Fine structure mapping and phenotypic analysis of five temperature-sensitive mutations in the second largest subunit of vaccinia virus DNA-dependent RNA polymerase

We have used plasmid clones spanning the region encoding the 132-kDa subunit of the cowpox virus RNA polymerase (CPV rpo 132) to marker rescue each of five vaccinia virus (VV) temperature sensitive (ts) mutants, ts 27, ts 29, ts 32, ts 47, and ts 62, which together constitute a single complementation group. The experiments fine-map the vaccinia mutations to a 1.3-kb region containing the 3' end of the CPV rpo 132 gene. Phenotypic characterization shows that all five mutants are affected to varying extents in their ability to synthesize late viral proteins at the nonpermissive temperature, similar to other ts mutants with lesions in the 22- and the 147-kDa subunits of the VV RNA polymerase. Two mutants, ts 27 and ts 32, exhibit a delay in the synthesis of late viral proteins at both the permissive and the nonpermissive temperatures. We conclude that the five VV mutants affect the 132-kDa subunit of the VV RNA polymerase. Additional genetic experiments demonstrate intragenic complementation between ts 62 and three other members of this complementation group, ts 27, ts 29, and ts 32.     

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.     

A study of TMV ts mutant Ni2519. I. Complementation experiments

Two distinct virus-specific functions, i.e., virus assembly and spreading of infection from cell to cell (transport function), are temperature-sensitive (ts) in TMV mutant Ni2519. Assembly of Ni2519 cannot be complemented by the temperature-resistant TMV strains used: A14 (a wild type strain from which Ni2519 was derived) and dolichos enation mosaic virus (DEMV, or cowpea strain of TMV), a thermophilic strain. On the other hand, Ni2519 can serve as a donor of the coat protein to complement is strain Ni118, which has a mutation in the coat protein gene. The genomic RNA can be produced by Ni2519 at a nonpermissive temperature; functionally active Ni2519 coat protein (capable of coating Ni118 RNA upon mixed infection) is produced at a nonpermissive temperature as well. The is phenotype of Ni2519 upon virus assembly probably results not from the ts behavior of any virus-coded protein(s) but is due to the ts properties of the genomic RNA molecule itself, so the possibility of the complementation of assembly of Ni2519 is ruled out. Thus, Ni2519 appears to represent a novel class of virus mutants with is virion RNA. The second is function of Ni2519 (transport of infection) can be complemented by a helper virus. The experimental system used for complementation of the transport function allowed Ni2519 to spread from cell to cell at a nonpermissive temperature. Obviously, Ni2519 infection spreads under these conditions in a form different from that in the mature virions, since its assembly cannot be complemented by the helper virus. Some aspects of the transport function are discussed.     

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.     

Functions of the major nonstructural DNA binding protein of a herpesvirus (pseudorabies)

Eight mutants of pseudorabies virus belonging to complementation group 3 and situated between 0.14 and 0.18 units on the physical map of the genome were analyzed. All the mutants tested in this respect (seven) recombined with one another, indicating that the mutations were located in different regions of the gene. All mutants were DNA-; the first round, as well as subsequent rounds, of DNA replication was completely blocked at the nonpermissive temperature in the mutant-infected cells. After shift-up from the permissive to the nonpermissive temperature, viral DNA synthesis continued for a short period of time only and viral DNA which had accumulated at the permissive temperature became degraded. Parental viral DNA, however, retained its integrity at the nonpermissive temperature and viral DNA synthesis started immediately after shift-down of the mutant-infected cells from the nonpermissive to the permissive temperature (even in the absence of protein synthesis). All mutants belonging to complementation group 3 tested (5 out of 8) produced a thermolabile nonstructural DNA binding protein (136K). In some of the mutant virus-infected cells this protein failed to migrate to the nucleus. We conclude that the pseudorabies virus mutants in complementation group 3 code for a defective 136K protein and that this protein is not only essential to the process of viral DNA synthesis but also plays a role in the stabilization of progeny DNA (but not of nonreplicating parental DNA) within the infected cells.     

Deoxyribonucleoprotein complexes and DNA synthesis of herpes simplex virus type 1

Two temperature-sensitive mutants of herpes simplex virus type 1 in complementation group 1-1 were analyzed to determine if the major DNA-binding protein they produced was thermolabile. Cells infected with these mutants were analyzed for deoxyribonucleoprotein complexes containing the DNA-binding protein. These complexes were found in cells infected at the permissive temperature but not at the nonpermissive temperature. In temperature shift-up experiments with mutant virus infected cells, the levels of the deoxyribonucleoprotein complexes decreased with time of incubation at the nonpermissive temperature. Viral DNA synthesis terminated in cells infected with these mutants after temperature shift-up. The kinetics of termination of viral DNA synthesis were similar to the kinetics of dissociation of the deoxyribonucleoprotein complexes. These results indicate that two mutants in complementation group 1-1 produce a thermolabile DNA-binding protein and that this protein is required for viral DNA synthesis. Furthermore, they suggest that the major DNA-binding protein of herpes simplex virus type 1 functions in viral DNA synthesis as a component of deoxyribonucleoprotein complexes.     

Biology of simian virus 40 (SV40) transplantation antigen (TrAg) II. Isolation and characterization of additional temperature-sensitive mutants of SV40.

Fourteen independent temperature-sensitive mutants of simian virus (SV40) were isolated following nitrous acid or hydroxylamine mutagenesis. Three mutants were assigned to the A group and seven to the BC group on the basis of standard qualitative and quantitative complementation assays. Three other mutants did not complement mutants of any complementation group well under standard conditions nor was delayed complementation observed in quantitative assays. However, these mutants were shown to complement members of the A and BC complementation groups but not members of the D group when the qualitative complementation test was modified by allowing the parental virions to uncoat at permissive temperature prior to incubation at 41°. The assignment of these mutants to the D group was substantiated by demonstrating the wild-type infectivity of DNA extracted from cells infected at 33° for growth at 41°. Thirteen of the mutants were tested for the production of tumor (T), capsid (C), virion (V), and major coat protein (VP1) antigens at permissive and nonpermissive temperature by immunofluorescence assays along with mutants which have been described previously by others for comparison. The temperature-sensitive (ts) mutants isolated in this study produced fully immunoreactive T antigen at both temperatures. None of the tsA mutants produced C, VPl, or V antigens at elevated temperature. The BC mutants isolated in this study all produced T antigen at 41°. These late mutants demonstrated two patterns of expression of virion antigens. One group synthesized C, V, and VP1 at 41° and were indistinguishable from wild type on the basis of antigenic phenotype. A second group showed cytoplasmic and nucleolar fluorescence for C and VPl antigens at the nonpermissive temperature similar to that observed for tsBCll previously. Mutants in this group did not produce V antigen at high temperature.     

Temperature-sensitive host-dependent mutants of Sindbis virus

Following chemical mutagenesis of Sindbis virus, two viral clones (clone 35 and clone 58) were isolated which at 34.5° had a relative plaquing efficiency on Aedes albopictus mosquito cells 10⁴- to 10⁵-fold lower than that of wild type virus. Standard growth curves showed that at 34.5° the viral mutants were restricted in mosquito cells but not in primary cultures of chick embryo fibroblasts (CEF). Adsorption of intact virus particles of clone 35 and clone 58 to mosquito cells was as efficient as that observed with wild type virions. However, transfection of mosquito cells with viral RNA of these mutant clones gave a significantly lower plaquing efficiency than infectious RNA from standard virus. Both mutants were temperature sensitive in CEF as well as in A. albopictus cells. At the nonpermissive temperature they were RNA^? and by complementation analysis were assigned to group F. Clones 35 and 58 did not complement each other.     

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.     

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, complementation and preliminary phenotypic characterization of temperature-sensitive mutants of herpes simplex virus type 2

Eight temperature-sensitive (ts) mutants of herpes simplex virus type 2, isolated following mutagenesis with 5-bromodeoxyuridine, were assigned to seven nonover-lapping complementation groups (A through G). Partial phenotypic characterization of the mutants at the nonpermissive temperature (38 °) revealed the following: (1) Mutants of 3 complementation groups had a DNA^? phenotype, the remaining being DNA⁺. (2) The DNA^? mutants were deficient in DNA polymerase activity, whereas the DNA⁺ mutants were not. (3) Two mutants, one DNA⁺ and one DNA^?, failed to induce thymidine kinase. (4) Except for one DNA^? mutant, all mutants synthesized physical particles; however, only DNA⁺ mutants synthesized enveloped particles. (5) Mutants belonging to 4 complementation groups were markedly more thermolabile than the wild-type virus, suggesting that they possessed alterations in viral structural proteins.     

Polyoma virus-transformed cells produce transforming growth factor(s) and grow in serum-free medium

Vaccinia virus temperature-sensitive (ts) mutants were isolated after nitrosoguanidine mutagenesis. A number of these mutants exhibited host range temperature sensitivity in that the efficiency of plaque formation at the nonpermissive temperature was poorer on chick cells than on hamster or human cells. Forty-two mutants were assigned to 23 different complementation groups on the basis of complementation and the efficiency of apparent recombination at the nonpermissive temperature. Recombination frequencies were also determined from mixed infections carried out at the permissive temperature and it was confirmed that mutants within the same complementation group recombined less efficiently with each other than mutants belonging to different groups. Mutants from two of the largest groups could be tentatively ordered on linear intragenic maps that spanned 0.8 and 2 recombination units. Moreover, from intergenic crosses between mutants in 14 different complementation groups, a linkage map spanning 66.3 recombination units, was derived. This study illustrates the feasibility of two-factor recombination mapping of poxvirus mutations and provides genetic data that should be of relevance in further analysis of the is mutations.     

Biochemical characteristics of fowl plague virus TS mutants

Five fowl plague virus ts mutants belonging to five different complementation groups were studied. In one mutant (ts 131) the virion RNA-polymerase was defective in vitro at the nonpermissive temperature and the later stages of replication in vivo (synthesis of RNA, polypeptides, and RNP) were found to be defective. One of the mutants studied (ts 5) under nonpermissive conditions showed no disorders in the function of virion RNA-polymerase or in vivo synthesis of RNA, polypeptides, functionally active hemagglutinin and neuraminidase or RNP production, however, no formation of infectious virions or virion-like structures occurred under these conditions. In three mutants (ts 43, ts 166, and ts29) the virion RNA-polymerase was capable of functioning in vitro at the nonpermissive temperature but no RNA synthesis in vivo was observed when tested 4 hr after infection. In these mutants, synthesis of virus-specific polypeptides at nonpermissive and optimal temperatures under the conditions of the experiment was similar both qualitatively and quantitatively. Despite the fact that in the cells infected with ts 43, ts 166, and ts 29 mutants synthesis of both polypeptides hemagglutinin precursors was observed at the nonpermissive temperature, no functionally active hemagglutinin was produced under these conditions. The data are discussed with reference to the mechanisms of reproduction of orthomyxoviruses.     

Further studies of the physical and metabolic properties of foot-and-mouth disease virus temperature-sensitive mutants.

Three temperature-sensitive (ts) mutants of foot-and-mouth disease virus were classified as ribonucleic acid negative and as belonging to the same complementation group when measured by virus yields and [3H] uridine incorporation in paired, mixed infections at the nonpermissive temperature (38.5C). Mutant ts-22, the only mutant able to produce plaques at 38.5 C, was more sensitive to acid than were the parental wild-type or other mutant viruses. Diethylaminoethyl-dextran did not enhance the plaque-forming ability of the mutant viruses at 38.5C. All of the viruses inhibited host cell protein syntehsis at both permissive (33C) and nonpermissive (38.5C) temperatures.     

Mutants of sindbis virus. I. Isolation and partial characterization of 89 new temperature-sensitive mutants.

More than 100 new temperature-sensitive mutants of Sindbis virus have been isolated, following mutagenesis with nitrous acid, N-methyl-N′-nitro-N-nitrosoguanidine, 5-azacytidine, and 5-fluorouridine. Thirty-six of these mutants synthesize at least 60% as much RNA at the nonpermissive temperature as does the parental strain and are designated RNA⁺; 23 mutants synthesize between 10 and 60% as much RNA as the parental strain at 40° and are designated RNA^±; 30 mutants make less than 10% as much RNA at 40° and are called RNA^?. The remaining mutants have not been tested for RNA incorporation.The thermal stability at 56° of most of the mutant particles has been examined. The majority of the RNA⁺ mutants is more sensitive to heating at 56° than the parental HR strain, and RNA⁺ mutations appear to reside primarily in genes coding for the structural proteins. Approximately 20% of either RNA^± or RNA^? mutants are thermosensitive, and these mutations thus appear to reside primarily in genes coding for the nonstructural proteins.Complementation assays have been performed with a number of these mutants and with those of Burge and Pfefferkorn (1966a, b). The existence of three complementation groups among the RNA⁺ mutants, which appear to encode the three major structural proteins, has been confirmed; no new complementation groups among RNA⁺ mutants have been identified. A total of four complementation groups has been identified among the RNA^? mutants. Thus, Sindbis virus contains at least seven complementation groups.     

Functional analysis of the A complementation group mutants of Sindbis HR virus

The 10 members of the A complementation group of temperature-sensitive (ts) mutants of SIN HR, the heat-resistant strain of Sindbis virus, were divided into two phenotypic subgroups. Subgroup I mutants (ts15, ts17, ts21, ts24, and ts133) demonstrated temperature-sensitive 26 S mRNA synthesis, whereas subgroup II mutants (ts4, ts14, ts16, ts19, and ts138) did not; both ts4 and ts19 demonstrated defective 26 S mRNA synthesis at 30 degrees. None of the A group mutants demonstrated temperature-sensitive 49 S plus-strand synthesis. Temperature-sensitive cleavage of ns230 was demonstrated by subgroup I mutants, except ts21, but not by subgroup II mutants. A revertant of ts133 that grew at 40 degrees retained temperature-sensitive 26 S mRNA synthesis but lost temperature-sensitive cleavage of ns230 and the RNA-negative phenotype. Only ts4, like ts11 of the B complementation group, demonstrated temperature-sensitive minus-strand RNA synthesis. In addition to ts24, cells infected with ts17 or ts133 continued to synthesize minus strands after shiftup in the absence of continued protein synthesis, and resumed synthesis of minus strands if shifted to the nonpermissive temperature after minus-strand synthesis had ceased at the permissive temperature.     

Defects in RNA+ temperature-sensitive mutants of Sindbis virus and evidence for a complex of PE2-E1 viral glycoproteins

Three temperature-sensitive mutants of Sindbis virus that are able to make viral RNA and belong to distinct complementation groups were found to share a common defect in the conversion of the PE2 precursor glycoprotein to E2 at the nonpermissive temperature. To resolve the conflict between the genetic data which suggest that the mutants have defects in different viral proteins with the biochemical data presented, we postulate the formation of a PE2-E1 complex in the infected cell. Further evidence for this complex was found in experiments showing that antiserum specific for E1 glycoprotein inhibits formation of E2 from PE2.