Discovering Novel Intrinsic Antiviral Responses To Arboviruses PDF
Preview Discovering Novel Intrinsic Antiviral Responses To Arboviruses
UUnniivveerrssiittyy ooff PPeennnnssyyllvvaanniiaa SScchhoollaarrllyyCCoommmmoonnss Publicly Accessible Penn Dissertations 2013 DDiissccoovveerriinngg NNoovveell IInnttrriinnssiicc AAnnttiivviirraall RReessppoonnsseess ttoo AArrbboovviirruusseess:: FFrroomm TTrraannssccrriippttiioonn ttoo IInntteessttiinnaall IInnnnaattee IImmmmuunniittyy Jie Xu University of Pennsylvania, [email protected] Follow this and additional works at: https://repository.upenn.edu/edissertations Part of the Allergy and Immunology Commons, Cell Biology Commons, Immunology and Infectious Disease Commons, Medical Immunology Commons, and the Microbiology Commons RReeccoommmmeennddeedd CCiittaattiioonn Xu, Jie, "Discovering Novel Intrinsic Antiviral Responses to Arboviruses: From Transcription to Intestinal Innate Immunity" (2013). Publicly Accessible Penn Dissertations. 723. https://repository.upenn.edu/edissertations/723 This paper is posted at ScholarlyCommons. https://repository.upenn.edu/edissertations/723 For more information, please contact [email protected]. DDiissccoovveerriinngg NNoovveell IInnttrriinnssiicc AAnnttiivviirraall RReessppoonnsseess ttoo AArrbboovviirruusseess:: FFrroomm TTrraannssccrriippttiioonn ttoo IInntteessttiinnaall IInnnnaattee IImmmmuunniittyy AAbbssttrraacctt Many (re) emerging pathogens are arthropod-borne, transmitted via an insect vector, and cause significant health and agricultural problems worldwide. Despite their significance, there are few vaccines and no targeted therapies that exist. This is at least in part due to our limited understanding of virus-host interactions and the mechanisms used by hosts to restrict infection. In particular, insect vectors play a critical role in the transmission and spread of these pathogens, but performing molecular and genetic studies has proven to be difficult. Drosophila is a model organism that shares a high degree of conservation with insect vectors and has a wealth of molecular and genetic tools for study. Hence, this thesis aims to provide a deeper understanding of the innate immune factors that restrict arthropod-borne viruses using this model organism. Using genetic approaches both in vitro and in vivo, two novel antiviral pathways are discovered and examined in this thesis. First, using RNA interference (RNAi) screening against disparate viruses in Drosophila, the transcriptional pausing pathway is found to be essential for antiviral insect immunity. This led to the characterization of a rapidly induced antiviral transcriptional program, half of which is genetically dependent on this regulatory mechanism and has pausing- associated chromatin features. These findings suggest transcriptional pausing primes virally induced genes by enhancing promoter accessibility to allow for rapid gene induction, thereby coordinating a robust and complex antiviral response. Subsequently, the ERK pathway is found to be part of this transcriptional response to viral infection. Not only is this nutrient responsive pathway induced by viral infection, but it also restricts disparate arboviral pathogens. Furthermore, ERK signaling is essential for antiviral defense in the insect intestinal epithelium. While wild type flies are refractory to oral infection by arboviruses, this innate restriction can be overcome chemically by oral administration of an ERK pathway inhibitor or genetically via the specific loss of ERK in the intestinal epithelial cells. In addition, vertebrate insulin that activates ERK signaling in the mosquito gut during a blood meal, can restrict viral infection in insect cells and protect against viral invasion of the gut epithelium. These studies collectively demonstrate that ERK signaling in the insect intestines potently restricts viral infection, suggesting that insects take advantage of signals in the meal to preemptively activate antiviral immunity. DDeeggrreeee TTyyppee Dissertation DDeeggrreeee NNaammee Doctor of Philosophy (PhD) GGrraadduuaattee GGrroouupp Cell & Molecular Biology FFiirrsstt AAddvviissoorr Sara Cherry SSeeccoonndd AAddvviissoorr Frederic Bushman KKeeyywwoorrddss Antiviral Immunity, Drosophila melanogaster, Innate Immunity SSuubbjjeecctt CCaatteeggoorriieess Allergy and Immunology | Cell Biology | Immunology and Infectious Disease | Medical Immunology | Microbiology This dissertation is available at ScholarlyCommons: https://repository.upenn.edu/edissertations/723 DISCOVERING NOVEL INTRINSIC ANTIVIRAL RESPONSES TO ARBOVIRUSES: FROM TRANSCRIPTION TO INTESTINAL INNATE IMMUNITY Jie Xu A DISSERTATION in Cell and Molecular Biology Presented to the Faculties of the University of Pennsylvania in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy 2013 Supervisor of Dissertation ________________________ Dr. Sara Cherry Associate Professor of Microbiology Graduate Group Chairperson ________________________ Dr. Daniel S. Kessler, Associate Professor of Cell and Developmental Biology Dissertation Committee Dr. Nancy Bonini, Professor of Biology Dr. Margaret Chou, Associate Professor of Pathology and Laboratory Medicine Dr. Gary Koretzky, Francis C. Wood Professor of Medicine Dr. Frederic Bushman, Professor of Microbiology (Chair) “DISCOVERING NOVEL INTRINSIC ANTIVIRAL RESPONSES TO ARBOVIRUSES: FROM TRANSCRIPTION TO INTESTINAL INNATE IMMUNITY” COPYRIGHT 2013 Jie Xu This work is licensed under the Creative Commons Attribution- NonCommercial-ShareAlike 3.0 License To view a copy of this license, visit http://creativecommons.org/licenses/by-ny-sa/2.0/ ii DEDICATION I would like to dedicate this dissertation to my grandparents: Xu Zhong Liang, Yang Jian Qun, Li Jian, and Gao Ke Qin. Each of one their lives is a complex story of hardship and perseverance. Most importantly, it is from each of their lives that inspire my own academic and life aspirations. iii ACKNOWLEDGMENTS I would like to thank the following individuals for their contributions to this dissertation: Dr. Sara Cherry for providing unequivocal mentorship throughout my graduate training, which has singlehandedly helped shape me as a scientific researcher in almost every aspect, as well as for her valuable guidance and support in my personal life. My thesis committee members Dr. Gary Koretzky, Dr. Nancy Bonini, Dr. Frederick Bushman, and Dr. Margaret Chou for their mentorship and guidance throughout my training. Dr. Gregory Grant for his kindness and forming a wonderful collaboration for the crucial bioinformatics analyses in Chapter III. Dr. Matt Tudor for performing all of the transcriptional profiling analyses discussed in Chapter III and the concluding remarks, along with critical reading of manuscripts. Dr. Gerd Blobel for helpful advice and support in Chapter III, as well as Rena Zheng for helping with the technical aspects of chromatin immunoprecipitation (ChIP) performed in Chapter III. Dr. Beth Gordesky-Gold for her contributions in fly husbandry and experiments for both Chapters III and IV. The undergraduates, particularly Veronica Schad and Kendrick Chow, for also helping to maintain the fly room and always willing to help others. Dr. Michele Markstein for critical reading of the manuscript in Chapter IV as well as providing important technical advice for manipulating fly guts for microscopy. Dr. Leah Sabin for performing the RNAi screen and validation of the initial factors identified for Chapters III and IV. Kaycie Hopkins and Ian Lamborn for helping screen the three MAPK pathways against disparate viruses for antiviral activity, as discussed in Chapter IV. Ari Yasunaga for providing crucial technical support in all of the phospho-ERK westerns in Chapter IV, the insulin experiments in cell culture and flies, as well as the receptor experiments in the concluding remarks. Gregory Osborn for helping screen the 65 MAPK-dependent genes for antiviral activity against disparate viruses, as discussed in the concluding remarks. All members of the Cherry lab, including Ryan Moy, Sheri Hanna, Debasis Panda, for helpful ideas and making the lab such a special place to do research. My family and friends for their encouragement, guidance, and love. The Howard Hughes Medical Institute (HHMI) for providing financial support in my graduate training, as well as providing opportunities to further explore my research interests. iv ABSTRACT DISCOVERING NOVEL INTRINSIC ANTIVIRAL RESPONSES TO ARBOVIRUSES: FROM TRANSCRIPTION TO INTESTINAL INNATE IMMUNITY Jie Xu Sara Cherry Many (re) emerging pathogens are arthropod-borne, transmitted via an insect vector, and cause significant health and agricultural problems worldwide. Despite their significance, there are few vaccines and no targeted therapies that exist. This is at least in part due to our limited understanding of virus-host interactions and the mechanisms used by hosts to restrict infection. In particular, insect vectors play a critical role in the transmission and spread of these pathogens, but performing molecular and genetic studies has proven to be difficult. Drosophila is a model organism that shares a high degree of conservation with insect vectors and has a wealth of molecular and genetic tools for study. Hence, this thesis aims to provide a deeper understanding of the innate immune factors that restrict arthropod-borne viruses using this model organism. Using genetic approaches both in vitro and in vivo, two novel antiviral pathways are discovered and examined in this thesis. First, using RNA interference (RNAi) screening against disparate viruses in Drosophila, the transcriptional pausing pathway is found to be essential for antiviral insect immunity. This led to the characterization of a rapidly induced antiviral transcriptional program, half of which is genetically dependent on this regulatory mechanism and has pausing-associated chromatin features. These findings suggest transcriptional pausing primes virally induced genes by enhancing promoter accessibility to allow for rapid gene induction, thereby coordinating a robust and complex antiviral response. Subsequently, the ERK pathway is found to be part of this transcriptional response to viral infection. Not only is this nutrient responsive pathway induced by viral infection, but it also restricts disparate arboviral pathogens. Furthermore, ERK signaling is essential for antiviral defense in the insect intestinal epithelium. While wild type flies are refractory to oral infection by arboviruses, this innate restriction can be overcome chemically by oral administration of an ERK pathway inhibitor or genetically via the specific loss of ERK in the intestinal epithelial cells. In v addition, vertebrate insulin that activates ERK signaling in the mosquito gut during a blood meal, can restrict viral infection in insect cells and protect against viral invasion of the gut epithelium. These studies collectively demonstrate that ERK signaling in the insect intestines potently restricts viral infection, suggesting that insects take advantage of signals in the meal to preemptively activate antiviral immunity. vi TABLE OF CONTENTS CHAPTER 1 Introduction 1. (Re) emerging Infectious Arboviral Pathogens ...................................................... 1 2. The Infection Cycle in Insect Vectors ..................................................................... 3 3. Antiviral Vector Immunity: Tissue Barriers and Immune Compartments ................ 4 4. Cellular Antiviral Immune Mechanisms in Insect Vectors ...................................... 7 5. Drosophila melanogaster as a Model Organism for Antiviral Innate Immunity ...... 14 6. Aims of the Present Studies ................................................................................... 18 CHAPTER 2 Materials and Methods 1. Cells and Cell Culture ............................................................................................ 27 2. Viruses ................................................................................................................... 27 3. Antibodies .............................................................................................................. 27 4. dsRNA Synthesis ................................................................................................... 28 5. RNA interference .................................................................................................... 28 6. Viral Infections for Cell Culture Experiments .......................................................... 28 7. Immunofluorescence .............................................................................................. 29 8. Immunoblotting ....................................................................................................... 29 9. Adult Fly Infections ................................................................................................. 30 10. RNA, Northern Blotting and RT-qPCR ................................................................... 31 11. Chromatin-Immunoprecipitation (ChIP) ................................................................. 32 12. DNA Microarray Analysis ...................................................................................... 32 13. Bioinformatics and Statistics .................................................................................. 33 14. Oligonucleotide Sequences for RT-qPCR .............................................................. 34 CHAPTER 3. Transcriptional Pausing Orchestrates a Rapid Antiviral Response in Drosophila melanogaster 1. Abstract .................................................................................................................. 36 2. Background ............................................................................................................ 36 3. Results ................................................................................................................... 38 4. Discussion .............................................................................................................. 46 5. Figures ................................................................................................................... 49 6. Supplemental Figures ............................................................................................ 56 7. Supplemental Tables ............................................................................................ 62 CHAPTER 4. Erk Signaling Couples Nutrient Status with Antiviral Defense in the Insect Gut 1. Abstract .................................................................................................................. 88 2. Background ............................................................................................................ 88 3. Results ................................................................................................................... 89 4. Discussion .............................................................................................................. 93 5. Figures ................................................................................................................... 95 6. Supplemental Tables ............................................................................................ 99 7. Supplemental Figures ............................................................................................ 100 CHAPTER 5. Concluding Remarks. ............................................................................ 113 CHAPTER 6. References ............................................................................................ 122 vii
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