在過去幾年中，魚類神經壞死病毒重創了台灣和其他亞洲國家的石斑魚養殖產業，因此探討魚類自然抵抗病原菌的機制是極為重要的工作。感染病毒的細胞可經由外來刺激產生干擾素以抵抗病毒。然而魚類產生干擾素拮抗神經壞死病毒的分子機制還尚未被瞭解，但是干擾素所誘發的一種表現於細胞質的蛋白質：魚類Mx基因，具有對抗核醣核酸病毒的活性。Mx基因是屬於GTPase的一成員，具有和GTPase相似的動力學且和廣泛性細胞內的傳送過程有關。在之前所做的研究中，實驗室已從感染神經壞死病毒的石斑魚身上選殖到點帶石斑的Mx基因，且在石斑魚實驗中證實經由注射神經壞死病毒能引導其Mx基因的表現，也發現石斑魚Mx蛋白具有降低病毒感染之能力。在本篇實驗中以螢光共振能量轉移(FRET)證實了石斑魚Mx蛋白與神經壞死病毒外鞘毒白(CP)有結合反應，並且推測石斑魚Mx蛋白的抗病毒能力是建立在細胞內與神經壞死病毒外鞘蛋白的結合之上，同時也發現帶有不同片段功能性區域的Mx蛋白會干擾病毒外鞘蛋白轉譯後運輸進入細胞核內的現象。Mx蛋白功能性區域分析方面則證實了Mx蛋白的effector domain區域對石斑魚Mx蛋白抗病毒能力有較大影響，且在Mx蛋白尾端的leucine zipper 區域會影響Mx蛋白的表現位置，其中胺基酸Leu609、Leu616、Leu623是影響Mx蛋白表現位置的關鍵氨基酸。此外在Mx基因啟動子序列分析方面證實經過virus genome-like double strand RNA (poly[I:C])的處理下可活化啟動子的表現情形，並且以細胞免疫螢光染色證實經過poly [I:c]誘發內生性Mx蛋白表現後可在病毒感染的早期明顯抑制神經壞死病毒的外鞘蛋白表現情形。

Over the past years, piscine nodavirus has devastated the grouper (Epinephelus coioides) culture industry in Taiwan and other Asian countries and so a better understanding of fish natural defense mechanisms against such pathogens is needed. If cell infected with a virus could be stimulated to produce and secrete interferon (IFNs) to against the virus infection. However, the mechanism of the piscine interferon protection against nodaviruses at the molecular level is still unknown. Piscine Mx is an IFN-induced cytoplasmic protein with antiviral activity against a number of RNA viruses. It belongs to a family of conserved large GTPase and to the superfamily of dynamin-like GTPase, which are involved in a wide range of intracellular transport processes. In previous studies, we have been reported the full sequences of an E. coioides Mx gene from nodavirus-infected grouper. The grouper Mx gene has been shown to be inducible in vivo by injection of live fish with nodavirus. We also found that grouper Mx (gMx) effectively prevented nodavirus infection. In this study, we use the fluorescence resonance energy transfer (FRET) to demonstrate that there is a binding interaction between the gMx protein and the nodavirus coat protein. And we suggest that the antiviral activity of gMx protein was due to the interaction of gMx with nodavirus coat protein in vivo. The transportation of viral coat protein to the nucleus was interrupted by the WT Mx and truncated Mx proteins with different functional domain. In the characterization of the functional domain of Mx protein we demonstrate that the effector domain of Mx protein is able to against the virus infection mainly, and the leucine zipper at the C-terminal of Mx can affect the localization of Mx protein, especially the Leu609、Leu616、Leu623 are the key amino acid. We also demonstrate that the Mx promoter expression can be up-regulated by treating virus genome-like double strand RNA (poly[I:C]), and the cell immunofluorescence staining was demonstrated that after the poly[I:C] induction, the endogenous Mx can inhibit the nodavirus coat protein expression at the early infection stage.

謝佩璇。抗病毒蛋白Mx在點帶石斑魚細胞之特性分析，國立成功大學生物科技研究所碩士論文，2008，台南。

蔡典融。石斑神經壞死病毒傳染途徑及宿主反應特性分析，國立成功大學生物科技研究所碩士論文，2008，台南。

蘇永霖。石斑魚抗病毒蛋白Mx之表現及特性分析，國立成功大學生物科技研究所碩士論文，2005，台南。

Altmann SM, Mellon MT, Distl DL, Kim CH. Molecular and functional analysis of an interferon gene from the zebrafish, Danio rerio. J Virol. 2003;77:1992-2002.

Altmann ST, Mellon, MT, Johnson MC, Paw BH, Trede NS, Zon LI, Kim CH. Cloning and characterization of an Mx gene and its corresponding promoter from the zebrafish, Danio rerio. Dev Comp Immunol. 2004;28:295-306.

Ball LA. Nodavirus RNA Recombination. Semin Virol. 1997;8:95-100.

Charles AJ, Travers P, Walport M, Shlomchik M. Immunobiology, 5th edition-The Immune System in Health and Disease. 2001;chapter 1, p.1-35.

Chen YM, Su YL, Lin JH, Yang HL, Chen TY. Cloning of an orange-spotted grouper (Epinephelus coioides) Mx cDNA and characterization of its expression in response to nodavirus. Fish Shellfish Immunol. 2006;20:58-71.

Chen YM, Su YL, Shie PS, Huang SL, Yang HL, Chen TY. Grouper Mx confers resistance to nodavirus and interacts with coat protein. Dev Comp Immunol. 2008;32:825-836.

Chi SC, Lin SC, Su HM, Hu WW. Temperature effect on nervous necrosis virus infection in grouper cell and gouper larvae. Virus Res. 1999;63:107- 114.

Christopher MA, Hirono I, Aoki T. In vitro inhibition of fish rhabdoviruses by Japanese flounder, Paralichthys olivaceus Mx. Virology. 2003;317:373- 382.

Danino D, Hinshaw JE. Dynamin family of mechanoenzymes. Curr Opini Cell Biol. 2001;13:454-460.

Goodbourn S, Didcock L, Randall RE. Interferon: cell signal, immune modulation, antiviral responses and virus countermeasures. J Gen Virol. 2000;81:2341-2364.

Gordien E, Rosmorduc O, Tekian CP, Garreall F, Brechot C, Kremsdorf D. Inhibition of hepatitis B virus replication by the interferon-inducible MxA protein. J Virol. 2001;75:2684-2691.

Guo YX, Dallmann K, Kwang J. Identification of nucleolus location signal of betanodavirus GGNNV protein α. Virology. 2003;306:225-235.

Haller O, Arnheiter H, Lindenmann J, Gresser I. Host gene influences sensitivity to interferon action selectively for virus. Nature. 1980;283:660- 662.

Hiscox JA, Ball LA. Cotranslational disassembly of flock house virus in a cell-free system. J Virol. 1997;10:7974-7977.

Hiscox JA, Wurm T, Wilson L, Britton P, Cavanagh D, Brooks G. The coronavirus infectious bronchitis virus nucleoprotein localizes to the nucleolus. J Virol. 2001;75:506-512.

Hu G, Yin X, Lou H, Xia J, Dong X, Zhang J, Liu Q. Interferon regulatory factor 3 (IRF-3) in Japanese flounder, Paralichthys olivaceus: sequencing, limited tissue distribution, inducible expression and induction of fish type I interferon promoter. Dev Comp Immunol. 2011;35:164-173.

Jensen I, Robertsen B. Effect of double-stranded RNA and interferon on the antiviral activity of Atlantic salmon cells against infectious salmon anemia virus and infectious pancreatic. Fish Shellfish Immunol. 2002;13:211-241.

Jensen I, Albuquerque A, Sommer AI, Robertsen B. Effect of poly I:C on the expression of Mx proteins and resistance against infection by infectious salmon anaemia virus in Atlantic salmon. Fish Shellfish Immunol. 2002;13: 311-326.

Jensen V, Robertsen B. Cloning of an Mx cDNA from Atlantic halibut (Hippoglossus hippoglossus) and characterization of Mx mRNA expression in responseto double-stranded RNA or infectious pancreatic necrosis virus. J Interferon Cytokine Res. 2000;20:701-710.

King MC, Raposo G, Lemmon MA. Inhibition of nuclear import and cell-cycle progression by mutated forms of the dynamin-like GTPase MxB. Proc Natl Acad Sci USA. 2004;101:8957-8962.

Kochs G, Haller O. Interferon-induced human MxA GTPase blocks nuclear import of Thogoto virus nucleocapsids. Proc Natl Acad Sci USA. 1999;96: 2082-2086.

Krister M, Tapani R, Rarbara B, Robert MK, Vonbonsdorff CH, Ilkka J. Interferon-induces Mx proteins form oligomers and contain a putative Leucine zipper. J Biol Chem. 1992;25:25898-25907.

Landolfo S, Gribaudo G, Angeretti A, Gariglio M. Mechanisms of viral inhibition by interferons. Pharmacol Ther. 1995;65:415-442.

Lee SH, Vidal SM. Functional diversity of Mx proteins: variations on a theme of host resistance to infection. Genome Res. 2002;12:527-530.

Lee JY, Hirono I, Aoki T. Cloning and analysis of expression of Mx cDNA in Japanese flounder, Paralichthys olivaceus. Dev Comp Immunol. 2000;24: 407-415.

Leong JC, Trobridge GD, Kim CH, Johnson M, Simon B. Interferon -inducible Mx proteins in fish. Immunol Rev. 1998;166:349-363.

Lin OE, Ohira T, Hirono I, Tatsuo ST, Aoki T. Immunoanalysis of antiviral Mx protein expression in Japanese Flounder (Paralichthys olivaceus) cells. Dev Comp Immunol. 2005;29:443-445.

Lindenmann J. Resistance of mice to mouse-adapted influenza A virus. Virology. 1962;16:203-204.

Liu YJ. IPC: professional type 1 interferon-producing cells and plasmacytoid dendritic cell precursors. Annu Rev Immunol. 2005;23:275-306.

Lu L, Zhou SY, Chen C, Weng SP, Chan SM, He JG. Complete genome sequence analysis of an iridovirus isolated from the orange-spotted grouper, Epinephelus coioides. Virology. 2005;339:81-100.

Mahardika K, Zafran, Yamamoto A, Miyazaki T. Susceptibility of juvenile humpback grouper Cromileptes altivelis to grouper sleepy disease iridovirus (GSDIV). Dis Aquat Organ. 2004;59:1-9.

Megan CK, Graca R, Lemmon MA. Inhibition of nuclear import and cell-cycle progression by mutated forms of the dynmun-like GTPase MxB. Proc Natl Acad Sci USA. 2004;101:8957-8962.

Melaine N, Lienard MO, Guillaume E, Ruffault A, Dejucq-Rainsford N, Jegou B. Production of the antiviral proteins 2’5’oligoadenylate synthetase, PKR and Mx in interstitial cells and spermatogonia. J Reprod Immunol. 2003;59:53-60.

Michienzi A, Cagnon L, Bahner I, Rossi JJ. Ribozyme-mediated inhibition of HIV 1 suggests nucleolar trafficking of HIV-1 RNA. Proc Natl Acad Sci USA. 2000;97:8955-8960.

Munday BL, Nakai T. Special topic review: nodaviruses as pathogens in larval and juvenile marine finfish. World J Microbiol Biotechnol. 1997;13: 375-381.

Nina A, Hans W, Christina B, Kristina E, Allan RT, Soren RP. Lambda interferon (IFN-λ), a type III IFN, is induced by viruses and IFNs and displays potent antiviral activity against select virus infections in vivo. J Virol. 2006;80:4501-4509.

DiPaolo C, Hefti HP, Meli M, Landis H, Pavlovic J. Intramolecular backfolding of the carboxylterminal end of MxA protein is a prerequisite for its oligomerization. J Biol Chem. 1999;274:32071-32078.

Rannveig L, Torunn PR, Borre R. Inhibition of infectious Pancreatic Necrosis Virus Replication by Altantic Salmon Mx1 protein. J Virol. 2004;78:7938- 7944.

Robertsen B, Bergan V, Rokenes T, Larsen R, Albuquerque A. Altantic salmon interferon genes: cloning, sequence, analysis, expression and biological activity. J Interferon Cytokine Res. 2003;23:601-612.

Robertsen B, Trobridg GD, Leong JA. Molecular cloning of double-stranded RNA inducible Mx genes from Atlantic salmon (Salmo salar L.). Dev Comp Immunol. 1997;15:397-412.

Robertsen B. The interferon system of teleost fish. Fish Shellfish Immunol. 2005;20:172-191.

Rikkonen M, Peränen J, Kääriäinen L. Nuclear and nucleolar targeting signals of Semliki Forest virus nonstructural protein nsP2. Virology. 1992;189:462- 473.

Samuel TW, Matthew LR, Molly JT, Frederick SB. The fight between the teleost fish immune response and aquatic viruses. Mol Immunol. 2010;47: 2525-2536.

Schumacher B, Staeheli P. Domains mediating intramolecular floding and oligomerization of Mx GTPase. J Biol Chem. 1998;273:28365-28370.

Schwemmle M, Richter MF, Herrmann C, Nassar N, Staeheli P. Unexpected structural requirements for GTPase activity of the interferon-induced MxA protein. J Biol Chem. 1995;270:13518-13523.

Shang D, Yang P, Liu Y, Song J, Zhang F, Tian Y. Interferon-α induces G1 cell-cycle arrest in renal cell carcinoma cells via activation of Jak-Stat signaling. Cancer Invest. 2011;29:347-352.

Siomi H, Shida H, Maki M, Hatanaka M. Effects of a highly basic region of human immunodeficiency virus Tat protein on nucleolar localization. J Virol. 1990;64:1803-1807.

Staeheli P, Yu Y-S, Grob R, Haller O. A double-stranded RNA-inducible fish gene homologous to the murine influenza virus resistance gene Mx. Mol Cell Biol. 1989;9:3117-3121.

Stark GR, Kerr IM, Williams BR, Silverman RH, Schreiber RD. How cell respond to interferons. Annu Rev Biochem. 1998;67:227-264.

Stephen MA, Mark TM, Daniel LD, Carol HK. Molecular and functional analysis of an interferon gene from the zebrafish, Danio rerio. J Virol. 2003;77:1992-2002.

Supriya P, Joshi CV, Ajitkumar P. IFN-gamma inhibits growth of WISH cells in a cell cycle phase-specific manner. J Interferon Cytokine Res. 1998;18: 215-217.

Iwamoto T, Mise K, Takeda A, Okinaka Y, Mori K, Arimoto M, Okuno T, Nakai T. Characterization of Striped jack nervous necrosis virus subgenomic RNA3 and biological activities of its encoded protein B2. J Gen Virol. 2005;86:2807-2816.

Trobridge GD, Leong JA. Characterization of a rainbow trout Mx gene. J Interferon Cytokine Res. 1995;15:691-702.

Trobridge GD, Chiou PP, Leong JC. Cloning of the rainbow trout (Oncohynchus mylciss) Mx2 and Mx3 cDNAs and characterization of trout Mx protein expression in salmon cells. J Virol. 1997;71:5304-5311.

Turan K, Mibayashi M, Sugiyama K, Saiyo S, Numajiri A, Negata K. Nuclear MxA proteins from a complex with influenza virus NP and inhibit the transcription of the enginerred influenza virus genome. Nucleic Acids Res. 2004;32:643-652.

Bergan V, Robertsen B. Characterization of Atlantic halibut (Hippoglossus hippoglossus) Mx protein expression. Dev Comp Immunol. 2004;28:1037- 1047.

Wengler G. Identification of a transfer of viral core protein to cellular ribosomes during the early stages of alphavirus infection. Virology. 1984;134: 435-442.

Wurm T, Chen H, Hodgson T, Britton P, Brooks G, Hiscox JA. Localization to the nucleolus is a common feature of coronavirus nucleoproteins, and the protein may disrupt host cell division. J Virol. 2001;75:9345-9356.

Yap WH, Tay A, Brenner S, Venkatesh B. Molecular cloning of the pufferfish (Takifugu rubripes) Mx gene and functional characterization of its promoter. Immunogenetics. 2003;54:705-713.