The Foot-and-Mouth Disease Virus Replication Complex: Dissecting the Role of the Viral Polymerase (3Dpol) and Investigating Interactions with Phosphatidylinositol-4-kinase (PI4K) Eleni-Anna Loundras Submitted in accordance with the requirements for the degree of Doctor of Philosophy The University of Leeds School of Molecular and Cellular Biology August 2017 The candidate confirms that the work submitted is her own, except where work which has formed part of jointly authored publications has been included. The contribution of the candidate and the other authors to this work has been explicitly indicated below. The candidate confirms that appropriate credit has been given within the thesis where reference has been made to the work of others. The work appearing in Chapter 3 and Chapter 4 of the thesis has appeared in publications as follow: • Employing transposon mutagenesis in investigate foot-and-mouth disease virus replication. Journal of Virology (2015), 96 (12), pp 3507-3518., DOI: 10.1099/jgv.0.000306. Morgan R. Herod (MRH), Eleni-Anna Loundras (EAL), Joseph C. Ward, Fiona Tulloch, David J. Rowlands (DJR), Nicola J. Stonehouse (NJS). The author (EAL) was responsible for assisting with preparation of experiments and production of experimental data. MRH, as primary author drafted the manuscript and designed the experiments. NJS and DJR conceived the idea, supervised the project, and edited the manuscript. • Both cis and trans activities of foot-and-mouth disease virus 3D polymerase are essential for viral replication. Journal of Virology (2016), 90 (15), pp 6864-688., DOI: 10.1128/JVI.00469-16. Morgan R. Herod, Cristina Ferrer-Orta, Eleni-Anna Loundras, Joseph C. Ward, Nuria Verdaguer, David J. Rowlands, Nicola J. Stonehouse. The author (EAL) was responsible for carrying out structural experimental analysis and production of data, and contributed equally to the work alongside Cristina Ferrer-Orta. EAL assisted in the drafting the manuscript. MRH, as primary author, drafted the manuscript and designed experimental work. NJS and DJR conceived the idea, supervised the project, and edited the manuscript. The work appearing in Chapter 5 of the thesis has appeared in publication as follows: • Foot-and-mouth disease virus genome replication is unaffected by inhibition of type III phosphatidyl-4-kinases. Journal of General Virology (2016). 97 (9), pp 2221-2230., DOI: 10.1099/jgv.0.00052. Eleni-Anna Loundras, Morgan R. Herod, Mark Harris (MH), Nicola J. Stonehouse. The author (EAL) was responsible for carrying out all experiments and drafting the manuscript. MRH assisted with some of the preliminary experimental work and the drafting of the manuscript. NJS and MH conceived the idea, supervised the project, prepared and edited the manuscript. 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 acknowledgement © 2017 The University of Leeds and ELENI-ANNA LOUNDRAS The right of ELENI-ANNA LOUNDRAS to be identified as Author of this work has been asserted by her in accordance with the Copyright, Designs and Patents Act 1988. ii Acknowledgements Firstly, I’d like to thank my supervisors Prof. Nicola Stonehouse and Prof. Mark Harris for their guidance and unwavering support throughout my PhD. I’d also like to thank them for pushing me to achieve more than I thought was possible and for having faith in my abilities. To Morgan, thank you for all your support and help with my research, keeping me thinking critically and remembering all my controls! Thank you for the opportunity to be part of some exciting research. To Joe, thank you for your help with assays and putting up with all my cloning questions (and for overseeing my “little pot of crazy!”). To the rest of the ‘Stonelands’ and Harris groups (past and present) for all our enlightening discussions and copious amount of coffee. To Kate Loveday, for helping me take my project to new heights, and to Sue Matthews for being my extra pair of hands. To Garstang 8.53, especially Chris: without you guys, coming in everyday to tackle science would have been nigh on impossible! Last but not least, I would like to thank Charlotte and Lorna, for keeping me sane and standing by me during some of my darkest days. To Mum and Dad, Alexia, and Natalie for believing in me and encouraging me through this endeavour. To Lana and Archer: woof-woof, meow. Finally, to Philip for being my rock: you absolute star, thank you, for everything. iii Abstract Replication of many positive-strand RNA viruses have been shown to occur within intracellular membrane-associated compartments termed replication complexes. Replication of viral RNA occurs within these intracellular compartments as a way for the virus to concentrate the structural and non-structural components into a small area to facilitate replication as well as protecting the virus components from host- cell pathogen recognition and innate immune responses. Using immunofluorescent confocal and electron microscopy, foot-and-mouth disease virus (FMDV) has been shown to dysregulate Golgi and ER-derived membranes, but to date, no distinct membrane-bound replication complex comprised of viral RNA, structural and non- structural proteins, and host-cell proteins have yet to be identified for FMDV. The FMDV RNA-dependent RNA polymerase, 3Dpol, is the primary protein involved in virus genome replication and has been previously shown to form higher-order fibril- like structures in vitro in the presence of RNA. These 3Dpol fibril structures could act to ‘scaffold’ replication complex formation. Here, several mutations were made in 3Dpol to assess their role in higher-order complex formation. The ability for the different 3Dpol mutations to function was assessed biochemically, structurally and in cell culture. The results point towards the necessity for a fully functional (catalytically active) polymerase in the formation of the higher-order structures. Furthermore, complementation studies indicate that 3Dpol has two distinct functions necessary for replication within cells. Additionally, it was pertinent to investigate the role of membrane-associated kinases, such as PI4K, as a number of related viruses utilise this cellular pathway to form an iv optimal environment within which viral replication can occur by upregulating the formation of lipids used in the building of intracellular membranes. Investigation of translation and replication of FMDV RNA within cells show that FMDV does not appear to utilise the PI4K pathway. These results highlight differences between FMDV and other related picornaviruses and provide a basis to investigate alternative methods for replication complex formation. v Table of Contents Acknowledgements ..................................................................................................... iii Abstract ....................................................................................................................... iv List of Figures ............................................................................................................... x List of Tables .............................................................................................................. xiii List of Abbreviations ................................................................................................. xiv Chapter 1 ...................................................................................................................... 1 Introduction ................................................................................................................. 1 1.1 Picornaviruses ................................................................................................... 1 1.1.1 Classification and genome structure .............................................................. 1 1.1.2 Aphthoviruses ................................................................................................. 5 1.2 Foot-and-Mouth Disease Virus .............................................................................. 7 1.2.1 History, Taxonomy and Epidemiology ............................................................ 7 1.2.2 Virus Transmission and Control ...................................................................... 8 1.2.3 Genome Structure ......................................................................................... 13 1.2.3.1 The 5ʹ UTR .............................................................................................. 16 1.2.3.2 The 3ʹ UTR .............................................................................................. 19 1.2.3.3 The Leader proteinase ........................................................................... 20 1.2.3.4 Structural proteins 1A-1D/2A ................................................................ 22 1.2.3.5 Non-structural proteins 2A-3Dpol ........................................................... 23 1.2.4 FMDV genome replication ............................................................................ 30 1.3 Replication Complex ............................................................................................ 36 1.3.1 FMDV Fibrils .................................................................................................. 36 1.3.2 Cellular Interacting Partners in Replication Complex Formation ................. 37 1.4 Aims of the Project ............................................................................................... 40 Chapter