Human parainfluenza virus fusion complexglycoproteins imaged in action

项目摘要 人类副流感病毒(HPIV)感染会导致重要的下呼吸道疾病，包括哮喘， 毛细支气管炎和肺炎，导致全世界数百万婴幼儿患病或死亡。 目前还没有疫苗或有效的治疗方法。在病毒进入期间，感染的第一步，病毒融合复合体 -由表面糖蛋白HN(受体结合蛋白；血凝素神经氨酸酶)和F组成 (融合蛋白)-在受体结合时介导融合。HN触发F发生构象变化 促进病毒进入的重排，这些关键步骤还不清楚。这个项目的总体目标是 建议在以下过程中捕获HPIV3 HN-F融合复合体在自然状态下的系列中间状态 激活并用冷冻电子断层扫描(ET)检测融合机制。Cryo-et将直接提供 结构证据与我们提出的糖蛋白复合体在病毒进入过程中的作用机制有关。 目标1将重点介绍HN在使用冷冻-ET防止F介导的融合过早激活方面的保护作用 比较HN-F特异性络合物(1.1)和与HN相互作用并诱导其激活F的小分子 (1.2)。在目标2中，我们将捕捉并可视化HN-F聚变复合体在 核聚变过程的一系列阶段。这些目标的实现以及概述的培训将为我做好准备 以一种将结构生物学方法应用于重要的机械问题的方式推进我的领域 病毒学。 我希望以前所未有的细节描述HN如何触发HPIV3的F-介导融合，并提供明确的 关于病毒融合过程的答案一直是中心和令人费解的-同时也导致了 发展冷冻技术中的尖端技术。使用这种方法，不仅可以测试 试图简化HN-F关系性质的假设，但在 病毒上存在的本机可信配置中的特定中间时间点。我们的实验 将解释必要的中间体是如何形成的，然后从我们的结果中收集的机械数据可以 指导抗病毒策略，因为这种理解是开发禁用融合/进入的方法的先决条件 感染阶段。该项目在技术和概念方面都是创新的，因此提供了一个理想的 培训工具，让我发展成为一名严谨、富有创造力的独立科学家。

Project Summary Infection by human parainfluenza viruses (HPIVs) causes important lower respiratory diseases including croup, bronchiolitis and pneumonia, that lead to illness or death in millions of infants and young children worldwide. There are no vaccines or effective treatments. During viral entry, the first step of infection, the viral fusion complex – comprised of the surface glycoproteins HN (receptor-binding protein; hemagglutinin-neuraminidase) and F (fusion protein) – mediates fusion upon receptor binding. HN triggers F to undergo conformational rearrangements that promote viral entry, and these key steps are not understood. The overall goal of this proposal is to capture serial intermediate states of the HPIV3 HN-F fusion complex in native state during activation and examine the fusion mechanism using cryo-electron tomography (ET). Cryo-ET will provide direct structural evidence related to our proposed mechanisms of action of the glycoprotein complex during viral entry. Aim 1 will focus on HN’s protective role in preventing premature activation of F-mediated fusion, using cryo-ET to compare HN-F specific complexes (1.1) and small molecules that interact with HN and induce it to activate F (1.2). In aim 2, we will capture and visualize the structural rearrangements in the HN-F fusion complex during serial stages of the fusion process. Execution of these aims along with the training as outlined will prepare me to advance my field in a fashion that applies structural biology methods to important mechanistic questions in virology. I expect to characterize in unprecedented detail how HN triggers F-mediated fusion for HPIV3 and provide clear answers about the process of viral fusion that have been central and puzzling – while at the same time leading to development of cutting-edge methods in cryo-ET. Using this approach, it will be possible not only to test hypotheses that attempt to simplify the nature of the HN-F relationship, but to examine the fusion complex at specific intermediate points in time in native, authentic configurations as they exist on the virus. Our experiments will explain how the necessary intermediates form, and the mechanistic data gleaned from our results can then guide antiviral strategies, as this understanding is a prerequisite for developing ways to disable the fusion/entry phase of infection. The project is innovative in both technical and conceptual aspects and thus provides an ideal training vehicle for me to develop into a rigorous and creative independent scientist.