THE E5 ONCOPROTEIN OF HUMAN PAPILLOMAVIRUS TYPE-16 TRANSFORMS FIBROBLASTS AND EFFECTS THE DOWN-REGULATION OF THE EPIDERMAL GROWTH-FACTOR RECEPTOR IN KERATINOCYTES

To determine the function of the E5 open reading frame (ORF) of the hu man papillomaviruses (HPVs), rodent fibroblast cell lines were transfe cted with the E5 ORF of HPV type 6 (HPV-6) and HPV-16 expressed from a n exogenous promoter. Transfected fibroblasts were transformed to colo ny formation in soft agar, and the transformation frequency was increa sed by epidermal growth factor (EGF) but not by platelet-derived growt h factor. In a transitory assay, the E5 ORFs from both HPV-6 and HPV-1 6 were mitogenic in primary human foreskin epithelial cells (keratinoc ytes) and acted synergistically with EGF. Investigation of keratinocyt es expressing HPV-16 E5 showed that the number of endogenous EGF recep tors (EGFRs) per cell was increased two- to fivefold. Immunofluorescen ce microscopy of HPV-16 E5-expressing keratinocytes indicated that the re was an apparent delay in the internalization and degradation of EGF Rs compared with controls. Kinetic studies with [I-125]EGF showed that the ligand underwent normal internalization and degradation in both H PV-16 E5-expressing and control keratinocytes, but in E5-expressing ce lls, a greater number of receptors recycled back to the cell surface w ithin 1 to 6 h of ligand binding. Finally, ligand-stimulated phosphory lation of the EGFR on tyrosine, an indication of receptor kinase activ ity, was of greater magnitude in the HPV-16 E5-expressing keratinocyte s than in control cells, although the basal level of receptor phosphor ylation was similar.