Binding of the human papillomavirus type 16 E7 oncoprotein and the adeno-associated virus Rep78 major regulatory protein in vitro and in yeast and the potential for downstream effects

Objective: Both human papillomavirus (HPV) and adenoassociated virus (AAV) are common anogenital viruses and likely co-infect the epithelium in vivo. However, whereas HPVs are positively associated with cervical cancer, AAV a ppears to be negatively associated. In tissue culture, AAV-encoded Rep78-wh ich is essential for AAV-inhibits gene expression and oncogenic transformat ion by HPV-16/18 and bovine papillomavirus type 1. Here we observed whether the HPV-16 E7 oncoprotein might recognize and bind Rep78. Further, upon fi nding Rep78-E7 binding, we investigated some of the potential downstream ef fects such an interaction might have. E7 is capable of recognizing a variet y of proteins, including RB105, TATA box-binding protein (TBP), TBP-associa ted factor (TAF)(11)110, E2F, cyclins A and D, and c-jun. Some of these int eractions are likely responsible for E7's cancer-promoting activity. Study Design/Methods: Rep78-E7 interaction was investigated in vitro by Wes t(far)-Western and affinity chromatography analysis and in vivo in living y east by the GAL4 two-hybrid cDNA assay. Mapping of the E7 binding domain wi thin Rep78 was carried out using a series of amino- and carboxy-truncated R ep78 proteins in a West(far)-Western assay. Downstream effects of the inter action were analyzed by competitive affinity chromatography (protein-protei n) and competitive electrophoretic mobility shift assay (protein-DNA). Resu lts: E7 and Rep78 were found to interact both in vitro and in vivo (yeast) in all assays attempted. The E7 binding domain within Rep78 was found to re side within amino acids 121- 370. Regarding downstream effects of this inte raction, Rep78 was found to mildly inhibit E7-TAF(II)110 and E7-RB105 inter action in vitro bur to have little affect on E7-TBP interaction. Finally, i t was found that E7 was able to affect Rep78's interaction with AhV's termi nal repeat (TR) DNA in vitro, reducing the formation of the largest sized R ep78-TR complexes in a dosage-dependent manner. Conclusions: These data suggest that the Rep78-E7 interaction may have repe rcussions for both viruses. The Rep78-E7 interaction may be a second mechan ism, in addition to Rep78 regulation of the p97 promoter, by which AAV inhi bits HPV-16 oncogenic transformation. These data also suggest that HPV-16 m ay affect the AAV life cycle by altering Rep78-TR interaction.