MECHANISM OF RECOMBINATION BY HIV REVERSE TRANSCRIPTASE

HIV逆转录酶的重组机制

• 批准号: 6342897
• 负责人: JEFFREY J DESTEFANO
• 金额: $ 19.27万
• 依托单位: UNIV OF MARYLAND, COLLEGE PARK
• 依托单位国家: 美国
• 财政年份: 1994
• 资助国家: 美国
• 起止时间: 1994-05-01 至 2003-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6342897
• 关键词: DNA directed DNA polymerase; DNA directed RNA polymerase; RNA directed DNA polymerase; active sites; chemical association; enzyme activity; enzyme inhibitors; enzyme mechanism; enzyme substrate; genetic recombination; human immunodeficiency virus; mutant; nucleic acid biosynthesis; nucleic acid structure; nucleocapsid; nucleoproteins; polymerase chain reaction; protein protein interaction; site directed mutagenesis; virus genetics; virus protein; virus replication

The human immunodeficiency virus (HIV), the Causative agent of Acquired Immune Deficiency Syndrome (AIDS), has been responsible for the deaths of millions of people in the last two decades. Attempts to combat HIV have been hampered due to the viruses ability to rapidly mutate and produce genetic variants that can circumvent the immune response and resist drug therapy. Recombination, which occurs by a process referred to as strand transfer, is an important mechanisms used by HIV to increase diversity. Two viral proteins, reverse transcriptase (RT) and nucleocapsid (NC) have been clearly implicated in recombination. The goal of this proposal is to answer key questions regarding the mechanism of recombination and the interaction of RT with nucleic acids. This will be accomplished by investigating 4 specific aims related directly to recombination: (1) Analysis of strand transfer "hotspots" that promote recombination events, (2) Defining the role of RT-directed base misincorporations in inducing recOmbination (strand transfer) events, (3) Analysis of RT binding and cleavage on structures mimicking replication or strand transfer intermediates, and (4) Probing the mechanism by which NC catalyzes strand-exchange to promote strand transfer. The goal of the first aim is to determine why particular sites promote transfer while others do not. A potential link between two of the major mechanisms for the generation of HIV mutants will be explored in the second aim while specific interactions between key viral proteins and unique strand transfer intermediates are studied in aims 3 and 4. The basic interaction of RT with nucleic acids will be investigated through 3 specific aims: (1) Quantitative assessment of the interaction of RT with nucleic acid substrates under physiological conditions, (2) Determining the role of different regions of RT in binding to nucleic acid using chimeric substrates, and (3) Searching for nucleic acids sequences that can bind RT with high affinity. The basic goal of these studies is to define RT and nucleic acid properties importantJor 'tight" binding between the two. Overall, the proposed experiments should help clarify some of the important unanswered questions and could also be important in developing and evaluating strategies to combat HIV. For example, an understanding of the interplay between RT fidelity and recombination could allow for better predictive models that help determine how therapy will impact HIV evolution. Perhaps drug combinations that tend to lead to a more or less accurate RT would be advantageous in the long run.

人类免疫缺陷病毒（HIV）是获得性免疫缺陷综合症（AIDS）的病原体，在过去二十年中已导致数百万人死亡。由于病毒能够快速突变并产生可以规避免疫反应并抵抗药物治疗的基因变异，因此对抗艾滋病毒的尝试受到了阻碍。重组是通过链转移过程发生的，是 HIV 用于增加多样性的重要机制。两种病毒蛋白，逆转录酶（RT）和核衣壳（NC）明显与重组有关。该提案的目标是回答有关重组机制以及 RT 与核酸相互作用的关键问题。这将通过研究与重组直接相关的 4 个具体目标来实现：(1) 分析促进重组事件的链转移“热点”，(2) 定义 RT 导向的碱基错误掺入在诱导重组（链转移）事件中的作用， （3）模拟复制或链转移中间体结构上的RT结合和切割分析，以及（4）探讨NC催化链交换促进链的机制 转移。第一个目标是确定为什么特定站点促进传输而其他站点不促进传输。第二个目标将探讨产生 HIV 突变体的两种主要机制之间的潜在联系，而目标 3 和 4 将研究关键病毒蛋白和独特链转移中间体之间的特定相互作用。 RT 与核酸的基本相互作用将通过 3 个具体目标进行研究：(1) 定量评估生理条件下 RT 与核酸底物的相互作用，(2) 使用嵌合底物确定 RT 不同区域在与核酸结合中的作用，以及 (3)寻找核酸可以高亲和力结合 RT 的序列。这些研究的基本目标是定义 RT 和核酸特性（重要的是两者之间的“紧密”结合）。总体而言，拟议的实验应有助于澄清一些重要的未解答的问题，并且对于制定和评估对抗 HIV 的策略也很重要例如，了解 RT 保真度和重组之间的相互作用可以建立更好的预测模型，帮助确定治疗将如何影响 HIV 的进化，从长远来看，可能导致或多或少准确的 RT 的药物组合将是有利的。跑步。