Regulation of splicing related SR proteins during the life cycle of human papillomavirus type 16

Mole, Sarah (2007) Regulation of splicing related SR proteins during the life cycle of human papillomavirus type 16. PhD thesis, University of Glasgow.

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Abstract

The aim of this project was to investigate the role of SF2/ASF in the life cycle of HPV-16, and to determine how the virus regulates this protein, as well as other SR proteins. As SF2/ASF is upregulated in cells expressing HPV-16 E2, experiments were undertaken to determine if SF2/ASF expression is transactivated by E2. Promoter transactivation studies were performed using the CAT reporter gene fused to the SF2/ASF promoter. Results demonstrate that the promoter is transactivated in cells expressing E2. Furthermore, to determine a direct role for E2 in transactivation, chromatin immunoprecipitation assays were used to show presence of E2 at the endogenous SF2/ASF promoter. However, a direct interaction could not be observed in vitro, using electrophoretic mobility shift assays. Whilst direct interaction is possible in vivo, it is likely that one or more cellular proteins aid the interaction. As the SF2/ASF promoter is as yet undefined, nothing is known about its cellular regulation. Therefore, further EMSAs were performed to determine if cellular partners of E2 could be found to associate with this promoter. In the case of Sp1, potential association of the protein with the SF2/ASF promoter was observed in vitro, using supershift assays. However, association of this protein with the promoter was not determined in vivo. This suggests Sp1 may be involved in E2-mediated regulation of SF2/ASF; however, further experiments are required to confirm this role. The next aim was to discover if HPV-16 regulates SR proteins, other than SF2/ASF, during the life cycle. This was achieved using western blot and immunofluorescence techniques, comparing undifferentiated and differentiated HPV-16 infected epithelial cells and cell extracts. Whilst elevated levels of a subset of SR proteins, including SF2/ASF, SRp20, SRp55, SRp40 and SC35 were observed upon differentiation, this was not the case for all SR proteins analysed. This suggests the SR proteins which are regulated during HPV-16 infection may be important for processing of late transcripts in differentiated cells. Furthermore, SF2/ASF and SRp20 were observed to redistribute from nuclear speckles to diffuse cytoplasmic localisation upon differentiation. However, redistribution was observed inconsistently, the reason for which is unclear. To determine if viral proteins E2 and E1.

Item Type: Thesis (PhD)
Qualification Level: Doctoral
Additional Information: Adviser: Sheila Graham
Keywords: Molecular biology, Virology
Date of Award: 2007
Depositing User: Enlighten Team
Unique ID: glathesis:2007-71016
Copyright: Copyright of this thesis is held by the author.
Date Deposited: 09 May 2019 14:28
Last Modified: 09 May 2019 14:28
URI: http://theses.gla.ac.uk/id/eprint/71016

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