Structural dynamics of single Ebolavirus GP molecules.

 描述(申请人提供)：当前疫情持续存在的部分原因是缺乏任何疫苗和任何经批准的暴露后治疗方法。由于对埃博拉病毒生命周期的了解有限，此类预防和治疗药物的开发进展缓慢。像所有被包膜的病毒一样，埃博拉病毒进入 靶细胞需要将病毒膜融合到细胞膜上。这一过程由包膜糖蛋白(GP)催化。GP是唯一暴露在埃博拉病毒粒子表面的病毒蛋白，使其成为疫苗和抗病毒治疗的诱人靶点。GP向低能构象的转变克服了病毒和细胞膜融合的重要能量障碍。但目前几乎没有关于GP的结构动力学、受体结合对GP构象的影响或GP催化病毒与细胞膜融合的机制的信息。鉴于包膜糖蛋白构象变化在包膜病毒进入机制中的核心作用，理解埃博拉病毒的进入需要深入了解GP的构象图景。在这里，我们提出了一个集成框架的应用，包括smFRET成像以及动力学和热力学分析。我们将应用这一框架来探索完整假病毒粒子表面单个三聚体GP分子的构象动力学，询问它们与细胞受体和抗体的相互作用，并阐明pH变化的影响。这一方法将为我们对埃博拉病毒进入特别是一般情况下的包膜病毒进入的理解带来新的曙光。此外，我们将设计稳定暴露抗原表面的GP变体，并使用我们基于smFRET的框架作为一种强大和高通量的手段来分析这些潜在免疫原的构象图景。因此，我们将开发smFRET作为结构性疫苗设计的工具。

 DESCRIPTION (provided by applicant): The persistence of the current epidemic is in part a result of the lack of any vaccine, and any approved post- exposure therapies. Progress in the development of such preventative and therapeutic agents has been slow due to a limited understanding of the Ebola viral life cycle. Like all enveloped viruses, entry of Ebolaviruses into target cells requires fusion of the viral membrane to a cell membrane. This process is catalyzed by the envelope glycoprotein (GP). GP is the only viral protein exposed on the surface of Ebola virions, making it an attractive target for vaccines and antiviral therapies. The significant energetic barrier to fusing viral and cell membranes is surmounted by transition of GP to successively lower energy conformations. But virtually no information presently exists on the structural dynamics of GP, the impact of receptor binding on GP conformation, or on the mechanism by which GP catalyzes the fusion of viral and cell membranes. Given the central role of envelope glycoprotein conformational changes in the mechanism of enveloped viral entry, an understanding of Ebolavirus entry requires a deep insight into the conformational landscape of GP. Here, we propose the application of an integrated framework involving smFRET imaging, and kinetic and thermodynamic analysis. We will apply this framework to probe the conformational dynamics of individual trimeric GP molecules on the surface of intact pseudovirions, interrogate their interaction with cellular receptors and antibodies, and elucidate the impact of changes in pH. This approach will bring new light to our understanding of Ebolavirus entry specifically, and enveloped virus entry in general. In addition, we will design GP variants that stably expose antigenic surfaces, and use our smFRET-based framework as a robust and high-throughput means of assaying the conformational landscape of these potential immunogens. We will thereby develop smFRET as a tool for structural vaccine design.