FIDELITY OF RETROVIRAL REVERSE TRANSCRIPTASES

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A critical feature of the life cycle of retroviruses, including the human immunodeficiency virus (HIV), is their ability to generate diversity. Retroviruses have 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 reverse transcriptase (RT). Our objective is to examine the mutagenic potential of RTs during DNA synthesis in vitro, using an M13mp2 mutagenesis assay that monitors a wide variety of base substitution, frameshift, deletion and complex errors. We began by measuring the error frequency of a single round of natural DNA synthesis with RTs isolated from avian myeloblastosis virus (MLV) and murine leukemia virus (MLV). Both enzymes exhibit low fidelity. DNA sequence analysis of mutants generated by the AMV RT demonstrate that it commits many different types of errors. Two specificities are unique; base substitution errors result exclusively from misinsertion of purine nucleotides and many -1 deletions occur at non-reiterated base sequences. Furthermore, in an assay employing a terminally mismatched template-primer, both AMV and MLV RTs exhibit an unexpected extension preference that is unique compared to normal cellular polymerases. These specificities may reflect a structural differences in the active site of RTs. Overall, these data support the concept that the observed high mutation rate of retroviruses indeed reflects low fidelity of reverse transcription. We have recently obtained a preparation of reverse transcriptase from HIV-1. Our initial results indicate that this enzyme is even more error prone than the AMV and MLV RTs. We are presently sequencing a large collection of HIV RT-generated mutants to determine the most common mutational events perpetrated by this enzyme. We also intend to use reversion assays to examine specific mutational pathways in order to elucidate the mechanisms by which the diversity of the HIV is generated.