MECHANISM OF CORONAVIRUS RNA TRANSCRIPTION

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Coronaviruses are enveloped RNA viruses that cause a variety of diseases in humans and animals. The long-term goal of the proposed research is to elucidate the mechanisms of coronavirus RNA synthesis by using mouse hepatitis virus (MHV), a prototype murine coronavirus, as a model system. Coronavirus RNA transcription involves the interaction between the leader RNA and intergenic sequence. It has been proposed that this interaction is probably mediated by direct RNA-RNA interactions between complementary sequences. Using a mutant MHV (JHM2c), we recently found that MHV RNA transcription does not strictly require sequence complementarity. A specific subgenomic mRNA species is initiated at various sites of the intergenic region where there is no sequence complementarity between the leader and the intergenic template. This finding leads us to hypothesize that protein-RNA and protein-protein interactions rather than direct RNA-RNA interactions are the driving force for the initiation of mRNA transcription. We also found that the heterogeneous nuclear ribonucleoprotein (hnRNP) A1 is a potential cellular factor in the regulation of MHV RNA transcription. We propose to use JHM2c as a unique means and to employ biochemical and molecular approaches to test this hypothesis with the two Specific Aims: (I) To define the interactions between hnRNP-A1 and the cis-acting sequences of MHV RNA both in vitro and in vivo, and to determine the functional significance of this protein-RNA interactions in regulation of MHV RNA transcription; (II) To characterize the interactions between hnRNP-A1 and the MHV RNA polymerase and nucleocapsid proteins both in vitro and in vivo, and to determine the functional significance of these protein- protein interactions in regulation of MHV RNA transcription. These studies will contribute to the fundamental understanding of the mechanisms of regulation of RNA synthesis in coronaviruses and other RNA viruses as well.