酵母双杂交系统筛选与禽流感病毒NS1相互作用的宿主因子
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摘要
禽流感病毒非结构蛋白1(NS1蛋白)全长约为230个氨基酸,理论分子量约为26KDa,主要包括两个功能结构域,即N-末端的RNA结合结构域和和C-末端的效应结构域。NS1蛋白是一个多功能的病毒蛋白,它不仅影响着该病毒其他基因的表达,更能通过与宿主多种细胞因子的相互作用干预宿主细胞的正常功能,抵抗宿主的抗病毒系统。NS1蛋白因此被认为是禽流感病毒的一个重要毒力因子。为了寻找与NS1相互作用的其它宿主蛋白以及试图探索NS1的新功能,最终阐明禽流感的致病性机制,我们利用酵母双杂交系统,以NS1为诱饵蛋白,从人胸腺cDNA文库中筛到了8个宿主因子可与NS1蛋白相互作用,我们选择U蛋白作为我们研究的目的蛋白,并作了点对点酵母双杂交回转实验。结果发现两者确实是互作的,并且互作的部位位于NS1蛋白的RNA结合结构域上。同时我们利用GST Pull-down进一步验证了二者的相互作用。在研究当中还发现,高致病性禽流感病毒H5N1亚型NS1蛋白的80-84位氨基酸缺失可能对禽流感的致病性很重要,这提示针对H5N1型高致病性禽流感病毒可能是一个新的药物靶点。
The NS1 protein, a non-structural protein of Avian Influenza Virus, is about 230 amino acids in length. Its molecular weight is about 26 KDa. The protein has two functional domains: an RNA binding domain in the amino terminal and an effector domain in the carboxyl terminal. As a multifunctional viral protein, the NS1 protein can not only influence the expression of other genes of the virus, but also counteract host defense system by interacting with many cellular proteins. Therefore, the NS1 protein is regarded as a crucial virulent factor for Avian Influenza Virus. To find other cellular proteins that interact with NS1, to explore new functions of NS1 and then to elucidate the possible mechanism of NS1 pathogenicity, a human thymus cDNA library was screened using yeast two-hybrid system and eight genes were found to interact with NS1. We chose U protein as our object protein and did yeast point to point back-hybrid to identify its interaction. We also found that U interacts with NS1 RNA binding domain. The interaction of these two proteins was further confirmed by GST pull down. From our study, we also found that the 80-84aa deletion of HPAIV NS1 may be very important for NS1 pathogenicity. This indicates a new drug target for lethal H5N1.
The NS1 protein, a non-structural protein of Avian Influenza Virus, is about 230 amino acids in length. Its molecular weight is about 26 KDa. The protein has two functional domains: an RNA binding domain in the amino terminal and an effector domain in the carboxyl terminal. As a multifunctional viral protein, the NS1 protein can not only influence the expression of other genes of the virus, but also counteract host defense system by interacting with many cellular proteins. Therefore, the NS1 protein is regarded as a crucial virulent factor for Avian Influenza Virus. To find other cellular proteins that interact with NS1, to explore new functions of NS1 and then to elucidate the possible mechanism of NS1 pathogenicity, a human thymus cDNA library was screened using yeast two-hybrid system and eight genes were found to interact with NS1. We chose U protein as our object protein and did yeast point to point back-hybrid to identify its interaction. We also found that U interacts with NS1 RNA binding domain. The interaction of these two proteins was further confirmed by GST pull down. From our study, we also found that the 80-84aa deletion of HPAIV NS1 may be very important for NS1 pathogenicity. This indicates a new drug target for lethal H5N1.
引文
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[2]陆承平,高致病性禽流感与流感病毒。中国病毒学,2004,19(2):204-207。
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[9]Wahle E,Kühn U.The mechanism of 3' cleavage and polyadenylation of eukaryotic pre-mRNA,Prog Nucleic Acid Res Mol Biol,1997,57:41-71.
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[12]Zhongying Chen,Yongzhong Li and Robert M.Krug.Influenza A virus NS1protein targets poly(A)-binding protein Ⅱ of the cellular 3'end processing machinery, EMBO, 1999,18:2273-2283.
[13] Leo L. M.Poon, et al Direct Evidence that the Poly(A) Tail of Influenza A Virus mRNA Is Synthesized by Reiterative Copying of a U Track in the Virion RNA Template, J Virol, 1999, 73:3473-3476.
[14] Tomas Aragon, et.al. Eukaryotic Translation Initiation Factor 4GI Is a Cellular Target for NS1 Protein,a Translational Activator of Influenza Virus, Mol.Cel.Bio, 2000,20:6259-6268.
[15]Murphy,B.R.,Baron,S.,Chalhub,E.G.,Uhlendorf,C.P.,Chanock,R.M.,Temperature -sensitive mutants of influenza virus. IV.Induction of interferon in the nasopharynx by wild-type and a temperature sensitive recombinant virus, J.Infect.Dis, 1973, 128: 488-493.
[16] Seo,S.H.,Webster,R.G.,Tumor necrosis factor alpha exerts powerful anti-influenza virus effects in lung epithelial cells, J.Virol, 2002, 76:1071-1076.
[17] Adolfo G.S.et.al. Influenza A Virus Lacking the NS1 Gene Replicates in Interferon-Deficient Systems, Virology, 1998,252:324-330.
[18] Diana LNoah, Karen Y.Twu,and Robert M.Krug.Cellular antiviral responses against influenza A virus are countered at the posttranscriptional level by the viral NS1A protein via its binding to a cellular protein required for the 3'end processing of cellular pre- mRNAs, Virology, 2003, 307:386-395.
[19] Salvatore M, Basler C F, Parisian J P, et al. Effects of influenza a virus NS1 protein on protein expression:the NS1 protein enhances translation and is not required for shut-off of host protein synthesis, J Virol, 2002, 76:1206-1212.
[20] Solorzano A, Webby RJ, Lager KM, et al. Mutations in the NS1 protein of swine influenza virus impair anti-interferon activity and confer attenuation in pigs, J Virol, 2005,79:7535-7543.
[21] Sen, G.C.Novel functions of interferon-induced proteins, Semin.Cancer Biol, 2000,10:93-101.
[22] Min, J.Y.,and R.M.Krug.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.USA 2006,103:7100-7105.
[23] Li, S., J.Y.Min,R.M.Krug,and G.C.Sen.Binding of the influenza A virus NS1 protein to PKR mediates the inhibition of activation by either PACT or double -stranded RNA, Virology, 2006, 349:13-21.
[24] Hiscott, J., R.Lin, P.Nakhaei, and S.Paz. MasterCARD:a priceless link to innate immunity, Trends Mol.Med, 2006,12:53-56.
[25] Mibayashi,M., L. Martinez-Sobrido, Y.M.Loo, W.B.Cardenas, M.Gale, Jr., and A.Garcia-Sastre. Inhibition of retinoic acid-inducible gene I-mediated induction of beta interferon by the NS1 protein of influenza A virus, J.Virol, 2007, 81:514-524.
[26] Sastre G A.Inhibition of interferon-mediated antiviral responses by Influenza a viruses and other negativestrand RNA viruses, J virol, 2001, 279:375-384.
[27] Ludwig S,Wang XY, Ehrhandt C, et al. The influenza a virus NS1 protein inhibits activation of Jun N-terminal kinase and AP-1 transcription factors, J Virol, 2002, 76:11166-11171.
[28] CEhrhardt, et.al.Influenza A Virus NS1 Protein Activates the PI3K/Akt Pathway To Mediate Antiapoptotic Signaling Responses, J.Virol, 2007, 81:3058-3067.
[29] Zhirnov O P, Konakova T E, Wolff T, et al. NS1 protein of influenza A virus down-regulates apoptosis, J Virol, 2002,76:1617-1625.
[30] Zhirnov O P, Klenk HD.Control of apoptosis in influenza virus-infected cells by up-regulation of Akt and p53 signaling, Apoptosis, 2007,12:1419-1432.
[31] R.Murayama, et.al.Influenza A virus non-structural protein 1 (NS1) interacts with cellular multifunctional protein nucleolin during infection, Biochem. Biophys. Res.Commun, 2007.
[2]陆承平,高致病性禽流感与流感病毒。中国病毒学,2004,19(2):204-207。
[3]Chien CY,et.al.Biophysical characterization of the complex between double stranded RNA and the N-terminal domain of the NS1 protein from influenza A virus:Evidence for a novel RNA-binding mode,Biochemistry,2004,43:1950-1962.
[4]Wang W,Riedel K,Lynch P,Chien CY,Montelione GT,Krug RM.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,1999,5:195-205.
[5]Melén K et.al.Nuclear and Nucleolar Targeting of Influenza A Virus NS1 Protein:Striking Differences between Different Virus Subtypes,J Virol,2007,81:5995-6006.
[6]Bornholdt ZA,Prasad BV.X-ray structure of influenza virus NS1 effector domain,Nat Struct Mol Biol,2006,13:559-560.
[7]Fortes P,Beloso A,Ortín J.Influenza virus NS1 protein inhibits pre-mRNA splicing and blocks mRNA nucleocytoplasmic transport,EMBO,1994,13:704-712.
[8]Yun Qiu,Martin Nemerff,and Robert M.Krug.The influenza virus NS1 protein binds to a specific region in human U6 snRNA and inhibits U6-U2 and U6-U4snRNA interanctions during splicing,RNA,1995,1:304-316.
[9]Wahle E,Kühn U.The mechanism of 3' cleavage and polyadenylation of eukaryotic pre-mRNA,Prog Nucleic Acid Res Mol Biol,1997,57:41-71.
[10]Martin E.Nemeroff et.al.Influenza virus NS1 protein interacts with the cellular 30 KDa subunit of CPSF and inhibits 3' end formation of cellular pre-mRNAs,Molecular Cell,1998,1:991-1000.
[11]Li,Y.,Chen,Z.Y.,Wang,W.,Baker,C.C.,Krug,R.M.The 3'-end-processing factor CPSF is required for the splicing of single intron pre-mRNAs in vivo,RNA,2001,7:920-931.
[12]Zhongying Chen,Yongzhong Li and Robert M.Krug.Influenza A virus NS1protein targets poly(A)-binding protein Ⅱ of the cellular 3'end processing machinery, EMBO, 1999,18:2273-2283.
[13] Leo L. M.Poon, et al Direct Evidence that the Poly(A) Tail of Influenza A Virus mRNA Is Synthesized by Reiterative Copying of a U Track in the Virion RNA Template, J Virol, 1999, 73:3473-3476.
[14] Tomas Aragon, et.al. Eukaryotic Translation Initiation Factor 4GI Is a Cellular Target for NS1 Protein,a Translational Activator of Influenza Virus, Mol.Cel.Bio, 2000,20:6259-6268.
[15]Murphy,B.R.,Baron,S.,Chalhub,E.G.,Uhlendorf,C.P.,Chanock,R.M.,Temperature -sensitive mutants of influenza virus. IV.Induction of interferon in the nasopharynx by wild-type and a temperature sensitive recombinant virus, J.Infect.Dis, 1973, 128: 488-493.
[16] Seo,S.H.,Webster,R.G.,Tumor necrosis factor alpha exerts powerful anti-influenza virus effects in lung epithelial cells, J.Virol, 2002, 76:1071-1076.
[17] Adolfo G.S.et.al. Influenza A Virus Lacking the NS1 Gene Replicates in Interferon-Deficient Systems, Virology, 1998,252:324-330.
[18] Diana LNoah, Karen Y.Twu,and Robert M.Krug.Cellular antiviral responses against influenza A virus are countered at the posttranscriptional level by the viral NS1A protein via its binding to a cellular protein required for the 3'end processing of cellular pre- mRNAs, Virology, 2003, 307:386-395.
[19] Salvatore M, Basler C F, Parisian J P, et al. Effects of influenza a virus NS1 protein on protein expression:the NS1 protein enhances translation and is not required for shut-off of host protein synthesis, J Virol, 2002, 76:1206-1212.
[20] Solorzano A, Webby RJ, Lager KM, et al. Mutations in the NS1 protein of swine influenza virus impair anti-interferon activity and confer attenuation in pigs, J Virol, 2005,79:7535-7543.
[21] Sen, G.C.Novel functions of interferon-induced proteins, Semin.Cancer Biol, 2000,10:93-101.
[22] Min, J.Y.,and R.M.Krug.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.USA 2006,103:7100-7105.
[23] Li, S., J.Y.Min,R.M.Krug,and G.C.Sen.Binding of the influenza A virus NS1 protein to PKR mediates the inhibition of activation by either PACT or double -stranded RNA, Virology, 2006, 349:13-21.
[24] Hiscott, J., R.Lin, P.Nakhaei, and S.Paz. MasterCARD:a priceless link to innate immunity, Trends Mol.Med, 2006,12:53-56.
[25] Mibayashi,M., L. Martinez-Sobrido, Y.M.Loo, W.B.Cardenas, M.Gale, Jr., and A.Garcia-Sastre. Inhibition of retinoic acid-inducible gene I-mediated induction of beta interferon by the NS1 protein of influenza A virus, J.Virol, 2007, 81:514-524.
[26] Sastre G A.Inhibition of interferon-mediated antiviral responses by Influenza a viruses and other negativestrand RNA viruses, J virol, 2001, 279:375-384.
[27] Ludwig S,Wang XY, Ehrhandt C, et al. The influenza a virus NS1 protein inhibits activation of Jun N-terminal kinase and AP-1 transcription factors, J Virol, 2002, 76:11166-11171.
[28] CEhrhardt, et.al.Influenza A Virus NS1 Protein Activates the PI3K/Akt Pathway To Mediate Antiapoptotic Signaling Responses, J.Virol, 2007, 81:3058-3067.
[29] Zhirnov O P, Konakova T E, Wolff T, et al. NS1 protein of influenza A virus down-regulates apoptosis, J Virol, 2002,76:1617-1625.
[30] Zhirnov O P, Klenk HD.Control of apoptosis in influenza virus-infected cells by up-regulation of Akt and p53 signaling, Apoptosis, 2007,12:1419-1432.
[31] R.Murayama, et.al.Influenza A virus non-structural protein 1 (NS1) interacts with cellular multifunctional protein nucleolin during infection, Biochem. Biophys. Res.Commun, 2007.