Mechanism of recombination by HIV reverse transcriptase

HIV逆转录酶重组机制

• 批准号: 7028364
• 负责人: JEFFREY J DESTEFANO
• 金额: $ 25.38万
• 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 2008-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7028364
• 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; genetic recombination; human immunodeficiency virus; mutant; nucleic acid biosynthesis; nucleic acid chemical synthesis; nucleic acid structure; nucleocapsid; polymerase chain reaction; protein protein interaction; protein structure; site directed mutagenesis; virus genetics; virus protein; virus replication

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.

描述（由申请人提供）：在过去的二十年中，人类免疫缺陷病毒（HIV）已导致数百万人死亡。由于病毒快速突变和产生可以避免免疫反应并抵抗药物治疗的遗传变异的能力，试图对抗HIV的尝试受到阻碍。重组是通过称为链转移的过程发生的，是艾滋病毒用于增加多样性的重要机制。两种病毒蛋白，逆转录酶（RT）和核素（NC）已明显与重组有关。该提案的目的是回答有关重组机制以及NC在此过程中的作用的关键问题。这将通过研究四个特定目的来实现：（1）探测NC蛋白在链转移中的两个锌手指的潜在不同作用； （2）确定受体结构的作用（DNA在RNA供体转移到链转移机理上合成的RNA的作用； （3）设计和分析能够生产非常长的DNA合成产物的体外系统； （4）分析在体内产生的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。这些实验将使用体外和体内方法的组合。例如，将在体外测定中以及在培养细胞感染期间的病毒基因组中分析用于AIM 1的突变NC蛋白。 AIM 4提出了可以瞥见RT在细胞感染过程中如何遍历病毒基因组的实验。目前，此过程的唯一知识来自体外分析。总体而言，拟议的实验应有助于阐明一些重要的未解决问题，并且对于制定和评估打击HIV的策略也可能很重要。例如，更好地了解NC蛋白的机制如何导致干扰NC的特定药物。同样，了解重组发生的机制可能允许设计特定的抑制剂。