University of California, Irvine

UNIVERSITY OF CALIFORNIA, IRVINE Deciphering the mechanism of TDP2/VPg unlinkase activity during picornavirus infections DISSERTATION Submitted in partial satisfaction of the requirements for the degree of DOCTOR OF PHILOSOPHY in Biomedical Sciences by Autumn Candace Holmes Dissertation Committee: Dr. Bert L. Semler, Chair Dr. Paul Gershon Dr. Michael McClelland Dr. Suzanne Sandmeyer 2019 © 2019 Autumn C. Holmes TABLE OF CONTENTS Page List of figures iii List of tables v Acknowledgements vi Curriculum vitae vii Abstract of the dissertation ix CHAPTER 1: Introduction Summary 1 Significance 2 Picornavirus translation, RNA synthesis, and role of VPg 9 5’ tyrosyl-DNA phosphodiesterase 2 as VPg unlinkase 20 Biological significance of VPg unlinkase during picornavirus infections 27 CHAPTER 2: Post-translational effects of TDP2 VPg unlinkase activity during picornavirus infection in a human cell model Summary 30 Introduction 31 Results 36 Discussion 71 Materials and Methods 77 CHAPTER 3: Differential patterns of TDP2 and VP1 subcellular localization during picornavirus infections of multiple human cell lines Summary 84 Introduction 85 Results 88 Discussion 106 Materials and Methods 110 CHAPTER 4: Final conclusions and overall significance 112 REFERENCES 120 ii LIST OF FIGURES Page Figure 1.1 Schematic of the picornavirus genome 11 Figure 1.2 Forms of the viral RNA that arise during picornavirus infections and their linkage to VPg 19 Figure 1.3 Cellular roles of TDP2 beyond DNA repair 26 Figure 2.1 Binding of PCBP and 3CDpro to the poliovirus 5’ cloverleaf structure 35 Figure 2.2 TDP2 protein expression and VPg unlinkase activity is absent in KO 39 RPE cells Figure 2.3 Virus growth kinetics are delayed in the absence of TDP2 for 41 poliovirus and CVB3 in hRPE-1 cells and the effect is MOI-dependent Figure 2.4 Poliovirus titers are comparable in WT and KO RPE cells after 42 multiple replication cycles Figure 2.5 eIF4G is cleaved in both WT and KO TDP2 RPE cells during poliovirus 44 infection Figure 2.6 An alternative source of VPg unlinkase activity is not activated 46 at late times of infection in TDP2 KO hRPE-1 cells Figure 2.7 All isoforms of TDP2 can be used by poliovirus during the replication 49 cycle Figure 2.8 TDP2 5’ phosphodiesterase activity is required for efficient 52 poliovirus replication Figure 2.9 Poliovirus protein production is delayed in the absence of TDP2 54 in hRPE-1 cells Figure 2.10 Polysomes can assemble efficiently on VPg-linked RNA after the 57 initial round of translation in cultured cells Figure 2.11 Viral RNA synthesis is delayed in the absence of TDP2 in hRPE cells 59 Figure 2.12 RF and/or RI formation is delayed in TDP2 KO hRPE-1 cells 61 Figure 2.13 The EMCV IRES driving P2 and P3 protein expression does 64 not rescue virus particle production or RNA synthesis iii Figure 2.14 Premature encapsidation is not responsible for observed 67 growth phenotype in the absence of TDP2 Figure 2.15 Proteinase K treatment of viral RNA partially rescues both 70 positive- and negative-strand RNA production in TDP2 KO cells Figure 3.1 TDP2 is re-localized to the cell periphery in poliovirus-infected 89-92 HeLa cells at 4 and 6 hpi Figure 3.2 TDP2 colocalizes with plasma membrane marker, PVR, at the 94-95 cell periphery in poliovirus-infected HeLa cells at 6 hpi Figure 3.3 TDP2 partially colocalizes with poliovirus VP1 but does not 96-97 relocalize to the cell periphery during infection of HEK-293 cells Figure 3.4 TDP2 forms cytoplasmic aggregates but does not relocalize to 99-100 the cell periphery during poliovirus infection of SK-N-SH cells Figure 3.5 Poliovirus and CVB3 VP1 translocates to the nucleus and 103-105 colocalizes with TDP2 in MCF7 and HeLa cells, respectively Figure 4.1 Model of viral RNA synthesis in the presence and absence of VPg 119 iv LIST OF TABLES Page Table 3.1 Summary of TDP2 and VP1 localization patterns 109 observed during either poliovirus or CVB3 infection of multiple human cell lines v ACKNOWLEDGMENTS I would like to acknowledge my thesis advisor Dr. Bert L. Semler for his guidance, mentorship, intellectual rigor, and support during my time in the PhD program. Having an excellent mentor makes all the difference in graduate school. I would like to also acknowledge my current and former lab mates, Dr. Alexis Bouin, Michelle Vu, Dr. Wendy Ullmer, Dr. Dylan Flather, Dr. Eric Baggs, and Dr. Sonia Maciejewski for their collegiality and camaraderie in the lab. I also appreciate our scientific discussions and their critical review of my work. Additionally, I would like to thank my dissertation committee, Dr. Paul Gershon, Dr. Michael McClelland, and Dr. Suzanne Sandmeyer for their keen scientific insights, which helped to drive my thesis project forward. I would also like to acknowledge my collaborators on this project, Dr. Keith Caldecott, Dr. George Belov, and Dr. Eckard Wimmer for critical reagents they generously provided. Finally, I would like to acknowledge my mother, Carol, my sister, Cheyna, and my fiancée, Damen for their love and support on this journey. It has meant the world to me. vi CURRICULUM VITAE Autumn C. Holmes EDUCATION: 2013-2019 PhD, Biomedical Sciences, University of California, Irvine (UC Irvine) 2008-2012 B.S., Microbiology, (Spanish minor), University of Michigan, Ann Arbor HONORS AND AWARDS: March 2018 American Society for Virology Travel Award February 2018 UC Irvine School of Medicine Dr. Lorna Carlin Scholar Fellowship June 2015 AAAS/Science Program for Excellence in Science March 2015 National Institutes of Health (NIH) Diversity Supplement Award March 2015 National Science Foundation Graduate Research Fellowship Program (Honorable Mention) March 2015 Ford Foundation Fellowship Program (Honorable Mention) October 2014 Initiative for Maximizing Student Development-Minority Biomedical Research Support Program Training Grant (NIH-funded) March 2015 Ford Foundation Fellowship Program (Honorable Mention) February 2013 UC Irvine Graduate Dean’s Recruitment Fellowship March 2011 University of Michigan University Honors September 2008 University of Michigan Tradition Scholarship PROFESSIONAL ASSOCIATIONS 2017-present American Society for Virology 2015-2018 AAAS (General Membership) 2014-2016 American Society for Microbiology 2014-present Graduate Women in Science (Orange County, CA Chapter) RESEARCH EXPERIENCE UC Irvine, Department of Microbiology and Molecular Genetics June 2014 – September 2019 Principal Investigator: Bert L. Semler, PhD vii University of Rochester Post-baccalaureate Research and Education Program (PREP), Department of Microbiology and Immunology July 2012 – June 2013 Principal Investigator: Luis Martinez-Sobrido, PhD Research Focus: Characterizing the interaction between the nucleoprotein and Z matrix protein of arenaviruses University of Michigan Medical School, Department of Human Genetics and Internal Medicine September 2011 – June 2012 Principal Investigator: David Ginsburg, MD Research Focus: Testing small molecule inhibitors for the treatment of antibiotic-resistant Group A Streptococcus PUBLICATIONS Autumn C. Holmes and Bert L. Semler. Picornaviruses and RNA Metabolism: Local and Global Effects of Infection. Journal of Virology. (In press) Autumn C. Holmes, Guido Zagnoli Vieira, Fernando Gomez-Herreros, Keith W. Caldecott, and Bert L. Semler. Post-translational effects of VPg unlinkase activity during picornavirus infections of human cells. (In prep) Bryan D. Yestrepsky, Colin A. Kretz, Yuanxi Xu, Autumn C. Holmes, Hongmin Sun, David Ginsburg, Scott D. Larsen. Development of tag-free photoprobes for studies aimed at identifying the target of novel Group A Streptococcus antivirulence agents. Bioorg. Med. Chem. Lett. 24, 1538–1544 (2014). PRESENTATIONS AND CONFERENCES July 2019 American Society for Virology Meeting, University of Minnesota (Oral Presentation) July 2018 American Society for Virology Meeting, University of Maryland, College Park (Oral Presentation) July 2017 International Union of Microbiological Societies (IUMS), Marina Bay Sands Conference Center, Singapore (Poster) March 2016 Models of Infectious Disease Agent Study Meeting May 2013 PREP program, University of Rochester (Poster) October 2012 The Institute on Teaching and Mentoring viii ABSTRACT OF THE DISSERTATION Deciphering the mechanism of TDP2/VPg unlinkase activity during picornavirus infections By Autumn Candace Holmes Doctor of Philosophy in Biomedical Sciences University of California, Irvine, 2019 Dr. Bert L. Semler, Chair Members of the Picornaviridae family are responsible for many highly prevalent human illnesses. These include: the common cold, hepatitis, myocarditis, and paralytic poliomyelitis, which are caused by human rhinoviruses, hepatitis A virus, coxsackieviruses, and poliovirus, respectively. Given their limited coding capacity, picornaviruses must repurpose host factors to carry out their replication cycles. One such host protein is the DNA repair enzyme, tyrosyl- DNA phosphodiesterase 2 (TDP2), also known as VPg unlinkase. Picornaviruses use TDP2 to cleave the bond between viral genomic RNA and VPg, the primer for RNA synthesis that is covalently linked to the 5’ end of virion RNA. Notably, viral RNA destined for RNA packaging must be VPg-linked, while VPg is absent from polysome-associated viral mRNAs and positive-strand RNA templates used to prime negative-strand RNA synthesis. Previous studies in cultured murine cells have revealed a significant dependence on TDP2 during ix infection by poliovirus, coxsackievirus, or human rhinovirus. However, the

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