Viruses are often potent oncogenes and are causally linked to approximately 20 percent of human malignancies. Human papillomavirus (HPV), in particular, is considered to be the etiological agent of human cervical cancer. HPV infections are also implicated in other malignancies including cancer of the vulva, vagina, penis, anus, skin, esophagus, and oropharyngeal region. Transformation by HPV is mediated by two relatively small viral oncoproteins, E6 and E7 that mediate their activities through their interaction with host proteins, which are normally involved in controlling cell growth and division. HPV-E6 abrogates the function of the p53 tumor suppressor gene by cooperating with E6AP to target p53 for ubiquitin-mediated degradation, and also inhibits the transactivation properties of the global transcriptional coactivators, CBP and p300. E7 binds to the Retinoblastoma (pRb) tumor suppressor and liberates E2F transcription factors causing premature activation of genes involved in DNA synthesis during the G1 to S cell cycle transition. E7 also interacts with the AP1 family of transcription factors for transactivation of AP1-mediated transcription, with Mi2 and histone deacetylases to perturb pRb-mediated transcriptional repression, and with the p21(WAF1/CIP1) family of cyclin-dependent kinase inhibitors to perturb cell cycle regulation. The functional homologue of HPV-E7, Adenovirus E1A (Ad-ElA), also inhibits the activity of pRb as well as other transcriptional cofactors such as the P/CAF and CBP/p300 histone acetyltransferases. In order to obtain mechanistic insights into the mode of cell transformation by HPV we propose to determine the X-ray crystal structures of HPV-E6 and E7 bound to relevant cellular protein targets and to biochemically characterize their respective protein-protein interactions. Specifically, we will (1) Determine the X-ray crystal structure of HPV-E7 alone and in complex with pRb, (2) Determine the X-ray crystal structure of Ad-E1A in complex with pRb, (3) Determine the structure of a pRb/E2F complex, characterize the binding properties of pRb to E2F in the presence or absence of HPV-E7 or Ad5-E1a, and use mutational analysis to probe the interactions of pRb with E2F, HPV-E7 and Ad-E1A, and (4) Determine the X-ray crystal structure of HPV-E6 alone and in complex with p53 and characterize the binding properties of the complex using mutational analysis. These studies will provide detailed mechanistic insights into the mode by which HPV-E6 and -E7 disrupt normal cellular processes for cell transformation and will lead to the structure-based design of small molecule E6- and E7- inhibitors to combat HPV-mediated cancers such as cervical cancer.
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