Mechanism of recombination by HIV reverse transcriptase

HIV逆转录酶重组机制

• 批准号: 6799077
• 负责人: JEFFREY J DESTEFANO
• 金额: $ 26.44万
• 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/6799077
• 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）：过去二十年，人类免疫缺陷病毒（HIV）导致数百万人死亡。由于该病毒能够快速突变并产生可以规避免疫反应并抵抗药物治疗的基因变异，因此对抗艾滋病毒的尝试受到了阻碍。重组是通过链转移过程发生的，是 HIV 用于增加多样性的重要机制。两种病毒蛋白，逆转录酶（RT）和核衣壳（NC）明显与重组有关。该提案的目标是回答有关重组机制和 NC 在此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）探讨 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(RNA供体上合成的DNA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很长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DNA合成在细胞中暂停的频率和位置。这些实验将结合体外和体内方法。例如，为目标 1 产生的突变 NC 蛋白将在体外测定中以及在培养细胞感染期间的病毒基因组背景下进行分析。目标 4 提出的实验可以让我们了解 RT 在细胞感染过程中如何穿越病毒基因组。目前，对该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答的问题，并且对于制定和评估对抗艾滋病毒的策略也很重要。例如，更好地了解 NC 蛋白的机制如何导致开发出干扰 NC 的特定药物。此外，了解重组发生的机制可能有助于设计针对该过程的特定抑制剂。