A cis-Acting Element Present within the gag Open Reading Frame Negatively Impacts on the Activity of the HIV-1 IRES
Figure 1
INS-1 sequence negatively impacts on HIV-1 IRES activity.
A) Schematic representation of the dual luciferase (dl) mRNAs used in vitro translation assay. As reporter genes the used bicistronic mRNAs harbor an upstream Renilla luciferase (RLuc) and a downstream firefly luciferase (FLuc). INS-1: sense orientation (5′to 3′) and SNI: antisense orientation (3′ to 5′). B) Results of in vitro translation assay in which luciferase activity values for each cistron corresponds to mean (+/− SEM) of three independent experiments performed each in triplicate. The relative RLuc and FLuc activities for dl HIV-1 IRES were arbitrarily set to 100%. C) The luciferase activity was determined 24 h after transfecting HeLa cells with 200 ng of each bicistronic plasmid. The figure shows the values of luciferase activities corresponding to the mean (+/− SEM) of three independent experiments performed each in triplicate. The relative RLuc and FLuc activities for dl HIV-1 IRES were arbitrarily set to 100%. D) Total RNA (200 ng) extracted from transfected HeLa cells was used as template in parallel RT-qPCR reactions designed to specifically detect the RLuc or FLuc containing RNAs. The RNA-FLuc concentration (pmoles)/RNA-RLuc (pmoles) ratio was calculated. Values are the means (+/− SEM) from three independent experiments. Statistical analysis was performed by ANOVA test followed by Dunnet multiple comparison. *p<0.05 v/s dlHIV-IRES.