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A Lectin Isolated from Bananas Is a Potent Inhibitor of HIV Replication*

  • Michael D. Swanson
    Affiliations
    Program in Immunology, University of Michigan Medical Center, Ann Arbor, Michigan 48109
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  • Harry C. Winter
    Affiliations
    Department of Biological Chemistry, University of Michigan Medical Center, Ann Arbor, Michigan 48109
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  • Irwin J. Goldstein
    Affiliations
    Department of Biological Chemistry, University of Michigan Medical Center, Ann Arbor, Michigan 48109
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  • David M. Markovitz
    Correspondence
    To whom correspondence should be addressed: University of Michigan Medical Center, 5220 MSRB III, 1150 West Medical Center Dr., Ann Arbor, MI 48109-5640. Tel.: 734-647-1786; Fax: 734-764-0101;
    Affiliations
    Program in Immunology, University of Michigan Medical Center, Ann Arbor, Michigan 48109

    Department of Internal Medicine, Division of Infectious Diseases, University of Michigan Medical Center, Ann Arbor, Michigan 48109

    Cellular and Molecular Biology Program, University of Michigan Medical Center, Ann Arbor, Michigan 48109
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  • Author Footnotes
    * This work was supported, in whole or in part, by National Institutes of Health Grant R01 AI062248 (of D. M. M.). This work was also supported by a Burroughs Wellcome Fund Clinical Scientist Award in Translational Research.
Open AccessPublished:January 15, 2010DOI:https://doi.org/10.1074/jbc.M109.034926
      BanLec is a jacalin-related lectin isolated from the fruit of bananas, Musa acuminata. This lectin binds to high mannose carbohydrate structures, including those found on viruses containing glycosylated envelope proteins such as human immunodeficiency virus type-1 (HIV-1). Therefore, we hypothesized that BanLec might inhibit HIV-1 through binding of the glycosylated HIV-1 envelope protein, gp120. We determined that BanLec inhibits primary and laboratory-adapted HIV-1 isolates of different tropisms and subtypes. BanLec possesses potent anti-HIV activity, with IC50 values in the low nanomolar to picomolar range. The mechanism for BanLec-mediated antiviral activity was investigated by determining if this lectin can directly bind the HIV-1 envelope protein and block entry of the virus into the cell. An enzyme-linked immunosorbent assay confirmed direct binding of BanLec to gp120 and indicated that BanLec can recognize the high mannose structures that are recognized by the monoclonal antibody 2G12. Furthermore, BanLec is able to block HIV-1 cellular entry as indicated by temperature-sensitive viral entry studies and by the decreased levels of the strong-stop product of early reverse transcription seen in the presence of BanLec. Thus, our data indicate that BanLec inhibits HIV-1 infection by binding to the glycosylated viral envelope and blocking cellular entry. The relative anti-HIV activity of BanLec compared favorably to other anti-HIV lectins, such as snowdrop lectin and Griffithsin, and to T-20 and maraviroc, two anti-HIV drugs currently in clinical use. Based on these results, BanLec is a potential component for an anti-viral microbicide that could be used to prevent the sexual transmission of HIV-1.

      Introduction

      Despite the development of more than 25 approved anti-HIV
      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 macrophages
      MTT
      (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
      PBS
      phosphate-buffered saline
      TCID50
      tissue culture infective dose 50%.
      drugs and improvements in the availability of antiretroviral drugs in low and middle income countries, the rate of new HIV-1 infections is outpacing the rate of new individuals receiving antiretroviral therapy by 2.5:1 (

      Executive Summary United Nations Programme on HIV/AIDS (UNAIDS) and World Health Organization (WHO) (2008) Report on the Global AIDS Epidemic

      ). At present, it appears that an efficacious HIV vaccine is still many years away. Therefore, other methods for halting the spread of HIV are vitally needed. This has raised the possibility of developing either intravaginally or intrarectally applied microbicides to halt the spread of HIV during sexual intercourse. This type of intervention is particularly needed in the developing world, such as sub-Saharan Africa, where more than 20 million people are living with HIV/AIDS (

      Executive Summary United Nations Programme on HIV/AIDS (UNAIDS) and World Health Organization (WHO) (2008) Report on the Global AIDS Epidemic

      ). Although abstinence has been suggested by some groups, campaigns to encourage this method of halting transmission have not been effective (
      • Underhill K.
      • Montgomery P.
      • Operario D.
      ). Although condoms are quite effective against the spread of HIV and some other sexually transmitted diseases, they are only effective if they are used consistently and correctly, which is often not the case (
      • Randolph M.E.
      • Pinkerton S.D.
      • Bogart L.M.
      • Cecil H.
      • Abramson P.R.
      ,
      • Crosby R.
      • Yarber W.L.
      • Sanders S.A.
      • Graham C.A.
      ). This is particularly true in the developing world, where women have relatively little control over sexual encounters and, thus, have not been able to enforce condom usage (
      • Gupta G.R.
      ), so the development of a long-lasting, self-applied, microbicide is very attractive. In fact, it is estimated that 20% coverage with a microbicide that is only 60% effective against HIV may prevent up to 2.5 million HIV infections over three years (
      • Watts C.
      ). Therefore, even modest success with microbicides could save millions of lives.
      Some of the most promising compounds for inhibiting vaginal or rectal HIV transmission are agents that block HIV before integration of the viral genome into the target cell. Thus, the viral entry step is one potential target for a microbicide. Entry inhibitors that have been proposed for use in a vaginal microbicide include long chain and ionic polymers (such as Pro 2000) as well as dendrimers, lipid membrane modifiers, and anti-CD4 antibodies. HIV-binding peptides and small molecule inhibitors have also been considered, including the fusion inhibitor T-20 (enfuvirtide) and the CCR5 blocker maraviroc, which are already in clinical use for the treatment of HIV infection. In addition, lectins are a growing class of HIV-1 inhibitors under consideration as microbicide candidates (
      • Balzarini J.
      • Van Damme L.
      ,
      • Balzarini J.
      ). Lectins inhibit HIV-1 entry by binding to carbohydrate structures found on the viral envelope. Examples of anti-HIV lectins include Cyanovirin-N (CV-N) (
      • Boyd M.R.
      • Gustafson K.R.
      • McMahon J.B.
      • Shoemaker R.H.
      • O'Keefe B.R.
      • Mori T.
      • Gulakowski R.J.
      • Wu L.
      • Rivera M.I.
      • Laurencot C.M.
      • Currens M.J.
      • Cardellina 2nd, J.H.
      • Buckheit Jr., R.W.
      • Nara P.L.
      • Pannell L.K.
      • Sowder 2nd, R.C.
      • Henderson L.E.
      ), Griffithsin (GRFT) (
      • Mori T.
      • O'Keefe B.R.
      • Sowder 2nd, R.C.
      • Bringans S.
      • Gardella R.
      • Berg S.
      • Cochran P.
      • Turpin J.A.
      • Buckheit Jr., R.W.
      • McMahon J.B.
      • Boyd M.R.
      ), and snowdrop lectin (GNA) (
      • Balzarini J.
      ,
      • Balzarini J.
      • Schols D.
      • Neyts J.
      • Van Damme E.
      • Peumans W.
      • De Clercq E.
      ,
      • Balzarini J.
      • Hatse S.
      • Vermeire K.
      • Princen K.
      • Aquaro S.
      • Perno C.F.
      • De Clercq E.
      • Egberink H.
      • Vanden Mooter G.
      • Peumans W.
      • Van Damme E.
      • Schols D.
      ).
      The HIV-1 envelope protein gp120 contains 20–30 possible N-linked glycosylation sites. These carbohydrate structures make up ∼50% of the molecular weight of the protein (
      • Matthews T.J.
      • Weinhold K.J.
      • Lyerly H.K.
      • Langlois A.J.
      • Wigzell H.
      • Bolognesi D.P.
      ,
      • Geyer H.
      • Holschbach C.
      • Hunsmann G.
      • Schneider J.
      ,
      • Allan J.S.
      • Coligan J.E.
      • Barin F.
      • McLane M.F.
      • Sodroski J.G.
      • Rosen C.A.
      • Haseltine W.A.
      • Lee T.H.
      • Essex M.
      ). Glycosylation affects aspects of the viral life cycle including protein folding (
      • Li Y.
      • Luo L.
      • Rasool N.
      • Kang C.Y.
      ), cellular transport, binding to cellular receptors (
      • Matthews T.J.
      • Weinhold K.J.
      • Lyerly H.K.
      • Langlois A.J.
      • Wigzell H.
      • Bolognesi D.P.
      ,
      • Clevestig P.
      • Pramanik L.
      • Leitner T.
      • Ehrnst A.
      ,
      • 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.
      ), trans-infection by dendritic cells (
      • 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.
      ), and shielding from the immune response (
      • Back N.K.
      • Smit L.
      • De Jong J.J.
      • Keulen W.
      • Schutten M.
      • Goudsmit J.
      • Tersmette M.
      ). Because glycosylation is essential to the virus, it presents an attractive therapeutic target.
      The lectin termed BanLec, isolated from the ripened fruit of the banana (Musa acuminata cultivars), exists as a dimer with a molecular mass of ∼30 kDa (
      • Peumans W.J.
      • Zhang W.
      • Barre A.
      • Houlès
      • Astoul C.
      • Balint-Kurti P.J.
      • Rovira P.
      • Rougé P.
      • May G.D.
      • Van Leuven F.
      • Truffa-Bachi P.
      • Van Damme E.J.
      ). It is a member of the jacalin-related lectin family and can recognize high mannose structures (
      • Koshte V.L.
      • van Dijk W.
      • van der Stelt M.E.
      • Aalberse R.C.
      ,
      • Nakamura-Tsuruta S.
      • Uchiyama N.
      • Peumans W.J.
      • Van Damme E.J.
      • Totani K.
      • Ito Y.
      • Hirabayashi J.
      ,
      • Mo H.
      • Winter H.C.
      • Van Damme E.J.
      • Peumans W.J.
      • Misaki A.
      • Goldstein I.J.
      ). Lectins in this family are characterized by the presence of a β-prism 1 structure composed of three Greek Key turn motifs. Greek Keys 1 and 2 are both involved in binding carbohydrates and contain a GXXXD binding motif, whereas Key 3 does not contain the binding motif (
      • Meagher J.L.
      • Winter H.C.
      • Ezell P.
      • Goldstein I.J.
      • Stuckey J.A.
      ,
      • Singh D.D.
      • Saikrishnan K.
      • Kumar P.
      • Surolia A.
      • Sekar K.
      • Vijayan M.
      ). However, this loop can assist ligand binding and determine lectin specificity (
      • Jeyaprakash A.A.
      • Srivastav A.
      • Surolia A.
      • Vijayan M.
      ). Because of its affinity for high mannose structures (
      • Geyer H.
      • Holschbach C.
      • Hunsmann G.
      • Schneider J.
      ), we sought to investigate whether BanLec might bind the mannose-rich envelope of HIV-1 and thereby block HIV infection. The results presented below demonstrate that BanLec is a potent inhibitor of HIV infection that markedly reduces the replication of a range of HIV-1 isolates and has potential to be further developed for use as a vaginal microbicide.

      DISCUSSION

      The primary mechanism of inhibition by BanLec appears to be blocking cellular attachment of HIV and, thus, viral entry. Our conclusion is based on the findings from our ELISA assays that BanLec can bind to high mannose structures found on HIV-1 gp120, including the high mannose structures that are recognized by the monoclonal antibody 2G12. This was corroborated by the finding that cells treated with BanLec had decreased amounts of an early HIV reverse transcription product, strong-stop DNA, that can be detected shortly after cellular entry of the virus and before viral uncoating. In addition, we performed an assay that took advantage of a temperature-arrested state (16 °C) that prevents HIV-1 fusion and compared the inhibitory activity of BanLec and other anti-HIV drugs pre- and post-cellular attachment of the virus, finding that most of the inhibitory activity of BanLec comes from blocking viral attachment. Interestingly, whereas most of the inhibitory activity of the CCR5 blocker maraviroc, used as a control in these experiments, was due to blocking viral attachment, when maraviroc was added post-attachment we still observed inhibition of HIV, albeit at a reduced level. A similar result was also seen with BanLec, suggesting that these two compounds could have additional inhibitory activity at a post-attachment step, such as fusion 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-1 infection through interactions with glycosylation sites found on the viral envelope. The inhibitory activity of BanLec against HIV-1 was broad, independent of tropism, and effective against several subtype B and C envelope sequences. HIV-1 pseudotyped with envelopes derived from primary isolates was inhibited by BanLec in the low nanomolar range. BanLec was also able to inhibit HIV-1 infection of primary cells, 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 glycosylation sites in their envelope sequences. The isolates used in our experiments differed in the number of predicted N-linked glycosylation sites, supporting the likelihood that BanLec will be effective against most HIV subtypes found in both the developing and developed world. Because glycosylation is not specific to HIV-1, lectins have the potential to inhibit the replication of a broad spectrum of viruses. Indeed, it has been shown that lectins can inhibit other enveloped viruses including Ebola (
      • Barrientos L.G.
      • O'Keefe B.R.
      • Bray M.
      • Sanchez A.
      • Gronenborn A.M.
      • Boyd M.R.
      ,
      • Barrientos L.G.
      • Lasala F.
      • Otero J.R.
      • Sanchez A.
      • Delgado R.
      ), Marburg (
      • Barrientos L.G.
      • Lasala F.
      • Otero J.R.
      • Sanchez A.
      • Delgado R.
      ), influenza (
      • Smee D.F.
      • Bailey K.W.
      • Wong M.H.
      • O'Keefe B.R.
      • Gustafson K.R.
      • Mishin V.P.
      • Gubareva L.V.
      ), severe acute respiratory syndrome coronavirus (
      • Keyaerts E.
      • Vijgen L.
      • Pannecouque C.
      • Van Damme E.
      • Peumans W.
      • Egberink H.
      • Balzarini J.
      • Van Ranst M.
      ), and hepatitis C virus (
      • Helle F.
      • Wychowski C.
      • Vu-Dac N.
      • Gustafson K.R.
      • Voisset C.
      • Dubuisson J.
      ,
      • Bertaux C.
      • Daelemans D.
      • Meertens L.
      • Cormier E.G.
      • Reinus J.F.
      • Peumans W.J.
      • Van Damme E.J.
      • Igarashi Y.
      • Oki T.
      • Schols D.
      • Dragic T.
      • Balzarini J.
      ).
      One potential benefit of the use of lectins as anti-HIV agents is their ability to target multiple different glycosylation sites on the virus, thus making it more difficult for resistance to develop. In support of this prediction, previous studies that determined the resistance profiles of HIV-1 treated with lectin showed that multiple mutations in the envelope sequence were needed for the development of resistance (
      • Witvrouw M.
      • Fikkert V.
      • Hantson A.
      • Pannecouque C.
      • O'keefe B.R.
      • McMahon J.
      • Stamatatos L.
      • de Clercq E.
      • Bolmstedt A.
      ). Furthermore, different mutations in N-linked glycosylation sites are required for the development of resistance to different lectins. This suggests that the combinatorial or simultaneous use of multiple lectins can reduce the likelihood of failure of a lectin-based anti-viral therapy due to resistance. If a population of virus develops resistance to BanLec or other anti-HIV lectins, one interesting possible consequence is that the virus will then be more susceptible to neutralization by the human immune response, as the carbohydrate structures found on the HIV-1 envelope are thought to act as a shield against neutralizing antibody responses (
      • Wei X.
      • Decker J.M.
      • Wang S.
      • Hui H.
      • Kappes J.C.
      • Wu X.
      • Salazar-Gonzalez J.F.
      • Salazar M.G.
      • Kilby J.M.
      • Saag M.S.
      • Komarova N.L.
      • Nowak M.A.
      • Hahn B.H.
      • Kwong P.D.
      • Shaw G.M.
      ). This glycan shield works by blocking access of epitopes to potentially neutralizing antibodies. Previously published data demonstrate that alterations in glycosylation that result in resistance to lectins can make the virus vulnerable to neutralizing antibody responses (
      • Clevestig P.
      • Pramanik L.
      • Leitner T.
      • Ehrnst A.
      ,
      • 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.
      ).
      Although several anti-HIV lectins have been described, it is highly unlikely that a majority of them can be developed for therapeutic use. Like all potential drugs, lectins can vary in their degrees of potency and toxicity (
      • Lis H.
      • Sharon N.
      ,
      • Huskens D.
      • Vermeire K.
      • Vandemeulebroucke E.
      • Balzarini J.
      • Schols D.
      ). Also, it has been shown that two anti-HIV lectins can significantly differ in their ability to block attachment of HIV to epithelial cells (
      • Saïdi H.
      • Nasreddine N.
      • Jenabian M.A.
      • Lecerf M.
      • Schols D.
      • Krief C.
      • Balzarini J.
      • Bélec L.
      ). Concerns have been raised about the potential toxicity of lectins, for example CV-N. This lectin has shown success as a microbicide in in vivo macaque vaginal and rectal transmission models (
      • Tsai C.C.
      • Emau P.
      • Jiang Y.
      • Tian B.
      • Morton W.R.
      • Gustafson K.R.
      • Boyd M.R.
      ,
      • Tsai C.C.
      • Emau P.
      • Jiang Y.
      • Agy M.B.
      • Shattock R.J.
      • Schmidt A.
      • Morton W.R.
      • Gustafson K.R.
      • Boyd M.R.
      ), but safety concerns exist. CV-N was found to have mitogenic activity when PBMC cultures were exposed to the lectin for 3 days (
      • Huskens D.
      • Vermeire K.
      • Vandemeulebroucke E.
      • Balzarini J.
      • Schols D.
      ,
      • Buffa V.
      • Stieh D.
      • Mamhood N.
      • Hu Q.
      • Fletcher P.
      • Shattock R.J.
      ). However, recombinant therapeutic proteins can be attached to polyethylene glycol (PEG) polymer chains to change bioavailability and reduce toxicity. This modification of CV-N has been shown to be effective in reducing mitogenic activity in vitro (
      • Zappe H.
      • Snell M.E.
      • Bossard M.J.
      ). Although BanLec has also been reported to possess mitogenic activity (
      • Gavrovic-Jankulovic M.
      • Poulsen K.
      • Brckalo T.
      • Bobic S.
      • Lindner B.
      • Petersen A.
      ), the relationship between mitogenic activity in vitro and microbicide efficacy has not been elucidated, so it remains possible that recombinant versions of BanLec and other lectins could be developed that retain efficacy but have minimal mitogenic activity. The anti-HIV lectin GRFT has recently been reported not to have a mitogenic effect when added to human PBMCs (
      • O'Keefe B.R.
      • Vojdani F.
      • Buffa V.
      • Shattock R.J.
      • Montefiori D.C.
      • Bakke J.
      • Mirsalis J.
      • d'Andrea A.L.
      • Hume S.D.
      • Bratcher B.
      • Saucedo C.J.
      • McMahon J.B.
      • Pogue G.P.
      • Palmer K.E.
      ). This observation is of interest, as GRFT is in the same jacalin-related lectin family and has a similar structure to BanLec (
      • Ziółkowska N.E.
      • O'Keefe B.R.
      • Mori T.
      • Zhu C.
      • Giomarelli B.
      • Vojdani F.
      • Palmer K.E.
      • McMahon J.B.
      • Wlodawer A.
      ). GRFT has also been shown to be non-inflammatory, non-toxic, and capable of being manufactured on a large scale. Although clinical testing of these newer lectins has yet to be performed, it appears that lectins have potential to be used as anti-HIV agents (
      • O'Keefe B.R.
      • Vojdani F.
      • Buffa V.
      • Shattock R.J.
      • Montefiori D.C.
      • Bakke J.
      • Mirsalis J.
      • d'Andrea A.L.
      • Hume S.D.
      • Bratcher B.
      • Saucedo C.J.
      • McMahon J.B.
      • Pogue G.P.
      • Palmer K.E.
      ). Because the binding, toxicity, and anti-HIV activity of lectins vary, the identification of novel anti-viral lectins, such as BanLec, will further increase the possibility of successful development of a lectin-based anti-HIV microbicide.

      Acknowledgments

      The following reagents were obtained through the AIDS Research and Reference Reagent Program, Division of AIDS, NIAID, National Institutes of Health: TZM-bl cells from Dr. John C. Kappes, Dr. Xiaoyun Wu, and Tranzyme Inc. (catalog #8129), MAGI-CCR5 cells from Dr. Julie Overbaugh (catalog #3522), Maraviroc (catalog #11580), T-20 fusion inhibitor from Roche Applied Science (catalog #9845), CD4-IgG2 from Progenics Pharmaceuticals (catalog #11780), HIS-Griffithsin from Drs. Barry O'Keefe and James McMahon (catalog #11610), pSG3Δenv from Drs. John C. Kappes and Xiaoyun Wu (catalog #11051), p81A-4 from Dr. Bruce Chesebro (catalog #11440), pAD8(NL4-3) from Dr. Eric O. Freed (catalog #11346), p89.6 from Ronald G. Collman (catalog #3552), pNL4-3 from Dr. Malcolm Martin (catalog #114), standard reference panel of Subtype B HIV-1 Env Clones (catalog #11227), standard reference panel of subtype C HIV-1 Env Clones (catalog #11326), pConC gp160-opt from Dr. Beatrice Hahn (catalog #11407), HIV-1BaL gp120 from Division of AIDS Acquired Immunodeficiency Syndrome, NIAID (catalog #4961), HIV-1 gp120 monoclonal antibody (2G12) from Dr. Hermann Katinger (catalog #1476).

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