Biochemical Mechanisms of Drug Resistance in HIV RT

HIV RT耐药的生化机制

基本信息

批准号：
7760783
负责人：
WALTER A SCOTT
金额：
$ 38.25万
依托单位：
UNIVERSITY OF MIAMI SCHOOL OF MEDICINE
依托单位国家：
美国
项目类别：
财政年份：
1997
资助国家：
美国
起止时间：
1997-01-15 至 2011-08-31
项目状态：
已结题

项目摘要

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.

描述（由申请人提供）：核苷逆转录酶（RT）抑制剂仍然是针对人类免疫缺陷病毒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，从而在病毒DNA合成过程中导致链终止。 HIV-1 RT突变体从新生DNA链中去除链末端核苷酸的能力（切除活性）是耐药性的重要机制，在治疗过程中通常会选择具有升高切除活性的突变体。我们建议开发一种分析方法，以确定受感染细胞中切除活性的影响，这将有助于我们了解驱动体内突变体选择的原因。 AIM 1是使用纯化的野生型和突变的HIV-1重组蛋白来评估RT的核糖核酸酶H活性和模板片段分离在调节封闭的DNA链的切除中的RNA裂解作用。目的2是确定HIV核素蛋白对次级核糖核酸酶H裂解片段的释放和链终止剂的切除的影响。目的3是开发测量细胞内切除活性并使用包装野生型或突变体RT包装的慢病毒载体来评估调节感染细胞切除的因素的方法。通过缺乏对细胞内反应的了解，使用HIV RT作为治疗靶靶的切除反应的使用受到限制抑制分子。这些问题将由拟议的研究解决。公共卫生相关性：针对HIV的新疗法的发展将是必要的，因为该病毒的快速突变率促进了耐药性的选择，并且由于需要在每位患者多年内继续治疗。提出了研究来表征细胞内过程，这些过程会影响HIV摆脱阻断病毒DNA合成的一类药物的能力，这将为这些药物在不同组织和代谢条件下的效力提供深入了解，这些组织的效力，抗药性突变体选择的组织部位，以及如何改变治疗策略，以避免使用抗药性抗药性，以避免使用抗药性突变剂。