This proposal seeks to investigate the mechanisms by which the E7 gene product of genital human papillomaviruses (HPV) contributes to the pathogenesis of viral infections. In anogenital cancers, the E6 and E7 genes of the high risk 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 and the inclusion of E6 leads to a significant increase in frequency. Epithelial cells immortalized by E6 and E7 also exhibit altered differentiation capabilities in rat 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 regulation of E2F mediated transcription. E7 also binds cyclin E and A indirectly through its association with p107 and that this complex exhibits kinase activity as well as several inhibitors of cyclin activity including p21 and p27 which act to regulate kinase function. It is not clear which of these activities is important in the productive life cycle. Recently, my laboratory has developed methods to synthesize HPV from transfected DNA templates which permits a genetic analysis of viral functions. In addition we have recently used growth in semi-solid media to induce late viral functions following less than 24 hours of growth. These techniques now permit a detailed analysis of E7 function in the productive life cycle. The following questions will be addressed: 1). What role do E7 binding partners play during epithelial differentiation? 2). What are the effects of E7 mutations on the viral life cycle? 3). How does E6 augment E7's role in viral pathogenesis? 4). Does E6/E7 induced Rb degradation play a role in the differentiation dependent viral life cycle?
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