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http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/57614完整後設資料紀錄
| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 陳振陽(Jen-Yang Chen) | |
| dc.contributor.author | Shih-Hsin Chiu | en |
| dc.contributor.author | 邱世昕 | zh_TW |
| dc.date.accessioned | 2021-06-16T06:54:10Z | - |
| dc.date.available | 2019-10-09 | |
| dc.date.copyright | 2014-10-09 | |
| dc.date.issued | 2014 | |
| dc.date.submitted | 2014-07-21 | |
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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/57614 | - |
| dc.description.abstract | EB病毒BALF3與第一型疱疹單純病毒溶裂複製有關的末端酶UL28具高度同源性，然而BALF3對於EB病毒生活史與宿主細胞的影響至今仍尚未清楚。為了釐清BALF3的特性，利用純化的BALF3蛋白來進行試管中酵素反應實驗，發現BALF3具有核酸內切酶的活性且可由鎂錳離子與ATP分子來調控。此外，當帶有EB病毒的鼻咽癌細胞中的EB病毒進入溶裂期時，BALF3會開始表現並由細胞質進入細胞核中。進一步將BALF3基因默化則明顯降低病毒DNA包裝作用與病毒顆粒的產生，同時也減少病毒DNA合成效率。另一方面，BALF3可造成EB病毒陰性的鼻咽癌細胞產生基因不穩定性如微核形成及DNA斷裂的增加，而細胞經過BALF3反覆表現影響後，出現顯著的基因拷貝數變異及細胞移行、細胞侵襲與球狀體形成等腫瘤特徵，同時將其細胞注射至具免疫缺陷的小鼠體中，與控制組相較之下則形成較大的腫瘤。最後，利用RNA微陣列發現BALF3反覆表現會影響許多癌症相關基因及致癌基因的表現，而其變化與基因變異相關。因此，由以上的實驗結果可得知EB病毒BALF3參與病毒DNA合成、切割與包裝進而影響病毒顆粒產生，以及也扮演EB病毒感染導致鼻咽癌發生的潛在角色。 | zh_TW |
| dc.description.abstract | Epstein-Barr virus (EBV) BALF3 is a homologue of the herpes simplex virus type 1 terminase UL28 that is responsible for viral lytic replication. However, the effects of BALF3 on the EBV life cycle and host cells are still unclear. In order to characterize BALF3, the purified protein was produced and examined in an enzymatic reaction in vitro, which determined that BALF3 acts as an endonuclease, and its activity is modulated by Mg2+, Mn2+, and ATP. Moreover, in EBV-positive nasopharyngeal carcinoma (NPC) cells, BALF3 was expressed and transported from the cytoplasm into the nucleus following lytic induction, and gene silencing of BALF3 caused a reduction of DNA packaging and virion release. Meanwhile, suppression of BALF3 expression also decreased the efficiency of DNA synthesis. On the other hand, BALF3 has the ability to induce host genomic instability, such as micronucleus formation and DNA strand breaks. After recurrent expression of BALF3 in EBV-negative NPC cells, genomic copy number aberrations had accumulated to a significant extent, and tumorigenic features such as cell migration, cell invasion, and spheroid formation increased with the rounds of induction. In parallel experiments, cells after highly recurrent induction developed into larger tumor nodules than control cells when inoculated into NOD/SCID mice. Furthermore, RNA microarrays showed that differential expression of multiple cancer capability-related genes and oncogenes increased with recurrent BALF3 expression, and these changes were correlated with genetic aberrations. Therefore, EBV BALF3 is involved simultaneously in DNA synthesis, cleavage, and packaging, which accounts for the production of mature virions, and it also is a potential factor that mediates the impact of EBV on nasopharyngeal carcinogenesis. | en |
| dc.description.provenance | Made available in DSpace on 2021-06-16T06:54:10Z (GMT). No. of bitstreams: 1 ntu-103-D96445003-1.pdf: 2367883 bytes, checksum: 12fb9dd2528905c9942a3f99e497ffa5 (MD5) Previous issue date: 2014 | en |
| dc.description.tableofcontents | 謝辭.......................................................i
摘要......................................................ii Abstract.................................................iii Chapter 1 Introduction.....................................1 1.1 Epstein-Barr virus (EBV)...............................1 1.1.1 Introduction.........................................1 1.1.2 EBV DNA replication..................................2 1.2 Viral DNA cleavage and packaging.......................3 1.2.1 Terminase............................................3 1.2.2 Terminal repeat of the viral genome..................4 1.3 Nasopharyngeal carcinoma (NPC).........................6 1.3.1 Epidemiology and etiology............................6 1.3.2 Histopathology and staging...........................7 1.3.3 Association between NPC and EBV......................8 1.3.4 Association between NPC and genomic instability......9 1.4 Aim...................................................10 Chapter 2 Materials and methods...........................12 2.1 Buffers...............................................12 2.1.1 Cell culture........................................12 2.1.2 Western blotting....................................12 2.1.3 Protein purification................................12 2.1.4 Subcellular fractionation...........................12 2.1.5 Detection of intracellular viral genomic DNA........13 2.1.6 Purification of viral capsids.......................13 2.1.7 Gardella gel analysis...............................13 2.1.8 Southern blotting and hybridization.................13 2.2 Plasmids..............................................13 2.2.1 pBacPAK8-MTGFP-His-BALF3............................13 2.2.2 pBS-TR..............................................14 2.2.3 pEGFP-C1-BALF3......................................14 2.2.4 pLenti4-BALF3.......................................14 2.3 Primers...............................................14 2.3.1 Polymerase chain reaction (PCR).....................15 2.3.2 Site-directed mutagenesis...........................15 2.4 Small interfering RNAs (siRNAs).......................16 2.5 Cell lines............................................16 2.5.1 Sf9 cells...........................................16 2.5.2 NPC-TW01 and NA cells...............................16 2.5.3 TW01TREx-VC and TW01TREx-BALF3 cells................17 2.6 Site-directed mutagenesis.............................17 2.7 Transfection..........................................18 2.7.1 Expression of the EBV BALF3 protein in the baculovirus expression system…...........................18 2.7.2 Expression of EBV BALF3 with GFP tag in TW01 cells..18 2.7.3 Establishment of tetracycline-regulated BALF3 inducible cell lines......................................18 2.7.4 Knockdown of EBV BALF3..............................18 2.8 Western blotting......................................19 2.9 Expression and purification of the EBV BALF3 protein..19 2.10 In vitro nuclease activity assay.....................20 2.11 PCR and quantitative reverse transcription-PCR (qRT-PCR)......................................................21 2.12 Indirect immunofluorescence staining.................21 2.13 Subcellular fractionation............................22 2.14 Detection and quantification of viral DNA............22 2.15 Ultra-thin sectioning and transmission electron microscopy................................................23 2.16 Purification of viral capsids........................23 2.17 Gardella gel analysis................................24 2.18 Southern blotting and hybridization..................24 2.19 Micronucleus formation assay.........................25 2.20 Cell proliferation assay.............................25 2.21 Recurrent expression of EBV BALF3....................26 2.22 Array-based comparative genomic hybridization (array CGH)......................................................26 2.23 Cell migration assay.................................26 2.24 Cell invasion assay..................................27 2.25 Spheroid assay.......................................27 2.26 In vivo tumorigenesis assay..........................27 2.27 RNA expression analysis..............................28 Chapter 3 Results.........................................29 3.1 The role of EBV BALF3 in the viral life cycle.........29 3.1.1 Expression and purification of EBV BALF3............29 3.1.2 Nuclease activity of EBV BALF3......................30 3.1.3 Expression of EBV BALF3 during the lytic cycle......31 3.1.4 Subcellular localization of EBV BALF3...............32 3.1.5 Contribution of EBV BALF3 to virion production......33 3.1.6 Contribution of EBV BALF3 to viral DNA replication..34 3.2 The role of EBV BALF3 in nasopharyngeal carcinogenesis............................................35 3.2.1 Effects of EBV BALF3 on genomic instability.........35 3.2.2 Accumulation of genomic instability after recurrent expression of EBV BALF3...................................36 3.2.3 Contribution of EBV BALF3 to tumorigenic properties of NPC cells..............................................38 3.2.4 Differential expression of cancer-related genes in NPC cells after recurrent EBV BALF3 expression............39 Chapter 4 Discussion......................................41 4.1 Regulation of the nuclease activity of EBV BALF3......41 4.2 Zinc-finger and leucine-zipper motifs in EBV BALF3....42 4.3 Non-specific nucleolytic reaction by EBV BALF3........43 4.4 Nuclear translocation of EBV BALF3....................44 4.5 Regulation of viral DNA synthesis by EBV BALF3........45 4.6 Abortive lytic cycle of EBV in NPC....................46 4.7 Possible mechanism for host genomic instability induced by EBV BALF3..............................................46 4.8 Design and comparison of experiments of recurrent EBV reactivation and recurrent EBV BALF3 expression...........47 4.9 Differentially expressed cancer-related genes involved in NPC after recurrent EBV BALF3 expression...............48 4.10 Contribution of EBV BALF3 to nasopharyngeal carcinogenesis............................................49 4.11 Conclusion...........................................51 References................................................52 Figures and Tables........................................76 Figure 1 Representative illustrations of plasmids used in this study................................................76 Figure 2 Sequence alignment of putative conserved motifs in human herpesvirus terminase subunits......................77 Figure 3 Expression and purification of EBV BALF3.........78 Figure 4 Nuclease activity of EBV BALF3...................79 Figure 5 Expression of EBV lytic genes in EBV-positive cells during the lytic cycle..............................81 Figure 6 Subcellular localization of EBV BALF3 in EBV-positive cells during the lytic cycle.....................82 Figure 7 Examination of the efficiency of knockdown by siBALF3s in EBV-positive cells during the lytic cycle.....84 Figure 8 Production of mature virions in EBV-positive cells with BALF3 knockdown......................................85 Figure 9 Maturation of capsids in EBV-positive cells with BALF3 knockdown...........................................86 Figure 10 Examination of viral DNA replication in EBV-positive cells with BALF3 knockdown.......................88 Figure 11 Induction of genomic instability in NPC cells with EBV BALF3 expression.................................89 Figure 12 Effect of EBV BALF3 expression on genomic instability and growth of NPC cells.......................90 Figure 13 Examination of EBV BALF3 expression in NPC cells.....................................................92 Figure 14 Accumulation of genomic instability in NPC cells after recurrent EBV BALF3 expression......................93 Figure 15 Examination of cell migration by NPC cells after recurrent EBV BALF3 expression............................95 Figure 16 Examination of cell invasion by NPC cells after recurrent EBV BALF3 expression............................96 Figure 17 Examination of spheroid formation by NPC cells after recurrent EBV BALF3 expression......................97 Figure 18 Examination of cell proliferation by NPC cells after recurrent EBV BALF3 expression......................98 Figure 19 Examination of in vivo tumor growth after recurrent EBV BALF3 expression............................99 Figure 20 Differentially expressed genes in NPC cells after recurrent EBV BALF3 expression...........................100 Figure 21 Examination of the efficiency of knockdown by siBALF3s in TW01TREx-BALF3 cells.........................101 Figure 22 Subcellular localization of EBV BALF3 in TW01TREx-BALF3 cells..............................................102 Figure 23 Expression of EBV BALF3 in NA and TW01TREx-BALF3 cells....................................................103 Figure 24 Roles of EBV BALF3 in viral life cycle and nasopharyngeal carcinogenesis............................104 Table 1 Types of capsids in EBV-positive cells with BALF3 knockdown................................................105 Table 2 Summary of array CGH data with high aberration score....................................................106 Table 3 Gene ontology of genes differentially expressed in NPC cells after recurrent BALF3 expression...............107 | |
| dc.language.iso | en | |
| dc.subject | 鼻咽癌發生 | zh_TW |
| dc.subject | EB病毒BALF3 | zh_TW |
| dc.subject | DNA包裝 | zh_TW |
| dc.subject | DNA切割 | zh_TW |
| dc.subject | DNA合成 | zh_TW |
| dc.subject | 末端? | zh_TW |
| dc.subject | 基因不穩定性 | zh_TW |
| dc.subject | Epstein-Barr virus BALF3 | en |
| dc.subject | nasopharyngeal carcinogenesis | en |
| dc.subject | genomic instability | en |
| dc.subject | DNA packaging | en |
| dc.subject | DNA cleavage | en |
| dc.subject | DNA synthesis | en |
| dc.subject | terminase | en |
| dc.title | EB病毒BALF3於病毒生活史與鼻咽癌發生之角色探討 | zh_TW |
| dc.title | Roles of Epstein-Barr Virus BALF3 in Viral Life Cycle and Nasopharyngeal Carcinogenesis | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 102-2 | |
| dc.description.degree | 博士 | |
| dc.contributor.oralexamcommittee | 董馨蓮(Shin-Lian Doong),陳美如(Mei-Ru Chen),林素芳(Su-Fang Lin),李財坤(Tsai-Kun Li) | |
| dc.subject.keyword | EB病毒BALF3,末端?,DNA合成,DNA切割,DNA包裝,基因不穩定性,鼻咽癌發生, | zh_TW |
| dc.subject.keyword | Epstein-Barr virus BALF3,terminase,DNA synthesis,DNA cleavage,DNA packaging,genomic instability,nasopharyngeal carcinogenesis, | en |
| dc.relation.page | 108 | |
| dc.rights.note | 有償授權 | |
| dc.date.accepted | 2014-07-21 | |
| 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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