Dissecting the early steps of the Nipah virus fusion cascade

描述（由申请方提供）：尼帕病毒（NiV）是最致命的副粘病毒，人类死亡率高达75%。NiV和Hendra病毒（HeV）（亨德拉病毒科属）是副粘病毒科中的新兴病毒，并且它们的感染引起人类和牲畜的呼吸道和脑炎疾病。大多数副粘病毒的进入需要附着（HN、H或G）和融合（F）糖蛋白的协调作用。附着蛋白（G代表NiV）与细胞受体结合，G受体结合（在NiV的情况下是ephrinB 2或B3）被认为触发F蛋白。然而，F和G相互作用如何将受体结合与F蛋白的触发和融合激活联系起来仍然知之甚少。副粘病毒F蛋白表现出I类融合蛋白共有的典型特征，其通过两相构象级联引起融合：第一阶段从亚稳态融合前状态发展为前发夹中间体（PHI），而第二阶段以从PHI转变为六螺旋束发夹为标志，在病毒进入过程中导致病毒和宿主细胞膜融合的最终构象变化。我们的总体驱动假设是，在NiV-F和-G融合调制基序可以通过不同的中间体的融合级联体现其效果。我们的目标是更好地了解调节融合级联的各个步骤的关键参数，以促进靶向进入过程的抗病毒治疗的发展。对于NiV，我们最近开发了一种定量和动力学F-触发测定，可以量化膜融合级联中不同中间体的半衰期。此外，我们已经使用了一个多方面的战略，以确定受体诱导的构象变化G的关键能力，触发F。我们已经开发了灵敏的细胞-细胞和病毒-细胞融合动力学测定来研究在低于BSL 4条件下的NiV进入，检测F/G相互作用的新测定，以及针对F和G的新型构象抗体，其帮助我们鉴定G中的变构F触发结构域。这些创新和有利的特征使我们能够使用尼帕病毒系统作为副粘病毒进入的启发性模型。此外，我们最近在NiV-F和-G中发现了新的促融合性决定因素，包括（a）NiV-F中调节膜融合的第三个七肽重复（HR 3）区域，（B）F和G相互作用的亲合力作为促融合性的主要决定因素，以及（c）G的头部和茎部区域中对F的变构触发重要的特异性结构域。我们将使用我们的创新检测方法来研究我们的新的促融合性决定因素。因此，我们提出了以下两个不同但互补的目标：（1）阐明新的融合调节作用的HR 3区的NiV融合级联。(2)了解F触发过程中F蛋白和G蛋白之间的协同作用。这些目标的成功完成不仅将促进靶向致命的新兴NiV病原体进入的治疗方法的开发，而且还将促进我们对控制NiV和副粘病毒以及I类病毒膜融合和进入的参数的理解。

DESCRIPTION (provided by applicant): Nipah virus (NiV) is the deadliest Paramyxovirus, with a mortality rate of up to 75% in humans. NiV and Hendra virus (HeV) (Henipaviridae genus) are emerging viruses within the Paramyxoviridae family, and their infections cause respiratory and encephalitic disease in humans and livestock. Entry of most paramyxoviruses requires the coordinated action of the attachment (HN, H, or G) and fusion (F) glycoproteins. The attachment protein (G for NiV) binds the cell receptor, and G-receptor-binding (ephrinB2 or B3 in the case of NiV) is thought to trigger the F protein. However, how F and G interactions link receptor binding to triggering and fusion-activation of the F protein remains poorly understood. Paramyxoviral F proteins exhibit canonical features common to class I fusion proteins, which cause fusion via a two-phase conformational cascade: the first phase progresses from a metastable pre-fusion state to a pre-hairpin intermediate (PHI), while the second phase is marked by transition from the PHI to a six-helix bundle hairpin, the final conformational change that leads to fusion of the viral and host cell membranes during viral entry. Our overall driving hypothesis is that fusion modulatory motifs in NiV-F and -G can manifest their effects through distinct intermediates of the fusion cascade. Our objective is to better understand the critical parameters that modulate the individual steps of the fusion cascade so as to facilitate the development of anti-viral therapeutics that target the entry process. For NiV, we recently developed a quantitative and kinetic F-triggering assay that can quantify the half-lives of distinct intermediates in the membrane fusion cascade. In addition, we have used a multi-faceted strategy to identify a receptor-induced conformational change in G critical to its ability to trigger F. We have developed sensitive cell-cell and virus-cell fusion kinetics assays to study NiV entry at less than BSL4 conditions, a new assay to detect F/G interactions, and novel conformational antibodies against F and G that helped us identify an allosteric F-triggering domain in G. These innovative and advantageous features allow us to use the Nipah virus system as an illuminative model for paramyxoviral entry. In addition, we have recently discovered novel determinants of fusogenicity in NiV-F and -G, including (a) a third heptad repeat (HR3) region in NiV-F that modulates membrane fusion, (b) the avidity of F and G interactions as a primary determinant of fusogenicity, and (c) specific domains in the head and stalk regions of G that are important for the allosteric triggering of F. We will use our innovative assays to study our novel determinants of fusogenicity. Thus, we propose the following two distinct but complementary aims: (1) To elucidate the novel fusion-modulatory role of the HR3 region in the NiV fusion cascade. (2) To understand the cooperativity between the F and G proteins during F triggering. The successful completion of these aims will not only facilitate the development of therapeutics that target entry of the deadly emerging NiV pathogen, but also advance our understanding of the parameters that govern NiV and paramyxovirus and class I viral membrane fusion and entry.