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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 陳培哲 | zh_TW |
| dc.contributor.advisor | Pei-Jer Chen | en |
| dc.contributor.author | 陳思瑜 | zh_TW |
| dc.contributor.author | Szu-Yu Chen | en |
| dc.date.accessioned | 2023-09-07T17:18:16Z | - |
| dc.date.available | 2023-11-09 | - |
| dc.date.copyright | 2023-09-07 | - |
| dc.date.issued | 2023 | - |
| dc.date.submitted | 2023-08-04 | - |
| dc.identifier.citation | 1. Abbas, Z. and R. Afzal, Life cycle and pathogenesis of hepatitis D virus: A review. World J Hepatol, 2013. 5(12): p. 666-75.
2. Riaz, M., et al., An overview of triple infection with hepatitis B, C and D viruses. Virol J, 2011. 8: p. 368. 3. Negro, F., Hepatitis D virus coinfection and superinfection. Cold Spring Harb Perspect Med, 2014. 4(11): p. a021550. 4. Oberhardt, V., et al., Adaptive Immune Responses, Immune Escape and Immune-Mediated Pathogenesis during HDV Infection. Viruses, 2022. 14(2). 5. Govindarajan, S., K.M. De Cock, and A.G. Redeker, Natural course of delta superinfection in chronic hepatitis B virus-infected patients: histopathologic study with multiple liver biopsies. Hepatology, 1986. 6(4): p. 640-4. 6. Farci, P. and G.A. Niro, Clinical features of hepatitis D. Semin Liver Dis, 2012. 32(3): p. 228-36. 7. Caviglia, G.P., A. Ciancio, and M. Rizzetto, A Review of HDV Infection. Viruses, 2022. 14(8). 8. Rizzetto, M., et al., Chronic hepatitis in carriers of hepatitis B surface antigen, with intrahepatic expression of the delta antigen. An active and progressive disease unresponsive to immunosuppressive treatment. Ann Intern Med, 1983. 98(4): p. 437-41. 9. Fattovich, G., et al., Influence of hepatitis delta virus infection on morbidity and mortality in compensated cirrhosis type B. The European Concerted Action on Viral Hepatitis (Eurohep). Gut, 2000. 46(3): p. 420-6. 10. Stockdale, A.J., et al., The global prevalence of hepatitis D virus infection: Systematic review and meta-analysis. J Hepatol, 2020. 73(3): p. 523-532. 11. Rizzetto, M., S. Hamid, and F. Negro, The changing context of hepatitis D. J Hepatol, 2021. 74(5): p. 1200-1211. 12. Niro, G.A., F. Rosina, and M. Rizzetto, Treatment of hepatitis D. J Viral Hepat, 2005. 12(1): p. 2-9. 13. Ye, J. and J. Chen, Interferon and Hepatitis B: Current and Future Perspectives. Front Immunol, 2021. 12: p. 733364. 14. Perrillo, R., Benefits and risks of interferon therapy for hepatitis B. Hepatology, 2009. 49(5 Suppl): p. S103-11. 15. Abbas, Z., et al., Co-treatment with pegylated interferon alfa-2a and entecavir for hepatitis D: A randomized trial. World J Hepatol, 2016. 8(14): p. 625-31. 16. Lefkowitz, E.J., et al., Virus taxonomy: the database of the International Committee on Taxonomy of Viruses (ICTV). Nucleic Acids Res, 2018. 46(D1): p. D708-d717. 17. Rizzetto, M., et al., Immunofluorescence detection of new antigen-antibody system (delta/anti-delta) associated to hepatitis B virus in liver and in serum of HBsAg carriers. Gut, 1977. 18(12): p. 997-1003. 18. Kuo, M.Y., et al., Molecular cloning of hepatitis delta virus RNA from an infected woodchuck liver: sequence, structure, and applications. J Virol, 1988. 62(6): p. 1855-61. 19. Le Gal, F., et al., Genetic diversity and worldwide distribution of the deltavirus genus: A study of 2,152 clinical strains. Hepatology, 2017. 66(6): p. 1826-1841. 20. Casey, J.L., RNA editing in hepatitis delta virus. Curr Top Microbiol Immunol, 2006. 307: p. 67-89. 21. Chao, M., S.Y. Hsieh, and J. Taylor, Role of two forms of hepatitis delta virus antigen: evidence for a mechanism of self-limiting genome replication. J Virol, 1990. 64(10): p. 5066-9. 22. Glenn, J.S., et al., Identification of a prenylation site in delta virus large antigen. Science, 1992. 256(5061): p. 1331-3. 23. Rizzetto, M., et al., delta Agent: association of delta antigen with hepatitis B surface antigen and RNA in serum of delta-infected chimpanzees. Proc Natl Acad Sci U S A, 1980. 77(10): p. 6124-8. 24. Gudima, S., et al., Assembly of hepatitis delta virus: particle characterization, including the ability to infect primary human hepatocytes. J Virol, 2007. 81(7): p. 3608-17. 25. Li, W. and S. Urban, Entry of hepatitis B and hepatitis D virus into hepatocytes: Basic insights and clinical implications. J Hepatol, 2016. 64(1 Suppl): p. S32-s40. 26. Yan, H., et al., Sodium taurocholate cotransporting polypeptide is a functional receptor for human hepatitis B and D virus. Elife, 2012. 1: p. e00049. 27. Gudima, S., et al., Origin of hepatitis delta virus mRNA. J Virol, 2000. 74(16): p. 7204-10. 28. Yamaguchi, Y., et al., Hepatitis delta antigen binds to the clamp of RNA polymerase II and affects transcriptional fidelity. Genes Cells, 2007. 12(7): p. 863-75. 29. Moraleda, G. and J. Taylor, Host RNA polymerase requirements for transcription of the human hepatitis delta virus genome. J Virol, 2001. 75(21): p. 10161-9. 30. Webb, C.H. and A. Lupták, HDV-like self-cleaving ribozymes. RNA Biol, 2011. 8(5): p. 719-27. 31. Polson, A.G., B.L. Bass, and J.L. Casey, RNA editing of hepatitis delta virus antigenome by dsRNA-adenosine deaminase. Nature, 1996. 380(6573): p. 454-6. 32. Chang, M.F., et al., Human hepatitis delta antigen is a nuclear phosphoprotein with RNA-binding activity. J Virol, 1988. 62(7): p. 2403-10. 33. Choi, S.H., K.J. Park, and S.B. Hwang, Large hepatitis delta antigen is phosphorylated at multiple sites and phosphorylation is associated with protein conformational change. Intervirology, 2002. 45(3): p. 142-9. 34. Mu, J.J., D.S. Chen, and P.J. Chen, The conserved serine 177 in the delta antigen of hepatitis delta virus is one putative phosphorylation site and is required for efficient viral RNA replication. J Virol, 2001. 75(19): p. 9087-95. 35. Mu, J.J., et al., The small delta antigen of hepatitis delta virus is an acetylated protein and acetylation of lysine 72 may influence its cellular localization and viral RNA synthesis. Virology, 2004. 319(1): p. 60-70. 36. Li, Y.J., M.R. Stallcup, and M.M. Lai, Hepatitis delta virus antigen is methylated at arginine residues, and methylation regulates subcellular localization and RNA replication. J Virol, 2004. 78(23): p. 13325-34. 37. Tseng, C.H., et al., Modification of small hepatitis delta virus antigen by SUMO protein. J Virol, 2010. 84(2): p. 918-27. 38. Tseng, C.H., K.S. Jeng, and M.M. Lai, Transcription of subgenomic mRNA of hepatitis delta virus requires a modified hepatitis delta antigen that is distinct from antigenomic RNA synthesis. J Virol, 2008. 82(19): p. 9409-16. 39. Bordier, B.B., et al., A prenylation inhibitor prevents production of infectious hepatitis delta virus particles. J Virol, 2002. 76(20): p. 10465-72. 40. Sureau, C., The role of the HBV envelope proteins in the HDV replication cycle. Curr Top Microbiol Immunol, 2006. 307: p. 113-31. 41. Branch, A.D. and H.D. Robertson, A replication cycle for viroids and other small infectious RNA's. Science, 1984. 223(4635): p. 450-5. 42. Lai, M.M., RNA replication without RNA-dependent RNA polymerase: surprises from hepatitis delta virus. J Virol, 2005. 79(13): p. 7951-8. 43. Schier, A.C. and D.J. Taatjes, Structure and mechanism of the RNA polymerase II transcription machinery. Genes Dev, 2020. 34(7-8): p. 465-488. 44. Fu, T.B. and J. Taylor, The RNAs of hepatitis delta virus are copied by RNA polymerase II in nuclear homogenates. J Virol, 1993. 67(12): p. 6965-72. 45. MacNaughton, T.B., et al., Hepatitis delta antigen is necessary for access of hepatitis delta virus RNA to the cell transcriptional machinery but is not part of the transcriptional complex. Virology, 1991. 184(1): p. 387-90. 46. Abrahem, A. and M. Pelchat, Formation of an RNA polymerase II preinitiation complex on an RNA promoter derived from the hepatitis delta virus RNA genome. Nucleic Acids Res, 2008. 36(16): p. 5201-11. 47. Greco-Stewart, V.S., et al., The human RNA polymerase II interacts with the terminal stem-loop regions of the hepatitis delta virus RNA genome. Virology, 2007. 357(1): p. 68-78. 48. Nedialkov, Y.A., et al., NTP-driven translocation by human RNA polymerase II. J Biol Chem, 2003. 278(20): p. 18303-12. 49. Yamaguchi, Y., et al., Stimulation of RNA polymerase II elongation by hepatitis delta antigen. Science, 2001. 293(5527): p. 124-7. 50. Cao, D., et al., Combined proteomic-RNAi screen for host factors involved in human hepatitis delta virus replication. Rna, 2009. 15(11): p. 1971-9. 51. Verrier, E.R., et al., Combined small molecule and loss-of-function screen uncovers estrogen receptor alpha and CAD as host factors for HDV infection and antiviral targets. Gut, 2020. 69(1): p. 158-167. 52. Paule, M.R. and R.J. White, Survey and summary: transcription by RNA polymerases I and III. Nucleic Acids Res, 2000. 28(6): p. 1283-98. 53. Turowski, T.W. and M. Boguta, Specific Features of RNA Polymerases I and III: Structure and Assembly. Front Mol Biosci, 2021. 8: p. 680090. 54. Macnaughton, T.B., et al., Rolling circle replication of hepatitis delta virus RNA is carried out by two different cellular RNA polymerases. J Virol, 2002. 76(8): p. 3920-7. 55. Modahl, L.E., et al., RNA-Dependent replication and transcription of hepatitis delta virus RNA involve distinct cellular RNA polymerases. Mol Cell Biol, 2000. 20(16): p. 6030-9. 56. Greco-Stewart, V.S., E. Schissel, and M. Pelchat, The hepatitis delta virus RNA genome interacts with the human RNA polymerases I and III. Virology, 2009. 386(1): p. 12-5. 57. Abraham, K.J., et al., Nucleolar RNA polymerase II drives ribosome biogenesis. Nature, 2020. 585(7824): p. 298-302. 58. Nesser, N.K., D.O. Peterson, and D.K. Hawley, RNA polymerase II subunit Rpb9 is important for transcriptional fidelity in vivo. Proc Natl Acad Sci U S A, 2006. 103(9): p. 3268-73. 59. Malik, S. and R.G. Roeder, Isolation and functional characterization of the TRAP/mediator complex. Methods Enzymol, 2003. 364: p. 257-84. 60. Hu, X., et al., A Mediator-responsive form of metazoan RNA polymerase II. Proc Natl Acad Sci U S A, 2006. 103(25): p. 9506-11. 61. Bernecky, C., et al., Structure of transcribing mammalian RNA polymerase II. Nature, 2016. 529(7587): p. 551-4. 62. Bichko, V.V. and J.M. Taylor, Redistribution of the delta antigens in cells replicating the genome of hepatitis delta virus. J Virol, 1996. 70(11): p. 8064-70. 63. Cimmino, C., F. Santori, and P. Donini, Ligation of nonmatching DNA molecule ends. Plasmid, 1995. 34(1): p. 1-10. 64. Wild, T. and P. Cramer, Biogenesis of multisubunit RNA polymerases. Trends Biochem Sci, 2012. 37(3): p. 99-105. 65. Tavanez, J.P., et al., Hepatitis delta virus ribonucleoproteins shuttle between the nucleus and the cytoplasm. Rna, 2002. 8(5): p. 637-46. 66. Lee, C.H., et al., The nucleolin binding activity of hepatitis delta antigen is associated with nucleolus targeting. J Biol Chem, 1998. 273(13): p. 7650-6. 67. Cunha, C., et al., Localization of hepatitis delta virus RNA in the nucleus of human cells. Rna, 1998. 4(6): p. 680-93. 68. Chou, H.C., et al., Hepatitis delta antigen mediates the nuclear import of hepatitis delta virus RNA. J Virol, 1998. 72(5): p. 3684-90. 69. Peng, Z.H., et al., Synthesis and application of a chain-terminating dinucleotide mRNA cap analog. Org Lett, 2002. 4(2): p. 161-4. 70. Kocmik, I., et al., Modified ARCA analogs providing enhanced translational properties of capped mRNAs. Cell Cycle, 2018. 17(13): p. 1624-1636. 71. Harichandran, K., et al., Hepatitis Delta Antigen Regulates mRNA and Antigenome RNA Levels during Hepatitis Delta Virus Replication. J Virol, 2019. 93(8). | - |
| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/89507 | - |
| dc.description.abstract | D型肝炎病毒是一種會導致人類D型肝炎的RNA病毒。D型肝炎病毒(HDV)和B型肝炎病毒 (HBV) 同時感染可能會引起急性肝炎或慢性病毒性肝炎，並導致肝損傷，且後續可能會發展為肝癌。根據統計，目前全球約有2.96億人患有慢性B型肝炎，其中近5%的患者合併感染HDV，並發展成更為嚴重的肝炎。在此情況下，有關HDV 分子生物學的研究對於預防和治療HDV感染具有重要的意義。
如今，關於HDV RNA在人體中的複製仍存在爭議。先前的研究分別提出了三種人類RNA聚合酶可能參與HDV生命週期中的HDV RNA複製。 在這項研究中，我們假設 RNA 聚合酶 II 主要負責進行 HDV RNA 的複製。 為了證實我們的假說，我們希望純化含有RNA 聚合酶 II (Pol II) 的HDV RNA複製複合物，並進行核連續測試 (Nuclear run on assay) 和體外轉錄 (In vitro transcription) 。我們選擇Pol II 的次單元RPB9作為純化含有 Pol II 的 HDV 複製複合物之目標，使用帶有FLAG-RPB9的質體和HeLa wild type cell進行DNA轉染 (Transfection) ，經過細胞株篩選後挑選出15支可表達FLAG-RPB9的HeLa穩定細胞株，並進行DNA轉染確認細胞株可以支持HDV RNA的複製。接著，我們使用HDV cDNA轉染後的細胞株進行核質分離 (Nucleus cytoplasm fractionation)，發現兩種不同的HDV抗原 (HDAg) 在細胞中位於不同位置。此外，我們還建構了可產生HDV mRNA、基因組或抗基因組串聯二聚體RNA的質體，進行體外轉錄生產這三種HDV RNA。我們將這三種HDV RNA分別和表達S-HDAg的質體於FLAG-RPB9 HeLa細胞中進行共轉染，以確認這些RNA的功能。我們確認了HDV基因組和抗基因組串聯二聚體RNA可以與S-HDAg進行RNA複製，並且HDV mRNA可以在FLAG-RPB9 HeLa細胞中被轉譯成S-HDAg。這些結果為後續研究中HDV複製複合物的純化提供了重要的資訊和材料。 在往後的研究將使用此研究中的HDV RNA 和HeLa-FLAG-RPB9細胞株進行RNA轉染，進行核質分離後進行核連續測試，確認分離的細胞核質是否可以進行HDV RNA 的複製，並使用蔗糖梯度(Sucrose Gradient)以及免疫沉澱(Immunoprecipitation)進一步純化含有Pol II 的HDV RNA複製複合物，最後再以體外轉錄測試是否可以進行HDV RNA 的複製。 | zh_TW |
| dc.description.abstract | Hepatitis D virus is a RNA virus which leads to the Hepatitis D in human. The co-infection of HDV and HBV causes acute hepatitis D or chronic viral hepatitis that may lead to liver damage and may develop into liver cancer. In this day, there have been about 296 million people living with chronic Hepatitis B, and nearly 5% of patients are co-infected with HDV and may develop into more severe form of hepatitis. Hence, the research of HDV molecular biology is important for prevention and treatment of HDV infection.
There is a dispute of HDV RNA replication. The three human RNA polymerases may be involved in the replication of HDV RNA in HDV life cycle. In this study, we hypothesize that RNA polymerase II (Pol II) is the major enzyme involving in HDV RNA replication. To confirm our hypothesis, we decided to purify the Pol II-containing HDV replication complex and performed the nuclear run-on assay and in vitro transcription. We selected the RPB9, a subunit of Pol II, as a purification target of Pol II-containing HDV replication complex. We first performed DNA transfection of HeLa wild type cell with the plasmid containing FLAG-RPB9 to establish a HeLa stable cell line expressing FLAG-RPB9 by subsequent single cell selection. We have confirmed three HeLa single cell clones S1, S2, and S3 capable of supporting the HDV RNA replication by subsequent transfection with pCR3.1_HDV-I(I)_221T/219C, a plasmid contains HDV genomic tandem dimer cDNA. Further, we have extracted the nuclei from HeLa single cell clones S1 and S2 both transfected by pCR3.1_HDV-I(I)_221T/219C. We have also observed that the two HDAg located in different subcellular localizations in transfected HeLa cells. Finally, we constructed the plasmid encoding the HDV mRNA, genomic, or anti-genomic tandem dimer RNA to produce the three types of RNAs by in vitro transcription. Also, we performed HDV RNA and DNA expressing S-HDAg co-transfection with FLAG-RPB9 HeLa cell to confirm the function of these RNAs. We confirm that HDV genomic and anti-genomic tandem dimer RNA could undergo the RNA replication with S-HDAg, and HDV mRNA could be translation into S-HDAg in FLAG-RPB9 HeLa cell. These results provide the important information and materials for the purification of HDV replication complex in the following research. For the future work, it is necessary to extract the nuclei from the HeLa cells transfected with HDV RNA. With the extracted nuclei, nuclear run-on assay will be performed to initially confirm if the replication of HDV RNA occurs in the nucleus. After confirming the HDV RNA is produced from the run-on assay, sucrose gradient and immunoprecipitation (IP) would be used to further purify the Pol II-containing HDV replication complex. Finally, the in vitro transcription would be performed to confirm if the pol II-containing complex supports the replication of HDV RNA. | en |
| dc.description.provenance | Submitted by admin ntu (admin@lib.ntu.edu.tw) on 2023-09-07T17:18:16Z No. of bitstreams: 0 | en |
| dc.description.provenance | Made available in DSpace on 2023-09-07T17:18:16Z (GMT). No. of bitstreams: 0 | en |
| dc.description.tableofcontents | 摘要................................................................3
Abstract ...........................................................5 List of abbreviation...............................................10 CHAPTER I. Introduction............................................12 1.1 Hepatitis D....................................................12 1.2 Hepatitis delta virus..........................................13 1.2.1 Virion genome and structure..................................13 1.2.2 Life cycle...................................................14 1.3 Dispute of Hepatitis delta virus replication...................16 1.3.1 RNA polymerase II............................................17 1.3.2 RNA polymerase I and III.....................................18 1.4 Hypothesis.....................................................19 1.5 Aim of our works...............................................20 CHAPTER II. Materials and methods..................................22 2.1 DNA and RNA constructs.........................................22 2.1.1 hRPB9-pVP5...................................................22 2.1.2 pCR3.1_HDV-I(I)_221T/219C....................................22 2.1.3 pCDAG-S......................................................23 2.1.4 pEGFP-N1.....................................................23 2.1.5 Cloning of pGEM4Z-D1T-dimer..................................23 2.1.6 Cloning of pGEM4Z-mRNA.......................................24 2.1.7 HDV RNA......................................................24 2.1.8 Nuclear cytoplasm fractionation..............................25 2.1.9 Plasmid isolation............................................26 2.2 Cell culture and transfection..................................28 2.2.1 Cell line....................................................28 2.2.2 DNA transfection.............................................29 2.2.3 RNA transfection.............................................32 2.3 Protein analysis...............................................32 2.3.1 Cell lysis...................................................32 2.3.2 BCA assay....................................................33 2.3.3 Western blot.................................................33 2.4 RNA analysis...................................................35 2.4.1 Cell lysis...................................................35 2.4.2 Northern blot................................................35 CHAPTER III. Results...............................................39 3.1 Establishment of HeLa stable cell expressing FLAG-hRPB9........39 3.2 HeLa stable cell supports the replication of HDV RNA...........40 3.3 Two HDAg locates in different subcellular localization.........43 3.4 Preparation of HDV RNA for RNA transfection....................44 CHAPTER IV. Conclusion and discussion..............................49 CHAPTER V Figures and tables.......................................56 Reference..........................................................74 | - |
| dc.language.iso | en | - |
| dc.subject | RNA聚合酶Ⅱ次單元RPB9 | zh_TW |
| dc.subject | 丁型肝炎病毒 | zh_TW |
| dc.subject | RNA聚合酶Ⅱ | zh_TW |
| dc.subject | 丁型肝炎病毒RNA複製 | zh_TW |
| dc.subject | Hepatitis D virus | en |
| dc.subject | HDV RNA replication | en |
| dc.subject | RNA polymerase II | en |
| dc.subject | RNA polymerase II subunit RPB9 | en |
| dc.title | D型肝炎病毒於表現FLAG-RPB9細胞系中的複製 | zh_TW |
| dc.title | Hepatitis D Virus Replication in FLAG-RPB9 Expressing Cell Line | en |
| dc.type | Thesis | - |
| dc.date.schoolyear | 111-2 | - |
| dc.description.degree | 碩士 | - |
| dc.contributor.oralexamcommittee | 趙玫;陳宏達 | zh_TW |
| dc.contributor.oralexamcommittee | Mei Chao;Hung-Ta Chen | en |
| dc.subject.keyword | 丁型肝炎病毒,RNA聚合酶Ⅱ,RNA聚合酶Ⅱ次單元RPB9,丁型肝炎病毒RNA複製, | zh_TW |
| dc.subject.keyword | Hepatitis D virus,HDV RNA replication,RNA polymerase II,RNA polymerase II subunit RPB9, | en |
| dc.relation.page | 78 | - |
| dc.identifier.doi | 10.6342/NTU202302848 | - |
| dc.rights.note | 同意授權(限校園內公開) | - |
| dc.date.accepted | 2023-08-04 | - |
| dc.contributor.author-college | 醫學院 | - |
| dc.contributor.author-dept | 微生物學研究所 | - |
| 顯示於系所單位： | 微生物學科所 | |
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