Aaron Diamond AIDS Research Center, The Rockefeller University, 455 First Avenue, 7th Floor, New York, NY 10016, USA
Correspondence
Amanda Brown
abrown76{at}jhmi.edu
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ABSTRACT |
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Present address: The Johns Hopkins University, School of Medicine, Department of Neurology, 600 North Wolfe Street, Meyer 6-181, Baltimore, MD 21287-7131, USA.
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MAIN TEXT |
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HIV-1-infected T-lymphocytes and the resulting destruction and impairment of these cells play a major role in the development of AIDS. Accordingly, most studies of Nef functions have focused on this cell type. However, macrophages are also a critical HIV-1 target cell, and play an important role in viral transmission and dissemination (Hirsch et al., 1998; Ignatius et al., 2002
) and in the development of HIV-associated dementia (Kaul et al., 2001
). Furthermore, the HIVmacrophage interaction differs in important ways from that of HIVT-cell biology. Although virus entry into T-cells requires the CD4CCR5 receptorcoreceptor interaction that allows fusion between Env and the plasma membrane, alternative and/or specialized entry pathways appear to be operative in macrophages (Marechal et al., 2001
; von Lindern et al., 2003
). Furthermore, virions were shown to accumulate in intracellular vesicles in HIV-infected macrophages (Orenstein et al., 1988
) that have recently been identified as late endosomes (Pelchen-Matthews et al., 2003
; Raposo et al., 2002
). These properties, as well as their increased resistance to anti-retroviral therapy (Aquaro et al., 1997
) and the cytopathic effects of HIV-1 infection (Gartner et al., 1986
; Ho et al., 1986
; Meltzer et al., 1990
), suggest that macrophages may serve as virus reservoirs (Gartner et al., 1986
; Meltzer et al., 1990
; Orenstein et al., 1997
). Therefore, understanding the role of Nef in the context of HIV-1 infection of macrophages will be important for developing theraputic strategies aimed at inhibiting virus replication via targeting this gene product.
To determine which function(s) of Nef contribute to efficient replication in MDM, single or double alanine point mutations in amino acid residues known to disrupt the ability of Nef to interact with PAK (Manninen et al., 1998; Sawai et al., 1995
; Wiskerchen & Cheng-Mayer, 1996
) or Src (Saksela et al., 1995
) family kinases, and to down-regulate CD4 (Grzesiek et al., 1996
; Hua et al., 1997
), were introduced into a bicistronic pIRES-EGFP plasmid (Clontech) expressing nef from the M-tropic viral isolate SF162 (Cheng-Mayer et al., 1990
). Before introducing the mutant genes into the proviral DNA backbone, the nef alleles were characterized for their ability to down-regulate CD4 in transient transfection assays performed using Jurkat JJK cells (D. Littman, Skirball Institute, New York, NY, USA). Approximately 6x105 JJK cells were transfected with 20 µg of the pCEF2Nef-IRES-EGFP plasmids using the lipofection agent DMRIE-C (Invitrogen) and harvested for flow cytometry (FACS) 40 h post-transfection. Cells were washed once in PBS containing 10 mM EDTA and stained with saturating amounts of anti-CD4 antibody conjugated to phycoerythrin (PE) (CalTag) in the same buffer with 10 mM sodium azide at room temperature for 30 min. The stained cells were washed and fixed in PBS/1 % formaldehyde and receptor levels were measured using a FACSCalibur flow cytometer and CellQuest analysis software (BD Biosciences). The mean fluoresence intensity (MFI) of the GFP-positive and hence the Nef-expressing cells was examined. In agreement with previous reports, the W59 mutant displayed a defect, while the proline mutants were competent for CD4 down-regulation (Fig. 1
A, B). The Nef 156EE157 mutant that should fail to target CD4 to a degradative compartment (Piguet et al., 1999
) displayed an intermediate phenotype (Fig. 1A, B
). Western analyses of transfected cell lysates revealed that the mutant Nefs were all expressed at comparable levels (Fig. 1C
).
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In contrast to the findings with the CD4 down-regulation mutants, the replication pattern of viruses harbouring mutations in the Nef PXXP motif appeared to be more variable and dependent on the donor macrophages. Furthermore, whereas a correlation between PAK kinase association and enhancement of infectivity was seen in single-cycle assays (Fig. 2), this was absent in infected MDM (Fig. 3
). This discordance between single-round MAGI and multi-round replication assays in assessing the requirement for specific residues with the PXXP motif of Nef was also reported for primary T-cells, perhaps reflecting the more variable activation states of primary cells in culture (Lundquist et al., 2002
). In T-cell and transgenic mouse models, Nef can cause T-lymphocyte activation (Alexander et al., 1997
; Skowronski et al., 1993
) and induce a gene transcription profile that resembles that of T-cells stimulated through CD3 (Simmons et al., 2001
). The PXXP motif of Nef has been implicated in interactions with MAP (Greenway et al., 1995
) and tyrosine kinase signalling pathways (Collette et al., 1996
; Saksela et al., 1995
). In addition, the P74 and P77 residues (P72 and P75 NL4-3 numbering), respectively, are required for interaction with Hck or with both Hck and a PAK-containing complex (Renkema & Saksela, 2000
). These kinases and associated signalling proteins may be present or activated to a varying extent at different stages of macrophage differentiation, explaining the donor-dependent requirement for residues within the PXXP motif for efficient replication in this cell type. Indeed, similar lack of consensus on the requirement for specific PXXP residues for efficient HIV-1 replication in T-cells (Craig et al., 1999
; Lundquist et al., 2002
; Saksela et al., 1995
), and in different mouse (Hanna et al., 2001
; Kawano et al., 1997
; Stoddart et al., 2003
) and rhesus macaque (Khan et al., 1998
; Lang et al., 1997
) models, has also been reported. Further studies are required to understand the biochemical and/or molecular pathways that regulate Nef function in primary macrophages.
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ACKNOWLEDGEMENTS |
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REFERENCES |
---|
![]() ![]() ![]() ![]() |
---|
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, 60946099.[Abstract]
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, 138143.[Abstract]
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, 283291.[Medline]
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) chain leads to TCR down-modulation. J Gen Virol 79, 27172727.[Abstract]
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, 98999907.
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, 43904398.[Medline]
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, 29062914.[Abstract]
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, 63336341.
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, 17701779.
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, 5563.[CrossRef][Medline]
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, 988991.[Abstract]
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, 729739.[CrossRef][Medline]
Freed, E. O. (2003). The HIV-TSG101 interface: recent advances in a budding field. Trends Microbiol 11, 5659.[CrossRef][Medline]
Garcia, J. V. & Miller, A. D. (1991). Serine phosphorylation-independent downregulation of cell-surface CD4 by nef. Nature 350, 508511.[CrossRef][Medline]
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, 215219.[Medline]
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, 171176.[Medline]
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, 834838.[CrossRef][Medline]
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, 1011310117.
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, 41124121.[Abstract]
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, 18421850.[Abstract]
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, 163175.
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, 266269.[CrossRef][Medline]
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, 93789392.
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, 14011408.[CrossRef][Medline]
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, 17121715.[Medline]
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, 98279834.
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, 231238.[CrossRef][Medline]
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, 97879797.
Kaul, M., Garden, G. A. & Lipton, S. A. (2001). Pathways to neuronal injury and apoptosis in HIV-associated dementia. Nature 410, 988994.[CrossRef][Medline]
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, 84568466.[Abstract]
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, 651662.[Medline]
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, 58205830.
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, 22322239.[Abstract]
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, 228232.
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, 622631.[CrossRef][Medline]
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, 860865.[Medline]
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, 17151722.
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, 50065015.[Abstract]
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, 46254633.
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, 273282.[CrossRef][Medline]
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, 1116611177.
Marsh, M. & Thali, M. (2003). HIV's great escape. Nat Med 9, 12621263.[CrossRef][Medline]
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, 967971.[Medline]
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, 579584.[Abstract]
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, 5234752354.
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, 61576161.[Abstract]
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, 25782586.[Medline]
Orenstein, J. M., Fox, C. & Wahl, S. M. (1997). Macrophages as a source of HIV during opportunistic infections. Science 276, 18571861.
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, 84558459.
Pelchen-Matthews, A., Kramer, B. & Marsh, M. (2003). Infectious HIV-1 assembles in late endosomes in primary macrophages. J Cell Biol 162, 443455.
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 -COP in endosomes. Cell 97, 6373.[Medline]
Pornillos, O., Garrus, J. E. & Sundquist, W. I. (2002). Mechanisms of enveloped RNA virus budding. Trends Cell Biol 12, 569579.[CrossRef][Medline]
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, 718729.[CrossRef][Medline]
Renkema, G. H. & Saksela, K. (2000). Interactions of HIV-1 NEF with cellular signal transducing proteins. Front Biosci 5, D268283.[Medline]
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, 613621.[CrossRef][Medline]
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, 484491.[Abstract]
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, 15391543.[Abstract]
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, 1530715314.
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, 40534059.[Abstract]
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, 338342.[Medline]
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, 763777.[CrossRef][Medline]
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, 703713.[Abstract]
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, 1675316757.
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, 21242133.
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, 36243633.
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, 292301.[CrossRef][Medline]
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 chain. J Exp Med 189, 14891496.
Received 4 January 2004;
accepted 19 February 2004.