In this study, we are examining the mechanism of poliovirus RNA replication. A soluble RNA dependent RNA polymerase has been isolated from poliovirus infected cells. The purified polymerase will copy poliovirion RNA when oligo(U) is used as a primer. Under the appropriate reaction conditions, the oligo(U) primed polymerase will synthesize full-sized copies of poliovirion RNA as well as other viral and cellular RNAs. Studies on the structure of this enzyme have shown that a single viral protein, p63 (NCVP4), is responsible for this activity. Studies are being proposed to determine how poliovirus RNA synthesis initiates. We plan to isolate and characterize and initiating form of the poliovirus RNA polymerase. The activity will be purified and the virus-coded and host-coded proteins that are required for this activity will be determined. The product RNA synthesized by the initiating polymerase will be characterized for its size and structure. The role that VPg plays in RNA replication will be investigated. We also plan studies to further characterize the oligo(U)- dependent elongating polymerase. The product RNA that is synthesized by this enzyme will be fully characterized by sequencing the 5 feet and 3 feet terminal ends of the product RNA. The fidelity of in vitro replication will be examined, and it will be determined if infectious RNA can be synthesized in vitro by the purified polymerase. The template requirements and template specificity of the initiating and elongating forms of the polymerase will be fully characterized. Using our in vitro replication system we hope to answer specific questions about the mechanism of poliovirus RNA replication.
