PROJECT SUMMARY Variants of SARS-CoV-2 continue to emerge with mutations in Spike that cause increased resistance to monoclonal antibodies and vaccines. These variants underscore the need for more universal antiviral approaches, such as targeting conserved regions in Spike and utilizing more broadly reactive Fc-mediated immune functions. Spike contains highly conserved regions in the S2 domain which may be an attractive target for the design of inhibitors. These regions are thought to be exposed when S2 undergoes large conformational changes to mediate membrane fusion and viral entry. Molecular dynamics simulations have modelled this process in silico, structural detail is lacking in situ which leaves a gap in our understanding of viral entry and may preclude further inhibitor development. Spike can also be targeted by antibody Fc effector functions such as antibody-dependent cellular cytotoxicity. Antibody Fc effector functions against Spike have been shown to be more broadly reactive and longer lasting in patients than virus neutralization. Eliciting stronger Fc-mediated immunity is therefore an important consideration in the design of immunogens and antibody therapies. However, Spike-IgG-Fc receptor complexes in native membranes have never been described at the molecular level which makes it difficult to define the structural correlates of Fc effector functions. The overarching goal of this proposal is to investigate the conserved Spike S2 domain and its inhibition during membrane fusion and Fc effector functions targeting Spike within native membranes. I hypothesize that Spike-host receptor interactions in native membranes are key vulnerabilities that can be targeted through antibody Fc effector functions and inhibitors to the conserved S2 domain. To test this hypothesis, I have developed a system to observe Spike-host receptor interactions in situ by presenting them on opposing virus-like particles (VLPs) and monitoring their interactions with cryo-electron tomography (cryoET). In Aim 1, I will investigate the conserved S2 domain during membrane fusion by arresting the membrane fusion process at different stages. My preliminary data show how temperature arrests and inhibitors stabilize prefusion Spike and S2 intermediate structures in situ to provide a unique window into viral entry. Further, my data suggests that multivalent inhibitor cross-linking of S2 intermediates may be a key antiviral strategy to disrupt the cooperative arrangements of S2 that orchestrate membrane fusion. In Aim 2, I will identify structural correlates of Fc effector functions by determining how antibody Fc accessibility and the ability to cluster Spikes and Fc receptors on membranes affect Spike-antibody-Fc receptor complex formation. In preliminary data, I have visualized these complexes in situ in unprecedented molecular detail using cryoET. I have also developed a method to directly visualize and quantify antibody-mediated Spike clustering on virion membranes. Col...