Biophysics of Protein-Mediated Membrane Fusion

描述(申请人提供)：HIV包膜蛋白(Env)通过调节病毒和细胞膜之间的融合，将核衣壳沉积到宿主细胞中。融合是通过Env的构象变化来促进的，这种变化是由与CD4和辅助受体的顺序相互作用触发的。由于有关环境病毒的结构信息有限，而且缺乏监测艾滋病毒-细胞融合的实时技术，药物和抗体可以靶向的艾滋病毒融合的中间步骤尚未完全了解。另一个并发症是，这种病毒的感染进入途径没有得到很好的定义。虽然艾滋病毒被认为是通过直接与质膜融合来感染细胞的，但越来越多的证据表明，感染也可以通过与胞体内隔膜的pH无关的融合而发生。该项目的重点是检查艾滋病毒的进入途径，并描绘中期阶段的融合进程。病毒-细胞融合将直接通过病毒核心相关酶递送试验进行监测。HIV-细胞融合的各个步骤将通过加入时间实验进行剖析，使用一组针对CD4和辅助受体结合步骤的进入抑制剂，以及膜融合步骤本身。细胞内融合的贡献将通过阻断病毒内吞作用或通过抑制外质和内质融合途径来检验。单病毒-细胞融合的时间分辨成像也将用于阐明艾滋病毒进入的途径。我们的成像数据表明，由单个HIV颗粒在细胞表面形成的融合孔并不完全扩大，而那些与内体膜形成的融合孔有效地扩张，允许病毒内容物释放到细胞质中。融合毛孔扩张倾向的这些差异导致了一种假设，即HIV已适应在指定位置形成相对较小的毛孔，但依赖内吞机制来扩大这些毛孔并启动感染。为了验证这一假说，我们将检查Env诱导的细胞表面和内小体融合孔的形成和扩大，并评估病毒和细胞因素在扩张这些孔中的作用。拟议的方法将有助于描绘最终导致病毒核衣壳释放的艾滋病毒融合的途径，并将阐明这一过程的关键中间步骤。与公共卫生相关：人类免疫缺陷病毒(HIV)通过将其基因组存放在宿主细胞中而引发感染--这一过程涉及病毒核心周围的膜与细胞膜的融合。膜融合是由专门的HIV包膜蛋白(Env)介导的复杂的多步骤反应。为了阐明Env诱导的膜融合的机制，将用时间分辨荧光显微镜观察单个HIV颗粒，并将监测它们与宿主细胞的融合从开始(病毒和细胞膜的局部合并)到完成(将病毒基因组释放到细胞质中)。这些研究将有助于确定艾滋病毒的进入机制，并将提出预防感染的新战略。

DESCRIPTION (provided by applicant): HIV envelope (Env) protein deposits the nucleocapsid into a host cell by mediating fusion between the viral and cell membranes. Fusion is promoted through conformational changes in Env, which are triggered upon sequential interactions with CD4 and coreceptors. The intermediate steps of HIV fusion that can be targeted by drugs and antibodies are not fully understood, owing to the limited structural information on Env and to the lack of real-time techniques to monitor HIV-cell fusion. An additional complication is that the infectious entry pathways of this virus are not well defined. Whereas HIV is thought to infect cells by fusing directly with a plasma membrane, an accumulating body of evidence suggests that infection can also occur via pH- independent fusion with endosomal compartments. This project focuses on examining HIV entry pathways and on delineating the progression of fusion through intermediate stages. Virus-cell fusion will be directly monitored by the viral core-associated enzyme delivery assay. Individual steps of HIV-cell fusion will be dissected by time-of-addition experiments, using a panel of entry inhibitors targeting CD4 and coreceptor binding steps, as well as the membrane fusion step itself. The contribution from intracellular fusion will be examined by blocking virus endocytosis or by inhibiting both exoplasmic and endoplasmic fusion pathways. Time-resolved imaging of single virus-cell fusion will also be employed to elucidate pathways of HIV entry. Our imaging data suggest that fusion pores formed by individual HIV particles at the cell surface do not fully enlarge, whereas those formed with endosomal membranes dilate efficiently, permitting the release of viral content into the cytosol. These differences in the propensity of fusion pores to dilate have led to the hypothesis that HIV has adapted to form relatively small pores at designated sites, but relies on endocytic machinery to enlarge these pores and initiate infection. In order to test this hypothesis, the formation and enlargement of Env-induced fusion pores at the cell surface and in endosomes will be examined and the roles of viral and cellular factors in dilating these pores will be evaluated. The proposed approaches will help delineate the pathways of HIV fusion that culminate in the release of viral nucleocapsid and will elucidate key intermediate steps of this process. PUBLIC HEALTH RELEVANCE: Human immunodeficiency virus (HIV) initiates infection by depositing its genome into a host cell - a process that involves fusion of the membrane surrounding the viral core with a cell membrane. Membrane fusion is a complex multi-step reaction mediated by specialized HIV envelope protein (Env). To elucidate the mechanism of Env-induced membrane fusion, single HIV particles will be visualized by time-resolved fluorescence microscopy and their fusion with a host cell will be monitored from its initiation (a local merger of viral and cellular membranes) to completion (release of the viral genome into the cytosol). These studies will help define the entry mechanisms of HIV and will suggest new strategies to prevent infection.