Molecular Mechanism of LEDGF/p75 in HIV Integration

描述（由申请人提供）：最近，我们已经证明了透镜上皮衍生生长因子（LEDGF/p75）在人类免疫缺陷病毒1型（HIV-1）DNA整合过程中的重要作用。缺乏这种染色质结合蛋白的T细胞在病毒整合步骤中对HIV-1感染具有抗性，这表明LEDGF/p75是抗HIV药物开发的靶点。虽然我们证明了LEDGF/p75染色质和整合酶结合结构域是必需的，但其在此过程中的确切作用仍然未知。本研究的目的是了解LEDGF/p75 HIV-1 DNA整合的分子机制。这些知识将对开发针对这一过程的药物产生直接影响。我们的研究结果将有助于设计高通量筛选小分子HIV整合抑制化合物的方法。 具体目标 (1)确定LEDGF/p75是否是HIV整合前复合物（PIC）染色质拴系所必需的。LEDGF/p75通过质粒转染在非感染细胞中确定整合酶表达为唯一蛋白的染色质定位。已经提出LEDGF/p75充当整合酶和染色质之间的分子系链。由于整合酶定位和HIV整合需要相同的LEDGF/p75结构域，因此也假定了LEDGF/p75在HIV整合中的束缚作用。然而，这一模式有待直接证据的证明。使用嵌合LEDGF蛋白，亚细胞分级分离和DNA损伤信号，我们将在HIV感染的细胞中评估该模型。 (2)研究LEDGF/p75与DNA修复蛋白的相互作用。通过GST下拉实验研究LEDGF/p75与DNA修复蛋白的相互作用。LEDGF/p75中的相关结构域将被突变，并评估其在HIV感染中的相关性。 (3)LEDGF/p75-整合酶相互作用的小抑制化合物的高通量筛选系统的开发。LEDGF/p75保护整合酶免于蛋白酶体介导的降解。整合酶-eGFP融合蛋白将在HeLa细胞中表达;干扰LEDGF/p75-整合酶相互作用的药物预计将降低这些细胞中的eGFP表达。

DESCRIPTION (provided by applicant): Recently we have demonstrated an essential role of the lens epithelium-derived growth factor (LEDGF/p75) in the human immunodeficiency virus type 1 (HIV-1) DNA integration process. T cells lacking this chromatin-bound protein are resistant to HIV-1 infection at the viral integration step suggesting LEDGF/p75 as a target for anti-HIV drug development. Although we demonstrated that LEDGF/p75 chromatin and integrase binding domains are required, its exact role in this process is still unknown. The goal of this proposal is to understand the molecular mechanism of LEDGF/p75 HIV-1 DNA integration. This knowledge will have a direct impact in the development of drugs targeting this process. Our results will help the design of high-throughput screening methods for small-molecule HIV integration inhibitory compounds. Specific Aims (1) To determine if LEDGF/p75 is required for chromatin tethering of HIV pre-integration complexes (PICs). LEDGF/p75 determines chromatin localization of integrase expressed as a sole protein by plasmid transfection in non-infected cells. It has been proposed that LEDGF/p75 acts as a molecular tether between integrase and chromatin. A tethering role of LEDGF/p75 in HIV integration was also postulated because of the requirement of the same LEDGF/p75 domains for integrase localization and HIV integration. However, this model awaits direct evidence for demonstration. Using chimeric LEDGF proteins, subcellular fractionation and DNA damage signaling, we will evaluate this model in HIV infected cells. (2) To evaluate the interaction of LEDGF/p75 with DNA repair proteins. The interaction of LEDGF/p75 with DNA repair proteins will be investigated by GST pull-down experiments. Relevant domains in LEDGF/p75 will be mutated and its relevance in HIV infection evaluated. (3) Development of a high-throughput screening system for small inhibitory compounds of LEDGF/p75-integrase interaction. LEDGF/p75 protects integrase from proteasomal-mediated degradation. Integrase-eGFP fusion protein will be expressed in HeLa cells; drugs interfering with LEDGF/p75-integrase interaction are expected to reduce eGFP expression in these cells.