Molecular Mechanism of LEDGF/p75 in HIV Integration

LEDGF/p75在HIV整合中的分子机制

• 批准号: 7585734
• 负责人: Manuel Llano
• 金额: $ 11.1万
• 依托单位: UNIVERSITY OF TEXAS EL PASO
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-03-12 至 2011-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7585734
• 关键词: Adenosine Diphosphate Ribose; Anti-HIV Agents; Binding; Binding Proteins; Cell Fractionation; Cells; Chimeric Proteins; Chromatin; Complex; DNA Damage; DNA Integration; DNA repair protein; Development; Drug Delivery Systems; Goals; HIV; HIV Infections; HIV-1; Hela Cells; In Vitro; Infection; Integrase; Knowledge; Maps; Mediating; Methods; Modeling; Molecular; Mutate; Pharmaceutical Preparations; Plasmids; Polymerase; Process; Proteins; Resistance; Role; Signal Transduction; System; T-Lymphocyte; Transfection; Viral; design; drug development; high throughput screening; lens epithelium-derived growth factor; research study; sensor; small molecule; transcriptional coactivator p75

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.

描述（由申请人提供）：最近，我们证明了晶状体上皮衍生的生长因子（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。缺乏这种染色质蛋白的T细胞在病毒整合步骤下对HIV-1感染具有抗性，这表明LEDGF/p75是抗HIV药物开发的靶标。尽管我们证明了LEDGF/p75染色质和整合酶结合域是必需的，但其在此过程中的确切作用仍然未知。该建议的目的是了解LEDGF/p75 HIV-1 DNA整合的分子机制。这些知识将直接影响针对该过程的药物的开发。我们的结果将有助于设计用于小分子HIV整合抑制化合物的高通量筛选方法。 具体目标 （1）确定HIV预共同络合物（PICS）的染色质绑定是否需要LEDGF/P75。 LEDGF/p75确定积分酶在未感染细胞中通过质粒转染表示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束缚。由于需要相同的LEDGF/p75域来进行整合酶定位和HIV整合，因此还假定了LEDGF/p75在HIV整合中的束缚作用。但是，该模型正在等待直接证明证明。使用嵌合LEDGF蛋白，亚细胞分馏和DNA损伤信号传导，我们将在HIV感染的细胞中评估该模型。 （2）评估LEDGF/P75与DNA修复蛋白的相互作用。 GST下拉实验将研究LEDGF/P75与DNA修复蛋白的相互作用。 LEDGF/P75中的相关域将被突变，并评估其在HIV感染中的相关性。 （3）开发用于LEDGF/P75-积极酶相互作用的小抑制化合物的高通量筛选系统。 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-积聚酶相互作用的药物有望降低这些细胞中的EGFP表达。