The E6 and E7 proteins of the high-risk human papillomaviruses (HPVs) act coordinately to immortalize human keratinocytes. These viral oncoproteins function by binding and altering the activity of cellular proteins which regulate cell cycle progression. Among the proteins bound by E7 are the retinoblastoma protein, Rb, as well as the related p107 and p130 proteins. In addition, E7 binds cyclin A, which regulates transit through the S and G2/M phases of the cell cycle. In this study, we demonstrate that HPV 18 E7 also associates with cyclin E which controls the G1/S transition. E7/cyclin E complexes were immunoprecipitated from E7-expressing cells as well as from cell extracts using GST-E7 fusion proteins. E7 was found to complex with a single form of cyclin E, and the binding was mediated through p107. Both E7/cyclin E and E7/cyclin A complexes exhibit kinase activity through associated cdk2 proteins which can contribute to phosphorylation of p107. The association of E7 with proteins which regulate transit through the cell cycle may provide an additional mechanism by which infection with human papillomaviruses results in cellular hyperproliferation.

Human papillomavirus type 18 (HPV-18) E7 proteins bind zinc through Cys-X-X-Cys repeats located at the C terminus of the protein. In order to examine the role of these cysteine motifs in E7 function, we expressed the HPV-18 E7 protein in bacteria and found that purified E7 forms a dimer through interactions with zinc. Mutants with single mutations within the Cys-X-X-Cys motifs bound a reduced level of zinc in a zinc blot assay, while a double mutant lost all zinc-binding activity. When expressed in vivo, none of the mutants cooperated with an activated ras oncogene to transform primary rat embryo fibroblasts, but all mutants retained nearly wild-type Rb-binding activity. The results indicate that the cysteine motifs play an important role in transformation by HPV-18 E7 but do not contribute to Rb binding. The E6 and E7 genes of human papillomavirus types 16 (HPV-16), HPV-18, and HPV-31 are selectively retained and expressed in cervical carcinomas (1, 7, 12, 13, 27, 31, 39-41). The E7 gene product can independently transform immortalized rodent cells as well as primary rat embryo fibroblasts (REFs) in cooperation with an activated ras oncogene (4, 8, 9, 15, 23, 24, 28, 43, 44). The E6 gene can also transform immortalized rodent cells and is required in conjunction with E7 for the high-frequency transformation of human keratinocytes, the natural host for HPV infection (3, 21, 22, 25, 29, 35, 38, 45). These studies thus implicate E6 and E7 as viral oncogenes.

P27, an inhibitor of cyclin-dependent kinases, plays an important role in the control of cell adhesion and contact inhibition-dependent cell cycle regulation. Hepatocytes, maintained in primary culture, oer a model of synchronized primary epithelial cells which retain a dierentiated pro®le while stimulated to proliferate. We therefore investigated the pattern of endogenous p27 expression in cyclin rat hepatocytes isolated by collagenase perfusion followed by mitogenic stimulation. P27 was expressed in whole normal liver and freshly isolated hepatocytes. We then observed a sharp decrease in p27 levels, concomitant with the progression in earlymid G1, followed by reaccumulation in late G1 and the G1/S transition. Immunochemistry and BrdU labelling demonstrated nuclear localization of p27 and its expression in cells engaged in both G1 and S phase. P27 was detected in late G1 in complexes containing cyclins D1, E and A. Cyclin E-and A-associated kinase activities, however, were detected at the G1/S transition and depletion experiments con®rmed that most active complexes were free of p27. Phosphorylated forms of p27 were detected in unstimulated and stimulated hepatocytes in both early-mid G1 and G1/S. Finally, two-dimensional gel electrophoresis showed evidence for several forms of p27 with a distinct pro®le of distribution in quiescent and stimulated hepatocytes. Collectively, our data oer a model in which p27 shows a biphasic pro®le of accumulation, with the early decrease possibly involved in the progression through early and mid G1. In contrast with most cell types tested so far, the late G1 accumulation did not impair formation of active cyclin E-and A associated kinases, and thus G1/S transition.