FIG 6.

The conserved S2′ site is the site of TMPRSS2-mediated activation of SARS2-S for cell-cell fusion. (A) Cell-cell fusion assay. Effector cells (293T cells transfected with expression plasmids for the indicated spike variants together with the Vp16-Gal4 expression plasmid) were added to target cells (293T cells transfected with expression plasmids for ACE2, ACE2/TMPRSS2, or TMPRSS2 and the Gal4-TurboGFP-Luc reporter plasmid). After 24 h, luciferase activity was measured. The data show fold values for the empty vector control, and the error bars represent the standard deviations of results from three independent experiments, each performed in triplicate. (B) Expression of analyzed spike variants in 293T cells. The unprocessed spike (S0) and the S1/S2 site-processed spike (S2) are indicated by arrows. The expression of GAPDH served as a loading control. (C) Cell surface expression and ACE2 binding. Cell surface expression and binding of soluble ACE2-Fc by the indicated spike variants was determined by flow cytometry. Analysis was performed as in Fig. 1C. (D) Cell-cell fusion assay. Effector cells (293T cells transfected with expression plasmids for the indicated spike variants together with the Vp16-Gal4 expression plasmid) were preincubated with the furin inhibitor CMK (10 μM) and after 16 h were added to target cells (293T cells transfected with expression plasmids for ACE2 and the Gal4-TurboGFP-Luc reporter plasmid) that had been preincubated for 30 min with the same inhibitor concentration. After the addition of effector cells, effector und target cells were cocultured in the presence of CMK. After 24 h, luciferase activity was measured. The data show values normalized to those for solvent treatment, which were set to 100%, and the error bars represent the standard deviations from two independent experiments, each performed in triplicate. (E) Cell-cell fusion assay. Effector cells (293T cells transfected with expression plasmids for the indicated spike variants together with the Vp16-Gal4 expression plasmid) were added to target cells (293T cells transfected with expression plasmids for ACE2, ACE2/TMPRSS2, or TMPRSS2 and the Gal4-TurboGFP-Luc reporter plasmid) that had been preincubated with batimastat and/or camostat for 30 min at twice the final concentration; final concentrations were 10 μM batimastat and/or 50 μM camostat. After 24 h, luciferase activity was measured. The data show values normalized to those for solvent treatment, which were set to 100%, and the error bars represent the standard deviations from two independent experiments, each performed in triplicate. Statistical significance in panels A, C, D, and E was determined by two-way ANOVA, and P values were corrected for multiple comparisons by Sidak’s method (P > 0.05, ns; P ≤ 0.05, *; P ≤ 0.01, **; P ≤ 0.001, ***; P ≤ 0.0001, ****).