Molecular Mechanism of LEDGF/p75 in HIV Integration

描述(申请人提供)：最近我们证明了晶状体上皮源性生长因子(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细胞在病毒整合阶段对HIV-1感染具有抵抗力，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区是必需的，但它在这一过程中的确切作用仍不清楚。这项建议的目的是了解LEDGF/p75 HIV-1 DNA整合的分子机制。这种知识将对针对这一过程的药物的开发产生直接影响。我们的结果将有助于设计高通量的小分子HIV整合抑制化合物的筛选方法。 具体目标 (1)确定HIV前整合复合体(PIC)的染色质连接是否需要LEDGF/p75。LEDGF/p75决定了整合酶在非感染细胞中的染色质定位，该整合酶通过质粒转染法以单一蛋白的形式表达。有研究认为，LEDGF/p75在整合酶和染色质之间起着分子纽带的作用。由于整合酶定位和HIV整合需要相同的LEDGF/p75结构域，因此也推测LEDGF/p75在HIV整合中起到了拴系作用。然而，这一模型还有待于直接的证据来证明。利用嵌合的LEDGF蛋白、亚细胞分裂和DNA损伤信号，我们将在HIV感染的细胞中评估这一模型。 (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的相关结构域将发生突变，并评估其与HIV感染的相关性。 (3)建立LEDGF/p75-整合酶相互作用小分子抑制物的高通量筛选体系。LEDGF/p75保护整合酶免受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有望降低这些细胞中EGFP的表达。

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.