Structure and dynamics of membrane microdomains used for viral entry and egress

用于病毒出入的膜微域的结构和动力学

• 批准号: 7999969
• 负责人: Kenneth A Jacobson
• 金额: $ 8万
• 依托单位: UNIV OF NORTH CAROLINA CHAPEL HILL
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-01-07 至 2010-12-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7999969
• 关键词: Antibodies; Antigens; Arts; Avidity; Beds; Binding; Biological Models; C Type Lectin Receptors; CD209 gene; Caliber; Cell membrane; Cell surface; Cell-Adhesion Molecule Receptors; Cells; Cellular Membrane; DC-specific ICAM-3 grabbing nonintegrin; Dendritic Cells; Electron Microscopy; Environment; Exhibits; Fibroblasts; Gagging; Goals; Grant; HIV; HIV Infections; HIV-1; Heterogeneity; Image; Imaging technology; Immune response; Individual; Infection; Influenza; Influenza Hemagglutinin; Integral Membrane Protein; Label; Lateral; Life; Ligand Binding; Ligands; Ligation; Light; Lipids; Maintenance; Measures; Membrane; Membrane Lipids; Membrane Microdomains; Methodology; Methods; Microbe; Microscopy; Molecular; Molecular Biology; Mutation; Nature; Photobleaching; Process; Property; Protein Precursors; Quantum Dots; Signal Transduction; Site; Small Interfering RNA; Staging; Structure; Surface; Techniques; Technology; Testing; Viral; Virion; Virus; Virus Assembly; Virus Diseases; Virus-like particle; base; cell fixing; cellular imaging; gag Gene Products; human PHEMX protein; molecular dynamics; mutant; nanoassay; new therapeutic target; particle; pathogen; public health relevance; receptor; tool; vaccine development

DESCRIPTION (provided by applicant): This proposal represents a shift in direction from basic membrane domain studies in model and biological membranes in the previous grant to a commanding health-related problem, HIV infection, with strong connections to membrane lateral heterogeneity. Our overall goal is to apply cutting edge imaging technologies for the living cell to elucidate those aspects of virus infectivity that depend crucially on dynamic domains in the plasma membrane. In Specific Aim I, using molecular biology and lipid manipulation techniques in combination with live cell imaging, we will ask what molecular factors promote formation and stabilization of microdomains of influenza hemeagglutinin, HA, expressed in fibroblasts. This combined approach will provide live cell nanoassays for molecular interactions important in domain assembly and stability. Aim II will focus on the C-type lectin receptor DC-SIGN (CD209) which is expressed on the surface of Dendritic Cells (DC) where it functions as an antigen capture receptor and cell adhesion molecule. Various microbes, including HIV-1, bind to DC-SIGN to gain entry into DC. Previous studies using fixed cells demonstrated that DC-SIGN forms discrete antigen-capture clusters of 100-200 nm diameter on immature DC in the absence of any receptor ligation. We will study DC-SIGN clusters on living cells where detailed analysis of their dynamic properties--when loaded with different cargos--can be pursued. We will test the hypothesis that a key initial step in viral infection involves DC- SIGN clusters that, when loaded with pathogen cargo, are triggered to undergo directed lateral transport to sites of further processing by the host cell. Aim III will focus on microdomains that contain multiple components including the HIV-1 Gag polyprotein and a class of transmembrane proteins, the tetraspanins. We will test the hypothesis that the initial stages of virus assembly require Gag multimers to localize to tetraspanin-enriched microdomains and, once there, form stable clusters that will become sites of viral budding. PUBLIC HEALTH RELEVANCE The health-related significance of this proposal is based on the fact that effective, long-lasting strategies to thwart productive HIV-1 infection have remained elusive. The overwhelming majority of vaccine development has focused on the traditional methods of modifying the virus to elicit an immune response but this has been problematic because HIV-1 maintains one of the highest mutation rates amongst all viruses. As a result, increasing efforts have been focused on understanding how the virus interacts with its host cell with the ultimate goal of therapeutically disrupting these interactions. Cellular membrane microdomains are critical to the life of HIV-1 in cells in that the virus relies on their specific spatial arrangements for both entry into and exit out of target cells. Detailed information obtained in this grant on such membrane domains in the host cell will provide a better understanding of certain steps in HIV infection, thereby leading to new therapeutic targets.

描述（由申请人提供）：该提案代表了从先前拨款中的模型和生物膜的基本膜域研究方向的转变，转向与膜横向异质性密切相关的与健康相关的重大问题，即艾滋病毒感染。我们的总体目标是应用尖端的活细胞成像技术来阐明病毒感染性的那些方面，这些方面主要取决于质膜中的动态域。在具体目标 I 中，我们将结合活细胞成像使用分子生物学和脂质操作技术，探究哪些分子因素促进成纤维细胞中表达的流感血凝素 (HA) 微结构域的形成和稳定。这种组合方法将为活细胞纳米分析提供对结构域组装和稳定性至关重要的分子相互作用。 Aim II 将重点关注 C 型凝集素受体 DC-SIGN (CD209)，它在树突细胞 (DC) 表面表达，充当抗原捕获受体和细胞粘附分子。包括 HIV-1 在内的各种微生物与 DC-SIGN 结合以进入 DC。先前使用固定细胞的研究表明，在没有任何受体连接的情况下，DC-SIGN 在未成熟的 DC 上形成直径为 100-200 nm 的离散抗原捕获簇。我们将研究活细胞上的 DC-SIGN 簇，详细分析它们在装载不同货物时的动态特性。我们将检验以下假设：病毒感染的关键初始步骤涉及 DC-SIGN 簇，当装载病原体货物时，这些簇会被触发进行定向横向运输，到达宿主细胞进一步加工的位点。 Aim III 将重点关注包含多种成分的微结构域，包括 HIV-1 Gag 多蛋白和一类跨膜蛋白（四跨膜蛋白）。我们将检验以下假设：病毒组装的初始阶段需要 Gag 多聚体定位于富含四跨膜蛋白的微结构域，一旦到达那里，就会形成稳定的簇，成为病毒出芽的位点。公共卫生相关性 该提案与健康相关的重要性是基于这样一个事实，即阻止生产性 HIV-1 感染的有效、持久的策略仍然难以捉摸。绝大多数疫苗开发都集中在修改病毒以引发免疫反应的传统方法上，但这一直是个问题，因为 HIV-1 是所有病毒中突变率最高的病毒之一。因此，越来越多的努力集中在了解病毒如何与其宿主细胞相互作用，最终目标是在治疗上破坏这些相互作用。细胞膜微区对于 HIV-1 在细胞中的生命至关重要，因为病毒依赖其特定的空间排列来进入和退出靶细胞。本次资助获得的有关宿主细胞膜结构域的详细信息将有助于更好地理解 HIV 感染的某些步骤，从而找到新的治疗靶点。