Retroviral reverse transcriptases contain a DNA polymerase activity and an RNase H activity that cooperate to copy the viral single-stranded RNA genome, producing a linear double-stranded DNA that is subsequently integrated into the genome of the host cell. The long term objective of this program is to understand in molecular detail all of the steps in the process of reverse transcription by both Moloney murine leukemia virus (M- MuLV) and human immunodeficiency virus type 1 (HIV-1). A combined biochemical and genetic approach is proposed to investigate (i) the multiple roles of RNase H in reverse transcription, and (ii) the capability of reverse transcription, and (ii) the capability of reverse transcriptase to displace the nontemplate strand during DNA synthesis on a nicked DNA template. Studies to elucidate the basis for the specificity of plus- strand priming will involve both a search for reverse transcriptase mutants that retain non-specific RNase H activity but are impaired for primer generation, and a characterization of the in vivo phenotype of mutations in the polypurine tract. The specificity of RNase H for removal of both the tRNA primer for minus strands and the polypurine tract primer for plus strands will be investigated in relation to the terminal structures required for integration. The strand displacement capability of the polymerase activity of HIV-1 reverse transcriptase will be determined and compared with recent results from the M-MuLV system. The ability of the polymerases to displace an RNA strand and the effects of the nucleocapsid protei on displacement synthesis will be investigated. The kinetics, processivity, and pausing patterns during displacement synthesis along with biochemical studies on the nature of the interaction between the polymerase and the nontemplate strand will provide data to distinguish a passive from an active mechanism for the displacement reaction. Experiments will be undertaken to isolate and characterize mutants that are defective in helix unwinding as a means to identify the domains of the reverse transcriptases responsible for displacement synthesis.
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