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| ???org.dspace.app.webui.jsptag.ItemTag.dcfield??? | Value | Language |
|---|---|---|
| dc.contributor.advisor | 陳美如 | |
| dc.contributor.author | Yen-Tzu Liao | en |
| dc.contributor.author | 廖晏慈 | zh_TW |
| dc.date.accessioned | 2021-06-17T01:57:58Z | - |
| dc.date.available | 2022-09-08 | |
| dc.date.copyright | 2017-09-08 | |
| dc.date.issued | 2017 | |
| dc.date.submitted | 2017-07-20 | |
| dc.identifier.citation | References
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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/67917 | - |
| dc.description.abstract | 屬於γ-皰疹病毒科 (γ-herpesviridae) 的EB病毒 (Epstein-Barr virus) 在細胞核內完成病毒DNA的複製後，其複製完成的DNA會被包裹形成核殼體(nucelocapsid)，當核殼體完成組裝後，會透過出核複合體幫助病毒核殼體離開細胞核進到細胞質，以便進行最後階段的外套膜組裝。EB病毒的出核複合體由BFLF2及BFRF1所組成，分別是單純皰疹病毒 (Herpes simplex virus-1) 的UL31, UL34同源物。目前已知BFLF2單獨表現時會位在細胞質，但當其與BFRF1共同表現時，BFLF2則與BFRF1共位在核膜及細胞質，並且有泡狀產生。因此我們要探討BFLF2及BFRF1之間如何互相調節來幫助病毒核殼體出核，本篇研究著重於探討BFLF2不同的功能區段 (functional domain) 對於病毒複製的影響。先前研究顯示BFLF2胺基酸序列2-102對其進核 (nuclear localization) 及與BFRF1交互作用有重要影響，而本篇研究針對此段胺基酸進行分段序列刪除。我們發現BFLF2胺基酸序列81-107為其與BFRF1的交互作用序列，並且發現BFLF2的進核序列 (nuclear localization signal) 是由三個重要的胺基酸 (47 Arginine, 50 Lysine, 52 Arginine) 及一段較為分散、帶有正電的胺基酸序列所組成，並且藉由輸入蛋白β (importin β) 幫助進核。另外，在帶有EB病毒基因組並剔除BFLF2的細胞 (293TetER/p2089ΔBFLF2) ，發現病毒的分泌程度會因為缺少BFLF2而下降，一旦回補BFLF2便會回復病毒分泌程度。也發現當缺少BFLF2進核序列以及與BFRF1的交互作用序列，病毒的分泌也會受到減縮。綜合結果而論，本篇研究發現了BFLF2的進核序列及其與BFRF1交互作用之序列，而這兩段序列對於病毒分泌有重要的影響。 | zh_TW |
| dc.description.abstract | Epstein-Barr virus is a human gamma herpes virus, which replicates its genomic DNA in the nucleus. Replicated viral DNA is packaged into procapsids as nucleocapsids before the transport from the nucleus into the cytoplasm for subsequent maturation. This nuclear egress process is facilitated by the function of viral nuclear egress complex (NEC) which consisted of BFLF2 and BFRF1, the homologs of UL31 and UL34 in HSV-1, respectively. It was known that BFLF2 localized in the nucleus when expressed alone. However, the distribution of BFLF2 changes to both nuclear rim and cytoplasm with vesicle formation once it was co-expressed with BFRF1. We were intrigued by the different functional domains of BFLF2 in EBV replication. Our previous study indicated that BFLF2 a.a. 2-102 was required for the nuclear targeting and the interaction with BFRF1. To know exact functional motifs of BFLF2 within a.a. 2-102, systemic deletion mutants of BFLF2 were generated. We found that a.a. 81-107 of BFLF2 was responsible for the interaction with BFRF1. In addition, non-canonical nuclear localization signal (NLS) of BFLF2, which consisted of three crucial amino acids (47R, 50K, 52R) and several separated arginines were identified. Although the nuclear localization of BFLF2 is still importin β-dependent, BFLF2 NLS is different from other conserved NLSs of α- and β-herpesviruses. Furthermore, virion secretion was diminished in BFLF2 knock out 293TetER/p2089 EBV bacmid cells, but it was rescued after BFLF2 trans-complementation. In addition, the absence of identified NLS or BFRF1-interacting domain of BFLF2 leads to a decrease on virion secretion. Altogether, these results revealed the nuclear targeting and BFRF1-interacting domains of BFLF2, which are important for virion secretion. | en |
| dc.description.provenance | Made available in DSpace on 2021-06-17T01:57:58Z (GMT). No. of bitstreams: 1 ntu-106-R04445113-1.pdf: 4053685 bytes, checksum: fec61aa28fdae2d8c56ef0fee6c9f28f (MD5) Previous issue date: 2017 | en |
| dc.description.tableofcontents | 口試委員會審定書 i
誌謝 ii 中文摘要 iii Abstract iv Contents v Chapter 1: Introduction 1 1.1. Epstein-Barr Virus 1 1.1.1. Classification, Characterization and Associated diseases 1 1.1.2. Life Cycle 2 1.2. Nucleocytoplasmic Transport 2 1.2.1. Nuclear Envelope 2 1.2.2. Nuclear Pore Complex 3 1.2.3. Nuclear localization signal (NLS) 3 1.3. Nuclear Egress of Herpesvirus 5 1.3.1. Nuclear Egress Complex of Herpesviruses 5 1.3.2. Crystal Structure of Nuclear Egress Complex 6 1.3.3. Primary and Secondary Envelopment 6 1.3.4. The differences of Nuclear Egress between EBV NEC and α-Herpesviruses 7 1.4. BFLF2 and its homolog proteins 8 1.4.1. BFLF2 and its homologous proteins in Herpesviruses 8 1.4.2. The Nuclear Localization Signals of BFLF2 homologous proteins 8 1.5. The interaction of BFLF2 and BFRF1 9 1.6. Specific Aims 9 Chapter 2: Materials & Methods 10 2.1. Cell culture 10 2.2. Transfection 10 2.3. Plasmids construction 10 2.4. Immunofluorescence assay 11 2.5 Sodium dodecyle sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and western blot analysis 12 2.6 Co-Immunoprecipitation assay 12 2.7 Construction of BFLF2 knockout EBV bacmid, and selection of doxycycline inducible cells containing EBV bacmid 13 2.8 Extraction of intracellular EBV DNA 14 2.9. Isolation of secreted EBV particles and DNA extraction 14 2.10. Quantitative real-time PCR (qPCR) analysis 15 2.11. Importin β inhibitor treatment 15 2.12. Time-lapse Microscopy 16 2.13. Subcellular fractionation 16 Chapter 3: Results 19 3.1. The prediction of BFLF2 putative nuclear targeting and BFRF1-interacting domains are in its N-terminus according to the alignment with BFLF2 homologs 19 3.2. The nuclear localization signal and BFRF1-interacitng domain of BFLF2 locates in the a.a 2-102 20 3.2.1. Amino acids 28-58 of BFLF2 are important for its nuclear localization 20 3.2.2 The region of a.a 81-107 in BFLF2 is required to co-localize and interact with GFP-BFRF1 21 3.2.3. Three amino acids, 47 Arginine, 50 Lysine, and 52 Arginine, are crucial for the nuclear localization of BFLF2 21 3.2.4. The three amino acids, 47R, 50K, 52R, and a.a 58-81 of BFLF2 coordinately regulate the nuclear targeting of BFLF2 22 3.3. Nuclear translocation of BFLF2 depends on importin β 23 3.4. The dynamic subcellular localization of CFP-BFLF2 with DsRed-BFRF1 in HeLa cells 23 3.5. The dynamic subcellular localization of GFP-BFLF2 or GFP-F2d4 (Δ81-107) with DsRed-BFRF1 in HeLa cells 24 3.6. The complementation of Flag-BFLF2 in 293TetER/p2089ΔBFLF2 bacmid cells 26 3.7. The levels of virion secretion were decreased after complementation of F2d4 or F2(3A,d3) mutant 27 Chapter 4 : Discussion 28 4.1. Amino acids 28-81 and 81-107 contribute to the nuclear targeting and BFRF1-interacting domains, respectively 28 4.2. The non-classical NLS in BFLF2 is different from other homologs 29 4.3. A probable nuclear export signal of BFLF2 may locate at CR4 and regulate the translocation of NEC 31 4.4. A distinct cytoplasmic localization of BFLF2 identified by subcellular fraction 33 Chapter 5 : Figures 35 Figure 1. Functional domain mapping for nuclear targeting and BFRF1-interacting domains of BFLF2 35 Figure 2. The protein sequence alignment of BFLF2 with herpesviral homologs 36 Figure 3. 3D structural prediction of EBV nuclear egress complex according to herpesviral homologs 38 Figure 4. The nuclear localization was defective in the absence of a.a 2-102 in BFLF2 39 Figure 5. F2d2 (Δ28-58 a.a) of BFLF2 was distributed in both cytoplasm and nucleus in HeLa cells 40 Figure 6. F2d4 (Δ81-107 a.a) was defective to localize and interact with BFRF1 41 Figure 7. Three amino acids (R47, K50, R52) were crucial for the nuclear localization of Flag-BFLF2 43 Figure 8. Flag-BFLF2 NLS mutant F2(3A,d3) was defective to localize in nucleus, but it could still co-localize and interact with BFRF1 44 Figure 9. Nuclear translocation of BFLF2 is sensitive to importazole treatment 46 Figure 10. The dynamic subcellular distribution of CFP-BFLF2 and DsRed-BFRF1 47 Figure 11. The dynamic subcellular distribution of GFP-BFLF2 and DsRed-BFRF1 48 Figure 12. The dynamic subcellular distribution of GFP-F2d4 (Δ81-107 a.a ) and DsRed-BFRF1 50 Figure 13. Construction and characterization of BFLF2 knockout EBV bacmid cells 52 Figure 14. The virion secretion was defected in 293TetER/p2089ΔBFLF2 cells after doxycycline induction 53 Figure 15. The complementation with F2(3A,d3) or F2d4 mutants in 293TetER/p2089ΔBFLF2 cells reduced the virion secretion 54 Chapter 6 : Supplemental Data and Tables 55 Supplemental data 1. The region of a.a 2-102 of BFLF2 was required for the co-localization and interaction with BFRF1 55 Supplemental data 2. The distribution of GFP-BFLF2, GFP-F2d4, and F2(3A,d3) in HeLa cells 56 Supplemental data 3. The nuclear fractionation of Flag-BFLF2 in HeLa cells 57 Supplemental data 4. The second repeated complementation of Flag-BFLF2 wild type, F2d4, and F2(3A,d3) in 293TetER/p2089ΔBFLF2 bacmid cells 58 Supplemental data 5. The third repeated complementation of Flag-BFLF2 wild type, F2d4, and F2(3A,d3) in 293TetER/p2089ΔBFLF2 bacmid cells 59 Table 1. Plasmids 60 Table 2. Primers 61 References 62 | |
| dc.language.iso | en | |
| dc.title | BFLF2蛋白之進核及與BFRF1交互作用序列對於EB病毒顆粒釋出之影響 | zh_TW |
| dc.title | The nuclear targeting and BFRF1-interacting domains of BFLF2 are required for Epstein-Barr Virus secretion | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 105-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 張麗冠,劉雅雯,李重霈 | |
| dc.subject.keyword | EB病毒,出核, | zh_TW |
| dc.subject.keyword | EBV,nuclear egress, | en |
| dc.relation.page | 66 | |
| dc.identifier.doi | 10.6342/NTU201701734 | |
| dc.rights.note | 有償授權 | |
| dc.date.accepted | 2017-07-20 | |
| dc.contributor.author-college | 醫學院 | zh_TW |
| dc.contributor.author-dept | 微生物學研究所 | zh_TW |
| Appears in Collections: | 微生物學科所 | |
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| File | Size | Format | |
|---|---|---|---|
| ntu-106-1.pdf Restricted Access | 3.96 MB | Adobe PDF |
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