Skip to main content
PMC home page
Journal of Virology logoLink to Journal of Virology
. 1994 Aug;68(8):5156–5163. doi: 10.1128/jvi.68.8.5156-5163.1994

Human immunodeficiency virus type 1 Nef-induced down-modulation of CD4 is due to rapid internalization and degradation of surface CD4.

S S Rhee 1, J W Marsh 1
PMCID: PMC236459  PMID: 8035515

Abstract

Human immunodeficiency virus type 1 (HIV-1) Nef is a myristylated protein with a relative molecular mass of 27 kDa, is localized to the cytoplasmic surfaces of cellular membranes, and has been reported to down-modulate CD4 in human T cells. To understand the mechanism of HIV-1 Nef-mediated down-modulation of cell surface CD4, we expressed Nef protein in human T-cell line VB. Expression of HIV-1 Nef protein down-modulated surface CD4 molecules. In pulse-chase experiments, CD4 molecules in Nef-expressing cells were synthesized at normal levels. However, the bulk of newly synthesized CD4 protein was degraded with a half-life of approximately 6 h, compared with the 24-h half-life in control cells. This Nef-induced acceleration of CD4 turnover was inhibited by lysosomotropic agents NH4Cl and chloroquine as well as by the protease inhibitor leupeptin. Surface CD4 biotinylation experiments demonstrated that CD4 molecules in Nef-expressing T cells are transported to the plasma membrane with normal kinetics but are then rapidly internalized. Therefore, HIV-1 Nef-induced down-modulation of CD4 is due to rapid internalization of surface CD4 and subsequent degradation by an acid-dependent process, potentially lysosomal. Additionally, in a Nef-expressing cell, we find accelerated dissociation of the T-cell tyrosine kinase p56lck and CD4 but only after the complex reaches the plasma membrane. This implies that HIV-1 Nef protein might play a role in triggering a series of T-cell activation-like events, which contribute to p56lck dissociation and internalization of surface CD4 molecules.

Full text

PDF
5156

Images in this article

5158Image
on p.5158
5159Image
on p.5159
5159Image
on p.5159
5160Image
on p.5160
5160Image
on p.5160

Selected References

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

  1. Acres R. B., Conlon P. J., Mochizuki D. Y., Gallis B. Rapid phosphorylation and modulation of the T4 antigen on cloned helper T cells induced by phorbol myristate acetate or antigen. J Biol Chem. 1986 Dec 5;261(34):16210–16214. [PubMed] [Google Scholar]
  2. 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]
  3. Aiken C., Konner J., Landau N. R., Lenburg M. E., Trono D. Nef induces CD4 endocytosis: requirement for a critical dileucine motif in the membrane-proximal CD4 cytoplasmic domain. Cell. 1994 Mar 11;76(5):853–864. doi: 10.1016/0092-8674(94)90360-3. [DOI] [PubMed] [Google Scholar]
  4. Allan J. S., Coligan J. E., Lee T. H., McLane M. F., Kanki P. J., Groopman J. E., Essex M. A new HTLV-III/LAV encoded antigen detected by antibodies from AIDS patients. Science. 1985 Nov 15;230(4727):810–813. doi: 10.1126/science.2997921. [DOI] [PubMed] [Google Scholar]
  5. Ameisen J. C., Guy B., Chamaret S., Loche M., Mouton Y., Neyrinck J. L., Khalife J., Leprevost C., Beaucaire G., Boutillon C. Antibodies to the nef protein and to nef peptides in HIV-1-infected seronegative individuals. AIDS Res Hum Retroviruses. 1989 Jun;5(3):279–291. doi: 10.1089/aid.1989.5.279. [DOI] [PubMed] [Google Scholar]
  6. Banda N. K., Bernier J., Kurahara D. K., Kurrle R., Haigwood N., Sekaly R. P., Finkel T. H. Crosslinking CD4 by human immunodeficiency virus gp120 primes T cells for activation-induced apoptosis. J Exp Med. 1992 Oct 1;176(4):1099–1106. doi: 10.1084/jem.176.4.1099. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Benson R. E., Sanfridson A., Ottinger J. S., Doyle C., Cullen B. R. Downregulation of cell-surface CD4 expression by simian immunodeficiency virus Nef prevents viral super infection. J Exp Med. 1993 Jun 1;177(6):1561–1566. doi: 10.1084/jem.177.6.1561. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Blue M. L., Hafler D. A., Craig K. A., Levine H., Schlossman S. F. Phosphorylation of CD4 and CD8 molecules following T cell triggering. J Immunol. 1987 Dec 15;139(12):3949–3954. [PubMed] [Google Scholar]
  9. Blumberg B. M., Epstein L. G., Saito Y., Chen D., Sharer L. R., Anand R. Human immunodeficiency virus type 1 nef quasispecies in pathological tissue. J Virol. 1992 Sep;66(9):5256–5264. doi: 10.1128/jvi.66.9.5256-5264.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Braun M., Waheed A., von Figura K. Lysosomal acid phosphatase is transported to lysosomes via the cell surface. EMBO J. 1989 Dec 1;8(12):3633–3640. doi: 10.1002/j.1460-2075.1989.tb08537.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Chen C., Okayama H. High-efficiency transformation of mammalian cells by plasmid DNA. Mol Cell Biol. 1987 Aug;7(8):2745–2752. doi: 10.1128/mcb.7.8.2745. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Crise B., Buonocore L., Rose J. K. CD4 is retained in the endoplasmic reticulum by the human immunodeficiency virus type 1 glycoprotein precursor. J Virol. 1990 Nov;64(11):5585–5593. doi: 10.1128/jvi.64.11.5585-5593.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Crise B., Rose J. K. Human immunodeficiency virus type 1 glycoprotein precursor retains a CD4-p56lck complex in the endoplasmic reticulum. J Virol. 1992 Apr;66(4):2296–2301. doi: 10.1128/jvi.66.4.2296-2301.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Delassus S., Cheynier R., Wain-Hobson S. Evolution of human immunodeficiency virus type 1 nef and long terminal repeat sequences over 4 years in vivo and in vitro. J Virol. 1991 Jan;65(1):225–231. doi: 10.1128/jvi.65.1.225-231.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. 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]
  16. 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]
  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. Hammes S. R., Dixon E. P., Malim M. H., Cullen B. R., Greene W. C. Nef protein of human immunodeficiency virus type 1: evidence against its role as a transcriptional inhibitor. Proc Natl Acad Sci U S A. 1989 Dec;86(23):9549–9553. doi: 10.1073/pnas.86.23.9549. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Hoxie J. A., Matthews D. M., Callahan K. J., Cassel D. L., Cooper R. A. Transient modulation and internalization of T4 antigen induced by phorbol esters. J Immunol. 1986 Aug 15;137(4):1194–1201. [PubMed] [Google Scholar]
  20. Hurley T. R., Luo K., Sefton B. M. Activators of protein kinase C induce dissociation of CD4, but not CD8, from p56lck. Science. 1989 Jul 28;245(4916):407–409. doi: 10.1126/science.2787934. [DOI] [PubMed] [Google Scholar]
  21. Jabbar M. A., Nayak D. P. Intracellular interaction of human immunodeficiency virus type 1 (ARV-2) envelope glycoprotein gp160 with CD4 blocks the movement and maturation of CD4 to the plasma membrane. J Virol. 1990 Dec;64(12):6297–6304. doi: 10.1128/jvi.64.12.6297-6304.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Kaminchik J., Bashan N., Pinchasi D., Amit B., Sarver N., Johnston M. I., Fischer M., Yavin Z., Gorecki M., Panet A. Expression and biochemical characterization of human immunodeficiency virus type 1 nef gene product. J Virol. 1990 Jul;64(7):3447–3454. doi: 10.1128/jvi.64.7.3447-3454.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. 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]
  24. Kim S., Ikeuchi K., Byrn R., Groopman J., Baltimore D. Lack of a negative influence on viral growth by the nef gene of human immunodeficiency virus type 1. Proc Natl Acad Sci U S A. 1989 Dec;86(23):9544–9548. doi: 10.1073/pnas.86.23.9544. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Klatzmann D., Champagne E., Chamaret S., Gruest J., Guetard D., Hercend T., Gluckman J. C., Montagnier L. T-lymphocyte T4 molecule behaves as the receptor for human retrovirus LAV. Nature. 1984 Dec 20;312(5996):767–768. doi: 10.1038/312767a0. [DOI] [PubMed] [Google Scholar]
  26. Letourneur F., Klausner R. D. A novel di-leucine motif and a tyrosine-based motif independently mediate lysosomal targeting and endocytosis of CD3 chains. Cell. 1992 Jun 26;69(7):1143–1157. doi: 10.1016/0092-8674(92)90636-q. [DOI] [PubMed] [Google Scholar]
  27. Libby P., Goldberg A. L. Leupeptin, a protease inhibitor, decreases protein degradation in normal and diseased muscles. Science. 1978 Feb 3;199(4328):534–536. doi: 10.1126/science.622552. [DOI] [PubMed] [Google Scholar]
  28. Lifson J. D., Reyes G. R., McGrath M. S., Stein B. S., Engleman E. G. AIDS retrovirus induced cytopathology: giant cell formation and involvement of CD4 antigen. Science. 1986 May 30;232(4754):1123–1127. doi: 10.1126/science.3010463. [DOI] [PubMed] [Google Scholar]
  29. Lippincott-Schwartz J., Fambrough D. M. Lysosomal membrane dynamics: structure and interorganellar movement of a major lysosomal membrane glycoprotein. J Cell Biol. 1986 May;102(5):1593–1605. doi: 10.1083/jcb.102.5.1593. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Lisanti M. P., Le Bivic A., Sargiacomo M., Rodriguez-Boulan E. Steady-state distribution and biogenesis of endogenous Madin-Darby canine kidney glycoproteins: evidence for intracellular sorting and polarized cell surface delivery. J Cell Biol. 1989 Nov;109(5):2117–2127. doi: 10.1083/jcb.109.5.2117. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. 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]
  32. Maddon P. J., Dalgleish A. G., McDougal J. S., Clapham P. R., Weiss R. A., Axel R. The T4 gene encodes the AIDS virus receptor and is expressed in the immune system and the brain. Cell. 1986 Nov 7;47(3):333–348. doi: 10.1016/0092-8674(86)90590-8. [DOI] [PubMed] [Google Scholar]
  33. Maitra R. K., Ahmad N., Holland S. M., Venkatesan S. Human immunodeficiency virus type 1 (HIV-1) provirus expression and LTR transcription are repressed in NEF-expressing cell lines. Virology. 1991 Jun;182(2):522–533. doi: 10.1016/0042-6822(91)90593-z. [DOI] [PubMed] [Google Scholar]
  34. Mariani R., Skowronski J. CD4 down-regulation by nef alleles isolated from human immunodeficiency virus type 1-infected individuals. Proc Natl Acad Sci U S A. 1993 Jun 15;90(12):5549–5553. doi: 10.1073/pnas.90.12.5549. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Marsh M., Armes J. E., Pelchen-Matthews A. Endocytosis and recycling of CD4. Biochem Soc Trans. 1990 Apr;18(2):139–143. doi: 10.1042/bst0180139. [DOI] [PubMed] [Google Scholar]
  36. Matsuura Y., Maekawa M., Hattori S., Ikegami N., Hayashi A., Yamazaki S., Morita C., Takebe Y. Purification and characterization of human immunodeficiency virus type 1 nef gene product expressed by a recombinant baculovirus. Virology. 1991 Oct;184(2):580–586. doi: 10.1016/0042-6822(91)90428-e. [DOI] [PubMed] [Google Scholar]
  37. McDougal J. S., Kennedy M. S., Sligh J. M., Cort S. P., Mawle A., Nicholson J. K. Binding of HTLV-III/LAV to T4+ T cells by a complex of the 110K viral protein and the T4 molecule. Science. 1986 Jan 24;231(4736):382–385. doi: 10.1126/science.3001934. [DOI] [PubMed] [Google Scholar]
  38. Miceli M. C., Parnes J. R. The roles of CD4 and CD8 in T cell activation. Semin Immunol. 1991 May;3(3):133–141. [PubMed] [Google Scholar]
  39. Miller A. D., Rosman G. J. Improved retroviral vectors for gene transfer and expression. Biotechniques. 1989 Oct;7(9):980-2, 984-6, 989-90. [PMC free article] [PubMed] [Google Scholar]
  40. 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]
  41. 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]
  42. 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]
  43. Parnes J. R. Molecular biology and function of CD4 and CD8. Adv Immunol. 1989;44:265–311. doi: 10.1016/s0065-2776(08)60644-6. [DOI] [PubMed] [Google Scholar]
  44. Pelchen-Matthews A., Armes J. E., Griffiths G., Marsh M. Differential endocytosis of CD4 in lymphocytic and nonlymphocytic cells. J Exp Med. 1991 Mar 1;173(3):575–587. doi: 10.1084/jem.173.3.575. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Pelchen-Matthews A., Armes J. E., Marsh M. Internalization and recycling of CD4 transfected into HeLa and NIH3T3 cells. EMBO J. 1989 Dec 1;8(12):3641–3649. doi: 10.1002/j.1460-2075.1989.tb08538.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Pelchen-Matthews A., Boulet I., Littman D. R., Fagard R., Marsh M. The protein tyrosine kinase p56lck inhibits CD4 endocytosis by preventing entry of CD4 into coated pits. J Cell Biol. 1992 Apr;117(2):279–290. doi: 10.1083/jcb.117.2.279. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Ranki A., Järvinen K., Valle S. L., Nurmilaakso P., Krohn K. Antibodies to recombinant HIV-1 nef protein detected in HIV-1 infection as well as in nonrisk individuals. J Acquir Immune Defic Syndr. 1990;3(4):348–355. [PubMed] [Google Scholar]
  48. Ratner L., Haseltine W., Patarca R., Livak K. J., Starcich B., Josephs S. F., Doran E. R., Rafalski J. A., Whitehorn E. A., Baumeister K. Complete nucleotide sequence of the AIDS virus, HTLV-III. Nature. 1985 Jan 24;313(6000):277–284. doi: 10.1038/313277a0. [DOI] [PubMed] [Google Scholar]
  49. Ratner L., Starcich B., Josephs S. F., Hahn B. H., Reddy E. P., Livak K. J., Petteway S. R., Jr, Pearson M. L., Haseltine W. A., Arya S. K. Polymorphism of the 3' open reading frame of the virus associated with the acquired immune deficiency syndrome, human T-lymphotropic virus type III. Nucleic Acids Res. 1985 Nov 25;13(22):8219–8229. doi: 10.1093/nar/13.22.8219. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Rhee S. S., Hui H. X., Hunter E. Preassembled capsids of type D retroviruses contain a signal sufficient for targeting specifically to the plasma membrane. J Virol. 1990 Aug;64(8):3844–3852. doi: 10.1128/jvi.64.8.3844-3852.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Robert-Guroff M., Popovic M., Gartner S., Markham P., Gallo R. C., Reitz M. S. Structure and expression of tat-, rev-, and nef-specific transcripts of human immunodeficiency virus type 1 in infected lymphocytes and macrophages. J Virol. 1990 Jul;64(7):3391–3398. doi: 10.1128/jvi.64.7.3391-3398.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. Schwartz O., Alizon M., Heard J. M., Danos O. Impairment of T cell receptor-dependent stimulation in CD4+ lymphocytes after contact with membrane-bound HIV-1 envelope glycoprotein. Virology. 1994 Jan;198(1):360–365. doi: 10.1006/viro.1994.1042. [DOI] [PubMed] [Google Scholar]
  53. Seglen P. O. Inhibitors of lysosomal function. Methods Enzymol. 1983;96:737–764. doi: 10.1016/s0076-6879(83)96063-9. [DOI] [PubMed] [Google Scholar]
  54. Shaw A. S., Amrein K. E., Hammond C., Stern D. F., Sefton B. M., Rose J. K. The lck tyrosine protein kinase interacts with the cytoplasmic tail of the CD4 glycoprotein through its unique amino-terminal domain. Cell. 1989 Nov 17;59(4):627–636. doi: 10.1016/0092-8674(89)90008-1. [DOI] [PubMed] [Google Scholar]
  55. Shin J., Doyle C., Yang Z., Kappes D., Strominger J. L. Structural features of the cytoplasmic region of CD4 required for internalization. EMBO J. 1990 Feb;9(2):425–434. doi: 10.1002/j.1460-2075.1990.tb08127.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  56. 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]
  57. Sleckman B. P., Shin J., Igras V. E., Collins T. L., Strominger J. L., Burakoff S. J. Disruption of the CD4-p56lck complex is required for rapid internalization of CD4. Proc Natl Acad Sci U S A. 1992 Aug 15;89(16):7566–7570. doi: 10.1073/pnas.89.16.7566. [DOI] [PMC free article] [PubMed] [Google Scholar]
  58. 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]
  59. Tsunetsugu-Yokota Y., Matsuda S., Maekawa M., Saito T., Takemori T., Takebe Y. Constitutive expression of the nef gene suppresses human immunodeficiency virus type 1 (HIV-1) replication in monocytic cell lines. Virology. 1992 Dec;191(2):960–963. doi: 10.1016/0042-6822(92)90272-q. [DOI] [PubMed] [Google Scholar]
  60. Turner J. M., Brodsky M. H., Irving B. A., Levin S. D., Perlmutter R. M., Littman D. R. Interaction of the unique N-terminal region of tyrosine kinase p56lck with cytoplasmic domains of CD4 and CD8 is mediated by cysteine motifs. Cell. 1990 Mar 9;60(5):755–765. doi: 10.1016/0092-8674(90)90090-2. [DOI] [PubMed] [Google Scholar]
  61. 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]
  62. Veillette A., Bookman M. A., Horak E. M., Samelson L. E., Bolen J. B. Signal transduction through the CD4 receptor involves the activation of the internal membrane tyrosine-protein kinase p56lck. Nature. 1989 Mar 16;338(6212):257–259. doi: 10.1038/338257a0. [DOI] [PubMed] [Google Scholar]
  63. Wain-Hobson S., Sonigo P., Danos O., Cole S., Alizon M. Nucleotide sequence of the AIDS virus, LAV. Cell. 1985 Jan;40(1):9–17. doi: 10.1016/0092-8674(85)90303-4. [DOI] [PubMed] [Google Scholar]
  64. Willey R. L., Maldarelli F., Martin M. A., Strebel K. Human immunodeficiency virus type 1 Vpu protein induces rapid degradation of CD4. J Virol. 1992 Dec;66(12):7193–7200. doi: 10.1128/jvi.66.12.7193-7200.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  65. Zúiga-Pflücker J. C., Jones L. A., Chin L. T., Kruisbeek A. M. CD4 and CD8 act as co-receptors during thymic selection of the T cell repertoire. Semin Immunol. 1991 May;3(3):167–175. [PubMed] [Google Scholar]
  66. de Ronde A., Klaver B., Keulen W., Smit L., Goudsmit J. Natural HIV-1 NEF accelerates virus replication in primary human lymphocytes. Virology. 1992 May;188(1):391–395. doi: 10.1016/0042-6822(92)90772-h. [DOI] [PubMed] [Google Scholar]

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

RESOURCES