Abstract
The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein binds to the cellular receptor-angiotensin-converting enzyme-2 (ACE2) as the first step in viral cell entry. SARS-CoV-2 spike protein expression in the ACE2-expressing cell surface induces cell-cell membrane fusion, thus forming syncytia. To exert its fusogenic activity, the spike protein is typically processed at a specific site (the S1/S2 site) by cellular proteases such as furin. The C488 residue, located at the spike-ACE2 interacting surface, is critical for the fusogenic and infectious roles of the SARS-CoV-2 spike protein. We have demonstrated that the C488 residue of the spike protein is involved in subcellular targeting and S1/S2 processing. C488 mutant spike localization to the Golgi apparatus and cell surface were impaired. Consequently, the S1/S2 processing of the spike protein, probed by anti-Ser-686-cleaved spike antibody, markedly decreased in C488 mutant spike proteins. Moreover, brefeldin-A-mediated endoplasmic-reticulum-to-Golgi traffic suppression also suppressed spike protein S1/S2 processing. As brefeldin A treatment and C488 mutation inhibited S1/S2 processing and syncytia formation, the C488 residue of spike protein is required for functional spike protein processing.