Functional analysis of mRNA export mediated by HIV-1 Rev

HIV-1 Rev介导的mRNA输出的功能分析

• 批准号: 6821915
• 负责人: Larry R. Gerace
• 金额: $ 21.5万
• 依托单位: SCRIPPS RESEARCH INSTITUTE, THE
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-09-30 至 2008-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6821915
• 关键词: antiviral agents; conformation; drug screening /evaluation; genetic regulatory element; human immunodeficiency virus 1; intracellular transport; messenger RNA; proteomics; transcription factor; virus protein; virus replication

Replication of the human immunodeficiency virus type-1 (HIV-1) in the nucleus requires the virus-encoded regulatory protein Rev. Because of its key role in the viral life cycle, Rev is an attractive therapeutic target for small molecules. Rev binds to a structured RNA element, termed the Rev Response Element (RRE), which is found in the unspliced and singly spliced HIV mRNAs that encode the Gag, Pol and Env proteins. The binding and multimerization of Rev at the RRE recruits the nuclear export receptor Crm1, and as a consequence induces export of the HIV mRNAs to the cytoplasm to allow their translation. It is known that Crm1 and the small GTPase Ran are involved in Rev-mediated viral mRNA export, and a number of other potential export factors have been described as well. However, the composition and assembly of the nuclear export complex based on the Rev/RRE interaction remain poorly understood. The goals of this project are to investigate these issues in detail, using a combination of biochemical and functional approaches. The specific aims are: 1) Proteomic and in vitro binding assays will be used to analyze the nuclear export complex formed on the Rev oligomer associated with the RRE; 2) A permeabilized cell assay will be used to analyze the soluble factors involved in nuclear export of an HIV-derived mRNA containing the RRE; 3) In vivo functional studies will be carried out to analyze potential factors involved in mRNA export mediated by the Rev-RRE complex. The other investigators in this program will study in detail the Rev-RRE interaction and Rev multimerization, and will attempt to identify small molecule inhibitors of these associations. In turn, we will test promising compounds for their effect on HIV mRNA export in cells. This work is expected to lead to a greater understanding of how Rev mediates HIV mRNA export, and could help to identify promising lead compounds for drug development.

人类免疫缺陷病毒1型(HIV-1)在细胞核内的复制需要病毒编码的调节蛋白Rev.由于其在病毒生命周期中的关键作用，Rev是一个有吸引力的小分子治疗靶点。REV与一种称为REV反应元件(RRE)的结构化RNA元件结合，该元件存在于编码Gag、Pol和Env蛋白的未剪接和单剪接的HIV mRNAs中。REV在RRE的结合和多聚体招募核输出受体CRM1，结果诱导HIV mRNAs输出到细胞质，使其能够翻译。已知CRM1和小的GTP酶RAN参与了REV介导的病毒mRNA输出，也描述了其他一些潜在的输出因子。然而，核的组成和组装 基于REV/RRE相互作用的出口复合体仍然知之甚少。这个项目的目标是使用生化和功能相结合的方法来详细研究这些问题。具体目标是：1)蛋白质组学和体外结合分析将用于分析与RRE相关的REV寡聚体上形成的核输出复合体；2)渗透细胞分析将用于分析含有RRE的HIV来源的mRNA核输出的可溶性因素；3)体内功能研究将分析REV-RRE复合体介导的mRNA输出的潜在因素。该项目中的其他研究人员将详细研究REV-RRE相互作用和REV多聚化，并试图识别这些关联的小分子抑制剂。反过来，我们 将测试有希望的化合物对细胞中HIV mRNA输出的影响。这项工作有望使人们更好地理解REV是如何介导HIV mRNA输出的，并可能有助于确定用于药物开发的有前景的先导化合物。