The mechanism of poliovirus RNA replication is being examined in this study. The poliovirus RNA dependent RNA polymerase was isolated as a soluble and template dependent enzyme from the cytoplasm of infected cells. Highly purified forms of the polymerase were found to synthesize full-sized copies of poliovirion RNA other polyadenylated RNAs when oligo(U) was added as a primer. In the presence of a cellular protein component or "host factor", the polymerase will initiate RNA synthesis in the absence of an oligo(U) primer. We found that the product RNA that was synthesized in the presence of the host-factor was about twice the size of the template RNA. This and other evidence indicated that the product RNA was covalently linked to the template RNA. This suggested that in the presence of the host-factor the polymerase used a template-priming mechanism to initiate RNA synthesis in vitro. We are continuing these studies to further characterize the in vitro polymerase reaction to determine if this model is correct. The activity of the polymerase on poliovirus minus-strand templates is being examined, and the product RNA from these reactions is being characterized for its size and polarity. The role of VPg in the replication of poliovirus RNA is also being investigated. To carry out these studies, VPg was prepared synthetically, and was used to prepare anti-VPg antibody. We hope to determine if VPg (or a VPg precursor protein) plays a role in the initiation of RNA synthesis. In addition to our studies on the initiation of RNA synthesis, we plan to further characterize the elongating activity of the polymerase. The fidelity of RNA replication in vitro will be measured, and it will be determined if infectious RNA can be synthesized using minus-strand RNA templates. To study viral RNA replication in vivo, we will characterize the structure of poliovirion RNA isolated from infected cells and will use a genetic approach to identify and define the functions of viral proteins required for poliovirus RNA replication. Poliovirus mutants will be constructed using infectious DNA plasmids containing the complete viral sequence.