The long-term goal of this renewal application is to understand the molecular basis of pathogenicity of ,viruses belonging to the negative strand RNA (nsRNA) family using vesicular stomatitis virus (VSV) as the prototype virus. The nsRNA viruses comprise a vast multitude of viruses that inflict profound damage and destruction to all living organisms including vertebrates, invertebrates, and plant kingdom. The viruses, such as rabies, measles, mumps, parainfluenza, respiratory syncitial, and many more fall in this nsRNA virus category. Recently, the emergence of novel pathogenic hanta and Ebola viruses has provided renewed impetus to delineate the molecular basis of pathogenicity of the nsRNA viruses. A thorough understanding of the mode of transcription and replication of these viruses is fundamental to develop reagents to combat these deadly pathogens. Our major emphasis toward this goal has been to establish the functions of the key viral proteins of VSV, such as L, the RNA polymerase, P, the transcription factor and N, the genome RNA-binding nucleocapsid protein. These proteins constitute the transcribing ribonucleoprotein (RNP) complex infection within the infected cells. We have been successful in expressing, in biologically active form, these polypeptides in procaryotic and eucaryotic cells using recombinant expression vectors. During the current granting period, we made several important discoveries specially with respect to the subunit composition of the L protein involving cellular translation elongation factors and the putative replicase subunit complex. In the current proposal, we intend to study in detail the structure and function of the transcriptase and replicase complexes of VSV using biochemical and reverse genetics approaches. We will investigate in detail (1) the structure and function of the L protein, specifically the role of translation elongation factor EF-1 alphabetagamma in L activity; (2) the structure and function of the putative replicase complex L-(N-P) and the roles of N-P complex and cellular functions in the replicase function; (3) the structure and function of the P protein with regard to the role of domain II phosphorylation in replication in vitro and using reverse genetics. We will undertake detail mutational studies to determine the precise functions of various domains in the P protein and characterize the unique protein kinase involved in rabies es virus P protein phosphorylation. These studies have the potential to gain insight into the biosynthetic pathways leading to transcription and replication of VSV genome RNA, which is fundamental to our understanding of the molecular basis of pathogenesis of VSV and other nsRNA viruses.
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