BPV-4 E8 TRANSFORMS NIH3T3 CELLS, UP-REGULATES CYCLIN-A AND CYCLIN-A-ASSOCIATED KINASE-ACTIVITY AND DE-REGULATES EXPRESSION OF THE CDK INHIBITOR P27(KIP1)

The E8 open reading frame of Bovine papillomavirus type 4 (BPV-4) enco des a small (42 amino acid) hydrophobic polypeptide localized to cellu lar membranes and capable of conferring an anchorage-independent (AI) growth phenotype on primary bovine cells co-transfected with BPV-4 E7 ORF and an activated ras gene. To further study the function of E8 ind ependently of other viral gene products, we have expressed it in the m urine fibroblast cell line, NIH3T3, Cells expressing E8 are capable of AI growth and escape growth arrest after serum withdrawal. E8 deregul ates cyclin A expression, induces transactivation of the human cyclin A gene promoter and increases endogenous protein levels in cells maint ained in short-term suspension culture and in low-serum (LS), Both the se culture conditions promote downregulation of cyclin A in control ce lls. In LS growth conditions E8 permits sustained cyclin A-associated kinase activity but not cyclin E-cdk2 activity, Cyclin A-cdk2 activity and, in part, cyclin A gene expression are regulated by the cdk inhib itor p27(Kip1). Expression of this cdk inhibitor is also de-regulated in E8 cells, with high levels being detected under all culture conditi ons tested. These data suggest that the ability of BPV-4 E8 to transfo rm NIH3T3 cells is associated with upregulation of cyclin A-associated kinase activity and de-regulated expression of the cdk inhibitor p27( Kip1) and does not rely on down-regulation of p27(Kip1) expression. An alysis of E8 mutants indicate that the hydrophilic 'tail' of the molec ule (residues 31 - 42) is required for cell transformation, as assesse d by anchorage-independent growth, while a form of E8 with expression restricted to the Endoplasmic Reticulum/cis-Golgi membranes by additio n of a 'KDEL' retention signal revealed that the sub-cellular localiza tion is an important determinant of E8 biological activity.