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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 陳培哲 | |
| dc.contributor.author | Hsiang-lin Lee | en |
| dc.contributor.author | 李湘琳 | zh_TW |
| dc.date.accessioned | 2021-06-15T02:22:01Z | - |
| dc.date.available | 2009-09-15 | |
| dc.date.copyright | 2009-09-15 | |
| dc.date.issued | 2009 | |
| dc.date.submitted | 2009-08-19 | |
| dc.identifier.citation | References
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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. 30. Beard, M.R., T.B. MacNaughton, and E.J. Gowans, Identification and characterization of a hepatitis delta virus RNA transcriptional promoter. J Virol, 1996. 70(8): p. 4986-95. 31. Haussecker, D., et al., Capped small RNAs and MOV10 in human hepatitis delta virus replication. Nat Struct Mol Biol, 2008. 15(7): p. 714-21. 32. Huang, W.H., Y.S. Chen, and P.J. Chen, Nucleolar targeting of hepatitis delta antigen abolishes its ability to initiate viral antigenomic RNA replication. J Virol, 2008. 82(2): p. 692-9. 33. Modahl, L.E. and M.M. Lai, Transcription of hepatitis delta antigen mRNA continues throughout hepatitis delta virus (HDV) replication: a new model of HDV RNA transcription and replication. J Virol, 1998. 72(7): p. 5449-56. 34. Rho, J., et al., Arginine methylation of Sam68 and SLM proteins negatively regulates their poly(U) RNA binding activity. Arch Biochem Biophys, 2007. 466(1): p. 49-57. 35. Osborne, T.C., et al., Protein arginine methyltransferase 1: positively charged residues in substrate peptides distal to the site of methylation are important for substrate binding and catalysis. Biochemistry, 2007. 46(46): p. 13370-81. 36. Cote, J., et al., Sam68 RNA binding protein is an in vivo substrate for protein arginine N-methyltransferase 1. Mol Biol Cell, 2003. 14(1): p. 274-87. 37. Chang, J., et al., Transcription of hepatitis delta virus RNA by RNA polymerase II. J Virol, 2008. 82(3): p. 1118-27. 38. Lehmann, E., F. Brueckner, and P. Cramer, Molecular basis of RNA-dependent RNA polymerase II activity. Nature, 2007. 450(7168): p. 445-9. 39. Liu, Y.V., et al., Role of C-terminal domain phosphorylation in RNA polymerase II transcription through the nucleosome. Biopolymers, 2003. 68(4): p. 528-38. 40. Xia, Y.P. and M.M. Lai, Oligomerization of hepatitis delta antigen is required for both the trans-activating and trans-dominant inhibitory activities of the delta antigen. J Virol, 1992. 66(11): p. 6641-8. 41. Moraleda, G., et al., Interactions between hepatitis delta virus proteins. J Virol, 2000. 74(12): p. 5509-15. 42. Bedford, M.T., Arginine methylation at a glance. J Cell Sci, 2007. 120(Pt 24): p. 4243-6. 43. Gary, J.D. and S. Clarke, RNA and protein interactions modulated by protein arginine methylation. Prog Nucleic Acid Res Mol Biol, 1998. 61: p. 65-131. 44. Fronz, K., et al., Promiscuous modification of the nuclear poly(A)-binding protein by multiple protein-arginine methyltransferases does not affect the aggregation behavior. J Biol Chem, 2008. 283(29): p. 20408-20. 45. Niu, L., et al., Redundant requirement for a pair of PROTEIN ARGININE METHYLTRANSFERASE4 homologs for the proper regulation of Arabidopsis flowering time. Plant Physiol, 2008. 148(1): p. 490-503. 46. 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. 47. Yamaguchi, Y., et al., Stimulation of RNA polymerase II elongation by hepatitis delta antigen. Science, 2001. 293(5527): p. 124-7. 48. Huang, W.H., R.T. Mai, and Y.H. Lee, Transcription factor YY1 and its associated acetyltransferases CBP and p300 interact with hepatitis delta antigens and modulate hepatitis delta virus RNA replication. J Virol, 2008. 82(15): p. 7313-24. 49. Huang, W.H., et al., The nucleolar phosphoprotein B23 interacts with hepatitis delta antigens and modulates the hepatitis delta virus RNA replication. J Biol Chem, 2001. 276(27): p. 25166-75. 50. 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. 51. Long, M., S.J. de Souza, and W. Gilbert, Delta-interacting protein A and the origin of hepatitis delta antigen. Science, 1997. 276(5313): p. 824-5. | |
| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/43463 | - |
| dc.description.abstract | D型肝炎病毒是一種缺陷病毒，需要有B型肝炎病毒的輔助才可以進行複製和感染，故D型肝炎必需同時B型肝炎病毒感染才可致病。根據此兩種病毒的相互關係，可分有同時感染和重疊感染兩種。前者常為急性肝炎，但絕大多數為良性過程。後者因先前B型肝炎引起的肝臟損害，百分之七十至九十的患者原有病情會加重，且容易進展至慢性肝炎和肝硬化。D型肝炎其基因體唯一具有的開放閱讀框架可表現兩個不同大小的D肝抗原，分別稱為大型D肝抗原和小型D肝抗原。小型D肝抗原是丁肝病毒之核糖核酸複製所必需的激活蛋白之一，目前己發現在小型D肝抗原上有很多轉譯後蛋白修飾的存在，例如絲胺酸177的磷酸化、精胺酸13的甲基化、以及賴胺酸72的乙醯化。這些修飾被認為和小型D肝抗原的幫助其病毒複製功能相關。先前研究指出，精胺酸13的點突變成丙胺酸，會造成其位點無法被甲基化，進而影響到小型D肝抗原在細胞內的表現位置和病毒複製。在本篇研究中，則利用了小型D肝抗原的突變質體在細胞內轉染表現，得到以下結果：
一) 精胺酸13的兩種突變型對於絲胺酸177的磷酸化均無明顯抑制作用。 二) 活體內甲基化試驗的結果，在該突變型沒有看到明顯甲基化被抑制之情形。因此我們無法確定小型D肝抗原精胺酸13個位點在活體內是否真的有甲基化。此外在轉染細胞實驗裡，野生型加入甲基化抑制藥後，甲基化有略為下降的情形。 三) 精胺酸13突變成丙胺酸的突變體，無法幫助病毒進行複製之原因，可能 並非因為其無法甲基化，而是其它原因。根據這點，我們利用精胺酸13突變成丙胺酸或賴胺酸的突變型，測試了幾種假設，雖然並未發現其影響複製之關鍵原因，但也得到下列發現： 甲) 精胺酸13兩種突變型蛋白，在細胞內的表現位置和野生型不同，突變型在百分之三十到四十的轉染細胞中主要分佈在細胞質。但賴胺酸突變型的複製能力未受影響，因此我們推論丙胺酸突變型無法進行病毒複製，和在細胞內表現位置以及該位點是否有甲基化均無重大關連。 乙) 和野生型比較，精胺酸13突變體的自體雙體化聚作用並未受到影響。 丙) 突變體抗原和細胞中的第二型核糖核酸聚合酶的結合力並未下降。 丁) 突變體其氮端前六十個胺基酸的蛋白二級結構無明顯改變。 根據我們的實驗結果，小型D肝抗原精胺酸13突變成丙胺酸後，病毒複製能力下降的原因，可能並不在於該突變造成的甲基化修飾缺乏，而是在於此突變影響到蛋白電荷或和其它相關蛋白質的結合等其它機制。 | zh_TW |
| dc.description.abstract | Hepatitis delta virus (HDV) is a defective satellite virus which is capable of replication in the presence of its helper virus, hepatitis B virus (HBV). The only open reading frame in its genome encodes hepatitis delta antigen (HDAg) expressing in 2 forms: the small delta antigen (S-HDAg, δAg-S) and the large delta antigen (L-HDAg, δAg-L). δAg-S is an essential factor for the initiation and maintenance of HDV RNA replication in vivo. So far some post-translational modifications (PTMs) in δAg-S such as phosphorylation, methylation, and acetylation have already been demonstrated to participate in modulating its functions and properties. A previous research showed that Arg13 of δAg-S could be methylated by protein arginine methyltransferase (PRMT1). Moreover, the R13A mutant of δAg-S could not facilitate HDV RNA replication through affecting its subcellular localization and thus the association with different components of the replication machineries. Here we use another mutant R13K as well as R13A to analyze the contribution of this amino acid residue of δAg-S in promoting HDV RNA replication. We have the following preliminary findings:
1) In spite of the opposite effects of δAg-S R13A and R13K mutants on HDV replication, the two R13 mutants both exerted little effect on S177 phosphorylation. 2) The in vivo methylation level of R13 mutants was not significantly affected. Therefore, we are not able to conclude whether R13 is methylated in vivo or not. We could see a slight but not significant decrease in wildtype-transfected cells treated with methylation inhibitor- S-AdoHcy (S-adenosyl-homocysteine). 3) Our IFA data of R13A and R13K mutants were not consistent with the previous research describing that the methylation on R13 affects its subcellular localization, and thus affects HDV RNA replication. In our results, R13K and R13A showed similar subcellular distribution pattern. However, R13K mutant could facilitate the HDV G-RNA replication, while R13A mutant could not. It revealed that the inability of δAg-S mutant R13A to promote HDV G-RNA replication may not lie in its deficiency of R13 methylation. 4) TheδAg-S R13A mutant may not affect: a) the protein secondary structure of its N-terminal amino acid 1-60 b) its binding ability to wild type δAg-S to form dimers c) its association with RNAPII According to our results, there might be some other mechanisms involved in the role of R13 on HDV RNA replication other than the methylation modification. | en |
| dc.description.provenance | Made available in DSpace on 2021-06-15T02:22:01Z (GMT). No. of bitstreams: 1 ntu-98-R94445116-1.pdf: 1462414 bytes, checksum: 5e84a9bcb072be1eae896d9433ccf97c (MD5) Previous issue date: 2009 | en |
| dc.description.tableofcontents | Abstract...…9
Chinese abstract……… .11 Introduction……… 13 Background knowledge of HDV…………………………….14 The functional domains of δAg-S………………… ...14 The HDV RNA replication…………………...14 Roles of δAg-S in HDV replication……………………….16 Post-translational modifications (PTMs) of proteins……...16 Post-translational modifications (PTMs) of δAg-S………..17 The interaction between δAg-S and RNAPII……………19 l Hypothesis and Specific Aim…… .………… ..…….20 l Materials and methods…………… ………………… ..…21 Cell culture……………………………………………...…21 Transfection………………………………………..…21 Plasmids………………………………………............22 Site-directed mutagenesis of δAg-S……………….....23 Partitioning of nuclear and cytoplasmic fractions….......23 RNA preparation……………………………………………...24 Northern blot analysis…………………………………..25 Protein extract…………………………………..25 Western blot analysis……………………………............26 Immunofluorescent stain………………………………..27 Immunoprecipitation…………………………………............28 Coimmunoprecipitation………………………………............29 Immunoaffinity purification of δAg complexes……….......30 The secondary protein structure prediction……….....….30 Results………………………………… ...……..31 The G-RNA synthesis is reduced in R13A and S177A mutants…………………….31 S177 phosphorylation level was not affected in R13 mutants…………………………….32 The δAg-S methylation level of R13A mutant was not obviously decreased in the in vivo methylation assay……………………………….…33 The subcellular localization of δAg-S of R13A mutant was still mainly in the nucleus ……………………………….…………35 The secondary structure of R13A was not dramatically changed according to the structure predication software PSIPRED PREDICTION…………………37 Immunoaffinity purification of complexes formed between Flag-δAg and δAg mutants showed no difference of dimerization degree in wild-type and mutants……………....…..38 The association between RNAPII and δAg-S was not decreased in R13 mutants……39 Discussion……………… ……………………..…………41 Tables………………………… ……………………………… .47 Figures………………………… ……………………………… 49 References…………………………………………………… ..61 | |
| dc.language.iso | en | |
| dc.title | D型肝炎病毒之小型D肝抗原位點精胺酸13對病毒複製影響之研究 | zh_TW |
| dc.title | The importance of small delta antigen Arg13 residue and its mutant effects on HDV replication | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 97-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 葉秀慧,陳美如 | |
| dc.subject.keyword | D型肝炎,小型D肝抗原,複製,精胺酸,甲基化,磷酸化, | zh_TW |
| dc.subject.keyword | HDV,hepatitis delta,small delta antigen,methylation,phosphorylation,post-translational modification,eplication, | en |
| dc.relation.page | 67 | |
| dc.rights.note | 有償授權 | |
| dc.date.accepted | 2009-08-19 | |
| dc.contributor.author-college | 醫學院 | zh_TW |
| dc.contributor.author-dept | 微生物學研究所 | zh_TW |
| 顯示於系所單位： | 微生物學科所 | |
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