臺灣博碩士論文加值系統

C型肝炎病毒帶有一單股正向的RNA基因體，可編碼出一條多蛋白質前驅物，藉由細胞蛋白酶和病毒NS2-3及NS3蛋白酶的切割，產生單一具功能性的結構性及非結構性蛋白質，其中非結構性蛋白質NS4A為NS3蛋白酶的一個共同因子。NS3蛋白質被報導過具有自身內部截切的現象。在本研究中，證明了NS3（基因型1b）在NS4A存在時會進行內部截切，而NS3的蛋白酶活性為內部截切所必需，此蛋白酶活性可藉由自身或另一蛋白酶分子提供。截切後的氨基端產物具有較全長NS3高的細胞轉型能力。IPT402|S為NS3三個內部截切中最主要的切位，而NS3(T402A)點突變的細胞轉型能力明顯下降。另外，NS4A在病毒多蛋白質切割及NS3內部截切上扮演著不同的角色。NS4A的Ile-25、Val-26、Ile-29三個胺基酸殘基對於NS3內部截切和細胞轉型能力都是重要的。然而這些位置的突變對於病毒基因體的複製效率僅有些微的增加。本研究亦針對基因型2a、4a、5a及6a的NS3及NS4A蛋白質進行NS3內部截切的分析。另一方面，NS4A蛋白質已被證實具有抑制蛋白質轉譯的能力。本實驗室先前已利用GST pull-down分析鑑定出與NS4A作用的真核細胞延長因子eEF1A。在本研究中，除了證明NS4A與eEF1A的專一性交互作用，也發現NS4A藉由中央區域21至34個胺基酸殘基與eEF1A產生交互作用。NS4A抑制細胞及病毒的蛋白質轉譯。在轉譯系統中，外加純化的重組eEF1A蛋白質，可部分回復NS4A(21-34)的轉譯抑制作用。NS4A的轉譯抑制作用可能和慢性C型肝炎病人血清中，病毒蛋白質的低表現量有關，此結果也顯示eEF1A在C型肝炎病毒慢性感染中可能參與的角色。本研究證明了NS3及NS4A蛋白質在C型肝炎病毒複製及致病機轉上扮演多重的角色，可成為理想的治療藥物標的。

The genomic RNA of hepatitis C virus (HCV) encodes the viral polyprotein precursor that undergoes proteolytic cleavage into structural and nonstructural proteins by cellular and the viral NS2-3 and NS3 proteases. NS4A is a cofactor of the NS3 protease. The NS3 protein was previously found to be internally cleaved but the mechanism is unclear. In this study, internal cleavages were demonstrated with the NS3 protein of genotype 1b in the presence of NS4A. Three potential cleavage sites were detected in the NS3 protein (genotype 1b) with IPT402|S being the major one. The internal cleavages required the polyprotein processing activity of NS3 protease that can be supplemented in trans. Mutational analysis demonstrated differential requirements of NS4A for the polyprotein processing and the internal cleavages of NS3, indicating that the mechanisms of NS4A involved in these two proteolytic cleavages are different. Furthermore, Ile-25, Val-26, and Ile-29 of the NS4A protein that were demonstrated to be involved in the interaction between NS3 and NS4A in culture cells are also important for the NS4A-dependent internal NS3 cleavages. These residues were also shown to be critical for the transforming activity of NS3, while mutations at these critical residues increased only slightly the efficiency of HCV replication. The internal cleavage-associated enhancement of the transforming activity of NS3 was significantly reduced when T402A substitution at the major internal cleavage site was introduced. Internal cleavage activity of the NS3-4A proteins of HCV genotypes 2a, 4a, 5a, and 6a were also examined. On the other hand, NS4A has been demonstrated to inhibit protein synthesis. In this study, a specific interaction between NS4A and eEF1A was demonstrated. The central domain from residues 21 to 34 of NS4A is the key player involved in the NS4A-mediated translation inhibition of host and the viral proteins. The translation inhibitory effect of NS4A(21-34) was relieved by the addition of purified recombinant eEF1A in an in vitro translation system. The study indicates a possible role of eEF1A in regulating HCV multiplication during a chronic infection. Taken together, the multiple roles of NS3 and NS4A involved in the viral multiplication and pathogenesis make them ideal molecular targets for HCV therapy.

口試委員會審定書.....i
中文摘要.....ii
Abstract.....iii
Contents.....v
List of Tables.....vii
List of Figures.....viii
Chapter 1: Introduction.....1
1.1 General introduction for HCV.....1
1.2 Possible roles of the NS3-4A protease in HCV pathogenesis.....15
Chapter 2: Specific aims.....20
Chapter 3: Materials and Methods.....22
3.1 Kits.....22
3.2 Antibodies.....22
3.3 Cell lines.....23
3.4 Plasmids.....24
3.5 Transformation.....34
3.6 Expression of GST fusion proteins.....35
3.7 Purification of GST fusion proteins.....36
3.8 GST pull-down assay.....37
3.9 In vitro transcription.....37
3.10 Translation inhibition assay.....38
3.11 Restoration of translational inhibition.....38
3.12 DNA transfection.....39
3.13 RNA extraction and reverse transcription.....39
3.14 PCR and real-time PCR.....40
3.15 Luciferase activity assay.....41
3.16 Flotation assay.....41
3.17 Immunoprecipitation.....42
3.18 Western blot analysis.....43
3.19 Soft agar assay.....44
Chapter 4: Results.....45
4.1 Effects of NS4A-dependent internal NS3 cleavage on cell
transformation and viral replication.....45
4.2 NS4A-dependent internal NS3 cleavage in various HCV genotypes.....57
4.3 Molcular mechanisms involved in translational inhibition of NS4A.....60
Chapter 5: Discussion.....69
5.1 Differential requirements of NS4A for internal NS3 cleavage and viral polyprotein processing.....69
5.2 Genotype-specific internal NS3 cleavage.....73
5.3 NS4A inhibits cap-dependent and HCV IRES-mediated translation through interacting with eEF1A.....74
Chapter 6: Tables and Figures.....79
Chapter 7: References.....114
Appendix: Curriculum Vitae.....133

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