This thesis has been submitted in fulfilment of the requirements for a postgraduate degree (e.g. PhD, MPhil, DClinPsychol) at the University of Edinburgh. Please note the following terms and conditions of use: • This work is protected by copyright and other intellectual property rights, which are retained by the thesis author, unless otherwise stated. • A copy can be downloaded for personal non-commercial research or study, without prior permission or charge. • 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. Systematic analysis of protein‐protein interactions of oncogenic human papillomavirus Ramya M. Gundurao A thesis presented for the degree of Doctor of Philosophy, The University of Edinburgh February 2013 Division of Pathway Medicine College of Medicine and Veterinary Medicine The University of Edinburgh DECLARATION I hereby declare that this thesis and the work presented in it are my own. I confirm that: This work was done wholly or mainly while in candidature for a research degree at this University. Where any part of this thesis has previously been submitted for a degree or any other qualification at this University or any other institution, this has been clearly stated. Where I have consulted the published work of others, this is always clearly attributed. Where I have quoted from the work of others, the source is always given. With the exception of such quotations, this thesis is entirely my own work. I have acknowledged all main sources of help. Where the thesis is based on work done by myself jointly with others, I have made clear exactly what was done by others and what I have contributed myself. Ramya M. Gundurao University of Edinburgh II ABSTRACT Human papilloma virus (HPV) is a ubiquitous virus implicated in a growing list of cancers, particularly cervical cancer‐ the second most common cancer among women worldwide. Although persistent infection with high‐risk oncogenic HPVs such as types ‐16 or ‐18 is necessary, additional factors like co‐infection with other viruses can play a role in cancer progression. Protein‐protein interactions play a central role in the infection, survival and proliferation of the virus in the host. Although some interactions of HPV proteins are well characterised, it is essential to discover other key viral interactions to further improve our understanding of the virus and to use this knowledge for the development of newer biomarkers and therapeutics. The aim of this study was to systematically analyse the interactions of HPV‐16 proteins using yeast two‐hybrid (Y2H). To achieve this, a clone collection of the viral proteome was generated by recombinatorial cloning and three independent Y2H screens were performed: (i) Intra‐viral screen to identify interactions among the HPV‐16 proteins; (ii) Inter‐viral screen to identify interactions with proteins of Herpes Simplex Virus (HSV) which is suggested to be a co‐factor; and (iii) Virus‐host screen to identify novel cellular binding partners. The intra‐viral Y2H screen confirmed some of the previously known interactions and also identified binding of the E1 and E7 proteins. Deletion mutagenesis was performed to map the interaction domains to the amino‐terminal 92 amino acids of E1 and carboxy‐terminal CxxC domain of E7. Replication assays suggest a possible repression of E1‐mediated episomal replication by direct binding of E7. The inter‐viral Y2H screen identified interactions of HPV proteins with seventeen HSV‐1 proteins including transcriptional regulator ICP4 and neurovirulance factor ICP34.5. The biological relevance of these interactions in the context of co‐infection is discussed. The virus‐host screen performed against a human cDNA library identified 54 interactions, a subset of which was validated by biochemical pull‐down assays. The III functional relevance of an interaction between E7 and a proto‐oncogene spermatogenic leucine zipper protein (SPZ1) was further investigated suggesting a role of SPZ1 in E7‐ mediated cell proliferation. The work presented in this thesis identifies several novel interactions of HPV proteins. Future work will involve the in‐depth elucidation of biological relevance of these interactions. In particular, the interactions of E7 with E1 and SPZ1 are of great interest to improve our understanding of the life cycle and pathogenesis of the virus which can be applied for improved strategies of prevention and treatment of malignancies caused by HPV. IV ACKNOWLEDGEMENTS Obtaining a PhD has been a goal ever since I was in high school. I am blessed to be surrounded by inspiring people who have played a huge role in achieving this. Mom and Dad, you have always encouraged me to do my best in everything, been there through all my small successes and failures, stood behind me in all the decisions I have taken and given me the confidence to achieve this, thank you. The credit for all my achievements goes to you both; I hope I have made you proud. My sister, Rashmi, thank you for always believing in me, for never letting me become overconfident, for correcting every mistake and inspiring me to become like you. Thanks to my brother‐ in‐law, Harish for all the encouragement and my lovely niece, Stuti who always brings a smile on my face. I would like to thank my two teachers who made me fall in love with Biology. Suja Ma’am, my Biology teacher in secondary school without who I would not be doing Biology, thank you very much. Prathibha Ma’am, my Biotechnology lecturer who has injected into me a bit of her passion for the subject and desire to keep learning and never stop aspiring and I am ever so thankful for that. I would like to thank my supervisor Prof Jürgen Haas for the opportunity to do my PhD and encouraging me to expand my horizons and be independent in thinking and planning my project. Thank you for all your critical support, advice and encouragement. Prof Heather Cubie, my second supervisor during my PhD, you are a tremendous inspiration and through every phase of my PhD, talking to you has always encouraged me. Your knowledge and support has been a valuable asset to me. Thank you for your constant support. V I would like to thank the current and old members of Haas group, Dr Samantha Griffiths for answering my never ending questions, for all your reagents that I have used and for taking the time to proof‐read my thesis. Thanks to Rui Chen and Dr Alessandro Ceroni for the much required pep talks, encouragement and thesis comments. Thanks to Dr Suzanne Esper, Lakshmi Narayan Kaza and Dr Ola Hassanin for all your help during my PhD. I would also like to thank the students who have provided valuable results towards my thesis, Jenna Schafers, Annette Murr, Joe Sherwan and Tracy Mak. Thanks to Clark Russell and Peter Thomas for being my English proof readers. I would like to say a special thanks to all my fellow PhD students who have sailed in the same boat as me, Senthil Gandi, Shahida Syed, Fairus Noor Hassim, Wayne Hsieh, Rui Chen and Miriam Kaatz. Thank you for welcoming me into the group, for all the lunch and coffee break craziness which kept me sane through the stressful PhD. I would have never enjoyed these years as much without you guys. Many thanks also to all the members of Division of Pathway Medicine for all the timely help throughout the course of my PhD. I would also like to thank the members of Scottish HPV reference lab, Kate Cushieri, Catherine Moore, Maurice Canham and Johanna Pedraza for all their advice and support. I would like to thank the members of Max von Pettenkofer institute, Munich, who trained me in yeast two‐hybrid, Markus Heinzemann, Hannah Streibinger, Verena Hofer, Georg Malterer, Rose Dietlind and especially Suzanne Bailer for all their help with the technique and making my stay in Munich wonderful. Thanks to Prof Iain Morgan and Dr Mary Donaldson, University of Glasgow for giving me an opportunity to do HPV replication assays in their lab and Dr Andrew Macdonald for all his valuable correspondence, suggestions and reagents. Lastly I would like to thank Abhishek, my fiancé, for encouraging me to pursue my dream, for listening to me jump with joy when things went well and more often than not, complain when things didn’t and mostly for being my biggest competition. Thank you for being my strength! VI Dedicated to my grandmother, Mrs Radha Bai VII CONTENTS DECLARATION ................................................................................................................ II ABSTRACT ....................................................................................................................... III ACKNOWLEDGEMENTS .............................................................................................. V LIST OF FIGURES ...................................................................................................... XIII LIST OF TABLES ......................................................................................................... XVI ABBREVIATIONS .................................................................................................... XVIII Chapter 1: ............................................................................................................................