臺灣博碩士論文加值系統

冠狀病毒 (coronavirus；CoV)為單鏈正股RNA病毒，其基因體長度約30 kilobases(kb)，冠狀病毒基因體結構具有5’端帽與3’端poly(A) tail。在病毒的複製過程中，以正股基因體為模板合成相對應的負股基因體是冠狀病毒複製的第一步。根據文獻指出，冠狀病毒基因體3’端poly(A) tail會影響病毒正股基因體合成效率，但是冠狀病毒3’端poly(A) tail是否會降低冠狀病毒負股基因體合成量目前仍未證實。為了探討冠狀病毒基因體3’端poly(A) tail是否會影響冠狀病毒負股基因體合成，我們首先構築具有不同poly(A) tail長度的defective interfering (DI) RNA，另外構築以poly(U)、poly(C)或poly(G) tail將DI RNA poly(A) tail進行取代突變的DI RNA，並且利用qRT-PCR分析病毒負股基因體合成量。結果發現: (i) poly(A) tail的長度增加，負股基因體合成效率也會隨之上升，但是DI RNA 3’端不具有poly(A) tail時，產生負股DI RNA的量會下降。(ii)利用poly(U)、poly(C)或poly(G) tail將DI RNA poly(A) tail進行取代突變後，突變的DI RNA仍能產生與未突變DI RNA相似的負股DI RNA合成量。(iii)負股的形成的起始位置會因為poly(A) tail的長短而有所不同。此結果顯示poly(A) tail長度會影響負股的合成效率以及負股形成的起始位置。此外，除了poly(A) tail之外，與病毒3’端poly(A) tail產生交互作用的蛋白質可能在病毒合成負股基因體當中扮演主要角色。由於冠狀病毒nucleocapsid protein (以下簡稱N protein)與poly(A) binding protein(PABP)具有與病毒基因體交互作用的能力，因此本次實驗選擇N protein與PABP以探討它在負股基因體形成上的角色。由電泳凝膠遷移率實驗electrophoretic mobility shift assay (EMSA)，冠狀病毒N protein與poly(A) poly(U)，poly(C)，poly(G) tail的結合效率與負股基因體合成量成正相關，而PABP與poly(A) poly(U)，poly(C)，poly(G) tail的結合效率與負股基因體合成量彼此不一致。因此，病毒N protein與病毒基因體的結合也被推測是影響病毒負股基因體合成的重要因素之一。更進一步的實驗也發現冠狀病毒3’端poly(A) tail與N protein的結合效率也和病毒負股基因體合成效率有關。另外，目前已知位於病毒基因體5’端的序列，3’ UTR中最後55個核苷酸以及其所構成的S3與S4結構的完整性會影響病毒負股基因體的合成或複製，而我們也證明N protein與這些結構的結合能力與病毒複製效率成正相關。除了病毒基因體3’ 端的序列與N protein的結合被認為與負股形基因體成正相關外，最後，在本次實驗中也證實冠狀病毒5’端與3’端的序列會同時與N protein產生交互作用，而此交互作用可能在形成病毒複製複合體的重要因子。綜合本研究的結果可知，冠狀病毒N protein與病毒3’端 poly(A) tail會影響負股的形成。而N protein與5’端及3’端序列之交互作用在病毒的複製上也扮演重要的角色。

Coronavirus is an enveloped, positive-sense, single-stranded RNA virus, with the genome size of ~30 kilobases (kb). Coronavirus genome is 5’ capped and 3’ polyadenylated. The synthesis of negative-strand coronaviral genome is a necessary early step for genome replication. However, whether poly(A) tail is involved in the (-)-strand RNA synthesis remains unknown. For this, a series of bovine coronavirus (BCoV) defective interfering (DI) RNA mutants were constructed and tested using qRT-PCR. The major findings are as follows: (i) synthesis of (-)-strand RNA was enhanced with the increase of poly(A) tail length, (ii) the initiation site for synthesis of (-)-strand depends on the length of poly(A) tail and (iii) substitution of poly(A) tail with poly(U), (C) or (G) only had slightly effect on (-)-strand synthesis. These findings suggests that the poly(A) tail is able to affect both the efficiency and initiation site of (-)-strand synthesis and that, in addition to poly(A) tail, other factors may also be involved in the (-)-strand synthesis. Due to RNA-binding activity, BCoV nucleocapsid (N) protein and poly(A) binding protein(PABP) were selected to test its involvement in the (-)-strand synthesis. Using electrophoretic mobility shift assay, it was found that, instead of various binding affinity with poly(A) binding protein (PABP), poly(A), (U), (C) and (G) had similar binding affinity with N protein, suggesting N is more correlated to (-)-strand synthesis than PABP. Such binding was also found between N protein and 5’-terminal sequence, poly(A) tail, the 3’-terminal 55 nts and its resulting structures, which have been shown to be required for (-)-strand synthesis or replication, suggesting further the correlation of N protein with (-)-strand synthesis. Furthermore, we also demonstrated that N protein can simultaneously bind to 5’- and 3’-terminal sequence of coronavirus genome, suggesting N protein may serve a scaffold for the constitution of replication complex for the initiation of (-)-strand synthesis. In conclusion, these findings suggest that both poly(A) tail and N protein are involved in the (-)-strand RNA synthesis of coronaviruses and the circularization of coronaviral genome mediated by N protein may also play an important role in the initiation of (-)-strand RNA synthesis.

目次

摘要 i
Abstract ii
目次 iii
第一章 前言 1
第二章 文獻探討 2
第一節 冠狀病毒之基本簡介 2
1.1 冠狀病毒之分類 2
1.2冠狀病毒之基因體結構 2
第二節 cis-acting RNA elements介紹 2
2.1 cis-acting RNA elements的特性 3
2.2冠狀病毒5’UTR之cis-acting RNA elements 3
2.3冠狀病毒3’UTR之cis-acting RNA elements 4
第三節 冠狀病毒的轉譯、複製及轉錄 4
3.1 冠狀病毒的生活史 4
3.2 冠狀病毒複製相關蛋白質之轉譯 5
3.3 冠狀病毒RNA基因體的複製 5
3.4 冠狀病毒次級基因體RNA的轉錄 6
第四節 Poly(A) tail在基因表達所扮演的角色 6
4.1 poly(A) tail在真核細胞messenger RNA的形成機轉與重要性 6
4.2 病毒3’端poly(A) tail與病毒基因表達的相關性 7
第五節 以冠狀病毒的Defect interfering RNA研究cis-acting RNA elements對於病毒複製之影響 7
5.1 冠狀病毒天然缺陷株RNA (DI RNA) 7
5.2 以Defect interfering RNA作為研究材料的原因 8
5.3 以牛冠狀病毒DI RNA為系統來研究負股RNA合成 8
第三章 材料與方法 10
第一節 野外株與突變株DIRNA 質體之構築 10
1.1 牛冠狀病毒天然缺陷株DI RNA 質體之構築 10
1.2 以pBM25A 構築DI RNA突變株質體 10
第二節　選殖及增殖具突變序列之DNA片段 11
2.1 利用XL-TOPO® Vector (invitrogenTM)系統將突變之 PCR DNA 片段進行選殖 11
2.2 利用 PCR 篩選含有突變序列的菌落 12
2.3 萃取質體DNA 12
2.4 利用限制酵素切割突變 DNA 片段 12
2.5 DNA片段純化 13
2.6 將突變 DNA 片段接入 pGEM®-3Zf(-)載體 13
2.7 利用 100%酒精沉澱 DNA 連接反應之產物 13
2.8 突變 DNA 片段之轉型作用及選殖 14
2.9 利用 PCR 篩選含有突變序列的菌落 14
2.10 萃取質體DNA 14
2.11 DNA濃度測定 14
第三節 體外轉錄反應以及定量 15
3.1利用單一限制酵素 Mlu I 使環形質體形成線狀質體 15
3.2 體外轉錄反應(In vitro transcription) 15
3.3 純化RNA 15
3.4 RNA濃度測定 16
第四節 野外株與突變株BCoV DI RNA之轉染 16
4.1 細胞及病毒 16
4.2 牛冠狀病毒DI RNA之轉染 16
第五節 利用即時定量聚合酶連鎖反應偵測BCoV負股DI RNA之合成 17
5.1 自HRT-18細胞萃取RNA 17
5.2 去除 RNA 5’端磷酸根的結構 17
5.3 利用 T4 RNA LigaseⅠ將 RNA 3’端與5’端頭尾相連 18
5.4 互補DNA (cDNA) 合成 18
5.5 即時定量聚合酶連鎖反應 18
5.6反轉錄聚合酵素連鎖反應 19
5.7反轉錄聚合酶鏈鎖反應產物確認 19
第六節 BCoV 負股DIRNA序列分析 19
6.1 將反轉錄聚合酵素連鎖反應的產物進行序列分析 19
第七節 以electrophoretic mobility shift assay (EMSA)來探討蛋白質與病毒poly(A) tail之結合效率 19
7.1合成具啟動子(promoter)及突變poly(A) tail之短片段DNA 20
7.2利用體外轉錄反應(In vitro transcription)以合成具放射性RNA探針 20
7.3 純化具放射性RNA探針 20
7.4 將RNA探針分別與nucleocapsid protein或poly(A) 結合蛋白進行結合效率分析 21
7.5 以軟體分析EMSA結果 21
第八節 以EMSA探討N protein與病毒3’ 端末端序列之間結合效率 21
8.1合成具啟動子(promoter)及突變3’ 端末端序列之短片段DNA 21
8.2利用體外轉錄反應(In vitro transcription)以合成具放射性RNA探針 22
8.3 純化具放射性RNA探針 22
8.4 將RNA探針分別與N protein進行結合效率分析 23
8.5 以軟體分析EMSA結果 23
第九節 以EMSA探討N protein與病毒5’ 端末端之間結合效率 24
9.1合成具啟動子(promoter)及突變3’ 端末端序列之短片段DNA 24
9.2利用體外轉錄反應(In vitro transcription)以合成具放射性RNA探針 24
9.3 純化具放射性RNA探針 24
9.4 將RNA探針分別與N protein進行結合效率分析 25
9.5 以軟體分析EMSA結果 25
第十節 統計分析 25
第四章 實驗結果 27
第一節 利用qRT-PCR 探討poly(A) tail對負股DI RNA的形成之重要性 27
第二節 利用序列之分析確認負股基因體合成起始位置 28
第三節 利用EMSA 比較N protein或PABP蛋白與冠狀病毒poly(A) tail結合效率是否與其負股基因體合成有關 28
第四節 利用EMSA 確認N protein與病毒基因體3’端最後55個核苷酸的結合效率，以評估N protein在負股形成的重要性 31
第五節 利用EMSA 確認N protein與病毒基因體3’端S3與S4結構的結合效率，以評估N protein在複製的重要性 31
第六節 利用EMSA 確認N protein能否與病毒基因體5’ 端序列的結合效率，以評估N protein在負股形成的重要性 32
第七節 利用EMSA 探討N protein能否同時與病毒基因體5’ 端以及3’端序列結合，進而將病毒基因體環型化 32
第五章 討論 34
參考文獻 66
















表次
Table 1. Oligonucleotides used in this study. 39




































圖次
Figure 1. The RNA structures in the 5’ and 3’ terminus of BCoV genome. 41
Figure 2. Analysis of the effect of poly(A) tail on the efficiency of (-)-strand DI RNA synthesis. 43
Figure 3. The relative efficiency of (-)-strand DI RNA synthesis between DI RNA with poly(A), poly(U), (C), or (G) tail 47
Figure 4. Identification of the initiation site of (-)-strand DI RNA synthesis 46
Figure 5. Binding affinity between N protein and RNA with 3’- terminal 95 nts and poly(A), (U), (C) or (G) tail is evaluated by electrophoretic mobility shift assay (EMSA) 49
Figure 6. Binding affinity between PABP and RNA with 3’- terminal 95 nts and poly(A), (U), (C) or (G) tail is evaluated by electrophoretic mobility shift assay (EMSA) 51
Figure 7. Binding affinity between N protein and RNA with β-actin sequence or Topo sequence is evaluated by electrophoretic mobility shift assay (EMSA) 53
Figure 8. Binding affinity between N protein and RNA with 3’- terminal 95 nts or 3’- terminal 95 nts with poly(A) tail is evaluated by electrophoretic mobility shift assay (EMSA) 55
Figure 9. Binding affinity between N protein and RNA with or without 3’- terminal 55 nts is evaluated by electrophoretic mobility shift assay (EMSA) 57
Figure 10. Binding affinity between N protein and RNA with S3 or S4 structures is evaluated by electrophoretic mobility shift assay (EMSA) 59
Figure 11. Binding affinity between N protein and RNA with 5’ terminal sequence is evaluated by electrophoretic mobility shift assay (EMSA) 61
Figure 12. Simultaneous binding of N protein with 5’ and 3’ terminal sequence of coronavirus genome 63
Figure 13. Model underlying the synthesis of negative-strand RNA. 65

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