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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 陳美如 | |
| dc.contributor.author | Wei-Yi Hsu | en |
| dc.contributor.author | 徐唯逸 | zh_TW |
| dc.date.accessioned | 2021-06-17T01:40:40Z | - |
| dc.date.available | 2022-09-12 | |
| dc.date.copyright | 2017-09-12 | |
| dc.date.issued | 2017 | |
| dc.date.submitted | 2017-07-28 | |
| dc.identifier.citation | References
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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/67621 | - |
| dc.description.abstract | EB病毒感染超過九成的全球人口，除了可能造成感染性單核球增多症之外，EB病毒和許多人類惡性腫瘤的發展具有高度的相關。因此，了解病毒如何進到細胞中並複製完成到離開細胞的過程是很重要的。先前的研究指出，在病毒複製期間表現的 EB 病毒蛋白激酶 BGLF4 除了可透過磷酸化細胞內許多蛋白來改變細胞內的環境以利病毒進行複製之外，也會促使沒有進核序列的病毒蛋白，例如病毒複製的過程中需要的引子酶 (BSLF1) ─解旋酶 (BBLF4) 複合體，從細胞質進到細胞核中執行作用。此外最近的研究也發現，細胞分子伴護子 40 (Hsp40s) 中的一員 DNAJB6a、DNAJB6b 會和許多的病毒蛋白有交互作用並且幫助蛋白在細胞質 / 細胞核中傳遞。然而上述這些病毒複製相關因子的作用機制仍有許多未知的部分，因此，本研究探討 (1) BGLF4 造成的病毒蛋白進核的過程是否有 DNAJB6 的參與 (2) DNAJB6 是否會影響 EB 病毒的複製和病毒顆粒的產生。研究結果顯示，利用免疫螢光染色技術在共同轉染 BGLF4、DNAJB6a 和病毒引子酶 BSLF1 的細胞中，發現 BGLF4 和 DNAJB6a 皆會和 BSLF1 共同表現在細胞核中，表示 BGLF4 和 DNAJB6a 都可能幫助 BSLF1 的進核。經由免疫沉澱實驗發現 DNAJB6a 和 DNAJB6b會和 BGLF4、BSLF1 有交互作用。而免疫螢光染色實驗也顯示 BGLF4 的激酶活性對於 BSLF1 的進核有著顯著的影響。此外 DNAJB6a 並不會幫助引子酶相關因子和解旋酶進核。在含有 EB病毒的表皮細胞和 B 細胞中發現，抑制 DNAJB6a 會使病毒複製效率和病毒顆粒產生的總量下降；而抑制 DNAJB6b 則會使病毒的複製速率上升並且產出更多的具感染性粒子。綜合以上實驗結果，我們發現病毒激酶 BGLF4 的激酶活性在引子酶的進核過程中可能扮演相當重要的角色，並且分子伴護子 DNAJB6a 和 DNAJB6b也可以調控病毒引子酶的進核，進而影響病毒複製的速率和病毒顆粒的產生。 | zh_TW |
| dc.description.abstract | Epstein-Barr virus, which is a world-spread virus, infects over 90% of mankind and is highly associated with several malignancies. As the only protein kinase encoded by EBV, BGLF4, phosphorylates several viral and cellular proteins to optimize the cellular environment for viral replication and nuclear egress of viral nucleocapsid. Previous findings demonstrated that EBV protein kinase, BGLF4, which expressed in EBV early lytic replication, can help the translocation of viral primase-helicase complex from the cytoplasm into nucleus, but the detailed mechanisms remained unknown. Recent studies revealed that a cellular heat shock protein 40 family member (Hsp40s), DNAJB6, is involved in diverse cellular functions including proteins transport and also implicated in numerous pathologies and infectious virus diseases. In this study, we showed that cytoplasmic EBV BSLF1 (primase) could translocate into the nucleus in the presence of DNAJB6a while BBLF2/3 (primase-associated factor) and BBLF4 (helicase) retained in cytoplasm by immuno-fluorescence assay. BSLF1 and BGLF4 was co-immunoprecipitated by DNAJB6a and DNAJB6b, suggesting that DNAJB6a may promote the translocation of EBV primase through direct interaction. Notably, we found that DNAJB6a and DNAJB6b showed different effects on EBV replication. When DNAJB6a was knockdown in EBV positive NA and Akata B cells, it decreased viral DNA replication and virion secretion; while knocking-down DNAJB6b, it increased both viral replication rate and secretion in Akata B cells. The results may emphasize the ratio of DNAJB6 isoforms in affecting viral replication. Moreover, we found that BGLF4 changed the distribution of DNAJB6b from cytoplasm to nucleus. Taken together, we suggested that DNAJB6 and the kinase activity of BGLF4 are required for the nuclear import of BSLF1 and DNAJB6 isoforms affect the viral DNA replication and virion secretion in NA and B cells. | en |
| dc.description.provenance | Made available in DSpace on 2021-06-17T01:40:40Z (GMT). No. of bitstreams: 1 ntu-106-R04445111-1.pdf: 2892907 bytes, checksum: f39a11ee888a82cf53d10dc862269ff5 (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 ………………………………………….…………………………………1 Chapter 1: Introduction …...…………………………………………………4 1.1 Epstein-Barr virus (EBV) ……………………………………………………4 1.1.1. EBV and associated diseases ……………………………………………..4 1.1.2. EBV genome and structure ……………………………………………….4 1.1.3. The life cycle of EBV …………………………………………………….5 1.1.3.1. Infection ……………………………………………………………...5 1.1.3.2. Latent infection ………………………………………………………6 1.1.3.3. Lytic replication ……………………………………………………...6 1.2 Nuclear and Cytoplasmic transport …………………………..……………..8 1.3 Herpesviruses protein kinase …………………………………..…………….8 1.3.1. Characteristics of conserved herpesviruses protein kinase (CHPK) …..…8 1.3.2. Functions of EBV BGLF4 protein kinase ………………………………..9 1.3.3. The nuclear targeting of BGLF4 ………………………………………...10 1.4 Molecular chaperones ……………………………………………………….11 1.4.1. Cellular chaperone-dependent machinery ………………………………11 1.4.2. Characteristics of heat shock protein 40s (Hsp40s) ……………………..12 1.4.3. Characterization of DNAJB6 isoforms ………………………………….13 1.5 Aims of this study ……………...…………………………………………….13 Chapter 2: Materials and Methods…………………………………….15 2.1 Plasmids information ……………………………………………………….15 2.2 Cell culture, transfection, and lentivirus infection ………………………..15 2.3 Immunoblotting assay and antibodies ……………………………………..16 2.4 Indirect Immunofluorescence assay ………………………………………..17 2.5 Fractionation assay ………………………………………………………….17 2.6 Co-immunoprecipitation assay ……………………………………………..18 2.7 shRNA lentivirus package …………………………………………………..19 2.8 Lentivirus infection ………………………………………………………….19 2.9 Genomic DNA extraction and quantitative real-time PCR for EBV copy Number ………………………………………………………………………20 2.10 Quantitation of extracellular EBV DNA copy numbers ………………...21 Chapter 3: Results ………….…………………………………………………22 3.1 Cytopalsmic Myc-BSLF1 is translocated into nucleus in the presence of BGLF4 or DNAJB6a. ……………………………………………………….22 3.2 BSLF1 and BGLF4 interact with DNAJB6 isoforms. …………………….23 3.3 The kinase activity of BGLF4 is important for the nuclear targeting of BSLF1. ……………………………………………………………………….23 3.4 Cytoplasmic Myc-BBLF2/3 and Myc-BBLF4 retained in cytoplasm in the presence of HA-DNAJB6a. ……………………………………………..24 3.5 DNAJB6a and DNAJB6b affect EBV DNA replication in EBV-positive Akata B cells and EBV-positive epithelial NA cells. ………………………..24 3.6 The protein level of DNAJB6 isoforms decreased in TSA-treated NA cells. ………………………………………………………………………………...25 3.7 The protein level of DNAJB6b in nucleus increased in the presence of BGLF4. ………………………………………………………………………26 Chapter 4: Discussion ……..…………………………………………………27 4.1 The mechanisms of nuclear import of EBV lytic replication components. ………………………………………………………………………………...27 4.2 The kinase activity of BGLF4 for DNAJB6 isoforms. …………………….27 4.3 The contribution of DNAJB6 isoforms on EBV lytic replication. ………..28 4.4 The anti-virus roles of DNAJB6 isoforms in other viral pathologies. ……30 4.5 The roles and mechanisms of DNAJB6 in cancers. ……………………….31 Figures and supplements ….………………………………………………..32 Fig. 1. Functional domains of DNAJB6 isoforms. …………………………….32 Fig. 2. Both GFP-BGLF4 and HA-DNAJB6a can promote the nuclear targeting of BSLF1-Myc. ……………………………………………….33 Fig. 3. HA-DNAJB6a and HA-DNAJB6b could interact with BSLF1-Myc and GFP-BGLF4. …………………………………………………………….34 Fig. 4. The kinase activity of GFP-BGLF4 is important for the nuclear import of BSLF1-Myc. …………………………………………………………..35 Fig. 5. HA-DNAJB6a cannot promote the nuclear targeting of Myc-BBLF2/3 and Myc-BBLF4. ………………………………………………………..36 Fig. 6. Knockdown DNAJB6a and DNAJB6b affect EBV replication and virion secretion in TSA-treated EBV positive NA cells. ………………37 Fig. 7. Knockdown DNAJB6a and DNAJB6b affect EBV replication and virions secretion in IgG cross-linked EBV positive Akata B cells. …...38 Fig. 8. The protein expression level decreased in TSA-treated NA cells and distribution of DNAJB6 isoforms changed in the presence of BGLF4. …………………………………………………………………………….39 Fig. S1. Protein sequence alignment of HSV-1 UL52, HCMV UL70, and EBV BSLF1. ………………………………………………………………….40 References ………………………………………………………………………...41 | |
| dc.language.iso | en | |
| dc.title | EB病毒BGLF4蛋白質激酶急DNAJB6對於病毒DNA引子酶/解旋酶複合體進核調控之探討 | zh_TW |
| dc.title | Epstein-Barr Virus BGLF4 kinase and DNAJB6 mediated regulation of nuclear targeting of viral DNA primase-helicase complex | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 105-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 楊宏志,林素芳,李重霈,黃立民 | |
| dc.subject.keyword | EB病毒,進核, | zh_TW |
| dc.subject.keyword | EBV,DNAJB6,nuclear targeting, | en |
| dc.relation.page | 48 | |
| dc.identifier.doi | 10.6342/NTU201702169 | |
| dc.rights.note | 有償授權 | |
| dc.date.accepted | 2017-07-28 | |
| 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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