Identification of cyclophilin A-dependent HIV-1 restriction factors

描述（由申请人提供）：HIV-1复制需要宿主细胞蛋白亲环蛋白A （CypA）与病毒衣壳结合。CypA- ca相互作用的遗传或药理学抑制导致人类T细胞中HIV-1复制受损，但CypA促进HIV-1感染的机制尚不清楚。最近的研究表明，CypA在靶细胞中保护进入的核心免受宿主的内在限制，但对CypA的需求独立于最近发现的宿主限制因子TRIMSa。靶细胞依赖CypA需求的证据存在于能够复制的HIV-1突变体中。环孢素A （CsA）的存在这些突变体在病毒CA蛋白的cypa结合环中含有替换。在一些（但不是全部）人类细胞系中，这些HIV-1突变体的复制实际上需要CsA的存在。我们假设未知的人类细胞蛋白决定了CypA与HIV-1衣壳结合对病毒感染的影响。在这个R21应用程序中，我们提出了实验来验证这一假设并确定相关的宿主因子。在Specific Aim 1中，我们将通过研究HIV-1感染中对CypA-CA相互作用需求不同的细胞产生的异核体，来确定细胞特异性表型是否与显性限制因子的表达一致。在Specific Aim 2中，我们将通过结合候选基因分析和筛选特定cDNA文库来确定相关因素。这些研究将有助于阐明CypA促进HIV-1复制的机制，并可能揭示治疗HIV感染的新策略。

DESCRIPTION (provided by applicant): HIV-1 replication requires binding of the host cell protein cyclophilin A (CypA) to the viral capsid. Genetic or pharmacologic inhibition of the CypA-CA interaction results in impaired HIV-1 replication in human T cells, yet the mechanism by which CypA promotes HIV-1 infection is unknown. Recent studies suggest that CypA protects the incoming core from intrinsic host restriction in target cells, yet the requirement for CypA is independent of the recently identified host restriction factor TRIMSa. Evidence for a target cell-dependent requirement for CypA exists in the form of HIV-1 mutants capable of replicating in .the presence of cyclosporin A (CsA). These mutants contain substitutions in the CypA-binding loop of the viral CA protein. In some, but not all, human cell lines the replication of these HIV-1 mutants actually requires the presence of CsA. We hypothesize that as-vet unidentified human cell proteins determine the consequences of CypA binding to the HIV-1 capsid for virus infection. In this R21 application, we propose experiments to test this hypothesis and identify the relevant host factor(s). In Specific Aim 1, we will determine whether the cell- specific phenotypes are consistent with the expression of dominant restriction factors by studying heterokaryons generated from cells that differ in their requirements for CypA-CA interactions in HIV-1 infection. In Specific Aim 2, we will identify the relevant factor(s) by a combination of candidate gene analysis and screening of specific cDNA libraries. These studies will help elucidate the mechanism by which CypA promotes HIV-1 replication and may reveal novel strategies for the treatment of HIV infection.