The non-structural protein NS2, also called nuclear export protein, of influenza A virus contains a leucine-rich nuclear-export signal that could guide viral ribonucleoproteins to cross the nuclear pore complex (NPC) and complete directional nucleocytoplasmic trafficking. In this study, human nucleoporin 98 (hNup98), an NPC protein, was identified as an NS2-binding protein by using yeast two-hybrid screening of a human cDNA library. Interaction between NS2 and hNup98 was confirmed in yeast and mammalian cells. Mapping tests further demonstrated that aa 22–53 in the N-terminal region of NS2 and the glycine-leucine-phenylalanine-glycine (GLFG) repeat domain (aa 1–511) of hNup98 are crucial for the interaction of these two proteins. Confocal microscopy showed that hNup98 could specifically recruit NS2 to the nucleoli and that this process was inhibited by leptomycin B, a specific inhibitor of human chromosomal region maintenance 1 protein. NS2 recruitment to the nucleoli was through the N-terminal GLFG repeat domain of hNup98, but not through the C-terminal domain. Moreover, influenza virus infection downregulated Nup98 levels significantly in 293T and Madin–Darby canine kidney cells. Overexpression of the GLFG repeat domain of hNup98 apparently inhibited virus propagation. Together, these findings reveal the interaction between hNup98 and NS2. The GLFG repeat domain of hNup98 might competitively inhibit the interaction between NS2 and endogenous hNup98, consequently inhibiting virus propagation.


Article metrics loading...

Loading full text...

Full text loading...



  1. Akarsu, H., Burmeister, W. P., Petosa, C., Petit, I., Müller, C. W., Ruigrok, R. W. & Baudin, F.(2003). Crystal structure of the M1 protein-binding domain of the influenza A virus nuclear export protein (NEP/NS2). EMBO J 22, 4646–4655.[CrossRef] [Google Scholar]
  2. Bayliss, R., Littlewood, T., Strawn, L. A., Wente, S. R. & Stewart, M.(2002). GLFG and FxFG nucleoporins bind to overlapping sites on importin-β. J Biol Chem 277, 50597–50606.[CrossRef] [Google Scholar]
  3. Bogerd, H. P., Echarri, A., Ross, T. M. & Cullen, B. R.(1998). Inhibition of human immunodeficiency virus Rev and human T-cell leukemia virus Rex function, but not Mason-Pfizer monkey virus constitutive transport element activity, by a mutant human nucleoporin targeted to Crm1. J Virol 72, 8627–8635. [Google Scholar]
  4. Boulo, S., Akarsu, H., Ruigrok, R. W. & Baudin, F.(2007). Nuclear traffic of influenza virus proteins and ribonucleoprotein complexes. Virus Res 124, 12–21.[CrossRef] [Google Scholar]
  5. Boyle, S. M., Ruvolo, V., Gupta, A. K. & Swaminathan, S.(1999). Association with the cellular export receptor CRM 1 mediates function and intracellular localization of Epstein–Barr virus SM protein, a regulator of gene expression. J Virol 73, 6872–6881. [Google Scholar]
  6. Brass, A. L., Huang, I. C., Benita, Y., John, S. P., Krishnan, M. N., Feeley, E. M., Ryan, B. J., Weyer, J. L., van der Weyden, L. & other authors(2009). The IFITM proteins mediate cellular resistance to influenza A H1N1 virus, West Nile virus, and dengue virus. Cell 139, 1243–1254.[CrossRef] [Google Scholar]
  7. Carmody, S. R. & Wente, S. R.(2009). mRNA nuclear export at a glance. J Cell Sci 122, 1933–1937.[CrossRef] [Google Scholar]
  8. Chakraborty, P., Seemann, J., Mishra, R. K., Wei, J. H., Weil, L., Nussenzveig, D. R., Heiber, J., Barber, G. N., Dasso, M. & other authors(2009). Vesicular stomatitis virus inhibits mitotic progression and triggers cell death. EMBO Rep 10, 1154–1160.[CrossRef] [Google Scholar]
  9. 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]
  10. Cros, J. F. & Palese, P.(2003). Trafficking of viral genomic RNA into and out of the nucleus: influenza, Thogoto and Borna disease viruses. Virus Res 95, 3–12.[CrossRef] [Google Scholar]
  11. Enninga, J., Levy, D. E., Blobel, G. & Fontoura, B. M.(2002). Role of nucleoporin induction in releasing an mRNA nuclear export block. Science 295, 1523–1525.[CrossRef] [Google Scholar]
  12. Floer, M. & Blobel, G.(1999). Putative reaction intermediates in Crm1-mediated nuclear protein export. J Biol Chem 274, 16279–16286.[CrossRef] [Google Scholar]
  13. Fritz, C. C., Zapp, M. L. & Green, M. R.(1995). A human nucleoporin-like protein that specifically interacts with HIV Rev. Nature 376, 530–533.[CrossRef] [Google Scholar]
  14. Griffis, E. R., Altan, N., Lippincott-Schwartz, J. & Powers, M. A.(2002). Nup98 is a mobile nucleoporin with transcription-dependent dynamics. Mol Biol Cell 13, 1282–1297.[CrossRef] [Google Scholar]
  15. Griffis, E. R., Xu, S. & Powers, M. A.(2003). Nup98 localizes to both nuclear and cytoplasmic sides of the nuclear pore and binds to two distinct nucleoporin subcomplexes. Mol Biol Cell 14, 600–610.[CrossRef] [Google Scholar]
  16. Gustin, K. E.(2003). Inhibition of nucleo-cytoplasmic trafficking by RNA viruses: targeting the nuclear pore complex. Virus Res 95, 35–44.[CrossRef] [Google Scholar]
  17. Hao, L., Sakurai, A., Watanabe, T., Sorensen, E., Nidom, C. A., Newton, M. A., Ahlquist, P. & Kawaoka, Y.(2008).Drosophila RNAi screen identifies host genes important for influenza virus replication. Nature 454, 890–893.[CrossRef] [Google Scholar]
  18. Hodel, A. E., Hodel, M. R., Griffis, E. R., Hennig, K. A., Ratner, G. A., Xu, S. & Powers, M. A.(2002). The three-dimensional structure of the autoproteolytic, nuclear pore-targeting domain of the human nucleoporin Nup98. Mol Cell 10, 347–358.[CrossRef] [Google Scholar]
  19. Huang, S., Chen, J., Wang, H., Sun, B., Wang, H., Zhang, Z., Zhang, X. & Chen, Z.(2009). Influenza A virus matrix protein 1 interacts with hTFIIIC102-s, a short isoform of the polypeptide 3 subunit of human general transcription factor IIIC. Arch Virol 154, 1101–1110.[CrossRef] [Google Scholar]
  20. Iwatsuki-Horimoto, K., Horimoto, T., Fujii, Y. & Kawaoka, Y.(2004). Generation of influenza A virus NS2 (NEP) mutants with an altered nuclear export signal sequence. J Virol 78, 10149–10155.[CrossRef] [Google Scholar]
  21. Karlas, A., Machuy, N., Shin, Y., Pleissner, K. P., Artarini, A., Heuer, D., Becker, D., Khalil, H., Ogilvie, L. A. & other authors(2010). Genome-wide RNAi screen identifies human host factors crucial for influenza virus replication. Nature 463, 818–822.[CrossRef] [Google Scholar]
  22. Kiss, A., Li, L., Gettemeier, T. & Venkatesh, L. K.(2003). Functional analysis of the interaction of the human immunodeficiency virus type 1 Rev nuclear export signal with its cofactors. Virology 314, 591–600.[CrossRef] [Google Scholar]
  23. Kutay, U. & Güttinger, S.(2005). Leucine-rich nuclear-export signals: born to be weak. Trends Cell Biol 15, 121–124.[CrossRef] [Google Scholar]
  24. Lim, R. Y., Ullman, K. S. & Fahrenkrog, B.(2008). Biology and biophysics of the nuclear pore complex and its components. Int Rev Cell Mol Biol 267, 299–342. [Google Scholar]
  25. Lommer, B. S. & Luo, M.(2002). Structural plasticity in influenza virus protein NS2 (NEP). J Biol Chem 277, 7108–7117.[CrossRef] [Google Scholar]
  26. Nayak, D. P., Hui, E. K. & Barman, S.(2004). Assembly and budding of influenza virus. Virus Res 106, 147–165.[CrossRef] [Google Scholar]
  27. Neumann, G., Hughes, M. T. & Kawaoka, Y.(2000). Influenza A virus NS2 protein mediates vRNP nuclear export through NES-independent interaction with hCRM1. EMBO J 19, 6751–6758.[CrossRef] [Google Scholar]
  28. Neville, M., Stutz, F., Lee, L., Davis, L. I. & Rosbash, M.(1997). The importin-β family member Crm1p bridges the interaction between Rev and the nuclear pore complex during nuclear export. Curr Biol 7, 767–775. [Google Scholar]
  29. Oka, M., Asally, M., Yasuda, Y., Ogawa, Y., Tachibana, T. & Yoneda, Y.(2010). The mobile FG nucleoporin Nup98 is a cofactor for Crm1-dependent protein export. Mol Biol Cell 21, 1885–1896.[CrossRef] [Google Scholar]
  30. O'Neill, R. E., Talon, J. & Palese, P.(1998). The influenza virus NEP (NS2 protein) mediates the nuclear export of viral ribonucleoproteins. EMBO J 17, 288–296.[CrossRef] [Google Scholar]
  31. Otero, G. C., Harris, M. E., Donello, J. E. & Hope, T. J.(1998). Leptomycin B inhibits equine infectious anemia virus Rev and feline immunodeficiency virus Rev function but not the function of the hepatitis B virus post-transcriptional regulatory element. J Virol 72, 7593–7597. [Google Scholar]
  32. Park, N., Katikaneni, P., Skern, T. & Gustin, K. E.(2007). Differential targeting of nuclear pore complex proteins in poliovirus-infected cells. J Virol 82, 1647–1655. [Google Scholar]
  33. Pemberton, L. F. & Paschal, B. M.(2005). Mechanisms of receptor-mediated nuclear import and nuclear export. Traffic 6, 187–198.[CrossRef] [Google Scholar]
  34. Powers, M. A., Forbes, D. J., Dahlberg, J. E. & Lund, E.(1997). The vertebrate GLFG nucleoporin, Nup98, is an essential component of multiple RNA export pathways. J Cell Biol 136, 241–250.[CrossRef] [Google Scholar]
  35. Pritchard, C. E., Fornerod, M., Kasper, L. H. & van Deursen, J. M.(1999). RAE1 is a shuttling mRNA export factor that binds to a GLEBS-like NUP98 motif at the nuclear pore complex through multiple domains. J Cell Biol 145, 237–254.[CrossRef] [Google Scholar]
  36. Radu, A., Moore, M. S. & Blobel, G.(1995a). The peptide repeat domain of nucleoporin Nup98 functions as a docking site in transport across the nuclear pore complex. Cell 81, 215–222.[CrossRef] [Google Scholar]
  37. Radu, A., Blobel, G. & Moore, M. S.(1995b). Identification of a protein complex that is required for nuclear protein import and mediates docking of import substrate to distinct nucleoporins. Proc Natl Acad Sci U S A 92, 1769–1773.[CrossRef] [Google Scholar]
  38. Reed, L. J. & Muench, H.(1938). A simple method of estimating fifty per cent endpoints. Am J Hyg 27, 493–497. [Google Scholar]
  39. Ricour, C., Delhaye, S., Hato, S. V., Olenyik, T. D., Michel, B., van Kuppeveld, F. J., Gustin, K. E. & Michiels, T.(2009). Inhibition of mRNA export and dimerization of interferon regulatory factor 3 by Theiler's virus leader protein. J Gen Virol 90, 177–186.[CrossRef] [Google Scholar]
  40. Sanz-Ezquerro, J. J., de la Luna, S., Ortín, J. & Nieto, A.(1995). Individual expression of influenza virus PA protein induces degradation of coexpressed proteins. J Virol 69, 2420–2426. [Google Scholar]
  41. Satterly, N., Tsai, P. L., van Deursen, J., Nussenzveig, D. R., Wang, Y., Faria, P. A., Levay, A., Levy, D. E. & Fontoura, B. M.(2007). Influenza virus targets the mRNA export machinery and the nuclear pore complex. Proc Natl Acad Sci U S A 104, 1853–1858.[CrossRef] [Google Scholar]
  42. Stutz, F., Izaurralde, E., Mattaj, I. W. & Rosbash, M.(1996). A role for nucleoporin FG repeat domains in export of human immunodeficiency virus type 1 Rev protein and RNA from the nucleus. Mol Cell Biol 16, 7144–7150. [Google Scholar]
  43. Terry, L. J. & Wente, S. R.(2007). Nuclear mRNA export requires specific FG nucleoporins for translocation through the nuclear pore complex. J Cell Biol 178, 1121–1132.[CrossRef] [Google Scholar]
  44. Terry, L. J. & Wente, S. R.(2009). Flexible gates: dynamic topologies and functions for FG nucleoporins in nucleocytoplasmic transport. Eukaryot Cell 8, 1814–1827.[CrossRef] [Google Scholar]
  45. Tran, E. J. & Wente, S. R.(2006). Dynamic nuclear pore complexes: life on the edge. Cell 125, 1041–1053.[CrossRef] [Google Scholar]
  46. Turner, J. G. & Sullivan, D. M.(2008). CRM1-mediated nuclear export of proteins and drug resistance in cancer. Curr Med Chem 15, 2648–2655.[CrossRef] [Google Scholar]
  47. Weis, K.(2002). Nucleocytoplasmic transport: cargo trafficking across the border. Curr Opin Cell Biol 14, 328–335.[CrossRef] [Google Scholar]
  48. Yasuda, J., Nakada, S., Kato, A., Toyoda, T. & Ishihama, A.(1993). Molecular assembly of influenza virus: association of the NS2 protein with virion matrix. Virology 196, 249–255.[CrossRef] [Google Scholar]
  49. Zheng, L., Wang, F., Yang, Z., Chen, J., Chang, H. & Chen, Z.(2009). A single immunization with HA DNA vaccine by electroporation induces early protection against H5N1 avian influenza virus challenge in mice. BMC Infect Dis 9, 17.[CrossRef] [Google Scholar]
  50. Zolotukhin, A. S. & Felber, B. K.(1999). Nucleoporins Nup98 and Nup214 participate in nuclear export of human immunodeficiency virus type 1 Rev. J Virol 73, 120–127. [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