Mechanisms of Nipah virus fusion and entry

副粘病毒科由全球流行的人类病原体组成，如麻疹、腮腺炎、人副流感以及致命的尼帕病毒和亨德拉病毒。NiV在人类中的死亡率约为75%，是NIAID研究议程中的BSL-4 C类优先病原体，并且被世界卫生组织列为可能导致未来大流行的病原体，需要“紧急行动”。NiV和HeV是一个快速增长的属，最近发现了约20种亨尼帕病毒；因此，有可能在人群中出现更多的亨尼帕病毒。对于NiV的动物-人传播和人与人之间的传播以及缺乏批准的疫苗或治疗方法，强调需要进行研究和开发治疗方法。细胞进入的过程是所有病毒感染的关键，为抗病毒治疗提供了靶点。在我们的第一个资助期，我们在建立新的概念和工具来剖析膜融合过程的步骤方面取得了重大进展。因此，我们准备建立和扩展这一进展，以机械地了解最致命的亨尼帕病毒的膜融合过程，对副粘病毒有更广泛的影响。副粘病毒进入细胞（病毒-细胞融合）和病理性合胞体形成（细胞-细胞融合）与感染相关，需要膜融合，这一过程由两种病毒蛋白协调：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触发和膜融合级联的后续步骤联系起来，仍然是包括NiV和HeV在内的副粘病毒的关键知识空白。在我们提出的研究中，我们将解决这些知识空白，并验证在NiV G和F中新发现的融合调节域调节膜融合级联的不同特定的早期和晚期中间产物的假设。为了验证这一假设，我们确定了许多有用的G和F突变体，包括能够结合受体但不能触发F的突变体，或者能够触发F但在F触发后的步骤中捕获融合级联。这些是令人兴奋和非常有用的副粘病毒表型，可以梳理出膜融合级联的步骤。此外，我们最近的技术进步包括：检测膜融合的不同早期和晚期中间产物的分析，以及通过流动病毒学检测病毒颗粒上G和F构象变化和相互作用的工具。因此，我们首次收集了识别单个膜融合中间体所需的概念和技术进展，并揭示了支配亨尼帕病毒膜融合的机制。我们将使用这些工具来实现：目标1。确定NiV-G头部和茎结构域如何调节受体诱导的膜融合；目标2。确定NiV-F如何调节f触发和后期膜融合步骤；和Aim 3。确定G/F相互作用如何调节膜融合和病毒进入。我们的目标的完成将为导致感染的亨尼帕病毒膜融合过程创建一个全面的机制模型，可能对副粘病毒产生更广泛的影响。

The Paramyxoviridae family is comprised of globally prevalent human pathogens such as measles, mumps, human parainfluenza, and the deadly henipaviruses Nipah (NiV) and Hendra (HeV). NiV has a mortality rate in humans of ~75%, is a BSL-4 Category C priority pathogen in the NIAID Research Agenda and is listed by the WHO as likely to cause future pandemics, requiring “urgent action.” NiV and HeV represent a rapidly growing genus with ~20 recently discovered henipaviruses; thus, it is possible that additional henipaviruses will emerge in the human population. For NiV animal-to-human and human-to-human transmission and the lack of approved vaccines or therapeutics, underscore the need for research and treatment development. The process of cell entry is key to infection of all viruses and provides targets for antiviral treatments. In our first funding period, we made significant progress in establishing novel concepts and tools to dissect the steps of the membrane fusion process. Thus, we are poised to build and expand upon this progress to mechanistically understand the membrane fusion process for the deadliest henipaviruses, with broader impact for the paramyxoviruses. Paramyxoviral entry into cells (viral-cell fusion) and the pathologic syncytia formation (cell-cell fusion) associated with infections, require membrane fusion, a process coordinated by two viral proteins: the attachment (HN, H, or G) and fusion (F) glycoproteins. How G/F interactions link cell receptor binding to F-triggering and later steps in the membrane fusion cascade remain critical knowledge gaps for the paramyxoviruses, including NiV and HeV. In our proposed studies, we will address these knowledge gaps and test the hypothesis that newly-discovered fusion-modulatory domains in NiV G and F modulate distinct specific early and late intermediates of the membrane fusion cascade. To test this hypothesis, we identified many useful G and F mutants, including mutants capable of receptor-binding but incapable of F-triggering, or capable of F-triggering but trapping the fusion cascade at post-F-triggering steps. These are exciting and highly-useful paramyxoviral phenotypes for teasing out the steps of the membrane fusion cascade. Further, our recent technical advances include: assays to measure the distinct early and late intermediates of membrane fusion, and tools to detect G and F conformational changes and interactions on viral particles in situ by flow virometry. Thus, for the first time, we have gathered the conceptual and technical advances needed to discern the individual membrane fusion intermediates and reveal mechanisms that govern henipaviral membrane fusion. We will use these tools to: Aim 1. Determine how the NiV-G head and stalk domains modulate receptor-induced membrane fusion; Aim 2. Determine how NiV-F modulates F-triggering and late membrane fusion steps; and Aim 3. Determine how G/F interactions modulate membrane fusion and viral entry. Completion of our Aims will create a comprehensive mechanistic model for the henipaviral membrane fusion process leading to infection, with likely broader impact for the paramyxoviruses.