Genetic analysis of murine hepatitis virus strain JHM.

We performed a genetic analysis of 37 temperature-sensitive mutants of murine hepatitis virus strain JHM. Of our mutants, 32 did not induce murine hepatitis virus-specific RNA synthesis in infected cells at the restrictive temperature, 39 degrees C. By complementation testing we have identified at least seven nonoverlapping complementation groups. Six of the genes identified in this way are required for murine hepatitis virus-specific RNA synthesis. The seventh complementation group is made up of five mutants which induced virus-specific RNA synthesis at 39 degrees C.     

Isolation and characterization of temperature-sensitive mutants of Streptomyces coelicolor A3(2) blocked in macromolecular synthesis.

A collection of temperature-sensitive mutants of Streptomyces coelicolor A3(2) was isolated. The majority of the mutants showed an osmotically remedial phenotype. Mutants defective in macromolecular synthesis were identified and characterized further. Four mutants were found in which DNA replication was defective, but which continued to synthesize RNA and protein at the restrictive temperature (39 degrees C). The kinetics of cessation of DNA synthesis allowed a tentative identification of slow (initiation) and fast (elongation) stop dna mutants. The inhibition of DNA replication in the four mutants was found to be reversible on returning to the permissive temperature (30 degrees C), but only after a delay of about 2 h. Three other mutants were identified which showed not only cessation of DNA replication at the restrictive temperature, but also defects in other macromolecular synthesis events.     

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.     

Decline in mutation frequency in temperature-sensitive SV40 viruses before viral DNA synthesis.

Lesions that promote reversion from a temperature-sensitive to a wild-type phenotype were induced in temperature-sensitive late mutants of SV40 virus by UV irradiation. When cultures infected with UV-irradiated temperature-sensitive mutants were grown for various times at permissive temperature (35 degrees C) and then at restrictive temperature (39 degrees C), the reversion frequency declined just before the onset of semiconservative DNA synthesis when DNA synthesis began at 32 degrees C. This can be explained by competition between reactions that lead to the onset of viral DNA synthesis and reactions that repair the lesions before the onset of viral DNA synthesis.     

Temperature-sensitive defect of vesicular stomatitis virus in complementation group II.

The prototype member of the complementation group II temperature-sensitive (ts) mutants of vesicular stomatitis virus, ts II 052, has been investigated. In ts II 052-infected HeLa cells at the restrictive temperature (39.5 degrees C), reduced viral RNA synthesis was observed by comparison with infections conducted at the permissive temperature (30 degrees C). It was found that for an infection conducted at 39.5 degrees C, no 38S RNA or intracytoplasmic nucleocapsids were present. For nucleocapsids isolated from ts II 052 purified virions or from ts II 052-infected cells at 30 degrees C, the RNA was sensitive to pancreatic RNase after an exposure at 39.5 degrees C in contrast to the resistance observed for wild-type virus. The nucleocapsid stability of wild-type virus when heated to 63 degrees C or submitted to varying pH was not found in nucleocapsids extracted from ts II 052 purified virions. The data suggest that for ts II 052 there is an altered relationship between the viral 38S RNA and the nucleocapsid protein(s) by comparison with wild-type virus. Such results argue for the complementation group II gene product being N protein, so that the ts defect in ts II 052 represents an altered N protein.     

Simian Virus 40-Host Cell Interactions II. Cytoplasmic and Nucleolar Accumulation of Simian Virus 40 Virion Protein

We have used immunofluorescence in parallel with transmission and scanning electron microscopy to characterize the unusual cytoplasmic and nucleolar accumulation of Simian virus 40 (SV40) virion protein (C antigen) at restrictive temperatures (39 to 41 C) in monkey cells infected with a temperature-sensitive mutant of SV40 defective in virion assembly, tsB11. Cytoplasmic and nucleolar accumulation of C antigen did not occur in wild-type-infected cells at any temperature. Wild-type- and tsBll-infected cells were not distinguishable at 33 C by immunofluorescence or electron microscopy. Temperature-shift experiments using metabolic inhibitors of DNA (cytosine arabinonucleoside, 20 mug/ml), RNA (actinomycin D, 5 mug/ml), and protein synthesis (cycloheximide, 2 x 10(-4) to 10 x 10(-4) M) were used to investigate the requirements for ongoing DNA, RNA, and protein synthesis in the distribution of virion protein between the nucleus, nucleolus, and cytoplasm. The transport of C antigen from the nucleolus and cytoplasm into the nucleus was complete after a temperature shift-down (41 and 39 to 33 C). Limited virus particle formation occurred after the shift-down in the presence of actinomycin D and cycloheximide, indicating some of the 39 to 41 C synthesized virion protein could be used for capsid assembly at 33 C in the absence of further virion protein synthesis. Nucleolar and cytoplasmic accumulations of C antigen occurred in the absence of drugs after a shift-up (33 to 39 C and 41 C) indicating a continuous requirement for the tsB11 mutant function. Furthermore, the virion protein synthesized at 33 C remained confined to the nucleus when the cells were shifted to 39 and 41 C in the presence of actinomycin D or cycloheximide. In the presence of cytosine arabinonucleoside, however, the virion protein accumulated in large aggregates in the nucleus and nucleolus after the shift-up, but did not migrate into the cytoplasm as it did in drug-free tsB11-infected control cells. Colchicine (10(-3) M) had no effect on the abnormal accumulation of C antigen during shift-up or shift-down experiments suggesting that microtubular transport plays little if any role in the abnormal transport of tsB11 virion protein from cytoplasm to nucleus. Although virus particles were never observed by electron microscopy and V antigen was not detected by immunofluorescence at 39 or 41 C in tsB11-infected cells, dense amorphous accumulations were formed in the nucleoli and cytoplasm. We suggest that the tsB11 function is continuously required for the normal transport of SV40 virion protein between the cytoplasm, nucleolus, and nucleus and for the assembly of capsids and virions. Several possible mechanisms for the altered tsB11 function or protein are discussed. One of the virion proteins may also be involved in some presently undetermined nucleolar function during SV40 productive infection.     

Identification of temperature-sensitive DNA- mutants of Chinese hamster cells affected in cellular and viral DNA synthesis.

We described a strategy which facilitates the identification of cell mutants which are restricted in DNA synthesis in a temperature-dependent manner. A collection of over 200 cell mutants temperature-sensitive for growth was isolated in established Chinese hamster cell lines (CHO and V79) by a variety of selective and nonselective techniques. Approximately 10% of these mutants were identified as ts DNA- based on differential inhibition of macromolecular synthesis at the restrictive temperature (39 degrees C) as assessed by incorporation of [3H]thymidine and [35S]methionine. Nine such mutants, selected for further study, demonstrated rapid shutoff of DNA replication at 39 degrees C. Infections with two classes of DNA viruses extensively dependent on host-cell functions for their replication were used to distinguish defects in DNA synthesis itself from those predominantly affecting other aspects of DNA replication. All cell mutants supported human adenovirus type 2 (Ad2) and mouse polyomavirus DNA synthesis at the permissive temperature. Five of the nine mutants (JB3-B, JB3-O, JB7-K, JB8-D, and JB11-J) restricted polyomavirus DNA replication upon transfection with viral sequences at 33 degrees C and subsequent shift to 39 degrees C either before or after the onset of viral DNA synthesis. Only one of these mutants (JB3-B) also restricted Ad2 DNA synthesis after virion infection under comparable conditions. No mutant was both restrictive for Ad2 and permissive for polyomavirus DNA synthesis at 39 degrees C. The differential effect of these cell mutants on viral DNA synthesis is expected to assist subsequent definition of the biochemical defect responsible.     

Preliminary Physiological Characterization of Temperature-Sensitive Mutants of Vesicular Stomatitis Virus

Different temperature-sensitive mutants of vesicular stomatitis virus have been characterized in terms of their ability to induce synthesis of viral ribonucleic acid (RNA) in BHK-21 cells at 39 C (the restrictive temperature for these mutants). Mutants belonging to complementation groups I and IV (and probably II) did not induce actinomycin-resistant RNA synthesis in infected cells incubated at 39 C. All three mutants comprising complementation group III induced viral RNA synthesis at 39 C. The temperature sensitivity of the defective viral functions has also been studied by temperature-shift experiments. The functions associated with the mutants of groups I, II, and IV were required early, whereas the function associated with the group III mutants was not required until a late stage of the viral cycle. The heat sensitivity of extracellular virion was not correlated with complementation group.     

Altered Protein Metabolism in Infection by the Late tsB11 Mutant of Simian Virus 40

The DNA of the temperature-sensitive mutant tsB11 is replicated at the same rate as the DNA of wild-type virus in infection at the restrictive temperature. The progeny mutant DNA cannot be distinguished from wild-type DNA by gel electrophoresis and is assembled into a nucleoprotein complex with the same velocity sedimentation characteristics as the wild-type complex. Analysis of in vivo protein synthesis by sodium dodecyl sulfate polyacrylamide gel electrophoresis and immunoprecipitation techniques demonstrated that the capsid components VP1, VP2, and VP3 of the mutant and wild-type virus are synthesized at a similar rate, but VP1 fails to accumulate within cells infected by tsB11. Furthermore, VP1 is located predominantly in the cytoplasmic rather than in the nuclear fraction of extracts from cells infected by the mutant. Immunofluorescent studies localized virion antigen within the nucleolus as well as the cytoplasm. The altered intracellular distribution and stability of VP1 suggest that it may be the mutant protein of tsB11. The synthesis of a 72,000 dalton protein is consistently induced in significant quantity in cells infected by tsB11 at the restrictive temperature. A protein of the same apparent molecular weight is present in smaller quantities in uninfected cells and is only slightly increased in quantity in cells infected by wild-type virus.     

Functional defects of RNA-negative temperature-sensitive mutants of Sindbis and Semliki Forest viruses.

Defects in RNA and protein synthesis of seven Sindbis virus and seven Semliki Forest virus RNA-negative, temperature-sensitive mutants were studied after shift to the restrictive temperature (39 degrees C) in the middle of the growth cycle. Only one of the mutants, Ts-6 of Sindbis virus, a representative of complementation group F, was clearly unable to continue RNA synthesis at 39 degrees C, apparently due to temperature-sensitive polymerase. The defect was reversible and affected the synthesis of both 42S and 26S RNA equally, suggesting that the same polymerase component(s) is required for the synthesis of both RNA species. One of the three Sindbis virus mutants of complementation group A, Ts-4, and one RNA +/- mutant of Semliki Forest virus, ts-10, showed a polymerase defect even at the permissive temperature. Seven of the 14 RNA-negative mutants showed a preferential reduction in 26S RNA synthesis. The 26S RNA-defective mutants of Sindbis virus were from two different complementation groups, A and G, indicating that functions of two viral nonstructural proteins ("A" and "G") are required in the regulation of the synthesis of 26S RNA. Since the synthesis of 42S RNA continued, these functions of proteins A and G are not needed for the polymerization of RNA late in infection. The RNA-negative phenotype of 26S RNA-deficient mutants implies that proteins regulating the synthesis of this subgenomic RNA must have another function vital for RNA synthesis early in infection or in the assembly of functional polymerase. Several of the mutants having a specific defect in the synthesis of 26S RNA showed an accumulation of a large nonstructural precursor protein with a molecular weight of about 200,000. One even larger protein was demonstrated in both Semliki Forest virus- and Sindbis virus-infected cells which probably represents the entire nonstructural polyprotein.     

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.     

Temperature-sensitive multicellular mutants of Wangiella dermatitidis.

Three temperature-sensitive morphological mutants of Wangiella dermatitidis were isolated and characterized. The mutants grew in the yeastlike morphology at the permissive temperature (25 degrees C) but expressed a multicellular (Mc) phenotype at the restrictive temperature (37 degrees C). Cultures of Mc 2 and 3 incubated at the restrictive temperature showed rapid reductions in the percentage of budded cells in the population. In contrast, budding continued for several generations in cultures of Mc 1. Incubation of cultures of Mc 2 and 3 at the restrictive temperature for 48 h resulted in nearly total conversion of yeastlike cells to the multicellular form; about 50% of the cells of Mc 1 had converted to multicellular forms after 48 h at the restrictive temperature. Studies using radiolabeled compounds documented that DNA, RNA, and protein synthesis continued at the restrictive temperature. The results suggest that multicellularity is the result of inhibition of bud emergence and cell separation without inhibition of growth nuclear division, and cytokinesis.     

Recombination Between Endogenous and Exogenous Simian Virus 40 Genes. I. Rescue of a Simian Virus 40 Temperature-Sensitive Mutant by Passage in Permissive Transformed Monkey Lines

Passage of the simian virus 40 (SV40) temperature-sensitive (ts) mutant tsD202 at the permissive temperature in each of three permissive lines of SV40-transformed monkey CV1 cells resulted in the emergence of temperature-insensitive virus, which plated like wild-type SV40 at the restrictive temperature on normal CV1 cells. In independent experiments, the amount of temperature-insensitive virus that appeared after passage on transformed cells was from 10(3)- to 10(6)-fold greater than the amount of ts-revertant virus that appeared after an equal number of passages in nontransformed CV1 cells. The virus rescued by passage on transformed cells bred true upon sequential plaque purification, plated on normal CV1 cells with single-hit kinetics at the restrictive temperature, and displayed no selective growth advantage on transformed cells compared to non-transformed cells. Hence, the reversion of the ts phenotype is neither due to complementation effects nor to the selection of preexisting revertants, which grow better on transformed cells. In the accompanying article (T. Vogel et al., J. Virol. 24:541-550, 1977), we present biochemical evidence that the rescue of tsD202 mediated by passage on transformed cells is due to recombination with the resident SV40 genome. Parallel experiments in which tsA, tsB, and tsC SV40 mutants were passaged in each of the three permissive lines of SV40-transformed monkey cells resulted in either only borderline levels of rescue (tsA mutants) or no detectable rescue (tsB and tsC mutants). Evidence is presented that the resident SV40 genome of the transformed monkey lines is itself a late ts mutant, and we suggest that this accounts for the lack of detectable rescue of the tsB and tsC mutants. We furthermore suggest that the borderline level of rescue observed with two tsA mutants is related to a previous finding (Y. Gluzman et al., J. Virol. 22:256-266, 1977) which indicated that the resident SV40 genome of the permissive transformed monkey cells is defective in the function required for initiation of viral DNA synthesis.     

Isolation and characterization of temperature-sensitive mutants of measles virus.

Nine temperature-sensitive (ts) mutants of nonattenuated Edmonston strain measles virus were isolated from wild-type virus which was grown in the presence of 5-fluorouracil. Adsorption, temperature shift, and complementation experiments indicated that all these mutants were restricted at an intracellular stage of infection. However, all the mutants were more rapidly inactivated at 41 C than was wild-type virus, suggesting that the ts product of each mutant either influences or is a structural component of the virus. Three complementation groups were found to be represented among the mutants. Group A contained one mutant and it did not induce synthesis of detectable amounts of viral antigen at the nonpermissive temperature (39 C). Group B consisted of six mutants which did not induce viral antigen synthesis at 39 C and one mutant which did. Group C was represented by one mutant and it induced viral antigen synthesis at 39 C. The two mutants which induced sythesis of viral antigen also induced synthesis of relatively small amounts of virus-specific RNA at 39 C. These mutants, while producing cytoplasmic and nuclear accumulations of viral antigen at 39 C, were restricted in production of syncytia and hemadsorption. All the mutants were less neurovirulent than wild-type virus, as indicated by their inability to produce acute disease in newborn hamsters.     

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.     

Respiratory syncytial virus ts mutants and nuclear immunofluorescence.

A replicated sector-plating procedure was used to isolate 35 induced temperature-sensitive (ts) mutants and one spontaneous ts mutant from a wild-type stock of respiratory syncytial (RS) virus cloned from recent clinical material. Seven of these mutants were ts for plaque formation at 37 degrees C as well as at the restrictive temperature of 39 degrees C. The wild-type strain did not differ markedly from standard laboratory strains of RS virus. It was dependent on exogenous arginine (84 mug/ml) for optimal growth, and was not significantly inhibited by mitomycin C (10 mug/ml). It was sensitive to actinomycin D (2.5 mug/ml) during the early part of the growth phase. A characteristic focal cytopathic effect was obtained in BS-C-1 cells. Staining of infected monolayers by an indirect immunofluorescence procedure revealed a profusion of filamentous processes extending from the plasma membrane, and a similar modification of the surface of infected cells could be visualized by scanning electron microscopy. Filament production was inhibited when certain ts mutants were incubated at 39 degrees C, confirming the virus-specific nature of the phenomenon. Thirty-four of the mutants were classified into three groups by immunofluorescence. Complementation was observed in mixed infection with a single mutant from each group. Nuclear, as well as cytoplasmic, immunofluorescence was detected in RS virus-infected cells using a high-titer bovine anti-bovine RS virus serum. Visualization of nuclear antigen was dependent on the inhibition of cytoplasmic fluorescence obtained when ts mutants in groups I and III were incubated at restrictive temperature.