Pardaxin, a hydrophobic toxin of the Red Sea flatfish, disassembles the intact membrane of vesicular stomatitis virus

Reaction of vesicular stomatitis virus with pardaxin, the hydrophobic toxin of the Red Sea flatfish, resulted in a profound morphological change of many virions and dissociation of their membrane and nucleocapsid into components readily separable by density gradient centrifugation. The basic matrix protein and acidic pardaxin segregated largely with the high density nucleocapsid. The dissociated virion membrane formed lipoprotein vesicles which retained glycoprotein spikes and a certain amount of N protein but no appreciable amounts of other nucleocapsid proteins and little if any RNA. Iodination of the tyrosine residue of the glycoprotein tail fragment provided supporting evidence that the COOH terminus of the glycoprotein extends beyond the inner layer of the membrane into the interior of the virion. These data indicate that pardaxin may serve as a probe for studying the organization of viral membranes, and, hopefully, other biological membranes.     

Association of vesicular stomatitis virus glycoprotein with virion membrane: characterization of the lipophilic tail fragment.

The proteolytic enzyme, thermolysin, degraded the external segment of the membrane glycoprotein of intact vesicular stomatitis (VS) virions but left behind a small nonglycosylated fragment, presumably embedded in the virion membrane. Other proteases generated membrane-associated glycoprotein fragments differing somewhat in molecular weight. The thermolysin-resistant, virion-associated fragment, which can be selectively solubilized by either Triton X-100 or chloroform/methanol, has a molecular weight of 5,200. Amino acid analysis of the glycoprotein fragment reveals a preponderance of hydrophobic amino acids (64% of the residues); the amino-terminal amino acid is alanine as determined by dansylation. Cyanogen bromide digestion of the tail fragment generated two peptides, confirming the presence of one methionine residue per thermolysin-resistant glycoprotein fragment. The secondary structure of this glycoprotein tail peptide is maintained by at least one disulfide bridge. Thermolysin treatment is isolated VS viral glycoprotein in the presence of Triton X-100 also generated a hydrophobic peptide fragment which is very similar to the virion-associated glycoprotein fragment. The amino acid terminus of intact glycoprotein was also found to be alanine as was its dansylated Triton-micellar fragment that resisted thermolytic degradation; this finding suggests that the amino-terminal end of the VS viral glycoprotein is embedded in the virion membrane. These results suggest that the VS viral glycoprotein is an amphipathic molecule, the hydrophilic portion of which contains all the carbohydrate and a lipophilic tail segment which forms lipid or detergent micelles, thus rendering it resistant to proteolysis.     

Heterogeneity of vesicular stomatitis virus particles: implications for virion assembly

Vesicular stomatitis virus (VSV) particles formed at early times after infection contain only one-third the amount of viral glycoportein (G protein), relative to the major internal structural proteins M and N, as is found in particles released later. These "early" particles also have a lower density in equilibrium sucrose gradients than do those formed later; however, the sedimentation velocity and specific infectivity of these two classes of particles are the same. VSV-infected cells also release virus-like particles which sediment considerably faster than authentic virions and contain a higher-than-normal proportion of the VSV G protein relative to internal VSV proteins. These particles have a reduced specific infectivity but a normal density in sucrose gradients. All classes of VSV virions contain a constant proportion of M and N polypeptides. The ratio of G protein to M or N protein, in contrast, can vary over a sixfold range; this implies that an interaction between a precise number of surface G proteins with either of the underlying M and N proteins is not a prerequisite for budding of infectious viral particles from the cell surface.     

Infectivity-deficient vesicular stomatitis virus produced in the presence of interferon has a functional virion core

Interferon induces two antiviral actions against vesicular stomatitis virus by (i) inhibiting viral protein synthesis which leads to a reduction in virion production, and (ii) producing progeny which are deficient in infectivity (VSVIF). At low or physiological concentrations of interferon, while the virion production was decreased by less than 10-fold, the virion infectivity yield was suppressed more than 1000-fold. The VSVIF was found to be deficient (quantitatively) in envelop glycoprotein G and protein M. Tryptic peptide mapping indicated that there was no detectable structural abnormality in the G, M, and N proteins of VSVIF. The virion cores, lacking only the envelop G protein, isolated from VSVIF and control VSV have essentially identical specific infectivity. This indicated that the virion proteins L, N, NS, and M, as well as viral RNA that make up the virion core, must be functionally normal, and the observed deficiency in G protein was likely to be the cause of the functional deficiency of the virion. Low concentrations of DEAE-dextran, which is known to partially overcome the virion's dependence on the G protein for adsorption to the cell during infection, were found to enhance the infectivity of VSVIF more than the control virion. These results together indicated that the loss of infectivity in the VSVIF was due to the deficiency of the surface glycoprotein G.     

Mutations in the PPPY motif of vesicular stomatitis virus matrix protein reduce virus budding by inhibiting a late step in virion release

The N terminus of the matrix (M) protein of vesicular stomatitis virus (VSV) and of other rhabdoviruses contains a highly conserved PPPY sequence (or PY motif) similar to the late (L) domains in the Gag proteins of some retroviruses. These L domains in retroviral Gag proteins are required for efficient release of virus particles. In this report, we show that mutations in the PPPY sequence of the VSV M protein reduce virus yield by blocking a late stage in virus budding. We also observed a delay in the ability of mutant viruses to cause inhibition of host gene expression compared to wild-type (WT) VSV. The effect of PY mutations on virus budding appears to be due to a block at a stage just prior to virion release, since electron microscopic examination of PPPA mutant-infected cells showed a large number of assembled virions at the plasma membrane trapped in the process of budding. Deletion of the glycoprotein (G) in addition to these mutations further reduced the virus yield to less than 1% of WT levels, and very few particles were assembled at the cell surface. This observation suggested that G protein aids in the initial stage of budding, presumably during the formation of the bud site. Overall, our results confirm that the PPPY sequence of the VSV M protein possesses L domain activity analogous to that of the retroviral Gag proteins.     

Pseudotypes of vesicular stomatitis virus with the mixed coat of reticuloendotheliosis virus and vesicular stomatitis virus.

Vesicular stomatitis virus (VSV) forms pseudotypes with envelope components of reticuloendotheliosis virus (REV). The VSV pseudotype possesses the limited host range and antigenic properties of REV. Approximately 70% of the VSV, Indiana serotype, and 45% of VSV, New Jersey serotype, produced from the REV strain T-transformed chicken bone marrow cells contain mixed envelope components of both VSV and REV. VSV pseudotypes with mixed envelope antigens can be neutralized with excess amounts of either anti-VSV antiserum or anti-REV antiserum.     

Interaction of vesicular stomatitis virus with lipid vesicles: depletion of cholesterol and effect on virion membrane fluidity and infectivity.

Interaction with excess unilamellar phosphatidylcholine (PC) vesicles resulted in depletion of as much as 90% of the cholesterol from the membrane of intact vesicular stomatitis (VS) virus. The cholesterol depletion was not significantly influenced by the proteolytic removal of virion glycoprotein spikes, but it was temperature dependent. Cholesterol depletion caused substantial reduction in anisotropy of the VS virion membrane as measured by fluorescence depolarization of the lipophilic probe 1,6-diphenyl-1,3,5-hexatriene; residual adsorbed vesicles represent a significant factor in this apparent increase in virion membrane fluidity. Interaction with PC vesicles resulted in a substantial loss of VS viral infectivity as measured by plating efficiency on L-cell monolayers. Reduction in infectivity appeared to be related to temperature-dependent depletion of virion cholesterol by PC vesicles. Interaction of VS virions with cholesterol-containing PC vesicles resulted in significantly less decline in infectivity, but attempts to restore cholesterol and infectivity to depleted VS virions were unsuccessful. Depletion of virion cholesterol apparently results through collision with PC vesicles rather than movement of cholesterol monomers or micelles through the aqueous phase, because PC vesicle-virion interaction in the presence of cholesterol oxidase did not result in substantial oxidation of translocated cholesterol.     

Asymmetric distribution of phosphatidylethanolamine in the membrane of vesicular stomatitis virus.

The membrane-impermeable reagent trinitrobenzenesulfonate has been shown to react only with the surface components of vesicular stomatitis virus (VSV) membranes. When the amount of phosphatidylethanolamine (PE) available to modification by trinitrobenzenesulfonate in intact virions was determined, it was found that 36% of the total membrane PE was converted to the trinitrophenyl derivative. The same proportion of the total membrane PE was reactive after removal of the surface glycoprotein by trypsin digestion, but disruption of the virus membrane by sonication rendered all of the PE reactive. These results indicate that PE is asymmetrically distributed in the VSV membrane; 36% is present in the outer lipid leaflet, whereas 64% is found on the inner layer.     

Expression of the vesicular stomatitis virus membrane glycoprotein gene in Bacillus subtilis

Abstract The molecularly cloned gene encoding the vesicular stomatitis virus (VSV) membrane glycoprotein G was modified and joined to a Bacillus subtilis secretion vector constructed from the plasmid pUB110 and containing the promoter and signal sequence regions of the α-amylase (a secretory protein) gene from Bacillus amyloliquefaciens . The regions encoding the NH₂-terminal signal peptide and the COOH-terminal hydrophobic transmembrane domains of the VSV gene were deleted to facilitate the secretion of the G protein in soluble form. The truncated G protein was found to be expressed in B. subtilis . The expression level was low, probably due to rapid proteolytic degradation of the protein and, contrary to what was expected, almost all of the protein remained cell-associated.     

Structural changes in BHK cell plasma membrane caused by the binding of vesicular stomatitis virus

Spin label electron spin resonance techniques using a nitroxide derivative of stearic acid were used to detect changes in plasma membrane structure caused by the binding of vesicular stomatitis virus (VSV) to cell plasma membranes of intact BHK-21 cells. The results indicate that binding of VSV to cell surface receptors causes an increase in the observed rigidity of the plasma membrane lipid bilayer. This change in membrane structure, which appears to be caused by the cross-linking of receptors in the plane of the plasma membrane, could be prevented by treating the cells with colchicine before addition of virus and could be reversed by treating the cells with colchicine after addition of virus. Cells treated with a monovalent, water-soluble derivative of VSV G-protein (Gs) did not show an increase in plasma membrane bilayer rigidity. However, addition of anti-VSV G-protein immunoglobulin G to cells pretreated with G8 caused an increase in plasma membrane bilayer rigidity. This increased rigidity could also be reversed by the addition of colchicine. Fluorescence microscopy was used to determine the distribution of fluorescein-labeled VSV particles on the cell surface after addition of virus. Approximately 30 min after addition of virus, discrete areas on the cell surface showed fluorescent staining, which coalesced to apical regions of the cell after approximately 40 min.     

Characterization of vesicular stomatitis virus mutants by partial proteolysis

Structural proteins of temperature-sensitive (ts) mutants of vesicular stomatitis virus, Indiana serotype, were compared with those of wild-type and revertant virions by electrophoresis on polyacrylamide gels of partial digests with Staphylococcus aureus V8 protease. Mutants of complementation groups III (tsG31 and tsG33), II (tsG22), and IV (tsG41) differed from the wild-type virion in peptide profiles of their M, NS, and N proteins, respectively. The differences were only detectable over a narrow range of enzyme-substrate ratios and were due to peptides transiently generated during incomplete digestion. Proteins of revertants to tsG31, tsG22, and tsG41 exhibited the wild-type virion peptide pattern, indicating that reversion had restored their original conformation. However, in the case of tsG22, the NS peptide profile reverted to the wild-type phenotype only partially, suggesting that a silent mutation might have taken place during either the original chemical mutagenesis or the following repeated laboratory passages. The apparent alteration in protein conformation and its restoration upon reversion of the mutants indicated that the lesions of groups III and IV were located in the M and N proteins, respectively. Moreover, for the first time, the site of mutation of group II could be positively identified as the NS protein cistron.