Skip to main content
PMC home page
RNA logoLink to RNA
. 1999 Feb;5(2):195–205. doi: 10.1017/s1355838299981621

RNA binding by the novel helical domain of the influenza virus NS1 protein requires its dimer structure and a small number of specific basic amino acids.

W Wang 1, K Riedel 1, P Lynch 1, C Y Chien 1, G T Montelione 1, R M Krug 1
PMCID: PMC1369752  PMID: 10024172

Abstract

The RNA-binding/dimerization domain of the NS1 protein of influenza A virus (73 amino acids in length) exhibits a novel dimeric six-helical fold. It is not known how this domain binds to its specific RNA targets, one of which is double-stranded RNA. To elucidate the mode of RNA binding, we introduced single alanine replacements into the NS1 RNA-binding domain at specific positions in the three-dimensional structure. Our results indicate that the dimer structure is essential for RNA binding, because any alanine replacement that causes disruption of the dimer also leads to the loss of RNA-binding activity. Surprisingly, the arginine side chain at position 38, which is in the second helix of each monomer, is the only amino-acid side chain that is absolutely required only for RNA binding and not for dimerization, indicating that this side chain probably interacts directly with the RNA target. This interaction is primarily electrostatic, because replacement of this arginine with lysine had no effect on RNA binding. A second basic amino acid, the lysine at position 41, which is also in helix 2, makes a strong contribution to the affinity of binding. We conclude that helix 2 and helix 2', which are antiparallel and next to each other in the dimer conformation, constitute the interaction face between the NS1 RNA-binding domain and its RNA targets, and that the arginine side chain at position 38 and possibly the lysine side chain at position 41 in each of these antiparallel helices contact the phosphate backbone of the RNA target.

Full Text

The Full Text of this article is available as a PDF (643.2 KB).

Selected References

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

  1. Alonso-Caplen F. V., Nemeroff M. E., Qiu Y., Krug R. M. Nucleocytoplasmic transport: the influenza virus NS1 protein regulates the transport of spliced NS2 mRNA and its precursor NS1 mRNA. Genes Dev. 1992 Feb;6(2):255–267. doi: 10.1101/gad.6.2.255. [DOI] [PubMed] [Google Scholar]
  2. Banner D. W., Kokkinidis M., Tsernoglou D. Structure of the ColE1 rop protein at 1.7 A resolution. J Mol Biol. 1987 Aug 5;196(3):657–675. doi: 10.1016/0022-2836(87)90039-8. [DOI] [PubMed] [Google Scholar]
  3. Battiste J. L., Mao H., Rao N. S., Tan R., Muhandiram D. R., Kay L. E., Frankel A. D., Williamson J. R. Alpha helix-RNA major groove recognition in an HIV-1 rev peptide-RRE RNA complex. Science. 1996 Sep 13;273(5281):1547–1551. doi: 10.1126/science.273.5281.1547. [DOI] [PubMed] [Google Scholar]
  4. Berglund H., Rak A., Serganov A., Garber M., Härd T. Solution structure of the ribosomal RNA binding protein S15 from Thermus thermophilus. Nat Struct Biol. 1997 Jan;4(1):20–23. doi: 10.1038/nsb0197-20. [DOI] [PubMed] [Google Scholar]
  5. Briedis D. J., Lamb R. A. Influenza B virus genome: sequences and structural organization of RNA segment 8 and the mRNAs coding for the NS1 and NS2 proteins. J Virol. 1982 Apr;42(1):186–193. doi: 10.1128/jvi.42.1.186-193.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Bycroft M., Grünert S., Murzin A. G., Proctor M., St Johnston D. NMR solution structure of a dsRNA binding domain from Drosophila staufen protein reveals homology to the N-terminal domain of ribosomal protein S5. EMBO J. 1995 Jul 17;14(14):3563–3571. doi: 10.1002/j.1460-2075.1995.tb07362.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Chien C. Y., Tejero R., Huang Y., Zimmerman D. E., Ríos C. B., Krug R. M., Montelione G. T. A novel RNA-binding motif in influenza A virus non-structural protein 1. Nat Struct Biol. 1997 Nov;4(11):891–895. doi: 10.1038/nsb1197-891. [DOI] [PubMed] [Google Scholar]
  8. Davies C., Gerstner R. B., Draper D. E., Ramakrishnan V., White S. W. The crystal structure of ribosomal protein S4 reveals a two-domain molecule with an extensive RNA-binding surface: one domain shows structural homology to the ETS DNA-binding motif. EMBO J. 1998 Aug 17;17(16):4545–4558. doi: 10.1093/emboj/17.16.4545. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Finerty P. J., Jr, Bass B. L. A Xenopus zinc finger protein that specifically binds dsRNA and RNA-DNA hybrids. J Mol Biol. 1997 Aug 15;271(2):195–208. doi: 10.1006/jmbi.1997.1177. [DOI] [PubMed] [Google Scholar]
  10. Fortes P., Beloso A., Ortín J. Influenza virus NS1 protein inhibits pre-mRNA splicing and blocks mRNA nucleocytoplasmic transport. EMBO J. 1994 Feb 1;13(3):704–712. doi: 10.1002/j.1460-2075.1994.tb06310.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Hatada E., Fukuda R. Binding of influenza A virus NS1 protein to dsRNA in vitro. J Gen Virol. 1992 Dec;73(Pt 12):3325–3329. doi: 10.1099/0022-1317-73-12-3325. [DOI] [PubMed] [Google Scholar]
  12. Hinck A. P., Markus M. A., Huang S., Grzesiek S., Kustonovich I., Draper D. E., Torchia D. A. The RNA binding domain of ribosomal protein L11: three-dimensional structure of the RNA-bound form of the protein and its interaction with 23 S rRNA. J Mol Biol. 1997 Nov 21;274(1):101–113. doi: 10.1006/jmbi.1997.1379. [DOI] [PubMed] [Google Scholar]
  13. Kharrat A., Macias M. J., Gibson T. J., Nilges M., Pastore A. Structure of the dsRNA binding domain of E. coli RNase III. EMBO J. 1995 Jul 17;14(14):3572–3584. doi: 10.1002/j.1460-2075.1995.tb07363.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Liu J., Lynch P. A., Chien C. Y., Montelione G. T., Krug R. M., Berman H. M. Crystal structure of the unique RNA-binding domain of the influenza virus NS1 protein. Nat Struct Biol. 1997 Nov;4(11):896–899. doi: 10.1038/nsb1197-896. [DOI] [PubMed] [Google Scholar]
  15. Lu Y., Qian X. Y., Krug R. M. The influenza virus NS1 protein: a novel inhibitor of pre-mRNA splicing. Genes Dev. 1994 Aug 1;8(15):1817–1828. doi: 10.1101/gad.8.15.1817. [DOI] [PubMed] [Google Scholar]
  16. Lu Y., Wambach M., Katze M. G., Krug R. M. Binding of the influenza virus NS1 protein to double-stranded RNA inhibits the activation of the protein kinase that phosphorylates the elF-2 translation initiation factor. Virology. 1995 Dec 1;214(1):222–228. doi: 10.1006/viro.1995.9937. [DOI] [PubMed] [Google Scholar]
  17. Markus M. A., Gerstner R. B., Draper D. E., Torchia D. A. The solution structure of ribosomal protein S4 delta41 reveals two subdomains and a positively charged surface that may interact with RNA. EMBO J. 1998 Aug 17;17(16):4559–4571. doi: 10.1093/emboj/17.16.4559. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Nagai K. RNA-protein complexes. Curr Opin Struct Biol. 1996 Feb;6(1):53–61. doi: 10.1016/s0959-440x(96)80095-9. [DOI] [PubMed] [Google Scholar]
  19. Nemeroff M. E., Barabino S. M., Li Y., Keller W., Krug R. M. Influenza virus NS1 protein interacts with the cellular 30 kDa subunit of CPSF and inhibits 3'end formation of cellular pre-mRNAs. Mol Cell. 1998 Jun;1(7):991–1000. doi: 10.1016/s1097-2765(00)80099-4. [DOI] [PubMed] [Google Scholar]
  20. Nemeroff M. E., Qian X. Y., Krug R. M. The influenza virus NS1 protein forms multimers in vitro and in vivo. Virology. 1995 Oct 1;212(2):422–428. doi: 10.1006/viro.1995.1499. [DOI] [PubMed] [Google Scholar]
  21. Qian X. Y., Alonso-Caplen F., Krug R. M. Two functional domains of the influenza virus NS1 protein are required for regulation of nuclear export of mRNA. J Virol. 1994 Apr;68(4):2433–2441. doi: 10.1128/jvi.68.4.2433-2441.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Qian X. Y., Chien C. Y., Lu Y., Montelione G. T., Krug R. M. An amino-terminal polypeptide fragment of the influenza virus NS1 protein possesses specific RNA-binding activity and largely helical backbone structure. RNA. 1995 Nov;1(9):948–956. [PMC free article] [PubMed] [Google Scholar]
  23. Qiu Y., Krug R. M. The influenza virus NS1 protein is a poly(A)-binding protein that inhibits nuclear export of mRNAs containing poly(A). J Virol. 1994 Apr;68(4):2425–2432. doi: 10.1128/jvi.68.4.2425-2432.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Qiu Y., Nemeroff M., Krug R. M. The influenza virus NS1 protein binds to a specific region in human U6 snRNA and inhibits U6-U2 and U6-U4 snRNA interactions during splicing. RNA. 1995 May;1(3):304–316. [PMC free article] [PubMed] [Google Scholar]
  25. Stebbins C. E., Borukhov S., Orlova M., Polyakov A., Goldfarb A., Darst S. A. Crystal structure of the GreA transcript cleavage factor from Escherichia coli. Nature. 1995 Feb 16;373(6515):636–640. doi: 10.1038/373636a0. [DOI] [PubMed] [Google Scholar]
  26. Stewart L., Redinbo M. R., Qiu X., Hol W. G., Champoux J. J. A model for the mechanism of human topoisomerase I. Science. 1998 Mar 6;279(5356):1534–1541. doi: 10.1126/science.279.5356.1534. [DOI] [PubMed] [Google Scholar]
  27. Wang W., Krug R. M. The RNA-binding and effector domains of the viral NS1 protein are conserved to different extents among influenza A and B viruses. Virology. 1996 Sep 1;223(1):41–50. doi: 10.1006/viro.1996.0453. [DOI] [PubMed] [Google Scholar]
  28. Wang W., Krug R. M. U6atac snRNA, the highly divergent counterpart of U6 snRNA, is the specific target that mediates inhibition of AT-AC splicing by the influenza virus NS1 protein. RNA. 1998 Jan;4(1):55–64. [PMC free article] [PubMed] [Google Scholar]
  29. Weeks K. M., Crothers D. M. Major groove accessibility of RNA. Science. 1993 Sep 17;261(5128):1574–1577. doi: 10.1126/science.7690496. [DOI] [PubMed] [Google Scholar]
  30. Xing Y., Guha Thakurta D., Draper D. E. The RNA binding domain of ribosomal protein L11 is structurally similar to homeodomains. Nat Struct Biol. 1997 Jan;4(1):24–27. doi: 10.1038/nsb0197-24. [DOI] [PubMed] [Google Scholar]

Articles from RNA are provided here courtesy of The RNA Society

RESOURCES