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完整後設資料紀錄
DC 欄位 | 值 | 語言 |
---|---|---|
dc.contributor.advisor | 王萬波(Won-Bo Wang) | |
dc.contributor.author | Wei-hsin Tsai | en |
dc.contributor.author | 蔡瑋欣 | zh_TW |
dc.date.accessioned | 2021-06-08T01:19:38Z | - |
dc.date.copyright | 2014-10-09 | |
dc.date.issued | 2014 | |
dc.date.submitted | 2014-08-08 | |
dc.identifier.citation | Beatriz B. Leitao (2011) The SUMO E3-ligase PIAS1 couples reactive oxygen speciesdependent JNK activation to oxidative cell death. FASEB J. 25, 3416-3425
Beigel J & Bray M (2008) Current and future antiviral therapy of severe seasonal and avian influenza. Antiviral Res 78, 91-102. Blaas D, Patzelt E & Kuechler E (1982) Identification of the cap binding protein of influenza virus. Nucleic Acids Res 10, 4803-4812. Chang LK, Lee YH, Cheng TS, Hong YR, Lu PJ, Wang JJ, Wang WH, Kuo CW, Li SS, and Liu ST (2004) Post-translational Modification of Rta of Epstein-Barr Virus by SUMO-1. J. Biol. Chem. 279, 38803–38812. Liu B, Yang R, Wong KA, Getman C, Stein N, Teitell MA, Cheng G, Wu H, Shuai K (2005) Negative regulation of NF-kappaB signaling by PIAS1. Mol Cell Biol 25, 1113-23. Li R, Pan Y, Shi DD, Zhang Y, Zhang J (2013) PIAS1 negatively modulates virus triggered type I IFN signaling by blocking the DNA binding activity of IRF3. Antiviral Res 100, 546-54. Cheung TK, Guan Y, Ng SS, Chen H, Wong CH, Peiris JS & Poon LL (2005) Generation of recombinant influenza A virus without M2 ion-channel protein by introduction of a point mutation at the 5' end of the viral intron. J Gen Virol 86, 1447-1454. De Clercq E (2006) Antiviral agents active against influenza A viruses. Nat Rev Drug Discov 5, 1015-1025. Delphine Guilligay, Franck Tarendeau, Patricia Resa-Infante, Rocio Coloma, Thibaut Crepin, Peter Sehr, Joe Lewis, Rob W H Ruigrok, Juan Ortin, Darren J Hart & Stephen Cusack (2008) The structural basis for cap binding by influenza virus polymerase subunit PB2. Nature Structural & Molecular Biology 15, 500 – 506. Deng T, Engelhardt OG, Thomas B, Akoulitchev AV, Brownlee GG & Fodor E (2006) Role of ran binding protein 5 in nuclear import and assembly of the influenza virus RNA polymerase complex. J Virol 80, 11911-11919. Perez JT, Varble A, Sachidanandam R, Zlatev I, Manoharan M, Garcia-Sastre A, tenOever BR (2010) Influenza A virus-generated small RNAs regulate the switch from transcription to replication. Proc Natl Acad Sci U S A 107, 11525-30 Perez JT, Zlatev I, Aggarwal S, Subramanian S, Sachidanandam R, Kim B, Manoharan M, tenOever BR.(2012) A small-RNA enhancer of viral polymerase activity. J Virol86, 13475-85. Dias A, Bouvier D, Crepin T, McCarthy AA, Hart DJ, Baudin F, Cusack S & Ruigrok RW (2009) The cap-snatching endonuclease of influenza virus polymerase resides in the PA subunit. Nature 458, 914-918. Engelhardt OG & Fodor E (2006) Functional association between viral and cellular transcription during influenza virus infection. Rev Med Virol 16, 329-345. Fechter P & Brownlee GG (2005) Recognition of mRNA cap structures by viral and cellular proteins. J Gen Virol 86, 1239-1249. Datta K, Wolkerstorfer A, Szolar OH, Cusack S, Klumpp K (2013) Characterization of PA-N terminal domain of Influenza A polymerase reveals sequence specific RNA cleavage. Nucleic Acids Res 17, 8289-99. Fechter P, Mingay L, Sharps J, Chambers A, Fodor E & Brownlee GG (2003) Two aromatic residues in the PB2 subunit of influenza A RNA polymerase are crucial for cap binding. J Biol Chem 278, 20381-20388. Fodor E, Crow M, Mingay LJ, Deng T, Sharps J, Fechter P & Brownlee GG (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, 8989-9001. Fodor E, Devenish L, Engelhardt OG, Palese P, Brownlee GG & Garcia-Sastre A (1999) Rescue of influenza A virus from recombinant DNA. J Virol 73, 9679-9682. Fodor E & Smith M (2004) The PA subunit is required for efficient nuclear accumulation of the PB1 subunit of the influenza A virus RNA polymerase complex. J Virol 78, 9144-9153. Fouchier RA, Munster V, Wallensten A, Bestebroer TM, Herfst S, Smith D, Rimmelzwaan GF, Olsen B & Osterhaus AD (2005) Characterization of a novel influenza A virus hemagglutinin subtype (H16) obtained from black-headed gulls. J Virol 79, 2814-2822. Fraser C, Donnelly CA, Cauchemez S, Hanage WP, Van Kerkhove MD, Hollingsworth TD, Griffin J, Baggaley RF, Jenkins HE, Lyons EJ, Jombart T, Hinsley WR, Grassly NC, Balloux F, Ghani AC, Ferguson NM, Rambaut A, Pybus OG, Lopez-Gatell H, Apluche-Aranda CM, Chapela IB, Zavala EP, Guevara DM, Checchi F, Garcia E, Hugonnet S & Roth C (2009) Pandemic Potential of a Strain of Influenza A (H1N1) : Early Findings. Science. Gabriel G, Herwig A & Klenk HD (2008) Interaction of polymerase subunit PB2 and NP with importin alpha1 is a determinant of host range of influenza A virus. PLoS Pathog 4, e11. Katy M. Graef, Frank T. Vreede, Yuk-Fai Lau, Amber W. McCall, Simon M. Carr, Kanta Subbarao, Ervin Fodor (2010) The PB2 Subunit of the Influenza Virus RNA Polymerase Affects Virulence by Interacting with the Mitochondrial Antiviral Signaling Protein and Inhibiting Expression of Beta Interferon. J Virol. 84, 8433–8445. Gallie DR (1998) A tale of two termini: a functional interaction between the termini of an mRNA is a prerequisite for efficient translation initiation. Gene 216, 1-11. Garaigorta U & Ortin J (2007) Mutation analysis of a recombinant NS replicon shows that influenza virus NS1 protein blocks the splicing and nucleo-cytoplasmic transport of its own viral mRNA. Nucleic Acids Res 35, 4573-4582. Gaush CR & Smith TF (1968) Replication and plaque assay of influenza virus in an established line of canine kidney cells. Appl Microbiol 16, 588-594. Graham FL, Smiley J, Russell WC & Nairn R (1977) Characteristics of a human cell line transformed by DNA from human adenovirus type 5. J Gen Virol 36, 59-74. Guilligay D, Tarendeau F, Resa-Infante P, Coloma R, Crepin T, Sehr P, Lewis J, Ruigrok RW, Ortin J, Hart DJ & Cusack S (2008) The structural basis for cap binding by influenza virus polymerase subunit PB2. Nat Struct Mol Biol 15, 500-506. Hara K, Schmidt FI, Crow M & Brownlee GG (2006) Amino acid residues in the N-terminal region of the PA subunit of influenza A virus RNA polymerase play a critical role in protein stability, endonuclease activity, cap binding, and virion RNA promoter binding. J Virol 80, 7789-7798. Hatta M, Gao P, Halfmann P & Kawaoka Y (2001) Molecular basis for high virulence of Hong Kong H5N1 influenza A viruses. Science 293, 1840-1842. Hatta M, Hatta Y, Kim JH, Watanabe S, Shinya K, Nguyen T, Lien PS, Le QM & Kawaoka Y (2007) Growth of H5N1 influenza A viruses in the upper respiratory tracts of mice. PLoS Pathog 3, 1374-1379. Honda A, Mizumoto K & Ishihama A (1999) Two separate sequences of PB2 subunit constitute the RNA cap-binding site of influenza virus RNA polymerase. Genes Cells 4, 475-485. Horimoto T & Kawaoka Y (2005) Influenza: lessons from past pandemics, warnings from current incidents. Nat Rev Microbiol 3, 591-600. Ish-Horowicz D & Burke JF (1981) Rapid and efficient cosmid cloning. Nucleic Acids Res 9, 2989-2998. Kalyan D, James MA, Ma LC, Krug RM, Eddy A (2010) Structures of influenza A proteins and insights into antiviral drug targets. Nature Structural & Molecular Biology 17, 530-538 Kobasa, D.,Jones, S.M., Shinya, K., Kash, J.C., Copps, J., Ebihara, H., Hatta, Y., Kim, J.H., Halfmann, P., Hatta, M., Feldmann, F., Alimonti J.B., Fernando, L., Li, Y., Katze, M.G., Feldmann, H., & Kawaoka, Y. (2007) Aberrant innate immune response in lethal infection of macaques with the 1918 influenza virus. Nature, 455, 319-323. Xu K, Klenk C, Liu B, Keiner B, Cheng J, Zheng BJ, Li L, Han Q, Wang C, Li T, Chen Z, Shu Y, Liu J, Klenk HD, Sun B. (2011) Modification of nonstructural protein 1 of influenza A virus by SUMO1.J Virol 85, 1086-98. Wu CY, Jeng KS, Lai MM. (2011) The SUMOylation of matrix protein M1 modulates the assembly and morphogenesis of influenza A virus. J Virol 85, 6618-28 Han Q, Chang C, Li L, Klenk C, Cheng J, Chen Y, Xia N, Shu Y, Chen Z, Gabriel G, Sun B, Xu K.(2014) Sumoylation of influenza a virus nucleoprotein is essential for intracellular trafficking and virus growth. J Virol 88, 9379-90. Boggio R, Chiocca S. (2005) Gam1 and the SUMO pathway. Cell Cycle 4, 533-5. Phillip R. Heaton, Adeline F. Deyrieux, Xue-Lin Bian, Van G. Wilson (2011) HPV E6 proteins target Ubc9, the SUMO conjugating enzyme. Virus Res 158, 199–208. Konig R, Stertz S, Zhou Y, Inoue A, Hoffmann HH, Bhattacharyya S, Alamares JG, Tscherne DM, Ortigoza MB, Liang Y, Gao Q, Andrews SE, Bandyopadhyay S, De Jesus P, Tu BP, Pache L, Shih C, Orth A, Bonamy G, Miraglia L, Ideker T, Garcia-Sastre A, Young JA, Palese P, Shaw ML, Chanda SK. (2010) Human host factors required for influenza virus replication. Nature 7282,813-7. Labadie K, Dos Santos Afonso E, Rameix-Welti MA, van der Werf S & Naffakh N (2007) Host-range determinants on the PB2 protein of influenza A viruses control the interaction between the viral polymerase and nucleoprotein in human cells. Virology 362, 271-282. Lee JM, Kang HJ, Lee HR, Choi CY, Jang WJ, Ahn JH (2003) PIAS1 enhances SUMO-1 modification and the transactivation activity of the major immediate earlyIE2 protein of human cytomegalovirus. FEBS Lett. 555, 322–328 Li ML, Rao P & Krug RM (2001) The active sites of the influenza cap-dependent endonuclease are on different polymerase subunits. EMBO J 20, 2078-2086. Liu B, Liao J, Rao X, Kushner SA, Chung CD, Chang DD, Shuai K (1998) Inhibition of Stat1-mediated gene activation by PIAS1. Proc Natl Acad Sci U S A. 95,10626-10631. Soares IN, Caetano FA, Pinder J, Rodrigues BR, Beraldo FH, Ostapchenko VG, Durette C, Pereira GS, Lopes MH, Queiroz-Hazarbassanov N, Cunha IW,Sanematsu PI, Suzuki S, Bleggi-Torres LF, Schild-Poulter C, Thibault P, Dellaire G, Martins VR, Prado VF, Prado MA(2013) Regulation of stress-inducible phosphoprotein 1 nuclear retention by protein inhibitor of activated STAT PIAS1 Mol Cell Proteomics12, 3253-70. Marongiu M1, Deiana M, Meloni A, Marcia L, Puddu A, Cao A, Schlessinger D, Crisponi L (2010) The forkhead transcription factor Foxl2 is sumoylated in both human and mouse: sumoylation affects its stability,localization, and activity. PLoS One 5, 10-1371 Luytjes W, Krystal M, Enami M, Parvin JD & Palese P (1989) Amplification, expression, and packaging of foreign gene by influenza virus. Cell 59, 1107-1113. Naito T, Momose F, Kawaguchi A & Nagata K (2007) Involvement of Hsp90 in assembly and nuclear import of influenza virus RNA polymerase subunits. J Virol 81, 1339-1349. Massin P, van der Werf S & Naffakh N (2001) Residue 627 of PB2 is a determinant of cold sensitivity in RNA replication of avian influenza viruses. J Virol 75, 5398-5404. Momose F, Basler CF, O'Neill RE, Iwamatsu A, Palese P & Nagata K (2001) Cellular splicing factor RAF-2p48/NPI-5/BAT1/UAP56 interacts with the influenza virus nucleoprotein and enhances viral RNA synthesis. J Virol 75, 1899-1908. Momose F, Naito T, Yano K, Sugimoto S, Morikawa Y & Nagata K (2002) Identification of Hsp90 as a stimulatory host factor involved in influenza virus RNA synthesis. J Biol Chem 277, 45306-45314. Ortega J, Martin-Benito J, Zurcher T, Valpuesta JM, Carrascosa JL, Ortin J. (2000) Ultrastructural and functional analyses of recombinant influenza virus ribonucleoproteins suggest dimerization of nucleoprotein during virus amplification. J Virol 1, 156-63. Pal S, Santos A, Rosas JM, Ortiz-Guzman J, Rosas-Acosta G (2011) Influenza A virus interacts extensively with the cellular SUMOylation system during infection. Virus Res 158, 12-27. Palese P & Young JF (1982) Variation of influenza A, B, and C viruses. Science 215, 1468-1474. Pleschka S, Jaskunas R, Engelhardt OG, Zurcher T, Palese P & Garcia-Sastre A (1996) A plasmid-based reverse genetics system for influenza A virus. J Virol 70, 4188-4192. Rytinki MM, Kaikkonen S, Pehkonen P, Jaaskelainen T, Palvimo JJ (2009) PIAS proteins: pleiotropic interactors associated with SUMO. Cell. Mol. Life Sci. 66, 3029-3041. Sangita Pal, Andres Santos, Juan M Rosas, Joshua Ortiz-Guzman, German Rosas-Acosta (2011) Influenza A virus interacts extensively with the cellular SUMOylation system during infection. Virus Research 158, 12-27. Shih SR & Krug RM (1996) Novel exploitation of a nuclear function by influenza virus: the cellular SF2/ASF splicing factor controls the amount of the essential viral M2 ion channel protein in infected cells. EMBO J 15, 5415-5427. Shih SR, Nemeroff ME & Krug RM (1995) The choice of alternative 5' splice sites in influenza virus M1 mRNA is regulated by the viral polymerase complex. Proc Natl Acad Sci U S A 92, 6324-6328. Shuai K, Lui B (2005) Regulation of gene-activation pathways by PIAS proteins in the immune system. Nat Rev Immunol. 8, 593-605. Sidorenko Y & Reichl U (2004) Structured model of influenza virus replication in MDCK cells. Biotechnol Bioeng 88, 1-14. Subbarao EK, London W & Murphy BR (1993) A single amino acid in the PB2 gene of influenza A virus is a determinant of host range. J Virol 67, 1761-1764. Tarendeau F, Boudet J, Guilligay D, Mas PJ, Bougault CM, Boulo S, Baudin F, Ruigrok RW, Daigle N, Ellenberg J, Cusack S, Simorre JP & Hart DJ (2007) Structure and nuclear import function of the C-terminal domain of influenza virus polymerase PB2 subunit. Nat Struct Mol Biol 14, 229-233. Ulmanen I, Broni BA & Krug RM (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, 7355-7359. Vassileva MT, Matunis MJ. (2004) SUMO modification of heterogeneous nuclear ribonucleoproteins. Mol Cell Biol 9, 3623-32. Vertegaal AC, Ogg SC, Jaffray E, Rodriguez MS, Hay RT, Andersen JS, Mann M, Lamond AI. (2004) A proteomic study of SUMO-2 target proteins. J Biol Chem 32,33791-8. Webster RG, Bean WJ, Gorman OT, Chambers TM & Kawaoka Y (1992) Evolution and ecology of influenza A viruses. Microbiol Rev 56, 152-179. Wolff T, O'Neill RE & Palese P (1998) NS1-Binding protein (NS1-BP): a novel human protein that interacts with the influenza A virus nonstructural NS1 protein is relocalized in the nuclei of infected cells. J Virol 72, 7170-7180. Wu CY, Jeng KS, Lai MM (2011) The SUMOylation of matrix protein M1 modulates the assembly and morphogenesis of influenza A virus. J Virol 85, 6616-6628. Xu K, Klenk C, Liu B, Keiner B, Cheng J, Zheng BJ, Li L, Han Q, Wang C, Li T, Chen Z, Shu Y, Liu J, Klenk HD, Sun B (2011) Modification of nonstructural protein 1 of influenza A virus by SUMO1. J Virol 85, 1086-1098. Zhang S, Wang Q, Wang J, Mizumoto K, Toyoda T (2012) Two mutations in the C-terminal domain of influenza virus RNA polymerase PB2 enhance transcription by enhancing cap-1 RNA binding activity. Biochim Biophys Acta. 1819, 78-83. Zhou B, Li Y, Speer SD, Subba A, Lin X, Wentworth DE (2012) Engineering temperature sensitive live attenuated influenza vaccines from emerging viruses. Vaccine 24, 3691-3702. Zebedee SL & Lamb RA (1988) Influenza A virus M2 protein: monoclonal antibody restriction of virus growth and detection of M2 in virions. J Virol 62, 2762-2772. | |
dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/18690 | - |
dc.description.abstract | PB2是流行性感冒的病毒蛋白,其功能是參與cap snatching,與PB1、PA、NP共同構成RNA-dependent RNA polymerase (RdRp),可進行virus transcription及replication。PB2蛋白在cap snatching的過程中具有結合到宿主pre-mRNA的帽蓋上的功能。
為了更深入研究流感病毒複製的機轉,及在流感病毒複製過程中可能參與的細胞內蛋白有哪些、它們參與或影響了流感病毒複製過程中的哪個部分,本實驗室利用酵母菌雙雜合系統(Yeast two-hybrid system),分別找出了胞內會與各流感病毒蛋白有交互作用的蛋白質。 PIAS1是其中一種會和流感病毒蛋白PB2有交互作用的細胞蛋白質,它的功能是SUMO E3 ligase,可將其他蛋白質做SUMOylation的修飾。我們想藉由對PB2和PIAS1之間交互作用的探討和研究,希望能更深入了解流感病毒轉錄和複製的機制。 SUMO是small ubiquitin like modifier的簡稱,它是一種後轉譯修飾,會影響及調控許多細胞內的生理功能。 先前實驗室已利用酵母菌雙雜合系統(Yeast two-hybrid system)及GST pull down assay再次確認PB2和PIAS1之間的交互作用關係。我們再利用co-immunoprecipitation確認PB2和PIAS1在細胞內確實有交互作用。 而由於PIAS1在細胞中為SUMO E3 Ligase,因此我們想知道PIAS1與PB2的交互作用的意義是否包括PIAS1會將PB2 SUMOylation。因此首先我們想要知道PB2是否會被SUMOylation修飾。在先前的研究中已發現PB2在in vitro有被SUMOyation的情形。我們使用293T細胞進行in vivo SUMOylation,並使用immunoprecipitation實驗分析。發現A型流感病毒WSN/33的PB2蛋白質的確有in vivo SUMOylation的現象。 確定了PB2有被SUMOylation的情形後,接著我們想要尋找PB2的SUMOylation sites。先前實驗室利用電腦分析尋找PB2上可能會有SUMOylation的位點,發現在lysine339與718附近的氨基酸序列符合SUMO consensus sequence,因此實驗室先前已利用site-directed mutagenesis建構了PB2 K339R, K718R, K339,718R mutants。我們利用in vivo SUMOylation測試這些mutants,發現lysin 339有可能是PB2的SUMOylation sites 之一。 接著,我們想知道PIAS1是否會在PB2的SUMOylation扮演角色。我們改變293T細胞中的PIAS1 protein 表現量,並分析PB2的 SUMOylation情形。實驗結果發現WSN/33 PB2蛋白的SUMOylation的情形在knockdown PIAS1時會下降,overexpress PIAS1時上升。 因此我們懷疑PB2可能是PIAS1的SUMOylation target,是PIAS1將PB2做了SUMO這個修飾。 為了釐清PIAS1及SUMO對PB2的影響,我們利用免疫螢光染色觀察大量表現PIAS1時PB2的入核情形。實驗發現PB2在正常的情況下是會入核的,但在大量表現PIAS1的情況下,PB2入核的情形會受到影響。另外,PB2 K339R,K718R, K339,718R mutants的入核情形在正常情況下和wild type PB2是相似的,但在大量表現PIAS1的情況下,mutants的入核情形,相較於wild type PB2其受PIAS1大量表現的影響較小。 另外,為了更進一步測試PIAS1對流感病毒複製的影響,我們分別使用knockdown PIAS1及正常細胞感染流感病毒並測試其病毒蛋白表現量。實驗發現,若將PIAS1 knockdown,流感病毒蛋白的表現情形會上升。反之如果大量表現PIAS1,使用流感病毒溶斑分析,可發現流感病毒的複製在PIAS1大量表現的細胞,有明顯的減少情形。依這些實驗的結果推斷,PIAS1的存在,對流感病毒的複製是抑制的效果。 總結這些研究結果,顯示PIAS1可能藉由將PB2 SUMOylation的方式,影響PB2的入核等等,進而影響influenza virus的複製。 | zh_TW |
dc.description.abstract | Influenza A viral PB2 protein, a cap binding protein which functions together with PB1, PA and NP in the process of cap snatching, composes the influenza A viral RNA-dependent RNA polymerase (RdRp). It plays the role of binding to the cap of pre-mRNA while undergoing cap snatching process. RdRp, which contains the PB2 protein, can transcribe and replicate RNA genome of Influenza A virus.
Our previous research team has make study on influenza A virus and its life cycle, which it used yeast two-hybrid system to identify PB2-interacting cellular proteins, and found that PIAS1, a SUMO E3 ligase, is one of the cellular proteins that can interact with PB2. By investigating the interaction between viral PB2 and cellular protein PIAS1, We would like to further explore the mechanism of influenza A viral transcription and replication. As we understand the interaction betweem PB2 and PIAS1 in vitro and in vivo, and PIAS1 is a SUMO E3 ligase, it is possible that through interacting with PB2, PIAS1 plays a role in PB2 SUMOylation. To begin with, we would like to discuss whether PB2 could be SUMOylated in cells. By using in vivo SUMOylation assay and immunoprepicitation, we found that PB2 was SUMOylated in 293T cells. Later on, more detailed studies confirmed that K339 might be one of the SUMOylation sites of PB2. Also, we would like to understand if PIAS1 can influence the SUMO status of PB2. The data shows that expression level of cellular PIAS1 protein can influence the amounts of SUMOylated PB2. According to the above research data, we deduced that PB2 might be a substrate of PIAS1. Furthermore, to investigate the influence of PIAS1 to PB2, we observed the PB2 nuclear import under normal or experimental condition by immunofluorescence staining. The data showed that PB2 will be imported into the nucleus under normal condition but this situation will be affected when PIAS1 was overexpressed. Besides, PB2 K339R,K718R, K339,718R mutants will be imported into nucleus, similar to the wild type ones under normal condiction. However, in the condition of overexpressing PIAS1, the intense of affection of nuclear import was less obvious compared to the wild type ones. Finally, to understand the effects of PIAS1 to influenza A viral transcription and replication, we tested the viral protein expression of infected normal and PIAS1 knocked down cells. The result showed that knocking down of PIAS1 enhanced the expression level of viral proteins. On the other hand, , plaque assay data also showed that overexpression of PIAS1 reduced the replication of influenza A virus. Research data indicates that PIAS1 plays an inhibitory role in influenza A virus transcription and replication. In a nutshell, we assumed that PIAS1 may SUMOylated PB2, and SUMOylation of PB2 by PIAS1 may influence the nuclear import of PB2, thereby affects the transcription and replication of influenza A virus. | en |
dc.description.provenance | Made available in DSpace on 2021-06-08T01:19:38Z (GMT). No. of bitstreams: 1 ntu-103-R01445120-1.pdf: 2358994 bytes, checksum: 0c5beab7f267d62abd806ef32de5f5fc (MD5) Previous issue date: 2014 | en |
dc.description.tableofcontents | 目錄
目錄………………………………………………………..……………………1 圖目錄………………………………………………………..…………………4 附圖目錄………………………………………………………..………………6 中文摘要…………………………………………………………..……………7 Abstract……………………………………………………………...……..……9 緒論…………………………………………………………………………… 11 研究目的………………………………………………………..…..………….19 實驗材料……………………………………………………………..…….….19 一、 化學藥品及試劑………………………………………..……..20 二、 套組試劑…………………………………………………..…..23 三、 抗體………………………………………………………..…..23 四、 酵素…………………………………………………………....24 五、 其他…………………………………………………………....24 六、 細胞株(Cell line)……………………………………………....24 七、 質體(Plasmid)……………………………………………….…25 實驗方法………………………………………………………………………29 一、 細菌轉型(Transformation)…………………………………….29 二、 小量質體製備(Mini preparation).............................................29 三、 大量質體製備(Large-scale plasmid isolation)…………………30 四、 質體轉染(Transfection)………………………………………...32 五、 點突變(Point mutation)…………………………………………34 六、 慢病毒製備……………………………………………………..35 七、 慢病毒定量……………………………………………………..36 八、 慢病毒感染……………………………………………………..38 九、 細胞全蛋白質之收取…………………………………………..38 十、 蛋白質定量……………………………………………………..39 十一、 西方墨點法(Western blot)……………...………………………39 十二、 免疫共沉澱法(Co-Immunoprepicitation)……………………...40 十三、 In vivo SUMOylation…………………………………………...40 十四、 免疫共沉澱法分析in vivo SUMOylation……………………..40 十五、 Glutathione S-transferase(GST) pull-down 分析……………...41 十六、 免疫螢光分析(Immunoflurorescence assay, IFA)……..….…...43 十七、 流感病毒感染及增殖(Influenza virus infection and amplification)…………………………………………………...43 十八、 流感病毒之溶斑分析法(Plaque assay of influenza virus)…….44 實驗結果………………………………………………………………………..46 一、 利用免疫共沉澱法(Co-Immunoprepicitation)確認PB2與PIAS1 在in vivo的情況下有交互作用。………………………..…..46 二、 利用GST pull down assay確認PB2與PIAS1交互作用的功能 域。…………………………………………………………….46 三、 A/WSN/33流感病毒蛋白PB2具有SUMO1 modification。………………………………….…………....…46 四、 A/WSN/33流感病毒蛋白PB2無論UBC9存在與否皆會被 SUMO modified。…………….…………………………….….47 五、 尋找Influenza A/WSN/33 PB2的SUMOylation site。….…...48 六、 PIAS1是否參與病毒蛋白PB2的SUMO1 modification。.....48 七、 病毒蛋白PB2的大量表現會影響PIAS1的SUMO情形…...49 八、 PIAS1的大量表現影響PB2的入核。………………………..50 九、 點突變的PB2入核情形與Wild type PB2相似。…………….50 十、 點突變的PB2入核情形較不受PIAS1大量表現之影響。….51 十一、 降低PIAS1的表現量會使流感病毒蛋白的複製情形增加。..51 十二、 PIAS1的大量表現影響流感病毒的複製。……………………51 討論……………………………………………………………………………..53 附表……………………………………………………………………………..74 參考文獻………………………………………………………………………..76 | |
dc.language.iso | zh-TW | |
dc.title | A型流行性感冒病毒PB2蛋白質與細胞蛋白質PIAS1之交互作用 | zh_TW |
dc.title | The interaction between Influenza A viral PB2 protein and cellular protein PIAS1 | en |
dc.type | Thesis | |
dc.date.schoolyear | 102-2 | |
dc.description.degree | 碩士 | |
dc.contributor.oralexamcommittee | 鄧述諄(Shu-Chun Teng),楊宏志(Hung-Chih Yang) | |
dc.subject.keyword | 流行性感冒病毒,PB2 蛋白質,PIAS1,SUMOylation, | zh_TW |
dc.subject.keyword | Influenza A virus,PB2,PIAS1,SUMOylation, | en |
dc.relation.page | 97 | |
dc.rights.note | 未授權 | |
dc.date.accepted | 2014-08-08 | |
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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