CONSTRUCTION AND ANALYSIS OF RETROVIRUS MUTANTS

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The long-term objective of the experiments described in this proposal is to increase our understanding of the replication of retroviruses, formerly called RNA tumor viruses. These agents, widely distributed in nature, cause a variety of leukemias, tumors and other diseases in animals and man. The specific goal of this work is to determine in as much detail as possible the structure and functions of the viral gene products encoded by the Moloney murine leukemia virus, a prototypical replication-competent retrovirus. Three major approaches will be taken toward that goal. First, mutagenesis of cloned DNA copies of the viral genome will be used to make specific alterations in selected regions, and the effects of these mutations on the viral life cycle will be determined by direct analyses of viral proteins and nucleic acids. Several domains of the viral gag, pol, and env genes, as well as various cis-acting regions, will be analyzed in this way. These experiments will determine the essential sequences in each gene or region, and will further define their roles in replication. Second, a major effort will be initiated to carry out reversion analysis of virus mutants. We will generate a series of replication-competent revertant viruses from several mutant parents, each arising by virtue of suppressing mutations that compensate for the effects of the original mutation. Mapping and analysis of these reversion mutations will help to locate domains of the viral gene products that interact with one another, and may help identify proteins that bind to viral DNA and RNA. Third, biochemical tests will be applied to three viral gene products expressed as fusion proteins in bacteria. Rapid assays will be developed to screen these proteins for various activities, such as DNA binding, tRNA binding, and nuclease activity. In each case, these tests will be applied to existing panels of mutants to help localize the regions needed for these activities.