Many enveloped viruses induce multinucleated cells (syncytia), reflective of membrane fusion events caused by the same machinery that underlies viral entry. These syncytia are thought to facilitate replication and evasion of the host immune response. Here, we report that co-culture of human cells expressing the receptor ACE2 with cells expressing SARS-CoV-2 spike, results in synapse-like intercellular contacts that initiate cell-cell fusion, producing syncytia resembling those we identify in lungs of COVID-19 patients. To assess the mechanism of spike/ACE2-driven membrane fusion, we developed a microscopy-based, cell-cell fusion assay to screen ~6000 drugs and >30 spike variants. Together with quantitative cell biology approaches, the screen reveals an essential role for biophysical aspects of the membrane, particularly cholesterol-rich regions, in spike-mediated fusion, which extends to replication-competent SARS-CoV-2 isolates. Our findings potentially provide a molecular basis for positive outcomes reported in COVID-19 patients taking statins and suggest new strategies for therapeutics targeting the membrane of SARS-CoV-2 and other fusogenic viruses.

SummaryMany enveloped viruses induce multinucleated cells (syncytia), reflective of membrane fusion events caused by the same machinery that underlies viral entry. These syncytia are thought to facilitate replication and evasion of the host immune response. Here, we report that co-culture of human cells expressing the receptor ACE2 with cells expressing SARS-CoV-2 spike, results in synapse-like intercellular contacts that initiate cell-cell fusion, producing syncytia resembling those we identify in lungs of COVID-19 patients. To assess the mechanism of spike/ACE2-driven membrane fusion, we developed a microscopy-based, cell-cell fusion assay to screen ∼6000 drugs and >30 spike variants. Together with cell biological and biophysical approaches, the screen reveals an essential role for membrane cholesterol in spike-mediated fusion, which extends to replication-competent SARS-CoV-2 isolates. Our findings provide a molecular basis for positive outcomes reported in COVID-19 patients taking statins, and suggest new strategies for therapeutics targeting the membrane of SARS-CoV-2 and other fusogenic viruses.HighlightsCell-cell fusion at ACE2-spike clusters cause pathological syncytia in COVID-19Drug screen reveals critical role for membrane lipid composition in fusionSpike’s unusual membrane-proximal cysteines and aromatics are essential for fusionCholesterol tunes relative infectivity of SARS-CoV-2 viral particles

Eukaryotic membranes can be partitioned into lipid-driven membrane microdomains called lipid rafts, which function to sort lipids and proteins in the plane of the membrane. As protein selectivity underlies all functions of lipid rafts, there has been significant interest in understanding the structural and molecular determinants of raft affinity. Such determinants have been described for lipids and single-spanning transmembrane proteins; however, how multi-pass transmembrane proteins (TMPs) partition between ordered and disordered phases has not been widely explored.Here we used cell-derived Giant Plasma Membrane Vesicles (GPMVs) to systematically measure multi-pass TMP partitioning to ordered membrane domains. Across a set of 24 structurally and functionally diverse multi-pass TMPs, the large majority (92%) had minimal raft affinity. The only exceptions were two myelin-associated four pass TMPs, Myelin and Lymphocyte protein (MAL) and ProteoLipid Protein (PLP). We characterized the potential mechanisms for their exceptional raft affinity and observed that PLP requires cholesterol and sphingolipids for optimal association with ordered membrane domains and that PLP and MAL appear to compete for cholesterolmediated raft affinity. These observations suggest broad conclusions about the composition of ordered membrane domains in cells and point to previously unrecognized drivers of raft affinity for multi-pass transmembrane proteins.

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During T cell activation, the transmembrane adaptor protein LAT (linker for activation of T cells) forms biomolecular condensates with Grb2 and Sos1, facilitating signaling. LAT has also been associated with cholesterol-rich condensed lipid domains; However, the potential coupling between protein condensation and lipid phase separation and its role in organizing T cell signaling were unknown. Here, we report that LAT/Grb2/Sos1 condensates reconstituted on model membranes can induce and template lipid domains, indicating strong coupling between lipid- and protein-based phase separation. Correspondingly, activation of T cells induces cytoplasmic protein condensates that associate with and stabilize raft-like membrane domains. Inversely, lipid domains nucleate and stabilize LAT protein condensates in both reconstituted and living systems. This coupling of lipid and protein assembly is functionally important, as uncoupling of lipid domains from cytoplasmic protein condensates abrogates T cell activation. Thus, thermodynamic coupling between protein condensates and ordered lipid domains regulates the functional organization of living membranes.