Structural polypeptides of simian rotavirus SA11 and the effect of trypsin

Analysis of purified simian rotavirus has shown that it contains fewer structural polypeptide classes than previously reported. Two polypeptides (molecular weights, 62,000 and 28,000) commonly found in purified rotaviruses were, in fact, produced by cleavage of a larger structural polypeptide (molecular weight, about 88,000) by trypsin, which is usually employed to increase the yield of rotaviruses in tissue culture. Trypsin-uncleaved, double-shelled rotaviruses are probably composed of only five polypeptide classes; three in the inner layer, and two in the outer layer.     

Characterization of temperature-sensitive mutants of simian rotavirus SA11: protein synthesis and morphogenesis.

The synthesis of viral polypeptides, distribution of viral antigens, and morphogenesis of viral structures have been examined in cells infected with temperature-sensitive (ts) mutants of SA11 representing 10 recombination groups. At the permissive temperature (31 degrees C) the synthesis of viral polypeptides and the distribution of viral antigens did not differ significantly from those of the wild type. At the nonpermissive temperature (39 degrees C) some mutants (tsB, -C, -E, -F, and -G) synthesized significantly smaller amounts of viral polypeptides and had a very diffuse distribution of viral antigen. Several of the mutants synthesized one or more electrophoretically aberrant polypeptide species at both 31 and 39 degrees C. All of the mutants, except tsF, assembled morphogenetic intermediates at 39 degrees C. Aberrant intermediates were assembled in all mutants at 31 and 39 degrees C. No specific morphogenic defect could be associated with any of the ts mutants.     

Replication of simian herpesvirus SA8 and identification of viral polypeptides in infected cells

The replication of the simian herpesvirus SA8 in Vero cells was examined. The time course of replication of the simian herpesvirus SA8 was found to be similar to that of the herpes simplex viruses. Infectious progeny virions were first detectable by 6 h postinfection and were readily released into the extracellular fluids beginning at 9 h postinfection. All cell lines tested, with the exception of Madin-Darby canine kidney cells, were permissive for SA8. Analysis of SA8-infected cells by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed over 40 infected cell polypeptides ranging in molecular weight from 158,000 to less than 10,000. Of these proteins, 23 were present in virions. Three classes of infected cell polypeptides could be identified based on the kinetics of their synthesis. Post-translational processing of several SA8-induced proteins was also observed in pulse-chase experiments. Six distinct SA8-specific glycoproteins ranging from 118,000 to 19,500 daltons were also identified in infected cells. Of these glycoproteins, five were present in virions.     

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.     

Inactivation of simian rotavirus SA11 by chlorine, chlorine dioxide, and monochloramine.

The kinetics of inactivation of simian rotavirus SA11 by chlorine, chlorine dioxide, and monochloramine were studied at 5 degrees C with a purified preparation of single virions and a preparation of cell-associated virions. Inactivation of the virus preparations with chlorine and chlorine dioxide was studied at pH 6 and 10. The monochloramine studies were done at pH 8. With 0.5 mg of chlorine per liter at pH 6, more than 4 logs (99.99%) of the single virions were inactivated in less than 15 s. Both virus preparations were inactivated more rapidly at pH 6 than at pH 10. With chlorine dioxide, however, the opposite was true. Both virus preparations were inactivated more rapidly at pH 10 than at pH 6. With 0.5 mg of chlorine dioxide per liter at pH 10, more than 4 logs of the single-virus preparation were inactivated in less than 15 s. The cell-associated virus was more resistant to inactivation by the three disinfectants than was the preparation of single virions. Chlorine and chlorine dioxide, each at a concentration of 0.5 mg/liter and at pH 6 and 10, respectively, inactivated 99% of both virus preparations within 4 min. Monochloramine at a concentration of 10 mg/liter and at pH 8 required more than 6 h for the same amount of inactivation.     

Inactivation of simian rotavirus SA11 by chlorine, chlorine dioxide, and monochloramine

The kinetics of inactivation of simian rotavirus SA11 by chlorine, chlorine dioxide, and monochloramine were studied at 5 degrees C with a purified preparation of single virions and a preparation of cell-associated virions. Inactivation of the virus preparations with chlorine and chlorine dioxide was studied at pH 6 and 10. The monochloramine studies were done at pH 8. With 0.5 mg of chlorine per liter at pH 6, more than 4 logs (99.99%) of the single virions were inactivated in less than 15 s. Both virus preparations were inactivated more rapidly at pH 6 than at pH 10. With chlorine dioxide, however, the opposite was true. Both virus preparations were inactivated more rapidly at pH 10 than at pH 6. With 0.5 mg of chlorine dioxide per liter at pH 10, more than 4 logs of the single-virus preparation were inactivated in less than 15 s. The cell-associated virus was more resistant to inactivation by the three disinfectants than was the preparation of single virions. Chlorine and chlorine dioxide, each at a concentration of 0.5 mg/liter and at pH 6 and 10, respectively, inactivated 99% of both virus preparations within 4 min. Monochloramine at a concentration of 10 mg/liter and at pH 8 required more than 6 h for the same amount of inactivation.     

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.     

Synthesis of the outer-capsid glycoprotein of the simian rotavirus SA11 in Escherichia coli

The major outer layer protein, VP7, of the simian rotavirus SA11 has been synthesized in Escherichia coli, under the control of the lac promoter, as a fusion polypeptide with beta-galactosidase (beta Gal). The viral protein in the hybrid polypeptide is missing its N-terminal hydrophobic region and 26 amino acids (aa) at its C-terminus; it is flanked at both ends by beta Gal sequences. We have purified the hybrid 145-kDa protein by affinity chromatography using a column specific for beta Gal. Unexpectedly, a second protein of 118-kDa was also specifically bound to the column. N-terminal aa sequence analysis of these two proteins showed that the 145-kDa protein represented the expected fusion product, whereas the 118-kDa protein was apparently the result of initiation of translation at an internal site close to the 3' end of the viral sequence, in the chimeric mRNA. Each of the two polypeptides represented about 2 to 3% of the total protein of the recombinant-plasmid-carrying bacteria. When a bacterial lysate enriched for the hybrid polypeptides was injected into mice, it induced neutralizing antibodies to SA11 rotavirus.     

Identification of herpesvirus sylvilagus-induced polypeptides in productively infected cells.

Polypeptides synthesized in cell cultures infected with high multiplicities of herpesvirus sylvilagus were identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of [35S]methionine-labeled cell extracts. Initiation of polypeptide synthesis was detected by 6 h after infection. The maximum intensity of many [35S]methionine-labeled viral bands was observed at 45 h after infection. Production of detectable infectious virus began between 18 and 24 h and reached a plateau at 48 h after infection. Immunoprecipitation of cell extracts identified a minimum of 45 virus-induced polypeptides ranging in molecular weight from 230,000 to 27,000. The major polypeptide appeared to have a molecular weight of 150,000. The pattern of these extracts suggested that the synthesis of host polypeptides is stimulated during the first 12 h and thereafter reduced, but not completely inhibited, during the remaining course of infection.     

Primary structure of the neutralization antigen of simian rotavirus SA11 as deduced from cDNA sequence.

DNA sequences complementary to the double-stranded RNA coding for the neutralization antigen (glycoprotein VP7) of simian rotavirus SA11 have been cloned. The VP7 gene consists of 1,062 nucleotides, containing an uninterrupted coding sequence of 978 nucleotides which specifies a glycoprotein of 326 amino acids. The significance of a second possible initiation site 30 nucleotides downstream from the first is discussed. Partial amino acid sequence of this glycoprotein showed unequivocally that the cloned segment (segment 9) codes for glycoprotein VP7 of SA11. The resulting amino acid sequence contained only one carbohydrate acceptor site. Possible sites of membrane interaction and antigenic determinants are discussed based on the analysis of the hydrophobicity and hydrophilicity profiles of VP7.     

Simian rotavirus SA11 replication in cell cultures.

Understanding the basic virology of rotavirus infections has been hampered by the fastidiousness of most isolates and by the lack of a rapid quantitative assay method. The growth characteristics of the simian rotavirus SA11 were studied because it grows to high titers in tissue culture and infectivity can be quantitated by plaque assay. SA11 replication was analyzed in a variety of primary cell cultures or continuous cell lines derived from both homologous and heterologous hosts. Viral replication was observed in each of the cell cultured examined. The individual cell cultures demonstrated marked variability in their susceptibility to rotavirus infection. The highest titers were obtained with MA104, BSC-1, CV-1, and BGM cells. Observable cytopathic effect was found to correlate with the percentage of infected cells in the culture. This study presents growth curves of the simian rotavirus in a variety of cell cultures.     

A murine model for oral infection with a primate rotavirus (simian SA11).

Simian rotavirus SA11 was shown to replicate in the gastrointestinal tracts of infant mice after oral inoculation. Clinical symptoms, histopathological changes in the small intestinal mucosa, and the type-specific humoral immune response were all characteristic of rotavirus-induced gastroenteritis. The availability of this small animal model for the study of a primate rotavirus infection should expedite analysis of the immune response necessary for protection against challenge.     

Functional and topographical analyses of epitopes on the hemagglutinin (VP4) of the simian rotavirus SA11.

An immunochemical analysis of the hemagglutinin (VP4) of the simian rotavirus SA11 was performed to better understand the structure and function of this molecule. Following immunization of mice with double-shelled virus particles and VP4-enriched fractions from CsCl gradients, a battery of anti-SA11 hybridomas was generated. A total of 13 clones secreting high levels of anti-VP4 monoclonal antibody (MAb) was characterized and compared with two cross-reactive anti-VP4 MAbs generated against heterologous rhesus (RRV) and porcine (OSU) rotavirus strains. These cross-reactive MAbs effectively neutralized SA11 infectivity in vitro. The epitopes recognized by these 15 MAbs were grouped into six antigenic sites on the SA11 hemagglutinin. These sites were identified following analysis of the MAbs by using a simple competitive binding enzyme-linked immunosorbent assay (ELISA) and biological assays. Three of the antigenic sites were involved in neutralization of virus infectivity in vitro. All the MAbs with neutralization activity and two nonneutralizing MAbs were able to inhibit viral hemagglutination of human erythrocytes. Competitive binding ELISA data showed a positive cooperative binding effect with some pairs of the anti-VP4 MAbs, apparently due to a conformational change induced by the binding of the first MAb. Some of the MAbs also bound better to trypsin-treated virus than to non-trypsin-treated virus. A topographic map for VP4 is proposed on the basis of the observed properties of each antigenic site.     

Antibodies specific for the carboxy-terminal region of the major surface glycoprotein of simian rotavirus (SA11) and human rotavirus (Wa)

Antibodies specific for the major outer capsid protein (VP7) of the simian rotavirus SA11 were obtained by immunization of rabbits with a synthetic peptide, Ser-Ala-Ala-Phe-Tyr-Tyr-Arg-Val, corresponding to the eight carboxy-terminal amino acids of the viral protein predicted from the nucleotide sequence of the gene segment 9 of the SA11 genome. As the carboxy-terminal region of the VP7 of human rotavirus Wa has an identical sequence, cross-reactivity of the raised antibodies was observed with this strain.