臺灣博碩士論文加值系統

腸病毒71型 (EV71) 是微小RNA病毒科(Picornaviridae)腸病毒屬(Enterovirus genus)的成員。若感染者是5歲以下的幼童，病毒有機會侵犯中樞神經系統，甚至造成死亡。EV71在亞太地區常有週期性的流行發生，目前仍沒有有效的抗病毒藥物或是疫苗上市。因此，EV71被視為新興的具神經向性(neurotropism)的病毒，屬於重要的公衛議題之一。為了尋找EV71新穎的參與在IRES轉譯作用的細胞因子，以做為抗病毒藥物標的。我們使用了EV71 IRES RNA做為探針，在試管內結合反應後，將與其交互作用的蛋白質複合體抓取下來，並以蛋白質體學的方式分析其組成，發現50個在感染情況下會與IRES RNA有交互作用的細胞因子。更進一步使用慢病毒攜帶shRNA的方式分別敲落這50個細胞因子，以IRES報導基因觀察這些細胞因子對於EV71轉譯活性的影響。我們發現大部分的細胞因子對於EV71 IRES轉譯活性都有程度不同的影響。更進一步分析這些細胞因子對於病毒蛋白表現的影響，結果雷同於報導基因的分析。現今有許多的報導指出熱休克蛋白(heat shock protein) 參與在許多病毒的生活史當中，從我們的實驗中也得知HSC70在EV71生活史中扮演正向調控者的角色。在未來我們想針對HSC70做更進一步的研究及探討。此外，在實驗室早期研究中得知RNA解螺旋酶DDX3透過其RNA解螺旋酶活性，解開IRES SL-VI二級結構，以提升病毒轉譯作用的效率，然而在試管內RNA競爭實驗中，我們發現DDX3對於IRES RNA的結合是沒有序列專一性的。利用RNA-蛋白質抓取以及免疫共沉澱法，我們認為DDX3可能是透過與截短的eIF4G*交互作用，而被帶到正確的位置上。另外，抗癌藥物Sorafenib被報導能削弱EV71的轉譯，我們推測它可能是透過調節DDX3蛋白的解螺旋酶活性，進而影響EV71的基因表現，在未來我們也會以純化DDX3蛋白來了解Sorafnib是否會影響DDX3的解螺旋酶活性。綜合上述結果，本論文除了篩選出新穎的病毒感染相關細胞因子，也對DDX3在EV71轉譯機制有更進一步的了解。

Enterovirus 71 (EV71) is a member of Enterovirus genus within Picornaviridae family. EV71 infection may cause severe neurological complications and even death in young patients, especially those who are under 5 years old. Several countries across the Asia-Pacific region experienced cyclic epidemics occurring every 2-3 years. There is no FDA approved antiviral therapy or vaccine to date. As a result, EV71 is regarded as an emerging neurotropic virus, and becomes a major issue of public health. In order to find out the potential antiviral targets, we are devoted to screening cellular factors that are associated with internal ribosome entry site (IRES)-mediated translation under EV71 infected condition. We conducted an in vitro RNA-protein binding assay, followed by proteomic analysis, and found that 50 cellular proteins may interact with IRES RNA under EV71 infection condition. Then we used shRNA-expressing lentivirus knockdown strategy to validate the importance of these 50 factors in viral protein translation. The results showed that most of these factors played positive roles in EV71 life cycle. We will choose HSC70 as a candidate for further investigation in the future. In addition, previous studies have shown that the RNA helicase DDX3 facilitats EV71 translation via its helicase activity to unwind IRES Stem-loop (SL)-VI. However, according to the in vitro RNA binding assay, DDX3 does not have sequence specificity for binding RNA. Using a co-immunoprecipitation experiment, we demonstrated that DDX3 may bind the SL-VI of IRES in vivo via its interaction with truncated eIF4G which binds to SL-V. Additionally, sorafenib, an anti-cancer drug, was shown to be able to inhibit EV71 IRES activity. Because its structure is similar to that of compound 6, a putative DDX3 inhibitor, we hypothesize that sorafenib may suppress EV71 via inhibiting DDX3 helicase activity, which remains to be investigated in the future. In conclusion, our study has identified several new factors that may be associated with EV71 IRES activity, and provide new insight into the role of DDX3 in EV71 translation.

誌謝 i
中文摘要 ii
Abstract iii
目錄 iv
圖表目錄 v
附圖表目錄 vi
第一章 緒論 1
第一節、腸病毒71型 (Enterovirus 71, EV71) 1
第二節、 腸病毒71型轉譯作用及相關細胞因子 (EV71 IRES-mediated translation and related cellular factors) 6
第三節、 DEAD-box Polypeptide 3 (DDX3) 8
第四節、 熱休克蛋白(Heat shock protein, HSP) 10
第五節、 前人研究與實驗目的 13
第二章 實驗材料與方法 15
第一節、 實驗材料 (Materials) 15
第二節、 實驗方法 (Methods) 23
第三章 實驗結果 32
第一節、 尋找在感染情況下與腸病毒71型IRES有交互作用之細胞蛋白 32
第二節、 熱休克蛋白70正向調控腸病毒71型生活史 35
第三節、 RNA解螺旋酶DDX3結合在腸病毒71型IRES上的專一性探討 37
第四節、 抗癌藥物Sorafenib抑制腸病毒71型的機轉之探討 40
第四章 討論 42
參考資料 49
圖表 59
附圖 69
附表 75

圖表目錄
圖一、測量IRES驅動之螢火蟲冷光酵素活性探討50個基因對於腸病毒71型轉譯作用之重要性。 60
圖二、 探討敲落20個感染相關的結合細胞因子對於腸病毒71型病毒蛋白的表現 62
圖三、 探討HSC70在腸病毒71型生活史中是否為正向調控者。 63
圖四、利用試管內RNA競爭實驗探討DDX3與IRES RNA結合是否有序列專一性 65
圖五、免疫共沉澱法探討DDX3與eIF4G*之交互作用 66
圖六、 Sorafenib對於腸病毒71型的影響 68


附圖表目錄
附圖一、 前人研究發現當病毒感染時，會顯著增加腸病毒71型IRES轉譯作用之活性 (本實驗由何岳峰完成) 69
附圖二、 尋找感染情況下才會與腸病毒71型IRES有交互作用之細胞因子(本實驗由黃馨儀完成) 70
附圖三、 DDX3對於腸病毒71型IRES 序列專一性的探討 71
附圖四、腸病毒71型之第一型IRES轉譯機制示意圖 72
附圖五、腸病毒71型IRES SL-VI 二級結構遭受破壞後，DDX3對於IRES轉譯作用便不再重要 (本實驗由何岳峰進行SL-VI結構破壞並由蔡艾璇進行轉譯作用活性的測試) 73
附圖六、基因本體分析 (Gene Ontology)分析50個腸病毒感染相關結合因子 (由林立峰學長協助完成) 74

附表一、 50個與腸病毒71型感染相關之細胞結合因子。 75

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