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.     

Rescue of vesicular stomatitis virus from interferon-induced resistance by superinfection with vaccinia virus: I. Rescue in cell cultures from different species

The replication of two DNA viruses, vaccinia and pseudorabies (PsRV), was not inhibited in three cell lines of rabbit origin (RK-13 and RK-1337, rabbit kidney; and RC-60, rabbit cornea) which had been pretreated with rabbit interferon. In contrast, the replication of vesicular stomatitis virus (VSV), an RNA virus, was susceptible to interferon-induced resistance in rabbit cell lines. These results reinforced the possibility that there are separate interferon-induced resistance factors for RNA and DNA viruses and that cultured rabbit cells are deficient in the synthesis of the resistance factors needed to inhibit DNA viruses.When cell cultures of rabbit origin were pretreated with homologous interferon and doubly infected with vaccinia and VSV, a variety of responses was observed. Vaccinia was able to rescue VSV from the inhibitory effects of interferon-induced resistance in two rabbit cell lines (RK-13 and RC-60), but not in RK-1337 cells. Similar experiments were carried out in mouse L cells and in primary chick embryo (CE) cell cultures; both RNA and DNA viruses were susceptible to inhibition by interferon-induced resistance in these cells. In L cells, double infection with vaccinia was able to rescue VSV; however, in CE cell cultures superinfection with vaccinia did not rescue VSV from the inhibitory effect of interferon. In these cells the synthetic pathways or virion factors furnished by vaccinia which are required for the rescue of VSV are sensitive to the action of interferon.     

Different Sensitivity of Ribonucleic and Deoxyribonucleic Acid Viruses to Resistance Induced in Rabbit Cells (RK-13) by Polyriboinosinic Acid·Polyribocytidilic Acid

When a continuous line of rabbit kidney cells (RK-13) was exposed to polyriboinosinic acid polyribocytidilic acid [poly(rI).poly(rC)], the cells became resistant to the replication of a ribonucleic acid (RNA) virus, vesicular stomatitis (VSV), but remained susceptible to the replication of two deoxyribonucleic acid (DNA) viruses, pseudorabies and vaccinia. These differences were identical to previous findings with RK-13 cells pretreated with exogenous rabbit interferon. Confirmation is thereby provided that RK-13 cells are deficient in the synthesis of resistance factors active against the DNA viruses tested and that there are separate resistance factors for RNA and DNA viruses in RK-13 cells. The resistance against VSV which developed in RK-13 cells exposed to poly(rI).poly(rC) could be significantly reversed by prior infection with infective vaccinia virus but not by vaccinia inactivated by ultraviolet irradiation or heat.     

Poxvirus specific cytopathic effect in interferon-treated L cells

L cells pretreated with homologous interferon preparations and infected with vaccinia strain WR or IHD develop a drastic cytopathic effect, which differs morphologically from the normal cytopathic effect observed in poxvirus-infected cells. In addition, the ⁵¹Cr bound to interferon-treated cells is released from the cells after 4 hr of infection. No comparable release occurs in L cells without interferon treatment or in interferon-treated infected chick embryo fibroblasts. Release of ⁵¹Cr depends on the multiplicity of infection, the amount of interferon used, and the time of exposure to interferon before infection. The release of ⁵¹Cr is not observed if cells are pretreated with heterologous or inactivated interferon. Infection of interferon-treated L cells with cowpox virus or inactivated vacciniavirus does not result in the increased destruction of cells or release of ⁵¹Cr. The addition of inhibitors of DNA or protein synthesis or actinomycin D after infection prevents both effects. It is suggested that release of ⁵¹Cr might be used to study quantitatively the poxvirus-specific destruction of interferon-treated cells. Several possibilities for the explanation of the degeneration of interferon treated L cells are discussed with respect to the documented inhibitory action of interferon on virus-specific processes.     

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.     

Rapid decrease in the levels of the double-stranded RNA-dependent protein kinase during virus infections

The double-stranded RNA-dependent protein kinase from human cells is a 68,000 molecular weight protein (p68 kinase), the level of which is enhanced significantly in cells treated with interferon. With a monoclonal antibody specific for p68 kinase, here we show the phosphorylation and steady-state levels of p68 kinase during virus infection. The p68 kinase is phosphorylated in interferon-treated cells during infection with encephalomyocarditis virus (EMCV), vesicular stomatitis virus (VSV), and vaccinia virus, thus indicating activation of p68 kinase during these virus infections, an essential step required for autophosphorylation of p68 kinase. However, in spite of this activation, the level of p68 kinase is rapidly decreased in virus-infected cells. The half-life of p68 kinase in uninfected cells is 6 to 7 hr, whereas in EMCV-infected cells it is 2 to 3 hr. This decrease in the level of p68 kinase is dependent on the multiplicity of virus infection and it seems to be specific since other cellular proteins as well as the activity of 2'-5'-oligoadenylate synthetase are not modified. Decreased levels of p68 kinase are also observed in cells infected with VSV and vaccinia virus. In the absence of virus infection, decreased levels of p68 kinase occur in cells following incubation with poly(I).poly(C).     

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.     

Vaccinia virus stimulates the growth of vesicular stomatitis virus at the level of protein synthesis in mouse L cells

Coinfection with vaccinia virus increases the growth of vesicular stomatitis virus (VSV) in mouse L cells by 10- to 20-fold. Although vaccinia has no significant effect on RNA synthesis by VSV, VSV protein synthesis is dramatically stimulated by double infection. The enhancement of VSV growth is correlated with the ability of vaccinia to inhibit the VSV-mediated damage to the host translational machinery. Coinfection with vaccinia fails to stimulate the growth of a VSV mutant which is deficient in its ability to shut off protein synthesis during infection.     

Vaccinia virus K1L protein mediates host-range function in RK-13 cells via ankyrin repeat and may interact with a cellular GTPase-activating protein

The K1L protein of vaccinia virus is required for its growth in certain cell lines (RK-13 and human). The cowpox host-range protein CP77 has been shown to complement K1L function in RK-13 cells, despite a lack of homology between the two proteins except for ankyrin repeats. We investigated the role of ankyrin repeats of K1L protein in RK-13 cells. The growth of a recombinant vaccinia virus, with K1L gene mutated in the most conserved ankyrin repeat, was severely impaired. Infection with the mutant virus caused shutdown of cellular and viral protein synthesis early in infection. We also investigated the interaction of K1L protein with cellular proteins and found that K1L interacts with the rabbit homologue of human ACAP2, a GTPase-activating protein with ankyrin repeats. Our result suggests the importance of ankyrin repeat for host-range function of K1L in RK-13 cells and identifies ACAP2 as a cellular protein, which may be interacting with K1L.     

Effect of interferon on the exogenous Friend murine leukemia virus infection

Interferon treatment (600 U/ml) of NIH/3T3 cells induced greater than 90% reduction in the de novo production of Friend MuLV when measured 24 hr postinfection. Early events in viral replication such as the synthesis of proviral DNA and its subsequent integration into the cell genome were not inhibited by interferon treatment indicating that the suppression of virus production by interferon appears to occur after synthesis of proviral DNA. Analysis of viral RNA species present in controls and interferon-treated cells 24 hr after infection show that the same RNA species were present in the presence and absence of interferon. Synthesis of viral polypeptides was reduced but not blocked in interferon-treated cells when measured within 24 hr after infection while processing of gag precursor, Pr65^gag, and glycoprotein precursor, gPr85^env, to viral proteins was not altered. Phosphorylation of viral protein p12 but not that of the precursor, Pr65^gag, was inhibited in newly infected interferon-treated cells. In contrast to the first replicative cycle, interferon did not inhibit synthesis of viral proteins, and phosphorylation of p12 in those cells chronically infected with F-MuLV.     

Mechanism of interferon action. Kinetics of interferon action in mouse L929 cells: translation inhibition, protein phosphorylation, and messenger RNA methylation and degradation

The effect of the duration of interferon treatment of mouse L929 fibroblasts on the expression of ribosome-associated and cell-sap-localized translation inhibition, protein phosphorylation, and messenger RNA (mRNA) methylation and degradation was investigated. The following results were obtained: (1) Ribosomal salt-wash fractions prepared from L929 cells treated for 12 or 18 hr significantly inhibited the translation of methylated reovirus mRNA catalyzed by the cell-free system prepared from untreated ascites tumor cells. The translation of reovirus mRNA was slightly stimulated by the ribosomal salt-wash fractions prepared from untreated cells and cells treated for 2 hr and was slightly inhibited by the salt-wash fraction from cells treated for 6 hr. (2) Cell-sap fractions prepared from untreated cells and from cells treated for 2 or 6 hr did not appreciably affect the translation of reovirus mRNA in the absence of double-stranded RNA (dsRNA); cell-sap fractions prepared from cells treated for 12 or 18 hr were slightly inhibitory. (3) Neither reovirus genome dsRNA nor polyriboinosinic acid-polyribocytidylic acid inhibited the translation of reovirus mRNA by the untreated ascites system. The inhibitory activity of ribosomal salt-wash fractions from interferon-treated cells was dsRNA independent, whereas dsRNA enhanced the inhibitory activity of cell-sap fractions from interferon-treated cells. (4) Interferon treatment significantly enhanced a cell-sap-independent phosphorylation of at least three proteins present in the ribosomal salt-wash fractions. Phosphorylation of an approximately 50,000-dalton component was maximally enhanced after 12 hr of interferon treatment and was increased by dsRNA; phosphorylation of 30,000- and <20,000-dalton components was enhanced earlier and was dsRNA independent. (5) An increase in the nuclease-mediated degradation of [3H]uridine-labeled methylated reovirus mRNA catalyzed by ribosomal salt-wash fractions was detectable after 6 hr of interferon treatment and was maximally enhanced after 12 hr of interferon treatment to about threefold the level of the untreated ribosomal salt-wash fraction. The degradation was not enhanced by dsRNA. The levels of reovirus mRNA degradation catalyzed by cell-sap fractions from untreated cells and from cells treated with interferon for 2 to 18 hr were comparable. (6) No significant difference was observed in the ability of cell-sap fractions from untreated cells or from cells treated with interferon for 2 to 18 hr to catalyze the methylation of unmethylated reovirus mRNA. In addition, none of the ribosomal salt-wash fractions significantly inhibited reovirus mRNA methylation catalyzed by untreated ascites cell-free extracts. These results suggest that interferon treatment may mediate multiple biochemical changes in murine cells, including the specific phosphorylation of protein(s) associated with ribosomes that possess interferon-mediated inhibitor(s) of viral mRNA translation.     

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 vaccinia virus replication in RK-13 cells by N,N'-bis(methylisatin-beta-thiosemicarbazone)-2-methylpiperazine

N,N'-bis(methylisatin-beta-thiosemicarbazone)-2-methylpiperazine in a 100 mumol/l concentration inhibited the reproduction of vaccinia virus in RK-13 cells by about 90%. This compound (bis-IBTMP) had no influence on virus adsorption and on early stages of virus multiplication, but affected virus reproduction from 12 to 24 hr post-infection (p.i.). The incorporation of 3H-thymidine into infected cells increased during first 10 hr p.i., decreasing gradually afterwards. In the infected cells treated with bis-IBTMP the same tendency was observed up to 10 hr p.i., but later on the incorporation level remained unchanged. The uptake of 14C-amino acids in the presence of bis-IBTMP was reduced both in vaccinia virus-infected and non-infected RK-13 cells.     

Polykaryocyte formation induced by VSV in mouse L cells.

Infection of mouse L cells with VSV leads to the formation of polykaryocytes about 4 to 12 h p.i. When anti-VSV immune serum was added during the course of infection, progression of cell fusion was soon suppressed. Cycloheximide completely suppressed the cell fusion when the drug was added within 1 h p.i., while the cell fusion was not suppressed at all when the drug was added at and after 3 h. Early polykaryocyte formation, 'fusion from without', was observed only at a low level in cells infected at very high multiplicities. The development of cell fusion induced by VSV was found to be different in several cell types, although all these cells produced a rather high yield of virus: L and C-243-3 mouse cell lines showed a high level of polykaryocytosis (80 to 100%), BHK and RK-13 cells responded at low level, and PS and Vero cells showed no cell fusion in response to VSV infection. In PS cells, however, cell fusion occurred when VSV-infected L cells were co-cultivated. From these observations, the mechanism of cell fusion induced by VSV was discussed.