Figure 9. Analysis of E8ˇE1 overexpression on HPV18 genome replication and transcription.

A: U2OS cells were transfected with 2 µg of the HPV18 E8 mutant minicircle alone or with different concentrations (10 ng, 50 ng and 250 ng) of the E8ˇE1 expression plasmid. The E8ˇE2 expression plasmid (250 ng) was added as a control. Genomic DNA was extracted 3 and 4 days after the transfection, and a qPCR-based analysis of the viral genome relative copy number (C_N) was performed as described in the Materials and Methods section. The value obtained from the HPV18 E8 mutant 3-day timepoint was set to 1, and the CN values of the other samples are expressed relative to this point. The averages of the results of two experiments are presented with standard deviations. B: U2OS cells were transfected with different concentrations (0.1 ng, 1 ng, 10 ng, 100 ng and 500 ng) of E8ˇE1 or 250 ng of the E8ˇE2 expression plasmid together with the reporter plasmid URR-Luc containing the native HPV18 URR and a non-specific reporter pRL-TK. Twenty-four hours after the transfection, luciferase activities were measured and normalized to those for Renilla luciferase expression from the thymidine kinase promoter of the co-transfected plasmid pRL-TK. These data indicate the effect of E8ˇE1 on promoter activity relative to the reporter alone. The averages of the results of two experiments are presented with standard deviations.