Acute gastroenteritis caused by rotaviruses is a leading cause of human infant mortality. Because of the multiple distinct serotypes of human rotaviruses and the lack of protection by different serotypes in cross-challenge experiment, prevention of rotavirus disease by vaccination may meet with oly limited success. Development of methods for disease prevention would be helped by information on the role that each rotavirus protein plays in replication. Towards this end, the establishment of an in vitro system that supports rotavirus RNA replication and particle assembly would provide a method for examining the role of viral proteins in these processes. Rotaviruses are segmented, double-stranded RNA virsuses whose replication is dependent upon protection synthesis. The purpose of this project is to optimize and characterize rotavirus SA11 replication and morphgensis in a cell-free system that was previously shown to support viral dsRNA, mRNA, and protein synthesis in vitro. The system is constructed from viral templates to direct RNA synthesis and a micrococcal nuclease-treated rabbit reticulocyte lysate to translate mRNAs into proteins. Various methods for preparing templates and mRNAs will be tried in the system to determine those which support maximum double-strand RNA synthesis. After optimization, the RNA replication, transcription and core particle assembly of rotavirus will be characterized in detail in the system. Studies will attempt to separate and characterize the different species of viral particles present in infected cells and will assay their ability to template RNA synthesis in vitro. RResults obtained from this project will provide a better understanding of the molecular biology of the rotaviruses as well as the role of viral proteins in the replication of these pathogenic viruses.
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