A major gap in the 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 efforts to develop an in vitro RNA replication system by reconstituting purified components have led us to question the relevance of the in vitro reaction to the mechanism of polio RNA replications in vitro. Thus, in this application, we propose several alternative approaches to improve our understanding of viral RNA replication, and, in particular, of the viral RNA dependent RNA polymerase. We have expressed this enzyme in clones of E. coli carrying plasmids with the polio cDNA gene sequence for the enzyme. This enzyme demonstrates RNA polymerase activity in vitro, and the clones can thus be used to analyze structure-function relationships in vitro. We will perform site-directed mutagenesis of the polymerase gene and characterize the effects of known mutations on enzymatic activity. We will generate conditional lethal polymerase virus mutants and determine the relationship between their selected phenotypes and their altered gene sequences. Protein-protein and protein-RNA interactions will be probed by cross-linking procedures, and efforts to reconstitute the initiation reaction in an active replication complex, with synthetic membranes will be explored. Ultimately, a complete understanding of the structure and function(s) of the RNA replication machinery will be sought.
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