Fig. 2

The SARS-CoV-2 M protein suppresses the activation of the luciferase reporters of type I and III IFNs and ISGs. The pcDNA6B empty vector and the SARS-CoV-2 M protein plasmids (100 ng) were transfected with the indicated combinations of plasmids expressing RIG-IN (100 ng), MDA-5 (100 ng), TBK1 (100 ng), IKKε (100 ng), IRF3-5D (100 ng, an active form of IRF3), TRIF (100 ng, component of TLR3-TRIF pathway), or STING (100 ng, component of cGAS-STING pathway) into HEK293T cells cultured in 48-well plates (0.5 × 10⁵ cells per well). The IFN-β-Luc (45 ng, the IFN-β luciferase reporter) (a), IFN-λ1-Luc (45 ng, the IFN-λ1 luciferase reporter) (b), or ISRE-Luc (45 ng, the IFN-stimulated response element luciferase reporter) (c) plasmids were also transfected to assess the activation of type I IFNs, type III IFNs, or ISGs, respectively. The pRL-TK (5 ng) was transfected into each well as an internal control. The pcDNA6B empty vector was used to normalize the total amount of transfected plasmid DNA. Dual‐luciferase assays were performed 36 h after transfection. d The pcDNA6B empty vector and the SARS-CoV-2 M protein plasmids (100 ng) were transfected into HEK293T cells as indicated. Twenty-four hours later, the cells were treated with recombinant human IFN-β (10 ng/mL) or IFN-λ1 (10 ng/mL). Three hours after simulation, the cells were harvested for RNA extraction and subsequent RT-qPCR analysis. Three independent biological replicates were analyzed, the results of one representative experiment are shown, and the error bars indicate the SD values. Statistical significance is shown as indicated. SARS-CoV-2 M protein SCV2-M