From the 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
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ABSTRACT |
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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.
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.
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 Treatment of Cells with Methyl- 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 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 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 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 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 ( 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).
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.
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).
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).
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 CD4 Redistribution into Nonraft Domains Does Not Impair
CCR5-dependent HIV-1 Entry--
The consequences of
expression of the CD4 double mutant
(Palm
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 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 CD8
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-
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.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
(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).
-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-
-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-
-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
-position (Roche
Molecular Biochemicals).
-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
-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
-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
-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.
-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).
)
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.
, 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
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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) ( 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%).
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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.
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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).
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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).
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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).
), 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.
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.
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
(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.
and SDF-1
, respectively (65, 74). Nonetheless, whether
diminished HIV-1 infection in cholesterol-depleted cells relates to
modulation of CCR5 or CXCR4 still remains speculative.
![]() |
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--cyclodextrin;
CTx, choleratoxin;
2-BP, 2-bromopalmitate;
WT, wild type;
DRM, detergent-resistant membrane.
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REFERENCES |
---|
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