Mechanism of recombination by HIV reverse transcriptase

描述（由申请人提供）：在过去二十年中，人类免疫缺陷病毒（HIV）已导致数百万人死亡。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一直受到阻碍。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。本提案的目标是回答有关重组机制和NC在该过程中的作用的关键问题。这将通过研究四个具体目标来实现：(1)探索NC蛋白的两个锌指在链转移中的潜在不同作用；(2)确定受体（dna在RNA供体上合成后转移到的RNA）结构在链转移机制中的作用；(3)设计和分析能够产生超长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，以确定细胞中DNA合成暂停的频率和位置。这些实验将采用体内和体外相结合的方法。例如，为目标1产生的突变NC蛋白将在体外试验中进行分析，也可以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提出的实验可以提供RT在细胞感染期间如何穿越病毒基因组的一瞥。目前，对这一过程的唯一了解来自体外分析。总的来说，拟议的实验应该有助于澄清一些重要的未解之谜，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，更好地了解NC蛋白的机制如何可能导致干扰NC的特定药物。此外，了解重组发生的机制可以设计针对该过程的特定抑制剂。

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.