請用此 Handle URI 來引用此文件:
http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/78570
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 | Pei-Chen Lin | en |
dc.contributor.author | 林佩箴 | zh_TW |
dc.date.accessioned | 2021-07-11T15:04:39Z | - |
dc.date.available | 2022-08-28 | |
dc.date.copyright | 2019-08-28 | |
dc.date.issued | 2019 | |
dc.date.submitted | 2019-08-15 | |
dc.identifier.citation | 1. Squires, J.E. and W.F. Balistreri, Hepatitis C virus infection in children and adolescents. Hepatology communications, 2017. 1(2): p. 87-98.
2. Chen, S.L. and T.R. Morgan, The natural history of hepatitis C virus (HCV) infection. International journal of medical sciences, 2006. 3(2): p. 47. 3. Pawlotsky, J.-M., et al., From non-A, non-B hepatitis to hepatitis C virus cure. Journal of hepatology, 2015. 62(1): p. S87-S99. 4. Falade-Nwulia, O., et al., Oral direct-acting agent therapy for hepatitis C virus infection: a systematic review. Annals of internal medicine, 2017. 166(9): p. 637-648. 5. Bartlett, C., et al., Visualisation and analysis of hepatitis C virus non-structural proteins using super-resolution microscopy. Scientific reports, 2018. 8(1): p. 13604. 6. Shoukry, N.H., Hepatitis C vaccines, antibodies and T cells. Frontiers in immunology, 2018. 9: p. 1480. 7. Simmonds, P., et al., ICTV virus taxonomy profile: Flaviviridae. The Journal of general virology, 2017. 98(1): p. 2. 8. Moradpour, D., F. Penin, and C.M. Rice, Replication of hepatitis C virus. Nat Rev Microbiol, 2007. 5(6): p. 453-63. 9. Hsu, S.C., et al., Circulating sphingosine-1-phosphate as a prognostic biomarker for community-acquired pneumonia. PLoS One, 2019. 14(5): p. e0216963. 10. 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. 11. 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. 12. Bartenschlager, R., et al., Kinetic and structural analyses of hepatitis C virus polyprotein processing. Journal of virology, 1994. 68(8): p. 5045-5055. 13. 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. 14. 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. 15. Kumar, M., et al., Crystallographic and biochemical analysis of rotavirus NSP2 with nucleotides reveals a nucleoside diphosphate kinase-like activity. Journal of virology, 2007. 81(22): p. 12272-12284. 16. Lohmann, V., et al., Biochemical properties of hepatitis C virus NS5B RNA-dependent RNA polymerase and identification of amino acid sequence motifs essential for enzymatic activity. J Virol, 1997. 71(11): p. 8416-28. 17. 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. 18. Foster, T.L., et al., All three domains of the hepatitis C virus nonstructural NS5A protein contribute to RNA binding. Journal of virology, 2010. 84(18): p. 9267-9277. 19. Masaki, T., et al., Involvement of hepatitis C virus NS5A hyperphosphorylation mediated by casein kinase I-α in infectious virus production. Journal of virology, 2014. 88(13): p. 7541-7555. 20. Hsu, S.C., et al., Sequential S232/S235/S238 Phosphorylation of the Hepatitis C Virus Nonstructural Protein 5A. J Virol, 2018. 92(20). 21. Masaki, T., et al., Involvement of hepatitis C virus NS5A hyperphosphorylation mediated by casein kinase I-α in infectious virus production. Journal of virology, 2014. 88(13): p. 7541-7555. 22. 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. 23. Hsu, S.C., et al., Serine 235 Is the Primary NS5A Hyperphosphorylation Site Responsible for Hepatitis C Virus Replication. J Virol, 2017. 91(14). 24. 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. 25. 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. 26. 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. Journal of virology, 1999. 73(12): p. 9984-9991. 27. Greenberg, H.B. and M.K. Estes, Rotaviruses: from pathogenesis to vaccination. Gastroenterology, 2009. 136(6): p. 1939-1951. 28. Vende, P., Z.F. Taraporewala, and J.T. Patton, RNA-binding activity of the rotavirus phosphoprotein NSP5 includes affinity for double-stranded RNA. J Virol, 2002. 76(10): p. 5291-9. 29. 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. 30. Tai, C.L., et al., Structure-based mutational analysis of the hepatitis C virus NS3 helicase. J Virol, 2001. 75(17): p. 8289-97. 31. Tai, C.-L., et al., Structure-based mutational analysis of the hepatitis C virus NS3 helicase. Journal of virology, 2001. 75(17): p. 8289-8297. 32. Chong, W.M., et al., Phosphoproteomics identified an NS5A phosphorylation site involved in hepatitis C virus replication. Journal of Biological Chemistry, 2016. 291(8): p. 3918-3931. 33. Hsu, S.-C., et al., Serine 235 is the primary NS5A hyperphosphorylation site responsible for hepatitis C virus replication. Journal of virology, 2017. 91(14): p. e00194-17. 34. Lee, K.-Y., et al., Phosphorylation of serine 235 of the hepatitis C virus non-structural protein NS5A by multiple kinases. PLoS One, 2016. 11(11): p. e0166763. 35. Allen, J.J., et al., A semisynthetic epitope for kinase substrates. Nature methods, 2007. 4(6): p. 511. 36. Ultanir, S.K., et al., MST3 kinase phosphorylates TAO1/2 to enable Myosin Va function in promoting spine synapse development. Neuron, 2014. 84(5): p. 968-982. 37. Quintavalle, M., et al., Hepatitis C virus NS5A is a direct substrate of casein kinase I-α, a cellular kinase identified by inhibitor affinity chromatography using specific NS5A hyperphosphorylation inhibitors. Journal of Biological Chemistry, 2007. 282(8): p. 5536-5544. 38. Toyoshima, C., et al., Crystal structure of the calcium pump of sarcoplasmic reticulum at 2.6 A resolution. Nature, 2000. 405(6787): p. 647-55. 39. Afrikanova, I., et al., Rotavirus NSP5 phosphorylation is up-regulated by interaction with NSP2. Journal of General Virology, 1998. 79(11): p. 2679-2686. 40. Lai, M.C., et al., DDX3 regulates cell growth through translational control of cyclin E1. Mol Cell Biol, 2010. 30(22): p. 5444-53. 41. Peters, D., et al., The DEAD-box RNA helicase DDX41 is a novel repressor of p21(WAF1/CIP1) mRNA translation. J Biol Chem, 2017. 292(20): p. 8331-8341. 42. Diot, C., et al., Influenza A Virus Polymerase Recruits the RNA Helicase DDX19 to Promote the Nuclear Export of Viral mRNAs. Scientific Reports, 2016. 6: p. 33763. 43. Montpetit, B., et al., A conserved mechanism of DEAD-box ATPase activation by nucleoporins and InsP6 in mRNA export. Nature, 2011. 472(7342): p. 238-42. 44. Cruciat, C.M., et al., RNA helicase DDX3 is a regulatory subunit of casein kinase 1 in Wnt-beta-catenin signaling. Science, 2013. 339(6126): p. 1436-41. 45. Lauinger, L., et al., The RNA helicase FRH is an ATP-dependent regulator of CK1a in the circadian clock of Neurospora crassa. Nature Communications, 2014. 5: p. 3598. 46. zur Wiesch, J.S., et al., The proteins of the Hepatitis C virus: their features and interactions with intracellular protein phosphorylation. Archives of virology, 2003. 148(7): p. 1247-1267. 47. McGivern, D.R. and S.M. Lemon, Virus-specific mechanisms of carcinogenesis in hepatitis C virus associated liver cancer. Oncogene, 2011. 30(17): p. 1969. | |
dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/78570 | - |
dc.description.abstract | C型肝炎病毒(Hepatitis C virus)的非結構蛋白5A(non-structural protein 5A, NS5A)是一個磷酸化蛋白,它有兩個磷酸化態:低度及高度磷酸化態。兩者都對HCV生命週期至關重要。 NS5A通過高度保守的絲氨酸位點(即S225,S229,S232和S235)以連續磷酸化方式從低磷酸化狀態轉變為高磷酸化狀態。然而S225的磷酸化是由哪個蛋白質磷酸化仍未知。許多文獻指出NS5A的高度磷酸化取決於NS3(non-structural protein 3, NS3),NS3為具有蛋白酶(protease)及解旋酶(helicase)雙重功能的蛋白質。首先,必須當NS3, NS4A, NS4B及NS5A轉譯在同一條多蛋白質上才會發生NS5A的高度磷酸化。再來,當NS3自行切割後NS5A才會高度磷酸化。最後,NS5A的高度磷酸化可以發生在沒有激酶的體外試驗中。這使我們推測是否當NS3蛋白酶在切割多蛋白質中的NS5A時,會使結合三磷酸腺苷的NS3解旋酶將其水解的磷酸根轉移至NS5A。在本篇的研究探討NS3解旋酶是否可以作為激酶磷酸化NS5A。我們利用突變的方式扼殺了NS3解旋酶三磷酸腺苷結合及水解的能力,結果顯示NS5A的高度磷酸化及NS5A上S225、S229、S232、S235和S238的磷酸化程度大幅降低。另外,NS5A的高度磷酸化及這些位點的磷酸化無法用正常功能的NS3解旋酶來補救。綜合上述結果,顯示NS3解旋酶對於NS5A的高度磷酸化是必要的。接著利用激酶不敏感性的N6-芐基-三磷酸腺苷-伽馬-硫(N6-benzyl-ATP-g-S)的三磷酸腺苷類似物作為三磷酸腺苷的材料,再透過特異性抗硫代磷酸化抗體進行磷酸根的追蹤,我們檢測到 NS5A 上的硫代磷酸化為高度磷 酸化。當 NS3 上的關鍵三磷腺苷結合位點發生突變時,硫代磷酸化顯著降低。這 些結果顯示 NS3 解旋酶參與在 NS5A S225/S229/S232/S235 的磷酸化的鏈鎖反應而 造成 NS5A 的高度磷酸化。總之,我們的結果提供了關於病毒如何在其生命週期中 調節蛋白質磷酸化以及解旋酶功能的新觀點。 | zh_TW |
dc.description.abstract | The non-structural protein 5A (NS5A) of the hepatitis C virus (HCV) is a phosphoprotein with hypo- or hyper-phosphorylation states. Both are critical to the HCV life cycle. NS5A transits from hypo- to hyper-phosphorylated state via phosphorylation of a cluster of highly conserved serine sites i.e. S225, S229, S232, and S235 in a cascade manner. How the initial phosphorylation occurs on S225 remains unknown. Several lines of evidence indicate that NS5A hyper-phosphorylation could be mediated by NS3, another non-structural protein that has both protease and helicase activities. First, hyper-phosphorylation only occurs when NS5A is encoded on a polyprotein alongside with NS3 and other viral non-structural proteins (NS4A and NS4B). Second, NS5A hyper-phosphorylation requires NS3-mediated intra-molecular auto-cleavage at the NS3-NS4A junction. Third, in vitro transcription/translation assay result suggests that NS5A hyper-phosphorylation occurs in the absence of host kinases. All these evidences prompted us to test whether the ATP-binding activity of the NS3 helicase may be involved in NS5A hyper-phosphorylation. By mutating key helicase sites involved in ATP-binding and ATP-hydrolysis, we found that NS5A hyper-phosphorylation and phosphorylation at serine sites S225, S229, S232, S235, and S238 were significantly reduced. Co-transfecting the helicase-defective NS3 helicase with a wild-type NS3 did not restore NS5A hyper-phosphorylation in trans. Using a kinase-insensitive N6-benzyl-ATP-g-S analog that allowed phosphate tracking by a specific anti-thiophosphorylation antibody, we detected thiophosphorylation on NS5A that corresponded to hyper-phosphorylation. The thiophosphorylation was significantly reduced when the key ATP-binding site on NS3 was mutated. We conclude that the NS3 helicase is involved in NS5A S225/S229/S232/S235 phosphorylation cascade that leads to NS5A hyper- phosphorylation. Our results provide new perspectives on how viruses regulate protein phosphorylation during their life cycle and on the yet fully unraveled functions of helicase. | en |
dc.description.provenance | Made available in DSpace on 2021-07-11T15:04:39Z (GMT). No. of bitstreams: 1 ntu-108-R05442012-1.pdf: 6072648 bytes, checksum: 5e833c33752a166bc2c05970c096fd69 (MD5) Previous issue date: 2019 | en |
dc.description.tableofcontents | 口試委員審定書. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . i
摘要. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ii Abstract. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . iv Contents. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . vi Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .1 Materials and Methods. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 Results Mutations that disrupt NS3 ATP binding or hydrolysis reduced NS5A hyper- phosphorylation at S225, S232, S235, and S238. . . . . . . . . . . . . . . . . . . . . . . . . . 10 Mutations that disrupt NS3 ATP binding or hydrolysis reduced NS5A hyper- phosphorylation at S225, S229, and S232 in NS5A S235A mutant. . . . . . . . . . . .11 NS5A hyper-phosphorylation required NS3 ATP-binding activity and NS3 encoded on the same polyprotein . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .12 NS3 helicase transferred -thiophosphate of N6-benzyl-ATP--S to NS5A. . . . . 14 Caesin Kinase I was unable to use N6-benzyl-ATP--S. . . . . . . . . . . . . . . . . . . .16 Discussion. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18 Figures. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .25 References. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .38 | |
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 解旋酶三磷酸腺苷結合之活性 | zh_TW |
dc.title | ATP-Binding Activity of Hepatitis C Virus NS3 Helicase Is Required for NS5A Hyper-phosphorylation | en |
dc.type | Thesis | |
dc.date.schoolyear | 107-2 | |
dc.description.degree | 碩士 | |
dc.contributor.oralexamcommittee | 林敬哲,詹迺立,劉旻禕 | |
dc.subject.keyword | 非結構蛋白5A,非結構蛋白3解旋?,高度磷酸化, | zh_TW |
dc.subject.keyword | NS5A,NS3 helicase,hyper-phosphorylation, | en |
dc.relation.page | 41 | |
dc.identifier.doi | 10.6342/NTU201903419 | |
dc.rights.note | 有償授權 | |
dc.date.accepted | 2019-08-15 | |
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-108-R05442012-1.pdf 目前未授權公開取用 | 5.93 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。