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. 2019 Jul 30;93(16):e00718-19. doi: 10.1128/JVI.00718-19

FIG 3.

FIG 3

hHB inhibits the MDA5-mediated antiviral signaling. (A and B) Overexpression of hHB suppressed MDA5-mediated activation of the IFN-β promoter. HEK293T cells were cotransfected with the indicated amounts of p3×Flag-hHB, pMyc-MDA5 (A) or long poly(I·C) (B), pRLuc-TK, and pIFN-β-FLuc for 24 h. The activation of IFN-β promoter was examined. (C to E) Overexpression of hHB decreased the transcription of IFN-β, GBP1, and ISG56. HEK293T cells were transfected with the p3×Flag-EV or p3×Flag-hHB and pMyc-MDA5 or long poly(I·C) for 24 h, and the IFN-β, GBP1, and ISG56 mRNA levels in cells were analyzed, as indicated. (F) The MDA5-mediated activation of the IFN-β promoter in hHB−/− cells. hHB−/− and WT cells were cotransfected with pMyc-MDA5 or long poly(I·C) as well as pRLuc-TK and pIFN-β-FLuc. At 24 hpt, the activation of IFN-β promoter was analyzed. (G to I) The MDA5-mediated transcription of IFN-β, GBP1, and ISG56 in hHB−/− cells. hHB−/− and WT cells were transfected with pMyc-MDA5 or long poly(I·C) for 24 h. Then the IFN-β, GBP1, and ISG56 mRNA abundances, as indicated, were determined. (J) The knockout efficiency of RIG-I in WT and hHB−/− cells. (K) Overexpression of hHB inhibited long-poly(I·C)-induced transcription of IFN-β in RIG-I-deficient HEK293T (RIG-I−/−) cells. RIG-I−/− cells were cotransfected with the indicated amounts of p3×Flag-hHB, long poly(I·C), pRLuc-TK, and pIFN-β-FLuc for 24 h. The IFN-β promoter activation was then assessed. Additionally, RIG-I−/− cells were cotransfected with p3×Flag-EV or p3×Flag-hHB and long poly(I·C) for 24 h, and the IFN-β mRNA levels were analyzed. (M) The effect of the deficiency of hHB on long-poly(I·C)-mediated transcription of the IFN-β. Double-knockout cells deficient in RIG-I and hHB (RIG-I−/− hHB−/−) and RIG-I−/− cells were cotransfected with long poly(I·C), pRLuc-TK, and pIFN-β-FLuc. At 24 hpt, the activation of IFN-β promoter was analyzed. In addition, RIG-I−/− hHB−/− and RIG-I−/− cells were transfected with long poly(I·C) for 24 h. The IFN-β mRNA abundance was determined. The data represent the means ± standard deviations from three independent experiments. Significant differences are denoted as follows: *, P < 0.05; **, P < 0.01; ***, P < 0.001; NS, not significant (P > 0.05).