The polypeptide compositions of single-shelled and double-shelled simian rotavirus particles were modified by exposure to proteolytic enzymes. Specificially, a major outer capsid polypeptide (VP3) having a molecular weight of 88,000 in double-shelled particles was cleaved by trypsin to yield two polypeptides, VP5* and VP8* (molecular weights, 60,000 and 28,000, respectively). The cleavage of VP3 by enzymes that enhanced infectivity (trypsin, elastase, and pancreatin) yielded different products compared to those detected when VP3 was cleaved by chymotrypsin, which did not enhance infectivity. The appearance of VP5* was correlated with an enhancement of infectivity. Cleavages of the major internal capsid polypeptide VP2 were also observed. The VP2 cleavage products had molecular weights similar to those ofknown structural and nonstructural rotavirus polypeptides. We confirmed the precursor-product relationships by comparing the peptide maps of the polypeptides generated by digestions with V-8 protease and chymotrypsin. The remaining rotavirus structural polypeptides, including the outer capsid glycoproteins (VP7 and 7a), were not altered by exposure to pancreatic enzymes. Cleavage of VP3 was not required for virus assembly, and specific cleavage of the polypeptides occurred only on assembled particles. We also discuss the role of cleavage activation in other virus-specific biological functions (e.g., hemagglutination and virulence).

Rotavirus gene products were examined, with the simian rotavirus SAll as a model. The endogenous viral RNA-dependent RNA polymerase associated with single-shelled virus particles or with activated double-shelled particles was used to synthesize viral RNA transcripts. Sedimentation velocity sucrose gradient analysis of the RNA transcripts revealed four peaks at 9S, 12S, 14S, and 18S, whereas agarose gel electrophoresis under partially denaturing conditions revealed eight groups of RNA species ranging in molecular weight from 2 x 105 to 1.2 x 106. The transcripts synthesized in vitro were active in an mRNA-dependent cell-free translation system derived from rabbit reticulocytes. The transcripts directed the synthesis of 11 polypeptides that had molecular weights ranging from 125,000 to 20,000 when analyzed by electrophoresis in sodium dodecyl sulfatepolyacrylamide gels. The products of in vitro translation were compared with polypeptides from purified virus and those synthesized in infected cells. Several of the polypeptides synthesized in vitro were designated as structural polypeptides by comparing the molecular weights determined by polyacrylamide gel electrophoresis analysis or by precipitation with hyperimmune serum prepared against purified virus. Three of the viral structural polypeptides (VP4,-5, and-5a) were not synthesized in vitro as primary gene products, demonstrating that processing must occur for the production of some structural polypeptides. Other in vitrosynthesized polypeptides were tentatively identified as either precursors to the viral glycoproteins or nonstructural polypeptides.

The synthesis and processing of simian rotavirus SA11 polypeptides was investigated after infection of MA104 cells. [35S]methionine- or 3H-amino acid-labeled cell extracts were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Viral protein synthesis was maximal 3 to 5 h postinfection, and 12 major viral polypeptides were detected. Immunoprecipitation and peptide mapping experiments, demonstrated five viral structural proteins (125,000 daltons [125K], 94K, 88K, 41K, and 38K). Three proteins (53K, 35K, and 34K) were identified as nonstructural by comparison of their partial proteolysis maps with those from polypeptides of similar molecular weight synthesized in vitro from viral RNA transcripts. Assignment as to structural or nonstructural status of two other primary gene products (26K and 20K) remains tentative. Pulse-chase experiments and tunicamycin blockage of glycosylation revealed cotranslational or post-translational modifications (or both) and precursor-product relationships of several of the polypeptides. Tunicamycin inhibition of glycosylation identified a 35.5K polypeptide which was proven to be the precursor to the 38K structural glycoprotein by immunoprecipitation and peptide mapping analyses. Tunicamycin treatment of infected cells also resulted in the disappearance of other glycoprotein species (23K to 29K) and in the concomitant build-up of an unglycosylated 20K polypeptide, suggesting a precursor-product relationship between those polypeptides. Labeling with [3H]glucosamine or [3H]mannose suggested that the rotavirus glycoproteins contained high mannose oligosaccharides. The effects of amino acid analogs on rotavirus polypeptide synthesis and processing were also investigated.

As a major cause of acute and chronic liver disease as well as hepatocellular carcinoma, hepatitis B virus (HBV) continues to pose significant health problems worldwide. Recombinant hepatitis B vaccines based on adenovirus vectors have been developed to address global needs for effective control of hepatitits B infection. Although considerable progress has been made in the construction ofrecombinant adenoviruses that express large amounts of HBV gene products, preclinical immunogenicity and efficacy testing of candidate vaccines has remained difficult due to the lack of a suitable animal model. We demonstrate here that chimpanzees are susceptible to enteric infection by human adenoviruses type 7 (Ad7) and type 4 (Ad4) following oral admiistrtion of live virus. Moreover, after sequential oral immunization with Ad7-and Ad4-vectored vaccines containing the hepatitis B surface antigen (HBsAg) gene, significant antibody responses to HBsAg (anti-HBs) were induced in two chimpanzees. After challenge with heterologous HBV, one chimpanzee was protected from acute hepatitis and the other chimpanzee experienced modified HBV-induced disease. These data demonstrate the feasibility of using orally administered recombinant adenoviruses as a general approach to vaccination.

No abstract