The goal of this investigation is to understand the mechanism of replication of picornaviruses typified by poliovirus. Proteins involved in viral RNA replication will be purified using various chromatographic techniques. Purified proteins as well as serological reagents will be used to determine the role of viral and host proteins in the initiation of poliovirus minus-stranded RNA synthesis. The hypothesis that poliovirus genome-linked protein, VPg (or its precursors) is used as a primer for initiating poliovirus plus- and minus-strand RNA synthesis will be tested using biochemcal techniques and an antibody to VPg. Roles of host factor (HF) and the virus-specific RNA polymerase (P63, replicase) in this reaction will be examined using biochemical and serological means. Specific agents, which selectively inhibit initiation of RNA synthesis as well as phosphorylation of a 63K protein in replicase preparation, will be used to study the involvement of protein phosphorylation in initiation of viral RNA replication. Biochemical and serological techniques will be used to determine if the kinase activity associated with viral polymerase is actually due to P63. Poliovirus ts-mutants that are defective in RNA synthesis will be used in complementation studies to furter define the roles of viral proteins in RNA replication. Molecular organization of the enzymatic machinery responsible for RNA replication will be studied by biochemical and immunochemical analysis of the isolated active replicase complex formed by mixing purified viral and host proteins in vitro. Also anti-VPg and anti-HF antibodies will be used to immunoprecipitate proteins complexed with HF and/or VPg from crude, soluble 7S replicase complex. Purified proteins will be used to study specific binding of polymerase to virion RNA using a recently developed RNA binding assay. Specificity of transcription of polio RNA (by the replicase) compared to other RNAs will be determined using biochemical and serological methods. The long-term goal of this research is to understand the mechanism of replication of picornaviruses. Studies will be continued to understand the mechanism of replication of poliovirus plus-strand RNA synthesis on minus-strand RNA template. Elucidation of the mechanism of replication of poliovirus will certainly fill the remaining large gap in knowledge concerning replication of this medically important virus group which includes those inducing common cold, infectious hepatitis, encephalomyocarditis and foot-to-mouth disease in humans and animals.
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