multifacetedmechanismsofhiv-1entryinhibitionbyhuman...

Multifaceted Mechanisms of HIV-1 Entry Inhibition by Human�-Defensin*□S �

Received for publication, April 26, 2012, and in revised form, June 13, 2012 Published, JBC Papers in Press, June 25, 2012, DOI 10.1074/jbc.M112.375949

Lusine H. Demirkhanyan‡, Mariana Marin‡, Sergi Padilla-Parra‡, Changyou Zhan§, Kosuke Miyauchi¶,Maikha Jean-Baptiste�, Gennadiy Novitskiy§, Wuyuan Lu§, and Gregory B. Melikyan‡**1

From the ‡Division of Pediatric Infectious Diseases, Emory University Children’s Center, Atlanta, Georgia 30322, the §Institute ofHuman Virology and Department of Biochemistry, University of Maryland School of Medicine, Baltimore, Maryland 21201, the¶Research Center for Allergy and Immunology, RIKEN Yokohama Institute, 1-7-22 Suehiro-cho, Tsurumi, Yokohama, Kanagawa230-0045, Japan, the �National Parkinson Foundation, Inc., Miami, Florida 33136, and the **Children’s Healthcare of Atlanta,Atlanta, Georgia 30322

Background: Human neutrophil peptide 1 (HNP-1) inhibits HIV-1 infection by a poorly defined mechanism.Results: Effects of HNP-1 on individual steps of virus-cell fusion were examined.Conclusion:HNP-1 interferes with all major steps of HIV-1 entry, from CD4/coreceptor binding to virus uptake and refoldingof Env glycoprotein.Significance: The ability of HNP-1 to block HIV-1 uptake suggests a novel universal mechanism for antiviral activity.

The human neutrophil peptide 1 (HNP-1) is known to blockthe human immunodeficiency virus type 1 (HIV-1) infection,but the mechanism of inhibition is poorly understood. Weexamined the effect of HNP-1 on HIV-1 entry and fusion andfound that, surprisingly, this �-defensin inhibited multiplesteps of virus entry, including: (i) Env binding to CD4 and core-ceptors; (ii) refolding of Env into the final 6-helix bundle struc-ture; and (iii) productive HIV-1 uptake but not internalizationof endocytic markers. Despite its lectin-like properties, HNP-1could bind to Env, CD4, and other host proteins in a glycan- andserum-independent manner, whereas the fusion inhibitoryactivity was greatly attenuated in the presence of human orbovine serum. This demonstrates that binding of �-defensin tomolecules involved in HIV-1 fusion is necessary but not suffi-cient for blocking the virus entry. We therefore propose thatoligomeric forms of defensin, whichmay be disrupted by serum,contribute to the anti-HIV-1 activity perhaps through cross-linking virus and/or host glycoproteins. This notion is sup-ported by the ability of HNP-1 to reduce the mobile fraction ofCD4 and coreceptors in the plasma membrane and to precipi-tate a core subdomainof Env in solution.The ability ofHNP-1 toblock HIV-1 uptake without interfering with constitutive endo-cytosis suggests a novel mechanism for broad activity againstthis and other viruses that enter cells through endocyticpathways.

Mammalian leukocytes and epithelial cells produce defensins,cysteine-rich cationic peptides exhibiting broad antimicrobial and

immunomodulatory activities (1–3). These peptides are classifiedinto three subfamilies,�-,�-, and �-defensins (4–7). All defensinscontain three disulfide bonds, which are critical for their anti-mi-crobial activities, including the inhibition of infection by a numberof enveloped and nonenveloped viruses (2, 8–14). Mammaliandefensins have been reported to inhibit HIV-1 replication in vitro(15–18) and in vivo (19, 20). However, themechanism underlyingthe anti-HIV-1 activity of defensins remains controversial. Retro-cyclin (a �-defensin) has been shown to bind to both CD4 andHIV-1gp120glycoprotein inaglycan-dependentmanner, and thisbinding is correlatedwith its anti-HIV-1activity (21).Basedonthislectin-like property of retrocyclin and its ability to reduce the lat-eral mobility of cell surface glycoproteins, Leikina et al. (22) pro-posed that �-defensin acts by reversibly cross-linking the plasmamembrane glycoproteins and thus erecting a barrier for virusfusion. Conversely, retrocyclin has been reported to inhibitHIV-1fusion by specifically binding to theHIV-1 gp41, but not toHIV-2or SIV gp41, in a glycan-independent manner and preventing theformation of the gp41 6-helix bundle structure (23, 24).The mechanism of anti-HIV-1 activity of �-defensins, also

known as human neutrophil peptides (HNPs),2 is also debated.These defensins have been implicated in inhibition of differentsteps of the HIV-1 replication cycle, from binding to cognatereceptors (4, 25) to post-reverse transcription and even post-integration processes (16, 17, 26). �-Defensins have also beenreported to inhibitHIV-1 infection by up-regulating expressionand secretion of chemokines (27) and by directly inactivating

* This work was supported, in whole or in part, by National Institutes of HealthGrants AI087453 (to G. B. M.) and AI072732 (to W. L.). This work was alsosupported by Emory and Children’s Pediatric Research Center FlowCytometry Core.

� This work was selected as a Paper of the Week.□S This article contains supplemental Figs. S1–S3 and additional references.1 To whom correspondence should be addressed: Dept. of Pediatric Infec-

tious Diseases, Emory University, 2015 Uppergate Dr., Atlanta, GA 30322.Tel.: 404-727-4652; Fax: 404-727-9223; E-mail: [email protected].

2 The abbreviations used are: HNP, human neutrophil peptide; BlaM, �-lacta-mase; C34 and C52L, peptides derived from the C-terminal heptad repeatdomain of HIV-1 gp41; DiI, 1,1�-dioctadecyl-3,3,3�,3�-tetramethylindocar-bocyanine perchlorate; DMA, 5-(N,N-dimethyl)amiloride hydrochloride;EcpH, ecliptic pHluorin; Env, envelope glycoprotein; FCS, fluorescence cor-relation spectroscopy; FRAP, fluorescence recovery after photobleaching;ICAM-1, intercellular adhesion molecule 1; PBMC, peripheral blood mono-nuclear cells; PMA, phorbol 12-myristate 13-acetate; TAS, temperature-ar-rested stage of HIV fusion; Trf, transferrin; TZM-bl, HeLa-derived HIV indi-cator cell line; PFA, paraformaldehyde; VSV, vesicular stomatitis virus;HBSS, Hanks’ balanced salt solution; FAM, 6-carboxyfluorescein; Abu,�-aminobutyric acid.

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 287, NO. 34, pp. 28821–28838, August 17, 2012© 2012 by The American Society for Biochemistry and Molecular Biology, Inc. Published in the U.S.A.

AUGUST 17, 2012 • VOLUME 287 • NUMBER 34 JOURNAL OF BIOLOGICAL CHEMISTRY 28821

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the virus in a serum-freemedium (17, 26, 28). At the same time,certain human �-defensins (HD5 and HD6) can enhance entryof HIV-1 and unrelated viruses (29–31). HNP-1, -2, and -3exhibiting lectin-like properties have been reported to bind toCD4 and toHIV-1 gp120with a relatively high affinity, a featurethat appears to correlate with their anti-HIV-1 activity (4).However, HNP-4, which exhibits weak glycan-independentbinding to gp120 and CD4, is a more potent inhibitor of HIV-1infection (32). Thus, the exact steps of HIV-1 replication tar-geted by �-defensins and the mechanisms of their action arenot well understood.To gain insight into the elusive mechanism of antiviral activ-

ity of human defensins, we focused on HIV-1 entry into cells.We have recently provided evidence that HIV enters suscepti-ble cell lines and primary CD4� T cells via endocytosis andfusion with endosomes (33, 34). We have also dissected keysteps of HIV-1 entry and fusion, using respective inhibitors(33–35). Here, by employing imaging, functional and biochem-ical assays, we examined the effect of an �-defensin, HNP-1, onHIV-1 fusion.Major steps of HIV-1 entry, from binding to CD4and coreceptors to productive endocytosis and gp41-mediatedfusion with endosomes, were analyzed.Our experiments revealed the striking ability of HNP-1 to

interfere with multiple steps of HIV-1 entry. This defensinbound to Env glycoprotein, as well as to CD4 and likely to core-ceptors, without inactivating the virus or compromising the cellviability. In addition, HNP-1 down-regulated the expression ofCD4 and CXCR4 and blocked weak interactions between Envand CD4 or coreceptors. Moreover, analysis of HIV-1 fusionintermediates downstream of CD4/coreceptor binding showedthat HNP-1 also inhibited late steps of fusion, apparently bytargeting intermediate conformations of Env. We also foundthat defensin was able to bind Env and CD4 in a glycan-inde-pendent manner and to reduce the mobile fraction of CD4 andcoreceptors in the plasma membrane. Perhaps the most unex-pected anti-HIV-1 activity of this defensin was the selectiveinhibition of HIV-1 uptake but not of the overall endocyticactivity of a target cell. These findings imply that, through aninherent ability to bind to multiple targets, HNP-1 mounts apowerful defense against HIV-1. However, despite poor inhib-itory activity of HNP-1 in the presence of serum, its binding tocellular and virus proteins was not affected by serum. The lackof correlation between the defensin binding and inhibitoryactivity suggests that, in addition to the ability to engage multi-ple targets, the antiviral effect may require defensin oligomeri-zation, which could be disrupted by serum.

EXPERIMENTAL PROCEDURES

Cells and Reagents—HEK 293T/17 cells (from ATCC,Manassas, VA) were grown in DMEM supplemented with 10%fetal bovine serum (FBS,HyCloneLaboratories, Logan,UT), 0.5mg/ml geneticin (Invitrogen), and penicillin/streptomycin(Sigma). HeLa-derived indicator TZM-bl cells expressing CD4,CXCR4, and CCR5 were grown in DMEM supplemented with10% FBS and penicillin/streptomycin. HeLa-ADA cells stablyexpressing Env and Tat from the HIV-1 ADA strain were a giftfromDr.MarcAlizon (Pasteur Institute, France) (36). Allmediaand buffers were obtained from HyClone (Thermo Scientific)

or Cellgro (Mediatech Inc., Manassas, VA). The following celllines and reagents were obtained from the AIDS Research andReferenceReagent Program,National Institutes ofHealth: indi-cator TZM-bl cells (donated by Drs. J. C. Kappes and X. Wu(37)); hybridoma cells secreting the anti-HIV-1 gp41 Chessie8antibody (from Dr. G. Lewis (38)); pBABE-Fusin (donated byDr. N. Landau (39)); HIV-1 immunoglobulin (HIV-IG) (Dr.Luiz Barbosa from the NHLBI, National Institutes of Health);TAK-779 (40); and anti-CXCR4 antibody 12G5 (donated byDr.James Hoxie (41)). The 2D7 antibody was purchased fromPharmingen. Human peripheral blood mononuclear cells(PBMCs) were isolated and activated with 10 ng/ml IL-2 (AIDSReference Reagent Program, National Institutes of Health,from Dr. M. Gately, Hoffmann-La Roche) and 2.5 �g/ml PHA(Sigma), as described previously (33).The pCAGGS plasmids encoding JRFL or HXB2 Env were

provided by Dr. J. Binley (Torrey Pines Institute). The pMDGvector expressing VSVGwas provided by Dr. John Young (SalkInstitute). TheHIV-1-based packaging vector pR8�Env lackingthe env genewas fromDr. D. Trono (Geneva, Switzerland). TheC52L recombinant peptide was a gift fromDr. Min Lu (CornellUniversity) (42). The C34 peptide was a gift from Dr. L. Wang(Institute of Human Virology, University of Maryland Balti-more). Anti-human CD4-Alexa Fluor488 conjugate and puri-fied mouse IgG1 and IgG2a, � isotype controls were obtainedfrom BioLegend (San Diego). AD101 was a kind gift from Dr. J.Strizki (Schering-Plough). Endocytic markers transferrin-Al-exa488, dextran-Alexa488 and DiI-low density lipoprotein(DiI-LDL) were from Invitrogen. BMS-806 was synthesized byChemPacific Corp. (Baltimore, MD), and phorbol 12-myristate13-acetate (PMA) and AMD3100 were purchased from Sigma.Protein G-horseradish peroxidase (HRP) conjugate was pur-chased from Bio-Rad, and streptavidin-HRP was from GEHealthcare. The 3,3�,5,5�-tetramethylbenzidine substrate kitfor chromogenic detection of the horseradish peroxidase (HRP)activity was purchased from Thermo Scientific. The enhancedluminol-based chemiluminescent (ECL) substrate for the HRPdetection on immunoblots was obtained from Pierce. Theenzymatic deglycosylation kit was fromProzyme (San Leandro,CA).Synthesis and Purification of HNP-1 and Its Derivatives—

The chemical synthesis, oxidative folding, and structural char-acterization of HNP-1 were described elsewhere (43–45). Thesix Cys residues in HNP-1 were all replaced by �-aminobutyricacid (Abu), isosteric to Cys, yielding the linear analog Abu-HNP. For biotinylation of HNP-1, an N-terminally acetylatedHNP-1 analog, N-acetyl-A11K-HNP-1, was synthesized, folded,and purified and subsequently reacted through its only aminogroup at Lys-11 with N-hydroxysulfosuccinimide ester-activatedbiotin as per the instructions provided by the manufacturer(Pierce/Thermo Scientific). Residue 11 was selected for labelingdue to its minimal interference with defensin folding anddimerization, as judged by structural analysis of wild type HNP-1.For biotinylation of Abu-HNP, the linear peptideN-acetyl-A11K-Abu-HNPwas used. All peptides were purified by reversed-phaseHPLC to homogeneity, and their molecular masses were ascer-tained by electrospray ionizationmass spectrometry.

Defensin Blocks HIV-1 Endocytosis and Key Steps of Fusion

28822 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 287 • NUMBER 34 • AUGUST 17, 2012


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HIV-1 gp41 Coiled Coil and 6-Helix Bundle FormationAssays—The N36 (HIV-1 gp160(546–581)) and C34 (HIV-1gp160(628–661)) peptides derived from the N- and C-terminalheptad repeat domains of gp41were synthesized using theO-ben-zotriazole-N,N�,N�-tetramethyluronium-hexafluorophosphate acti-vation/N,N-diisopropylethylamine in situ neutralization proto-col developed by Kent and co-workers for t-butoxycarbonylchemistry solid phase peptide synthesis (46). The N36 peptidecontains one Lys residue (Lys-574) and was chosen for covalentattachment of succinimidyl ester-activated carboxyfluorescein(FAM) as per the instructions provided by the manufacturer(Invitrogen). An N-terminally acetylated N36 peptide was pre-pared for site-specific conjugation of FAM at Lys-574. All pep-tides were purified by reversed-phase HPLC to homogeneity,and their molecular masses were ascertained by electrosprayionization mass spectrometry. Peptide concentration wasquantified spectroscopically at 280 nm using molar extinctioncoefficients calculated according to the algorithm of Pace et al.(47).Size-exclusion chromatographic analysis of N36, C34, and

N36-C34 complexes was performed on a Superdex 75 10/300GL column (GEHealthcare) running PBS, pH 7.4, at a flow rateof 0.5 ml/min. The peptides were prepared in PBS at 10 �M

each, from which 100 �l was injected. Gel filtration molecularweight standardswere used for calibration. Fluorescence polar-ization assays were conducted in 384-well plates on a TecanInfiniteM1000multimode plate reader. The FAM-labeled N36peptide at 50 nM in PBS was incubated at room temperature for30 min with a 2-fold dilution series of unlabeled N36 or N36/C34 (0–25 �M, 100 �l total volume per well), and the polariza-tion values were determined at �ex � 470 nm and �em � 530nm. For defensin-induced N36 dissociation assays, 10 �M

N36 and 50 nM N-acetyl-N36-FAM in PBS were incubated atroom temperature with a 2-fold dilution series of HNP-1 orAbu-HNP (0–100 �M) for 30 min before polarizationmeasurements.Peptide precipitation induced by defensin was quantified

spectroscopically. Briefly, 40 �M soluble C34 or N36/C34 pre-pared in PBSwas incubated at room temperature with differentconcentrations of HNP-1 or Abu-HNP (0–120 �M) for 5 min,followed by mixing and turbidity measurements at 600 nm.Circular dichroism spectroscopic analysis of N36, C34, andN36/C34, each at 40�M,was performed in a 0.1-cmpath lengthcuvette on a Jasco-810 spectrometer. All peptideswere dialyzedseparately against 5 mM phosphate buffer, pH 7.2, overnightbefore CD measurements at room temperature. The N36/C346-helix bundle structures formed after equal volumes of N36and C34 peptides (80 �M) were mixed. Upon addition of differ-ent concentrations of HNP-1, the N36/C34 solution was pre-cipitated, and the supernatants were subjected to CD analysisafter centrifugation at 12,000 rpm.Virus Production andCharacterization—For the BlaM assay,

pseudoviruses were produced by cotransfecting a 100-mmdishof 293T cells using PolyFect transfection reagent (Qiagen Sci-ences). The transfectionmixture included the following: 2�g ofpR8�Env, 2 �g of BlaM-Vpr expressing pMM310 vector, 1 �gof pcRev, and 3 �g of pCAGGS encoding HIV-1 JRFL, BaL, orHXB2 Env. Control influenza or VSV G pseudoviruses were

produced as above, but using 2 �g of the pCAGGS vectorsexpressing the WSN H1N1 influenza hemagglutinin andneuraminidase or 3 �g of pMDG vector expressing VSV Ginstead of HIV-1 Env plasmids. After 12 h, the transfectionreagent was removed, and cells were further cultivated in phe-nol red-free media. Cell culture supernatant was collected at48 h post-transfection, passed through a 0.45-�m filter, ali-quoted, and stored at �80 °C. When required, viruses wereconcentrated by pelleting onto a 20% sucrose cushion. Thevirus pellet was resuspended in culture medium and stored at�80 °C in aliquots. The infectious titer of virus stocks wasdetermined using TZM-bl cells, as described previously (34).Virus-Cell and Cell-Cell Fusion Assays—HIV-1 fusion with

target cells was measured using the BlaM assay based on thetransfer of viral core-incorporated enzyme to the cytosol (48),as described previously (34). Briefly, HIV-1 pseudoviruses withdifferent coreceptor tropism (HXB2, JRFL, and BaL) werebound to TZM-bl cells by centrifugation at 4 °C for 30 min at1550 � g. PBMCs were resuspended in Hanks’ buffer andallowed to adhere to a poly-L-lysine-coated 96-well plate (2 �105 cells/well) for 30 min at room temperature and blockedwith 10% FBS-supplemented Hanks’ buffer. HXB2 pseudovi-ruses (4 � 105 IU/well) were pre-bound to PBMCs by centrifu-gation at 4 °C for 30 min at 1550 � g. After the virus bindingstep, TZM-bl cells or PBMCs were washed and incubated at37 °C for 90 min to allow virus entry. Fusion was stopped byplacing cells on ice and loading with the BlaM substrateCCF4-AM (Invitrogen). Following the overnight incubation at12 °C, the BlaM activity was determined from the ratio of blueand green fluorescence signals, using the Synergy HT fluores-cence plate reader (Bio-Tek Instruments, Germany).Fusion between indicator TZM-bl cells and HeLa-ADA cells

was measured using Tat-driven luciferase expression by theindicator cells. HeLa-ADA cells were detached from cultureplates using a nonenzymatic cell dissociation solution andadded to TZM-bl culture. After coculture for 2.5 h at 22 °C tocreate temperature-arrested stage (TAS) or at 4 °C in controlexperiments, cells were exposed to fusion inhibitors or leftuntreated and shifted to 37 °C for 1 h to initiate fusion. Cellfusion was stopped by adding C52L (5�M), and cells were incu-bated in the presence of the inhibitory peptide for 12 h at 37 °C.The extent of fusion was evaluated by a luciferase assay, usingSteady-Glo system (Promega, Madison, WI).CD4 and Coreceptor Expression—Surface expression of CD4

and coreceptors was determined by immunofluorescencestaining. For detection of CD4 and CCR5 expression, TZM-blcells were seeded into a 96-well plate (4.5�104 cells/well) on theday before the experiment. Cells were washed and pretreatedwith 5.8 �M HNP-1 (or Abu-HNP) or left untreated. In controlexperiments, the CD4 and CXCR4 expression was down-regu-lated by treatment with 0.2�g/ml PMA for 30–60min (49, 50),and CCR5 expression was reduced by treatment with CCR5agonist, regulated on activation normal T cell expressed andsecreted (400 nM). After preincubation with or without defen-sin, cells were washed with HBSS, 10% FBS and placed on ice.Cells were next washed with PBS supplemented with 0.1%sodium azide and 5% calf serum and incubated with primaryantibodies toCD4 (OKT4 conjugatedwithAlexa Fluor488, Bio-




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Legend), CCR5 (2D7) or with isotype-matched antibodies for1 h at 4 °C. Cells were washed and fixed with 3% PFA. Cellsincubated with unconjugated primary antibodies were furtherincubatedwith FITC-labeled secondary antibody for 1 h at 4 °C.Cell surface expression of CXCR4 in TZM-bl cells stably over-expressing this coreceptor (see above) were analyzed by immu-nofluorescence with allophycocyanin-conjugated anti-CXCR4antibodies (12G5). As a negative control for subtracting thebackground allophycocyanin signal, we used anti-CD8-allo-phycocyanin antibody. The overall fluorescence signal fromcells was measured by a plate reader, using a standard fluores-cein filter set. Parental HeLa cells lacking CD4 and CCR5 wereused as an additional control for expression of these receptors.HNP-1 Binding to Cells and Proteins—HNP-1 binding to

cells was measured using biotinylated HNP-1 (B-HNP) or itslinear analog (Abu-B-HNP). TZM-bl or parental HeLa cellswere incubated with B-HNP or its linear analog for 10 min onice, washed, and fixed with 2% PFA. After blocking with 5% FBSin PBS for 30 min, cells were incubated with streptavidin-HRPat 12 °C for 1 h. The amount of cell-associated B-HNP wasmeasured by cell ELISA using a chromogenic HRP substrate.HNP-1 binding to the tetrameric CD4-IgG2 protein (51) was

carried out by incubating 10 �g of native or deglycosylatedCD4-IgG2 with B-HNP or Abu-B-HNP for 1 h at 37 °C. Degly-cosylation was carried out using the endoglycosidase mixturefrom the enzymatic deglycosylation kit (Prozyme) consisting ofN-glycanase F (24 microunits/�l), O-glycanase (6 �units/ �l),and sialidase A (24 microunits/�l) in the final mixture. Thereaction was allowed to proceed for 24 h at 37 °C.Mouse IgG2a(BioLegend)was used as a negative control. Defensin binding toHIV-1 Env on virus particles was measured using a concen-tratedHXB2pseudovirus stock. Viruseswere allowed to adhereto polylysine-coated plates in the cold.Where indicated, viruseswere enzymatically deglycosylated (using a 3-fold higher con-centration of the enzymemixture comparedwith theCD4-IgG2deglycosylation protocol above) for 24 h at 37 °C before attachingthe virus to a plate. Immobilized viruses were incubated withB-HNP orAbu-B-HNP in the presence or absence of 10% FBS for1hat 37 °C.Unbounddefensinwas removedbywashingwithPBS,and viruses were lysed using 2mMEDTA and 1%Nonidet P-40 inPBS, pH 7.4, buffer for 30 min at 4 °C. The virus-bound B-HNPwas immunoprecipitated with streptavidin beads. Immunopre-cipitatedproteinswere separatedbySDS-PAGEunderdenaturingconditions and detected by immunoblotting with the anti-gp41Chessie8 monoclonal antibody (38).Internalization of Endocytic Markers and Viruses—To mea-

sure cellular uptake of endocytic markers, TZM-bl cells grownon 96-well plateswere preincubatedwith 80�Mdynasore for 30min or with 200 �M 5-(N,N-dimethyl)amiloride hydrochlorideinHBSS for 1 h at 37 °C or left untreated. Cells werewashed andincubated on ice for 20 min with Alexa488-conjugated Trf (20�g/ml) or DiI-labeled LDL (20 �g/ml). Cells were washed oncewith HBSS and, where indicated, exposed to 5.8 �M of HNP-1for 5min on ice prior to shifting to 37 °C for 10min to allowTrfuptake and 40min for LDL uptake in the presence or in absenceof HNP-1. Uptake of a fluid-phase marker, dextran-Alexa488(200 �g/ml), was assessed by adding the dextran to cells justbefore shifting to 37 °C and incubating for 60 min. Cells were

then chilled, washed, and treatedwith Pronase (2mg/ml) for 10min on ice to remove noninternalized markers. Cells were har-vested by an additional incubation with trypsin/EDTA (3 min at37 °C), washed, and analyzed by flow cytometry. HIV-1 uptake bycells was measured as described previously (52, 53). Briefly, pH-sensing pseudoparticles were produced by incorporating theEcliptic pHluorin-ICAM-1construct (EcpH-ICAM) into the virusmembrane and HIV-1 Gag-mCherry into their core. The EcpHquenching atmildly acidic pHpermitsmonitoring of virus uptakeand entry into acidic intracellular compartments.FRAP and FCS Measurements—Fluorescence recovery after

photobleaching (FRAP) was used to assess the changes in thelateral mobility of membrane proteins after HNP-1 treatment.HeLa cells grown on glass-bottomPetri dishes were transfectedwith plasmids encoding CD4-YFP, CCR5-GFP, or CXCR4-GFP. Twenty four hours post-transfection, cells were trans-ferred into Hanks’ buffer, mounted on the Zeiss LSM780 laserscanning confocal microscope, and visualized with the�63/1.4NA oil immersion objective. Experiments were carried out atroom temperature tominimize cell movement. Flat cells express-ing low tomoderate amounts of fluorescent proteins localizedpri-marily at the plasmamembranewere selected for photobleaching.Measurements were performed using the FRAPmodule availablethrough the Zen software (Carl Zeiss MicroImaging). Small rec-tangular regions of interest along the cell plasmamembrane werebleached by a brief exposure to a 488 laser at 100% intensity. Wedid not use the conventional circular regions or long strips acrossthe cell (54, 55) for photobleaching to avoid the contribution froman intracellular pool of fluorescent proteins. Instead, we selectedsmall rectangular regions (�1� 2�m) encompassing the plasmamembrane (see below) andparameterized the fluorescence recov-ery using the mobile fraction and half-time, as determined by theZen software.For FCS (56, 57) measurements, we used a Zeiss LSM780

microscope equipped with hybrid GaAsP detectors and a Con-Focor �40 (1.2 NA) water immersion objective. CCR5-GFPwas excitedwith a continuous 488 nmargon laser. Zen softwarecontrolled the data acquisition and displayed autocorrelationcurves. Collected data were processed off-line using the Originsoftware package (OriginLab, Northampton, MA). The waist(�0) of the excitation beam was calibrated before experimentsby measuring the autocorrelation function of different concen-trations of fluorescein in solution (supplemental Fig. 1), using apredetermined diffusion coefficient of 300�m2/s (58, 59). Typ-ical�0 values obtained for our setup were 0.25–0.30�m.Auto-correlation functions were fitted assuming a two-dimensionalBrownian diffusion with fast triplet state kinetics and autofluo-rescent species exhibiting fast three-dimensional diffusion, asdescribed previously (60) and in Equation 1,

G�� 1 1

N�F1��1

�

�D1� ��1

�o

�z

�

�D1��1

�1 � F1�1 � �

�D2��1�1

Te��

�T

1 � T�� offset (Eq. 1)

where N is the number of particles; F1 is the fraction of three-dimensional species; �0 is the waist of the confocal excitation




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volume;�z is the axial radius of the excitation volume; �D1 is theresidence time for three-dimensional diffusing species; �D2 isthe residence time for two-dimensional species; T is the ampli-tude of the triplet state, and �t the triplet lifetime. Autocorrela-tion curves were analyzed assuming a Gaussian-Lorentzianexcitation volume. The beamwas positioned at the periphery ofCCR5-expressing cells, and the average autocorrelation func-tion from 10measurements with an acquisition time of 5 s eachwas obtained and analyzed. Abnormal individual traces due tophotobleaching or cell movement were not taken into account.

RESULTS

Defensin Inhibits HIV-1 Env-mediated Fusion—We exam-ined the ability of the human neutrophil peptide 1 (HNP-1,hereafter also referred to as defensin) to block HIV-1 fusion,using a direct virus-cell fusion assay based on the cytosolicdelivery of the �-lactamase (BlaM) incorporated into the pseu-dovirus core (34, 48). Pseudoviruses bearing different HIV-1Env glycoproteins were pre-bound in the cold to HeLa-derivedTZM-bl cells expressingCD4,CCR5, andCXCR4, and the virusentry/fusion was triggered by shifting to 37 °C in the presence

of varied concentrations of defensin. HNP-1 inhibited virus-cell fusion in a dose-dependent manner, irrespective of thecoreceptor-tropism (Fig. 1A). However, R5-tropic viruses(JRFL and BaL) were somewhat more resistant to inhibitionthan the laboratory-adapted X4-tropic HXB2 strain. Inhibitionof HIV-1 fusion was not due to compromised cell viability (Fig.1A). In control experiments, we tested the activity of the linear-ized version of defensin (Abu-HNP), in which all cysteine resi-dues were replaced with aminobutyric acid. The unstructuredpeptide, which possessed the same net charge as HNP-1, didnot noticeably affect HIV-1 fusion, even at high concentrations(Fig. 1B). Thus, it is the structure of defensin, but not its primarysequence, that is essential for the antiviral activity. Note that,because HNP-1 was applied to cells after virus binding, it couldnot affect the virus binding step. Also, pretreatment of pseudo-viruses with a fully inhibitory concentration of defensin prior toaddition to target cells did not interfere with the virus binding(data not shown).It has been previously observed that bovine and human sera

strongly attenuate the anti-HIV-1 activity of HNP-1 and otherdefensins (4, 22, 25, 26, 61). This attenuation is thought to be

FIGURE 1. Inhibition of HIV-1 fusion by HNP-1. A, HXB2, JRFL, and BaL envelope-pseudotyped viruses were fused with TZM-bl cells in the presence ofincreasing concentrations of defensin. Cell viability (black stars) was measured by a 3-(4,7-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2H-tetrazoliumassay using Promega CellTiter 96� AQueous One Solution Cell Proliferation Assay according to the manufacturer’s instructions. Unless stated otherwise, datapoints are means S.E. for at least two independent experiments performed in triplicate. B, titration curves of HNP-1 were obtained in the presence of 10%human (HS) or bovine (BS) serum in Hank’s buffer or in serum-free buffer after incubation of cell-bound viruses with defensin at 37 °C for 90 min. The linearanalog of HNP-1 (Abu-HNP, filled circles) was used as a control. Data are means S.D. for representative experiments. C, HXB2 pseudoviruses were pre-boundto activated PBMCs adhered to a polylysine-coated 96-well plate by centrifugation in the cold. Virus fusion was initiated by shifting to 37 °C and incubating for90 min in the absence (control) or in the presence of varied concentrations of HNP-1 in a serum-free medium. In control experiments, 2 �M C52L was added tocells to fully block fusion. The extent of fusion was measured by the BlaM assay. Data for 5.8 �M HNP-1 are from a single experiment performed in triplicate.D, time course of HXB2 pseudovirus escape from fusion inhibitors. Viruses were pre-bound to cells in the cold, and their entry was initiated by shifting to 37 °C.Fully inhibitory concentrations of HNP-1 and inhibitors of CD4-binding (BMS-806), coreceptor-binding (AMD3100), and six-helix bundle formation (C52L) wereadded at indicated time points. Following the addition of fusion inhibitors, cells were loaded with the BlaM substrate, and the extent of virus-cell fusion wasquantified after overnight incubation at 12 °C. Larger error bars reflect experimental variability when using different batches of the virus.




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due to defensin binding to serum glycoproteins (32, 62). In fullagreement with these studies, we found that the inhibitoryactivity of HNP-1 was markedly diminished in media contain-ing human or bovine serum (Fig. 1B). The IC50 value againstHXB2 pseudoviruses increased from 1.2 to�10�M in the pres-ence of bovine serum. For this reason, experiments were con-ducted in the absence of serum, unless indicated otherwise.Next, we examined the effect of this defensin on HIV-1

fusion with human PBMCs. As with HeLa-derived target cells,pseudoviruses were pre-bound to PBMCs in the cold, andfusion was initiated by raising the temperature in the presenceor in the absence of varied concentrations of HNP-1. HIV-1fusion with PBMCs was inhibited by this defensin (Fig. 1C),albeit at higher concentrations compared with those inhibitingfusion with TZM-bl cells. Even at this high concentration,HNP-1 did not affect cell viability (data not shown).HIV-1 entry is a multistep process that progresses through

CD4 binding, coreceptor engagement, and endocytosis, whichculminates in virus-endosome fusion (34, 35). It has been pre-viously reported that �-defensin inhibits Env-receptor interac-tions by binding to both CD4 and gp120 and, at the same time,by down-modulating the CD4 expression on the cell surface(25). To assess which step(s) of HIV-1 entry are inhibited bydefensin, the time course of virus escape from inhibition bydefensin was measured and compared with the rate of escapefrom inhibitors targeting distinct steps of HIV-cell fusion (35).Fusion was stopped at the indicated time points by adding fullyinhibitory concentrations of BMS-806 (to assess the receptorbinding (63)). The kinetics of CXCR4 binding was determinedby adding a high concentration of AMD3100 (64, 65) or T22(66). Finally, the recombinant C52L peptide derived from thegp41 second heptad repeat domain was used to monitor theprogression through late steps of fusion. C52L inhibits Env-mediated fusion by blocking the formation of the final gp416-helix bundle structure (42, 67). Because HIV-1 enters HeLa-derived cells by fusing with endosomes (34), escape from themembrane-impermeant C52L should reflect the kinetics ofproductive endocytosis and not the kinetics of virus fusion atthe cell surface (35).We have previously found that HXB2 binding to CD4 pre-

ceded its binding to CXCR4, which in turn was faster than pro-ductive endocytosis (escape from C52L, see Fig. 1D) (35). Thetime course of HXB2 escape from a fully inhibitory concentra-tion of defensin clearly lagged behind the CD4 and coreceptorbinding steps and was close to virus escape from C52L. Similarresults were obtained for the R5-tropic HIV-1 pseudoviruses(data not shown). Thus, both C52L and defensin appear toinhibit HIV-1 fusion at the stage just prior to productive endo-cytosis. It must be stressed, however, that the sustained sensi-tivity to defensin throughout the surface-accessible stages offusion does not rule out inhibition of earlier steps of this processby defensin. The time-of-addition method reveals only the lat-est step of fusion blocked by an inhibitor. To assess the effect ofdefensin on early steps of HIV-1 entry, we employed alternativestrategies described below.HIV-1 Remains Fusion-competent in the Presence of Defensin—

The effect of HNP-1 on the ability of HIV-1 to undergo fusionwas tested by pre-exposing the cell-bound virus to defensin for

different time intervals, washing with a serum-containingmedium, and incubating for 90min at 37 °C to allow fusion (Fig.2,diagram). Remarkably,HIV-1 retained the ability to fusewithTZM-bl cells in the presence of a fully inhibitory concentrationof defensin for at least 1 h at 37 °C. The extent of HXB2 fusionwas only slightly reduced following a prolonged incubationwith defensin, whereas the BaL fusion even tended to increasewithin the first 30 min of incubation (Fig. 2, A–C, open circles).In contrast, pretreatment of cell-bound viruses with conca-navalin A resulted in a quick loss of fusion activity (data notshown). A small molecule dynamin inhibitor, dynasore, whichblocked HIV-1 endocytosis, also irreversibly inactivated thevirus (33). Because infectivity assays widely employed to assessthe effect of�-defensins onHIV-1 require returning target cellsto a complete growth medium, the inhibitory effect of thisdefensin on virus entry could be grossly underestimated. Ourfunctional experiments provide the first demonstration ofreversibility of the HNP-1 block of HIV-1 fusion.HIV-1 Env Can Bind CD4 and CCR5 in the Presence of

Defensin—To identify the defensin-sensitive step(s) of HIV-1entry, we examined the ability of the virus to engage CD4 andcoreceptors and to undergo fusion in the presence of defensin.Viruses were pre-bound to cells in the cold and incubated witha fully inhibitory concentration of defensin for varied times at37 °C to allow Env to engage the receptor and coreceptor.HNP-1was removed bywashingwith FBS-containingmedium,and viruseswere further incubatedwith cells for 90min at 37 °Cin the presence of high doses of HIV-1 fusion inhibitors. Anyfusion observed under these conditions could only occur as aresult of irreversible engagement of CD4 or bothCD4 and core-ceptor (depending on the fusion inhibitor employed) by HIV-1Env in the presence of defensin. As in the time-of-additionexperiment (Fig. 1D), Env-CD4 binding was assessed by replac-ing defensin with BMS-806, whereas the formation of ternaryEnv-CD4-coreceptor complexes was derived from the extent offusion after addition of AMD3100 or TAK-779.As expected, fusion was not detected when HNP-1 was

replaced with fusion inhibitors before shifting to 37 °C (Fig. 2,t � 0). In some experiments, however, the inhibition of HXB2fusion upon replacing defensin with BMS-806 was incomplete,consistent with the limited CD4 engagement at 4 °C, which wasalso apparent in the time-of addition experiments (Fig. 1D)(35). Upon incubation with defensin at 37 °C, HXB2 particlesefficiently engagedCD4, as evidenced by�60%of fusion occur-ring upon substituting HNP-1 with BMS-806 compared withcontrol experiments using HBSS instead of the fusion inhibitor(Fig. 2A). However, both the small molecule (AMD3100) andpeptide (T22) inhibitors of CXCR4 binding blocked fusion,irrespective of the time of preincubation with defensin (Fig. 2A,squares and upright triangles). Thus, HNP-1 allowed partialformation of HXB2-CD4 complexes but blocked the subse-quent binding of CXCR4. Overexpressing CXCR4 in TZM-blcells did not rescue the ability of HIV-1 to engage this corecep-tor in the presence of defensin (supplemental Fig. 2). To con-clude, defensin blocks HXB2 entry at the coreceptor bindingstep while permitting partial engagement of CD4.Next, we examined the defensin-sensitive stage(s) of fusion

induced by the CCR5-tropic BaL pseudovirus. In accordance




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with a somewhat lower apparent binding affinity of BaL to CD4compared with HXB2 (35), only �20% of viruses engaged CD4in the presence of defensin (Fig. 2B). However, CCR5 bindingwas relatively efficient, as evidenced by the comparable extentsof fusion in the presence of BMS-806 and TAK-779 after a pro-longed incubation with defensin (Fig. 2B, triangles versussquares). Note, however, that the CCR5 binding was delayedrelative to CD4 binding, whichwas in contrast to the fast rate ofCCR5 engagement observed in the time of inhibitor additionexperiments (35). Delayed CCR5 binding could be indicative ofcompetition between the virus and defensin for coreceptorbinding.

To further assess whether defensin selectively interferes withEnv-CXCR4 interactions, we examined the fusion activity ofthe chimeric HXB2 Env, in which the gp120 V3-loop was sub-stituted by that of BaL (designated V3BaL) (68). This chimerauses CCR5 for infection and fusion and binds this coreceptorwith a higher affinity compared with the HXB2-CXCR4 bind-ing (68, 69). V3BaL engaged both CD4 and CCR5 in the pres-ence of HNP-1 (Fig. 2C). Nearly 60% of viruses that formedfunctional complexes with CD4 also bound CCR5 (Fig. 2C, tri-angles versus squares), but defensin markedly slowed down theCCR5 binding step compared with experiments in the absenceof HNP-1 (35). Taken together, our findings show that HNP-1

FIGURE 2. HNP-1 interferes with multiple steps of HIV-1 entry. To identify the defensin-sensitive stages of HIV-1 fusion, HXB2 (A), BaL (B), and chimeric V3BaL(C) pseudoviruses were pre-bound to TZM-bl in the cold and incubated at 37 °C in the presence of 5.8 �M HNP-1. After varied times of incubation, cells werewashed with a serum-supplemented Hanks’ buffer and overlaid with the same buffer lacking (circles) or containing HIV-1 fusion inhibitors, BMS-806 (downwardtriangles), AMD3100 (A, squares) or TAK-779 (B and C, squares), T22 (upright triangles), and C52L (diamonds). Cells were incubated for additional 90 min at 37 °C,and the resulting fusion was measured by the BlaM assay. The experimental protocol is shown schematically on the top panel. Data are means S.E. for twoindependent experiments performed in duplicate. D, down-modulation of CD4 expression by HNP-1. After the indicated times of pretreatment with HNP-1(black circles) or Abu-HNP (open circles), TZM-bl cells were incubated with Alexa488-conjugated anti-CD4 antibody OKT4 for 1 h on ice, washed, and fixed with3% paraformaldehyde. The relative surface expression of CD4 was determined based on the overall fluorescent signal from the cells in a microplate well. As apositive control for CD4 down-regulation, cells were treated with PMA (black triangles). Kinetics of HXB2 and BaL binding to CD4 are shown with dashed andsolid lines, respectively.




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targets both CD4 and coreceptor binding steps. It slows downhigh affinity interactions (BaL-CCR5 binding), while attenuat-ing or even blocking weak interactions (BaL-CD4 and HXB2-CXCR4 binding).The block of BaL fusion, despite its limited binding to CD4

and CCR5, shows that defensin also targets downstream stepsof virus entry. Indeed, following a 1-h incubation with HNP-1,all pseudoviruses tested in our experiments remained sensitiveto C52L (Fig. 2, A–C, diamonds). The lack of resistance to thisgp41 6-helix bundle inhibitor suggests that all fusion-compe-tent viruses remained at the cell surface, accessible to C52L. Inother words, defensin appears to prevent HIV-1 endocytosiswithout significantly inactivating the virus. To gain furtherinsights into the mechanism of HNP-1 action, we assessed itseffect on the surface expression of CD4/coreceptors, as well ason the late steps of HIV-1 entry and fusion.HNP-1 Diminishes Cell Surface Expression of CD4 and

CXCR4 but Not CCR5—The reduced or delayed virus bindingto CD4 and CCR5 and failure to engage CXCR4 in the presenceof defensin (Fig. 2) could be due, at least in part, to induction ofCD4 and/or coreceptor endocytosis. For PBMCs and HOS-de-rived cells engineered to express CD4 and coreceptors, pro-longed incubation with HNP-1 or other defensins has beenreported to slightly down-modulate the CXCR4 but not CD4 orCCR5 expression (17). In contrast, another study (25) has foundthatHNP-1 andHNP-2 down-regulate theCD4 expression, butnot CCR5 or CXCR4 expression in PM-1 cells. We thereforesought to determine whether HNP-1 reduced the surface levelsof CD4 or coreceptors in TZM-bl cells on a time scale relevantto HIV-1 entry.Attempts to measure expression levels by flow cytometry

were not successful, because, surprisingly, defensin strength-ened the cell attachment to plates. We thus had difficulty har-vesting defensin-treated TZM-bl cells with a nonenzymaticsolution without damaging them. For this reason, surfaceexpression of CD4/coreceptors was measured using a fluores-cence plate reader. To rule out competition between defensinand antibodies for binding to CD4, we used the monoclonalantibody OKT4 against the membrane-proximal domain ofCD4, because binding of this antibody to CD4 is not affected by�-defensins (25). We found that HNP-1, but not Abu-HNP,quickly (half-time �18 min) decreased the CD4 expression(Fig. 2D andTable 1). The defensin effect onCXCR4 expressionwas determined using TZM-CXCR4 cells overexpressing thiscoreceptor. Pretreatment with HNP-1 or PMA (as a positivecontrol (50)) markedly reduced the surface density of this core-ceptor (Table 1). Down-regulation of CXCR4 expression maybe the reason for the block of HIV-CXCR4 binding by defensin

(Fig. 2A). By comparison, theCCR5 expressionwas not affectedby defensin, whereas the CCR5 agonist, regulated on activationnormal T cell expressed and secreted, significantly reduced thenumber of coreceptor molecules on the cell surface (Table 1).It is striking that HXB2 and, to some extent, BaL were able to

engage a requisite number of CD4 and undergo fusion (Fig. 2,A–C), despite rapid CD4 clearance from the cell surface (Fig.2D). The HIV-CD4 binding in the presence of defensin likelyoccurs in kinetic competition with receptor endocytosis. Thegreater extent of CD4 engagement by HXB2 compared withBaL is consistent with the faster receptor binding by the formerEnv, as measured by the rate of virus escape from fusion inhib-itors (Fig. 2D) (35). To conclude, defensin inhibits HIV-1 bind-ing to CD4 and CXCR4 through down-regulating their expres-sion and potentially through interfering with Env-coreceptorinteractions, as suggested by delayed CCR5 engagement by Envin the presence of HNP-1 (Fig. 2, B and C).HNP-1 Binds to Viral and Host Glycoproteins in Glycan- and

Serum-independent Manner—We measured the defensinbinding to cells and viruses using B-HNP. In control experi-ments, B-HNP inhibited HIV fusion even somewhat more effi-ciently than wild type defensin, and its activity was grosslydiminished in the presence of serum (Fig. 3A). To measuredefensin binding to cells, TZM-bl cells were incubated withB-HNP, washed with serum-containing medium to removeunbound peptide, fixed, and incubated with streptavidin-HRP.The amount of cell-associated B-HNP was determined by acell-based ELISA. Although the fusion inhibitory effect ofdefensin was strongly attenuated by serum (Figs. 1B and 3A),B-HNP bound to cells equally well in the presence and in theabsence of serum (Fig. 3B). Once bound, B-HNP could not beremoved from cells by multiple washes with serum-supple-mentedmedium. By comparison, the linear biotinylated defen-sin (designated Abu-B-HNP) bound poorly to cells even inserum-freemedium (Fig. 3C). This demonstrates that the struc-ture of the defensin is essential for binding to cells and thatbiotinylation does not significantly alter this activity. The over-all binding of B-HNP was not affected by expression of CD4-and CCR5, as evidenced by its equal association with TZM-bl and parental HeLa cells (Fig. 3C). The B-HNP-cell bindingappeared specific, because it was saturable (Fig. 3C) and couldbe displaced by an excess of nonbiotinylated HNP-1 but not byAbu-HNP (Fig. 3D).The fact that theB-HNPbinding to cells is strikingly different

from its reversible effect on virus fusion (Figs. 1 and 2) impliesthat this defensin either interacts with cell surface moleculesother thanCD4or coreceptors or its binding is not sufficient forinhibition of HIV-1 entry. We therefore tested whether defen-

TABLE 1Effect of HNP-1 on expression of CD4, CXCR4, and CCR5 on TZM-bl and TZM-CXCR4 cellsSurface expression of CD4 and coreceptors was measured as described under “Experimental Procedures” using a fluorescence plate reader after 45 min (CD4) or 60min (coreceptors) of HNP-1 pretreatment (5.8 �M) at 37 °C. In control experiments, cells were pretreated with 0.2 �g/ml PMA. Data are means S.E. of triplicatemeasurements.

Treatment CD4 (% of untreated) CXCR4a (% of untreated) CCR5 (% of untreated)

HNP-1 28.6 3.3 (n � 3) 22.1 6.8 (n � 6) 93.7 0.8 (n � 3)

PMA or RANTESb 23.4 6.7 (n � 3) 57.9 5.3 (n � 5) 64.7b 0.8 (n � 3)a Effect of defensin on the CXCR4 expression was determined on TZM-bl cells overexpressing this coreceptor (see supplemental Fig. 2).b Cells were preincubated with 400 nM regulated on activation normal T cell expressed and secreted (RANTES) for 60 min.




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sin binds to CD4 and gp41 in the presence of serum andwhether this binding was glycan-dependent, as has been shownfor another defensin, retrocyclin (21, 22). First, we examinedbinding to the recombinant tetrameric CD4-IgG2 protein con-taining the N-terminal D1 and D2 domains of CD4 (51), ofwhich the first domain interacts with HIV-1 gp120 (70).Although D1-D2 domains of CD4 are not glycosylated (51), wenonetheless tested the B-HNP binding to both untreated andenzymatically deglycosylated CD4-IgG2. As expected, no sig-nificant shift in the mobility of the recombinant protein couldbe observed following deglycosylation (Fig. 4A). The B-HNPbinding to CD4-IgG2 was then assessed by immunoprecipita-tion with streptavidin-agarose beads followed by immunoblot-ting. The ability of defensin to effectively immunoprecipitateboth untreated and glycosidase-treated CD4-IgG2 (Fig. 4A)demonstrated that its binding to the N-terminal domains ofCD4 was glycan-independent.Next, we examined the defensin binding to a native or enzy-

matically deglycosylated HIV-1 Env on virus particles. Theappearance of a lower band (�37 kDa) on Western blots fol-lowing a prolonged enzymatic treatment implies that a fraction

of gp41 was fully deglycosylated (Fig. 4B, lanes 4 and 6). Tomeasure Env-HNP binding, viruses (treated or untreated) wereimmobilized on microplate wells, incubated with B-HNP,washed, and lysed. Defensin-Env complexes were precipitatedwith streptavidin beads and subjected to SDS-PAGE. Blottingwith an anti-gp41 antibody revealed that HNP-1 efficientlybound glycan-free gp41, as evidenced by similar relative inten-sities of upper and lower bands in lysate and in immunoprecipi-tated samples (Fig. 4B, lanes 4 and 6). In control experiments,linear Abu-B-HNP poorly precipitated either native or glycan-free Env (Fig. 4B, lanes 7–9). Interestingly, a combination ofgp41 deglycosylation with the defensin pulldown in the pres-ence of serum enhanced the band intensity for bothHNP-1 andAbu-HNP samples (Fig. 4B, lanes 6 and 10). This effect could bedue to the better binding of the Chessie8 antibody to gp41.Together, these data imply that glycans are not essential forHNP-1 binding to gp41 andCD4 and that serumdoes not inter-fere with defensin-gp41 interactions.The lack of efficient inhibition of HIV-1 fusion by defensins

in the presence of serumhas been thought to reflect diminishedbinding to relevant targets. Our results revealed a striking dis-

FIGURE 3. HNP-1 binding to cells. A, HXB2 pseudovirus fusion to TZM-bl cells was measured in the presence of different concentrations of HNP-1 in theabsence of serum (open circles) or with B-HNP in the absence (open triangles) or in the presence of 10% bovine serum (filled circles). Data are means S.D. fora representative experiment performed in triplicate. B, B-HNP binding to TZM-bl cells is not inhibited by 10% bovine serum. Data are means S.D. for twoindependent experiments performed in duplicate. C, binding of B-HNP or Abu-B-HNP to TZM-bl cells and to parental HeLa cells. Cells were incubated withvaried concentrations of defensins for 10 min on ice, washed once, fixed with 2% paraformaldehyde for 15 min, and blocked with 5% FBS in PBS for 30 min.B-HNP binding was quantified using streptavidin-HRP. D, untagged HNP-1, but not Abu-HNP, competes with B-HNP binding to TZM-bl cells. Cells wereincubated with increasing concentrations of HNP-1 or Abu-HNP in the presence of 0.5 �M of B-HNP on ice for 10 min. Cells were washed, and the amount ofcell-associated B-HNP was measured by whole-cell ELISA, using a chromogenic HRP substrate. Data in C and D are means S.D. for a representative experimentperformed in triplicate.




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cordance between the defensin binding to proteins involved inHIV-1 fusion and its ability to block virus entry. Whereas inhi-bition of HIV-1 fusion was attenuated in the presence of serumand could be readily reversed bywashing off HNP-1, its bindingto cellular and virus proteins was virtually irreversible. Thus,defensin binding to Env and CD4 (and likely to coreceptors) isnot sufficient for its entry inhibitory activity. Because theHNP-1 oligomerization might be essential for the ability toblock fusion (71), we surmised that the formation of function-ally important higher order oligomers could be inhibited byserum.HNP-1 Reduces the Mobile Fraction of CD4 and Coreceptors

on the Cell Surface—We next asked whether defensin couldinhibit HIV-1 fusion by cross-linking the cell surface glycopro-teins. Cross-linking would “freeze” glycoproteins on the cellsurface and interfere with the virus fusion (22). To test this

notion, wemeasured themobility of proteins on the cell surfaceby FRAP (55, 72). Lateral diffusion of cross-linked membraneproteins is slower and/or more restricted than that of free pro-teins. For these experiments, fluorescently labeledCD4or core-ceptors were individually expressed in HeLa cells by transienttransfection. Small rectangular regions along the plasmamem-brane (Fig. 5A) were photobleached, and fluorescence recoveryover time was monitored. The mobile fraction of fluorophoresand the half-time of fluorescence recovery (Table 2) werederived from the experimental curves as described previously(54). Because of the CD4 and CXCR4 down-regulation byHNP-1 (Table 1), incubation with defensin was carried out atroom temperature (to slow down endocytosis) and was limitedto 20–25 min. We found that concanavalin A diminished therate of fluorescence recovery (Fig. 5B). By contrast, defensinsignificantly reduced themobile fraction of CD4 and both core-ceptors, consistent with its ability to cross-link a fraction ofthese glycoproteins, but it did not reduce their lateral diffusion.It is difficult to assess whether the modest reduction of themobile fraction of CD4 and coreceptors in the absence of sig-nificant mobility changes could account for the potent inhibi-tion of HIV-1 fusion by this defensin.To more quantitatively assess the effect of HNP-1 on mobil-

ity of membrane proteins, we employed FCS (56, 57), whichmeasures diffusion coefficients/residence time and the concen-tration of a fluorophore based on fluctuations of fluorescenceintensity in a small confocal volume. The 488 nm laser beamwas parked at selected locations on the plasma membrane ofTZM-bl cells transiently expressing CCR5-GFP (Fig. 5C), andthe resulting fluorescence signal was acquired and analyzed.The decay of the autocorrelation functions (Fig. 5, D–F) wasbest fitted with a two-component model (described under“Experimental Procedures”) accounting to fast three-dimen-sional diffusion in the cytosol (an average residence time of�200 �s) and much slower lateral diffusion within the plasmamembrane (an average residence time of �30 ms). In agree-ment with the FRAP data, preincubation with HNP-1 did notsignificantly alter the lateral mobility of this coreceptor (Fig. 5,D and F). As expected, mild fixation of cells with PFAmarkedlydiminished the CCR5-GFP mobility, as evidenced by a muchhigher residence time following the fixation step (Fig. 5E). TheFCS data are summarized in Table 3. To conclude, both FCSand FRAP measurements showed that HNP-1 did not signifi-cantly alter the mobility of CD4 or coreceptors, while reducingthe mobile fraction of these proteins. The latter effect is con-sistent with clustering and/or immobilization of a fraction ofCD4 and coreceptor molecules on the cell surface.Defensin Inhibits HIV-1 Fusion at a Post-coreceptor Binding

Stage—Having demonstrated that HNP-1 down-regulates CD4and CXCR4 expression and slows down Env-CCR5 binding(Fig. 2, B and C), we sought to determine whether this peptidecan also act downstream of the ternary Env-CD4-CXCR4 com-plex formation, as suggested by the time-of-addition data (Fig.1D). Toward this goal, a temperature-arrested stage (TAS) wascreated by pre-binding the virus to cells in the cold and incu-bating at a temperature just below the threshold for fusion (33,34). HIV-1 Env-pseudoviruses were bound to TZM-bl cells inthe cold and incubated at 23 or at 4 °C (control samples) for 3 h.

FIGURE 4. Defensin binding to CD4 and HIV-1 envelope glycoprotein.CD4-IgG2 chimeric protein (51) (A) or HXB2 pseudoviruses (B) were leftuntreated or subjected to enzymatic deglycosylation, as described under“Experimental Procedures.” A, 10 �g of CD4-IgG2 was incubated with 30 �M

B-HNP or Abu-B-HNP for 1 h at 37 °C, and biotin-bound proteins were immu-noprecipitated (IP) by streptavidin-agarose resin. B, 7�104 IU of concentratedHXB2 pseudoviruses were allowed to adhere to polylysine-coated plates,washed, and incubated with 28.7 �M B-HNP or Abu-B-HNP in the presence orabsence of 10% FBS for 1 h at 37 °C. Immobilized viruses were washed twicewith PBS and lysed in PBS supplemented with 2 mM EDTA and 1% NonidetP-40 for 30 min at 4 °C. Biotinylated defensins were precipitated with strepta-vidin-agarose beads for 2 h in the cold. For both CD4-IgG2 and HXB2 samples,the beads were washed, and the precipitated proteins were analyzed byWestern blotting using protein G-HRP for CD4-IgG2 or the Chessie8 monoclo-nal antibody against gp41. Mouse IgG2a was used as a control for CD4-IgG2.




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The formation of ternary Env-CD4-coreceptor complexes atTAS was ascertained based on the acquisition of resistance tohigh concentrations of CD4 and coreceptor binding inhibitors(40 �M BMS-806 and 10 �M AMD3100, respectively) uponshifting cells to 37 °C. Under these conditions, any fusion signal(measured by the BlaM assay) must originate from viruses thathave formed functional ternary complexes with CD4 andcoreceptors.Fig. 6A shows that preincubation at 23 °C rendered a fraction

of viruses resistant to receptor and coreceptor binding inhibi-

tors added prior to raising the temperature. In control experi-ments, when viruses and cells were preincubated at 4 °C for 3 h,a partial protection against BMS-806 inhibition was observed,consistent with the ability of Env to slowly bind CD4 in the cold(Figs. 1D and 2D) (33–35). The cold-incubated virus remainedfully sensitive to AMD3100. By contrast, almost half of thefusion-competent viruses engaged both CD4 and CXCR4 atTAS, as evidenced by considerable fusion in the presence ofAMD3100. The nearly complete inhibition of fusion by C52Ladded at TAS rules out the possibility that viruses were pro-

FIGURE 5. Effect of HNP-1 on the lateral mobility of CD4 and coreceptors. A, image illustrating the FRAP measurements for HeLa cells transfected withCCR5-GFP. B, CCR5-GFP fluorescence recovery for untreated (black line), as well as HNP-1-treated (red line) and concanavalin A-treated (dotted line) cells.Determination of the mobile fraction and t1⁄2 (shown for untreated cells) is illustrated. C–F, FCS measurements of CCR5-GFP mobility in the plasma membrane.C, confocal image of a TZM-bl cell expressing CCR5-GFP merged with the differential interference contrast channel. The white cross marks the position of thelaser beam during one of the FCS measurements. D, normalized autocorrelation curve (green dots) for control cells. The characteristic residence times deter-mined by fitting (solid line) are �1 � 200 �s and �2 � 30 ms (assuming a Gaussian volume). E, normalized autocorrelation curve (red dots) for cells pretreated withPFA (0.8%). Optimal fitting (solid line) was obtained for �1 � 400 �s and �2 � 403 ms. F, normalized autocorrelation curve (black dots) for cells pretreated with8.7 �M HNP-1 for 30 min at room temperature. Two characteristic residence times, �1 � 270 �s and �2 � 34 ms, were obtained by curve fitting (solid line). Insetsto D–F show the curve fitting residuals.




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tected from receptor/coreceptor binding inhibitors by under-going productive endocytosis (33, 34). Importantly, defensinadded at TAS potently inhibited fusion to the extent compara-ble with that of C52L (Fig. 6A), demonstrating that HNP-1 canact at a post-coreceptor binding steps of HIV-1 entry. We fur-ther tested the ability of defensin to block fusion from TASusing a cell-cell fusion model. HIV-1 ADA Env-expressingeffector cells were mixed with TZM-bl cells and preincubatedat 2.5 h at 22 °C, a temperature that was not permissive forfusion (73, 74). After establishing TAS, a large fraction of cellsbecame resistant to high concentrations of BMS-806 and of theCCR5 antagonist AD101. By contrast, �-defensin and C52Lblocked fusion when added after the formation of ternary Env-CD4-CCR5 complexes at 22 °C prior to raising the temperature(Fig. 6B).Collectively, our data imply that defensin not only interferes

with the CD4 and/or coreceptor binding steps, but also targetsdownstream steps of HIV-1 fusion. The ability to inhibit cell-cell fusion from TAS and to bind gp41 (Fig. 4B) suggests that�-defensin may interfere with the gp41 refolding into the final6-helix bundle in a manner similar to the �-defensin, retrocy-clin (23).HNP-1 Inhibits Trimerization of the gp41 N-terminal Coiled

Coil Domain and Binds to Both the C-terminal Domain and the6-Helix Bundle Structure—We first examined the ability ofdefensin to bind to the heptad repeat domains of gp41 thatcompose the final 6-helix bundle structure. The N36 and C34peptides derived from the N- and C-terminal heptad repeatdomains of gp41, when mixed at a 1:1 molar ratio, form thestable post-fusion core, where three helices of N36 associateinto the central coiled coil with hydrophobic grooves on itssurface occupied by three helices ofC34 in a antiparallel fashion(for review see Ref. 67). Consistent with these structural fea-tures, an equalmolar (10�M)mixture of syntheticN36 andC34peptides eluted predominantly as a hexamer on size-exclusionchromatography (Fig. 7A). By contrast, whereas N36 had a

tendency to trimerize by itself, C34 existed only as a monomerat the same concentration in solution (Fig. 7A). Concentration-dependent N36 trimerization and N36/C34 formation of the6-helix bundle structure were independently verified by fluo-rescence polarization studies, using a FAM-labeled N36 pep-tide as a fluorescent indicator (Fig. 7B).To assess the ability of defensin to interfere with gp41 refold-

ing into the 6-helix bundle structure, we incubated differentconcentrations of HNP-1 or Abu-HNPwith trimeric N36 lacedwith the fluorescent indicator peptide N-acetyl-N36-FAM. Asthe HNP-1 concentration increased from 0 to 100 �M, fluores-cence polarization decreased in a sigmoidal manner from 320.5to 254.5 milli-polarizations (Fig. 7C), indicative of an HNP-1-induced dissociation of N36 trimers. By contrast, Abu-HNPcaused a marginal decrease in fluorescence polarization of 13milli-polarizations, suggesting that destabilization of the N36trimer by defensin was structure-dependent, as is the case withmany other functionalities of HNP-1 (13). Importantly, incuba-tion of HNP-1 at different concentrations with either C34 orN36/C34 triggered a defensin dose-dependent aggregation ofthe virus peptide(s), as analyzed by UV-visible spectrophotom-etry and CD spectroscopy (Fig. 7, D and E). The tertiary struc-ture of HNP-1 was also required for the induction of C34 andN36/C34 aggregation in solution as Abu-HNP had no effect onthe solubility of C34 and N36/C34 peptides. Taken together,these results strongly indicate that HNP-1 is capable of inter-acting with both N36 and C34 peptides as well as the N36/C34bundle structure, thus contributing to the inhibition of fusion,presumably by interfering with the formation of the fusogenicgp41 structure.Defensin Blocks HIV-1 Uptake without Interfering with Inter-

nalization of Endocytic Markers—The sensitivity of HIV-1 toinhibition by C52L after preincubation with defensin (Fig. 2)shows that fusion-competent viruses remained at the cell sur-face, accessible to the 6-helix bundle inhibitor. This findingimplies that HNP-1 can inhibit HIV-1 entry and fusion, at leastin part, by blocking the virus endocytosis. To test whetherdefensin prevented the virus uptake, we employed the fluores-cence quenching assay previously developed by our group (52,53). This assay employs double-labeled pseudoviruses contain-ing HIV-1 Gag-mCherry and carrying a pH-sensitive GFP var-iant, referred to as EcpH (75), in their membrane. EcpH, whichis anchored to the virus membrane by the ICAM-1 transmem-brane domain, is virtually nonfluorescent at pH � 6.0 (53, 75).The pH-insensitive mCherry within the virus interior serves as

TABLE 2Lateral mobility of CD4 and coreceptors in the presence of HNP-1 or concanavalin A (ConA) determined by FRAP

Control ConA HNP-1M.F.a t1⁄2 M.F. t1⁄2 M.F. t1⁄2% s % s % s

CD4-YFP 54.5 5.2 28.3 4.6 NDb ND 26.7 2.1c 50.0 13.3(n � 4) (n � 4)

CCR5-GFP 74.6 6.0 18.4 1.1 59.3 4.0 26.1 3.3c 56.9 3.3c 17.5 1.4(n � 11) (n � 6) (n � 22)

CXCR4-GFP 49.3 6.2 36.8 4.5 ND ND 23.7 2.6c 38.8 5.7(n � 8) (n � 13)

a M.F. means mobile fraction.b NDmeans not determined.c Statistically significant difference (p � 0.05) was found. Data are means S.E.

TABLE 3FCS analysis of HNP-1 effect on the CCR5-GFP mobilityA two-component model based on three- and two-dimensional Gaussian diffusionwas used to fit the experimental curves. Means S.E. are from five independentexperiments with TZM-bl cells are shown.

Untreated HNP-1 PFA

�D1 (�s) fast component 211 39 249 53 460 100�D2 (ms) slow component 20 9 33 3 244 92F1 (%) fast component 0.5 0.1 0.5 0.1 0.4 0.1F2 (%) slow component 0.5 0.1 0.5 0.1 0.6 0.1




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a reference signal to track the virus particles and perform realtime ratiometric pH measurements.JRFL pseudoviruses colabeled with EcpH-ICAM and Gag-

mCherry were pre-bound to TZM-bl cells in the cold, and their

internalization was initiated by shifting to 37 °C. In the absence ofdefensin, the majority of virions entered acidic compartmentswithin 1–1.5 h, as evidenced by the nearly complete loss of theEcpHsignalwithoutconsiderable changes in themCherry fluores-cence (Fig. 8,A andB). In sharp contrast, when viruses were incu-bated with cells in the presence of a fully inhibitory concentrationof defensin, EcpH fluorescence was not noticeably quenched, andthe virus particles remained peripherally bound to cells. Analysisof the ratio of green to red fluorescence confirmed the strongEcpH quenching in control experiments, although this ratioremained nearly constant throughout the experiments withHNP-1 (Fig. 8B). The inhibition of HIV-1 endocytosis was inde-pendent of coreceptor tropism, becauseHXB2 pseudoviruses alsoremained on the cell surface in the presence of HNP-1 (supple-mental Fig. 3, A and B). These striking results demonstrate thatinhibition of virus endocytosis could be a major mechanism bywhich HNP-1 blocks infection.In control experiments, defensin efficiently blocked internal-

ization of particles pseudotypedwith theVSVGglycoprotein inTZM-bl cells (Fig. 8C). Because VSV fuses with acidic endo-somes, the inhibition of its uptake by defensin was further ver-ified by measuring virus-cell fusion that was abrogated byHNP-1 (Fig. 8D). Interestingly, uptake of the HIV-1 core pseu-dotyped with influenza hemagglutinin and neuraminidase gly-coproteins appeared to occur in the presence of defensin (Fig.8E). Yet HNP-1 inhibited influenza pseudovirus fusion (Fig.8F). In agreement with this observation, imaging experimentswith bona fide influenza virus did not show strong inhibition ofinternalization by HNP-1 (supplemental Fig. 3C). Takentogether, these data show that inhibition of virus endocytosis byHNP-1 was not specific to HIV-1 or its receptors and was thusunlikely to be related to down-regulation of CD4 or CXCR4expression by defensin.Inhibition of HIV-1 uptake prompted us to assess the ability

of defensin to interfere with constitutive endocytosis in HeLa-derived cells. Uptake of fluorescently labeled markers of clath-rin-dependent endocytosis, transferrin-Alexa488 (Trf), andLDL labeled with DiI, was measured by flow cytometry. Cellsincubated at 4 °C or pretreated with 80 �M dynasore, a smallmolecule inhibitor of dynamin (76), served as negative controls.We found that Trf and LDL were effectively internalized in thepresence of defensin, whereas their uptake was markedlydiminished by dynasore (Table 4). In fact, endocytosis of Trfwas even somewhat more efficient in the presence of defensin,apparently due to its enhanced binding to cells. We also foundthat endocytosis of a fluid-phase marker, dextran-Alexa488,was slightly enhanced in the presence of defensin. These resultsdemonstrate the previously unappreciated ability of �-defensinto selectively block HIV-1 and VSV uptake without inhibitingthe endocytic activity of the cell.

DISCUSSION

Although the anti-HIV-1 activity of HNP-1 is well docu-mented, the mechanism by which this peptide blocks virusentry and replication remained poorly characterized. Here, wedissected the effects of �-defensin on key steps of HIV-1 entryand fusion and demonstrated that this peptide targets all majorsteps of the process. Key novel findings of this study include the

FIGURE 6. HNP-1 inhibits fusion downstream of Env-CD4-coreceptorcomplex formation. A, fusion of HXB2 pseudoviruses with TZM-bl cells wasmonitored after creating a TAS by incubating viruses with TZM-bl cells at 23 orat 4 °C (control samples) for 3 h. Fusion inhibitors (40 �M BMS-806; 10 �M

AMD3100; 5.8 �M HNP-1, and 1 �M C52L) were added to cells at TAS andincubated for 5 min prior to raising the temperature to 37 °C for 1 h. Theresulting virus-cell fusion was measured by the BlaM assay. Engagement ofCD4 and coreceptors at TAS was manifested by resistance of fusion to CD4and coreceptor binding inhibitors (BMS-806 and AMD3100, respectively).Data are means S.E. for a representative experiment performed in triplicate.B, HeLa cells constitutively expressing HIV-1 ADA Env and Tat were mixedwith indicator TZM-bl cells and preincubated for 2.5 h at 22 °C to create TAS(or at 4 °C, data not shown). Cell-cell fusion was then triggered by shifting to37 °C and measured by the Tat-dependent luciferase expression in TZM-blcells. When preincubation at 22 °C was carried out in the presence of 20 �M

BMS-806 or 3 �M AD101, subsequent fusion at 37 °C was fully blocked (2ndand 3rd columns). Partial inhibition of cell-cell fusion by the same concentra-tions of BMS-806 and AD101 added at TAS (4th and 5th columns) shows theformation of Env-CD4 and Env-CD4-CCR5 complexes, respectively. TheAD101-resistant fusion observed after creating TAS was completely blockedby 5.8 �M HNP-1 or 1 �M C52L (last two columns). In these experiments, HNP-1or C52L were added together with AD101 just prior to raising the tempera-ture from TAS.




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ability of HNP-1 to: (i) interfere with functional engagement ofCD4 and coreceptors by fusion-competent Env; (ii) reduce themobile fraction of these receptors in the plasmamembrane; (iii)bind to gp41 and host proteins in glycan- and serum-indepen-dentmanner; (iv) block post-coreceptor binding steps of fusion;and (v) inhibit HIV-1 and VSV uptake, but not constitutiveendocytosis. Finally, the major new finding of this study is thelack of correlation between the binding and the fusion inhibi-tory activity of defensin.It should be pointed out that another group has not observed

significant inhibition of HIV-1 fusion with PBMCs by HNP-1(17). The lack of effect on fusion is likely due to a lower concen-

tration of defensin used in these studies compared with thoseemployed in our experiments (Fig. 1C). Another study reportedstrong promotion of HIV-1 infection when the virus was pre-treated with HNP-1 (29). Here, we avoided complications aris-ing from the effects of HNP-1 on the virus attachment step bypre-binding the virus to cells prior to adding defensin.The observation that HNP-1 binds to the nonglycosylated

N-terminal domains of CD4 and to deglycosylated trimeric Envon viruses is in contrast to the previous report of glycan- andserum-dependent binding to monomeric gp120 (4). On theother hand, our data are consistent with the studies showingthat�-defensin does not compete with the broadly neutralizing

FIGURE 7. Interactions of N36 and/or C34 peptides with defensins. A, size-exclusion chromatographic analysis of N36, C34, and N36/C34. C34 elutes as amonomer, and N36 elutes mainly as a trimer. (The lower UV absorbance of N36 results from its stronger column retention compared with C34.) As expected,an equal molar mixture of N36 and C34 elutes predominantly as a hexamer. B, concentration-dependent N36 trimerization and N36/C34 formation of thesix-helix bundle structure confirmed by fluorescence polarization in a triplicate assay. C, HNP-1 destabilizes N36 trimer formation in a dose- and structure-de-pendent fashion as measured by fluorescence polarization in a triplicate assay. A decrease in fluorescence polarization is indicative of partial N36 trimerdissociation as a result of increased HNP-1. D, HNP-1 causes C34 and N36/C34 to aggregate in a dose- and structure-dependent fashion as determined byturbidity measurements at 600 nm. An increase in the absorbance at 600 nm directly correlates to C34 or N36/C34 peptide precipitation. E, HNP-1 causesN36/C34 to aggregate in a dose-dependent manner as analyzed by CD spectroscopy. C34 alone displays a random coil in solution, whereas N36 alone showsa distinct �-helical conformation with significantly weak signals, in contrast to the six-helix bundle structure of N36/C34 characterized by strong doublenegative peaks at 208 and 222 nm and a single positive peak at 195 nm. Addition of HNP-1 to N36/C34 causes peptide precipitation in solution, yieldingdecreased signal intensity after centrifugation.




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monoclonal antibody 2G12 for binding to amannose-rich clus-ter on gp120 (25). Also, the ability of �-defensins to competewith antibodies against the N-terminal domain of CD4 (25)lacking sugar residues (51) supports the notion of glycan-inde-pendent binding. Interestingly, the retrocyclin-resistant HIV-1BaL escape mutant acquired three mutations, all of which weresubstitutions for positively charged amino acids (24). Theseresults indicate that retrocyclinmay also interact with Env elec-trostatically, perhaps in addition to glycan binding. Finally,HNP-1 is a potent ligand of many bacterial toxins that arenonglycosylated.Although HNP-1 can bind to glycan-free proteins, glycan

binding could be essential for cross-linking cell surface glyco-proteins in a manner similar to retrocyclin (22). Such cross-

linking could be responsible for inhibition of virus endocytosis(Figs. 5 and 8). The ability of HNP-1 to reduce the mobile frac-tion of CD4 and coreceptors and the lack of correlationbetween binding to viral/cellular targets and fusion inhibitoryactivity (Figs. 1–4) are consistent with cross-linking as a pre-requisite for anti-HIV-1 activity.We showed that HNP-1 selec-tively inhibits internalization of HIV-1 and VSV pseudoviruses,while allowing constitutive endocytosis. Notably, retrocyclinalso does not inhibit transferrin uptake (22), but its effect onvirus endocytosis has not been explicitly tested. Our resultsimply that interference with virus internalization could be themajormechanismbywhichHNP-1 blocks fusion of viruses thatenter cells via endocytosis (33, 34). Influenza virus appears to bean exception, because HNP-1 blocks its fusion at a post-inter-

FIGURE 8. HNP-1 blocks HIV-1 and VSV G-pseudovirus endocytosis but not influenza uptake. Top, diagram of pseudovirus colabeling with the pH-sensi-tive membrane marker, EcpH-ICAM (green), and virus core marker, Gag-mCherry (red); the EcpH signal is quenched by mildly acidic pH. A and B, uptake of JRFLpseudoviruses by TZM-bl cells in the presence or in absence of HNP-1. Viruses were pre-bound to cells by centrifugation at 4 °C, and their uptake was initiatedby shifting to 37 °C for 90 min. Endocytosis and delivery into mildly acidic endosomes was monitored based on the drop of the EcpH-ICAM signal, although theGag-mCherry signal remained relatively constant. The EcpH/mCherry signal did not change significantly in the presence of 8.7 �M of HNP-1. The graph showsanalysis of representative images shown in A. C, HNP-1 blocks endocytosis of VSV G-pseudotyped particles colabeled with EcpH-ICAM and Gag-mCherry, as inA. VSV G-pseudotyped particles tended to aggregate around TZM-bl cells upon pre-binding in the cold, making it more difficult to visualize the transition fromvery bright colabeled particles (yellow) to far less intense small red puncta forming after the virus entry into acidic endosomes after 1 h at 37 °C. Apparently, thevirus aggregates disassociated before or during internalization. The bright peripherally located aggregates remained virtually intact when incubation wascarried out in the presence of 8.7 �M defensin. D, inhibition of VSV G-pseudotype fusion with TZM-bl cells by defensin following a 1.5-h incubation at 37 °C wasmeasured by the BlaM assay. E and F, effect of HNP-1 (14.5 �M) on endocytosis of influenza HA/NA-pseudotyped particles by HeLa cells (E) and virus-cell fusionmeasured after 90 min at 37 °C (F). The lower number of cell-bound particles reflects the lower titer of this pseudovirus compared with HIV-1 Env- and VSVG-pseudotypes.




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nalization step (Fig. 8, E and F, and supplemental Fig. 3C). Sim-ilarly, defensins do not inhibit the binding or uptake of nonen-veloped viruses but prevent their escape from endosomes,which results in virus accumulation within these compart-ments (9, 77). The selective block of HIV-1 uptake by �-defen-sin is proof-of-principle for novel approaches to fight infection.Wehypothesize that this unique feature ofHNP-1 (and likely ofother �-defensins) could potentiate the antiviral activity of theexisting fusion inhibitors as well as of neutralizing antibodies.Althoughbroad anti-microbial activity of defensins is incom-

patible with specific high affinity interactions with diverse tar-gets, defensins appear to bind glycanmoieties of proteins with arelatively high affinity (4). Our data reveal that HNP-1 boundrelatively tightly to key players in HIV-1 entry and fusion (Env,CD4, and coreceptors). More strikingly, we found that HNP-1bound to cellular and virus targets in a glycan- and serum-independent manner, which is in contrast with its markedlyattenuated inhibition of HIV-1 fusion in the presence of serum.This novel observation shows that the defensin binding torespective targets is necessary but not sufficient for blockingHIV-1 fusion and that an additional step, perhaps defensin olig-omerization (71), must occur to inhibit virus entry/fusion. Ourfindings are entirely consistent with a growing consensus thatthe ability of HNP-1 to dimerize, oligomerize, and multimerizeupon target binding endows this �-defensin with additionalmolecular complexity and structural diversity necessary for itsfunctional versatility against a broad array of bacterial, viral,and cellular targets (13).

Acknowledgments—We thank Weirong Yuan and Rokas Judeska formaking peptides, Dr. Min Lu for providing the C52L peptide, Dr. J.Strizki for providing the AD101 peptide, and Drs. Michelle de la Vegaand Naoyuki Kondo for helpful discussions.

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TABLE 4Effect of HNP-1 on endocytosis of transferrin, LDL, and dextran byTZM-bl cellsAverage mean fluorescence intensities from two independent experiments areshown. Cells were preincubated with endocytic markers for 20 min in the cold andtreated with defensin (5.8 �M) for 5 min in serum-free medium or left untreated.Cells were washed and shifted to 37 °C or kept on ice as a negative control. Dextranwas added to cells immediately prior to shifting to 37 °C. Alternatively, cells werepretreated with 80 �M dynasore for 30 min or, to inhibit dextran uptake, with 200�M of DMA for 1 h at 37 °C before carrying out endocytosis experiments in thecontinued presence of these compounds. Internalization of transferrin, LDL, anddextran was allowed to proceed at 37 °C for 10, 40, and 60 min, respectively. Cellswere then treated with Pronase (2 mg/ml) for 10 min on ice to remove cell surface-bound markers, harvested with trypsin-EDTA, and analyzed by flow cytometry.DMA is 5-(N,N-dimethyl)amiloride hydrochloride.

TreatmentTransferrin

(% of untreated)LDL

(% of untreated)Dextran

(% of untreated)

Dynasore 2.3 15.2DMA 31.0HNP-1 110.0 70.0 112.6




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MelikyanMiyauchi, Maikha Jean-Baptiste, Gennadiy Novitskiy, Wuyuan Lu and Gregory B.

Lusine H. Demirkhanyan, Mariana Marin, Sergi Padilla-Parra, Changyou Zhan, Kosuke-DefensinαMultifaceted Mechanisms of HIV-1 Entry Inhibition by Human

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