A cowpox virus gene required for multiplication in Chinese hamster ovary cells.

Cowpox virus, in contrast to vaccinia virus, can multiply in Chinese hamster ovary cells. To study the genetic basis for this difference in host range, recombinants between vaccinia and cowpox viruses were isolated and their DNA restriction patterns were examined. The ability to multiply in Chinese hamster ovary cells could be correlated with the conservation of cowpox virus sequences mapping at the left end of the genome. This was further demonstrated by marker rescue of the host range phenotype with restricted cowpox virus DNA. Marker rescue with cloned restriction fragments of decreasing size enabled the fine localization of the host range function to a 2.3-kilobase-pair fragment. Nucleotide sequencing revealed that the fragment encoded a single major polypeptide of approximately 77,000 daltons. It is suggested that the role of the host range gene from cowpox virus is to prevent the early and extensive shutoff of protein synthesis that normally occurs in Chinese hamster ovary cells infected by vaccinia virus.     

Irreversible Effects of Cycloheximide During the Early Period of Vaccinia Virus Replication

The presence of cycloheximide, an inhibitor of protein synthesis, during the period 30 to 60 min after vaccinia infection produced an irreversible block in virus replication. In contrast (i) cycloheximide given at earlier or later times, even for prolonged periods, did not prevent continuation of the infectious cycle after removal of the drug, and (ii) treatment with cycloheximide during the first 2 hr did not prevent virus growth when the early stages of replication proceeded more slowly due to infection with a low multiplicity of virus. These findings were interpreted as an indication that protein synthesis is required at a critical time in the virus growth cycle. Under the conditions in which brief cycloheximide treatment prevented virus growth, ribonucleic acid (RNA) synthesis continued at an undiminished rate for at least 2 hr after removal of the drug. Although this RNA appeared identical by polyacrylamide gel electrophoresis to "early" viral messenger RNA, it was not found associated with ribosomes or polyribosomes. Failure to observe viral protein synthesis was consistent with the latter finding. It appeared unlikely that the translational block resulted from inadequate removal of cycloheximide, since the effects of the drug were shown to be reversible at earlier or later times in infection or even at the same time when a lower multiplicity of virus was used. Interference with the normal synthesis of specific viral protein factors required for translation was postulated to explain the results.     

Foot-and-Mouth Disease Virus-Induced Alterations of Baby Hamster Kidney Cell Macromolecular Biosynthesis: Inhibition of Ribonucleic Acid Methylation and Stimulation of Ribonucleic Acid Synthesis

The kinetics of ribonucleic acid (RNA) and protein synthesis and RNA methylation were examined after foot-and-mouth disease virus (FMDV) infection of baby hamster kidney cells. The synthesis of RNA extracted from the whole cells was stimulated two- to threefold above the control level of synthesis. This increased rate was attributed to viral RNA synthesis. The inhibition of host RNA methylation was concomitant with but more pronounced than protein synthesis inhibition. The methylation of transfer RNA was initially inhibited by virus infection, but rose to within 70 to 80% of the control level just prior to the production of maximal amounts of virus-specific RNA polymerase. Cycloheximide studies showed that rapid cessation of protein synthesis did not result in the immediate cessation of RNA methylation. A comparison between the kinetics of inhibition of these processes by cycloheximide and FMDV infection suggests that FMDV selectively inhibits RNA methylation.     

Inhibition of host protein synthesis in vaccinia virus-infected cells in the presence of cordycepin (3''-deoxyadenosine).

Cordycepin inhibited efficiently viral mRNA and polyadenylic acid syntheses in vaccinia virus-infected cells, but allowed the shutoff of host protein synthesis to occur. Therefore, cordycepin was used to study this shutoff in the absence of gene expression. Ribosome transit time was increased in infected cells, revealing an inhibition at the level of elongation and/or release of polypeptide chains. However, the disappearance of heavy polysomes in vaccinia virus-infected cells showed that the inhibition of host protein synthesis resulted predominantly from a block at the stage of initiation. This conclusion was confirmed by the recovery of heavy polyribosomes when low levels of cycloheximide were added to slow down ribosome release from the mRNA. Similar amounts of cellular mRNA (present in the polyribosomes) were found in vaccinia virus-infected cells and in mock-infected cels (exposed to cordycepin), showing that the cellular mRNA was not inactivated in these conditions. It was concluded that a component of the vaccinia virion inhibits, in the absence of viral RNA and polyadenylic acid syntheses, host protein synthesis at the level of initiation and, to a lesser extent, at the level of elongation (and/or release) of polypeptide chains.     

Nuclear protein synthesis in a temperature-sensitive Chinese hamster ovary cell line at 40 degrees C

We examined the incorporation of radioactive amino acids into nuclear proteins occurring at nonpermissive conditions in tsH1 Chinese hamster ovary cells with a temperature-sensitive defect in cytosol nonmitochondrial protein synthesis. In leucine-free medium at 40 degrees C, total cellular protein synthesis declined by 1-1.5%/min. As reported by others, preincubating these cells at 42 degrees C for 5-10 min sharply increased the rate of decline. The synthesis of acidic nuclear proteins at nonpermissive conditions (40 degrees C + 300 micrograms/ml cycloheximide) was demonstrated by the nuclear incorporation of 3H-tryptophan. Radioactivity, seen by autoradiography to be associated with these isolated Triton-X-100-washed nuclei, was released after incubating labelled nuclei with proteolytic enzymes. During incubation of tsH1 cells at nonpermissive conditions, pulse/chase experiments were consistent with the loss of some nuclear radioactivity into the cytoplasm. The distribution of cytosol and nuclear proteins, labelled at permissive or nonpermissive conditions and separated by isoelectric focusing, differed quantitatively and probably qualitatively, confirming the residual synthesis of acidic nuclear proteins at 40 degrees C in the presence of cycloheximide. Most newly synthesized acidic proteins retained by nuclei from cells labeled at nonpermissive conditions were present in a transciptionally active chromatin fraction. Although under these conditions the apparent rate of cellular RNA synthesis was unchanged, inhibiting residual cycloheximide-resistant nuclear protein synthesis with puromycin proportionately reduced RNA synthesis. Preincubating cells with 20 micrograms/ml of actinomycin D did not inhibit residual labelling of nuclear proteins; effects on residual nuclear labelling of impaired mitochondrial respiration were ambiguous. Nuclear proteins labelled under nonpermissive conditions probably included some of the 'prompt' heat shock proteins recently described. Provided certain assumptions are correct, our results are consistent with very limited protein synthesis associated with and even intrinsic to cell nuclei. They also suggest that this residual cycloheximide-resistant protein synthesis could be concerned with optimum synthesis or processing of certain nuclear RNA species.     

Mechanism of vesicular stomatitis virus mRNA decay

The chemical and functional stability of the five vesicular stomatitis virus (VSV) messenger RNAs during infection of Chinese hamster ovary (CHO) cells was studied using the temperature-sensitive mutant, tsG114. By incubating infected cells at the nonpermissive temperature (39 °C), RNA synthesis was blocked and the five VSV mRNAs decayed chemically and functionally with a half-life of 1 to 1.5 h. However, all five VSV mRNAs were stable in vivo at 39 °C when protein synthesis was blocked with either cycloheximide or emetine. In contrast, when pactamycin was used to inhibit protein synthesis, the chemical and functional decay rates of the VSV mRNAs were indistinguishable from those observed in the absence of antibiotic. On the basis of the mode of action of each of the antibiotic inhibitors, these data imply that (a) ribosome movement along VSV mRNAs plays no role in their stabilities, and (b) each VSV mRNA contains a nuclease-sensitive site, at its 5′ end at or near the initiation site, which regulates its decay in vivo.     

Coronavirus minus-strand RNA synthesis and effect of cycloheximide on coronavirus RNA synthesis.

The temporal sequence of coronavirus plus-strand and minus-strand RNA synthesis was determined in 17CL1 cells infected with the A59 strain of mouse hepatitis virus (MHV). MHV-induced fusion was prevented by keeping the pH of the medium below pH 6.8. This had no effect on the MHV replication cycle, but gave 5- to 10-fold-greater titers of infectious virus and delayed the detachment of cells from the monolayer which permitted viral RNA synthesis to be studied conveniently until at least 10 h postinfection. Seven species of poly(A)-containing viral RNAs were synthesized at early and late times after infection, in nonequal but constant ratios. MHV minus-strand RNA synthesis was first detected at about 3 h after infection and was found exclusively in the viral replicative intermediates and was not detected in 60S single-stranded form in infected cells. Early in the replication cycle, from 45 to 65% of the [3H]uridine pulse-labeled RF core of purified MHV replicative intermediates was in minus-strand RNA. The rate of minus-strand synthesis peaked at 5 to 6 h postinfection and then declined to about 20% of the maximum rate. The addition of cycloheximide before 3 h postinfection prevented viral RNA synthesis, whereas the addition of cycloheximide after viral RNA synthesis had begun resulted in the inhibition of viral RNA synthesis. The synthesis of both genome and subgenomic mRNAs and of viral minus strands required continued protein synthesis, and minus-strand RNA synthesis was three- to fourfold more sensitive to inhibition by cycloheximide than was plus-strand synthesis.     

Vaccinia virus induces cellular mRNA degradation.

The infection of mouse L cells with vaccinia virus induced a rapid inhibition of cellular polypeptide synthesis and a diversion of protein synthesis to the exclusive production of viral polypeptides. This shutoff of cell-specific protein synthesis was achieved by a novel mechanism by which the virus induced the rapid degradation of cellular mRNAs. Concurrent with the degradation of cellular mRNA, the virus proceeds in the orderly temporal expression of its own genetic information. The effect of vaccinia virus infection upon two abundant L-cell mRNAs was assessed by using the highly conserved cDNA sequences that encode chicken beta-actin and rat alpha-tubulin. Hybridization analyses demonstrated that throughout infection there is a rapid and progressive degradation of both of these mRNAs. In fact, after 3 h of infection they are reduced to less than 50% of their concentration in uninfected L cells, and between 8 to 10 h they are almost entirely degraded. This observation explains in part the mechanism by which vaccinia virus inhibits host cell protein synthesis.     

Isolation and characterization of a Chinese hamster ovary mutant cell line with altered sensitivity to vaccinia virus killing.

The Chinese hamster ovary (CHO) cell line is nonpermissive for vaccinia virus, and translation of viral intermediate genes was reported to be blocked (A. Ramsey-Ewing and B. Moss, Virology 206:984-993, 1995). However, cells are readily killed by vaccinia virus. A vaccinia virus-resistant CHO mutant, VV5-4, was isolated by retroviral insertional mutagenesis. Parental CHO cells, upon infection with vaccinia virus, die within 2 to 3 days, whereas VV5-4 cells preferentially survive this cytotoxic effect. The survival phenotype of VV5-4 is partial and in inverse correlation with the multiplicity of infection used. In addition, viral infection fails to shut off host protein synthesis in VV5-4. VV5-4 was used to study the relationship of progression of the virus life cycle and cell fate. We found that in parental CHO cells, vaccinia virus proceeds through expression of viral early genes, uncoating, viral DNA replication, and expression of intermediate and late promoters. In contrast, we detect only expression of early genes and uncoating in VV5-4 cells, whereas viral DNA replication appears to be blocked. Consistent with the cascade regulation model of viral gene expression, we detect little intermediate- and late-gene expression in VV5-4 cells. Since vaccinia virus is known to be cytolytic, isolation of this mutant therefore demonstrates a new mode of the cellular microenvironment that affects progression of the virus life cycle, resulting in a different cell fate. This process appears to be mediated by a general mechanism, since VV5-4 is also resistant to Shope fibroma virus and myxoma virus killing. On the other hand, VV5-4 remains sensitive to cowpox virus killing. To examine the mechanism of VV5-4 survival, we investigated whether apoptosis is involved. DNA laddering and staining of apoptotic nuclei with Hoechst 33258 were observed in both CHO and VV5-4 cells infected with vaccinia virus. We concluded that the cellular pathway, which blocks viral DNA replication and allows VV5-4 to survive, is independent of apoptosis. This mutant also provides evidence that an inductive signal for apoptosis upon vaccinia virus infection occurs prior to viral DNA replication.     

Interferon-mediated, double-stranded RNA-dependent protein kinase is inhibited in extracts from vaccinia virus-infected cells

The interferon-inducible, double-stranded RNA (dsRNA)-dependent protein kinase which phosphorylates an endogenous HeLa 69 kilodalton polypeptide or exogenous initiation factor eIF2 was inhibited during vaccinia virus infection. High interferon doses (20,000 reference units per ml) did not prevent this inhibition. The inhibition required protein synthesis but not viral DNA synthesis during infection, suggesting that an early vaccinia virus gene function was responsible. An active dsRNA-dependent protein kinase could be recovered from an inactive extract by purification on polyinosinate X polycytidylate-cellulose. An inhibitor of the protein kinase, therefore, must be present in the inactive extract. Similar results have been obtained with mouse L929 cells. At early time points of infection, the protein kinase in cell extracts required exogenous dsRNA for activity. This argues against endogenous viral dsRNA and activation of the kinase in the intact cell. At late time points of infection (when vaccinia virus dsRNA was almost certainly formed), the inhibitor of the kinase is present. Accordingly, it seems unlikely that the kinase played any role in the interferon-mediated inhibition of virus growth observed in these cells under these particular conditions.     

Delay of vaccinia virus-induced apoptosis in nonpermissive Chinese hamster ovary cells by the cowpox virus CHOhr and adenovirus E1B 19K genes.

The infection of vaccinia virus in Chinese hamster ovary (CHO) cells produces a rapid shutdown in protein synthesis, and the infection is abortive (R.R. Drillien, D. Spehner, and A. Kirn, Virology 111:488-499, 1978; D.E. Hruby, D.L. Lynn, R. Condit, and J.R. Kates, J. Gen. Virol. 47:485-488, 1980). Cowpox virus, which can productively infect CHO cells, had previously been shown to contain a host range gene, CHOhr, which confers on vaccinia virus the ability to replicate in CHO cells (D. Spehner, S. Gillard, R. Drillien, and A. Kirn, J. Virol. 62:1297-1304, 1988). We found that CHO cells underwent apoptosis when infected with vaccinia virus. The expression of the CHOhr gene in vaccinia virus allowed for the expression of late virus genes. CHOhr also delayed or prevented vaccinia virus-induced apoptosis in CHO cells such that there was sufficient time for replication of the virus before the cell died. The E1B 19K gene from adenovirus also delayed vaccinia virus-induced apoptosis; however, there was no detectable expression of late virus genes. Furthermore, E1B 19K also delayed cell death in CHO cells which had been productively infected with vaccinia virus. This study identifies a new antiapoptotic gene from cowpox virus, CHOhr, for which the protein contains an ankyrin-like repeat and shows no significant homology to other proteins. This work also indicates that an antiapoptotic gene from one virus family can delay cell death in an infection of a virus from a different family.     

Characterization of two RNA polymerase activities induced by mouse hepatitis virus

RNA-dependent RNA polymerase activity was found in mouse hepatitis virus strain A59 (MHV-A59)-infected cells. The enzyme was induced in the infected cells and could not be detected in the MHV-A59 virion. Two peaks of RNA polymerase activity, one early and the other late in infection, were detected. These polymerase activities were in temporal sequence with early and late virus-specific RNA synthesis. Both of them were found to be associated with membrane fractions. There were significant differences in the enzymatic properties of the two polymerases. The early polymerase, but not the late polymerase, could be activated by potassium ions in the absence of magnesium ions and also had a lower optimum pH than the late polymerase. It was therefore probable that the enzymes represent two different species of RNA polymerase and perform different roles in virus-specific RNA synthesis. The effects of cycloheximide on MHV-specific RNA synthesis were determined. Continuous protein synthesis was required for both early and late RNA synthesis and might also be required for shutoff of early RNA synthesis.     

Mechanism of Inhibition of Vaccinia Virus Replication in Adenovirus-Infected HeLa Cells

The ability of vaccinia virus to replicate in HeLa cells which had been previously infected with adenovirus type 2 (Ad2) was studied in order to gain insight into the mechanism by which adenovirus inhibits the expression of host cell functions. Vaccinia virus was employed in these studies because it replicates in the cytoplasm, whereas Ad2 replicates in the nucleus of the cell. It was found that vaccinia deoxyribonucleic acid (DNA) synthesis is greatly inhibited in adeno-preinfected HeLa cells provided that vaccinia superinfection does not occur before 18 hr after adeno infection. The inhibition of vaccinia DNA synthesis can be traced to an inhibition of vaccinia protein synthesis and viral uncoating. Vaccinia ribonucleic acid (RNA) synthesis is not inhibited in adeno-preinfected cells, but the vaccinia RNA does not become associated with polysomes.