Please use this identifier to cite or link to this item:
標題: A型流感病毒H1N1之NS1蛋白其二聚體化對其功能之影響
The effect of dimeric structure of NS1 of influenza A virus (H1N1) on its functions
作者: 鄭雅心
Cheng, Ya-Hsin
關鍵字: influenza A virus;A型流感病毒;NS1;dimeric structure;NS1;二聚體化
出版社: 生物化學研究所
引用: Akira, S., Uematsu, S. and Takeuchi, O. (2006) Pathogen recognition and innate immunity. Cell, 124, 783-801. Aragon, T., de la Luna, S., Novoa, I., Carrasco, L., Ortin, J. and Nieto, A. (2000) Eukaryotic translation initiation factor 4GI is a cellular target for NS1 protein, a translational activator of influenza virus. Mol Cell Biol, 20, 6259-6268. Barabino, S.M., Hubner, W., Jenny, A., Minvielle-Sebastia, L. and Keller, W. (1997) The 30-kD subunit of mammalian cleavage and polyadenylation specificity factor and its yeast homolog are RNA-binding zinc finger proteins. Genes Dev, 11, 1703-1716. Belshe, R.B. (2005) The origins of pandemic influenza--lessons from the 1918 virus. N Engl J Med, 353, 2209-2211. Bienroth, S., Wahle, E., Suter-Crazzolara, C. and Keller, W. (1991) Purification of the cleavage and polyadenylation factor involved in the 3''-processing of messenger RNA precursors. J Biol Chem, 266, 19768-19776. Bornholdt, Z.A. and Prasad, B.V. (2006) X-ray structure of influenza virus NS1 effector domain. Nat Struct Mol Biol, 13, 559-560. Bornholdt, Z.A. and Prasad, B.V. (2008) X-ray structure of NS1 from a highly pathogenic H5N1 influenza virus. Nature, 456, 985-988. Bucher, E., Hemmes, H., de Haan, P., Goldbach, R. and Prins, M. (2004) The influenza A virus NS1 protein binds small interfering RNAs and suppresses RNA silencing in plants. J Gen Virol, 85, 983-991. Burgui, I., Aragon, T., Ortin, J. and Nieto, A. (2003) PABP1 and eIF4GI associate with influenza virus NS1 protein in viral mRNA translation initiation complexes. J Gen Virol, 84, 3263-3274. Chen, Z., Li, Y. and Krug, R.M. (1999) Influenza A virus NS1 protein targets poly(A)-binding protein II of the cellular 3''-end processing machinery. Embo J, 18, 2273-2283. Cheng, A., Wong, S.M. and Yuan, Y.A. (2009) Structural basis for dsRNA recognition by NS1 protein of influenza A virus. Cell Res, 19, 187-195. Das, K., Ma, L.C., Xiao, R., Radvansky, B., Aramini, J., Zhao, L., Marklund, J., Kuo, R.L., Twu, K.Y., Arnold, E., Krug, R.M. and Montelione, G.T. (2008) Structural basis for suppression of a host antiviral response by influenza A virus. Proc Natl Acad Sci U S A, 105, 13093-13098. Enami, M. (1997) [Structure and function of influenza virus NS1 and NS2 proteins]. Nippon Rinsho, 55, 2605-2609. Fodor, E., Pritlove, D.C. and Brownlee, G.G. (1994) The influenza virus panhandle is involved in the initiation of transcription. J Virol, 68, 4092-4096. Galabru, J. and Hovanessian, A. (1987) Autophosphorylation of the protein kinase dependent on double-stranded RNA. J Biol Chem, 262, 15538-15544. Geiss, G.K., Salvatore, M., Tumpey, T.M., Carter, V.S., Wang, X., Basler, C.F., Taubenberger, J.K., Bumgarner, R.E., Palese, P., Katze, M.G. and Garcia-Sastre, A. (2002) Cellular transcriptional profiling in influenza A virus-infected lung epithelial cells: the role of the nonstructural NS1 protein in the evasion of the host innate defense and its potential contribution to pandemic influenza. Proc Natl Acad Sci U S A, 99, 10736-10741. Ghedin, E., Sengamalay, N.A., Shumway, M., Zaborsky, J., Feldblyum, T., Subbu, V., Spiro, D.J., Sitz, J., Koo, H., Bolotov, P., Dernovoy, D., Tatusova, T., Bao, Y., St George, K., Taylor, J., Lipman, D.J., Fraser, C.M., Taubenberger, J.K. and Salzberg, S.L. (2005) Large-scale sequencing of human influenza reveals the dynamic nature of viral genome evolution. Nature, 437, 1162-1166. Gilmartin, G.M. and Nevins, J.R. (1991) Molecular analyses of two poly(A) site-processing factors that determine the recognition and efficiency of cleavage of the pre-mRNA. Mol Cell Biol, 11, 2432-2438. Hale, B.G., Barclay, W.S., Randall, R.E. and Russell, R.J. (2008a) Structure of an avian influenza A virus NS1 protein effector domain. Virology, 378, 1-5. Hale, B.G., Randall, R.E., Ortin, J. and Jackson, D. (2008b) The multifunctional NS1 protein of influenza A viruses. J Gen Virol, 89, 2359-2376. Hogg, J.R. and Collins, K. (2007) RNA-based affinity purification reveals 7SK RNPs with distinct composition and regulation. Rna, 13, 868-880. Hornung, V., Ellegast, J., Kim, S., Brzozka, K., Jung, A., Kato, H., Poeck, H., Akira, S., Conzelmann, K.K., Schlee, M., Endres, S. and Hartmann, G. (2006) 5''-Triphosphate RNA is the ligand for RIG-I. Science, 314, 994-997. Hovanessian, A.G. (1989) The double stranded RNA-activated protein kinase induced by interferon: dsRNA-PK. J Interferon Res, 9, 641-647. Jenny, A., Hauri, H.P. and Keller, W. (1994) Characterization of cleavage and polyadenylation specificity factor and cloning of its 100-kilodalton subunit. Mol Cell Biol, 14, 8183-8190. Kalderon, D., Roberts, B.L., Richardson, W.D. and Smith, A.E. (1984) A short amino acid sequence able to specify nuclear location. Cell, 39, 499-509. Karimova, G., Pidoux, J., Ullmann, A. and Ladant, D. (1998) A bacterial two-hybrid system based on a reconstituted signal transduction pathway. Proc Natl Acad Sci U S A, 95, 5752-5756. Kato, H., Takeuchi, O., Sato, S., Yoneyama, M., Yamamoto, M., Matsui, K., Uematsu, S., Jung, A., Kawai, T., Ishii, K.J., Yamaguchi, O., Otsu, K., Tsujimura, T., Koh, C.S., Reis e Sousa, C., Matsuura, Y., Fujita, T. and Akira, S. (2006) Differential roles of MDA5 and RIG-I helicases in the recognition of RNA viruses. Nature, 441, 101-105. Kawai, T. and Akira, S. (2006) Innate immune recognition of viral infection. Nat Immunol, 7, 131-137. Keller, W., Bienroth, S., Lang, K.M. and Christofori, G. (1991) Cleavage and polyadenylation factor CPF specifically interacts with the pre-mRNA 3'' processing signal AAUAAA. Embo J, 10, 4241-4249. Krug, R.M., Yuan, W., Noah, D.L. and Latham, A.G. (2003) Intracellular warfare between human influenza viruses and human cells: the roles of the viral NS1 protein. Virology, 309, 181-189. Lanford, R.E. and Butel, J.S. (1984) Construction and characterization of an SV40 mutant defective in nuclear transport of T antigen. Cell, 37, 801-813. Li, W.X., Li, H., Lu, R., Li, F., Dus, M., Atkinson, P., Brydon, E.W., Johnson, K.L., Garcia-Sastre, A., Ball, L.A., Palese, P. and Ding, S.W. (2004) Interferon antagonist proteins of influenza and vaccinia viruses are suppressors of RNA silencing. Proc Natl Acad Sci U S A, 101, 1350-1355. Ligon, B.L. (2005) Avian influenza virus H5N1: a review of its history and information regarding its potential to cause the next pandemic. Semin Pediatr Infect Dis, 16, 326-335. Liu, J., Lynch, P.A., Chien, C.Y., Montelione, G.T., Krug, R.M. and Berman, H.M. (1997) Crystal structure of the unique RNA-binding domain of the influenza virus NS1 protein. Nat Struct Biol, 4, 896-899. Malathi, K., Dong, B., Gale, M., Jr. and Silverman, R.H. (2007) Small self-RNA generated by RNase L amplifies antiviral innate immunity. Nature, 448, 816-819. Melen, K., Kinnunen, L., Fagerlund, R., Ikonen, N., Twu, K.Y., Krug, R.M. and Julkunen, I. (2007) Nuclear and nucleolar targeting of influenza A virus NS1 protein: striking differences between different virus subtypes. J Virol, 81, 5995-6006. Meurs, E., Chong, K., Galabru, J., Thomas, N.S., Kerr, I.M., Williams, B.R. and Hovanessian, A.G. (1990) Molecular cloning and characterization of the human double-stranded RNA-activated protein kinase induced by interferon. Cell, 62, 379-390. Min, J.Y. and Krug, R.M. (2006) The primary function of RNA binding by the influenza A virus NS1 protein in infected cells: Inhibiting the 2''-5'' oligo (A) synthetase/RNase L pathway. Proc Natl Acad Sci U S A, 103, 7100-7105. Min, J.Y., Li, S., Sen, G.C. and Krug, R.M. (2007) A site on the influenza A virus NS1 protein mediates both inhibition of PKR activation and temporal regulation of viral RNA synthesis. Virology, 363, 236-243. Murthy, K.G. and Manley, J.L. (1992) Characterization of the multisubunit cleavage-polyadenylation specificity factor from calf thymus. J Biol Chem, 267, 14804-14811. Nemeroff, M.E., Barabino, S.M., Li, Y., Keller, W. and Krug, R.M. (1998) Influenza virus NS1 protein interacts with the cellular 30 kDa subunit of CPSF and inhibits 3''end formation of cellular pre-mRNAs. Mol Cell, 1, 991-1000. Neumann, G., Noda, T. and Kawaoka, Y. (2009) Emergence and pandemic potential of swine-origin H1N1 influenza virus. Nature, 459, 931-939. Noton, S.L., Medcalf, E., Fisher, D., Mullin, A.E., Elton, D. and Digard, P. (2007) Identification of the domains of the influenza A virus M1 matrix protein required for NP binding, oligomerization and incorporation into virions. J Gen Virol, 88, 2280-2290. O''Neill, R.E., Talon, J. and Palese, P. (1998) The influenza virus NEP (NS2 protein) mediates the nuclear export of viral ribonucleoproteins. Embo J, 17, 288-296. Opitz, B., Rejaibi, A., Dauber, B., Eckhard, J., Vinzing, M., Schmeck, B., Hippenstiel, S., Suttorp, N. and Wolff, T. (2007) IFNbeta induction by influenza A virus is mediated by RIG-I which is regulated by the viral NS1 protein. Cell Microbiol, Vol. 9, pp. 930-938. Pichlmair, A., Schulz, O., Tan, C.P., Naslund, T.I., Liljestrom, P., Weber, F. and Reis e Sousa, C. (2006) RIG-I-mediated antiviral responses to single-stranded RNA bearing 5''-phosphates. Science, 314, 997-1001. Reid, A.H., Janczewski, T.A., Lourens, R.M., Elliot, A.J., Daniels, R.S., Berry, C.L., Oxford, J.S. and Taubenberger, J.K. (2003) 1918 influenza pandemic caused by highly conserved viruses with two receptor-binding variants. Emerg Infect Dis, 9, 1249-1253. Robb, N.C., Smith, M., Vreede, F.T. and Fodor, E. (2009) NS2/NEP protein regulates transcription and replication of the influenza virus RNA genome J Gen Virol, 90, 1398-1407. Sadler, A.J. and Williams, B.R. (2008) Interferon-inducible antiviral effectors. Nat Rev Immunol, 8, 559-568. Samuel, C.E. (2007) Innate immunity minireview series: making biochemical sense of nucleic acid sensors that trigger antiviral innate immunity. J Biol Chem, 282, 15313-15314. Satterly, N., Tsai, P.L., van Deursen, J., Nussenzveig, D.R., Wang, Y., Faria, P.A., Levay, A., Levy, D.E. and Fontoura, B.M. (2007) Influenza virus targets the mRNA export machinery and the nuclear pore complex. Proc Natl Acad Sci U S A, 104, 1853-1858. Silverman, R.H. (2007) Viral encounters with 2'',5''-oligoadenylate synthetase and RNase L during the interferon antiviral response. J Virol, 81, 12720-12729. Smith, G.J., Naipospos, T.S., Nguyen, T.D., de Jong, M.D., Vijaykrishna, D., Usman, T.B., Hassan, S.S., Nguyen, T.V., Dao, T.V., Bui, N.A., Leung, Y.H., Cheung, C.L., Rayner, J.M., Zhang, J.X., Zhang, L.J., Poon, L.L., Li, K.S., Nguyen, V.C., Hien, T.T., Farrar, J., Webster, R.G., Chen, H., Peiris, J.S. and Guan, Y. (2006) Evolution and adaptation of H5N1 influenza virus in avian and human hosts in Indonesia and Vietnam. Virology, 350, 258-268. Suzuki, Y., Ito, T., Suzuki, T., Holland, R.E., Jr., Chambers, T.M., Kiso, M., Ishida, H. and Kawaoka, Y. (2000) Sialic acid species as a determinant of the host range of influenza A viruses. J Virol, 74, 11825-11831. Taubenberger, J.K., Reid, A.H. and Fanning, T.G. (2000) The 1918 influenza virus: A killer comes into view. Virology, 274, 241-245. Twu, K.Y., Noah, D.L., Rao, P., Kuo, R.L. and Krug, R.M. (2006) The CPSF30 binding site on the NS1A protein of influenza A virus is a potential antiviral target. J Virol, 80, 3957-3965. Uematsu, S. and Akira, S. (2007) Toll-like receptors and Type I interferons. J Biol Chem, 282, 15319-15323. Wagner, R., Matrosovich, M. and Klenk, H.D. (2002) Functional balance between haemagglutinin and neuraminidase in influenza virus infections. Rev Med Virol, 12, 159-166. Wang, W., Riedel, K., Lynch, P., Chien, C.Y., Montelione, G.T. and Krug, R.M. (1999) RNA binding by the novel helical domain of the influenza virus NS1 protein requires its dimer structure and a small number of specific basic amino acids. Rna, 5, 195-205. Yin, C., Khan, J.A., Swapna, G.V., Ertekin, A., Krug, R.M., Tong, L. and Montelione, G.T. (2007) Conserved surface features form the double-stranded RNA binding site of non-structural protein 1 (NS1) from influenza A and B viruses. J Biol Chem, 282, 20584-20592. Yoneyama, M. and Fujita, T. (2007) Function of RIG-I-like receptors in antiviral innate immunity. J Biol Chem, 282, 15315-15318. Yoneyama, M., Kikuchi, M., Natsukawa, T., Shinobu, N., Imaizumi, T., Miyagishi, M., Taira, K., Akira, S. and Fujita, T. (2004) The RNA helicase RIG-I has an essential function in double-stranded RNA-induced innate antiviral responses. Nat Immunol, 5, 730-737.
A型流行性感冒病毒之非結構蛋白 (nonstructural protein,NS1) 是一個多功能蛋白,同時也是病毒的毒性因子,然而其各種功能與毒性機轉間之關係仍不十分清楚。NS1可區分為兩個區域,分別為N端雙股核醣核酸結合區域 (double-stranded RNA binding domain,RBD) 及C端因子結合區域 (effector domain) ,NS1蛋白雙股核醣核酸結合區域與雙股核醣核酸結合可抑制RNaseL系統;此外,NS1蛋白還會與一些宿主細胞因子結合,認為能抵抗宿主的抗病毒反應。例如,NS1蛋白與CPSF30結合會影響pre-mRNA processing,因此抑制細胞mRNA 出核。由於NS1蛋白會形成二聚體結構,我們想探討二聚體與NS1的功能之相關性。因此本實驗室利用定點突變構築數種NS1突變株試著破壞NS1的二聚體以研究二聚體對其功能性之影響。首先把不同NS1質體DNA轉染入人類胚胎腎細胞進行 luciferase 分析,以分析突變後對干擾素分泌的影響。之後再利用GST pulldown與bacterial two hybrid方式雙重檢驗不同突變對NS1形成二聚體的影響。除了檢驗突變點對二聚體的影響,我們也利用 EMSA 去檢查突變點對NS1與雙股核醣核酸結合之能力。此外本實驗室亦利用GST pulldown檢驗NS1蛋白上不同突變點對於NS1與宿主因子 (僅針對CPSF30) 的結合是否有顯著的影響。從已有實驗結果來看,S103F/I106M/R35A/R46A突變株抑制干擾素的分泌強於S103F/I106M突變株,不僅如此,S103F/I106M/R35A/R46A亦喪失二聚體化能力以及和雙股核醣核酸結合之能力;而且對與CPSF30結合能力明顯大於其他突變株。此結果暗示了,當我們試著破壞 NS1 的二聚體結構,希望能降低對細胞的影響,有可能得到適得其反的結果。

The influenza A virus non-structural protein 1 (NS1) is a multifunctional protein and a virulence factor. It is considered to antagonize host innate immune responses. However, there is no clear correlation between the function of NS1 and its virulence. NS1 structure can be divided into two distinct functional domains. An N-terminal double-stranded RNA binding domain (RBD), which binds double-strand RNA and inhibit the activity of RNase L system and a C-terminal effector domain. NS1 has been shown to associate with several cellular proteins via the effector domain, including CPSF 30, a cellular factor required for 3´ end processing of cellular pre-mRNA, thereby inhibiting the production of all cellular mRNAs. As NS1 can dimerize and is coupled with dsRNA binding activity, we want to understand the correlation between its dimeric structure and functions by constructing several NS1 mutants disrupting the NS1 dimeric interface. We then examine the dimerization activity of NS1 mutants via GST pulldown assay and bacteria two hybrid assay, we use EMSA technique to analyze the dsRNA binding activity. Unexpectedly, we found the S103F/I106M/R35A/R46A mutant represses interferon-beta expression stronger than the S103F/I106M mutant, although the dimerization and dsRNA binding activity of S103F/I106M/R35A/R46A are impaired severely. Therefore, it should be cautious in the attempt of disrupting NS1 dimeric interface to decrease its virulence toward host cell.
其他識別: U0005-1208200915475600
Appears in Collections:生物化學研究所

Show full item record

Google ScholarTM


Items in DSpace are protected by copyright, with all rights reserved, unless otherwise indicated.