In-vitro analysis of HSV-1 membrane fusion mechanism

项目摘要/摘要 单纯疱疹病毒1型(HSV-1)需要四种糖蛋白才能进入细胞和膜融合--gB，gD， Gh和Gl-除细胞受体外。对膜融合机制的透彻了解是 对于了解疱疹病毒如何穿透细胞并找到抑制这一过程的方法至关重要。由于 尽管经过几十年的研究，但这一过程的复杂性，许多基本问题仍然存在。此外， 传统的进入试验依赖于下游报告，不直接测量融合。细胞-细胞的研究 表达四种基本HSV进入糖蛋白的未感染受体承载细胞的融合已产生 目前膜融合的许多机制知识，包括普遍存在的调节级联 模特。然而，细胞-细胞融合系统虽然提供了信息，但未能捕捉到病毒-细胞的适当背景 核聚变。因此，准确、系统地剖析HSV介导的膜融合机制需要一种 实验上易于处理的系统，使病毒融合的直接可视化和动力学测量成为可能。 这一探索性方案的目标是在体外重建HSV-1融合，并在单个细胞上对其进行表征。 通过使用全内反射将单个病毒粒子与液体、支持的脂质双层进行成像融合的病毒粒子水平 显微镜。这种方法将用于可视化融合的不同阶段，测量它们的动力学 参数，确定动力学中间体，并将它们与GB中的结构重排相关联。此外 对于HSV-1，拟议的研究将使用缺乏天然Fuso基因的水疱性口炎病毒(VSV)病毒粒子 G和与HSV-1进入糖蛋白Gb、Gd、Gh和Gl的假型(VSVDG-BHLD)。这个的用法 简化的“基本”系统将排除其他12个信封的潜在影响，从而使这项工作受益 蛋白质。融合的体外重建及其在单颗粒水平上的表征将解决 HSV介导的膜融合机制中挥之不去的问题，最终目标是重建 HSV-1介导的融合途径更加充分。

PROJECT SUMMARY/ABSTRACT Herpes Simplex virus type 1 (HSV-1) requires four glycoproteins for cell entry and membrane fusion – gB, gD, gH, and gL – in addition to a cellular receptor. A thorough knowledge of membrane fusion mechanisms is essential for understanding how herpesviruses penetrate cells and finding ways to inhibit this process. Due to the complexity of this process, many fundamental questions endure despite decades of research. Moreover, traditional entry assays rely on downstream reporters and do not measure fusion directly. Studies of cell-cell fusion of uninfected receptor-bearing cells expressing the four essential HSV entry glycoproteins have yielded much of the current mechanistic knowledge of membrane fusion, including the prevalent regulatory cascade model. However, the cell-cell fusion system, while informative, fails to capture the proper context of virus-cell fusion. Accurate, systematic dissection of the HSV-mediated membrane fusion mechanism thus requires an experimentally tractable system that enables direct visualization and kinetic measurements of the viral fusion. The goal of this exploratory proposal is to reconstitute HSV-1 fusion in vitro and to characterize it at a single- virion level by imaging fusion of individual virions with fluid, supported lipid bilayers using total internal reflection microscopy. This approach will be used to visualize different stages in fusion, measure their kinetic parameters, identify kinetic intermediates, and correlate them with structural rearrangements in gB. In addition to HSV-1, the proposed studies will employ Vesicular Stomatitis Virus (VSV) virions lacking the native fusogen G and pseudotyped with HSV-1 entry glycoproteins gB, gD, gH, and gL (VSVDG-BHLD). The use of this simplified, “bare-bones” system will benefit this work by excluding the potential effects of the 12 other envelope proteins. In-vitro reconstitution of fusion and its characterization at a single particle level will address the lingering questions in HSV-mediated membrane fusion mechanism, with the ultimate goal of reconstructing the HSV-1-mediated fusion pathway more fully.