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Activity of - and -Defensins against Primary Isolates of HIV-1¹

Wei Wang^2,*, Sherry M. Owen^2,, Donna L. Rudolph, Alexander M. Cole^*, Teresa Hong^*, Alan J. Waring^*, Renu B. Lal and Robert I. Lehrer^3,*

^* Department of Medicine, David Geffen School of Medicine at University of California, Los Angeles, CA 90095; and HIV Immunology and Diagnostics Branch, Division of AIDS, STD, and TB Laboratory Research, National Center for Infectious Diseases, Centers for Disease Control and Prevention, Atlanta, GA 30333

	Abstract

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-Defensins are lectin-like, cyclic octadecapeptides found in the leukocytes of nonhuman primates. They are also homologues of the more familiar -defensins expressed by humans and certain other mammals. This study compares the ability of six -defensins (hominid retrocyclins 1–3 and rhesus -defensins 1–3) and four human -defensins (human neutrophil peptides (HNPs) 1–4) to bind gp120 and CD4. In addition, we compared the ability of these -defensins and HNP-1 to protect J53-BL cells (an indicator cell line) from primary HIV-1 isolates that varied in subtype and coreceptor usage. The most potent -defensin, retrocyclin-2, bound with exceptionally high affinity to gp120 (K_D, 9.4 nM) and CD4 (K_D, 6.87 nM), and its effectiveness against subtype B isolates (IC₅₀, 1.05 ± 0.28 µg/ml; 520 ± 139 nM) was approximately twice as great as that of HNP-1 on a molar basis. We also show, for the first time, that human -defensins, HNPs 1–3, are lectins that bind with relatively high affinity to gp120 (K_D range, 15.8–52.8 nM) and CD4 (K_D range, 8.0–34.9 nM). Proteins found in human and FBS bound exogenous HNP-2 and retrocyclin-1, and competed with their ability to bind gp120. However, even the low concentrations of -defensins found in normal human serum suffice to bind over half of the gp120 spikes on HIV-1 and a higher percentage of cell surface CD4 molecules. Although this report principally concerns the relationship between carbohydrate-binding and the antiviral properties of - and -defensins, the lectin-like behavior of defensins may contribute to many other activities of these multifunctional peptides.

	Introduction

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Antimicrobial peptides, such as cathelicidins and defensins, are components of the innate immune system (1, 2). Whereas cathelicidins are structurally diverse, all defensins have six cysteine residues and a largely -sheet structure that is stabilized by three intramolecular disulfide bonds. Three subfamilies of defensins exist in vertebrates, and are known as -, -, and -defensins (3). Because -defensins have been found in mammals, birds, and certain reptiles (4), they are believed to be the oldest family members. To date, -defensin peptides have been isolated only from mammals, and -defensin peptides only from rhesus macaques (5, 6, 7). In addition to their antibacterial and antifungal activities, some -defensins have in vitro activity against viruses, including herpes simplex (8, 9), vesicular stomatitis virus (8), CMV (8), influenza A (8), adenovirus (10, 11), and HIV-1 (12, 13, 14). It was recently suggested that -defensins (human neutrophil peptides (HNPs)⁴ 1–3) in human CD8⁺ T cells may play a role in delaying HIV-1 disease progression, and that they contribute to the anti-HIV-1 activity of "cell antiviral factor", a noncytolytic, protective factor released by cytotoxic CD8 T lymphocytes (12). However, this interpretation was disputed (12, 13, 15), and later withdrawn (16).

This study examines the binding properties of - and -defensins and their activities against HIV-1. -Defensins are circular octadecapeptides formed via the posttranslational splicing of two nonapeptides, each of which contributes three cysteines that form an intramolecular, tri-disulfide ladder in the mature, cyclic peptide (6). Because identical or nonidentical nonapeptides can be combined, two -defensin genes suffice to form three different -defensin peptides (5, 7). We identified six -defensin pseudogenes in the human genome database (17). Five were situated near the - and -defensin genes on chromosome 8, and the other had translocated to chromosome 1. Five of the pseudogenes encoded the identical nonapeptide, CICGRGICR. The other, DEFT-4, encoded a nonapeptide with an additional arginine residue, CICGRRICR. All six pseudogenes had the same stop codon in their signal sequence, and this premature stop codon was also found in human bone marrow mRNA (18).

Although endogenous -defensin peptides were lost before modern-day humans evolved, ancestral hominids whose -defensin genes lacked these stop codons may have produced three -defensins: retrocyclins-1, -2, and -3. Retrocyclin-1 is a -defensin composed of tandem repeats of the CICGRGICR nonapeptide, which is also encoded by human DEFT-pseudogene, mRNA, expressed in human bone marrow (18). Retrocyclin-2 (CICGRGICRCICGRRICR) contains one copy of the nonapeptide encoded by the DEFT-4 pseudogene.

We showed recently that synthetic retrocyclin-1 was a lectin (19) that protected human peripheral blood leukocytes from T- and M-tropic laboratory-adapted strains of HIV-1 (18) by preventing viral entry (20). The word lectin comes from the Latin lectus (the past participle of legere) and means to choose or select. Lectins have been formally defined as "carbohydrate-binding proteins, excluding sugar-specific Abs, receptors of free mono- or di-saccharides for transport or chemotaxis, and enzymes modifying the bound carbohydrate" (21). Other lectins that take part in innate immunity, including collectins (e.g., mannan-binding lectin and surfactant proteins A and D), ficolins, siglecs, and DC-SIGN were reviewed recently (22, 23, 24).

Studies with analogues of retrocyclin-1 revealed that their protective activity and their ability to bind gp120 and CD4 were correlated (19). For the present study, we synthesized all three retrocyclins and compared their binding and antiviral properties to the -defensins of rhesus macaques (RTDs 1–3) and to native -defensins (HNPs) purified from human leukocytes.

	Materials and Methods

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Viral isolates

Primary HIV-1 isolates were obtained from Institutional Review Board-approved Centers for Disease Control and Prevention studies, or from the National Institutes of Health Research and Reference Reagent Program via the World Health Organization collaborative network. Subtype determination and coreceptor usage was obtained from the National Institutes of Health Research and Reference Reagent Program, or determined as described elsewhere (25). Viral stocks were generated by infecting CD8-depleted normal human PBMC, passed through 0.22-µm filters, and stored at –70°C until used.

Peptides

The retrocyclin peptides used in this study were prepared in our laboratory by solid phase peptide synthesis, and then cyclized and purified as previously described (5, 18). Human neutrophil -defensins HNPs 1–4 were purified from peripheral blood granulocytes as previously described (26) and stored frozen until used. Table I shows the net charge, mass, and sequence of each -defensin used in this study. Note that human -defensins, HNPs 1–3, have almost identical sequences, and that HNP-4 shares little primary sequence homology to HNPs 1–3. RTDs 1–3 and retrocyclins 1–3 are similar, in that they contain 13 of 18 (72.2%) identical residues, and all are cyclic molecules with three intramolecular disulfide bonds, and a net charge of ⁺4 to ⁺6 (Table I).

JC53-BL cells are HeLa cell-derived indicator cells that express high levels of CD4, CXCR4, and CCR5, and contain reporter cassettes for luciferase and -galactosidase, both driven by the HIV-1 long terminal repeat (27). The JC53-BL cells were a gift from Tranzyme (Birmingham, AL). HIV infection (Tat production) was detected by measuring luciferase activity. We counted blue foci after staining with 5-bromo-4-chloro-3-indolyl -D-galactoside to titer virus stocks and calculate the multiplicity of infection (27). Infections were conducted in the presence or absence of - or -defensins, at final concentrations of 1.25, 2.5, 5, and 10 µg/ml. Briefly, JC53-BL cells were removed from T-150 flasks with EDTA 18 h before infection, and transferred to sterile white 96-well plates (Thermo Environmental Instruments, Franklin, MA), to provide 20,000 cells in 50 µl of C-DMEM per well. Three hours before adding the virus, the defensins were added to the wells. Virus (multiplicity of infection range, 0.009–0.65) was added in medium containing 40 µg/ml DEAE-dextran to give a final volume of 200 µl per well. DEAE-dextran enhances the in vitro infectivity of some HIV-1 strains (28), and its use in this system was suggested by Transzyme. After incubating the plates for 48 h at 37°C in room air containing 5% CO₂, the cells were lysed using the manufacturer’s lysis protocol and luciferase activity was measured with the Steady-Glo Luciferase Assay System (Promega, Madison, WI) using a Tecan luminometer running Magellan software (Tecan, Research Triangle Park, NC). Assays were run in duplicate on plates with the following control wells, in quadruplicate: medium only (background), virus only (no peptide), and recombinant luciferase. Relative light units (RLU) values were recorded, and percent of inhibition was calculated as percent of protection = (1 – (mean RLU in peptide-containing wells per average RLU of virus-only wells)) x 100.

Surface plasmon resonance experiments

Glycosylated HIV-1 gp120 envelope glycoprotein and recombinant soluble human CD4 (full-length 45-kDa glycosylated ectodomain) were purchased from Protein Sciences (Meriden, CT). BSA was from Sigma-Aldrich (St. Louis, MO), and glycosidases (PNGase F, GE23 sialidase A, and GE43 endo-O-glycosidase) were purchased from ProZyme (San Leandro, CA). Surface plasmon resonance experiments were performed on CM5 sensor chips with a Biacore 2000 system (Biacore, Piscataway, NJ) as previously described (19). Unless otherwise noted, all binding experiments were performed in HEPES-buffered saline (HBS; 150 mM NaCl, 10 mM HEPES, pH 7.4). In some experiments, gp120 was immobilized on a CM5 chip and deglycosylated in the BiaCore by perfusing the chambers for 3 h at 37°C with PNGase F (5 U), sialidase A (0.005 U), and endo-O-glycosidase (0.00125 U). These enzymes were delivered in 450 µl of 50 mM sodium phosphate buffer, pH 7.0. Data were analyzed and ² analyses were performed with BIAevaluation 3.1 software (Biacore). Curve-fitting was also done with BIAevaluation 3.1 software, with an assumption of one-to-one binding.

	Results

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Activity against primary isolates of HIV-1

We tested primary isolates that belonged to subtype A (five isolates), B (seven isolates), or C (eight isolates). The IC₅₀ of retrocyclin-2 for 19 of these primary isolates (one subtype B isolate was not tested) was 2.33 ± 0.42 µg/ml (mean ± SEM). As can be seen in Table II, the IC₅₀ of retrocyclin-2 was 1.05 ± 0.28 µg/ml (520 ± 139 nM) for the 6 subtype B isolates and 3.32 ± 0.76 µg/ml for the 8 subtype C isolates. The difference between subtype B and -C isolates was not statistically significant (P_exact = 0.108, Mann-Whitney U rank sum test). The five subtype A isolates showed intermediate susceptibility to retrocyclin-2, with an IC₅₀ of 2.30 ± 0.63 µg/ml. Fourteen of these 19 primary isolates in this group were R5 strains, and 5 were X4 strains. The mean IC₅₀ values of retrocyclin-2 against the R5 strains and X4 strains were 2.81 ± 0.50 µg/ml and 1.01 ± 0.34 µg/ml, respectively.

Comparative activity of retrocyclins and RTDs

Table II also compares the activity of RTDs 1–3 and retrocyclin-2 against these primary isolates and additional isolates of subtypes CRF01_AE, D, and G. In general, the -defensins were most potent against the subtype B strains, with all but retrocyclin-1 having mean IC₅₀ values below 2.0 µg/ml. Retrocyclin-2 had lower IC₅₀ values (p < 0.02) than RTD-1 or -3 against subtypes A and C. RTD-2 was less effective against subtypes A and C, with three subtype C strains having IC₅₀ values above 10 µg/ml.

Comparative activity of - and -defensins

Because -defensin genes are mutated -defensin genes (6), and because human -defensins may play a role in delaying progression of human HIV-1 infections (12), we compared the activities of retrocyclin-2 and human -defensin HNP-1 against 16 primary HIV-1 isolates (Table III). Both peptides showed concentration-dependent activity. HNP-1 was protective at 10 µg/ml and 5.0 µg/ml, but was ineffective at 2.5 µg/ml against all of the isolates. In contrast, retrocyclin-2 was protective at 10, 5, and 2.5 µg/ml. The mean percent of protection afforded by 10 µg/ml (2.90 µM) HNP-1 was significantly less than that provided by 5.0 µg/ml (2.48 µM) of retrocyclin-2 (P_exact = 0.009, Wilcoxon signed rank test, n = 16), and slightly less than that afforded by 2.5 µg/ml (1.24 µM) retrocyclin-1. Fig. 1 shows that, on a molar basis, retrocyclin-2 was approximately twice as potent as HNP-1 in protecting cells from primary isolates of HIV-1.

View larger version (19K): [in this window] [in a new window]   FIGURE 1. Activity against primary isolates of HIV-1. The activity of retrocyclin-2 and HNP-1 against the entire group (n = 16) of primary isolates shown in Table III is compared.

Some -defensins are also lectins

As noted above, -defensins contain two spliced nonapeptides, each part of an -defensin-like propeptide (6). To see whether -defensins might also be lectins, we performed binding experiments (Fig. 2) which showed that HNPs 1–3 bound glycosylated gp120 and CD4 (HNP-2 > HNP-1 > HNP-3). In contrast, HNP-4 showed very little binding to these glycoproteins. When immobilized gp120 was treated with enzymes that removed both its N-linked and O-linked carbohydrates, the binding of HNP 1–3 (but not that of HNP-4) was virtually abrogated. Thus, some, but not all, human -defensins have lectin-like properties.

View larger version (21K): [in this window] [in a new window]   FIGURE 2. Binding of human -defensins. The isotherms show binding of 1 µg/ml HNPs 1, 2, 3, and 4 to two immobilized recombinant glycoproteins, gp120 () and CD4 (—). Also shown is the binding isotherm for deglycosylated gp120 (gly gp120, •). The N- and O-linked sugars of gp120 were removed by immobilizing gp120 on the biosensor chip, and then perfusing the chip with endoglycosidases, as previously described (17 ). Each defensin was introduced into the flow cell containing the CM5 biosensor chip after 1 min, and the defensin-free washout buffer began flowing at 4 min.

The affinities of retrocyclin-2 for gp120 and CD-4 were 3.76-fold and 4.65-fold higher, respectively, than those of retrocyclin-1 (Table IV). The kinetic-binding constants (k_on and k_off) and the equilibrium binding constant (K_D) for all six -defensins and four -defensins used in this study are also shown in Table IV.

Whereas our binding studies were routinely performed in HBS at pH 7.4, the antiviral studies were done in DMEM, a complex, isotonic tissue culture medium that contains 15 aa, 6 vitamins, 10 inorganic ions (Ca²⁺, Mg²⁺, Na⁺, K⁺, Cl^–, HCO₃^–, NO₃^–, SO₄⁼, Fe²⁺, and HPO₄⁼/H₂PO₄^–) plus glucose, pyruvate, and choline. In other experiments (data not shown), we determined that binding of retrocyclin-1 or HNP-2 to immobilized gp120 was unaffected by the addition of 2 mM Ca²⁺ or 2 mM Mg²⁺ to the HBS, and that it was virtually identical in DMEM and HBS (data not shown).

However, serum markedly reduced binding as shown in Fig. 3. The dotted lines in each panel represent the binding isotherm of 1 µg/ml retrocyclin-1 (RC100) or HNP-2 to immobilized gp120 in HBS. The solid lines show the binding of these peptides in DMEM containing FCS (Fig. 3, a and c) or normal human serum (NHS; Fig. 3, b and d). When present, the final serum concentration was 10% v/v. Note that serum greatly reduced the binding of retrocyclin-1 to gp120, such that it required >10 µg/ml RC100 to obtain the binding seen when serum was absent. There was relatively little difference between human and FCS in their ability to inhibit binding by retrocyclin-1.

View larger version (38K): [in this window] [in a new window]   FIGURE 3. Effect of serum on binding to gp120. In each panel, the dotted line shows the binding of 1 µg/ml defensin to immobilized gp120 in HBS. The solid lines show the binding isotherms for RC100 (a and b) or HNP-2 (c and d) in DMEM containing 10% FCS (a and c) or 10% NHS (b and d). The number adjacent to each isotherm represents the peptide concentration in micrograms per milliliter.

FCS differs from human serum in many respects, including the absence of immunoglobulins and the presence of fetuin, a glycoprotein that typically comprises 20% or more of its total protein. Having previously determined that retrocyclin-1 and RTDs bound fetuin with high affinity, we suspected that human -defensins might do so as well. This suspicion was borne out by the experiments shown in Table V. HNP-1 and HNP-2 bound immobilized fetuin with a K_d of 3–5 nM, and both retrocyclin-2 and HNP-3 had a K_d of 15 nM. In contrast, HNP-4 had a much lower low affinity for fetuin (K_d > 1000 nM). The lower affinity of HNP-4 was attributable primarily to the instability of the HNP-4/fetuin complex, as indicated by its very high off-rate.

	Discussion

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We interpret the inhibitory effects of serum on binding to gp120 as resulting from the binding of - and -defensins by serum proteins and glycoproteins. For example, it has been shown that human -defensins, HNP 1–3, are bound by activated ₂-macroglobulin (29), and by several other serum proteins, including C1q, ₁-proteinase inhibitor, ₁-antichymotrypsin, ₂-antiplasmin, and antithrombin III (30, 31). It remains to be determined whether these defensins bind to glycan moieties, or whether any of the proteins also bind -defensins. The remarkably high affinity of HNP-1, -2, and -3 for fetuin, a principal component of FCS, was shown in Table V.

The inhibitory effects of serum help explain why our EC₅₀ values for antiviral activity by the peptides were considerably higher than their K_d values for binding gp120 under serum-free conditions. The K_d represents the defensin concentration that occupies half of the binding sites of the immobilized ligand at equilibrium, and the EC₅₀ represents the concentration of peptide that inhibits HIV-1 infection by 50%. It has been estimated that 40,000–50,000 CD4 molecules are present on the surface of a normal CD4⁺ T cell (32) and that 216 gp120 molecules (contained on 72 gp spikes) are present on the surface of each HIV-1 virus (33). HIV-1 neutralization by 2G12, a broad-spectrum mAb directed against carbohydrate epitopes of gp120 (34, 35) requires the attachment of 70 IgG molecules — about one per spike. Retrocyclins and HNP 1–3 bind both gp120 and CD4 with high affinity (Table IV), and it is not known whether binding both sites is required to prevent HIV-1 entry or if binding only one of them would suffice.

If it is necessary for defensins to occupy at least 90% of both surface molecules, gp120 and CD4, to prevent infection, this would require a peptide concentration 10-fold higher than the K_d for gp120, i.e., 354 nM retrocyclin-1 and 158 nM HNP-2. As 10% FCS inhibited retrocyclin-1’s binding to gp120 by 10-fold, it would require 3.54 µM retrocyclin-1 (6.79 µg/ml) to occupy 90% of the gp120 sites on HIV-1. This value, although based on speculations, is in reasonable agreement with the experimental data shown in Table II.

What do these data imply about the conditions and anatomic sites that might allow - or -defensins to block HIV infection in vivo? Given the prominent inhibitory effects of serum on their binding to gp120, it seems unlikely that they would be effective in serum-containing anatomical compartments unless: 1) they were present in sufficient concentration to bind surface receptors (e.g., CD4 and gp120) involved in viral entry; or 2) their local concentrations in proximity to such primary receptors was enhanced by "nonspecific" binding to cell surface glycoproteins and/or glycosaminoglycans. A situation analogous to 2) exists for certain chemokines, (e.g., RANTES, MCP-1, IL-8, MIP-1) with an affinity for cell surface glycosaminoglycans (36). Reversible binding to these glycosaminoglycans concentrates the chemokines on and near the cell surface, thereby enhancing their presentation to their cognate-specific receptors (36). Since the lectin-like properties of - and -defensins allow them to bind glycolipids and many glycoproteins "nonspecifically", their concentration on or near cell surfaces could be enhanced as a consequence of such interactions.

Although substantially higher serum concentrations of extracellular defensins are present in the serum of patients with infection or sepsis (37), carefully separated normal human serum contains only 50–75 ng/ml HNP 1–3, about 15–22 nM (38). However, since HNP-2 binds CD4 with a K_d of 8 nM and gp120 with a K_d of 15.8 nM (Table IV) even these low concentrations would occupy at least 50% of the gp120-containing spikes and a higher percentage of CD4 molecules. Thus, HNP 1–3 might be effective even at the levels of defensins present in normal serum.

There is little information about the composition of normal human peripheral lymph, and almost none about the composition of the lymph that permeates human lymph nodes. Rabbit limb lymph contains less than half of the total protein concentration of rabbit plasma (1.9 ± 0.4 g/dl vs 4.1 ± 0.2 g/dl) and about one-quarter of its fibrinogen, prothrombin, or factor X (39). To the extent that lymph-node lymph lacks the various defensin-binding molecules of serum, low concentrations of - and -defensins could show greater activity against HIV-1 in lymph nodes than within the vascular compartment.

Mucous membranes are protected by -defensins produced by local epithelial cells (40, 41, 42). Consequently, human -defensins (hBDs) may defend against HIV-1 at mucosal sites that are intimately involved in the sexual transmission of HIV-1 infection. HIV-1 was recently reported to induce the expression of hBD-2 and -3 mRNA in normal human oral epithelial cells, and recombinant hBD-2 and -3 peptides were shown to bind HIV-1 virions and cause concentration-dependent inhibition of HIV-1 replication without causing cytotoxicity (43). These investigators noted that CXCR4-tropic HIV-1 isolates were more susceptible to hBD-2 and -3 than were CCR5-tropic isolates. Both hBD-2 and -3 down-modulated surface CXCR4 coreceptors in PBMC and T lymphocytes, but did not affect CCR5. It remains to be seen if -defensins are also lectins, and, if they are, what part glycan binding might play in their activity against HIV-1. Because substantial concentrations of -defensins (HNPs) are present in mucus plugs that occlude the uterine cervix (44), even HNPs that arrive by leukocyte carriage may contribute to mucosal defenses against HIV-1.

In this report, the carbohydrate-binding properties of - and -defensins were viewed in the context of HIV-1 and the interactions of these defensins with key cellular and viral glycoproteins involved in HIV-1 entry. Although important, this focus should not obscure the likelihood that the lectin-like properties of defensins contribute to other activities of these multifunctional peptides. Finally, even if endogenous -defensins no longer exist in Homo sapiens, their synthetic counterparts (e.g., retrocyclins) could prove useful as broad-spectrum, topical microbicides to prevent sexually transmitted viral infections.

	Acknowledgments

 
We thank Prof. Ron Kaback for generous access to his BiaCore 2000 instrument.

	Footnotes

 
¹ This work was supported by grants from the National Institutes of Health: AI-22839 and AI-37945 (to R.I.L.) and AI-52017 (to A.M.C.). Use of trade names is for identification only, and does not constitute endorsement by the U. S. Department of Health and Human Services, the Public Health Service, or the Centers for Disease Control and Prevention.

² W.W. and S.M.O. contributed equally to this work.

³ Address correspondence and reprint requests to Dr. Robert I. Lehrer, Center for Health Sciences, Center for Health Sciences 37-062, University of California, Los Angeles School of Medicine, 10833 LeConte Avenue, Los Angeles, CA 90095. E-mail address: rlehrer{at}mednet.ucla.edu

⁴ Abbreviations used in this paper: HNP, human neutrophil peptide; RTD, rhesus -defensin; RLU, relative light unit; HBS, HEPES-buffered saline; NHS, normal human serum; hBD, human -defensin.

Received for publication July 15, 2003. Accepted for publication April 19, 2004.

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