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Towards the Identification of Novel Interferon-Alpha Induced Anti TOWARDS THE IDENTIFICATION OF NOVEL INTERFERON-ALPHA INDUCED ANTI-HEPATITIS C VIRUS EFFECTORS by Filip BEBEK Submitted for the degree of Doctor of Philosophy (Ph.D.) School of Biotechnology and Biomolecular Sciences UNSW, Australia APRIL, 2012 ii Author: Filip Bebek ([email protected], [email protected]) Supervisor: Associate Prof Laurent P. Rivory ([email protected]) Co-supervisor: Associate Prof Peter A White ([email protected]) Submitted for the degree of Doctor of Philosophy (PhD), April, 2012 The School of Biotechnology and Biomolecular Sciences (BABS) The University of New South Wales (UNSW), Sydney, NSW, 2052, Australia Previously in association with Johnson and Johnson Research Pty. Ltd. (JJR) Level 4, Biomedical Building, 1 Central Ave., Australian Technology Park, Eveleigh, NSW, 1430, Australia iii Originality Statement ‘I hereby declare that this submission is my own work and to the best of my knowledge it contains no materials previously published or written by another person, or substantial proportions of material which have been accepted for the award of any other degree or diploma at UNSW or any other educational institution, except where due acknowledgement is made in the thesis. Any contribution made to the research by others, with whom I have worked at UNSW or elsewhere, is explicitly acknowledged in the thesis. I also declare that the intellectual content of this thesis is the product of my own work, except to the extent that assistance from others in the project's design and conception or in style, presentation and linguistic expression is acknowledged.’ Filip Bebek ……………………………………. Date 27-Nov-2012……………………. iv Copyright Statement ‘I hereby grant the University of New South Wales or its agents the right to archive and to make available my thesis or dissertation in whole or part in the University libraries in all forms of media, now or here after known, subject to the provisions of the Copyright Act 1968. I retain all proprietary rights, such as patent rights. I also retain the right to use in future works (such as articles or books) all or part of this thesis or dissertation. I also authorise University Microfilms to use the 350 word abstract of my thesis in Dissertation Abstract International (this is applicable to doctoral theses only). I have either used no substantial portions of copyright material in my thesis or I have obtained permission to use copyright material; where permission has not been granted I have applied/will apply for a partial restriction of the digital copy of my thesis or dissertation.' Signed .............................................................................. Date 27-Nov-2012........................................................ Authenticity Statement ‘I certify that the Library deposit digital copy is a direct equivalent of the final officially approved version of my thesis. No emendation of content has occurred and if there are any minor variations in formatting, they are the result of the conversion to digital format.’ Signed .............................................................................. Date 27-Nov-2012………............................................. v Abstract Hepatitis C virus (HCV) infection is primarily treated with regimens that contain pegylated interferon alpha (PEG-IFN-!). IFN induces antiviral effects through the up-regulation of many interferon-stimulated genes (ISGs). While many ISGs have previously been identified, limitations of screening approaches employed to date raise the possibility that other anti-HCV ISGs are yet to be discovered. In the present study, a novel screening strategy combined suppression subtractive hybridisation (SSH) and recombinant Dicer generated siRNA pools to screen for anti-HCV ISGs - utilising a replicon model of HCV which is sensitive to IFN-!, and in which the impact on HCV replication can be readily assessed through the incorporation of a bicistronic luciferase reporter gene. This approach does not require a priori gene sequence data, and thereby opens up the possibility of detecting the anti-HCV activity of novel genes, functional polymorphisms and splice variants. SSH and its related technique mirror orientation selection (MOS) were employed to isolate differentially expressed genes from IFN-! treated Huh-7 cells. The isolated SSH and MOS genes were cross-referenced with microarray data to identify likely mediators of the anti- HCV replicon effects of IFN-! treatment. Subsequently, SSH/MOS cDNA clones were used to produce complementary dsRNA, which was digested with recombinant Dicer to generate target-specific siRNA pools, that were then screened for their ability to suppress the effects of IFN (using luciferase activity as a measure of replicon RNA copy number) following transfection into the stable HCV-replicon cell line (Huh-7 Luc). The list of positive screen hits included ZC3HAV1 and IFIT1. Further validation experiments showed the long isoform of ZC3HAV1 to be a new bona fide anti-replicon ISG. Thus, this study demonstrates the utility of unbiased screening approaches in better understanding how IFN-! limits HCV replication. vi Acknowledgements First and foremost, I would like to thank my supervisor A/Prof Laurent P. Rivory whose guidance, understanding, assistance and determination to stick with this project were always present and supplied in generous quantities. I would also like to sincerely thank all of my past colleagues at JJR for their moral and scientific support. Notably, I would like to acknowledge Dr Toby Passioura for his unwavering patience in answering my many questions, and for his assistance in editing this thesis. Additionally I would like to extend my gratitude to Dr Gaurav Gupta for his assistance in the editing this thesis. I would also like to thank A/Prof Peter White, and the remainder of EMI group at UNSW – Dr Rowena Bull, J-S Eden, Sean Pham and Auda Eltahla – for both your acceptance and support. To my family, I want to say thank you for the never ending support, understanding, care and prayers that have all contributed to help me realize my dreams. Additionally, I want to expresses my very sincere thanks to all of my extended family and friends who have been constant sources of encouragement and support throughout my PhD. Finally, I would like to gratefully acknowledge the financial support received from both the Australian Postgraduate Award and from Johnson and Johnson Research Pty Ltd throughout the duration of my postgraduate studies. vii Table of Contents Originality Statement............................................................................................. iii! Copyright Statement ............................................................................................. iv! Authenticity Statement.......................................................................................... iv! Abstract..................................................................................................................v! Acknowledgements............................................................................................... vi! Table of Contents................................................................................................. vii! List of Tables......................................................................................................... xi! List of Figures and Illustrations ............................................................................ xii! List of Symbols, Abbreviations and Nomenclature ............................................. xiii! CHAPTER 1 – GENERAL INTRODUCTION.................................................................. 1! Hepatitis C Virus ................................................................................................1! Structure and Virology...................................................................................1! HCV Therapy.................................................................................................2! (i) Interferon Alpha...................................................................................2! (ii) Ribavirin .............................................................................................3! (iii) Directly Acting Anti-virals – First Generation Protease Inhibitors ......3! (iv) Future Therapy Directions.................................................................4! HCV Molecular Biology .................................................................................4! In Vitro Models ........................................................................................5! The Interferons.................................................................................................10! The Type I IFN Signalling Cascade.............................................................11! IFN-! and HCV Infection .............................................................................13! ISG Identification..............................................................................................14! RNA-Specific Adenosine Deaminase (ADAR) ............................................16! Eukaryotic Translation Initiation Factor 2-alpha Kinase 2 (EIF2AK2) .........16! Interferon-Inducible Guanylate Binding Protein 1 (GBP1)...........................17! Interferon-Induced Tetratricopeptide (IFIT) Family members – IFIT1 and IFITM1 ..........................................................................................18! Interferon-Induced Protein with Tetratricopeptide Repeats 1 (IFIT1) ....18! Interferon
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