Recruitment of a SAP18-HDAC1 complex into HIV-1 virions and its requirement for viral replication.

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Masha Sorin

2009-06-01

Full Text Available HIV-1 integrase (IN is a virally encoded protein required for integration of viral cDNA into host chromosomes. INI1/hSNF5 is a component of the SWI/SNF complex that interacts with HIV-1 IN, is selectively incorporated into HIV-1 (but not other retroviral virions, and modulates multiple steps, including particle production and infectivity. To gain further insight into the role of INI1 in HIV-1 replication, we screened for INI1-interacting proteins using the yeast two-hybrid system. We found that SAP18 (Sin3a associated protein 18 kD, a component of the Sin3a-HDAC1 complex, directly binds to INI1 in yeast, in vitro and in vivo. Interestingly, we found that IN also binds to SAP18 in vitro and in vivo. SAP18 and components of a Sin3A-HDAC1 complex were specifically incorporated into HIV-1 (but not SIV and HTLV-1 virions in an HIV-1 IN-dependent manner. Using a fluorescence-based assay, we found that HIV-1 (but not SIV virion preparations harbour significant deacetylase activity, indicating the specific recruitment of catalytically active HDAC into the virions. To determine the requirement of virion-associated HDAC1 to HIV-1 replication, an inactive, transdominant negative mutant of HDAC1 (HDAC1(H141A was utilized. Incorporation of HDAC1(H141A decreased the virion-associated histone deacetylase activity. Furthermore, incorporation of HDAC1(H141A decreased the infectivity of HIV-1 (but not SIV virions. The block in infectivity due to virion-associated HDAC1(H141A occurred specifically at the early reverse transcription stage, while entry of the virions was unaffected. RNA-interference mediated knock-down of HDAC1 in producer cells resulted in decreased virion-associated HDAC1 activity and a reduction in infectivity of these virions. These studies indicate that HIV-1 IN and INI1/hSNF5 bind SAP18 and selectively recruit components of Sin3a-HDAC1 complex into HIV-1 virions. Furthermore, HIV-1 virion-associated HDAC1 is required for efficient early post

Lymphotropic Virions Affect Chemokine Receptor-Mediated Neural Signaling and Apoptosis: Implications for Human Immunodeficiency Virus Type 1-Associated Dementia

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Zheng, Jialin; Ghorpade, Anuja; Niemann, Douglas; Cotter, Robin L.; Thylin, Michael R.; Epstein, Leon; Swartz, Jennifer M.; Shepard, Robin B.; Liu, Xiaojuan; Nukuna, Adeline; Gendelman, Howard E.

1999-01-01

Chemokine receptors pivotal for human immunodeficiency virus type 1 (HIV-1) infection in lymphocytes and macrophages (CCR3, CCR5, and CXCR4) are expressed on neural cells (microglia, astrocytes, and/or neurons). It is these cells which are damaged during progressive HIV-1 infection of the central nervous system. We theorize that viral coreceptors could effect neural cell damage during HIV-1-associated dementia (HAD) without simultaneously affecting viral replication. To these ends, we studied the ability of diverse viral strains to affect intracellular signaling and apoptosis of neurons, astrocytes, and monocyte-derived macrophages. Inhibition of cyclic AMP, activation of inositol 1,4,5-trisphosphate, and apoptosis were induced by diverse HIV-1 strains, principally in neurons. Virions from T-cell-tropic (T-tropic) strains (MN, IIIB, and Lai) produced the most significant alterations in signaling of neurons and astrocytes. The HIV-1 envelope glycoprotein, gp120, induced markedly less neural damage than purified virions. Macrophage-tropic (M-tropic) strains (ADA, JR-FL, Bal, MS-CSF, and DJV) produced the least neural damage, while 89.6, a dual-tropic HIV-1 strain, elicited intermediate neural cell damage. All T-tropic strain-mediated neuronal impairments were blocked by the CXCR4 antibody, 12G5. In contrast, the M-tropic strains were only partially blocked by 12G5. CXCR4-mediated neuronal apoptosis was confirmed in pure populations of rat cerebellar granule neurons and was blocked by HA1004, an inhibitor of calcium/calmodulin-dependent protein kinase II, protein kinase A, and protein kinase C. Taken together, these results suggest that progeny HIV-1 virions can influence neuronal signal transduction and apoptosis. This process occurs, in part, through CXCR4 and is independent of CD4 binding. T-tropic viruses that traffic in and out of the brain during progressive HIV-1 disease may play an important role in HAD neuropathogenesis. PMID:10482576

Human Immunodeficiency Virus type-1 reverse transcriptase exists as post-translationally modified forms in virions and cells

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Warrilow David

2008-12-01

Full Text Available Abstract Background HIV-1 reverse transcriptase (RT is a heterodimer composed of p66 and p51 subunits and is responsible for reverse transcription of the viral RNA genome into DNA. RT can be post-translationally modified in vitro which may be an important mechanism for regulating RT activity. Here we report detection of different p66 and p51 RT isoforms by 2D gel electrophoresis in virions and infected cells. Results Major isoforms of the p66 and p51 RT subunits were observed, with pI's of 8.44 and 8.31 respectively (p668.44 and p518.31. The same major isoforms were present in virions, virus-infected cell lysates and intracellular reverse transcription complexes (RTCs, and their presence in RTCs suggested that these are likely to be the forms that function in reverse transcription. Several minor RT isoforms were also observed. The observed pIs of the RT isoforms differed from the pI of theoretical unmodified RT (p668.53 and p518.60, suggesting that most of the RT protein in virions and cells is post-translationally modified. The modifications of p668.44 and p518.31 differed from each other indicating selective modification of the different RT subunits. The susceptibility of RT isoforms to phosphatase treatment suggested that some of these modifications were due to phosphorylation. Dephosphorylation, however, had no effect on in vitro RT activity associated with virions, infected cells or RTCs suggesting that the phospho-isoforms do not make a major contribution to RT activity in an in vitro assay. Conclusion The same major isoform of p66 and p51 RT is found in virions, infected cells and RTC's and both of these subunits are post-translationally modified. This post-translational modification of RT may be important for the function of RT inside the cell.

Suboptimal inhibition of protease activity in human immunodeficiency virus type 1: Effects on virion morphogenesis and RNA maturation

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Moore, Michael D.; Fu, William; Soheilian, Ferri; Nagashima, Kunio; Ptak, Roger G.; Pathak, Vinay K.; Hu, Wei-Shau

2008-01-01

Protease activity within nascently released human immunodeficiency virus type 1 (HIV-1) particles is responsible for the cleavage of the viral polyproteins Gag and Gag-Pol into their constituent parts, which results in the subsequent condensation of the mature conical core surrounding the viral genomic RNA. Concomitant with viral maturation is a conformational change in the packaged viral RNA from a loosely associated dimer into a more thermodynamically stable form. In this study we used suboptimal concentrations of two protease inhibitors, lopinavir and atazanavir, to study their effects on Gag polyprotein processing and on the properties of the RNA in treated virions. Analysis of the treated virions demonstrated that even with high levels of inhibition of viral infectivity (IC 90 ), most of the Gag and Gag-Pol polyproteins were processed, although slight but significant increases in processing intermediates of Gag were detected. Drug treatments also caused a significant increase in the proportion of viruses displaying either immature or aberrant mature morphologies. The aberrant mature particles were characterized by an electron-dense region at the viral periphery and an electron-lucent core structure in the viral center, possibly indicating exclusion of the genomic RNA from these viral cores. Intriguingly, drug treatments caused only a slight decrease in overall thermodynamic stability of the viral RNA dimer, suggesting that the dimeric viral RNA was able to mature in the absence of correct core condensation

A complex of seven vaccinia virus proteins conserved in all chordopoxviruses is required for the association of membranes and viroplasm to form immature virions

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Szajner, Patricia; Jaffe, Howard; Weisberg, Andrea S.; Moss, Bernard

2004-01-01

Early events in vaccinia virus (VAC) morphogenesis, particularly the formation of viral membranes and their association with viroplasm, are poorly understood. Recently, we showed that repression of A30 or G7 expression results in the accumulation of normal viral membranes that form empty-looking immature virions (IV), which are separated from large masses of electron-dense viroplasm. In addition, A30 and G7 physically and functionally interact with each other and with the F10 protein kinase. To identify other proteins involved in early morphogenesis, proteins from cells that had been infected with vaccinia virus expressing an epitope-tagged copy of F10 were purified by immunoaffinity chromatography and analyzed by gel electrophoresis. In addition to F10, A30, and G7, viral proteins A15, D2, D3, and J1 were identified by mass spectrometry of tryptic peptides. Further evidence for the complex was obtained by immunopurification of proteins associated with epitope-tagged A15, D2, and D3. The previously unstudied A15, like other proteins in the complex, was expressed late in infection, associated with virus cores, and required for the stability and kinase activity of F10. Biochemical and electron microscopic analyses indicated that mutants in which A15 or D2 expression was regulated by the Escherichia coli lac operator system exhibited phenotypes characterized by the presence of large numbers of empty immature virions, similar to the results obtained with inducible A30 and G7 mutants. Empty immature virions were also seen by electron microscopy of cells infected with temperature-sensitive mutants of D2 or D3, though the numbers of membrane forms were reduced perhaps due to additional effects of high temperature

Selective dansylation of M protein within intact influenza virions

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Robertson, B.H.; Bennett, J.C.; Compans, R.W.

1982-12-01

Exposure of purified influenza virions to (/sup 14/C)dansyl chloride resulted in the covalent attachment of the dansyl chromophore to the virion. Gel electrophoresis revealed that the dansyl chromophore was specifically coupled to the internal membrane (M) protein. Purification of the M protein by gel filtration followed by cyanogen bromide cleavage and peptide fractionation revealed that four of six peptide peaks contained dansyl label. Acid hydrolysis of the separated peptide peaks followed by thin-layer chromatography revealed that dansyl label was coupled to lysine residues present in these peptides. The results of these investigations have demonstrated that the M protein molecule is the major viral polypeptide labeled when intact virions are exposed to dansyl chloride.

Selective dansylation of M protein within intact influenza virions

International Nuclear Information System (INIS)

Robertson, B.H.; Bennett, J.C.; Compans, R.W.

1982-01-01

Exposure of purified influenza virions to [ 14 C]dansyl chloride resulted in the covalent attachment of the dansyl chromophore to the virion. Gel electrophoresis revealed that the dansyl chromophore was specifically coupled to the internal membrane (M) protein. Purification of the M protein by gel filtration followed by cyanogen bromide cleavage and peptide fractionation revealed that four of six peptide peaks contained dansyl label. Acid hydrolysis of the separated peptide peaks followed by thin-layer chromatography revealed that dansyl label was coupled to lysine residues present in these peptides. The results of these investigations have demonstrated that the M protein molecule is the major viral polypeptide labeled when intact virions are exposed to dansyl chloride

Reduction of virion-associated σ1 fibers on oncolytic reovirus variants promotes adaptation toward tumorigenic cells.

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Mohamed, Adil; Teicher, Carmit; Haefliger, Sarah; Shmulevitz, Maya

2015-04-01

Wild-type mammalian orthoreovirus serotype 3 Dearing (T3wt) is nonpathogenic in humans but preferentially infects and kills cancer cells in culture and demonstrates promising antitumor activity in vivo. Using forward genetics, we previously isolated two variants of reovirus, T3v1 and T3v2, with increased infectivity toward a panel of cancer cell lines and improved in vivo oncolysis in a murine melanoma model relative to that of T3wt. Our current study explored how mutations in T3v1 and T3v2 promote infectivity. Reovirions contain trimers of σ1, the reovirus cell attachment protein, at icosahedral capsid vertices. Quantitative Western blot analysis showed that purified T3v1 and T3v2 virions had ∼ 2- and 4-fold-lower levels of σ1 fiber than did T3wt virions. Importantly, using RNA interference to reduce σ1 levels during T3wt production, we were able to generate wild-type reovirus with reduced levels of σ1 per virion. As σ1 levels were reduced, virion infectivity increased by 2- to 5-fold per cell-bound particle, demonstrating a causal relationship between virion σ1 levels and the infectivity of incoming virions. During infection of tumorigenic L929 cells, T3wt, T3v1, and T3v2 uncoated the outer capsid proteins σ3 and μ1C at similar rates. However, having started with fewer σ1 molecules, a complete loss of σ1 was achieved sooner for T3v1 and T3v2. Distinct from intracellular uncoating, chymotrypsin digestion, as a mimic of natural enteric infection, resulted in more rapid σ3 and μ1C removal, unique disassembly intermediates, and a rapid loss of infectivity for T3v1 and T3v2 compared to T3wt. Optimal infectivity toward natural versus therapeutic niches may therefore require distinct reovirus structures and σ1 levels. Wild-type reovirus is currently in clinical trials as a potential cancer therapy. Our molecular studies on variants of reovirus with enhanced oncolytic activity in vitro and in vivo now show that distinct reovirus structures promote

Picornavirus RNA is protected from cleavage by ribonuclease during virion uncoating and transfer across cellular and model membranes.

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Groppelli, Elisabetta; Levy, Hazel C; Sun, Eileen; Strauss, Mike; Nicol, Clare; Gold, Sarah; Zhuang, Xiaowei; Tuthill, Tobias J; Hogle, James M; Rowlands, David J

2017-02-01

Picornaviruses are non-enveloped RNA viruses that enter cells via receptor-mediated endocytosis. Because they lack an envelope, picornaviruses face the challenge of delivering their RNA genomes across the membrane of the endocytic vesicle into the cytoplasm to initiate infection. Currently, the mechanism of genome release and translocation across membranes remains poorly understood. Within the enterovirus genus, poliovirus, rhinovirus 2, and rhinovirus 16 have been proposed to release their genomes across intact endosomal membranes through virally induced pores, whereas one study has proposed that rhinovirus 14 releases its RNA following disruption of endosomal membranes. For the more distantly related aphthovirus genus (e.g. foot-and-mouth disease viruses and equine rhinitis A virus) acidification of endosomes results in the disassembly of the virion into pentamers and in the release of the viral RNA into the lumen of the endosome, but no details have been elucidated as how the RNA crosses the vesicle membrane. However, more recent studies suggest aphthovirus RNA is released from intact particles and the dissociation to pentamers may be a late event. In this study we have investigated the RNase A sensitivity of genome translocation of poliovirus using a receptor-decorated-liposome model and the sensitivity of infection of poliovirus and equine-rhinitis A virus to co-internalized RNase A. We show that poliovirus genome translocation is insensitive to RNase A and results in little or no release into the medium in the liposome model. We also show that infectivity is not reduced by co-internalized RNase A for poliovirus and equine rhinitis A virus. Additionally, we show that all poliovirus genomes that are internalized into cells, not just those resulting in infection, are protected from RNase A. These results support a finely coordinated, directional model of viral RNA delivery that involves viral proteins and cellular membranes.

Picornavirus RNA is protected from cleavage by ribonuclease during virion uncoating and transfer across cellular and model membranes.

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Elisabetta Groppelli

2017-02-01

Full Text Available Picornaviruses are non-enveloped RNA viruses that enter cells via receptor-mediated endocytosis. Because they lack an envelope, picornaviruses face the challenge of delivering their RNA genomes across the membrane of the endocytic vesicle into the cytoplasm to initiate infection. Currently, the mechanism of genome release and translocation across membranes remains poorly understood. Within the enterovirus genus, poliovirus, rhinovirus 2, and rhinovirus 16 have been proposed to release their genomes across intact endosomal membranes through virally induced pores, whereas one study has proposed that rhinovirus 14 releases its RNA following disruption of endosomal membranes. For the more distantly related aphthovirus genus (e.g. foot-and-mouth disease viruses and equine rhinitis A virus acidification of endosomes results in the disassembly of the virion into pentamers and in the release of the viral RNA into the lumen of the endosome, but no details have been elucidated as how the RNA crosses the vesicle membrane. However, more recent studies suggest aphthovirus RNA is released from intact particles and the dissociation to pentamers may be a late event. In this study we have investigated the RNase A sensitivity of genome translocation of poliovirus using a receptor-decorated-liposome model and the sensitivity of infection of poliovirus and equine-rhinitis A virus to co-internalized RNase A. We show that poliovirus genome translocation is insensitive to RNase A and results in little or no release into the medium in the liposome model. We also show that infectivity is not reduced by co-internalized RNase A for poliovirus and equine rhinitis A virus. Additionally, we show that all poliovirus genomes that are internalized into cells, not just those resulting in infection, are protected from RNase A. These results support a finely coordinated, directional model of viral RNA delivery that involves viral proteins and cellular membranes.

Structural lability of Barley stripe mosaic virus virions.

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Valentin V Makarov

Full Text Available Virions of Barley stripe mosaic virus (BSMV were neglected for more than thirty years after their basic properties were determined. In this paper, the physicochemical characteristics of BSMV virions and virion-derived viral capsid protein (CP were analyzed, namely, the absorption and intrinsic fluorescence spectra, circular dichroism spectra, differential scanning calorimetry curves, and size distributions by dynamic laser light scattering. The structural properties of BSMV virions proved to be intermediate between those of Tobacco mosaic virus (TMV, a well-characterized virus with rigid rod-shaped virions, and flexuous filamentous plant viruses. The BSMV virions were found to be considerably more labile than expected from their rod-like morphology and a distant sequence relation of the BSMV and TMV CPs. The circular dichroism spectra of BSMV CP subunits incorporated into the virions, but not subunits of free CP, demonstrated a significant proportion of beta-structure elements, which were proposed to be localized mostly in the protein regions exposed on the virion outer surface. These beta-structure elements likely formed during virion assembly can comprise the N- and C-terminal protein regions unstructured in the non-virion CP and can mediate inter-subunit interactions. Based on computer-assisted structure modeling, a model for BSMV CP subunit structural fold compliant with the available experimental data was proposed.

TIM1 (HAVCR1) Is Not Essential for Cellular Entry of Either Quasi-enveloped or Naked Hepatitis A Virions.

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Das, Anshuman; Hirai-Yuki, Asuka; González-López, Olga; Rhein, Bethany; Moller-Tank, Sven; Brouillette, Rachel; Hensley, Lucinda; Misumi, Ichiro; Lovell, William; Cullen, John M; Whitmire, Jason K; Maury, Wendy; Lemon, Stanley M

2017-09-05

Receptor molecules play key roles in the cellular entry of picornaviruses, and TIM1 (HAVCR1) is widely accepted to be the receptor for hepatitis A virus (HAV), an unusual, hepatotropic human picornavirus. However, its identification as the hepatovirus receptor predated the discovery that hepatoviruses undergo nonlytic release from infected cells as membrane-cloaked, quasi-enveloped HAV (eHAV) virions that enter cells via a pathway distinct from naked, nonenveloped virions. We thus revisited the role of TIM1 in hepatovirus entry, examining both adherence and infection/replication in cells with clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9-engineered TIM1 knockout. Cell culture-derived, gradient-purified eHAV bound Huh-7.5 human hepatoma cells less efficiently than naked HAV at 4°C, but eliminating TIM1 expression caused no difference in adherence of either form of HAV, nor any impact on infection and replication in these cells. In contrast, TIM1-deficient Vero cells showed a modest reduction in quasi-enveloped eHAV (but not naked HAV) attachment and replication. Thus, TIM1 facilitates quasi-enveloped eHAV entry in Vero cells, most likely by binding phosphatidylserine (PtdSer) residues on the eHAV membrane. Both Tim1 -/- Ifnar1 -/- and Tim4 -/- Ifnar1 -/- double-knockout mice were susceptible to infection upon intravenous challenge with infected liver homogenate, with fecal HAV shedding and serum alanine aminotransferase (ALT) elevations similar to those in Ifnar1 -/- mice. However, intrahepatic HAV RNA and ALT elevations were modestly reduced in Tim1 -/- Ifnar1 -/- mice compared to Ifnar1 -/- mice challenged with a lower titer of gradient-purified HAV or eHAV. We conclude that TIM1 is not an essential hepatovirus entry factor, although its PtdSer-binding activity may contribute to the spread of quasi-enveloped virus and liver injury in mice. IMPORTANCE T cell immunoglobulin and mucin-containing domain protein 1 (TIM1) was reported more than

cGAS-Mediated Innate Immunity Spreads Intercellularly through HIV-1 Env-Induced Membrane Fusion Sites.

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Xu, Shuting; Ducroux, Aurélie; Ponnurangam, Aparna; Vieyres, Gabrielle; Franz, Sergej; Müsken, Mathias; Zillinger, Thomas; Malassa, Angelina; Ewald, Ellen; Hornung, Veit; Barchet, Winfried; Häussler, Susanne; Pietschmann, Thomas; Goffinet, Christine

2016-10-12

Upon sensing cytoplasmic retroviral DNA in infected cells, cyclic GMP-AMP (cGAMP) synthase (cGAS) produces the cyclic dinucleotide cGAMP, which activates STING to trigger a type I interferon (IFN) response. We find that membrane fusion-inducing contact between donor cells expressing the HIV envelope (Env) and primary macrophages endogenously expressing the HIV receptor CD4 and coreceptor enable intercellular transfer of cGAMP. This cGAMP exchange results in STING-dependent antiviral IFN responses in target macrophages and protection from HIV infection. Furthermore, under conditions allowing cell-to-cell transmission of HIV-1, infected primary T cells, but not cell-free virions, deliver cGAMP to autologous macrophages through HIV-1 Env and CD4/coreceptor-mediated membrane fusion sites and induce a STING-dependent, but cGAS-independent, IFN response in target cells. Collectively, these findings identify an infection-specific mode of horizontal transfer of cGAMP between primary immune cells that may boost antiviral responses, particularly in infected tissues in which cell-to-cell transmission of virions exceeds cell-free infection. Copyright © 2016 Elsevier Inc. All rights reserved.

RAB1A promotes Vaccinia virus replication by facilitating the production of intracellular enveloped virions

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Pechenick Jowers, Tali; Featherstone, Rebecca J.; Reynolds, Danielle K.; Brown, Helen K. [The Roslin Institute and Royal (Dick) School of Veterinary Studies, University of Edinburgh, Roslin, Midlothian EH25 9RG, Scotland (United Kingdom); James, John; Prescott, Alan [Division of Cell Signalling and Immunology, College of Life Sciences, University of Dundee, Dundee DD1 5EH, Scotland (United Kingdom); Haga, Ismar R. [The Roslin Institute and Royal (Dick) School of Veterinary Studies, University of Edinburgh, Roslin, Midlothian EH25 9RG, Scotland (United Kingdom); Beard, Philippa M., E-mail: pip.beard@roslin.ed.ac.uk [The Roslin Institute and Royal (Dick) School of Veterinary Studies, University of Edinburgh, Roslin, Midlothian EH25 9RG, Scotland (United Kingdom)

2015-01-15

Vaccinia virus (VACV) is a large double-stranded DNA virus with a complex cytoplasmic replication cycle that exploits numerous cellular proteins. This work characterises the role of a proviral cellular protein, the small GTPase RAB1A, in VACV replication. Using siRNA, we identified RAB1A as required for the production of extracellular enveloped virions (EEVs), but not intracellular mature virions (IMVs). Immunofluorescence and electron microscopy further refined the role of RAB1A as facilitating the wrapping of IMVs to become intracellular enveloped virions (IEVs). This is consistent with the known function of RAB1A in maintenance of ER to Golgi transport. VACV can therefore be added to the growing list of viruses which require RAB1A for optimal replication, highlighting this protein as a broadly proviral host factor. - Highlights: • Characterisation of the role of the small GTPase RAB1A in VACV replication. • RAB1A is not required for production of the primary virion form (IMV). • RAB1A is required for production of processed virion forms (IEVs, CEVs and EEVs). • Consistent with known role of RAB1A in ER to Golgi transport.

TIM1 (HAVCR1 Is Not Essential for Cellular Entry of Either Quasi-enveloped or Naked Hepatitis A Virions

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Anshuman Das

2017-09-01

Full Text Available Receptor molecules play key roles in the cellular entry of picornaviruses, and TIM1 (HAVCR1 is widely accepted to be the receptor for hepatitis A virus (HAV, an unusual, hepatotropic human picornavirus. However, its identification as the hepatovirus receptor predated the discovery that hepatoviruses undergo nonlytic release from infected cells as membrane-cloaked, quasi-enveloped HAV (eHAV virions that enter cells via a pathway distinct from naked, nonenveloped virions. We thus revisited the role of TIM1 in hepatovirus entry, examining both adherence and infection/replication in cells with clustered regularly interspaced short palindromic repeat (CRISPR/Cas9-engineered TIM1 knockout. Cell culture-derived, gradient-purified eHAV bound Huh-7.5 human hepatoma cells less efficiently than naked HAV at 4°C, but eliminating TIM1 expression caused no difference in adherence of either form of HAV, nor any impact on infection and replication in these cells. In contrast, TIM1-deficient Vero cells showed a modest reduction in quasi-enveloped eHAV (but not naked HAV attachment and replication. Thus, TIM1 facilitates quasi-enveloped eHAV entry in Vero cells, most likely by binding phosphatidylserine (PtdSer residues on the eHAV membrane. Both Tim1−/− Ifnar1−/− and Tim4−/− Ifnar1−/− double-knockout mice were susceptible to infection upon intravenous challenge with infected liver homogenate, with fecal HAV shedding and serum alanine aminotransferase (ALT elevations similar to those in Ifnar1−/− mice. However, intrahepatic HAV RNA and ALT elevations were modestly reduced in Tim1−/−Ifnar1−/− mice compared to Ifnar1−/− mice challenged with a lower titer of gradient-purified HAV or eHAV. We conclude that TIM1 is not an essential hepatovirus entry factor, although its PtdSer-binding activity may contribute to the spread of quasi-enveloped virus and liver injury in mice.

BST2/CD317 counteracts human coronavirus 229E productive infection by tethering virions at the cell surface

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Wang, Shiu-Mei [Department of Medical Research and Education, Taipei Veterans General Hospital and Institute of Clinical Medicine, Taipei 11217, Taiwan (China); Institute of Clinical Medicine, National Yang-Ming University School of Medicine, Taipei, Taiwan (China); Huang, Kuo-Jung [Department of Medical Research and Education, Taipei Veterans General Hospital and Institute of Clinical Medicine, Taipei 11217, Taiwan (China); Wang, Chin-Tien, E-mail: chintien@ym.edu.tw [Department of Medical Research and Education, Taipei Veterans General Hospital and Institute of Clinical Medicine, Taipei 11217, Taiwan (China); Institute of Clinical Medicine, National Yang-Ming University School of Medicine, Taipei, Taiwan (China)

2014-01-20

Bone marrow stromal antigen 2 (BST2), an interferon-inducible antiviral factor, has been shown to block the release of various enveloped viruses from cells. It has also been identified as an innate immune system component. Most enveloped viruses subject to BST2 restriction bud at the plasma membrane. Here we report our findings that (a) the production of human coronavirus 229E (HCoV-229E) progeny viruses, whose budding occurs at the ER-Golgi intermediate compartment (ERGIC), markedly decreases in the presence of BST2; and (b) BST2 knockdown expression results in enhanced HCoV-229E virion production. Electron microscopy analyses indicate that HCoV-229E virions are tethered to cell surfaces or intracellular membranes by BST2. Our results suggest that BST2 exerts a broad blocking effect against enveloped virus release, regardless of whether budding occurs at the plasma membrane or intracellular compartments. - Highlights: • BST2 knockdown expression results in enhanced HCoV-229E egress. • HCoV-229E virions are tethered to cell surfaces or intracellular membranes by BST2. • HCoV-229E infection at high MOI can significantly downregulate HeLa BST2 and rescue HIV-1 egress.

Solution Structure and Membrane Interaction of the Cytoplasmic Tail of HIV-1 gp41 Protein.

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Murphy, R Elliot; Samal, Alexandra B; Vlach, Jiri; Saad, Jamil S

2017-11-07

The cytoplasmic tail of gp41 (gp41CT) remains the last HIV-1 domain with an unknown structure. It plays important roles in HIV-1 replication such as mediating envelope (Env) intracellular trafficking and incorporation into assembling virions, mechanisms of which are poorly understood. Here, we present the solution structure of gp41CT in a micellar environment and characterize its interaction with the membrane. We show that the N-terminal 45 residues are unstructured and not associated with the membrane. However, the C-terminal 105 residues form three membrane-bound amphipathic α helices with distinctive structural features such as variable degree of membrane penetration, hydrophobic and basic surfaces, clusters of aromatic residues, and a network of cation-π interactions. This work fills a major gap by providing the structure of the last segment of HIV-1 Env, which will provide insights into the mechanisms of Gag-mediated Env incorporation as well as the overall Env mobility and conformation on the virion surface. Copyright © 2017 Elsevier Ltd. All rights reserved.

Identification of the 15FRFG domain in HIV-1 Gag p6 essential for Vpr packaging into the virion

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Zhu Henghu

2004-09-01

Full Text Available Abstract The auxiliary regulatory protein Vpr of HIV-1 is packaged in the virion through interaction with the Gag C-terminal p6 domain. Virion packaging of Vpr is critical for Vpr to exert functions in the HIV-1 life cycle. Previous studies suggest that Vpr interacts with a (Lxx4 domain in p6 for virion packaging. In the present study, mutational analysis of HIV-1 Gag p6 domain was performed in the context of the HIV-1 genome to examine the effect on virion packaging of Vpr. Surprisingly, Ala substitutions for Leu44 and Phe45 in the (Lxx4 domain or deletion of the whole (Lxx4 domain (amino acid #35–52 of the Gag p6 domain did not affect Vpr virion packaging. Vpr virion packaging was normal when amino acid #1–23 of the Gag p6 domain was preserved. Most importantly, Ala substitutions for Phe15, Arg16 and Phe17 in the context of amino acid #1–23 of the Gag p6 domain abolished Vpr virion packaging. Single Ala substitutions for Phe15 and Phe17 also abolished Vpr virion packaging, whereas Ala substitution for Arg16 had no effect. Our studies have revealed a novel signal sequence for Vpr packaging into the HIV-1 virion. The 15FRFG domain in p6 resembles the FxFG repeat sequences commonly found in proteins of the nuclear pore complex. These results have provided novel insights into the process of virion packaging of Vpr and suggest for the first time that Vpr may recognize the FxFG domain for both virion packaging and association with nuclear pores.

Proteasome-independent degradation of HIV-1 in naturally non-permissive human placental trophoblast cells

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Barré-Sinoussi Françoise

2009-05-01

Full Text Available Abstract Background The human placenta-derived cell line BeWo has been demonstrated to be restrictive to cell-free HIV-1 infection. BeWo cells are however permissive to infection by VSV-G pseudotyped HIV-1, which enters cells by a receptor-independent mechanism, and to infection by HIV-1 via a cell-to-cell route. Results Here we analysed viral entry in wild type BeWo (CCR5+, CXCR4+ and BeWo-CD4+ (CD4+, CCR5+, CXCR4+ cells. We report that HIV-1 internalisation is not restricted in either cell line. Levels of internalised p24 antigen between VSV-G HIV-1 pseudotypes and R5 or X4 virions were comparable. We next analysed the fate of internalised virions; X4 and R5 HIV-1 virions were less stable over time in BeWo cells than VSV-G HIV-1 pseudotypes. We then investigated the role of the proteasome in restricting cell-free HIV-1 infection in BeWo cells using proteasome inhibitors. We observed an increase in the levels of VSV-G pseudotyped HIV-1 infection in proteasome-inhibitor treated cells, but the infection by R5-Env or X4-Env pseudotyped virions remains restricted. Conclusion Collectively these results suggest that cell-free HIV-1 infection encounters a surface block leading to a non-productive entry route, which either actively targets incoming virions for non-proteasomal degradation, and impedes their release into the cytoplasm, or causes the inactivation of mechanisms essential for viral replication.

Antigenic properties of the human immunodeficiency virus envelope glycoprotein gp120 on virions bound to target cells.

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Meron Mengistu

2015-03-01

Full Text Available The HIV-1 envelope glycoprotein, gp120, undergoes multiple molecular interactions and structural rearrangements during the course of host cell attachment and viral entry, which are being increasingly defined at the atomic level using isolated proteins. In comparison, antigenic markers of these dynamic changes are essentially unknown for single HIV-1 particles bound to target cells. Such markers should indicate how neutralizing and/or non-neutralizing antibodies might interdict infection by either blocking infection or sensitizing host cells for elimination by Fc-mediated effector function. Here we address this deficit by imaging fluorescently labeled CCR5-tropic HIV-1 pseudoviruses using confocal and superresolution microscopy to track the exposure of neutralizing and non-neutralizing epitopes as they appear on single HIV-1 particles bound to target cells. Epitope exposure was followed under conditions permissive or non-permissive for viral entry to delimit changes associated with virion binding from those associated with post-attachment events. We find that a previously unexpected array of gp120 epitopes is exposed rapidly upon target cell binding. This array comprises both neutralizing and non-neutralizing epitopes, the latter being hidden on free virions yet capable of serving as potent targets for Fc-mediated effector function. Under non-permissive conditions for viral entry, both neutralizing and non-neutralizing epitope exposures were relatively static over time for the majority of bound virions. Under entry-permissive conditions, epitope exposure patterns changed over time on subsets of virions that exhibited concurrent variations in virion contents. These studies reveal that bound virions are distinguished by a broad array of both neutralizing and non-neutralizing gp120 epitopes that potentially sensitize a freshly engaged target cell for destruction by Fc-mediated effector function and/or for direct neutralization at a post-binding step

Liver/kidney microsomal antibody type 1 targets CYP2D6 on hepatocyte plasma membrane.

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Muratori, L; Parola, M; Ripalti, A; Robino, G; Muratori, P; Bellomo, G; Carini, R; Lenzi, M; Landini, M P; Albano, E; Bianchi, F B

2000-04-01

Liver/kidney microsomal antibody type 1 (LKM1) is the marker of type 2 autoimmune hepatitis (AIH) and is detected in up to 6% of patients with hepatitis C virus (HCV) infection. It recognises linear and conformational epitopes of cytochrome P450IID6 (CYP2D6) and may have liver damaging activity, provided that CYP2D6 is accessible to effector mechanisms of autoimmune attack. The presence of LKM1 in the plasma membrane was investigated by indirect immunofluorescence and confocal laser microscopy of isolated rat hepatocytes probed with 10 LKM1 positive sera (five from patients with AIH and five from patients with chronic HCV infection) and a rabbit polyclonal anti-CYP2D6 serum. Serum from both types of patient stained the plasma membrane of non-permeabilised cells, where the fluorescent signal could be visualised as discrete clumps. Conversely, permeabilised hepatocytes showed diffuse submembranous/cytoplasmic staining. Adsorption with recombinant CYP2D6 substantially reduced plasma membrane staining and LKM1 immunoblot reactivity. Plasma membrane staining of LKM1 colocalised with that of anti-CYP2D6. Immunoprecipitation experiments showed that a single 50 kDa protein recognised by anti-CYP2D6 can be isolated from the plasma membrane of intact hepatocytes. AIH and HCV related LKM1 recognise CYP2D6 exposed on the plasma membrane of isolated hepatocytes. This observation supports the notion that anti-CYP2D6 autoreactivity may be involved in the pathogenesis of liver damage.

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.

Herpesvirus glycoproteins undergo multiple antigenic changes before membrane fusion.

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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.

Characterization of retrokeratoprosthetic membranes in the Boston type 1 keratoprosthesis.

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Stacy, Rebecca C; Jakobiec, Frederick A; Michaud, Norman A; Dohlman, Claes H; Colby, Kathryn A

2011-03-01

To evaluate retroprosthetic membranes that can occur in 25% to 65% of patients with the Boston type 1 keratoprosthesis (KPro). Two patients with Peter anomaly and 2 with neurotrophic scarred corneas underwent revisions of their type 1 KPros because of visually compromising retroprosthetic membranes. The excised membranes were studied by light microscopy with hematoxylin-eosin, periodic acid-Schiff, and toluidine blue stains. Immunohistochemical and transmission electron microscopic examination were also used. Light microscopic examination revealed that the retro-KPro fibrous membranes originated from the host's corneal stroma. These mildly to moderately vascularized membranes grew through gaps in the Descemet membrane to reach behind the KPro back plate and adhere to the anterior iris surface, which had undergone partial lysis. In 2 cases, the fibrous membranes merged at the pupil with matrical portions of metaplastic lens epithelium, forming a bilayered structure that crossed the optical axis. Retro-KPro membranes stained positively for α-smooth muscle actin but negatively for pancytokeratin. Electron microscopy confirmed the presence of actin filaments within myofibroblasts and small surviving clusters of metaplastic lens epithelial cells. Stromal downgrowth, rather than epithelial downgrowth, was the major element of the retro-KPro membranes in this series. Metaplastic lens epithelium also contributed to opacification of the visual axis. Florid membranous inflammation was not a prominent finding and thus probably not a requisite stimulus for membrane development. Further advances in prosthetic design and newer antifibroproliferative agents may reduce membrane formation.

Mixed genotype transmission bodies and virions contribute to the maintenance of diversity in an insect virus

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Clavijo, Gabriel; Williams, Trevor; Muñoz, Delia; Caballero, Primitivo; López-Ferber, Miguel

2010-01-01

An insect nucleopolyhedrovirus naturally survives as a mixture of at least nine genotypes. Infection by multiple genotypes results in the production of virus occlusion bodies (OBs) with greater pathogenicity than those of any genotype alone. We tested the hypothesis that each OB contains a genotypically diverse population of virions. Few insects died following inoculation with an experimental two-genotype mixture at a dose of one OB per insect, but a high proportion of multiple infections were observed (50%), which differed significantly from the frequencies predicted by a non-associated transmission model in which genotypes are segregated into distinct OBs. By contrast, insects that consumed multiple OBs experienced higher mortality and infection frequencies did not differ significantly from those of the non-associated model. Inoculation with genotypically complex wild-type OBs indicated that genotypes tend to be transmitted in association, rather than as independent entities, irrespective of dose. To examine the hypothesis that virions may themselves be genotypically heterogeneous, cell culture plaques derived from individual virions were analysed to reveal that one-third of virions was of mixed genotype, irrespective of the genotypic composition of the OBs. We conclude that co-occlusion of genotypically distinct virions in each OB is an adaptive mechanism that favours the maintenance of virus diversity during insect-to-insect transmission. PMID:19939845

Selective incorporation of vRNP into influenza A virions determined by its specific interaction with M1 protein

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Chaimayo, Chutikarn [Department of Microbiology and Immunology, University of Rochester Medical Center, Rochester, NY 14642 (United States); Department of Microbiology, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok 10700 (Thailand); Hayashi, Tsuyoshi; Underwood, Andrew; Hodges, Erin [Department of Microbiology and Immunology, University of Rochester Medical Center, Rochester, NY 14642 (United States); Takimoto, Toru, E-mail: toru_takimoto@urmc.rochester.edu [Department of Microbiology and Immunology, University of Rochester Medical Center, Rochester, NY 14642 (United States)

2017-05-15

Influenza A viruses contain eight single-stranded, negative-sense RNA segments as viral genomes in the form of viral ribonucleoproteins (vRNPs). During genome replication in the nucleus, positive-sense complementary RNPs (cRNPs) are produced as replicative intermediates, which are not incorporated into progeny virions. To analyze the mechanism of selective vRNP incorporation into progeny virions, we quantified vRNPs and cRNPs in the nuclear and cytosolic fractions of infected cells, using a strand-specific qRT-PCR. Unexpectedly, we found that cRNPs were also exported to the cytoplasm. This export was chromosome region maintenance 1 (CRM1)-independent unlike that of vRNPs. Although both vRNPs and cRNPs were present in the cytosol, viral matrix (M1) protein, a key regulator for viral assembly, preferentially bound vRNPs over cRNPs. These results indicate that influenza A viruses selectively uptake cytosolic vRNPs through a specific interaction with M1 during viral assembly. - Highlights: •Influenza cRNPs are exported from the nucleus of an infected cell via a CRM1-independent pathway. •Influenza A viruses selectively incorporate cytosolic vRNPs through a specific interaction with M1 during viral assembly. •M1 dissociates from vRNP export complex after nuclear export, and is re-associated with vRNPs at the plasma membrane.

Selective incorporation of vRNP into influenza A virions determined by its specific interaction with M1 protein

International Nuclear Information System (INIS)

Chaimayo, Chutikarn; Hayashi, Tsuyoshi; Underwood, Andrew; Hodges, Erin; Takimoto, Toru

2017-01-01

Influenza A viruses contain eight single-stranded, negative-sense RNA segments as viral genomes in the form of viral ribonucleoproteins (vRNPs). During genome replication in the nucleus, positive-sense complementary RNPs (cRNPs) are produced as replicative intermediates, which are not incorporated into progeny virions. To analyze the mechanism of selective vRNP incorporation into progeny virions, we quantified vRNPs and cRNPs in the nuclear and cytosolic fractions of infected cells, using a strand-specific qRT-PCR. Unexpectedly, we found that cRNPs were also exported to the cytoplasm. This export was chromosome region maintenance 1 (CRM1)-independent unlike that of vRNPs. Although both vRNPs and cRNPs were present in the cytosol, viral matrix (M1) protein, a key regulator for viral assembly, preferentially bound vRNPs over cRNPs. These results indicate that influenza A viruses selectively uptake cytosolic vRNPs through a specific interaction with M1 during viral assembly. - Highlights: •Influenza cRNPs are exported from the nucleus of an infected cell via a CRM1-independent pathway. •Influenza A viruses selectively incorporate cytosolic vRNPs through a specific interaction with M1 during viral assembly. •M1 dissociates from vRNP export complex after nuclear export, and is re-associated with vRNPs at the plasma membrane.

Measles virus polypeptides in purified virions and in infected cells

International Nuclear Information System (INIS)

Vainionpaeae, R.; Ziola, B.; Salmi, A.

1978-01-01

A wild-type measles virus was radiolabeled during growth in VERO cells and purified by two successive potassium tartrate gradient centrifugations. The virion polypeptide composition was determined by SDS-polyacrylamide gel electrophoresis employing two different buffer systems. Six virus-specific polypeptides were consistently detected. The largest (L) had a molecular weight (MW) of greater than 150,000. The second largest polypeptide, G (MW 79,000), was the only glycoprotein found. The proteins designated polypeptide 2 (MW 66 to 70,000) and nucleocapsid protein or NP (MW 61,000) were phosphorylated. The remaining virus-coded proteins were polypeptide 5 (MW 40,000) and the matrix or M protein (MW 37,000). Measles virions also contained a polypeptide (MW 42,000) thought to be actin due to co-migration with this component of uninfected cells. Analysis of in vitro 3 H-acetic anhydride radiolabeled virions confirmed the presence of these seven polypeptides. Acetic anhydride also labeled a protein designated polypeptide 4 (MW 53,000) which was not consistently radiolabeled in vivo, as well as several other minor proteins believed to be cellular in origin. Synthesis of the six virus-specific structural polypeptides was detected in lysates of infected cells by SDS-polyacrylamide slab gel electrophoresis. Virus specificity of polypeptide 4 could not be confirmed due to the similar MW of several cellular polypeptides. Two non-virion, but virus-specified polypeptides, of MW 38,000 and 18,000 were also detected. Synthesis of the virus structural proteins was in the same proportions as the polypeptides found in virions except for under production of polypeptide G and over production of polypeptide 2. (author)

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

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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.

Herpes Simplex Virus 1 Us3 Deletion Mutant is Infective Despite Impaired Capsid Translocation to the Cytoplasm

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Peter Wild

2015-01-01

Full Text Available Herpes simplex virus 1 (HSV-1 capsids are assembled in the nucleus bud at the inner nuclear membrane into the perinuclear space, acquiring envelope and tegument. In theory, these virions are de-enveloped by fusion of the envelope with the outer nuclear membrane and re-enveloped by Golgi membranes to become infective. Us3 enables the nucleus to cytoplasm capsid translocation. Nevertheless, Us3 is not essential for the production of infective progeny viruses. Determination of phenotype distribution by quantitative electron microscopy, and calculation per mean nuclear or cell volume revealed the following: (i The number of R7041(∆US3 capsids budding at the inner nuclear membrane was significantly higher than that of wild type HSV-1; (ii The mean number of R7041(∆US3 virions per mean cell volume was 2726, that of HSV-1 virions 1460 by 24 h post inoculation; (iii 98% of R7041(∆US3 virions were in the perinuclear space; (iv The number of R7041(∆US3 capsids in the cytoplasm, including those budding at Golgi membranes, was significantly reduced. Cell associated R7041(∆US3 yields were 2.37 × 108 and HSV-1 yields 1.57 × 108 PFU/mL by 24 h post inoculation. We thus conclude that R7041(∆US3 virions, which acquire envelope and tegument by budding at the inner nuclear membrane into the perinuclear space, are infective.

Liver/kidney microsomal antibody type 1 targets CYP2D6 on hepatocyte plasma membrane

Science.gov (United States)

Muratori, L; Parola, M; Ripalti, A; Robino, G; Muratori, P; Bellomo, G; Carini, R; Lenzi, M; Landini, M; Albano, E; Bianchi, F

2000-01-01

BACKGROUND—Liver/kidney microsomal antibody type 1 (LKM1) is the marker of type 2 autoimmune hepatitis (AIH) and is detected in up to 6% of patients with hepatitis C virus (HCV) infection. It recognises linear and conformational epitopes of cytochrome P450IID6 (CYP2D6) and may have liver damaging activity, provided that CYP2D6 is accessible to effector mechanisms of autoimmune attack.
METHODS—The presence of LKM1 in the plasma membrane was investigated by indirect immunofluorescence and confocal laser microscopy of isolated rat hepatocytes probed with 10 LKM1 positive sera (five from patients with AIH and five from patients with chronic HCV infection) and a rabbit polyclonal anti-CYP2D6 serum.
RESULTS—Serum from both types of patient stained the plasma membrane of non-permeabilised cells, where the fluorescent signal could be visualised as discrete clumps. Conversely, permeabilised hepatocytes showed diffuse submembranous/cytoplasmic staining. Adsorption with recombinant CYP2D6 substantially reduced plasma membrane staining and LKM1 immunoblot reactivity. Plasma membrane staining of LKM1 colocalised with that of anti-CYP2D6. Immunoprecipitation experiments showed that a single 50 kDa protein recognised by anti-CYP2D6 can be isolated from the plasma membrane of intact hepatocytes.
CONCLUSIONS—AIH and HCV related LKM1 recognise CYP2D6 exposed on the plasma membrane of isolated hepatocytes. This observation supports the notion that anti-CYP2D6 autoreactivity may be involved in the pathogenesis of liver damage.


Keywords: liver/kidney microsomal antibody type 1; autoimmunity; autoimmune hepatitis; hepatitis C virus infection; confocal microscopy PMID:10716687

The allosteric HIV-1 integrase inhibitor BI-D affects virion maturation but does not influence packaging of a functional RNA genome.

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Nikki van Bel

Full Text Available The viral integrase (IN is an essential protein for HIV-1 replication. IN inserts the viral dsDNA into the host chromosome, thereby aided by the cellular co-factor LEDGF/p75. Recently a new class of integrase inhibitors was described: allosteric IN inhibitors (ALLINIs. Although designed to interfere with the IN-LEDGF/p75 interaction to block HIV DNA integration during the early phase of HIV-1 replication, the major impact was surprisingly found on the process of virus maturation during the late phase, causing a reverse transcription defect upon infection of target cells. Virus particles produced in the presence of an ALLINI are misformed with the ribonucleoprotein located outside the virus core. Virus assembly and maturation are highly orchestrated and regulated processes in which several viral proteins and RNA molecules closely interact. It is therefore of interest to study whether ALLINIs have unpredicted pleiotropic effects on these RNA-related processes. We confirm that the ALLINI BI-D inhibits virus replication and that the produced virus is non-infectious. Furthermore, we show that the wild-type level of HIV-1 genomic RNA is packaged in virions and these genomes are in a dimeric state. The tRNAlys3 primer for reverse transcription was properly placed on this genomic RNA and could be extended ex vivo. In addition, the packaged reverse transcriptase enzyme was fully active when extracted from virions. As the RNA and enzyme components for reverse transcription are properly present in virions produced in the presence of BI-D, the inhibition of reverse transcription is likely to reflect the mislocalization of the components in the aberrant virus particle.

The allosteric HIV-1 integrase inhibitor BI-D affects virion maturation but does not influence packaging of a functional RNA genome.

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van Bel, Nikki; van der Velden, Yme; Bonnard, Damien; Le Rouzic, Erwann; Das, Atze T; Benarous, Richard; Berkhout, Ben

2014-01-01

The viral integrase (IN) is an essential protein for HIV-1 replication. IN inserts the viral dsDNA into the host chromosome, thereby aided by the cellular co-factor LEDGF/p75. Recently a new class of integrase inhibitors was described: allosteric IN inhibitors (ALLINIs). Although designed to interfere with the IN-LEDGF/p75 interaction to block HIV DNA integration during the early phase of HIV-1 replication, the major impact was surprisingly found on the process of virus maturation during the late phase, causing a reverse transcription defect upon infection of target cells. Virus particles produced in the presence of an ALLINI are misformed with the ribonucleoprotein located outside the virus core. Virus assembly and maturation are highly orchestrated and regulated processes in which several viral proteins and RNA molecules closely interact. It is therefore of interest to study whether ALLINIs have unpredicted pleiotropic effects on these RNA-related processes. We confirm that the ALLINI BI-D inhibits virus replication and that the produced virus is non-infectious. Furthermore, we show that the wild-type level of HIV-1 genomic RNA is packaged in virions and these genomes are in a dimeric state. The tRNAlys3 primer for reverse transcription was properly placed on this genomic RNA and could be extended ex vivo. In addition, the packaged reverse transcriptase enzyme was fully active when extracted from virions. As the RNA and enzyme components for reverse transcription are properly present in virions produced in the presence of BI-D, the inhibition of reverse transcription is likely to reflect the mislocalization of the components in the aberrant virus particle.

Nef decreases HIV-1 sensitivity to neutralizing antibodies that target the membrane-proximal external region of TMgp41.

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Rachel P J Lai

2011-12-01

Full Text Available Primate lentivirus nef is required for sustained virus replication in vivo and accelerated progression to AIDS. While exploring the mechanism by which Nef increases the infectivity of cell-free virions, we investigated a functional link between Nef and Env. Since we failed to detect an effect of Nef on the quantity of virion-associated Env, we searched for qualitative changes by examining whether Nef alters HIV-1 sensitivity to agents that target distinct features of Env. Nef conferred as much as 50-fold resistance to 2F5 and 4E10, two potent neutralizing monoclonal antibodies (nAbs that target the membrane proximal external region (MPER of TMgp41. In contrast, Nef had no effect on HIV-1 neutralization by MPER-specific nAb Z13e1, by the peptide inhibitor T20, nor by a panel of nAbs and other reagents targeting gp120. Resistance to neutralization by 2F5 and 4E10 was observed with Nef from a diverse range of HIV-1 and SIV isolates, as well as with HIV-1 virions bearing Env from CCR5- and CXCR4-tropic viruses, clade B and C viruses, or primary isolates. Functional analysis of a panel of Nef mutants revealed that this activity requires Nef myristoylation but that it is genetically separable from other Nef functions such as the ability to enhance virus infectivity and to downregulate CD4. Glycosylated-Gag from MoMLV substituted for Nef in conferring resistance to 2F5 and 4E10, indicating that this activity is conserved in a retrovirus that does not encode Nef. Given the reported membrane-dependence of MPER-recognition by 2F5 and 4E10, in contrast to the membrane-independence of Z13e1, the data here is consistent with a model in which Nef alters MPER recognition in the context of the virion membrane. Indeed, Nef and Glycosylated-Gag decreased the efficiency of virion capture by 2F5 and 4E10, but not by other nAbs. These studies demonstrate that Nef protects lentiviruses from one of the most broadly-acting classes of neutralizing antibodies. This newly

Broad target cell selectivity of Kaposi's sarcoma-associated herpesvirus glycoprotein-mediated cell fusion and virion entry

International Nuclear Information System (INIS)

Kaleeba, Johnan A.R.; Berger, Edward A.

2006-01-01

The molecular mechanism of Kaposi's sarcoma-associated herpesvirus (KSHV, human herpesvirus 8) entry is poorly understood. We tested a broad variety of cell types of diverse species and tissue origin for their ability to function as targets in a quantitative reporter gene assay for KSHV-glycoprotein-mediated cell fusion. Several human, non-human primate, and rabbit cell lines were efficient targets, whereas rodent and all human lymphoblastoid cell lines were weak targets. Parallel findings were obtained with a virion entry assay using a recombinant KSHV encoding a reporter gene. No correlation was observed between target cell activity and surface expression of α3β1 integrin, a proposed KSHV receptor. We hypothesize that target cell permissiveness in both the cell fusion and virion entry assays reflects the presence of a putative KSHV fusion-entry receptor

Endophilins interact with Moloney murine leukemia virus Gag and modulate virion production

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De Camilli Pietro

2003-12-01

Full Text Available Abstract Background The retroviral Gag protein is the central player in the process of virion assembly at the plasma membrane, and is sufficient to induce the formation and release of virus-like particles. Recent evidence suggests that Gag may co-opt the host cell's endocytic machinery to facilitate retroviral assembly and release. Results A search for novel partners interacting with the Gag protein of the Moloney murine leukemia virus (Mo-MuLV via the yeast two-hybrid protein-protein interaction assay resulted in the identification of endophilin 2, a component of the machinery involved in clathrin-mediated endocytosis. We demonstrate that endophilin interacts with the matrix or MA domain of the Gag protein of Mo-MuLV, but not of human immunodeficiency virus, HIV. Both exogenously expressed and endogenous endophilin are incorporated into Mo-MuLV viral particles. Titration experiments suggest that the binding sites for inclusion of endophilin into viral particles are limited and saturable. Knock-down of endophilin with small interfering RNA (siRNA had no effect on virion production, but overexpression of endophilin and, to a lesser extent, of several fragments of the protein, result in inhibition of Mo-MuLV virion production, but not of HIV virion production. Conclusions This study shows that endophilins interact with Mo-MuLV Gag and affect virion production. The findings imply that endophilin is another component of the large complex that is hijacked by retroviruses to promote virion production.

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

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

2018-01-01

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

The p2 domain of human immunodeficiency virus type 1 Gag regulates sequential proteolytic processing and is required to produce fully infectious virions.

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Pettit, S C; Moody, M D; Wehbie, R S; Kaplan, A H; Nantermet, P V; Klein, C A; Swanstrom, R

1994-12-01

The proteolytic processing sites of the human immunodeficiency virus type 1 (HIV-1) Gag precursor are cleaved in a sequential manner by the viral protease. We investigated the factors that regulate sequential processing. When full-length Gag protein was digested with recombinant HIV-1 protease in vitro, four of the five major processing sites in Gag were cleaved at rates that differ by as much as 400-fold. Three of these four processing sites were cleaved independently of the others. The CA/p2 site, however, was cleaved approximately 20-fold faster when the adjacent downstream p2/NC site was blocked from cleavage or when the p2 domain of Gag was deleted. These results suggest that the presence of a C-terminal p2 tail on processing intermediates slows cleavage at the upstream CA/p2 site. We also found that lower pH selectively accelerated cleavage of the CA/p2 processing site in the full-length precursor and as a peptide primarily by a sequence-based mechanism rather than by a change in protein conformation. Deletion of the p2 domain of Gag results in released virions that are less infectious despite the presence of the processed final products of Gag. These findings suggest that the p2 domain of HIV-1 Gag regulates the rate of cleavage at the CA/p2 processing site during sequential processing in vitro and in infected cells and that p2 may function in the proper assembly of virions.

Glutamic Acid Residues in HIV-1 p6 Regulate Virus Budding and Membrane Association of Gag.

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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.

Kinetics of proton transport into influenza virions by the viral M2 channel.

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Tijana Ivanovic

Full Text Available M2 protein of influenza A viruses is a tetrameric transmembrane proton channel, which has essential functions both early and late in the virus infectious cycle. Previous studies of proton transport by M2 have been limited to measurements outside the context of the virus particle. We have developed an in vitro fluorescence-based assay to monitor internal acidification of individual virions triggered to undergo membrane fusion. We show that rimantadine, an inhibitor of M2 proton conductance, blocks the acidification-dependent dissipation of fluorescence from a pH-sensitive virus-content probe. Fusion-pore formation usually follows internal acidification but does not require it. The rate of internal virion acidification increases with external proton concentration and saturates with a pK(m of ∼4.7. The rate of proton transport through a single, fully protonated M2 channel is approximately 100 to 400 protons per second. The saturating proton-concentration dependence and the low rate of internal virion acidification derived from authentic virions support a transporter model for the mechanism of proton transfer.

Interactions Between HIV-1 Gag and Viral RNA Genome Enhance Virion Assembly

DEFF Research Database (Denmark)

Dilley, Kari A; Nikolaitchik, Olga A; Galli, Andrea

2017-01-01

between Gag and viral RNA are required for the enhancement of particle production. Taken together, these studies are consistent with our previous hypothesis that specific dimeric viral RNA:Gag interactions are the nucleation event of infectious virion assembly, ensuring that one RNA dimer is packaged......Most HIV-1 virions contain two copies of full-length viral RNA, indicating that genome packaging is efficient and tightly regulated. However, the structural protein Gag is the only component required for the assembly of noninfectious virus-like particles and the viral RNA is dispensable...... in this process. The mechanism that allows HIV-1 to achieve such high efficiency of genome packaging when a packageable viral RNA is not required for virus assembly is currently unknown. In this report, we examined the role of HIV-1 RNA in virus assembly and found that packageable HIV-1 RNA enhances particle...

Analysis of contributions of herpes simplex virus type 1 UL43 protein ...

African Journals Online (AJOL)

Tropical Journal of Pharmaceutical Research June 2016; 15 (6): 1137-1144 ... Revised accepted: 21 May 2016. Abstract ... membrane fusion and efficient entry of virion into target cells. ..... viral envelope with cellular plasma membranes.

Proteomic characterization of murid herpesvirus 4 extracellular virions.

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Sarah Vidick

Full Text Available Gammaherpesvirinae, such as the human Epstein-Barr virus (EBV and the Kaposi's sarcoma associated herpesvirus (KSHV are highly prevalent pathogens that have been associated with several neoplastic diseases. As EBV and KSHV are host-range specific and replicate poorly in vitro, animal counterparts such as Murid herpesvirus-4 (MuHV-4 have been widely used as models. In this study, we used MuHV-4 in order to improve the knowledge about proteins that compose gammaherpesviruses virions. To this end, MuHV-4 extracellular virions were isolated and structural proteins were identified using liquid chromatography tandem mass spectrometry-based proteomic approaches. These analyses allowed the identification of 31 structural proteins encoded by the MuHV-4 genome which were classified as capsid (8, envelope (9, tegument (13 and unclassified (1 structural proteins. In addition, we estimated the relative abundance of the identified proteins in MuHV-4 virions by using exponentially modified protein abundance index analyses. In parallel, several host proteins were found in purified MuHV-4 virions including Annexin A2. Although Annexin A2 has previously been detected in different virions from various families, its role in the virion remains controversial. Interestingly, despite its relatively high abundance in virions, Annexin A2 was not essential for the growth of MuHV-4 in vitro. Altogether, these results extend previous work aimed at determining the composition of gammaherpesvirus virions and provide novel insights for understanding MuHV-4 biology.

A structural model of the genome packaging process in a membrane-containing double stranded DNA virus.

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Chuan Hong

2014-12-01

Full Text Available Two crucial steps in the virus life cycle are genome encapsidation to form an infective virion and genome exit to infect the next host cell. In most icosahedral double-stranded (ds DNA viruses, the viral genome enters and exits the capsid through a unique vertex. Internal membrane-containing viruses possess additional complexity as the genome must be translocated through the viral membrane bilayer. Here, we report the structure of the genome packaging complex with a membrane conduit essential for viral genome encapsidation in the tailless icosahedral membrane-containing bacteriophage PRD1. We utilize single particle electron cryo-microscopy (cryo-EM and symmetry-free image reconstruction to determine structures of PRD1 virion, procapsid, and packaging deficient mutant particles. At the unique vertex of PRD1, the packaging complex replaces the regular 5-fold structure and crosses the lipid bilayer. These structures reveal that the packaging ATPase P9 and the packaging efficiency factor P6 form a dodecameric portal complex external to the membrane moiety, surrounded by ten major capsid protein P3 trimers. The viral transmembrane density at the special vertex is assigned to be a hexamer of heterodimer of proteins P20 and P22. The hexamer functions as a membrane conduit for the DNA and as a nucleating site for the unique vertex assembly. Our structures show a conformational alteration in the lipid membrane after the P9 and P6 are recruited to the virion. The P8-genome complex is then packaged into the procapsid through the unique vertex while the genome terminal protein P8 functions as a valve that closes the channel once the genome is inside. Comparing mature virion, procapsid, and mutant particle structures led us to propose an assembly pathway for the genome packaging apparatus in the PRD1 virion.

A structural model of the genome packaging process in a membrane-containing double stranded DNA virus.

Science.gov (United States)

Hong, Chuan; Oksanen, Hanna M; Liu, Xiangan; Jakana, Joanita; Bamford, Dennis H; Chiu, Wah

2014-12-01

Two crucial steps in the virus life cycle are genome encapsidation to form an infective virion and genome exit to infect the next host cell. In most icosahedral double-stranded (ds) DNA viruses, the viral genome enters and exits the capsid through a unique vertex. Internal membrane-containing viruses possess additional complexity as the genome must be translocated through the viral membrane bilayer. Here, we report the structure of the genome packaging complex with a membrane conduit essential for viral genome encapsidation in the tailless icosahedral membrane-containing bacteriophage PRD1. We utilize single particle electron cryo-microscopy (cryo-EM) and symmetry-free image reconstruction to determine structures of PRD1 virion, procapsid, and packaging deficient mutant particles. At the unique vertex of PRD1, the packaging complex replaces the regular 5-fold structure and crosses the lipid bilayer. These structures reveal that the packaging ATPase P9 and the packaging efficiency factor P6 form a dodecameric portal complex external to the membrane moiety, surrounded by ten major capsid protein P3 trimers. The viral transmembrane density at the special vertex is assigned to be a hexamer of heterodimer of proteins P20 and P22. The hexamer functions as a membrane conduit for the DNA and as a nucleating site for the unique vertex assembly. Our structures show a conformational alteration in the lipid membrane after the P9 and P6 are recruited to the virion. The P8-genome complex is then packaged into the procapsid through the unique vertex while the genome terminal protein P8 functions as a valve that closes the channel once the genome is inside. Comparing mature virion, procapsid, and mutant particle structures led us to propose an assembly pathway for the genome packaging apparatus in the PRD1 virion.

The herpes simplex virus 1 U{sub S}3 regulates phospholipid synthesis

Energy Technology Data Exchange (ETDEWEB)

Wild, Peter, E-mail: pewild@access.uzh.ch [Institute of Veterinary Anatomy, University of Zuerich (Switzerland); Institute of Virology, University of Zuerich (Switzerland); Oliveira, Anna Paula de [Institute of Virology, University of Zuerich (Switzerland); Sonda, Sabrina [Institute for Parasitology, University of Zuerich (Switzerland); Schraner, Elisabeth M. [Institute of Veterinary Anatomy, University of Zuerich (Switzerland); Institute of Virology, University of Zuerich (Switzerland); Ackermann, Mathias; Tobler, Kurt [Institute of Virology, University of Zuerich (Switzerland)

2012-10-25

Herpes simplex virus type 1 capsids bud at nuclear and Golgi membranes for envelopment by phospholipid bilayers. In the absence of U{sub S}3, nuclear membranes form multiple folds harboring virions that suggests disturbance in membrane turnover. Therefore, we investigated phospholipid metabolism in cells infected with the U{sub S}3 deletion mutant R7041({Delta}U{sub S}3), and quantified membranes involved in viral envelopment. We report that (i) [{sup 3}H]-choline incorporation into nuclear membranes and cytoplasmic membranes was enhanced peaking at 12 or 20 h post inoculation with wild type HSV-1 and R7041({Delta}U{sub S}3), respectively, (ii) the surface area of nuclear membranes increased until 24 h of R7041({Delta}U{sub S}3) infection forming folds that equaled {approx}45% of the nuclear surface, (iii) the surface area of viral envelopes between nuclear membranes equaled {approx}2400 R7041({Delta}U{sub S}3) virions per cell, and (iv) during R7041({Delta}U{sub S}3) infection, the Golgi complex expanded dramatically. The data indicate that U{sub S}3 plays a significant role in regulation of membrane biosynthesis.

Enzymatic treatment of duck hepatitis B virus: Topology of the surface proteins for virions and noninfectious subviral particles

International Nuclear Information System (INIS)

Franke, Claudia; Matschl, Urte; Bruns, Michael

2007-01-01

The large surface antigen L of duck hepatitis B virus exhibits a mixed topology with the preS domains of the protein alternatively exposed to the particles' interior or exterior. After separating virions from subviral particles (SVPs), we compared their L topologies and showed that both particle types exhibit the same amount of L with the following differences: 1-preS of intact virions was enzymatically digested with chymotrypsin, whereas in SVPs only half of preS was accessible, 2-phosphorylation of L at S118 was completely removed by phosphatase treatment only in virions, 3-iodine-125 labeling disclosed a higher ratio of exposed preS to S domains in virions compared to SVPs. These data point towards different surface architectures of virions and SVPs. Because the preS domain acts in binding to a cellular receptor of hepatocytes, our findings implicate the exclusion of SVPs as competitors for the receptor binding and entry of virions

Epiretinal membrane: a treatable cause of visual disability in myotonic dystrophy type 1.

Science.gov (United States)

Kersten, Hannah M; Roxburgh, Richard H; Child, Nicholas; Polkinghorne, Philip J; Frampton, Chris; Danesh-Meyer, Helen V

2014-01-01

A wide range of ocular abnormalities have been documented to occur in patients with myotonic dystrophy type 1. The objectives of this study were to investigate the macular and optic nerve morphology using optical coherence tomography in patients with myotonic dystrophy type 1. A total of 30 myotonic dystrophy type 1 patients and 28 controls were recruited for participation. All participants underwent a thorough ophthalmologic examination, including spectral-domain optical coherence tomography of the macula and retinal nerve fibre layer. Images were reviewed by a retinal specialist ophthalmologist, masked to the diagnosis of the participants. Average macular thickness was significantly greater in the myotonic dystrophy group compared to controls [327.3 μm vs. 308.5 μm (p Visual acuity was reduced due to the presence of epiretinal membrane in six patient eyes and none of the control eyes. The presence of an epiretinal membrane was significantly correlated with increasing age in the patient group. We report an increased prevalence of epiretinal membrane in the myotonic dystrophy type 1 group. This may be a previously under-recognised form of visual impairment in this group. Epiretinal membranes can be treated surgically. We suggest that, in addition to a comprehensive clinical examination, optical coherence tomography examination is implemented as part of an ophthalmological assessment for the myotonic dystrophy type 1 patient with reduced visual acuity.

Syncytial Hepatitis of Tilapia ( Oreochromis niloticus L.) is Associated With Orthomyxovirus-Like Virions in Hepatocytes.

Science.gov (United States)

Del-Pozo, J; Mishra, N; Kabuusu, R; Cheetham, S; Eldar, A; Bacharach, E; Lipkin, W I; Ferguson, H W

2017-01-01

Using transmission electron microscopy (TEM), the presented work expands on the ultrastructural findings of an earlier report on "syncytial hepatitis," a novel disease of tilapia (SHT). Briefly, TEM confirmed the presence of an orthomyxovirus-like virus within the diseased hepatocytes but not within the endothelium. This was supported by observing extracellular and intracellular (mostly intraendosomal), 60-100 nm round virions with a trilaminar capsid containing up to 7 electron-dense aggregates. Other patterns noted included enveloped or filamentous virions and virion-containing cytoplasmic membrane folds, suggestive of endocytosis. Patterns atypical for orthymyxovirus included the formation of syncytia and the presence of virions within the perinuclear cisternae (suspected to be the Golgi apparatus). The ultrastructural morphology of SHT-associated virions is similar to that previously reported for tilapia lake virus (TiLV). A genetic homology was investigated using the available reverse transcriptase polymerase chain reaction (RT-PCR) probes for TiLV and comparing clinically sick with clinically normal fish and negative controls. By RT-PCR analysis, viral nucleic acid was detected only in diseased fish. Taken together, these findings strongly suggest that a virus is causally associated with SHT, that this virus shares ultrastructural features with orthomyxoviruses, and it presents with partial genetic homology with TiLV (190 nucleotides).

A membrane-anchored E-type endo-1,4-beta-glucanase is localized on Golgi and plasma membranes of higher plants.

Science.gov (United States)

Brummell, D A; Catala, C; Lashbrook, C C; Bennett, A B

1997-04-29

Endo-1,4-beta-D-glucanases (EGases, EC 3.2.1.4) are enzymes produced in bacteria, fungi, and plants that hydrolyze polysaccharides possessing a 1,4-beta-D-glucan backbone. All previously identified plant EGases are E-type endoglucanases that possess signal sequences for endoplasmic reticulum entry and are secreted to the cell wall. Here we report the characterization of a novel E-type plant EGase (tomato Cel3) with a hydrophobic transmembrane domain and structure typical of type II integral membrane proteins. The predicted protein is composed of 617 amino acids and possesses seven potential sites for N-glycosylation. Cel3 mRNA accumulates in young vegetative tissues with highest abundance during periods of rapid cell expansion, but is not hormonally regulated. Antibodies raised to a recombinant Cel3 protein specifically recognized three proteins, with apparent molecular masses of 93, 88, and 53 kDa, in tomato root microsomal membranes separated by sucrose density centrifugation. The 53-kDa protein comigrated in the gradient with plasma membrane markers, the 88-kDa protein with Golgi membrane markers, and the 93-kDa protein with markers for both Golgi and plasma membranes. EGase enzyme activity was also found in regions of the density gradient corresponding to both Golgi and plasma membranes, suggesting that Cel3 EGase resides in both membrane systems, the sites of cell wall polymer biosynthesis. The in vivo function of Cel3 is not known, but the only other known membrane-anchored EGase is present in Agrobacterium tumefaciens where it is required for cellulose biosynthesis.

Organization of cellular receptors into a nanoscale junction during HIV-1 adhesion.

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Terrence M Dobrowsky

2010-07-01

Full Text Available The fusion of the human immunodeficiency virus type 1 (HIV-1 with its host cell is the target for new antiretroviral therapies. Viral particles interact with the flexible plasma membrane via viral surface protein gp120 which binds its primary cellular receptor CD4 and subsequently the coreceptor CCR5. However, whether and how these receptors become organized at the adhesive junction between cell and virion are unknown. Here, stochastic modeling predicts that, regarding binding to gp120, cellular receptors CD4 and CCR5 form an organized, ring-like, nanoscale structure beneath the virion, which locally deforms the plasma membrane. This organized adhesive junction between cell and virion, which we name the viral junction, is reminiscent of the well-characterized immunological synapse, albeit at much smaller length scales. The formation of an organized viral junction under multiple physiopathologically relevant conditions may represent a novel intermediate step in productive infection.

COMPARISON OF DETERGENTS FOR EXTRACTION AND ION-EXCHANGE HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHY OF SENDAI VIRUS MEMBRANE-PROTEINS

NARCIS (Netherlands)

WELLING, GW; FEIJLBRIEF, M; ORVELL, C; VANEDE, J; WELLINGWESTER, S

1992-01-01

The integral membrane proteins of Sendai virus, haemagglutinin-neuraminidase (HN) and fusion protein (F) were extracted from purified virions with a non-ionic and two zwitterionic detergents, i.e.. pentaethylene glycol monolauryl ether (C-12E5), lauryldimethylamine oxide (LDAO) and

Identification of carbohydrate-binding domains in the attachment proteins of type 1 and type 3 reoviruses.

Science.gov (United States)

Chappell, J D; Duong, J L; Wright, B W; Dermody, T S

2000-09-01

The reovirus attachment protein, sigma1, is responsible for strain-specific patterns of viral tropism in the murine central nervous system and receptor binding on cultured cells. The sigma1 protein consists of a fibrous tail domain proximal to the virion surface and a virion-distal globular head domain. To better understand mechanisms of reovirus attachment to cells, we conducted studies to identify the region of sigma1 that binds cell surface carbohydrate. Chimeric and truncated sigma1 proteins derived from prototype reovirus strains type 1 Lang (T1L) and type 3 Dearing (T3D) were expressed in insect cells by using a baculovirus vector. Assessment of expressed protein susceptibility to proteolytic cleavage, binding to anti-sigma1 antibodies, and oligomerization indicates that the chimeric and truncated sigma1 proteins are properly folded. To assess carbohydrate binding, recombinant sigma1 proteins were tested for the capacity to agglutinate mammalian erythrocytes and to bind sialic acid presented on glycophorin, the cell surface molecule bound by type 3 reovirus on human erythrocytes. Using a panel of two wild-type and ten chimeric and truncated sigma1 proteins, the sialic acid-binding domain of type 3 sigma1 was mapped to a region of sequence proposed to form the more amino terminal of two predicted beta-sheet structures in the tail. This unit corresponds to morphologic region T(iii) observed in computer-processed electron micrographs of sigma1 protein purified from virions. In contrast, the homologous region of T1L sigma1 sequence was not implicated in carbohydrate binding; rather, sequences in the distal portion of the tail known as the neck were required. Results of these studies demonstrate that a functional receptor-binding domain, which uses sialic acid as its ligand, is contained within morphologic region T(iii) of the type 3 sigma1 tail. Furthermore, our findings indicate that T1L and T3D sigma1 proteins contain different arrangements of receptor

The herpes simplex virus 1 UL51 protein interacts with the UL7 protein and plays a role in its recruitment into the virion.

Science.gov (United States)

Roller, Richard J; Fetters, Rachel

2015-03-01

The alphaherpesvirus UL51 protein is a tegument component that interacts with the viral glycoprotein E and functions at multiple steps in virus assembly and spread in epithelial cells. We show here that pUL51 forms a complex in infected cells with another conserved tegument protein, pUL7. This complex can form in the absence of other viral proteins and is largely responsible for recruitment of pUL7 to cytoplasmic membranes and into the virion tegument. Incomplete colocalization of pUL51 and pUL7 in infected cells, however, suggests that a significant fraction of the population of each protein is not complexed with the other and that they may accomplish independent functions. The ability of herpesviruses to spread from cell to cell in the face of an immune response is critical for disease and shedding following reactivation from latency. Cell-to-cell spread is a conserved ability of herpesviruses, and the identification of conserved viral genes that mediate this process will aid in the design of attenuated vaccines and of novel therapeutics. The conserved UL51 gene of herpes simplex virus 1 plays important roles in cell-to-cell spread and in virus assembly in the cytoplasm, both of which likely depend on specific interactions with other viral and cellular proteins. Here we identify one of those interactions with the product of another conserved herpesvirus gene, UL7, and show that formation of this complex mediates recruitment of UL7 to membranes and to the virion. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Intracellular HIV-1 Gag localization is impaired by mutations in the nucleocapsid zinc fingers

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Muriaux Delphine

2007-08-01

Full Text Available Abstract Background The HIV-1 nucleocapsid protein (NC is formed of two CCHC zinc fingers flanked by highly basic regions. HIV-1 NC plays key roles in virus structure and replication via its nucleic acid binding and chaperoning properties. In fact, NC controls proviral DNA synthesis by reverse transcriptase (RT, gRNA dimerization and packaging, and virion assembly. Results We previously reported a role for the first NC zinc finger in virion structure and replication 1. To investigate the role of both NC zinc fingers in intracellular Gag trafficking, and in virion assembly, we generated series of NC zinc fingers mutations. Results show that all Zinc finger mutations have a negative impact on virion biogenesis and maturation and rendered defective the mutant viruses. The NC zinc finger mutations caused an intracellular accumulation of Gag, which was found either diffuse in the cytoplasm or at the plasma membrane but not associated with endosomal membranes as for wild type Gag. Evidences are also provided showing that the intracellular interactions between NC-mutated Gag and the gRNA were impaired. Conclusion These results show that Gag oligomerization mediated by gRNA-NC interactions is required for correct Gag trafficking, and assembly in HIV-1 producing cells and the release of infectious viruses.

Computational study of the RGD-peptide interactions with perovskite-type BFO-(1 1 1) membranes under aqueous conditions

Science.gov (United States)

Li, Hai-long; Bian, Liang; Hou, Wen-ping; Dong, Fa-Qin; Song, Mian-Xin; Zhang, Xiao-yan; Wang, Li-sheng

2016-07-01

We elucidated a number of facets regarding arginine-glycine-aspartate (RGD)-bismuth ferrite (BFO)-(1 1 1) membrane interactions and reactivity that have previously remained unexplored on a molecular level. Results demonstrate the intra-molecular interaction facilitates a ;horseshoe; structure of RGD adsorbed onto the BFO-(1 1 1) membrane, through the electrostatic (Asp-cation-Fe) and water-bridge (Osbnd H2O and H2Osbnd NH2) interactions. The effect of structural and electron-transfer interactions is attributed to the cation-valences, indicating that the divalent cations are electron-acceptors and the monovalent cations as electron-donors. Notably, the strongly bound Ca2+ ion exerts a ;gluing; effect on the Asp-side-chain, indicating a tightly packed RGD-BFO configuration. Thus, modulating the biological response of BFO-(1 1 1) membrane will allow us to design more appropriate interfaces for implantable diagnostic and therapeutic perovskite-type micro-devices.

UV-Sensitivity of Shiga Toxin-Converting Bacteriophage Virions Φ24B, 933W, P22, P27 and P32

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Sylwia Bloch

2015-09-01

Full Text Available Shiga toxin-converting bacteriophages (Stx phages are present as prophages in Shiga toxin-producing Escherichia coli (STEC strains. Theses phages can be transmitted to previously non-pathogenic E. coli cells making them potential producers of Shiga toxins, as they bear genes for these toxins in their genomes. Therefore, sensitivity of Stx phage virions to various conditions is important in both natural processes of spreading of these viruses and potential prophylactic control of appearance of novel pathogenic E. coli strains. In this report we provide evidence that virions of Stx phages are significantly more sensitive to UV irradiation than bacteriophage λ. Following UV irradiation of Stx virions at the dose of 50 J/m2, their infectivity dropped by 1–3 log10, depending on the kind of phage. Under these conditions, a considerable release of phage DNA from virions was observed, and electron microscopy analyses indicated a large proportion of partially damaged virions. Infection of E. coli cells with UV-irradiated Stx phages resulted in significantly decreased levels of expression of N and cro genes, crucial for lytic development. We conclude that inactivation of Stx virions caused by relatively low dose of UV light is due to damage of capsids that prevents effective infection of the host cells.

Biliary Secretion of Quasi-Enveloped Human Hepatitis A Virus

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Asuka Hirai-Yuki

2016-12-01

Full Text Available Hepatitis A virus (HAV is an unusual picornavirus that is released from cells cloaked in host-derived membranes. These quasi-enveloped virions (eHAV are the only particle type circulating in blood during infection, whereas only nonenveloped virions are shed in feces. The reason for this is uncertain. Hepatocytes, the only cell type known to support HAV replication in vivo, are highly polarized epithelial cells with basolateral membranes facing onto hepatic (blood sinusoids and apical membranes abutting biliary canaliculi from which bile is secreted to the gut. To assess whether eHAV and nonenveloped virus egress from cells via vectorially distinct pathways, we studied infected polarized cultures of Caco-2 and HepG2-N6 cells. Most (>99% progeny virions were released apically from Caco-2 cells, whereas basolateral (64% versus apical (36% release was more balanced with HepG2-N6 cells. Both apically and basolaterally released virions were predominantly enveloped, with no suggestion of differential vectorial release of eHAV versus naked virions. Basolateral to apical transcytosis of either particle type was minimal (<0.02%/h in HepG2-N6 cells, arguing against this as a mechanism for differences in membrane envelopment of serum versus fecal virus. High concentrations of human bile acids converted eHAV to nonenveloped virions, whereas virus present in bile from HAV-infected Ifnar1−/−Ifngr1−/− and Mavs−/− mice banded over a range of densities extending from that of eHAV to that of nonenveloped virions. We conclude that nonenveloped virions shed in feces are derived from eHAV released across the canalicular membrane and stripped of membranes by the detergent action of bile acids within the proximal biliary canaliculus.

Retroviral Gag protein-RNA interactions: Implications for specific genomic RNA packaging and virion assembly.

Science.gov (United States)

Olson, Erik D; Musier-Forsyth, Karin

2018-03-31

Retroviral Gag proteins are responsible for coordinating many aspects of virion assembly. Gag possesses two distinct nucleic acid binding domains, matrix (MA) and nucleocapsid (NC). One of the critical functions of Gag is to specifically recognize, bind, and package the retroviral genomic RNA (gRNA) into assembling virions. Gag interactions with cellular RNAs have also been shown to regulate aspects of assembly. Recent results have shed light on the role of MA and NC domain interactions with nucleic acids, and how they jointly function to ensure packaging of the retroviral gRNA. Here, we will review the literature regarding RNA interactions with NC, MA, as well as overall mechanisms employed by Gag to interact with RNA. The discussion focuses on human immunodeficiency virus type-1, but other retroviruses will also be discussed. A model is presented combining all of the available data summarizing the various factors and layers of selection Gag employs to ensure specific gRNA packaging and correct virion assembly. Copyright © 2018 Elsevier Ltd. All rights reserved.

Enterovirus 71 virion-associated galectin-1 facilitates viral replication and stability.

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Pei-Huan Lee

Full Text Available Enterovirus 71 (EV71 infection causes a myriad of diseases from mild hand-foot-and-mouth disease or herpangina to fatal brain stem encephalitis complicated with pulmonary edema. Several severe EV71 endemics have occurred in Asia-Pacific region, including Taiwan, and have become a serious threat to children's health. EV71 infection is initiated by the attachment of the virion to the target cell surface. Although this process relies primarily upon interaction between viruses and cell surface receptors, soluble factors may also influence the binding of EV71 to host cells. Galectin-1 has been reported to participate in several virus infections, but is not addressed in EV71. In this study, we found that the serum levels of galectin-1 in EV71-infected children were higher than those in non-infected people. In EV71 infected cells, galectin-1 was found to be associated with the EV71 VP1 and VP3 via carbohydrate residues and subsequently released and bound to another cell surface along with the virus. EV71 propagated from galectin-1 knockdown SK-N-SH cells exhibited lower infectivity in cultured cells and less pathogenicity in mice than the virus propagated from parental cells. In addition, this galectin-1-free EV71 virus was sensitive to high temperature and lost its viability after long-term storage, which could be restored following supplement of recombinant galectin-1. Taken together, our findings uncover a new role of galectin-1 in facilitating EV71 virus infection.

Glycoprotein H of herpes simplex virus type 1 requires glycoprotein L for transport to the surfaces of insect cells

NARCIS (Netherlands)

Westra, DF; Glazenburg, KL; Harmsen, MC; Tiran, A; Scheffer, AJ; Welling, GW; The, TH; WellingWester, S

In mammalian cells, formation of heterooligomers consisting of the glycoproteins H and L (gH and gL) of herpes simplex virus type 1 is essential for the cell-to-cell spread of virions and for the penetration of virions into cells. We examined whether formation of gH1/gL1 heterooligomers and cell

Deletion of the AcMNPV core gene ac109 results in budded virions that are non-infectious

International Nuclear Information System (INIS)

Fang Minggang; Nie, Yingchao; Theilmann, David A.

2009-01-01

Autographa californica multiple nucleopolyhedrovirus (AcMNPV) ac109 is a core gene and its function in the virus life cycle is unknown. To determine its role in the baculovirus life cycle, we used the AcMNPV bacmid system to generate an ac109 deletion virus (vAc 109KO ). Fluorescence and light microscopy showed that transfection of vAc 109KO results in a single-cell infection phenotype. Viral DNA replication is unaffected and the development of occlusion bodies in vAc 109KO -transfected cells evidenced progression to the very late phases of viral infection. Western blot and confocal immunofluorescence analysis showed that AC109 is expressed in the cytoplasm and nucleus throughout infection. In addition, AC109 is a structural protein as it was detected in both budded virus (BV) and occlusion derived virus in both the envelope and nucleocapsid fractions. Titration assays by qPCR and TCID 50 showed that vAc 109KO produced BV but the virions are non-infectious. The vAc 109KO BV were indistinguishable from the BV of repaired and wild type control viruses as determined by negative staining and electron microscopy.

P2X7 receptors regulate multiple types of membrane trafficking responses and non-classical secretion pathways.

Science.gov (United States)

Qu, Yan; Dubyak, George R

2009-06-01

Activation of the P2X7 receptor (P2X7R) triggers a remarkably diverse array of membrane trafficking responses in leukocytes and epithelial cells. These responses result in altered profiles of cell surface lipid and protein composition that can modulate the direct interactions of P2X7R-expressing cells with other cell types in the circulation, in blood vessels, at epithelial barriers, or within sites of immune and inflammatory activation. Additionally, these responses can result in the release of bioactive proteins, lipids, and large membrane complexes into extracellular compartments for remote communication between P2X7R-expressing cells and other cells that amplify or modulate inflammation, immunity, and responses to tissue damages. This review will discuss P2X7R-mediated effects on membrane composition and trafficking in the plasma membrane (PM) and intracellular organelles, as well as actions of P2X7R in controlling various modes of non-classical secretion. It will review P2X7R regulation of: (1) phosphatidylserine distribution in the PM outer leaflet; (2) shedding of PM surface proteins; (3) release of PM-derived microvesicles or microparticles; (4) PM blebbing; (5) cell-cell fusion resulting in formation of multinucleate cells; (6) phagosome maturation and fusion with lysosomes; (7) permeability of endosomes with internalized pathogen-associated molecular patterns; (8) permeability/integrity of mitochondria; (9) exocytosis of secretory lysosomes; and (10) release of exosomes from multivesicular bodies.

Amphipathic alpha-helices and putative cholesterol binding domains of the influenza virus matrix M1 protein are crucial for virion structure organisation.

Science.gov (United States)

Tsfasman, Tatyana; Kost, Vladimir; Markushin, Stanislav; Lotte, Vera; Koptiaeva, Irina; Bogacheva, Elena; Baratova, Ludmila; Radyukhin, Victor

2015-12-02

The influenza virus matrix M1 protein is an amphitropic membrane-associated protein, forming the matrix layer immediately beneath the virus raft membrane, thereby ensuring the proper structure of the influenza virion. The objective of this study was to elucidate M1 fine structural characteristics, which determine amphitropic properties and raft membrane activities of the protein, via 3D in silico modelling with subsequent mutational analysis. Computer simulations suggest the amphipathic nature of the M1 α-helices and the existence of putative cholesterol binding (CRAC) motifs on six amphipathic α-helices. Our finding explains for the first time many features of this protein, particularly the amphitropic properties and raft/cholesterol binding potential. To verify these results, we generated mutants of the A/WSN/33 strain via reverse genetics. The M1 mutations included F32Y in the CRAC of α-helix 2, W45Y and W45F in the CRAC of α-helix 3, Y100S in the CRAC of α-helix 6, M128A and M128S in the CRAC of α-helix 8 and a double L103I/L130I mutation in both a putative cholesterol consensus motif and the nuclear localisation signal. All mutations resulted in viruses with unusual filamentous morphology. Previous experimental data regarding the morphology of M1-gene mutant influenza viruses can now be explained in structural terms and are consistent with the pivotal role of the CRAC-domains and amphipathic α-helices in M1-lipid interactions. Copyright © 2015 Elsevier B.V. All rights reserved.

The host outer membrane proteins OmpA and OmpC are associated with the Shigella phage Sf6 virion

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Zhao Haiyan; Sequeira, Reuben D.; Galeva, Nadezhda A.; Tang Liang

2011-01-01

Assembly of dsDNA bacteriophage is a precisely programmed process. Potential roles of host cell components in phage assembly haven't been well understood. It was previously reported that two unidentified proteins were present in bacteriophage Sf6 virion (Casjens et al, 2004, J.Mol.Biol. 339, 379-394, Fig. 2A). Using tandem mass spectrometry, we have identified the two proteins as outer membrane proteins (OMPs) OmpA and OmpC from its host Shigella flexneri. The transmission electron cryo-microscopy structure of Sf6 shows significant density at specific sites at the phage capsid inner surface. This density fit well with the characteristic beta-barrel domains of OMPs, thus may be due to the two host proteins. Locations of this density suggest a role in Sf6 morphogenesis reminiscent of phage-encoded cementing proteins. These data indicate a new, OMP-related phage:host linkage, adding to previous knowledge that some lambdoid bacteriophage genomes contain OmpC-like genes that express phage-encoded porins in the lysogenic state.

Phosphatidylserine Lateral Organization Influences the Interaction of Influenza Virus Matrix Protein 1 with Lipid Membranes.

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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

[Effect of extracted ZG from gardenia on Hep-2 cell membrane post infected with parainfluenza virus type 1 (PIV-1)].

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Guo, Shan-Shan; Huang, Yang; Zhao, Ye; Gao, Ying-Jie; Gong, Wen-Feng; Cui, Xiao-Lan

2007-09-01

In order to study the anti-viral mechanism of extracted ZG from Gardenia, the effect of extracted ZG on Hep-2 cell membrane potential, Na -K+-ATPase activity and membrane fluidity post infected with parainfluenza virus type 1 (PIV-1) was observed. Acetylcholine which was fluorescent labeled with DiBAC4 (3) was taken as positive control to observe the changes of membrane potential and was measured by flow cytometer. The phosphorus determination method and spectrophotometer were used to measure the Na+-K+-ATPase activity of Hep-2 cell membrane post PIV-1 infection. Hep-2 cell membrane phospholipids was labeled with fluorescent NBD-C6-HPC and membrane fluidity was measured by confocal laser scanning microscope. The results demonstated that after PIV-1 infection the Hep-2 cell membrane potential decreased significantly and the membrane was in the state of hyperpolarization, Na+-K+-ATPase activity increased and membrane fluidity decreased significantly. There was no apparent interferring effect of extracted ZG on the changes of membrane potential and Na+-K+-ATPase activity post PIV-1 infection, while membrane fluidity was improved significantly. Acetylcholine improved the state of hyperpolarization. The changes of membrane potential, Na -K+-ATPase activity and membrane fluidity might be the biomechanism of PIV-1 infectoin. The extracted ZG improved membrane fluidity to prevent from PIV-1 infection by protecting the cell membrane, which was probably the mechanism of anti-PIV-1 activity of the extracted ZG, but ZG probably had nothing to do with membrane potential and Na+-K+-ATPase activity.

Type-1 cannabinoid receptors reduce membrane fluidity of capacitated boar sperm by impairing their activation by bicarbonate.

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Barbara Barboni

Full Text Available BACKGROUND: Mammalian spermatozoa acquire their full fertilizing ability (so called capacitation within the female genital tract, where they are progressively exposed to inverse gradients of inhibiting and stimulating molecules. METHODOLOGY/PRINCIPAL FINDINGS: In the present research, the effect on this process of anandamide, an endocannabinoid that can either activate or inhibit cannabinoid receptors depending on its concentration, and bicarbonate, an oviductal activatory molecule, was assessed, in order to study the role exerted by the type 1 cannabinoid receptor (CB1R in the process of lipid membrane remodeling crucial to complete capacitation. To this aim, boar sperm were incubated in vitro under capacitating conditions (stimulated by bicarbonate in the presence or in the absence of methanandamide (Met-AEA, a non-hydrolysable analogue of anandamide. The CB1R involvement was studied by using the specific inhibitor (SR141716 or mimicking its activation by adding a permeable cAMP analogue (8Br-cAMP. By an immunocytochemistry approach it was shown that the Met-AEA inhibits the bicarbonate-dependent translocation of CB1R from the post-equatorial to equatorial region of sperm head. In addition it was found that Met-AEA is able to prevent the bicarbonate-induced increase in membrane disorder and the cholesterol extraction, both preliminary to capacitation, acting through a CB1R-cAMP mediated pathway, as indicated by MC540 and filipin staining, EPR spectroscopy and biochemical analysis on whole membranes (CB1R activity and on membrane enriched fraction (C/P content and anisotropy. CONCLUSIONS/SIGNIFICANCE: Altogether, these data demonstrate that the endocannabinoid system strongly inhibits the process of sperm capacitation, acting as membrane stabilizing agent, thus increasing the basic knowledge on capacitation-related signaling and potentially opening new perspectives in diagnostics and therapeutics of male infertility.

The Human ABCG1 Transporter Mobilizes Plasma Membrane and Late Endosomal Non-Sphingomyelin-Associated-Cholesterol for Efflux and Esterification

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Edward B. Neufeld

2014-12-01

Full Text Available We have previously shown that GFP-tagged human ABCG1 on the plasma membrane (PM and in late endosomes (LE mobilizes sterol on both sides of the membrane lipid bilayer, thereby increasing cellular cholesterol efflux to lipid surfaces. In the present study, we examined ABCG1-induced changes in membrane cholesterol distribution, organization, and mobility. ABCG1-GFP expression increased the amount of mobile, non-sphingomyelin(SM-associated cholesterol at the PM and LE, but not the amount of SM-associated-cholesterol or SM. ABCG1-mobilized non-SM-associated-cholesterol rapidly cycled between the PM and LE and effluxed from the PM to extracellular acceptors, or, relocated to intracellular sites of esterification. ABCG1 increased detergent-soluble pools of PM and LE cholesterol, generated detergent-resistant, non-SM-associated PM cholesterol, and increased resistance to both amphotericin B-induced (cholesterol-mediated and lysenin-induced (SM-mediated cytolysis, consistent with altered organization of both PM cholesterol and SM. ABCG1 itself resided in detergent-soluble membrane domains. We propose that PM and LE ABCG1 residing at the phase boundary between ordered (Lo and disordered (Ld membrane lipid domains alters SM and cholesterol organization thereby increasing cholesterol flux between Lo and Ld, and hence, the amount of cholesterol available for removal by acceptors on either side of the membrane bilayer for either efflux or esterification.

Importance of the short cytoplasmic domain of the feline immunodeficiency virus transmembrane glycoprotein for fusion activity and envelope glycoprotein incorporation into virions

International Nuclear Information System (INIS)

Celma, Cristina C.P.; Paladino, Monica G.; Gonzalez, Silvia A.; Affranchino, Jose L.

2007-01-01

The mature form of the envelope (Env) glycoprotein of lentiviruses is a heterodimer composed of the surface (SU) and transmembrane (TM) subunits. Feline immunodeficiency virus (FIV) possesses a TM glycoprotein with a cytoplasmic tail of approximately 53 amino acids which is unusually short compared with that of the other lentiviral glycoproteins (more than 100 residues). To investigate the relevance of the FIV TM cytoplasmic domain to Env-mediated viral functions, we characterized the biological properties of a series of Env glycoproteins progressively shortened from the carboxyl terminus. All the mutant Env proteins were efficiently expressed in feline cells and processed into the SU and TM subunits. Deletion of 5 or 11 amino acids from the TM C-terminus did not significantly affect Env surface expression, fusogenic activity or Env incorporation into virions, whereas removal of 17 or 23 residues impaired Env-mediated cell-to-cell fusion. Further truncation of the FIV TM by 29 residues resulted in an Env glycoprotein that was poorly expressed at the cell surface, exhibited only 20% of the wild-type Env fusogenic capacity and was inefficiently incorporated into virions. Remarkably, deletion of the TM C-terminal 35 or 41 amino acids restored or even enhanced Env biological functions. Indeed, these mutant Env glycoproteins bearing cytoplasmic domains of 18 or 12 amino acids were found to be significantly more fusogenic than the wild-type Env and were efficiently incorporated into virions. Interestingly, truncation of the TM cytoplasmic domain to only 6 amino acids did not affect Env incorporation into virions but abrogated Env fusogenicity. Finally, removal of the entire TM cytoplasmic tail or deletion of as many as 6 amino acids into the membrane-spanning domain led to a complete loss of Env functions. Our results demonstrate that despite its relatively short length, the FIV TM cytoplasmic domain plays an important role in modulating Env-mediated viral functions

Influence of membrane fluidity on human immunodeficiency virus type 1 entry

International Nuclear Information System (INIS)

Harada, Shinji; Yusa, Keisuke; Monde, Kazuaki; Akaike, Takaaki; Maeda, Yosuke

2005-01-01

For penetration of human immunodeficiency virus type 1 (HIV-1), formation of fusion-pores might be required for accumulating critical numbers of fusion-activated gp41, followed by multiple-site binding of gp120 with receptors, with the help of fluidization of the plasma membrane and viral envelope. Correlation between HIV-1 infectivity and fluidity was observed by treatment of fluidity-modulators, indicating that infectivity was dependent on fluidity. A 5% decrease in fluidity suppressed the HIV-1 infectivity by 56%. Contrarily, a 5% increase in fluidity augmented the infectivity by 2.4-fold. An increased temperature of 40 deg C or treatment of 0.2% xylocaine after viral adsorption at room temperature enhanced the infectivity by 2.6- and 1.5-fold, respectively. These were inhibited by anti-CXCR4 peptide, implying that multiple-site binding was accelerated at 40 deg C or by xylocaine. Thus, fluidity of both the plasma membrane and viral envelope was required to form the fusion-pore and to complete the entry of HIV-1

Proteomic analysis of the herpes simplex virus 1 virion protein 16 transactivator protein in infected cells.

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Suk, Hyung; Knipe, David M

2015-06-01

The herpes simplex virus 1 virion protein 16 (VP16) tegument protein forms a transactivation complex with the cellular proteins host cell factor 1 (HCF-1) and octamer-binding transcription factor 1 (Oct-1) upon entry into the host cell. VP16 has also been shown to interact with a number of virion tegument proteins and viral glycoprotein H to promote viral assembly, but no comprehensive study of the VP16 proteome has been performed at early times postinfection. We therefore performed a proteomic analysis of VP16-interacting proteins at 3 h postinfection. We confirmed the interaction of VP16 with HCF-1 and a large number of cellular Mediator complex proteins, but most surprisingly, we found that the major viral protein associating with VP16 is the infected cell protein 4 (ICP4) immediate-early (IE) transactivator protein. These results raise the potential for a new function for VP16 in associating with the IE ICP4 and playing a role in transactivation of early and late gene expression, in addition to its well-documented function in transactivation of IE gene expression. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Structural evolution of the P22-like phages: Comparison of Sf6 and P22 procapsid and virion architectures

Energy Technology Data Exchange (ETDEWEB)

Parent, Kristin N. [University of California, San Diego, Department of Chemistry and Biochemistry, La Jolla, CA 92093 (United States); Gilcrease, Eddie B. [University of Utah School of Medicine, Division of Microbiology and Immunology, Department of Pathology, Salt Lake City, UT 84112 (United States); Casjens, Sherwood R., E-mail: sherwood.casjens@path.utah.edu [University of Utah School of Medicine, Division of Microbiology and Immunology, Department of Pathology, Salt Lake City, UT 84112 (United States); Baker, Timothy S., E-mail: tsb@ucsd.edu [University of California, San Diego, Department of Chemistry and Biochemistry, La Jolla, CA 92093 (United States); University of California, San Diego, Division of Biological Sciences, La Jolla, CA 92093 (United States)

2012-06-05

Coat proteins of tailed, dsDNA phages and in herpesviruses include a conserved core similar to the bacteriophage HK97 subunit. This core is often embellished with other domains such as the telokin Ig-like domain of phage P22. Eighty-six P22-like phages and prophages with sequenced genomes share a similar set of virion assembly genes and, based on comparisons of twelve viral assembly proteins (structural and assembly/packaging chaperones), these phages are classified into three groups (P22-like, Sf6-like, and CUS-3-like). We used cryo-electron microscopy and 3D image reconstruction to determine the structures of Sf6 procapsids and virions ({approx} 7 A resolution), and the structure of the entire, asymmetric Sf6 virion (16-A resolution). The Sf6 coat protein is similar to that of P22 yet it has differences in the telokin domain and in its overall quaternary organization. Thermal stability and agarose gel experiments show that Sf6 virions are slightly less stable than those of P22. Finally, bacterial host outer membrane proteins A and C were identified in lipid vesicles that co-purify with Sf6 particles, but are not components of the capsid.

Membrane potential and ion transport in lung epithelial type II cells

International Nuclear Information System (INIS)

Gallo, R.L.

1986-01-01

The alveolar type II pneumocyte is critically important to the function and maintenance of pulmonary epithelium. To investigate the nature of the response of type II cells to membrane injury, and describe a possible mechanism by which these cells regulate surfactant secretion, the membrane potential of isolated rabbit type II cells was characterized. This evaluation was accomplished by measurements of the accumulation of the membrane potential probes: [ 3 H]triphenylmethylphosphonium ([ 3 H]TPMP + ), rubidium 86, and the fluorescent dye DiOC 5 . A compartmental analysis of probe uptake into mitochondrial, cytoplasmic, and non-membrane potential dependent stores was made through the use of selective membrane depolarizations with carbonycyanide M-chlorophenylhydrazone (CCCP), and lysophosphatidylcholine (LPC). These techniques and population analysis with flow cytometry, permitted the accurate evaluation of type II cell membrane potential under control conditions and under conditions which stimulated cell activity. Further analysis of ion transport by cells exposed to radiation or adrenergic stimulation revealed a common increase in Na + /K + ATPase activity, and an increase in sodium influx across the plasma membrane. This sodium influx was found to be a critical step in the initiation of surfactant secretion. It is concluded that radiation exposure as well as other pulmonary toxicants can directly affect the membrane potential and ionic regulation of type II cells. Ion transport, particularly of sodium, plays an important role in the regulation of type II cell function

Virion assembly factories in the nucleus of polyomavirus-infected cells.

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Kimberly D Erickson

Full Text Available Most DNA viruses replicate in the cell nucleus, although the specific sites of virion assembly are as yet poorly defined. Electron microscopy on freeze-substituted, plastic-embedded sections of murine polyomavirus (PyV-infected 3T3 mouse fibroblasts or mouse embryonic fibroblasts (MEFs revealed tubular structures in the nucleus adjacent to clusters of assembled virions, with virions apparently "shed" or "budding" from their ends. Promyelocytic leukemia nuclear bodies (PML-NBs have been suggested as possible sites for viral replication of polyomaviruses (BKV and SV40, herpes simplex virus (HSV, and adenovirus (Ad. Immunohistochemistry and FISH demonstrated co-localization of the viral T-antigen (Tag, PyV DNA, and the host DNA repair protein MRE11, adjacent to the PML-NBs. In PML⁻/⁻ MEFs the co-localization of MRE11, Tag, and PyV DNA remained unchanged, suggesting that the PML protein itself was not responsible for their association. Furthermore, PyV-infected PML⁻/⁻ MEFs and PML⁻/⁻ mice replicated wild-type levels of infectious virus. Therefore, although the PML protein may identify sites of PyV replication, neither the observed "virus factories" nor virus assembly were dependent on PML. The ultrastructure of the tubes suggests a new model for the encapsidation of small DNA viruses.

Viral membrane fusion

International Nuclear Information System (INIS)

Harrison, Stephen C.

2015-01-01

Membrane fusion is an essential step when enveloped viruses enter cells. Lipid bilayer fusion requires catalysis to overcome a high kinetic barrier; viral fusion proteins are the agents that fulfill this catalytic function. Despite a variety of molecular architectures, these proteins facilitate fusion by essentially the same generic mechanism. Stimulated by a signal associated with arrival at the cell to be infected (e.g., receptor or co-receptor binding, proton binding in an endosome), they undergo a series of conformational changes. A hydrophobic segment (a “fusion loop” or “fusion peptide”) engages the target-cell membrane and collapse of the bridging intermediate thus formed draws the two membranes (virus and cell) together. We know of three structural classes for viral fusion proteins. Structures for both pre- and postfusion conformations of illustrate the beginning and end points of a process that can be probed by single-virion measurements of fusion kinetics. - Highlights: • Viral fusion proteins overcome the high energy barrier to lipid bilayer merger. • Different molecular structures but the same catalytic mechanism. • Review describes properties of three known fusion-protein structural classes. • Single-virion fusion experiments elucidate mechanism

Viral membrane fusion

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Harrison, Stephen C., E-mail: harrison@crystal.harvard.edu

2015-05-15

Membrane fusion is an essential step when enveloped viruses enter cells. Lipid bilayer fusion requires catalysis to overcome a high kinetic barrier; viral fusion proteins are the agents that fulfill this catalytic function. Despite a variety of molecular architectures, these proteins facilitate fusion by essentially the same generic mechanism. Stimulated by a signal associated with arrival at the cell to be infected (e.g., receptor or co-receptor binding, proton binding in an endosome), they undergo a series of conformational changes. A hydrophobic segment (a “fusion loop” or “fusion peptide”) engages the target-cell membrane and collapse of the bridging intermediate thus formed draws the two membranes (virus and cell) together. We know of three structural classes for viral fusion proteins. Structures for both pre- and postfusion conformations of illustrate the beginning and end points of a process that can be probed by single-virion measurements of fusion kinetics. - Highlights: • Viral fusion proteins overcome the high energy barrier to lipid bilayer merger. • Different molecular structures but the same catalytic mechanism. • Review describes properties of three known fusion-protein structural classes. • Single-virion fusion experiments elucidate mechanism.

Inhibition of Non Canonical HIV-1 Tat Secretion Through the Cellular Na+,K+-ATPase Blocks HIV-1 Infection

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Silvia Agostini

2017-07-01

Full Text Available Besides its essential role in the activation of HIV-1 gene expression, the viral Tat protein has the unusual property of trafficking in and out of cells. In contrast to Tat internalization, the mechanism involved in extracellular Tat release has so far remained elusive. Here we show that Tat secretion occurs through a Golgi-independent pathway requiring binding of Tat with three short, non-consecutive intracytoplasmic loops at the C-terminus of the cellular Na+,K+-ATPase pump alpha subunit. Ouabain, a pump inhibitor, blocked this interaction and prevented Tat secretion; virions produced in the presence of this drug were less infectious, consistent the capacity of virion-associated Tat to increase HIV-1 infectivity. Treatment of CD4+ T-cells with short peptides corresponding to the Tat-binding regions of the pump alpha subunit impaired extracellular Tat release and blocked HIV-1 replication. Thus, non canonical, extracellular Tat secretion is essential for viral infectivity.

Rational site-directed mutations of the LLP-1 and LLP-2 lentivirus lytic peptide domains in the intracytoplasmic tail of human immunodeficiency virus type 1 gp41 indicate common functions in cell-cell fusion but distinct roles in virion envelope incorporation.

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Kalia, Vandana; Sarkar, Surojit; Gupta, Phalguni; Montelaro, Ronald C

2003-03-01

Two highly conserved cationic amphipathic alpha-helical motifs, designated lentivirus lytic peptides 1 and 2 (LLP-1 and LLP-2), have been characterized in the carboxyl terminus of the transmembrane (TM) envelope glycoprotein (Env) of lentiviruses. Although various properties have been attributed to these domains, their structural and functional significance is not clearly understood. To determine the specific contributions of the Env LLP domains to Env expression, processing, and incorporation and to viral replication and syncytium induction, site-directed LLP mutants of a primary dualtropic infectious human immunodeficiency virus type 1 (HIV-1) isolate (ME46) were examined. Substitutions were made for highly conserved arginine residues in either the LLP-1 or LLP-2 domain (MX1 or MX2, respectively) or in both domains (MX4). The HIV-1 mutants with altered LLP domains demonstrated distinct phenotypes. The LLP-1 mutants (MX1 and MX4) were replication defective and showed an average of 85% decrease in infectivity, which was associated with an evident decrease in gp41 incorporation into virions without a significant decrease in Env expression or processing in transfected 293T cells. In contrast, MX2 virus was replication competent and incorporated a full complement of Env into its virions, indicating a differential role for the LLP-1 domain in Env incorporation. Interestingly, the replication-competent MX2 virus was impaired in its ability to induce syncytia in T-cell lines. This defect in cell-cell fusion did not correlate with apparent defects in the levels of cell surface Env expression, oligomerization, or conformation. The lack of syncytium formation, however, correlated with a decrease of about 90% in MX2 Env fusogenicity compared to that of wild-type Env in quantitative luciferase-based cell-cell fusion assays. The LLP-1 mutant MX1 and MX4 Envs also exhibited an average of 80% decrease in fusogenicity. Altogether, these results demonstrate for the first time that

Human Cytomegalovirus Exploits Interferon-Induced Transmembrane Proteins To Facilitate Morphogenesis of the Virion Assembly Compartment

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Xie, Maorong; Xuan, Baoqin; Shan, Jiaoyu; Pan, Deng; Sun, Yamei; Shan, Zhao; Zhang, Jinping; Yu, Dong

2014-01-01

ABSTRACT Recently, interferon-induced transmembrane proteins (IFITMs) have been identified to be key effector molecules in the host type I interferon defense system. The invasion of host cells by a large range of RNA viruses is inhibited by IFITMs during the entry step. However, the roles of IFITMs in DNA virus infections have not been studied in detail. In this study, we report that human cytomegalovirus (HCMV), a large human DNA virus, exploits IFITMs to facilitate the formation of the virion assembly compartment (vAC) during infection of human fibroblasts. We found that IFITMs were expressed constitutively in human embryonic lung fibroblasts (MRC5 cells). HCMV infection inhibited IFITM protein accumulation in the later stages of infection. Overexpression of an IFITM protein in MRC5 cells slightly enhanced HCMV production and knockdown of IFITMs by RNA interference reduced the virus titer by about 100-fold on day 8 postinfection, according to the findings of a virus yield assay at a low multiplicity of infection. Virus gene expression and DNA synthesis were not affected, but the typical round structure of the vAC was not formed after the suppression of IFITMs, thereby resulting in defective virion assembly and the production of less infectious virion particles. Interestingly, the replication of herpes simplex virus, a human herpesvirus that is closely related to HCMV, was not affected by the suppression of IFITMs in MRC5 cells. These results indicate that IFITMs are involved in a specific pathway required for HCMV replication. IMPORTANCE HCMV is known to repurpose the interferon-stimulated genes (ISGs) viperin and tetherin to facilitate its replication. Our results expand the range of ISGs that can be exploited by HCMV for its replication. This is also the first report of a proviral function of IFITMs in DNA virus replication. In addition, whereas previous studies showed that IFITMs modulate virus entry, which is a very early stage in the virus life cycle, we

Properties of virion transactivator proteins encoded by primate cytomegaloviruses

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Barry Peter A

2009-05-01

Full Text Available Abstract Background Human cytomegalovirus (HCMV is a betaherpesvirus that causes severe disease in situations where the immune system is immature or compromised. HCMV immediate early (IE gene expression is stimulated by the virion phosphoprotein pp71, encoded by open reading frame (ORF UL82, and this transactivation activity is important for the efficient initiation of viral replication. It is currently recognized that pp71 acts to overcome cellular intrinsic defences that otherwise block viral IE gene expression, and that interactions of pp71 with the cell proteins Daxx and ATRX are important for this function. A further property of pp71 is the ability to enable prolonged gene expression from quiescent herpes simplex virus type 1 (HSV-1 genomes. Non-human primate cytomegaloviruses encode homologs of pp71, but there is currently no published information that addresses their effects on gene expression and modes of action. Results The UL82 homolog encoded by simian cytomegalovirus (SCMV, strain Colburn, was identified and cloned. This ORF, named S82, was cloned into an HSV-1 vector, as were those from baboon, rhesus monkey and chimpanzee cytomegaloviruses. The use of an HSV-1 vector enabled expression of the UL82 homologs in a range of cell types, and permitted investigation of their abilities to direct prolonged gene expression from quiescent genomes. The results show that all UL82 homologs activate gene expression, and that neither host cell type nor promoter target sequence has major effects on these activities. Surprisingly, the UL82 proteins specified by non-human primate cytomegaloviruses, unlike pp71, did not direct long term expression from quiescent HSV-1 genomes. In addition, significant differences were observed in the intranuclear localization of the UL82 homologs, and in their effects on Daxx. Strikingly, S82 mediated the release of Daxx from nuclear domain 10 substructures much more rapidly than pp71 or the other proteins tested. All

Mechanism of Human Influenza Virus RNA Persistence and Virion Survival in Feces: Mucus Protects Virions From Acid and Digestive Juices.

Science.gov (United States)

Hirose, Ryohei; Nakaya, Takaaki; Naito, Yuji; Daidoji, Tomo; Watanabe, Yohei; Yasuda, Hiroaki; Konishi, Hideyuki; Itoh, Yoshito

2017-07-01

Although viral RNA or infectious virions have been detected in the feces of individuals infected with human influenza A and B viruses (IAV/IBV), the mechanism of viral survival in the gastrointestinal tract remains unclear. We developed a model that attempts to recapitulate the conditions encountered by a swallowed virus. While IAV/IBV are vulnerable to simulated digestive juices (gastric acid and bile/pancreatic juice), highly viscous mucus protects viral RNA and virions, allowing the virus to retain its infectivity. Our results suggest that virions and RNA present in swallowed mucus are not inactivated or degraded by the gastrointestinal environment, allowing their detection in feces. © The Author 2017. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail: journals.permissions@oup.com.

Target-dependent enrichment of virions determines the reduction of high-throughput sequencing in virus discovery.

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Randi Holm Jensen

Full Text Available Viral infections cause many different diseases stemming both from well-characterized viral pathogens but also from emerging viruses, and the search for novel viruses continues to be of great importance. High-throughput sequencing is an important technology for this purpose. However, viral nucleic acids often constitute a minute proportion of the total genetic material in a sample from infected tissue. Techniques to enrich viral targets in high-throughput sequencing have been reported, but the sensitivity of such methods is not well established. This study compares different library preparation techniques targeting both DNA and RNA with and without virion enrichment. By optimizing the selection of intact virus particles, both by physical and enzymatic approaches, we assessed the effectiveness of the specific enrichment of viral sequences as compared to non-enriched sample preparations by selectively looking for and counting read sequences obtained from shotgun sequencing. Using shotgun sequencing of total DNA or RNA, viral targets were detected at concentrations corresponding to the predicted level, providing a foundation for estimating the effectiveness of virion enrichment. Virion enrichment typically produced a 1000-fold increase in the proportion of DNA virus sequences. For RNA virions the gain was less pronounced with a maximum 13-fold increase. This enrichment varied between the different sample concentrations, with no clear trend. Despite that less sequencing was required to identify target sequences, it was not evident from our data that a lower detection level was achieved by virion enrichment compared to shotgun sequencing.

Detergent extraction of herpes simplex virus type 1 glycoprotein D by zwitterionic and non-ionic detergents and purification by ion-exchange high-performance liquid chromatography

NARCIS (Netherlands)

Welling-Wester, S; Feijlbrief, M; Koedijk, DGAM; Welling, GW

1998-01-01

Detergents (surfactants) are the key reagents in the extraction and purification of integral membrane proteins. Zwitterionic and non-ionic detergents were used for the extraction of recombinant glycoprotein D (gD-1) of herpes simplex virus type 1 (HSV-1) from insect cells infected with recombinant

Endoplasmic reticulum-to-Golgi transitions upon herpes virus infection [version 2; referees: 1 approved, 3 approved with reservations

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Peter Wild

2018-02-01

Full Text Available Background: Herpesvirus capsids are assembled in the nucleus, translocated to the perinuclear space by budding, acquiring tegument and envelope, or released to the cytoplasm via impaired nuclear envelope. One model proposes that envelopment, “de-envelopment” and “re-envelopment” is essential for production of infectious virus. Glycoproteins gB/gH were reported to be essential for de-envelopment, by fusion of the “primary” envelope with the outer nuclear membrane. Yet, a high proportion of enveloped virions generated from genomes with deleted gB/gH were found in the cytoplasm and extracellular space, suggesting the existence of alternative exit routes. Methods: We investigated the relatedness between the nuclear envelope and membranes of the endoplasmic reticulum and Golgi complex, in cells infected with either herpes simplex virus 1 (HSV-1 or a Us3 deletion mutant thereof, or with bovine herpesvirus 1 (BoHV-1 by transmission and scanning electron microscopy, employing freezing technique protocols. Results:  The Golgi complex is a compact entity in a juxtanuclear position covered by a membrane on the cis face. Golgi membranes merge with membranes of the endoplasmic reticulum forming an entity with the perinuclear space. All compartments contained enveloped virions. After treatment with brefeldin A, HSV-1 virions aggregated in the perinuclear space and endoplasmic reticulum, while infectious progeny virus was still produced. Conclusions: The data suggest that virions derived by budding at nuclear membranes are intraluminally transported from the perinuclear space via Golgi -endoplasmic reticulum transitions into Golgi cisternae for packaging. Virions derived by budding at nuclear membranes are infective like Us3 deletion mutants, which  accumulate in the perinuclear space. Therefore, i de-envelopment followed by re-envelopment is not essential for production of infective progeny virus, ii the process taking place at the outer nuclear

Herpes simplex virus 1 induces de novo phospholipid synthesis

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Sutter, Esther [Electron Microscopy, Institute of Veterinary Anatomy, University of Zuerich (Switzerland); Oliveira, Anna Paula de; Tobler, Kurt [Electron microscopy, Institute of Virology, University of Zuerich (Switzerland); Schraner, Elisabeth M. [Electron Microscopy, Institute of Veterinary Anatomy, University of Zuerich (Switzerland); Sonda, Sabrina [Institute of Parasitology, University of Zuerich (Switzerland); Kaech, Andres [Center for Microscopy and Image Analysis, University of Zuerich (Switzerland); Lucas, Miriam S. [Electron Microscopy ETH Zuerich (EMEZ), Swiss Federal Institute of Technology, Zuerich (Switzerland); Ackermann, Mathias [Electron microscopy, Institute of Virology, University of Zuerich (Switzerland); Wild, Peter, E-mail: pewild@access.uzh.ch [Electron Microscopy, Institute of Veterinary Anatomy, University of Zuerich (Switzerland)

2012-08-01

Herpes simplex virus type 1 capsids bud at nuclear membranes and Golgi membranes acquiring an envelope composed of phospholipids. Hence, we measured incorporation of phospholipid precursors into these membranes, and quantified changes in size of cellular compartments by morphometric analysis. Incorporation of [{sup 3}H]-choline into both nuclear and cytoplasmic membranes was significantly enhanced upon infection. [{sup 3}H]-choline was also part of isolated virions even grown in the presence of brefeldin A. Nuclei expanded early in infection. The Golgi complex and vacuoles increased substantially whereas the endoplasmic reticulum enlarged only temporarily. The data suggest that HSV-1 stimulates phospholipid synthesis, and that de novo synthesized phospholipids are inserted into nuclear and cytoplasmic membranes to i) maintain membrane integrity in the course of nuclear and cellular expansion, ii) to supply membrane constituents for envelopment of capsids by budding at nuclear membranes and Golgi membranes, and iii) to provide membranes for formation of transport vacuoles.

Expression of collagen in ovular membranes of pregnant smokers and non-smokers: a pilot study.

Science.gov (United States)

Negrini, Romulo; Araujo Júnior, Edward; Piato, Sebastião; Chade, Milca Cezar; Rios, Adriana Ribeiro Santos; Silva, Maria Antonieta Galvão; Aldrighi, José Mendes

2015-09-01

Our study compared the amount of total collagen and type I collagen in ovular membranes of pregnant smokers and non-smokers. The study group consisted of 14 pregnant smokers at 24-36 weeks of gestation; 39 pregnant non-smokers between 24-36 weeks of gestation comprised the control group. The expressions of total collagen and type I collagen were analyzed using two histological sections of the fetal membranes. The assessment of total collagen was performed using the Picro-Cirius red stain, and type I collagen expression was determined by means of immunohistochemistry The Mann-Whitney test was applied to verify possible differences between the groups. The average area covered by total collagen was lower in smokers (20630.45 microm2) as compared to non-smokers (24058.61 microm2), although the difference was not statistically significant (p = 0.454). Comparison involving collagen type I deemed similar results (20001.33 microm2 vs. 25328.29 microm2, p = 0.158). The amount of total collagen and type I collagen was lower in ovular membranes of pregnant smokers as compared to non-smokers, although the difference was not statistically significant.

Dynamics of HIV-1 RNA Near the Plasma Membrane during Virus Assembly.

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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

Crystallographic characterization of the radixin FERM domain bound to the cytoplasmic tail of membrane-type 1 matrix metalloproteinase (MT1-MMP)

International Nuclear Information System (INIS)

Terawaki, Shin-ichi; Kitano, Ken; Aoyama, Miki; Hakoshima, Toshio

2008-01-01

The radixin FERM domain was shown to bind the MT1-MMP cytoplasmic peptide and crystals of the complex were obtained. ERM proteins play a role in the cross-linking found between plasma membranes and actin filaments. The N-terminal FERM domains of ERM proteins are responsible for membrane association through direct interaction with the cytoplasmic tails of integral membrane proteins. During cell migration and movement, membrane-type 1 matrix metalloproteinase (MT1-MMP) on plasma membranes sheds adhesion molecule CD44 in addition to degrading the extracellular matrix. Here, the interaction between the radixin FERM domain and the MT1-MMP cytoplasmic tail is reported and preliminary crystallographic characterization of crystals of the radixin FERM domain bound to the cytoplasmic tail of MT1-MMP is presented. The crystals belong to space group P6 1 22, with unit-cell parameters a = b = 122.7, c = 128.3 Å, and contain one complex in the crystallographic asymmetric unit. The diffraction data were collected to a resolution of 2.4 Å

The coronavirus spike protein : mechanisms of membrane fusion and virion incorporation

NARCIS (Netherlands)

Bosch, B.J.

2004-01-01

The coronavirus spike protein is a membrane-anchored glycoprotein responsible for virus-cell attachment and membrane fusion, prerequisites for a successful virus infection. In this thesis, two aspects are described regarding the molecular biology of the coronavirus spike protein: its membrane fusion

Two-color fluorescence analysis of individual virions determines the distribution of the copy number of proteins in herpes simplex virus particles.

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Clarke, Richard W; Monnier, Nilah; Li, Haitao; Zhou, Dejian; Browne, Helena; Klenerman, David

2007-08-15

We present a single virion method to determine absolute distributions of copy number in the protein composition of viruses and apply it to herpes simplex virus type 1. Using two-color coincidence fluorescence spectroscopy, we determine the virion-to-virion variability in copy numbers of fluorescently labeled tegument and envelope proteins relative to a capsid protein by analyzing fluorescence intensity ratios for ensembles of individual dual-labeled virions and fitting the resulting histogram of ratios. Using EYFP-tagged capsid protein VP26 as a reference for fluorescence intensity, we are able to calculate the mean and also, for the first time to our knowledge, the variation in numbers of gD, VP16, and VP22 tegument. The measurement of the number of glycoprotein D molecules was in good agreement with independent measurements of average numbers of these glycoproteins in bulk virus preparations, validating the method. The accuracy, straightforward data processing, and high throughput of this technique make it widely applicable to the analysis of the molecular composition of large complexes in general, and it is particularly suited to providing insights into virus structure, assembly, and infectivity.

Identification of membrane-type 1 matrix metalloproteinase tyrosine phosphorylation in association with neuroblastoma progression

International Nuclear Information System (INIS)

Nyalendo, Carine; Sartelet, Hervé; Barrette, Stéphane; Ohta, Shigeru; Gingras, Denis; Béliveau, Richard

2009-01-01

Neuroblastoma is a pediatric tumor of neural crest cells that is clinically characterized by its variable evolution, from spontaneous regression to malignancy. Despite many advances in neuroblastoma research, 60% of neuroblastoma, which are essentially metastatic cases, are associated with poor clinical outcome due to the lack of effectiveness of current therapeutic strategies. Membrane-type 1 matrix metalloproteinase (MT1-MMP, MMP-14), an enzyme involved in several steps in tumor progression, has previously been shown to be associated with poor clinical outcome for neuroblastoma. Based on our recent demonstration that MT1-MMP phosphorylation is involved in the growth of fibrosarcoma tumors, we examined the potential role of phosphorylated MT1-MMP in neuroblastoma progression. Tyrosine phosphorylated MT1-MMP was immunostained on tissue microarray samples from 55 patients with neuroblastoma detected by mass screening (known to be predominantly associated with favourable outcome), and from 234 patients with standard diagnosed neuroblastoma. In addition, the effects of a non phosphorylable version of MT1-MMP on neuroblastoma cell migration and proliferation were investigated within three-dimensional collagen matrices. Although there is no correlation between the extent of tyrosine phosphorylation of MT1-MMP (pMT1-MMP) and MYCN amplification or clinical stage, we observed greater phosphorylation of pMT1-MMP in standard neuroblastoma, while it is less evident in neuroblastoma from mass screening samples (P = 0.0006) or in neuroblastoma samples from patients younger than one year (P = 0.0002). In vitro experiments showed that overexpression of a non-phosphorylable version of MT1-MMP reduced MT1-MMP-mediated neuroblastoma cell migration and proliferation within a three-dimensional type I collagen matrix, suggesting a role for the phosphorylated enzyme in the invasive properties of neuroblastoma cells. Overall, these results suggest that tyrosine phosphorylated MT1-MMP

CMV-Promoter Driven Codon-Optimized Expression Alters the Assembly Type and Morphology of a Reconstituted HERV-K(HML-2

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Oliver Hohn

2014-11-01

Full Text Available The HERV-K(HML-2 family contains the most recently integrated and best preserved endogenized proviral sequences in the human genome. All known elements have nevertheless been subjected to mutations or deletions that render expressed particles non-infectious. Moreover, these post-insertional mutations hamper the analysis of the general biological properties of this ancient virus family. The expression of consensus sequences and sequences of elements with reverted post-insertional mutations has therefore been very instrumental in overcoming this limitation. We investigated the particle morphology of a recently reconstituted HERV-K113 element termed oriHERV-K113 using thin-section electron microscopy (EM and could demonstrate that strong overexpression by substitution of the 5'LTR for a CMV promoter and partial codon optimization altered the virus assembly type and morphology. This included a conversion from the regular C-type to an A-type morphology with a mass of cytoplasmic immature cores tethered to the cell membrane and the membranes of vesicles. Overexpression permitted the release and maturation of virions but reduced the envelope content. A weaker boost of virus expression by Staufen-1 was not sufficient to induce these morphological alterations.

CMV-promoter driven codon-optimized expression alters the assembly type and morphology of a reconstituted HERV-K(HML-2).

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Hohn, Oliver; Hanke, Kirsten; Lausch, Veronika; Zimmermann, Anja; Mostafa, Saeed; Bannert, Norbert

2014-11-11

The HERV-K(HML-2) family contains the most recently integrated and best preserved endogenized proviral sequences in the human genome. All known elements have nevertheless been subjected to mutations or deletions that render expressed particles non-infectious. Moreover, these post-insertional mutations hamper the analysis of the general biological properties of this ancient virus family. The expression of consensus sequences and sequences of elements with reverted post-insertional mutations has therefore been very instrumental in overcoming this limitation. We investigated the particle morphology of a recently reconstituted HERV-K113 element termed oriHERV-K113 using thin-section electron microscopy (EM) and could demonstrate that strong overexpression by substitution of the 5'LTR for a CMV promoter and partial codon optimization altered the virus assembly type and morphology. This included a conversion from the regular C-type to an A-type morphology with a mass of cytoplasmic immature cores tethered to the cell membrane and the membranes of vesicles. Overexpression permitted the release and maturation of virions but reduced the envelope content. A weaker boost of virus expression by Staufen-1 was not sufficient to induce these morphological alterations.

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

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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.

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

International Nuclear Information System (INIS)

González, Silvia A.; Paladino, Mónica G.; Affranchino, José L.

2012-01-01

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.

Non-Native Metal Ion Reveals the Role of Electrostatics in Synaptotagmin 1-Membrane Interactions.

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Katti, Sachin; Nyenhuis, Sarah B; Her, Bin; Srivastava, Atul K; Taylor, Alexander B; Hart, P John; Cafiso, David S; Igumenova, Tatyana I

2017-06-27

C2 domains are independently folded modules that often target their host proteins to anionic membranes in a Ca 2+ -dependent manner. In these cases, membrane association is triggered by Ca 2+ binding to the negatively charged loop region of the C2 domain. Here, we used a non-native metal ion, Cd 2+ , in lieu of Ca 2+ to gain insight into the contributions made by long-range Coulombic interactions and direct metal ion-lipid bridging to membrane binding. Using X-ray crystallography, NMR, Förster resonance energy transfer, and vesicle cosedimentation assays, we demonstrate that, although Cd 2+ binds to the loop region of C2A/B domains of synaptotagmin 1 with high affinity, long-range Coulombic interactions are too weak to support membrane binding of individual domains. We attribute this behavior to two factors: the stoichiometry of Cd 2+ binding to the loop regions of the C2A and C2B domains and the impaired ability of Cd 2+ to directly coordinate the lipids. In contrast, electron paramagnetic resonance experiments revealed that Cd 2+ does support membrane binding of the C2 domains in full-length synaptotagmin 1, where the high local lipid concentrations that result from membrane tethering can partially compensate for lack of a full complement of divalent metal ions and specific lipid coordination in Cd 2+ -complexed C2A/B domains. Our data suggest that long-range Coulombic interactions alone can drive the initial association of C2A/B with anionic membranes and that Ca 2+ further augments membrane binding by the formation of metal ion-lipid coordination bonds and additional Ca 2+ ion binding to the C2 domain loop regions.

Mutations in matrix and SP1 repair the packaging specificity of a Human Immunodeficiency Virus Type 1 mutant by reducing the association of Gag with spliced viral RNA

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Ristic Natalia

2010-09-01

Full Text Available Abstract Background The viral genome of HIV-1 contains several secondary structures that are important for regulating viral replication. The stem-loop 1 (SL1 sequence in the 5' untranslated region directs HIV-1 genomic RNA dimerization and packaging into the virion. Without SL1, HIV-1 cannot replicate in human T cell lines. The replication restriction phenotype in the SL1 deletion mutant appears to be multifactorial, with defects in viral RNA dimerization and packaging in producer cells as well as in reverse transcription of the viral RNA in infected cells. In this study, we sought to characterize SL1 mutant replication restrictions and provide insights into the underlying mechanisms of compensation in revertants. Results HIV-1 lacking SL1 (NLΔSL1 did not replicate in PM-1 cells until two independent non-synonymous mutations emerged: G913A in the matrix domain (E42K on day 18 postinfection and C1907T in the SP1 domain (P10L on day 11 postinfection. NLΔSL1 revertants carrying either compensatory mutation showed enhanced infectivity in PM-1 cells. The SL1 revertants produced significantly more infectious particles per nanogram of p24 than did NLΔSL1. The SL1 deletion mutant packaged less HIV-1 genomic RNA and more cellular RNA, particularly signal recognition particle RNA, in the virion than the wild-type. NLΔSL1 also packaged 3- to 4-fold more spliced HIV mRNA into the virion, potentially interfering with infectious virus production. In contrast, both revertants encapsidated 2.5- to 5-fold less of these HIV-1 mRNA species. Quantitative RT-PCR analysis of RNA cross-linked with Gag in formaldehyde-fixed cells demonstrated that the compensatory mutations reduced the association between Gag and spliced HIV-1 RNA, thereby effectively preventing these RNAs from being packaged into the virion. The reduction of spliced viral RNA in the virion may have a major role in facilitating infectious virus production, thus restoring the infectivity of NLΔSL1

Deficient incorporation of spike protein into virions contributes to the lack of infectivity following establishment of a persistent, non-productive infection in oligodendroglial cell culture by murine coronavirus

International Nuclear Information System (INIS)

Liu Yin; Herbst, Werner; Cao Jianzhong; Zhang Xuming

2011-01-01

Infection of mouse oligodendrocytes with a recombinant mouse hepatitis virus (MHV) expressing a green fluorescence protein facilitated specific selection of virus-infected cells and subsequent establishment of persistence. Interestingly, while viral genomic RNAs persisted in infected cells over 14 subsequent passages with concomitant synthesis of viral subgenomic mRNAs and structural proteins, no infectious virus was isolated beyond passage 2. Further biochemical and electron microscopic analyses revealed that virions, while assembled, contained little spike in the envelope, indicating that lack of infectivity during persistence was likely due to deficiency in spike incorporation. This type of non-lytic, non-productive persistence in oligodendrocytes is unique among animal viruses and resembles MHV persistence previously observed in the mouse central nervous system. Thus, establishment of such a culture system that can recapitulate the in vivo phenomenon will provide a powerful approach for elucidating the mechanisms of coronavirus persistence in glial cells at the cellular and molecular levels.

Membrane remodeling by amyloidogenic and non-amyloidogenic proteins studied by EPR.

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Varkey, Jobin; Langen, Ralf

2017-07-01

The advancement in site-directed spin labeling of proteins has enabled EPR studies to expand into newer research areas within the umbrella of protein-membrane interactions. Recently, membrane remodeling by amyloidogenic and non-amyloidogenic proteins has gained a substantial interest in relation to driving and controlling vital cellular processes such as endocytosis, exocytosis, shaping of organelles like endoplasmic reticulum, Golgi and mitochondria, intracellular vesicular trafficking, formation of filopedia and multivesicular bodies, mitochondrial fusion and fission, and synaptic vesicle fusion and recycling in neurotransmission. Misregulation in any of these processes due to an aberrant protein (mutation or misfolding) or alteration of lipid metabolism can be detrimental to the cell and cause disease. Dissection of the structural basis of membrane remodeling by proteins is thus quite necessary for an understanding of the underlying mechanisms, but it remains a formidable task due to the difficulties of various common biophysical tools in monitoring the dynamic process of membrane binding and bending by proteins. This is largely since membranes generally complicate protein structure analysis and this problem is amplified for structural analysis in the presence of different types of membrane curvatures. Recent EPR studies on membrane remodeling by proteins show that a significant structural information can be generated to delineate the role of different protein modules, domains and individual amino acids in the generation of membrane curvature. These studies also show how EPR can complement the data obtained by high resolution techniques such as X-ray and NMR. This perspective covers the application of EPR in recent studies for understanding membrane remodeling by amyloidogenic and non-amyloidogenic proteins that is useful for researchers interested in using or complimenting EPR to gain better understanding of membrane remodeling. We also discuss how a single

Membrane remodeling by amyloidogenic and non-amyloidogenic proteins studied by EPR

Science.gov (United States)

Varkey, Jobin; Langen, Ralf

2017-07-01

The advancement in site-directed spin labeling of proteins has enabled EPR studies to expand into newer research areas within the umbrella of protein-membrane interactions. Recently, membrane remodeling by amyloidogenic and non-amyloidogenic proteins has gained a substantial interest in relation to driving and controlling vital cellular processes such as endocytosis, exocytosis, shaping of organelles like endoplasmic reticulum, Golgi and mitochondria, intracellular vesicular trafficking, formation of filopedia and multivesicular bodies, mitochondrial fusion and fission, and synaptic vesicle fusion and recycling in neurotransmission. Misregulation in any of these processes due to an aberrant protein (mutation or misfolding) or alteration of lipid metabolism can be detrimental to the cell and cause disease. Dissection of the structural basis of membrane remodeling by proteins is thus quite necessary for an understanding of the underlying mechanisms, but it remains a formidable task due to the difficulties of various common biophysical tools in monitoring the dynamic process of membrane binding and bending by proteins. This is largely since membranes generally complicate protein structure analysis and this problem is amplified for structural analysis in the presence of different types of membrane curvatures. Recent EPR studies on membrane remodeling by proteins show that a significant structural information can be generated to delineate the role of different protein modules, domains and individual amino acids in the generation of membrane curvature. These studies also show how EPR can complement the data obtained by high resolution techniques such as X-ray and NMR. This perspective covers the application of EPR in recent studies for understanding membrane remodeling by amyloidogenic and non-amyloidogenic proteins that is useful for researchers interested in using or complimenting EPR to gain better understanding of membrane remodeling. We also discuss how a single

Role of the DIS hairpin in replication of human immunodeficiency virus type 1

NARCIS (Netherlands)

Berkhout, B.; van Wamel, J. L.

1996-01-01

The virion-associated genome of human immunodeficiency virus type 1 consists of a noncovalently linked dimer of two identical, unspliced RNA molecules. A hairpin structure within the untranslated leader transcript is postulated to play a role in RNA dimerization through base pairing of the

Crystal structure of an orthomyxovirus matrix protein reveals mechanisms for self-polymerization and membrane association.

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Zhang, Wenting; Zheng, Wenjie; Toh, Yukimatsu; Betancourt-Solis, Miguel A; Tu, Jiagang; Fan, Yanlin; Vakharia, Vikram N; Liu, Jun; McNew, James A; Jin, Meilin; Tao, Yizhi J

2017-08-08

Many enveloped viruses encode a matrix protein. In the influenza A virus, the matrix protein M1 polymerizes into a rigid protein layer underneath the viral envelope to help enforce the shape and structural integrity of intact viruses. The influenza virus M1 is also known to mediate virus budding as well as the nuclear export of the viral nucleocapsids and their subsequent packaging into nascent viral particles. Despite extensive studies on the influenza A virus M1 (FLUA-M1), only crystal structures of its N-terminal domain are available. Here we report the crystal structure of the full-length M1 from another orthomyxovirus that infects fish, the infectious salmon anemia virus (ISAV). The structure of ISAV-M1 assumes the shape of an elbow, with its N domain closely resembling that of the FLUA-M1. The C domain, which is connected to the N domain through a flexible linker, is made of four α-helices packed as a tight bundle. In the crystal, ISAV-M1 monomers form infinite 2D arrays with a network of interactions involving both the N and C domains. Results from liposome flotation assays indicated that ISAV-M1 binds membrane via electrostatic interactions that are primarily mediated by a positively charged surface loop from the N domain. Cryoelectron tomography reconstruction of intact ISA virions identified a matrix protein layer adjacent to the inner leaflet of the viral membrane. The physical dimensions of the virion-associated matrix layer are consistent with the 2D ISAV-M1 crystal lattice, suggesting that the crystal lattice is a valid model for studying M1-M1, M1-membrane, and M1-RNP interactions in the virion.

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

Science.gov (United States)

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

2013-01-01

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

Herpes simplex virus type 1 strain KOS carries a defective US9 and a mutated US8A gene.

Science.gov (United States)

Negatsch, Alexandra; Mettenleiter, Thomas C; Fuchs, Walter

2011-01-01

The membrane protein encoded by the US9 gene of alphaherpesviruses plays an important role during virion assembly and transport in neurons. Here, we demonstrate that in herpes simplex virus type 1 (HSV-1) strain KOS, due to base substitutions, the predicted TATA-box of US9 is mutated, and a premature stop is present at codon 58 of US9, which contains 91 codons in other HSV-1 strains. The TATA-box mutation also removes the native stop codon of the adjacent US8A gene, leading to extension of the coding region from 160 to 191 codons. Northern blot analyses revealed reduced transcription of US9 in cells infected with HSV-1 KOS. Moreover, a US9-specific antiserum did not detect any gene products in Western blot and immunofluorescence analyses of KOS-infected cells, indicating that the truncated protein is not stable. In contrast, Western blot reactions of a pUS8A-specific antiserum confirmed enlargement of this protein in HSV-1 KOS.

Proteomic and Functional Analyses of the Virion Transmembrane Proteome of Cyprinid Herpesvirus 3.

Science.gov (United States)

Vancsok, Catherine; Peñaranda, M Michelle D; Raj, V Stalin; Leroy, Baptiste; Jazowiecka-Rakus, Joanna; Boutier, Maxime; Gao, Yuan; Wilkie, Gavin S; Suárez, Nicolás M; Wattiez, Ruddy; Gillet, Laurent; Davison, Andrew J; Vanderplasschen, Alain F C

2017-11-01

Virion transmembrane proteins (VTPs) mediate key functions in the herpesvirus infectious cycle. Cyprinid herpesvirus 3 (CyHV-3) is the archetype of fish alloherpesviruses. The present study was devoted to CyHV-3 VTPs. Using mass spectrometry approaches, we identified 16 VTPs of the CyHV-3 FL strain. Mutagenesis experiments demonstrated that eight of these proteins are essential for viral growth in vitro (open reading frame 32 [ORF32], ORF59, ORF81, ORF83, ORF99, ORF106, ORF115, and ORF131), and eight are nonessential (ORF25, ORF64, ORF65, ORF108, ORF132, ORF136, ORF148, and ORF149). Among the nonessential proteins, deletion of ORF25, ORF132, ORF136, ORF148, or ORF149 affects viral replication in vitro , and deletion of ORF25, ORF64, ORF108, ORF132, or ORF149 impacts plaque size. Lack of ORF148 or ORF25 causes attenuation in vivo to a minor or major extent, respectively. The safety and efficacy of a virus lacking ORF25 were compared to those of a previously described vaccine candidate deleted for ORF56 and ORF57 (Δ56-57). Using quantitative PCR, we demonstrated that the ORF25 deleted virus infects fish through skin infection and then spreads to internal organs as reported previously for the wild-type parental virus and the Δ56-57 virus. However, compared to the parental wild-type virus, the replication of the ORF25-deleted virus was reduced in intensity and duration to levels similar to those observed for the Δ56-57 virus. Vaccination of fish with a virus lacking ORF25 was safe but had low efficacy at the doses tested. This characterization of the virion transmembrane proteome of CyHV-3 provides a firm basis for further research on alloherpesvirus VTPs. IMPORTANCE Virion transmembrane proteins play key roles in the biology of herpesviruses. Cyprinid herpesvirus 3 (CyHV-3) is the archetype of fish alloherpesviruses and the causative agent of major economic losses in common and koi carp worldwide. In this study of the virion transmembrane proteome of CyHV-3, the

Baculovirus virions displaying Plasmodium berghei circumsporozoite protein protect mice against malaria sporozoite infection

International Nuclear Information System (INIS)

Yoshida, Shigeto; Kondoh, Daisuke; Arai, Eriko; Matsuoka, Hiroyuki; Seki, Chisato; Tanaka, Takao; Okada, Masaji; Ishii, Akira

2003-01-01

The display of foreign proteins on the surface of baculovirus virions has provided a tool for the analysis of protein-protein interactions and for cell-specific targeting in gene transfer applications. To evaluate the baculovirus display system as a vaccine vehicle, we have generated a recombinant baculovirus (AcNPV-CSPsurf) that displays rodent malaria Plasmodium berghei circumsporozoite protein (PbCSP) on the virion surface as a fusion protein with the major baculovirus envelope glycoprotein gp64. The PbCSP-gp64 fusion protein was incorporated and oligomerized on the virion surface and led to a 12-fold increase in the binding activity of AcNPV-CSPsurf virions to HepG2 cells. Immunization with adjuvant-free AcNPV-CSPsurf virions induced high levels of antibodies and gamma interferon-secreting cells against PbCSP and protected 60% of mice against sporozoite challenge. These data demonstrate that AcNPV-CSPsurf displays sporozoite-like PbCSP on the virion surface and possesses dual potentials as a malaria vaccine candidate and a liver-directed gene delivery vehicle

Double anisotropic electrically conductive flexible Janus-typed membranes.

Science.gov (United States)

Li, Xiaobing; Ma, Qianli; Tian, Jiao; Xi, Xue; Li, Dan; Dong, Xiangting; Yu, Wensheng; Wang, Xinlu; Wang, Jinxian; Liu, Guixia

2017-12-07

Novel type III anisotropic conductive films (ACFs), namely flexible Janus-typed membranes, were proposed, designed and fabricated for the first time. Flexible Janus-typed membranes composed of ordered Janus nanobelts were constructed by electrospinning, which simultaneously possess fluorescence and double electrically conductive anisotropy. For the fabrication of the Janus-typed membrane, Janus nanobelts comprising a conductive side and an insulative-fluorescent side were primarily fabricated, and then the Janus nanobelts are arranged into parallel arrays using an aluminum rotary drum as the collector to obtain a single anisotropically conductive film. Subsequently, a secondary electrospinning process was applied to the as-prepared single anisotropically conductive films to acquire the final Janus-typed membrane. For this Janus-typed membrane, namely its left-to-right structure, anisotropic electrical conduction synchronously exists on both sides, and furthermore, the two electrically conductive directions are perpendicular. By modulating the amount of Eu(BA) 3 phen complex and conducting polyaniline (PANI), the characteristics and intensity of the fluorescence-electricity dual-function in the membrane can be tuned. The high integration of this peculiar Janus-typed membrane with simultaneous double electrically conductive anisotropy-fluorescent dual-functionality is successfully realized in this study. This design philosophy and preparative technique will provide support for the design and construction of new types of special nanostructures with multi-functionality.

Determination of avian influenza A (H9N2) virions by inductively coupled plasma mass spectrometry based magnetic immunoassay with gold nanoparticles labeling

Science.gov (United States)

Xiao, Guangyang; Chen, Beibei; He, Man; Shi, Kaiwen; Zhang, Xing; Li, Xiaoting; Wu, Qiumei; Pang, Daiwen; Hu, Bin

2017-12-01

Avian influenza viruses are the pathogens of global poultry epidemics, and may even cause the human infections. Here, we proposed a novel inductively coupled plasma mass spectrometry (ICP-MS) based immunoassay with gold nanoparticles (Au NPs) labeling for the determination of H9N2 virions. Magnetic-beads modified with anti-influenza A H9N2 hemagglutinin mono-antibody (mAb-HA) were utilized for the capture of H9N2 virions in complex matrix; and Au NPs conjugated with mAb-HA were employed for the specific labeling of H9N2 virions for subsequent ICP-MS detection. With a sandwich immunoassay strategy, this method exhibited a high specificity for H9N2 among other influenza A virions such as H1N1 and H3N2. Under the optimized conditions, this method could detect as low as 0.63 ng mL- 1 H9N2 virions with the linear range of 2-400 ng mL- 1, the relative standard deviation for seven replicate detections of H9N2 virions was 7.2% (c = 10 ng mL- 1). The developed method was applied for the detection of H9N2 virions in real-world chicken dung samples, and the recovery for the spiking samples was 91.4-116.9%. This method is simple, rapid, sensitive, selective, reliable and has a good application potential for virions detection in real-world samples.

The Peri-islet Basement Membrane, a Barrier to Infiltrating Leukocytes in Type 1 Diabetes in Mouse and Human

DEFF Research Database (Denmark)

Korpos, Eva; Kadri, Nadir; Kappelhoff, Reinhild

2013-01-01

We provide the first comprehensive analysis of the extracellular matrix (ECM) composition of peri-islet capsules, composed of the peri-islet basement membrane (BM) and subjacent interstitial matrix (IM), in development of type 1 diabetes in NOD mice and in human type 1 diabetes. Our data demonstr...... IM are reconstituted once inflammation subsides, indicating that the peri-islet BM-producing cells are not lost due to the inflammation, which has important ramifications to islet transplantation studies.......We provide the first comprehensive analysis of the extracellular matrix (ECM) composition of peri-islet capsules, composed of the peri-islet basement membrane (BM) and subjacent interstitial matrix (IM), in development of type 1 diabetes in NOD mice and in human type 1 diabetes. Our data...... demonstrate global loss of peri-islet BM and IM components only at sites of leukocyte infiltration into the islet. Stereological analyses reveal a correlation between incidence of insulitis and the number of islets showing loss of peri-islet BM versus islets with intact BMs, suggesting that leukocyte...

The structures of bovine herpesvirus 1 virion and concatemeric DNA: implications for cleavage and packaging of herpesvirus genomes

International Nuclear Information System (INIS)

Schynts, Frederic; McVoy, Michael A.; Meurens, Francois; Detry, Bruno; Epstein, Alberto L.; Thiry, Etienne

2003-01-01

Herpesvirus genomes are often characterized by the presence of direct and inverted repeats that delineate their grouping into six structural classes. Class D genomes consist of a long (L) segment and a short (S) segment. The latter is flanked by large inverted repeats. DNA replication produces concatemers of head-to-tail linked genomes that are cleaved into unit genomes during the process of packaging DNA into capsids. Packaged class D genomes are an equimolar mixture of two isomers in which S is in either of two orientations, presumably a consequence of homologous recombination between the inverted repeats. The L segment remains predominantly fixed in a prototype (P) orientation; however, low levels of genomes having inverted L (I L ) segments have been reported for some class D herpesviruses. Inefficient formation of class D I L genomes has been attributed to infrequent L segment inversion, but recent detection of frequent inverted L segments in equine herpesvirus 1 concatemers [Virology 229 (1997) 415-420] suggests that the defect may be at the level of cleavage and packaging rather than inversion. In this study, the structures of virion and concatemeric DNA of another class D herpesvirus, bovine herpesvirus 1, were determined. Virion DNA contained low levels of I L genomes, whereas concatemeric DNA contained significant amounts of L segments in both P and I L orientations. However, concatemeric termini exhibited a preponderance of L termini derived from P isomers which was comparable to the preponderance of P genomes found in virion DNA. Thus, the defect in formation of I L genomes appears to lie at the level of concatemer cleavage. These results have important implications for the mechanisms by which herpesvirus DNA cleavage and packaging occur

VP3 is crucial for the stability of Nora virus virions.

Science.gov (United States)

Sadanandan, Sajna Anand; Ekström, Jens-Ola; Jonna, Venkateswara Rao; Hofer, Anders; Hultmark, Dan

2016-09-02

Nora virus is an enteric virus that causes persistent, non-pathological infection in Drosophila melanogaster. It replicates in the fly gut and is transmitted via the fecal-oral route. Nora virus has a single-stranded positive-sense RNA genome, which is translated in four open reading frames. Reading frame three encodes the VP3 protein, the structure and function of which we have investigated in this work. We have shown that VP3 is a trimer that has an α-helical secondary structure, with a functionally important coiled-coil domain. In order to identify the role of VP3 in the Nora virus life cycle, we constructed VP3-mutants using the cDNA clone of the virus. Our results show that VP3 does not have a role in the actual assembly of the virus particles, but virions that lack VP3 or harbor VP3 with a disrupted coiled coil domain are incapable of transmission via the fecal-oral route. Removing the region downstream of the putative coiled coil appears to have an effect on the fitness of the virus but does not hamper its replication or transmission. We also found that the VP3 protein and particularly the coiled coil domain are crucial for the stability of Nora virus virions when exposed to heat or proteases. Hence, we propose that VP3 is imperative to Nora virus virions as it confers stability to the viral capsid. Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.

Perforated membrane-type acoustic metamaterials

International Nuclear Information System (INIS)

Langfeldt, F.; Kemsies, H.; Gleine, W.; Estorff, O. von

2017-01-01

This letter introduces a modified design of membrane-type acoustic metamaterials (MAMs) with a ring mass and a perforation so that an airflow through the membrane is enabled. Simplified analytical investigations of the perforated MAM (PMAM) indicate that the perforation introduces a second anti-resonance, where the effective surface mass density of the PMAM is much higher than the static value. The theoretical results are validated using impedance tube measurements, indicating good agreement between the theoretical predictions and the measured data. The anti-resonances yield high low-frequency sound transmission loss values with peak values over 25 dB higher than the corresponding mass-law. - Highlights: • A new membrane-type acoustic metamaterial exhibiting negative density is presented. • The metamaterial design contains a ring mass with a perforation through the membrane. • The sound transmission loss exhibits narrow-band peaks much higher than the mass-law. • The emergence of the peaks is explained using a simple theoretical model. • Impedance tube measurements are used to validate the theoretical predictions.

Perforated membrane-type acoustic metamaterials

Energy Technology Data Exchange (ETDEWEB)

Langfeldt, F., E-mail: Felix.Langfeldt@haw-hamburg.de [Department of Automotive and Aeronautical Engineering, Hamburg University of Applied Sciences, Berliner Tor 9, D-20099 Hamburg (Germany); Kemsies, H., E-mail: Hannes.Kemsies@haw-hamburg.de [Department of Automotive and Aeronautical Engineering, Hamburg University of Applied Sciences, Berliner Tor 9, D-20099 Hamburg (Germany); Gleine, W., E-mail: Wolfgang.Gleine@haw-hamburg.de [Department of Automotive and Aeronautical Engineering, Hamburg University of Applied Sciences, Berliner Tor 9, D-20099 Hamburg (Germany); Estorff, O. von, E-mail: estorff@tu-harburg.de [Institute of Modelling and Computation, Hamburg University of Technology, Denickestr. 17, D-21073 Hamburg (Germany)

2017-04-25

This letter introduces a modified design of membrane-type acoustic metamaterials (MAMs) with a ring mass and a perforation so that an airflow through the membrane is enabled. Simplified analytical investigations of the perforated MAM (PMAM) indicate that the perforation introduces a second anti-resonance, where the effective surface mass density of the PMAM is much higher than the static value. The theoretical results are validated using impedance tube measurements, indicating good agreement between the theoretical predictions and the measured data. The anti-resonances yield high low-frequency sound transmission loss values with peak values over 25 dB higher than the corresponding mass-law. - Highlights: • A new membrane-type acoustic metamaterial exhibiting negative density is presented. • The metamaterial design contains a ring mass with a perforation through the membrane. • The sound transmission loss exhibits narrow-band peaks much higher than the mass-law. • The emergence of the peaks is explained using a simple theoretical model. • Impedance tube measurements are used to validate the theoretical predictions.

Mechanisms for lymphocytic choriomeningitis virus glycoprotein cleavage, transport, and incorporation into virions

International Nuclear Information System (INIS)

Kunz, Stefan; Edelmann, Kurt H.; Torre, Juan-Carlos de la; Gorney, Robert; Oldstone, Michael B.A.

2003-01-01

The glycoprotein (GP) of lymphocytic choriomeningitis virus (LCMV) serves as virus attachment protein to its receptor on host cells and is a key determinant for cell tropism, pathogenesis, and epidemiology of the virus. The GP of LCMV is posttranslationally cleaved by the subtilase SKI-1/S1P into two subunits, the peripheral GP1, which is implicated in receptor binding, and the transmembrane GP2 that is structurally similar to the fusion active membrane proximal portions of the glycoproteins of other enveloped viruses. The present study shows that cleavage by SKI-1/S1P is not required for cell surface expression of LCMVGP on infected cells but is essential for its incorporation into virions and for the production of infectious virus particles. In absence of SKI-1/S1P cleavage, cell-to-cell propagation of the virus was markedly reduced. Further, proteolytic processing of LCMVGP depends on the presence of a cluster of basic amino acids at the C-terminus of the cytoplasmic domain of GP2, a structural motif that is conserved in Old World arenaviruses. The effect of the truncation of the cytoplasmic tail on cleavage suggests a structural interdependence between the cytoplasmic domain and the ectodomains of LCMVGP

Liver/kidney microsomal antibody type 1 targets CYP2D6 on hepatocyte plasma membrane

OpenAIRE

Muratori, L; Parola, M; Ripalti, A; Robino, G; Muratori, P; Bellomo, G; Carini, R; Lenzi, M; Landini, M; Albano, E; Bianchi, F

2000-01-01

BACKGROUND—Liver/kidney microsomal antibody type 1 (LKM1) is the marker of type 2 autoimmune hepatitis (AIH) and is detected in up to 6% of patients with hepatitis C virus (HCV) infection. It recognises linear and conformational epitopes of cytochrome P450IID6 (CYP2D6) and may have liver damaging activity, provided that CYP2D6 is accessible to effector mechanisms of autoimmune attack.
METHODS—The presence of LKM1 in the plasma membrane was investigated by indirect immunofluorescence and confo...

HIV-1 Infection of T Cells and Macrophages Are Differentially Modulated by Virion-Associated Hck: A Nef-Dependent Phenomenon

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Gilda Tachedjian

2013-09-01

Full Text Available The proline repeat motif (PxxP of Nef is required for interaction with the SH3 domains of macrophage-specific Src kinase Hck. However, the implication of this interaction for viral replication and infectivity in macrophages and T lymphocytes remains unclear. Experiments in HIV-1 infected macrophages confirmed the presence of a Nef:Hck complex which was dependent on the Nef proline repeat motif. The proline repeat motif of Nef also enhanced both HIV-1 infection and replication in macrophages, and was required for incorporation of Hck into viral particles. Unexpectedly, wild-type Hck inhibited infection of macrophages, but Hck was shown to enhance infection of primary T lymphocytes. These results indicate that the interaction between Nef and Hck is important for Nef-dependent modulation of viral infectivity. Hck-dependent enhancement of HIV-1 infection of T cells suggests that Nef-Hck interaction may contribute to the spread of HIV-1 infection from macrophages to T cells by modulating events in the producer cell, virion and target cell.

The C-terminal HRET sequence of Kv1.3 regulates gating rather than targeting of Kv1.3 to the plasma membrane.

Science.gov (United States)

Voros, Orsolya; Szilagyi, Orsolya; Balajthy, András; Somodi, Sándor; Panyi, Gyorgy; Hajdu, Péter

2018-04-12

Kv1.3 channels are expressed in several cell types including immune cells, such as T lymphocytes. The targeting of Kv1.3 to the plasma membrane is essential for T cell clonal expansion and assumed to be guided by the C-terminus of the channel. Using two point mutants of Kv1.3 with remarkably different features compared to the wild-type Kv1.3 (A413V and H399K having fast inactivation kinetics and tetraethylammonium-insensitivity, respectively) we showed that both Kv1.3 channel variants target to the membrane when the C-terminus was truncated right after the conserved HRET sequence and produce currents identical to those with a full-length C-terminus. The truncation before the HRET sequence (NOHRET channels) resulted in reduced membrane-targeting but non-functional phenotypes. NOHRET channels did not display gating currents, and coexpression with wild-type Kv1.3 did not rescue the NOHRET-A413V phenotype, no heteromeric current was observed. Interestingly, mutants of wild-type Kv1.3 lacking HRET(E) (deletion) or substituted with five alanines for the HRET(E) motif expressed current indistinguishable from the wild-type. These results demonstrate that the C-terminal region of Kv1.3 immediately proximal to the S6 helix is required for the activation gating and conduction, whereas the presence of the distal region of the C-terminus is not exclusively required for trafficking of Kv1.3 to the plasma membrane.

The roles of the cyclo-oxygenases types one and two in prostaglandin synthesis in human fetal membranes at term.

Science.gov (United States)

Sawdy, R J; Slater, D M; Dennes, W J; Sullivan, M H; Bennett, P R

2000-01-01

The aim of this study was to determine the relative contributions of cyclo-oxygenase (COX) types 1 and 2 to prostaglandin synthesis at term. Fetal membranes were collected from 6 pregnancies after elective caesarean section at term, prior to labour. The presence of COX-1 and COX-2 protein was determined using Western analysis. The relative contributions of the two isoforms of COX to prostaglandin synthesis were determined by incubation of fetal membrane discs with either a COX-2 selective inhibitor, SC236, or a COX-1 selective inhibitor, SC560, and measurement of prostaglandin release during 24 h using enzyme-linked immuno-sorbent assay (ELISA). Both COX-1 and COX-2 protein were demonstrated in amnion and chorion-decidua. The COX-2 selective inhibitor, SC-236, significantly reduced prostaglandin synthesis, both in its COX-2 specific and higher, non-specific concentration ranges. The COX-1 selective inhibitor, SC-560, had no effect upon prostaglandin synthesis in its COX-1 specific concentration range, but did significantly reduce prostaglandin synthesis at higher, non-selective concentrations. Fetal membranes contain both COX-1 and COX-2 at term, but only COX-2 contributes towards prostaglandin synthesis. COX-2 selective NSAI drugs will be as effective as non-selective agents in inhibition of fetal membrane prostaglandin synthesis and may represent a new strategy for tocolysis. Copyright 2000 Harcourt Publishers Ltd.

Histological evaluation of different biodegradable and non-biodegradable membranes implanted subcutaneously in rats

DEFF Research Database (Denmark)

Zhao, S; Pinholt, E M; Madsen, J E

2000-01-01

Different types of biodegradable membranes have become available for guided tissue regeneration. The purpose of this study was to evaluate histologically three different biodegradable membranes (Bio-Gide, Resolut and Vicryl) and one non-biodegradable membrane (expanded polytetrafluoroethylene/e-PTFE...... that e-PTFE was well tolerated and encapsulated by a fibrous connective tissue capsule. There was capsule formation around Resolut and Vicryl and around Bio-Gide in the early phase there was a wide inflammatory zone already. e-PTFE and Vicryl were stable materials while Resolut and Bio-Gide fragmented...

Equine herpesvirus type 1 (EHV-1) glycoprotein K is required for efficient cell-to-cell spread and virus egress

International Nuclear Information System (INIS)

Neubauer, Antonie; Osterrieder, Nikolaus

2004-01-01

The function of the equine herpesvirus type 1 (EHV-1) glycoprotein K (gK) homologue was investigated. Deletion of 88% of the UL53-homologous open reading frame in EHV-1 strain RacH resulted in a severe growth defect of the gK-negative virus (HΔgK) as reflected by a significant decrease in the production of infectious virus progeny on RK13 cells. The HΔgK virus induced only minute plaques, was unable to form syncytia, and its penetration efficiency into RK13 cells was reduced by approximately 40%. To further analyze gK function and intracellular trafficking, gK of strain RacH was replaced by a C-terminally truncated gK-green fluorescent protein fusion protein (gK-GFP). The generated recombinant virus was shown to replicate well on non-complementing cells, and virus penetration and syncytium formation were comparable to parental RacH. A reduction in plaque size and slightly decreased intra- and extracellular virus titers, however, were observed. The gK-GFP fusion protein was expressed with early-late kinetics, and multiple forms of the protein exhibiting M r s between 50,000 and 85,000 were detected by Western blot analysis. The various gK-GFP forms were shown to be N-glycosylated, associated with membranes of the Golgi apparatus, and were incorporated into extracellular virions. Complete processing of gK-GFP was only observed within the context of viral infection. From the results, we concluded that EHV-1 gK is required for efficient virus growth in vitro and that the carboxy-terminal amino acids are not required for its function, because the gK-GFP fusion protein was able to complement for EHV-1 growth in the absence of authentic gK

Protein 4.1, a component of the erythrocyte membrane skeleton and its related homologue proteins forming the protein 4.1/FERM superfamily.

Directory of Open Access Journals (Sweden)

Aleksander F Sikorski

2007-01-01

Full Text Available The review is focused on the domain structure and function of protein 4.1, one of the proteins belonging to the membrane skeleton. The protein 4.1 of the red blood cells (4.1R is a multifunctional protein that localizes to the membrane skeleton and stabilizes erythrocyte shape and membrane mechanical properties, such as deformability and stability, via lateral interactions with spectrin, actin, glycophorin C and protein p55. Protein 4.1 binding is modulated through the action of kinases and/or calmodulin-Ca2+. Non-erythroid cells express the 4.1R homologues: 4.1G (general type, 4.1B (brain type, and 4.1N (neuron type, and the whole group belongs to the protein 4.1 superfamily, which is characterized by the presence of a highly conserved FERM domain at the N-terminus of the molecule. Proteins 4.1R, 4.1G, 4.1N and 4.1B are encoded by different genes. Most of the 4.1 superfamily proteins also contain an actin-binding domain. To date, more than 40 members have been identified. They can be divided into five groups: protein 4.1 molecules, ERM proteins, talin-related molecules, protein tyrosine phosphatase (PTPH proteins and NBL4 proteins. We have focused our attention on the main, well known representatives of 4.1 superfamily and tried to choose the proteins which are close to 4.1R or which have distinct functions. 4.1 family proteins are not just linkers between the plasma membrane and membrane skeleton; they also play an important role in various processes. Some, such as focal adhesion kinase (FAK, non-receptor tyrosine kinase that localizes to focal adhesions in adherent cells, play the role in cell adhesion. The other members control or take part in tumor suppression, regulation of cell cycle progression, inhibition of cell proliferation, downstream signaling of the glutamate receptors, and establishment of cell polarity; some are also involved in cell proliferation, cell motility, and/or cell-to-cell communication.

Generation of a human immunodeficiency virus type 1 chronically infected monkey B cell line expressing low levels of endogenous TRIM5alpha.

Science.gov (United States)

Ridolfi, Barbara; Catone, Stefania; Sgarbanti, Marco; Sernicola, Leonardo; Battistini, Angela; Parolin, Cristina; Titti, Fausto; Borsetti, Alessandra

2009-12-01

Several innate cellular antiviral factors exist in mammalian cells that prevent the replication of retroviruses. Among them, the tripartite motif protein (TRIM)5alpha has been shown to block human immunodeficiency virus type 1 (HIV-1) infection in several types of Old World monkey cells. Here we report a novel HIV-1 chronically infected monkey B cell line, F6/HIV-1, characterized by very low levels of TRIM5alpha expression that allows HIV-1 to overcome the restriction. Virus produced by F6/HIV-1 cells fails to infect monkey cells but retains the ability to infect human peripheral blood mononuclear cells (PBMCs) and T cell lines, although with a reduced infectivity compared to the input virus. Ultrastructural analyses revealed the presence of budding virions at the F6/HIV-1 cells plasma membrane characterized by a typical conical core shell. To our knowledge F6/HIV-1 is the first monkey cell line chronically infected by HIV-1 and able to release infectious particles thus representing a useful tool to gain further insights into the molecular mechanisms of HIV-1 pathogenesis.

Empty virions in AAV8 vector preparations reduce transduction efficiency and may cause total viral particle dose-limiting side effects

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Kai Gao

2014-01-01

Full Text Available Empty virions are inadvertent by-products of recombinant adeno-associated virus (rAAV packaging process, resulting in vector lots with mixtures of full and empty virions at variable ratios. Impact of empty virions on the efficiency and side effects of rAAV transduction has not been well characterized. Here, we generated partially and completely empty AAV8 virions, fully packaged rAAV8 lots, and mixtures of empty and fully packaged virions with variable ratios of empty virions. The aforementioned dosing formulations of rAAV8 expressing either cellular (EGFP (enhanced green fluorescent protein or nuclear-targeted (n LacZ or secreted (human α1-antitrypsin (hA1AT reporter genes were intravenously injected into two different mouse strains, followed by analyses of transgene expressions and serum alanine aminotransferase (ALT levels at different time points. We found that addition of empty particles to the fixed doses of rAAV8 preparations repressed liver transduction up to 64% (serum hA1AT and 44% (nLacZ in C57BL/6 mice, respectively. The similar trend in inhibiting EGFP expression together with concurrent elevations of serum ALT levels were observed in the BALB/c mice, indicating that empty particles may also exacerbate side effects of rAAV8 EGFP transduction. Our results suggest that removal of empty particles from rAAV preparations may improve efficacy and safety of AAV in clinical applications.

The pancreatic zymogen granule membrane protein, GP2, binds Escherichia coli type 1 Fimbriae

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Lowe Anson W

2009-07-01

Full Text Available Abstract Background GP2 is the major membrane protein present in the pancreatic zymogen granule, and is cleaved and released into the pancreatic duct along with exocrine secretions. The function of GP2 is unknown. GP2's amino acid sequence is most similar to that of uromodulin, which is secreted by the kidney. Recent studies have demonstrated uromodulin binding to bacterial Type 1 fimbria. The fimbriae serve as adhesins to host receptors. The present study examines whether GP2 also shares similar binding properties to bacteria with Type 1 fimbria. Commensal and pathogenic bacteria, including E. coli and Salmonella, express type 1 fimbria. Methods An in vitro binding assay was used to assay the binding of recombinant GP2 to defined strains of E. coli that differ in their expression of Type 1 fimbria or its subunit protein, FimH. Studies were also performed to determine whether GP2 binding is dependent on the presence of mannose residues, which is a known determinant for FimH binding. Results GP2 binds E. coli that express Type 1 fimbria. Binding is dependent on GP2 glycosylation, and specifically the presence of mannose residues. Conclusion GP2 binds to Type 1 fimbria, a bacterial adhesin that is commonly expressed by members of the Enterobacteriacae family.

Conformational changes in Sindbis virions resulting from exposure to low pH and interactions with cells suggest that cell penetration may occur at the cell surface in the absence of membrane fusion

International Nuclear Information System (INIS)

Paredes, Angel M.; Ferreira, Davis; Horton, Michelle; Saad, Ali; Tsuruta, Hiro; Johnston, Robert; Klimstra, William; Ryman, Kate; Hernandez, Raquel; Chiu Wah; Brown, Dennis T.

2004-01-01

Alphaviruses have the ability to induce cell-cell fusion after exposure to acid pH. This observation has served as an article of proof that these membrane-containing viruses infect cells by fusion of the virus membrane with a host cell membrane upon exposure to acid pH after incorporation into a cell endosome. We have investigated the requirements for the induction of virus-mediated, low pH-induced cell-cell fusion and cell-virus fusion. We have correlated the pH requirements for this process to structural changes they produce in the virus by electron cryo-microscopy. We found that exposure to acid pH was required to establish conditions for membrane fusion but that membrane fusion did not occur until return to neutral pH. Electron cryo-microscopy revealed dramatic changes in the structure of the virion as it was moved to acid pH and then returned to neutral pH. None of these treatments resulted in the disassembly of the virus protein icosahedral shell that is a requisite for the process of virus membrane-cell membrane fusion. The appearance of a prominent protruding structure upon exposure to acid pH and its disappearance upon return to neutral pH suggested that the production of a 'pore'-like structure at the fivefold axis may facilitate cell penetration as has been proposed for polio (J. Virol. 74 (2000) 1342) and human rhino virus (Mol. Cell 10 (2002) 317). This transient structural change also provided an explanation for how membrane fusion occurs after return to neutral pH. Examination of virus-cell complexes at neutral pH supported the contention that infection occurs at the cell surface at neutral pH by the production of a virus structure that breaches the plasma membrane bilayer. These data suggest an alternative route of infection for Sindbis virus that occurs by a process that does not involve membrane fusion and does not require disassembly of the virus protein shell

Characterization of a non-fouling ultrafiltration membrane

DEFF Research Database (Denmark)

Wei, J.; Helm, G.S.; Corner-Walker, N.

2006-01-01

This report describes the properties of surface-modified poly(vinylidene fluoride) (PVDF) membranes. These membranes were created by coating hydrophilic polymers on the support PVDF membrane to reduce the tendency to protein fouling. The modified membranes with different molecular weight cut......-off (MWCO) were characterized by filtration studies using bovine serum albumin (BSA) and an enzyme solution as test media, and the membranes exhibited the non-fouling property. The surface chemistry of the unmodified and modified PVDF membranes was characterized by X-ray photoelectron spectroscopy (XPS......) and time-of-flight secondary ion mass spectroscopy (ToF-SIMS). These surface sensitive techniques were used to confirm the successful surface modification. ToF-SIMS imaging visualizes the distribution of the coating layer on the PVDF membrane. Furthermore, the amount of protein adsorption onto the membrane...

Dominant-negative effect of hetero-oligomerization on the function of the human immunodeficiency virus type 1 envelope glycoprotein complex

International Nuclear Information System (INIS)

Herrera, Carolina; Klasse, Per Johan; Kibler, Christopher W.; Michael, Elizabeth; Moore, John P.; Beddows, Simon

2006-01-01

The human immunodeficiency virus type 1 (HIV-1) envelope (Env) glycoprotein forms trimers that mediate interactions with the CD4 receptor and a co-receptor on the target cell surface, thereby triggering viral fusion with the cell membrane. Cleavage of Env into its surface, gp120, and transmembrane, gp41, moieties is necessary for activation of its fusogenicity. Here, we produced pseudoviruses with phenotypically mixed wild-type (Wt) and mutant, cleavage-incompetent Env in order to quantify the effects of incorporating uncleaved Env on virion infectivity, antigenicity and neutralization sensitivity. We modeled the relative infectivity of three such phenotypically mixed viral strains, JR-FL, HXBc2 and a derivative of the latter, 3.2P, as a function of the relative amount of Wt Env. The data were fit very closely (R 2 > 0.99) by models which assumed that only Wt homotrimers were functional, with different approximate thresholds of critical numbers of functional trimers per virion for the three strains. We also produced 3.2P pseudoviruses containing both a cleavage-competent Env that is defective for binding the neutralizing monoclonal antibody (NAb) 2G12, and a cleavage-incompetent Env that binds 2G12. The 2G12 NAb was not able to reduce the infectivity of these pseudoviruses detectably. Their neutralization by the CD4-binding site-directed agents CD4-IgG2 and NAb b12 was also unaffected by 2G12 binding to uncleaved Env. These results further strengthen the conclusion that only homotrimers consisting of cleaved Env are functional. They also imply that the function of a trimer is unaffected sterically by the binding of an antibody to an adjacent trimer

Herpes simplex virus types 1 and 2 induce shutoff of host protein synthesis by different mechanisms in Friend erythroleukemia cells

International Nuclear Information System (INIS)

Hill, T.M.; Sinden, R.R.; Sadler, J.R.

1983-01-01

Herpes simplex virus type 1 (HSV-1) and HSV-2 disrupt host protein synthesis after viral infection. We have treated both viral types with agents which prevent transcription of the viral genome and used these treated viruses to infect induced Friend erythroleukemia cells. By measuring the changes in globin synthesis after infection, we have determined whether expression of the viral genome precedes the shutoff of host protein synthesis or whether the inhibitor molecule enters the cells as part of the virion. HSV-2-induced shutoff of host protein synthesis was insensitive to the effects of shortwave (254-nm) UV light and actinomycin D. Both of the treatments inhibited HSV-1-induced host protein shutoff. Likewise, treatment of HSV-1 with the cross-linking agent 4,5',8-trimethylpsoralen and longwave (360-nm) UV light prevented HSV-1 from inhibiting cellular protein synthesis. Treatment of HSV-2 with 4,5',8-trimethylpsoralen did not affect the ability of the virus to interfere with host protein synthesis, except at the highest doses of longwave UV light. It was determined that the highest longwave UV dosage damaged the HSV-2 virion as well as cross-linking the viral DNA. The results suggest that HSV-2 uses a virion-associated component to inhibit host protein synthesis and that HSV-1 requires the expression of the viral genome to cause cellular protein synthesis shutoff

Estimating the fraction of progeny virions that must incorporate APOBEC3G for suppression of productive HIV-1 infection

International Nuclear Information System (INIS)

Thangavelu, Pulari U.; Gupta, Vipul; Dixit, Narendra M.

2014-01-01

The contest between the host factor APOBEC3G (A3G) and the HIV-1 protein Vif presents an attractive target of intervention. The extent to which the A3G–Vif interaction must be suppressed to tilt the balance in favor of A3G remains unknown. We employed stochastic simulations and mathematical modeling of the within-host dynamics and evolution of HIV-1 to estimate the fraction of progeny virions that must incorporate A3G to render productive infection unsustainable. Using three different approaches, we found consistently that a transition from sustained infection to suppression of productive infection occurred when the latter fraction exceeded ∼0.8. The transition was triggered by A3G-induced hypermutations that led to premature stop codons compromising viral production and was consistent with driving the basic reproductive number, R 0 , below unity. The fraction identified may serve as a quantitative guideline for strategies targeting the A3G–Vif axis. - Highlights: • We perform simulations and mathematical modeling of the role of APOBEC3G in suppressing HIV-1 infection. • In three distinct ways, we estimate that when over 80% of progeny virions carry APOBEC3G, productive HIV-1 infection would be suppressed. • Our estimate of this critical fraction presents quantitative guidelines for strategies targeting the APOBEC3G–Vif axis

Human endogenous retrovirus K Gag coassembles with HIV-1 Gag and reduces the release efficiency and infectivity of HIV-1.

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Monde, Kazuaki; Contreras-Galindo, Rafael; Kaplan, Mark H; Markovitz, David M; Ono, Akira

2012-10-01

Human endogenous retroviruses (HERVs), which are remnants of ancestral retroviruses integrated into the human genome, are defective in viral replication. Because activation of HERV-K and coexpression of this virus with HIV-1 have been observed during HIV-1 infection, it is conceivable that HERV-K could affect HIV-1 replication, either by competition or by cooperation, in cells expressing both viruses. In this study, we found that the release efficiency of HIV-1 Gag was 3-fold reduced upon overexpression of HERV-K(CON) Gag. In addition, we observed that in cells expressing Gag proteins of both viruses, HERV-K(CON) Gag colocalized with HIV-1 Gag at the plasma membrane. Furthermore, HERV-K(CON) Gag was found to coassemble with HIV-1 Gag, as demonstrated by (i) processing of HERV-K(CON) Gag by HIV-1 protease in virions, (ii) coimmunoprecipitation of virion-associated HERV-K(CON) Gag with HIV-1 Gag, and (iii) rescue of a late-domain-defective HERV-K(CON) Gag by wild-type (WT) HIV-1 Gag. Myristylation-deficient HERV-K(CON) Gag localized to nuclei, suggesting cryptic nuclear trafficking of HERV-K Gag. Notably, unlike WT HERV-K(CON) Gag, HIV-1 Gag failed to rescue myristylation-deficient HERV-K(CON) Gag to the plasma membrane. Efficient colocalization and coassembly of HIV-1 Gag and HERV-K Gag also required nucleocapsid (NC). These results provide evidence that HIV-1 Gag heteromultimerizes with HERV-K Gag at the plasma membrane, presumably through NC-RNA interaction. Intriguingly, HERV-K Gag overexpression reduced not only HIV-1 release efficiency but also HIV-1 infectivity in a myristylation- and NC-dependent manner. Altogether, these results indicate that Gag proteins of endogenous retroviruses can coassemble with HIV-1 Gag and modulate the late phase of HIV-1 replication.

Derangement of cellular plasma membranes due to non-lethal radiation doses

International Nuclear Information System (INIS)

Koeteles, G.J.; Kubasova, T.; Somosy, Z.; Horvath, L.

1983-01-01

Earlier observations in the laboratory on fibroblasts and various blood cells of animal and human origins pointed to alteration of concanavalin A binding sites of plasma membranes as well as to concomitant morphological changes and scanning electron microscopic appearance of cell surfaces following sub-lethal doses of X-, fission neutron and beta irradiations. The effects appeared early and existed temporarily; their intensities and the restitution of membrane function depended on radiation doses, types and conditions of cells. In the present paper further aspects of structural and functional derangements of plasma membranes are introduced which were provoked by X- and tritium beta irradiation in the dose range up to 2.5 Gy and in the concentration range from 3.7 kBq/mL, respectively. The state of membrane structure was followed by bindings of various ligands of different receptor requirements, concanavalin A, cationized ferritin and polio virus. In the case of X-irradiation the binding conditions suggest the shift of overall negative surface charges to less negative ones. It was also found that radiation-induced phenomena appear on the cell surface unevenly. Long- and short-term treatments of cells with 3 H-thymidine and 3 H-water also perturb the plasma membrane; beta irradiation affects it directly. Membrane structure and function are suggested to offer good biological models to study correlation of energy deposition and biological effects, both restricted to domains of nanometre range. The data give evidence for radiation-induced membrane alterations in the sub-lethal or non-lethal ranges which might have consequences in the development of stochastic and non-stochastic effects. (author)

Biliary Secretion of Quasi-Enveloped Human Hepatitis A Virus.

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Hirai-Yuki, Asuka; Hensley, Lucinda; Whitmire, Jason K; Lemon, Stanley M

2016-12-06

Hepatitis A virus (HAV) is an unusual picornavirus that is released from cells cloaked in host-derived membranes. These quasi-enveloped virions (eHAV) are the only particle type circulating in blood during infection, whereas only nonenveloped virions are shed in feces. The reason for this is uncertain. Hepatocytes, the only cell type known to support HAV replication in vivo, are highly polarized epithelial cells with basolateral membranes facing onto hepatic (blood) sinusoids and apical membranes abutting biliary canaliculi from which bile is secreted to the gut. To assess whether eHAV and nonenveloped virus egress from cells via vectorially distinct pathways, we studied infected polarized cultures of Caco-2 and HepG2-N6 cells. Most (>99%) progeny virions were released apically from Caco-2 cells, whereas basolateral (64%) versus apical (36%) release was more balanced with HepG2-N6 cells. Both apically and basolaterally released virions were predominantly enveloped, with no suggestion of differential vectorial release of eHAV versus naked virions. Basolateral to apical transcytosis of either particle type was minimal (work reveals that it has an unusual life cycle. Virus is found in cell culture supernatant fluids in two mature, infectious forms: one wrapped in membranes (quasi-enveloped) and another that is nonenveloped. Membrane-wrapped virions circulate in blood during acute infection and are resistant to neutralizing antibodies, likely facilitating HAV dissemination within the liver. On the other hand, virus shed in feces is nonenveloped and highly stable, facilitating epidemic spread and transmission to naive hosts. Factors controlling the biogenesis of these two distinct forms of the virus in infected humans are not understood. Here we characterize vectorial release of quasi-enveloped virions from polarized epithelial cell cultures and provide evidence that bile acids strip membranes from eHAV following its secretion into the biliary tract. These results

[Computer modeling the hydrostatic pressure characteristics of the membrane potential for polymeric membrane, separated non-homogeneous electrolyte solutions].

Science.gov (United States)

Slezak, Izabella H; Jasik-Slezak, Jolanta; Rogal, Mirosława; Slezak, Andrzej

2006-01-01

On the basis of model equation depending the membrane potential deltapsis, on mechanical pressure difference (deltaP), concentration polarization coefficient (zetas), concentration Rayleigh number (RC) and ratio concentration of solutions separated by membrane (Ch/Cl), the characteristics deltapsis = f(deltaP)zetas,RC,Ch/Cl for steady values of zetas, RC and Ch/Cl in single-membrane system were calculated. In this system neutral and isotropic polymeric membrane oriented in horizontal plane, the non-homogeneous binary electrolytic solutions of various concentrations were separated. Nonhomogeneity of solutions is results from creations of the concentration boundary layers on both sides of the membrane. Calculations were made for the case where on a one side of the membrane aqueous solution of NaCl at steady concentration 10(-3) mol x l(-1) (Cl) was placed and on the other aqueous solutions of NaCl at concentrations from 10(-3) mol x l(-1) to 2 x 10(-2) mol x l(-1) (Ch). Their densities were greater than NaCl solution's at 10(-3) mol x l(-1). It was shown that membrane potential depends on hydrodynamic state of a complex concentration boundary layer-membrane-concentration boundary layer, what is controlled by deltaP, Ch/Cl, RC and zetas.

Orsay virus utilizes ribosomal frameshifting to express a novel protein that is incorporated into virions

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Jiang, Hongbing; Franz, Carl J.; Wu, Guang; Renshaw, Hilary; Zhao, Guoyan [Departments of Molecular Microbiology and Pathology and Immunology, Washington University in St. Louis School of Medicine, St. Louis, MO 63110 (United States); Firth, Andrew E. [Department of Pathology, University of Cambridge, Cambridge CB2 1QP (United Kingdom); Wang, David, E-mail: davewang@borcim.wustl.edu [Departments of Molecular Microbiology and Pathology and Immunology, Washington University in St. Louis School of Medicine, St. Louis, MO 63110 (United States)

2014-02-15

Orsay virus is the first identified virus that is capable of naturally infecting Caenorhabditis elegans. Although it is most closely related to nodaviruses, Orsay virus differs from nodaviruses in its genome organization. In particular, the Orsay virus RNA2 segment encodes a putative novel protein of unknown function, termed delta, which is absent from all known nodaviruses. Here we present evidence that Orsay virus utilizes a ribosomal frameshifting strategy to express a novel fusion protein from the viral capsid (alpha) and delta ORFs. Moreover, the fusion protein was detected in purified virus fractions, demonstrating that it is most likely incorporated into Orsay virions. Furthermore, N-terminal sequencing of both the fusion protein and the capsid protein demonstrated that these proteins must be translated from a non-canonical initiation site. While the function of the alpha–delta fusion remains cryptic, these studies provide novel insights into the fundamental properties of this new clade of viruses. - Highlights: • Orsay virus encodes a novel fusion protein by a ribosomal frameshifting mechanism. • Orsay capsid and fusion protein is translated from a non-canonical initiation site. • The fusion protein is likely incorporated into Orsay virions.

Formation of virions is strictly required for turnip yellows virus long-distance movement in plants.

Science.gov (United States)

Hipper, Clémence; Monsion, Baptiste; Bortolamiol-Bécet, Diane; Ziegler-Graff, Véronique; Brault, Véronique

2014-02-01

Viral genomic RNA of the Turnip yellows virus (TuYV; genus Polerovirus; family Luteoviridae) is protected in virions formed by the major capsid protein (CP) and the minor component, the readthrough (RT*) protein. Long-distance transport, used commonly by viruses to systemically infect host plants, occurs in phloem sieve elements and two viral forms of transport have been described: virions and ribonucleoprotein (RNP) complexes. With regard to poleroviruses, virions have always been presumed to be the long-distance transport form, but the potential role of RNP complexes has not been investigated. Here, we examined the requirement of virions for polerovirus systemic movement by analysing CP-targeted mutants that were unable to form viral particles. We confirmed that TuYV mutants that cannot encapsidate into virions are not able to reach systemic leaves. To completely discard the possibility that the introduced mutations in CP simply blocked the formation or the movement of RNP complexes, we tested in trans complementation of TuYV CP mutants by providing WT CP expressed in transgenic plants. WT CP was able to facilitate systemic movement of TuYV CP mutants and this observation was always correlated with the formation of virions. This demonstrated clearly that virus particles are essential for polerovirus systemic movement.

Transcriptome analysis of the Spodoptera frugiperda ascovirus in vivo provides insights into how its apoptosis inhibitors and caspase promote increased synthesis of viral vesicles and virion progeny.

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Zaghloul, Heba; Hice, Robert; Arensburger, Peter; Federici, Brian A

2017-09-27

Ascoviruses are ds DNA viruses that attack caterpillars and differ from all other viruses by inducing nuclear lysis followed by cleavage of host cells into numerous anucleate vesicles in which virus replication continues as these grow in the blood. Ascoviruses are also unusual in that most encode apoptosis inhibitors and caspase or caspase-like proteins. A robust cell line to study the novel molecular biology of ascovirus replication in vitro is lacking. Therefore, we used strand-specific RNA-Seq to study transcription in vivo in third instars of Spodoptera frugiperda infected with the Spodoptera frugiperda ascovirus, a member of the type species, Spodoptera frugiperda ascovirus (SfAV-1a), sampling transcripts at different time points after infection. We targeted transcription of two types of SfAV-1a genes; first, 44 core genes that occur in several ascovirus species, and second, 26 genes predicted in silico to have metabolic functions likely involved in synthesizing viral vesicle membranes. Gene cluster analysis showed differences in temporal expression of SfAV-1a genes, enabling their assignment to three temporal classes; early, late and very late. Inhibitors of apoptosis (IAP-like proteins; ORF016, ORF025 and ORF074) were expressed early, whereas its caspase (ORF073) was expressed very late, which correlated with apoptotic events leading to viral vesicle formation. Expression analysis revealed that a Diedel gene homolog (ORF121), the only known "virokine," was highly expressed, implying this ascovirus protein helps evade innate host immunity. Lastly, single-nucleotide resolution of RNA-Seq data revealed 15 bicistronic and tricistronic messages along the genome, an unusual occurrence for large ds DNA viruses. IMPORTANCE Unlike all other DNA viruses, ascoviruses code for an executioner caspase, apparently involved in a novel cytopathology in which viral replication induces nuclear lysis followed by cell cleavage yielding numerous large anucleate viral vesicles

Morphogenesis of Pestiviruses: New Insights from Ultrastructural Studies of Strain Giraffe-1

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Mast, Jan; Thiel, Heinz-Jürgen; König, Matthias

2014-01-01

Knowledge on the morphogenesis of pestiviruses is limited due to low virus production in infected cells. In order to localize virion morphogenesis and replication sites of pestiviruses and to examine intracellular virion transport, a cell culture model was established to facilitate ultrastructural studies. Based on results of virus growth kinetic analysis and quantification of viral RNA, pestivirus strain Giraffe-1 turned out to be a suitable candidate for studies on virion generation and export from culture cells. Using conventional transmission electron microscopy and single-tilt electron tomography, we found virions located predominately in the lumen of the endoplasmic reticulum (ER) in infected cells and were able to depict the budding process of virions at ER membranes. Colocalization of the viral core protein and the envelope glycoprotein E2 with the ER marker protein disulfide isomerase (PDI) was demonstrated by immunogold labeling of cryosections. Moreover, pestivirions could be shown in transport vesicles and the Golgi complex and during exocytosis. Interestingly, viral capsid protein and double-stranded RNA (dsRNA) were detected in multivesicular bodies (MVBs), which implies that the endosomal compartment plays a role in pestiviral replication. Significant cellular membrane alterations such as those described for members of the Flavivirus and Hepacivirus genera were not found. Based on the gained morphological data, we present a consistent model of pestivirus morphogenesis. PMID:24352462

Hierarchical, domain type-specific acquisition of antibodies to Plasmodium falciparum erythrocyte membrane protein 1 in Tanzanian children

DEFF Research Database (Denmark)

Cham, Gerald K K; Turner, Louise; Kurtis, Jonathan D

2010-01-01

Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) is a variant antigen expressed on the surface of malaria-infected erythrocytes. PfEMP1 attaches to the vascular lining and allows infected erythrocytes to avoid filtration through the spleen. Each parasite genome encodes about 60 diffe...... and play a major role in limiting parasite multiplication in the blood.......Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) is a variant antigen expressed on the surface of malaria-infected erythrocytes. PfEMP1 attaches to the vascular lining and allows infected erythrocytes to avoid filtration through the spleen. Each parasite genome encodes about 60...... different PfEMP1 variants, each PfEMP1 comprises several domains in its extracellular region, and the PfEMP1 repertoire in different parasites contains domain types that are serologically cross-reactive. In this longitudinal study, we followed 672 children living in an area of high malaria transmission...

Tropism, Cytotoxicity, and Inflammatory Properties of Two Envelope Genes of Murine Leukemia Virus Type-Endogenous Retroviruses of C57BL/6J Mice

Directory of Open Access Journals (Sweden)

Young-Kwan Lee

2011-01-01

Full Text Available Envelope (env proteins of certain endogenous retroviruses (ERVs participate in various pathophysiological processes. In this study, we characterized pathophysiologic properties of two murine leukemia virus-type ERV (MuLV-ERV env genes cloned from the ovary of C57BL/6J mice. The two env genes (named ENVOV1 and ENVOV2, with 1,926\\,bp coding region, originated from two MuLV-ERV loci on chromosomes 8 and 18, respectively. ENVOV1 and ENVOV2 were ~75 kDa and predominantly expressed on the cell membrane. They were capable of producing pseudotype murine leukemia virus virions. Tropism trait and infectivity of ENVOV2 were similar to the polytropic env; however, ENVOV1 had very low level of infectivity. Overexpression of ENVOV2, but not ENVOV1, exerted cytotoxic effects and induced expression of COX-2, IL-1β, IL-6, and iNOS. These findings suggest that the ENVOV1 and ENVOV2 are capable of serving as an env protein for virion assembly, and they exert differential cytotoxicity and modulation of inflammatory mediators.

Patents on Membranes Based on Non-Fluorinated Polymers for Vanadium Redox Flow Batteries.

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Choi, So-Won; Kim, Tae-Ho; Cha, Sang-Ho

2017-07-10

Vanadium redox flow batteries (VRFBs) have received considerable attention as large-scale electrochemical energy storage systems. In particular, VRFBs offer a higher power and energy density than other RFBs and mitigate undesirable performance fading, such as inevitable ion crossover, because of the unique advantage that only the vanadium ion is employed as the active species in the two electrolytes. The key constituent of VRFBs is a separator to conduct protons and prevent cross-mixing of the positive and negative electrolytes. For this purpose, ion exchange membranes like sulfonated polymer membranes can be used. Although this type of membrane does not have ion exchange groups, it can achieve an ion exchange capacity by the formation of pores. This review highlights the patents on the preparation of non-fluorinated membranes (sulfonated aromatic polymer membranes and porous membranes) as alternatives to high-cost perfluorinated polymers and their VRFB performance. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

Imaging Herpes Simplex Virus Type 1 Amplicon Vector–Mediated Gene Expression in Human Glioma Spheroids

OpenAIRE

Christine Kaestle; Alexandra Winkeler; Raphaela Richter; Heinrich Sauer; Jürgen Hescheler; Cornel Fraefel; Maria Wartenberg; Andreas H. Jacobs

2011-01-01

Vectors derived from herpes simplex virus type 1 (HSV-1) have great potential for transducing therapeutic genes into the central nervous system; however, inefficient distribution of vector particles in vivo may limit their therapeutic potential in patients with gliomas. This study was performed to investigate the extent of HSV-1 amplicon vector–mediated gene expression in a three-dimensional glioma model of multicellular spheroids by imaging highly infectious HSV-1 virions expressing green fl...

A riboswitch regulates RNA dimerization and packaging in human immunodeficiency virus type 1 virions

NARCIS (Netherlands)

Ooms, Marcel; Huthoff, Hendrik; Russell, Rodney; Liang, Chen; Berkhout, Ben

2004-01-01

The genome of retroviruses, including human immunodeficiency virus type I (HIV-1), consists of two identical RNA strands that are packaged as noncovalently linked dimers. The core packaging and dimerization signals are located in the downstream part of the untranslated leader of HIV-1 RNA-the Psi

ALS-causing profilin-1-mutant forms a non-native helical structure in membrane environments.

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Lim, Liangzhong; Kang, Jian; Song, Jianxing

2017-11-01

Despite having physiological functions completely different from superoxide dismutase 1 (SOD1), profilin 1 (PFN1) also carries mutations causing amyotrophic lateral sclerosis (ALS) with a striking similarity to that triggered by SOD1 mutants. Very recently, the C71G-PFN1 has been demonstrated to cause ALS by a gain of toxicity and the acceleration of motor neuron degeneration preceded the accumulation of its aggregates. Here by atomic-resolution NMR determination of conformations and dynamics of WT-PFN1 and C71G-PFN1 in aqueous buffers and in membrane mimetics DMPC/DHPC bicelle and DPC micelle, we deciphered that: 1) the thermodynamic destabilization by C71G transforms PFN1 into coexistence with the unfolded state, which is lacking of any stable tertiary/secondary structures as well as restricted ps-ns backbone motions, thus fundamentally indistinguishable from ALS-causing SOD1 mutants. 2) Most strikingly, while WT-PFN1 only weakly interacts with DMPC/DHPC bicelle without altering the native structure, C71G-PFN1 acquires abnormal capacity in strongly interacting with DMPC/DHPC bicelle and DPC micelle, energetically driven by transforming the highly disordered unfolded state into a non-native helical structure, similar to what has been previously observed on ALS-causing SOD1 mutants. Our results imply that one potential mechanism for C71G-PFN1 to initiate ALS might be the abnormal interaction with membranes as recently established for SOD1 mutants. Copyright © 2017 Elsevier B.V. All rights reserved.

The morphogenesis of herpes simplex virus type 1 in infected parental mouse L fibroblasts and mutant gro29 cells

DEFF Research Database (Denmark)

Jensen, Helle Lone; Norrild, Bodil

2003-01-01

Mutants of cell lines and viruses are important biological tools. The pathway of herpesvirus particle maturation and egress are contentious issues. The mutant gro29 line of mouse L cells is defective for egress of herpes simplex virus type 1 (HSV-1) virions, and a candidate for studies of virus...

The enzymatic activity of CEM15/Apobec-3G is essential for the regulation of the infectivity of HIV-1 virion but not a sole determinant of its antiviral activity.

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Shindo, Keisuke; Takaori-Kondo, Akifumi; Kobayashi, Masayuki; Abudu, Aierken; Fukunaga, Keiko; Uchiyama, Takashi

2003-11-07

Human immunodeficiency virus, type 1 (HIV-1) Vif protein plays an essential role in the regulation of the infectivity of HIV-1 virion. Vif functions to counteract an anti-HIV-1 cellular factor in non-permissive cells, CEM15/Apobec-3G, which shares a cytidine deaminase motif. CEM15/Apobec-3G deaminates dC to dU in the minus strand DNA of HIV-1, resulting in G to A hypermutation in the plus strand DNA. In this study, we have done the mutagenesis analysis on two cytidine deaminase motifs in CEM15/Apobec-3G and examined their antiviral functions as well as the DNA editing activity. Point mutations in the C-terminal active site such as E259Q and C291A almost completely abrogated the antiviral function, while those in the N-terminal active site such as E67Q and C100A retained this activity to a lesser extent as compared with that of the wild type. The DNA editing activities of E67Q and E259Q mutants were both retained but impaired to the same extent. This indicates that the enzymatic activity of this protein is essential but not a sole determinant of the antiviral activity. Furthermore, all the deletion mutants tested in this study lost the antiviral activity because of the loss of the activity for dimerization, suggesting that the entire protein structure is necessary for the antiviral function.

Cholesterol trafficking and raft-like membrane domain composition mediate scavenger receptor class B type 1-dependent lipid sensing in intestinal epithelial cells.

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Morel, Etienne; Ghezzal, Sara; Lucchi, Géraldine; Truntzer, Caroline; Pais de Barros, Jean-Paul; Simon-Plas, Françoise; Demignot, Sylvie; Mineo, Chieko; Shaul, Philip W; Leturque, Armelle; Rousset, Monique; Carrière, Véronique

2018-02-01

Scavenger receptor Class B type 1 (SR-B1) is a lipid transporter and sensor. In intestinal epithelial cells, SR-B1-dependent lipid sensing is associated with SR-B1 recruitment in raft-like/ detergent-resistant membrane domains and interaction of its C-terminal transmembrane domain with plasma membrane cholesterol. To clarify the initiating events occurring during lipid sensing by SR-B1, we analyzed cholesterol trafficking and raft-like domain composition in intestinal epithelial cells expressing wild-type SR-B1 or the mutated form SR-B1-Q445A, defective in membrane cholesterol binding and signal initiation. These features of SR-B1 were found to influence both apical cholesterol efflux and intracellular cholesterol trafficking from plasma membrane to lipid droplets, and the lipid composition of raft-like domains. Lipidomic analysis revealed likely participation of d18:0/16:0 sphingomyelin and 16:0/0:0 lysophosphatidylethanolamine in lipid sensing by SR-B1. Proteomic analysis identified proteins, whose abundance changed in raft-like domains during lipid sensing, and these included molecules linked to lipid raft dynamics and signal transduction. These findings provide new insights into the role of SR-B1 in cellular cholesterol homeostasis and suggest molecular links between SR-B1-dependent lipid sensing and cell cholesterol and lipid droplet dynamics. Copyright © 2017 Elsevier B.V. All rights reserved.

Cholesterol-dependent energy transfer between fluorescent proteins-insights into protein proximity of APP and BACE1 in different membranes in Niemann-Pick type C disease cells.

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von Einem, Bjoern; Weber, Petra; Wagner, Michael; Malnar, Martina; Kosicek, Marko; Hecimovic, Silva; Arnim, Christine A F von; Schneckenburger, Herbert

2012-11-26

Förster resonance energy transfer (FRET) -based techniques have recently been applied to study the interactions between β-site APP-cleaving enzyme-GFP (BACE1-GFP) and amyloid precursor protein-mRFP (APP-mRFP) in U373 glioblastoma cells. In this context, the role of APP-BACE1 proximity in Alzheimer's disease (AD) pathogenesis has been discussed. FRET was found to depend on intracellular cholesterol levels and associated alterations in membrane stiffness. Here, NPC1 null cells (CHO-NPC1-/-), exhibiting increased cholesterol levels and disturbed cholesterol transport similar to that observed in Niemann-Pick type C disease (NPC), were used to analyze the influence of altered cholesterol levels on APP-BACE1 proximity. Fluorescence lifetime measurements of whole CHO-wild type (WT) and CHO-NPC1-/- cells (EPI-illumination microscopy), as well as their plasma membranes (total internal reflection fluorescence microscopy, TIRFM), were performed. Additionally, generalized polarization (GP) measurements of CHO-WT and CHO-NPC1-/- cells incubated with the fluorescence marker laurdan were performed to determine membrane stiffness of plasma- and intracellular-membranes. CHO-NPC1-/- cells showed higher membrane stiffness at intracellular- but not plasma-membranes, equivalent to cholesterol accumulation in late endosomes/lysosomes. Along with higher membrane stiffness, the FRET efficiency between BACE1-GFP and APP-mRFP was reduced at intracellular membranes, but not within the plasma membrane of CHO-NPC1-/-. Our data show that FRET combined with TIRF is a powerful technique to determine protein proximity and membrane fluidity in cellular models of neurodegenerative diseases.

Capsid protein VP4 of human rhinovirus induces membrane permeability by the formation of a size-selective multimeric pore.

Directory of Open Access Journals (Sweden)

Anusha Panjwani

2014-08-01

Full Text Available Non-enveloped viruses must deliver their viral genome across a cell membrane without the advantage of membrane fusion. The mechanisms used to achieve this remain poorly understood. Human rhinovirus, a frequent cause of the common cold, is a non-enveloped virus of the picornavirus family, which includes other significant pathogens such as poliovirus and foot-and-mouth disease virus. During picornavirus cell entry, the small myristoylated capsid protein VP4 is released from the virus, interacts with the cell membrane and is implicated in the delivery of the viral RNA genome into the cytoplasm to initiate replication. In this study, we have produced recombinant C-terminal histidine-tagged human rhinovirus VP4 and shown it can induce membrane permeability in liposome model membranes. Dextran size-exclusion studies, chemical crosslinking and electron microscopy demonstrated that VP4 forms a multimeric membrane pore, with a channel size consistent with transfer of the single-stranded RNA genome. The membrane permeability induced by recombinant VP4 was influenced by pH and was comparable to permeability induced by infectious virions. These findings present a molecular mechanism for the involvement of VP4 in cell entry and provide a model system which will facilitate exploration of VP4 as a novel antiviral target for the picornavirus family.

Feline immunodeficiency virus envelope glycoproteins antagonize tetherin through a distinctive mechanism that requires virion incorporation.

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Morrison, James H; Guevara, Rebekah B; Marcano, Adriana C; Saenz, Dyana T; Fadel, Hind J; Rogstad, Daniel K; Poeschla, Eric M

2014-03-01

BST2/tetherin inhibits the release of enveloped viruses from cells. Primate lentiviruses have evolved specific antagonists (Vpu, Nef, and Env). Here we characterized tetherin proteins of species representing both branches of the order Carnivora. Comparison of tiger and cat (Feliformia) to dog and ferret (Caniformia) genes demonstrated that the tiger and cat share a start codon mutation that truncated most of the tetherin cytoplasmic tail early in the Feliformia lineage (19 of 27 amino acids, including the dual tyrosine motif). Alpha interferon (IFN-α) induced tetherin and blocked feline immunodeficiency virus (FIV) replication in lymphoid and nonlymphoid feline cells. Budding of bald FIV and HIV particles was blocked by carnivore tetherins. However, infectious FIV particles were resistant, and spreading FIV replication was uninhibited. Antagonism mapped to the envelope glycoprotein (Env), which rescued FIV from carnivore tetherin restriction when expressed in trans but, in contrast to known antagonists, did not rescue noncognate particles. Also unlike the primate lentiviral antagonists, but similar to the Ebola virus glycoprotein, FIV Env did not reduce intracellular or cell surface tetherin levels. Furthermore, FIV-enveloped FIV particles actually required tetherin for optimal release from cells. The results show that FIV Envs mediate a distinctive tetherin evasion. Well adapted to a phylogenetically ancient tetherin tail truncation in the Felidae, it requires functional virion incorporation of Env, and it shields the budding particle without downregulating plasma membrane tetherin. Moreover, FIV has evolved dependence on this protein: particles containing FIV Env need tetherin for optimal release from the cell, while Env(-) particles do not. HIV-1 antagonizes the restriction factor tetherin with the accessory protein Vpu, while HIV-2 and the filovirus Ebola use their envelope (Env) glycoproteins for this purpose. It turns out that the FIV tetherin antagonist is

Herpesvirus papio 2 encodes a virion host shutoff function.

Science.gov (United States)

Bigger, John E; Martin, David W

2002-12-05

Infection of baboons with herpesvirus papio 2 (HVP-2) produces a disease that is similar to human infection with herpes simplex viruses (HSV). Molecular characterization of HVP-2 has demonstrated that the virion contains a factor which rapidly shuts off host cell protein synthesis after infection. Reduction of host cell protein synthesis occurs in parallel with the degradation of mRNA species. A homolog of the HSV virion host shutoff (vhs) gene was identified by Southern and DNA sequence analysis. The sequence of the HVP-2 vhs gene homolog had greater than 70% identity with the vhs genes of HSV 1 and 2. Disruption of the HVP-2 vhs open reading frame diminished the ability of the virus to shut off protein synthesis and degrade cellular mRNA, indicating that this gene was responsible for the vhs activity. The HVP-2 model system provides the opportunity to study the biological role of vhs in the context of a natural primate host. Further development of this system will provide a platform for proof-of-concept studies that will test the efficacy of vaccines that utilize vhs-deficient viruses.

Non-insulin drugs to treat hyperglycaemia in type 1 diabetes mellitus

DEFF Research Database (Denmark)

Frandsen, Christian Seerup; Dejgaard, Thomas Fremming; Madsbad, Sten

2016-01-01

Insulin treatment of individuals with type 1 diabetes has shortcomings and many patients do not achieve glycaemic and metabolic targets. Consequently, the focus is on novel non-insulin therapeutic approaches that reduce hyperglycaemia and improve metabolic variables without increasing the risk...... with few participants; evidence for the efficacy of concomitant treatments is scarce and largely clinically insignificant. A subgroup of patients with type 1 diabetes for whom non-insulin antidiabetic drugs could significantly benefit glycaemic control cannot yet be defined, but we suggest that obese...... of hypoglycaemia or other adverse events. Several therapies given in conjunction with insulin have been investigated in clinical trials, including pramlintide, glucagon-like peptide-1 receptor agonists, dipeptidyl peptidase-4 inhibitors, sodium-glucose co-transporter inhibitors, metformin, sulfonylureas...

Structure of Hepatitis E Virion-Sized Particle Reveals an RNA-Dependent Viral Assembly Pathway

Energy Technology Data Exchange (ETDEWEB)

Xing, L.; Wall, J.; Li, T.-C.; Mayazaki, N.; Simon, M. N.; Moore, M.; Wang, C.-Y.; Takeda, N.; Wakita, T.; Miyamura, T.; Cheng, R. H.

2010-10-22

Hepatitis E virus (HEV) induces acute hepatitis in humans with a high fatality rate in pregnant women. There is a need for anti-HEV research to understand the assembly process of HEV native capsid. Here, we produced a large virion-sized and a small T=1 capsid by expressing the HEV capsid protein in insect cells with and without the N-terminal 111 residues, respectively, for comparative structural analysis. The virion-sized capsid demonstrates a T=3 icosahedral lattice and contains RNA fragment in contrast to the RNA-free T=1 capsid. However, both capsids shared common decameric organization. The in vitro assembly further demonstrated that HEV capsid protein had the intrinsic ability to form decameric intermediate. Our data suggest that RNA binding is the extrinsic factor essential for the assembly of HEV native capsids.

Human Papillomavirus (HPV) virion induced cancer and subfertility, two sides of the same coin.

Science.gov (United States)

Depuydt, C E; Beert, J; Bosmans, E; Salembier, G

2016-12-01

In the natural history of HPV infections, the HPV virions can induce two different pathways, namely the infec- tious virion producing pathway and the clonal transforming pathway. An overview is given of the burden that is associated with HPV infections that can both lead to cervical cancer and/or temporal subfertility. That HPV infections cause serious global health burden due to HPV-associated cancers is common knowledge, but that it is also responsible for a substantial part of idiopathic subfertility is greatly underestimated. The bulk of the detected HPV DNA whether in men or women is however infectious from origin. Because the dissociation between HPV viruses and HPV virions or infection and disease remains difficult for clinicians as well as for HPV detection, we propose a review of the different effects caused by the two different HPV virion induced pathways, and highlight the mechanisms that are responsible for causing transient subfertility and cancer.

Structural requirements for the binding of tRNA Lys3 to reverse transcriptase of the human immunodeficiency virus type 1

NARCIS (Netherlands)

Oude Essink, B. B.; Das, A. T.; Berkhout, B.

1995-01-01

Reverse transcription of the human immunodeficiency virus type 1 (HIV-1) RNA genome is primed by the cellular tRNA Lys3 molecule. Packaging of this tRNA primer during virion assembly is thought to be mediated by specific interactions with the reverse transcriptase (RT) protein. Portions of the tRNA

Genetic reconstitution of the human Adenovirus type 2 temperature-sensitive 1 mutant defective in endosomal escape

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Gastaldelli Michele

2009-10-01

Full Text Available Abstract Human Adenoviruses infect the upper and lower respiratory tracts, the urinary and digestive tracts, lymphoid systems and heart, and give rise to epidemic conjunctivitis. More than 51 human serotypes have been identified to-date, and classified into 6 species A-F. The species C Adenoviruses Ad2 and Ad5 (Ad2/5 cause upper and lower respiratory disease, but how viral structure relates to the selection of particular infectious uptake pathways is not known. An adenovirus mutant, Ad2-ts1 had been isolated upon chemical mutagenesis in the past, and shown to have unprocessed capsid proteins. Ad2-ts1 fails to package the viral protease L3/p23, and Ad2-ts1 virions do not efficiently escape from endosomes. It had been suggested that the C22187T point mutation leading to the substitution of the conserved proline 137 to leucine (P137L in the L3/p23 protease was at least in part responsible for this phenotype. To clarify if the C22187T mutation is necessary and sufficient for the Ad2-ts1 phenotype, we sequenced the genes encoding the structural proteins of Ad2-ts1, and confirmed that the Ad2-ts1 DNA carries the point mutation C22187T. Introduction of C22187T to the wild-type Ad2 genome in a bacterial artificial chromosome (Ad2-BAC gave Ad2-BAC46 virions with the full Ad2-ts1 phenotype. Reversion of Ad2-BAC46 gave wild-type Ad2 particles indicating that P137L is necessary and sufficient for the Ad2-ts1 phenotype. The kinetics of Ad2-ts1 uptake into cells were comparable to Ad2 suggesting similar endocytic uptake mechanisms. Surprisingly, infectious Ad2 or Ad5 but not Ad2-ts1 uptake required CALM (clathrin assembly lymphoid myeloid protein, which controls clathrin-mediated endocytosis and membrane transport between endosomes and the trans-Golgi-network. The data show that no other mutations than P137L in the viral protease are necessary to give rise to particles that are defective in capsid processing and endosomal escape. This provides a basis for

Effect of non-solvents used in the coagulation bath on morphology of PVDF membranes

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Mônica Beatriz Thürmer

2012-12-01

Full Text Available The aim of this paper was to prepare a poly (vinylidene fluoride (PVDF membrane using different non-solvents in the coagulation bath for the phase inversion method. In order to increase the mechanical strength of membranes, facing the pressure of work, was used a macro-porous polyester support. The morphology and structure of the resulting membranes were evaluated by scanning electron microscopy, porosity measurements, water and 1-octanol uptake, contact angle, pure water flux, hydraulic permeability and hydraulic resistance. The morphology and pure water flux changed significantly using ethanol (symmetric membrane and/or water (asymmetric membrane as the non-solvent. The symmetric membrane presented a high hydrophobic surface (water contact angle ~136º and a higher pure water flux and porosity than the asymmetric membrane, which presented a lower hydrophobicity surface (water contact angle ~90º. The morphologies obtained suggest different applications.

Purification of infectious human herpesvirus 6A virions and association of host cell proteins

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Garoff Henrik

2007-10-01

Full Text Available Abstract Background Viruses that are incorporating host cell proteins might trigger autoimmune diseases. It is therefore of interest to identify possible host proteins associated with viruses, especially for enveloped viruses that have been suggested to play a role in autoimmune diseases, like human herpesvirus 6A (HHV-6A in multiple sclerosis (MS. Results We have established a method for rapid and morphology preserving purification of HHV-6A virions, which in combination with parallel analyses with background control material released from mock-infected cells facilitates qualitative and quantitative investigations of the protein content of HHV-6A virions. In our iodixanol gradient purified preparation, we detected high levels of viral DNA by real-time PCR and viral proteins by metabolic labelling, silver staining and western blots. In contrast, the background level of cellular contamination was low in the purified samples as demonstrated by the silver staining and metabolic labelling analyses. Western blot analyses showed that the cellular complement protein CD46, the receptor for HHV-6A, is associated with the purified and infectious virions. Also, the cellular proteins clathrin, ezrin and Tsg101 are associated with intact HHV-6A virions. Conclusion Cellular proteins are associated with HHV-6A virions. The relevance of the association in disease and especially in autoimmunity will be further investigated.

Membrane Targeting of P-type ATPases in Plant Cells

International Nuclear Information System (INIS)

Harper, Jeffrey F.

2004-01-01

How membrane proteins are targeted to specific subcellular locations is a very complex and poorly understood area of research. Our long-term goal is to use P-type ATPases (ion pumps), in a model plant system Arabidopsis, as a paradigm to understand how members of a family of closely related membrane proteins can be targeted to different subcellular locations. The research is divided into two specific aims. The first aim is focused on determining the targeting destination of all 10 ACA-type calcium pumps (Arabidopsis Calcium ATPase) in Arabidopsis. ACAs represent a plant specific-subfamily of plasma membrane-type calcium pumps. In contrast to animals, the plant homologs have been found in multiple membrane systems, including the ER (ACA2), tonoplast (ACA4) and plasma membrane (ACA8). Their high degree of similarity provides a unique opportunity to use a comparative approach to delineate the membrane specific targeting information for each pump. One hypothesis to be tested is that an endomembrane located ACA can be re-directed to the plasma membrane by including targeting information from a plasma membrane isoform, ACA8. Our approach is to engineer domain swaps between pumps and monitor the targeting of chimeric proteins in plant cells using a Green Fluorescence Protein (GFP) as a tag. The second aim is to test the hypothesis that heterologous transporters can be engineered into plants and targeted to the plasma membrane by fusing them to a plasma membrane proton pump. As a test case we are evaluating the targeting properties of fusions made between a yeast sodium/proton exchanger (Sod2) and a proton pump (AHA2). This fusion may potentially lead to a new strategy for engineering salt resistant plants. Together these aims are designed to provide fundamental insights into the biogenesis and function of plant cell membrane systems

Fluorescent sensor systems based on nanostructured polymeric membranes for selective recognition of Aflatoxin B1.

Science.gov (United States)

Sergeyeva, Tetyana; Yarynka, Daria; Piletska, Elena; Lynnik, Rostyslav; Zaporozhets, Olga; Brovko, Oleksandr; Piletsky, Sergey; El'skaya, Anna

2017-12-01

Nanostructured polymeric membranes for selective recognition of aflatoxin B1 were synthesized in situ and used as highly sensitive recognition elements in the developed fluorescent sensor. Artificial binding sites capable of selective recognition of aflatoxin B1 were formed in the structure of the polymeric membranes using the method of molecular imprinting. A composition of molecularly imprinted polymer (MIP) membranes was optimized using the method of computational modeling. The MIP membranes were synthesized using the non-toxic close structural analogue of aflatoxin B1, ethyl-2-oxocyclopentanecarboxylate as a dummy template. The MIP membranes with the optimized composition demonstrated extremely high selectivity towards aflatoxin B1 (AFB1). Negligible binding of close structural analogues of AFB1 - aflatoxins B2 (AFB2), aflatoxin G2 (AFG2), and ochratoxin A (OTA) was demonstrated. Binding of AFB1 by the MIP membranes was investigated as a function of both type and concentration of the functional monomer in the initial monomer composition used for the membranes' synthesis, as well as sample composition. The conditions of the solid-phase extraction of the mycotoxin using the MIP membrane as a stationary phase (pH, ionic strength, buffer concentration, volume of the solution, ratio between water and organic solvent, filtration rate) were optimized. The fluorescent sensor system based on the optimized MIP membranes provided a possibility of AFB1 detection within the range 14-500ngmL -1 demonstrating detection limit (3Ϭ) of 14ngmL -1 . The developed technique was successfully applied for the analysis of model solutions and waste waters from bread-making plants. Copyright © 2017 Elsevier B.V. All rights reserved.

Vaccinia protein F12 has structural similarity to kinesin light chain and contains a motor binding motif required for virion export.

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Gareth W Morgan

2010-02-01

Full Text Available Vaccinia virus (VACV uses microtubules for export of virions to the cell surface and this process requires the viral protein F12. Here we show that F12 has structural similarity to kinesin light chain (KLC, a subunit of the kinesin-1 motor that binds cargo. F12 and KLC share similar size, pI, hydropathy and cargo-binding tetratricopeptide repeats (TPRs. Moreover, molecular modeling of F12 TPRs upon the crystal structure of KLC2 TPRs showed a striking conservation of structure. We also identified multiple TPRs in VACV proteins E2 and A36. Data presented demonstrate that F12 is critical for recruitment of kinesin-1 to virions and that a conserved tryptophan and aspartic acid (WD motif, which is conserved in the kinesin-1-binding sequence (KBS of the neuronal protein calsyntenin/alcadein and several other cellular kinesin-1 binding proteins, is essential for kinesin-1 recruitment and virion transport. In contrast, mutation of WD motifs in protein A36 revealed they were not required for kinesin-1 recruitment or IEV transport. This report of a viral KLC-like protein containing a KBS that is conserved in several cellular proteins advances our understanding of how VACV recruits the kinesin motor to virions, and exemplifies how viruses use molecular mimicry of cellular components to their advantage.

Ebola virion attachment and entry into human macrophages profoundly effects early cellular gene expression.

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Victoria Wahl-Jensen

2011-10-01

Full Text Available Zaire ebolavirus (ZEBOV infections are associated with high lethality in primates. ZEBOV primarily targets mononuclear phagocytes, which are activated upon infection and secrete mediators believed to trigger initial stages of pathogenesis. The characterization of the responses of target cells to ZEBOV infection may therefore not only further understanding of pathogenesis but also suggest possible points of therapeutic intervention. Gene expression profiles of primary human macrophages exposed to ZEBOV were determined using DNA microarrays and quantitative PCR to gain insight into the cellular response immediately after cell entry. Significant changes in mRNA concentrations encoding for 88 cellular proteins were observed. Most of these proteins have not yet been implicated in ZEBOV infection. Some, however, are inflammatory mediators known to be elevated during the acute phase of disease in the blood of ZEBOV-infected humans. Interestingly, the cellular response occurred within the first hour of Ebola virion exposure, i.e. prior to virus gene expression. This observation supports the hypothesis that virion binding or entry mediated by the spike glycoprotein (GP(1,2 is the primary stimulus for an initial response. Indeed, ZEBOV virions, LPS, and virus-like particles consisting of only the ZEBOV matrix protein VP40 and GP(1,2 (VLP(VP40-GP triggered comparable responses in macrophages, including pro-inflammatory and pro-apoptotic signals. In contrast, VLP(VP40 (particles lacking GP(1,2 caused an aberrant response. This suggests that GP(1,2 binding to macrophages plays an important role in the immediate cellular response.

LIM kinase1 modulates function of membrane type matrix metalloproteinase 1: implication in invasion of prostate cancer cells

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Chakrabarti Ratna

2011-01-01

Full Text Available Abstract Background LIM kinase 1 (LIMK1 is an actin and microtubule cytoskeleton modulatory protein that is overexpressed in a number of cancerous tissues and cells and also promotes invasion and metastasis of prostate and breast cancer cells. Membrane type matrix metalloproteinase 1 (MT1-MMP is a critical modulator of extracellular matrix (ECM turnover through pericellular proteolysis and thus plays crucial roles in neoplastic cell invasion and metastasis. MT1-MMP and its substrates pro-MMP-2 and pro-MMP-9 are often overexpressed in a variety of cancers including prostate cancer and the expression levels correlate with the grade of malignancy in prostate cancer cells. The purpose of this study is to determine any functional relation between LIMK1 and MT1-MMP and its implication in cell invasion. Results Our results showed that treatment with the hydroxamate inhibitor of MT1-MMP, MMP-2 and MMP-9 ilomastat inhibited LIMK1-induced invasion of benign prostate epithelial cells. Over expression of LIMK1 resulted in increased collagenolytic activity of MMP-2, and secretion of pro-MMP2 and pro-MMP-9. Cells over expressing LIMK1 also exhibited increased expression of MT1-MMP, transcriptional activation and its localization to the plasma membrane. LIMK1 physically associates with MT1-MMP and is colocalized with it to the Golgi vesicles. We also noted increased expression of both MT1-MMP and LIMK1 in prostate tumor tissues. Conclusion Our results provide new information on regulation of MT1-MMP function by LIMK1 and showed for the first time, involvement of MMPs in LIMK1 induced cell invasion.

Understanding the Process of Envelope Glycoprotein Incorporation into Virions in Simian and Feline Immunodeficiency Viruses

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José L. Affranchino

2014-01-01

Full Text Available The lentiviral envelope glycoproteins (Env mediate virus entry by interacting with specific receptors present at the cell surface, thereby determining viral tropism and pathogenesis. Therefore, Env incorporation into the virions formed by assembly of the viral Gag polyprotein at the plasma membrane of the infected cells is a key step in the replication cycle of lentiviruses. Besides being useful models of human immunodeficiency virus (HIV infections in humans and valuable tools for developing AIDS therapies and vaccines, simian and feline immunodeficiency viruses (SIV and FIV, respectively are relevant animal retroviruses; the study of which provides important information on how lentiviral replication strategies have evolved. In this review, we discuss the molecular mechanisms underlying the incorporation of the SIV and FIV Env glycoproteins into viral particles.

Membrane versus shell type elements in F.E. analysis of box type buildings

International Nuclear Information System (INIS)

Canetta, G.

1979-01-01

Finite element analysis of box-type buildings is discussed under typical loading conditions - gravity, seismic and temperature loads. The computation effort is recognized to be noticeably different, according to whether membrane or shell type elements are used. The relevance of membrane and bending stress components to the total stress distribution is outlined in the table below; the different role of the typical members under the various loading conditions is emphasized. (orig.)

ROCK1-directed basement membrane positioning coordinates epithelial tissue polarity.

Science.gov (United States)

Daley, William P; Gervais, Elise M; Centanni, Samuel W; Gulfo, Kathryn M; Nelson, Deirdre A; Larsen, Melinda

2012-01-01

The basement membrane is crucial for epithelial tissue organization and function. However, the mechanisms by which basement membrane is restricted to the basal periphery of epithelial tissues and the basement membrane-mediated signals that regulate coordinated tissue organization are not well defined. Here, we report that Rho kinase (ROCK) controls coordinated tissue organization by restricting basement membrane to the epithelial basal periphery in developing mouse submandibular salivary glands, and that ROCK inhibition results in accumulation of ectopic basement membrane throughout the epithelial compartment. ROCK-regulated restriction of PAR-1b (MARK2) localization in the outer basal epithelial cell layer is required for basement membrane positioning at the tissue periphery. PAR-1b is specifically required for basement membrane deposition, as inhibition of PAR-1b kinase activity prevents basement membrane deposition and disrupts overall tissue organization, and suppression of PAR-1b together with ROCK inhibition prevents interior accumulations of basement membrane. Conversely, ectopic overexpression of wild-type PAR-1b results in ectopic interior basement membrane deposition. Significantly, culture of salivary epithelial cells on exogenous basement membrane rescues epithelial organization in the presence of ROCK1 or PAR-1b inhibition, and this basement membrane-mediated rescue requires functional integrin β1 to maintain epithelial cell-cell adhesions. Taken together, these studies indicate that ROCK1/PAR-1b-dependent regulation of basement membrane placement is required for the coordination of tissue polarity and the elaboration of tissue structure in the developing submandibular salivary gland.

[Computer modeling the dependences of the membrane potential for polymeric membrane separated non-homogeneous electrolyte solutions on concentration Rayleigh number].

Science.gov (United States)

Slezak, Izabella H; Jasik-Slezak, Jolanta; Bilewicz-Wyrozumska, Teresa; Slezak, Andrzej

2006-01-01

On the basis of model equation describing the membrane potential delta psi(s) on concentration Rayleigh number (R(C)), mechanical pressure difference (deltaP), concentration polarization coefficient (zeta s) and ratio concentration of solutions separated by membrane (Ch/Cl), the characteristics delta psi(s) = f(Rc)(delta P, zeta s, Ch/Cl) for steady values of zeta s, R(C) and Ch/Cl in single-membrane system were calculated. In this system neutral and isotropic polymeric membrane oriented in horizontal plane, the non-homogeneous binary electrolytic solutions of various concentrations were separated. Nonhomogeneity of solutions is results from creations of the concentration boundary layers on both sides of the membrane. Calculations were made for the case where on a one side of the membrane aqueous solution of NaCl at steady concentration 10(-3) mol x l(-1) (Cl) was placed and on the other aqueous solutions of NaCl at concentrations from 10(-3) mol x l(-1) to 2 x 10(-2) mol x l(-1) (Ch). Their densities were greater than NaCl solution's at 10(-3) mol x l(-1). It was shown that membrane potential depends on hydrodynamic state of a complex concentration boundary layer-membrane-concentration boundary layer, what is controlled by deltaP, Ch/Cl, Rc and Zeta(s).

Gp120 stability on HIV-1 virions and Gag-Env pseudovirions is enhanced by an uncleaved Gag core

International Nuclear Information System (INIS)

Hammonds, Jason; Chen Xuemin; Ding Lingmei; Fouts, Timothy; De Vico, Anthony; Megede, Jan zur; Barnett, Susan; Spearman, Paul

2003-01-01

Human immunodeficiency virus type-1 (HIV-1) particles incorporate a trimeric envelope complex (Env) made of gp120 (SU) and gp41 (TM) heterodimers. It has been previously established that soluble CD4 (sCD4) interaction leads to shedding of gp120 from viral particles, and that gp120 may also be easily lost from virions during incubation or particle purification procedures. In the design of HIV particle or pseudovirion-based HIV vaccines, it may be important to develop strategies to maximize the gp120 content of particles. We analyzed the gp120 retention of HIV-1 laboratory-adapted isolates and primary isolates following incubation with sCD4 and variations in temperature. NL4-3 shed gp120 readily in a temperature- and sCD4-dependent manner. Surprisingly, inactivation of the viral protease led to markedly reduced shedding of gp120. Gp120 shedding was shown to vary markedly between HIV-1 strains, and was not strictly determined by whether the isolate was adapted to growth on immortalized T cell lines or was a primary isolate. Pseudovirions produced by expression of codon-optimized gag and env genes also demonstrated enhanced gp120 retention when an immature core structure was maintained. Pseudovirions of optimal stability were produced through a combination of an immature Gag protein core and a primary isolate Env. These results support the feasibility of utilizing pseudovirion particles as immunogens for the induction of humoral responses directed against native envelope structures

Phase separation of the plasma membrane in human red blood cells as a potential tool for diagnosis and progression monitoring of type 1 diabetes mellitus.

Science.gov (United States)

Maulucci, Giuseppe; Cordelli, Ermanno; Rizzi, Alessandro; De Leva, Francesca; Papi, Massimiliano; Ciasca, Gabriele; Samengo, Daniela; Pani, Giovambattista; Pitocco, Dario; Soda, Paolo; Ghirlanda, Giovanni; Iannello, Giulio; De Spirito, Marco

2017-01-01

Glycosylation, oxidation and other post-translational modifications of membrane and transmembrane proteins can alter lipid density, packing and interactions, and are considered an important factor that affects fluidity variation in membranes. Red blood cells (RBC) membrane physical state, showing pronounced alterations in Type 1 diabetes mellitus (T1DM), could be the ideal candidate for monitoring the disease progression and the effects of therapies. On these grounds, the measurement of RBC membrane fluidity alterations can furnish a more sensitive index in T1DM diagnosis and disease progression than Glycosylated hemoglobin (HbA1c), which reflects only the information related to glycosylation processes. Here, through a functional two-photon microscopy approach we retrieved fluidity maps at submicrometric scale in RBC of T1DM patients with and without complications, detecting an altered membrane equilibrium. We found that a phase separation between fluid and rigid domains occurs, triggered by systemic effects on membranes fluidity of glycation and oxidation. The phase separation patterns are different among healthy, T1DM and T1DM with complications patients. Blood cholesterol and LDL content are positively correlated with the extent of the phase separation patterns. To quantify this extent a machine learning approach is employed to develop a Decision-Support-System (DSS) able to recognize different fluidity patterns in RBC. Preliminary analysis shows significant differences(pBlood cells is a potential tool for diagnosis and progression monitoring of type 1 diabetes mellitus, and could allow customization and the selection of medical treatments in T1DM in clinical settings, and enable the early detection of complications.

Phase separation of the plasma membrane in human red blood cells as a potential tool for diagnosis and progression monitoring of type 1 diabetes mellitus.

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Giuseppe Maulucci

Full Text Available Glycosylation, oxidation and other post-translational modifications of membrane and transmembrane proteins can alter lipid density, packing and interactions, and are considered an important factor that affects fluidity variation in membranes. Red blood cells (RBC membrane physical state, showing pronounced alterations in Type 1 diabetes mellitus (T1DM, could be the ideal candidate for monitoring the disease progression and the effects of therapies. On these grounds, the measurement of RBC membrane fluidity alterations can furnish a more sensitive index in T1DM diagnosis and disease progression than Glycosylated hemoglobin (HbA1c, which reflects only the information related to glycosylation processes. Here, through a functional two-photon microscopy approach we retrieved fluidity maps at submicrometric scale in RBC of T1DM patients with and without complications, detecting an altered membrane equilibrium. We found that a phase separation between fluid and rigid domains occurs, triggered by systemic effects on membranes fluidity of glycation and oxidation. The phase separation patterns are different among healthy, T1DM and T1DM with complications patients. Blood cholesterol and LDL content are positively correlated with the extent of the phase separation patterns. To quantify this extent a machine learning approach is employed to develop a Decision-Support-System (DSS able to recognize different fluidity patterns in RBC. Preliminary analysis shows significant differences(p<0.001 among healthy, T1DM and T1DM with complications patients. The development of an assay based on Phase separation of the plasma membrane of the Red Blood cells is a potential tool for diagnosis and progression monitoring of type 1 diabetes mellitus, and could allow customization and the selection of medical treatments in T1DM in clinical settings, and enable the early detection of complications.

Proposal for a histopathological consensus classification of the periprosthetic interface membrane.

Science.gov (United States)

Morawietz, L; Classen, R-A; Schröder, J H; Dynybil, C; Perka, C; Skwara, A; Neidel, J; Gehrke, T; Frommelt, L; Hansen, T; Otto, M; Barden, B; Aigner, T; Stiehl, P; Schubert, T; Meyer-Scholten, C; König, A; Ströbel, P; Rader, C P; Kirschner, S; Lintner, F; Rüther, W; Bos, I; Hendrich, C; Kriegsmann, J; Krenn, V

2006-06-01

The introduction of clearly defined histopathological criteria for a standardised evaluation of the periprosthetic membrane, which can appear in cases of total joint arthroplasty revision surgery. Based on histomorphological criteria, four types of periprosthetic membrane were defined: wear particle induced type (detection of foreign body particles; macrophages and multinucleated giant cells occupy at least 20% of the area; type I); infectious type (granulation tissue with neutrophilic granulocytes, plasma cells and few, if any, wear particles; type II); combined type (aspects of type I and type II occur simultaneously; type III); and indeterminate type (neither criteria for type I nor type II are fulfilled; type IV). The periprosthetic membranes of 370 patients (217 women, 153 men; mean age 67.6 years, mean period until revision surgery 7.4 years) were analysed according to the defined criteria. Frequency of histopathological membrane types was: type I 54.3%, type II 19.7%, type III 5.4%, type IV 15.4%, and not assessable 5.1%. The mean period between primary arthroplasty and revision surgery was 10.1 years for type I, 3.2 years for type II, 4.5 years for type III and 5.4 years for type IV. The correlation between histopathological and microbiological diagnosis was high (89.7%), and the inter-observer reproducibility sufficient (85%). The classification proposed enables standardised typing of periprosthetic membranes and may serve as a tool for further research on the pathogenesis of the loosening of total joint replacement. The study highlights the importance of non-infectious, non-particle induced loosening of prosthetic devices in orthopaedic surgery (membrane type IV), which was observed in 15.4% of patients.

Deconstructing the DGAT1 enzyme: membrane interactions at substrate binding sites.

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Jose L S Lopes

Full Text Available Diacylglycerol acyltransferase 1 (DGAT1 is a key enzyme in the triacylglyceride synthesis pathway. Bovine DGAT1 is an endoplasmic reticulum membrane-bound protein associated with the regulation of fat content in milk and meat. The aim of this study was to evaluate the interaction of DGAT1 peptides corresponding to putative substrate binding sites with different types of model membranes. Whilst these peptides are predicted to be located in an extramembranous loop of the membrane-bound protein, their hydrophobic substrates are membrane-bound molecules. In this study, peptides corresponding to the binding sites of the two substrates involved in the reaction were examined in the presence of model membranes in order to probe potential interactions between them that might influence the subsequent binding of the substrates. Whilst the conformation of one of the peptides changed upon binding several types of micelles regardless of their surface charge, suggesting binding to hydrophobic domains, the other peptide bound strongly to negatively-charged model membranes. This binding was accompanied by a change in conformation, and produced leakage of the liposome-entrapped dye calcein. The different hydrophobic and electrostatic interactions observed suggest the peptides may be involved in the interactions of the enzyme with membrane surfaces, facilitating access of the catalytic histidine to the triacylglycerol substrates.

Compartmentalized cAMP Signaling Associated With Lipid Raft and Non-raft Membrane Domains in Adult Ventricular Myocytes.

Science.gov (United States)

Agarwal, Shailesh R; Gratwohl, Jackson; Cozad, Mia; Yang, Pei-Chi; Clancy, Colleen E; Harvey, Robert D

2018-01-01

Aim: Confining cAMP production to discrete subcellular locations makes it possible for this ubiquitous second messenger to elicit unique functional responses. Yet, factors that determine how and where the production of this diffusible signaling molecule occurs are incompletely understood. The fluid mosaic model originally proposed that signal transduction occurs through random interactions between proteins diffusing freely throughout the plasma membrane. However, it is now known that the movement of membrane proteins is restricted, suggesting that the plasma membrane is segregated into distinct microdomains where different signaling proteins can be concentrated. In this study, we examined what role lipid raft and non-raft membrane domains play in compartmentation of cAMP signaling in adult ventricular myocytes. Methods and Results: The freely diffusible fluorescence resonance energy transfer-based biosensor Epac2-camps was used to measure global cytosolic cAMP responses, while versions of the probe targeted to lipid raft (Epac2-MyrPalm) and non-raft (Epac2-CAAX) domains were used to monitor local cAMP production near the plasma membrane. We found that β-adrenergic receptors, which are expressed in lipid raft and non-raft domains, produce cAMP responses near the plasma membrane that are distinctly different from those produced by E-type prostaglandin receptors, which are expressed exclusively in non-raft domains. We also found that there are differences in basal cAMP levels associated with lipid raft and non-raft domains, and that this can be explained by differences in basal adenylyl cyclase activity associated with each of these membrane environments. In addition, we found evidence that phosphodiesterases 2, 3, and 4 work together in regulating cAMP activity associated with both lipid raft and non-raft domains, while phosphodiesterase 3 plays a more prominent role in the bulk cytoplasmic compartment. Conclusion: These results suggest that different membrane

Members of the HCMV US12 family of predicted heptaspanning membrane proteins have unique intracellular distributions, including association with the cytoplasmic virion assembly complex

International Nuclear Information System (INIS)

Das, Subhendu; Pellett, Philip E.

2007-01-01

The human cytomegalovirus (HCMV) US12 gene family is a group of 10 predicted seven-transmembrane domain proteins that have some features in common with G-protein-coupled receptors. Little is known of their patterns of expression, localization, or functional interactions. Here, we studied the intracellular localization of three US12 family members, US14, US17, and US18, with respect to various intracellular markers and the cytoplasmic virion assembly compartment (AC). The three proteins have distinct patterns of expression, which include associations with the AC. US14 is often distributed in a uniform granular manner throughout the cytoplasm, concentrating in the AC in some cells. US17 is expressed in a segmented manner, with its N-terminal domain localizing to the periphery of what we show here to be the AC and the C-terminal domain localizing to nuclei and the cytoplasm [Das, S., Skomorovska-Prokvolit, Y., Wang, F. Z., Pellett, P.E., 2006. Infection-dependent nuclear localization of US17, a member of the US12 family of human cytomegalovirus-encoded seven-transmembrane proteins. J. Virol. 80, 1191-1203]. Here, we show that the C-terminal domain is present at the center of the AC, in close association with markers of early endosomes; the N-terminal staining corresponds to an area stained by markers for the Golgi and trans-Golgi. US18 is distributed throughout the cytoplasm, concentrating in the AC at later stages of infection; it is localized more to the periphery of the AC than are US14 and US17C, in association with markers of the trans-Golgi. Although not detected in virions, their structures and localization in various zones within the AC suggest possible roles for these proteins in the process of virion maturation and egress

Completely assembled virus particles detected by transmission electron microscopy in proximal and mid-axons of neurons infected with herpes simplex virus type 1, herpes simplex virus type 2 and pseudorabies virus

International Nuclear Information System (INIS)

Huang Jialing; Lazear, Helen M.; Friedman, Harvey M.

2011-01-01

The morphology of alphaherpesviruses during anterograde axonal transport from the neuron cell body towards the axon terminus is controversial. Reports suggest that transport of herpes simplex virus type 1 (HSV-1) nucleocapsids and envelope proteins occurs in separate compartments and that complete virions form at varicosities or axon termini (subassembly transport model), while transport of a related alphaherpesvirus, pseudorabies virus (PRV) occurs as enveloped capsids in vesicles (assembled transport model). Transmission electron microscopy of proximal and mid-axons of primary superior cervical ganglion (SCG) neurons was used to compare anterograde axonal transport of HSV-1, HSV-2 and PRV. SCG cell bodies were infected with HSV-1 NS and 17, HSV-2 2.12 and PRV Becker. Fully assembled virus particles were detected intracellularly within vesicles in proximal and mid-axons adjacent to microtubules after infection with each virus, indicating that assembled virions are transported anterograde within axons for all three alphaherpesviruses.

Non-Brownian diffusion in lipid membranes: Experiments and simulations.

Science.gov (United States)

Metzler, R; Jeon, J-H; Cherstvy, A G

2016-10-01

The dynamics of constituents and the surface response of cellular membranes-also in connection to the binding of various particles and macromolecules to the membrane-are still a matter of controversy in the membrane biophysics community, particularly with respect to crowded membranes of living biological cells. We here put into perspective recent single particle tracking experiments in the plasma membranes of living cells and supercomputing studies of lipid bilayer model membranes with and without protein crowding. Special emphasis is put on the observation of anomalous, non-Brownian diffusion of both lipid molecules and proteins embedded in the lipid bilayer. While single component, pure lipid bilayers in simulations exhibit only transient anomalous diffusion of lipid molecules on nanosecond time scales, the persistence of anomalous diffusion becomes significantly longer ranged on the addition of disorder-through the addition of cholesterol or proteins-and on passing of the membrane lipids to the gel phase. Concurrently, experiments demonstrate the anomalous diffusion of membrane embedded proteins up to macroscopic time scales in the minute time range. Particular emphasis will be put on the physical character of the anomalous diffusion, in particular, the occurrence of ageing observed in the experiments-the effective diffusivity of the measured particles is a decreasing function of time. Moreover, we present results for the time dependent local scaling exponent of the mean squared displacement of the monitored particles. Recent results finding deviations from the commonly assumed Gaussian diffusion patterns in protein crowded membranes are reported. The properties of the displacement autocorrelation function of the lipid molecules are discussed in the light of their appropriate physical anomalous diffusion models, both for non-crowded and crowded membranes. In the last part of this review we address the upcoming field of membrane distortion by elongated membrane

Highly porous polytriazole ion exchange membranes cast from solutions in non-toxic cosolvents

KAUST Repository

Chisca, Stefan

2017-04-04

The development of highly functionalized porous materials for protein separation is important for biotech processes. We report the preparation of highly porous polytriazole with sulfonic acid functionalization. The resulting ion exchange membranes are selective for protein adsorption. The starting material was a hydroxyl-functionalized polytriazole, which is an advantageous platform for further modification. The polymer was dissolved in a mixture of 1-ethyl-3-methylimidazolium acetate ([C2mim]OAc) and dimethyl carbonate (DMC), which can be both considered green solvents. The polymer solubilization was only possible due to an interesting effect of cosolvency, which is discussed, based in phase diagrams. Membranes were prepared by solution casting, followed by immersion in a non-solvent bath. We then grafted sulfone groups on the membranes, by reacting the hydroxyl groups with 1,3-propane sultone and 1,4-butane sultone. Lysozyme adsorption was successfully evaluated. Membranes modified with 1,4-butane sultone adsorbed more protein than those with 1,3-propane sultone.

Viral RNA annealing activities of human immunodeficiency virus type 1 nucleocapsid protein require only peptide domains outside the zinc fingers.

Science.gov (United States)

De Rocquigny, H; Gabus, C; Vincent, A; Fournié-Zaluski, M C; Roques, B; Darlix, J L

1992-07-15

The nucleocapsid (NC) of human immunodeficiency virus type 1 consists of a large number of NC protein molecules, probably wrapping the dimeric RNA genome within the virion inner core. NC protein is a gag-encoded product that contains two zinc fingers flanked by basic residues. In human immunodeficiency virus type 1 virions, NCp15 is ultimately processed into NCp7 and p6 proteins. During virion assembly the retroviral NC protein is necessary for core formation and genomic RNA encapsidation, which are essential for virus infectivity. In vitro NCp15 activates viral RNA dimerization, a process most probably linked in vivo to genomic RNA packaging, and replication primer tRNA(Lys,3) annealing to the initiation site of reverse transcription. To characterize the domains of human immunodeficiency virus type 1 NC protein necessary for its various functions, the 72-amino acid NCp7 and several derived peptides were synthesized in a pure form. We show here that synthetic NCp7 with or without the two zinc fingers has the RNA annealing activities of NCp15. Further deletions of the N-terminal 12 and C-terminal 8 amino acids, leading to a 27-residue peptide lacking the finger domains, have little or no effect on NC protein activity in vitro. However deletion of short sequences containing basic residues flanking the first finger leads to a complete loss of NC protein activity. It is proposed that the basic residues and the zinc fingers cooperate to select and package the genomic RNA in vivo. Inhibition of the viral RNA binding and annealing activities associated with the basic residues flanking the first zinc finger of NC protein could therefore be used as a model for the design of antiviral agents.

Inspirations on Virus Replication and Cell-to-Cell Movement from Studies Examining the Cytopathology Induced by Lettuce infectious yellows virus in Plant Cells

Directory of Open Access Journals (Sweden)

Wenjie Qiao

2017-09-01

Full Text Available Lettuce infectious yellows virus (LIYV is the type member of the genus Crinivirus in the family Closteroviridae. Like many other positive-strand RNA viruses, LIYV infections induce a number of cytopathic changes in plant cells, of which the two most characteristic are: Beet yellows virus-type inclusion bodies composed of vesicles derived from cytoplasmic membranes; and conical plasmalemma deposits (PLDs located at the plasmalemma over plasmodesmata pit fields. The former are not only found in various closterovirus infections, but similar structures are known as ‘viral factories’ or viroplasms in cells infected with diverse types of animal and plant viruses. These are generally sites of virus replication, virion assembly and in some cases are involved in cell-to-cell transport. By contrast, PLDs induced by the LIYV-encoded P26 non-virion protein are not involved in replication but are speculated to have roles in virus intercellular movement. These deposits often harbor LIYV virions arranged to be perpendicular to the plasma membrane over plasmodesmata, and our recent studies show that P26 is required for LIYV systemic plant infection. The functional mechanism of how LIYV P26 facilitates intercellular movement remains unclear, however, research on other plant viruses provides some insights on the possible ways of viral intercellular movement through targeting and modifying plasmodesmata via interactions between plant cellular components and viral-encoded factors. In summary, beginning with LIYV, we review the studies that have uncovered the biological determinants giving rise to these cytopathological effects and their importance in viral replication, virion assembly and intercellular movement during the plant infection by closteroviruses, and compare these findings with those for other positive-strand RNA viruses.

Mutations in type 3 reovirus that determine binding to sialic acid are contained in the fibrous tail domain of viral attachment protein sigma1.

Science.gov (United States)

Chappell, J D; Gunn, V L; Wetzel, J D; Baer, G S; Dermody, T S

1997-03-01

The reovirus attachment protein, sigma1, determines numerous aspects of reovirus-induced disease, including viral virulence, pathways of spread, and tropism for certain types of cells in the central nervous system. The sigma1 protein projects from the virion surface and consists of two distinct morphologic domains, a virion-distal globular domain known as the head and an elongated fibrous domain, termed the tail, which is anchored into the virion capsid. To better understand structure-function relationships of sigma1 protein, we conducted experiments to identify sequences in sigma1 important for viral binding to sialic acid, a component of the receptor for type 3 reovirus. Three serotype 3 reovirus strains incapable of binding sialylated receptors were adapted to growth in murine erythroleukemia (MEL) cells, in which sialic acid is essential for reovirus infectivity. MEL-adapted (MA) mutant viruses isolated by serial passage in MEL cells acquired the capacity to bind sialic acid-containing receptors and demonstrated a dependence on sialic acid for infection of MEL cells. Analysis of reassortant viruses isolated from crosses of an MA mutant virus and a reovirus strain that does not bind sialic acid indicated that the sigma1 protein is solely responsible for efficient growth of MA mutant viruses in MEL cells. The deduced sigma1 amino acid sequences of the MA mutant viruses revealed that each strain contains a substitution within a short region of sequence in the sigma1 tail predicted to form beta-sheet. These studies identify specific sequences that determine the capacity of reovirus to bind sialylated receptors and suggest a location for a sialic acid-binding domain. Furthermore, the results support a model in which type 3 sigma1 protein contains discrete receptor binding domains, one in the head and another in the tail that binds sialic acid.

Automatic design of deterministic and non-halting membrane systems by tuning syntactical ingredients.

Science.gov (United States)

Zhang, Gexiang; Rong, Haina; Ou, Zhu; Pérez-Jiménez, Mario J; Gheorghe, Marian

2014-09-01

To solve the programmability issue of membrane computing models, the automatic design of membrane systems is a newly initiated and promising research direction. In this paper, we propose an automatic design method, Permutation Penalty Genetic Algorithm (PPGA), for a deterministic and non-halting membrane system by tuning membrane structures, initial objects and evolution rules. The main ideas of PPGA are the introduction of the permutation encoding technique for a membrane system, a penalty function evaluation approach for a candidate membrane system and a genetic algorithm for evolving a population of membrane systems toward a successful one fulfilling a given computational task. Experimental results show that PPGA can successfully accomplish the automatic design of a cell-like membrane system for computing the square of n ( n ≥ 1 is a natural number) and can find the minimal membrane systems with respect to their membrane structures, alphabet, initial objects, and evolution rules for fulfilling the given task. We also provide the guidelines on how to set the parameters of PPGA.

Non-porous membrane-assisted liquid-liquid extraction of UV filter compounds from water samples.

Science.gov (United States)

Rodil, Rosario; Schrader, Steffi; Moeder, Monika

2009-06-12

A method for the determination of nine UV filter compounds [benzophenone-3 (BP-3), isoamyl methoxycinnamate, 4-methylbenzylidene camphor, octocrylene (OC), butyl methoxydibenzoylmethane, ethylhexyl dimethyl p-aminobenzoate (OD-PABA), ethylhexyl methoxycinnamate (EHMC), ethylhexyl salicylate and homosalate] in water samples was developed and evaluated. The procedure includes non-porous membrane-assisted liquid-liquid extraction (MALLE) and LC-atmospheric pressure photoionization (APPI)-MS/MS. Membrane bags made of different polymeric materials were examined to enable a fast and simple extraction of the target analytes. Among the polymeric materials tested, low- and high-density polyethylene membranes proved to be well suited to adsorb the analytes from water samples. Finally, 2 cm length tailor-made membrane bags were prepared from low-density polyethylene in order to accommodate 100 microL of propanol. The fully optimised protocol provides recoveries from 76% to 101% and limits of detection (LOD) between 0.4 ng L(-1) (OD-PABA) and 16 ng L(-1) (EHMC). The interday repeatability of the whole protocol was below 18%. The effective separation of matrix molecules was proved by only marginal matrix influence during the APPI-MS analysis since no ion suppression effects were observed. During the extraction step, the influence of the matrix was only significant when non-treated wastewater was analysed. The analysis of lake water indicated the presence of seven UV filter compounds included in this study at concentrations between 40 ng L(-1) (BP-3) and 4381 ng L(-1) (OC). In non-treated wastewater several UV filters were also detected at concentration levels as high as 5322 ng L(-1) (OC).

Image Restoration and Analysis of Influenza Virions Binding to Membrane Receptors Reveal Adhesion-Strengthening Kinetics.

Directory of Open Access Journals (Sweden)

Donald W Lee

Full Text Available With the development of single-particle tracking (SPT microscopy and host membrane mimics called supported lipid bilayers (SLBs, stochastic virus-membrane binding interactions can be studied in depth while maintaining control over host receptor type and concentration. However, several experimental design challenges and quantitative image analysis limitations prevent the widespread use of this approach. One main challenge of SPT studies is the low signal-to-noise ratio of SPT videos, which is sometimes inevitable due to small particle sizes, low quantum yield of fluorescent dyes, and photobleaching. These situations could render current particle tracking software to yield biased binding kinetic data caused by intermittent tracking error. Hence, we developed an effective image restoration algorithm for SPT applications called STAWASP that reveals particles with a signal-to-noise ratio of 2.2 while preserving particle features. We tested our improvements to the SPT binding assay experiment and imaging procedures by monitoring X31 influenza virus binding to α2,3 sialic acid glycolipids. Our interests lie in how slight changes to the peripheral oligosaccharide structures can affect the binding rate and residence times of viruses. We were able to detect viruses binding weakly to a glycolipid called GM3, which was undetected via assays such as surface plasmon resonance. The binding rate was around 28 folds higher when the virus bound to a different glycolipid called GD1a, which has a sialic acid group extending further away from the bilayer surface than GM3. The improved imaging allowed us to obtain binding residence time distributions that reflect an adhesion-strengthening mechanism via multivalent bonds. We empirically fitted these distributions using a time-dependent unbinding rate parameter, koff, which diverges from standard treatment of koff as a constant. We further explain how to convert these models to fit ensemble-averaged binding data

Transmission-Blocking Antibodies against Mosquito C-Type Lectins for Dengue Prevention

Science.gov (United States)

Liu, Yang; Zhang, Fuchun; Liu, Jianying; Xiao, Xiaoping; Zhang, Siyin; Qin, Chengfeng; Xiang, Ye; Wang, Penghua; Cheng, Gong

2014-01-01

C-type lectins are a family of proteins with carbohydrate-binding activity. Several C-type lectins in mammals or arthropods are employed as receptors or attachment factors to facilitate flavivirus invasion. We previously identified a C-type lectin in Aedes aegypti, designated as mosquito galactose specific C-type lectin-1 (mosGCTL-1), facilitating the attachment of West Nile virus (WNV) on the cell membrane. Here, we first identified that 9 A. aegypti mosGCTL genes were key susceptibility factors facilitating DENV-2 infection, of which mosGCTL-3 exhibited the most significant effect. We found that mosGCTL-3 was induced in mosquito tissues with DENV-2 infection, and that the protein interacted with DENV-2 surface envelop (E) protein and virions in vitro and in vivo. In addition, the other identified mosGCTLs interacted with the DENV-2 E protein, indicating that DENV may employ multiple mosGCTLs as ligands to promote the infection of vectors. The vectorial susceptibility factors that facilitate pathogen invasion may potentially be explored as a target to disrupt the acquisition of microbes from the vertebrate host. Indeed, membrane blood feeding of antisera against mosGCTLs dramatically reduced mosquito infective ratio. Hence, the immunization against mosGCTLs is a feasible approach for preventing dengue infection. Our study provides a future avenue for developing a transmission-blocking vaccine that interrupts the life cycle of dengue virus and reduces disease burden. PMID:24550728

Inhibition of Early Stages of HIV-1 Assembly by INI1/hSNF5 Transdominant Negative Mutant S6 ▿

Science.gov (United States)

Cano, Jennifer; Kalpana, Ganjam V.

2011-01-01

INI1/hSNF5 is an HIV-1 integrase (IN) binding protein specifically incorporated into virions. A truncated mutant of INI1 (S6, amino acids 183 to 294) harboring the minimal IN binding Rpt1 domain potently inhibits HIV-1 particle production in a transdominant manner. The inhibition requires interaction of S6 with IN within Gag-Pol. While INI1 is a nuclear protein and harbors a masked nuclear export signal (NES), the transdominant negative mutant S6 is cytoplasmic, due to the unmasking of NES. Here, we examined the effects of subcellular localization of S6 on HIV-1 inhibition and further investigated the stages of assembly that are affected. We found that targeting a nuclear localization signal-containing S6 variant [NLS-S6(Rpt1)] to the nucleoplasm (but not to the nucleolus) resulted in complete reversal of inhibition of particle production. Electron microscopy indicated that although no electron-dense particles at any stage of assembly were seen in cells expressing S6, virions were produced in cells expressing the rescue mutant NLS-S6(Rpt1) to wild-type levels. Immunofluorescence analysis revealed that p24 exhibited a diffuse pattern of localization within the cytoplasm in cells expressing S6 in contrast to accumulation along the membrane in controls. Pulse-chase analysis indicated that in S6-expressing cells, although Gag(Pr55gag) protein translation was unaffected, processing and release of p24 were defective. Together, these results indicate that expression of S6 in the cytoplasm interferes with trafficking of Gag-Pol/Gag to the membrane and causes a defective processing leading to inhibition of assembly at an early stage prior to particle formation and budding. PMID:21159874

A palmitoylation switch mechanism regulates Rac1 function and membrane organization

Science.gov (United States)

Navarro-Lérida, Inmaculada; Sánchez-Perales, Sara; Calvo, María; Rentero, Carles; Zheng, Yi; Enrich, Carlos; Del Pozo, Miguel A

2012-01-01

The small GTPase Rac1 plays important roles in many processes, including cytoskeletal reorganization, cell migration, cell-cycle progression and gene expression. The initiation of Rac1 signalling requires at least two mechanisms: GTP loading via the guanosine triphosphate (GTP)/guanosine diphosphate (GDP) cycle, and targeting to cholesterol-rich liquid-ordered plasma membrane microdomains. Little is known about the molecular mechanisms governing this specific compartmentalization. We show that Rac1 can incorporate palmitate at cysteine 178 and that this post-translational modification targets Rac1 for stabilization at actin cytoskeleton-linked ordered membrane regions. Palmitoylation of Rac1 requires its prior prenylation and the intact C-terminal polybasic region and is regulated by the triproline-rich motif. Non-palmitoylated Rac1 shows decreased GTP loading and lower association with detergent-resistant (liquid-ordered) membranes (DRMs). Cells expressing no Rac1 or a palmitoylation-deficient mutant have an increased content of disordered membrane domains, and markers of ordered membranes isolated from Rac1-deficient cells do not correctly partition in DRMs. Importantly, cells lacking Rac1 palmitoylation show spreading and migration defects. These data identify palmitoylation as a mechanism for Rac1 function in actin cytoskeleton remodelling by controlling its membrane partitioning, which in turn regulates membrane organization. PMID:22157745

Filament-producing mutants of influenza A/Puerto Rico/8/1934 (H1N1 virus have higher neuraminidase activities than the spherical wild-type.

Directory of Open Access Journals (Sweden)

Jill Seladi-Schulman

Full Text Available Influenza virus exhibits two morphologies - spherical and filamentous. Strains that have been grown extensively in laboratory substrates are comprised predominantly of spherical virions while clinical or low passage isolates produce a mixture of spheres and filamentous virions of varying lengths. The filamentous morphology can be lost upon continued passage in embryonated chicken eggs, a common laboratory substrate for influenza viruses. The fact that the filamentous morphology is maintained in nature but lost in favor of a spherical morphology in ovo suggests that filaments confer a selective advantage within the infected host that is not necessary for growth in laboratory substrates. Indeed, we have recently shown that filament-producing variant viruses are selected upon passage of the spherical laboratory strain A/Puerto Rico/8/1934 (H1N1 [PR8] in guinea pigs. Toward determining the nature of the selective advantage conferred by filaments, we sought to identify functional differences between spherical and filamentous particles. We compared the wild-type PR8 virus to two previously characterized recombinant PR8 viruses in which single point mutations within M1 confer a filamentous morphology. Our results indicate that these filamentous PR8 mutants have higher neuraminidase activities than the spherical PR8 virus. Conversely, no differences were observed in HAU:PFU or HAU:RNA ratios, binding avidity, sensitivity to immune serum in hemagglutination inhibition assays, or virion stability at elevated temperatures. Based on these results, we propose that the pleomorphic nature of influenza virus particles is important for the optimization of neuraminidase functions in vivo.

Cross-linked PAN-based thin-film composite membranes for non-aqueous nanofiltration

KAUST Repository

Pérez-Manríquez, Liliana

2015-01-01

A new approach on the development of cross-linked PAN based thin film composite (TFC) membranes for non-aqueous application is presented in this work. Polypropylene backed neat PAN membranes fabricated by phase inversion process were cross-linked with hydrazine to get excellent solvent stability toward dimethylformamide (DMF). By interfacial polymerization a selective polyamide active layer was coated over the cross-linked PAN using N,N′-diamino piperazine (DAP) and trimesoyl chloride (TMC) as monomers. Permeation and molecular weight cut off (MWCO) experiments using various dyes were done to evaluate the performance of the membranes. Membranes developed by such method show excellent solvent stability toward DMF with a permeance of 1.7 L/m2 h bar and a molecular weight cut-off of less than 600 Da.

A Method for Coupling a Direct Current Power Source Across a Dielectric Membrane or Other Non-Conducting Membrane

National Research Council Canada - National Science Library

Steinbrecher, Donald H

2008-01-01

.... A second set of plates on the second side of the membrane form a set of coupling capacitors wherein the non-conducting dielectric membrane becomes part of the coupling-capacitor dielectric material...

A virus of hyperthermophilic archaea with a unique architecture among DNA viruses.

Science.gov (United States)

Rensen, Elena Ilka; Mochizuki, Tomohiro; Quemin, Emmanuelle; Schouten, Stefan; Krupovic, Mart; Prangishvili, David

2016-03-01

Viruses package their genetic material in diverse ways. Most known strategies include encapsulation of nucleic acids into spherical or filamentous virions with icosahedral or helical symmetry, respectively. Filamentous viruses with dsDNA genomes are currently associated exclusively with Archaea. Here, we describe a filamentous hyperthermophilic archaeal virus, Pyrobaculum filamentous virus 1 (PFV1), with a type of virion organization not previously observed in DNA viruses. The PFV1 virion, 400 ± 20 × 32 ± 3 nm, contains an envelope and an inner core consisting of two structural units: a rod-shaped helical nucleocapsid formed of two 14-kDa major virion proteins and a nucleocapsid-encompassing protein sheath composed of a single major virion protein of 18 kDa. The virion organization of PFV1 is superficially similar to that of negative-sense RNA viruses of the family Filoviridae, including Ebola virus and Marburg virus. The linear dsDNA of PFV1 carries 17,714 bp, including 60-bp-long terminal inverted repeats, and contains 39 predicted ORFs, most of which do not show similarities to sequences in public databases. PFV1 is a lytic virus that completely disrupts the host cell membrane at the end of the infection cycle.

Discriminating lysosomal membrane protein types using dynamic neural network.

Science.gov (United States)

Tripathi, Vijay; Gupta, Dwijendra Kumar

2014-01-01

This work presents a dynamic artificial neural network methodology, which classifies the proteins into their classes from their sequences alone: the lysosomal membrane protein classes and the various other membranes protein classes. In this paper, neural networks-based lysosomal-associated membrane protein type prediction system is proposed. Different protein sequence representations are fused to extract the features of a protein sequence, which includes seven feature sets; amino acid (AA) composition, sequence length, hydrophobic group, electronic group, sum of hydrophobicity, R-group, and dipeptide composition. To reduce the dimensionality of the large feature vector, we applied the principal component analysis. The probabilistic neural network, generalized regression neural network, and Elman regression neural network (RNN) are used as classifiers and compared with layer recurrent network (LRN), a dynamic network. The dynamic networks have memory, i.e. its output depends not only on the input but the previous outputs also. Thus, the accuracy of LRN classifier among all other artificial neural networks comes out to be the highest. The overall accuracy of jackknife cross-validation is 93.2% for the data-set. These predicted results suggest that the method can be effectively applied to discriminate lysosomal associated membrane proteins from other membrane proteins (Type-I, Outer membrane proteins, GPI-Anchored) and Globular proteins, and it also indicates that the protein sequence representation can better reflect the core feature of membrane proteins than the classical AA composition.

The herpes simplex virus 2 virion-associated ribonuclease vhs interferes with stress granule formation.

Science.gov (United States)

Finnen, Renée L; Hay, Thomas J M; Dauber, Bianca; Smiley, James R; Banfield, Bruce W

2014-11-01

In a previous study, it was observed that cells infected with herpes simplex virus 2 (HSV-2) failed to accumulate stress granules (SGs) in response to oxidative stress induced by arsenite treatment. As a follow-up to this observation, we demonstrate here that disruption of arsenite-induced SG formation by HSV-2 is mediated by a virion component. Through studies on SG formation in cells infected with HSV-2 strains carrying defective forms of UL41, the gene that encodes vhs, we identify vhs as a virion component required for this disruption. Cells infected with HSV-2 strains producing defective forms of vhs form SGs spontaneously late in infection. In addition to core SG components, these spontaneous SGs contain the viral immediate early protein ICP27 as well as the viral serine/threonine kinase Us3. As part of these studies, we reexamined the frameshift mutation known to reside within the UL41 gene of HSV-2 strain HG52. We demonstrate that this mutation is unstable and can rapidly revert to restore wild-type UL41 following low-multiplicity passaging. Identification of the involvement of virion-associated vhs in the disruption of SG formation will enable mechanistic studies on how HSV-2 is able to counteract antiviral stress responses early in infection. In addition, the ability of Us3 to localize to stress granules may indicate novel roles for this viral kinase in the regulation of translation. Eukaryotic cells respond to stress by rapidly shutting down protein synthesis and storing mRNAs in cytoplasmic stress granules (SGs). Stoppages in protein synthesis are problematic for all viruses as they rely on host cell machinery to synthesize viral proteins. Thus, many viruses target SGs for disruption or modification. Infection by herpes simplex virus 2 (HSV-2) was previously observed to disrupt SG formation induced by oxidative stress. In this follow-up study, we identify virion host shutoff protein (vhs) as a viral protein involved in this disruption. The

Membrane Estrogen Receptor-α Interacts with Metabotropic Glutamate Receptor Type 1a to Mobilize Intracellular Calcium in Hypothalamic Astrocytes

Science.gov (United States)

Kuo, John; Hariri, Omid R.; Bondar, Galyna; Ogi, Julie; Micevych, Paul

2009-01-01

Estradiol, acting on a membrane-associated estrogen receptor-α (mERα), induces an increase in free cytoplasmic calcium concentration ([Ca2+]i) needed for progesterone synthesis in hypothalamic astrocytes. To determine whether rapid estradiol signaling involves an interaction of mERα with metabotropic glutamate receptor type 1a (mGluR1a), changes in [Ca2+]i were monitored with the calcium indicator, Fluo-4 AM, in primary cultures of female postpubertal hypothalamic astrocytes. 17β-Estradiol over a range of 1 nm to 100 nm induced a maximal increase in [Ca2+]i flux measured as a change in relative fluorescence [ΔF Ca2+ = 615 ± 36 to 641 ± 47 relative fluorescent units (RFU)], whereas 0.1 nm of estradiol stimulated a moderate [Ca2+]i increase (275 ± 16 RFU). The rapid estradiol-induced [Ca2+]i flux was blocked with 1 μm of the estrogen receptor antagonist ICI 182,780 (635 ± 24 vs. 102 ± 11 RFU, P estradiol-induced membrane signaling in astrocytes. PMID:18948402

Pressure balanced type membrane covered polarographic oxygen detectors for use in high temperature-high pressure water, (1)

International Nuclear Information System (INIS)

Nakayama, Norio; Uchida, Shunsuke

1984-01-01

A pressure balanced type membrane covered polarographic oxygen detector was developed to determine directly oxygen concentrations in high temperature, high pressure water without cooling and pressure reducing procedures. The detector is characterized by the following features: (1) The detector body and the membrane for oxygen penetration are made of heat resistant resin. (2) The whole detector body is contained in a pressure chamber where interior and exterior pressures of the detector are balanced. (3) Thermal expansion of the electrolyte is absorbed by deformation of a diaphragm attached to the detector bottom. (4) The effect of dissolved Ag + on the signal current is eliminated by applying a guard electrode. As a result of performance tests at elevated temperature, it was demonstrated that a linear relationship between oxygen concentration and signal current was obtained up to 285 0 C, which was stabilized by the guard electrode. The minimum O 2 concentration detectable was 0.03ppm (9.4 x 10 -7 mol/kg). (author)

A Novel Non-Planar Transverse Stretching Process for Micro-Porous PTFE Membranes and Resulting Characteristics

KAUST Repository

Chang, Y.-H.

2018-02-26

Polytetrafluoroethylene (PTFE) micro-porous membranes were prepared from PTFE fine powder through extruding, rolling, and uniaxial longitudinally stretching. In contrast to conventional planar transverse stretching, a novel 3D mold design of non-planar transverse stretching process was employed in this study to produce micro-porous structure. The morphology, membrane thickness, mean pore size, and porosity of the PTFE membrane were investigated. The results show that the non-planar transverse stretched membranes exhibit more uniform average pore diameter with thinner membrane thickness. Morphological changes induced by planar and non-planar transverse stretching for pore characteristics were investigated. The stretching conditions, stretching temperature and rate, affect the stretched membrane. Increasing temperature facilitated the uniformity of pore size and uniformity of membrane thickness. Moreover, increase in stretching rate resulted in finer pore size and thinner membrane.

Quantitative Proteome Analysis Reveals Increased Content of Basement Membrane Proteins in Arteries from Patients with Type 2 Diabetes and Lower Levels among Metformin Users

DEFF Research Database (Denmark)

Rørdam Preil, Simone; Kristensen, Lars P; Beck, Hans C

2015-01-01

hypothesized that metformin intake influences the protein composition. METHODS AND RESULTS: -We analyzed non-atherosclerotic repair arteries gathered at coronary by-pass operations from 30 patients with type 2 diabetes, as well as from 30 age- and gender-matched non-diabetic individuals. Quantitative proteome......BACKGROUND: -The increased risk of cardiovascular diseases (CVD) in type 2 diabetes has been extensively documented, but the origins of the association remain largely unknown. We sought to determine changes in protein expressions in arterial tissue from patients with type 2 diabetes and moreover...... analysis was done by iTRAQ-labelling and LC-MS/MS analysis on individual arterial samples. The amounts of the basement membrane (BM) components, alpha-1- and alpha-2- type IV collagen, gamma-1- and beta-2-laminin were significantly increased in patients with diabetes. Moreover, the expressions of basement...

Membrane re-modelling by BAR domain superfamily proteins via molecular and non-molecular factors.

Science.gov (United States)

Nishimura, Tamako; Morone, Nobuhiro; Suetsugu, Shiro

2018-04-17

Lipid membranes are structural components of cell surfaces and intracellular organelles. Alterations in lipid membrane shape are accompanied by numerous cellular functions, including endocytosis, intracellular transport, and cell migration. Proteins containing Bin-Amphiphysin-Rvs (BAR) domains (BAR proteins) are unique, because their structures correspond to the membrane curvature, that is, the shape of the lipid membrane. BAR proteins present at high concentration determine the shape of the membrane, because BAR domain oligomers function as scaffolds that mould the membrane. BAR proteins co-operate with various molecular and non-molecular factors. The molecular factors include cytoskeletal proteins such as the regulators of actin filaments and the membrane scission protein dynamin. Lipid composition, including saturated or unsaturated fatty acid tails of phospholipids, also affects the ability of BAR proteins to mould the membrane. Non-molecular factors include the external physical forces applied to the membrane, such as tension and friction. In this mini-review, we will discuss how the BAR proteins orchestrate membrane dynamics together with various molecular and non-molecular factors. © 2018 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.

Potency of whole virus particle and split virion vaccines using dissolving microneedle against challenges of H1N1 and H5N1 influenza viruses in mice.

Science.gov (United States)

Nakatsukasa, Akihiro; Kuruma, Koji; Okamatsu, Masatoshi; Hiono, Takahiro; Suzuki, Mizuho; Matsuno, Keita; Kida, Hiroshi; Oyamada, Takayoshi; Sakoda, Yoshihiro

2017-05-15

Transdermal vaccination using a microneedle (MN) confers enhanced immunity compared with subcutaneous (SC) vaccination. Here we developed a novel dissolving MN patch for the influenza vaccine. The potencies of split virion and whole virus particle (WVP) vaccines prepared from A/Puerto Rico/8/1934 (H1N1) and A/duck/Hokkaido/Vac-3/2007 (H5N1), respectively, were evaluated. MN vaccination induced higher neutralizing antibody responses than SC vaccination in mice. Moreover, MN vaccination with a lower dose of antigens conferred protective immunity against lethal challenges of influenza viruses than SC vaccination in mice. These results suggest that the WVP vaccines administered using MN are an effective combination for influenza vaccine to be further validated in humans. Copyright © 2017 Elsevier Ltd. All rights reserved.

Theoretical investigation of the sound attenuation of membrane-type acoustic metamaterials

International Nuclear Information System (INIS)

Zhang, Yuguang; Wen, Jihong; Xiao, Yong; Wen, Xisen; Wang, Jianwei

2012-01-01

Membrane-type acoustic metamaterials have been recently shown to exhibit good performance of sound attenuation in a low frequency range. An analytical approach for the fast calculation of sound transmission loss of the membrane-type acoustic metamaterials is presented here. The discussion indicate that the first transmission loss valley and the transmission loss peak depend strongly on the attaching mass, while the second transmission loss valley is mainly influenced by the membrane properties. The effects of membrane tension and mass position on the transmission loss and characteristic frequencies are also discussed in detail. -- Highlights: ► An analytical approach was presented here. ► First TL valley and peak depend strongly on attaching mass. ► Second TL valley is mainly influenced by properties of membrane. ► TL peak move to low frequency at a special position keeping TL valley motionless.

Numerical analysis on centrifugal compressor with membrane type dryer

Science.gov (United States)

Razali, M. A.; Zulkafli, M. F.; Mat Isa, N.; Subari, Z.

2017-09-01

Moisture content is a common phenomenon in industrial processes especially in oil and gas industries. This contaminant has a lot of disadvantages which can lead to mechanical failure DEC (Deposition, Erosion & Corrosion) problems. To overcome DEC problem, this study proposed to design a centrifugal compressor with a membrane type dryer to reduce moisture content of a gas. The effectiveness of such design has been analyzed in this study using Computational Fluid Dynamics (CFD) approach. Numerical scheme based on multiphase flow technique is used in ANSYS Fluent software to evaluate the moisture content of the gas. Through this technique, two kind of centrifugal compressor, with and without membrane type dryer has been tested. The results show that the effects of pressure on dew point temperature of the gas change the composition of its moisture content, where high value lead more condensation to occur. However, with the injection of cool dry gas through membrane type dryer in the centrifugal compressor, the pressure and temperature of moisture content as well as mass fraction of H2O in centrifugal compressor show significant reduction.

Preparation and Characterization of Polypropylene Non-woven Fabrics Prepared by Melt-blown Spinning for Filtration Membranes

Energy Technology Data Exchange (ETDEWEB)

Chu, Konghee; Park, Mira; Kim, Hakyong [Chonbuk National Univ., Jeonju (Korea, Republic of); Jin, Fanlong; Park, Soojin [Inha Univ., Incheon (Korea, Republic of)

2014-06-15

PP non-woven fabrics were prepared by melt-blown spinning, followed by heat and plasma treatments. After heat treatment, the PP non-woven fabrics displayed decreased water flux, increased tensile strength, decreased elongation, and an average pore size of 0.7 μm. The hydrophilicity of the PP non-woven fabrics was improved by plasma treatment. The water flux of the PP non-woven fabrics increased about two fold after the plasma treatment. The particle removal efficiency was determined to be 97.2-99.4% for 1-3 μm sized particles, demonstrating a high particle removal efficiency. Polypropylene (PP) non-woven fabrics have been widely used as filtration membranes in wastewater purification with industrial applications due to their low cost, good mechanical strength, and high thermal and chemical stability. The membrane fouling behavior depends strongly on the physical and mechanical properties of the membrane, including pore size, porosity, morphology, and hydrophilicity. In general, PP non-woven fabrics have poor hydrophilicity; this has limited their application in the biomedical field. It is therefore necessary to develop PP non-woven fabrics with improved surface hydrophilicity to increase the scope of their use. Plasma treatment, an environmentally friendly alternative to traditional chemical activation, only changes the uppermost atomic layers of a membrane surface without affecting the bulk properties of the polymer.

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

Science.gov (United States)

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

HIV-1 incorporates and proteolytically processes human NDR1 and NDR2 serine-threonine kinases

International Nuclear Information System (INIS)

Devroe, Eric; Silver, Pamela A.; Engelman, Alan

2005-01-01

Mammalian genomes encode two related serine-threonine kinases, nuclear Dbf2 related (NDR)1 and NDR2, which are homologous to the Saccharomyces cerevisiae Dbf2 kinase. Recently, a yeast genetic screen implicated the Dbf2 kinase in Ty1 retrotransposition. Since several virion-incorporated kinases regulate the infectivity of human immunodeficiency virus type 1 (HIV-1), we speculated that the human NDR1 and NDR2 kinases might play a role in the HIV-1 life cycle. Here we show that the NDR1 and NDR2 kinases were incorporated into HIV-1 particles. Furthermore, NDR1 and NDR2 were cleaved by the HIV-1 protease (PR), both within virions and within producer cells. Truncation at the PR cleavage site altered NDR2 subcellular localization and inhibited NDR1 and NDR2 enzymatic activity. These studies identify two new virion-associated host cell enzymes and suggest a novel mechanism by which HIV-1 alters the intracellular environment of human cells

Non-genomic actions of aldosterone: From receptors and signals to membrane targets.

LENUS (Irish Health Repository)

2012-02-01

In tissues which express the mineralocorticoid receptor (MR), aldosterone modulates the expression of membrane targets such as the subunits of the epithelial Na(+) channel, in combination with important signalling intermediates such as serum and glucocorticoid-regulated kinase-1. In addition, the rapid \\'non-genomic\\' activation of protein kinases and secondary messenger signalling cascades has also been detected in aldosterone-sensitive tissues of the nephron, distal colon and cardiovascular system. These rapid actions are variously described as being coupled to MR or to an as yet unidentified, membrane-associated aldosterone receptor. The rapidly activated signalling cascades add a level of fine-tuning to the activity of aldosterone-responsive membrane transporters and also modulate the aldosterone-induced changes in gene expression through receptor and transcription factor phosphorylation.

Non-genomic actions of aldosterone: From receptors and signals to membrane targets.

LENUS (Irish Health Repository)

Dooley, Ruth

2011-07-26

In tissues which express the mineralocorticoid receptor (MR), aldosterone modulates the expression of membrane targets such as the subunits of the epithelial Na(+) channel, in combination with important signalling intermediates such as serum and glucocorticoid-regulated kinase-1. In addition, the rapid \\'non-genomic\\' activation of protein kinases and secondary messenger signalling cascades has also been detected in aldosterone-sensitive tissues of the nephron, distal colon and cardiovascular system. These rapid actions are variously described as being coupled to MR or to an as yet unidentified, membrane-associated aldosterone receptor. The rapidly activated signalling cascades add a level of fine-tuning to the activity of aldosterone-responsive membrane transporters and also modulate the aldosterone-induced changes in gene expression through receptor and transcription factor phosphorylation.

Resonant modal group theory of membrane-type acoustical metamaterials for low-frequency sound attenuation

Science.gov (United States)

Ma, Fuyin; Wu, Jiu Hui; Huang, Meng

2015-09-01

In order to overcome the influence of the structural resonance on the continuous structures and obtain a lightweight thin-layer structure which can effectively isolate the low-frequency noises, an elastic membrane structure was proposed. In the low-frequency range below 500 Hz, the sound transmission loss (STL) of this membrane type structure is greatly higher than that of the current sound insulation material EVA (ethylene-vinyl acetate copo) of vehicle, so it is possible to replace the EVA by the membrane-type metamaterial structure in practice engineering. Based on the band structure, modal shapes, as well as the sound transmission simulation, the sound insulation mechanism of the designed membrane-type acoustic metamaterials was analyzed from a new perspective, which had been validated experimentally. It is suggested that in the frequency range above 200 Hz for this membrane-mass type structure, the sound insulation effect was principally not due to the low-level locally resonant mode of the mass block, but the continuous vertical resonant modes of the localized membrane. So based on such a physical property, a resonant modal group theory is initially proposed in this paper. In addition, the sound insulation mechanism of the membrane-type structure and thin plate structure were combined by the membrane/plate resonant theory.

Non-reciprocal elastic wave propagation in 2D phononic membranes with spatiotemporally varying material properties

Science.gov (United States)

Attarzadeh, M. A.; Nouh, M.

2018-05-01

One-dimensional phononic materials with material fields traveling simultaneously in space and time have been shown to break elastodynamic reciprocity resulting in unique wave propagation features. In the present work, a comprehensive mathematical analysis is presented to characterize and fully predict the non-reciprocal wave dispersion in two-dimensional space. The analytical dispersion relations, in the presence of the spatiotemporal material variations, are validated numerically using finite 2D membranes with a prescribed number of cells. Using omnidirectional excitations at the membrane's center, wave propagations are shown to exhibit directional asymmetry that increases drastically in the direction of the material travel and vanishes in the direction perpendicular to it. The topological nature of the predicted dispersion in different propagation directions are evaluated using the computed Chern numbers. Finally, the degree of the 2D non-reciprocity is quantified using a non-reciprocity index (NRI) which confirms the theoretical dispersion predictions as well as the finite simulations. The presented framework can be extended to plate-type structures as well as 3D spatiotemporally modulated phononic crystals.

MgO as a non-pyrolyzable pore former in porous membrane supports

DEFF Research Database (Denmark)

Haugen, A. B.; Geffroy, A.; Kaiser, Andreas

2018-01-01

the performance of oxygen transport membranes or other membranes relying on gas transport to the active membrane surface. Thermoplastic feedstocks for extrusion of tubular 3Y-TZP supports were prepared with four different amounts of pyrolyzable pore formers and/or MgO as non-pyrolyzable pore former. The Mg...

Virology: The Next Generation from Digital PCR to Single Virion Genomics

Energy Technology Data Exchange (ETDEWEB)

White, Richard A.; Brazelton De Cardenas, Jessica N.; Hayden, Randall T.

2015-10-01

In the past 25 years, virology has had major technology breakthroughs stemming first from the introduction of nucleic acid amplification testing, but more recently from the use of next-generation sequencing, digital PCR, and the possibility of single virion genomics. These technologies have and will improve diagnosis and disease state monitoring in clinical settings, aid in environmental monitoring, and reveal the vast genetic potential of viruses. Using the principle of limiting dilution, digital PCR amplifies single molecules of DNA in highly partitioned endpoint reactions and reads each of those reactions as either positive or negative based on the presence or absence of target fluorophore. In this review, digital PCR will be highlighted along with current studies, advantages/disadvantages, and future perspectives with regard to digital PCR, viral load testing, and the possibility of single virion genomics.

Action potentials and ion conductances in wild-type and CALHM1-knockout type II taste cells

Science.gov (United States)

Saung, Wint Thu; Foskett, J. Kevin

2017-01-01

Taste bud type II cells fire action potentials in response to tastants, triggering nonvesicular ATP release to gustatory neurons via voltage-gated CALHM1-associated ion channels. Whereas CALHM1 regulates mouse cortical neuron excitability, its roles in regulating type II cell excitability are unknown. In this study, we compared membrane conductances and action potentials in single identified TRPM5-GFP-expressing circumvallate papillae type II cells acutely isolated from wild-type (WT) and Calhm1 knockout (KO) mice. The activation kinetics of large voltage-gated outward currents were accelerated in cells from Calhm1 KO mice, and their associated nonselective tail currents, previously shown to be highly correlated with ATP release, were completely absent in Calhm1 KO cells, suggesting that CALHM1 contributes to all of these currents. Calhm1 deletion did not significantly alter resting membrane potential or input resistance, the amplitudes and kinetics of Na+ currents either estimated from action potentials or recorded from steady-state voltage pulses, or action potential threshold, overshoot peak, afterhyperpolarization, and firing frequency. However, Calhm1 deletion reduced the half-widths of action potentials and accelerated the deactivation kinetics of transient outward currents, suggesting that the CALHM1-associated conductance becomes activated during the repolarization phase of action potentials. NEW & NOTEWORTHY CALHM1 is an essential ion channel component of the ATP neurotransmitter release mechanism in type II taste bud cells. Its contribution to type II cell resting membrane properties and excitability is unknown. Nonselective voltage-gated currents, previously associated with ATP release, were absent in cells lacking CALHM1. Calhm1 deletion was without effects on resting membrane properties or voltage-gated Na+ and K+ channels but contributed modestly to the kinetics of action potentials. PMID:28202574

Molecular dynamics simulations of membrane deformation induced by amphiphilic helices of Epsin, Sar1p, and Arf1

Science.gov (United States)

Li, Zhen-Lu

2018-03-01

The N-terminal amphiphilic helices of proteins Epsin, Sar1p, and Arf1 play a critical role in initiating membrane deformation. The interactions of these amphiphilic helices with the lipid membranes are investigated in this study by combining the all-atom and coarse-grained simulations. In the all-atom simulations, the amphiphilic helices of Epsin and Sar1p are found to have a shallower insertion depth into the membrane than the amphiphilic helix of Arf1, but remarkably, the amphiphilic helices of Epsin and Sar1p induce higher asymmetry in the lipid packing between the two monolayers of the membrane. The insertion depth of amphiphilic helix into the membrane is determined not only by the overall hydrophobicity but also by the specific distributions of polar and non-polar residues along the helix. To directly compare their ability to deform the membrane, the coarse-grained simulations are performed to investigate the membrane deformation under the insertion of multiple helices. Project supported by the National Natural Science Foundation of China (Grant Nos. 91427302 and 11474155).

Bovine herpesvirus type-1 glycoprotein K (gK) interacts with UL20 and is required for infectious virus production

Energy Technology Data Exchange (ETDEWEB)

Haque, Muzammel; Stanfield, Brent; Kousoulas, Konstantin G.

2016-12-15

We have previously shown that the HSV-1 gK and UL20 proteins interact and function in virion envelopment, membrane fusion, and neuronal entry. Alignment of the predicted secondary structures of gKs encoded by BoHV-1, HSV-1, HSV-2, EHV-1 and VZV indicated a high degree of domain conservation. Two BoHV-1 gK-null mutant viruses were created by either gK gene deletion or stop codon insertion. In addition, a V5 epitope-tag was inserted at the carboxyl terminus of gK gene to detect gK. The engineered gK-null mutant viruses failed to replicate and produce viral plaques. Co-immunoprecipitation of gK and UL20 expressed via different methods revealed that gK and UL20 physically interacted in the presence or absence of other viral proteins. Confocal microscopy showed that gK and UL20 colocalized in infected cells. These results indicate that BoHV-1 gK and UL20 may function in a similar manner to other alphaherpesvirus orthologues specified by HSV-1, PRV and EHV-1. -- Highlights: •Glycoprotein K(gK) is conserved among alphaherpesviruses and serves similar functions. •The bovine herpesvirus-1 gK and UL20 proteins physically interact in a similar manner to herpes simplex virus type 1 and equine herpesvirus-1. •The bovine herpesvirus-1 (BoHV-1) gK interacts with UL20 and is essential for virus replication and spread.

Bovine herpesvirus type-1 glycoprotein K (gK) interacts with UL20 and is required for infectious virus production

International Nuclear Information System (INIS)

Haque, Muzammel; Stanfield, Brent; Kousoulas, Konstantin G.

2016-01-01

We have previously shown that the HSV-1 gK and UL20 proteins interact and function in virion envelopment, membrane fusion, and neuronal entry. Alignment of the predicted secondary structures of gKs encoded by BoHV-1, HSV-1, HSV-2, EHV-1 and VZV indicated a high degree of domain conservation. Two BoHV-1 gK-null mutant viruses were created by either gK gene deletion or stop codon insertion. In addition, a V5 epitope-tag was inserted at the carboxyl terminus of gK gene to detect gK. The engineered gK-null mutant viruses failed to replicate and produce viral plaques. Co-immunoprecipitation of gK and UL20 expressed via different methods revealed that gK and UL20 physically interacted in the presence or absence of other viral proteins. Confocal microscopy showed that gK and UL20 colocalized in infected cells. These results indicate that BoHV-1 gK and UL20 may function in a similar manner to other alphaherpesvirus orthologues specified by HSV-1, PRV and EHV-1. -- Highlights: •Glycoprotein K(gK) is conserved among alphaherpesviruses and serves similar functions. •The bovine herpesvirus-1 gK and UL20 proteins physically interact in a similar manner to herpes simplex virus type 1 and equine herpesvirus-1. •The bovine herpesvirus-1 (BoHV-1) gK interacts with UL20 and is essential for virus replication and spread.

Non-bilayer structures in mitochondrial membranes regulate ATP synthase activity.

Science.gov (United States)

Gasanov, Sardar E; Kim, Aleksandr A; Yaguzhinsky, Lev S; Dagda, Ruben K

2018-02-01

Cardiolipin (CL) is an anionic phospholipid at the inner mitochondrial membrane (IMM) that facilitates the formation of transient non-bilayer (non-lamellar) structures to maintain mitochondrial integrity. CL modulates mitochondrial functions including ATP synthesis. However, the biophysical mechanisms by which CL generates non-lamellar structures and the extent to which these structures contribute to ATP synthesis remain unknown. We hypothesized that CL and ATP synthase facilitate the formation of non-bilayer structures at the IMM to stimulate ATP synthesis. By using 1 H NMR and 31 P NMR techniques, we observed that increasing the temperature (8°C to 37°C), lowering the pH (3.0), or incubating intact mitochondria with CTII - an IMM-targeted toxin that increases the formation of immobilized non-bilayer structures - elevated the formation of non-bilayer structures to stimulate ATP synthesis. The F 0 sector of the ATP synthase complex can facilitate the formation of non-bilayer structures as incubating model membranes enriched with IMM-specific phospholipids with exogenous DCCD-binding protein of the F 0 sector (DCCD-BPF) elevated the formation of immobilized non-bilayer structures to a similar manner as CTII. Native PAGE assays revealed that CL, but not other anionic phospholipids, specifically binds to DCCD-BPF to promote the formation of stable lipid-protein complexes. Mechanistically, molecular docking studies identified two lipid binding sites for CL in DCCD-BPF. We propose a new model of ATP synthase regulation in which CL mediates the formation of non-bilayer structures that serve to cluster protons and ATP synthase complexes as a mechanism to enhance proton translocation to the F 0 sector, and thereby increase ATP synthesis. Copyright © 2017 Elsevier B.V. All rights reserved.

B cell lymphomas express CX3CR1 a non-B cell lineage adhesion molecule

DEFF Research Database (Denmark)

Andreasson, U.; Ek, S.; Merz, H.

2008-01-01

normally is not expressed on B cells, is expressed both at the mRNA and protein level in several subtypes of lymphoma. CX3CR1 has also shown to be involved in the homing to specific tissues that express the ligand, CX3CL1, in breast and prostate cancer and may thus be involved in dissemination of lymphoma......To study the differential expression of cell membrane-bound receptors and their potential role in growth and/or survival of the tumor cells, highly purified follicular lymphoma cells were analyzed, using gene expression analysis, and compared to non-malignant B cell populations. Filtering...... the genome for overexpressed genes coding for cell membrane-bound proteins/receptors resulted in a hit list of 27 identified genes. Among these, we have focused on the aberrant over expression of CX3CR1, in different types of B cell lymphoma, as compared to non-malignant B cells. We show that CX3CR1, which...

Non-abelian gauge bosons in the compactified bosonic membrane theory

International Nuclear Information System (INIS)

Kubo, J.

1988-01-01

We consider the bosonic membrane compactified on a torus. The membrane motion is stabilized by a topologically non-trivial background. We find that, in the narrow membrane limit, the mass formula to O(ℎ) reduces to exactly the same form as that of the compactified closed bosonic string theory, and we obtain (almost) massless vector bosons in the adjoint representation of a simply laced Lie group in D=27. This is only dimension at which the graviton and gauge bosons may coexist in that background. (orig.)

Different collagen types define two types of idiopathic epiretinal membranes

OpenAIRE

Kritzenberger , Michaela; Junglas , Benjamin; Framme , Carsten; Helbig , Horst; Gabel , Veit-Peter; Fuchshofer , Rudolf; Tamm , Ernst R; Hillenkamp , Jost

2011-01-01

Abstract Aims: To identify differences in extracellular matrix contents between idiopathic epiretinal membranes (IEM) of cellophane macular reflex (CMRM) or preretinal macular fibrosis (PMFM) type. Methods and results: IEM were analyzed by light and quantitative transmission electron microscopy, immunohistochemistry, and Western blotting. Substantial differences between CMRM and PMFM were observed regarding the nature of extracellular fibrils. In CMRM, the fibrils were thin with...

Recent developments on ion-exchange membranes and electro-membrane processes.

Science.gov (United States)

Nagarale, R K; Gohil, G S; Shahi, Vinod K

2006-02-28

Rapid growth of chemical and biotechnology in diversified areas fuels the demand for the need of reliable green technologies for the down stream processes, which include separation, purification and isolation of the molecules. Ion-exchange membrane technologies are non-hazardous in nature and being widely used not only for separation and purification but their application also extended towards energy conversion devices, storage batteries and sensors etc. Now there is a quite demand for the ion-exchange membrane with better selectivities, less electrical resistance, high chemical, mechanical and thermal stability as well as good durability. A lot of work has been done for the development of these types of ion-exchange membranes during the past twenty-five years. Herein we have reviewed the preparation of various types of ion-exchange membranes, their characterization and applications for different electro-membrane processes. Primary attention has been given to the chemical route used for the membrane preparation. Several general reactions used for the preparation of ion-exchange membranes were described. Methodologies used for the characterization of these membranes and their applications were also reviewed for the benefit of readers, so that they can get all information about the ion-exchange membranes at one platform. Although there are large number of reports available regarding preparations and applications of ion-exchange membranes more emphasis were predicted for the usefulness of these membranes or processes for solving certain type of industrial or social problems. More efforts are needed to bring many products or processes to pilot scale and extent their applications.

Amino acid differences in glycoproteins B (gB, C (gC, H (gH and L(gL are associated with enhanced herpes simplex virus type-1 (McKrae entry via the paired immunoglobulin-like type-2 receptor α

Directory of Open Access Journals (Sweden)

Chowdhury Sona

2012-06-01

Full Text Available Abstract Background Herpes simplex virus type-1 (HSV-1 enters into cells via membrane fusion of the viral envelope with plasma or endosomal membranes mediated by viral glycoproteins. HSV-1 virions attach to cell surfaces by binding of viral glycoproteins gC, gD and gB to specific cellular receptors. Here we show that the human ocular and highly neurovirulent HSV-1 strain McKrae enters substantially more efficiently into cells via the gB-specific human paired immunoglobulin-like type-2 receptor-α (hPILR-α. Comparison of the predicted amino acid sequences between HSV-1(F and McKrae strains indicates that amino acid changes within gB, gC, gH and gL may cause increased entry via the hPILR- α receptor. Results HSV-1 (McKrae entered substantially more efficiently than viral strain F in Chinese hamster ovary (CHO cells expressing hPIRL-α but not within CHO-human nectin-1, -(CHO-hNectin-1, CHO-human HVEM (CHO-hHVEM or Vero cells. The McKrae genes encoding viral glycoproteins gB, gC, gD, gH, gL, gK and the membrane protein UL20 were sequenced and their predicted amino acid (aa sequences were compared with virulent strains F, H129, and the attenuated laboratory strain KOS. Most aa differences between McKrae and F were located at their gB amino termini known to bind with the PILRα receptor. These aa changes included a C10R change, also seen in the neurovirulent strain ANG, as well as redistribution and increase of proline residues. Comparison of gC aa sequences revealed multiple aa changes including an L132P change within the 129-247 aa region known to bind to heparan sulfate (HS receptors. Two aa changes were located within the H1 domain of gH that binds gL. Multiple aa changes were located within the McKrae gL sequence, which were preserved in the H129 isolate, but differed for the F strain. Viral glycoproteins gD and gK and the membrane protein UL20 were conserved between McKrae and F strains. Conclusions The results indicate that the observed

Human Ubc9 is involved in intracellular HIV-1 Env stability after trafficking out of the trans-Golgi network in a Gag dependent manner.

Directory of Open Access Journals (Sweden)

Christopher R Bohl

Full Text Available The cellular E2 Sumo conjugase, Ubc9 interacts with HIV-1 Gag, and is important for the assembly of infectious HIV-1 virions. In the previous study we demonstrated that in the absence of Ubc9, a defect in virion assembly was associated with decreased levels of mature intracellular Envelope (Env that affected Env incorporation into virions and virion infectivity. We have further characterized the effect of Ubc9 knockdown on HIV Env processing and assembly. We found that gp160 stability in the endoplasmic reticulum (ER and its trafficking to the trans-Golgi network (TGN were unaffected, indicating that the decreased intracellular mature Env levels in Ubc9-depleted cells were due to a selective degradation of mature Env gp120 after cleavage from gp160 and trafficked out of the TGN. Decreased levels of Gag and mature Env were found to be associated with the plasma membrane and lipid rafts, which suggest that these viral proteins were not trafficked correctly to the assembly site. Intracellular gp120 were partially rescued when treated with a combination of lysosome inhibitors. Taken together our results suggest that in the absence of Ubc9, gp120 is preferentially degraded in the lysosomes likely before trafficking to assembly sites leading to the production of defective virions. This study provides further insight in the processing and packaging of the HIV-1 gp120 into mature HIV-1 virions.

R5 HIV-1 envelope attracts dendritic cells to cross the human intestinal epithelium and sample luminal virions via engagement of the CCR5.

Science.gov (United States)

Cavarelli, Mariangela; Foglieni, Chiara; Rescigno, Maria; Scarlatti, Gabriella

2013-05-01

The gastrointestinal tract is a principal route of entry and site of persistence of human immunodeficiency virus type 1 (HIV-1). The intestinal mucosa, being rich of cells that are the main target of the virus, represents a primary site of viral replication and CD4(+) T-cell depletion. Here, we show both in vitro and ex vivo that HIV-1 of R5 but not X4 phenotype is capable of selectively triggering dendritic cells (DCs) to migrate within 30 min between intestinal epithelial cells to sample virions and transfer infection to target cells. The engagement of the chemokine receptor 5 on DCs and the viral envelope, regardless of the genetic subtype, drive DC migration. Viruses penetrating through transient opening of the tight junctions likely create a paracellular gradient to attract DCs. The formation of junctions with epithelial cells may initiate a haptotactic process of DCs and at the same time favour cell-to-cell viral transmission. Our findings indicate that HIV-1 translocation across the intestinal mucosa occurs through the selective engagement of DCs by R5 viruses, and may guide the design of new prevention strategies. Copyright © 2013 The Authors. Published by John Wiley and Sons, Ltd on behalf of EMBO.

The pestivirus Erns glycoprotein interacts with E2 in both infected cells and mature virions

International Nuclear Information System (INIS)

Lazar, Catalin; Zitzmann, Nicole; Dwek, Raymond A.; Branza-Nichita, Norica

2003-01-01

E rns is a pestivirus envelope glycoprotein indispensable for virus attachment and infection of target cells. Unlike the other two envelope proteins E1 and E2, E rns lacks a transmembrane domain and a vast quantity is secreted into the medium of infected cells. The protein is also present in fractions of pure pestivirus virions, raising the important and intriguing question regarding the mechanism of its attachment to the pestivirus envelope. In this study a direct interaction between E rns and E2 glycoproteins was demonstrated in both pestivirus-infected cells and mature virions. By co- and sequential immunoprecipitation we showed that an E rns -E2 heterodimer is assembled very early after translation of the viral polyprotein and before its processing is completed. Our results suggest that E rns is attached to the pestivirus envelope via a direct interaction with E2 and explain the role of E rns in the initial virus-target cell interaction

The virion RNA species of the Kirsten murine sarcoma-leukemia virus complex released from a clonally related series of mouse cells

International Nuclear Information System (INIS)

Clewley, J.P.; Avery, R.J.

1982-01-01

We have characterized the virion RNA species of Kirsten sarcoma (KiSV) and Kirsten leukemia (KiLV) viruses released from a clonally related series of mouse cells (14). We have identified the KiLV and KiSV genome RNAs. In addition to the viral RNA species we find large amounts of a virus-like RNA (VL30 RNA), which is heterogeneous and shows variability in its expression. The amount of VL30 RNA in virions does not correlate with the state of transformation of the cells releasing the virus or the ability of the virus to transform other cells. Characterization of RNA rescued from non-producer cells has revealed a sarcoma virus (KiSVsub(SB3) with an oligonucleotide fingerprint different from that of a standard KiSV RNA, suggesting that it has lost some viral sequences. The oligonucleotide fingerprints of KiLV and VL30 RNAs are distinct from each other and from those reported for other murine leukemia virus RNAs. (Author)

Expression of Plasmodium falciparum erythrocyte membrane protein 1 in experimentally infected humans

DEFF Research Database (Denmark)

Lavstsen, Thomas; Magistrado, Pamela; Hermsen, Cornelus C

2005-01-01

-encoded Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) family, which is expressed on the surface of infected erythrocytes where it mediates binding to endothelial receptors. Thus, severe malaria may be caused by parasites expressing PfEMP1 variants that afford parasites optimal sequestration...... in immunologically naive individuals and high effective multiplication rates. METHODS: var gene transcription was analysed using real time PCR and PfEMP1 expression by western blots as well as immune plasma recognition of parasite cultures established from non-immune volunteers shortly after infection with NF54...... compared to parasites expressing other var genes. The differential expression of PfEMP1 was confirmed at the protein level by immunoblot analysis. In addition, serological typing showed that immune sera more often recognized second and third generation parasites than first generation parasites. CONCLUSION...

Characterization of modified PVDF membrane by gamma irradiation for non-potable water reuse.

Science.gov (United States)

Lim, Seung Joo; Kim, Tak-Hyun; Shin, In Hwan

2015-01-01

Poly(vinylidene fluorine) (PVDF) membranes were grafted by gamma-ray irradiation and were sulfonated by sodium sulfite to modify the surface of the membranes. The characteristics of the modified PVDF membranes were evaluated by the data of Fourier transform infrared (FT-IR), X-ray photoelectron spectroscopy (XPS), field-emission scanning electron microscope (FE-SEM), the contact angle of the membrane surface and the water permeability. From the results of FT-IR, XPS and FE-SEM, it was shown that the modified membranes were successfully grafted by gamma-ray irradiation and were sulfonated. The content of oxygen and sulfur increased with the monomer concentration, while the content of fluorine sharply decreased. The pore size of the modified membranes decreased after gamma-ray irradiation. The contact angle and the water permeability showed that the hydrophilicity of the modified membranes played a role in determining the membrane performance. The feasibility study of the modified PVDF membranes for using non-potable water reuse were carried out using a laboratory-scale microfiltration system. Grey wastewater was used as the influent in the filtration unit, and permeate quality satisfied non-potable water reuse guidelines in the Republic of Korea.

Histological study of SlNPV infection on body weight and peritrophic membrane damage of Spodoptera litura larvae

Directory of Open Access Journals (Sweden)

NANIN DIAH KURNIAWATI

2010-11-01

Full Text Available Sanjaya, Machmudin D, Kurniawati ND. 2010. Histological study of SlNPV infection on body weight and peritrophic membrane damage of Spodoptera litura larvae. Nusantara Bioscience 2: 135-140. The effect of SlNPV infection on body weight and peritrophic membrane damage of Spodoptera litura Fab. larvae has been carried out. The method was used Probit analysis, and based on LD 50 the virus was infected to know body weight and post infection damage.The damage of histological structure caused by SlNPV (0, 315, 390, 465, 540 dan 615 PIB/mL was investigated after 0, 12, 24, 72 and 96 hours post infection. The histological material was prepared by using parafin method after fixation with Bouin Solution, then slice into 7 um and colored with Hematoxilin-Eosin. The result showed that the exposure SlNPV cause decreasing food consumption especially on 540 PIB/mL give average rate as amount of 0.1675 mg. The descriptive obsevation on structural intact of peritrophic membrane histology caused by SlNPV infection shows a tendency to decrease, while in control, there was no damage at all. The longer the exposition of virion in the midgut lumen the more damage on peritrophic membrane occurred. The severest damage occurred 96 hour after infection. The result prove that haNPV virion can destroy hystological structure of midgut.

Frog virus 3 ORF 53R, a putative myristoylated membrane protein, is essential for virus replication in vitro

International Nuclear Information System (INIS)

Whitley, Dexter S.; Yu, Kwang; Sample, Robert C.; Sinning, Allan; Henegar, Jeffrey; Norcross, Erin; Chinchar, V. Gregory

2010-01-01

Although previous work identified 12 complementation groups with possible roles in virus assembly, currently only one frog virus 3 protein, the major capsid protein (MCP), has been linked with virion formation. To identify other proteins required for assembly, we used an antisense morpholino oligonucleotide to target 53R, a putative myristoylated membrane protein, and showed that treatment resulted in marked reductions in 53R levels and a 60% drop in virus titers. Immunofluorescence assays confirmed knock down and showed that 53R was found primarily within viral assembly sites, whereas transmission electron microscopy detected fewer mature virions and, in some cells, dense granular bodies that may represent unencapsidated DNA-protein complexes. Treatment with a myristoylation inhibitor (2-hydroxymyristic acid) resulted in an 80% reduction in viral titers. Collectively, these data indicate that 53R is an essential viral protein that is required for replication in vitro and suggest it plays a critical role in virion formation.

Transcriptome and membrane fatty acid analyses reveal different strategies for responding to permeating and non-permeating solutes in the bacterium Sphingomonas wittichii

Directory of Open Access Journals (Sweden)

Johnson David R

2011-11-01

Full Text Available Abstract Background Sphingomonas wittichii strain RW1 can completely oxidize dibenzo-p-dioxins and dibenzofurans, which are persistent contaminants of soils and sediments. For successful application in soil bioremediation systems, strain RW1 must cope with fluctuations in water availability, or water potential. Thus far, however, little is known about the adaptive strategies used by Sphingomonas bacteria to respond to changes in water potential. To improve our understanding, strain RW1 was perturbed with either the cell-permeating solute sodium chloride or the non-permeating solute polyethylene glycol with a molecular weight of 8000 (PEG8000. These solutes are assumed to simulate the solute and matric components of the total water potential, respectively. The responses to these perturbations were then assessed and compared using a combination of growth assays, transcriptome profiling, and membrane fatty acid analyses. Results Under conditions producing a similar decrease in water potential but without effect on growth rate, there was only a limited shared response to perturbation with sodium chloride or PEG8000. This shared response included the increased expression of genes involved with trehalose and exopolysaccharide biosynthesis and the reduced expression of genes involved with flagella biosynthesis. Mostly, the responses to perturbation with sodium chloride or PEG8000 were very different. Only sodium chloride triggered the increased expression of two ECF-type RNA polymerase sigma factors and the differential expression of many genes involved with outer membrane and amino acid metabolism. In contrast, only PEG8000 triggered the increased expression of a heat shock-type RNA polymerase sigma factor along with many genes involved with protein turnover and repair. Membrane fatty acid analyses further corroborated these differences. The degree of saturation of membrane fatty acids increased after perturbation with sodium chloride but had the

Herpes simplex virus types 1 and 2 modulate autophagy in SIRC ...

Indian Academy of Sciences (India)

2014-06-29

Jun 29, 2014 ... production of infectious virions severely when it was present throughout the ... drug control (figure 2D, lanes 3, 5 and 1, respectively). Thus, .... Methods. Data are ... Ocular therapeutics handbook: A clinical manual 2nd edition.

Mini-review: novel non-destructivein situbiofilm characterization techniques in membrane systems

KAUST Repository

Valladares Linares, Rodrigo; Fortunato, Luca; Farhat, Nadia; Bucs, Szilard; Staal, M.; Fridjonsson, E.O.; Johns, M.L.; Vrouwenvelder, Johannes S.; Leiknes, TorOve

2016-01-01

Membrane systems are commonly used in the water industry to produce potable water and for advanced wastewater treatment. One of the major drawbacks of membrane systems is biofilm formation (biofouling), which results in an unacceptable decline in membrane performance. Three novel in situ biofouling characterization techniques were assessed: (i) optical coherence tomography (OCT), (ii) planar optodes, and (iii) nuclear magnetic resonance (NMR). The first two techniques were assessed using a biofilm grown on the surface of nanofiltration (NF) membranes using a transparent membrane fouling simulator that accurately simulates spiral wound modules, modified for in situ biofilm imaging. For the NMR study, a spiral wound reverse osmosis membrane module was used. Results show that these techniques can provide information to reconstruct the biofilm accurately, either with 2-D (OCT, planar optodes and NMR), or 3-D (OCT and NMR) scans. These non-destructive tools can elucidate the interaction of hydrodynamics and mass transport on biofilm accumulation in membrane systems. Oxygen distribution in the biofilm can be mapped and linked to water flow and substrate characteristics; insights on the effect of crossflow velocity, flow stagnation, and feed spacer presence can be obtained, and in situ information on biofilm structure, thickness, and spatial distribution can be quantitatively assessed. The combination of these novel non-destructive in situ biofilm characterization techniques can provide real-time observation of biofilm formation at the mesoscale. The information obtained with these tools could potentially be used for further improvement in the design of membrane systems and operational parameters to reduce impact of biofouling on membrane performance. © 2016 Balaban Desalination Publications. All rights reserved.

Mini-review: novel non-destructivein situbiofilm characterization techniques in membrane systems

KAUST Repository

Valladares Linares, R.

2016-05-12

Membrane systems are commonly used in the water industry to produce potable water and for advanced wastewater treatment. One of the major drawbacks of membrane systems is biofilm formation (biofouling), which results in an unacceptable decline in membrane performance. Three novel in situ biofouling characterization techniques were assessed: (i) optical coherence tomography (OCT), (ii) planar optodes, and (iii) nuclear magnetic resonance (NMR). The first two techniques were assessed using a biofilm grown on the surface of nanofiltration (NF) membranes using a transparent membrane fouling simulator that accurately simulates spiral wound modules, modified for in situ biofilm imaging. For the NMR study, a spiral wound reverse osmosis membrane module was used. Results show that these techniques can provide information to reconstruct the biofilm accurately, either with 2-D (OCT, planar optodes and NMR), or 3-D (OCT and NMR) scans. These non-destructive tools can elucidate the interaction of hydrodynamics and mass transport on biofilm accumulation in membrane systems. Oxygen distribution in the biofilm can be mapped and linked to water flow and substrate characteristics; insights on the effect of crossflow velocity, flow stagnation, and feed spacer presence can be obtained, and in situ information on biofilm structure, thickness, and spatial distribution can be quantitatively assessed. The combination of these novel non-destructive in situ biofilm characterization techniques can provide real-time observation of biofilm formation at the mesoscale. The information obtained with these tools could potentially be used for further improvement in the design of membrane systems and operational parameters to reduce impact of biofouling on membrane performance. © 2016 Balaban Desalination Publications. All rights reserved.

Nephritogenic antigen determinants in epidermal and renal basement membranes of kindreds with Alport-type familial nephritis.

Science.gov (United States)

Kashtan, C; Fish, A J; Kleppel, M; Yoshioka, K; Michael, A F

1986-10-01

We probed epidermal basement membranes (EBM) of acid-urea denatured skin from members of kindreds with Alport-type familial nephritis (FN) for the presence of antigens reactive with Goodpasture sera (GPS) and serum (FNS) from an Alport patient who developed anti-glomerular basement membrane (GBM) nephritis in a renal allograft. By immunoblotting, GPS reacted primarily with the 28,000 molecular weight (mol wt) monomer but also the 24,000 mol wt and 26,000 mol wt monomers of the noncollagenous globular domain (NC1) of type IV collagen from normal human GBM, while FNS identified only the 26,000-mol wt monomer. FNS reacted with EBM of 12 controls and nine unaffected male kindred members but not EBM of eight affected males. Five affected females exhibited interrupted reactivity of FNS with EBM. GPS showed variable reactivity with EBM and was not discriminating with respect to Alport-type FN. FNS did not stain renal basement members of five affected males. However, the EBM, tubular basement membrane, and Bowman's capsules of affected males contained antigens reactive with GPS. These immunochemical studies suggest that the FNS antigen is distinct from Goodpasture antigen(s). The expression of FNS antigen located on the NC1 domain of type IV collagen is altered in basement membranes of patients with Alport-type FN, and the distribution of this antigenic anomaly within kindreds suggests X-linked dominant transmission of a defective gene.

Three-dimensional structure of the human immunodeficiency virus type 1 matrix protein.

Science.gov (United States)

Massiah, M A; Starich, M R; Paschall, C; Summers, M F; Christensen, A M; Sundquist, W I

1994-11-25

The HIV-1 matrix protein forms an icosahedral shell associated with the inner membrane of the mature virus. Genetic analyses have indicated that the protein performs important functions throughout the viral life-cycle, including anchoring the transmembrane envelope protein on the surface of the virus, assisting in viral penetration, transporting the proviral integration complex across the nuclear envelope, and localizing the assembling virion to the cell membrane. We now report the three-dimensional structure of recombinant HIV-1 matrix protein, determined at high resolution by nuclear magnetic resonance (NMR) methods. The HIV-1 matrix protein is the first retroviral matrix protein to be characterized structurally and only the fourth HIV-1 protein of known structure. NMR signal assignments required recently developed triple-resonance (1H, 13C, 15N) NMR methodologies because signals for 91% of 132 assigned H alpha protons and 74% of the 129 assignable backbone amide protons resonate within chemical shift ranges of 0.8 p.p.m. and 1 p.p.m., respectively. A total of 636 nuclear Overhauser effect-derived distance restraints were employed for distance geometry-based structure calculations, affording an average of 13.0 NMR-derived distance restraints per residue for the experimentally constrained amino acids. An ensemble of 25 refined distance geometry structures with penalties (sum of the squares of the distance violations) of 0.32 A2 or less and individual distance violations under 0.06 A was generated; best-fit superposition of ordered backbone heavy atoms relative to mean atom positions afforded root-mean-square deviations of 0.50 (+/- 0.08) A. The folded HIV-1 matrix protein structure is composed of five alpha-helices, a short 3(10) helical stretch, and a three-strand mixed beta-sheet. Helices I to III and the 3(10) helix pack about a central helix (IV) to form a compact globular domain that is capped by the beta-sheet. The C-terminal helix (helix V) projects away

Association of faecal elastase 1 with non-fasting triglycerides in type 2 diabetes.

Science.gov (United States)

Rathmann, Wolfgang; Haastert, Burkhard; Oscarsson, Jan; Berglind, Niklas; Lindkvist, Björn; Wareham, Nicholas J

2016-01-01

Intestinal absorption of esterified fatty acids depends on exocrine pancreatic function and influences plasma triglycerides levels. The aim was to investigate the association of reduced exocrine pancreatic function (low fecal elastase-1; FE1) with plasma triglycerides in type 2 diabetes and controls without diabetes. FE1 (μg/g stool) and non-fasting plasma triglyceride measurements were undertaken in 544 type 2 diabetes patients (age: 63 ± 8 years) randomly selected from diabetes registers in Cambridgeshire (UK), and 544 matched controls (age, sex, practice) without diabetes. Linear regression models were fitted using FE1 as dependent and log-triglycerides as independent variable adjusting for sex, age, body mass index, alcohol consumption, serum lipase, HbA1c, and smoking. FE1 concentrations were lower (mean ± SD: 337 ± 204 vs. 437 ± 216 μg/g, p triglycerides were higher (geometric mean */: standard deviation factor: 2.2*/:1.9 vs. 1.6*/:1.8 mmol/l, p triglycerides was associated with 4.5 μg/g higher FE1 concentrations (p triglycerides (significant only in controls). Non-fasting triglycerides were positively related to FE1 in both type 2 diabetes and controls suggesting that impairment of exocrine pancreas function is influencing plasma triglycerides. Marked loss of exocrine pancreatic function had the opposite effect, resulting in higher levels of plasma triglycerides. Copyright © 2016 IAP and EPC. Published by Elsevier B.V. All rights reserved.

Early non-destructive biofouling detection in spiral wound RO Membranes using a mobile earth's field NMR

KAUST Repository

Fridjonsson, E.O.; Vogt, S.J.; Vrouwenvelder, Johannes S.; Johns, M.L.

2015-01-01

We demonstrate the use of Earth's field (EF) Nuclear Magnetic Resonance (NMR) to provide early non-destructive detection of active biofouling of a commercial spiral wound reverse osmosis (RO) membrane module. The RO membrane module was actively biofouled to different extents, by the addition of biodegradable nutrients to the feed stream, as revealed by a subtle feed-channel pressure drop increase. Easily accessible EF NMR parameters (signal relaxation parameters T1, T2 and the total NMR signal modified to be sensitive to stagnant fluid only) were measured and analysed in terms of their ability to detect the onset of biofouling. The EF NMR showed that fouling near the membrane module entrance significantly distorted the flow field through the whole membrane module. The total NMR signal is shown to be suitable for non-destructive early biofouling detection of spiral wound membrane modules, it was readily deployed at high (operational) flow rates, was particularly sensitive to flow field changes due to biofouling and could be deployed at any position along the membrane module axis. In addition to providing early fouling detection, the mobile EF NMR apparatus could also be used to (i) evaluate the production process of spiral wound membrane modules, and (ii) provide an in-situ determination of module cleaning process efficiency.

Early non-destructive biofouling detection in spiral wound RO Membranes using a mobile earth's field NMR

KAUST Repository

Fridjonsson, E.O.

2015-04-20

We demonstrate the use of Earth\\'s field (EF) Nuclear Magnetic Resonance (NMR) to provide early non-destructive detection of active biofouling of a commercial spiral wound reverse osmosis (RO) membrane module. The RO membrane module was actively biofouled to different extents, by the addition of biodegradable nutrients to the feed stream, as revealed by a subtle feed-channel pressure drop increase. Easily accessible EF NMR parameters (signal relaxation parameters T1, T2 and the total NMR signal modified to be sensitive to stagnant fluid only) were measured and analysed in terms of their ability to detect the onset of biofouling. The EF NMR showed that fouling near the membrane module entrance significantly distorted the flow field through the whole membrane module. The total NMR signal is shown to be suitable for non-destructive early biofouling detection of spiral wound membrane modules, it was readily deployed at high (operational) flow rates, was particularly sensitive to flow field changes due to biofouling and could be deployed at any position along the membrane module axis. In addition to providing early fouling detection, the mobile EF NMR apparatus could also be used to (i) evaluate the production process of spiral wound membrane modules, and (ii) provide an in-situ determination of module cleaning process efficiency.

Amino-terminal sequence of glycoprotein D of herpes simplex virus types 1 and 2

International Nuclear Information System (INIS)

Eisenberg, R.J.; Long, D.; Hogue-Angeletti, R.; Cohen, G.H.

1984-01-01

Glycoprotein D (gD) of herpes simplex virus is a structural component of the virion envelope which stimulates production of high titers of herpes simplex virus type-common neutralizing antibody. The authors caried out automated N-terminal amino acid sequencing studies on radiolabeled preparations of gD-1 (gD of herpes simplex virus type 1) and gD-2 (gD of herpes simplex virus type 2). Although some differences were noted, particularly in the methionine and alanine profiles for gD-1 and gD-2, the amino acid sequence of a number of the first 30 residues of the amino terminus of gD-1 and gD-2 appears to be quite similar. For both proteins, the first residue is a lysine. When we compared out sequence data for gD-1 with those predicted by nucleic acid sequencing, the two sequences could be aligned (with one exception) starting at residue 26 (lysine) of the predicted sequence. Thus, the first 25 amino acids of the predicted sequence are absent from the polypeptides isolated from infected cells

Allergic reaction to polysulphone membrane dialyser masquerading as infection.

Science.gov (United States)

Mukaya, Japheth E; Jacobson, Mitchell S; Esprit, Don; Ajayi, Tokunbo

2015-02-18

Hypersensitivity reactions during haemodialysis are well documented in the literature. Most dialysis membrane reactions occur with ethylene oxide or non-biocompatible membranes and less frequently with biocompatible membranes. The symptoms are non-specific except for the temporal relationship typically occurring during the haemodialysis. Patients who present with the non-classic type of reaction pose a diagnostic challenge and clinicians must have a high index of suspicion. We present a non-classic presentation of a hypersensitivity reaction to a polysulfone membrane dialyser with recurrent fevers during and after haemodialysis, initially thought to be due to infection. After a series of negative work ups for infection, dialysis membrane reaction was considered and the patient improved after change of the membrane. 2015 BMJ Publishing Group Ltd.

Developmental mechanisms of the tympanic membrane in mammals and non-mammalian amniotes.

Science.gov (United States)

Takechi, Masaki; Kitazawa, Taro; Hirasawa, Tatsuya; Hirai, Tamami; Iseki, Sachiko; Kurihara, Hiroki; Kuratani, Shigeru

2016-01-01

The tympanic membrane is a thin layer that originates from the ectoderm, endoderm, and mesenchyme. Molecular-genetic investigations have revealed that interaction between epithelial and mesenchymal cells in the pharyngeal arches is essential for development of the tympanic membrane. We have recently reported that developmental mechanisms underlying the tympanic membrane seem to be different between mouse and chicken, suggesting that the tympanic membrane evolved independently in mammals and non-mammalian amniotes. In this review, we summarize previous studies of tympanic membrane formation in the mouse. We also discuss its formation in amniotes from an evolutionary point of view. © 2015 Japanese Teratology Society.

Enzyme immobilization by fouling in ultrafiltration membranes: Impact of membrane configuration and type on flux behavior and biocatalytic conversion efficacy

DEFF Research Database (Denmark)

Luo, Jianquan; Meyer, Anne S.; Jonsson, Gunnar Eigil

2014-01-01

Enzyme-immobilization in membranes accomplished by fostering membrane fouling was evaluated. Four different membrane configurations and five membranes were compared for immobilization of alcohol dehydrogenase (ADH) in terms of enzyme loading, permeate flux and final biocatalytic conversion...... and PLGC regenerated cellulose membranes. With these two highly hydrophilic membranes, the ADH enzyme activity was fully retained even after 24h of storage of the membrane. Filtration blocking and resistance models were used to analyze the fouling/immobilization mechanisms and give explanations...... for the different results. The work confirms that fouling-induced enzyme immobilization is a promising option for enhancing biocatalytic productivity, and highlights the significance of the membrane type and configuration for optimal performance....

Adenovirus type 2 endopeptidase: an unusual phosphoprotein enzyme matured by autocatalysis

Energy Technology Data Exchange (ETDEWEB)

Chatterjee, P.K.; Flint, S.J.

1987-02-01

A 19-kDa protein, present in low copy number in purified adenovirus type 2, has been characterized. Several criteria were used to establish that this protein is neither a degradation product of the known structural proteins of the virion nor a minor, unusually modified, form of protein VII. This 19-kDa protein, unlike other virion proteins, possesses alkali-resistant phosphoamino acids. Analysis by partial proteolysis indicated that it is related to a 23-kDa phosphoprotein present in H2ts-1 virions assembled in infected cells maintained at 39/sup 0/C. Affinity labeling with (/sup 3/H)diisopropyl fluorophosphate showed that the 19-kDa protein contains the active site for a serine protease. The authors, therefore, conclude that the 19-kDa protein is the active form of the adenovirus-encoded endopeptidase, defined by the H2ts-1 mutation, and is synthesized as a 23-kDa precursor that appears to mature by autocatalysis.

Sexual transmission of human T-cell lymphotropic virus type 1

Directory of Open Access Journals (Sweden)

Arthur Paiva

2014-06-01

Full Text Available Human T-cell lymphotropic virus type 1 (HTLV-1 is endemic in many parts of the world and is primarily transmitted through sexual intercourse or from mother to child. Sexual transmission occurs more efficiently from men to women than women to men and might be enhanced by sexually transmitted diseases that cause ulcers and result in mucosal ruptures, such as syphilis, herpes simplex type 2 (HSV-2, and chancroid. Other sexually transmitted diseases might result in the recruitment of inflammatory cells and could increase the risk of HTLV-1 acquisition and transmission. Additionally, factors that are associated with higher transmission risks include the presence of antibodies against the viral oncoprotein Tax (anti-Tax, a higher proviral load in peripheral blood lymphocytes, and increased cervicovaginal or seminal secretions. Seminal fluid has been reported to increase HTLV replication and transmission, whereas male circumcision and neutralizing antibodies might have a protective effect. Recently, free virions were discovered in plasma, which reveals a possible new mode of HTLV replication. It is unclear how this discovery might affect the routes of HTLV transmission, particularly sexual transmission, because HTLV transmission rates are significantly higher from men to women than women to men.

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.

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

International Nuclear Information System (INIS)

Paquette, Stéphane G.; Banner, David; Chi, Le Thi Bao; Leon, Alberto J.; Xu, Luoling; Ran, Longsi; Huang, Stephen S.H.; Farooqui, Amber

2014-01-01

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

Membrane topology analysis of HIV-1 envelope glycoprotein gp41

Directory of Open Access Journals (Sweden)

Xiao Dan

2010-11-01

Full Text Available Abstract Background The gp41 subunit of the HIV-1 envelope glycoprotein (Env has been widely regarded as a type I transmembrane protein with a single membrane-spanning domain (MSD. An alternative topology model suggested multiple MSDs. The major discrepancy between the two models is that the cytoplasmic Kennedy sequence in the single MSD model is assigned as the extracellular loop accessible to neutralizing antibodies in the other model. We examined the membrane topology of the gp41 subunit in both prokaryotic and mammalian systems. We attached topological markers to the C-termini of serially truncated gp41. In the prokaryotic system, we utilized a green fluorescent protein (GFP that is only active in the cytoplasm. The tag protein (HaloTag and a membrane-impermeable ligand specific to HaloTag was used in the mammalian system. Results In the absence of membrane fusion, both the prokaryotic and mammalian systems (293FT cells supported the single MSD model. In the presence of membrane fusion in mammalian cells (293CD4 cells, the data obtained seem to support the multiple MSD model. However, the region predicted to be a potential MSD is the highly hydrophilic Kennedy sequence and is least likely to become a MSD based on several algorithms. Further analysis revealed the induction of membrane permeability during membrane fusion, allowing the membrane-impermeable ligand and antibodies to cross the membrane. Therefore, we cannot completely rule out the possible artifacts. Addition of membrane fusion inhibitors or alterations of the MSD sequence decreased the induction of membrane permeability. Conclusions It is likely that a single MSD model for HIV-1 gp41 holds true even in the presence of membrane fusion. The degree of the augmentation of membrane permeability we observed was dependent on the membrane fusion and sequence of the MSD.

A general theory of non-equilibrium dynamics of lipid-protein fluid membranes

DEFF Research Database (Denmark)

Lomholt, Michael Andersen; Hansen, Per Lyngs; Miao, L.

2005-01-01

We present a general and systematic theory of non-equilibrium dynamics of multi-component fluid membranes, in general, and membranes containing transmembrane proteins, in particular. Developed based on a minimal number of principles of statistical physics and designed to be a meso...

Design and optimization of membrane-type acoustic metamaterials

Science.gov (United States)

Blevins, Matthew Grant

One of the most common problems in noise control is the attenuation of low frequency noise. Typical solutions require barriers with high density and/or thickness. Membrane-type acoustic metamaterials are a novel type of engineered material capable of high low-frequency transmission loss despite their small thickness and light weight. These materials are ideally suited to applications with strict size and weight limitations such as aircraft, automobiles, and buildings. The transmission loss profile can be manipulated by changing the micro-level substructure, stacking multiple unit cells, or by creating multi-celled arrays. To date, analysis has focused primarily on experimental studies in plane-wave tubes and numerical modeling using finite element methods. These methods are inefficient when used for applications that require iterative changes to the structure of the material. To facilitate design and optimization of membrane-type acoustic metamaterials, computationally efficient dynamic models based on the impedance-mobility approach are proposed. Models of a single unit cell in a waveguide and in a baffle, a double layer of unit cells in a waveguide, and an array of unit cells in a baffle are studied. The accuracy of the models and the validity of assumptions used are verified using a finite element method. The remarkable computational efficiency of the impedance-mobility models compared to finite element methods enables implementation in design tools based on a graphical user interface and in optimization schemes. Genetic algorithms are used to optimize the unit cell design for a variety of noise reduction goals, including maximizing transmission loss for broadband, narrow-band, and tonal noise sources. The tools for design and optimization created in this work will enable rapid implementation of membrane-type acoustic metamaterials to solve real-world noise control problems.

Membranes having aligned 1-D nanoparticles in a matrix layer for improved fluid separation

Science.gov (United States)

Revanur, Ravindra; Lulevich, Valentin; Roh, Il Juhn; Klare, Jennifer E.; Kim, Sangil; Noy, Aleksandr; Bakajin, Olgica

2015-12-22

Membranes for fluid separation are disclosed. These membranes have a matrix layer sandwiched between an active layer and a porous support layer. The matrix layer includes 1-D nanoparticles that are vertically aligned in a porous polymer matrix, and which substantially extend through the matrix layer. The active layer provides species-specific transport, while the support layer provides mechanical support. A matrix layer of this type has favorable surface morphology for forming the active layer. Furthermore, the pores that form in the matrix layer tend to be smaller and more evenly distributed as a result of the presence of aligned 1-D nanoparticles. Improved performance of separation membranes of this type is attributed to these effects.

Adaptive silicone-membrane lenses: planar vs. shaped membrane

CSIR Research Space (South Africa)

Schneider, F

2009-08-01

Full Text Available Engineering, Georges-Koehler-Allee 102, Freiburg 79110, Germany florian.schneider@imtek.uni-freiburg.de ABSTRACT We compare the performance and optical quality of two types of adaptive fluidic silicone-membrane lenses. The membranes feature either a...-membrane lenses: planar vs. shaped membrane Florian Schneider1,2, Philipp Waibel2 and Ulrike Wallrabe2 1 CSIR, Materials Science and Manufacturing, PO Box 395, Pretoria 0001, South Africa 2 University of Freiburg – IMTEK, Department of Microsystems...

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

International Nuclear Information System (INIS)

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

2008-01-01

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

Predicting membrane protein types by fusing composite protein sequence features into pseudo amino acid composition.

Science.gov (United States)

Hayat, Maqsood; Khan, Asifullah

2011-02-21

Membrane proteins are vital type of proteins that serve as channels, receptors, and energy transducers in a cell. Prediction of membrane protein types is an important research area in bioinformatics. Knowledge of membrane protein types provides some valuable information for predicting novel example of the membrane protein types. However, classification of membrane protein types can be both time consuming and susceptible to errors due to the inherent similarity of membrane protein types. In this paper, neural networks based membrane protein type prediction system is proposed. Composite protein sequence representation (CPSR) is used to extract the features of a protein sequence, which includes seven feature sets; amino acid composition, sequence length, 2 gram exchange group frequency, hydrophobic group, electronic group, sum of hydrophobicity, and R-group. Principal component analysis is then employed to reduce the dimensionality of the feature vector. The probabilistic neural network (PNN), generalized regression neural network, and support vector machine (SVM) are used as classifiers. A high success rate of 86.01% is obtained using SVM for the jackknife test. In case of independent dataset test, PNN yields the highest accuracy of 95.73%. These classifiers exhibit improved performance using other performance measures such as sensitivity, specificity, Mathew's correlation coefficient, and F-measure. The experimental results show that the prediction performance of the proposed scheme for classifying membrane protein types is the best reported, so far. This performance improvement may largely be credited to the learning capabilities of neural networks and the composite feature extraction strategy, which exploits seven different properties of protein sequences. The proposed Mem-Predictor can be accessed at http://111.68.99.218/Mem-Predictor. Copyright © 2010 Elsevier Ltd. All rights reserved.

Dengue Virus Non-structural Protein 1 Modulates Infectious Particle Production via Interaction with the Structural Proteins.

Directory of Open Access Journals (Sweden)

Pietro Scaturro

Full Text Available Non-structural protein 1 (NS1 is one of the most enigmatic proteins of the Dengue virus (DENV, playing distinct functions in immune evasion, pathogenesis and viral replication. The recently reported crystal structure of DENV NS1 revealed its peculiar three-dimensional fold; however, detailed information on NS1 function at different steps of the viral replication cycle is still missing. By using the recently reported crystal structure, as well as amino acid sequence conservation, as a guide for a comprehensive site-directed mutagenesis study, we discovered that in addition to being essential for RNA replication, DENV NS1 is also critically required for the production of infectious virus particles. Taking advantage of a trans-complementation approach based on fully functional epitope-tagged NS1 variants, we identified previously unreported interactions between NS1 and the structural proteins Envelope (E and precursor Membrane (prM. Interestingly, coimmunoprecipitation revealed an additional association with capsid, arguing that NS1 interacts via the structural glycoproteins with DENV particles. Results obtained with mutations residing either in the NS1 Wing domain or in the β-ladder domain suggest that NS1 might have two distinct functions in the assembly of DENV particles. By using a trans-complementation approach with a C-terminally KDEL-tagged ER-resident NS1, we demonstrate that the secretion of NS1 is dispensable for both RNA replication and infectious particle production. In conclusion, our results provide an extensive genetic map of NS1 determinants essential for viral RNA replication and identify a novel role of NS1 in virion production that is mediated via interaction with the structural proteins. These studies extend the list of NS1 functions and argue for a central role in coordinating replication and assembly/release of infectious DENV particles.

Injectible candidate sealants for fetal membrane repair: Bonding and toxicity in vitro

Science.gov (United States)

Bilic, Grozdana; Brubaker, Carrie; Messersmith, Phillip B.; Mallik, Ajit S.; Quinn, Thomas M.; Haller, Claudia; Done, Elisa; Gucciardo, Leonardo; Zeisberger, Steffen M.; Zimmermann, Roland; Deprest, Jan; Zisch, Andreas H.

2010-01-01

Objective This study was undertaken to test injectible surgical sealants that are biocompatible with fetal membranes, eventually for closure of iatrogenic membrane defects. Study Design Dermabond, Histoacryl, Tissucol fibrin glue, and three types of in situ forming poly(ethylene glycol)-based polymer hydrogels were tested for acute toxicity upon direct contact with fetal membranes for 24h. For determination of elution toxicity, extracts of sealants were incubated on amnion cell cultures for 72h. Bonding and toxicity was assessed through morphological and/or biochemical analysis. Results Extracts of all adhesives were non-toxic for cultured cells. However, only Tissucol and one type of poly(ethylene glycol)-based hydrogel, mussel-mimetic tissue adhesive, showed efficient, non-disruptive, non-toxic bonding to fetal membranes. Mussel-mimetic tissue adhesive applied over membrane defects created with a 3.5 mm trocar accomplished leak-proof closure that withstood membrane stretch in an in vitro model. Conclusion A synthetic hydrogel-type tissue adhesive emerged as potential sealing modality for iatrogenic membrane defects that merits further evaluation in vivo. PMID:20096254

Non-woven PET fabric reinforced and enhanced the performance of ultrafiltration membranes composed of PVDF blended with PVDF-g-PEGMA for industrial applications

Science.gov (United States)

Wang, Shuai; Li, Tong; Chen, Chen; Chen, Sheng; Liu, Baicang; Crittenden, John

2018-03-01

Ultrafiltration (UF) membranes composed of poly(vinylidene fluoride) (PVDF) blended with poly(vinylidene fluoride)-graft-poly(ethylene glycol) methyl ether methacrylate (PVDF-g-PEGMA) can present high flux and excellent foulant removal efficiencies under suitable preparation conditions. However, these PVDF/PVDF-g-PEGMA blended membranes cannot be applied industrially because of the insufficient mechanical strength (strength-to-break value of 8.4 ± 0.6 MPa). We incorporated two types of non-woven polyethylene terephthalate (PET) fabrics (thin hydrophobic and thick hydrophilic fabrics) as support layers to improve the mechanical properties of the blended membranes. The thin and thick PET fabrics were able to significantly improve the tensile strength to 23.3 ± 3.7 MPa and 30.1 ± 1.4 MPa, respectively. The PET fabrics had a limited impact on the separation-related membrane performance such as hydrophilicity, foulant rejection, whereas the mechanical strength and pure water flux was improved several folds. The enhanced flux was attributed to the higher surface porosity and wider finger-like voids in the cross-section. The thin PET fabric with larger porosity was able to maintain a consistent toughness simultaneously; thus it is recommended as a support material for this blended membrane.

Kir2.1 channels set two levels of resting membrane potential with inward rectification.

Science.gov (United States)

Chen, Kuihao; Zuo, Dongchuan; Liu, Zheng; Chen, Haijun

2018-04-01

Strong inward rectifier K + channels (Kir2.1) mediate background K + currents primarily responsible for maintenance of resting membrane potential. Multiple types of cells exhibit two levels of resting membrane potential. Kir2.1 and K2P1 currents counterbalance, partially accounting for the phenomenon of human cardiomyocytes in subphysiological extracellular K + concentrations or pathological hypokalemic conditions. The mechanism of how Kir2.1 channels contribute to the two levels of resting membrane potential in different types of cells is not well understood. Here we test the hypothesis that Kir2.1 channels set two levels of resting membrane potential with inward rectification. Under hypokalemic conditions, Kir2.1 currents counterbalance HCN2 or HCN4 cation currents in CHO cells that heterologously express both channels, generating N-shaped current-voltage relationships that cross the voltage axis three times and reconstituting two levels of resting membrane potential. Blockade of HCN channels eliminated the phenomenon in K2P1-deficient Kir2.1-expressing human cardiomyocytes derived from induced pluripotent stem cells or CHO cells expressing both Kir2.1 and HCN2 channels. Weakly inward rectifier Kir4.1 or inward rectification-deficient Kir2.1•E224G mutant channels do not set such two levels of resting membrane potential when co-expressed with HCN2 channels in CHO cells or when overexpressed in human cardiomyocytes derived from induced pluripotent stem cells. These findings demonstrate a common mechanism that Kir2.1 channels set two levels of resting membrane potential with inward rectification by balancing inward currents through different cation channels such as hyperpolarization-activated HCN channels or hypokalemia-induced K2P1 leak channels.

Development of the sulphonated poly(2,6-Dimethyl-1,4-Phenylene Oxide) membranes for proton exchange membranes fuel cells

International Nuclear Information System (INIS)

Ebrasu, Daniela; Petreanu, Irina; Iordache, Ioan; Stefanescu, Ioan; Gaspar, Costinela-Laura; Militaru, Daniela

2008-01-01

Full text: Fuel cells have the potential to become an important energy conversion technology. Research efforts directed toward the widespread commercialization of fuel cells have accelerated the developing of new types of Proton Exchange Membranes (also termed 'polymer electrolyte membranes') (PEM). Common issues critical to all high performance proton exchange membranes include: - high protonic conductivity; - low electronic conductivity; - low permeability to fuel and oxidant; - low water transport through diffusion and electro-osmosis; - oxidative and hydrolytic stability; - good mechanical properties in both the dry and hydrated states; - cost; and capability for fabrication into Membrane Electrode Assemblies (MEAs). In this sense we choose to use poly(2,6-Dimethyl-1,4-Phenylene Oxide) (PPO) as basis for development of new PEM membranes. The membranes were prepared by lamination from solution (Doctor Balde) method in controlled atmosphere (preliminary vacuum 0.003 Torr and/or nitrogen). FTIR spectra of the sulphonated polymers prove the sulphonic groups presence according the literature. Ionic exchange capacity (IEC) have the values 1.15-3.6 meq/g. TGA-DSC analysis put in evidence the thermal degradation of the sulphonated polymers at about 120 deg. C. These properties of the sulphonated PPO are in accordance of the requirements for PEM membranes and indicate that this polymer is suitable for PEM Fuel cells. (authors)

Integrity of the Linker of Nucleoskeleton and Cytoskeleton Is Required for Efficient Herpesvirus Nuclear Egress.

Science.gov (United States)

Klupp, Barbara G; Hellberg, Teresa; Granzow, Harald; Franzke, Kati; Dominguez Gonzalez, Beatriz; Goodchild, Rose E; Mettenleiter, Thomas C

2017-10-01

Herpesvirus capsids assemble in the nucleus, while final virion maturation proceeds in the cytoplasm. This requires that newly formed nucleocapsids cross the nuclear envelope (NE), which occurs by budding at the inner nuclear membrane (INM), release of the primary enveloped virion into the perinuclear space (PNS), and subsequent rapid fusion with the outer nuclear membrane (ONM). During this process, the NE remains intact, even at late stages of infection. In addition, the spacing between the INM and ONM is maintained, as is that between the primary virion envelope and nuclear membranes. The linker of nucleoskeleton and cytoskeleton (LINC) complex consists of INM proteins with a luminal SUN (Sad1/UNC-84 homology) domain connected to ONM proteins with a KASH (Klarsicht, ANC-1, SYNE homology) domain and is thought to be responsible for spacing the nuclear membranes. To investigate the role of the LINC complex during herpesvirus infection, we generated cell lines constitutively expressing dominant negative (dn) forms of SUN1 and SUN2. Ultrastructural analyses revealed a significant expansion of the PNS and the contiguous intracytoplasmic lumen, most likely representing endoplasmic reticulum (ER), especially in cells expressing dn-SUN2. After infection, primary virions accumulated in these expanded luminal regions, also very distant from the nucleus. The importance of the LINC complex was also confirmed by reduced progeny virus titers in cells expressing dn-SUN2. These data show that the intact LINC complex is required for efficient nuclear egress of herpesviruses, likely acting to promote fusion of primary enveloped virions with the ONM. IMPORTANCE While the viral factors for primary envelopment of nucleocapsids at the inner nuclear membrane are known to the point of high-resolution structures, the roles of cellular components and regulators remain enigmatic. Furthermore, the machinery responsible for fusion with the outer nuclear membrane is unsolved. We show here

Reverse-Bumpy-Ball-Type-Nanoreactor-Loaded Nylon Membranes as Peroxidase-Mimic Membrane Reactors for a Colorimetric Assay for H₂O₂.

Science.gov (United States)

Tong, Ying; Jiao, Xiangyu; Yang, Hankun; Wen, Yongqiang; Su, Lei; Zhang, Xueji

2016-04-01

Herein we report for the first time fabrication of reverse bumpy ball (RBB)-type-nanoreactor-based flexible peroxidase-mimic membrane reactors (MRs). The RBB-type nanoreactors with gold nanoparticles embedded in the inner walls of carbon shells were loaded on nylon membranes through a facile filtration approach. The as-prepared flexible catalytic membrane was studied as a peroxidase-mimic MR. It was found that the obtained peroxidase-mimic MR could exhibit several advantages over natural enzymes, such as facile and good recyclability, long-term stability and easy storage. Moreover, the RBB NS-modified nylon MRs as a peroxidase mimic provide a useful colorimetric assay for H₂O₂.

Cholesterol is required for stability and infectivity of influenza A and respiratory syncytial viruses.

Science.gov (United States)

Bajimaya, Shringkhala; Frankl, Tünde; Hayashi, Tsuyoshi; Takimoto, Toru

2017-10-01

Cholesterol-rich lipid raft microdomains in the plasma membrane are considered to play a major role in the enveloped virus lifecycle. However, the functional role of cholesterol in assembly, infectivity and stability of respiratory RNA viruses is not fully understood. We previously reported that depletion of cellular cholesterol by cholesterol-reducing agents decreased production of human parainfluenza virus type 1 (hPIV1) particles by inhibiting virus assembly. In this study, we analyzed the role of cholesterol on influenza A virus (IAV) and respiratory syncytial virus (RSV) production. Unlike hPIV1, treatment of human airway cells with the agents did not decrease virus particle production. However, the released virions were less homogeneous in density and unstable. Addition of exogenous cholesterol to the released virions restored virus stability and infectivity. Collectively, these data indicate a critical role of cholesterol in maintaining IAV and RSV membrane structure that is essential for sustaining viral stability and infectivity. Copyright © 2017 Elsevier Inc. All rights reserved.

Crosslinked anion exchange membranes prepared from poly(phenylene oxide) (PPO) for non-aqueous redox flow batteries

Science.gov (United States)

Li, Yun; Sniekers, Jeroen; Malaquias, João C.; Van Goethem, Cedric; Binnemans, Koen; Fransaer, Jan; Vankelecom, Ivo F. J.

2018-02-01

A stable and eco-friendly anion-exchange membrane (AEM) was prepared and applied in a non-aqueous all-copper redox flow battery (RFB). The AEM was prepared via a simple procedure, leading to a cross-linked structure containing quaternary ammonium groups without involvement of harmful trimethylamine. A network was thus constructed which ensured both ion transport and solvent resistance. The ion exchange capacity (IEC) of the membrane was tuned from 0.49 to 1.03 meq g-1 by varying the content of the 4, 4‧-bipyridine crosslinking agent. The membrane showed a good anion conductivity and retention of copper ions. As a proof of principle, a RFB single cell with this crosslinked membrane yielded a coulombic efficiency of 89%, a voltage efficiency of 61% and an energy efficiency of 54% at 7.5 mA cm-2.

Phosphate barrier on pore-filled cation-exchange membrane for blocking complexing ions in presence of non-complexing ions

Science.gov (United States)

Chavan, Vivek; Agarwal, Chhavi; Shinde, Rakesh N.

2018-06-01

In present work, an approach has been used to form a phosphate groups bearing surface barrier on a cation-exchange membrane (CEM). Using optimized conditions, the phosphate bearing monomer bis[2-(methacryloyloxy)ethyl] phosphate has been grafted on the surface of the host poly(ethersulfone) membranes using UV light induced polymerization. The detailed characterizations have shown that less than a micron layer of phosphate barrier is formed without disturbing the original microporous structure of the host membrane. The pores of thus formed membrane have been blocked by cationic-gel formed by in situ UV-initiator induced polymerization of 2-acrylamido-2-methyl-1-propane sulphonic acid along with crosslinker ethylene glycol dimethacrylate in the pores of the membrane. UV-initiator is required for pore-filling as UV light would not penetrate the interior matrix of the membrane. The phosphate functionalized barrier membrane has been examined for permselectivity using a mixture of representative complexing Am3+ ions and non-complexing Cs+ ions. This experiment has demonstrated that complex forming Am3+ ions are blocked by phosphate barrier layer while non-complexing Cs+ ions are allowed to pass through the channels formed by the crosslinked cationic gel.

Generation and Characterization of a Defective HIV-1 Virus as an Immunogen for a Therapeutic Vaccine

Science.gov (United States)

García-Pérez, Javier; García, Felipe; Blanco, Julia; Escribà-García, Laura; Gatell, Jose Maria; Alcamí, Jose; Plana, Montserrat; Sánchez-Palomino, Sonsoles

2012-01-01

Background The generation of new immunogens able to elicit strong specific immune responses remains a major challenge in the attempts to obtain a prophylactic or therapeutic vaccine against HIV/AIDS. We designed and constructed a defective recombinant virus based on the HIV-1 genome generating infective but non-replicative virions able to elicit broad and strong cellular immune responses in HIV-1 seropositive individuals. Results Viral particles were generated through transient transfection in producer cells (293-T) of a full length HIV-1 DNA carrying a deletion of 892 base pairs (bp) in the pol gene encompassing the sequence that codes for the reverse transcriptase (NL4-3/ΔRT clone). The viral particles generated were able to enter target cells, but due to the absence of reverse transcriptase no replication was detected. The immunogenic capacity of these particles was assessed by ELISPOT to determine γ-interferon production in a cohort of 69 chronic asymptomatic HIV-1 seropositive individuals. Surprisingly, defective particles produced from NL4-3/ΔRT triggered stronger cellular responses than wild-type HIV-1 viruses inactivated with Aldrithiol-2 (AT-2) and in a larger proportion of individuals (55% versus 23% seropositive individuals tested). Electron microscopy showed that NL4-3/ΔRT virions display immature morphology. Interestingly, wild-type viruses treated with Amprenavir (APV) to induce defective core maturation also induced stronger responses than the same viral particles generated in the absence of protease inhibitors. Conclusions We propose that immature HIV-1 virions generated from NL4-3/ΔRT viral clones may represent new prototypes of immunogens with a safer profile and stronger capacity to induce cellular immune responses than wild-type inactivated viral particles. PMID:23144996

C-type lectins do not act as functional receptors for filovirus entry into cells

Energy Technology Data Exchange (ETDEWEB)

Matsuno, Keita; Nakayama, Eri; Noyori, Osamu [Department of Global Epidemiology, Hokkaido University Research Center for Zoonosis Control, Sapporo (Japan); Marzi, Andrea; Ebihara, Hideki [Laboratory of Virology, Division of Intramural Research, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Rocky Mountain Laboratories, Hamilton, MT (United States); Irimura, Tatsuro [Graduate School of Pharmaceutical Science, University of Tokyo, Tokyo (Japan); Feldmann, Heinz [Laboratory of Virology, Division of Intramural Research, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Rocky Mountain Laboratories, Hamilton, MT (United States); Takada, Ayato, E-mail: atakada@czc.hokudai.ac.jp [Department of Global Epidemiology, Hokkaido University Research Center for Zoonosis Control, Sapporo (Japan)

2010-12-03

Research highlights: {yields} Filovirus glycoprotein (GP) having a deficient receptor binding region were generated. {yields} Mutant GPs mediated virus entry less efficiently than wild-type GP. {yields} Mutant GPs bound to C-type lectins but not mediated entire steps of cellular entry. {yields} C-type lectins do not independently mediate filovirus entry into cells. {yields} Other molecule(s) are required for C-type lectin-mediated entry of filoviruses. -- Abstract: Cellular C-type lectins have been reported to facilitate filovirus infection by binding to glycans on filovirus glycoprotein (GP). However, it is not clearly known whether interaction between C-type lectins and GP mediates all the steps of virus entry (i.e., attachment, internalization, and membrane fusion). In this study, we generated vesicular stomatitis viruses pseudotyped with mutant GPs that have impaired structures of the putative receptor binding regions and thus reduced ability to infect the monkey kidney cells that are routinely used for virus propagation. We found that infectivities of viruses with the mutant GPs dropped in C-type lectin-expressing cells, parallel with those in the monkey kidney cells, whereas binding activities of these GPs to the C-type lectins were not correlated with the reduced infectivities. These results suggest that C-type lectin-mediated entry of filoviruses requires other cellular molecule(s) that may be involved in virion internalization or membrane fusion.

C-type lectins do not act as functional receptors for filovirus entry into cells

International Nuclear Information System (INIS)

Matsuno, Keita; Nakayama, Eri; Noyori, Osamu; Marzi, Andrea; Ebihara, Hideki; Irimura, Tatsuro; Feldmann, Heinz; Takada, Ayato

2010-01-01

Research highlights: → Filovirus glycoprotein (GP) having a deficient receptor binding region were generated. → Mutant GPs mediated virus entry less efficiently than wild-type GP. → Mutant GPs bound to C-type lectins but not mediated entire steps of cellular entry. → C-type lectins do not independently mediate filovirus entry into cells. → Other molecule(s) are required for C-type lectin-mediated entry of filoviruses. -- Abstract: Cellular C-type lectins have been reported to facilitate filovirus infection by binding to glycans on filovirus glycoprotein (GP). However, it is not clearly known whether interaction between C-type lectins and GP mediates all the steps of virus entry (i.e., attachment, internalization, and membrane fusion). In this study, we generated vesicular stomatitis viruses pseudotyped with mutant GPs that have impaired structures of the putative receptor binding regions and thus reduced ability to infect the monkey kidney cells that are routinely used for virus propagation. We found that infectivities of viruses with the mutant GPs dropped in C-type lectin-expressing cells, parallel with those in the monkey kidney cells, whereas binding activities of these GPs to the C-type lectins were not correlated with the reduced infectivities. These results suggest that C-type lectin-mediated entry of filoviruses requires other cellular molecule(s) that may be involved in virion internalization or membrane fusion.

Plasma disappearance of glycated and non-glycated albumin in type 1 (insulin-dependent) diabetes mellitus

DEFF Research Database (Denmark)

Bent-Hansen, L; Feldt-Rasmussen, B; Kverneland, Arne

1993-01-01

transport ratio) was determined from the disappearance data. The index was high in control subjects (1.021 +/- 0.0057 (SEM)). This reflects a mean difference between the two escape rates of 2.1% per hour (for comparison the mean of the fractional escape rate of non-glycated albumin of the normal control......The fractional plasma escape rates of glycated and non-glycated albumin have earlier been measured in groups of Type 1 (insulin-dependent) diabetic patients and control subjects. The escape of non-glycated albumin was similar in control subjects and normoalbuminuric patients, but elevated...... in patients with micro or macroalbuminuria. In all groups the escape rate of glycated albumin was lower than that of non-glycated albumin. Glycation increases the anionic charge of albumin. To assay for charge-dependent alterations of transport a selectivity index (non-glycated albumin/glycated albumin...

Single-Molecule FISH Reveals Non-selective Packaging of Rift Valley Fever Virus Genome Segments.

Directory of Open Access Journals (Sweden)

Paul J Wichgers Schreur

2016-08-01

Full Text Available The bunyavirus genome comprises a small (S, medium (M, and large (L RNA segment of negative polarity. Although genome segmentation confers evolutionary advantages by enabling genome reassortment events with related viruses, genome segmentation also complicates genome replication and packaging. Accumulating evidence suggests that genomes of viruses with eight or more genome segments are incorporated into virions by highly selective processes. Remarkably, little is known about the genome packaging process of the tri-segmented bunyaviruses. Here, we evaluated, by single-molecule RNA fluorescence in situ hybridization (FISH, the intracellular spatio-temporal distribution and replication kinetics of the Rift Valley fever virus (RVFV genome and determined the segment composition of mature virions. The results reveal that the RVFV genome segments start to replicate near the site of infection before spreading and replicating throughout the cytoplasm followed by translocation to the virion assembly site at the Golgi network. Despite the average intracellular S, M and L genome segments approached a 1:1:1 ratio, major differences in genome segment ratios were observed among cells. We also observed a significant amount of cells lacking evidence of M-segment replication. Analysis of two-segmented replicons and four-segmented viruses subsequently confirmed the previous notion that Golgi recruitment is mediated by the Gn glycoprotein. The absence of colocalization of the different segments in the cytoplasm and the successful rescue of a tri-segmented variant with a codon shuffled M-segment suggested that inter-segment interactions are unlikely to drive the copackaging of the different segments into a single virion. The latter was confirmed by direct visualization of RNPs inside mature virions which showed that the majority of virions lack one or more genome segments. Altogether, this study suggests that RVFV genome packaging is a non-selective process.

The F-BAR domains from srGAP1, srGAP2 and srGAP3 regulate membrane deformation differently

Science.gov (United States)

Coutinho-Budd, Jaeda; Ghukasyan, Vladimir; Zylka, Mark J.; Polleux, Franck

2012-01-01

Summary Coordination of membrane deformation and cytoskeletal dynamics lies at the heart of many biological processes critical for cell polarity, motility and morphogenesis. We have recently shown that Slit-Robo GTPase-activating protein 2 (srGAP2) regulates neuronal morphogenesis through the ability of its F-BAR domain to regulate membrane deformation and induce filopodia formation. Here, we demonstrate that the F-BAR domains of two closely related family members, srGAP1 and srGAP3 [designated F-BAR(1) and F-BAR(3), respectively] display significantly different membrane deformation properties in non-neuronal COS7 cells and in cortical neurons. F-BAR(3) induces filopodia in both cell types, though less potently than F-BAR(2), whereas F-BAR(1) prevents filopodia formation in cortical neurons and reduces plasma membrane dynamics. These three F-BAR domains can heterodimerize, and they act synergistically towards filopodia induction in COS7 cells. As measured by fluorescence recovery after photobleaching, F-BAR(2) displays faster molecular dynamics than F-BAR(3) and F-BAR(1) at the plasma membrane, which correlates well with its increased potency to induce filopodia. We also show that the molecular dynamic properties of F-BAR(2) at the membrane are partially dependent on F-Actin. Interestingly, acute phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] depletion in cells does not interfere with plasma membrane localization of F-BAR(2), which is compatible with our result showing that F-BAR(2) binds to a broad range of negatively-charged phospholipids present at the plasma membrane, including phosphatidylserine (PtdSer). Overall, our results provide novel insights into the functional diversity of the membrane deformation properties of this subclass of F-BAR-domains required for cell morphogenesis. PMID:22467852

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

Energy Technology Data Exchange (ETDEWEB)

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

2012-10-10

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

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

International Nuclear Information System (INIS)

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

2012-01-01

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

Functional role of EF-hands 3 and 4 in membrane-binding of KChIP1

Indian Academy of Sciences (India)

Prakash

type KChIP1, truncated KChIP1 exhibited lower lipid-binding capability. Compared with wild-type KChIP1, increasing membrane permeability by the use of digitonin caused a marked .... with sonication, the supernatant (soluble protein fractions).

Probing the HIV-1 genomic RNA trafficking pathway and dimerization by genetic recombination and single virion analyses.

Directory of Open Access Journals (Sweden)

Michael D Moore

2009-10-01

Full Text Available Once transcribed, the nascent full-length RNA of HIV-1 must travel to the appropriate host cell sites to be translated or to find a partner RNA for copackaging to form newly generated viruses. In this report, we sought to delineate the location where HIV-1 RNA initiates dimerization and the influence of the RNA transport pathway used by the virus on downstream events essential to viral replication. Using a cell-fusion-dependent recombination assay, we demonstrate that the two RNAs destined for copackaging into the same virion select each other mostly within the cytoplasm. Moreover, by manipulating the RNA export element in the viral genome, we show that the export pathway taken is important for the ability of RNA molecules derived from two viruses to interact and be copackaged. These results further illustrate that at the point of dimerization the two main cellular export pathways are partially distinct. Lastly, by providing Gag in trans, we have demonstrated that Gag is able to package RNA from either export pathway, irrespective of the transport pathway used by the gag mRNA. These findings provide unique insights into the process of RNA export in general, and more specifically, of HIV-1 genomic RNA trafficking.

Envelope proteins of bovine herpesvirus 1: immunological and biochemical studies

International Nuclear Information System (INIS)

Rodriguez Roque, L.L.

1986-01-01

The authors studied immunological and biochemical properties of the bovid herpesvirus 1 (BHV-1) envelope proteins in order to understand the pathogenesis of BHV-1 infection and to provide basic information for the production of effective subunit vaccines against BHV-1. Ten glycoproteins MW 180, 150, 130, 115, 97, 77, 74, 64, 55, and 45 kilodaltons (K), and a single non-glycosylated 108 K protein were quantitatively removed from purified BHV-1 virions by detergent treatment. These glycoproteins were present on the virion envelope and on the surface of BHV-1 infected cells. The quantitative removal from virions by treatment with nonionic detergents and their presence on the surface of infected cells indicate that 180/97, 150/77, and 130/74/55 K are major components of the BHV-1 envelope and are also the targets of virus neutralizing humoral immune response. Envelope glycoproteins of herpes simplex type 1 (HSV-1) bind immunoglobulin by the Fc end and it is suggested this may increase pathogenicity of this virus. They searched for a similar function in BVH-1 by measuring the ability of BHV-1 infected cells and viral envelope proteins to bind radiolabelled rabbit and bovine IgG. Binding activity for rabbit IgG or bovine IgG-Fc could not be demonstrated by BHV-1 infected MDBK cells, whereas, MDBK cells infected with HSV-1 bound rabbit IgG and bovine IgG-Fc. None of the three major envelope proteins of BHV-1 bound to rabbit or bovine IgG. The results of this study indicate that BHV-1, unlike some other herpesviruses, lack Fc binding activity

N-terminal arginines modulate plasma-membrane localization of Kv7.1/KCNE1 channel complexes.

Directory of Open Access Journals (Sweden)

Zenawit Girmatsion

Full Text Available BACKGROUND AND OBJECTIVE: The slow delayed rectifier current (I(Ks is important for cardiac action potential termination. The underlying channel is composed of Kv7.1 α-subunits and KCNE1 β-subunits. While most evidence suggests a role of KCNE1 transmembrane domain and C-terminus for the interaction, the N-terminal KCNE1 polymorphism 38G is associated with reduced I(Ks and atrial fibrillation (a human arrhythmia. Structure-function relationship of the KCNE1 N-terminus for I(Ks modulation is poorly understood and was subject of this study. METHODS: We studied N-terminal KCNE1 constructs disrupting structurally important positively charged amino-acids (arginines at positions 32, 33, 36 as well as KCNE1 constructs that modify position 38 including an N-terminal truncation mutation. Experimental procedures included molecular cloning, patch-clamp recording, protein biochemistry, real-time-PCR and confocal microscopy. RESULTS: All KCNE1 constructs physically interacted with Kv7.1. I(Ks resulting from co-expression of Kv7.1 with non-atrial fibrillation '38S' was greater than with any other construct. Ionic currents resulting from co-transfection of a KCNE1 mutant with arginine substitutions ('38G-3xA' were comparable to currents evoked from cells transfected with an N-terminally truncated KCNE1-construct ('Δ1-38'. Western-blots from plasma-membrane preparations and confocal images consistently showed a greater amount of Kv7.1 protein at the plasma-membrane in cells co-transfected with the non-atrial fibrillation KCNE1-38S than with any other construct. CONCLUSIONS: The results of our study indicate that N-terminal arginines in positions 32, 33, 36 of KCNE1 are important for reconstitution of I(Ks. Furthermore, our results hint towards a role of these N-terminal amino-acids in membrane representation of the delayed rectifier channel complex.

The requirements for herpes simplex virus type 1 cell-cell spread via nectin-1 parallel those for virus entry.

Science.gov (United States)

Even, Deborah L; Henley, Allison M; Geraghty, Robert J

2006-08-01

Herpes simplex virus type 1 (HSV-1) spreads from an infected cell to an uninfected cell by virus entry, virus-induced cell fusion, and cell-cell spread. The three forms of virus spread require the viral proteins gB, gD, and gH-gL, as well as a cellular gD receptor. The mutual requirement for the fusion glycoproteins and gD receptor suggests that virus entry, cell fusion, and cell-cell spread occur by a similar mechanism. The goals of this study were to examine the role of the nectin-1alpha transmembrane domain and cytoplasmic tail in cell-cell spread and to obtain a better understanding of the receptor-dependent events occurring at the plasma membrane during cell-cell spread. We determined that an intact nectin-1alpha V-like domain was required for cell-cell spread, while a membrane-spanning domain and cytoplasmic tail were not. Chimeric forms of nectin-1 that were non-functional for virus entry did not mediate cell-cell spread regardless of whether they could mediate cell fusion. Also, cell-cell spread of syncytial isolates was dependent upon nectin-1alpha expression and occurred through a nectin-1-dependent mechanism. Taken together, our results indicate that nectin-1-dependent events occurring at the plasma membrane during cell-cell spread were equivalent to those for virus entry.

Identification of immunogenic outer membrane proteins of Haemophilus influenzae type b in the infant rat model system

International Nuclear Information System (INIS)

Hansen, E.J.; Frisch, C.F.; McDade, R.L. Jr.; Johnston, K.H.

1981-01-01

Outer membrane proteins of Haemophilus influenzae type b which are immunogenic in infant rats were identified by a radioimmunoprecipitation method. Intact cells of H. influenzae type b were radioiodinated by a lactoperoxidase-catalyzed procedure, and an outer membrane-containing fraction was prepared from these cells. These radioiodinated outer membranes were mixed with sera obtained from rats convalescing from systemic H. influenzae type b disease induced at 6 days of age, and the resultant (antibody-outer membrane protein antigen) complexes were extracted from these membranes by treatment with nonionic detergent and ethylenediaminetetraacetic acid. These soluble antibody-antigen complexes were isolated by means of adsorption to protein A-bearing staphylococci, and the radioiodinated protein antigens were identified by gel electrophoresis followed by autoradiography. Infant rats were shown to mount a readily detectable antibody response to several different proteins present in the outer membrane of H. influenzae type b. Individual infant rats were found to vary both qualitatively and quantitatively in their immune response to these immunogenic outer membrane proteins

Preparation of conductive membranes using poly pyrrole

International Nuclear Information System (INIS)

Madaeni, S.; Khavaran, B.

2003-01-01

Conductive membranes show many benefits including fouling reduction for feeds containing ionic species. These membranes may be prepared either by conductive polymers or coating of the surfaces of non-conductive membranes with conductive polymer. In this research, the commercial micro filtration GVHP membrane manufactured from PVDF was coated with poly pyrrole using two different techniques. The conductivity of the prepared membranes was measured. In this paper, effects of various factors including concentration of the solutions, oxidizing agents, time for leaving the support in the solutions, support type and temperature on membrane conductivity were investigated

A novel branched side-chain-type sulfonated polyimide membrane with flexible sulfoalkyl pendants and trifluoromethyl groups for vanadium redox flow batteries

Science.gov (United States)

Li, Jinchao; Liu, Suqin; He, Zhen; Zhou, Zhi

2017-04-01

A novel branched side-chain-type sulfonated polyimide (6F-s-bSPI) membrane with accessible branching agents of melamine, hydrophobic trifluoromethyl groups (sbnd CF3), and flexible sulfoalkyl pendants is prepared by a high-temperature polycondensation and post-sulfonation method for use in vanadium redox flow batteries (VRFBs). The chemical structure of the 6F-s-bSPI membrane is confirmed by ATR-FTIR and 1H NMR spectra. The physico-chemical properties of the as-prepared 6F-s-bSPI membrane are systematically investigated and found to be strongly related to the specially designed structure. The 6F-s-bSPI membrane offers a reduced cost and possesses a significantly lowered vanadium ion permeability (1.18 × 10-7 cm2 min-1) compared to the linear SPI (2.25 × 10-7 cm2 min-1) and commercial Nafion 115 (1.36 × 10-6 cm2 min-1) membranes, prolonging the self-discharge duration of the VRFBs. In addition, the VRFB assembled with a 6F-s-bSPI membrane shows higher coulombic (98.3%-99.7%) and energy efficiencies (88.4%-66.12%) than that with a SPI or Nafion 115 membrane under current densities ranging from 20 to 100 mA cm-2. Moreover, the VRFB with a 6F-s-bSPI membrane delivers a stable cycling performance over 100 cycles with no decline in coulombic and energy efficiencies. These results show that the branched side-chain-type structure is a promising design to prepare excellent proton conductive membranes.

CD4- and dynamin-dependent endocytosis of HIV-1 into plasmacytoid dendritic cells

Energy Technology Data Exchange (ETDEWEB)

Pritschet, Kathrin; Donhauser, Norbert; Schuster, Philipp; Ries, Moritz; Haupt, Sabrina; Kittan, Nicolai A.; Korn, Klaus [Institute of Clinical and Molecular Virology, National Reference Centre for Retroviruses, Friedrich-Alexander-Universitaet Erlangen-Nuernberg, 91054 Erlangen (Germany); Poehlmann, Stefan [Institute of Virology, Hannover Medical School, 30625 Hannover (Germany); Holland, Gudrun; Bannert, Norbert [Robert Koch-Institute, Center for Biological Security 4, 13353 Berlin (Germany); Bogner, Elke [Institute of Virology, Charite University Hospital, 10117 Berlin (Germany); Schmidt, Barbara, E-mail: baschmid@viro.med.uni-erlangen.de [Institute of Clinical and Molecular Virology, National Reference Centre for Retroviruses, Friedrich-Alexander-Universitaet Erlangen-Nuernberg, 91054 Erlangen (Germany)

2012-02-20

Chronic immune activation, triggered by plasmacytoid dendritic cell (PDC) interferon (IFN)-alpha production, plays an important role in HIV-1 pathogenesis. As the entry of HIV-1 seems to be important for the activation of PDC, we directly characterized the viral entry into these cells using immuno-electron microscopy, cellular fractionation, confocal imaging, and functional experiments. After attachment to PDC, viruses were taken up in an energy-dependent manner. The virions were located in compartments positive for caveolin; early endosomal antigen 1; Rab GTPases 5, 7 and 9; lysosomal-associated membrane protein 1. PDC harbored more virus in endocytic vesicles than CD4+ T cells (p < 0.05). Blocking CD4 inhibited the uptake of virions into cytosolic and endosomal compartments. Dynasore, an inhibitor of dynamin-dependent endocytosis, not the fusion inhibitor T-20, reduced the HIV-1 induced IFN-alpha production. Altogether, our morphological and functional data support the role of endocytosis for the entry and IFN-alpha induction of HIV-1 in PDC.

The virion N protein of infectious bronchitis virus is more phosphorylated than the N protein from infected cell lysates

International Nuclear Information System (INIS)

Jayaram, Jyothi; Youn, Soonjeon; Collisson, Ellen W.

2005-01-01

Because phosphorylation of the infectious bronchitis virus (IBV) nucleocapsid protein (N) may regulate its multiple roles in viral replication, the dynamics of N phosphorylation were examined. 32 P-orthophosphate labeling and Western blot analyses confirmed that N was the only viral protein that was phosphorylated. Pulse labeling with 32 P-orthophosphate indicated that the IBV N protein was phosphorylated in the virion, as well as at all times during infection in either chicken embryo kidney cells or Vero cells. Pulse-chase analyses followed by immunoprecipitation of IBV N proteins using rabbit anti-IBV N polyclonal antibody demonstrated that the phosphate on the N protein was stable for at least 1 h. Simultaneous labeling with 32 P-orthophosphate and 3 H-leucine identified a 3.5-fold increase in the 32 P: 3 H counts per minute (cpm) ratio of N in the virion as compared to the 32 P: 3 H cpm ratio of N in the cell lysates from chicken embryo kidney cells, whereas in Vero cells the 32 P: 3 H cpm ratio of N from the virion was 10.5-fold greater than the 32 P: 3 H cpm ratio of N from the cell lysates. These studies are consistent with the phosphorylation of the IBV N playing a role in assembly or maturation of the viral particle

In-situ Non-destructive Studies on Biofouling Processes in Reverse Osmosis Membrane Systems

KAUST Repository

Farhat, Nadia

2016-12-01

Reverse osmosis (RO) and nanofiltration (NF) membrane systems are high-pressure membrane filtration processes that can produce high quality drinking water. Biofouling, biofilm formation that exceeds a certain threshold, is a major problem in spiral wound RO and NF membrane systems resulting in a decline in membrane performance, produced water quality, and quantity. In practice, detection of biofouling is typically done indirectly through measurements of performance decline. Existing direct biofouling detection methods are mainly destructive, such as membrane autopsies, where biofilm samples can be contaminated, damaged and resulting in biofilm structural changes. The objective of this study was to test whether transparent luminescent planar oxygen sensing optodes, in combination with a simple imaging system, can be used for in-situ, non-destructive biofouling characterization. Aspects of the study were early detection of biofouling, biofilm spatial patterning in spacer filled channels, and the effect of feed cross-flow velocity, and feed flow temperature. Oxygen sensing optode imaging was found suitable for studying biofilm processes and gave detailed spatial and quantitative biofilm development information enabling better understanding of the biofouling development process. The outcome of this study attests the importance of in-situ, non-destructive imaging in acquiring detailed knowledge on biofilm development in membrane systems contributing to the development of effective biofouling control strategies.

Increased risk of severe hypoglycemic events with increasing frequency of non-severe hypoglycemic events in patients with Type 1 and Type 2 diabetes.

LENUS (Irish Health Repository)

Sreenan, Seamus

2014-07-15

Severe hypoglycemic events (SHEs) are associated with significant morbidity, mortality and costs. However, the more common non-severe hypoglycemic events (NSHEs) are less well explored. We investigated the association between reported frequency of NSHEs and SHEs among patients with type 1 diabetes mellitus (T1DM) and type 2 diabetes mellitus (T2DM) in the PREDICTIVE study.

Growth and morphogenesis of embryonic mouse organs on non-coated and extracellular matrix-coated Biopore membrane

Science.gov (United States)

Hardman, P.; Klement, B. J.; Spooner, B. S.

1993-01-01

Embryonic mouse salivary glands, pancreata, and kidneys were isolated from embryos of appropriate gestational age by microdissection, and were cultured on Biopore membrane either non-coated or coated with type I collagen or Matrigel. As expected, use of Biopore membrane allowed high quality photomicroscopy of the living organs. In all organs extensive mesenchymal spreading was observed in the presence of type I collagen or Matrigel. However, differences were noted in the effects of extracellular matrix (ECM) coatings on epithelial growth and morphogenesis: salivary glands were minimally affected, pancreas morphogenesis was adversely affected, and kidney growth and branching apparently was enhanced. It is suggested that these differences in behaviour reflect differences in the strength of interactions between the mesenchymal cells and their surrounding endogenous matrix, compared to the exogenous ECM macromolecules. This method will be useful for culture of these and other embryonic organs. In particular, culture of kidney rudiments on ECM-coated Biopore offers a great improvement over previously used methods which do not allow morphogenesis to be followed in vitro.

Role of Gag and lipids during HIV-1 assembly in CD4 T cells and Macrophages

Directory of Open Access Journals (Sweden)

Charlotte eMariani

2014-06-01

Full Text Available HIV-1 is an RNA enveloped virus that preferentiallyinfects CD4+ T lymphocytes andalso macrophages. In CD4+ T cells, HIV-1mainly buds from the host cell plasma membrane.The viral Gag polyprotein targets theplasma membrane and is the orchestrator ofthe HIV assembly as its expression is sufficientto promote the formation of virus-likeparticles particles carrying a lipidic envelopederiving from the host cell membrane. Certainlipids are enriched in the viral membraneand are thought to play a key role in theassembly process and the envelop composition.A large body of work performed oninfected CD4+ T cells has provided importantknowledge about the assembly process andthe membrane virus lipid composition. WhileHIV assembly and budding in macrophages isthought to follow the same general Gag-drivenmechanism as in T-lymphocytes, the HIV cyclein macrophage exhibits specific features.In these cells, new virions bud from the limitingmembrane of seemingly intracellular compartments,where they accumulate while remaininginfectious. These structures are now oftenreferred to as Virus Containing Compartments(VCCs. Recent studies suggest that VCCsrepresent intracellularly sequestered regionsof the plasma membrane, but their precisenature remains elusive. The proteomic andlipidomic characterization of virions producedby T cells or macrophages has highlightedthe similarity between their composition andthat of the plasma membrane of producercells, as well as their enrichment in acidiclipids, some components of raft lipids andin tetraspanin-enriched microdomains. Greatchances are that Gag promotes the coalescenceof these components into an assemblyplatform from which viral budding takesplace. How Gag exactly interacts with membranelipids and what are the mechanisms involvedin the interaction between the differentmembrane nanodomains within the assemblyplatform remains unclear. Here we review recentliterature regarding the role of Gag andlipids

Reduced incorporation of the influenza B virus BM2 protein in virus particles decreases infectivity

International Nuclear Information System (INIS)

Jackson, David; Zuercher, Thomas; Barclay, Wendy

2004-01-01

BM2 is the fourth integral membrane protein encoded by the influenza B virus genome. It is synthesized late in infection and transported to the plasma membrane from where it is subsequently incorporated into progeny virus particles. It has recently been reported that BM2 has ion channel activity and may be the functional homologue of the influenza A virus M2 protein acting as an ion channel involved in viral entry. Using a reverse genetic approach it was not possible to recover virus which lacked BM2. A recombinant influenza B virus was generated in which the BM2 AUG initiation codon was mutated to GUG. This decreased the efficiency of translation of BM2 protein such that progeny virions contained only 1/8 the amount of BM2 seen in wild-type virus. The reduction in BM2 incorporation resulted in a reduction in infectivity although there was no concomitant decrease in the numbers of virions released from the infected cells. These data imply that the incorporation of sufficient BM2 protein into influenza B virions is required for infectivity of the virus particles

Membrane compartment of Can1 (MCC): specialized functional microdomain of the yeast plasma membrane

OpenAIRE

Doudová, Lenka

2017-01-01

Membrane compartment of Can1 (MCC): specialized functional microdomain of the yeast plasma membrane Yeast plasma membrane is divided into several different compartments. Membrane compartment of Can1 is specific for its protein and lipid composition, furthermore it creates furrow-like invaginations on the plasma membrane. These invaginations are made by multiprotein complexes called eisosomes, which are located in the cytosolic side of MCCs. It was established that this domain plays an importa...

Two barcodes encoded by the type-1 PDZ and by phospho-Ser312 regulate retromer/WASH-mediated sorting of the ß1-adrenergic receptor from endosomes to the plasma membrane.

Science.gov (United States)

Nooh, Mohammed M; Bahouth, Suleiman W

2017-01-01

Recycling of the majority of agonist-internalized GPCR is dependent on a type I-PDZ "barcode" in their C-tail. The recycling of wild-type (WT) ß 1 -AR is also dependent on its default "type-1 PDZ barcode", but trafficking of the ß 1 -AR is inhibited when PKA or its substrate serine at position 312 (Ser 312 ) are inactivated. We tested the hypothesis that phospho-Ser 312 provided a second barcode for ß 1 -AR sorting from endosomes to the plasma membrane by determining the role of retromer/WASH complexes in ß 1 -AR trafficking. Recycling of WT ß 1 -AR or WT ß 2 -AR was dependent on targeting the retromer to endosomal membranes via SNX3 and rab7a, and on complexing the retromer to the WASH pentamer via the C-tail of FAM21 (FAM21 C ). These maneuvers however, did not inhibit the recycling of a phospho-Ser 312 ß 1 -AR mimic ((S312D) ß 1 -AR). Knockdown of the trans-acting PDZ protein sorting nexin27 (SNX27) inhibited the recycling of WT ß 1 -AR and WT ß 2 -AR, but had no effect on (S312D) ß 1 -AR∆PDZ or on phosphorylation of WT ß 1 -AR by PKA at Ser 312 . However, depletion of FKBP15, a FAM21 C -binding endosomal protein, selectively inhibited WT ß 1 -AR but not ß 2 -AR recycling, suggesting divergence might exist in GPCR trafficking roadmaps. These results indicate that two barcodes are involved in sorting WT ß 1 -AR out of early endosomes. The first and antecedent "barcode" was the "type-1 PDZ", followed by a second reversible "phospho-Ser 312 " verification "barcode". This organization allows tight regulation of ß 1 -AR density to signaling intensity in conditions associated with aberrant ß 1 -AR signaling such as in hypertension and heart failure. Copyright © 2016 Elsevier Inc. All rights reserved.

Envelope conformational changes induced by human immunodeficiency virus type 1 attachment inhibitors prevent CD4 binding and downstream entry events.

Science.gov (United States)

Ho, Hsu-Tso; Fan, Li; Nowicka-Sans, Beata; McAuliffe, Brian; Li, Chang-Ben; Yamanaka, Gregory; Zhou, Nannan; Fang, Hua; Dicker, Ira; Dalterio, Richard; Gong, Yi-Fei; Wang, Tao; Yin, Zhiwei; Ueda, Yasutsugu; Matiskella, John; Kadow, John; Clapham, Paul; Robinson, James; Colonno, Richard; Lin, Pin-Fang

2006-04-01

BMS-488043 is a small-molecule human immunodeficiency virus type 1 (HIV-1) CD4 attachment inhibitor with demonstrated clinical efficacy. The compound inhibits soluble CD4 (sCD4) binding to the 11 distinct HIV envelope gp120 proteins surveyed. Binding of BMS-488043 and that of sCD4 to gp120 are mutually exclusive, since increased concentrations of one can completely block the binding of the other without affecting the maximal gp120 binding capacity. Similarly, BMS-488043 inhibited virion envelope trimers from binding to sCD4-immunoglobulin G (IgG), with decreasing inhibition as the sCD4-IgG concentration increased, and BMS-488043 blocked the sCD4-induced exposure of the gp41 groove in virions. In both virion binding assays, BMS-488043 was active only when added prior to sCD4. Collectively, these results indicate that obstruction of gp120-sCD4 interactions is the primary inhibition mechanism of this compound and that compound interaction with envelope must precede CD4 binding. By three independent approaches, BMS-488043 was further shown to induce conformational changes within gp120 in both the CD4 and CCR5 binding regions. These changes likely prevent gp120-CD4 interactions and downstream entry events. However, BMS-488043 could only partially inhibit CD4 binding to an HIV variant containing a specific envelope truncation and altered gp120 conformation, despite effectively inhibiting the pseudotyped virus infection. Taken together, BMS-488043 inhibits viral entry primarily through altering the envelope conformation and preventing CD4 binding, and other downstream entry events could also be inhibited as a result of these induced conformational changes.

A lightweight low-frequency sound insulation membrane-type acoustic metamaterial

Directory of Open Access Journals (Sweden)

Kuan Lu

2016-02-01

Full Text Available A novel membrane-type acoustic metamaterial with a high sound transmission loss (STL at low frequencies (⩽500Hz was designed and the mechanisms were investigated by using negative mass density theory. This metamaterial’s structure is like a sandwich with a thin (thickness=0.25mm lightweight flexible rubber material within two layers of honeycomb cell plates. Negative mass density was demonstrated at frequencies below the first natural frequency, which results in the excellent low-frequency sound insulation. The effects of different structural parameters of the membrane on the sound-proofed performance at low frequencies were investigated by using finite element method (FEM. The numerical results show that, the STL can be modulated to higher value by changing the structural parameters, such as the membrane surface density, the unite cell film shape, and the membrane tension. The acoustic metamaterial proposed in this study could provide a potential application in the low-frequency noise insulation.

A lightweight low-frequency sound insulation membrane-type acoustic metamaterial

Science.gov (United States)

Lu, Kuan; Wu, Jiu Hui; Guan, Dong; Gao, Nansha; Jing, Li

2016-02-01

A novel membrane-type acoustic metamaterial with a high sound transmission loss (STL) at low frequencies (⩽500Hz) was designed and the mechanisms were investigated by using negative mass density theory. This metamaterial's structure is like a sandwich with a thin (thickness=0.25mm) lightweight flexible rubber material within two layers of honeycomb cell plates. Negative mass density was demonstrated at frequencies below the first natural frequency, which results in the excellent low-frequency sound insulation. The effects of different structural parameters of the membrane on the sound-proofed performance at low frequencies were investigated by using finite element method (FEM). The numerical results show that, the STL can be modulated to higher value by changing the structural parameters, such as the membrane surface density, the unite cell film shape, and the membrane tension. The acoustic metamaterial proposed in this study could provide a potential application in the low-frequency noise insulation.

Genome and infection characteristics of human parechovirus type 1: the interplay between viral infection and type I interferon antiviral system.

Directory of Open Access Journals (Sweden)

Jenn-Tzong Chang

Full Text Available Human parechoviruses (HPeVs, members of the family Picornaviridae, are associated with severe human clinical conditions such as gastrointestinal disease, encephalitis, meningitis, respiratory disease and neonatal sepsis. A new contemporary strain of HPeV1, KVP6 (accession no. KC769584, was isolated from a clinical specimen. Full-genome alignment revealed that HPeV1 KVP6 shares high genome homology with the German strain of HPeV1, 7555312 (accession no. FM178558 and could be classified in the clade 1B group. An intertypic recombination was shown within the P2-P3 genome regions of HPeV1. Cell-type tropism test showed that T84 cells (colon carcinoma cells, A549 cells (lung carcinoma cells and DBTRG-5MG cells (glioblastoma cells were susceptible to HPeV1 infection, which might be relevant clinically. A facilitated cytopathic effect and increased viral titers were reached after serial viral passages in Vero cells, with viral genome mutation found in later passages. HPeV1 is sensitive to elevated temperature because 39C incubation impaired virion production. HPeV1 induced innate immunity with phosphorylation of interferon (IFN regulatory transcription factor 3 and production of type I IFN in A549 but not T84 cells. Furthermore, type I IFN inhibited HPeV1 production in A549 cells but not T84 cells; T84 cells may be less responsive to type I IFN stimulation. Moreover, HPeV1-infected cells showed downregulated type I IFN activation, which indicated a type I IFN evasion mechanism. The characterization of the complete genome and infection features of HPeV1 provide comprehensive information about this newly isolated HPeV1 for further diagnosis, prevention or treatment strategies.

Deletions in the fifth alpha helix of HIV-1 matrix block virus release

International Nuclear Information System (INIS)

Sanford, Bridget; Li, Yan; Maly, Connor J.; Madson, Christian J.; Chen, Han; Zhou, You; Belshan, Michael

2014-01-01

The matrix (MA) protein of HIV-1 is the N-terminal component of the Gag structural protein and is critical for the early and late stages of viral replication. MA contains five α-helices (α1–α5). Deletions in the N-terminus of α5 as small as three amino acids impaired virus release. Electron microscopy of one deletion mutant (MA∆96-120) showed that its particles were tethered to the surface of cells by membranous stalks. Immunoblots indicated all mutants were processed completely, but mutants with large deletions had alternative processing intermediates. Consistent with the EM data, MA∆96-120 retained membrane association and multimerization capability. Co-expression of this mutant inhibited wild type particle release. Alanine scanning mutation in this region did not affect virus release, although the progeny virions were poorly infectious. Combined, these data demonstrate that structural ablation of the α5 of MA inhibits virus release. - Highlights: • Deletions were identified in the C-terminus of matrix that block virus release. • These deletion mutants still multimerized and associated with membranes. • TEM showed the mutant particles were tethered to the cell surface. • Amino acid mutagenesis of the region did not affect release. • The data suggests that disruption of matrix structure blocks virus release

Deletions in the fifth alpha helix of HIV-1 matrix block virus release

Energy Technology Data Exchange (ETDEWEB)

Sanford, Bridget; Li, Yan; Maly, Connor J.; Madson, Christian J. [Department of Medical Microbiology and Immunology, Creighton University, 2500 California Plaza, Omaha, NE 68178 (United States); Chen, Han [Center for Biotechnology, University of Nebraska-Lincoln, Lincoln, NE (United States); Zhou, You [Center for Biotechnology, University of Nebraska-Lincoln, Lincoln, NE (United States); Nebraska Center for Virology, Lincoln, NE (United States); Belshan, Michael, E-mail: michaelbelshan@creighton.edu [Department of Medical Microbiology and Immunology, Creighton University, 2500 California Plaza, Omaha, NE 68178 (United States); Nebraska Center for Virology, Lincoln, NE (United States)

2014-11-15

The matrix (MA) protein of HIV-1 is the N-terminal component of the Gag structural protein and is critical for the early and late stages of viral replication. MA contains five α-helices (α1–α5). Deletions in the N-terminus of α5 as small as three amino acids impaired virus release. Electron microscopy of one deletion mutant (MA∆96-120) showed that its particles were tethered to the surface of cells by membranous stalks. Immunoblots indicated all mutants were processed completely, but mutants with large deletions had alternative processing intermediates. Consistent with the EM data, MA∆96-120 retained membrane association and multimerization capability. Co-expression of this mutant inhibited wild type particle release. Alanine scanning mutation in this region did not affect virus release, although the progeny virions were poorly infectious. Combined, these data demonstrate that structural ablation of the α5 of MA inhibits virus release. - Highlights: • Deletions were identified in the C-terminus of matrix that block virus release. • These deletion mutants still multimerized and associated with membranes. • TEM showed the mutant particles were tethered to the cell surface. • Amino acid mutagenesis of the region did not affect release. • The data suggests that disruption of matrix structure blocks virus release.

Single-particle tracking: applications to membrane dynamics.

Science.gov (United States)

Saxton, M J; Jacobson, K

1997-01-01

Measurements of trajectories of individual proteins or lipids in the plasma membrane of cells show a variety of types of motion. Brownian motion is observed, but many of the particles undergo non-Brownian motion, including directed motion, confined motion, and anomalous diffusion. The variety of motion leads to significant effects on the kinetics of reactions among membrane-bound species and requires a revision of existing views of membrane structure and dynamics.

Retroviral gene transfer of an antisense construct against membrane type 1 matrix metalloproteinase reduces the invasiveness of rheumatoid arthritis synovial fibroblasts.

Science.gov (United States)

Rutkauskaite, Edita; Volkmer, Dagmar; Shigeyama, Yukio; Schedel, Jörg; Pap, Geza; Müller-Ladner, Ulf; Meinecke, Ingmar; Alexander, Dorothea; Gay, Renate E; Drynda, Susanne; Neumann, Wolfram; Michel, Beat A; Aicher, Wilhelm K; Gay, Steffen; Pap, Thomas

2005-07-01

Membrane type 1 matrix metalloproteinase (MT1-MMP) is expressed prominently in rheumatoid arthritis synovial fibroblasts (RASFs), but the specific contribution of MT1-MMP to fibroblast-mediated destruction of articular cartilage is incompletely understood. This study used gene transfer of an antisense expression construct to assess the effects of MT1-MMP inhibition on the invasiveness of RASFs. Retroviral gene transfer of a pLXIN vector-based antisense RNA expression construct (MT1-MMPalphaS) to MT1-MMP was used to stably transduce RASFs. Levels of MT1-MMP RNA and protein were determined by quantitative polymerase chain reaction, Western blotting, and immunocytochemistry in MT1-MMPalphaS-transduced RASFs as well as in control cells, with monitoring for 60 days. The effects of MT1-MMPalphaS on the invasiveness of RASFs were analyzed in the SCID mouse co-implantation model of RA. MT1-MMPalphaS-transduced RASFs produced high levels of antisense RNA that exceeded endogenous levels of MT1-MMP messenger RNA by 15-fold and resulted in a down-regulation of MT1-MMP at the protein level. Inhibition of MT1-MMP production was maintained for 60 days and significantly reduced the invasiveness of RASFs in the SCID mouse model. Whereas prominent invasion into cartilage by non-transduced and mock-transduced RASFs was observed (mean invasion scores 3.0 and 3.1, respectively), MT1-MMPalphaS-transduced cells showed only moderate invasiveness (mean invasion score 1.8; P < 0.05). The data demonstrate that an antisense RNA expression construct against MT1-MMP can be generated and expressed in RASFs for at least 60 days. Inhibition of MT1-MMP significantly reduces the cartilage degradation by RASFs.

Parainfluenza virus type 5 (PIV-5) morphology revealed by cryo-electron microscopy.

Science.gov (United States)

Terrier, Olivier; Rolland, Jean-Paul; Rosa-Calatrava, Manuel; Lina, Bruno; Thomas, Daniel; Moules, Vincent

2009-06-01

The knowledge of parainfluenza type 5 (PIV-5) virion morphology is essentially based on the observation of negatively stained preparations in conventional transmission electron microscopy (CTEM). In this study, the ultrastructure of frozen-hydrated intact PIV-5 was examined by cryo-electron microscopy (cryo-EM). Cryo-EM revealed a majority of spherical virions (70%), with a lower pleiomorphy than originally observed in CTEM. Phospholipid bilayer thickness, spike length and glycoprotein spikes density were measured. About 2000 glycoprotein spikes were present in an average-sized spherical virion. Altogether, these data depict a more precise view of PIV-5 morphology.

Neutrophil glycoprotein Mo1 is an integral membrane protein of plasma membranes and specific granules

International Nuclear Information System (INIS)

Stevenson, K.B.; Nauseef, W.M.; Clark, R.A.

1987-01-01

The glucoprotein Mo1 has previously been demonstrated to be on the cell surface and in the specific granule fraction of neutrophils and to be translocated to the cell surface during degranulation. It is not known, however, whether Mo1 is an integral membrane protein or a soluble, intragranular constituent loosely associated with the specific granule membrane. Purified neutrophils were disrupted by nitrogen cavitation and separated on Percoll density gradients into four fractions enriched for azurophilic granules, specific granules, plasma membrane, and cytosol, respectively. The glycoproteins in these fractions were labeled with 3 H-borohydride reduction, extracted with Triton X-114, and immunoprecipitated with 60.3, an anti-Mo1 monoclonal antibody. Mo1 was detected only in the specific granule and plasma membrane fractions and partitioned exclusively into the detergent-rich fraction consistent with Mo1 being an integral membrane protein. In addition, treatment of specific granule membranes with a high salt, high urea buffer to remove adsorbed or peripheral proteins failed to dissociate Mo1. These data support the hypothesis that Mo1 is an integral membrane protein of plasma and specific granule membranes in human neutrophils

Modification of the poly(ethylene) terephthalate track membrane structure and surface in the plasma of non-polymerized gases

International Nuclear Information System (INIS)

Kravets, L.I.; Dmitriev, S.N.; Apel, P.Y.

1999-01-01

An investigation of the properties of poly(ethylene) terephthalate track membranes (PETTMs) treated with a plasma RF-discharge in non-polymerized gases has been performed. The influence of the plasma treatment conditions on the basic properties of the membranes has been studied. It was arranged that the effect of non-polymerized gases plasma on the PETTMs results to etching a membrane's surface layer. The membranes' pore size and the form in this case change. It is shown that it is possible to change the structure of track membranes directly by gas discharge etching

The dimer interface of the membrane type 1 matrix metalloproteinase hemopexin domain: crystal structure and biological functions.

Science.gov (United States)

Tochowicz, Anna; Goettig, Peter; Evans, Richard; Visse, Robert; Shitomi, Yasuyuki; Palmisano, Ralf; Ito, Noriko; Richter, Klaus; Maskos, Klaus; Franke, Daniel; Svergun, Dmitri; Nagase, Hideaki; Bode, Wolfram; Itoh, Yoshifumi

2011-03-04

Homodimerization is an essential step for membrane type 1 matrix metalloproteinase (MT1-MMP) to activate proMMP-2 and to degrade collagen on the cell surface. To uncover the molecular basis of the hemopexin (Hpx) domain-driven dimerization of MT1-MMP, a crystal structure of the Hpx domain was solved at 1.7 Å resolution. Two interactions were identified as potential biological dimer interfaces in the crystal structure, and mutagenesis studies revealed that the biological dimer possesses a symmetrical interaction where blades II and III of molecule A interact with blades III and II of molecule B. The mutations of amino acids involved in the interaction weakened the dimer interaction of Hpx domains in solution, and incorporation of these mutations into the full-length enzyme significantly inhibited dimer-dependent functions on the cell surface, including proMMP-2 activation, collagen degradation, and invasion into the three-dimensional collagen matrix, whereas dimer-independent functions, including gelatin film degradation and two-dimensional cell migration, were not affected. These results shed light on the structural basis of MT1-MMP dimerization that is crucial to promote cellular invasion.

Diversity of phage infection types and associated terminology: the problem with 'Lytic or lysogenic'.

Science.gov (United States)

Hobbs, Zack; Abedon, Stephen T

2016-04-01

Bacteriophages, or phages, are viruses of members of domain Bacteria. These viruses play numerous roles in shaping the diversity of microbial communities, with impact differing depending on what infection strategies specific phages employ. From an applied perspective, these especially are communities containing undesired or pathogenic bacteria that can be modified through phage-mediated bacterial biocontrol, that is, through phage therapy. Here we seek to categorize phages in terms of their infection strategies as well as review or suggest more descriptive, accurate or distinguishing terminology. Categories can be differentiated in terms of (1) whether or not virion release occurs (productive infections versus lysogeny, pseudolysogeny and/or the phage carrier state), (2) the means of virion release (lytic versus chronic release) and (3) the degree to which phages are genetically equipped to display lysogenic cycles (temperate versus non-temperate phages). We address in particular the use or overuse of what can be a somewhat equivocal phrase, 'Lytic or lysogenic', especially when employed as a means of distinguishing among phages types. We suggest that the implied dichotomy is inconsistent with both modern as well as historical understanding of phage biology. We consider, therefore, less ambiguous terminology for distinguishing between 'Lytic' versus 'Lysogenic' phage types. © FEMS 2016. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Maize plasma membrane aquaporin ZmPIP2;5, but not ZmPIP1;2, facilitates transmembrane diffusion of hydrogen peroxide.

Science.gov (United States)

Bienert, Gerd P; Heinen, Robert B; Berny, Marie C; Chaumont, François

2014-01-01

Plant aquaporins play important roles in transmembrane water transport processes, but some also facilitate the diffusion of other small uncharged solutes ranging from gases to metalloids. Recent evidence suggests that the transmembrane movement of hydrogen peroxide, an intra- and intercellular multifunctional signaling and defense compound, can be regulated by aquaporins. We addressed the question whether maize aquaporins belonging to the plasma membrane intrinsic protein (PIP) subfamily facilitate hydrogen peroxide diffusion using heterologous expression in the yeast Saccharomyces cerevisiae. We showed that ZmPIP proteins belonging to the PIP1 and PIP2 groups were significantly expressed in yeast cells only after codon optimization of their cDNA. In accordance with previous localization studies in oocytes and plants, ZmPIP1;2 was mainly retained in intracellular membranes, while ZmPIP2;5 was localized to the plasma membrane. However, upon co-expression with ZmPIP2;5, ZmPIP1;2 was re-localized to the plasma membrane. Using a non-functional plasma membrane-localized ZmPIP2;5 mutant to deliver ZmPIP1;2 to the plasma membrane, we demonstrated that, in contrast to wild type ZmPIP2;5, ZmPIP1;2 was not permeable to hydrogen peroxide. Our study further highlighted the fact that, when using the yeast system, which is widely employed to study substrates for plant aquaporins and other transporters, although positive transport assay results allow direct conclusions to be drawn regarding solute permeability, negative results require additional control experiments to show that the protein is expressed and localized correctly before concluding on the lack of transport activity. © 2013.

Analysis of nucleotide sequence variations in herpes simplex virus types 1 and 2, and varicella-zoster virus

International Nuclear Information System (INIS)

Chiba, A.; Suzutani, T.; Koyano, S.; Azuma, M.; Saijo, M.

1998-01-01

To analyze the difference in the degree of divergence between genes from identical herpes virus species, we examined the nucleotide sequence of genes from the herpes simplex virus type 1 (HSV-l ) strains VR-3 and 17 encoding thymidine kinase (TK), deoxyribonuclease (DNase), protein kinase (PK; UL13) and virion-associated host shut off (vhs) protein (UL41). The frequency of nucleotide substitutions per 1 kb in TK gene was 2.5 to 4.3 times higher than those in the other three genes. To prove that the polymorphism of HSV-1 TK gene is common characteristic of herpes virus TK genes, we compared the diversity of TK genes among eight HSV-l , six herpes simplex virus type 2 (HSV-2) and seven varicella-zoster virus (VZV) strains. The average frequency of nucleotide substitutions per 1 kb in the TK gene of HSV-l strains was 4-fold higher than that in the TK gene of HSV-2 strains. The VZV TK gene was highly conserved and only two nucleotide changes were evident in VZV strains. However, the rate of non-synonymous substitutions in total nucleotide substitutions was similar among the TK genes of the three viruses. This result indicated that the mutational rates differed, but there were no significant differences in selective pressure. We conclude that HSV-l TK gene is highly diverged and analysis of variations in the gene is a useful approach for understanding the molecular evolution of HSV-l in a short period. (authors)

Prediction of power consumption and performance in ultrafiltration of simulated latex effluent using non-uniform pore sized membranes

Energy Technology Data Exchange (ETDEWEB)

Abdelrasoul, Amira; Doan, Huu; Lohi, Ali; Cheng, Chil-Hung [Ryerson University, 350 Victoria Street, Toronto (Canada)

2016-03-15

Tha aim of the present study was to develop a series of numerical models for an accurate prediction of the power consumption in ultrafiltration of simulated latex effluent. The developed power consumption model incorporated fouling attachment, as well as chemical and physical factors in membrane fouling, in order to ensure accurate prediction and scale-up. This model was applied to heterogeneous membranes with non-uniform pore sizes at a given operating conditions and membrane surface charges. Polysulfone flat membrane, with a membrane molecular weight cutoff (MWCO) of 60,000 dalton, at different surface charges was used under a constant flow rate and cross-flow mode. In addition, the developed models were examined using various membranes at a variety of surface charges so as to test the overall reliability and accuracy of these models. The power consumption predicted by the models corresponded to the calculated values from the experimental data for various hydrophilic and hydrophobic membranes with an error margin of 6.0% up to 19.1%.

Tumor cell invasion of collagen matrices requires coordinate lipid agonist-induced G-protein and membrane-type matrix metalloproteinase-1-dependent signaling

Directory of Open Access Journals (Sweden)

Anthis Nicholas J

2006-12-01

Full Text Available Abstract Background Lysophosphatidic acid (LPA and sphingosine 1-phosphate (S1P are bioactive lipid signaling molecules implicated in tumor dissemination. Membrane-type matrix metalloproteinase 1 (MT1-MMP is a membrane-tethered collagenase thought to be involved in tumor invasion via extracellular matrix degradation. In this study, we investigated the molecular requirements for LPA- and S1P-regulated tumor cell migration in two dimensions (2D and invasion of three-dimensional (3D collagen matrices and, in particular, evaluated the role of MT1-MMP in this process. Results LPA stimulated while S1P inhibited migration of most tumor lines in Boyden chamber assays. Conversely, HT1080 fibrosarcoma cells migrated in response to both lipids. HT1080 cells also markedly invaded 3D collagen matrices (~700 μm over 48 hours in response to either lipid. siRNA targeting of LPA1 and Rac1, or S1P1, Rac1, and Cdc42 specifically inhibited LPA- or S1P-induced HT1080 invasion, respectively. Analysis of LPA-induced HT1080 motility on 2D substrates vs. 3D matrices revealed that synthetic MMP inhibitors markedly reduced the distance (~125 μm vs. ~45 μm and velocity of invasion (~0.09 μm/min vs. ~0.03 μm/min only when cells navigated 3D matrices signifying a role for MMPs exclusively in invasion. Additionally, tissue inhibitors of metalloproteinases (TIMPs-2, -3, and -4, but not TIMP-1, blocked lipid agonist-induced invasion indicating a role for membrane-type (MT-MMPs. Furthermore, MT1-MMP expression in several tumor lines directly correlated with LPA-induced invasion. HEK293s, which neither express MT1-MMP nor invade in the presence of LPA, were transfected with MT1-MMP cDNA, and subsequently invaded in response to LPA. When HT1080 cells were seeded on top of or within collagen matrices, siRNA targeting of MT1-MMP, but not other MMPs, inhibited lipid agonist-induced invasion establishing a requisite role for MT1-MMP in this process. Conclusion LPA is a

Assaying the proton transport and regulation of UCP1 using solid supported membranes.

Science.gov (United States)

Blesneac, Iulia; Ravaud, Stéphanie; Machillot, Paul; Zoonens, Manuela; Masscheylen, Sandrine; Miroux, Bruno; Vivaudou, Michel; Pebay-Peyroula, Eva

2012-08-01

The uncoupling protein 1 (UCP1) is a mitochondrial protein that carries protons across the inner mitochondrial membrane. It has an important role in non-shivering thermogenesis, and recent evidence suggests its role in human adult metabolism. Using rapid solution exchange on solid supported membranes, we succeeded in measuring electrical currents generated by the transport activity of UCP1. The protein was purified from mouse brown adipose tissue, reconstituted in liposomes and absorbed on solid supported membranes. A fast pH jump activated the ion transport, and electrical signals could be recorded. The currents were characterized by a fast rise and a slow decay, were stable over time, inhibited by purine nucleotides and activated by fatty acids. This new assay permits direct observation of UCP1 activity in controlled cell-free conditions, and opens up new possibilities for UCP1 functional characterization and drug screening because of its robustness and its potential for automation.

Quantitation of human immunodeficiency virus type 1 in breast milk.

Science.gov (United States)

Ghosh, M K; Kuhn, L; West, J; Semrau, K; Decker, D; Thea, D M; Aldrovandi, G M

2003-06-01

The distribution and stability of human immunodeficiency virus type 1 (HIV-1) in breast milk (BM) components remain largely unknown. Inhibitory effects, if any, of BM on HIV RNA and DNA PCR amplification are poorly understood. We have addressed these issues by using virus-spiked BM samples from HIV-negative women. BM samples from HIV-negative women were spiked with HIV-1 virions or cells containing a single integrated copy of HIV DNA (8E5/LAV). After incubation under different experimental conditions, viral RNA was detected by the Roche Amplicor UltraSensitive assay in whole-milk, skim milk, and lipid fractions. We found excellent correlation between HIV-1 input copy and recovery in whole milk (r = 0.965, P milk (r = 0.972, P 0.982). The effects of incubation duration and temperature and repeated freeze-thaw cycles on HIV RNA recovery were analyzed. HIV RNA levels were remarkably stable in whole milk after three freeze-thaw cycles and for up to 30 h at room temperature. Our findings improve the understanding of the dynamics of HIV detection in BM and the conditions for BM sample collection, storage, and processing.

Low-frequency and multiple-bands sound insulation using hollow boxes with membrane-type faces

Science.gov (United States)

Yu, Wei-wei; Fan, Li; Ma, Ren-hao; Zhang, Hui; Zhang, Shu-yi

2018-04-01

Hollow boxes with their faces made up of elastic membranes are used to block acoustic waves. It is demonstrated that placing a cuboid membrane-type box inside a pipe can effectively insulate acoustic waves even if the box is smaller than the cross-section of the pipe. The sound insulation is achieved within multiple frequency-bands below 500 Hz based on different mechanisms, which originate from the coaction of the cavity, membrane-type faces, and the intervals between the box and pipe walls. Furthermore, by adjusting the structural parameters and establishing an array of boxes, we can achieve better sound insulation at more frequency-bands.

Capillarity-induced disassembly of virions in carbon nanotubes

Energy Technology Data Exchange (ETDEWEB)

Fan Xiaobin; Peng Wenchao; Li Yang; Li Xianyu; Zhang Guoliang; Zhang Fengbao [School of Chemical Engineering and Technology, Tianjin University, Tianjin (China); Barclay, J Elaine; Evans, David J [Department of Biological Chemistry, John Innes Centre, Norwich Research Park, Norwich NR4 7UH (United Kingdom)], E-mail: fbzhang@tju.edu.cn

2008-04-23

Studying the transport and fate of viruses through nanochannels is of great importance. By using the nanochannel of a carbon nanotube (CNT) as an ideal model, we evaluated the possibility of capillarity-induced viral transport through a closely fitting nanochannel and explored the mechanisms involved. It is shown both experimentally and theoretically that Cowpea mosaic virus can enter CNTs by capillarity. However, when introduced into a nanotube the protein capsid may disassemble. During the initial capillary filling stage, anomalous needle-shaped high pressure exists in the centre of the nanotube's entrance. This high pressure, combining with the significant negative pressure within the nanotube, may account for the disassembly of the virions.

Genetic heterogeneity in type 1 Gaucher disease: Multiple genotypes in Ashkenazic and non-Ashkenazic individuals

International Nuclear Information System (INIS)

Tsuji, Shoji; Martin, B.M.; Stubblefield, B.K.; LaMarca, M.E.; Ginns, E.I.; Barranger, J.A.

1988-01-01

Nucleotide sequence analysis of a genomic clone from an Ashkenazic Jewish patient with type 1 Gaucher disease revealed a single-base mutation (adenosine to guanosine transition) in exon 9 of the glucocerebrosidase gene. This change results in the amino acid substitution of serine for asparagine. Transient expression studies following oligonucleotide-directed mutagenesis of the normal cDNA confirmed that the mutation results in loss of glucocerebrosidase activity. Allele-specific hybridization with oligonucleotide probes demonstrated that this mutation was found exclusively in type 1 phenotype. None of the 6 type 2 patients, 11 type 3 patients, or 12 normal controls had this allele. In contrast, 15 of 24 type 1 patients had one allele with this mutation, and 3 others were homozygous for the mutation. Furthermore, some of the Ashkenazic Jewish type 1 patients had only one allele with this mutation, suggesting that even in this population there is allelic heterozygosity. These findings indicate that there are multiple allelic mutations responsible for type 1 Gaucher disease in both the Jewish and non-Jewish populations. Allelic-specific hybridization demonstrating this mutation in exon 9, used in conjunction with the Nci I restriction fragment length polymorphism described as a marker for neuronopathic Gaucher disease, provides a tool for diagnosis and genetic counseling that is ∼80% informative in all Gaucher patients studied

Genetic heterogeneity in type 1 Gaucher disease: Multiple genotypes in Ashkenazic and non-Ashkenazic individuals

Energy Technology Data Exchange (ETDEWEB)

Tsuji, Shoji; Martin, B.M.; Stubblefield, B.K.; LaMarca, M.E.; Ginns, E.I. (National Institute of Mental Health, Bethesda, MD (USA)); Barranger, J.A. (Childrens Hospital of Los Angeles, CA (USA))

1988-04-01

Nucleotide sequence analysis of a genomic clone from an Ashkenazic Jewish patient with type 1 Gaucher disease revealed a single-base mutation (adenosine to guanosine transition) in exon 9 of the glucocerebrosidase gene. This change results in the amino acid substitution of serine for asparagine. Transient expression studies following oligonucleotide-directed mutagenesis of the normal cDNA confirmed that the mutation results in loss of glucocerebrosidase activity. Allele-specific hybridization with oligonucleotide probes demonstrated that this mutation was found exclusively in type 1 phenotype. None of the 6 type 2 patients, 11 type 3 patients, or 12 normal controls had this allele. In contrast, 15 of 24 type 1 patients had one allele with this mutation, and 3 others were homozygous for the mutation. Furthermore, some of the Ashkenazic Jewish type 1 patients had only one allele with this mutation, suggesting that even in this population there is allelic heterozygosity. These findings indicate that there are multiple allelic mutations responsible for type 1 Gaucher disease in both the Jewish and non-Jewish populations. Allelic-specific hybridization demonstrating this mutation in exon 9, used in conjunction with the Nci I restriction fragment length polymorphism described as a marker for neuronopathic Gaucher disease, provides a tool for diagnosis and genetic counseling that is {approx}80% informative in all Gaucher patients studied.

Separation of FFA from Partially Hydrogenated Soybean Oil Hydrolysate by Means of Membrane Processing

DEFF Research Database (Denmark)

Jala, Ram Chandra Reddy; Guo, Zheng; Xu, Xuebing

2011-01-01

Different types of commercial porous and non-porous polymeric membranes have been investigated for their capabilities to separate free fatty acids (FFA) from hydrolysate of partially hydrogenated soybean oil. A regenerated cellulose (RC, PLAC) membrane exhibited the most prominent difference...... in rejection between FFA and glycerides and the highest flux (27 kg h−1 m−2) in hydrolysate ethanol solution. The results also showed that, besides the pore size of membrane, the membrane flux depended largely on the property matching between membrane and solvent, as observed (40 kg h−1 m−2) flux was achieved...... with methanol but no flux detected with hexane for PLAC. The polyvinyl alcohol (PVA, NTR-729 HF) and Polyamide (PA, NTR-759HR) membranes gave the second and third highest flux (10.1 and 5.7 kg h−1 m−2, respectively), where solute rejections for NTR-759HR were 95.9% for triacylglycerols (TG), 83...

High levels of γ-H2AX foci and cell membrane oxidation in adolescents with type 1 diabetes

Energy Technology Data Exchange (ETDEWEB)

Giovannini, Caterina [Unità di Genetica, Dipartimento di Biologia, Pisa University, Pisa (Italy); Piaggi, Simona [Sezione di Patologia Sperimentale, Dipartimento di Ricerca Traslazionale e delle Nuove Tecnologie in Medicina e Chirurgia, Pisa University, Pisa (Italy); Federico, Giovanni [Unità di Endocrinologia Pediatrica e Diabete, Dipartimento di Medicina Clinica e Sperimentale Pisa University, Pisa (Italy); Scarpato, Roberto, E-mail: roberto.scarpato@unipi.it [Unità di Genetica, Dipartimento di Biologia, Pisa University, Pisa (Italy)

2014-12-15

Highlights: • We aimed to detect signs of very early damage in peripheral cells of T1DM adolescents. • T1DM patients had high levels of oxidized cells and reduced expression of iNOS and NO. • Highly mutagenic lesions were markedly increased in the diabetic group, mainly in females. • The observed damage might increase the risk of cancer in the patients later in life. - Abstract: Oxidative stress caused by an excess of free radicals is implicated in the pathogenesis and development of type 1 diabetes mellitus (T1DM) and, in turn, it can lead to genome damage, especially in the form of DNA double-strand break (DSB). The DNA DSB is a potentially carcinogenic lesion for human cells. Thus, we aimed to evaluate whether the level of oxidative stress was increased in peripheral blood lymphocytes of a group of affected adolescents. In 35 T1DM adolescents and 19 healthy controls we assessed: (1) spontaneous and H{sub 2}O{sub 2}-induced oxidation of cell membrane using a fluorescence lipid probe; (2) spontaneous and LPS-induced expression of iNOS protein and indirect NO determination via cytofluorimetric analysis of O{sub 2}{sup −}; (3) immunofluorescent detection of the basal level of histone H2AX phosphorylation (γ-H2AX foci), a well-validated marker of DNA DSB. In T1DM, the frequencies of oxidized cells, both spontaneous and H{sub 2}O{sub 2}-induced (47.13 ± 0.02) were significantly higher than in controls (35.90 ± 0.03). Patients showed, in general, both a reduced iNOS expression and production of NO. Furthermore, the level of spontaneous nuclear damage, quantified as γ-H2AX foci, was markedly increased in T1DM adolescents (6.15 ± 1.08% of γ-H2AX{sup +} cells; 8.72 ± 2.14 γ-H2AXF/n; 9.26 ± 2.37 γ-H2AXF/np), especially in females. In the present study, we confirmed the role that oxidative stress plays in the disease damaging lipids of cell membrane and, most importantly, causing genomic damage in circulating white blood cells of affected adolescents

High levels of γ-H2AX foci and cell membrane oxidation in adolescents with type 1 diabetes

International Nuclear Information System (INIS)

Giovannini, Caterina; Piaggi, Simona; Federico, Giovanni; Scarpato, Roberto

2014-01-01

Highlights: • We aimed to detect signs of very early damage in peripheral cells of T1DM adolescents. • T1DM patients had high levels of oxidized cells and reduced expression of iNOS and NO. • Highly mutagenic lesions were markedly increased in the diabetic group, mainly in females. • The observed damage might increase the risk of cancer in the patients later in life. - Abstract: Oxidative stress caused by an excess of free radicals is implicated in the pathogenesis and development of type 1 diabetes mellitus (T1DM) and, in turn, it can lead to genome damage, especially in the form of DNA double-strand break (DSB). The DNA DSB is a potentially carcinogenic lesion for human cells. Thus, we aimed to evaluate whether the level of oxidative stress was increased in peripheral blood lymphocytes of a group of affected adolescents. In 35 T1DM adolescents and 19 healthy controls we assessed: (1) spontaneous and H 2 O 2 -induced oxidation of cell membrane using a fluorescence lipid probe; (2) spontaneous and LPS-induced expression of iNOS protein and indirect NO determination via cytofluorimetric analysis of O 2 − ; (3) immunofluorescent detection of the basal level of histone H2AX phosphorylation (γ-H2AX foci), a well-validated marker of DNA DSB. In T1DM, the frequencies of oxidized cells, both spontaneous and H 2 O 2 -induced (47.13 ± 0.02) were significantly higher than in controls (35.90 ± 0.03). Patients showed, in general, both a reduced iNOS expression and production of NO. Furthermore, the level of spontaneous nuclear damage, quantified as γ-H2AX foci, was markedly increased in T1DM adolescents (6.15 ± 1.08% of γ-H2AX + cells; 8.72 ± 2.14 γ-H2AXF/n; 9.26 ± 2.37 γ-H2AXF/np), especially in females. In the present study, we confirmed the role that oxidative stress plays in the disease damaging lipids of cell membrane and, most importantly, causing genomic damage in circulating white blood cells of affected adolescents. This also indicates that

Non-Hookean Mechanics of Crystalline Membranes

Science.gov (United States)

Nicholl, Ryan J. T.

The goal of the thesis is to explore the effect of crumpling on the mechanics of graphene--the ultimate thin membrane. The effect due to crumpling on the mechanical response of 2D materials is almost universally ignored in prior experiments. This is because the most widely used measurement schemes require high and non-uniform applied stress that suppresses crumpling. Experiments that do probe the interplay between crumpling and graphene mechanics remain highly challenging. To measure the mechanical effects of crumpling we need to develop a new measurement scheme which can apply low and uniform stress, allow non-invasive topography measurements, and be applicable at cryogenic temperatures. The motivating questions of this thesis are the following: • How does out-of plane crumpling affect the mechanical constants of 2D materials? • How do we implement measurement techniques sensitive to crumpling? • Can we identify sources of crumpling and distinguish between static and dynamic crumpling? • Can we tune the mechanical properties of 2D materials by controlling crumpling?

The conserved structures of the 5' nontranslated region of Citrus tristeza virus are involved in replication and virion assembly

International Nuclear Information System (INIS)

Gowda, Siddarame; Satyanarayana, Tatineni; Ayllon, Maria A.; Moreno, Pedro; Flores, Ricardo; Dawson, William O.

2003-01-01

The genomic RNA of different isolates of Citrus tristeza virus (CTV) reveals an unusual pattern of sequence diversity: the 3' halves are highly conserved (homology >90%), while the 5' halves show much more dissimilarity, with the 5' nontranslated region (NTR) containing the highest diversity (homology as low as 42%). Yet, positive-sense sequences of the 5' NTR were predicted to fold into nearly identical structures consisting of two stem-loops (SL1 and SL2) separated by a short spacer region. The predicted most stable secondary structures of the negative-sense sequences were more variable. We introduced mutations into the 5' NTR of a CTV replicon to alter the sequence and/or the predicted secondary structures with or without additional compensatory changes designed to restore predicted secondary structures, and examined their effect on replication in transfected protoplasts. The results suggested that the predicted secondary structures of the 5' NTR were more important for replication than the primary structure. Most mutations that were predicted to disrupt the secondary structures fail to replicate, while compensatory mutations were allowed replication to resume. The 5' NTR mutations that were tolerated by the CTV replicon were examined in the full-length virus for effects on replication and production of the multiple subgenomic RNAs. Additionally, the ability of these mutants to produce virions was monitored by electron microscopy and by passaging the progeny nucleocapsids to another batch of protoplasts. Some of the mutants with compensatory sequence alterations predicted to rebuild similar secondary structures allowed replication at near wild-type levels but failed to passage, suggesting that the 5' NTR contains sequences required for both replication and virion assembly

Mechanics of membrane-cytoskeleton attachment in Paramecium

Science.gov (United States)

Campillo, C.; Jerber, J.; Fisch, C.; Simoes-Betbeder, M.; Dupuis-Williams, P.; Nassoy, P.; Sykes, C.

2012-12-01

In this paper we assess the role of the protein MKS1 (Meckel syndrome type 1) in the cortical membrane mechanics of the ciliated protist Paramecium. This protein is known to be crucial in the process of cilium formation, and we investigate its putative role in membrane-cytoskeleton attachment. Therefore, we compare cells where the gene coding for MKS1 is silenced to wild-type cells. We found that scanning electron microscopy observation of the cell surface reveals a cup-like structure in wild-type cells that is lost in silenced cells. Since this structure is based on the underlying cytoskeleton, one hypothesis to explain this observation is a disruption of membrane attachment to the cytoskeleton in the absence of MKS1 that should affect plasma membrane mechanics. We test this by probing the mechanics of wild-type and silenced cells by micropipette aspiration. Strikingly, we observe that, at the same aspiration pressure, the membrane of silenced cells is easily aspirated by the micropipette whereas that of wild-type cells enters only at a moderate velocity, an effect that suggests a detachment of the membrane from the underlying cytoskeleton in silenced cells. We quantify this detachment by measuring the deformation of the cell cortex and the rate of cell membrane entry in the micropipette. This study offers a new perspective for the characterization of membrane-cytoskeleton attachment in protists and paves the way for a better understanding of the role of membrane-cortex attachment in cilium formation.

Nocturnal antihypertensive treatment in patients with type 1 diabetes with autonomic neuropathy and non-dipping

DEFF Research Database (Denmark)

Hjortkjær, Henrik Øder; Jensen, Tonny; Kofoed, Klaus F

2016-01-01

to test if bedtime dosing (BD) versus morning dosing (MD) of the ACE inhibitor enalapril would affect the 24-hour BP profile in patients with type 1 diabetes (T1D), CAN and non-dipping. SETTING: Secondary healthcare unit in Copenhagen, Denmark. PARTICIPANTS: 24 normoalbuminuric patients with T1D with CAN...... and non-dipping were included, consisting of mixed gender and Caucasian origin. Mean±SD age, glycosylated haemoglobin and diabetes duration were 60±7 years, 7.9±0.7% (62±7 mmol/mol) and 36±11 years. INTERVENTIONS: In this randomised, placebo-controlled, double-blind cross-over study, the patients were......OBJECTIVES: Cardiovascular autonomic neuropathy (CAN) and abnormal circadian blood pressure (BP) rhythm are independent cardiovascular risk factors in patients with diabetes and associations between CAN, non-dipping of nocturnal BP and coronary artery disease have been demonstrated. We aimed...

Imaging with radiolabelled anti-membrane type 1 matrix metalloproteinase (MT1-MMP) antibody: potentials for characterizing atherosclerotic plaques

International Nuclear Information System (INIS)

Kuge, Yuji; Takai, Nozomi; Ogawa, Yuki; Temma, Takashi; Nishigori, Kantaro; Ishino, Seigo; Kamihashi, Junko; Saji, Hideo; Zhao, Yan; Kiyono, Yasushi; Shiomi, Masashi

2010-01-01

Membrane type 1 matrix metalloproteinase (MT1-MMP) activates pro-MMP-2 and pro-MMP-13 to their active forms and plays important roles in the destabilization of atherosclerotic plaques. This study sought to determine the usefulness of 99m Tc-labelled monoclonal antibody (mAb), recognizing MT1-MMP, for imaging atherosclerosis in a rabbit model (WHHLMI rabbits). Anti-MT1-MMP monoclonal IgG 3 and negative control IgG 3 were radiolabelled with 99m Tc after derivatization with 6-hydrazinonicotinic acid (HYNIC) to yield 99m Tc-MT1-MMP mAb and 99m Tc-IgG 3 , respectively. WHHLMI and control rabbits were injected with these radio-probes. The aorta was removed and radioactivity was measured at 24 h after the injection. Autoradiography and histological studies were performed. 99m Tc-MT1-MMP mAb accumulation in WHHLMI rabbit aortas was 5.4-fold higher than that of control rabbits. Regional 99m Tc-MT1-MMP mAb accumulation was positively correlated with MT1-MMP expression (r = 0.59, p 99m Tc-IgG 3 accumulation was independent of MT1-MMP expression (r = 0.03, p = NS). The highest 99m Tc-MT1-MMP mAb accumulation was found in atheromatous lesions (4.8 ± 1.9, %ID x BW/mm 2 x 10 2 ), followed in decreasing order by fibroatheromatous (1.8 ± 1.3), collagen-rich (1.6 ± 1.0) and neointimal lesions (1.5 ± 1.5). In contrast, 99m Tc-IgG 3 accumulation was almost independent of the histological grade of lesions. Higher 99m Tc-MT1-MMP mAb accumulation in grade IV atheroma was shown in comparison with neointimal lesions or other more stable lesions. Nuclear imaging with 99m Tc-MT1-MMP mAb, in combination with CT and MRI, could provide new diagnostic imaging capabilities for detecting vulnerable plaques, although further investigations to improve target to blood ratios are strongly required. (orig.)

Virion Glycoprotein-Mediated Immune Evasion by Human Cytomegalovirus: a Sticky Virus Makes a Slick Getaway

Science.gov (United States)

Gardner, Thomas J.

2016-01-01

SUMMARY The prototypic herpesvirus human cytomegalovirus (CMV) exhibits the extraordinary ability to establish latency and maintain a chronic infection throughout the life of its human host. This is even more remarkable considering the robust adaptive immune response elicited by infection and reactivation from latency. In addition to the ability of CMV to exist in a quiescent latent state, its persistence is enabled by a large repertoire of viral proteins that subvert immune defense mechanisms, such as NK cell activation and major histocompatibility complex antigen presentation, within the cell. However, dissemination outside the cell presents a unique existential challenge to the CMV virion, which is studded with antigenic glycoprotein complexes targeted by a potent neutralizing antibody response. The CMV virion envelope proteins, which are critical mediators of cell attachment and entry, possess various characteristics that can mitigate the humoral immune response and prevent viral clearance. Here we review the CMV glycoprotein complexes crucial for cell attachment and entry and propose inherent properties of these proteins involved in evading the CMV humoral immune response. These include viral glycoprotein polymorphism, epitope competition, Fc receptor-mediated endocytosis, glycan shielding, and cell-to-cell spread. The consequences of CMV virion glycoprotein-mediated immune evasion have a major impact on persistence of the virus in the population, and a comprehensive understanding of these evasion strategies will assist in designing effective CMV biologics and vaccines to limit CMV-associated disease. PMID:27307580

Basement membrane abnormalities in human eyes with diabetic retinopathy

DEFF Research Database (Denmark)

Ljubimov, A V; Burgeson, R E; Butkowski, R J

1996-01-01

Vascular and parenchymal basement membranes (BMs) are thickened in diabetes, but alterations in individual BM components in diabetic eyes, especially in diabetic retinopathy (DR), are obscure. To identify abnormalities in the distribution of specific constituents, we analyzed cryostat sections...... of human eyes obtained at autopsy (seven normal, five diabetic without DR, and 13 diabetic with DR) by immunofluorescence with antibodies to 30 BM and extracellular matrix components. In non-DR eyes, no qualitative changes of ocular BM components were seen. In some DR corneas, epithelial BM was stained...... discontinuously for laminin-1, entactin/nidogen, and alpha3-alpha4 Type IV collagen, in contrast to non-DR corneas. Major BM alterations were found in DR retinas compared to normals and non-DR diabetics. The inner limiting membrane (retinal BM) of DR eyes had accumulations of fibronectin (including cellular...

Estimation of phase separation temperatures for polyethersulfone/solvent/non-solvent systems in RTIPS and membrane properties

DEFF Research Database (Denmark)

Liu, Min; Liu, Sheng-Hui; Skov, Anne Ladegaard

2018-01-01

was observed. When the membrane-forming temperature was higher than the cloud point, membranes with a bi-continuous structure were acquired and showed a higher pure water permeation flux than that of membranes prepared with the non-solvent induced phase separation (NIPS) process. The pure water permeation flux...... and the mean pore size of membranes prepared with the RTIPS process decreased in line with an increase of PES molecular weight. When the membrane formation mechanism was the RTIPS process, the mechanical properties were better than those of the corresponding membranes prepared with the NIPS process....

Fangchinoline inhibits human immunodeficiency virus type 1 replication by interfering with gp160 proteolytic processing.

Directory of Open Access Journals (Sweden)

Zhitao Wan

Full Text Available The introduction of highly active antiretroviral therapy has led to a significant reduction in the morbidity and mortality of acquired immunodeficiency syndrome patients. However, the emergence of drug resistance has resulted in the failure of treatments in large numbers of patients and thus necessitates the development of new classes of anti-HIV drugs. In this study, more than 200 plant-derived small-molecule compounds were evaluated in a cell-based HIV-1 antiviral screen, resulting in the identification of a novel HIV-1 inhibitor (fangchinoline. Fangchinoline, a bisbenzylisoquinoline alkaloid isolated from Radix Stephaniae tetrandrae, exhibited antiviral activity against HIV-1 laboratory strains NL4-3, LAI and BaL in MT-4 and PM1 cells with a 50% effective concentration ranging from 0.8 to 1.7 µM. Mechanism-of-action studies showed that fangchinoline did not exhibit measurable antiviral activity in TZM-b1 cells but did inhibit the production of infectious virions in HIV-1 cDNA transfected 293T cells, which suggests that the compound targets a late event in infection cycle. Furthermore, the antiviral effect of fangchinoline seems to be HIV-1 envelope-dependent, as the production of infectious HIV-1 particles packaged with a heterologous envelope, the vesicular stomatitis virus G glycoprotein, was unaffected by fangchinoline. Western blot analysis of HIV envelope proteins expressed in transfected 293T cells and in isolated virions showed that fangchinoline inhibited HIV-1 gp160 processing, resulting in reduced envelope glycoprotein incorporation into nascent virions. Collectively, our results demonstrate that fangchinoline inhibits HIV-1 replication by interfering with gp160 proteolytic processing. Fangchinoline may serve as a starting point for developing a new HIV-1 therapeutic approach.

Multifaceted counter-APOBEC3G mechanisms employed by HIV-1 Vif.