Human immunodeficiency virus 1 downregulates cell surface expression of the non-classical major histocompatibility class I molecule HLA-G1

Muriel Derrien1,{dagger}, Nathalie Pizzato2,{dagger}, Guillermina Dolcini1, Elisabeth Menu1,3, Gérard Chaouat3, Françoise Lenfant2, Françoise Barré-Sinoussi1 and Philippe Le Bouteiller2

1 Unité de Biologie des Rétrovirus, Département de Virologie, Institut Pasteur, 25 rue du Dr Roux, 75724 Paris Cedex 15, France
2 INSERM U563, Centre de Physiopathologie de Toulouse Purpan, 31059 Toulouse Cedex 3, France
3 INSERM U131, 32 rue des Carnets, 92140 Clamart, France

Correspondence
Elisabeth Menu
emenu{at}pasteur.fr


   ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Human immunodeficiency virus 1 (HIV-1) downregulates cell surface expression of HLA-A and HLA-B but not HLA-C or HLA-E to ultimately escape immune defences. Here, it is shown that cell surface expression of the non-classical HLA-G1 is also downregulated by HIV-1, by using co-transfection experiments and infection with cell-free HIV-1 of HLA-G1-expressing U87 glioma cells or macrophages in primary culture. Moreover, co-transfection experiments using proviruses deleted in either nef or vpu or plasmids encoding HIV-1 Nef and Vpu mixed together with a HLA-G1-expressing construct demonstrated that HLA-G1 downregulation is Nef-independent and Vpu-dependent, contrasting with the Nef- and Vpu-dependent HLA-A2 downregulation. Together, these results show that the decrease of HLA-A2 and HLA-G1 caused by HIV-1 occurs through distinct mechanisms.

{dagger}Both authors contributed equally to this work.


   INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Expression of major histocompatibility class I (MHC-I) molecules is required to initiate and sustain an efficient anti-viral immunity (Tortorella et al., 2000). Human immunodeficiency virus 1 (HIV-1) has evolved multiple immunoevasive strategies to escape the host immune response (Collins & Baltimore, 1999). This includes downregulation of cell surface MHC-I, which could induce the decline of HIV-specific CD8+ T cells and modulation of the function of natural killer cells (Collins & Baltimore, 1999). MHC-I downregulation was shown to be dependent on HIV Nef mainly, affecting endocytosis of classical HLA-A and HLA-B, but not that of HLA-C or of non-classical HLA-E (Cohen et al., 1999; Blagoveshchenskaya et al., 2002). Unlike other MHC-I molecules, HLA-G is characterized by a low polymorphism and a restricted tissue distribution, being found mostly in placental trophoblasts, thymic epithelial cells (Le Bouteiller & Blaschitz, 1999) and macrophages (M{phi}) (Yang et al., 1996). Moreover, HLA-G is transcribed and translated into multiple membrane-bound and soluble isoforms, as a result of alternative splicing (Ishitani & Geraghty, 1992). The soluble HLA-G1 isoform was shown to induce apoptosis of activated CD8+ T cells (Fournel et al., 2000) and to suppress the allo-proliferative response of CD4+ T cells (Lila et al., 2001). Recent studies suggest that, in pathological conditions, HLA-G1 expression could be either induced in cells that do not usually express this MHC-I, such as in certain tumours (Lefebvre et al., 2002; Wiendl et al., 2002), or upregulated in monocytes/M{phi} during the chronic phase of viral infections (Onno et al., 2000; Lozano et al., 2002). In addition, HLA-G1 has the ability to present viral peptides to cytotoxic CD8+ T cells (Lenfant et al., 2003), suggesting that this class Ib molecule could also play a role during early phases of viral infections.

In this report, we asked whether acute HIV-1 infection could modulate cell surface expression of HLA-G1. We found that HIV-1 decreases cell surface expression of HLA-G1 through a Nef-independent and Vpu-dependent mechanism, whereas both proteins affect HLA-A2 expression.


   METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Cells and cell lines.
A U87 cell line that was stably expressing CD4 and the chemokine receptor CXCR4 (Deng et al., 1997) was maintained in Dulbecco's modified Eagle's medium (DMEM)/4·5g glucose l–1/glutamax/sodium pyruvate (Gibco-BRL) containing 10 % fetal calf serum (FCS), 10 mM HEPES and 1 % antibiotics (penicillin/streptomycin). The human choriocarcinoma cell lines JEG-3 and JAR were maintained in DMEM and RPMI 1640 (Bio Whittaker) with Glutamax (Invitrogen), respectively, supplemented with 10 % FCS, 1 mM sodium pyruvate and antibiotics.

The human CD4+ lymphoblastoid cell line (CEM) was cultured in RPMI 1640 supplemented with 10 % FCS, 1 % glutamine and antibiotics. Peripheral blood mononuclear cells (PBMCs) or mononuclear cells isolated from umbilical cord blood were from HIV-1- and hepatitis B virus-negative healthy volunteers (Centre de Transfusion Sanguine d'Ile-de France, Rungis, France). Cells were obtained by density-gradient centrifugation using Ficoll Hypaque (Pharmacia Biotech). Phytohaemagglutinin (PHA)-stimulated PBMCs or mononuclear cells isolated from umbilical cord blood were cultured in RPMI 1640 containing 10 % heat-inactivated fetal calf serum, 20 mM glutamine, antibiotics and 5 U IL2 ml–1. Cell supernatants of PHA-IL2-stimulated PBMCs were used for mock infections.

Monocyte-derived M{phi} were obtained as described previously (Perez-Bercoff et al., 2003). Briefly, monocytes were cultured for 7 days in RPMI 1640 containing 20 mM glutamine, 100 U penicillin ml–1, 100 µg streptomycin ml–1, 10 mM HEPES, 10 mM sodium pyruvate, 50 µM {beta}-mercaptoethanol, 1 % MEM vitamins, 1 % essential amino acids (all from Life Technology) and 15 % heat-inactivated human AB serum (Valbiotech). Matured cells were harvested by centrifugation, washed, infected with HIV-1 Bal (m.o.i.=0·001) and cultured for various periods of time in hydrophobic Teflon pockets in media containing 10 % FCS. The purity of the isolated population used in the experiments was greater than 95 %, as determined by M{phi}-specific markers, including CD11b, CD14, CD32 and CD64 (Perez-Bercoff et al., 2003). LPS content in media and working solutions was less than 10 pg ml–1.

Viruses and inoculations.
HIV-1 Bal and LAI use CCR5 and CXCR4, respectively, as a co-receptor and were inoculated onto M{phi} and HLA-G1-expressing U87.CD4-CXCR4 cells, respectively. Viruses were grown from laboratory stocks using mononuclear cells isolated from umbilical cord blood for HIV-1 Bal and CEM cells, for HIV-1 LAI. Culture supernatants containing HIV LAI were clarified at 2500 r.p.m. for 20 min and viral particles were concentrated by centrifugation at 17 000 r.p.m. for 3 h. Pellets were resuspended in NTE buffer (0·1 M NaCl, 0·01 M Tris, 0·001 M EDTA pH 7·4) and frozen at –80 °C until use. The infectious titre of each virus was determined by limiting dilution assay on PHA-activated PBMCs and is expressed as 50 % tissue culture infectious dose (TCID50) ml–1. TCID50 values calculated for HIV-1 Bal and LAI were 105 ml–1 and 1010 ml–1, respectively.

Transfections.
Effectene reagent (Qiagen) was used for transfection experiments. In all experiments, endotoxin-free DNAs (1–2 µg per 106 cells) were used. U87.CD4-CXCR4 glioma cells were seeded in T25 or T75 flasks 2–3 days before transfection. The following constructs and backbones were used: pcDNA3, pcDNA3-G1 encoding HLA-G*01011, and pLUMC9901 encoding HLA-A*02011 (gift of Dr G. C. Hassink, Leiden, The Netherlands) (Kikkert et al., 2001), pBR322, pNL4-3 HIV-1 provirus (Adachi et al., 1986) deleted in the nef or vpu gene (pNL4-3/vpuDEL-1; Klimkait et al., 1990) (referred to as pNL4-3{Delta}nef and pNL4-3{Delta}vpu), and pcDNA3.1/myc-HuDC-SIGN (Halary et al., 2002). Plasmid pVpuHAGFP was a personal gift from R. Benarous (Besnard-Guérin et al., 2004). pCMV-nef has been obtained by subcloning Nef-Lai from pSG-nef (gift from G. Sutter, Munchen, Germany) into the BamHI–NotI site of pCMV-IntronA (gift from J. Barr, Canada). Plasmid pN3-EGFP (Clontech) was used as a control.

Two different ratios (1 : 1 and 1 : 5) of DNA constructs corresponding to pcDNA3-G1 plus empty construct (control cells) or pcDNA3-G1 plus pNL4-3 HIV-1 provirus (HIV-1+ cells) were tested. As a marker of co-transfection efficiency, cells were transfected with one pcDNA3-G1 for every five pcDNA3.1/myc-HuDC-SIGN, a construct encoding another molecule whose expression was detectable at the cell surface. Cell surface expression of HLA-G1, HLA-A2, DC-SIGN (CD209) or control adhesion molecule CD56 was determined by flow cytometry, using specific monoclonal antibodies (mAbs) (Table 1), 24 and 48 h post-transfection (p.t.). For double transfection in JAR cells, combinations of expression vectors in a ratio of 2 : 1 corresponding to pcDNA3-G1 or pLUMC9901 and pVpuHAGFP or pN3-EGFP (control) were used. For triple transfection, JAR cells were transfected with combinations of expression vectors in a ratio of 2 : 2 : 1 corresponding to pcDNA-3-G1 or pLUMC9901 and pCMV-IA-nef and pVpuHAGFP or pN3-EGFP, respectively. Two days later, HLA-A2 or HLA-G1 cell surface expression was analysed by flow cytometry, using specific mAbs (Table 1).


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Table 1. Antibodies used in this study

Each of these Abs was used at 5–10 µg ml–1.

 
p24 antigen determination.
HIV-1 expression was determined by measuring the amount of HIV-1 p24 antigen in cell culture supernatants using a commercial kit (Coulter). Each supernatant was tested for p24 content in duplicate.

Flow cytometry analysis.
U87 and JEG-3 cells were detached using either PBS containing 5 mM EDTA or trypsin EDTA (Gibco-BRL). U87 cells, JEG-3 cells or M{phi} were blocked (for 30 min at 4 °C) with human immunoglobulins (Tegeline, Institut Français du Sang, France). After one washing step, the labelled or unlabelled primary antibody (Table 1) was added at a final concentration of 5 or 10 µg ml–1. Isotype control mAbs were used at the same concentration as the primary antibody. Incubation was done on ice for 30 min, followed by one wash. Goat anti-mouse IgG [F(ab')2-PE; 5 µg ml–1; DAKO] or the F(ab')2 goat anti-mouse IgG antibody conjugated with cy5-RPE (Coulter-Immunotech) was used as secondary antibody. Samples were analysed by flow cytometry, using either a XL2 EPICS or an EPICS-Elite flow cytometer (Coulter), gated to exclude non-viable cells and to include EGFP-positive cells. Ten-thousand events were analysed for each sample in duplicate. Mean fluorescence intensity (m.f.i.) represents the m.f.i. of all cells. When indicated, m.f.i. represents the m.f.i. of positively stained cells.

Western blot analysis.
Cellular extracts were prepared by incubating 106 cells in 50 µl of chilled (4 °C) lysis buffer containing 500 mM NaCl, 1 mM EDTA, 0·2 % Triton X-100, 200 mM PMSF, 110 mM {beta}-mercaptoethanol, a mixture (4 µg ml–1) of pepstatin A, aprotinin, chymostatin and antipain, 500 mM sucrose, 0·15 mM spermine, 0·5 mM spermidine and 10 mM HEPES pH 8 for 5 min. Cell extracts were submitted to centrifugation (15 000 r.p.m. for 15 min at 4 °C). Supernatants were collected and frozen at –80 °C with 10 % (v/v) glycerol before use. Protein content in each cellular extract was measured by using Bradford reagent (Bio-Rad). Cellular extracts (20 µg protein per lane) prepared from M{phi} were run through a 12·5 % SDS-polyacrylamide gel next to cellular extracts (20 µg per lane) from choriocarcinoma JEG-3 cells, used as membrane-bound HLA-G1-positive control, and to molecular mass markers. After electrophoretic transfer of proteins on to Immobilon-P (Millipore), blots were incubated in the presence of MEM-G1 at a dose of 5 µg ml–1 (primary mAb specific for denatured forms of HLA-G1), followed by horseradish peroxidase (HRP)-conjugated goat anti-mouse antibodies (secondary antibodies). Blots were developed by using enhanced chemiluminescence (ECL) (SuperSignal West Pico Chemiluminescent Substrate; Pierce).

Statistical analyses.
Statistical significance was determined using the non-parametric Mann–Whitney test or t-test; P values of less than 0·05 were regarded as significant.


   RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Expression of HIV-1 downregulates HLA-G1 cell surface expression
To analyse cell surface expression of HLA-G1 in cells expressing HIV-1, we set up a transient co-transfection assay using human CD4- and CXCR4-expressing U87 glioma cells. Optimal conditions of co-transfection were obtained using pcDNA3-G1 plus empty construct ({Delta}HLA-G1 transfectant), pNL4-3 (HIV-1+ cells) or pcDNA3.1/myc-HuDC-SIGN (*HLA-G1/CD209 transfectant) in a ratio of 1 : 5. Under these experimental conditions, the efficiency of transfection evaluated by measuring cell surface expression of HLA-G1 and CD209 was 20±9 % at 24 h p.t. (Fig. 1A profiles a–d) and 40±13·9 % at 48 h p.t. (Fig. 1A profile e and Fig. 1B–D). Moreover, HLA-G1 expression was stable even when it was expressed with CD209 (Fig. 1A profiles b, c). In contrast, HLA-G1 expression (number of cells expressing HLA-G1 and m.f.i.) was significantly (P<0·01) and reproducibly (n=9) decreased in cells co-transfected with pcDNA3-G1 and pNL4-3 (Fig. 1B upper panel; Table 2; Fig. 1C, D). In the different individual experiments performed, a 25–45 % decrease in HLA-G1 expression was detected, and the amount of HIV-1 p24 antigen detected in the cell culture supernatants of these double transfectants ranged between 0·7 and 1. 9 pg ml–1. As expected, a parallel and significant (P<0·05) decrease in HLA-A2 expression was detected in HIV-1-expressing cells, whereas CD56 labelling remained stable regardless of whether HIV-1 was expressed (Fig. 1B lower panel and Fig. 1D).



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Fig. 1. HIV-1 downregulates cell surface expression of HLA-G1. (A) Expression of HLA-G1 and co-expression marker DC-SIGN (CD209), after co-transfection. U87.CD4-CXCR4 glioma cells were co-transfected either with an equivalent amount of pcDNA3-G1 plus either empty construct ({Delta}) or pcDNA3.1/myc-HuDC-SIGN (*) mixed together in a ratio of 1 : 5. Cell surface expression of HLA-G1, determined by flow cytometry using MEM-G9, in pcDNA3-G1 plus empty construct (profile b) and pcDNA3-G1 plus pcDNA3.1/myc-HuDC-SIGN (profile c) is compared to expression of CD209 (8A5 labelling, profile d) and to HLA-G1 expression at 48 h p.t. (profile e). Staining obtained with IgG isotype control of either HLA-G1- or HLA-G1/CD209-expressing cells is represented in grey. a, IgGs 3·86 %, m.f.i. 0·64; b, MEM-G9 19·1 %, m.f.i. 1·97; c, MEM-G9 20·9 %, m.f.i. 2·34; d, MEM-G9 (t=48 h) 30·3 %, m.f.i. 8·07; and e, 8A5 32·0 %, m.f.i. 8·39. (B) Decrease of HLA-G1 and HLA-A2 but not of CD56 cell surface expression in U87 cells co-transfected with pcDNA3-G1 and pNL4-3. U87.CD4-CXCR4 glioma cells were co-transfected with pcDNA3-G1 and either pNL4-3 (HIV+) or empty construct (HIV). At 48 h p.t., cell surface expression of transfected HLA-G1, endogenous HLA-A2 and CD56 was measured by flow cytometry using 87G or MEM-G9 (for HLA-G1), BB7.2 (for HLA-A2), CD56 mAbs or IgG-matched isotype control, followed by PE-labelled anti-mouse IgG F(ab')2. Overlaid profiles of HIV mock-transfected (in grey) and of HIV-1-transfected (in black) cells are shown. Percentage of cells stained positively and m.f.i. are described in Table 2. The staining obtained with IgG isotype control is represented in white. Fluorescence profiles shown are representative of nine independent experiments. (C) HLA-G1 expression in cells expressing HIV-1 defective in either the nef or the vpu gene. CD4/CXCR4 U87 glioma cells were co-transfected with pcDNA3-G1 and either empty construct (control), pNL4-3 or pNL4-3{Delta}nef (left panel), or empty construct (control), pNL4-3 or pNL4-3{Delta}vpu (right panel). Cell surface expression of HLA-G1 in each transfectant (versus labelling with IgG isotype control) was determined by flow cytometry using MEM-G9. Numbers represent the percentage of cells labelled with MEM-G9; the percentage of decrease as compared to control cells is also shown (in parentheses). (D) Comparison of HLA-G1, HLA-A2 and CD56 expression in cells expressing HIV-1 defective or not in the nef or vpu gene. U87.CD4-CXCR4 glioma cells were co-transfected with pcDNA3-G1 and pNL4-3, pNL4-3{Delta}nef, pNL4-3{Delta}vpu or empty construct (control). Cells were stained with mAbs specific for HLA-G1 (MEM-G9), HLA-A2 (BB7.2) or CD56 and analysed by flow cytometry. Data represent mean values±SD of the percentage of m.f.i. as compared to the control (100 % m.f.i.=10·70±0·50 for MEM-G9, 36·80±3·03 for BB7.2 and 11·00±0·96 for CD56) from four independent experiments. **P<0·01, *P<0·05, as compared to the control (Mann–Whitney test).

 

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Table 2. Percentage of cells stained positively and m.f.i. corresponding to FACS plots in Fig. 1(B)

 
Taken together, these results confirmed that HIV-1 downregulates expression of the classical MHC-I molecule HLA-A2 and indicated that the non-classical MHC-I molecule HLA-G1 can undergo similar downregulation in HIV-1-expressing cells.

HIV-1-mediated downregulation of HLA-A2 is Nef- and Vpu-dependent, whereas HIV-1-mediated downregulation of HLA-G1 is Vpu-dependent and Nef-independent
Next, we wanted to determine which HIV-1-encoded protein was responsible for downregulating cell surface expression of HLA-G1. Several viral proteins, including Nef and Vpu, were shown to attenuate the expression of classical MHC-I molecules (Schwartz et al., 1996; Kerkau et al., 1997). To determine whether these HIV proteins could decrease HLA-G1 expression, we first co-transfected U87 cells with combinations of expression vectors in a ratio of 1 : 5 corresponding to pcDNA-3-G1 and empty construct (control), wild-type pNL4-3, pNL4-3{Delta}nef or pNL4-3{Delta}vpu. Flow cytometry analysis of two representative experiments are presented in Fig. 1(C) (left and right panels) and the results obtained in different independent experiments are summarized in Fig. 1(D). The results showed that HLA-G1 expression (number of cells expressing HLA-G1 and m.f.i.) was reduced in U87 cells transfected with pNL4-3 or pNL4-3{Delta}nef (P<0·001 and P<0·05 as compared to control, respectively) (Fig. 1C, D). In contrast, HLA-G1 expression in pNL4-3{Delta}vpu-transfected cells was similar to that of control cells (Fig. 1C left panel and 1D). In these experiments, the amount of HIV-1 p24 antigen detected in the supernatant of the different transfectants ranged between 0·2 and 2·1 ng ml–1, with comparable amounts of p24 antigen detected in the supernatants of pNL4-3{Delta}vpu- (2·1 ng ml–1±0·2) and pNL4-3-expressing cells (1·6 ng ml–1±0·7). By comparison, analysis of HLA-A2 surface expression in cells transfected with pNL4-3 showed a decrease (P<0·02 as compared to control cells; Fig. 1D), whereas overall expression of HLA-A2 was similar in control, pNL4-3{Delta}nef- and pNL4-3{Delta}vpu-transfected cells (Fig. 1D). Control CD56 surface expression remained unchanged irrespective of the construct transfected and expressed in U87 cells (Fig. 1D).

Then, JAR cells were co-transfected with plasmids encoding HLA-G1 or HLA-A2 and EGFP, Vpu/EGFP, Nef/EGFP or Nef/Vpu/EGFP. At 48 h p.t., cell surface expression of HLA-G1 or HLA-A2 was analysed by flow cytometry in EGFP-positive cells expressing either Vpu or Nef or both Vpu and Nef, and compared to cell surface expression of HLA-G1 or HLA-A2 in EGFP control positive cells. Results indicated that Vpu downregulated both HLA-G1 and HLA-A2 cell surface expression, in contrast to Nef, which only downregulated HLA-A2 expression (Fig. 2). When cells co-expressed both Vpu and Nef, we observed that the modulation of HLA-G1 was similar to that observed with Vpu only (~40 % decrease). HLA-A2 downregulation was slightly more important in cells expressing both Vpu and Nef than in cells expressing either Nef or Vpu (Fig. 2).



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Fig. 2. Vpu but not Nef decreases cell surface expression of HLA-G1, whereas both Vpu and Nef decrease cell surface expression of HLA-A2. JAR cells were co-transfected with pcDNA3-G1 (encoding HLA-G1) or pLUMC9901 (encoding HLA-A2) plus pN3-EGFP (EGFP) or plus pVpuHAGFP (Vpu) or plus pIA-nef plus pN3-EGFP (Nef) or plus pIA-nef plus pVpuHAGFP (Nef/Vpu). After 48 h, cell surface expression of HLA-G1 or HLA-A2 was analysed by flow cytometry in EGFP-positive cells, using 87G or BB7.2 mAbs specific for HLA-G or HLA-A2, respectively. Then, cell surface expression of HLA-G1 (solid bars) or HLA-A2 (open bars) in Vpu-, Nef- or Nef/Vpu-positive cells was compared to HLA-G1 or HLA-A2 cell surface expression in EGFP-positive cells without Nef or Vpu. Results are the mean±SD of three independent experiments. *P<0·05, as compared to the EGFP control (t-test).

 
Taken together, these results demonstrated that, whereas Nef and Vpu could co-operate to decrease HLA-A2 expression, Vpu alone is involved in the downregulation of HLA-G1 expression.

Cell-free HIV-1 downregulates HLA-G1 cell surface expression in U87 glioma cells expressing HLA-G1 and in primary culture of M{phi}
We then investigated whether cell-free HIV-1 could similarly downregulate the surface expression of HLA-G1. U87.CD4-CXCR4 cells were transfected with pcDNA3-G1 and inoculated, 24 h later, with HIV-1 LAI at an m.o.i. of 1. The results showing that both CD4 and HLA-A2 expression decreased rapidly after viral inoculation, to reach a level below the level of detection for CD4 expression at 48 h after viral inoculation (Fig. 3A), indicated that the majority of cells were infected. At that time after infection (48 h), parallel examination of cell surface expression of HLA-G1 showed that HLA-G1 was significantly downregulated (P<0·05, as compared to mock infection) (Fig. 3B, C).



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Fig. 3. Cell-free HIV-1 downregulates HLA-G1 expression in U87 cells expressing HLA-G1. U87.CD4-CXCR4 glioma cells transfected 24 h earlier with pcDNA3-G1 were inoculated with HIV-1 (LAI, m.o.i. of 1) or with the supernatant of uninfected cells (HIV-1). Cells were then immunostained with anti-HLA-G (MEM-G9), anti-HLA-A2 (BB7.2), anti-CD4 mAbs or a mixture of IgG2b/IgG1 followed by PE-labelled anti-mouse IgG F(ab')2. Cells were analysed by flow cytometry. (A) Kinetics of CD4 and HLA-A2 expression determined in HIV-1 and HIV-1+ cells. (B) HLA-G1 expression determined by using MEM-G9 and 87G in HIV-1 and HIV-1+ cells, at 48 h post-infection. Numbers represent the percentage of cells stained with either MEM-G9 or 87G and the m.f.i. (corresponding to the percentage of positive cells). (C) HLA-G1, HLA-A2 and CD4 expression (relative cell counts and m.f.i.) (from the left to the right: labelling with MEM-G9, 87G, BB7.2 and anti-CD4) determined in different independent experiments (n=5) in control (HIV-1; open bars) and HIV-1+ (LAI-infected cells; solid bars), 48 h after virus inoculation. *P<0·05 (Mann–Whitney test).

 
Several studies have reported that M{phi} can express HLA-G1 (Yang et al., 1996; Onno et al., 2000). To investigate whether HLA-G1 expression was downregulated during HIV-1 infection of primary cultures of human M{phi} derived from PBMCs, we first analysed HLA-G1 expression in seven different primary cultures of human M{phi}. Flow cytometry analysis indicated that, after blockage of non-specific binding using human IgGs, 15–25 % of the cells expressed HLA-G1 with a low m.f.i. of 1·31±0·40 versus 0·3±0·05 for control IgG1 (data not shown). This low level of expression was confirmed by Western blot analysis (Fig. 4A). Several bands reacting with HLA-G1-specific MEM-G1 mAb were detected in cell extracts prepared with primary M{phi} (lane M{phi}). Among these bands, one was running at the level of the band corresponding to the membrane-bound form of HLA-G (HLA-G1) of 39 kDa expressed in control JEG-3 trophoblast cells (lane JEG-3). A lower band that could correspond to a soluble isoform of HLA-G1 was also detected. Cell surface HLA-G1 expression was also increased in M{phi} treated with IFN-{gamma} at a dose of 500 UI ml–1 and higher (data not shown).



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Fig. 4. (A) Detection of HLA-G1 by Western blot analysis in monocyte-derived M{phi}. Cell lysates from M{phi} and human choriocarcinoma JEG-3 cells (JEG-3) (20 µg each) were subjected to SDS-PAGE and blotted with MEM-G1 mAb specific for HLA-G followed by an HRP-conjugated antiserum and ECL development. Molecular mass markers (kDa) are shown on the right. (B) Cell-free HIV-1 downregulates expression of HLA-G1 in M{phi} in primary culture. Monocyte-derived M{phi} were inoculated with HIV-1 Bal (m.o.i. of 0·001) or supernatant of PHA-IL2-activated PBMCs (HIV-1). At different times after virus inoculation, cells were immunostained using (B) anti-HLA-G (MEM-G9) or (C) anti-HLA-B/C (B1.23.2) mAbs or a mixture of IgG2a/IgG1 followed by PE-labelled anti-mouse IgG F(ab')2. (B) Data are expressed as percentage of positively stained cells and m.f.i. of MEM-G9 (background IgG1: HIV-1 cells 4·5 %, m.f.i. 0·492; HIV-1+ cells 4·3 %, m.f.i. 0·690) (upper left and right panel, respectively) obtained after analysis by flow cytometry and are representative of four independent experiments. *P<0·05 (Mann–Whitney test). Lower panels represents HLA-G1 staining obtained 24 h post-infection in HIV-1 (left) and HIV-1+ (right) cells. Numbers represent the percentage of HIV-1 and HIV-1+ cells stained with control IgG1 or MEM-G9 and the m.f.i. (C) HLA-B/C expression (percentage of positively stained cells and m.f.i. values) in HIV-1 (open bars) and HIV-1+ (solid bars) cells.

 
Using these cells, we then analysed whether expression of HLA-G1 could be modulated by cell-free HIV-1. M{phi} were inoculated with HIV-1 Bal at a dose of 1000 TCID50, or with an equivalent volume of supernatant from uninfected activated PBMCs (as a control). At different times after viral inoculation, cells were collected and HLA-G1 expression was assessed by flow cytometry using MEM-G9 anti-HLA-G1 mAb. Expression of HLA-B/C was also quantified. Fig. 4(B, C) shows representative results obtained using four different cell preparations. HLA-G1 expression was downregulated in M{phi}, as compared to control cells as soon as 24 h p.t. In different independent experiments, the decrease of HLA-G1 expression was observed in 25–40 % of the cells [percentage of cells labelled with anti-HLA-G1 and m.f.i.: HIV-1 46·73±4·10, m.f.i. 0·99±0·39; HIV-1+ 29·66±7·50, m.f.i. 0·495±0·160 (P<0·05, as compared to control group)]. By comparison, staining of HIV-1-infected M{phi}, using antibodies recognizing HLA-B and C, did not significantly differ from that detected in non-infected cells (Fig. 4C). Taken together, these data indicated that acute HIV-1 infection downregulated HLA-G1 surface expression in both primary M{phi} and a U87 glioma cell line expressing HLA-G1.


   DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Data obtained in this study demonstrate that expression of the membrane-bound isoform HLA-G1 is selectively downregulated by HIV-1. First, we showed that expression of HIV-1 (but not expression of CD209) in U87-HLA-G1 glioma cell line induces a significant decrease in HLA-G1 expression. Second, cell-free HIV-1 infection either of U87-HLA-G1-expressing cells or of primary M{phi} naturally expressing cell surface HLA-G1 also resulted in a downregulation of HLA-G1. The downregulation of HLA-G1 expression was not immediate after exposure of cells to HIV-1. Furthermore, it was parallel to endogenous HLA-A2 downregulation. These observations suggest that (1) anti-HLA-G antibodies did not compete with HIV-1-binding sites and (2) HIV-1 has to be expressed in infected cells for HLA-G1 expression to be attenuated. More precisely, the present results showing that HLA-G1 downregulation is linked to vpu and is nef-independent further indicate that expression of a particular HIV-1 protein, namely Vpu, is required for HLA-G1 expression to be modulated. This was demonstrated in experiments showing that expression of Vpu but not of Nef downregulated HLA-G1 expression.

These latter results were expected since Nef triggers endocytosis of classical MHC-I molecules by direct binding to specific residues present in the cytoplasmic tail (Schwartz et al., 1996; Cohen et al., 1999; Williams et al., 2002; Blagoveshchenskaya et al., 2002) and HLA-G1 lacks these residues due to a short cytoplasmic tail (Geraghty et al., 1987). In contrast, the present results suggest that HLA-G1 molecules may be degraded before their transport to the membrane, reducing HLA-G1 expression at the cell surface. Indeed, Vpu interacts with various molecules within the endoplasmic reticulum, including CD4, and alters their transport to the membrane by directing them to the degradation pathway (Lenburg & Landau, 1993; Bour et al., 2001). In the case of Vpu-mediated CD4 degradation, Vpu interacts with a discrete amino acid sequence in the cytoplasmic tail of CD4 containing a dilysine motif (EKKT) (Lenburg & Landau, 1993; Bour et al., 1995). Interestingly, HLA-G contains a dilysine residue (RKKSSD) in positions –4 and –5 from the C terminus (Geraghty et al., 1987) with which Vpu could interact. Furthermore, this particular motif is determinant for HLA-G trafficking within the different intracellular compartments, regulating its transport from the endoplasmic reticulum to the cis-Golgi network and its export to the membrane upon binding of high-affinity peptides to HLA-G1 (Park et al., 2001). Alternatively, Vpu could affect an early step in the biosynthesis of HLA-G1, resulting in a decrease of cell surface expression, as described previously for classical MHC-I molecules (Kerkau et al., 1997).

The present results indicate that both Nef and Vpu can reduce the expression level of HLA-A2 at the cell surface. Discordant results have been described in the literature. Cohen et al. (1999) reported that Nef is a main determinant whereas Vpu and Vpr have minor influence on the downregulation of HLA-A and HLA-B surface expression. However, in the study of Kerkau et al. (1997), it appears that Vpu is crucial for downregulating MHC-I. Major differences in the experimental procedures, such as use of recombinant vaccinia virus (Kerkau et al., 1997) versus reporter viruses (Cohen et al., 1999), can explain this apparent discrepancy. Considering that Nef and Vpu can affect the expression of the same host molecule, for example CD4, HLA-A2 (present study), through distinct mechanisms (Vincent et al., 1993; Buonocore et al., 1994; Blagoveshchenskaya et al., 2002), it is possible that both HIV proteins co-operate to downregulate the expression of MHC-I molecules by interfering with different regulatory pathways responsible for proper MHC-I expression (Gromme & Neefjes, 2002). This would indicate that HIV-1 has evolved several strategies to ultimately downregulate the same molecule and escape immune defences.

As observed here in cells infected with HIV, acute infection with other viruses, such as human cytomegalovirus (HCMV) and herpes simplex virus, was shown to decrease cell surface expression of HLA-G1 (Schust et al., 1999; Tortorella et al., 2000; Fisher et al., 2000). Nevertheless, HCMV was also found to increase HLA-G1 expression, either upon viral reactivation (Onno et al., 2000) or through indirect mechanisms, involving cytokines, in particular HCMV IL10 (Spencer et al., 2002). Likewise, HLA-G1 is decreased in cells acutely infected with HIV-1 (present study) and might be increased in cells surviving HIV infection, in latently and/or chronically infected cells that constitute a reservoir for HIV (Lozano et al., 2002). Modulations of HLA-G1 expression during HIV-1 infection may have functional consequences. Thus, recognition of HIV-1-infected cells by CD8+ CTL and/or natural killer cells (Lopez-Botet et al., 2000) and survival of these cells may, at least in part, depend on HLA-G1 expression levels. Future experiments will determine whether changes in the expression of different isoforms of HLA-G1, including soluble, could limit or favour HIV spreading through the host or the placenta, where HLA-G1 is highly expressed (Le Bouteiller & Blaschitz, 1999; Solier et al., 2002; Ishitani et al., 2003).

In conclusion, our results show that acute HIV-1 infection results in the downregulation of the cell surface expression of membrane-bound HLA-G1. They also indicate that HIV-1 uses distinct mechanisms to alter the surface expression of this non-classical MHC-I molecule and of HLA-A2. Further investigations are required to understand the functional significance of this HLA-G1 downregulation after acute HIV-1 infection.


   ACKNOWLEDGEMENTS
 
This work was funded by grants from ANRS (492/99) to F. B. S. and P. L. B., Sidaction Ensemble Contre le SIDA, Association Journée Mondiale Sida, ASUPS (F. L.), Etablissement français des greffes (P. L. B.). M. D. was supported by a postdoctoral fellowship from ANRS and SIDACTION, G. D. by a fellowship (SeCyT – UNCPBA and CIC, Argentina), N. P. by a grant from Ministère de la Recherche et de la Technologie (MENRT). We thank Olivier Schwartz (Institut Pasteur, Paris) for providing various HIV constructs and for helpful discussion, Stephan Bour (NIH, Bethesda) for providing pNL4-3/vpuDEL-1, Vaclav Horejsi (Praha, Czech Republic) for the gift of MEM-G1 and MEM-G9 mAbs, and Maryse Aguerre-Girr for technical help.


   REFERENCES
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Adachi, A., Gendelman, H. E., Koenig, S., Folks, T., Willey, R., Rabson, A. & Martin, M. A. (1986). Production of acquired immunodeficiency syndrome-associated retrovirus in human and nonhuman cells transfected with an infectious molecular clone. J Virol 59, 284–291.[Medline]

Besnard-Guérin, C., Belaïdouni, N., Lassot, I., Segaral, E., Jobart, A., Marchal, C. & Benarous, R. (2004). HIV-1 Vpu sequesters {beta}-transducin repeat-containing protein ({beta}TrCP) in the cytoplasm and provokes the accumulation of {beta}-catenin and other SCF{beta}TrCP substrates. J Biol Chem 279, 788–795.[Abstract/Free Full Text]

Blagoveshchenskaya, A. D., Thomas, L., Feliciangeli, S. F., Hung, C. H. & Thomas, G. (2002). HIV-1 Nef downregulates MHC-I by a PACS-1- and PI3K-regulated ARF6 endocytic pathway. Cell 111, 853–866.[Medline]

Bour, S., Schubert, U. & Strebel, K. (1995). The human immunodeficiency virus type 1 Vpu protein specifically binds to the cytoplasmic domain of CD4: implications for the mechanism of degradation. J Virol 69, 1510–1520.[Abstract]

Bour, S., Perrin, C., Akari, H. & Strebel, K. (2001). The human immunodeficiency virus type 1 Vpu protein inhibits NF-kappa B activation by interfering with beta TrCP-mediated degradation of IkappaB. J Biol Chem 276, 15920–15928.[Abstract/Free Full Text]

Buonocore, L., Turi, T. G., Crise, B. & Rose, J. K. (1994). Stimulation of heterologous protein degradation by the Vpu protein of HIV-1 requires the transmembrane and cytoplasmic domains of CD4. Virology 204, 482–486.[CrossRef][Medline]

Cohen, G. B., Gandhi, R. T., Davis, D. M., Mandelboim, O., Chen, B. K., Strominger, J. L. & Baltimore, D. (1999). The selective downregulation of class I major histocompatibility complex proteins by HIV-1 protects HIV-infected cells from NK cells. Immunity 10, 661–671.[Medline]

Collins, K. L. & Baltimore, D. (1999). HIV's evasion of the cellular immune response. Immunol Rev 168, 65–74.[Medline]

Corbeau, P., Benkirane, M., Weil, R., David, C., Emiliani, S., Olive, D., Mawas, C., Serre, A. & Devaux, C. (1993). Ig CDR3-like region of the CD4 molecule is involved in HIV-induced syncytia formation but not in viral entry. J Immunol 150, 290–301.[Abstract/Free Full Text]

Deng, H., Unutmaz, D., KewalRamani, V. N. & Littman, D. R. (1997). Expression cloning of new receptors used by simian and human immunodeficiency viruses. Nature 388, 296–300.[CrossRef][Medline]

Fisher, S., Genbacev, O., Maidji, E. & Pereira, L. (2000). Human cytomegalovirus infection of placental cytotrophoblasts in vitro and in utero: implications for transmission and pathogenesis. J Virol 74, 6808–6820.[Abstract/Free Full Text]

Fournel, F., Aguerre-Girr, M., Huc, X., Lenfant, F., Alam, A., Toubert, A., Bensussan, A. & Le Bouteiller, P. (2000). Cutting edge: soluble HLA-G1 triggers CD95/CD95 ligand-mediated apoptosis in activated CD8+ cells by interacting with CD8. J Immunol 164, 6100–6104.[Abstract/Free Full Text]

Geraghty, D. E., Koller, B. H. & Orr, H. T. (1987). A human major histocompatibility complex class I gene that encodes a protein with a shortened cytoplasmic segment. Proc Natl Acad Sci U S A 84, 9145–9149.[Abstract]

Gromme, M. & Neefjes, J. (2002). Antigen degradation or presentation by MHC class I molecules via classical and non-classical pathways. Mol Immunol 39, 181–202.[CrossRef][Medline]

Halary, F., Amara, A., Lortat-Jacob, H. & 7 other authors (2002). Human cytomegalovirus binding to DC-SIGN is required for dendritic cell infection and target cell trans-infection. Immunity 17, 653–664.[Medline]

Ishitani, A. & Geraghty, D. E. (1992). Alternative splicing of HLA-G transcripts yields proteins with primary structures resembling both class I and class II antigens. Proc Natl Acad Sci U S A 88, 3947–3951.

Ishitani, A., Sageshima, N., Lee, N., Dorofeeva, N., Hatake, K., Marquardt, H. & Geraghty, D. E. (2003). Protein expression and peptide binding suggest unique and interacting functional roles for HLA-E, F, and G in maternal-placental immune recognition. J Immunol 171, 1376–1384.[Abstract/Free Full Text]

Kerkau, T., Bacik, I., Bennink, J. R., Yewdell, J. W., Hunig, T., Schimpl, A. & Schubert, U. (1997). The human immunodeficiency virus type 1 (HIV-1) Vpu protein interferes with an early step in the biosynthesis of major histocompatibility complex (MHC) class I molecules. J Exp Med 185, 1295–1305.[Abstract/Free Full Text]

Kikkert, M., Hassink, G., Barel, M., Hirsch, C., Van Der Wal, F. J. & Wiertz, E. (2001). Ubiquitination is essential for human cytomegalovirus US11-mediated dislocation of MHC class I molecules from the endoplasmic reticulum to the cytosol. Biochem J 358, 369–377.[CrossRef][Medline]

Klimkait, T., Strebel, K., Hoggan, M. D., Martin, M. A. & Orenstein, J. M. (1990). The human immunodeficiency virus type 1-specific protein Vpu is required for efficient virus maturation and release. J Virol 64, 621–629.[Medline]

Le Bouteiller, P. & Blaschitz, A. (1999). The functionality of HLA-G is emerging. Immunol Rev 167, 233–244.[Medline]

Lee, N., Malacko, A. R., Ishitani, A., Chen, M. C., Bajorath, J., Marquardt, H. & Geraghty, D. E. (1995). The membrane-bound and soluble forms of HLA-G bind identical sets of endogenous peptides but differ with respect to TAP association. Immunity 3, 591–600.[Medline]

Lefebvre, S., Antoine, M., Uzan, S., McMaster, M., Dausset, J., Carosella, E. D. & Paul, P. (2002). Specific activation of the non-classical class I histocompatibility HLA-G antigen and expression of the ILT2 inhibitory receptor in human breast cancer. J Pathol 196, 266–274.[CrossRef][Medline]

Lemonnier, F. A., Le Bouteiller, P., Olive, D. & 7 other authors (1984). Transformation of LMTK- cells with purified class I genes. V. Antibody-induced structural modification of HLA class I molecules results in potentiation of the fixation of a second monoclonal antibody. J Immunol 132, 1176–1182.[Abstract/Free Full Text]

Lenburg, M. E. & Landau, N. R. (1993). Vpu-induced degradation of CD4: requirement for specific amino acid residues in the cytoplasmic domain of CD4. J Virol 67, 7238–7245.[Abstract]

Lenfant, F., Pizzato, N., Liang, S., Davrinche, C., Le Bouteiller, P. & Horuzsko, A. (2003). Induction of HLA-G-restricted human cytomegalovirus pp65 (UL83)-specific cytotoxic T lymphocytes in HLA-G transgenic mice. J Gen Virol 84, 307–317.[Abstract/Free Full Text]

Lila, N., Rouas-Freiss, N., Dausset, J., Carpentier, A. & Carosella, E. D. (2001). Soluble HLA-G protein secreted by allo-specific CD4+ T cells suppresses the allo-proliferative response: a CD4+ T cell regulatory mechanism. Proc Natl Acad Sci U S A 98, 12150–12155.[Abstract/Free Full Text]

Lopez-Botet, M., Llano, M., Navarro, F. & Bellon, T. (2000). NK cell recognition of non-classical HLA class I molecules. Semin Immunol 12, 109–119.[CrossRef][Medline]

Lozano, J. M., Gonzalez, R., Kindelan, J. M., Rouas-Freiss, N., Caballos, R., Dausset, J., Carosella, E. D. & Pena, J. (2002). Monocytes and T lymphocytes in HIV-1-positive patients express HLA-G molecule. AIDS 16, 347–351.[CrossRef][Medline]

Onno, M., Pangault, C., Le Friec, G., Guilloux, V., Andre, P. & Fauchet, R. (2000). Modulation of HLA-G antigens expression by human cytomegalovirus: specific induction in activated macrophages harboring human cytomegalovirus infection. J Immunol 164, 6426–6434.[Abstract/Free Full Text]

Park, B., Lee, S., Kim, E., Chang, S., Jin, M. & Ahn, K. (2001). The truncated cytoplasmic tail of HLA-G serves a quality-control function in post-ER compartments. Immunity 15, 213–224.[Medline]

Perez-Bercoff, D., David, A., Sudry, H., Barré-Sinoussi, F. & Pancino, G. (2003). Fc{gamma} receptor-mediated suppression of HIV replication in primary human macrophages. J Virol 77, 4081–4094.[Abstract/Free Full Text]

Schust, D. J., Tortorella, D. & Ploegh, H. L. (1999). HLA-G and HLA-C at the feto-maternal interface: lessons learned from pathogenic viruses. Semin Cancer Biol 9, 37–46.[CrossRef][Medline]

Schwartz, O., Marechal, V., Le Gall, S., Lemonnier, F. & Heard, J. M. (1996). Endocytosis of major histocompatibility complex class I molecules is induced by the HIV-1 Nef protein. Nat Med 2, 338–342.[Medline]

Solier, C., Aguerre-Girr, M., Lenfant, F., Campan, A., Berrebi, A., Rebmann, V., Grosse-Wilde, H. & Le Bouteiller, P. (2002). Secretion of pro-apoptotic intron 4-retaining soluble HLA-G1 by human villous trophoblast. Eur J Immunol 32, 3576–3586.[CrossRef][Medline]

Spencer, J. V., Lockridge, K. M., Barry, P. A., Lin, G., Tsang, M., Penfold, M. E. & Schall, T. J. (2002). Potent immunosuppressive activities of cytomegalovirus-encoded interleukin-10. J Virol 76, 1285–1292.[Abstract/Free Full Text]

Tortorella, D., Gewurz, B. E., Furman, M. H., Schust, D. J. & Ploegh, H. L. (2000). Viral subversion of the immune system. Annu Rev Immunol 18, 861–926.[CrossRef][Medline]

Vincent, M. J., Raja, N. U. & Jabbar, M. A. (1993). Human immunodeficiency virus type 1 Vpu protein induces degradation of chimeric envelope glycoproteins bearing the cytoplasmic and anchor domains of CD4: role of the cytoplasmic domain in Vpu-induced degradation in the endoplasmic reticulum. J Virol 67, 5538–5549.[Abstract]

Wiendl, H., Mitsdoerffer, M., Hofmeister, V., Wischhusen, J., Bornemann, A., Meyermann, R., Weiss, E. H., Melms, A. & Weller, M. A. (2002). Functional role of HLA-G expression in human gliomas: an alternative strategy of immune escape. J Immunol 168, 4772–4780.[Abstract/Free Full Text]

Williams, M., Roeth, J. F., Kasper, M. R., Fleis, R. I., Przybycin, C. G. & Collins, K. L. (2002). Direct binding of human immunodeficiency virus type 1 Nef to the major histocompatibility complex class I (MHC-I) cytoplasmic tail disrupts MHC-I trafficking. J Virol 76, 12173–12184.[Abstract/Free Full Text]

Yang, Y., Chu, W., Geraghty, D. E. & Hunt, J. S. (1996). Expression of HLA-G in human mononuclear phagocytes and selective induction by IFN-gamma. J Immunol 156, 4224–4231.[Abstract]

Received 2 December 2003; accepted 23 February 2004.



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