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研究生：	林淑娟 Lin, Shu-Chuan
論文名稱：	研究C型肝炎病毒非結構蛋白2的致病機轉 Study on the Pathogenesis of Non-structural Protein 2 of Hepatitis C Virus
指導教授：	楊孔嘉 Young, Kung-Chia 張定宗 Chang, Ting-Tsung
學位類別：	碩士 Master
系所名稱：	醫學院 - 分子醫學研究所 Institute of Molecular Medicine
論文出版年：	2004
畢業學年度：	92
語文別：	英文
論文頁數：	140
中文關鍵詞：	C型肝炎病毒、非結構蛋白2
外文關鍵詞：	CD9, CD81, HCV, tetraspanin, NS2
相關次數：	點閱：108 下載：1
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　　C型肝炎病毒(hepatitis C virus, HCV)是造成輸血後肝炎的重要致病原，到目前為止，已有超過一億七千萬人感染HCV，持續感染HCV通常會發展成慢性肝炎、肝硬化、以及肝癌。但是現在對於HCV持續性感染的機制及病毒的致病機轉都還不甚清楚，為了了解何種病毒蛋白參與在HCV致病機轉中，於是我們針對HCV非結構蛋白2(non-structural protein 2, NS2)作為研究對象。NS2蛋白在HCV生活史中所扮演的角色並非十分清楚，它含有較多的厭水性氨基酸，可以藉此而與由膜所形成的結構結合。最近研究也證實，NS2蛋白參與在肝細胞的抗細胞凋亡(anti-apoptosis)過程中，以及調控細胞基因的轉錄(transcription)。因為NS2蛋白是第一個被切割而從多蛋白前趨物(polyprotein precursor)釋出的非結構蛋白，因此我們假設NS2蛋白對於宿主細胞的功能和影響可能位於較上游的位置。在本實驗室先前的研究中利用微陣列(microarray)分析結果發現：NS2蛋白會調升細胞CD9的表現量。CD9在分類上屬於四穿膜蛋白家族(tetraspanin superfamily)，研究顯示這個家族中的成員與細胞移行(cell mobility)、細胞生長(cell growth)、貼附(adherence)、癌症的生成(carcinogenesis)以及病毒感染(viral infection)相關。在本篇的研究中，主要的目標是了解NS2蛋白對於CD9及其家族中相關蛋白的調控。流式細胞儀結果顯示：不管是在肝癌細胞中或是非肝癌細胞株中，NS2蛋白都可以增加CD9以及CD81蛋白的表現量；然而，同屬於四穿膜家族蛋白的CD151其表現量卻不受NS2蛋白的影響。加強型綠螢光蛋白(enhanced green fluorescence protein, EGFP)所標定的NS2蛋白則位於被轉染細胞(transfected cells)的細胞質中。利用溶菌斑試驗(plaque assay)來分析細胞對於小兒麻痺病毒(poliovirus)的敏感性，結果顯示表現NS2蛋白之細胞感染小兒麻痺病毒後，會產生較大的溶菌斑，且溶菌斑的數目也比表現載體的對照組細胞多7倍。為了解NS2蛋白的功能性區域，將NS2蛋白切成N端前半段及C端後半段，流式細胞儀及溶菌斑試驗結果皆顯示NS2蛋白的功能性區域位於C端後半段。為研究NS2蛋白在HCV生活史中所扮演的角色，於是我們分析NS2蛋白對於HCV replicon細胞中HCV RNA複製的影響，結果證明當replicon細胞被轉染的NS2愈多時，HCV RNA的量也愈多，顯示NS2蛋白可能可以幫助HCV RNA的複製。由本篇研究結果推知，NS2蛋白會增加細胞對於病毒感染的敏感性，同時促進HCV RNA的複製，或許藉此幫助HCV在宿主細胞中感染及繁殖。

　　Hepatitis C virus (HCV) is an important pathogen of post-transfusion hepatitis. To date, more than 170 million people have been infected with HCV. Persistent HCV infection often progresses to chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma. The mechanism involved in the development of persistent infection and the pathogenesis is not fully understood. In order to determine any viral factor potentially implied in viral pathogenesis, we focus our work on non-structural (NS) protein-2, a viral protein with less-known function in HCV. HCV NS2 protein, rich in hydrophobic amino acids might associate with membranous compartment. Recently, NS2 has been demonstrated to participate in hepatic anti-apoptosis and transcriptional regulation. Since NS2 protein is first released from the HCV polyprotein precursor, we predict that the function and influence of NS2 may have an earlier onset than the other mature HCV proteins. In our previous study, the microarray screening resulted in up-regulation of CD9 expression level by NS2. CD9 protein belongs to tetraspanin superfamily, in which the members are considered to be related to cell mobility, cell growth, adherence, carcinogenesis and viral infection. In this study, we aimed to characterize the NS2-mediated modulation of CD9 and related tetraspanin family. FACS analysis showed that HCV NS2 increased CD9 and CD81 expressions in both hepatic and non-hepatic cell lines. However, the expression of another tetraspanin protein, CD151, showed no significant difference. EGFP-tagged NS2 protein was found to localize in the cytoplasm of the transfected cells. We determined the susceptibility to poliovirus infection with plaque assay, resulting in larger plaque size and more plaque number by 7 folds in NS2-expressing cells as compared to the control cells bearing vector only. In order to elucidate the functional domain of NS2, NS2 was chopped into N- and C-terminal parts. The functional motif of NS2 protein was mapped to the carboxyl-terminal half which acts more likely as full-length NS2 than does the amino-terminal fraction of NS2 in CD9 regulation and plaque assay. We also examined the influence of HCV NS2 protein on the replication of replicon HCV RNA to clarify the role of NS2 in HCV life cycle. HCV RNA was increased by NS2 expression in a dose-dependent manner, indicating that HCV NS2 protein might facilitate the replication of HCV RNA. In conclusion, this study demonstrated that NS2 protein rendered cells more susceptible to viral infection and promoted the replication of HCV RNA by which NS2 protein might facilitate the infection and propagation of HCV in host cells.

Index
  
Abstract (in Chinese)                                         I
Abstract (in English)                                       III
Acknowledgements                                              V
Index                                                        VI
Figures / Tables / Appendixes Index                          IX
Reagents and Instruments                                     XI

Chapter 1: Introduction                                       1
  1. Hepatitis C virus (HCV)                                  2
  2. Review of HCV NS2                                       18
  3. Tetraspanin superfamily                                 19
  4. Goal and strategy of this study                         22

Chapter 2: Materials and Methods                             25
  1. Analysis of CD9 and CD81 expression level in different 
     cell lines with HCV NS2 gene
  (1) Cell culture                                           27
  (2) Preparation of competent cells                         30
  (3) Purification of plasmids                               32
  (4) Transient transfection                                 34
  (5) Detection of CD9 and CD81 expression level on the 
      hepatic and non-hepatic cellular surface with FACS 
      analysis                                               36
  (6) Establishment of stably transfected cell lines with 
      HCV NS2 gene                                           37
  (7) Quantification of CD9 and CD81 RNA by real-time PCR    40
  2. Functional assay of viral infection
  (1) Plaque assay                                           45
  3. Investigation of functional elements of HCV NS2 protein
  (1) Plasmids construction containing N-terminal half and 
      C-terminal half of NS2 sequence                        47
  (2) Establishment of cell lines expressing HCV NS2 
      (827-926) and HCV NS2 (926-1026) fragments             53
  (3) Comparison of CD9 expression regulated by different 
      NS2 fragments                                          59
  (4) Plaque assay with poliovirus                           59
  (5) The subcellular distribution of truncated NS2-EGFP 
      fusion proteins                                        60
  4. Mechanism of enhanced CD81 expression regulated by HCV
     NS2 protein
  (1) Luciferase assay                                       61
  5. The effects of HCV NS2 protein on the replication of 
     replicon HCV RNA
  (1) Reverse transcription of HCV                           63
  (2) Quantitation of HCV RNA with real-time PCR             64

Chapter 3: Results                                           67
  1. Analysis of CD9 and CD81 expression level in different 
     cell lines with HCV NS2 gene
  (1) Purification of plasmids                               69
  (2) Transient transfection                                 70
  (3) Detection of CD9 and CD81 expression level on the 
      hepatic and non-hepatic cellular surface with FACS 
      analysis                                               71
  (4) Establishment of stably transfected cell lines with 
      HCV NS2 gene                                           72
  (5) Quantification of CD9 and CD81 RNA by real-time PCR    74
  2. Functional assay of viral infection
  (1) Plaque assay                                           75
  3. Investigation of functional elements of HCV NS2 protein
  (1) Plasmids construction containing N-terminal half and 
      C-terminal half of NS2 sequence                        76
  (2) Establishment of cell lines expressing HCV NS2 
      (827-926) and HCV NS2 (926-1026) fragments             77
  (3) Comparison of CD9 expression regulated by different 
      NS2 fragments                                          79
  (4) Plaque assay with poliovirus                           80
  (5) The subcellular distribution of truncated NS2-EGFP 
      fusion proteins                                        81
  4. Mechanism of enhanced CD81 expression regulated by HCV 
     NS2 protein
  (1) Luciferase assay                                       81
  5. The effects of HCV NS2 protein on the replication of 
     replicon HCV RNA
  (1) Quantitation of HCV RNA with real-time PCR             82

Chapter 4: Discussion                                        84
  1. Previous remark of HCV NS2 protein                      85
  2. Analysis of CD9 and CD81 expression level in different 
     cell lines with HCV NS2 gene                            85
  3. Functional assay of viral infection                     87
  4. Investigation of functional elements of HCV NS2 protein 88                                  
  5. Mechanism of enhanced CD81 expression regulated by HCV 
     NS2 protein                                             89
  6. The effects of HCV NS2 protein on the replication of 
     replicon HCV RNA                                        90

References                                                   92
Figures/Tables                                              101
Appendixes                                                  130
Author                                                      140   
                                    

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校內：2005-07-29公開
校外：2005-07-29公開

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