Function of the membrane proximal domains of the SARS-CoV-2 Spike protein in membrane fusion – FUMESPIME

Cellular infection by SARS-CoV-2 begins with the fusion of its lipid envelope with the host cell membrane, a process mediated by the Spike (S) protein of the viral membrane. The S protein is composed of two subunits: S1, responsible for docking to the host cell and S2, which drives fusion. Fusion by S2 starts with the insertion of its fusion peptide FP1 into the target cell membrane, forming a molecular bridge. S2 then folds back onto itself, which brings the viral and cellular membranes in close apposition and triggers fusion. The final stages of S2-mediated fusion, leading to lipid bilayer merging and fusion pore opening, are less understood but likely involve membrane perturbation by FP1, possibly acting in conjunction with the TM domain and its adjacent pTM and Cyto domains. We have recently found that FP1 mediates membrane fusion in vitro and that the presence of cholesterol and ceramide in the membrane strongly enhances FP1-induced fusion. The membrane proximal domains of S2, pTM and Cyto, were also proposed to interact with cholesterol but the exact role of these protein-lipid interactions in S2-mediated fusion remains to be established. In this project, we will study the molecular mechanisms of S2-mediated membrane fusion, with a particular focus on how its FP1 and membrane proximal domains, pTM and Cyto, cooperate with each other and with specific lipids to induce fusion. To achieve this, we will use a combination of in vitro cell-free membrane imaging and docking/fusion assays (partner 1) along with in situ observations of cell-cell fusion events (partner 2). We will generate protein constructs of increasingly complex sequence and structure to progressively mimic the molecular architecture of S2. We also aim to purify the full-length S protein (S1+S2) and its cellular receptors to reconstitute S-mediated membrane docking and fusion in vitro and characterize the protein determinants crucial for viral infection. Notably, we will investigate the optimal concentration of S required for docking and fusion, as well as the influence of receptor presence on the cellular membrane.