The objectives of this research proposal are to continue my work with a model system developed to study the in vivo mutation rate of human immunodeficiency virus type 1 (HIV-1) in a single cycle of retrovirus replication. This system uses a genetically engineered HIV-1 vector containing the lacZa peptide gene as a reporter for mutations. The long term goal in studying this model system is to identify variables that could have profound effects on HIV-1 genetic variation, evolution, and progression to AIDS. As a first step, I will focus may efforts on three specific aims to extend my work. First, I propose experiments to characterize extragenic determinants of HIV-1 reverse transcription accuracy. The determinants responsible for the influence of Vpr on the HIV-1 mutation rate be mapped. In addition, variants of the HIV-1 nucleocapsid (NC) protein will be tested for their influence on the in vivo mutation rate. Second, I will determine the influence of HIV-1 reverse transcriptase (RT) variants on the in vivo mutation rate. Mutations in HIV-1 RT that confer resistance to the antiviral drugs AZT and 3TC will be analyzed for their influence on the in vivo mutation rate. In addition, RT mutants that have altered fidelities in cell-free systems will also be analyzed. Third, I will determine how combined variants of RT and/or other HIV-1 proteins influence the in vivo mutation rate. Specifically, HIV-1 RT variants will be tested together in one combined HIV-1 RT variant, or HIV-1 RT variants will be tested in the presence of a variant Vpr to determine how simultaneous mutation of both RT and Vpr influence the accuracy of reverse transcription. Combinations will also include NC proteins, if variants are characterized that influence the mutation rate.
