FIG 4.

NTD loops control protease-triggered membrane fusion. (a) D614G-2 VLPs were incubated with hACE2-negative or hACE2-positive EVs for 30 min at 37°C in the presence of the indicated trypsin concentrations, and trypsin cleavage products were identified by Western blotting. Uncleaved S (S-unc), S2, and S2′ cleavage products are indicated. (b) The indicated SARS-CoV-2 VLPs were evaluated in cell-free VLP-EV fusion assays at the indicated trypsin concentrations. Fusion readouts were taken after 3 h at 37°C, and plotted data trendlines were normalized to the highest measured fusion levels. Error bars present standard errors (SE) of the means. (c) Cell-free VLP-EV fusion assays were repeated three times and trypsin EC₅₀ values calculated from the fitted normalized response trendlines. Statistical analyses were assessed by an unpaired Student t test (***, P < 0.001; ns, not significant).