DETERMINANTS OF ARCHETYPE BK POLYOMAVIRUS REPLICATION by Nicole Marie Broekema A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy (Microbiology and Immunology) in The University of Michigan 2013 Doctoral Committee: Professor Michael J. Imperiale, Chair Professor Gregory R. Dressler Professor Katherine R. Spindler Associate Professor David Miller ACKNOWLEDGMENTS I want to thank my Ph.D. advisor, Dr. Michael Imperiale, first and foremost. Thank you for always taking time to listen to my ideas and constantly challenging me to be a better scientist. To my fellow labmates, especially Shauna Bennett and Dr. Mengxi Jiang: thank you for all the good times and advice about lab and life. I'm grateful for having such great people to share in my scientific journey. Also, I would like to thank Nico Conti for his help and enthusiasm on the model of archetype persistence in urothelial cells. I am grateful for my undergraduate and post-baccalaureate mentors: Dr. Robert Goodman, Dr. Mark Liles, Dr. Stacey Schultz-Cherry, Dr. David Warshauer, and Steve Marshall. It takes a village. Thank you for your patience and guidance. Thank you to all my committee members and other Microbiology and Immunology department faculty members for your time, advice, and guidance of my dissertation work. I want to thank Kathy Spindler. Christaine Wobus, Beth Moore, Billy Tsai, and members of the Imperiale laboratory for comments, discussion, and critical reading of manuscripts. Thanks to Amanda Pattridge and Dorothy Sorenson for technical assistance with TEM. We thank Richard Frisque for the pJC-CY plasmid and VP1 antibody, David Wang for the pBlu-KI and pBlu-WU plasmids and VP1 antibodies, Chris Sullivan and Gil Ju Seo for the miRNA luciferase reporter assay constructs and advice, and Hans Hirsch and Rainer Gosert for the phRG promoter construct. We thank Peter Gergics and the Sally Camper laboratory for use of their Promega GloMax 96 Microplate luminometer. ii This work would not have been possible without these funding sources: NIH grant AI060584 awarded to M.J.I and in part by NIH grant CA046592 awarded to the University of Michigan Cancer Center. N.M.B. was supported in part by the NIH National Research Service Award T32-GM07544 from the National Institute of General Medicine Sciences and a Rackham Predoctoral Fellowship from the University of Michigan. Finally, I am grateful for the love and support of friends and family, especially Jacob Notbohm and my parents. iii TABLE OF CONTENTS Acknowledgments ii List of Figures vi List of Tables viii Abstract ix Chapter I Introduction BK polyomavirus.............................................................................. 1 Viral persistence............................................................................... 3 Transmission and epidemiology....................................................... 5 Reactivation and clinical disease...................................................... 5 The intracellular viral lifecycle........................................................ 10 Archetype BKPyV............................................................................ 12 Host immune responses to BKPyV.................................................. 13 The polyomavirus miRNA............................................................... 16 Summary and chapter outline........................................................... 18 Notes................................................................................................ 19 References........................................................................................ 20 Chapter II A system for the analysis of BKPyV non-coding control regions: application to clinical isolates from an HIV/AIDS patient Abstract.................................. .......................................................... 27 Introduction...................................................................................... 27 Results.............................................................................................. 31 Discussion........................................................................................ 36 Materials and Methods..................................................................... 41 Notes................................................................................................ 45 References........................................................................................ 46 Chapter III Efficient propagation of archetype BK and JC polyomaviruses Abstract.................................. .......................................................... 50 Introduction...................................................................................... 50 Results.............................................................................................. 55 Discussion........................................................................................ 65 iv Materials and Methods..................................................................... 70 Notes................................................................................................ 74 References........................................................................................ 75 Chapter IV miRNA regulation of BK polyomavirus replication during early infection Abstract.................................. .......................................................... 79 Introduction...................................................................................... 79 Results.............................................................................................. 84 Discussion........................................................................................ 98 Materials and Methods..................................................................... 103 References........................................................................................ 110 Chapter V Discussion Chapter Summary............................................................................ 113 Future areas of study........................................................................ 120 Conclusions...................................................................................... 131 References........................................................................................ 135 Appendix Preliminary Studies of archetype virus persistence in urothelial cells Introduction...................................................................................... 140 Materials and Methods..................................................................... 143 Results and Discussion..................................................................... 144 References........................................................................................ 149 v List of Figures Figure 2.1 The NCCR determines replication efficiency of BKPyV........................................ 30 2.2 Schematic of the NCCRs.......................................................................................... 34 2.3 Clones isolated from a clinical specimen display variable replication abilities....... 35 2.4 The O block polymorphism does not affect replication ability................................ 37 3.1 TAg expression limits archetype BKPyV replication in RPTE cells....................... 56 3.2 Archetype BKPyV and JCPyV replicate efficiently in 293TT cells........................ 58 3.3 KI, WU, and MCPyV do not replicate efficiently in 293TT cells........................... 60 3.4 Archetype BKPyV and JCPyV produce infectious progeny in 293TT cells............ 62 3.5 Morphology of BKPyV produced in 293TT cells.................................................... 64 3.6 Purified archetype BKPyV is infectious in 293TT cells.......................................... 66 4.1 Schematic of the BKPyV genome............................................................................ 81 4.2 The BKPyV miRNA mutant is unable to regulate its target early mRNA............... 85 4.3 The wt Northern blot probe recognizes the mutant miRNA sequence..................... 87 4.4 Schematic of the miRNA stem-loop qPCR assay.................................................... 88 4.5 The BKPyV miRNA limits viral replication in RPTE cells..................................... 90 4.6 Promoter activity of archetype virus vs. rearranged variants................................... 93 4.7 Balance of promoter activity in archetype virus vs. rearranged variants................. 95 4.8 miRNA expression is partially regulated by sequences in the enhancer region....... 97 4.9 Model of miRNA control of archetype virus replication.......................................... 100 vi 5.1 Naturally occuring mutations in the BKPyV miRNA region................................... 128 5.2 Overview model of potential factors contributing to BKPyV persistence............... 132 A.1 Archetype virus DNA is maintained as an episome in urothelial cells..................... 146 A.2 Archetype virus DNA is specifically maintained as an episome in urothelial cells under selection.......................................................................................................... 147 vii List of Tables Tables 2.1 List of primers used for site-directed mutagenesis and SOE PCR........................... 42 4.1 List of primers used for cloning, site-directed mutagenesis, and real-time PCR...... 104 6.1 BK polyomavirus 5P miRNA [bkv-miR-B1-5p] predicted targets.........................