FIG. 5.

HPV16 E6 expression causes as increase in cap-dependent translation, which is sensitive to rapamycin treatment. (A) Diagram of bicistronic firefly Renilla reporter plasmid, pFR_CrPV_xb, used for these experiments. Firefly luciferase is translated through a cap-dependent mechanism, whereas Renilla luciferase is expressed from an internal ribosomal entry site (IRES) through a cap-independent mechanism (top). HPV16 E6 expression causes an increase in firefly but not Renilla luciferase activity (bottom). U2OS cells were transfected with control or HPV16 E6 expression vector, and lysates were processed for Renilla and firefly luciferase assays at 48 h posttransfection. The data are presented as the change of firefly and Renilla luciferase activities normalized to control vector-transfected cells (left and middle) and the fold change of normalized firefly compared to normalized Renilla luciferase activity (FF/Ren) (right). The bar graphs represent averages and standard deviations of four experiments, each performed in triplicate. The asterisk denotes statistical significance (P < 0.0001). (B) Western blot analysis of firefly and Renilla luciferase expression in U2OS cells transiently transfected with the indicated plasmids. U, untransfected cells. (C) Western blot analysis of eIF4G binding to a synthetic 7-methyl-GTP (7MeGTP) mRNA cap upon transient transfection of HPV16 E6 or control vector in U2OS cells. (D) HPV16 E6-mediated increase in cap-dependent translation is rapamycin sensitive. U2OS cells were transfected with pFR_CrPV_xb and the indicated plasmids; 18 h prior to lysis, cells were treated with dimethyl sulfoxide (DMSO) or 100 nM rapamycin (Rap). The graph represents averages and standard deviations of four experiments, each performed in triplicate. (E) Western blot analysis of S6K phosphorylation in U2OS cells transiently transfected with HPV16 E6 or control vector. One hour prior to lysis, cells were treated with DMSO or 100 nM rapamycin (Rap). Decreases in p53 levels are shown to document HPV16 E6 expression, and an actin blot is included as a loading control. (F) Transient transfection of HPV16 E6 activates cap-dependent translation in primary HFKs. Cells were transfected with pFR_CrPV_xb and the indicated plasmids and processed for Renilla and firefly luciferase assays at 48 h posttransfection. Firefly and Renilla luciferase activities were normalized to control vector-transfected cells and are presented as fold changes of normalized firefly relative to normalized Renilla luciferase activity. The bar graph represents the average and standard deviation of four experiments, each performed in triplicate; asterisks indicate statistical significance (P = 0.0001).