University of Pennsylvania ScholarlyCommons Publicly Accessible Penn Dissertations 2019 Innate Immune Antagonism By Diverse Coronavirus Phosphodiesterases Stephen Goldstein University of Pennsylvania, [email protected] Follow this and additional works at: https://repository.upenn.edu/edissertations Part of the Allergy and Immunology Commons, Immunology and Infectious Disease Commons, Medical Immunology Commons, and the Virology Commons Recommended Citation Goldstein, Stephen, "Innate Immune Antagonism By Diverse Coronavirus Phosphodiesterases" (2019). Publicly Accessible Penn Dissertations. 3363. https://repository.upenn.edu/edissertations/3363 This paper is posted at ScholarlyCommons. https://repository.upenn.edu/edissertations/3363 For more information, please contact [email protected]. Innate Immune Antagonism By Diverse Coronavirus Phosphodiesterases Abstract Coronaviruses comprise a large family of viruses within the order Nidovirales containing single-stranded positive-sense RNA genomes of 27-32 kilobases. Divided into four genera (alpha, beta, gamma, delta) and multiple newly defined subgenera, coronaviruses include a number of important human and livestock pathogens responsible for a range of diseases. Historically, human coronaviruses OC43 and 229E have been associated with up to 30% of common colds, while the 2002 emergence of severe acute respiratory syndrome- associated coronavirus (SARS-CoV) first raised the specter of these viruses as possible pandemic agents. Although the SARS-CoV pandemic was quickly contained and the virus has not returned, the 2012 discovery of Middle East respiratory syndrome-associated coronavirus (MERS-CoV) once again elevated coronaviruses to a list of global public health threats. The eg netic diversity of these viruses has resulted in their utilization of both conserved and unique mechanisms of interaction with infected host cells. Like all viruses, coronaviruses encode multiple mechanisms for evading, suppressing, or otherwise circumventing host antiviral responses. Specifically, our lab has studied coronavirus interactions with antiviral pathways activated by the presence of cytoplasmic viral double-stranded RNA (dsRNA) such as OAS-RNase L and interferons (IFN). Previous work from our lab demonstrated that the murine coronavirus mouse hepatitis virus (MHV) uses a phosphodiesterase (PDE) to suppress RNase L activation. We have also now shown that additional viruses within Nidovirales encode similar PDEs that suppress RNase L activation in the context of chimeric MHV, and that a PDE encoded by MERS-CoV, the NS4b accessory protein, inhibits RNase L in its native context. I have further shown that MERS-CoV NS4b is a unique PDE with additional functions inhibiting the IFN response, a role dependent on both nuclear localization and its catalytic activity. Degree Type Dissertation Degree Name Doctor of Philosophy (PhD) Graduate Group Cell & Molecular Biology First Advisor Susan R. Weiss Keywords coronavirus, innate immunity, interferon, MERS-CoV, phosphodiesterase, RNase L Subject Categories Allergy and Immunology | Immunology and Infectious Disease | Medical Immunology | Microbiology | Virology This dissertation is available at ScholarlyCommons: https://repository.upenn.edu/edissertations/3363 INNATE IMMUNE ANTAGONISM BY DIVERSE CORONAVIRUS PHOSPHODIESTERASES Stephen A. Goldstein A DISSERTATION in Cell and Molecular Biology Presented to the Faculties of the University of Pennsylvania in Partial Fulfillment for the Degree of Doctor of Philosophy 2019 Supervisor of Dissertation Susan R. Weiss, Ph.D. Professor of Microbiology Graduate Group Chairperson Daniel Kessler, Ph.D. Associate Professor of Cell and Developmental Biology Dissertation Committee: Paul Bates, Professor of Microbiology Sara Cherry, Professor of Microbiology Matthew Weitzman, Professor of Microbiology Carolina Lopez, Associate Professor of Microbiology and Immunology ACKNOWLEDGEMENTS I have been fortunate and privileged to do this work under the mentorship of Dr. Susan Weiss. Together I think we have struck a perfect balance of close guidance and support while allowing me a great deal of latitude to pursue my ideas. Aside from support for my work at the bench, Susan has provided invaluable mentorship in grant and manuscript writing. Most importantly perhaps, Susan fosters a collegial, healthy work environment conducive to scientific progress. My thesis committee has been indispensable as well and I’m sure will remain valuable mentors in the future. Thank you to Dr. Paul Bates, Dr. Sara Cherry, Dr. Matthew Weitzman, and Dr. Carolina Lopez who have been available to me at any time throughout my years here. The other members of the Weiss lab also have my thanks for their scientific support and valuable personal relationships. Ruth Elliott taught me many of the things I’ve needed to have success and Yize (Henry) Li has been an unparalleled sounding board and source of ideas. Every member of the lab during my time here has in some way contributed to my success. This is especially true of my co-authors Dr. Joshua Thornbrough, Dr. Rong Zhang, and Courtney Comar. I’m also grateful for valuable collaborations with Dr. Robert Silverman and Dr. Ralph Baric. Looking further back, Dr. Diane Griffin at the Johns Hopkins University gave me my first research opportunity as a fairly clueless Masters student. ii Without my mentors in her lab, this dissertation wouldn’t exist. I’m eternally grateful to Dr. Kimberly Schultz, Dr. Kirsten Kulcsar, and Dr. Victoria Baxter. I’ve also been fortunate enough to have supportive and generous friends, especially Dr. Katie and Chris Wetzel, Dr. Adam SanMiguel and Dr. Jen Myers SanMiguel, and Dr. Suzi Shapira and Dr. Jeffrey Rosa. Despite conventional wisdom, in some ways grad school gets harder as it goes on. Thank you as well to frequent running partners Sophie Trefely and Katie Strelau who helped make my time outside the lab conducive to success in the lab over this last challenging year of graduate school (and in one instance alerted me to a rattlesnake). My family has been enormously supportive since I made what must have seemed a very strange decision to pursue a career in scientific research. My parents Michael and Linda Goldstein and my sister Melissa Goldstein have nevertheless never doubted me and instead supported me in whatever way I needed. I wouldn’t be here without them and I am lucky to be able to count on their continued support. Last but most significantly, my wife Jaclyn has been a better partner than I’d ever thought I would find. She has backed me from the very beginning of my scientific journey and never doubted for a second that I would have success or failed to tell me that, despite confronting enormous personal challenges of her own. There is no way to express what she and her support have meant to me or the degree to which they’ve enabled my success until now and will foster it going forward. I am so looking forward to our next adventure together and hope I will continue to be as good a partner to her as she has been to me. iii ABSTRACT Coronaviruses comprise a large family of viruses within the order Nidovirales containing single-stranded positive-sense RNA genomes of 27-32 kilobases. Divided into four genera (alpha, beta, gamma, delta) and multiple newly defined subgenera, coronaviruses include a number of important human and livestock pathogens responsible for a range of diseases. Historically, human coronaviruses OC43 and 229E have been associated with up to 30% of common colds, while the 2002 emergence of severe acute respiratory syndrome- associated coronavirus (SARS-CoV) first raised the specter of these viruses as possible pandemic agents. Although the SARS-CoV pandemic was quickly contained and the virus has not returned, the 2012 discovery of Middle East respiratory syndrome-associated coronavirus (MERS-CoV) once again elevated coronaviruses to a list of global public health threats. The genetic diversity of these viruses has resulted in their utilization of both conserved and unique mechanisms of interaction with infected host cells. Like all viruses, coronaviruses encode multiple mechanisms for evading, suppressing, or otherwise circumventing host antiviral responses. Specifically, our lab has studied coronavirus interactions with antiviral pathways activated by the presence of cytoplasmic viral double-stranded RNA (dsRNA) such as OAS-RNase L and interferons (IFN). Previous work from our lab demonstrated that the murine coronavirus mouse hepatitis virus (MHV) uses a phosphodiesterase (PDE) to suppress RNase L activation. We have also now shown that additional viruses within Nidovirales encode similar PDEs that suppress RNase L activation in the iv context of chimeric MHV, and that a PDE encoded by MERS-CoV, the NS4b accessory protein, inhibits RNase L in its native context. I have further shown that MERS-CoV NS4b is a unique PDE with additional functions inhibiting the IFN response, a role dependent on both nuclear localization and its catalytic activity. v TABLE OF CONTENTS ACKNOWLEDGEMENTS ...................................................................................... ii ABSTRACT .......................................................................................................... iv LIST OF FIGURES ............................................................................................... vii CHAPTER 1: GENERAL INTRODUCTION ........................................................... 1 CHAPTER 2: LINEAGE A BETACORONAVIRUS NS2 PROTEINS