Document Type

Theses, Ph.D

Rights

Available under a Creative Commons Attribution Non-Commercial Share Alike 4.0 International Licence

Disciplines

1.6 BIOLOGICAL SCIENCES

Publication Details

Successfully submitted for the award of Doctor of Philosophy (Ph.D.) to the Dublin Insitute of Technolgy, 2012

Abstract

Human papillomaviruses (HPVs) are present in 99.7% of all cervical cancers and HPV type 16 (HPV-16) is the major cause of cervical cancer. Expression of the viral capsid gene L1 and L2 can be detected only in the terminally epithelial cells and we speculate that inhibition of HPV-16 late gene expression in the early stage of the life cycle is probably a prerequisite for persistence of infection. The products of the late genes, L1 and L2, are highly immunogenic and expression of these proteins in the lower layers of the cervical epithelium could lead to clearance of the virus. Therefore, it is of interest to understand how HPV late gene expression is regulated. The goal of this thesis was to examine the regulation of late genes in HPV-16. To this end we wished to generate reporter plasmids based on the HPV-16 genome with the L1 gene replaced by an easily measurable reporter gene, such as chloramphenicol acetyltransferase (CAT), green fluorescent protein (GFP), secreted alkaline phosphatase (SEAP) or luciferase, and to establish reporter stable cell lines useful for large scale screening of small molecules or cellular factors that influence RNA processing events during late gene expression. CAT and GFP proved to be functional surrogate markers of late gene expression and their expression was dependent on the levels of known inducers of HPV-16 late gene expression such as adenovirus E4orf4 protein (E4orf4), polypyrimidine tract binding protein (PTB), arginine/serine-rich SRp30c protein (SRp30c) or alternative splicing factor/splicing factor 2 (ASF/SF2). Functional stable cell lines with CAT reporter plasmids, separately integrated into the HeLa cellular genome, were also generated allowing the identification of a number of small molecules capable of modulating CAT expression. Phorbol 12-myristate 13-acetate (TPA), valproic acid and tannic acid were identified as inducers of HPV-16 late gene expression. Further experiments identified the TPA inducible, hnRNP A2/B1 protein as a novel regulator of HPV-16 late gene expression. Immunohistochemical analysis of this protein in cervical epithelium at the different stages of the development of cervical cancer demonstrated that hnRNP A2/B1 is highly expressed in normal cervical epithelium and low-grade squamous intraepithelial lesion (LSIL) and decreased in highgrade squamous intraepithelial lesion (HSIL) and squamous cell carcinoma (SCC). In conclusion, the HPV-16 reporter plasmids and reporter cell lines described herein are functional and can be used for the investigations of HPV-16 late gene expression.

DOI

https://doi.org/10.21427/D7JG68

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