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. 1997 Apr;71(4):2591–2599. doi: 10.1128/jvi.71.4.2591-2599.1997

Dominant-negative mutants of human MxA protein: domains in the carboxy-terminal moiety are important for oligomerization and antiviral activity.

A Ponten 1, C Sick 1, M Weeber 1, O Haller 1, G Kochs 1
PMCID: PMC191379  PMID: 9060610

Abstract

Human MxA protein is an interferon-induced 76-kDa GTPase that exhibits antiviral activity against several RNA viruses. Wild-type MxA accumulates in the cytoplasm of cells. TMxA, a modified form of wild-type MxA carrying a foreign nuclear localization signal, accumulates in the cell nucleus. Here we show that MxA protein is translocated into the nucleus together with TMxA when both proteins are expressed simultaneously in the same cell, demonstrating that MxA molecules form tight complexes in living cells. To define domains important for MxA-MxA interaction and antiviral function in vivo, we expressed mutant forms of MxA together with wild-type MxA or TMxA in appropriate cells and analyzed subcellular localization and interfering effects. An MxA deletion mutant, MxA(359-572), formed heterooligomers with TMxA and was translocated to the nucleus, indicating that the region between amino acid positions 359 and 572 contains an interaction domain which is critical for oligomerization of MxA proteins. Mutant T103A with threonine at position 103 replaced by alanine had lost both GTPase and antiviral activities. T103A exhibited a dominant-interfering effect on the antiviral activity of wild-type MxA rendering MxA-expressing cells susceptible to infection with influenza A virus, Thogoto virus, and vesicular stomatitis virus. To determine which sequences are critical for the dominant-negative effect of T103A, we expressed truncated forms of T103A together with wild-type protein. A C-terminal deletion mutant lacking the last 90 amino acids had lost interfering capacity, indicating that an intact C terminus was required. Surprisingly, a truncated version of MxA representing only the C-terminal half of the molecule exerted also a dominant-negative effect on wild-type function, demonstrating that sequences in the C-terminal moiety of MxA are necessary and sufficient for interference. However, all MxA mutants formed hetero-oligomers with TMxA and were translocated to the nucleus, indicating that physical interaction alone is not sufficient for disturbing wild-type function. We propose that dominant-negative mutants directly influence wild-type activity within hetero-oligomers or else compete with wild-type MxA for a cellular or viral target.

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Selected References

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  1. Aebi M., Fäh J., Hurt N., Samuel C. E., Thomis D., Bazzigher L., Pavlovic J., Haller O., Staeheli P. cDNA structures and regulation of two interferon-induced human Mx proteins. Mol Cell Biol. 1989 Nov;9(11):5062–5072. doi: 10.1128/mcb.9.11.5062. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Albanese M., Bruno-Smiraglia C., Di di Cuonzo G., Lavagnino A., Srihongse S. Isolation of Thogoto virus from Rhipicephalus bursa ticks in western Sicily. Acta Virol. 1972 May;16(3):267–267. [PubMed] [Google Scholar]
  3. Chen M. S., Obar R. A., Schroeder C. C., Austin T. W., Poodry C. A., Wadsworth S. C., Vallee R. B. Multiple forms of dynamin are encoded by shibire, a Drosophila gene involved in endocytosis. Nature. 1991 Jun 13;351(6327):583–586. doi: 10.1038/351583a0. [DOI] [PubMed] [Google Scholar]
  4. Dreiding P., Staeheli P., Haller O. Interferon-induced protein Mx accumulates in nuclei of mouse cells expressing resistance to influenza viruses. Virology. 1985 Jan 15;140(1):192–196. doi: 10.1016/0042-6822(85)90460-x. [DOI] [PubMed] [Google Scholar]
  5. Frese M., Kochs G., Feldmann H., Hertkorn C., Haller O. Inhibition of bunyaviruses, phleboviruses, and hantaviruses by human MxA protein. J Virol. 1996 Feb;70(2):915–923. doi: 10.1128/jvi.70.2.915-923.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Frese M., Kochs G., Meier-Dieter U., Siebler J., Haller O. Human MxA protein inhibits tick-borne Thogoto virus but not Dhori virus. J Virol. 1995 Jun;69(6):3904–3909. doi: 10.1128/jvi.69.6.3904-3909.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Garber E. A., Hreniuk D. L., Scheidel L. M., van der Ploeg L. H. Mutations in murine Mx1: effects on localization and antiviral activity. Virology. 1993 Jun;194(2):715–723. doi: 10.1006/viro.1993.1312. [DOI] [PubMed] [Google Scholar]
  8. Gautier C., Mehtali M., Lathe R. A ubiquitous mammalian expression vector, pHMG, based on a housekeeping gene promoter. Nucleic Acids Res. 1989 Oct 25;17(20):8389–8389. doi: 10.1093/nar/17.20.8389. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Gu X., Verma D. P. Phragmoplastin, a dynamin-like protein associated with cell plate formation in plants. EMBO J. 1996 Feb 15;15(4):695–704. [PMC free article] [PubMed] [Google Scholar]
  10. Haller O., Frese M., Rost D., Nuttall P. A., Kochs G. Tick-borne thogoto virus infection in mice is inhibited by the orthomyxovirus resistance gene product Mx1. J Virol. 1995 Apr;69(4):2596–2601. doi: 10.1128/jvi.69.4.2596-2601.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Horisberger M. A. Interferon-induced human protein MxA is a GTPase which binds transiently to cellular proteins. J Virol. 1992 Aug;66(8):4705–4709. doi: 10.1128/jvi.66.8.4705-4709.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Huang T., Pavlovic J., Staeheli P., Krystal M. Overexpression of the influenza virus polymerase can titrate out inhibition by the murine Mx1 protein. J Virol. 1992 Jul;66(7):4154–4160. doi: 10.1128/jvi.66.7.4154-4160.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Israël A. Preliminary characterization of the particles from productive and abortive infections of L cells by fowl plague virus. Ann Microbiol (Paris) 1979 Jul;130B(1):85–100. [PubMed] [Google Scholar]
  14. Ito W., Ishiguro H., Kurosawa Y. A general method for introducing a series of mutations into cloned DNA using the polymerase chain reaction. Gene. 1991 Jun 15;102(1):67–70. doi: 10.1016/0378-1119(91)90539-n. [DOI] [PubMed] [Google Scholar]
  15. Johannes L., Arnheiter H., Meier E. Switch in antiviral specificity of a GTPase upon translocation from the cytoplasm to the nucleus. J Virol. 1993 Mar;67(3):1653–1657. doi: 10.1128/jvi.67.3.1653-1657.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Jones B. A., Fangman W. L. Mitochondrial DNA maintenance in yeast requires a protein containing a region related to the GTP-binding domain of dynamin. Genes Dev. 1992 Mar;6(3):380–389. doi: 10.1101/gad.6.3.380. [DOI] [PubMed] [Google Scholar]
  17. Jones L. D., Nuttall P. A. The effect of virus-immune hosts on Thogoto virus infection of the tick, Rhipicephalus appendiculatus. Virus Res. 1989 Oct;14(2):129–139. doi: 10.1016/0168-1702(89)90034-8. [DOI] [PubMed] [Google Scholar]
  18. Kanerva M., Melén K., Vaheri A., Julkunen I. Inhibition of puumala and tula hantaviruses in Vero cells by MxA protein. Virology. 1996 Oct 1;224(1):55–62. doi: 10.1006/viro.1996.0506. [DOI] [PubMed] [Google Scholar]
  19. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  20. Melén K., Julkunen I. Mutational analysis of murine Mx1 protein: GTP binding core domain is essential for anti-influenza A activity. Virology. 1994 Nov 15;205(1):269–279. doi: 10.1006/viro.1994.1643. [DOI] [PubMed] [Google Scholar]
  21. Melén K., Keskinen P., Ronni T., Sareneva T., Lounatmaa K., Julkunen I. Human MxB protein, an interferon-alpha-inducible GTPase, contains a nuclear targeting signal and is localized in the heterochromatin region beneath the nuclear envelope. J Biol Chem. 1996 Sep 20;271(38):23478–23486. doi: 10.1074/jbc.271.38.23478. [DOI] [PubMed] [Google Scholar]
  22. Melén K., Ronni T., Broni B., Krug R. M., von Bonsdorff C. H., Julkunen I. Interferon-induced Mx proteins form oligomers and contain a putative leucine zipper. J Biol Chem. 1992 Dec 25;267(36):25898–25907. [PubMed] [Google Scholar]
  23. Mulligan R. C., Berg P. Selection for animal cells that express the Escherichia coli gene coding for xanthine-guanine phosphoribosyltransferase. Proc Natl Acad Sci U S A. 1981 Apr;78(4):2072–2076. doi: 10.1073/pnas.78.4.2072. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Nakayama M., Yazaki K., Kusano A., Nagata K., Hanai N., Ishihama A. Structure of mouse Mx1 protein. Molecular assembly and GTP-dependent conformational change. J Biol Chem. 1993 Jul 15;268(20):15033–15038. [PubMed] [Google Scholar]
  25. Obar R. A., Collins C. A., Hammarback J. A., Shpetner H. S., Vallee R. B. Molecular cloning of the microtubule-associated mechanochemical enzyme dynamin reveals homology with a new family of GTP-binding proteins. Nature. 1990 Sep 20;347(6290):256–261. doi: 10.1038/347256a0. [DOI] [PubMed] [Google Scholar]
  26. Pavlovic J., Arzet H. A., Hefti H. P., Frese M., Rost D., Ernst B., Kolb E., Staeheli P., Haller O. Enhanced virus resistance of transgenic mice expressing the human MxA protein. J Virol. 1995 Jul;69(7):4506–4510. doi: 10.1128/jvi.69.7.4506-4510.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Pavlovic J., Haller O., Staeheli P. Human and mouse Mx proteins inhibit different steps of the influenza virus multiplication cycle. J Virol. 1992 Apr;66(4):2564–2569. doi: 10.1128/jvi.66.4.2564-2569.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Pavlovic J., Zürcher T., Haller O., Staeheli P. Resistance to influenza virus and vesicular stomatitis virus conferred by expression of human MxA protein. J Virol. 1990 Jul;64(7):3370–3375. doi: 10.1128/jvi.64.7.3370-3375.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Pitossi F., Blank A., Schröder A., Schwarz A., Hüssi P., Schwemmle M., Pavlovic J., Staeheli P. A functional GTP-binding motif is necessary for antiviral activity of Mx proteins. J Virol. 1993 Nov;67(11):6726–6732. doi: 10.1128/jvi.67.11.6726-6732.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Richter M. F., Schwemmle M., Herrmann C., Wittinghofer A., Staeheli P. Interferon-induced MxA protein. GTP binding and GTP hydrolysis properties. J Biol Chem. 1995 Jun 2;270(22):13512–13517. [PubMed] [Google Scholar]
  31. Rothman J. H., Raymond C. K., Gilbert T., O'Hara P. J., Stevens T. H. A putative GTP binding protein homologous to interferon-inducible Mx proteins performs an essential function in yeast protein sorting. Cell. 1990 Jun 15;61(6):1063–1074. doi: 10.1016/0092-8674(90)90070-u. [DOI] [PubMed] [Google Scholar]
  32. Schneider-Schaulies S., Schneider-Schaulies J., Schuster A., Bayer M., Pavlovic J., ter Meulen V. Cell type-specific MxA-mediated inhibition of measles virus transcription in human brain cells. J Virol. 1994 Nov;68(11):6910–6917. doi: 10.1128/jvi.68.11.6910-6917.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Schnorr J. J., Schneider-Schaulies S., Simon-Jödicke A., Pavlovic J., Horisberger M. A., ter Meulen V. MxA-dependent inhibition of measles virus glycoprotein synthesis in a stably transfected human monocytic cell line. J Virol. 1993 Aug;67(8):4760–4768. doi: 10.1128/jvi.67.8.4760-4768.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Schuster A., Johnston I. C., Das T., Banerjee A. K., Pavlovic J., ter Meulen V., Schneider-Schaulies S. Expression of the human MxA protein is associated with hyperphosphorylation of VSV P protein in human neural Cells. Virology. 1996 Jun 1;220(1):241–245. doi: 10.1006/viro.1996.0308. [DOI] [PubMed] [Google Scholar]
  35. Schwemmle M., Richter M. F., Herrmann C., Nassar N., Staeheli P. Unexpected structural requirements for GTPase activity of the interferon-induced MxA protein. J Biol Chem. 1995 Jun 2;270(22):13518–13523. [PubMed] [Google Scholar]
  36. Schwemmle M., Weining K. C., Richter M. F., Schumacher B., Staeheli P. Vesicular stomatitis virus transcription inhibited by purified MxA protein. Virology. 1995 Jan 10;206(1):545–554. doi: 10.1016/s0042-6822(95)80071-9. [DOI] [PubMed] [Google Scholar]
  37. Staeheli P., Dreiding P., Haller O., Lindenmann J. Polyclonal and monoclonal antibodies to the interferon-inducible protein Mx of influenza virus-resistant mice. J Biol Chem. 1985 Feb 10;260(3):1821–1825. [PubMed] [Google Scholar]
  38. Staeheli P., Haller O., Boll W., Lindenmann J., Weissmann C. Mx protein: constitutive expression in 3T3 cells transformed with cloned Mx cDNA confers selective resistance to influenza virus. Cell. 1986 Jan 17;44(1):147–158. doi: 10.1016/0092-8674(86)90493-9. [DOI] [PubMed] [Google Scholar]
  39. Staeheli P., Haller O. Interferon-induced human protein with homology to protein Mx of influenza virus-resistant mice. Mol Cell Biol. 1985 Aug;5(8):2150–2153. doi: 10.1128/mcb.5.8.2150. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Staeheli P., Pitossi F., Pavlovic J. Mx proteins: GTPases with antiviral activity. Trends Cell Biol. 1993 Aug;3(8):268–272. doi: 10.1016/0962-8924(93)90055-6. [DOI] [PubMed] [Google Scholar]
  41. Stranden A. M., Staeheli P., Pavlovic J. Function of the mouse Mx1 protein is inhibited by overexpression of the PB2 protein of influenza virus. Virology. 1993 Dec;197(2):642–651. doi: 10.1006/viro.1993.1639. [DOI] [PubMed] [Google Scholar]
  42. Toyoda T., Asano Y., Ishihama A. Role of GTPase activity of murine Mx1 protein in nuclear localization and anti-influenza virus activity. J Gen Virol. 1995 Jul;76(Pt 7):1867–1869. doi: 10.1099/0022-1317-76-7-1867. [DOI] [PubMed] [Google Scholar]
  43. Vater C. A., Raymond C. K., Ekena K., Howald-Stevenson I., Stevens T. H. The VPS1 protein, a homolog of dynamin required for vacuolar protein sorting in Saccharomyces cerevisiae, is a GTPase with two functionally separable domains. J Cell Biol. 1992 Nov;119(4):773–786. doi: 10.1083/jcb.119.4.773. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Weber F., Haller O., Kochs G. Nucleoprotein viral RNA and mRNA of Thogoto virus: a novel "cap-stealing" mechanism in tick-borne orthomyxoviruses? J Virol. 1996 Dec;70(12):8361–8367. doi: 10.1128/jvi.70.12.8361-8367.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Zürcher T., Pavlovic J., Staeheli P. Mechanism of human MxA protein action: variants with changed antiviral properties. EMBO J. 1992 Apr;11(4):1657–1661. doi: 10.1002/j.1460-2075.1992.tb05212.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Zürcher T., Pavlovic J., Staeheli P. Nuclear localization of mouse Mx1 protein is necessary for inhibition of influenza virus. J Virol. 1992 Aug;66(8):5059–5066. doi: 10.1128/jvi.66.8.5059-5066.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. van der Bliek A. M., Meyerowitz E. M. Dynamin-like protein encoded by the Drosophila shibire gene associated with vesicular traffic. Nature. 1991 May 30;351(6325):411–414. doi: 10.1038/351411a0. [DOI] [PubMed] [Google Scholar]
  48. van der Bliek A. M., Redelmeier T. E., Damke H., Tisdale E. J., Meyerowitz E. M., Schmid S. L. Mutations in human dynamin block an intermediate stage in coated vesicle formation. J Cell Biol. 1993 Aug;122(3):553–563. doi: 10.1083/jcb.122.3.553. [DOI] [PMC free article] [PubMed] [Google Scholar]

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