臺灣博碩士論文加值系統

A 型流行性感冒病毒感染通常會造成呼吸道疾病。我們發現 NP 是與 hnRNP A2/B1 的 glycine rich domain (GRM) 直接結合，且當病 毒感染 A549 細胞 3 至 6 小時後 NP 與 hnRNP A2/B1 分佈於細胞核內 相同的位置。與 hnRNP A2/B1 序列相似的 hnRNP A1 已被報導參與 pri-miRNA processing，而 hnRNP A2/B1 已知與 miR-7-1 terminal loop 結合。流感病毒感染會造成 miRNA的改變，進而影響病毒的複製， 已有文獻指出 miR-7可藉由抑制免疫基因進而正向調控病毒。基於以 上文獻進一步探討 NP與 hnRNPA2/B1之間的交互作用是否對感染後 的 miRNA 進行調控。本研究發現： (1) hnRNP A2/B1 可正向調控 miR-7-1 的生合成。藉由抑制 hnRNP A2/B1 表現導致 miR-7-1 的表現 量下降，而過度表現 hnRNP A2 時，miR-7-1 的表現量上升。 (2) NP 可能藉由與 hnRNP A2/B1 的結合正向調控 miR-7-1 的生合成。顯示在 過度表現 NP-Flag 的細胞中，pri-miR-7-1 可與較多的 NP 及 hnRNP A2/B1 結合。 (3) 當 A549 細胞被 A/WSN/33 病毒株感染時，hnRNP A2/B1 可正向調控 miR-7-1 生合成去影響病毒的複製。當 A/WSN/33 感染 hnRNP A2/B1 被抑制的細胞時，miR-7-1 的表現量下降。

Influenza A virus infection frequently causes respiratory disease. Our research group observed that NP can interact directly with a glycine- rich motif of heterogeneous nuclear ribonucleoproteins (hnRNP A2/B1). In addition, NP and hnRNP A2/B1 colocalize in the nuclei of virus-infected cells early in the infection process. The RNA recognition motif of hnRNP A2/B1 exhibits greater than 80% similarity with hnRNP A1, which can modulate miRNA biogenesis. A previous study indicated that hnRNP A2/B1 can interact with the terminal loop of miR-7-1. Another research demonstrated that miR-7-1 positively regulate virus replication by targeting immune genes. In view of previous researches, we predicted NP modulated miRNA by interacting with hnRNP A2/B1. Thus, we chose miR-7-1 as our taget based on previous reports. Our research indicated that： (1) hnRNP A2/B1 may positively regulate
miR-7-1 biogenesis. MiR-7-1 will decrease when knockdown hnRNP A2/B1, and miR-7-1 will increase when over express hnRNP A2/B1. (2) NP may positively regulate miR-7-1 through hnRNP A2/B1. A pri-miR-7-1 RNA pull-down assay revealed that NP and hnRNP A2/B1 were enhanced as the NP-Flag plasmid expressed. (3) hnRNP A2/B1 may positively regulate miR-7-1 in A/WSN/33 virus-infected cell. MiR-7-1 decreased when hnRNP A2/B1-inhibited A549 cells were infected with A/WSN/33.

指導教授推薦書
論文口試委員會審定書
致謝 iii
中文摘要 iv
Abstract v
目錄 vi
圖目錄 x
附圖目錄 xii
第一章 前言 1
1.1 A型流感病毒性質簡介 1
1.2 NP簡介 2
1.2.1 NP之RNA結合活性 2
1.2.2 NP在viral ribonucleoprotein中扮演的角色 3
1.2.3 NP與宿主蛋白質的交互作用 3
1.2.4 NP對於病毒複製的重要性 3
1.3 Group A heterogeneous nuclear ribonucleoprotein簡介 4
1.3.1 hnRNP A2/B1簡介 4
1.3.2 hnRNP A2/B1與hnRNP A1的相似性 5
1.3.3 hnRNP A/B family 5
1.3.4 Group A hnRNPs調控某些正股病毒的複製 7
1.4 小分子核醣核酸 (MicroRNA, miRNA) 8
1.5 證實NP與hnRNP A2/B1的交互作用 10
1.5.1 hnRNPA2/B1為NP結合的宿主蛋白質之一 10
1.5.2 NP與hnRNP A2/B1在病毒感染不同時期的細胞內分佈位置 11
第二章 研究目的 13
第三章 材料與方法 16
3.1 A549細胞株的培養 16
3.2 microRNA萃取 16
3.3 轉染作用 18
3.3.1 應用於過量表現hnRNP A2 18
.3.3.2 應用於抑制hnRNPA2/B1表現 18
3.4 蛋白質萃取 19
3.5 蛋白質定量 19
3.6 西方墨點法 19
3.7 microRNA之Reverse transcription PCR 20
3.8 利用real-time PCR監控miRNA precursor之轉染效率 21
3.9 In Vitro Transcripition試管內轉錄 21
3.10 RNA pull down assay 21
第四章 實驗結果 23
4.1 在A549細胞中抑制hnRNP A2/B1觀察miR-7-1、miR-132、miR-146a、miR-187、miR-200c和miR-1275的表現 23
4.2 在A549細胞中過度表現hnRNP A2觀察miR-7-1、miR-132、miR-146a、miR-187、miR-200c和miR-1275的表現 24
4.3 在A549細胞中單獨表現hnRNP A2或NP-Flag及共同表現hnRNP A2和NP對於miR-7-1、miR-132和miR-146a生合成的影響 24
4.4 hnRNP A2/B1與pri-let-7a、pri-miR-7-1、pri-miR-132和pri-miR-146a在Hela細胞及A549細胞中的結合情形 25
4.5 hnRNP A2/B1和NP 與pri-let-7a、pri-miR-7-1、pri-miR-132和pri-miR-146a在過度表現NP-Flag的A549細胞中的結合情形 26
4.6 於A549細胞中過度表現glycine rich domain (GRM) 缺失的HA-hnRNP A2和NP-Flag，比較hnRNP A2和NP與pri-let-7a、pri-miR-7-1和pri-miR-146a的細胞中的結合情形 27
4.7 探討當A/WSN/33感染A549細胞時，hnRNP A2/B1如何調控let-7a、miR-7-1及miR-146a的表現 28
第五章 結果與討論 29
5.1 結論 29
5.2 hnRNP A2/B1正向調控miR-7-1生合成 30
5.3 NP藉由與hnRNP A2/B1結合正向調控miR-7-1生合成 32
5.4 當A型流感病毒感染細胞時，病毒核蛋白質nucleoprotein (NP)能夠藉由與hnRNP A2/B1直接結合正向調控miR-7-1生合成進而抑制先天免疫基因表現以促進病毒複製速率 33
第六章 參考資料 35
第七章 圖 42
第八章 附圖 51

圖目錄
圖 1、利用轉染將hnRNP A2/B1 siRNA送入A549細胞中抑制hnRNP A2/B1表現，miR-7-1、miR-132、miR-146a、miR-187、miR-200c和miR-1275的表現量 42
圖 2、利用轉染將HA-hnRNP A2質體送入A549細胞中過度表現hnRNP A2，miR-7-1、miR-132、miR-146a、miR-187、miR-200c和miR-1275的表現量 43
圖 3、利用轉染將HA-hnRNP A2和NP-Flag質體送入A549細胞中單獨或共同表現hnRNP A2和NP，miR-7-1、miR-132和miR-146a的表現量 44
圖 4、在Hela細胞和A549細胞，pri-let-7a和pri-miR-7-1皆會與hnRNP A2/B1直接或間接結合 45
圖 5、在過度表現NP-Flag的A549細胞中，NP和hnRNP A2/B1皆會與pri-let-7a、pri-miR-7-1和pri-miR-146a結合將A549細胞細胞萃取液與Biotin標定的pri-miRNA於試管中反應後，以Strepavidin沉澱。 46
圖 6、在共同表現缺少glycine rich domain (GRM) 的HA-hnRNP A2和NP-Flag的A549細胞中， NP和hnRNP A2/B1與 pri-let-7a和pri-miR-7-1結合的表現量減少將A549細胞細胞萃取液與Biotin標定的pri-miRNA於試管中反應後，以Strepavidin沉澱。 48
圖 7、當A/WSN/33感染hnRNP A2/B1被抑制的A549細胞時，let-7a、miR-7-1和miR-146a的表現量 50

附圖目錄
附圖 1、流感病毒顆粒卡通示意圖 51
附圖 2、流感病毒複製週期 52
附圖 3、hnRNPs 結構圖 53
附圖 4、hnRNP A2與hnRNP B1來源為同一基因，經替代性剪接而產生的兩種isoforms 54
附圖 5、以Clustal W比對hnRNP A1與hnRNP A2序列 55
附圖 6、利用EMBOSS軟體比對hnRNP A0、hnRNP A1、hnRNP A2、hnRNP B1和hnRNP A3序列 56
附圖 7、調控microRNA processing的蛋白群 57
附圖 8、成熟miRNA的生成過程 58
附圖 9、miRNA影響病毒複製的可能機制 59
附圖 10、hnRNP A2/B1為可與NP結合的宿主蛋白質之一 60
附圖 11、NP與hnRNPA2/B1在病毒感染不同時期的細胞內分佈位置 62
附圖 12、當細胞被A/WSN/33病毒株感染初期，NP可能藉由與hnRNP A2/B1在細胞核內直接結合正調控miR-7-1生合成，且miR-7-1能夠結合先天免疫基因抑制基因表現進而增加病毒的複製的模型圖。 63
附圖 13、在過度表現NP-Flag的293T細胞中做Flag-IP，利用anti-Flag免疫沉澱NP-Flag及其結合的蛋白 64

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