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Title: Identification of high-risk human papillomavirus type 16 E5 oncoprotein as a novel viroporin
Author: Wetherill, Laura Faye
ISNI:       0000 0004 2746 6572
Awarding Body: University of Leeds
Current Institution: University of Leeds
Date of Award: 2012
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High-risk human papillomavirus type 16 (HPV16) is the primary causative agent of cervical cancer and is therefore associated with high mortality worldwide. Cellular transformation of host cells is directly mediated by the expression of three viral oncoproteins, of which HPV16 E5 (16E5) is the least characterised. The hydrophobic protein consists of 83 amino acids, which constitute three, alpha helical transmembrane domains. The protein is able to enhance proliferative signalling and subvert recognition processes in host cells. A number of 16E5-host interactions and SDS-resistant 16E5 oligomers have been identified, however the molecular basis of 16E5 function remains unclear. Often disregarded as an artefact of over-expression, we show that the 16E5-16E5 interaction, in mammalian cell Iysates, is lipid and not concentration dependent. Biochemical analysis of 16E5 oligomers has been hindered by the lack of a robust purification system. Here, we describe several, novel E5 expression and purification systems, used to purify significant quantities of detergent-free recombinant protein, for study in membrane mimetic systems. Purified 16E5 was able to associate with and integrate into liposomal membranes and formed ring-like structures visible by transmission electron microscopy (TEM). Complexes owned a defined lumenal diameter, allowing fluorescent dye release from carboxyfluorescein (CF) loaded liposomes and a hexameric stoichiometry was inferred by native PAGE. Furthermore, 16E5 channel activity was activated upon lowered hydrogen ion concentration, suggesting a potential pH dependent gating mechanism. These data identified a novel function for 16E5, as a member of the viroporin family of virally encoded channels. Known viroporin inhibitors, rimantadine and NN-DNJ, prevented 16E5 channel function in a dose-dependent manner and in the absence of high- resolution structural data, established de novo molecular modelling and rational drug design were used to develop the first specific 16E5 inhibitors. Compound MV006 did not prevent 16E5 association with liposomes or oligomerisation, validating docking studies. Functional determinants of channel activity and 16E5 oligomerisation were predicted using an in silico model. Identification of 16E5 viroporin function will enhance the understanding of the physiological role of 16E5 in HPV infections, persistence and carcinogenesis. Furthermore, 16E5 channel activity may represent an important target for antiviral intervention.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available