Mechanisms of Nipah virus fusion and entry

描述（由申请人提供）：副粘病毒是全球流行的人类病原体，包括麻疹、腮腺炎、呼吸道合胞体、人类副流感和亨尼帕病毒。以尼帕病毒和亨德拉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%。已报告了NiV的动物-人传播和人-人传播，这突出了研究和开发治疗方法的必要性。因此，在NIAID研究议程中，NiV被列为风险组4和C类优先病原体。解剖感染和传播所需的机制将为抗病毒药物阻断疾病发病机制的关键早期步骤提供新的靶点。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，仍然是包括NiV和HeV在内的副粘病毒的关键知识空白。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结构域（头部）通过一个茎结构域连接到它的跨膜锚点上。F是一类三聚体融合蛋白，具有同类蛋白的典型结构/功能特征。I类融合蛋白被合成为三聚体前体，被切割激活成亚稳的预融合构象，为实现膜融合做好准备。裂解产生一种新的疏水n端融合肽（FP），它被埋在分子内，直到f触发和预发夹中间体（PHI）。

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.