Figure 4.

eIF2A binds to the IIId domain of the HCV IRES. (A) The predicted secondary structure of the HCV IRES (upper panel) and RNA segments used in RNA pull-down experiments (lower panel). Biotinylated RNAs corresponding to the lines shown in this panel were synthesized by in vitro transcription. (B) RNA pull-down experiments were performed using biotinylated RNAs and purified His₆-tagged eIF2A proteins. The RNA-bound proteins were precipitated with streptavidin-conjugated agarose resin and visualized by western blotting using an anti-His antibody. (C) RNA pull-down experiments were performed using cell extracts and biotinylated 18–331 (WT) and 18–331 (ΔIIId) RNAs corresponding to nts 18–331 of wild-type and IIId domain-deleted HCV IRES, respectively. The RNA-bound proteins were precipitated with streptavidin-conjugated agarose resin and visualized by western blotting using anti-eIF2A and anti-eIF3b antibodies.