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Virology 371 (200

Galectin-1 promotes HIV-1 infectivity in macrophagesthrough stabilization of viral adsorption

Simon Mercier, Christian St-Pierre, Isabelle Pelletier, Michel Ouellet,Michel J. Tremblay ⁎, Sachiko Sato ⁎

Research Center for Infectious Diseases, CHUL Research Center, Quebec, CanadaFaculty of Medicine, Laval University, Quebec, Canada

Received 26 June 2007; returned to author for revision 23 July 2007; accepted 22 September 2007Available online 29 October 2007

Abstract

Following primary infection with human immunodeficiency virus type-1 (HIV-1), macrophages are thought to play an important role, as theyare one of the first target cells the virus encounters and can also sustain a significant production of viruses over extended periods of time. While theinteraction between the primary cellular receptor CD4 and the virus-encoded external envelope glycoprotein gp120 initiates the infection process,it has been suggested that various host factors are exploited by HIV-1 to facilitate adsorption onto the cell surface. Macrophages and other cellsfound at the infection site can secrete a soluble mammalian lectin, galectin-1, which binds to β-galactoside residues through its carbohydraterecognition domain. Being a dimer, galectin-1 can cross-link ligands expressed on different constituents to mediate adhesion between cells orbetween cells and pathogens. We report here that galectin-1, but not galectin-3, increased HIV-1 infectivity in monocyte-derived macrophages(MDMs). This phenomenon was likely due to an enhancement of virus adsorption kinetics, which facilitates HIV-1 entry. The fusion inhibitors T-20and TAK779 remained effective at reducing infection even in the presence of galectin-1, indicating that the galectin-1-mediated effect is occurring at astep prior to fusion. Together, our data suggest that galectin-1 can facilitate HIV-1 infection in MDMs by promoting early events of the virusreplicative cycle (i.e. adsorption).© 2007 Elsevier Inc. All rights reserved.

Keywords: Galectin-1; HIV-1; Macrophages

Introduction

It has been estimated that the human immunodeficiencyvirus type-1 (HIV-1) pandemic now affects more than 40 millionpeople and has caused more than 20 million deaths worldwide.This retrovirus preferentially infects activated CD4+ T lym-phocytes and macrophages through a pH-independent fusionprocess involving an association between the viral transmem-brane and surface proteins gp41 and gp120 with a complexmade of the cellular surface protein CD4 and a coreceptor of thechemokine receptor family, generally CCR5 or CXCR4. Fol-

⁎ Corresponding authors. Laboratory of Human Immuno-Retrovirology andLaboratory of Glycobiology, Research Center for Infectious Diseases, RC709,CHUL Research Center, 2705 Laurier Blvd., Quebec (QC), Canada G1V 4G2.

E-mail addresses: michel.j.tremblay@crchul.ulaval.ca (M.J. Tremblay),sachiko.sato@crchul.ulaval.ca (S. Sato).

0042-6822/$ - see front matter © 2007 Elsevier Inc. All rights reserved.doi:10.1016/j.virol.2007.09.034

lowing primary infection, continuous viral replication and itsassociated CD4+ T cell toxicity slowly but relentlessly cripplethe host's ability to mount an efficient immune response,leading to the appearance of a large variety of symptoms, betterknown as acquired immunodeficiency syndrome (AIDS).

The majority of mammalian cell surface molecules, as well asthose of enveloped virus, are heavily glycosylated. Specificsugar sequences presented by the glycans of these glycoproteinscan be recognized by a variety of glycan-binding proteins calledlectins. One of such lectin families, galectins, has been recentlysuggested to play functional roles in various immune responseprocesses through binding to host surface glycoproteins(Almkvist et al., 2002; Barrionuevo et al., 2007; Baum et al.,1995; Correa et al., 2003; Gauthier et al., 2002; He and Baum,2004; Perillo et al., 1995; Rabinovich et al., 1998, 2002a,b; Sato,2002; Sato and Nieminen, 2004). For example, galectin-1belongs to the galectin family, β-galactoside-binding proteins,

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defined by conserved peptide sequence elements of carbohy-drate recognition domain (CRD). Up to 14 galectins (galectin-1–14) have been found in mammals so far, which can be sub-divided into three categories depending on the presentation ofCRD domains: prototype, tandem-repeat and chimera (Hira-bayashi and Kasai, 1993). Some galectins contain one CRD(prototype), and exist as monomers (galectin-5, 7, 10) or dimers(galectin-1, 2, 11, 13, 14), other galectins, such as galectin-4, 6,8, 9 and 12, contain two CRD connected by a short linker region(tandem repeat) (Hirabayashi and Kasai, 1993). In contrast,galectin-3 uniquely occurs as a chimeric protein with one CRDand an additional non-CRD domain, which is involved in theoligomerization of galectin-3 (Hirabayashi and Kasai, 1993;Nieminen et al., 2007). Galectin-1 expression has been reportedin thymus and lymphoid parenchymal epithelial cells, endothe-lial cells, trophoblasts, activated T cells, macrophages, activatedB cells, follicular dendritic cells and CD4+CD25+ regulatory Tcells (Baum et al., 1995; Blaser et al., 1998; Dettin et al., 2003;Garin et al., 2007; Jeschke et al., 2004; Rabinovich et al., 1996;Stillman et al., 2006; Zuniga et al., 2001a). We recently foundthat human tonsil lymphoid tissues contain∼16 μMof galectin-1 (Ouellet et al., 2005). Galectins are synthesized as cytosolicproteins, thereby the export of galectins into extracellular spaceis regulated (Cooper and Barondes, 1990; Hughes, 1999; Sato etal., 1993; Sato and Hughes, 1994), while their biologicalsignificance remains speculative (Hughes, 1999; Rabinovich etal., 2007; Sato, 2002; Sato and Nieminen, 2004). Indeed,galectin-1 is actively secreted by activated B cells, T cells andmacrophages and certain epithelial cells (without compromisingmembrane integrity) through a “leaderless” secretory pathway(Baum et al., 1995; Blaser et al., 1998; Garin et al., 2007;Gauthier et al., 2002; Rabinovich et al., 1998; Zuniga et al.,2001b), which is also used by fibroblast growth factors and IL-1(Hughes, 1999; Nickel, 2003, 2005; Oppenheim et al., 2007).Due to their multivalent nature, galectins can cross-link differentconstituents, acting as adhesion molecules (Baum et al., 1995;Gauthier et al., 2002; Hughes, 2001; Rabinovich et al., 2002a;Rabinovich and Gruppi, 2005; Sato, 2002; Sato and Nieminen,2004). We have previously found that one member of thegalectin family, galectin-1 (Gal-1), can potentiate infection ofhuman tonsilar tissues and primary human peripheral CD4+ Tcells by X4-tropic isolates of HIV-1 (Ouellet et al., 2005). Gal-1can stabilize attachment of HIV-1 particles onto CD4+ T cellsthereby facilitating the infection of such susceptible cells. Ourprevious study was the first demonstration of the impact of Gal-1in the HIV-1 life cycle, but it mainly reflected the role of Gal-1 inthe later stages of the infection, when the virus has reached thesecondary lymphoid organs. However, there is still no inform-ative data on the possible involvement of Gal-1 during primaryinfection. The value of such results resides in the possibility ofdeveloping strategies aimed at reducing the occurrence of initialinfection.

Infection of macrophages by R5-tropic isolates of HIV-1may play an important role during the early stages of infection(Noursadeghi et al., 2006). Indeed, macrophages found in thegenital and intestinal tracts are mainly exposed to R5-tropicvirions during primary infection (Brumme et al., 2005), pos-

sibly because the mucosal barrier restricts the transmission ofX4 strains (Margolis and Shattock, 2006; Meng et al., 2002).Some studies also revealed that recently infected individualspredominantly harbor R5 isolates of HIV-1 (Koot et al., 1996;Zhang et al., 1998). Macrophages are also thought to play a rolein later stages of infection because they are inherently resistantto the cytopathic effects of the virus, and are thus considered tobe a stable and long-lived cellular reservoir (Verani et al., 2005).Moreover, their interplay with CD4+ T lymphocytes as profes-sional antigen presenting cells may facilitate trans or cis infec-tion of this cell type through their direct contact and activation(Verani et al., 2005). As macrophages are thought to be im-portant actors in the pathogenesis of HIV-1 infection, and asthey also express and secrete Gal-1 following their activation(Correa et al., 2003), it is essential to extend our initial obser-vations to this particular cell type.

This study thus focuses on the role of Gal-1 in the infectionprocess of monocyte-derived macrophages (MDMs) by R5-tropic variants of HIV-1. We report here that exogenous additionof Gal-1 to MDMs enhances HIV-1 infection possibly throughan increase in viral binding kinetics. Interestingly, the antiviralpotency of fusion inhibitors targeting gp41 (T-20) and CCR5(TAK779) was not affected by Gal-1, thus indicating that thissoluble factor is affecting the HIV-1 life cycle at a step prior tofusion. The possible implications of this set of results couldprove to be important for the efficient use of the already availableantiretroviral drugs.

Results

Galectin-1, but not galectin-3, promotes HIV-1 infection inMDMs

MDMs were initially infected with recombinant luciferase-encoding reporter viruses that were pseudotyped with the R5-tropic JR-FL envelope. This experimental setup allows a rapid,accurate and quantitative evaluation of single-cycle infectionevents through measurement of cell-associated luciferaseactivity following infection. As shown in Fig. 1A, the presenceof Gal-1 significantly increased virus-induced luciferaseactivity. This Gal-1-mediated augmentation in virus replicationwas dose-dependent up to a 4 μM final concentration of Gal-1(data not shown). In order to assess the specificity of Gal-1action, further infection experiments were performed in thepresence of another member of the galectin family, namelygalectin-3 (Gal-3). Interestingly, infection of MDMs by thereporter virus remained unaffected when it was performed in thepresence of Gal-3 (Fig. 1A). These results suggest that the Gal-1-mediated enhancing effect on HIV-1 infection is specific andis not a general phenomenon that can take place with all mem-bers of the galectin family.

Although the initial series of investigations is informative, itis important to emphasize that such experiments have beenperformed with pseudotyped, replication-incompetent, reporterviruses produced in a human cell line, which is not consideredas a natural cellular reservoir for HIV-1 (i.e. 293T cells). More-over, it is well established that HIV-1 acquires a vast array of

Fig. 1. Gal-1 increases HIV-1 infection in MDMs. (A) MDMs (5×104) wereincubated for 1 h at 37 °Cwith reporter viruses pseudotypedwith JR-FL envelope(1×104 TCID50) produced in 293T cells in the presence of 2 μM of Gal-1, Gal-3or equal volumes of the appropriate control. Cells where then washed andincubated for 5 days at 37 °C before luciferase activity was assayed as describedin Materials and methods. (B) Similar experiments were carried out using fullycompetent NL4-3/BaL virions produced in MDMs (5×102 TCID50). Cell-freesupernatants were harvested every 3 days and the p24 content was assayed asdescribed inMaterials andmethods. Data shown correspond to themeans±SD oftriplicate samples and are representative of three independent experiments.(C) MDMs were infected 3 days with reporter viruses pseudotyped with VSV-Genv. Cells were then washed twice prior to adding of 2 μM of Gal-1 or equalvolumes of the appropriate control. Cells were then incubated for 1 to 24 h beforeluciferase activity was assayed as described in Materials and methods.

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host-derived surface molecules in its bilayer lipid envelope(Tremblay et al., 1998). Since the majority of surface proteinson mammalian cells are glycosylated to increase their stabilityand lock-in their conformation, viruses budding from these cellswill carry such host-derived glycosylated proteins on theirsurface. While the glycosylation patterns of surface proteinsare protein-specific, their patterns can vary, to some extent,depending on the cell-type and cell status (e.g. resting versusactivated, apoptotic, etc.) (Ohtsubo and Marth, 2006). Thus,viruses produced in different cell types might acquire different

glycosylation patterns that could, in turn, have an impact on theability of Gal-1 to affect viral replication. Acknowledging thispossibility, the experimental setup described above was re-peated, but this time using replication-competent R5-tropicvirions (i.e. NL4-3/BaL) harvested from acutely infectedMDMs. Data shown in Fig. 1B demonstrate that replication offully competent virus was augmented by the presence of Gal-1.Together, the data suggest that Gal-1 facilitates infection ofMDMs by R5-tropic viruses. Additional studies revealed thatGal-1 was unable to induce transcription from the HIV-1 regu-latory elements (i.e. long terminal region/LTR) (Fig. 1C).Therefore it can be concluded that the Gal-1-dependent effect onHIV-1 replication is not due to signal transduction events thatcan in turn promote HIV-1 LTR-driven activity.

Given the reported capacity of Gal-1 to cross-link ligandsexpressed on different constituents (Baum et al., 1995; Gauthieret al., 2002; Hughes, 2001; Rabinovich et al., 2002a;Rabinovich and Gruppi, 2005; Sato, 2002; Sato and Nieminen,2004) and its inability to drive HIV-1 LTR-driven geneexpression, it can be proposed that Gal-1 favors the first stepsin the HIV-1 replicative cycle by promoting the initialinteractions between the virus and the surface of target cells,thereby facilitating viral entry. Adsorption of HIV-1 particles onMDMs was thus assessed by measuring the cell-associated p24levels following incubation for 1 h at 37 °C in the presence orabsence of 2 μM of Gal-1 or Gal-3 as described in Materials andmethods. As our experiments are performed at 37 °C, we referto it as an adsorption experiment. Adsorption includes virus thatbind and enter the cell through fusion or endocytosis. We choseto proceed this way to better approximate the initial steps ofHIV-1 infection in vivo. As shown in Fig. 2A, virus adsorptionwas significantly enhanced upon addition of Gal-1 (i.e. 9-foldincrease) but not Gal-3 (data not shown). The Gal-1-mediatedenhancement of HIV-1 adsorption was totally abolished bylactose, which competes for the CRD of Gal-1 and acts as anantagonist of this soluble β-galactoside-binding protein. Whenthe incubation period was extended to 6 h, cell-associated p24levels in the absence of Gal-1 became comparable to those inthe presence of Gal-1 (Fig. 2B). This observation suggests thatGal-1 accelerates the kinetics of the initial steps of HIV-1replication. The Gal-1-mediated enhancement of HIV-1 adsorp-tion was also observed when experiments were conducted withX4-using virus (i.e. NL4-3 strain) (data not shown). This resultsuggests that the increased adsorption observed is independentof specific co-receptors. Given that our observations led us topropose that virus adsorption occurred with faster kinetics in thepresence of Gal-1, we next investigated the level of viralinternalization in these cells in the presence or absence of Gal-1.MDMs were incubated with NL4-3/BaL and increasing concen-trations of Gal-1 (0 to 4 μM) for 1 h at 37 °C, in the presence orabsence of 50 mM of lactose. Unbound virus particles and thosethat bound but did not enter the cells were removed by a trypsintreatment as published previously (Tardif and Tremblay,2005a,b). Results depicted in Fig. 2C demonstrate that virusinternalization was promoted in a dose-dependent manner byGal-1 (i.e. 2.5-fold increase in presence of 4 μM Gal-1; 1070±75 versus 440±34 pg/ml). As seen with adsorption assays, this

Fig. 2. Adsorption and internalization of HIV-1 are promoted by Gal-1. (A) MDMs (5×104) were incubated for 1 h at 37 °C with fully competent NL4-3/BaL viruses(10 ng of p24) in the presence of Gal-1 (2 μM) or mock preparations. Lactose (50 mM) was also added to some samples. Cells were then washed twice and the p24content was assayed in lysed cells as described in Materials and methods. (B) MDMs (5×105) were incubated for 6 h at 37 °C with fully competent NL4-3/BaL viruses(10 ng of p24) in the presence of Gal-1 (2 μM) or mock preparations. Cells were then washed twice and the p24 content was assayed in lysed cells as described inMaterials and methods. (C) MDMs (5×104) were incubated for 1 h at 37 °C with fully competent NL4-3/BaL viruses (10 ng of p24) in the presence of increasingconcentrations of Gal-1 or mock preparations. Lactose (50 mM) was also added to some samples. Next, cells were washed twice, treated with trypsin to removeuninternalized virions and washed again twice. Finally, the p24 content was estimated as described in Materials and methods. (D) Similar studies were performed withthe fusion inhibitors T-20 (10 μg/ml) and TAK779 (100 μM). Data shown correspond to the means±SD of triplicate samples and are representative of threeindependent experiments.

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Gal-1-dependent increase is lost upon addition of lactose. Inorder to confirm these results using a different experimentalstrategy to remove bound but uninternalized viruses, we alsoperformed acid washes instead of trypsin treatment as describedpreviously (Vidricaire and Tremblay, 2005). Similar observa-tions were made when using this method (data not shown),which confirm that Gal-1 is affecting the earliest step in the HIV-1 replicative cycle (i.e. adsorption).

The Gal-1-mediated increase in virus internalization couldbe due to an enhancement in virus-mediated fusion and/or anaugmentation in HIV-1 entry through an endocytic processsuch as macropinocytosis. We thus performed additional entryassays, but this time in the presence of the fusion inhibitors T-20and TAK779, which are targeting different actors of theinfection process. T-20 prevents fusion between virus and cellmembranes by binding to the viral fusion peptide of gp41,whereas TAK779 is a chemical antagonist of CCR5 that inhibitsits interaction with the V3-loop of gp120 after it has bound toCD4. These two inhibitors act directly on the essential inter-actions required for the fusion of viral and cellular membranes.Results illustrated in Fig. 2D indicate that, at the doses whichgave maximal inhibition of HIV-1 entry in CD4+ T cells (Tardifand Tremblay, 2005a), HIV-1 internalization within MDMs,

regardless of the presence or the absence of Gal-1, was notaffected by either T-20 or TAK779. These data suggest that, atleast under these experimental conditions, the gp41-mediatedmembrane fusion events do not contribute significantly to theprocess of HIV-1 internalization into MDMs.

Galectin-1 does not reduce the overall sensitivity of HIV-1 tofusion inhibitors

Our previous report in CD4+ T lymphocytes indicates thatT-20 efficiently inhibits Gal-1-promoted viral replication,suggesting that Gal-1 facilitates initial adsorption of X4-tropicHIV-1 to CD4+ T lymphocytes, but not the following membranefusion step (Ouellet et al., 2005). In the case of MDMs, the datapresented above suggest that the fusion inhibitors T-20 andTAK779 exhibit a limited capacity to reduce viral internaliza-tion, which was significantly increased by Gal-1, possiblythrough an increase of adsorption kinetics of the virus. There-fore, we next studied whether these fusion inhibitors caneffectively limit infection ofMDMs byHIV-1. In the presence ofGal-1, HIV-1 infection of MDMs was still detectable even athigh doses of T-20 or TAK779 due to the Gal-1-mediatedincrease in HIV-1 infection (Figs. 3A and B). However, the

Fig. 3. Gal-1 does not decrease HIV-1 sensitivity to neutralization by fusion inhibitors. MDMs (5×104) were incubated for 1 h at 37 °C with reporter virusespseudotyped with JR-FL envelope (1×104 TCID50) in the presence of Gal-1 (2 μM) or PBS in combination with increasing concentrations of either T-20 (A and C) orTAK779 (B and D). Cells were then washed twice and incubated for 5 days at 37 °C before luciferase activity was assayed as described in Materials and methods. Theresults are presented as raw infection levels measured by luciferase activity assays (A, B) or as percentages of inhibition by a given concentration of the tested fusioninhibitors with respect to the virus-infected samples that were left untreated with T-20 or TAK779 (C, D). The data shown represent the means±SD of triplicatesamples and are representative of three independent experiments.

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percentages of inhibition of virus infection in MDMs achievedby T-20 were comparable whether the cells were treated or notwith 2 μM of Gal-1 (Fig. 3C). Similar observations were madewhen experiments were carried out in the presence of TAK779(Fig. 3D). Indeed, the calculated IC50 are 37 and 41 ng/ml forT-20 and 0.028 and 0.030 nM for TAK779, in the presence andabsence of 2 μM of Gal-1. Together, the data suggest thatalthough Gal-1 significantly increased viral internalization andinfection in MDMs, the relative efficiency of the tested fusioninhibitors was unaffected by Gal-1, even if they fail to limitviral entry.

Discussion

Several lines of evidence imply that galectins participate inthe immune response, both as immunomodulators and mole-cules that facilitate pathogen-host cell interactions (Kohatsuet al., 2006; Mey et al., 1996; Rabinovich and Gruppi, 2005;Sato, 2002; Sato and Nieminen, 2004). In addition, recentworks suggest that galectins could facilitate pathogen internal-ization in phagocytic cells, such as macrophages (Pelletier et al.,2003; van den Berg et al., 2004). However, there is very few, ifany, information concerning the roles of galectins in viral in-fection, even though many enveloped virus express glycopro-teins on their surface. We have recently reported that Gal-1 can

increase adsorption of X4-using isolates of HIV-1 onto CD4+ Tlymphocytes, thus enhancing the overall infection process(Ouellet et al., 2005). This observation is in line with previousstudies showing that some galectins can act as adhesion mole-cules by cross-linking surface ligands expressed on pathogensand their target cells (Ouellet et al., 2005; Pelletier et al., 2003;Rabinovich and Gruppi, 2005; Sato, 2002). Results depicted inthe present work therefore represent additional evidence of thespecific cross-linking capacity of galectins and suggest thatGal-1 can modulate sexual transmission of HIV-1 throughenhancement of viral adsorption kinetics on diverse targetcells' surface. Interestingly, another type of galectin, Gal-3,displayed no effect on HIV-1 adsorption, entry and infection ina similar context, even though both Gal-1 and Gal-3 bindefficiently to macrophages (Camby et al., 2006; Dumic et al.,2006). This is in contrast to its ability to facilitate the in-teraction between parasites and macrophages (Pelletier et al.,2003; van den Berg et al., 2004). Likely due to structuraldifferences between Gal-1 and Gal-3, both in their CRDs and inits presentation, some (but not all) of the ligands ofmacrophages preferentially bind to galectin-1. In addition, itis possible that the affinities of those galectins for HIV-1 aredifferent. Such ligand specificity might be responsible for thisdifferential effect (Rabinovich et al., 2002a; Rabinovich andGruppi, 2005).

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An increase in HIV-1 adsorption mediated by Gal-1 mightbear some significant relevance with respect to primary infec-tion, considering that intracellular Gal-1 could be released bysheared fibroblasts and epithelial cells following sex-relatedtrauma such as micro-abrasions (Akimoto et al., 1995; Baumet al., 1995). In the context of sexual transmission of HIV-1, thepresence of Gal-1 in the ejaculate could also be an importantfactor since Gal-1 has been shown to be present on the headsand tails of late spermatids in rat testes (Dettin et al., 2003). Inaddition, Gal-1 is actively secreted by activated macrophages,Langerhans cells, dendritic cells, and activated CD4+ and CD8+

T lymphocytes (Blaser et al., 1998; Rabinovich et al., 1996,1998, 2002b; Reynolds et al., 2007; Zuniga et al., 2001a).Fibroblasts present at the infection site could also be anothersource of Gal-1. Thus, mucosal macrophages could be exposedto both Gal-1 and R5-tropic HIV-1 particles (Verani et al.,2005), likely creating a situation that might facilitate and accel-erate primary infection. Concerning our infection model, eventhrough Gal-1 is secreted by macrophages, tissular concentra-tions of Gal-1 could never be reached simply by maintainingMDMs in culture due to the frequent addition of fresh culturemedium and the fact that the differentiation process does notlead to the activation of MDMs so that they do not secrete Gal-1themselves. Moreover, as mentioned earlier, other sources (cellsor sperm) of Gal-1 are normally present in vivo but are absent inthe context of cell culture. Therefore, addition of exogenousGal-1 to our infection model is physiologically relevant, inorder to partially reconstitute the initial mucosal infection sitewhere the presence of extracellular Gal-1 is expected.

This study suggests that Gal-1 increases viral adsorptionkinetics on MDMs, thereby shortening the time required toestablish an infection. During primary infection, the number ofHIV-1 particles is limited. In addition, complement, naturalantibodies and other soluble factors at the primary infectionsites could reduce the infectious potential of the virus (Neilet al., 2005). Thus, shortening the exposure time required for thevirus to establish an infection would significantly increase thepossibility of a successful HIV-1 infection. Moreover an in-crease in viral adsorption and entry kinetics reduces the timerequired to establish infection and significantly shortens thecrucial period in which post-exposure HAART treatment couldprevent it.

Even in the presence of Gal-1, fusion inhibitors retain theirantiviral potency and display similar IC50 values. These resultsconfirm that Gal-1 acts mainly at the initial adsorption step, andnot at the fusion-dependent process that would allow HIV-1 togain access to the cytoplasm.While fusion inhibitors were potentinhibitors of HIV-1 infection in MDMs, these drugs could notefficiently inhibit the inherent ability of MDMs to internalizelarge amount of viruses. It has been suggested that followinginternalization of HIV-1 within MDMs, HIV-1 could still gainaccess to the cytoplasm through fusion of its membrane with theendosomal membrane. This step remained dependent on gp41(Marechal et al., 2001) and sensitive to fusion inhibitors such asT-20. Our data also support the notion that MDMs mainlyinternalize HIV-1 through endocytosis, a step that is insensitiveto fusion inhibitors, and are then infected by HIV-1 when it

accesses the cytosol from endosomes in a fusion-dependentmanner. Importantly, despite of the presence of fusion inhibitors,Gal-1 was still efficient at increasing the amount of internalizedHIV-1 by enhancing initial viral adsorption onto MDMs. In asituation where the virus is unable to perform fusion (T-20treated individual), Gal-1 could thus act as a soluble scavengerreceptor and enhance the uptake of the virus bymacrophages. Asthose accumulated virions in endosomes are likely to bedegraded and presented in the MHC-II context, the corollaryof these results is that early treatment of patients with fusioninhibitors could lead to the development of a stronger immuneresponse towards HIV-1 while limiting viral replication andspread.

Further investigations are necessary to define whether Gal-1might act as DC-SIGN, a C-type lectin involved in the endo-cytosis of HIV-1 that is known to facilitate both HIV-1 antigen-presentation and viral transmission to CD4+ T lymphocytes(Geijtenbeek et al., 2000, 2004; Turville et al., 2002, 2004). Itcould otherwise be possible that, in the presence of fusioninhibitors, Gal-1 could act as Langerin, another C-type lectinfound on Langerhans cells, which promotes endocytosis anddegradation of HIV-1, thereby preventing its spread (de Witteet al., 2007).

Materials and methods

Cell lines

Peripheral blood mononuclear cells (PBMCs) were purifiedfrom blood of healthy donors by Ficoll centrifugation (Lym-phocyte Separation Medium; Wisent Inc.; St-Bruno, QC).MDMs were produced by incubating PBMCs at a concentrationof 1.25×106 cells in a T-75 flask for 2 h at 37 °C, thus allowingadhesion of monocytes to the bottom of the flask. Non-adherentcells were then removed and monocytes were washed twice withRPMI 1640 containing 5% autologous heat-inactivated plasmaand cultured for 1 week in medium supplemented with 100 ng/ml macrophage-colony stimulating factor (M-CSF) to allowdifferentiation into MDMs. Next, MDMs were detached usingAccutase® (Sigma-Aldrich Inc.; St-Louis, MO) as recom-mended by the manufacturer and gently scraped with a cellscraper. MDMs were then dispensed in a 48-well plate at a finalconcentration of 5×104 cells per well. Isolation, differentiationand assays described below were all performed in RPMI 1640medium containing 5% autologous heat-inactivated plasma.

Viral preparations

Virus particles (NL4-3/BaL) were produced by transientlytransfecting human embryonic kidney 293T cells with the R5-tropic infectious molecular clone NL4.3BaLenv (kindly sup-plied by Dr. R.J. Pomerantz, Thomas Jefferson University,Philadelphia, PA) (Dornadula et al., 1999). The infectiousmolecular clone pNL4-3 (X4-tropic) was also used in someexperiments. Recombinant luciferase-encoding reporter R5-tropic viruses were generated using pNL4-3Luc+Env− (AIDSRepository Reagent Program, Germantown, MD) and a vector

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coding for JR-FL env (kind gift by Dr. N.R. Landau, The SalkInstitute, La Jolla, CA) or VSV-G env. Briefly, 2×106 cells wereseeded in a T-75 flask for 24 h before transient transfection,which was carried out by adding a total of 30 μg of vectors(pNL4-3/BaL or pNL4-3Luc+Env− and a vector coding for JR-FL env) to the cells as a calcium phosphate precipitate. At 2 dayspost-transfection, the virus-containing supernatant was filteredand frozen at −85 °C until used. Infectious virus particles werealso prepared from the culture supernatant ofMDMs infected for3 to 4 weeks using NL4-3/BaL. Titers of virus particles werenormalized by their content of the capsid protein p24 asdetermined by a sandwich ELISA or by TCID50 as determinedby testing virus dilution performed on the TZM-BL reporter cellline (Derdeyn et al., 2000; Platt et al., 1998; Wei et al., 2002)(obtained through the NIH AIDS Repository Reagent Program;TZM-bl from Dr. John C. Kappes, Dr. Xiaoyun Wu andTranzyme, Inc.).

Recombinant galectins

Recombinant galectins were produced and purified as pre-viously described (Nieminen et al., 2007, 2005; Ouellet et al.,2005; Pelletier et al., 2003) with the following modifications ofthe protocol: lactose was first removed either by using Hiprep26/10 desalting column (GE Healthcare, Fairfield, Connecticut)or by extensive dialysis against PBS and the purified galectinswere then passed through Acticlean ETOX endotoxin removinggels (Sterogene, Carlsbad, California). The purified galectinpreparations were then sterilized by filtration through a 0.22 μmfilter. Mock preparations were also prepared using E. coliJM109 that does not express Gal-1 using the same purificationprotocol. The bioactivities of galectins were estimated by usinga hemagglutinin assay as described previously (Giguere et al.,2006a,b).

Infection assays

MDMs were infected for 1 h with HIV-1 (1×104 TCID50 ofNL4-3 Luc+Env- pseudotyped with JR-FL env) in the presenceof various concentrations of Gal-1 (ranging from 0 to 4 μM).Cells were then washed twice with cell culture medium andincubated for 5 days at 37 °C prior to lysis with 5× lysis buffer(125 mM Tris phosphate pH 7.8, 10 mM dithiothreitol, 5%Triton X-100 and 50% glycerol) before measuring luciferaseactivity as described previously (Ouellet et al., 1999). For someexperiments, 500 TCID50 of NL4-3/BaL produced by MDMswas used and p24 levels were estimated by a sandwich ELISA.Inhibition studies were performed using the fusion inhibitorsT-20 and TAK779 (obtained through the NIH AIDS Repos-itory Reagent Program).

Virus adsorption and entry assays

MDMs were incubated for 1 h at 37 °C with HIV-1 NL4-3/Bal (10 ng of p24) in the presence or absence of 2 μMGal-1 andthe fusion inhibitor T-20 or TAK779. Cells were then washedtwice with PBS prior to lysis with 5× lysis buffer (phosphate

buffered saline pH 7.2, 0.05% Tween-20, 2.5% Triton X-100and 0.02% thimerosal) and the p24 content associated withMDMs was estimated by ELISA. Entry assays were performedin a similar manner, except that MDMs were treated withtrypsin (Invitrogen, Carlsbad, CA) for 5 min at 37 °C to removeuninternalized viruses and washed twice with PBS prior to lysisas described previously (Tardif and Tremblay, 2005a,b).

LTR activation assay

MDMs were infected 3 days with HIV-1 (5 ng of NL4-3Luc+Env− pseudotyped with VSV-G env). Cells were thenwashed twice prior to adding of 2 μM Gal-1 for 1 to 24 h. Cellswere then lysed with 5× lysis buffer (125 mM Tris phosphatepH 7.8, 10 mM dithiothreitol, 5% Triton X-100 and 50%glycerol) before measuring luciferase activity as describedpreviously (Ouellet et al., 1999). Integrated LTR will respond toexternal stimuli and lead to a dose-dependent increase inluciferase gene transcription and, therefore, activity. TNF-α isused as a positive control. It is important to note that time courseexperiments were performed since gene integration requires an,as yet, undetermined amount of time.

Acknowledgments

We thank Ms. Sylvie Méthot and Julie Nieminen for editorialassistance. We also want to thank Dr. Jun Hirabayashi from theResearch Centre for Glycoscience (National Institute ofAdvanced Industrial Science and Technology, Tsukuba, Ibaraki,Japan) for giving us the Gal-1-encoding plasmid. This study wasmade possible by an operating grant to M.J.T. and S.S. from theCanadian Institutes of Health Research (CIHR) HIV/AIDSResearch Program (grant #HOP-75351). This work was per-formed by S.M. in partial fulfillment of a Ph.D. degree from theMicrobiology-Immunology Program, Faculty of Medicine,Laval University. S.M. and C.S.-P. each hold a CIHR DoctoralAward. M.J.T. is the recipient of a Tier 1 Canada Research Chairin Human Immuno-Retrovirology and S.S. holds a ScholarshipAward (senior level) from the Fonds de la Recherche en Santé duQuébec.

References

Akimoto, Y., Hirabayashi, J., Kasai, K., Hirano, H., 1995. Expression of theendogenous 14-kDa beta-galactoside-binding lectin galectin in normalhuman skin. Cell Tissue Res. 280 (1), 1–10.

Almkvist, J., Dahlgren, C., Leffler, H., Karlsson, A., 2002. Activation of theneutrophil nicotinamide adenine dinucleotide phosphate oxidase bygalectin-1. J. Immunol. 168 (8), 4034–4041.

Barrionuevo, P., Beigier-Bompadre, M., Ilarregui, J.M., Toscano, M.A., Bianco,G.A., Isturiz, M.A., Rabinovich, G.A., 2007. A novel function for galectin-1at the crossroad of innate and adaptive immunity: galectin-1 regulatesmonocyte/macrophage physiology through a nonapoptotic ERK-dependentpathway. J. Immunol. 178 (1), 436–445.

Baum, L.G., Pang, M., Perillo, N.L., Wu, T., Delegeane, A., Uittenbogaart, C.H.,Fukuda, M., Seilhamer, J.J., 1995. Human thymic epithelial cells express anendogenous lectin, galectin-1, which binds to core 2 O-glycans onthymocytes and T lymphoblastoid cells. J. Exp. Med. 181 (3), 877–887.

Blaser, C., Kaufmann, M., Muller, C., Zimmermann, C., Wells, V., Mallucci, L.,Pircher, H., 1998. Beta-galactoside-binding protein secreted by activated T

128 S. Mercier et al. / Virology 371 (2008) 121–129

cells inhibits antigen-induced proliferation of Tcells. Eur. J. Immunol. 28 (8),2311–2319.

Brumme, Z.L., Goodrich, J., Mayer, H.B., Brumme, C.J., Henrick, B.M.,Wynhoven, B., Asselin, J.J., Cheung, P.K., Hogg, R.S., Montaner, J.S.,Harrigan, P.R., 2005. Molecular and clinical epidemiology of CXCR4-usingHIV-1 in a large population of antiretroviral-naive individuals. J. Infect. Dis.192 (3), 466–474.

Camby, I., Le Mercier, M., Lefranc, F., Kiss, R., 2006. Galectin-1: a smallprotein with major functions. Glycobiology 16 (11), 137R–157R.

Cooper, D.N., Barondes, S.H., 1990. Evidence for export of a muscle lectin fromcytosol to extracellular matrix and for a novel secretory mechanism. J. CellBiol. 110 (5), 1681–1691.

Correa, S.G., Sotomayor, C.E., Aoki, M.P., Maldonado, C.A., Rabinovich,G.A., 2003. Opposite effects of galectin-1 on alternative metabolic pathwaysof l-arginine in resident, inflammatory, and activated macrophages.Glycobiology 13 (2), 119–128.

de Witte, L., Nabatov, A., Pion, M., Fluitsma, D., de Jong, M.A., de Gruijl, T.,Piguet, V., van Kooyk, Y., Geijtenbeek, T.B., 2007. Langerin is a naturalbarrier to HIV-1 transmission by Langerhans cells. Nat. Med. 13 (3), 367–371.

Derdeyn, C.A., Decker, J.M., Sfakianos, J.N., Wu, X., O'Brien, W.A., Ratner,L., Kappes, J.C., Shaw, G.M., Hunter, E., 2000. Sensitivity of humanimmunodeficiency virus type 1 to the fusion inhibitor T-20 is modulated bycoreceptor specificity defined by the V3 loop of gp120. J. Virol. 74 (18),8358–8367.

Dettin, L., Rubinstein, N., Aoki, A., Rabinovich, G.A., Maldonado, C.A., 2003.Regulated expression and ultrastructural localization of galectin-1, a pro-apoptotic beta-galactoside-binding lectin, during spermatogenesis in rattestis. Biol. Reprod. 68 (1), 51–59.

Dornadula, G., Zhang, H., Shetty, S., Pomerantz, R.J., 1999. HIV-1 virionsproduced from replicating peripheral blood lymphocytes are more infectiousthan those from nonproliferating macrophages due to higher levels ofintravirion reverse transcripts: implications for pathogenesis and transmis-sion. Virology 253 (1), 10–16.

Dumic, J., Dabelic, S., Flogel, M., 2006. Galectin-3: an open-ended story.Biochim. Biophys. Acta 1760 (4), 616–635.

Garin, M.I., Chu, C.C., Golshayan, D., Cernuda-Morollon, E., Wait, R., Lechler,R.I., 2007. Galectin-1: a key effector of regulation mediated by CD4+CD25+T cells. Blood 109 (5), 2058–2065.

Gauthier, L., Rossi, B., Roux, F., Termine, E., Schiff, C., 2002. Galectin-1 is astromal cell ligand of the pre-B cell receptor (BCR) implicated in synapseformation between pre-B and stromal cells and in pre-BCR triggering. Proc.Natl. Acad. Sci. U. S. A. 99 (20), 13014–13019.

Geijtenbeek, T.B., Kwon, D.S., Torensma, R., van Vliet, S.J., van Duijnhoven,G.C., Middel, J., Cornelissen, I.L., Nottet, H.S., KewalRamani, V.N.,Littman, D.R., Figdor, C.G., van Kooyk, Y., 2000. DC-SIGN, a dendriticcell-specific HIV-1-binding protein that enhances trans-infection of T cells.Cell 100 (5), 587–597.

Geijtenbeek, T.B., van Vliet, S.J., Engering, A., t Hart, B.A., van Kooyk, Y.,2004. Self- and nonself-recognition by C-type lectins on dendritic cells.Annu. Rev. Immunol. 22, 33–54.

Giguere, D., Patnam, R., Bellefleur, M.A., St-Pierre, C., Sato, S., Roy, R.,2006a. Carbohydrate triazoles and isoxazoles as inhibitors of galectins-1 and-3. Chem. Commun. (Camb.) 22, 2379–2381.

Giguere, D., Sato, S., St-Pierre, C., Sirois, S., Roy, R., 2006b. Aryl O- and S-galactosides and lactosides as specific inhibitors of human galectins-1 and-3: role of electrostatic potential at O-3. Bioorg. Med. Chem. Lett. 16 (6),1668–1672.

He, J., Baum, L.G., 2004. Presentation of galectin-1 by extracellular matrixtriggers T cell death. J. Biol. Chem. 279 (6), 4705–4712.

Hirabayashi, J., Kasai, K., 1993. The family of metazoan metal-independentbeta-galactoside-binding lectins: structure, function and molecular evolu-tion. Glycobiology 3 (4), 297–304.

Hughes, R.C., 1999. Secretion of the galectin family of mammaliancarbohydrate-binding proteins. Biochim. Biophys. Acta 1473 (1), 172–185.

Hughes, R.C., 2001. Galectins as modulators of cell adhesion. Biochimie 83 (7),667–676.

Jeschke, U., Reimer, T., Bergemann, C., Wiest, I., Schulze, S., Friese, K.,Walzel, H., 2004. Binding of galectin-1 (gal-1) on trophoblast cells and

inhibition of hormone production of trophoblast tumor cells in vitro by gal-1.Histochem. Cell Biol. 121 (6), 501–508.

Kohatsu, L., Hsu, D.K., Jegalian, A.G., Liu, F.T., Baum, L.G., 2006. Galectin-3induces death of Candida species expressing specific beta-1,2-linkedmannans. J. Immunol. 177 (7), 4718–4726.

Koot, M., van 't Wout, A.B., Kootstra, N.A., de Goede, R.E., Tersmette, M.,Schuitemaker, H., 1996. Relation between changes in cellular load, evo-lution of viral phenotype, and the clonal composition of virus populations inthe course of human immunodeficiency virus type 1 infection. J. Infect. Dis.173 (2), 349–354.

Marechal, V., Prevost, M.C., Petit, C., Perret, E., Heard, J.M., Schwartz, O.,2001. Human immunodeficiency virus type 1 entry into macrophagesmediated by macropinocytosis. J. Virol. 75 (22), 11166–11177.

Margolis, L., Shattock, R., 2006. Selective transmission of CCR5-utilizingHIV-1: the ‘gatekeeper’ problem resolved? Nat. Rev., Microbiol. 4 (4),312–317.

Meng, G., Wei, X., Wu, X., Sellers, M.T., Decker, J.M., Moldoveanu, Z.,Orenstein, J.M., Graham, M.F., Kappes, J.C., Mestecky, J., Shaw, G.M.,Smith, P.D., 2002. Primary intestinal epithelial cells selectively transfer R5HIV-1 to CCR5+ cells. Nat. Med. 8 (2), 150–156.

Mey, A., Leffler, H., Hmama, Z., Normier, G., Revillard, J.P., 1996. The animallectin galectin-3 interacts with bacterial lipopolysaccharides via twoindependent sites. J. Immunol. 156 (4), 1572–1577.

Neil, S.J., McKnight, A., Gustafsson, K., Weiss, R.A., 2005. HIV-1 incorporatesABO histo-blood group antigens that sensitize virions to complement-mediated inactivation. Blood 105 (12), 4693–4699.

Nickel, W., 2003. The mystery of nonclassical protein secretion. A current viewon cargo proteins and potential export routes. Eur. J. Biochem. 270 (10),2109–2119.

Nickel, W., 2005. Unconventional secretory routes: direct protein export acrossthe plasma membrane of mammalian cells. Traffic 6 (8), 607–614.

Nieminen, J., Kuno, A., Hirabayashi, J., Sato, S., 2007. Visualization ofgalectin-3 oligomerization on the surface of neutrophils and endothelial cellsusing fluorescence resonance energy transfer. J. Biol. Chem. 282 (2),1374–1383.

Nieminen, J., St-Pierre, C., Sato, S., 2005. Galectin-3 interacts with naive andprimed neutrophils, inducing innate immune responses. J. Leukoc. Biol. 78(5), 1127–1135.

Noursadeghi, M., Katz, D.R., Miller, R.F., 2006. HIV-1 infection ofmononuclear phagocytic cells: the case for bacterial innate immunedeficiency in AIDS. Lancet, Infect. Dis. 6 (12), 794–804.

Ohtsubo, K., Marth, J.D., 2006. Glycosylation in cellular mechanisms of healthand disease. Cell 126 (5), 855–867.

Oppenheim, J.J., Tewary, P., de la Rosa, G., Yang, D., 2007. Alarmins initiatehost defense. Adv. Exp. Med. Biol. 601, 185–194.

Ouellet, M., Barbeau, B., Tremblay, M.J., 1999. p56(lck), ZAP-70, SLP-76, andcalcium-regulated effectors are involved in NF-kappaB activation bybisperoxovanadium phosphotyrosyl phosphatase inhibitors in human Tcells. J. Biol. Chem. 274 (49), 35029–35036.

Ouellet, M., Mercier, S., Pelletier, I., Bounou, S., Roy, J., Hirabayashi, J., Sato,S., Tremblay, M.J., 2005. Galectin-1 acts as a soluble host factor thatpromotes HIV-1 infectivity through stabilization of virus attachment to hostcells. J. Immunol. 174 (7), 4120–4126.

Pelletier, I., Hashidate, T., Urashima, T., Nishi, N., Nakamura, T., Futai, M.,Arata, Y., Kasai, K., Hirashima, M., Hirabayashi, J., Sato, S., 2003. Specificrecognition of Leishmania major poly-beta-galactosyl epitopes by galectin-9: possible implication of galectin-9 in interaction between L. major andhost cells. J. Biol. Chem. 278 (25), 22223–22230.

Perillo, N.L., Pace, K.E., Seilhamer, J.J., Baum, L.G., 1995. Apoptosis of T cellsmediated by galectin-1. Nature 378 (6558), 736–739.

Platt, E.J., Wehrly, K., Kuhmann, S.E., Chesebro, B., Kabat, D., 1998. Effects ofCCR5 and CD4 cell surface concentrations on infections by macrophage-tropic isolates of human immunodeficiency virus type 1. J. Virol. 72 (4),2855–2864.

Rabinovich, G.A., Gruppi, A., 2005. Galectins as immunoregulators duringinfectious processes: from microbial invasion to the resolution of thedisease. Parasite Immunol. 27 (4), 103–114.

Rabinovich, G., Castagna, L., Landa, C., Riera, C.M., Sotomayor, C., 1996.

129S. Mercier et al. / Virology 371 (2008) 121–129

Regulated expression of a 16-kd galectin-like protein in activated ratmacrophages. J. Leukoc. Biol. 59 (3), 363–370.

Rabinovich, G.A., Iglesias, M.M., Modesti, N.M., Castagna, L.F., Wolfenstein-Todel, C., Riera, C.M., Sotomayor, C.E., 1998. Activated rat macrophagesproduce a galectin-1-like protein that induces apoptosis of T cells: bio-chemical and functional characterization. J. Immunol. 160 (10), 4831–4840.

Rabinovich, G.A., Baum, L.G., Tinari, N., Paganelli, R., Natoli, C., Liu, F.T.,Iacobelli, S., 2002a. Galectins and their ligands: amplifiers, silencers ortuners of the inflammatory response? Trends Immunol. 23 (6), 313–320.

Rabinovich, G.A., Rubinstein, N., Toscano, M.A., 2002b. Role of galectins ininflammatory and immunomodulatory processes. Biochim. Biophys. Acta1572 (2–3), 274–284.

Rabinovich, G.A., Liu, F.T., Hirashima, M., Anderson, A., 2007. An emergingrole for galectins in tuning the immune response: lessons from experimentalmodels of inflammatory disease, autoimmunity and cancer. Scand. J.Immunol. 66 (2–3), 143–158.

Reynolds, J.L., Mahajan, S.D., Sykes, D.E., Schwartz, S.A., Nair, M.P., 2007.Proteomic analyses of methamphetamine (METH)-induced differentialprotein expression by immature dendritic cells (IDC). Biochim. Biophys.Acta 1774 (4), 433–442.

Sato, S., 2002. Galectin as a molecule of danger signal, which could evokeimmune response to infection. Trends Glycosci. Glycotechnol. 14 (79),285–301.

Sato, S., Burdett, I., Hughes, R.C., 1993. Secretion of the baby hamster kidney30-kDa galactose-binding lectin from polarized and nonpolarized cells: apathway independent of the endoplasmic reticulum-Golgi complex. Exp.Cell Res. 207 (1), 8–18.

Sato, S., Hughes, R.C., 1994. Regulation of secretion and surface expression ofMac-2, a galactoside-binding protein of macrophages. J. Biol. Chem. 269(6), 4424–4430.

Sato, S., Nieminen, J., 2004. Seeing strangers or announcing “danger”: galectin-3 in two models of innate immunity. Glycoconj. J. 19 (7–9), 583–591.

Stillman, B.N., Hsu, D.K., Pang, M., Brewer, C.F., Johnson, P., Liu, F.T., Baum,L.G., 2006. Galectin-3 and galectin-1 bind distinct cell surface glycoproteinreceptors to induce T cell death. J. Immunol. 176 (2), 778–789.

Tardif, M.R., Tremblay, M.J., 2005a. LFA-1 is a key determinant for preferentialinfection of memory CD4+ T cells by human immunodeficiency virus type1. J. Virol. 79 (21), 13714–13724.

Tardif, M.R., Tremblay, M.J., 2005b. Regulation of LFA-1 activity throughcytoskeleton remodeling and signaling components modulates the efficiencyof HIV type-1 entry in activated CD4+ T lymphocytes. J. Immunol. 175 (2),926–935.

Tremblay, M.J., Fortin, J.F., Cantin, R., 1998. The acquisition of host-encodedproteins by nascent HIV-1. Immunol. Today 19 (8), 346–351.

Turville, S.G., Cameron, P.U., Handley, A., Lin, G., Pohlmann, S., Doms, R.W.,Cunningham, A.L., 2002. Diversity of receptors binding HIV on dendriticcell subsets. Nat. Immunol. 3 (10), 975–983.

Turville, S.G., Santos, J.J., Frank, I., Cameron, P.U., Wilkinson, J., Miranda-Saksena, M., Dable, J., Stossel, H., Romani, N., Piatak Jr., M., Lifson, J.D.,Pope, M., Cunningham, A.L., 2004. Immunodeficiency virus uptake,turnover, and 2-phase transfer in human dendritic cells. Blood 103 (6),2170–2179.

van den Berg, T.K., Honing, H., Franke, N., van Remoortere, A., Schiphorst, W.E., Liu, F.T., Deelder, A.M., Cummings, R.D., Hokke, C.H., van Die, I.,2004. LacdiNAc-glycans constitute a parasite pattern for galectin-3-mediatedimmune recognition. J. Immunol. 173 (3), 1902–1907.

Verani, A., Gras, G., Pancino, G., 2005. Macrophages and HIV-1: dangerousliaisons. Mol. Immunol. 42 (2), 195–212.

Vidricaire, G., Tremblay, M.J., 2005. Rab5 and Rab7, but not ARF6, govern theearly events of HIV-1 infection in polarized human placental cells.J. Immunol. 175 (10), 6517–6530.

Wei, X., Decker, M., Liu, H., Zhang, Z., Arani, R.B., Kilby, J.M., Saag, M.S.,Wu, X., Shaw, G.M., Kappes, J.C., 2002. Emergence of resistant humanimmunodeficiency virus type 1 in patients receiving fusion inhibitor (T-20)monotherapy. Antimicrob. Agents Chemother. 46 (6), 1896–1905.

Zhang, Y.J., Dragic, T., Cao, Y., Kostrikis, L., Kwon, D.S., Littman, D.R.,KewalRamani, V.N., Moore, J.P., 1998. Use of coreceptors other than CCR5by non-syncytium-inducing adult and pediatric isolates of human immuno-deficiency virus type 1 is rare in vitro. J. Virol. 72 (11), 9337–9344.

Zuniga, E., Gruppi, A., Hirabayashi, J., Kasai, K.I., Rabinovich, G.A., 2001a.Regulated expression and effect of galectin-1 on Trypanosoma cruzi-infected macrophages: modulation of microbicidal activity and survival.Infect. Immun. 69 (11), 6804–6812.

Zuniga, E., Rabinovich, G.A., Iglesias, M.M., Gruppi, A., 2001b. Regulatedexpression of galectin-1 during B-cell activation and implications for T-cellapoptosis. J. Leukoc. Biol. 70 (1), 73–79.