Studies of shrimp white spot syndrome virus by protein array platform and proteomics approaches

STUDIES OF SHRIMP WHITE SPOT SYNDROME VIRUS BY PROTEIN ARRAY PLATFORM AND PROTEOMICS APPROACHES CHEN JING (B. Sc, M. Sc., Ocean University of Qingdao, China) A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY DEPARTMENT OF BIOLOGICAL SCIENCES NATIONAL UNIVERSITY OF SINGAPORE 2008 Dedicated to My Family ２ Acknowledgements I thank my supervisor, Professor Hew Choy Leong, from the bottom of my heart for his invaluable guidance, advice and mentorship. Many thanks for providing me the opportunity to pursue my PhD degree in a motivating and enthusiastic research environment in the Department of Biological Sciences, National University of Singapore. I am deeply indebted to Dr. Li Zhengjun, for her continuous support and constructive comments on our research project. She also gave me many valuable suggestions and help about the writing of manuscripts and thesis. I acknowledge Dr. Lin Qingsong for his collaboration on the proteomics work, assistance on protein array experiment work and reviewing of manuscripts. I appreciate Dr Wu Jinlu for his instruction and assistance on electron microscopy and virus purification and the former colleagues Dr. Zhang Xiaobo, Dr. Song Wenjun for their guidance on experimental work. I thank Mr. Shashikant Joshi for reviewing of manuscripts and all the staff in the Proteins and Proteomics Centre (PPC) for their technical supports on mass spectrometry. I would like to extend my thanks to Ms. Low Siew See and previous colleague Ms. Qiu Jin for their kind help on the research project work. I thank all my lab colleagues Liu Yang, Wang Fan, Chen Liming, Tran Bich Ngoc for their support and help on the experimental work. Thanks specially go to Ms. Tang Xuhua and Ms. Zhuang Ying for their sincerity and friendship. Finally, I would like to express my heart-felt gratitude to my dear parents for their selfless love and support all the way. I would also like to thank my husband, Wang Xingang, for his help, understanding and encouragement throughout my graduate studies. i Table of Contents Acknowledgements i Table of Contents ii Summary . vi List of Tables viii List of Figures . ix List of Abbreviations . xi Literature Review 1.1 Overview of White Spot Syndrome Virus 1.1.1 Shrimp and Crayfish Viruses 1.1.2 Characteristics of WSSV . 1.2 Research Progress of WSSV 10 1.2.1 Isolation and Propagation of WSSV . 10 1.2.2 Complete Genomic Study of WSSV 10 1.2.3 Proteomics Study of WSSV . 11 1.2.4 Localization of Structural Proteins in the WSSV Virion . 11 1.2.5 Structural Study of WSSV 13 1.2.6 Functional Study of WSSV 14 1.3 Approaches for WSSV Functional Study . 15 1.3.1 Gateway Technology 15 1.3.2 Protein Array Technology 18 1.3.3 Common Techniques for Protein-Protein Interaction Studies 21 1.3.3.1 Protein Overlay Assay 21 1.3.3.2 Pull Down Assay 22 1.3.3.3 Co-immunoprecipitation . 22 1.3.4 Proteomics Approaches 23 1.4 Objectives of the Study 29 1.5 Significance of the Study 29 1.6 Scope of the Study 29 ii Construction of WSSV Entry and Expression Clones Using Gateway System . 30 2.1 Introduction 31 2.2 Material and Methods . 31 2.2.1 Extraction of WSSV Genomic DNA 31 2.2.2 Producing attB-PCR Products of WSSV ORFs . 31 2.2.2.1 Design of WSSV attB PCR Primers 31 2.2.2.2 Amplifying attB-PCR Products 32 2.2.2.3 Agarose Gel Electrophoresis and Purification of attB-PCR Products . 32 2.2.3 Construction of WSSV Entry Clones 33 2.2.3.1 Preparation of E.coli Competent Cells . 33 2.2.3.2 BP Recombination Reaction . 34 2.2.3.3 Entry Clones Sequencing . 35 2.2.4 Construction of WSSV Expression Clones 35 2.3 Results 36 2.3.1 Producing attB-PCR Products of WSSV ORFs . 36 2.3.2 Construction of WSSV Entry Clones . 43 2.3.3 Construction of WSSV Expression Clones 48 2.4 Discussion and Conclusions . 50 Expression and Purification of Recombinant WSSV Proteins 51 3.1 Introduction 52 3.2 Material and Methods . 52 3.2.1 Protein Analytical Techniques . 52 3.2.1.1 SDS-PAGE Gel Electrophoresis 52 3.2.1.2 Western Blot Analysis 53 3.2.1.3 MALDI TOF Spectrometry to Identify Proteins 54 3.2.2 Expression of Recombinant WSSV Proteins . 55 3.2.2.1 Transformation of BL21Cells . 55 3.2.2.2 Expression and Solubility Test of Recombinant WSSV Proteins 56 3.2.3 Large Scale Culture and Purification of Recombinant WSSV Proteins . 57 iii 3.2.4 Desalting and Lyophilization of the Purified Proteins . 58 3.3 Results and Discussion . 59 3.3.1 The WSSV Recombinant Protein Expression in E. coli System 59 3.3.2 Large Scale Cultured and Purified Recombinant WSSV Proteins . 61 Study of White Spot Syndrome Virus by Protein Array Platform . 65 4.1 Introduction 66 4.2 Material and Methods . 66 4.2.1 Protein Array 66 4.2.1.1 Labeling Probe Protein Samples with Dye . 66 4.2.1.2 Protein Array Procedure . 66 4.2.2 Pull Down Assay 67 4.2.3 Protein Overlay Assay 67 4.2.4 Co-immunoprecipitation . 68 4.3 Results and Discussion . 69 4.3.1 Interaction of WSSV Proteins with Actin 69 4.3.2 Interaction of WSSV Proteins with Shrimp Cytoskeleton and Nuclear Proteins75 Study of White Spot Syndrome Virus by Proteomics Approaches . 79 5.1 Introduction 80 5.2 Material and Methods . 80 5.2.1 Proliferation and Isolation of WSSV Virions . 80 5.2.2 Shotgun Proteomics Analysis of WSSV Structural Proteins . 81 5.2.3 Separation of Viral Envelope and Nucleocapsid Proteins 82 5.2.4 Western Blot Analysis of Envelope and Nucleocapsid Fractions 83 5.2.5 ITRAQ Labeling and Two Dimensional (2D) LC-MALDI MS to Determine Viral Protein Localization 83 5.2.6 Antibody Preparation of Novel WSSV Structural Proteins . 85 5.2.7 Localization Study by Western Blot Analysis and Immunogold Electron Microscopy Technique . 86 5.2.8 Protein-Protein Interaction Studies of Novel WSSV Structural Proteins 87 5.3 Results 87 iv 5.3.1 Identification of Virion-associated Proteins by Shotgun Proteomics . 87 5.3.2 Separation of WSSV Envelope and Nucleocapsid Proteins . 90 5.3.3 Localization of WSSV Structural Proteins by iTRAQ . 92 5.3.4 Localization of Novel WSSV Structural Proteins by Western Blot Analysis and Immunogold Electron Microscopy Observation 97 5.3.5 Protein-Protein Interaction Studies of Novel WSSV Structural Proteins 101 5.4 Discussion and Conclusions . 104 General Conclusion and Future Studies 109 Bibliography 113 Appendices . 121 List of Publications . 123 v Summary White spot syndrome virus (WSSV) is the most serious pathogen in shrimp aquaculture recently. One of the objectives of this study is to apply protein array platform for high-throughput screening of WSSV protein functions. Gateway cloning technique was applied to construct entry and expression clones of the open reading frames of WSSV. The expressed and purified WSSV recombinant proteins were used to screen protein-protein interactions by protein array technology. Potential interactions of WSSV proteins with actin, shrimp cytoskeleton and nuclear proteins were screened. It was found that wsv006, wsv077, wsv254, wsv407, wsv477 and wsv076 interacted with actin. The interaction of wsv006 with actin was confirmed by protein overlay assay and the interaction of wsv254 with actin was further confirmed by co-immmunoprecipitation. By interacting with actin, both of the structural proteins may help the viral nucleocapsid to move toward the host nucleus. Several viral proteins were found to interact with shrimp cytoskeleton and nuclear proteins separately by the protein array screening. Pull down assay of wsv254, wsv407 with shrimp cytoskeleton proteins and wsv254, wsv493 with shrimp nuclear proteins were carried out, but no specific binding was found. Meanwhile, shotgun proteomics was applied to investigate the WSSV proteome and 45 viral proteins were identified and 13 of them were reported for the first time. Furthermore, 23 envelope proteins and nucleocapsid proteins were identified by iTRAQ (isobaric tags for relative and absolute quantification). Among them, 12 envelope proteins and nucleocapsid proteins were identified for the first time. Two novel proteins vi wsv010 and wsv432 identified in the shotgun proteomics study were shown to be viral envelope proteins by Western blot and immunoelectron microscopy. Furthermore, pulldown assay revealed that wsv010 could interact with VP24, a major WSSV envelope protein. Previous studies indicated that VP24 could also interact with another two major WSSV structural proteins VP26 and VP28. Therefore, we proposed that VP24 may act as a linker protein to associate these envelope proteins together to form a complex, which may play an important role in viral morphogenesis and viral infection. This comprehensive study of WSSV proteins should facilitate the studies of the WSSV assembly and mechanism of infection. It should also provide the foundation for the development of drugs to control this virus disease. vii List of Tables Table 1.1 The DNA and RNA viruses of penaeid shrimp Table 2.1 WSSV ORF database in our laboratory 38 Table 3.1 Summary of purified recombinant WSSV proteins 62 Table 5.1 Structural Proteins of WSSV Identified by Shotgun Proteomics . 89 Table 5.2 Envelope Proteins and Nucleocapsid Proteins of WSSV Identified by iTRAQ . 95 Table 5.3 The Localization of Structural Proteins in WSSV 96 Table 5.4 Measured and calculated molecular masses of tryptic peptides which match VP24 of shrimp white spot syndrome virus 102 viii study could provide new information for investigation of the molecular mechanisms of virus assembly and virus entry. Among the novel structural proteins, the localizations of wsv010 and wsv432 were confirmed by routine western blot analysis and IEM technique. The pull-down assay of wsv010 showed the interaction of wsv010 with VP24. This result suggests that the association of wsv010 with VP24 either directly or indirectly may allow wsv010 to anchor to the envelope. A previous study indicated that VP24 could also interact with important structural proteins VP28 and VP26 to form a complex that plays a role in virus infection (Xie and Yang, 2006). Thus, we postulate that VP24 may play a role as a linker protein to link up the structural proteins VP28, VP26, wsv010, and probably other envelope proteins, to form a complex on the viral envelope. The possibility of existence of this complex will be tested in future study. Since both VP26 and VP28 are important for virus infection, it will be interesting to determine if wsv010 is also critical for virus entry. The characterization of the interaction of wsv010 with VP24 will enrich our understanding of viral morphogenesis and viral infection. 108 CHAPTER SIX General Conclusion and Future Studies 109 Due to the low homology of WSSV genome with other genes of known functions and the lack of a suitable shrimp cell line, we applied protein array platform for highthroughput screening of WSSV protein functions and proteomics approaches for investigation of the WSSV structural proteins. Firstly, Gateway cloning technique was applied to construct entry and expression clones of the ORFs of WSSV in a high-throughput format. Entry clones of nearly all the WSSV ORFs were constructed and sequenced. Expression clones of most WSSV ORFS for E. coli expression system were constructed. Totally 65 WSSV proteins were expressed as soluble proteins and 45 were expressed as insoluble proteins by western blotting identification. The recombinant proteins expressed as soluble proteins and the recombinant proteins expressed as insoluble proteins containing few cysteine residues were chosen for large scale culture and purification. Several recombinant WSSV proteins were produced with high yield and purity. But many of the recombinant WSSV proteins were poorly produced. Until now, 33 purified proteins were obtained and 20 of them were purified as soluble proteins. Except wsv146, wsv321 and wsv386, all of the purified proteins were confirmed by MALDI-TOF mass spectrometry analysis. To understand the mechanism of the virus infection, it is very important to study the interactions of virus proteins with host proteins. The purified recombinant WSSV proteins were used for protein array experiments to screen potential virus-host proteinprotein interactions. Potential interactions of WSSV proteins with actin, shrimp cytoskeleton and nuclear proteins were screened in this study. The result of protein array probed with actin showed that wsv006, wsv077, wsv254, wsv407, wsv477 and wsv076 have binding signal with actin. The interaction of wsv006 with actin was confirmed by 110 protein overlay assay and the interaction of wsv254 with actin was confirmed by coimmmunoprecipitation. By interacting with actin, both of the structural proteins may help the viral nucleocapsid to move toward the host nucleus. From the protein array results probed with shrimp cytoskeleton and nuclear proteins, several WSSV proteins were found to interact with shrimp cytoskeleton and nuclear proteins separately. Pull down assay of wsv254, wsv407 with shrimp cytoskeleton proteins and wsv254, wsv493 with shrimp nuclear proteins were carried out, but no specific binding was found. The interaction conditions for pull down assay will be optimized and other protein-protein interaction methods will be applied to validate the protein array results. Meantime, we applied shotgun proteomics using offline coupling of LC system with MALDI TOF/TOF MS/MS to investigate the WSSV proteome. By this approach, 45 structural proteins were identified and 13 of them were reported for the first time. Furthermore, iTRAQ was employed to distinguish WSSV envelope proteins and nucleocapsid proteins. Based on iTRAQ ratios, 23 envelope proteins and nucleocapsid proteins were successfully identified. Our results validated 15 structural proteins with previously known localization and determined localization of additional 12 envelope proteins and nucleocapsid proteins. iTRAQ was demonstrated to be an effective approach for high-throughput viral protein localization determination. In summary, WSSV was assembled by at least 58 structural proteins. The localization of 43 structural proteins has been determined, 34 of which were envelope proteins and as nucleocapsid proteins. Two novel proteins wsv010 and wsv432 identified in the shotgun proteomics study were shown to be viral envelope proteins by western blotting and IEM technology. 111 Furthermore, the pull-down assay revealed that wsv010 could interact with VP24, which is a major WSSV envelope protein. Since wsv010 lacks a transmembrane domain, these results suggest that wsv010 may anchor to the envelope through interaction with VP24. Previous studies indicated that VP24 could also interact with another two major WSSV structural proteins VP26 (wsv311) and VP28 (wsv421) (Xie, Xu, and Yang, 2006; Xie and Yang, 2006). Therefore, we proposed that VP24 may act as a linker protein to associate these envelope proteins together to form a complex, which may play an important role in viral morphogenesis and viral infection. In conclusion, protein array of WSSV proteins will be continuously applied to analyze virus protein-protein interactions, virus-host protein-protein interactions. 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Identification of an envelope protein (VP39) gene from shrimp white spot syndrome virus. Arch Virol 151(1), 7182. Zieske, L. R. (2006). A perspective on the use of iTRAQTM reagent technology for protein complex and profiling studies Journal of Experimental Botany 57, 1501-1508. 120 Appendices Media and solutions LB broth (per liter) To 950 mL of MQ H2O, add: Bacto-tryptone 10 g Bacto-yeast extract 5g NaCl 10 g Dissolve solutes. Adjust pH to 7.0 with M NaOH. Adjust volume to 1L with MQ H2O. Sterilize by autoclaving. SOC medium (per liter) To 950 mL of MQ H2O, add: Bacto-tryptone 20 g Bacto-yeast extract 5g NaCl 0.58 g Dissolve solutes. Add 10 mL of 250 mM KCl. Adjust pH to 7.0 with M NaOH. Adjust volume to 980 mL with MQ water. Sterilize by autoclaving. Allow to cool to 60°C or less, and then add 20 mL of sterile 1M glucose. Add 10 mL of sterile M MgCl2 just prior to use. Tfb I (per liter) To 950 mL of MQ H2O, add: CH3COOK 2.94 g RbCl 12.1 g CaCl2·2H2O 14.7 g MnCl2·4H2O 9.9 g Glycerol 150 mL 121 Adjust pH to 5.8 with dilute acetic acid, add MQ H2O to L and autoclave. Tfb II (per liter) To 950 mL of MQ H2O, add: MOPS 2.1 g CaCl2 11.1 g RbCl 1.21 g Glycerol 150 mL Adjust pH to 6.5 with M NaOH, add MQ H2O to L and autoclave. Kits Compartmental protein extraction Kit Chemicon PCR Cloning System with Gateway Technology Invitrogen QIAquick PCR purification kit QIAGEN QIAprep spin miniprep kit QIAGEN QIAquick gel extraction kit QIAGEN BigDye® terminator v3.1 cycle sequencing kit Applied Biosystems Hi-Di formamide Applied Biosystems Equipments High Speed Centrifuge Beckman, J2-21 Ultracentrifuge Beckman, XL-90 MicroPulser® Electroporator Bio-Rad Thermal Cycler Bio-Rad ABI PRISM™ 3100 Genetic Analyzer Applied Biosystems Voyager-DE™ STR BioSpectrometry Workstation Applied Biosystems 4700 Proteomics Analyzer with TOF/TOF™ optics Applied Biosystems Ultimate™ LC system Dionex LC Packings Typhoon 9410 instrument Amersham Bioscience 122 List of Publications 1. Jing Chen*, Zhengjun Li *, Choy-Leong Hew. (2007) Characterization of a novel envelope protein WSV010 of shrimp white spot syndrome virus and its interaction with a major viral structural protein VP24. Virology. 20; 364(1):20813. 2. Zhengjun Li*, Qingsong Lin*, Jing Chen*, Jin Lu Wu, Teck Kwang Lim, Siew See Loh, Xuhua Tang, and Choy-Leong Hew. (2007) Shotgun identification of structural proteome of shrimp white spot syndrome virus and iTRAQ differentiation of envelope and nucleocapsid subproteomes Mollecular & Cellular Proteomics. (9): 1609-1620. * equal contribution. 123 [...]... With protein array, the biochemical activities of proteins can be systematically analyzed by probing proteins in a high-throughput fashion Another advantage of protein array is that it is very sensitive and only requires a small quantity of protein in each assay Currently, there are mainly two classes of protein arrays: analytical and functional protein arrays (David A Hall, 2007; LaBaer and Ramachandran,... Protein array of WSSV proteins with shrimp cytoskeleton proteins 76 Figure 4.5 Protein array of WSSV proteins with shrimp nuclear proteins 77 Figure 5.1 Electron micrographs of negatively stained WSSV 88 ix Figure 5.2 Western blot analysis of total proteins, envelope proteins and nucleocapsid proteins 91 Figure 5.3 The iTRAQ labeling workflow and 2D LC MS for the localization of structural... kinase WSSV white spot syndrome virus xi CHAPTER ONE Literature Review 1 1.1 Overview of White Spot Syndrome Virus 1.1.1 Shrimp and Crayfish Viruses Shrimp aquaculture has been an important industry for several decades in many countries worldwide However, since 1992, shrimp diseases have emerged as a major constraint to the continual expansion of this industry Many diseases are caused by environmental... knowledge of WSSV structural proteins A total of 38 proteins were identified by these three proteomics studies Most recently, 11 additional WSSV proteins were identified for the first time in infected shrimp epithelium by shotgun proteomics and were tentatively postulated as potential candidates of non-structural proteins (Wu et al., 2007) 1.2.4 Localization of Structural Proteins in the WSSV Virion... Representatives of identification of WSSV recombinant protein expression by Western Blot assay 60 Figure 3.2 Purification of wsv069 63 Figure 3.3 MALDI-TOF result of wsv069 64 Figure 4.1 Protein array of WSSV proteins with actin 70 Figure 4.2 Protein overlay assay of WSSV proteins with actin 72 Figure 4.3 Coimmunoprecipitation of wsv254 with actin 74 Figure 4.4 Protein. .. biochemical activities of an entire proteome in a single experiment They are used to study numerous protein interactions, such as protein protein, protein DNA, protein phospholipid, and protein small molecule interactions (Fig 1.7) (Zhu and Snyder, 2003) An example of protein array experiment is shown in Fig 1.8 (Zhu et al., 2001) 18 Fig 1.7 Applications of protein microarrays (Zhu and Snyder, 2003) There... wide range of biochemical activities (e.g protein protein, protein lipid, protein nucleic-acid, and enzyme–substrate interactions), as well as drug and drug target identification Small molecule and carbohydrate microarrays are other types of analytical microarrays that have been demonstrated to be capable of studying protein binding activities to ligands and carbohydrates 19 Fig1.8 Examples of different... deterioration and intensive aquaculture Among all the pathogens, viruses are the biggest threat to the shrimp aquaculture industry Roughly 20 shrimp viruses have been found in penaeid shrimp (Table 1.1) (Chen, 1997; Lightner, 1998) It should be pointed out that systemic ectodermal and mesodermal baculovirus (SEMBV), rod-shaped virus of Penaeus japonicus (RV-PJ), white spot baculovirus (WSBV) and hypodermal and. .. general types of protein microarray: analytical and functional protein microarrays Analytical microarrays involve a high-density array of affinity reagents (e.g antibodies or antigens) that are used for detecting proteins in a complex mixture Functional protein chips are constructed by immobilizing large numbers of purified proteins on a solid surface Unlike the antibody–antigen chips, protein chips... syndrome virus was from the distinctive feature of white spots in the cuticle of the acutely infected shrimp (Fig 1.1) (Kiatpathomchai et al., 2001; Wang et al., 1999) The white spots are abnormal deposits of calcium, which probably caused by the disruption of exudates transfering from epithelial cells to the cuticle via cuticular pore canals 2 Table 1.1 The DNA and RNA viruses of penaeid shrimp (Lightner, . STUDIES OF SHRIMP WHITE SPOT SYNDROME VIRUS BY PROTEIN ARRAY PLATFORM AND PROTEOMICS APPROACHES CHEN JING (B. Sc, M. Sc., Ocean University of Qingdao, China). Interaction of WSSV Proteins with Actin 69 4.3.2 Interaction of WSSV Proteins with Shrimp Cytoskeleton and Nuclear Proteins75 5 Study of White Spot Syndrome Virus by Proteomics Approaches. Cultured and Purified Recombinant WSSV Proteins 61 4 Study of White Spot Syndrome Virus by Protein Array Platform 65 4.1 Introduction 66 4.2 Material and Methods 66 4.2.1 Protein Array 66