. ICAM-1-independent attachment in the cold was readily reversible upon subsequent incubation at elevated temperature, whereas ICAM-1-bearing particles were largely retained by cells. The better virus retention resulted in a proportional increase in HIV-1 internalization and fusion, suggesting that ICAM-1 did not specifically accelerate endocytosis or fusion steps. We also measured the rates of CD4 engagement, productive endocytosis and HIV-endosome fusion using specific fusion inhibitors. These rates were virtually independent of the presence of ICAM-1 in viral particles. Importantly, irrespective of the presence of ICAM-1, HIV-1 escaped from the low temperature block, which stopped virus endocytosis and fusion, much later than from a membrane-impermeant fusion inhibitor targeting surface-accessible particles. This result, along with the complete inhibition of HIV-1 fusion by a small molecule dynamin inhibitor, implies this virus enters lymphoid cells used in this study via endocytosis and that this pathway is not altered by the viral ICAM-1. Our data highlight the role of ICAM-1 in stabilizing the HIV-1 attachment to LFA-1 expressing cells, which leads to a proportional enhancement of the receptor-mediated uptake and fusion with endosomes.

  9. Attenuated Human Parainfluenza Virus Type 1 (HPIV1) Expressing the Fusion Glycoprotein of Human Respiratory Syncytial Virus (RSV) as a Bivalent HPIV1/RSV Vaccine

    Science.gov (United States)

    Mackow, Natalie; Amaro-Carambot, Emérito; Liang, Bo; Surman, Sonja; Lingemann, Matthias; Yang, Lijuan; Collins, Peter L.

    2015-01-01

    ABSTRACT Live attenuated recombinant human parainfluenza virus type 1 (rHPIV1) was investigated as a vector to express the respiratory syncytial virus (RSV) fusion (F) glycoprotein, to provide a bivalent vaccine against RSV and HPIV1. The RSV F gene was engineered to include HPIV1 transcription signals and inserted individually into three gene locations in each of the two attenuated rHPIV1 backbones. Each backbone contained a single previously described attenuating mutation that was stabilized against deattenuation, specifically, a non-temperature-sensitive deletion mutation involving 6 nucleotides in the overlapping P/C open reading frames (ORFs) (CΔ170) or a temperature-sensitive missense mutation in the L ORF (LY942A). The insertion sites in the genome were pre-N (F1), N-P (F2), or P-M (F3) and were identical for both backbones. In vitro, the presence of the F insert reduced the rate of virus replication, but the final titers were the same as the final titer of wild-type (wt) HPIV1. High levels of RSV F expression in cultured cells were observed with rHPIV1-CΔ170-F1, -F2, and -F3 and rHPIV1-LY942A-F1. In hamsters, the rHPIV1-CΔ170-F1, -F2, and -F3 vectors were moderately restricted in the nasal turbinates, highly restricted in lungs, and genetically stable in vivo. Among the CΔ170 vectors, the F1 virus was the most immunogenic and protective against wt RSV challenge. The rHPIV1-LY942A vectors were highly restricted in vivo and were not detectably immunogenic or protective, indicative of overattenuation. The CΔ170-F1 construct appears to be suitably attenuated and immunogenic for further development as a bivalent intranasal pediatric vaccine. IMPORTANCE There are no vaccines for the pediatric respiratory pathogens RSV and HPIV. We are developing live attenuated RSV and HPIV vaccines for use in virus-naive infants. Live attenuated RSV strains in particular are difficult to develop due to their poor growth and physical instability, but these obstacles could be

  10. Hepatitis C virus suppresses C9 complement synthesis and impairs membrane attack complex function.

    Science.gov (United States)

    Kim, Hangeun; Meyer, Keith; Di Bisceglie, Adrian M; Ray, Ranjit

    2013-05-01

    Hepatitis C virus (HCV) proteins inhibit complement component expression, which may attenuate immunity against infection. In this study, we examined whether HCV regulates the membrane attack complex (MAC) via complement component C9. MAC is composed of C5b to C9 (C5b-9) and mediates cell lysis of invaded pathogens. Liver biopsy specimens from chronically HCV-infected patients exhibited a lower level of C9 mRNA expression than liver biopsy specimens from unrelated disease or healthy control human liver RNA. Hepatocytes infected with cell culture-grown HCV or expressing HCV core protein also displayed significant repression of C9 mRNA and protein levels. Promoter analysis suggested that the T cell factor-4 (TCF-4E) transcription factor is responsible for HCV core-mediated C9 promoter regulation. Sera from chronically HCV-infected patients displayed a lower level of C5b-9 and a reduced antimicrobial effect on model organisms compared to unrelated patient sera or sera from healthy volunteers. Together, these results for C9 regulation by HCV core protein coupled with functional impairment of the membrane attack complex underscore HCV-mediated attenuation of immune mechanisms.

  11. The yeast vacuolar Rab GTPase Ypt7p has an activity beyond membrane recruitment of the homotypic fusion and protein sorting-Class C Vps complex.

    Science.gov (United States)

    Stroupe, Christopher

    2012-04-01

    A previous report described lipid mixing of reconstituted proteoliposomes made using lipid mixtures that mimic the composition of yeast vacuoles. This lipid mixing required SNARE {SNAP [soluble NSF (N-ethylmaleimide-sensitive factor)-attachment protein] receptor} proteins, Sec18p and Sec17p (yeast NSF and α-SNAP) and the HOPS (homotypic fusion and protein sorting)-Class C Vps (vacuole protein sorting) complex, but not the vacuolar Rab GTPase Ypt7p. The present study investigates the activity of Ypt7p in proteoliposome lipid mixing. Ypt7p is required for the lipid mixing of proteoliposomes lacking cardiolipin [1,3-bis-(sn-3'-phosphatidyl)-sn-glycerol]. Omission of other lipids with negatively charged and/or small head groups does not cause Ypt7p dependence for lipid mixing. Yeast vacuoles made from strains disrupted for CRD1 (cardiolipin synthase) fuse to the same extent as vacuoles from strains with functional CRD1. Disruption of CRD1 does not alter dependence on Rab GTPases for vacuole fusion. It has been proposed that the recruitment of the HOPS complex to membranes is the main function of Ypt7p. However, Ypt7p is still required for lipid mixing even when the concentration of HOPS complex in lipid-mixing reactions is adjusted such that cardiolipin-free proteoliposomes with or without Ypt7p bind to equal amounts of HOPS. Ypt7p therefore must stimulate membrane fusion by a mechanism that is in addition to recruitment of HOPS to the membrane. This is the first demonstration of such a stimulatory activity--that is, beyond bulk effector recruitment--for a Rab GTPase.

  12. Resistance of Human Immunodeficiency Virus Type 1 to a Third-Generation Fusion Inhibitor Requires Multiple Mutations in gp41 and Is Accompanied by a Dramatic Loss of gp41 Function

    NARCIS (Netherlands)

    Eggink, Dirk; Bontjer, Ilja; Langedijk, Johannes P. M.; Berkhout, Ben; Sanders, Rogier W.

    2011-01-01

    HIV-1 entry into target cells requires the fusion of viral and cellular membranes. This process is an attractive target for therapeutic intervention, and a first-generation fusion inhibitor, T20 (Enfuvirtide; Fuzeon), was approved for clinical use in 2003. Second-generation (T1249) and

  13. IFITM proteins incorporated into HIV-1 virions impair viral fusion and spread.

    Science.gov (United States)

    Compton, Alex A; Bruel, Timothée; Porrot, Françoise; Mallet, Adeline; Sachse, Martin; Euvrard, Marine; Liang, Chen; Casartelli, Nicoletta; Schwartz, Olivier

    2014-12-10

    The interferon-induced transmembrane (IFITM) proteins protect cells from diverse virus infections by inhibiting virus-cell fusion. IFITM proteins also inhibit HIV-1 replication through mechanisms only partially understood. We show that when expressed in uninfected lymphocytes, IFITM proteins exert protective effects during cell-free virus infection, but this restriction can be overcome upon HIV-1 cell-to-cell spread. However, when present in virus-producing lymphocytes, IFITM proteins colocalize with viral Env and Gag proteins and incorporate into nascent HIV-1 virions to limit entry into new target cells. IFITM in viral membranes is associated with impaired virion fusion, offering additional and more potent defense against virus spread. Thus, IFITM proteins act additively in both productively infected cells and uninfected target cells to inhibit HIV-1 spread, potentially conferring these proteins with greater breadth and potency against enveloped viruses. Copyright © 2014 Elsevier Inc. All rights reserved.

  14. Engineering of papaya mosaic virus (PapMV nanoparticles through fusion of the HA11 peptide to several putative surface-exposed sites.

    Directory of Open Access Journals (Sweden)

    Gervais Rioux

    Full Text Available Papaya mosaic virus has been shown to be an efficient adjuvant and vaccine platform in the design and improvement of innovative flu vaccines. So far, all fusions based on the PapMV platform have been located at the C-terminus of the PapMV coat protein. Considering that some epitopes might interfere with the self-assembly of PapMV CP when fused at the C-terminus, we evaluated other possible sites of fusion using the influenza HA11 peptide antigen. Two out of the six new fusion sites tested led to the production of recombinant proteins capable of self assembly into PapMV nanoparticles; the two functional sites are located after amino acids 12 and 187. Immunoprecipitation of each of the successful fusions demonstrated that the HA11 epitope was located at the surface of the nanoparticles. The stability and immunogenicity of the PapMV-HA11 nanoparticles were evaluated, and we could show that there is a direct correlation between the stability of the nanoparticles at 37°C (mammalian body temperature and the ability of the nanoparticles to trigger an efficient immune response directed towards the HA11 epitope. This strong correlation between nanoparticle stability and immunogenicity in animals suggests that the stability of any nanoparticle harbouring the fusion of a new peptide should be an important criterion in the design of a new vaccine.

  15. Structural basis for immunization with postfusion respiratory syncytial virus fusion F glycoprotein (RSV F) to elicit high neutralizing antibody titers

    Energy Technology Data Exchange (ETDEWEB)

    Swanson, Kurt A.; Settembre, Ethan C.; Shaw, Christine A.; Dey, Antu K.; Rappuoli, Rino; Mandl, Christian W.; Dormitzer, Philip R.; Carfi, Andrea (Novartis)

    2012-02-07

    Respiratory syncytial virus (RSV), the main cause of infant bronchiolitis, remains a major unmet vaccine need despite more than 40 years of vaccine research. Vaccine candidates based on a chief RSV neutralization antigen, the fusion (F) glycoprotein, have foundered due to problems with stability, purity, reproducibility, and potency. Crystal structures of related parainfluenza F glycoproteins have revealed a large conformational change between the prefusion and postfusion states, suggesting that postfusion F antigens might not efficiently elicit neutralizing antibodies. We have generated a homogeneous, stable, and reproducible postfusion RSV F immunogen that elicits high titers of neutralizing antibodies in immunized animals. The 3.2-{angstrom} X-ray crystal structure of this substantially complete RSV F reveals important differences from homology-based structural models. Specifically, the RSV F crystal structure demonstrates the exposure of key neutralizing antibody binding sites on the surface of the postfusion RSV F trimer. This unanticipated structural feature explains the engineered RSV F antigen's efficiency as an immunogen. This work illustrates how structural-based antigen design can guide the rational optimization of candidate vaccine antigens.

  16. Human Immunodeficiency Virus Type 1 Nef protein modulates the lipid composition of virions and host cell membrane microdomains

    Directory of Open Access Journals (Sweden)

    Geyer Matthias

    2007-10-01

    Full Text Available Abstract Background The Nef protein of Human Immunodeficiency Viruses optimizes viral spread in the infected host by manipulating cellular transport and signal transduction machineries. Nef also boosts the infectivity of HIV particles by an unknown mechanism. Recent studies suggested a correlation between the association of Nef with lipid raft microdomains and its positive effects on virion infectivity. Furthermore, the lipidome analysis of HIV-1 particles revealed a marked enrichment of classical raft lipids and thus identified HIV-1 virions as an example for naturally occurring membrane microdomains. Since Nef modulates the protein composition and function of membrane microdomains we tested here if Nef also has the propensity to alter microdomain lipid composition. Results Quantitative mass spectrometric lipidome analysis of highly purified HIV-1 particles revealed that the presence of Nef during virus production from T lymphocytes enforced their raft character via a significant reduction of polyunsaturated phosphatidylcholine species and a specific enrichment of sphingomyelin. In contrast, Nef did not significantly affect virion levels of phosphoglycerolipids or cholesterol. The observed alterations in virion lipid composition were insufficient to mediate Nef's effect on particle infectivity and Nef augmented virion infectivity independently of whether virus entry was targeted to or excluded from membrane microdomains. However, altered lipid compositions similar to those observed in virions were also detected in detergent-resistant membrane preparations of virus producing cells. Conclusion Nef alters not only the proteome but also the lipid composition of host cell microdomains. This novel activity represents a previously unrecognized mechanism by which Nef could manipulate HIV-1 target cells to facilitate virus propagation in vivo.

  17. MEPPitope: spatial, electrostatic and secondary structure perturbations in the post-fusion Dengue virus envelope protein highlights known epitopes and conserved residues in the Zika virus [version 1; referees: 3 approved

    Directory of Open Access Journals (Sweden)

    Sandeep Chakraborty

    2016-06-01

    Full Text Available The dramatic transformation of the Zika virus (ZIKV from a relatively unknown virus to a pathogen generating global-wide panic has exposed the dearth of detailed knowledge about this virus. Decades of research in the related Dengue virus (DENV, finally culminating in a vaccine registered for use in endemic regions (CYD-TDV, provides key insights in developing strategies for tackling ZIKV. The previously established MEPP methodology compares two conformations of the same protein and identifies residues with significant spatial and electrostatic perturbations. In the current work, MEPP analyzed the pre-and post-fusion DENV type 2 envelope (E protein, and identified several known epitopes (His317, Tyr299, Glu26, Arg188, etc. (MEPPitope. These residues are overwhelmingly conserved in ZIKV and all DENV serotypes. Characterization of α-helices in E-proteins show that α1 is not conserved in the sequence space of ZIKV and DENV. Furthermore, perturbation of α1 in the post-fusion DENV structure includes a known epitope Asp215, a residue absent in the pre-fusion α1. A cationic β-sheet in the GAG-binding domain that is stereochemically equivalent in ZIKV and all DENV serotypes is also highlighted due to a residue pair (Arg286-Arg288 that has a significant electrostatic polarity reversal upon fusion. Finally, two highly conserved residues (Thr32 and Thr40, with little emphasis in existing literature, are found to have significant electrostatic perturbation. Thus, a combination of different computational methods enable the rapid and rational detection of critical residues that can be made the target of small drugs, or as epitopes in the search for an elusive therapy or vaccine that neutralizes multiple members of the Flaviviridae family.

  18. Measurement of antibodies to varicella-zoster virus using a virus-free fluorescent-antibody-to-membrane-antigen (FAMA) test.

    Science.gov (United States)

    Park, Rackhyun; Hwang, Ji Young; Lee, Kang Il; Namkoong, Sim; Choi, Seuk-Keun; Park, Songyong; Park, Hosun; Park, Junsoo

    2015-02-01

    The fluorescent-antibody-to-membrane-antigen (FAMA) test is regarded as the "gold standard" to detect protective antibodies to varicella-zoster virus (VZV) because of its high sensitivity and specificity. Because the classic FAMA test uses an infectious virus for detection of antibodies to VZV, it is labor-intensive, and also requires special equipment for handling the virus. For this reason, we attempted to develop a simple and safe FAMA assay. Because VZV glycoprotein E (gE) is one of the major VZV glycoproteins, we used the gE protein for the FAMA test (gE FAMA). Here, we demonstrate that overexpression of gE in HEK293T cells can be used to measure antibodies in human serum, and that gE FAMA titers are closely correlated with gpEIA ELISA data. These results indicate that our gE FAMA test has the potential to measure antibodies to VZV.

  19. Morphogenesis and release of fowlpox virus.

    Science.gov (United States)

    Boulanger, D; Smith, T; Skinner, M A

    2000-03-01

    Release of fowlpox virus (FWPV) as extracellular enveloped virus (EEV) appears to proceed both by the budding of intracellular mature virus (IMV) through the plasma membrane and by the fusion of intracellular enveloped virus (IEV) with the plasma membrane. Based on the frequency of budding events compared to wrapping events observed by electron microscopy, FWPV FP9 strain seems to exit chick embryo fibroblast cells predominantly by budding. In contrast to vaccinia virus (VV), the production of FWPV extracellular virus particles is not affected by N(1)-isonicotinoyl-N(2)-3-methyl-4-chlorobenzoylhydrazine (IMCBH). Comparison of the sequence of the VV F13L gene product with its FWPV orthologue showed a mutation, in the fowlpox protein, at the residue involved in IMCBH resistance in a mutant VV. Glucosamine, monensin or brefeldin A did not have any specific effect on FWPV extracellular virus production. Cytochalasin D, which inhibits the formation of actin filaments, reduces the production of extracellular virus particles by inhibiting the release of cell-associated enveloped virus (CEV) particles from the plasma membrane. Involvement of actin filaments in this mechanism is further supported by the co-localization of actin with viral particles close to the plasma membrane in the absence of cytochalasin D. Actin is also co-localized with virus factories.

  20. The Role of the MHV Receptor and Related Glycoproteins in Murine Hepatitis Virus Infection of Murine Cell Lines

    Science.gov (United States)

    1995-04-13

    colipids, as does that of bovine coronavirus (Vlasak, et al ., 1988) . The interaction of HE protein wi th its cellular l igand is not sufficient to...glycoprotein of human parainfluenza virus type 3 is required for virus spread and virus -mediated membrane fusion (Moscona, et al., 1992). The relative...affinity for sialic acid of the hemagglutinin- neuraminidase glycoproteins of different parainfluenza virus type 3 variants correlates with their

  1. Fate of the inner nuclear membrane protein lamin B receptor and nuclear lamins in herpes simplex virus type 1 infection.

    Science.gov (United States)

    Scott, E S; O'Hare, P

    2001-09-01

    During herpesvirus egress, capsids bud through the inner nuclear membrane. Underlying this membrane is the nuclear lamina, a meshwork of intermediate filaments with which it is tightly associated. Details of alterations to the lamina and the inner nuclear membrane during infection and the mechanisms involved in capsid transport across these structures remain unclear. Here we describe the fate of key protein components of the nuclear envelope and lamina during herpes simplex virus type 1 (HSV-1) infection. We followed the distribution of the inner nuclear membrane protein lamin B receptor (LBR) and lamins A and B(2) tagged with green fluorescent protein (GFP) in live infected cells. Together with additional results from indirect immunofluorescence, our studies reveal major morphologic distortion of nuclear-rim LBR and lamins A/C, B(1), and B(2). By 8 h p.i., we also observed a significant redistribution of LBR-GFP to the endoplasmic reticulum, where it colocalized with a subpopulation of cytoplasmic glycoprotein B by immunofluorescence. In addition, analysis by fluorescence recovery after photobleaching reveals that LBR-GFP exhibited increased diffusional mobility within the nuclear membrane of infected cells. This is consistent with the disruption of interactions between LBR and the underlying lamina. In addition to studying stably expressed GFP-lamins by fluorescence microscopy, we studied endogenous A- and B-type lamins in infected cells by Western blotting. Both approaches reveal a loss of lamins associated with virus infection. These data indicate major disruption of the nuclear envelope and lamina of HSV-1-infected cells and are consistent with a virus-induced dismantling of the nuclear lamina, possibly in order to gain access to the inner nuclear membrane.

  2. [Incorporation of glycoproteins of the Aujeszky's disease virus ( Suid herpesvirus 1) into artificial liposome membranes and their interaction with cells].

    Science.gov (United States)

    Vrublevskaia, V V; Vinokurov, M G; Kholodkov, O A; Kornev, A N; Morenkov, O S

    2004-01-01

    The purpose of the case study was to investigate the interplay between liposomes, containing the in-built glycoproteins of the Aujeszky disease