臺灣博碩士論文加值系統

C 型肝炎病毒 (hepatitis C virus, HCV) 為輸血後非 A 非 B 型肝炎的重要致病原，感染後易轉變成慢性肝炎，並與肝癌形成具有密切的關係，然而有關C型肝炎病毒的致病機轉並不清楚，臨床上也尚未找到有效的治療及預防的方式。本論文希望藉由探討與病毒複製相關的病毒基因著手，嘗試藉由對這些基因生物活性的瞭解，勾勒病毒複製的可能機轉並藉以尋求治療 C 型肝炎病毒感染的對策。
C 型肝炎病毒的基因體為一正向單股線狀的RNA分子，約 9.5 kb ，除5''端及3''端非轉譯區外，只含有一個開放編閱架構，可轉譯出一條約 3000 個胺基酸的多蛋白質先驅分子，經由宿主和病毒自身蛋白梅切割成結構性蛋白質(core, E1, E2, p7)與非結構性蛋白質(NS2, NS3, NS4A, NS4B, NS5A, NS5B)。其中 NS5B 含有與一般核糖核酸病毒複製梅 (replicase)所共有之胺基酸序列 -GlyAspAsp-， NS5A 為一磷酸化蛋白質，NS3 具有解螺旋的酵素活性功能區，推測均與病毒複製有關。為探討 HCV 非結構性蛋白質之生物特性與病毒複製間的相關性，本論文首先以反轉錄聚合連鎖反應 (RT-PCR) 得到 NS5A 與 NS5B 基因 cDNA 片段進行擇殖，並與實驗室先前已擇殖得到的 NS3 表現質體，於原核系統進行表現，再利用純化後的蛋白質在體外進行測試。
在 NS5A 與 NS5B 的研究中，以真核系統進行表現時，由間接免疫螢光染色法的結果顯示兩者皆分佈於受轉染細胞的細胞質中。由於病毒複製應具有與 RNA 結合的能力以進行病毒 RNA 複製，因此本研究採用原核系統表現的 NS5B 及其刪除突變蛋白質，經純化後，以北西方墨點分析法及 EMSA (electrophoresis mobility shift assay) 探討 NS5B 蛋白質與 C 型肝炎病毒基因體 3* 端結合的能力。結果發現 NS5B 與病毒基因體 3'' 端，包含 3'' 非轉譯區中具 genotype 特異性的區域以及病毒蛋白質轉譯區相當於 NS5B 羧基端的區域有結合能力，且其結合能力甚至強於最 3'' 端的 98-nucleotide 的保守序列。由 NS5B刪除突變的研究定出，此結合的序列位於 NS5B 胺基酸 83-194 的 RBD1 與 196-298 的 RBD2 兩個區域，其中 RBD2 包含了所有 RNA 聚合梅共有的 220-DxxxxD-225 A motif 與 282-S/TGxxxTxxxNS/T-292 B motif 保守序列。此 RNA 結合能力與其複製活性的相關性，則有待進一步的探討。至於 NS5A因不具與病毒基因體 3'' 端結合的能力，其參與病毒複製機轉的角色有待進一步的研究。
NS3 蛋白質為 RNA 解螺旋，具有結合 ATP, 水解 ATP, 解開RNA雙股螺旋及結合RNA 等四個保守序列。本研究以聚合連鎖反應將點突變引入 NS3 與解螺旋家族所共同具有的結合 ATP (1230-AX4GKS-1237) 與結合 RNA (1486-QRRGRTGR-1493) 的兩個保守序列中心，探討此二功能區在 HCV NS3 蛋白質的生化特性。結果顯示在 1230-AX4GKS-1237 功能區的突變，不但影響 NS3 蛋白質與 ATP 結合的能力，也會因此影響 NS3 蛋白質水解 ATP，與病毒 RNA 結合以及解開 RNA 雙股螺旋的能力。在 1486-QRRGRTGR-1493 功能區的突變不但影響 NS3 蛋白質與 RNA 結合的能力，也影響了 NS3 蛋白質水解 ATP 及解開 RNA 雙股螺旋的能力，但不影響其與 ATP 結合的能力。
由於 NS3, NS5A 與 NS5B 可能彼此間或與細胞因子共同形成複合體參與病毒的複製，因此本研究也藉由免疫沉殿法分析其間之交互作用，結果發現 NS3, NS5A 及 NS5B 彼此間確實可互相作用，但是否有其他細胞因子參與則需進一步的研究。

Hepatitis C virus (HCV) is the major etiology agent of posttransfusion and sporadic, community acquired non-A, non-B hepatitis. It plays a major role in the development of chronic hepatitis and hepatocellular carcinoma. The viral particle possesses a positive-sense RNA genome that contains a large open reading frame encoding a polyprotein about 3000 amino acid residues. Generation of mature proteins from the polyprotein precursor is mediated by cellular and viral proteases.
Amino acid sequence analysis revealed highly conserved function motifs within the NS5 and NS3 proteins of HCV. To elucidate biological functions of NS5 and NS3 proteins, the cDNA fragments of NS5B, NS5A and NS3 were generated by reverse transcriptase-nested polymerase chain reaction from sera of HCV patients and expressed in both prokaryotic and eukaryotic systems.
HCV NS5B protein possesses the conserved -GDD- motif and the viral RNA-dependent RNA polymerase. It is generally believed that the NS5B protein binds to the 3'' terminus of the viral genome to initiate viral replication. In this study, electrophoretic mobility shift assay was performed to analyze the RNA binding activity of the NS5B protein. The results demonstrated that HCV NS5B protein was capable of interacting with a 3'' viral genomic RNA with or without the 98-nucleotide at sequences extreme 3'' terminus. Both of the 3'' viral RNAs contain 295 nucleotides of the 3''coding region of the HCV polyprotein. The NS5B-RNA complexes were specifically competed away by the unlabeled homologous RNA, but not by the viral 5'' noncoding region and very poorly by the 3*''conserved 98-nucleotide tail. A 3'' coding region with conserved stem-loop structures rather than the 3'' noncoding region of the HCV genome is critical for the specific binding of NS5B. In addition, the binding activity was not observed with the HCV NS5A protein. Furthermore, two independent RNA-binding domains (RBDs) of NS5B were identified, RBD1 from amino acid residues 83 to 194 and RBD2 from 196 to 298. Interestingly, the conserved motifs of RNA dependent RNA polymerase for putative RNA-binding (220-DxxxxD-225) and template/primer position (282-S/TGxxxTxxxNS/T-292) are present in the RBD2.
HCV NS3 protein possesses NTPase and helicase activities. Although the enzymatic activities have been extensively studied, the ATP- and RNA-binding domains of the NS3 helicase were not well-characterized. In this study, NS3 proteins with point mutations were analyzed for their activities on ATP binding, RNA binding, ATP hydrolysis, and RNA unwinding. UV crosslinking experiments demonstrated that the lysine residue in the 1230-AX4GKS-1237 motif was directly involved in ATP binding, whereas an NS3 mutant in which the arginine-rich motif (1486-QRRGRTGR-1493) was changed to QRRDTTGR bound ATP as well as the wild type. The binding activity of HCV NS3 helicase to the viral RNA was drastically reduced with the mutations in the arginine-rich motif and was also affected by a substitution of the lysine residue in the AX4GKS motif. Previously, Arg-1490 in the arginine-rich motif of the HCV NS3 was suggested, based on the crystal structure of an NS3-deoxyuridine octamer complex, to directly interact with the g-phosphate group of ATP. Nevertheless, our functional analysis demonstrated the critical roles of Arg-1490 in binding to the viral RNA, ATP hydrolysis, and RNA unwinding, but not in ATP binding.
To further elucidate possible interactions among the nonstructural proteins of HCV to form a replicative complex, co-immunoprecipitation assay was performed. The results indicated that the HCV NS3, NS5A and NS5B proteins interacted with each other, either directly or indirectly. Although the roles of cellular factors involved in the complex formation and HCV replication remained to be analyzed, the present studies have provided the information on the molecular mechanisms of the nonstructural proteins involved in the replication of HCV.

封面
目錄
中文摘要
英文摘要
縮寫/代號
藥品來源
圖表目錄(List of Tables and Figrues)
質體構築流程圖目錄(List of Plasmid Constructions)
第一章 導論
1.1.C型肝炎病毒(HCV)的發現
1.2.疾病與可能的致病機轉
1.3.病毒的結構與分類
1.4.病毒的基因體構造與病毒蛋白質的功能
1.5.病毒的複製及與宿主細胞的關係
1.6.本論文的主旨及目的
第二章 C型肝炎病毒非結構性蛋白質NS5A與NS5B的表現與功能分析
2.1.本章概要
2.2.材料與方法
2.2.1.HCVNS5A與NS5B的製備
2.2.2.實驗室提供的質體
2.2.3.質體的構築
2.2.3.1.NS5AcNDA的選殖
2.2.3.2.NS5BcDNA的選殖
2.2.3.3.NS5A與NS5B刪除突變質體的構築
2.2.3.4.HCV3''端基因體cDNA質體的構築
2.2.4.質體DNA的製備序列分析
2.2.4.1.勝任細胞的製備
2.2.4.2.細菌轉形
2.2.4.3.小量質體的製備
2.2.4.4.大量質體的製備
2.2.4.5.DNA定序法
2.2.5.NS5A與NS5B蛋白質在原核系統的表現
2.2.5.1.TrpE融合蛋白質的表現
2.2.5.2.His-tagged 融合蛋白質的表現
2.2.5.3.NS5A與NS5B蛋白質的純化
2.2.6.蛋白質表現的相關分析系統
2.2.6.1.正十二烷硫酸鈉-聚丙烯醯胺凝膠電泳
2.2.6.2.活體外轉錄反應
2.2.6.3.活體外轉譯反應
2.2.6.4.抗體的製備
2.2.6.5.免疫沈澱法
2.2.6.6.西方墨點分析法
2.2.7.NS5A與NS5B蛋白質在真核系統的表現
2.2.7.1.細胞株及DNA轉染試驗
2.2.8.HCV NS5B蛋白質功能的分析
2.2.8.1.RNA受質的製備
2.2.8.3.HCV NS5B RNA複製？功能的分析
2.2.8.3.1.HCV NS5B RdRp 活性分析-細胞試驗
2.2.8.3.2.HCV NS5B RdRp 活性分析-體外試驗
2.2.8.4.HCV NS5B與 RNA結合能力的分析
2.2.8.4.1.北西方墨點分析(Northwestern blot analysis)
2.2.8.4.2.Electrophoretic mobility shift assay(EMSA)
2.3.結果
2.3.1.NS5A與NS5B蛋白質在原核系統的表現
2.3.2.NS5A與NS5B蛋白質在真核系統的表現
2.3.3.NS5A與NS5B蛋白質分佈於受轉染細胞的細胞質中
2.3.4.HCV NS5B具有RNA聚合？的活性
2.3.5.NS5B蛋白質專一性結合於HCV3''端基因體
2.3.6.HCV3'' CNU RNA與NS5B蛋白質結合需要保留屬於NS5B羧基諯轉譯區的基因序列
2.3.7.NS5B蛋白質具有兩處與RNA結合的區域
2.4.討論
2.4.1.NS5A與NS5B蛋白質的表現
2.4.2.NS5B RNA聚合？功能的分析
2.4.3.HCV3''端基因體與NS5B的專一性結合區
2.4.4.NS5B 蛋白質具有兩處與RNA結合的區域
2.4.5.藉由對NS5B生化特性的瞭解推測HCV可能的複製機轉
2.5.本章總結
第三章 C型肝炎病毒非結構性蛋白質NS3的表現與功能分析
3.1.本章概要
3.2.材料與方法
3.2.1.實驗室提供的質體
3.2.2.HCV NS3及其變異株蛋白質的表現與純化
3.2.2.1.NS3蛋白質的表現與純化
3.2.2.2.HCV NS3抗體的製備
3.2.2.3.西方墨點法分析
3.2.3.
3.2.3.1.ATP水解？活性的測試
3.2.3.2.蛋白質解螺旋？活性的測試
3.2.3.3.蛋白質ATP結合能力的測試
3.2.3.4.蛋白質RNA結合能力的測試
3.2.3.4.1.Filter binding結合分析
3.2.3.4.2.北西方墨點分析(Northwestern blotting)
3.3.結果
3.3.1.HCV NS3蛋白質的表現與純化
3.3.2.突變株蛋白質對ATP水解？及解螺旋？活性的影響
3.3.3.突變株蛋白質與ATP結合能力的分析
3.3.4.突變株蛋白質與RNA結合能力的分析
3.4.討論
3.4.1.1230-AX4GKS-1237-保守序列中Lys-1236對於NS3蛋白質解螺旋？的重要性
3.4.2.1486-QRRGRTGR-1493-保守列中Arg-1490對於NS3蛋白質解螺旋？的重要性
3.4.3.NS5各保守序列胺基酸間的交互作用在NS3蛋白質活性上所扮演的角色
3.5.本章總結
第三章 C型肝炎病毒非結構性蛋白質間及其與細胞蛋白間交互作用的探討
4.1.本章概述
4.2.材料與方法
4.2.1.質體
4.2.2.活體外蛋白質轉譯
4.2.3.免疫沈澱法
4.2.4.西方墨點分析
4.3.結果與討論
4.4.本章總結
第五章 結論與未來展望
圖表
質體構築流程圖
參考文獻
發表的相關論文

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