Mutational Analysis of the Transforming Protein E8 of Bovine Papillomavirus Type-4 (BPV-4)

Ashrafi, G. Hossein (1998) Mutational Analysis of the Transforming Protein E8 of Bovine Papillomavirus Type-4 (BPV-4). PhD thesis, University of Glasgow.

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The transforming genes of bovine papillomavirus type 4 (BPV-4) are encoded by the E7 and E8 open reading frames (ORFs). E7 is the transforming gene of BPV-4, in that, in co-operation with activated ras, it induces morphological transformation of primary bovine fibroblasts (PalF) in the absence of other viral genes, but the acquisition of anchorage independent growth requires the additional presence of E8. The BPV-4 E8 is a small transforming protein localized to cellular membrane. It consists of two domains: a very hydro'phobic region encompassing the first 30 amino acids of the protein, and a second region of mainly hydrophilic amino acids comprising the C terminal 12 residues. The results in this thesis demonstrated that in addition to the ability of E8 to grow independently of anchorage, PalF cells expressing E8 lose gap junction intercellular communication (GJIC), can grow in low serum, and are not contact inhibited. E8 also transactivates the cyclin A promoter in PalF cells. Mutant forms of E8 were generated to establish if the transforming functions of the protein could be segregated and therefore to define its functional domains. Mutations were introduced both in the hydrophobic domain and in the hydrophilic C-terminal tail, and chimeras with BPV-1 E5 were constructed. Cells expressing either E8 wild type or its mutants were analysed for their ability to: morphological transformation, anchorage independent growth, focus formation, cell population growth in low serum, tumorigenicity in nude mice, trans-activation of the cyclin A promoter, binding to ductin and down regulation of GJIC. The analysis of E8 mutants and chimeras constructed with BPV-1 E5 show that the multiple transforming function of E8 can be segregated and that both the hydrophobic domain and the hydrophilic C-terminal tail of E8 are critical for its functions and for the transactivation of the cyclin A promoter. These results support the hypothesis that the different aspects of cellular transformation produced by E8 might be due to interaction with different cellular targets. The observation from the analysis of the transformation parameters of E8 and BPV-1 E5 expressing cells suggest that E8 acts differently from E5. This study also demonstrates that the separate domains of E5 and E8 are not functionally interchangeable. The short term co-transfection analysis of E8 mutants suggest that substitution of alanine with proline, which is expected to alter the conformation of the hydrophobic domain, may have an effect on cell transformation. Also the short-term co-transfection experiments of E8 mutants in the putative casein kinase II site support the possibility that BPV-4 E8 might be phosphorylated by CKII and that this phosphorylation could have an effect on the biological activities of this protein.

Item Type: Thesis (PhD)
Qualification Level: Doctoral
Additional Information: Adviser: Saveria M Campo
Keywords: Virology, Genetics
Date of Award: 1998
Depositing User: Enlighten Team
Unique ID: glathesis:1998-75918
Copyright: Copyright of this thesis is held by the author.
Date Deposited: 19 Nov 2019 17:36
Last Modified: 19 Nov 2019 17:36

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