Binding of the human papillomavirus type 16 p97 promoter by the adeno- associated virus Rep78 major regulatory protein correlates with inhibition

Human papillomavirus type 16 (HPV-16) infection is positively associated with cervical cancer, whereas adeno-associated virus (AAV) infection is negatively associated with this same cancer. In earlier studies these two virus types have been shown to directly interact, with AAV inhibiting or enhancing papillomavirus functions depending upon the specific circumstances. One defined interaction between these two viruses is the ability of the AAV Rep78 major regulatory protein to inhibit gene expression of the E6 promoter of BPV-1 (bovine papillomavirus type 1) and HPV types 16 and 18. As Rep78 is a DNA binding transcription factor, we considered whether Rep78 might bind HPV-16 DNA. Here, Rep78 is demonstrated to bind a 44-base pair region (nucleotides 14-56) within the HPV-16 p97 promoter using the electrophoretic mobility shift assay. This region is important for HPV-16 because it includes functional Sp1 and E2 protein binding motifs as well as part of the origin of replication. Furthermore, two Rep78 amino acid substitution mutants, at positions 77 or 64-65, were identified that did not recognize p97 DNA. Both of these Rep78 mutants were found to be defective for inhibition of p97 promoter activity in HeLa and T-47D nuclear extracts in vitro, in a transient chloramphenicol acetyltransferase assay, as well as defective for full inhibition of HPV-16-directed focus formation. These data, taken together, strongly suggest that the Rep78-p97 promoter interaction is at least partially responsible for Rep78-mediated inhibition of HPV-16. Finally, the finding that Rep78 specifically recognizes p97 DNA is surprising because the p97 promoter region contains no GAGC motifs, the core motif for Rep78 recognition. These data suggest that the p97 promoter may represent a new prototypical DNA target type for Rep78.