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.     

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.     

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.     

Assignment of simian rotavirus SA11 temperature-sensitive mutant groups A, C, F, and G to genome segments

Crosses were performed between prototype temperature-sensitive (ts) mutants of simian rotavirus SA11 representing reassortment groups A, C, F, and G and ts mutants of rhesus rotavirus RRV that belonged to different reassortment groups. Wild-type (ts+) reassortant progeny were identified by plaque formation at nonpermissive temperature (39 degrees), picked, and grown to high titer. The ts+ phenotype of the resulting progeny clones was verified by titration at 39 degrees and 31 degrees. The electropherotypes of the ts+ clones were determined by electrophoresis, and parental origin of each genome segment was assigned by comparison of segment mobility to parental markers. Analysis of the parental origin of genome segments in the ts+ reassortants derived from SA11 ts X RRV ts crosses revealed the following map locations of the SA11 prototype ts mutants: tsA(778), segment 4; tsC(606), segment 1; tsF(2124), segment 2; and tsG(2130), segment 6. The assignment of tsA was made on the basis of genome segment segregation in two independent crosses with each of two independent RRV ts mutants. The assignment of tsC was made on the basis of segregation in only a single cross with an RRV ts mutant; however, a larger number of progeny clones were examined from this cross. The lesion of tsF was mapped with data from three independent crosses using two different RRV ts mutants. The assignment of tsG was made on the basis of segregation in three independent crosses, two with RRV ts mutants and one with Wa. The assignments of tsA, tsC, and tsF were confirmed in crosses between RRV ts mutants representing those reassortment groups, and SA11 ts mutants in other reassortment groups.     

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 mutants of fowl plague virus: isolation and genetic characterization.

Forty-nine temperature-sensitive mutants of fowl plague virus (FPV) strain Rostock and four is mutants of FPV-strain Dobson were isolated by utilizing two methods of plaque screening, after either spontaneous or chemically induced mutagenesis. Twenty-nine of the FPV-Rostock mutants were further characterized by genetic recombination studies and were found to fall into six high frequency recombination groups. The genome segment carrying the is mutation in each group was identified by analyzing the gene composition of ts⁺ recombinants generated from crosses between representatives of each group and ts mutants of FPV-Dobson. It was concluded that the six groups correspond to mutations in six different genome segments, namely, those coding for the P₁, P₂, P₃, HA, NP, and NS proteins.     

Isolation and biochemical characterization of intertypic recombinants of foot-and-mouth disease virus

Recombinants were isolated between two European serotypes (O and A) and between two of the most distantly related serotypes (O from Europe and SAT2 from Africa) using appropriate ts mutants in an infectious centre assay. The recombinants were characterised by electrofocusing of their induced proteins and by RNase-T1 fingerprinting of their RNA. The approximate location of the cross-over event in each recombinant was determined by sequencing the unique distinguishable O or A oligonucleotides and locating them within the known genome sequence. Nine different types of recombinant were identified from the two types of cross (O X A and O X SAT) and all had a single cross-over in the middle or 3' half of the genome, i.e. in the nonstructural coding region. Recombination between the most distantly related viruses (O X SAT2) appeared to occur at a lower frequency than recombination between serotypes of the same group (O X A). A higher incidence of recombinant proteins with unique pI was also observed in the O X SAT2 crosses.     

Polarity in adenovirus recombination

The distributions of the crossovers necessary to generate ts+ genomes have been examined in a collection of clonally unrelated ts+ recombinants from a set of ts X ts adenovirus crosses. In a cross between two parents that are grossly heterologous between map units 80.2 and 91.5, the distribution of crossovers was significantly skewed toward the left-hand end of the genome, with a declining frequency proceeding rightward. This gradient of recombination was modified by the removal of the right-hand heterology and by the presence of another region of heterology between map units 3.67 and 10.11. In a cross where the ts markers were flanked by both heterologies, no gradient was observed and ts+ recombinants were characterized by a higher rate of supernumerary crossovers. In a cross designed so that one ts marker was internal to two heterologies, crossovers were found disproportionately between the second ts marker and the nearby heterology. In addition, ts+ recombinants formed by crossing over internal to the heterologies again were accompanied by a high frequency of supernumerary crossovers. Finally, ts+ recombinant frequencies in crosses identical except for the presence of either one or two flanking heterologies were markedly lower in the latter case. These data, taken together, suggest that a major pathway of adenovirus recombination initiates at, or near, the molecular termini and is perhaps driven by the displaced single strands produced during DNA replication. Internal initiation, on the other hand, may employ these single strands to form genetic "patches."     

Temperature-sensitive mutants of herpes simplex virus type 2: a provisional linkage map based on recombination analysis.

Thirteen ts mutants of type 2 herpes simplex virus were backcrossed to a syncytial but not temperature-sensitive mutant of wild-type virus. This was an attempt to introduce a third marker, syncytial plaque morphology or syn, into at least some of the ts mutants. Three ts mutants carrying the syn marker were obtained but only one, ts 9, was satisfactory for genetic experiments. Three-factor crosses were carried out between ts 9 syn and the mutants which determined the order of eleven ts mutations relative to both the ts 9 mutation and the syn mutation. A provisional linkage map based both on the order derived from the three-factor crosses and on map distances from recombination frequencies has been prepared: it contains nine ts mutations and the syn mutation.     

The kinetics of adenovirus recombination in homotypic and heterotypic genetic crosses

The kinetics of recombination between temperature-sensitive mutants has been studied in both homotypic (Ad5 x Ad5) and heterotypic (Ad5 x Ad2+NDI) crosses. There is a significant increase in the recombination frequency during the rise period of viral replication in a single-step replication cycle. This observation suggests that adenovirus DNA molecules can undergo progressive rounds of recombination before assembly into virions. To examine this possibility further, advantage was taken of the serotype-specific restriction endonuclease sites and polypeptide sizes of Ad5 and Ad2+ND1 to enumerate and to localize the sites of crossing over in ts+ recombinants isolated early and late in a heterotypic infection. The late recombinants exhibited, on average, more crossovers per genome than did the early recombinants. This finding is predicted if multiple rounds of recombination take place in some genomes. Using blot hybridization with specific probes, the production of recombinant molecules in the intracellular DNA replicating pool has been followed. Recombinants were found before the rise in infectious virus and increased in frequency relative to the parental molecules throughout the exponential period. These data confirm and extend the genetic observations, which were made on a selected set of infectious virus.     

A genetic map of reovirus. II. Assignment of the double-stranded RNA-negative mutant groups C, D, and E to genome segments

The double-stranded RNA (dsRNA) of recombinants derived from crosses of the dsRNA-negative, temperature-sensitve (ts) mutants of reovirus type 3 and reovirus serotypes 1 or 2 were examined by polyacrylamide gel electrophoresis. Analysis of deletions and replacements in the recombinants allowed identification of genome segments containing the ts lesions. In this way the location of the mutation of the group C prototype mutant tsC(447) os genome segment S2, that of the group D prototype mutant tsD(357) is genome segment L1, and that of the group E prototype mutant tsE(320) is genome segment S3. In addition the location of the temperature-sensitive lesion of serotype 2 is genome segment S1.     

The brome mosaic virus-based recombination vector triggers a limited gene silencing response depending on the orientation of the inserted sequence

In some RNA viruses (e.g. in brome mosaic virus, BMV), the same factor (intra- or intermolecular hybridization between viral RNA molecules) is capable of inducing two different processes: RNA silencing and RNA recombination. To determine whether there is some interplay between these two phenomena, we have examined if the BMV-based recombination vector containing a plant-genome-derived sequence can function as a gene-silencing vector. Surprisingly, we found that neither dsRNA forming during the replication of the BMV-based vector nor highly structured regions of its genome were effective RNAi triggers. Only mutants carrying a sequence complementary to the target mRNA functioned as gene silencing vectors and were steadily maintained in the infected plant. The constructs containing a sense sequence or inverted repeats did not induce gene silencing but instead were eliminated from the plant cells.     

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.     

Assignment of simian rotavirus SA11 temperature-sensitive mutant groups B and E to genome segments

Recombinant (reassortant) viruses were selected from crosses between temperature-sensitive (ts) mutants of simian rotavirus SA11 and wild-type human rotavirus Wa. The double-stranded genome RNAs of the reassortants were examined by electrophoresis in Tris-glycine-buffered polyacrylamide gels and by dot hybridization with a cloned DNA probe for genome segment 2. Analysis of replacements of genome segments in the reassortants allowed construction of a map correlating genome segments providing functions interchangeable between SA11 and Wa. The reassortants revealed a functional correspondence in order of increasing electrophoretic mobility of genome segments. Analysis of the parental origin of genome segments in ts+ SA11/Wa reassortants derived from the crosses SA11 tsB(339) X Wa and SA11 tsE(1400) X Wa revealed that the group B lesion of tsB(339) was located on genome segment 3 and the group E lesion of tsE(1400) was on segment 8.     

The organization and rate of evolution of wheat genomes are correlated with recombination rates along chromosome arms

Genes detected by wheat expressed sequence tags (ESTs) were mapped into chromosome bins delineated by breakpoints of 159 overlapping deletions. These data were used to assess the organizational and evolutionary aspects of wheat genomes. Relative gene density and recombination rate increased with the relative distance of a bin from the centromere. Single-gene loci present once in the wheat genomes were found predominantly in the proximal, low-recombination regions, while multigene loci tended to be more frequent in distal, high-recombination regions. One-quarter of all gene motifs within wheat genomes were represented by two or more duplicated loci (paralogous sets). For 40 such sets, ancestral loci and loci derived from them by duplication were identified. Loci derived by duplication were most frequently located in distal, high-recombination chromosome regions whereas ancestral loci were most frequently located proximal to them. It is suggested that recombination has played a central role in the evolution of wheat genome structure and that gradients of recombination rates along chromosome arms promote more rapid rates of genome evolution in distal, high-recombination regions than in proximal, low-recombination regions.     

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.     

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.     

Assignment of avian reovirus temperature-sensitive mutant recombination groups B, C, and D to genome segments

We recently generated a new set of avian orthoreovirus (ARV) temperature-sensitive (ts) mutants after chemical mutagenesis of wild-type strain ARV138 and described mutants in the A recombination group. Here, each prototype ts mutant from ARV recombination groups B, C, and D was crossed with wild-type ARV strain 176 to generate reassortant clones that were used to map the ts lesions in the respective mutants. Reassortant clones were identified by comparison of segment mobility to parental markers in polyacrylamide gels. An efficiency of plating (EOP) value, which measures the capacity of a virus clone to grow under non-permissive conditions, was used to assign reassortant clones to either a ts group or non-ts group. Analysis of EOP values and parental origin of genome segments in the reassortant clones revealed that the group B lesion in tsB31 was located on the M2 genome segment; the group C lesion in tsC37 was on the S3 genome segment; and the group D lesion in tsD46 was on the L2 genome segment. The assignments of tsB31 and tsC37 were further confirmed by sequence analysis and amino acid substitutions in the corresponding muB and sigmaB proteins localized within the recently determined homologous mammalian reovirus mu1/sigma3 heterohexameric crystal structure.