Biochemical Mechanisms of Drug Resistance in HIV-1 RT

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DESCRIPTION (provided by applicant): Nucleoside reverse transcriptase (RT) inhibitors play an important role in therapy for HIV-1 infection and AIDS; however, long-term use of these drugs is limited by the selection of resistant mutants. These compounds give rise to chain-terminating nucleotides that are incorporated by HIV-1 RT and block further DNA chain elongation. Work from this laboratory was instrumental in showing that HIV-1 RT can catalyze the excision in vitro of chain-terminating nucleotides by transfer to a nucleotide acceptor and that a major class of nucleoside resistant mutants of HIV-1 encodes RT with increased nucleotide-dependent excision activity. Research is proposed to (a) define factors that determine the efficiency of the excision reaction in vitro including structural features of the acceptor substrate, structure of the chain-terminating residue, and sequence elements in the primer-template, (b) investigate the mechanism of inhibition of excision and DNA synthesis by foscarnet (PFA), a structural analog of pyrophosphate, and to determine the mechanism of resistance to PFA by PFA-resistant mutants, (c) investigate the effect of non-nucleoside RT inhibitors on excision and on dNTP inhibition of excision, and (d) to identify factors that determine which intracellular compounds serve as acceptor substrates for excision in vivo -- i.e., in lymphocytes from uninfected and HIV-1 infected individuals. In addition, research is proposed to extend these studies to identify factors that control excision in vivo using an enzymatic assay to detect the products of excision. The goal of this research is to define the role of the excision activity in HIV-1 infection, which will be crucial for the development of more effective drugs and therapeutic strategies against HIV, particularly against the emergence of resistant virus.