Fig. 3. TMPRSS2, TMPRSS4 but not ST14 mediate SARS-CoV-2 S mediated entry.

(A) Bulk RNA-sequencing results of intestine-specific serine protease expression in HEK293, Huh7.5, H1-Hela, HT-29 cells and human ileum enteroids. (B) HEK293 cells were transfected with pcDNA3.1-V5-ACE2, DDP4, or ANPEP for 24 hours (left panel), or transfected with indicated plasmid combination for 24 hours (right panel), and infected with 1.5X10⁵ PFU of VSV-SARS-CoV-2 for 24 hours. The expression of VSV-N was measured by RT-qPCR and normalized to that of GAPDH. (C) HEK293 cells stably expressing human ACE2 were transfected with SARS-CoV-2 S and TMPRSS2 or TMPRSS4 or 48 hours. Cells were treated with trypsin at 0.5 μg/ml for 10 min. The levels of S and GAPDH were measured by Western blot. The intensity of bands was quantified by ImageJ and shown as percentage of the bottom band versus the top band in each lane. (D) HEK293 cells stably expressing human ACE2 were transfected with TMPRSS2 or TMPRSS4 for 24 hours, incubated with 5.8X10⁵ PFU of VSV-SARS-CoV-2 on ice for 1 hour, washed with cold PBS for 3 times, and shifted to 37°C for another hour. The expression of VSV-N was measured by RT-qPCR and normalized to that of GAPDH. (E) Wild-type or human ACE2 expressing HEK293 cells were transfected with SARS-CoV-2 S and GFP, with or without TMPRSS2 or TMPRSS4 or 24 hours. The red arrows highlight the formation of large syncytia. Scale bar: 100 μm. For all figures except A, experiments were repeated at least three times with similar results. RNA-seq in Fig. 3A was performed once with duplicate samples. Data are represented as mean ± SEM. Statistical significance is indicated (*p≤0.05; **p≤0.01; ***p≤0.001).