Using label free proteomics and RNA sequencing to investigate the human respiratory syncytial virus and the effects of the antiviral ribavirin Thesis submitted in accordance with the requirements of the University of Liverpool for the degree of Doctor in Philosophy by Waleed A. Aljabr November 2016 i AUTHOR’S DECLARATION Apart from the help and advice acknowledged, this thesis represents the unaided work of the author ………………………………………………. Waleed Abdurhaman Suliman Aljabr November 2016 This research was carried out in the Department of Infection Biology and Institute of Infection and Global Health, University of Liverpool ii DEDICATION I dedicate this work to my parents who pray me constantly and inspire me. A dedication also goes to my brothers, sisters and my daughters (Leen, Almas and Yara) and my son (Luay) who were always encouraging and supportive throughout my PhD. Finally, special thanks go to my wife (Amal) for her encouragement and support during this time and for looking after my children. iii ACKNOWLEDGEMENTS In the name of Allah, the Most Gracious, the Most Merciful. All Thanks to Allah the Creator of the universe and the mastermind of everything. I would like to express my sincere gratitude to my supervisor Prof Julian Hiscox for the continuous support of my PhD study and research, for his motivation, enthusiasm, patience and wide knowledge. This work would not have been possible without the help and support of my principle supervisor. Very special thanks to my second supervisor Prof James Stuart for his support and encouragement. I am also indebted to my PhD advisors, Prof Stuart Carter and Dr Jane Hodgkinson who have been invaluable on both an academic and a personal level, for which I am extremely grateful. Appreciation also goes out to Dr Olivier for his technical support and the assistance he provided at all levels during this thesis. I must acknowledge Dr Weining Wu and Dr Diane Monday for all the instances in which their assistance helped me along the way. I would like to thank Mrs Catherine Hartley and Jenna Dawson for their support in the laboratory. Special thanks for Dr Stuart Armstrong for his help on MS-proteomic analysis. I wish to thank Centre for Genomic Research, University of Liverpool for RNA sequencing. I am also grateful to all members of the staff of Department of Infection Biology and all people in IGH, Liverpool University for being helpful during this course. I would like to thank all my colleagues back home and in the UK for encouragements. Also, I wish to thanks Hiscox’s group mates, Dr Simon Clegg and all members in Infection of Biology for being supportive and encouragements. Thanks to my sponsorship, Prince Mohammed Medical City, Ministry of Health, the Ministry of Education, in Saudi Arabia for financial support to complete the project. I appreciate the kindness for all members of the Saudi Arabia Cultural Bureau and Saudi Embassy for guidance. I would like to thanks parents, brothers and sisters for their love, care and encouragement. All my successes would not have been possible without the prayers of my mother and may Allah bless her and I ask Allah for her the paradise. Last but not least, unlimited thanks go to my wife, daughters and son for all your love, encouragement and constant support throughout this PhD journey. I would never have got through it without you. iv ABSTRACT Using label free proteomics and RNA sequencing to investigate the human respiratory syncytial virus and the effects of the antiviral ribavirin Waleed A. Aljabr Human respiratory syncytial virus (HRSV) is a known cause of severe lower respiratory tract infection (LRTI) in infants and young children worldwide. HRSV can cause illness in all ages especially those people at high risk including the immunocompromised and the elderly. Globally, HRSV infection leads to a significant healthcare and economic burden due to the lack of an approved vaccine and costly antiviral therapies that are potentially ineffective in some cases. Ribavirin is the only therapeutic licensed for the treatment of severe HRSV infection. It is a synthetic nucleoside with broad spectrum of antiviral activity encompassing both DNA and RNA viruses. The mechanism of action of ribavirin is unclear. It is thought to inhibit the replication of HRSV and lead to a reduction in viral load. How it does this is unknown and the subject of this thesis. This study focused on investigating the effect of the anti-viral ribavirin on cells in general and then infected with HRSV using both label free quantitative proteomics and transcriptomics. This allowed the investigation of the mutation frequency in HRSV and cellular protein abundance, which encompass several mechanisms by which ribavirin is postulated to work. This study was demonstrated that treatment of cells with ribavirin resulted in the increased transcription of selected cellular mRNAs including those involved in mediating anti-viral signalling. Additionally, ribavirin treatment caused a decrease in viral mRNA and proteins. In the absence of ribavirin, HRSV specific transcripts accounted for up to one third of total RNA reads from the infected cell RNA population. Ribavirin treatment resulted in a greater than 90% reduction in reads mapping to viral mRNA, while at the same time no such drastic reduction was detected for the abundance of cellular transcripts. The presented data revealed that ribavirin significantly increased the frequency of HRSV-specific RNA mutations in the viral genome, suggesting direct influence on the fidelity of the HRSV polymerase. The presented data shows transition and transversion substitutions occur during HRSV replication, and that these changes occurred in ‘hot spots’ along the HRSV genome. Examination of nucleotide substitution rates in the viral genome indicated an increase in the frequency of transition but not transversion mutations in the presence of ribavirin. In addition, the data indicated that in the continuous cell types used, and at the time points analyzed, the abundance of some HRSV mRNAs did not reflect the order in which the mRNAs were transcribed. Overall, the work describes a mechanism of action for ribavirin in the context of viral infection, that has not previously been elucidated for HRSV. v TABLE OF CONTENTS AUTHOR’S DECLARATION ii Dedication iii Acknowledgements iv Abstract v Table of contents vi List of figures x List of tables xiii List of abbreviations xiv Chapter 1: introduction 1 1.1. Classification, discovery and epidemiology 1 1.1.1. Classification 1 1.1.2. Discovery and epidemiology 3 1.2. HRSV structure, genome organisation and genomic rna 5 1.2.1 HRSV structure 5 1.2.2. Paramyxoviridae genomic organisation 7 1.2.3. The HRSV genome 8 1.3. Proteins 10 1.3.1. Envelope proteins: G, F, SH and M 10 1.3.1.1. G protein 11 1.3.1.2. F protein 11 1.3.1.3. SH protein 12 1.3.1.4. M protein 13 1.3.2. Nucleocapsid proteins: N, P, L and M2 proteins 13 1.3.3. Accessory proteins: NS1 and NS2 15 1. 4. Infectious cycle 16 1.4.1. Binding and entry 16 1.4.2. Transcription and replication of the viral genome 18 1.4.3. Assembly and budding 19 1.5. Disease pathogenesis and host-cell interactions 20 1.5.2. Disease pathogenesis 20 1.5.2.1. Factors playing a role in HRSV pathogenesis and disease outcome 21 1.5.2.1.1.Environmental and social factors 22 1.5.2.1.2 Viral factors 23 1.5.2.1.3 Host factors 24 1.5.2.1.4 Genetic factors 24 1.6. Treatments and prevention 25 1.6.1. Treatment 25 1.6.1.1. Ribavirin 25 1.6.1.2. Mechanism of action (five mechanisms) 28 1.6.1.2.1.Immunomodulatory effect 28 vi 1.6.1.2.2.Inhibition of IMPDH 28 1.6.1.2.3.Inhibition of RNA capping activity 29 1.6.1.2.4.Inhibition of viral polymerase 30 1.6.1.2.5.Loss of fitness of RNA virus genomes 30 1.6.2. Preventative therapy 31 1.7. High throughput approaches 32 1.7.1 An introduction to label free proteomics 32 1.7.2. An introduction to rna-seq 35 1.8. Research objectives 37 Chapter 2: materials and methods 39 2.1. Tissue cell culture methods 39 2.1.1. Continuous cell culture 39 2.1.2. Freezing and thawing cells 41 2.1.3. Inhibitor study 41 2.1.3.1. Ribavirin 41 2.1.3.2. 17-AAG & 17-DMAG 42 2.1.3.3. Etoposide 42 2.1.4. MTT assay 42 2.1.5. HRSV strains 43 2.1.6. Virus propagation: supernatant 43 2.1.7. Virus purification by sucrose gradient 44 2.1.8. HRSV titre determination 45 2.1.8.1. Plaque assay 45 2.1.8.2. Tissue culture infectious dose 50 (TCID50) 48 2.2. Molecular biological techniques 49 2.2.1. RNA extraction 49 2.2.2. First-strand cDNA synthesis and reverse transcription 51 2.2.3. Oligonucleotides 52 2.2.4. Measurement of DNA concentraion 54 2.2.5. Polymerase chain reachtion (PCR) for HRSV-G and GAPDH detection 54 2.2.6. Quantitative real-time RT-PCR 55 2.2.7. Agarose gel electrophoresis 56 2.2.8. Purification of DNA from gel 57 2.2.9. Total RNA-seq 58 2.3. Protein methods 59 2.3.1. Preparation of whole cell lysates 59 2.3.2. Determining protein concentration by BCA assay DETERMINING PROTEIN CONCENTRATION BY BCA ASSAY 60 2.3.3. Sodium-dodecyl sulphate polyacrylamide gel electrophoresis (SDS- PAGE) SODIUM-DODECYL SULPHATE POLYACRYLAMIDE GEL ELECTROPHORESIS (SDS-PAGE) 61 2.3.4.