The overall objective of this grant application is to understand the mechanism of regulation of the murine coronavirus mouse hepatitis virus (MHV). This virus has the largest RNA genome (31 kb) and a complex mechanism of RNA transcription; thus, it potentially serves as a model for RNA viruses in general. Dr. Lai has previously identified two cellular proteins, heterogeneous nuclear ribonuclear protein (hnRNP A1) and polypyrimidine tract-binding protein (PTB), which bind to the cis-acting regulatory sequences of MHV RNA. In this project, the roles of these cellular proteins in MHV RNA transcription and the mechanism of formation of the transcription complex will be studied.
The specific aims are: 1.Determination of the functional roles of PTB and hnRNP A1 in MHV RNA synthesis: Using permanent cell lines expressing wild-type and dominant negative mutants of PTB and hnRNP A1, the composition of RNP complex involving cellular and viral proteins will be characterized. 2.Characterization of additional cellular factors involved in MHV RNA synthesis: Other cellular proteins interacting with components of viral transcription complex will be examined by yeast two hybrid screening. 3.Characterization of proteins involved in MHV RNA synthesis: The role of viral polymerases will be determined by expressing dominant negative mutants of various gene 1 protein products and by performing protein depletion studies. To complement these studies, targeted recombination within gene 1 will be performed. 4.Study of the effects of hnRNP A1 and PTB on viral translation and cellular RNA splicing: The potential effects of these cellular proteins on regulation of viral translation will be studied. Finally, the potential effects of viral RNA synthesis on the cellular RNA processing, with particular emphasis on the possible alterations of alternative RNA splicing will be examined.
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