BIOCHEMICAL MECHANISMS OF DRUG RESISTANCE IN HIV-1 RT

链终止核苷类似物如3 '-叠氮胸苷（AZT）在治疗HIV-1感染患者中的应用受到治疗期间耐药突变病毒选择的限制。 该实验室最近描述了HIV-1逆转录酶（RT）的一种新的核苷酸依赖性焦磷酸解相关活性（NPP活性），该活性能够在存在生理浓度的三磷酸核糖核苷的情况下从生长的DNA链的3 '末端去除链终止残基，并且在AZT抗性HIV中升高。 NPP活性受到下一个互补dNTP的生理水平的抑制，该互补dNTP与RT和链终止的引物/模板形成死端复合物。提出研究以检验NPP活性及其被下一个互补dNTP抑制是决定链终止核苷酸抑制HIV-1 RT的DNA合成的能力的主要因素的假设。具体目的：（i）确定NPP活性在具有临床和/或机理重要性的链终止核苷酸抑制RT依赖性DNA合成中的作用;（ii）评估引物末端、下一个互补dNTP和突变型或野生型HIV-1 RT在NPP活性中的作用及其被dNTP抑制;（iii）鉴定和/或设计抑制NPP活性的新化合物并阐明其抑制机制;和（iv）开发一种灵敏的检测NPP活性产物的方法，并使用该测定来监测感染HIV-1突变体的细胞中的NPP活性。HIV-1 RT的NPP活性可能是决定核苷类似物治疗HIV-1感染和AIDS有效性的主要因素。 拟议的研究可能会导致改善艾滋病毒和艾滋病抗逆转录病毒疗法的战略，并合理开发针对这一活动的新药。

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.