Investigating the roles of the JC virus agnogene and regulatory region using a naturally occurring, pathogenic viral isolate The Harvard community has made this article openly available. Please share how this access benefits you. Your story matters Citation Ellis, Laura Christine. 2014. Investigating the roles of the JC virus agnogene and regulatory region using a naturally occurring, pathogenic viral isolate. Doctoral dissertation, Harvard University. Citable link http://nrs.harvard.edu/urn-3:HUL.InstRepos:12274559 Terms of Use This article was downloaded from Harvard University’s DASH repository, and is made available under the terms and conditions applicable to Other Posted Material, as set forth at http:// nrs.harvard.edu/urn-3:HUL.InstRepos:dash.current.terms-of- use#LAA Investigating the roles of the JC virus agnogene and regulatory region using a naturally occurring, pathogenic viral isolate A dissertation presented by Laura Christine Ellis to The Division of Medical Sciences in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the subject of Virology Harvard University Cambridge, Massachusetts April 2014 © 2014 Laura Christine Ellis All rights reserved Dissertation Advisor: Dr. Igor Koralnik Laura Christine Ellis Investigating the roles of the JC virus agnogene and regulatory region using a naturally occurring, pathogenic viral isolate Abstract Progressive Multifocal Leukoencephalopathy (PML) is caused by lytic infection of oligodendrocytes by JC Virus (JCV). JCV Encephalopathy (JCVE) is a newly identified disease characterized by JCV infection of cortical pyramidal neurons. JCVCPN was isolated from the brain of a JCVE patient. JCVCPN contains a unique 143 base pair deletion in the agnogene and has an archetype-like regulatory region (RR), of the type typically found in the kidneys. In this dissertation, we studied the JCVCPN virus to better understand the role of the agnogene and the RR in JCV replication. We used kidney, glial and neuronal cell lines to compare the replication of JCVCPN to the prototype virus JCVMad-1. JCVCPN was able to replicate viral DNA in all cell lines tested, but was unable to establish the high level of infection seen with JCVMad-1. Levels of VP1 capsid protein were undetectable in JCVCPN transfected cells, and few infectious virions were produced. JCVCPN did not have a replication advantage in the neuronal cell line tested. To determine if the agnogene deletion or the archetype-like RR was responsible for the observed phenotype of JCVCPN, we generated a series of chimeric viruses between JCVCPN and JCVMad-1. We found that the phenotype of JCVCPN was due predominantly to the deletion in the agnogene, in particular the loss of the DNA and not the lack of a iii full length agnoprotein. To further study the role of the agnogene DNA in JCV replication, we introduced a series of small agnogene deletions into a virus with a start codon mutation which prevents agnoprotein expression. We characterized the replication of these additional mutants and found that nucleotides 376-396 are crucial for the expression of VP1 capsid protein. Previous studies have provided evidence for the binding of host cell proteins to the agnogene DNA. We used DNA- Immunoprecipitations with the agnogene to identify candidate binding proteins, but were unable to confirm any candidate proteins as binding specifically to the JCV agnogene. Studying this naturally occurring pathogenic variant of JCV provided a valuable tool for understanding the functions of the agnogene and RR form in JCV replication. iv ACKNOWLEDGMENTS There are many people who I need to thank for support and encouragement throughout my time in graduate school. First, thank you to my advisor Igor Koralnik, for supporting me throughout my dissertation research and for valuable discussion and advice. I also need to thank my dissertation advisory committee, Jim DeCaprio, Dana Gabuzda and Fred Wang, for helpful advice, discussions and intellectual input. Thank you to the past and present members of the Koralnik lab: Shruti Agnihotri, Stephanie Batson, Evelyn Bord, Tom Broge, Spyridon Chalkias, Xin Dang, Sarah Gheuens, Michael Khoury, Gabriel Lerner, Matt McKenzie, Liz Norton, Sabrina Tan and Chris Wuthrich. You offered helpful and interesting conversations, both scientific and otherwise, and made the lab a fun place to work. A special thanks to Liz, for helping with protocol troubleshooting and sample processing. Thank you to the Virology Program and my fellow Virology graduate students, in particular my Vironaut cohort, for support and friendship. You’ve made my time here at Harvard a lot of fun. v Thank you to my previous mentors, Brian Murphy, Ann-Marie Cruz, Anne Fausto- Sterling, Jürgen Kreyling and Randy Morse, for getting me started and supporting me early in my research career. A big thanks to my friends here in Boston for filling my free time with fun activities. To Kevin McCarthy and Victor Lum for being great roommates. To my climbing partners and friends for great times on the cliffs and elsewhere. To Erik Guldbech for being supportive and encouraging, and for going on awesome adventures with me. Finally, I need to thank my family. To my parents for unconditional love, support and encouragement throughout my entire education, and for getting me interested in science. Thanks Mom, for the encouragement throughout my years here, and for all the food and baked goods! To my sister, Karen, and brother, Joe, for being the best siblings anyone could ask for. vi TABLE OF CONTENTS ABSTRACT……………...………………………………………………………………………iii ACKNOWLEDGEMENTS……………………………………………………………………...v TABLE OF CONTENTS……………………………………………………………………….vi FIGURES AND TABLES……………………………………………………………………….x CHAPTER 1: Introduction…………………………………………………………………….1 JC Virus…………………………………………………………………………………..2 Genome Organization, Proteins and Virion Structure……………………….2 Replication Cycle………………………………………………………………..5 JCV Tropism and RR Variation………………………………………………..7 miRNAs in JCV Replication…………………………………………………….9 JCV and Human Disease……………………………………………………………..10 Progressive Multifocal Leukoencephalopathy………………………………10 JCV Granule Cell Neuronopathy……………………………………………..13 JCV Encephalopathy………………………………………………………….14 JCV Cortical Pyramidal Neuron 1……………………………………………………18 Isolation and Sequencing……………………………………………………..18 Molecular Biology Studies…………………………………………………….18 Histological Studies of JCVE brain samples………………………………..21 Agnoprotein…………………………………………………………………………….25 Roles in Viral Replication……………………………………………………..26 Alterations of Host Cell Processes…………………………………………..27 Agnogene Cis-Acting Regulatory Element………………………………….28 vii Scope of the Dissertation……………………………………………………………..29 References……………………………………………………………………………..31 CHAPTER 2: Agnogene deletion in a novel pathogenic JC Virus isolate impairs VP1 expression and virion production……………………………………………...……41 Abstract………………………………………………………………………………....42 Introduction……………………………………………………………………………..43 Materials and Methods………………………………………………………………..46 Results……………………………………………………………………………….....51 Discussion……………………………………………………………………………...68 Acknowledgements…………………………………………………..………………..74 References……………………………………………………………………………..75 CHAPTER 3: Nucleotides 376-396 are critical for VP1 capsid protein expression……………………………………………………………………………………..79 Abstract…………………………………………………………………………………80 Introduction……………………………………………………………………………..81 Materials and Methods………………………………………………………………..84 Results……………………………………………………………………………….....90 Discussion.……………………………………………………………………………103 References……………………………………………………………………...…….107 CHAPTER 4: Discussion………………………………………………………………..…110 Summary………………………………………………………………………….…..111 Discussion…………………………………………………………………………….114 JCV infection in neurons………………………………………………….....114 viii JCVCPN and JCVE………………………………………………………...….117 JCVCPN biology and the function of the agnogene……………………….120 Challenges of studying JCV in cell culture……………………………......125 Conclusion……………………………………………………………………………128 References……………………………………………………………………………130 Appendix A (copy)………………………………………………………………………….134 Agnogene Deletion in a Novel Pathogenic JC Virus Isolate Impairs VP1 Expression and Virion Production Appendix B (copy)………………………………………………………………………….146 JC Virus Latency in the Brain and Extraneural Organs of Patients with and without Progressive Multifocal Leukoencephalopathy ix FIGURES AND TABLES Figure 1.1 Schematic of JCV genome organization…………………………………………3 Figure 1.2 The JCV Virion……………………………………………………………………...5 Figure 1.3 JCV Replication Cycle…………………………………………………………..…6 Figure 1.4 Schematic of Mad-1 versus Archetype RR……………………………………...8 Figure 1.5 MRI of PML, JCV GCN and JCVE brains……………………………………...11 Figure 1.6 JCV infection in astrocytes and neurons in the cortex and GWJ in a JCVE patient…………………………………………………………………………………………...16 Figure 1.7 Confirmation of JCV infection in neuronal cells………………………………..17 Figure 1.8 JCVCPN1 Genome map and RR……………………………………….….……...20 Figure 1.9 Structure of JCVCPN1.1 and JCVCPN1.2…………………………………………...21 Figure 1.10 Double IF staining of classic PML patient for JCV VP1 and Agnoprotein…………………………………………………………………………………….22 Figure 1.11 Double IF staining of JCVE patient for JCV VP1 and Agnoprotein…...…...23 Figure 1.12 Double IF of JCV Agnoprotein and neuronal marker in JCVE patient……..24 Figure 1.13 Structure of the JCV agnoprotein……………………………………………...26 Table 1.1 Single nucleotide mutations in JCVCPN1…………………………………….......19 Figure 2.1 JCVCPN replicates viral DNA, but at lower levels than JCVMad-1……………...52 Figure