Dissecting the early steps of the Nipah virus fusion cascade

描述（由申请人提供）：尼帕病毒（NiV）是最致命的副粘病毒，人类死亡率高达75%。NiV和亨德拉病毒（HeV）（亨帕病毒科）是副粘病毒科中的新出现病毒，它们的感染可引起人类和牲畜的呼吸道和脑病。大多数副粘病毒的进入需要附着（HN、H或G）和融合(F)糖蛋白的协同作用。附着蛋白（NiV为G）结合细胞受体，G受体结合（NiV为ephrinB2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蛋白表现出与I类融合蛋白共同的典型特征，通过两阶段的构象级联引起融合：第一阶段从亚稳态预融合状态发展到预发夹中间体（PHI），而第二阶段的标志是从PHI过渡到六螺旋束发夹，最终的构象变化导致病毒和宿主细胞膜在病毒进入时融合。我们的总体驱动假设是，NiV-F和-G中的融合调节基序可以通过融合级联的不同中间体来表现它们的作用。我们的目标是更好地了解调节融合级联各个步骤的关键参数，从而促进针对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态的f触发分析，可以量化膜融合级联中不同中间体的半衰期。此外，我们使用了多方面的策略来确定受体诱导的G的构象变化对其触发F的能力至关重要。我们开发了敏感的细胞-细胞和病毒-细胞融合动力学分析，以研究在低于BSL4条件下NiV的进入，这是一种检测F/G相互作用的新方法。以及针对F和G的新型构象抗体，这些构象抗体帮助我们确定了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，我们最近发现了NiV-F和-G中融合性的新决定因素，包括(a) NiV-F中调节膜融合的第三个七tad重复（HR3）区域，(b) F和G相互作用的亲疏度是融合性的主要决定因素，以及(c) G头部和茎部区域的特定区域对F的变弹性触发很重要。我们将使用我们的创新分析来研究我们的新的融合性决定因素。因此，我们提出以下两个不同但互补的目标：(1)阐明HR3区域在NiV融合级联中的新型融合调节作用。(2)了解F触发过程中F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针对新出现的致命NiV病原体进入的治疗方法的发展，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. PUBLIC HEALTH RELEVANCE: The paramyxoviruses include serious human pathogens, such as measles, mumps, human parainfluenza, Hendra, and Nipah viruses. The latter is the deadliest of the known paramyxoviruses. Understanding how Nipah virus infects cells, specifically the early events of membrane fusion and entry, will provide new approaches for prevention and therapy of not only Nipah virus, but more broadly for paramyxoviruses.