Fig. 3.

A single amino acid modulates NS1 to subvert IFN-β activation. a IFN-β promoter luciferase activity assay. HEK-293T cells were co-transfected with IFN-β promoter firefly luciferase reporter plasmid, renilla luciferase plasmid (for transfection normalization), non-structural proteins encoding plasmid from different ZIKV strains or control plasmid, and RIG-I (2CARD) expression plasmid as a stimulator. Cells were harvested and lysed at 24 h post-transfection, followed by luciferase assay as described in the legend to Fig. 1, and western blotting with indicated antibodies, the full blots can be found in Supplementary Fig. 6. b Amino acid sequence alignment between FSS13025 NS1 and PRVABC-59 NS1, residue 188 is highlighted with a red box. c–f HEK-293T cells were co-transfected with IFN-β promoter firefly luciferase reporter plasmid, renilla luciferase plasmid, ZIKV non-structural protein-encoding plasmid, or empty control plasmid, cells were stimulated by MAVS (c), IKKε (d), TBK1 (e), or IRF3/5D (f), followed by luciferase assay as described above. Results in (a, c–f) were normalized first by renilla luciferase values, and then normalized by none stimulated samples to obtain fold induction. Empty vector samples were set as 100% fold induction. Data are mean ± SD from three independent replicates, each experiment in triplicate. P-values were determined by unpaired Student’s t-test, *P < 0.05, **P < 0.01, ***P < 0.001, or no significance (n.s.)