Archives of Virology

, Volume 156, Issue 6, pp 1031–1040 | Cite as

Atypical characteristics of nucleoprotein of pandemic influenza virus H1N1 and their roles in reassortment restriction

  • Asawin Wanitchang
  • Prasatha Patarasirin
  • Juggragarn Jengarn
  • Anan JongkaewwattanaEmail author
Original Article


Sequence analysis of the nucleoprotein (NP) of swine-origin influenza virus H1N1 (S-OIV) reveals a number of atypical characteristics including an early start codon and a highly conserved, non-aromatic residue at position 313. Using an in vitro viral polymerase reconstitution assay, we found that the polymerase complex containing the NP of S-OIV (NPS-OIV) yielded substantially lower activity than those assayed with NP derived from other influenza virus strains. Moreover, alteration of the early start codon or introduction of an aromatic residue at position 313 (V313Y) did not increase but instead exacerbated the poor polymerase activity. Interestingly, when NPS-OIV was allowed to compete with that of a mouse-adapted influenza virus (A/PR/8/34) to form progeny virions, only progeny bearing NPS-OIV were produced, despite the low polymerase activity associated with NPS-OIV. Our results indicated that NPS-OIV requires both the early start codon and the V313 residue for its optimal function. These characteristics are required for a strong compatibility between the S-OIV polymerase subunits and its indigenous NP over that of other strains, which might explain why productive reassortment between S-OIV and seasonal influenza viruses has yet to occur in nature.


Influenza Virus Polymerase Activity Swine Influenza Virus Human Influenza Virus Polymerase Complex 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.



We are grateful to Drs. R.G. Webster and E. Hoffmann (St. Jude Children’s Research Hospital) for providing the plasmids for reverse genetics of the PR8 strain; and to Drs. Pathom Sawanpanyalert, Sathit Pichyangkul and Arunee Thititanyanont for providing viral samples. This work was supported in part by the National Science Development Agency (NSTDA) (CPMO-P-00-20386 grant).


  1. 1.
    Faruqui F, Mukundan D (2009) Pandemic influenza: a review. Curr Opin Pediatr 22:530CrossRefGoogle Scholar
  2. 2.
    Halasa NB (2010) Update on the 2009 pandemic influenza A H1N1 in children. Curr Opin Pediatr 22:83–87PubMedCrossRefGoogle Scholar
  3. 3.
    Siston AM, Rasmussen SA, Honein MA, Fry AM, Seib K, Callaghan WM, Louie J, Doyle TJ, Crockett M, Lynfield R, Moore Z, Wiedeman C, Anand M, Tabony L, Nielsen CF, Waller K, Page S, Thompson JM, Avery C, Springs CB, Jones T, Williams JL, Newsome K, Finelli L, Jamieson DJ (2010) Pandemic 2009 influenza A(H1N1) virus illness among pregnant women in the United States. JAMA 303:1517–1525PubMedCrossRefGoogle Scholar
  4. 4.
    Herfst S, Chutinimitkul S, Ye J, de Wit E, Munster VJ, Schrauwen EJ, Bestebroer TM, Jonges M, Meijer A, Koopmans M, Rimmelzwaan GF, Osterhaus AD, Perez DR, Fouchier RA (2010) Introduction of virulence markers in PB2 of pandemic swine-origin influenza virus does not result in enhanced virulence or transmission. J Virol 84:3752–3758PubMedCrossRefGoogle Scholar
  5. 5.
    Jagger BW, Memoli MJ, Sheng ZM, Qi L, Hrabal RJ, Allen GL, Dugan VG, Wang R, Digard P, Kash JC, Taubenberger JK (2010) The PB2-E627K mutation attenuates viruses containing the 2009 H1N1 influenza pandemic polymerase. mBio 1:e00067-10Google Scholar
  6. 6.
    Zhu H, Wang J, Wang P, Song W, Zheng Z, Chen R, Guo K, Zhang T, Peiris JS, Chen H, Guan Y (2010) Substitution of lysine at 627 position in PB2 protein does not change virulence of the 2009 pandemic H1N1 virus in mice. Virology 401:1–5PubMedCrossRefGoogle Scholar
  7. 7.
    Mehle A, Doudna JA (2009) Adaptive strategies of the influenza virus polymerase for replication in humans. Proc Natl Acad Sci USA 106:21312–21316PubMedCrossRefGoogle Scholar
  8. 8.
    Yamada S, Hatta M, Staker BL, Watanabe S, Imai M, Shinya K, Sakai-Tagawa Y, Ito M, Ozawa M, Watanabe T, Sakabe S, Li C, Kim JH, Myler PJ, Phan I, Raymond A, Smith E, Stacy R, Nidom CA, Lank SM, Wiseman RW, Bimber BN, O’Connor DH, Neumann G, Stewart LJ, Kawaoka Y (2010) Biological and structural characterization of a host-adapting amino acid in influenza virus. PLoS Pathog 6:e1001034Google Scholar
  9. 9.
    Vijaykrishna D, Poon LL, Zhu HC, Ma SK, Li OT, Cheung CL, Smith GJ, Peiris JS, Guan Y (2010) Reassortment of pandemic H1N1/2009 influenza A virus in swine. Science 328:1529PubMedCrossRefGoogle Scholar
  10. 10.
    Chen GW, Shih SR (2009) Genomic signatures of influenza A pandemic (H1N1) 2009 virus. Emerg Infect Dis 15:1897–1903PubMedGoogle Scholar
  11. 11.
    Pan C, Cheung B, Tan S, Li C, Li L, Liu S, Jiang S Genomic signature and mutation trend analysis of pandemic (H1N1) 2009 influenza A virus. PLoS One 5:e9549Google Scholar
  12. 12.
    Hoffmann E, Neumann G, Kawaoka Y, Hobom G, Webster RG (2000) A DNA transfection system for generation of influenza A virus from eight plasmids. Proc Natl Acad Sci USA 97:6108–6113PubMedCrossRefGoogle Scholar
  13. 13.
    Hoffmann E, Stech J, Guan Y, Webster RG, Perez DR (2001) Universal primer set for the full-length amplification of all influenza A viruses. Arch Virol 146:2275PubMedCrossRefGoogle Scholar
  14. 14.
    Hoffmann E, Stech J, Guan Y, Webster RG, Perez DR (2001) Universal primer set for the full-length amplification of all influenza A viruses. Arch Virol 146:2275–2289PubMedCrossRefGoogle Scholar
  15. 15.
    Wanitchang A, Jengarn J, Jongkaewwattana A (2011) The N terminus of PA polymerase of swine-origin influenza virus H1N1 determines its compatibility with PB2 and PB1 subunits through a strain-specific amino acid serine 186. Virus Res 155:325–333PubMedCrossRefGoogle Scholar
  16. 16.
    Yongkiettrakul S, Boonyapakron K, Jongkaewwattana A, Wanitchang A, Leartsakulpanich U, Chitnumsub P, Eurwilaichitr L, Yuthavong Y (2009) Avian influenza A/H5N1 neuraminidase expressed in yeast with a functional head domain. J Virol Methods 156:44–51PubMedCrossRefGoogle Scholar
  17. 17.
    Kozak M (1992) Regulation of translation in eukaryotic systems. Annu Rev Cell Biol 8:197–225PubMedCrossRefGoogle Scholar
  18. 18.
    Wise HM, Foeglein A, Sun J, Dalton RM, Patel S, Howard W, Anderson EC, Barclay WS, Digard P (2009) A complicated message: Identification of a novel PB1-related protein translated from influenza A virus segment 2 mRNA. J Virol 83:8021–8031PubMedCrossRefGoogle Scholar
  19. 19.
    Ozawa M, Fujii K, Muramoto Y, Yamada S, Yamayoshi S, Takada A, Goto H, Horimoto T, Kawaoka Y (2007) Contributions of two nuclear localization signals of influenza A virus nucleoprotein to viral replication. J Virol 81:30–41PubMedCrossRefGoogle Scholar
  20. 20.
    Wu WW, Sun YH, Pante N (2007) Nuclear import of influenza A viral ribonucleoprotein complexes is mediated by two nuclear localization sequences on viral nucleoprotein. Virol J 4:49PubMedCrossRefGoogle Scholar
  21. 21.
    Belshe RB (2005) The origins of pandemic influenza—lessons from the 1918 virus. N Engl J Med 353:2209–2211PubMedCrossRefGoogle Scholar
  22. 22.
    Schnitzler SU, Schnitzler P (2009) An update on swine-origin influenza virus A/H1N1: a review. Virus Genes 39:279–292PubMedCrossRefGoogle Scholar
  23. 23.
    Li C, Hatta M, Nidom CA, Muramoto Y, Watanabe S, Neumann G, Kawaoka Y (2010) Reassortment between avian H5N1 and human H3N2 influenza viruses creates hybrid viruses with substantial virulence. Proc Natl Acad Sci USA 107:4687–4692PubMedCrossRefGoogle Scholar
  24. 24.
    Li C, Hatta M, Watanabe S, Neumann G, Kawaoka Y (2008) Compatibility among polymerase subunit proteins is a restricting factor in reassortment between equine H7N7 and human H3N2 influenza viruses. J Virol 82:11880–11888PubMedCrossRefGoogle Scholar
  25. 25.
    Octaviani CP, Ozawa M, Yamada S, Goto H, Kawaoka Y (2010) High genetic compatibility between swine-origin H1N1 and highly pathogenic avian H5N1 influenza viruses. J Virol 84:10918–10922PubMedCrossRefGoogle Scholar
  26. 26.
    Naffakh N, Tomoiu A, Rameix-Welti MA, van der Werf S (2008) Host restriction of avian influenza viruses at the level of the ribonucleoproteins. Annu Rev Microbiol 62:403–424PubMedCrossRefGoogle Scholar
  27. 27.
    Ye Q, Krug RM, Tao YJ (2006) The mechanism by which influenza A virus nucleoprotein forms oligomers and binds RNA. Nature 444:1078–1082PubMedCrossRefGoogle Scholar
  28. 28.
    Biswas SK, Boutz PL, Nayak DP (1998) Influenza virus nucleoprotein interacts with influenza virus polymerase proteins. J Virol 72:5493–5501PubMedGoogle Scholar
  29. 29.
    Medcalf L, Poole E, Elton D, Digard P (1999) Temperature-sensitive lesions in two influenza A viruses defective for replicative transcription disrupt RNA binding by the nucleoprotein. J Virol 73:7349–7356PubMedGoogle Scholar
  30. 30.
    Poole E, Elton D, Medcalf L, Digard P (2004) Functional domains of the influenza A virus PB2 protein: identification of NP- and PB1-binding sites. Virology 321:120–133PubMedCrossRefGoogle Scholar
  31. 31.
    Gabriel G, Herwig A, Klenk HD (2008) Interaction of polymerase subunit PB2 and NP with importin alpha1 is a determinant of host range of influenza A virus. PLoS Pathog 4:e11PubMedCrossRefGoogle Scholar

Copyright information

© Springer-Verlag 2011

Authors and Affiliations

  • Asawin Wanitchang
    • 1
  • Prasatha Patarasirin
    • 2
  • Juggragarn Jengarn
    • 1
  • Anan Jongkaewwattana
    • 1
    Email author
  1. 1.Virology and Cell Technology LaboratoryNational Center for Genetic Engineering and Biotechnology (BIOTEC)PathumthaniThailand
  2. 2.Faculty of EngineeringChulalongkorn UniversityBangkokThailand

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