1887

Abstract

The human immunodeficiency virus type 1 (HIV-1) Nef protein has been shown to accelerate viral growth kinetics in primary human T-lymphocytes and macrophages; however, the specific function(s) of Nef responsible for this phenotype in macrophages is unknown. To address this issue, mutants of a molecularly cloned macrophage-tropic isolate, HIV-1, were generated expressing single point mutations that abrogate the ability of Nef to interact with cellular kinases or mediate CD4 down-regulation. Infection of primary monocyte-derived macrophages (MDM) with these mutant viruses revealed that residues in the PXXP motif contribute to efficient replication. Interestingly, viruses expressing alleles of Nef defective in CD4 down-modulation activity retain wild-type levels of infectivity in single-round assays but exhibited delayed replication kinetics and grew to lower titres compared to the wild-type virus in MDM. These data suggest that efficient HIV-1 replication is dependent on the ability of Nef to interact with cellular kinases and remove CD4 from the surface of infected macrophages.

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2004-06-01
2024-03-28
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References

  1. Aiken C., Trono D. 1995; Nef stimulates human immunodeficiency virus type 1 proviral DNA synthesis. J Virol 69:5048–5056
    [Google Scholar]
  2. Alexander L., Du Z., Rosenzweig M., Jung J. U., Desrosiers R. C. 1997; A role for natural simian immunodeficiency virus and human immunodeficiency virus type 1 nef alleles in lymphocyte activation. J Virol 71:6094–6099
    [Google Scholar]
  3. Aquaro S., Perno C. F., Balestra E. 7 other authors 1997; Inhibition of replication of HIV in primary monocyte/macrophages by different antiviral drugs and comparative efficacy in lymphocytes. J Leukoc Biol 62:138–143
    [Google Scholar]
  4. Baur A. S., Sass G., Laffert B., Willbold D., Cheng M. C., Peterlin B. M. 1997; The N-terminus of Nef from HIV-1/SIV associates with a protein complex containing Lck and a serine kinase. Immunity 6:283–291 [CrossRef]
    [Google Scholar]
  5. Bell I., Ashman C., Maughan J., Hooker E., Cook F., Reinhart T. A. 1998; Association of simian immunodeficiency virus Nef with the T-cell receptor (TCR) ζ chain leads to TCR down-modulation. J Gen Virol 79:2717–2727
    [Google Scholar]
  6. Brown A., Wang X., Sawai E., Cheng-Mayer C. 1999; Activation of the PAK-related kinase by human immunodeficiency virus type 1 Nef in primary human peripheral blood lymphocytes and macrophages leads to phosphorylation of a PIX-p95 complex. J Virol 73:9899–9907
    [Google Scholar]
  7. Cheng-Mayer C., Quiroga M., Tung J. W., Dina D., Levy J. A. 1990; Viral determinants of human immunodeficiency virus type I T-cell and macrophage tropism, cytopathogenicity, and CD4 antigen modulation. J Virol 64:4390–4398
    [Google Scholar]
  8. Chowers M. Y., Spina C. A., Kwoh T. J., Fitch N. J. S., Richman D. D., Guateli J. C. 1994; Optimal infectivity in vitro of human immunodeficiency virus type 1 requires an intact nef gene. J Virol 68:2906–2914
    [Google Scholar]
  9. Collette Y., Dutartre H., Benziane A., Ramos-Morales F., Benarous R., Harris M., Olive D. 1996; Physical and functional interaction of Nef with Lck. J Biol Chem 271:6333–6341 [CrossRef]
    [Google Scholar]
  10. Cortes M., Wong-Staal F., Lama J. 2002; Cell surface CD4 interferes with the infectivity of HIV-1 particles released from T cells. J Biol Chem 277:1770–1779 [CrossRef]
    [Google Scholar]
  11. Craig H., Pandori M., Riggs N. L., Richman D. D., Guatelli J. 1999; Analysis of the SH3-binding region of HIV-1 Nef: Partial functional defects introduced by mutations in the polyproline helix and the hydrophobic pocket. Virology 262:55–63 [CrossRef]
    [Google Scholar]
  12. Deacon N. J., Tsykin A., Solomon A. 17 other authors 1995; Genomic structure of an attenuated quasi species from a blood transfusion donor and recipients. Science 270:988–991 [CrossRef]
    [Google Scholar]
  13. Fackler O. T., Luo W., Geyer M., Alberts A. S., Peterlin B. M. 1999; Activation of Vav by Nef induces cytoskeletal rearrangements and downstream effector functions. Mol Cell 3:729–739 [CrossRef]
    [Google Scholar]
  14. Freed E. O. 2003; The HIV-TSG101 interface: recent advances in a budding field. Trends Microbiol 11:56–59 [CrossRef]
    [Google Scholar]
  15. Garcia J. V., Miller A. D. 1991; Serine phosphorylation-independent downregulation of cell-surface CD4 by nef . Nature 350:508–511 [CrossRef]
    [Google Scholar]
  16. Gartner S., Markovits P., Markovits D. M., Kaplan M. H., Gallo R. C., Popovic M. 1986; The role of mononuclear phagocytes in HTLV-III/LAV infection. Science 233:215–219 [CrossRef]
    [Google Scholar]
  17. Gelderblom H. R., Hausmann E. H., Ozel M., Pauli G., Koch M. A. 1987; Fine structure of human immunodeficiency virus (HIV) and immunolocalization of structural proteins. Virology 156:171–176 [CrossRef]
    [Google Scholar]
  18. Geleziunas R., Xu W., Tkeda K., Ichijo H., Greene W. C. 2001; HIV-1 Nef inhibits ASK1-dependent death signalling providing a potential mechanism for protecting the infected host cell. Nature 410:834–838 [CrossRef]
    [Google Scholar]
  19. Glushakova S., Munch J., Carl S., Greenough T. C., Sullivan J. L., Margolis L., Kirchhoff F. 2001; CD4 down-modulation by human immunodeficiency virus type 1 Nef correlates with the efficiency of viral replication and with CD4+ T-cell depletion in human lymphoid tissue ex vivo. J Virol 75:10113–10117 [CrossRef]
    [Google Scholar]
  20. Goldsmith M. A., Warmerdam M. T., Atchison R. E., Miller M. D., Greene W. C. 1995; Dissociation of the CD4 downregulation and viral infectivity enhancement functions of human immunodeficiency virus type 1 Nef. J Virol 69:4112–4121
    [Google Scholar]
  21. Greenway A. L., Azad A., McPhee D. A. 1995; Human immunodeficiency virus type 1 Nef protein inhibits activation pathways in peripheral blood mononuclear cells and T-cell lines. J Virol 69:1842–1850
    [Google Scholar]
  22. Grzesiek S., Stahl S. J., Wingfield P. T., Bax A. 1996; The CD4 determinant for downregulation by HIV-1 Nef directly binds to Nef: mapping of the Nef binding surface by NMR. Biochemistry 35:163–175
    [Google Scholar]
  23. 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 [CrossRef]
    [Google Scholar]
  24. Hanna Z., Weng X., Kay D. G., Poudrier J., Lowell C., Jolicoeur P. 2001; The pathogenicity of human immunodeficiency virus (HIV) type 1 Nef in CD4C/HIV transgenic mice is abolished by mutation of its SH3-binding domain, and disease development is delayed in the absence of Hck. J Virol 75:9378–9392 [CrossRef]
    [Google Scholar]
  25. Hirsch V. M., Sharkey M. E., Brown C. R. 8 other authors 1998; Vpx is required for dissemination and pathogenesis of SIV(SM) PBj: evidence of macrophage-dependent viral amplification. Nat Med 4:1401–1408 [CrossRef]
    [Google Scholar]
  26. Ho D. D., Rota T. R., Hirsch M. S. 1986; Infection of monocyte/macrophages by human T lymphotrophic virus type III. J Clin Invest 77:1712–1715 [CrossRef]
    [Google Scholar]
  27. Howe A. Y., Jung J. U., Desrosiers R. C. 1998; Zeta chain of the T-cell receptor interacts with Nef of simian immunodeficiency virus and human immunodeficiency virus type 2. J Virol 72:9827–9834
    [Google Scholar]
  28. Hua J., Blair W., Truant R., Cullen B. R. 1997; Identification of regions in HIV-1 Nef required for efficient downregulation of cell surface CD4. Virology 231:231–238 [CrossRef]
    [Google Scholar]
  29. Ignatius R., Tenner-Racz K., Messmer D. 9 other authors 2002; Increased macrophage infection upon subcutaneous inoculation of rhesus macaques with simian immunodeficiency virus-loaded dendritic cells or T cells but not with cell-free virus. J Virol 76:9787–9797 [CrossRef]
    [Google Scholar]
  30. Kaul M., Garden G. A., Lipton S. A. 2001; Pathways to neuronal injury and apoptosis in HIV-associated dementia. Nature 410:988–994 [CrossRef]
    [Google Scholar]
  31. Kawano Y., Tanaka Y., Misawa N. 10 other authors 1997; Mutational analysis of human immunodeficiency virus type 1 (HIV-1) accessory genes: requirement of a site in the nef gene for HIV-1 replication in activated CD4+ T cells in vitro and in vivo. J Virol 71:8456–8466
    [Google Scholar]
  32. Kestler H. W. I., 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 [CrossRef]
    [Google Scholar]
  33. Khan I. H., Sawai E. T., Antonio E., Weber C. J., Mandell C. P., Montbriand P., Luciw P. A. 1998; Role of the SH3-ligand domain of simian immunodeficiency virus Nef in interaction with Nef-associated kinase and simian AIDS in rhesus macaques. J Virol 72:5820–5830
    [Google Scholar]
  34. Kimpton J., Emerman M. 1992; Detection of replication-competent and pseudotyped human immunodeficiency virus with a sensitve cell line on the basis of activation of an integrated beta-galactosidase gene. J Virol 66:2232–2239
    [Google Scholar]
  35. Kirchhoff F., Geenough T. C., Brettler D. B., Sullivan J. L., Desrosiers R. C. 1995; Absence of intact nef sequences in a long-term survivor with nonprogressive HIV-1 infection. N Engl J Med 332:228–232 [CrossRef]
    [Google Scholar]
  36. Lama J., Mangasarian A., Trono D. 1999; Cell-surface expression of CD4 reduces HIV-1 infectivity by blocking Environ incorporation in a Nef- and Vpu-inhibitable manner. Curr Biol 9:622–631 [CrossRef]
    [Google Scholar]
  37. Lang S. M., Iafrate A. J., Stahl-Henning C. 7 other authors 1997; Association of simian immunodeficiency virus Nef with cellular serine/threonine kinases is dispensable for the development of AIDS in rhesus macaques. Nat Med 3:860–865 [CrossRef]
    [Google Scholar]
  38. Learmont J. C., Geczy A. F., Mills J. 9 other authors 1999; Immunologic and virologic status after 14 to 18 years of infection with an attenuated strain of HIV-1. N Engl J Med 340:1715–1722 [CrossRef]
    [Google Scholar]
  39. Lee C.-H., Leung B., Lemmon M. A., Zheng J., Cowburn D., Kuriyan J., Saksela K. 1995; A single amino acid in the SH3 domain of Hck determines its high affinity and specificity in binding to HIV-1 Nef protein. EMBO J 14:5006–5015
    [Google Scholar]
  40. Lundquist C. A., Tobiume M., Zhou J., Unutmaz D., Aiken C. 2002; Nef-mediated downregulation of CD4 enhances human immunodeficiency virus type 1 replication in primary T lymphocytes. J Virol 76:4625–4633 [CrossRef]
    [Google Scholar]
  41. Manninen A., Hiipakkam M., Vihinen M., Lu W., Mayer B. J., Saksela K. 1998; SH3-domain binding function of HIV-1 Nef is required for association with PAK-related kinase. Virology 250:273–282 [CrossRef]
    [Google Scholar]
  42. Marechal V., Prevost M.-C., Petit C., Perret E., Heard J.-M., Schwartz O. 2001; Human immunodeficiency virus type 1 entry into macrophages mediated by macropinocytosis. J Virol 75:11166–11177 [CrossRef]
    [Google Scholar]
  43. Marsh M., Thali M. 2003; HIV's great escape. Nat Med 9:1262–1263 [CrossRef]
    [Google Scholar]
  44. Meltzer M. S., Nakamura M., Hansen B. D., Turpin J. A., Kalter D. C., Gendelman H. E. 1990; Macrophages as susceptible targets for HIV infection, persistent viral reservoirs in tissue, and key immunoregulatory cells that control levels of virus replication and extent of disease. AIDS Res Hum Retrovir 6:967–971
    [Google Scholar]
  45. Miller M. D., Warmerdam M. T., Page K. A., Feinberg M. B., Greene W. C. 1995; Expression of human immunodeficiency virus type I (HIV-1) nef gene during HIV-1 production increases progeny particle infectivity independently of gp120 or viral entry. J Virol 69:579–584
    [Google Scholar]
  46. Nguyen D. G., Booth A., Gould S. J., Hildreth J. E. K. 2003; Evidence that HIV budding in primary macrophages occurs through the exosome release pathway. J Biol Chem 278:52347–52354 [CrossRef]
    [Google Scholar]
  47. Nunn M. F., Marsh J. W. 1996; Human immunodeficiency virus type 1 Nef associates with a member of the p21-activated kinase family. J Virol 70:6157–6161
    [Google Scholar]
  48. Orenstein J. M., Meltzer M. S., Phipps T., Gendelman H. E. 1988; Cytoplasmic assembly and accumulation of human immunodeficiencyvirus types 1 and 2 in recombinant human colony-stimulating factor-1-treated human monocytes: an ultrastructural study. J Virol 62:2578–2586
    [Google Scholar]
  49. Orenstein J. M., Fox C., Wahl S. M. 1997; Macrophages as a source of HIV during opportunistic infections. Science 276:1857–1861 [CrossRef]
    [Google Scholar]
  50. Papkalla A., Munch J., Otto C., Kirchhoff F. 2002; Nef enhances human immunodeficiency virus type 1 infectivity and replication independently of viral coreceptor tropism. J Virol 76:8455–8459 [CrossRef]
    [Google Scholar]
  51. Pelchen-Matthews A., Kramer B., Marsh M. 2003; Infectious HIV-1 assembles in late endosomes in primary macrophages. J Cell Biol 162:443–455 [CrossRef]
    [Google Scholar]
  52. Piguet V., Gu F., Foti M., Demaurex N., Gruenberg J., Carpentier J.-L., Trono D. 1999; Nef-induced CD4 degradation: a diacidic-based motif in Nef functions as a lysosomal targeting signal through the binding of β -COP in endosomes. Cell 97:63–73 [CrossRef]
    [Google Scholar]
  53. Pornillos O., Garrus J. E., Sundquist W. I. 2002; Mechanisms of enveloped RNA virus budding. Trends Cell Biol 12:569–579 [CrossRef]
    [Google Scholar]
  54. Raposo G., Moore M., Innes D., Leijendekker R., Leigh-Brown A., Benaroch P., Geuze H. 2002; Human macrophages accumulate HIV-1 particles in MHC II compartments. Traffic 3:718–729 [CrossRef]
    [Google Scholar]
  55. Renkema G. H., Saksela K. 2000; Interactions of HIV-1 NEF with cellular signal transducing proteins. Front Biosci 5:D268–283 [CrossRef]
    [Google Scholar]
  56. Ross T., Oran A., Cullen B. 1999; Inhibition of HIV-1 progeny virion release by cell-surface CD4 is relieved by expression of the viral Nef protein. Curr Biol 9:613–621 [CrossRef]
    [Google Scholar]
  57. Saksela K., Cheng G., Baltimore D. 1995; Proline-rich (PxxP) motifs in HIV-1 Nef bind to SH3 domains of a subset of Src kinases and are required for the enhanced growth of Nef+ viruses but not for down-regulation of CD4. EMBO J 14:484–491
    [Google Scholar]
  58. 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. Proc Natl Acad Sci U S A 91:1539–1543 [CrossRef]
    [Google Scholar]
  59. Sawai E., Baur A. S., Peterlin B. M., Levy J. A., Cheng-Mayer C. 1995; A conserved domain and membrane targeting of Nef from HIV and SIV are required for association with a cellular serine kinase activity. J Biol Chem 270:15307–15314 [CrossRef]
    [Google Scholar]
  60. Schwartz O., Marechal V., Danos O., Heard J.-M. 1995; Human immunodeficiency virus type 1 Nef increases the efficiency of reverse transcription in the infected cell. J Virol 69:4053–4059
    [Google Scholar]
  61. Schwartz O., Marechal V., Le Gall S., Lemonnier F., Heard J. M. 1996; Endocytosis of MHC-1 molecules is induced by HIV-1 Nef. Nature Med 2:338–342 [CrossRef]
    [Google Scholar]
  62. Simmons A., Aluvihare V., McMichael A. 2001; Nef triggers a transcriptional program in T cells imitating single-signal T cell activation and inducing HIV virulence mediators. Immunity 14:763–777 [CrossRef]
    [Google Scholar]
  63. Skowronski J., Parks D., Mariani R. 1993; Altered T cell activation and development in transgenic mice expressing the HIV-1 nef gene. EMBO J 12:703–713
    [Google Scholar]
  64. Smith B. L., Krushelnycky B. W., Mochly R. D., Berg P. 1996; The HIV Nef protein associates with protein kinase C theta. J Biol Chem 271:16753–16757 [CrossRef]
    [Google Scholar]
  65. Stoddart C. A., Geleziunas R., Ferrell S. 8 other authors 2003; Human immunodeficiency virus type 1 Nef-mediated downregulation of CD4 correlates with Nef enhancement of viral pathogenesis. J Virol 77:2124–2133 [CrossRef]
    [Google Scholar]
  66. von Lindern J. J., Rojo D., Grovit-Ferbas K. 9 other authors 2003; Potential role for CD63 in CCR5-mediated human immunodeficiency virus type 1 infection of macrophages. J Virol 77:3624–3633 [CrossRef]
    [Google Scholar]
  67. Wiskerchen M., Cheng-Mayer C. 1996; HIV-1 Nef association with cellular serine kinase correlates with enhanced virion infectivity and efficient proviral DNA synthesis. Virology 224:292–301 [CrossRef]
    [Google Scholar]
  68. Xu X.-N., Laffert B., Screaton G. R., Kraft M., Wolf D., Kolanus W., Mongkolsapay J., McMichael A. J., Baur A. S. 1999; Induction of fas ligand expression by HIV involves the interaction of Nef with the T cell receptor ζ chain. J Exp Med 189:1489–1496 [CrossRef]
    [Google Scholar]
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