Mechanisms of Nipah virus fusion and entry

描述(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、呼吸道合胞病毒、人类副流感和埃尼帕病毒。以Nipah(Niv)和Hendra(HEV)病毒为代表的海尼帕病毒是最致命的副粘病毒。尼罗河病毒在人类中的死亡率为40%-92%，在最近的疫情中平均为75%。已经报告了新城疫的动物传人和人传人，强调了研究和治疗开发的必要性。因此，新城疫病毒在NIAID研究议程中被列为第4风险组和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，以阻止疾病发病的关键早期步骤。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，仍然是包括新城疫和戊型肝炎病毒在内的副粘病毒的一个关键知识缺口。副粘病毒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(Head)结构域，通过茎结构域连接到它的跨膜锚上。F是一种三聚体I类融合蛋白，具有与其类相同的典型结构/功能特征。I类融合蛋白被合成为三聚体前体，被切割激活为亚稳定的融合前构象，为膜融合做好准备。裂解产生一种新的疏水性N末端融合肽(FP)，它被埋在分子内直到F-触发和前发夹中间体(PHI) 形成，当FP插入到靶细胞膜中时。PHI含有两个螺旋区(HR1和HR2)，它们有很高的相互结合的倾向，形成一个六螺旋束(6HB)，使膜融合成为可能。我们的初步研究表明，受体结合导致NIV-G头部结构变化的机制暴露了NIV-G柄C-末端结构域，从而触发NIV-F。在目标1中，我们将确定NIV-G头和柄中的特定结构域是否对于NIV-F触发和病毒进入是必要的和充分的。我们在NIV-F中发现了三个新的融合调节区：HR3、N1和N4。在目标2中，我们将确定在NIV-G信号转导时，这些融合调节区是否破坏融合前NIV-F构象的稳定和/或调节融合过程中的后续步骤。我们发现Niv G/F解离在膜融合过程中起着重要作用。在目标3中，我们将确定融合前G/F复合体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膜融合过程中是否发生移动。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.