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

描述（由申请人提供）：该提案代表了一个方向的转变，从以前的资助中对模型和生物膜的基本膜域研究转向了一个与健康相关的重大问题，HIV感染，与膜侧异质性有很强的联系。我们的总体目标是应用最先进的活细胞成像技术来阐明病毒传染性的那些方面，这些方面在很大程度上取决于质膜中的动态结构域。在Specific Aim I中，使用分子生物学和脂质处理技术结合活细胞成像，我们将询问哪些分子因子促进成纤维细胞中表达的流感血凝素（HA）微域的形成和稳定。这种结合的方法将为在结构域组装和稳定性中重要的分子相互作用提供活细胞纳米测定。Aim II将重点关注c型凝集素受体DC- sign (CD209)，它在树突状细胞（DC）表面表达，在那里它作为抗原捕获受体和细胞粘附分子起作用。包括HIV-1在内的各种微生物结合DC- sign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- 200nm的离散抗原捕获簇。我们将在活细胞上研究DC-SIGN集群，在那里可以对它们的动态特性进行详细分析——当装载不同的货物时。我们将验证一个假设，即病毒感染的关键初始步骤涉及DC- SIGN集群，当装载病原体货物时，该集群被触发进行定向横向运输到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，四跨蛋白。我们将验证病毒组装的初始阶段需要Gag多聚子定位到富含四聚氰胺的微域，一旦在那里，形成稳定的簇，将成为病毒出芽的地点。这一建议的健康相关意义是基于这样一个事实，即有效、持久的战略，以阻止生产性艾滋病毒-1感染仍然难以捉摸。绝大多数疫苗开发都集中在修改病毒以引起免疫反应的传统方法上，但这一直存在问题，因为HIV-1在所有病毒中保持着最高的突变率之一。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上，最终目标是在治疗上破坏这些相互作用。细胞膜微结构域对HIV-1在细胞中的生存至关重要，因为病毒依靠其特定的空间安排进入和退出靶细胞。在这项资助中获得的关于宿主细胞中这些膜结构域的详细信息将提供对HIV感染的某些步骤的更好理解，从而导致新的治疗靶点。

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.