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UG ETD Template Characterization of Autographa californica nucleopolyhedrovirus immediate early protein ME53: The role of conserved domains in BV production, viral gene transcription, and evidence for ME53 presence at the ribosome by Robyn Ralph A Thesis presented to The University of Guelph In partial fulfilment of requirements for the degree of Master of Science in Molecular and Cellular Biology Guelph, Ontario, Canada © Robyn Ralph, December, 2018 ABSTRACT CHARACTERIZATION OF AUTOGRAPHA CALIFORNICA NUCLEOPOLYHEDROVIRUS IMMEDIATE EARLY PROTEIN ME53: THE ROLE OF CONSERVED DOMAINS IN BV PRODUCTION, VIRAL GENE TRANSCRIPTION, AND EVIDENCE FOR ME53 PRESENCE AT THE RIBOSOME Robyn Ralph Advisors: University of Guelph, 2018 Dr. Peter Krell Dr. Sarah Wooton The baculovirus AcMNPV early/late gene me53 is required for efficient BV production and is conserved in all alpha and betabaculoviruses. The 449-amino acid protein contains several highly conserved functionally important domains including two putative C4 zinc finger domains (ZnF-N and ZnF-C) whose cysteine residues are 100% conserved. One purpose of this study is to confirm the presence of two zinc binding domains in ME53, as well as determine their role in virus infection and viral gene transcription. Interestingly, deletion of ZnF-C results in an early delay of BV production from 12 to 18 hours post transfection correlating to ME53's cytoplasmic localization. Cytoplasmic functions at early times post-transfection may include translational regulation, which is supported by yeast-2-hybrid data that ME53 interacts with the host 40S ribosomal subunit protein RACK1. In this study the association of ME53 with the ribosomes of virus infected cells was also investigated. iii DEDICATION I dedicate this thesis to my father, Ronald James Ralph. Through him I learned that in life there are always choices. Choices to learn, choices to succeed, and choices to love. The tenacity he showed throughout his life to ensure that his time was well spent in his favourite places with the people he loved, is a quality I hope to emulate. Time is the most valuable possession and I am blessed to have learned that lesson early in life. To my dad, I love you. In the Big Rock Candy Mountains, All the cops have wooden legs And the bulldogs all have rubber teeth And the hens lay soft-boiled eggs. The farmers' trees are full of fruit And the barns are full of hay, Oh I'm bound to go Where there ain't no snow Where the rain don't fall The wind don't blow In the Big Rock Candy Mountains. - “Big Rock Candy Mountain”, Harry McClintock iv ACKNOWLEDGEMENTS The research I completed during this thesis would not have been possible without the guidance and support of Dr. Peter J. Krell. Thank you for introducing me to virology, and providing your expertise throughout my undergraduate and Master’s degrees, it has been an invaluable experience. I would also like to acknowledge Dr. Sarah Wootton, for the advice and valuable discussions during my research and thesis writing. In addition, I would like to thank Dr. Baozhong Meng and Dr. Steffen Graether for the support and suggestions that kept me focused and on track. Past and present members of the Krell and Meng labs provided endless help throughout my research. To Dr. Emine Ozsahin, thank you for always having time for my questions, and for the friendship that made long days and nights in the lab all that more enjoyable. Thank you as well to Sunny for the friendship and support, especially towards the end of my research where you were vital to the completion of several experiments. To Dr. Huogen Xiao, thank you for the guidance and help, and Clayton Moore for the friendship and laughs through two and a half years. Finally, I need to thank my family and friends who were my cheerleaders through thick and thin. Specifically, my mother Anita for your love and patience while I wrote in your living room, and my husband Josh for taking everything in stride and never failing to support me. As this chapter closes, I look forward to where we will go. v TABLE OF CONTENTS ABSTRACT ......................................................................................................................... II DEDICATION ..................................................................................................................... III ACKNOWLEDGEMENTS ................................................................................................. IV TABLE OF CONTENTS ..................................................................................................... V LIST OF TABLES ............................................................................................................ VIII LIST OF FIGURES ............................................................................................................ IX ABBREVIATIONS .............................................................................................................. XI CHAPTER 1: LITERATURE REVIEW ............................................................................ 1 Baculoviruses ........................................................................................................ 1 Baculovirus virion phenotypes ........................................................................... 3 Viral life cycle ......................................................................................................... 3 Immediate early gene expression regulation and function ............................. 6 Viral DNA replication and late gene expression ............................................. 10 Baculovirus strategies to evade the host anti-viral response ...................... 11 AcMNPV ME53 ..................................................................................................... 15 ME53 intracellular localization........................................................................... 19 The association of ME53 with the capsid and its role in DNA replication............ 21 ME53 and the transcription of viral genes ....................................................... 22 C4 zinc finger proteins ........................................................................................ 23 Thesis Objectives ................................................................................................ 26 vi CHAPTER 2: BIOINFORMATIC ANALYSIS OF ME53 SECONDARY STRUCTURE AND CONFIRMATION OF ZINC FINGER DOMAINS, AND ME53 OVEREXPRESSION 29 Introduction .......................................................................................................... 29 Methods ................................................................................................................ 31 2.2.1 Bioinformatic analysis of ME53 disorder, binding prediction, and redox potential ......................................................................................................................... 31 2.2.2 Cell lines ............................................................................................................. 32 2.2.3 Peptide resuspension ........................................................................................ 32 2.2.4 Circular dichroism .............................................................................................. 34 2.2.5 Analysis of CD data ........................................................................................... 34 2.2.6 Construction of ME53 overexpression bacmid ................................................. 35 2.2.7 Bacmid DNA isolation ........................................................................................ 36 2.2.8 Virus amplification .............................................................................................. 36 2.2.9 Estimation of viral titre using the end-point dilution assay ............................... 37 2.2.10 Multiplicity of infection calculation.................................................................. 38 2.2.11 His-ME53 purification ..................................................................................... 38 2.2.12 SDS-PAGE and Western blot analysis.......................................................... 39 Results .................................................................................................................. 40 2.3.1 Structural Analysis of ME53 .............................................................................. 40 2.3.2 Circular dichroism analysis of ME53 peptides .................................................. 46 2.3.3 Construction of His-ME53 overexpression bacmid ........................................... 48 2.3.4 Purification of His-ME53 .................................................................................... 50 Discussion ............................................................................................................ 54 CHAPTER 3: THE ROLE OF ME53 DOMAINS IN BV PRODUCTION AND VIRAL GENE TRANSCRIPTION ................................................................................................. 60 Introduction ............................................................................................................ 60 Methods ................................................................................................................. 62 3.2.1 Bioinformatic analysis of ME53 ......................................................................... 62 3.2.2 ME53 knockout bacmid construction ................................................................ 63 3.2.3 Transfections for growth curve analysis
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