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DC 欄位 | 值 | 語言 |
---|---|---|
dc.contributor.advisor | 陳培哲 | |
dc.contributor.author | Kai-Yi Lin | en |
dc.contributor.author | 林凱一 | zh_TW |
dc.date.accessioned | 2021-07-11T15:08:32Z | - |
dc.date.available | 2021-08-28 | |
dc.date.copyright | 2019-08-28 | |
dc.date.issued | 2019 | |
dc.date.submitted | 2019-08-13 | |
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dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/78631 | - |
dc.description.abstract | 全球約有1500-2000萬人同時感染B型肝炎病毒及D型肝炎病毒,受到B型肝炎及D型肝炎病毒感染的病人,將會有更高的機率發展成猛爆性肝炎。D型肝炎是B型肝炎病毒的衛星病毒,它需要B型肝炎的表面抗原來形成自己的外套膜。D型肝炎病毒是一負股、單股的RNA病毒,其RNA只有1.7kb大,其中大約有74%的鹼基對,D型肝炎病毒只會產生一種蛋白, delta 抗原。然而,delta抗原是有大小之分的,其主要是由D型肝炎病毒RNA藉由宿主蛋白ADAR的修飾,使得原本會製作小的delta 抗原的mRNA,改為製作大的delta 抗原。鑑於D型肝炎病毒只表現大小delta抗原兩種蛋白,而其生命週期又如此的繁雜,病毒應有利用宿主細胞內其他因子來幫助病毒完成其生命週期。N6-甲基腺苷(m6A)是mRNA中最多的修飾,由於近年來m6A去甲基酶的發現,使得m6A修飾被認為是可逆的,也因此激起了科學家研究的興趣。近年來有研究指出m6A修飾不只會影響細胞中RNA的代謝、癌症、神經的發展,也會影響如人類免疫人類免疫缺失病毒、C型肝炎病毒及流感病毒的生命週期。因此,我想知道D型肝炎病毒的RNA上是否有m6A的修飾,並且了解它在D型肝炎病毒的生命週期中所扮演的角色。首先,我使用CRISPR/Cas9基因剔除系統建立了剔除m6A的甲基酶METTL3的細胞,在D型肝炎病毒感染剔除N6-甲基腺苷的甲基酶METTL3的細胞中,D型肝炎病毒的delta 抗原及RNA皆顯著增加,表示m6A為HDV整體生命週期之負調控因子,為了了解m6A是影響病毒生命週期中的哪一個步驟,我以剔除m6A的甲基酶METTL3的細胞生產D型肝炎病毒,並感染剔除METTL3的細胞株,結果顯示,以此病毒感染後,相較於正常的病毒,原本應在剔除m6A的甲基酶METTL3的細胞中增加的S-HDAg的趨勢消失了,因此,我推測D型肝炎病毒的virion RNA上的m6A可能會影響D型肝炎病毒生活史中早期的階段。 | zh_TW |
dc.description.abstract | There are 15-20 million people worldwide chronically coinfected with Hepatitis D virus (HDV) and Hepatitis B virus (HBV). Simultaneous infection with HBV and HDV increases the risk to progress into fulminant hepatitis. HDV is a satellite virus of HBV and requires HBV surface proteins (HBs) to form viral envelopes. HDV is a negative sense single strand RNA virus, contains only 1.7kb viral genome with about 74% complementary base-pairing, and encodes only one open reading frame for delta antigen. HDV 0.8kb mRNA is translated into small delta antigen, S-HDAg and large delta antigen, L-HDAg. The production of L-HDAg is the consequence of A-to-I editing on HDV antigenomic RNA by ADAR. Since HDV only process two proteins, it must utilize host cellular regulatory machinery to fulfill its life cycle. RNA modification has been reported to influence cell function and also present on viral RNA. Among RNA modifications, N6-methyladenosine (m6A) is the most abundant RNA modification on mRNA. The responsible demethylase for m6A was not identified until recently. The discoveries of FTO and ALKBH5 as the m6A demethylases reveal that m6A is reversible and regain the researchers’ attention and interest in m6A. Recent papers show that m6A not only affect cellular function including RNA metabolism, cancer progression and neuronal development, but also some steps of virus life cycle including Human Immunodeficiency virus (HIV-1), Hepatitis C Virus (HCV) and Influenza A virus (IAV). We would like to know whether m6A is present on HDV RNA and the role of m6A in HDV life cycle. In order to understand how m6A affect HDV. I knockout the major methyltransferase of m6A, METTL3 in cell line by using CRISPR/Cas9 knockout system and infect these cells with HDV. The result shows that the HDV RNAs and HDAgs are significantly increased, indicating that m6A serves as a negative regulator to HDV. To further dissect which step of HDV life cycle is affected by m6A, m6A-depleted HDV is derived from METTL3-knocked out cells or control cells and infected into METTL3-knocked out cells. The result shows that comparing to wile type HDV infection, the increased level of S-HDAg disappears. Thus, I hypothesize that m6A may affect the initial step of HDV infection. | en |
dc.description.provenance | Made available in DSpace on 2021-07-11T15:08:32Z (GMT). No. of bitstreams: 1 ntu-108-R06445128-1.pdf: 206150835 bytes, checksum: 4e01e201012651971bd4435549f1c5b5 (MD5) Previous issue date: 2019 | en |
dc.description.tableofcontents | 中文摘要…………………………………………….............................7
ABSTRACT…………………………………………............................9 LIST OF ABBREVIATIONS…………………………………………..11 CHAPTER 1 INTRODUCTION 1.1 Clinical significance of HDV…………………………………..12 1.2 History of HDV………………………………………………...12 1.3 Virion structure of HDV………………………………………..13 1.4 HDV life cycle…………………………………………………13 1.4.1 Attachment and entry…………………………………..14 1.4.2 Transcription and translation……………………………15 1.4.3 Replication………………………………………………16 1.4.4 Assembly and release…………………………………...16 1.5 N6-methyladenosine(m6A) modification ……………………..16 1.6 Discovery of m6A ……………………………………………..18 1.7 The effect of m6A modification on virus ……………………...20 1.8 The interaction between m6A and A-to-I editing………………21 1.9 Hypothesis……………………………………………………..21 CHAPTER 2 MATERIALS AND METHONS 2.1 Plasmid construct……………………………………………….23 2.2 Cell culture and cell line………………………………………..26 2.3 In vitro transcription……………………………………………30 2.4 HDV preparation and quantification…………………………..31 2.5 HDV infection………………………………………………….33 2.6 Immunofluorescence assay…………………………………….34 2.7 Western blot……………………………………………………35 2.8 Northern blot…………………………………………………36 2.9 Sequence -based N6-methyladenosine(m6A) modification site predictor (SRAMP)…………………………………………..38 2.10 ESI-MS/MS…………………………………………………38 CHAPTER 3 RESULTS 3.1 No significant effect on HDV infection in METTL3 knockdown cells……………………………………………………………40 3.2 Different results were observed in HDV-infected HuH7-hNTCP- D8 knockout bulk cells……………………………………….40 3.3 No significant effect on HDV in HDV(I)-1 221T/219C transfected HuH7-hNTCP-D8 knockout bulk cells……………….41 3.4 The infection of HDV increased significantly in HuH-hNTCP- D8-K4 and K6 cell line…………………………………………….42 3.5 The transfection of HDV increased significantly in HuH7-hNTCP -D8-K4………………………………………………………..43 3.6 The level of m6A on HDV virion RNA affected the HDAg expression in HDV-infected HuH7-hNTCP-D8-K4………………43 3.7 Detection of m6A/A ration in HDV virion RNA……………..45 3.8 The m6A prediction of HDV genomic RNA, antigenomic RNA and mRNA sequence by SRAMP………………………………...45 CHAPTAR4 DISCUSSION…………………………………………..47 CHAPTAR 5 FIGURES Figure 1. No significant difference was observed in HDV infected METTL3 knockdown HepG2-hNTCP-C4………………………...50 Figure 2. No significant difference was observed in HDV-infected METTL3 knockdown HuH7-hNTCP-D8………………………….52 Figure 3. The HDAg expression level was significantly decreased in HDV-infected METTL3 knockout HuH7-hNTCP-D8 bulk cells…………………………………………………………………54 Figure 4. The HDV mRNA and HDAg were significantly increased in HDV-infected METTL3 knockout HuH7-hNTCP-D8 bulk cells.56 Figure 5. The HDV antigenomic RNA, mRNA and L-HDAg increased in PCR3.1-HDV-1(I) 221T/219C transfected METTL3 knockout HuH7-hNTCP-D8 bulk cells……………………………58 Figure 6. HDV RNAs and HDAgs significantly increased in PCR3.1- HDV-1(I) 221T/219C transfected METTL3 knockout HuH7-hNTCP- D8 bulk cells.……………………………………………………....60 Figure 7. The HDV RNAs and HDAg expression level were significantly increased in HDV-infected HuH7-hNTCP-D8-K4 cells.……………….……………………………………………….62 Figure 8. The HDV mRNA and HDAg were significantly increased in PCR3.1-HDV-1(I) 221T/219C transfected HuH7-hNTCP-D8-K4 cells……………………………………………………………….64 Figure 9. The HDV mRNA and S-HDAg expression level was significantly increased in HuH7-hNTCP-D8-K6 cells……………66 Figure 10. The HDV RNAs and S-HDAg expression level were significantly increased in HuH7-VC derived HDV-infected HuH7- hNTCP-D8-K4 cells...……………….……………………………..68 Figure 11. The HDV RNAs significantly increased in HuH7-KO* derived HDV-infected HuH7-hNTCP-D8-K4 cells.……….…….70 Figure 12. Confirmation of m6A depletion in METTL3 knockout cell line by ESI-MS/MS………………………………………………72 Figure 13. The m6A prediction of HDV genomic RNA, antigenomic RNA and mRNA by SRAMP………………………………………74 Figure 14. Detection of m6A/A ratio in HDV virion RNA………..76 SUPPLEMENTARY INFORMATION…………………………………79 REFERENCE…………………………………………………………...83 | |
dc.language.iso | en | |
dc.title | 探討 N6-甲基腺苷修飾對於 D 型肝炎病毒生命週期中所扮演的角色 | zh_TW |
dc.title | The Effect of N6-Methyladenosine Modification in Hepatitis Delta Virus Life Cycle | en |
dc.type | Thesis | |
dc.date.schoolyear | 107-2 | |
dc.description.degree | 碩士 | |
dc.contributor.oralexamcommittee | 葉秀惠,趙玫 | |
dc.subject.keyword | D型肝炎病毒,衛星病毒,B型肝炎病毒,N6-甲基腺?,去甲基?, | zh_TW |
dc.subject.keyword | Hepatitis Delta Virus,N6-methyladenine,FTO (fat mass and obesity-associated protein), | en |
dc.relation.page | 92 | |
dc.identifier.doi | 10.6342/NTU201901229 | |
dc.rights.note | 有償授權 | |
dc.date.accepted | 2019-08-13 | |
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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