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). A careful examination of such a co-complex will highlight areas where suboptimal interactions are present or where selectivity may be affected. 113 Coordinates The NMR structures and X-ray structure VP9 were deposited in the Protein Data Bank, with the PDB ID 2GJI and 2GJ2, respectively. 114 References Banci, L., Bertini, I., Ciofi-Baffoni, S., Finney, L.A., Outten, C.E., O`Halloran, T.V. (2002). a new zinc-protein coordination site in intracellular metal trafficking, solution structure of the apo and Zn (II) forms of ZntA (46-118) J.Mol.Biol, 323, 883-897. Bax, A. (2003).Weak alignment offers new NMR opportunities to study protein structure and dynamics, Protein Sci, 1, 1-16. Bergfors, T., ed. 1999). Protein Crystallization, Techniques, Strategies, and Tips, International University Line, La Jolla, CA. Blissard, G.W. (1996). Baculovirus—insect cell interactions. Cytotechnology, 20, 73-93. Blissard, G.W., Rohrmann, G.F. (1990). Baculovirus diversity and molecular biology. Annu. Rev. Entomol, 35, 127-155. Bloch F., Hansen W. W., and Packard M. (1946). Nuclear induction. Phys. Rev, 70, 460-474. Blow DM. (2002). Rearrangement of Cruickshank's formulae for the diffraction-component precision index. Acta Crystallogr D Biol Crystallogr, 58, 792-7. Brunger, A. T., Adams, P. D., Clore, G. M., DeLano, W. L., Gros, P., Grosse-Kunstleve, R. W., Jiang, J. S., Kuszewski, J., Nilges, M., Pannu, N. S., Read, R. J., Rice, L. M., Simonson, T., and Warren, G. L. (1998). Crystallography & NMR system, a new software suite for macromolecular structure determination, Acta Crystallogr. Sect. D, 54, 905-921. Bustin SA, Mueller R. (2005). Real-time reverse transcription PCR (qRT-PCR) and its potential use in clinical diagnosis. Clin Sci (Lond), 109, 365-79. Chang, P., C. F. Lo, Y. Wang, and G. H. Kou. (1996). Identification of white spot syndrome associated baculovirus (WSBV) target organs in the shrimp Penaeus monodon by in situ hybridization. Dis. Aquat. Org, 27, 131-139. Chaturvedi UC, Shrivastava R. (2005). Interaction of viral proteins with metal ions, role in maintaining the structure and functions of viruses. FEMS Immunol Med Microbiol, 43, 105-14. 115 Chayen, N.E., Turning. (2004). Protein crystallization from an art into a science. Curr. Opinions Struct Biol, 14, 577-583. Chen, L. L., C. F. Lo, Y. L. Chiu, C. F. Chang, and G. H. Kou. (2000). Natural and experimental infection of white spot syndrome virus (WSSV) in benthic larvae of mud crab Scylla serrata. Dis. Aquat. Org, 40, 157-161. Chen LL, Wang HC, Huang CJ, Peng SE, Chen YG, Lin SJ, Chen WY, Dai CF, Yu HT, Wang CH, Lo CF, Kou GH. (2002).Transcriptional analysis of the DNA polymerase gene of shrimp white spot syndrome virus. Virology, 301, 136-147. Clore, G. M. and Gronenborn, A. M. (1991). Prog. NMR Spectrosc, 23, 43-92. Cornilescu, G., Delaglio, F., and Bax, A. (1999). Protein backbone angle restraints from searching a database for chemical shift and sequence homology. J. Biomol. NMR, 13, 289–302. Daniel Figeys (2005). Industry Proteomics: Applications for Biotechnology and Pharmaceuticals. Publisher: Wiley-Liss. Delaglio, F., Grzesiek, S., Vuister, G. W., Zhu, G., Pfeifer, J., and Bax, A. (1995). NMRPipe, a multidimensional spectral processing system based on UNIX pipes. J. Biomol. NMR, 6, 277–293. de La Fortelle, E. & Bricogne, G (1997). Maximum-likelihood heavy-atom parameter refinement for multiple isomorphous replacement and multiwavelength anomalous diffraction methods. Methods Enzymol, 276, 472–494. Donald Lightner. (1999). Report to OIE of "notifiable" disease in shrimp. Dubochet, J., M. Adrian, J. J. Chang, J. C. Homo, J. Lepault, A. W. McDowall, and P. Schultz . (1988). Cryo-electron microscopy of vitrified specimens. Q. Rev. Biophys, 21, 129-228. Durand, S., D. V. Lightner, R. M. Redman, and J. R. Bonami. (1997). Ultrastructure and morphogenesis of white spot syndrome baculovirus (WSSV). Dis. Aquat. Organ, 29, 205-211. Emiliani, C. (1993). Extinction and viruses. BioSystems, 31, 155-159. Farrow NA, Zhang O, Forman-Kay JD, Kay LE. (1995). Comparison of the backbone dynamics of a folded and an unfolded SH3 domain existing in equilibrium in 116 aqueous buffer. Biochemistry, 34, 868-78. Fesik S. W., and Zuiderweg, E. R. P., Quart. (1990). Rev. Biophys, 23, 97-131. Freeman, R. and Anderson, W. A. (1962). J. Chem. Phys, 37, 2053-2073. Fischer MW, Zeng L, Majumdar A, Zuiderweg ER. (1998). Characterizing semilocal motions in proteins by NMR relaxation studies. Proc Natl Acad Sci U S A, 95, 8016-9. Flegel, T. W. (1997). Special topic review, major viral diseases of black tiger prawn (Penaeus monodon) in Thailand. World J. Microbiol. Biotechnol, 13, 443-42. Friesen, P.D. (1997). Regulation of Baculovirus early gene expression. In, Miller, L.K. (Ed.). The Baculoviruses, Plenum Press, New York, pp. 141-170. Friesen, P.D., Miller, L.K. (1986). The regulation of baculovirus gene expression. Curr. Top. Microbiol. Immunol, 131, 31-49. Greber, U. F., Suomalainen, M., Stidwill, R. P., Boucke, K., Ebersold, M. W., and Helenius, A. (1997). The role of the nuclear pore complex in adenovirus DNA entry. EMBO J, 16, 5998–6007. Greber, U. F., Webster, P., Weber, J., and Helenius, A. (1996). The role of the adenovirus protease in virus entry into cells. EMBO J, 15, 1766–1777. Hameed, A. S. S., M. Anilkumar, M. L. Stephen Raj, and K. Jayaraman. (1998). Studies on the pathogenicity of systemic ectodermal and mesodermal baculovirus and its detection in shrimp by immunological methods. Aquaculture, 160, 31-45. Hegde RS, Grossman SR, Laimins LA, Sigler PB. (1992). Crystal structure at 1.7 A of the bovine papillomavirus-1 E2 DNA-binding domain bound to its DNA target. Nature, 359, 505-12. Herrmann, T., Guntert, P & Wuthrich, K. (2002). Protein NMR structure determination with automated NOE assignment using the new software CANDID and the torsion angle dynamics algorithm DYANA. J. Mol. Biol, 319, 209-227. Holm, L. and Sander, C. (1995). Trends Biochem. Sci, 20, 478–480. Honess, R.W., Roizman, B. (1974). Regulation of herpesvirus macromolecular synthesis, I. Cascade regulation of the synthesis of three groups of viral proteins. J. Virol, 14, 8-19. Huang, C., X. Zhang, Q. Lin, X. Xu, and C. L. Hew. (2002a). Characterization of a 117 novel envelope protein (VP281) of shrimp white spot syndrome virus by mass spectrometry. J. Gen. Virol, 83, 2385-2392. Huang, C., X. Zhang, Q. Lin, X. Xu, Z. Hu, and C. L. Hew. (2002b). Proteomic analysis of shrimp white spot syndrome viral proteins and characterization of a novel envelope protein VP466. Mol. Cell. Proteomics, 1, 223-31. Huang, C., Zhang, L., Zhang, J., Xiao, L., Wu, Q., Chen, D., and Li, J. K. (2001). Purification and characterization of white spot syndrome virus (WSSV) produced in an alternate host, crayfish, Cambarus clarkii. Virus Res, 76, 115–125. Hsu, Y., Y. Yang, Y.Chen, M. Tung, J. Wu, M. Engeking and J. Leong. (1995). Development of an invitro subcuture system for the oka organ (Lymphoid tissue) of Penaeus monodon. Aquaculture, 136, 43-55. James L. Cole and Jeffrey C. (1999). Hansen Analytical Ultracentrifugation as a Contemporary Biomolecular Research Tool. J Biomol Tech, 10, 163-176. Jean-Luc DARLIX, Branka HORVAT, Viktor VOLCHKOV and all the participants. (2005). "Virus & Immunity" Course, Lyon, France. http,//vandi.ens-lyon.fr/virus_and_immunity. Johnson B.A. (2004). Using NMRView to visualize and analyze the NMR spectra of macromolecules. Methods Mol Biol, 278, 313-52. Jones, T. A., Zou, J. -Y., Cowan, S. W., and Kjeldgaard, M. (1991). Improved methods for building models in electron density maps and the location of errors in these models. Acta Crystallogr. Sect. A, 47,110-119. Kann, M., Sodeik, B., Vlachou, A., Gerlich, W. H., and Helenius, A. (1999). Phosphorylation-dependent binding of hepatitis B virus core particles to the nuclear pore complex. J. Cell Biol. 145, 45–55. Kasamatsu, H., and Nakanishi, A. (1998). How animal DNA viruses get to the nucleus? Annu. Rev. Microbiol. 52, 627–686. Kenneth H. Lundstrom. (2006). Structural genomics on membrane proteins. CRC Press, Taylor & Francis Group. 6000 Broken Sound Parkway NW, Suite 300. Boca Raton, FL 33487-2742. Khadijah S, Neo SY, Hossain MS, Miller LD, Mathavan S, Kwang J. (2003). Identification of white spot syndrome virus latency-related genes in specific-pathogen-free shrimps by use of a microarray. J Virol. 77, 10162-7. Koradi, R., Billeter, M., and Wüthrich, K. (1996). MOLMOL, a program for display 118 and analysis of macromolecular structures. J Mol Graphics 14, 51-55. Kyte, J. and Doolittle, R. F. (1982). A simple method for displaying the hydropathic character of a protein. J. Mol. Bio, 157, 105-132. Laemmli, U.K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature, 227, 680-685. Lamitina T. (2006). Functional genomic approaches in C. elegans. Methods Mol Biol, 351, 127-38. Laskowski, R. A., MacArthur, M. W., Moss, D. S., and Thornton, J. M. (1993). PROCHECK, a program to check the stereochemical quality of protein structures. J. Appl. Crystallogr, 26, 283-291. Leu, J. H., J. M. Tsai, H. C. Wang, A. H. Wang, C. H. Wang, G. H. Kou, and C. F. Lo. (2005). The unique stacked rings in the nucleocapsid of the white spot syndrome virus virion are formed by the major structural protein VP664, the largest viral structural protein ever found. J. Virol. 79, 140-149. Li, Q., Y. Chen, and F. Yang. (2004). Identification of a collagen-like protein gene from white spot syndrome virus. Arch. Virol. 149,215-223. Li, Y., Lu, Z., Sun, L., Ropp, S., Kutish, G. F., Rock, D. L., and Van Etten, J. L. (1997). Analysis of 74 kb of DNA located at the right end of the 330-kb chlorella virus PBCV-1 genome. Virology, 237, 360-377. Lightner, D. V. (1996). A handbook of pathology and diagnostic procedures for diseases of penaeid shrimp. World Aquaculture Society, Baton Rouge, LA. Liu, W. J., H. T. Yu, S. E. Peng, Y. S. Chang, H. W. Pien, C. J. Lin, C. J. Huang, M. F. Tsai, C. H. Wang, J. Y. Lin, C. F. Lo, and G. H. Kou. (2001). Cloning, characterization, and phylogenetic analysis of a shrimp white spot syndrome virus gene that encodes a protein kinase. Virology, 289, 362-377. Liu WJ, Chang YS, Wang CH, Kou GH, Lo CF. (2005). Microarray and RT-PCR screening for white spot syndrome virus immediate-early genes in cycloheximide-treated shrimp. Virology, 10, 327-41. Liu WJ, Chang YS, Wang AH, Kou GH, Lo CF. (2006). WSSV has Successfully Annexed a Shrimp STAT To Enhance the Expression of the Immediate Early Gene (ie1). J Virol, Nov 1; [Epub ahead of print] Liu, Y, Wu JL, Song JX, Sivaraman1 J, Hew CL. (2006). Identification of a novel 119 nonstructural protein VP9 from white spot syndrome virus, its structure reveals a ferredoxin fold with specific metal binding sites. J Virol, 80, 10419-27. Lo, C. F. (2005). One step ahead of the emerging crustacean viruses. http,//www.iq2000kit.com/news_019a.htm. Lo, C.F., C.T. Cheng, ad G. H. kou. (1998). PCR monitoring of cultured shrimp for white spot syndrome virus (WSSV) infection in grow out ponds In T.W. flegel (ed.). Advances in shrimp biotechnology. National Center for Genetic Engineering and Biotechnology, Bangkok, Thailand, P281-286. Lu, R., Maduro, M., Li, F., Li, H.W., Broitman-Maduro, G., Li, W.X., Ding, S.W. (2005). Animal virus replication and RNAi-mediated antiviral silencing in Caenorhabditis elegans. Nature, 436, 1040–1043. Luedman, R.A and D.V. Lightner. (1992). Development of an in vitro primary cell culture system from the penaeid shrimp, Penaeus stylirostris and Penaeus vannamei. Aquaculture, 101, 205-211. Magbanua, F. O., K. T. Natividad, V. P. Migo, C. G. Alfafara, F. O. de la Pena, R. O. Miranda, J. D. Albaladejo, E. C. Nadala, Jr., P. C. Loh, and L. Nahilum-Tapay. (2000). White spot syndrome virus (WSSV) in cultured Penaeus monodom in the Philippines. Dis. Aquat. Org, 42, 77-82. Mandel AM, Akke M, Palmer AG III. (1996). Dynamics of ribonuclease H, temperature dependence of motions on multiple time scales. Biochemistry, 35, 16009-23. Marks, H., R. W. Goldbach, J. M. Vlak, and M. C. W. van Hulten. 2004. Genetic variation among isolates of white spot syndrome virus. Arch. Virol, 149, 673-697. Marsh, M., and Helenius, A. (1989). Virus entry into animal cells. Adv. Virus Res, 36, 107–151. Mayo, M. A. (2002). A summary of taxonomic changes recently approved by ICTV. Arch. Virol, 147, 1655-63. McPherson A. (1993). Effects of a microgravity environment on the crystallization of biological macromolecules. Microgravity Sci Technol, 6, 101-9. Moradian-Oldak, J., Leung, W., and Fincham, A.G (1998). Temperature and pH-dependent supramolecular self-assembly of amelogenin molecules, a dynamic light-scattering analysis. J. Struct Biol, 122, 320-327. 120 Nadala, E. C. B., L. M. Tapay, and P. C. Loh. (1998). Characterization of a non-occluded baculovirus-like agent pathogenic to penaeid shrimp. Dis. Aquat. Org, 33, 221-229. Nakanishi, A., Clever J., Yamada. M., Li, P. P., and Kasamatsu, H. (1996). Association with capsid proteins promotes nuclear targeting of simian virus 40 DNA. Proc. Natl. Acad. Sci. USA, 93, 96–100. Naylor, R. L., R. J. Goldburg, H. Mooney, M. Beveridge, J. Clay, C. Folke, N. Kautsky, J. Lubchenco, J. Primavera, and M. Williams. (1998). ECOLOGY, Nature's 10 Subsidies to Shrimp and Salmon Farming. Science, 282, 883-4. Oppenheimer, N. J., Meth. (1989). Enzymol, 176, 78-89. Oschkinat, H., Grieinger, C., Kraulis, P. J., Sorensen, O. W., Ernst, R. R., Gronenborn, A. M., and Clore, G. M. (1998). Nature, 332, 374-376. Otwinowski, Z., and W. Minor. (1997). Processing of X-ray diffraction data collected in oscillation mode. Methods Enzymol, 276, 307–326. Primrose, W. U. (1993). “NMR of Macromolecules, a Practical Approach” (G. C. K. Roberts, ed.), IRL Press, Oxford, pp. 7-34, Purcell, E. M., Torrey, H. C. and Pound, R. V. (1946). Phys. Rev, 69, 37-38. Purushothaman, V., K. Sankaranarayananad, R.M. Ravikumar and P. Ramasamy. (1998). Development of in vitro primary cell culture system from penaeid shrimp, Penaeus indicus, Penaeus monodon and sand crab Emerita asiatica. Indian Journal of Animal Sciences, 68, 1097-1099. Randall, G., Grakoui, A., Rice, C.M. (2003). Clearance of replicating hepatitis C virus replicon RNAs in cell culture by small interfering RNAs. Proc. Natl. Acad. Sci. USA, 100, 235–240. Rajni Hatti-Kaul, Bo Mattiasson. (2003). Isolation and purification of proteins. New York, Marcel Dekker. Robalino, J., Bartlett, T., Shepard, E., Prior, S., Jaramillo, G., Scura, E., Chapman, R.W., Gross, P.S., Browdy, C.L., Warr, G.W. (2005). Double-stranded RNA induces sequence-specific antiviral silencing in addition to nonspecific immunity in a marine shrimp, convergence of RNA interference and innate immunity in the invertebrate antiviral response. J. Virol, 79, 13561–13571. Robalino, J., Browdy, C.L., Prior, S., Metz, A., Parnell, P., Gross, P., Warr, G (2004). 121 Induction of antiviral immunity by double-stranded RNA in a marine invertebrate. J. Virol, 78, 10442–10448. Roper, K.G., L. Owens and L. West. (2001). The media used in primary cell cultures of prawn tissues , A review and a comparative study. Asian Fish Science, 14, 61-75. Rosenzweig, A.C., Huffman, D.L., Hou, M.Y., Wernimont, A.K., Pufahl, R.A., O`Halloran, T.V. (1999). Crystal structure of the Atx1 metallochaperone protein at 1.02 A resolution. Structure Fold. Des, 7, 605-617. Schachman, H.K. (1992). Is there a future for the ultracentrifuge? In Analytical ultracentrifugation in biochemistry and polymer science (eds. S.E. Harding et al.). The Royal Society of Chemistry, Cambridge, UK , pp. 3–15. Shi W, Zhan C, Ignatov A, Manjasetty BA, Marinkovic N, Sullivan M, Huang R, Chance MR. (2005). Metalloproteomics, high-throughput structural and functional annotation of proteins in structural genomics. Structure, 13, 1473-86. Sivaraman J, Myers RS, Boju L, Sulea T, Cygler M, Jo Davisson V, Schrag JD. (2005). Crystal structure of Methanobacterium thermoautotrophicum phosphoribosyl-AMP cyclohydrolase HisI. Biochemistry, 44, 10071-80. Sritunyalucksana K, Wannapapho W, Lo CF, Flegel TW. (2006). PmRab7 is a VP28-binding protein involved in white spot syndrome virus infection in shrimp. J Virol, 80, 10734-42. Sodeik, B., Ebersold, M. W., and Helenius, A. (1997). Microtubulemediated transport of incoming herpes simplex virus capsids to the nucleus. J. Cell Biol, 136, 1007–1021. Song, J., and F. Ni. (1998). NMR for the design of functional mimetics of protein-protein interactions, one key is in the building of bridges. Biochem Cell Biol, 76, 177-88. Song WJ, Qin QW, Qiu J, Huang CH, Wang F, Hew CL. (2004). Functional genomics analysis of Singapore grouper iridovirus, complete sequence determination and proteomic analysis. J Virol, 78, 12576-90. Stewart, L., Clark, R., and Behnke, C. (2002). High-throughput crystallization and structure determination in drug discovery. Drug Discov. Today, 7, 187-196. Suomalainen, M., Nakano, M. Y., Keller, S., Boucke, K., Stidwill, R. P., and Greber, U. F. (1999). Microtubule-mediated plus- and minus enddirected motilities are competing processes for nuclear targeting of adenoviruses. J. Cell 122 Biol, 144, 657–672. Terwilliger, T.C. (2002). Automated structure solution, density modification and model building. Acta Crystallogr. D Biol. Crystallogr, 58, 1937–1940. Thakur, P. C., F. Corsin, J. F. Turnbull, K. M. Shankar, N. V. Hao, P.A. Padiyar, M. Madhusudhan, K. L. Morgan, and C. V. Mohan. (2002). Estimation of prevalence of white spot syndrome virus (WSSV) by polymerase chain reaction in Penaeus monodom postlarvae at time of stocking in shrimp farms of Karnataka, India, a population-based study. Dis. Aquat. Org, 49, 235-243. Tidona, C. A., and Darai, G (1997). Molecular anatomy of lymphocystis disease virus. Arch. Virol. Suppl, 13, 49–56. Toullec, J.Y., Y. Crozat, J. Patrois and P. Porcheron. (1996). Development of primary cell culture from the penaeid shrimps Penaeus vannamei and P. indicus. Journal of Crustacean Biology, 16, 643 – 649. Tsai, M.F., Kou, G.H., Liu, H.C., Liu, K.F., ea al. (1999). Long-term presence of white spot syndrome virus (WSSV) in a cultivated shrimp population without disease outbreaks. Dis. Aquat. Org, 38, 107-114. Tsai, M. F., C. F. Lo, M. C. van Hulten, H. F. Tzeng, C. M. Chou, C. J. Huang, C. H. Wang, J. Y. Lin, J. M. Vlak, and G. H. Kou. (2000a). Transcriptional analysis of the ribonucleotide reductase genes of shrimp white spot syndrome virus. Virology, 277, 92-99. Tsai, M. F., H. T. Yu, H. F. Tzeng, J. H. Leu, C. M. Chou, C. J. Huang, C. H. Wang, J. Y. Lin, G. H. Kou, and C. F. Lo. (2000b). Identification and characterization of a shrimp white spot syndrome virus (WSSV) gene that encodes a novel chimeric polypeptide of cellular-type thymidine kinase and thymidylate kinase. Virology, 277, 100-110. Tsai, J. M., H. C. Wang, J. H. Leu, H. H. Hsiao, A. H. Wang, G. H. Kou, and C. F. Lo. (2004). Genomic and proteomic analysis of thirty-nine structural proteins of shrimp white spot syndrome virus. J Virol, 78, 11360-70. van Hulten, M. C., J. Witteveldt, S. Peters, N. Kloosterboer, R. Tarchini, M. Fiers, H. Sandbrink, R. K. Lankhorst, and J. M. Vlak. (2001). The white spot syndrome virus DNA genome sequence. Virology, 286, 7-22. van Hulten, M. C. W., M. F. Tsai, C. A. Schipper, C. F. Lo, G. H. Kou, and J. M. Vlak. (2000a). Analysis of a genomic segment of white spot syndrome virus of shrimp containing ribonucleotide reductase genes and repeat regions. J. Gen. Virol, 123 81, 307-316. van Hulten MCW, Westernberg M, Goodall SD, Vlak JM. (2000b). Identification of two major virion protein genes of white spot syndrome virus of shrimp. Virology, 266, 227-236. van Hulten, M. C., M. Reijns, A. M. Vermeesch, F. Zandbergen, and J. M. Vlak. (2002). Identification of VP19 and VP15 of white spot syndrome virus (WSSV) and glycosylation status of the WSSV major structural proteins. J. Gen. Virol, 83, 257-265. Vihinen-Ranta, M., Yuan, W., and Parrish, C. R. (2000). Cytoplasmic trafficking of the canine parvovirus capsid and its role in infection and nuclear transport. J Virol， 74, 4853–4859. Vlak, J.M., Bonami, J.R., Flegel, T.W., Kou, G.H., Lightner, D.V., Lo, C.F., Loh P.C. and Walker P.W. (2004). Nimaviridae. In, VIIIth Report of the International Committee on Taxonomy of Viruses (C.M. Fauquet, M.A. Mayo, J. Maniloff, U. Desselberger and L.A. Ball, Eds.), Elsevier, p. 187-192. Wang, C. H., C. F. Lo, J. H. Leu, C. M. Chou, P. Y. Yeh, H. Y. Chou, M. C. Tung, C. F. Chang, M. S. Su, and G. H. Kou. (1995). Purification and genomic analysis of baculovirus associated with white spot syndrome (WSBV) of Penaeus monodon. Dis. Aquat. Organ, 23, 239-242. Wang YG, Hassan MD, Shariff M, Zamri SM, Chen X. (1999). Histopathology and cytopathology of white spot syndrome virus (WSSV) in cultured Penaeus monodon from peninsular Malaysia with emphasis on pathogenesis and the mechanism of white spot formation. Dis Aquat Organ, 39, 1-11. Wang CH, Wu CY, Lo CF. (1999). A new picorna-like virus, PnPV, isolated from ficus transparent wing moth, Perina nuda (Fabricius). J Invertebr Pathol. 74, 62-8. Westenberg, M., Heinhuis, B., Zuidema, D., Vlak, J.M. (2005). siRNA injection induces sequence-independent protection in Penaeus monodon against white spot syndrome virus. Virus Res, 114, 133–139. Whittaker, G. R., and Helenius, A. (1998). Nuclear import and export of viruses and virus genomes. Virology, 246, 1–23. Wilkins, C., Dishongh, R., Moore, S.C., Whitt, M.A., Chow, M., Machaca, K. (2005). RNAinterference is an antiviral defence mechanism in Caenorhabditis elegans. Nature, 436, 1044–1047. 124 Wittek., R. (1982). Organization and expression of the poxvirus genome. Experientia, 38, 285–297. Wongteerasupaya, C., J. E. Vickers, S. Sriurairatana, G. L. Nash, A. Akarajamorn, V. Boonsaeng, S. Panyim, A. Tassanakajon, B. Withyachumnarnkul, and T. W. Flegel. (1995). A non-occluded, systemic baculovirus that occurs in cells of ectodermal and mesodermal origin and causes high mortality in the black tiger prawn Penaeus monodon. Dis. Aquat. Organ, 21, 69-77. Wu J., Arimoto M, Nishizawa T and Muroga K. (2002). Preparation of an inoculum of white spot syndrome virus for challenge tests in Penaeu japonicus. Fish Pathol, 37, 65–69. Wychowski, C., Benichou, D., and Girard, M. (1986). A domain of SV40 capsid polypeptide VP1 that specifies migration into the cell nucleus. EMBO J, 5, 2569–2576. Wychowski, C., Benichou, D., and Girard, M. (1987). The intranuclear localization of simian virus 40 polypeptides Vp2 and Vp3 depends on a specific amino acid sequence. J Virol, 61, 3862–3869. Yang, F., J. He, X. Lin, Q. Li, D. Pan, X. Zhang, and X. Xu. (2001). Complete genome sequence of the shrimp white spot bacilliform virus. J Virol, 75, 11811-20. Zhang, X., C. Huang, X. Xu, and C. L. Hew. (2002a). Transcription and identification of an envelope protein gene (p22) from shrimp white spot syndrome virus. J. Gen. Virol, 83, 471-477. Zhang, X., C. Huang, X. Xu, and C. L. Hew. (2002b). Identification and localization of a prawn white spot syndrome virus gene that encodes an envelope protein. J. Gen. Virol, 83, 1069-1074. Zhang, X., X. Xu, and C. L. Hew. (2001). The structure and function of a gene encoding a basic peptide from prawn white spot syndrome virus. Virus Res, 79, 137-144. Zhang X, Huang C, Tang X, Zhuang Y, Hew CL. (2004). Identification of structural proteins from shrimp white spot syndrome virus (WSSV) by 2DE-MS. Proteins, 55, 229-35. Zhou D, He QS, Wang C, Zhang J, Wong-Staal F. (2006). RNA interference and potential applications. Curr Top Med Chem, 6, 901-11. 125 Appendices Name Company Restriction endonucleases, T4-DNA ligase New England Biolabs pfuturbo DNA polymerase, pfu buffer and Dpn I. Statagene Tag-DNA polymerase New England Biolabs Hen egg white lysozyme Sigma RNase A Sigma DNase I Sigma Thrombin Sigma Complete protease inhibitors cocktail Roche Applied Science Phenylmethylsulfonyl fluorid (PMSF) Roche Applied Science List of various kits and reagents used QIAquick PCR purification kit QIAGEN QIAprep Spin Miniprep kit QIAGEN QIAquick Gel Extraction kit QIAGEN Coomassie Protein Assay kit PIERCE Crystallization kits SuperScript III 1st Strand Synthesis System Hampton Research Invitrogen 126 Publications 1. Liu Y, Sivaraman J, Hew CL. (2006). Expression, purification and crystallization of a novel nonstructural protein VP9 from white spot syndrome virus. Acta Crystallograph Sect F Struct Biol Cryst Commun. 62, 802-4. 2. Liu Y, Wu JL, Song JX, Sivaraman1 J, Hew CL. (2006). Identification of a novel nonstructural protein VP9 from white spot syndrome virus, its structure reveals a ferredoxin fold with specific metal binding sites. J Virol. 80, 10419-27. 3. Liu, Y.*, Li, ZJ*, Lin QS, Jan, K., Seetharaman J., Janusz M. B., Sivaraman, J., Hew CL. (2007). Structure and evolutionary origin of herring type II antifreeze protein. 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