Fig. 3.

SUMOylation activates the MDA5-mediated IFN response. (A) Ubc9 increases MDA5-driven IFN-β reporter activity. HEK293T cells were transfected with a Renilla luciferase internal control plasmid, IFN-β reporter, Flag-MDA5, HA-SUMO-1, and increasing amounts of Myc-Ubc9 or Ubc9 (C93S) as indicated for 24 h. Luciferase activities were determined and normalized against renilla luciferase activity. Fold activation expressed for the ratio of relative luciferase activity (RLU) in the presence or absence of Myc-Ubc9 and HA-SUMO-1. Data represent the average of three independent experiments (mean ± SD). (B) Ubc9 expression inhibits VSV replication. HEK293T cells were transfected with the plasmids indicated for 16 h in 24-well plates. Cells were then infected with VSV (MOI = 0.1) for an additional 1 h. Supernatants were collected 9 h post infection and standard plaque assays in HeLa cells were used to determine virus titers. The data represent at least 3 independent experiments. (C) Downregulation of endogenous Ubc9 expression inhibits IFN production. HeLa cells (1 × 10⁶) were infected with sham lentivirus, lentivirus containing Ubc9-shRNA or scrambled Ubc9 shRNA (MOI = 2) for 12 h. Cells were then co-transfected with a Renilla luciferase internal control plasmid, IFN-β reporter and Flag-MDA5 or a control vector. Dual luciferase activities were determined and normalized as in (A), and fold activation was derived by RLU in the presence or absence of Flag-MDA5 and HA-SUMO-1. Data represent the average of three independent experiments (mean ± SD). Efficiency of gene silencing was detected by RT-PCR, with GAPDH gene expression as an internal control (right panel).