Structure and Function of HIV-1 Reverse Transcriptase

HIV-1逆转录酶的结构和功能

• 批准号: 6952066
• 负责人: stephen h hughes
• 金额: --
• 依托单位: DIVISION OF BASIC SCIENCES - NCI
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/6952066
• 关键词: RNA directed DNA polymerase; X ray crystallography; antiAIDS agent; enzyme inhibitors; enzyme structure; human immunodeficiency virus 1; multidrug resistance; murine leukemia virus; nucleoside analog; protein structure function; recombinant proteins

HIV-1 is the causative agent of AIDS. Three viral enzymes - RT, integrase (IN), and protease (PR) - have essential roles in the replication of HIV-1. We are engaged in a long-term effort to study HIV-1 RT, with the expectation that this information will be useful in the development of more effective anti-RT drugs. Our strategy has involved the synthesis, using recombinant DNA techniques, of both wild-type and mutant HIV-1 RTs, including drug-resistant mutants. Some of this purified RT has been used by our long-term collaborator, Edward Arnold (Rutgers University), for structural studies. We have used purified HIV-1 RT to study the biochemical properties of RT mutants, including drug-resistant mutants, and in some cases, have compared the properties of HIV-1 RT to other retroviral RTs, primarily murine leukemia virus (MLV) RT. A major focus of our recent work on HIV-1 RT is the mechanism(s) of nucleoside RT inhibitor (NRTI) resistance. NRTIs are widely used to treat HIV-1 infections; however, there are serious problems with drug toxicity and with the development of resistance. Two of the commonly used NRTIs, 3TC and AZT, have elements ("handles") that project beyond the corresponding positions of normal dNTPs. 3TC- and AZT-resistant HIV-1 RTs take advantage of these handles and use steric hindrance to block the incorporation of 3TC and to enhance the excision of AZT. Unfortunately, there are HIV-1 RTs that are resistant to NRTIs that do not have obvious handles. We plan to continue to investigate the mechanisms that underlie drug resistance - in particular, we want to understand the mechanisms that underlie resistance to multiple NRTIs. We will ask whether the newly approved drug, tenofovir, will be efficiently excised by RTs that can excise a number of NRTIs. We have started a collaboration with a skilled nucleoside chemist, Victor Marquez (Laboratory of Medicinal Chemistry, NCI); we will ask if it is possible to develop a class of nucleoside analogs that are relatively resistant to excision by HIV-1 RTs that can excise multiple NRTIs. All of these experiments involve a combined structural/biochemical approach; what we have learned from RT structure has been an invaluable guide for planning the biochemical studies, while the results and hypothesis generated in the biochemical experiments have inspired new rounds of structural experiments. NRTI resistance is only one aspect of the behavior of RT. We also want to understand how RT carries out reverse transcription in an infected cell, and to correlate the wealth of structural and biochemical data on HIV-1 RT with the actual process of reverse transcription. These experiments are part of Project Z01 CCR 010482 (Retroviral Replication and Vector Design). In some cases, it will not be possible to explain the in vivo data with the available structural and biochemical results. In such cases, we will do additional biochemical and structural experiments to complement (and better understand) the in vivo results.

HIV-1是艾滋病的病因。三种病毒酶 - RT，整合酶（IN）和蛋白酶（PR） - 在HIV -1的复制中具有重要作用。我们从事长期研究HIV-1 RT的努力，期望此信息可用于开发更有效的抗RT药物。我们的策略涉及使用重组DNA技术的野生型和突变型HIV-1 RT（包括耐药突变体）的合成。我们的长期合作者爱德华·阿诺德（Rutgers University）使用了其中一些纯化的RT进行结构研究。我们已经使用纯化的HIV-1 RT来研究RT突变体的生化特性，包括抗药性突变体，在某些情况下，将HIV-1 RT的性质与其他逆转录病毒RT进行了比较，主要是鼠白血病病毒（MLV）RT。我们最近在HIV-1 RT工作的主要重点是核苷RT抑制剂（NRTI）抗性的机制。 NRTI被广泛用于治疗HIV-1感染；但是，药物毒性和抗药性发展存在严重问题。两个常用的NRTI，3TC和AZT具有超出正常DNTP相应位置的元素（“ handles”）。 3TC和抗AZT的HIV-1 RTS利用这些手柄，并使用空间障碍阻止3TC的掺入并增强AZT的切除。不幸的是，有一些对NRTI具有抗药性的HIV-1 RT没有明显的手柄。 我们计划继续研究抗药性基础的机制 - 特别是我们要了解对多个NRTI的抗性的机制。我们将询问新批准的药物Tenofovir是否会被RTS有效切除，可以切除许多NRTI。我们已经与熟练的核苷化学家Victor Marquez（NCI药物化学实验室）合作；我们将询问是否有可能开发一类核苷类似物，这些核苷类似物相对抗HIV-1 RTs的抗性，可以切除多个NRTI。所有这些实验都涉及结构/生化方法。我们从RT结构中学到的是计划生化研究的宝贵指南，而生化实验中产生的结果和假设激发了新的结构实验。 NRTI抗性只是RT行为的一个方面。我们还想了解RT如何在受感染的细胞中进行逆转录，并将HIV-1 RT上的结构和生化数据的财富与逆转录的实际过程相关联。这些实验是项目Z01 CCR 010482（逆转录病毒复制和矢量设计）的一部分。在某些情况下，不可能用可用的结构和生化结果来解释体内数据。在这种情况下，我们将进行其他生化和结构实验，以补充体内结果（并更好地理解）。