Fig 2.

EWSR1 is required for HCV efficient RNA replication and infectious virus production. (A) HCV reporter constructs used to measure replication (sg-neo-luciferase) or input translation (the polymerase defective HCV-luciferase GDD→GND). Huh-7.5 cells were electroporated with the indicated siRNAs, and maintained for 48 h. (B to D) Protein was harvested, separated by SDS-PAGE, and immunoblotted for EWSR1 or actin (B) or transfected with polymerase-defective HCV RNAs (C) for 4 h or subgenomic HCV-luciferase RNAs (D) and assayed for luciferase activity at 4 h for input RNA translation and 48 h for replication-associated reporter activity. (E to G) Alternatively, the siRNA-treated cells were infected with HCV for 48 h, and intracellular HCV RNA (E) or infectious HCV (F) production was quantified. (G and H) Huh-7.5 or Huh-7.5-EWSR1 cells were treated with the indicated siRNAs for 72 h, and then protein lysates were harvested, separated by SDS-PAGE, and immunoblotted for EWSR1 or actin (G) or infected with HCV for 48 h (H), and the HCV RNA was quantified. *, P ≤ 0.05.