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The Effects of HIV-1 Nef on CD4 Surface Expression and Viral Infectivity in Lymphoid Cells Are Independent of Rafts

2004; Elsevier BV; Volume: 279; Issue: 30 Linguagem: Inglês

10.1074/jbc.m401621200

ISSN

1083-351X

Autores

Nathalie Sol‐Foulon, Cécile Esnault, Yann Percherancier, Françoise Porrot, Patricia Metais-Cunha, Françoise Bachelerie, Olivier Schwartz,

Tópico(s)

Immunotherapy and Immune Responses

Resumo

The HIV-1 Nef protein is a critical virulence factor that exerts multiple effects during viral replication. Nef modulates surface expression of various cellular proteins including CD4 and MHC-I, enhances viral infectivity, and affects signal transduction pathways. Nef has been shown to partially associate with rafts, where it can prime T cells for activation. The contribution of rafts during Nef-induced CD4 down-regulation and enhancement of viral replication remains poorly understood. We show here that Nef does not modify the palmitoylation state of CD4 or its partition within rafts. Moreover, CD4 mutants lacking palmitoylation or unable to associate with rafts are efficiently down-regulated by Nef. In HIV-infected cells, viral assembly and budding occurs from rafts, and Nef has been suggested to increase this process. However, using T cells acutely infected with wild-type or nef-deleted HIV, we did not observe any impact of Nef on raft segregation of viral structural proteins. We have also designed a palmitoylated mutant of Nef (NefG3C), which significantly accumulates in rafts. Interestingly, the efficiency of NefG3C to down-regulate CD4 and MHC-I, and to promote viral replication was not increased when compared with the wild-type protein. Altogether, these results strongly suggest that rafts are not a key element involved in the effects of Nef on trafficking of cellular proteins and on viral replication. The HIV-1 Nef protein is a critical virulence factor that exerts multiple effects during viral replication. Nef modulates surface expression of various cellular proteins including CD4 and MHC-I, enhances viral infectivity, and affects signal transduction pathways. Nef has been shown to partially associate with rafts, where it can prime T cells for activation. The contribution of rafts during Nef-induced CD4 down-regulation and enhancement of viral replication remains poorly understood. We show here that Nef does not modify the palmitoylation state of CD4 or its partition within rafts. Moreover, CD4 mutants lacking palmitoylation or unable to associate with rafts are efficiently down-regulated by Nef. In HIV-infected cells, viral assembly and budding occurs from rafts, and Nef has been suggested to increase this process. However, using T cells acutely infected with wild-type or nef-deleted HIV, we did not observe any impact of Nef on raft segregation of viral structural proteins. We have also designed a palmitoylated mutant of Nef (NefG3C), which significantly accumulates in rafts. Interestingly, the efficiency of NefG3C to down-regulate CD4 and MHC-I, and to promote viral replication was not increased when compared with the wild-type protein. Altogether, these results strongly suggest that rafts are not a key element involved in the effects of Nef on trafficking of cellular proteins and on viral replication. The human immunodeficiency virus type-1 (HIV-1) 1The abbreviations used are: HIV-1, human immunodeficiency virus type-1; gp, glycoproteins; WT, wild-type; MHC-I, major histocompatibility class I complex; PBMC, peripheral blood mononuclear cells; IL, interleukin; mAb, monoclonal antibody; PHA, phytohemagglutinin; ELISA, enzyme-linked immunosorbent assay; MOI, multiplicity of infection; GFP, green fluorescent protein. 1The abbreviations used are: HIV-1, human immunodeficiency virus type-1; gp, glycoproteins; WT, wild-type; MHC-I, major histocompatibility class I complex; PBMC, peripheral blood mononuclear cells; IL, interleukin; mAb, monoclonal antibody; PHA, phytohemagglutinin; ELISA, enzyme-linked immunosorbent assay; MOI, multiplicity of infection; GFP, green fluorescent protein. Nef protein is a critical player of viral pathogenesis (1Kestler H.W. Ringler D.J. Mori K. Panicali D.L. Sehgal P.K. Daniel M.D. Desrosiers R.C. Cell. 1991; 65: 651-662Google Scholar, 2Hanna Z. Kay D. 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Le Gall S. Raposo G. Schwartz O. Benaroch P. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 12144-12149Google Scholar, 10Swigut T. Shohdy N. Skowronski J. EMBO J. 2001; 20: 1593-1604Google Scholar, 11Sol-Foulon N. Moris A. Nobile C. Boccaccio C. Engering A. Abastado J.P. Heard J.M. van Kooyk Y. Schwartz O. Immunity. 2002; 16: 145-155Google Scholar). One of the best characterized function of Nef is the down-regulation of CD4, which occurs by a rapid endocytosis of surface molecules (12Garcia J.V. Miller A.D. Nature. 1991; 350: 508-511Google Scholar, 13Aiken C. Konner J. Landau N.R. Lenburg M.E. Trono D. Cell. 1994; 76: 853-864Google Scholar, 14Schwartz O. Dautry-Varsat A. Goud B. Maréchal V. Subtil A. Heard J.M. Danos O. J. Virol. 1995; 69: 528-533Google Scholar). Reduction of CD4 surface expression may facilitate viral release and infectivity of virions, and may prevent Env-associated cytopathic effects, resulting in an optimal viral replication (6Schwartz O. Rivière Y. Heard J.M. Danos O. J. Virol. 1993; 67: 3274-3280Google Scholar, 15Lama J. Mangasarian A. Trono D. Curr. Biol. 1999; 9: 622-631Google Scholar, 16Ross T.M. Oran A.E. Cullen B.R. Curr. Biol. 1999; 9: 613-621Google Scholar, 17Lundquist C.A. Tobiume M. Zhou J. Unutmaz D. Aiken C. J. Virol. 2002; 76: 4625-4633Google Scholar). Nef misroutes CD4 and other proteins through an interaction with a number of sorting proteins, including β-COP, AP complexes, and PACS-1 (18Benichou S. Bomsel M. Bodeus M. Durand H. Doute M. Letourneur F. Camonis J. Benarous R. J. Biol. Chem. 1994; 269: 30073-30076Google Scholar, 19Le Gall S. Erdtmann L. Benichou S. Berlioz-Torrent C. Liu L.X. Benarous R. Heard J.M. Schwartz O. Immunity. 1998; 8: 483-495Google Scholar, 20Piguet V. Schwartz O. Le Gall S. Trono D. Immunol. Rev. 1999; 168: 51-63Google Scholar, 21Piguet V. Wan L. Borel C. Mangasarian A. Demaurex N. Thomas G. Trono D. Nat. Cell Biol. 2000; 2: 163-167Google Scholar). Overexpression of Nef also provokes pleiotropic effects on sorting organelles, inducing the accumulation of clathrin-coated pits, endosomes, lysosomes, and multivesicular bodies (22Foti M. Mangasarian A. Piguet V. Lew D.P. Krause K.H. Trono D. Carpentier J.L. J. Cell Biol. 1997; 139: 37-47Google Scholar, 23Sanfridson A. Hester S. Doyle C. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 873-878Google Scholar, 24Stumptner-Cuvelette P. Jouve M. Helft J. Dugast M. Glouzman A.S. Jooss K. Raposo G. Benaroch P. Mol. Biol. Cell. 2003; 14: 4857-4870Google Scholar). Nef facilitates viral propagation by different means. Viral particles produced in the presence of Nef are more infectious (25Schwartz O. Maréchal V. Danos O. Heard J.M. J. Virol. 1995; 69: 4053-4059Google Scholar, 26Aiken C. Trono D. J. Virol. 1995; 69: 5048-5056Google Scholar), performing more efficiently the early steps of the replicative cycle (25Schwartz O. Maréchal V. Danos O. Heard J.M. J. Virol. 1995; 69: 4053-4059Google Scholar, 26Aiken C. Trono D. J. Virol. 1995; 69: 5048-5056Google Scholar, 27Tobiume M. Lineberger J.E. Lundquist C.A. Miller M.D. Aiken C. J. Virol. 2003; 77: 10645-10650Google Scholar, 28Schaeffer E. Geleziunas R. Greene W.C. J. Virol. 2001; 75: 2993-3000Google Scholar). Nef also exerts direct and indirect positive effects on virus replication in primary human lymphocytes (26Aiken C. Trono D. J. Virol. 1995; 69: 5048-5056Google Scholar, 29de Ronde A. Klaver B. Keulen W. Smit L. Goudsmit J. Virology. 1992; 188: 391-395Google Scholar, 30Spina C.A. Kwoh T.J. Chowers M.Y. Guatelli J.C. Richman D.D. J. Exp. Med. 1994; 179: 115-123Google Scholar, 31Miller M.D. Warmerdam M.T. Gaston I. Greene W.C. Feinberg M.B. J. Exp. Med. 1994; 179: 101-113Google Scholar). Direct effects are particularly visible in lymphocytes activated a few days after infection, in which replication of nef-deleted HIV (HIVΔnef) is barely detectable. Nef also indirectly impacts lymphocytes, when expressed in macrophages or in dendritic cells: HIV-infected macrophages or dendritic cells release paracrine factors (chemokines, soluble CD23, and soluble ICAM), which permit the infection of resting T cells (32Swingler S. Mann A. Jacque J. Brichacek B. Sasseville V.G. Williams K. Lackner A.A. Janoff E.N. Wang R. Fisher D. Stevenson M. Nat. Med. 1999; 5: 997-1003Google Scholar, 33Messmer D. Jacque J.M. Santisteban C. Bristow C. Han S.Y. Villamide-Herrera L. Mehlhop E. Marx P.A. Steinman R.M. Gettie A. Pope M. J. Immunol. 2002; 169: 4172-4182Google Scholar, 34Swingler S. Brichacek B. Jacque J.M. Ulich C. Zhou J. Stevenson M. Nature. 2003; 424: 213-219Google Scholar). The capacity of HIV to replicate in non-activated lymphocytes is likely linked to the numerous described effects of Nef on T cell activation. Nef interacts with a number of kinases or other cellular proteins involved in signal transduction pathways (4Fackler O.T. Baur A.S. Immunity. 2002; 16: 493-497Google Scholar, 35Saksela K. Front Biosci. 1997; 2: d606-d618Google Scholar, 36Witte V. Laffert B. Rosorius O. Lischka P. Blume K. Galler G. Stilper A. Willbold D. D'Aloja P. Sixt M. Kolanus J. Ott M. Kolanus W. Schuler G. Baur A.S. Mol. Cell. 2004; 13: 179-190Google Scholar, 37Janardhan A. Swigut T. Hill B. Myers M.P. Skowronski J. PLoS Biol. 2004; 2: E6Google Scholar), potentially priming infected cells for activation and protecting them from apoptosis (38Simmons A. Aluvihare V. McMichael A. Immunity. 2001; 14: 763-777Google Scholar, 39Geleziunas R. Xu W. Takeda K. Ichijo H. Greene W.C. Nature. 2001; 410: 834-838Google Scholar, 40Wolf D. Witte V. Laffert B. Blume K. Stromer E. Trapp S. d'Aloja P. Schurmann A. Baur A.S. Nat. Med. 2001; 7: 1217-1224Google Scholar). Nef has been suggested to act at the level of rafts to trigger cell activation (41Wang J.K. Kiyokawa E. Verdin E. Trono D. Proc. Nat. Acad. Sci. U. S. A. 2000; 97: 394-399Google Scholar). Rafts are involved in many biological events, including intracellular protein trafficking, signal transduction pathways, entry and release of various virus species (42Simons K. Toomre D. Nat. Rev. 2000; 1: 31-39Google Scholar, 43Suomalainen M. Traffic. 2002; 3: 705-709Google Scholar, 44Chazal N. Gerlier D. Microbiol. Mol. Biol. Rev. 2003; 67: 226-237Google Scholar, 45Briggs J.A. Wilk T. Fuller S.D. J. Gen. Virol. 2003; 84: 757-768Google Scholar). Regarding HIV, rafts are thought to operate at multiple steps of the viral life cycle. Rafts were initially proposed to act as platforms for virus entry, facilitating interactions between CD4 co-receptors, and incoming virions (44Chazal N. Gerlier D. Microbiol. Mol. Biol. Rev. 2003; 67: 226-237Google Scholar, 46Manes S. del Real G. Lacalle R.A. Lucas P. Gomez-Mouton C. Sanchez-Palomino S. Delgado R. Alcami J. Mira E. Martinez A.C. EMBO Rep. 2000; 1: 190-196Google Scholar, 47Del Real G. Jimenez-Baranda S. Lacalle R.A. Mira E. Lucas P. Gomez-Mouton C. Carrera A.C. Martinez A.C. Manes S. J. Exp. Med. 2002; 196: 293-301Google Scholar, 48Graham D.R. Chertova E. Hilburn J.M. Arthur L.O. Hildreth J.E. J. Virol. 2003; 77: 8237-8248Google Scholar). This role has been, however, recently questioned, because CD4 molecules unable to associate with rafts still allow virus entry (49Percherancier Y. Lagane B. Planchenault T. Staropoli I. Altmeyer R. Virelizier J.L. Arenzana-Seisdedos F. Hoessli D.C. Bachelerie F. J. Biol. Chem. 2003; 278: 3153-3161Google Scholar, 50Popik W. Alce T.M. J. Biol. Chem. 2004; 279: 704-712Google Scholar). There is also mounting evidence that rafts are important for HIV-1 assembly and budding. HIV-1 Gag and Env viral structural components are concentrated in rafts or in raft-like complexes (51Nguyen D.H. Hildreth J.E. J. Virol. 2000; 74: 3264-3272Google Scholar, 52Ono A. Freed E.O. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 13925-13930Google Scholar, 53Lindwasser O.W. Resh M.D. J. Virol. 2001; 75: 7913-7924Google Scholar, 54Holm K. Weclewicz K. Hewson R. Suomalainen M. J. Virol. 2003; 77: 4805-4817Google Scholar, 55Ding L. Derdowski A. Wang J.J. Spearman P. J. Virol. 2003; 77: 1916-1926Google Scholar, 56Rousso I. Mixon M.B. Chen B.K. Kim P.S. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 13523-13525Google Scholar, 57Guyader M. Kiyokawa E. Abrami L. Turelli P. Trono D. J. Virol. 2002; 76: 10356-10364Google Scholar). Viral particles are enriched in cellular raft proteins and lipids, and cholesterol-depleting agents decrease viral infectivity. Interestingly, Nef has been proposed to increase viral infectivity via lipid rafts. In this model, Nef induces accumulation of Gag within rafts, thus facilitating viral release from these membrane domains (58Zheng Y.H. Plemenitas A. Linnemann T. Fackler O.T. Peterlin B.M. Curr. Biol. 2001; 11: 875-879Google Scholar). Nef may also increase synthesis and transport of cholesterol to rafts and progeny virions (59Zheng Y.H. Plemenitas A. Fielding C.J. Peterlin B.M. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 8460-8465Google Scholar). Recently, raft targeting by Nef has been reported to be functionally important for CD4 and MHC-I down-regulation as well as infectivity enhancement (60Alexander M. Bor Y. Ravichandran K.S. Hammarskjold M.L. Rekosh D. J. Virol. 2004; 78: 1685-1696Google Scholar). These conclusions were drawn by comparing the activity of LAT-Nef chimeras accumulating in rafts or excluded from these domains. Association of cellular and viral proteins with rafts is often regulated by palmitoylation. Palmitoylation is a reversible post-translational fatty acylation of many proteins, such as transmembrane receptors, G-proteins, tyrosine kinases (61Resh M.D. Biochim. Biophys. Acta. 1999; 1451: 1-16Google Scholar, 62Bijlmakers M.J. Marsh M. Trends Cell Biol. 2003; 13: 32-42Google Scholar, 63Qanbar R. Bouvier M. Pharmacol. Ther. 2003; 97: 1-33Google Scholar), as well as viral envelope glycoproteins (64Olsen K.E. Andersen K.B. J. Virol. 1999; 73: 8975-8981Google Scholar, 65Nozawa N. Daikoku T. Koshizuka T. Yamauchi Y. Yoshikawa T. Nishiyama Y. J. Virol. 2003; 77: 3204-3216Google Scholar, 66Li M. Yang C. Tong S. Weidmann A. Compans R.W. J. Virol. 2002; 76: 11845-11852Google Scholar, 67Ochsenbauer-Jambor C. Miller D.C. Roberts C.R. Rhee S.S. Hunter E. J. Virol. 2001; 75: 11544-11554Google Scholar, 68Zhang J. Pekosz A. Lamb R.A. J. Virol. 2000; 74: 4634-4644Google Scholar). Palmitoylation generally occurs on cysteine residues located close to cellular membranes. The reversible nature of the thioester bound that links palmitate to cysteine allows dynamic changes in protein palmitoylation (63Qanbar R. Bouvier M. Pharmacol. Ther. 2003; 97: 1-33Google Scholar, 69el-Husseini Ael D. Bredt D.S. Nat. Rev. Neurosci. 2002; 3: 791-802Google Scholar). Palmitoylation regulates protein trafficking and association with plasma membranes and with rafts. It also potentially modulates protein activity, oligomerization, and interaction with other proteins. At least two proteins involved in HIV replication are known to be palmitoylated, the cellular receptor CD4 and the viral Env glycoprotein. Efficient CD4 association with rafts requires palmitoylation, but also interaction with the tyrosine kinase p56Lck (49Percherancier Y. Lagane B. Planchenault T. Staropoli I. Altmeyer R. Virelizier J.L. Arenzana-Seisdedos F. Hoessli D.C. Bachelerie F. J. Biol. Chem. 2003; 278: 3153-3161Google Scholar, 50Popik W. Alce T.M. J. Biol. Chem. 2004; 279: 704-712Google Scholar, 70Fragoso R. Ren D. Zhang X. Su M.W. Burakoff S.J. Jin Y.J. J. Immunol. 2003; 170: 913-921Google Scholar). CD4 palmitoylation occurs on two membrane proximal residues (49Percherancier Y. Lagane B. Planchenault T. Staropoli I. Altmeyer R. Virelizier J.L. Arenzana-Seisdedos F. Hoessli D.C. Bachelerie F. J. Biol. Chem. 2003; 278: 3153-3161Google Scholar, 71Crise B. Rose J.K. J. Biol. Chem. 1992; 267: 13593-13597Google Scholar) and also requires a short cluster of positively charged residues within the cytoplasmic tail (50Popik W. Alce T.M. J. Biol. Chem. 2004; 279: 704-712Google Scholar). HIV-1 Env glycoproteins are palmitoylated on two intracellular cysteines (56Rousso I. Mixon M.B. Chen B.K. Kim P.S. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 13523-13525Google Scholar, 72Yang C. Spies C.P. Compans R.W. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 9871-9875Google Scholar). Mutating these residues did not affect cell fusion activity or surface expression of HIV-1 Env, but abrogated raft association, virion incorporation, and viral infectivity. In general, palmitoylation is a reversible and dynamic process. Two major classes of palmitoylthioesterases have been described. One family is lysosomal; the second is cytosolic and removes palmitate moieties from membrane-associated proteins (73Linder M.E. Deschenes R.J. Biochemistry. 2003; 42: 4311-4320Google Scholar). Nef induces CD4 endocytosis and disrupts the association between CD4 and Lck (13Aiken C. Konner J. Landau N.R. Lenburg M.E. Trono D. Cell. 1994; 76: 853-864Google Scholar, 14Schwartz O. Dautry-Varsat A. Goud B. Maréchal V. Subtil A. Heard J.M. Danos O. J. Virol. 1995; 69: 528-533Google Scholar, 74Salghetti S. Mariani R. Skowronski J. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 349-353Google Scholar). Of note, Nef also efficiently binds to a thioesterase enzyme, although the role of this enzyme in Nef function remains unknown (75Liu L.X. Margottin F. Le Gall S. Schwartz O. Selig L. Benarous R. Benichou S. J. Biol. Chem. 1997; 272: 13779-13785Google Scholar, 76Cohen G.B. Rangan V.S. Chen B.K. Smith S. Baltimore D. J. Biol. Chem. 2000; 275: 23097-23105Google Scholar). The central role of rafts in many cellular events parallels the multiple activity of Nef on cellular trafficking, signal transduction pathways, and viral replication. We therefore asked whether rafts intervene in the effects of Nef on CD4 down-regulation, and examined whether the down-regulating activity of Nef on CD4 is associated with a modification of the palmitoylation state of CD4. We also asked whether Nef improves viral infectivity by increasing the association of Gag with detergent-resistant membranes. We report here that Nef-induced CD4 down-regulation is independent of CD4 palmitoylation or raft association. We also show that Nef is associated at rather low levels with rafts. We have designed a palmitoylated Nef mutant, which strongly accumulates within rafts. However, targeting Nef to rafts does not improve CD4 or MHC-I down-regulation and does not facilitate viral replication in non-activated primary lymphocytes. Furthermore, Nef does not modify the raft segregation of Env and Gag proteins. Therefore, rafts are not a key element involved in the effects of Nef on trafficking of cellular proteins and on viral replication. Cells, Viruses, and Infections—A2.01 T cells, Jurkat T cells, and human peripheral blood mononuclear cells (PBMCs) were cultured in RPMI 1640 with Glutamax (Invitrogen) supplemented with 10% fetal calf serum (Sigma) and penicillin-streptomycin (100 IU/ml each, Sigma). A2.01 cells expressing WT and mutant CD4 have been described (49Percherancier Y. Lagane B. Planchenault T. Staropoli I. Altmeyer R. Virelizier J.L. Arenzana-Seisdedos F. Hoessli D.C. Bachelerie F. J. Biol. Chem. 2003; 278: 3153-3161Google Scholar). HeLa cells were maintained in Dulbecco's modified Eagle's medium with Glutamax supplemented with 10% fetal calf serum and penicillin-streptomycin. PBMCs were isolated from healthy donors using Ficoll-Hypaque (Amersham Biosciences) density gradient centrifugation. The production and use of WT and mutant HIV (from NL4.3 or NLAD8 strains) have been described (77Maréchal V. Clavel F. Heard J.M. Schwartz O. J. Virol. 1998; 72: 2208-2212Google Scholar, 78Petit C. Buseyne F. Boccaccio C. Abastado J.P. Heard J.M. Schwartz O. Virology. 2001; 286: 225-236Google Scholar). For HIV replication experiments, freshly prepared PBMCs were exposed to the indicated virus stocks (viral input ranging from 20 to 0.02 ng of p24/106 cells) for 2 h 30 min, washed twice with phosphate-buffered saline, and cultured in 96-well plates (2 × 105 cells/well, in triplicates). Four days after infection, cells were activated by phytohemagglutinin (PHA, 1 μg/ml, Abbott) and grown in the presence of recombinant interleukin 2 (IL-2, 50 international units/ml, Chiron). Alternatively, HIV replication experiments were performed with PBMC activated (with PHA and IL-2) 3 days prior to infection. Viral replication was monitored by measuring HIV-1 Gag p24 release in supernatants by ELISA (PerkinElmer Life Sciences). Jurkat cells (30 × 106 cells per point) were infected with the indicated virus stocks, (50 ng of p24/106 cells), washed with phosphate-buffered saline, and cultured for 1 or 2 days before proceeding with further experiments. Single cycle infections of P4C5 cells were performed as described earlier (77Maréchal V. Clavel F. Heard J.M. Schwartz O. J. Virol. 1998; 72: 2208-2212Google Scholar). Viral infectivity was measured 24 h after viral exposure. Design and Use of Plasmids and Lentiviral Vectors—Nef WT and Nef mock plasmids carry the nef gene (from the HIV LAI isolate) under the control of the cytomegalovirus promoter, in a sense and antisense orientation, respectively (19Le Gall S. Erdtmann L. Benichou S. Berlioz-Torrent C. Liu L.X. Benarous R. Heard J.M. Schwartz O. Immunity. 1998; 8: 483-495Google Scholar). pHR′Nef lentiviral vector encoding the HIV LAI (also termed R7) nef gene (under the control of the elongation factor-1α promoter) was a kind gift from Didier Trono (41Wang J.K. Kiyokawa E. Verdin E. Trono D. Proc. Nat. Acad. Sci. U. S. A. 2000; 97: 394-399Google Scholar). nef G3C and G2A mutants were generated using the QuickChange kit (Stratagene). Mutations were introduced in Nef WT plasmid, in pHR′Nef vector, or in pNL4.3 and pNLAD8 proviruses. The accuracy of the mutations was verified by sequencing. The CD4 LL/AA mutant was introduced in the pTRIP CD4 lentiviral vector (49Percherancier Y. Lagane B. Planchenault T. Staropoli I. Altmeyer R. Virelizier J.L. Arenzana-Seisdedos F. Hoessli D.C. Bachelerie F. J. Biol. Chem. 2003; 278: 3153-3161Google Scholar). Lentiviral vector particle production and transduction of A2.01 CD4 T-cells or Jurkat T cells were performed as described (11Sol-Foulon N. Moris A. Nobile C. Boccaccio C. Engering A. Abastado J.P. Heard J.M. van Kooyk Y. Schwartz O. Immunity. 2002; 16: 145-155Google Scholar, 41Wang J.K. Kiyokawa E. Verdin E. Trono D. Proc. Nat. Acad. Sci. U. S. A. 2000; 97: 394-399Google Scholar, 49Percherancier Y. Lagane B. Planchenault T. Staropoli I. Altmeyer R. Virelizier J.L. Arenzana-Seisdedos F. Hoessli D.C. Bachelerie F. J. Biol. Chem. 2003; 278: 3153-3161Google Scholar). The stable integration of the vector into the host DNA allows efficient and long term transgene expression, without selection of cell clones. HeLa cells were cotransfected with Nef, CD4 or HLA-A2 and GFP expression vectors as described (19Le Gall S. Erdtmann L. Benichou S. Berlioz-Torrent C. Liu L.X. Benarous R. Heard J.M. Schwartz O. Immunity. 1998; 8: 483-495Google Scholar). Flow Cytometry and Immunofluorescence Analysis—A2.01 and Jurkat cells were stained with anti-CD4 (SK3-PE, BD Biosciences) and anti-MHC-I (W6.32-FITC, Sigma) mAbs, 6 days after transduction with lentiviral vectors. Anti-Gag staining was performed on permeabilized HIV-infected Jurkat cells using an anti-p24 mAb (KC57-RDE, Beckman Coulter). HeLa cells were stained with anti-CD4 (SK3-PE) or with anti-HLA-A2 (BB7.2) (19Le Gall S. Erdtmann L. Benichou S. Berlioz-Torrent C. Liu L.X. Benarous R. Heard J.M. Schwartz O. Immunity. 1998; 8: 483-495Google Scholar) mAbs, 24 h after transfection. Surface levels of CD4 and HLA-A2 were measured in GFP+ cells, which represented the fraction of the cell population (∼30–50% of cells) that was transfected. Cells were analyzed by flow cytometry with a FACScalibur apparatus (BD Biosciences). Isotype-matched mAbs were used as negative controls in all experiments. For immunofluorescence studies, Jurkat cells were fixed, permeabilized, and stained with anti-Nef mAb (MATG020) as described (19Le Gall S. Erdtmann L. Benichou S. Berlioz-Torrent C. Liu L.X. Benarous R. Heard J.M. Schwartz O. Immunity. 1998; 8: 483-495Google Scholar). Confocal microscopy was performed on a Leica TCS4D instrument. Series of optical sections at ∼0.5-μm intervals were recorded and mounted using Adobe Photoshop software. Detergent-resistant membranes (DRMs) isolation. DRMs were obtained by sucrose flotation after Triton X-100 cell lysis as previously described (49Percherancier Y. Lagane B. Planchenault T. Staropoli I. Altmeyer R. Virelizier J.L. Arenzana-Seisdedos F. Hoessli D.C. Bachelerie F. J. Biol. Chem. 2003; 278: 3153-3161Google Scholar). Briefly, 30 × 106 cells were washed twice in ice-cold TKM buffer (50 mm Tris, pH 7.4, 25 mm KCL, 5 mm MgCl2, 1 mm EGTA) containing phosphatase and protease inhibitors (Roche Applied Science). Cells were then incubated for 1 h on ice in TKM containing 1% Triton X-100 (v/v), in a final volume of 0.375 ml. Cell lysates were loaded onto a 5-ml discontinuous sucrose gradient (5 to 35–40% in w/v) and then centrifuged at equilibrium for 18 h at 4 °C and 200,000 × g (Beckman L70-ultracentrifuge). Fractions were collected from the top of the gradient, and their protein contents were estimated using the Nano-Orange quantification kit (Molecular Probes). Immunoblotting and Immunoprecipitation—Equal volumes of each fraction of the gradient were analyzed by SDS-PAGE using a 4–12% NuPage Gel (Invitrogen) under reducing (for the analysis of CD4, p56Lck, Gag, Env, and Nef) or non-reducing (for the analysis of CD46) conditions. For immunodetection, the following antibodies were used: CD4 (1F6, Novocastra), p56Lck (sc-13, Santa Cruz Biotechnology), CD46 (J4–48 Immunotech), Gag p24 and p17 (25A+18A mAbs) (79Petit C. Schwartz O. Mammano F. J. Virol. 1999; 73: 5079-5088Google Scholar), Env gp120 and gp41 (160A+41A+110H mAbs Hybridolabs, Institut Pasteur, Paris), Nef (MATG020, Ref. 19Le Gall S. Erdtmann L. Benichou S. Berlioz-Torrent C. Liu L.X. Benarous R. Heard J.M. Schwartz O. Immunity. 1998; 8: 483-495Google Scholar). Immobilized antigen-antibody complexes were detected with secondary IgG-horseradish peroxidase conjugates (Pierce), revealed using enhanced chemiluminescence (ECL+, Amersham Biosciences), and quantified using an electronically cooled LAS-1000 plus charge-coupled device (CCD) camera system (Image Gauge 3.4 software, Fuji Photo Film Co., Tokyo, Japan). Ganglioside GM1 detection by slot-blot was performed using peroxidase-coupled cholera toxin (Sigma). [3H]palmitate or [35S]Met/Cys metabolic labeling were performed as previously described (49Percherancier Y. Lagane B. Planchenault T. Staropoli I. Altmeyer R. Virelizier J.L. Arenzana-Seisdedos F. Hoessli D.C. Bachelerie F. J. Biol. Chem. 2003; 278: 3153-3161Google Scholar). Briefly, radiolabeled cells were lysed in TKM buffer containing 1% Triton (w/v), and CD4 and Nef were immunoprecipitated using OKT4 and Nef MATG 020 mAbs, respectively. Detection of [3H]palmitate was performed using the enhanced autoradiography kit (EABiotech). Non-palmitoyated CD4 Is Efficiently Down-regulated by Nef—Preferential lipid raft localization of Nef (41Wang J.K. Kiyokawa E. Verdin E. Trono D. Proc. Nat. Acad. Sci. U. S. A. 2000; 97: 394-399Google Scholar, 80Walk S.F. Alexander M. Maier B. Hammarskjold M.L. Rekosh D.M. Ravichandran K.S. J. Virol. 2001; 75: 834-843Google Scholar) and CD4 (49Percherancier Y. Lagane B. Planchenault T. Staropoli I. Altmeyer R. Virelizier J.L. Arenzana-Seisdedos F. Hoessli D.C. Bachelerie F. J. Biol. Chem. 2003; 278: 3153-3161Google Scholar, 50Popik W. Alce T.M. J. Biol. Chem. 2004; 279: 704-712Google Scholar, 81Cinek T. Hilgert I. Horejsi V. Immunogenetics. 1995; 41: 110-116Google Scholar) suggested that Nef-induced CD4 down-regulation may be linked to distribution of both molecules within these membrane microdomains. To test this hypothesis, we first studied the sensitivity to Nef of CD4 mutants, which were targeted to non-raft domains. Both CD4 palmitoylation and its association with p56Lck contribute to the presence of CD4 in rafts (49Percherancier Y. Lagane B. Planchenault T. Staropoli I. Altmeyer R. Virelizier J.L. Arenzana-Seisdedos F. Hoessli D.C. Bachelerie F. J. Biol. Chem. 2003; 278: 3153-3161Google Scholar, 50Popik W. Alce T.M. J. Biol. Chem. 2004; 279: 704-712Google Scholar, 70Fragoso R. Ren D. Zhang X. Su M.W. Burakoff S.J. Jin Y.J. J. Immunol. 2003; 170: 913-921Google Scholar, 81Cinek T. Hilgert I. Horejsi V. Immunogenetics. 1995; 41: 110-116Google Scholar). Palmitoylation of CD4 occurs on two cysteine residues (Cys-419 and Cys-422, Fig. 1A) located in the membrane-proximal region of the CD4 cytoplasmic tail (49Percherancier Y. Lagane B. Planchenault T. Staropoli I. Altmeyer R. Virelizier J.L. Arenzana-Seisdedos F. Hoessli D.C. Bachelerie F. J. Biol. Chem. 2003; 278: 3153-3161Google Scholar, 71Crise B. Rose J.K. J. Biol. Chem. 1992; 267: 13593-13597Google Scholar). We previously described the design of A2.01 T cells stably expressing either CD4 WT or CD4 palm– (49Percherancier Y. Lagane B. Planchenault T. Star

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