The overall objective of this study is to characterize the basic molecular mechanisms of coronavirus transcription utiizing mouse hepatitis virus (MHV) as a model. These viruses replicate and cause significant economic losses in a wide variety of animal species, including humans. Coronaviruses replicate by a unique mode of discontinuous RNA synthesis in which a free leader RNA is transcribed independently, and is utlized as a primer for transcription of 6 subgenomic mRNAs. This proposal is designed to provide additional understanding into the basic mechanism of coronavirus replication. Specifically, we intend to: (a) Clone and sequence the leader RNA sequences encoded at the 5' end of the genome. (b) Develop an entirely new approach for analyzing the sequences which function in priming transcription of the subgenomic mRNAs. This approach utilizes expression vectors which synthesize virus-like (-)-sensed RNAs that can be recognized and transcribed by the viral polymerase. Such an approach will allow us to prove that leader RNA acts in trans to prime subgenomic RNA synthesis; (b) Utilize this approach to map the functional sequences at the intergenic """"""""start"""""""" sites of mRNAs 5, 6, and 7, and determine what sequences regulate the initition and synthesis of the subgenomic mRNAs: (c) Analyze the origin, structure and synthesis of the leader-containing RNAs to determine if transcriptional """"""""pausing"""""""" occurs during MHV RNA synthesis at regions of 2 degree-structure in the product or template RNAs. Such findings will shed considerable light on the mechanism of MHV RNA synthesis, RNA recombination, and generation of defective interfering RNAs; and (d) clone and sequence the MHV encapsidation signal, and determine the sequences which bind the pp60 nucleocapsid protein.
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