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Stephan Becker - One of the best experts on this subject based on the ideXlab platform.
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phosphorylation of marburg virus matrix protein vp40 triggers assembly of nucleocapsids with the Viral Envelope at the plasma membrane
Cellular Microbiology, 2012Co-Authors: Larissa Kolesnikova, Gordian Schudt, Evamaria Mittler, Hosam Shamseldin, Stephan BeckerAbstract:Summary Marburg virus (MARV) matrix protein VP40 plays a key role in virus assembly, recruiting nucleocapsids and the surface protein GP to filopodia, the sites of Viral budding. In addition, VP40 is the only MARV protein able to induce the release of filamentous virus-like particles (VLPs) indicating its function in MARV budding. Here, we demonstrated that VP40 is phosphorylated and that tyrosine residues at positions 7, 10, 13 and 19 represent major phosphorylation acceptor sites. Mutagenesis of these tyrosine residues resulted in expression of a non-phosphorylatable form of VP40 (VP40mut). VP40mut was able to bind to cellular membranes, produce filamentous VLPs, and inhibit interferon-induced gene expression similarly to wild-type VP40. However, VP40mut was specifically impaired in its ability to recruit nucleocapsid structures into filopodia, and released infectious VLPs (iVLPs) had low infectivity. These results indicated that tyrosine phosphorylation of VP40 is important for triggering the recruitment of nucleocapsids to the Viral Envelope.
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Role of the Transmembrane Domain of Marburg Virus Surface Protein GP in Assembly of the Viral Envelope
Journal of virology, 2007Co-Authors: Eva Mittler, Larissa Kolesnikova, Thomas Strecker, Wolfgang Garten, Stephan BeckerAbstract:The major protein constituents of the filoViral Envelope are the matrix protein VP40 and the surface transmembrane protein GP. While VP40 is recruited to the sites of budding via the late retrograde endosomal transport route, GP is suggested to be transported via the classical secretory pathway involving the endoplasmic reticulum, Golgi apparatus, and trans-Golgi network until it reaches the plasma membrane where most filoViral budding takes place. Since both transport routes target the plasma membrane, it was thought that GP and VP40 join there to form the Viral Envelope. However, it was recently shown that, upon coexpression of both proteins, GP is partially recruited into peripheral VP40-enriched multivesicular bodies, which contained markers of the late endosome. Accumulation of GP and VP40 in this compartment was presumed to play an important role in the formation of the filoViral Envelope. Using a domain-swapping approach, we were able to show that the transmembrane domain of GP was essential and sufficient for (i) partial recruitment of chimeric glycoproteins into VP40-enriched multivesicular bodies and (ii) incorporation into virus-like particles (VLPs) that were released upon expression of VP40. Only those chimeric glycoproteins which were targeted to VP40-enriched endosomal multivesicular bodies were subsequently recruited into VLPs. These data show that the transmembrane domain of GP is critical for the mixing of VP40 and GP in multivesicular bodies and incorporation of GP into the Viral Envelope. Results further suggest that trapping of GP in the VP40-enriched late endosomal compartment is important for the formation of the Viral Envelope.
Xinzhen Yang - One of the best experts on this subject based on the ideXlab platform.
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An Unrelated Monoclonal Antibody Neutralizes Human Immunodeficiency Virus Type 1 by Binding to an Artificial Epitope Engineered in a Functionally Neutral Region of the Viral Envelope Glycoproteins
Journal of virology, 2005Co-Authors: Xinping Ren, Joseph Sodroski, Xinzhen YangAbstract:Neutralizing antibodies often recognize regions of Viral Envelope glycoproteins that play a role in receptor binding or other aspects of virus entry. To address whether this is a necessary feature of a neutralizing antibody, we identified the V4 region of the gp120 Envelope glycoprotein of human immunodeficiency virus type 1 (HIV-1) as a sequence that is tolerant of drastic change and thus appears to play a negligible role in Envelope glycoprotein function. An artificial epitope tag was inserted into the V4 region without a significant effect on virus entry or neutralization by antibodies that recognize HIV-1 Envelope glycoprotein sequences. An antibody directed against the artificial epitope tag was able to neutralize the modified, but not the wild-type, HIV-1. Thus, the specific target of a neutralizing antibody need not contribute functionally to the process of virus entry.
L. Haas - One of the best experts on this subject based on the ideXlab platform.
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Canine distemper virus infection requires cholesterol in the Viral Envelope.
Journal of Virology, 2007Co-Authors: Heidi Imhoff, Veronika Von Messling, Georg Herrler, L. HaasAbstract:Cholesterol is known to play an important role in stabilizing particular cellular membrane structures, so-called lipid or membrane rafts. For several viruses, a dependence on cholesterol for virus entry and/or morphogenesis has been shown. Using flow cytometry and fluorescence microscopy, we demonstrate that infection of cells by canine distemper virus (CDV) was not impaired after cellular cholesterol had been depleted by the drug methyl-beta-cyclodextrin. This effect was independent of the multiplicity of infection and the cellular receptor used for infection. However, cholesterol depletion of the Viral Envelope significantly reduced CDV infectivity. Replenishment by addition of exogenous cholesterol restored infectivity up to 80%. Thus, we conclude that CDV entry is dependent on cholesterol in the Viral Envelope. Furthermore, reduced syncytium formation was observed when the cells were cholesterol depleted during the course of the infection. This may be related to the observation that CDV Envelope proteins H and F partitioned into cellular detergent-resistant membranes. Therefore, a role for lipid rafts during virus assembly and release as well is suggested.
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Canine Distemper Virus Infection Requires Cholesterol in the Viral Envelope
Journal of virology, 2007Co-Authors: Heidi Imhoff, Veronika Von Messling, Georg Herrler, L. HaasAbstract:Cholesterol is known to play an important role in stabilizing particular cellular membrane structures, so-called lipid or membrane rafts. For several viruses, a dependence on cholesterol for virus entry and/or morphogenesis has been shown. Using flow cytometry and fluorescence microscopy, we demonstrate that infection of cells by canine distemper virus (CDV) was not impaired after cellular cholesterol had been depleted by the drug methyl-β-cyclodextrin. This effect was independent of the multiplicity of infection and the cellular receptor used for infection. However, cholesterol depletion of the Viral Envelope significantly reduced CDV infectivity. Replenishment by addition of exogenous cholesterol restored infectivity up to 80%. Thus, we conclude that CDV entry is dependent on cholesterol in the Viral Envelope. Furthermore, reduced syncytium formation was observed when the cells were cholesterol depleted during the course of the infection. This may be related to the observation that CDV Envelope proteins H and F partitioned into cellular detergent-resistant membranes. Therefore, a role for lipid rafts during virus assembly and release as well is suggested.
Robert Blumenthal - One of the best experts on this subject based on the ideXlab platform.
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Inactivation of Retroviruses with Preservation of Structural Integrity by Targeting the Hydrophobic Domain of the Viral Envelope
Journal of virology, 2005Co-Authors: Yossef Raviv, Mathias Viard, Julian W. Bess, Elena Chertova, Robert BlumenthalAbstract:We describe a new approach for the preparation of inactivated retroviruses for vaccine application. The lipid domain of the Viral Envelope was selectively targeted to inactivate proteins and lipids therein and block fusion of the virus with the target cell membrane. In this way, complete elimination of the infectivity of human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV) could be achieved with preservation of antigenic determinants on the surface of the Viral Envelope. Inactivation was accomplished by modification of proteins and lipids in the Viral Envelope using the hydrophobic photoinduced alkylating probe 1,5 iodonaphthylazide (INA). Treatment of HIV and SIV isolates with INA plus light completely blocked fusion of the Viral Envelope and abolished infectivity. The inactivated virus remained structurally unchanged, with no detectable loss of Viral proteins. Modifications to Envelope and nucleocapsid proteins were detected by changes in their elution pattern on reverse-phase high-performance liquid chromatography. These modifications had no effect on primary and secondary structure epitopes as determined by monoclonal antibodies. Likewise, the inactivated HIV reacted as well as the live virus with the conformation-sensitive and broadly neutralizing anti-HIV type 1 monoclonal antibodies 2G12, b12, and 4E10. Targeting the lipid domain of biological membranes with hydrophobic alkylating compounds could be used as a general approach for inactivation of Enveloped viruses and other pathogenic microorganisms for vaccine application.
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Role of Viral Envelope sialic acid in membrane fusion mediated by the vesicular stomatitis virus Envelope glycoprotein.
Biochemistry, 1992Co-Authors: Anu Puri, Settimio Grimaldi, Robert BlumenthalAbstract:Fusion of vesicular stomatitis virus (VSV) with cells and liposomes before and after treatment with neuraminidase was studied using the R18 dequenching assay. Desialylation of VSV significantly enhanced the extent of fusion with Vero cells but affected neither the pH dependence nor the binding of VSV to Vero cells. The enhanced fusion of asialo-VSV was observed both at the plasma membrane as well as via the endocytic pathway. Both VSV and asialo-VSV fused with liposomes made of neutral phospholipid, but only asialo-VSV fused with liposomes containing a 1:1 mixture of neutral and negatively charged phospholipid. To examine factors which contribute to the extent of fusion, we analyzed the various activation and inactivation reactions that take place as a result of low-pH triggering of VSV prebound to the target membrane. Lag times for the onset of fusion were similar for VSV and asialo-VSV, indicating that desialylation did not affect the activation reactions. However, exposure of VSV bound to target membranes at pH 6.5 for 400 s led to considerable inactivation, whereas little inactivation was seen after desialylation of VSV. These results are analyzed in terms of a model which allows us to determine which components of the overall fusion process are dominated by Viral Envelope sialic acid.
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A dissection of steps leading to Viral Envelope protein-mediated membrane fusion.
Annals of the New York Academy of Sciences, 1991Co-Authors: Robert Blumenthal, Christian Schoch, Anu Puri, Michael J. ClagueAbstract:increase in [Ca”] at the presynaptic nerve terminal sets a complex set of biochemical events in motion that finally terminates in the specific fusion of the membrane of the secretory vesicle with the plasma membrane.’ In studying the action of CaZ+ at the presynaptic terminal a distinction has to be made between processes that occur in the cytosol that result in the triggering of the fusion event, and the final membrane fusion event itself. Although specific fusogenic proteins have been postulated to mediate exocytotic membrane fusion, the molecular components of exocytotic membrane fusion machinery remain to be identified. Our own studies have focused on pH-regulated Viral fusion for which the participatory proteins have been identified.’ The first steps in infection of animal cells by Enveloped virus are binding and fusion of the cell and Viral membranes? Those events are mediated by Viral Envelope glycoproteins. Some virus strains infect cells by acid-activated fusion following endocytosis
Hanneke Schuitemaker - One of the best experts on this subject based on the ideXlab platform.
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autologous neutralizing humoral immunity and evolution of the Viral Envelope in the course of subtype b human immunodeficiency virus type 1 infection
Journal of Virology, 2008Co-Authors: Evelien M. Bunnik, Linaida Pisas, Ad C Van Nuenen, Hanneke SchuitemakerAbstract:Most human immunodeficiency virus type 1 (HIV-1)-infected individuals develop an HIV-specific neutralizing antibody (NAb) response that selects for escape variants of the virus. Here, we studied autologous NAb responses in five typical CCR5-using progressors in relation to Viral NAb escape and molecular changes in the Viral Envelope (Env) in the period from seroconversion until after AIDS diagnosis. In sera from three patients, high-titer neutralizing activity was observed against the earliest autologous virus variants, followed by declining humoral immune responses against subsequent Viral escape variants. Autologous neutralizing activity was undetectable in sera from two patients. Patients with high-titer neutralizing activity in serum showed the strongest positive selection pressure on Env early in infection. In the initial phase of infection, gp160 length and the number of potential N-linked glycosylation sites (PNGS) increased in viruses from all patients. Over the course of infection, positive selection pressure declined as the NAb response subsided, coinciding with reversions of changes in gp160 length and the number of PNGS. A number of identical amino acid changes were observed over the course of infection in the Viral quasispecies of different patients. Our results indicate that although neutralizing autologous humoral immunity may have a limited effect on the disease course, it is an important selection pressure in virus evolution early in infection, while declining HIV-specific humoral immunity in later stages may coincide with reversion of NAb-driven changes in Env.
