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• W2138746585 abstract "Retrocyclin-1, a θ-defensin, protects target cells from human immunodeficiency virus, type 1 (HIV-1) by preventing viral entry. To delineate its mechanism, we conducted fusion assays between susceptible target cells and effector cells that expressed HIV-1 Env. Retrocyclin-1 (4 μm) completely blocked fusion mediated by HIV-1 Envs that used CXCR4 or CCR5 but had little effect on cell fusion mediated by HIV-2 and simian immunodeficiency virus Envs. Retrocyclin-1 inhibited HIV-1 Env-mediated fusion without impairing the lateral mobility of CD4, and it inhibited the fusion of CD4-deficient cells with cells bearing CD4-independent HIV-1 Env. Thus, it could act without cross-linking membrane proteins or inhibiting gp120-CD4 interactions. Retrocyclin-1 acted late in the HIV-1 Env fusion cascade but prior to 6-helix bundle formation. Surface plasmon resonance experiments revealed that retrocyclin bound the ectodomain of gp41 with high affinity in a glycan-independent manner and that it bound selectively to the gp41 C-terminal heptad repeat. Native-PAGE, enzyme-linked immunosorbent assay, and CD spectroscopic analyses all revealed that retrocyclin-1 prevented 6-helix bundle formation. This mode of action, although novel for an innate effector molecule, resembles the mechanism of peptidic entry inhibitors based on portions of the gp41 sequence. Retrocyclin-1, a θ-defensin, protects target cells from human immunodeficiency virus, type 1 (HIV-1) by preventing viral entry. To delineate its mechanism, we conducted fusion assays between susceptible target cells and effector cells that expressed HIV-1 Env. Retrocyclin-1 (4 μm) completely blocked fusion mediated by HIV-1 Envs that used CXCR4 or CCR5 but had little effect on cell fusion mediated by HIV-2 and simian immunodeficiency virus Envs. Retrocyclin-1 inhibited HIV-1 Env-mediated fusion without impairing the lateral mobility of CD4, and it inhibited the fusion of CD4-deficient cells with cells bearing CD4-independent HIV-1 Env. Thus, it could act without cross-linking membrane proteins or inhibiting gp120-CD4 interactions. Retrocyclin-1 acted late in the HIV-1 Env fusion cascade but prior to 6-helix bundle formation. Surface plasmon resonance experiments revealed that retrocyclin bound the ectodomain of gp41 with high affinity in a glycan-independent manner and that it bound selectively to the gp41 C-terminal heptad repeat. Native-PAGE, enzyme-linked immunosorbent assay, and CD spectroscopic analyses all revealed that retrocyclin-1 prevented 6-helix bundle formation. This mode of action, although novel for an innate effector molecule, resembles the mechanism of peptidic entry inhibitors based on portions of the gp41 sequence. Three structurally distinct subfamilies of defensins, α, β, and θ, exist in primates (1Lehrer R.I. Nat. Rev. Microbiol. 2004; 2: 727-738Crossref PubMed Scopus (448) Google Scholar). θ-Defensins, the smallest of these and the only known cyclic peptides of animal origin, contain only 18 residues (2Selsted M.E. Curr. Protein Pept. Sci. 2004; 5: 365-371Crossref PubMed Scopus (99) Google Scholar). Three θ-defensin peptides have been isolated from rhesus macaque leukocytes (3Tang Y.Q. Yuan J. Osapay G. Osapay K. Tran D. Miller C.J. Ouellette A.J. Selsted M.E. Science. 1999; 286: 498-502Crossref PubMed Scopus (614) Google Scholar) and bone marrow (4Leonova L. Kokryakov V.N. Aleshina G. Hong T. Nguyen T. Zhao C. Waring A.J. Lehrer R.I. J. Leukocyte Biol. 2001; 70: 461-464PubMed Google Scholar), and intact θ-defensin genes exist in other non-human primates (5Nguyen T.X. Cole A.M. Lehrer R.I. Peptides. 2003; 24: 1647-1654Crossref PubMed Scopus (159) Google Scholar). Humans have multiple θ-defensin genes and express θ-defensin mRNA transcripts in bone marrow. However, these genes and transcripts harbor a premature stop codon, and neither humans nor their closest primate relatives (chimpanzees and gorillas) produce θ-defensin peptides (5Nguyen T.X. Cole A.M. Lehrer R.I. Peptides. 2003; 24: 1647-1654Crossref PubMed Scopus (159) Google Scholar, 6Cole A.M. Hong T. Boo L.M. Nguyen T. Zhao C. Bristol G. Zack J.A. Waring A.J. Yang O.O. Lehrer R.I. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 1813-1818Crossref PubMed Scopus (264) Google Scholar).Retrocyclin-1 (RC-1), 3The abbreviations used are: RC-1, retrocyclin-1; HIV, human immunodeficiency virus; SIV, simian immunodeficiency virus; 6HB, 6-helix bundle; N-HR, N helical region; C-HR, C-helical region; FRAP, fluorescence recovery after photobleaching; sCD4, soluble CD4; BSA, bovine serum albumin. 3The abbreviations used are: RC-1, retrocyclin-1; HIV, human immunodeficiency virus; SIV, simian immunodeficiency virus; 6HB, 6-helix bundle; N-HR, N helical region; C-HR, C-helical region; FRAP, fluorescence recovery after photobleaching; sCD4, soluble CD4; BSA, bovine serum albumin. a synthetic cyclic octadecapeptide, represents a θ-defensin peptide that humans could produce if the corresponding gene had not been silenced by mutation. Retrocyclins and other θ-defensins exert broad spectrum antiviral properties in vitro and can protect cells from infection by HIV-1 (6Cole A.M. Hong T. Boo L.M. Nguyen T. Zhao C. Bristol G. Zack J.A. Waring A.J. Yang O.O. Lehrer R.I. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 1813-1818Crossref PubMed Scopus (264) Google Scholar, 7Owen S.M. Rudolph D.L. Wang W. Cole A.M. Waring A.J. Lal R.B. Lehrer R.I. AIDS Res. Hum. Retroviruses. 2004; 20: 1157-1165Crossref PubMed Scopus (82) Google Scholar, 8Owen S.M. Rudolph D. Wang W. Cole A.M. Sherman M.A. Waring A.J. Lehrer R.I. Lal R.B. J. Pept. Res. 2004; 63: 469-476Crossref PubMed Scopus (40) Google Scholar, 9Wang W. Owen S.M. Rudolph D.L. Cole A.M. Hong T. Waring A.J. Lal R.B. Lehrer R.I. J. Immunol. 2004; 173: 515-520Crossref PubMed Scopus (171) Google Scholar), herpes simplex (10Yasin B. Wang W. Pang M. Cheshenko N. Hong T. Waring A.J. Herold B.C. Wagar E.A. Lehrer R.I. J. Virol. 2004; 78: 5147-5156Crossref PubMed Scopus (202) Google Scholar), and influenza A viruses (11Leikina E. Delanoe-Ayari H. Melikov K. Cho M.S. Chen A. Waring A.J. Wang W. Xie Y. Loo J.A. Lehrer R.I. Chernomordik L.V. Nat. Immunol. 2005; 6: 995-1001Crossref PubMed Scopus (215) Google Scholar).HIV enters a target cell after its gp120/gp41 glycoprotein (Env) binds CD4 (12Maddon P.J. Dalgleish A.G. McDougal J.S. Clapham P.R. Weiss R.A. Axel R. Cell. 1986; 47: 333-348Abstract Full Text PDF PubMed Scopus (1502) Google Scholar) and a co-receptor, CCR5 or CXCR4 (13Berger E.A. Murphy P.M. Farber J.M. Annu. Rev. Immunol. 1999; 17: 657-700Crossref PubMed Scopus (1876) Google Scholar). The ensuing conformational changes result in a 6-helix bundle (6HB) core structure wherein three N-helical regions (N-HR) pair with three C-helical regions (C-HR) and drive membrane fusion (14Weissenhorn W. Dessen A. Harrison S.C. Skehel J.J. Wiley D.C. Nature. 1997; 387: 426-428Crossref PubMed Scopus (1457) Google Scholar, 15Chan D.C. Fass D. Berger J.M. Kim P.S. Cell. 1997; 89: 263-273Abstract Full Text Full Text PDF PubMed Scopus (1830) Google Scholar, 16Tan K. Liu J. Wang J. Shen S. Lu M. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 12303-12308Crossref PubMed Scopus (517) Google Scholar, 17Caffrey M. Cai M. Kaufman J. Stahl S.J. Wingfield P.T. Covell D.G. Gronenborn A.M. Clore G.M. EMBO J. 1998; 17: 4572-4584Crossref PubMed Scopus (368) Google Scholar). Peptides that mimic N-HR or C-HR inhibit fusion by binding their opposite counter-part and preventing 6HB formation (18Jiang S. Lin K. Strick N. Neurath A.R. Nature. 1993; 365: 113Crossref PubMed Scopus (474) Google Scholar, 19Wild C. Greenwell T. Matthews T. AIDS Res. Hum. Retroviruses. 1993; 9: 1051-1053Crossref PubMed Scopus (371) Google Scholar, 20Wild C. Oas T. McDanal C. Bolognesi D. Matthews T. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 10537-10541Crossref PubMed Scopus (479) Google Scholar, 21Chan D.C. Chutkowski C.T. Kim P.S. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 15613-15617Crossref PubMed Scopus (483) Google Scholar, 22Louis J.M. Bewley C.A. Clore G.M. J. Biol. 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Wang W. Hong T. Lehrer R.I. Landau N.R. Cole A.M. AIDS Res. Hum. Retroviruses. 2003; 19: 875-881Crossref PubMed Scopus (122) Google Scholar).Retrocyclin-1 has lectin-like properties and binds glycosylated molecules such as CD4, HIV gp120, and galactosylceramide with high (Kd∼ 20-30 nm) affinity (29Wang W. Cole A.M. Hong T. Waring A.J. Lehrer R.I. J. Immunol. 2003; 170: 4708-4716Crossref PubMed Scopus (176) Google Scholar). Under certain conditions, this allows retrocyclins to prevent viral entry by cross-linking cell surface molecules, as recently shown for influenza A (11Leikina E. Delanoe-Ayari H. Melikov K. Cho M.S. Chen A. Waring A.J. Wang W. Xie Y. Loo J.A. Lehrer R.I. Chernomordik L.V. Nat. Immunol. 2005; 6: 995-1001Crossref PubMed Scopus (215) Google Scholar). Despite its activity against HIV-1, retrocyclin-1 is considerably less effective against HIV-2 or SIV. Because the cross-linked barrier mechanism would not explain such selectivity, we examined the inhibitory effects of retrocyclin-1 on fusion mediated by HIV-1 Env. We found that retrocyclin-1 prevents HIV-1 entry by binding the C-heptad repeat of gp41 in a lectin-independent manner that prevents 6HB formation.EXPERIMENTAL PROCEDURESCell-Cell Fusion Assay—Dye transfer assays, which involve observing the transfer of fluorescent cytosolic dyes from the target cell type to a non-labeled envelope expressing cell type, were used to quantify the amount on envelope-induced cell-cell fusion. HIV/SIV Env-expressing HeLa cells labeled with CMTMR and target cells labeled with calcein were co-cultured in suspension at 37 °C. Inhibitors were added at the onset of incubation or at various times thereafter. Phase and fluorescent images were collected with a 10× objective lens (30Munoz-Barroso I. Durell S. Sakaguchi K. Appella E. Blumenthal R. J. Cell Biol. 1998; 140: 315-323Crossref PubMed Scopus (269) Google Scholar, 31Gallo S.A. Puri A. Blumenthal R. Biochemistry. 2001; 40: 12231-12236Crossref PubMed Scopus (120) Google Scholar). Normalized results were expressed as percent of the control fusion.Binding Studies—Surface plasmon resonance studies were performed on a Biacore 3000 instrument (Biacore, Uppsala, Sweden) in two modes, standard and competitive (see the supplemental “Methods” section and supplemental Table 3).Effect on 6-Helix Bundle Formation—Native-PAGE was done as previously described (32Liu S. Zhao Q. Jiang S. Peptides. 2003; 24: 1303-1313Crossref PubMed Scopus (69) Google Scholar). Inhibition of 6HB formation was also determined by a modified ELISA using the mAb NC-1 (33Jiang S. Lin K. Lu M. J. Virol. 1998; 72: 10213-10217Crossref PubMed Google Scholar, 34Jiang S. Lin K. Zhang L. Debnath A.K. J. Virol. Methods. 1999; 80: 85-96Crossref PubMed Scopus (110) Google Scholar). Percent inhibition was calculated as previously described (35Jiang S. Lu H. Liu S. Zhao Q. He Y. Debnath A.K. Antimicrob. Agents Chemother. 2004; 48: 4349-4359Crossref PubMed Scopus (256) Google Scholar). The IC50 was calculated with Calcusyn software (36Chou T.C. Hayball M.P. CalcuSyn: Windows Software for Dose Effect Analysis, Version 2.0. 1999; (BIOSOFT, Cambridge, UK)Google Scholar), kindly provided by Dr. T. C. Chou (Sloan-Kettering Cancer Center, New York, NY).CD Spectroscopy—Spectra were obtained on a Jasco J-715 instrument at 25 °C. Samples, diluted in 10 mm HEPES, pH 7.4, were placed in a 0.1 cm path length CD cell (Hellma, Plainview, NY). The spectra were averaged from four scans, smoothed, and expressed as the mean residue ellipticity [θ]MRE. Additional information about cells, recombinants, and methods is provided in supplemental “Methods and Materials,” on-line.Fluorescence Recovery after Photobleaching (FRAP)—FRAP was performed using a Zeiss LSM 510 (Carl Zeiss, Jena, Germany) confocal laser scanning microscope. HeLa cells were plated on 35-mm glass bottom dishes (MatTek, Ashland, MA) and transfected 24 h prior to confocal analysis with CD4-GFP. These constructs were generous gifts from W. Popik, and have been described previously (37Popik W. Alce T.M. J. Biol. Chem. 2004; 279: 704-712Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar). During FRAP analysis cells were kept at physiological conditions of 37 °C and 5% CO2 in a stage-incubation system (“Incubator S,” PeCon GmbH, Erbach, Germany). Retrocyclin was added directly into the medium and incubated for 10 min before commencing FRAP measurements. A 488 nm Ar+ laser line was used for GFP excitation and emission light was collected with a 500-550 bandpass filter. A 40×/1.3 NA oil immersion objective lens was used with a zoom factor of 4. The detector pinhole was opened slightly to acquire an optical section of 2 μm thickness, allowing more light to be collected for better quantification. Three prebleach images were acquired to determine the rate of non-purposeful photobleaching. Photobleaching was performed by increasing the transmission of the laser to 100% for 20-50 iterations to get as complete a bleach as possible without overbleaching. After photobleaching, 8-10 images were acquired at 1-s intervals. Then the time resolution was changed to 10 s to follow recovery to completion. A total of 20-40 data points was acquired for image analysis.FRAP analysis was performed using the Medical Imaging Processing, Analysis, and Visualization (MIPAV; CIT/NIH, Bethesda, MD) software package. Data were automatically corrected with background subtraction, and normalization for the non-purposeful photobleaching rate was calculated from the whole cell. Data were analyzed using the one-dimensional FRAP model.RESULTSInhibition of Fusion—Retrocyclin-1 inhibited HIV-1 Env-mediated membrane fusion in a concentration-dependent manner (Fig. 1a), with an IC50 of ∼1.5 μm, and complete inhibition at ∼4 μm. Retrocyclin-1 inhibited fusion mediated by HIV-1IIIB (X4) and HIV-1BAL (R5) Env but not HIV-2ROD Env (Fig. 1b). Fusion mediated by HIV-2SBL and SIVMAC Env was slightly inhibited by retrocyclin-1. For comparison, Fig. 1 also shows inhibition of HIV/SIV Env-mediated fusion by cyanovirin-N (CV-N), an 11-kDa mannose-binding protein isolated from a Cyanobacterium, Nostoc ellipsosporum, which potently inactivates diverse strains of HIV-1, HIV-2, and SIV (38Boyd M.R. Gustafson K.R. McMahon J.B. Shoemaker R.H. O'Keefe B.R. Mori T. Gulakowski R.J. Wu L. Rivera M.I. Laurencot C.M. Currens M.J. Cardellina J.H. Buckheit R.W. Nara Jr., P.L. Pannell Sowder L.K. R. C. Henderson L.E. Antimicrob. Agents Chemother. 1997; 41: 1521-1530Crossref PubMed Google Scholar).Excluding Potential Membrane Targets—As retrocyclin binds CD4 and glycosphingolipids (29Wang W. Cole A.M. Hong T. Waring A.J. Lehrer R.I. J. Immunol. 2003; 170: 4708-4716Crossref PubMed Scopus (176) Google Scholar), its inhibition of HIV-1 Env-mediated fusion might reflect interactions with these membrane components. We examined the fusion of CD4-independent 8x Env (39Hoffman T.L. LaBranche C.C. Zhang W. Canziani G. Robinson J. Chaiken I. Hoxie J.A. Doms R.W. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 6359-6364Crossref PubMed Scopus (247) Google Scholar) with mouse fibroblast 3T3 target cells bearing CD4 and CXCR4, or bearing CXCR4 alone. Retrocyclin-1 blocked 8x Env-mediated fusion with CXCR4+ cells with or without CD4, indicating that its effects were independent of gp120-CD4 interactions (Fig. 1c). The ∼30% residual fusion of 3T3CD4CXCR4 and 3T3CXCR4 in the presence of retrocyclin is likely because of nonspecific background dye transfer, because cyanovirin-N, which completely blocks HIV-1 Env-mediated fusion with various cell types, showed the same background. Because retrocyclin-1 also inhibited HIV-1 Env-mediated fusion with glycosphingolipid-deficient mouse skin fibroblasts (GM95 cells) that expressed CD4 and CXCR4 (40Rawat S.S. Eaton J. Gallo S.A. Martin T.D. Ablan S. Ratnayake S. Viard M. KewalRamani V.N. Wang J.M. Blumenthal R. Puri A. Virology. 2004; 318: 55-65Crossref PubMed Scopus (21) Google Scholar), interactions with glycosphingolipids were not essential for its inhibitory effects on HIV-1 (Fig. 1c).Excluding Membrane Cross-linking—A recently described mechanism (11Leikina E. Delanoe-Ayari H. Melikov K. Cho M.S. Chen A. Waring A.J. Wang W. Xie Y. Loo J.A. Lehrer R.I. Chernomordik L.V. Nat. Immunol. 2005; 6: 995-1001Crossref PubMed Scopus (215) Google Scholar), based largely on studies with influenza A under serum-free conditions, could account for the activity of retrocyclins against HIV-1. In those studies, retrocyclin inhibited viral fusion by erecting a barricade of cross-linked and immobilized surface glycoproteins. When FRAP experiments were performed to assess the effect of retrocyclin-1 on the lateral mobility of CD4 in medium that contained 10% fetal calf serum, we found the lateral mobility of CD4 to be intact (Table 1) and concluded that cross-linked surface barricades on the target membrane did not form under the experimental conditions used in our fusion experiments.TABLE 1Effect of retrocyclin-1 on the lateral mobility of CD4 HeLa cells were transfected with CD4-GFP 1 day before measuring FRAP. The medium contained 10% fetal bovine serum. Retrocyclin was added directly into the medium and incubated at 37 °C for 10 minutes before FRAP measurements began. The mobile fraction and diffusion coefficients are shown as mean ± S.E.RetrocyclinDiffusion coefficientMobile fractionnμg/mlμm2/s ( × 102)02.5 ± 0.394 ± 226104.6 ± 0.298 ± 521203.6 ± 0.497 ± 122 Open table in a new tab Temporal Analysis of the Retrocyclin Target—To dissect the kinetics of the fusion reaction and determine at which point retrocyclin-1 loses its inhibitory potency, we have utilized a time-of-addition experiment, in which inhibitors are added that act at the various steps of the fusion reaction at various times following co-culture of Env-expressing cells with target cells. All reactions were run for 120 min, from the initial co-culture to fusion quantification.Time-of-addition studies were done with Leu3A, retrocyclin-1, and C34 to assess the availability of the target of retrocyclin during the fusion process (31Gallo S.A. Puri A. Blumenthal R. Biochemistry. 2001; 40: 12231-12236Crossref PubMed Scopus (120) Google Scholar). Leu3A inhibits attachment of gp120 to CD4, and C34, a 34-residue peptide whose sequence matches the C-HR of gp41 (supplemental Fig. 1) prevents 6HB formation. Previously we had shown that C34 operates after Leu3A (31Gallo S.A. Puri A. Blumenthal R. Biochemistry. 2001; 40: 12231-12236Crossref PubMed Scopus (120) Google Scholar). Fig. 2a shows that at 30 min, by which time 50% of the HIV-1 Envs had interacted with CD4, fusion remained 100% inhibitable by retrocyclin-1 and C34. Thereafter, retrocyclin-1 and C34 had identical inhibition kinetics, indicating that the target of retrocyclin disappeared late, in a time frame similar to that of 6HB formation.FIGURE 2Retrocyclin-1 inhibits HIV-1 Env-mediated fusion in a late stage of the fusion cascade. a, time of Addition. Inhibitors Leu3a (3 μg/ml), RC-1 (4 μm), and C34 (2 μm) were added at different times after the initial co-culture of HIV-1 Env-expressing CV-1 cells with SupT1 target cells and the fusion was monitored using a dye redistribution assay as described under “Experimental Procedures.” b, soluble CD4-primed cells. Fusion of HIV-1 Env-expressing CV-1 cells (E) with SupT1 target cells (T) was monitored using a dye redistribution assay after pretreatment of targets or effectors. The following conditions were examined: no treatment (E0, T0); co-culture in the presence of inhibitor (E+, T+); pretreatment with inhibitor of effectors, followed by washing and co-culture with untreated targets (E+W & T0); pretreatment with inhibitor of targets followed by washing and co-culture with untreated effectors (T+W/E0); incubation of effectors for one h at 37 °C with sCD4 (40 μg/ml) followed by washing and incubation with targets (sCD4 E0 T0); incubation of effectors for one h at 37 °C with sCD4 + inhibitor, followed by washing and incubation with targets (sCD4 E+ T+). The inhibitors were retrocyclin-1 (4 μm) (black bars), sCD4 and C34 (2 μm) (gray bars).View Large Image Figure ViewerDownload Hi-res image Download (PPT)Effect of Priming—Binding HIV-1 Env to CD4 and either CXCR4 or CCR5 triggers conformational changes that lead to viral hairpin (6HB) formation and membrane fusion (27Gallo S.A. Finnegan C.M. Viard M. Raviv Y. Dimitrov A. Rawat S.S. Puri A. Durell S. Blumenthal R. Biochim. Biophys. Acta. 2003; 1614: 36-50Crossref PubMed Scopus (337) Google Scholar, 41Melikyan G.B. Markosyan R.M. Hemmati H. Delmedico M.K. Lambert D.M. Cohen F.S. J. Cell Biol. 2000; 151: 413-423Crossref PubMed Scopus (478) Google Scholar). In the prehairpin state that follows CD4 binding and precedes 6HB formation, N-terminal ectodomain regions are exposed (42Dimitrov A.S. Louis J.M. Bewley C.A. Clore G.M. Blumenthal R. Biochemistry. 2005; 44: 12471-12479Crossref PubMed Scopus (55) Google Scholar), later becoming inaccessible about when membrane fusion occurs (43Markosyan R.M. Cohen F.S. Melikyan G.B. Mol. Biol. Cell. 2003; 14: 926-938Crossref PubMed Scopus (167) Google Scholar). Throughout the period of accessibility, HIV-1 Env-mediated fusion can be inhibited by peptides derived from the N-HR or C-HR regions of gp41 (31Gallo S.A. Puri A. Blumenthal R. Biochemistry. 2001; 40: 12231-12236Crossref PubMed Scopus (120) Google Scholar, 41Melikyan G.B. Markosyan R.M. Hemmati H. Delmedico M.K. Lambert D.M. Cohen F.S. J. Cell Biol. 2000; 151: 413-423Crossref PubMed Scopus (478) Google Scholar, 44Gallo S.A. Clore G.M. Louis J.M. Bewley C.A. Blumenthal R. Biochemistry. 2004; 43: 8230-8233Crossref PubMed Scopus (19) Google Scholar).As the time-of-addition studies (Fig. 2a) had implicated N- or C-HR as targets for retrocyclin, we primed HIV-1 Env-expressing cells with 1 μg/ml of soluble CD4 (sCD4) and either retrocyclin-1 or C34 for 1 h at 37 °C before washing and then co-culturing them with target cells. Exposure to this suboptimal sCD4 concentration caused no inhibition, alone or in combination with C34 or retrocyclin-1 (Fig. 2b). However, incubating sCD4-primed cells with retrocyclin-1 or C34 inhibited fusion, lending support to the hypothesis that retrocyclin targets the N-HR and/or C-HR. Fig. 2b also shows that incubating either effectors or targets with RC-1 followed by washing resulted in no fusion inhibition, indicating that the RC-1 binding to targets and unprimed effectors is reversible.Binding of Retrocyclin-1 to gp41—Fig. 3 compares binding of retrocyclin-1 to gp41HXB2, gp120LAV, and bovine serum albumin (BSA). Per unit of mass, gp41 and gp120 bound retrocyclin-1 to an equal extent. However, as the mass of gp120 is ∼8 times larger than that of the gp41 ectodomain (∼16.4 kDa, exclusive of glycans), 8 times more retrocyclin-1 molecules bound a molecule of gp120 than a molecule of gp41. supplemental Table 1 shows four experiments comparing binding of retrocyclin-1 to gp120 and gp41. The Kd of retrocyclin-1 (mean ± S.E., n = 4) for gp41HXB2 was 67.6 ± 9.1 nm, and the Kd for gp120LAV was 33.0 ± 4.7 nm. These differences were significant (p = 0.021, paired t test).FIGURE 3Binding of retrocyclin-1. Binding to gp41HXB2 ectodomain, gp120LAV, and BSA was studied by surface plasmon resonance. Each data point is the mean of values from two separate complete dose-response curves. The biosensor chip contained the following amounts of attached protein: gp41, 2221 relative units (RU); gp120, 5746 RU; BSA, 5605 RU. Each data set has been normalized to show binding to 5000 RU of immobilized protein.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Because retrocyclin-1 has lectin-like properties (29Wang W. Cole A.M. Hong T. Waring A.J. Lehrer R.I. J. Immunol. 2003; 170: 4708-4716Crossref PubMed Scopus (176) Google Scholar) and the ectodomain of gp41HXB2 contains N-linked glycosylation sites, we did the experiments shown in supplemental Table 2 to learn whether retrocyclin-1 bound gp41 via its N-linked glycans. By using lectins, we determined that only high mannose glycans were present in the immobilized gp41 ectodomain. Removing these glycans with endoglycosidase H did not decrease the binding of retrocyclin to gp41, showing that the binding of retrocyclin-1 to gp41 was not carbohydrate-related.We next used a library of synthetic peptide fragments of HIV-1MN gp41 to determine where retrocyclin-1 might bind the gp41 ectodomain. Two C-terminal peptides, gp41(633-650) (EREIDNYTSLIYSLLEKS) and gp41(675-685) (DITNWLWYIK) showed the greatest binding, and gp41(651-674) bound retrocyclin-1 to a lesser extent (supplemental Table 3). We ran these surface plasmon resonance binding experiments in a competitive mode, using three different biosensors: BSA, gp120LAV, and gp41HXB2. Fig. 3 shows that binding of retrocyclin-1 to immobilized BSA is highly linear with peptide concentration, and its binding to the gp120 and gp41 biosensors is reasonably linear. Consequently, any of these biosensors can be used to assess the concentration of free (unbound) retrocyclin-1 in the presence of a potential binder, as long as the potential binder does not itself bind significantly to the biosensor. Indeed, with only one exception, biosensors containing BSA, gp120LAV, or gp41HXB2 gave similar results (supplemental Table 3).Fig. 4a shows that retrocyclin-1 bound the C34 domain of HIV-1 with greater affinity than the C34 domains of HIV-2 or SIV. Fig. 4b shows that BSA and gp120 biosensors detected binding between retrocyclin-1 and gp41(633-650) equally well, with an estimated IC50 of ∼15 μg/ml (7 μm). Fig. 4c shows that retrocyclin bound more extensively to gp41 (HIV-1HXB2) than to gp36 (HIV-2, Biodesign R5B220). Both recombinant Env glycoproteins had been expressed in yeast, Pichia pastoris. The net anionicity of a gp41 peptide did not fully account for its ability to bind retrocyclin-1, because gp41(651-674) (net charge, -3) bound less well than either gp41(633-650) or gp41(675-684), with respective net charges of -2 and 0. Nor was the net positive charge of retrocyclin-1 sufficient, because retrocyclin-1 that had been reduced and then alkylated with iodoacetamide no longer bound gp41, despite its undiminished net charge of +4 (data not shown). Thus, topological factors involving the placement and accessibility of the charged residues in gp41 and retrocyclin-1 could play key roles in binding.FIGURE 4Binding of retrocyclin-1 to C34 peptides. a, retrocyclin-1 (1 μg/ml) was mixed with various concentrations of C34 peptides from HIV-1, HIV-2, and SIV. This biosensor chip contained 5545 RU of immobilized BSA, and showed negligible binding of C34. b, biosensor chips on which either BSA or gp120 had been immobilized gave very similar results, here illustrated by gp41(633-650) from HIV-1. In a and b, binding (percent of control) is expressed relative to binding by retrocyclin-1 in the absence of C34 peptide. The concentrations of the C34 peptides were established by quantitative amino acid analyses. c, binding of retrocyclin-1 to recombinant gp41 from HIV-1 greatly exceeded its binding to immobilized gp36 from HIV-2. Both recombinant glycoproteins were expressed in a yeast, P. pastoris, and were immobilized to a similar density (∼2000 RU) on the biosensor chips.View Large Image Figure ViewerDownload Hi-res image Download (PPT)The gp41 ectodomains from HIV-1 strains MN, HXB2, and IIIB are shown in supplemental Fig. 1. The HXB2 strain used in our surface plasmon resonance experiments and the IIIB strain used in the cell fusion experiments have identical sequences that are similar to the MN sequence used for the gp41 peptide library. gp41(633-650), the peptide with the greatest binding to retrocyclin-1, comprises about half of the C34 sequence.Retrocyclin-1 Inhibits gp41 6HB Formation—Because retrocyclin-1 bound the C-peptides derived from the HIV-1 gp41 C-HR region, we considered that it might also block C-peptide/N-peptide interactions that form the 6HB of gp41. We tested this (Fig. 5 inset) by a native-PAGE method (32Liu S. Zhao Q. Jiang S. Peptides. 2003; 24: 1303-1313Crossref PubMed Scopus (69) Google Scholar). Neither N36 (lane 1) nor retrocyclin-1 (lane 3) nor a mixture of the two (lane 5) show up as a band, because their net positive charge cau" @default.
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• W2138746585 title "θ-Defensins Prevent HIV-1 Env-mediated Fusion by Binding gp41 and Blocking 6-Helix Bundle Formation" @default.
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