BIOCHEMICAL MECHANISMS OF DRUG RESISTANCE IN HIV-1 RT

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The use of chain-terminating nucleoside analogues such as 3'-azidothymidine (AZT) in the treatment of HIV-1 infected patients is limited by the selection of drug-resistance mutant virus during therapy. This laboratory has recently described a novel nucleotide-dependent, pyrophosphorolysis-related activity (NPP activity) of HIV-1 reverse transcriptase (RT) that is capable of removing a chain-terminating residue from the 3'- terminus of the growing DNA chain in the presence of physiological concentrations of ribonucleoside triphosphates and is elevated in AZT-resistant HIV. NPP activity is inhibited by physiological levels of the next complementary dNTP which forms a dead-end complex with RT and chain-terminated primer/template. Research is proposed to test the hypothesis that NPP activity and its inhibition by the next complementary dNTP are major factors in determining the ability of chain-terminating nucleotides to inhibit DNA synthesis by HIV-1 RT. Specific Aims: (i) To determine the role of NPP activity in the inhibition of RT-dependent DNA synthesis by chain-terminating nucleotides that are of clinical and/or mechanistic importance; (ii) To evaluate the role of the primer terminus, the next complementary dNTP, and mutant or wild type HIV-1 RT in NPP activity and its inhibition by dNTPs; (iii) To identify and/or design new compounds that inhibit NPP activity and to elucidate the mechanism of their inhibition; and (iv) To develop a sensitive method to detect the products of NPP activity and to use this assay to monitor NPP activity in cells infected with mutants of HIV-1. The NPP activity of HIV-1 RT may be a major factor determining the effectiveness of nucleoside analogues in the treatment of HIV-1 infection and AIDS. The proposed research may lead to improved strategies for antiretroviral therapy against HIV and AIDS and the rational development of new drugs that target this activity.