Inhibition of RNA and protein synthesis in interferon-treated HeLa cells infected with vesicular stomatitis virus

Synthesis of vesicular stomatitis virus RNA and protein is almost completely inhibited in HeLa cells treated with relatively high doses of human fibroblast interferon. With lower concentrations of interferon virus replication is inhibited, but near normal amounts of virus RNA are found in cells infected at a m.o.i. of 10. All the virus RNA species are found in these cells with the exception of genomic size RNA. In contrast, synthesis of all the virus proteins is equally inhibited in proportion to the interferon concentration used to treat the cells. This inhibition is due to a decline in the rate of protein synthesis, which occurs in interferon-treated cells sooner after infection than in untreated cells. The decreased rate of protein synthesis is accompanied by a change in the polysome pattern of infected cells, characterized by polysome run-off and increase in 80S ribosomes. At the same time, a larger proportion of the virus poly(A)-containing RNA is not associated with polysomes in interferon-treated cells than in control cells. The non-polysomal virus RNA has a sedimentation rate identical with that of polysomal virus RNA. Possible causes for the decline in the rate of protein synthesis observed in interferon-treated cells are discussed.     

RNA synthesis in BHK 21 cells persistently infected with vesicular stomatitis virus and rabies virus.

Virus-induced RNA synthesis was studied in BHK 21 cells persistently infected with vesicular stomatitis virus (VSV) and rabies virus by labelling RNA synthesized in the presence of antinomycin D. During persistent infection the species of messenger RNA synthesized were similar in size and relative proportions to those seen during acute infection, but there were some minor differences. Full-sized B virion RNA was generally not detected during persistent infection, and new species (probably DI virion RNA) appeared.     

Control of vesicular stomatitis virus protein synthesis.

The five structural polypeptides of vesicular stomatitis virus are synthesized in infected cells at nonequimolar rates which correspond to their relative amounts in the virus particle. These experiments are concerned with the mechanism(s) responsible for the relative rates of viral polypeptide synthesis.Treatment of infected cells with amino acid analogs in order to prevent formation of functional viral proteins resulted in inhibition of viral genome replication and virion assembly. This treatment, however, did not alter the relative rates of viral polypeptide synthesis. This suggested that the mechanisms determining these rates do not require the function of newly-synthesized viral proteins and do not involve direct coupling between protein synthesis and virion assembly.Relative translation frequencies of viral messenger RNA molecules coding for each polypeptide were compared by treating cells with low levels of cycloheximide and anisomycin, inhibitors of protein synthesis that interfere preferentially with polypeptide chain elongation. Under these conditions the rate of synthesis of each viral polypeptide should be proportional only to the amount of its messenger RNA available for translation. The relative rates of viral polypeptide synthesis were not altered by this treatment, suggesting that viral messenger RNA molecules coding for each polypeptide are translated with similar average frequencies.Rates of peptide chain growth were compared by following the incorporation of ³H-amino acids into completed viral polypeptides following a pulse-label. The time necessary to achieve maximum incorporation of isotope into each completed viral polypeptide was taken as its translation time, the time necessary to synthesize and release a completed molecule. We were able to determine translation times for the M and G polypeptides by this method and found that they correlated directly with their molecular weights, suggesting that nascent chains of these two polypeptides are propagated at similar rates.     

Distribution of M protein and nucleocapsid protein of vesicular stomatitis virus in infected cell plasma membranes

The association of M protein and the nucleocapsid (N) protein of vesicular stomatitis virus (VSV) with the cytoplasmic surface of plasma membranes prepared from infected cells was examined by double label immunofluorescence. M protein in association with the cytoplasmic surface of the plasma membrane was distributed in two distinct labeling patterns. Punctate labeling of M protein in the plasma membrane was observed in association with corresponding labeling for the nucleocapsid protein. Diffusely labeled M protein was distributed in areas of the plasma membrane that were devoid of any detectable labeling for the nucleocapsid protein. Similar results were obtained with two different cell types at 4 h and later times postinfection. The diffuse label for M protein was present in membranes prepared from cells infected with a temperature-sensitive M protein mutant at the nonpermissive temperature, but neither the punctate label for M protein nor labeling for the nucleocapsid protein was observed. Upon shift to permissive temperature, both the punctate label for M protein and labeling for the nucleocapsid protein began to reappear in membranes prepared from cells infected with the M protein mutant. These results indicate that M protein can associate with the plasma membrane without prior binding to nucleocapsids and that association of functional M protein with the plasma membrane is required for the stable association of nucleocapsids with the membrane during the process of viral budding.     

Lack of correlation between the accumulation of plus-strand leader RNA and the inhibition of protein and RNA synthesis in vesicular stomatitis virus infected mouse L cells

The inhibition of protein synthesis in mouse L cells infected by vesicular stomatitis virus (VSV) requires expression of two regions (one large and one small) of the viral genome, as determined by target size analysis. The inhibition of host RNA synthesis was also shown to be dependent on expression of two regions of the VSV genome, most likely the same ones. In some cases, such as in cells infected by mutants T1026R1, or tsG41 at 40 degrees, or moderately uv irradiated VSV, only one of the two regions was expressed, yet cellular protein and RNA synthesis was decreased. This suggests that the product of each region of the viral genome can act independently. In these instances the severity of the inhibition was dependent on both the length of the infection period and the multiplicity of infection. The identity of neither gene product is known, but it has been suggested that small product is plus-strand leader RNA. As shown herein, however, there was no correlation between the extent of host macromolecular synthesis inhibition and the quantity of leader RNA in infected cells.     

Studies on tumorigenicity of cells persistently infected with vesicular stomatitis virus in nude mice

Previous work has demonstrated that persistent infection of cultured cells (normally tumorigenic in the nude mouse) by any of a number of enveloped RNA viruses results in the loss of their ability to form tumors in nude mice. From the BHK 21/VSV persistently infected cell line which did not form tumors in these nude mice, we selected in vivo a subline which is tumorigenic even though it is still persistently infected. We report here a preliminary characterization of this subline. These virus-infected cells were consistently tumorigenic for more than six sequential passages in nude mice. They shed large amounts of virus and DI particles into the bloodstream and tissues of the mice; however, we found no evidence of the virus establishing a persistent infection in the mouse tissues. We demonstrated that virus isolated from this tumorigenic cell line was able to establish new persistently infected cell lines which were also tumorigenic, indicating that this is a characteristic of the virus. T₁ oligonucleotide mapping of the virion RNA recovered from this tumorigenic carrier subline demonstrates that the viral genome is undergoing continual mutation in vivo. This is similar to mutational changes occurring in vitro in the parental persistently infected cells. Very recently, the BHK 21 cell line persistently infected with VSV for over 5 years has spontaneously (with no in vivo selection) acquired the ability to form tumors in nude mice. In contrast to the other tumorigenic subline, this carrier line produces almost no mature virus or DI particles at present, and its tumorigenicity appears to be associated with very low virus expression in infected cells. Apparently, viruses can escape the natural killer (NK) cell response by a variety of means.     

Sequential disassembly of the cytoskeleton in BHK21 cells infected with vesicular stomatitis virus

The cytopathic effects of vesicular stomatitis virus (VSV) that result in the rounding of BHK21 cells have been studied. The results indicate that they are mediated by a sequential alteration in the distribution of the components of the cytoskeleton, an effect that requires the expression of the viral L protein. The constituents of the cytoskeleton of BHK21 cells were analyzed by fluorescence microscopy. Actin filaments were the first component to become disorganized, so that disassembly of stress fibers were detected 1 hr after infection. The distribution of microtubules and intermediate filaments was unchanged at 2 hr after infection; however, both these cytoskeletal elements exhibited an altered distribution at 3-4 hr after infection. Actinomycin D and cycloheximide did not cause the same effects as infection with VSV, suggesting that inhibition of host-cell gene expression was not responsible. However, viral gene expression was required, since cells infected with uv-irradiated VSV showed the same distribution of cytoskeletal constituents as mock-infected controls. Cells infected at 39.5 degrees (the nonpermissive temperature) with mutants of VSV temperature sensitive in the viral NS (ts G22), N(ts G41), M(ts 0 23), and G(ts 0 45) proteins showed the same changes in the cytoskeleton as those detected with wild-type virus. In contrast, cells infected with ts G11 (L-) showed the characteristic effect of VSV on the cytoskeleton when incubated at 34 degrees (the permissive temperature), but not when incubated at 39.5 degrees. The T-1026 R1 mutant of VSV, which has a much less dramatic effect on cell morphology than wild-type virus, also caused a less marked disruption of the cytoskeleton.     

Inhibition of SV40 DNA synthesis by vesicular stomatitis virus in doubly infected monkey kidney cells

Vesicular stomatitis virus (VSV) inhibited SV40 DNA synthesis in doubly infected synchronized Vero cells. Gel-electrophoretic profiles demonstrated that SV40 DNA monomers accumulated in all stages of supercoiling, regardless of whether cells were superinfected with VSV early or late in the S phase. These gel profiles were indistinguishable from ones obtained from SV40-infected, cycloheximide-treated cells in the absence of VSV infection. Radiolabel in the partial supercoils could be chased into supercoils, but only by restoring protein synthesis. The relative rates of SV40 DNA chain elongation were determined in VSV-superinfected and nonsuperinfected cells. The gradients of 3H incorporation as a function of distance from the origin of replication in pulse-labeled form I DNA were unaffected by VSV. It is concluded that VSV inhibition of SV40 DNA synthesis is an indirect result of inhibition of host cell protein synthesis and it is suggested that incompletely supercoiled SV40 chromatin is not a good template for DNA synthesis.     

Double-layered membrane vesicles released from mammalian cells infected with Sendai virus expressing the matrix protein of vesicular stomatitis virus.

The matrix (M) protein of vesicular stomatitis virus (VSV) was reported to form vesicles on the cell surface and subsequently to be released into the cultured medium when expressed from cDNA by virus vectors. To further investigate VSV M activity, we generated a recombinant Sendai virus (SeV) expressing the VSV M protein (SeV-M(VSV)). When cells were infected with SeV-M(VSV), VSV M was found abundantly in the culture medium. Electron microscopy demonstrated the budding of two-membraned vesicles (>/= 0.8 microm in diameter) from the infected cells. The outer membrane of the vesicle was derived from the plasma membrane and the inner one possibly derived from the membrane of an intracellular vesicle. Immuno-gold labeling showed that VSV M was exclusively located in a double-layered region. The released membranes were divided into three parts: the VSV M vesicles with SeV F and HN glycoproteins, SeV particles, and vesicles associated with the cytosolic components. The last abundantly contained phosphorylated SeV matrix (M) protein, which is not released in a usual SeV infection. Furthermore the VSV M protein expressed without using a virus vector was efficiently released into the culture medium. These results suggest that the VSV M protein has a budding activity per se and that SeV proteins are passively involved in the release of VSV M.     

Role of clathrin-mediated endocytosis during vesicular stomatitis virus entry into host cells

Vesicular stomatitis virus (VSV) is well established to enter cells by pH-dependent endocytosis, but mechanistic aspects of its internalization have remained unclear. Here, we examined the functional role of clathrin in VSV entry by expression of a dominant-negative mutant of Eps15 (GFP-Eps15Delta95/295), a protein essential for clathrin-mediated endocytosis. Whereas expression of GFP alone had no effect on VSV infection, expression of GFP-Eps15Delta95/295 severely limited infection. As independent ways to examine clathrin function, we also examined cells that had been treated with chlorpromazine and utilized small interfering RNA (siRNA) techniques. Inhibition of clathrin-mediated endocytosis by chlorpromazine treatment, as well as clathrin knock-down using siRNA duplexes directed against the clathrin heavy chain, also prevented VSV infection. In combination with previous morphological approaches, these experiments establish clathrin as an essential component needed for endocytosis of VSV.