A major gap in knowledge of the replication cycle of the picornaviruses lies in the area of viral RNA synthesis from RNA templates. The human virus, poliovirus, offers a useful prototype to study this process. Recent progress in the identification of the enzyme system utilized for viral RNA synthesis and in the characterization of replicating RNA structures in the infected cell has provided an appropriate base upon which to initiate biochemical studies of the process of RNA replication. This proposal is directed toward these goals. The viral polymerase subunit has been purified to apparent homogeneity. It catalyzes the synthesis of complementary RNA from a template in the presence of a primer. A host factor has been isolated from uninfected HeLa cells which permits initiation by the viral polymerase on polio RNA template. We propose to isolate clones of E. coli which express the viral polymerase as well as clones which express the polymerase precursor protein. We will also prepare monoclonal antibodies to each component of the RNA synthesis activity. With these reagents, protein-RNA binding and protein-protein interactions of the various components of the reaction will be analyzed by crosslinking procedures, and the structural features of the RNA template which are essential for recognition by the enzyme and host factor will be investigated by hybrid-arrested transcription and template specificity studies. A comparison between the utilization of plus and minus strand templates will be made. In addition, the effect of cellular membranes on the RNA replication process will be examined, and studies will be initiated to determine the possible role of the uncharacterized viral protein X on RNA replication. Ultimately, reconstruction of the viral RNA replication reaction in vitro will be sought.
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