The overall goal of the proposed research is to test a series of hypotheses relating to the molecular details of human immunodeficiency virus type 1 (HIV-1) and Moloney murine leukemia virus (M-MuLV) replication. Biochemical and genetic approaches will be employed to study the DNA polymerase and RNase H activities of reverse transcriptases. The work will focus on the roles and specificities of RNase H during reverse transcription and on the mechanism and fidelity of DNA displacement synthesis by the DNA polymerase. Recent findings on the nucleotide specificity of RNase H at internal cleavage sites will be extended to determine the relative importance of each of the positions where base preferences are observed and how nucleotide sequence preferences influence both RNA 5' end-directed and recessed DNA primer-directed cleavages. Since an RNA 5' end at a nick is ignored by RNases H, the gap size that is required for 5' end-directed cleavage will be determined. The hypothesis that DNA primer-dependent cleavage at a pause site during minus-strand synthesis, rather than internal cleavage, is responsible for generating the HIV-1 plus-strand primer will be tested. A comprehensive series of biochemical and genetic assays will be employed to determine whether the fidelity of HIV-1 reverse transcriptase during displacement synthesis is different from the fidelity of the enzyme during nondisplacement synthesis. Our in vitro finding that hairpin-induced pausing by reverse transcriptase leads to an increase in strand transfers will be extended to an analysis of the effect of hairpins on direct repeat recombination during reverse transcription in vivo. The same in vivo system will be utilized to examine the ability of select reverse transcriptase mutants to carry out RNA displacement synthesis. An in vitro system based on an RNA that contains the essential viral R-U5- PBS-PPT-U3-R sequences will be developed to test the hypothesis that reverse transcriptase in combination with the nucleocapsid protein is capable of carrying out all of the steps of reverse transcription in vitro. These studies will enhance our understanding of key molecular steps of HIV-1 replication. Such findings are crucial to the identification of novel drug targets and to the development and testing of new drugs for the treatment of AIDS. The analysis of reverse transcriptase fidelity during displacement synthesis will directly contribute to our understanding of how the virus rapidly evolves during progression to AIDS.
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