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

描述(由申请者提供)：这项建议代表着方向的转变，从上一次拨款中对模型膜和生物膜的基本膜域研究，转向与健康有关的主要问题-艾滋病毒感染，与膜的横向异质性有很强的联系。我们的总体目标是应用活细胞的尖端成像技术来阐明病毒感染性的那些方面，这些方面关键取决于质膜中的动态结构域。具体目标一，利用分子生物学和脂质操纵技术，结合活细胞成像，我们将探讨哪些分子因素促进了流感血凝素在成纤维细胞中表达的微区的形成和稳定。这种结合的方法将为在结构域组装和稳定性中至关重要的分子相互作用提供活的细胞纳米分析。目的研究表达于树突状细胞(DC)表面的C型凝集素受体DC-SIGN(CD209)，它具有抗原捕获受体和细胞黏附分子的功能。各种微生物，包括HIV-1，与DC-SIGN结合以进入DC。以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100-200 nm的离散抗原捕获簇。我们将研究活细胞上的DC-SIGN簇，在那里可以对它们在装载不同货物时的动态特性进行详细的分析。我们将测试病毒感染的关键初始步骤涉及DC-SIGN簇的假设，当DC-SIGN簇装载病原体货物时，被触发进行直接的横向运输到宿主细胞进一步处理的位置。目的III将专注于包含多种成分的微域，包括HIV-1 Gag多蛋白和一类跨膜蛋白，即Tetraspanins。我们将测试这样的假设，即病毒组装的初始阶段需要Gag多聚体定位于富含Tetraspanin的微域，一旦有了微域，就形成了稳定的簇，这些簇将成为病毒萌芽的地点。公共卫生相关性这项建议与健康有关的意义是基于这样一个事实，即有效、长期的战略，以挫败生产性艾滋病毒-1感染仍然难以捉摸。绝大多数疫苗开发都集中在传统的修改病毒以引起免疫反应的方法上，但这一直是有问题的，因为艾滋病毒-1保持着所有病毒中最高的变异率之一。因此，越来越多的努力集中在了解病毒如何与宿主细胞相互作用，最终目标是从治疗上扰乱这些相互作用。细胞膜微区对HIV-1在细胞中的生命至关重要，因为病毒依赖于其进入和离开目标细胞的特定空间安排。在这笔赠款中获得的关于宿主细胞中这种膜结构域的详细信息将有助于更好地了解艾滋病毒感染的某些步骤，从而导致新的治疗目标。

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.