The biosynthesis of RNA directed by an RNA template is a reaction that is unique to RNA viruses. The viral RNA-dependent RNA polymerases that catalyze this reaction all appear to share a major structural motif, suggesting that at least some aspect of their reaction mechanism is similar. Since mammalian host cells do not perform RNA-dependent RNA synthesis, this reaction presents an attractive target for the development of anti-viral chemotherapy. To pursue this strategy, it is essential to understand the structure and function of the enzyme that mediates transcription from RNA templates. The poliovirus RNA polymerase has been studied in several laboratories for a number of years, and it represents perhaps the best understood system among all of the positive strand RNA viruses. Nevertheless, the biochemistry of RNA replication for this virus has not been described. In this grant application, we propose to perform a detailed structural and functional analysis of the poliovirus enzyme, and to determine the roles that additional viral proteins contribute to the reaction mechanism. We have cloned the polymerase in two recombinant expression systems, and we have purified it to near homogeneity. We now propose to define its biochemical properties and have arranged a collaboration to determine its atomic structure by X-ray crystallographic analysis. We will also compare the structure and functional activities of a mutant enzyme that is encoded by an attenuated strain of virus. We plan to utilize both biochemical and genetic approaches to examine individual steps in the synthesis of minus strand RNA and in the addition of protein covalently linked to newly-synthesized RNA Chains. Lastly, studies will be initiated to identify the helicase activity responsible for unwinding RNA strands during synthesis, and to determine the function(s) of viral protein 2C in RNA synthesis.
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