MECHANISM OF RECOMBINATION BY HIV REVERSE TRANSCRIPTASE

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The human immunodeficiency virus (HIV), the Causative agent of Acquired Immune Deficiency Syndrome (AIDS), 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 viruses 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 interaction of RT with nucleic acids. This will be accomplished by investigating 4 specific aims related directly to recombination: (1) Analysis of strand transfer "hotspots" that promote recombination events, (2) Defining the role of RT-directed base misincorporations in inducing recOmbination (strand transfer) events, (3) Analysis of RT binding and cleavage on structures mimicking replication or strand transfer intermediates, and (4) Probing the mechanism by which NC catalyzes strand-exchange to promote strand transfer. The goal of the first aim is to determine why particular sites promote transfer while others do not. A potential link between two of the major mechanisms for the generation of HIV mutants will be explored in the second aim while specific interactions between key viral proteins and unique strand transfer intermediates are studied in aims 3 and 4. The basic interaction of RT with nucleic acids will be investigated through 3 specific aims: (1) Quantitative assessment of the interaction of RT with nucleic acid substrates under physiological conditions, (2) Determining the role of different regions of RT in binding to nucleic acid using chimeric substrates, and (3) Searching for nucleic acids sequences that can bind RT with high affinity. The basic goal of these studies is to define RT and nucleic acid properties importantJor 'tight" binding between the two. Overall, the proposed experiments should help clarify some of the important unanswered questions and could also be important in developing and evaluating strategies to combat HIV. For example, an understanding of the interplay between RT fidelity and recombination could allow for better predictive models that help determine how therapy will impact HIV evolution. Perhaps drug combinations that tend to lead to a more or less accurate RT would be advantageous in the long run.