University of Nebraska - Lincoln DigitalCommons@University of Nebraska - Lincoln Dissertations and Theses in Biological Sciences Biological Sciences, School of 4-2019 Regulation of Vaccinia Virus Replication: a Story of Viral Mimicry and a Novel Antagonistic Relationship Between Vaccinia Kinase and Pseudokinase Annabel T. Olson University of Nebraska-Lincoln, [email protected] Follow this and additional works at: https://digitalcommons.unl.edu/bioscidiss Part of the Biology Commons Olson, Annabel T., "Regulation of Vaccinia Virus Replication: a Story of Viral Mimicry and a Novel Antagonistic Relationship Between Vaccinia Kinase and Pseudokinase" (2019). Dissertations and Theses in Biological Sciences. 106. https://digitalcommons.unl.edu/bioscidiss/106 This Article is brought to you for free and open access by the Biological Sciences, School of at DigitalCommons@University of Nebraska - Lincoln. It has been accepted for inclusion in Dissertations and Theses in Biological Sciences by an authorized administrator of DigitalCommons@University of Nebraska - Lincoln. REGULATION OF VACCINIA VIRUS REPLICATION: A STORY OF VIRAL MIMICRY AND A NOVEL ANTAGONISTIC RELATIONSHIP BETWEEN VACCINIA KINASE AND PSEUDOKINASE by Annabel T. Olson A DISSERTATION Presented to the Faculty of The Graduate College at the University of Nebraska In Partial Fulfillment of Requirements For the Degree of Doctor of Philosophy Major: Biological Sciences (Genetics, Cell, and Molecular Biology) Under the Supervision of Professor Matthew S. Wiebe Lincoln, Nebraska April, 2019 SUPERVISORY COMMITTEE SIGNATURES SUPERVISORY COMMITTEE: Matt Wiebe, Ph.D. (Primary Advisor) Deb Brown, Ph.D. Audrey Atkin, Ph.D. Luwen Zhang, Ph.D. Asit Pattnaik, Ph.D. APPROVED Regulation of vaccinia virus replication: A story of viral mimicry and a novel antagonistic relationship between vaccinia kinase and pseudokinase Annabel T. Olson, Ph.D. University of Nebraska, 2019 Advisor: Matthew S. Wiebe Poxviruses employ sophisticated signaling pathways that thwart cellular defense mechanisms and simultaneously ensure viral factors are modulated properly. Yet, our understanding of these complex signaling networks are incomplete. For example, the vaccinia B1 kinase plays a vital role in inactivating the cellular antiviral factor BAF, and is suggested to orchestrate other pathways. B1 is highly conserved among poxviruses and exhibits a remarkable degree of similarity to VRKs, a family of cellular kinases, suggesting that the viral enzyme has evolved to mimic VRK activity. Indeed, B1 and VRKs have been demonstrated to target a shared substrate, the DNA binding protein BAF, elucidating a signaling pathway important for mitosis and the antiviral response. Our research further characterized the role of B1 during vaccinia infection to gain novel insights into its regulation and integration with cellular signaling pathways. We began by constructing and characterizing the first B1 deletion virus (ΔB1). Then using this virus, we tested the hypothesis that cellular VRKs can complement B1 function, and discovered a VRK2 role in facilitating DNA replication in the absence of B1. Study of the VRK2 mechanism revealed that B1 and VRK2 mediate DNA replication via an additional pathway that is BAF independent. We also utilized the ΔB1 virus in an experimental evolution assay to perform an unbiased search for suppressor mutations and identify novel pathways involving B1. Interestingly, our characterization of the adapted viruses reveals that mutations correlating with a loss of function of the vaccinia B12 pseudokinase provide a striking fitness enhancement to this virus. Next, B12 characterization showed a nuclear localization, unique for poxvirus proteins, that is related to its repressive function. Our data indicate that B12 is not a global repressor, but inhibits vaccinia replication in the absence of the B1 kinase. The mechanism of B12 partially depends on suppression of BAF antiviral activity. However, the parallel B12 pathway to restrict virus replication is less clear. Together, our studies of B1 and B12 present novel evidence that a paralogous kinase-pseudokinase pair can exhibit a unique epistatic relationship in a virus, and orchestrate yet-to-be-discovered nuclear events during infection. DEDICATION This doctoral dissertation thesis is dedicated to my parents David and Diana Olson who support me constantly through their love and prayers. By the graces of God my mom is with us today to see me graduate. My mom was diagnosed with stage IV pancreatic cancer in November 2015 and is now in remission since November 2018. She is truly our miracle. God is good. ACKNOWLEDGMENTS When they say it takes a village to raise a child, the same can be said for a doctoral recipient. This achievement was not obtained without the support and guidance of many mentors, colleagues/friends and family members. I feel extremely fortunate to have been advised by Matt Wiebe over my graduate education. His guidance in my personal life, professional development and training as a researcher has greatly contributed to my success as a virologist and growth as an individual. In addition to my training in virology research, learning from Matt’s leadership roles in our lab and at the faculty and university levels has been an outstanding example of what a good mentor and colleague looks like. From his example, I will strive to emulate his kindness and insistent drive in order to be a good mentor and colleague to others. Recognition of my graduate committee members Deb Brown, Audrey Atkin, Asit Pattnaik, and Luwen Zhang is also a necessity for their contributions to my development as a researcher. They challenged my critical thinking skills and ability to articulate my research achievements and proposals. Deb recognized my talent in soft skills and encouraged me to develop this area. She also provided an excellent example of a passionate and skilled educator and researchers who knows the importance of lab community. Each of my committee members welcomed informal meetings and these discussions were some of the best conversations, as I maneuvered through the challenges during my graduate degree. The previous and current members of the Wiebe lab have been extremely supportive over my time here. I am grateful for the training and patience of Augusta Jamin and April Wicklund during my first years of research in the Wiebe lab and the fellowship of Zhigang Wang, Angela Mercurio, Shawna Fitzwater, Amber Rico, Allie Linville, and Luisa Valencia. I am grateful for the guidance and friendship of my colleagues Jessica Hargarten, Yen Ahmad Hussin, Kay Kimpston-Burkgren, Nacho Correas, Dane Bowder, Alex Vogel, Anna Lampe, Nicole Bacheller, Eric Noel, Sara Privatt, and the graduate students at the NCV. The staff members at the NCV have been wonderful to work with, especially Jan Edwards who always greeted me with a warm smile and words of encouragement. My friends outside of graduate school have also been extremely supportive during this time in graduate school including Grace Regan Reid, Tim Reid, Kelsey Nelson, Allie Klug Dunham, Alicia Wagner Gotto, Katie Johnson Harmelink, Kairsten Nelson, Christie Gleason Lewandoski, and Connor Johnson. Lastly, my family has been my strong pillar throughout graduate school. I am so thankful for their many visits, long phone conversations and many prayers. The pilgrimages we took together and discussions of God have led me down this current path. Thank you Dad, Mom, Chris, Hailey, Logan, Sophia, Marty, Kathy, and Cordelia for bringing so much happiness and light into my life. Thank you Lord for all of these people and the ones that are not mentioned, but still impacted by successful achievement of becoming a doctoral recipient. PREFACE Chapters 3 and 4, expect section 4.1, have been published in Journal of Virology. (Olson, AT. Rico, AB. Wang, Z. Delhon, G. Wiebe, MS. Deletion of the Vaccinia B1 Kinase Reveals Essential Function of this Enzyme Complemented Partly by the Homologous Cellular Kinase VRK2. J Virol. 2017 Jul 12; 91(15). pii: e00635-17. doi: 10.1128/JVI.00635-17) Chapters 5 and 6, except parts of 5.3, 6.2, and 6.4-6.7, have been published in PLOS Pathogens. (Olson, AT. Wang, Z. Rico, AB. Wiebe, MS. A poxvirus pseudokinase represses viral DNA replication via a pathway antagonized by its paralog kinase. PLoS Pathog. 2019 Feb 15;15(2):e1007608. doi: 10.1371/journal.ppat.1007608) TABLE OF CONTENTS TITLE…………………………………………………………………………………......i SUMPERVISORY COMMITTEE SIGNATURES……………………………………ii ABSTRACT…………………………………………………………………………......iii DEDICATION…………………………………………………………………………...v ACKNOWLEGEMENTS………………………………………………………………vi PREFACE………………………………………………………………………………vii ABBREVIATIONS LIST……………………………………………………………...xiii FIGURES LIST…………………………………………………………………………xv CHAPTER 1 INTRODUCTION…………………………………………………………………….....1 1.1. POXVIRIDAE FAMILY…………………………………………………………...1 1.1.A. History…………………………………………………………..............................2 1.1.B. Genome………………………………………………………….............................3 1.1.C. Vaccinia virus life cycle…………………………………………………………....6 1.1.D. Innate immunity………………………………………………………………….10 1.1.E. Viral mimicry elicited for counteracting innate immunity………………………..14 1.2. VACCINIA B1 KINASE VIRUS MIMICRY OF HOST VACCINIA-RELATED KINASES…………………………………………………………..........................15 1.2.A. Vaccinia virus B1 kinase…………………………………………………………17 1.2.B. Cellular vaccinia-related kinases (VRKs) ……………………………………….19 1.2.C. Cellular barrier-to-autointegration factor (BAF) substrate……………………….21 1.3. ENZYME / PSEUDOENZYME