Identification of Trim5alpha cofactors

描述(申请人提供)：TRIM5a是一种抗病毒宿主蛋白，其作用机制尚不清楚。恒河猴TRIM5a可以限制HIV-1，但不能限制SIV。遗传和生化证据表明，当病毒进入靶细胞时，TRIM5a直接与病毒衣壳结合。最近有证据表明，TRIM5a与传入衣壳的结合会导致病毒快速脱壳，可能导致逆转录中断。TRIM5的结构表明，它可能作为E3连接酶发挥作用，尽管尚未证实其对泛素化或衣壳降解的作用。这项拟议的项目试图通过确定其抗病毒功能所需的细胞蛋白质来了解TRIM5a发挥作用的机制。TRIM5a抗病毒功能所需的细胞蛋白将通过全基因组筛选和针对人类基因组中大多数表达序列的siRNA文库进行鉴定。该屏幕将检测干扰恒河猴TRIM5a功能的siRNA。作为第二种方法，将对与TRIM5a形成复合体的细胞蛋白质进行生化鉴定。筛选中确定的蛋白质在调节TRIM5a功能中的作用将通过细胞和分子实验进行研究。该项目的具体目标如下：1.通过RNAi筛选鉴定TRIM5a功能所需的基因。2.确定与TRIM5a物理相关的蛋白质。3.确定基因产物在TRIM5a抗病毒功能中的作用。人类细胞含有一种名为TRIM5a的蛋白质，它具有预防感染艾滋病毒的能力。TRIM5a被认为是在病毒进入细胞时通过直接与细胞结合来攻击它，尽管确切地说这是如何工作的还不清楚。该项目将使用最近开发的一项名为RNAi的技术，该技术允许研究人员选择性地关闭细胞的各种功能，以发现TRIM5a抑制艾滋病毒感染的机制。这一认识可能有助于艾滋病新药的开发。

DESCRIPTION (provided by applicant): TRIM5a is an antiviral host protein the mechanism of which is not yet understood. Rhesus macaque TRIM5a restricts HIV-1 but not SIV. Genetic and biochemical evidence suggest that TRIM5a binds directly to the viral capsid as the virus enters a target cell. Evidence was recently reported suggesting that the binding of TRIM5a to the incoming capsid causes a rapid uncoating of the virus, possibly resulting in a disruption to reverse transcription. The structure of TRIM5 suggests that it may act as an E3 ligase although a role for ubiquitination or capsid degradation has not been demonstrated. The proposed project seeks to derive understanding of the mechanism by which TRIM5a functions by identifying cellular proteins that are required for its antiviral function. Cellular proteins that are required for TRIM5a antiviral function will be identified by a genome-wide screen with an siRNA library that targets most of the expressed sequences in the human genome. The screen will detect siRNAs that interfere with rhesus TRIM5a function. As a secondary approach, cell proteins that form a complex with TRIM5a will be identified biochemically. The role of the proteins identified in the screen in mediating TRIM5a function will be studied by cell and molecular experiments. The specific aims of the project are the following: 1. Identify genes that are required for TRIM5a function by RNAi screening. 2. Identify proteins that physically associate with TRIM5a. 3. Determine the role of the gene products in TRIM5a antiviral function. Human cells contain a protein called TRIM5a that has the ability to prevent infection with HIV. TRIM5a is thought to attack the virus as it enters a cell by binding directly to it, although precisely how this works is not understood. The project will use a recently developed technology termed RNAi which allows researchers to selectively shut-off various functions of the cell, to discover the mechanism by which TRIM5a inhibits HIV infection. This understanding can be useful for the development of new drugs for AIDS.