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The Role of Type I Interferon in the Immunobiology of Chikungunya Virus

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The role of type I interferon in the immunobiology of chikungunya virus Jane Amelia Clare Wilson B. App. Sc. (Human Biology), B. App. Sc. (Hons) A thesis submitted for the degree of Doctor of Philosophy at The University of Queensland in 2015 School of Medicine & QIMR Berghofer Medical Research Institute I Abstract Chikungunya virus (CHIKV) is a mosquito-transmitted alphavirus that can cause explosive outbreaks of a febrile, arthritic/arthralgic disease usually lasting weeks to months, and in rare cases, more than a year. In 2004, the largest ever CHIKV outbreak began in Kenya, spreading to islands of the Indian Ocean, India, South East Asia and major outbreaks have recently occurred in the South Pacific Islands and the Caribbean. The host type I interferon (IFN) response is crucial for effective control of CHIKV infection. Herein, the dynamics, source and responses generated by the type I IFNs following CHIKV infection were investigated. Interferon regulatory factors 3 (IRF3) and IRF7 are key transcription factors for the type I IFN response. While CHIKV infection of wild-type mice is non-lethal, infection of mice deficient in both IRF3 and IRF7 (IRF3/7-/-) resulted in mortality, illustrating that these factors are essential for protection. Using knockout mice for the adaptor molecules upstream of IRF3 and 7, IPS1 was found to be the most important for type I IFN production, with TRIF and MyD88 also contributing to the response. Mortality in IRF3/7-/- mice was also associated with type I IFN suppression of pathological levels of IFNγ and haemorrhagic shock. Heterozygous reporter mice, in which eGFP was expressed under the control of either the IFNβ or the IFNα6 promoter on one chromosome, were employed to try and identify the cellular source of type I IFN production following CHIVK infection. However, eGFP production was found to be insufficient for detection, limiting the utility of this approach. Investigations of type I IFN production in tissues using RT-PCR revealed a strong correlation between type I IFN induction and viral tissue titres, with feet and lymph nodes producing the most robust type I IFN responses. RNASeq was undertaken to examine, in detail, the nature of the type I IFN responses generated in the feet and inguinal lymph nodes of mice following CHIKV infection at days 2 and 7 post-infection. High quality sequencing data was generated from II host and viral poly-adenylated RNA, permitting investigation of low level gene transcription from both sources, and the identification of novel genes/transcripts, in addition to analysis of differential gene expression. Investigation of host transcriptional activity revealed a type I IFN dominated immune response, in spite of a low induction of type I IFN mRNA transcripts at day 2 and an absence of type I IFN mRNA induction on day 7. Many type I IFN-regulated genes, including IRF7, remained up-regulated in the feet at day 7, suggesting type I IFN- independent mechanisms for maintenance of type I IFN-regulated gene expression. Six novel IRF3 isoforms and a potentially novel protein coding gene were also identified. RNA-Seq analysis of CHIKV transcriptional activity reflected the known CHIKV replication kinetics, in which virus replicates in the joints, and spreads to the draining lymph nodes, with viral titres peaking at day 2 and largely resolving at day 7. Assessment of mutations within the CHIKV genome showed an accumulation with time, with error accumulation higher in the lymph nodes than in the feet. These studies represent the first deep sequencing analysis of CHIKV genomes in tissues. The findings in this thesis substantially add to our knowledge of the role and function of the immune response after CHIKV, illustrating for the first time that type I IFNs protect against haemorrhagic shock and that expression of type I IFN inducible genes is maintained well after type I IFN induction has waned. III Declaration by author This thesis is composed of my original work, and contains no material previously published or written by another person except where due reference has been made in the text. I have clearly stated the contribution by others to jointly-authored works that I have included in my thesis. I have clearly stated the contribution of others to my thesis as a whole, including statistical assistance, survey design, data analysis, significant technical procedures, professional editorial advice, and any other original research work used or reported in my thesis. The content of my thesis is the result of work I have carried out since the commencement of my research higher degree candidature and does not include a substantial part of work that has been submitted to qualify for the award of any other degree or diploma in any university or other tertiary institution. I have clearly stated which parts of my thesis, if any, have been submitted to qualify for another award. I acknowledge that an electronic copy of my thesis must be lodged with the University Library and, subject to the policy and procedures of The University of Queensland, the thesis be made available for research and study in accordance with the Copyright Act 1968 unless a period of embargo has been approved by the Dean of the Graduate School. I acknowledge that copyright of all material contained in my thesis resides with the copyright holder(s) of that material. Where appropriate I have obtained copyright permission from the copyright holder to reproduce material in this thesis. IV Publications during candidature Peer-reviewed papers: Wilson JAC, Rudd PA,, Gardner J, Larcher T, Babarit C, Le TT, Anraku I, Kumagai Y, Loo Y-M, Gale Jr M, Akira S, Khromykh AA, Suhrbier A. 2012. Interferon Response Factors 3 and 7 Protect against Chikungunya Virus Hemorrhagic Fever and Shock. Journal of Virology 86:9888-9898. Poo YS, Rudd PA, Gardner J, Wilson JAC, Larcher T, Colle M-A, Le TT, Nakaya HI, Warrilow D, Allcock R. 2014. Multiple Immune Factors Are Involved in Controlling Acute and Chronic Chikungunya Virus Infection. PLoS neglected tropical diseases 8:e3354. Conference abstracts: Wilson JAC, Ellis J, Schroder WA, Suhrbier A. 2014. RNA-Seq analysis of chikungunya virus-induced arthritis reveals significant similarity to gene expression in rheumatoid arthritis. 9th International Congress on Autoimmunity. Nice, France. Wilson JAC, Ellis J, Schroder WA, Suhrbier A. 2013. RNASeq analysis of temporal and tissue difference in virus and host gene expression during chikungunya virus infection. Brisbane Immunology Group Meeting. Surfers Paradise, Qld. Wilson JAC, Rudd PA,, Gardner J, Larcher T, Babarit C, Le TT, Anraku I, Kumagai Y, Loo Y-M, Gale Jr M, Akira S, Khromykh AA, Suhrbier A. 2012. Interferon Response Factors 3 and 7 Protect against Chikungunya Virus Hemorrhagic Fever and Shock. Brisbane Immunology Group 13th Annual Retreat. Kingscliff, NSW, December 2012 Wilson JAC, Rudd PA,, Gardner J, Larcher T, Babarit C, Le TT, Anraku I, Kumagai Y, Loo Y-M, Gale Jr M, Akira S, Khromykh AA, Suhrbier A. 2012. Interferon Response V Factors 3 and 7 Protect against Chikungunya Virus Hemorrhagic Fever and Shock. 6th Australasian Virology Society Meeting. Kingscliff, NSW, December 2012. Publications included in this thesis No publications included. Contributions by others to the thesis Prof. Andreas Suhrbier supervised and conceptualised the project, assisted with interpretation of results and reviewed this thesis. Dr Wayne Shroder provided day to day supervision, assisted with interpretation of results and reviewed this thesis. Dr Jonathan Ellis provided bioinformatics support for Chapters 4-6. Prof Thibaut Larcher performed the in situ hybridisation and assisted with interpretation and analysis of histological findings in Chapter 2. Dr Penny Rudd contributed to non-routine technical work and data analysis in Chapter 2. Statement of parts of the thesis submitted to qualify for the award of another degree None. VI Acknowledgements I extend my sincere gratitude to my supervisors Andreas Suhrbier, Wayne Shroder and Alex Khromykh for their guidance throughout my PhD. I thank Andreas Suhrbier for allowing me the opportunity to undertake my PhD within his laboratory, and for his knowledge, advice, generosity and humour, inter alia. I thank Wayne Schroder for his dedication and thoroughness, and for his patience, humour, resolute confidence in my abilities, and genuine understanding and kindness during problematic(al) times. I thank Alex Khromykh for his encouragement, advice and outsider perspectives. I acknowledge the University of Queensland for my Australian Postgraduate Award and QIMR Berghofer Medical Research Institute for my Student Top-Up Scholarship, and Student Travel Award. I also wish to thank my fellow members of the Inflammation Biology Lab, past and present, for their ever present willingness to teach me and share their expertise, and for making my day to day work life (especially lunchtimes) enjoyable and often hilarious. Thank you very much Lee Major, Thuy Le, Itaru Anraku, Yee-Suan Poo, Joy Gardner, Penny Rudd, Tom Partridge, Christiana Agyei-Yeboah, Dilan Pathirana, Vinay Venugopalan and Natalie Prow - I couldn't have asked for a better group to work with! I am also very grateful to the many excellent people I have met while completing my PhD, who I now call friends. In particular, I thank Michelle Neller, Haran Sivakumaran, Tamara Powell, Patrick Dwyer and Claire Foldi for their genuine kindness, empathy and reassuring wisdom. I also owe a very big thanks to all of my friends who encouraged me to undertake this and saw me through to the end, especially Charlie Baker, Sam Vincent and Tara Edwards. VII Thanks also to my beautiful family Sarah, Ben, Chrissy, Jem and Noah, Pete, Janka and Emil, Si and Bron, Nick and Lucy, for their interest, encouragement and high entertainment value. Finally, I thank my mum and dad, Clare and Michael Wilson, for encouraging a love of learning, for their relentless enthusiasm, love and generous support and for knowing me better than anyone else.
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  • Translation Factors and Ribosomal Proteins Control Tumor Onset and Progression: How?

    Translation Factors and Ribosomal Proteins Control Tumor Onset and Progression: How?

    Oncogene (2014) 33, 2145–2156 & 2014 Macmillan Publishers Limited All rights reserved 0950-9232/14 www.nature.com/onc REVIEW Translation factors and ribosomal proteins control tumor onset and progression: how? F Loreni1, M Mancino2,3 and S Biffo2,3 Gene expression is shaped by translational control. The modalities and the extent by which translation factors modify gene expression have revealed therapeutic scenarios. For instance, eukaryotic initiation factor (eIF)4E activity is controlled by the signaling cascade of growth factors, and drives tumorigenesis by favoring the translation of specific mRNAs. Highly specific drugs target the activity of eIF4E. Indeed, the antitumor action of mTOR complex 1 (mTORc1) blockers like rapamycin relies on their capability to inhibit eIF4E assembly into functional eIF4F complexes. eIF4E biology, from its inception to recent pharmacological targeting, is proof-of-principle that translational control is druggable. The case for eIF4E is not isolated. The translational machinery is involved in the biology of cancer through many other mechanisms. First, untranslated sequences on mRNAs as well as noncoding RNAs regulate the translational efficiency of mRNAs that are central for tumor progression. Second, other initiation factors like eIF6 show a tumorigenic potential by acting downstream of oncogenic pathways. Third, genetic alterations in components of the translational apparatus underlie an entire class of inherited syndromes known as ‘ribosomopathies’ that are associated with increased cancer risk. Taken together, data suggest that in spite of their evolutionary conservation and ubiquitous nature, variations in the activity and levels of ribosomal proteins and translation factors generate highly specific effects. Beside, as the structures and biochemical activities of several noncoding RNAs and initiation factors are known, these factors may be amenable to rational pharmacological targeting.