Revealing determinants for SARS-CoV-2 spike-mediated syncytia formation

Abstract

Multinucleated syncytial pneumonocytes, induced by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), are commonly observed in the postmortem lung tissues of severe COVID-19 cases. The syncytia formation is primarily determined by the fusogenicity of the spike protein of SARS-CoV-2. Infected cells express spikes on their cell surface, which leads to cell-cell fusion with neighboring cells, forming syncytia. Syncytia formation potentially contributes to pathology by facilitating viral transmission, immune evasion, inflammatory response, and cytopathicity. Since its emergence, SARS-CoV-2 has been continuously evolving. Mutations in spike protein result in numerous variants with different degrees of infectivity and pathogenicity. Functionally annotating the syncytia-forming potential of spike variants could help identify variants that may cause severe pathological consequences and should be tracked in the future. Additionally, the in-depth investigation of the host factors crucial for syncytia formation may facilitate the development of novel therapeutic strategies. In this study, we conducted deep mutational scanning on the cytoplasmic tail of the SARS-CoV-2 spike in the context of full-length spike protein under human cell environments. The K1255F mutation was identified and validated to enhance the spike’s fusogenicity and ACE2 binding capacity. Mechanistically, it was demonstrated that the K1255F substitution creates a diaromatic FF motif, which improves endoplasmic reticulum (ER) exit and thus increases spike’s availability at the cell surface for triggering fusion with neighboring cells and forming syncytia. In addition, we developed a size-exclusion selection-based strategy and combined it with the genome-wide CRISPR knockout screen to systematically identify host factors crucial for SARS-CoV-2 spike-mediated syncytia formation. Apart from the known receptor ACE2, two key regulators of clathrin-mediated endocytosis (CME), AP2M1 and FCHO2, were identified as crucial factors for SARS-CoV-2 spike-mediated syncytia formation. It was validated that AP2M1 or FCHO2 knockout in receiver cells significantly reduced the syncytia formation mediated by both the D614G and Omicron spikes. Furthermore, the involvement of CME machinery in driving syncytia formation was confirmed by CHC knockdown and the treatment with CME inhibitors. Moreover, it was demonstrated that the treatment of CME-inhibiting drugs, Chlorpromazine and Fluvoxamine, significantly inhibits SARS-CoV-2 replication and reduces the presence of syncytium-like multinucleated cells in the lung tissues of hamsters infected with SARS-CoV-2. Taken together, these findings demonstrate the crucial role of the CME machinery in driving SARS-CoV-2-induced syncytia formation, providing support for the repurposing of Chlorpromazine and Fluvoxamine to alleviate COVID-19 severity in patients.