Figure 3

Identification of SADS-CoV proteins antagonizing IFN-λ1. A Expression of individual SADS-CoV proteins in IPI-FX cells detected by IFA. B Suppression of IFN-λ1 promoter activity by individual SADS-CoV proteins. IPI-FX cells were co-transfected with plasmids expressing individual SADS-CoV proteins, IFN-λ1-luc and pRL-TK at a ratio of 1:0.1:0.01. At 24 h post-transfection, cells were transfected with or without 1 μg/mL poly(I:C) for 12 h. Cell lysates were prepared, and luciferase activity was measured. The IFN-λ1 promoter activity induced by poly(I:C) stimulation was normalized to that without stimulation. C Dose-dependent inhibition of poly(I:C)-induced IFN-λ1 promoter activity by SADS-CoV nsp1. IPI-FX cells were transfected with different amounts (0.25/0.5/0.75 μg) of pEGFP-nsp1 or control plasmid pEGFP-N1, along with IFN-λ1-luc and pRL-TK at a ratio of 1:0.1:0.01 for 24 h. Subsequently, cells were transfected with or without 1 μg/mL poly(I:C) for 12 h. Cell lysates were prepared, and luciferase activity was measured. The IFN-λ1 promoter activity induced by poly(I:C) stimulation was normalized to that without stimulation. D Suppression of poly(I:C)-induced IFN-λ1 promoter activity by SADS-CoV nsp1. IPI-FX cells were transfected with pcDNA3.1-nsp1 or control plasmid pcDNA3.1, along with IFN-λ1-luc and pRL-TK at a ratio of 1:0.1:0.01 for 24 h. Subsequently, cells were transfected with or without 1 μg/mL poly(I:C) for 12 h. Cell lysates were prepared, and luciferase activity was measured. E Suppression of poly(I:C)-induced IFN-λ1 mRNA expression by SADS-CoV nsp1. IPI-FX cells were transfected with pcDNA3.1-nsp1 or the control plasmid pcDNA3.1 for 24 h, followed by transfection with or without 1 μg/mL poly(I:C) for 12 h. Total cellular RNA was extracted, and the expression of IFN-λ1 mRNA was determined using RT-qPCR. *P < 0.05; **P < 0.01; ***P < 0.001.