Viral DNA synthesis in cells infected with temperature-sensitive mutants of herpes simplex virus type 1.

Temperature-sensitive mutants of herpes simplex virus type 1 representing eight DNA-negative complementation groups were grouped into the following three categories based on the viral DNA synthesis patterns after shift-up from the permissive to the nonpermissive temperature and after shift-down from the nonpermissive to the permissive temperature in the presence and absence of inhibitors of RNA and protein synthesis. (i) Viral DNA synthesis was inhibited after shift-up in cells infected with tsB, tsH, and tsJ. After shift-down, tsB- and tsH-infected cells synthesized viral DNA in the absence of de novo RNA and protein synthesis whereas tsJ-infected cells synthesized no viral DNA in the absence of protein synthesis. The B, H, and J proteins appear to be continuously required for the synthesis of viral DNA. (ii) Viral DNA synthesis continued after shift-up in cells infected with tsD and tsK whereas no viral DNA was synthesized after shift-down in the absence of RNA and protein synthesis. Mutants tsD and tsK appear to be defective in early regulatory functions. (iii) Cells infected with tsL, tsS, and tsU synthesized viral DNA after shift-up and after shift-down in the absence of RNA and protein synthesis. The functions of the L, S, and U proteins cannot yet be determined.     

Molecular genetics of herpes simplex virus. VIII. further characterization of a temperature-sensitive mutant defective in release of viral DNA and in other stages of the viral reproductive cycle.

Previous studies have shown that cells infected with the herpes simplex virus 1(HFEM) mutant tsB7 and maintained at the nonpermissive temperature fail to accumulate viral polypeptides. Analyses of intertypic recombinants generated by marker rescue of tsB7 with herpes simplex virus 2 DNA fragments localized the mutation between 0.46 and 0.52 map units on the viral genome (Knipe et al., J. Virol. 38:539-547, 1981). In this paper we report that the mutation in tsB7 affects several aspects of the reproductive cycle of the virus at the nonpermissive temperature. Thus, (i) viral capsids accumulate at the nuclear pores and do not release viral DNA for at least 6 h postinfection at 39 degrees C. The DNA was released within 30 min after a shift to the permissive temperature. (ii) Experiments involving shifts from the permissive to the nonpermissive temperature indicated that viral protein synthesis was not sustained in cells maintained at the permissive temperature for less than 4 h. (iii) Viral DNA synthesis was delayed at the permissive temperature for as long as 8 h. Once initiated, it continued at 39 degrees C. (iv) Marker rescue of tsB7 by transfection with herpes simplex virus 1(F) DNA fragments localized the mutation to between 0.501 and 0.503 map units on the viral genome. These results are consistent with the tsB7 lesion being in a gene coding for a virion component which affects release of viral DNA from capsids and onset of viral DNA synthesis.     

Studies of two temperature-sensitive mutants of Mengo virus.

Studies of the synthesis of viral ribonucleates and polypeptides in cells infected with two RNA- ts mutants of Mengo virus (ts 135 and ts 520) have shown that when ts 135 infected cells are shifted from the permissive (33 degrees C) to the nonpermissive (39 degrees C) temperature: (i) the synthesis of all three species of viral RNA (single stranded, replicative form, and replicative intermediate) is inhibited to about the same extent, and (ii) the posttranslational cleavage of structural polypeptide precursors A and B is partially blocked. Investigations of the in vivo and in vitro stability of the viral RNA replicase suggest that the RNA- phentotype reflects a temperature-sensitive defect in the enzyme. The second defect does not appear to result from the inhibition of viral RNA synthesis at 39 degrees C, since normal cleavage of polypeptides A and B occurs in wt Mengo-infected cells in which viral RNA synthesis is blocked by cordycepin, and at the nonpermissive temperature in ts 520 infected cells. Considered in toto, the evidence suggests that ts 135 is a double mutant. Subviral (53S) particles have been shown to accumulate in ts 520 (but not ts 135) infected cells when cultures are shifted from 33 to 39 degrees C. This observation provides supporting evidence for the proposal that this recently discovered particle is an intermediate in the assembly pathway of Mengo virions.     

Specific Sindbis virus-coded function for minus-strand RNA synthesis.

The synthesis of minus-strand RNA was studied in cell cultures infected with the heat-resistant strain of Sindbis virus and with temperature-sensitive (ts) belonging to complementation groups A, B, F, and G, all of which exhibited an RNA-negative (RNA-) phenotype when infection was initiated and maintained at 39 degrees C, the nonpermissive temperature. When infected cultures were shifted from 28 degrees C (the permissive temperature) to 39 degrees C at 3 h postinfection, the synthesis of viral minus-strand RNA ceased in cultures infected with ts mutants of complementation groups B and F, but continued in cultures infected with the parental virus and mutans of complementation groups A and G. In cultures infected with ts11 of complementation group B, the synthesis of viral minus-strand RNA ceased, whereas the synthesis of 42S and 26S plus-strand RNAs continued for at least 5 h after the shift to 39 degrees C. However, when ts11-infected cultures were returned to 28 degrees C 1 h after the shift to 39 degrees C, the synthesis of viral minus-strand RNA resumed, and the rate of viral RNA synthesis increased. The recovery of minus-strand synthesis translation of new proteins. We conclude that at least one viral function is required for alphavirus minus-strand synthesis that is not required for plus-strand synthesis. In cultures infected with ts6 of complementation group F, the syntheses of both viral plus-strand and minus-strand RNAs were drastically reduced after the shift to 39 degrees C. Since ts6 failed to synthesize both plus-strand and minus-strand RNAs after the shift to 39 degrees C, at least one common viral component appears to be required for the synthesis of both minus-strand and plus-strand RNAs.     

Temperature-Sensitive Mutants of Vesicular Stomatitis Virus: Synthesis of Virus-Specific Proteins

Viral proteins synthesized in L cells infected with temperature-sensitive (ts) mutants of vesicular stomatitis (VS) virus at permissive (31 C) and nonpermissive (39 C) temperatures were compared by polyacrylamide gel electrophoresis. Mutant ts 5, deficient in synthesis of viral ribonucleic acid (RNA⁻), failed to synthesize any of the five identifiable viral proteins at 39 C. Each of three RNA⁺ mutants, representing three separate complementation groups, showed distinctive patterns of viral protein synthesis at nonpermissive temperature. Equivalent amounts of ³H-amino acids were incorporated into the five viral proteins made in cells infected with RNA⁺ mutant ts 45 at 31 and 39 C. Complete virions of ts 45 could be identified by electron microscopy of infected cells incubated at the nonpermissive temperature; the defect in ts 45 appeared to be due in part to greater thermolability of virions as compared with the wild-type. RNA⁺ mutant ts 23 was deficient in synthesis of viral envelope protein S and failed to make detectable virions at the nonpermissive temperature. Infection of cells at 39 C with the third RNA⁺ mutant, ts 52, resulted in synthesis of all five viral proteins, but the peak of radioactivity representing the viral membrane glycoprotein migrated more rapidly on gels than coelectrophoresed authentic virion ¹⁴C-glycoprotein or viral ³H-glycoprotein extracted from cells infected at 31 C. These data and results of experiments on incorporation of radioactive glucosamine suggest that the primary defect in mutant ts 52 at nonpermissive temperature is failure of glycosylation of the viral glycoprotein. The viral structural proteins made in cells infected with ts 52 at the nonpermissive temperature did not assemble into sedimentable components as they did at permissive temperature; this observation indicates failure of insertion of the nonglycosylated protein (G′) into cell membrane. In support of this hypothesis was the finding that antiviral-antiferritin hybrid antibody did not detect VS viral antigen on the plasma membrane of L cells infected at 39 C with ts 52. In contrast, VS viral antigen localized in plasma membrane of L cells infected at 39 C with mutants ts 23 and ts 45 was readily detected by electron microscopy and fluorescence microscopy.     

An X-linked gene affecting mouse cell DNA synthesis also affects production of unintegrated linear and supercoiled DNA of murine leukemia virus.

To identify specific cellular factors which could be required during the synthesis of retroviral DNA, we have studied the replication of murine leukemia virus in mouse cells temperature sensitive for cell DNA synthesis (M. L. Slater and H. L. Ozer, Cell 7:289-295, 1976) and in several of their revertants. This mutation has previously been mapped on the X chromosome. We found that a short incubation of mutant cells at a nonpermissive temperature (39 degrees C) during the early part of the virus cycle (between 0- to 20-h postinfection) greatly inhibited virus production. This effect was not observed in revertant or wild-type cells. Molecular studies by the Southern transfer procedure of the unintegrated viral DNA synthesized in these cells at a permissive (33 degrees C) or nonpermissive temperature revealed that the levels of linear double-stranded viral DNA (8.8 kilobase pairs) were nearly identical in mutant or revertant cells incubated at 33 or 39 degrees C. However, the levels of two species of supercoiled viral DNA (with one or two long terminal repeats) were significantly lower in mutant cells incubated at 39 degrees C than in mutant cells incubated at 33 degrees C or in revertant cells incubated at 39 degrees C. Pulse-chase experiments showed that linear viral DNA made at 39 degrees C could not be converted into supercoiled viral DNA in mutant cells after a shift down to 33 degrees C. In contrast, such conversion was observed in revertant cells. Restriction endonuclease analysis did not detect differences in the structure of linear viral DNA made at 39 degrees C in mutant cells as compared to linear viral DNA isolated from the same cells at 33 degrees C. However, linear viral DNA made at 39 degrees C in mutant cells was poorly infectious in transfection assays. Taken together, these results strongly suggest that this X-linked gene, affecting mouse cell DNA synthesis, is operating in the early phase of murine leukemia virus replication. It seems to affect the level of production of unintegrated linear viral DNA only slightly while greatly reducing the infectivity of these molecules. In contrast, the accumulation of supercoiled viral DNA and subsequent progeny virus production are greatly reduced. Our pulse-chase experiments suggest that the apparent, but not yet identified, defect in linear viral DNA molecules might be responsible for their subsequent impaired circularization.     

Control of protein synthesis by a temperature-sensitive mutant of reovirus 3. I. Temperature-sensitive function of ts261-b mutant.

The ability of a temperature-sensitive (ts) mutant of reovirus, ts261-b, to synthesize virus-specific RNAs and proteins during infection at the nonpermissive temperature (37 degrees C) was investigated. The relative amounts of the mutant virus-specific single-stranded (ss) RNA''s and double-stranded (ds) RNA''s synthesized in cells at 37 degrees C were 20 to 25% as much as those synthesized in the wild-type virus-infected cells. The 10 segments of the mutant ds RNAs and the three size classes of the ss RNAs were synthesized in the usual proportions. The methylation of the mutant viral mRNA''s (ss RNAs) was not blocked at 37 degrees C in infected cells. A striking temperature-sensitive restricted function of the ts261-b mutant was expressed in the synthesis of the viral proteins. This study, which uses an in vitro protein-synthesizing system reconstituted with an endogenous polysomal fraction and a postribosomal supernatant from reovirus-infected cells, has demonstrated that the endogenous polysomes obtained from ts261-b mutant-infected cells at 37 degrees C are not active in the synthesis of the viral polypeptides of known molecular weights, and the amounts of the mutant viral polypeptides synthesized in vitro by these polysomes are 5 to 9% of those synthesized by the corresponding fraction from wild-type-infected cells. The impaired protein-synthesizing capacity of the mutant virus-specific polysomes can be restored during maintenance of the infected cells at 30 degrees C after shift-down from 37 degrees C. The in vitro synthesis of viral polypeptides of known size by the active endogenous polysomes derived from cells infected at the permissive temperature is accelerated by the addition of the postribosomal supernatant obtained from cells infected at the permissive temperature. The postribosomal supernatant from mutant-infected cells at 37 degrees C did not have a stimulatory effect, but rather, it inhibited in vitro viral protein synthesis.     

Thermosensitive Block of the Sabin Strain of Poliovirus Type I

The thermosensitive defect of the Sabin LSc2ab strain of poliovirus type I was studied. Transfer of infected KB cells from 36 to 38.5 C resulted in 30% inhibition of viral RNA replication but in 90% inhibition of formation of virions. Neither 74S procapsids nor 14S particles were detected in the cells transferred to the non-permissive temperature. However, procapsids, once accumulated at 36 C, were normally stable at 38.5 C and could transform into virions at that temperature. Viral proteins synthesized at the nonpermissive temperature were not different from those synthesized at permissive temperature, as judged from their pattern in polyacrylamide gel electrophoresis and from the fact that they normally matured into virions when the infected cells were brought back to permissive temperature, even under conditions of inhibition of protein synthesis. This leads to the conclusion that the defect in the Sabin strain studied lies in the assembly of its viral capsid proteins into capsomeres.     

Herpes Simplex Virus Glycoproteins: Isolation of Mutants Resistant to Immune Cytolysis

Immune cytolysis mediated by antibody and complement is directed against components of the major herpes simplex virus (HSV) glycoprotein complex (molecular weight, 115,000 to 130,000), comprised of gA, gB, and gC, and against glycoprotein gD-all present on the surfaces of infected cells. Tests with a temperature-sensitive (ts) mutant of HSV-1 (tsA1) defective in glycoprotein synthesis at the nonpermissive temperature (39 degrees C) demonstrated that over 90% of mutant-infected cells maintained at 39 degrees C and treated with antibody and complement were not lysed, presumably due to the absence of viral glycoproteins on the surface of infected cells at this temperature. Furthermore, a small number of tsA1-infected cells could be detected among a large excess of wild-type virus-infected cells by virtue of their failure to be lysed at 39 degrees C by antibody and complement. Making use of the involvement of viral glycoproteins in immune cytolysis and the ability of cells infected with glycoprotein-defective mutants to escape cytolysis, we sought mutants defective in the expression of individual viral glycoproteins. For this purpose, antisera directed against the VP123 complex and against the gC and combined gA and gB glycoprotein subcomponents of this complex were first tested for their ability to lyse wild-type virus-infected cells in the presence of complement. Wild-type virus-infected cells were lysed after treatment with each of the three antisera, demonstrating that the gC glycoprotein and the combined gA and gB glycoproteins can act as targets in the immune cytolysis reaction. Next, these antisera were used to select for mutants which were resistant to immune cytolysis. Cells infected with wild-type virus which had been mutagenized with 2-aminopurine and incubated at 39 degrees C were treated with one of the three types of antisera (anti-VP123 complex, anti-gC, or anti-gAgB) and lysed by the addition of complement. Cells which survived immune cytolysis were plated, and virus in the resulting plaques was isolated. Plaque isolates were tested for temperature sensitivity of growth and altered cytopathic effects in cell culture at 34 degrees C (the permissive temperature) and 39 degrees C. A total of 73 mutants was isolated in this manner. Selection with glycoprotein-specific antisera resulted in a 2- to 16-fold enrichment for mutants compared with "mock" -selected mutants using normal rabbit serum. Phenotypically, 24 mutants were temperature sensitive for growth, 27 were partially temperature sensitive, and 22 were not temperature sensitive but exhibited markedly altered cytopathic effects at both permissive and nonpermissive temperatures. Nine mutants of each phenotype (temperature sensitive, partially temperature sensitive, and non-temperature sensitive) were selected at random for confirmatory immune cytolysis tests with the antisera used in their selection. Cells infected with eight of the nine mutants were shown to be significantly more resistant to immune cytolysis at the nonpermissive temperature than were the mock-selected mutants or the wild-type virus from which they were derived.     

Characterization of temperature-sensitive mutants of simian rotavirus SA11: protein synthesis and morphogenesis.

The synthesis of viral polypeptides, distribution of viral antigens, and morphogenesis of viral structures have been examined in cells infected with temperature-sensitive (ts) mutants of SA11 representing 10 recombination groups. At the permissive temperature (31 degrees C) the synthesis of viral polypeptides and the distribution of viral antigens did not differ significantly from those of the wild type. At the nonpermissive temperature (39 degrees C) some mutants (tsB, -C, -E, -F, and -G) synthesized significantly smaller amounts of viral polypeptides and had a very diffuse distribution of viral antigen. Several of the mutants synthesized one or more electrophoretically aberrant polypeptide species at both 31 and 39 degrees C. All of the mutants, except tsF, assembled morphogenetic intermediates at 39 degrees C. Aberrant intermediates were assembled in all mutants at 31 and 39 degrees C. No specific morphogenic defect could be associated with any of the ts mutants.     

Inhibition of Interjacent Ribonucleic Acid (26S) Synthesis in Cells Infected by Sindbis Virus

The interrelationship of viral ribonucleic acid (RNA) and protein synthesis in cells infected by Sindbis virus was investigated. When cultures were treated with puromycin early in the course of infection, the synthesis of interjacent RNA (26S) was preferentially inhibited. A similar result was obtained by shifting cells infected by one temperature-sensitive mutant defective in RNA synthesis from the permissive (29 C) to the nonpermissive (41.5 C) temperature. Under both conditions, the viral RNA produced appeared to be fully active biologically. Once underway, the synthesis of viral RNA in wild-type Sindbis infections did not require concomitant protein synthesis.     

Impaired processing of precursor polypeptides of temperature-sensitive mutants of Rauscher murine leukemia virus.

The synthesis and processing of virus-specific precursor polypeptides in NIH/3T3 cells infected at the permissive temperature (31 degrees C) with temperature-sensitive (ts) mutants of Rauscher murine leukemia virus was studied in pulse-chase experiments at the permissive and nonpermissive (39 degrees C) temperatures. The newly synthesized virus-specific polypeptides were analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis after immunoprecipitation with polyvalent and monospecific antisera against Rauscher murine leukemia virus proteins. In cells infected with ts mutants defective in early replication steps (the early mutants ts17 and ts29), and ts mutants defective in postintegration steps (the late mutants ts25 and ts26), the processing of the primary gag gene product was impaired at the nonpermissive temperature. gag-pr75 of all four mutants was converted into gag-pr65; however, gag-pr65 accumulated at the nonpermissive temperature, and the main internal virion polypeptide p30 was not formed. Therefore, the proteolytic cleavage is blocked beyond gag-pr65. Concomitantly, the formation of the env gene-related polypeptide p12(E) of all four mutants was blocked at the restrictive temperature. In contrast, cells infected with the late mutant ts28, which produced noninfectious virions at 39 degrees C, showed a normal turnover of the gag and env precursor polypeptides.