Fig. 1.

The fusogenic and infective functions of the spike protein are disrupted by mutation of Cys-488.

(A) Amino acid sequence similarity around Cys-488 in SARS-CoV-2 RBD among ACE2-interactive spikes of SARS-related coronaviruses. The number at right position of each alignment is the amino acid position of the final Tyr (Y). (B) The structure model of ACE2 and RBD interaction from PDB. Disulfide bridges between Cys-480 and -488 are indicated.(C) C488A mutant spike expression did not induce syncytium formation. Expression of Wuhan type and C488A mutant spike proteins (Green signal) in Vero cells were probed in non-permeabilized cells. (D) Expression of mutant spike proteins detected by Western blot analysis Wuhan type (WT) and mutant spike proteins were expressed in 293T cells. GAPDH is shown as a loading control. (E) The infectivities of the pseudotyped viruses expressing each mutant spike protein were assessed by a reporter luciferase activities in VeroE6/TMPRSS2 cells. Luciferase activity is shown in arbitrary units (AU). Data from triplicated samples were expressed as the means ± standard deviation (SD). One-way ANOVA was performed to assess statistical significance. ∗ indicates p < 0.05, and ∗∗ indicates p < 0.01. (F) The assays for spike-induced syncytium formation and cell-cell fusion were illustrated. (G) Syncytium formation by spike expression. Each mutant spike protein was co-expressed with GFP in VeroE6/TMPRSS2 cells. At 12 h after transfection, syncytium formation was visualized by green signal from GFP. Giant green cells indicate syncytium formation. (H) Spike-mediated cell-cell fusion. 293T cells transiently co-expressing each spike and GFP were overlaid on VeroE6/TMPRSS2 cells, and images of cell-cell fused giant adherent cells were captured after 3 h incubation. The lack of giant green adherent cells after treatment with the C488A mutant spike indicate a lack of cell-cell fusion between C488A mutant spike-expressing 293T cells and VeroE6/TMPRSS2 cells. Experiments were repeated independently twice and representative images are shown.