Effects of human papillomavirus type 16 E5 deletion mutants on epithelial morphology: functional characterization of each transmembrane domain

Human papillomavirus type 16 (HPV-16) is the cause of cervical cancer. The HPV genome encodes three transforming proteins, E5, E6 and E7, E6 and E7 are the main transforming proteins of HPV, while the role of E5 is still poorly understood. Using three dimensional organotypic raft cultures we show that HaCaT human keratinocytes expressing HPV-16 E5 form a very perturbed epithelium, with simultaneous hyperkeratinization of some cells and defective differentiation of other cells. The basal layer is disturbed and many cells invade the collagen matrix. Many cells among the differentiated layers show characteristics of basal cells: progression through the cell cycle, expression of cytokeratin 14, lack of cytokeratin 1 and production of matrix metalloproteases (MMP). Using deletion mutants which encompass the three hydrophobic domains of E5, we have assigned the ability to promote invasion of the matrix to the first hydrophobic domain, and the capacity to induce MMP9 to the C-terminal four amino acids. We also show that invasion and production of MMP9 can be dissociated, as mutants that are still capable of invasion do not produce MMP9 and vice versa.