Gauson, Elaine J. (2015) Regulation of viral and host genomes by high risk human papillomavirus E2 protein in association with cellular factors. PhD thesis. http://theses.gla.ac.uk/7020/ Copyright and moral rights for this thesis are retained by the author A copy can be downloaded for personal non-commercial research or study This thesis cannot be reproduced or quoted extensively from without first obtaining permission in writing from the Author The content must not be changed in any way or sold commercially in any format or medium without the formal permission of the Author When referring to this work, full bibliographic details including the author, title, awarding institution and date of the thesis must be given Glasgow Theses Service http://theses.gla.ac.uk/ [email protected] Regulation of viral and host genomes by high risk human papillomavirus E2 protein in association with cellular factors Elaine J Gauson A thesis submitted to the University of Glasgow, College of Medical, Veteri- nary and Life Sciences for the degree of Doctor of Philosophy Centre for Virus Research 2 Abstract Human papillomaviruses (HPVs) are the causative agent in cervical cancer and have been implicated in a rising number of head and neck cancers in both men and women. There are two licensed prophylactic vaccines, both target HPV 16 and 18, the two most common, oncogenic types. However, there are no antiviral drugs for the treatment of HPV infection and disease. Papillomaviruses encode two DNA binding proteins, E1 and E2, which interact with host cell proteins to induce replication. Two essential cellular factors for viral transcription and replication are TopBP1 and Brd4. TopBP1 is a known interactor of HPV16 E2, and is essential for the initiation of DNA replication in eukaryotic cells. Previous studies with E2 mutants have shown that Brd4 is essential for transactivation properties of E2. Brd4 has also been implicated in the regulation of E1-E2 mediated viral DNA replication. However, the role of E2 in regulating the host genome has been less well studied. As attempts at developing a therapeutic vaccine have failed, and current small molecule inhibitors which block the interaction of replication factors, E1 and E2, are not effective across all HPV types due to slight differences in E1-E2 interactions, Brd4 and TopBP1 may present themselves as pan-type specific targets. Blocking the interaction of the host proteins Brd4 and TopBP1 with viral E2 is a viable target for HPV related cancers. This thesis set out to understand how E2 interacts with TopBP1 and Brd4 to regulate the cellular genome as well as the viral genome, to better understand how to terminate the viral life cycle. Two E2 mutants, E2-TopBP1 and E2-Brd4, which fail to bind TopBP1 and Brd4 respectively, were made to address this question. Functional assays with E2 wildtype and the two E2 mutants have enhanced our understanding of viral replication. TopBP1 and Brd4 are present at the viral origin of replication in an E1-E2 dependent manner, and optimal initiation of DNA replication is dependent on the interaction with E2. TopBP1 and Brd4 locate into E1-E2 foci and shRNA targeting these proteins destroys these replication foci. However, this has no effect on E1-E2 mediated levels of DNA replication. The results suggest a role for TopBP1 and Brd4 in the initiation of HPV16 E1-E2 DNA replication but not continuing replication, which may be mediated by alternative processes such as rolling circle amplification and/or homologous recombination. 3 To address the question of how E2 regulates the host, we stably expressed HPV16 and 18E2 wildtype, E2-TopBP1 and E2-Brd4, in U2OS cells, a p53 positive cell line that tolerates E2 expression. These cells were used in human exon array analysis, to determine which host cellular genes E2 regulates. We determined that HPV16 and 18E2 can regulate cellular genes and a failure to bind either TopBP1 or Brd4 increases the number of cellular genes altered. Overall the re- sults suggest that the levels of TopBP1 and Brd4, which can interact, regulate E2 function and therefore could regulate viral infection. 4 Table of contents Abstract .................................................................................... 2-3 List of tables ................................................................................ 9 List of figures .......................................................................... 10-11 Acknowledgements ........................................................................ 12 Author’s declaration ...................................................................... 13 Definitions/abbreviations ............................................................ 14-18 Chapter 1 – Introduction .............................................................. 19-87 History of HPV ................................................................ 19-21 HPV involvement in cancer..................................................... 21 1.2.1 Cervical cancer ......................................................... 21-24 1.2.2 Head and neck cancer ................................................. 24-25 1.2.3 Anal cancer .................................................................. 25 1.2.4 Penile cancer ................................................................ 26 1.2.5 Vulvar cancer ............................................................ 26-27 HPV vaccine ...................................................................... 29 1.3.1 Clinical trials ............................................................ 29-32 1.3.2 Biology of the HPV vaccine ................................................ 32 1.3.3 Implementing HPV vaccination ....................................... 33-34 HPV life cycle .................................................................... 34 1.4.1 HPV genome ................................................................. 34 1.4.2 Viral entry ............................................................... 37-38 1.4.3 Transcriptional regulation of the HPV long control region (LCR).39-40 Host transcription factors ........................................... 40-41 1.4.4 Three stages of viral DNA replication .................................... 42 1.4.4.1 Establishment ........................................................... 42-43 1.4.4.2 Genome maintenance ...................................................... 43 1.4.4.3 Genome amplification ................................................. 44-45 1.4.4.4 Mode of DNA replication ............................................... 45-46 1.4.5 Virus synthesis .............................................................. 47 The viral oncoproteins .......................................................... 48 1.5.1 E7 protein ................................................................ 48-49 1.5.2 E6 protein ................................................................ 49-50 The roles of E1 and E2 in replication ......................................... 51 1.6.1 Replication protein E1 ................................................. 51-54 1.6.2 Replication protein E2 ................................................. 55-56 Additional E2 functions and cellular partners ............................... 56 5 1.7.1 Transcriptional repression ............................................. 56-57 1.7.2 Growth inhibition and E2-mediated Apoptosis ......................... 59 1.7.3 Mitosis and genome segregation ...................................... 59-60 HPV E2 interaction with host proteins ........................................ 61 1.8.1 E2 regulation of the host genome .................................... 61-62 E5 protein ..................................................................... 62-63 E4 protein .................................................................. 63-65 Caspid proteins ............................................................ 65-66 1.11.1 L1 protein ................................................................. 66 1.11.2 L2 protein ............................................................. 66-67 E2 interactors TopBP1 and Brd4 ............................................ 67 1.12.1 TopBP1 structure and function ..................................... 68-70 1.12.2 TopBP1 and DNA damage response ................................ 70-71 1.12.3 TopBP1 and DNA replication ........................................ 71-72 1.12.4 The role of TopBP1 in the viral life cycle ........................ 72-74 Bromodomain proteins and disease .................................... 78-78 1.13.1 Structure and function of Brd4 ..................................... 81-79 1.13.2 The role of Brd4 in the viral life cycle ............................ 81-82 Purpose of study ............................................................. 855 Chapter 2 – Materials and Methods ................................................ 86-107 Materials ...................................................................... 86-95 2.1.1 Antibodies ................................................................... 86 2.1.2 Bacteriology ................................................................. 87 2.1.3 Cell Lines .................................................................... 87 2.1.4 Chemical and Reagents ................................................ 88-90 2.1.5 Enzymes ...................................................................... 90 2.1.6 Kits ............................................................................ 90 2.1.7 Miscellaneous...........................................................