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Innate Immune Responses to Rotavirus and Viral Countermeasures in Infected Macrophages and Intestinal Cells

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Innate Immune Responses to Rotavirus and Viral Countermeasures in Infected Macrophages and Intestinal Cells Innate immune responses to rotavirus and viral countermeasures in infected macrophages and intestinal cells Izabel Julien Martini Di Fiore Submitted in total fulfillment of the requirements of the degree of Doctor of Philosophy August 2016 Department of Microbiology and Immunology The University of Melbourne ABSTRACT Rotavirus infections are a major cause of life-threatening gastroenteritis. The innate immune system provides an immediate mechanism of suppressing viral replication and is required for an effective adaptive immunity. The induction of an innate immune response involves the viral detection by the host, which initiates intracellular signaling events and culminates in the activation of transcription factors, such as IRF3 and NF- κB. NF-κB activation requires the degradation of NF-κB-inhibitor protein, IκB, by the β-TrCP protein. In the nucleus, these transcription factors mediate the expression of antiviral cytokines, including type I interferon and proinflammatory cytokines. Rotavirus NSP1 can antagonize immune responses by inducing the degradation of IRF proteins or by blocking NF-κB action through the degradation of β-TrCP. Macrophages are the front-line cells of innate immunity and have a central role in controlling dissemination of microbial pathogens. In response to viral infection, they can produce several interferon (IFN) types and inflammatory cytokines. However, the role of macrophages during rotavirus infection is not completely understood. Intestinal epithelial cells, the main target of rotavirus infection, also produce antiviral cytokines in response to virus infection, which modulate both innate and adaptative immune responses. This study firstly showed that rotavirus is capable to infect macrophages, inducing the expression of type I IFN and proinflammatory cytokines. Cytokine production relied on the MAVS adaptor, and rotavirus triggered antiviral responses through RIG-I and/or MDA-5 viral recognition. Rotavirus infection did not elicit activation of kinases JNK/p38 or inflammasomes, suggesting that these innate immune signaling pathways might not be involved in rotavirus control in macrophages. These findings increased our understanding of how macrophages mediated innate immunity to rotavirus infection. Secondly, this study addressed the ability of human rotavirus strains to counteract innate immune responses in epithelial cells. It was identified that several human rotaviruses stabilize IκB during infection of epithelial cell and effectively degrade β-TrCP, blocking NF-κB activation. It was demonstrated that this antagonism of NF-κB signaling is mediated by viral NSP1, and that a C-terminal motif, conserved amongst human NSP1s, ii is required for NSP1 ability to disrupt NF-κB activation. The same motif was observed in porcine rotavirus strains. It resembles the motif used by β-TrCP to recognize IκB and mediate its degradation. Thus, these data revealed a primary mechanism used by human rotavirus to inhibit NF-κB signaling. Finally, studies were conducted to analyse the ability of rotavirus strains that can selectively interfere with IRF3 or NF-κB signaling to differentially modulate cytokine expression in intestinal epithelial cells. Indeed, infection of cells with a rotavirus strain able to degrade IRF3, but not NF-κB, resulted in reduced expression of cytokines that depend on IRF3 activation. Conversely, the expression of NF-κB-mediated cytokines was diminished following cellular infection with a rotavirus expressing an NSP1 with NF-κB antagonist activity. This indicates that cytokine modulation occurs during rotavirus infection of intestinal cells, which is driven by viral NSP1. This effect possibly plays a role in delaying both innate and adaptative immune responses against rotavirus in a strain-specific manner. Overall, these studies have improved the current understanding of the rotavirus-host interaction. iii DECLARATION This is to certify that: i. the thesis comprises only my original work towards the PhD except where indicated in the Preface, ii. due acknowledgement has been made in the text to all other material used, iii. the thesis is fewer than 100,000 words in length, exclusive of tables, maps, bibliographies and appendices Izabel Julien Martini Di Fiore August 2016 iv PREFACE All work is the candidate’s as part of this PhD except for elements presented in Figure 4.7 and Figure 5.7, which were produced by Dr G. Holloway (Department of Microbiology and Immunology, The University of Melbourne, Australia), who performed experiments as described in Sections 4.2.8 and 5.2.5. Sections of work presented in this thesis have been published in peer-review journals. The production of two manuscripts was performed through a collaborative effort amongst the authors, and the candidate is the primary author in both papers. The nature and proportion of author contributions was as follows; Paper 1. Di Fiore, I.J.M., Holloway, G., Coulson, B.S., 2015a. Innate immune responses to rotavirus infection in macrophages depend on MAVS but involve neither the NLRP3 inflammasome nor JNK and p38 signaling pathways. Virus Res 208, 89-97. - Proportion of candidate’s contribution: 75% - Authors’ contributions: GH and BSC contributed equally. IJMDF and GH designed the experiments and interpreted the data. IJMDF performed all the experiments, wrote the first manuscript’s draft and executed all the subsequent editing. IJMDF, GH and BSC edited the manuscript. Paper 2. Di Fiore, I.J.M., Pane, J.A., Holloway, G., Coulson, B.S., 2015b. NSP1 of human rotaviruses commonly inhibits NF-kappaB signalling by inducing beta-TrCP degradation. J Gen Virol 96, 1768-1776. - Proportion of candidate’s contribution: 40% - Authors’ contributions: IJMDF and GH designed the experiments. IJMDF performed most of the experiments and interpreted the data. GH wrote the manuscript and performed a proportion of infection and Western blotting assays. JAP assisted with experiments using flow cytometry analysis. IJMDF, GH and BSC edited the manuscript. The work presented in this thesis was supported by Project Grant 1023786 from the National Health and Medical Research Council of Australia. v ACKNOWLEDGEMENTS Firstly, I would like to express my sincere gratitude to my supervisors, A/Prof Barbara Coulson and Dr Gavan Holloway. To Barbara, thank you for accepting me as a PhD student in your laboratory and for your support and encouragement. To Gavan, thank you for all your help, support and understanding spirit. Your guidance throughout the years has enabled me to successfully complete this thesis. I also would like to thank the members of my committee, A/Prof Patrick Reading and Prof Elizabeth Hartland. Your advice and assistance has been greatly appreciated. I gratefully acknowledge the University of Melbourne, for providing the funding sources that made this work possible. The past and present members of Coulson Lab have contributed greatly to my personal and professional time at Unimelb. The group has been a source of friendships, support and collaboration. Special thanks to Fiona Fleming, Jessica Pane and Nicole Webster for teaching me cell culture and flow cytometry techniques. A big thank you to Vi Dang who have helped with rotavirus genome extraction and Christel Zufferey for helping me analysing data. I am very grateful to have you all as close friends. I would like to thank my collaborators, Douglas Golenbock, Ashley Mansel and Carl Kirkwood for recommendation and provision of cell lines used in this work. Also, thanks to Fanxiu Zhu and Luke O’Neill for providing the requested plasmids, and Malcolm McCrae and John Patton for some rotavirus strains. My sincere thanks also goes to all my friends that helped me endure and conclude this venture, particularly Glaucia and Guilherme Benevides, Sarah Mason and family, Angela and Fabiano Dall’Aqua, Vinz Roh, and my two sisters from another mother, Denise Gomes and Luciana Pimenta. I am extremely grateful to my entire family; my mom and dad, who raised me with a love for science; my siblings Carolina and Bruno, and my in-laws, Helena and Flavio. You have encouraged, loved, helped and supported me in any possible way. Special vi thanks to my little goddaughters, my niece Camila, Eloise and Isabel. You have been an endless source of joy, particularly during the stressful times. Finally, I would like to dedicate this thesis to my beloved husband, Rodrigo. You have support me fully and without your assistance I am not sure whether I would have been able to accomplish this project. You are my home. For your countless love, thank you! vii TABLE OF CONTENTS ABSTRACT ......................................................................................................... ii DECLARATION ................................................................................................. iv PREFACE ............................................................................................................ v ACKNOWLEDGMENTS ................................................................................... vi TABLE OF CONTENTS .................................................................................... viii LIST OF TABLES AND FIGURES .................................................................. xiii COMMONLY USED ABBREVIATIONS ........................................................ xvii CHAPTER 1: Introduction ................................................................................ 1 1.1 Rotavirus discovery and importance ........................................................... 2 1.2
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