) Specific genotypes of human papillomaviruses (HPV) are known to cause the majority of cervical cancers as well as a high percentage of anal and penile cancers. HIV infection and the immunocompromised state in AIDS place HPV-infected individuals at increased risk for developing these HPV-induced malignancies. HPV type 16 is the most common isolate from anogenital cancers, and its E6 and E7 proteins are selectively retained and expressed in these tumors. This application examines the mechanism of transformation by the HPV 16 E6 protein with emphasis on its interactions with cellular factors and pathways. HPV 16 E6 binds the human protein E6-AP, and formation of this complex allows subsequent binding to p53. When bound to E6, E6-AP acts as an ubiquitin ligase that directly catalyzes the ubiquitination of p53, and in this manner E6 selectively targets p53 for degradation. Nonetheless, several lines of evidence imply that the association of HPV E6 with p53 does not explain its full spectrum of oncogenic effects. The applicant has isolated a new E6 binding protein, E6BP, using the yeast two-hybrid screen. A large series of HPV 16 E6 mutations, characterized for their ability to induce p53 degradation and bind E6BP, will be tested for immortalization of primary human keratinocytes and mammary epithelial cells and transformation of murine cell lines. Because E6BP is a calcium binding protein and HPV 16 E6 blocks calcium induced terminal differentiation of keratinocytes, the applicant hypothesizes that the E6-E6BP interaction maintains the infected epithelial cell competent to support viral DNA replication, and this will be experimentally tested using the applicant s E6 mutants. The normal functions of E6BP and the consequences of its association with E6 will be investigated in a series of cell models, and cellular targets of E6BP will be sought in order to decipher its mechanism of action. The applicant has identified a small peptide in E6BP that mediates binding to E6 and have obtained a preliminary structure by NMR spectroscopy. Studies to test the ability of this E6BP peptide to inhibit E6 interactions with E6BP and p53 in vivo will be performed.
