Biochemical Mechanisms of Drug Resistance in HIV RT

描述（由申请方提供）：核苷逆转录酶（RT）抑制剂仍然是抗人类免疫缺陷病毒1型（HIV-1）治疗的重要组成部分。这些化合物被细胞酶磷酸化，并通过HIV-1 RT掺入DNA中，导致病毒DNA合成期间的链终止。HIV-1 RT突变体从新生DNA链中去除链终止核苷酸（切除活性）的能力是耐药性的重要机制，在治疗过程中经常选择切除活性升高的突变体。我们建议开发一种分析方法来确定是什么影响了受感染细胞的切除活性，这将有助于我们了解是什么驱动体内突变体选择。目的1是利用纯化的野生型和突变型HIV-1重组蛋白来评估RT的核糖核酸酶H活性切割RNA和模板片段解离在调节阻断的DNA链的切除拯救中的作用。目的2是确定HIV核衣壳蛋白对二级核糖核酸酶H切割片段释放和链终止子切除的影响。目的3是开发测量细胞内切除活性的方法，并使用包装有野生型或突变型RT的慢病毒载体来评估受感染细胞中调节切除的因素。使用HIV RT的切除反应作为治疗靶点受到我们对细胞内反应缺乏了解的限制，包括影响反应时间和速率的因素，进行二级核糖核酸酶H切割和链终止子切除的酶-底物复合物的稳定性，病毒和细胞因子对这些反应的贡献，以及病毒复制机制对受体底物和抑制分子的可接近性。这些问题将通过拟议的研究加以解决。公共卫生相关性：由于这种病毒的快速突变率促进了耐药性的选择，并且由于需要在每个患者中持续多年治疗，因此仍然需要开发针对HIV的新疗法。研究拟表征影响HIV从一类阻断病毒DNA合成的药物中逃逸的能力的细胞内过程，这将提供对这些药物在不同组织和代谢条件下的效力的深入了解，选择抗性突变体发生的组织部位，以及如何改变治疗策略以优化药物疗效并避免选择抗性突变体。

DESCRIPTION (provided by applicant): Nucleoside reverse transcriptase (RT) inhibitors continue to be an important component of therapy against human immunodeficiency virus type 1 (HIV-1). These compounds are phosphorylated by cellular enzymes and are incorporated into DNA by HIV-1 RT leading to chain termination during viral DNA synthesis. The ability of mutants of HIV-1 RT to remove chain-terminating nucleotides from nascent DNA chains (excision activity) is an important mechanism of drug resistance, and mutants with elevated excision activity are often selected during therapy. We propose to develop an analytical approach to determine what influences excision activity in infected cells that will help us understand what drives mutant selection in vivo. Aim 1 is to use purified wild type and mutant HIV-1 recombinant proteins to evaluate the roles of RNA cleavage by the ribonuclease H activity of RT and template fragment dissociation in regulating excision rescue of blocked DNA chains. Aim 2 is to determine the effect of HIV nucleocapsid proteins on the release of secondary ribonuclease H cleavage fragments and excision of chain terminators. Aim 3 is to develop methods to measure intracellular excision activity and to use lentivirus vectors packaged with wild type or mutant RTs to evaluate factors that regulate excision in infected cells. The use of the excision reaction of HIV RT as a therapeutic target is limited by our lack of understanding of the intracellular reaction, including factors that influence timing and rate of the reaction, stability of the enzyme-substrate complexes that carry out secondary ribonuclease H cleavage and chain terminator excision, contribution of viral and cellular factors to these reactions, and accessibility of the viral replication machinery to acceptor substrates and inhibitory molecules. These questions will be addressed by the proposed studies. PUBLIC HEALTH RELEVANCE: Developoment of new therapies against HIV will continue to be necessary because of the rapid mutation rate of this virus that facilitates selection of drug resistance and because of the need to continue therapy in each patient over many years. Research is proposed to characterize intracellular processes that influence the ability of HIV to escape from a class of drugs that blocks viral DNA synthesis, which will provide insight into the potency of these drugs in different tissues and metabolic conditions, the tissue sites where selection of resistance mutants takes place, and how therapeutic strategies can be changed to optimize drug efficacy and avoid selection of resistant mutants.