The molecular mechanisms involved in the replication of poliovirus RNA will be examined in this study. We plan to identify viral and cellular proteins and viral RNA sequences that are important in the initiation of RNA synthesis, the binding of the polymerase, and the linkage of VPg to the RNA. The poliovirus RNA polymerase requires an oligonucleotide primer or a cellular host factor to initiate RNA synthesis. Dimer-sized product RNA is synthesized by the polymerase in the presence of the host factor. At least two host factor activities have been identified. The host factor associated with terminal uridylyl transferase activity will be extensively purified and used to study the initiation of RNA synthesis in vitro. Poliovirus cDNA clones will be constructed in transcription vectors to prepare viral RNA transcripts of different polarities and containing various deletions and other mutations. We will characterize the structure of the product RNAs synthesized by the polymerase and host factor (TUT, etc.) on both (+) and (-) strand specific templates and will determine the sequence in the joint region of dimer-sized product RNAs. The linkage of VPg to poliovirus RNA will be studied to determine if this involves a cleavage reaction and if specific viral RNA sequences are required for this reaction. Modified VPg with an altered amino acid sequence and VPg precursor proteins will be assayed for linkage activity. The synthesis of dimer-sized poliovirus RNA in infected cells will be further characterized. We will determine if the synthesis of dimer-sized RNA is increased in cells infected with certain viral mutants and if guanidine HCl affects the synthesis of dimer-sized RNA. The binding of the polymerase to viral RNA will be examined to determine if high affinity binding sites for the polymerase exist in (+) and (-) strand viral RNAs. The optimal conditions for binding will be determined and other viral and cellular proteins will be tested for their ability to affect binding. A final objective of the study is to quantitate the polymerase error frequency both in vitro and in vivo. The polymerase error frequency will be measured at various sites in poliovirus RNA under different reaction conditions. The results of this study should give us a better understanding of the molecular basis of poliovirus RNA replication.
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