Characterization of Shrimp White Spot Syndrome Virus (WSSV) Structural Protein VP51A (ORF294)
周宗錄、張雲祥
E-mail: [email protected]
ABSTRACT
White spot syndrome virus (WSSV) is an important crustacean virus causing high mortality in cultured shrimp. WSSV is a
double-stranded DNA virus with a genome size of about 300 kbp. So far, 58 viral structural proteins were identified. In this research, one of the structural protein translated form ORF294 (GeneBank accession no. AF440570), the VP51A, was studied. Gene structure analysis showed that the transcription initiation site of vp51A was located 135 bp upstream of the translation start codon ATG.
TATA box, or its related consensus sequence was not recognized is 5’ untranslated region of this gene. The poly-A addition signal was overlapped with the translation stop codon TAA and the poly-A tail was added 23 bp downstream of the stop codon.The vp51A transcripts was observed 6 hours after virus infection and the expression levels increasing with the infection time course. Computer software anlysis discovered a conserved sequence of the nuclear localization signal (NLS) between 37 and 43 of VP51A coding region, but such prediction wasn’t confirmed by the following in vitro analysis performed in Sf9 cells. Immunoelectron microscopy analysis and Western blot hybridization performed on intact virus particle and separated viral components showed that the VP51A is an envelope protein. Furthermore, Western blot analysis of WSSV virion also demonstrated that except the expected 53 kDa band, there were another protein bands such as an obvious signal around 72 kDa and some other small molecular weight proteins exist.
Similar result was found in the Western blot results performed on WSSV infect shrimp tissues and recombinant VP51A expressed insect cells. But when using the in vitro transcription and translation system to express the recombinant VP51A it demonstrated a 72 kDa protein only. This result showed that the VP51A gene might expresses a large molecular weight protein first and it will then be processed into another lower molecular weight ones. Other experiments, including the predict protein cutting site mutation of VP51A and Western blot hybridization by VP51A different region fragments derived antibadies suggested that most of the cutting sites of VP51A might distribute closer to the N-terminal region. The is processed and what are the biological meanings of there different types of VP51A proteins, still left to be elocudate.
Keywords : WSSV ; structural protein ; envelope protein
Table of Contents
封面內頁 簽名頁 授權書... iii 中文摘要... iv 英文摘要... vi 誌 謝... viii 目錄... x 圖目錄... xiii 表目
錄... xv 1. 前言... 1 1.1 白點症病毒... 1 1.2 結構性蛋白質之重要 性... 5 1.3 白點症病毒結構性蛋白質之發現與應用... 5 1.4 研究目的... 7 2. 材料方
法... 9 2.1 基因結構分析... 9 2.1.1 樣品來源... 9 2.1.2 total RNA萃
取... 9 2.1.3 反轉錄聚合?鏈反應(reverse transcription polymerase chain reaction, RT-PCR)... 10 2.1.4 基因表現時 序分析... 11 2.1.5 快速擴增cDNA 5’/3’端... 11 2.2 蛋白質表現及特性分析... 14 2.2.1 VP51A 之細胞內表現位置分析... 14 2.2.2 抗體製備... 16 2.2.3 VP51A於病毒顆粒之定位分析... 18 2.2.3.1 白點症病毒純化... 18 2.2.3.2 白點症病毒之抗VP51A C端抗體西方轉漬 分析... 19 2.2.3.3 以西方轉漬分析VP51A於白點症病毒顆粒 上之定位... 20 2.2.3.4 以免疫電顯分析VP51A於白點症病毒顆粒 上 之定位... 20 2.2.4 VP51A分子量變異之分析... 21 2.2.4.1 VP51A試管內轉錄及轉譯 (Transcription/
TranslationSystems, TNT)分析... 21 2.2.4.2 昆蟲細胞Sf9表現重組VP51A之抗V5抗體西 方轉漬分析... 22 2.2.4.3 白點症病毒顆粒、病毒感染蝦組織及昆蟲 細胞Sf9抗VP51A C端抗體西方轉漬分析... 23 2.2.4.4 白點症病毒顆粒、病 毒感染蝦組織及昆蟲 細胞Sf9抗VP51A中段片段抗體西方轉漬分析23 2.2.4.5 VP51A切割位分析... 24 3. 結果與討 論... 25 3.1 基因結構分析... 25 3.1.1 基因表現時序分析... 25 3.1.2 快速擴增cDNA 5’/3’端... 26 3.2 蛋白質表現及特性分析... 26 3.2.1 VP51A之細胞內表現位置分析... 26 3.2.2 VP51A於病毒顆粒之定位分析... 27 3.2.3 VP51A分子量變異之分析... 28 3.2.3.1 VP51A試管內轉錄及轉譯分 析... 28 3.2.3.2 昆蟲細胞Sf9表現重組VP51A之抗V5抗體西方 轉漬分析... 29 3.2.3.3 白點症病毒顆粒、病 毒感染蝦組織及昆蟲細 胞Sf9抗VP51A C端抗體西方轉漬分析... 29 3.2.3.4 白點症病毒顆粒、病毒感染蝦組織及昆蟲細 胞Sf9抗VP51A中段片段抗體西方轉漬分析. 30 3.2.3.5 VP51A切割位分析... 30 4. 結論... 31 參
考文獻... 59 REFERENCES
1. 王涵青，2002。蝦白點症病毒四種基因轉錄分析之研究。國立台灣大學海洋研究所碩士論文。 2. Bartosch, B., Vitelli, A., and Granier, C. (2003) Cell entry of hepatitis C virus requires a set of co-receptors that include the CD81 tetraspanin and the SR-B1 scavenger receptor. J Biol Chem. 278:41624-41630. 3. Bartenschlager, R., and Lohmann, V. (2000) Replication of hepatitis C virus. J Gen Virol 81:1631-1648. 4. Bieleski, L., Hindley, C., and Talbot, S. J. (2004) A polypyrimidine tract facilitates the expression of Kaposi's sarcoma-associated herpesvirus vFLIP through an internal ribosome entry site. J Gen Virol 85:615-620. 5. Bieleski, L., and Talbot, S. J. (2001) Kaposi's sarcoma-associated herpesvirus vCyclin open reading frame contains an internal ribosome entry site. J Virol 75:1864-1869. 6. Bravo, D. A., Gleason, J. B., Sanchez, R. I., Roth, R. A., and Fuller, R. S. (1994) Accurate and efficient cleavage of the human insulin proreceptor by the human proprotein-processing protease furin.
Characterization and kinetic parameters using the purified, secreted soluble protease expressed by a recombinant baculovirus. J Biol Chem 269:25830-25837 7. Chang, P. S., Lo, C. F., Wang, Y. C., and Kou, G. H. (1996) Identification of white spot syndrome associated baculovirus (WSSV) target organs in shrimp, Penaeus monodon, by in situ hybridization. Dis Aquat Org 27:131-139. 8. Chang, J., Yang, S. H., Cho, Y. G., Hwang, S. B., Hahn, Y. S., and Song, Y. C. (1998) Hepatitis C virus core from two different genotypes has an oncogenic potential but is not sufficient for transforming primary rat embryo fibroblasts in cooperation with the H-ras oncogene. J Virol 72:3060-3065. 9. Chen, L. L., Leu, J. H., Huang, C. J., Chou, C. M., Chen, S. M., Wang, C. H., Lo, C. F., and Kou, G. H. (2002a) Identification of a nucleocapsid protein (VP35) gene of shrimp white spot syndrome virus and characterization of the motif important for targeting VP35 to the nuclei of transfected insect cells. Virology 293:44-53. 10. Chen, L. L., Wang, H. C., Huang, C. J., Peng, S. E., Chen, Y. G., Lin, S. J., Chen, W. Y., Dai, C. F., Yu, H. T., Wang, C. H., Lo, C. F., and Kou, G. H. (2002b) Transcriptional analysis of the DNA polymerase gene of shrimp white spot syndrome virus. Virology 301:136-147.
11. Chou, H. Y., Huang, C.Y., Wang, C.H., Chiang, H. C., and Lo, C. F. (1995) Pathogenicity of a baculovirus infection causing white spot syndrome in cultured penaeid shrimp in Taiwan. Dis Aquat Org 23:165-173. 12. Couch, J. A. (1974) An enzootic nuclear polyhedrosis virus of pink shrimp: ultrastructure, prevalence, and enhancement. J Invertebr Pathol. 24:311-31. 13. Flegel, T. M., Sriurairatana, S., Wongteerasupaya, C., Boonsaeng, V., Panyim, S., and Withyachumnarnkul, W. (1995) Progress in characterization and control of yellow head virus of Penaeus monodon.
In: Browdy CL, Hopkins JS, eds. Swimming Through Troubled Water, Proceedings of the Special Session on Shrimp Farming. World Aquaculture Society, Baton Rouge, LA, USA, 76-83. 14. Flegel TW. (1997) Special topic review: Major viral diseases of the black tiger prawn ( Penaeus monodon ) in Thailand. World J Microbiol Biotech 13: 422-433. 15. Flint, M., Thomas, J. M., Maidens, C. M., Shotton, C., Levy, S., Barclay, W.
S., and Mckeating, J. A. (1999) Functional analysis of cell surfaceexpresed hepatitis C virus E2 glycoprotein. J Virol 73:6782-6790. 16. Gale, M. Jr., Tan, S. L., and Katze, M. G. (2000) Translational control of viral gene expression in eukaryotes. Microbiol Mol Biol Rev 64:239-280. 17. Geballe, A. P., and Mocarski, E. S., (1988) Translational control of cytomegalovirus gene expression is mediated by upstream AUG codons. J Virol 62:3334-3340 18. Gesteland, R. F., and Atkins, J. F. (1996) Recoding: dynamic reprogramming of translation. Annu Rev Biochem 65:741-768. 19.
Hasson, K. W., Lightner, D. V., Poulos, B. T., Redman, R. M., White, B. L., and Brock, J. A. (1995) Taura syndrome in Penaeus vannamei:
demonstration of a viral etiology. Dis Aquat Org 23:115-126. 20. Huang, J., Song, X. L., Yu, J., and Yang, C. H. (1995a) Baculoviral hypodermal and hematopoietic necrosis - study on the pathogen and pathology of the explosive epidemic disease of shrimp. Mar Fish Res 16:1-10. 21. Huang, C. C., Song, Y. L. (1999) Maternal transmission of immunity to white spot syndrome associated virus (WSSV) in shrimp (Penaeus monodon). Dev Comp Immunol 23:545-52. 22. Huang, J., Yu, J., Song, X. L., Kong, J., and Yang, C.H. (1995b) Studies on fine structure, nucleic acid,
polypeptide and serology of hypodermal and hematopoietic necrosis baculovirus of penaeid shrimp. Mar Fish Res 16:11-23. 23. Inouye, K., Miwa, S., Oseko, N., Nakano, H., and Kimura, T. (1994) Mass mortalities of cultured kuruma shrimp, Penaeus japonicus, in Japan in 1993: Electron microscopic evidence of the causative virus. Fish Pathol 29:149-158. 24. Jean, F., Stella, K., Thomas, L., Liu, G., Xiang, Y., Reason, A. J., and Thomas, G. (1998) Alpha1-Antitrypsin Portland, a bioengineered serpin highly selective for furin: application as an antipathogenic agent. Proc Natl Acad Sci USA. 95:7293-7298. 25. Karunasagar, I., Otta, S. K., and Karunasagar, I. (1997) Histopathological and bacteriological study of white spot syndrome of Penaeus monodon along the west coast of India. Aquaculture 153:9-13. 26. Kurosaki, M., Enomoto, N., Marumo, F., and Sato, C. (1993) Rapid sequence variation of the hypervariable region of hepatitis C virus during the course of chronic infection. Hepatology
18:1293-1299. 27. Leu, J. H., Tsai, J. M., Wang, H. C., Wang, A. H., Wang, C. H., Kou, G. H., and Lo, C. F. (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. 28. Lightner, D. V., Poulos, B. T., Redman, R. M., Mari, J., and Bonami, J. R. (1992) New developmentsin penaeid virology: Application of biotechnology in research and disease diagnosisfor shrimp viruses of concern in the Americas. In: Fulks, W., and Main, K.
(eds) Proceedings of the Asian Interchange Program Workshop on the Diseases of Cultured Penaeid Shrimp, Asian Interchange Program, The Oceanic Institute, Oahu,HI, 233-253. 29 Lightner DV. (1996) A Handbook of Shrimp Pathology and Diagnostic Procedures for Diseases of Culturd Penaeid Shrimp. Society, Baron Rouge, LA.:The World Aquaculture. 304 30.2Lindenbach, B. D., and Rice, C. M. (2001) Flaviviridae: the viruses and their replication, pp. 991–1041 in D.M. Knipe, P.M. Howley, D.E. Griffin: Fields Virology, 4th ed. Lippincott–Williams & Wilkins, Philadelphia (USA). 31. Liu, W. J., Yu, H. T., Peng, S. E., Chang, Y. S., Pien, H. W., Lin, C. J., Huang, C. J., Tsai, M. F., Huang, C. J., Wang, C.
H., Lin, J. Y., Lo, C. F., and Kou, G. H. (2001) Cloning, characterization, and phylogenetic analysis of a shrimp white spot syndrome virus gene
that encodes a protein kinase. Virology 289:362-377. 32. Liu, W., Wang, Y. T., Tian, D. S., Yin. Z. C., and Kwang, J. (2002) Detection of white spot syndrome virus (WSSV) of shrimp by means of monoclonal antibodies (MAbs) specific to an envelope protein (28 kDa). Dis Aquat Organ.
49:11-8. 33. Li, Z., Lin, Q., Chen, J., Wu, J. L., Lim, T. K., Loh, S. S., Tang, X., and Hew, C. L. (2007) Shotgun identification of structural proteome of shrimp white spot syndrome virus and iTRAQ differentiation of envelope and nucleocapsid subproteomes. Mol Cell Proteomics. *:*-*
34. Lo, C. F., Ho, C. H., Peng, S. E., Chen, C. H., Hsu, H. C., Chiu, Y. L., Chen, Y. T., Chang, C. F., Liu, K. F., Su, M. S., Wang, C. H., and Kou, G. H. (1996a) Infection of white spot syndrome associated virus (WSBV) in cultured and wild-caught shrimps, crabs and other arthropods.
Dis Aquat Org 27:215-225. 35. Lo, C. F., Leu, J. H., Ho, C. H., Chen, C. H., Peng, S. E., Chen, Y. T., Chou, C. M., Yeh, P. Y., Huang, C. J., Chou, H. Y., Wang, C. H., and Kou, G. H. (1996b) Detection of baculovirus associated with white spot syndrome (WSBV) in penaeid shrimps using polymerase chain reaction. Dis Aquat Org 25:133-141. 36. Lo, C. F., Ho, C. H., Chen, C. H., Liu, K. F., Chiu, Y. L., Yeh, P. Y., Peng, S. E., Hsu, H. C., Liu, H. C., Chang, C. F., Su, M. S., Wang, C. H., and Kou, G. H. (1997) Detection and tissue tropism of white spot syndrome baculovirus (WSBV) in captured brooders of Penaeus monodon with a special emphasis on reproductive organs. Dis Aquat Org 30:53-72. 37. Lo, C. F., and Kou, G. H. (1998) Virus associated white spot syndrome of shrimp in Taiwan: a review. Fish Pathol 33:365-371. 38. Lotz, J. M., Browdy, C. L., Carr, W. H., Frelier, P. F., and Lightner, D. V. (1995) USMSFP suggested procedures and guidelines for assuring the specific pathogen status of shrimp broodstock and seed. In: Browdy CL, Hopkins JS (eds) Swimming Through Troubled Water, Proceedings the Special Session on Shrimp Farming, Aquaculture '95. World Aquaculture Society, Baton Rouge, Louisina, USA, 66-75. 39. Lo, S. Y., Masiarz, F., Hwang, S. B., Lai, H. H., and Ou, J. H. (1995) Differential subcellular localization of hepatitis C virus core gene products. Virology 213:455-461. 40.
Marks, H., Mennens, M., Vlak, J. M., and van Hulten, M. C. (2003) Transcriptional analysis of the white spot syndrome virus major virion protein genes. J Gen Virol 84:1517-1523. 41. Mclauchlan, J. (2000) Properties of the hepatitis C virus core protein: a structural protein that modulates cellular processes. J Viral Hepatitis 7:2-14. 42. Momoyama, K., Hiraoka, M., Nakano, H., Koube, H., Inouye, A. K., and Oseko, N. (1994) Mass mortalities of cultured kuruma shrimp, Penaeus japonicus, in Japan in 1993: Histopathological study. Fish Pathol 29:141-148. 43. Moon, C. H., Do, J. W., Cha, S. J., Yoon, W. J., Kim, S. B., Ko, M. S., Park, M. A., Kim, J. W., Sohn, S. K., Lee, J. H., and Park, J. W. (2003) Highly conserved sequences of three major virion proteins of a Korean isolate of white spot syndrome virus (WSSV). Dis Aquat Organ 53:11-13. 44. Nadala, E. C.
B., Tappy, L. M., and Loh, P. C. (1997) Yellow-head virus: a rhabdovirus-like pathogen of penaeid shrimp. Dis Aquat Org 31:141–146. 45.
Nakano, H., Koube, H., Umezawa, S., Momoyama, K., Hiraoka, M., Inouye, K., and Oseko, N. (1994) Mass mortalities of cultured kuruma shrimp, in Japan in 1993: epizootiological survey and infection trials. Fish Pathol 29:135-139. 46. Pileri, P., Uematsu, Y., Campagnoli, S., Galli, G., Falugi, F., Petracca, R., Weiner A. J., Houghton, M., Rosa, D., Grandi, G., and Abrignani, S. (1998) Binding of hepatitis C virus to CD 81. Science 282:938-941. 47. Robalino, J., Payne, C., Parnell, P., Shepard, E., Grimes, A. C., Metz, A., Prior, S., Witteveldt, J., Vlak, J. M., Gross, P. S., Warr, G., and Browdy, C. L. (2006) Inactivation of White Spot Syndrome Virus (WSSV) by normal rabbit serum: implications for the role of the envelope protein VP28 in WSSV infection of shrimp.Virus Res 118:55-61. 48. Seok, S. H., Park, J. H., Cho, S. A., Baek, M. W., Lee, H. Y., Kim, D. J., and Park, J. H. (2004) Cloning and sequencing of envelope proteins (VP19, VP28) and nucleocapsid proteins (VP15, VP35) of a white spot syndrome virus isolate from Korean shrimp. Dis Aquat Organ 60:85-88. 49. Shimizu, Y. K., Igarashi, H., Kiyohara, T., Cabezon, T., Farci, P., Purcell, R.
H., and Yoshikura, H. (1996) A hyperimmune serum against a synthetic peptide corresponding to the hypervariable region 1 of hepatitis C virus can prevent viral infection in cell cultures. Virology 223:409-412. 50. Tang, X., Wu, J., Sivaraman, J., and Hew, C. L.(2007) Crystal structures of major envelope proteins VP26 and VP28 from white spot syndrome virus shed light on their evolutionary relationship. J Virol. 81:6709-17. 51.
Takahashi, Y., Itami, T., Kondo, M., Maeda, M., Fujii, R., Tomonaga, S., Supamattaya, K., and Boonyaratpalin, S. (1994) Electron microscopic evidence of bacilliform virus infection in kuruma shrimp ( Penaeus japonicas ). Fish Pathol 29:121-125. 52. Taylor, D. R., Shi, S. T., Romano, P.
R., Barber, G. N., and Lai, M. M. C. (1999) Inhibition of the interferon inducible protein kinase PKR by HCV E2 protein. Science 285:107-110.
53. Tsai, J. M., Wang, H. C., Leu, J. H., Hsiao, H. H., Wang, A. H., Kou, G. H., and Lo, C. F. (2004) Genomic and proteomic analysis of thirty-nine structural proteins of shrimp white spot syndrome virus. J Virol 78:11360-11370. 54. Tsai, M. F., Lo, CF, van Hulten, M. C., Tzeng, H.
F., Chou, C. M., Huang, C. J., Wang, C. H., Lin, J. Y., Vlak, J. M., and Kou, G. H. (2000a) Transcriptional analysis of the ribonucleotide reductase genes of shrimp white spot syndrome virus. Virology 277:92-99. 55. Tsai, M. F., Yu, H. T., Tzeng. H. F., Leu, J. H., Chou, C. M., Huang, C. J., Wang, C. H, Lin, J.Y., Kou, G. H., and Lo, C. F. (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 56. Van Hulten, M. C., Goldbach, R. W., and Vlak, J. M.. (2000) Three functionally diverged major structural proteins of white spot syndrome virus evolved by gene duplication. J Gen Virol. 81:2525-2529. 57. Van Etten, J. L., Graves, M. V., Muller, D. G., Boland, W., and Delaroque, N. (2002) Phycodnaviridae--large DNA algal viruses. Arch Virol 147:1479-1516. 58. Van Hulten, M. C., Reijns, M., Vermeesch, AM., Zandbergen, F., Vlak, J. M. (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. 59. Van Hulten, M. C., and Vlak, J. M. (2001) Identification and phylogeny of a protein kinase gene of white spot syndrome virus. Virus Genes 22:201-207. 60. Van Hulten, M. C., Witteveldt, J., Snippe, M., and Vlak, J. M.. (2001) White spot syndrome virus envelope protein VP28 is involved in the systemic infection of shrimp. Virology. Jul 285:228-233. 61. Wang, C. H., Lo, C. F., Leu, J. H., Chou, C.
M., Yeh, P. Y., Chou, H.Y., E. T. M., Chang, C. F., Su, M. S., and Kou, G. H. (1995) Purification and genomic analysis of baculovirus associated with white spot syndrome (WSBV) of Penaeus monodon. Dis Aquat Org 23:239-242. 62. Weiner, A. J., Geysen, H. M., Christopherson, C., Hall, J.
E., Mason, T. J., Saracco, G., Bonino, F., Crawford, K., Marion, C. D., and Crawford, K. A. (1992) Evidence for immune selection of hepatitis C
virus (HCV) putative envelope glycoprotein variants: potential role in chronic HCV infections. Proc Nat Acad Sci USA 89:3468-3472. 63.
Witteveldt, J., Vermeesch, A. M., Langenhof, M., de Lang, A., Vlak, J. M., and van Hulten, M. C. (2005) Nucleocapsid protein VP15 is the basic DNA binding protein of white spot syndrome virus of shrimp. Arch Virol. 150:1121-33. 64. Witteveldt, J., Vlak, J. M., and van Hulten, M. C.
(2006) Increased tolerance of Litopenaeus vannamei to white spot syndrome virus (WSSV) infection after oral application of the viral envelope protein VP28. Dis Aquat Organ 70:167-70. 65. Wongteerasupaya, C., Vickers, J. E., Sriurairatana, S., Nash, G. L., Akarajamorn, A., Boonsaeng, V., Panyim, S., Tassankajon, A., Withyanchumnarnkul, B., and Flegel, T. W. (1995) A non-occluded, systemic baculovirus that occurs in cells of ectodermal and mesodermal origin and causes high mortality in black tiger prawn Penaeus monodon. Dis Aquat Org 21:69-77. 66. Xu, Z., Choj, J., Yen, T. S. B., Lu, W., Strohecker, A., Govindarajan, S., Chien, D., Selby, M. J. and Ou, J.H. (2001) Synthesis of a novel hepatitis C virus protein by ribosomal frameshift. EMBO J 20:3840-3848. 67. Yang, F., He, J., Lin, X., Li, Q., Pan, D., Zhang, X., and Xu, X. (2001) Complete genome sequence of the shrimp white spot bacilliform virus. J Virol 75:11811-11820. 68. Yi, G., Wang, Z., Qi, Y., Yao, L., Qian, J., and Hu, L.
(2004) Vp28 of shrimp white spot syndrome virus is involved in the attachment and penetration into shrimp cells. J Biochem Mol Biol 37:726-734.
69. Yoganandhan, K., Syed, M. S., Narayanan, R. B., and Sahul, H. A. S. (2004) Production of polyclonal antiserum against recombinant VP28 protein and its application for the detection of white spot syndrome virus in crustaceans. J Fish Dis 27:517-522.
