臺灣博碩士論文加值系統

在冠狀病毒感染細胞的過程中，正股的基因體需要同時被當作轉譯以及複製的模板。然而，目前在冠狀病毒的領域中，還沒有文獻針對這兩個機制的調節進行探討。本研究針對poly(A) tail、冠狀病毒核鞘蛋白質(nucleocapsid protein，簡稱N protein)和poly(A) binding protein (PABP)三者之間的交互作用進而調節冠狀病毒基因表達的機制進行探討。經由解離常數(dissociation constant，Kd)評估N蛋白質質對poly(A) tail的結合效率後，我們得知N 蛋白對poly(A) tail有高度親和性，能夠和poly(A) tail結合，因此N protein為一個poly(A) binding protein。我們也透過UV cross-linking以及免疫沉澱法(immunoprecipitation)證明N protein能夠在感染的細胞中和poly(A) tail結合。本研究進一步證明了N蛋白質和poly(A) tail的結合在活體外及細胞中能抑制病毒基因體及細胞中mRNA的轉譯。從研究的結果中發現，雖然N 蛋白質能和poly(A) tail以及真核轉譯起始因子eIF4G結合，但N 蛋白與poly(A) tail的結合，使得與eIF4E的結合效率變差造成轉譯效率的降低。N 蛋白質除了和真核細胞轉譯起始因子eIF4G結合，也能夠和參與冠狀病毒椱製作用的nsp9結合。因此綜合本篇的研究成果，我們發現在活體外及感染細胞中poly(A) tail、N protein和PABP三者之間存在交互作用的關係。此外，poly(A) tail和N 蛋白的結合會降低poly(A) tail和eIF4E的結合效率，因而造成抑制轉譯作用。根據以上的結果，我們認為冠狀病毒基因體3’端poly(A) tail在轉換轉譯(和PABP結合)及複製(和N蛋白質結合)上扮演調節的角色，而使基因表達更有效率。

During infection, the positive-sense genome in RNA virus functions as a template for both translation and replication; however, the strategy for coordinating the use of positive-sense genome for cap-dependent translation or replication remains unclear. In the present study, we investigated the roles of interactions among poly(A) tail, coronavirus nucleocapsid (N) protein and poly(A)-binding protein (PABP) in the regulation of coronavirus gene expression. Through dissociation constant (Kd) comparison, we found that the coronavirus N protein can bind to the poly(A) tail with high affinity, demonstrating that N protein is a PABP. Subsequent analyses with UV cross-linking and immunoprecipitation revealed that the N protein is able to bind to the poly(A) tail in infected cells. Further examination demonstrated that poly(A) tail bound to the N protein negatively regulates translation of coronaviral RNA and host mRNA both in vitro and in cells. Although the N protein can interact with PABP and eIF4G, the poor interaction efficiency between the poly(A)-bound N protein and eIF4E may explain the observed decreased translation efficiency. In addition to interaction with translation factor eIF4G, the N protein is able to interact with coronavirus nonstructural protein 9 (nsp9), one of the replicase proteins required for replication. The study demonstrates interactions among the poly(A) tail, N protein and PABP both in vitro and in infected cells. Of the interactions, binding of poly(A) tail to N protein decreases the interaction efficiency between the poly(A) tail and eIF4E, leading to translation inhibition. Taken together, the results indicate that the 3’-poly(A) tail of coronavirus genome may function as a riboswitch to coordinate the utilization of genome for translation (binding to PABP) or replication (binding to N protein).

摘要 i
Abstract ii
目次 iii
第一章 文獻探討 1
第一節 冠狀病毒之基本簡介 1
1.1 冠狀病毒之分類 1
1.2冠狀病毒造成之疾病 1
1.3冠狀病毒基因體結構 1
1.4冠狀病毒的生活史 2
第二節 冠狀病毒核殼蛋白(nucleocapsid protein，N protein) 2
2.1 冠狀病毒核殼蛋白(以下簡稱N蛋白質) 2
2.2冠狀病毒N蛋白質的結構 3
2.3冠狀病毒N蛋白質的功能 3
第三節 利用冠狀病毒天然缺陷株(DI RNA)研究冠狀病毒的基因表達 4
3.1冠狀病毒天然缺陷株(DI RNA) 4
3.2 利用BCoV DI RNA探討冠狀病毒的基因表達 4
第四節 冠狀病毒之基因表達 4
4.1冠狀病毒基因體的複製 4
4.2冠狀病毒次級基因體的轉錄 5
4.3 冠狀病毒基因體的轉譯 5
第五節 正股RNA病毒轉譯與複製之調控機轉 5
5.1正股RNA病毒轉譯與複製之調控 5
第二章 材料與方法 8
第一節　病毒、細胞株及抗體 8
1.1病毒株 8
1.2細胞株 8
1.3抗體 8
第二節 質體之構築 8
2.1 牛冠狀病毒天然缺陷株pGEM DI RNA 質體之構築 8
2.2 pGEM DI EGFP及TOPO β-actin質體之構築 8
第三節 RNA transcript及RNA探針 (RNA probe)之合成 9
3.1 RNA transcript及RNA probe 之DNA模板合成 9
3.2 將RNA transcript及RNA probe之DNA模板純化 9
第四節 In vitro transcription及定量 10
4.1 將合成RNA transcript的DNA模板序列進行In vitro transcription 10
4.2 將合成RNA probe所需的DNA模板序列進行In vitro transcription 10
4.3 純化RNA及濃度測定 10
第五節　以大腸桿菌表達系統表達蛋白 11
5.1 利用大腸桿菌表現系統表達重組N及PABP蛋白 11
第六節　以電泳凝膠遷移試驗(electrophoretic mobility shift assay, EMSA)探討冠狀病毒基因體poly(A) tail對冠狀病毒N蛋白質及細胞PABP之結合 11
6.1 RNA 探針之純化 11
6.2 將RNA 探針分別與N protein及PABP以EMSA進行結合效率分析 12
6.3 以軟體分析EMSA結果 12
第七節　利用UV cross- linking方法探討N蛋白質和RNA的專一性結合 12
7.1 轉染RNA探針進細胞 12
7.2 將轉染進細胞的RNA探針和結合的蛋白質進行UV cross- linking 13
7.3 將經過UV cross- linking的核酸蛋白質複合體進行蛋白質電泳分析 13
第八節　免疫沉澱法(immunoprecipitation)和pulldown assay 13
8.1 PABP和N蛋白質之交互作用 13
8.2 西方墨點法 13
8.2.1 蛋白質電泳 13
8.2.2 西方墨點法 14
第九節　以生物素標定RNA探討RNA及蛋白結合之效力 14
9.1 合成生物素標定之RNA 14
9.2 Biotinylated RNA pull down assay 15
第十節 在in vitro和細胞內(in vivo)探討冠狀病毒N蛋白質對病毒及宿主細胞轉譯之影響 15
10.1 在In vitro探討N蛋白質對DI EGFP轉譯之影響 15
10.1.1 探討N是否透過和poly(A) tail結合影響DI-EGFP轉譯效率 15
10.1.2 探討N蛋白質是否透過和poly(A) tail結合影響細胞mRNA(例如β-actin mRNA)的轉譯效率 15
10.2 在細胞內(in vivo)探討N蛋白質對DI EGFP轉譯之影響 16
10.2.1 細胞轉染實驗 16
10.3 在細胞內(in vivo)探討N蛋白質對冠狀病毒mRNA轉譯之影響 16
10.3.1 細胞轉染實驗 16
10.4 在細胞內(in vivo)探討N蛋白質對宿主細胞mRNA轉譯之影響 16
10.4.1 細胞轉染實驗 16
第十一節 探討細胞膜及細胞質PABP及N蛋白質在病毒感染的相對量 16
11.1 細胞感染實驗 17
11.2 萃取細胞質及細胞膜蛋白 17
第十二節 統計分析 17
第三章 實驗結果 18
第一節 冠狀病毒N蛋白質對poly(A) tail的結合具有高度親和性 18
1.1 冠狀病毒N蛋白質對冠狀病毒poly(A) tail結合的專一性 18
1.2 冠狀病毒N蛋白質對poly(A) tail結合效率之評估 18
1.3冠狀病毒N蛋白質為一poly(A) binding protein (PABP) 19
第二節 冠狀病毒N蛋白質和PABP同時存在時會競爭和poly(A) tail結合 19
2.1 在in vitro探討冠狀病毒N蛋白質及PABP同時存在對冠狀病毒poly(A) tail的結合 19
2.2冠狀病毒N蛋白質和PABP與poly(A) tail在細胞內結合之探討 20
第三節 評估冠狀病毒N蛋白質和PABP在不同胞器中的相對量 20
3.1評估冠狀病毒N蛋白質和PABP在細胞膜及細胞質的相對量 20
第四節 冠狀病毒N蛋白質和poly(A) tail的結合對於冠狀病毒轉譯的影響 20
4.1冠狀病毒N蛋白質和poly(A) tail的結合對DI EGFP在in vitro translation的影響 20
4.2冠狀病毒N蛋白質對poly(A) tail的結合對冠狀病毒基因體在細胞中(in vivo)轉譯的影響 21
第五節 冠狀病毒N蛋白質和poly(A) tail的結合對於細胞mRNA轉譯效率的影響 21
5.1冠狀病毒N蛋白質和poly(A) tail結合對細胞蛋白β-actin RNA在in vitro中轉譯的影響 21
5.2冠狀病毒N蛋白質和poly(A) tail結合對整個細胞mRNAs轉譯的影響 22
第六節 探討冠狀病毒N蛋白質、PABP和poly(A) tail三者之間的交互作用 22
6.1 探討poly(A) tail對細胞中PABP及冠狀病毒N蛋白質的交互作用 22
6.2 以pull down assay探討PABP及冠狀病毒N蛋白質的交互作用 22
第七節 冠狀病毒N蛋白質和poly(A) tail對於轉譯及複製相關因子的交互作用 23
7.1探討poly(A) tail與細胞轉譯因子及病毒複製因子的交互作用 23
7.2 探討冠狀病毒N蛋白質與細胞轉譯因子eIF4G及病毒複製因子nsp9的交互作用 23
第八節 冠狀病毒N蛋白質和poly(A) tail結合後對於轉譯及複製相關因子結合能力的影響 24
8.1 在未感染的細胞中探討poly(A) tail和N蛋白質結合後是否對轉譯相關蛋白的交互作用造成影響 24
8.2 探討poly(A) tail在不同病毒感染時期是否對轉譯和複製相關蛋白的交互作用造成影響 24
第四章 討論 26
Table 1. Oligonucleotides used for this study 31
Fig. 1. Coronavirus N protein binds to poly(A) tail with high affinity. 32
Fig. 2. N protein competes with PABP for binding to the poly(A) tail. 34
Fig. 3. Molar ratio of N protein to PABP in subcellular fractions during infection. 35
Fig. 4. Translation inhibition of coronaviral RNA by N protein. 36
Fig. 5. Translation inhibition of cellular mRNA by N protein. 38
Fig. 6. Interactions between poly(A) tail, N protein and PABP. 40
Fig. 7. Interactions of poly(A) tail and N protein with cellular eIF4G and coronavirus nsp9. 42
Fig. 8. Interactions of the poly(A) tail and N protein with cellular eIF4E. 44
Fig. 9. Proposed model for the regulation of gene expression in coronaviruses and host cells (I). 46
Fig. 10. Proposed model for the regulation of gene expression in coronaviruses and host cells (II). 48
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