臺灣博碩士論文加值系統

中文摘要

EB病毒為人類皰疹病毒的一員，大約全球90%的成人曾受其感染，它被報導與包含Burkitt's淋巴瘤以及鼻咽癌等許多腫瘤的發病有關連性。如同其它皰疹病毒，EB病毒的生活史可分為潛伏期與溶裂期兩個階段，而從潛伏期進入溶裂期的過程被稱為再活化，近年相研究顯示再活化的發生率可能影響一些EB病毒相關腫瘤的發病。

先前實驗室發現蛋白激酶MSK1調控Burkitt's淋巴瘤Raji細胞中病毒的再活化。在透過shRNA降低MSK1表現的鼻咽癌NA細胞中，再活化刺激後的BZLF1、BRLF1以及BMRF1表現皆受抑制。在MSK1減量表現細胞中表現極早期基因，將使病毒再活化從受抑制的狀態下恢復。組蛋白去甲基酶KDM3A近期被發現受MSK1所調控，它的主要功能為移除Histone3 K9單、雙甲基化，而在過去的文獻中Histone3 K9上的甲基修飾被移除認為能增加病毒極早期基因BZLF1啟動子的活躍程度。在以KDM3A抑制劑IOX-1進行的實驗中，觀察到受IOX-1處理的細胞中再活化受到顯著抑制。CREB為MSK1所調控的轉錄因子，病毒極早期基因BZLF1啟動子上具有結合位，兩者的結合被認為能促進BZLF1的表現。再活化刺激24小時後可觀察到MSK1減量表現細胞中CREB磷酸化上升幅度略遜於MSK1正常表現細胞。最後，為了後續實驗，更進一步了解CREB對於EB病毒再活化的影響，製備了三個質體，分別表現野生型CREB、無法被磷酸化的CREB(S133A)以及提升其受PKA磷酸化程度的CREB(Y134F)。

Abstract

Epstein-Barr virus (EBV), a member of human Herpesviridae family, is implicated with several malignancies including Burkitt's lymphoma and nasopharyngeal carcinoma (NPC), about 90% adults worldwide has been infected with it. The life cycle of EBV can be divided into two stage, latency and lytic replication, a switch from latency to lytic replication is called reactivation. Recent studies had demonstrated that the incidence of reactivation can influence the pathogenesis of some EBV-associated malignancies.
In our previous study, MSK1, a protein kinase, had been found to regulate EBV reactivation in Raji cells, a Burkitt's lymphoma cell line. In NA cell, a NPC cell line, knockdown MSK1 with shRNA resulted in suppressed expression of BZLF1, BRLF1 and BMRF1 after lytic induction. Expressing BZLF1 or BRLF1 could launch unsuppressed reactivation in MSK1 knockdown cells. KDM3A, a histone demethylase, which removes mono-, di- methyl modification from histone3 lysine residue 9, had recently been found to be a downstream target of MSK1. The elimination of methyl modification on histone3 lysine residue 9 has been demonstrated to increase promoter activity of BZLF1. EBV reactivation was suppressed when treating cells with IOX-1, an inhibitor of KDM3A. CREB, a MSK1-regulated transcription factor, can bind to BZLF1 promotor and be suggested to enhance BZLF1 expression. Compared with shcontrol cell, a minor increase in CREB phosphorylation occurred in MSK1 knockdown cells. For further studying the effect of CREB on EBV reactivation, we also produce three plasmids which expressing wild type CREB, CREB(S133A), a non-phosphorylatable mutated form, and CREB(Y134F), a mutated form with enhanced phosphorylatability by PKA, respectively.

目次
致謝…………………………………………………………………i
中文摘要…………………………………………………………...ii
Abstract…………………………………………………………….iii
目錄………………………………………………………………...iv
圖次………………………………………………………………..vii
附表次…………………………………………………………….viii
第一章 緒論
1. EB病毒簡介…………………………………………………………….1
2. EB病毒生活史………………………………………………………….1
2.1. 潛伏期………………………………………………………...2
2.2. 溶裂期………………………………………………………...2
2.3. BZLF1 promotor的調控……………………………………...3
2.4. 造成再活化的環境刺激……………………………………...5
3. MSK1……………………………………………………………………...5
3.1. MSK1簡介…………………………………………………….6
3.2. MSK1對於EB病毒再活化的影響…………………………..7
第二章 實驗材料及方法…………………………………………………………..8
1. 實驗材料………………………………………………………………...8
1.1. 細胞培養液以及相關試劑……………………………………8
1.2. 蛋白質電泳與西方墨點法……………………………………8
2. 實驗方法………………………………………………………………...9
2.1. 細胞培養………………………………………………………9
2.2. 誘導EB病毒再活化………………………………………….9
2.3. 轉染…………………………………………..………………10
2.4. 慢病毒製備…………………………………………………..10
2.5. 慢病毒感染…………………………………………………..10
2.6. 西方墨點法…………………………………………………..10
2.7. 質體小量萃取………………………………………...……...11
2.8. 質體中量萃取……………………………….…………..…...12
2.9.Bacteria transformation………………………………………..13
2.10. 細胞DNA萃取…………………………………………….13
2.11. 半定量聚合酶反應…………………………………………13
2.12. 細胞RNA萃取……………………………………………..14
2.13. 反轉錄……………………………………………………….14
2.14. CREB表現質體製備………………………………………...15
2.15. 聚合酶反應點突變………………………………………….15
第三章 實驗結果…………………………………………………….17
1. MSK1協助EB病毒再活化…………………………………………….17
1.1. MSK1減量表現NA細胞的製備…………………………….17
1.2.MSK1 減量表現NA細胞中EB病毒再活化受到抑制……..17
1.3. 表現Zta以及Rta可以回復EB病毒的再活化……………..18
1.4. 在MSK1 knockdown NA細胞中表現MSK1可以回復EB病
毒再活化……………………………………………………....19
1.5. H89對於EB病毒再活化的影響……………………………..20
1.6. 利用H89尋找MSK1參與EB病毒再活化的時間點……...21
2. IOX-1抑制EB病毒再活化…………………………………………….21
3. MSK1對於EBV 基因體數量維持的影響…………………………….22
4. CREB表現質體製備……………………………………………………22
第四章 結論及討論…………………………………………………………..24
1. 結論…………………………………………………………………...24
2. 討論……………………………………………………………….…..26
2.1. MSK1如何影響EB病毒再活化……………………….…….26
2.2. KDM3A在病毒再活化上扮演的角色………………………..27
參考文獻…………………………………………………………....28
圖……………………………………………………………………………31
附表一……………………………………………………………………....44
附表二……………………………………………………………………....45

圖次
圖一 MSK1減量表現NA細胞的製備…………………………………………...31
圖二 MSK1 減量表現NA細胞中EB病毒再活化受到抑制…………………..32
圖三 表現Zta以及Rta可以回復EB病毒的再活化…………………………....34
圖四 在MSK1 knockdown NA細胞中表現MSK1可以回復EB病毒再活化...35
圖五 H89對於EB病毒再活化的影響…………………………………………...36
圖六 利用H89尋找MSK1參與EB病毒再活化的時間點……………………..39
圖七 IOX-1抑制EB病毒再活化…………………………………………………40
圖八 MSK1對於EBV 基因體數量維持的影響…………………………………42
圖九 CREB表現質體製備……………………………………………...43

附表次
附表一 shRNA與引子序列…………………………………………………….44
附表二 抗體……………………………………………………………………..45

參考文獻

1. Odumade, O.A., K.A. Hogquist, and H.H. Balfour, Jr., Progress and problems in understanding and managing primary Epstein-Barr virus infections. Clin Microbiol Rev, 2011. 24(1): p. 193-209.
2. Rickinson, A.B., Co-infections, inflammation and oncogenesis: future directions for EBV research. Semin Cancer Biol, 2014. 26: p. 99-115.
3. Connolly, S.A., et al., Fusing structure and function: a structural view of the herpesvirus entry machinery. Nat Rev Microbiol, 2011. 9(5): p. 369-81.
4. Hutt-Fletcher, L.M., Epstein-Barr virus entry. J Virol, 2007. 81(15): p. 7825-32.
5. Lieberman, P.M., Keeping it quiet: chromatin control of gammaherpesvirus latency. Nat Rev Microbiol, 2013. 11(12): p. 863-75.
6. Smeenk, G. and H. van Attikum, The chromatin response to DNA breaks: leaving a mark on genome integrity. Annu Rev Biochem, 2013. 82: p. 55-80.
7. Nanbo, A., A. Sugden, and B. Sugden, The coupling of synthesis and partitioning of EBV's plasmid replicon is revealed in live cells. EMBO J, 2007. 26(19): p. 4252-62.
8. Ali, A.S., et al., Epstein- Barr Virus: Clinical and Epidemiological Revisits and Genetic Basis of Oncogenesis. Open Virol J, 2015. 9: p. 7-28.
9. Rowe, M., et al., Burkitt's lymphoma: the Rosetta Stone deciphering Epstein-Barr virus biology. Semin Cancer Biol, 2009. 19(6): p. 377-88.
10. Gruffat, H., R. Marchione, and E. Manet, Herpesvirus Late Gene Expression: A Viral-Specific Pre-initiation Complex Is Key. Front Microbiol, 2016. 7: p. 869.
11. Liu, P. and S.H. Speck, Synergistic autoactivation of the Epstein-Barr virus immediate-early BRLF1 promoter by Rta and Zta. Virology, 2003. 310(2): p. 199-206.
12. Mahot, S., et al., A novel function for the Epstein-Barr virus transcription factor EB1/Zta: induction of transcription of the hIL-10 gene. J Gen Virol, 2003. 84(Pt 4): p. 965-74.
13. Borras, A.M., J.L. Strominger, and S.H. Speck, Characterization of the ZI domains in the Epstein-Barr virus BZLF1 gene promoter: role in phorbol ester induction. J Virol, 1996. 70(6): p. 3894-901.
14. Flemington, E. and S.H. Speck, Evidence for coiled-coil dimer formation by an Epstein-Barr virus transactivator that lacks a heptad repeat of leucine residues. Proc Natl Acad Sci U S A, 1990. 87(23): p. 9459-63.
15. Liu, S., et al., Cyclosporin A-sensitive induction of the Epstein-Barr virus lytic switch is mediated via a novel pathway involving a MEF2 family member. EMBO J, 1997. 16(1): p. 143-53.
16. Lu, J., et al., Upregulation of tyrosine kinase TKT by the Epstein-Barr virus transactivator Zta. J Virol, 2000. 74(16): p. 7391-9.
17. Kraus, R.J., J.G. Perrigoue, and J.E. Mertz, ZEB negatively regulates the lytic-switch BZLF1 gene promoter of Epstein-Barr virus. J Virol, 2003. 77(1): p. 199-207.
18. Montalvo, E.A., et al., Negative regulation of the BZLF1 promoter of Epstein-Barr virus. J Virol, 1991. 65(7): p. 3647-55.
19. Li, H., et al., Epstein-Barr virus lytic reactivation regulation and its pathogenic role in carcinogenesis. Int J Biol Sci, 2016. 12(11): p. 1309-1318.
20. Bergbauer, M., et al., CpG-methylation regulates a class of Epstein-Barr virus promoters. PLoS Pathog, 2010. 6(9): p. e1001114.
21. Woellmer, A. and W. Hammerschmidt, Epstein-Barr virus and host cell methylation: regulation of latency, replication and virus reactivation. Curr Opin Virol, 2013. 3(3): p. 260-5.
22. Buisson, M., et al., The C-terminal region but not the Arg-X-Pro repeat of Epstein-Barr virus protein EB2 is required for its effect on RNA splicing and transport. J Virol, 1999. 73(5): p. 4090-100.
23. Cayrol, C. and E.K. Flemington, The Epstein-Barr virus bZIP transcription factor Zta causes G0/G1 cell cycle arrest through induction of cyclin-dependent kinase inhibitors. EMBO J, 1996. 15(11): p. 2748-59.
24. Gao, Z., et al., The Epstein-Barr virus lytic transactivator Zta interacts with the helicase-primase replication proteins. J Virol, 1998. 72(11): p. 8559-67.
25. Gradoville, L., et al., Protein kinase C-independent activation of the Epstein-Barr virus lytic cycle. J Virol, 2002. 76(11): p. 5612-26.
26. Westphal, E.M., et al., Activation of lytic Epstein-Barr virus (EBV) infection by radiation and sodium butyrate in vitro and in vivo: a potential method for treating EBV-positive malignancies. Cancer Res, 2000. 60(20): p. 5781-8.
27. Adamson, A.L., et al., Epstein-Barr virus immediate-early proteins BZLF1 and BRLF1 activate the ATF2 transcription factor by increasing the levels of phosphorylated p38 and c-Jun N-terminal kinases. J Virol, 2000. 74(3): p. 1224-33.
28. Fixman, E.D., G.S. Hayward, and S.D. Hayward, trans-acting requirements for replication of Epstein-Barr virus ori-Lyt. J Virol, 1992. 66(8): p. 5030-9.
29. Reyskens, K.M. and J.S. Arthur, Emerging Roles of the Mitogen and Stress Activated Kinases MSK1 and MSK2. Front Cell Dev Biol, 2016. 4: p. 56.
30. McCoy, C.E., et al., MSK1 activity is controlled by multiple phosphorylation sites. Biochem J, 2005. 387(Pt 2): p. 507-17.
31. Hauge, C. and M. Frodin, RSK and MSK in MAP kinase signalling. J Cell Sci, 2006. 119(Pt 15): p. 3021-3.
32. McCoy, C.E., et al., Identification of novel phosphorylation sites in MSK1 by precursor ion scanning MS. Biochem J, 2007. 402(3): p. 491-501.
33. Thomson, S., et al., The nucleosomal response associated with immediate-early gene induction is mediated via alternative MAP kinase cascades: MSK1 as a potential histone H3/HMG-14 kinase. EMBO J, 1999. 18(17): p. 4779-93.
34. Vermeulen, L., et al., The versatile role of MSKs in transcriptional regulation. Trends Biochem Sci, 2009. 34(6): p. 311-8.
35. Kew, V.G., et al., Mitogen and stress activated kinases act co-operatively with CREB during the induction of human cytomegalovirus immediate-early gene expression from latency. PLoS Pathog, 2014. 10(6): p. e1004195.
36. Hancock, R.L., et al., Epigenetic regulation by histone demethylases in hypoxia. Epigenomics, 2015. 7(5): p. 791-811.
37. Thinnes, C.C., et al., Targeting histone lysine demethylases - progress, challenges, and the future. Biochim Biophys Acta, 2014. 1839(12): p. 1416-32.
38. Kim, K.Y., et al., Kaposi's sarcoma-associated herpesvirus (KSHV) latency-associated nuclear antigen regulates the KSHV epigenome by association with the histone demethylase KDM3A. J Virol, 2013. 87(12): p. 6782-93.
39. Wille, C.K., et al., Viral genome methylation differentially affects the ability of BZLF1 versus BRLF1 to activate Epstein-Barr virus lytic gene expression and viral replication. J Virol, 2013. 87(2): p. 935-50.