Activation of SARS-CoV-2 S protein by cathepsin and trypsin.
a Effects of cathepsin inhibitors on entry of SARS-CoV-2 S pseudovirions on 293/hACE2 cells. HEK 293/hACE2 cells were pretreated with broad-spectrum cathepsin inhibitor E64D, cathepsin L-specific inhibitor (SID 26681509), or cathepsin B-specific inhibitor (CA-074) and then transduced with SARS-CoV-2 S and VSV-G pseudovirions. Pseudoviral transduction was measured at 40 h post inoculation. Experiments were done in triplicates and repeated at least three times. One representative is shown. Error bars indicate SEM of technical triplicates. b Cell–cell fusion mediated by SARS-CoV-2 S protein. HEK 293T cells were transiently expressing eGFP and either SARS-CoV-2 or SARS-CoV S protein were detached with either trypsin or EDTA, and co-cultured with 293/hACE2 or 293 cells for 4 h at 37 °C. The scale bar indicates 250 µm. c Quantitative analysis of syncytia in panel b. d, e Thermostability analysis of SARS-CoV-2 S protein. d SARS-CoV and SARS-CoV-2 S pseudovirons were incubated at 37 °C for the specified times (0 to 4 h) in the absence of serum, and then assayed on 293/hACE2 cells. The results from infection at 0 h were set as 100%, and the experiments were repeated four times, and means with standard deviations are shown. e SARS-CoV and SARS-CoV-2 S pseudovirions without serum were incubated at the indicated temperature (37 to 51 °C) for 2 h and then assayed on 293/hACE2 cells. The results are reported as the percentage of transduction at 37 °C. The experiments were repeated four times, and means with standard deviations are shown. Source data are provided as a Source Data file.