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.     

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.     

Analysis of adenovirus 12 temperature-sensitive mutants defective in viral DNA replication

Ten temperature-sensitive mutants of adenovirus 12 defective in viral DNA replication at the nonpermissive temperature (40°) were selected and seven of them were classified into three groups (A, B, and C) by complementation tests in viral DNA synthesis. The mutants in Groups A, B, and C were defective in initiation of viral DNA synthesis at 40°, as revealed by viral DNA chain elongation or ligation and density label in temperature shift-up experiments. Analysis by the M band technique indicated that the mutants in Groups B and C were less defective than the mutant in Group A in formation of viral DNA replication complex at the nonpermissive temperature. Additional differences among mutants in Groups A, B, and C were also shown by temperature shift-down experiments. All these mutants were not defective in formation of T antigen in permissive cells and in induction of cellular DNA synthesis in nonpermissive cells at the nonpermissive temperature. These observations suggest that at least three viral genes are involved at different steps in initiation of viral DNA replication.     

Isolation and genetic characterization of temperature-sensitive mutants that define five additional recombination groups in simian rotavirus SA11

Nineteen independent temperature-sensitive (ts) mutants were isolated from SA11 following mutagenesis with proflavin or 5-azacytidine. Fourteen of the ts mutants fell into one or another of five mutant groups previously defined by recombination. Five of the ts mutants defined five recombination groups (F, G, H, I, and J) that had not been previously identified. Thus, 10 of the 11 expected mutant groups have been identified in SA11. The prototype mutants of the 10 mutant groups were tested for recombination at nonpermissive temperature to determine if any group had a lesion affecting recombination. Most mutant pairs recombined efficiently; however, the tsH mutant was restricted in its recombination with the tsB and tsI mutants and the tsG and tsJ mutants failed to recombine at detectable levels at nonpermissive temperature. The mutants of groups F-J did not complement, or did so inefficiently, and interfered with the growth of wild type at both permissive and nonpermissive temperatures. The growth properties of the mutants of groups F-J are described.     

Temperature-sensitive mutants of influenza virus

Summary Six temperature-sensitive (ts) mutants were isolated from the progeny of wildtype influenza A virus grown in the presence of 5-fluorouracil. All mutants had the efficiency of plating lower than 10^–4 at 38° C compared with 31° C. One mutant (ts- 5) failed to produce hemagglutinin at the nonpermissive temperature. Temperature-shift experiments revealed that the temperature-sensitive step ofts-5 resides in the process normally taking place at 4–6 hours post infection. After mixed infection ofts-5 andts-9, different from each other in physiological defect, the occurrence of both complementation and recombination was demonstrated, confirming that they carry genetic defects at different genes.     

Temperature-sensitive mutants of influenza A/Udorn/72 (H3N2) virus. III. Genetic analysis of temperature-dependent host range mutants

One hundred thirty-three ts mutants of influenza A/Udorn/72 virus were arranged into eight complementation groups, A-H, on Madin-Darby canine kidney (MDCK) monolayer cultures at the restrictive temperature of 40 degrees. The eight complementation groups, A-H, on MDCK cells corresponded to the eight recombination groups, A-H, on rhesus monkey kidney (RMK) cells, respectively, and this suggested that each MDCK complementation group represented one of the eight influenza A RNA gene segments. These ts viruses were used to identify the locus of the ts mutation in temperature-dependent host range (td-hr) mutants of the A/Udorn/72 virus. Sixteen of the 133 ts mutants exhibited distinct host (MDCK)-dependent restriction of plaque formation at 40 degrees but not at 34 degrees and were referred to as td-hr mutants. These 16 td-hr mutants were ts+ (not ts) on RMK cells but ts on MDCK cells. The td-hr mutants did not share a common lesion and the ts lesions were distributed among the eight complementation groups, A-H, when tested on MDCK cells. An analysis of one of the td-hr mutants indicated that an extrageneic RMK-dependent suppressor mutation did not account for the td-hr phenotype. These data suggested that a host-dependent ts mutation was responsible for the td-hr restriction of this mutant. Representation of td-hr mutations in each of the eight complementation groups indicates that the influenza A virus genome can undergo mutation leading to an altered host range in any of its eight RNA segments.     

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.     

Genetic studies of respiratory syneytial virus temperature-sensitive mutants

Summary Seven temperature-sensitive mutants of RS virus were analyzed genetically for evidence of complementation and recombination. The results of these tests suggested that there were 3 mutually exclusive complementation groups. No evidence of recombination was obtained; however, mixed infection of cells with mutants belonging to 2 different complementation groups yielded viruses which appeared to be complementing heterozygotes.     

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.     

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.     

Analysis of ts mutations of cold-adapted influenza A/Leningrad/134/57 virus variants

By recombination of ts mutants of fowl plague virus belonging to different complementation groups with two cold-adapted variants of human influenza virus, the number and gene localization of ts mutations occurring in these variants was determined. In the course of passaging of human influenza virus at lowered temperature, the number of genes with ts mutations increased.     

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.     

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.     

Synthesis and processing of the nonstructural polyproteins of several temperature-sensitive mutants of Sindbis virus

We have examined the synthesis and processing of nonstructural polyproteins by several temperature-sensitive mutants of Sindbis virus, representing the four known RNA-minus complementation groups. Four mutants that possess mutations in the C-terminal domain of nonstructural protein nsP2 all demonstrated aberrant processing patterns when cells infected with these mutants were shifted from a permissive (30 degrees) to a nonpermissive (40 degrees) temperature. Mutants ts17, ts18, and ts24 showed severe defects in processing of nonstructural polyproteins at 40 degrees, whereas ts7 showed only a minor defect. In each case, cleavage of the bond between nsP2 and nsP3 was greatly reduced whereas cleavage between nsP1 and nsP2 occurred almost normally, giving rise to a set of polyprotein precursors not seen in wild-type-infected cells at this stage of infection. The nsP1 produced by these mutants was unstable and only small amounts could be detected in infected cells at the nonpermissive temperature. Submolar quantities of nsP2 were also present. We suggest that nsP1 and nsP2 may function as a complex and that free nsP1, and possibly nsP2, is degraded. Cleavage between nsP3 and nsP4 appeared to be normal in the mutants except in the case of ts17, where upon shift to 40 degrees P34 was unstable and nsP4 accumulated. We propose that the change in the P34/nsP4 ratio upon shift is responsible for the previously observed temperature sensitivity of subgenomic 26 S RNA synthesis in ts17 and for the failure of the mutant to regulate minus strand synthesis at 40 degrees. Other mutations tested, including ts21, which is found in the N-terminal half of nsP2, ts11, which has a mutation in nsP1, and ts6, which has a mutation in nsP4, all demonstrated nonstructural polyprotein processing indistinguishable from that in wild-type-infected cells. These results support our conclusion, based upon deletion mapping studies, that the C-terminal domain of nsP2 contains the nonstructural proteinase activity.     

The formation of arenaviruses that are genetically diploid

Analyses of RNA extracted from preparations of arenaviruses indicate that the relative molar proportions of the genomic L and S RNA species are frequently far from equal. In order to investigate the genetic significance of this observation temperature-sensitive (ts) mutants of two lymphocytic choriomeningitis (LCM) virus strains (ARM and WE) have been recovered and categorized into recombination groups (Groups I and II). Fingerprint analyses of wild-type progeny viruses obtained from dual infections with ARM Group II and WE Group I ts viruses indicate that they have L/S RNA genotypes of WE/ARM. It is concluded that the ARM Group II ts viruses have mutations in their L RNA species and that the WE Group I ts viruses have mutations in their S RNA species. Correspondingly it is deduced that the ARM Group I ts viruses have S RNA mutations and the WE Group II ts viruses mutations in their L RNA species. Cells coinfected with certain WE Group I mutants, or an ARM Group I and certain WE Group I ts mutants, have also yielded wild-type viruses. Fingerprint analyses have shown that the wild-type viruses obtained from the latter crosses are diploid with respect to their S RNA species. On subsequent passage these wild-type viruses shed high proportions of ts mutants. We interpret the data to indicate that the original Group I ts mutants that yielded the diploid viruses have mutations in different S RNA gene products so that the progeny produce plaques at the nonpermissive temperature by gene product complementation. No wild-type recombinant viruses have been obtained from crosses involving Pichinde and LCM ts mutants.     

Three new complementation groups of temperature-sensitive Chinese hamster ovary cell mutants defective in the endocytic pathway

We describe here the results of complementation studies with six mutant Chinese hamster ovary cells expressing temperature-sensitive lesions affecting the endocytic pathway. The mutants were crossed with representatives of the End1 and End2 complementation groups identified previously by Robbins et al. (J. Cell Biol. 99:1296–1308, 1984). Two mutants, G.8.1 and 31.1, were members of the End1 complementation group. One mutant, 25.2, was a member of the End2 complementation group. The other three mutants each defined new complementation groups, which we have designated End3 (mutant G.7.1), End4 (mutant V.24.1), and End5 (mutant 42.2). Previous work on mutants of the Endl, End2, and End3 classes had shown that these mutants were defective in endosomal acidification. We prepared postnuclear supernatants from mutants harvested at the nonpermissive temperature and compared their acidification activities, assessed by ATP-stimulated quenching of acridine orange. Members of the End1, End2, and End2 groups had reduced acidification activity, correlating with the acidification defects known to be expressed by these mutants. Strain V.24.1 (End4) also expressed a 40% reduction in acidification activity, while strain 42.2 (End5) had no reduction of acidification activity.     

Factors that affect genetic interaction during mixed infection with temperature-sensitive mutants of simian rotavirus SA11

A number of factors that affect genetic interaction during mixed infection with temperature-sensitive mutants of simian rotavirus SA11 have been examined. (1) Statistical analyses of recombination frequency (RF) indicated that (a) the variability noted in RF was not related to variations in experimental conditions and (b) a linear map of the mutations could not be drawn. (2) The wild phenotype of recombinant progeny was stable on passage. (3) Aggregates of progeny virus or heterozygous progeny virus particles did not contribute significantly to the observed RF. (4) RF increased in parallel with multiplicity of infection. (5) A maximal, or near maximal, RF was obtained at the earliest time significant recombinants could be detected. (6) Recombination was efficient at nonpermissive temperature. (7) Complementation did not occur or was inefficient. (8) Mutants from all recombination groups interfered with the growth of wild-type virus at both permissive and nonpermissive temperatures.     

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.