Figure 6

SADS-CoV nsp1 degrades IRF1 through the ubiquitin–proteasome pathway. A SADS-CoV nsp1 does not directly interact with IRF1. IPI-FX cells were co-transfected with pcDNA3.1-HA-IRF1 and pcDNA3.1-nsp1-FLAG for 24 h. Cell extracts were prepared and subjected to Co-IP analysis. B Endogenous IRF1 protein abundance is reduced by SADS-CoV nsp1. IPI-FX cells were transfected with pcDNA3.1 or pcDNA3.1-nsp1 for 24 h. Cell extracts were prepared and subjected to Western blot analysis. C Endogenous IRF1 protein abundance is reduced by SADS-CoV. IPI-FX cells were mock-infected or infected with SADS-CoV at a MOI of 1 for 24 h. Cell extracts were prepared and subjected to Western blot analysis. D SADS-CoV nsp1 does not regulate the expression of IRF1 at the transcriptional level. IPI-FX cells were transfected with pcDNA3.1 or pcDNA3.1-nsp1 for 24 h, and with or without 1 μg/mL poly(I:C) for 12 h. Total cellular RNA was prepared to detect IRF1 mRNA levels by RT-qPCR. E SADS-CoV nsp1 shortens the half-life of IRF1. 293-T cells were co-transfected with pcDNA3.1-HA-IRF1 and pcDNA3.1-nsp1-FLAG for 24 h, and treated with or without 25 μg/mL CHX for 30/60/120 min. Cell extracts were prepared and subjected to Western blot analysis. F MG132 treatment blocks IRF1 degradation caused by nsp1. 293-T cells were co-transfected with pcDNA3.1-HA-IRF1 and pcDNA3.1-nsp1-FLAG for 24 h, and treated with 50 μM MG132 for 3 h. The equal amount of DMSO was used as a control. Cell extracts were prepared and subjected to Western blot analysis. G CQ treatment does not block IRF1 degradation caused by nsp1. 293-T cells were co-transfected with pcDNA3.1-HA-IRF1 and pcDNA3.1-nsp1-FLAG for 24 h, and treated with 50 μM CQ for 10 h. The equal amount of DMSO was used as a control. Cell extracts were prepared and subjected to Western blot analysis. The average intensity of the band of endogenous or exogenous IRF1 are normalized to GAPDH by Image J software.