The overall objective of this renewal grant application is to understand the mechanism of coronavirus RNA synthesis using mouse hepatitis virus (MHV) as a model. This virus group is responsible for several severe diseases in livestocks and humans, including a possible etiological association with multiple sclerosis. The molecular biology of this virus group includes several unique features, including a novel mechanism of RNA synthesis, which involves leader RNA-primed transcription. This proposal is designed to attempt to provide further understanding into this unique mechanism.
The specific aims i n this proposal include the following: 1) To characterize the properties, structure and biological functions of the MHV leader RNA by determining the protein species bound and the sequence heterogeneity of the leader RNA. We also want to determine whether the leader RNA can be freely reassorted and attempt to characterize the negative-stranded leader RNA. 2) To compare the sequences of intergenic regions in the genomic RNA and 5'end sequences of mRNAs by cDNA cloning of the genomic and mRNAs. This approach will determine how the leader RNA is joined to the body sequences of mRNAs. 3) To further characterize the structure of the negative-stranded RNA template by hybridization analysis using a positive-stranded leader DNA probe. 4) To characterize the RNA recombinant viruses by cDNA cloning and sequencing of the recombinant viruses. We will also attempt to isolate more recombinant viruses in order to characterize the mechanism of RNA recombination and to determine the sequence requirement of the leader-body fusion in various mRNAs. 5) To study the genomic sequences of defective-interfering (DI) particles to gain further understanding of mRNA synthesis.
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