Fig. 4.

Furin cleavage in the S1/S2 Site of SARS-CoV-2 spike is required for efficient cell-cell fusion butisnot essential. A. 293 T-S-GFP/293 T-ACE2 mediated cell-cell fusion with the four variants. The effector cell was identified as co-expressing S and GFP proteins, 293 T-ACE2 was the target cell. Mock-fusion between target cells and 293 T/GFP effector cells without S-expression as control. (Scale bar = 100 µm). B. Detection of SARS-CoV-2 S subcellular localization in 293 T/293 T-ACE2-GFP cells fusion by confocal microscopy. Red represented high SARA-Cov-2 antibody binding. GFP (green) represented GFP over-expressed 293 T-ACE2 cells. Nuclei (blue) were counter-stained with DAPI. The nucleus was observed to fuse to form multinucleated syncytia and the adjacent cells grew and fused into pieces. (Scale bar = 20 µm). C. Statistical analysis of fusion rates mediated by wild-type or mutated S protein after co-culture for 4 h. Experiments were repeated at least three times independently, and the data are expressed as means ± SD. p ≤ 0.001[***] indicates significant differences; ns: no significance. D. Detection of S variant proteins in cells lysate by western blot. E. Furin-mediated S protein cleavage with PVs subjected to western blot analysis. F. The virus-like particles (VLPs) with S, M, E were subjected to western blot analysis. All experiments were biologically repeated at least three times.