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.
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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.
Banerjee, I. et al. Influenza A virus uses the aggresome processing machinery for host cell entry. Science 346, 473–477 (2014).
Lee, B. J. et al. Rules for nuclear localization sequence recognition by karyopherin β2. Cell 126, 543–558 (2006).
Noda, T. & Kawaoka, Y. Packaging of influenza virus genome: robustness of selection. Proc. Natl Acad. Sci. USA 109, 8797–8798 (2012).
Yamauchi, Y. & Greber, U. F. Principles of virus uncoating: cues and the snooker ball. Traffic 17, 569–592 (2016).
Helenius, A. Virus entry: looking back and moving forward. J. Mol. Biol. 430, 1853–1862 (2018).
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).
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).
White, J., Kartenbeck, J. & Helenius, A. Membrane fusion activity of influenza virus. EMBO J. 1, 217–222 (1982).
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).
Eisfeld, A. J., Neumann, G. & Kawaoka, Y. At the centre: influenza A virus ribonucleoproteins. Nat. Rev. Microbiol. 13, 28–41 (2015).
Whittaker, G., Bui, M. & Helenius, A. Nuclear trafficking of influenza virus ribonuleoproteins in heterokaryons. J. Virol. 70, 2743–2756 (1996).
Martin, K. & Helenius, A. Transport of incoming influenza virus nucleocapsids into the nucleus. J. Virol. 65, 232–244 (1991).
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).
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).
Kemler, I., Whittaker, G. & Helenius, A. Nuclear import of microinjected influenza virus ribonucleoproteins. Virology 202, 1028–1033 (1994).
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).
Hao, R. et al. Proteasomes activate aggresome disassembly and clearance by producing unanchored ubiquitin chains. Mol. Cell 51, 819–828 (2013).
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).
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).
Fried, H. & Kutay, U. Nucleocytoplasmic transport: taking an inventory. Cell. Mol. Life Sci. 60, 1659–1688 (2003).
Konig, R. et al. Human host factors required for influenza virus replication. Nature 463, 813–817 (2010).
Pollard, V. W. et al. A novel receptor-mediated nuclear protein import pathway. Cell 86, 985–994 (1996).
Siomi, H. & Dreyfuss, G. A nuclear localization domain in the hnRNP A1 protein. J. Cell Biol. 129, 551–560 (1995).
Bogerd, H. P. et al. Definition of a consensus transportin-specific nucleocytoplasmic transport signal. J. Biol. Chem. 274, 9771–9777 (1999).
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).
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).
Soniat, M. & Chook, Y. M. Karyopherin-β2 recognition of a PY-NLS variant that lacks the proline-tyrosine motif. Structure 24, 1802–1809 (2016).
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).
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).
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).
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).
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).
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).
Nohinek, B., Gerhard, W. & Schulze, I. T. Characterization of host cell binding variants of influenza virus by monoclonal antibodies. Virology 143, 651–656 (1985).
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).
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).
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).
Kabsch, W. Integration, scaling, space-group assignment and post-refinement. Acta Crystallogr. D Biol. Crystallogr. 66, 133–144 (2010).
McCoy, A. J. et al. Phaser crystallographic software. J. Appl. Crystallogr. 40, 658–674 (2007).
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).
Bricogne G. et al. BUSTER version X.Y.Z (Global Phasing, 2017).
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.
The authors declare no competing interests.
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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). https://doi.org/10.1038/s41564-018-0332-2
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