Figure 6

SADS-CoV N protein suppresses IFN-β production in HEK293T. A and B SADS-CoV N inhibited the SeV and poly(I:C)-mediated IFN-β promoter activity in HEK293T cells. HEK293T cells were transfected with plasmid for SADS-CoV N expression (Myc-N) and IFN-β-Luc plasmid for 24 h. Cells were infected with SeV (A) or stimulated with poly(I:C) (B) for 12 h. Luciferase assays were performed with a dual luciferase reporter assay kit. C and D SADS-CoV N inhibits the transcription of IFN-β, CXCL10, and ISG56 after SeV infection and poly(I:C) transfection in HEK293T cells. HEK293T cells were transfected with empty vector or Myc-N for 24 h. Cells were stimulated with SeV infection C or poly(I:C) transfection (D) for 12 h. The relative mRNA levels of IFN-β, CXCL10, and ISG56 were evaluated by qRT-PCR. E and F SADS-CoV N inhibits phosphorylation of IRF3 and TBK1 stimulated by SeV and poly(I:C) in HEK293T cells. HEK293T cells were transfected with Myc-N or empty vector for 24 h. Cells were stimulated with SeV infection (E) or poly(I:C) transfection (F) for 12 h. The cell lysates were subjected to Western blotting analysis. G SADS-CoV N suppresses the RIG-I-induced activation of luciferase reporters of IFN-β. HEK293T cells were co-transfected with IFN-β-Luc, Flag-RIG-I, pRL-TK plasmid, and Myc-N for 24 h. Luciferase activity was assessed. H SADS-CoV N inhibits mRNA levels of IFN-β induced by RIG-I. HEK293T cells were co-transfected with Flag-RIG-I and Myc-N for 24 h. Cells were harvested for qRT-PCR analysis. I and J SADS-CoV N inhibits TRIM25-mediated RIG-I signaling enhancement. I HEK293T cells were co-transfected with IFN-β-Luc, and Myc-N, HA-TRIM25 or Flag-RIG-I expression plasmid for 24 h. Luciferase assays were performed with a dual luciferase reporter assay kit. J HEK293T cells were transfected with Myc-N, Flag-RIG-I, or HA-TRIM25 expression plasmid for 24 h. The IFN-β mRNA levels were evaluated with qRT-PCR. K–M SADS-CoV N facilitates the replication of VSV-GFP. HEK293T cells were transfected with Myc-N and then infected with VSV-GFP for 12 h. Fluorescence was analyzed through Western blotting (K), fluorescence microscopy (L), and flow cytometry (M). P values were calculated using two-tailed unpaired Student’s t-test.