The arsenal of antiviral agents that may be useful for treatment of infections by RNA viruses classified as either category A, B or C pathogens consists of one compound, ribavirin. Ribavirin is the only broad-spectrum antiviral nucleoside employed clinically. Development of compounds with antiviral activity greater than that of ribavirin has been thwarted by the absence of a clear understanding of the mechanism of action of this compound. Current dogma states that ribavirin functions by inhibiting inosine monophosphate dehydrogenase (IMPDH), a cellular enzyme required for de novo synthesis of GTP. Our preliminary studies with a model RNA virus suggest an alternative mechanism of action for ribavirin. Briefly, RTP is a substrate for viral RNA-dependent RNA polymerases (RdRP) and is incorporated opposite both cytidine and uridine. The lack of specific incorporation of ribavirin causes an increase in the mutation frequency of the virus beyond that capable of supporting viability of the virus population. These data suggest that ribavirin is a lethal mutagen of RNA virus genomes. The long-term goal of the collaborative research presented in this proposal is the development of lethal mutagenesis into a useful therapeutic regimen for the treatment of RNA virus infections. This goal will be accomplished by pursuing the following specific aims: ? 1. Identify leads for development ? (a) by evaluating the mutagenic activity of a subset of compounds from appropriate libraries provided to us by Ribapharm, Inc. (a subsidiary of ICN Pharmaceuticals, Inc.) and the Medical Research Council (United Kingdom), and ? (b) by synthesizing and characterizing ribonucleoside analogs containing pseudo bases that are known or predicted to pair ambiguously; ? 2. Evaluate the ability of ribonucleotide reductase to utilize mutagens as substrates and/or effectors in order to obtain information on potential issues of safety; ? 3. Establish dengue virus type 2 as a model system for evaluation of lethal mutagenesis by characterizing the mechanism of action of ribavirin against this virus; ? 4. Develop cell-based assays to screen for inhibitors and lethal mutagens of dengue virus. ? Validation of lethal mutagenesis as an antiviral strategy by identifying novel mutagens and demonstrating efficacy against important pathogens without heritable genetic consequences should provide the impetus for pursuit of this strategy by the pharmaceutical industry. The availability of cell-based assays to screen for lethal mutagens should greatly facilitate the discovery effort. Lethal mutagens should have broad-spectrum activity against RNA viruses, thus better equipping this nation to deal with the potential use of RNA viruses as biological weapons. ? ?
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