Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/36207
標題: 煙草宿主蛋白Hsp70和Hsp90參與竹嵌紋病毒感染週期之研究
Identification of Host Factors Hsp70 and Hsp90 of Nicotiana benthamiana Participated in the Infection Cycle of Bamboo Mosaic Virus
作者: 陸曉慧
Lu, Shiau-Huei
關鍵字: Hsp70;熱休克蛋白70;Hsp90;Bamboo mosaic virus;熱休克蛋白90;竹嵌紋病毒
出版社: 生物科技學研究所
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Cheng, C. P. & Tsai, C. H. (1999). Structural and functional analysis of the 3'' untranslated region of bamboo mosaic potexvirus genomic RNA. J Mol Biol 288, 555-565. Cheng, J. H., Ding, M. P., Hsu, Y. H. & 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 Res 80, 41-52. Cruz, S. S., Roberts, A. G., Prior, D. A., Chapman, S. & Oparka, K. J. (1998). Cell-to-cell and phloem-mediated transport of potato virus X. The role of virions. The Plant cell 10, 495-510. Hartl, F. U. & Hayer-Hartl, M. (2002). Molecular chaperones in the cytosol: from nascent chain to folded protein. Science (New York, NY 295, 1852-1858. Huang, C. Y., Huang, Y. L., Meng, M., Hsu, Y. H. & Tsai, C. H. (2001). Sequences at the 3'' untranslated region of bamboo mosaic potexvirus RNA interact with the viral RNA-dependent RNA polymerase. J Virol 75, 2818-2824. Huang, Y. L., Han, Y. T., Chang, Y. T., Hsu, Y. H. & Meng, M. (2004). Critical residues for GTP methylation and formation of the covalent m7GMP-enzyme intermediate in the capping enzyme domain of bamboo mosaic virus. J Virol 78, 1271-1280. Kalantidis, K., Denti, M. A., Tzortzakaki, S., Marinou, E., Tabler, M. & Tsagris, M. (2007). Virp1 is a host protein with a major role in Potato spindle tuber viroid infection in Nicotiana plants. Journal of virology 81, 12872-12880. Li, Y. I., Chen, Y. J., Hsu, Y. H. & Meng, M. (2001a). Characterization of the AdoMet-dependent guanylyltransferase activity that is associated with the N terminus of bamboo mosaic virus replicase. J Virol 75, 782-788. Li, Y. I., Cheng, Y. M., Huang, Y. L., Tsai, C. H., Hsu, Y. H. & Meng, M. (1998). Identification and characterization of the Escherichia coli-expressed RNA-dependent RNA polymerase of bamboo mosaic virus. J Virol 72, 10093-10099. Li, Y. I., Shih, T. W., Hsu, Y. H., Han, Y. T., Huang, Y. L. & 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. J Virol 75, 12114-12120. Lin, J. W., Chiu, H. N., Chen, I. H., Chen, T. C., Hsu, Y. H. & Tsai, C. H. (2005). Structural and functional analysis of the cis-acting elements required for plus-strand RNA synthesis of Bamboo mosaic virus. J Virol 79, 9046-9053. Lin, J. W., Ding, M. P., Hsu, Y. H. & Tsai, C. H. (2007). Chloroplast phosphoglycerate kinase, a gluconeogenetic enzyme, is required for efficient accumulation of Bamboo mosaic virus. Nucleic Acids Res 35, 424-432. Lin, M. K., Chang, B. Y., Liao, J. T., Lin, N. S. & Hsu, Y. H. (2004). Arg-16 and Arg-21 in the N-terminal region of the triple-gene-block protein 1 of Bamboo mosaic virus are essential for virus movement. J Gen Virol 85, 251-259. Lin, M. K., Hu, C. C., Lin, N. S., Chang, B. Y. & Hsu, Y. H. (2006). Movement of potexviruses requires species-specific interactions among the cognate triple gene block proteins, as revealed by a trans-complementation assay based on the bamboo mosaic virus satellite RNA-mediated expression system. J Gen Virol 87, 1357-1367. Lin, N. S., Lin, B. Y., Lo, N. W., Hu, C. C., Chow, T. Y. & Hsu, Y. H. (1994). Nucleotide sequence of the genomic RNA of bamboo mosaic potexvirus. J Gen Virol 75 ( Pt 9), 2513-2518. Vijaya Palani, P., Kasiviswanathan, V., Chen, J. C., Chen, W., Hsu, Y. H. & Lin, N. S. (2006). The arginine-rich motif of Bamboo mosaic virus satellite RNA-encoded P20 mediates self-interaction, intracellular targeting, and cell-to-cell movement. Mol Plant Microbe Interact 19, 758-767. Weeks, S. A. & 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, 2004-2012. Young, J. 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摘要: 
竹嵌紋病毒(Bamboo mosaic virus, BaMV) 為一長絲狀植物病毒顆粒,隸屬於Flexiviridae科中之Potexvirus屬,擁有單股正極核醣核酸基因體。由於病毒屬於絕對寄主,因此寄主蛋白在病毒複製過程中,乃是不可或缺的因子,因此我們想探討在竹嵌紋病毒進行正股複製時,是否也有寄主蛋白參與其中。在前人的研究中,利用竹嵌紋病毒負股3''端的啟動子序列(Ba-77 RNA)為探針,進行紫外光誘發核醣核酸-蛋白質交互作用,來偵測在竹嵌紋病毒複製複合體的蔗糖梯度離心萃取液,是否含有能和這段探針進行交互作用的宿主蛋白,結果發現,有兩個蛋白質分子量分別是72kDa和81kDa能和這兩段探針進行專一性的結合,經由蛋白質層析純化及質譜儀鑑定顯示72kDa及81kDa蛋白質可能是Hsp70及Hsp90。另外,利用病毒誘導式基因沉默系統 (virus-induced gene silencing) 抑制Hsp70或Hsp90的表現,會抑制竹嵌紋病毒鞘蛋白的累積量。因此為了進一步證實這兩個蛋白質與病毒感染的關係,我們分別將Hsp70和Hsp90全長基因選殖出來並接上不同HA與T7-Tag。我們將這兩個蛋白質分別在感染竹嵌紋病毒的煙草中進行短暫的表現(transient expression)後再感染病毒,發現對竹嵌紋病毒在細胞內的累積量並沒有很大的影響。我們則進一步追蹤發現這兩個短暫性表現的蛋白質確實會出現於竹嵌紋病毒複製複合體內,然而利用Ba-77 RNA當探針,進行紫外光誘發核醣核酸-蛋白質交互作用再進行免疫沉澱法後,並沒有看到預期的NbHsp70和NbHsp90-2,初步猜測可能是,植物內生性的NbHsp70和NbHsp90量太多,而短暫表現具有tag的蛋白相對太少或者因為抗體辨識tag的效率不盡理想,以至於沒有看到預期的結果;因此,另外嘗試可以辨識NbHsp90-2的抗體進行免疫沉澱,沉澱效率不如預期,需進一步調整實驗條件。

Bamboo mosaic virus (BaMV), belonging to genus Potexvirus of the family Flexiviridae, is a plus-strand RNA virus. Previously, the promoter sequence at the 3-end of minus-strand for plus-strand genomic RNA synthesis (Ba-77) was found to crosslinked with two specific host factors derived from RdRp extracts of BaMV-infected N. benthamiana plants. These two host factors with Mr 72 and 81kDa were identified as Hsp70 and Hsp90, respectively, by LC/MS/MS. We also found that a reduction of gene expression of Hsp70 or Hsp90 in N. benthamiana could cause a defect in BaMV coat protein accumulation. Therefore, we would like to further analyze the relationship of these two genes with the infection cycle of BaMV. First, we cloned the full-length of these two genes from N. benthamiana plant by polymerase chain reaction. The full-length of NbHsp70 and NbHsp90-2 fused with HA- and T7-tag at its N-terminus are transiently expressed in N. benthamiana leaves by agroinfiltration. We have found that these two transiently expressed proteins can be recruited into the BaMV functional RdRp preparations. However, through UV-crosslinking with Ba-77 and immunoprecipitation assays we can not detect these two transiently expressed proteins in the functional RdRp preparation. It is possible that the antibody against the fusion tag is not efficient enough to pull down the crosslinked NbHsp70 or NbHsp90-2 in the crosslinked products. We then tried to use the polyclonal IgG against E. coli-expressed Hsp90 to pull down the crosslinked products from one of the RdRp activity fractions but shown inefficient.
URI: http://hdl.handle.net/11455/36207
其他識別: U0005-1408200916151500
Appears in Collections:生物科技學研究所

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