FIG. 6.
Sequences in the 3′ NTR do not affect RNA translation from the HCV IRES. (A) Structure of the bicistronic reporter construct carrying the luciferase genes from Renilla (Ren-luc) or the firefly (Ffl-luc) under control of the HCV or the EMCV IRES, respectively. Bars, reporter genes; solid line, EMCV IRES. The HCV NTRs are given with their secondary structures. (B) In vitro translation of bicistronic reporter constructs carrying the given mutations in the HCV 3′ NTR in rabbit reticulocyte lysate. Results obtained with RNAs encoding only the firefly or Renilla luciferase are shown in the two left lanes. Numbers below the lanes are the ratios between the amounts of the two reporters as determined by phospho imaging. The ratio found with the replicon carrying a homopolymeric uridine tract of 46 nucleotides was set at 1. Luciferases are identified at the right; the sizes of protein molecular mass standards are given at the left. (C) Translation efficiencies from the HCV IRES after transfection of given bicistronic RNAs into Huh-7 cells. Luciferase activities in lysates prepared from cells 3 h post transfection were measured. Values refer to relative light units (RLU) of Renilla luciferase after normalization for transfection efficiency by using the firefly luciferase activity.