2001, 75(1):439. DOI: 10.1128/JVI.75.1.439-447.2001. J. Virol. 

HazanRené Olivier, Stephano Marullo, Pascale Briand and Uriel Susana T. Valente, Chantal Chanel, Julie Dumonceaux, m7NDKImmunodeficiency Virus Type 1 Isolatewith the CD4-Independent X4 Human CXCR4 Is Down-Regulated in Cells Infected

http://jvi.asm.org/content/75/1/439Updated information and services can be found at:

These include:

REFERENCEShttp://jvi.asm.org/content/75/1/439#ref-list-1at:

This article cites 40 articles, 22 of which can be accessed free

CONTENT ALERTS more»articles cite this article),

Receive: RSS Feeds, eTOCs, free email alerts (when new

http://journals.asm.org/site/misc/reprints.xhtmlInformation about commercial reprint orders: http://journals.asm.org/site/subscriptions/To subscribe to to another ASM Journal go to:

on October 27, 2012 by 00308377

http://jvi.asm.org/

Dow

nloaded from

JOURNAL OF VIROLOGY,0022-538X/01/$04.0010 DOI: 10.1128/JVI.75.1.439–447.2001

Jan. 2001, p. 439–447 Vol. 75, No. 1

Copyright © 2001, American Society for Microbiology. All Rights Reserved.

CXCR4 Is Down-Regulated in Cells Infected with theCD4-Independent X4 Human Immunodeficiency

Virus Type 1 Isolate m7NDKSUSANA T. VALENTE,1* CHANTAL CHANEL,1,2 JULIE DUMONCEAUX,1 RENE OLIVIER,3

STEPHANO MARULLO,4 PASCALE BRIAND,1 AND URIEL HAZAN1,2

INSERM Unite 380 Laboratoire de Pathologie et Genetique Experimentales, Institut Cochin de Genetique Moleculaire,75014 Paris,1 Unite d’Oncologie Virale, Institut Pasteur, 75015 Paris,3 Unite UPRES A-8068,

Centre National de la Recherche Scientifique, 75014 Paris,4 and UFR de Biochimie,Universite de Paris VII-Denis Diderot, 75251 Paris,2 France

Received 24 May 2000/Accepted 29 September 2000

Macrophages and T cells infected in vitro with CD4-dependent human immunodeficiency virus type 1 (HIV-1)isolates have reduced levels of CD4 protein, a phenomenon involved in retroviral interference. We have previouslycharacterized the first CD4-independent HIV-1 X4 isolate m7NDK, which directly interacts with CXCR4through its mutated gp120. We thus investigate CXCR4 expression in cells infected with this m7NDK CXCR4-dependent HIV-1 mutant. We present evidence of the down-regulation of CXCR4 membrane expression in CD4-positive or -negative cells chronically infected with the HIV-1 m7NDK, a phenomenon which is not observed in theCD4-dependent HIV-1 NDK parental strain. This down-regulation of CXCR4 was demonstrated by fluores-cence-activated cell sorter analysis and was confirmed by the absence of CXCR4 functionality in m7NDK-infectedcells, independently of the presence of CD4 protein. Furthermore, a drastic reduction of the intracellular levelof CXCR4 protein was also observed. Reduced levels of CXCR4 mRNA transcripts were found in m7NDK-infected HeLa and CEM cells, reduced levels that could not be attributed to a reduced stability of CXCR4 mRNA.Down-regulation of CXCR4 on m7NDK-infected cells may thus be explained by transcriptional regulation.

Expression of CD4 (20, 22) and the chemokine receptorsCCR5 and CXCR4 at the target cell surface is essential forhuman immunodeficiency virus (HIV) entry (2, 24). HIV type1 (HIV-1) cell entry is mediated by a first interaction betweenenvelope (Env) glycoprotein gp120 and CD4, which induces aconformational change in gp120, exposing the coreceptor bind-ing site or creating the conformational coreceptor binding site,leading to membrane fusion (6, 21, 23, 32).

Macrophages and T cells infected with HIV in vitro havereduced surface CD4 expression (8, 13, 16). The reduction ofCD4 surface expression is due to the combined action of threeviral proteins: Env, Vpu, and Nef. The HIV envelope proteinprecursor gp160 forms a complex with CD4 in the endoplasmicreticulum (ER) of infected cells (7, 18, 36), and Vpu triggersthe degradation of ER-retained CD4 molecules (37, 38). Theauxiliary Nef protein triggers the accelerated internalization ofCD4 molecules that have already reached the cell surface (1,30, 33).

We have previously reported the characterization of the firstHIV-1 strain that no longer requires the presence of CD4 toenter its target cells (10). This CD4-independent isolate wasderived spontaneously from the X4 HIV-1 isolate NDK after along-term culture (average of 200 days) in the CD41 T-cell lineCEM and has been named m7NDK. This new tropism hasbeen shown to correlate with seven specific amino acid changes

in critical regions of gp120, C2, V3, and C3. We have postu-lated that this mutant envelope subunit has either a predis-posed conformation or a greater binding affinity for CXCR4and overcomes the need for CD4-induced conformationalmodifications (10).

Our interest focused on CXCR4 receptor expression, incells infected with the CD4-independent CXCR4-dependentm7NDK HIV-1. Down-regulation of CXCR4 has been de-scribed in CD41 T cells following infection with the humanherpesvirus 6 (HHV-6) and HHV-7 (34, 39); however, it isworth noting that these viruses do not use CXCR4 as areceptor (40). It has been established that CXCR4 is down-regulated by a few HIV-2 isolates which use CXCR4 as theirprimary receptor (12), although down-regulation of the core-ceptor CXCR4 by X4 CD4-dependent HIV-1 viruses has neverbeen characterized. A variant of HIV-1/IIIB termed HIV-1/IIIBx has been characterized (17) that is both replication com-petent and fusogenic for a CD4-negative subclone of SupT1cell line. However, it failed to down-regulate CXCR4 in chron-ically infected cells (17).

Recently, several studies have shown that regulation ofCXCR4 mRNA expression depends on cell activation andoxidative stress, as well as cell type (5, 26, 31). Furthermore,signaling and internalization of CXCR4 protein can be regu-lated by receptor phosphorylation-dependent and -indepen-dent mechanisms (15), and alternative trafficking of CXCR4can be induced by several chemical or pathogenic agents (3,35). Nevertheless, regulation of CXCR4 membrane expressionhas not yet been described after infection with a CXCR4-dependent virus.

Here we present evidence of CXCR4 down-regulation in the

* Corresponding author. Mailing address: INSERM Unite 380Laboratoire de Pathologie et Genetique Experimentales, Institut Co-chin de Genetique Moleculaire, 22 Rue Mechain, 75014 Paris, France.Phone: 33-1-40516484. Fax: 33-1-40516407. E-mail: valente@cochin.inserm.fr.

439

on October 27, 2012 by 00308377

http://jvi.asm.org/

Dow

nloaded from

CD41 T-cell line CEM, as well as in the nonlymphocytic CD42

HeLa cell line, infected with the m7NDK mutant. We demon-strate the absence of CXCR4 surface expression and function-ality in these cells. Analysis of CXCR4 mRNA transcriptsrevealed a decreased CXCR4 mRNA steady-state level whichwas not observed in the CD4-dependent parental strain oruninfected cells. However, these results did not correlate witha reduction of CXCR4 mRNA transcript stability. In addition,we demonstrate that it is an active phenomenon since we ob-serve CXCR4 down-regulation upon acute infection of CEMcells.

Taking all of these findings together, our results suggest thatdown-regulation of CXCR4 upon m7NDK infection might beexplained by transcriptional regulations and may provide amean for the m7NDK isolate to monitor viral interference.

MATERIALS AND METHODS

Cell lines and viruses. The CD4-positive human lymphoid cell line CEM wasa gift from J. L. Virelizier (Institut Pasteur, Paris, France) and was grown inRPMI 1640 (Life Technologies) medium supplemented with 5% fetal calf serum,antibiotics, and glutamine (Life Technologies). The NDK isolate was a gift fromF. Barre-Sinousi (Institut Pasteur) (11) and was propagated in CEM cells. Thepreviously described NDK mutant m7NDK was obtained after a long-term cul-ture in CEM cells (10).

HeLa, P42 cells (HeLa CD41 long terminal repeat [LTR]-lacZ) and Z24(HeLa LTR-lacZ) have been previously described (9) and were kindly providedby M. Alizon, ICGM, Paris, France. Adherent cell lines were grown in Dulbeccomodified Eagle medium supplemented with 5% fetal calf serum, antibiotics, andglutamine (Life Technologies).

Three clones (14, 48, and 108) of HeLa cells chronically infected with m7NDKisolate were kept for further analysis, after endpoint dilution cloning of chron-ically infected HeLa cells.

Infection of CEM cells. Virus were added to CEM cells at 16 ng per 106 cellsand incubated at 37°C for 4 h in a minimum volume. After this period of time,supplemented RPMI medium was added to attain a final concentration of 106

cells/ml. Infection was then followed by fluorescence-activated cell sorter (FACS)analysis, as well as by cell fusion assays.

Flow cytometry analysis. Aliquots of 106 cells were subjected to direct orindirect label staining to analyze the surface and/or intracellular expression ofantigens. Nonadherent cells were washed with ice-cold Cell Wash (Becton Dick-inson) and stained with the primary antibodies for 1 h at 4°C. Adherent cells werefirst harvested using 13 phosphate-buffered saline (PBS; Life Technologies)–citrate (0.01 M) and then treated as the nonadherent cell lines. They werewashed with Cell Wash to remove unbound antibody and stained with thesecondary antibody for 1 h at 4°C. The cells were then washed and resuspendedin Cell Wash containing 1% formaldehyde (Merck), kept at 4°C, and analyzed.The chemokine receptor CXCR4 was detected by indirect staining with anti-CXCR4 MAb12G5, MAb171, MAb172, or MAb173 (R & D Systems) monoclo-nal antibodies (MAbs), followed by treatment with phycoerythrin (PE)-conju-gated rabbit anti-mouse immunoglobulin G (IgG) (Dako) or by direct stainingwith PE-conjugated anti-CXCR4 MAb173 or with fluorescein isothiocyanate(FITC)-conjugated anti-CXCR4 MAb12G5.

For the intracellular detection of CXCR4, cells were first saturated withunconjugated anti-CXCR4 (MAb173), followed by staining with the secondaryantibody FITC-conjugated rabbit anti-mouse IgG (Amersham). After staining,cells were fixed with 3% formaldehyde, washed with Cell Wash (Pharmingen)and quenched to saturate free radicals with glycine (20 mM). Cells were perme-abilized in Cell Wash containing 0.05% saponin and then stained with PE-conjugated anti-CXCR4 MAb173. Nonspecific fluorescence was determined byusing irrelevant isotype-matched MAbs (Dako).

Transferrin receptor was detected using a MAb R-Trf (Roche), CD4 wasdetected using MAb MT310 (Dako) or MAb OKT4, and the HIV-1 Env proteinwas detected using a seropositive serum.

A FACSCalibur (Becton Dickinson Immunocytometry Systems, San Jose,Calif.) or a FACScan (Epics Elite; Coulter, Miami, Fla.) was used for cytometryanalysis. The excitement radius was 488 nm, and the emission radius band-passwas at 575 nm for 10,000 cell events.

Immunofluorescence microscopy. Adherent cells were plated in Lab-Tekchamber slides (Polylabo). Characterization of intracellular chemokine receptor

expression was achieved by fixation of cells in 3% formaldehyde (15 min) at roomtemperature (RT), quenching in 0.1 M glycine-PBS, and saturation with PBScontaining 0.2% bovine serum albumin and 0.05% saponin (Sigma). In the samebuffer, an overnight incubation at 4°C was performed with MAb against CXCR4(MAb173). The cells were washed and subsequently incubated with anti-mousecyanin 3 (Cy3)-conjugated secondary antibody (Caltag) in the same buffer for 1 hat RT, followed by extensive washing prior to mounting. The staining of cellsurface proteins was as described above except for the use of saponin, which wasexcluded from the buffer. Cells were double stained for CXCR4 and Env protein,with MAb173 and a seropositive serum, followed by treatment with anti-mouseantibody–Cy3 and anti-human antibody–FITC.

Omission of the primary antibody and substitution with an isotype-matchedMAb served as a control. After mounting, the cells were observed with a laserconfocal microscope (MRC 1000; Bio-Rad, Hercules, Calif.).

Cell fusion assays. Cell fusion assays were performed between adherent ornonadherent cells chronically infected by HIV-1, as previously described (9, 10).Fusion efficiency was analyzed 11 h later. Measurements of b-galactosidaseenzyme activities were done as previously described by staining in situ with5-bromo-4-chloro-3-indolyl-b-D-galactopyranoside (X-Gal; Life Technologies)as a substrate or by using a quantitative assay using chlorophenol red–b-D-galactopyranoside (CPRG; Roche) as a substrate (10).

Measurement of calcium mobilization. Changes in the cytosolic free-calciumconcentration were measured in cells loaded with 1 mM Fura 2-acetoxymethyl-ester (Fura 2-AM) (Sigma) at 37°C for 1 h (14, 27, 29). Cells were washed andresuspended in 20 mM HEPES-Hanks balanced salt solution (HBSS; Life Tech-nologies). Fura-2 fluorescence assays were performed with aliquots of 4 3 107

cells in 2 ml of HBSS, using a fluorimeter (Jobin Yvon 3D; Jobin Yvon, Lon-jumeau, France) equipped with a thermally controlled cuve holder and a mag-netic stirrer. After we recorded the baseline [Ca21]i levels, stromal-cell-derivedfactor 1a (SDF-1a) at 10 nM (R & D Systems) was added. The excitation andemission wavelengths for Fura-2 fluorescence assays were 340 and 510 nm,respectively. Cytosolic calcium concentrations were calculated as described pre-viously (14). Tracings were reproduced and scanned using an Agfa Snap CAM,with version F-3.0 Color It software (Apple).

Chemotaxis assays. Cell migration in response to SDF-1a (R & D Systems)was measured in 3.0-mm-pore-size Transwell cell culture chambers (Costar). Inthe upper chamber, 106 cells were suspended in 100 ml of complete RPMI 1640and placed on top of the lower chamber containing 500 ml of complete RPMI1640 with different concentrations of SDF-1a. Plates were incubated at 37°C inCO2 for 5 h. The upper chamber was then carefully removed, and the numbersof viable cells present on the lower chamber were counted using trypan blueexclusion. The percentages of the transmigrations were determined for eachconcentration of SDF-1a.

Northern blot analysis. Total cellular RNA extraction and purification wasperformed using an RNA B isolation system (Bioprobe) according to the man-ufacturer’s protocol. RNA was extracted from uninfected or chronically infectedcells, and 10 mg of each preparation was denatured with formaldehyde and sizefractionated by electrophoresis on a 1% agarose gel. The RNAs were thentransferred to a hybridization transfer membrane and hybridized with a 32P-labeled CXCR4 cDNA or glyceraldehyde-3-phosphate dehydrogenase (GAPDH)cDNA probes. To determine the posttranscriptional stability of CXCR4 mRNA,actinomycin D (Sigma) was added at 5 mg/ml to uninfected or infected cells toblock transcription. Cells were collected after various incubation periods andwere used for RNA extraction. The half-life of CXCR4 mRNA was estimated byplotting the densitometric ratios of CXCR4 mRNA versus GAPDH mRNA,determined using Image Quant Tools, version 1.0, for Power Macintosh.

RESULTS

Surface CXCR4 protein expression. The level of cell sur-face expression of CXCR4 in uninfected, Wild-type NDK (wt-NDK)-infected, or m7NDK chronically infected CEM cellswas analyzed by flow cytometry (Fig. 1A). Two different MAbswere used, directed against different CXCR4 epitopes,MAb12G5 and MAb173. Binding of MAb12G5 and MAb173to m7NDK-infected cells was negative compared to that foruninfected or wtNDK-infected cells. The same result was ob-tained with two other antibodies directed to different CXCR4epitopes (data not shown). The expression of CD4 by flowcytometry was also monitored to insure that down-modulation

440 VALENTE ET AL. J. VIROL.

on October 27, 2012 by 00308377

http://jvi.asm.org/

Dow

nloaded from

of CD4 was observed on wtNDK-and m7NDK-infected CEMcells (Fig. 1B). Expression levels of the transferrin receptorand the HIV envelope protein were also measured by flowcytometry in order to verify the integrity and the infected stateof these cells, respectively (Fig. 1B).

Surface expression of CXCR4 was also determined on aCD4-negative cell line to determine whether down-regulationof CXCR4 upon m7NDK infection was a CD4-dependent phe-nomenon. CXCR4 expression was evaluated on uninfectedHeLa cells and three different clones of m7NDK chronicallyinfected HeLa cells, by flow cytometry using MAb12G5 andMAb173 (Fig. 1C). Surface expression of CXCR4 was detected

on uninfected HeLa cells but was not detected on any of thethree different HeLa m7NDK clones, thus indicating that theCXCR4 modulation is independent of CD4 cellular expressionand is not specific to the CEM CD4-positive lymphocytic cellline.

A coculture test using LTR-lacZ indicator cells either posi-tive or negative for CD4 expression (9, 10) was also performedin parallel to the FACS assays to confirm Env protein expres-sion and fusion ability (data not shown).

Functionality of CXCR4. The natural ligand for CXCR4 isSDF-1a (4, 28). To test CXCR4 functionality at the cell sur-face, we analyzed both the intracellular Ca21 flux and chemo-

FIG. 1. Analysis of cell surface expression of CXCR4. (A) Flow cytometric analysis of CXCR4 at the surface of uninfected, wtNDK-infected,or m7NDK chronically infected CEM cells. Cells were incubated with two different MAbs directed against CXCR4, MAb12G5 and MAb173, orwith an isotype-matched control (CTRL; shaded area). (B) Uninfected, wtNDK-infected, and m7NDK-infected CEM cells were stained for flowcytometric analysis of CD4 (MAb MT310), transferrin receptor (MAb R-Trf), or the HIV-1 Env protein (serum from seropositive patient). (C)Flow cytometric analysis of surface CXCR4 of uninfected or three different HIV-1 m7NDK-infected HeLa cell clones using MAb12G5 andMAb173. The control represents each one of the cell lines stained with a respective isotype-matched control.

VOL. 75, 2001 CXCR4 DOWN-REGULATION BY X4 CD4-INDEPENDENT HIV-1 441

on October 27, 2012 by 00308377

http://jvi.asm.org/

Dow

nloaded from

tactic response following SDF-1a stimulation on m7NDK-in-fected CEM cells compared to uninfected or wtNDK-infectedCEM cells.

As shown in Fig. 2A, m7NDK-infected CEM cells presentedan elevation of [Ca21]i in response to SDF-1a that was .60%reduced in comparison to uninfected or wtNDK-infected CEMcells. These last two cell lines presented a similar response toSDF-1a, excluding the possibility that the chronically infectedcondition might alter cell responsiveness to SDF-1a. Each ofthese three cell lines responded strongly and in a similar fash-ion to the nonspecific Ca21 ionophore, ionomycin (14; data notshown).

We next verified the capacity of surface CXCR4 to initiatecell migration in response to an SDF-1a gradient (Fig. 2B)(19). Uninfected, wtNDK-infected, or m7NDK chronically in-fected CEM cells were layered on a transwell upper chamber,and their migration, induced by two different doses of SDF-1a

(10 and 100 nM), was evaluated 5 h later. No migration wasobserved with m7NDK CEM cells even at the highest SDF-1aconcentration (100 nM). In contrast, wtNDK-infected or un-infected CEM cells presented a similar pattern of migrationwhatever SDF-1a concentration was used. This finding indi-cates that the chronic status of infection is not responsible form7NDK-infected cell unresponsiveness.

In summary, CXCR4 expressed at the cell surface of un-infected or wtNDK-infected CEM cells displayed equally thecharacteristics of functional receptors. In contrast, on m7NDK-infected cells, CXCR4 functionality for chemotaxis, as well asfor intracellular calcium flux responses, was severely reduced.This clearly correlates with previous FACS analyses and isindicative of the absence of CXCR4 membrane expression.

Intracellular CXCR4 protein expression. The intracellularCXCR4 level was determined by flow cytometry in m7NDK-infected cells, as well as in uninfected or wtNDK-infected cells.Figure 3 shows the intracellular versus surface presence ofCXCR4 detected by MAb12G5. This experiment allowed thedetection of intracellular CXCR4 in m7NDK-infected CEMcells, although its expression was drastically reduced comparedto those in uninfected or wtNDK-infected cells (Fig. 3A). Sim-ilar results were observed for CD4-negative HeLa cells, sincethe cellular clones of m7NDK chronically infected HeLa cellsshowed a drastic intracellular CXCR4 reduction compared touninfected HeLa cells (Fig. 3B).

Immunofluorescent microscopy experiments were carriedout in both uninfected (Fig. 4A) and m7NDK-infected (Fig.4B) HeLa cells to analyze intracellular CXCR4 protein. Cellswere permeabilized and stained with anti-CXCR4 MAb173,followed by treatment with anti-mouse antibody–Cy3 (red). Ininfected cells the intracellular expression of CXCR4 presenteda pattern of distribution similar to that for the uninfectedHeLa cells, although only scarce amounts could be detected.

As a control to verify surface CXCR4 expression and theinfected condition of these cells, cells were left intact anddouble stained for CXCR4 and Env, using MAb173 followedby treatment with anti-mouse antibody–Cy3 (red) and a sero-positive serum followed by treatment with anti-human anti-body–fluorescein isothiocyanate (FITC) (green), respectively.In uninfected HeLa cells (Fig. 4C), CXCR4 is clearly observedand no viral proteins were detected; as expected, CXCR4 wasnot detected on the membrane of m7NDK-infected HeLa cells(Fig. 4D), and thus only the staining for viral Env proteins wasdetermined.

These results, along with the intracellular FACS analysis re-sults, confirm the intracellular down-modulation of the CXCR4protein in m7NDK-infected HeLa and CEM cells.

CXCR4 mRNA analysis. Since a clear reduction of intracel-lular CXCR4 protein was observed in m7NDK-infected CEMand HeLa cells, we performed CXCR4 mRNA transcript anal-ysis (Fig. 5). Northern blot experiments, using a cDNA CXCR4-specific 1-kb probe, revealed an almost undetectable level ofCXCR4 mRNA transcripts on both CEM m7NDK-infected(Fig. 5) and HeLa m7NDK-infected (data not shown) cells,while CXCR4 transcripts were present in similar amounts ei-ther in uninfected HeLa cells or in uninfected and wtNDK-infected CEM cells. Nevertheless, a more sensitive assay, re-verse transcription-PCR, confirmed that a specific CXCR4cDNA could be identified (data not shown).

FIG. 2. Analysis of CXCR4 functionality in uninfected, wtNDK-infected, and m7NDK-infected CEM cells. (A) Measure of intracellu-lar calcium concentration in response to 10 nM SDF-1a. Cells wereloaded with Fura 2-AM and incubated in HEPES-buffered Na1 solu-tion, and the [Ca21]i was determined fluorimetrically. After the base-line [Ca21]i levels were recorded, SDF-1a (10 nM) was added asindicated. The cytosolic calcium concentrations were calculated asdescribed previously (14). Tracings are shown for one population ofcells and are representative of at least three independent experiments.(B) Chemotaxis response to SDF-1a. Chemotaxis assays were per-formed in transwell chemotaxis chambers in the absence or presence ofSDF-1a (10 and 100 nM). The results are shown as the number ofmigrated cells related to the SDF-1a concentration. These results arerepresentative of at least three independent experiments.

442 VALENTE ET AL. J. VIROL.

on October 27, 2012 by 00308377

http://jvi.asm.org/

Dow

nloaded from

We next assessed the posttranscriptional stability of CXCR4mRNA in m7NDK-infected CEM cells (Fig. 6). ActinomycinD was added to uninfected and wtNDK-or m7NDK-infectedCEM cells to block transcription, and mRNAs were extractedfrom each sample at different times (Fig. 6A). Northern blot-ting experiments were performed using a CXCR4-specificcDNA probe. The half-life of CXCR4 mRNA was estimatedby plotting the densitometric ratio of the CXCR4 mRNA lev-els to the GAPDH mRNA levels. The estimated mean half-lives of the CXCR4 mRNAs of three independent experimentswere 3.7, 3.8, and 3.9 h in uninfected, wtNDK-infected, andm7NDK-infected CEM cells, respectively (Fig. 6). Similarly, ahalf-life of 3 h was found for both uninfected and m7NDK-infected HeLa cells (data not shown). These data demonstratethat infection by m7NDK virus in either CEM or HeLa cells

does not reduce the posttranscriptional stability of CXCR4mRNA.

Kinetics of CXCR4 down-regulation. To preclude the pos-sibility of positive selection of CXCR4 low-expressing cells and

FIG. 3. Analysis of intracellular versus surface expression ofCXCR4 protein. Flow cytometric analysis of surface expression ofCXCR4 and intracellular expression of CXCR4 in uninfected, wt-NDK-infected, and m7NDK chronically infected CEM cells (A) and inuninfected and m7NDK-infected HeLa cells (B). Surface CXCR4expression was determined by measuring the FITC fluorescenceintensity, and intracellular CXCR4 expression was determined bymeasuring the PE fluorescence intensity. In both cases, anti-CXCR4MAb12G5 (black tracings) and an isotype-matched control (shadedarea) were used.

FIG. 4. Detection of intracellular CXCR4 by immunofluorescencemicroscopy. Uninfected (A) and m7NDK chronically infected (B) HeLacells were grown on LabTek coverslips, fixed, permeabilized, andstained for CXCR4. Staining for anti-CXCR4 antibody (MAb173),followed by staining for Cy3-conjugated secondary antibody (red), wasphotographed at 380 magnification. Double staining was performed asa control in uninfected (C) and m7NDK-infected (D) HeLa cells. Cellswere left intact and double stained for CXCR4 and Env protein usingMAb173 and a seropositive serum, respectively. Staining withanti-CXCR4 antibody was followed by staining with Cy3-conjugatedsecondary antibody (red) and Env, followed by staining with FITC-conjugated secondary antibody (green). Photographs were then ob-tained at 332 magnification. Cells were photographed after analysisusing a confocal microscope (MRC 1240; Bio-Rad).

FIG. 5. Northern blot analysis of CXCR4 mRNA in uninfected,wtNDK-infected, and m7NDK-infected CEM cells. Samples of totalcellular RNA were hybridized with a 32P-labeled CXCR4 cDNA probeor a GAPDH cDNA probe.

VOL. 75, 2001 CXCR4 DOWN-REGULATION BY X4 CD4-INDEPENDENT HIV-1 443

on October 27, 2012 by 00308377

http://jvi.asm.org/

Dow

nloaded from

in order to verify that CXCR4 down-regulation is an activeprocess induced by the m7NDK isolate, CEM cells were in-fected either with m7NDK or its wild-type counterpart isolate.The expression kinetics of CD4, CXCR4, and HIV Env proteinwere monitored by FACS analysis for 29 days postinfection(Fig. 7). Cells were infected with 16 ng/106 cells of m7NDK(CEM1m7NDK) and wtNDK (CEM1wtNDK) isolates. As acontrol, uninfected cells (CEM) or m7NDK chronically in-fected cells (CEM1m7NDK) were subjected to the sameFACS analysis. Fusion test assays were also performed to en-sure HIV Env protein expression and fusion ability (data notshown).

The antibodies used for this analysis were MAb OKT4 forCD4, which does not compete with gp120 for CD4 binding,and MAb12G5 for CXCR4 detection. HIV Env protein ex-pression was determined using serum from a seropositive pa-tient. Upon infection with the m7NDK isolate, Env proteinexpression could be detected on the surface of infected cells atday 1 postinfection (Fig. 7A). This drastic increase was accom-panied by a marked decrease in CD4 expression (Fig. 7B). Asexpected, CXCR4 expression presented a drastic reduction,reaching an undetectable level by approximately 7 days postin-fection (Fig. 7C), which was maintained until chronic infectionwas established.

Cells infected with wtNDK isolate did not present the sametype of infection pattern, since the envelope expression in-creased slowly upon infection to reach a maximum at approx-imately 5 days postinfection. This progressive increase in theenvelope expression was accompanied by a progressive de-crease in CD4 expression. Furthermore, CXCR4 expression

was not affected by infection with this isolate, as was expected.Fusion tests were performed along throughout the kineticanalysis to ensure correct HIV Env protein expression andfusion ability (data not shown).

During this kinetics analysis and independent of the viralisolate, cells behaved as a whole, and no subpopulations wereobserved by FACS analysis for CXCR4 expression (data notshown). This result clearly precludes the coexistence of heter-ogeneous high- and low-CXCR4-expressing cells in the CEMpopulation.

DISCUSSION

We present here evidence of CXCR4 down-regulation inm7NDK-infected cells, a phenomenon that is independent ofcellular CD4 expression status. Surface expression of CXCR4was not detected on these cells using two antibodies directedagainst different epitopes of CXCR4, MAb12G5 and MAb173(Fig. 1). This was confirmed using two other antibodies di-rected to two other different CXCR4 epitopes (data not shown).

The functionality of CXCR4 was analyzed by its ability torespond to SDF-1a-induced signalization. Chemotactic assaysdemonstrated a complete insensitivity of m7NDK CEM cellsto SDF-1a-induced migration (Fig. 2B), and the intracellularcalcium elevation was 60% reduced compared to that for un-infected or wtNDK-infected CEM cells (Fig. 2A). This reduc-tion, given the cascade nature of this type of signalization,might be correlated with a loss of more than 90% of the cellsurface receptor level. These results, together with the FACS

FIG. 6. Stability of CXCR4 mRNA transcripts in uninfected, wtNDK-infected, and m7NDK-infected CEM cells. (A) Actinomycin D was addedat a concentration of 5 mg/ml for the indicated times, and CXCR4 mRNA expression was determined by Northern blot analysis. Since the CXCR4mRNA transcriptional level in m7NDK-infected CEM cells was low, longer exposure times were used to estimate the half-life (data not shown).(B) The half-life of CXCR4 mRNA was determined by plotting the densitometric CXCR4 mRNA/GAPDH mRNA ratio versus time (in hours).These results are representative of three independent experiments.

444 VALENTE ET AL. J. VIROL.

on October 27, 2012 by 00308377

http://jvi.asm.org/

Dow

nloaded from

analysis of surface CXCR4 expression, strongly indicate theabsence of CXCR4 at the surface of m7NDK-infected cells.

Surface expression of CXCR4 was also determined on aCD4-negative cell line to verify if down-regulation of CXCR4

was a phenomenon dependent on surface CD4 expression.CXCR4 expression was evaluated by flow cytometry on unin-fected HeLa cells and on three HeLa cell clones chronicallyinfected with m7NDK (Fig. 1B). Surface expression of CXCR4was not detected in any of these three different HeLa m7NDKclones (Fig. 2B), thus indicating that the phenomenon is inde-pendent of CD4 cellular expression and is not a particularity ofa CD4-positive lymphocyte cell line.

The intracellular content of CXCR4 in m7NDK-infectedHeLa and CEM cells was determined both by intracellularFACS analyses and immunofluorescent microscopy (Fig. 3 and4). In both cases, a very reduced level of intracellular CXCR4was found in m7NDK-infected HeLa and CEM cells comparedto that in uninfected or wild-type-infected cells.

Analyses of CXCR4 mRNA transcripts in m7NDK-infectedHeLa and CEM cells were performed. A Northern blot assayrevealed a drastic reduction of the steady-state level of CXCR4mRNA compared with that of uninfected or wtNDK-infectedcells (Fig. 5). This drastic reduction was clearly not a conse-quence of decreased CXCR4 mRNA transcript stability (Fig.6), since their half-life was not altered in m7NDK-infectedcells. The transcriptional activity of the CXCR4 gene mayprobably be affected in these cells, even though no regulatorysequences in the CXCR4 gene promoter has yet been de-scribed as a target site for viral protein inhibition of transcrip-tion initiation (5, 25).

The hypothesis that, during acute infection, cells with ab-normally low CXCR4 gene expression are positively selectedfor m7NDK isolate infection could be raised. In order to verifythat the phenomenon of CXCR4 down-regulation is actuallyan active process and not a selection of CXCR4 low-express-ing cells, we performed infections of CEM cells with eitherm7NDK or wtNDK viral isolates (Fig. 7). We observed a rapidappearance of HIV envelope protein expression on the surfaceof infected cells (Fig. 7A). This increase was followed by areduction of CD4 expression also from the first day postinfec-tion (Fig. 7B). A total loss of CXCR4 surface expression wasobserved approximately 7 days after infection, and this loss wasconserved thereafter (Fig. 7C). The parental isolate wtNDKpresented a slower process of expression; the envelope proteinexpression appeared gradually on infected cells, and the loss ofCD4 was also gradual, with a total loss by 4 days postinfection.The expression of CXCR4 was unaltered in wtNDK-infectedcells compared to uninfected cells. The loss of CXCR4 inm7NDK-infected cells occurred rapidly, and the whole popu-lation behaved similarly, which means that no subpopulationsappeared with lower amounts of CXCR4 surface expression.This clearly precludes the hypothesis of positive selection of alow-CXCR4-expressing CEM cell clone after m7NDK infec-tion. Furthermore, lower-CXCR4-expressing cells, in order tobe positively selected for m7NDK virus infection, shouldpresent growth advantages to overcome high-CXCR4-express-ing cell growth.

We performed cocultures between wtNDK- or m7NDK-infected CEM cells and uninfected HeLa CD41 CXCR41

cells. We then measured specific cell fusion inhibition in thepresence of three different MAbs to CXCR4. Fusions of CEMm7NDK-infected cells were less inhibited by the three anti-bodies than was wtNDK-induced fusion (data not shown). Theinhibitory effect was dose dependent, which is consistent with a

FIG. 7. Kinetics of CXCR4 down-modulation upon m7NDK infec-tion. Cells were infected with 16 ng of each of the indicated viralsupernatants per 106 cells. The expression levels of CD4, CXCR4, andHIV-1 Env protein were measured postinfection by FACS analysis asindicated. CD4 was detected using MAb OKT4, CXCR4 detection wasbased on MAb12G5, and HIV-1 Env protein was detected using aseropositive serum. The results are presented as a percentage of thepositive cells. The results of one of two representative experiments areshown.

VOL. 75, 2001 CXCR4 DOWN-REGULATION BY X4 CD4-INDEPENDENT HIV-1 445

on October 27, 2012 by 00308377

http://jvi.asm.org/

Dow

nloaded from

reversible, competitive inhibition. This suggests a greater af-finity of the m7NDK isolate Env protein for CXCR4, allowingit to strongly compete with the antibodies for CXCR4 binding,and correlates with kinetic infection data in which m7NDKEnv expression is detectable as early as 1 day postinfection.Moreover, we can then suppose that its affinity for CXCR4 orits favorable conformation enables it to bypass the CD4-in-duced conformational change necessary for target cell entry(23, 32).

Down-regulation of receptor expression is a classical mech-anism used to allow viral interference. An example of this is theability of HIV and simian immunodeficiency virus to down-regulate the cell surface expression of CD4, their primary re-ceptor. Down-regulation of the coreceptor CXCR4 by X4CD4-dependent HIV-1 has never been characterized, althoughit has been established that CXCR4 is down-regulated by a fewHIV-2 isolates which use CXCR4 as their primary receptor(12). A CD4-independent HIV-1 isolate, HIV-1/IIIBx, has re-cently been derived from the parental isolate. However, itfailed to down-regulate CXCR4 on chronically infected cells(17). Other viral families induce down-regulation of CXCR4,as is the case with HHV-6 and HHV-7. These viruses induce amarkedly decreased level of CXCR4 gene transcription, with-out any significant alteration of the posttranscriptional stabilityof CXCR4 mRNA (34, 39). Nevertheless, unlike the m7NDKHIV-1 isolate, these viruses do not use CXCR4 as a receptorfor viral entry (40).

A down-regulation of CXCR4 in cells chronically infectedwith m7NDK isolate was expected to occur to allow cell sur-vival. If this coreceptor, or main receptor in this case, waspresent on the surface of infected cells, syncytium formationwould result, leading to cell death.

The results here presented support the concept of retroviralinterference. They show that a virus, which derived spontane-ously and which uses CXCR4 as a primary receptor, mustdown-regulate this receptor to maintain chronic expressionin the infected cell line. However, the down-regulation ofCXCR4 here described brings new insights into the mecha-nisms used by the viruses to achieve this. While CD4 is down-regulated in CD4-dependent HIV-1 by a number of differentproteins that interfere with its stability and subcellular local-ization, CXCR4 is down-regulated by the m7NDK isolate pri-marily at the transcriptional level. We do not exclude thehypothesis of a retention of gp120-CXCR4 complex in the ERfollowed by degradation of CXCR4. However, since the steadystates of CXCR4 mRNA and proteins levels are very muchdiminished, this mechanism, although possible, would thus oc-cur with relatively low or undetectable efficiency.

Besides the relevance of these findings in relation to newaspects of CXCR4 expression regulation, the future identifi-cation of the mechanism used by the m7NDK HIV-1 isolate toachieve this modulation may provide new insights into HIV-1-cell interactions and could be a useful tool for the developmentof new prophylaxis concepts against HIV-1 infection.

ACKNOWLEDGMENTS

We are grateful for the technical support of Isabelle Bouchaert andMichelle Tissot. We thank Lena Brydon for editing the English andVeronique Joliot and Arielle Rosenberg for critical reading of themanuscript. We thank Valerie Marechal for technical support.

S.T.V. is supported by a grant from the Portuguese Education Min-istry, Praxis XXI. J.D. has a fellowship from the French NationalEducation Ministry. This work was supported by grants from AgenceNationale de la Recherche contre le SIDA (ANRS), Sidaction AO11and AO2 “Lute anti-Sida” from the University of Paris VII.

REFERENCES

1. Aiken, C., J. Konner, N. R. Landau, M. E. Lenburg, and D. Trono. 1994. Nefinduces CD4 endocytosis: requirement for a critical dileucine motif in themembrane-proximal CD4 cytoplasmic domain. Cell 76:853–864.

2. Berger, E. A., P. M. Murphy, and J. M. Farber. 1999. Chemokine receptorsas HIV-1 coreceptors: roles in viral entry, tropism, and disease. Annu. Rev.Immunol. 17:657–700.

3. Bermejo, M., J. Martin-Serrano, E. Oberlin, M. A. Pedraza, A. Serrano, B.Santiago, A. Caruz, P. Loetscher, M. Baggiolini, F. Arenzana-Seisdedos, andJ. Alcami. 1998. Activation of blood T lymphocytes down-regulates CXCR4expression and interferes with propagation of X4 HIV strains. Eur. J. Im-munol. 28:3192–3204.

4. Bleul, C. C., R. C. Fuhlbrigge, J. M. Casasnovas, A. Aiuti, and T. A.Springer. 1996. A highly efficacious lymphocyte chemoattractant, stromalcell-derived factor 1 (SDF-1). J. Exp. Med. 184:1101–1109.

5. Caruz, A., M. Samsom, J. M. Alonso, J. Alcami, F. Baleux, J. L. Virelizier,M. Parmentier, and F. Arenzana-Seisdedos. 1998. Genomic organizationand promoter characterization of human CXCR4 gene. FEBS Lett. 426:271–278.

6. Clapham, P. R., J. D. Reeves, G. Simmons, N. Dejucq, S. Hibbitts, and A.McKnight. 1999. HIV coreceptors, cell tropism and inhibition by chemokinereceptor ligands. Mol. Membr. Biol. 16:49–55.

7. Crise, B., L. Buonocore, and J. K. Rose. 1990. CD4 is retained in theendoplasmic reticulum by the human immunodeficiency virus type 1 glyco-protein precursor. J. Virol. 64:5585–5593.

8. Dalgleish, A. G., P. C. L. Beverley, P. R. Clapham, D. H. Crawford, M. F.Greaves, and R. A. Weiss. 1984. The CD4 (T4) antigen is an essentialcomponent of the receptor for the AIDS retrovirus. Nature 312:763–766.

9. Dragic, T., P. Charneau, F. Clavel, and M. Alizon. 1992. Complementationof murine cells for human immunodeficiency virus envelope/CD4-mediatedfusion in human-murine heterokaryons. J. Virol. 66:4794–4802.

10. Dumonceaux, J., S. Nisole, C. Chanel, L. Quivet, A. Amara, F. Baleux, P.Briand, and U. Hazan. 1998. Spontaneous mutations in the env gene of thehuman immunodeficiency virus type 1 NDK isolate are associated with aCD4-independent entry phenotype. J. Virol. 72:512–519.

11. Ellrodt, A., F. Barre-Sinoussi, P. Le Bras, M. T. Nugeyre, L. Palazzo, F. Rey,F. Brun-Vezinet, C. Rouzioux, P. Segond, R. Caquet, L. Montagnier, andJ.-C. Chermann. 1984. Isolation of a new human T-lymphotropic retrovirus(LAV) from a married couple of Zairians, one with AIDS, the other withprodromes. Lancet i:1383–1385.

12. Endres, M. J., P. R. Clapham, M. Marsh, M. Ahuja, J. Davis-Turner, A.McKnight, J. F. Thomas, B. Stoebenau-Haggarty, S. Choe, P. J. Vance,T. N. C. Wells, C. A. Power, S. S. Sutterwala, R. W. Doms, N. R. Landau, andJ. A. Hoxie. 1996. CD4-independent infection by HIV-2 is mediated byfusin/CXCR-4. Cell 87:745–756.

13. Geleziunas, R., S. Bour, F. Boulerice, J. Hiscott, and M. A. Wainberg. 1991.Diminution of CD4 surface protein but not CD4 messenger RNA levels inmonocytic cells infected by HIV-1. AIDS 5:29–33. (Erratum, 5:1281.)

14. Grynkiewicz, G., M. Poenie, and R. Y. Tsien. 1985. A new generation of Ca21

indicators with greatly improved fluorescence properties. J. Biol. Chem.260:3440–3450.

15. Haribabu, B., R. M. Richardson, I. Fisher, S. Sozzani, S. C. Peiper, R.Horuk, H. Ali, and R. Snyderman. 1997. Regulation of human chemokinereceptors CXCR4. Role of phosphorylation in desensitization and internal-ization. J. Biol. Chem. 272:28726–28731.

16. Hoxie, J. A., J. D. Alpers, J. L. Rackowski, K. Huebner, B. S. Haggarty, A. J.Cedarbaum, and J. C. Reed. 1986. Alterations in T4 (CD4) protein andmRNA synthesis in cells infected with HIV. Science 234:1123–1127.

17. Hoxie, J. A., C. C. LaBranche, M. J. Endres, J. D. Turner, J. F. Berson, R. W.Doms, and T. J. Matthews. 1998. CD4-independent utilization of theCXCR4 chemokine receptor by HIV-1 and HIV-2. J. Reprod. Immunol.41:197–211.

18. Jabbar, M. A., and D. P. Nayak. 1990. Intracellular interaction of humanimmunodeficiency virus type 1 (ARV-2) envelope glycoprotein gp160 withCD4 blocks the movement and maturation of CD4 to the plasma membrane.J. Virol. 64:6297–6304.

19. Jinquan, T., S. Quan, H. H. Jacobi, H. O. Madsen, C. Glue, P. S. Skov, H.Malling, and L. K. Poulsen. 2000. CXC chemokine receptor 4 expressionand stromal cell-derived factor-1a-induced chemotaxis in CD41 T lympho-cytes are regulated by interleukin-4 and interleukin-10. Immunology 99:402–410.

20. Klatzmann, D., E. Champagne, S. Chamaret, J. Gruest, D. Guetard, T.Hercend, J.-C. Gluckman, and L. Montagnier. 1984. T-lymphocyte T4 mol-ecule behaves as the receptor for human retrovirus LAV. Nature 312:767–768.

446 VALENTE ET AL. J. VIROL.

on October 27, 2012 by 00308377

http://jvi.asm.org/

Dow

nloaded from

21. Kwong, P. D., R. Wyatt, Q. J. Sattentau, J. Sodroski, and W. A. Hendrickson.2000. Oligomeric modeling and electrostatic analysis of the gp120 envelopeglycoprotein of human immunodeficiency virus. J. Virol. 74:1961–1972.

22. Landau, N. R., M. Warton, and D. R. Littman. 1988. The envelope glyco-protein of the human immunodeficiency virus binds to the immunoglobulin-like domain of CD4. Nature 334:159–162.

23. Moore, J. P., and J. Binley. 1998. HIV. Envelope’s letters boxed into shape.Nature 393:630–631.

24. Moore, J. P., A. Trkola, and T. Dragic. 1997. Co-receptors for HIV-1 entry.Curr. Opin. Immunol. 9:551–562.

25. Moriuchi, M., H. Moriuchi, D. M. Margolis, and A. S. Fauci. 1999. USF/c-Myc enhances, while Yin-Yang 1 suppresses, the promoter activity ofCXCR4, a coreceptor for HIV-1 entry. J. Immunol. 162:5986–5992.

26. Moriuchi, M., H. Moriuchi, W. Turner, and A. S. Fauci. 1997. Cloning andanalysis of the promoter region of CXCR4, a coreceptor for HIV-1 entry.J. Immunol. 159:4322–4329.

27. Nasmith, P. E., and S. Grinstein. 1987. Phorbol ester-induced changes incytoplasmic Ca21 in human neutrophils. Involvement of a pertussis toxin-sensitive G protein. J. Biol. Chem. 262:13558–13566.

28. Oberlin, E., A. Amara, F. Bachelerie, C. Bessia, J. L. Virelizier, S. F.Arenzana, O. Schwartz, J. M. Heard, L. I. Clark, D. F. Legler, M. Loetscher,M. Baggiolini, and B. Moser. 1996. The CXC chemokine SDF-1 is the ligandfor LESTR/fusin and prevents infection by T-cell-line-adapted HIV-1. Na-ture 382:833–835. (Erratum, 384:288.)

29. Pozzan, T., D. P. Lew, C. B. Wollheim, and R. Y. Tsien. 1983. Is cytosolicionized calcium regulating neutrophil activation? Science 221:1413–1415.

30. Rhee, S. S., and J. W. Marsh. 1994. Human immunodeficiency virus type 1Nef-induced down-modulation of CD4 is due to rapid internalization anddegradation of surface CD4. J. Virol. 68:5156–5163.

31. Saccani, A., S. Saccani, S. Orlando, M. Sironi, S. Bernasconi, P. Ghezzi, A.Mantovani, and A. Sica. 2000. Redox regulation of chemokine receptorexpression. Proc. Natl. Acad. Sci. USA 97:2761–2766.

32. Sattentau, Q. J. 1998. HIV gp120: double lock strategy foils host defences.Structure 6:945–949.

33. Schwartz, O., V. A. Dautry, B. Goud, V. Marechal, A. Subtil, J. M. Heard,and O. Danos. 1995. Human immunodeficiency virus type 1 Nef inducesaccumulation of CD4 in early endosomes. J. Virol. 69:528–533.

34. Secchiero, P., D. Zella, O. Barabitskaja, M. S. Reitz, S. Capitani, R. C. Gallo,and G. Zauli. 1998. Progressive and persistent downregulation of surfaceCXCR4 in CD41 T cells infected with human herpesvirus 7. Blood 92:4521–4528.

35. Signoret, N., J. Oldridge, A. Pelchen-Matthews, P. J. Klasse, T. Tran, L. F.Brass, M. M. Rosenkilde, T. W. Schwartz, W. Holmes, W. Dallas, M. A.Luther, T. N. C. Wells, J. A. Hoxie, and M. Marsh. 1997. Phorbol esters andSDF-1 induce rapid endocytosis and down modulation of the chemokinereceptor CXCR4. J. Cell Biol. 139:651–664.

36. Stevenson, M., C. Meier, A. M. Mann, N. Chapman, and A. Wasiak. 1988.Envelope glycoprotein of HIV induces interference and cytolysis resistancein CD41 cells: mechanism for persistence in AIDS. Cell 53:483–496.

37. Tiganos, E., X. J. Yao, J. Friborg, N. Daniel, and E. A. Cohen. 1997. Putativealpha-helical structures in the human immunodeficiency virus type 1 Vpuprotein and CD4 are involved in binding and degradation of the CD4 mol-ecule. J. Virol. 71:4452–4460.

38. Willey, R. L., F. Maldarelli, M. A. Martin, and K. Strebel. 1992. Humanimmunodeficiency virus type 1 Vpu protein induces rapid degradation ofCD4. J. Virol. 66:7193–7200.

39. Yasukawa, M., A. Hasegawa, I. Sakai, H. Ohminami, J. Arai, S. Kaneko, Y.Yakushijin, K. Maeyama, H. Nakashima, R. Arakaki, and S. Fujita. 1999.Down-regulation of CXCR4 by human herpesvirus 6 (HHV-6) and HHV-7.J. Immunol. 162:5417–5422.

40. Zhang, Y., S. Hatse, E. De Clercq, and D. Schols. 2000. CXC-chemokinereceptor 4 is not a coreceptor for human herpesvirus 7 entry into CD41 Tcells. J. Virol. 74:2011–2016.

VOL. 75, 2001 CXCR4 DOWN-REGULATION BY X4 CD4-INDEPENDENT HIV-1 447

on October 27, 2012 by 00308377

http://jvi.asm.org/

Dow

nloaded from