Single-Molecule Studies of Host Factor Function in HIV-1 Rev-RRE Complex Assembly

HIV-1 Rev-RRE 复合物组装中宿主因子功能的单分子研究

• 批准号: 7623274
• 负责人: Rae Marie Robertson-Anderson
• 金额: $ 3.41万
• 依托单位: SCRIPPS RESEARCH INSTITUTE, THE
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-01-01 至 2009-08-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7623274
• 关键词: Acquired Immunodeficiency Syndrome; Advanced Development; Back; Binding; Biochemical; Cell Nucleus; Cells; Color; Complex; Cytoplasm; Dissociation; Ensure; Epidemic; Fluorescence; Fluorescence Microscopy; Fluorescent Dyes; Goals; HIV-1; Image; Imaging Techniques; Integration Host Factors; Kinetics; Label; Mediating; Messenger RNA; Methods; Monitor; Nuclear; Nuclear Export; Nuclear Pore Complex; Pathway interactions; Play; Production; Proteins; RNA; RNA Splicing; Recruitment Activity; Research; Response Elements; Ribonucleoproteins; Role; Staging; Structural Protein; Structure; Techniques; Time; Translations; Viral; Viral Packaging; Viral Proteins; Virion; Virus; base; cofactor; fight against; fluorescence imaging; genetic regulatory protein; mRNA Export; monomer; novel; public health relevance; single molecule; stoichiometry; therapeutic target; trafficking; viral RNA

DESCRIPTION (provided by applicant): The proposed research will consist of biophysical studies that probe the interactions between the HIV-1 regulatory protein, Rev, and the viral RNA structure, the Rev Response Element (RRE). The goal of the proposed research is to elucidate the kinetic pathway of Rev, which activates nuclear export of unspliced and partially spliced RNAs, and thus plays an essential role in HIV-1 replication. Novel single-molecule fluorescence imaging techniques will be developed and utilized to dissect the assembly and disassembly pathways of the Rev-RRE complex, and to illuminate the role of various host cell factors in assembly and dissociation. Specifically, total internal reflection fluorescence microscopy and two-color colocalization imaging of fluorescently-labeled Rev monomers and host factors will be employed to achieve the stated goals. Due to the complex kinetic pathway of Rev, including features such as Rev oligomerization, Rev- RRE complex formation and dissociation, and direct and mediated interactions with host cell factors, traditional bulk biochemical techniques have failed to provide a detailed picture of the Rev and Rev-RRE kinetic pathways. Therefore, while Rev is a promising therapeutic target, due to its essential role in HIV-1 replication, effective Rev-based therapies have yet to be developed. This research will directly probe the interactions between Rev, the RRE, and various host factors on a single-molecule level, and thus will be able to define detailed kinetic pathway(s) associated with each step in the Rev cycle, as well as determine the stoichiometries of Rev, RRE, and host factors at each step. Consequently, this research will answer many unsolved questions regarding the Rev kinetic pathway and be able to advance progress towards the implementation of HIV-1 therapies that target Rev. PUBLIC HEALTH RELEVANCE: The HIV-1 regulatory protein, Rev, is essential to HIV-1 replication, and thus it is a promising therapeutic target in the fight against the worldwide AIDS epidemic. However, due to the complex kinetic pathway of Rev, therapies that target Rev have yet to be actualized. This research will elucidate many details about Rev by directly probing the interactions between Rev, RNA and various host cell proteins on a single- molecule level, and thus will be able to advance the development of Rev-based HIV-1 therapies.

描述(申请人提供)：拟议的研究将包括生物物理研究，探索HIV-1调节蛋白REV和病毒RNA结构REV反应元件(RRE)之间的相互作用。这项研究的目的是阐明REV的动力学途径，REV激活未剪接和部分剪接的RNA的核输出，从而在HIV-1复制中发挥重要作用。新的单分子荧光成像技术将被开发和利用来剖析REV-RRE复合体的组装和分解途径，并阐明各种宿主细胞因子在组装和解离中的作用。具体地说，将使用全内反射荧光显微镜和荧光标记的REV单体和宿主因子的双色共聚焦成像来实现所述目标。由于REV的复杂动力学途径，包括REV齐聚、REV-RRE复合体的形成和解离以及与宿主细胞因子的直接和中介相互作用，传统的整体生化技术无法提供REV和REV-RRE动力学途径的详细图景。因此，虽然REV是一个很有前途的治疗靶点，但由于其在HIV-1复制中的重要作用，基于REV的有效疗法尚未开发出来。这项研究将直接在单分子水平上探索REV、RRE和各种宿主因子之间的相互作用，从而能够确定REV循环中每一步的详细动力学路径(S)，以及每一步REV、RRE和宿主因子的化学计量比。因此，这项研究将回答许多关于REV动力学途径的悬而未决的问题，并能够推动针对REV公共卫生相关性的HIV-1疗法的实施进展：HIV-1调节蛋白REV是HIV-1复制所必需的，因此它是抗击全球艾滋病流行的一个有希望的治疗靶点。然而，由于REV的复杂动力学途径，针对REV的治疗尚未实现。本研究将通过在单分子水平上直接探测REV、RNA与多种宿主细胞蛋白之间的相互作用来阐明REV的许多细节，从而能够推动基于REV的HIV-1治疗的发展。