Fig. 3: CTSL cleavage sites are essential for SARS-CoV-2 infection and efficient cell–cell fusion.

a Overview of SARS-CoV-2 S proteins with mutations in CS-1, CS-2, and the S1/S2 cleavage site. b Infectivity of PsVs with different point mutations in CS-1 and CS-2 was assessed in LLC-MK2, Vero, Huh7, 293 T/ACE2 cells and in 293 T/ACE2 cells with CTSL (293 T/CTSL), TMPRSS2 (293 T/TMPRSS2), and FURIN (293 T/FURIN) genes overexpression. PsV infectivity was measured by a luciferase assay and is shown as the raw luciferase activity (n = 3–4). Statistical significance was assessed by one-way ANOVA with Tukey’s post-hoc test. c–e Quantitative analysis of syncytium formation induced by CS mutant SARS-CoV-2 S proteins (see also Supplementary Fig. S9). c Luciferase gene expression was driven by the ERE promoter, and ESR1 (activator) bound and activated the ERE promoter to upregulate luciferase expression. d, e Effector Huh7 cells were cotransfected with plasmids expressing ERE-luciferase and different S proteins as indicated, and another plate of target Huh7 cells was transfected with plasmid expressing ESR1. After 24 h, the effector cells were detached and added to the target cells for 30–60 min. Then, the supernatant was removed and treated with PBS or CTSL (8 μg/mL) for 20 min. The reaction was stopped by adding 500 μL of medium, and culture was continued for another 24 h to allow cell–cell fusion. When a target cell and effector cell fused to form a syncytium, ESR1 bound and activated the ERE promoter to upregulate luciferase expression. Luciferase activity was then measured as a proxy for the fusion rate. The data were normalized to the WT-PBS group (n = 3). Statistical significance was assessed between the indicated group and the WT-PBS group by two-way ANOVA with Dunnett’s post-hoc test. f Images of syncytium formation induced by CS mutant SARS-CoV-2 S proteins. Huh7 cells were transfected with plasmids to express the WT, CS-1M, CS-2M, or CS-1M + 2 M S protein. Cells were treated in the absence (PBS, pH = 5.8) or presence of CTSL (4 μg/mL, pH = 5.8). Images were acquired after an additional 10–16 h of incubation in medium (scale bars, 50 μm). The black arrowheads indicate syncytia. Representative data from three independent experiments are shown. g Overexpression or knockdown of the CTSL gene dose-dependently promoted or inhibited, respectively, infection with WT (Wuhan-1) and three mutant SARS-CoV-2 PsVs with different point mutations in the furin cleavage site (FM-delta, FM-ARAA, and FM-GSAS). PsV infectivity in Huh7 cells was measured by a luciferase assay and is shown as the relative luciferase activity (n = 3). Statistical significance was assessed by one-way ANOVA with Tukey’s post-hoc test. h CTSL promoted syncytium formation induced by the FM-ARAA mutant SARS-CoV-2 S protein. Effector cells were cotransfected with ERE-luciferase plasmids and either FM-ARAA S or scramble vectors (Control). Target cells were transfected with ESR1 expression plasmid. After the effector cells and target cells were mixed, the supernatant was removed and treated with PBS or CTSL (8 μg/mL and 16 μg/mL). Luciferase activity was then measured and normalized to that in the control group (n = 4). Statistical significance was assessed by one-way ANOVA with Tukey’s post-hoc test. i Huh7 cells were transfected with scramble vector (Control) or FM-ARAA S protein expression plasmid. Cells were treated in the absence or presence of CTSL (2 or 4 μg/mL). Images were acquired after an additional 10–16 h of incubation in medium (scale bars, 50 μm). The black arrowheads indicate syncytia. Representative data from four independent experiments are shown. The data are presented as the means ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, ***P < 0.0001.