Viral gastroenteritis is a worldwide problem, and the rotaviruses are now recognized as the major cause of diarrhea in children and animals in all countries. The significance of these newly recognized human and veterinary pathogens has established a need for effective methods of disease prevention and control. We propose studies to investigate the molecular biology and replication strategies of the rotaviruses. First, studies of the simian rotavirus SA11 will be performed to elucidate the structure and function of the individual rotavirus genes and their protein products. The nucleotide sequence of DNA copies of SA11 genes will be determined and analyzed to deduce the amino acid sequence of the proteins and to identify possible functional domains of the proteins. The protein sequence data will also be utilized to design synthetic peptides for the production of antibodies to probe the functions of the nonstructural proteins. Second, methods to express and manipulate cloned rotavirus genes will be developed. Full length gene 6 cloned DNA will be used as a model to obtain gene expression, to produce specific alterations in the gene, and to determine if manipulated genes can be rescued in progeny virus particles. These experiments will evaluate whether wild-type rotaviruses can be recovered from cells coinfected with cloned gene 6 DNA (or a synthetic mRNA made to this DNA) and with temperature-sensitive (ts) SA11 or bovine rotavirus mutants that contain their ts lesions in gene 6. Finally, the functions and interactions of the SA11 polypeptides in virus structure, replication and morphogenesis will be evaluated. Denaturation of the particles will be used to investigate whether conformation of the outer capsid polypeptides is important for antigenicity and biologic activities. Immune electron microscopy and immunocytochemistry experiments using monoclonal or monospecific antibodies will localize specific polypeptides in infected cells and in immature (enveloped) and mature virus particles. Enveloped particles will be purified and characterized biochemically and biologically. The role of polypeptide interactions in outer capsid assembly will also be examined in an in vitro system. These studies will provide fundamental knowledge of the structure and functions of rotavirus genes and their gene products that should help develop strategies to prevent this important disease in children and animals.
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