Fig. 1

Diverse mechanisms utilized by SARS-CoV-2 M^pro in antagonizing type I IFN production and JAK-STAT signaling. a Immunoblot analysis of extracts of Huh7 cells infected with SARS-CoV-2 (MOI = 0.1) for the indicated time points. b Luciferase activity in 293T cells transfected with IFNβ luciferase reporter, together with empty vector (EV) or increasing amounts of M^pro of SARS-CoV-2. Then the cells were transfected with poly(I:C) for 12 h. **p < 0.01. c Luciferase activity in 293T cells transfected with IFNβ luciferase reporter and vectors for RIG-I (2CARD), MAVS, TBK1, and IRF3 (5D), along with empty vector or expression vectors for M^pro. d 293T cells were transfected with plasmids encoding HA-M^pro and Flag-tagged key proteins in type I IFN signaling (Flag-RIG-I, Flag-MAVS, Flag-TBK1, Flag-IKKi, and Flag-IRF3) and treated with SeV (MOI = 0.1) for 12 h, followed by immunoprecipitation with anti-Flag beads and immunoblot analysis with anti-HA. Asterisk (*) indicates the nonspecific bands. e Lysates of 293T cells transfected with Flag-RIG-I, Myc-TRIM25, and HA-ubiquitin (K63 only) together with empty vector or expression vectors for M^pro, followed with SeV infection (MOI = 0.1) for 12 h, were immunoprecipitated after SDS denaturation with anti-Flag and immunoblotted with the indicated antibodies. f Luciferase activity (above) in 293T cells transfected with IFNβ luciferase reporter and RIG-I (2CARD) along with empty vector or expression vectors for M^pro of SARS-CoV-2 and SARS-CoV. **p < 0.01. Immunoblot analysis (below) of extracts of 293T cells transfected with expression vectors for M^pro of SARS-CoV-2 and SARS-CoV. g Quantitative PCR with reverse transcription analysis of IFIT1 and ISG15 mRNA in Huh7 cells infected with SARS-CoV-2 (MOI = 0.1) for 24 h, followed by IFNβ treatment (1000 U ml⁻¹) for 3 h. **p < 0.01. h Immunoblot analysis of extracts of Huh7 cells infected with SARS-CoV-2 (MOI = 0.1) for 48 h, followed with IFN treatment for 30 min. i Co-immunoprecipitation and immunoassay of extracts of M^pro-inducible A549 cells were treated with doxycycline (Dox; 200 ng ml⁻¹) for 24 h, followed by IFNβ treatment (1000 U ml⁻¹) for 3 h. j Immunoassay of extracts of M^pro-inducible A549 cells were treated with doxycycline (Dox; 200 ng ml⁻¹) for 24 h, followed by IFNβ treatment (1000 U ml⁻¹) for the indicated time points. Below, RT-PCR analysis of STAT1 mRNA; RPL13A mRNA serves as a loading control. k Confocal microscopic analysis (left) of STAT1 localization in Huh7 cells transfected with empty vector or Flag-M^pro for 24 h, followed by IFNβ treatment (1000 U ml⁻¹) for 30 min or left untreated (UT). Scale bars, 10 μm. Quantitative analysis (right) of the colocalization (30 cells per sample). l Quantitative PCR with reverse transcription analysis of IFIT1 mRNA in Huh7 cells transfected with empty vector or expression vectors for M^pro of SARS-CoV-2 and SARS-CoV for 36 h, followed by IFNβ treatment (1000 U ml⁻¹) for 3 h. **p < 0.01. m Schematic representation of SARS-CoV-2 M^pro antagonizing antiviral immunity. All the experiments are representatives of three independent biological experiments with similar results