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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 陳美如 | zh_TW |
| dc.contributor.author | 黃暐涵 | zh_TW |
| dc.contributor.author | Wei-Han Huang | en |
| dc.date.accessioned | 2021-07-11T15:29:07Z | - |
| dc.date.available | 2024-02-28 | - |
| dc.date.copyright | 2018-10-09 | - |
| dc.date.issued | 2018 | - |
| dc.date.submitted | 2002-01-01 | - |
| dc.identifier.citation | Ahmad, S.S., J. Glatzle, K. Bajaeifer, S. Buhler, T. Lehmann, I. Konigsrainer, J.P. Vollmer, B. Sipos, S.S. Ahmad, H. Northoff, A. Konigsrainer, and D. Zieker. 2013. Phosphoglycerate kinase 1 as a promoter of metastasis in colon cancer. International journal of oncology. 43:586-590.
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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/78918 | - |
| dc.description.abstract | 當EB病毒在細胞核完成DNA複製，帶有病毒DNA的核殼體需要被運送到細胞質進一步組裝披膜蛋白、外套膜並釋出細胞。然而目前EB 病毒細胞質組裝過程的機制並不清楚。實驗室先前的研究中已知在EB病毒複製的晚期，病毒的結構蛋白和重新分佈的細胞胞器會共同聚集在細胞核凹陷處並且與EB病毒激酶BGFL4有共位的現象。因此，本研究試圖進一步觀察當EB病毒再活化時，細胞質組裝區域 (cytoplasmic assembly compartment) 的形成及探討參與幫助病毒顆粒組裝包裹的病毒及細胞因子。已知EB病毒激酶BGLF4能夠誘導細胞骨架重排排列，當EB 病毒在鼻咽癌上皮NA細胞株中再活化時，細胞骨架蛋白IQGAP1會重新分布至核凹陷處，並與BGLF4有共位現象。本研究透過共軛焦顯微鏡，也發現BGLF4能夠直接改變內生性IQGAP1在細胞中的分布情形，並且BGLF4與IQGAP1相互作用並非透過直接磷酸化IQGAP1的方式進行。當利用siRNA降低IQGAP1表現量時，EB病毒的DNA複製和病毒顆粒的釋出也跟著降低，代表IQGAP1在EB病毒DNA複製時扮演重要的角色。此外，在質譜分析中，也發現表現GFP-BGLF4時，與IQGAP1相互作用的蛋白質發生變化，顯示BGLF4能夠調控IQGAP1的功能，例如，磷酸甘油酸激酶 (Phosphoglycerate kinase 1, PGK1) 會與IQGAP1被共同免疫沉澱。PGK1為參與在醣解路徑當中一個能產生ATP能量的蛋白質，並且能與許多細胞內胞器的膜有交互作用。當EB病毒再活化時，PGK1會重新分佈到核凹陷處並與IQGAP1有部分共位現象。當利用shRNA降低PGK1表現量時，病毒顆粒的釋出量隨之減低，顯示PGK1在EB病毒成熟過程中扮演重要的角色。綜言之，當EB病毒再活化時，會形成EB病毒特別的細胞質組裝區域，並且IQGAP1與PGK1等細胞因子會聚集至此區域當中。至於PGK1細胞質組裝區域形成的區域中所扮演的角色，有待未來進一步澄清。 | zh_TW |
| dc.description.abstract | After viral genome replication and encapsidation in the nucleus, EBV nucleocapsids needed to translocate into the cytoplasm for subsequent tegumentation and maturation. In our previous study, the viral capsid, tegument proteins, glycoproteins, and redistributed cytoplasmic organelles were clustered at the perinuclear concave region and colocalized with EBV viral kinase BGFL4 during the late stage of viral replication. Therefore, we aimed to further observed the specialized viral cytoplasmic assembly compartment in EBV replicating cells and to characterize the key viral and cellular factors involved in the virus maturation process. It is known that the BGLF4 induces cytoskeleton rearrangement and IQGAP1 redistribution to the perinuclear concave region in EBV reactivated nasopharyngeal carcinoma NA cells. Here, we further show that expression of BGLF4 induces the redistribution endogenous of IQGAP1 to perinuclear region directly in confocal images. BGLF4 does not alter the SP/TP phosphorylation on IQGAP1 in transient transfected cells. Knockdown of IQGAP1 by siRNA shows downregulated EBV DNA replication and virion secretion, suggesting IQGAP1 plays a crucial role in EBV lytic DNA replication. In Mass analysis, the interactome of IQGAP1 is changed in the presence of BGLF4, suggesting BGLF4 modulates the function of IQGAP1. Among the interacting proteins, Phosphoglycerate kinase 1 (PGK1), which is an ATP-generating enzyme in the glycolytic pathway and partitions in various cellular organelle membranes, is found to be associated with IQGAP1. PGK1 is redistributed to perinuclear concave region and partially colocalized with IQGAP1 at late stage of virus replication. Furthermore, knockdown of PGK1 by shRNA shows downregulation of EBV virion secretion. Results here reveal that a specialized viral cytoplasmic assembly compartment is formed after EBV reactivation, and IQGAP1 and PGK1 redistribute to this site. Thus, the function of PGK1 in viral assembly compartment needs be further clarified. | en |
| dc.description.provenance | Made available in DSpace on 2021-07-11T15:29:07Z (GMT). No. of bitstreams: 1 ntu-107-R05445116-1.pdf: 3785515 bytes, checksum: 1a3430f98c026b31f47bdf45082f9c12 (MD5) Previous issue date: 2018 | en |
| dc.description.tableofcontents | 口試委員審定書 ………………………………….……………….…………………….I
誌謝 ………………………………………………………………….…………….……II 中文摘要 ……………………………………………………………..…………….…..III Abstract …………………………………………………………….……………….…..IV Contents …………………………………………………………………………………V 1. Introduction ……..…………………………….……………………………………..1 1.1. Epstein-Barr Virus………………………………………………………………...1 1.1.1. The classification, characterization and associated diseases of EBV………..1 1.1.2. The life cycle of EBV………………………………………………………..2 1.1.3. EBV viral protein kinase BGLF4…………………………………………….3 1.2. The assembly strategy of herpesvirus viral particles……………………………5 1.2.1. Primary and secondary envelopment process………………………………...5 1.2.2. The viral cytoplasmic assembly compartment formation……………………5 1.3. The cytoskeleton scaffold protein IQGAP1………………………………………6 1.3.1. IQGAPs family……………………………………………………………….6 1.3.2. The distribution and general functions of IQGAP1…………………………..7 1.3.3. The interplay of IQGAP1 and phosphoinositide………………………..…….9 1.4. PGK1 protein……………………………………………………………………...9 1.4.1. Characteristics of PGK1………………………………….……………...…..9 1.4.2. The association of PGK1 and virus………………………………………….10 1.5. Specific aims………………………………………………………………...……10 2. Materials and Methods……………………………………………………………..12 2.1. Cell culture………………………………………………………………………...12 2.2. Plasmid…………………………………………………………………………….12 2.3. Cell transfection…………………………………………………………………..13 2.4. Western blot analysis……………………………………………………………..13 2.5. Co-immunoprecipitation assay…………………………………………………..14 2.6. Immunofluorescence assay……………………………………………………….15 2.7. Subcellular fractionation…………………………………………………………16 2.7.1. Hypotonic buffer fractionation………………………………….…………..16 2.7.2. Qproteome Cell compartment kit fractionation…………………………….17 2.8. Proteomics analysis……………………………………………………………….18 2.9. shRNA lentivirus infection………………………………………………………..18 2.10. Quantitative real-time PCR (q-PCR) analysis……..…………………………..19 2.10.1. EBV intracellular DNA extraction……..………………………………….19 2.10.2. Secreted EBV particles isolation and DNA extraction….…………………19 2.10.3. Quantitative real-time PCR……………..…………………………………20 2.11. Transmission electron microscopy (TEM) analysis……………………………20 3. Results……………………………………………………………………………….22 3.1. A specialized viral cytoplasmic assembly compartment is formed upon EBV reactivation………………………………………………………………………..22 3.2. EBV nucleocapsids appear in a vesicle-like structure at the perinuclear concave region in Rta reactivated NA cells………………………………………………….23 3.3. EBV reactivation induces the redistribution of IQGAP1 to the perinuclear concave region……………………………………………………………………………....23 3.4. BGLF4 kinase induces the change of endogenous IQGAP1 redistribution in transiently transfected HeLa cells…………………………………………..……...24 3.5. BGLF4 does not change SP/TP phosphorylation on IQGAP1……….……………..26 3.6. Knockdown of IQGAP1 represses EBV DNA replication and virion secretion in NA cells………………………………………………………………………………...27 3.7. Knockdown of IQGAP1 does not change the clustering of BBLF1 at the perinuclear concave region upon EBV reactivation…………………………………………….27 3.8. A proteomic analysis of IQGAP1 interactome in GFP-BGLF4 transiently transfected HeLa cells…………………………………………………………………….…….28 3.9. PGK1 does not co-immunoprecipitate with IQGAP1……………………………...29 3.10. IQGAP1 and PGK1 are partially colocalized in the perinuclear concave region in Rta reactivation NA cells……………...………………………………………….30 3.11. Knockdown of PGK1 represses EBV virion secretion in transfected NA cells……31 3.12. BGLF4 induces PGK1 redistribution in transiently transfected HeLa cells.…..…31 3.13. Recruitment of PGK1 is independent of IQGAP1 in IQGAP1-knockdown (IQGAP1-KD) NA cells…………………………………………………...……….32 4. Discussion…………………………………………………………………….……...33 4.1. The EBV viral cytoplasmic assembly compartment is similar to that of HCMV…...33 4.2. The role of IQGAP1 in EBV replicating cells……………………………………..34 4.3. The possible role of PGK1 in viral cytoplasmic assembly compartment…………..35 5. Figures and Tables……………………………………………………………….....38 Fig. 1. EBV reactivation induces the gradual changes of nuclear shapes and clustering of viral proteins and cis-Golgi marker GM130 in the perinuclear concave region………..38 Fig. 2. EBV reactivation induces viral proteins and GM130 redistribution to the perinuclear concave region……………………………………………………………..40 Fig. 3. EBV nucleocapsids appear in a vesicle-like structure at the perinuclear concave region in Rta reactivated NA cells…….……………………………………..………….41 Fig. 4. EBV reactivation induces the redistribution of IQGAP1 to the perinuclear concave region…………………………………………………………….……………………..42 Fig. 5. BGLF4 induces the redistribution of endogenous IQGAP1 in HeLa cells...........43 Fig. 6. BGLF4 does not change the subcellular distribution of IQGAP1 in HeLa cells…44 Fig. 7. There is no significant MPM-2 signal on IQGAP1 in GFP-BGLF4 expressing HeLa cells………………………………………………………………………………45 Fig. 8. Knockdown of IQGAP1 represses the viral replication in Rta reactivated cells…46 Fig. 9. Knockdown of IQGAP1 does not change the clustering of BBLF1 at the perinuclear concave region…………………………………………….……………….47 Fig. 10. BGLF4 does not induces PGK1 to be co-immunoprecipitate with IQGAP1…...48 Fig. 11. EBV reactivation induces the redistribution of PGK1 to the perinuclear concave region…………………………………………………………………………………...49 Fig. 12. IQGAP1 and PGK1 are partially colocalized in the perinuclear concave region in EBV reactivated NA cells……………………………………………………………….50 Fig. 13. Knockdown of PGK1 represses EBV virion secretion in transfected NA cells…51 Fig. 14. BGLF4 induces PGK1 redistribution in transiently transfected HeLa cells…….52 Fig. 15. Knockdown of IQGAP1 does not change the clustering of PGK1 at perinuclear concave region upon EBV reactivation………………………………...……………….53 Fig. 16. A hypothetic model for the viral and cellular components in EBV cytoplasmic assembly compartment component……………………………………………………..54 Table 1. The functional pathway analysis of IQGAP1 interactome in GFP-BGLF4 expressing HeLa cells…………………………………………………………………..55 6. Supplemental Data………………………………………………………………….58 Supplementary Fig. 1. Protein structure of and specific domain-interacting partners of IQGAP1…………………………………………………………………………….…..58 Supplementary Fig. 2. SDS-PAGE preparation for Mass spectrum analysis…………..59 References………………………………………………...……………………………60 | - |
| dc.language.iso | en | - |
| dc.subject | 磷酸甘油酸激? | zh_TW |
| dc.subject | EB病毒 | zh_TW |
| dc.subject | 細胞質組裝區域 | zh_TW |
| dc.subject | PGK1 | en |
| dc.subject | EBV | en |
| dc.subject | cytoplasmic assembly compartment | en |
| dc.title | 探討病毒蛋白質與細胞因子參與在EB病毒細胞質組裝區域之研究 | zh_TW |
| dc.title | Characterization of viral proteins and cellular factors involved in EBV cytoplasmic assembly compartment | en |
| dc.type | Thesis | - |
| dc.date.schoolyear | 106-2 | - |
| dc.description.degree | 碩士 | - |
| dc.contributor.oralexamcommittee | 張麗冠;李重霈;張鑫 | zh_TW |
| dc.contributor.oralexamcommittee | ;; | en |
| dc.subject.keyword | EB病毒,細胞質組裝區域,磷酸甘油酸激?, | zh_TW |
| dc.subject.keyword | EBV,cytoplasmic assembly compartment,PGK1, | en |
| dc.relation.page | 66 | - |
| dc.identifier.doi | 10.6342/NTU201803945 | - |
| dc.rights.note | 未授權 | - |
| dc.date.accepted | 2018-08-17 | - |
| dc.contributor.author-college | 醫學院 | - |
| dc.contributor.author-dept | 微生物學研究所 | - |
| dc.date.embargo-lift | 2023-10-09 | - |
| 顯示於系所單位： | 微生物學科所 | |
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