host-soluble galectin-1 promotes hiv-1 replication through...

JOURNAL OF VIROLOGY, Nov. 2011, p. 11742–11751 Vol. 85, No. 220022-538X/11/$12.00 doi:10.1128/JVI.05351-11Copyright © 2011, American Society for Microbiology. All Rights Reserved.

Host-Soluble Galectin-1 Promotes HIV-1 Replication through a DirectInteraction with Glycans of Viral gp120 and Host CD4�

Christian St-Pierre,1 Hiroshi Manya,3 Michel Ouellet,2 Gary F. Clark,4 Tamao Endo,3Michel J. Tremblay,2* and Sachiko Sato1*

Glycobiology and Bioimaging Laboratory1 and Laboratory of Human ImmunoRetrovirology,2 Research Centre for Infectious Diseases,Faculty of Medicine, Laval University, 2705 Boul. Laurier, Quebec, Canada G1V 4G2; Glycobiology Research Group,Tokyo Metropolitan Institute of Gerontology, Foundation for Research on Aging and Promotion of Human Welfare,

35-2 Sakaecho, Itabashi-ku, Tokyo 173-0015, Japan3; and Division of Reproductive andPerinatal Research, University of Missouri, Columbia, Missouri4

Received 9 June 2011/Accepted 25 August 2011

Sexual transmission of HIV-1 requires virus adsorption to a target cell, typically a CD4� T lymphocyteresiding in the lamina propria, beneath the epithelium. To escape the mucosal clearance system and reach itstarget cells, HIV-1 has evolved strategies to circumvent deleterious host factors. Galectin-1, a soluble lectinfound in the underlayers of the epithelium, increases HIV-1 infectivity by accelerating its binding to susceptiblecells. By comparison, galectin-3, a family member expressed by epithelial cells and part of the mucosalclearance system, does not perform similarly. We show here that galectin-1 directly binds to HIV-1 in a�-galactoside-dependent fashion through recognition of clusters of N-linked glycans on the viral envelopegp120. Unexpectedly, this preferential binding of galectin-1 does not rely on the primary sequence of anyparticular glycans. Instead, glycan clustering arising from the tertiary structure of gp120 hinders its bindingby galectin-3. Increased polyvalency of a specific ligand epitope is a common strategy for glycans to increasetheir avidity for lectins. In this peculiar occurrence, glycan clustering is instead exploited to prevent bindingof gp120 by galectin-3, which would lead to a biological dead-end for the virus. Our data also suggest thatgalectin-1 binds preferentially to CD4, the host receptor for gp120. Together, these results suggest that HIV-1exploits galectin-1 to enhance gp120-CD4 interactions, thereby promoting virus attachment and infectionevents. Since viral adhesion is a rate-limiting step for HIV-1 entry, modulation of the gp120 interaction withgalectin-1 could thus represent a novel approach for the prevention of HIV-1 transmission.

The frequency of human immunodeficiency virus type 1(HIV-1) transmission following unprotected sexual intercourseis relatively low compared to other sexually transmitted vi-ruses, such as hepatitis B virus (3, 44, 55). However, oncetransmission occurs, HIV-1 efficiently replicates and rapidlyexpands to deplete more than 90% of gut-associated CD4� Tcells within the first few weeks (4, 30, 63). One of the rate-limiting steps of HIV-1 infection involves its early interactionwith virus-susceptible cells (62). Prevention of this initial at-tachment should be exploited to further reduce transmissionevents, therefore avoiding chronic infection, life-long monitor-ing, and costly antiretroviral therapies.

The attachment to the surface of the target cell is mediatedthrough binding of the external viral envelope glycoprotein(Env) to the major cellular receptor CD4 (52). This physicalcontact triggers conformational changes in Env, which leads tofusion of the viral and host plasma membranes. Thus, Env-CD4 interactions are critical to initiate fusion of membranesunder optimal conditions. These conditions include high ex-

pression levels of surface CD4 and coreceptors (e.g., CCR5and CXCR4) as well as significant amounts of infectious virusparticles. However, such optimal conditions are rarely metunder in vivo situations, especially during the initial stages ofinfection. Even if Env molecules have a relatively high affinityfor CD4, the general avidity of Env is unexpectedly low, and itexhibits slow equilibrium binding kinetics at 37°C (14, 36, 37,51). In addition, only a few Env spikes (i.e., about 20 spikes pervirion) are sparsely distributed on the viral surface (65). Thus,during a transmission event, when host cell surface levels ofCD4 are far from optimal and when innate mucosal clearancemechanisms are active, the formation of a stable associationbetween Env and CD4 becomes a significant limiting factor toinfection (35, 62). Interestingly, HIV-1 has elaborated numer-ous strategies to compensate for this limiting factor. One ofthese involves the alteration of the shell-like glycan layer,called the glycocalyx, on the virus surface. This phenomenonresults in the formation of interactions between HIV-1 andhost lectins expressed as membrane-anchored proteins on thesurface of target cells (11, 15, 23, 24, 27, 31, 61).

The glycocalyx of HIV-1 is composed of glycan chains thatare covalently linked to host membrane glycoproteins acquiredby the virus while emerging from an infected cell (13, 60). Inaddition, Env itself is densely glycosylated (i.e., from 13 to 33chains per single molecule). Even though a single virion carriesvery few Env spikes on its surface (65), Env glycans have beenshown to mediate numerous and biologically relevant interac-tions with host lectins (53). Despite high genetic variability

* Corresponding author. Mailing address for S. Sato: Glycobiologyand Bioimaging Laboratory, Faculty of Medicine, Laval University,2705 Boul. Laurier, Quebec G1V 4G2, Canada. Phone: (418) 656-4141,ext. 48647. Fax: (418) 654-2715. E-mail: [email protected] address for M. J. Tremblay: Laboratory of HumanImmunoRetrovirology, Faculty of Medicine, Laval University, 2705 Boul.Laurier, Quebec G1V 4G2, Canada. Phone: (418) 656-4141, ext. 48274.Fax: (418) 654-2715. E-mail: [email protected].

� Published ahead of print on 31 August 2011.

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among different groups of HIV-1, the N-glycosylation sites ofgp120 are spatially conserved. Two types of N-linked glycansare found on gp120, namely, oligomannose-type (OM) glycans,which are rich in mannose residues, and complex-type (CX)glycans, which carry 2 to 6 �-galactoside residues (i.e., lac-tosamine residue [LacNAc]) (25, 26, 41, 64). Glycosylation ofgp120 exhibits two unique features that distinguish it fromglycosylation patterns normally found on host membrane pro-teins (53). First, gp120 displays unusually high levels of OMglycans, which are considered incomplete processed forms ofglycan chains and are therefore rarely found in the extracellu-lar space. Second, OM and CX glycans are spatially distributedon the surface of gp120 to form distinct homogenous patches(53). For example, OM glycans are clustered in an area distalfrom the CD4 binding site, while CX glycan patches are foundproximal to the CD4 binding site. The functionality of OMglycan has already been established, as multiple C-type mem-brane-anchored lectin receptors (CLRs), such as DC-SIGN,CD206, and DCIR, recognize OM glycans and enhance bothHIV-1 attachment and infection (11, 15, 23, 24, 27, 31, 61). Incontrast, despite the determination that CX glycan patches arefound proximal to the CD4 binding site of gp120, their involve-ment in HIV-1 pathogenesis remains unclear.

We have recently found that a non-CLR, soluble, host gal-actoside binding protein, galectin-1 (Gal-1), greatly accelerates(as high as 40-fold) the binding kinetics of HIV-1 to susceptiblecells, leading to faster viral entry and a more robust viralreplication (33, 40, 59). As of yet, the mechanism as well aspotential ligands found on susceptible cells or HIV-1 thatwould be responsible for this effect of Gal-1 remain unknown.Importantly, this effect was observed with clinical strains ofHIV-1 produced in both primary lymphocytes and macro-phages (33, 40), suggesting that glycans specific for Gal-1 couldbe naturally found on the surface of field isolates of HIV-1.Gal-1 belongs to the family of galectins, the most widely ex-pressed class of lectins in multicellular organisms. All membersof the galectin family share a primary structure homology intheir glycan recognition domains, which have an affinity forglycans containing �-galactosides (2, 21). In humans, Gal-1 andGal-3 are expressed by various cell types, most of them in-volved in acquired and innate immunity. Their expression isincreased in the context of inflammation or infection and canbe found at high concentrations (as high as 10 �M) in lym-phoid tissues and their associated mucosa, where active HIV-1replication has been reported to occur (28, 40, 45, 48–50, 57).Being multivalent, Gal-1 or Gal-3 cross-links at least two dif-ferent glycans either on the same cell surface or on differentconstituents. In the latter case, this cross-linking activityconfers to galectins the properties of adhesion molecules bydirectly mediating either cell-to-cell or cell-to-pathogen in-teractions despite being soluble proteins (28, 45, 48–50).Importantly, even though Gal-1 and Gal-3 often share sim-ilar ligands on host cells, only Gal-1, but not Gal-3, canpromote binding of HIV-1 to susceptible cells in a �-galac-toside-dependent manner (33, 40, 59).

Upon transmission, HIV-1 must penetrate across an epithe-lium that is covered with heavily glycosylated mucins. Gal-3 isabundantly expressed at the apical surface of these mucosalmembranes (38, 39) and is believed to be an active participantof the mucosal clearance system (1). Thus, mucins and Gal-3

are most likely among the first molecules that HIV-1 encoun-ters, and they would severely reduce its penetration efficiency.In contrast to Gal-3, Gal-1 is highly expressed in the laminamuscularis mucosae, just beneath the epithelium and its laminapropria, where susceptible CD4� T cells can be found (12, 39,56). Both of these galectins normally exhibit similar glycanbinding properties, but our results suggest that they nonethe-less differentially affect HIV-1 binding to susceptible cells (33,40, 59). Considering the clearance role of Gal-3 and the pos-itive influence of Gal-1 for viral attachment and entry, thisdifferential binding could be advantageous for efficient viraltransmission and early viral replication. The molecular mech-anisms responsible for this differential specificity are currentlyunknown, but their elucidation could pave the way for thedevelopment of new therapies seeking to undermine the abilityof HIV-1 to infect founder cells during transmission events.We now report here for the first time that both gp120 and CD4are specifically bound by Gal-1. We provide evidence thatGal-1 binds preferentially to clustered CX glycans of gp120 onHIV-1 and to CX glycans of CD4 to drastically increase theavidity of HIV-1 for susceptible CD4-expressing T cells, there-fore enhancing virus infectivity. Our results also suggest thatclustering of CX glycans, resulting from structural constraintsof the peptide backbone of gp120, effectively prevents Gal-3binding. Thus, the differential binding of HIV-1 gp120 to Gal-1and Gal-3 does not rely on the presence of any particular CXglycan sequence (primary structure), as is normally the case formost lectins, but relies instead on the peculiar arrangementand clustering of CX glycans attached to gp120 (tertiary struc-ture).

MATERIALS AND METHODS

Reagents. Chemicals and other reagents were obtained from Sigma ChemicalCo. (St. Louis, MO). Recombinant HIV-1 Bal gp120 (R5), 96ZM651 gp120 (X4),recombinant soluble human CD4 (produced by a mammalian [CHO cell] ex-pression system), interleukin-2, and antiserum to gp120 were provided by theNIH AIDS Research and Reference Reagent Program (Germantown, MD). Apolyclonal antibody against human gp120 was purified from sheep antiserum(18). Peroxidase-AffiniPure rabbit anti-sheep IgG was obtained from JacksonImmunoResearch Laboratories Inc. (West Grove, PA).

Recombinant galectins were produced and purified as previously described(40, 59). In some experiments, Gal-1 was S-carboxyamidomethylated with iodo-acetamide (58, 59) to prevent its oxidation. This stabilized form of Gal-1 exhibitssimilar activity for HIV-1 binding and infection (59). The stabilized Gal-1 wasused for Gal-1–agarose, while all Gal-1 column chromatographies were per-formed with both native and stabilized Gal-1, and both Gal-1 forms gave similarresults. Alexa 488-labeled Gal-1 and Gal-3 were prepared following the manu-facturer’s instructions (Molecular Probes, Eugene, OR) with slight modifications(42, 43).

Viral preparations and binding and infection assays. Virus particles wereprepared from the culture medium of HEK 293T cells that were transientlytransfected with the molecular clones pNL4-3, pNL4-3Balenv, or pNL4-3�env aspreviously described (59). Alternatively HIV-1 (X4R5 or R5) was purified fromthe medium of peripheral blood mononuclear cells (PBMCs) infected withHIV-1 as previously described (59). For lectin affinity chromatography, virusparticles were inactivated either by a 30-min incubation with 0.5% Triton X-100(for virus lysates) or by overnight incubation with 2-aldrithiol (AT-2; 2,2�-dithio-dipyridine; for whole virus particles) (9, 46). HIV-1 binding and infection exper-iments were carried out as previously published (59). Briefly, for viral binding,activated CD4� T lymphocytes were first incubated with HIV-1 in the presenceor absence of Gal-1 or -3 for 1 h. Cells were then washed with phosphate-buffered saline (PBS) to remove unbound HIV-1 and lysed with a disruptionbuffer (PBS [pH 7.4] and Triton X-100 at 0.5%). Levels of HIV-1 binding wereestimated by a double-antibody sandwich enzyme-linked immunosorbent assay(ELISA) directed against p24. For Raji cells, either a Raji cell line or Raji

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derivative stably expressing CD4 (6) was incubated with HIV-1 (10 ng of p24 per1 � 105 cells) for 10 min at room temperature. For infection studies, target cellswere incubated with HIV-1 (10 ng of p24 per 1 � 105 cells) for 3 days in thepresence or absence of Gal-1, and HIV-1 production was estimated by measuringp24 levels in cell-free supernatants.

Microscale affinity chromatography. Gal-1, Gal-3, and CD4-agarose columnswere prepared using AminoLink Plus coupling resin (Pierce Biotechnology,Rockford, IL) with some modifications (43). Briefly, 1 to 2 mg/ml of purifiedprotein was coupled to AminoLink Plus coupling gel through a 4-h incubation atroom temperature. For galectin-agarose, lactose was added when immobilized toprotect the glycan binding domains. AT-2-treated virus particles (containing 1 to1.5 �g of p24), viral lysates prepared from HIV-1 (p24, 500 ng) treated with 0.5%Triton X-100–Tris-HCl (pH 7.2)–0.12 M NaCl, gp120 (2 �g), or labeled glycanswere applied to the galectin column (0.5-ml bed volume). For viral lysates, thefractionation was done in the presence of 0.5% Triton X-100. For whole virusparticles, the fractionation was done in the absence of such detergent. Columnswere then washed with PBS (0.5 ml, 3 fractions), followed by elution with 100mM mannose (Man; 0.5 ml, 3 fractions) and finally with 100 mM lactose (Lac; 0.3ml, 6 fractions). Levels of HIV-1, gp120, and 2-aminobenzamide (2-AB)-labeledglycans in the fractions were estimated by using p24 ELISA, Western blottingwith anti-gp120 antibody, and 2-AB fluorescence, respectively. In the case ofCD4, galectins were applied to a CD4-agarose column (25-�l bed volume), andthe presence of galectins in the eluted fractions was estimated by the level ofAlexa 488 fluorescence associated with galectins. For the binding of gp120 toCD4, gp120 (R5) in the presence or absence of Gal-1 was incubated withCD4-agarose (25-�l bed volume) for 15 min with or without Lac (100 mM) atroom temperature. After unbound material was removed by two washes with 100�l of Tris-buffered saline (TBS)–0.1% Triton X-100, beads were treated withSDS-PAGE sample buffer at 65°C for 5 min to recover bound material, whichwas subjected to Western blotting with anti-gp120 antibody.

Western blotting. To detect gp120, samples were subjected to a 7.5% poly-acrylamide gel followed by electrotransfer on a 0.45 �m polyvinylidene difluoridemembrane. Membranes were then blocked overnight at 37°C with 5% gelatin inTBS–1% Tween 20 (TBST) and incubated with a sheep IgG anti-gp120 (1:1,000)for 4 h at room temperature in 0.5% gelatin–TBST. After incubation withperoxidase-labeled rabbit anti-sheep IgG (1:10,000) for 2 h at room temperature,gp120 bands were detected by using the Western lighting chemiluminescencereagent (Perkin-Elmer LAS, Inc.).

Glycan purification and labeling. N-linked glycans attached to recombinantgp120 (X4 and R5) were removed either by PNGase F (glycerol free; NewEngland BioLabs, Pickering, ON, Canada) or by hydrazinolysis as previouslydescribed (29). Recovered glycans were purified with LudgerClean E cartridges(QA-bio, San Mateo, CA) and then dried and labeled with 2-AB using theLudgerTag 2-AB labeling kit (QA-bio). Free dyes were removed using Ludger-Clean S (QA-bio).

Glycan structure analyses. As previously described, 2-AB-labeled glycans wereanalyzed by anion-exchange chromatography, normal-phase high-performanceliquid chromatography, and sequential glycosidase digestion (29).

RESULTS

Gal-1, but not Gal-3, binds to HIV-1 in a �-galactoside-dependent manner. Our previous studies suggest that Gal-1accelerates the kinetics of HIV-1 attachment to the target cellsurface, and this effect is dependent on its �-galactoside bind-ing activity, since the galectin antagonist lactose efficiently in-hibits virus binding (33, 40, 59). In contrast to Gal-1, Gal-3does not enhance HIV-1 infection (X4, R5, or X4R5 variants)in host cells. Since both galectins exhibit relatively similar gly-can binding specificity, the molecular mechanism for this Gal-1specificity in the context of HIV-1 infection remains unknown.As shown in Fig. 1, when HIV-1 was exposed to CD4� T cellsin the presence or absence of the studied galectins, Gal-1, butnot Gal-3, significantly enhanced HIV-1 binding to susceptiblecells (Fig. 1), suggesting that Gal-1-specific HIV-1 bindingcontributes to the increase of virus infection. Therefore, eventhough HIV-1 virus particles are covered with a thick glycoca-lyx (53), only Gal-1 participates in increasing virus attachment

to susceptible cells. To shed light on the precise mechanism(s)by which Gal-1 increases virus adsorption, intact infectiousHIV-1 particles produced by HEK cells were subjected togalectin affinity chromatography to determine whether Gal-1directly binds to HIV-1 or not (Fig. 2A and B). After removingunbound virus, a first step of elution of galectin-associatedvirus was performed with mannose (Man). This first elutionstep thus represented a specificity control for �-galactoside-dependent binding of Gal-1 to HIV-1, since Man does notinhibit the Gal-1-mediated enhancement of HIV-1 infectivity(40, 59). A second elution step was then performed with Lac,which binds to galectins and competes with viral particles as-sociated with Gal-1 in a �-galactoside-dependent manner. Asshown in Fig. 2A and B, as much as 75% of HIV-1 was elutedfrom the Gal-1 column by Lac, suggesting that Gal-1 directlybinds to HIV-1 particles through �-galactoside-dependent in-teractions. No significant level of virus was eluted by Man,confirming that Gal-1 does not bind to HIV-1 through OMglycans. In contrast, only 30% of HIV-1 applied to the Gal-3column was eluted by Lac, and the majority of virus particleswere found in the washing steps, prior to Man elution. Sincesome peripheral modification of glycans can be altered de-pending on cell type or lineage, it is possible that HIV-1 pro-duced by primary cells displays a different pattern of surfaceprotein glycosylation from those glycans that arise from estab-lished cell lines. Such a difference might have an impact onGal-1-specific recognition of HIV-1. To address this possibility,we next investigated binding of Gal-1 to X4R5 and R5 virusparticles that were prepared from HIV-1-infected PBMCs. Asshown in Fig. 2C, X4R5 HIV-1 preferentially binds to Gal-1(69.8% of total input virus) compared to Gal-3 (28.7%). Thistrend was more evident with R5 virus from infected PBMCs,since as little as 5% of HIV-1 bound to Gal-3 while Gal-1captured close to 69% of input virus (Fig. 2D). Thus, thesedata suggest that Gal-1-specific binding is consistent whetherHIV-1 is produced by PBMCs or HEK cells.

HIV-1 is an enveloped virus that acquires both host glyco-

FIG. 1. Gal-1 but not Gal-3 facilitates HIV-1 adsorption to CD4�

T cells. Activated CD4� T cells were incubated with NL4-3 HIV-1 withor without Gal-1 or Gal-3 (2 �M) for 1 h at 4°C. After unbound virionswere removed, cells were lysed and HIV-1 binding was estimated bymeasuring the levels of p24 associated with cells in an ELISA. Datashown represent the means � standard errors of the means of fourdeterminations. The representative results from three different exper-iments are shown. P values were determined from comparisons toHIV-1 binding in the absence of galectin. **, P � 0.01.

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proteins and virus-encoded Env glycoproteins during its bud-ding from the infected cell. Since glycans can be found on all ofthese glycoproteins (7, 13), it is not clear whether such differ-ences in retention of the virus by galectins are due to specificrecognition of Env or host-derived glycoprotein incorporatedwithin virions. Thus, the affinities of virus particles either lack-ing (i.e., NL4-3�env) or bearing Env (i.e., NL4-3) for Gal-1

and Gal-3 were next compared (Fig. 3). Regardless of thepresence or absence of Env, comparable amounts of virusparticles were retained by the Gal-3 column, suggesting thatGal-3 binds to virus-associated host glycoproteins rather thanEnv. In contrast to Gal-3, elimination of Env from the viriondramatically reduced its binding to the Gal-1 column, there-fore indicating that Gal-1 uniquely recognizes the presence ofEnv on HIV-1.

Viral gp120 of both X4 and R5 is recognized by Gal-1. Tofurther elucidate Gal-1-specific recognition of Env, a deter-gent-soluble lysate of X4-tropic HIV-1 particles was applied toa galectin-agarose column. Significant levels of HIV-1 gp120were retained by the Gal-1 column and recovered in the Lacfractions, while very low levels, if any, could be recovered fromthe Gal-3 column (Fig. 4A). These results demonstrate thatviral gp120 indeed binds to Gal-1 and also that specific bindingof gp120 by Gal-1 is not likely due to an architectural presen-tation of gp120 on the virus surface. Since the above-men-tioned experiments were performed with an X4 strain ofHIV-1, we verified whether this Gal-1-specific recognition andenhancement of HIV-1 infectivity could also be observed withan R5-tropic variant. As shown in Fig. 4B, Gal-1 enhancedHIV-1 infectivity of both X4- and R5-using strains followingacute infection of primary human CD4� T cells. Furthermore,consistent with the results of HIV-1 lysates, the binding pref-erence of Gal-1 for gp120 was also observed when recombinantgp120 from either X4 and R5 variants was used for galectinaffinity chromatography (Fig. 4C). In contrast, recombinant

FIG. 3. Gal-1 binds specifically to Env-bearing HIV-1. AT-2-treated virions either bearing or lacking Env were applied to Gal-1– orGal-3–agarose columns. The representative results from two differentexperiments are shown.

FIG. 2. Gal-1 but not Gal-3 binds to HIV-1 in a �-galactoside-dependent manner. (A) AT-2-treated HIV-1 virus particles were applied to aGal-1– or Gal-3–agarose column (0.5-ml bed volume), and each column was first washed with PBS (0.5 ml, 3 fractions), followed by Man (0.5 ml,3 fractions) and Lac (0.3 ml, 6 fractions) to elute bound HIV-1. The levels of virus in each fraction were estimated by p24 ELISA. Data shownrepresent the means � standard errors of the means of three determinations. The representative results from two different experiments are shown.P values were determined by comparison of the percentages of virus in each fraction collected from the galectin-1 column to those collected onthe galectin-3 column. ***, P � 0.005. (B) Elution profile of HIV-1 by galectin affinity chromatography. Representative results from two differentexperiments are shown. (C) HIV-1 virus (X4R5 and R5), which were prepared from HIV-infected PBMC, were applied to a Gal-1– orGal-3–agarose column as described for panel A.


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gp120 molecules failed to be retained by a Gal-3 column andtheir retention was rather retarded throughout elution withoutforming any significant peaks when lactose was introduced.Together, these data suggest that Gal-1, but not Gal-3, specif-ically recognizes both soluble and virus-bound gp120.

Structural analysis of N-linked glycans of gp120. Previousstudies on the structure of the glycans of gp120 from varioussources suggest that it contains bi-, tri-, and tetra-antennaryCX glycans, which carry 2, 3, and 4 LacNAc groups, respec-tively (16, 34, 41). Separate studies on the binding specificitiesof galectins have established that Gal-3 displays a higher af-finity and robust avidity for CX glycans carrying a higher num-ber of LacNAc groups than does Gal-1 (8, 20, 47, 58). Thus, theresults presented so far are not easily reconciled with theestablished glycan specificities of Gal-1 and Gal-3. In order tofurther study this specific recognition of gp120 by Gal-1, N-linked glycan structures of the X4 and R5 gp120s were char-acterized in details. N-linked glycans were released from gp120by hydrazinolysis as previously described (29). These glycanswere first separated by anion-exchange (MonoQ) column chro-

matography into neutral (N) and acidic (A) fractions, and theratio of N to A was approximately 7 to 3 (Fig. 5A). Whensubjected to normal-phase (GlycoSepN) column chromatogra-phy, the majority of the neutral fractions were eluted at posi-tions corresponding to the 2-AB-labeled authentic OM glycans(Fig. 5B, triangles M5, M6, M7, M8, and M9; their structuresare shown in E). These data suggest that, irrespective of viraltropism, 70% of gp120 glycans are OM glycans (Table 1). Theacidic fractions were further studied by treating the acidicglycans with sialidase to remove sialic acid residues beforebeing run on a GlycoSepN column. Most of the glycans wereeluted at positions that corresponded to the authentic CXglycan standards (Fig. 5C, triangles Bi, Bi-F, Tri, and Tetra-F;their structures are shown in E). Further confirmation of theseglycan structures (fractions f, g, h, and i) were performed asshown in Fig. 5D. Fractions f and h, which were eluted atpositions corresponding to authentic biantennary and trianten-nary CX glycans, respectively, were incubated with �-galacto-sidase. This digestion removed 2 and 3 galactose residues fromfractions f and h, respectively, as indicated by the position atwhich they were eluted from the GlycoSepN column. In addi-tion, the digested glycans were eluted at positions correspond-ing to the authentic bi- and triantennary glycan standards thatlack terminal galactose residues (Fig. 5F, structures I and III),respectively. Together, these analyses suggest that the glycanstructures in fractions f and h were bi- and triantennary CXglycans, respectively (Fig. 5E, Bi and Tri). As fractions g and ieluted at the positions of monofucosylated bi- and tetra-anten-nary glycans, these fractions were first treated with -fucosi-dase. Once digested, glycans were eluted at positions repre-senting one less monosaccharide than their original glycan size(Fig. 5D, peaks II and V) and also corresponded to the defu-cosylated bi- and tetra-antennary chain (Fig. 5F, structures IIand V). Those defucosylated glycans were further incubatedwith �-galactosidase, resulting in a reduction of 2 and 4 mono-saccharide residues, and they eluted at the positions of stan-dard structures I and IV of Fig. 5F. Thus, the glycan structuresof fractions g and i are monofucosylated bi- and tetra-anten-nary glycans, respectively. Structures and their percent ratiosof N-linked gylcans from gp120 are summarized in Table 1.Together, detailed analysis of the glycan structure of the re-combinant gp120 proteins used in the previous section showedthat 30% of glycans of both X4 and R5 gp120 contain CXglycans (Fig. 5 and Table 1).

Gal-1, but not Gal-3, recognizes clustered CX glycanpatches of gp120. Data from previous glycan binding specificitystudies indicated that such glycans display similar affinity forboth Gal-1 and Gal-3. For example, Gal-3 has a higher affinity(at least 3- to 5-fold-higher Kd) for tri- and tetra-antennary CXglycans, while biantennary CX glycans show a persistent avidityfor Gal-1 under low concentrations (8, 20, 47, 58). This glycananalysis thus suggests that the observed preference of Gal-1 forgp120, compared to that of Gal-3, is not likely to be due to ade facto preference of one galectin toward specific glycansdisplayed by gp120. Indeed, when glycans released from bothX4 and R5 recombinant gp120 were subjected to galectin af-finity chromatography, both the Gal-1 and Gal-3 columns cap-tured similar proportions of gp120-derived glycans (Fig. 6A).Thus, the released glycans did not show any binding preferencefor Gal-1 over Gal-3, which is consistent with the established

FIG. 4. Viral gp120s of both the X4 and R5 variants are recognizedby Gal-1. (A) HIV-1 lysate was applied to a Gal-1– or Gal-3–agarosecolumn, and PBS-, Man-, and Lac-eluted fractions were collected.Levels of gp120 in Lac-eluted fractions were detected with anti-gp120antibody. Representative results from three different experiments areshown. (B) Activated CD4� T cells were infected with either X4 or R5HIV-1 with or without Gal-1 (2 �M) for 1 h. After unbound virionswere removed, cells were further incubated for 48 h. The levels of p24in the cell-free medium were used to estimate virus infection. Repre-sentative results from two different experiments are shown. (C) Re-combinant gp120 (X4 or R5) was subjected to galectin affinity chro-matography as described for Fig. 1. The presence of gp120 in eachfraction was revealed by using anti-gp120 antibody. Results shown arerepresentative of three different experiments.


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FIG. 5. Characterization of N-linked glycans of gp120. (A) 2-AB-labeled N-linked glycan of gp120 was first separated into neutral and acidic fractions byusing anion-exchange chromatography (MonoQ HR5/5). N, neutral fraction; A, acidic fraction. (B) Neutral fractions were further subjected to normal-phasecolumn chromatography by using a GlycoSep N column. The open triangles (M5 to M9, peaks a, b, c, and d, respectively) and their structures are shown in panelE and indicate the elution positions of the 2AB-labeled authentic OM N-glycan standards. The 2-AB-labeled glucose oligomer ladder (gu) was used to estimatethe size change of glycans after glycosidase digestion and was shown at the top. The molar ratios of each component are summarized in Table 1. (C) The acidicfraction from panel A was first treated with sialidase to remove sialic acid residues and then subjected to normal-phase column chromatography (GlycoSep Ncolumn). The open triangles indicate the elution positions of the 2-AB-labeled authentic CX N-linked glycan standards, as shown in panel F. The structures andmolar ratios of each component are summarized in Table 1. (D) Sequential glycosidase digestion of each component in the acidic fraction. Each component (fto i) in panel C was fractionated as a single peak by reverse-phase and normal-phase column chromatography. After digestion by a particular glycosidase, eachcomponent was then analyzed and recovered by reverse-phase and normal-phase column chromatography. The displacements of the elution positions on thenormal-phase column by sequential glycosidase digestion are shown. The arrows indicate displacements of elution positions by digestion with the followingglycosidases: �-G’ase, �-galactosidase (Streptococcus 6646K); -F’ase, -fucosidase (bovine kidney). The open triangles indicate the elution positions of thefollowing 2AB-labeled authentic N-glycan standards as shown in panel E. (E) Glycan structures of gp120 glycans. (F) Glycan structures of the authentic2-AB-labeled glycan standards. M, mannose; GN, N-acetylglucosamine; G, galactose; F, fucose; Bi, biantennary CX glycan; Bi-F, fucosylated biantennary glycan;Tri, triantennary glycan; Tetra-F, fucosylated triantennary glycan; gu, glucose unit.

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glycan binding specificity of Gal-1 and Gal-3. Thus, while Gal-3can recognize the free glycans of gp120, it is unable to do sowhen these same glycans are attached to gp120. We next stud-ied whether or not the CX glycan clusters are involved in theinability of Gal-3 to recognize these glycans in the specificcontext of an intact gp120 glycoprotein tertiary structure. Thus,gp120 disulfide bonds were reduced by dithiothreitol (DTT)treatment, and the reduced protein was applied to Gal-1 andGal-3 columns. As shown in Fig. 6B, the reduced form of gp120was retained efficiently by the Gal-3 column, and its elutionpattern became similar to that of the Gal-1 column. Together,these results suggest that the spatial organization of CX gly-cans on gp120, rather than their composition, contributesmostly to its specific recognition by Gal-1 and the repulsion ofGal-3.

CD4 is a ligand for Gal-1. The above data suggest that Gal-1binds to CX glycans found on native gp120, which are adjacentto the CD4 binding pocket. Gal-1 is a homodimer and thusharbors two glycan binding sites. Since Gal-1 rapidly stabilizesHIV-1 attachment onto susceptible target cells (40), it is pos-sible that Gal-1 also binds to glycans attached to the primarycellular receptor of gp120, the CD4 molecule. CD4 carries two

N-linked glycans, both of which are CX biantennary chains(64). Thus, to test this possibility, Gal-1 or Gal-3 protein wassubjected to CD4 affinity chromatography. As shown in Fig.7A, Gal-1, but not Gal-3, bound to CD4 in a �-galactoside-dependent manner. Furthermore, in the presence of Gal-1, theamount of gp120 retained by the CD4 column was higher andwas specifically inhibited by Lac (Fig. 7B), suggesting thatGal-1 stabilizes the interaction between gp120 and CD4. Toconfirm the ability of Gal-1 to cross-link virus-associated gp120and cell surface CD4, experiments were performed with aCD4-negative Raji cell line and a Raji derivative stably ex-pressing CD4 (Raji-CD4). Results depicted in Fig. 7C demon-strate that binding of HIV-1 was significantly enhanced inRaji-CD4 cells in the presence of Gal-1, in contrast to what wasseen in Raji cells (CD4 negative), for which virus adsorptionwas not affected by the lectin. Altogether, these data suggestthat Gal-1 can cross-link gp120 and CD4 (Fig. 7D). Therefore,the results raise the possibility that CD4 represents one of theligands targeted by Gal-1 to promote HIV-1 attachment on thecell surface.

DISCUSSION

Sexual transmission of HIV-1 is severely limited by multipleanatomical, chemical, and immunological barriers so that,

TABLE 1. Percent molar ratios of N-linked glycans attached togp120 of HIV-1a

Fraction Elutionposition

Chromatographypeak

HIV-1gp120 molar

ratio (%)

X4 R5

Neutral, OM type M5 a 48 10M6 b 22 14M7 c 0 18M8 d 0 18M9 e 0 10

Acidic, CX type Bi f 6 0Bi-F g 10 7Tri h 10 0Tetra-F i 0 18

a Structures and chromatography peaks for these data are provided in Fig. 5.

FIG. 6. Both Gal-1 and Gal-3 recognize glycans of gp120 once theyare released from glycan patches or gp120 is partially denatured.(A) N-linked glycans were first released from gp120 and then labeledwith 2-AB. The glycans were subjected to galectin affinity chromatog-raphy. Representative results from three different experiments areshown. (B) DTT-treated (overnight, 1 mM) gp120 (R5) was applied togalectin columns. Results shown are representative of three differentexperiments.

FIG. 7. CD4 is a ligand for Gal-1. (A) Gal-1 was applied to aCD4-agarose column, and bound lectin was eluted with Lac as de-scribed for Fig. 1. Results shown are representative of three differentexperiments. (B) In the presence or absence of Gal-1, gp120 wasincubated with CD4-agarose. After unbound materials were removedby a series of PBS washes, gp120 that was bound to CD4 was releasedby adding SDS-PAGE sample buffer and detected by anti-gp120 anti-body. Representative results from two different experiments areshown. (C) HIV-1 (10 ng of p24) was incubated with Raji and Raji-CD4 cells at room temperature for 10 min in the absence or presenceof Gal-1 (2 �M). Controls consisted of cells incubated with HIV-1 inthe absence of Gal-1. The representative results from two differentexperiments are shown. P values were created by comparison to HIV-1binding in the absence of galectin 1. *, P � 0.05. (D) Proposed modelof the interaction of gp120 and CD4 together with Gal-1.


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when it occurs, only a small number of infectious viral particlessuccessfully reach the underlying mucosa to create a foundercell population that rapidly expands and establishes a self-propagating infection (17, 19, 22, 32, 54). Mucosa-associatedCD4� T cells found just beneath the vaginal and cervical ep-ithelium are known to be somewhat less competent than fullyactivated peripheral blood CD4� T cells (17, 22, 32). Theirefficient infection is further complicated by the fact that HIV-1must compensate in some way for the paucity of functional Envspikes on its surface, which results in a low avidity for itscellular receptor, CD4. HIV-1 must therefore evade and/orexploit host mucosal immune defenses, such as glycan-lectininteractions, to facilitate its specific interaction with suscepti-ble cells in the underlying lamina propria. These initial stepsthus represent a significant bottleneck of HIV-1 infection thatremains to be exploited in order to further reduce the fre-quency of transmission events.

Glycosylation of gp120 has been the subject of intense stud-ies since the discovery of HIV-1 as the etiologic agent of AIDS.This interest arises from the large amount of glycans attachedto gp120 as well as from their unusual composition and theirpeculiar arrangement in specific clusters. Three clusters ofglycans are found on the surface of gp120: one is formed fromOM glycans, and two are formed from CX glycans (53). HIV-1uses the host’s own glycosylation machinery for glycosylation ofgp120. As such, the arrangement of glycans on gp120 is con-sidered unique, since glycans attached to host glycoproteinsnormally exhibit considerable conformational freedom be-cause of their highly hydrophilic nature. Recognition of OMglycans of gp120 by CLRs of immune cells is exploited byHIV-1 to enhance infection of susceptible cells (11, 15, 23, 24,27, 31, 61). The present study now suggests a role for the CXglycans of gp120. Recognition of gp120 CX patches by non-CLR soluble lectin, Gal-1, but not Gal-3, explains the specificenhancement of HIV-1 binding to susceptible cells mediatedby the former and not the latter. While the recent report byDoores and colleagues suggested a relatively low abundance ofCX glycans on native HIV-1 (10), our previously publishedwork and the present study demonstrate that Gal-1 promotesattachment of native HIV-1 produced by both lymphocytes andmacrophages through a specific interaction with CX glycans(33, 40). Thus, regardless of levels of CX glycans attached tonative virus, Gal-1, but not Gal-3, binds to the CX glycans,facilitating HIV-1 infection. It is likely that the anatomicallocalization of cells expressing each galectin could explain howHIV-1 has evolved its glycan shield to recruit Gal-1 and notGal-3. Indeed, Gal-3 is highly expressed at the apical surface ofmucosal membranes, while Gal-1 is highly expressed in thelamina muscularis mucosae, just beneath the epithelia and itslamina propria, where abundant susceptible CD4� T cells canbe found (12, 39, 56).

Recognition of HIV-1 by Gal-1 but not Gal-3 may arise froma unique binding preference (or a positional selection) of eachgalectin for the position of LacNAc displayed by CX glycans.Loss of differential binding to each galectin following enzy-matic cleavage of gp120 glycans or partially denatured gp120suggest that specific binding of Gal-1 to gp120 largely dependson the peculiar presentation of CX glycans by the peptidebackbone itself. Indeed, those glycans are constrained withintight clusters on gp120, where sugar-sugar interactions are

formed between neighboring glycans (53). Thus, for a lectinsuch as Gal-3, which has been shown to bind to internal Lac-NAc residues (8, 20, 47, 58), tightly clustered glycans mayprevent efficient binding. In contrast, Gal-1 preferentiallybinds to the peripheral LacNAc of CX glycans (8, 20, 58), andit is thus possible that Gal-1 efficiently recognizes LacNAcresidues of even densely packed glycans, as indicated by thisstudy (Fig. 8). Such glycan clustering-dependent exclusion oflectin binding, for which the biological significance had notbeen recognized previously, may also be beneficial for HIV-1to escape from being captured by Gal-3 at the surface ofmucosal membranes, where no susceptible cells can be foundand where multiple mucosal clearance mechanisms are active.Conversely, HIV-1 binding by Gal-1, which is found proximalto the lamina propria, may allow rapid and efficient attachmentof HIV-1 to susceptible founder cells and prevent entrapmentand dilution of the initial viral inoculum. Together with itslocalization and its potential to accelerate viral attachment andentry, the soluble lectin Gal-1 can be proposed as a host im-mune factor exploited by HIV-1 for the eclipse and initialphase of infection.

Our previous studies suggested that Gal-1 facilitates viralinfection even under suboptimal conditions, such as in thepresence of anti-CD4 antibody or fusion inhibitors, althoughvirus entry remains entirely dependent on CD4-gp120 interac-tions. Thus, it seems that it is the ability of Gal-1 to greatlyaccelerate the binding kinetics of HIV-1 on susceptible cellsthat severely cripples the efficiency of entry inhibitors. Gal-1 isa homodimer, and its glycan binding domain is of a size thataccommodates only one LacNAc of a glycan. Thus, when Gal-1cross-links a virus particle to a susceptible cell, at least one

FIG. 8. Glycan density-dependent gp120 recognition by Gal-1.


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LacNAc of a glycan of gp120 and one LacNAc of a glycan ofthe cell are expected to simultaneously bind to one dimerof Gal-1 (Fig. 7D). This rapid HIV-1 fixation on the surface ofcells leads to a significant increase in HIV-1 infectivity, sug-gesting that Gal-1 could help the viral particle via efficientinitiation of fusion. Indeed, our present data suggest that Gal-1recognizes CD4 and increases the interaction between CD4and gp120. Together, the present data indicate that the pecu-liar spatial arrangement of CX glycans of gp120 would allow itscross-linking with a cellular ligand, such as CD4, by dimericGal-1 molecules without interfering with the gp120/CD4 bind-ing site and later fusion events (Fig. 7D). Although Gal-1 couldbind to other cellular ligands (5), CD4 can be considered animportant ligand in the context of HIV-1 infection. Theseresults further suggest that HIV-1 exploits the Gal-1 cross-linking ability not only to increase its avidity toward highlysusceptible CD4-expressing cells but also to directly target itsgp120 spikes toward its cognate cellular receptor.

In summary, our results provide evidence that Gal-1 is thefirst soluble host lectin to be exploited by HIV-1 to improve itschances of being efficiently transmitted through its glycanstructures. Our study has unveiled the unexpected role of clus-tered CX glycans in the specific recognition of gp120 by Gal-1.Furthermore, we have demonstrated specific binding of CD4by Gal-1, raising the possibility that the Gal-1-mediated cross-linking ability could direct viral spikes to their cognate recep-tors. Modulation of Gal-1 expression or activity at mucosalsites where susceptible cells initially encounter HIV-1 thusrepresents an interesting therapeutic avenue to decrease thelikelihood of sexual transmission of HIV-1.

ACKNOWLEDGMENTS

We acknowledge Isabelle Pelletier for her preliminary results ofGal-1 binding to HIV-1, Sean Stowell for the method of iodoacetami-dation of Gal-1, Yukiko Sato for purification of galectins, and JulieNieminen for her editorial work on the manuscript.

This work was supported by an operating grant from the CanadianInstitutes of Health Research (CIHR) to S.S. and M.J.T. (MOP-89743). C.S.-P. holds a Graduate Scholarship Award from CIHR, S.S.holds a Scholarship Award (senior level) from the Fonds de la Re-cherche en Sante du Quebec, and M.J.T. is the recipient of a Tier 1Canada Research Chair in Human Immuno-Retrovirology.

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