Figure 6.

ORF3 regulates IKBKB or RIG-I mediated IFN-β promoter activation and type I IFN production via distinct pathways. (A) and (B) HEK293T cells were grown in 24-well plates and transfected with plasmids expressing ORF3, IKBKB or RIG-I, firefly luciferase driven IFN-β promoter and Renilla luciferase. At 24 hpt, undiluted and serial 2-fold dilutions (1/2, 1/4 and 1/8) of supernatants (n = 3) were collected and added into 96-well plate following with HEK-Blue^TM IFN-α/β cell suspension. After incubation for 24 hrs, induced HEK-Blue^TM IFN-α/β cell supernatant was used to measure type I IFN induction through JAK-STAT pathway activation by a SEAP detection following the manufacturer’s protocol. The SEAP levels were quantified using a spectrophotometer at 655 nm (A and B, right panels). Cells were lysed, and the firefly luciferase and Renilla luciferase activities were measured. Data were shown as the relative firefly luciferase activities normalized to the Renilla luciferase activities (A and B, left panels). *, p < 0.05; ***, p < 0.0001; ns, no statistical difference.