1887

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Volume 87, Issue 3

Role of myristoylation and N-terminal basic residues in membrane association of the human immunodeficiency virus type 1 Nef protein Free

Abstract

Human immunodeficiency virus type 1 Nef protein is N-terminally myristoylated, a modification reported to be required for the association of Nef with cytoplasmic membranes. As myristate alone is not sufficient to anchor a protein stably into a membrane, it has been suggested that N-terminal basic residues contribute to Nef membrane association via electrostatic interactions with acidic phospholipids. Here, data are presented pertaining to the role of the myristate and basic residues in Nef membrane association, subcellular localization and function. Firstly, by using a biochemical assay for membrane association it was shown that, whereas myristoylation of Nef was not essential, mutation of a cluster of four arginines between residues 17 and 22 reduced membrane association dramatically. Mutation of two lysines at residues 4 and 7 had negligible effect alone, but when combined with the arginine substitutions, abrogated membrane association completely. By using indirect immunofluorescence, it was demonstrated that mutation of either of the two basic clusters altered the subcellular distribution of Nef dramatically. Thirdly, the requirement of the arginine and lysine clusters for Nef-mediated CD4 downmodulation was shown to correlate precisely with membrane association. These data suggest that membrane localization and subcellular targeting of Nef are controlled by a complex interplay of signals at the N terminus of the protein.

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2006-03-01
2024-04-16
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References

  1. Aiken C., Konner J., Landau N. R., Lenburg M. E., Trono D. 1994; Nef induces CD4 endocytosis: requirement for a critical dileucine motif in the membrane-proximal CD4 cytoplasmic domain. Cell 76:853–864 [CrossRef]
     [Google Scholar]
  2. Aiken C., Krause L., Chen Y.-L., Trono D. 1996; Mutational analysis of HIV-1 Nef: identification of two mutants that are temperature-sensitive for CD4 downregulation. Virology 217:293–300 [CrossRef]
     [Google Scholar]
  3. Arold S., Hoh F., Domergue S., Birck C., Delsuc M.-A., Jullien M., Dumas C. 2000; Characterization and molecular basis of the oligomeric structure of HIV-1 Nef protein. Protein Sci 9:1137–1148 [CrossRef]
     [Google Scholar]
  4. Arora V. K., Fredericksen B. L., Garcia J. V. 2002; Nef: agent of cell subversion. Microbes Infect 4:189–199 [CrossRef]
     [Google Scholar]
  5. Bentham M., Mazaleyrat S., Harris M. 2003; The di-leucine motif in the cytoplasmic tail of CD4 is not required for binding to human immunodeficiency virus type 1 Nef, but is critical for CD4 down-modulation. J Gen Virol 84:2705–2713 [CrossRef]
     [Google Scholar]
  6. Carl S., Greenough T. C., Krumbiegel M., Greenberg M., Skowronski J., Sullivan J. L., Kirchhoff F. 2001; Modulation of different human immunodeficiency virus type 1 Nef functions during progression to AIDS. J Virol 75:3657–3665 [CrossRef]
     [Google Scholar]
  7. Coates K., Cooke S. J., Mann D. A., Harris M. P. G. 1997; Protein kinase C-mediated phosphorylation of HIV-I Nef in human cell lines. J Biol Chem 272:12289–12294 [CrossRef]
     [Google Scholar]
  8. Fackler O. T., Baur A. S. 2002; Live and let die: Nef functions beyond HIV replication. Immunity 16:493–497 [CrossRef]
     [Google Scholar]
  9. Fauré J., Stalder R., Borel C., Sobo K., Piguet V., Demaurex N., Gruenberg J., Trono D. 2004; ARF1 regulates Nef-induced CD4 degradation. Curr Biol 14:1056–1064 [CrossRef]
     [Google Scholar]
  10. Geyer M., Munte C. E., Schorr J., Kellner R., Kalbitzer H. R. 1999; Structure of the anchor-domain of myristoylated and non-myristoylated HIV-1 Nef protein. J Mol Biol 289:123–138 [CrossRef]
     [Google Scholar]
  11. Green S., Issemann I., Sheer E. 1988; A versatile in vivo and in vitro eukaryotic expression vector for protein engineering. Nucleic Acids Res 16:369 [CrossRef]
     [Google Scholar]
  12. Griffin S. D. C., Harvey R., Clarke D. S., Barclay W. S., Harris M., Rowlands D. J. 2004; A conserved basic loop in hepatitis C virus p7 protein is required for amantadine-sensitive ion channel activity in mammalian cells but is dispensable for localization to mitochondria. J Gen Virol 85:451–461 [CrossRef]
     [Google Scholar]
  13. Harris M. P. G., Neil J. C. 1994; Myristoylation-dependent binding of HIV-1 Nef to CD4. J Mol Biol 241:136–142 [CrossRef]
     [Google Scholar]
  14. Iafrate A. J., Bronson S., Skowronski J. 1997; Separable functions of Nef disrupt two aspects of T cell receptor machinery: CD4 expression and CD3 signaling. EMBO J 16:673–684 [CrossRef]
     [Google Scholar]
  15. Kaminchik J., Margalit R., Yaish S., Drummer H., Amit B., Sarver N., Gorecki M., Panet A. 1994; Cellular distribution of HIV type 1 Nef protein: identification of domains in Nef required for association with membrane and detergent-insoluble cellular matrix. AIDS Res Hum Retroviruses 10:1003–1010 [CrossRef]
     [Google Scholar]
  16. Krautkrämer E., Giese S. I., Gasteier J. E., Muranyi W., Fackler O. T. 2004; Human immunodeficiency virus type 1 Nef activates p21-activated kinase via recruitment into lipid rafts. J Virol 78:4085–4097 [CrossRef]
     [Google Scholar]
  17. McCabe J. B., Berthiaume L. G. 1999; Functional roles for fatty acylated amino-terminal domains in subcellular localization. Mol Biol Cell 10:3771–3786 [CrossRef]
     [Google Scholar]
  18. McLaughlin S., Aderem A. 1995; The myristoyl-electrostatic switch: a modulator of reversible protein-membrane interactions. Trends Biochem Sci 20:272–276 [CrossRef]
     [Google Scholar]
  19. Murray D., Hermida-Matsumoto L., Buser C. A., Tsang J., Sigal C. T., Ben-Tal N., Honig B., Resh M. D., McLaughlin S. 1998; Electrostatics and the membrane association of Src: theory and experiment. Biochemistry 37:2145–2159 [CrossRef]
     [Google Scholar]
  20. Niederman T. M. J., Hastings W. R., Ratner L. 1993; Myristoylation-enhanced binding of the HIV-1 Nef protein to T cell skeletal matrix. Virology 197:420–425 [CrossRef]
     [Google Scholar]
  21. Ono A., Freed E. O. 2001; Plasma membrane rafts play a critical role in HIV-1 assembly and release. Proc Natl Acad Sci U S A 98:13925–13930 [CrossRef]
     [Google Scholar]
  22. Pitcher C., Höning S., Fingerhut A., Bowers K., Marsh M. 1999; Cluster of differentiation antigen 4 (CD4) endocytosis and adaptor complex binding require activation of the CD4 endocytosis signal by serine phosphorylation. Mol Biol Cell 10:677–691 [CrossRef]
     [Google Scholar]
  23. Sol-Foulon N., Esnault C., Percherancier Y., Porrot F., Metais-Cunha P., Bachelerie F., Schwartz O. 2004; The effects of HIV-1 Nef on CD4 surface expression and viral infectivity in lymphoid cells are independent of rafts. J Biol Chem 279:31398–31408 [CrossRef]
     [Google Scholar]
  24. Song J., Hirschman J., Gunn K., Dohlman H. G. 1996; Regulation of membrane and subunit interactions by N -myristoylation of a G protein α subunit in yeast. J Biol Chem 271:20273–20283 [CrossRef]
     [Google Scholar]
  25. Spearman P., Wang J.-J., Vander Heyden N., Ratner L. 1994; Identification of human immunodeficiency virus type 1 Gag protein domains essential to membrane binding and particle assembly. J Virol 68:3232–3242
     [Google Scholar]
  26. Tang C., Loeliger E., Luncsford P., Kinde I., Beckett D., Summers M. F. 2004; Entropic switch regulates myristate exposure in the HIV-1 matrix protein. Proc Natl Acad Sci U S A 101:517–522 [CrossRef]
     [Google Scholar]
  27. Tuthill T. J., Papadopoulos N. G., Jourdan P. & 12 other authors 2003; Mouse respiratory epithelial cells support efficient replication of human rhinovirus. J Gen Virol 84:2829–2836 [CrossRef]
     [Google Scholar]
  28. Wang J.-K., Kiyokawa E., Verdin E., Trono D. 2000; The Nef protein of HIV-1 associates with rafts and primes T cells for activation. Proc Natl Acad Sci U S A 97:394–399 [CrossRef]
     [Google Scholar]
  29. Welker R., Harris M., Cardel B., Kräusslich H.-G. 1998; Virion incorporation of human immunodeficiency virus type 1 Nef is mediated by a bipartite membrane-targeting signal: analysis of its role in enhancement of viral infectivity. J Virol 72:8833–8840
     [Google Scholar]
  30. Yu G., Felsted R. L. 1992; Effect of myristoylation on p27 nef subcellular distribution and suppression of HIV-LTR transcription. Virology 187:46–55 [CrossRef]
     [Google Scholar]
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Role of myristoylation and N-terminal basic residues in membrane association of the human immunodeficiency virus type 1 Nef protein
J. Gen. Virol. 87, 563 (2006); https://doi.org/10.1099/vir.0.81200-0
/content/journal/jgv/10.1099/vir.0.81200-0
/content/journal/jgv/10.1099/vir.0.81200-0
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