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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 王萬波(Won-Bo Wang) | |
| dc.contributor.author | Pin-Ching Pu | en |
| dc.contributor.author | 蒲品靜 | zh_TW |
| dc.date.accessioned | 2021-06-16T02:32:31Z | - |
| dc.date.available | 2020-09-25 | |
| dc.date.copyright | 2015-09-25 | |
| dc.date.issued | 2015 | |
| dc.date.submitted | 2015-07-29 | |
| dc.identifier.citation | Beigel, J. and M. Bray (2008). 'Current and future antiviral therapy of severe seasonal and avian influenza.' Antiviral Res 78(1): 91-102. Blaas, D., E. Patzelt and E. Kuechler (1982). 'Identification of the cap binding protein of influenza virus.' Nucleic Acids Res 10(15): 4803-4812. Blin, J. and K. A. Fitzgerald (2015). 'Perspective: The RNA exosome, cytokine gene regulation and links to autoimmunity.' Cytokine 74(2): 175-180. Boivin, S., S. Cusack, R. W. Ruigrok and D. J. Hart (2010). 'Influenza A virus polymerase: structural insights into replication and host adaptation mechanisms.' J Biol Chem 285(37): 28411-28417. Brass, A. L., I. C. Huang, Y. Benita, S. P. John, M. N. Krishnan, E. M. Feeley, B. J. Ryan, J. L. Weyer, L. van der Weyden, E. Fikrig, D. J. Adams, R. J. Xavier, M. Farzan and S. J. Elledge (2009). 'The IFITM proteins mediate cellular resistance to influenza A H1N1 virus, West Nile virus, and dengue virus.' Cell 139(7): 1243-1254. Chen, W., P. A. Calvo, D. Malide, J. Gibbs, U. Schubert, I. Bacik, S. Basta, R. O'Neill, J. Schickli, P. Palese, P. Henklein, J. R. Bennink and J. W. Yewdell (2001). 'A novel influenza A virus mitochondrial protein that induces cell death.' Nat Med 7(12): 1306-1312. Chlebowski, A., M. Lubas, T. H. Jensen and A. Dziembowski (2013). 'RNA decay machines: the exosome.' Biochim Biophys Acta 1829(6-7): 552-560. Das, K., J. M. Aramini, L. C. Ma, R. M. Krug and E. Arnold (2010). 'Structures of influenza A proteins and insights into antiviral drug targets.' Nat Struct Mol Biol 17(5): 530-538. De Clercq, E. (2006). 'Antiviral agents active against influenza A viruses.' Nat Rev Drug Discov 5(12): 1015-1025. Deleault, K. M., S. J. Skinner and S. A. Brooks (2008). 'Tristetraprolin regulates TNF-alpha mRNA stability via a proteasome dependent mechanism involving the combined action of the ERK and p38 pathways.' Mol Immunol 45(1): 13-24. Diamond, M. S. and M. Farzan (2013). 'The broad-spectrum antiviral functions of IFIT and IFITM proteins.' Nat Rev Immunol 13(1): 46-57. Eckard, S. C., G. I. Rice, A. Fabre, C. Badens, E. E. Gray, J. L. Hartley, Y. J. Crow and D. B. Stetson (2014). 'The SKIV2L RNA exosome limits activation of the RIG-I-like receptors.' Nat Immunol 15(9): 839-845. Engelhardt, O. G. and E. Fodor (2006). 'Functional association between viral and cellular transcription during influenza virus infection.' Rev Med Virol 16(5): 329-345. Everitt, A. R., S. Clare, T. Pertel, S. P. John, R. S. Wash, S. E. Smith, C. R. Chin, E. M. Feeley, J. S. Sims, D. J. Adams, H. M. Wise, L. Kane, D. Goulding, P. Digard, V. Anttila, J. K. Baillie, T. S. Walsh, D. A. Hume, A. Palotie, Y. Xue, V. Colonna, C. Tyler-Smith, J. Dunning, S. B. Gordon, I. I. Gen, M. Investigators, R. L. Smyth, P. J. Openshaw, G. Dougan, A. L. Brass and P. Kellam (2012). 'IFITM3 restricts the morbidity and mortality associated with influenza.' Nature 484(7395): 519-523. Fodor, E., M. Crow, L. J. Mingay, T. Deng, J. Sharps, P. Fechter and G. G. Brownlee (2002). 'A single amino acid mutation in the PA subunit of the influenza virus RNA polymerase inhibits endonucleolytic cleavage of capped RNAs.' J Virol 76(18): 8989-9001. Fodor, E., L. Devenish, O. G. Engelhardt, P. Palese, G. G. Brownlee and A. Garcia-Sastre (1999). 'Rescue of influenza A virus from recombinant DNA.' J Virol 73(11): 9679-9682. Fouchier, R. A., V. Munster, A. Wallensten, T. M. Bestebroer, S. Herfst, D. Smith, G. F. Rimmelzwaan, B. Olsen and A. D. Osterhaus (2005). 'Characterization of a novel influenza A virus hemagglutinin subtype (H16) obtained from black-headed gulls.' J Virol 79(5): 2814-2822. Fraser, C., C. A. Donnelly, S. Cauchemez, W. P. Hanage, M. D. Van Kerkhove, T. D. Hollingsworth, J. Griffin, R. F. Baggaley, H. E. Jenkins, E. J. Lyons, T. Jombart, W. R. Hinsley, N. C. Grassly, F. Balloux, A. C. Ghani, N. M. Ferguson, A. Rambaut, O. G. Pybus, H. Lopez-Gatell, C. M. Apluche-Aranda, I. B. Chapela, E. P. Zavala, D. M. Guevara, F. Checchi, E. Garcia, S. Hugonnet and C. Roth (2009). 'Pandemic potential of a strain of influenza A (H1N1) : Early findings.' Science 324(5934): 1557-1561. Gabriel, G., A. Herwig and H. D. Klenk (2008). 'Interaction of polymerase subunit PB2 and NP with importin alpha1 is a determinant of host range of influenza A virus.' PLoS Pathog 4(2): e11. Gaush, C. R. and T. F. Smith (1968). 'Replication and plaque assay of influenza virus in an established line of canine kidney cells.' Appl Microbiol 16(4): 588-594. Graham, F. L., J. Smiley, W. C. Russell and R. Nairn (1977). 'Characteristics of a human cell line transformed by DNA from human adenovirus type 5.' J Gen Virol 36(1): 59-74. Guilligay, D., F. Tarendeau, P. Resa-Infante, R. Coloma, T. Crepin, P. Sehr, J. Lewis, R. W. Ruigrok, J. Ortin, D. J. Hart and S. Cusack (2008). 'The structural basis for cap binding by influenza virus polymerase subunit PB2.' Nat Struct Mol Biol 15(5): 500-506. Hatta, M., P. Gao, P. Halfmann and Y. Kawaoka (2001). 'Molecular basis for high virulence of Hong Kong H5N1 influenza A viruses.' Science 293(5536): 1840-1842. Hatta, M., Y. Hatta, J. H. Kim, S. Watanabe, K. Shinya, T. Nguyen, P. S. Lien, Q. M. Le and Y. Kawaoka (2007). 'Growth of H5N1 influenza A viruses in the upper respiratory tracts of mice.' PLoS Pathog 3(10): 1374-1379. Horimoto, T. and Y. Kawaoka (2005). 'Influenza: lessons from past pandemics, warnings from current incidents.' Nat Rev Microbiol 3(8): 591-600. Hornung, V., R. Hartmann, A. Ablasser and K. P. Hopfner (2014). 'OAS proteins and cGAS: unifying concepts in sensing and responding to cytosolic nucleic acids.' Nat Rev Immunol 14(8): 521-528. Houseley, J., J. LaCava and D. Tollervey (2006). 'RNA-quality control by the exosome.' Nat Rev Mol Cell Biol 7(7): 529-539. Hutchinson, E. C. and E. Fodor (2012). 'Nuclear import of the influenza A virus transcriptional machinery.' Vaccine 30(51): 7353-7358. Ish-Horowicz, D. and J. F. Burke (1981). 'Rapid and efficient cosmid cloning.' Nucleic Acids Res 9(13): 2989-2998. Iwai, A., T. Shiozaki, T. Kawai, S. Akira, Y. Kawaoka, A. Takada, H. Kida and T. Miyazaki (2010). 'Influenza A virus polymerase inhibits type I interferon induction by binding to interferon beta promoter stimulator 1.' J Biol Chem 285(42): 32064-32074. Jagger, B. W., H. M. Wise, J. C. Kash, K. A. Walters, N. M. Wills, Y. L. Xiao, R. L. Dunfee, L. M. Schwartzman, A. Ozinsky, G. L. Bell, R. M. Dalton, A. Lo, S. Efstathiou, J. F. Atkins, A. E. Firth, J. K. Taubenberger and P. Digard (2012). 'An overlapping protein-coding region in influenza A virus segment 3 modulates the host response.' Science 337(6091): 199-204. Lykke-Andersen, S., D. E. Brodersen and T. H. Jensen (2009). 'Origins and activities of the eukaryotic exosome.' J Cell Sci 122(Pt 10): 1487-1494. Massin, P., S. van der Werf and N. Naffakh (2001). 'Residue 627 of PB2 is a determinant of cold sensitivity in RNA replication of avian influenza viruses.' J Virol 75(11): 5398-5404. Melchjorsen, J., H. Kristiansen, R. Christiansen, J. Rintahaka, S. Matikainen, S. R. Paludan and R. Hartmann (2009). 'Differential regulation of the OASL and OAS1 genes in response to viral infections.' J Interferon Cytokine Res 29(4): 199-207. Mitchell, P., E. Petfalski, A. Shevchenko, M. Mann and D. Tollervey (1997). 'The exosome: a conserved eukaryotic RNA processing complex containing multiple 3'-->5' exoribonucleases.' Cell 91(4): 457-466. Momose, F., T. Naito, K. Yano, S. Sugimoto, Y. Morikawa and K. Nagata (2002). 'Identification of Hsp90 as a stimulatory host factor involved in influenza virus RNA synthesis.' J Biol Chem 277(47): 45306-45314. Naito, T., F. Momose, A. Kawaguchi and K. Nagata (2007). 'Involvement of Hsp90 in assembly and nuclear import of influenza virus RNA polymerase subunits.' J Virol 81(3): 1339-1349. Nayak, D. P., E. K. Hui and S. Barman (2004). 'Assembly and budding of influenza virus.' Virus Res 106(2): 147-165. Newcomb, L. L., R. L. Kuo, Q. Ye, Y. Jiang, Y. J. Tao and R. M. Krug (2009). 'Interaction of the influenza a virus nucleocapsid protein with the viral RNA polymerase potentiates unprimed viral RNA replication.' J Virol 83(1): 29-36. Noah, D. L. and R. M. Krug (2005). 'Influenza virus virulence and its molecular determinants.' Adv Virus Res 65: 121-145. Noda, T., H. Sagara, A. Yen, A. Takada, H. Kida, R. H. Cheng and Y. Kawaoka (2006). 'Architecture of ribonucleoprotein complexes in influenza A virus particles.' Nature 439(7075): 490-492. Palese, P. and J. F. Young (1982). 'Variation of influenza A, B, and C viruses.' Science 215(4539): 1468-1474. Plotch, S. J., M. Bouloy, I. Ulmanen and R. M. Krug (1981). 'A unique cap(m7GpppXm)-dependent influenza virion endonuclease cleaves capped RNAs to generate the primers that initiate viral RNA transcription.' Cell 23(3): 847-858. Poole, E., D. Elton, L. Medcalf and P. Digard (2004). 'Functional domains of the influenza A virus PB2 protein: identification of NP- and PB1-binding sites.' Virology 321(1): 120-133. Raijmakers, R., W. V. Egberts, W. J. van Venrooij and G. J. Pruijn (2002). 'Protein-protein interactions between human exosome components support the assembly of RNase PH-type subunits into a six-membered PNPase-like ring.' J Mol Biol 323(4): 653-663. Sidorenko, Y. and U. Reichl (2004). 'Structured model of influenza virus replication in MDCK cells.' Biotechnol Bioeng 88(1): 1-14. Stumpo, D. J., W. S. Lai and P. J. Blackshear (2010). 'Inflammation: cytokines and RNA-based regulation.' Wiley Interdiscip Rev RNA 1(1): 60-80. Subbarao, E. K., W. London and B. R. Murphy (1993). 'A single amino acid in the PB2 gene of influenza A virus is a determinant of host range.' J Virol 67(4): 1761-1764. Tarendeau, F., J. Boudet, D. Guilligay, P. J. Mas, C. M. Bougault, S. Boulo, F. Baudin, R. W. Ruigrok, N. Daigle, J. Ellenberg, S. Cusack, J. P. Simorre and D. J. Hart (2007). 'Structure and nuclear import function of the C-terminal domain of influenza virus polymerase PB2 subunit.' Nat Struct Mol Biol 14(3): 229-233. Ulmanen, I., B. A. Broni and R. M. Krug (1981). 'Role of two of the influenza virus core P proteins in recognizing cap 1 structures (m7GpppNm) on RNAs and in initiating viral RNA transcription.' Proc Natl Acad Sci U S A 78(12): 7355-7359. Wang, Y. and M. Colonna (2014). 'RNA exosomes keep endogenous RNA under the radar.' Nat Immunol 15(9): 830-831. Yamada, H., R. Chounan, Y. Higashi, N. Kurihara and H. Kido (2004). 'Mitochondrial targeting sequence of the influenza A virus PB1-F2 protein and its function in mitochondria.' FEBS Lett 578(3): 331-336. Yan, N. and Z. J. Chen (2012). 'Intrinsic antiviral immunity.' Nat Immunol 13(3): 214-222. | |
| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/53889 | - |
| dc.description.abstract | PB2是流行性感冒的病毒蛋白，其功能是參與cap snatching，在cap snatching的過程中具有結合到宿主pre-mRNA的帽蓋上的功能。PB2與PB1及PA構成RNA-dependent RNA polymerase (RdRp)，參與流感病毒的轉錄及複製。 為了找出能與A型流行性感冒病毒PB2蛋白交互作用的細胞蛋白，本實驗室利用酵母菌雙雜合系統 (yeast two-hybrid system) 找出細胞蛋白質hRrp43 (exosome complex的組成蛋白之一) 會和流感病毒PB2進行交互作用，並利用免疫共沉澱法以及GST pull-down方法，證明細胞蛋白質hRrp43與流感病毒蛋白質PB2具有交互作用。而在本研究中，利用不同的hRrp43-specific shRNAs在H1299細胞株中knockdown細胞蛋白質hRrp43，證明了knockdown hRrp43會抑制A型流感病毒之複製，並且排除此現象是由shRNA所造成的off-target現象。更進一步證明感染不同病毒株 (WSN33及PR8病毒株) 或是在不同細胞株 (H1299及A549細胞株) 中，knockdown細胞蛋白hRrp43皆會抑制流感病毒的複製。此外，當knockdown exosome中的其他組成蛋白，亦能抑制流感病毒的複製，因此可以推定exosome complex會參與調控A型流感病毒之複製繁殖。 為了瞭解hRrp43是如何參與調控A型流感病毒之複製繁殖，利用GST pull-down方法，找出病毒蛋白質PB2主要利用271-522胺基酸序列與hRrp43進行交互作用，而胺基酸序列511-759則有較弱的交互作用，此兩區域皆有細胞核定位訊號 (nuclear localization signal, NLS)，因此利用免疫螢光染色觀察病毒入核情形，證明knockdown hRrp43會影響病毒蛋白質vRNP入核的情形。此外，利用qRT-PCR證實knockdown hRrp43會影響流感病毒mRNA、cRNA及vRNA之表現量。 另外，發現hRrp43-knockdown細胞可以釋放出某些物質去抑制流感病毒的複製，利用cDNA微陣列分析發現hRrp43-knockdown細胞中有21個基因的表現量會高於GFP-knockdown細胞兩倍。在這些表現量提高的基因中，很多被發現是interferon-stimulated genes。利用qRT-PCR再次確認後，證實knockdown hRrp43會使得IFN-β1表現量較高，導致下游ISGs包括IFIT1、IFIT2、IFIT3、IFITM2、IFITM3以及OASL的表現量均較控制組高，因此，由上述的結果中找到一個A型流行感冒病毒複製繁殖需要的host factor，並且提供一個控制流感病毒感染的新標靶。 | zh_TW |
| dc.description.abstract | Influenza A virus (IAV) PB2 protein, a cap binding protein, is a subunit of viral RNA-dependent RNA polymerase which transcribes and replicates RNA genome of influenza A virus. Our previous studies demonstrated that hRrp43 could interact with PB2 by using GST pull-down and co-immunoprecipitation assays. In this study, we used different hRrp43-specific shRNAs to knockdown hRrp43 in H1299 cells. Our data showed that the viral protein production and viral propagation were dramatically decreased in H1299 cells transduced with different hRrp43 shRNAs, indicating that knockdown of hRrp43 indeed can lead to inhibition of influenza A viral propagation and these phenotype is not caused by off-target effect of shRNA. We also showed that the replication of different strains (including WSN33 and PR8 viruses) of IAV was inhibited in hRrp43-knockdown H1299 and A549 cells, indicating that the knockdown phenotype can be observed in different cells or using different viral strains. hRrp43 is a subunit of exosome, a large complex involved in RNA processing. To study whether an intact exosome is required for influenza A viral replication, we knocked down hRrp45, which is another subunit of exosome. We found that viral replication was also down-regulated in hRrp45-knockdown cells, indicating that an intact exosome is required for influenza A viral replication. The hRrp43-knockdown cells could secrete factors that can inhibit IAV replication. Finally we showed that 21 cellular genes were up-regulated more than 2 fold in hRrp43- knockdown H1299 cells compared to control cells by using cDNA microarray analysis. Among these up-regulated genes, many of them were found to be interferon-stimulated genes. By using qRT-PCR, we confirmed that IFN-β1 and the ISGs including IFIT1, IFIT2, IFIT3, IFITM2, IFITM3 and OASL were significantly up-regulated in H1299-shhRrp43 cells. Together, these results not only identify an intrinsic host factor that can facilitate IAV multiplication but also provide a new target for controlling IAV infection. | en |
| dc.description.provenance | Made available in DSpace on 2021-06-16T02:32:31Z (GMT). No. of bitstreams: 1 ntu-104-R02445107-1.pdf: 1961552 bytes, checksum: 78c60b72f21ab2e1654fa188ba7aa26f (MD5) Previous issue date: 2015 | en |
| dc.description.tableofcontents | 中文摘要 2 Abstract4 目錄 6 圖目錄 9 表目錄 10 緒論 11 研究目的 19 材料與方法20 實驗材料 20 一、化學藥品及試劑 20 二、套組試劑23 三、抗體 24 四、酵素 24 五、其它 24 六、細胞株（Cell line）24 七、質體 (Plasmid)26 實驗方法 28 一、細菌轉形 (Transformation)28 二、勝任細胞的製備 (Preparation of competent cells)28 三、小量質體製備 (Mini-preparation)29 四、大量質體製備 (Large-scale plasmid isolation)30 五、質體轉染 (Transfection)32 六、慢病毒製備 (Preparation of Lentivirus)32 七、慢病毒定量 (Quantification of Lentivirus)34 八、慢病毒感染 (Lentivirus infection)35 九、細胞核糖核酸萃取 (RNA extraction)35 十、反轉錄反應 (Reverse transcription)36 十一、即時聚合酶鏈鎖反應 (Real-time PCR)36 十二、cDNA微陣列分析 (cDNA microarray)37 十三、細胞全蛋白質之收取38 十四、蛋白質定量38 十五、西方墨點法 (Western blot)38 十六、流感病毒感染及增殖(Influenza virus infection and amplification) 39 十七、流感病毒之溶斑分析法 (Plaque assay of influenza virus) 40 十八、Glutathione S-transferase (GST) pull-down 分析41 實驗結果 44 一、利用不同的hRrp43-specific shRNA於H1299細胞內knockdown hRrp43造成A型流感病毒的複製受到抑制 44 二、於不同細胞株中knockdown hRrp43造成A型流感病毒的複製受到抑制45 三、knockdown細胞蛋白質hRrp45抑制A型流感病毒A/WSN/33的複製45 四、利用GST pull down assay確認PB2與hRrp43交互作用的功能域46 五、利用免疫螢光染色法確認hRrp43影響vRNP的入核46 六、knockdown細胞蛋白質hRrp43影響流感病毒之mRNA、cRNA及vRNA的表現量46 七、H1299-shhRrp43細胞株會釋放某些物質，進而抑制流感病毒的複製 47 八、利用cDNA微陣列分析找出H1299-shhRrp43細胞株所釋放的物質 47 九、利用qRT-PCR進一步確認H1299-shhRrp43細胞株中六個ISGs的表現量確實較高48 十、H1299-shhRrp43細胞株其IFN-β1之表現量較高48 十一、流感病毒蛋白質PB2影響IFN-β1之表現量49 討論 50 表格 70 附圖 73 參考文獻 79 | |
| dc.language.iso | zh-TW | |
| dc.subject | 病毒轉錄與複製 | zh_TW |
| dc.subject | A型流行性感冒病毒 | zh_TW |
| dc.subject | 干擾素刺激基因 | zh_TW |
| dc.subject | PB2蛋白質 | zh_TW |
| dc.subject | hRrp43 | zh_TW |
| dc.subject | hRrp43 | en |
| dc.subject | Influenza A virus | en |
| dc.subject | interferon-stimulated gene | en |
| dc.subject | transcription and replication | en |
| dc.subject | PB2 | en |
| dc.title | 細胞蛋白質hRrp43在A型流行性感冒病毒感染中所扮演的角色 | zh_TW |
| dc.title | The regulatory role of hRrp43 in Influenza A virus multiplication | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 103-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 張鑫(Shin Chang),楊宏志(Hung-Chih Yang) | |
| dc.subject.keyword | A型流行性感冒病毒,PB2蛋白質,hRrp43,病毒轉錄與複製,干擾素刺激基因, | zh_TW |
| dc.subject.keyword | Influenza A virus,PB2,hRrp43,transcription and replication,interferon-stimulated gene, | en |
| dc.relation.page | 85 | |
| dc.rights.note | 有償授權 | |
| dc.date.accepted | 2015-07-29 | |
| dc.contributor.author-college | 醫學院 | zh_TW |
| dc.contributor.author-dept | 微生物學研究所 | zh_TW |
| 顯示於系所單位： | 微生物學科所 | |
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