Identification of Trim5alpha cofactors

描述（由申请人提供）：TRIM 5a是一种抗病毒宿主蛋白，其机制尚不清楚。恒河猴TRIM 5a限制HIV-1，但不限制SIV。遗传和生物化学证据表明，TRIM 5a在病毒进入靶细胞时直接与病毒衣壳结合。最近报道的证据表明，TRIM 5a与进入的衣壳的结合导致病毒的快速脱壳，可能导致逆转录的破坏。TRIM 5的结构表明，它可能作为一个E3连接酶，尽管泛素化或衣壳降解的作用尚未得到证实。该拟议项目旨在通过识别其抗病毒功能所需的细胞蛋白质来了解TRIM 5a的作用机制。TRIM 5a抗病毒功能所需的细胞蛋白将通过利用靶向人类基因组中大部分表达序列的siRNA文库进行全基因组筛选来鉴定。该筛选将检测干扰恒河猴TRIM 5a功能的siRNA。作为第二种方法，与TRIM 5a形成复合物的细胞蛋白将被生物化学鉴定。将通过细胞和分子实验研究在筛选中鉴定的蛋白质在介导TRIM 5a功能中的作用。该项目的具体目标如下：1.通过RNAi筛选鉴定TRIM 5a功能所需的基因。2.识别与TRIM 5a物理相关的蛋白质。3.确定基因产物在TRIM 5a抗病毒功能中的作用。人类细胞中含有一种名为TRIM 5a的蛋白质，它具有预防艾滋病毒感染的能力。TRIM 5a被认为在病毒进入细胞时通过直接结合病毒来攻击病毒，尽管还不清楚它是如何工作的。该项目将使用最近开发的称为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TRIM 5a抑制HIV感染的机制。这种理解可能有助于开发治疗艾滋病的新药。

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.