Mechanism of recombination by HIV reverse transcriptase

描述(申请人提供)：人类免疫缺陷病毒(HIV)在过去20年里造成了数百万人的死亡。抗击艾滋病毒的努力一直受到阻碍，因为该病毒能够迅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。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。这项提案的目标是回答有关重组机制和NC在这一进程中的作用的关键问题。这将通过研究四个特定的目标来实现：(1)探测NC蛋白质的两个锌指在链转移中的潜在不同作用；(2)确定受体(在RNA供体上合成的DNA被转移到的RNA)的结构在链转移机制中的作用；(3)设计和分析能够产生非常长的DNA合成产物的体外系统；(4)分析体内产生的HIV DNA合成产物的性质，以确定DNA合成在细胞中暂停的频率和位置。这些实验将采用体外和体内相结合的方法。例如，为AIM 1产生的突变NC蛋白将在体外分析，并在培养细胞感染期间在病毒基因组的背景下进行分析。Aim 4提出了一些实验，可以让人们一瞥RT在细胞感染过程中是如何穿越病毒基因组的。目前，对这一过程的唯一了解来自于体外分析。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战略方面发挥重要作用。例如，更好地理解NC蛋白的机制如何导致干扰NC的特定药物。此外，了解重组发生的机制(S)可能有助于设计这一过程的特定抑制剂。

DESCRIPTION (provided by applicant): Human immunodeficiency virus (HIV) 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 virus's 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 role of NC in the process. This will be accomplished by investigating four specific aims: (1) Probing the potentially different roles of the two zinc fingers of NC protein in strand transfer; (2) Determining the roles of the structure of the acceptor (RNA to which DNAs synthesized on the RNA donor transfer to) in the mechanism of strand transfer; (3) Designing and analyzing in vitro systems capable of producing very long DNA synthesis products; (4) Analysis of the nature of HIV DNA synthesis products produced in vivo to determine how frequently and at what locations DNA synthesis pauses in the cell. A combination of in vitro and in vivo approaches will be used for these experiments. For example, mutant NC proteins produced for aim 1 will be analyzed in in vitro assays and also in the context of the viral genome during infection of culture cells. Aim 4 proposes experiments that could provide a glimpse of how RT traverses the viral genome during cellular infections. Currently, the only knowledge of this process comes from in vitro analysis. Overall, the proposed experiments should help clarify some important unanswered questions and could also be important in developing and evaluating strategies to combat HIV. For example, a better understanding of how the mechanism of NC proteins could lead to specific drugs that interfere with NC. Also, understanding the mechanism(s) by which recombination occurs may allow the design of specific inhibitors to this process.