Mechanism of recombination by HIV reverse transcriptase

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DESCRIPTION (provided by applicant): Human immunodeficiency virus (HIV) has been responsible for the deaths of millions of people in the last two decades. Attempts to combat HIV have been hampered due to the virus's ability to rapidly mutate and produce genetic variants that can circumvent the immune response and resist drug therapy. Recombination, which occurs by a process referred to as strand transfer, is an important mechanisms used by HIV to increase diversity. Two viral proteins, reverse transcriptase (RT) and nucleocapsid (NC) have been clearly implicated in recombination. The goal of this proposal is to answer key questions regarding the mechanism of recombination and the role of NC in the process. This will be accomplished by investigating four specific aims: (1) Probing the potentially different roles of the two zinc fingers of NC protein in strand transfer; (2) Determining the roles of the structure of the acceptor (RNA to which DNAs synthesized on the RNA donor transfer to) in the mechanism of strand transfer; (3) Designing and analyzing in vitro systems capable of producing very long DNA synthesis products; (4) Analysis of the nature of HIV DNA synthesis products produced in vivo to determine how frequently and at what locations DNA synthesis pauses in the cell. A combination of in vitro and in vivo approaches will be used for these experiments. For example, mutant NC proteins produced for aim 1 will be analyzed in in vitro assays and also in the context of the viral genome during infection of culture cells. Aim 4 proposes experiments that could provide a glimpse of how RT traverses the viral genome during cellular infections. Currently, the only knowledge of this process comes from in vitro analysis. Overall, the proposed experiments should help clarify some important unanswered questions and could also be important in developing and evaluating strategies to combat HIV. For example, a better understanding of how the mechanism of NC proteins could lead to specific drugs that interfere with NC. Also, understanding the mechanism(s) by which recombination occurs may allow the design of specific inhibitors to this process.