All RNA viruses besides retroviruses encode an RNA-dependent RNA polymerase to amplify their genomes. Study of the poliovirus RNA-dependent RNA polymerase has recently become a structure-based problem with the solution of the three-dimensional structure of 3Dpol. The poliovirus polymerase is homologous to RNA-dependent RNA polymerases from other picornaviruses such as rhinoviruses and echoviruses for which good vaccines are not available. Several interesting features of poliovirus polymerase biochemistry demand further investigation, both to acquire detailed structural and functional understanding of the mechanism of RNA-dependent RNA polymerization and to develop targets for pharmaceutical disruption. The applicant has shown that both RNA binding and RNA polymerization are cooperative with respect to polymerase concentration. To define the path of bound RNA substrates, the homology of the polymerase with HIV reverse transcriptase and U1 snRNP binding protein will be used to guide site-directed mutagenesis experiments. Binding affinities of mutant polymerases for RNA will be tested to distinguish effects on intrinsic RNA binding affinity from effects on the protein-protein interactions that give rise to cooperative RNA binding. Polymerase-polymerase interactions that may be involved in cooperative RNA binding are seen in the three-dimensional structure, which shows head-to-tail oligomerization of polymerase molecules in long fibers within the crystal. Gel-shift RNA binding experiments with wild type and mutant polymerases, site-directed mutagenesis of potential protein-protein interfaces and atomic force microscopy will be used to determine the stoichiometry of RNA polymerase bound to RNA substrates. Mutations that specifically affect intrinsic RNA binding and protein-protein interactions will be tested for their effects on the poliovirus replicative cycle both in infected cells and in cell-free extracts. Peptide inhibitors of polymerase-polymerase interactions will be sought using the yeast two-hybid system and tested for their effects on cooperative RNA binding and the poliovirus replicative cycle in cell-free extracts. Inhibitors of interactions within multiprotein complexes have the theoretical advantage that mutations that confer resistance to such inhibitors should be recessive or only partially dominant. They should be inefficiently selected even in RNA viruses, with their notoriously high mutation rate.
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