Lees, Emma Margaret; (1990) The E2 protein of human papillomavirus type 16. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Gene expression within HPV16 is regulated, at least in part, by the product of the E2 gene. These studies describe physical and functional dissection of this DNA-binding transactivator protein. 1) Overexpression of HPV16 E2 in primary epithelial cells, in the presence of the intact viral genome, has given us an important insight into the activities of the E2 protein. The presence of E2 within these cells markedly increases the levels of E7 protein produced from the viral genome, thereby potentiating the ability of the virus to cooperate with an activated ras oncogene in the transformation of primary baby rat kidney cells. These experiments show that E2 is a potent transactivator of early gene expression. 2) E2 has been overexpressed in E.coli to a level approaching 15% of total cell protein. The protein has been purified to homogeneity and retains its ability to bind DNA in vitro and to transactive viral promoters in vivo. 3) Computer modelling has been used to predict contact points between the E2 protein and its palindromic recognition sequence. The predicted model was used to direct mutagenesis of the protein, to abolish DNA-binding. Site- directed mutagenesis has shown that two conserved arginines within the DNA-binding domain of the protein are essential contacts in the binding of E2 to DNA. 4) Monoclonal antibodies have been raised against an E2 galactosidase fusion protein. These antibodies have been used to examine E2 expression in cervical lesions. The prevalence of the E2 protein appears to be closely correlated with the severity of lesion. Control of early gene expression is likely to be important in tumorigenesis, given that the E6 and E7 genes encode proteins essential and sufficient for transformation. Therefore knowledge of the activities and expression patterns of any transcriptional modulators will be critical to our understanding of cervical carcinogenesis.

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