DNA SYNTHESIS AND RECOMBINATION BY HIV DNA POLYMERASE

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Description (Adapted from Applicant's abstract): The proposed studies focus on the polymerase and RNase H functions of HIV-1 reverse transcriptase (RT) in viral replication and recombination. In HIV infected individuals, 5 to 10 percent of HIV clinical isolates are identified as recombinant. This recombining of sequences contributes to viral adaptation. Examining recombination mechanisms using purified components, we found that the hairpin structures at the 5'-ends of retroviral genomes, such as the HIV-1 TAR, facilitate homologous recombination through template exchange by the DNA primer. We have proposed a mechanism whereby hairpin interactions can bring homologous regions of the RNA genomes together, promoting crossovers. Recent cell culture studies using MLV highlight the kissing loop, a well characterized stem loop structure within the dimerization domain of the retroviral genome, as a hot spot for recombination. In kissing loops, the loop sequence allows base pairing between two identical copies of the stem loops. We now suspect that there is a hierarchy in the ability to promote crossovers in natural stem-loops, based on specific structural features that enhance template interactions, and create a favorable geometry for primer transfer. We propose to determine how template switches are facilitated by interacting hairpins, and whether structural features of the hairpin and interaction stability correlate directly with crossover frequency. Changes in sequence that alter stem and loop size, complementarity, and kissing stability will be analyzed. The RNase H function of RT cuts the genomic RNA into oligomers in the course of minus strand synthesis. Non-PPT RNA oligomers are degraded by RT through an ordered series of primary and secondary cuts made from the RNA 5'-end. From clinical isolates, we found non-nucleoside drug resistant RT mutants, e.g. P237L, that are defective in this mode of RNA 5'-end directed cleavage. We propose an analysis of the mechanism of 5'-end cleavage, and experiments to determine how defects in this process could impair viral replication.