Inhibition of serum- and calcium-induced terminal differentiation of human keratinocytes by HPV 16 E6: Study of the association with p53 degradation, inhibition of p53 transactivation, and binding to E6BP

Transfection of the E6 gene of human papillovirus (HPV) 16 into primary human keratinocytes (PHKs) generates proliferating cell colonies which are resistant to serum- and calcium-induced terminal differentiation. The extreme C-terminus of E6 was shown to be dispensable for this activity. To map further the amino acid sequences required for inducing resistance to serum and calcium, and to address the functional significance of E6 interactions with p53 and E6BP (ERC-55) in this function, we evaluated the activities of a series of E6 mutants. Small deletions within the central portion of the second putative zinc-finger abolished, or markedly reduced, E6 biological activity, while mutations affecting the cysteine residues in the base of the finger were less effective in this respect. When these mutants were assayed for their ability to degrade p53 in vitro and in vivo and to inhibit p53 transcriptional activation (TA), we found that there was a dissociation of these activities in some mutants. We mapped one mutant which was highly efficient in p53 degradation and inhibition of p53 TA, yet displayed severely reduced activity in the biological assay, and conversely, a subset of mutants that showed moderate activities in the colony assay while being severely impaired in p53 degradation and inhibition of p53 TA. These data argue that p53 inactivation or even elimination are not sufficient, and may not be essential, for altering the response of PHKs to serum and calcium. When these E6 mutants were evaluated for E6BP binding in vitro, there was a similar dissociation between the biological and biochemical activities of several mutants. We mapped mutants with moderate activity in the biological assay that lacked the ability to bind to E6BP and a mutant that showed high biological activity with only marginal binding to E6BP. Thus, there is no absolute correlation between the ability of E6 mutant proteins to induce alterations in keratinocyte differentiation responses to calcium and serum and to induce p53 degradation, inhibit p53 mediated transactivation, or bind E6BP. Evidently there are additional cellular targets for E6 which mediate this alteration in cellular differentiation.