Protein-DNA Interactions of pUL34, an Essential Human Cytomegalovirus DNA- Binding Protein A dissertation presented to the faculty of the College of Arts and Sciences of Ohio University In partial fulfillment of the requirements for the degree Doctor of Philosophy Mark D. Slayton August 2018 © 2018 Mark D. Slayton. All Rights Reserved. 2 This dissertation titled Protein-DNA Interactions of pUL34, an Essential Human Cytomegalovirus DNA- Binding Protein by MARK D. SLAYTON has been approved for the Department of Molecular and Cellular Biology and the College of Arts and Sciences by Bonita J. Biegalke Associate Professor of Biomedical Sciences Joseph Shields Interim Dean, College of Arts and Sciences 3 ABSTRACT SLAYTON, MARK D., Ph.D., August 2018, Molecular and Cellular Biology Protein-DNA Interactions of pUL34, an Essential Human Cytomegalovirus DNA- Binding Protein Director of Dissertation: Bonita J. Biegalke Human cytomegalovirus (HCMV) is primarily an opportunistic pathogen in human, causing significant disease in immunocompromised individuals. A large, double- stranded DNA genome (~230 kilobases) provides the coding capacity for over 200 genes, of which only 25% are required for viral replication in cell culture. The viral UL34 gene encodes sequence-specific DNA-binding proteins (pUL34) which are essential for replication, and viruses lacking the proper expression of pUL34 cannot replicate in cell culture. Interactions of pUL34 with DNA binding sites (US3 and US9?) represses transcription of (these) two viral immune evasion genes that are dispensable for replication in cell culture. There are 12 additional predicted pUL34-binding sites present in the HCMV genome (strain AD169), with three of them concentrated near the HCMV origin of lytic replication (oriLyt). Analysis of 47 clinical isolates of HCMV confirmed that the predicted UL34-binding sites were highly conserved. Protein-DNA interactions were analyzed during infection with ChIP-seq and confirmed that pUL34 binds to the human and viral genome during infection, including at the three predicted UL34-binding sites in the oriLyt region. Mutagenesis of the UL34-binding sites in an oriLyt-containing plasmid significantly reduced viral-mediated, oriLyt-dependent DNA replication. Subsequently, mutagenesis of these same sites in the HCMV genome reduced the 4 replication efficiencies of the resulting viruses. Protein-protein interaction analyses demonstrated that pUL34 interacts with 3 virus proteins that are essential for viral DNA replication - IE2, UL44, and UL84, suggesting that pUL34-DNA interactions in the oriLyt region are involved in the DNA replication cascade. Lastly, mutagenesis of the predicted UL34-binding site in the third exon of another essential viral gene, UL37, demonstrated that some UL34-binding sites are not important for viral replication. 5 DEDICATION This work is dedicated to my father, Daniel Earl Slayton, who wanted nothing more than to see his children succeed at their goals. Even though he was unable to see this accomplishment, I was motivated and encouraged by his memory. 6 ACKNOWLEDGMENTS I am thankful to a great number of individuals who assisted me during my thesis research in a variety of ways. First and foremost, I would like to thank my dissertation advisor, Dr. Bonita Biegalke, who made this entire project possible. She accepted me into her lab and set me off on a well-designed project, giving me invaluable advice and assistance along the way. Her bold and direct personality helped to shape me into the scientist that I am today. I would also like to thank the members of my dissertation committee, Drs. Calvin James, Mark Berryman, and Justin Holub, who examined my work and asked me challenging questions which were critical to refining both my research and my knowledge. From the laboratory, I would like to thank my colleagues Ms. Janet Hammer, who kept the lab running efficiently and helped me perform several experiments, as well as Dr. Tanvir Hossain, who created an important virus and assisted in several experiments. Both Janet and Tanvir encouraged me when I was frustrated with the results of a long but failed experiment, telling me to “keep moving forward”. I am thankful to several undergraduate students who rotated through the laboratory and provided time-saving assistance through laboratory maintenance – in particular, Ms. Allison Marsh, Ms. Gianna Montague and Mr. Jim Wilson. I thank my mother and my three sisters for believing in me and offering continual words of encouragement. Finally, I thank my wife, Liza Zimmerman-Slayton, for her devotion, love, encouragement, and support by making my life outside the laboratory as wonderful as possible. 7 TABLE OF CONTENTS Page Abstract ............................................................................................................................... 3 Dedication ........................................................................................................................... 5 Acknowledgments............................................................................................................... 6 List of Tables ...................................................................................................................... 9 List of Figures ................................................................................................................... 10 Chapter 1: Introduction ..................................................................................................... 11 Medical Significance .................................................................................................. 11 Human Cytomegalovirus ............................................................................................ 12 Classification......................................................................................................... 12 Structural organization .......................................................................................... 12 Genome organization ............................................................................................ 13 Viral infection and replication .............................................................................. 14 Lytic-Phase Genome Replication ............................................................................... 16 The origin of lytic replication ............................................................................... 16 Viral genes essential to DNA replication .............................................................. 17 DNA-Binding Protein UL34 ....................................................................................... 21 Summary ..................................................................................................................... 22 Chapter 2: Materials and Methods .................................................................................... 24 Human cell culture. ............................................................................................... 24 Oligonucleotides. .................................................................................................. 24 Plasmid mutagenesis and cloning. ........................................................................ 27 Transient transfection and total DNA isolation. ................................................... 28 Southern blotting. .................................................................................................. 29 Quantitative polymerase chain reactions. ............................................................. 29 RT-qPCR............................................................................................................... 30 Generation of mutant viruses. ............................................................................... 30 One-step growth curve. ......................................................................................... 31 Chromatin immunoprecipitation. .......................................................................... 32 Next-generation sequencing.................................................................................. 33 Genomics data analysis. ........................................................................................ 34 8 Protein-protein interactions. .................................................................................. 35 Statistical analysis. ................................................................................................ 36 Data availability. ................................................................................................... 36 Chapter 3: Conservation of UL34 Binding Sites and ChIP-Seq Analysis ........................ 37 Background ................................................................................................................. 37 Aims ............................................................................................................................ 39 Results ......................................................................................................................... 39 Discussion ................................................................................................................... 67 Chapter 4: UL34 Binding Sites in the Origin of Lytic Replication .................................. 70 Background ................................................................................................................. 70 Aims ...........................................................................................................................