Fate of interferon-treated cells.

Interferon-treated cultures of Ly cells survived initial infection with high multiplicities of vesicular stomatitis (VSV) or herpes simplex virus (HSV). In the case of HSV, infectious virus and intracellular viral antigen were rapidly eliminated from the interferon-treated cultures, and the cells grew out to form apparently normal monolayers that could be cultured indefinitely. In the VSV-infected Ly cultures, virus titers remained at low levels in interferon-treated cells but after about 14 days rapidly rose and the culture was destroyed. If interferon was added to the medium on days 4 and 6 after infection, virus titers rapidly declined but again recovered and the cells were destroyed. If, however, interferon treatment was resumed 9 days after initial infection, detectable infectious VSV was eliminated from the medium. Several methods, including cocultivation and molecular hybridization, failed to demonstrate persistence of a significant portion of the VSV genome in these cultures.     

Persistence of vesicular stomatitis virus in interferon-treated cell cultures.

A significant difference was observed in the functional stability of the vesicular stomatitis virus (VSV) genome in mouse L cells and the RK-13 line of rabbit kidney cells pretreated with homologous interferon. By utilizing the ability of vaccinia to rescue VSV from the inhibitory effects of interferon in these cell lines, it was demonstrated that there was a loss of a rescuable form of VSV genome in RK-13 cells pretreated with interferon; superinfection with vaccinia 24 hr after VSV infection did not result in a significant increase in VSV yield. In contrast, significant rescue of VSV occurred in interferon-treated L cells superinfected with vaccinia as late as 72 hr after VSV infection.The present study also provides evidence that in interferon-treated RK-13 cells doubly infected with VSV and vaccinia there was a correlation of the rescuability of the VSV genome and its ability to direct RNA synthesis.     

Induction of interferon in L cells by defective-interfering (DI) particles of vesicular stomatitis virus: Lack of correlation with content of [±] snapback RNA

The ability of defective-interfering (DI) particles of vesicular stomatitis virus (VSV) to induce interferon was studied in relation to the amount of snapback [±] double-stranded sequences in their RNA. Five DI particles propagated in BHK-21 cells were analyzed: two DI particles generated by undiluted passages of cloned wild-type VSV (Indiana); two DI particles generated by serial undiluted passages of culture fluid from L cells persistently infected with VSV; and DI-011, a DI particle with [±] snapback RNA, which is known to be a potent inducer of interferon. Induction of interferon in L cells by these DI particles was not proportional to the amount of [±] sequences in their RNA. DI-011 (26 to 37% [±] RNA sequences) induced a significant amount of interferon at a multiplicity of infection of one DI particle per cell. In contrast, the two DI particles from wild-type VSV (43 to 54% [±] RNA sequences) were 20- to 30-fold less efficient inducers of interferon than DI-011. Furthermore, the two DI particles (1 to 4% [±] RNA sequences) generated from L cell carrier cultures were only slightly less efficient inducers of interferon than the wild-type DI particles. The data also indicate that a population of DI particles which contains [±] RNA is not selected in L cells persistently infected with VSV.     

Rescue of vesicular stomatitis virus from interferon-induced resistance by superinfection with vaccinia virus. II. Effect of UV-inactivated vaccinia and metabolic inhibitors

The rescue of vesicular stomatitis virus (VSV) from interferon-induced resistance in rabbit (RK-13) and mouse (L) cell cultures superinfected with vaccinia requires early (up to 2 hr) vaccinia DNA-dependent RNA synthesis. The inability of hydroxyurea to inhibit rescue of VSV in interferon-treated RK-13 and L cells, whether the drug is added at the time of or after infection, suggests that vaccinia DNA synthesis is not required for the rescue of VSV in cells superinfected with vaccinia.In both RK-13 and L cells pretreated with homologous interferon and then doubly infected with vaccinia and VSV, there was a significant increase (up to 8 hr) in the lag period before infective VSV progeny appeared. It appears that a product of vaccinia synthesis must accumulate before VSV replication can begin in cells pretreated with interferon. This product could be vaccinia-directed early RNA or a translation product of this RNA.In RK-13 cells pretreated with interferon, the ability of vaccinia to rescue VSV is much more resistant to UV-irradiation than the infectivity of the virus; in L cells there is a close correspondence in the inactivation rates of infectivity and the ability of vaccinia to rescue VSV. These results suggest a difference in the efficiency of uncoating of UV-irradiated vaccinia in RK-13 and L cells. In L cells it is possible that UV-irradiated vaccinia is not uncoated efficiently and the early vaccinia RNA product required for rescue of VSV is not synthesized.     

Studies of L cells persistently infected with VSV: factors involved in the regulation of persistent infection.

Infection of interferon-treated L cells with VSV led frequently to the establishment of L cells persistently infected with VSV (LVSV cells). These cells were characterized by the following properties; (I) no supplement of antiviral factors such as anti-VSV antiserum, interferon, was required for their maintenance; (2) virus antigens were detected in about 5 to 30% of the cells by immunofluorescence staining; (3) the cells were not only resistant to superinfection by homologous virus, but also resistant to challenge by heterologous viruses such as Mengo virus; (4) the cells were destroyed by co-cultivation with heterologous cells susceptible to VSV infection; (5) the cells could be cured by serial cultivation in medium containing antiviral antibody, and the cured cells were as susceptible to VSV as normal L cells. It was shown that at least three factors (interferon, defective interfering [DI] particles and a selection of small-plaque temperature-sensitive [ts] mutants) took part in the maintenance of LVSV cells although it was difficult to evaluate exactly the relative importance of these factors. The effect of antiviral antibody, interferon and incubation temperature upon the maintenance of LVSV cells are discussed further.     

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.     

VSV replication in neurons is inhibited by type I IFN at multiple stages of infection

Vesicular stomatitis virus (VSV) is a rhabdovirus which causes acute encephalitis in mice after intranasal infection. Because type I interferon (IFN) has been shown to be a potent inhibitor of VSV, we investigated the role of type I IFN in viral replication in neurons in culture. Pre-treatment of NB41A3 neuroblastoma cells or primary neuron cultures with IFN-beta or IFN-alpha strongly inhibits virus replication, with 1000-fold inhibition of infectious virus release occurring at 7 h post-infection, and maximum inhibition of 14,000-fold occurring at 14 h. Type I IFN inhibited both viral protein and RNA synthesis, but not enough to account for the inhibition of infectious virus yield. The influenza virus protein NS1 binds dsRNA and antagonizes induction of PKR activity, an IFN-inducible antiviral protein which phosphorylates and inactivates the elongation factor eIF-2alpha, resulting in cessation of translation. In NS1-expressing neuroblastoma cells, VSV replication was inhibited by IFN-beta as well as in control NB41A3 cells, and eIF-2alpha phosphorylation was blocked, suggesting that PKR activity was not involved in inhibition of viral protein synthesis. Similarly, inhibition of VSV by IFN-beta was not affected by addition of inhibitors of nitric oxide synthase, indicating that IFN-beta activity is not mediated by nitric oxide or superoxide. This contrasts with the essential role of NOS-1 in inhibition of VSV replication when neurons are treated with IFN-gamma. Analysis of cell culture supernatants revealed suppression of release of VSV particles from both NB41A3 cells and primary neurons treated with IFN. The inhibition of virion release closely matched the overall suppression of infectious VSV particle release, suggesting that type I IFN plays a role in inhibition of VSV assembly.     

Interferon-mediated inhibition of production of Gazdar murine sarcoma virus, a retrovirus lacking env proteins and containing an uncleaved gag precursor

HTG2 cells are murine sarcoma virus-transformed hamster cells. These cells continuously produce Gazdar sarcoma virus particles which are devoid of viral envelope proteins and which contain the uncleaved gag precursor polyprotein, Pr65, as their major protein constituent. Human interferon-alpha elicited an antiviral response in these cells as shown by the inhibition of replication of vesicular stomatitis virus in interferon-treated cells. Extracellular production of the retroviral particles by these cells was also inhibited by interferon in a dose-dependent manner and this inhibition was abolished by a specific antiserum to interferon. The intracellular level of Pr65 was not lowered in the interferon-treated cells, indicating that inhibition of viral protein synthesis was not responsible for inhibition of virus production. The present study suggests that interferon-mediated inhibition of retrovirus production, in general, is not a consequence of either a defective interaction between viral nucleoprotein cores and viral envelope proteins or a defect in the proteolytic processing of the gag polyprotein, since neither of these processes occurs during the morphogenesis of Gazdar particles and their production is nonetheless inhibited by interferon.     

Viral messenger RNA unmethylated in the 5'-terminal guanosine in interferon-treated HeLa cells infected with vesicular stomatitis virus

The distribution of viral mRNA between polysomal and nonpolysomal fractions was investigated in interferon-treated cells infected with vesicular stomatitis virus (VSV). More than half of the viral mRNA synthesized by these cells is in the nonpolysomal fraction, whereas less than one-third of the mRNA synthesized by control cells is found in this fraction. Polysomal and nonpolysomal mRNA sediment identically on sucrose density gradients, but the nonpolysomal mRNA from interferon-treated cells in under-methylated, as shown by labeling experiments with [methyl-³H]methionine. Analysis of VSV mRNA after digestion with nucleases shows that all molecules are capped but about 60% of the nonpolysomal mRNA isolated from interferon-treated cells is unmethylated in the 5′-terminal G. When tested in an assay for initiation of protein synthesis, only 45% of this mRNA binds to ribosomes, as compared to 80% binding for polysomal mRNA from either control or interferon-treated cells.     

Assembly of membrane glycoproteins studied by phenotypic mixing between mutants of vesicular stomatitis virus and retroviruses

Temperature sensitive (ts) mutations of vesicular stomatitis virus (VSV), Indiana serotype, which belong to complementation group V (tsV) have been shown to affect the viral envelope glycoprotein, or G protein. When ts V mutants are grown in cells producing avian leukosis viruses, the titers of infectious VSV obtained at the nonpermissive temperature are 10⁴-fold higher than in control cells. Cells releasing murine leukemia viruses or avian reticuloendotheliosis virus rescue VSV ts V mutants much less efficiently. The rescued virions have the properties of envelope pseudotypes in that their host range is restricted to that of the helper retrovirus, they are neutralized by anti-retrovirus antibodies but not anti-VSV antibodies, and they are not thermolabile. Sensitive serological techniques, including the use of complement-mediated virolysis, immunoprecipitation, and monoclonal antibody reacting with G protein, show that VSV pseudotypes produced at the nonpermissive temperature have no detectable G protein, whereas VSV particles released from retrovirus infected cells at the permissive temperature have mosaic envelopes bearing both VSV G protein and retrovirus glycoprotein. In mixed infections of Rous sarcoma virus (RSV) and VSV ts V mutants, pseudotype particles with RSV genomes and VSV envelope antigens are produced only at the permissive temperature. In contrast, substantial yields of RSV(VSV) pseudotypes but no VSV(RSV) pesudotypes are obtained at the nonpermissive temperature with VSV carrying mutations in complementation group III, which affect M protein. Thermolabile VSV tsV mutants form RSV(VSV) pseudotypes which also are thermolabile. The kinetics of heat inactivation of G protein function in tsV mutants is the same in VSV particles with unmixed envelopes and with mosaic envelopes. From these studies of phenotypic mixing we draw the following conclusions: (i) The synthesis of functional M protein but not G protein is essential for the maturation of VSV virions. (ii) VSV M protein is not required for the assembly of G protein into retrovirus virions. (iii) The thermolabile nature of tsV VSV mutants is an intrinsic property of the G protein, independent of the type of virion into which it is incorporated and of other viral glycoproteins which may be assembled into the envelope of the same virion.     

Vaccinia rescue of VSV from interferon-induced resistance: reversal of translation block and inhibition of protein kinase activity

Coinfection with vaccinia virus rescues vesicular stomatitis virus (VSV) from the inhibitory effects of interferon (IFN) in mouse L cells. While vaccinia infection does not significantly affect VSV RNA synthesis, coinfection with vaccinia dramatically increases VSV protein synthesis in IFN-treated cells. Evidence is provided that vaccinia inhibits the activity of the IFN-induced dsRNA-dependent protein kinase.