HIV-1 Nef Stabilizes the Association of Adaptor Protein Complexes with Membranes*

Katy JanvierDagger §, Heather Craig§, Douglas Hitchin, Ricardo MadridDagger , Nathalie Sol-Foulon||, Louis Renault**, Jacqueline Cherfils**, Dan CasselDagger Dagger , Serge BenichouDagger §§, and John Guatelli¶¶||||

From the Dagger  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 Dagger Dagger  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

    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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.

    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 gamma  chain are competitive for binding to the beta  subunit of AP-2, the direct binding of HIV-1 Nef to intact AP-2 is extremely weak (9, 24).

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.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 alpha  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).

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-gamma -adaptin: murine monoclonal antibodies (mAbs) 100.3 (Sigma) and 88 (Transduction Laboratories); anti-alpha -adaptin: murine mAb AP6 (provided by A. Benmerah); anti-delta -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.

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-delta -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-gamma -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.

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: [Delta 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).

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-gamma - and anti-delta -adaptin antibodies and chemiluminescent detection; the signals were quantified using NIH Image Software.

Guanine Nucleotide Binding, Exchange, and GTP-hydrolysis Assays

GTP Loading of ARF1-- [Delta 17]ARF1 was loaded with [gamma -32P]GTP in the presence of purified ARNO-Sec7 as the guanine nucleotide exchange factor. [Delta 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 [gamma -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.

GDP/GTP Exchange on [Delta 17]ARF1-- Nucleotide exchange on [Delta 17]ARF1 was measured as an increase in tryptophan fluorescence as described (51). Experiments were performed at 37 °C in the presence 500 nM [Delta 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.

ARF GAP Assays-- Hydrolysis of GTP on [Delta 17]ARF1 was assayed by a modification of the assay described (52). GAP assays contained 150 nM [gamma -32P]GTP-loaded [Delta 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
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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.


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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 gamma -adaptin (a specific subunit of the AP-1 complex), then examined by confocal microscopy. Green, GFP; red, gamma -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. alpha -Adaptin is a specific subunit of AP-2; delta -adaptin is a specific subunit of AP-3. Scale bar, 10 µm.


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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.


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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 (gamma -adaptin, red) or AP-3 (delta -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.

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).


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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 (gamma -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 (gamma -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.

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, [Delta 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 Delta 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).


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Fig. 5.   Nef does not bind GTP, nor does it modulate ARF1 or its regulators. A, Nef does not bind GTP. GST-Nef or [Delta 17]ARF1 (used as a positive control) were incubated with [gamma -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 [Delta 17]ARF1. Nucleotide exchange on [Delta 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 [gamma -32P]GTP-loaded [Delta 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 [gamma -32P]GTP-loaded [Delta 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.

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.


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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 (gamma -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.

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.


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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 (gamma -adaptin, red) or AP-3 (delta -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-gamma -adaptin (AP-1) or anti-delta -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 gamma - and delta -adaptin (left lanes). The Coomassie Blue-stained gel documents the equal relative loading of the GST fusion proteins on the beads.

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.

    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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).

    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.

    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

    ABBREVIATIONS

The abbreviations used are: MHC-1, class I major histocompatibility complex; AMP-PNP, 5'-adenylyl-beta ,gamma -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.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

1. Deacon, N. J., Tsykin, A., Solomon, A., Smith, K., Ludford-Menting, M., Hooker, D. J., McPhee, D. A., Greenway, A. L., Ellett, A., Chatfield, C., Lawson, V. A., Crowe, S., Maerz, A., Sonza, S., Learmont, J., Sullivan, J. S., Cunningham, A., Dwyer, D., Dowton, D., and Mills, J. (1995) Science 270, 988-991[Abstract]
2. Aiken, C., Konner, J., Landau, N. R., Lenburg, M., and Trono, D. (1994) Cell 76, 853-864[Medline] [Order article via Infotrieve]
3. Mangasarian, A., Foti, M., Aiken, C., Chin, D., Carpentier, J.-L., and Trono, D. (1997) Immunity 6, 67-77[Medline] [Order article via Infotrieve]
4. Schwartz, O., Marechal, V., Le Gall, S., Lemonnier, F., and Heard, J.-M. (1996) Nat. Med. 2, 338-342[Medline] [Order article via Infotrieve]
5. Bresnahan, P. A., Yonemoto, W., Ferrell, S. S., Williams-Herman, D. G. R., and Greene, W. C. (1998) Curr. Biol. 8, 1235-1238[Medline] [Order article via Infotrieve]
6. Benichou, S., Bomsel, M., Bodeus, M., Durand, H., Doute, M., Letourneur, F., Camonis, J., and Benarous, R. (1994) J. Biol. Chem. 269, 30073-30076[Abstract/Free Full Text]
7. Le Gall, S., Erdtmann, L., Benichou, S., Berlioz-Torrent, C., Liu, L., Benarous, R., Heard, J.-M., and Schwartz, O. (1998) Immunity 8, 483-495[Medline] [Order article via Infotrieve]
8. Piguet, V., Chen, Y.-L., Mangasarian, A., Foti, M., Carpentier, J.-L., and Trono, D. (1998) EMBO J. 17, 2472-2481[Abstract/Free Full Text]
9. Greenberg, M., DeTulleo, L., Rapoport, I., Skowronski, J., and Kirchhausen, T. (1998) Curr. Biol. 8, 1239-1242[Medline] [Order article via Infotrieve]
10. Lama, J., and Ware, C. F. (2000) J. Virol. 74, 9396-9402[Abstract/Free Full Text]
11. Swigut, T., Shohdy, N., and Skowronski, J. (2001) EMBO J. 20, 1593-1604[Abstract/Free Full Text]
12. Stumptner-Cuvelette, P., Morchoisne, S., Dugast, M., Le Gall, S., Raposo, G., Schwartz, O., and Benaroch, P. (2001) Proc. Natl. Acad. Sci. U. S. A. 98, 12144-12149[Abstract/Free Full Text]
13. Sol-Foulon, N., Moris, A., Nobile, C., Boccaccio, C., Engering, A., Abastado, J. P., Heard, J. M., van Kooyk, Y., and Schwartz, O. (2002) Immunity 16, 145-155[Medline] [Order article via Infotrieve]
14. Erdtmann, L., Janvier, K., Raposo, G., Craig, H. M., Benaroch, P., Berlioz-Torrent, C., Guatelli, J. C., Bernarous, R., and Benichou, S. (2000) Traffic 1, 871-883[CrossRef][Medline] [Order article via Infotrieve]
15. Sanfridson, A., Hester, S., and Doyle, C. (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 873-878[Abstract/Free Full Text]
16. Hirst, J., and Robinson, M. S. (1998) Biochim. Biophys. Acta 1404, 173-193[Medline] [Order article via Infotrieve]
17. Le Borgne, R., and Hoflack, B. (1998) Curr. Opin. Cell Biol. 10, 499-503[CrossRef][Medline] [Order article via Infotrieve]
18. Kirchhausen, T., Bonifacino, J. S., and Riezman, H. (1997) Curr. Opin. Cell Biol. 9, 488-495[CrossRef][Medline] [Order article via Infotrieve]
19. Trowbridge, I. S., Collawn, J. F., and Hopkins, C. R. (1993) Annu. Rev. Cell Biol. 9, 129-161[CrossRef]
20. Letourneur, F., and Klausner, R. D. (1992) Cell 69, 1143-1157[Medline] [Order article via Infotrieve]
21. Pond, L., Kuhn, L. A., Teyton, L., Schutze, M.-P., Tainer, J. A., Jackson, M. R., and Peterson, P. A. (1995) J. Biol. Chem. 270, 19989-19997[Abstract/Free Full Text]
22. Craig, H. M., Pandori, M. W., and Guatelli, J. C. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 11229-11234[Abstract/Free Full Text]
23. Craig, H. M., Reddy, T. R., Riggs, N. L., Dao, P. P., and Guatelli, J. (2000) Virology 271, 9-17[CrossRef][Medline] [Order article via Infotrieve]
24. Bresnahan, P. A., Yonemoto, W., and Greene, W. C. (1999) J. Immunol. 163, 2977-2981[Abstract/Free Full Text]
25. Honing, S., Sandoval, I. V., and von Figura, K. (1998) EMBO J. 17, 1304-1314[Abstract/Free Full Text]
26. Darsow, T., Burd, C. G., and Emr, S. D. (1997) J. Cell Biol. 142, 913-922[Abstract/Free Full Text]
27. Stamnes, M. A., and Rothman, J. E. (1993) Cell 73, 999-1005[Medline] [Order article via Infotrieve]
28. Traub, L. M., Ostrom, J. A., and Kornfeld, S. (1993) J. Cell Biol. 123, 561-573[Abstract]
29. Ooi, C. E., Dell'Angelica, E. C., and Bonifacino, J. S. (1998) J. Cell Biol. 142, 291-402
30. Simpson, F., Peden, A. A., Christopoulou, L., and Robinson, M. S. (1997) J. Cell Biol. 137, 835-845[Abstract/Free Full Text]
31. Donaldson, J. G., and Klausner, R. D. (1994) Curr. Opin. Cell Biol. 6, 527-532[Medline] [Order article via Infotrieve]
32. Helms, J. B., and Rothman, J. E. (1992) Nature 360, 352-354[CrossRef][Medline] [Order article via Infotrieve]
33. Donaldson, J. G., Finazzi, D., and Klausner, R. D. (1992) Nature 360, 350-352[CrossRef][Medline] [Order article via Infotrieve]
34. Robinson, M. S., and Kreis, T. E. (1992) Cell 69, 129-138[Medline] [Order article via Infotrieve]
35. Morinaga, N., Tsai, S. C., Moss, J., and Vaughan, M. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 12856-12860[Abstract/Free Full Text]
36. Cukierman, E., Huber, I., Rotman, M., and Cassel, D. (1995) Science 270, 1999-2002[Abstract]
37. Tanigawa, G., Orci, L., Amherdt, M., Ravazzola, M., Helms, J. B., and Rothman, J. E. (1993) J. Cell Biol. 123, 1365-1371[Abstract]
38. Zhu, Y., Traub, L. M., and Kornfeld, S. (1998) Mol. Biol. Cell 9, 1323-1337[Abstract/Free Full Text]
39. Seaman, M. N., Sowerby, P. J., and Robinson, M. S. (1996) J. Biol. Chem. 271, 25446-25451[Abstract/Free Full Text]
40. Dittie, A. S., Hajibagheri, N., and Tooze, S. A. (1996) J. Cell Biol. 132, 523-536[Abstract]
41. Zhu, Y., Drake, M. T., and Kornfeld, S. (1999) Proc. Natl. Acad. Sci. U. S. A. 96, 5013-5018[Abstract/Free Full Text]
42. Godi, A., Pertile, P., Meyers, R., Marra, P., Di Tullio, G., Iurisci, C., Luini, A., Corda, D., and De Matteis, M. A. (1999) Nat. Cell Biol. 1, 280-287[CrossRef][Medline] [Order article via Infotrieve]
43. Austin, C., Hinners, I., and Tooze, S. A. (2000) J. Biol. Chem. 275, 21862-21869[Abstract/Free Full Text]
44. Boehm, M., Aguilar, R. C., and Bonifacino, J. S. (2001) EMBO J. 20, 6265-6276[Abstract/Free Full Text]
45. Peters, P. J., Hsu, V. W., Ooi, C. E., Finazzi, D., Teal, S. B., Oorschot, V., Donaldson, J. G., and Klausner, R. D. (1995) J. Cell Biol. 128, 1003-1017[Abstract]
46. Aoe, T., Cukierman, E., Lee, A., Cassel, D., Peters, P. J., and Hsu, V. W. (1997) EMBO J. 16, 7305-7316[Abstract/Free Full Text]
47. Paris, S., Beraud-Dufour, S., Robineau, S., Bigay, J., Antonny, B., Chabre, M., and Chardin, P. (1997) J. Biol. Chem. 272, 22221-22226[Abstract/Free Full Text]
48. Antonny, B., Beraud-Dufour, S., Chardin, P., and Chabre, M. (1997) Biochemistry 36, 4675-4684[CrossRef][Medline] [Order article via Infotrieve]
49. Chardin, P., Paris, S., Antonny, B., Robineau, S., Beraud-Dufour, S., Jackson, C. L., and Chabre, M. (1996) Nature 384, 481-484[CrossRef][Medline] [Order article via Infotrieve]
50. Huber, I., Rotman, M., Pick, E., Makler, V., Rothem, L., Cukierman, E., and Cassel, D. (2001) Methods Enzymol. 329, 307-316[CrossRef][Medline] [Order article via Infotrieve]
51. Beraud-Dufour, S., Robineau, S., Chardin, P., Paris, S., Chabre, M., Cherfils, J., and Antonny, B. (1998) EMBO J. 17, 3651-3659[Abstract/Free Full Text]
52. Goldberg, J. (1999) Cell 96, 893-902[Medline] [Order article via Infotrieve]
53. Greenberg, M. E., Bronson, S., Lock, M., Neumann, M., Pavlakis, G. N., and Skowronski, J. (1997) EMBO J. 16, 6964-6976[Abstract/Free Full Text]
54. Baur, A. S., Sawai, E. T., Dazin, P., Fantl, W. J., Cheng-Mayer, C., and Peterlin, B. M. (1994) Immunity 1, 373-384[Medline] [Order article via Infotrieve]
55. Le Borgne, R., Alconada, A., Bauer, U., and Hoflack, B. (1998) J. Biol. Chem. 273, 29451-29461[Abstract/Free Full Text]
56. Dascher, C., and Balch, W. E. (1994) J. Biol. Chem. 269, 1437-1448[Abstract/Free Full Text]
57. Huber, I., Cukierman, E., Rotman, M., Aoe, T., Hsu, V. W., and Cassel, D. (1998) J. Biol. Chem. 273, 24786-24791[Abstract/Free Full Text]
58. Guy, B., Kieny, M.-P., Reviere, Y., La Peuch, C., Dott, K., Girand, M., Montagnier, L., and Lecocq, J.-P. (1987) Nature 330, 266-269[CrossRef][Medline] [Order article via Infotrieve]
59. Kaminchik, J., Bashan, N., Pinchasi, D., Amit, B., Sarver, N., Johnston, M. I., Fischer, M., Yavin, Z., Gorecki, M., and Panet, A. (1990) J. Virol. 64, 3447-3454[Medline] [Order article via Infotrieve]
60. Backer, J. M., Mendola, C. E., Fairhurst, J. L., and Kovesdi, I. (1991) AIDS Res. Hum. Retroviruses 7, 1015-1020[Medline] [Order article via Infotrieve]
61. Szafer, E., Pick, E., Rotman, M., Zuck, S., Huber, I., and Cassel, D. (2000) J. Biol. Chem. 275, 23615-23619[Abstract/Free Full Text]
62. Linnemann, T., Zheng, Y. H., Mandic, R., and Peterlin, B. M. (2002) Virology 294, 246-255[CrossRef][Medline] [Order article via Infotrieve]
63. Teal, S. B., Hsu, V. W., Peters, P. J., Klausner, R. D., and Donaldson, J. G. (1994) J. Biol. Chem. 269, 3135-3138[Abstract/Free Full Text]
64. Piguet, V., Gu, F., Foti, M., Demaurex, N., Gruenberg, J., Carpentier, J.-L., and Trono, D. (1999) Cell 97, 63-73[Medline] [Order article via Infotrieve]
65. Pandori, M. W., Fitch, N. J. S., Craig, H. M., Richman, D. D., Spina, C. A., and Guatelli, J. C. (1996) J. Virol. 70, 4283-4290[Abstract]
66. Ohno, H., Aguilar, R. C., Fournier, M. C., Hennecke, S., Cosson, P., and Bonifacino, J. S. (1997) Virology 238, 305-315[CrossRef][Medline] [Order article via Infotrieve]
67. Wyss, S., Berlioz-Torrent, C., Boge, M., Blot, G., Honing, S., Benarous, R., and Thali, M. (2001) J. Virol. 75, 2982-2992[Abstract/Free Full Text]


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