FIG 4.

SUMOylation of DENV NS5 is required for the replication of DENV replicon and for the antagonism of IFN signaling. (A) Endogenous Ubc9 protein expression level in Huh7 cells stably expressing shRNA targeting control (Ctrl) or Ubc9 (Ubc9) were analyzed by WB. (B) Huh7 cells stably expressing shRNA targeting Ctrl or Ubc9 were transfected with DENV replicon RNA harboring WT or SIM-mutated NS5 as indicated. The cell lysates were harvested and analyzed by luciferase assay after 24 h of transfection. The error bars represent the means and SD (n = 3 per group) and were compared by Student's t test. (C) A549 cells were cotransfected with Vip-Luc (0.3 μg) and pRL-TK (0.1 μg) plus LacZ control, WT, or SIM-mutated DENV NS5 (0.5 μg) for 16 h. Subsequently, transfected cells were treated with IFN (500 U) or left untreated (0 U) for 8 h. The cells were harvested and analyzed by dual-luciferase assay. Data are expressed as means and SD (n = 3 per group) and were compared by two-tailed Student's t test. (D) A549 cells were transfected with LacZ control, WT, or SIM-mutated NS5 expression plasmids for 16 h, followed by another 8-h treatment of IFN (500 U). The cells were collected and analyzed by WB with the indicated antibodies. (E) A549 cells were transfected with LacZ control, WT, or SIM-mutated NS5 expression plasmids for 24 or 40 h and then analyzed by WB using the indicated antibodies.