Biophysics of Protein-Mediated Membrane Fusion

描述（由申请人提供）：长期以来，人们一直认为人类免疫缺陷病毒（HIV）通过与质膜融合进入靶细胞。这一概念部分地基于HIV Env糖蛋白与细胞表面上的CD 4和辅助受体CCR 5或CXCR 4接合，然后通过经历pH非依赖性构象变化来促进膜融合的事实。我们最近的研究通过提供HIV通过内吞途径进入的直接证据挑战了这一观点。这种进入途径的论点是基于：（i）相对于获得对膜不渗透融合抑制剂的抗性，HIV内容物延迟释放到胞质溶胶中;和（ii）单一HIV成像显示与内体完全融合，但在细胞表面仅部分融合。然而，HIV通过内吞进入的概念在该领域尚未被广泛接受。因此，我们建议仔细评估艾滋病毒进入途径在不同的细胞类型，并确定艾滋病毒融合的网站的病毒和细胞的决定因素。我们的中心假设是，HIV只能促进融合的早期步骤，而依赖于宿主细胞来完成这一过程。这一假设源于这样一种想法，即HIV粒子上的少量Env可能不足以克服与在融合过程中产生高度不利的脂质中间体相关的巨大能量障碍。所提出的模型做出了可检验的预测，这将指导我们寻求可以帮助HIV融合的宿主因素。我们将：1.检查HIV进入途径的Env和细胞类型依赖性。将使用改进的单病毒成像方法确定淋巴细胞系和原代CD 4 + T细胞中的HIV融合位点。2.确定HIV与质膜的完全融合是否需要外力。我们将评估HIV-细胞融合和HIV介导的细胞-细胞融合对肌动蛋白重塑的依赖性，肌动蛋白重塑可以产生侧向膜张力，从而促进融合孔的扩张。3.探索负责HIV与核内体融合的细胞因子。我们将跟进我们的试点数据，涉及几个宿主蛋白质在艾滋病毒的贩运和融合。所选宿主因子的共同特征是它们产生膜曲率或改变脂质组成，从而有利于HIV-内体融合。拟议的研究将确定艾滋病毒进入和融合途径。我们期望阐明病毒对宿主因子的依赖程度，并描述以核衣壳释放为高潮的完全HIV融合的机制。

DESCRIPTION (provided by applicant): The Human Immunodeficiency Virus (HIV) has long been thought to enter target cells by fusing with the plasma membrane. This notion is based, in part, on the fact that the HIV Env glycoprotein engages CD4 and coreceptors, CCR5 or CXCR4, on the cell surface and then promotes membrane fusion by undergoing pH- independent conformational changes. Our recent study has challenged this view by presenting direct evidence for HIV entry via an endocytic pathway. The arguments for this entry route are based on: (i) the delayed release of HIV content into the cytosol relative to the acquisition of resistane to a membrane-impermeant fusion inhibitor; and (ii) single HIV imaging which reveals complete fusion with endosomes but only partial fusion at the cell surface. However, the notion of HIV entry via endocytosis has not been widely accepted in the field. We therefore propose to carefully evaluate the HIV entry routes in different cell types and define the viral and cellular determinants of the sites of HIV fusion. Our central hypothesis is that HIV can promote only the early steps of fusion, while relying on the host cell to complete this process. This hypothesis stems from the idea that a handful of Env on HIV particles may not be sufficient to overcome a large energy barrier associated with creating the highly unfavorable lipid intermediates en route to fusion. The proposed model makes testable predictions that will guide our quest for the host factors that can aid the HIV fusion. We will: 1. Examine the Env- and cell type-dependence of HIV entry routes. The HIV fusion sites in lymphoid cell lines and primary CD4+ T cells will be defined using an improved single virus imaging approach. 2. Determine whether complete HIV fusion with the plasma membrane requires an external force. We will evaluate the dependence of HIV-cell fusion and of HIV-mediated cell-cell fusion on actin remodeling which can generate lateral membrane tension and thereby promote the dilation of a fusion pore. 3. Explore cellular factors responsible for HIV fusion with endosomes. We will follow up on our pilot data implicating several host proteins in HIV trafficking and fusion. A common feature of the selected host factors is that they generate a membrane curvature or modify the lipid composition and can thus favor HIV-endosome fusion. The proposed studies will define the HIV entry and fusion pathways. We expect to elucidate the extent of virus' reliance on host factors and delineate the mechanism of complete HIV fusion that culminates in the release of the nucleocapsid.