Gene protein products of SA11 simian rotavirus genome

When MA104 cells were infected with SA11 rotavirus, 12 protein classes, absent in mock-infected cells, could be distinguished by polyacrylamide gel electrophoresis. At least two of these proteins were glycosylated, and their synthesis could be blocked with tunicamycin. The oligosaccharides of both glycoproteins were cleaved by endo-beta-N-acetylglucosaminidase H, suggesting that they were residues of the "high-mannose" type. Of the 12 viral polypeptides observed in infected cells, 1 was probably the apoprotein of one of these glycoproteins; 5, including 1 glycoprotein, were structural components of the virions, whereas the other 6, including a second and possibly third glycoprotein, were nonstructural viral proteins. When the 11 double-stranded RNA genome segments of SA11 were translated, after denaturation, in an RNA-dependent cell-free translation system, at least 11 different polypeptides were synthesized. Ten of these polypeptides had electrophoretic migration patterns equal to those of viral proteins observed in tunicamycin-treated infected cells. Nine of the 11 double-stranded RNA genome segments were resolved by polyacrylamide gel electrophoresis and were translated individually. Two were not resolved from each other and therefore were translated together. Correlation of each synthesized polypeptide with an individual RNA segment allowed us to make a probable gene-coding assignment for the different SA11 genome segments.     

Completion of the gene coding assignments of SA11 rotavirus: gene products of segments 7, 8, and 9.

Improved fractionation of double-stranded RNA segments 7, 8, and 9 of simian rotavirus SA11 has permitted their isolation and individual translation in vitro. Segment 7 codes for p31 (NS2), segment 8 codes for p33 (NS1), and the segment 9 gene product resembles the gp34 precursor observed in SA11 virus-infected cells. In vitro glycosylation of translation products of segments 5 and 10 was also observed.     

Mapping of the influenza virus genome. III. Identification of genes coding for nucleoprotein, membrane protein, and nonstructural protein.

In previous communications we reported that the eight RNA segments of influenza A/PR/8/34 (HON1) virus could be distinguished from corresponding segments of influenza A/Hong Kong/8/68 (H3N2) virus by migration on polyacrylamide-urea gels. Examination of the RNA patterns of the two parent viruses and recombinants derived from them in concert with serological identification of surface proteins and analysis of the other proteins on sodium dodecyl sulfate gradient gels permitted the identification of the genes coding for hemagglutinin, neuraminidase, and the P1, P2, and P3 proteins (Palese and Schulman, 1976; P. Palese et al., Virology, in press). In the present report we have extended these observations using similar techniques to examine other recombinants and have identified the genes coding for the remaining virus-specific moving RNA segment as 1) and segment 6 of Hong Kong virus coding for the respective nucleoproteins, and that segment 7 of both viruses codes for the membtane protein and RNA segment 8 codes for the nonstructural protein. This completes the mapping of the influenza A virus genome.     

Respiratory syncytial virus mRNA coding assignments.

The polypeptide coding assignments for six of the respiratory syncytial virus-specific mRNAs were determined by translation of the individual mRNAs in vitro. The coding assignments of the RNAs are as follows. RNA band 1 is complex and can be separated into at least two components on the basis of electrophoretic mobility (molecular weights [MWs] approximately equal to 0.21 X 10(6) and 0.31 X 10(6), respectively) that code for three polypeptides of 9.5, 11, and 14 kilodaltons (K). RNA 2 (MW, 0.39 X 10(6)) codes for a 34K polypeptide; RNA 3 (MW, 0.40 X 10(6)) codes for a 26K polypeptide; RNA 4 (MW, 0.47 X 10(6)) codes for a 42K polypeptide; and RNA 5 (MW, 0.74 X 10(6)) codes for a 59K polypeptide. By limited-digest peptide mapping, the 34, 26, and 42K polypeptides synthesized in vitro appeared to be unique. Additionally, peptide mapping showed that the 34, 26, and 42K polypeptides synthesized in vitro were indistinguishable from their counterparts synthesized in infected cells. Thus, the 34, 26, and 42K polypeptides coded for by mRNAs 2, 3, and 4, respectively, were identified as the respiratory syncytial virus phosphoprotein (34K), matrix protein (26K), and nucleocapsid protein (42K), respectively. RNA 5 was shown to code for a 59K polypeptide. The 59K polypeptide synthesized in vitro did not comigrate with any polypeptide specific to infected cells, suggesting that it is a candidate for co- or post-translational modification.     

Comparison of the genomes of simian, bovine, and human rotaviruses by gel electrophoresis and detection of genomic variation among bovine isolates.

By co-electrophoresis in polyacrylamide gels, the segmented double-standed RNA genome of the simian rotavirus, SA 11, was compared with those of human and bovine rotaviruses. A comparison between SA 11 virus and the Northern Ireland cell culture adapted bovine virus showed that the electrophoretic mobilities of each of the 11 corresponding segments differed. In other comparisons, four to seven segment variations were more common. When the genomes of various bovine rotaviruses were compared, eight different electropherotypes were detected. Four of these electropherotypes were obtained from one property during a single outbreak of disease. In view of such genetic diversity, a scheme for the systematic designation of different rotavirus samples is proposed. The significance of the variations in relation to the molecular epidemiology of bovine rotavirus infections is discussed.     

Homologous terminal sequences of the genome double-stranded RNAs of bluetongue virus.

The 3'-terminal sequences of the 10 double-stranded RNA genome segments of bluetongue virus (serotypes 10 and 11) were determined. The double-stranded RNAs were 3' labeled with [5'-32P]pCp and resolved into 10 segments by electrophoresis. After denaturation, the two complementary strands of segments 4 through 10 were resolved into fast- and slow-migrating species by polyacrylamide gel electrophoresis, and their 3' end sequences were determined. Complete RNase T1 digestion of the individual 3'-labeled double-stranded RNA segments yielded two labeled oligonucleotides, one of which migrated faster than the other on 20% polyacrylamide-7 M urea gels. Sequence analyses of the two oligonucleotides of segments 4 through 10 confirmed the corresponding RNA sequence data. For RNA segments 1 through 3 the oligonucleotide analyses gave comparable results. The 3'-terminal sequences of the fast-migrating RNA species were HOCAAUUU. . . ; those of the slow-migrating RNA species were HOCAUUCACA. . . . Similar results were obtained for double-stranded RNA from bluetongue virus serotypes 10 and 11. Beyond the common termini, the sequences for each segment varied considerably.     

Molecular characterization of a rotaviruslike virus isolated from striped bass (Morone saxatilis).

The characteristics of a rotaviruslike (SBR) virus isolated from striped bass (Morone saxatilis) were examined following purification of viruses from infected cell cultures. Virions had a double-layered capsid of icosahedral symmetry and a diameter of 75 nm. Purified viruses contained five polypeptides ranging in molecular mass from 130 to 35 kDa. None of the structural proteins were glycosylated. Treatment with EDTA did not remove the outer capsid. By using enzymes and a chaotropic agent, it was shown that VP5 was the most external polypeptide. The genome of SBR virus was composed of 11 segments of double-stranded RNA (dsRNA). The electrophoretic pattern of the dsRNA of SBR virus was different from that of reovirus type 1 (Lang) and rotavirus (SA11) dsRNA. The SBR virus was compared with reovirus type 1 and SA11 virus by RNA-RNA blot hybridization. There was no cross-hybridization between any of the genome segments of the SBR, reovirus type 1, or SA11 viruses. Antigenic comparison of SBR virus and SA11 virus by cross-immunoprecipitation and cross-immunofluorescence tests did not show any relationship. These results suggest that SBR virus could represent a new genus within the family Reoviridae.     

Reconciliation of rotavirus temperature-sensitive mutant collections and assignment of reassortment groups D, J, and K to genome segments

Four rotavirus SA11 temperature-sensitive (ts) mutants and seven rotavirus RRV ts mutants, isolated at the National Institutes of Health (NIH) and not genetically characterized, were assigned to reassortment groups by pairwise crosses with the SA11 mutant group prototypes isolated and characterized at Baylor College of Medicine (BCM). Among the NIH mutants, three of the RRV mutants and all four SA11 mutants contained mutations in single reassortment groups, and four RRV mutants contained mutations in multiple groups. One NIH mutant [RRVtsK(2)] identified the previously undefined 11th reassortment group (K) expected for rotavirus. Three NIH single mutant RRV viruses, RRVtsD(7), RRVtsJ(5), and RRVtsK(2), were in reassortment groups not previously mapped to genome segments. These mutants were mapped using classical genetic methods, including backcrosses to demonstrate reversion or suppression in reassortants with incongruent genotype and temperature phenotype. Once located to specific genome segments by genetic means, the mutations responsible for the ts phenotype were identified by sequencing. The reassortment group K mutant RRVtsK(2) maps to genome segment 9 and has a Thr280Ileu mutation in the capsid surface glycoprotein VP7. The group D mutant RRVtsD(7) maps to segment 5 and has a Leu140Val mutation in the nonstructural interferon (IFN) antagonist protein NSP1. The group J mutant RRVtsJ(5) maps to segment 11 and has an Ala182Gly mutation affecting only the NSP5 open reading frame. Rotavirus ts mutation groups are now mapped to 9 of the 11 rotavirus genome segments. Possible segment locations of the two remaining unmapped ts mutant groups are discussed.     

Reovirus type 3 genome segment S4: nucleotide sequence of the gene encoding a major virion surface protein

A full-length cDNA copy of reovirus double-stranded RNA genome segment S4 which codes for a major virion structural polypeptide, sigma 3, has been completely sequenced. The 1,196-nucleotide cDNA contains a single long open reading frame in the plus strand extending 1,095 nucleotides from the 5'-proximal A-T-G to a single stop codon. This corresponds to translation of 92% of the S4 gene. The inferred sigma 3 polypeptide is hydrophilic and consists of 365 amino acids, totalling 41,164 daltons.     

Differences in RNA patterns of influenza A viruses.

Analysis of the segmented RNAs of influenza A viruses by electrophoresis on polyacrylamide urea slab gels has provided a method for sharper resolution of the number and migration rates of different segments than previously has been possible. Using this system, the RNA genome of influenza A/WSN (HON1) virus can be separated into seven to nine separate bands, depending on whether virus is obtained after high or low multiplicity of infection, and the genome of influenza A/PR/8 (HON1) virus can be resolved into eight bands, six of which migrate differently from comparable RNA bands of WSN virus. Comparision of the RNA patterns produced by influenza A/PR/8 (HON1) and A/England/42/72 (H8n2) virus also reveals major differences in migration speeds of different bands, and analysis of the RNAs of the RNAs of an HON2 recombinant virus derived from these two strains permits the identification of RNA segments which have been derived from one particular parent. By extension of these techniques, it may be possible to define which RNA segment codes for each viral protein and to analyze recombinant strains to identify which genes have been derived from each of its parents.     

A comparison of simian rotavirus SA11 preparations maintained in different laboratories

Preparations of simian rotavirus SA11 maintained in different laboratories were compared with each other by polyacrylamide gel electrophoresis of genomic RNA. Differences in the migration of genome segments 4, 5 and 7 allowed the classification of eight virus preparations into four electrophoretic types.     

Cloning and sequence of UK bovine rotavirus gene segment 7: marked sequence homology with simian rotavirus gene segment 8.

The genome of the UK bovine rotavirus, which consists of eleven segments of dsRNA was polyadenylated and reverse-transcribed into cDNA. Complementary cDNA strands were annealed and the termini of the duplexes completed using DNA polymerase I. Full-length DNA copies of RNA segments 7, 8 and 9 were cloned into the Pst I site of pBR322 and a clone containing the entire gene 7 was identified and sequenced. Gene 7 is 1059 nucleotides in length and contains a single long open reading frame capable of coding for a protein of 317 amino-acids. The known gene product of segment 7 is a protein with an estimated molecular weight of 33,000 daltons. When the UK bovine rotavirus gene 7 sequence was compared with the published data for the homologous gene (segment 8) of the simian rotavirus SA11, it was found to be identical to it in size and the arrangement of the proposed coding and non-coding regions, and very similar in nucleotide sequence (88% homology). Most of the base changes are silent and the predicted amino-acid sequences are almost identical (96% homology).     

Translation of Sindbis virus 26 S RNA and 49 S RNA in lysates of rabbit reticulocytes

Sindbis virus-specific polypeptides were synthesized in lysates of rabbit reticulocytes in response to added 26 S or 49 S RNA. Sindbis 26 S RNA was translated into as many as three polypeptides which co-migrate in acrylamide gels with proteins found in infected cells.Wild type 26 S RNA was translated primarily into two polypeptides, which appear to be the Sindbis nucleocapsid protein (mol. wt 30,000) and the precursor of the two glycoproteins of the virion (mol. wt 100,000). A larger polypeptide (mol. wt 130,000) was synthesized in response to ts2 26 S RNA, a species of RNA which was isolated from cells infected with the ts2 mutant of Sindbis virus. This large polypeptide is apparently the protein which accumulates in cells infected with the mutant virus and which is thought to be a precursor of all three viral structural proteins.These results support the hypothesis that 26 S RNA is the messenger for the three structural proteins of the virion and that the RNA codes for one large polypeptide precursor. The precursor may then be cleaved at a specific site to yield the nucleocapsid protein and a second polypeptide which, in infected cells, is cleaved in a series of steps to yield the two glycoproteins of the virion.Sindbis 49 S RNA was translated into eight or nine polypeptides ranging from 60,000 to 180,000 molecular weights. The viral structural proteins, as such, were not synthesized in response to the added 49 S RNA.     

Influenza B virus genome: assignment of viral polypeptides to RNA segments.

It was shown that all eight RNA segments of influenza B viruses are most likely monocistronic and code for eight virus-specific polypeptides. A genetic map of the influenza B virus genome was established, and six polypeptides (P1 protein, nucleoprotein, hemagglutinin, neuraminidase, M protein, and nonstructural protein) were unambiguously assigned to specific RNA segments. Molecular weight estimates of the eight individual genes are obtained by using the glyoxal method. These results suggest that each influenza B virus RNA segment has a greater molecular weight than the influenza A virus RNA segment which codes for the analogous gene product.     

The S RNA segment of lymphocytic choriomeningitis virus codes for the nucleoprotein and glycoproteins 1 and 2.

The lymphocytic choriomeningitis virus (LCMV) genome consists of a large RNA segment and a small RNA segment. The three major structural proteins of this virus are an internal nucleoprotein and two surface glycoproteins. Intertypic reassortants between the Armstrong and WE strains of LCMV were made to map proteins encoded by the LCMV genome segments. Using monoclonal antibodies specific for the nucleoprotein and the glycoproteins of WE and Armstrong, we showed that the small RNA segment of LCMV codes for the three major structural polypeptides.     

The genome of Uukuniemi virus consists of three unique RNA segments

The three RNA species isolated from virions of Uukuniemi virus, a proposed member of the newly defined Bunyaviridae family, have been characterized by analysis of ³²P-labeled ribonuclease T1 oligonucleotides separated on two-dimensional polyacrylamide gels. Each RNA species contains unique oligonucleotides not present in the two others, indicating that the genome of this virus is segmented. Each segment appears to contain a unique primary sequence with little or no overlapping among the segments. The complexities of the RNA segments as calculated from the radioactivity in unique oligonucleotides of defined lengths are about 8000 (L RNA), 3500 (M) and 1900 (S) nucleotides. Since these values are similar to the molecular weights determined by other methods, each size class of RNA corresponds to a single molecular species. The presence of a 5′ terminal pppAp … structure in each RNA segment confirms indications from electron microscopy that the apparently circular RNA segments are not covalently closed. The absence of either a 5′ terminal “cap” or 3′ terminal poly(A) supports the concept that Uukuniemi virus is a negative strand virus.