Downloaded from orbit.dtu.dk on: Oct 06, 2021 Conserved elements within the genome of foot-and-mouth disease virus; their influence on viral replication Kjær, Jonas Publication date: 2017 Document Version Publisher's PDF, also known as Version of record Link back to DTU Orbit Citation (APA): Kjær, J. (2017). Conserved elements within the genome of foot-and-mouth disease virus; their influence on viral replication. Danmarks Tekniske Universitet (DTU). General rights Copyright and moral rights for the publications made accessible in the public portal are retained by the authors and/or other copyright owners and it is a condition of accessing publications that users recognise and abide by the legal requirements associated with these rights. Users may download and print one copy of any publication from the public portal for the purpose of private study or research. You may not further distribute the material or use it for any profit-making activity or commercial gain You may freely distribute the URL identifying the publication in the public portal If you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediately and investigate your claim. Conserved elements within the genome of foot-and-mouth disease virus; their influence on viral replication PhD Thesis · 2017 Jonas Kjær Technical University of Denmark National Veterinary Institute Lindholm Supervisors Graham J. Belsham, Professor National Veterinary Institute, Technical University of Denmark, Kalvehave, Denmark Thomas B. Rasmussen, Senior Researcher National Veterinary Institute, Technical University of Denmark, Kalvehave, Denmark Assessment committee Lars E. Larsen, Professor National Veterinary Institute, Technical University of Denmark, Lyngby, Denmark Martin D. Ryan, Professor School of Biology, University of St. Andrews, St. Andrews, United Kingdom Jens Bukh, Professor Department of Immunology and Microbiology, University of Copenhagen, Copenhagen, Denmark Conserved elements within the genome of foot-and-mouth disease virus; their influence on viral replication PhD thesis © Jonas Kjær. 2017 Contents Preface ....................................................................................................................................... I Acknowledgements ................................................................................................................. II Objectives and Summary ...................................................................................................... IV Formål og Sammenfatning .................................................................................................... VI Part 1 · Introduction ................................................................................................................ 1 Foot-and-mouth disease ....................................................................................................... 2 Foot-and-mouth disease: from past to present .................................................................... 2 Genome organization of foot-and-mouth disease virus .................................................... 5 Genome organisation .......................................................................................................... 5 The 5ʹ- and 3ʹ-untranslated regions .................................................................................... 5 Leader protein ..................................................................................................................... 6 The structural proteins VP1-VP4 ....................................................................................... 7 2A ....................................................................................................................................... 7 2B........................................................................................................................................ 8 2C........................................................................................................................................ 8 3A ....................................................................................................................................... 9 3B........................................................................................................................................ 9 3C...................................................................................................................................... 10 3D ..................................................................................................................................... 10 Quasispecies and genetic diversity ................................................................................... 11 Reverse genetics system ................................................................................................... 12 Strategies to study viral replication .................................................................................. 12 Replication of FMDV ......................................................................................................... 16 FMDV life cycle ............................................................................................................... 16 Adsorption and entry ........................................................................................................ 16 Translation and polyprotein processing ............................................................................ 18 Replication of the viral genome........................................................................................ 19 Assembly and release of the FMDV virion ...................................................................... 21 2A mediated ribosomal skipping at the 2A/2B junction .................................................. 22 Functional structural RNA elements within the FMDV genome .................................. 28 The multi-functional RNA molecule ................................................................................ 28 RNA structure and function .............................................................................................. 28 RNA secondary structure probing .................................................................................... 29 Conserved RNA structures in FMDV .............................................................................. 31 SHAPE probing of the FMDV genome ............................................................................ 35 In silico prediction of RNA secondary structures ............................................................ 36 Part 2 · Manuscripts .............................................................................................................. 37 Manuscript 1: Modifications to the foot-and-mouth disease virus 2A peptide; influence on polyprotein processing and virus replication ....................................................................... 38 Manuscript 2: Selection of functional 2A sequences within foot-and-mouth disease virus; requirements for the NPGP motif with a distinct codon bias ............................................... 74 Manuscript 3: The influence of conserved RNA structures in the 2B, 3C and 3D coding regions of the foot-and-mouth disease virus genome on virus replication .......................... 93 Part 3 · Conclusions and perspectives ................................................................................ 110 Conclusions and perspectives .......................................................................................... 111 Abbreviations ....................................................................................................................... 117 References ............................................................................................................................. 119 Appendix 1 ............................................................................................................................ 141 Preface This PhD thesis is based on the experimental work performed at the island of Lindholm which is a part of the National Veterinary Institute at the Technical University of Denmark (DTU- Vet). It was conducted from December 2014 to December 2017 under the supervision of Professor Graham J. Belsham with co-supervision from Senior Researcher Thomas B. Rasmussen. The studies were supported by core funds within DTU-Vet. The thesis is organized in three parts: Part 1 consist of a general introduction which provides a review surrounding the different aspects of the molecular biology of FMDV with a focus on the viral life cycle and the conserved elements within the viral genome which influence it. Part 2 describes the results achieved during this study presented as three manuscripts listed below: I. Kjær J., Belsham G. J. (2018) Modifications to the foot-and-mouth disease virus 2A peptide; influence on polyprotein processing and virus replication. Submitted. II. Kjær J., Belsham G. J. (2017). Selection of functional 2A sequences within foot-and- mouth disease virus; requirements for the NPGP motif with a distinct codon bias. RNA. DOI: 10.1261/rna.063339.117 III. Kjær J., Belsham G. J. The influence of conserved RNA structures in the 2B, 3C and 3D coding region of the foot-and-mouth disease virus on virus replication. Manuscript in preparation. Part 3 provides a Discussion of the achieved results and future perspectives for continued research. I Acknowledgements The last three