Biochemical Mechanisms of Drug Resistance in HIV RT

说明(申请人提供)：核苷逆转录酶(RT)抑制剂仍然是人类免疫缺陷病毒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-1RT结合到DNA中，导致在病毒DNA合成过程中链终止。HIV-1RT的突变株从新生的DNA链上去除末端核苷酸的能力(切除活性)是耐药的一个重要机制，在治疗过程中经常选择具有高切除活性的突变株。我们建议开发一种分析方法来确定是什么影响受感染细胞的切除活性，这将帮助我们了解是什么驱动了体内的突变选择。目的1利用纯化的野生型和突变型HIV-1重组蛋白，评价RT核糖核酸酶H活性的RNA切割和模板片段解离在调节被阻断的DNA链的切除挽救中的作用。目的2确定HIV核衣壳蛋白对次级核糖核酸酶H裂解片段的释放和链终止子的切除的影响。目的3是建立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RTS的慢病毒载体来评估感染细胞中调节切除的因素。HIV RT的切除反应作为治疗靶点的使用受到限制，因为我们缺乏对细胞内反应的了解，包括影响反应的时间和速度的因素，进行次级核糖核酸酶H裂解和链终止子切除的酶-底物复合体的稳定性，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及性。这些问题将在拟议的研究中得到解决。与公共卫生相关：由于这种病毒的快速变异率有助于选择抗药性，而且需要对每个患者进行多年持续治疗，因此仍有必要开发针对艾滋病毒的新疗法。有人提议进行一项研究，以表征影响HIV从阻止病毒DNA合成的一类药物中逃脱的细胞内过程，这将提供对这些药物在不同组织和代谢条件下的效力、发生耐药突变选择的组织部位以及如何改变治疗策略以优化药物疗效和避免选择耐药突变的洞察。

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.