Influenza A/Hong Kong/156/1997(H5N1) virus NS1 gene mutations F103L and M106I both increase IFN antagonism, virulence and cytoplasmic localization but differ in binding to RIG-I and CPSF30

Virology Journal

Earl G Brown²  Jianjun Jia¹  Jihui Ping²  Mohammed Selman²  Ali Tavassoli³  Martin Pelchat¹  Nicole E Forbes²  Elena Miranda⁴  Samar K Dankar² 

[1] Department of Biochemistry, Microbiology and Immunology, Faculty of Medicine, University of Ottawa, 451 Smyth Rd, Ottawa, Ontario K1H 8M5, Canada;Emerging Pathogens Research Centre, Faculty of Medicine, University of Ottawa, 451 Smyth Rd, Ottawa, Ontario K1H 8M5, Canada;Cancer Sciences, Faculty of Medicine, University of Southampton, Southampton SO16 6YD, UK;Chemistry, University of Southampton, Southampton SO17 1BJ, UK

关键词: tropism;    Alveoli;    H6N1;    H5N1;    H7N9;    Interferon;    Virulence;    Host gene expression;    Nuclear export signal;    Cytoplasmic localization;    Nuclear;    Host switch;    Influenza A virus;    Nonstructural protein 1;    NS1;   

Others  :  828679
DOI  :  10.1186/1743-422X-10-243

 received in 2013-05-01, accepted in 2013-07-23,  发布年份 2013

【 摘 要 】

Background

The genetic basis for avian to mammalian host switching in influenza A virus is largely unknown. The human A/HK/156/1997 (H5N1) virus that transmitted from poultry possesses NS1 gene mutations F103L + M106I that are virulence determinants in the mouse model of pneumonia; however their individual roles have not been determined. The emergent A/Shanghai/patient1/2013(H7N9)-like viruses also possess these mutations which may contribute to their virulence and ability to switch species.

Methods

NS1 mutant viruses were constructed by reverse genetics and site directed mutagenesis on human and mouse-adapted backbones. Mouse infections assessed virulence, virus yield, tissue infection, and IFN induction. NS1 protein properties were assessed for subcellular distribution, IFN antagonism (mouse and human), CPSF30 and RIG-I domain binding, host transcription (microarray); and the natural prevalence of 103L and 106I mutants was assessed.

Results

Each of the F103L and M106I mutations contributes additively to virulence to reduce the lethal dose by >800 and >3,200 fold respectively by mediating alveolar tissue infection with >100 fold increased infectious yields. The 106I NS1 mutant lost CPSF binding but the 103L mutant maintained binding that correlated with an increased general decrease in host gene expression in human but not mouse cells. Each mutation positively modulated the inhibition of IFN induction in mouse cells and activation of the IFN-β promoter in human cells but not in combination in human cells indicating negative epistasis. Each of the F103L and M106I mutations restored a defect in cytoplasmic localization of H5N1 NS1 in mouse cells. Human H1N1 and H3N2 NS1 proteins bound to the CARD, helicase and RD RIG-I domains, whereas the H5N1 NS1 with the same consensus 103F and 106M mutations did not bind these domains, which was totally or partially restored by the M106I or F103L mutations respectively.

Conclusions

The F103L and M106I mutations in the H5N1 NS1 protein each increased IFN antagonism and mediated interstitial pneumonia in mice that was associated with increased cytoplasmic localization and altered host factor binding. These mutations may contribute to the ability of previous HPAI H5N1 and recent LPAI H7N9 and H6N1 (NS1-103L+106M) viruses to switch hosts and cause disease in humans.

【 授权许可】

   
2013 Dankar et al.; licensee BioMed Central Ltd.

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【 参考文献 】

• [1]Guan Y, Shortridge KF, Krauss S, Webster RG: Molecular characterization of H9N2 influenza viruses: were they the donors of the “internal” genes of H5N1 viruses in Hong Kong? Proc Natl Acad Sci USA 1999, 96:9363-9367.
• [2]Kageyama T, Fujisaki S, Takashita E, Xu H, Yamada S, Uchida Y, et al.: Genetic analysis of novel avian A(H7N9) influenza viruses isolated from patients in China, February to April 2013. Euro Surveill 2013, 18(15):20453.
• [3]Liu D, Shi W, Shi Y, Wang D, Xiao H, Li W, et al.: Origin and diversity of novel avian influenza A H7N9 viruses causing human infection: phylogenetic, structural, and coalescent analyses. Lancet 2013, 381(9881):1926-1932.
• [4]Claas EC, de Jong JC, van Beek R, Rimmelzwaan GF, Osterhaus AD: Human influenza virUSA/HongKong/156/97 (H5N1) infection. Vaccine 1998, 16:977-978.
• [5]Gao R, Cao B, Hu Y, Feng Z, Wang D, Hu W, et al.: Human Infection with a Novel Avian-Origin Influenza A (H7N9) Virus. N Engl J Med 2013, 368:1888-1897.
• [6]Yang F, Wang J, Jiang L, Jin J, Shao L, Zhang Y, et al.: A fatal case caused by novel H7N9 avian influenza A virus in China. Emerg Microbes Infect 2013, 2:e19.
• [7]Bi J, Deng G, Dong J, Kong F, Li X, Xu Q, et al.: Phylogenetic and molecular characterization of H9N2 influenza isolates from chickens in Northern China from 2007–2009. PLoS One 2010, 5(9):e13063.
• [8]Deng G, Bi J, Kong F, Li X, Xu Q, Dong J, et al.: Acute respiratory distress syndrome induced by H9N2 virus in mice. Arch Virol 2010, 155:187-195.
• [9]Guo YJ, Krauss S, Senne DA, Mo IP, Lo KS, Xiong XP, et al.: Characterization of the pathogenicity of members of the newly established H9N2 influenza virus lineages in Asia. Virology 2000, 267:279-288.
• [10]Selman M, Dankar SK, Forbes NE, Jia JJ, Brown EG: Adaptive mutation in influenza A virus non-structural gene is linked to host switching and induces a novel protein by alternative splicing. Emerg Microbes Infect 2012, 1:e42.
• [11]Influenza at the Human-Animal Interface (HAI). WHO; 2013. Ref Type: Electronic Citation. http://www.who.int/influenza/human_animal_interface/en/ webcite. publisher: World Health Organization, Geneva, Switzerland
• [12]Herfst S, Schrauwen EJ, Linster M, Chutinimitkul S, De WE, Munster VJ, et al.: Airborne transmission of influenza A/H5N1 virus between ferrets. Science 2012, 336:1534-1541.
• [13]Dankar SK, Wang S, Ping J, Forbes NE, Keleta L, Li Y, et al.: Influenza A virus NS1 gene mutations F103L and M106I increase replication and virulence. Virol J 2011, 8:13. BioMed Central Full Text
• [14]Fukushi M, Ito T, Oka T, Kitazawa T, Miyoshi-Akiyama T, Kirikae T, et al.: Serial histopathological examination of the lungs of mice infected with influenza A virus PR8 strain. PLoS One 2011, 6:e21207.
• [15]Forbes NE, Ping J, Dankar SK, Jia JJ, Selman M, Keleta L, et al.: Multifunctional Adaptive NS1 Mutations Are Selected upon Human Influenza Virus Evolution in the Mouse. PLoS One 2012, 7:e31839.
• [16]Spesock A, Malur M, Hossain MJ, Chen LM, Njaa BL, Davis CT, et al.: The virulence of 1997 H5N1 influenza viruses in the mouse model is increased by correcting a defect in their NS1 proteins. J Virol 2011, 85:7048-7058.
• [17]Bloom JD, Gong LI, Baltimore D: Permissive secondary mutations enable the evolution of influenza oseltamivir resistance. Science 2010, 328:1272-1275.
• [18]Kryazhimskiy S, Dushoff J, Bazykin GA, Plotkin JB: Prevalence of epistasis in the evolution of influenza a surface proteins. PLoS Genet 2011, 7:e1001301.
• [19]Rimmelzwaan GF, Berkhoff EG, Nieuwkoop NJ, Smith DJ, Fouchier RA, Osterhaus AD: Full restoration of viral fitness by multiple compensatory co-mutations in the nucleoprotein of influenza A virus cytotoxic T-lymphocyte escape mutants. J Gen Virol 2005, 86:1801-1805.
• [20]Elena SF, Sole RV, Sardanyes J: Simple genomes, complex interactions: epistasis in RNA virus. Chaos 2010, 20:026106.
• [21]Handel A, Regoes RR, Antia R: The role of compensatory mutations in the emergence of drug resistance. PLoS Comput Biol 2006, 2:e137.
• [22]Hale BG, Randall RE, Ortin J, Jackson D: The multifunctional NS1 protein of influenza A viruses. J Gen Virol 2008, 89:2359-2376.
• [23]Hatada E, Fukuda R: Binding of influenza A virus NS1 protein to dsRNA in vitro. J Gen Virol 1992, 73(Pt 12):3325-3329.
• [24]Qian XY, Chien CY, Lu Y, Montelione GT, Krug RM: An amino-terminal polypeptide fragment of the influenza virus NS1 protein possesses specific RNA-binding activity and largely helical backbone structure. RNA 1995, 1:948-956.
• [25]Pichlmair A, Schulz O, Tan CP, Naslund TI, Liljestrom P, Weber F, et al.: RIG-I-mediated antiviral responses to single-stranded RNA bearing 5′-phosphates. Science 2006, 314:997-1001.
• [26]Akira S: TLR signaling. Curr Top Microbiol Immunol 2006, 311:1-16.
• [27]Yoneyama M, Kikuchi M, Natsukawa T, Shinobu N, Imaizumi T, Miyagishi M, et al.: The RNA helicase RIG-I has an essential function in double-stranded RNA-induced innate antiviral responses. Nat Immunol 2004, 5:730-737.
• [28]Talon J, Horvath CM, Polley R, Basler CF, Muster T, Palese P, et al.: Activation of interferon regulatory factor 3 is inhibited by the influenza A virus NS1 protein. J Virol 2000, 74:7989-7996.
• [29]Wang X, Li M, Zheng H, Muster T, Palese P, Beg AA, et al.: Influenza A virus NS1 protein prevents activation of NF-kappaB and induction of alpha/beta interferon. J Virol 2000, 74:11566-11573.
• [30]Ludwig S, Wang X, Ehrhardt C, Zheng H, Donelan N, Planz O, 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.
• [31]Mibayashi M, Martinez-Sobrido L, Loo YM, Cardenas WB, Gale M Jr, Garcia-Sastre A: 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.
• [32]Gack MU, Albrecht RA, Urano T, Inn KS, Huang IC, Carnero E, et al.: Influenza A virus NS1 targets the ubiquitin ligase TRIM25 to evade recognition by the host viral RNA sensor RIG-I. Cell Host Microbe 2009, 5:439-449.
• [33]Kato H: Cell type-specific involvement of RIG-I in antiviral response. Immunity 2005, 23:19-28.
• [34]Rajsbaum R, Albrecht RA, Wang MK, Maharaj NP, Versteeg GA, Nistal-Villan E, et al.: Species-specific inhibition of RIG-I ubiquitination and IFN induction by the influenza A virus NS1 protein. PLoS Pathog 2012, 8:e1003059.
• [35]Miranda E, Forafonov F, Tavassoli A: Deciphering interactions used by the influenza virus NS1 protein to silence the host antiviral sensor protein RIG-I using a bacterial reverse two-hybrid system. Mol Biosyst 2011, 7:1042-1045.
• [36]Nemeroff ME, Barabino SM, Li Y, Keller W, Krug RM: Influenza virus NS1 protein interacts with the cellular 30 kDa subunit of CPSF and inhibits 3′end formation of cellular pre-mRNAs. Mol Cell 1998, 1:991-1000.
• [37]Twu KY, Noah DL, Rao P, Kuo RL, Krug RM: The CPSF30 binding site on the NS1A protein of influenza A virus is a potential antiviral target. J Virol 2006, 80:3957-3965.
• [38]Chen Z, Li Y, Krug RM: Influenza A virus NS1 protein targets poly(A)-binding protein II of the cellular 3′-end processing machinery. EMBO J 1999, 18:2273-2283.
• [39]Wang W, Krug RM: U6atac snRNA, the highly divergent counterpart of U6 snRNA, is the specific target that mediates inhibition of AT-AC splicing by the influenza virus NS1 protein. RNA 1998, 4:55-64.
• [40]Fortes P, Beloso A, Ortin J: Influenza virus NS1 protein inhibits pre-mRNA splicing and blocks mRNA nucleocytoplasmic transport. EMBO J 1994, 13:704-712.
• [41]Qiu Y, Krug RM: The influenza virus NS1 protein is a poly(A)-binding protein that inhibits nuclear export of mRNAs containing poly(A). J Virol 1994, 68:2425-2432.
• [42]Satterly N, Tsai PL, Van DJ, Nussenzveig DR, Wang Y, Faria PA, et al.: Influenza virus targets the mRNA export machinery and the nuclear pore complex. Proc Natl Acad Sci USA 2007, 104:1853-1858.
• [43]Jia D, Rahbar R, Chan RW, Lee SM, Chan MC, Wang BX, et al.: Influenza virus non-structural protein 1 (NS1) disrupts interferon signaling. PLoS One 2010, 5:e13927.
• [44]Li S, Min JY, Krug RM, Sen GC: Binding of the influenza A virus NS1 protein to PKR mediates the inhibition of its activation by either PACT or double-stranded RNA. Virology 2006, 349:13-21.
• [45]Min JY, Krug RM: 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.
• [46]Min JY, Li S, Sen GC, Krug RM: A site on the influenza A virus NS1 protein mediates both inhibition of PKR activation and temporal regulation of viral RNA synthesis. Virology 2007, %20;363:236-243.
• [47]Han H, Cui ZQ, Wang W, Zhang ZP, Wei HP, Zhou YF, et al.: New regulatory mechanisms for the intracellular localization and trafficking of influenza A virus NS1 protein revealed by comparative analysis of A/PR/8/34 and A/Sydney/5/97. J Gen Virol 2010, 91:2907-2917.
• [48]la LS D, Fortes P, Beloso A, Ortin J: Influenza virus NS1 protein enhances the rate of translation initiation of viral mRNAs. J Virol 1995, 69:2427-2433.
• [49]Burgui I, Aragon T, Ortin J, Nieto A: PABP1 and eIF4GI associate with influenza virus NS1 protein in viral mRNA translation initiation complexes. J Gen Virol 2003, 84:3263-3274.
• [50]Shapira SD, Gat-Viks I, Shum BO, Dricot A, de Grace MM, Wu L, et al.: A physical and regulatory map of host-influenza interactions reveals pathways in H1N1 infection. Cell 2009, 139:1255-1267.
• [51]Lin L, Li Y, Pyo HM, Lu X, Thulasi Raman SN, Liu Q, et al.: Identification of RNA helicase A as a cellular factor that interacts with influenza A NS1 protein and its role in virus life cycle. J Virol 2011, 86(4):1942-1954.
• [52]Ping J, Dankar SK, Forbes NE, Keleta L, Zhou Y, Tyler S, et al.: PB2 and HA Mutations are Major Determinants of Host Range and Virulence in Mouse-Adapted Influenza A Virus. J Virol 2010, 84:10606-10618.
• [53]Ping J, Keleta L, Forbes NE, Dankar S, Stecho W, Tyler S, et al.: Genomic and protein structural maps of adaptive evolution of human influenza A virus to increased virulence in the mouse. PLoS One 2011, 6:e21740.
• [54]Lipatov AS, Andreansky S, Webby RJ, Hulse DJ, Rehg JE, Krauss S, et al.: Pathogenesis of Hong Kong H5N1 influenza virus NS gene reassortants in mice: the role of cytokines and B- and T-cell responses. J Gen Virol 2005, 86:1121-1130.
• [55]Seo SH, Hoffmann E, Webster RG: Lethal H5N1 influenza viruses escape host anti-viral cytokine responses. Nat Med 2002, 8:950-954.
• [56]Jackson D, Hossain MJ, Hickman D, Perez DR, Lamb RA: A new influenza virus virulence determinant: the NS1 protein four C-terminal residues modulate pathogenicity. Proc Natl Acad Sci USA 2008, 105:4381-4386.
• [57]Jiao P, Tian G, Li Y, Deng G, Jiang Y, Liu C, et al.: A single-amino-acid substitution in the NS1 protein changes the pathogenicity of H5N1 avian influenza viruses in mice. J Virol 2008, 82:1146-1154.
• [58]Li Z, Jiang Y, Jiao P, Wang A, Zhao F, Tian G, et al.: The NS1 gene contributes to the virulence of H5N1 avian influenza viruses. J Virol 2006, 80:11115-11123.
• [59]Zhu Q, Yang H, Chen W, Cao W, Zhong G, Jiao P, et al.: A naturally occurring deletion in its NS gene contributes to the attenuation of an H5N1 swine influenza virus in chickens. J Virol 2008, 82:220-228.
• [60]Liu Q, Wang S, Ma G, Pu J, Forbes NE, Brown EG, et al.: Improved and simplified recombineering approach for influenza virus reverse genetics. J Mol Genet Med 2009, 3:225-231.
• [61]Wang S, Liu Q, Pu J, Li Y, Keleta L, Hu YW, et al.: Simplified recombinational approach for influenza A virus reverse genetics. J Virol Methods 2008, 151:74-78.
• [62]Brown EG, Liu H, Kit LC, Baird S, Nesrallah M: Pattern of mutation in the genome of influenza A virus on adaptation to increased virulence in the mouse lung: identification of functional themes. Proc Natl Acad Sci USA 2001, 98:6883-6888.
• [63]Hamilton MA: Trimmed Spearman-Karber Method for Estimating Median Lethal Concentrations in Toxicity Bioassays. Environ Sci Technol 1977, 11:714-719.
• [64]Suzuki K, Bose P, Leong-Quong RY, Fujita DJ, Riabowol K: REAP: A two minute cell fractionation method. BMC Res Notes 2010, 3:294. BioMed Central Full Text
• [65]Edgar R, Domrachev M, Lash AE: Gene Expression Omnibus: NCBI gene expression and hybridization array data repository. Nucleic Acids Res 2002, 30:207-210.
• [66]Murakami S, Horimoto T, Ito M, Takano R, Katsura H, Shimojima M, et al.: Enhanced growth of influenza vaccine seed viruses in vero cells mediated by broadening the optimal pH range for virus membrane fusion. J Virol 2012, 86:1405-1410.
• [67]Twu KY, Kuo RL, Marklund J, Krug RM: The H5N1 influenza virus NS genes selected after 1998 enhance virus replication in mammalian cells. J Virol 2007, 81:8112-8121.
• [68]Li Y, Yamakita Y, Krug RM: Regulation of a nuclear export signal by an adjacent inhibitory sequence: the effector domain of the influenza virus NS1 protein. Proc Natl Acad Sci USA 1998, 95:4864-4869.
• [69]Fulcher AJ, Jans DA: Regulation of nucleocytoplasmic trafficking of viral proteins: an integral role in pathogenesis? Biochim Biophys Acta 1813, 2011:2176-2190.
• [70]Kosugi S, Hasebe M, Tomita M, Yanagawa H: Nuclear export signal consensus sequences defined using a localization-based yeast selection system. Traffic 2008, 9:2053-2062.
• [71]Meng J, Zhang Z, Zheng Z, Liu Y, Wang H: Methionine-101 from one strain of H5N1 NS1 protein determines its IFN-antagonizing ability and subcellular distribution pattern. Sci China Life Sci 2012, 55:933-939.
• [72]McLane LM, Corbett AH: Nuclear localization signals and human disease. IUBMB Life 2009, 61:697-706.
• [73]Ivan FX, Rajapakse JC, Welsch RE, Rozen SG, Narasaraju T, Xiong GM, et al.: Differential pulmonary transcriptomic profiles in murine lungs infected with low and highly virulent influenza H3N2 viruses reveal dysregulation of TREM1 signaling, cytokines, and chemokines. Funct Integr Genomics 2012, 12:105-117.
• [74]Mukherjee S, Vipat VC, Mishra AC, Pawar SD, Chakrabarti AK: Pandemic (H1N1) 2009 influenza virus induces weaker host immune responses in vitro: a possible mechanism of high transmissibility. Virol J 2011, 8:140. BioMed Central Full Text
• [75]Cheng X, Xu Q, Song E, Yang CF, Kemble G, Jin H: The hemagglutinin protein of influenza A/Vietnam/1203/2004 (H5N1) contributes to hyperinduction of proinflammatory cytokines in human epithelial cells. Virology 2010, 406:28-36.
• [76]Kochs G, Garcia-Sastre A, Martinez-Sobrido L: Multiple anti-interferon actions of the Influenza A virus NS1 protein. J Virol 2007, 81(13):7011-7021.
• [77]Iyekdh IK: Study of Medical Supplies. 19th edition. publisher: Shibuya, Shibuya-ku, Tokyo 150, Japan: ; 1988:164. [Nippon Koteisho Kyokai, 12–15, 2-chome]
• [78]Matthaei M, Budt M, Wolff T: Highly Pathogenic H5N1 Influenza A Virus Strains Provoke Heterogeneous IFN-+¦/+¦ Responses That Distinctively Affect Viral Propagation in Human Cells. PLoS One 2013, 8:e56659.
• [79]To KF, Chan PK, Chan KF, Lee WK, Lam WY, Wong KF, et al.: Pathology of fatal human infection associated with avian influenza A H5N1 virus. J Med Virol 2001, 63:242-246.
• [80]Cheung CY, Poon LLM, Lau AS, Luk W, Lau YL, Shortridge KF, et al.: Induction of proinflammatory cytokines in human macrophages by influenza A (H5N1) viruses: a mechanism for the unusual severity of human disease? Lancet 2002, 360:1831-1837.
• [81]Steidle S, Martinez-Sobrido L, Mordstein M, Lienenklaus S, Garcia-Sastre A, Staheli P, et al.: Glycine 184 in nonstructural protein NS1 determines the virulence of influenza A virus strain PR8 without affecting the host interferon response. J Virol 2010, 84:12761-12770.
• [82]De J, Simmons CP, Thanh TT, Hien VM, Smith GJ, Chau TN, et al.: Fatal outcome of human influenza A (H5N1) is associated with high viral load and hypercytokinemia. Nat Med 2006, 12:1203-1207.