請用此 Handle URI 來引用此文件:
http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/38085
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 | Zhi-Cheng Hong | en |
dc.contributor.author | 洪志成 | zh_TW |
dc.date.accessioned | 2021-06-13T16:26:14Z | - |
dc.date.available | 2016-10-07 | |
dc.date.copyright | 2011-10-07 | |
dc.date.issued | 2011 | |
dc.date.submitted | 2011-07-19 | |
dc.identifier.citation | 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. Carstens RP, McKeehan WL & Garcia-Blanco MA (1998) An intronic sequence element mediates both activation and repression of rat fibroblast growth factor receptor 2 pre-mRNA splicing. Mol Cell Biol 18, 2205-2217. 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. 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. Datar KV, Dreyfuss G & Swanson MS (1993) The human hnRNP M proteins: identification of a methionine/arginine-rich repeat motif in ribonucleoproteins. Nucleic Acids Res 21, 439-446. De Clercq E (2006) Antiviral agents active against influenza A viruses. Nat Rev Drug Discov 5, 1015-1025. 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. 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. 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. Ferre F (1992) Quantitative or semi-quantitative PCR: reality versus myth. PCR Methods Appl 2, 1-9. 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. 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. Hovhannisyan RH & Carstens RP (2007) Heterogeneous ribonucleoprotein m is a splicing regulatory protein that can enhance or silence splicing of alternatively spliced exons. J Biol Chem 282, 36265-36274. Ish-Horowicz D & Burke JF (1981) Rapid and efficient cosmid cloning. Nucleic Acids Res 9, 2989-2998. Jain RA & Gavis ER (2008) The Drosophila hnRNP M homolog Rumpelstiltskin regulates nanos mRNA localization. Development 135, 973-982. Jorba N, Juarez S, Torreira E, Gastaminza P, Zamarreno N, Albar JP & Ortin J (2008) Analysis of the interaction of influenza virus polymerase complex with human cell factors. Proteomics 8, 2077-2088. 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 JH, Kim SH, Pascua PN, Song MS, Baek YH, Jin X, Choi JK, Kim CJ, Kim H, Choi YK. (2010) Direct interaction of cellular hnRNP-F and NS1 of influenza A virus accelerates viral replication by modulation of viral transcriptional activity and host gene expression. Virology 1, 89-99. 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. 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. 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. 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. Neubauer G, King A, Rappsilber J, Calvio C, Watson M, Ajuh P, Sleeman J, Lamond A & Mann M (1998) Mass spectrometry and EST-database searching allows characterization of the multi-protein spliceosome complex. Nat Genet 20, 46-50. 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. 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. Poole E, Elton D, Medcalf L & Digard P (2004) Functional domains of the influenza A virus PB2 protein: identification of NP- and PB1-binding sites. Virology 321, 120-133. 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. 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. 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/123456789/38085 | - |
dc.description.abstract | 流感病毒的 PB2 蛋白質為構成其病毒特有的RNA-dependent RNA polymerase (RdRp) 的重要一員,對於病毒複製或是RNA 轉錄都是不可或缺的。本實驗室在找尋可能與PB2 進行交互作用的細胞蛋白質時,利用共免疫沈澱法(co-IP) 發現細胞中的hnRNP M 可與PB2 結合,並且在真實病毒感染之下,也能夠發現到hnRNP M 可與PB2 結合,而在前人的研究中也證實hnRNP M 可能會結合上PA-PB2- PB1-TAP tag 複合體。
在之前的研究中,我們利用GST pull-down assay觀察到PB2可能透過其中間片段(胺基酸281-511)與hnRNP M結合,而此結合片段恰包含了PB2的cap-binding位置。為了反向找出hnRNP M是利用本身哪一個位置與PB2來結合,我們將hnRNP M分為許多片段刪除組,同樣使用GST pull-down assay來觀察,發現到hnRNP M可能是透過其中間Met-Arg-Gly rich片段來結合PB2。 為了測試hnRNP M結合了PB2對於流感病毒的生長所造成之影響,利用hnRNP M shRNA分別在H1299與NPC-TW04細胞株內篩選出hnRNP M knock-down之穩定細胞株,而發現流感病毒在hnRNP M-knockdown之穩定細胞株內的複製效率會有增加的情形,顯示hnRNP M可能會抑制流感病毒的複製機轉。而在螢光酶報導系統中觀察到了hnRNP M可能不利於流感病毒的轉錄或複製。 更進一步探討hnRNP M是否參與在流感病毒內M(matrix) mRNA splicing之調控,以達到影響流感病毒生長的作用,使用流感病毒感染NPC-TW04 hnRNP M-knockdown混合穩定細胞株,並使用semi-qPCR 來偵測M1 mRNA、M2 mRNA 及mRNA3的含量,經過定量後發現hnRNP M並無影響在流感病毒M mRNA splicing的過程,但是卻觀察到在hnRNP M-knockdown穩定細胞株中的M mRNA含量較母細胞株來的更高,而在M vRNA的含量,雖然在hnRNP M-knockdown細胞中較多,但可能是由轉錄作用的二次效應而來,因此推測hnRNP M可能是透過抑制流感病毒的轉錄來達到減少病毒複製的效果。 為了測試hnRNP M是否透過阻擋了PB2的cap-binding片段去接觸mRNA來抑制病毒的轉錄作用,我們純化出流感病毒的聚合酶複合體(vRNPs)與hnRNP M蛋白質,並將兩者與含有5’-cap的微粒子一起混合,而在結果中發現到hnRNP M的確會降低PB2與5’-cap微粒子之間的結合,因此我們才會在之前的實驗中觀察到流感病毒的轉錄作用會受到hnRNP M的抑制。 為了觀察PB2對於hnRNP M本身RNA選擇性剪接功能之影響,將FGFR2 minigene 質體送入293T 細胞中,利用semi-qPCR來觀察splicing 產物,結果發現到在表現PB2時,hnRNP M的splicing功能會受到抑制。另外,我們在病毒感染後的H1299細胞內觀察到,hnRNP M的表現量在感染初期會有短暫性增加的現象,而hnRNP M的活性與表現量在病毒感染期間是如何被調控仍需要更進一步的探討。 最後我們想知道hnRNP M的MAG-rich region是否能夠因為結合了PB2的cap-binding domain而使得流感病毒的複製能力受到影響,於是我們將MAG-rich region放入細胞內,發現到流感病毒的聚合酶活性會受到抑制,使得病毒的轉錄作用有所減少,因此降低了流感病毒的生長能力,未來可以找出結合上cap-binding domain的主要胺基酸片段,利用合成peptide的方式來將此胺基酸片段成為可實際應用的流感治劑。 | zh_TW |
dc.description.abstract | PB2, a component of influenza A RNA polymerase complex, plays an important role in influenza A viral transcription and replication. In the process of searching for cellular proteins that interact with PB2, we accidentally found that PB2 could interact with cellular hnRNP M protein, an mRNA splicing factor. This interaction was confirmed by co-immunoprecipitation and GST pull-down assays. More importantly, we also found that hnRNP M could interact with PB2 during influenza A virus infection of human H1299 cells. This finding is consistent with the previous report that hnRNP M can be pull-down by PA-PB2-PB1-TAP tag complex.
In the previous study, our lab found that PB2 can interact with hnRNP M through a region containing amino acids 281-511 which overlaps the crucial cap-binding domain of PB2. To study which regions of hnRNP M interact with PB2, a series of truncated hnRNP M proteins were used in GST pull-down assays. We found that hnRNP M used its Met-Arg-Gly rich domain to interact with PB2. To study the effect of hnRNP M on influenza A viral replication, we generated hnRNP M knock-down cells by using lentivirus expressing shRNA against hnRNP M in H1299 and NPC-TW04 cell lines. We found that the production of influenza A virus was significantly increased in hnRNP M knock-down cells, suggesting that hnRNP M can inhibit influenza A viral replication. This conclusion was further supported by the following two results. First, overexpression of hnRNP M could inhibit influenza A virus replication. Second, Influenza replication-reporter assays showed that the viral RNA-dependent RNA polymerase activity was increased in hnRNP M knock-down cells. To study the mechanism underlying hnRNP M inhibition of influenza replication, we first tested whether hnRNP M could affect the splicing of influenza M mRNA. We infected hnRNP M-knockdown NPC-TW04 cells and the control cells with influenza A virus and quantified the splicing products of M pre-mRNA. We found that relative amounts of M splicing products were similar in hnRNP M-knockdown cells and control cells, suggesting that hnRNP M may not be involved in the regulation of M mRNA splicing. However, we did find that the level of total M mRNA was increased in hnRNP M-knockdown cells when compared to control cells. The level of NP mRNA was also higher in hnRNP M-knockdown cells than in control cells. These data suggest that hnRNP M can inhibit the transcription of influenza genes. We also tested whether hnRNP M would affect the production of influenza vRNA. Our data indicated that the expression level of M vRNA was up-regulated in hnRNP M-knockdown cells, which is in consistent with the above data that influenza viral replication was increased in hnRNP M-knockdown cells. Together, these data suggest that hnRNP M may inhibit influenza viral replication through repressing viral transcription. To study the mechanism by which hnRNP M repress influenza viral transcription, we tested whether hnRNP M could inhibit the cap-snatching activity of PB2 by performing in vitro cap-binding assays. PB2, in complex with PB1 and PA, is known to be able to bind the cap structure of mRNA. We thus tested whether purified hnRNP M would affect viral polymerase complex (vRNPs) to bind its substrate 7-methyl-GTP. Our data indicated the capability of PB2 to bind 7-methyl-GTP was decreased in the presence of hnRNP M protein, suggesting that hnRNP M can inhibit the cap-snatching activity of PB2. We also tested whether PB2, through interacting with hnRNP M, would affect hnRNP M’s regulatory function on RNA splicing. hnRNP M is known to promote alternative splicing of the FGFR2 minigene. By quantifying the splicing products of the FGFR2 minigene in the presence or absence of PB2, we found that PB2 could dose-dependently inhibit the activity of hnRNP M to promote alternative splicing of the FGFR2 minigene. Knowing that hnRNP M can inhibit PB2 cap-snatching activity and uses its Met-Arg-Gly rich domain to interact with PB2’s cap-binding domain, we next tested whether the peptide containing Met-Arg-Gly rich domain of hnRNP M could inhibit influenza A viral transcription and replication. We found that both viral transcription and replication was reduced in cells expressing the Met-Arg-Gly rich domain, suggesting that peptides that can bind to the cap-binding domain of PB2 may be used to inhibit influenza A replication. Finally, we found that the expression level of hnRNP M was up-regulated transiently during the early stage of influenza A virus infection. This data together with the above data strongly suggest that hnRNP M is a molecule host uses to defend influenza A virus infection. | en |
dc.description.provenance | Made available in DSpace on 2021-06-13T16:26:14Z (GMT). No. of bitstreams: 1 ntu-100-R98445125-1.pdf: 8364483 bytes, checksum: f94604b98b065d7eb67745b19dcd6b35 (MD5) Previous issue date: 2011 | en |
dc.description.tableofcontents | 中文摘要 ……………………………….…………………………………………………………………... i
Abstract ……………………………………………………..……………….……………………………… iii 目錄 …………………………………………………………………………………………………………... vi 圖目錄 ……………………………………………..………………………………………………...…….. ix 緒論 …………………………………………………………………………………………………………... 1 研究目的 …………………………………………………………………………………………………... 8 材料與方法 ◆ 實驗材料 ……………………….…………………………………………………………............. 9 一、化學藥品及試劑 ….………………………………………………................................ 9 二、套組試劑 ………………………………………………………………………………………….. 12 三、抗體 .…………………………………………………………………………………………………. 13 四、酵素 .…………………………………………………………………………………………………. 13 五、其它 .…………………………………………………………………………………………………. 14 六、細胞株 (Cell line) ………………………………………………………………………..…….. 14 七、質體 (Plasmid) …………………………………………………………………………………... 15 ◆ 實驗方法……………………………………………………………..................................... 21 一、質體建構 (Construction) ………………………………………………………….……….. 21 二、細菌轉形 (Transformation) ……………………………………………………………….. 21 三、勝任細胞的製備 (Preparation of competent cells) ……………………..…….. 21 四、小量質體製備 (Mini-preparation) ……………………………………………………… 23 五、大量質體製備 (Large-scale plasmid isolation) ……………………................. 24 六、質體轉染 (Transfection) …………………………..………………………………………… 26 七、慢病毒製備 (Preparation of Lentivirus) ……………………………………………... 27 八、慢病毒定量 (quantification of Lentivirus)…………………............................ 28 九、慢病毒感染 (Lentivirus infection) ……………….......................................... 30 十、細胞核糖核酸萃取 (RNA extraction)……………………………………………..…... 30 十一、 反轉錄反應 (Reverse transcription)…………………………...……………..…. 31 十二、 即時聚合酶鏈鎖反應 (Real-time PCR) …………………………………………. 31 十三、 半定量聚合酶鏈鎖反應(semi-quantitative PCR)……………………….…… 32 十四、 螢光酶分析 (Luciferase assay)…………………………………....................... 33 十五、 細胞全蛋白質之收取……………………………………………………………..……... 33 十六、 蛋白質定量………………………………........................................................ 34 十七、 西方墨點法 (Western blot) ..…………………………………………………….….. 34 十八、 MTT assay ……………………………………………………………………..….….………… 35 十九、 流感病毒感染及增殖 (Influenza virus infection and amplification).. 35 二十、 流感病毒之溶斑分析法 (Plaque assay of Influenza virus)………........ 36 二十一、Glutathione S-transferase (GST) pull-down 分析….......................... 37 二十二、免疫共沈澱法 (Co-Immunoprecipitation)………............................... 39 二十三、流感病毒聚合酶蛋白(vRNP)純化………………………….…………………..… 39 二十四、 hnRNP M蛋白質純化 .…………………….………….…………………………….. 40 二十五、Cap結合分析(Cap-bindnig assay) …………………………………………………. 40 二十六、vRNA定量法(vRNA quantification) ………………………………….…………... 40 實驗結果 一、透過免疫共沈澱(Co-immunoprecipitation)確認PB2蛋白質與hnRNP M蛋白質之間有交互作用。…...……………………………………………………………………….… 42 二、透過免疫共沈澱(Co-immunoprecipitation)確認PB2蛋白質與hnRNP M蛋白質之間在病毒感染的細胞內有交互作用。…………………………………….……… 42 三、透過不同長度的hnRNP M片段,確認了PB2可能是結合在hnRNP M的400到654胺基酸片段之間。……………………………………………………………......... 43 四、確認PB2對於hnRNP M的結合位置可能是在PB2的305到515胺基酸片段之間。………………………………………………………….…………………….………...…….. 43 五、流感病毒在大量表現hnRNP M的細胞中,其複製能力與聚合酶活性會受到抑制。……................................................................................................. 44 六、利用hnRNP M shRNA抑制H1299細胞內的hnRNP M會造成流感病毒螢光酶報導系統表現較佳且流感病毒的複製能力增加。 ……………….…….…… 44 七、hnRNP M的knock-down可能是影響到流感病毒的mRNA transcription,而不是影響到M mRNA splicing。……………………………………………………………... 45 八、將hnRNP M補回hnRNP M-knockdown細胞株,使原本受到活化的流感病毒轉錄與複製皆被再次被抑制回去。…………………………………………..….…… 47 九、hnRNP M可能不會影響到流感病毒的基因組複製。…………………........ 48 十、hnRNP M可能會阻擋PB2與mRNA的5’cap之間的結合。…………..…. 49 十一、PB2可能會干擾hnRNP M的alternative splicing功能。…................ 50 十二、在真實病毒感染之下,細胞內的hnRNP M表現量,在感染的初期會有短暫性的上升,而在感染的後期會逐漸下降。…..................................... 51 十三、hnRNP M蛋白質的MAG region具有抑制流感病毒轉錄作用的能力 。…………………………………………………………………………………………………………….…… 51 討論 ..…………………………….………………………………………….................................... 53 附圖 ..…………………………………………………………………….………………………….….……. 58 附表 ..………………………………………………………………………………….……….………..…... 85 參考文獻 ..……………………………………………………………………….………….……….….... 87 | |
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hnRNP M之交互作用 | zh_TW |
dc.title | The interaction between Influenza A viral PB2 protein and cellular protein hnRNP M | en |
dc.type | Thesis | |
dc.date.schoolyear | 99-2 | |
dc.description.degree | 碩士 | |
dc.contributor.oralexamcommittee | 鄧述諄(Shu-Chun Teng),楊宏志(Hung-Chih Yang),施信如(Shin-Ru Shih) | |
dc.subject.keyword | 流行性感冒病毒,PB2 蛋白質,hnRNP M,RNA 選擇性剪接,流感病毒轉錄與複製, | zh_TW |
dc.subject.keyword | Influenza A virus,PB2,hnRNP M,RNA alternative splicing,influenza A viral transcription and replication,cap-snatching, | en |
dc.relation.page | 91 | |
dc.rights.note | 有償授權 | |
dc.date.accepted | 2011-07-19 | |
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 |
文件中的檔案:
檔案 | 大小 | 格式 | |
---|---|---|---|
ntu-100-1.pdf 目前未授權公開取用 | 8.17 MB | Adobe PDF |
系統中的文件,除了特別指名其著作權條款之外,均受到著作權保護,並且保留所有的權利。