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完整後設資料紀錄
DC 欄位 | 值 | 語言 |
---|---|---|
dc.contributor.advisor | 陳美如 | zh_TW |
dc.contributor.advisor | en | |
dc.contributor.author | 阮憶婷 | zh_TW |
dc.contributor.author | Yi-Ting Juan | en |
dc.date.accessioned | 2021-07-11T15:42:30Z | - |
dc.date.available | 2024-02-28 | - |
dc.date.copyright | 2018-10-11 | - |
dc.date.issued | 2018 | - |
dc.date.submitted | 2002-01-01 | - |
dc.identifier.citation | Adell, A.Y., S.M. Migliano, and D. Teis. 2016. ESCRT‐III and Vps4: a dynamic multipurpose tool for membrane budding and scission. The FEBS journal. 283:3288-3302.
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dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/79082 | - |
dc.description.abstract | 當EB病毒進入溶裂期 (lytic replication) 後會開始在核內進行DNA複製,複製完成後的DNA會被包裹形成核殼體(nucelocapsid),核殼體會透過出核複合體(nuclear egress complex, NEC)的幫助離開細胞核進到細胞質。EB病毒的出核複合體是由BFRF1及BFLF2兩個病毒蛋白所組成,已知BFRF1會透過和細胞中的Alix進行交互作用進而吸引ESCRT (endosomal sorting complex required for transport)相關分子進行核膜剪切,幫助BFRF1產生核膜衍生液泡(nuclear envelope-derived vesicle)以加速病毒核殼體出核。本篇研究著重在探討病毒釋出過程中,BFRF1是透過哪些功能區域 (functional domains) 和Alix以及BFLF2進行交互作用,並透過初步篩檢找出哪些ESCRT-III分子可能參與在釋出過程中。之前實驗室研究發現BFRF1的Late domain 1 (LD1) 區域可以和Alix的Bro區域產生交互作用。本研究進一步發現BFRF1的EBV specific region (ESR) 區域可以和Alix的PRR區域產生交互作用,若將ESR區域上的正電荷進行點突變或是藉由核酸酶處理皆會影響兩者之間的交互作用,推測ESR和PRR之間的交互作用需要透過核酸的參與。另外在帶有EB病毒B95-8 strain bacmid (p2089)且剔除BFRF1的細胞 (293TetEZ/p2089BFRF1) 中,透過qPCR偵測發現病毒釋出可能會因為缺少這些和Alix進行交互作用的區域而下降。此外也發現BFRF1上的多段功能區域皆能和BFLF2產生交互作用,但刪除掉任何一段皆會影響BFRF1將BFLF2帶出核到核膜並形成共位的現象,顯示BFRF1可能需要依賴多段功能區域來執行其出核複合體的功能。而在BFLF2上觀察到胺基酸序列81-107為和BFRF1進行交互作用及共位的主要胺基酸序列。最後也篩選出可能參與在病毒釋出過程中的ESCRT-III分子,像是CHMP7以及CHMP2A。本研究除了闡明BFRF1的功能區域如何和Alix及BFLF2進行交互作用以利病毒核殼體出核,也篩選到在釋出過程中可能參與的ESCRT-III分子,以供未來之研究。 | zh_TW |
dc.description.abstract | During lytic replication in the nucleus, Epstein-Barr virus genome is replicated and packaged into capsids. Subsequently, the assembled nucleocapsids egress from the nucleus through a nuclear envelope-derived vesicle dependent manner, which is mediated by the nuclear egress complex (NEC) composed of viral BFRF1 and BFLF2 proteins. Previously, we demonstrated that cellular endosomal sorting complex required for transport (ESCRT) machinery is involved in the nuclear egress through the interaction between BFRF1 and the ESCRT-adaptor protein Alix. In this study, it is aimed to dissect the functional domains in BFRF1 required for the recruitment of Alix and BFLF2 and to identify the ESCRT-III components participated in the process. Previous study revealed that the Late Domain 1 (LD1) of BFRF1 interacts with Bro domain of Alix. Here, we further showed that EBV-specific region (ESR) interacts with PRR domain of Alix and the interaction is nucleic acid-dependent. Remarkably, virion secretion is diminished in BFRF1 knock-out 293TetEZ/p2089 EBV bacmid inducible cells after complemented with interacting domain-defective mutants. In addition, in the part of dissecting BFLF2-interacting domains, multiple regions of BFRF1 interact with BFLF2. All BFRF1 mutants with deletion of individual domain fail to recruit BFLF2 out of the nucleus, indicating that multiple regions of BFRF1 are required for its function. By contrast, a.a. 81-107 of BFLF2 is the major region responsible for the interaction with BFRF1. On the other hand, screening with various ESCRT-III dominant negative clones indicates that CHMP2A and CHMP7 may be required in the maturation process. Altogether, we characterize the functional domains of BFRF1 involved in the recruitment of Alix and BFLF2. The candidates of ESCRT-III components participated in the maturation process will be further accessed. | en |
dc.description.provenance | Made available in DSpace on 2021-07-11T15:42:30Z (GMT). No. of bitstreams: 1 ntu-107-R05445110-1.pdf: 4002998 bytes, checksum: ab6a304619402a685b6a0a8d7233801b (MD5) Previous issue date: 2018 | en |
dc.description.tableofcontents | 口試委員會審定書…………………………………………………………………….i
致謝……………………………………………………………………………………ii 中文摘要……………………………………………………………………………...iii Abstract ……………………………………………………………………………….iv Contents ……………………………………………………………………………... v 1. INTRODUCTION 1 1.1. Epstein-Barr Virus 1 1.1.1. Classification, characterization and associated diseases 1 1.1.2. Life cycle 1 1.2. Nuclear egress of herpesvirus 2 1.2.1. Nuclear egress of herpesviruses 2 1.2.2. Nuclear egress complex of herpesviruses 3 1.2.3. Crystal structure of nuclear egress complex 3 1.2.4. EBV nuclear membrane-associated protein BFRF1 and its interaction with the BFLF2 4 1.2.5. The differences of nuclear egress between EBV and α-Herpesviruses 5 1.3. The ESCRT machinery 5 1.3.1. The compositions in ESCRT machinery 5 1.3.2. The functions of ESCRTs in cellular processes 6 1.3.3. ESCRTs in viral budding 7 1.3.4. The relationship between ESCRTs and herpesvirus 7 1.3.5. ESCRRT-associated protein Alix and its relationship with BFRF1 8 1.4. Specific aim of this study 9 2. MATERIAL & METHODS 10 2.1. Cell culture 10 2.2. Transfection 10 2.3. Plasmids construction 11 2.4. Sodium dodecyle sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and western blot analysis 12 2.5. GST pull down assay 13 2.6. Co-Immunoprecipitation assay 14 2.7. Indirect immunofluorescence assay 14 2.8. Selection of doxycycline-inducible EBV bacmid containing cells 15 2.9. Extraction of intracellular EBV DNA 15 2.10. Isolation of secreted EBV particles and DNA extraction 16 2.11. Quantitative real-time PCR (qPCR) analysis 16 2.12. His tag protein purification 17 2.13. Purification of rabbit anti-BFRF1 antibody in serum 18 3. RESULTS 19 3.1. Mapping of Alix interacting domains on EBV BFRF1 19 3.1.1. The positive-charged amino acids of the ESR domain in BFRF1 are important for the interaction with the PRR domain of Alix 19 3.1.2. The ESR domain of BFRF1 interacts with the PRR domain of Alix in a nucleic acid dependent manner 20 3.1.3. Deletion or mutation of domains required for the interaction with Alix affects the EBV virion secretion 21 3.1.4. Establishment of the fresh TetEZp2089 WT and BFRF1-KO stable cells and detection of the lytic protein expression in different pools of stable cells 22 3.2. Mapping of BFLF2-interacting domains on EBV BFRF1 23 3.2.1. Multiple regions of BFRF1 interact with BFLF2 23 3.2.2. Serial deletion mutants of BFRF1 fail to bring BFLF2 out of the nucleus and to co-localize with BFLF2 23 3.3. Mapping of BFRF1-interacting domains on EBV BFLF2 24 3.3.1. Amino acids 2-102 of BFLF2 are required for the interaction with BFRF1 24 3.3.2. Amino acids 81-107 of BFLF2 are required for co-localization and interaction with BFRF1 24 3.4. Screening of the ESCRT-III components involved in EBV maturation 25 3.5. The production of anti-BFRF1 antibody 26 3.5.1. Purification and concentration of recombinant 6x-His-BFRF1TM protein 26 3.5.2. Detection of rabbit anti-6x-His-BFRF1TM antibody with different dilute titration in immunoblotting 27 3.5.3. Detection of BFRF1 expression with rabbit anti-6x-His-BFRF1TM antibody in immunofluorescence assay 27 4. DISCUSSION 29 4.1. BFRF1 interacts with Alix via its multiple regions 29 4.2. BFRF1 and BFLF2 interact with each other via multiple domains in more than one interface 30 4.3. Multiple regions of BFRF1 are required for bringing BFLF2 out of the nucleus 32 4.4. ESCRT-III components involve in virion secretion 32 4.5. The effectiveness of rabbit anti-6x-His-BFRF1TM antibody to detect BFRF1 is not optimal 33 5. FIGURES 35 Figure 1. Mutants of BFRF1 were presented to map the functional domains for interaction with Alix and BFLF2 35 Figure 2. Mutants of BFLF2 were presented to map the functional domains for interaction with BFRF1 36 Figure 3. The mutation of positive-charged amino acids in ESR domain of BFRF1 affects the interaction with the PRR domain of Alix 37 Figure 4. The ESR domain of BFRF1 interacts with the PRR domain of Alix in a nucleic acid-dependent manner 38 Figure 5. Deletion or mutation of domains required for the interaction with Alix affects the EBV virion secretion 39 Figure 6. Establishment of the fresh TetEZp2089 WT and BFRF1-KO stable cells and detection of the lytic protein expression in different pools of stable cells 40 Figure 7. Serial deletion mutants of BFRF1 are co-immunoprecipitated with BFLF2. 41 Figure 8. Different domains of BFRF1 interacts with BFLF2 42 Figure 9. Serial deletion mutants of BFRF1 fail to bring BFLF2 out of the nucleus and co-localize with BFLF2 43 Figure 10. Amino acids 2-102 of BFLF2 are required for interaction with BFRF1 44 Figure 11. Amino acids 81-107 of BFLF2 are required for co-localization with BFRF1 45 Figure 12. Amino acids 81-107 in BFLF2 are required to interact with BFRF1 47 Figure 13. The effect of dominant negative forms of ESCRT-III components to expression of lytic proteins, viral replication and viral release 48 Figure 14. Recombinant 6x-His-BFRF1TM protein is purified and concentrated. 49 Figure 15. The diluted titration of rabbit anti-6x-His-BFRF1TM antibody in serum is examined by immunoblotting 50 Figure 16. Detection of BFRF1 expression pattern with rabbit antiserum in immunofluorescence assay in HeLa cells overexpressing HA-BFRF1 51 Figure 17. Endogenous BFRF1 in NA cells is not detected by mouse anti-BFRF1 antibody (E10), rabbit anti-6x-His-BFRF1TM generated antibody and Commercial Ab in immunofluorescence assay 52 Figure 18. Schematic representation of the hypothetical model of the interaction between EBV BFRF1 and cellular Alix 53 Figure 19. 3D structural prediction of EBV nuclear egress complex according to herpesviral homologs 54 6. SUPPLEMENTAL FIGURES & TABLES 55 Supplemental figure 1. Crystal structures of NEC from HCMV and HSV-1 55 Supplemental figure 2. The LD1 domain of BFRF1 directly interacts with the Bro domain of Alix 56 Supplemental figure 3. The interaction between BFRF1 and the PRR domain of ALIX is defective after nuclease treatment 57 Table 1. Plasmids-1 58 Table 2. Plasmids-2 59 Table 3. Primers 60 REFERENCE 61 | - |
dc.language.iso | en | - |
dc.title | 探討EB病毒釋出過程中BFRF1蛋白之功能區域及ESCRT模組的參與 | zh_TW |
dc.title | Characterization of the functional domains in EBV BFRF1 and the roles of cellular ESCRT components in virus maturation process | en |
dc.type | Thesis | - |
dc.date.schoolyear | 106-2 | - |
dc.description.degree | 碩士 | - |
dc.contributor.oralexamcommittee | 楊宏志;李明學;李重霈 | zh_TW |
dc.contributor.oralexamcommittee | ;; | en |
dc.subject.keyword | EB病毒,BFRF1,釋出過程,Alix,BFLF2,ESCRT-III, | zh_TW |
dc.subject.keyword | Epstein-Barr virus,BFRF1,virus release,Alix,BFLF2,ESCRT-III, | en |
dc.relation.page | 66 | - |
dc.identifier.doi | 10.6342/NTU201802932 | - |
dc.rights.note | 未授權 | - |
dc.date.accepted | 2018-08-13 | - |
dc.contributor.author-college | 醫學院 | - |
dc.contributor.author-dept | 微生物學研究所 | - |
dc.date.embargo-lift | 2023-10-11 | - |
顯示於系所單位: | 微生物學科所 |
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