Protein-mediated membrane fusion is essential for a multitude of fundamental biological processes. Despite intensive study, at present we have a limited mechanistic understanding of how fusion protein machinery manipulates lipid membranes in order to induce their fusion. This lack of knowledge is particularly acute regarding the structure of membrane intermediates, the extent to which their leaflets are bent or disrupted into nonbilayer structures, and how they are coordinated and remodeled by fusogens. Similarly, in terms of the structure of the fusion proteins themselves, very little structural information is available to describe how they change as they drive membrane fusion. These are processes that are targeted by therapeutics such as fusion inhibitors or neutralizing antibodies in the case of preventing virus infection, and they are processes that can go awry as a result of disease mutations for cellular fusogens. The proposed studies will expand our understanding of these fundamental processes and reveal general principles employed by divergent fusion machines. Cryo-electron microscopy and structural mass spectrometry provide powerful complementary methods to directly image and probe membrane fusion because they allow us to trigger a fusion reaction under native conditions then trap and then image or analyze intermediate states over the course of the reaction. Cryo-electron tomography in particular can resolve individual fusion machines and membrane leaflets captured in the process of fusing and can discern when the proteins and membranes have adopted non-canonical intermediate structures. Hydrogen/deuterium- exchange mass spectrometry complements cryo-EM by enabling us to monitor local backbone dynamics under native conditions. This approach is particularly effective for tracking conformational changes and for comparing protein structure in different states. Building on our work with influenza virus, we will apply these methods to investigate pathways of membrane fusion in two Class I viral fusion systems: the Env fusion protein used by HIV and the S spike protein used by SARS-CoV-2. These fusion machines employ sequential modes of activation and triggering involving receptor priming followed by either coreceptor binding (Env) or a proteolytic cleavage event (S). These systems thus offer the opportunity to analyze in detail the fusion system arrested at an intermediate, primed stage. For each of these systems, our goal is to image the architecture and progression of membrane remodeling leading to formation of fusion pores and to understand the means by which the protein machinery induces two separate membrane bilayers to join into one. By performing such an analysis, we will gain novel insight into general, obligatory events in Class I protein-mediated membrane fusion, while also revealing system-specific mechanisms. Our study should thus advance our structural and mechanistic understanding of the fundamental process of biological membran...