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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 王萬波(Won-Bo Wang) | |
| dc.contributor.author | Cheng-Cheng Yu | en |
| dc.contributor.author | 余正晟 | zh_TW |
| dc.date.accessioned | 2021-06-15T01:30:03Z | - |
| dc.date.available | 2014-09-15 | |
| dc.date.copyright | 2009-09-15 | |
| dc.date.issued | 2009 | |
| dc.date.submitted | 2009-07-21 | |
| dc.identifier.citation | Al-Maghrebi, M., Brule, H., Padkina, M., Allen, C., Holmes, W.M. and Zehner, Z.E. (2002) The 3' untranslated region of human vimentin mRNA interacts with protein complexes containing eEF-1gamma and HAX-1. Nucleic Acids Res, 30, 5017-5028.
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(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, O.G., Palese, P., Brownlee, G.G. and Garcia-Sastre, A. (1999) Rescue of influenza A virus from recombinant DNA. J Virol, 73, 9679-9682. Gallagher, A.R., Cedzich, A., Gretz, N., Somlo, S. and Witzgall, R. (2000) The polycystic kidney disease protein PKD2 interacts with Hax-1, a protein associated with the actin cytoskeleton. Proc Natl Acad Sci U S A, 97, 4017-4022. Gastaminza, P., Perales, B., Falcon, A.M. and Ortin, J. (2003) Mutations in the N-terminal region of influenza virus PB2 protein affect virus RNA replication but not transcription. J Virol, 77, 5098-5108. Graham, F.L., Smiley, J., Russell, W.C. and Nairn, R. (1977) Characteristics of a human cell line transformed by DNA from human adenovirus type 5. J Gen Virol, 36, 59-74. Hatta, M., Gao, P., Halfmann, P. and Kawaoka, Y. (2001) Molecular basis for high virulence of Hong Kong H5N1 influenza A viruses. Science, 293, 1840-1842. Hinshaw, V.S., Olsen, C.W., Dybdahl-Sissoko, N. and Evans, D. (1994) Apoptosis: a mechanism of cell killing by influenza A and B viruses. J Virol, 68, 3667-3673. Honda, A., Mizumoto, K. and 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. James, P., Halladay, J. and Craig, E.A. (1996) Genomic libraries and a host strain designed for highly efficient two-hybrid selection in yeast. Genetics, 144, 1425-1436. 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. and Kawaoka, Y. (2007) Aberrant innate immune response in lethal infection of macaques with the 1918 influenza virus. Nature, 445, 319-323. Lee, A.Y., Lee, Y., Park, Y.K., Bae, K.H., Cho, S., Lee do, H., Park, B.C., Kang, S. and Park, S.G. (2008) HS 1-associated protein X-1 is cleaved by caspase-3 during apoptosis. Mol Cells, 25, 86-90. Luytjes, W., Krystal, M., Enami, M., Parvin, J.D. and Palese, P. (1989) Amplification, expression, and packaging of foreign gene by influenza virus. Cell, 59, 1107-1113. Modem, S. and Reddy, T.R. (2008) An anti-apoptotic protein, Hax-1, inhibits the HIV-1 rev function by altering its sub-cellular localization. J Cell Physiol, 214, 14-19. Mukaigawa, J. and Nayak, D.P. (1991) Two signals mediate nuclear localization of influenza virus (A/WSN/33) polymerase basic protein 2. J Virol, 65, 245-253. Naito, T., Momose, F., Kawaguchi, A. and Nagata, K. (2007) Involvement of Hsp90 in assembly and nuclear import of influenza virus RNA polymerase subunits. J Virol, 81, 1339-1349. Palese, P. and Young, J.F. (1982) Variation of influenza A, B, and C viruses. Science, 215, 1468-1474. Peiris, J.S., de Jong, M.D. and Guan, Y. (2007) Avian influenza virus (H5N1): a threat to human health. Clin Microbiol Rev, 20, 243-267. Pleschka, S., Jaskunas, R., Engelhardt, O.G., Zurcher, T., Palese, P. and Garcia-Sastre, A. (1996) A plasmid-based reverse genetics system for influenza A virus. J Virol, 70, 4188-4192. Sarnowska, E., Grzybowska, E.A., Sobczak, K., Konopinski, R., Wilczynska, A., Szwarc, M., Sarnowski, T.J., Krzyzosiak, W.J. and Siedlecki, J.A. (2007) Hairpin structure within the 3'UTR of DNA polymerase beta mRNA acts as a post-transcriptional regulatory element and interacts with Hax-1. Nucleic Acids Res, 35, 5499-5510. Sharp, T.V., Wang, H.W., Koumi, A., Hollyman, D., Endo, Y., Ye, H., Du, M.Q. and Boshoff, C. (2002) K15 protein of Kaposi's sarcoma-associated herpesvirus is latently expressed and binds to HAX-1, a protein with antiapoptotic function. J Virol, 76, 802-816. Shaw, J. and Kirshenbaum, L.A. (2006) HAX-1 represses postmitochondrial caspase-9 activation and cell death during hypoxia-reoxygenation. Circ Res, 99, 336-338. Sidorenko, Y. and Reichl, U. (2004) Structured model of influenza virus replication in MDCK cells. Biotechnol Bioeng, 88, 1-14. Subbarao, E.K., London, W. and Murphy, B.R. (1993) A single amino acid in the PB2 gene of influenza A virus is a determinant of host range. J Virol, 67, 1761-1764. Suzuki, Y., Demoliere, C., Kitamura, D., Takeshita, H., Deuschle, U. and Watanabe, T. (1997) HAX-1, a novel intracellular protein, localized on mitochondria, directly associates with HS1, a substrate of Src family tyrosine kinases. J Immunol, 158, 2736-2744. Takizawa, T., Matsukawa, S., Higuchi, Y., Nakamura, S., Nakanishi, Y. and Fukuda, R. (1993) Induction of programmed cell death (apoptosis) by influenza virus infection in tissue culture cells. J Gen Virol, 74 ( Pt 11), 2347-2355. Tarendeau, F., Boudet, J., Guilligay, D., Mas, P.J., Bougault, C.M., Boulo, S., Baudin, F., Ruigrok, R.W., Daigle, N., Ellenberg, J., Cusack, S., Simorre, J.P. and Hart, D.J. (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, B.A. and Krug, R.M. (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. Webster, R.G., Bean, W.J., Gorman, O.T., Chambers, T.M. and Kawaoka, Y. (1992) Evolution and ecology of influenza A viruses. Microbiol Rev, 56, 152-179. Wurzer, W.J., Planz, O., Ehrhardt, C., Giner, M., Silberzahn, T., Pleschka, S. and Ludwig, S. (2003) Caspase 3 activation is essential for efficient influenza virus propagation. Embo J, 22, 2717-2728. Yedavalli, V.S., Shih, H.M., Chiang, Y.P., Lu, C.Y., Chang, L.Y., Chen, M.Y., Chuang, C.Y., Dayton, A.I., Jeang, K.T. and Huang, L.M. (2005) Human immunodeficiency virus type 1 Vpr interacts with antiapoptotic mitochondrial protein HAX-1. J Virol, 79, 13735-13746. | |
| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/42949 | - |
| dc.description.abstract | Influenza A virus的RNA聚合脢，是一個由三個蛋白質所組成的聚合體，分別是PB1,PB2以及PA。我們在這裡試著找出會和PB2作用的細胞內因子，並且研究它們互相做用的生理意義為何。
將病毒的PB2蛋白之C端蛋白質序列305-759作餌，利用酵母菌雙雜合系統 (Yeast two hybrid)，去篩選HeLa細胞的cDNA library，找出可能會和PB2作用的細胞內因子。篩選後得到8個基因，我們選擇其中幾個有興趣的候選人，往下做更深入的研究，其中包括HAX-1。 利用可表現帶有Flag的PB2蛋白質體，及融合其他tag的候選基因，共同轉染入細胞株293 FT大量表現，利用抗FLAG及其他的抗體，來進行免疫沉澱、GST pull-down，來更加的確認找到的細胞因子與PB2的作用是否真實。並且初步找出PB2與HAX-1結合的片段主要在中間第271-522的胺基酸序列，而C端的第636-759胺基酸序列有較弱的結合。在目前的結果中，初步證明細胞因子HAX-1會與PB2結合。 再往下做更近一步的分析，將細胞內的HAX-1利用RNAi knockdown後，病毒的複製有上升的情形，代表HAX-1可能會抑制病毒的生長。而在類病毒基因體的冷光報導蛋白系統中，大量表現HAX-1會使得冷光蛋白酶的表現下降，則暗示HAX-1也許是影響病毒RNA聚合酶的功能。因為HAX-1是已知的會對抗細胞凋亡 (apoptosis) 的蛋白，所以我們想看PB2的表現是否會影響HAX-1抗apoptosis的效果，但這方面的研究目前還沒有結論，必須要再多加改進。另外，在實驗的過程中，我們意外地發現共同表現HAX-1及PB2後，HAX-1會進入細胞核，而入核是否為PB2將HAX-1帶入核內或利用其它的機制以及HAX-1入核的功能為何，也有待釐清。 | zh_TW |
| dc.description.abstract | PB2 is a component of influenza A RNA polymerase complex and has been shown to play an important role in influenza A viral transcription and replication. To study the role of PB2 involved in influenza A viral replication, we set to fish out cellular factors that interact with PB2 by using yeast two-hybrid system. We found that eight cellular proteins could interact with PB2 C-terminal region. Among them, HAX-1 was confirmed to interact with PB2 by co-immunoprecipitation and GST pull-down assays.
HAX-1, a multifunction protein, has been shown to have an anti-apoptotic function and play roles in regulating mRNA transport. To study the role of HAX-1 in influenza A viral replication, HAX-1 knock-down H1299 cells were established by using lentivirus expressing shRNA against HAX-1. We found that influenza A viral production was significantly increased in HAX-1 knock-down cells, indicating that HAX1 plays an inhibitory role in influenza A viral replication. To study the mechanism underlying HAX-1 inhibition of influenza A replication, we transfected PB2-Flag alone, HA-HAX-1 alone, or PB2-Flag + HA-HAX-1 into 293FT cells and detected the location of PB2-Flag and HA-HAX-1 by confocal microscope. HA-HAX-1 localized in the cytoplasm in the absence of PB2. This data is consistent with the previous reports that HAX-1 mainly localized in the mitochondria. Interestingly, when HA-HAX-1 was co-expressed with PB2-Flag, a protein known to localize in the nucleus, a significant proportion of HA-HAX-1 was found in the nucleus. These data indicate that PB2 may translocate HAX-1 into nucleus by interacting with the latter. The significance of this finding and how PB2 interaction with HAX-1 may facilitate influenza A viral production is discussed. | en |
| dc.description.provenance | Made available in DSpace on 2021-06-15T01:30:03Z (GMT). No. of bitstreams: 1 ntu-98-R96445104-1.pdf: 3140417 bytes, checksum: 7798bd3aabfd0e27acb7fc123761c9ae (MD5) Previous issue date: 2009 | en |
| dc.description.tableofcontents | 目錄
口試委員審定書……………………………………………………………i 致謝…………………………………………………………………………ii 中文摘要……………………………………………………………………iii Abstract………………………………………………………………..……iv 目錄…………………………………………………………………....…...v 圖目錄…………………………………………………………………...…viii 表格目錄………………………………………………………………...…viii 序論………………………………………………………………….………1 研究目的…………………………………………………………….………5 實驗材料…………………………………………………………….………6 一、 化學藥品…………………………………………………………….………6 二、 套組試劑……………………………………………………………….……9 三、 抗體…………………………………………………………………………10 四、 酵素…………………………………………………………………………10 五、 質體…………………………………………………………………………11 六、 細胞株………………………………………………………………………15 七、 其它…………………………………………………………………………16 實驗方法……………………………………………………………………17 一、 質體建構 (Construction)…………………………………………….…17 二、 細菌轉形 (Transformation)……………………………………….……17 三、 勝任細胞的製備 (Preparation of competent cells)………………….…17 四、 小量質體製備 (Mini-preparation)………………………………..……18 五、 大量質體製備 (Large-scale plasmid isolation)………………………...19 六、 酵母菌雙雜合篩選 (Yeast two-hybrid screening)………………..……21 七、 酵母菌轉形 (Yeast transformation)……………………………………22 八、 酵母菌大量轉形 (Large-scale yeast transformation)……………….…23 九、 酵母菌質體DNA抽取 (Plasmid isolation from Yeast)…………….…24 十、 免疫染色 (Immunofluorescence assay)…………………………..……24 十一、 免疫沉澱 (Immunoprecipitation)………………………………..…….25 十二、 質體轉染 (Transfection)………………………………….……...….…25 十三、 慢病毒製備 (Preparation of Lentivirus)……………………………….26 十四、 慢病毒定量 (quantification of Lentivirus)…………………..…………27 十五、 慢病毒感染 (Lentivirus infection)……………………………..………28 十六、 細胞核糖核酸萃取 (RNA extraction)……………………….…………28 十七、 反轉錄反應 (Reverse transcription)…………………………………....29 十八、 即時聚合酶鏈鎖反應 (Real-time PCR)…………………………….….29 十九、 冷光酶分析 (Luciferase assay)…………………………………………30 二十、 細胞全蛋白質之收取………………………………………..………….31 二十一、 蛋白質定量……………………………………………….……………31 二十二、 西方墨點法 (Western blot)……………………………………………31 二十三、 流感病毒感染及增殖 (Influenza virus infection and amplification)………………………………………………………….32 二十四、 流感病毒之溶斑分析法 (Plaque assay of Influenza virus)…………...32 二十五、 Glutathione S-transferase (GST) pull-down 分析……………………..33 實驗結果…………………………………………………………………..…35 一、 利用酵母菌雙雜合篩選找出與PB2有交互作用的細胞因子…………….35 二、 利用共同免疫沉澱法來確認HAX-1與病毒PB2間的結合………………35 三、 利用GST pull-down確認HAX-1與PB2的結合……………………….…36 四、 HAX-1與PB2的結合是蛋白間直接的作用，並非RNA從中 媒介的結果……………….…….….................................................................37 五、 利用冷光蛋白報導系統確定HAX-1是否會影響病毒聚合酶的功能…….37 六、 利用RNAi將HAX-1 knockdown造成流感病毒的複製上升……..……....37 七、 Knockdown HAX-1造成病毒誘發的細胞apoptosis些微地上升…………38 八、 PB2對HAX-1功能的影響………………………………………………….38 九、 共同表現PB2及HAX-1使得HAX-1進入細胞核增加………………..…40 討論………………………………………………………………………..….41 圖…………………………………………………………………………..….45 表格………………………………………………………………………..….54 附圖………………………………………………………………………..….57 參考文獻…………………………………………………………………..….60 圖目錄 圖一、利用共同免疫沉澱確認HAX-1與PB2的結合。………………………45 圖二、利用GST pull-down確認HAX-1與PB2的結合及找出作用的片段….46 圖三、確認HAX-1與PB2的結合，不是因為RNA作為媒介的結果。……..48 圖四、病毒vRNP功能報導系統顯示，大量表現HAX-1會抑制vRNP合 成病RNA。……………………………………………………………..49 圖五、將細胞內HAX-1 knockdown，會增加病毒的複製。………………...…50 圖六、Knockdown HAX-1會些微增加病毒所誘發apoptosis………………..…51 圖七、PB2不會影響HAX-1 anti-apoptosis的功能……………………………..52 圖八、共同表現PB2及HAX-1使得HAX-1入核增加………………………...53 表格目錄 表一、A型流感所表現蛋白質之功能……………………………………………54 表二、引子序列……………………………………………………………………55 表三、酵母菌雙雜合篩選的結果…………………………………………………56 | |
| dc.language.iso | zh-TW | |
| dc.subject | 入核 | zh_TW |
| dc.subject | 聚合脢 | zh_TW |
| dc.subject | 細胞凋亡 | zh_TW |
| dc.subject | 病毒複製 | zh_TW |
| dc.subject | 流感 | zh_TW |
| dc.subject | localization | en |
| dc.subject | Influenza virus | en |
| dc.subject | PB2 | en |
| dc.subject | HAX-1 | en |
| dc.subject | vRNP | en |
| dc.subject | virus replication | en |
| dc.title | 鑑定並分析與A形流行性感冒病毒之PB2蛋白作用的細胞因子 | zh_TW |
| dc.title | Identification and characterization of cellular proteins that interact with Influenza A virus PB2 protein | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 97-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 鄧述諄(Shu-Chun Teng),施信如(Shin-Ru Shih) | |
| dc.subject.keyword | 流感,聚合脢,細胞凋亡,病毒複製,入核, | zh_TW |
| dc.subject.keyword | Influenza virus,PB2,HAX-1,vRNP,virus replication,localization, | en |
| dc.relation.page | 64 | |
| dc.rights.note | 有償授權 | |
| dc.date.accepted | 2009-07-21 | |
| 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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