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Volume 76, Issue 6

binding and phosphorylation of human immunodeficiency virus type 1 Nef protein by serine/threonine protein kinase Free

Abstract

Although the human immunodeficiency virus type 1 (HIV-1) gene still has no precisely defined function, studies have demonstrated that Nef is an important pathogenic determinant of HIV. In order to identify cellular proteins capable of binding to Nef, the HIV-1 gene product was expressed in the bacterial vector pGEX-2T as a glutathione -transferase (GST)-Nef fusion protein. Deletion mutants corresponding to 86 and 35 N-terminal residues of the Nef protein were prepared. The GST-Nef constructs were used to identify cellular kinases capable of interacting with Nef. After incubation with a Jurkat cell lysate, the GST-Nef constructs immobilized on glutathione-agarose beads bound to cellular kinase(s) and were phosphorylated at three sites : one on threonine at position 15, one on serine between residues 1 and 35, and one on threonine between residues 36 and 86. The Nef-phosphorylating activity was inhibited by protein kinase C (PKC)-selective inhibitors. Cell fractionation showed that this Nef-binding kinase was mainly in the membrane-associated fraction. These results suggest that kinase(s) of the PKC family are specifically bound to and phosphorylate Nef . The interaction of Nef with cellular kinases and its phosphorylation may be important in mediating the effects of Nef in HIV-1 pathogenesis.

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  • Accepted:
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© Journal of General Virology 1995
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1995-06-01
2024-04-24
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References

  1. Bachelerie F., Alcami J., Hazan U., Israel N., Goud B., Arenzana-Seisdedos F., Virelizier J. L. 1990; Constitutive expression of human immunodeficiency virus (HIV) nef protein in human astrocytes does not influence basal or induced HIV long terminal repeat activity. Journal of Virology 64:3059–3062
     [Google Scholar]
  2. Backer J. M., Mendola C. E., Fairhurst J. L., Kovesdi I. 1991; The HIV-1 nef protein does not have guanine nucleotide binding, GTPase, or autophosphorylation activities. AIDS Research and Human Retroviruses 12:1015–1020
     [Google Scholar]
  3. Bandres J. C., Ratner L. 1994; Human immunodeficiency virus type 1 Nef protein down-regulates transcription factors nf-k b and AP-1 in human T-cells in vitro after T-cell receptor stimulation. Journal of Virology 68:3243–3249
     [Google Scholar]
  4. Bandres J. C., Luria S., Ratner L. 1994; Regulation of human immunodeficiency virus Nef protein by phosphorylation. Virology 201:157–161
     [Google Scholar]
  5. Benichou S., Bomsel M., Bodeus M., Durand H., Doute M., Letourneur F., Camonis J., Benarous R. 1994; Physical interaction of the HIV-1 Nef protein with β-COP, a component of non-clathrin-coated vesicles essential for membrane traffic. Journal of Biological Chemistry 269:30073–30076
     [Google Scholar]
  6. Bougeret C., Rothhut B., Jullien P., Fischer S., Benarous R. 1993; Recombinant Csk expressed in Escherichia coli is auto-phosphorylated on tyrosine residue(s). Oncogene 8:1241–1247
     [Google Scholar]
  7. Chowers M. Y., Spina C. A., Kwoh T. J., Fitch N. J. S., Richman D. D., Guatelli J. C. 1994; Optimal infectivity of human immunodeficiency virus type 1 requires an intact nef gene. Journal of Virology 68:2906–2914
     [Google Scholar]
  8. Delassus S., Cheynier R., Wain-Hobson S. 1991; Evolution of human immunodeficiency virus type 1 nef and long terminal repeat sequences over four years in vivo and in vitro . Journal of Virology 65:225–231
     [Google Scholar]
  9. De Ronde A., Klaver B., Keulen W., Smit L., Goudsmit J. 1992; Natural HIV-1 NEF accelerates virus replication in primary human lymphocytes. Virology 188:391–395
     [Google Scholar]
  10. Duden R., Griffiths G., Frank R., Argos P., Kreis E. 1991; β-COP, a 110 kd protein associated with non-clathrin-coated vesicles and the Golgi complex, shows homology to β-adaptin. Cell 64:649–665
     [Google Scholar]
  11. Garcia J. V., Miller A. D. 1991; Serine phosphorylation-independent downregulation of cell-surface CD4 by nef. Nature 350:508–510
     [Google Scholar]
  12. Guy B., Kieny M. P., Riviere Y., Le Peuch C., Dott K., Girard M., Montagnier L., Lecocq J. P. 1987; HIV F/3′ orf encodes a phosphorylated GTP-binding protein resembling an oncogene product. Nature 330:266–269
     [Google Scholar]
  13. Guy B., Riviere Y., Dott K., Regnault A., Kieny M. P. 1990; Mutational analysis of the HIV nef protein. Virology 176:413–125
     [Google Scholar]
  14. Hammes S. R., Dixon E. P., Malim M. H., Cullen B. R., Greene W. C. 1989; Nef protein of human immunodeficiency virus type 1: evidence against its role as a transcriptional inhibitor. Proceedings of the National Academy of Sciences, USA 86:9549–9553
     [Google Scholar]
  15. Harris M., Coates K. 1993; Identification of cellular proteins that bind to the human immunodeficiency virus type 1 nef gene product in vitro: a role for myristylation. Journal of General Virology 74:1581–1589
     [Google Scholar]
  16. Harris M., Hislop S., Patsilinacos P., Neil J. C. 1992; In vivo derived HIV-1 nef gene products are heterogeneous and lack detectable nucleotide binding activity. AIDS Research and Human Retroviruses 8:537–543
     [Google Scholar]
  17. Hidaka H., Inagaki M., Kawamoto S., Sasaki Y. 1984; Isoquinolinesulfonamide, novel and potent inhibitors of cyclic nucleotide dependent protein kinase and protein kinase C. Biochemistry 23:5036–5041
     [Google Scholar]
  18. Hunter T., Sefton B. M. 1980; Transforming gene product of Rous sarcoma virus phosphorylates tyrosine. Proceedings of the National Academy of Sciences, USA 77:1311–1315
     [Google Scholar]
  19. Jamieson B. D., Aldrovandi G. M., Planelles V., Jowett J. B. M., Gao L., Bloch L. M., Chen I. S. Y., Zack J. A. 1994; Requirement of human immunodeficiency virus type 1 nef for in vivo replication and pathogenicity. Journal of Virology 68:3478–3485
     [Google Scholar]
  20. Jullien P., Bougeret C., Camoin L., Bodeus M., Durand H., Disanto J. P., Fisher S., Benarous R. 1994; Tyr394 and Tyr505 are autophosphorylated in recombinant Lck protein-tyrosine kinase expressed in Escherichia coli . European Journal of Biochemistry 224:589–596
     [Google Scholar]
  21. Kaminchik J., Bashan N., Pinchasi D., Amit B., Sarver N., Johnston M. I., Fischer M., Yavin Z., Gorecki M., Panet A. 1990; Expression and biochemical characterization of human immunodeficiency virus type 1 nef gene product. Journal of Virology 64:3447–3454
     [Google Scholar]
  22. Kamps M. P., Sefton B. M. 1989; Acid and base hydrolysis of phosphoproteins bound to immobilon facilitates analysis of phosphoamino acids in gel-fractionated proteins. Analytical Biochemistry 176:22–27
     [Google Scholar]
  23. Kestler J. H. III, Ringler D. J., Mori K., Panicali D. L., Sehgal P. K., Daniel M. D., Desrosiers R. C. 1991; Importance of the nef gene for maintenance of high virus loads and for the development of AIDS. Cell 65:651–662
     [Google Scholar]
  24. Kim S., Ikeuchi K., Byrn R., Groopman J., Baltimore D. 1989; Lack of negative influence on viral growth by the nef gene of human immunodeficiency virus type 1. Proceedings of the National Academy of Sciences, USA 86:9544–9548
     [Google Scholar]
  25. Laurent A. G., Hovanessian A. G., Riviere Y., Krust B., Regnault A., Montagnier L., Findeli A., Kieny M. P., Guy B. 1990; Production of a non-functional nef protein in human immunodeficiency virus type 1-infected CEM cells. Journal of General Virology 71:2273–2281
     [Google Scholar]
  26. Luciw P. A., Cheng-Mayer C., Levy J. A. 1987; Mutational analysis of the human immunodeficiency virus: the orf-B region downregulates virus replication. Proceedings of the National Academy of Sciences, USA 84:1434–1438
     [Google Scholar]
  27. Luria S., Chambers I., Berg P. 1991; Expression of the type 1 human immunodeficiency virus Nef protein in T cells prevents antigen receptor-mediated induction of interleukin 2 mRNA. Proceedings of the National Academy of Sciences, USA 88:5326–5330
     [Google Scholar]
  28. Matsuura Y., Maekawa M., Hattori S., Ikegami N., Hayashi A., Yamazaki S., Morita C., Takebe Y. 1991; Purification and characterization of human immunodeficiency virus type 1 nef gene product expressed by a recombinant baculovirus. Virology 184:580–586
     [Google Scholar]
  29. Nebrada A. R., Bryan T., Segade F., Wingfield P., Venkatesan S., Santos E. 1991; Biochemical and biological comparison of HIV-1 nef and ras gene products. Virology 183:151–159
     [Google Scholar]
  30. Niederman T. M. J., Garcia J. V., Hastings W. R., Luria S., Ratner L. 1992; Human immunodeficiency virus type 1 Nef protein inhibits nf-k b induction in human T cells. Journal of Virology 66:6213–6219
     [Google Scholar]
  31. Niederman T. M. J., Hastings W. R., Luria S., Bandres J. C., Ratner L. 1993; hiv-1 Nef protein inhibits the recruitment of AP-1 DNA-binding activity in human T-cells. Virology 197:338–344
     [Google Scholar]
  32. Peden K., Emerman M., Montagnier L. 1991; Changes in growth properties on passage in tissue culture of viruses derived from infectious molecular clones of HIV-1LAI, HIV-1MAL and HIV-1ELI . Virology 185:661–672
     [Google Scholar]
  33. Poulin L., Levy J. A. 1992; The HIV-1 Nef gene product is associated with phosphorylation of a 46 kD cellular protein. AIDS 6:787–791
     [Google Scholar]
  34. Sawai E. T., Baur A., Struble H., Peterlin B. M., Levy J. A., Cheng-Mayer C. 1994; Human immunodeficiency virus type 1 Nef associates with a cellular serine kinase in T lymphocytes. Proceedings of the National Academy of Sciences, USA 91:1539–1543
     [Google Scholar]
  35. Schwartz O., Riviere Y., Heard J. M., Danos O. 1993; Reduced cell surface expression of processed human immunodeficiency virus type 1 envelope glycoprotein in the presence of Nef. Journal of Virology 67:3274–3280
     [Google Scholar]
  36. Skowronski J., Parks D., Mariani R. 1993; Altered T cell activation and development in transgenic mice expressing the HIV-1 nef gene. EMBO Journal 12:703–713
     [Google Scholar]
  37. Smith D. B., Johnson K. S. 1988; Single-step purification of polypeptides in Escherichia coli as fusions with glutathione S- transferase. Gene 67:31–40
     [Google Scholar]
  38. Terwilliger E. F., Sodroski J. G., Rosen C. A., Haseltine W. A. 1986; Effects of mutations within the 3′orf open reading frame region of human T-lymphotropic virus type III (HTLVIII/LAV) on replication and cytopathogenicity. Journal of Virology 60:7545–7560
     [Google Scholar]
  39. Toullec D., Pianetti P., Coste H., Bellevergue P., Grand-Perret T., Ajakane M., Baudet V., Boissin P., Boursier E., Loriolle F., Duhamel L., Charon D., Kirilovsky J. 1991; The bisindolylmaleimide GF 109203X is a potent and selective inhibitor of protein kinase C. Journal of Biological Chemistry 266:15771–15781
     [Google Scholar]
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In vitro binding and phosphorylation of human immunodeficiency virus type 1 Nef protein by serine/threonine protein kinase
J. Gen. Virol. 76, 1337 (1995); https://doi.org/10.1099/0022-1317-76-6-1337
/content/journal/jgv/10.1099/0022-1317-76-6-1337
/content/journal/jgv/10.1099/0022-1317-76-6-1337
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