During influenza A virus infection, the NS1 protein is engaged in different functions in different intracellular compartments. In this study, we showed that the NS1 of A/PR/8/34 localized in different positions from that of A/Sydney/5/97 when transiently expressed in Madin–Darby canine kidney cells. Residue 221 of NS1 was identified to be a new key residue involved in the C-terminal nuclear localization signal (NLS) and nucleolar localization signal (NoLS) of NS1 from A/Sydney/5/97. Analysis of chimeric NS1 and further mutants showed that residues responsible for the binding between NS1 and the cleavage and polyadenylation specificity factor (CPSF) are correlated with the intracellular localization of transiently expressed NS1 proteins. Fluorescence loss in photobleaching imaging revealed that the NS1 protein with both functional NLSs and nuclear export signal (NES) was able to shuttle between the nucleus and cytoplasm. Drug inhibition experiments and fluorescence resonance energy transfer analysis suggested that NS1 was exported out of the cell nuclei via a Crm1-independent pathway. Moreover, it is likely that another cytoplasmic localization-related sequence exists in the NS1 protein other than the leucine-rich NES. These findings provide new insights into the mechanism of intracellular localization and trafficking of influenza A virus NS1 protein, which is important for understanding its function.


Article metrics loading...

Loading full text...

Full text loading...



  1. Askjaer, P., Jensen, T. H., Nilsson, J., Englmeier, L. & Kjems, J.(1998). The specificity of the Crm1-Rev nuclear export signal interaction is mediated by RanGTP. J Biol Chem 273, 33414–33422.[CrossRef] [Google Scholar]
  2. Bornholdt, Z. A. & Prasad, B. V.(2006). X-ray structure of influenza virus NS1 effector domain. Nat Struct Mol Biol 13, 559–560.[CrossRef] [Google Scholar]
  3. Chen, Z., Li, Y. & Krug, R. M.(1999). Influenza A virus NS1 protein targets poly(A)-binding protein II of the cellular 3′-end processing machinery. EMBO J 18, 2273–2283.[CrossRef] [Google Scholar]
  4. Chen, I. H., Sciabica, K. S. & Sandri-Goldin, R. M.(2002). ICP27 interacts with the RNA export factor Aly/REF to direct herpes simplex virus type 1 intronless mRNAs to the TAP export pathway. J Virol 76, 12877–12889.[CrossRef] [Google Scholar]
  5. Daelemans, D., Costes, S. V., Lockett, S. & Pavlakis, G. N.(2005). Kinetic and molecular analysis of nuclear export factor Crm1 association with its cargo in vivo. Mol Cell Biol 25, 728–739.[CrossRef] [Google Scholar]
  6. Das, K., Ma, L.-C., Xiao, R., Aramini, J., Marklund, J., Kuo, R.-L., Arnold, E., Krug, R. M. & Montelione, G. T.(2008). Structural basis for suppression of a host antiviral response by influenza A virus. Proc Natl Acad Sci U S A 105, 13093–13098.[CrossRef] [Google Scholar]
  7. Dong, X., Biswas, A. & Chook, Y. M.(2009a). Structural basis for assembly and disassembly of the Crm1 nuclear export complex. Nat Struct Mol Biol 16, 558–560.[CrossRef] [Google Scholar]
  8. Dong, X., Biswas, A., Süel, K. E., Jackson, L. K., Martinez, R., Gu, H. & Chook, Y. M.(2009b). Structural basis for leucine-rich nuclear export signal recognition by Crm1. Nature 458, 1136–1141.[CrossRef] [Google Scholar]
  9. Fornerod, M., Ohno, M., Yoshida, M. & Mattaj, I. W.(1997). Crm1 is an export receptor for leucine-rich nuclear export signals. Cell 90, 1051–1060.[CrossRef] [Google Scholar]
  10. Fukuda, M., Asano, S., Nakamura, T., Adachi, M., Yoshida, M., Yanagida, M. & Nishida, E.(1997). Crm1 is responsible for intracellular transport mediated by the nuclear export signal. Nature 390, 308–311.[CrossRef] [Google Scholar]
  11. Hayman, A., Comely, S., Lackenby, A., Murphy, S., McCauley, J., Goodbourn, S. & Barclay, W.(2006). Variation in the ability of human influenza A viruses to induce and inhibit the IFN-β pathway. Virology 347, 52–64.[CrossRef] [Google Scholar]
  12. Kochs, G., García-Sastre, A. & Martínez-Sobrido, L.(2007). Multiple anti-interferon actions of the influenza A virus NS1 protein. J Virol 81, 7011–7021.[CrossRef] [Google Scholar]
  13. Kudo, N., Matsumori, N., Taoka, H., Fujiwara, D., Schreiner, E. P., Wolff, B., Yoshida, M. & Horinouchi, S.(1999). Leptomycin B inactivates Crm1/exportin 1 by covalent modification at a cysteine residue in the central conserved region. Proc Natl Acad Sci U S A 96, 9112–9117.[CrossRef] [Google Scholar]
  14. Kutay, U. & Güttinger, S.(2005). Leucine-rich nuclear-export signals: born to be weak. Trends Cell Biol 15, 121–124.[CrossRef] [Google Scholar]
  15. Li, Y., Yamakita, Y. & Krug, R. M.(1998). Regulation of a nuclear export signal by an adjacent inhibitory sequence: the effector domain of the influenza virus NS1 protein. Proc Natl Acad Sci U S A 95, 4864–4869.[CrossRef] [Google Scholar]
  16. Li, Y., Chen, Z. Y., Wang, W., Baker, C. C. & Krug, R. M.(2001). The 3′-end-processing factor CPSF is required for the splicing of single-intron pre-mRNAs in vivo. RNA 7, 920–931.[CrossRef] [Google Scholar]
  17. Melén, K., Kinnunen, L., Fagerlund, R., Ikonen, N., Twu, K. Y., Krug, R. M. & Julkunen, I.(2007). Nuclear and nucleolar targeting of influenza A virus NS1 protein: striking differences between different virus subtypes. J Virol 81, 5995–6006.[CrossRef] [Google Scholar]
  18. Monecke, T., Güttler, T., Neumann, P., Dickmanns, A., Görlich, D. & Ficner, R.(2009). Crystal structure of the nuclear export receptor Crm1 in complex with Snurportin1 and RanGTP. Science 324, 1087–1091.[CrossRef] [Google Scholar]
  19. Nemeroff, M. E., Barabino, S. M. L., Li, Y., Keller, W. & Krug, R. M.(1998). 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.[CrossRef] [Google Scholar]
  20. Neville, M., Stutz, F., Lee, L., Davis, L. I. & Rosbash, M.(1997). The importin-beta family member Crm1p bridges the interaction between Rev and the nuclear pore complex during nuclear export. Curr Biol 7, 767–775. [Google Scholar]
  21. Noah, D. L., Twu, K. Y. & Krug, R. M.(2003). 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.[CrossRef] [Google Scholar]
  22. Volmer, R., Mazel-Sanchez, B., Volmer, C., Soubies, S. M. & Guérin, J. L.(2010). Nucleolar localization of influenza A NS1: striking differences between mammalian and avian cells. Virol J 7, 63.[CrossRef] [Google Scholar]

Data & Media loading...

This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error