FUNCTIONAL AND STRUCTURAL GENOMICS STUDY OF SINGAPORE GROUPER IRIDOVIRUS CHEN LI MING (B.SC., XIAMEN UNIVERSITY) A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY DEPARTMENT OF BIOLOGICAL SCIENCES NATIONAL UNIVERSITY OF SINGAPORE 2008 Acknowledgements First of all, I would like to thank my supervisor, Professor Hew Choy Leong, for giving me the opportunity to pursue my PhD study and for his guidance, and mentorship. Second, I would like to thank Dr Lance Miller for the discussion on the chip set-up. I also appreciate Dr Jin Hua Han for helpful discussions on the data analysis. I also appreciate Dr. Jayaraman Sivaraman for his advice on crystallization, Dr. Song Jianxing and Dr. Yang Daiwen for their advices on NMR. I thank Dr. Lin Qingsong for his advice on iTRAQ and proteome related works. I thank Dr. Gong Zhiyuan for his advice on transgenetic fish studies. Third, I appreciate the Genomic Institute of Singapore for the provision of the facilities for DNA microarray and real-time RT-PCR work. I am grateful for Kun Yan's assistance with chip spotting. I thank Dr Zhen Jun Li for her advice on the molecular biological techniques. I thank Dr Yan Liu for his useful suggestions and kind helps on the structural study of my project. I thank Mr. Wang Fan for his suggestions on my project. Finally, I would like to thank my other lab mates: Dr. Song Wenjun, Dr. Wu Jinlu, Dr. Tang Xuhua, Ms Chen Jing, and Ms Tran Bich Ngoc for their valuable discussion and friendship. I Summary Singapore grouper iridovirus (SGIV), an iridovirus in the genus Ranavirus, is a major pathogen that results in significant economic losses in grouper aquaculture. In this thesis, first, we report the temporal and differential gene expression of SGIV using SGIV viral DNA microarray; Second, we report the first proteomics study of grouper embryonic cells (GEC) infected by Singapore grouper iridovirus (SGIV) to take an insight into the interaction of SGIV and its host cell at proteome scale by iTRAQ; third, we report that a novel viral coding protein ORF158L is involved in the regulation of host histone H3 K79 methylation; finally, we report the structural study of ORF158L. Our work provides important insights into the pathogenesis of iridoviruses. II Table of Contents Acknowledgements………………………………………………… … I Summary………………………………………………………………………II Table of Contents………………………………………………….…………III List of Tables………………………………………………………………….VIII List of Figures……………………………………………………………… IX Chapter Introduction & Literature Review…………………………… 1.1 Overview…………………………………………………………………… 1.2 Introduction of Iridovirus………………………………………………… 1.3 Introduction of Singapore grouper iridovirus (SGIV)………………… 1.4 Transcriptional regulation & Replication cycle of the iridovirus……… 1.5 Functional genomics………………………………………………………. .5 1.5.1 Introduction of Functional genomics……………………………… .5 1.5.2 Gene expression profile and differential gene expression of the iridovirus…………………………………………………………………… 1.5.3 Functional genomic studies at proteomic scale by using iTRAQ… 1.6 Structural genomics……………………………………………………… .10 1.6.1 Introduction of Structural genomics……………………………… .10 1.6.2 NMR spectroscopy………………………………………………….…10 1.6.3 X-Ray crystallography…………………………………………… 11 1.7 Scope of thesis……………………………………………………………….12 III Chapter An investigation of temporal and differential gene expression of Singapore grouper iridovirus by DNA microarray……………….….16 2.1 Summary………………………………………………………………… .17 2.2 Introduction……………………………………………………………… 18 2.3 Materials and Methods…………………………………………………… 20 2.3.1 Cell lines……………………………………………………………….20 2.3.2 Positive controls for the SGIV DNA microarray………………… 20 2.3.3 Preparation of amplicons for the SGIV DNA microarray………….20 2.3.4 Virus infection and CHX and aphidicoline treatments………….…21 2.3.5 Total RNA preparation, reverse transcription and labeling….… .21 2.3.6 Real-time PCR………………………………………………….…… 22 2.4 Results…………………………………………………………………….….23 2.4.1 Viral microarray for grouper iridovirus………………………….…23 2.4.2 Temporal gene-expression analysis of the SGIV genome…… ……23 2.4.3 SGIV viral gene expression with different concentrations of CHX…………………………………………………………………….……24 2.4.4 SGIV viral gene expression with aphidicoline treatment…….…….24 2.5 Discussions …………………………………………………………….……27 Chapter iTRAQ study of Grouper embryonic cells infected by Singapore grouper Iridovirus: an insight into the Singapore grouper iridovirus and host cell interaction……………………………….… 45 3.1 Summary……………………………………………………………….……46 3.2 Introduction……………………………………………………………… 47 3.3 Materials and Methods…………………………………………………… 47 3.3.1 Cell and virus infection……………………………………………….47 IV 3.3.2 iTRAQ Labeling and Two Dimensional (2D) LC-MALDI MS….…47 3.3.3 RT PCR………………………………………………………….….….49 3.3.4 Western blotting………………………………………………….……49 3.4 Results………………………………………………………………….….…50 3.4.1 Identification of viral proteins by using iTRAQ………………….…50 3.4.2 Identification of differential expression host proteins by using iTRAQ………………………………………………………………….……50 3.4.3 RT-PCR and western blot analysis of the viral proteins………… .51 3.4.4 Up regulation of host Histone H3 Lysine 79 (K79) methylation upon SGIV infection……………………………………………………….…… 52 3.5 Discussions …………………………………………………………… .….53 Chapter A novel SGIV coding protein ORF158L is involved in the regulation of K79 methylation of histione H3…………………… …81 4.1 Summary……………………………………………………………….……82 4.2 Introduction…………………………………………………………….……82 4.3 Materials and Methods………………………………………………….… 82 4.3.1 Cell and virus infection……………………………………….…….…82 4.3.2 Antibodies……………………………………………………….…… 82 4.3.3 Knockdown of ORF158L……………………………………….…… 83 4.3.4 Electron Microscopy……………………………………………….….83 4.3.5 DNA Microarray and Real Time RT PCR…………………….…….83 4.3.6 Immunofluorescence …………………………………………….……83 4.4 Results……………………………………………………………………85 4.4.1 Western blot against ORF158L………………………………………85 4.4.2 Knockdown of ORF158L…………………………………………… 85 V 4.4.3 DNA Microarray and Real time RT-PCR investigation of transcriptsomes of viral genes when ORF158L was knocked down….….85 4.4.4 Subcellular localization of ORF158L and ORF158L & Histone H3 colocalization……………………………………………………………… .86 4.4.5 iTRAQ study the effect of knockdown ORF158L at proteome scale 86 4.4.6 ORF158L involved in Histone H3 lysine 79 (K79) methylation regulation…………………………………………………………………….86 4.5 Discussions………………………………………………………………… .88 Chapter Structural study of ORF158L…………………………….97 5.1 Summary & Introduction……………………………………………………98 5.2 Materials and Methods………………………………………………………99 5.2.1 Construction of the expression plasmid…………………………….…99 5.2.2 Expression and purification of ORF158L………………………… …99 5.2.3 Mass spectrometry analysis………………………………………….…99 5.2.4 Dynamic light scattering (DLS) study……………………………….…99 5.2.5 Circular dichroism (CD) study…………………………………………99 5.2.6 NMR sample preparation…………………………………………… .100 5.2.7 NMR Experiments and data process. ………………………… .100 5.2.8 SeMet ORF158L preparation………………………………………….100 5.2.9 Crystallization …………………….……………………………………100 5.2.10 Data collection, structure solution and refinement ………… .101 5.2.11 Expression and purification of histone H3 and H4 complex…….…102 5.2.11 Surface Plasmon Resonance (SPR)…………………………… .102 5.3 Results…………………………………………………………………………103 VI 5.3.1 Protein purification profiles of ORF158L ……………………………103 5.3.2 Mass spectrometry analysis……………………………………………103 5.3.3 Dynamic light scattering (DLS) study…………………………………103 5.3.4 Circular dichroism study…………………………………………….…103 5.3.5 1D NMR…………………………………………………………… …104 5.3.6 1H-15N HSQC study of ORF158L……………………………… 104 5.3.7 Overall Structure of ORF158L…………………………………………105 5.3.8 Sequence and structural homology….…………………………………106 5.3.9 Putative Histone binding region….…………………………………….106 Chapter Achievements & Future experiments………………… .119 6.1 Achievements……………………………………………………………….…120 6.2 Future experiments……………………………………………………………120 References………………………………………………………………………….122 VII List of Tables Table 2.1. Kinetic class of SGIV ORF expression. ………………………… .31 Table 2.2 SGIV primers……………………………………………… .36 Table 2.3. Partial cDNA sequences of β-actin and GAPDH…………… .39 Table 2.4. Primers for Real time PCR……………………………………… 40 Table 2.5. SGIV genes with temporal expression on array……………… …41 Table 3.1. 38 viral proteins were consistent with those that were reported…60 Table 3.2. 11 viral proteins were newly identified and first reported……… .62 Table 3.3. 12 host proteins were up-regulated during SGIV infection……… 63 Table 3.4. host proteins were down-regulated during SGIV infection… … 64 Table 3.5. GEC host proteins identified from iTRAQ analysis……………… 65 Table 3.6 Primers for the 11 newly identified viral proteins………………… 80 Table 5.1 Data collection and refinement statistics of ORF158L…………… 118 VIII List of Figures Figure 1.1 The diagram of iridovirus replication cycle…………………… 13 Figure 1.2 The chemical structure of iTRAQ reagent…………………… …14 Figure 1.3 Workflows of iTRAQ experiments……………………………… .15 Figure 2.1. Hierarchical clustering gene tree of SGIV temporal gene expression data……………………………………………………………………………….32 Figure 2.2. Validation DNA microarray result with real time RT-PCR…….33 Figure 2.3. Effect of CHX treatment on SGIV gene expression…………….34 Figure 2.4. SGIV gene expression profiles with aphidicoline treatment……35 Figure 3.1 Full length amplification of 11 novel genes of SGIV via RTPCR………………………………………………………………………………57 Figure 3.2 Presence of SGIV proteins expressed by ORF018R, 026R, 093L and newly identified proteins encoded by ORF135L and 140R………………… 58 Figure 3.3 Histone H3 K79 methylation analysis of SGIV-infected GECs compared to mock SGIV-infected GECs by western blot……………………59 Figure 4.1 Identification and knockdown of ORF158L in cell culture………90 Figure 4.2 Comparison of transcriptional profiles of SGIV with and without ORF158L knock down………………………………………………………… 91 Figure 4.3 Subcellular localization of ORF158L………………………………92 IX Figure 5.10 SPR study of ORF158L binding to histone H3 & H4 complex. 117 Table 5.1 Data collection and refinement statistics of ORF158L Data collection and refinement statistics Space group Unit cell dimensions (Å) Data set Resolution range (Å) Wavelength (Å) Observed hkl Unique hkl Completeness (%) Overall (I/oI) Rsym a (%) Refinement and quality Resolution range [I>o(I)] Rwork b (no. of reflections) Rfree c (no. of reflections) rmsd bond lengths (A) rmsd bond angles (deg) Ramachandran plot Most favored regions (%) Additional allowed regions (%) Generously allowed regions (%) Disallowed regions (%) C2 a=59.98, b= 41.01, c= 52.33 α=90, β= 95.32, γ=90.0 Peak 50-1.8 0.979 86250 11855 99.9 14.8 6.2 Inflection 0.9792 73264 10223 99.9 12.3 1.8 -20.0 0.2074(21617) 0.2447(1093) 0.006 1.3 86 13.2 0.9 118 Chapter Six Achievements & Future experiments 119 6.1 Achievements In this thesis, we have presented the following achievements: 1) We are the first to study the temporal and differential gene expression of Singapore grouper iridovirus (SGIV) by DNA microarray. To our knowledge, this is one of the first studies where DNA microarray technique and also realtime RT-PCR were used for the investigation of fish viruses. (Chen et al., 2006) 2) We are the first to study the SGIV and host cell interaction at proteomics scale by using iTRAQ. (Chen et al., 2008) 3) We are the first to demonstrate that ORF158L is a real protein encoded by SGIV. 4) We are the first to characterize the function of ORF158L and show that ORF158L is involved in the regulation of histone H3 K79 methylation. 5) We are the first to elucidate the crystal structure of ORF158L. 6.2 Future experiments In future, we are going to further investigate the following scientific questions: 1) We are going to study the mechanism about how ORF158L regulates host histone H3 K79 methylation and the consequences of the regulation of host histone H3 K79 methylation. 2) By combining knockdown, DNA microarray and iTRAQ, we are going to carry functional studies of SGIV genes. 3) With the help of NMR and X-ray crystallography, we are going to continue the structural studies of both viral structural proteins and viral non structural proteins. 120 4) Based on the functional genomics and structural genomics studies of SGIV, we are going to uncover the SGIV pathogenesis and provide valuable knowledge. 5) Based on the functional genomics and structural genomics studies of SGIV, we are going to design new drugs and raise new strategies in the treatment of SGIV infection on grouper. 121 References: Aparicio, S., J. 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Exp Bot 57:1501-1508. 132 [...]... first iridovirus was discovered by Smith and Xeros (Smith and Xeros, 1954) To date, more than 100 iridoviruses belonging to the four genera of the iridovirus family have been isolated The four genera of the iridovirus family are Iridovirus, Chloriridovirus and, Lymphocystivirus and Ranavirus (Chinchar et al., 2005) 1.3 Introduction of Singapore grouper iridovirus (SGIV) In 1994, a novel member of Ranavirus,... investigation of temporal and differential gene expression of Singapore grouper iridovirus by DNA microarray Published: Chen et al 2006 Temporal and differential gene expression of Singapore grouper iridovirus J Gen Virol 87:2907-15 16 2.1 Summary Singapore grouper iridovirus (SGIV), an iridovirus in the genus Ranavirus, is a major pathogen that results in significant economic losses in grouper aquaculture... protein target and what changes might be advisable to improve it (Scapin, 2006) 1.7 Scope of thesis In this thesis, we will present 1) The study of temporal and differential gene expression of SGIV using DNA microarray 2) The proteomics study of grouper embryonic cells infected by SGIV using iTRAQ 3) The functional study of ORF158L, a novel protein coded by SGIV genome 4) The structural study of ORF158L... transcriptional profile of RSIV using DNA microarray and found that 97-99% of the RSIV ORFs were expressed (Lua et al., 2007) However, the gene expression and transcriptional program of SGIV has not been investigated either by Northern blot or DNA microarray The study of the transcriptional profile of SGIV may provide a profound insight into the replication and pathogenesis of the iridovirus family... expression of the immediate early genes, the early genes are expressed, and they encode proteins which play important roles in the iridovirus genome replication After the onset of the iridovirus genome replication, the late genes are expressed, and they encode structural proteins of iridovirus particles Given the different functions of the three groups of iridovirus genes, the study on the temporal and differential... transcription, translation, and protein-protein interactions 1.5.2 Gene expression profile and differential gene expression of the iridovirus In the iridovirus replication cycle, the iridovirus genes are differentially expressed (Williams, 1996) On one hand, some iridovirus genes are expressed at the first stage of iridovirus genome replication in the nucleus of host cells On the other hand, some genes are... transcribed, and the iridovirus genome is transported to the cytoplasm of the cell and the iridovirus commences its second stage genome replication in the cytoplasm Besides, the early and late genes of the iridoviruse are transcripted and translated The genomes and structural proteins of the iridoviruse begin to assemble to form new iridoviruses, and are released from the host cells The virus is now ready to... the entry, the iridovirus particles are delivered to the lysosomes of host cells, and they are uncoated inside the lysosomes As a result, the genome of iridoviruses is released The released iridovirus genome is transported to the host nucleus and the first stage of iridovirus genome replication is initiated At the same time, the iridovirus immediate early genes are transcribed, and the iridovirus genome... 1.1) (Murti et al., 1985) 4 1.5 Functional genomics 1.5.1 Introduction of Functional genomics Genomic projects, such as genome sequencing projects, have produced a vast wealth of data Functional genomics is a field of molecular biology that attempts to describe large scale gene/protein functions and interactions by using the data produced by genomic projects Functional genomics focuses on the dynamic... systematic diseases in farms of both feral and cultured groupers So far, genomic sequences of two grouper iridoviruses have been published: SGIV (Song et al., 2004 )and grouper iridovirus (GIV) (Tsai et al., 2005), with whole-genomic sequence similarity of >90 % Willis et al (1977) designated 10 ‘early’ RNAs (of 47 mRNAs), expressed from 1 to 1.5 h after frog virus 3 (FV-3) infection of fathead minnow cells . FUNCTIONAL AND STRUCTURAL GENOMICS STUDY OF SINGAPORE GROUPER IRIDOVIRUS CHEN LI MING (B.SC., XIAMEN UNIVERSITY) A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY. …………………………………………………………….……27 Chapter 3 iTRAQ study of Grouper embryonic cells infected by Singapore grouper Iridovirus: an insight into the Singapore grouper iridovirus and host cell interaction ……………………………….…. are Iridovirus, Chloriridovirus and, Lymphocystivirus and Ranavirus (Chinchar et al., 2005). 1.3 Introduction of Singapore grouper iridovirus (SGIV) In 1994, a novel member of Ranavirus, Singapore