Single-particle 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。这一数字远远超过人类利用的糖蛋白 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 要知道，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FusoGen，通过与其他病毒进行类比 Fusogens，被认为通过经历大规模的折叠而促进膜合并，从而带来病毒和 细胞膜结合在一起。然而，GB的不同寻常之处在于需要额外的几种蛋白质来发挥作用。 因此，要了解Gb在细胞进入过程中如何调节融合，需要直接测量它的 融合活性。近年来，单粒子跟踪(SPT)已经成为一种强有力的定量检测工具 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方法使病毒融合途径的直接可视化和动力学测量成为可能，并具有 已经成功地与pH触发和受体触发的融合原一起使用。这一探索性的目标是 建议开发HSV-1融合的单粒子成像，并支持脂质双层以显示不同的 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 英国的结构调整。鉴于SPT方法尚未应用于使用 &gt；1糖蛋白，并可能难以应用于具有15种包膜蛋白的HSV-1，Aim 1将利用 水疱性口炎病毒(VSV)病毒颗粒缺乏天然FusoGen G，并与HSV-1进入假型 糖蛋白Gb、Gd、Gh和gl(VSVDG-BHLD)，它们保留了HSV-1进入的关键特征。在目标2中， SPT方法将扩展到HSV-1，这是一个更复杂但更具生物学相关性的系统。这个 拟议工作的科学前提是利用病毒粒子聚变的单粒子成像的能力 为解决HSV介导的膜融合中挥之不去的问题提供了一个独特的机会 机制，最终目标是更全面地重建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.