HIV Nucleocapsid Protein Nucleic Acid Chaperone Activity

描述(申请人提供)：HIV-1核衣壳蛋白(NC)是一种 核酸伴侣，它催化核酸重排成 大概是它们在热力学上最稳定的结构。多数 逆转录病毒使用宿主细胞转移rna(TRNA)来启动负链dna。 综合。人tRNA(Lys，3)被选为HIV-1逆转录酶(RT)的天然引物。在其众多的陪伴功能中，NC已经显示出来 以促进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。这个tRNA 退火步是一系列复杂事件的第一步，最终 导致双链前病毒DNA的合成。这项建议是基于 关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 然而，NC？S的核酸伴侣功能的作用机制尚不清楚 很好理解。由人tRNA(Lys，3)、RNA基因组，NC， 和其他逆转录病毒的中间产物很有吸引力。 新疗法的靶点。因此，我们建议：a)探讨其发病机制。 HIV-1NC介导的tRNA引物/RNA模板起始复合体的形成 Tb切割分析，体外选择(SELEX)，核苷酸类似物 干扰图谱(NAIM)实验和动力学分析，B)以探索 用荧光技术研究HIV-1 NC介导的负链转移机制 共振能量转移(FRET)，以及C)探索HIV-1 NC的机制？S 单分子DNA和RNA拉伸对核酸伴侣活性的影响 一种光学镊子装置。

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.