FMDV L protease negatively impacts on the translation of cap-dependent translation but not on translation initiation mediated by the downstream IRES element.

<p>(A) Schematic representation of the capped and polyadenylated bicistronic mRNAs used in this study. Vectors dlΔEMCV and dl HIV-1 IRES have been previously described <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0068108#pone.0068108-Brasey1" target="_blank">[16]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0068108#pone.0068108-Wilson1" target="_blank">[70]</a>. The 5′UTR recovered from the HIV-1 infectious clone pNL4.3, dl HIV-1 IRES, was mutated as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0068108#pone-0068108-g001" target="_blank">figure 1</a> to add an additional initiation codon in an optimal context at position 77 (with respect to pNL4.3), AUG 77. The mutated HIV-1 5′UTR was cloned into dual luciferase bicistronic (dl) vectors harboring RLuc and FLuc luciferase as reporter genes to generate the dl HIV-1 IRES AUG 77 vector. The 5′UTR recovered from the HIV-1 infectious clone pNL4.3, dl HIV-1 IRES, was used as the reference IRES <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0068108#pone.0068108-Brasey1" target="_blank">[16]</a>, while the RLuc/FLuc bicistronic vector harboring a defective encephalomyocarditis virus (ΔEMCV) IRES, dlΔEMCV, was used as a negative control <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0068108#pone.0068108-Brasey1" target="_blank">[16]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0068108#pone.0068108-Wilson1" target="_blank">[70]</a>. As indicated by the arrows and dead end lines the AUG 77 initiation codon is out of frame with respect to the FLuc ORF. (B) <i>In vitro</i> transcribed capped and polyadenylated (+/+) RNA were translated in absence (−; black bar) or presence (+; white bar) of FMDV-L protease <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0068108#pone.0068108-Vallejos3" target="_blank">[33]</a>. RLuc (left panel) and FLuc (middle panel) activities, as well as the relative translation activity (right panel) which corresponds to the [(FLuc/RLuc)] ratio, normally used as an index of IRES activity, are shown. Values are the means ± SEM from three independent experiments each conducted in triplicate. Statistical analysis was performed by Student <i>t</i>-test (*p<0.05; **p<0.01; ***p<0.001).