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DC 欄位 | 值 | 語言 |
---|---|---|
dc.contributor.advisor | 陳培哲(Pei-Jer Chen) | |
dc.contributor.author | Cheng-Hui Tien | en |
dc.contributor.author | 田政輝 | zh_TW |
dc.date.accessioned | 2021-07-10T21:42:04Z | - |
dc.date.available | 2021-07-10T21:42:04Z | - |
dc.date.copyright | 2020-09-10 | |
dc.date.issued | 2020 | |
dc.date.submitted | 2020-08-03 | |
dc.identifier.citation | 1. Su, T.S., et al., Hepatitis B virus transcript produced by RNA splicing. Journal of virology, 1989. 63(9): p. 4011-4018.
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Braaten, D., E.K. Franke, and J. Luban, Cyclophilin A is required for an early step in the life cycle of human immunodeficiency virus type 1 before the initiation of reverse transcription. J Virol, 1996. 70(6): p. 3551-60. | |
dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/76973 | - |
dc.description.abstract | 慢性HBV感染仍是全球性的公共衛生問題。迄今為止,全長3.2 kb的HBV病毒可經由轉錄出四個主要的未剪接型unspliced RNA以及十六種剪接型spliced RNA (SpRNA) 。而spliced RNA裡發現最多的spliced 產物為SP1,其功能對於HBV 病毒複製仍然不清楚。研究發現SP1 所編碼出的新蛋白中,C端半胱胺酸(cysteine) 缺少之core 蛋白質 (HBc-cys) 會干擾HBV蛋白質外殼 (nucleocapsid) 的形成,這與感染過程中HBV核殼分解和病毒複製有關。從而發現增加HBc-cys的表現在HBV複製過程中扮演了一定的角色。先前的研究已報導這些spliced RNA對於轉染系統中的病毒複製並不重要。在動物實驗的初步結果指出,在人肝嵌合鼠中(chimera mice) ,缺乏splicing的HBV與野生型 HBV相比顯示出感染力受損。此外,在處理 β-巰基乙醇 (mercaptoethanol) 之下,相較野生型HBV病毒顆粒中單分子型式的HBc明顯多於剪接位點487突變的病毒顆粒。這個結果可能顯示蛋白質外殼之形成會受到胱胺酸之間的雙硫建差異而影響整個外殼的完整性。 在本篇研究中,我們試圖去探討HBV spliced RNA產物SP1所編碼的HBc-cys的功能作用。利用細胞實驗去了解缺乏HBc-cys的HBV是否會影響病毒初期感染進程中進入細胞或脫去蛋白質外殼的步驟。因此我們的結果顯示與spliced-deficient HBV相比,野生型HBV PF-rcDNA透過DNA脫蛋白以及蛋白質外殼結構變化在細胞質中測到的量比較多。 | zh_TW |
dc.description.abstract | Chronic HBV infection still remains a global public health problem. Up to date, the 3.2 kb HBV transcribes four major unspliced RNA as well as 16 types of spliced RNAs (SpRNAs) which have been identified. The most abundant spliced variant Sp1, which its functions still remain obscure. Among the novel protein it encodes, HBV core minus one cysteine (HBc-Cys) has been reported to interfere with nucleocapsid formation which is associated with HBV capsid disassembly and replication during infection, raising the possibility that with the increased expression, it may play a functional role in HBV replication [1]. Previous studies have reported these spliced variants are not important for viral replication in transfection system. However, in our preliminary results from in vivo experiment indicated that splicing-deficient HBV shows impaired infectivity compared to HBV wild-type in humanized liver chimera mice. In this study, we sought to explore the functional role of the spliced RNA specifically the HBc-Cys protein encoded by the SP1 spliced RNA species. In addition, the capsid integrity of wild-type encoding the HBc-Cys is different from the A487C splicing-deficient mutant under treatment with β-mercaptoethanol. This result may suggest the cysteine-mediated disulfide bond linkages regulate the formation of the capsid integrity. Thus, the possibilities of which it may influence the steps of viral entry and uncoating process at the early phase of HBV infection in vitro. Our result thus indicates wild type HBV through capsid disassembly and DNA deproteinization, PF-rcDNA is detected in the cytoplasmic fraction with more amount compared to spliced-deficient mutant. | en |
dc.description.provenance | Made available in DSpace on 2021-07-10T21:42:04Z (GMT). No. of bitstreams: 1 U0001-3007202016032800.pdf: 2892797 bytes, checksum: 9632118be1cef0a2016e637079a37150 (MD5) Previous issue date: 2020 | en |
dc.description.tableofcontents | Acknowledgement i 摘要 ii Abstract iii Contents v 1. Introduction 1 1.1. Introduction of HBV 1 1.1.1. HBV Genome and Structure 2 1.1.2. HBV Life Cycle 2 1.1.3. HBV Viral Entry 4 1.1.4. HBV viral capsid disassembly 5 1.1.5. Formation of cccDNA 6 1.2. Alternative Splicing of HBV Transcripts 7 1.2.1. Novel Proteins encoded by HBV spliced Transcripts 8 1.3. HBV viral capsid formation 9 1.4. Hypothesis 11 2. Material and Methods 11 2.1. Cell culture and Transfection 12 2.2. Plasmids, Reagents and Antibodies 12 2.3. HBV virus preparation 14 2.4. Quantification of HBV DNA 15 2.5. In Vitro Infection 15 2.6. Dephosphorylation of HBV core protein 16 2.7. Isolation of viral DNA and Protein 17 2.8. Protein-free and Protein-associated viral DNA extract 17 2.9. Western blotting 18 2.10. Southern blotting 19 3. Results 20 3.1. Capsid Integrity were different between HBV wild-type and A487C mutant virion. 20 3.2. Capsid integrity rescued by SP1 cDNA complementation 21 3.3. Capsid integrity rescued by C182 cDNA complementation. 22 3.4. Internalization of HBc protein monomer were similar between wild-type and mutant virus 23 3.5. Phosphorylation of HBV capsid in early time point infection. 23 3.6. Internalization of HBV core protein in non-reducing state. 24 3.7. Intracellular PF-rcDNA of HBV WT was detected at early time-point infection. 24 3.8. Intracellular HBV core protein are internalized into the nucleus fraction. 25 3.9. Nucleus/Cytoplasmic fraction analysis of intracellular HBV DNA after infection. 26 3.10. Intracellular protein-associated rcDNA are majorly localized in nucleus compartment. 27 3.11. Intracellular protein-free rcDNA are majorly localized in cytoplasmic compartment… 28 3.12. Restoration of early phase infectivity by SP1 complementation. 29 3.13. Restoration of early phase infection by C182 complementation. 30 4. Discussion 31 5. Figures 38 Figure 1. Sensitivity of β-mercaptoethnol between pCMV-HBV (D) WT and A487C mutant viruses. 38 Figure 2. Complementation with SP1 cDNA into A487C mutant rescued capsid integrity. 40 Figure 3. Complementation with C182 into A487C mutant rescued capsid integrity in a dose-dependent manner. 42 Figure 4. Internalization of HBV capsid protein in early time point infection. 43 Figure 5. Analysis of intracellular HBV capsid phosphorylation in early phase infection in HepG2-NTCP-C4 cells. 44 Figure 6. Analysis of intracellular HBV protein in non-reducing SDS-PAGE in early phase infection in HepG2-NTCP-C4 cells. 45 Figure 7. pCMV-HBV (D) Wild-type virus releases PF-rcDNA in an earlier time-point in HBV infection compared to A487C mutant. 46 Figure 8. Localization of intracellular total HBc protein analysis after HBV infection. 47 Figure 9. Analysis of intracellular HBV DNA in early time point infection. 48 Figure 10. Localization of intracellular HBV protein-associated DNA after HBV infection. 51 Figure 11. Localization of intracellular HBV protein-free DNA after HBV infection. 53 Figure 12. Restoration of early phase Infectivity by SP1 complement in protein-free intracellular HBV DNA in HepG2-NTCP-C4 cells. 54 Figure 13. Restoration of early phase Infectivity by C182 complement in protein-free intracellular HBV DNA in HepG2-NTCP-C4 cells. 56 6. Reference 57 7. Appendix 61 7.1. Frequency of spliced variants produced from 3.5 kb pregenomic RNA (pgRNA). 61 7.2. Plasmid map : pCMV-HBV (D). 62 7.3. Conclusion 63 | |
dc.language.iso | en | |
dc.title | 探討B型肝炎病毒細野生型和剪接突變型病毒顆粒在細胞培養中的完整性及感染力 | zh_TW |
dc.title | Integrity and Infectivity of HBV Wild-type and Sp1-deficient Mutant Virion in Cell Culture | en |
dc.type | Thesis | |
dc.date.schoolyear | 108-2 | |
dc.description.degree | 碩士 | |
dc.contributor.oralexamcommittee | 葉秀慧(Shiou-Hwei Yeh),陶秘華(Mi-Hua Tao) | |
dc.subject.keyword | B型肝炎病毒,剪接型RNA,C端半胱胺酸,cysteine 缺少之core 蛋白質, | zh_TW |
dc.subject.keyword | hepatitis B virus,HBV,spliced RNA,spRNA,HBV core minus cysteine protein,HBc-Cys, | en |
dc.relation.page | 63 | |
dc.identifier.doi | 10.6342/NTU202002111 | |
dc.rights.note | 未授權 | |
dc.date.accepted | 2020-08-04 | |
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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