The Experts below are selected from a list of 321 Experts worldwide ranked by ideXlab platform
Dennis Brown - One of the best experts on this subject based on the ideXlab platform.
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Mutations in the endodomain of Sindbis Virus glycoprotein E2 define sequences critical for Virus Assembly.
Journal of virology, 2006Co-Authors: John West, Raquel Hernandez, Davis Fernandes Ferreira, Dennis BrownAbstract:Envelopment of Sindbis Virus at the plasma membrane is a multistep process in which an initial step is the association of the E2 protein via a cytoplasmic endodomain with the preassembled nucleocapsid. Sindbis Virus is vectored in nature by blood-sucking insects and grows efficiently in a number of avian and mammalian vertebrate hosts. The Assembly of Sindbis Virus, therefore, must occur in two very different host cell environments. Mammalian cells contain cholesterol which insect membranes lack. This difference in membrane composition may be critical in determining what requirements are placed on the E2 tail for Virus Assembly. To examine the interaction between the E2 tail and the nucleocapsid in Sindbis Virus, we have produced substitutions and deletions in a region of the E2 tail (E2 amino acids 408 to 415) that is initially integrated into the endoplasmic reticulum. This sequence was identified as being critical for nucleocapsid binding in an in vitro peptide protection assay. The effects of these mutations on Virus Assembly and function were determined in both vertebrate and invertebrate cells. Amino acid substitutions (at positions E2: 408, 410, 411, and 413) reduced infectious Virus production in a position-dependent fashion but were not efficient in disrupting Assembly in mammalian cells. Deletions in the E2 endodomain (406-407, 409-411, and 414-417) resulted in the failure to assemble virions in mammalian cells. Electron microscopy of BHK cells transfected with these mutants revealed Assembly of nucleocapsids that failed to attach to membranes. However, introduction of these deletion mutants into insect cells resulted in the Assembly of Virus-like particles but no assayable infectivity. These data help define protein interactions critical for Virus Assembly and suggest a fundamental difference between Sindbis Virus Assembly in mammalian and insect cells.
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In vivo processing and isolation of furin protease-sensitive alphaVirus glycoproteins: a new technique for producing mutations in Virus Assembly.
Virology, 2005Co-Authors: Steevenson Nelson, Raquel Hernandez, Davis Fernandes Ferreira, Dennis BrownAbstract:Sindbis Virus particles are composed of three structural proteins (Capsid/E2/E1). In the mature virion the E1 glycoprotein is organized in a highly constrained, energy-rich conformation. It is hypothesized that this energy is utilized to drive events that deliver the viral genome to the cytoplasm of a host cell. The extraction of the E1 glycoprotein from Virus membranes with detergent results in disulfide-bridge rearrangement and the collapse of the protein to a number of low-energy, non-native configurations. In a new approach to the production of membrane-free membrane glycoproteins, furin protease recognition motifs were installed at various positions in the E1 glycoprotein ectodomain. Proteins containing the furin-sensitive sites undergo normal folding and Assembly in the endoplasmic reticulum and only experience the consequence of the mutation during transport to the cell surface. Processing by furin in the Golgi results in the release of the protein from the membrane. Processing of the proteins also impacts the envelopment of the nucleocapsid in the modified plasma membrane. This technique provides a unique method for studying the mechanism of Virus Assembly and protein structure without altering crucial early events in protein Assembly, folding, and maturation.
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A single deletion in the membrane-proximal region of the Sindbis Virus glycoprotein E2 endodomain blocks Virus Assembly.
Journal of virology, 2000Co-Authors: Raquel Hernandez, Heuiran Lee, Christine Nelson, Dennis BrownAbstract:The envelopment of the Sindbis Virus nucleocapsid in the modified cell plasma membrane involves a highly specific interaction between the capsid (C) protein and the endodomain of the E2 glycoprotein. We have previously identified a domain of the Sindbis Virus C protein involved in binding to the E2 endodomain (H. Lee and D. T. Brown, Virology 202:390–400, 1994). The C-E2 binding domain resides in a hydrophobic cleft with C Y180 and W247 on opposing sides of the cleft. Structural modeling studies indicate that the E2 domain, which is proposed to bind the C protein (E2 398T, 399P, and 400Y), is located at a sufficient distance from the membrane to occupy the C protein binding cleft (S. Lee, K. E. Owen, H. K. Choi, H. Lee, G. Lu, G. Wengler, D. T. Brown, M. G. Rossmann, and R. J. Kuhn, Structure 4:531–541, 1996). To measure the critical spanning length of the E2 endodomain which positions the TPY domain into the putative C binding cleft, we have constructed a deletion mutant, ΔK391, in which a nonconserved lysine (E2 K391) at the membrane-cytoplasm junction of the E2 tail has been deleted. This mutant was found to produce very low levels of Virus from BHK-21 cells due to a defect in an unidentified step in nucleocapsid binding to the E2 endodomain. In contrast, ΔK391 produced wild-type levels of Virus from tissue-cultured mosquito cells. We propose that the phenotypic differences displayed by this mutant in the two diverse host cells arise from fundamental differences in the lipid composition of the insect cell membranes which affect the physical and structural properties of membranes and thereby Virus Assembly. The data suggest that these Viruses have evolved properties adapted specifically for Assembly in the diverse hosts in which they grow.
Robert A Lamb - One of the best experts on this subject based on the ideXlab platform.
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the influenza Virus m2 protein cytoplasmic tail interacts with the m1 protein and influences Virus Assembly at the site of Virus budding
Journal of Virology, 2008Co-Authors: Benjamin J Chen, George P Leser, David Jackson, Robert A LambAbstract:The cytoplasmic tail of the influenza A Virus M2 proton-selective ion channel has been shown to be important for Virus replication. Previous analysis of M2 cytoplasmic tail truncation mutants demonstrated a defect in incorporation of viral RNA (vRNA) into virions, suggesting a role for M2 in the recruitment of M1-vRNA complexes. To further characterize the effect of the M2 cytoplasmic tail mutations on Virus Assembly and budding, we constructed a series of alanine substitution mutants of M2 with mutations in the cytoplasmic tail, from residues 71 to 97. Mutant proteins M2-Mut1 and M2-Mut2, with mutations of residues 71 to 73 and 74 to 76, respectively, appeared to have the greatest effect on Virus-like particle and Virus budding, showing a defect in M1 incorporation. Mutant Viruses containing M2-Mut1 and M2-Mut2 failed to replicate in multistep growth analyses on wild-type (wt) MDCK cells and were able to form plaques only on MDCK cells stably expressing wt M2 protein. Compared to wt M2 protein, M2-Mut1 and M2-Mut2 were unable to efficiently coimmunoprecipitate with M1. Furthermore, statistical analysis of planar sheets of membrane from cells infected by Virus containing M2-Mut1 revealed a reduction in M1-hemagglutinin (HA) and M2-HA clustering as well as a severe loss of clustering between M1 and M2. These results suggest an essential, direct interaction between the cytoplasmic tail of M2 and M1 that promotes the recruitment of the internal viral proteins and vRNA to the plasma membrane for efficient Virus Assembly to occur.
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Palmitylation of the influenza Virus hemagglutinin (H3) is not essential for Virus Assembly or infectivity.
Journal of virology, 1996Co-Authors: Hong Jin, George P Leser, Kanta Subbarao, Sangeeta Bagai, Brian R. Murphy, Robert A LambAbstract:The C terminus of the influenza Virus hemagglutinin (HA) contains three cysteine residues that are highly conserved among HA subtypes, two in the cytoplasmic tail and one in the transmembrane domain. All of these C-terminal cysteine residues are modified by the covalent addition of palmitic acid through a thio-ether linkage. To investigate the role of HA palmitylation in Virus Assembly, we used reverse genetics technique to introduce substitutions and deletions that affected the three conserved cysteine residues into the H3 subtype HA. The rescued Viruses contained the HA of subtype H3 (A/Udorn/72) in a subtype H1 helper Virus (A/WSN/33) background. Rescued Viruses which do not contain a site for palmitylation (by residue substitution or substitution combined with deletion of the cytoplasmic tail) were obtained. Rescued virions had a normal polypeptide composition. Analysis of the kinetics of HA low-pH-induced fusion of the mutants showed no major change from that of Virus with wild-type (wt) HA. The PFU/HA ratio of the rescued Viruses grown in eggs ranged from that of Virus with wt HA to 16-fold lower levels, whereas the PFU/HA ratio of the rescued Viruses grown in MDCK cells varied only 2-fold from that of Virus with wt HA. However, except for one rescued mutant Virus (CAC), the mutant Viruses were attenuated in mice, as indicated by a > or = 400-fold increase in the 50% lethal dose. Interestingly, except for one mutant Virus (CAC), all of the rescued mutant Viruses were restricted for replication in the upper respiratory tract but much less restricted in the lungs. Thus, the HA cytoplasmic tail may play a very important role in the generation of Virus that can replicate in multiple cell types.
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Effects of Antibody to the Influenza A Virus M2 Protein on M2 Surface Expression and Virus Assembly
Virology, 1995Co-Authors: P G Hughey, Robert A Lamb, Paul C. Roberts, Leslie J. Holsinger, Suzanne Zebedee, Richard W. CompansAbstract:We have investigated the effect of a monoclonal antibody on influenza Virus release and the cell surface expression of M2, comparing Virus strains which were observed previously to be sensitive (A/Udorn) or resistant (A/WSN and A/Udorn variants) to growth inhibition by M2 antibody 14C2. Incubation of A/Udorn Virus-infected cells in the presence of the inhibitory M2 antibody resulted in a significant reduction in the yield of Virus, as measured by infectivity assays as well as by the release of purified virions. The release of A/Udorn Virus was not inhibited by the presence of monovalent 14C2 Fab, in contrast to IgG, indicating that a bivalent structure is essential for 14C2 antibody-mediated viral growth restriction. The level of M2 surface expression in A/Udorn Virus-infected MDCK cells was found to be reduced to approximately 60% of control levels in cells incubated with the 14C2 antibody. In contrast, M2 surface expression levels in A/WSN Virus-infected cells were decreased by only approximately 5-15%, and A/WSN Virus Assembly appeared to be unaffected by the M2 antibody treatment. M2 antigen associated with cell membranes and Virus particles was redistributed into clusters after M2 antibody treatment in infected cells. Incubation in the presence of the 14C2 antibody also reduced M2 surface expression by approximately 40-50% in cells infected with a recombinant vaccinia Virus that expresses the M2 A/Udorn protein. These results demonstrate that M2 antibody reduces the level of influenza Virus particle formation in a single cycle of infection and suggest that inhibition of A/Udorn Virus replication by the 14C2 antibody is related to the reduced cell surface expression and redistribution of the M2 protein induced by the antibody treatment.
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The influenza Virus hemagglutinin cytoplasmic tail is not essential for Virus Assembly or infectivity.
The EMBO Journal, 1994Co-Authors: Hong Jin, George P Leser, Robert A LambAbstract:The influenza A Virus hemagglutinin (HA) glycoprotein contains a cytoplasmic tail which consists of 10-11 amino acids, of which five residues re conserved in all subtypes of influenza A Virus. As the cytoplasmic tail is not needed for intracellular transport to the plasma membrane, it has become virtually dogma that the role of the cytoplasmic tail is in forming protein-protein interactions necessary for creating an infectious budding Virus. To investigate the role of the HA cytoplasmic tail in Virus replication, reverse genetics was used to obtain an influenza Virus that lacked an HA cytoplasmic tail. The rescued Virus contained the HA of subtype A/Udorn/72 in a helper Virus (subtype A/WSN/33) background. Biochemical analysis indicated that only the introduced tail- HA was incorporated into virions and these particles lacked a detectable fragment of the helper Virus HA. The tail- HA rescued Virus assembled and replicated almost as efficiently as virions containing wild-type HA, suggesting that the cytoplasmic tail is not essential for the Virus Assembly process. Nonetheless, a revertant Virus was isolated, suggesting that possession of a cytoplasmic tail does confer an advantage.
Raquel Hernandez - One of the best experts on this subject based on the ideXlab platform.
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Mutations in the endodomain of Sindbis Virus glycoprotein E2 define sequences critical for Virus Assembly.
Journal of virology, 2006Co-Authors: John West, Raquel Hernandez, Davis Fernandes Ferreira, Dennis BrownAbstract:Envelopment of Sindbis Virus at the plasma membrane is a multistep process in which an initial step is the association of the E2 protein via a cytoplasmic endodomain with the preassembled nucleocapsid. Sindbis Virus is vectored in nature by blood-sucking insects and grows efficiently in a number of avian and mammalian vertebrate hosts. The Assembly of Sindbis Virus, therefore, must occur in two very different host cell environments. Mammalian cells contain cholesterol which insect membranes lack. This difference in membrane composition may be critical in determining what requirements are placed on the E2 tail for Virus Assembly. To examine the interaction between the E2 tail and the nucleocapsid in Sindbis Virus, we have produced substitutions and deletions in a region of the E2 tail (E2 amino acids 408 to 415) that is initially integrated into the endoplasmic reticulum. This sequence was identified as being critical for nucleocapsid binding in an in vitro peptide protection assay. The effects of these mutations on Virus Assembly and function were determined in both vertebrate and invertebrate cells. Amino acid substitutions (at positions E2: 408, 410, 411, and 413) reduced infectious Virus production in a position-dependent fashion but were not efficient in disrupting Assembly in mammalian cells. Deletions in the E2 endodomain (406-407, 409-411, and 414-417) resulted in the failure to assemble virions in mammalian cells. Electron microscopy of BHK cells transfected with these mutants revealed Assembly of nucleocapsids that failed to attach to membranes. However, introduction of these deletion mutants into insect cells resulted in the Assembly of Virus-like particles but no assayable infectivity. These data help define protein interactions critical for Virus Assembly and suggest a fundamental difference between Sindbis Virus Assembly in mammalian and insect cells.
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In vivo processing and isolation of furin protease-sensitive alphaVirus glycoproteins: a new technique for producing mutations in Virus Assembly.
Virology, 2005Co-Authors: Steevenson Nelson, Raquel Hernandez, Davis Fernandes Ferreira, Dennis BrownAbstract:Sindbis Virus particles are composed of three structural proteins (Capsid/E2/E1). In the mature virion the E1 glycoprotein is organized in a highly constrained, energy-rich conformation. It is hypothesized that this energy is utilized to drive events that deliver the viral genome to the cytoplasm of a host cell. The extraction of the E1 glycoprotein from Virus membranes with detergent results in disulfide-bridge rearrangement and the collapse of the protein to a number of low-energy, non-native configurations. In a new approach to the production of membrane-free membrane glycoproteins, furin protease recognition motifs were installed at various positions in the E1 glycoprotein ectodomain. Proteins containing the furin-sensitive sites undergo normal folding and Assembly in the endoplasmic reticulum and only experience the consequence of the mutation during transport to the cell surface. Processing by furin in the Golgi results in the release of the protein from the membrane. Processing of the proteins also impacts the envelopment of the nucleocapsid in the modified plasma membrane. This technique provides a unique method for studying the mechanism of Virus Assembly and protein structure without altering crucial early events in protein Assembly, folding, and maturation.
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A single deletion in the membrane-proximal region of the Sindbis Virus glycoprotein E2 endodomain blocks Virus Assembly.
Journal of virology, 2000Co-Authors: Raquel Hernandez, Heuiran Lee, Christine Nelson, Dennis BrownAbstract:The envelopment of the Sindbis Virus nucleocapsid in the modified cell plasma membrane involves a highly specific interaction between the capsid (C) protein and the endodomain of the E2 glycoprotein. We have previously identified a domain of the Sindbis Virus C protein involved in binding to the E2 endodomain (H. Lee and D. T. Brown, Virology 202:390–400, 1994). The C-E2 binding domain resides in a hydrophobic cleft with C Y180 and W247 on opposing sides of the cleft. Structural modeling studies indicate that the E2 domain, which is proposed to bind the C protein (E2 398T, 399P, and 400Y), is located at a sufficient distance from the membrane to occupy the C protein binding cleft (S. Lee, K. E. Owen, H. K. Choi, H. Lee, G. Lu, G. Wengler, D. T. Brown, M. G. Rossmann, and R. J. Kuhn, Structure 4:531–541, 1996). To measure the critical spanning length of the E2 endodomain which positions the TPY domain into the putative C binding cleft, we have constructed a deletion mutant, ΔK391, in which a nonconserved lysine (E2 K391) at the membrane-cytoplasm junction of the E2 tail has been deleted. This mutant was found to produce very low levels of Virus from BHK-21 cells due to a defect in an unidentified step in nucleocapsid binding to the E2 endodomain. In contrast, ΔK391 produced wild-type levels of Virus from tissue-cultured mosquito cells. We propose that the phenotypic differences displayed by this mutant in the two diverse host cells arise from fundamental differences in the lipid composition of the insect cell membranes which affect the physical and structural properties of membranes and thereby Virus Assembly. The data suggest that these Viruses have evolved properties adapted specifically for Assembly in the diverse hosts in which they grow.
Douglas S. Lyles - One of the best experts on this subject based on the ideXlab platform.
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Role of Residues 121 to 124 of Vesicular Stomatitis Virus Matrix Protein in Virus Assembly and Virus-Host Interaction
Journal of virology, 2006Co-Authors: John H. Connor, Margie O. Mckenzie, Douglas S. LylesAbstract:The recent solution of the crystal structure of a fragment of the vesicular stomatitis Virus matrix (M) protein suggested that amino acids 121 to 124, located on a solvent-exposed loop of the protein, are important for M protein self-association and association with membranes. These residues were mutated from the hydrophobic AVLA sequence to the polar sequence DKQQ. Expression and purification of this mutant from bacteria showed that it was structurally stable and that the mutant M protein had self-association kinetics similar to those of the wild-type M protein. Analysis of the membrane association of M protein in the context of infection with isogenic recombinant Viruses showed that both wild-type and mutant M proteins associated with membranes to the same extent. Virus expressing the mutant M protein did show an approximately threefold-lower binding affinity of M protein for nucleocapsid-M complexes. In contrast to the relatively minor effects of the M protein mutation on Virus Assembly, the mutant Virus exhibited growth restriction in MDBK but not BHK cells, a slower induction of apoptosis, and lower viral-protein synthesis. Despite translating less viral protein, the mutant Virus produced more viral mRNA, showing that the mutant Virus could not effectively promote viral translation. These results demonstrate that the 121-to-124 region of the VSV M protein plays a minor role in Virus Assembly but is involved in Virus-host interactions and VSV replication by augmenting viral-mRNA translation.
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Activity of Vesicular Stomatitis Virus M Protein Mutants in Cell Rounding Is Correlated with the Ability to Inhibit Host Gene Expression and Is Not Correlated with Virus Assembly Function
Virology, 1997Co-Authors: Douglas S. Lyles, Margie O. MckenzieAbstract:Abstract In addition to its role in Virus Assembly, the matrix (M) protein of vesicular stomatitis Virus (VSV) is involved in Virus-induced cell rounding and inhibition of host-directed gene expression. Previous experiments have shown that two M protein mutants genetically dissociate the ability of M protein to inhibit host-directed gene expression from its function in Virus Assembly: M protein from tsO82 Virus is fully functional in Virus Assembly but defective in the inhibition of host-directed gene expression, while the MN1 deletion mutant, which lacks amino acids 4–21, inhibits host-directed gene expression but cannot function in Virus Assembly. Experiments presented here compared cell rounding induced by these two mutant M proteins to that of wt M protein. BHK cells were transfected with M protein mRNA transcribedin vitro,and the extent of cell rounding was evaluated at 24 hr posttransfection. The MN1 protein was nearly as effective as wt M protein in the induction of cell rounding, while tsO82 M protein expressed from transfected RNA was not able to induce cell rounding above that observed in negative controls without M protein, although it did cause BHK cells to have a less elongated shape. These results indicate that the ability of MN1 and tsO82 M proteins to induce cell rounding is not correlated with their Virus Assembly function. Instead the cell rounding activity of these mutants is correlated with their ability to inhibit host-directed gene expression. Previous data suggesting that these two cytopathic activities could be dissociated can be readily accounted for by quantitative differences in M protein expression required. Infection of either BHK cells or L cells with tsO82 Virus induced cell rounding, although cell rounding was delayed relative to that following infection with wt VSV, suggesting that tsO82 M protein retains some cytopathic activity. The distribution of actin, vimentin, and tubulin in transfected cells was determined by fluorescence microscopy. In cells transfected with tsO82 M mRNA, these cytoskeletal elements were indistinguishable from those of negative control transfected cells. In cells rounded as a result of transfection with wt M or MN1 mRNA, actin-containing filaments were reorganized into a thick perinuclear ring but were not depolymerized. In contrast, tubulin and vimentin appeared to be diffusely distributed throughout the cytoplasm of rounded cells. These results support the idea that cell rounding induced by M protein results from the depolymerization of microtubules and/or intermediate filaments.
Michael A Whitt - One of the best experts on this subject based on the ideXlab platform.
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The protease-sensitive loop of the vesicular stomatitis Virus matrix protein is involved in Virus Assembly and protein translation.
Virology, 2011Co-Authors: Chad E Mire, Michael A WhittAbstract:To study the contribution of the protease-sensitive loop of the VSV M protein in Virus Assembly we recovered recombinant VSV (rVSV) with mutations in this region and examined Virus replication. Mutations in the highly conserved LXD motif (aa 123-125) resulted in reduced virion budding, reduced Virus titers and enhanced M protein exchange with M-ribonucleocapsid complexes (M-RNPs), suggesting that the mutant M proteins were less tightly associated with RNP skeletons. In addition, viral protein synthesis began to decrease at 4h post-infection (hpi) and was reduced by ~80% at 8 hpi for the mutant rVSV-D125A. The reduced protein synthesis was not due to decreased VSV replication or transcription; however, translation of a reporter gene with an EMCV IRES was not reduced, suggesting that cap-dependent, but not cap-independent translation initiation was affected in rVSV-D125A infected cells. These results indicate that the LXD motif is involved in both Virus Assembly and VSV protein translation.
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The Membrane-Proximal Stem Region of Vesicular Stomatitis Virus G Protein Confers Efficient Virus Assembly
Journal of virology, 2000Co-Authors: Clinton S. Robison, Michael A WhittAbstract:In this report, we show that the glycoprotein of vesicular stomatitis Virus (VSV G) contains within its extracellular membrane-proximal stem (GS) a domain that is required for efficient VSV budding. To determine a minimal sequence in GS that provides for high-level Virus Assembly, we have generated a series of recombinant ΔG-VSVs which express chimeric glycoproteins having truncated stem sequences. The recombinant Viruses having chimeras with 12 or more membrane-proximal residues of the G stem, and including the G protein transmembrane-cytoplasmic tail domains, produced near-wild-type levels of particles. In contrast, Viruses encoding chimeras with shorter or no G-stem sequences produced ∼10- to 20-fold less. This budding domain when present in chimeric glycoproteins also promoted their incorporation into the VSV envelope. We suggest that the G-stem budding domain promotes Virus release by inducing membrane curvature at sites where Virus budding occurs or by recruiting condensed nucleocapsids to sites on the plasma membrane which are competent for efficient Virus budding.
