FIGURE 6.

Effect of 3′X domain mutations on HCV translation and replication. (A) Huh-7 cells were cotransfected with a mixture of wild-type or mutant ICU-Fluc RNA and cap-Rluc-mRNA, and IRES activity was measured as relative luciferase activity (Fluc/Rluc) as previously described (Romero-López and Berzal-Herranz 2012). The value obtained for each mutant was referred to that obtained for the wild-type molecule, which was assigned a value of 1. The bars represent the average and standard deviation of three independent experiments. (B,C) Huh-7.5 cells were electroporated with a mixture of wild-type or mutant 3′X subgenomic HCV replicon and cap-Rluc-mRNA as previously described (Fernández-Sanlés et al. 2015). Relative luciferase activity values (Fluc/Rluc) were determined at 5 h (B) and 72 h (C) after transfection, representing translation and replication activities, respectively. The Fluc/Rluc values of each mutant were referred to that obtained with the wild-type molecule, which was assigned a value of 1, and the bars represent the mean and standard deviation of an average of two independent experiments. In A–C, mutants experimentally verified to adopt the wild-type two-stem conformation are represented in green, mutants adopting a different structure are indicated in red, and the dimerization-defective C₂₉G/A₃₁U mutant is plotted in blue. The scatter plots shown in the insets display the individual values of two-stem, three-stem, and dimerization mutants, together with mean and standard deviation values.