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.