Fig. 1

Elastase facilitates RaTG13 and Khosta-2 S protein-mediated cell entry Elastase, but not trypsin or thermolysin, facilitates cell entry of pseudotyped RaTG13 (A) and Khosta-2 (B) viruses. Particles bearing S proteins were preincubated for 5 min at room temperature with or without each protease and then added to VeroE6/TMPRSS2 cells. S protein-mediated cell entry was analyzed by measuring the activity of virus-encoded luciferase in the cell lysate at 3 days post inoculation. Experiments were independently repeated three times, and similar results were observed. Data from a representative experiment are shown (mean ± SD). Statistical analysis was performed using one-way ANOVA and subsequent Dunnett’s test. **** indicates p < 0.0001, ns: no significance. (C, D) Elastase treatment of viral particles but not target cells facilitates entry, nor does it facilitate post inoculation. VeroE6/TMPRSS2 cells or pseudotyped particles bearing RaTG13 (panel C) or Khosta-2 (panel D) S proteins were treated with elastase (final concentration: 50 µg/mL). In the “Pre” condition, cells were treated with elastase for 5 min at room temperature before virus inoculation. In the “Simultaneous” condition, pseudotyped virus particles were pretreated with elastase and immediately added to the cells. In the “Post” condition, cells were first inoculated with virus and then treated with elastase 24 h later under the same conditions. S protein-mediated cell entry was analyzed as described in panels (A, B). Experiments were independently repeated three times, and similar results were observed. Data from a representative experiment are shown (mean ± SD). Statistical analysis was performed using the Student’s t-test or Welch’s t-test. * indicates p < 0.05, **** indicates p < 0.0001, ns: no significance. Trypsin and elastase treatments facilitate cell entry of pseudotyped SARS-CoV-2, as shown in panels (E) and (F), respectively. Protease treatments were performed in the same manner as described in panels (A and B). Experiments were independently repeated twice, and similar results were observed. Data from a representative experiment are shown (mean ± SD). Statistical analysis was performed using one-way ANOVA and subsequent Dunnett’s test. * indicates p < 0.05, ** indicates p < 0.01, **** indicates p < 0.0001, ns: no significance. (G) Elastase treatment has no effect on VSV-G-mediated cell entry. Particles bearing the VSV-G were pre-incubated for 5 min with or without elastase and then added to VeroE6/TMPRSS2 cells. The experiments were independently repeated twice, and similar results were observed. Data from a representative experiment are shown (mean ± SD). Statistical analysis was performed using one-way ANOVA and subsequent Dunnett’s test. * indicates p < 0.05, **** indicates p < 0.0001, ns: no significance.