Skip to main content
PMC home page
Journal of Virology logoLink to Journal of Virology
. 1997 Dec;71(12):9690–9700. doi: 10.1128/jvi.71.12.9690-9700.1997

Nuclear import and export of influenza virus nucleoprotein.

G Neumann 1, M R Castrucci 1, Y Kawaoka 1
PMCID: PMC230279  PMID: 9371635

Abstract

Influenza virus nucleoprotein (NP) shuttles between the nucleus and the cytoplasm. A nuclear localization signal (NLS) has been identified in NP at amino acids 327 to 345 (J. Davey et al., Cell 40:667-675, 1985). However, some NP mutants that lack this region still localize to the nucleus, suggesting an additional NLS in NP. We therefore investigated the nucleocytoplasmic transport of NP from influenza virus A/WSN/33 (H1N1). NP deletion constructs lacking the 38 N-terminal amino acids, as well as those lacking the 38 N-terminal amino acids and the previously identified NLS, localized to both the cytoplasm and the nucleus. Nuclear localization of a protein containing amino acids 1 to 38 of NP fused to LacZ proved that these 38 amino acids function as an NLS. Within this region, we identified two basic amino acids, Lys7 and Arg8, that are crucial for NP nuclear import. After being imported into the nucleus, the wild-type NP and the NP-LacZ fusion construct containing amino acids 1 to 38 of NP were both transported back to the cytoplasm, where they accumulated. These data indicate that NP has intrinsic structural features that allow nuclear import, nuclear export, and cytoplasmic accumulation in the absence of any other viral proteins. Further, the information required for nuclear import and export is located in the 38 N-terminal amino acids of NP, although other NP nuclear export signals may exist. Treatment of cells with a protein kinase C inhibitor increased the amounts of nuclear NP, whereas treatment of cells with a phosphorylation stimulator increased the amounts of cytoplasmic NP. These findings suggest a role of phosphorylation in nucleocytoplasmic transport of NP.

Full Text

The Full Text of this article is available as a PDF (3.5 MB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Almond J. W., Felsenreich V. Phosphorylation of the nucleoprotein of an avian influenza virus. J Gen Virol. 1982 Jun;60(Pt 2):295–305. doi: 10.1099/0022-1317-60-2-295. [DOI] [PubMed] [Google Scholar]
  2. Arrese M., Portela A. Serine 3 is critical for phosphorylation at the N-terminal end of the nucleoprotein of influenza virus A/Victoria/3/75. J Virol. 1996 Jun;70(6):3385–3391. doi: 10.1128/jvi.70.6.3385-3391.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Borer R. A., Lehner C. F., Eppenberger H. M., Nigg E. A. Major nucleolar proteins shuttle between nucleus and cytoplasm. Cell. 1989 Feb 10;56(3):379–390. doi: 10.1016/0092-8674(89)90241-9. [DOI] [PubMed] [Google Scholar]
  4. Bui M., Whittaker G., Helenius A. Effect of M1 protein and low pH on nuclear transport of influenza virus ribonucleoproteins. J Virol. 1996 Dec;70(12):8391–8401. doi: 10.1128/jvi.70.12.8391-8401.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Chang M. F., Chang S. C., Chang C. I., Wu K., Kang H. Y. Nuclear localization signals, but not putative leucine zipper motifs, are essential for nuclear transport of hepatitis delta antigen. J Virol. 1992 Oct;66(10):6019–6027. doi: 10.1128/jvi.66.10.6019-6027.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Davey J., Dimmock N. J., Colman A. Identification of the sequence responsible for the nuclear accumulation of the influenza virus nucleoprotein in Xenopus oocytes. Cell. 1985 Mar;40(3):667–675. doi: 10.1016/0092-8674(85)90215-6. [DOI] [PubMed] [Google Scholar]
  7. Davis L. I. The nuclear pore complex. Annu Rev Biochem. 1995;64:865–896. doi: 10.1146/annurev.bi.64.070195.004245. [DOI] [PubMed] [Google Scholar]
  8. Dingwall C., Laskey R. A. Nuclear targeting sequences--a consensus? Trends Biochem Sci. 1991 Dec;16(12):478–481. doi: 10.1016/0968-0004(91)90184-w. [DOI] [PubMed] [Google Scholar]
  9. FRANKLIN R. M., BREITENFELD P. M. The abortive infection of Earle's L-cells by fowl plague virus. Virology. 1959 Jul;8(3):293–307. doi: 10.1016/0042-6822(59)90031-5. [DOI] [PubMed] [Google Scholar]
  10. Fischer U., Huber J., Boelens W. C., Mattaj I. W., Lührmann R. The HIV-1 Rev activation domain is a nuclear export signal that accesses an export pathway used by specific cellular RNAs. Cell. 1995 Aug 11;82(3):475–483. doi: 10.1016/0092-8674(95)90436-0. [DOI] [PubMed] [Google Scholar]
  11. Fischer U., Michael W. M., Lührmann R., Dreyfuss G. Signal-mediated nuclear export pathways of proteins and RNAs. Trends Cell Biol. 1996 Aug;6(8):290–293. doi: 10.1016/0962-8924(96)20030-3. [DOI] [PubMed] [Google Scholar]
  12. Gerace L. Molecular trafficking across the nuclear pore complex. Curr Opin Cell Biol. 1992 Aug;4(4):637–645. doi: 10.1016/0955-0674(92)90083-o. [DOI] [PubMed] [Google Scholar]
  13. Gerace L. Nuclear export signals and the fast track to the cytoplasm. Cell. 1995 Aug 11;82(3):341–344. doi: 10.1016/0092-8674(95)90420-4. [DOI] [PubMed] [Google Scholar]
  14. Görlich D., Mattaj I. W. Nucleocytoplasmic transport. Science. 1996 Mar 15;271(5255):1513–1518. doi: 10.1126/science.271.5255.1513. [DOI] [PubMed] [Google Scholar]
  15. HENLE G., GIRARDI A., HENLE W. A non-transmissible cytopathogenic effect of influenza virus in tissue culture accompanied by formation of non-infectious hemagglutinins. J Exp Med. 1955 Jan 1;101(1):25–41. doi: 10.1084/jem.101.1.25. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Hidaka H., Inagaki M., Kawamoto S., Sasaki Y. Isoquinolinesulfonamides, novel and potent inhibitors of cyclic nucleotide dependent protein kinase and protein kinase C. Biochemistry. 1984 Oct 9;23(21):5036–5041. doi: 10.1021/bi00316a032. [DOI] [PubMed] [Google Scholar]
  17. Huddleston J. A., Brownlee G. G. The sequence of the nucleoprotein gene of human influenza A virus, strain A/NT/60/68. Nucleic Acids Res. 1982 Feb 11;10(3):1029–1038. doi: 10.1093/nar/10.3.1029. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Izaurralde E., Mattaj I. W. RNA export. Cell. 1995 Apr 21;81(2):153–159. doi: 10.1016/0092-8674(95)90323-2. [DOI] [PubMed] [Google Scholar]
  19. Kalderon D., Roberts B. L., Richardson W. D., Smith A. E. A short amino acid sequence able to specify nuclear location. Cell. 1984 Dec;39(3 Pt 2):499–509. doi: 10.1016/0092-8674(84)90457-4. [DOI] [PubMed] [Google Scholar]
  20. Kistner O., Müller K., Scholtissek C. Differential phosphorylation of the nucleoprotein of influenza A viruses. J Gen Virol. 1989 Sep;70(Pt 9):2421–2431. doi: 10.1099/0022-1317-70-9-2421. [DOI] [PubMed] [Google Scholar]
  21. Kunkel T. A. Rapid and efficient site-specific mutagenesis without phenotypic selection. Proc Natl Acad Sci U S A. 1985 Jan;82(2):488–492. doi: 10.1073/pnas.82.2.488. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Kurihara T., Hori M., Takeda H., Inoue M., Yoneda Y. Partial purification and characterization of a protein kinase that is activated by nuclear localization signal peptides. FEBS Lett. 1996 Feb 19;380(3):241–245. doi: 10.1016/0014-5793(96)00010-5. [DOI] [PubMed] [Google Scholar]
  23. Laskey R. A., Dingwall C. Nuclear shuttling: the default pathway for nuclear proteins? Cell. 1993 Aug 27;74(4):585–586. doi: 10.1016/0092-8674(93)90505-k. [DOI] [PubMed] [Google Scholar]
  24. Leukel M., Jost E. Two conserved serines in the nuclear localization signal flanking region are involved in the nuclear targeting of human lamin A. Eur J Cell Biol. 1995 Oct;68(2):133–142. [PubMed] [Google Scholar]
  25. Li R. A., Palese P., Krystal M. Complementation and analysis of an NP mutant of influenza virus. Virus Res. 1989 Feb;12(2):97–111. doi: 10.1016/0168-1702(89)90057-9. [DOI] [PubMed] [Google Scholar]
  26. Liao W., Ou J. H. Phosphorylation and nuclear localization of the hepatitis B virus core protein: significance of serine in the three repeated SPRRR motifs. J Virol. 1995 Feb;69(2):1025–1029. doi: 10.1128/jvi.69.2.1025-1029.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Martin K., Helenius A. Nuclear transport of influenza virus ribonucleoproteins: the viral matrix protein (M1) promotes export and inhibits import. Cell. 1991 Oct 4;67(1):117–130. doi: 10.1016/0092-8674(91)90576-k. [DOI] [PubMed] [Google Scholar]
  28. Martin K., Helenius A. Transport of incoming influenza virus nucleocapsids into the nucleus. J Virol. 1991 Jan;65(1):232–244. doi: 10.1128/jvi.65.1.232-244.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Mears W. E., Lam V., Rice S. A. Identification of nuclear and nucleolar localization signals in the herpes simplex virus regulatory protein ICP27. J Virol. 1995 Feb;69(2):935–947. doi: 10.1128/jvi.69.2.935-947.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Meier U. T., Blobel G. Nopp140 shuttles on tracks between nucleolus and cytoplasm. Cell. 1992 Jul 10;70(1):127–138. doi: 10.1016/0092-8674(92)90539-o. [DOI] [PubMed] [Google Scholar]
  31. Melchior F., Gerace L. Mechanisms of nuclear protein import. Curr Opin Cell Biol. 1995 Jun;7(3):310–318. doi: 10.1016/0955-0674(95)80084-0. [DOI] [PubMed] [Google Scholar]
  32. Michael W. M., Choi M., Dreyfuss G. A nuclear export signal in hnRNP A1: a signal-mediated, temperature-dependent nuclear protein export pathway. Cell. 1995 Nov 3;83(3):415–422. doi: 10.1016/0092-8674(95)90119-1. [DOI] [PubMed] [Google Scholar]
  33. Michaud N., Goldfarb D. S. Multiple pathways in nuclear transport: the import of U2 snRNP occurs by a novel kinetic pathway. J Cell Biol. 1991 Jan;112(2):215–223. doi: 10.1083/jcb.112.2.215. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Moll T., Tebb G., Surana U., Robitsch H., Nasmyth K. The role of phosphorylation and the CDC28 protein kinase in cell cycle-regulated nuclear import of the S. cerevisiae transcription factor SWI5. Cell. 1991 Aug 23;66(4):743–758. doi: 10.1016/0092-8674(91)90118-i. [DOI] [PubMed] [Google Scholar]
  35. Mukaigawa J., Nayak D. P. Two signals mediate nuclear localization of influenza virus (A/WSN/33) polymerase basic protein 2. J Virol. 1991 Jan;65(1):245–253. doi: 10.1128/jvi.65.1.245-253.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Mullis K., Faloona F., Scharf S., Saiki R., Horn G., Erlich H. Specific enzymatic amplification of DNA in vitro: the polymerase chain reaction. Cold Spring Harb Symp Quant Biol. 1986;51(Pt 1):263–273. doi: 10.1101/sqb.1986.051.01.032. [DOI] [PubMed] [Google Scholar]
  37. Nadler S. G., Tritschler D., Haffar O. K., Blake J., Bruce A. G., Cleaveland J. S. Differential expression and sequence-specific interaction of karyopherin alpha with nuclear localization sequences. J Biol Chem. 1997 Feb 14;272(7):4310–4315. doi: 10.1074/jbc.272.7.4310. [DOI] [PubMed] [Google Scholar]
  38. Nakielny S., Dreyfuss G. The hnRNP C proteins contain a nuclear retention sequence that can override nuclear export signals. J Cell Biol. 1996 Sep;134(6):1365–1373. doi: 10.1083/jcb.134.6.1365. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Nath S. T., Nayak D. P. Function of two discrete regions is required for nuclear localization of polymerase basic protein 1 of A/WSN/33 influenza virus (H1 N1). Mol Cell Biol. 1990 Aug;10(8):4139–4145. doi: 10.1128/mcb.10.8.4139. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Newmeyer D. D., Lucocq J. M., Bürglin T. R., De Robertis E. M. Assembly in vitro of nuclei active in nuclear protein transport: ATP is required for nucleoplasmin accumulation. EMBO J. 1986 Mar;5(3):501–510. doi: 10.1002/j.1460-2075.1986.tb04239.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Nieto A., de la Luna S., Bárcena J., Portela A., Ortín J. Complex structure of the nuclear translocation signal of influenza virus polymerase PA subunit. J Gen Virol. 1994 Jan;75(Pt 1):29–36. doi: 10.1099/0022-1317-75-1-29. [DOI] [PubMed] [Google Scholar]
  42. Niwa H., Yamamura K., Miyazaki J. Efficient selection for high-expression transfectants with a novel eukaryotic vector. Gene. 1991 Dec 15;108(2):193–199. doi: 10.1016/0378-1119(91)90434-d. [DOI] [PubMed] [Google Scholar]
  43. 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. 1995 Sep 29;270(39):22701–22704. doi: 10.1074/jbc.270.39.22701. [DOI] [PubMed] [Google Scholar]
  44. O'Neill R. E., Palese P. NPI-1, the human homolog of SRP-1, interacts with influenza virus nucleoprotein. Virology. 1995 Jan 10;206(1):116–125. doi: 10.1016/s0042-6822(95)80026-3. [DOI] [PubMed] [Google Scholar]
  45. Obrig T. G., Culp W. J., McKeehan W. L., Hardesty B. The mechanism by which cycloheximide and related glutarimide antibiotics inhibit peptide synthesis on reticulocyte ribosomes. J Biol Chem. 1971 Jan 10;246(1):174–181. [PubMed] [Google Scholar]
  46. Pines J., Hunter T. The differential localization of human cyclins A and B is due to a cytoplasmic retention signal in cyclin B. EMBO J. 1994 Aug 15;13(16):3772–3781. doi: 10.1002/j.1460-2075.1994.tb06688.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Piñol-Roma S., Dreyfuss G. Shuttling of pre-mRNA binding proteins between nucleus and cytoplasm. Nature. 1992 Feb 20;355(6362):730–732. doi: 10.1038/355730a0. [DOI] [PubMed] [Google Scholar]
  48. Pollard V. W., Michael W. M., Nakielny S., Siomi M. C., Wang F., Dreyfuss G. A novel receptor-mediated nuclear protein import pathway. Cell. 1996 Sep 20;86(6):985–994. doi: 10.1016/s0092-8674(00)80173-7. [DOI] [PubMed] [Google Scholar]
  49. Robbins J., Dilworth S. M., Laskey R. A., Dingwall C. Two interdependent basic domains in nucleoplasmin nuclear targeting sequence: identification of a class of bipartite nuclear targeting sequence. Cell. 1991 Feb 8;64(3):615–623. doi: 10.1016/0092-8674(91)90245-t. [DOI] [PubMed] [Google Scholar]
  50. Schmidt-Zachmann M. S., Dargemont C., Kühn L. C., Nigg E. A. Nuclear export of proteins: the role of nuclear retention. Cell. 1993 Aug 13;74(3):493–504. doi: 10.1016/0092-8674(93)80051-f. [DOI] [PubMed] [Google Scholar]
  51. Simos G., Hurt E. C. Nucleocytoplasmic transport: factors and mechanisms. FEBS Lett. 1995 Aug 1;369(1):107–112. doi: 10.1016/0014-5793(95)00674-x. [DOI] [PubMed] [Google Scholar]
  52. Stegmann T., White J. M., Helenius A. Intermediates in influenza induced membrane fusion. EMBO J. 1990 Dec;9(13):4231–4241. doi: 10.1002/j.1460-2075.1990.tb07871.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  53. Szilvay A. M., Brokstad K. A., Kopperud R., Haukenes G., Kalland K. H. Nuclear export of the human immunodeficiency virus type 1 nucleocytoplasmic shuttle protein Rev is mediated by its activation domain and is blocked by transdominant negative mutants. J Virol. 1995 Jun;69(6):3315–3323. doi: 10.1128/jvi.69.6.3315-3323.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  54. Takemoto Y., Tashiro S., Handa H., Ishii S. Multiple nuclear localization signals of the B-myb gene product. FEBS Lett. 1994 Aug 15;350(1):55–60. doi: 10.1016/0014-5793(94)00733-0. [DOI] [PubMed] [Google Scholar]
  55. Vandromme M., Cavadore J. C., Bonnieu A., Froeschlé A., Lamb N., Fernandez A. Two nuclear localization signals present in the basic-helix 1 domains of MyoD promote its active nuclear translocation and can function independently. Proc Natl Acad Sci U S A. 1995 May 9;92(10):4646–4650. doi: 10.1073/pnas.92.10.4646. [DOI] [PMC free article] [PubMed] [Google Scholar]
  56. Wang P., Palese P., O'Neill R. E. The NPI-1/NPI-3 (karyopherin alpha) binding site on the influenza a virus nucleoprotein NP is a nonconventional nuclear localization signal. J Virol. 1997 Mar;71(3):1850–1856. doi: 10.1128/jvi.71.3.1850-1856.1997. [DOI] [PMC free article] [PubMed] [Google Scholar]
  57. Wen S. T., Jackson P. K., Van Etten R. A. The cytostatic function of c-Abl is controlled by multiple nuclear localization signals and requires the p53 and Rb tumor suppressor gene products. EMBO J. 1996 Apr 1;15(7):1583–1595. [PMC free article] [PubMed] [Google Scholar]
  58. Wen W., Meinkoth J. L., Tsien R. Y., Taylor S. S. Identification of a signal for rapid export of proteins from the nucleus. Cell. 1995 Aug 11;82(3):463–473. doi: 10.1016/0092-8674(95)90435-2. [DOI] [PubMed] [Google Scholar]
  59. Whittaker G., Bui M., Helenius A. Nuclear trafficking of influenza virus ribonuleoproteins in heterokaryons. J Virol. 1996 May;70(5):2743–2756. doi: 10.1128/jvi.70.5.2743-2756.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  60. Whittaker G., Bui M., Helenius A. The role of nuclear import and export in influenza virus infection. Trends Cell Biol. 1996 Feb;6(2):67–71. doi: 10.1016/0962-8924(96)81017-8. [DOI] [PubMed] [Google Scholar]
  61. Ye Z., Robinson D., Wagner R. R. Nucleus-targeting domain of the matrix protein (M1) of influenza virus. J Virol. 1995 Mar;69(3):1964–1970. doi: 10.1128/jvi.69.3.1964-1970.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  62. Ylikomi T., Bocquel M. T., Berry M., Gronemeyer H., Chambon P. Cooperation of proto-signals for nuclear accumulation of estrogen and progesterone receptors. EMBO J. 1992 Oct;11(10):3681–3694. doi: 10.1002/j.1460-2075.1992.tb05453.x. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Journal of Virology are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES