Mechanisms that determine subcellular sites of HIV-1 assembly

决定 HIV-1 组装亚细胞位点的机制

• 批准号: 8068079
• 负责人: Akira Ono
• 金额: $ 8.57万
• 依托单位: UNIVERSITY OF MICHIGAN AT ANN ARBOR
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-05-17 至 2011-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8068079
• 关键词: Accounting; Address; Affect; Amino Acid Substitution; Amino Acids; Anti-HIV Agents; Antiviral Agents; Binding; Binding Proteins; Biological Assay; Cell membrane; Cells; Clone Cells; Coupled; Data; Defect; Dependence; Development; Drug Delivery Systems; Enzymes; Factor Analysis; Fluorescence Microscopy; Future; Gagging; Goals; HIV-1; Hela Cells; In Vitro; Infection; Intervention; Lead; Lipid Binding; Lipids; Liposomes; Location; Measurement; Mediating; Membrane; Membrane Lipids; Molecular; Multivesicular Body; Pathway interactions; Phosphatidylinositol Phosphates; Phosphatidylinositols; Phospholipids; Phosphoric Monoester Hydrolases; Play; Process; Production; Proteins; Recycling; Reporting; Research Proposals; Resistance; Role; Series; Site; System; T-Lymphocyte; Tertiary Protein Structure; Testing; Translations; Transmembrane Transport; Vesicle; Viral; Viral Structural Proteins; Virion; Virus; Virus Assembly; Virus-like particle; base; cell type; genetic regulatory protein; instrument; late endosome; macrophage; mutant; overexpression; particle; protein transport; research study; trafficking

DESCRIPTION (provided by applicant): HIV-1 particle production, a process driven by the viral structural protein Gag, takes place predominantly at the plasma membrane in cell types including T cells, whereas in macrophages a majority of virus particles appears to assemble in the late endosomes. The molecular mechanisms that determine the sites of virus assembly remain to be elucidated. The observed cell type difference is consistent with the possibility that a cellular factor(s) is involved in determining the subcellular location of HIV-1 assembly. We recently reported that depletion of the plasma membrane lipid PI(4,5)P2 in HeLa cells relocates Gag from the plasma membrane to late endosomes and markedly reduces virus particle production. Preliminary results show that Gag binding to total membrane is also impaired upon PI(4,5)P2 depletion. Recent structural data suggest that PI(4,5)P2 interacts directly with the matrix domain of Gag. These results suggest that a Gag-PI(4,5)P2 interaction is essential for virus assembly at the plasma membrane. Our goal in this proposal is to elucidate the molecular mechanisms by which PI(4,5)P2 regulates HIV-1 assembly in different cell types. Our specific aims are: [Aim 1] To identify Gag regions that are responsible for PI(4,5)P2 dependence. This will be achieved by a) characterization of Gag derivatives and b) isolation and characterization of viruses adapted to low PI(4,5)P2 levels. [Aim 2] To determine if PI(4,5)P2 regulates Gag localization by directly binding to Gag or through a PI(4,5)P2-dependent cellular trafficking pathway, or both. To address the first possibility, we will develop in vitro Gag-lipid binding assays. To examine the second possibility, we will analyze the impact of inhibition of membrane transport pathways and compare it with the effects of cellular PI(4,5)P2 depletion. [Aim 3] To examine, the role of PI(4,5)P2 in Gag localization to late endosomes and virus particle production in macrophages. We will perform quantitative analyses of PI(4,5)P2-Gag colocalization and virus release from macrophages with or without PI(4,5)P2 perturbation. The subcellular sites of virus assembly play key roles in the efficiency of virus production and viral persistence. Therefore, analyses of factors that determine the assembly sites have major implications in strategies for intervention in HIV-1 infection. The experiments proposed here will determine the roles played by PI(4,5)P2 in HIV-1 particle production and will contribute to the future development of antiviral drugs that target HIV-1 assembly and release.

描述（由申请人提供）：HIV-1颗粒的产生是由病毒结构蛋白GAG驱动的过程，主要发生在包括T细胞在内的细胞类型的质膜上，而在巨噬细胞中，大多数病毒颗粒似乎在晚期内体中聚集。确定病毒组装部位的分子机制仍有待阐明。观察到的细胞类型差异与细胞因子（S）在确定HIV-1组装的亚细胞位置有关的可能性一致。我们最近报道说，HeLa细胞中质膜脂质Pi（4,5）P2的耗竭可将GAG从质膜转移到晚期内体，并显着减少了病毒颗粒的产生。初步结果表明，PI（4,5）P2耗竭也会受损与总膜的结合。最近的结构数据表明，PI（4,5）P2与GAG的基质结构域直接相互作用。这些结果表明，GAG-PI（4,5）P2相互作用对于质膜的病毒组装至关重要。我们在此提案中的目标是阐明PI（4,5）P2通过不同细胞类型调节HIV-1组装的分子机制。我们的具体目的是：[AIM 1]确定负责PI（4,5）P2依赖性的插科打式区域。这将通过a）a）表征插孔衍生物的表征和b）适应低PI（4,5）P2水平的病毒的隔离和表征。 [AIM 2]确定PI（4,5）P2是否通过直接与GAG结合或通过PI（4,5）P2依赖性的细胞运输途径来调节GAG定位。为了解决第一个可能性，我们将开发体外插入脂质结合测定法。为了检查第二种可能性，我们将分析抑制膜转运途径的影响，并将其与细胞PI（4,5）P2耗竭的影响进行比较。 [AIM 3]要检查Pi（4,5）P2在巨噬细胞中晚期内体和病毒颗粒产生中的作用。我们将对有或没有PI（4,5）P2扰动的巨噬细胞对PI（4,5）P2-GAG共定位和病毒释放进行定量分析。病毒组装的亚细胞位点在病毒生产和病毒持久性的效率中起关键作用。因此，对决定组装位点的因素的分析对HIV-1感染干预策略具有重大影响。此处提出的实验将确定PI（4,5）P2在HIV-1颗粒产生中扮演的角色，并将有助于靶向HIV-1组装和释放的抗病毒药药的未来开发。