Dose-dependent regulation of the early promoter of human papillomavirus type 18 by the viral E2 protein

The activity of the E6/E7 promoter of genital human papillomaviruses (HPVs) is positively and negatively modulated by a complex interplay between a variety of cellular transcription factors and the virally encoded E2 protein. The long control region of genital HPVs contains four E2 binding sites in conserved positions, two of which are very close to the TATA box. Binding of E2 to these two sites has been shown to repress the promoter. To carefully analyze the effect of E2 on the activity of the early promoter P105 of HPV18, we used an in vitro transcription system, which allowed titration of the amount of E2 protein. We found that low amounts of HPV18 E2 stimulated the promoter, whereas increasing amounts resulted in promoter repression. When the affinity was analyzed, it became obvious that E2 bound with highest affinity to E2 binding site 4 (BS-4), located 500 bp upstream of the promoter. The promoter most proximal binding site (BS-1) was the weakest site. Transient transfection assays confirmed that small amounts of BPV type (HPV18) E2 and also of bovine papillomavirus type 1 (BPV1) E2 were able to activate the P105, which was dependent on an intact BS-4. The positive role of BS-4 was also obvious at higher E2 concentrations, since mutation of BS-4 enhanced repression, In contrast to HPV18 E2, BPVI E2 bound better to BS-1 and, in correlation, was able to more strongly repress the P105 in vivo. Our results suggest a dose-dependent regulation of the HPV18 E6/E7 promoter bs E2 due to variable occupancy of its binding sites, which have antagonizing effects on the activity of the E6/E7 promoter.