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Vol. 14, Issue 12, 4857-4870, December 2003
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* Institut National de la Santé et de la Recherche Médicale U520, Institut Curie, Section de recherche. 75005 Paris, France;
Centre National de la Recherche Scientifique Unité Mixte Recherche 144, Institut Curie, Section de recherche. 75005 Paris, France; and
Genethon III, 91002 Evry, France
Submitted April 8, 2003;
Revised August 1, 2003;
Accepted August 22, 2003
Monitoring Editor: Juan Bonifacino
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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; Hanna et al., 1998a,b). However, the underlying cellular and molecular mechanisms of its contribution to disease progression and pathogenesis remain elusive. Nef also enhances virion infectivity and viral production (Miller et al., 1994; Spina et al., 1994; Schwartz et al., 1995; Dorfman et al., 2002). This phenomenon is possibly related to one of the best characterized functions of Nef, the down-regulation of the CD4 cell surface receptor by a posttranslational mechanism (Garcia and Miller, 1991; Aiken et al., 1994; Lundquist et al., 2002). The reduction in cell surface CD4 facilitates viral release by preventing sequestration of the HIV gp120 protein through CD4 and increases the infectivity of viral particles (Lama et al., 1999; Ross et al., 1999).
Nef interferes with the immune system by disrupting antigen recognition at different levels. In addition to the accelerated endocytosis of CD4, part of the T cell receptor machinery, Nef also enhances internalization of the costimulatory CD28 molecule, impeding effective T cell activation (Swigut et al., 2001). Furthermore, Nef induces apoptosis of virus-specific cytotoxic T-lymphocytes via expression of CD95L, while protecting infected cells by modifying the signal transduction pathway regulating apoptosis (Fackler and Baur, 2002). This modification involves a phosphatidylinositol 3-kinase (PI 3-kinase) and a p21-activated kinase, which are both bound and activated by Nef in a sequential manner (Wolf et al., 2001). The fate of cell surface major histocompatibility complex (MHC) class I is also altered in Nef-expressing or HIV-infected cells, leading to HLA-A and -B sequestering in perinuclear compartments (Schwartz et al., 1996; Greenberg et al., 1998; Le Gall et al., 1998). Nef induces, together with PACS-1, MHC I endocytosis from the plasma membrane to the trans-Golgi network (TGN) through the PI3 kinase-mediated ARF6 endosomal pathway (Piguet et al., 2000; Blagoveshchenskaya et al., 2002). The reduced density of HLA-A and -B at the cell surface may allow infected cells to escape recognition and lysis by virus specific CD8+ T cells (Collins et al., 1998; Collins and Baltimore, 1999). Interestingly the surface expression of HLA-C is not modified, protecting infected cells from lysis by NK cells (Le Gall et al., 1998; Cohen et al., 1999). Infection of rhesus macaques with simian immunodeficiency virus carrying a point mutation in its nef gene that selectively disrupts MHC I down-regulation resulted in rapid reversion to wild-type nef, indicating a strong selective advantage of Nef-mediated MHC I down-regulation for viral replication in vivo (Munch et al., 2001).
In the presence of Nef, we demonstrated an increase in the cell surface expression of invariant (Ii) chain-associated, and therefore nonfunctional, MHC II. In contrast, the cell surface expression of peptide-loaded mature MHC II was reduced (Stumptner-Cuvelette et al., 2001). We also showed that up-regulation of surface Ii chain complexes requires lower amounts of Nef than the down-regulation of mature MHC II and that both functions are genetically separable. A recent study has confirmed these results and further shows that both activities are well conserved among nef-alleles from primary HIV-1, HIV-2, and simian immunodeficiency virus (Schindler et al., 2003). Importantly, Ii surface up-regulation was observed for nef-alleles derived from all progressing HIV-1–infected individuals studied but for two of four long-term nonprogressors (Schindler et al., 2003). This suggests that this function of Nef participates to viral immune escape in vivo. Indeed, HIV-specific helper T cell responses are impaired in progressing HIV-1–infected individuals and are strong in long-term nonprogressors (Rosenberg et al., 1997; Pitcher et al., 1999). The capacity of Nef to affect MHC II antigen presentation and therefore the generation of virus-specific T cell help may be important for the control of the infection. To achieve their function of antigen presentation, MHC II traffic through a specific intracellular pathway. Newly synthesized MHC II is made of 
and 
chains, which associate with the Ii chain in the endoplasmic reticulum. The Ii complexes have to traffic through the Golgi apparatus and the endocytic pathway to undergo complete maturation before presenting their antigenic peptide cargo at the cell surface (Hiltbold and Roche, 2002). During this journey, the associated Ii chain is degraded in a sequential manner, and Ii and its fragments drive associated complexes to specialized endocytic compartments, where peptides are loaded onto MHC II (Stumptner-Cuvelette and Benaroch, 2002).
Here, we show that Nef impedes MHC II function through a novel posttranslational mechanism modifying the MHC II intracellular trafficking. Nef expression led to an increased access of Ii complexes at the cell surface and an impairment of the degradation of the Ii chain. We observed by electron microscopy (EM) that Nef expression induced a striking increase in the number of multivesicular bodies (MVBs) in two different cell lines. These structures contained MHC II and large amounts of apparently intact Ii chain. The Nef-induced up-regulation of Ii chain was dependent on a PI 3-kinase activity, which could be recruited and activated by Nef at the MVB level as Nef seemed enriched at the MVB outer membrane. Because phosphoinositides are involved in MVB biogenesis, the Nef-induced increase of MVB numbers might be related to the PI 3-kinase activation. Our results indicate that Nef affects transport of immature MHC II complexes (Ii) to lysosomal compartments, which rather accumulate in MVBs and at the plasma membrane.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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), the mAb L243 (Lampson and Levy, 1980), and the mAb 1B5 (Adams et al., 1983). ICC5 and ICN1 antibodies specific for the C-terminal and N-terminal domains of the Ii chain, respectively, have been generated by P. Morton (Monsanto, St. Louis, MO) and were used for immuno-EM. A rabbit immunesera directed against Ii (Salamero et al., 1996), and three Ii chain-reactive mouse mAbs were also used: PIN1 (Lamb and Cresswell, 1992) directed against the N terminus of Ii, BU45 (Wraight et al., 1990) and LN-2 (BD PharMingen, San Diego, CA) directed against the lumenal part of Ii. In addition, we used mAb W6/32 (Parham et al., 1979) that recognizes mature MHC I complexes, mAb 66Ig10 (van de Rijn et al., 1983) directed against the human transferrin receptor (TfR), mAb MATG 020 directed against Nef, mAb H37/38 directed against hemagglutinin (HA) (Sarukhan et al., 2001), mAb anti-Lamp-1 from BD PharMingen, and an IgG1 mAb anti-CD63 clone CLB-gran (Caltag Laboratories, Burlingame, CA), a sheep immunesera anti-TGN46 (kind gift of V. Ponnambalam, University of Dundee, Dundee, United Kingdom). Secondary antibodies were from Jackson ImmunoResearch Laboratories (West Grove, PA).
Plasmids
We used cytomegalovirus-driven vectors carrying the nef gene from LAI: Nef-FT (Bachelerie et al., 1990), a control plasmid Nef-mock carrying the nef A01 gene in an antisense orientation (Le Gall et al., 1998).
Cell Culture
HeLa and HeLa-CIITA cells (Stumptner-Cuvelette et al., 2001) and Mel JuSo cells were cultured in DMEM containing 4.5 g glucose/l (Invitrogen, Paisley, Scotland) supplemented with 10% fetal calf serum (Dutscher, Brumath, France), 2 mM glutamine, penicillin/streptomycin (100 IU/ml and 100 µg/ml, respectively), 1 mM sodium pyruvate, and 1% nonessential amino acids. HeLa-CIITA cells were grown in the presence of 300 µg/ml hygromycin B (Calbiochem, La Jolla, CA).
Transfections and Infections
For transient transfection experiments, cells were electroporated using a Bio-Rad gene pulser II as described previously (Stumptner-Cuvelette et al., 2001). Briefly, 8 x 106 cells were transfected at 200 V and 975 µF with up to 25 µg of the different expression vectors mixed with 1 µg of pEGFP and completed with the pSP72 plasmid to reach a total of 30 µg of DNA per electroporation. Two recombinant adenoviral vectors were used. rAd-HA codes for the influenza virus HA has been described previously (Sarukhan et al., 2001). For the generation of rAd-Nef-IRES-HA, nef was isolated from pSP73-Nef (kindly provided by O. Schwartz, Pasteur Institute, Paris, France) by SalI/EcoRI digestion and cloned into the XhoI/SalI sites of pIRESeGFP (BD Biosciences Clontech, Palo Alto, CA). The eGFP sequence from pIRESeGFP was excised by BstXI/NotI digestion and replaced by a polylinker harboring the following sites: BstXI-SpeI-PmeI-PvuI-NotI, resulting in NEF-IRES-mcs. The HA cDNA was isolated from HApcDNA3 (kindly provided by D. Pardoll, Johns Hopkins School of Medicine, Baltimore, MD) and cloned into the SpeI/NotI sites of NEF-IRES-mcs. The NEF-IRES-HA cassette was excised by NheI/NotI and cloned into the same sites in pTG6600 (Transgene, Strasbourg, France). The E1-deleted rAd-NEF-IRES-HA vector was generated via homologous recombination as described previously (Chartier et al., 1996).
Flow Cytometry
For surface staining cells were detached 24 h after transfection and stained with various mouse mAbs in phosphate-buffered saline (PBS) containing 3% fetal calf serum and 0.05% azide on ice. Phycoerythrine (PE)-conjugated goat anti-mouse F(ab)'2 fragments were used as secondary reagents. Dead cells were excluded by a gate in foward light scatter/standard saline citrate. Events corresponding to at least 3000 live cells positive for green fluorescent protein (GFP) were accumulated per sample. Flow cytometry analysis was performed with a FACScan using CellQuest software (BD Biosciences, Mountain View, CA).
For permeabilized fluorescence-activated cell sorting (FACS) experiments cells were detached 24 h after transfection, fixed with 1% paraformaldehyde, before quenching with 0.1 M glycine in PBS, and permeabilizing the cells in PBS, 0.2% bovine serum albumin (BSA), 0.05% saponin. Cells were then stained as described above except that the permeabilization buffer was used for dilution of the antibodies and all washing steps.
Internalization experiments were performed with cells recovered 24 h after transfection and incubated in parallel with LN-2 and 66Ig10 mAbs for 30 min on ice. The cells were then washed in ice-cold PBS and incubated at 37°C in complete medium supplemented with 20 mM HEPES for different periods of time. Antibodies remaining at the cell surface were revealed with PE-conjugated anti-mouse F(ab)'2 fragments. Samples were analyzed with a FACScan as described above. Mean fluorescence intensity (MFI) of FL2 on GFP+ cells, from which the background value obtained with a control mAb was deduced, was plotted as a function of time.
Pulse-Chase Analysis, Cell Surface Biotinylation, and Immunoprecipitation
Twenty-four hours after transfection cells were detached, washed, and starved in RPMI met–cys– supplemented with 2 mM L-glutamine and 20 mM HEPES for 45 min at 37°C. Cells were then pulsed for 15 min at 0.5 mCi/ml before incubation in complete RPMI supplemented with 1 mM L-methionine for different periods of time. At the end of the chase, cells were biotinylated for 5 min with NHS-SS-biotin (2 mg/ml in PBS at 4°C; Pierce Chemical, Rockford, IL). Lysis and immunoprecipitations were carried out as described previously (Benaroch et al., 1995). Ten percent of the eluate was kept for subsequent SDS-PAGE analysis (total molecules), and the remaining 90% was incubated with strepavidin-agarose beads (Pierce Chemical) to precipitate the biotinylated fraction. All samples were run on 12% polyacrylamide-SDS gels, analyzed by autoradiography, and quantified with a PhosphorImager (Amersham Biosciences, Piscataway, NJ) by using the ImageQuant software.
Immunofluorescence Microscopy
Cells (1 x 105) were seeded immediately after transfection on glass coverslips in 24-well plates and allowed to adhere for 24 h. Rabbit anti-Ii immune serum, LN-2 mAb, or as a control the irrelevant mAb 9E10 were internalized for 1 h at 19.5°C and then chased for 90 min at 37°C. Lysosomes were identified by incubating the cells during 90 min with LysoTracker Red DND99 (Molecular Probes, Eugene, OR) at 37°C. All subsequent steps were carried out at room temperature. Cells were fixed for 30 min with 4% (wt/vol) paraformaldehyde in PBS followed by quenching with 0.1 M glycine in PBS. Permeabilization and saturation were performed in PBS containing 0.2% BSA and 0.05% (wt/vol) saponin. Primary antibodies were incubated for 1 h. Secondary antibodies coupled to fluorescein isothiocyanate (FITC), Cy-3, or Cy-5 were allowed to bind for 30 min. The coverslips were mounted with Mowiol on glass slides and analyzed using a TCS4D scanning laser confocal microscope (Leica Microscopy and Scientific Instruments, Heerbrugg, Switzerland). Uptake of the irrelevant 9E10 mAb gave only very faint staining showing that given the low mAb concentration used (1 µg/ml), uptake by fluid phase endocytosis was neglectable compared with uptake through Ii chain endocytosis. For conventional microscopy, cells were fixed, permeabilized, and stained as described above. Images were captured using a 100x oil immersion objective on a Leica DMRB microscope using a Nikon ACT-1 version 2.00 software.
Subcellular Fractionation
Cells (16 x 106) in 3.2 ml of 1% fetal calf serum in DMEM were infected with Ad-HA or with Ad-Nef-IRES-HA with a multiplicity of infection of 75 infectious viral particles for 90 min followed by two washes in PBS. Twenty-four hours after infection, cells were detached, washed, and subjected to subcellular fractionation, adapted from Bonnerot et al. (1998). In brief, 3 x 107 cell/ml were incubated in 1x tetraethylammonium (TEA) buffer (10 mM triethanolamine, 1 mM EDTA, 10 mM acetic acid, 250 mM sucrose, pH 7.4) during 10 min at 0°C. Cells were then disrupted in presence of 1 mM phenylmethylsulfonyl fluoride by a ball-bearing homogenizer at 0°C. Nuclei were eliminated by two sequential centrifugations of 7 min at 1600 x g. Protein contents of postnuclear supernatants were determined with a Bradford test and for each cell population, the same amount of proteins was added under a volume of 400 µl in 1x TEA buffer to a mix of Percoll at 90% in 10x TEA buffer (1.6 ml) and 3.1 ml of 1x TEA buffer, in a Beckman Quick Seal centrifuge tube. Tubes were ultracentrifuged for 35 min at 4°C at 33,000 x g. 16 fractions of 300 µl were harvested from which Percoll was eliminated by a NaOH precipitation before analysis by Western blotting for their contents in TfR, Erp57, HLA-DM, HLA-DR, Ii, and Lamp-1. Anti-immunoglobulin secondary antibodies (Jackson ImmunoResearch Laboratories) used to reveal these proteins were coupled to alkaline phosphatase, whose activity was measured with ECF substrate (Amersham Biosciences) allowing quantification on a PhosphorImager (Amersham Biosciences) by using the ImageQuant software. In addition, -hexosaminidase activity was directly measured in the fractions as follows: 40 µl of each fraction was mixed with 12 µl of substrate 10x (4-methyl-umbelliferyl-N-acetyl--D-glucosaminide; Sigma-Aldrich, St. Louis, MO) and 60 µl of 2x buffer (22.4 mM acid citric, 35.2 mM Na2HPO4, pH 4.5, 0.2% Triton X-100). After 3 h of incubation at 37°C, the reaction was read on a multiplate reader (Victor, Wallac, Finland) at 355 nm.
Immunoelectron Microscopy
HeLa-CIITA cells were recovered 24 h after transfection and fixed with a mixture of 2% paraformaldehyde and 0.125% glutaraldehyde in 0.2 M phosphate buffer pH 7.4 for 2 h at room temperature. Mel JuSo cells were infected with Ad-HA or with Ad-Nef-IRES-HA as described for subcellular fractionation experiments. Twenty-four hours after infection, cells were recovered and processed as described above. Fixed cells were processed for ultrathin sectioning and immunolabeling as described previously (Raposo et al., 1997). Quantification of the numbers of MVBs and lysosomes in both Nef-expressing cells and mock-transfected HeLa-CIITA cells was directly performed under the electron microscope. Similar quantifications were achieved in Mel JuSo cells infected with Ad-HA or with Ad-Nef-IRES-HA. In each case, 30 profiles of ultrathin-cryosectionned cells, randomly taken, were analyzed.
For analyzing the uptake and transport of a fluid phase marker of endocytosis HeLa-CIITA cells were recovered 24 h after transfection, washed once in RPMI, and then incubated with BSA coupled to 5-nm colloidal gold particles (OD520 = 5) for 90 min at 37°C in RPMI before fixing the cells and proceeding as described above.
Inhibition of PI 3-Kinase
HeLa-CIITA cells transfected 24 h earlier with Nef-FT or Nef-mock, and pEGFP were processed for either flow cytometry or immunofluorescence. For flow cytometry analysis, the last 15 h of transfection cells were incubated with 50 µM LY294002 (BIOMOL Research Laboratories, Plymouth Meeting, PA) or its solvent, dimethyl sulfoxide (DMSO). For immunofluorescence, the inhibitor or its solvent were added during the last 3 h of transfection.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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and were immunoprecipitated from cells expressing Nef and from control cells (Figure 1A, see 0-h chase lanes and the corresponding histogram). Similar results were obtained in another cell system with a shorter 5-min pulse. This suggests that Nef does not affect the rate of synthesis of Ii, DR, and chains. However, degradation of the Ii chain was slower in cells expressing Nef than in control cells, because both DR and Ii immunoprecipitates contained higher amounts of Ii chain at 4, 8, and 22 h of chase than in control cells in which Ii was hardly detectable at the 8-h chase point (Figure 1A, DR [95°C] and Ii, and the histogram of Ii quantification).
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Depending on the DR alleles, Ii can remain stable in SDS at room temperature, generating complexes of apparent molecular mass of 
100 kDa, which dissociate upon boiling (Stumptner and Benaroch, 1997). Such complexes were observed in immunoprecipitates performed with 1B5, an anti-DR mAb (Figure 1) or with PiN1, an anti-Ii mAb when eluted at room temperature. Therefore, these SDS-stable complexes were in all likelihood Ii complexes. They clearly stayed associated for longer time in the presence of Nef [Figure 1A, DR (RT); see arrowhead]. This indicated that the pool of Ii chain, which was degraded more slowly in the presence of Nef, remained associated with DR molecules. The disappearance of Ii was accompanied by the appearance of SDS-stable complexes, representative of a peptide-loaded state [Figure 1A, DR (RT); see arrowhead and star, respectively]. Quantification of these peptide-loaded SDS-stable complexes at the different chase times gave rise to only slightly reduced values in Nef-FT versus Nef-mock transfected cells (our unpublished data). This may be due in part to the chase times chosen, which may prevent visualization of a delay in the appearance of mature MHC II. In addition, Nef expression led to only a twofold reduction of the surface level of mature MHC II (Stumptner-Cuvelette et al., 2001). To control the experimental conditions, TfR was sequentially immunoprecipitated from the supernatants of Ii chain immunoprecipitations. The levels of synthesis, the rate of acquisition of complex sugars and the degradation of this receptor were very similar in both cell populations (Figure 1A, TfR panel and corresponding histogram), suggesting that the secretory pathway was not affected by Nef expression.
To investigate surface access of the proteins of interest, our pulse-chase experiments included a cell surface biotinylation step before cell lysis. The biotinylated fraction of the immunoprecipitated molecules was further reprecipitated with streptavidin-agarose beads. Under these conditions, streptavidin-precipitated material exclusively represents cell surface-expressed molecules. Whereas both cell populations contained similar total levels of Ii complexes after 4 h of chase (Figure 1A, DR and Ii), a much larger fraction of these complexes had reached the cell surface in the case of Nef expressing cells (Figure 1B; see corresponding panels). Ii chain at the cell surface remained associated with MHC II chains (Figure 1B). In Nef-expressing cells, the disappearance of the total population of Ii complexes paralleled the situation observed at the cell surface (compare Ii histograms Figure 1, A and B). This disappearance was concomitant with the appearance at the cell surface of SDS-stable -peptide complexes whose production was not significantly altered by Nef (Figure 1B). The transport kinetics of the TfR to the cell surface remained similar in both cell types (Figure 1B).
We conclude that expression of Nef does not affect the synthesis of MHC II and Ii but reduces the rate of degradation of Ii associated to MHC II. Newly synthesized Ii complexes accumulate at the cell surface in the presence of Nef, but neither the formation nor the transport to the cell surface of mature MHC II are significantly impaired.
Nef Induces Intracellular Accumulation of Mature MHC II
Next, the effect of Nef expression on intracellular versus surface MHC II levels was evaluated. HeLa-CIITA cells cotransfected with pEGFP and either Nef-FT or Nef-mock were analyzed by FACS analysis for cell surface staining on intact cells and total staining on permeabilized cells. Note that MFI values obtained on permeabilized cells cannot be directly compared with those obtained with intact cells due to the permeabilization procedure, which partially affects the surface levels. Although Nef reduced surface levels of mature MHC II by 50%, total levels of mature MHC II were not affected by Nef (Figure 2A). This indicated that Nef expression induced an intracellular accumulation of mature MHC II. Similar conclusions could be drawn from our data regarding MHC I (Figure 2A) in accordance with the Nef-induced accumulation of MHC I observed at the Golgi level (Le Gall et al., 1998; Piguet et al., 2000). Surface and total levels of Ii chain as detected with a mAb specific for the Ii lumenal domain were significantly increased in Nef-expressing cells. Therefore, the increase of total Ii chain might reflect at least in part the increase of surface Ii chain but may also mean that Ii containing complexes accumulate intracellularly.
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Because more Ii complexes seemed to gain access to the cell surface in the presence of Nef, their fate was investigated in normal HeLa-CIITA cells and in cells transfected with Nef-FT or Nef-mock. Cells exposed to 20 nM ConB (concanamycin B) overnight were used as a positive control for surface Ii chain expression, because this potent v-ATPase inhibitor induces expression of Ii complexes at the surface of Epstein-Barr virus-transformed B cells (Benaroch et al., 1995). The various cell populations were incubated on ice with LN2, a mAb specific for the lumenal side of the Ii chain. After washing, cells were shifted to 37°C for various lengths of time and then fixed and stained with secondary antibodies specific for mouse IgG. As described above, transfected cells were identified and gated on the basis of their GFP expression. In Nef-expressing cells, 50% of the surface bound Ii mAb was removed within 20 min, whereas the kinetics observed in ConB-treated cells was slightly slower (Figure 2B). Disappearance of surface bound anti-Ii mAb correlated with its intracellular appearance by confocal microscopy (see below). Low levels of surface Ii expressed on control cells (either Nef-mock transfected cells or nontransfected cells) prevented accurate determination of the uptake (Figure 2B). As a control, we followed the fate of a surface bound mAb specific for the TfR, which was rapidly internalized in both cell populations (80% in 10 min), in agreement with previous studies showing that Tf uptake was not affected by Nef expression in T cells (Foti et al., 1997) and in HeLa cells (Johannes et al., 2003). These data together with the pulse-chase surface biotinylation experiments (Figure 1B) suggest that in Nef-expressing cells, surface Ii complexes are internalized.
Surface Immature MHC II Are Delayed on Their Way to Lysosomal Compartments after Internalization in Nef-expressing Cells
To follow the fate of Ii complexes after their internalization, HeLa-CIITA cells transfected with Nef-FT or with Nef-mock were incubated first for 60 min at 19.5°C in the presence of appropriate anti-Ii antibodies. At this temperature, endocytosed material is blocked in the early endosome in HeLa cells (Dunn et al., 1980; Mallard et al., 1998). Cells were then washed and chased at 37°C for 90 min before fixation, permeabilization, and staining with labeled secondary antibodies to reveal the internalized antibodies. Using this procedure, even control cells internalized detectable levels of mAb despite their low level of surface Ii. We first established by three-color confocal microscopy analysis that only transfected cells, expressing GFP and Nef, were heavily labeled with internalized anti-Ii mAb. The latter mainly consisted of a vesicular staining, concentrated in a perinuclear area where it partially but significantly codistributed with Nef but not with TGN46 (Figure 3A). In Nef-expressing cells, internalized anti-Ii mAb poorly codistributed with lysosomal/acidic structures labeled with LysoTracker (Figure 3B) or with antibodies specific for Lamp-1, despite the long chase time used. In contrast, in Nef-mock transfected cells, a significant codistribution of stainings was observed (Figure 3B), showing that the antibodies bound to the Ii chain can reach acidic compartments and remain detectable. Note that in control cells, both markers localized in small dots scattered all over the cells, whereas in cells expressing Nef, they were retained in larger but distinct structures polarized on one side of the cells (Figure 3). Additional stainings were performed under similar conditions, by using TfR-specific mAb instead of LysoTracker (Figure 3B) or by exposing cells to Tf-Cy3 for 1 h. Both procedures gave identical results. As expected in control cells, no codistribution was seen, indicating that anti-Ii chain antibodies and probably the Ii-containing complexes had left TfR+ compartments to reach lysosomes. In contrast, in Nef-expressing cells both markers exhibited a partial codistribution in a perinuclear area (Figure 3B). Finally, the use of rab11-specific antibodies in Nef-expressing cells resulted in very limited colocalization with internalized anti-Ii antibodies (our unpublished data). These data suggest that in the presence of Nef, internalized anti-Ii antibodies, and therefore probably Ii chain-containing complexes are delayed on their way to lysosomal compartments and accumulate in perinuclear compartments, partially colocalizing with TfR+ structures.
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Nef Effect on Transport to Lysosomes
To further establish the Nef-induced delay in transport of immature MHC II to lysosomes and to evaluate the specificity of this inhibition, subcellular fractionation experiments were carried out. Such biochemical analysis requires homogenous cell populations expressing Nef. For this purpose, we made a recombinant adenovirus encoding Nef-Ires-HA (see MATERIALS AND METHODS). The HA protein can be detected by FACS at the cell surface of infected cells, and these HA-positive cells also express Nef. Cells infected with a virus encoding HA alone were used as controls. Infection efficiency exceeded 90% in both cell populations as judged by HA expression monitored by FACS. The Nef-induced modifications of cell surface markers such as Ii and MHC I and II were checked in parallel and gave results similar to the ones depicted in Figure 2A. Control and Nef-expressing cells were disrupted using a ball-bearing homogenizer, and their postnuclear supernatants were subjected to Percoll density gradient fractionation. Western blotting of the fractions showed that this procedure successfully separated light organelles such as the endoplasmic reticulum (ER) from heavy ones such as lysosomes. ER-containing fractions (number 10–15) were identified using calreticulin and Erp57-specific antibodies (our unpublished data). Lysosome-containing fractions (number 2–5) were identified by monitoring -hexosaminidase activity and reactivity for Lamp-1 and HLA-DM-specific antibodies. In control cells, MHC II was distributed in two peaks. The first one (fractions 10–15) is probably composed of MHC II associated with plasma membrane, endosomes as well as ER membranes. The second one (fractions 2–5) is enriched in lysosomes. Nef expression profoundly modified the distribution of Ii and MHC II chains. In the presence of Nef, dense fractions contained much lower levels (almost undetectable) of Ii chain and lower amounts of MHC II. In light fractions the reverse was true, i.e., both Ii chain and MHC II levels were increased compared with control cells. As previously observed (Stumptner-Cuvelette et al., 2001) and in agreement with the pulse-chase experiments (Figure 1), the total amount of Ii chain found in Nef-expressing cells was much higher than in control cells (Figure 4). In contrast, Nef expression did not alter the distribution of Lamp-1, HLA-DM, and -hexosaminidase (Figure 4), indicating that protein transport to lysosomes normally occurs in the presence of Nef. This conclusion is supported by our observation that EGF degradation was not affected by Nef expression (Johannes et al., 2003).
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Together, our data suggest that Nef expression leads to a specific inhibition of transport to lysosomal compartments of Ii complexes.
Nef-expressing Cells Accumulate MVBs
To gain insights into the Nef-induced modifications of the endocytic pathway, we performed immunogold labeling on ultrathin cryosections of Nef or mock-transfected HeLa-CIITA cells. Nef labeling was mainly associated with intracellular membranes, such as the inner face of the plasma membrane (our unpublished data), in the Golgi area, and all along endosomal compartments (Figure 5). The association of Nef with tubulovesicular elements in the TGN area seemed particularly evident (Figure 5) and might be related to the capacity of Nef to interact with AP-1 and PACS-1 complexes. Interestingly, Nef labeling was often observed at the limiting membrane of MVBs (Figure 5, top inset) and also in tubulovesicular structures closely apposed to these organelles (Figure 5, bottom inset). These elements often exhibited electron-dense areas suggestive of cytosolic coats (Figure 5, arrow, bottom inset). Nef expression did not affect the morphology of the Golgi apparatus (Figure 5) but induced a striking accumulation of MHC II-positive multivesicular endosomes or MVBs (Figure 6). These MVBs were often accumulated near the nucleus (our unpublished data) and were distinct from the Lamp-1-positive lysosomal compartments containing both internal vesicles and multilaminar membranes (Figure 6, B and C). Our immunoelectron microscopy observations indicated that in the presence of Nef, MVBs were more abundant compared with lysosomes than in mock-transfected cells. Moreover, they often seemed increased in size (with a diameter up to 700 nm, whereas in control cells it is closer to 300 nm) and contained tightly packed intralumenal vesicles (Figures 6 and 8). We have therefore estimated the numbers of MVBs and lysosomes (defined on the basis of their morphological appearance and content; Figure 6) in Nef-expressing HeLa-CTIIA and Mel JuSo cells compared with control cells (Figure 7). In both cell lines, Nef expression induced a substantial and similar increase in the numbers of MVBs compared with the numbers of lysosomes. These MVBs contained MHC II and CD63, which is a classical marker for late endosomes also present in MHC II-rich compartments (Figure 8A). Invariant chain detected by cytosolic-specific (ICN1) or lumenal-specific (ICC5) antibodies was also abundant in these MVBs (Figure 8, B and C, respectively). ICC5 antibodies decorated 10% of the MVBs in control cells, whereas the majority of the MVBs were positive for ICC5 in Nef-expressing cells. Moreover, Ii chain seemed more concentrated on internal vesicles than on the limiting membrane (Figure 8, B and C). In agreement with FACS and biochemical data, Ii chain was clearly seen by immuno-EM at the plasma membrane of Nef-expressing cells (our unpublished data). Finally, access of BSA-gold, a marker of fluid phase endocytosis to these compartments was monitored. Cells transfected with Nef-FT or Nef-mock were exposed to BSA-gold for 2 h at 37°C. BSA-gold was detected in the Nef-induced MVBs, further confirming their endocytic origin (Figure 8C). However, the number of gold particles per compartment was smaller in Nef-expressing cells, possibly due to the increased number of MVBs. Accumulation of LysoTracker during 1 h at 37°C measured by FACS (Stumptner-Cuvelette et al., 2001), as well as accumulation of the weak base DAMP measured by immuno-EM, were comparable in cells expressing Nef versus control cells (our unpublished data), indicating that acidification of the endosomal pathway was not affected by Nef expression.
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We concluded that Nef expression induced a strong increase in the number of MVBs, which were filled with numerous internal vesicles. These MVBs belong to the endocytic pathway because they were accessible to a marker of fluid phase endocytosis. Finally, the MVBs were surrounded by Nef, seemed to contain high amounts of Ii chain and MHC II and thus may represent the location, where immature and mature MHC II complexes accumulate in the presence of Nef.
Nef-induced Up-Regulation of Surface Ii Chain Is Inhibited by LY294002 Exposure
Proteins that regulate intracellular trafficking and that interact with Nef are candidates for cellular effectors of Nef. Interestingly, Nef has been shown to be able to bind and activate a PI 3-kinase (Schibeci et al., 2000; Wolf et al., 2001; Linnemann et al., 2002). This enzyme is involved in Nef-induced down-regulation of MHC I (Swann et al., 2001; Blagoveshchenskaya et al., 2002). To test whether a PI 3-kinase is involved in Nef's effect on MHC II, we treated Nef- or mock-transfected HeLa-CIITA cells with LY294002, a specific inhibitor of PI 3-kinases. This agent blocked the ability of Nef to induce Ii chain surface expression but had no effect on Nef's effects on mature MHC II nor on MHC I as assayed by flow cytometry (Figure 9A). This latter result is in agreement with the published observation that Nef induces down-regulation of HLA-A2 is inhibited by LY294002 in T cells and astrocytic cells but not in HeLa cells (Kasper and Collins, 2003). The modifications induced by the inhibitor were further examined on samples stained for Ii chain and examined by immunofluorescence (Figure 9B). As previously shown in Mel JuSo cells (Fernandez-Borja et al., 1999), LY294002 exposure of control cells induced large vacuoles. Ii chain staining became exclusively concentrated in these vacuoles. Nef-expressing cells treated with DMSO showed the expected Ii chain surface staining, whereas LY294002 exposure drastically reduced Ii chain surface staining, which seemed concentrated in intracellular compartments of intermediate size. Confirming our previous mutational analysis of Nef effects, these data indicate that effects of Nef on mature and immature MHC II probably involve different cellular effectors. The PI 3-kinase activated by Nef seems to be required for the effects of Nef on Ii chain and therefore on immature MHC II.
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DISCUSSION |
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). One of the many effects of Nef is to modulate cell surface expression of important immunoreceptors. In the presence of Nef, peptide presentation by MHC II is impaired (Stumptner-Cuvelette et al., 2001). Here, we have followed the intracellular trafficking of MHC II in Nef-expressing cells to understand the cellular events causing the Nef-induced down-regulation of mature MHC II (-peptide) and up-regulation of immature MHC II (Ii). Nef expression did not affect the rate of synthesis of the MHC II , and Ii chains, indicating that it exerts its effects at a posttranslational level. Regarding Ii immature complexes, metabolic pulse-chase analysis combined with surface biotinylation experiments, showed that Nef expression 1) increases access of Ii complexes at the cell surface and 2) delays Ii chain degradation. -peptide complexes accumulated intracellularly in the presence of Nef as shown by cytometry on permeabilized cells, but pulse-chase analysis failed to reveal any significant inhibition of the generation of SDS-stable complexes. Antibody internalization experiments suggest that surface Ii are internalized in the presence of Nef but cannot reach lysosomes. This interpretation is supported by results from subcellular fractionation experiments showing that in the presence of Nef, dense lysosomal-enriched fractions contained low levels of Ii chain, whereas the levels of three other lysosomal proteins remained unchanged. Compared with control cells, Nef-expressing cells contained larger amounts of Ii chain, which were rather distributed in light fractions. Therefore, Nef's effects on Ii complexes specifically lead to both an increased cell surface expression of Ii, as documented previously (Stumptner-Cuvelette et al., 2001), and limited access of these complexes to lysosomes (this study).
Newly synthesized Ii complexes are sorted at the level of the TGN toward the endocytic pathway (Hiltbold and Roche, 2002). This sorting step relies on signals carried by the Ii cytoplasmic tail, which also function as endocytosis signals. However, depending on the cell type considered, a variable fraction of newly synthesized Ii complexes rapidly transits via the cell surface, is internalized, and thereby reaches the endocytic pathway (Roche et al., 1993; Benaroch et al., 1995; Saudrais et al., 1998; Davidson, 1999). In our cell system, low expression levels of cell surface Ii chain in control cells prevented accurate determination of the rate of endocytosis in this cell population. Nef expression does not affect endocytosis in general, because internalization of Tf (Foti et al., 1997), the B subunit of the Shiga toxin (Johannes et al., 2003) and the TfR were not modified by Nef, nor acidification of the endosomal pathway (Stumptner-Cuvelette et al., 2001; this study). We also confirmed that in our cell system, Nef expression did not affect uptake of lucifer yellow (Foti et al., 1997), a marker of fluid phase endocytosis, nor dextran-FITC (40 kDa) or mannosylated-BSA-FITC (our unpublished data).
In Nef-expressing cells, surface bound anti-Ii antibodies were internalized but delayed in reaching late acidic compartments labeled with LysoTracker or anti-Lamp mAb. The anti-Ii antibodies accumulated in vesicular compartments that were negative for TGN46 and Lamp-1, and concentrated in a perinuclear area. There was some overlap between endocytosed anti-Ii antibodies and TfR, whereas the extent of the overlap was larger with Nef. These observations further illustrate the Nef-induced alterations of the endocytic pathway at the dynamic level. We also analyzed these alterations at the steady state by immuno-EM. Our analysis reveals that Nef expression induces an increase in size and number of MVBs. We estimated MVB numbers by comparison with the number of lysosomes encountered in randomly chosen ultrathin cryosections of control and Nef-expressing HeLa-CIITA cells. Nef expression induced a significant increase (almost a threefold factor) of the numbers of MVBs compared with those of lysosomes. We observed a similar Nef-induced accumulation of MVBs in the Mel JuSo melanoma cell line. As expected for such compartments, the MVBs observed in Nef-expressing cells contained internal vesicles positively labeled for CD63 and were accessible to the endocytic tracer BSA-gold after 20 min at 37°C. Antibodies specific for MHC II and Ii chain heavily decorated these compartments, whose limiting membrane was rich in the Nef protein. An unusual high reactivity with antibodies directed against the lumenal part of the Ii chain was associated with these MVBs, suggesting that in the presence of Nef large amounts of intact Ii chain have reached these compartments. Exposure of Nef expressing cells to an anti-Ii mAb led to its internalization from the surface and its access to MVBs (our unpublished data). The accumulation of MVBs observed at the EM level near the nucleus might therefore correspond to the large vesicular structures labeled with internalized anti-Ii chain antibodies and observed by confocal microscopy in Nef-expressing cells. Each vacuolar structure observed by confocal microscopy may thus correspond to several MVBs concentrated in a restricted area. Together, our data suggest that Nef induces an accumulation of MVBs where immature and mature MHC II complexes accumulate. By preventing Ii access to lysosomal compartments rich in proteases, Ii chain degradation would be reduced. Cysteine proteases are involved in Ii chain degradation in the endocytic pathway. We analyzed the cysteine protease activity present in total lysates from Nef-expressing cells compared with control cells by using an active site-directed probe (Lennon-Dumenil et al., 2002). We did not find any significant difference (our unpublished data), suggesting that Nef does not affect cysteine protease activity in general. This conclusion is supported by our observation that EGF degradation normally occurs in Nef-expressing cells (Johannes et al., 2003).
Strikingly, the effects of Nef expression on MVBs seem opposite to the effects induced by the PI 3-kinase inhibitors wortmannin and LY294002 (Fernandez-Borja et al., 1999). Indeed, exposure of MHC II-expressing cells to PI 3-kinase inhibitors induces swelling of MVBs, which contain much less internal vesicles. Moreover, Nef is known to bind and activate a PI 3-kinase (Schibeci et al., 2000; Wolf et al., 2001; Linnemann et al., 2002), which is involved in Nef-induced MHC I down-regulation (Swann et al., 2001; Blagoveshchenskaya et al., 2002). We therefore tested the possible involvement of PI 3-kinase in the Nef-induced effects on MHC II. We observed that exposure of Nef-expressing cells to LY294002 abolished Nef-induced up-regulation of Ii chain at the cell surface as estimated by FACS and by immunofluorescence. In contrast, the inhibitor had no effect on Nef-induced down-regulation of surface mature MHC II and MHC I. These data support our previous conclusion based on mutational analysis that Nef acts through different mechanisms on mature and immature MHC II (Stumptner-Cuvelette et al., 2001). In addition, these data suggest that the increased surface expression of Ii induced by Nef involves a PI 3-kinase activity sensitive to LY294002. Phosphoinositides produced by PI 3-kinases direct the localization and the activity of effector proteins critical for cellular transport processes such as endocytosis and biosynthetic trafficking (Odorizzi et al., 2000; Simonsen et al., 2001). Thus, Nef could increase the access of Ii complexes at the cell surface either from endosomes or from the TGN, via a PI 3-kinase–dependent pathway. Interestingly, several studies indicate that a PI 3-kinase activity is involved in controlling a recycling route from endosomes to the plasma membrane (Spiro et al., 1996; Siddhanta et al., 1998; Hunyady et al., 2002). In HeLa cells, this PI 3-kinase–dependent pathway rapidly occurs for the TfR from sorting endosomes and bypasses recycling endosomes (van Dam et al., 2002). However, we previously showed that Tf endocytosis and recycling are not affected by Nef expression (Johannes et al., 2003), indicating that recycling from sorting endosomes is not particularly active in Nef-expressing cells. In addition, recycling from late compartments, including MVBs, was also reported to be dependent on a wortmannin-sensitive PI 3-kinase activity in fibroblast cells (Bright et al., 2001). We localized at the ultrastructural level Nef in different compartments but a great proportion of the immunogold labeling was associated with MVB outer membrane. Nef could recruit a PI 3-kinase at this location and thus induce a local production of phosphoinositides, which have been shown to play a key role in the inward budding of MVBs and consequently, in MVB biogenesis (Simonsen et al., 2001; Katzmann et al., 2002). Therefore, the Nef-induced increase in MVB numbers might be related to the activation of a PI 3-kinase by Nef. Finally, the Nef-mediated activation of the PI 3-kinase could promote recycling of Ii complexes to the cell surface due to their accumulation in MVBs and in agreement with their reduced capacity to access lysosomes. However, more work is required to estimate whether these hypotheses are correct.
One of the many consequences of Nef's presence is a major remodeling of the endocytic pathway as documented by this study. The significance of this modification in regard to the viral cycle is still unknown. We recently reported that in HIV-1–infected macrophages, HIV assembly seems to occur at the limiting membrane of MVB compartments, rich in MHC II (Raposo et al., 2002). After they pinch off, the newly formed virus particles accumulate in the lumen of these MVBs, which can fuse with the plasma membrane, releasing their viral content to the extracellular medium. This assembly process explains the particular composition of the HIV membrane, which is enriched in certain lipids such as (PI3)P (phosphatidylinositol 3-phosphate) and in host proteins such as MHC II and CD63. A very recent study has further established these points showing that in primary macrophages most of infectious HIV-1 indeed assembles in late endosomes (Pelchen-Matthews et al., 2003). In addition, it is now recognized that the late domain of Gag from many retroviruses, including HIV, is able to recruit the MVB machinery for viral assembly, budding, and fission processes. Mimicking Hrs during vesicles formation in MVBs, Gag late p6 domain assembles in oligomeric and monoubiquitinated complexes, which are then recognized by Tsg101, thereby initiating the assembly process (Pornillos et al., 2002). In T cells, this recruitment seems to occur at the plasma membrane in raft-enriched microdomains. In this respect, it is important to note that Nef not only associates with rafts (Wang et al., 2000) but also increases infectivity of HIV through this association, promoting incorporation of the ganglioside GM1 in the viral membrane (Zheng et al., 2001). Moreover, Nef-induced down-regulation of surface envelope results in its transport to the endocytic pathway (Schwartz et al., 1993). Therefore, HIV-1 infection may lead to the accumulation of viral and nonviral proteins in endosomal compartments. We propose that the remodeling of the endocytic pathway induced by Nef expression reflects the role of Nef in the viral program for viral assembly. There is no doubt that future studies on Nef, a small but very gifted protein, will go on providing exciting insights into the biology of host–virus interactions.
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ACKNOWLEDGMENTS |
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Footnotes |
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Abbreviations used: EM, electron microscopy; HA, hemagglutinin; Ii, Ii chain; MFI, mean fluorescence intensity; MHC, major histocompatibility complex; MVB, multivesicular body; PI 3-kinase, phosphatidylinositol 3-kinase; TfR, transferrin receptor; TGN, trans-Golgi network.
Corresponding author. E-mail address: benaroch{at}curie.fr or graposo{at}curie.fr.
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REFERENCES |
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Benaroch, P., Yilla, M., Raposo, G., Ito, K., Miwa, K., Geuze, H.J., and Ploegh, H.L. (1995). How MHC class II molecules reach the endocytic pathway. EMBO J. 14, 37–49.[Medline]
Blagoveshchenskaya, A.D., Thomas, L., Feliciangeli, S.F., Hung, C.H., and Thomas, G. (2002). HIV-1 Nef downregulates MHC-I by a PACS-1- and PI3K-regulated ARF6 endocytic pathway. Cell 111, 853–866.
