MOLECULAR MECHANISMS IN REOVIRUS TRANSCRIPTION & CAPPING

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The proposed studies will provide fundamental information about how the transcription and capping enzymes of mammalian reoviruses are organized within the subviral "core" particle. The general approach involves expression of the five core proteins (gamma1, gamma2, gamma3, mu2, and sigma2) for studies of their structure, function, and assembly into particles and more classical genetic and biochemical studies to associate the different activities in transcription, capping, and export of the viral messenger RNAs with specific core proteins and their domains. The long- term goal of this work is to characterize in a full molecular, structural, and mechanical sense how reovirus cores mediate these activities. The findings can be applied to understanding and controlling these processes in viruses and other pathogens, as well as in diseased and normal eukaryotic cells. The specific aims of the project are (1) to determine the structural positions and domain organizations of all five core proteins, (2) to investigate the RNA-dependent nucleoside triphosphatase activity in cores and to identify its role in transcription or capping, (3) to affiliate the four capping enzyme activities (RNA triphosphatase, RNA guanylyltransferase, RNA [guanine-7-N]-methyltransferase, and RNA [ribose- 2'-O]-methyltransferase) with specific proteins or protein regions in the core, and (4) to determine how the genomic dsRNA segments are organized and interact with proteins in the core.