The long-term goal of this proposal is to understand the mechanisms by which the E7 gene product of the high risk human papillomaviruses (HPV) contributes to the development of anogenital malignancies as well as to the pathogenesis of viral infections. In anogenital cancers, the E6 and E7 genes of the high risk viruses are selectively retained and expressed implicating their products as oncoproteins. Furthermore, studies in tissue culture have demonstrated that the E7 gene product alone can immortalize human keratinocytes, but the inclusion of E6 leads to a dramatic increase in frequency. The B6 protein by itself has no immortalizing activity and appears to augment the action of E7. Epithelial cells immortalized by E6 and E7 also exhibit altere differentiation capabilities in raft cultures similar to those seen in neoplasias in vivo. The mechanism by which these viral oncoproteins immortalize and alter the differentiation capabilities of epithelial cells involves the binding of host proteins involved in cell-cycle regulation. The E6 proteins bind and accelerate the degradation of p53 by the ubiquitin-mediated pathway. The E7 protein binds the retinoblastoma gene product (Rb) as well as the related p107 and p130 proteins. The binding of E7 to Rb and p107 interferes with the attachment of other cellular proteins such as E2F and ATF-2. We have recently demonstrated that E7 also binds cyclin E and A indirectly through its association with p107 and that this complex exhibits kinase activity. Several inhibitor of cyclin activity including p21 and p27 which act to regulate kinase function have also been identified. Expression of p21 normally increases following exposure to radiation and is dependent upon p53 transcriptional activation. In HPV positive cells, p53 levels are often reduced resulting in a failure to activate p21 expression. Little information is currently available regarding the normal role of p27. In this application, we propose to examine the interaction of E7 with cyclin kinase complexes and study E7's role immortalization and in viral pathogenesis. The following questions will be addressed: I). What is the interaction of the cyclin associated proteins p21 p27 and Cdi with cyclin /E7 complexes or How are these E7/cyclin complexes altered following exposure to DNA damaging agents? II). Are similar cell cycle regulatory proteins associated with E7 from the low risk viruses? Ill). Is the substrate specificity of cyclin complexes altered by the presence of E7 or is the primary function of E7 to prevent association with cyclin inhibitory proteins? IV). Can mutants of E7 discriminate between these various activities and provide insight into the mechanism of E7 action? V). What is the role of E7 and, in particular, cyclin /E7 complexes in the HPV life cycle?
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