Human papillomavirus type 16 (HPV-16) is the commonest cause of cervical cancer worldwide, yet until recently little was known of the mechanism of action of this leading cause of female disease and death. We have shown that three genes of HPV-16, E6, E7, and E5 are involved in the immortalization and transformation of mammalian cells and this proposal wishes to determine the mechanism of action of E7 and E5. E7 has been shown to bind to the retinoblastoma family of proteins, pRB, p107, and p130, which are involved in cell cycle control during G1. We have shown that E7 also binds to the AP-1 family of transcription factors, and inhibits the DNA binding of c-Jun, a member of the family. AP-1 factors have an important role in the differentiation of human keratinocytes, and family members can form homo- and hetero-dimers, affording them a range of binding and transcriptional activities. It is noteworthy that HPV-16 inhibits keratinocyte differentiation, both in vivo and in vitro and this may partly be explained by E7/Jun interactions. We plan to investigate the effect of E7 on transcription of the various AP-1 family members and how this changes with keratinocyte differentiation. We found that c-Jun also binds to pRB, so we will investigate the functional significance of this complex and determine how E7 effects the interaction. We have shown that HPV-l6 E5 acts with E6 and E7 to optimism keratinocyte immortalization. One mechanism of action is its ability to inhibit degradation and down- regulation of the epidermal growth factor receptor (EGFR), resulting in a strong mitogenic signal. This signal is augmented by the fact that E5 also causes an increase in phosphorylation of the EGFR. The inhibition of degradation is due to a delay in acidification of EGFR containing endosomes through the binding of E5 to the vacuolar ATPase. We wish to carry out, for the first time, a mutagenesis study of E5 to determine functional domains, investigate the mechanism of increased receptor phosphorylation, and determine the effect of E5 on other tyrosine kinase receptors. E5 also effects secretory pathways, and we found that E5 causes instability of the TAP-1 protein, which transports immune peptides to class I molecules as part of the process for the immune recognition of foreign antigens. This results in a down-regulation of the class I molecules on the cell surface, and an increased chance that infected cells will not be recognized by the immune response cells. This could be one reason why HPV lesions persist, and in part may explain the malignant potential of these viruses. We will investigate the mechanism by which E5 increases the instability of the TAP-1 protein, and whether E5 binds directly to TAP-1 proteins.
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