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研究生:賴景郁
研究生(外文):Ching-Yu Lai
論文名稱:A型流行性感冒病毒PB2蛋白質與細胞蛋白質hRrp43之交互作用
論文名稱(外文):The Interaction Between Influenza A Viral PB2 Protein and Cellular Protein hRrp43
指導教授:王萬波
指導教授(外文):Wong-Bo Wang
口試委員:楊宏志張鑫
口試委員(外文):Hong-Chih YangHsin Chang
口試日期:2013-07-29
學位類別:碩士
校院名稱:國立臺灣大學
系所名稱:微生物學研究所
學門:生命科學學門
學類:微生物學類
論文種類:學術論文
論文出版年:2013
畢業學年度:101
語文別:中文
論文頁數:75
中文關鍵詞:A型流行性感冒病毒PB2蛋白質hRrp43病毒轉錄與複製
外文關鍵詞:Influenza virusPB2proteinhRrp43 proteinvirus transcription and replication
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流感病毒的PA、PB1、PB2蛋白質構成病毒特有的RNA-dependent RNA polymerase (RdRp),其中PB2蛋白質具有結合至宿主pre-mRNA的帽蓋上,對於病毒複製或是轉錄都是不可或缺的。本實驗室找尋可能與PB2進行交互作用的細胞蛋白質時,藉由酵母菌雙雜合系統(Yeast two-hybrid system),找出細胞蛋白質hRrp43會與PB2進行交互作用。
我們利用GST pull-down assay和co-immunoprecipitiation確認PB2會與hRrp43結合。由於hRrp43與PB2都可以與RNA結合,為了避免hRrp43與PB2之間的交互作用是透過RNA,因此在進行co-immunoprecipitation之前,使用RNaseA去除細胞萃取物中的RNA,結果GFP-PB2仍然可以與Flag-hRrp43結合,顯示PB2與hRrp43的結合不是透過RNA。此外,使用共軛聚焦顯微鏡也觀察到GFP-PB2與Flag-hRrp43都可以共同定位在細胞核中。
為了測試hRrp43對於流感病毒複製所造成之影響,利用hRrp43的 shRNA在H1299細胞內篩選出hRrp43 knockdown之穩定細胞株H1299-shhRrp43。流感病毒在H1299-shhRrp43細胞株內的複製效率下降,顯示hRrp43可能影響到流感病毒的複製。此外在病毒轉錄與複製的螢光酶報導系統中觀察到hRrp43可能影響流感病毒的轉錄或複製。
有趣的是,比較H1299-shhRrp43與控制組H1299-shGFP之穩定細胞株被病毒感染後四十八小時的情況,H1299-shhRrp43之病毒數目較少、細胞型態有明顯差異且其生長速度較慢。為了確認此型態改變是否影響H1299-shhRrp43穩定細胞株走向細胞凋亡(apoptosis),使用流式細胞儀(FACS, Cytoflowmetry)做分析,發現H1299-shhRrp43細胞株被感染後較不易走向細胞凋亡。
更進一步探討H1299-shhRrp43細胞株為何產生較少病毒產量,我們發現H1299-shhRrp43細胞被病毒感染後,病毒蛋白質及病毒mRNA, cRNA,及vRNA的表現量均較控制組低很多。透過免疫螢光分析的技術,觀察到H1299-shhRrp43細胞被感染後2小時,病毒NP蛋白質會在細胞核內出現, 但量較少,因此病毒感染H1299-shhRrp43細胞後可以入核,但效率較差,綜合以上結果,我們推測hRrp43除了會影響病毒入核外也會影響病毒RNA的轉錄, 而hRrp43如何影響轉錄過程仍需更深入探討。


Influenz A virus uses an RNA-dependent RNA polymerase (RdRp)consisting of PA, PB1 and PB2 proteins to transcribe and replicate its RNA genome. PB2, which is a cap binding protein, plays an important role in influenza A viral transcription and replication. Our lab previously used yeast two-hybrid system to identify cellular proteins that interact with PB2. The data indicated that hRrp43 is one of the cellular proteins that may interact with PB2.
In this study, we confirmed the interaction between PB2 and hRrp43 by GST pull-down assays and co-immunoprecipitation assays. Since both PB2 and hRrp43 can bind RNA, it is important to rule out the possibility that the interaction is mediated through RNA. We thus treated the cell extract with RNaseA before performing co-immunoprecipitation assays. We found that GFP-PB2 could still interact with Flag-hRrp43, indicating that PB2 can interact with hRrp43 even in the absence of RNA.
In addition, we found that hRrp43 and PB2 were co-localized in the nucleus in the immunofluorescence assay.
To test the effect of hRrp43 on influenza A viral replication, we generated hRrp43 knock-down cell line by using lentivirus expressing shRNA against hRrp43. We found that influenza A viral multiplication was dramatically decreased in the hRrp43 knockdown cell line H1299-shhRrp43, indicating that hRrp43 is required for influenza A viral multiplication. This conclusion was further supported by finding that the viral RNA-dependent RNA transcription and replication rate was decreased in the viral minigenome reporter assay.
Interestingly, when H1299-shhRrp43 cells and control H1299-shGFP cells were infected with influenza A virus for 48 hours, the infected H1299-shhRrp43 cells exhibited different morphology and slower growth rate than the infected H1299-shGFP cells. To investigate whether the above phenotypes of infected H1299-shhRrp43 cells was caused by virus-induced apoptosis, we analysed cellular DNA content by flow cytometry. Our data indicated that the extent of apoptosis was lower in virus-infected H1299-shhRrp43 cells than in virus-infected H1299-shGFP cells. It will be of interest to study why hRrp43-knockdown cells, upon influenza A virus infection, undergo apoptosis less severely than control cells in the future.
To further study why influenza A virus produced much less progeny in H1299-shhRrp43 cells than in control cells, we measured the amount of viral proteins and viral mRNA, cRNA, and vRNA synthesized in the infected H1299-shhRrp43 and control cells. We found that the expression of viral proteins, including PB2, PA, and NP, was much less in H1299-shhRrp43 cells than in control cells. Moreover, the expression levels of NP mRNA, NP cRNA, and NP vRNA were also much lower in H1299-shhRrp43 cells than in control cells. To study whether the lower virus production in the hRrp43-knockdown cells was due to deficiency in viral entry, we detected the presence of NP protein in the nucleus at 2 h post virus infection by immunoflorescence assays. We found that NP could enter the nucleus after virus infection of H1299-shhRrp43 cells. vRNP entered nucleus slightly less efficiently in H1299-shhRrp43 cells than in control cells. Thus, lower virus entry could not completely account for the extremely low virus production in hRrp43-knockdown cells. We think that deficiency in virus transcription is the main reason that influenza A virus cannot multiplicate well in hRrp43-knockdown cells. Further study is required to prove this hypothesis.


誌謝 i
中文摘要 ii
ABSTRACT iv
目錄 vi
圖目錄 ix
附圖目錄 x
緒論 1
研究目的 9
材料與方法
◆實驗材料 10
一、化學藥品及試劑 10
二、套組試劑 14
三、抗體 15
四、其他 15
五、細胞株 16
六、質體 17
◆實驗方法 21
一、細菌轉型(Transformation) 21
二、勝任細胞的製備(Preparation of competent cells) 22
三、小量質體製備(Mini-preparation) 23
四、大量質體製備(Large-scale plasmid isolation) 24
五、質體轉染(Transfection) 27
六、慢病毒製備(Preparation of lentivirus) 28
七、慢病毒定量(Quantification of lentivirus) 30
八、慢病毒感染(Lentivirus infection) 31
九、細胞核醣核酸萃取(RNA extraction) 32
十、反轉錄反應(Reverse transcription) 33
十一、即時聚合酶鏈鎖反應(Real-time PCR) 33
十二、螢光酶分析(Luciferase assay) 34
十三、細胞全蛋白質之收取 35
十四、蛋白質定量 35
十五、西方墨點法(Western blot) 36
十六、流感病毒感染及增值(Influenza virus infection and amplification) 36
十七、流感病毒之溶斑分析法(Plaque assay of influenza virus) 37
十八、Glutathione S-transferase(GST)pull-down分析 38
十九、免疫共沉澱法(Co-immunoprecipitation) 40
二十、免疫螢光分析(Immunofluorescence assay, IFA) 41
二十一、流式細胞儀分析(Flow cytometry analysis) 42
二十二、SRB assay 42
◆實驗結果 44
一、透過免疫共沉澱(Co-immunoprecipitation)確認病毒PB2蛋白質與細胞hRrp43蛋白質之間有交互作用 44
二、PB2蛋白質與hRrp43蛋白質之間的交互作用不透過RNA 44
三、透過GST pull-down assay的方式,再次確認PB2與hRrp43蛋白質會進行交互作用 45
四、透過共軛聚焦顯微鏡觀察到PB2蛋白質與hRrp43蛋白質共同位於(Co-localize)細胞核中 45
五、利用不同長度片段之PB2來找尋與hRrp43進行結合的區域 45
六、利用Rrp43的shRNA抑制H1299細胞內的hRrp43會造成流感病毒的轉錄複製能力降低 46
七、將細胞hRrp43 knockdown會使流感病毒複製受到抑制 47
八、hRrp43的knock-down會抑制病毒蛋白及病毒RNA的表現 47
九、證實influenza virus結合至H1299-shhRrp43細胞株與H1299、H1299-shGFP細胞株的數量是相近的 47
十、H1299-shhRrp43細胞被病毒感染後,病毒vRNP可以入核 48
十一、hRrp43-knockdown細胞被病毒感染後,其細胞型態(morphology)與控制組有差異且較不易走向細胞凋亡(apoptosis) 48
討論 49
附表 66
參考文獻 67





圖目錄
圖一、透過免疫共沉澱法(co-immunoprecipitation)法,顯示PB2蛋白質與hRrp43蛋白質之間具有交互作用且不透過RNA 52
圖二、透過GST pull-down assay分析亦顯示PB2與hRrp43具有交互作用 54
圖三、透過共軛聚焦顯微鏡觀察到PB2蛋白質與hRrp43蛋白質共同位於(co-localize)細胞核中 56
圖四、PB2對於hRrp43的結合位置可能是在PB2的271到759胺基酸片段之間 57
圖五、挑選H1299-shhRrp43穩定細胞株,且此細胞株生長速率較佳 58
圖六、hRrp43 knockdown會使流感病毒複製能力與聚合酶活性受到抑制 59
圖七、H1299-shhRrp43穩定細胞株被病毒感染後,病毒蛋白質及RNA表現量均較低,但病毒貼附至三種細胞的數量是相近的 60
圖八、H1299-shhRrp43被病毒感染後,病毒vRNP可以入核 62
圖九、hRrp43-knockdown細胞被病毒感染後,其細胞型態(morphology)與控制組有差異且較不易走向細胞凋亡(apoptosis) 64







附圖目錄
圖一、A型流行性感冒病毒結構圖 71
圖二、A型流感病毒的生活史 72
圖三、流感病毒PB2蛋白質的功能區域(functional domain) 73
圖四、細胞蛋白質hRrp43(OIP2)的功能性區域 74
圖五、似流感病毒複製之螢光酶報導系統 75



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