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

FIG 2.

FIG 2

hHB enhances the RIG-I-mediated antiviral signaling. (A and B) Overexpression of hHB upregulated RIG-I-mediated activation of the IFN-β promoter. HEK293T cells were cotransfected with the indicated amounts of p3×Flag-hHB (hHB), pMyc-RIG-I (A) or short poly(I·C) (B), TK-Renilla luciferase (RLuc) internal reference reporter plasmid (pRLuc-TK), and IFN-β promoter firefly luciferase (FLuc) reporter plasmid (pIFN-β-FLuc) for 24 h. The activation of the IFN-β promoter was presented as luciferase reporter gene induction mediated by the IFN-β promoter and calculated as the relative levels of FLuc/RLuc. (C) The effect of hHB on the IFN-β promoter activation. HEK293T cells were cotransfected with the indicated amounts of p3×Flag-hHB, pRLuc-TK, and pIFN-β-FLuc for 24 h. The IFN-β promoter activation was tested. (D to F) Overexpression of hHB enhanced the transcription of IFN-β, GBP1, and ISG56. HEK293T cells were cotransfected with p3×Flag-EV (EV) or p3×Flag-hHB (hHB) and pMyc-RIG-I or short poly(I·C) for 24 h, and the IFN-β, GBP1, and ISG56 mRNA levels in cells were analyzed using real-time RT-PCR, as indicated. (G) The RIG-I-mediated activation of the IFN-β promoter in hHB−/− cells. hHB−/− and WT cells were cotransfected with pMyc-RIG-I or short poly(I·C) as well as pRLuc-TK and pIFN-β-FLuc. At 24 h posttransfection (hpt), the activation of IFN-β promoter was tested. (H to J) The RIG-I-mediated transcription of IFN-β, GBP1, and ISG56 in hHB−/− cells. hHB−/− and WT cells were transfected with pMyc-RIG-I or short poly(I·C). At 24 hpt, the IFN-β, GBP1, and ISG56 mRNA abundances, as indicated, were determined. (K) The knockout efficiency of MDA5 in WT and hHB−/− cells. (L) Overexpression of hHB upregulated the short-poly(I·C)-induced transcription of IFN-β in MDA5-deficient HEK293T (MDA5−/−) cells. MDA5−/− cells were cotransfected with the indicated amounts of p3×Flag-hHB, short poly(I·C), pRLuc-TK, and pIFN-β-FLuc for 24 h. The IFN-β promoter activation was tested. In addition, MDA5−/− cells were cotransfected with p3×Flag-EV or p3×Flag-hHB and short poly(I·C) for 24 h, and the IFN-β mRNA levels were analyzed. (M) The effect of the deficiency of hHB on the short-poly(I·C)-mediated transcription of the IFN-β. Double-knockout cells deficient in MDA5 and hHB (MDA5−/− hHB−/−) and MDA5−/− cells were cotransfected with short poly(I·C) as well as pRLuc-TK and pIFN-β-FLuc for 24 h. Then the activation of IFN-β promoter was tested. In addition, MDA5−/− hHB−/− and MDA5−/− cells were transfected with short poly(I·C). At 24 hpt, the IFN-β mRNA abundances were measured. 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).