Lecitina de Bananas

A Lectin Isolated from Bananas Is a Potent Inhibitor ofHIV Replication*

Received for publication, June 24, 2009, and in revised form, January 15, 2010 Published, JBC Papers in Press, January 15, 2010, DOI 10.1074/jbc.M109.034926

Michael D. Swanson‡, Harry C. Winter§, Irwin J. Goldstein§, and David M. Markovitz‡¶�1

From the ¶Department of Internal Medicine, Division of Infectious Diseases, ‡Program in Immunology, �Cellular and MolecularBiology Program, and §Department of Biological Chemistry, University of Michigan Medical Center, Ann Arbor, Michigan 48109

BanLec is a jacalin-related lectin isolated from the fruit ofbananas, Musa acuminata. This lectin binds to high mannosecarbohydrate structures, including those found on viruses con-taining glycosylated envelope proteins such as human immuno-deficiency virus type-1 (HIV-1). Therefore, we hypothesizedthat BanLec might inhibit HIV-1 through binding of the glyco-sylated HIV-1 envelope protein, gp120. We determined thatBanLec inhibits primary and laboratory-adapted HIV-1 isolatesof different tropisms and subtypes. BanLec possesses potentanti-HIV activity, with IC50 values in the lownanomolar to pico-molar range. The mechanism for BanLec-mediated antiviralactivity was investigated by determining if this lectin candirectly bind the HIV-1 envelope protein and block entry of thevirus into the cell.Anenzyme-linked immunosorbent assay con-firmed direct binding of BanLec to gp120 and indicated thatBanLec can recognize the highmannose structures that are rec-ognized by the monoclonal antibody 2G12. Furthermore, Ban-Lec is able to block HIV-1 cellular entry as indicated by temper-ature-sensitive viral entry studies and by the decreased levels ofthe strong-stop product of early reverse transcription seen inthe presence of BanLec. Thus, our data indicate that BanLecinhibits HIV-1 infection by binding to the glycosylated viralenvelope and blocking cellular entry. The relative anti-HIVactivity of BanLec compared favorably to other anti-HIV lectins,such as snowdrop lectin and Griffithsin, and to T-20 and mara-viroc, two anti-HIV drugs currently in clinical use. Based onthese results, BanLec is a potential component for an anti-viralmicrobicide that could be used to prevent the sexual transmis-sion of HIV-1.

Despite the development of more than 25 approved anti-HIV2 drugs and improvements in the availability of antiretro-viral drugs in low andmiddle income countries, the rate of newHIV-1 infections is outpacing the rate of new individuals

receiving antiretroviral therapy by 2.5:1 (1). At present, itappears that an efficaciousHIV vaccine is still many years away.Therefore, other methods for halting the spread of HIV arevitally needed. This has raised the possibility of developingeither intravaginally or intrarectally applied microbicides tohalt the spread of HIV during sexual intercourse. This type ofintervention is particularly needed in the developing world,such as sub-Saharan Africa, wheremore than 20million peopleare living with HIV/AIDS (1). Although abstinence has beensuggested by some groups, campaigns to encourage thismethod of halting transmission have not been effective (2).Although condoms are quite effective against the spread ofHIVand some other sexually transmitted diseases, they are onlyeffective if they are used consistently and correctly, which isoften not the case (3, 4). This is particularly true in the devel-oping world, where women have relatively little control oversexual encounters and, thus, have not been able to enforce con-dom usage (5), so the development of a long-lasting, self-ap-plied, microbicide is very attractive. In fact, it is estimated that20% coverage with a microbicide that is only 60% effectiveagainst HIV may prevent up to 2.5 million HIV infections overthree years (6). Therefore, even modest success with microbi-cides could save millions of lives.Some of the most promising compounds for inhibiting vagi-

nal or rectal HIV transmission are agents that blockHIV beforeintegration of the viral genome into the target cell. Thus, theviral entry step is one potential target for a microbicide. Entryinhibitors that have been proposed for use in a vaginal micro-bicide include long chain and ionic polymers (such as Pro 2000)as well as dendrimers, lipidmembranemodifiers, and anti-CD4antibodies. HIV-binding peptides and small molecule inhibi-tors have also been considered, including the fusion inhibitorT-20 (enfuvirtide) and the CCR5 blocker maraviroc, which arealready in clinical use for the treatment of HIV infection. Inaddition, lectins are a growing class of HIV-1 inhibitors underconsideration as microbicide candidates (7, 8). Lectins inhibitHIV-1 entry by binding to carbohydrate structures found onthe viral envelope. Examples of anti-HIV lectins include Cya-novirin-N (CV-N) (9), Griffithsin (GRFT) (10), and snowdroplectin (GNA) (11–13).The HIV-1 envelope protein gp120 contains 20–30 possible

N-linked glycosylation sites. These carbohydrate structuresmake up �50% of the molecular weight of the protein (14–16).Glycosylation affects aspects of the viral life cycle includingprotein folding (17), cellular transport, binding to cellularreceptors (14, 18, 19), trans-infection by dendritic cells (19),and shielding from the immune response (20). Because glyco-

* This work was supported, in whole or in part, by National Institutes of HealthGrant R01 AI062248 (of D. M. M.). This work was also supported by a Bur-roughs Wellcome Fund Clinical Scientist Award in Translational Research.

1 To whom correspondence should be addressed: University of Michigan Medi-cal Center, 5220 MSRB III, 1150 West Medical Center Dr., Ann Arbor, MI 48109-5640. Tel.: 734-647-1786; Fax: 734-764-0101; E-mail: [email protected].

2 The abbreviations used are: HIV, human immunodeficiency virus; ELISA,enzyme-linked immunosorbent assay; GRFT, Griffithsin; HEK-293 cells,human embryonic kidney cells; PBL, peripheral blood lymphocytes; PBMC,peripheral blood mononuclear cell; MDM, monocyte-derived macro-phages; MTT, (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bro-mide; PBS, phosphate-buffered saline; TCID50, tissue culture infective dose50%.

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 285, NO. 12, pp. 8646 –8655, March 19, 2010© 2010 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A.

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sylation is essential to the virus, it presents an attractive thera-peutic target.The lectin termed BanLec, isolated from the ripened fruit of

the banana (Musa acuminata cultivars), exists as a dimerwith amolecular mass of �30 kDa (21). It is a member of the jacalin-related lectin family and can recognize high mannose struc-tures (22–24). Lectins in this family are characterized by thepresence of a�-prism 1 structure composed of three Greek Keyturn motifs. Greek Keys 1 and 2 are both involved in bindingcarbohydrates and contain a GXXXD binding motif, whereasKey 3 does not contain the bindingmotif (25, 26). However, thisloop can assist ligand binding and determine lectin specificity(27). Because of its affinity for highmannose structures (15), wesought to investigate whether BanLecmight bind themannose-rich envelope of HIV-1 and thereby block HIV infection. Theresults presented below demonstrate that BanLec is a potentinhibitor of HIV infection that markedly reduces the replica-tion of a range of HIV-1 isolates and has potential to be furtherdeveloped for use as a vaginal microbicide.

MATERIALS AND METHODS

Proteins and Anti-HIV Compounds—BanLec was isolatedfrom bananas bymodification of previously describedmethods(21, 22).3 Snowdrop lectin (GNA) was isolated from crudeextracts of snowdrop bulbs and purified over a mannose-aga-rose column as described previously (28). Recombinant HIV-1gp120, human monoclonal antibody 2G12, recombinant His-tagged GRFT, T-20, maraviroc, and CD4-IgG2 were obtainedfrom the NIH AIDS Research and Reference Reagent Program(10). Recombinant, glycosylated gp120 was produced in HEK-293 cells (human embryonic kidney cells), the recombinanthuman antibody 2G12 was produced in Chinese hamster ovarycells, and recombinant GRFT was produced in Escherichia coli.The purity of all of the proteins was found to be�95% as deter-mined by SDS-PAGE.HIV-1 Production—The HIV-1 isolate Bru was produced in

peripheral blood lymphocytes (PBL), whereas the HIV-1 BaL(29) isolatewas produced inmacrophages. Primary, dual-tropicisolates ASM44 andASM54were expanded in peripheral bloodmononuclear cells (PBMCs) containing both lymphocytes andmacrophages. Production of pseudotyped HIV-1 was per-formed by transfecting HEK-293FT cells (a fast growing cellline derived fromHEK-293 that contains the SV40 large T anti-gen) with plasmids coding for an HIV-1 envelope from eithersubtype B (30) or C along with an envelope-deleted proviralclone, pSG3�env (31). Proviral plasmid DNA clones pNL4-3(32), pNL(AD8) (33), p81A-4 (34–36), and p89.6 (37) weretransfected into HEK-293FT cells with Lipofectamine 2000(Invitrogen). The media were changed 24 h post-transfection,and at 48 h post-transfection the supernatants were harvestedand frozen at �80 °C. The concentration of virus in the stockswas determined by the HIV-1 p24 Antigen Capture AssayELISA (AIDS andCancerVirus Program) or by determining theinfectious titer. HIV-1 Bru was treated with 10 units/�l ofRNase-free DNase I (Roche Applied Science) before use in the

experiments in which the products of early reverse transcrip-tion were assayed.HIV-1 Indicator Assays—HIV-1 infection was quantified

using TZM-bl cells, which express a luciferase and �-galacto-sidase gene under the control of the HIV-1 LTR promoter (31,38–40). The day before infection of 5000 TZM-bl cells/ml in100 �l of Dulbecco’s modified Eagle’s medium containing 10%fetal bovine serum, 25 mM HEPES, and 50 �g/ml Geneticinwere added to the wells of white, opaque, 96-well tissue cultureplates (Falcon). Cells were pretreated with BanLec for 30 minbefore infection with 100 TCID50 units of virus (�15,000 rela-tive luminescence units) to a final volume of 200 �l/well. Cellswere exposed to virus and lectin for either 2 days with replica-tion competent viruses or for 3 days with pseudotyped, replica-tion defective virus. A Steady-Glo� luciferase assay system(Promega) and a plate reader containing a luminometer(Tecan) were used to measure luminescence, which was indic-ative of viral infection.MAGI-CCR5 cells (41, 42) were plated in 24-well tissue cul-

ture plates with 40,000 cells per well in Dulbecco’s modifiedEagle’s medium containing 10% fetal bovine serum, penicillin,and streptomycin. The cells were pretreated with lectin for 30min and then infected with different viral isolates at concentra-tions that yielded �100 positively infected cells per well. Fortyhours post-infection, cells were stained for �-galactosidaseactivity as described within the reagent data sheet, and positivecells were counted visually.Isolation and Culture of Primary Cells—PBMCs were iso-

lated from healthy donors by venipuncture. Briefly, blood wasdrawn into a 60-ml syringe containing 7 ml of 250 mM sodiumcitrate and 10 ml of 6% dextran solution and mixed by inver-sion. After 30 min, to allow for the sedimentation of red bloodcells, the supernatant was separated using Hypaque-Ficoll, andthe buffy coat layer was removed, washed twice with cold PBScontaining 0.2% bovine serum albumin, and centrifuged at350 � g for 10 min. The cell pellet was resuspended in RPMI1640 media at a concentration of 5 � 106 cells per ml andseeded into non-tissue culture-treated plates. PBL wereremoved from the adherentmonocytes andwashed three timeswith PBS. For the differentiation of monocytes to macrophagesfor HIV-1 infection, the monocytes were cultured with Iscove’smodified Dulbecco’s media containing 10% heat-inactivatedhuman AB sera for 7 days.Infection of Monocyte-derived Macrophages (MDM)—MDM

were washed with PBS three times followed by the addition offresh media containing BanLec or PBS 30 min before infection.Cells were infectedwith�100TCID50 ofNL(AD8) for 24 h, andthe residual viruswas removed by three PBSwashes followed bythe addition of freshmedia. Every 3 days, a samplewas removedand replaced with fresh media containing the appropriateamount of BanLec for 15 days. The samples were stored at�80 °C until viral replication was determined by theHIV-1 p24Antigen Capture Assay ELISA (AIDS and Cancer Virus Pro-gram). A similar experiment was done in which samples werenot removed until the end of the experiment on day 7. For bothexperiments, an MTT ((3-(4,5-Dimethylthiazol-2-yl)-2,5-di-phenyltetrazolium bromide) reduction assay was performed onthe final day to assess cellular viability.3 H. C. Winter, K. A. Wearne, and I. J. Goldstein, manuscript in preparation.

Inhibition of HIV-1 Replication by BanLec

MARCH 19, 2010 • VOLUME 285 • NUMBER 12 JOURNAL OF BIOLOGICAL CHEMISTRY 8647


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Detection of Early Products of HIV-1 Reverse Transcription(Strong-Stop DNA) in Peripheral Blood Lymphocytes—PBLwere stimulated with phytohemagglutinin for 3 days in RPMImedia containing 10% heat-inactivated fetal bovine serum andinterleukin-2. The cellswerewashedwith PBS and resuspendedin RPMI media containing 10% heat-inactivated fetal bovineserum. Lectins were added 30 min before centrifuge-mediatedinfection (spin-infection) with DNase I-treatedHIV-1 Bru (43).Three hours post-infection the cells were harvested, washedwith PBS, and then stored at �80 °C. Cellular DNA, includinggenomic and viral DNA products, was isolated using theQIAamp DNA Blood Mini kit (Qiagen). Strong-stop DNA,the first product of HIV-1 reverse transcription, is used for theassessment of viral entry (13, 44). This reverse transcriptionproduct was quantified by performing real-time PCR withprimers specific for strong-stop DNA, the DNA concentrationof the each samplewas normalized, and equal DNA loadingwasconfirmed with primers for �-tubulin (44).Determination of BanLec and Glycosylated HIV-1 gp120

Interaction by ELISA—96-Well ELISA plates were coated byadding 50 �l of 5 �g/ml BanLec per well and incubated over-night at room temperature. The next day plates were blockedfor 1.5 h at room temperature with PBS containing 1% bovineserum albumin, 5% sucrose, and 0.05% sodium azide and thenrinsed with wash buffer (PBS containing 0.05% Tween 20, pH7.4) 3 times before the addition of recombinant, glycosylatedgp120 protein diluted in blocking buffer. After a 1-h incubationat room temperature, the plates were washed 3 times before theaddition of the detection antibodies. A sheep anti-gp120 anti-body (AIDS Research and Reference Reagent Program) wasdiluted 1:2000 in dilution buffer (wash buffer containing 0.1%bovine serum albumin) and added to the wells and incubatedfor 1 h. The platewaswashed again before a 1-h incubationwithan anti-sheep antibody conjugated to alkaline phosphatase(Sigma) diluted 1:40,000 in dilution buffer. After the plate waswashed, p-nitrophenyl phosphate (Sigma) was added for color-imetric analysis, and the absorbance was measured at 405 nm.To determinewhether BanLec could block the recognition of

gp120 by the anti-HIV monoclonal antibody 2G12, ELISAplates were coated overnight with 100 �l of a 1 �g/ml solutionof recombinant gp120 diluted in PBS. The plates were blockedas described above, washed, and then treated with serial dilu-tions of BanLec in dilution buffer. After a 1-h incubation theplates were washed to remove unbound BanLec. The 2G12antibodywas added at a concentration of 100 ng/ml to allow forbinding to the gp120 protein. One hour later the plates werewashed and incubated with biotinylated anti-human antibody(Jackson ImmunoResearch) followed by another series ofwashes and the addition of streptavidin-conjugated alkalinephosphatase (Jackson ImmunoResearch). The alkaline phos-phatase dilution buffer contained 500 mM methyl-�-D-man-nopyranoside, which was added to reduce any potential non-specific binding of the 2G12 antibody to BanLec. The plateswere washed again, and the substrate p-nitrophenyl phosphate(Sigma) was added for colorimetric analysis of 2G12 binding.The amount of antibody that remained bound was quantifiedby comparison to a standard curve consisting of serial 2-folddilutions of 2G12.

Determination of Anti-HIVActivity Post-cellular Attachment—For measuring the inhibition of HIV-1 infection post-cellularattachment, TZM-bl cells were plated in 96-well plates asdescribed above and spin-infected with a pseudotyped viruswith pConCgp160-opt, a consensus subtype C envelopesequence, at 1250� g for 2 h at 16 °C 1 day after plating (43, 45).The cells were placed on ice, the supernatant was removed, andunbound virus was removed by washing 2 times with PBS con-taining 0.2% bovine serum albumin. Cell culture media con-taining inhibitors (CD4-IgG2, T-20, maraviroc, or BanLec) wasadded to the cells and incubated for 30 min on ice. The cellswere then moved to a 37 °C incubator, and luciferase activitywas measured 3 days later. These results were compared withcells that were infectedwith virus that had been pretreatedwiththe same inhibitors described above on ice for 30 min and thenincubated at 37 °C (i.e. standard infection conditions).

RESULTS

BanLec Is a Potent Inhibitor of Multiple HIV-1 Isolates—Todetermine the anti-HIV activity of BanLec, different concentra-tions of the lectin were incubated with TZM-bl indicator cellsbefore infection with various HIV-1 isolates. Because a micro-

FIGURE 1. BanLec has antiviral activity against multiple HIV-1 isolateswith different tropisms. A, TZM-bl cells were pretreated with different con-centrations of BanLec before infection with the R5 tropic isolates NL(AD8) and81A-4, dual tropic 89.6, and X4 tropic NL4-3. Forty-eight hours after exposureto virus, luciferase activity was determined by measuring relative lumines-cent units (RLU). The averages from three separate experiments were used forthe calculation of IC50 values, which were determined by nonlinear regres-sion. The IC50 for viral inhibition were as follows: NL(AD8) � 2.06 nM, 81A-4 �0.69 nM, 89.6 � 0.48 nM, NL4-3 � 0.49 nM. B, Magi-CCR5 indicator cells wereused to determine anti-viral activity of BanLec against multiple strains ofHIV-1. Forty hours after exposure to virus, infected cells were quantified bystaining for �-galactosidase activity. Infectivity of BanLec-treated virus is pre-sented as a percent of positively infected cells as compared with the PBScontrol. Error bars represent S.D. from three separate experiments.




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bicide would need to inhibit HIV-1 of different tropisms andbecause glycosylation can play a role in determining viral tro-pism (18), we tested the ability of BanLec to inhibit severaldifferent HIV-1 isolates. The viral clones 81A-4 and NL(AD8)are both derivatives ofNL4-3 inwhich a portion of the envelopeis swapped with the envelope region from either the R5 HIV-1isolates BaL or ADA, respectively. 81A-4 and NL(AD8) useCCR5 as a cellular co-receptor (R5 tropic), whereas NL4-3 usesCXCR4 (X4 tropic). These isolates allow for the assessment ofdifferent HIV-1 envelope sensitivity to BanLec while keepingthe remainder of the NL4-3 viral components unchanged. Thedual-tropic isolate 89.6 was also assessed for susceptibility toBanLec. We observed dose-dependent inhibition of viral infec-tion with IC50 values calculated in the low nanomolar rangeagainst viral isolates with different tropisms (Fig. 1A). Theseresults suggest that sensitivity of HIV-1 isolates to BanLec isindependent of viral tropism.We further confirmed the anti-HIV activity of BanLec with

the HIV-1 indicator cell line, MAGI-CCR5. With this cell line,we tested the ability of BanLec to inhibit infection by the labo-ratory-adapted isolates BaL (R5) and Bru (X4) and the primaryisolates ASM 44 (R5X4) and ASM 54 (R5X4) and determinedthat all were inhibited byBanLec (Fig. 1B). The virus used in this

experiment was generated by infection of PBMC, whereas theexperiment shown in Fig. 1A used virus produced by transfec-tion of HEK-293FT cells with a proviral plasmid clone. Theseresults further support our initial studies by showing thatBanLec can inhibit HIV isolates both independent of viral tro-pism and of the cell type used to produce virus.

FIGURE 2. BanLec inhibits infection of HIV-1 pseudotyped with envelopesfrom multiple primary isolates. TZM-bl cells were infected with HIV-1pseudotyped with primary HIV-1 envelope proteins from subtype B (A) andsubtype C (B) in the presence of different concentrations of BanLec. Forty-eight hours later, luciferase activity was assessed. The IC50 values were deter-mined as in Fig. 1 and are shown in Table 1. Results shown are the average ofthree independent experiments, and error bars represent the S.D. RLU, rela-tive luminescent units.

FIGURE 3. BanLec inhibits HIV-1 infection of MDM. A, MDM were pretreatedwith BanLec for 30 min before the addition of 100 TCID50 of HIV-1 NL(AD8).Twenty-four hours later, the media was removed, and the cells were washedwith PBS to eliminate remaining virus. Fresh media containing BanLec or PBSwas added to the cells. A sample of culture supernatant was taken every 3days for p24 quantification by ELISA and replaced with new media containinglectin in PBS or PBS alone as a control. On day 15, viability was assessed by anMTT assay, which indicated no cellular toxicity (data not shown). B, MDM werepretreated and infected with HIV-1 as described above. 24 h post-infectionthe cells were washed with PBS to remove residual virus and cultured inmedia containing BanLec or PBS. Seven days post-infection, supernatantswere removed for determination of p24 antigen as detected by ELISA. Theconcentration for a 50% reduction in p24 production was calculated to be9.72 nM. Cellular viability was assessed by an MTT assay, and no toxicity wasobserved (data not shown). Results shown in panels A and B are representa-tive of three and two separate experiments, respectively.

TABLE 1Summary of the calculated IC50 values for BanLec inhibition of HIV-1pseudotyped with HIV-1 envelopes from subtypes B and C

Subtype B Subtype C

Envelope IC5095% Confidence

intervals Envelope IC5095% Confidence

intervals

nM nMSVPB5 0.30 0.27–0.34 SVPC3 0.57 0.51–0.63SVPB6 0.85 0.74–0.98 SVPC5 0.30 0.21–0.42SVPB11 0.71 0.65–0.77 SVPC6 0.28 0.25–0.31SVPB17 0.33 0.26–0.41 SVPC7 2.3 1.8–2.9




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R5 tropic viruses are the dominant form found in sexuallytransmittedHIV-1 and, therefore, would have to be neutralizedby microbicides. In addition, anti-HIV microbicides will needto inhibit infection by viral isolates from different subtypes.Although onewould assume that all clades could be neutralizeddue to the conservation of gp120 glycosylation, a difference inthe susceptibility of the viral subtypes B and C to the anti-HIV,high mannose-recognizing antibody 2G12 has been observed(46). To determine whether BanLec could inhibit additionalprimary isolates from different clades, we tested BanLec forinhibition of HIV-1 pseudotyped with envelopes derived fromprimary isolates of subtypes B and C. These subtypes are com-monly found in North and Central America (subtype B) andparts of Africa and India (subtype C).We observed potent, sub-nanomolar inhibition of viral replication by BanLec (Fig. 2 andTable 1), and no significant difference was observed when theaverage IC50 values from the two different subtypes were com-pared by Student’s t test (p � 0.56). This suggests that BanLeccan effectively inhibit infection by viral isolates prominent inregions where a microbicide would be most valuable.BanLec Blocks Infection of MDM—Macrophages are suscep-

tible to HIV-1 infection and can become viral reservoirs thatcannot be eliminated by highly active antiretroviral therapy.The role of vaginalmacrophages inHIV-1 pathogenesis has notbeen fully characterized, but recent evidence indicates thatthese cells are permissive for HIV-1 infection (47). We testedthe ability of BanLec to inhibit HIV-1 infection of MDM. Asshown in Fig. 3A, nanomolar concentrations of BanLec inhib-ited HIV replication inMDMover a period of 15 days. Further-more, BanLec had no effect on cellular viability as determinedbyMTTassay performed on day 15 (data not shown); therefore,this effect was not due to cellular toxicity. When BanLecremained in the culture supernatant for 7 days without chang-

ing the media or adding additionallectin, the IC50 value for BanLecinhibition of HIV-1 replication was9.72 nM (Fig. 3B), demonstratingthat BanLec remains a potent andstable inhibitor in a long term cul-ture system at 37 °C.BanLec Appears to Block HIV-1

Infection at the Viral Entry Step—Having shown that BanLec caninhibit HIV replication inMDM,wetested the ability of BanLec to blockcellular entry of HIV-1 in PBL. Wehypothesized that BanLec binds tohigh mannose structures found onthe HIV-1 envelope, preventingentry and, thus, infection. If so, littleor none of the strong-stop DNAproduct of early HIV-1 reverse tran-scription (see “Materials and Meth-ods”) should be detected when cellsare exposed to HIV-1 in the pres-ence of BanLec (44, 48). To test thishypothesis, we incubated PBL withthe HIV-1 Bru isolate in the pres-

ence of different concentrations of BanLec. As a positive con-trol and for comparison, a similar experiment with the lectinGNA was performed in parallel. Real-time PCR was used todetect strong-stop DNA, which is a reverse transcription prod-uct that can be detected early after viral entry before viraluncoating takes place (49–51). Strong-stopDNA thatmay havebeen present in the virus stock was removed by treatment withDNase I to eliminate false detection of reverse transcriptionproducts. Treatment with BanLec resulted in a marked de-crease in strong-stop DNA at low lectin concentrations (Fig. 4)indicating that, in addition to inhibiting viral replication inMDM, BanLec blocks HIV-1 infection in PBL. Furthermore,this inhibition occurs at a step before early replication events,apparently at the level of viral entry. Although it appearsunlikely that BanLec inhibits reverse transcription, these PCRresults do not exclude this possibility, as similar results could beproduced by an inhibitor of HIV-1 reverse transcriptase. Thisissue is addressed further below.BanLec Binds to Glycosylated gp120—BanLec is known to

bind to mannose, and thus, we hypothesized that BanLec bindsthe high mannose structures found on the glycosylated gp120envelope protein and blocks entry of HIV-1 into cells. We pre-pared a BanLec-based ELISA to measure binding of glycosy-lated HIV-1 gp120 to BanLec. We observed that BanLec doesindeed bind to gp120 in a concentration-dependent manner(Fig. 5A). Furthermore, a known BanLec ligand, methyl-�-D-mannopyranoside, inhibited such binding in a concentration-dependent manner (Fig. 5B). Not surprisingly, a high concen-tration of methyl-�-D-mannopyranoside ligand was needed tocompete for binding to gp120 because of the high density ofcarbohydrate residues on the HIV-1 envelope protein. Theseresults corroborate our hypothesis that BanLec inhibits HIV-1

FIGURE 4. BanLec inhibits production of early HIV-1 reverse transcription products in peripheral bloodlymphocytes. Peripheral blood lymphocytes were treated with different lectin concentrations 30 min beforeinfection with HIV-1 Bru. Three hours post-infection, cellular DNA of the infected cells was harvested, andstrong-stop DNA was quantified by real-time PCR. The number of copies was normalized to a PBS-treatedcontrol (100%). The known anti-HIV lectin GNA (circles) was used as a positive control and to assess the relativemolar potency of BanLec (squares).




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cellular entry by binding to high mannose structures found onthe virus.To explore the BanLec binding sites on gp120, we deter-

mined the ability of BanLec to block binding by themonoclonalantibody 2G12 using the ELISA-based assay. 2G12 recognizes acluster of N-linked glycosylation structures at positions Asn-295, -332, and -392 (position numbering is of the HXB2 refer-ence sequence) that are crucial for antibody recognition (52–54). We found that pretreatment of gp120 with BanLecinhibited recognition by 2G12 in a dose-dependent manner,suggesting that BanLec is capable of binding to this antibody’sepitope consisting of high mannose structures (Fig. 6).BanLec Inhibits HIV-1 Infection before Viral Fusion—To fur-

ther investigate at which point in the viral life cycle BanLecinhibits HIV-1 infection, we tested BanLec for its ability toinhibit HIV-1 infection post-attachment. To do so, we tested ifBanLec could inhibit HIV-1 that was already bound to the cellbut could not complete fusion due to temperature restriction(55). As controls, we took advantage of the fact that CD4-IgG2inhibits HIV-1 infection by blocking attachment, whereas T-20works by blocking fusion. As anticipated, we observed a large

decrease in the inhibitory activity of the HIV-1 attachmentinhibitor CD4-IgG2 in the post-attachment assay, whereas thebound virus was still essentially completely susceptible to thefusion inhibitor T-20 (Fig. 7 and Table 2). This demonstratesthat the assay works as expected, with viral attachment, but notfusion, taking place at 16 °C. Both the CCR5 binding inhibitormaraviroc and BanLec primarily blocked viral replication byinhibiting HIV-1 attachment, but each does appear to also havea modest effect on viral fusion (Fig. 7). Importantly, when wecompare the IC50 value of BanLec to those of other anti-HIVcompounds, we see that BanLec potency compares quitewell tothe clinically approved anti-virals T-20 and maraviroc (Table2). Thus, we conclude that BanLec potently inhibits the attach-ment of HIV-1 to cells and has a more modest effect on viralfusion.The Anti-HIV Activity of BanLec Compares Well to Other

Anti-HIV Lectins—Several different lectins have been found toinhibit HIV-1 infection. However, they vary in their degree ofanti-viral activity. To assess the relativemolar-based potency ofBanLec, we compared the anti-HIV activity of BanLec to thatof two previously described anti-HIV lectins, GNA and GRFT.Upon comparison, all three lectins showed activity in the nano-molar range (Fig. 8). Taken together, our data suggest thatBanLec inhibits infection with a broad range of HIV-1 isolatesby blocking viral entry, compares favorably with the potency ofpreviously described lectins and clinically available anti-HIVdrugs, and is a potential component for future anti-HIV vaginalmicrobicides.

DISCUSSION

The primary mechanism of inhibition by BanLec appears tobe blocking cellular attachment of HIV and, thus, viral entry.Our conclusion is based on the findings from our ELISA assaysthat BanLec can bind to high mannose structures found on

FIGURE 5. BanLec binds to glycosylated gp120. A, shown is dose-depen-dent binding of BanLec to glycosylated gp120. BanLec was used to coat a96-well ELISA plate. Serial dilutions of gp120 were added in duplicate to thewells. gp120 was detected with an anti-gp120 antibody. Results are repre-sentative of four independent experiments. B, methyl �-D-mannopyranosideinhibits interaction of BanLec with gp120. ELISA plates were coated withBanLec as in panel A. Serial dilutions of methyl �-D-mannopyranosidewere added to wells along with a constant amount of gp120. The amountof gp120 bound was determined using the standard curve produced inpanel A. Abs, absorbance.

FIGURE 6. Binding of gp120 by BanLec blocks access to the anti-HIVmonoclonal antibody 2G12. Recombinant gp120-coated ELISA plates weretreated with different concentrations of BanLec before incubation with the2G12 antibody, which recognizes the high mannose structures found at posi-tions Asn-295, -332, and -392 of the gp120 protein. Unbound antibody wasremoved, and the amount of antibody remaining was determined by com-parison to a standard curve. The results shown represent the average fromthree separate experiments. Error bars represent S.E. The effect of increasingamounts of BanLec was determined to be significant by 1-way analysis ofvariance testing (p � 0.01).




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HIV-1 gp120, including the high mannose structures that arerecognized by themonoclonal antibody 2G12. Thiswas corrob-orated by the finding that cells treated with BanLec haddecreased amounts of an early HIV reverse transcription prod-uct, strong-stopDNA, that can be detected shortly after cellularentry of the virus and before viral uncoating. In addition, weperformed an assay that took advantage of a temperature-ar-rested state (16 °C) that prevents HIV-1 fusion and comparedthe inhibitory activity of BanLec and other anti-HIV drugs pre-and post-cellular attachment of the virus, finding that most ofthe inhibitory activity of BanLec comes from blocking viralattachment. Interestingly, whereasmost of the inhibitory activ-ity of the CCR5 blocker maraviroc, used as a control in these

experiments, was due to blocking viral attachment, whenmara-viroc was added post-attachment we still observed inhibition ofHIV, albeit at a reduced level. A similar result was also seenwithBanLec, suggesting that these two compounds could have addi-

FIGURE 7. BanLec primarily inhibits binding of HIV to the cellular membrane. TZM-bl cells were spin-infected at 16 °C, a temperature that allows forattachment of virus but does not allow fusion events to occur. The unbound virus was removed, and the cells were incubated with media containing inhibitors(CD4-IgG2, T-20, maraviroc, or BanLec) on ice for 30 min, and then the plates were shifted to 37 °C to allow for fusion and infection to be completed (E). Theresults were compared with a standard infection procedure (pre-attachment) in which the virus and inhibitors were incubated together on ice for 30 min andthen added to TZM-bl cells and incubated at 37 °C (F). The results shown are the averages from three separate experiments. Nonlinear regression analysis wasused for curve fitting and calculation of IC50 values (Table 2). RLU, relative luminescent units.

FIGURE 8. Comparison of the anti-HIV activity of BanLec to the anti-HIVlectins GNA and GRFT. TZM-bl cells were pretreated with BanLec, GRFT, orGNA diluted in PBS or PBS alone, as a control, for 30 min before infection bythe R5 tropic HIV-1 virus 81-A. Forty-eight hours later, luciferase activity wasmeasured. The results are normalized to infected cells treated with PBS alone.The average of three separate experiments is shown and was used to calcu-late IC50 values by nonlinear regression. The calculated IC50 values are thefollowing: GNA � 34.3 nM, BanLec 3.18 nM, and GRFT 0.42 nM. RLU, relativeluminescent units.

TABLE 2Summary of the calculated IC50 values for inhibition of HIV-1infection with the addition of drug either pre- or post-attachment toTZM-bl cells

CompoundPost-attachment Pre-attachment

IC5095% Confidence

intervals IC5095% Confidence

intervals

nM nMCD4-IgG2 –a 1.39 0.667–2.89T-20 2.78 1.96–3.95 1.90 0.958–3.77Maraviroc 10.2 7.5–13.8 1.23 0.584–2.58BanLec 13.1 9.61–17.7 0.224 0.168–0.300

a Too high to calculate.




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tional inhibitory activity at a post-attachment step, such asfusion of the virus to the cell.Our studies indicate that BanLec is a new and promising

member of the group of lectins that are able to inhibit HIV-1infection through interactions with glycosylation sites foundon the viral envelope. The inhibitory activity of BanLecagainst HIV-1 was broad, independent of tropism, and effec-tive against several subtype B and C envelope sequences.HIV-1 pseudotyped with envelopes derived from primaryisolates was inhibited by BanLec in the low nanomolar range.BanLec was also able to inhibit HIV-1 infection of primarycells, and thus, our results are not limited to cell lines.Based on our findings, it is likely that BanLec will be able to

inhibit other HIV-1 subtypes, as they all contain glycosylationsites in their envelope sequences. The isolates used in ourexperiments differed in the number of predicted N-linked gly-cosylation sites, supporting the likelihood that BanLec will beeffective against most HIV subtypes found in both the develop-ing and developed world. Because glycosylation is not specificto HIV-1, lectins have the potential to inhibit the replication ofa broad spectrum of viruses. Indeed, it has been shown thatlectins can inhibit other enveloped viruses including Ebola (56,57), Marburg (57), influenza (58), severe acute respiratory syn-drome coronavirus (59), and hepatitis C virus (60, 61).One potential benefit of the use of lectins as anti-HIV agents is

their ability to target multiple different glycosylation sites on thevirus, thus making it more difficult for resistance to develop. Insupport of this prediction, previous studies that determined theresistance profiles of HIV-1 treated with lectin showed that mul-tiple mutations in the envelope sequence were needed for thedevelopment of resistance (62). Furthermore, different mutationsinN-linked glycosylation sites are required for the development ofresistance todifferent lectins.This suggests that the combinatorialor simultaneoususeofmultiple lectinscanreduce the likelihoodoffailure of a lectin-based anti-viral therapy due to resistance. If apopulation of virus develops resistance to BanLec or other anti-HIV lectins, one interesting possible consequence is that the viruswill then be more susceptible to neutralization by the humanimmune response, as the carbohydrate structures found on theHIV-1 envelope are thought to act as a shield against neutralizingantibody responses (63). This glycan shield works by blockingaccessofepitopes topotentiallyneutralizingantibodies.Previouslypublished data demonstrate that alterations in glycosylation thatresult in resistance to lectins canmake the virus vulnerable toneu-tralizing antibody responses (18, 19).Although several anti-HIV lectins have been described, it is

highly unlikely that a majority of them can be developed fortherapeutic use. Like all potential drugs, lectins can vary in theirdegrees of potency and toxicity (64, 65). Also, it has been shownthat two anti-HIV lectins can significantly differ in their abilityto block attachment of HIV to epithelial cells (66). Concernshave been raised about the potential toxicity of lectins, forexample CV-N. This lectin has shown success as a microbicidein in vivomacaque vaginal and rectal transmission models (67,68), but safety concerns exist. CV-N was found to have mito-genic activity when PBMC cultures were exposed to the lectinfor 3 days (65, 69). However, recombinant therapeutic proteinscan be attached to polyethylene glycol (PEG) polymer chains to

change bioavailability and reduce toxicity. This modification ofCV-N has been shown to be effective in reducing mitogenicactivity in vitro (70). AlthoughBanLec has also been reported topossess mitogenic activity (71), the relationship between mito-genic activity in vitro and microbicide efficacy has not beenelucidated, so it remains possible that recombinant versions ofBanLec and other lectins could be developed that retain efficacybut haveminimalmitogenic activity. The anti-HIV lectinGRFThas recently been reported not to have a mitogenic effect whenadded to human PBMCs (72). This observation is of interest, asGRFT is in the same jacalin-related lectin family and has a sim-ilar structure to BanLec (73). GRFT has also been shown to benon-inflammatory, non-toxic, and capable of being manufac-tured on a large scale. Although clinical testing of these newerlectins has yet to be performed, it appears that lectins havepotential to be used as anti-HIV agents (72). Because the bind-ing, toxicity, and anti-HIV activity of lectins vary, the identifi-cation of novel anti-viral lectins, such as BanLec, will furtherincrease the possibility of successful development of a lectin-based anti-HIV microbicide.

Acknowledgments—The following reagents were obtained through theAIDS Research and Reference Reagent Program, Division of AIDS,NIAID, National Institutes of Health: TZM-bl cells from Dr. John C.Kappes, Dr. XiaoyunWu, andTranzyme Inc. (catalog #8129),MAGI-CCR5 cells fromDr. JulieOverbaugh (catalog #3522),Maraviroc (cat-alog #11580), T-20 fusion inhibitor from Roche Applied Science (cat-alog #9845), CD4-IgG2 from Progenics Pharmaceuticals (catalog#11780), HIS-Griffithsin from Drs. Barry O’Keefe and JamesMcMahon (catalog #11610), pSG3�env from Drs. John C. Kappes andXiaoyunWu (catalog #11051), p81A-4 fromDr. Bruce Chesebro (cat-alog #11440), pAD8(NL4-3) from Dr. Eric O. Freed (catalog #11346),p89.6 from Ronald G. Collman (catalog #3552), pNL4-3 fromDr.MalcolmMartin (catalog #114), standard reference panel of Sub-type B HIV-1 Env Clones (catalog #11227), standard reference panelof subtype C HIV-1 Env Clones (catalog #11326), pConC gp160-optfromDr. Beatrice Hahn (catalog #11407), HIV-1BaL gp120 fromDivi-sion of AIDS Acquired Immunodeficiency Syndrome, NIAID (catalog#4961), HIV-1 gp120 monoclonal antibody (2G12) from Dr. Her-mann Katinger (catalog #1476).

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