Identification of Cellular Interaction Partners of Human Papillomavirus Minor Capsid Protein L2 Dissertation Submitted to the Combined Faculties for the Natural Sciences and for Mathematics of the Ruperto-Carola University of Heidelberg, Germany for the degree of Doctor of Natural Sciences presented by Caroline Odenwald, M.Sc. August 2015 Dissertation submitted to the Combined Faculties for the Natural Sciences and for Mathematics of the Ruperto-Carola University of Heidelberg, Germany for the degree of Doctor of Natural Sciences presented by Caroline Odenwald, M.SC. Born in Heidelberg, Germany Oral-examination:: ................................................ Identification of Cellular Interaction Partners of Human Papillomavirus Minor Capsid Protein L2 Referees: Prof. Dr. Martin Müller PD Dr. Dirk Nettelbeck Thesis Declaration Thesis Declaration Declaration according to §8 of the doctoral degree regulations I hereby declare that I have written the submitted dissertation independently and have not used other sources and materials than those particularly indicated. Further, I declare that I have not applied to be examined at any other institution, nor have I presented this dissertation to any other faculty, nor have I used this dissertation in any form in another examination. Heidelberg, Caroline Odenwald Acknowledgments Acknowledgments I would like to express my gratitude to all of those who supported me in any conceivable way during my time as a PhD student. Above all I would like to thank Prof. Dr. Martin Müller for giving me the opportunity to accomplish my PhD thesis in his lab and for providing this very interesting and challenging project. Further I would like to thank Prof. Dr. Müller for the excellent supervision and always providing scientific input and advice. I thank PD Dr. Dirk Nettelbeck for being my second examiner and his support and scientific advice during my TAC meetings. I would also like to thank PD Dr. Suat Özbek and Dr. Steeve Boulant for agreeing to participate in my thesis evaluation committee. I am thankful to Dr. Dirk Grimm who joined my TAC committee and always participated with good scientific discussion and advice. Special thanks, also goes to Dr. Damir Krunic who supported me a lot, teaching me how to handle the fluorescence microscopes and always being willing to help with all the questions and problems I had. Furthermore I would like to thank Dr. Martina Schnölzer and Dr. Tore Kempf for the MS analysis of my samples and the supporting advice on the sample preparation. I like to thank all current and former lab members of F035 for the nice working atmosphere, the helpful discussions and their unconfined readiness to help with every problem and also mental support in hard times. In this respect special thanks goes to Dr. Ivonne Rubio, Dr. Anna Sacher, Dr. Lis Ribeiro, Dr. Frank Burkart, Dr. Somayeh Pouyanfard, Qingxin Chen, Xueer Zao, Liesa-Marie Schreiber, Katharina Henrich, Michael Bartolff-Kopp, Federica Savini, Irene Daverio, Almira Henic, Tobias Schulze. The time in the lab was a great experience and I became acquainted with a lot of very nice people and new friend. In particular, I like to thank Dr. Hanna Seitz who supported me through my whole PhD with very good scientific input and help whenever it was needed but rather became a very good and close friend. I am more than grateful that I had the privilege to meet her and I am very happy that our friendship persists even across the continents. Acknowledgments I also want to give a special thanks to Petra Galmbacher who was always willing to support me in the lab and was always a good interlocutor concerning scientific but also personal topics. She was a really great and entertaining bench neighbor and I will for sure miss the time standing next to her. I am grateful to all the colleagues on the 2nd floor of the ATV for all the kindness, support, scientific discussion and valuable input in and outside of the Monday Seminars over the last years. In this respect a special thanks goes to the assi room crew, Dr. Angelika Michel, Dr. Daniela Höfler, Lea Schoeder, Juliane Schröter, Dr. Axel Szabowski. I will really miss the time with you, especially the great conversations about every conceivable topic concerning science, future or whatever was worth to discuss. I like to thank Madeleine Sporleder, Susanne Latzko and Diana Sandel for always having an open ear and becoming really good friends. I want to express gratitude and deepest respect for my parents, Gisela and Hans-Jürgen Odenwald for the continuous support and appreciation of my work but rather for the fact that they are always there being the greatest support I can imagine throughout my life. My sister and her husband, Katrin and Christoph Teusch, I want to thank for being my best and most trusted friends. I am really thankful for them being my “rock in the surf” and that I can always count on them. Additionally, I want to thank my two gorgeous nieces, Clara Sophie and Charlotte Marie, which I love more than I could have ever expected, for giving me the necessary distraction whenever I saw them. My friends I thank for their support and consideration, especially during the time of writing and for not being offended by never-returned phone calls. I am grateful to have friends like this, always willing to listen to my problems and concerns but also participating in the joyful moments making my life much better at all times. Finally, I like to thank everybody who supported me in the execution of my PhD and thus accounted for the success of this thesis. Table of Contents Table of Contents Summary ..................................................................................................................................................................... 1 Zusammenfassung .................................................................................................................................................. 2 1. Introduction...................................................................................................................................................... 3 1.1. Human Papillomavirus (HPV) ......................................................................................................... 3 1.2. The HPV Capsid ...................................................................................................................................... 6 1.3. Minor Capsid Protein L2 .................................................................................................................... 9 1.3.1. The role of L2 in capsid assembly and genome encapsidation ................................. 9 1.3.2. HPV entry – from the cell surface to the nucleus .........................................................10 1.4. Objective..................................................................................................................................................16 2. Material ............................................................................................................................................................18 2.1. Biological Material .............................................................................................................................18 2.1.1. Prokaryotic Cells ........................................................................................................................18 2.1.2. Eukaryotic Cells ..........................................................................................................................18 2.1.3. Pseudoviruses (PSV) .................................................................................................................19 2.1.4. Adeno-associated Viruses (AAV) .........................................................................................19 2.1.5. Lentiviruses ..................................................................................................................................19 2.2. Media and Supplements ...................................................................................................................19 2.2.1. Procaryotic Cells .........................................................................................................................19 2.2.2. Eukaryotic Cells ..........................................................................................................................20 2.3. Molecular Cloning ...............................................................................................................................20 2.3.1. Oligonucleotides for Cloning of HPV16L2 Fragments for TAP ...............................20 2.3.2. Oligonucleotides for siRNA knockdown ...........................................................................20 2.3.3. Oligonucleotides for shRNA knockdown ..........................................................................21 2.3.4. Plasmids .........................................................................................................................................22 2.3.5. Enzymes .........................................................................................................................................23 2.3.6. Buffers and Solutions for DNA Purification and Analysis .........................................24 2.3.6.1. Purification of plasmid DNA ..................................................................................24 2.3.6.2. Agarose Gel Electrophoresis ..................................................................................24