Please use this identifier to cite or link to this item:
標題: 熱休克蛋白70參與竹嵌紋病毒複製之研究
The involvement of NbHsp70 in BaMV replication
作者: 楊孟佳
Yang, Meng-Ja
關鍵字: 竹嵌紋病毒;Bamboo mosaic virus;熱休克蛋白 70;複製酵素;Heat shock protein 70;RdRp
出版社: 生物科技學研究所
引用: 柒、 參考文獻 林銘裕。2004。利用竹嵌紋病毒載體表現口蹄疫病毒抗原決定基。國立中興大學 生物科技研究所碩士論文。 何筱嬋。2006。鑑定參與竹嵌紋病毒正股複製的寄主蛋白。國立中興大學生物科技學研究所碩士論文。 李雅倩。2009。發展轉殖植物表現竹嵌紋病毒及其衛星核酸並應用其於輔助病毒 -衛星核酸交互作用之研究。國立中興大學生物科技學研究所博士論文。 陳宗賢。2012。利用竹嵌紋病毒載體表現疫苗蛋白。國立中興大學生物科技研究 所博士論文。 陳盈君。2007。參與竹嵌紋病毒核酸基因體正股複製的寄主蛋白鑑定與探討。國立中興大學生物科技學研究所碩士論文。 陸曉慧。2008。煙草宿主蛋白Hsp70和Hsp90參與竹嵌紋病毒感染週期之研究。 國立中興大學生物科技學研究所碩士論文。 Ahlquist, P. (2006). Parallels among positive-strand RNA viruses, reverse-transcribing viruses and double-stranded RNA viruses. Nature Reviews Microbiology 4(5), 371-382. Ahlquist, P., Noueiry, A. O., Lee, W. M., Kushner, D. B., and Dye, B. T. (2003). Host factors in positive-strand RNA virus genome replication. Journal of Virology 77(15), 8181-8186. Aparicio, F., Thomas, C. L., Lederer, C., Niu, Y., Wang, D. W., and Maule, A. J. (2005). Virus induction of heat shock protein 70 reflects a general response to protein accumulation in the plant cytosol. Plant Physiology 138(1), 529-536. Aranda, M. A., Escaler, M., Wang, D. W., and Maule, A. J. (1996). Induction of HSP70 and polyubiquitin expression associated with plant virus replication. Proceedings of the National Academy of Sciences of the United States of America 93(26), 15289-15293. Brennan, F. R., Jones, T. D., Longstaff, M., Chapman, S., Bellaby, T., Smith, H., Xu, F., Hamilton, W. D. O., and Flock, J. I. (1999). Immunogenicity of peptides derived from a fibronectin-binding protein of S-aureus expressed on two different plant viruses. Vaccine 17(15-16), 1846-1857. Brown, G., Rixon, H. W. M., Steel, J., McDonald, T. P., Pitt, A. R., Graham, S., and Sugrue, R. J. (2005). Evidence for an association between heat shock protein 70 and the respiratory syncytial virus polymerase complex within lipid-raft membranes during virus infection. Virology 338(1), 69-80. Bukau, B., and Horwich, A. L. (1998). The Hsp70 and Hsp60 chaperone machines. Cell 92(3), 351-366. Canizares, M. C., Nicholson, L., and Lomonossoff, G. P. (2005). Use of viral vectors for vaccine production in plants. Immunology and Cell Biology 83(3), 263-270. Chang, B. Y., Lin, N. S., Liou, D. Y., Chen, J. P., Liou, G. G., and Hsu, Y. H. (1997). Subcellular localization of the 28 kDa protein of the triple-gene-block of bamboo mosaic potexvirus. Journal of General Virology 78 , 1175-1179. Chen, Z. R., Zhou, T., Wu, X. H., Hong, Y. G., Fan, Z. F., and Li, H. F. (2008). Influence of cytoplasmic heat shock protein 70 on viral infection of Nicotiana benthamiana. Molecular Plant Pathology 9(6), 809-817. Cheng, C. W., Hsiao, Y. Y., Wu, H. C., Chuang, C. M., Chen, J. S., Tsai, C. H., Hsu, Y. H., Wu, Y. C., Lee, C. C., and Meng, M. S. A. (2009). Suppression of Bamboo Mosaic Virus Accumulation by a Putative Methyltransferase in Nicotiana benthamiana. Journal of Virology 83(11), 5796-5805. Cheng, J. H., Ding, M. P., Hsu, Y. H., and Tsai, C. H. (2001). The partial purified RNA-dependent RNA polymerases from bamboo mosaic potexvirus and potato virus X infected plants containing the template-dependent activities. Virus Research 80(1-2), 41-52. Cheng, S. F., Huang, Y. P., Wu, Z. R., Hu, C. C., Hsu, Y. H., and Tsai, C. H. (2010). Identification of differentially expressed genes induced by Bamboo mosaic virus infection in Nicotiana benthamiana by cDNA-amplified fragment length polymorphism. BMC Plant Biology 10:286. Conley, A. J., Joensuu, J. J., Menassa, R., and Brandle, J. E. (2009a). Induction of protein body formation in plant leaves by elastin- like polypeptide fusions. BMC Biology 7:48. Conley, A. J., Mohib, K., Jevnikar, A. M., and Brandle, J. E. (2009b). Plant recombinant erythropoietin attenuates inflammatory kidney cell injury. Plant Biotechnology Journal 7(2), 183-199. Dufresne, P. J., Thivierge, K., Cotton, S., Beauchemin, C., Ide, C., Ubalijoro, E., Laliberte, J. F., and Fortin, M. G. (2008). Heat shock 70 protein interaction with Turnip mosaic virus RNA-dependent RNA polymerase within virus-induced membrane vesicles. Virology 374(1), 217-227. Fields, S. and Song. O. (1989). A novel genetic system to detect protein-protein interactions. Nature 340, 245-246. Floss, D. M., Sack, M., Stadlmann, J., Scheller, J., Stoger, E., Fischer, R., and Conrad, U. (2008). Biochemical and functional characterization of anti-HIV antibody-ELP fusion proteins from transgenic plants (vol 6, pg 379, 2008). Plant Biotechnology Journal 6(4), 424-424. Hafren, A., Hofius, D., Ronnholm, G., Sonnewald, U., and Makinen, K. (2010). HSP70 and Its Cochaperone CPIP Promote Potyvirus Infection in Nicotiana benthamiana by Regulating Viral Coat Protein Functions. Plant Cell 22(2), 523-535. Hahn, A., Bublak, D., Schleiff, E., and Scharf, K. D. (2011). Crosstalk between Hsp90 and Hsp70 chaperones and heat stress transcription factors in tomato. Plant Cell 23(2), 741-755. Hakanpaa, J., Paananen, A., Askolin, S., Nakari-Setala, T., Parkkinen, T., Penttila, M., Linder, M. B., and Rouvinen, J. (2004). Atomic resolution structure of the HFBII hydrophobin, a self-assembling amphiphile. Journal of Biological Chemistry 279(1), 534-539. Hu, J. M., Flores, D., Toft, D., Wang, X. T., and Nguyen, D. (2004). Requirement of heat shock protein 90 for human hepatitis B virus reverse transcriptase function. Journal of Virology 78(23), 13122-13131. Huang, X. Q., and Madan, A. (1999). CAP3: A DNA sequence assembly program. Genome Research 9(9), 868-877. Huang, Y. L., Han, Y. T., Chang, Y. T., Hsu, Y. H., and Meng, M. S. (2004). Critical residues for GTP methylation and formation of the covalent m(7)GMP-enzyme intermediate in the capping enzyme domain of Bamboo mosaic virus. Journal of Virology 78(3), 1271-1280. Huang, Y. W., Hu, C. C., Lin, N. S., Tsai, C. H., and Hsu, Y. H. (2008). In vitro replication of Bamboo mosaic virus satellite RNA. Virus Research 136(1-2), 98-106. Huang, Y. W., Hu, C. C., Liou, M. R., Chang, B. Y., Tsai, C. H., Meng, M., Lin, N. S., and Hsu, Y. H. (2012). Hsp90 Interacts Specifically with Viral RNA and Differentially Regulates Replication Initiation of Bamboo mosaic virus and Associated Satellite RNA. PLoS Pathogens 8(5), e1002726. Jockusch, H., Wiegand, C., Mersch, B., and Rajes, D. (2001). Mutants of Tobacco mosaic virus with temperature-sensitive coat proteins induce heat shock response in tobacco leaves. Molecular Plant-Microbe Interactions 14(7), 914-917. Joensuu, J. J., Conley, A. J., Lienemann, M., Brandle, J. E., Linder, M. B., and Menassa, R. (2010). Hydrophobin Fusions for High-Level Transient Protein Expression and Purification in Nicotiana benthamiana. Plant Physiology 152(2), 622-633. Johnson, B. D., Schumacher, R. J., Ross, E. D., and Toft, D. O. (1998). Hop modulates hsp70/hsp90 interactions in protein folding. Journal of Biological Chemistry 273(6), 3679-3686. Jungkunz, I., Link, K., Vogel, F., Voll, L. M., Sonnewald, S., and Sonnewald, U. (2011). AtHsp70-15-deficient Arabidopsis plants are characterized by reduced growth, a constitutive cytosolic protein response and enhanced resistance to TuMV. Plant Journal 66(6), 983-995. Kampmueller, K. M., and Miller, D. J. (2005). The cellular chaperone heat shock protein 90 facilitates flock house virus RNA replication in Drosophila cells. Journal of Virology 79(11), 6827-6837. Kanazawa, S., Driscoll, M., and Struhl, K. (1988). ATR1, a Saccharomyces cerevisiae Gene Encoding a Transmembrane Protein Requiared for Aminotriazole Resistance. Molecular and Cellular Biology 8, 664-673. Kimura, Y., Matsumoto, S., and Yahara, I. (1994). Temperature-sensitive mutants of hsp82 of the budding yeast Saccharomyces cerevisiae. Molecular and General Genetics 242(5), 517-527. Lee, Y. S., Lin, B. Y., Hsu, Y. H., Chang, B. Y., and Lin, N. S. (1998). Subgenomic RNAs of bamboo mosaic potexvirus-V isolate are packaged into virions. Journal of General Virology 79, 1825-1832. Leu, J. I. J., Pimkina, J., Frank, A., Murphy, M. E., and George, D. L. (2009). A Small Molecule Inhibitor of Inducible Heat Shock Protein 70. Molecular Cell 36(1), 15-27. Li, P. P., Itoh, N., Watanabe, M., Shi, Y. F., Liu, P. N., Yang, H. J., and Kasamatsu, H. (2009). Association of Simian Virus 40 Vp1 with 70-Kilodalton Heat Shock Proteins and Viral Tumor Antigens. Journal of Virology 83(1), 37-46. Li, Y. I., Chen, Y. J., Hsu, Y. H., and Meng, M. H. (2001a). Characterization of the AdoMet-dependent guanylyltransferase activity that is associated with the N terminus of bamboo mosaic virus replicase. Journal of Virology 75(2), 782-788. Li, Y. I., Cheng, Y. M., Huang, Y. L., Tsai, C. H., Hsu, Y. H., and Meng, M. H. (1998). Identification acid characterization of the Escherichia coli-expressed RNA-dependent RNA polymerase of bamboo mosaic virus. Journal of Virology 72(12), 10093-10099. Li, Y. I., Shih, T. W., Hsu, Y. H., Han, Y. T., Huang, Y. L., and Meng, M. (2001b). The helicase-like domain of plant potexvirus replicase participates in formation of RNA 5'' cap structure by exhibiting RNA 5''-triphosphatase activity. Journal of Virology 75(24), 12114-12120. Lin, B. L., Wang, J. S., Liu, H. C., Chen, R. W., Meyer, Y., Barakat, A., and Delseny, M. (2001). Genomic analysis of the Hsp70 superfamily in Arabidopsis thaliana. Cell Stress Chaperones 6(3), 201-208. Lin, J. W., Ding, M. P., Hsu, Y. H., and Tsai, C. H. (2007). Chloroplast phosphoglycerate kinase, a gluconeogenetic enzyme, is required for efficient accumulation of Bamboo mosaic virus. Nucleic Acids Research 35(2), 424-432. Lin, M. T., Kitajima, E. W., Cupertino, F. P. and Costa, C. L. (1977). Partial purification and some properties of bamboo mosaic virus. Phytopathology 67, 1439-1443. Lin, N. S., Lin, B. Y., Lo, N. W., Hu, C. C., Chow, T. Y., and Hsu, Y. H. (1994). Nucleotide sequence of the genomic RNA of bamboo mosaic potexvirus. Journal of General Virology 75, 2513-2518. Linder, M. B., Qiao, M. Q., Laumen, F., Selber, K., Hyytia, T., Nakari-Setala, T., and Penttila, M. E. (2004). Efficient purification of recombinant proteins using hydrophobins as tags in surfactant-based two-phase systems. Biochemistry 43(37), 11873-11882. Lomonossoff, G., and Johnson, J. E. (1995). Eukaryotic Viral Expression Systems for Polypeptides. Seminars in Virology 6(4), 257-267. Lomonossoff, G. P., and Hamilton, W. D. O. (1999). Cowpea mosaic virus-based vaccines. Plant Biotechnology-Bk 240, 177-189. Marusic, C., Rizza, P., Lattanzi, L., Mancini, C., Spada, M., Belardelli, F., Benvenuto, E., and Capone, I. (2001). Chimeric plant virus particles as immunogens for inducing murine and human immune responses against human immunodeficiency virus type 1. Journal of Virology 75(18), 8434-8439. Mayer, M. P. (2005). Recruitment of Hsp70 chaperones: a crucial part of viral survival strategies. Reviews of Physiology, Biochemistry & Pharmacology153, 1-46. Mayer, M. P., and Bukau, B. (2005). Hsp70 chaperones: cellular functions and molecular mechanism. Cellular and Molecular Life Sciences 62(6), 670-684. Millson, S. H., Vaughan, C. K., Zhai, C., Ali, M. M. U., Panaretou, B., Piper, P. W., Pearl, L. H., and Prodromou, C. (2008). Chaperone ligand-discrimination by the TPR-domain protein Tah1. Biochemical Journal 413, 261-268. Mine, A., Takeda, A., Taniguchi, T., Taniguchi, H., Kaido, M., Mise, K., and Okuno, T. (2010). Identification and Characterization of the 480-Kilodalton Template-Specific RNA-Dependent RNA Polymerase Complex of Red Clover Necrotic Mosaic Virus. Journal of Virology 84(12), 6070-6081. Mishra, S., Yadav, D. K., and Tuli, R. (2006). Ubiquitin fusion enhances cholera toxin B subunit expression in transgenic plants and the plant-expressed protein binds GM1 receptors more efficiently. Journal of Biotechnology 127(1), 95-108. Mitra, R., and Bhatia, C. R. (2008). Bioenergetic cost of heat tolerance in wheat crop. Current Science 94(8), 1049-1053. Molina, A., Hervas-Stubbs, S., Daniell, H., Mingo-Castel, A. M., and Veramendi, J. (2004). High-yield expression of a viral peptide animal vaccine in transgenic tobacco chloroplasts. Plant Biotechnology Journal 2(2), 141-153. Momose, F., Naito, T., Yano, K., Sugimoto, S., Morikawa, Y., and Nagata, K. (2002). Identification of Hsp90 as a stimulatory host factor involved in influenza virus RNA synthesis. Journal of Biological Chemistry 277(47), 45306-45314. Nagy, P. D. (2008). Yeast as a model host to explore plant virus-host interactions. Annual Review of Phytopathology 46, 217-242. NakariSetala, T., Aro, N., Kalkkinen, N., Alatalo, E., and Penttila, M. (1996). Genetic and biochemical characterization of the Trichoderma reesei hydrophobin HFBI. European Journal of Biochemistry 235(1-2), 248-255. Obregon, P., Chargelegue, D., Drake, P. M. W., Prada, A., Nuttall, J., Frigerio, L., and Ma, J. K. C. (2006). HIV-1 p24-immunoglobulin fusion molecule: a new strategy for plant-based protein production. Plant Biotechnology Journal 4(2), 195-207. Patel, J., Zhu, H., Menassa, R., Gyenis, L., Richman, A., and Brandle, J. (2007). Elastin-like polypeptide fusions enhance the accumulation of recombinant proteins in tobacco leaves. Transgenic Research 16(2), 239-249. Phillips, A. C., and Vousden, K. H. (1997). Analysis of the interaction between human papillomavirus type 16 E7 and the TATA binding protein, TBP. Journal of General Virology 78, 905-909. Pogue, G. P., Lindbo, J. A., Garger, S. J., and Fitzmaurice, W. P. (2002). Making an ally from an enemy: Plant virology and the new agriculture. Annual Review of Phytopathology 40, 45-74. Prasanth, K. R., Huang, Y. W., Liou, M. R., Wang, R. Y. L., Hu, C. C., Tsai, C. H., Meng, M. H., Lin, N. S., and Hsu, Y. H. (2011). Glyceraldehyde 3-Phosphate Dehydrogenase Negatively Regulates the Replication of Bamboo Mosaic Virus and Its Associated Satellite RNA. Journal of Virology 85(17), 8829-8840. Santos, M. J. D., Wigdorovitz, A., Trono, K., Rios, R. D., Franzone, P. M., Gil, F., Moreno, J., Carrillo, C., Escribano, J. M., and Borca, M. V. (2002). A novel methodology to develop a foot and mouth disease virus (FMDV) peptide-based vaccine in transgenic plants. Vaccine 20(7-8), 1141-1147. Schmidt, T. G., Koepke, J., Frank, R., and Skerra, A. (1996). Molecular interaction between the Strep-tag affinity peptide and its cognate target, streptavidin. Journal of Molecular Biology 255(5), 753-766. Scholthof, H. B., Scholthof, K. B. G., and Jackson, A. O. (1996). Plant virus gene vectors for transient expression of foreign proteins in plants. Annual Review of Phytopathology 34, 299-323. Serva, S., and Nagy, P. D. (2006). Proteomics analysis of the tombusvirus replicase: Hsp70 molecular chaperone is associated with the replicase and enhances viral RNA replication. Journal of Virology 80(5), 2162-2169. Sugio, A., Dreos, R., Aparicio, F., and Maule, A. J. (2009). The Cytosolic Protein Response as a Subcomponent of the Wider Heat Shock Response in Arabidopsis. Plant Cell 21(2), 642-654. Swindell, W. R., Huebner, M., and Weber, A. P. (2007). Transcriptional profiling of Arabidopsis heat shock proteins and transcription factors reveals extensive overlap between heat and non-heat stress response pathways. BMC Genomics 8:125. Thivierge, K., Nicaise, V., Dufresne, P. J., Cotton, S., Laliberte, J. F., Le Gall, O., and Fortin, M. G. (2005). Plant virus RNAs. Coordinated recruitment of conserved host functions by (+) ssRNA viruses during early infection events. Plant Physiology 138(4), 1822-1827. Tissieres A, Mitchell H. K. and Tracey U. M. (1974). Protein synthesis in the salivary glands of D. Melanogaster . Relation to chromosome puffs. Journal of Molecular Biology 84 389–398 Tomita, Y., Mizuno, T., Diez, J., Naito, S., Ahlquist, P., and Ishikawa, M. (2003). Mutation of host DnaJ homolog inhibits brome mosaic virus negative-strand RNA synthesis. Journal of Virology 77(5), 2990-2997. Torrent, M., Llompart, B., Lasserre-Ramassamy, S., Llop-Tous, I., Bastida, M., Marzabal, P., Westerholm-Parvinen, A., Saloheimo, M., Heifetz, P. B., and Ludevid, M. D. (2009). Eukaryotic protein production in designed storage organelles. BMC Biology 7:5. Valle, J. R. D., Chavez-Salinas, S., Medina, F., and del Angel, R. M. (2005). Heat shock protein 90 and heat shock protein 70 are components of dengue virus receptor complex in human cells. Journal of Virology 79(8), 4557-4567. Wahid, A., Gelani, S., Ashraf, M., and Foolad, M. R. (2007). Heat tolerance in plants: An overview. Environmental and Experimental Botany 61(3), 199-223. Wainberg, Z., Oliveira, M., Lerner, S., Tao, Y. Z., and Brenner, B. G. (1997). Modulation of stress protein (hsp27 and hsp70) expression in CD4+ lymphocytic cells following acute infection with human immunodeficiency virus type-1. Virology 233(2), 364-373. Wang, R. Y. L., Stork, J., and Nagy, P. D. (2009). A Key Role for Heat Shock Protein 70 in the Localization and Insertion of Tombusvirus Replication Proteins to Intracellular Membranes. Journal of Virology 83(7), 3276-3287. Weeks, S. A., and Miller, D. J. (2008). The heat shock protein 70 cochaperone YDJ1 is required for efficient membrane-specific flock house virus RNA replication complex assembly and function in Saccharomyces cerevisiae. Journal of Virology 82(4), 2004-2012. Whitham, S. A., Quan, S., Chang, H. S., Cooper, B., Estes, B., Zhu, T., Wang, X., and Hou, Y. M. (2003). Diverse RNA viruses elicit the expression of common sets of genes in susceptible Arabidopsis thaliana plants. Plant Journal 33(2), 271-283. Whitham, S. A., and Wang, Y. Z. (2004). Roles for host factors in plant viral pathogenicity. Current Opinion in Plant Biology 7(4), 365-371. Wung, C. H., Hsu, Y. H., Liou, D. Y., Huang, W. C., Lin, N. S., and Chang, B. Y. (1999). Identification of the RNA-binding sites of the triple gene block protein 1 of bamboo mosaic potexvirus. Journal of General Virology 80 , 1119-1126. Yang, C. C., Liu, J. S., Lin, C. P., and Lin, N. S. (1997). Nucleotide sequence and phylogenetic analysis of a bamboo mosaic potexvirus isolate from common bamboo (Bambusa vulgaris McClure). Botanical Bulletin of Academia Sinica 38(2), 77-84. Yeh, C.C., Cheng, A.H., and Hwang, H.Y. (1993). Indexing of bamboo mosaicvirus and propagation of virus-free bamboo. In: Chiu RJ, Yeh Y (eds) Proceedings of the symposium plant virus and virus-like diseases. Council of Agriculture, Plant Protection Series No. 1., 275-282
熱休克蛋白70 (Heat shock protein 70, Hsp70)在細胞內的功能主要是幫助蛋白質正確的折疊,並促進蛋白質之間的交互作用和組裝。在前人的研究中發現,Hsp70和竹嵌紋病毒(Bamboo mosaic virus, BaMV)負股核酸3’端的啟動子有交互作用,利用基因靜默降低菸草(Nicotiana benthamiana)內Hsp70的表現量之後發現BaMV的感染力也跟著下降,猜測Hsp70會參與BaMV的複製。為了證實此假說,我們利用Hsp70的抑制劑(Pifithrin-μ , PES)抑制菸草原生質體細胞中Hsp70的功能後進行病毒複製效率的分析,結果發現BaMV核酸複製能力會隨著PES的劑量增加而降低,以同樣的實驗測試Potato virus X (PVX)和Cucumber mosaic virus所得到的結果並不明顯,顯示出Hsp70對於BaMV的複製具有正調控的作用。
為了進一步分析Hsp70參與BaMV複製的分子機制,首先,我們由菸草中選殖出Hsp70的全長序列,設計探針,並利用北方墨點法實驗觀察到當菸草經過熱休克處理或病毒感染後,細胞內Hsp70基因的表現量會大量增加。從酵母菌雙雜交法我們證明了Hsp70和BaMV複製酵素之間的交互作用,接著希望藉由免疫共沉澱分析方法觀察到細胞內Hsp70和BaMV複製酵素之間的交互作用,以證實Hsp70分子伴護功能參與BaMV複製的過程。然而,此實驗需要製備能夠專一辨認BaMV 複製酵素的抗體,因此利用由大腸桿菌表現的蛋白質(ORF1 RdRp domain)當作抗原,免疫紐西蘭大白兔後取得抗體,此抗體可以成功辨認由大腸桿菌表現的蛋白質,但尚無法在BaMV感染的植物組織中偵測到BaMV ORF1產物,推測病毒在複製過程中不需要太多的複製酵素,導致在植物細胞內所表達的量並不足夠被抗體所辨認。為了增加BaMV複製酵素的表現量,構築融合HFBI胜肽在BaMV複製酵素C端的質體,此質體雖然仍具有感病活性但卻不如預期可提高活性的現象,所以最後仍無法測得複製酵素。在HFBI胜肽中含有StrepII序列,期待利用StrepII抗體偵測複製酵素(ORF1-HFBI),因此利用實驗室建立的狐尾草嵌紋病毒(Foxtail mosaic virus)和馬鈴薯病毒X(Potato virus X)胜肽展示病毒載體,以嵌合病毒型態在病毒顆粒表面展示StrepII胜肽分子並做為抗原以生產偵測StrepII的專一性抗體。結果發現雖然嵌合病毒質體可以成功地感染植物葉片,但無法大量製備病毒顆粒以當作抗原製造StrepII抗體。本實驗未來將進一步分析Hsp70 異構體參與BaMV複製的可能功能。

Heat shock protein 70 (Hsp70) is a highly conserved molecular chaperone for assisting proper folding of cellular proteins and facilitating protein-protein interaction and complex assembly. In the previous work, Hsp70 was found in association with partially purified replication complexes from Bamboo mosaic virus (BaMV)-infected Nicotiana benthamiana, and shown to interact with the 3’ untranslated region of BaMV minus-strand RNA. The knockdown of NbHsp70 by virus-induced gene silencing (VIGS) suppressed BaMV infectivity, suggesting that NbHsp70 involved in BaMV replication. To test this possibility, we used Pifithrin-μ (PES), a small molecule inhibitor of Hsp70, to inhibit Hsp70 function and then inoculated BaMV into N. benthamiana protoplasts. The results revealed that BaMV RNA accumulation was significantly reduced for PES treatments in a dose-dependent manner, suggesting that accumulation of BaMV RNA depends on the Hsp70 function.
To further analyze the molecular mechanism of Hsp70 involved in BaMV replication, we have cloned six isoforms of Hsp70 ORF from N. benthamiana and observed that the Hsp70 gene expression was significantly increased after heat shock treatment and virus infection. Next, to understand the recruitment of Hsp70 chaperone function in BaMV replication, Yeast-two-hybrid assay result showed that there are interaction between Hsp70 and BaMV RNA-dependent RNA polymerase (RdRp). Co-immunoprecipitation was another way to define the interaction, and would be performed with BaMV RdRp specific antibody. However, there is no such antibody in our lab so far. To this end, BaMV RdRp, over-expressed and purified from E. coli, was used to immunize rabbit for antibody production. The BaMV RdRp antibody prepared in this study could react to recombinant BaMV RdRp, but not to BaMV ORF1 product in BaMV infected tissue. This result revealed that the BaMV ORF1 expression is beyond the detection limit by BaMV RdRp antibody. To increase BaMV replicase accumulation, a HFBI peptide was fused to the C-terminal of BaMV ORF1 and constructed as an infectious clone, pCBP1HFBI. No increment of infectivity of pCBP1HFBI was shown in the infected N. benthamiana and ORF1-HFBI was still undetectable by BaMV RdRp antibody. Since HFBI comprises StrepII peptide,we therefore used FoMV- and PVX-based epitope-presentation system for antibody production by fusing the StrepII peptide to the surface of the chimeric virion. The FoMV and PVX chimeric clones were able to infect plant leaves. However, the infectivities were dramatically decreased, and in consequence we could not get large scale material for virion purification. In the future, we will continue to analyze the involvement of Hsp70 isoforms in BaMV replication.
其他識別: U0005-1207201213241900
Appears in Collections:生物科技學研究所

Show full item record

Google ScholarTM


Items in DSpace are protected by copyright, with all rights reserved, unless otherwise indicated.