Membrane fusion during HSV-1 entry

描述（由申请人提供）：单纯疱疹病毒（HSV）和可能所有的疱疹病毒，使用两种方法进入细胞：(1)直接融合：病毒将其脂质双分子层与细胞表面的质膜融合；(2)受体介导的内吞作用（RME）：病毒在内吞作用和ph降低后与内体膜融合。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病毒与细胞融合过程中的融合中间体（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介导膜融合的病毒的融合中间体。我们的初步数据与在需要内吞作用和低pH值以供HSV-1进入的细胞表面发生的半灌注一致。本应用的重点是在我们新提出的疱疹病毒RME模型中检查膜融合。该模型的一个独特方面是提出的半灌注事件的细胞表面位置。我们在这里概述了通过与细胞标记物共同定位半融合部位来确定HSV-1 RME期间半融合的细胞位置的实验。我们还将使用阻断内吞作用和内体pH值降低的药物来确定这些过程是否需要发生半灌注。由于疱疹病毒在中性pH下发生在细胞表面，并且假定在后期内体的低pH下发生，一个可测试的预测是，HSV-1融合机制必须在中性和低pH下都起作用。为了验证这一预测，我们将检查低pH下的HSV-1膜融合，并将其与中性pH下的融合进行比较。从这个应用中获得的结果将建立HSV-1 RME模型。详细了解直接融合和RME过程中诱导的膜融合对于设计阻断半融合、完全融合和病毒传播的抑制剂是必要的。

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.