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(Bkpyv) Genome.. . 26 Figure 1 Role of cellular ion channels in the BK polyomavirus life cycle Margarita-Maria Panou Submitted in accordance with the requirements for the degree of Doctor of Philosophy The University of Leeds School of Molecular and Cellular Biology August, 2018 The candidate confirms that the work submitted is her own and that appropriate credit has been given where reference has been made to the work of others. This copy has been supplied on the understanding that it is copyright material and that no quotation from the thesis may be published without proper acknowledgment. © 2018 The University of Leeds, Margarita-Maria Panou The right of Margarita-Maria Panou to be identified as Author of this work has been asserted by her in accordance with the Copyright, Designs and Patents Act 1988. I Acknowledgement I would like to thank my two supervisors Dr Andrew Macdonald and Dr Jamel Mankouri for the opportunity to study for a PhD and for their invaluable support and guidance throughout this project. I would also like to thank all past and present members of the Macdonald and Mankouri labs for their helpful discussions. In particular, thank you to Dr Emma Prescott for the advice and the time spent training me up in the lab and Dr Christopher Wasson for invaluable support throughout my studies. Thank you to Ethan Morgan, David Kealy, Gemma Swinscoe and Dan Hurdiss for your advice and interesting perspectives. I would also like to thank Kidney Research UK for funding my PhD. Δεν θα μπορούσα να παραλείψω την ανεκτίμητη βοήθεια και στήριξη της Μικαέλλας Αντώνη, διδακτορικής φοιτήτριας στην ομάδα του Dr Andrew Macdonald. Εκτός από στήριγμα στο εργαστήριο, ήταν και θα είναι εξαιρετική φίλη. Θα ήθελα επίσης να ευχαριστήσω τους γονείς μου τόσο για την ψυχολογική αλλά και την υλική υποστήριξη κατά τη διάρκεια των σπουδών μου. Ένα μεγάλο ευχαριστώ σε όλους τους ανθρώπους του στενού μου περιβάλλοντος που με στήριξαν στις δύσκολες στιγμές μου και χάρηκαν με τις επιτυχίες μου. II Abstract BK polyomavirus (BKPyV) is a human pathogen that infects the majority of the population, worldwide, establishing a lifelong infection. Immunocompromised patients following renal transplantation, are likely to suffer from severe clinical complications, including polyomavirus-associated nephropathy (PVAN), which can ultimately lead to kidney graft failure. Currently, there are no direct acting anti-viral compounds targeting BKPyV and the number of renal transplants is increasing significantly. Therefore, there is an urgent need to understand the viral life cycle in order to identify potential targets that can be exploited for therapeutic development. Ion channels play a critical role in kidney physiology by controlling several processes, implicating them as candidate proteins required for BKPyV infection. A pharmacological analysis was performed in which human primary renal epithelial cells were treated with a range of pharmacological modulators of host ion channels and the effect on BKPyV production assayed using a fluorescence-based technique. From this approach, it was identified that the clinically available drug, Glibenclamide is a potent inhibitor of BKPyV infection. Biochemical analysis and molecular-based techniques revealed that the cystic fibrosis transmembrane conductance regulator (CFTR) was the target of Glibenclamide and time-of-addition experiments indicated that CFTR might be required during the entry and trafficking of BKPyV through the cytoplasm. These studies provide the first reported requirement for host ion channels in the BKPyV life cycle. Studies on other related polyomaviruses, including JCPyV, SV40 and Merkel cell polyomavirus determined a cell type-dependent requirement of CFTR in the viruses’ life cycle, highlighting the importance of understanding the role of host ion channels in polyomaviruses’ life cycle. Ion channels are an emerging target for many medical conditions and such compounds that target these may represent a novel strategy for developing therapeutics to treat PVAN and/or other polyomavirus-associated clinical complications. III Table of Contents Acknowledgement ...................................................................................... II Abstract ..................................................................................................... III Table of Contents ..................................................................................... IV List of Tables ........................................................................................... XII List of illustrative materials ...................................................................... XIII Abbreviations .......................................................................................... XVI Introduction ........................................................................... 1 1.1 Taxonomy, Classification and Characteristics of Polyomaviridae 1 1.2 Human polyomaviruses .................................................................. 5 1.3 Polyomavirus-associated nephropathy (PVAN) ......................... 10 1.3.1 History and background of the disease .................................... 10 1.3.2 Clinical manifestation of PVAN ................................................. 12 1.3.3 Diagnosis of PVAN ................................................................... 15 1.3.4 Therapeutic interventions for PVAN ......................................... 18 1.4 BK Polyomavirus (BKPyV) ........................................................... 20 1.4.1 Epidemiology and transmission of BKPyV ................................ 20 1.4.2 Cellular tropism of BKPyV ........................................................ 21 1.5 Molecular Virology of BK Polyomavirus (BKPyV) ...................... 22 1.5.1 Structure and Genome Organization ........................................ 22 1.5.2 Non-coding control region (NCCR) ........................................... 27 1.5.3 BKPyV proteins ........................................................................ 29 1.5.3.1 Early proteins .................................................................................29 IV 1.5.3.2 Structural late proteins ...................................................................30 1.5.3.3 Non-structural late protein ..............................................................35 1.5.3.4 Putative VP4 protein ......................................................................40 1.5.4 BKPyV microRNA molecules .................................................... 41 1.6 BK Polyomavirus (BKPyV) life cycle ........................................... 42 1.6.1 BKPyV-host cell receptor attachment ....................................... 42 1.6.2 Internalization of BKPyV ........................................................... 46 1.6.3 Trafficking of BKPyV through the Endoplasmic Reticulum ....... 50 1.6.4 Release from the ER and nuclear entry .................................... 54 1.6.5 BKPyV Gene expression and Genome replication ................... 58 1.6.6 BKPyV assembly and progeny release .................................... 62 1.7 Renal Ion Channels ....................................................................... 66 1.8 CFTR ion channels........................................................................ 67 1.8.1 CFTR localization ..................................................................... 67 1.8.2 CFTR as an intracellular chloride channel ................................ 68 1.8.3 Structure and function of the CFTR ion channel ....................... 71 1.8.4 CFTR and regulation of ROMK ion channel activity in kidneys 76 1.8.5 CFTR ion channel inhibitory compounds .................................. 78 1.9 Viral modulation of host ion channels ........................................ 80 1.9.1 Host ion channels and virus trafficking ..................................... 80 1.9.2 Host ion channels and virus persistence .................................. 81 1.9.3 Viral modulation of host ion channels in excitable cells ............ 82 1.10 Aims and Objectives ..................................................................... 84 Materials and Methods ........................................................ 85 V 2.1 Bacterial cell culture ..................................................................... 85 2.1.1 Preparation of competent bacteria cells ................................... 85 2.1.2 Transformation of plasmid DNA into bacteria ........................... 85 2.1.3 Preparation of plasmid DNA ..................................................... 86 2.1.3.1 Small scale bacterial culture ..........................................................86 2.1.3.2 Large scale bacterial culture ..........................................................86 2.2 Mammalian cell culture ................................................................. 86 2.2.1 Growing, maintaining and passaging mammalian cells ............ 86 2.2.2 Cell counting ............................................................................. 88 2.2.3 Freezing and thawing mammalian cells .................................... 88 2.2.4 Transient transfections with NanoJuice .................................... 88 2.2.5 Transfecting siRNA into RPTE cells ......................................... 89 2.2.6 Use of ion channel modulators ................................................. 90 2.2.7 Cell viability (MTT) assay ......................................................... 92 2.2.8 Resting membrane potential assay .........................................
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