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Transcriptomic and proteomic analysis of arbovirus-infected tick cells Sabine Weisheit Thesis submitted for the degree of Doctor of Philosophy The Roslin Institute and Royal (Dick) School of Veterinary Studies, University of Edinburgh 2014 Declaration .................................................................................................... i Acknowledgements ..................................................................................... ii Abstract of Thesis ....................................................................................... iii List of Figures .............................................................................................. v List of Tables .............................................................................................. vii Abbreviations .............................................................................................. ix 1 Chapter 1 Introduction .......................................................................... 1 2 Chapter 2 Materials and Methods ...................................................... 52 3 Chapter 3 Characterisation of viral infection in tick cells ................ 95 4 Chapter 4: Transcriptomic and proteomic analysis of tick cell lines infected with TBEV .................................................................................. 142 5 Chapter 5 Functional role of genes differentially regulated in tick cells during arbovirus infection .............................................................. 227 6 Chapter 6 Concluding Remarks ....................................................... 269 Reference list ........................................................................................... 274 Appendix .................................................................................................. 310 Declaration I declare that all the work included in this thesis is my own except where otherwise stated. No part of this work has been, or will be submitted for any other degree or professional qualification. Sabine Weisheit 2014 The Roslin Institute and Royal (Dick) School of Veterinary Studies University of Edinburgh Easter Bush Midlothian EH25 9RG Scotland, UK The Pirbright Institute Ash Road Pirbright, Woking Surrey GU24 0NF England, UK i Acknowledgements I would like to thank my supervisors John Fazakerley and Lesley Bell-Sakyi for their guidance during my PhD. Especially Lesley was always there for answering questions about ticks and tick cell lines, providing fresh tick cells whenever needed and she also tirelessly read and corrected my thesis - thank you. During my PhD project I had the special opportunity to do TBEV work in the laboratory of Libor Grubhoffer at the University of South Bohemia in Budweis, Czech Republic and to do proteomics work in the laboratory of José de la Fuente at the University of Castilla-La Mancha in Ciudad Real, Spain. I want to thank them for giving me the opportunity to do essential experiments for my PhD, as well as for their constant support whenever needed. Special thanks also go to members of Libor’s lab, including Hana Tykalová and Daniel Růžek who grew wildtype TBEV and to Marie Vancová who processed and helped with interpretation of my EM samples. Special thanks go to members of José’s lab, including Margarita M Villar Rayo and Marina Popara for training me in processing samples for MS analysis, as well as to Margarita for conducting the MS experiments and analysing the 2D-DIGE images. Thanks go to Ulrike Munderloh for the provision of the IDE8 cell line, Franz X. Heinz for TBEV NS1 antibody and plasmids encoding TBEV and TBEV replicons, Andres Merits for SFV reporter viruses, Sonja Best for LGTV NS5 antibodies, Mayuri Sharma and Richard Kuhn for the LGTV replicon, Steve Mitchell for preparing SFV EM samples, Houssam Attoui for training me to work in the CL3 facility, Jan Kim and Mick Watson for advice concerning bioinformatics, Claudia Rückert for sharing her LGTV data, Gerald Barry and Claire Donald for propagating SFV reporter viruses, Rennos Fragkoudis for the SFV4(3F)-ZsGreen plasmid and Esther Schnettler for all her invaluable input. Thanks also go to the POSTICK project in the Marie Curie FP7- PEOPLE-ITN programme for funding. Thank you to the other current and previous members of our lab: Mhairi Ferguson, Karen Sherwood, Stacey Human, Julio Rodriguez, Joana Ferrolho and Adrian Zagrajek. On a more personal note I want to thank my family Michael, Gunda, Stefanie, Markus, Tim and Ben without whose support and love I would not have achieved as much as I have. Lastly I also want to thank the amazing student community at the Pirbright Institute and my friends, who have helped me through the last year of my PhD - I do not know what I would have done without your constant encouragement. ii Abstract of Thesis Ticks are important vectors of a wide variety of pathogens including protozoa, bacteria and viruses. Many of the viruses transmitted by ticks are of medical or veterinary importance including tick-borne encephalitis virus (TBEV) and Crimean- Congo hemorrhagic fever virus causing disease in humans, and African swine fever virus and Nairobi sheep disease virus affecting livestock. Although several studies have elucidated tick antimicrobial mechanisms including cellular immune responses such as nodulation, encapsulation and phagocytosis and humoral immune responses such as the JAK/STAT pathway, complement-like proteins, antimicrobial peptides, lectin like pattern-recognition molecules and lysozymes, very little is known about the innate immune response of ticks towards viral infection. This study therefore aimed to identify molecules that might be involved in the response of ticks to viral infection. The hypothesis was that TBEV infection leads to changes in the expression of immunity-related transcripts and proteins in Ixodes spp. tick cells and that at least some of these might be antiviral. Ixodes scapularis-derived cell lines IDE8 and ISE6 were chosen since I. scapularis is currently the only tick species with a sequenced genome and an Ixodes ricinus-derived cell line, IRE/CTVM19, was used because I. ricinus is the natural vector of TBEV. Basic parameters required to study the responses of tick cells to infection were determined, including levels of virus infection, kinetics of virus replication and production, formation of replication complexes and uptake of dsRNA or siRNA. The cell lines IDE8, ISE6 and IRE/CTVM19 were infected with either of two tick-borne flaviviruses, TBEV and Langat virus (LGTV), or with the mosquito-borne alphavirus Semliki Forest virus (SFV). Infection was characterised using techniques including plaque assay, luciferase assay, immunostaining and conventional, confocal and electron microscopy. Two time points for transcriptomics and proteomics analysis of TBEV- infected IDE8 and IRE/CTVM19 cells were selected: day 2 post-infection (p.i.) when virus production was increasing and day 6 p.i. when virus production was decreasing. RNA and protein were isolated from TBEV-infected and mock-infected tick cells at iii days 2 and 6 p.i. and RNA-Seq and mass spectrometric technologies were used to identify changes in, respectively, transcript and protein abundance. Differential expression of transcripts was determined using the data analysis package DESeq resulting in a total of 43 statistically significantly differentially expressed transcripts in IDE8 cells and 83 in IRE/CTVM19 cells, while differential protein representation using Χ2 test statistics with Bonferroni correction in IDEG6 software resulted in 76 differentially represented proteins in IDE8 cells and 129 in IRE/CTVM19 cells. These included transcripts and proteins which could affect stages of the virus infection, including virus entry, replication, maturation and protein trafficking, and also innate immune responses such as phagocytosis, RNA interference (RNAi), the complement system, the ubiquitin-proteasome pathway, cell stress and the endoplasmic reticulum (ER) stress response. After verification of sequencing data by qRT-PCR, the ability of several of the identified transcripts or proteins to affect virus infection was determined by knockdown experiments in IDE8 and IRE/CTVM19 cells using wild type LGTV, LGTV replicons or TBEV replicons. Knockdown of genes encoding proteins including the ER chaperone gp96 and the heat-shock protein HSP90 resulted in increased virus production in both cell lines, hinting at an antiviral role. In contrast, knockdown of calreticulin, another ER chaperone, resulted in a decrease in virus production in IRE/CTVM19 cells but not in IDE8 cells, implying a requirement for virus production. This functional