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Structural basis for influenza virus NS1 protein block of mRNA nuclear export


Influenza viruses antagonize key immune defence mechanisms via the virulence factor non-structural protein 1 (NS1). A key mechanism of virulence by NS1 is blocking nuclear export of host messenger RNAs, including those encoding immune factors1,2,3; however, the direct cellular target of NS1 and the mechanism of host mRNA export inhibition are not known. Here, we identify the target of NS1 as the mRNA export receptor complex, nuclear RNA export factor 1–nuclear transport factor 2-related export protein 1 (NXF1–NXT1), which is the principal receptor mediating docking and translocation of mRNAs through the nuclear pore complex via interactions with nucleoporins4,5. We determined the crystal structure of NS1 in complex with NXF1–NXT1 at 3.8 Å resolution. The structure reveals that NS1 prevents binding of NXF1–NXT1 to nucleoporins, thereby inhibiting mRNA export through the nuclear pore complex into the cytoplasm for translation. We demonstrate that a mutant influenza virus deficient in binding NXF1–NXT1 does not block host mRNA export and is attenuated. This attenuation is marked by the release of mRNAs encoding immune factors from the nucleus. In sum, our study uncovers the molecular basis of a major nuclear function of influenza NS1 protein that causes potent blockage of host gene expression and contributes to inhibition of host immunity.

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Data availability

The coordinates of the NS1–NXF1–NXT1 structure have been deposited in the Protein Data Bank under accession number 6E5U. The data for the transcriptome analysis has been deposited in the Gene Expression Omnibus under accession number GSE129318. All other data that support the findings of this study are available from the corresponding authors upon request.


  1. 1.

    Satterly, N. et al. Influenza virus targets the mRNA export machinery and the nuclear pore complex. Proc. Natl Acad. Sci. USA 104, 1853–1858 (2007).

  2. 2.

    Zhang, L. et al. Inhibition of pyrimidine synthesis reverses viral virulence factor-mediated block of mRNA nuclear export. J. Cell Biol. 196, 315–326 (2012).

  3. 3.

    Qiu, Y. & Krug, R. M. The influenza virus NS1 protein is a poly(A)-binding protein that inhibits nuclear export of mRNAs containing poly(A). J. Virol. 68, 2425–2432 (1994).

  4. 4.

    Carmody, S. R. & Wente, S. R. mRNA nuclear export at a glance. J. Cell Sci. 122, 1933–1937 (2009).

  5. 5.

    Stewart, M. Nuclear export of mRNA. Trends Biochem. Sci. 35, 609–617 (2010).

  6. 6.

    Nair, H. et al. Global burden of respiratory infections due to seasonal influenza in young children: a systematic review and meta-analysis. Lancet 378, 1917–1930 (2011).

  7. 7.

    Reed, C. et al. Estimating influenza disease burden from population-based surveillance data in the United States. PLoS ONE 10, e0118369 (2015).

  8. 8.

    Fact sheet Number 211. WHO http://www.who.int/mediacentre/factsheets/fs211/en/index.html (2009).

  9. 9.

    Kim, S. J. et al. Integrative structure and functional anatomy of a nuclear pore complex. Nature 555, 475–482 (2018).

  10. 10.

    Ayllon, J. & Garcia-Sastre, A. The NS1 protein: a multitasking virulence factor. Curr. Top. Microbiol. Immunol. 386, 73–107 (2015).

  11. 11.

    Chen, Z., Li, Y. & Krug, R. M. Influenza A virus NS1 protein targets poly(A)-binding protein II of the cellular 3′-end processing machinery. EMBO J. 18, 2273–2283 (1999).

  12. 12.

    Nemeroff, M. E., Barabino, S. M., Li, Y., Keller, W. & Krug, R. M. 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).

  13. 13.

    Bornholdt, Z. A. & Prasad, B. V. X-ray structure of NS1 from a highly pathogenic H5N1 influenza virus. Nature 456, 985–988 (2008).

  14. 14.

    Carrillo, B. et al. The influenza A virus protein NS1 displays structural polymorphism. J. Virol. 88, 4113–4122 (2014).

  15. 15.

    Aibara, S., Katahira, J., Valkov, E. & Stewart, M. The principal mRNA nuclear export factor NXF1:NXT1 forms a symmetric binding platform that facilitates export of retroviral CTE-RNA. Nucleic Acids Res. 43, 1883–1893 (2015).

  16. 16.

    Fribourg, S., Braun, I. C., Izaurralde, E. & Conti, E. Structural basis for the recognition of a nucleoporin FG repeat by the NTF2-like domain of the TAP/p15 mRNA nuclear export factor. Mol. Cell 8, 645–656 (2001).

  17. 17.

    Blevins, M. B., Smith, A. M., Phillips, E. M. & Powers, M. A. Complex formation among the RNA export proteins Nup98, Rae1/Gle2, and TAP. J. Biol. Chem. 278, 20979–20988 (2003).

  18. 18.

    Faria, P. A. et al. VSV disrupts the Rae1/mrnp41 mRNA nuclear export pathway. Mol. Cell 17, 93–102 (2005).

  19. 19.

    Bachi, A. et al. The C-terminal domain of TAP interacts with the nuclear pore complex and promotes export of specific CTE-bearing RNA substrates. RNA 6, 136–158 (2000).

  20. 20.

    Ullman, K. S., Shah, S., Powers, M. A. & Forbes, D. J. The nucleoporin nup153 plays a critical role in multiple types of nuclear export. Mol. Biol. Cell 10, 649–664 (1999).

  21. 21.

    Lelek, M. et al. Chromatin organization at the nuclear pore favours HIV replication. Nat. Commun. 6, 6483 (2015).

  22. 22.

    Twu, K. Y., Kuo, R. L., Marklund, J. & Krug, R. M. The H5N1 influenza virus NS genes selected after 1998 enhance virus replication in mammalian cells. J. Virol. 81, 8112–8121 (2007).

  23. 23.

    Das, K. et al. Structural basis for suppression of a host antiviral response by influenza A virus. Proc. Natl Acad. Sci. USA 105, 13093–13098 (2008).

  24. 24.

    Chow, K. T., Gale, M. Jr. & Loo, Y. M. RIG-I and other RNA sensors in antiviral immunity. Annu. Rev. Immunol. 36, 667–694 (2018).

  25. 25.

    Fensterl, V. & Sen, G. C. Interferon-induced Ifit proteins: their role in viral pathogenesis. J. Virol. 89, 2462–2468 (2015).

  26. 26.

    Yarbrough, M. L., Mata, M. A., Sakthivel, R. & Fontoura, B. M. Viral subversion of nucleocytoplasmic trafficking. Traffic 15, 127–140 (2014).

  27. 27.

    Mor, A. et al. Influenza virus mRNA trafficking through host nuclear speckles. Nat. Microbiol. 1, 16069 (2016).

  28. 28.

    Solorzano, A. et al. Mutations in the NS1 protein of swine influenza virus impair anti-interferon activity and confer attenuation in pigs. J. Virol. 79, 7535–7543 (2005).

  29. 29.

    Radu, A., Moore, M. S. & Blobel, G. The peptide repeat domain of nucleoporin Nup98 functions as a docking site in transport across the nuclear pore complex. Cell 81, 215–222 (1995).

  30. 30.

    Tsai, P. L. et al. Cellular RNA binding proteins NS1-BP and hnRNP K regulate influenza A virus RNA splicing. PLoS Pathog. 9, e1003460 (2013).

  31. 31.

    Fodor, E. et al. Rescue of influenza A virus from recombinant DNA. J. Virol. 73, 9679–9682 (1999).

  32. 32.

    Fontoura, B. M., Blobel, G. & Matunis, M. J. A conserved biogenesis pathway for nucleoporins: proteolytic processing of a 186-kilodalton precursor generates Nup98 and the novel nucleoporin, Nup96. J. Cell Biol. 144, 1097–1112 (1999).

  33. 33.

    Otwinowski, Z. & Minor, W. Processing of X-ray diffraction data collected in oscillation mode. Methods Enzym. 276, 307–326 (1997).

  34. 34.

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

  35. 35.

    Emsley, P., Lohkamp, B., Scott, W. G. & Cowtan, K. Features and development of Coot. Acta Crystallogr. D 66, 486–501 (2010).

  36. 36.

    Afonine, P. V. et al. Towards automated crystallographic structure refinement with phenix.refine. Acta Crystallogr. D 68, 352–367 (2012).

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We thank Z. Wawrzak at Advanced Photon Source (APS) beamline 21-ID-F for assistance with X-ray data collection; P. Jeffrey for suggestions for data processing; and Y. M. Chook for reagents and assistance with protein purification. Y.R. and Y.X. are supported by funds from Vanderbilt University School of Medicine. Funding was provided by NIH R01 GM113874 to B.M.A.F.; R01 AI125524 to B.M.A.F. and A.G.-S. This work is also partially funded by CRIP (Center for Research on Influenza Pathogenesis), an NIAID funded Center of Excellence for Influenza Research and Surveillance (CEIRS, contract no. HHSN272201400008C) and by NIAID grant U19AI135972.

Author information

B.M.A.F. and Y.R. conceived the study. K.Z., Y.X., R.M.-M., J.W., L.Z., M.E. and Y.R. performed the experiments. K.Z., Y.X., R.M.-M., J.W., L.Z., M.E., A.G.-S., B.M.A.F. and Y.R. analysed the data. K.Z., Y.X., B.M.A.F. and Y.R. wrote the manuscript with input from all authors.

Correspondence to Beatriz M. A. Fontoura or Yi Ren.

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Supplementary information

Supplementary Information

Supplementary Figs. 1–7, Supplementary Tables 1 and 2, raw image figures and raw image Supplementary Figures.

Reporting Summary

Supplementary Table 3

Transcriptome profiling of A549 cells infected with influenza virus shows a subset of cellular mRNAs blocked in the nucleus.

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Fig. 1: Influenza A virus NS1 protein binds the nucleoporin-binding domain of NXF1 to block host mRNA nuclear export.
Fig. 2: Structure of a 2:2:2 complex of *NS1–NXF1(117–619)–NXT1.
Fig. 3: NS1 blocks binding of the FG nucleoporin Nup98 to NXF1–NXT1 in vitro and in influenza-infected cells.
Fig. 4: Influenza virus mutant on the NXF1-binding site of NS1 allows nuclear export of host mRNAs and is attenuated.