Functional characterization of a novel ENU‐induced mutation in Unc93b1 and its role in single‐stranded RNA virus infection Erin Isabel Lafferty Division of Experimental Medicine McGill University, Montréal April 2014 A thesis submitted to McGill University in partial fulfillment of the requirements of the degree of Doctor of Philosophy © Erin Isabel Lafferty, 2014 Acknowledgements I would like to thank my supervisor Dr. Salman Qureshi for providing me with the opportunity, tools, and guidance to tackle this stimulating project and for always challenging me to achieve excellence. In addition, I would like to thank the members of my supervisory committee, Dr. Silvia Vidal, Dr. Ciriaco Piccirillo, and Dr. Danuta Radzioch, for helpful advice both during and between my yearly committee meetings. I would also like to thank all manuscript co‐authors, in particular Adam Flaczyk and Sean Wiltshire, for their technical, intellectual, and editorial contributions. A big thank you to past and present members of the Qureshi Lab for experimental and general support and to members of the MGH L11 for smiles in the hall and lunchtime conversations. In particular, thank you to Dr. Scott Carroll for teaching me how to be a PhD student and Isabelle Angers for her technical expertise, friendship, and French translation skills. Thank you to the people I have met and worked with through SKILLSETS, Let’s Talk Science, and the PGSS Equity Committee for your friendship and advice on life, careers, and graduate school; with special thanks to my thesis writing buddy Heather. To my incredible friends who live near and far; Carolyn, Monique, Sophia, Marie‐Claire, Iona, and many more; you have been beside me through so much, both in person and on the phone/over Skype. Your support, words of encouragement, amazing personal successes, and outlook on life inspire me everyday. A special thank you to Colin for your genuine interest in what I have spent the last six years of my life studying (even if you did not always understand it!), and for reminding me that a Doctor of Philosophy is, in its most basic definition, an advanced degree in the ‘love of wisdom’. Your belief in my abilities, calm and rational outlook, and constant support and love through the successes and challenges of thesis and manuscript writing has been truly incredible. I look forward to a bright future together! Finally, thank you to my wonderful family – Mom, Dad, and Megan. You have always been so proud of my accomplishments and picked me up and dusted me off every time I stumbled in life. Thank you for your unwavering belief that I could do this. You are my role models of hard work and determination and your love and support keep me strong. II Table of Contents Acknowledgements ................................................................................................................. II List of Figures and Tables ........................................................................................................ VI Author Contributions ............................................................................................................ VIII Original scholarship and contributions ............................................................................. IX Other contributions ........................................................................................................... X List of Abbreviations .............................................................................................................. XII Abstract ................................................................................................................................. XV Résumé ............................................................................................................................... XVII CHAPTER 1. Introduction ...................................................................................................... 1 I. Studying host‐pathogen interactions ................................................................................. 2 Reverse genetics ................................................................................................................ 3 Forward genetics ............................................................................................................... 4 II. ENU mutagenesis .............................................................................................................. 4 Mutagenesis and screening ............................................................................................... 5 Designing phenotypic screens for ENU mutagenesis ........................................................ 8 Advantages and challenges of ENU mutagenesis .............................................................. 9 III. The immune response to viral infection ............................................................................ 9 Toll‐like receptors ............................................................................................................ 10 Endosomal TLRs ........................................................................................................... 10 Cytosolic receptors .......................................................................................................... 12 RLRs .............................................................................................................................. 12 NLRs.............................................................................................................................. 13 Innate immunity .............................................................................................................. 16 Adaptive immunity .......................................................................................................... 16 IV. Influenza ......................................................................................................................... 18 The virus ........................................................................................................................... 19 Viral mechanisms of entry and immune evasion ......................................................... 20 Clinical features and diagnosis ........................................................................................ 20 Treatment ........................................................................................................................ 21 Vaccines ........................................................................................................................... 21 Animal models ................................................................................................................. 22 Immune recognition of the influenza virus ..................................................................... 22 Endosomal TLR signaling ............................................................................................. 22 Cytosolic PRR signaling – RIG‐I ..................................................................................... 24 Cytosolic PRR signaling – NLRP3 .................................................................................. 25 Conflict on PRR significance ......................................................................................... 26 Immune mechanisms of protection and pathogenesis ................................................... 26 Macrophages ............................................................................................................... 27 Dendritic cells ............................................................................................................... 28 T cells ............................................................................................................................ 29 V. Coxsackievirus‐induced myocarditis ............................................................................... 31 The virus ........................................................................................................................... 32 III Viral mechanisms of entry and immune evasion ......................................................... 32 Diagnosis .......................................................................................................................... 33 Treatment ........................................................................................................................ 33 Animal models ................................................................................................................. 34 Immune recognition of the CVB3 virus ........................................................................... 35 Endosomal TLR signaling ............................................................................................. 35 Cytosolic PRR signaling ................................................................................................ 36 Immune mechanisms of protection and pathogenesis ................................................... 37 NK cells ......................................................................................................................... 37 T cells ............................................................................................................................ 38 Proinflammatory mediators ......................................................................................... 38 VI. Unc93b1 ........................................................................................................................