Replication-associated activities of purified human papillomavirus type 11E1 helicase

Replication of human papillomavirus type11 (HPV11) requires both the E1 and the E2 proteins. E1 is structurally and functionally similar to SV40 large T-antigen and is a DNA helicase/NTPase that binds to the origin of replica tion and initiates viral DNA replication. The biochemical characterization of HPV E1 is incompletely documented in the literature in part because of d ifficulties in expressing and purifying the protein. Herein, we report a me thod for the overexpression of full-length, untagged E1 (73.5 kDa) in bacul ovirus-infected Trichoplusia ni insect cells and the purification to homoge neity using a two-step procedure. The purified protein is a nonspecific NTP ase that hydrolyzes ATP, dATP, UTP, or GTP equally well. Point mutations we re made in the putative NTPase domain to verify that the activities observe d were encoded by E1. Purified mutant D523N had negligible ATPase and helic ase activities but retained DNA-binding activity. Sedimentation equilibrium ultracentrifugation and glycerol gradient centrifugation demonstrated that the wild-type protein is primarily a hexamer in its purified form. Seconda ry structure determination by circular dichroism revealed a large percentag e of alpha-helical structure consistent with secondary structure prediction s. These data define a fundamental set of biochemical and kinetic parameter s for HPV E1 which are a critical prerequisite to future mechanistic studie s of the enzyme. (C) 2000 Academic Press.