Mechanisms of Nipah virus fusion and entry

描述（由申请方提供）：副粘病毒是全球流行的人类病原体，包括麻疹、腮腺炎、呼吸道合胞病毒、人副流感病毒和亨尼帕病毒。以尼帕（Nipah，NiV）和亨德拉（Hendra，HeV）病毒为代表的亨尼帕病毒是最致命的副粘病毒。NiV在人类中的死亡率为40- 92%，在最近的疫情中平均为75%。据报道，NiV可发生动物与人类之间和人与人之间的传播，这强调了研究和治疗开发的必要性。因此，NiV在NIAID研究议程中被列为风险组4和C类优先病原体。剖析感染和传播所需的机制将为抗病毒药物提供新的靶点，以阻断疾病发病机制中的关键早期步骤。副粘病毒进入哺乳动物细胞（病毒-细胞膜融合）和合胞体形成（细胞-细胞膜融合）都需要膜融合，这需要两种病毒蛋白的协调作用：附着（HN，H或G）和融合（F）糖蛋白。G/F相互作用如何将细胞受体结合与F触发联系起来仍然是副粘病毒（包括NiV和HeV）的关键知识缺口。G/HN/H和F的许多结构和功能特征在副粘病毒中是保守的。G/HN/H有一个受体结合的球状（头部）结构域，通过茎结构域连接到其跨膜锚。F是一种三聚体I类融合蛋白，具有与其类别相同的典型结构/功能特征。I类融合蛋白被合成为三聚体前体，其被切割以活化成亚稳定的融合前构象，准备用于实现膜融合。切割产生一个新的疏水性N-末端融合肽（FP），其被埋在分子内，直到F-触发和前发夹中间体（PHI） 当FP插入到靶细胞膜中时，PHI包含两个螺旋区域（HR 1和HR 2），其具有彼此结合以形成六螺旋束（6 HB）的高倾向，从而实现膜融合。我们的初步研究提出了一种机制，通过该机制受体结合导致NiV-G头部的结构变化，暴露NiV-G茎C-末端结构域以触发NiV-F。在目标1中，我们将确定NiV-G头部和茎部中的特定结构域是否是NiV-F触发和病毒进入所必需和充分的。我们在NiV-F中发现了三个新的融合调节区域：HR 3，N1和N4。在目标2中，我们将确定在NiV-G信号传导后，这些融合调节区域是否使融合前NiV-F构象不稳定和/或调节融合中的后续步骤。我们发现NiV G/F解离在膜融合期间是重要的。在目标3中，我们将鉴定融合前G/F复合物中最关键的相互作用结构域，并确定这些G/F相互作用结构域在NiV膜融合期间是否发生变化。了解调节膜融合的G/F决定因素可以为抗副粘病毒治疗设计提供新的靶点。

DESCRIPTION (provided by applicant): Paramyxoviruses are globally prevalent human pathogens and include measles, mumps, respiratory syncytial, human parainfluenza, and the henipaviruses. The henipaviruses, represented by Nipah (NiV) and Hendra (HeV) viruses are the deadliest paramyxoviruses. NiV's mortality rate in humans is 40-92%, averaging 75% in the latest outbreaks. Animal-to-human and human-to-human transmission have been reported for NiV, underscoring the need for research and treatment development. NiV is thus classified as a Risk Group 4 and a Category C priority pathogen in the NIAID Research Agenda. Dissecting the mechanisms required for infection and spread will provide new targets for antivirals to block critical early steps in disease pathogenesis. Both paramyxovirus entry into mammalian cells (viral-cell membrane fusion) and syncytia formation (cell-cell membrane fusion) require membrane fusion, which necessitates the coordinated actions of two viral proteins: the attachment (HN, H, or G) and fusion (F) glycoproteins. How G/F interactions link cell receptor binding to F-triggering remains a critical knowledge gap for the paramyxoviruses, including NiV and HeV. Numerous structural and functional features of G/HN/H and F are conserved among the paramyxoviruses. G/HN/H has a receptor-binding globular (head) domain connected to its transmembrane anchor via a stalk domain. F is a trimeric class I fusion protein with canonical structural/functional features common to its class. Class I fusion proteins are synthesized as trimeric precursors that are cleaved for activation into a metastable pre-fusion conformation, poised for enabling membrane fusion. Cleavage generates a new hydrophobic N-terminal fusion peptide (FP) that is buried intra-molecularly until F-triggering and pre-hairpin intermediate (PHI) formation, when the FP is inserted into the target cell membrane. The PHI contains two helical regions (HR1 and HR2) with high propensity to bind each other to form a six-helix bundle (6HB), enabling membrane fusion. Our preliminary studies suggest a mechanism by which receptor binding causes structural changes in the NiV-G head that expose a NiV-G stalk C-terminal domain to trigger NiV-F. In Aim 1 we will determine if specific domains in the NiV-G head and stalk are necessary and sufficient for NiV-F triggering and viral entry. We uncovered three new fusion-modulatory regions in NiV-F: HR3, N1, and N4. In Aim 2 we will determine if upon NiV-G signaling, these fusion-modulatory regions destabilize the pre-fusion NiV-F conformation and/or modulate later steps in fusion. We discovered that NiV G/F dissociation is important during membrane fusion. In Aim 3 we will identify the most critical interactive domains in the pre-fusion G/F complex, and determine if these G/F interactive domains shift during NiV membrane fusion. Understanding the determinants of G/F that modulate membrane fusion can offer new targets for anti-paramyxoviral therapeutic design.