A point mutation in the poliovirus polymerase gene determines a complementable temperature-sensitive defect of RNA replication

We have previously described the isolation of a RNA- temperature-sensitive (ts) mutant of poliovirus type 1, ts035, after chemical mutagenesis by 5-fluorouracil. The ts defect of ts035 correlated with defective RNA replication, since the two characters corevert in the case of spontaneous revertants. The alteration of a trans-acting replication function of ts035 was suggested by significant rescue following mixed infection with another ts mutant, ts221, or with wild-type virus. Protein synthesis appeared normal at 39 degrees (nonpermissive temperature) in shift-up experiments and no defect of RNA elongation was evidenced when the activity of replication complexes or purified polymerase was measured at 39 degrees. These results provide circumstantial evidence that the initiation of ts035 RNA synthesis at 39 degrees is impaired. Molecular cloning of the ts035 genome allowed us to construct a recombinant virus with the same ts phenotype as ts035, by the transfer of a fragment of the mutant polymerase gene into the wild-type genome. Two mutations were present in this region of the ts035 genome but the determination of nucleotide sequences in the case of ts035 revertants indicated that only the substitution from A to G at nucleotide 7256 was necessary for the ts phenotype. This mutation replaces Asn 426 by an Asp in polypeptide 3D, the viral polymerase.     

Extrachromosomal recombination in vaccinia-infected cells requires a functional DNA polymerase participating at a level other than DNA replication

Homologous recombination was measured in vaccinia-infected cells cotransfected with two plasmid recombination substrates. One plasmid contains a vaccinia protein lacZ coding region bearing a 1.1 kb 3' terminal deletion while the other plasmid contains a non-promoted lacZ coding region bearing a 1.1 kb 5' terminal deletion. Homologous recombination occurring between the 825 bp of lacZ common to both plasmids regenerates a functional lacZ gene from which B-galactosidase expression was measured. The entire 3 kb lacZ gene was used as a positive control. A panel of thermosensitive mutants was screened in cells either transfected with the positive control plasmid or cotransfected with the recombination substrates. A DNA - mutant, ts42, known to map to the viral DNA polymerase gene was found to be defective in recombination. Significantly, other DNA - mutants, ts17 or ts25, or other DNA polymerase mutants did not exhibit a defect in recombination similar to ts42. Inhibitors of viral DNA synthesis did not uniformly affect recombination. Cytosine arabinoside and aphidicolin inhibited B-galactosidase expression from the recombination substrates but not from the positive control plasmid, whereas hydroxyurea enhanced expression from both. Marker rescue with the cloned wildtype DNA polymerase gene repaired the defect in ts42. Southern and western analyses demonstrated that B-galactosidase activity was consistent with a recombined lacZ gene and unit size 116 kDa protein. Measurement of plasmid and viral DNA replication in cells infected with the different DNA - mutants indicated that recombination was independent of plasmid and viral DNA replication. Together these results suggest that the vaccinia DNA polymerase participates in homologous recombination at a level other than that of DNA replication.     

A genetic recombination map of foot-and-mouth disease virus.

Sixty ts mutants were isolated from the Pacheco strain of type O foot-and-mouth disease virus after treatment with either 5-fluorouracil or hydroxylamine. The conditions affecting recombination and assay of the ts+ recombinants were standardized. Using two ts mutants resistant to guanidine, three-factor crosses, supported by two-factor crosses, located 34 of the mutations in a linear arrangement. The recombination frequencies between certain pairs of mutations were additive. The guanidine character of the two resistant mutants mapped as a single site mutation and was located near the middle of the map.     

Temperature-sensitive (ts) mutants of an influenza A virus for which genetic synergism is required to express their ts phenotypes

Temperature-sensitive (ts) mutants of fowl plague virus (FU 67 and ts 7/36) have been isolated. They can be rescued to wild type by double infection with all standard ts mutants having single ts defects in any of the eight RNA segments. Rescue of FU 67 to wild type was not possible when ts mutants having simultaneous mutations in one of the RNA segments coding for the P proteins, the haemagglutinin, and nonstructural protein genes were used. After rescue with various prototype influenza A strains different RNA segments of FU 67 were replaced, depending on the strain used for rescue. The results are compatible with the idea that in FU 67 and ts the ts phenotype is caused by the synergism of mutations in several RNA segments, each of which by itself is not sufficient to exhibit the ts phenotype.     

An extended genetic recombination map for foot-and-mouth diseases virus.

The original foot-and-mouth diseases virus recombination map (Lake, Priston & Slade, 1975), which include 35 mutagen-induced ts mutants, has been extended both in detail and size by the mapping of a further 33 ts mutants (9 mutagen-induced and 24 spontaneous). The size increase from 0.57% to 3.27% maximum recombination fequency was principally due to the use of a new standardization technique for recombination fequencies but, in addition, the original map distance was increased by approx. 30% due to the mapping of new mutations. As in the original map, there was a marked concentration of mutations near the guanidine (gs) locus, i.e. 83% of the mutants had mutations in the third of the map adjacent to the gs locus.     

Multiple sites of recombination within the RNA genome of foot-and-mouth disease virus

Recombinant foot-and-mouth disease viruses were isolated from cells infected with a mixture of temperature-sensitive (ts) mutants belonging to different subtype strains. In order to select for recombination events in many different regions of the genome, crosses were performed between various pairs of mutants, with ts mutations in different regions of the genome. ts+ progeny were analysed by electrofocusing virus-induced proteins and RNase T1 fingerprinting of their RNA. All but 5 out of 43 independent isolates, from nine crosses, proved to have recombinant RNA genomes. Maps of these genomes, based on a knowledge of the locations of the unique oligonucleotides, were constructed. Most could be interpreted as being the products of single genetic cross-overs, although three recombinants were formed by two cross-overs each. Cross-overs in at least twelve distinct regions of the genome were identified. This evidence of a large number of recombination sites suggests that RNA recombination in picornaviruses is a general, as opposed to a site-specific, phenomenon.     

Chemically-induced temperature sensitive mutants of dengue virus type 2

Temperature sensitive (ts) mutants of dengue virus type 2 (DEN-2, TH-36 isolate) were induced by replication in primary hamster kidney cells treated with 5-azacytidine. Seven ts mutants were obtained from 138 clones isolated by an immunofluorescent cloning technique. Of these 7 ts mutants, 5 were sufficiently stable to permit partial characterization. Complementation was detected at very low but statistically significant levels between some ts mutants at 40 degrees C. Viral double-stranded RNA production was evaluated in LLC-MK2 cells at 30 degrees and 40 degrees C by micro-quantitative complement fixation. The results of complementation tests and RNA production tests indicated that the 4 of 5 stable ts mutants constitute 3 separate complementation groups (2 RNA+ and 1 RNA-groups), while a fifth ts mutant was RNA- but non-complementable. The data presented here indicate that a genetic system can be developed without employing traditional plaque or cytopathology methods. Further, the 5 DEN-2 ts mutants are believed to represent the only set of complementation-positive flavivirus mutants so far isolated.     

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.     

Mutations in the RNA-binding domains of tombusvirus replicase proteins affect RNA recombination in vivo

RNA recombination, which is thought to occur due to replicase errors during viral replication, is one of the major driving forces of virus evolution. In this article, we show evidence that the replicase proteins of Cucumber necrosis virus, a tombusvirus, are directly involved in RNA recombination in vivo. Mutations within the RNA-binding domains of the replicase proteins affected the frequency of recombination observed with a prototypical defective-interfering (DI) RNA, a model template for recombination studies. Five of the 17 replicase mutants tested showed delay in the formation of recombinants when compared to the wild-type helper virus. Interestingly, two replicase mutants accelerated recombinant formation and, in addition, these mutants also increased the level of subgenomic RNA synthesis (Virology 308 (2003), 191-205). A trans-complementation system was used to demonstrate that mutation in the p33 replicase protein resulted in altered recombination rate. Isolated recombinants were mostly imprecise (nonhomologous), with the recombination sites clustered around a replication enhancer region and a putative cis-acting element, respectively. These RNA elements might facilitate the proposed template switching events by the tombusvirus replicase. Together with data in the article cited above, results presented here firmly establish that the conserved RNA-binding motif of the replicase proteins is involved in RNA replication, subgenomic RNA synthesis, and RNA recombination.     

Studies of two temperature-sensitive mutants of Moloney murine leukemia virus

Events associated with the replication of two spontaneous temperature-sensitive mutants (ts1 and ts3) of Moloney murine leukemia virus were studied under non-permissive and permissive conditions of infection. From temperature-shift experiments ts1 appears to have a temperature-sensitive step occurring early in the growth cycle and in an unidentified function. The temperature-sensitive defect of ts3 occurs late in the replicative cycle. Electron microscope studies showed that although budding partially occurs at the nonpermissive temperature there is no release of virus particles from the cell membrane. The defect of ts3 was also found to be in one of the factors required for the rescue of MuSV.     

Multiple recombination sites at the 5'-end of murine coronavirus RNA

Mouse hepatitis virus (MHV), a murine coronavirus, contains a nonsegmented RNA genome. We have previously shown that MHV could undergo RNA-RNA recombination in crosses between temperature-sensitive mutants and wild-type viruses at a very high frequency (S. Makino, J.G. Keck, S.A. Stohlman, and M.M.C. Lai (1986) J. Virol. 57, 729-737). To better define the mechanism of RNA recombination, we have performed additional crosses involving different sets of MHV strains. Three or possibly four classes of recombinants were isolated. Recombinants in the first class, which are similar to the ones previously reported, contain a single crossover in either gene A or B, which are the 5'-most genes. The second class of recombinants contain double crossovers in gene A. The third class of recombinants have crossovers within the leader sequence located at the 5'-end of the genome. The crossover sites of the third class have been located between 35 and 60 nucleotides from the 5'-end of the leader RNA. One of these recombinants has double crossovers within the short region comprising the leader sequences. Finally, we describe one recombinant which may contain a triple crossover. The presence of so many recombination sites within the 5'-end of the genome of murine coronaviruses confirms that RNA recombination is a frequent event during MHV replication and is consistent with our proposed model of "copy-choice" recombination in which RNA replication occurs in a discontinuous and nonprocessive manner.     

Isolation and characterization of temperature-sensitive mutants of fowl plague virus.

Twenty-five temperature-sensitive (ts) mutants of fowl plague virus have been isolated and tentatively characterized. Twenty-three of them can be arranged into six recombination groups. The classification of the two other ts mutants is still uncertain. RNA-negative mutants have been found in four recombination groups. One group harbors ts mutants with a lesion in the hemagglutinin gene, another group consists of mutants with a lesion in the neuraminidase gene. One group seems to have a maturation defect. Cells infected with still another group retain the RNA polymerase-template complex within the nucleus. The biological properties of the ts mutants of the various recombination groups suggest that at least some of the gene products might have more than one function.     

Effects of Bloom's syndrome fibroblasts on genetic recombination and mutagenesis of herpes simplex virus type 1

The effects of Bloom's syndrome (BS) fibroblasts on genetic recombination and the mutation frequency of herpes simplex virus type 1 (HSV-1) was determined by employing two factor crosses of selected temperature-sensitive (ts)mutants. A significant increase in the recombination frequency (RF) was observed in seven of nine crosses when multiple BS fibroblast lines were compared to normal human fibroblasts. The RF of HSV-1 ts mutants increased following 12-O-tetradecanoylphorbol-13-acetate (TPA) treatment of normal, but not BS fibroblasts, suggesting that BS fibroblasts express higher constitutive levels of genetic recombination activity. HSV-1 ts mutants demonstrated significantly higher reversion frequencies to the nontemperature sensitive (ts⁺)phenotype following growth in BS rather than normal fibroblasts, indicating that exogenous viral DNA encoding many of the enzymes necessary for its own replication is affected by the mutator phenotype of BS.Presented in part at the American Association for Cancer Research, May 1986, Los Angeles, California.     

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.