請用此 Handle URI 來引用此文件：
http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/19304完整後設資料紀錄
| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 余明俊(Ming-Jiun Yu) | |
| dc.contributor.author | Yen-Ling Lai | en |
| dc.contributor.author | 賴彥伶 | zh_TW |
| dc.date.accessioned | 2021-06-08T01:52:53Z | - |
| dc.date.copyright | 2016-08-26 | |
| dc.date.issued | 2016 | |
| dc.date.submitted | 2016-07-20 | |
| dc.identifier.citation | 1. Messina, J.P., et al., Global distribution and prevalence of hepatitis C virus genotypes. Hepatology, 2015. 61(1): p. 77-87.
2. Chen, S.L. and T.R. Morgan, The natural history of hepatitis C virus (HCV) infection. Int J Med Sci, 2006. 3(2): p. 47-52. 3. Pybus, O.G., et al., The hepatitis C virus epidemic among injecting drug users. Infect Genet Evol, 2005. 5(2): p. 131-39. 4. Simmonds, P., The origin of hepatitis C virus. Curr Top Microbiol Immunol, 2013. 369: p. 1-15. 5. Hajarizadeh, B., J. Grebely, and G.J. Dore, Epidemiology and natural history of HCV infection. Nat Rev Gastroenterol Hepatol, 2013. 10(9): p. 553-62. 6. Gower, E., et al., Global epidemiology and genotype distribution of the hepatitis C virus infection. J Hepatol, 2014. 61(1 Suppl): p. S45-57. 7. Salam, K.A. and N. Akimitsu, Hepatitis C virus NS3 inhibitors: current and future perspectives. Biomed Res Int, 2013. 2013: p. 467869. 8. Bartenschlager, R., V. Lohmann, and F. Penin, The molecular and structural basis of advanced antiviral therapy for hepatitis C virus infection. Nat Rev Microbiol, 2013. 11(7): p. 482-96. 9. Li, H.C. and S.Y. Lo, Hepatitis C virus: Virology, diagnosis and treatment. World J Hepatol, 2015. 7(10): p. 1377-89. 10. Lindenbach, B.D. and C.M. Rice, Unravelling hepatitis C virus replication from genome to function. Nature, 2005. 436(7053): p. 933-38. 11. Scheel, T.K. and C.M. Rice, Understanding the hepatitis C virus life cycle paves the way for highly effective therapies. Nat Med, 2013. 19(7): p. 837-49. 12. Boulant, S., et al., Hepatitis C virus core protein is a dimeric alpha-helical protein exhibiting membrane protein features. J Virol, 2005. 79(17): p. 11353-65. 13. Vieyres, G., et al., Characterization of the envelope glycoproteins associated with infectious hepatitis C virus. J Virol, 2010. 84(19): p. 10159-68. 14. StGelais, C., et al., Determinants of hepatitis C virus p7 ion channel function and drug sensitivity identified in vitro. J Virol, 2009. 83(16): p. 7970-81. 15. Jirasko, V., et al., Structural and functional characterization of nonstructural protein 2 for its role in hepatitis C virus assembly. J Biol Chem, 2008. 283(42): p. 28546-62. 16. Raney, K.D., et al., Hepatitis C virus non-structural protein 3 (HCV NS3): a multifunctional antiviral target. J Biol Chem, 2010. 285(30): p. 22725-31. 17. Gu, M. and C.M. Rice, Structures of hepatitis C virus nonstructural proteins required for replicase assembly and function. Curr Opin Virol, 2013. 3(2): p. 129-36. 18. Tomei, L., et al., NS3 is a serine protease required for processing of hepatitis C virus polyprotein. J Virol, 1993. 67(7): p. 4017-26. 19. Bartenschlager, R., et al., Substrate determinants for cleavage in cis and in trans by the hepatitis C virus NS3 proteinase. J Virol, 1995. 69(1): p. 198-205. 20. Lundin, M., et al., Topology of the membrane-associated hepatitis C virus protein NS4B. J Virol, 2003. 77(9): p. 5428-38. 21. Aligo, J., et al., Formation and function of hepatitis C virus replication complexes require residues in the carboxy-terminal domain of NS4B protein. Virology, 2009. 393(1): p. 68-83. 22. Tellinghuisen, T.L., K.L. Foss, and J. Treadaway, Regulation of hepatitis C virion production via phosphorylation of the NS5A protein. PLoS Pathog, 2008. 4(3): p. e1000032. 23. Moradpour, D., F. Penin, and C.M. Rice, Replication of hepatitis C virus. Nat Rev Microbiol, 2007. 5(6): p. 453-63. 24. Choi, K.H. and M.G. Rossmann, RNA-dependent RNA polymerases from Flaviviridae. Curr Opin Struct Biol, 2009. 19(6): p. 746-51. 25. Fridell, R.A., et al., Intragenic complementation of hepatitis C virus NS5A RNA replication-defective alleles. J Virol, 2013. 87(4): p. 2320-29. 26. Miyanari, Y., et al., The lipid droplet is an important organelle for hepatitis C virus production. Nat Cell Biol, 2007. 9(9): p. 1089-97. 27. Appel, N., T. Pietschmann, and R. Bartenschlager, Mutational analysis of hepatitis C virus nonstructural protein 5A: potential role of differential phosphorylation in RNA replication and identification of a genetically flexible domain. J Virol, 2005. 79(5): p. 3187-94. 28. Tanji, Y., et al., Phosphorylation of hepatitis C virus-encoded nonstructural protein NS5A. J Virol, 1995. 69(7): p. 3980-86. 29. Lemay, K.L., et al., A hepatitis C virus NS5A phosphorylation site that regulates RNA replication. J Virol, 2013. 87(2): p. 1255-60. 30. Masaki, T., et al., Involvement of hepatitis C virus NS5A hyperphosphorylation mediated by casein kinase I-alpha in infectious virus production. J Virol, 2014. 88(13): p. 7541-55. 31. Huang, Y., et al., Phosphorylation of hepatitis C virus NS5A nonstructural protein: a new paradigm for phosphorylation-dependent viral RNA replication? Virology, 2007. 364(1): p. 1-9. 32. Chong, W.M., et al., Phosphoproteomics Identified an NS5A Phosphorylation Site Involved in Hepatitis C Virus Replication. J Biol Chem, 2016. 291(8): p. 3918-31. 33. Quintavalle, M., et al., Hepatitis C virus NS5A is a direct substrate of casein kinase I-alpha, a cellular kinase identified by inhibitor affinity chromatography using specific NS5A hyperphosphorylation inhibitors. J Biol Chem, 2007. 282(8): p. 5536-44. 34. Chen, Y.C., et al., Polo-like kinase 1 is involved in hepatitis C virus replication by hyperphosphorylating NS5A. J Virol, 2010. 84(16): p. 7983-93. 35. Koch, J.O. and R. Bartenschlager, Modulation of hepatitis C virus NS5A hyperphosphorylation by nonstructural proteins NS3, NS4A, and NS4B. J Virol, 1999. 73(9): p. 7138-46. 36. Neddermann, P., A. Clementi, and R. De Francesco, Hyperphosphorylation of the hepatitis C virus NS5A protein requires an active NS3 protease, NS4A, NS4B, and NS5A encoded on the same polyprotein. J Virol, 1999. 73(12): p. 9984-91. 37. Tai, C.L., et al., The helicase activity associated with hepatitis C virus nonstructural protein 3 (NS3). J Virol, 1996. 70(12): p. 8477-84. 38. Kim, J.L., et al., Hepatitis C virus NS3 RNA helicase domain with a bound oligonucleotide: the crystal structure provides insights into the mode of unwinding. Structure, 1998. 6(1): p. 89-100. 39. Tai, C.L., et al., Structure-based mutational analysis of the hepatitis C virus NS3 helicase. J Virol, 2001. 75(17): p. 8289-97. 40. Hou, X., et al., Internal cleavages of hepatitis C virus NS3 induced by P1 mutations at the NS3/4A cleavage site. Virology, 2009. 383(2): p. 271-78. 41. Kim, D.-j. and S. Forst, Genomic analysis of the histidine kinase family in bacteria and archaea. Microbiology, 2001. 147(5): p. 1197-1212. 42. Tanaka, T., et al., NMR structure of the histidine kinase domain of the E. coli osmosensor EnvZ. Nature, 1998. 396(6706): p. 88-92. 43. Canova, M.J. and V. Molle, Bacterial serine/threonine protein kinases in host-pathogen interactions. J Biol Chem, 2014. 289(14): p. 9473-79. 44. Tanji, Y., et al., Hepatitis C virus polyprotein processing: kinetics and mutagenic analysis of serine proteinase-dependent cleavage. J Virol, 1994. 68(12): p. 8418-22. 45. Flotow, H., et al., Phosphate groups as substrate determinants for casein kinase I action. J Biol Chem, 1990. 265(24): p. 14264-69. | |
| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/19304 | - |
| dc.description.abstract | C型肝炎病毒(Hepatitis C virus)的非結構蛋白質5A( non-structural protein 5A, NS5A)是一個磷酸化蛋白質並且有著兩種磷酸化態，包含：低度(Hypo)以及高度(Hyper)磷酸化態，其中高度磷酸化的NS5A在病毒的生活史中扮演重要的角色。先前研究已指出，NS5A的高度磷酸化會受到非結構蛋白質3( non-structural protein 3, NS3)的影響，然而NS5A上哪些位點會受到NS3的影響至今未知。我的研究目標是利用我們實驗室所擁有的專一性抗體針對S2198、S2208、S2211和S2214的磷酸化，來探討NS5A上哪些磷酸化位點會受到NS3的影響。我們利用突變的方式扼殺了NS3蛋白酶(protease)的活性，結果顯示多蛋白(polyprotein)無法被切割，而且NS5A上S2208、S2209和S2211這三個位點皆無法被磷酸化。另外，這三個位點的磷酸化以及NS3和4A間的切割無法用蛋白酶功能正常的NS3來補救，顯示NS3和4A之間的切割對於NS5A的高度磷酸化是必要的。接著，我們進一步利用點突變的方法阻擋NS3、4A、4B和5A之間的切割，發現只要NS3和4A之間無法進行正常的切割，NS5A就無法有高度磷酸化。因此我們可以推論NS5A高度磷酸化依賴同分子上NS3和4A之間的自行切割。這個結果也驅使我們研究NS3與三磷酸腺苷(ATP)的結合活性是否會影響NS5A的高度磷酸化。結果顯示，當我們利用突變去降低NS3與ATP的結合能力時，NS5A上的S2208、 S2211和S2214的磷酸化程度大幅降低，由此可以推論出NS5A高度磷酸化需要NS3的自行切割能力以及與ATP的結合活性。 | zh_TW |
| dc.description.abstract | The non-structural protein 5A (NS5A) is a hepatitis C virus (HCV) protein required for the viral life cycle. It has hypo- and hyper-phosphorylation states; the latter has been implicated in viral replication and assembly. NS5A hyper-phosphorylation is influenced by the non-structural protein 3 (NS3); however, the exact phosphorylation sites affected by NS3 are unknown. In this thesis, I used four phospho-specific antibodies to NS5A S2198, S2208, S2211 and S2214 to study how NS3 participates in NS5A hyper-phosphorylation. Mutation at the NS3 catalytic serine residue resulted in an un-cleaved NS3-5A polyprotein that was not phosphorylated at S2208, S2211 or S2214. This defect in phosphorylation could not be rescued by providing an active NS3 in trans, suggesting that NS5A phosphorylation at these sites requires cleavage of the polyprotein by NS3 in cis. Mutations that block cleavage at the NS3-4A junction, not the NS4A-4B or NS4B-5A junction, abolished S2208, S2211 and S2214 phosphorylation. These results suggest that NS5A hyper-phosphorylation depends on successful intramolecular auto-cleavage at the NS3-4A junction and prompted us to examine whether ATP-binding ability of NS3 affects NS5A hyper-phosphorylation. Indeed, mutations that reduce NS3 ATP-binding ability reduced NS5A hyper-phosphorylation at S2208, S2211 and S2214. We conclude that auto-proteolytic and ATP-binding activities of NS3 are required for NS5A hyper-phosphorylation. | en |
| dc.description.provenance | Made available in DSpace on 2021-06-08T01:52:53Z (GMT). No. of bitstreams: 1 ntu-105-R02442017-1.pdf: 2996068 bytes, checksum: 71eafd4ae60bd9d38a7e0ed28841e71f (MD5) Previous issue date: 2016 | en |
| dc.description.tableofcontents | 口試委員會審定書 i
誌謝 ii 摘要 iii Abstract iv Introduction 1 Materials & Methods 6 Results 15 NS5A hyper-phosphorylation requires NS3-5A encoded on the same protein molecule and NS3 protease activity 15 NS5A hyper-phosphorylation requires auto-cleavage at the NS3-NS4A junction by NS3 16 NS5A hyper-phosphorylation at S2208, S2211 and S2214 require NS3 activity in cis 17 NS5A hyper-phosphorylation at S2208, S2211 and S2214 requires cleavage between NS3 and 4A junction 19 NS5A hyper-phosphorylation at S2208, S2211 and S2214 correlates with the cleavage efficiency at the NS3-4A junction 21 Mutation at NS3 catalytic serine residue or cleavage junction sites affects HCV reporter viral replication 22 NS3-5A polyprotein cannot be processed correctly when expressed in the E. coli 23 NS3 ATP-binding ability correlates with NS5A hyper-phosphorylation at S2208, S2211 and S2214 24 Mutation at NS3 ATP binding, ATP hydrolysis or RNA binding domain affects HCV reporter virus activity 26 Discussion 27 Figures 32 References 45 | |
| dc.language.iso | en | |
| dc.title | C型肝炎病毒非結構蛋白質5A高度磷酸化需要非結構蛋白質3的自我切割以及與三磷酸腺苷結合之能力 | zh_TW |
| dc.title | Both Auto-Proteolytic and ATP-Binding Activities of Hepatitis C Virus Non-Structural Protein 3 are Required for Non-Structural Protein 5A Hyper-Phosphorylation | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 104-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 林敬哲(Jing-Jer Lin),張明富(Ming-Fu Chang) | |
| dc.subject.keyword | C型肝炎,非結構蛋白質3,非結構蛋白質5A,磷酸化,自我切割,與三磷酸腺?結合之能力, | zh_TW |
| dc.subject.keyword | HCV,Non-structural protein 3,Non-structural 5A,phosphorylation,auto-proteolytic,ATP-binding activitiy, | en |
| dc.relation.page | 47 | |
| dc.identifier.doi | 10.6342/NTU201601076 | |
| dc.rights.note | 未授權 | |
| dc.date.accepted | 2016-07-20 | |
| dc.contributor.author-college | 醫學院 | zh_TW |
| dc.contributor.author-dept | 生物化學暨分子生物學研究所 | zh_TW |
| 顯示於系所單位： | 生物化學暨分子生物學科研究所 | |
文件中的檔案：
| 檔案 | 大小 | 格式 | |
|---|---|---|---|
| ntu-105-1.pdf 未授權公開取用 | 2.93 MB | Adobe PDF |
系統中的文件，除了特別指名其著作權條款之外，均受到著作權保護，並且保留所有的權利。