Biochemical Mechanisms of Drug Resistance in HIV RT

HIV RT耐药的生化机制

• 批准号: 8317534
• 负责人: Yanbin Zhang
• 金额: $ 37.69万
• 依托单位: UNIVERSITY OF MIAMI SCHOOL OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 1997
• 资助国家: 美国
• 起止时间: 1997-01-15 至 2014-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8317534
• 关键词: 3' -azido-3' -deoxythymidine 5' phosphate; Active Sites; Address; Affect; Binding; Biochemical; Cells; DNA; DNA biosynthesis; Development; Dissociation; Drug resistance; Effectiveness; Enzymes; Excision; HIV; HIV-1; Health; In Vitro; Laboratories; Length; Lentivirus Vector; Life; Measures; Metabolic; Methods; Mutation; N-terminal; Nucleocapsid Proteins; Nucleosides; Nucleotides; Patients; Pharmaceutical Preparations; Positioning Attribute; Process; RNA; RNA-Directed DNA Polymerase; Reaction; Recombinant Proteins; Research; Resistance; Reverse Transcriptase Inhibitors; Ribonuclease H; Role; Site; Therapeutic; Thymidine; Time; Tissues; Viral; Virus; Work; Zidovudine; analog; drug discovery; drug efficacy; enzyme substrate complex; improved; in vivo; insight; mutant; nucleotide analog; public health relevance; reaction rate; research study; therapeutic target; viral DNA

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.

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。

项目成果

A role of template cleavage in reduced excision of chain-terminating nucleotides by human immunodeficiency virus type 1 reverse transcriptase containing the M184V mutation.

模板切割在减少含有 M184V 突变的人类免疫缺陷病毒 1 型逆转录酶切除链终止核苷酸中的作用。

• DOI: 10.1128/jvi.05767-11
• 发表时间: 2012
• 期刊: Journal of virology
• 影响因子: 5.4
• 作者: Acosta-Hoyos,AntonioJ;Matsuura,SuzanneE;Meyer,PeterR;Scott,WalterA
• 通讯作者: Scott,WalterA

Effects of specific zidovudine resistance mutations and substrate structure on nucleotide-dependent primer unblocking by human immunodeficiency virus type 1 reverse transcriptase.

特定齐多夫定抗性突变和底物结构对人类免疫缺陷病毒 1 型逆转录酶解封闭核苷酸依赖性引物的影响。

• DOI: 10.1128/aac.46.5.1540-1545.2002
• 发表时间: 2002
• 期刊: Antimicrobial agents and chemotherapy
• 影响因子: 4.9
• 作者: Meyer,PeterR;Matsuura,SuzanneE;Tolun,AdviyeA;Pfeifer,Ingrid;So,AnteroG;Mellors,JohnW;Scott,WalterA
• 通讯作者: Scott,WalterA

Intracellular substrates for the primer-unblocking reaction by human immunodeficiency virus type 1 reverse transcriptase: detection and quantitation in extracts from quiescent- and activated-lymphocyte subpopulations.

人类免疫缺陷病毒 1 型逆转录酶进行引物解封闭反应的细胞内底物：静态和活化淋巴细胞亚群提取物的检测和定量。

• DOI: 10.1128/aac.49.5.1761-1769.2005
• 发表时间: 2005
• 期刊: Antimicrobial agents and chemotherapy
• 影响因子: 4.9
• 作者: Smith,AnthonyJ;Meyer,PeterR;Asthana,Deshratn;Ashman,MargaritaR;Scott,WalterA
• 通讯作者: Scott,WalterA

Structures of reverse transcriptase pre- and post-excision complexes shed new light on HIV-1 AZT resistance.

• DOI: 10.3390/v3010020
• 发表时间: 2011-01
• 期刊: Viruses
• 作者: Scott WA

Differential removal of thymidine nucleotide analogues from blocked DNA chains by human immunodeficiency virus reverse transcriptase in the presence of physiological concentrations of 2'-deoxynucleoside triphosphates.

在生理浓度的 2-脱氧核苷三磷酸存在下，通过人免疫缺陷病毒逆转录酶从封闭的 DNA 链中差异性去除胸苷核苷酸类似物。

• DOI: 10.1128/aac.44.12.3465-3472.2000
• 发表时间: 2000
• 期刊: Antimicrobial agents and chemotherapy
• 影响因子: 4.9
• 作者: Meyer,PR;Matsuura,SE;Schinazi,RF;So,AG;Scott,WA
• 通讯作者: Scott,WA