The murine cytomegalovirus immunoevasin gp40 binds to MHC class I molecules to retain them in the early secretory pathway by Linda Ellen Janßen A thesis submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Biochemistry Approved: Dissertation Committee Prof. Dr. Sebastian Springer Jacobs University Bremen Dr. Susanne Illenberger Jacobs University Bremen Prof. Dr. Matthias Ullrich Jacobs University Bremen Prof. Dr. Hartmut Hengel Universitätsklinikum Freiburg Date of Defense: November 4th, 2015 Statutory Declaration (Declaration on Authorship of a Dissertation) I, Linda Ellen Janßen, hereby declare, under penalty of perjury, that I am aware of the consequences of a deliberately or negligently wrongly submitted affidavit, in particular the punitive provisions of § 156 and § 161 of the Criminal Code (up to 1 year imprisonment or a fine at delivering a negligent or 3 years or a fine at a knowingly false affidavit). Furthermore I declare that I have written this PhD thesis independently, unless where clearly stated otherwise. I have used only the sources, the data and the support that I have clearly mentioned. This PhD thesis has not been submitted for the conferral of a degree elsewhere. Bremen October, 1st Place Date Signature 1 This work was funded by Tönjes Vagt Foundation of Bremen (grant XXIX to S.Sp.). 2 Parts of this work will be published in: Janßen L, Ramnarayan V, Aboelmagd M, Iliopoulou M, Majoul I, Fritzsche S, Halenius A, Springer S. The mCMV protein m152/gp40 retains MHC class I molecules in the early secretory pathway by direct interaction. Manuscript in revision at Journal of Cell Science. 3 Abstract To recognize the viral infection of a cell, the adaptive immune system depends on Major Histocompatibility Complex (MHC) class I molecules, which present viral peptides (antigens) at the surface of an infected cell to patrolling immune cells. MHC class I molecules are assembled and loaded with viral peptides in the endoplasmic reticulum (ER), and, after passing a thourough quality control, are transported to the cell surface. Viruses have evolved mechanisms to impair the so-called antigen presentation, as this reduces viral dissemination. Herpesviruses, which usually chronically infect their host, dedicate a big part of their ample genome to manipulate the antigen presentation pathway at all possible levels. In the presence of the murine cytomegalovirus (mCMV) gp40 (m152) protein, murine MHC class I molecules do not reach the cell surface but are retained in an early compartment of the secretory pathway by an unknown mechanism. In this work, I found that gp40 does not hijack any known cellular factors to retain MHC class I molecules, but rather binds to them and most likely circulates with them in the early secretory pathway, which consists of the endoplasmic reticulum (ER), the ER-Golgi intermediate compartment (ERGIC), and the cis- Golgi. A flexible sequence in the lumenal domain of gp40 appears to be responsible for the circulation, as destruction of this sequence releases both MHC class I molecules and gp40 from the early secretory pathway without impairing their interaction. Furthermore, I could show that the expression of gp40 influences the transcription of genes of the antigen presenting machinery (APM) in some, but not in all, cell lines, and that factors that influence the folding and export speed of MHC class I molecules (protein sequence, β2m abundance) decide on the effectiveness of gp40 function. 1 Acknowledgments After almost five years working at this inspiring small university, I finish the adventurous journey of my PhD thesis. It was a truly great time and I will miss it. First of all, I want to thank Sebastian Springer for giving me the opportunity to do this exciting project. Thank you so much for all your support, experience, and, most importantly, your undying passion for good science. I want to also thank my thesis committee, Dr. Susanne Illenberger, Prof. Dr. Hartmut Hengel and Prof. Dr. Matthias Ullrich, for your intellectual input throughout this project and for reviewing my research work. Apart from the science, the former and present members of the Springer laboratory contributed largely to make this time a particularly joyful one: many thanks to Britta Borchert, Cindy Kroll, Esam Abualrous, Peter Reinink, Sebastiàn Montealegre, Sunil Kumar, Sujit Verma, Susi Fritzsche, Venkat Ramnarayan, and Zeynep Hein, for being so positive, supportive, and smart. I am very spoiled now. Special thanks to ‘Super-Uschi’ Ursula Wellbrock – this lab would not function without you, you are certainly irreplaceable. I want to also thank the students that have worked with me during these years, Ina Huppertz, Maria Bottermann, Sharon Versteeg, Nikki Dimitrova Atanasova, Vaishnavi Venugopalan, Mohamed Aboelmagd, and Maria Iliopoulou, for contributing to this thesis with your hard work and good ideas. Many thanks to Anne Halenius, for inspiring discussions and for investing so much time and energy into teaching me the methods that made many of our data possible. I would like to thank the Tönjes-Vagt-Foundation for their funding in this project. Ich möchte mich bei meiner Familie, meiner Mutter Rita, meiner Schwester Evelyn, meinem Vater Kalle, und Marion, dafür bedanken, dass ihr mich bedingungslos bei all meinen Vorhaben unterstützt. Many thanks to Mehdi, my partner, for being the awesomest and most supportive person. Kheili dusset daram! 2 Table of Contents 1 Introduction ........................................................................................................................................ 11 1.1 The immune response to viruses ................................................................................................ 11 1.1.1 The innate immune system ................................................................................................. 11 1.1.2 Natural killer cells ................................................................................................................ 12 1.1.3 The adaptive immune system ............................................................................................. 13 1.1.4 Interferons .......................................................................................................................... 13 1.2 MHC class I trafficking ................................................................................................................. 15 1.2.1 Synthesis and assembly ...................................................................................................... 16 1.2.2 MHC class I retention and retrieval .................................................................................... 18 1.3 Transcription of APM genes ........................................................................................................ 21 1.4 ER stress ...................................................................................................................................... 22 1.5 Viral inhibition of the immune response .................................................................................... 24 1.5.1 General examples and herpesviruses ................................................................................. 24 1.6 Cytomegalovirus ......................................................................................................................... 27 1.6.1 HCMV from a medical perspective ..................................................................................... 27 1.6.2 Infection and latency .......................................................................................................... 27 1.7 gp40 ............................................................................................................................................ 28 1.7.1 Discovery of gp40 ................................................................................................................ 28 1.7.2 Background information about gp40 .................................................................................. 28 1.7.3 Comparison of gp40 sequence of different MCMV strains ................................................ 30 1.7.4 gp40 in the viral context ..................................................................................................... 32 1.7.5 The gp40 mystery ................................................................................................................ 35 1.8 The aim of this study ................................................................................................................... 36 2 Materials and Methods ....................................................................................................................... 38 2.1 Constructs ................................................................................................................................... 38 2.2 Domain swaps ............................................................................................................................. 43 2.3 Cell culture .................................................................................................................................. 44 2.4 Protein expression in mammalian cells ...................................................................................... 45 2.4.1 Lentiviral transduction ........................................................................................................ 45 2.4.2 Preparation of lentiviral particles ......................................................................................