A vaccinia virus isatin-beta-thiosemicarbazone resistance mutation maps in the viral gene encoding the 132-kDa subunit of RNA polymerase

The mutation in a vaccinia virus mutant resistant to inhibition by isatin-beta-thiosemicarbazone was mapped by marker rescue. DNA from the resistant mutant was cloned into cosmid and plasmid vectors and tested for its ability to convert wild-type vaccinia virus to IBT resistant virus in a helper-mediated marker rescue protocol. Resistance was mapped in this way to a 0.9-kb DNA fragment derived from the HindIII A fragment of vaccinia genome. Southern blot hybridization using this DNA as a probe demonstrated that the 0.9-kb fragment is contained within the DNA sequence encoding the second largest subunit of vaccinia RNA polymerase, rpo132. Thus, mutation of rpo132 can cause resistance to IBT in vaccinia virus.     

Isolation, characterization, and physical mapping of temperature-sensitive mutants of vaccinia virus

Thirty-nine new temperature-sensitive mutants of vaccinia virus have been isolated, expanding a previously reported collection of mutants (R. C. Condit and A. Motyczka, Virology 113, 224-241, 1981) to a total of 65. The 65 mutants have been assigned to 32 complementation groups, based primarily on a qualitative spot test described previously (Condit and Motyczka, 1981). Representatives of each complementation group have been assayed for DNA and protein synthesis at the nonpermissive temperature, revealing one new DNA-negative complementation group, three new groups which contain mutants defective in late protein synthesis, and ten new groups containing mutants which synthesize DNA and protein in a normal fashion. Marker rescue has been achieved with 29 of the 65 mutants using cloned DNA fragments from wild-type virus. These 29 mutants together represent 20 of the 32 complementation groups. A preliminary physical map of the mutants is presented.     

Isolation and preliminary characterization of temperature-sensitive mutants of vaccinia virus

Methods have been developed for rapid isolation and genetic analysis of vaccinia virus mutants. These methods include: (1) monitoring mutagenesis by measuring conversion of wild-type, phosphonoacetic acid-sensitive virus to phosphonoacetic acid-resistant virus, (2) screening for mutants by plaque enlargement, and (3) a qualitative spot test for complementation. Twenty-six temperature-sensitive mutants of vaccinia virus have been isolated. All have reversion indices of 10^?4 or less. One-step growth experiments have been done at 40° and 31° with all the mutants and in all cases the virus yield at 40° is less than 8% of the yield observed at 31°. Complementation analysis has been completed on all 26 mutants, showing that these mutants together comprise 16 complementation groups. Twenty-four of the mutants have been analyzed for their ability to synthesize viral DNA at the nonpermissive temperature. The results show that 3 of the 24 have a DNA-negative phenotype. These three mutants fall into two complementation groups. Twenty-four of the mutants have been analyzed for their ability to synthesize early and late viral proteins at the nonpermissive temperature. From this analysis, four phenotypes appear: (1) normal, (2) a phenotype associated with DNA-negative mutants characterized by prolonged synthesis of early proteins and the absence of late protein synthesis, (3) weak or slow late protein synthesis, (4) abortive late protein synthesis.     

Macromolecular synthesis in cells infected with frog virus 3. III. Virus-specific protein synthesis by temperature-sensitive mutants.

Six temperature-sensitive (ts) mutants of frog virus 3, (five DNA⁺ and one DNA^?) representing six separate complementation groups, were examined for their intracellular patterns of virus-specific protein synthesis both at permissive (23°) and nonpermissive (30°) temperature. At permissive temperature protein synthesis and its regulation by each mutant was similar to wild type. At nonpermissive temperature all proteins were detected but some had altered rates of synthesis, indicating defective regulation by three of the six ts mutants.The six mutants can be divided into three categories based upon the time and nature of the ts defect during the replication cycle. The first category includes three mutants, each representing a separate complementation group in which virus-specific protein synthesis and its regulation is apparently normal at nonpermissive temperature. These mutants have defects in the virus maturation and assembly process suggesting the participation of several frog virus 3 genes in the assembly process. The second category consists of a single mutant that has an early defect in the regulation of viral protein synthesis; consequently a late pattern of virus-specific protein (VSP) synthesis is absent in cells infected with this mutant at nonpermissive temperature. The third category includes two mutants; these mutants are unable to regulate the rate of synthesis of certain VSP but have some features of the late pattern of virus-specific protein synthesis.The data presented in this report confirm and extend the evidence for temporal control of the rate of viral protein synthesis during frog virus 3 infection. This control appears to be mediated through several viral regulatory proteins.     

Isolation and basic properties of temperature--sensitive mutants of Venezuelan equine encephalomyelitis virus

Fifteen temperature-sensitive (ts) mutants were isolated from 4 Venezuelan equine encephalomyelitis virus clones by nitrous acid treatment or by growing the virus in the presence of 5-fluoruracil. Three of them were classified as RNA- mutants by their inability to synthesize RNA at nonpermissive temperature (1.5-3.3% in respect to the wild type). The remaining 11 mutants showed a slight decrease of RNA synthesis at the nonpermissive temperature (32-73+) and were referred to the RNA+ phenotype. One mutant possessed RNA +/- phenotype (18%). Five complementation groups were determined by complementation analysis of the mutants.     

Studies on temperature-sensitive events in synthesis of poliovirus ts mutants

Summary Studies on temperature-sensitive events of four poliovirus ts mutants indicated that the temperature-sensitive stage of ts2 and ts5 mutants occurs late in the reproduction cycle, but for ts 11 and tsM/23 it occurs both at early and at late stages of the cycle. RNA production by ts2, ts11 and tsM/23 mutants (RNA^–) under nonpermissive conditions (40° C) is 13–14 per cent as compared with RNA production at 36° C. All the mutants under study form both 35S and 20S RNA under nonpermissive conditions, but the relative amount of 35S RNA synthesized at 40° C is considerably lower than at 36° C. A degradation of viral RNA under nonpermissive conditions is detected. Obtained data suggest that ts5 mutant (RNA⁺) under nonpermissive conditions is incapable of assembly of virus particles from synthesized components, but the multiplication of ts2, ts11 and tsM/23 mutants does not occur because of strong inhibition of RNA synthesis. Protein subvirion structures induced by ts5 mutant under nonpermissive conditions after subsequent transfer of the infected cells to permissive conditions, are incorporated into mature virus particles. Subvirion structures induced by ts M/23 mutant do not participate in further stages of morphogenesis after the infected cells are shifted to permissive conditions.     

Novel acylation of poxvirus A-type inclusion proteins

Myristylation is one of several post-translational modifications that occur on vaccinia virus (VV) proteins. Previously, time course labeling of VV-infected cells with myristic acid had indicated that five late proteins (17, 25, 36, 38 and 92 kDa) are myristylated. Four of these proteins were mapped to the E7R, L1R, AI6L and G9R open-reading frames, respectively, because of the predicted presence of the N-myristyltransferase recognition sequence (M-G-X-X-X-S/T/A) at their amino termini. In contrast, computer analyses of large (80–100 kDa) VV open reading frames did not reveal any predicted species with this N-terminal motif. By immunoprecipitation with monospecific sera and transient expression of cloned gene products, the myristylated 92-kDa protein has been demonstrated to be the A-type inclusion protein encoded by the Western Reserve (WR) strain of VV. Labeling of cowpox virus (CPV) infected cells with myristic acid indicated that the 160-kDa A-type inclusion protein appears to be myristylated as well. Both the VV 92-kDa and the CPV 160-kDa A-type inclusion proteins labeled with myristic acid were stable to hydroxylamine treatment, suggesting an amide linkage between the fatty acid and the acceptor protein. HPLC analysis confirmed that the 92-kDa protein was in fact myristylated. This data suggests that poxvirus ATI proteins may be subject to a novel type of internal myristylation modification, and the roles such modifications may play in the replication cycles of these viruses is discussed.     

Phenotypic characterization of a vaccinia virus temperature-sensitive complementation group affecting a virion component

Genetic and biochemical evidence is presented which shows that the product of the vaccinia virus gene 18R is a virion protein. Western blot analysis of virion proteins using anti-18R serum detects a 78,000-Da protein, localized in the virus core. Of five ts mutants which map to gene 18R, two mutants, ts 10 and ts 44, possess thermolabile virions. Temperature shifts performed during single-step growth of ts 44 suggest that precursors required for virion maturation accumulate during nonpermissive infections with ORF 18R mutants and that protein synthesis is required for recovery from nonpermissive condition.     

St. Louis encephalitis virus temperature-sensitive mutants

Nine temperature-sensitive (ts) mutants of St. Louis encephalitis virus were isolated after "forced mutagenesis" with 5-fluorouracil or 5-azacytidine. The ts mutants could be grouped on the basis of RNA synthesis at 40 degrees C, the nonpermissive temperature and complementation analysis. Four complementation groups were identified. Members of two of the groups were negative for RNA synthesis at 40 degrees C while the remainder were positive.     

Mouse mammary tumor virus can mediate cell fusion at reduced pH

Four different temperature-sensitive (ts) mutants derived from the Mahoney strain of poliovirus type 1, were crossed in an infectious center recombination test. Evidence for recombination was obtained in three crosses, with a different segregation of an unselected marker, resistance to guanidine, in each case. Evidence for genetic complementation between ts mutants was not found, except with one set of RNA- mutants, ts 221 and ts 035. The marked virus yield enhancement which was observed in cells mixedly infected by these two mutants resulted from a nonreciprocal rescue of ts 035 by ts 221. The effects of ts 221 input multiplicity and of guanidine inhibition of viral RNA replication on the rescue were analyzed. The results showed that yield enhancement of ts 035 in mixed infection could be correlated to the low level RNA replication of ts 221 at the nonpermissive temperature.     

Identification of the point mutations in two vaccinia virus nucleoside triphosphate phosphohydrolase I temperature-sensitive mutants and role of this DNA-dependent ATPase enzyme in virus gene expression

The biological function of the nucleoside triphosphate phosphohydrolase I (NTPase I) enzyme of vaccinia virus is not yet known. In this investigation we have identified the genetic lesion of two temperature-sensitive mutants of vaccinia virus, ts50 and ts36, as single point mutations contained within the 5'615 nucleotides of the NTPase I gene (ts50, G to A at position 131; ts36, C to T at position 556). The point mutations result in amino acid substitutions of Gly to Glu-44 (ts50) and Pro to Ser-186 (ts36). In monkey BSC-40 cells, ts50 and ts36 behave phenotypically like wild-type virus with respect to replication and synthesis of viral DNA but are defective in late polypeptide synthesis. However, these two ts mutants displayed a drastically different phenotype in virus-infected human HeLa cells at the restrictive temperature; viral DNA replication did not occur and late polypeptide synthesis was absent. Moreover, if the early block was overcome by a temperature shift-up, then HeLa cells infected with the ts mutants displayed a profile characteristic of defective late viral polypeptide synthesis. Our results reveal that vaccinia NTPase I enzyme functions early and late in the viral replication cycle and that the phenotype of these ts mutants is dependent upon the cell type.     

The white pock mutants of rabbit poxvirus IV. The late white pock (μ) host range (hr) mutants of rabbit poxvirus are blocked in morphogenesis

The white pock (μ) host range (hr) mutants of rabbit poxvirus (RPμhr mutants) can be divided into two subclasses (early and late) based on their ability to synthesize viral DNA in nonpermissive pig kidney (PK) cells. We have previously shown that the infection of PK cells by two early (DNA minus) mutants is abortive due to an inhibition of translation early in the infectious cycle. Here we have examined the abortive infection of PK cells by three late (DNA plus) mutants. All three late mutants synthesize DNA and late proteins (at reduced levels) in PK cells but each of the three late mutants is missing a late viral protein of 72,000 daltons. The three late mutants appear to process the precursor structural proteins in a normal fashion suggesting that viral morphogenesis begins. No induction of particulate viral RNA polymerase indicative of internal core differentiation is observed, however, and little, if any, mature virus is formed. Instead, infection of nonpermissive cells leads to the accumulation of DNA-containing, primitive immature principles (PIPS) which do not differentiate further into virus.