Fig. 2.

The SVV IRES functions in the presence of cleaved eIF4G and an eIF4A inhibitor. (A) Dicistronic plasmids (2 μg) of the form CAT/IRES/LUC were transfected into 293T cells in the absence (−) or presence (+) of the plasmid pAΔ802 (0.5 μg), which expresses the PV 2A protease. After 20 h, cell extracts were prepared and analyzed for CAT and LUC expression as described in Fig. 1. LUC and CAT assays were also performed on extracts from three separate experiments, and the mean LUC values are shown, standardized to the LUC expression values from the EMCV IRES-containing construct, which was set at 100%. LUC activities were normalized to CAT expression as described in the legend to Fig. 1. (B) The same dicistronic plasmids were transfected into 293T cells in the absence (−) or presence (+) of the eIF4A inhibitor, hippuristanol (Hipp.). Cells were harvested 20 h after transfection, and the hippuristanol (0.5 μM) was added for the last 10 h of the incubation. Cell extracts were analyzed for CAT and LUC expression as described in the legend to Fig. 1. The data shown are representative of results from two independent experiments.