Structural and functional studies of VP9, a novel nonstructural protein from white spot syndrome virus

STRUCTURAL AND FUNCTIONAL STUDIES OF VP9, A NOVEL NONSTRUCTURAL PROTEIN FROM WHITE SPOT SYNDROME VIRUS LIU YANG (B.Sc., Xiamen University) A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY DEPARTMENT OF BIOLOGICAL SCIENCES NATIONAL UNIVERSITY OF SINGAPORE 2007 ii Dedicated to My Family Table of Contents Table of Contents i Acknowledgement viii Abstract x List of Figures xi List of Tables xiii List of Abbreviations xiv Chapter Literature Review Introduction 1.1 Introduction to virus 1.2 Introduction to crustacean virus 1.3 Introduction to WSSV 1.3.1 Background 1.3.2 Structural features of WSSV 1.4 1.3.3 Classification of WSSV Research progress of WSSV 1.4.1 Sequence determination and analysis 1.4.2 Viral proteins identification 1.4.2.1 Latency-related genes identification 1.4.2.2 Immediate-early genes identification i 1.5 1.6 1.4.2.3 Structural genes identification 10 1.4.2.4 Nonstructural genes identification 11 Introduction to methodology 14 1.5.1 Protein purification techniques 14 1.5.1.1 Affinity chromatography 14 1.5.1.2 Ion exchange chromatography 15 1.5.1.3 Size exclusion chromatography 15 1.5.2 Quantitative real-time RT-PCR 16 1.5.3 X-ray crystallography 17 1.5.4 NMR spectroscopy 21 Objectives of this project Chapter Materials and Methods 2.1 26 27 Materials 28 2.1.1 Enzyme and other proteins 28 2.1.2 Kit and reagents 28 2.1.3 Media 28 2.1.3.1 LB medium 28 2.1.3.2 M9 medium 29 Stock solutions and buffers 29 2.1.4.1 IPTG stock solution 29 2.1.4.2 Ampicillin stock solution 29 2.1.4 ii 2.1.4.3 Buffers for Ni-NTA purification under native conditions 2.1.5 2.2 30 E.coli strains 2.1.6 Plasmid for protein expression 2.1.7 NMR chemicals and sample tube 30 30 Methods 31 2.2.1 Molecular biology techniques (DNA related) 31 2.2.1.1 PCR 31 2.2.1.2 Agarose gel electrophoresis 31 2.2.1.3 PCR products purification 31 2.2.1.4 Enzyme digestion, dephosphorylation and purification 2.2.1.5 Ligation and transformation 31 32 2.2.1.6 Positive clone screening and plasmid preparation 2.2.2 32 2.2.1.7 Cycle Sequencing Reaction 33 2.2.1.8 Sequence determination 33 2.2.1.9 Transformation 34 Protein manipulation techniques 34 2.2.2.1 Small scale test 34 2.2.2.2 Large scale production of recombinant protein 35 iii 2.2.2.3 SDS-PAGE 35 2.2.2.4 Cell storage 36 2.2.2.5 Production of polyclonal antibodies 36 2.2.2.6 Western blot 36 2.2.2.7 Silver staining 37 Chapter Characterization of VP9 38 3.1 Introduction 39 3.2 Materials and methods 39 3.3 3.4 3.2.1 Materials 39 3.2.2 Construction of the expression plasmid 41 3.2.3 Expression and purification of VP9 41 3.2.4 Mass spectrometry analysis 42 3.2.5 Dynamic light scattering study 42 3.2.6 Circular dichroism study 43 Results 43 3.3.1 Hydrophobicity plot 43 3.3.2 Protein purification profiles of VP9 43 3.3.3 Mass spectrometry analysis 44 3.3.4 Dynamic light scattering study 44 3.3.5 Circular dichroism study 44 Discussion 52 iv Chapter Functional Studies of VP9 53 4.1 Introduction 54 4.2 Materials and methods 55 4.2.1 Materials 55 4.2.2 Shrimp infection with WSSV 55 4.2.3 WSSV purification 55 4.2.4 Real-time RT-PCR 56 4.2.4.1 RNA extraction 56 4.2.4.2 Reverse transcription 57 4.2.4.3 Real-time PCR 57 4.2.5 Localization by Western blot 58 4.2.6 Localization by immuno-electron microscopy 59 4.2.7 Pull down assay 60 4.2.7.1 Bait protein preparation 60 4.2.7.2 Prey protein preparation 61 4.2.7.3 Pull down by Ni-NTA agarose beads 61 4.3 Results and discussions 62 4.3.1 Real-time RT-PCR 62 4.3.2 Localization 63 4.3.3 Pull down assay 63 v Chapter Structural Studies of VP9 75 5.1 Introduction 76 5.2 Materials and methods 76 5.2.1 Materials 76 5.2.2 X-ray studies 77 5.2.2.1 SeMet VP9 preparation 77 5.2.2.2 Crystallization 77 5.2.2.3 Data collection 77 NMR studies 78 5.2.3.1 Sample preparation 78 5.2.3.2 NMR experiments and data process 78 5.2.3.3 NMR relaxation studies 79 5.2.3.4 NMR metal titration 80 5.2.3 5.3 Results and discussions 5.3.1 5.3.2 80 X-ray studies 80 5.3.1.1 SeMet VP9 preparation 80 5.3.1.2 Crystallization 81 5.3.1.3 Data collection 81 5.3.1.4 Structure solution and refinement 82 5.3.1.5 Crystal structure of VP9 83 NMR studies 89 5.3.2.1 Sample preparation 89 vi 5.3.3 5.3.2.2 NMR structure 89 5.3.2.3 NMR relaxation studies 90 VP9 interacts with metals 95 5.3.3.1 Metal binding sites 95 5.3.3.2 NMR metal titration 99 5.3.4 Comparison of crystal structure vs. NMR structure 101 5.3.5 Sequence and structural homology 101 5.3.6 Functional implications 104 Chapter Summary and Future Studies 107 6.1 Summary 108 6.2 Future studies 109 6.2.1 Establishment of cell line 109 6.2.2 RNAi 109 6.2.3 Structural genomics 110 6.2.4 Structure-based drug design 112 Coordinates 114 References 115 Appendices 126 Publications 127 vii immuno-EM can be used to determine the location of individual particles in the virus particles. With the available X-ray structures of component proteins, these can be combined with the overall EM structure to provide a more complete structural description. Structural genomics aims to determine the structures of all structured proteins. Besides elucidating the functions of the presumptive proteins based on the determined structure, such study should provide the essential structural insight for the designing of inhibitors or small molecules. Ultimately, drugs can be developed to keep the WSSV infection under control and therefore boost the shrimp aquaculture industry. 6.2.4 Structure-based drug design The analysis of protein structure is often performed with one goal in mind: to design a ligand capable of binding to it and moderating its activity (Stewart, 2002). Our X-ray and NMR-based structural studies revealed that VP9 possesses a DNA recognition fold with specific metal binding sites (coordinating residues include Asp9, Glu31, and Cys46). The specific DNA sequence for recognition by VP9 has not yet been established. However, based on homology modeling, a possible DNA binding region located at α1 (Thr17-Thr26) and the β-turn (Ser36-Asp40) has been proposed (Liu et al., 2006). Thus, the metal binding sites, α1 and β-turn form three presumably active sites for de novo VP9-structure-based drug design. Algorithms are available to design ligands in silico and approach the task in different ways (Bohm, 1992; Gillet et al., 112 1993). Efforts to dock potential chemical groups and subsequently accept those groups that appear to bind well to the active site may be attempted; otherwise, we can dock a starting group or a substructure, and then add groups to this starting point, effectively “evolving” a ligand in the active site. Once identified, crystallization of lead structures in the protein target will be performed in order to enable further designing (Daniel, 2005). 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PLoS ONE (accepted) 127 [...]... to consist of a wide range of viral families, including Baculoviridae, Birnaviridae, Bunyaviridae, Herpesviridae, Piconaviridae, Parvoviridae, Reoviridae, Rhabdoviridae, Togaviridae, Iridoviridae, Nodaviridae and Nimaviridae Crustacean viral diseases listed by the OIE (Office International Des Epizooties; the World Organization for Animal Health) include Taura Syndrome (TS), White Spot Disease (WSD),... prey protein samples 69 Table 6 Real-time RT-PCR analysis of vp9, vp28 and 70 dnapol from 0 to 72 h.p.i Table 7 Data collection and refinement statistics 87 Table 8 NMR structural statistics 93 xiii List of Abbreviations 1D one-dimensional 2D two-dimensional 3D three-dimensional Å Ångstrom (10-10m) a. a amino acid ATPase adenosine triphosphatase AUC analytical ultracentrifugation bp base pair B0 magnetic... exchange chromatography and gel filtration chromatography The following introduction is adapted from Rajni (2003) 1.5.1.1 Affinity chromatography Affinity chromatography (AF) separates proteins based on reversible interaction between a protein and a specific ligand coupled to a chromatographic matrix One of the most common applications of AF is to purify recombinant proteins Proteins that are genetically... to any other known proteins 1.4.2 Viral proteins identification The availability of the complete WSSV sequence facilitated the global molecular characterization of the virus by genomic and proteomic approaches and has recently led to the discovery of many important WSSV genes (Sritunyalucksana et al., 2006) including latency-associated genes, immediate-early genes, structural genes and nonstructural. .. (Emiliani, 1993) Viruses are ubiquitous and abundant in nature and can infect and parasitize all living organisms from bacteria to mammals They are considered to be simple biological entities composed of a small number of macromolecules produced by, and thus derived from, the organism they infect There are more than 3000 families of viruses Viruses can differ greatly in their physical form They can be... sequence of WSSV (Yang et al., 2001) and viral particles consist of a relatively narrow range of proteins that have constant stable profiles Due to the introduction of proteomic methods, the total number of known WSSV viral structural proteins has been increased to 39 (Huang et al., 200 2a; Huang et al., 2002b, Li et al., 2004; Tsai et al., 2004), and Li et al further increased to 55 (unpublished data) However,... products of viruses generally are comprised of structural proteins 3 (SPs), which are the components of virus particles and nonstructural proteins (NPs), which act as regulators or cofactors controlling the viral infection process 1.2 Introduction to crustacean virus The first report of a crustacean virus was in the crab Macropipus depurator by Vago in 1966 (Vlak et al., 2004) Crustacean viruses are currently... that WSSV could exist in an asymptomatic carrier state Certain stress conditions such as transportation and poor water quality can induce the virus from a carrier state to infective state and initiate an outbreak (Tsai et al., 1999) Other researchers have also observed the symptoms of WSSV infection in normal shrimps that were thought to result from environmental stress rather than viral contamination... present and former members of Functional Genomics Laboratory as well as Structural Biology Laboratory Special thanks to Lim Daina and Thomas Hegendoerfer (Munich, Germany) for their contribution to the functional studies of VP9 I would like to thank Professor Wong Sek Man and A/ P Lin Tianwei (Scripps Research Institute, USA) for the guidance on Cowpea Mosaic Virus project Special thanks go to Mr Shashi... known about the functions of these structural proteins except that there was an identification of PmRab that binds directly to VP28 (Sritunyalucksana et al., 2006) 1.4.2.4 Nonstructural genes identification Besides structural proteins, nonstructural proteins are also required for replication of the viral genome, production of virus particles and inhibition of certain host cell functions These proteins are . Dr Asha, Dr Huang Canhua, Dr Wu Jinlu, Ms Tang Xuhua, Ms Sunita and, Mr. Jobi and the rest of the lab mates for the valuable discussion and friendship and the present and former members of Functional. STRUCTURAL AND FUNCTIONAL STUDIES OF VP9, A NOVEL NONSTRUCTURAL PROTEIN FROM WHITE SPOT SYNDROME VIRUS LIU YANG (B.Sc., Xiamen University) A THESIS SUBMITTED. Functional Genomics Laboratory as well as Structural Biology Laboratory. Special thanks to Lim Daina and Thomas Hegendoerfer (Munich, Germany) for their contribution to the functional studies of