TMPRSS2 and furin are both essential for proteolytic activation of SARS-CoV-2 in human airway cells

(A) Fluorescence resonance energy transfer substrates of the S protein S1/S2 sites of the indicated CoVs. M1 and M2 are mutants of the SARS-CoV-2 S1/S2 site with substitution of A → K or A → R in P2 position. IBV, avian infectious bronchitis virus strain Beaudette. Cleavage by furin is indicated in red. (B) Cleavage of the fluorescence resonance energy transfer substrates (20 μM) by furin (0.5 nM). Cleavage efficiency of SARS-CoV-2_M2 was set as 100%. (C) Cleavage of SARS-CoV-2 S by furin and TMPRSS2 in HEK293 cells. Cells were co-transfected with pCAGGS-S-Myc-6xHis and either empty vector or pCAGGS-TMPRSS2. Cells were then incubated in the absence or presence of aprotinin or furin inhibitor MI-1851 (50 μM each) for 48 h. Cell lysates were subjected to SDS–PAGE and Western blot analysis using antibodies against the C-terminal Myc-tag. For each Western blot lanes are spliced together from one immunoblot from one experiment. β-actin was used as loading control.

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(A) Multicycle replication of SARS-CoV-2 in T-ex5–treated Calu-3 cells. Cells were treated with 25 μM T-ex5 or control PPMO (scramble) for 24 h or remained without treatment (w/o). Cells were then inoculated with SARS-CoV-2 at a MOI of 0.001 for 1 h 30 min, the inoculum was removed and the cells further incubated in the absence of PPMO for 72 h. Cells were fixed and immunostained using a serum against SARS-CoV. Virus-positive cells are stained in blue. Scale bars indicate 500 μm. (B) Calu-3 cells were treated with PPMO for 24 h and then infected with SARS-CoV-2 for 72 h as described above. Virus titers in supernatants were determined by tissue culture infection dose 50% (TCID₅₀) end point dilution at indicated time points. Results are mean values ± SD of three independent experiments. (C) Analysis of TMPRSS2-mRNA in PPMO-treated Calu-3 cells. Cells were treated with 25 μM T-ex5, scramble PPMO or remained untreated (w/o) for 24 h (lanes 1–4). T-ex5–treated cells were inoculated with SARS-CoV-2 as described above and incubated in the absence of PPMO for 72 h (lane 4). Total RNA was isolated and analyzed by RT-PCR using primers designed to amplify 1,228 nt of full-length TMPRSS2-mRNA. Full-length and truncated PCR products lacking exon 5 are indicated by filled and open arrow heads, respectively. (D) Effect of PPMO treatment on Calu-3 cell viability. Calu-3 cells were treated with scramble or T-ex5 PPMO (25 μM) for 24 h. Cell viability of untreated (w/o) cells was set as 100%. Results are mean values ± SD (n = 3).

Source data are available for this figure.

Calu-3 cells were inoculated with SARS-CoV-2 at a low MOI of 0.001 and then incubated in the presence of inhibitors of TMPRSS2 (aprotinin, MI-432, and MI-1900), furin (MI-1851), and endosomal cathepsins (E64d), respectively, at the indicated concentrations. Cells were fixed and immunostained using a rabbit serum against SARS-CoV at 72 h p.i. Virus-positive cells are stained in blue or dark gray depending on the staining intensity. Cells infected in the absence of inhibitors (w/o), in the presence of DMSO (0.5%) and noninfected cells (mock) were used as controls. Scale bars indicate 500 μm. Images are representatives of three independent experiments.

(A) Calu-3 cells were inoculated with SARS-CoV-2 at a low MOI of 0.001 and then incubated in the absence (w/o) or presence of inhibitors of TMPRSS2 (aprotinin, MI-432, and MI-1900), furin (MI-1851), and endosomal cathepsins (E64d), respectively, or DMSO (0.5%), at the indicated concentrations. At 16, 24, 48, and 72 h postinfection (p.i.), supernatants were collected, and virus replication was determined by tissue culture infection dose 50% (TCID₅₀) titration at indicated time points. Data are mean values ± SD of three to five independent experiments. (B) Effect of inhibitor treatment on cell viability. Calu-3 cells were treated with the indicated protease inhibitor (50 μM) for 72 h. Untreated cells (w/o) and DMSO treated cells were used as controls. Cell viability of untreated cells was set as 100%. Results are mean values ± SD (n = 3). (C) Antiviral activity of combinations of TMPRSS2 and furin inhibitors against SARS-CoV-2 in human airway epithelial cells. Calu-3 cells were inoculated with SARS-CoV-2 at an MOI of 0.001 as described above and then incubated in the presence of single protease inhibitors or inhibitor combinations at the indicated concentrations. Virus titers in supernatants were determined by TCID₅₀ at 16, 24, 48, and 72 h p.i. Data are mean values ± SD of three independent experiments. (D) Calu-3 cells were treated with PPMO for 24 h, then infected with SARS-CoV-2 as described above and incubated in the absence of PPMO (w/o, scramble and T-ex5) and with or without 50 μM of furin inhibitor treatment (MI-1851) for 72 h. At 16, 24, 48, and 72 h p.i., supernatants were collected, and viral titers were determined by TCID₅₀ at indicated time points. Data are mean values ± SD (n = 2).