Viral products in cells infected with Vesicular Stomatitis virus and superinfected with Rous Sarcoma virus (original) (raw)
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Journal of Virology, 1977
We report here an in vitro system designed to study the interactions of vesicular stomatitis virus (VSV) proteins with cellular membranes. We have synthesized the VSV nucleocapsid (N) protein, nonstructural (NS) protein, glycoprotein (G protein), and membrane (M) protein in a wheat germ, cell-free, protein-synthesizing system directed by VSV 12 to 18S RNA. When incubated at low salt concentrations with purified cytoplasmic membranes derived from Chinese hamster ovary cells, the VSV M and G proteins bind to membranes, whereas the VSV N and NS proteins do not. The VSV M protein binds to membranes in low or high divalent cation concentrations, whereas binding of significant amounts of G protein requires at least 5 mM magnesium acetate concentrations. Vesicular stomatitis virus (VSV) is a simple, lar membranes, whereas the VSV N and NS enveloped virus that contains two membrane proteins do not. proteins: the glycoprotein (G protein), which forms the spikes of the virion (4, 23), and the MATERIALS AND METHODS membrane (M) protein, which lines the inner Cells and viruses. Membranes were prepared surface of the viral membrane (3). There are from CHO cells. Stocks of VSV (pure B particles of three other known viral proteins, the VSV nuthe Indiana serotype) were grown in CHO cells and cleocapsid (N) protein, the nonstructural (NS) purified as described previously (20). protein, and the viral transcriptase (L) protein. Preparation of VSV 12-186 polyribosomal RNA. These three proteins are associated with the The procedure described by Palmiter (15) was used core ofthe virus particle (16, 23, 24). Each of the with several modifications for the preparation of five viral proteins is synthesized from a mono-VSV 12-18S polyribosomal RNA. CHO cells growing cistronic mRNA (9, 13, 14). at 37°C were infected with VSV at a multiplicity of 3 risngtic m arNAl(9 13, 14). PFU/cell as described previously (20), except that 5 During the early stages in the maturation Of~.tg of actinomycin D per ml was added at the beginthis virus, host cell membranes are modified ning of infection. [3H]uridine (70 Ci/mmol, 25 ,uCi/ with the VSV G and M proteins. Cell fractionaml; New England Nuclear Corp.) was added 2 h tion studies of VSV-infected cells have shown postinfection. Infected cells were harvested at 4.5 h that the VSV M and G proteins rapidly become postinfection, suspended in sucrose-TKM buffer associated with the membrane fraction of the (0.05 M Tris [pH 7.5], 0.025 M KCl, 0.005 M magnecells after their synthesis (5, 11, 12, 24). Nucleo-sium acetate, 0.25 M sucrose), and disrupted with 10 capsid structures containing the genome RNA strokes of a tight-fitting Dounce homogenizer. Nuas well as the viral N, NS, and L proteins are clei were removed by centrifugation (1,000 x g for 2 asswemlld as the vira plaNsm. andsLptroteisures min). The resulting cytoplasmic extract was centriassembled in the cytoplasm. These structures fuged at 20,000 x g for 20 min, and the pellet (mem
Virology, 1996
The matrix (M) protein of vesicular stomatitis virus (VSV) functions in virus assembly and also appears to be involved in the inhibition of host gene expression that is a characteristic cytopathic effect of VSV infection. Previous studies have shown that expression of M protein inhibits host-directed transcription in the absence of other viral gene products and have suggested that only small amounts of M protein are required for the inhibition. In experiments described here, the potency of M protein in inhibition of host-directed gene expression was determined by cotransfecting different amounts of in vitro-transcribed M protein mRNA together with a target gene encoding chloramphenicol acetyl transferase (CAT) into BHK cells or PC12 cells that had been cultured in the presence or the absence of nerve growth factor. The results of these experiments showed that the potency of M protein was similar in the two cell types and was not affected by the extent of differentiation of PC12 cells. Inhibition of CAT gene expression by M protein was also independent of the nature of the promoter activating sequences of several different RNA polymerase II-dependent promoters. The amount of M protein needed to give 50% inhibition of CAT expression was estimated to be 6700-11,000 copies per cell. Earlier data that temperature-sensitive (ts) M gene mutants of VSV inhibit host transcription had been interpreted to indicate that M protein was not involved in the inhibition. When the amount of M protein expressed was taken into account, ts M protein was as effective as wild-type M protein in the inhibition of host-directed transcription at the nonpermissive temperature. Thus, inhibition of host transcription by ts M mutants of VSV is due to the potent activity of M protein, which is evident even at the low levels produced at the nonpermissive temperature. ᭧
Journal of virology, 1977
Maturation of viral proteins in cells infected with mutants of vesicular stomatitis virus was studied by surface iodination and cell fractionation. The movement of G, M, and N proteins to the virion bud appeared to be interdependent. Mutations thought to be in G protein prevented its migration to the cell surface, allowed neither M nor N protein to become membrane bound, and blocked formation of viral particles. Mutant G protein appeared not to leave the endoplasmic reticulum at the nonpermissive temperature, but this defect was partially reversible. In cells infected with mutants that caused N protein to be degraded rapidly or prevented its assembly into nucleocapsids, M protein did not bind to membranes and G protein matured to the cell surface, but never entered structures with the density of virions. Mutations causing M protein to be degraded prevented virion formation, and G protein behaved as in cells infected by mutants in N protein. These results are consistent with a model ...
Archives of Virology, 1987
Infection of L929 routine cells with vesicular stomatitis virus (VSV) results in inhibition of host protein synthesis and appearance of membrane alterations at a time when cells are still actively engaged in viral protein synthesis. VSV temperature-sensitive (ts) mutants have been used to explore the role(s) played by the virus-coded proteins in the genesis of these effects. Cells were injected with each of five ts mutants representing the known complementation groups of VSV Indiana serotype, and incubated at permissive (32 °C) and non-permissive temperatures (39 °C). Protein synthesis in the presence and absence of Hygromycin B (Hyg.B) was analyzed during virus infection via incorporation of aSS-methionine in acid-precipitable materiM and SDS-polyacrylamide gel electrophoresis.