HIV Nucleocapsid Protein Nucleic Acid Chaperone Activity

描述（由申请人提供）：HIV-1核衣壳蛋白（NC）是一种 核酸分子伴侣，其催化核酸重排成 可能是它们最稳定的结构。最 逆转录病毒使用宿主细胞转移RNA（tRNA）来引发负链DNA 合成.选择人tRNA（Lys，3）作为HIV-1逆转录酶（RT）的天然引物。在其众多的伴侣功能，数控已被证明是 以促进退火的18个核苷酸在3？tRNA末端（Lys，3） 与HIV RNA基因组上的互补引物结合位点（PBS）结合。该tRNA 退火步骤是一系列复杂事件的第一步， 导致双链前病毒DNA的合成。该提案基于 观察到特定的分子相互作用发生在 tRNA（Lys，3）引物、HIV RNA基因组和NC。这些互动的细节 以及NC的作用机制。然而，核酸分子伴侣功能不是 很好理解。由人tRNA（Lys，3），RNA基因组，NC， 和其他逆转录病毒逆转录的中间体是有吸引力的 新疗法的目标。因此，我们建议：A）探讨 HIV- 1 NC介导的tRNA引物/RNA模板起始复合物的形成 铽切割试验，体外选择（SELEX），核苷酸类似物 干扰作图（NAIM）实验和动力学测定，B）为了探测 荧光法研究HIV-1 NC介导的负链转移机制 共振能量转移（FRET），和C）探讨HIV-1 NC？S 通过使用单分子DNA和RNA拉伸的核酸伴侣活性 一种光镊装置，

DESCRIPTION (Provided by applicant): The HIV-1 nucleocapsid protein (NC) is a nucleic acid chaperone, which catalyzes the rearrangement of nucleic acids into what is presumably their most thermodynamically stable structure. Most retroviruses use a host cell transfer RNA (tRNA) to prime minus-strand DNA synthesis. Human tRNA (Lys,3) is selected as the natural primer for HIV-1 reverse transcriptase (RT). Among its numerous chaperone functions, NC has been shown to facilitate the annealing of the 18 nucleotides at the 3? end of tRNA (Lys,3) to the complementary primer binding site (PBS) on the HIV RNA genome. This tRNA annealing step is the first step in a complex series of events that ultimately result in the synthesis of double-stranded proviral DNA. This proposal is based on the observation that specific molecular interactions occur between the tRNA (Lys,3) primer, the HIV RNA genome, and NC. The details of these interactions and the mechanism of NC?s nucleic acid chaperone function, however, are not well understood. The complexes formed by human tRNA (Lys,3), the RNA genome, NC, and other intermediates in retroviral reverse transcription are attractive targets for new therapies. Therefore, we propose: A) To probe the mechanism of HIV- 1 NC-mediated tRNA primer/RNA template initiation complex formation by terbium cleavage assays, in vitro selection (SELEX), nucleotide analog interference mapping (NAIM) experiments, and kinetic assays, B) To probe the mechanism of minus-strand transfer mediated by HIV-1 NC using fluorescence resonance energy transfer (FRET), and C) To probe the mechanism of HIV-1 NC?s nucleic acid chaperone activity by single molecule DNA and RNA stretching using an optical tweezers apparatus.