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完整後設資料紀錄
DC 欄位 | 值 | 語言 |
---|---|---|
dc.contributor.advisor | 王萬波 | |
dc.contributor.author | Wei-Bin Hsu | en |
dc.contributor.author | 徐維彬 | zh_TW |
dc.date.accessioned | 2021-06-16T16:30:55Z | - |
dc.date.available | 2018-03-04 | |
dc.date.copyright | 2013-03-04 | |
dc.date.issued | 2012 | |
dc.date.submitted | 2012-12-20 | |
dc.identifier.citation | REFERENCE
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dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/63254 | - |
dc.description.abstract | A型流感病毒的基因體是RNA,其轉譯及複製的過程是透過病毒本身的RNA-dependent RNA polymerase在宿主細胞核中進行,而這RNA-dependent RNA polymerase 是由PB1,PB2,PA等蛋白所組成。此外宿主細胞的分子在病毒的轉譯及複製過程中也扮演重要的角色。因此,為了找尋可能會與A型流感病毒蛋白PA交互作用的宿主細胞分子,我們利用HeLa cDNA庫於酵母菌雙雜交系統中做篩選,結果釣到6個可能與PA交互作用的細胞蛋白。在本篇研究裡我們專注其中一個細胞蛋白HAX1,其已知的功能是抑制細胞凋亡。我們利用GST-pull down與Co-IP的實驗方法證實,無論在in vitro和in vivo下,PA都會與HAX1交互作用,進一步我們也證實了HAX1是透過結合上PA的NLS序列作交互作用。在HAX1 knock-down細胞株裡,發現PA進入細胞核的現象有明顯增加,且在HAX1表現回補時,此現象即消失。因此我們認為HAX1會抑制PA的核內聚集。另一方面,我們利用minigenome方法,分析病毒聚合酶的活性,發現在HAX1 knock-down細胞裡,聚合酶的活性有明顯增加,同樣地HAX1回補時,此現象即消失。最後在測定病毒的複製能力時,也發現相同的結果。綜合所有結果,我們不只是提供一個解釋PA進核機制的新觀點,且也發現一個細胞內生性的分子能抑制A型流感的感染的藥物研發的潛力。 | zh_TW |
dc.description.abstract | Transcription and replication of the influenza A virus RNA genome occurs in the nucleus through the viral RNA-dependent RNA polymerase consisting of PB1, PB2, and PA. Cellular factors that associate with the viral polymerase complex play important roles in these processes. To look for cellular factors that could associate with influenza A virus PA protein, we have carried out a yeast two-hybrid screen using a HeLa cell cDNA library. We identified six cellular proteins that may interact with PA. In this report, we focused our study on one of the new PA-interacting proteins HAX1, a protein with anti-apoptotic function. By using Glutathione S-transferase pull-down and coimmunoprecipitation assays, we demonstrated that HAX1 specifically interacted with PA in vitro and in vivo, and that HAX1 interacted with the nuclear localization signal domain of PA. Nuclear accumulation of PA was increased in HAX1-knockdown cells and this phenotype could be reversed by reexpression of HAX1, indicating that HAX1 can impede nuclear transport of PA. As a consequence, knockdown of HAX1 resulted in a significant increase on virus yield and polymerase activity in a minigenome assay and this phenotype could be reversed by reexpression of HAX1, indicating that HAX1 can inhibit influenza A virus propagation. Together, these results not only provide an insight about the mechanism underlying nuclear transport of PA but also identify an intrinsic host factor that restricts influenza A virus infection. | en |
dc.description.provenance | Made available in DSpace on 2021-06-16T16:30:55Z (GMT). No. of bitstreams: 1 ntu-101-D94445002-1.pdf: 3049220 bytes, checksum: c19ed0833939d4acee111f64ac72e012 (MD5) Previous issue date: 2012 | en |
dc.description.tableofcontents | Index
中文摘要 I Abstract II Index III Table and Figures Index V INTRODUCTION 1 Influenza A virus 1 Life cycle of Influenza A virus 3 Functions of the RNP in influenza virus 5 Biological functions of HAX1 7 Aims 9 MATERIALS AND METHODS 11 Materials 11 Chemical and Reagents 11 Others 14 Cell culture 14 Methods 15 Plasmids. 15 Antibodies. 17 Yeast two-hybrid screen. 17 GST pull-down assay. 18 Western blot analysis. 19 Coimmunoprecipitation. 20 Construction of lentivirus expressing HAX1 shRNA or Luc shRNA. 21 Lentiviral production 21 Establishment of HAX1-knockdown, non-specific knockdown, and HAX1-reexpressing H1299 cell clones. 23 Immunofluorescence. 24 Fractionation of cytoplasmic and nuclear proteins. 24 RNA extraction and quantitative reverse transcription-polymerase chain reaction (RT-PCR). 25 Minigenome replication and transcription assay. 27 Viral propagation and titration. 27 Plaque assay. 28 Statistical analyses. 28 RESULTS 29 Identification of cellular HAX1 as a novel PA-interacting protein. 29 PA specifically interacts with HAX1 in vitro. 30 PA specifically interacts with HAX1 in mammalian cells. 30 HAX1 interacts with the NLS domain of PA. 31 HAX1 inhibits nuclear accumulation of PA. 32 The binding of HAX1 to PA is reduced in the presence of PB1. 34 HAX1 inhibits the propagation of influenza A virus. 34 Knockdown of HAX1 leads to increase of mRNA, vRNA, and cRNA synthesis during influenza A virus infection. 35 HAX1 down-regulates the activity of the influenza A virus polymerase complex. 36 PA does not affect the anti-apoptotic function of HAX1. 37 HAX1 cannot inhibit apoptosis induced by influenza A virus infection. 38 DISCUSSION 39 TABLE AND FIGURES 43 REFERENCE 69 | |
dc.language.iso | en | |
dc.title | 細胞蛋白HAX1與A型流行性感冒病毒之PA蛋白交
互作用並阻礙PA入核 | zh_TW |
dc.title | Cellular Protein HAX1 Interacts With Influenza A Virus Polymerase PA Subunit and Impedes Its Nuclear
Translocation | en |
dc.type | Thesis | |
dc.date.schoolyear | 101-1 | |
dc.description.degree | 博士 | |
dc.contributor.oralexamcommittee | 鄧述諄,張鑫,楊宏志,施信如 | |
dc.subject.keyword | A型流感病毒,PA,酵母菌雙雜交系統,GST pull-down,HAX1, | zh_TW |
dc.subject.keyword | Influenza A virus,PA,Yeast two-hybrid screen,GST pull-down,HAX1, | en |
dc.relation.page | 79 | |
dc.rights.note | 有償授權 | |
dc.date.accepted | 2012-12-21 | |
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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