Molecular Mechanisms in Reovirus mRNA Synthesis

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DESCRIPTION (provided by applicant): The molecular mechanisms by which mammalian orthoreoviruses (reoviruses) and other dsRNA viruses mediate synthesis of their mRNA molecules using virally encoded enzymes packaged within infectious virus particles is a subject of active inquiry because they promise insight into how the steps in mRNA synthesis - plus-strand RNA synthesis (transcription), RNA 5' capping, and RNA transport - occur within the delimited three-dimensional setting of the icosahedral virus particle. A better understanding of these mechanisms in dsRNA viruses should be useful for understanding how related processes are mediated by analogous other viral, microbial, or cellular enzymes and for designing new antiviral, antimicrobial, or anticellular agents directed at them. The long-term objective of our work with reoviruses is to define structure-function relationships for the roles of reovirus proteins in viral replication and effects on host cells and animals. In the current proposal, emphasis is placed on the proteins within the reovirus core (subviral) particle that mediates mRNA synthesis in vitro. Three specific aims are identified, which reflect where further progress appears most promising or necessary to advance understanding in this system. These aims are (1) to determine the structure of the reovirus transcriptase complexes and their arrangement in cores, (2) to define the assembly pathway of the reovirus core shell, and (3) to dissect the functions of the reovirus core proteins in RNA synthesis, capping, and transport. Reovirus particles reconstituted from recombinant proteins expressed using baculovirus vectors play a prominent role in the proposed experiments because of their broad applicability to studies of particle structure, assembly, and functions. Recently determined crystal structures of the transcription- and capping-competent reovirus core particle and the reovirus RNA-dependent RNA polymerase also figure heavily in this proposal by having focused attention on particular areas where more structure information is needed, suggested specific new hypotheses about steps in mRNA synthesis and assembly, and identified specific amino acids in the core proteins to subject to mutagenesis for structure-function testing. The results of these studies will enhance our understanding of the reovirus core as an elegantly designed molecular machine for mRNA synthesis.