Membrane fusion during HSV-1 entry

描述(申请人提供)：单纯疱疹病毒(HSV)，可能是所有疱疹病毒，使用两种方法进入细胞：(1)直接融合：病毒将其脂双层与细胞表面的质膜融合；(2)受体介导的内吞作用(RME)：病毒在内吞和pH降低后与内吞体膜融合。我们的长期目标是了解HSV-1诱导的膜融合过程中发生的分子事件，包括直接融合和RME。我们最近发现了HSV-1在病毒到细胞融合过程中的一种融合中间产物(半融合)。这是首次描述任何疱疹病毒或任何使用两种以上蛋白质来介导膜融合的病毒的融合中间体。我们的初步数据与细胞表面发生的半融合一致，这些细胞需要内吞作用和低pH值才能进入HSV-1。这项应用的重点是检查我们新提出的疱疹病毒RME模型中的膜融合。这个模型的一个独特之处是提出了半融合事件的细胞表面位置。我们概述了在HSV-1RME期间通过与细胞标志物共同定位半融合部位来确定半融合细胞位置的实验。我们还将使用阻止内吞作用和内体pH降低的药理学药物来确定这些过程是否是发生半血所必需的。由于疱疹病毒在中性pH下发生在细胞表面，并被认为发生在晚期内体的低pH下，因此一个可测试的预测是HSV-1融合机制必须在中性和低pH下都能工作。为了验证这一预测，我们将检测HSV-1在低pH下的膜融合，并将其与中性pH下的融合进行比较。这一应用结果将为HSV-1 RME建立模型。对直接融合和RME过程中诱导的膜融合有详细的了解，这对于设计阻止半融合、完全融合和病毒传播的抑制剂是必要的。 公共卫生相关性：该项目的目的是描述单纯疱疹病毒1型(HSV-1)通过受体介导的内吞作用进入细胞期间的膜融合事件。HSV-1通过多条途径进入细胞，因此需要对每条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.