The long-term goal of this renewal application is to understand the molecular mechanism of replication of negative strand RNA viruses using vesicular stomatitis virus (VSV) as the prototype virus. This virus is the paradigm of all nonsegmented negative strand RNA viruses, such as rabies, measles, mumps, parainfluenza, respiratory syntitial, and Sendai, to mention a few. This group of viruses comprises some of the most serious human pathogens in terms of both mortality and morbidity. A clear understanding of the mode of transcription and replication of these viruses would ultimately help the investigator to delineate the molecular basis of pathogenesis of these viruses. The investigator has approached this problem by attempting to establish the functions of the key viral proteins of VSV such as L, the RNA polymerase, P, the transcription factor, and N, the RNA-binding nucleocapsid protein. These proteins constitute the transcribing RNP complex of VSV which initiates infection within the infected cell.The investigator has been successful in expressing, in biologically active form, these polypeptides in procaryotic and eucaryotic cells using recombinant expression vectors. This achievement has helped the investigator to elucidate the unique role of cellular casein kinase II in phosphorylating the P protein for its activation.In the current proposal the investigator intends to study in detail the functions of these three key polypeptides in the life- cycle of VSV. The investigator proposes to investigate in detail 1) the functions of the P protein in transcription/replication, specifically the role of phosphorylation in these processes, 2) the functions of the N protein in relation to establishing the domains that control encapsidation and replication of the genomic RNA. In addition, the investigator will study the roles of N-P complex in these processes, 3) the functions of the L protein with special emphasis on the role of the protein kinase activity associated with it and its interaction with the P protein. The functional domains involved in binding to nucleotides, protein kinase and capping activities will also be established. These studies have the potential to unravel the important biosynthetic pathways in the transcription/replication steps in the life-cycle of the virus as well as characterizing the functional domains within these three polypeptides which would be the potential targets for eventual designing of antiviral drugs.
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