目前已經研發出的抗黃質病毒的活疫苗只有黃熱病毒 (Yellow fever virus) (17D strain) 與日本腦炎病毒 (JEV) (SA-14-14-2 strain)，目前已利用反向基因 (reverse genetic) 和重組DNA技術，生產嵌合黃質病毒，作為活性減毒疫苗 (live attenuated vaccine) 來對抗其他黃質病毒感染。人類對黃熱病毒來說是擴增宿主，但日本腦炎病毒是終點宿主 (dead-end host)；所以人類感染日本腦炎病毒時並不會產生高量的病毒血症。在本論文中，藉由JEV感染性質體 (infectious clone) 表達可誘發感染宿主免疫反應的登革病毒 (Dengue virus) 基因，例如登革病毒結構性基因prME或非結構性基因NS1置換至JEV上。經轉染細胞生產JEV/DEN-2嵌合病毒 (chimeric viruses)，並評估是否適合作為活性減毒疫苗藉以對抗登革病毒之感染。此外，為防止已突變完成之病毒回復成野生株，本論文利用定點突變 (site-directed mutagenesis) 的方式，結合至少兩個以上已被報導之黃質病毒相關的弱毒標誌 (attenuated markers)，作為活性減毒疫苗的材料，以期得到更穩定之減弱病毒株，增加此類活性減毒疫苗之安全性。但截至目前為止，尚無生產出嵌合病毒及含有兩個以上弱毒標誌的病毒。
本論文另一個研究重點是藉由突變之日本腦炎病毒E和NS1蛋白質對細胞ER stress的影響。對黃質病毒來說，細胞的內質網是黃質病毒RNA複製、醣化蛋白質 (glycoprotein) 合成以及病毒顆粒成熟的主要場所。日本腦炎病毒感染細胞後所引發細胞凋亡，也是與內質網壓力 (ER stress) 的訊息傳遞有關。過去研究均指出黃質病毒感染細胞時，會引發ER stress及未摺疊蛋白質反應 (UPR)。我們藉由RT-PCR及Dual-
Luciferase Assay偵測細胞中X-box binding protein 1 splicing form (XBP1s)，分析細胞ER stress的程度。結果顯示：E蛋白質S331R與Q52K之單點突變病毒株所引發之ER stress明顯較野生株病毒小，然而NS1蛋白質P250L與T132A之單點突變病毒株則引發較強的ER stress。為區隔其他病毒蛋白對ER stress的干擾，進一步分析單獨表現E與NS1對細胞ER stress的影響。所有突變E蛋白質引發之ER stress均強於野生株E蛋白質。而當NS1蛋白質無法形成dimer或無法醣化時均會造成強的ER stress。綜合上述可知JEV的醣化蛋白E和NS1在病毒感染細胞所引發之ER stress扮演著重要的角色，減弱病毒株的E或NS1蛋白質會造成病毒引發細胞XBP1s程度的不同，推測病毒藉由E或NS1蛋白質調控UPR，影響細胞修復或凋亡，進而影響病毒本身在細胞中複製、包裝等特性。

In recent years, mosquito-borne infections are reemerging or emerging worldwide at an alarming rate. Thus, their vaccine development remains a top priority on the public health agenda. The current licensed live attenuated vaccines against flaviviral infection include yellow fever virus YF-17D and Japanese encephalitis virus (JEV) 14-14-2. Characteristically, human are the dead-end host for JEV infection, but not YFV, because of insufficient viremia. Therefore, we use JEV genomic RNA as a backbone to develop chimeric live attenuated vaccine against other flaviviral infection. Hopefully, the chimeric viruses may be able to trigger a protective immunity but incapable of transmitting from human back to mosquitoes. We have replaced the structural or non-structural proteins from other flavivirus into JEV genomic RNA, and then evaluate the vaccine capability of these chimeric viruses. To prevent the given mutant viruses revert to wild-type strain, we combine at least two attenuated markers into one backbone of JEV infectious clone by site-directed mutagenesis. So far, we have not generated any such chimeric viruses, and the construction process is currently underway.
ER is the primary site of flaviviral glycoprotein synthesis, viral genomic RNA replication, and virus particle maturation. ER stress-mediated signaling has been implicated in the JEV-induced apoptosis. We in this study investigate how the ER stress could be induced by mutant E or NS1 proteins. ER stress was monitored by analysis XBP1 splicing signal using RT-PCR and Luciferase reporter system. We found mutant viruses P0004 (E, S331R) and P0006 (E, Q52K) could obviously induce less ER stress compared with other mutants and the wild-type RP9/X. Additionally, mutant viruses P0011 (NS1, P250L) and P0016 (NS1, T132A) induce stronger ER stress than wild-type RP9/X. To exclude other viral proteins affect ER stress, we have individually expressed E or NS1 protein to clarify the correlation between attenuate makers and ER stress. We found the E mutant protein induce stronger ER stress than the wild-type E, whereas NS1 mutants of glycosylation (P0016) or dimerisation (P0011) induce stronger ER stress than the wild-type. The result suggests that JEV glycoproteins E and NS1 have important role of virus-induced ER stress. Virus may regulate UPR by E or NS1 protein to influence cell repair or apoptosis, and further to influence replication and assembly of virus itself in cell.

誌謝
目錄 I
圖表目錄 III
中文摘要 V
Abstract VI
第一章 前言 1
第二章 材料與方法 7
一、細胞培養及病毒株 8
二、本論文所使用的抗體 8
三、本論文所使用之引子 (Primers) 9
四、微量質體DNA的製備 10
五、質體DNA的大量製備 10
六、勝任細胞 (Competent cell) 製備及其轉型作用 (Transformation) 11
七、利用LipofectamineTM 2000進行細胞的轉染反應 (Transfection) 11
八、建立持續表現蛋白質之細胞株 (Permanent cell line) 12
九、西方墨點法 (Western blot) 12
十、質體於試管中進行轉錄合成RNA (In vitro transcription) 14
十一、RNA 轉染細胞反應 (RNA transfection) 14
十二、病毒之擴增 (Virus amplification) 15
十三、病毒效價測定 (Plaque assay) 15
十四、間接免疫螢光染色法 (Indirect immunofluorescent assay) 16
十五、DNA 聚合酵素連鎖反應 (Polymerase chain reaction; PCR) 16
十六、單一引子DNA聚合酵素連鎖反應 (Single primer PCR) 17
十七、萃取細胞之RNA 17
十八、反轉錄聚合酵素連鎖反應 (RT-PCR) 18
十九、Dual-Luciferase assay 18
第三章 實驗結果 19
壹、嵌合感染性質體之構築與嵌合病毒之生產 20
一、日本腦炎病毒 (JEV) 感染性質體之構築 (圖一) 20
二、JEV/DEN-2嵌合感染性質體之構築 20
1.構築嵌合感染性質體RP9-D2prME/pBR322 21
2.構築嵌合感染性質體RP9-D2NS1/pBR322 22
三、生產JEV/DEN-2之嵌合病毒 (圖六) 23
四、建立持續表現JEV病毒蛋白prM/M+E或NS1的細胞株 24
貳、發展更穩定之抗日本腦炎活性減毒疫苗 24
一、構築點突變之感染性質體以降低病毒毒力 24
二、結合至少兩個以上之病毒弱毒標誌 25
參、日本腦炎病毒醣化蛋白E和NS1引發細胞ER stress之研究 26
一、不同突變之JEV引發ER stress的差異 26
1.利用RT-PCR分析細胞中XBP內含子 (intron) 是否移除 26
2.利用Luciferase活性定量XBP1 intron被移除的程度 27
3.病毒突變蛋白質表現和病毒的一步生長曲線 (one-step growth curve) 28
二、JEV之不同突變醣化蛋白質對細胞ER stress之影響 29
1.構築突變JEV E蛋白質的表現質體並分析表現 29
2.比較不同突變的E蛋白質對細胞ER stress的影響 30
3.構築突變JEV NS1蛋白質的表現質體並分析表現 30
4.比較不同突變的NS1對細胞ER stress的影響 31
第四章 討論 32
一、發展以日本腦炎作為骨架的活性減毒疫苗 33
二、探討野生株與突變株JEV對於細胞ER stress的影響 35
三、探討JEV醣化蛋白質E和NS1對於細胞ER stress的影響 37
1.JEV醣化蛋白E對於細胞ER stress的影響 37
2.JEV醣化蛋白NS1對於細胞ER stress的影響 38
第五章 參考文獻 41
第六章 圖與表 48
第七章 附錄 73

1.Barrett, A. D. 2001. Current status of flavivirus vaccines. Ann N Y Acad Sci 951:262-71.
2.Bertolotti, A., Y. Zhang, L. M. Hendershot, H. P. Harding, and D. Ron. 2000. Dynamic interaction of BiP and ER stress transducers in the unfolded-protein response. Nat Cell Biol 2:326-32.
3.Boyce, M., and J. Yuan. 2006. Cellular response to endoplasmic reticulum stress: a matter of life or death. Cell Death Differ 13:363-73.
4.Burke, D. S., and T. P. Monath. 2001. Flaviviruses, p. 1043-1125. In D. M. Knipe and P. M. Howley (ed.), Fields Virology, 4th ed, vol. 1. Lippincott-Williams & Wilkins, Philadelphia, Pa., USA.
5.Calfon, M., H. Zeng, F. Urano, J. H. Till, S. R. Hubbard, H. P. Harding, S. G. Clark, and D. Ron. 2002. IRE1 couples endoplasmic reticulum load to secretory capacity by processing the XBP-1 mRNA. Nature 415:92-6.
6.Chambers, T. J., C. S. Hahn, R. Galler, and C. M. Rice. 1990. Flavivirus genome organization, expression, and replication. Annu Rev Microbiol 44:649-88.
7.Chambers, T. J., Y. Liang, D. A. Droll, J. J. Schlesinger, A. D. Davidson, P. J. Wright, and X. Jiang. 2003. Yellow fever virus/dengue-2 virus and yellow fever virus/dengue-4 virus chimeras: biological characterization, immunogenicity, and protection against dengue encephalitis in the mouse model. J Virol 77:3655-68.
8.Chapman, R., C. Sidrauski, and P. Walter. 1998. Intracellular signaling from the endoplasmic reticulum to the nucleus. Annu Rev Cell Dev Biol 14:459-85.
9.Chen, L. K., Y. L. Lin, C. L. Liao, C. G. Lin, Y. L. Huang, C. T. Yeh, S. C. Lai, J. T. Jan, and C. Chin. 1996. Generation and characterization of organ-tropism mutants of Japanese encephalitis virus in vivo and in vitro. Virology 223:79-88.
10.Duncan, B. K. 1985. Isolation of insertion, deletion, and nonsense mutations of the uracil-DNA glycosylase (ung) gene of Escherichia coli K-12. J Bacteriol 164:689-95.
11.Fiedler, K., and K. Simons. 1995. The role of N-glycans in the secretory pathway. Cell 81:309-12.
12.Flamand, M., V. Deubel, and M. Girard. 1992. Expression and secretion of Japanese encephalitis virus nonstructural protein NS1 by insect cells using a recombinant baculovirus. Virology 191:826-36.
13.Guirakhoo, F., J. Arroyo, K. V. Pugachev, C. Miller, Z. X. Zhang, R. Weltzin, K. Georgakopoulos, J. Catalan, S. Ocran, K. Soike, M. Ratterree, and T. P. Monath. 2001. Construction, safety, and immunogenicity in nonhuman primates of a chimeric yellow fever-dengue virus tetravalent vaccine. J Virol 75:7290-304.
14.Hall, R. A., A. A. Khromykh, J. M. Mackenzie, J. H. Scherret, T. I. Khromykh, and J. S. Mackenzie. 1999. Loss of dimerisation of the nonstructural protein NS1 of Kunjin virus delays viral replication and reduces virulence in mice, but still allows secretion of NS1. Virology 264:66-75.
15.Harding, H. P., M. Calfon, F. Urano, I. Novoa, and D. Ron. 2002. Transcriptional and translational control in the Mammalian unfolded protein response. Annu Rev Cell Dev Biol 18:575-99.
16.Hase, T., P. L. Summers, P. Ray, and E. Asafo-Adjei. 1992. Cytopathology of PC12 cells infected with Japanese encephalitis virus. Virchows Arch B Cell Pathol Incl Mol Pathol 63:25-36.
17.He, B. 2006. Viruses, endoplasmic reticulum stress, and interferon responses. Cell Death Differ (in press).
18.Hetz, C., P. Bernasconi, J. Fisher, A. H. Lee, M. C. Bassik, B. Antonsson, G. S. Brandt, N. N. Iwakoshi, A. Schinzel, L. H. Glimcher, and S. J. Korsmeyer. 2006. Proapoptotic BAX and BAK modulate the unfolded protein response by a direct interaction with IRE1alpha. Science 312:572-6.
19.Holden, K. L., and E. Harris. 2004. Enhancement of dengue virus translation: role of the 3' untranslated region and the terminal 3' stem-loop domain. Virology 329:119-33.
20.Huang, C. Y., S. J. Silengo, M. C. Whiteman, and R. M. Kinney. 2005. Chimeric dengue 2 PDK-53/West Nile NY99 viruses retain the phenotypic attenuation markers of the candidate PDK-53 vaccine virus and protect mice against lethal challenge with West Nile virus. J Virol 79:7300-10.
21.Kaufman, R. J. 1999. Stress signaling from the lumen of the endoplasmic reticulum: coordination of gene transcriptional and translational controls. Genes Dev 13:1211-33.
22.Keelapang, P., R. Sriburi, S. Supasa, N. Panyadee, A. Songjaeng, A. Jairungsri, C. Puttikhunt, W. Kasinrerk, P. Malasit, and N. Sittisombut. 2004. Alterations of pr-M cleavage and virus export in pr-M junction chimeric dengue viruses. J Virol 78:2367-81.
23.Kozutsumi, Y., M. Segal, K. Normington, M. J. Gething, and J. Sambrook. 1988. The presence of malfolded proteins in the endoplasmic reticulum signals the induction of glucose-regulated proteins. Nature 332:462-4.
24.Kuno, G., G. J. Chang, K. R. Tsuchiya, N. Karabatsos, and C. B. Cropp. 1998. Phylogeny of the genus Flavivirus. J Virol 72:73-83.
25.Lai, C. J., B. T. Zhao, H. Hori, and M. Bray. 1991. Infectious RNA transcribed from stably cloned full-length cDNA of dengue type 4 virus. Proc Natl Acad Sci U S A 88:5139-43.
26.Lee, A. H., N. N. Iwakoshi, K. C. Anderson, and L. H. Glimcher. 2003. Proteasome inhibitors disrupt the unfolded protein response in myeloma cells. Proc Natl Acad Sci U S A 100:9946-51.
27.Lee, E., and M. Lobigs. 2000. Substitutions at the putative receptor-binding site of an encephalitic flavivirus alter virulence and host cell tropism and reveal a role for glycosaminoglycans in entry. J Virol 74:8867-75.
28.Lidbury, B. A., and S. Mahalingam. 2000. Specific ablation of antiviral gene expression in macrophages by antibody-dependent enhancement of Ross River virus infection. J Virol 74:8376-81.
29.Lindenbach, B. D., and C. M. Rice. 2001. Flaviviridae: The viruses and their replication, p. 991-1041. In D. M. Knipe and P. M. Howley (ed.), Fields Virology, 4th ed, vol. 1. Lippincott-Williams & Wilkins, Philadelphia, Pa., USA.
30.Liu, C. Y., M. Schroder, and R. J. Kaufman. 2000. Ligand-independent dimerization activates the stress response kinases IRE1 and PERK in the lumen of the endoplasmic reticulum. J Biol Chem 275:24881-5.
31.Liu, C. Y., Z. Xu, and R. J. Kaufman. 2003. Structure and intermolecular interactions of the luminal dimerization domain of human IRE1alpha. J Biol Chem 278:17680-7.
32.Lorenz, I. C., S. L. Allison, F. X. Heinz, and A. Helenius. 2002. Folding and dimerization of tick-borne encephalitis virus envelope proteins prM and E in the endoplasmic reticulum. J Virol 76:5480-91.
33.Ma, K., K. M. Vattem, and R. C. Wek. 2002. Dimerization and release of molecular chaperone inhibition facilitate activation of eukaryotic initiation factor-2 kinase in response to endoplasmic reticulum stress. J Biol Chem 277:18728-35.
34.Mackenzie, J. S., D. J. Gubler, and L. R. Petersen. 2004. Emerging flaviviruses: the spread and resurgence of Japanese encephalitis, West Nile and dengue viruses. Nat Med 10:S98-109.
35.Mori, K. 2003. Frame switch splicing and regulated intramembrane proteolysis: key words to understand the unfolded protein response. Traffic 4:519-28.
36.Mukhopadhyay, S., R. J. Kuhn, and M. G. Rossmann. 2005. A structural perspective of the flavivirus life cycle. Nat Rev Microbiol 3:13-22.
37.Ng, D. T., S. W. Hiebert, and R. A. Lamb. 1990. Different roles of individual N-linked oligosaccharide chains in folding, assembly, and transport of the simian virus 5 hemagglutinin-neuraminidase. Mol Cell Biol 10:1989-2001.
38.Ng, D. T., E. D. Spear, and P. Walter. 2000. The unfolded protein response regulates multiple aspects of secretory and membrane protein biogenesis and endoplasmic reticulum quality control. J Cell Biol 150:77-88.
39.Okada, T., H. Yoshida, R. Akazawa, M. Negishi, and K. Mori. 2002. Distinct roles of activating transcription factor 6 (ATF6) and double-stranded RNA-activated protein kinase-like endoplasmic reticulum kinase (PERK) in transcription during the mammalian unfolded protein response. Biochem J 366:585-94.
40.Pahl, H. L. 1999. Signal transduction from the endoplasmic reticulum to the cell nucleus. Physiol Rev 79:683-701.
41.Peiris, J. S., and J. S. Porterfield. 1979. Antibody-mediated enhancement of Flavivirus replication in macrophage-like cell lines. Nature 282:509-11.
42.Rutkowski, D. T., and R. J. Kaufman. 2004. A trip to the ER: coping with stress. Trends Cell Biol 14:20-8.
43.Shen, J., X. Chen, L. Hendershot, and R. Prywes. 2002. ER stress regulation of ATF6 localization by dissociation of BiP/GRP78 binding and unmasking of Golgi localization signals. Dev Cell 3:99-111.
44.Shen, X., R. E. Ellis, K. Lee, C. Y. Liu, K. Yang, A. Solomon, H. Yoshida, R. Morimoto, D. M. Kurnit, K. Mori, and R. J. Kaufman. 2001. Complementary signaling pathways regulate the unfolded protein response and are required for C. elegans development. Cell 107:893-903.
45.Shen, X., K. Zhang, and R. J. Kaufman. 2004. The unfolded protein response--a stress signaling pathway of the endoplasmic reticulum. J Chem Neuroanat 28:79-92.
46.Sidrauski, C., J. S. Cox, and P. Walter. 1996. tRNA ligase is required for regulated mRNA splicing in the unfolded protein response. Cell 87:405-13.
47.Simmonds, P., and D. B. Smith. 1999. Structural constraints on RNA virus evolution. J Virol 73:5787-94.
48.Su, H. L., C. L. Liao, and Y. L. Lin. 2002. Japanese encephalitis virus infection initiates endoplasmic reticulum stress and an unfolded protein response. J Virol 76:4162-71.
49.Su, H. L., Y. L. Lin, H. P. Yu, C. H. Tsao, L. K. Chen, Y. T. Liu, and C. L. Liao. 2001. The effect of human bcl-2 and bcl-X genes on dengue virus-induced apoptosis in cultured cells. Virology 282:141-53.
50.Thurner, C., C. Witwer, I. L. Hofacker, and P. F. Stadler. 2004. Conserved RNA secondary structures in Flaviviridae genomes. J Gen Virol 85:1113-24.
51.Tilgner, M., and P. Y. Shi. 2004. Structure and function of the 3' terminal six nucleotides of the west nile virus genome in viral replication. J Virol 78:8159-71.
52.Tirasophon, W., A. A. Welihinda, and R. J. Kaufman. 1998. A stress response pathway from the endoplasmic reticulum to the nucleus requires a novel bifunctional protein kinase/endoribonuclease (Ire1p) in mammalian cells. Genes Dev 12:1812-24.
53.Travers, K. J., C. K. Patil, L. Wodicka, D. J. Lockhart, J. S. Weissman, and P. Walter. 2000. Functional and genomic analyses reveal an essential coordination between the unfolded protein response and ER-associated degradation. Cell 101:249-58.
54.Tuplin, A., J. Wood, D. J. Evans, A. H. Patel, and P. Simmonds. 2002. Thermodynamic and phylogenetic prediction of RNA secondary structures in the coding region of hepatitis C virus. Rna 8:824-41.
55.van Der Most, R. G., K. Murali-Krishna, R. Ahmed, and J. H. Strauss. 2000. Chimeric yellow fever/dengue virus as a candidate dengue vaccine: quantitation of the dengue virus-specific CD8 T-cell response. J Virol 74:8094-101.
56.Varnavski, A. N., P. R. Young, and A. A. Khromykh. 2000. Stable high-level expression of heterologous genes in vitro and in vivo by noncytopathic DNA-based Kunjin virus replicon vectors. J Virol 74:4394-403.
57.Wang, X. Z., H. P. Harding, Y. Zhang, E. M. Jolicoeur, M. Kuroda, and D. Ron. 1998. Cloning of mammalian Ire1 reveals diversity in the ER stress responses. Embo J 17:5708-17.
58.Wu, J., and R. J. Kaufman. 2006. From acute ER stress to physiological roles of the Unfolded Protein Response. Cell Death Differ 13:374-84.
59.Wu, S. C., and S. C. Lee. 2001. Complete nucleotide sequence and cell-line multiplication pattern of the attenuated variant CH2195LA of Japanese encephalitis virus. Virus Res 73:91-102.
60.Wu, S. F., C. L. Liao, Y. L. Lin, C. T. Yeh, L. K. Chen, Y. F. Huang, H. Y. Chou, J. L. Huang, M. F. Shaio, and H. K. Sytwu. 2003. Evaluation of protective efficacy and immune mechanisms of using a non-structural protein NS1 in DNA vaccine against dengue 2 virus in mice. Vaccine 21:3919-29.
61.Ye, J., R. B. Rawson, R. Komuro, X. Chen, U. P. Dave, R. Prywes, M. S. Brown, and J. L. Goldstein. 2000. ER stress induces cleavage of membrane-bound ATF6 by the same proteases that process SREBPs. Mol Cell 6:1355-64.
62.Yoshida, H., T. Matsui, N. Hosokawa, R. J. Kaufman, K. Nagata, and K. Mori. 2003. A time-dependent phase shift in the mammalian unfolded protein response. Dev Cell 4:265-71.
63.Yoshida, H., T. Matsui, A. Yamamoto, T. Okada, and K. Mori. 2001. XBP1 mRNA is induced by ATF6 and spliced by IRE1 in response to ER stress to produce a highly active transcription factor. Cell 107:881-91.
64.Yu, L., and L. Markoff. 2005. The topology of bulges in the long stem of the flavivirus 3' stem-loop is a major determinant of RNA replication competence. J Virol 79:2309-24.
65.Zhao, Z., T. Date, Y. Li, T. Kato, M. Miyamoto, K. Yasui, and T. Wakita. 2005. Characterization of the E-138 (Glu/Lys) mutation in Japanese encephalitis virus by using a stable, full-length, infectious cDNA clone. J Gen Virol 86:2209-20.