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Reversing HIV latency via sphingosine-1-phosphate receptor 1 signaling

Duquenne, Charlinea,*; Gimenez, Sandrinea,*; Guigues, Adelinea,*; Viala, Benjamina; Boulouis, Carolinea; Mettling, Clémenta; Maurel, Damienb; Campos, Noëliec; Doumazane, Etienned; Comps-Agrar, Laetitiad; Tazi, Jamale; Prézeau, Laurentd; Psomas, Christinaa; Corbeau, Pierrea,f,g,*; François, Vincenta,*

doi: 10.1097/QAD.0000000000001649
BASIC SCIENCE
Free

Objective: In this study, we looked for a new family of latency reversing agents.

Design: We searched for G-protein-coupled receptors (GPCR) coexpressed with the C-C chemokine receptor type 5 (CCR5) in primary CD4+ T cells that activate infected cells and boost HIV production.

Methods: GPCR coexpression was unveiled by reverse transcriptase-PCR. We used fluorescence resonance energy transfer to analyze the dimerization with CCR5 of the expressed GPCR. Viral entry was measured by flow cytometry, reverse transcription by quantitative PCR, nuclear factor-kappa B translocation by immunofluorescence, long terminal repeat activation using a gene reporter assay and viral production by p24 quantification.

Results: Gαi-coupled sphingosine-1-phophate receptor 1 (S1P1) is highly coexpressed with CCR5 on primary CD4+ T cells and dimerizes with it. The presence of S1P1 had major effects neither on viral entry nor on reverse transcription. Yet, S1P1 signaling induced NFκB activation, boosting the expression of the HIV LTR. Consequently, in culture medium containing sphingosine-1-phophate, the presence of S1P1 enhanced the replication of a CCR5−, but also of a CXCR4-using HIV-1 strain. The S1P1 ligand FTY720, a drug used in multiple sclerosis treatment, inhibited HIV-1 productive infection of monocyte-derived dendritic cells and of severe combined immunodeficiency mice engrafted with human peripheral blood mononuclear cells. Conversely, S1P1 agonists were able to force latently infected peripheral blood mononuclear cells and lymph node cells to produce virions in vitro.

Conclusion: Altogether these data indicate that the presence of S1P1 facilitates HIV-1 replicative cycle by boosting viral genome transcription, S1P1 antagonists have anti-HIV effects and S1P1 agonists are HIV latency reversing agents.

aInstitut de Génétique Humaine, CNRS-Université de Montpellier UMR9002

bARPEGE Pharmacology Screening Interactome platform facility, Institut de Génomique Fonctionnelle, Montpellier

cLaboratoire coopératif SPLICOS SAS, IGMM-CNRS-UMR5535, Montpellier

dInstitut de Génomique Fonctionnelle, CNRS-UMR5203, INSERM-U661, Université de Montpellier

eInstitut de Génétique Moléculaire de Montpellier, CNRS-UMR 5535, Université de Montpellier

fUniversité de Montpellier, Montpellier

gCentre Hospitalier Universitaire Carémeau, UF d’Immunologie, Nîmes, France.

Correspondence to Pierre Corbeau, MD, PhD, Institut de Géneétique Humaine, CNRS-Université de Montpellier UMR9002, 141 rue de la Cardonille, 34396 Montpellier Cedex 5, France. Tel: +33 434 359 932; fax: +33 434 359 901; e-mail: pcorbeau@igh.cnrs.fr

Received 4 July, 2017

Revised 23 August, 2017

Accepted 4 September, 2017

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Introduction

HAART do not eradicate HIV because of the ability of this retrovirus to integrate its genome in a quiescent form into target cells with a long lifespan. As long as they are not activated, these so-called reservoir cells do not express viral components and are therefore eliminated neither by HIV cytopathogenicity, nor by the antiviral immune response. Hence, latency reversing agents (LRA), able to force reservoir cells to produce virions, and thereby to induce their destruction, have been looked for. Various LRA candidates have been tested, but the ability of these LRA to reactivate HIV genome expression is limited. Some latent proviruses seem to even be refractory to LRA. Moreover, none of these LRA is so far specific for reservoir cells, and many of them present with dose-dependent toxicity. It is therefore of importance to identify new classes of LRA.

After binding to CD4+, the main HIV-1 strains (R5) utilize the C-C chemokine receptor type 5 (CCR5) as a coreceptor to enter and activate target cells. CCR5 belongs to the large family of G protein-coupled receptors (GPCR). GPCR may form homo-oligomers as well as hetero-oligomers [1]. Hetero-oligomerization can modulate receptor cell surface density, ligand specificity and/or affinity, coupling to G protein or other intracellular mediators and/or the nature and intensity of the signaling pathway [2–7]. GPCR can also influence each other by heterodesensitization. As we and others have shown that CCR5 signaling boosts HIV replication [8,9], we hypothesized that other GPCR coexpressed with CCR5 at the CD4+ T-cell surface could deliver signals able to reactivate HIV gene expression in reservoir cells. This could happen via at least three potential mechanisms. If such a GPCR dimerized with CCR5, ligands for this GPCR might trigger CCR5 signaling and thereby HIV DNA transcription. Reciprocally, the fact that CCR5 dimerized with this GPCR might modify the signaling of this GPCR resulting in HIV long terminal repeat (LTR) activation. Finally, even if such a GPCR did not dimerize with CCR5, ligands for this GPCR might trigger a signaling pathway able to activate HIV LTR.

To test this hypothesis, we screened the GPCR family for receptors expressed in CCR5+CD4+ T cells and able to interfere with HIV infection. Our approach yielded sphingosine-1-phophate (S1P) receptor 1 (S1P1) as a potential candidate. S1P1 is expressed in most immune cell types including T and B lymphocytes, natural killer cells, dendritic cells, macrophages and neutrophils [10,11]. It plays a critical role in T-cell and B-cell egress from thymus and lymph nodes toward high S1P concentrations present in body fluid compartments [10–16]. Investigating the influence of S1P1 triggering on HIV infection, we observed that S1P1 agonists are LRA.

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Methods

Cells

CD4+CCR5+ T cells were sorted from peripheral blood mononuclear cells (PBMC) with specific antibody-coupled magnetic beads (Dynabeads Pan Mouse IgG; Invitrogen, Thermo Fisher Scientific, Villebon sur Yvette, France) by positive selection. To obtain dendritic cells, monocytes were first isolated from PBMC with CD14 antibody-coated magnetic beads (Milteny, Thermo Fisher Scientific), and cultivated in supplemented RPMI 1640 for 7 days in presence of 10 ng/ml granulocyte-macrophage colony-stimulating factor (GM-CSF) and 50 ng/ml IL4 (Immunotools, Friesoythe, Germany). Macrophages were obtained by in-vitro differentiation with GM-CSF of PBMC isolated by low-density gradient centrifugation.

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Quantitative multi-reverse transcriptase-PCR

RNA was extracted with QIAmp RNA blood (Qiagen, Les Ulis, France). cDNA was synthetized with the cDNA archive kit (Applied Biosystems, Thermo Fisher Scientific). DNA from three donors were mixed and 750 ng of this mixture was loaded on a 384-well microfluidic cards preloaded with primers for 361 different GPCR and 14 housekeeping genes as positive control (TaqMan Low Density Arrays from Applied Biosystems). Quantitative PCR (qPCR) was realized on an ABI Prism 7900HT Sequence Detection System (Thermo Fisher Scientific).

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Time-resolved fluorescence resonance energy transfer

The 293T cells were cotransfected with plasmids expressing SNAP-tagged or CLIP-tagged receptors, plated at 105 cells/well in poly-ornithine-coated 96-well plates. After 30 h, SNAP and CLIP tags were labeled with the fluorophores BC-Lumi4-Tb (1 μmol/l) and BG-Green (0.3 μmol/l) in TagLite (TL) buffer (Cisbio Bioassays, Codolet, France) for 2 h at 37 °C. The emission signal from the BC-Lumi4-Tb and the Green fluorophore were recorded at 620 and 520 nm, respectively, after excitation at 337 and 485 nm on a time-resolved fluorimeter (PHERAstar FS; BMG Labtechnologies, Champigny sur Marne, France). In the same wells, time-resolved fluorescence resonance energy transfer (tr-FRET) signal was measured at 520 nm between 60 and 400 μs after laser excitation at 337 nm.

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Flow cytometry

Cells (2 × 105) were incubated with PE-Cy5-conjugated anti-CCR5 mAb 2D7 (Pharmingen BD, Le Pont de Claix, France), or antihuman S1P1 mAb 21813 (R&D Systems, Lille, France) for 1 h on ice at 10 μg/ml, followed by a 1 : 100 dilution of FITC-conjugated F(ab′)2 fragment goat antimouse IgG (H+L, Beckman Coulter, Vilepinte, France) after washing, or with an isotype control. Cells were then washed, fixed in CellFIX (Becton Dickinson) and analyzed on a FACScalibur (Becton Dickinson).

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Transduction

HIV transfer vectors that express CCR5, CXCR4, LacZ and S1P1 (pWPXL-S1P1) were produced as previously described [17]. The pWPXL-S1P1 plasmid was obtained by cloning a BamH1-Spe1 fragment after PCR amplification of an S1P1 cDNA (clone EDG0100000 from Missouri cDNA Resource Center) into the pWPXL lentiviral vector.

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Infection assays with replicative virus

Human osteosarcoma (HOS) cells were exposed to 10 ng/ml of p24 equivalent of the R5 strain Ad8 or of the X4 strain NL4.3, washed, and cultured in supplemented DMEM medium. MT4 cells were exposed to 10 ng/ml of p24 equivalent of the R5 strain Ad8 or to 40 ng/ml of p24 equivalent of the X4 strain NL4.3, washed, and cultured in supplemented RPMI-1640 medium. Dendritic cells were preincubated with Vpx-containing virus-like particles to neutralize sterile alpha motif and HD domain-containing protein 1 (SAMHD1) [18]. Dendritic cells were then exposed or not to 100 nmol/l of FTY720-P 1 day prior infection, plated at 105 cells/ml, infected overnight with 5 ng/ml of p24 equivalent of the R5 strain Ad8, washed and cultured in supplemented RPMI-1640 medium. FTY720-P was added at each passage. Cell number was adjusted twice a week, and HIV p24 production was measured in cell supernatant using Innotest HIV Antigen mAb kit (Fujirebio, Ingen, Chilly Mazarin, France).

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In-vitro model of HIV latency

PBMC from healthy patients (2 × 106 cells/ml) were exposed to 5 ng/ml of p24 equivalent of the R5 strain Ad8, washed five times and cultured in X-VIVO 15 (Lonza, Levallois-Perret, France) medium supplemented with 100 IU/ml of IL-2. At the indicated time and further on, ligands were added to the culture medium. Cells extracted from the lymph node of an organ donor (2 × 106 cells/ml) were exposed to 14 ng/ml of p24 equivalent of the primary R5 strain MP578, washed five times and cultured in X-VIVO 15 medium supplemented with 10 IU/ml of IL-2. At the indicated time and further on, S1P was added to the culture medium.

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Generation of hu-PBL-severe combined immunodeficiency mice

Severe combined immunodeficiency (SCID) mice are from cb17/Icr-Prkdcscid/Crl genotype. Animals were engrafted by intraperitoneal injection of 30 × 106 PBMC obtained from a healthy donor. Mice with human immunoglobulin plasma concentration above 100 μg/ml were infected with 1000 TCID50 of JR-CSF HIV-1 virus in 100 μl. Daily force-feeding with 100 μl FTY720 (Cayman, Montluçon, France) at 60 μg/ml dissolved in distilled water or vehicle was begun 1 day before infection and continued for 12 days. Viral load was measured by qPCR with mpliPrep/COBAS TaqMan HIV-1 test (Roche, Boulogne-Billancourt, France).

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HIV entry assay

We used the previously described Vpr–β-lactamase (Vpr–Blam) assay [19] to measure the efficiency of HIV core penetration into the cytosol of target cells. Virus stocks were produced by cotransfecting 3 millions of 293T cells with 15 μg of the HIV proviral DNA pNL4.3 env-luc+, an R5 or X4 envelope plasmid (pCMV-Ad8 or pVB34, respectively, 6 μg), a packaging plasmid (pAX2, 2 μg), and the plasmid encoding the Vpr gene fused to the β-lactamase gene (pMM310). Virus preparations were concentrated by ultracentrifugation on 4% sucrose (1 h 30 min, 26 000 rpm, 4 °C). MT4 cells were exposed for 3.5 h to 500 ng of p24 of R5 or to 1 μg of p24 of X4 Vpr–Blam virus. Cells were then washed and loaded with the CCF2-AM LiveBLAzer FRET-B/G Loading Kit (Life Technologies, Thermo Fisher Scientific) in the presence of 15 mmol/l Probenecid (Sigma, Merck, Lyon, France). Cells were incubated for 1 h at room temperature, washed and fixed. CCF2-AM and cleaved CCF2-AM fluorescence (excitation at 405 nm, emission at 520 nm and 447 nmol/l, respectively) was measured on a MACSQuant analyzer 10 (Mylteni, Paris, France).

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Real-time PCR detection of HIV transcripts

To produce replication-defective HIV virions, 293T cells were cotransfected with the pNL4.3-env-luc and pCMV-Ad8-Env plasmids. HOS cells were exposed to 70 ng of p24 equivalent of defective virions and lysed 24 h postinfection. DNA from early and late transcripts was amplified as described [17].

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Infection assays with replicative nuclear factor-kappa B+/− and NFAT+/− viruses

Nuclear factor-kappa B (NFkB)+ and NFKB− viruses were obtained from plasmid pILIC, a derivative of pNL4.3 with only one LTR or NFKB− pILIC in which both NFKB binding sites present in the LTR promoter are mutated (GGGactttccgctgGGG is replaced by CTCactttccgctgCTC). NFAT+ and NFAT− viruses were obtained from pNL4–3 mutated at the NFAT binding site [20]. MT4 cells were infected in triplicate with 20 ng/ml of these X4 viruses.

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Long terminal repeat activation assays

HeLa–LTR–Luc cells [21] were exposed for 10 h to 100 nmol/l of FTY720-P, to SEW2871 at the indicated concentrations, or to 30 μg/ml of the anti-S1P1 mAb 21813 (R&D Systems) and lysed. Luciferase activity was measured in cell extracts using a commercial assay (Promega, Charbonnières-les-Bains, France). Alternatively, HeLa cells were transiently transfected by using lipofectamine Plus (Invitrogen) with the LTR-luc reporter plasmid pL274 [22], either wild-type or mutated in the NFκB binding sites (pL274 with the same mutations as in the NFKB− pILIC). FTY720-P (100 nmol/l) or 10 ng/ml TNFα were added 24 h later for 6 h and luciferase activity was measured.

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Immunofluorescence assay

HeLa cells were exposed for 2 h to 100 nmol/l FTY720-P or to 10 ng/ml TNFα, labeled for 45 min with an NFκB p65 antibody (F-6, Santa Cruz, Merck) followed by a secondary fluorescent antimouse antibody, and with Dapi (5 μg/ml).

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Statistics

Values are expressed as the mean ± SEM. Differences were analyzed with unpaired Student's t test.

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Study approval

Animal studies were approved by the local Animal Care Committee. Lymph node extractions from organ donors were approved by the Agence de la Biomédecine.

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Results

Identification of the GPCR genes coexpressed with CCR5 in CD4+ T cells

We looked for GPCR expression in CD4+CCR5+ T cells sorted from the PBMC of healthy donors. Out of 361 GPCR genes, we detected the expression of 250 of them in CD4+CCR5+ T lymphocytes by multi-RT-PCR. In particular, S1P1 mRNA was 33-fold more abundant in these cells than CCR5 mRNA.

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Sphingosine-1-phosphate receptor 1 forms heteromers with CCR5 in the outer cell membrane

We then tested the ability of S1P1 to oligomerize with CCR5 by tr-FRET. Both receptors were fused at their N-terminal extracellular domain with the genetically encoded tags SNAP and CLIP, that allow specific labeling with nonpermeant fluorophores. SNAP-S1P1 and CLIP-CCR5 encoding plasmids were cotransfected in 293T cells, and the tr-FRET signal between cell surface tagged-receptors was measured [23]. We observed a superposition of the saturation curves with the two lowest CCR5 amounts (Fig. 1a), indicating that under these conditions the interactions between CCR5 and S1P1 are specific [24]. The beta-2 adrenergic receptor, known not to interact with CCR5 [25], gave a nonsaturating tr-FRET signal [26] with CLIP-CCR5 (Fig. 1b). As a positive control, CLIP-CCR5 was found to form homomers with SNAP-CCR5 as previously shown [27]. Conversely, no interaction was found between S1P1 and the other HIV coreceptor, CXCR4, whereas CXCR4 formed homomers (Fig. 1c).

Fig. 1

Fig. 1

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Sphingosine-1-phosphate receptor 1 expression increases HIV replication

To analyze the consequences of S1P1 coexpression on HIV infection, we transduced HOS cells expressing CD4 and CCR5 with either an S1P1 or a negative control lacZ transgene. The two cell lines thus obtained displayed the same cell surface CCR5 densities (Fig. 2a). We also checked that the former, but not the latter cell line expressed S1P1 (Fig. 2b). We then infected both cell lines with an R5 or an X4 strain, and observed that S1P1-positive cells produced much more virus than the lacZ-expressing cells (Fig. 2c and d). At day 11, the mean p24 concentrations in the supernatant of S1P1-expressing cells were 4441 and 879 ng/ml, whereas they were 189 and 2 ng/ml in the supernatant of lacZ-expressing cells after R5 and X4 infection, respectively. We obtained similar results with the lymphoid cell line MT4 (Fig. 2e–h).

Fig. 2

Fig. 2

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Sphingosine-1-phosphate receptor 1 expression boosts HIV genome transcription via NFκB

To examine whether the presence of S1P1 influenced HIV entry, we compared viral entry into MT4 cells transduced or not with S1P1. Cells were infected with R5 or X4 envelope-pseudotyped virions harboring the enzyme β-lactamase fused to the Vpr protein [28]. β-lactamase activity of the exposed cells was then measured by flow cytometry as a marker of viral entry. S1P1 expression increased by 30.2 ± 7.4% R5 (Fig. 3a), but not X4 (Fig. 3b) entry. Preincubation of target cells with a soluble form of CD4+ prevented viral entry (Fig. 3a and b).

Fig. 3

Fig. 3

To study whether the presence of S1P1 modified reverse transcription efficiency, we exposed S1P1-transduced or lacZ-transduced HOS cells to R5 virions, and quantified the presence of early and late reverse transcripts in the infected cells. As for the entry, we observed a slight increase in HIV DNA content in S1P1-expressing cells, but this difference was not significant (Fig. 3c and d). Thus, the presence of S1P1 in target cells did not modify reverse transcription efficiency.

As S1P1 triggering activates NFκB via Gαi proteins [29–31], we questioned whether S1P1 expression could increase HIV transcription. HeLa cells stably transfected with an LTR fused to a luciferase reporter gene [21] were transduced with S1P1 or lacZ gene. We observed that S1P1-expressing HeLa cells displayed a two-fold increase in luciferase expression compared with lacZ-expressing HeLa cells (Fig. 4a). As shown in Fig. 4a, preincubation of the S1P1-transduced cells with an anti-S1P1 antibody abolished LTR activation, suggesting that this activation was triggered via S1P1 by S1P present in the bovine serum added to the culture medium [32]. Conversely, addition to the cell culture of FTY720-P, a compound with an initial S1P receptor agonist effect [33], further increased luciferase expression (Fig. 4a). The same phenomenon was observed with the specific S1P1 agonist SEW2871 [34], in a dose-dependent manner (Fig. 4b).

Fig. 4

Fig. 4

We next explored whether S1P1 expression resulted in NFκB activation. Actually, we observed the accumulation of NFκB p65 protein at perinuclear and nuclear sites in S1P1-expressing cells (Fig. 4c). This translocation was increased in these cells by FTY720-P. TNFα did the same in S1P1-positive and S1P1-negative cells. To further assess the role of NFκB in S1P1-driven LTR activation, HeLa cells transduced or not with S1P1 were transiently transfected with plasmids carrying the luciferase gene under the control of an LTR deleted or not of the NFκB binding sites, and exposed to FTY720-P. As expected, FTY720-P induced the expression of the luciferase gene driven by the wild-type LTR in S1P1-positive cells. By contrast, the reporter gene driven by the NFκB-deleted LTR was transcribed in S1P1-expressing cells neither under FTY720-P nor under TNFα stimulus (Fig. 4d). We confirmed that NFκB is involved in S1P1-induced HIV-1 genome activation by infecting S1P1-transduced or lacZ-transduced MT4 cells with X4 HIV strains harboring an LTR deleted (NFκB−) or not (NFκB+) of its NFκB binding sites. As expected NFκB+ virus production was higher in S1P1-positive than in S1P1-negative cell cultures. And this difference was abrogated in cell cultures infected with the NFκB− virus (Fig. 4e). As a control we performed the same experiment with viruses harboring an LTR deleted (NFAT−) or not (NFAT+) of the NFAT binding site. This time, we observed no consequence of this deletion on the effect of the presence of S1P1 on viral production (Fig. 4f). Altogether, our data show that the major effect of S1P1 expression is an increase in viral genome transcription mediated by NFκB.

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A sphingosine-1-phosphate receptor 1 antagonist reduced HIV production in primary immune cells in vitro and in vivo

FTY720-P has an initial S1P1 agonist effect, as observed in Fig. 4a, but it can also display an antagonist effect on the long term due to S1P1 internalization and degradation. We reasoned that FTY720-P could inhibit HIV production in culture. Therefore, we decided to test the effect of FTY720-P on the infection of primary immune cells. We chose to study this effect on in-vitro infection of monocyte-derived dendritic as these cells display S1P1 receptors at their surface (Fig. 4g). We neutralized the HIV restriction factor SAMHD1 by exposing these dendritic cells to pseudo viral particles delivering Vpx, and infected them with an R5 strain in presence or absence of FTY720-P. FTY720-P reduced viral production (Fig. 4h). We also tested the effect of FTY720 on HIV-1 R5 infection in vivo. SCID mice were reconstituted by intraperitoneal injection of human PBMC. The animals were then force-fed with FTY720, that is converted into its phosphorylated active form FTY720-P in vivo, or with vehicle, and infected with an HIV-1 R5 strain. We observed a 13-fold decrease in plasma viral load in FTY720-treated mice as compared with the negative controls (31 ± 31 HIV versus 398 ± 106 HIV RNA copies/ml, P = 0.014, Fig. 4i).

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Sphingosine-1-phosphate receptor 1 agonists induce reactivation of HIV-1 in peripheral blood mononuclear cells and in lymph node cells

We surmised that S1P1 agonists might reverse HIV latency in reservoir cells. To generate an in-vitro model of reservoir cells, we infected PBMC or lymph node cells with a primary R5 strain in absence of stimulation. Under these conditions, cells transiently produce viral particles and return to a nonproductive quiescent state. At this stage, CD3 and CD28 stimulation induced a second round of viral production (Fig. 5a). As compared with nontreated cultures (26.6 ± 8.7 pg/ml of p24), exposition of quiescent infected PBMC to S1P (118.1 ± 41.9 pg/ml, P = 0.029, Fig. 5b and c) or to the S1P1 agonist SEW2871 (112.1 ± 38.5 pg/ml of p24, P = 0.028, Fig. 5b and c) provoked a rebound in HIV p24 production, 2 and 6 days after the S1P1 ligand addition. FTY720-P also induced p24 production, but this de-novo production was not significant (Fig. 5b). S1P addition to a culture of lymph node cells infected in nonactivating conditions after the peak of viral production also provoked a viral rebound (Fig. 5d). The amount of virus produced 72 h after S1P stimulation was significantly higher than in absence of stimulation (27.3 ± 9.1 and 0.9 ± 0.2 pg/ml of p24, respectively, P = 0.050, Fig. 5e).

Fig. 5

Fig. 5

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Discussion

In an effort to identify new GPCR that interfere with HIV replication in immune target cells, we analyzed GPCR genes expression in CCR5+CD4+ T cells by RT-PCR. We found that the S1P1 gene is highly transcribed in CCR5+CD4+ T cells. S1P1 is required for the exit of B and T lymphocytes from secondary lymphoid organs [11]. At the nervous system level, S1P1 is involved in myelination, astrogliosis and microglial production of proinflammatory cytokines [35]. S1P1 also regulates angiogenesis, cardiac development and vascular barrier integrity [36,37]. We show that S1P1 is able to form heteromers with CCR5 on the outer cellular membrane. It has been previously reported that CCR5 forms homo-oligomers and coimmunoprecipitates with the opioid receptors [38] independently of ligand binding [27]. Furthermore, mu opioid receptor and CCR5 heterodimerize and cross desensitize each other [39]. CCR5 has also been found to heterodimerize with CCR2 [40], CXCR3 [41], and CXCR4 [42]. It is also known that interaction of CCR5 with the mu and delta opioid receptors [43–46] and with other nonchemokine [47–54] or chemokine [40–42] GPCR modulate HIV infectability via heterodimerization or heterodesensitization. Here we show that the major consequence of S1P1 expression in HIV infectible cells is an increase in viral gene expression via the transcription factor NFκB. The fact that the early steps of HIV life cycle are not strongly modified by the presence of S1P1, and that its presence boosts X4 as well as R5 strain replication whereas S1P1 and CXCR4 do not dimerize, argues for an absence of role for S1P1-CCR5 heteromerization in S1P1 effect on HIV life cycle. In particular, S1P1 ability to boost HIV genome transcription appears no to be linked to its dimerization with CCR5. For in-vitro infection experiments, cells are grown into culture medium containing fetal bovine serum, a source of S1P. Moreover, S1P is produced by several cell types and may act in an autocrine or paracrine manner [55]. It is therefore possible that the effects we observed result from S1P1 stimulation by S1P present in the medium and/or produced by the cells.

We observed an HIV RNA plasma level decrease in FTY720-treated humanized mice, consistently with the results obtained by Murooka et al.[56] in a similar experiment. These authors attributed this phenomenon to the blocking of migratory T cells egress from the lymph nodes into efferent lymph vessels, and to the interruption of T-cell recirculation, hence limiting HIV dissemination [56]. Given our data, it may now be added that there is an additive inhibitory effect of this functional S1P1 antagonist on HIV infection. Two studies reported the effect of FTY720 administration on simian HIV (SHIV) infection. Kersh et al.[57] injected the drug into infected rhesus macaques, whereas Morris et al.[58] challenged FTY720-treated pigtail macaques by repeat vaginal exposures to the virus. In these experiments, FTY720 was administrated weekly rather than continuously. Given the bimodal action of the drug on S1P1 activation, this difference may be important. The authors reported no inhibitory effect of the S1P1 ligand, neither on SHIV susceptibility, nor on viremia. Nonetheless, it is interesting to note that in the former study, SHIV plasma levels raised after most of the injections of FTY720 at a functional dose of 0.1 mg/kg. This in-vivo effect in a primate model of HIV infection is consistent with our data.

It is possible that CCR5-S1P1 dimerization alters S1P1 functions. CCR5 ligands might further modify the response of S1P1 to S1P, and thereby hinder the T-cell egress from thymus and lymph nodes. This raises the interesting possibility that gp120, by binding to CCR5, might be responsible for the effects of HIV-1 on T-cell production via S1P1 [59], and for impaired T-cell responses to S1P in HIV-1-infected lymph nodes [60]. Such a mechanism might also be involved in the adenopathies observed in viremic patients [61].

It could be of interest to study the effect of FTY720, or of more recent S1P1 antagonists [35], in persons living with HIV, in as much as FTY720 is already used to treat multiple sclerosis [62]. In addition to the inhibitory effect on HIV DNA transcription we describe here, S1P1 antagonists could have additional beneficial effects. Actually, S1P1 signaling has been reported to enhance Th17 polarization [63] and to decrease the differentiation of thymic regulatory T cells (Treg) precursors as well as the function of mature Treg cells [64,65]. Thus, blocking this signaling could downregulate the immune activation that is deleterious even in patients aviremic under treatment [66].

HIV latency is the consequence of combined mechanisms. Therefore, the reversion of this phenomenon may need drugs acting on different levels. Moreover, antilatency agent association should allow to reduce the posology of each agent and thereby their toxicity. It will be of interest to look for a synergistic effect between S1P1 agonists and other LRA.

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Acknowledgements

We thank Fabrizio Mammano and Blandine Monel for sharing materials, protocole and advice with the Blam–Vpr assay, Mingce Zhang and Randy Cron for sharing the NFAT+/− viruses, Ke Zhang and Monsef Benkirane for the NFKB+/− constructs and advice, Mathieu Ringeard for advice with obtention of dendritic cells and Yamina Bannasser for sharing Vpx-containing virus-like particles. MT4 cells are a gift from Martine Biard.

The work was funded by Sidaction (grant no. 021325 A118-1-01232), by the ‘Association Française d’Epargne et de Retraite’, and by the ‘Agence Nationale de Recherche sur le SIDA et les Hépatites Virales’ (ANRS, decision no. 11125). C.D. was funded by a fellowship from ANRS (NM/no. 3099 ICSS 1/AO 2011-1).

Author contributions: C.D. and S.G. transduced the cells, acquired, analyzed and interpreted flow cytometry data, LTR activation and infection assays. A.G. performed HIV entry assays and quantified HIV transcripts. B.V. and C.B. carried out HIV infection assays. D.M., E.D., L.C.-A., L.P. and V.F. were in charge of the FRET analyses. N.C. and J.T. managed the infection of SCID mice. V.F. and P.C. contributed to the conception and design of the study, analyzed and interpreted data, and wrote the article.

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Conflicts of interest

There are no conflicts of interest.

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* Charline Duquenne, Sandrine Gimenez, Adeline Guigues, Pierre Corbeau and Vincent François contributed equally to the article.

Keywords:

CCR5; G protein-coupled receptor; HIV transcription; NFκB; signaling

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