FIG 2 .

Characterization of the late-promoter region of HPV18. (A) The strong activity of a P₈₁₁ promoter region is dependent on CCW orientation of the HPV18 Ori. HPV18-infected human foreskin keratinocytes (HFK18), differentiated by adding 2 mM calcium, were transfected for 48 h with each of the indicated plasmids. (A) Renilla luciferase plasmid pRL-TS was cotransfected as an internal control. The supernatant of the cell lysates from each transfection was examined for dual luciferase activities. Relative promoter activity levels were calculated by dividing the value representing the light unit readings from a testing promoter-firefly luciferase reporter by the value representing the light unit readings from the Renilla luciferase reporter (left panel). Plasmid pXHW61 directly derived from pXHW21 has the HPV18 Ori flipped into a CCW orientation (middle panel). Plasmid pXHW21-derived pXHW49 and pXHW22-derived pXHW50 have their corresponding promoter regions replaced by the SV40 early promoter derived from the pGL3 control vector (right panel). The data shown are means ± standard deviations (SD) of results from two to three independent experiments. P values were calculated using Student’s t test. (B) The P₈₁₁ promoter activity depends on keratinocyte differentiation. HFK18 cells with (+) or without (−) 2.0 mM calcium treatment were transfected with pXHW22 for 48 h and then analyzed for their luciferase activity. The data shown are from results from one of two experiments, with means ± SD calculated from triplicate samples. (C) Opposite orientations of the HPV18 Ori do not affect plasmid DNA replication in HFK18 cells. HPV18-infected HFK cells, differentiated by 2 mM calcium, were transfected with pXHW21 (Ori in CW orientation) or pXHW22 (Ori in CCW orientation) for 48 h. Replicated plasmid DNA isolated from the cells and the original input bacterial plasmid DNA were compared for their sensitivity to DpnI (digesting only methylated bacterial plasmid DNA) and MboI (digesting only unmethylated bacterial and replicated plasmid DNA). Because the input bacterial DNA is methylated at the adenine of GATC sequences, it is sensitive to digestion by DpnI but resistant to digestion by MboI (right panel), and because human cells lack adenine methylase activity, the newly replicated DNA lacking adenine methylation is thus resistant to DpnI digestion but susceptible to MboI digestion (left panel). ND, no digestion with a restriction enzyme. The digested DNA samples were then resolved in a 1% agarose gel and imaged by ethidium bromide staining. Lanes 1 and 8 represent DNA markers (M). (D) Aphidicolin, a DNA polymerase inhibitor, blocks CCW orientation-dependent HPV18 late promoter activity. The sensitivity of Ori-directed DNA replication and HPV18 late promoter activity in plasmid pXHW22 to aphidicolin at different doses was analyzed in the HFK18 cells cotransfected with plasmids pXHW22 and pRL-SV40 (an internal control). The supernatant of the cell lysates was examined for dual luciferase activities, and the relative promoter activity levels were calculated as described for panel A.