BIOCHEMICAL MECHANISMS OF DRUG RESISTANCE IN HIV-1 RT

链终止核苷类似物如3'-氮杂thymidine （AZT）在治疗HIV-1感染患者中的使用受到治疗期间耐药性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的焦磷酸裂解相关活性（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'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生理水平的抑制，后者与RT和链端引物/模板形成死端复合物。研究人员提出了一项假设，即NPP活性及其对下一个互补dNTP的抑制是决定链终止核苷酸抑制HIV-1 rt DNA合成能力的主要因素。具体目标：(i)确定NPP活性在抑制rt依赖性DNA合成中的作用，这些链终止核苷酸具有临床和/或机制重要性；（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活性及其dNTP抑制中的作用；鉴定和（或）设计抑制NPP活性的新化合物并阐明其抑制机制；（iv）开发一种检测NPP活性产物的灵敏方法，并利用该测定法监测感染HIV-1突变体的细胞中NPP的活性。HIV-1 RT的NPP活性可能是决定核苷类似物治疗HIV-1感染和艾滋病有效性的主要因素。这项研究可能会改善抗逆转录病毒治疗艾滋病毒和艾滋病的策略，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.