臺灣博碩士論文加值系統

外吐小體（exosomes）是一種從細胞質當中多泡體（multivesicular endosomes）形成而分泌出來具有雙層膜結構的微小膜泡，並且在細胞間的溝通扮演著不可或缺的角色。近年來已經有研究發現受EB病毒感染的細胞所分泌出的外吐小體可以包裹EB病毒潛伏期所表現病毒蛋白和miRNA，並且對其他細胞造成影響。然而外吐小體在EB病毒所感染細胞進入溶解期時所扮演的角色仍然是未知的。因此本研究是為了解當EB病毒近入缺損型型溶解期，外吐小體是否包裹EB病毒的溶解期所表現的病毒蛋白。Raji 細胞是帶有EB病毒的Burkitt’s 淋巴瘤細胞株，當Raji 細胞被刺激進入溶裂期時會表現溶裂期的病毒蛋白，但卻因為缺少了single-stranded-DNA binding protein (BALF2)而無法產生病毒。而我們發現Raji 細胞進入部分溶解期時所分泌出的外吐小體裡面包含了BGLF4 蛋白激酶、BFRF1/BFLF2病毒出核複合體蛋白、BMFR1 DNA聚合酶輔助因子。利用細胞轉染的方式，發現單獨表現的BGLF4 蛋白激酶可以被包裹在外吐小體當中，更有趣的是K102I也就是失去激酶活性的 BGLF4不會被包裹在外吐小體裡，因此BGLF4的激酶活性可以影響本身是否被包裹進去外吐小體當中，此外透過試管內激酶活性分析發現在外吐小體當中的BGLF4仍然保有激酶活性，接著我們發現HeLa細胞可以吸收帶有GFP-BGLF4的螢光外吐小體，外吐小體中的GFP-BGLF4可以促使細胞骨架重新排列而導致細胞皺縮圓起。Raji 細胞所分泌出的外吐小體被螢光標記後也會被HeLa細胞吸收。在我們的研究當中發現，當EB病毒進入溶裂時期所分泌出的外吐小體包裹著BGLF4，可以被細胞所吸收並造成細胞型態的改變。

Exosomes are membrane nano-vesicles secreted by most cells from multivesicular endosomes and participate in intercellular communications. A number of recent studies indicate that exosomes secreted from EBV latent cells contain latent viral proteins and miRNAs which induce various cellular changes in target cells. However, the characteristics of exosomes released from the lytic cycle of Epstein-Barr virus (EBV) infected cells are still unclear. Raji cells which is an EBV-genome positive Burkitt’s lymphoma cell line with the deletion of single-stranded-DNA binding protein (BALF2) express viral lytic proteins, but do not produce EBV virion after chemical induction into abortive lytic replication. Thus, in this study, we aimed to investigate whether EBV lytic proteins are packaged within exosomes secreted from Raji cells during abortive lytic cycle. Here, we showed that multiple EBV lytic viral proteins such as protein kinase BGLF4, the DNA polymerase processivity factors BMRF1, nuclear envelope-associated BFRF1 and intranuclear BFLF2 are packaged in exosomes secreted from Raji cells during abortive lytic replication. In transiently transfected HeLa cells, BGLF4 but not K102I (kinase dead mutant) is detected in exosomes. BGLF4 presented in exosomes is a kinase activity dependent manner. Furthermore, exosomal BGLF4 kinase carries kinase activity as demonstrated by in vitro kinase assay. We further show that fluorescently labelled exosomes containing GFP-BGLF4 are uptaken by HeLa cells and induce the reorgainzation of cytoskeleton which lead to cell rounding. Fluorescently labelled exosomes derived from non-induced and induced Raji cells were also uptaken by HeLa cells. Our findings suggest that exosome-carried active BGLF4 are taken up by the recipient cells and cause the rearrangement of cytoskeleton and cell morphology changes.

Contents
口試委員會審定書 ..................................................................................................... I
誌謝 ............................................................................................................................ II
中文摘要 ................................................................................................................... III
Abstract ..................................................................................................................... IV
Contents ..................................................................................................................... VI
1. Introduction ......................................................................................................... 1
1.1. Epstein-Barr virus ........................................................................................ 1
1.1.1. The classification, viral structure and genome of EBV ........................ 1
1.1.2. The life cycle of EBV ............................................................................ 2
1.1.3. Herpesviral homologues of BGLF4 kinase .......................................... 4
1.1.4. EBV BGLF4 protein kinase ................................................................. 6
1.2. Exosomes ........................................... 7
1.2.1. Characteristics of exosomes ................................................................. 8
1.2.2. Exosome biogenesis ............................................................................. 8
1.2.3. Functions of exosomes ........................................................................ 10
1.2.4. EBV and exosomes ............................................................................. 11
1.3. Aim of the study .......................................................................................... 12
2. Materials & Methods ........................................................................................ 14
2.1. Cell culture .................................................................................................. 14
2.2. Plasmids ...................................................................................................... 14
2.3. Cell transfection .......................................................................................... 15
2.4. Western blot analysis ................................................................................. 15
2.5. Indirect immunofluorescence assay (IFA) ............................................... 16
2.6. Exosome purification ................................................................................. 17
2.6.1. Exosome purification by ultracentrifugation ...................................... 17
2.6.2. Exosome purification by ExoQuick-TCTM Exosome Precipitation Solution .............................. 18
2.6.3. Exosomes-depleted medium ............................................................... 18
2.6.4. Nanoparticle tracking analysis (NTA) ................................................. 19
2.6.5. Fluorescent Exosomes Labeling and Exosome uptake assay .............. 19
2.7. Purification of bacterially expressed GST and GST-lamin A tail 2 fusion proteins ........................................................................................................ 20
2.8. Immunoprecipitation kinase assay ........................................................... 21
3. Result ................................................................................................................ 23
3.1. The size distributions of exosomes isolated from Raji cells by ultracentrifugation. ................................... 23
3.2. Multiple EBV lytic proteins are packaged in the exosomes. ................... 24
3.3. Package of BGLF4 in exosomes was kinase activity dependent. ............. 25
3.4. BGLF4 retains kinase activity in exosomes. .............................................. 26
3.5. Exosomal BGLF4 induces the cytoskeleton rearrangement of recipient cells. ............................................................................................................. 27
3.6. Exosomes isolated from chemical induced Raji cells led to less effective cell contraction. .......................................................................................... 28
4. Discussion ............................................................................................................. 29
5. Figures .................................................................................................................. 32
Fig.1. Nanoparticle tracking analysis (NTA) of exosomes released from EBV positive Raji cells. .................................... 32
Fig.2. Multiple EBV lytic proteins are packaged in the exosomes released from Raji cells during abortive lytic replication.............. 33
Fig.3. BGLF4 is packaged within exosomes secreted from transfected HeLa cells. ............................................................................................................ 34
Fig.4. BGLF4 is packaged within exosomes released from TW01 tet-on BGLF4 cells after doxycycline induction............................ 35
Fig.5. Exosomal BGLF4 and K102I in the exosomes purified from transfected HeLa cells. ............................................................................................................ 36
Fig.6. Purification of bacterially expressed GST and GST-lamin A Tail 2. ........ 37
Fig. 7. BGLF4 kinase in exosomes phosphorylate lamin A tail 2 in vitro. ............ 38
Fig. 8. BGLF4 induces cell contraction in HeLa cells. .......................................... 39
Fig. 9. GFP, GFP-BGLF4 and GFP-K102I was packaged within exosome purified by ultracentrifugation. .................................... 40
Fig. 10. Exosome derived GFP-BGLF4 is uptaken by HeLa cells and induces cell contraction. ................................................................................................. 41
Fig. 11. Exosome from non-induced and induced Raji cells labelled with green fluorescence PKH67 are uptaken by HeLa cells and induces cell contraction. ............. 42
Fig. 12. The protein levels of exosomal BGLF4 from Raji cells and transiently transfected HeLa cells. ................................ 43
Fig. 13. A hypothetical model of BGLF4 in exosomes. ....................................... 44
Reference ................................................................. 45

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