Structural Proteins of Vesicular Stomatitis Viruses

Three major and three minor structural proteins were identified by polyacrylamide gel electrophoresis of purified infectious virions of the Indiana serotype of vesicular stomatitis (VS) virus disrupted with acetic acid, 0.5 m urea, sodium dodecyl sulfate (SDS), and 2-mercaptoethanol. Molecular weights of the six virion proteins were estimated by comparative electrophoretic migration of known marker proteins in the presence of SDS. The following values were obtained: major proteins P6 congruent with 34,500, P5 congruent with 59,500, and P4 congruent with 81,500; minor proteins P3 congruent with 140,000, P2 congruent with 186,000, and P1 congruent with 275,000. P1 did not disaggregate in 8 m urea, but P2 and P3 did. The possibility that P1 is an uncleaved large polypeptide chain could not be ruled out. Six identical protein components were dissociated from Indiana VS virions grown in chick and mouse cells; no cellular proteins could be detected in purified virions. Of six proteins identified in virions of the New Jersey serotype, only the smallest protein (P6) could be distinguished from any of the six proteins of the Indiana serotype on the basis of migration in SDS gels. The defective T particles of Indiana VS virus contained the same six proteins in essentially the same proportions as those of the infectious B virions. Only P6 and P5 could be cleanly separated by preparative gel electrophoresis.     

Proteins of Vesicular Stomatitis Virus and of Phenotypically Mixed Vesicular Stomatitis Virus-Simian Virus 5 Virions

The identity of the glycoprotein of vesicular stomatitis virus (VSV) as the spike protein has been confirmed by the removal of the spikes with a protease from Streptomyces griseus, leaving bullet-shaped particles bounded by a smooth membrane. This treatment removes the glycoprotein but does not affect the other virion proteins, apparently because they are protected from the enzyme by the lipids in the viral membrane. The proteins of phenotypically mixed, bullet-shaped virions produced by cells mixedly infected with VSV and the parainfluenza virus simian virus 5 (SV5) have been analyzed by polyacrylamide gel electrophoresis. These virions contain all the VSV proteins plus the two SV5 spike proteins, both of which are glycoproteins. The finding of the SV5 spike glycoproteins on virions with the typical morphology of VSV indicates that there is not a stringent requirement that only the VSV glycoprotein can be used to form the bullet-shaped virion. On the other hand, the SV5 nucleocapsid protein and the major non-spike protein of the SV5 envelope were not detected in the phenotypically mixed virions, and this suggests that a specific interaction between the VSV nucleocapsid and regions of the cell membrane which contain the nonglycosylated VSV envelope protein is necessary for assembly of the bullet-shaped virion.     

Serological Characterization of the Three Major Proteins of Vesicular Stomatitis Virus

The three major proteins of vesicular stomatitis virus-Indiana, glycoprotein (G), nucleoprotein (N), and membrane protein (M), were isolated and characterized by means of specific monocomponent antisera. G, N, and M proteins are distinct, nonrelated antigens with specific serological properties. The G protein is the only antigen inducing the formation of virus-neutralizing antibodies and was shown to confer immunity to mice. Specific complement-fixing and precipitating activity was demonstrated for each of the three antisera. The future use of isolated rhabdovirus components and of monospecific antisera is considered for therapeutic and diagnostic purposes as well as for virus strain differentiation and classification work.     

Impact of Vesicular Stomatitis Virus M Proteins on Different Cellular Functions

Three different matrix (M) proteins termed M1, M2 and M3 have been described in cells infected with vesicular stomatitis virus (VSV). Individual expression of VSV M proteins induces an evident cytopathic effect including cell rounding and detachment, in addition to a partial inhibition of cellular protein synthesis, likely mediated by an indirect mechanism. Analogous to viroporins, M1 promotes the budding of new virus particles; however, this process does not produce an increase in plasma membrane permeability. In contrast to M1, M2 and M3 neither interact with the cellular membrane nor promote the budding of double membrane vesicles at the cell surface. Nonetheless, all three species of M protein interfere with the transport of cellular mRNAs from the nucleus to the cytoplasm and also modulate the redistribution of the splicing factor. The present findings indicate that all three VSV M proteins share some activities that interfere with host cell functions.     

Protein Composition of the Structural Components of Vesicular Stomatitis Virus

Digitonin, a sterol glycoside which complexes with cholesterol, stripped off the envelope of vesicular stomatitis (VS) virions and liberated two viral structural proteins, 83% of P6 and 53% of P4. Deoxycholate also disrupted VS virions but released nucleocapsid cores which could be identified by higher buoyant density, ratio of incorporated (3)H-uridine to (14)C-protein, and electron microscopy. The major nucleocapsid protein was P5 but varying amounts of the minor protein aggregate P2 were present, depending on the concentration of urea used for extraction. P2 appeared to be a polymer of P5. Two other minor structural proteins, P1 and P3, could not be located in the virion. From these data, we conclude that the three microscopically identifiable structures of VS virions are each composed primarily of a single major protein, as follows: P6 = envelope protein, P4 = protein of underlying "shell," and P5 = nucleocapsid protein.     

Temperature-Sensitive Mutants of Vesicular Stomatitis Virus: Synthesis of Virus-Specific Proteins

Viral proteins synthesized in L cells infected with temperature-sensitive (ts) mutants of vesicular stomatitis (VS) virus at permissive (31 C) and nonpermissive (39 C) temperatures were compared by polyacrylamide gel electrophoresis. Mutant ts 5, deficient in synthesis of viral ribonucleic acid (RNA⁻), failed to synthesize any of the five identifiable viral proteins at 39 C. Each of three RNA⁺ mutants, representing three separate complementation groups, showed distinctive patterns of viral protein synthesis at nonpermissive temperature. Equivalent amounts of ³H-amino acids were incorporated into the five viral proteins made in cells infected with RNA⁺ mutant ts 45 at 31 and 39 C. Complete virions of ts 45 could be identified by electron microscopy of infected cells incubated at the nonpermissive temperature; the defect in ts 45 appeared to be due in part to greater thermolability of virions as compared with the wild-type. RNA⁺ mutant ts 23 was deficient in synthesis of viral envelope protein S and failed to make detectable virions at the nonpermissive temperature. Infection of cells at 39 C with the third RNA⁺ mutant, ts 52, resulted in synthesis of all five viral proteins, but the peak of radioactivity representing the viral membrane glycoprotein migrated more rapidly on gels than coelectrophoresed authentic virion ¹⁴C-glycoprotein or viral ³H-glycoprotein extracted from cells infected at 31 C. These data and results of experiments on incorporation of radioactive glucosamine suggest that the primary defect in mutant ts 52 at nonpermissive temperature is failure of glycosylation of the viral glycoprotein. The viral structural proteins made in cells infected with ts 52 at the nonpermissive temperature did not assemble into sedimentable components as they did at permissive temperature; this observation indicates failure of insertion of the nonglycosylated protein (G′) into cell membrane. In support of this hypothesis was the finding that antiviral-antiferritin hybrid antibody did not detect VS viral antigen on the plasma membrane of L cells infected at 39 C with ts 52. In contrast, VS viral antigen localized in plasma membrane of L cells infected at 39 C with mutants ts 23 and ts 45 was readily detected by electron microscopy and fluorescence microscopy.     

Cellular Proteins Associated with the Interior and Exterior of Vesicular Stomatitis Virus Virions

Virus particles (virions) often contain not only virus-encoded but also host-encoded proteins. Some of these host proteins are enclosed within the virion structure, while others, in the case of enveloped viruses, are embedded in the host-derived membrane. While many of these host protein incorporations are likely accidental, some may play a role in virus infectivity, replication and/or immunoreactivity in the next host. Host protein incorporations may be especially important in therapeutic applications where large numbers of virus particles are administered. Vesicular stomatitis virus (VSV) is the prototypic rhabdovirus and a candidate vaccine, gene therapy and oncolytic vector. Using mass spectrometry, we previously examined cell type dependent host protein content of VSV virions using intact (“whole”) virions purified from three cell lines originating from different species. Here we aimed to determine the localization of host proteins within the VSV virions by analyzing: i) whole VSV virions; and ii) whole VSV virions treated with Proteinase K to remove all proteins outside the viral envelope. A total of 257 proteins were identified, with 181 identified in whole virions and 183 identified in Proteinase K treated virions. Most of these proteins have not been previously shown to be associated with VSV. Functional enrichment analysis indicated the most overrepresented categories were proteins associated with vesicles, vesicle-mediated transport and protein localization. Using western blotting, the presence of several host proteins, including some not previously shown in association with VSV (such as Yes1, Prl1 and Ddx3y), was confirmed and their relative quantities in various virion fractions determined. Our study provides a valuable inventory of virion-associated host proteins for further investigation of their roles in the replication cycle, pathogenesis and immunoreactivity of VSV.     

Model for Vesicular Stomatitis Virus

Vesicular stomatitis virus contains single-stranded ribonucleic acid of molecular weight 3.6 x 10(6) and three major proteins with molecular weights of 75 x 10(3), 57 x 10(3), and 32.5 x 10(3). The proteins have been shown to be subunits of the surface projections, ribonucleoprotein, and matrix protein, respectively. From these values and from estimates of the proportions of the individual proteins, it has been calculated that the virus has approximately 500 surface projections, 1,100 protein units on the ribonucleoprotein strand, and 1,600 matrix protein units. Possible models of the virus are proposed in which the proteins are interrelated.     

Infective Virus Substructure from Vesicular Stomatitis Virus

Treatment of suspensions of vesicular stomatitis virus with Tween-ether results in a rapid and considerable loss of infectivity (ca. 4 logs in 2 min), but the residual infectivity is comparatively stable to further treatment with ether. The infectivity remaining after the short exposure to Tween-ether is not due to virus for the following reasons. (i) It is much less infective for tissue cultures than for mice, whereas the intact virion is equally infective for both hosts. (ii) The residual infectivity is much less stable than virus infectivity in both sucrose and tartrate gradients. (iii) Virus immune serum does not neutralize its activity. (iv) The infectivity is associated with material which sediments further in sucrose gradients and has a greater buoyant density in tartrate gradients than the virion. Experiments with (32)P-labeled virion showed that the infective substructure contains ribonucleic acid with the same sedimentation characteristics as that extracted from the virion. Electron microscopy shows that the infective component has the same overall bullet-like structure as the virion but lacks the outer envelope and fringe structure.     

In Vitro Synthesis of Proteins by Membrane-Bound Polyribosomes from Vesicular Stomatitis Virus-Infected HeLa Cells

Membrane-bound polysomes from vesicular stomatitis virus (VSV)-infected HeLa cells synthesize predominantly three proteins in an in vitro protein synthesizing system. These three proteins have different molecular weights than the viral structural proteins, i.e., 115,000, 88,000, and 72,000. Addition of preincubated L or HeLa cell S10 or HeLa cell crude initiation factors stimulates amino acid incorporation and, furthermore, alters the pattern of proteins synthesized. Stimulated membrane-bound polysomes synthesize predominantly viral protein G and lesser amounts of N, NS, and M. In vitro synthesized proteins G and N are very similar to virion proteins G and N based on analysis of tryptic methionine-labeled peptides. Most methionine-labeled tryptic peptides of virion G protein contain no carbohydrate moieties, since about 90% of sugar-labeled peptides co-chromatograph with only about 10% of methionine-labeled peptides. Sucrose gradient analysis of the labeled RNA present in VSV-infected membrane-bound polysomes reveals a relative enrichment in a class of viral RNA sedimenting slightly faster than the total population of the 13 to 15S mRNA, as compared to a VSV-infected crude cytoplasmic extract. A number of proteins, other than the viral structural proteins, are synthesized in the cytoplasm of five lines of VSV-infected cells. One of these proteins has the same molecular weight as the major in vitro synthesized protein, P(88). In vitro synthesized protein P(88) does not appear to be a precursor of viral structural proteins G, N, or M based on pulse-chase experiments and tryptic peptide mapping. Nonstimulated membrane-bound polysomes from uninfected HeLa cells synthesize the same size distribution of proteins as nonstimulated VSV-infected membrane-bound polysomes.     

Carbohydrate Composition of Vesicular Stomatitis Virus

Analysis by gas-liquid chromatography of the trimethylsilylated sugar residues of purified vesicular stomatitis virus grown in L cells or chick embryo cells revealed the presence in the whole virion of four hexoses (glucose, galactose, mannose, and fucose), two hexosamines (glucosamine and galactosamine), and 34 to 40% neuraminic acid. The isolated viral glycoprotein was devoid of galactosamine and fucose, both of which sugars were present in whole virions presumably as part of the membrane glycolipids.     

Phenotypic Mixing of Envelope Proteins of the Parainfluenza Virus SV5 and Vesicular Stomatitis Virus

Cells mixedly infected with parainfluenza virus SV5 and vesicular stomatitis virus (VSV) yield phenotypically mixed virions, in addition to both parental types. Two types of phenotypically mixed virions have been identified: 0.6 to 1.2% of the VSV plaque formers were neutralized by SV5 antiserum, but not by VSV antiserum, suggesting the presence of a VSV genome in an SV5 envelope; 9 to 45% of the VSV plaque formers were neutralized by both antisera, indicating the presence of both SV5 and VSV antigens in their envelopes. The presence of SV5 antigen in virions with the typical bullet-shaped appearance of VSV was confirmed with ferritin-labeled anti-SV5 antibody. In contrast to standard VSV, phenotypically mixed virions adsorbed to and eluted from chicken erythrocytes, indicating that these virions contained in their envelopes SV5 hemagglutinin, and possibly neuraminidase. Thus, the VSV nucleocapsid can interact with membranes which contain SV5 proteins in the manner which leads to virus maturation, and the production of a high yield of phenotypically mixed virions with the morphology of VSV indicates that this process can function efficiently. No evidence of genetic recombination between the two viruses was found. These results raise the possibility of an evolutionary relatedness between the paramyxoviruses and the rhabdoviruses.     

Proteins of Vesicular Stomatitis Virus: II. Immunological Comparisons of Viral Antigens

The antigens of the nucleoprotein core and the coat of vesicular stomatitis virus (VSV) particles of the Indiana serotype were prepared and purified by sucrose gradient fractionation. Antibody was prepared separately to each of the two antigen fractions. By immunological procedures, it was shown that soluble antigens of VSV preparations sedimenting at 20S and in the leading edge of the 6S region are antigenically related to VP3, the protein of the virus core, whereas the 6S soluble antigen cross-reacts only with viral coat antibodies. These results confirm previous results obtained by polyacrylamide gel analysis of the antigens. It has further been demonstrated that the 6S antigen is a glycoprotein. Comparing antigens of the New Jersey and Indiana serotype showed that the coat antigens of virus particles and the 6S antigen are immunologically distinct in the two serotypes. In complement-fixation tests, the core antigens and the soluble 20S antigens from one serotype showed a cross-reaction with antiserum prepared against core proteins of the other serotype.     

Association of Ribosomes with Intracellular Vesicular Stomatitis Virus Particles

Ribosomes are observed intimately associated with nucleocapsids of vesicular, stomatitis virus, especially those that line structures that are either cytoplasmic vesicles or invaginations of the plasma membrane.     

Inactivation of Vesicular Stomatitis Virus by Disinfectants

Twenty-four chemical disinfectants considered to be viricidal were tested. Ten disinfectants were not viricidal for vesicular stomatitis virus within 10 min at 20 C when an LD(50) titer of 10(8.5) virus units per 0.1 ml were to be inactivated. Quantitative inactivation experiments were done with acid, alkaline, and a substituted phenolic disinfectant to determine the kinetics of the virus inactivation. Substituted phenolic disinfectants, halogens, and cresylic and hydrochloric acids were viricidal. Basic compounds such as lye and sodium metasilicate were not viricidal.     

Morphogenesis of the Nucleoprotein of Vesicular Stomatitis Virus

Accumulation of the nucleoprotein of vesicular stomatitis virus (VSV) in the cytoplasm of BHK-21 cells and in two of four human cell lines was demonstrated. Appearance and progression of the nucleoprotein inclusions paralleled development of virus-specific immunofluorescence and production of virus progeny. The inclusions appeared early as discrete foci of filamentous material which eventually increased in size to form large masses which replaced normal cytoplasmic constituents. The filamentous strands were found in close proximity to budding virions. The inclusion material was extracted from infected cells and purified in cesium chloride gradients. The isolated filaments resembled the ribonucleoprotein isolated from purified virions. They incorporated (3)H-uridine, exhibited virus-specific complement-fixing activity, had a buoyant density of 1.32 g/cm(3), and appeared as single wavy strands the width of which varied from 2.5 to 8.5 nm, depending on the angle of viewing.