FIG. 2.

siRNA-mediated knockdown of GBF1, but not of Arf1, inhibits CVB3 RNA replication in HeLa cells. (A) Cells were treated with siRNA against GBF1 for 40 h and then stained with an antibody against GBF1. The siRNA treatment inhibited GBF1 expression in about 80 to 90% of the cells. (B) Cells were cotransfected with a plasmid encoding EYFP-GBF1 and siRNA against GBF1 (left) or cotransfected with a plasmid encoding Arf1-EYFP and siRNA against Arf1. Fluorescence was monitored at 2 days posttransfection. (C) Cells were treated with siRNAs directed against GBF1 or Arf1 or a scrambled siRNA (control). At 40 h posttransfection, the cells were infected with GFP-expressing CVB3. GFP fluorescence, which is indicative of replication, was monitored at 7 h postinfection. (D) Similar to panel C, but the cells were infected with a Renilla luciferase-expressing CVB3. Luciferase production was measured at 7 h postinfection and is expressed as a percentage of that observed with the no-siRNA control (which was set at 100%). (E) Cells were treated with siRNAs against GBF1 or a scrambled siRNA and then transfected with in vitro transcripts from firefly luciferase-expressing replicons of CVB3 or EMCV. The amount of luciferase produced at 8 h posttransfection was measured and is expressed as a percentage of that observed with the siRNA control (which was set at 100%).