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.     

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.     

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.     

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.     

Enhanced Gene Transfer with Fusogenic Liposomes Containing Vesicular Stomatitis Virus G Glycoprotein

Exposure of Lipofectin-DNA complexes to the partially purified G glycoprotein of the vesicular stomatitis virus envelope (VSV-G) results in loss of serum-mediated inhibition and in enhanced efficiency of gene transfer. Sucrose density gradient sedimentation analysis indicated that the VSV-G associates physically with the DNA-lipid complex to produce a VSV-G liposome. The ability to incorporate surrogate viral or cellular envelope components such as VSV-G into liposomes may allow more-efficient and possibly targeted gene delivery by lipofection, both in vitro and in vivo.     

Protein Kinase and Phosphoproteins of Vesicular Stomatitis Virus

Protein kinases of similar but not identical activity were found associated with vesicular stomatitis (VS) virions grown in mouse L cells, primary chicken embryo (CE) cells, and BHK-21 cells, as well as being present in VS virions grown in HeLa and Aedes albopictus cells. The virion kinase preferentially phosphorylated the nucleocapsid NS protein in vitro and to a lesser extent the envelope M protein. Other virion proteins were phosphorylated in vitro only after drastic detergent treatment. Partial evidence that the virion kinase is of cellular origin was obtained by finding reduced enzyme activity in virions released from cells pretreated with actinomycin D and cycloheximide. Selective detergent and detergent-salt fractionation of VS virions revealed that the kinase activity was present in the envelope but not the spikes. The virion kinase activity in a Triton-salt-solubilized envelope fraction could be separated from M and G proteins and partially purified by phosphocellulose column chromatography. Virions released from L, CE, and BHK-21 cells infected in the presence of [(32)P]orthophosphate were labeled almost exclusively in the NS protein. Both soluble and nucleocapsid-associated NS phosphoprotein were present in cytoplasmic extracts of VS viral-infected L cells. The origin and function of the NS phosphoprotein remain to be elucidated.     

Entry of Vesicular Stomatitis Virus into L Cells

Early stages of the entry of vesicular stomatitis (VS) virus into L cells were followed by electron microscopy with the aid of ferritin antibody labeling. Cells which were infected at 0 C and incubated for 10 min at 37 C were reacted first with antiviral-antiferritin hybrid antibody and then with ferritin or fluorescein-labeled apoferritin. Extensive ferritin labeling of the cell surface was detected by both electron and fluorescence microscopy. The labeled regions of the cell surface were continuous with and indistinguishable from the rest of the host cell membrane, suggesting incorporation of viral antigens into the cell surface during viral penetration. Fusion of parental viral membrane with host cell membrane was further demonstrated by examining the localization of (3)H-labeled viral structural proteins in cells infected at 0 C and incubated for short periods at 37 C. Viral nucleoprotein was found in a soluble fraction of the cells which was derived primarily from the cytoplasm, whereas a particulate fraction from the cells was enriched in viral envelope proteins. Cytoplasmic membrane was isolated from these cells, and this membrane contained viral envelope proteins. These results suggest that penetration by VS virus occurs by fusion of the viral and cellular membranes followed by release of nucleo-protein into the cytoplasm.