Molecular basis for paramyxovirus entry

描述(申请人提供)：副粘病毒是被包裹的病毒，通过直接与细胞膜融合进入细胞。在进入过程中，两种病毒表面糖蛋白HN(受体结合分子)和F(病毒融合蛋白)以高度特异的方式协作，在受体结合时介导融合。然而，在病毒复制和在细胞质中组装的过程中，融合过程是静默的，在传入的病毒到达其目标细胞之前，融合机器是不活动的。融合过程在时间和地点上是如何协调的？为了了解这些机制并阐明副粘病毒是如何进入细胞的，我们研究了人类副流感病毒(HPIV)，这是导致儿童呼吸道疾病的重要原因。我们的结果揭示了受体结合蛋白在副粘病毒融合中的基本作用，以及病毒生命周期中糖蛋白之间协调相互作用的原理。我们已经开发了新的跨学科工具来剖析副粘病毒控制进入的复杂过程，现在可以解决该领域的核心问题。具体目的1.副粘病毒受体结合分子不同功能的协调机制。HN的主要受体结合部位的附着如何转化为由HN的第二结合/触发部位促进的F-触发？HN两个功能结合位点的突变将被用来研究HN如何触发F。这些实验将定义HN的活动是如何与HPIV相协调的，以及其他副粘病毒中的不同策略如何实现同样的目的。具体目的2.受体结合分子和融合分子在融合和进入过程中的通讯方式。在融合和进入过程中，HN和F之间的交流是什么性质，这种交流是如何调节的？一系列创新策略将检验这一假设，即特定的HN-F相互作用调节F-激活。这些实验将首次定义组成副粘病毒融合机器的分子之间的生物学相关对话。具体目标3.呼吸道细胞副粘病毒生命周期中融合的调节。F-激活的正确时机是进入细胞的关键；对于感染，只有当F与靶细胞膜接触时才能触发。这些蛋白在病毒生命周期中的不同功能是如何调节的，我们能否颠覆这种调节作为抗病毒药物的策略？我们将在一个代表人类肺上皮细胞的系统中，测试F-触发的失调阻止成功感染的假设。我们将确定化合物是否可以过早地触发F，以及这是否可以预防呼吸道感染，从而提供一种新的抗病毒策略。这里揭示的基本原理将提供对需要变构激活的复杂分子机器的见解。这一结果可能适用于其他病原体，这些病原体必须仅在启动感染的特定时间和地点触发这些关键进入活动。与公共卫生相关：这项研究建议将使人们更好地理解副粘病毒如何进入人类细胞开始感染。新确定的参与进入过程的机制可能成为抗病毒药物治疗或预防人类呼吸道疾病的潜在新目标。

DESCRIPTION (provided by applicant): Paramyxoviruses are enveloped viruses that enter cells by fusing directly with the cell membrane. During entry the two viral surface glycoproteins HN (the receptor-binding molecule) and F (the viral fusion protein) cooperate in a highly specific way to mediate fusion upon receptor binding. However, during virus replication and assembly in the cytoplasm the fusion process is silent, and until an incoming virus meets its target cell, the fusion machine is inactive. How is the fusion process coordinated in time and place? To understand these mechanisms and elucidate how paramyxoviruses enter cells, we study the human parainfluenza virus (HPIV), an important cause of respiratory disease in children. Our results have uncovered fundamental roles of the receptor binding protein in paramyxovirus fusion, and principles of coordinated interaction between the glycoproteins during the viral life cycle. We have developed new, interdisciplinary tools to dissect the complex process whereby paramyxoviruses control entry, and central questions in the field can now be addressed. Specific Aim 1. Mechanisms by which the diverse functions of the paramyxovirus receptor-binding molecule are coordinated. How does attachment by the primary receptor binding site of HN translate into the F-triggering that is promoted by HN's second binding/triggering site? Mutations at HN's two functional binding sites will be used to study how HN works to trigger F. These experiments will define how the activities of HN are coordinated for HPIV, and how distinct strategies in other paramyxoviruses accomplish the same ends. Specific Aim 2. How the receptor-binding molecule and the fusion molecule communicate during fusion and entry. What is the nature of the communication between HN and F during fusion and entry, and how is this communication regulated? A series of innovative strategies will test the hypothesis that specific HN-F interactions regulate F-activation. These experiments will for the first time define the biologically relevant dialog between the molecules that comprise the paramyxovirus fusion machine. Specific Aim 3. Regulation of fusion during the paramyxoviral life cycle in airway cells. Correct timing of F-activation is essential for entry; for infection, triggering must occur only when F is in contact with the target cell membrane. How are the diverse functions of these proteins regulated during the viral life cycle, and can we subvert this regulation as a strategy for antivirals? We will test the hypothesis that dysregulation of F-triggering precludes successful infection, in a system that represents human lung epithelium. We will determine whether compounds can prematurely trigger F, and whether this may prevent airway infection, providing a new antiviral strategy. The underlying principles revealed here will offer insights into complex molecular machines that require allosteric activation. The results will likely apply to other pathogens that must trigger these key entry activities only at a specific time and place to initiate infection. PUBLIC HEALTH RELEVANCE: This research proposal will lead to a better understanding of how paramyxoviruses enter human cells to initiate infection. Newly identified mechanisms involved in the entry process could serve as potential new targets for antivirals to treat or prevent human respiratory diseases.