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、人类副流感以及致命的海尼帕病毒Nipah(Niv)和Hendra(HEV)。新城疫病毒在人类中的死亡率约为75%，是NIAID研究议程中BSL-4 C类优先病原体，世界卫生组织将其列为可能导致未来大流行的疾病，需要“紧急行动”。新城疫病毒和戊型肝炎病毒代表了一个快速增长的属，最近发现了约20种海尼帕病毒；因此，有可能在人类种群中出现更多的海尼帕病毒。对于新城疫病毒在动物与人之间以及人与人之间的传播，以及缺乏已获批准的疫苗或疗法，强调了研究和治疗开发的必要性。细胞进入过程是感染所有病毒的关键，并为抗病毒治疗提供靶点。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，我们准备在这一进展的基础上扩大和发展，从机械上了解最致命的海尼帕病毒的膜融合过程，并对副粘病毒产生更广泛的影响。副粘病毒进入细胞(病毒-细胞融合)和与感染相关的病理性合胞体形成(细胞-细胞融合)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-触发以及膜融合级联的后期步骤联系起来，仍然是副粘病毒，包括新城疫和戊型肝炎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差距，并检验新发现的NIV G和F中的融合调节结构域调节膜融合级联的不同特定早期和晚期中间产物的假设。为了验证这一假设，我们发现了许多有用的G和F突变体，包括能够与受体结合但不能触发F-的突变体，或者能够触发F-但在F-触发后捕获融合级联的突变体。这些都是令人兴奋和高度有用的副粘病毒表型，用于梳理膜融合级联的步骤。此外，我们最近的技术进步包括：测量不同的膜融合早期和晚期中间体的方法，以及通过流动病毒计量学原位检测病毒颗粒上G和F构象变化和相互作用的工具。因此，我们第一次收集了识别单个膜融合中间体并揭示支配病毒膜融合的机制所需的概念和技术进展。我们将使用这些工具来：目的1.确定Niv-G头和柄结构域如何调控受体诱导的膜融合；目的2.确定Niv-F如何调控F触发和晚期膜融合步骤；以及目的3.确定G/F相互作用如何调控膜融合和病毒进入。我们的目标的完成将为导致感染的埃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.