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X-ray structure of NS1 from a highly pathogenic H5N1 influenza virus

Nature volume 456, pages 985–988 (2008)Cite this article

Abstract

The recent emergence of highly pathogenic avian (H5N1) influenza viruses, their epizootic and panzootic nature, and their association with lethal human infections have raised significant global health concerns1,2. Several studies have underlined the importance of non-structural protein NS1 in the increased pathogenicity and virulence of these strains3,4. NS1, which consists of two domains—a double-stranded RNA (dsRNA) binding domain5,6 and the effector domain7, separated through a linker—is an antagonist of antiviral type-I interferon response in the host8,9. Here we report the X-ray structure of the full-length NS1 from an H5N1 strain (A/Vietnam/1203/2004) that was associated with 60% of human deaths in an outbreak in Vietnam1,2. Compared to the individually determined structures of the RNA binding domain and the effector domain from non-H5N1 strains, the RNA binding domain within H5N1 NS1 exhibits modest structural changes, while the H5N1 effector domain shows significant alteration, particularly in the dimeric interface. Although both domains in the full-length NS1 individually participate in dimeric interactions, an unexpected finding is that these interactions result in the formation of a chain of NS1 molecules instead of distinct dimeric units. Three such chains in the crystal interact with one another extensively to form a tubular organization of similar dimensions to that observed in the cryo-electron microscopy images of NS1 in the presence of dsRNA. The tubular oligomeric organization of NS1, in which residues implicated in dsRNA binding face a 20-Å-wide central tunnel, provides a plausible mechanism for how NS1 sequesters varying lengths of dsRNA, to counter cellular antiviral dsRNA response pathways, while simultaneously interacting with other cellular ligands during an infection.

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Figure 1: H5N1 NS1 structure.
Figure 2: RBD and ED dimer formation, and the NS1 chain.
Figure 3: NS1 tubular structure.

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Accession codes

Primary accessions

Protein Data Bank

Data deposits

Atomic coordinates and structure factors for the reported crystal structure have been deposited with the Protein Data Bank under the accession number 3EU6.

References

  1. Abdel-Ghafar, A. N. et al. Update on avian influenza A (H5N1) virus infection in humans. N. Engl. J. Med. 358, 261–273 (2008)

    Article  CAS  Google Scholar 

  2. Beigel, J. H. et al. Avian influenza A (H5N1) infection in humans. N. Engl. J. Med. 353, 1374–1385 (2005)

    Article  Google Scholar 

  3. Seo, S. H., Hoffmann, E. & Webster, R. G. The NS1 gene of H5N1 influenza viruses circumvents the host anti-viral cytokine responses. Virus Res. 103, 107–113 (2004)

    Article  CAS  Google Scholar 

  4. Seo, S. H., Hoffmann, E. & Webster, R. G. Lethal H5N1 influenza viruses escape host anti-viral cytokine responses. Nature Med. 8, 950–954 (2002)

    Article  CAS  Google Scholar 

  5. Chien, C. Y. et al. A novel RNA-binding motif in influenza A virus non-structural protein 1. Nature Struct. Biol. 4, 891–895 (1997)

    Article  CAS  Google Scholar 

  6. Liu, J. et al. Crystal structure of the unique RNA-binding domain of the influenza virus NS1 protein. Nature Struct. Biol. 4, 896–899 (1997)

    Article  CAS  Google Scholar 

  7. Bornholdt, Z. A. & Prasad, B. V. X-ray structure of influenza virus NS1 effector domain. Nature Struct. Mol. Biol. 13, 559–560 (2006)

    Article  CAS  Google Scholar 

  8. Garcia-Sastre, A. et al. Influenza A virus lacking the NS1 gene replicates in interferon-deficient systems. Virology 252, 324–330 (1998)

    Article  CAS  Google Scholar 

  9. Wang, X. et al. Influenza A virus NS1 protein prevents activation of NF-kappaB and induction of alpha/beta interferon. J. Virol. 74, 11566–11573 (2000)

    Article  CAS  Google Scholar 

  10. Kochs, G., Garcia-Sastre, A. & Martinez-Sobrido, L. Multiple anti-interferon actions of the influenza A virus NS1 protein. J. Virol. 81, 7011–7021 (2007)

    Article  CAS  Google Scholar 

  11. Min, J. Y. & Krug, R. M. 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 103, 7100–7105 (2006)

    Article  ADS  CAS  Google Scholar 

  12. Lu, Y., Wambach, M., Katze, M. G. & Krug, R. M. Binding of the influenza virus NS1 protein to double-stranded RNA inhibits the activation of the protein kinase that phosphorylates the elF-2 translation initiation factor. Virology 214, 222–228 (1995)

    Article  CAS  Google Scholar 

  13. Hale, B. G. et al. Influenza A virus NS1 protein binds p85beta and activates phosphatidylinositol-3-kinase signaling. Proc. Natl Acad. Sci. USA 103, 14194–14199 (2006)

    Article  ADS  CAS  Google Scholar 

  14. Nemeroff, M. E. et al. 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 (1998)

    Article  CAS  Google Scholar 

  15. Twu, K. Y. et al. The CPSF30 binding site on the NS1A protein of influenza A virus is a potential antiviral target. J. Virol. 80, 3957–3965 (2006)

    Article  CAS  Google Scholar 

  16. Wang, W. et al. 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 (1999)

    Article  CAS  Google Scholar 

  17. Hale, B. G., Barclay, W. S., Randall, R. E. & Russell, R. J. Structure of an avian influenza A virus NS1 protein effector domain. Virology 378, 1–5 (2008)

    Article  CAS  Google Scholar 

  18. Li, K. S. et al. Genesis of a highly pathogenic and potentially pandemic H5N1 influenza virus in eastern Asia. Nature 430, 209–213 (2004)

    Article  ADS  CAS  Google Scholar 

  19. Noah, D. L., Twu, K. Y. & Krug, R. M. 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 307, 386–395 (2003)

    Article  CAS  Google Scholar 

  20. Diebold, S. S. et al. Viral infection switches non-plasmacytoid dendritic cells into high interferon producers. Nature 424, 324–328 (2003)

    Article  ADS  CAS  Google Scholar 

  21. Wang, Q. & Carmichael, G. G. Effects of length and location on the cellular response to double-stranded RNA. Microbiol. Mol. Biol. Rev. 68, 432–452 (2004)

    Article  CAS  Google Scholar 

  22. Li, W. X. et al. Interferon antagonist proteins of influenza and vaccinia viruses are suppressors of RNA silencing. Proc. Natl Acad. Sci. USA 101, 1350–1355 (2004)

    Article  ADS  CAS  Google Scholar 

  23. Melen, K. et al. Nuclear and nucleolar targeting of influenza A virus NS1 protein: Striking differences between different virus subtypes. J. Virol. 81, 5995–6006 (2007)

    Article  CAS  Google Scholar 

  24. Pflugrath, J. W. The finer things in X-ray diffraction data collection. Acta Crystallogr. D 55, 1718–1725 (1999)

    Article  CAS  Google Scholar 

  25. Evans, P. Scaling and assessment of data quality. Acta Crystallogr. D 62, 72–82 (2006)

    Article  Google Scholar 

  26. McCoy, A. J. et al. Phaser crystallographic software. J. Appl. Crystallogr. 40, 658–674 (2007)

    Article  CAS  Google Scholar 

  27. Murshudov, G. N., Vagin, A. A. & Dodson, E. J. Refinement of macromolecular structures by the maximum-likelihood method. Acta Crystallogr. D 53, 240–255 (1997)

    Article  CAS  Google Scholar 

  28. The. CCP4 suite: Programs for protein crystallography. Acta Crystallogr. D 50, 760–763 (1994)

  29. Brunger, A. T. et al. Crystallography & NMR system: A new software suite for macromolecular structure determination. Acta Crystallogr. D 54, 905–921 (1998)

    Article  CAS  Google Scholar 

  30. Emsley, P. & Cowtan, K. Coot: Model-building tools for molecular graphics. Acta Crystallogr. D 60, 2126–2132 (2004)

    Article  Google Scholar 

  31. Brunger, A. T. Free R value: A novel statistical quantity for assessing the accuracy of crystal structures. Nature 355, 472–475 (1992)

    Article  ADS  CAS  Google Scholar 

  32. Laskowski, R. A., MacArthur, M. W., Moss, D. S. & Thornton, J. M. PROCHECK: A program to check the stereochemical quality of protein structures. J. Appl. Crystallogr. 26, 283–291 (1993)

    Article  CAS  Google Scholar 

  33. Dubochet, J. et al. Cryo-electron microscopy of vitrified specimens. Q. Rev. Biophys. 21, 129–228 (1988)

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We thank P. Palese for providing us clones of A/Vietnam/1203/2004 H5N1 and A/PR8/34 (H1N1) NS1. This work was supported by the NIH (AI36040) and the Robert Welch Foundation (to B.V.V.P.). Z.A.B. acknowledges support from an NIH virology training grant (AI07471). We thank P. Palese, A. Rice, M. Schmid and B. Carrillo for discussions and comments on the manuscript and H. Chen for technical assistance with cryo-EM. We acknowledge the use of cryo-EM facilities at the National Center for Macromolecular Imaging (Baylor College of Medicine) supported by the National Institutes of Health (RR002250 to W. Chiu); we also acknowledge the Center for Advanced Microstructures & Devices (CAMD), Baton Rouge, Los Angeles, and H. Bellamy, and the SBC-CAT 19ID beam line at the Advanced Photon Source (supported by the US Department of Energy, Basic Energy Sciences, Office of Science, under contract no.W-31-109-Eng-38) and its staff for their help during data collection.

Author information

Authors and Affiliations

  1. Department of Molecular Virology and Microbiology,,

    Zachary A. Bornholdt & B. V. Venkataram Prasad

  2. Department of Biochemistry and Molecular Biology, Baylor College of Medicine, One Baylor Plaza, Houston, Texas 77030, USA,

    B. V. Venkataram Prasad

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Correspondence to B. V. Venkataram Prasad.

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Bornholdt, Z., Prasad, B. X-ray structure of NS1 from a highly pathogenic H5N1 influenza virus. Nature 456, 985–988 (2008). https://doi.org/10.1038/nature07444

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