From the Institut Cochin, Department of Infectious
Diseases, INSERM U567-CNRS UMR8104, Universite Paris V, 24 Rue du
Faubourg Saint-Jacques, 75014 Paris, France, the ¶ San Diego
Veterans Affairs Healthcare System, San Diego, California 92121, the
Institut Pasteur, 25 rue du Dr Roux, 75015 Paris, France,
the ** Laboratoire d'Enzymologie et Biochimie Structurales,
CNRS, 91198 Gif/Yvette, France, the
Institute Curie, UMR 144, 26 rue d'Ulm,
75248 Paris Cedex 05, France, and the ¶¶ Department of
Medicine, University of California,
La Jolla, California 92093-0679
Received for publication, October 2, 2002, and in revised form, December 9, 2002
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ABSTRACT |
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The maximal virulence of HIV-1
requires Nef, a virally encoded peripheral membrane protein. Nef binds
to the adaptor protein (AP) complexes of coated vesicles, inducing an
expansion of the endosomal compartment and altering the surface
expression of cellular proteins including CD4 and class I major
histocompatibility complex. Here, we show that Nef stabilizes
the association of AP-1 and AP-3 with membranes. These complexes
remained with Nef on juxtanuclear membranes despite the treatment of
cells with brefeldin A, which induced the release of ADP-ribosylation
factor 1 (ARF1) from these membranes to the cytosol. Nef also induced a
persistent association of AP-1 and AP-3 with membranes despite the
expression of dominant-negative ARF1 or the overexpression of an
ARF1-GTPase activating protein. Mutational analysis indicated that the
direct binding of Nef to the AP complexes is essential for this
stabilization. The leucine residues of the EXXXLL
motif found in Nef were required for binding to AP-1 and AP-3 in
vitro and for the stabilization of these complexes on membranes
in vivo, whereas the glutamic acid residue of this motif
was required specifically for the binding and stabilization of AP-3.
These data indicate that Nef mediates the persistent attachment of AP-1
and AP-3 to membranes by an ARF1-independent mechanism. The
stabilization of these complexes on membranes may underlie the
pleiotropic effects of Nef on protein trafficking within the endosomal system.
Nef is a 27-kDa, myristoylated, peripheral membrane protein that
alters the trafficking of transmembrane proteins within the endosomal
system and is required for the maximal virulence of HIV-1 (1). Nef
misdirects CD4 from the plasma membrane and the Golgi to lysosomes, and
it retains class I major histocompatibility complex
(MHC)1 in the
trans-Golgi region (2-4). When fused to the transmembrane domains of integral membrane proteins such as CD4 or CD8, Nef increases
the rate of internalization of these chimeras from the cell surface (3,
5). These observations led to the hypothesis that Nef functions as a
connector between the cytoplasmic domains of cellular proteins and the
membrane trafficking machinery. This hypothesis was supported by the
observations that Nef binds vesicle coat proteins including the adaptor
protein (AP) complexes (6-9). However, proteins whose trafficking is
affected by HIV-1 Nef now include mature class II MHC, the immature
class II MHC/invariant chain complex, the membrane-anchored cytokines
TNF and LIGHT, CD28, and DC-SIGN (4, 10-13). These data indicate a
general effect of Nef on the trafficking of membrane proteins within
the endosomal system. This general effect is supported by the
observation that Nef induces a morphologic expansion of endosomal
membranes (14, 15).
Membrane trafficking within the endosomal system is mediated in large
part by vesicles coated with AP complexes (16, 17). Of the four members
of the AP complex family, AP-1, AP-3, and AP-4 coat vesicles that
mediate transport between the trans-Golgi, endosomes, and
lysosomes, whereas AP-2 coats vesicles that mediate endocytosis. The
complexes are involved both in the formation and budding of coated
vesicles as well as in their selection of cargo. This selection
requires the recognition of specific sequences in the cytoplasmic
domains of transmembrane proteins (18). These sequences are tyrosine-
or leucine-based motifs, which mediate direct binding to the AP
complexes (19-21).
HIV-1 Nef contains a canonical leucine-based AP-binding motif
(EXXXLL) within a solvent exposed, unstructured loop near
the C terminus of the protein (5, 9, 22). However, the role of this
motif in the specific interactions of Nef with the various AP complexes
is incompletely defined (5, 9, 22-24). Leucines of the
EXXXLL motif are required for the binding of Nef to AP-1 (5). These residues are also required for the binding of Nef to the
medium subunit of AP-3 (23), but binding of Nef to intact AP-3 has not
been demonstrated. The role of the glutamic acid residue in the Nef
EXXXLL motif is untested, but analogous residues have been
implicated in the binding of several mammalian and yeast proteins to
AP-3 (25, 26). Although peptides containing the EXXXLL
sequence of Nef or the DKQTLL sequence of the T cell receptor With the exception of AP-2, the association of the AP complexes with
membranes is regulated by ADP-ribosylation factor 1 (ARF1) (27-30).
ARF1 undergoes a cycle of membrane association and dissociation controlled by a myristoyl-switch mechanism, which is in turn regulated by guanine-nucleotide exchange and GTP hydrolysis (27, 31). ARF1
associates with membranes when bound to GTP. The GTP-bound state of
ARF1 is induced by guanine nucleotide exchange proteins (GEPs), which
catalyze the exchange of GDP for GTP. Certain Golgi-associated ARF1-GEPs are inhibited by the fungal metabolite brefeldin A (BFA) (32-35). By inhibiting these GEPs, BFA induces the GDP-bound state of
ARF1 and causes the dissociation of ARF1 and ARF1-dependent coats from membranes. ARF1 also dissociates from membranes in response
to GTPase-activating proteins (GAPs), which cause the hydrolysis of GTP
to GDP on ARF (36). The physiologic action of ARF1-GAPs allows the
dissociation of ARF1-dependent vesicle coats from membranes
during vesicular transport (37). When membrane-associated, ARF1
generates a high-affinity binding site for the AP complexes (38). The
nature of this binding site is not known. ARF1 may activate an
unidentified "docking protein" for the AP complexes (38-41), or it
may induce a modification of membrane phospholipids that enables AP
binding (42). Alternatively, ARF1 may itself be the docking protein, an
hypothesis supported by its ability to bind directly to subunits of the
AP-1 and AP-4 complexes (43, 44).
To understand the basis of the pleiotropic effects of Nef on protein
trafficking within the endosomal system, we tested the hypothesis that
Nef regulates the association of AP complexes with membranes. We report
that Nef causes the persistent attachment of AP-1 and AP-3 to
juxtanuclear membranes despite experimental perturbations of the
ARF1-based regulatory system. Nef neither displayed ARF-like activity
nor did it modulate ARF1 or its regulators in vitro.
Instead, the direct binding of Nef to AP complexes appears crucial for
this membrane stabilization, because mutations in the leucine-based
motif of Nef affected both the binding to AP-1 and AP-3 in
vitro and the stabilization of these complexes on membranes
in vivo. These findings support the hypothesis that a
persistent attachment of AP complexes to membranes underlies a general
effect of Nef on the endosomal system.
Plasmid Constructions
Plasmid vectors for the expression of wild-type or mutated
HIV-1Lai Nef fused to the extracellular and transmembrane domains of
the human CD8 Cell Lines and Transfections
HeLa cells were grown in Dulbecco's modified Eagle's
medium with Glutamax (Invitrogen) supplemented with 10%
fetal calf serum (Invitrogen), 100 units/ml penicillin (Invitrogen),
and 100 µg/ml streptomycin (Invitrogen). Cells were transfected
either by electroporation as described with 12 µg of plasmid DNA (14)
or lipid-mediated transfer using 4 µg of DNA and FuGENE reagent
(Roche Molecular Biochemicals) or Lipofectin (Invitrogen).
Antibodies
The following primary antibodies were used:
anti- Indirect Immunofluorescence Microscopy
After transfection with the indicated plasmids, HeLa
cells were spread on glass coverslips in 24-well plates (8 × 104 cells/well) and then stained for immunofluorescence
24 h later as described (14). When indicated, the cells were
treated with BFA (Sigma or Epicentre) at 37 °C before fixation; the
concentration and duration of BFA treatment are indicated in the figure
legends. For anti- Protein Expression and Purification
Wild-type or mutated GST-Nef proteins were produced in E. coli as described (4), and eluted from Sepharose-glutathione beads
in 50 mM Tris-HCl (pH 7.5), containing 10 mM
reduced GSH. Coatomer was purified from rabbit liver as described
previously (47). The following proteins were expressed in E. coli: [ In Vitro Assay for Binding between Nef and AP Complexes
HeLa cells (107) were lysed in 50 mM
Tris-HCl (pH 8), 5 mM EDTA, 150 mM NaCl, and
1% Triton X-100. The cytoplasmic lysates were incubated overnight at
4 °C with 2 µg of GST or GST-Nef proteins immobilized on
GSH-Sepharose beads (Amersham Biosciences). Beads were washed five
times in lysis buffer. Bound cellular proteins were analyzed by Western
blotting using anti- Guanine Nucleotide Binding, Exchange, and GTP-hydrolysis
Assays
GTP Loading of ARF1--
[ GDP/GTP Exchange on [ ARF GAP Assays--
Hydrolysis of GTP on [ HIV-1 Nef Co-localizes with AP-1 and AP-3 Complexes on Juxtanuclear
Membranes--
The subcellular distribution of Nef in relation to AP
complexes was examined by confocal immunofluorescence microscopy (Fig. 1). For these studies, we used two fusion
proteins: one in which the green fluorescent protein (GFP) was appended
to the C terminus of Nef (Nef-GFP (23, 53)), and the other in which the
entire Nef sequence was appended to the lumenal and transmembrane
domains of CD8, generating a membrane protein containing Nef as the
cytoplasmic domain (CD8-Nef (14, 54)). These chimeras are functional
for the down-regulation of CD4 and class I MHC (data not shown and Fig.
2) and have been extensively used to
analyze the interaction of Nef with the endocytic machinery (3, 5, 8,
14, 24, 54). In HeLa cells, Nef-GFP was concentrated in a juxtanuclear region near the cell center, the region in which AP-1 complexes were
also concentrated (Fig. 1A). CD8-Nef was also concentrated in a juxtanuclear region (Fig. 1B), and its distribution
overlapped extensively with AP-1 and AP-3, but not with AP-2. Notably,
the distribution of Nef-GFP included a cytoplasmic component, but that
of CD8-Nef did not (Figs. 1 and 3),
because CD8-Nef is exclusively membrane-associated, whereas Nef-GFP,
which associates with membranes via its N-terminal myristoyl group, is
in part cytoplasmic. Furthermore, in most cells that expressed CD8-Nef,
the intensity of staining for AP-1 and AP-3 in the juxtanuclear region
was greater than in cells that did not express the chimera. This
supraphysiologic recruitment of AP-1 and AP-3 by CD8-Nef was
reminiscent of the recruitment of AP-1 or AP-3 to juxtanuclear
membranes caused by the overexpression of transmembrane chimeras
containing the cytoplasmic domains of the mannose 6-phosphate receptor,
Lamp I, or Limp II (55). These data confirm that the CD8-Nef chimera
provides an optimal experimental system for studying the relationship
between Nef and the AP complexes.
Nef Sequesters CD4 and Class I MHC in a Condensed Transferrin
Receptor-positive Compartment--
Ultrastructural analysis by
electron microscopy revealed that Nef expression severely affects the
morphology of the endosomal compartment (14, 15). To identify more
precisely the membrane systems affected by Nef, we characterized the
compartment in which CD8-Nef resides by co-staining for cellular
markers of the endocytic pathway (Fig. 2). CD8-Nef co-localized
extensively with transferrin receptor in the juxtanuclear region near
the cell center. Strikingly, CD8-Nef induced a marked increase in
transferrin receptor staining in this juxtanuclear region. These data
indicate that Nef induces an expansion of this compartment, presumably
via condensation of peripheral endosomal membranes into this region.
Notably, both CD4 and class I MHC co-localized with Nef in the
juxtanuclear region (Fig. 2). In contrast, little to no co-localization
was observed between Nef and CD63, a marker of late endosomes and lysosomes (Fig. 2), or between Nef and sialyltransferase, a Golgi marker (data not shown). Together, these observations suggest that Nef
sequesters CD4 and class I MHC in a perinuclear transferrin receptor-positive endosomal compartment at steady state.
Nef Renders the Juxtanuclear Distribution of AP-1 and AP-3
Resistant to BFA--
We hypothesized that Nef might alter the
endosomal system by regulating the membrane association of AP
complexes. To test this, we treated Nef-expressing cells with BFA, a
fungal metabolite that inhibits Golgi-associated ARF1 guanine
nucleotide exchange proteins (GEPs) and causes the release of ARF1 and
ARF1-dependent coat components from membranes (Fig. 3). In
cells that did not express Nef, AP-1 and AP-3 became diffusely
cytoplasmic because of dissociation from membranes. In contrast, AP-1
and AP-3 remained concentrated with Nef in a juxtanuclear region
despite BFA treatment in cells expressing either Nef-GFP (panel
A) or CD8-Nef (panel B). The juxtanuclear distribution
of AP-1 and AP-3 was not preserved in BFA-treated cells that expressed
a CD8 construct lacking a cytoplasmic domain, which localized
predominantly to the plasma membrane (data not shown). These data
indicate that Nef stabilizes the association of AP-1 and AP-3 with
membranes. This stabilization was also observed following infection of
CD4-positive HeLa cells with wild-type virus, indicating that it occurs
at physiological levels of Nef expression (data not shown).
The Nef-mediated Association of AP-1 and AP-3 with Juxtanuclear
Membranes Is Resistant to the Expression of Dominant Negative ARF1 and
the Overexpression of ARF1-GAP--
The resistance to BFA suggested
that the Nef-mediated membrane stabilization of the AP complexes was
independent of ARF1 activity. To test this hypothesis, we first
expressed a dominant negative ARF1 mutant (ARF1/T31N), either with or
without co-expression of CD8-Nef (Fig.
4A). ARF1/T31N is defective in
guanine nucleotide binding and causes the dissociation of
ARF1-dependent coats from membranes, presumably as a
consequence of sequestration of ARF1-GEPs (56). In cells expressing
ARF1/T31N alone, the AP-1 staining was diffuse and faint (Fig.
4A, upper panel), similar to that observed in
cells treated with BFA. In contrast, AP-1 remained concentrated in the
juxtanuclear region in cells co-expressing ARF1/T31N and CD8-Nef (Fig.
4A, lower panels). Similarly, AP-3 remained
concentrated in the juxtanuclear region of cells co-expressing ARF1/T31N and CD8-Nef (data not shown).
The hypothesis that the Nef-mediated stabilization was independent of
ARF1 was tested further by overexpression of ARF-GAP1 (Fig.
4B). Overexpression of ARF-GAP1 causes the release from membranes of the ARF1-dependent vesicle coat components as
a consequence of the hydrolysis of ARF1-bound GTP (57). In cells
overexpressing ARF-GAP1 alone, the AP-1 staining was diffuse and faint
(Fig. 4B, upper panel), similar to that observed
in cells treated with BFA or expressing ARF1/T31N. In contrast, in
cells co-expressing exogenous ARF-GAP1 and CD8-Nef, AP-1 remained
concentrated in the juxtanuclear region (Fig. 4B,
lower panel). Similarly, AP-3 remained concentrated in the
juxtanuclear region of Nef-expressing cells despite the overexpression
of ARF-GAP (data not shown). Altogether, these results support the
hypothesis that the expression of Nef induces membrane stabilization of
AP-1 and AP-3 via an ARF1-independent mechanism.
Nef Does Not Modulate the GTPase Cycle of ARF1--
Because
mimicry or modulation of the ARF1-based regulatory system could explain
the stabilization of AP complexes induced by Nef, we assessed using
in vitro assays whether Nef might display ARF1-like or
ARF1-GEP activities, or alternatively might stimulate ARF1-GEPs or
inhibit ARF-GAP (Fig. 5). First, we
tested whether Nef might be an ARF-like protein and bind GTP. Although
no similarity between Nef and ARF1 in primary or tertiary structure is
apparent, and Nef does not contain any canonical GTP-binding motifs,
controversial GTP-binding properties of Nef were initially reported
(58-60). As indicated in Fig. 5A, GST-Nef did not bind GTP,
even in the presence of a highly active ARF-GEP (the Sec7 domain of
ARNO), whereas a truncated form of ARF, [
These in vitro data indicated that the stabilization of AP
complexes in vivo is unlikely to be mediated by an ARF-like
or ARF-GEP-like activity of Nef or by modulation of the activity of the
proteins involved in the regulation of ARF1. In addition, we did not
detect any binding between Nef and ARF1 in either yeast two-hybrid or
GST pull-down assays (data not shown), suggesting that Nef is unlikely
to mediate the stabilization of AP complexes by recruiting ARF1 itself
to membranes.
Nef Maintains the Juxtnuclear Concentration of AP-1 in BFA-treated
Cells Despite the Dispersal of ARF1 into the Cytosol--
To further
exclude a role for ARF1 in the Nef-mediated stabilization of AP
complexes, we compared the distributions of ARF1, AP-1, and Nef in
BFA-treated cells. Cells were transfected with a vector expressing an
HA-tagged wild-type ARF1 (ARF1-HA), in combination with the CD8-Nef
expression vector, and were then examined by immunofluorescence
microscopy after BFA treatment (Fig. 6).
In the absence of Nef, ARF1-HA and AP-1 were concentrated in the
juxtanuclear region in untreated cells, and both became dispersed
throughout the cytoplasm when the cells were treated with BFA (Fig. 6,
top and middle panels). However, in BFA-treated cells that expressed CD8-Nef (Fig. 6, lower panels), AP-1
remained concentrated in a punctate, juxtanuclear distribution, whereas the distribution of ARF1-HA became diffuse and cytosolic. These data
definitively confirm that the membrane stabilization of AP complexes
induced by Nef in BFA-treated cells is independent of ARF1.
The Stabilization of AP-1 and AP-3 on Juxtanuclear Membranes
Requires the Leucine-based motif in Nef--
HIV-1 Nef contains a
leucine-based motif (EXXXLL) required for the association
with AP complexes (see Fig.
7B, and Refs. 5 and 22). To
determine whether the membrane-stabilization effect required AP-binding
mediated by this motif, alanine substitutions were introduced in place
of the leucine residues of the EXXXLL sequence. The
resulting CD8-NefL164A/L165A mutant was tested for the ability
to maintain the juxtanuclear concentration of AP-1 and AP-3 in the
presence of BFA (Fig. 7A). As previously reported (14),
mutation of these leucine residues caused a significant fraction of
CD8-Nef to localize to the periphery of the cytoplasm and plasma
membrane. Strikingly, this mutation completely abolished the ability of
Nef to maintain the juxtanuclear concentration of either AP-1 or AP-3
in cells treated with BFA. We checked that the leucine-based motif of
Nef was also required for direct recruitment of both AP-1 and AP-3
complexes. Recombinant GST-Nef fusions were used to analyze the
interaction between Nef and the intact AP complexes from cell lysates
(Fig. 7B). The dependence of the binding between Nef and
AP-1 on the leucine-based motif was confirmed (5). In addition, we
documented that Nef binds the intact AP-3 complex, and this binding was
also leucine-dependent.
We next examined the role of the glutamic acid residue in the
EXXXLL motif, because the presence of an acidic residue at
this location in leucine-based motifs has been associated with the ability to bind AP-3 complexes in vitro and to utilize
AP-3-based sorting pathways in vivo (25, 26). A mutation
encoding alanine substitution E160A was introduced into the CD8-Nef
chimera, and the mutant was tested for the ability to stabilize the
membrane association of AP-1 and AP-3 in the presence of BFA (Fig.
7A). This mutant was concentrated in the juxtanuclear
region, but in non-BFA-treated cells it did not co-localize
significantly with AP-3 (Fig. 7A and data not shown). In
BFA-treated cells, NefE160A failed to stabilize AP-3 on membranes, and
it was less effective than wild-type Nef in stabilizing AP-1 (Fig.
7A). In GST pull-down assays, NefE160A bound AP-1 with
reduced efficiency (~38% of wild-type activity), but it did not bind
at all to AP-3 (Fig. 7B).
Together, these data indicate that the EXXXLL motif is
important for the binding of Nef to AP-1 and AP-3 in vitro
and for the Nef-induced stabilization of these complexes on membranes in vivo. The data also indicate that the acidic residue of
the Nef EXXXLL motif is especially critical for the binding
to AP-3 and the stabilization of AP-3 on membranes.
The pleiotropic effects of the HIV-1 Nef protein on the
trafficking of cellular transmembrane proteins indicate a general perturbation in the regulation of vesicular transport throughout the
endosomal system. The data herein support the hypothesis that this
perturbation occurs at the level of the regulation of the membrane
attachment of vesicle coat components. Specifically, Nef stabilizes the
association of AP-1 and AP-3 complexes on juxtanuclear membranes by a
direct binding mechanism that is independent of ARF1. First, Nef
conferred resistance of AP-1 and AP-3 to the membrane-dissociating
effects of BFA, dominant negative ARF1 (ARF1/T31N), and ARF-GAP1.
Second, Nef lacked ARF-like or ARF-GEP-like activity, and it neither
stimulated ARF-GEPs nor inhibited ARF-GAP1. Third, Nef maintained the
juxtanuclear membrane-association of AP-1 despite the cytosolic
dispersal of ARF1 in BFA-treated cells. These data indicate that Nef
causes the persistent association of AP complexes with membranes, even
when their physiologic attachment as mediated by ARF1 is inhibited.
The leucine-based motif in Nef was required for both the binding of Nef
to AP-1 and AP-3 in vitro and the membrane stabilization of
these complexes in vivo. This motif has been associated with most of the perturbations in protein trafficking induced by Nef, including the down-regulation of CD4 and CD28, and the up-regulation of
DC-SIGN, the immature class II MHC/invariant chain complex, and the
membrane-anchored cytokines TNF and LIGHT (9-13).
The correlation between the ability of Nef to interact with specific AP
complexes and to stabilize these complexes on membranes was supported
further by the selective importance of the acidic residue within the
Nef EXXXLL motif for binding and membrane stabilization of
AP-3. The role of this glutamic acid residue for the specific recruitment of AP-3 was predicted based on the requirement of analogous
acidic residues within the leucine-based sorting motifs of Limp II and
tyrosinase for the binding to AP-3 in vitro (25). Similarly,
the AP-3-mediated transport of the Vam3p protein to the vacuole in
yeast requires an analogous acidic residue (26).
Two models can explain the persistent membrane association of AP
complexes induced by Nef. First, Nef may mediate de novo attachment events. As a peripheral membrane protein that binds AP-1 and
AP-3, Nef may be a constitutively active viral homologue of a putative
cellular docking protein for these complexes (38-41). Alternatively,
Nef and ARF1 may be similar insofar as each is able to constitute or
activate the membrane attachment sites for AP complexes. In support of
such a similarity, constitutively active ARF1 (ARF1/Q71L), which is
locked in the GTP-bound state and constitutively associates
with membranes, induces a BFA-resistant association of AP complexes
with membranes similar to that induced by Nef (Refs. 29 and 63, and
data not shown). Mechanistically, both Nef and ARF1 bind AP complex
subunits, so either may directly bind AP complexes to membranes (43,
44). Alternatively, both Nef and ARF1 may generate AP binding sites
indirectly by recruiting phospholipid-modifying enzymes to membranes
(42, 62).
The second model is that Nef causes the persistent attachment of AP
complexes by inhibiting their release from membranes. In this model,
the initial attachment of the complexes to membranes remains mediated
by ARF1, but the subsequent interaction with Nef causes a decrease in
the rate at which the complexes cycle off membranes. This model is
particularly compatible with the experiments using BFA, in which the
block to ARF1-mediated attachment of the complexes is introduced after
the expression of Nef. The model is potentially less compatible with
the experiments in which dominant negative ARF1 or ARF-GAP1 are
expressed concurrently with Nef, but it remains formally possible. The
regulation of the dissociation of AP complexes from membranes is not
well understood. However, this dissociation is presumably a
prerequisite to the fusion of transport vesicles with target membranes.
Whether caused by de novo attachment or a block to release,
how would the membrane stabilization of AP complexes by Nef relate to
its effects on protein trafficking? We observe herein that Nef expands
a perinuclear endosomal compartment and sequesters CD4 and class I MHC
in this membrane system (see Fig. 2). The membrane stabilization of
AP-1 and AP-3 may induce the expansion of this compartment either by
blocking the formation of donor vesicles on endosomal membranes or by
inhibiting coat dissociation and the subsequent fusion of vesicles
derived from endosomal membranes with their targets. Either effect
would cause the accumulation of AP-binding cargoes within endosomal
structures. This model predicts that Nef would sequester
intracellularly receptors that normally return to the cell surface
after endocytosis and transit through recycling endosomes. Such a block
to recycling has been proposed as part of the mechanism of Nef-mediated
down-regulation of surface CD4 (8, 64). This model accounts for a
general down-regulation of AP-binding cell-surface proteins by Nef, but how can the up-regulation of certain molecules be explained (10, 12,
13)? We hypothesize that the sequestration of AP complexes by Nef leads
to a competitive inhibition of access to the complexes for some
cellular proteins, causing their rerouting by default to the plasma
membrane (22). Consequently, Nef may either up-regulate or
down-regulate the surface expression of a specific protein, depending
on whether the protein in question is competitive with Nef for AP
complexes and is excluded from AP-coated transport vesicles, or whether
it is noncompetitive with Nef and is trapped in an expanded endosomal compartment.
Notably, the de novo attachment model leads to the question
of why HIV-1 would induce the membrane association of AP complexes independently of the physiologic regulatory mechanism. One possibility is that such an activity enables the recruitment of AP complexes to
nonphysiologic locations. For example, Nef is a virion-associated protein (65), and it may recruit AP complexes to sites of virion assembly along the plasma membrane. This could facilitate an aspect of
viral morphogenesis such as the incorporation of the envelope glycoprotein, the cytoplasmic domain of which contains AP-binding motifs (66, 67). In support of this scenario, the leucine-based AP-binding motif in Nef is required not only for the membrane stabilization of AP complexes described here but also for the optimal
infectivity of HIV-1 virions (22).
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
chain
are competitive for binding to the
subunit of AP-2, the direct
binding of HIV-1 Nef to intact AP-2 is extremely weak (9, 24).
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
chain were constructed in the pRcCMV plasmid (Invitrogen) as described (14). Nef point mutants were generated by
PCR-directed mutagenesis using appropriate primers as described (14).
The pCNstop plasmid used for expression of CD8Stop, which lacks a
cytoplasmic tail, was provided by A. Baur (Erlangen, Germany). The
plasmid vector for the expression of the Nef-GFP fusion has been
described (23). Plasmids for the expression in Escherichia coli of the wild-type or mutated Nef fused to GST were constructed in the pGEX-4T2 plasmid (Amersham Biosciences) as described (6). Plasmids expressing ARF1/T31N-HA and ARF1-HA were provided by Julie
Donaldson (45). The plasmid expressing ARF1-GAP-His was provided by
Victor Hsu (36, 46).
-adaptin: murine monoclonal antibodies (mAbs) 100.3 (Sigma) and
88 (Transduction Laboratories); anti-
-adaptin: murine mAb AP6
(provided by A. Benmerah); anti-
-adaptin: rabbit
polyclonal Ab provided by B. Hoflack and M. Robinson;
anti-HA epitope: rat mAb 3F10 (Roche Molecular Biochemicals), or rabbit
polyclonal antibody HA.11 (BabCO); fluorescein
isothiocyanate-conjugated anti-CD8: murine mAb CD8-fluorescein isothiocyanate (Coulter Coultronics); anti-poly-His (Covance); anti-transferrin receptor: murine mAb anti-CD71 (Sigma); anti-CD63: murine mAb CLB (Diagnostic Research); anti-CD4: murine mAb Leu3A (BD
Biosciences); and anti-class I MHC: murine mAb W6/32 (Sera Lab).
Secondary antibodies against the mouse, rabbit, and rat IgGs and
conjugated to Cy3, Cy5, rhodamine-X, or Texas Red were from Jackson
ImmunoResearch. A secondary antibody against rabbit IgG and conjugated
to Alexa 647 was from Molecular Probes. Horseradish peroxidase-conjugated anti-mouse and rabbit IgG were from Dako.
-adaptin staining, cells were fixed in 4%
paraformaldehyde in PBS, quenched for 10 min with 0.1 M
glycine in PBS and permeabilized for 10 min with 0.1% Triton in PBS.
For anti-
-adaptin staining, cells were either fixed with methanol
for 10 min at
20 °C and then permeabilized as described above or
fixed with 3% paraformaldehyde and permeablized with 0.1% Nonidet
P-40. After permeabilization, cells were incubated for 30 min with
0.2% bovine serum albumin in PBS, then successively incubated for 30 min at room temperature with primary and secondary antibody mixtures to
stain the adaptins and HA or poly-His epitope tags. Cells were then
washed, blocked for 10 min with 10% mouse serum in PBS, and stained
for 30 min with CD8-fluorescein isothiocyanate mAb. Cells transfected
with plasmid expressing Nef-GFP were stained only by indirect
immunofluorescence for the indicated adaptin. Coverslips were mounted
on slides using immunofluor mounting medium (ICN). Confocal microscopy
was performed with a Bio-Rad MRC1000 instrument or a Zeiss microscope
with a Bio-Rad laser scanning confocal attachment. Images were
collected using single fluorescence excitation and acquisition; the
absence of crossover between the signals from the doubly and triply
labeled cells was confirmed using appropriate controls. Images were
processed using Adobe Photoshop software.
17]ARF1, a truncated form of ARF1 lacking the first 17 N-terminal amino acids, was purified by gel filtration (Sephacryl S200
HR, Amersham Biosciences) (48); ARNO Sec7 domain (residues 50-252 of
ARNO) was isolated by anion exchange on QAE-Sepharose and gel
filtration on Sephacryl S-100 HR (Amersham Biosciences) as described
previously (49); and ARF-GAP1 (50). Full-length His-ARNO and
His-Cytohesin1 were purified by nickel affinity chromatography on
POROS-MC20 (Perceptive Biosystem).
- and anti-
-adaptin antibodies and
chemiluminescent detection; the signals were quantified using NIH Image Software.
17]ARF1 was loaded with
[
-32P]GTP in the presence of purified ARNO-Sec7 as the
guanine nucleotide exchange factor. [
17]ARF1 (500 nM)
was incubated in the presence of 15 µM ARNO-Sec7, 50 mM Hepes (pH 7.5), 100 mM KCl, 1 mM
dithiothreitol, 1 mM MgCl2, 1 mM
AMP-PNP, 1 µCi/ml [
-32P]GTP (6000 Ci/mmol), and 10 µM unlabeled GTP. Loading proceeded for 15 min at
25 °C, and free GTP was removed by gel filtration. The amount of GTP
that became ARF-bound was determined following nitrocellulose filtration.
17]ARF1--
Nucleotide
exchange on [
17]ARF1 was measured as an increase in tryptophan
fluorescence as described (51). Experiments were performed at 37 °C
in the presence 500 nM [
17]ARF1, 50 mM
Tris (pH 7.5), 100 mM KCl, 1 mM dithiothreitol,
and 1 mM MgCl2. The reaction was initiated by
the addition of 10 µM GTP.
17]ARF1 was
assayed by a modification of the assay described (52). GAP assays
contained 150 nM [
-32P]GTP-loaded
[
17]ARF1, 25 mM Hepes (pH 7.4), 5 mM
MgCl2, 100 mM KCl, 1 mM
dithiothreitol, and 0.5 mM AMP-PNP, and GAP1 as indicated in a final volume of 40 µl. Reactions were incubated at 25 °C in
the absence of coatomer with the indicated amounts of ARF-GAP1, or in
the presence of coatomer (100 nM) and 300 nM
ARF-GAP1. Reactions were terminated by the addition of 0.5 ml of cold
charcoal suspension (5% charcoal in 50 mM
NaH2PO4). Following centrifugation, the amount
of 32Pi in the supernatant was determined.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (29K):
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Fig. 1.
Nef co-localizes with AP-1 and AP-3 in a
juxtanuclear region near the cell center. A,
subcellular distribution of Nef-GFP. HeLa cells were transfected with a
plasmid expressing Nef with GFP appended to its C terminus
(Nef-GFP). Twenty-four hours later, the cells were fixed and
stained using indirect immunofluorescence for -adaptin (a specific
subunit of the AP-1 complex), then examined by confocal microscopy.
Green, GFP; red,
-adaptin. B,
subcellular distribution of CD8-Nef. HeLa cells were transfected with a
plasmid expressing Nef as the cytoplasmic domain of the transmembrane
protein, CD8 (CD8-Nef). Twenty-four hours later, the cells were fixed
and stained using indirect immunofluorescence for the indicated
adaptins (red), followed by direct immunofluorescence for
CD8-Nef (green). The cells were visualized using confocal
microscopy.
-Adaptin is a specific subunit of AP-2;
-adaptin is
a specific subunit of AP-3. Scale bar, 10 µm.
View larger version (27K):
[in a new window]
Fig. 2.
Nef expands the perinuclear recycling
endosomal compartment, where it sequesters CD4 and class I MHC.
HeLa cells were transfected with the plasmid expressing CD8-Nef, then
stained by indirect immunofluorescence for the indicated cellular
proteins (middle column, red) and by direct
immunofluorescence for CD8-Nef (left column,
green). Overlaps are shown in the right column.
Tf-R, transferrin receptor. Scale bar, 10 µm.
View larger version (25K):
[in a new window]
Fig. 3.
Nef confers resistance of AP-1 and AP-3 to
the membrane-dissociating effect of brefeldin A. HeLa cells were
transfected with the plasmid that expresses Nef-GFP (panel
A) or CD8-Nef (panel B), then examined by confocal
immunofluorescence microscopy to detect Nef-GFP and CD8-Nef
(green) and either AP-1 ( -adaptin, red) or
AP-3 (
-adaptin, red). Where indicated in panel
A, cells were treated with 5 µg/ml BFA for 30 min before
fixation and staining; where indicated in panel B, cells
were treated with 10 µg/ml BFA for 15 min before fixation and
staining. Scale bar, 10 µm.
View larger version (19K):
[in a new window]
Fig. 4.
Nef overcomes the membrane-dissociating
effects of the dominant negative ARF1/T31N mutant and the
overexpression of ARF-GAP1. A, HeLa cells were transfected
with a plasmid expressing the epitope-tagged, dominant negative mutant
ARF1/T31N-HA, either with or without the plasmid that expresses
CD8-Nef. Twenty-four hours later, the cells were fixed and stained
using indirect immunofluorescence for AP-1 ( -adaptin,
red) and for ARF1/T31N-HA (blue), followed by
direct immunofluorescence for CD8 (green). The upper
row of images shows cells that were transfected only with the
plasmid expressing ARF1/T31N-HA, and the lower row of images
shows cells co-transfected with both the plasmids expressing
ARF1/T31N-HA and CD8-Nef. Cells expressing ARF1/T31N-HA are indicated
by arrows. Scale bar, 10 µm. B, HeLa
cells were transfected with a plasmid expressing ARF1-GAP
(polyhistidine-tagged), either with or without the plasmid expressing
CD8-Nef. Twenty-four hours later, the cells were fixed and stained
using indirect immunofluorescence for AP-1 (
-adaptin,
red) and for ARF1-GAP (blue), followed by direct
immunofluorescence for CD8 (green). The upper row
of images shows cells that were transfected only with the plasmid
expressing ARF1-GAP, and the lower row of images shows cells
that were co-transfected with both plasmids expressing ARF1-GAP and
CD8-Nef. Cells expressing ARF1-GAP are indicated by arrows.
Scale bar, 10 µm.
17]ARF1, could be
activated by the Sec7 domain of ARNO and bound GTP efficiently under
the same conditions. Second, stoichiometric amounts of GST-Nef had no
effect on the kinetics of spontaneous nucleotide exchange on
17-ARF1, measured by tryptophan fluorescence (Fig. 5B).
Nef also had no effect on the exchange activity of catalytic amounts of the ARF1-GEPs ARNO (Fig. 5B) or cytohesin-1 (data not
shown). Third, GST-Nef did not affect the activity of ARF-GAP1, either at high ARF1-GAP concentrations (Fig. 5C) or in the presence
of coatomer (Fig. 5D), which strongly increases ARF-GAP1
activity (52, 61).
View larger version (19K):
[in a new window]
Fig. 5.
Nef does not bind GTP, nor does it modulate
ARF1 or its regulators. A, Nef does not bind GTP. GST-Nef or
[ 17]ARF1 (used as a positive control) were incubated with
[
-32P]GTP for 15 min with ARNO-Sec7 domain under the
conditions described for ARF1 loading (see "Experimental
Procedures"). The percentage of bound [32P]GTP was
determined by filtration onto nitrocellulose membranes. B,
Nef has no effect on spontaneous or ARNO-activated nucleotide exchange
on [
17]ARF1. Nucleotide exchange on [
17]ARF1 (0.5 µM) was assayed by fluorescence over time. GST-Nef or GST
(0.5 µM) were added at t = 300 s,
and ARNO (0.125 µM) at t = 450 s.
C, Nef does not affect GAP1 activity in the absence of
coatomer. Assays of GAP activity contained
[
-32P]GTP-loaded [
17]ARF1 and ARF-GAP1 as
described under "Experimental Procedures." GST and GST-Nef were
included at 25 µM. Reactions were stopped after 20 min of
incubation. Data are the percentage of ARF-bound GTP hydrolyzed.
D, Nef does not affect GAP1 activity in the presence of
coatomer. GAP activity was measured as above using
[
-32P]GTP-loaded [
17]ARF1, ARF-GAP1 (300 nM), and coatomer (100 nM). GST-Nef or GST (5 µM) were included together with GTP-loaded ARF, ARF-GAP1,
and coatomer as indicated. Open circles, GTP hydrolysis
under these conditions without coatomer; filled circles, GTP
hydrolysis with coatomer but without GST or GST-Nef. Data are the
percentage of ARF-bound GTP hydrolyzed.
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[in a new window]
Fig. 6.
Nef preserves the juxtanuclear membrane
association of AP-1 in BFA-treated cells despite the cytosolic
dispersal of ARF1. HeLa cells were transfected with a plasmid
expressing wild-type ARF1 (HA-tagged), either with or without the
plasmid expressing CD8-Nef. Twenty-four hours later, the cells were
fixed and stained using indirect immunofluorescence for AP-1
( -adaptin, red) and ARF1-HA (green), followed
by direct immunofluorescence for CD8-Nef (blue).
Upper and middle panels, cells were transfected
only with the plasmid expressing ARF1-HA and treated (middle
panels) or not (upper panels) with 10 µg/ml BFA for
15 min before fixation and staining. Lower panels, cells
were transfected with both plasmids expressing ARF1-HA and CD8-Nef, and
then treated with 10 µg/ml BFA for 15 min before fixation and
staining. Scale bar, 10 µm.
View larger version (27K):
[in a new window]
Fig. 7.
The BFA-resistant membrane association of
AP-1 and AP-3 requires the leucine-based AP-binding motif in Nef.
A, HeLa cells were transfected with plasmids expressing
either the wild-type Nef (CD8-Nef wt) or mutated CD8-Nef chimeras
(NefL164A/L165A and Nef E160A), then fixed and stained 24 h later
for CD8-Nef (green) and either AP-1 ( -adaptin,
red) or AP-3 (
-adaptin, red). The cells were
treated with 10 µg/ml BFA for 15 min before fixation. Scale
bar, 10 µm. B, GST pull-down assays of the
interaction of Nef with intact AP-1 and AP-3 complexes. HeLa cell
lysates were incubated with equal amounts of purified GST, GST-Nef,
GST-NefL164A/L165A, or GST-NefE160A previously immobilized on
GSH-agarose beads. Bound proteins were resolved by SDS-PAGE and the
association of AP complexes was analyzed by Western blotting with
anti-
-adaptin (AP-1) or anti-
-adaptin
(AP-3); the bands were quantified using NIH Image software
and the data expressed as the percent of binding relative to wild-type
Nef. Unfractionated HeLa cell lysates were run as a control for the
detection of
- and
-adaptin (left lanes). The
Coomassie Blue-stained gel documents the equal relative loading of the
GST fusion proteins on the beads.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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ACKNOWLEDGEMENTS |
---|
We thank Alexandre Benmerah, Bernard Hoflack, Margaret Robinson, Julie Donaldson, and Victor Hsu for the provision of antibodies and plasmids; B. Guibert (LEBS) for protein purifications; M. Franco and P. Chardin for the gift of the ARF1 and ARNO plasmids; W. Kolanus for the gift of the Cytohesin1 plasmid; and Mary MacCaffrey, Roland Le Borgne, and Margaret Robinson for helpful discussions.
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FOOTNOTES |
---|
* This work was supported by grants from Agence Nationale de Recherche sur le SIDA and SIDACTION, Association pour la Recherche contre le Cancer subvention number 4244, National Institutes of Health Grant AI38201, Universitywide AIDS Research Program of the University of California Grant RD98-SD-051, University of California, San Diego, Center for AIDS Research National Institutes of Health Grant AI36214, the Research Center of AIDS and HIV Infection of the San Diego Veterans Affairs Medical Center, and National Center for Microscopy and Imaging Resource at the University of California, San Diego, National Institutes of Health Grant RR04050.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.
§ Contributed equally to the results of this work.
§§ Co-senior author. To whom correspondence may be addressed: INSERM U567, CNRS UMR8104, Universite Paris V, 27 Rue du Faubourg Saint-Jacques, 75014 Paris, France. Tel.: 33-1-40-51-65-78; Fax: 33-1-40-51-65-70; E-mail: benichou@cochin.inserm.fr.
Co-senior author. To whom correspondence may be
addressed: University of California, San Diego, 9500 Gilman Drive, La
Jolla, CA 92093-0679. Tel.: 858-552-7439; Fax: 858-552-7445; E-mail: jguatelli@ucsd.edu.
Published, JBC Papers in Press, December 16, 2002, DOI 10.1074/jbc.M210115200
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ABBREVIATIONS |
---|
The abbreviations used are:
MHC-1, class
I major histocompatibility complex;
AMP-PNP, 5'-adenylyl-,
-imidodiphosphate;
ARF1, ADP-ribosylation factor 1;
GEP, guanine nucleotide exchange proteins;
BFA, brefeldin A;
GAP, GTPase-activating protein;
GST, glutathione S-transferase;
HA, hemagglutinin;
mAb, monoclonal antibody;
PBS, phosphate-buffered
saline;
GFP, green fluorescent protein.
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REFERENCES |
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