Show on the host cell plasma membrane, which enables neighboring cells to fuse into multinucleated `syncytia’ (Ciechonska and Duncan, 2014; Compton and Schwartz, 2017; Duelli and Lazebnik, 2007). Past research of respiratory syncytial virus (RSV), human immunodeficiency virus (HIV), and other folks recommend that cell-cell fusion can play crucial roles in pathogenicity, whether it be in viral replication, or evasion in the host immune response (Frankel et al., 1996; Johnson et al., 2007; Maudgal and Missotten, 1978). Pioneering function on SARS-CoV-1 (Li et al., 2003) at the same time as current research on SARS-CoV-2 identified similar syncytia (Buchrieser et al., 2020; Cattin-Ortola et al., 2020; Adiponectin Receptor Agonist custom synthesis Hoffmann et al., 2020a; Ou et al., 2020; Papa et al., 2020; Xia et al., 2020; Zang et al., 2020b), which may possibly or might not be relevant to patient pathology (Bryce et al., 2020; Giacca et al., 2020; Rockx et al., 2020; Tian et al., 2020). It remains an open query if syncytia are related to viral and host cell membrane composition, and no matter whether their formation delivers mechanistic insights into cholesterol-targeting therapeutics repurposed for COVID-19 therapy (Daniels et al., 2020; Zhang et al., 2020). Here, we address these important gaps in our understanding of COVID-19 pathobiology by employing a suite of microscopy-based approaches built around the acquiring that co-cultures of ACE2- and spike-expressing cells amass widespread syncytia. Mechanistically, ACE2-spike clusters assemble at transcellular, synapse-like contacts, which precede fusion pore formation and multinucleation. A high-throughput screen for modulators of cell-cell fusion, involving 6000 compounds and 30 spike variants, collectively underscore an crucial part of biophysical characteristics on the membrane, particularly spike-associated mTOR Inhibitor Species cholesterol, for SARS-CoV-2 infection. Our final results recommend that modulation of membrane composition may well inhibit viral propagation, and further informs important lipid-protein assemblies in physiological syncytia and cell adhesion.ResultsSyncytia derive from fusion events at synapse-like, spike-ACE2 protein clustersGiven the central function of your ACE2-spike interaction in viral infection (Hoffmann et al., 2020b; Li et al., 2003; Mittal et al., 2020), we sought to develop a live cell microscopy assay of binding and membrane fusion. We generated pooled populations of human osteosarcoma (U2OS) cells,Sanders, Jumper, Ackerman, et al. eLife 2021;10:e65962. DOI: https://doi.org/10.7554/eLife.2 ofResearch articleCell Biologychosen for their flat morphology and lack of essential fusion machinery (Beck et al., 2011), which stably express fluorescently tagged ACE2 or spike (full-length, `FL’ vs. receptor-binding domain, `RBD’; see Figure 1A for domain organization), applying the B7 transmembrane (`TM’) domain (Liao et al., 2001; Lin et al., 2013) as a control. Upon co-culture, ACE2 and spike RBD cluster at cell-cell interfaces in a binding-dependent manner (Figure 1B). By contrast, and in agreement with other people (Buchrieser et al., 2020; Cattin-Ortola et al., 2020; Hoffmann et al., 2020a; Ou et al., 2020; Xia et al., 2020; Zang et al., 2020b), spike FL/ACE2 interactions drove membrane fusion, with all the vast majority of cells joining multinucleated syncytia soon after a day of co-culture (Figure 1C). We reasoned that if this co-culture system recapitulates established findings with regards to spike/ ACE2-mediated viral entry, it may serve as a helpful high-throughput proxy assay for infection, with no.