FUNCTIONAL GENOMICS STUDY OF SINGAPORE GROUPER IRIDOVIRUS WANG FAN (M.Sc., Tsinghua University) A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY DEPARTMENT OF BIOLOGICAL SCIENCES NATIONAL UNIVERSITY OF SINGAPORE 2008 Dedicated to My Family Acknowledgements I would like to thank all the people who help me in this project in the past four years. First I would like to express my deepest appreciation to my supervisor, Professor HEW Choy Leong, for giving me this opportunity to pursue my PhD in the Department of Biological Sciences, National University of Singapore. With his sapiential guidance, meticulous care and invaluable advice, I have fully prepared for my future career. I am grateful to Mr. Shashikant JOSHI, for his invaluable help in my paper preparation and selfless assistance in Mass Spectrometry technical problem; Dr. Bi Xue Zhi, for his invaluable help on 2D PAGE and Mass Spectrometry; Dr. Wu Jin Lu and Loy Gek Luan for their help on Transmission Electron Microscopy. I would like to show special thanks to my senior, Dr. Song Wen Jun, for his crucial help in the initiation of my project. I really acknowledge Dr. Lin Qingsong for his collaboration on the mass spectrometric work and his selfless help in my difficult period. I will to also extend my thanks and gratitude to Ms. Wang Xianhui, Ms. Koh Say Tin, and Mr. Lim Teck Kwang for their assistance in the fields of proteomics. Thanks also to the other members of PPC for your friendships and encouragement. Special thanks to my parents and my wife for their encouragement in my difficult period. i Table of Contents Acknowledgements i Table of Contents ii Summary vi List of Tables vii List of Figures viii List of Abbreviations . ix Literature Review . 1.1 Overview of the DNA virus Family Iridoviridae . 1.1.1 Classification of the DNA virus Family Iridoviridae . 1.1.2 Structure of the Iridoviruses . 1.1.3 Previous Functional Genomics Studies of the Iridoviruses 1.1.4 Replication Strategy of the Iridovirus 13 1.2 Application of Antisense Technology in Anti-Virus Research 15 1.3 Proteomics Approach for Function Study of Proteins 17 1.3.1 What is Proteomics . 17 1.3.2 The Branches of Proteomics . 17 1.3.3 Application of Proteomics in SGIV Gene Function Study 19 1.4 Viral Infection and Phosphorylation/dephosphorylation 20 1.4.1 Virus-related Phosphatases . 20 1.4.1.1 Virus Encoded Phosphatases 20 1.4.1.2 Virus Regulated Phosphatases 21 1.4.2 Virus-related Protein Kinases . 22 1.4.2.1 Virus Encoded Protein Kinases 22 1.4.2.2 Host Protein Kinase Utilized by Virus . 23 1.4.3 Virus Infection and Phosphorylation/Dephosphorylation 23 1.5 Treatment of Viral Diseases from Protein Phosphorylation . 24 1.5.1 Overview of Viral Diseases 24 1.5.2 Kinases and Phosphatases as Anti-virus Drug Targets 25 1.5.2.1 Cyclin-dependent Kinases (CDKs) as Novel Targets for Antiviral Drugs 25 1.5.2.2 PP1/PP2A as Antiviral Drug Targets . 26 1.5.2.3 Virus-encoded Kinases and Phosphatases as Antivirus Drug Targets . 27 ii 1.5.2.4 Iridovirus-encoded Kinases and Related Regulators as Anti-iridovirus Drug Targets…………… . …… 28 1.6 Objectives and Significance of the Project . 29 Knockdown Platform Technology for the Studies of SGIV 30 2.1 Introduction 31 2.2 Materials and Methods . 33 2.2.1 Cells and Viruses 33 2.2.2 Antibodies . 33 2.2.3 AsMO Design and Transfection . 34 2.2.4 Radiolabeling of Newly Synthetic Proteins from Virus Infected Cells . 35 2.2.5 Viral Protein Analysis 35 2.2.6 Detection of Viral Proteins by Immunofluorescence (IF) 36 2.2.7 2D Gel Electrophoresis . 36 2.2.8 Gel Staining and Image Analysis . 37 2.2.9 Trypsin Digestion and MALDI-TOF/TOF MS and MS/MS Analyses 37 2.3 Results 38 2.3.1 AsMO Delivery Efficiency . 38 2.3.2 Viral Protein Expression Analysis with Autoradiography . 40 2.3.3 Viral Protein Expression Analysis with Western Blot Assay 43 2.3.4 Viral Protein Expression Analysis with Immunofluorescence . 43 2.3.5 Comparison between Uninfected and Infected Cell 2D Maps and the Identification of Some SGIV Viral Proteins with MS Analysis . 45 2.4 Discussion . 48 2.4.1 The Limitation of This Platform . 48 2.4.2 Detection of Phenotypic Changes 51 2.4.3 Significances of This Platform . 51 2.5 Conclusion 51 Serine/Threonine Phosphorylation in SGIV Late-Stage Infected Cells . 53 3.1 Introduction 54 3.2 Materials and Methods . 56 3.2.1 Preparation of Purified Truncated rORF039L 56 3.2.2 Preparation of Purified rORF075R . 57 3.2.3 Antibodies . 57 3.2.4 Detection of ORF018R by Immunofluorescence (IF) 57 iii 3.2.5 Titration of Viral Progeny and Light Microscopy 58 3.2.6 Electron Microscopy Observation of SGIV Infected Cells 58 3.2.7 Detection of Enhanced Serine Phosphorylation by Phospho-serine MAb . 58 3.2.8 Circular Dichroism (CD) Spectrum of rORF018R 59 3.2.9 Dynamic Light Scattering (DLS) Study of rORF018R 59 3.2.10 Viral Endogenous Kinases Activity Assay . 59 3.2.11 Purified Viral Kinase Activity Assay . 60 3.2.12 Trypsin Digestion and Linear Ion Trap Orbitrap (Thermo)-MS and MS/MS Analyses 60 3.3 Results 61 3.3.1 Intracellular Distribution of ORF018R in SGIV Infected Cells . 61 3.3.2 Effect of ORF018R Knockdown on Particle Formation 63 3.3.3 Effect of ORF018R Knockdown on Virus Infectivity and Viral Protein Synthesis . 65 3.3.4 Enhanced Serine Phosphorylations in ORF018R Knockdown Infected GE Cells 67 3.3.5 Standard 2DE-map of SGIV-infected Cells with AsMOctrl Transfected 67 3.3.6 Identification of Enhanced Phosphorylated Proteins in ORF018R Knockdown Infected GE Cells . 70 3.3.7 Further Confirmation of the Enhanced Phosphorylated Proteins with Linear Ion Trap Orbitrap (Thermo) 76 3.3.8 In Vitro Rescue Experiment with Recombinant ORF018R . 82 3.3.9 Protein Characteristics of rORF018R . 84 3.3.10 In Vitro Phosphorylation by the Endogenous Protein Kinase 87 3.4 Discussion . 90 3.5 Implications 93 Functional Study of ORF082L . 94 4.1 Introduction 95 4.2 Materials and Methods . 98 4.2.1 Primers Design . 98 4.2.2 Purification of Recombinant Viral Proteins and Antibodies Preparation 99 4.2.3 Delivery of pEGFP Vectors 99 4.2.4 Immunofluorescence (IF) Analyses for ORF082L . 100 4.2.5 GST Pull Down Assay and Mass Spectrometry Analyses for ORF082L 100 iv 4.3 Results 101 4.3.1 Overexpression of EGFP Fusion Proteins in GE Cells 101 4.3.2 Localization Study of ORF082L 103 4.3.3 ORF082L Pull Down Assay . 105 4.3.4 Time Course of ORF082L Localization Study in SGIV-infected GE Cells 109 4.4 Discussion . 111 4.4.1 Bioinformatics Analyses of ORF082L and Its Potential Function . 111 4.4.2 Some Indirect Evidence for ORF082L Function 113 4.5 Conclusion 114 General Conclusion and Future Studies . 115 5.1 General Conclusion 116 5.2 Future Studies . 117 5.2.1 Screening for More Essential Genes for Anti-iridovirus Drug Design 117 5.2.2 Explanation of how ORF018R acts in SGIV-infected late stage cells . 117 5.2.3 Structure Studies for Certain Essential Enzymes . 118 5.2.4 Functional Studies of ORF075R 118 Bibliography . 119 Appendices 134 Publications . 135 v Summary Iridovirus is a pathogen causing serious systemic diseases among feral, cultured and ornamental fish. Our lab aims to explore iridovirus infection mechanism and anti-iridovirus drug discovery. A knockdown platform has been built up for an effective knockdown of certain genes from SGIV. With phenotype screening a functional important SGIV encoded protein - ORF018R was successfully identified. Immunofluorescence staining showed that ORF018R expressed in high abundance in SGIV-infected cells. Knockdown of ORF018R expression resulted in reduction of expression of viral late genes, distortion of viral particle assembly and inhibition of SGIV infection in grouper embryonic cells. Immunoblot and 2DE-MS showed five enhanced phosphorylated proteins including three viral proteins: ORF049L (dUTPase), ORF075R and ORF086R; two host proteins: subunit 12 of eukaryotic translation factor (eIF3) and natural killer enhancing factor (NKEF). Furthermore, LTQ-Orbitrap experiment identified that the peptide - ORF049L (aa142-152) was phosphorylated by ORF018R knockdown. Moreover, One SGIV encoded kinaseORF039L was identified and another viral protein ORF075R could be phosphorylated by ORF039L in vitro. Thus ORF018R was probably a kinase inhibitory protein in SGIV infected cells. EGFP fusion viral proteins were expressed in host cell line to screen virus-host interaction. Only ORF082L-EGFP showed special localization. Pull down assay with GST-ORF082L revealed that this protein probably had two binding partners, namely enolase and actin. Based on this study, the mechanism of SGIV infection was explored which would facilitate anti-iridovirus research. vi List of Tables Table 1.1 Current classification of the Family Iridoviridae Table 1.2 Comparisons of iridovirus genomes Table 1.3 Core components of iridovirus genes .9 Table 1.4 Identified viral encoded phosphatases 21 Table 1.5 Identified viral encoded kinases 22 Table 1.6 Some pathogenic viruses and their vectors 24 Table 2.1 Bioinformatics analyses of 17 major structural components 32 Table 2.2 Designed asMOs .35 Table 2.3 The proteins from SGIV-infected cells identified by 2-DE coupled with mass spectrometry 47 Table 3.1 Significantly altered proteins from SGIV-infected cells identified by 2-DE coupled with mass spectrometry 74 Table 4.1 Bioinformatics analysis of eight SGIV ORFs .96 Table 4.2 Proteomics analysis of eight SGIV ORFs .97 Table 4.3 MS identification of protein bands in GST-ORF082L pull down assay . 107 Table 4.4 Function prediction of ORF082L with PFP . 112 Table 4.5 The comparisons of MW and pI between ORF082L and other iridovirus encoded DNA methyltransferase 114 vii List of Figures Figure 1.1 Current state of DNA viruses taxonomy Figure 1.2 Summary of SGIV proteins identified by MS 12 Figure 1.3 Iridovirus infection process .14 Figure 2.1 AsMO delivery efficiency in GE cell line .39 Figure 2.2 AsMO knockdown effects examined by autoradiography 41 Figure 2.3 AsMO knockdown effects checked by Western blot assay .42 Figure 2.4 AsMO93 knockdown effects checked by immunofluorescence .44 Figure 2.5 Comparison between uninfected cells and infected cells and identification of some proteins in infected cell lysates with mass spectrometry 46 Figure 2.6 Flowchart of the knockdown platform for SGIV . 49 Figure 3.1 Intracellular distribution of ORF018R . 62 Figure 3.2 Effects of ORF018R knockdown on SGIV assembly 64 Figure 3.3 Reduction of SGIV infectivity in asMO18 transfected cells . 66 Figure 3.4 Enhanced serine phosphorylations with ORF018R knockdown 68 Figure 3.5 Demonstration of SGIV-infected GE cells 2D gel reproducibility between runs…… 69 Figure 3.6 Detailed 2-DE maps of normal SGIV-infected cells (A) and ORF018R knockdown SGIV-infected cells (B) . 73 Figure 3.7 ORF075R isoform cluster changes between control (A-a) and ORF018R knockdown (A-b) on 2DE . 75 Figure 3.8 Identification of phosphorylated peptides with LTQ-Orbit trap .81 Figure 3.9 In vitro rescue experiments with recombinant ORF018R .83 Figure 3.10 DLS result of rORF018R 85 Figure 3.11 CD profile of rORF018R upon metal ion changes 86 Figure 3.12 Electrophoretic separations of phosphorylated SGIV polypeptides in vitro .88 Figure 3.13 In vitro viral encoded kinases assays 89 Figure 4.1 Overexpression of EGFP fusion viral proteins in GE cells 102 Figure 4.2 Localizations of ORF082L-EGFP fusion proteins 104 Figure 4.3 GST pull down assay with uninfected GE cell lysates 106 Figure 4.4 Comparison of MS coverages between full-length ORF082L and truncated ORF082L 108 Figure 4.5 Time course of ORF082L localization in SGIV-infected GE cells . 110 Figure 5.1 A proposed method to verify ORF018R function . 117 viii Berry, E. 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Virol. 2:393-399. 133 Appendices Name Company Minimal essential medium complex Sigma Fetal bovine serum Sigma Penicillin GIBCO Streptomycin GIBCO Restriction endonucleases, T4-DNA ligase New England Biolabs Antisense morpholino Gene tools Lab-Tek® chambered coverglass Nalge Nunc Glutathione-Sepharose 4B beads Pharmacia PrecissionTM protease Pharmacia Ni-NTA agarose beads Qiagen Hoechst Invitrogen AlexaFluo conjugated antibody Invitrogen Sequencing grade modified porcine trypsin Promega ProQ DiamondTM Invitrogen List of various kits and software used Company Nucleofactor® kit Amaxa Coomassie Protein Assay kit Pierce The Supersignal West Pico Luminol Pierce Enhancer Solution The Supersignal West Pico Stable Pierce Peroxide Solution PDQUEST 7.3 Bio-Rad MASCOT 2.0 Matrix Science The spur kit Sigma 134 Publications 1. Wang, F., X. Z. Bi, L. M. Chen, and C. L. Hew. 2008. ORF018R, a highly abundant virion protein from Singapore Grouper Iridovirus, is involved in serine/threonine phosphorylation and virion assembly. J Gen Virol. 89:11691178. 2. Chen, L. M., F. Wang, W. J. Song, and C. L. Hew. 2006. Temporal and differential gene expression of Singapore grouper iridovirus. J Gen Virol. 87:2907-15. 3. Song, W. J., Q. W. Qin, J. Qiu, C. H. Huang, F. Wang, and C. L. Hew. 2004. Functional genomics analysis of Singapore grouper iridovirus: complete sequence determination and proteomic analysis. J Virol. 78:12576-90. 135 [...]... vulgaris disease virus Red Sea bream iridovirus Taiwan grouper iridovirus Olive flounder iridovirus Rock bream iridovirus White sturgeon iridovirus Unassigned 5 1.1.2 Structure of the Iridoviruses Iridoviruses are icosahedral viruses, 120 to 300 nm in diameter The genome of iridoviruses is a linear, double-stranded DNA molecule with the size is between 100 and 210 kbp All of iridoviral genomes are circularly... prediction p i post infection PMF peptide mass fingerprinting Q-TOF quadrupole-TOF RACE rapid amplification of cDNA ends RBIV rock bream iridovirus RNAi RNA interference RNase ribonuclease SGIV Singapore grouper iridovirus SOR hydroxysteroid oxidoreductase TCID tissue culture infecting dose TFV tiger frog virus TNF tumor necrosis factor TOF time -of- flight TRAF the TNF-receptor associated factor Tris tris... for Function Study of Proteins 1.3.1 What is Proteomics (http://en.wikipedia.org/wiki/Proteomics) Proteomics is the large-scale study of proteins, particularly their structures and functions The term "proteomics" was created to make an analogy with genomics, the study of the genes The word "proteome" is a portmanteau (a word combined with two words) of "protein" and "genome" The proteome of an organism... Fibrillar structures surrounding the outer capsid of Chilo iridescent virus have been reported (Yan et al., 2000) The fibrils are up to 200 nm length, while they appear as a fringe around the edge of the capsid in other iridoviruses (Zwillenberg and Wolf, 1968; Willison and Cocking, 1972) 1.1.3 Previous Functional Genomic Studies of the Iridoviruses Currently, ten iridovirus genomes have been sequenced and... vertebrate iridoviruses (Berry et al., 1983; Speare and Smith, 1992) The lipid content of iridovirus virion has been reported from 5.2% to 17% (Williams, 1996) Phospholipid is the main component of the total lipids The composition of viral phospholipid is identical to the host cell in vertebrate iridovirus such as FV3 (Willis and Granoff, 1974), but is obviously different in the invertebrate iridovirus. .. organism is the set of proteins produced by it during its life, and its genome is its set of genes Proteomics is often regarded as the next step in the study of biological systems, after genomics It is much more complex than genomics, because while an organism's genome is constant, a proteome differs from cell to cell Even within a single cell it varies with the different environment of this cell This... One of the most effective strategies is to control phosphorylation and dephosphorylation which covers all aspects of virus infection processes Disruption of such controls is probably one of the best ways to treat viral diseases (Mohr, 2007) 23 1.5 Treatment of Viral Diseases from Protein Phosphorylation 1.5.1 Overview of Viral Diseases Some of the viruses will remain benign with their host but most of. .. 2005) Hosts of the former two genera come from invertebrates such as flies, silkworms, and mosquitoes Nomenclature of invertebrate isolates is based on the host species and is given a type number on the basis of the sequence of discovery following a recommended interim system (Tinsley and Kelly, 1970) The small iridoviruses, 120-140 nm in diameter, are classified into genus iridovirus The large iridoviruses,... have been decided since the publication of the Sixth Report of the ICTV (Murphy et al., 1995), they are indicated by asterisks 3 Iridoviruses are large, icosahedral viruses with a linear, double-stranded DNA genome (Chinchar et al., 2005) As one of the 17 families in dsDNA virus, the family Iridoviridae has been classified into four genuses – Iridovirus, Chloriridovirus, Ranavirus and Lymphocystivirus... frog virus 3 GE grouper embroynic GIV grouper iridovirus GFP green fluorescent protein GST glutathione S-transferase HBV hepatitis B virus HEPES 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid HIV human immunodeficiency virus IAA iodoacetamide LC liquid chromatography Ig immunoglobulins ICTV the International Committee on Taxonomy of Virus IF immunofluorescence IIV-6 invertebrate iridovirus 6 IPTG . FUNCTIONAL GENOMICS STUDY OF SINGAPORE GROUPER IRIDOVIRUS WANG FAN (M.Sc., Tsinghua University) A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY DEPARTMENT OF. Classification of the DNA virus Family Iridoviridae 2 1.1.2 Structure of the Iridoviruses 6 1.1.3 Previous Functional Genomics Studies of the Iridoviruses 7 1.1.4 Replication Strategy of the Iridovirus. bream iridovirus Taiwan grouper iridovirus Olive flounder iridovirus Rock bream iridovirus Unassigned White sturgeon iridovirus 6 1.1.2 Structure of the Iridoviruses Iridoviruses