Tài liệu hạn chế xem trước, để xem đầy đủ mời bạn chọn Tải xuống
1
/ 220 trang
THÔNG TIN TÀI LIỆU
Thông tin cơ bản
Định dạng
Số trang
220
Dung lượng
5,09 MB
Nội dung
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. Du L, Kao RY, Zhou Y, He Y, Zhao G, et al. (2007) Cleavage of spike protein of SARS coronavirus by protease factor Xa is associated with viral infectivity. Biochem Biophys Res Commun 359: 174-179. 21. Perlman S, Netland J (2009) Coronaviruses post-SARS: update on replication and pathogenesis. Nat Rev Microbiol 7: 439-450. 22. Yamada Y, Liu DX (2009) Proteolytic activation of the spike protein at a novel RRRR/S motif is implicated in furin-dependent entry, syncytium formation, and infectivity of coronavirus infectious bronchitis virus in cultured cells. J Virol 83: 8744-8758. 23. Follis KE, York J, Nunberg JH (2006) Furin cleavage of the SARS coronavirus spike glycoprotein enhances cell-cell fusion but does not affect virion entry. Virology 350: 358-369. 24. Smits SL, Gerwig GJ, van Vliet AL, Lissenberg A, Briza P, et al. (2005) Nidovirus sialate-Oacetylesterases: evolution and substrate specificity of coronaviral and toroviral receptor-destroying enzymes. J Biol Chem 280: 6933-6941. 25. Vlasak R, Luytjes W, Leider J, Spaan W, Palese P (1988) The E3 protein of bovine coronavirus is a receptor-destroying enzyme with acetylesterase activity. J Virol 62: 4686-4690. 26. Gagneten S, Gout O, Dubois-Dalcq M, Rottier P, Rossen J, et al. (1995) Interaction of mouse hepatitis virus (MHV) spike glycoprotein with receptor glycoprotein MHVR is required for infection with an MHV strain that expresses the hemagglutininesterase glycoprotein. J Virol 69: 889-895. 27. Popova R, Zhang X (2002) The spike but not the hemagglutinin/esterase protein of bovine coronavirus is necessary and sufficient for viral infection. Virology 294: 222236. 28. Zuniga S, Sola I, Moreno JL, Sabella P, Plana-Duran J, et al. (2007) Coronavirus nucleocapsid protein is an RNA chaperone. Virology 357: 215-227. 29. Cologna R, Spagnolo JF, Hogue BG (2000) Identification of nucleocapsid binding sites within coronavirus-defective genomes. Virology 277: 235-249. 30. Molenkamp R, Spaan WJ (1997) Identification of a specific interaction between the coronavirus mouse hepatitis virus A59 nucleocapsid protein and packaging signal. Virology 239: 78-86. 31. Nelson GW, Stohlman SA, Tahara SM (2000) High affinity interaction between nucleocapsid protein and leader/intergenic sequence of mouse hepatitis virus RNA. J Gen Virol 81: 181-188. 32. Huang Q, Yu L, Petros AM, Gunasekera A, Liu Z, et al. (2004) Structure of the N-terminal RNA-binding domain of the SARS CoV nucleocapsid protein. Biochemistry 43: 60596063. 33. Zhou M, Collisson EW (2000) The amino and carboxyl domains of the infectious bronchitis virus nucleocapsid protein interact with 3' genomic RNA. Virus Res 67: 31-39. 34. Masters PS (1992) Localization of an RNA-binding domain in the nucleocapsid protein of the coronavirus mouse hepatitis virus. Arch Virol 125: 141-160. 35. Masters PS (2006) The molecular biology of coronaviruses. Adv Virus Res 66: 193-292. 36. Parker MM, Masters PS (1990) Sequence comparison of the N genes of five strains of the coronavirus mouse hepatitis virus suggests a three domain structure for the nucleocapsid protein. Virology 179: 463-468. 184 37. Verma S, Bednar V, Blount A, Hogue BG (2006) Identification of functionally important negatively charged residues in the carboxy end of mouse hepatitis coronavirus A59 nucleocapsid protein. J Virol 80: 4344-4355. 38. Almazan F, Galan C, Enjuanes L (2004) The nucleoprotein is required for efficient coronavirus genome replication. J Virol 78: 12683-12688. 39. Zuniga S, Cruz JL, Sola I, Mateos-Gomez PA, Palacio L, et al. (2010) Coronavirus nucleocapsid protein facilitates template switching and is required for efficient transcription. J Virol 84: 2169-2175. 40. Baric RS, Nelson GW, Fleming JO, Deans RJ, Keck JG, et al. (1988) Interactions between coronavirus nucleocapsid protein and viral RNAs: implications for viral transcription. J Virol 62: 4280-4287. 41. Tahara SM, Dietlin TA, Bergmann CC, Nelson GW, Kyuwa S, et al. (1994) Coronavirus translational regulation: leader affects mRNA efficiency. Virology 202: 621-630. 42. Lu X, Pan J, Tao J, Guo D (2011) SARS-CoV nucleocapsid protein antagonizes IFN-beta response by targeting initial step of IFN-beta induction pathway, and its C-terminal region is critical for the antagonism. Virus Genes 42: 37-45. 43. Ye Y, Hauns K, Langland JO, Jacobs BL, Hogue BG (2007) Mouse hepatitis coronavirus A59 nucleocapsid protein is a type I interferon antagonist. J Virol 81: 2554-2563. 44. Wang J, Fang S, Xiao H, Chen B, Tam JP, et al. (2009) Interaction of the coronavirus infectious bronchitis virus membrane protein with beta-actin and its implication in virion assembly and budding. PLoS One 4: e4908. 45. Siu KL, Kok KH, Ng MH, Poon VK, Yuen KY, et al. (2009) Severe acute respiratory syndrome coronavirus M protein inhibits type I interferon production by impeding the formation of TRAF3.TANK.TBK1/IKKepsilon complex. J Biol Chem 284: 1620216209. 46. Ruch TR, Machamer CE (2012) The coronavirus e protein: assembly and beyond. Viruses 4: 363-382. 47. Liu DX, Yuan Q, Liao Y (2007) Coronavirus envelope protein: a small membrane protein with multiple functions. Cell Mol Life Sci 64: 2043-2048. 48. Madan V, Garcia Mde J, Sanz MA, Carrasco L (2005) Viroporin activity of murine hepatitis virus E protein. FEBS Lett 579: 3607-3612. 49. Liao Y, Lescar J, Tam JP, Liu DX (2004) Expression of SARS-coronavirus envelope protein in Escherichia coli cells alters membrane permeability. Biochem Biophys Res Commun 325: 374-380. 50. DeDiego ML, Nieto-Torres JL, Jimenez-Guardeno JM, Regla-Nava JA, Alvarez E, et al. (2011) Severe acute respiratory syndrome coronavirus envelope protein regulates cell stress response and apoptosis. PLoS Pathog 7: e1002315. 51. Ziebuhr J, Snijder EJ, Gorbalenya AE (2000) Virus-encoded proteinases and proteolytic processing in the Nidovirales. J Gen Virol 81: 853-879. 52. Snijder EJ, Bredenbeek PJ, Dobbe JC, Thiel V, Ziebuhr J, et al. (2003) Unique and conserved features of genome and proteome of SARS-coronavirus, an early split-off from the coronavirus group lineage. J Mol Biol 331: 991-1004. 53. Mazumder R, Iyer LM, Vasudevan S, Aravind L (2002) Detection of novel members, structure-function analysis and evolutionary classification of the 2H phosphoesterase superfamily. Nucleic Acids Res 30: 5229-5243. 54. Luytjes W, Bredenbeek PJ, Noten AF, Horzinek MC, Spaan WJ (1988) Sequence of mouse hepatitis virus A59 mRNA 2: indications for RNA recombination between coronaviruses and influenza C virus. Virology 166: 415-422. 185 55. Inberg A, Linial M (2004) Evolutional insights on uncharacterized SARS coronavirus genes. FEBS Lett 577: 159-164. 56. Sapats SI, Ashton F, Wright PJ, Ignjatovic J (1996) Novel variation in the N protein of avian infectious bronchitis virus. Virology 226: 412-417. 57. Yount B, Roberts RS, Sims AC, Deming D, Frieman MB, et al. (2005) Severe acute respiratory syndrome coronavirus group-specific open reading frames encode nonessential functions for replication in cell cultures and mice. J Virol 79: 1490914922. 58. de Haan CA, Masters PS, Shen X, Weiss S, Rottier PJ (2002) The group-specific murine coronavirus genes are not essential, but their deletion, by reverse genetics, is attenuating in the natural host. Virology 296: 177-189. 59. Youn S, Leibowitz JL, Collisson EW (2005) In vitro assembled, recombinant infectious bronchitis viruses demonstrate that the 5a open reading frame is not essential for replication. Virology 332: 206-215. 60. Haijema BJ, Volders H, Rottier PJ (2004) Live, attenuated coronavirus vaccines through the directed deletion of group-specific genes provide protection against feline infectious peritonitis. J Virol 78: 3863-3871. 61. Tan YJ, Lim SG, Hong W (2006) Understanding the accessory viral proteins unique to the severe acute respiratory syndrome (SARS) coronavirus. Antiviral Res 72: 78-88. 62. Narayanan K, Huang C, Makino S (2008) SARS coronavirus Accessory Proteins. Virus Res 133: 113-121. 63. Kopecky-Bromberg SA, Martinez-Sobrido L, Frieman M, Baric RA, Palese P (2007) Severe acute respiratory syndrome coronavirus open reading frame (ORF) 3b, ORF 6, and nucleocapsid proteins function as interferon antagonists. J Virol 81: 548-557. 64. Ito N, Mossel EC, Narayanan K, Popov VL, Huang C, et al. (2005) Severe acute respiratory syndrome coronavirus 3a protein is a viral structural protein. J Virol 79: 3182-3186. 65. Huang C, Ito N, Tseng CT, Makino S (2006) Severe acute respiratory syndrome coronavirus 7a accessory protein is a viral structural protein. J Virol 80: 7287-7294. 66. Schaecher SR, Mackenzie JM, Pekosz A (2007) The ORF7b protein of severe acute respiratory syndrome coronavirus (SARS-CoV) is expressed in virus-infected cells and incorporated into SARS-CoV particles. J Virol 81: 718-731. 67. Xu K, Zheng BJ, Zeng R, Lu W, Lin YP, et al. (2009) Severe acute respiratory syndrome coronavirus accessory protein 9b is a virion-associated protein. Virology 388: 279285. 68. Guan Y, Zheng BJ, He YQ, Liu XL, Zhuang ZX, et al. (2003) Isolation and characterization of viruses related to the SARS coronavirus from animals in southern China. Science 302: 276-278. 69. Li W, Moore MJ, Vasilieva N, Sui J, Wong SK, et al. (2003) Angiotensin-converting enzyme is a functional receptor for the SARS coronavirus. Nature 426: 450-454. 70. Hofmann H, Pyrc K, van der Hoek L, Geier M, Berkhout B, et al. (2005) Human coronavirus NL63 employs the severe acute respiratory syndrome coronavirus receptor for cellular entry. Proc Natl Acad Sci U S A 102: 7988-7993. 71. Schultze B, Gross HJ, Brossmer R, Herrler G (1991) The S protein of bovine coronavirus is a hemagglutinin recognizing 9-O-acetylated sialic acid as a receptor determinant. J Virol 65: 6232-6237. 72. Kunkel F, Herrler G (1993) Structural and functional analysis of the surface protein of human coronavirus OC43. Virology 195: 195-202. 186 73. Williams RK, Jiang GS, Holmes KV (1991) Receptor for mouse hepatitis virus is a member of the carcinoembryonic antigen family of glycoproteins. Proc Natl Acad Sci U S A 88: 5533-5536. 74. Belouzard S, Millet JK, Licitra BN, Whittaker GR (2012) Mechanisms of coronavirus cell entry mediated by the viral spike protein. Viruses 4: 1011-1033. 75. Asanaka M, Lai MM (1993) Cell fusion studies identified multiple cellular factors involved in mouse hepatitis virus entry. Virology 197: 732-741. 76. Mohandas DV, Dales S (1991) Endosomal association of a protein phosphatase with high dephosphorylating activity against a coronavirus nucleocapsid protein. FEBS Lett 282: 419-424. 77. Brockway SM, Clay CT, Lu XT, Denison MR (2003) Characterization of the expression, intracellular localization, and replication complex association of the putative mouse hepatitis virus RNA-dependent RNA polymerase. J Virol 77: 10515-10527. 78. Snijder EJ, van der Meer Y, Zevenhoven-Dobbe J, Onderwater JJ, van der Meulen J, et al. (2006) Ultrastructure and origin of membrane vesicles associated with the severe acute respiratory syndrome coronavirus replication complex. J Virol 80: 5927-5940. 79. Gosert R, Kanjanahaluethai A, Egger D, Bienz K, Baker SC (2002) RNA replication of mouse hepatitis virus takes place at double-membrane vesicles. J Virol 76: 36973708. 80. Oostra M, Hagemeijer MC, van Gent M, Bekker CP, te Lintelo EG, et al. (2008) Topology and membrane anchoring of the coronavirus replication complex: not all hydrophobic domains of nsp3 and nsp6 are membrane spanning. J Virol 82: 1239212405. 81. Enjuanes L, Almazan F, Sola I, Zuniga S (2006) Biochemical aspects of coronavirus replication and virus-host interaction. Annu Rev Microbiol 60: 211-230. 82. Jendrach M, Thiel V, Siddell S (1999) Characterization of an internal ribosome entry site within mRNA of murine hepatitis virus. Arch Virol 144: 921-933. 83. Klumperman J, Locker JK, Meijer A, Horzinek MC, Geuze HJ, et al. (1994) Coronavirus M proteins accumulate in the Golgi complex beyond the site of virion budding. J Virol 68: 6523-6534. 84. Lim KP, Liu DX (2001) The missing link in coronavirus assembly. Retention of the avian coronavirus infectious bronchitis virus envelope protein in the pre-Golgi compartments and physical interaction between the envelope and membrane proteins. J Biol Chem 276: 17515-17523. 85. Corse E, Machamer CE (2003) The cytoplasmic tails of infectious bronchitis virus E and M proteins mediate their interaction. Virology 312: 25-34. 86. Raamsman MJ, Locker JK, de Hooge A, de Vries AA, Griffiths G, et al. (2000) Characterization of the coronavirus mouse hepatitis virus strain A59 small membrane protein E. J Virol 74: 2333-2342. 87. Ye R, Montalto-Morrison C, Masters PS (2004) Genetic analysis of determinants for spike glycoprotein assembly into murine coronavirus virions: distinct roles for charge-rich and cysteine-rich regions of the endodomain. J Virol 78: 9904-9917. 88. Kuo L, Masters PS (2002) Genetic evidence for a structural interaction between the carboxy termini of the membrane and nucleocapsid proteins of mouse hepatitis virus. J Virol 76: 4987-4999. 89. Hurst KR, Kuo L, Koetzner CA, Ye R, Hsue B, et al. (2005) A major determinant for membrane protein interaction localizes to the carboxy-terminal domain of the mouse coronavirus nucleocapsid protein. J Virol 79: 13285-13297. 187 90. Luo H, Wu D, Shen C, Chen K, Shen X, et al. (2006) Severe acute respiratory syndrome coronavirus membrane protein interacts with nucleocapsid protein mostly through their carboxyl termini by electrostatic attraction. Int J Biochem Cell Biol 38: 589599. 91. Narayanan K, Maeda A, Maeda J, Makino S (2000) Characterization of the coronavirus M protein and nucleocapsid interaction in infected cells. J Virol 74: 8127-8134. 92. Narayanan K, Makino S (2001) Cooperation of an RNA packaging signal and a viral envelope protein in coronavirus RNA packaging. J Virol 75: 9059-9067. 93. Shen H, Fang SG, Chen B, Chen G, Tay FP, et al. (2009) Towards construction of viral vectors based on avian coronavirus infectious bronchitis virus for gene delivery and vaccine development. J Virol Methods 160: 48-56. 94. Dreyfuss G, Matunis MJ, Pinol-Roma S, Burd CG (1993) hnRNP proteins and the biogenesis of mRNA. Annu Rev Biochem 62: 289-321. 95. Hamilton BJ, Burns CM, Nichols RC, Rigby WF (1997) Modulation of AUUUA response element binding by heterogeneous nuclear ribonucleoprotein A1 in human T lymphocytes. The roles of cytoplasmic location, transcription, and phosphorylation. J Biol Chem 272: 28732-28741. 96. Hamilton BJ, Nagy E, Malter JS, Arrick BA, Rigby WF (1993) Association of heterogeneous nuclear ribonucleoprotein A1 and C proteins with reiterated AUUUA sequences. J Biol Chem 268: 8881-8887. 97. Li HP, Zhang X, Duncan R, Comai L, Lai MM (1997) Heterogeneous nuclear ribonucleoprotein A1 binds to the transcription-regulatory region of mouse hepatitis virus RNA. Proc Natl Acad Sci U S A 94: 9544-9549. 98. Furuya T, Lai MM (1993) Three different cellular proteins bind to complementary sites on the 5'-end-positive and 3'-end-negative strands of mouse hepatitis virus RNA. J Virol 67: 7215-7222. 99. Huang P, Lai MM (2001) Heterogeneous nuclear ribonucleoprotein a1 binds to the 3'untranslated region and mediates potential 5'-3'-end cross talks of mouse hepatitis virus RNA. J Virol 75: 5009-5017. 100. Zhang X, Li HP, Xue W, Lai MM (1998) Cellular protein hnRNP-A1 interacts with the 3'end and the intergenic sequence of mouse hepatitis virus negative-strand RNA to form a ribonucleoprotein complex. Adv Exp Med Biol 440: 227-234. 101. Zhang X, Lai MM (1995) Interactions between the cytoplasmic proteins and the intergenic (promoter) sequence of mouse hepatitis virus RNA: correlation with the amounts of subgenomic mRNA transcribed. J Virol 69: 1637-1644. 102. Shi ST, Huang P, Li HP, Lai MM (2000) Heterogeneous nuclear ribonucleoprotein A1 regulates RNA synthesis of a cytoplasmic virus. EMBO J 19: 4701-4711. 103. Zhang X, Li HP, Xue W, Lai MM (1999) Formation of a ribonucleoprotein complex of mouse hepatitis virus involving heterogeneous nuclear ribonucleoprotein A1 and transcription-regulatory elements of viral RNA. Virology 264: 115-124. 104. Wang Y, Zhang X (1999) The nucleocapsid protein of coronavirus mouse hepatitis virus interacts with the cellular heterogeneous nuclear ribonucleoprotein A1 in vitro and in vivo. Virology 265: 96-109. 105. Luo H, Chen Q, Chen J, Chen K, Shen X, et al. (2005) The nucleocapsid protein of SARS coronavirus has a high binding affinity to the human cellular heterogeneous nuclear ribonucleoprotein A1. FEBS Lett 579: 2623-2628. 188 106. Shi ST, Yu GY, Lai MM (2003) Multiple type A/B heterogeneous nuclear ribonucleoproteins (hnRNPs) can replace hnRNP A1 in mouse hepatitis virus RNA synthesis. J Virol 77: 10584-10593. 107. Li HP, Huang P, Park S, Lai MM (1999) Polypyrimidine tract-binding protein binds to the leader RNA of mouse hepatitis virus and serves as a regulator of viral transcription. J Virol 73: 772-777. 108. Huang P, Lai MM (1999) Polypyrimidine tract-binding protein binds to the complementary strand of the mouse hepatitis virus 3' untranslated region, thereby altering RNA conformation. J Virol 73: 9110-9116. 109. Sawicka K, Bushell M, Spriggs KA, Willis AE (2008) Polypyrimidine-tract-binding protein: a multifunctional RNA-binding protein. Biochem Soc Trans 36: 641-647. 110. Niepmann M (1996) Porcine polypyrimidine tract-binding protein stimulates translation initiation at the internal ribosome entry site of foot-and-mouth-disease virus. FEBS Lett 388: 39-42. 111. Kaminski A, Hunt SL, Patton JG, Jackson RJ (1995) Direct evidence that polypyrimidine tract binding protein (PTB) is essential for internal initiation of translation of encephalomyocarditis virus RNA. RNA 1: 924-938. 112. Ali N, Siddiqui A (1995) Interaction of polypyrimidine tract-binding protein with the 5' noncoding region of the hepatitis C virus RNA genome and its functional requirement in internal initiation of translation. J Virol 69: 6367-6375. 113. Spagnolo JF, Hogue BG (2000) Host protein interactions with the 3' end of bovine coronavirus RNA and the requirement of the poly(A) tail for coronavirus defective genome replication. J Virol 74: 5053-5065. 114. Lin YJ, Liao CL, Lai MM (1994) Identification of the cis-acting signal for minus-strand RNA synthesis of a murine coronavirus: implications for the role of minus-strand RNA in RNA replication and transcription. J Virol 68: 8131-8140. 115. Tarun SZ, Jr., Sachs AB (1996) Association of the yeast poly(A) tail binding protein with translation initiation factor eIF-4G. EMBO J 15: 7168-7177. 116. Sachs AB, Sarnow P, Hentze MW (1997) Starting at the beginning, middle, and end: translation initiation in eukaryotes. Cell 89: 831-838. 117. Nanda SK, Leibowitz JL (2001) Mitochondrial aconitase binds to the 3' untranslated region of the mouse hepatitis virus genome. J Virol 75: 3352-3362. 118. Kuhn LC, Hentze MW (1992) Coordination of cellular iron metabolism by posttranscriptional gene regulation. J Inorg Biochem 47: 183-195. 119. Hentze MW, Kuhn LC (1996) Molecular control of vertebrate iron metabolism: mRNAbased regulatory circuits operated by iron, nitric oxide, and oxidative stress. Proc Natl Acad Sci U S A 93: 8175-8182. 120. Nanda SK, Johnson RF, Liu Q, Leibowitz JL (2004) Mitochondrial HSP70, HSP40, and HSP60 bind to the 3' untranslated region of the Murine hepatitis virus genome. Arch Virol 149: 93-111. 121. Tanner NK, Linder P (2001) DExD/H box RNA helicases: from generic motors to specific dissociation functions. Mol Cell 8: 251-262. 122. Chen JY, Chen WN, Poon KM, Zheng BJ, Lin X, et al. (2009) Interaction between SARSCoV helicase and a multifunctional cellular protein (Ddx5) revealed by yeast and mammalian cell two-hybrid systems. Arch Virol 154: 507-512. 123. Xu L, Khadijah S, Fang S, Wang L, Tay FP, et al. (2010) The cellular RNA helicase DDX1 interacts with coronavirus nonstructural protein 14 and enhances viral replication. J Virol 84: 8571-8583. 189 124. Eckerle LD, Lu X, Sperry SM, Choi L, Denison MR (2007) High fidelity of murine hepatitis virus replication is decreased in nsp14 exoribonuclease mutants. J Virol 81: 1213512144. 125. Minskaia E, Hertzig T, Gorbalenya AE, Campanacci V, Cambillau C, et al. (2006) Discovery of an RNA virus 3'->5' exoribonuclease that is critically involved in coronavirus RNA synthesis. Proc Natl Acad Sci U S A 103: 5108-5113. 126. Eckerle LD, Becker MM, Halpin RA, Li K, Venter E, et al. (2010) Infidelity of SARS-CoV Nsp14-exonuclease mutant virus replication is revealed by complete genome sequencing. PLoS Pathog 6: e1000896. 127. Sun L, Xing Y, Chen X, Zheng Y, Yang Y, et al. (2012) Coronavirus papain-like proteases negatively regulate antiviral innate immune response through disruption of STINGmediated signaling. PLoS One 7: e30802. 128. Xiao H, Xu LH, Yamada Y, Liu DX (2008) Coronavirus spike protein inhibits host cell translation by interaction with eIF3f. PLoS One 3: e1494. 129. Bhardwaj K, Liu P, Leibowitz JL, Kao CC (2012) The coronavirus endoribonuclease Nsp15 interacts with retinoblastoma tumor suppressor protein. J Virol 86: 42944304. 130. Tan YW, Hong W, Liu DX (2012) Binding of the 5'-untranslated region of coronavirus RNA to zinc finger CCHC-type and RNA-binding motif enhances viral replication and transcription. Nucleic Acids Res 40: 5065-5077. 131. Yang Y, Xiong Z, Zhang S, Yan Y, Nguyen J, et al. (2005) Bcl-xL inhibits T-cell apoptosis induced by expression of SARS coronavirus E protein in the absence of growth factors. Biochem J 392: 135-143. 132. Teoh KT, Siu YL, Chan WL, Schluter MA, Liu CJ, et al. (2010) The SARS coronavirus E protein interacts with PALS1 and alters tight junction formation and epithelial morphogenesis. Mol Biol Cell 21: 3838-3852. 133. Fang X, Gao J, Zheng H, Li B, Kong L, et al. (2007) The membrane protein of SARS-CoV suppresses NF-kappaB activation. J Med Virol 79: 1431-1439. 134. Wei WY, Li HC, Chen CY, Yang CH, Lee SK, et al. (2012) SARS-CoV nucleocapsid protein interacts with cellular pyruvate kinase protein and inhibits its activity. Arch Virol 157: 635-645. 135. Zhang YP, Zhang RW, Chang WS, Wang YY (2010) Cxcl16 interact with SARS-CoV N protein in and out cell. Virol Sin 25: 369-374. 136. Wang Q, Li C, Zhang Q, Wang T, Li J, et al. (2010) Interactions of SARS coronavirus nucleocapsid protein with the host cell proteasome subunit p42. Virol J 7: 99. 137. Zhou B, Liu J, Wang Q, Liu X, Li X, et al. (2008) The nucleocapsid protein of severe acute respiratory syndrome coronavirus inhibits cell cytokinesis and proliferation by interacting with translation elongation factor 1alpha. J Virol 82: 6962-6971. 138. Li Q, Xiao H, Tam JP, Liu DX (2006) Sumoylation of the nucleocapsid protein of severe acute respiratory syndrome coronavirus by interaction with Ubc9. Adv Exp Med Biol 581: 121-126. 139. Chen Z, Mi L, Xu J, Yu J, Wang X, et al. (2005) Function of HAb18G/CD147 in invasion of host cells by severe acute respiratory syndrome coronavirus. J Infect Dis 191: 755760. 140. Surjit M, Liu B, Chow VT, Lal SK (2006) The nucleocapsid protein of severe acute respiratory syndrome-coronavirus inhibits the activity of cyclin-cyclin-dependent kinase complex and blocks S phase progression in mammalian cells. J Biol Chem 281: 10669-10681. 190 141. Zhao X, Nicholls JM, Chen YG (2008) Severe acute respiratory syndrome-associated coronavirus nucleocapsid protein interacts with Smad3 and modulates transforming growth factor-beta signaling. J Biol Chem 283: 3272-3280. 142. Li Q, Wang L, Dong C, Che Y, Jiang L, et al. (2005) The interaction of the SARS coronavirus non-structural protein 10 with the cellular oxido-reductase system causes an extensive cytopathic effect. J Clin Virol 34: 133-139. 143. Cornillez-Ty CT, Liao L, Yates JR, 3rd, Kuhn P, Buchmeier MJ (2009) Severe acute respiratory syndrome coronavirus nonstructural protein interacts with a host protein complex involved in mitochondrial biogenesis and intracellular signaling. J Virol 83: 10314-10318. 144. Devaraj SG, Wang N, Chen Z, Tseng M, Barretto N, et al. (2007) Regulation of IRF-3dependent innate immunity by the papain-like protease domain of the severe acute respiratory syndrome coronavirus. J Biol Chem 282: 32208-32221. 145. Lin CW, Tsai FJ, Wan L, Lai CC, Lin KH, et al. (2005) Binding interaction of SARS coronavirus 3CL(pro) protease with vacuolar-H+ ATPase G1 subunit. FEBS Lett 579: 6089-6094. 146. Vasilenko N, Moshynskyy I, Zakhartchouk A (2010) SARS coronavirus protein 7a interacts with human Ap4A-hydrolase. Virol J 7: 31. 147. Varshney B, Agnihothram S, Tan YJ, Baric R, Lal SK (2012) SARS coronavirus 3b accessory protein modulates transcriptional activity of RUNX1b. PLoS One 7: e29542. 148. Frieman M, Yount B, Heise M, Kopecky-Bromberg SA, Palese P, et al. (2007) Severe acute respiratory syndrome coronavirus ORF6 antagonizes STAT1 function by sequestering nuclear import factors on the rough endoplasmic reticulum/Golgi membrane. J Virol 81: 9812-9824. 149. Gramberg T, Hofmann H, Moller P, Lalor PF, Marzi A, et al. (2005) LSECtin interacts with filovirus glycoproteins and the spike protein of SARS coronavirus. Virology 340: 224-236. 150. Marzi A, Gramberg T, Simmons G, Moller P, Rennekamp AJ, et al. (2004) DC-SIGN and DC-SIGNR interact with the glycoprotein of Marburg virus and the S protein of severe acute respiratory syndrome coronavirus. J Virol 78: 12090-12095. 151. Padhan K, Tanwar C, Hussain A, Hui PY, Lee MY, et al. (2007) Severe acute respiratory syndrome coronavirus Orf3a protein interacts with caveolin. J Gen Virol 88: 30673077. 152. Fielding BC, Gunalan V, Tan TH, Chou CF, Shen S, et al. (2006) Severe acute respiratory syndrome coronavirus protein 7a interacts with hSGT. Biochem Biophys Res Commun 343: 1201-1208. 153. Tan YX, Tan TH, Lee MJ, Tham PY, Gunalan V, et al. (2007) Induction of apoptosis by the severe acute respiratory syndrome coronavirus 7a protein is dependent on its interaction with the Bcl-XL protein. J Virol 81: 6346-6355. 154. Pfefferle S, Schopf J, Kogl M, Friedel CC, Muller MA, et al. (2011) The SARS-coronavirushost interactome: identification of cyclophilins as target for pan-coronavirus inhibitors. PLoS Pathog 7: e1002331. 155. Hogan PG, Chen L, Nardone J, Rao A (2003) Transcriptional regulation by calcium, calcineurin, and NFAT. Genes Dev 17: 2205-2232. 156. Pines J (1999) Four-dimensional control of the cell cycle. Nat Cell Biol 1: E73-79. 157. Li FQ, Tam JP, Liu DX (2007) Cell cycle arrest and apoptosis induced by the coronavirus infectious bronchitis virus in the absence of p53. Virology 365: 435-445. 191 158. Dove B, Brooks G, Bicknell K, Wurm T, Hiscox JA (2006) Cell cycle perturbations induced by infection with the coronavirus infectious bronchitis virus and their effect on virus replication. J Virol 80: 4147-4156. 159. Zhou BB, Elledge SJ (2000) The DNA damage response: putting checkpoints in perspective. Nature 408: 433-439. 160. Xu LH, Huang M, Fang SG, Liu DX (2011) Coronavirus infection induces DNA replication stress partly through interaction of its nonstructural protein 13 with the p125 subunit of DNA polymerase delta. J Biol Chem 286: 39546-39559. 161. Yu GY, Lai MM (2005) The ubiquitin-proteasome system facilitates the transfer of murine coronavirus from endosome to cytoplasm during virus entry. J Virol 79: 644648. 162. Raaben M, Posthuma CC, Verheije MH, te Lintelo EG, Kikkert M, et al. (2010) The ubiquitin-proteasome system plays an important role during various stages of the coronavirus infection cycle. J Virol 84: 7869-7879. 163. Tanida I (2011) Autophagy basics. Microbiol Immunol 55: 1-11. 164. Prentice E, Jerome WG, Yoshimori T, Mizushima N, Denison MR (2004) Coronavirus replication complex formation utilizes components of cellular autophagy. J Biol Chem 279: 10136-10141. 165. Cottam EM, Maier HJ, Manifava M, Vaux LC, Chandra-Schoenfelder P, et al. (2011) Coronavirus nsp6 proteins generate autophagosomes from the endoplasmic reticulum via an omegasome intermediate. Autophagy 7: 1335-1347. 166. Reggiori F, Monastyrska I, Verheije MH, Cali T, Ulasli M, et al. (2010) Coronaviruses Hijack the LC3-I-positive EDEMosomes, ER-derived vesicles exporting short-lived ERAD regulators, for replication. Cell Host Microbe 7: 500-508. 167. Zhao Z, Thackray LB, Miller BC, Lynn TM, Becker MM, et al. (2007) Coronavirus replication does not require the autophagy gene ATG5. Autophagy 3: 581-585. 168. de Haan CA, Reggiori F (2008) Are nidoviruses hijacking the autophagy machinery? Autophagy 4: 276-279. 169. Cali T, Galli C, Olivari S, Molinari M (2008) Segregation and rapid turnover of EDEM1 by an autophagy-like mechanism modulates standard ERAD and folding activities. Biochem Biophys Res Commun 371: 405-410. 170. Sato K, Nakano A (2007) Mechanisms of COPII vesicle formation and protein sorting. FEBS Lett 581: 2076-2082. 171. Barlowe C (2000) Traffic COPs of the early secretory pathway. Traffic 1: 371-377. 172. Budnik A, Stephens DJ (2009) ER exit sites--localization and control of COPII vesicle formation. FEBS Lett 583: 3796-3803. 173. Oostra M, te Lintelo EG, Deijs M, Verheije MH, Rottier PJ, et al. (2007) Localization and membrane topology of coronavirus nonstructural protein 4: involvement of the early secretory pathway in replication. J Virol 81: 12323-12336. 174. D'Souza-Schorey C, Chavrier P (2006) ARF proteins: roles in membrane traffic and beyond. Nat Rev Mol Cell Biol 7: 347-358. 175. Verheije MH, Raaben M, Mari M, Te Lintelo EG, Reggiori F, et al. (2008) Mouse hepatitis coronavirus RNA replication depends on GBF1-mediated ARF1 activation. PLoS Pathog 4: e1000088. 176. Knoops K, Swett-Tapia C, van den Worm SH, Te Velthuis AJ, Koster AJ, et al. (2010) Integrity of the early secretory pathway promotes, but is not required for, severe acute respiratory syndrome coronavirus RNA synthesis and virus-induced remodeling of endoplasmic reticulum membranes. J Virol 84: 833-846. 192 177. Barber GN (2001) Host defense, viruses and apoptosis. Cell Death Differ 8: 113-126. 178. Levine AJ (1997) p53, the cellular gatekeeper for growth and division. Cell 88: 323-331. 179. Kaminskyy V, Zhivotovsky B (2010) To kill or be killed: how viruses interact with the cell death machinery. J Intern Med 267: 473-482. 180. Danthi P (2011) Enter the kill zone: initiation of death signaling during virus entry. Virology 411: 316-324. 181. Lamkanfi M, Dixit VM (2010) Manipulation of host cell death pathways during microbial infections. Cell Host Microbe 8: 44-54. 182. Zhong Y, Liao Y, Fang S, Tam JP, Liu DX (2012) Up-regulation of Mcl-1 and Bak by coronavirus infection of human, avian and animal cells modulates apoptosis and viral replication. PLoS One 7: e30191. 183. Liu C, Xu HY, Liu DX (2001) Induction of caspase-dependent apoptosis in cultured cells by the avian coronavirus infectious bronchitis virus. J Virol 75: 6402-6409. 184. Padhan K, Minakshi R, Towheed MA, Jameel S (2008) Severe acute respiratory syndrome coronavirus 3a protein activates the mitochondrial death pathway through p38 MAP kinase activation. J Gen Virol 89: 1960-1969. 185. Ye Z, Wong CK, Li P, Xie Y (2008) A SARS-CoV protein, ORF-6, induces caspase-3 mediated, ER stress and JNK-dependent apoptosis. Biochim Biophys Acta 1780: 1383-1387. 186. Tan YJ, Fielding BC, Goh PY, Shen S, Tan TH, et al. (2004) Overexpression of 7a, a protein specifically encoded by the severe acute respiratory syndrome coronavirus, induces apoptosis via a caspase-dependent pathway. J Virol 78: 14043-14047. 187. Diemer C, Schneider M, Seebach J, Quaas J, Frosner G, et al. (2008) Cell type-specific cleavage of nucleocapsid protein by effector caspases during SARS coronavirus infection. J Mol Biol 376: 23-34. 188. Stetson DB, Medzhitov R (2006) Type I interferons in host defense. Immunity 25: 373381. 189. McBride KM, McDonald C, Reich NC (2000) Nuclear export signal located within theDNA-binding domain of the STAT1transcription factor. EMBO J 19: 6196-6206. 190. Imada K, Leonard WJ (2000) The Jak-STAT pathway. Mol Immunol 37: 1-11. 191. Schoggins JW, Rice CM (2011) Interferon-stimulated genes and their antiviral effector functions. Curr Opin Virol 1: 519-525. 192. de Veer MJ, Holko M, Frevel M, Walker E, Der S, et al. (2001) Functional classification of interferon-stimulated genes identified using microarrays. J Leukoc Biol 69: 912920. 193. Stark GR, Kerr IM, Williams BR, Silverman RH, Schreiber RD (1998) How cells respond to interferons. Annu Rev Biochem 67: 227-264. 194. Samuel S (2007) Interferons, Interferon Receptors, Signal Transducer and Transcriptional Activators and Interferon Regulatory Factors. J Biol Chem 282: 20045-20046. 195. Kumagai Y, Takeuchi O, Akira S (2008) Pathogen recognition by innate receptors. J Infect Chemother 14: 86-92. 196. Kumar H, Kawai T, Akira S (2009) Pathogen recognition in the innate immune response. Biochem J 420: 1-16. 197. Kumar H, Kawai T, Akira S (2009) Toll-like receptors and innate immunity. Biochem Biophys Res Commun 388: 621-625. 193 198. Yoneyama M, Kikuchi M, Matsumoto K, Imaizumi T, Miyagishi M, et al. (2005) Shared and unique functions of the DExD/H-box helicases RIG-I, MDA5, and LGP2 in antiviral innate immunity. J Immunol 175: 2851-2858. 199. Kato H, Takeuchi O, Sato S, Yoneyama M, Yamamoto M, et al. (2006) Differential roles of MDA5 and RIG-I helicases in the recognition of RNA viruses. Nature 441: 101105. 200. Loo YM, Fornek J, Crochet N, Bajwa G, Perwitasari O, et al. (2008) Distinct RIG-I and MDA5 signaling by RNA viruses in innate immunity. J Virol 82: 335-345. 201. Wilkins C, Gale M, Jr. (2010) Recognition of viruses by cytoplasmic sensors. Curr Opin Immunol 22: 41-47. 202. Kawai T, Takahashi K, Sato S, Coban C, Kumar H, et al. (2005) IPS-1, an adaptor triggering RIG-I- and Mda5-mediated type I interferon induction. Nat Immunol 6: 981-988. 203. Seth RB, Sun L, Ea CK, Chen ZJ (2005) Identification and characterization of MAVS, a mitochondrial antiviral signaling protein that activates NF-kappaB and IRF 3. Cell 122: 669-682. 204. Fitzgerald KA, McWhirter SM, Faia KL, Rowe DC, Latz E, et al. (2003) IKKepsilon and TBK1 are essential components of the IRF3 signaling pathway. Nat Immunol 4: 491496. 205. Takeuchi O, Akira S (2008) MDA5/RIG-I and virus recognition. Curr Opin Immunol 20: 17-22. 206. Minakshi R, Padhan K, Rani M, Khan N, Ahmad F, et al. (2009) The SARS Coronavirus 3a protein causes endoplasmic reticulum stress and induces ligand-independent downregulation of the type interferon receptor. PLoS One 4: e8342. 207. Cruz JL, Sola I, Becares M, Alberca B, Plana J, et al. (2011) Coronavirus gene counteracts host defenses and modulates virus virulence. PLoS Pathog 7: e1002090. 208. Zheng D, Chen G, Guo B, Cheng G, Tang H (2008) PLP2, a potent deubiquitinase from murine hepatitis virus, strongly inhibits cellular type I interferon production. Cell Res 18: 1105-1113. 209. Wang G, Chen G, Zheng D, Cheng G, Tang H (2011) PLP2 of mouse hepatitis virus A59 (MHV-A59) targets TBK1 to negatively regulate cellular type I interferon signaling pathway. PLoS One 6: e17192. 210. Zhong Y, Tan YW, Liu DX (2012) Recent progress in studies of arterivirus- and coronavirus-host interactions. Viruses 4: 980-1010. 211. Liu DX, Inglis SC (1991) Association of the infectious bronchitis virus 3c protein with the virion envelope. Virology 185: 911-917. 212. Le TM, Wong HH, Tay FP, Fang S, Keng CT, et al. (2007) Expression, post-translational modification and biochemical characterization of proteins encoded by subgenomic mRNA8 of the severe acute respiratory syndrome coronavirus. FEBS J 274: 42114222. 213. Shen S, Wen ZL, Liu DX (2003) Emergence of a coronavirus infectious bronchitis virus mutant with a truncated 3b gene: functional characterization of the 3b protein in pathogenesis and replication. Virology 311: 16-27. 214. Liu DX, Inglis SC (1992) Identification of two new polypeptides encoded by mRNA5 of the coronavirus infectious bronchitis virus. Virology 186: 342-347. 215. Shen S, Law YC, Liu DX (2004) A single amino acid mutation in the spike protein of coronavirus infectious bronchitis virus hampers its maturation and incorporation into virions at the nonpermissive temperature. Virology 326: 288-298. 194 216. Fang S, Chen B, Tay FP, Ng BS, Liu DX (2007) An arginine-to-proline mutation in a domain with undefined functions within the helicase protein (Nsp13) is lethal to the coronavirus infectious bronchitis virus in cultured cells. Virology 358: 136-147. 217. Tan YW, Fang S, Fan H, Lescar J, Liu DX (2006) Amino acid residues critical for RNAbinding in the N-terminal domain of the nucleocapsid protein are essential determinants for the infectivity of coronavirus in cultured cells. Nucleic Acids Res 34: 4816-4825. 218. Ashburner M, Ball CA, Blake JA, Botstein D, Butler H, et al. (2000) Gene ontology: tool for the unification of biology. The Gene Ontology Consortium. Nat Genet 25: 25-29. 219. Rebhan M, Chalifa-Caspi V, Prilusky J, Lancet D (1998) GeneCards: a novel functional genomics compendium with automated data mining and query reformulation support. Bioinformatics 14: 656-664. 220. Huang da W, Sherman BT, Lempicki RA (2009) Systematic and integrative analysis of large gene lists using DAVID bioinformatics resources. Nat Protoc 4: 44-57. 221. Jensen LJ, Kuhn M, Stark M, Chaffron S, Creevey C, et al. (2009) STRING 8--a global view on proteins and their functional interactions in 630 organisms. Nucleic Acids Res 37: D412-416. 222. Sawicki SG, Sawicki DL, Siddell SG (2007) A contemporary view of coronavirus transcription. J Virol 81: 20-29. 223. Weiss SR, Leibowitz JL (2011) Coronavirus pathogenesis. Adv Virus Res 81: 85-164. 224. Tan YW, Hong W, Liu DX (2012) Binding of the 5'-untranslated region of coronavirus RNA to zinc finger CCHC-type and RNA-binding motif enhances viral replication and transcription. Nucleic Acids Res. 225. Galan C, Sola I, Nogales A, Thomas B, Akoulitchev A, et al. (2009) Host cell proteins interacting with the 3' end of TGEV coronavirus genome influence virus replication. Virology 391: 304-314. 226. Sessions OM, Barrows NJ, Souza-Neto JA, Robinson TJ, Hershey CL, et al. (2009) Discovery of insect and human dengue virus host factors. Nature 458: 1047-1050. 227. Tai AW, Benita Y, Peng LF, Kim SS, Sakamoto N, et al. (2009) A functional genomic screen identifies cellular cofactors of hepatitis C virus replication. Cell Host Microbe 5: 298-307. 228. Krishnan MN, Ng A, Sukumaran B, Gilfoy FD, Uchil PD, et al. (2008) RNA interference screen for human genes associated with West Nile virus infection. Nature 455: 242245. 229. Bushman FD, Malani N, Fernandes J, D'Orso I, Cagney G, et al. (2009) Host cell factors in HIV replication: meta-analysis of genome-wide studies. PLoS Pathog 5: e1000437. 230. Nelson HB, Tang H (2006) Effect of cell growth on hepatitis C virus (HCV) replication and a mechanism of cell confluence-based inhibition of HCV RNA and protein expression. J Virol 80: 1181-1190. 231. Shi ST, Lai MM (2005) Viral and cellular proteins involved in coronavirus replication. Curr Top Microbiol Immunol 287: 95-131. 232. Sola I, Mateos-Gomez PA, Almazan F, Zuniga S, Enjuanes L (2011) RNA-RNA and RNAprotein interactions in coronavirus replication and transcription. RNA Biol 8: 237248. 233. He Y, Smith R (2009) Nuclear functions of heterogeneous nuclear ribonucleoproteins A/B. Cell Mol Life Sci 66: 1239-1256. 195 234. Heiner M, Hui J, Schreiner S, Hung LH, Bindereif A (2010) HnRNP L-mediated regulation of mammalian alternative splicing by interference with splice site recognition. RNA Biol 7: 56-64. 235. Hahm B, Kim YK, Kim JH, Kim TY, Jang SK (1998) Heterogeneous nuclear ribonucleoprotein L interacts with the 3' border of the internal ribosomal entry site of hepatitis C virus. J Virol 72: 8782-8788. 236. Hwang B, Lim JH, Hahm B, Jang SK, Lee SW (2009) hnRNP L is required for the translation mediated by HCV IRES. Biochem Biophys Res Commun 378: 584-588. 237. Zhao L, Jha BK, Wu A, Elliott R, Ziebuhr J, et al. (2012) Antagonism of the InterferonInduced OAS-RNase L Pathway by Murine Coronavirus ns2 Protein Is Required for Virus Replication and Liver Pathology. Cell Host Microbe 11: 607-616. 238. Banerjee S, An S, Zhou A, Silverman RH, Makino S (2000) RNase L-independent specific 28S rRNA cleavage in murine coronavirus-infected cells. J Virol 74: 8793-8802. 239. Zhang N, Kaur R, Akhter S, Legerski RJ (2009) Cdc5L interacts with ATR and is required for the S-phase cell-cycle checkpoint. EMBO Rep 10: 1029-1035. 240. Hsu NY, Ilnytska O, Belov G, Santiana M, Chen YH, et al. (2010) Viral reorganization of the secretory pathway generates distinct organelles for RNA replication. Cell 141: 799-811. 241. Peyroche A, Antonny B, Robineau S, Acker J, Cherfils J, et al. (1999) Brefeldin A acts to stabilize an abortive ARF-GDP-Sec7 domain protein complex: involvement of specific residues of the Sec7 domain. Mol Cell 3: 275-285. 242. Saenz JB, Sun WJ, Chang JW, Li J, Bursulaya B, et al. (2009) Golgicide A reveals essential roles for GBF1 in Golgi assembly and function. Nat Chem Biol 5: 157-165. 243. Shin HW, Nakayama K (2004) Guanine nucleotide-exchange factors for arf GTPases: their diverse functions in membrane traffic. J Biochem 136: 761-767. 244. Casanova JE (2007) Regulation of Arf activation: the Sec7 family of guanine nucleotide exchange factors. Traffic 8: 1476-1485. 245. Popoff V, Langer JD, Reckmann I, Hellwig A, Kahn RA, et al. (2011) Several ADPribosylation factor (Arf) isoforms support COPI vesicle formation. J Biol Chem 286: 35634-35642. 246. Kudelko M, Brault JB, Kwok K, Li MY, Pardigon N, et al. (2012) Class II ADP-ribosylation factors are required for efficient secretion of dengue viruses. J Biol Chem 287: 767777. 247. Wessels E, Duijsings D, Niu TK, Neumann S, Oorschot VM, et al. (2006) A viral protein that blocks Arf1-mediated COP-I assembly by inhibiting the guanine nucleotide exchange factor GBF1. Dev Cell 11: 191-201. 248. Lanke KH, van der Schaar HM, Belov GA, Feng Q, Duijsings D, et al. (2009) GBF1, a guanine nucleotide exchange factor for Arf, is crucial for coxsackievirus B3 RNA replication. J Virol 83: 11940-11949. 249. Wessels E, Duijsings D, Lanke KH, van Dooren SH, Jackson CL, et al. (2006) Effects of picornavirus 3A Proteins on Protein Transport and GBF1-dependent COP-I recruitment. J Virol 80: 11852-11860. 250. Goueslain L, Alsaleh K, Horellou P, Roingeard P, Descamps V, et al. (2010) Identification of GBF1 as a cellular factor required for hepatitis C virus RNA replication. J Virol 84: 773-787. 251. Pintard L, Willems A, Peter M (2004) Cullin-based ubiquitin ligases: Cul3-BTB complexes join the family. EMBO J 23: 1681-1687. 196 252. Plafker KS, Singer JD, Plafker SM (2009) The ubiquitin conjugating enzyme, UbcM2, engages in novel interactions with components of cullin-3 based E3 ligases. Biochemistry 48: 3527-3537. 253. Merlet J, Burger J, Gomes JE, Pintard L (2009) Regulation of cullin-RING E3 ubiquitinligases by neddylation and dimerization. Cell Mol Life Sci 66: 1924-1938. 254. Wolf DH, Stolz A (2012) The Cdc48 machine in endoplasmic reticulum associated protein degradation. Biochim Biophys Acta 1823: 117-124. 255. Ye Y, Meyer HH, Rapoport TA (2001) The AAA ATPase Cdc48/p97 and its partners transport proteins from the ER into the cytosol. Nature 414: 652-656. 256. Ye Y (2006) Diverse functions with a common regulator: ubiquitin takes command of an AAA ATPase. J Struct Biol 156: 29-40. 257. Tang TK, Wu MP, Chen ST, Hou MH, Hong MH, et al. (2005) Biochemical and immunological studies of nucleocapsid proteins of severe acute respiratory syndrome and 229E human coronaviruses. Proteomics 5: 925-937. 258. Wang Y, Wu X, Li B, Zhou H, Yuan G, et al. (2004) Low stability of nucleocapsid protein in SARS virus. Biochemistry 43: 11103-11108. 259. Fan H, Ooi A, Tan YW, Wang S, Fang S, et al. (2005) The nucleocapsid protein of coronavirus infectious bronchitis virus: crystal structure of its N-terminal domain and multimerization properties. Structure 13: 1859-1868. 260. Ramanathan HN, Ye Y (2012) The p97 ATPase associates with EEA1 to regulate the size of early endosomes. Cell Res 22: 346-359. 261. Lass A, McConnell E, Fleck K, Palamarchuk A, Wojcik C (2008) Analysis of Npl4 deletion mutants in mammalian cells unravels new Ufd1-interacting motifs and suggests a regulatory role of Npl4 in ERAD. Exp Cell Res 314: 2715-2723. 262. McConnell E, Lass A, Wojcik C (2007) Ufd1-Npl4 is a negative regulator of cholera toxin retrotranslocation. Biochem Biophys Res Commun 355: 1087-1090. 263. Nowis D, McConnell E, Wojcik C (2006) Destabilization of the VCP-Ufd1-Npl4 complex is associated with decreased levels of ERAD substrates. Exp Cell Res 312: 29212932. 264. Arita M, Wakita T, Shimizu H (2012) Valosin-Containing Protein (VCP/p97) Is Required for Poliovirus Replication and Is Involved in Cellular Protein Secretion Pathway in Poliovirus Infection. J Virol 86: 5541-5553. 265. Huotari J, Meyer-Schaller N, Hubner M, Stauffer S, Katheder N, et al. (2012) Cullin-3 regulates late endosome maturation. Proc Natl Acad Sci U S A 109: 823-828. 266. Chu VC, McElroy LJ, Chu V, Bauman BE, Whittaker GR (2006) The avian coronavirus infectious bronchitis virus undergoes direct low-pH-dependent fusion activation during entry into host cells. J Virol 80: 3180-3188. 267. Simmons G, Gosalia DN, Rennekamp AJ, Reeves JD, Diamond SL, et al. (2005) Inhibitors of cathepsin L prevent severe acute respiratory syndrome coronavirus entry. Proc Natl Acad Sci U S A 102: 11876-11881. 268. Eifart P, Ludwig K, Bottcher C, de Haan CA, Rottier PJ, et al. (2007) Role of endocytosis and low pH in murine hepatitis virus strain A59 cell entry. J Virol 81: 10758-10768. 269. van Boxel-Dezaire AH, Rani MR, Stark GR (2006) Complex modulation of cell typespecific signaling in response to type I interferons. Immunity 25: 361-372. 270. Dediego ML, Pewe L, Alvarez E, Rejas MT, Perlman S, et al. (2008) Pathogenicity of severe acute respiratory coronavirus deletion mutants in hACE-2 transgenic mice. Virology 376: 379-389. 197 271. Taniguchi T, Ogasawara K, Takaoka A, Tanaka N (2001) IRF family of transcription factors as regulators of host defense. Annu Rev Immunol 19: 623-655. 272. Hiscott J (2007) Triggering the innate antiviral response through IRF-3 activation. J Biol Chem 282: 15325-15329. 273. Onomoto K, Yoneyama M, Fujita T (2007) Regulation of antiviral innate immune responses by RIG-I family of RNA helicases. Curr Top Microbiol Immunol 316: 193205. 274. Lin R, Heylbroeck C, Pitha PM, Hiscott J (1998) Virus-dependent phosphorylation of the IRF-3 transcription factor regulates nuclear translocation, transactivation potential, and proteasome-mediated degradation. Mol Cell Biol 18: 2986-2996. 275. Suhara W, Yoneyama M, Kitabayashi I, Fujita T (2002) Direct involvement of CREBbinding protein/p300 in sequence-specific DNA binding of virus-activated interferon regulatory factor-3 holocomplex. J Biol Chem 277: 22304-22313. 276. Thanos D, Maniatis T (1995) Virus induction of human IFN beta gene expression requires the assembly of an enhanceosome. Cell 83: 1091-1100. 277. Goodbourn S, Didcock L, Randall RE (2000) Interferons: cell signalling, immune modulation, antiviral response and virus countermeasures. J Gen Virol 81: 23412364. 278. Weber F, Kochs G, Haller O, Staeheli P (2003) Viral evasion of the interferon system: old viruses, new tricks. J Interferon Cytokine Res 23: 209-213. 279. Versteeg GA, Bredenbeek PJ, van den Worm SH, Spaan WJ (2007) Group coronaviruses prevent immediate early interferon induction by protection of viral RNA from host cell recognition. Virology 361: 18-26. 280. Raychoudhuri A, Shrivastava S, Steele R, Kim H, Ray R, et al. (2011) ISG56 and IFITM1 proteins inhibit hepatitis C virus replication. J Virol 85: 12881-12889. 281. Huang IC, Bailey CC, Weyer JL, Radoshitzky SR, Becker MM, et al. (2011) Distinct patterns of IFITM-mediated restriction of filoviruses, SARS coronavirus, and influenza A virus. PLoS Pathog 7: e1001258. 282. Brass AL, Huang IC, Benita Y, John SP, Krishnan MN, et al. (2009) The IFITM proteins mediate cellular resistance to influenza A H1N1 virus, West Nile virus, and dengue virus. Cell 139: 1243-1254. 283. Felicia P.L.Tay MH, Li Wang, Yoshiyuki Yamada, Ding Xiang Liu (2012) Characterization of cellular furin content as a potential factor determining the susceptibility of cultured human and animal cells to coronavirus infectious bronchitis virus infection. Virology http://dx.doi.org/10.1016/j.virol.2012.08.037. 284. Zhou H, Perlman S (2007) Mouse hepatitis virus does not induce Beta interferon synthesis and does not inhibit its induction by double-stranded RNA. J Virol 81: 568574. 285. Zust R, Cervantes-Barragan L, Habjan M, Maier R, Neuman BW, et al. (2011) Ribose 2'O-methylation provides a molecular signature for the distinction of self and nonself mRNA dependent on the RNA sensor Mda5. Nat Immunol 12: 137-143. 286. Daffis S, Szretter KJ, Schriewer J, Li J, Youn S, et al. (2010) 2'-O methylation of the viral mRNA cap evades host restriction by IFIT family members. Nature 468: 452-456. 287. Roth-Cross JK, Martinez-Sobrido L, Scott EP, Garcia-Sastre A, Weiss SR (2007) Inhibition of the alpha/beta interferon response by mouse hepatitis virus at multiple levels. J Virol 81: 7189-7199. 288. Spiegel M, Pichlmair A, Martinez-Sobrido L, Cros J, Garcia-Sastre A, et al. (2005) Inhibition of Beta interferon induction by severe acute respiratory syndrome 198 coronavirus suggests a two-step model for activation of interferon regulatory factor 3. J Virol 79: 2079-2086. 289. Zheng B, He ML, Wong KL, Lum CT, Poon LL, et al. (2004) Potent inhibition of SARSassociated coronavirus (SCOV) infection and replication by type I interferons (IFNalpha/beta) but not by type II interferon (IFN-gamma). J Interferon Cytokine Res 24: 388-390. 290. Spiegel M, Pichlmair A, Muhlberger E, Haller O, Weber F (2004) The antiviral effect of interferon-beta against SARS-coronavirus is not mediated by MxA protein. J Clin Virol 30: 211-213. 291. Wathelet MG, Orr M, Frieman MB, Baric RS (2007) Severe acute respiratory syndrome coronavirus evades antiviral signaling: role of nsp1 and rational design of an attenuated strain. J Virol 81: 11620-11633. 292. Kamitani W, Narayanan K, Huang C, Lokugamage K, Ikegami T, et al. (2006) Severe acute respiratory syndrome coronavirus nsp1 protein suppresses host gene expression by promoting host mRNA degradation. Proc Natl Acad Sci U S A 103: 12885-12890. 293. Narayanan K, Huang C, Lokugamage K, Kamitani W, Ikegami T, et al. (2008) Severe acute respiratory syndrome coronavirus nsp1 suppresses host gene expression, including that of type I interferon, in infected cells. J Virol 82: 4471-4479. 294. Collins SE, Noyce RS, Mossman KL (2004) Innate cellular response to virus particle entry requires IRF3 but not virus replication. J Virol 78: 1706-1717. 295. Clement JF, Bibeau-Poirier A, Gravel SP, Grandvaux N, Bonneil E, et al. (2008) Phosphorylation of IRF-3 on Ser 339 generates a hyperactive form of IRF-3 through regulation of dimerization and CBP association. J Virol 82: 3984-3996. 296. Lin R, Mamane Y, Hiscott J (1999) Structural and functional analysis of interferon regulatory factor 3: localization of the transactivation and autoinhibitory domains. Mol Cell Biol 19: 2465-2474. 297. Keng CT, Choi YW, Welkers MR, Chan DZ, Shen S, et al. (2006) The human severe acute respiratory syndrome coronavirus (SARS-CoV) 8b protein is distinct from its counterpart in animal SARS-CoV and down-regulates the expression of the envelope protein in infected cells. Virology 354: 132-142. 298. Law PY, Liu YM, Geng H, Kwan KH, Waye MM, et al. (2006) Expression and functional characterization of the putative protein 8b of the severe acute respiratory syndrome-associated coronavirus. FEBS Lett 580: 3643-3648. 299. Sung SC, Chao CY, Jeng KS, Yang JY, Lai MM (2009) The 8ab protein of SARS-CoV is a luminal ER membrane-associated protein and induces the activation of ATF6. Virology 387: 402-413. 300. Peiris JS, Chu CM, Cheng VC, Chan KS, Hung IF, et al. (2003) Clinical progression and viral load in a community outbreak of coronavirus-associated SARS pneumonia: a prospective study. Lancet 361: 1767-1772. 301. Roth-Cross JK, Bender SJ, Weiss SR (2008) Murine coronavirus mouse hepatitis virus is recognized by MDA5 and induces type I interferon in brain macrophages/microglia. J Virol 82: 9829-9838. 302. Li J, Liu Y, Zhang X (2010) Murine coronavirus induces type I interferon in oligodendrocytes through recognition by RIG-I and MDA5. J Virol 84: 6472-6482. 199 [...]... 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……………………………………………………………………