FIGURE 1.
MERS-CoV ORF8b inhibits IFN-β expression and IRF3 activation. (A) V5-tagged ORF8b expression construct (ORF8b-V5) was transfected into HEK293 cells at an increasing dose (100, 200, and 400 ng per well) together with a firefly luciferase reporter plasmid driven by IFN-β promoter (IFN-β-luc) and a control Renilla luciferase reporter plasmid. Cells were infected with SeV (100 hemagglutinating U/ml) at 24 h posttransfection of ORF8b-V5 for 16 h. At 16 hpi, cells were harvested for dual-luciferase reporter assay. The expression levels of the three doses introduced were detected by Western blotting with anti-V5 Ab (inset). β-Actin was included as an internal loading control. (B) V5-tagged ORF8b expression construct (ORF8b-V5) was transfected into HEK293 cells at an increasing dose (100, 200, and 400 ng per well). IFN-β expression was induced by poly(I:C) (1 μg/ml) at 24 h posttransfection, and cells were harvested for RT-qPCR at 16 hpi. GAPDH mRNA was used as an internal control. Relative expression of IFN-β transcripts was determined by ΔCT against GAPDH. (C) V5-tagged ORF8b expression construct (ORF8b-V5) was transfected into Huh-7 cells at an increasing dose (100, 200, and 400 ng per well) together with an expression vector for RIG-IN, a firefly luciferase reporter plasmid driven by IFN-β promoter (IFN-β-luc), and a control Renilla luciferase reporter plasmid. At 40 h posttransfection, cells were harvested for dual-luciferase reporter assay. The expression levels of the three doses introduced were detected by Western blotting with anti-V5 Ab (inset). β-Actin was included as an internal loading control. (D and E) ORF8b-V5 expression construct was transfected into HEK293 cells at an increasing dose (100, 200, and 400 ng) together with firefly luciferase reporter plasmid driven by either tandem copies of IRF3-binding elements (D) or κB elements (E) and a control Renilla luciferase reporter plasmid. Expression of IFN-β was induced by RIG-IN. HEK293 cells were harvested for dual-luciferase assay at 40 h posttransfection. (F) IRF3 and V5-ORF8b expression constructs were transfected into HEK293 cells at the dose mentioned above. RIG-IN was used to induce IRF3 phosphorylation. Cells were harvested 40 h posttransfection. The protein samples were then resolved with SDS-PAGE, and the indicated proteins were detected by Western blotting. The relative band intensity of phospho-IRF3 at serine 396 versus total IRF3 (#/*) and phospho-IRF3 at serine 386 versus total IRF3 (&/*) were measured using ImageJ software. Three independent experiments were performed. All bars represent the mean (n = 3), and error bars indicate their SD. The statistical significance between selected samples was evaluated by a two-tailed Student t test for unpaired samples with equal variance, and p values (P) were indicated. AU, arbitrary unit.