Rotaviruses, members of the family Reoviridae, are the major cause of acute gastroenteritis in young children. Despite the pathogenic importance of these viruses, the basic molecular biology of the rotaviruses is not well understood. The genome of these viruses consists of eleven segments of double-stranded RNA (dsRNA). The segmented nature of the genome allows different strains of the rotaviruses to undergo reassortment in nature producing new unique variants. The replication of each segment of the genome proceeds in an asymmetrical manner with viral messenger RNA (mRNA; plus- strand RNA) serving as the template for minus-strand RNA to produce dsRNA. To understand the mechanism of rotavirus RNA replication, we developed a cell-free system that supports the synthesis of simian rotavirus SAll dsRNA, mRNA and protein. Subsequently, this system allowed us to identify, isolate and provide a basic descrip- tion of rotavirus subviral particles (SVPs) that synthesize the viral genome. These data indicate that replicase particles contain cores that may be only partially surrounded by VP6, contain two nonstructural proteins (NS34 and NS35), and draw the template for replication into the core during dsRNA synthesis. The focus of this proposal is to characterize more completely the structure of the replicase particle, the function of proteins associated with these particles, and the recognition signals of viral mRNAs that allow their replication by replicase particles. Specifically, protein-proteins crosslinking, immunoelectron microscopy, and limited proteolytic digestion will be used to study the overall protein structure of the replicase particle. Site-specific cleavage, nuclease digestion and other methods will be used to examine the orientation of the mRNA template with replicase particles and how the template moves during replication. To establish possible function of viral proteins in replication, viral proteins that bind RNA and nucleotides will be identified. The role of VP6, NS34, NS35, and other viral proteins in rotavirus RNA replication, the assembly of single-shelled particles from replicase particles, and in mRNA translation will be studies using the cell-free system. Core particles will be disrupted and reassembled from subunits so as to understand better the morphogenesis of rotavirus SVPs. Initiation of minus-strand RNA synthesis on exogenous viral mRNAs in the cell-free system will be optimized and quantitated. Modified viral mRNAs will be synthesized in vitro by transcription vectors containing cDNAs and added to the system to identify recognition signals on viral mRNAs required for the initiation of minus-strand synthesis. These results should provide valuable information on events in the replication of the rotaviruses that may be useful for developing strategies of controlling diseases caused by these viruses.
