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 be tween a variety of cellular transcription factors and the virally enco ded E2 protein. The long control region of genital HPVs contains four E2 binding sites in conserved positions, two of which are very close t o the TATA box. Binding of E2 to these two sites has been shown to rep ress the promoter. To carefully analyze the effect of E2 on the activi ty of the early promoter P105 of HPV18, we used an in vitro transcript ion system, which allowed titration of the amount of E2 protein. We fo und that low amounts of HPV18 E2 stimulated the promoter, whereas incr easing 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 pr omoter most proximal binding site (BS-1) was the weakest site. Transie nt transfection assays confirmed that small amounts of BPV type (HPV18 ) E2 and also of bovine papillomavirus type 1 (BPV1) E2 were able to a ctivate the P105, which was dependent on an intact BS-4. The positive role of BS-4 was also obvious at higher E2 concentrations, since mutat ion of BS-4 enhanced repression, In contrast to HPV18 E2, BPVI E2 boun d better to BS-1 and, in correlation, was able to more strongly repres s 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 bindi ng sites, which have antagonizing effects on the activity of the E6/E7 promoter.