Isolation and genetic characterization of temperature-sensitive mutants of simian rotavirus SA 11

Ten stable temperature-sensitive (ts) mutants have been isolated from the simian rotavirus SA11 following mutagenesis with nitrous acid or hydroxylamine. These mutants define five recombination groups between which recombination occurs at high frequency (>1.0%) and within which recombination does not occur at detectable levels (<0.2%). These observations support the interpretation that recombination in rotaviruses occurs by reassortment of genome segments during mixed infection. Four of the is mutants are members of the mutant group designated A, three is mutants are members of group B, and groups C, D, and E are each represented by single is mutants. The is mutants were unable to efficiently complement during mixed infection. Growth studies indicated that the ts lesions conditionally, and strongly, inhibit both virus yield and plaque formation.     

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.     

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.     

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.     

Two types of glycoprotein precursors are produced by the simian rotavirus SA11

The rotavirus genome codes for two glycoproteins: an outer capsid structural glycoprotein (VP7, apparent molecular weight 38,000 (38K)) and a nonstructural glycoprotein (NS28K). The synthesis of these glycoproteins was analyzed in infected cells and in a cell-free system derived from rabbit reticulocyte lysates supplemented with dog pancreatic microsomes. The data showed a 37K product synthesized in the cell-free system is the precursor to the 38K glycoprotein and that the 37K polypeptide contains a cleavable signal sequence (apparent molecular weight 1.5K). The 37K polypeptide was glycosylated in vitro in the presence of microsomal membranes to a polypeptide of 38K that was immunoprecipitated by monospecific antiserum to VP7. Endo H digestion of the 38K polypeptides from either infected cells or the cell-free system produced polypeptides of identical molecular weight, 35.5K (the glycoprotein precursor lacking the signal sequence). These results were confirmed by comparative studies with a variant of SA11 that is defective in glycosylation of VP7. Similar experiments with the 20K precursor to the 29K nonstructural glycoprotein showed the 20K polypeptide contains a noncleavable signal sequence. Both glycoproteins were inserted into microsomal membranes and were processed via oligosaccharide trimming.     

Identification of the simian rotavirus SA11 genome segment 3 product

Previous studies on rotavirus gene-coding assignments failed to clearly identify the simian rotavirus SA11 genome segment 3 protein product. This question was reexamined by using new conditions of electrophoresis with improved resolution of proteins in the high-molecular-weight range. Our results showed that the SA11 genome segment 3 codes for a protein with an apparent mol wt of 88,000. This protein normally comigrates with the protein product of genome segment 4. The gene 3 protein was located in viral core particles by comparing the electrophoretic patterns of [35S]methionine-labeled viral polypeptides from infected cells and from purified double-shelled, single-shelled, and core particles. To confirm the identity of the gene 3 product, we also studied two reassortant viruses in which genome segment 3 was reassorted from each of two parental virus strains (SA11 and NCDV). The gene 3 and gene 4 products of these viruses were identified by (i) their separation by two different polyacrylamide gel systems, (ii) their location in distinct particle types, (iii) their differential sensitivity to trypsin digestion, and (iv) their distinctive protease peptide maps. We propose that the genome segment 3 product be called VP3 and that the gene 4 product be named VP4 from now on.     

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.     

The synthesis of A-rich RNA by temperature-sensitive mutants of reovirus

The synthesis of adenine-rich RNA has been examined in cells infected with temperature-sensitive mutants of Reovirus type 3. Prototypes of all mutant groups produce adenine-rich RNA at the permissive temperature. However, cells similarly infected at the nonpermissive temperature fail to produce measurable amounts of adenine-rich RNA under these same conditions.     

Heterogeneity in the structural glycoprotein (VP7) of simian rotavirus SA11

The polypeptides of cells infected with a series of plaque isolates of the simian rotavirus SA11 were analyzed by SDS-PAGE. Altered electrophoretic migration of the major outer capsid glycoprotein (VP7) was found with independent virus stocks exhibiting gene products of VP7 ranging in apparent molecular weight from 35.5K to 38K. Similar differences in electrophoretic migration in the polypeptide precursor of the glycoprotein (pVP7) suggested that the heterogeneity resulted from mutations in the gene encoding the glycoprotein. The glycoprotein phenotype was stable on passage; the phenotypes were unchanged for 10 passages at high and low multiplicity. The biologic consequences of heterogeneity in the polypeptide are discussed.     

The major surface glycoprotein of simian rotavirus (SA11) contains distinct epitopes

The polypeptide specificities on monoclonal antibodies previously derived against the SA11 simian, NIC bovine, and Wa human strains of rotavirus were determined by radioimmunoprecipitation of infected cell lysates. All the monoclonal antibodies derived using NIC and Wa were found to be directed against the major component of the inner capsid, while most of the SA11 monoclones were directed against the major outer capsid glycoprotein. When several SA11 glycoprotein-specific monoclonal antibodies were used in competitive binding studies, four distinct epitopes, which correlated with the functional activities of the antibodies, were defined. One epitope appeared most critical for virus neutralization, another was involved to a lesser extent, and the remaining two epitopes seemed to have no role. A possible topographical arrangement of these epitopes is suggested.     

Recombination between temperature-sensitive mutants of simian virus 40

Seven temperature-sensitive (ts) SV40 mutants have been isolated and characterized. All of the mutants were defective in a late function. Four of the mutants were assigned to complementation group B, one to a new group designated C and two could not be assigned to a complementation group. Recombination occurred between mutants in the B and C complementation groups and between mutants in the B group. The recombination frequency (RF) between tsB302 and tsB306 was about 2.0 × 10^?4 when virus was used for infection, but was 10-fold higher when infectious ts SV40 DNAs were used. Treatment of doubly infected cells with 1-β-d-arabinofuranosylcytosine (ara-C) to interrupt DNA synthesis increased the RF 3- to 14-fold. Ultraviolet irradiation of viral inocula to 1–5% survival resulted in a 25- to 40-fold increase in RF. However, only a 2-fold increase in RF was obtained when uv-irradiated ts SV40 DNAs were used for infection. Ultraviolet irradiation of host CV-1 cells or pretreatment of host CV-1 cells with nonirradiated or uv-irradiated CV-1 DNA prior to infection failed to increase the RF between tsB302 and tsB306.     

Study of fowl plague virus RNA synthesis in temperature-sensitive mutants.

A temperature-sensitive clear mutant of the temperate cyanophage SPI was isolated. This mutant, SPIcts1, enters a stable lysogenic state following infection of its blue-green algal host Plectonema boryanum. Induction takes place upon transfer of the culture to 40°.Prophage induction requires light energy during its early stage. Induction is prevented by the photosynthetic inhibitor 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) but can take place in the absence of CO₂. Infection by SPIcts1, on the other hand, can proceed in the dark. Shortly after infection there is extensive leakage of macromolecules from the host cell accompanied by cessation of CO₂ fixation. In contrast, the induced prophage does not cause early cellular leakage or early shutoff of CO₂ fixation of the host.     

RNA synthesis by temperature-sensitive mutants of vesicular stomatitis virus, New Jersey serotype.

We report the results of studies using temperature-sensitive mutants of Rous sarcoma virus (ts RSV) to study the alterations in surface proteins occurring on transformation. A large external transformation-sensitive (LETS) protein is detected by lactoperoxidase-catalyzed iodination on normal chicken embryo fibroblasts (CEF) but is reduced on RSV-transformed cells. The LETS protein is temperature sensitive in ts RSV-infected CEF and the kinetics of the alterations occurring on shifts between permissive and restrictive temperature are reported. During reversion to normality on shift-up, the LETS protein appears at the surface quite rapidly and in parallel with the morphological changes, although the change is less rapid than that in glucose transport. On transformation during shift-down, the disappearance of the LETS protein lags behind the morphological change. Cycloheximide does not inhibit reappearance of iodinatable LETS protein on shift-up, suggesting that the LETS protein is synthesized in transformed cells, although it is not present at the surface. Hypotheses to explain the absence of the LETS protein at the surface are discussed and evidence is presented for an increased rate of turnover after transformation.     

Mapping temperature-sensitive mutants of simian virus 40: Rescue of mutants by fragments of viral DNA

Five temperature-sensitive (ts) mutants of simian virus 40 (SV40), isolated and characterized by Tegtmeyer and Ozer (1971), have been mapped by marker rescue using endo R fragments of wild-type SV40 DNA. For each mutant a specific fragment corrected the ts defect, from which we infer that the mutation is within the genome segment corresponding to the active fragment. Since the position of each fragment in the SV40 cleavage map is known, the mutational sites could be localized. Of the five ts mutants examined, two were “early” mutants and were in complementation group A of Tegtmeyer; these mapped in contiguous Hin fragments H (tsA30) and I (tsA28), which had been shown previously to be part of the “early” region of the SV40 genome. Three ts mutants were “late” mutants and were in complementation group B of Tegtmeyer; these mapped in contiguous Hin fragments F (tsB8), J (tsB4), and G (tsB11), which had been shown previously to be part of the “late” region of the SV40 genome.     

Biological and immunological characterization of a simian rotavirus SA11 variant with an altered genome segment 4

We have studied a variant virus isolated from a stock of SA11 virus (H. G. Pereira, R. S. Azeredo, A. M. Fialho, and M. N. P. Vidal, 1984, J. Gen. Virol. 65, 815-818). This virus, designated 4F, was initially identified by its faster electrophoretic mobility for genome segment 4. The variant was analyzed to determine if the altered electrophoretic mobility of genome segment 4 could be correlated with phenotypic changes. Comparison of our standard laboratory SA11 virus (clone 3) with the 4F variant showed the following: (i) The 4F variant possesses a viral hemagglutinin (VP4) with a higher apparent molecular weight than clone 3. (ii) The 4F variant produces large plaques when assayed in vitro, as compared to clone 3. (iii) The 4F variant produces plaques in the absence of proteolytic enzymes, whereas clone 3 does not. (iv) The 4F variant reacts with serotype-specific neutralizing monoclonal antibodies to VP7, but fails to react with several neutralizing anti-VP4 monoclonal antibodies generated to SA11 clone 3. (v) The 4F variant grows to a higher titer and is more stable than clone 3. (vi) The 4F variant produces a VP4 that appears to be more susceptible to cleavage by trypsin than is the VP4 of clone 3. Further analyses with the 4F variant may lead to an understanding of the molecular basis for these altered phenotypes that appear to be related, at least in part, to the product of genome segment 4.     

A map of temperature-sensitive mutants of simian virus 40.

Temperature-sensitive mutants of simian virus 40 (SV40), representing each complementation class, have been mapped by marker rescue with Endo R fragments of wild-type SV40-DNA. The 41 mutants mapped cover most of the genome, but there is a notable absence of mutants mapping between 0.43 and 0.85 map-units. Mutants which are defective in viral DNA synthesis (tsA mutants) all map in the “early” region of the genome; 12 of 13 such mutants are clustered in about one-fourth of the “early” region between 0.32 and 0.43 map-units. Mutants in complementation classes defective in a late function map in the “late” region and also show clustering: eight of nine B mutants map between 0.94 and 0.06 map-units, all C mutants map between 0.06 and 0.11 map-units, and seven of eight BC mutants map between 0.11 and 0.17 map-units. D mutants, which are thought to be defective in virus uncoating at high temperature, all map between 0.85 and 0.94 map-units, i.e., in a small segment of the “late” region. Therefore, the genomic segment associated with the defect of D mutants likely codes for a virion protein. One C mutant proved to be a double mutant with one mutation in the B region and another in the C region, both mutations being required for the tsC phenotype. On the basis of the mapping data and prior complementation tests we suggest that B, C, and BC mutations are in one cistron, complementation occurring at the protein level.