HIV-1 Entry into T-cells Is Not Dependent on CD4 and CCR5 Localization to Sphingolipid-enriched, Detergent-resistant, Raft Membrane Domains*

Yann PercherancierDagger §, Bernard LaganeDagger §, Thierry PlanchenaultDagger , Isabelle StaropoliDagger , Ralf AltmeyerDagger , Jean-Louis VirelizierDagger , Fernando Arenzana-SeisdedosDagger , Daniel C. Hoessli, and Françoise BachelerieDagger ||

From the Dagger  Unité d'Immunologie Virale, Institut Pasteur, Paris 75724, France and the  Department of Pathology, Centre Medical Universitaire, CH-1211, Geneva 4, Switzerland

Received for publication, July 23, 2002, and in revised form, October 30, 2002

    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The contribution of raft domains to human immunodeficiency virus (HIV) 1 entry was assessed. In particular, we asked whether the CD4 and CCR5 HIV-1 receptors need to associate with sphingolipid-enriched, detergent-resistant membrane domains (rafts) to allow viral entry into primary and T-cell lines. Based on Triton X-100 solubilization and confocal microscopy, CD4 was shown to distribute partially to rafts. In contrast, CCR5 did not associate with rafts and localized in nonraft plasma membrane domains. HIV-1-receptor partitioning remained unchanged upon viral adsorption, suggesting that viral entry probably takes place outside rafts. To directly investigate this possibility, we targeted CD4 to nonraft domains of the membrane by preventing CD4 palmitoylation and interaction with p56lck. Directed mutagenesis of both targeting signals significantly prevented association of CD4 with rafts, but did not suppress the HIV-1 receptor function of CD4. Collectively, these results strongly suggest that the presence of HIV-1 receptors in rafts is not required for viral infection. We show, however, that depleting plasma membrane cholesterol inhibits HIV-1 entry. We therefore propose that cholesterol modulates the HIV-1 entry process independently of its ability to promote raft formation.

    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Numerous studies dealing with biological membrane organization and composition have emphasized the nonrandom distribution of lipids and proteins into distinct membrane domains (1). Domains composed of cholesterol and saturated lipids, e.g. sphingolipids, or rafts have recently been shown to support a wide range of cellular events, including signal transduction, sorting, and cellular trafficking of proteins and lipids, as well as pathogen entry into cells (2). Nonionic detergent insolubility of these domains at 4 °C was found to result from tight packing of cholesterol and sphingolipids in a liquid-ordered state (3). This property allows recovery of rafts as low-density, floating membranes by gradient centrifugation and makes it possible to characterize raft lipids and proteins (4).

Entry of human immunodeficiency virus type 1 (HIV-1)1 into host cells relies primarily upon interaction of the viral glycoprotein envelope (Env) gp120 subunit with cell surface CD4 (5). Conformational changes of gp120 upon CD4 binding trigger interactions of the Env with HIV-1 coreceptors CCR5 or CXCR4 (6). Subsequently, this binding to coreceptors exposes the Env gp41 transmembrane subunit and promotes fusion of viral and cellular membranes (7). It has been proposed that oligomeric assembly of Env proteins (8, 9) facilitates the recruitment of viral receptor (CD4 and coreceptors) molecules and ultimately HIV-1 entry (10). Because a correlation between the cell surface density of HIV-1 receptors and efficiency of infection has been emphasized (10), the clustering of CD4 and the coreceptor in delimited plasma membrane domains would be expected to favor HIV-1 entry.

The CD4 antigen is among the few membrane-spanning proteins found to partition into raft domains enriched in sphingolipids (e.g. the GM1 ganglioside, a prototypic marker of these domains) (11-13). To establish the contribution of rafts to HIV-1 entry, inhibition of glycosphingolipid synthesis (14, 15) and depletion of cell plasma membrane cholesterol have been investigated (16-18). The inhibition of HIV-1 infection was shown in both instances and believed to result from disruption of raft integrity. However, considering that inhibition of glycosphingolipids synthesis is not detrimental to raft domain formation (19), and that cholesterol is distributed throughout plasma membranes (20-22), inhibition of HIV-1 entry following these two means of lipid perturbation (14-17) cannot be attributed solely to disruption of rafts.

In the present work, a requirement for an association of CCR5 and CD4 with rafts to support HIV-1 entry was specifically addressed. A large body of evidence points to fatty acylation (specially, post-translational palmitoylation of cysteine residues) as a critical signal for targeting several inner leaflet signaling proteins to rafts (23) and for a few raft-seeking transmembrane proteins (24-26). It is conceivable that C-terminal palmitoylation of CD4 (27) might account for localization of this protein to rafts. This prompted us to investigate whether interference with CD4 palmitoylation would alter the distribution of the CD4 receptor to rafts. Such a noninvasive approach led us to further investigate if CD4 maintains its HIV-1 receptor function when localized outside rafts.

Here we show that palmitoylation of CD4 and its interaction with the tyrosine kinase p56lck are important for the distribution of CD4 to rafts. In contrast to CD4, we present evidence that the CCR5 coreceptor preferentially partitions to nonraft domains, despite its palmitoylation on three C-terminal cysteine residues (28, 29), both in T-cell lines and primary T-cells. Importantly, when predominantly redistributed to nonraft domains, CD4 still displays full receptor function for monocytotropic (R5)-HIV-1 strains. Together, our results indicate that CCR5-dependent HIV-1 infection does not depend upon the presence of CD4 and CCR5 receptors in rafts. Furthermore, we show that depleting plasma membrane cholesterol in target cells inhibits viral entry, suggesting that cholesterol-dependent membrane properties other than raft formation come into play to promote efficient HIV-1 infection.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Cell Cultures and Primary T-cell Blast Preparation-- T-cell lines were grown in RPMI medium (Invitrogen), supplemented with 10% fetal calf serum (FCS) and 100 units/ml penicillin, 100 µg/ml streptomycin (growth medium). Human embryonic kidney cells (HEK-293T), baby hamster kidney cells (BHK), and human HeLa cells were cultured in Dulbecco's modified Eagle's growth medium with the same additives. A3.01, a CD4 hypoxanthine/aminopterin/thymidine-sensitive variant of the CEM T-cell line (30) and its derivatives, CD4- (A2.01) and CCR5+ (A2.01R5, A3.01R5) cells, were obtained from H-T He (Centre d'Immunologie INSERM/CNRS de Marseille-Luminy, France). Stable ectopic CCR5 expression was maintained by culturing CCR5+ T-cell lines in RPMI growth medium supplemented with 1 mg/ml geneticin (G418) (Roche Molecular Biochemicals). Both A3.01 and A2.01 T-cell lines have a defect in cholesterol biosynthesis and accumulate lanosterol, a cholesterol precursor, in FCS-depleted growth medium (31). However, we confirmed others' data (31) that in 10% FCS growth medium, the sterol contained in plasma membranes of these cells is mainly cholesterol, as assessed by thin layer chromatography (TLC) (data not shown). Human peripheral blood mononuclear cells were isolated from healthy donors using Ficoll-Hypaque (Amersham Biosciences AB) density gradient centrifugation. For lymphocyte experiments, freshly prepared peripheral blood mononuclear cells at 2-3 × 106 cells per ml were cultured in growth medium containing phytohemagglutinin (Sigma) at 1 mg/ml for 3 days and afterward growth medium was supplemented with recombinant interleukin 2 at 150 units/ml (Chiron) for 2 weeks. Cells were mostly CD14 negative and more than 70% CD4 positive, as assessed by FACscan analysis (results not shown).

Treatment of Cells with Methyl-beta -cyclodextrin-- In cholesterol extraction experiments, 30 × 106 cells were rinsed once with phosphate-buffered saline and incubated for 30 min with (cholesterol-depleted cells) or without (untreated cells) 5 mM methyl-beta -cyclodextrin (MBCD, Sigma) in 25 ml of RPMI medium supplemented with 1% FCS. Experiments were carried out at 37 °C under continuous, gentle stirring. Cells were washed twice with a Hepes-based saline buffer (Hepes 20 mM, NaCl 150 mM). To restore the cholesterol content of cholesterol-depleted cells, cholesterol-methyl-beta -cyclodextrin inclusion complexes were first prepared by adding small aliquots of a 180 mM propanol-2 solution of cholesterol to an aqueous solution of MBCD (1 g/10 ml). This was performed at 50-80 °C under continuous shaking up to a 1/10 MBCD to cholesterol final molar ratio. This cholesterol-MBCD stock solution was further diluted in RMPI medium supplemented with 1% FCS to yield a 2.6 mM MBCD solution, stirred for 40 min at 40 °C, then filtered through a Millipore filter (0.22 µm) prior to use. Loading of cholesterol was achieved following incubation of cholesterol-depleted cells for 30 min in a 25-ml final volume of the latter filtered solution. This procedure was performed at 37 °C under continuous gentle stirring. Cells were then rinsed twice (20 mM Hepes, 150 mM NaCl). The amount of cholesterol in treated cells was normalized to both their protein and phospholipid contents estimated in the whole cell extracts and in some cases in plasma membranes extracted according to Refs. 31 and 32. Protein concentration was determined with the bicinchoninic acid protein assay reagent (Pierce) with bovine serum albumin as a standard. Total lipids were extracted according to Bligh and Dyer (33). The amount of phospholipid was estimated by a phosphate assay after total digestion in the presence of perchloric acid (34). Cholesterol was quantified using a colorimetric method based on the oxidation of the hydroxyl group at the carbon atom 3 in the beta -position (Roche Molecular Biochemicals).

HIV-1 Infection, Luciferase Assays, and Real-time Quantitative PCR (Taq-Man)-- HIV-1 particles were produced and their concentration estimated by measuring both the HIV-1 Gagp24 antigen (enzyme-linked immunosorbent assay detection kit (PerkinElmer Life Sciences)) and the beta -galactosidase activity in infected HeLa CD4LTRLacZ indicator cells, as previously described (29). Briefly, HEK-293T cells were transiently transfected either with the WT R5-HIV-1YU2 proviral DNA or the R5-HIV-1JR-CSF-luc, which carries the firefly luciferase (luc) reporter gene instead of nef (a gift from Dr. V. Planelles, University of Utah School of Medicine, Salt Lake City, UT), or were co-transfected with an HIV-1pNL4-3-luc envelope-deficient (env(-)), proviral DNA containing luc instead of nef and the R5-HIV-1-Ba-L Env-expressing vector. Luciferase activity was measured at the times indicated in the figure legends using a luminometer as described (29). Infection of T-cells with the indicated HIV-1 Gagp24 quantities is detailed in the figure legends. Genomic and HIV-1 reverse-transcribed DNAs were extracted 4 h post-infection after extensive cell washes, using a DNA isolation kit according to the manufacturer's indications (Roche Molecular Biochemicals). HIV-1 primers and probe were designed to detect reverse-transcribed HIV-1 DNA by real-time quantitative PCR using the Taq-Man procedure. These were chosen according to Zack et al. (35) to detect either all HIV-1 reverse-transcribed DNA (R/U5 region of the long terminal repeat) or the full-length DNA (long terminal repeat/gag) and modified according to the Primer ExpressedTM program (PerkinElmer Life Sciences). The forward primer was the same for the both sets: 5'-GCTAGCTAGGGAACCCACTGCT-3'. Reverse primers were 5'-CACTGCTAGAGATTTTCCACACTGAC-3' and [5'-GTCCTGCGTCGAGAGATCTCCT-3' mapping to the R/U5 and R/gag regions, respectively. The probe 5'-FAM-CCTCAATAAAGCTTGCCTTGAGTGCTTCA-TAMRA-3' carried the 6-carboxyfluorescein (FAM) fluorescent dye and the 6-carboxytetramethylrhodamine (TAMRA) quencher at the 5' and 3' ends, respectively. Quantification of HIV-1 DNA was normalized according to the amount of genomic DNA by measuring the alpha -L-iduronidase cellular gene with 5'-ACTTGGACCTTCTCAGGGAGAAC-3' and 5'-CACCTGCTTGTCCTCAAAGTCA-3' as forward and reverse primers, respectively, and the probe 5'-FAM-CAGCGCCTCGGGCCACTTCA-TAMRA-3' (a gift from Dr. J-M. Heard, Unité des Rétrovirus et Transfert Génétique, Institut Pasteur, Paris). Amplification was performed in a total volume of 50 µl containing 2 µl of DNA solution, 1 µM probe, 50 nM of each primer, and 25 µl of Taq-Man Universal PCR Mastermix Mix (PerkinElmer Life Sciences). Amplification and fluorescence detection were conducted in a 5700 Sequence Detector (PerkinElmer Life Sciences) with a program including first, 2 min at 50 °C and 10 min at 95 °C then 40 cycles, each one consisting of 15 s at 95 °C followed by 1 min at 60 °C. Intensity was related to the initial number of DNA copies. Standards, consisting of HIV-1YU2 and human alpha -L-iduronidase cDNA-containing plasmids (seven dilutions from 4 to 4 × 105 and 38 to 6 × 105 copies, respectively), and an HIV-1 minus template control (four dilutions of noninfected cells DNA, in ng: 100, 20, 2, and 0.2) were included in each assay, which were performed in duplicate. Copy numbers of alpha -L-iduronidase gene and HIV-1 genomes were determined for the four dilutions of DNA samples and by reporting the Ct (the minimum cycle number at which fluorescence was detected) for each assay on the respective standard curves. Mean quantities and standard deviation over the four dilutions of DNA samples were then determined.

Detergent-resistant Membranes (DRMs) Isolation-- DRMs were obtained by sucrose floatation after Triton X-100 cell lysis according to Ref. 36. Briefly, 30 or 60 × 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 a mixture of phosphatase (5 mM NAF, 10 mM p-nitrophenyl phosphate, 10 mM beta -glycerophosphate, 1 mM orthovanadate, 200 nM okadaic acid) and protease (Roche Molecular Biochemical) inhibitors. Cells were then incubated for 1 h on ice in TKM containing 1% Triton X-100 (v/v). Cell lysates were loaded on a sucrose step gradient (in w/v: 5-35 to 40%, 5- or 11-ml gradients for 30 or 60 × 106 cell, respectively) and were centrifuged to equilibrium for 20 h at 4 °C and 200,000 × g (Beckman L70 ultracentrifuge). Fractions were collected from the top of the gradient and protein contents were estimated using the NanoOrangeTM quantification kit (Molecular Probes).

Immunoblotting and Immunoaffinity Purification-- Equal volumes of each gradient fraction were resolved by SDS-PAGE using a 4-12% NuPage (Invitrogen) in reducing (CD4, CCR5, p56lck, and LAT) or nonreducing (CD46 and CD55) conditions. For immunodetection, the following antibodies were used: CD4 (1f6, Novocastra), CCR5 (MC5, a gift from Dr. M. Mack, Medical Policlinic, University of Munich, Germany), p56lck (sc-13, Santa Cruz), LAT (Transduction Laboratories), CD46 (J4-48 Immunotech), and CD55 (sc-9156 Santa Cruz). Immobilized antigen-antibody complexes were detected with secondary horseradish peroxidase-conjugated anti-species IgG (Pierce), developed by enhanced chemiluminescence (ECL+, Amersham Biosciences), and quantified using a LAS-1000 CCD camera (Image Gauge 3.4 software, Fuji Photo Film Co., Tokyo, Japan). GM1 ganglioside was detected by slot-blot using peroxidase-coupled choleratoxin (CTx) (Sigma). For immunoprecipitation, DRM fractions (pooled fractions 3-5) were centrifuged at 150,000 × g for 1 h at 4 °C, pellets were resuspended in 50 µl of SDS (2% w/v) for 15 min at room temperature, then diluted with 450 µl of TKM containing 1% Triton X-100 before adding p56lck or CD4 antibodies (OKT4, a gift from Dr. F. Lemonnier, Unité d'Immunité Cellulaire Antivirale, Institut Pasteur, Paris) and overnight incubation at 4 °C. Protein G/A-Sepharose-coupled beads (Calbiochem) were added for 2 h at 4 °C and immunoprecipitates were resolved by SDS-PAGE. [14C]Palmitate and [35S]Met metabolic labeling were performed as previously described (29). Radiolabeled cells were lysed in TKM buffer containing 1% Brij 96 (w/v) and CD4 was immunoprecipitated using OKT4 antibody. Where indicated, cells were incubated in the presence of 2-bromopalmitate (2-BP) (100 µM) for 16 h throughout the labeling time.

Immunofluorescence Microscopy-- A3.01 cells (1 × 105) were treated to aggregate GM1 as described (26). Briefly, tetramethylrhodamine isothiocyanate-labeled CTx (Interchim) was applied for 30 min at 4 °C, followed after washing by an anti-CTx monoclonal antibody (Sigma) for 30 min at 4 °C, then for 15 min at 37 °C. After three washes in 0.5% bovine serum albumin-containing RPMI (blocking buffer), cells were fixed for 10 min in 4% paraformaldehyde in phosphate-buffered saline. Cells were then incubated in phosphate-buffered saline containing 0.1 M glycine in blocking buffer for 30 min and fluorescein isothiocyanate-conjugated anti-human-CD4 (SK3, BD Pharmingen) or -CCR5 (2D7, BD Pharmingen) for 30 min. Coverslips were mounted in Mowiol (Hoechst) and fluorescence observed with a confocal laser microscope (Leica TCS4D instrument) using a PL APO ×63 oil immersion objective.

Plasmid Constructs and Stable Expression in T-cell Lines-- CD4 cDNA carrying mutations in cysteines (Cys) interacting with p56lck (Cys445-Ser/Cys447-Ser) (CD4 Lck-) was a gift from Dr. M. Marsh (MRC Laboratory for Molecular Cell Biology, University College, London, UK). Mutagenesis by PCR of the WT- or -Lck- CD4 cDNAs was performed to substitute Cys residues at position 419 and 422 with Ala residues (Palm- and Palm-Lck-, respectively) using overlap extension with T7, Sp6, and two internal primers containing the mutation, as described previously (29). The forward and reverse primers were, respectively: 5'-GCTGTCAGGGCCCGGCACCGAAGGCGCCAAGCAGAG-3' and 5'-GGCCCTGACAGCGAAGAAGATGCCTAGCCCAATGAAAAG-3'. The entire coding region of each construct (Palm-, Lck-, or Palm-Lck-) was confirmed by sequence analysis, before being cloned into the HIV-1-based lentiviral pTRIP vector (a gift from Dr. P. Charneau, Groupe de Virologie moléculaire et de Vectorologie, Institut Pasteur, Paris). The efficiency of the pTRIP vector relies on the presence of a triple-stranded DNA structure that acts as a cis-determinant of HIV-1 DNA import (37). Stable integration of the transgene into the host DNA permits efficient and long term transgene expression without clone selection. Virus stock production and infection were as described in Ref. 37 to transduce and express CD4 variants (WT, Palm-, Lck-, or Palm-Lck-) in the A2.01 CD4- T-cell line. Briefly, virus particles were produced after transient co-transfection of HEK-293T cells using a standard calcium phosphate method, with the p8.91 encapsidation plasmid, the pHCMV-G vector encoding the vesicular stomatitis virus envelope and the pTRIP vector encoding CD4 variants (WT, Palm-, Lck-, or Palm-Lck-). Viral titers were estimated as described above.

Fluorescence-based Assay of Syncitia Induced by HIV-1 Envelope-- The fluorescence-based assay of syncytia formation induced by HIV-1 Env (FLASH method) is described elsewhere (38). Briefly, BHK cells were infected for 8 h using a Semliki forest virus-based strategy permitting surface expression of the R5-HIV-1-BX08 Env. Infected BHK cells were labeled with SNARFTM-1 and A2.01R5 target cells expressing various CD4 variants (WT, Palm-, Lck-, or Palm-Lck-) with CellTrackerTM Green (CMFDA) for 30 min. Both cells were co-cultured in suspension at a 1:5 BHK to A2.01R5 cell ratio for 12 h at 37 °C. Fused cells were detected by FACscan analysis as doubly labeled syncitia, using CellQuest software. The dependence of this fusion assay upon the interaction between the HIV-1 Env and receptors was assessed using the following inhibitors, added at the start of the co-cultures: TAK779 (200 nM) (NIH AIDS reagent program) and AMD3100 (200 ng/ml) (AnorMed, Langley, Canada) that bind to CCR5 and CXCR4, respectively, and the CD4 monoclonal antibody, Q4120, that inhibits CD4-dependent fusion (5 µg/ml) (MRC AIDS reagent program).

    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Role of Plasma Membrane Cholesterol in HIV-1 Entry-- The effect of the modulation of plasma membrane cholesterol on HIV-1 entry was assessed using real-time quantitative PCR in a CD4 lymphoid T-cell line (A3.01) that stably expressed a functional CCR5 transgene (A3.01R5) and in interleukin-2-expanded primary T-cells that are mostly CD4+. Measurement of newly reverse-transcribed HIV-1 proviral DNA reflects the extent of viral fusion and internalization into target cells.

Modifying the membrane cholesterol content was performed using MBCD (39), known to remove plasma membrane sterol more specifically (40). We found that incubating cells with 5 mM MBCD resulted in a decrease in cholesterol to protein ratios: 10.1 ± 2 µg of cholesterol/mg of protein to 4.1 ± 1.5 µg of cholesterol/mg of protein in A3.01R5 cells and 19.5 ± 3 µg of cholesterol/mg of protein to 7.4 ± 0.9 µg of cholesterol per/mg of protein in primary T-cells. Phospholipid to protein ratios were unaltered in cells treated with MBCD (124 ± 18 nmol of phospholipid/mg of proteins) as compared with untreated cells (116 ± 9). The same was observed in plasma membranes of MBCD-treated cells (1470 compared with 1365 nmol of phospholipid/mg of proteins in plasma membrane of untreated cells), thus demonstrating the specificity of this reagent for sterols. We controlled that the cholesterol deficit was maintained throughout the 4-h post-infection experiment and was not detrimental to cell viability or plasma membrane expression of HIV-1 receptors CD4 and CCR5 (data not shown). Plasma membrane cholesterol extraction resulted in a 50 and 60% decrease in newly reverse-transcribed HIV-1-proviral DNA in A3.01R5 and in primary T-cells, respectively, compared with untreated, infected control cells (Fig. 1, left and right panels, respectively). The results were the same using primers designed to detect either all HIV-1 reverse-transcribed DNA (R/U5 region of the LTR) (Fig. 1) or only the full-length reverse-transcribed HIV-1 proviral DNA (R/gag region) (data not shown). The minute amounts of HIV-1 DNA detected in untreated cells infected in the presence of a CD4 monoclonal antibody and the nucleotide analog azidothymidine (alpha CD4 + azidothymidine) indicate that this assay allows detection of newly synthesized viral DNA. We observed that re-loading cholesterol (cholesterol/protein ratios = 22.1 ± 5 and 40.3 µg/mg, respectively, in cholesterol-loaded A3.01R5 and primary T-cells) restored the capacity of cells to support HIV-1 entry. This is illustrated by the increase of proviral DNA to levels close to those detected in untreated infected cells (Fig. 1, MBCD-cholesterol). Such near complete reversion again validates MBCD as a specific and nondeleterious tool. Collectively, these results confirm recent observations regarding the importance of target cell plasma membrane cholesterol in regulating viral entry (16-18) and extend them to primary CD4+ T-cells, the natural and major target of HIV-1. The question remains whether the observed regulatory effect of plasma membrane cholesterol relates to perturbation of raft domain integrity.


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Fig. 1.   Effect of cholesterol on R5-HIV-1 entry. Untreated or MBCD-treated A3.01R5 and primary T-cells were tested for R5-HIV-1 entry. Depleted cells were cholesterol back-loaded (MBCD-Cholesterol). Cells (5 × 106) were infected for 4 h with HIV-1YU2 (75 or 300 ng of HIV-1 Gagp24 for A3.01 and primary T-cells, respectively). Untreated control cells were also infected in the presence of CD4 monoclonal antibody Q4120 (5 µg/ml) and the nucleoside analog azidothymidine (AZT) (50 µM) (alpha CD4 + AZT) throughout infection. Following infection, genomic and reverse-transcribed HIV-1 DNAs were quantified by fluorescence-monitored, real-time PCR analysis. Values calculated as number of HIV-1 genomes/cell genome are expressed relative to values obtained for untreated cells (taken as 100%).

CD4 but Not CCR5 Localizes in Rafts of T Lymphocytes-- The insolubility of some lipids and proteins in cold nonionic detergent Triton X-100 correlates well with their partitioning into ordered phases of biological membranes (3, 41). This makes it possible to recover DRM components as low density material after centrifugation to equilibrium on a sucrose density gradient. Accordingly, the partitioning of CD4 and CCR5 was investigated in primary T (Fig. 2A) and A3.01R5 cells (Fig. 2B). Low density fractions of primary T cells (Fig. 2A, fractions 3-5) collected from the top of the gradient were enriched in gangliosides like GM1, as detected by specific binding of the CTx subunit B. By loading equal volumes of each fraction, the relative quantities of the marker studied can be assessed, regardless of the total protein content of each fraction. Indeed, a minor amount of the total protein content was recovered in the low-density fractions and the bulk (90-95%) was present in the fractions of higher density (data not shown and Ref. 42). We found that both the tyrosine kinase p56lck, which is known to associate with the cytoplasmic leaflet (Fig. 2A, Lck), and the exoplasmic CD55 leaflet glycophosphatidylinositol-anchored protein float with insoluble material at the top of the gradient (Fig. 2A, fractions 3-5), as reported by others (43, 44). CD4 was observed to segregate partially within the insoluble low-density fractions (fractions 3-5) (Fig. 2A), along with a marker of these fractions, the LAT transmembrane protein (Fig. 2A) (24). In sharp contrast, CCR5 was fully solubilized in 1% Triton X-100 (Fig. 2A, fractions 9-11). The same was observed for the membrane-spanning protein, CD46 (Fig. 2A), a measle virus receptor also known to associate with Triton X-100-soluble membranes (45).


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Fig. 2.   CCR5 does not localize into rafts in primary T cells, in contrast to CD4. A, primary T-cell lysates (1% Triton X-100) were loaded onto a step sucrose gradient (11 ml). Equal volume fractions were analyzed for the presence of CD4, CCR5, Lck, LAT, CD46, CD55, and GM1. B, A3.01R5 cell were lysed in 1, 0.3, or 0.1% Triton X-100 (TX-100) and loaded onto a small gradient (5 ml). Equal volume fractions were collected and analyzed for the presence of CD4, CCR5, CD46, and GM1. C, A3.01R5 cells were subjected (MBCD-treated cells) or not (untreated cells) to cholesterol depletion. Following an additional incubation of cells for 1 h at room temperature in 1% FCS-supplemented RPMI medium, cells were extracted with 1% Triton X-100 and sucrose gradient fractions were analyzed for the presence of CD4, CCR5, and GM1. D, CD4, CCR5, and CD46 content of low-density fractions 3 to 5 (i.e. DRMs) collected from the gradient shown in B were quantified. Protein enrichment was expressed as % of the quantities present in DRMs fractions over the total protein recovered. Standard deviations from four determinations are shown.

In A3.01R5 cells (Fig. 2B, upper panel), CCR5 partitioning was similar to that of the endogenous CCR5 protein in primary T-cells (Fig. 2A), i.e. CCR5 remained fully solubilized in 1% Triton X-100 in both cell types. Moreover, in both A3.01R5 (Fig. 2B) and A2.01R5 cells transduced with the wild-type (WT) CD4 molecule (Figs. 4 and 5), the relative distribution of CD4 in both low and high density fractions was similar to that observed in primary T-cells (Fig. 2A). One may thus conclude that the behavior of CCR5 and CD4 after stable ectopic expression in T-cell lines is similar to that of their endogenous counterparts in primary T lymphocytes.

The above results agree with previously reported partitioning of CD4 to sphingolipid-enriched, detergent-resistant domains (11-13). This contrasts strongly with the distribution pattern of CCR5, which was recovered in 1% Triton X-100-soluble domains where GM1 remains only marginally localized. Sphingolipids (i.e. glycosphingolipids and sphingomyelin) differ from the other class of membrane lipids, glycerolipids, in that they contain long, highly saturated fatty acyl chains. In appropriate admixture with cholesterol, sphingolipids form detergent-resistant domains in the plasma membrane (i.e. rafts) (46) that are sensitive to cholesterol depletion (22).

The nature of CD4- and GM1-enriched Triton X-100-resistant domains was further characterized following extraction of cholesterol-depleted A3.01R5 cells. As shown in Fig. 2C, decreasing the cholesterol content of plasma membranes resulted in a sharp enrichment of CD4 in 1% Triton X-100-soluble membranes, where CCR5 resides. This correlated with a parallel redistribution of GM1 throughout the sucrose gradient (Fig. 2C). These results illustrate the role that cholesterol plays in structuring the DRM domains where GM1 and CD4 are found. Moreover, compared with published phase diagrams of cholesterol:phospholipid binary mixtures (47), the sterol content found in our isolated Triton X-100-resistant domains (24 mol %) is consistent with the lipid phase described in DRMs (48). These domains are henceforth referred to as rafts, in accordance with the previously proposed nomenclature for DRM domains composed of cholesterol and sphingolipids (49).

Although the above results suggest that CCR5 and CD4 do not colocalize to 1% Triton X-100-resistant rafts in T-cells, the hypothesis of a colocalization to raft domains of lesser detergent resistance was envisaged. This possibility was based on the structural heterogeneity of membrane lipids (including cholesterol) that form a mosaic of domains in membranes (1, 50). The fact that the solubility of membrane proteins depends on the concentration of the nonionic detergent used is in keeping with this postulate (50, 51). We thus investigated the possibility that CCR5 and CD4 colocalize to rafts of lesser detergent resistance. A3.01R5 cells were lysed in decreasing Triton X-100 concentrations. At low Triton X-100 concentrations (0.1%), relatively less CD4 was found in the soluble pool (Fig. 2B, fractions 8-10) and more was recovered from the low-density insoluble fractions (Figs. 2, B (fractions 3-5), and D). In contrast, the bulk of CCR5 always remained soluble and sedimented in the gradient independently of the Triton X-100 concentration (Fig. 2B, middle and lower panels). CCR5 was not detected significantly in 0.3% Triton X-100-resistant membrane domains (Fig. 2B, middle panel, fractions 3-5) and only a minor part was detected in 0.1% Triton X-100-resistant membrane domains (Fig. 2, B, lower panel, fractions 3-5, and D). Moreover, quantification of CCR5 in low density fractions expressed as a function of Triton X-100 concentration revealed a distribution (Fig. 2D) identical to that of CD46 (Fig. 2, B and D), which is representative of Triton X-100-soluble membrane proteins (45). These observations further support our previous conclusion that CCR5 is excluded from rafts and highlight that only a very small amount of the receptor is found in membrane domains of lesser detergent resistance.

To rule out that CCR5 might transiently distribute into CD4-containing rafts, we forced stabilization of these domains into visible patches by specific clustering of GM1 by cross-linking with CTx. The coalescence of influenza hemagglutinin- and GM1-containing domains following GM1-CTx cross-linking was taken to reflect the propensity of hemagglutinin to localize to GM1-enriched rafts (26). Prior to CTx cross-linking, both CD4 and CCR5 receptors were found evenly distributed over the plasma membrane (Fig. 3, A and B, left panels). Cross-linking of GM1-CTx complexes with a rhodamine-labeled CTx antibody induced redistribution of CD4 into patches (Fig. 3A, second panel) that overlapped GM1 patches (Fig. 3A, GM1 and overlay panels). In marked contrast, CCR5 distribution remained unchanged and did not significantly overlap with GM1-patches following CTx-dependent cross-linking (Fig. 3B, GM1 and overlay panels). This clearly shows that CCR5 is mainly excluded from GM1-enriched domains and behaves differently from CD4 in this respect.


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Fig. 3.   Patching of GM1 with cholera toxin does not induce clustering of CCR5, in contrast to CD4. Aggregation of GM1 clusters was initiated by incubation of the rhodamine-labeled CTx labeled A3.01R5 cells with CTx antibody (GM1). CD4 (A) and CCR5 (B) staining before (left-hand panels) and after GM1 clustering (GM1-CTx-cross-linking) are shown. The merge of GM1 images with those of CD4 (A) or CCR5 (B) are also presented (overlay).

As concerns the requirement of HIV-1 entry on rafts, several possibilities remain. It was proposed that HIV-1 binding to CD4 might occur within rafts and that, subsequently, CD4-Env complexes redistribute to coreceptor-containing nonraft domains (52). Alternately, supramolecular complexes of CD4, the viral Env, and the coreceptor (53-58) could dock in raft domains (16-18). To test these possibilities, we investigated whether receptor cross-linking induced by R5-HIV-1 Env caused membrane redistribution of either CD4 or CCR5. A2.01R5 CD4 WT cells, exposed to R5-HIV-1-Ba-L virions for 45 or 90 min, were lysed in 1% Triton X-100, and fractioned on a sucrose gradient. As shown in Fig. 4 adsorption of virions onto these cells did not modify CCR5 or CD4 distribution compared with uninfected control cells (Fig. 4).


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Fig. 4.   R5-HIV-1 binding does not redistribute CD4 and CCR5. Binding of R5-HIV-1-Ba-L to A2.01-CD4R5 cells (24 µg of Gagp24/60 × 106 cells) was performed at 4 °C for 45 min, then at 37 °C for an additional 45 or 90 min accordingly to Ref. 54. Uninfected control cells were treated in parallel. DRMs were isolated from sucrose gradients of cell lysates and CD4 and CCR5 were detected for each condition. CD46 and GM1 representative profiles are shown. Luciferase activity (50 × 103 units/µg of protein), detected 48 h post-infection, controlled for the efficiency of HIV-1 entry and replication (data not shown).

These results emphasize the distinct propensities of HIV-1 receptors to partition into raft domains where GM1 is enriched. Taking into account both the resting distribution of HIV-1 receptors at the cell membrane and the lack of redistribution upon adsorption of HIV-1 virions, we conclude that the necessary colocalization of CD4 and CCR5 required for viral entry (57, 58) most likely occurs outside rafts. To directly address this issue we decided to force CD4 outside rafts and investigate the consequences of such CD4 redistribution on HIV-1 entry.

Palmitoylation and Interaction with p56lck Anchor CD4 in Rafts of the Membrane-- The possibility that palmitoylation of cysteine residues (Cys419 and Cys422) at the boundary of the cytoplasmic and the membrane spanning regions of CD4 (27) could affect its distribution at the plasma membrane was explored. We stabilized expression of human CD4 WT in the CD4-negative A2.01R5 T-cells using a lentivirus-mediated strategy. CD4 WT-expressing cells were then treated with 2-BP, a palmitic acid analog that inhibits palmitoylation (29, 59). The partition of CD4 in detergent-resistant and detergent-soluble fractions of untreated- and 2-BP-treated cells was compared following extraction in 1% Triton X-100 and fractionation on sucrose gradients. Fig. 5A shows that CD4 (CD4 WT, upper panel) distributed into low (3-5) and high density (9-11) fractions enriched in GM1 and CCR5, respectively, as previously observed (Fig. 2). Importantly, blocking the metabolic incorporation of palmitate (Fig. 5B, left panel, lane 3) resulted in an important redistribution of CD4 into high-density, nonraft fractions (9-11) (Fig. 5A, CD4 WT + 2-BP).


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Fig. 5.   CD4 palmitoylation and distribution into rafts are affected by 2-bromopalmitate (2BP). A, A2.01R5 cells expressing CD4 WT were either left untreated or were treated overnight with 2-BP (CD4 WT + 2-BP). DRMs were isolated on sucrose gradients and CD4, CCR5, CD46, and GM1 were detected. B, CD4 palmitoylation was monitored in A2.01R5 cells expressing either the WT (CD4 WT) or the palmitoylation-deficient (CD4 Palm-) CD4 proteins after incorporation of labeled [14C]palmitate (left panel). Cells were also metabolically labeled with [35S]Met (right panel).

This nonraft distribution of CD4 in cells treated with 2-BP indicated that palmitoylation is a key factor in regulating the distribution of this receptor within the cell membrane. Interestingly, 2-BP also inhibited p56lck palmitoylation and prevented its localization to rafts (59). CD4 interacts noncovalently with p56lck through its C-terminal cysteine residues (Cys445 and Cys447) (60) and it was proposed that this association occurs early in the secretory pathway, allowing both proteins to reach the plasma membrane together (61). Aside from blocking CD4 palmitoylation, 2-BP treatment may indirectly affect CD4 distribution by preventing p56lck localization to rafts.

To explore these possibilities, CD4 cDNAs carrying mutations in cysteine residues involved in CD4-palmitoylation (CD4 Palm-), in its interaction with p56lck (CD4 Lck-) or both (CD4 Palm-Lck-), were expressed in CD4-negative A2.01R5 T-cells. Membrane distributions of these molecules were compared with that in CD4 WT cells following 1% Triton X-100 solubilization (Fig. 6A). Similar to the effect of 2-BP (Fig. 5), direct prevention of CD4 palmitoylation by mutation (CD4 Palm-, Fig. 5B, left panel, lane 2) shifted CD4 to high density fractions (Fig. 6A). This strongly suggests that palmitoylation per se directs membrane localization of CD4 without affecting its biosynthesis (Fig. 5B, right panel, [35S]Met labeling) and cell surface expression (data not shown). Similar results obtained for the CD4 Lck- mutant show that, in addition, CD4/p56lck interaction also directs CD4 to rafts (Fig. 6A (CD4 Lck-)). In keeping with these observations, the double mutation (CD4 Palm-Lck-) almost abolished CD4 association with low density fractions (Fig. 6, A and B, 6 ± 2% remaining in the low density fractions). The distribution of the WT and mutant CD4 proteins in 1% Triton X-100-soluble and resistant fractions are compared in Fig. 6B. The lower recovery of the CD4 Palm-Lck- mutant protein in Triton X-100-resistant low density fractions (3-5) is consistent with an additive effect of palmitoylation and lack of interaction with p56lck in the targeting of CD4 to rafts. In agreement with previous reports (61), the distribution of p56lck at the plasma membrane in raft and nonraft domains (Fig. 6C, D and S fractions, respectively) is independent of its interaction with CD4 (Fig. 6D). The reduced cell surface expression of the CD4 protein in the Lck- and Palm-Lck- cells, compared with its expression in CD4 WT or Palm- cells (data not shown), is in keeping with CD4 down-regulation observed in cells lacking p56lck (62).


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Fig. 6.   Disruption of palmitoylation and p56lck interaction sites determines exclusion of CD4 from rafts. A, distribution pattern in sucrose gradients of CD4 WT protein and its variants (Palm-, Lck-, or Palm-Lck-) stably expressed into A2.01R5 cells. B, CD4 was quantified in DRMs (3-5) or soluble (9-11) fractions and is expressed as % of the total CD4 in the whole gradient. Data represent the mean values of six independent experiments. C, p56lck detection in pooled DRMs (3-5) and soluble fractions (9-11) of gradients shown in A. D, interaction of p56lck with CD4 WT and its mutants was monitored in DRMs (fractions 3-5) by co-immunoprecipitation and Western blot.

CD4 Redistribution into Nonraft Domains Does Not Impair CCR5-dependent HIV-1 Entry-- The consequences of expression of the CD4 double mutant (Palm-Lck-) on R5-HIV-1 entry were explored. We first used an HIV-Env glycoprotein-mediated cell fusion assay that is independent of the post-entry steps of the viral cycle and allows accurate quantification of fusion events (38). This method relies on syncytia formation between cells expressing the HIV-1 R5 BX08 Env glycoprotein and CD4-stabilized A2.01R5 target cells (Fig. 7A). CD4-WT and CD4-Palm-Lck- cells showed the same capacity to fuse with R5-Env expressing cells. Cell fusion was prevented by blocking with either monoclonal antibody Q4120 that recognizes the gp120-binding domain of CD4 (63), or TAK779 that binds to CCR5 (64).


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Fig. 7.   Extensive CD4 delocalization from rafts neither impairs fusion nor replication of R5-HIV-1. A, parental A2.01 cells (CD4 negative) or A2.01R5 cells stably expressing either CD4 WT or CD4 Palm-Lck- mutants were co-cultured with BHK cells expressing the BX08 R5-Env. Syncitia formation was monitored by FACscan analysis. When indicated, inhibitors of HIV-1 entry were added 30 min before starting the fusion assay. B, these cell lines were infected with R5-HIV-1-Ba-L (75 ng of p24 for 106 cells). Luciferase activity was analyzed 65 h after infection. Standard deviations of two independent determinations done in triplicate are shown.

Another alternative assay was used to verify the apparent independence of the HIV-1-R5 entry on the presence of CD4/CCR5 in rafts. CD4 WT and CD4 Palm-Lck--expressing cells were inoculated with cell-free virions generated upon trans-complementation of a luciferase reporter HIV-1 provirus (Env-) with an R5-HIV-1 Ba-L Env (Fig. 7B). This pseudotyped virus yields only a single round of infection, thereby allowing evaluation of HIV-1 entry by quantifying viral replication. Consistent with our findings in the cell-cell fusion assays (Fig. 7A), R5-HIV-1 virus replicated to the same levels in both CD4 WT and Palm-Lck- cells (Fig. 7B). Similar results were obtained with HIV-1 pseudotyped with an R5-strain Env (CSF) or the WT HIV-1YU2 R5-strain (data not shown). These results demonstrate that CD4 delocalization outside rafts does impair neither HIV-1 Env-mediated cell-cell fusion, nor entry and replication of HIV-1 cell-free particles. We conclude that adsorption of R5-viruses, which exposes fusogenic HIV-1 gp41 Env transmembrane subunits allowing subsequent virus entry, does not require localization of viral receptors to detergent-resistant raft domains.

    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Given the critical structural role played by cholesterol in promoting raft domain formation, it was postulated that removal of cholesterol would interfere with HIV-1 infection by disrupting rafts. The aim of this work was to critically evaluate the contribution of membrane rafts to HIV-1 entry. In particular, we investigated whether the HIV-1 receptors, CD4 and CCR5, need to reside in rafts to promote viral entry into CD4 T lymphocytes.

CCR5 Does Not Partition into Rafts-- Upon stable expression in T-cell lines, CCR5 did not associate with rafts. This result is in keeping with a recent observation by Nguyen and Taub (65). Fluorescent microscope findings fully corroborated flotation studies, showing that the bulk of CCR5 localizes to nonraft domains of plasma membranes. Furthermore, in biologically relevant primary CD4 T-cells, the endogenous CCR5 receptor was found exclusively in detergent-soluble, nonraft domains.

It is likely that the recovery of CCR5 in 0.2% Triton X-100-resistant membranes previously reported in adenocarcinoma cells transiently overexpressing a CCR5 transgene (66) reflects spillover of overexpressed CCR5 in rafts. In the latter, the relatively high recovery of CCR5 (11-18%) in 0.2% Triton X-100-resistant membranes could have resulted from loading equal amounts of protein rather than equal volumes of each fraction of the gradient. Indeed, with most of the cellular proteins concentrated in soluble fractions (Ref. 42 and data not shown), hence equalizing the amounts of protein may lead to an overestimation of the amount of CCR5 in detergent-resistant fractions.

Although CCR5 appears to be present in DRM domains, we envisaged that HIV-1 receptor engagement by viral Env might recruit CCR5 to rafts, to which CD4 and GM1 localize. This explanation was recently advanced to account for redistribution of CXCR4 by soluble X4-HIV-1 Env (16, 18). However, the finding by Kozak et al. (52) that CXCR4 coreceptor distribution into detergent-soluble domains is not modified by virion adsorption challenges previous observations (16, 18). HIV-1 entry is expected to be a cooperative process requiring the assembly of HIV-1 virions with CD4 and coreceptors. We therefore investigated whether R5-HIV-1 virion binding, which facilitates initial CD4 cross-linking unlike soluble monomeric viral Env, would induce CCR5 redistribution and colocalization with CD4 in T-cell rafts. In flotation studies, bridging of viral receptors by R5-HIV-1 virions adsorbed on T-cells did not shift CCR5 to raft fractions. Taken together, our data strongly suggest that recruitment of CCR5 to rafts is not a prerequisite for virus entry.

One could speculate that CCR5-enriched detergent-soluble membrane domains may form a ring around a raft area where virus could interact with the "raft" fraction of CD4. This hypothetical situation would be similar to the membrane organization described in T lymphocytes interacting with antigen-presenting cells (67), where the T-cell receptor interacts with an MHC II-bound cognate peptide not included in rafts. The role played by CD4 residing in rafts on HIV-1 infection was explored expressly to test this hypothesis.

Redistribution of CD4 Outside Raft Domains Does Not Affect HIV-1 Infection-- In contrast to CCR5, CD4 was equally distributed in both Triton X-100-resistant and Triton X-100-soluble membranes. The nonraft redistribution of the nonpalmitoylated CD4 protein strongly suggests that palmitoylation determines the targeting of this receptor to rafts. The role of fatty acylation in targeting membrane proteins to rafts (68, 69) has been established for a number of integral palmitoylated proteins, such as LAT and CD8alpha beta (24, 25). However, palmitoylation per se does not obligatorily direct the modified protein to rafts, as illustrated in our work showing exclusion of palmitoylated CCR5 (29) from rafts; this further illustrates that other molecular features of the protein determine its raft/nonraft distribution. For instance, both CD4 palmitoylation and its interaction with p56lck contribute to the presence of CD4 in raft domains. The association of p56lck with the nonpalmitoylated CD4 outside rafts is actually decreased, as observed previously for p56lck and CD8 (25). In agreement with previous studies (61, 70) we showed that p56lck partitioning into raft domains occurs independently of its association with CD4.

We used different mutants of CD4 to analyze the impact of CD4 delocalization from raft domains on infection of T-cells by R5-HIV-1 strains. Importantly, we observed that excluding most of the CD4 from rafts does not alter its capacity to support R5-Env-mediated cell-cell fusion and entry of R5-HIV-1 viruses. These data are fully consistent with previous observations suggesting that CD4-p56lck association is not necessary for HIV-1 infection (71, 72). Our results reinforce the view that rafts are not sites where virus binding and entry actually take place. This conclusion is in keeping with biophysical considerations suggesting that membrane fusion is more favorable in nonraft membrane environments of lipids, such as glycerophospholipids (73).

Membrane Cholesterol Is Required for Efficient HIV-1 Entry in Host Cells-- MBCD extraction of cholesterol inhibits entry of R5-HIV-1 into primary CD4 T-cells, its natural host cell. This confirms and extends recent observations in transformed cell lines stressing the requirement for cholesterol in HIV-1 infection (16-18, 74). However, taking into account that cholesterol is present everywhere on the plasma membrane (21, 22), inhibition of HIV-1 infection by cholesterol depletion does not necessarily implicate disturbances of rafts integrity. We showed that R5-HIV-1 fusion and entry are independent of CD4/CCR5 localization in membrane rafts. Consequently, it is unlikely that the inhibitory effect of cholesterol depletion on HIV-1 entry results from disorganization of rafts.

The role of cholesterol in HIV-1 entry can be interpreted differently. The membrane lipid environment, into which the HIV-1 gp41 fusion peptide partitions, plays an important role in the fusion of viral and cell membranes (75); plasma membrane cholesterol may be a critical component in this process. Membrane fusion is purported to set off local bending of lipid bilayers converging to a common type of nonlamellar, highly curved, stalk intermediates (73, 76). Indeed, fusion depends very much on lipids of spontaneous negative curvature (i.e. unsaturated phosphatidylethanolamine, cholesterol) to promote membrane bending (77, 78). Likewise, synthetic peptides mimicking the HIV-1 gp41 fusion domain were found to promote negative curvature that facilitates formation of stalk intermediates (79). The fusogenic activity of these peptides was stimulated by insertion of cholesterol into a nonraft environment (80). Therefore, the observed interference of the lack of cholesterol with HIV-1 entry might reflect the altered capacity of plasma membranes to undergo viral-induced fusion.

Furthermore, the role played by cellular cholesterol in HIV-1 entry may be related to its capacity to modulate the activity of membrane proteins. This has been shown for several G-protein-coupled receptors (81, 82) and also applies to the HIV-1 coreceptors, CCR5 and CXCR4. Effectively, depletion of cell membrane cholesterol has been shown to modify binding of CCR5 and CXCR4 to their natural chemokine ligands, MIP1-beta and SDF-1alpha , respectively (65, 74). Nonetheless, whether diminished HIV-1 infection in cholesterol-depleted cells relates to modulation of CCR5 or CXCR4 still remains speculative.

In conclusion, our findings exclude a significant participation of membrane rafts in HIV-1 binding to host cells and point to an important role for cholesterol in the mechanisms of viral entry into primary CD4 T lymphocytes. Further study, using real-time and quantitative approaches are needed to address the particularly intriguing modulator effect of plasma membrane cholesterol on the HIV-1 entry process.

    ACKNOWLEDGEMENTS

We thank E. Perret (Centre d'Imagerie Dynamique, Institut Pasteur, Paris, France) for help with confocal microscopy. We are grateful to Dr. P. Charneau for providing the pTRIP HIV-1-based lentiviral expression vector. We thank Dr. M. Mack and Dr. M. Marsh for providing MC-5 anti-CCR5 antibody and the CD4 cDNA encoding a mutation into cysteine residues interacting with p56lck-, respectively. We are grateful to Dr. S. Michelson for carefully reading and commenting on the manuscript.

    FOOTNOTES

* This work was supported in part by a grant from Ensemble contre le SIDA (Sidaction).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§ Supported by fellowships from the Agence Nationale pour la Recherche sur le SIDA (ANRS).

|| To whom correspondence should be addressed: Françoise Bachelerie, Institut Pasteur, Unité d'immunologie Virale, 25-28 rue du Dr Roux, 75724 Paris cedex, 15, France. E-mail: fbachele@pasteur.fr.

Published, JBC Papers in Press, November 12, 2002, DOI 10.1074/jbc.M207371200

    ABBREVIATIONS

The abbreviations used are: HIV-1, human immunodeficiency virus type 1; gp, glycoprotein; FCS, fetal calf serum; BHK, baby hamster ovary; MBCD, methyl-beta -cyclodextrin; CTx, choleratoxin; 2-BP, 2-bromopalmitate; WT, wild type; DRM, detergent-resistant membrane.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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