Membrane fusion during HSV-1 entry

描述（由申请人提供）：单纯疱疹病毒（HSV）和可能的所有疱疹病毒使用两种方法进入细胞：（1）直接融合：病毒在细胞表面将其脂质双层与质膜融合，以及（2）受体介导的内吞作用（RME）：病毒在内吞作用和pH值降低后与内体膜融合。术语的目标是了解在HSV-1诱导的膜融合过程中发生的直接融合和RME的分子事件。我们最近已经确定了一个融合中间体（半融合）在病毒细胞融合HSV-1。这是第一次描述任何疱疹病毒或任何使用两种以上蛋白质介导膜融合的病毒的融合中间体。我们的初步数据与发生在细胞表面的半融合一致，这些细胞需要内吞作用和低pH值才能进入HSV-1。本申请的重点是研究我们新提出的疱疹病毒RME模型中的膜融合。该模型的一个独特的方面是所提出的半融合事件的细胞表面位置。我们在此概述了通过将半融合位点与细胞标记物共定位来建立HSV-1 RME期间半融合的细胞定位的实验。我们还将使用阻断内吞作用和内体pH降低的药理学试剂，以确定这些过程是否是发生半融合所必需的。因为疱疹病毒进入发生在中性pH下的细胞表面，并且假定发生在晚期内体的低pH下，所以可测试的预测是HSV-1融合机器必须在中性和低pH下起作用。为了测试该预测，我们将研究HSV-1在低pH下的膜融合，并将其与中性pH下的膜融合进行比较。从该应用获得的结果将建立HSV-1的模型。1 RME。对直接融合和RME过程中诱导的膜融合的详细了解对于促进抑制剂的设计以阻断半融合、完全融合和病毒传播是必要的。 公共卫生相关性：本项目的目的是表征单纯疱疹病毒1型（HSV-1）通过受体介导的内吞作用进入细胞过程中的膜融合事件。HSV-1通过多种途径进入细胞，因此需要对每种途径的机制进行复杂的理解，以促进抗病毒药物的设计，以阻止病毒的进入和传播。

DESCRIPTION (provided by applicant): Herpes simplex virus (HSV), and likely all herpesviruses, use two methods to enter cells: (1) direct fusion: the virus fuses its lipid bilayer with the plasma membrane at the cell surface and (2) receptor-mediated endocytosis (RME): the virus fuses with an endosomal membrane after endocytosis and a reduction in pH. Our long-term goal is to understand the molecular events occurring during HSV-1-induced membrane fusion for both direct fusion and RME. We have recently identified a fusion intermediate (hemifusion) during virus-to- cell fusion for HSV-1. This is the first description of a fusion intermediate for any herpesvirus or any virus that uses more than two proteins to mediate membrane fusion. Our preliminary data are consistent with hemifusion occurring at the cell surface in cells that require endocytosis and low pH for HSV-1 entry. The focus of this application is to examine membrane fusion in our newly proposed model for herpesvirus RME. A unique aspect of this model is the proposed cell-surface location of the hemifusion event. We outline experiments here to establish the cellular location of hemifusion during HSV-1 RME by co- localizing the site of hemifusion with cellular markers. We will also use pharmacological agents that block endocytosis and endosomal pH reduction to determine if those processes are required for hemifusion to occur. Because herpesvirus entry occurs at the cell surface at neutral pH and is postulated to occur at the low pH of a late endosome, a testable prediction is that the HSV-1 fusion machinery must function at both neutral and low pH. To test that prediction, we will examine HSV-1 membrane fusion at low pH and compare it to that at neutral pH. The results obtained from this application will establish a model for HSV-1 RME. A detailed understanding of membrane fusion induced during direct fusion and RME is necessary to facilitate the design of inhibitors to block hemifusion, full fusion, and virus spread. PUBLIC HEALTH RELEVANCE: The aim of this project is to characterize the membrane fusion events during herpes simplex virus type 1 (HSV-1) entry into cells via receptor-mediated endocytosis. HSV-1 enters cells by multiple routes so the intricate understanding of the mechanism of each route is required to facilitate the design of anti-virals to block virus entry and spread.