Human papillomaviruses (HPVs) are small DNA viruses which infect only epithelial cells and are associated with a range of diseases from benign papillomas to dysplasia to cancer. A specific subset of HPVs has been associated with the development of the majority of cervical cancers as well as numerous other epithelial malignancies. The two major transforming proteins associated with these viruses, E6 and E7, have each been shown to interact with and disrupt the function of a cellular anti- oncogene. The oncogenic E6 proteins have been shown to bind to and mediate degradation of p53 in vitro via the ubiquitin protein degradation pathway. It has been shown that p53 functions as a transcripted regulator, exerting both activating and repressive effects at the promoters of numerous growth related genes. Co-expression of E6 with p53 inhibits transcriptional regulation by p53. We have used this transcriptional function now identified for p53 to further explore how various E6 proteins interact with p53. Using a series of HPV E6 mutants, we have found a close correlation between E6 inhibition of p53 transcription, in vitro binding and degradation and cellular immortalization. Thus, by making observations on the effect of E6 on this important functional activity of p53 we have a method to correlate in vivo E6/p53 interactions with in vitro observations. In a domain analysis of p53, we have found that the central region of p53 is required for binding E6, while degradation required the N-terminus of p53. Neither function specifically requires the C-terminal oligomerization domain of p53. In order to extend our observations on the physical requirements for p53 interaction with E6, we propose to make numerous deletion and fusion protein constructs of p53 suitable for testing in these experimental systems. Our goal is to more finely map the regions of p53 required for E6 binding, E6-mediated degradation, p53 DNA binding and normal metabolism of p53. We will use these observations to compare and contrast the requirements for p53 transcriptional activation vs. repression and the mechanisms through which the oncogenic and non-oncogenic E6 proteins interfere with these functions. There is accumulating evidence that p53 is important to epithelial growth regulation with levels of p53 increasing after UV exposure of skin and the detection of p53 mutations in numerous non- melanoma skin cancers. Thus, through a greater understanding of the mechanism through which E6 disrupts p53 function, we hope to gain insight into viral pathogenesis and also develop a model which may be exploited in studies of normal epithelial growth regulation.
