Figure 4

The effects of sub-sections of the PRE on reporter activity. (A) Schematic diagrams of Luc+ reporter constructs used in the study, the splicing Luc+ reporter constructs (pSpliceLuc) and the intronless luciferase reporter construct (pBasic (-IN)). Sub-sections of the PRE were indicated. Each HBV PRE sub-section was inserted into the pSpliceLuc vector at the NheI and XhoI sites. The numbering in the scheme corresponds to nucleotide number of HBV adw2 genotype A (EMBL:AM282986). (B) The ratios of test firefly luciferase and control Renilla luciferase proteins from the splicing luc+ reporter system (pSpliceLuc). (C) The ratios of firefly luciferase and Renilla luciferase proteins from the intronless luc+ reporter system pBasic (-IN)). The pSpliceLuc constructs used in B require PRE dependent prevention of splicing out the reporter from the intron, and thus have lower ratios than the intronless constructs used in C. In B and C, Cells were co-transfected with 195 ng of luciferase reporter plasmids, 5 ng of Renilla reporter. Forty-eight h post-transfection, HuH-7 cells were harvested and 10 μL of these cell lysates were assayed for expression of luciferase proteins using POLARstar OPTIMA (BMG Labtech). Each assay was repeated in triplicate. The mean values and standard deviations of three independent experiments of the normalized luciferase activities are shown. Error bars represent standard deviation. '*', '***' indicate significant differences of luciferase activities comparing to the control vector (pSpliceLuc) with p < 0.05 and p < 0.001 respectively.