MECHANISM OF RECOMBINATION BY HIV REVERSE TRANSCRIPTASE

人类免疫缺陷病毒（艾滋病毒）是获得性免疫缺陷综合症（艾滋病）的病原体，在过去二十年中造成了数百万人的死亡。由于这种病毒能够迅速变异并产生能够规避免疫反应和抵抗药物治疗的遗传变异，抗击艾滋病毒的努力受到了阻碍。重组发生在一个称为链转移的过程中，是HIV用来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）已明确参与重组。本提案的目的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个具体目标来完成：(1)分析促进重组事件的链转移“热点”，(2)定义RT导向的碱基错结合在诱导重组（链转移）事件中的作用，(3)分析RT结合和切割模拟复制或链转移中间体的结构，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网站促进迁移，而其他网站却没有。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和目标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)生理条件下RT与核酸底物的相互作用的定量评估；(2)利用嵌合底物确定RT不同区域与核酸结合的作用；(3)寻找能够高亲和力结合RT的核酸序列。这些研究的基本目标是确定RT和核酸的重要性质，即两者之间的“紧密”结合。总的来说，拟议的实验应该有助于澄清一些重要的未解决的问题，并且在制定和评估抗击艾滋病毒的战略方面也可能很重要。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，帮助确定治疗如何影响HIV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。

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.