Subunit interactions of vesicular stomatitis virus envelope glycoprotein stabilized by binding to viral matrix protein.

The mechanism by which viral glycoproteins are incorporated into virus envelopes during budding from host membranes is a major question of virus assembly. Evidence is presented here that the envelope glycoprotein (G protein) of vesicular stomatitis virus binds to the viral matrix protein (M protein) in vitro with the specificity, reversibility, and affinity necessary to account for virus assembly in vivo. The assay for the interaction is based on the ability of M protein to stabilize the interaction of G protein subunits, which exist as trimers of identical subunits in the virus envelope. The interaction with M protein was shown by using G proteins labeled with fluorescent probes capable of detecting subunit dissociation and reassociation in vitro. The results show that the M protein isolated from virions either as purified soluble protein or as nucleocapsid-M protein complexes interacts with the G protein in vitro and that the reaction is reversible. The interaction between the G and M proteins was not serotype specific, but no interaction between the vesicular stomatitis virus M protein and the influenza virus hemagglutinin could be detected. These results support the conclusion that the interactions described here are the ones that govern assembly of G protein into virus envelopes in vivo.     

Mutational analysis of the vesicular stomatitis virus glycoprotein G for membrane fusion domains.

The spike glycoprotein G of vesicular stomatitis virus (VSV) induces membrane fusion at low pH. We used linker insertion mutagenesis to characterize the domain(s) of G glycoprotein involved in low-pH-induced membrane fusion. Two or three amino acids were inserted in frame into various positions in the extracellular domain of G, and 14 mutants were isolated. All of the mutants expressed fully glycosylated proteins in COS cells. However, only seven mutant G glycoproteins were transported to the cell surface. Two of these mutants, D1 and A6, showed wild-type fusogenic properties. The mutant A2 had a temperature-sensitive defect in the transport of the mutant G glycoprotein to the cell surface. The other four mutants, H2, H5, H10, and A4, although present in cell surface, failed to induce cell fusion when cells expressing these mutant glycoproteins were exposed to acidic pH. These four mutant G proteins could form trimers, indicating that the defect in fusion was not due to defective oligomerization. One of these mutations, H2, is within a region of conserved, uncharged amino acids that has been proposed as a possible fusogenic sequence. The mutation in H5 was about 70 amino acids downstream of the mutation in H2, while mutations in H10 and A4 were about 300 amino acids downstream of the mutation in H2. Conserved sequences were also noted in the H10 and A4 segment. The results suggest that in the case of VSV G glycoprotein, the fusogenic activity may involve several spatially separated regions in the extracellular domain of the protein.     

Hepatocyte-specific gene expression by baculovirus pseudotyped with vesicular stomatitis virus envelope glycoprotein.

We have developed the recombinant baculovirus pseudotyped with vesicular stomatitis virus (VSV) G protein. The VSV-G gene was under the control of the polyhedrin promoter so that it was expressed at high levels in infected insect cells but not in mammalian cells. The presence of VSV-G protein in purified baculovirus preparations was confirmed by Western analysis. This recombinant baculovirus also carried human AFP (alpha-fetoprotein) promoter for hepatocyte-specific gene expression. After an in vitro infection by a recombinant baculovirus carrying the luciferase gene under the control of human AFP promoter/enhancer (BacG-AFP-Luc(+)), the luciferase gene was expressed in AFP-producing Huh7, Hep3B, and HepG2 cell lines, but not in AFP-nonproducing cell lines. BacG-AFP-Luc(+) transduced with human hepatoma cells in vitro at an efficiency about fivefold greater than the recombinant baculovirus lacking VSV-G (the virus Bac-AFP-Luc(+)). The utilization of the AFP promoter/enhancer in a baculovirus vector could provide benefits in gene therapy applications.     

Photoinactivation and kinetics of membrane fusion mediated by the human immunodeficiency virus type 1 envelope glycoprotein.

The fusion kinetics of cells expressing the human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein with CD4 target cells was continuously monitored by image-enhanced Nomarski differential interference contrast optics. The analysis of the videotape recordings showed that (i) cells made contact relatively rapidly (within minutes), in many cases by using microspikes to "touch" and adhere to adjoining cells; (ii) the adhered cells fused after a relatively long waiting period, which varied from 15 min to hours; (iii) the morphological changes after membrane fusion, which led to disappearance of the interface separating the two cells, were rapid (less than 1 min); and (iv) the process of syncytium formation involved subsequent fusion with other cells and not simultaneous fusion of many cells. To measure the kinetics of early stages of cell fusion, we used the recently developed very stable membrane-soluble dye, PKH26, which redistributes between labeled and unlabeled membranes after fusion but does not exchange spontaneously between membranes for prolonged periods. We found that photoactivation of this dye by illumination with green light inhibits fusion of cell membranes as indicated by the lack of dye transfer from the labeled HIV-1 envelope-expressing cells to unlabeled CD4 cells. The inhibitory effect was localized in space and time, which allowed us to develop a new assay for measuring the kinetics of membrane fusion by illuminating the cell mixture at different times after coculture. This assay has also been used to monitor the fusion kinetics of HIV-1 and recombinant vaccinia virus. The photoactivation of nonexchangeable membrane-soluble fluorescent dyes may be useful for development of new assays for measuring the kinetics of membrane fusion and could also be important in designing new antiviral approaches.     

pH-dependent fusion induced by vesicular stomatitis virus glycoprotein reconstituted into phospholipid vesicles

Purified G-protein from vesicular stomatitis virus was reconstituted into egg phosphatidylcholine vesicles by detergent dialysis of octyl glucoside. A homogeneous population of reconstituted vesicles could be obtained, provided the protein to lipid ratio was high (about 0.3 mol % protein) and the detergent removal was slow. The reconstituted vesicles were assayed for fusion activity using electron microscopy and fluorescence energy transfer. The fusion activity mediated by the viral envelope protein was dependent upon pH, temperature, and target membrane lipid composition. Incubation of reconstituted vesicles at low pH with small unilamellar vesicles containing negatively charged lipids resulted in the appearance of large cochleate structures, as shown by electron microscopy using negative stain. This process did not cause leakage of a vesicle-encapsulated aqueous marker. The rate of fusion was pH-dependent with a pK of about 4 and the apparent energy of activation for the fusion was 16 +/- 1 kcal/mol. G-protein-mediated fusion showed a large preference for target membranes which contain phosphatidylserine or phosphatidic acid. Inclusion of 36% cholesterol in any of the lipid compositions had no effect on the rate of fusion. These reconstituted vesicles provide a system to study the mechanism of pH-dependent fusion induced by a viral spike protein.     

Isolation of the envelope of vesicular stomatitis virus.

Vesicular stomatitis virus was disrupted by a combination of freezing and thawing, osmotic shock, and sonic treatment. Subviral components were separated by isopycnic centrifugation. The low-density, lipid-rich fractions were pooled and shown to contain primarily viral glycoprotein. Further purification of this material resulted in the isolation of a preparation of vesicles which contained only the G protein and the same phospholipids as in the intact virions and exhibited spikelike structures similar to those on intact vesicular stomatitis virions. We conclude that we have isolated fragments of native vesicular stomatitis virus envelopes.     

Dynamic nature of the quaternary structure of the vesicular stomatitis virus envelope glycoprotein

The envelope glycoprotein (G protein) of vesicular stomatitis virus probably exists in the viral envelope as a trimer of identical subunits. Depending on the conditions of solubilization, G protein may dissociate into monomers. G protein solubilized with the detergent octyl glucoside was shown to exist as oligomeric forms by sedimentation velocity analysis and chemical cross-linking. G protein was modified with either fluorescein isothiocyanate or rhodamine isothiocyanate. Resonance energy transfer between fluorescein and rhodamine labels was observed upon mixing the two labeled G proteins in octyl glucoside. This result provided further evidence that G protein in octyl glucoside is oligomeric and indicated that the subunits are capable of exchange to form mixed oligomers. Resonance energy transfer was independent of G protein concentration in the range examined (10-80 nM) and was not observed when labeled G proteins were mixed with fluorescein or rhodamine that was not conjugated to protein. Resonance energy transfer decreased upon incorporation of G protein into Triton X-100, consistent with sedimentation velocity data that G protein in Triton X-100 is primarily monomeric. Kinetic analysis showed that the subunit exchange reaction had a half-time of about 3 min at 27 degrees C that was independent of G protein concentration. These data indicate that the exchange occurs through dissociation of G protein trimers into monomers and dimers followed by reassociation into timers. Thus, in octyl glucoside, G protein must exist as an equilibrium between monomers and oligomers. This implies that monomers are capable of self-assembly into trimers.     

Assembly of the vesicular stomatitis virus envelope: incorporation of viral polypeptides into the host plasma membrane

Incorporation of viral polypeptides into the host plasma membrane is an essential step in the formation of the lipoprotein envelope of vesicular stomatitis virus. A quantitative study of this process was carried out using a double-isotope labeling procedure. Infected cells were incubated for two hours with ¹⁴C-labeled amino acids, pulse-labeled with [³H]leucine and incubated for various times with an excess of non-radioactive leucine. The ${}^3\text{H}{}^{14}\text{C}$ ratio was determined for each viral polypeptide in isolated plasma membranes and in the whole cell by polyacrylamide gel electrophoresis. It was found that [³H]leucine-labeled viral polypeptides could be detected in the plasma membranes immediately following a 30-second pulse but that the ${}^3\text{H}{}^{14}\text{C}$ ratios of polypeptides in the plasma membrane did not reach the ${}^3\text{H}{}^{14}\text{C}$ ratios in the whole cells until the end of a two-minute chase period. The addition of puromycin to the cultures at the end of the pulse period did not affect subsequent incorporation of [³H]leucine-labeled polypeptides into the plasma membrane. The incorporation of various amino acid analogs into the viral polypeptides did not affect the efficiency with which they were incorporated into the plasma membranes. It is proposed that viral polypeptides are selected for incorporation into the plasma membrane from a small interior pool of completed molecules.     

Organization of the vesicular stomatitis virus glycoprotein into membrane microdomains occurs independently of intracellular viral components

The glycoprotein (G protein) of vesicular stomatitis virus (VSV) is primarily organized in plasma membranes of infected cells into membrane microdomains with diameters of 100 to 150 nm, with smaller amounts organized into microdomains of larger sizes. This organization has been observed in areas of the infected-cell plasma membrane that are outside of virus budding sites as well as in the envelopes of budding virions. These observations raise the question of whether the intracellular virion components play a role in organizing the G protein into membrane microdomains. Immunogold-labeling electron microscopy was used to analyze the distribution of the G protein in arbitrarily chosen areas of plasma membranes of transfected cells that expressed the G protein in the absence of other viral components. Similar to the results with virus-infected cells, the G protein was organized predominantly into membrane microdomains with diameters of approximately 100 to 150 nm. These results indicate that internal virion components are not required to concentrate the G protein into membrane microdomains with a density similar to that of virus envelopes. To determine if interactions between the G protein cytoplasmic domain and internal virion components were required to create a virus budding site, cells infected with recombinant VSVs encoding truncation mutations of the G protein cytoplasmic domain were analyzed by immunogold-labeling electron microscopy. Deletion of the cytoplasmic domain of the G protein did not alter its partitioning into the 100- to 150-nm microdomains, nor did it affect the incorporation of the G protein into virus envelopes. These data support a model for virus assembly in which the G protein has the inherent property of partitioning into membrane microdomains that then serve as the sites of assembly of internal virion components.     

Fatty acid binding to vesicular stomatitis virus glycoprotein: a new type of post-translational modification of the viral glycoprotein.

The glycoprotein (G) of vesicular stomatitis virus (VSV) binds 1–2 moles of fatty acid per mole of protein. The fatty acids cannot be released by repeated extractions of the protein with organic solvents, nor can they be released by denaturing the protein with ionic or nonionic detergents. Pronase digestion of G yields an organic extractable fragment that contains bound fatty acid. The fatty acid is quantitatively released from this fragment and from intact G by mild alkali treatment in methanol and is identified by gas-liquid and thin-layer chromatography as, predominantly, the methyl ester of palmitic acid. Insignificant amounts of phosphate are found in G, thus ruling out the presence of bound phospholipid. Chicken embryo fibroblast pre-labeled with ³H-palmitate and then infected with VSV for 4 hr show the presence of ³H label in G but not in other viral structural proteins. The ³H label is present only in the fatty acid moiety of the protein. Much smaller amounts of ³H fatty acid are bound to G protein formed by the VSV mutant ts045 grown at the nonpermissive temperature, and no ³H fatty acid is bound to G synthesized at 37°C in cells pretreated with tunicamycin, an inhibitor of glycosylation. However, infection with the VSV-Orsay strain at 30°C in the presence of tunicamycin allows for production of VSV particles with nonglycosylated G (Gibson, Schlesinger and Kornfeld, 1979), and this G has the same proportion of the fatty acid as does the normal glycosylated G. These data indicate that fatty acids become covalently attached to the G polypeptide chain during maturation of the protein—perhaps as the glycoprotein moves to the cell's plasma membrane.     

Subunit interactions of vesicular stomatitis virus envelope glycoprotein influenced by detergent micelles and lipid bilayers.

The envelope glycoprotein (G protein) of vesicular stomatitis virus is a transmembrane protein that exists as a trimer of identical subunits in the virus envelope. We have examined the effect of modifying the environment surrounding the membrane-spanning sequence on the association of G protein subunits using resonance energy transfer. G protein subunits were labeled with either fluorescein isothiocyanate or rhodamine isothiocyanate. When the labeled G proteins were mixed in the presence of the detergent octyl glucoside, mixed trimers containing both fluorescent labels were formed as a result of subunit exchange, as shown by resonance energy transfer between the two labels. In contrast when fluorescein- and rhodamine-labeled G proteins were mixed in the presence of Triton X-100, no resonance energy transfer was observed, indicating that subunit exchange did not occur in Triton X-100 micelles. However, if labeled G proteins were first mixed in the presence of octyl glucoside, energy transfer persisted after dilution with buffer containing Triton X-100. This result indicates that the G protein subunits remained associated in Triton X-100 micelles and that the failure to undergo subunit exchange was due to lack of dissociation of G protein subunits. Chemical cross-linking experiments confirmed that G protein was trimeric in the presence of Triton X-100. The efficiency of resonance energy transfer between labeled G protein was higher when G proteins were incorporated into dimyristoylphosphatidylcholine liposomes compared to detergent micelles. This result indicates that the labels exist in a more favorable environment for energy transfer in membranes than in detergent micelles.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Molecular architecture of the bipartite fusion loops of vesicular stomatitis virus glycoprotein G, a class III viral fusion protein

The glycoprotein of vesicular stomatitis virus (VSV G) mediates fusion of the viral envelope with the host cell, with the conformational changes that mediate VSV G fusion activation occurring in a reversible, low pH-dependent manner. Based on its novel structure, VSV G has been classified as class III viral fusion protein, having a predicted bipartite fusion domain comprising residues Trp-72, Tyr-73, Tyr-116, and Ala-117 that interacts with the host cell membrane to initiate the fusion reaction. Here, we carried out a systematic mutagenesis study of the predicted VSV G fusion loops, to investigate the functional role of the fusion domain. Using assays of low pH-induced cell-cell fusion and infection studies of mutant VSV G incorporated into viral particles, we show a fundamental role for the bipartite fusion domain. We show that Trp-72 is a critical residue for VSV G-mediated membrane fusion. Trp-72 could only tolerate mutation to a phenylalanine residue, which allowed only limited fusion. Tyr-73 and Tyr-116 could be mutated to other aromatic residues without major effect but could not tolerate any other substitution. Ala-117 was a less critical residue, with only charged residues unable to allow fusion activation. These data represent a functional analysis of predicted bipartite fusion loops of VSV G, a founder member of the class III family of viral fusion proteins.     

Glycosylation sites of vesicular stomatitis virus glycoprotein.

Detailed analysis on DEAE-Sephadex of the tryptic digestion products of the glycoprotein from vesicular stomatitis virus grown in HeLa suspension cultures revealed the presence of two major and several minor sugar-labeled species. The minor tryptic glycopeptides were converted to one of the two major glycopeptide species by treatment with neuraminidase. Thus, vesicular stomatitis virus glycoprotein contains only two oligosaccharide side chains that are heterogeneous in their sialic acid content.     

Effect of certain inhibitors of glycoprotein synthesis on cell fusion induced by vesicular stomatitis virus.

The effect of certain metabolic inhibitors on the fusion of BHK-21 cells induced by vesicular stomatitis virus (VSV) was studied. The polykaryocyte formation in infected cells and virus growth were inhibited by 2-deoxy-D-glucose and D-glucosamine. Host-cell proteins synthesis was suppressed profoundly in both BHK-21-KB and B cells infected with VSV. On the other hand, glycoprotein synthesis was significantly enhanced during the polykaryocyte formation in BHK-21-KB cells, while it was suppressed in BHK-21-B cells which were not sensitive to cell fusion by VSV.     

Selective isolation of mutants of vesicular stomatitis virus defective in production of the viral glycoprotein.

We describe a procedure that enriches for temperature-sensitive (ts) mutants of vesicular stomatitis virus (VSV), Indiana serotype, which are conditionally defective in the biosynthesis of the viral glycoprotein. The selection procedure depends on the rescue of pseudotypes of known ts VSV mutants in complementation group V (corresponding to the viral G protein) by growth at 39.5 degrees C in cells preinfected with the avian retrovirus Rous-associated virus 1 (RAV-1). Seventeen nonleaky ts mutants were isolated from mutagenized stocks of VSV. Eight induced no synthesis of VSV proteins at the nonpermissive temperature and hence were not studied further. Four mutants belonged to complementation group V and resembled other ts (V) mutations in their thermolability, production at 39.5 degrees C of noninfectious particles specifically deficient in VSV G protein, synthesis at 39.5 degrees C of normal levels of viral RNA and protein, and ability to be rescued at 39.5 degrees C by preinfection of cells by avian retroviruses. Five new ts mutants were, unexpectedly, in complementation group IV, the putative structural gene for the viral nucleocapsid (N) protein. At 39.5 degrees C these mutants also induced formation of noninfectious particles relatively deficient in G protein, and production of infectious virus at 39.5 degrees C was also enhanced by preinfection with RAV-1, although not to the same extent as in the case of the group V mutants. We believe that the primary effect of the ts mutation is a reduced synthesis of the nucleocapsid and thus an inhibition of synthesis of all viral proteins; apparently, the accumulation of G protein at the surface is not sufficient to envelope all the viral nucleocapsids, or the mutation in the nucleocapsid prevents proper assembly of G into virions. The selection procedure, based on pseudotype formation with glycoproteins encoded by an unrelated virus, has potential use for the isolation of new glycoprotein mutants of diverse groups of enveloped viruses.     

Amino-terminal mutation of the vesicular stomatitis virus glycoprotein does not affect its fusion activity.

Earlier studies demonstrated that synthetic peptides corresponding to the amino terminus of the vesicular stomatitis virus glycoprotein (G protein) have a pH-dependent hemolytic activity that is thought to be related to the fusion activity of G protein (R. Schlegel and M. Wade, J. Biol. Chem. 259: 4691-4694, 1984; R. Schlegel and M. Wade, J. Virol. 53: 319-323, 1985). A single amino acid change (lysine to glutamic acid at the amino terminus) abolishes the hemolytic activity of the peptide. Here we used oligonucleotide-directed mutagenesis to create a DNA encoding G protein with this same amino acid change at its amino terminus. The mutant protein encoded by this gene was expressed transiently in a monkey fibroblast cell line (COS) and was found to have a pH-dependent fusion activity indistinguishable from wild-type G protein. This result indicates that the hemolytic activity of the synthetic peptides was not related to the fusion activity of the G protein.     

Charged residues are involved in membrane fusion mediated by a hydrophilic peptide located in vesicular stomatitis virus G protein

Membrane fusion is an essential step of the internalization process of the enveloped animal viruses. Vesicular stomatitis virus (VSV) infection is mediated by virus spike glycoprotein G, which induces membrane fusion at the acidic environment of the endosomal compartment. In a previous work, we identified a specific sequence in VSV G protein, comprising the residues 145 to 164, directly involved in membrane interaction and fusion. Unlike fusion peptides from other viruses, this sequence is very hydrophilic, containing six charged residues, but it was as efficient as the virus in catalyzing membrane fusion at pH 6.0. Using a carboxyl-modifying agent, dicyclohexylcarbodiimide (DCCD), and several synthetic mutant peptides, we demonstrated that the negative charges of peptide acidic residues, especially Asp¹⁵³ and Glu¹⁵⁸, participate in the formation of a hydrophobic domain at pH 6.0, which is necessary to the peptide-induced membrane fusion. The formation of the hydrophobic region and the membrane fusion itself were dependent on peptide concentration in a higher than linear fashion, suggesting the involvement of peptide oligomerization. His¹⁴⁸ was also necessary to hydrophobicity and fusion, suggesting that peptide oligomerization occurs through intermolecular electrostatic interactions between the positively-charged His and a negatively-charged acidic residue of two peptide molecules. Oligomerization of hydrophilic peptides creates a hydrophobic region that is essential for the interaction with the membrane that results in fusion.     

Charged residues are involved in membrane fusion mediated by a hydrophilic peptide located in vesicular stomatitis virus G protein

Membrane fusion is an essential step of the internalization process of the enveloped animal viruses. Vesicular stomatitis virus (VSV) infection is mediated by virus spike glycoprotein G, which induces membrane fusion at the acidic environment of the endosomal compartment. In a previous work, we identified a specific sequence in VSV G protein, comprising the residues 145 to 164, directly involved in membrane interaction and fusion. Unlike fusion peptides from other viruses, this sequence is very hydrophilic, containing six charged residues, but it was as efficient as the virus in catalyzing membrane fusion at pH 6.0. Using a carboxyl-modifying agent, dicyclohexylcarbodiimide (DCCD), and several synthetic mutant peptides, we demonstrated that the negative charges of peptide acidic residues, especially Asp153 and Glu158, participate in the formation of a hydrophobic domain at pH 6.0, which is necessary to the peptide-induced membrane fusion. The formation of the hydrophobic region and the membrane fusion itself were dependent on peptide concentration in a higher than linear fashion, suggesting the involvement of peptide oligomerization. His148 was also necessary to hydrophobicity and fusion, suggesting that peptide oligomerization occurs through intermolecular electrostatic interactions between the positively-charged His and a negatively-charged acidic residue of two peptide molecules. Oligomerization of hydrophilic peptides creates a hydrophobic region that is essential for the interaction with the membrane that results in fusion.