Positive-strand RNA viruses represent an existing and emerging threat to the US public health. These viruses cause diseases ranging from the common cold and summer flu to hepatitis and myocarditis. Currently, there is no effective treatment for positive-strand RNA virus infection. At the heart of replication of these viruses is the RNA-dependent RNA polymerase (RdRP). This enzyme represents a very attractive target for the design of antiviral agents as RdRP activity is only found in virus-infected cells. However, a large gap exists in our understanding of the biochemical mechanism of the RdRP relative to that of cellular, DNA-dependent DNA and RNA polymerases. A better understanding of the RdRP should permit this enzyme to be distinguished from cellular polymerases, thus facilitating the design of specific inhibitors for treatment of RNA virus infection. The primary goal of this proposal is to elucidate the thermodynamic, kinetic and structural basis for correct and incorrect nucleotide incorporation catalyzed by the RdRP from poliovirus. Studies with poliovirus polymerase (3Dpol) are proposed to exploit the wealth of existing information available for this system as a result of decades of effort by many laboratories that has culminated in the discovery of most, if not all, of the virus-encoded replication proteins and the solution of the poliovirus polymerase crystal structure.
The specific aims are as follows: (1) to characterize the substrate and cofactor specificity of 3Dpol by using steady state kinetic and steady-state fluorescence approaches; (2) to elaborate the minimal kinetic mechanism for correct and incorrect nucleotide incorporation catalyzed by 3Dpol by using pre-steady-state kinetic methods (chemical quench-flow and stopped-flow fluorescence); and (3) to evaluate the function of amino acid residues implicated in catalysis and/or binding of nucleic acid and nucleotide substrates by using kinetic analysis of site-directed mutants of 3Dpol. Successful completion of these studies will represent a systematic, quantitative characterization of an animal virus RdRP and should provide information which will facilitate the development and evaluation of inhibitors of this enzyme. Moreover, the availability of a minimal kinetic mechanism will also provide a framework in which to evaluate the effect, if any, of viral and host proteins on RdRP-catalyzed nucleotide incorporation. The identification of host and viral factors which modulate RdRP function will not only expand our understanding of the molecular mechanism of RNA virus genome replication but also suggest additional targets for chemotherapeutic intervention.
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