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完整後設資料紀錄
DC 欄位 | 值 | 語言 |
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dc.contributor.advisor | 余明俊(Ming-Jiun Yu) | |
dc.contributor.author | Weng-Man Chong | en |
dc.contributor.author | 鍾穎文 | zh_TW |
dc.date.accessioned | 2021-06-07T17:57:07Z | - |
dc.date.copyright | 2012-09-19 | |
dc.date.issued | 2012 | |
dc.date.submitted | 2012-08-14 | |
dc.identifier.citation | 1 Alter, M. J. Epidemiology of Hepatitis C Virus Infection. World Journal of Gastroenterology 13 2436-2441, (2007).
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EMBO J 21, 5017-5025, (2002). 8 Pileri, P., Uematsu, Y., Campagnoli, S., Galli, G., Falugi, F., Petracca, R., Weiner, A. J., Houghton, M., Rosa, D., Grandi, G. & Abrignani, S. Binding of Hepatitis C Virus to Cd81. Science 282, 938-941, (1998). 9 Moradpour, D., Penin, F. & Rice, C. M. Replication of Hepatitis C Virus. Nat Rev Micro 5, 453-463, (2007). 10 Reed, K. E., Xu, J. & Rice, C. M. Phosphorylation of the Hepatitis C Virus NS5A Protein in Vitro and in Vivo: Properties of the NS5A-Associated Kinase. Journal of Virology 71, 7187-7197, (1997). 11 Gosert, R., Egger, D., Lohmann, V., Bartenschlager, R., Blum, H. E., Bienz, K. & Moradpour, D. Identification of the Hepatitis C Virus RNA Replication Complex in Huh-7 Cells Harboring Subgenomic Replicons. Journal of Virology 77, 5487-5492, (2003). 12 Bartenschlager, R., Ahlborn-Laake, L., Mous, J. & Jacobsen, H. Nonstructural Protein 3 of the Hepatitis C Virus Encodes a Serine-Type Proteinase Required for Cleavage at the NS3/4 and Ns4/5 Junctions. J Virol 67, 3835-3844, (1993). 13 Egger, D., Wolk, B., Gosert, R., Bianchi, L., Blum, H. E., Moradpour, D. & Bienz, K. Expression of Hepatitis C Virus Proteins Induces Distinct Membrane Alterations Including a Candidate Viral Replication Complex. J Virol 76, 5974-5984, (2002). 14 He, Y., Staschke, K. A. & Tan, S. L. HCV NS5A: A Multifunctional Regulator of Cellular Pathways and Virus Replication. (2006). 15 Wu, S. C., Chang, S. C., Wu, H. Y., Liao, P. J. & Chang, M. F. Hepatitis C Virus NS5A Protein Down-Regulates the Expression of Spindle Gene Aspm through Pkr-P38 Signaling Pathway. J Biol Chem 283, 29396-29404, (2008). 16 Park, C.-Y., Choi, S.-H., Kang, S.-M., Kang, J.-I., Ahn, B.-Y., Kim, H., Jung, G., Choi, K.-Y. & Hwang, S. B. Nonstructural 5a Protein Activates [Beta]-Catenin Signaling Cascades: Implication of Hepatitis C Virus-Induced Liver Pathogenesis. Journal of hepatology 51, 853-864, (2009). 17 Evans, M. J., Rice, C. M. & Goff, S. P. Phosphorylation of Hepatitis C Virus Nonstructural Protein 5a Modulates Its Protein Interactions and Viral RNA Replication. Proc Natl Acad Sci U S A 101, 13038-13043, (2004). 18 Huang, Y., Staschke, K., De Francesco, R. & Tan, S. L. Phosphorylation of Hepatitis C Virus NS5A Nonstructural Protein: A New Paradigm for Phosphorylation-Dependent Viral RNA Replication? Virology 364, 1-9, (2007). 19 Tellinghuisen, T. L., Foss, K. L. & Treadaway, J. Regulation of Hepatitis C Virion Production Via Phosphorylation of the NS5A Protein. PLoS pathogens 4, e1000032, (2008). 20 Popescu, C. I., Callens, N., Trinel, D., Roingeard, P., Moradpour, D., Descamps, V., Duverlie, G., Penin, F., Heliot, L., Rouille, Y. & Dubuisson, J. NS2 Protein of Hepatitis C Virus Interacts with Structural and Non-Structural Proteins Towards Virus Assembly. PLoS Pathog 7, e1001278, (2011). 21 Lohmann, V., Korner, F., Herian, U. & Bartenschlager, R. Biochemical Properties of Hepatitis C Virus NS5B RNA-Dependent RNA Polymerase and Identification of Amino Acid Sequence Motifs Essential for Enzymatic Activity. J Virol 71, 8416-8428, (1997). 22 Francois, C., Castelain, S., Duverlie, G., Capron, D. & Nguyen-Khac, E. Optimizing the Treatment of Chronic Viral Hepatitis C. Expert Rev Gastroenterol Hepatol 3, 607-613, (2009). 23 Tanaka, Y., Nishida, N., Sugiyama, M., Kurosaki, M., Matsuura, K., Sakamoto, N., Nakagawa, M., Korenaga, M., Hino, K., Hige, S., Ito, Y., Mita, E., Tanaka, E., Mochida, S., Murawaki, Y., Honda, M., Sakai, A., Hiasa, Y., Nishiguchi, S., Koike, A., Sakaida, I., Imamura, M., Ito, K., Yano, K., Masaki, N., Sugauchi, F., Izumi, N., Tokunaga, K. & Mizokami, M. Genome-Wide Association of Il28b with Response to Pegylated Interferon-Alpha and Ribavirin Therapy for Chronic Hepatitis C. Nat Genet 41, 1105-1109, (2009). 24 Lin, C., Kwong, A. D. & Perni, R. B. Discovery and Development of Vx-950, a Novel, Covalent, and Reversible Inhibitor of Hepatitis C Virus NS3.4a Serine Protease. Infect Disord Drug Targets 6, 3-16, (2006). 25 Chen, K. X. & Njoroge, F. G. The Journey to the Discovery of Boceprevir: An NS3-Ns4 HCV Protease Inhibitor for the Treatment of Chronic Hepatitis C. Prog Med Chem 49, 1-36, (2010). 26 Brass, V., Bieck, E., Montserret, R., Wolk, B., Hellings, J. A., Blum, H. E., Penin, F. & Moradpour, D. An Amino-Terminal Amphipathic Alpha-Helix Mediates Membrane Association of the Hepatitis C Virus Nonstructural Protein 5a. J Biol Chem 277, 8130-8139, (2002). 27 T. Kaneko, Y. T., S. Satoh, M. Hijikata, S. Asabe, K. Kimura and K. Shimotohno. Production of Two Phosphoproteins from the NS5A Region of the Hepatitis C Viral Genome. Biochem. Biophys. Res. Commun. 205, 6, (1994). 28 Tu, H., Gao, L., Shi, S. T., Taylor, D. R., Yang, T., Mircheff, A. K., Wen, Y., Gorbalenya, A. E., Hwang, S. B. & Lai, M. M. Hepatitis C Virus RNA Polymerase and NS5A Complex with a Snare-Like Protein. Virology 263, 30-41, (1999). 29 Shirota, Y., Luo, H., Qin, W., Kaneko, S., Yamashita, T., Kobayashi, K. & Murakami, S. Hepatitis C Virus (HCV) NS5A Binds RNA-Dependent RNA Polymerase (Rdrp) NS5B and Modulates RNA-Dependent RNA Polymerase Activity. J Biol Chem 277, 11149-11155, (2002). 30 Appel, N., Pietschmann, T. & Bartenschlager, R. Mutational Analysis of Hepatitis C Virus Nonstructural Protein 5a: Potential Role of Differential Phosphorylation in RNA Replication and Identification of a Genetically Flexible Domain. J Virol 79, 3187-3194, (2005). 31 Bukh, J., Pietschmann, T., Lohmann, V., Krieger, N., Faulk, K., Engle, R. E., Govindarajan, S., Shapiro, M., St Claire, M. & Bartenschlager, R. Mutations That Permit Efficient Replication of Hepatitis C Virus RNA in Huh-7 Cells Prevent Productive Replication in Chimpanzees. Proc Natl Acad Sci U S A 99, 14416-14421, (2002). 32 Neddermann, P., Quintavalle, M., Di Pietro, C., Clementi, A., Cerretani, M., Altamura, S., Bartholomew, L. & De Francesco, R. Reduction of Hepatitis C Virus NS5A Hyperphosphorylation by Selective Inhibition of Cellular Kinases Activates Viral RNA Replication in Cell Culture. J. Virol. 78, 13306-13314, (2004). 33 Koch, J. O. & Bartenschlager, R. Modulation of Hepatitis C Virus NS5A Hyperphosphorylation by Nonstructural Proteins NS3, NS4A, and NS4B. J Virol 73, 7138-7146, (1999). 34 Tanji, Y., Kaneko, T., Satoh, S. & Shimotohno, K. Phosphorylation of Hepatitis C Virus-Encoded Nonstructural Protein NS5A. J Virol 69, 3980-3986, (1995). 35 Blight, K. J., Kolykhalov, A. A. & Rice, C. M. Efficient Initiation of HCV RNA Replication in Cell Culture. Science 290, 1972-1974, (2000). 36 Reed, K. E. & Rice, C. M. Identification of the Major Phosphorylation Site of the Hepatitis C Virus H Strain NS5A Protein as Serine 2321. J Biol Chem 274, 28011-28018, (1999). 37 Katze, M. G., Kwieciszewski, B., Goodlett, D. R., Blakely, C. M., Neddermann, P., Tan, S. L. & Aebersold, R. Ser(2194) Is a Highly Conserved Major Phosphorylation Site of the Hepatitis C Virus Nonstructural Protein NS5A. Virology 278, 501-513, (2000). 38 Lindenbach, B. D., Evans, M. J., Syder, A. J., Wolk, B., Tellinghuisen, T. L., Liu, C. C., Maruyama, T., Hynes, R. O., Burton, D. R., McKeating, J. A. & Rice, C. M. Complete Replication of Hepatitis C Virus in Cell Culture. Science 309, 623-626, (2005). 39 Moore, R. E., Young, M. K. & Lee, T. D. Protein Identification Using a Quadrupole Ion Trap Mass Spectrometer and Sequest Database Matching. Curr Protoc Protein Sci Chapter 16, Unit 16 10, (2001). 40 Barsnes, H., Huber, S., Sickmann, A., Eidhammer, I. & Martens, L. Omssa Parser: An Open-Source Library to Parse and Extract Data from Omssa Ms/Ms Search Results. Proteomics 9, 3772-3774, (2009). 41 Tanner, S., Shu, H., Frank, A., Wang, L. C., Zandi, E., Mumby, M., Pevzner, P. A. & Bafna, V. Inspect: Identification of Posttranslationally Modified Peptides from Tandem Mass Spectra. Anal Chem 77, 4626-4639, (2005). 42 Beausoleil, S., Villen, J., Gerber, S., Rush, J. & Gygi, S. A Probability-Based Approach for High-Throughput Protein Phosphorylation Analysis and Site Localization. Nature biotechnology 24, 1285-1292, (2006). 43 Beausoleil, S. A., Jedrychowski, M., Schwartz, D., Elias, J. E., Villen, J., Li, J., Cohn, M. A., Cantley, L. C. & Gygi, S. P. Large-Scale Characterization of Hela Cell Nuclear Phosphoproteins. Proceedings of the National Academy of Sciences of the United States of America 101, 12130-12135, (2004). 44 Ferraris, P., Blanchard, E. & Roingeard, P. Ultrastructural and Biochemical Analyses of Hepatitis C Virus-Associated Host Cell Membranes. Journal of General Virology 91, 2230-2237, (2010). 45 Moradpour, D., Evans, M. J., Gosert, R., Yuan, Z., Blum, H. E., Goff, S. P., Lindenbach, B. D. & Rice, C. M. Insertion of Green Fluorescent Protein into Nonstructural Protein 5a Allows Direct Visualization of Functional Hepatitis C Virus Replication Complexes. J. Virol. 78, 7400-7409, (2004). 46 Quintavalle, M., Sambucini, S., Di Pietro, C., De Francesco, R. & Neddermann, P. The Α Isoform of Protein Kinase Cki Is Responsible for Hepatitis C Virus NS5A Hyperphosphorylation. Journal of Virology 80, 11305-11312, (2006). 47 Quintavalle, M., Sambucini, S., Summa, V., Orsatti, L., Talamo, F., De Francesco, R. & Neddermann, P. Hepatitis C Virus NS5A Is a Direct Substrate of Casein Kinase I-Α, a Cellular Kinase Identified by Inhibitor Affinity Chromatography Using Specific NS5A Hyperphosphorylation Inhibitors. Journal of Biological Chemistry 282, 5536-5544, (2007). 48 Chen, Y. C., Su, W. C., Huang, J. Y., Chao, T. C., Jeng, K. S., Machida, K. & Lai, M. M. Polo-Like Kinase 1 Is Involved in Hepatitis C Virus Replication by Hyperphosphorylating NS5A. J Virol 84, 7983-7993, (2010). 49 Xue, Y., Li, A., Wang, L., Feng, H. & Yao, X. Ppsp: Prediction of Pk-Specific Phosphorylation Site with Bayesian Decision Theory. BMC Bioinformatics 7, 163, (2006). 50 Bain, J., Plater, L., Elliott, M., Shpiro, N., Hastie, C. J., McLauchlan, H., Klevernic, I., Arthur, J. S. C., Alessi, D. R. & Cohen, P. The Selectivity of Protein Kinase Inhibitors: A Further Update. Biochem J 408, 297-315, (2007). 51 Nordle Gilliver, A., Griffin, S. & Harris, M. Identification of a Novel Phosphorylation Site in Hepatitis C Virus NS5A. The Journal of general virology 91, 2428-2432, (2010). 52 Tellinghuisen, T. L., Marcotrigiano, J., Gorbalenya, A. E. & Rice, C. M. The NS5A Protein of Hepatitis C Virus Is a Zinc Metalloprotein. J Biol Chem 279, 48576-48587, (2004). 53 Shimakami, T., Hijikata, M., Luo, H., Ma, Y. Y., Kaneko, S., Shimotohno, K. & Murakami, S. Effect of Interaction between Hepatitis C Virus NS5A and NS5B on Hepatitis C Virus RNA Replication with the Hepatitis C Virus Replicon. Journal of Virology 78, 2738-2748, (2004). 54 Coito, C., Diamond, D. L., Neddermann, P., Korth, M. J. & Katze, M. G. High-Throughput Screening of the Yeast Kinome: Identification of Human Serine/Threonine Protein Kinases That Phosphorylate the Hepatitis C Virus NS5A Protein. J Virol 78, 3502-3513, (2004). 55 Kim, J., Lee, D. & Choe, J. Hepatitis C Virus NS5A Protein Is Phosphorylated by Casein Kinase Ii. Biochem Biophys Res Commun 257, 777-781, (1999). 56 Geer, L. Y., Markey, S. P., Kowalak, J. A., Wagner, L., Xu, M., Maynard, D. M., Yang, X., Shi, W. & Bryant, S. H. Open Mass Spectrometry Search Algorithm. J Proteome Res 3, 958-964, (2004). 57 Elias, J. E. & Gygi, S. P. Target-Decoy Search Strategy for Increased Confidence in Large-Scale Protein Identifications by Mass Spectrometry. Nat Methods 4, 207-214, (2007). 58 Kuiken, C., Yusim, K., Boykin, L. & Richardson, R. The Los Alamos Hepatitis C Sequence Database. Bioinformatics 21, 379-384, (2005). | |
dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/15981 | - |
dc.description.abstract | C型肝炎為一慢性的肝發炎疾病,也是引發肝癌的原因之一。其致病原 ─ C型肝炎病毒(Hepatitis C virus, HCV)為單一正股RNA病毒。HCV之基因體可轉譯出一條多蛋白 (Polyprotein),透過蛋白酶的切割,此多蛋白能產生具有功能的結構性(Structural protein)及非結構性蛋白(Nonstructural protein)。其中,C型肝炎病毒的非結構性蛋白5A (Non-structural protein 5A, NS5A)為一重要的抗病毒藥物標的,但NS5A在HCV生命周期中的詳細功能尚不清楚。過去報導顯示NS5A會受磷酸化調控,而其磷酸化會影響C型肝病毒的複製能力。但NS5A的磷酸化位點尚未清楚。
利用液相層析串聯式質譜術 (Liquid chromatography coupled with tandem mass spectrometry),我們鑑定出一個全新的NS5A磷酸化位點 (Serine 2207, S2207),並發現此位點存在於各基因型的C型肝炎病毒中。為了探討此位點功能,我們把帶有報導基因之HCV載體的serine 2207突變為alanine,以抑制該位點的磷酸化。將此載體表現於人類肝腫瘤細胞株(Huh7.5.1),結果顯示,HCV突變株的報導基因活性無法隨感染時間增加。在進一步的研究中,我們發現NS5A蛋白S2207位點的磷酸化程度隨病毒感染時間增加,亦觀察到S2207磷酸化的NS5A與另一參與病毒複製的蛋白─非結構性蛋白3 (NS3)有共位現象(co-localization)。這些結果確認S2207的磷酸化存在於HCV生命周期中,並暗示S2207的磷酸化在HCV複製之重要性。透過利用casein kinase I (CKI)的抑制劑進行實驗,我們發現S2207的磷酸化隨抑制劑劑量增加而下降,顯示CKI或許參與在S2207的磷酸化過程中。 綜上所述,本研究發現一個全新的NS5A磷酸化位點,並證明此位點之磷酸化在病毒複製中的重要性。這些發現增進我們對NS5A的認識,並將有助於我們探討調控HCV複製的詳細機轉。 | zh_TW |
dc.description.abstract | Phosphorylation of the hepatitis C virus (HCV) nonstructural protein 5A (NS5A) is implicated in HCV life cycle. Disrupting NS5A phosphorylation has been reported to affect viral replication and hence may be of clinical relevance; however, specific sites of NS5A phosphorylation have been elusive.
In this study, we identified 1,776 unique phosphorylation sites in 1,088 proteins of HCV (J6/JFH1 strain, genotype 2a) infected Huh7.5.1 cells via liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). NS5A protein was found to be phosphorylated at serine 2194, a previously reported site, and serine 2207 (S2207), a new site. Mutation of S2207 to a phosphorylation-ablative residue in an HCV reporter construct abolished HCV replication. Immunoblotting showed a time-dependent increase in S2207 phosphorylated NS5A upon HCV infection. Immunofluorescence staining revealed that S2207 phosphorylated NS5A co-localized with an HCV replication complex component, nonstructural protein 3 (NS3) and localized in cytoplasmic membranous structures, where viral replication takes place. These findings suggest a role of NS5A S2207 phosphorylation in HCV RNA replication. Furthermore, we discovered that a casein kinase I inhibitor, D4476, reduced S2207 phosphorylation in a dose-dependent manner, thus suggesting a role of casein kinase in S2207 phosphorylation. In conclusion, our study identifies a new casein kinase inhibitor sensitive NS5A phosphorylation site (serine 2207) which is involved in HCV replication. | en |
dc.description.provenance | Made available in DSpace on 2021-06-07T17:57:07Z (GMT). No. of bitstreams: 1 ntu-101-R99442036-1.pdf: 2752666 bytes, checksum: 20c13f808a65aab1c900d73ceae2e1e1 (MD5) Previous issue date: 2012 | en |
dc.description.tableofcontents | Certificate 1
Abstract (in Chinese) 2 Abstract 3 Introduction 8 Materials and Methods 13 Cells and Reagents 13 Phosphoproteomic Analysis 14 Site-Directed Mutagenesis 15 In Vitro Transcription of Viral RNAs 15 Viral RNAs Transfection and Luciferase Assay 15 Immunofluorescence Staining and Confocal Microscopy 16 NS5A Expression Constructs 16 Kinase Inhibition Assay 17 MTT Assay 17 Results 19 Phosphoproteomic Approach Identified a Novel HCV NS5A Phosphorylation Site 19 NS5A Phosphorylation at Serine 2207 Plays a Potential Regulatory Role in HCV RNA Replication 20 NS5A Phosphorylation at Serine 2207 Increases in HCV Propagation 21 Serine 2207 Phosphorylated NS5A Localizes in Vesicle-Like Compartments and Co-localizes with NS3, a Replication Complex Component 22 Inhibition of Casein Kinase I Reduces NS5A Phosphorylation at Serine 2207 23 Discussion 24 Figures and Tables 28 Figure Legends 39 Supplemental Figures 44 References 47 | |
dc.language.iso | en | |
dc.title | C型肝炎病毒非結構性蛋白5A磷酸化位點之鑑定與功能探討 | zh_TW |
dc.title | Identification and Functional Studies of a Novel Nonstructural Protein 5A Phosphorylation Site in Hepatitis C Virus | en |
dc.type | Thesis | |
dc.date.schoolyear | 100-2 | |
dc.description.degree | 碩士 | |
dc.contributor.oralexamcommittee | 鄧述諄(Shu-Chun Teng),楊宏志(Hung-Chih Yang) | |
dc.subject.keyword | C型肝炎病毒,HCV複製機轉,非結構性蛋白5A,NS5A磷酸化,NS5A磷酸化位點, | zh_TW |
dc.subject.keyword | Hepatitis C virus,HCV replication,Nonstructural protein 5A,NS5A phosphorylation,NS5A phosphorylation sites, | en |
dc.relation.page | 54 | |
dc.rights.note | 未授權 | |
dc.date.accepted | 2012-08-14 | |
dc.contributor.author-college | 醫學院 | zh_TW |
dc.contributor.author-dept | 生物化學暨分子生物學研究所 | zh_TW |
顯示於系所單位: | 生物化學暨分子生物學科研究所 |
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