MECHANISM OF RECOMBINATION BY HIV REVERSE TRANSCRIPTASE

人类免疫缺陷病毒(HIV)是获得性免疫缺陷综合征(AIDS)的病原体，在过去20年里造成了数百万人的死亡。由于病毒能够快速变异并产生基因变异，从而绕过免疫反应并抵抗药物治疗，抗击艾滋病毒的努力受到了阻碍。重组是艾滋病毒用来增加多样性的一种重要机制，它通过一种称为链转移的过程发生。两种病毒蛋白，逆转录酶(RT)和核衣壳蛋白(NC)已清楚地与重组有关。这项提议的目标是回答有关重组机制和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个特定目标来实现：(1)分析促进重组事件的链转移“热点”，(2)确定RT-定向碱基错结合在诱导重组(链转移)事件中的作用，(3)分析模拟复制或链转移中间产物的结构上的RT结合和切割，以及(4)探索NC催化链交换促进链转移的机制。第一个目标的目标是确定为什么某些站点会促进传输，而其他站点则不会。第二个目标将探讨HIV突变产生的两个主要机制之间的潜在联系，而AIMS 3和4将研究关键病毒蛋白和独特的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三个特定目标来研究：(1)定量评估在生理条件下RT与核酸底物的相互作用；(2)利用嵌合底物确定RT的不同区域在与核酸结合中的作用；(3)寻找与RT具有高亲和力的核酸序列。这些研究的基本目标是定义RT和核酸的重要性质，它们之间的紧密联系。总体而言，拟议的实验应该有助于澄清一些重要的悬而未决的问题，并可能在制定和评估抗击艾滋病毒的策略时发挥重要作用。例如，了解RT保真度和重组之间的相互作用可以帮助建立更好的预测模型，帮助确定治疗将如何影响HIV的进化。也许从长远来看，倾向于导致或多或少准确的RT的药物组合将是有利的。

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.