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; 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）：该提案代表了从模型和先前授予的生物膜中的基本膜域研究转变为指挥健康相关的问题，HIV感染，与膜侧向异质性有很强的联系。我们的总体目标是将最先进的成像技术应用于活细胞，以阐明质膜中动态域的至关重要的病毒感染性方面。在特定的目标I中，使用分子生物学和脂质操纵技术与活细胞成像结合使用，我们将询问哪些分子因素促进了在成纤维细胞中表达的流感heageglutinin，HA的微域的形成和稳定。这种合并的方法将为在域组装和稳定性中重要的分子相互作用提供活细胞纳米测定。 AIM II将集中在C型凝集素受体DC-sign（CD209）上，该c型在树突状细胞表面（DC）表达，在该表面，它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，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形成了未成熟DC的离散抗原捕获簇，直径为100-200 nm。我们将在活细胞上研究DC-SIGN簇，其中详细分析了它们的动态特性，当时使用了不同的cargos- can。我们将检验以下假设：病毒感染的关键初步步骤涉及DC-标志簇，该簇在装有病原体货物时会触发，以直接进行侧向运输到宿主细胞进一步处理的部位。 AIM III将集中在包含多个组分的微域上，包括HIV-1 GAG多蛋白和一类跨膜蛋白，即四叠腺苷。我们将检验以下假设：病毒组装的初始阶段需要插入多聚体来定位于四叠蛋白蛋白含有的微区，并且一旦那里形成稳定的簇，这些簇将成为病毒萌芽的位置。公共卫生相关性该提案与健康相关的意义是基于这样一个事实，即挫败生产性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传统的修饰病毒以引起免疫反应的方法上，但这是有问题的，因为HIV-1在所有病毒中保持最高的突变率之一。结果，越来越多的努力集中在理解病毒与宿主细胞相互作用的最终目的是在治疗上破坏这些相互作用的目标。细胞膜微区域对于细胞中HIV-1的寿命至关重要，因为该病毒依赖于其特定的空间排列以进入和退出靶细胞。在本赠款中获得的有关宿主细胞中此类膜域的详细信息将更好地理解HIV感染的某些步骤，从而导致新的治疗靶标。