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Title: Cell transformation by the E5 protein of bovine papillomavirus type 4
Author: Zago, Manola
ISNI:       0000 0001 3576 4789
Awarding Body: University of Glasgow
Current Institution: University of Glasgow
Date of Award: 2002
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The E5 protein of Bovine Papillomavirus type 4 (BPV-4) is, at only 42 amino acids, the smallest transforming protein identified to date. E5 consists of a N-terminal putative ?-helical transmembrane body and a hydrophilic C-terminal tail and is localised to cellular membranes. E5 transforms fibroblasts, its expression allows established NIH3T3 fibroblasts to proliferate in suspension and in the absence of mitogens. Here we analysed the cellular localisation of BPV-4 E5 showing that a form of E5 fused with the green fluorescent protein (GFP-E5) is distributed mainly to the Golgi apparatus and to a lesser extent to the Endoplasmic Reticulum but could not be detected at the plasma membrane. GFP-E5 was used in transient and stable transfection assays in NIH3T3 cells. These studies revealed that, contrary to E5 wt, GFP-E5 expressing cells are not morphologically transformed, and cannot proliferate in absence of mitogens, however these cells do not exit the cell cycle when maintained in suspension. We concluded that GFP-E5 could segregate the two transforming phenotypes of E5 wt. Here we investigated at what level of the cell cycle machinery the segregation occurs. Biochemical analysis revealed that GFP-E5 expressing cells do not sustain cyclin D1 and cyclin A expression after serum withdrawal, show an active hypophosphorylated form of pRb and pi07 and consequently exit from the cell cycle and become quiescent in these growth conditions. Moreover GFP-E5 ability to promote transcriptional activation of a heterologous cyclin A promoter in normal growth conditions is not maintained in low serum, contrary to E5 wt, and although the cyclin A-associated kinase activity in GFP-E5 cells was 1.5 times higher that the control in low serum, it was clearly not sufficient to sustain cells growth. However when maintained in suspension GFP-E5 cells, similarly to E5 wt cells, present an up-regulated cyclin Dl, can transactivate cyclin A promoter and sustain cyclin A expression level and cyclin A-associated kinase activity. Interestingly only cdc2, but not CDK2, can be detected in cyclin A immunoprecipitates. The use of inhibitors of the MAPK pathway revealed that in E5 wt cells the level of cyclin D1 and cyclin A expression is independent from this pathway. In GFP-E5 cells, however, cyclin D1 is down-regulated but cyclin A is still elevated after treatment with the inhibitors. From these data we conclude that GFP-E5 cells ability to grow in suspension correlates with the ability to up-regulate cyclin A expression and cyclin A-associated kinase activity and that the up-regulation is independent from MAPK pathway and cyclin D1 expression level. Moreover, we show here, in both E5 and GFP-E5 cells, that the principal cyclin-dependent kinase associated with cyclin A is cdc2 suggesting that the viral protein may act on the G2/M phase transition of the cell cycle.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available