DEAD-Box Helicase DDX6 Facilitated RIG-I-Mediated Type-I Interferon Response to EV71 Infection

Figure 1. EV71 reduced P-body component DDX6 but did not affect de novo expression. (A, B) EV71 infection reduces endogenous DDX6. HeLa and HT-29 cells were infected with EV71 under different MOIs for the indicated times, respectively. Cell lysates were determined by Western blot using antibodies against DDX6, VP1, and GAPDH. The value of DDX6 in uninfected cells (Lane1) is set as 1.00. The representative data are shown. (C, D) The mRNA of DDX6 showed no change after EV71 infection. The total cell RNA was extracted, and DDX6 mRNA was analyzed by real-time PCR assay, GAPDH as control. All these experiments were performed three times, and representative results were shown. ns indicate no significant difference. (E) HeLa cells grown on 12-well slides were infected with EV71 (MOI=2) or control treatment. After the corresponding time treatment, the cells were fixed and stained with antibodies against DDX6 (red), VP1 (green), and DAPI (blue); the images were acquired using a confocal microscope, and a representative of three independent experiments was shown.

Figure 2. Knocking down DDX6 facilitated EV71 replication. (A) Efficiency of DDX6 downregulation by si-DDX6 in HeLa cells. HeLa cells were treated with either si-NC (a scrambled sequence) or si-DDX6. The total protein extract from infected cells was analyzed by Western blot using antibodies against DDX6 and GAPDH (loading control). (B) Cells transfected with either si-NC or si-DDX6 grown in 12-well plates were infected with EV71 (MOI =3) for 36 h. The EV71-induced CPE in the DDX6+/+ and DDX6-/- cells were photographed using microscopy (scale bar: 50 µm). (C) After si-NC or si-DDX6 transfection for 36 h, the relative VP1 expression of HeLa cells 12 or 24 hpi was shown, respectively. Densitometry was performed, and lane 2 is set as 1.00. (D) Analysis of progeny virus in culture supernatants by a standard plaque assay. The values shown are the means ± SD of three determinations (*P < 0.05, **P < 0.01).

Figure 3. DDX6 conferred cellular resistance to EV71 infection. (A) The plasmids expressing GFP-NC or GFP-DDX6 were expressed in HeLa cells, and the cells were infected with EV71 (MOI=2) for 24 h. The infected HeLa were fixed and stained by staining with anti-VP1 antibody (red), and the cell nuclei stained with DAPI (blue). The images were taken with an Olympus FV3000 confocal microscope, scale bar: 50 µm. (B) HeLa cells were transfected with a plasmid expressing DDX6 (pcDNA3.1-Flag-DDX6) or a control plasmid (pcDNA3.1-Flag). After 24 h of transfection, the cells were treated with EV71 (MOI=2). At 24 hpi, the cell lysates were analyzed by western blot using antibodies specific for DDX6, VP1, and GAPDH. The value for lane 2 was 1.00. (C) The amount of progeny virus in the supernatant from treated cells was determined through a standard plaque assay. The values represented three independent experiments (n = 3, **P < 0.01). The error bar represented mean with SD. (D) The relative VP1 expression in vector or DDX6 over-expressed cells infected with EV71 (MOI = 1 or 3) was determined by Western blot.

Figure 4. Knockdown of DDX6 reduced I-IFN activation during EV71 infection. (A) HeLa cells were transfected with si-NC or si-DDX6 for 48 h and then infected with EV71 6 h p.i., then was analyzed by western blot with the indicated antibodies (RIG-I, P-IRF3, IRF3, DDX6, and VP1). GAPDH was used as a control. (B) After Poly (I: C) and EV71 stimulation, the relative expression of exogenous IFN-β was detected by real time-PCR in si-DDX6 and si-NC transfected cells. (C–E) The mRNA levels of IFN-β, IFI-27, and ISG15 were analyzed by real-time PCR and normalized to GAPDH. (F) HeLa cells were treated with si-NC or si-DDX6 for 48 h and treated with or without poly (I:C) (+/++ represent 200 ng and 500 ng, respectively) for 12 h, western blot analysis of cell lysates with antibodies specific for RIG-I, MAVS, P-IRF3, IRF3, DDX6, and GAPDH. (G–I) The relative mRNA expression of IFN-β, IFI-27, and ISG15 was determined by a real-time PCR assay. The results are presented as means with SD (*P < 0.05, **P < 0.01).

Figure 5. DDX6 positively regulates RIG-I and promotes the activation of type-I IFN during EV71 infection. (A) HeLa cells were transiently transfected with a plasmid expressing DDX6 or control plasmid (pcDNA3.1-flag). After 24 h transfection, the cells were infected with EV71 at (MOI = 3) for 6 h and 12 h, respectively. The nucleus and cytoplasm extracts were determined by western blot using antibodies specific for DDX6, VP1, RIG-I, MAVS, IRF3, P-IRF3, Lamin B, and GAPDH were used as the control for nucleus and cytoplasm, respectively. (B) The relative mRNA of IFN-β in HEK-293T was cotransfected with the plasmid vector or Flag-DDX6 and IFN-β for 24 h and stimulated by poly (I: C) or EV71 for 12 h, respectively. Data from three independent experiments were averaged. (C) Vectors expressing RIG-I, DDX6 were transfected into HEK-293T cells together with p125Luc (the IFN-β-luciferase reporter plasmid). After 24 h, the cells were unstimulated or stimulated with poly (I: C) for 12 h. Cell lysates were prepared, and luciferase activity was measured. +/++/+++ represents an increasing quantity of plasmid. (D) Vectors expressing MDA5, DDX6 were transfected into HEK-293T cells together with the…

Figure 6. The endogenous DDX6 was degraded by viral-2Apro but not 3Cpro. (A) HeLa cells were transfected with an empty vector (pEGFP-C1) or plasmid encoding EV71 protease 3C for 36 h. The whole-cell lysate was analyzed by immunoblotting using specific antibodies (GAPDH, DDX6, and GFP). (B) HeLa cells were cultured in 6-well dishes, transfected with equal amounts of Flag-NC, Flag-2A, and Flag-2A-mutant (2500 ng), respectively. The cells were lysed and analyzed by Western blot. (C) 293T cells transfected with Flag-DDX6 and then transfected with equal amounts of Flag-NC, Flag-2A, or Flag-2A-mutant plasmids for 24 h, respectively. Western blot was performed using various specific antibodies (eIF4G, DDX6, and GAPDH) (**P < 0.01; ***P < 0.001, ns indicate no significant difference). (D) The plasmid encoding 2A-mutant was constructed by mutating the amino acid at the 110th position of the wild-type 2A, and the amino acid comparison diagram of wild-type and mutant 2A was produced by DNAMAN.