A C-TERMINAL HELICASE DOMAIN OF THE HUMAN-PAPILLOMAVIRUS EL PROTEIN BINDS E2 AND THE DNA-POLYMERASE ALPHA-PRIMASE P68 SUBUNIT

The human papillomavirus (HPV) El and E2 proteins bind cooperatively t o the viral origin of replication (ori), forming an E1-E2-ori complex that is essential for initiation of DNA replication, All other replica tion proteins, including DNA polymerase alpha-primase (pol alpha-prima se), are derived from the host cell. We have carried out a detailed an alysis of the interactions of HPV type 16 (HPV-16) El with E2, ori, an d the four pol alpha-primase subunits, Deletion analysis showed that a C-terminal region of El (amino acids [aa] 432 to 583 or 617) is requi red for E2 binding, HPV 16 El was unable to bind the ori in the absenc e of E2, but the same C-terminal domain of El was sufficient to tether El to the ori via E2, Of the pol alpha-primase subunits, only p68 bou nd El, and binding was competitive with E2, The El region required (aa 397 to 583) was the same as that required for E2 binding but addition ally contained 34 N-terminal residues. In confirmation of these differ ences, we found that a monoclonal antibody, mapping adjacent to the N- terminal junction of the p68 binding region, blocked E1-p68 but not E1 -E2 binding, Sequence alignments and secondary-structure prediction fo r HPV-16 El and other superfamily 3 (SF3) viral helicases closely para llel the mapping data in suggesting that aa 439 to 623 constitute a di screte helicase domain. Assuming a common nucleoside triphosphate-bind ing fold, we have generated a structural model of this domain based on the X-ray structures of the hepatitis C virus and Bacillus stearother mophilus (SF2) helicases, The modelling closely matches the deletion a nalysis in suggesting that this region of El is indeed a structural do main, and our results suggest that it is multifunctional and critical to several stages of HPV DNA replication.