Epstein-Barr virus (EBV) 為人類皰疹病毒第四型，又稱為 EB病毒，為常見引發人類產生疾病的病毒之一。其核抗原 2 (EBNA2) 及前導蛋白 (EBNALP)，在 EB 病毒感染宿主細胞後幾小時就開始表現，並起始 EB 病毒的轉錄子及細胞當中基因的轉錄機制，使人類 B 細胞永生化 (immortalization) 避開宿主細胞凋亡，使自身成功建立永久性的感染。本研究探討 EBNA2 及 p53 之間的交互作用， p53 可能會透過抑制 EBNA2 所主導的轉錄機制保護宿主細胞免於病毒的感染。初步 reporter assay 結果顯示 p53 可以抑制 EBNA2 所起始的 EB 病毒啟動子活性，在 EBNALP共同激活 (co-activation) EBNA2 的情況下， p53 同樣具有抑制能力。接著探討 p53 基因不同區間對於 EBNA2 的抑制情形，分別為 p53 基因 N 端的 Transcription activation domain (AD), proline rich (Pro), 中間的 DNA binding domain (DBD), oligmerlization (Olg) 及 C 端的調控區域。 AD 及 Olg 對於抑制 EBNA2 是必須的， Pro 及 Olg 則參與在 EBNA2 及 EBNALP 抑制，以上結果顯示 p53 對於抑制 EBNA2 及 EBNALP 可能是透過不同的分子機轉。由 reporter assay 推斷 AD 胺基酸 60-70 區域對於 EBNA2 的抑制很重要，免疫沉澱法 (IP) 結果顯示 p53AD 與 EBNA2 之間有相互作用， p53AD1 (aa 1-39) 及 AD2 (aa 40-70) 為 AD2 與 EBNA2 的相互作用較為明顯。以上結果使我們推測， p53 可能會透過 AD 區間與 EBNA2 之間有相互作用，影響 EBNA2 所主導的 EB 病毒啟動子活性。

Epstein-Barr virus (EBV), also known as human herpes virus 4, is a member of the herpes virus family. This virus is one of the most common viruses which cause human disease. The nuclear antigen 2 (EBNA2) and the leader protein (EBNALP) of EBV will start to express after few hours when it infects the host cell, which also initiates the transcription of EBV nuclear antigen (EBNA) and cell gene. This reaction makes human B-cells immortalization, which help host cell avoid apoptosis and make it establish persistent infection. This study mainly investigates on the interaction of EBNA2 and p53. p53 may inhibit viral infection of the cells through the inhibition of EBNA2 transcriptional activation. From the reporter assay, we can know that p53 can inhibit the activity of EB viral promoters which initiate by EBNA2, even under the circumstances of the co-activation of EBNALP and EBNA2, several p53 deletion plasmids were analyzed for essential inhibition of EBNA2. Including the N-terminal transcription activation domain (AD), proline rich (Pro), central DNA binding domain (DBD), oligmerlization (Olg), and the C-terminal regulatory region (C) . AD is necessary for suppressing EBNA2. The Pro and Olg domain were implicated in down-regulation of EBNALP co-activation with EBNA2 specifically. From the results above, we can infer that p53 may have different molecular mechanisms for inhibiting EBNA2 and EBNALP.
AD ( aa 60-70) is important for down-regulation of EBNA2 by reporter assay. The immunoprecipitation assay showed that p53 AD was interacting with EBNA2, the other two deletion plasmids p53 AD1 (aa 1-39) and AD2 (aa 40-70) was AD2 and EBNA2 in interaction is more obvious. This result allows us to infer that p53 may inhibit EBNA2 dominated EB virus promoter activity through interaction with EBNA2 by AD region.

致謝詞 II
中文摘要 III
英文摘要 IV
緒論 1
材料與方法 7
結果 10
討論 15
圖表與說明 19
參考文獻 31

1.Bajaj, B.G., Murakami, M., and Robertson, E.S. (2007). Molecular biology of EBV in relationship to AIDS-associated oncogenesis. Cancer Treat. Res. 133, 141–162.
2.Buck, M., Cross, S., Krauer, K., Kienzle, N., and Sculley, T.B. (1999). A-type and B-type Epstein-Barr virus differ in their ability to spontaneously enter the lytic cycle. J. Gen. Virol. 80 ( Pt 2), 441–445.
3.Bullock, A.N., Henckel, J., DeDecker, B.S., Johnson, C.M., Nikolova, P.V., Proctor, M.R., Lane, D.P., and Fersht, A.R. (1997). Thermodynamic stability of wild-type and mutant p53 core domain. Proc. Natl. Acad. Sci. 94, 14338–14342.
4.Burkitt, D. (1958). A sarcoma involving the jaws in african children. Br. J. Surg. 46, 218–223.
5.Canadillas, J.M.P., Tidow, H., Freund, S.M.V., Rutherford, T.J., Ang, H.C., and Fersht, A.R. (2006). Solution structure of p53 core domain: Structural basis for its instability. Proc. Natl. Acad. Sci. U. S. A. 103, 2109–2114.
6.Candau, R., Scolnick, D.M., Darpino, P., Ying, C.Y., Halazonetis, T.D., and Berger, S.L. (1997). Two tandem and independent sub-activation domains in the amino terminus of p53 require the adaptor complex for activity. Oncogene 15, 807–816.
7.Carbone, A., Gloghini, A., and Dotti, G. (2008). EBV-associated lymphoproliferative disorders: classification and treatment. The Oncologist 13, 577–585.
8.Chabot, P.R., Raiola, L., Lussier-Price, M., Morse, T., Arseneault, G., Archambault, J., and Omichinski, J.G. (2014). Structural and Functional Characterization of a Complex between the Acidic Transactivation Domain of EBNA2 and the Tfb1/p62 Subunit of TFIIH. PLoS Pathog 10, e1004042.
9.Chang, J., Kim, D.H., Lee, S.W., Choi, K.Y., and Sung, Y.C. (1995). Transactivation ability of p53 transcriptional activation domain is directly related to the binding affinity to TATA-binding protein. J. Biol. Chem. 270, 25014–25019.
10.Cohen, J.I., and Kieff, E. (1991). An Epstein-Barr virus nuclear protein 2 domain essential for transformation is a direct transcriptional activator. J. Virol. 65, 5880–5885.
11.Epstein, M.., Achong, B.., and Barr, Y.. (1964a). Virus particles in cultured Lymphoblasts from Burkitt’s Lymphoma. The Lancet 283, 702–703.
12.Evans, T.J., Farrell, P.J., and Swaminathan, S. (1996). Molecular genetic analysis of Epstein-Barr virus Cp promoter function. J. Virol. 70, 1695–1705.
13.FRED, H.K., Sf, T., T.K., and Kieff. (1991). Epstein-Barr virus nuclear protein 2 transactivates a cis-acting CD23 DNA element. J. Virol. 65, 4101–4106.
14.Ferreon, J.C., Lee, C.W., Arai, M., Martinez-Yamout, M.A., Dyson, H.J., and Wright, P.E. (2009). Cooperative regulation of p53 by modulation of ternary complex formation with CBP/p300 and HDM2. Proc. Natl. Acad. Sci. 106, 6591–6596.
15.Flavell, K.J., and Murray, P.G. (2000). Hodgkin’s disease and the Epstein-Barr virus. Mol. Pathol. 53, 262–269.
16.Gaglia, G., Guan, Y., Shah, J.V., and Lahav, G. (2013). Activation and control of p53 tetramerization in individual living cells. Proc. Natl. Acad. Sci. U. S. A. 110, 15497–15501.
17.Grossman, S.R., Johannsen, E., Tong, X., Yalamanchili, R., and Kieff, E. (1994). The Epstein-Barr virus nuclear antigen 2 transactivator is directed to response elements by the J kappa recombination signal binding protein. Proc. Natl. Acad. Sci. U. S. A. 91, 7568–7572.
18.Harada, S., and Kieff, E. (1997). Epstein-Barr virus nuclear protein LP stimulates EBNA-2 acidic domain-mediated transcriptional activation. J. Virol. 71, 6611–6618.
19.Henkel, T., Ling, P.D., Hayward, S.D., and Peterson, M.G. (1994). Mediation of Epstein-Barr virus EBNA2 transactivation by recombination signal-binding protein J kappa. Science 265, 92–95.
20.Holowaty, M.N., Sheng, Y., Nguyen, T., Arrowsmith, C., and Frappier, L. (2003). Protein interaction domains of the ubiquitin-specific protease, USP7/HAUSP. J. Biol. Chem. 278, 47753–47761.
21.Jiménez-Ramírez, C., Brooks, A.J., Forshell, L.P., Yakimchuk, K., Zhao, B., Fulgham, T.Z., and Sample, C.E. (2006). Epstein-Barr virus EBNA-3C is targeted to and regulates expression from the bidirectional LMP-1/2B promoter. J. Virol. 80, 11200–11208.
22.Johannsen, E., Koh, E., Mosialos, G., Tong, X., Kieff, E., and Grossman, S.R. (1995a). Epstein-Barr virus nuclear protein 2 transactivation of the latent membrane protein 1 promoter is mediated by J kappa and PU.1. J. Virol. 69, 253–262.
23.Kaiser, C., Laux, G., Eick, D., Jochner, N., Bornkamm, G.W., and Kempkes, B. (1999). The proto-oncogene c-myc is a direct target gene of Epstein-Barr virus nuclear antigen 2. J. Virol. 73, 4481–4484.
24.Kelly, R.D.W., and Cowley, S.M. (2013). The physiological roles of histone deacetylase (HDAC) 1 and 2: complex co-stars with multiple leading parts. Biochem. Soc. Trans. 41, 741–749.
25.Lane, D.P., and Crawford, L.V. (1979). T antigen is bound to a host protein in SY40-transformed cells. Nature 278, 261–263.
26.Larsen, S., Yokochi, T., Isogai, E., Nakamura, Y., Ozaki, T., and Nakagawara, A. (2010). LMO3 interacts with p53 and inhibits its transcriptional activity. Biochem. Biophys. Res. Commun. 392, 252–257.
27.Lin, J., Johannsen, E., Robertson, E., and Kieff, E. (2002). Epstein-Barr virus nuclear antigen 3C putative repression domain mediates coactivation of the LMP1 promoter with EBNA-2. J. Virol. 76, 232–242.
28.Ling, P.D., Peng, R.S., Nakajima, A., Yu, J.H., Tan, J., Moses, S.M., Yang, W.-H., Zhao, B., Kieff, E., Bloch, K.D., et al. (2005). Mediation of Epstein–Barr virus EBNA‐LP transcriptional coactivation by Sp100. EMBO J. 24, 3565–3575.
29.Meek, D.W., and Anderson, C.W. (2009). Posttranslational Modification of p53: Cooperative Integrators of Function. Cold Spring Harb. Perspect. Biol. 1, a000950.
30.Middeldorp, J.M., Brink, A.A.T.P., van den Brule, A.J.C., and Meijer, C.J.L.M. (2003). Pathogenic roles for Epstein-Barr virus (EBV) gene products in EBV-associated proliferative disorders. Crit. Rev. Oncol. Hematol. 45, 1–36.
31.Moll, U.M., and Petrenko, O. (2003). The MDM2-p53 Interaction. Mol. Cancer Res. 1, 1001–1008.
32.Murphy, M., Ahn, J., Walker, K.K., Hoffman, W.H., Evans, R.M., Levine, A.J., and George, D.L. (1999). Transcriptional repression by wild-type p53 utilizes histone deacetylases, mediated by interaction with mSin3a. Genes Dev. 13, 2490–2501.
33.Nag, S., Qin, J., Srivenugopal, K.S., Wang, M., and Zhang, R. (2013). The MDM2-p53 pathway revisited. J. Biomed. Res. 27, 254–271.
34.Nikitin, P.A., Yan, C.M., Forte, E., Bocedi, A., Tourigny, J.P., White, R.E., Allday, M.J., Patel, A., Dave, S.S., Kim, W., et al. (2010). An ATM/Chk2-mediated DNA damage-responsive signaling pathway suppresses Epstein-Barr virus transformation of primary human B cells. Cell Host Microbe 8, 510–522.
35.Olivier, M., Eeles, R., Hollstein, M., Khan, M.A., Harris, C.C., and Hainaut, P. (2002). The IARC TP53 database: new online mutation analysis and recommendations to users. Hum. Mutat. 19, 607–614.
36.Peng, C.-W., Zhao, B., and Kieff, E. (2004a). Four EBNA2 Domains Are Important for EBNALP Coactivation. J. Virol. 78, 11439–11442.
37.Peng, C.-W., Xue, Y., Zhao, B., Johannsen, E., Kieff, E., and Harada, S. (2004b). Direct interactions between Epstein–Barr virus leader protein LP and the EBNA2 acidic domain underlie coordinate transcriptional regulation. Proc. Natl. Acad. Sci. U. S. A. 101, 1033–1038.
38.Peng, C.-W., Zhao, B., Chen, H.-C., Chou, M.-L., Lai, C.-Y., Lin, S.-Z., Hsu, H.-Y., and Kieff, E. (2007). Hsp72 up-regulates Epstein-Barr virus EBNALP coactivation with EBNA2. Blood 109, 5447–5454.
39.Portal, D., Zhou, H., Zhao, B., Kharchenko, P.V., Lowry, E., Wong, L., Quackenbush, J., Holloway, D., Jiang, S., Lu, Y., et al. (2013). Epstein-Barr virus nuclear antigen leader protein localizes to promoters and enhancers with cell transcription factors and EBNA2. Proc. Natl. Acad. Sci. U. S. A. 110, 18537–18542.
40.Sadowski, I., Ma, J., Triezenberg, S., and Ptashne, M. (1988). GAL4-VP16 is an unusually potent transcriptional activator. Nature 335, 563–564.
41.Saha, A., and Robertson, E.S. (2011). Epstein-Barr virus-associated B-cell lymphomas: pathogenesis and clinical outcomes. Clin. Cancer Res. Off. J. Am. Assoc. Cancer Res. 17, 3056–3063.
42.Sample, J., Hummel, M., Braun, D., Birkenbach, M., and Kieff, E. (1986). Nucleotide sequences of mRNAs encoding Epstein-Barr virus nuclear proteins: a probable transcriptional initiation site. Proc. Natl. Acad. Sci. U. S. A. 83, 5096–5100.
43.Sample, J., Young, L., Martin, B., Chatman, T., Kieff, E., Rickinson, A., and Kieff, E. (1990). Epstein-Barr virus types 1 and 2 differ in their EBNA-3A, EBNA-3B, and EBNA-3C genes. J. Virol. 64, 4084–4092.
44.Saridakis, V., Sheng, Y., Sarkari, F., Holowaty, M.N., Shire, K., Nguyen, T., Zhang, R.G., Liao, J., Lee, W., Edwards, A.M., et al. (2005). Structure of the p53 binding domain of HAUSP/USP7 bound to Epstein-Barr nuclear antigen 1 implications for EBV-mediated immortalization. Mol. Cell 18, 25–36.
45.Shi, Y., Mosser, D.D., and Morimoto, R.I. (1998). Molecular chaperones as HSF1-specific transcriptional repressors. Genes Dev. 12, 654–666.
46.Shortt, S.E.D., and Haynes, E.R. (1986). Chronic Mononucleosis Syndrome. Can. Fam. Physician 32, 1125–1129.
47.Sinclair, A.J., Palmero, I., Peters, G., and Farrell, P.J. (1994). EBNA-2 and EBNA-LP cooperate to cause G0 to G1 transition during immortalization of resting human B lymphocytes by Epstein-Barr virus. EMBO J. 13, 3321–3328.
48.Sjöblom, A., Nerstedt, A., Jansson, A., and Rymo, L. (1995). Domains of the Epstein-Barr virus nuclear antigen 2 (EBNA2) involved in the transactivation of the latent membrane protein 1 and the EBNA Cp promoters. J. Gen. Virol. 76 ( Pt 11), 2669–2678.
49.Sung, N.S., Kenney, S., Gutsch, D., and Pagano, J.S. (1991). EBNA-2 transactivates a lymphoid-specific enhancer in the BamHI C promoter of Epstein-Barr virus. J. Virol. 65, 2164–2169.
50.Szekely, L., Selivanova, G., Magnusson, K.P., Klein, G., and Wiman, K.G. (1993). EBNA-5, an Epstein-Barr virus-encoded nuclear antigen, binds to the retinoblastoma and p53 proteins. Proc. Natl. Acad. Sci. U. S. A. 90, 5455–5459.
51.Tanikawa, J., Nomura, T., Macmillan, E.M., Shinagawa, T., Jin, W., Kokura, K., Baba, D., Shirakawa, M., Gonda, T.J., and Ishii, S. (2004). p53 suppresses c-Myb-induced trans-activation and transformation by recruiting the corepressor mSin3A. J. Biol. Chem. 279, 55393–55400.
52.Thorley-Lawson, D.A., and Gross, A. (2004). Persistence of the Epstein–Barr Virus and the Origins of Associated Lymphomas. N. Engl. J. Med. 350, 1328–1337.
53.Tong, X., Wang, F., Thut, C.J., and Kieff, E. (1995a). The Epstein-Barr virus nuclear protein 2 acidic domain can interact with TFIIB, TAF40, and RPA70 but not with TATA-binding protein. J. Virol. 69, 585–588.
54.Tong, X., Drapkin, R., Yalamanchili, R., Mosialos, G., and Kieff, E. (1995b). The Epstein-Barr virus nuclear protein 2 acidic domain forms a complex with a novel cellular coactivator that can interact with TFIIE. Mol. Cell. Biol. 15, 4735–4744.
55.Tsui, S., and Schubach, W.H. (1994). Epstein-Barr virus nuclear protein 2A forms oligomers in vitro and in vivo through a region required for B-cell transformation. J. Virol. 68, 4287–4294.
56.Unger, T., Mietz, J.A., Scheffner, M., Yee, C.L., and Howley, P.M. (1993). Functional domains of wild-type and mutant p53 proteins involved in transcriptional regulation, transdominant inhibition, and transformation suppression. Mol. Cell. Biol. 13, 5186–5194.
57.Venot, C., Maratrat, M., Dureuil, C., Conseiller, E., Bracco, L., and Debussche, L. (1998). The requirement for the p53 proline-rich functional domain for mediation of apoptosis is correlated with specific PIG3 gene transactivation and with transcriptional repression. EMBO J. 17, 4668–4679.
58.Venot, C., Maratrat, M., Sierra, V., Conseiller, E., and Debussche, L. (1999). Definition of a p53 transactivation function-deficient mutant and characterization of two independent p53 transactivation subdomains. Oncogene 18, 2405–2410.
59.Virdis, F., Tacci, S., Messina, F., and Varcada, M. (2013). Hemophagocytic lymphohistiocytosis caused by primary Epstein-Barr virus in patient with Crohn’s disease. World J. Gastrointest. Surg. 5, 306–308.
60.Woisetschlaeger, M., Jin, X.W., Yandava, C.N., Furmanski, L.A., Strominger, J.L., and Speck, S.H. (1991). Role for the Epstein-Barr virus nuclear antigen 2 in viral promoter switching during initial stages of infection. Proc. Natl. Acad. Sci. U. S. A. 88, 3942–3946.
61.Wong, K.-B., DeDecker, B.S., Freund, S.M.V., Proctor, M.R., Bycroft, M., and Fersht, A.R. (1999). Hot-spot mutants of p53 core domain evince characteristic local structural changes. Proc. Natl. Acad. Sci. 96, 8438–8442.
62.Zhao, B., Zou, J., Wang, H., Johannsen, E., Peng, C., Quackenbush, J., Mar, J.C., Morton, C.C., Freedman, M.L., Blacklow, S.C., et al. (2011). Epstein-Barr virus exploits intrinsic B-lymphocyte transcription programs to achieve immortal cell growth. Proc. Natl. Acad. Sci. U. S. A. 108, 14902–14907.
63.Zhu, J., Zhou, W., Jiang, J., and Chen, X. (1998). Identification of a novel p53 functional domain that is necessary for mediating apoptosis. J. Biol. Chem. 273, 13030–13036.
64.Zilfou, J.T., Hoffman, W.H., Sank, M., George, D.L., and Murphy, M. (2001). The corepressor mSin3a interacts with the proline-rich domain of p53 and protects p53 from proteasome-mediated degradation. Mol. Cell. Biol. 21, 3974–3985.
65.Zimber-Strobl, U., and Strobl, L.J. (2001). EBNA2 and Notch signalling in Epstein-Barr virus mediated immortalization of B lymphocytes. Semin. Cancer Biol. 11, 423–434.