Skip to main content
PMC home page
Journal of Virology logoLink to Journal of Virology
. 1995 Mar;69(3):1842–1850. doi: 10.1128/jvi.69.3.1842-1850.1995

Human immunodeficiency virus type 1 Nef protein inhibits activation pathways in peripheral blood mononuclear cells and T-cell lines.

A Greenway 1, A Azad 1, D McPhee 1
PMCID: PMC188795  PMID: 7853525

Abstract

Human immunodeficiency virus type 1 (HIV-1) Nef protein causes the loss of cell surface CD4 and interleukin-2 (IL-2) receptor (Tac) from peripheral blood mononuclear cells (PBMC) and CD4+ T-cell lines. As both CD4 and the IL-2 receptor play crucial roles in antigen-driven helper T-cell signalling and T-cell proliferation, respectively, the role of Nef in the viral life cycle may be to perturb signalling pathways emanating from these receptors. However, the intracellular targets for Nef that result in receptor down-regulation are unknown. Using a recombinant glutathione S-transferase-full-length 27 kDa Nef (Nef27) fusion protein, produced in Escherichia coli by translation from the first start codon of HIV-1 nef clone pNL4-3, as an affinity reagent to probe cytoplasmic extracts of MT-2 cells and PBMC, we have shown interaction with at least seven host cell protein species ranging from 24 to 75 kDa. Immunoblotting identified four of these proteins as p56lck, CD4, p53, and p44mapk/erk1, all of which are intimately involved in intracellular signalling. To assess the relevance of these interactions and further define the biochemical activity of Nef in signal transduction pathways, highly purified Nef27 protein was introduced directly into PBMC by electroporation. Nef27-treated PBMC showed reduced proliferative responsiveness to exogenous recombinant IL-2. Normally, stimulation of T-cells by IL-2 or phorbol 12-myristate 13-acetate provokes both augmentation of p56lck activity and corresponding posttranslational modification of p56lck. These changes were also inhibited by treatment of PBMC with Nef, suggesting that Nef interferes with activation of p56lck and as a consequence of signalling via the IL-2 receptor. Further evidence for Nef interfering with cell proliferation was the decreased production of the proto-oncogene c-myb, which is required for cell cycle progression, in Nef-treated MT-2 cells. In contrast to the binding characteristics and biological effects of Nef27, the alternate 25-kDa isoform of Nef (Nef25) produced by translation from the second start codon of HIV nef pNL4-3 (57 nucleotide residues downstream) was shown to interact with only three cellular proteins of approximately 26, 28, and 56 kDa from PBMC and MT-2 cells, one of which was identified as p56lck. Also, proliferation and posttranslational modification of p56lck in response to IL-2 stimulation were not profoundly affected by treatment of PBMC with Nef25 compared with Nef27.(ABSTRACT TRUNCATED AT 400 WORDS)

Full Text

The Full Text of this article is available as a PDF (594.5 KB).

Selected References

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

  1. Ahmad N., Venkatesan S. Nef protein of HIV-1 is a transcriptional repressor of HIV-1 LTR. Science. 1988 Sep 16;241(4872):1481–1485. doi: 10.1126/science.3262235. [DOI] [PubMed] [Google Scholar]
  2. Angel P., Allegretto E. A., Okino S. T., Hattori K., Boyle W. J., Hunter T., Karin M. Oncogene jun encodes a sequence-specific trans-activator similar to AP-1. Nature. 1988 Mar 10;332(6160):166–171. doi: 10.1038/332166a0. [DOI] [PubMed] [Google Scholar]
  3. Azad A. A., Failla P., Lucantoni A., Bentley J., Mardon C., Wolfe A., Fuller K., Hewish D., Sengupta S., Sankovich S. Large-scale production and characterization of recombinant human immunodeficiency virus type 1 Nef. J Gen Virol. 1994 Mar;75(Pt 3):651–655. doi: 10.1099/0022-1317-75-3-651. [DOI] [PubMed] [Google Scholar]
  4. Backer J. M., Mendola C. E., Fairhurst J. L., Kovesdi I. The HIV-1 nef protein does not have guanine nucleotide binding, GTPase, or autophosphorylating activities. AIDS Res Hum Retroviruses. 1991 Dec;7(12):1015–1020. doi: 10.1089/aid.1991.7.1015. [DOI] [PubMed] [Google Scholar]
  5. Barber E. K., Dasgupta J. D., Schlossman S. F., Trevillyan J. M., Rudd C. E. The CD4 and CD8 antigens are coupled to a protein-tyrosine kinase (p56lck) that phosphorylates the CD3 complex. Proc Natl Acad Sci U S A. 1989 May;86(9):3277–3281. doi: 10.1073/pnas.86.9.3277. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Bolen J. B., Thompson P. A., Eiseman E., Horak I. D. Expression and interactions of the Src family of tyrosine protein kinases in T lymphocytes. Adv Cancer Res. 1991;57:103–149. doi: 10.1016/s0065-230x(08)60997-5. [DOI] [PubMed] [Google Scholar]
  7. Casnellie J. E., Gentry L. E., Rohrschneider L. R., Krebs E. G. Identification of the tyrosine protein kinase from LSTRA cells by use of site-specific antibodies. Proc Natl Acad Sci U S A. 1984 Nov;81(21):6676–6680. doi: 10.1073/pnas.81.21.6676. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Cheng-Mayer C., Iannello P., Shaw K., Luciw P. A., Levy J. A. Differential effects of nef on HIV replication: implications for viral pathogenesis in the host. Science. 1989 Dec 22;246(4937):1629–1632. doi: 10.1126/science.2531920. [DOI] [PubMed] [Google Scholar]
  9. Chowers M. Y., Spina C. A., Kwoh T. J., Fitch N. J., Richman D. D., Guatelli J. C. Optimal infectivity in vitro of human immunodeficiency virus type 1 requires an intact nef gene. J Virol. 1994 May;68(5):2906–2914. doi: 10.1128/jvi.68.5.2906-2914.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Cullen B. R., Greene W. C. Functions of the auxiliary gene products of the human immunodeficiency virus type 1. Virology. 1990 Sep;178(1):1–5. doi: 10.1016/0042-6822(90)90373-y. [DOI] [PubMed] [Google Scholar]
  11. Cullen B. R. Regulation of human immunodeficiency virus replication. Annu Rev Microbiol. 1991;45:219–250. doi: 10.1146/annurev.mi.45.100191.001251. [DOI] [PubMed] [Google Scholar]
  12. Ferris D. K., Willette-Brown J., Ortaldo J. R., Farrar W. L. IL-2 regulation of tyrosine kinase activity is mediated through the p70-75 beta-subunit of the IL-2 receptor. J Immunol. 1989 Aug 1;143(3):870–876. [PubMed] [Google Scholar]
  13. Franchini G., Robert-Guroff M., Ghrayeb J., Chang N. T., Wong-Staal F. Cytoplasmic localization of the HTLV-III 3' orf protein in cultured T cells. Virology. 1986 Dec;155(2):593–599. doi: 10.1016/0042-6822(86)90219-9. [DOI] [PubMed] [Google Scholar]
  14. Garcia J. V., Alfano J., Miller A. D. The negative effect of human immunodeficiency virus type 1 Nef on cell surface CD4 expression is not species specific and requires the cytoplasmic domain of CD4. J Virol. 1993 Mar;67(3):1511–1516. doi: 10.1128/jvi.67.3.1511-1516.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Garcia J. V., Miller A. D. Serine phosphorylation-independent downregulation of cell-surface CD4 by nef. Nature. 1991 Apr 11;350(6318):508–511. doi: 10.1038/350508a0. [DOI] [PubMed] [Google Scholar]
  16. Greenway A. L., McPhee D. A., Grgacic E., Hewish D., Lucantoni A., Macreadie I., Azad A. Nef 27, but not the Nef 25 isoform of human immunodeficiency virus-type 1 pNL4.3 down-regulates surface CD4 and IL-2R expression in peripheral blood mononuclear cells and transformed T cells. Virology. 1994 Jan;198(1):245–256. doi: 10.1006/viro.1994.1027. [DOI] [PubMed] [Google Scholar]
  17. Guy B., Kieny M. P., Riviere Y., Le Peuch C., Dott K., Girard M., Montagnier L., Lecocq J. P. HIV F/3' orf encodes a phosphorylated GTP-binding protein resembling an oncogene product. Nature. 1987 Nov 19;330(6145):266–269. doi: 10.1038/330266a0. [DOI] [PubMed] [Google Scholar]
  18. Harris M., Coates K. Identification of cellular proteins that bind to the human immunodeficiency virus type 1 nef gene product in vitro: a role for myristylation. J Gen Virol. 1993 Aug;74(Pt 8):1581–1589. doi: 10.1099/0022-1317-74-8-1581. [DOI] [PubMed] [Google Scholar]
  19. Hatakeyama M., Kono T., Kobayashi N., Kawahara A., Levin S. D., Perlmutter R. M., Taniguchi T. Interaction of the IL-2 receptor with the src-family kinase p56lck: identification of novel intermolecular association. Science. 1991 Jun 14;252(5012):1523–1528. doi: 10.1126/science.2047859. [DOI] [PubMed] [Google Scholar]
  20. Horak I. D., Gress R. E., Lucas P. J., Horak E. M., Waldmann T. A., Bolen J. B. T-lymphocyte interleukin 2-dependent tyrosine protein kinase signal transduction involves the activation of p56lck. Proc Natl Acad Sci U S A. 1991 Mar 1;88(5):1996–2000. doi: 10.1073/pnas.88.5.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Kaminchik J., Bashan N., Itach A., Sarver N., Gorecki M., Panet A. Genetic characterization of human immunodeficiency virus type 1 nef gene products translated in vitro and expressed in mammalian cells. J Virol. 1991 Feb;65(2):583–588. doi: 10.1128/jvi.65.2.583-588.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Kestler H. W., 3rd, Ringler D. J., Mori K., Panicali D. L., Sehgal P. K., Daniel M. D., Desrosiers R. C. Importance of the nef gene for maintenance of high virus loads and for development of AIDS. Cell. 1991 May 17;65(4):651–662. doi: 10.1016/0092-8674(91)90097-i. [DOI] [PubMed] [Google Scholar]
  23. Kobayashi N., Kono T., Hatakeyama M., Minami Y., Miyazaki T., Perlmutter R. M., Taniguchi T. Functional coupling of the src-family protein tyrosine kinases p59fyn and p53/56lyn with the interleukin 2 receptor: implications for redundancy and pleiotropism in cytokine signal transduction. Proc Natl Acad Sci U S A. 1993 May 1;90(9):4201–4205. doi: 10.1073/pnas.90.9.4201. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Laurent-Crawford A. G., Krust B., Muller S., Rivière Y., Rey-Cuillé M. A., Béchet J. M., Montagnier L., Hovanessian A. G. The cytopathic effect of HIV is associated with apoptosis. Virology. 1991 Dec;185(2):829–839. doi: 10.1016/0042-6822(91)90554-o. [DOI] [PubMed] [Google Scholar]
  25. Luciw P. A., Cheng-Mayer C., Levy J. A. Mutational analysis of the human immunodeficiency virus: the orf-B region down-regulates virus replication. Proc Natl Acad Sci U S A. 1987 Mar;84(5):1434–1438. doi: 10.1073/pnas.84.5.1434. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Luria S., Chambers I., Berg P. Expression of the type 1 human immunodeficiency virus Nef protein in T cells prevents antigen receptor-mediated induction of interleukin 2 mRNA. Proc Natl Acad Sci U S A. 1991 Jun 15;88(12):5326–5330. doi: 10.1073/pnas.88.12.5326. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Marth J. D., Lewis D. B., Cooke M. P., Mellins E. D., Gearn M. E., Samelson L. E., Wilson C. B., Miller A. D., Perlmutter R. M. Lymphocyte activation provokes modification of a lymphocyte-specific protein tyrosine kinase (p56lck). J Immunol. 1989 Apr 1;142(7):2430–2437. [PubMed] [Google Scholar]
  28. Miller M. D., Warmerdam M. T., Gaston I., Greene W. C., Feinberg M. B. The human immunodeficiency virus-1 nef gene product: a positive factor for viral infection and replication in primary lymphocytes and macrophages. J Exp Med. 1994 Jan 1;179(1):101–113. doi: 10.1084/jem.179.1.101. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Mills G. B., Lee J. W., Cheung R. K., Gelfand E. W. Characterization of the requirements for human T cell mitogenesis by using suboptimal concentrations of phytohemagglutinin. J Immunol. 1985 Nov;135(5):3087–3093. [PubMed] [Google Scholar]
  30. Minami Y., Kono T., Yamada K., Kobayashi N., Kawahara A., Perlmutter R. M., Taniguchi T. Association of p56lck with IL-2 receptor beta chain is critical for the IL-2-induced activation of p56lck. EMBO J. 1993 Feb;12(2):759–768. doi: 10.1002/j.1460-2075.1993.tb05710.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Morla A. O., Draetta G., Beach D., Wang J. Y. Reversible tyrosine phosphorylation of cdc2: dephosphorylation accompanies activation during entry into mitosis. Cell. 1989 Jul 14;58(1):193–203. doi: 10.1016/0092-8674(89)90415-7. [DOI] [PubMed] [Google Scholar]
  32. Morrison D. K., Kaplan D. R., Rapp U., Roberts T. M. Signal transduction from membrane to cytoplasm: growth factors and membrane-bound oncogene products increase Raf-1 phosphorylation and associated protein kinase activity. Proc Natl Acad Sci U S A. 1988 Dec;85(23):8855–8859. doi: 10.1073/pnas.85.23.8855. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Nebreda A. R., Bryan T., Segade F., Wingfield P., Venkatesan S., Santos E. Biochemical and biological comparison of HIV-1 NEF and ras gene products. Virology. 1991 Jul;183(1):151–159. doi: 10.1016/0042-6822(91)90128-x. [DOI] [PubMed] [Google Scholar]
  34. Niederman T. M., Garcia J. V., Hastings W. R., Luria S., Ratner L. Human immunodeficiency virus type 1 Nef protein inhibits NF-kappa B induction in human T cells. J Virol. 1992 Oct;66(10):6213–6219. doi: 10.1128/jvi.66.10.6213-6219.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Niederman T. M., Hastings W. R., Ratner L. Myristoylation-enhanced binding of the HIV-1 Nef protein to T cell skeletal matrix. Virology. 1993 Nov;197(1):420–425. doi: 10.1006/viro.1993.1605. [DOI] [PubMed] [Google Scholar]
  36. Niederman T. M., Thielan B. J., Ratner L. Human immunodeficiency virus type 1 negative factor is a transcriptional silencer. Proc Natl Acad Sci U S A. 1989 Feb;86(4):1128–1132. doi: 10.1073/pnas.86.4.1128. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Perper R. J., Zee T. W., Mickelson M. M. Purification of lymphocytes and platelets by gradient centrifugation. J Lab Clin Med. 1968 Nov;72(5):842–848. [PubMed] [Google Scholar]
  38. Purcell D. F., Martin M. A. Alternative splicing of human immunodeficiency virus type 1 mRNA modulates viral protein expression, replication, and infectivity. J Virol. 1993 Nov;67(11):6365–6378. doi: 10.1128/jvi.67.11.6365-6378.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Ramsay R. G., Ishii S., Nishina Y., Soe G., Gonda T. J. Characterization of alternate and truncated forms of murine c-myb proteins. Oncogene Res. 1989;4(4):259–269. [PubMed] [Google Scholar]
  40. Resh M. D. Myristylation and palmitylation of Src family members: the fats of the matter. Cell. 1994 Feb 11;76(3):411–413. doi: 10.1016/0092-8674(94)90104-x. [DOI] [PubMed] [Google Scholar]
  41. Robb R. J., Greene W. C., Rusk C. M. Low and high affinity cellular receptors for interleukin 2. Implications for the level of Tac antigen. J Exp Med. 1984 Oct 1;160(4):1126–1146. doi: 10.1084/jem.160.4.1126. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Rudd C. E. CD4, CD8 and the TCR-CD3 complex: a novel class of protein-tyrosine kinase receptor. Immunol Today. 1990 Nov;11(11):400–406. doi: 10.1016/0167-5699(90)90159-7. [DOI] [PubMed] [Google Scholar]
  43. Sawai E. T., Baur A., Struble H., Peterlin B. M., Levy J. A., Cheng-Mayer C. Human immunodeficiency virus type 1 Nef associates with a cellular serine kinase in T lymphocytes. Proc Natl Acad Sci U S A. 1994 Feb 15;91(4):1539–1543. doi: 10.1073/pnas.91.4.1539. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Shugars D. C., Smith M. S., Glueck D. H., Nantermet P. V., Seillier-Moiseiwitsch F., Swanstrom R. Analysis of human immunodeficiency virus type 1 nef gene sequences present in vivo. J Virol. 1993 Aug;67(8):4639–4650. doi: 10.1128/jvi.67.8.4639-4650.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Siegel J. P., Sharon M., Smith P. L., Leonard W. J. The IL-2 receptor beta chain (p70): role in mediating signals for LAK, NK, and proliferative activities. Science. 1987 Oct 2;238(4823):75–78. doi: 10.1126/science.3116668. [DOI] [PubMed] [Google Scholar]
  46. Siu G., Wurster A. L., Lipsick J. S., Hedrick S. M. Expression of the CD4 gene requires a Myb transcription factor. Mol Cell Biol. 1992 Apr;12(4):1592–1604. doi: 10.1128/mcb.12.4.1592. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Skowronski J., Parks D., Mariani R. Altered T cell activation and development in transgenic mice expressing the HIV-1 nef gene. EMBO J. 1993 Feb;12(2):703–713. doi: 10.1002/j.1460-2075.1993.tb05704.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Spina C. A., Kwoh T. J., Chowers M. Y., Guatelli J. C., Richman D. D. The importance of nef in the induction of human immunodeficiency virus type 1 replication from primary quiescent CD4 lymphocytes. J Exp Med. 1994 Jan 1;179(1):115–123. doi: 10.1084/jem.179.1.115. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Taniguchi T., Matsui H., Fujita T., Hatakeyama M., Kashima N., Fuse A., Hamuro J., Nishi-Takaoka C., Yamada G. Molecular analysis of the interleukin-2 system. Immunol Rev. 1986 Aug;92:121–133. doi: 10.1111/j.1600-065x.1986.tb01497.x. [DOI] [PubMed] [Google Scholar]
  50. Taniguchi T., Minami Y. The IL-2/IL-2 receptor system: a current overview. Cell. 1993 Apr 9;73(1):5–8. doi: 10.1016/0092-8674(93)90152-g. [DOI] [PubMed] [Google Scholar]
  51. Terwilliger E., Sodroski J. G., Rosen C. A., Haseltine W. A. Effects of mutations within the 3' orf open reading frame region of human T-cell lymphotropic virus type III (HTLV-III/LAV) on replication and cytopathogenicity. J Virol. 1986 Nov;60(2):754–760. doi: 10.1128/jvi.60.2.754-760.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. Trahey M., McCormick F. A cytoplasmic protein stimulates normal N-ras p21 GTPase, but does not affect oncogenic mutants. Science. 1987 Oct 23;238(4826):542–545. doi: 10.1126/science.2821624. [DOI] [PubMed] [Google Scholar]
  53. Veillette A., Bookman M. A., Horak E. M., Bolen J. B. The CD4 and CD8 T cell surface antigens are associated with the internal membrane tyrosine-protein kinase p56lck. Cell. 1988 Oct 21;55(2):301–308. doi: 10.1016/0092-8674(88)90053-0. [DOI] [PubMed] [Google Scholar]
  54. Veillette A., Horak I. D., Bolen J. B. Post-translational alterations of the tyrosine kinase p56lck in response to activators of protein kinase C. Oncogene Res. 1988 May;2(4):385–401. [PubMed] [Google Scholar]
  55. Weiss A., Littman D. R. Signal transduction by lymphocyte antigen receptors. Cell. 1994 Jan 28;76(2):263–274. doi: 10.1016/0092-8674(94)90334-4. [DOI] [PubMed] [Google Scholar]
  56. Yonish-Rouach E., Grunwald D., Wilder S., Kimchi A., May E., Lawrence J. J., May P., Oren M. p53-mediated cell death: relationship to cell cycle control. Mol Cell Biol. 1993 Mar;13(3):1415–1423. doi: 10.1128/mcb.13.3.1415. [DOI] [PMC free article] [PubMed] [Google Scholar]

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

RESOURCES