HOST-VIRAL INTERACTIONS: HOST FACTORS IN CORONAVIRUS REPLICATION AND CORONAVIRAL STRATEGUES OF IMMUNE EVASION WONG HUI HUI (B. SC. (Hons), NUS) A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY DEPARTMENT OF BIOCHEMISTRY NATIONAL UNIVERSITY OF SINGAPORE 2012 Declaration I hereby declare that this thesis is my original work and it has been written by me in its entirety. I have duly acknowledged all the sources of information which have been used in the thesis. __________________________ Wong Hui Hui 22nd October 2012 i Acknowledgements I would like to extend my heartfelt gratitude to my supervisors, Dr Frederic Bard and Associate Professor Liu Ding Xiang, for their mentorship, guidance and advice over the years. I will also like to thank Dr. Manoj, Dr Slvie Alonso for their advice and critical feedback during the thesis committee meeting. Special thanks to all the wonderful co-workers from both FB and LDX laboratories, especially Felicia, Yanxin, Ronghua, Violette, Joanne, Jasmine, for their friendship and encouragement and Dr. Fang Shouguo, Dr. Yoshiyuki Yamada, Dr. Nasirudeen AMA, Dr. Pankaj Kumar, Dr. Samuel Wang, Dr. Alexandre Chaumet and Dr Germaine Goh for their help and advice. I would also like to thank IMCB (A*STAR) for awarding me the research scholarship under the Scientific Staff Development Scheme. This work would not have been possible without the unfailing support of my family –my husband Alvin, my mum, Stella and my two brothers, Chen Wei and Yong Long. ii Table of Contents Summary xii List of Figures xiv List of Tables xv Publications xix CHAPTER ONE: A LITERATURE REVIEW OF THE BIOLOGY OF CORONAVIRUS 1.1 CORONAVIRUS: AN OVERVIEW 1.1.1 Taxonomy……………………………………………………………………………… .2 1.1.2 Diseases of Coronaviruses.……………………………………………………………….4 1.1.3 Morphology and Structure of Coronavirus.………………………………………………6 1.1.4 Genome Organization of Coronavirus……………………………………………………7 1.1.5 Proteins encoded by Coronavirus…………………………………………………… .…9 1.1.6 1.1.5.1.1 Structural proteins…………………………………………………………….9 1.1.5.1.2 Replicase and Non-structural proteins……………………………………….12 1.1.5.1.3 Accessory proteins………………………………………………………… .14 1.1.5.1.4 SARS Accessory proteins……………………………………………………14 The coronavirus life cycle……………………………………………………………….16 1.1.6.1 Attachment and Entry……………………………………………………… 16 1.1.6.2 Translation and assembly of replicase……………………………………….18 iii 1.1.7 1.1.6.3 Replication and Transcription………………………………………………19 1.1.6.4 Translation………………………………………………………………… 20 1.1.6.5 Virion assembly and Release……………………………………………… 22 Reverse Genetics and Genetic Manipulation of coronavirus……………………………23 1.2 VIRUS-HOST INTERACTIONS (I) 1.2.1 1.2.2 Host factors in coronavirus replication…………………………………………… … 24 1.2.1.1 Heterogenous nuclear ribonucleoprotein A1 (hnRNPA1)…………….….….25 1.2.1.2 Polypyrimidine-tract binding (PTB)……………………………………… 26 1.2.1.3 Poly (A) binding protein (PABP)………………………….…………….… .27 1.2.1.4 Mitochondria aconitase…………………………………………………… 28 1.2.1.5 DEAD box helicases……………………………………….……………… .29 1.2.1.6 Other cellular proteins…………………………………………………….….30 Cellular processes in coronavirus infection………………………………………….… 33 1.2.2.1 The role of cell cycle regulation in coronavirus replication…………………34 1.2.2.2 Ubiquitin-proteasome system and coronavirus infection……………………35 1.2.2.3 Autophagy, ERAD and early secretory pathway in DMV biogenesis……….36 1.2.2.4 Apoptosis and coronavirus infection……………………………………… 39 1.3 HOST-VIRAL INTERACTIONS (II): CORONAVIRUS AND THE INNATE IMMUNE RESPONSE 1.3.1 Interferons………………………………………………………………………….……40 iv 1.3.2 Pattern recognition receptors (PRRs)………………………………………….……… .43 1.3.3 The RIG-I-Like Helicase signaling pathway…………………………………….…… .46 1.3.4 Modulation of the innate immune pathways by coronaviruses………………………….48 1.4 OBJECTIVES 1.4 Objectives…… …………………………………………………………………………50 CHAPTER TWO: MATERIALS AND METHODS 2.1 MATERIALS 2.1.1 General reagents and chemicals……………………………………………….….…53 2.1.2 Enzymes…………………………………………………………………….………54 2.1.3 Antibodies………………………………………………………………………… 54 2.2 CELLS & VIRUSES 2.2.1 Cell culture………………………………………………………………………….55 2.2.2 Preparation of cell stock ……………………………………………………………55 2.2.3 Viruses…………………………………………………………………………… 57 2.2.4 Virus infection………………………………………………………………………58 2.2.5 Virus titration……………………………………………………………………….59 2.3 MOLECULAR CLONING 2.3.1 Preparation of competent cells………………………………………………………60 2.3.2 Polymerase chain reaction………………………………………………………… 60 2.3.3 DNA Agarose gel electrophoresis………………………………………………… 62 v 2.3.4 Gel purification…………………………………………………………………….62 2.3.5 Recombinant DNA technique – construction of plasmids…………………………63 2.3.6 Plasmid purification……………………………………………………………… 64 2.3.7 DNA sequencing……………………………………………………………….… 64 2.3.8 Plasmids……………………………………………………………………………65 2.4 RNA MANIPULATION 2.4.1 Extraction of total RNA from mammalian cells…………………………………….66 2.4.2 Reverse transcription……………………………………………………………… 67 2.4.3 Quantitative real-time PCR (qPCR)……………………………………………… .68 2.4.4 RNA interference (RNAI)………………………………………………………… 69 2.5 GENOME WIDE RNAI SCREEN 2.5.1 Screen setup…………………………………………………………………………70 2.5.2 Data formatting, normalization and screen quality control…………………………70 2.5.3 Z score calculation………………………………………………………………… 71 2.5.4 Deconvoluted screen……………………………………………………………… 72 2.5.5 Bioinformatics Analysis: Gene annotation and protein networks………………….72 2.6 PROTEIN EXPRESSION AND ANALYSIS 2.6.1 Transient expression of plasmid DNA in mammalian cells……………………….73 2.6.2 SDS-PAGE……………………………………………………………………… 74 2.6.3 Western Blot Analysis…………………………………………………………… 74 2.6.4 Native-PAGE…………………………………………………………………… .74 2.6.5 Co-immunoprecipitation………………………………………………………… .75 vi 2.7 LUCIFERASE ASSAYS 2.7.1 IFN-β reporter assay……………………………………………………………… 76 2.7.2 Luciferase assay with IBV-Luc………………………………… .…………….….77 2.8 IMMUNOFLUORESCENCE…………………………………………………………….77 2.9 SUBCELLULAR FRACTIONATION……………………………………………….… 78 CHAPTER THREE: GENOME WIDE RNAI SCREEN FOR CELLULAR FACTORS IN CORONAVIRUS INFECTION 3.1 INTRODUCTION…………………………………………………………………………80 3.2 RESULTS (I): GENOME WIDE RNAI SCREEN REVEAL CELLULAR FACTORS INVOLVED IN CORONAVIRUS INFECTION 3.2.1 Optimization of genome wide RNAi screen………………………………… ……82 3.2.2 86 cellular cofactors of coronavirus validated by at least two independent RNA……………………………………………………………………………… 87 3.2.3 Bioinformatics analysis of screen hits………… ………………………………….91 3.2.4 Comparison of screen with SARS interactome………….…………………………93 3.2.5 Screen recovered genes associated with cellular processes/molecular functions known to modulate coronavirus infection……….…………………………………96 3.2.6 Ubiquitin-proteasome pathway and ER and associated degradation in coronavirus replication…………………………………………………………………………103 vii 3.3 RESULTS (II): CHARACTERIZATION OF THE ROLE OF VCP IN IBV REPLICATION 3.3.1 VCP is involved in the early stages of virus replication………………………….106 3.3.2 VCP is not required for viral attachment to cell surface and virus entry…………108 3.3.3 Silencing of VCP inhibits disassembly of virus particles…………………………110 3.3.4 Silencing of VCP results in accumulation of virus in early endosomal fractions .112 3.3.5 N protein degradation as an assay to detect genes involved in early replication….114 3.4 DISCUSSION CHAPTER FOUR: HOST-VIRUS INTERACTION (II): CHARACTERISATION OF HOST ANTIVIRAL MECHANISMS AGAINST CORONAVIRUS INFECTION AND CORONAVIRAL STRATEGIES OF IMMUNE EVASION 4.1 CHARACTERIZATION OF HOST ANTIVIRAL MECHANISMS AGAINST CORONAVIRUS INFECTION 4.1.1 INTRODUCTION………….……………………… .………………………….122 4.1.2 RESULTS .……………………………… ………………………………… .…124 4.1.2.1 IBV is sensitive to IFN activation………………………………… …………124 4.1.2.2 IBV infection weakly induce IFN and IFN-stimulated genes…………………125 4.1.2.3 IBV infection is associated with inefficient IRF3 activation………………….126 viii 4.1.2.4 Lack of IFN induction in IBV-infected cells was not due to defective inherent IFN signaling in host cells……………………………………………………128 4.1.2.5 Overexpression of cytoplasmic dsRNA receptors RIG-I, Mda5 and TLR3 failed to suppress viral infection……………………………………………….……132 4.1.2.6 IBV infection was not modulated by over-expression of TLR3…………… .136 4.1.2.7 IBV replication up-regulates low levels of RIG-I, Mda5…………………….138 4.1.2.8 IBV replication partially inhibits IFN response at late stages of infection… .139 4.1.2.9 Expression of IFN and ISGs during IBV infection is cell-type dependent… 141 4.1.2.10 IBV infection did not lead to establishment of an effective anti-viral state in infected cells where IFNs are transcriptionally activated…………………145 4.1.3 DISCUSSION……………………………………………………………………147 4.2 SARS ORF8B AND ORF8AB ARE NOVEL INTERFERON ANTAGONISTS 4.2.1 INTRODUCTION………………………………………………………………151 4.2.2 RESULTS……………………………………………………………………… 153 4.2.2.1 SARS protein 8b and 8ab interacts physically with IRF3…………………….153 4.2.2.2 The DNA binding domain of IRF3 is dispensable for its interaction with protein 8b……………………………………………… …………………………….155 4.2.2.3 Suppression of IRF3 activation by protein 8b and 8ab……………………… 157 4.2.2.4 Expression of SARS8b and 8ab decreased IFN-β promoter activation in response to poly (I:C) and VISA stimulation……………………………………………158 ix 20. 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RNA interference screen, we identified critical cellular cofactors that have roles in modulating coronavirus replication In the second part of the thesis, we focused on understanding the interplay between coronavirus infection and host cell innate immune response Coronaviral strategies of immune evasion were also examined (151 words) CHAPTER ONE: LITERATURE REVIEW OF BIOLOGY OF CORONAVIRUS 1 1.1: CORONAVIRUS: ... between coronavirus infection and host cell innate immune response Through examining the status of interferon (IFN) activation in different cell lines susceptible to IBV infection, it was observed that IBV counteracts the effective activation of the host anti -viral mechanisms via distinct strategies that xii include passive evasion of immune detection, as well as active inhibition of events leading to... cellular host factors identified, the role of valosin containing protein (VCP) in the modulation of coronavirus replication was examined in greater detail From our study, it was found that VCP is required for the efficient transfer of viral particles from early endosomal compartments to the host cytosol where viral replication occur In the second part of the thesis, we focused on understanding the interplay... CORONAVIRUS INFECTION 5.1.1 Main conclusions……………………………………………………………………….170 5.1.2 General discussions…………………………………………………………………….171 5.1.2.1 The use of RNAi screen to identify cellular cofactors involved in coronavirus replication ……,,…………………………………………………………… 171 5.1.2.2 The role of VCP in virus and host endosomal trafficking………………….…172 5.1.2.3 Involvement of ERAD and UPS players in coronavirus replication and. .. of coronaviruses Alphacoronaviruses and betacoronaviruses comprise of diverse coronavirus species infecting a wide range of mammalian hosts (pigs, cattle, cats, dogs, bats) including human HCoV-229E and HCoV-NL63 are alphacoronaviruses while HCoV-HKU9, 2 HCoV-OC43 and severe respiratory syndrome coronavirus (SARS-CoV) belongs to the latter In contrast, the gammacoronaviruses group is made up of mainly... prototypic IBV as our model coronavirus, we explored two key aspects of virus -host interactions in this dissertation Firstly, through a genome-wide RNA interference screen, we identified critical cellular cofactors that have roles in modulating coronavirus replication While some of these factors are already known to be implicated in coronavirus replication, many others are novel cofactors that have yet to... Taxonomy Coronaviruses are species of enveloped RNA virus belonging to the subfamily of Coronavirinae in the family of Coronaviridae Together with the Arteviridae and Roniviridae family of viruses, they are classified in the order of Nidovirales [1] The word ‘nidus’, meaning ‘nest’ in Latin is in reference to the production of 3’ nested set of subgenomic mRNAs by these families of viruses during transcription... virus and its host is fundamental to the determination of virulence An improved understanding of these events will not only allows us to gain new insights into the complicated field of coronavirus biology, but may also provide new directions for therapeutic interventions Therefore, using primarily the prototypic IBV as our model coronavirus, we aspire to explore two aspects of virus host interactions in. .. carboxy (S2) domains, which correspond to the receptor binding and transmembrane domain respectively By harbouring the receptor-binding site, divergence in the S1 region is a major determinant of host specificity and cell tropism [21] The S2 domain on the other hand is more conserved across species and mediates viral and cellular membrane fusion via an internal peptide fusion sequence For some coronaviruses,... envelope, M protein is the most abundantly expressed protein in coronaviruses A triple membrane spanning protein accompanied by a short amino ectodomain and a large carboxy cytosolic domain, M protein is pivotal to virion morphogenesis and assembly by virtue of the extensive interactions made between itself and other structural components: Homotypic oligomerisation of M protein results in a protein lattice . HOST- VIRAL INTERACTIONS: HOST FACTORS IN CORONAVIRUS REPLICATION AND CORONAVIRAL STRATEGUES OF IMMUNE EVASION WONG HUI HUI (B. SC genes involved in early replication .114 3.4 DISCUSSION CHAPTER FOUR: HOST- VIRUS INTERACTION (II): CHARACTERISATION OF HOST ANTIVIRAL MECHANISMS AGAINST CORONAVIRUS INFECTION AND CORONAVIRAL. 174 xi 5.2 CHARACTERIZATION OF HOST INNATE RESPONSE TOWARDS IBV INFECTION AND CORONAVIRAL COUNTERSTRATEGIES OF IMMUNE EVASION FINAL REMARKS 5.2.1 Main conclusions……………………………………………………………………