Biophysics of Protein-Mediated Membrane Fusion

描述（由申请人提供）：HIV包膜（Env）蛋白通过介导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，这是在与CD4和辅助受体的连续相互作用下触发的。由于有限的Env结构信息和缺乏实时监测HIV细胞融合的技术，目前还不完全了解HIV融合的中间步骤，这些步骤可以被药物和抗体靶向。另一个并发症是，这种病毒的感染进入途径没有很好地确定。虽然HIV被认为是通过直接与质膜融合来感染细胞，但越来越多的证据表明，感染也可以通过与内体腔室的pH无关的融合发生。这个项目的重点是检查艾滋病毒的进入途径，并通过中间阶段描绘融合的进展。病毒-细胞融合将通过病毒核相关酶递送试验直接监测。hiv细胞融合的各个步骤将通过添加时间实验进行剖析，使用一组靶向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。细胞内融合的作用将通过阻断病毒内吞作用或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HIV的进入途径。我们的成像数据表明，单个HIV颗粒在细胞表面形成的融合孔不会完全扩大，而那些与内体膜形成的融合孔会有效地扩张，从而允许病毒内容物释放到细胞质中。融合孔扩张倾向的这些差异导致了一种假设，即HIV已经适应在指定位置形成相对较小的孔，但依赖于内吞机制来扩大这些孔并引发感染。为了验证这一假设，我们将研究环境诱导的细胞表面和核内体融合孔的形成和扩大，并评估病毒和细胞因子在扩大这些孔中的作用。所提出的方法将有助于描述以病毒核衣壳释放为高潮的HIV融合途径，并将阐明这一过程的关键中间步骤。公共卫生相关性：人类免疫缺陷病毒（HIV）通过将其基因组沉积到宿主细胞中引发感染，这一过程涉及围绕病毒核心的膜与细胞膜的融合。膜融合是由HIV特异性包膜蛋白介导的一个复杂的多步骤反应。为了阐明env诱导的膜融合机制，将通过时间分辨荧光显微镜观察单个HIV颗粒，并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（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策略。

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.