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Manipulation of the Host Cell Cycle by Murine Norovirus Vpg

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Manipulation of the Host Cell Cycle by Murine Norovirus Vpg Manipulation of the Host Cell Cycle by Murine Norovirus VPg Alice McSweeney A thesis submitted for the degree of Doctor of Philosophy At the University of Otago, Dunedin, New Zealand January 2020 Abstract Human norovirus (HuNV) is a leading cause of acute gastroenteritis worldwide. Despite advances in HuNV cell culture the virus remains difficult to study in the laboratory. Murine norovirus (MNV) is routinely utilised as a model virus for the study of HuNV replication and pathogenesis. MNV has been shown to manipulate the host cell to induce a G0/G1 cell cycle arrest and gain a beneficial replication environment. Expression of the viral protein genome-linked (VPg) protein of MNV is sufficient to induce cell cycle arrest. Manipulation of the cell cycle is independent of the known translation and replication functions of VPg and can be induced with the first 62 N-terminal amino acids. This research aimed to further characterise the interaction between norovirus VPg proteins and the cell cycle. All viruses in the Caliciviridae family encode a VPg protein however it is not known if the G0/G1 arrest induced by MNV VPg is conserved in other calicivirus VPg proteins. The cell cycle effect of representative VPg proteins from five norovirus genogroups and four other Caliciviridae genera were analysed. A G0/G1 cell cycle arrest was observed for all norovirus VPg proteins and representative VPg proteins from the Lagovirus and Sapovirus genera. These findings show that manipulation of the host cell cycle is a highly conserved function of the VPg proteins of caliciviruses and consequently is likely to be important for the viral lifecycle. Alignment of VPg protein sequences revealed a conserved KGKxKxGRG N- terminal motif with high levels of positively charged amino acids. To test the importance of this region for inducing a G0/G1 arrest, truncated or mutated MNV VPg constructs were tested. Removal of the N-terminal motif prevented an accumulation of cells in the G0/G1 phase confirming the importance of this region. Single point or triple mutations of lysine and arginine significantly reduced the accumulation of cells in the G0/G1 phase but did not completely inhibit the arrest confirming the role of positively charged amino acids in this activity. However, expression of the N-terminal motif fused to EGFP was not able to manipulate the cell cycle, indicating that there are additional elements of MNV VPg that contribute to an arrest. ii Bioinformatic and literature analysis of the N-terminal motif of MNV VPg indicated three activities that could have a role in inducing a G0/G1 phase arrest. The N-terminal region was predicted to have RNA binding activity, function as a nuclear localisation sequence and interact with eIF4E. Gel shift analysis showed that MNV VPg and HuNV VPg bind to RNA in a non-specific manner and the lysine and arginine residues within the N-terminal motif were shown to be important for this activity. No evidence was found for nuclear localisation of MNV VPg. Infection of cells with MNV resulted in a dysregulation of eIF4E leading to an increase in nuclear eIF4E levels. Changes in the nuclear/cytoplasmic ratios of eIF4E is a possible mechanism by which VPg could induce a G0/G1 phase arrest. This thesis provides evidence that manipulation of the host cell cycle by VPg proteins is a common feature of caliciviruses that involves a positively charged motif at the N-terminus of VPg. Both RNA binding and dysregulation of eIF4E were identified and remain as possible mechanisms by which MNV VPg may manipulate the cell cycle. iii Acknowledgments I would like to start by expressing my gratitude to my supervisor, Vernon Ward. You have helped keep me on track throughout this project and developed my skills as a scientist over the many years that I’ve been in the lab. Thank-you to Andy Mercer for co-supervising my project, your input at progress meetings was always valuable and much appreciated. I would like to acknowledge the University of Otago for my PhD scholarship, without which I would not have been able to undertake this project. Over the course of my time in the lab I have had the pleasure of attending numerous conferences both in New Zealand and overseas. This was made possible by generous travel grants from the Department of Microbiology and Immunology, the Division of Health Sciences, the Webster Centre for Infectious Diseases and a Professor Sandy Smith scholarship. To the past and present members of the Ward lab; you have helped to create a warm and inviting environment making it a pleasure to come in every day. You guys have listened to me complain and then provided encouragement and advice to keep me going. A special thank-you to Vivienne Young for teaching me about insect cell culture, protein expression, calculations and just generally everything. Zabeen Lateef, I greatly appreciate your infectious enthusiasm for science as well as the time and patience you have shown me especially in the beginning when I was learning the ropes. Colin Davies set this project in motion and established many of the assays for which I am very thankful. Finally, a huge thank-you to my friends and family. Your support during these years has been significant and I would not be finishing if it were not for you. You’ve listened to my highs and commiserated in my lows even if you didn’t always know what I was talking about. iv Table of Contents 1 Introduction .......................................................................................................... 1 1.1 Caliciviruses ................................................................................................... 1 1.2 Norovirus ....................................................................................................... 2 1.3 Murine norovirus .......................................................................................... 6 1.4 Viral protein genome-linked ..................................................................... 14 1.5 The mammalian cell cycle .......................................................................... 19 1.6 Viral manipulation of the cell cycle .......................................................... 25 1.7 Manipulation of the cell cycle by MNV VPg .......................................... 29 1.8 Focus of my research .................................................................................. 32 2 Methods ............................................................................................................... 33 2.1 Analysis of the role of VPg proteins on the cell cycle ............................ 33 2.2 Analysis of protein expression .................................................................. 40 2.3 Construction of norovirus VPg vectors for protein expression in insect cells ................................................................................................................ 43 2.4 Expression and purification of NT* tagged norovirus VPg proteins .. 45 2.5 RNA binding experiments: ........................................................................ 48 2.6 Immunofluorescence microscopy ............................................................. 50 2.7 Nuclear cytoplasmic splits ......................................................................... 52 2.8 Alignments ................................................................................................... 53 2.9 Statistical analysis ....................................................................................... 53 3 Results .................................................................................................................. 54 3.1 Conservation of a G0/G1 cell cycle arrest among diverse VPg proteins ........................................................................................................................ 54 3.2 Identification of a conserved N-terminal region important for cell cycle arrest .................................................................................................... 63 3.3 RNA binding potential of MNV VPg ....................................................... 82 3.4 Nuclear localisation sequence of MNV VPg ........................................... 98 v 3.5 Interaction of MNV VPg with host factor eIF4E ................................... 102 4 Discussion ......................................................................................................... 108 4.1 Induction of a G0/G1 cell cycle arrest by VPg proteins is conserved across a diverse range of noroviruses .................................................... 108 4.2 A conserved N-terminal motif is required for a G0/G1 cell cycle arrest ...................................................................................................................... 109 4.3 Functions of the N-terminal region of MNV VPg ................................ 113 4.4 Conclusions ................................................................................................ 119 5 Future Directions ............................................................................................. 121 6 References .......................................................................................................... 125 7 Appendix I: Recipes ......................................................................................... 154 8 Appendix II: Pentaprobe sequences ............................................................. 158 9 Appendix III: Expression of FCV VPg in CRFK cells ............................... 160 9.1 Methods
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