Single-particle analysis of HSV-1 membrane fusion mechanism

项目总结/摘要 单纯疱疹病毒1型（HSV-1）需要四种糖蛋白用于细胞进入和膜融合- gB，gD， gH和gL -除了细胞受体之外。这一数量远远超过了糖蛋白的数量， 大多数其他包膜病毒使HSV-1介导的进入和融合的机制研究复杂化。我们现在 我们知道，在HSV-1中，与其他疱疹病毒一样，受体结合、调节和融合功能是 分布在几种糖蛋白中。gB是保守的融合子，与其他病毒类似， 被认为是通过大规模的重折叠来促进膜的合并， 细胞膜在一起。然而，gB是不寻常的，需要几个额外的蛋白质的功能。 因此，了解gB如何在细胞进入过程中介导融合需要直接测量其 融合活性最近，单粒子跟踪（SPT）已经成为定量分析的有力工具。 使用全内反射显微镜研究单个病毒粒子与液体支持的脂质双层的融合。 SPT方法能够直接可视化和动力学测量病毒融合途径， 已经成功地与pH触发的和受体触发的融合子一起使用。这次探索的目的是 建议是开发HSV-1与支持的脂质双层融合的单粒子成像，以可视化不同的 在融合阶段，测量它们的动力学参数，识别动力学中间体，并将它们与 结构重排（gB）。鉴于SPT方法尚未应用于利用 >1糖蛋白，并且应用于具有15个包膜蛋白的HSV-1可能具有挑战性，Aim 1将利用 水泡性口炎病毒（VSV）病毒粒子缺乏天然融合原G，并与HSV-1进入假型 糖蛋白gB、gD、gH和gL（VSVDG-BHLD），其保留HSV-1进入的关键特征。在目标2中， SPT方法将被扩展到HSV-1，一个更复杂但生物学上更相关系统。的 这项工作的科学前提是，利用病毒融合的单粒子成像能力， 为解决HSV介导的膜融合中挥之不去的问题提供了独特的机会 机制，最终目标是更充分地重建HSV-1介导的融合途径。

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. This number far exceeds the number of glycoproteins utilized by most other enveloped viruses complicating mechanistic studies of HSV-1-mediated entry and fusion. We now know that in HSV-1, as in other herpesviruses, the receptor-binding, regulatory, and fusogenic functions are distributed among several glycoproteins. gB is the conserved fusogen that, by analogy with other viral fusogens, is thought to facilitate membrane merger by undergoing large-scale refolding that brings the viral and cellular membranes together. However, gB is unusual in requiring several additional proteins for function. Therefore, understanding how gB works to mediate fusion during cell entry requires direct measurements of its fusogenic activity. Recently, single-particle tracking (SPT) has emerged as a powerful tool for quantitative studies of fusion of individual virions with fluid, supported lipid bilayers using total internal reflection microscopy. The SPT approach enables direct visualization and kinetic measurements of the viral fusion pathway and has been successfully used with both pH-triggered and receptor-triggered fusogens. The goal of this exploratory proposal is to develop single-particle imaging of HSV-1 fusion with supported lipid bilayers to visualize different stages in fusion, measure their kinetic parameters, identify kinetic intermediates, and correlate them with structural rearrangements in gB. Given that the SPT approach has not yet been applied to viruses that utilize >1 glycoprotein and may be challenging to apply to HSV-1 that has 15 envelope proteins, Aim 1 will utilize Vesicular Stomatitis Virus (VSV) virions lacking the native fusogen G and pseudotyped with HSV-1 entry glycoproteins gB, gD, gH, and gL (VSVDG-BHLD), which retain key characteristics of HSV-1 entry. In Aim 2, the SPT approach will be extended to HSV-1, a more complicated yet more biologically relevant system. The scientific premise of the proposed work is that harnessing the power of single-particle imaging of virion fusion provides a unique opportunity to address the lingering questions in HSV-mediated membrane fusion mechanism, with the ultimate goal of reconstructing the HSV-1-mediated fusion pathway more fully.