Skip to main content

Thank you for visiting You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

Influenza virus uses transportin 1 for vRNP debundling during cell entry


Influenza A virus is a pathogen of great medical impact. To develop novel antiviral strategies, it is essential to understand the molecular aspects of virus–host cell interactions in detail. During entry, the viral ribonucleoproteins (vRNPs) that carry the RNA genome must be released from the incoming particle before they can enter the nucleus for replication. The uncoating process is facilitated by histone deacetylase 6 (ref.1). However, the precise mechanism of shell opening and vRNP debundling is unknown. Here, we show that transportin 1, a member of the importin-β family proteins, binds to a PY-NLS2 sequence motif close to the amino terminus of matrix protein (M1) exposed during acid priming of the viral core. It promotes the removal of M1 and induces disassembly of vRNP bundles. Next, the vRNPs interact with importin-α/β and enter the nucleus. Thus, influenza A virus uses dual importin-βs for distinct steps in host cell entry.

This is a preview of subscription content, access via your institution

Relevant articles

Open Access articles citing this article.

Access options

Rent or buy this article

Prices vary by article type



Prices may be subject to local taxes which are calculated during checkout

Fig. 1: TNPO1 is required for IAV infection.
Fig. 2: TNPO1 promotes M1 uncoating from incoming cytoplasmic vRNPs.
Fig. 3: TNPO1 binds to incoming IAV cores via a M1 N-terminal PY-NLS.
Fig. 4: Model of stepwise IAV uncoating by HDAC6 and TNPO1 during cell entry.

Data availability

The data that support the findings of this study are available from the corresponding author upon request. Atomic coordinates and structure factors of G18A M1-N have been deposited in the Protein Data Bank under accession code 6I3H.


  1. Banerjee, I. et al. Influenza A virus uses the aggresome processing machinery for host cell entry. Science 346, 473–477 (2014).

    Article  CAS  Google Scholar 

  2. Lee, B. J. et al. Rules for nuclear localization sequence recognition by karyopherin β2. Cell 126, 543–558 (2006).

    Article  CAS  Google Scholar 

  3. Noda, T. & Kawaoka, Y. Packaging of influenza virus genome: robustness of selection. Proc. Natl Acad. Sci. USA 109, 8797–8798 (2012).

    Article  CAS  Google Scholar 

  4. Yamauchi, Y. & Greber, U. F. Principles of virus uncoating: cues and the snooker ball. Traffic 17, 569–592 (2016).

    Article  CAS  Google Scholar 

  5. Helenius, A. Virus entry: looking back and moving forward. J. Mol. Biol. 430, 1853–1862 (2018).

    Article  CAS  Google Scholar 

  6. Stauffer, S. et al. Stepwise priming by acidic pH and a high K+ concentration is required for efficient uncoating of influenza A virus cores after penetration. J. Virol. 88, 13029–13046 (2014).

    Article  Google Scholar 

  7. Matlin, K. S., Reggio, H., Helenius, A. & Simons, K. Infectious entry pathway of influenza virus in a canine kidney cell line. J. Cell Biol. 91, 601–613 (1981).

    Article  CAS  Google Scholar 

  8. White, J., Kartenbeck, J. & Helenius, A. Membrane fusion activity of influenza virus. EMBO J. 1, 217–222 (1982).

    Article  CAS  Google Scholar 

  9. Maeda, T., Kawasaki, K. & Ohnishi, S. Interaction of influenza virus hemagglutinin with target membrane lipids is a key step in virus-induced hemolysis and fusion at pH 5.2. Proc. Natl Acad. Sci. USA 78, 4133–4137 (1981).

    Article  CAS  Google Scholar 

  10. Eisfeld, A. J., Neumann, G. & Kawaoka, Y. At the centre: influenza A virus ribonucleoproteins. Nat. Rev. Microbiol. 13, 28–41 (2015).

    Article  CAS  Google Scholar 

  11. Whittaker, G., Bui, M. & Helenius, A. Nuclear trafficking of influenza virus ribonuleoproteins in heterokaryons. J. Virol. 70, 2743–2756 (1996).

    CAS  PubMed  PubMed Central  Google Scholar 

  12. Martin, K. & Helenius, A. Transport of incoming influenza virus nucleocapsids into the nucleus. J. Virol. 65, 232–244 (1991).

    CAS  PubMed  PubMed Central  Google Scholar 

  13. Martin, K. & Helenius, A. Nuclear transport of influenza virus ribonucleoproteins: the viral matrix protein (M1) promotes export and inhibits import. Cell 67, 117–130 (1991).

    Article  CAS  Google Scholar 

  14. O‘Neill, R. E., Jaskunas, R., Blobel, G., Palese, P. & Moroianu, J. Nuclear import of influenza virus RNA can be mediated by viral nucleoprotein and transport factors required for protein import. J. Biol. Chem. 270, 22701–22704 (1995).

    Article  Google Scholar 

  15. Kemler, I., Whittaker, G. & Helenius, A. Nuclear import of microinjected influenza virus ribonucleoproteins. Virology 202, 1028–1033 (1994).

    Article  CAS  Google Scholar 

  16. Chou, Y. Y. et al. Colocalization of different influenza viral RNA segments in the cytoplasm before viral budding as shown by single-molecule sensitivity FISH analysis. PLoS Pathog. 9, e1003358 (2013).

    Article  CAS  Google Scholar 

  17. Hao, R. et al. Proteasomes activate aggresome disassembly and clearance by producing unanchored ubiquitin chains. Mol. Cell 51, 819–828 (2013).

    Article  CAS  Google Scholar 

  18. Wild, T. et al. A protein inventory of human ribosome biogenesis reveals an essential function of exportin 5 in 60S subunit export. PLoS Biol. 8, e1000522 (2010).

    Article  Google Scholar 

  19. Badertscher, L. et al. Genome-wide RNAi screening identifies protein modules required for 40S subunit synthesis in human cells. Cell Rep. 13, 2879–2891 (2015).

    Article  CAS  Google Scholar 

  20. Fried, H. & Kutay, U. Nucleocytoplasmic transport: taking an inventory. Cell. Mol. Life Sci. 60, 1659–1688 (2003).

    Article  CAS  Google Scholar 

  21. Konig, R. et al. Human host factors required for influenza virus replication. Nature 463, 813–817 (2010).

    Article  Google Scholar 

  22. Pollard, V. W. et al. A novel receptor-mediated nuclear protein import pathway. Cell 86, 985–994 (1996).

    Article  CAS  Google Scholar 

  23. Siomi, H. & Dreyfuss, G. A nuclear localization domain in the hnRNP A1 protein. J. Cell Biol. 129, 551–560 (1995).

    Article  CAS  Google Scholar 

  24. Bogerd, H. P. et al. Definition of a consensus transportin-specific nucleocytoplasmic transport signal. J. Biol. Chem. 274, 9771–9777 (1999).

    Article  CAS  Google Scholar 

  25. Banerjee, I., Yamauchi, Y., Helenius, A. & Horvath, P. High-content analysis of sequential events during the early phase of influenza A virus infection. PLoS ONE 8, e68450 (2013).

    Article  CAS  Google Scholar 

  26. White, J., Kartenbeck, J. & Helenius, A. Fusion of Semliki forest virus with the plasma membrane can be induced by low pH. J. Cell Biol. 87, 264–272 (1980).

    Article  CAS  Google Scholar 

  27. Soniat, M. & Chook, Y. M. Karyopherin-β2 recognition of a PY-NLS variant that lacks the proline-tyrosine motif. Structure 24, 1802–1809 (2016).

    Article  CAS  Google Scholar 

  28. Harris, A., Forouhar, F., Qiu, S., Sha, B. & Luo, M. The crystal structure of the influenza matrix protein M1 at neutral pH: M1–M1 protein interfaces can rotate in the oligomeric structures of M1. Virology 289, 34–44 (2001).

    Article  CAS  Google Scholar 

  29. Hoffmann, E., Neumann, G., Kawaoka, Y., Hobom, G. & Webster, R. G. A DNA transfection system for generation of influenza A virus from eight plasmids. Proc. Natl Acad. Sci. USA 97, 6108–6113 (2000).

    Article  CAS  Google Scholar 

  30. Arzt, S. et al. Combined results from solution studies on intact influenza virus M1 protein and from a new crystal form of its N-terminal domain show that M1 is an elongated monomer. Virology 279, 439–446 (2001).

    Article  CAS  Google Scholar 

  31. Chiang, M. J. et al. Maintaining pH-dependent conformational flexibility of M1 is critical for efficient influenza A virus replication. Emerg. Microbes Infect. 6, e108 (2017).

    Article  Google Scholar 

  32. Zhang, Y. et al. Mice lacking histone deacetylase 6 have hyperacetylated tubulin but are viable and develop normally. Mol. Cell Biol. 28, 1688–1701 (2008).

    Article  CAS  Google Scholar 

  33. Hurd, T. W., Fan, S. & Margolis, B. L. Localization of retinitis pigmentosa 2 to cilia is regulated by importin β2. J. Cell Sci. 124, 718–726 (2011).

    Article  CAS  Google Scholar 

  34. Nohinek, B., Gerhard, W. & Schulze, I. T. Characterization of host cell binding variants of influenza virus by monoclonal antibodies. Virology 143, 651–656 (1985).

    Article  CAS  Google Scholar 

  35. Singh, I. R., Suomalainen, M., Varadarajan, S., Garoff, H. & Helenius, A. Multiple mechanisms for the inhibition of entry and uncoating of superinfecting Semliki forest virus. Virology 231, 59–71 (1997).

    Article  CAS  Google Scholar 

  36. Guttinger, S., Muhlhausser, P., Koller-Eichhorn, R., Brennecke, J. & Kutay, U. Transportin2 functions as importin and mediates nuclear import of HuR. Proc. Natl Acad. Sci. USA 101, 2918–2923 (2004).

    Article  Google Scholar 

  37. Berrow, N. S., Alderton, D. & Owens, R. J. The precise engineering of expression vectors using high-throughput In-Fusion PCR cloning. Methods Mol. Biol. 498, 75–90 (2009).

    Article  CAS  Google Scholar 

  38. Kabsch, W. Integration, scaling, space-group assignment and post-refinement. Acta Crystallogr. D Biol. Crystallogr. 66, 133–144 (2010).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  40. Afonine, P. V. et al. phenix.model_vs_data: A high-level tool for the calculation of crystallographic model and data statistics. J. Appl. Crystallogr. 43, 669–676 (2010).

    Article  CAS  Google Scholar 

  41. Bricogne G. et al. BUSTER version X.Y.Z (Global Phasing, 2017).

Download references


We thank T. Wild for help with siRNA screen preparation, D. Alibhai for image analysis, T. Schwarz for super-resolution microscopy and E. Onischenko for protein purification. This work was supported by the European Research Council (2-73905-09, Cellular biology of virus infection to A.H.); and the Swiss National Science Foundation (2-77478-12, Regulation of early to late endosomal traffic to A.H.; 31003A 166565, NCCR RNA&Disease to U.K.; and SystemsX VirX—a host-directed approach against viral disease to Y.Y. and H.G.). The Friedrich Miescher Institute for Biomedical Research is supported by the Novartis Research Foundation (J.K. and H.G.). Y.M. was funded by the Japan Society for the Promotion of Science (Research Fellowship for Young Scientists). Part of this work was performed at beamline X10SA of the Swiss Light Source.

Author information

Authors and Affiliations



This study was conceptualized by Y.Y. and A.H., and investigated by Y.M., J.K., L.D., H.H.-X., S.I., H.G. and Y.Y. Resources were provided by U.K. The manuscript was written by Y.Y., Y.M., J.K., H.G., U.K. and A.H. and reviewed by all authors.

Corresponding author

Correspondence to Yohei Yamauchi.

Ethics declarations

Competing interests

The authors declare no competing interests.

Additional information

Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary information

Supplementary Information

Supplementary Figures 1–8, Supplementary Table 1 and Supplementary References.

Reporting Summary

Rights and permissions

Reprints and Permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Miyake, Y., Keusch, J.J., Decamps, L. et al. Influenza virus uses transportin 1 for vRNP debundling during cell entry. Nat Microbiol 4, 578–586 (2019).

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI:

This article is cited by


Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing