LOW-AFFINITY E2-BINDING SITE MEDIATES DOWN-MODULATION OF E2-TRANSACTIVATION OF THE HUMAN PAPILLOMAVIRUS TYPE-8 LATE PROMOTER

The constitutively active promoter P-7535 of the epidermodysplasia verruciformis-associated human papillomavirus type 8 (HPV8) is transactivated by the viral E2 protein. The distribution of potential E2-binding sites (ACCN(6)GGT) in the viral transcription control region is highly conserved among epidermodysplasia verruciformis-associated human papillomaviruses and differs completely from that of other papillomaviruses. To investigate the role of E2-binding sites PO to P4 in P-7535 regulation,we analyzed their binding affinities in gel retardation experiments using a full-length HPV8 E2 protein expressed from a recombinant baculovirus. Binding site P1 within a transcriptional silencer showed the highest affinity, followed by PO within the L1 gene and P3 downstream of P-7535. P2,33 nucleotides upstream of the mRNA cap site, and P4 were very weak binders. There is some indication that the number of A/T pairs in the nonconserved core of the recognition sequence is critical for the binding of HPV8 E2. Transient transfection experiments were carried out with an HPV8 E2 expression vector and reporter plasmids containing mutated E2-binding sites in the context of the HPV8 regulatory region. The knockout of the strongest binding site P1 sufficed to clearly diminish transactivation. P0, P3, and P4 mutations had little effect on their own, whereas double mutations P01 and P34 strongly reduced E2 inducibility. Both mutations in P2 severely affected constitutive promoter activity but had opposite effects on transactivation. They revealed an inverse correlation between E2-binding strength and the extent of E2 transactivation. This finding suggests that P2 mediates a negative control of P-7535 by E2, counteracting E2 transactivation exerted via the four distal E2 target sequences.