FIG 7.

The inhibitory effect of NS4B on STING accumulation is primarily mapped to the B domain. (A) Schematic representation of parental and chimeric NS4B constructs. A, B, and C denote the three domains of the NS4B protein. (B) Effects of NS4B chimeric constructs on STING accumulation. Huh7.5 cells were transfected with fixed amounts of STING and increasing concentrations of NS4B chimeras for 30 h. The cell lysate was separated by SDS-PAGE followed by Western blotting. Tubulin level served as a loading control. The relative level of STING was quantified and normalized to α-tubulin with ChemiDoc software. (C) Effects of NS4B subdomain chimeras on STING accumulation. The experiment was performed as in panel B, except that 293T cells were transfected. (D) Effects of NS4B domain B helices on STING accumulation in 293T cells. (E) Effect of 1b/2a-B NS4B expression on STING accumulation. (F) Effects of NS4Bs and chimera 2a/1b-B on the endogenous STING accumulation in 293 cells. 293 cells were transfected with increasing concentrations of either 2a NS4B, 1b NS4B, or 2a/1b-B plasmid. The endogenous STING, TBK1, and IRF3 levels were detected by Western blotting. (G) Effects of NS4B chimera on the inhibition of IFN-β-Luc reporter expression. 293T cells were transfected with fixed amounts of STING, IFN-β-Luc, and TK-Luc along with increasing concentrations of either NS4B or its chimeras. After 24 h, luciferase activity was determined and the data were plotted as IFN-β fold induction relative to the cells treated with 1b/Con1 NS4b. Data were expressed as an average of triplicates with standard deviation of the mean. (H) Effects of 1b/2a-B on the IFN-β reporter expression. The asterisk denotes a P value of <0.05.