Figure 7.

DDX21 and DDX60 were identified as important anti-hantaviral molecules that could positively regulate IFN responses after HTNV infection. (A) Differentially expressed DExD/H box helicases after HTNV infection shown by heat map from DGE analysis. (B) The altered expression levels of DDX3, DDX5, DDX6, DDX21, DDX50, and DDX60 at the different time intervals post-HTNV infection in HUVECs as determined by qRT-PCR (n = 3). ^*P < 0.05, ^**P < 0.01, ^***P < 0.001 vs. mock by Student's t-test. (C) The overexpression efficiency of DDX3, DDX5, DDX6, DDX21, DDX50, and DDX60 in HUVECs was detected by observing the inflorescence of ZsGreen at 2 days post-infection with the related lentivirus. (D,E) HUVECs ectopically expressing control plasmids, DDX3, DDX5, DDX6, DDX21, DDX50, and DDX60 were mock infected or infected with HTNV at an MOI of 0.1 and then fixed at 2 dpi for the ICW assay to screen antiviral molecules. Imaging results (D) and the intensity ratio analyzed by software (E) are presented. ^*P < 0.05, ^**P < 0.01, ^***P < 0.001 vs. vector by Student's t-test. (F) The viral loads of HUVECs with the identical treatment described in (D) were assessed by qRT-PCR (n = 3). ^*P < 0.05, ^**P < 0.01, ^***P < 0.001 vs. vector by Student's t-test. (G,H) Vero E6 cells ectopically expressing control plasmids, DDX3, DDX21, and DDX60 or pretreated with IFN-β (2 ng/ml) were mock infected or infected with HTNV at an MOI of 0.1 and then fixed at 2 dpi for the ICW assay to screen antiviral molecules. Imaging results (G) and the intensity ratio analyzed by software (H) are presented. (I) Viral loads in E6 cells with the identical treatment described in (G) were assessed by qRT-PCR (n = 3). (J and K) Dual-luciferase assays of IFN-β promoter activation following HTNV infection of HUVECs. HUVECs ectopically expressing control plasmids, DDX3, DDX21, and DDX60 were transfected with 100 ng of pGL3 basic or the reporter plasmid for the IFN-β promoter (pIFΔ (−116) lucter) together with 10 ng of the pRL-TK Renilla luciferase reporter. Twenty-four hours after transfection, the HUVECs were not challenged (J) or were challenged (K) with HTNV at an MOI of 0.1 for 1 h at 37°C. Luciferase assays were performed 24 h after infection, and the results were expressed as the comparative ratio of firefly luciferase to Renilla luciferase activity compared to the untreated group. ^*P < 0.05, ^**P < 0.01, ^***P < 0.001 vs. vector by Student's t-test. The experiments were performed independently at least three times with similar results.