A critical feature of the life cycle of retroviruses, including the human immuno-deficiency virus (HIV), is their ability to generate diversity. Retroviruses have exceptionally high mutation rates, permitting rapid evolution of new forms of the virus that are better able to escape host defense mechanisms. A major source of this diversity may be the infidelity of the viral-encoded reverse transcriptase. Our objective is to elucidate the molecular mechanisms responsible for the genetic diversity of the HIV and other retroviral genomes. We have begun to examine the mutagenic potential of reverse transcriptase using our recently developed M13mp2 mutagenesis assay. This assay measures the error frequency of a single round of natural DNA synthesis in system that permits analysis of a wide variety of mutational events, each precisely defined at the nucleotide sequence level. Our initial results with reverse transcriptases isolated from avian myeloblastosis virus and murine leukemia virus demonstrate a high error frequency for reverse transcriptase during copying of natural DNA. This supports the concept that the observed high mutation rate of retroviruses indeed reflects, at least in part, low reverse transcription fidelity. We plan to purify various forms of viral reverse transcriptase and examine their error frequency and the specificity of mutations produced during synthesis. We will then use reversion assays to monitor specific mutational pathways to test models of mutagenesis by reverse transcriptase. Ultimately we would like to focus on the reverse transcriptase encoded by the HIV genome, to elucidate the mechanisms by which diversity is generated by this enzyme.
