HGV) in the Selection

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Synthetic Peptides of Hepatitis G Virus (GBV-C/HGV) in the Selection of Putative Peptide Inhibitors of the HIV-1 Fusion Peptide Elena Herrera,† Maria J. Gomara,† Stefania Mazzini,‡ Enzio Ragg,‡ and Isabel Haro*,† Unit of Synthesis and Biomedical Applications of Peptides, IQAC-CSIC, Jordi Girona 18-26, 08034 Barcelona, Spain, and Department of Agri-Food Molecular Sciences, UniVersita` degli Studi, Via Celoria 2, 20133 Milano, Italy ReceiVed: January 23, 2009; ReVised Manuscript ReceiVed: April 6, 2009

The GB virus C or hepatitis G virus (GBV-C/HGV) is a single-stranded positive sense RNA virus that belongs to the Flaviviridae family. Recent years have seen the publication of numerous works in which coinfection with GBV-C/HGV and HIV has been associated with slower progression of the illness and a higher survival rate of patients once AIDS has developed. The mechanism by which the GBV-C/HGV virus has a “protective effect” in patients with HIV has still not been defined. Study of the interaction of the GBV-C/HGV and HIV viruses could lead to the development of new therapeutic agents for the treatment of AIDS. Given that the mechanism responsible for the beneficial effect exercised by the GBV-C/HGV virus in the course of HIV infection has not been defined, the present work is intended as a study of the structure and interactions between the fusion peptide of HIV-1, gp41(1-23), and synthetic peptide sequences of the E2 envelope protein of GBV-C/HGV using biophysical techniques. Our results highlight that the E2(269-286) sequence interacts with the target fusion peptide of HIV-1 and modifies its conformation. Introduction The GB virus C (GBV-C) or hepatitis G (HGV) virus is a single-stranded positive sense RNA virus that belongs to the Flaviviridae family. It is related to the hepatitis C virus (HCV) as it has a genomic structure and homology in the sequence very close to this hepatotropic virus. However, unlike HCV, it appears neither to cause hepatitis nor any other type of pathology. In recent years, numerous studies have been published in which coinfection with GBV-C/HGV and the human immunodeficiency virus (HIV) has been associated with slower progression of the illness and a higher survival rate of patients once AIDS has developed.1-3 Although numerous biological reports have studied the interference between GBV-C/HGV and HIV, the mechanism responsible for the beneficial effect that the GBV-C/HGV virus has on the course of infection caused by HIV has not still been defined. Recently, the HIV entry inhibition by the E2 envelope glycoprotein of GBV-C/HGV has been described.4 The results reported by this group demonstrated that GBV-C/HGV E2 protein inhibits early replication steps, such as binding or membrane fusion. On the basis of this purpose, the research initially proposed in our work group is based on the design of HIV fusion inhibitors using synthetic peptides of the GBV-C/ HGV E2 protein. Previous work of our group included identification, based on prediction studies, of possible internal fusion peptides corresponding to the E2 protein of the GBV-C/HGV virus using the selection of sequences that showed a capacity to insert themselves in membranes and to adopt β-turn structures. The chemical synthesis of the selected peptides and subsequent study of their interaction with model membranes enabled us to define * To whom correspondence should be addressed. Tel: +34934006109. Fax: +34932045904. E-mail: [email protected]. † Unit of Synthesis and Biomedical Applications of Peptides. ‡ Universita` degli Studi.

diverse sequences of the E2 structural protein that could be involved in the fusion process of the GBV-C/HGV virus. Specifically, the sequence (279-298) of the E2 protein of GBVC/HGV has been defined as a possible internal fusion peptide.4,5 J. H. McLinden et al. have characterized a single immunodominant antigenic site on GB virus C glycoprotein E2 that is involved in specific cellular binding.6 The minimal epitope characterized is the 9-mer sequence E2(285-293) (FYEPLVRRC), which is included in the E2 peptide sequence previously defined by us by means of biophysical studies as a putative fusion peptide. Thus, these authors suggested that this region of E2 is involved in cell binding and/or fusion and that structural changes associated with the addition of lipids to E2 protein may be involved in the formation of the antigenic site. Regarding that the E2 protein could be involved in the inhibition of HIV as it has been indicated by different groups,4,7 in the present work, we studied the inhibitory capacity of the interaction and destabilization process of membranes induced by the fusion peptide (FP) of the HIV-1 glycoprotein by GBVC/HGV E2 peptide sequences, including part of the minimal epitope involved in cellular-specific binding. The results indicate the capacity of certain peptide sequences of the (259-287) amino acid region of the GBV-C/HGV E2 protein in inhibiting the fusion process of liposomes induced by the FP of the HIV-1 glycoprotein, gp41. Moreover, peptide-peptide interactions have been characterized by means of conformational studies and isothermal titration calorimetry analysis. Experimental Section Peptides Synthesis. The multiple synthesis of 23 linear peptides related to the domain (259-287) of the E2 envelope protein of GBV-C/HGV was carried out in parallel in the solid phase using a semiautomatic synthesizer (Multisyntech GmbH). The 18-mer peptides are overlapped sequences by three residues. A Rink amide resin (TentaGel R RAM, RAPP Polymere GmbH)

10.1021/jp900707t CCC: $40.75  2009 American Chemical Society Published on Web 04/29/2009

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TABLE 1: E2(259-287) GBV-C/HGV Synthesized Peptide Sequences P1 P2 P3 P4 P5 P6 P7 P8 P9 P10 P11 P12 P13 P14 P15 P16 P17 P18 P19 P20 P21 P22 P23

multiple synthesis E2(259-287)

HPLC

ES-MS

TEVSEALGGAGLTGGFYE GTEVSEALGGAGLTGGFY LGTEVSEALGGAGLTGGF LLGTEVSEALGGAGLTGG LLLGTEVSEALGGAGLTG CLLLGTEVSEALGGAGLT NCLLLGTEVSEALGGAGL NNCLLLGTEVSEALGGAG LNNCLLLGTEVSEALGGA PLNNCLLLGTEVSEALGG LLLGTEVSEALGGAGLTGG CLLLGTEVSEALGGAGLTGG NCLLLGTEVSEALGGAGLTGG NNCLLLGTEVSEALGGAGLTGG LNNCLLLGTEVSEALGGAGLTGG PLNNCLLLGTEVSEALGGAGLTGG PPLNNCLLLGTEVSEALGGAGLTGG CLLLGTEVSEALGGAGLTG NCLLLGTEVSEALGGAGLT NNCLLLGTEVSEALGGAGL NNCLLLGTEVSEALGGAGLT PLNNCLLLGTEVSEALGGAG VPPLNNCLLLGTEVSEALGG VPPLNNCLLLGTEVSEALGGAGLTGGFYE

K′ ) 4,8 K′ ) 3,9 K′ ) 3,8 K′ ) 3,7 K′ ) 4,3 K′ ) 4,5 K′ ) 4,7 K′ ) 4,1 K′ ) 4,5 K′ ) 4,6 K′ ) 4,1 K′ ) 4,3 K′ ) 4,6 K′ ) 4,4 K′ ) 4,7 K′ ) 5,0 K′ ) 5,5 K′ ) 4,4 K′ ) 4,4 K′ ) 4,5 K′ ) 4,4 K′ ) 4,8 K′ ) 5,0

[M+] ) 1756,7 [M+] ) 1684,7 [M+] ) 16347 [M+] ) 1600,7 [M+] ) 1656,7 [M+] ) 1702,7 [M+] ) 1715,7 [M+] ) 1716,7 [M+] ) 1772,7 [M+] ) 1799,7 [M+] ) 1713,8 [M+] ) 1817,8 [M+] ) 1931,7 [M+] ) 2045,6 [M+] ) 2159,1 [M+] ) 2256,2 [M+] ) 2351,6 [M+] ) 1760,7 [M+] ) 1817,8 [M+] ) 1830,7 [M+] ) 1931,7 [M+] ) 1927,7 [M+] ) 1995,8

of functionalization 0.2 mmol/g, which allows carboxamide peptides to be obtained at the C-terminal end, was used. The Fmoc/tBut strategy based on the use of the 9-fluorenylmethoxycarbonyl (Fmoc) group, labile to piperidine/DMF, was used as temporary protection for the R-amino functions and tert-butyl (tBut) groups, labile to 95% TFA, and as semipermanent protection for the different functions of the side chains of the amino acids (tBut esters or ethers). The reagent groups were activated essentially by means of treatment with N,N′-diisopropylcarbodiimide (DIPCDI) and hydroxybenzotriazole (HOBt). The coupling reaction was carried out in duplicate using an excess of reagents three times. For the second coupling, the carboxyl group was activated by means of addition of a phosphonium salt, benzotriazole-1-yl-oxy-tris(pyrrolidino)phosphonium hexafluorophosphate (PyBop), in the presence of HOBt and a base such as diisopropylethylamine (DIEA). Once synthesis was complete, the cleavage and deprotection process of the peptidyl resins was carried out in the semiautomatic synthesizer using the Multisyntech accessories available for this purpose. This reaction took place by means of treatment with 95% trifluoroacetic acid (TFA) in the presence of scavengers, basically H2O and triisopropylsilane (TIS). The gp41 FP/AVGIGALFLGFLGAAGSTMGAAS was successfully synthesized in a 100% polyethylenglycol-based resin, the ChemMatrix, which has proved to be a superior support for the solid-phase synthesis of hydrophobic and highly structured peptides.8,9 The final characterization of synthetic peptides was carried out by analytical HPLC and electrospray mass spectrometry (Table 1). Liposomes. L-R-Palmitoyloleoylphosphatidylglicerol (POPG) was purchased from Avanti Polar-Lipids, Inc., and was used without further purification. POPG was dissolved in a chloroform/ methanol (2:1) mixture, and the lipid solution was dried by slow evaporation under a constant flow of nitrogen. The last traces of solvents were removed under vacuum at room temperature. Large unilamellar vesicles (LUVs) were prepared by hydration of the lipid film with Hepes buffer followed by 10 freeze-thaw cycles.10 This preparation was extruded 10 times through two

100 nm pore-size polycarbonate filters (Nucleopore, Pleasanton, CA) in a high-pressure extruder (Lipex, Biomembranes, Vancouver, Canada). The lipid concentration of liposome suspensions was determined by phosphate analysis.11 Vesicles size was determined from the measurement of the sample diffusion coefficient by photon correlation spectroscopy. Leakage of Vesicular Contents: ANTS/DPX Assay. For the ANTS (8-aminonaphtalene-1,3,6-trisulfonic acid)-DPX (N,N′p-xylenebis(pyridinium bromide) leakage assay,12 about 15 mg of POPG was dissolved in a mixture of chloroform and methanol (2:1) that was subsequently removed under a stream of nitrogen. About 2 mL of buffer containing 12.5 mM ANTS and 45 mM DPX from Molecular Probes (Eugene, OR) and 20 mM NaCl and 5 mM Hepes was added to the dry lipid. The osmolarity of the ANTS/DPX solution was adjusted to be equal to that of the buffer in a cryoscopic osmometer (Fiske One-ten). The suspension was frozen and thawed 10 times to ensure maximum entrapment prior to extrusion. A stock solution of LUV of approximately 0.1 µm in diameter was formed by extrusion pressure through Nucleopore polycarbonate membranes. The vesicles were separated from unencapsulated material on Sephadex G-75 (Pharmacia, Uppsala, Sweden), equilibrated with 100 mM NaCl/5 mM Hepes buffer (pH 7.4). The final lipid concentration was 0.1 mM. In order to select the E2 GBV-C/HGV peptide sequences that have the capacity to inhibit the interaction and destabilization process of membranes induced by the HIV fusion peptide, the biophysical assay on the vesicle contents release was used.13 In order to carry out screening of the synthesized peptides, the concentration of gp41(1-23) fusion peptide (HIV-1 FP) providing approximately half of the total vesicle contents release was selected. Each GBV-C/HGV peptide sequence corresponding to E2 protein was premixed with the HIV-1 FP in dimethylsulfoxide (DMSO) prior to its addition to the suspension of LUVs’ liposomes. Dequenching of coencapsulated ANTS and DPX fluorescence resulting from dilution was measured to assess the leakage of aqueous contents from vesicles.14

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ANTS/DPX leakage out of the LUVs (100 µM lipids) was measured after 30 min of incubation at room temperature in a Aminco Bowman AB2 (Microbeam, SA). Leakage was monitored by measuring the increase in the ANTS/DPX fluorescence intensity at 520 nm, with an excitation of 355 nm and slits of 8 nm. HIV-1 FP/E2 peptide ratios ranged from 1/1 to 1/40. The percentage of leakage was calculated as

% leakage ) [(F - F0) ⁄ (F100 - F0)] × 100 where F0 is the initial fluorescence of LUVs, F is the fluorescence intensity after incubation with the peptide, and F100 is fluorescence intensity after addition of 10 µL of a 10% (v/v) Triton-100 solution (complete lysis of the LUV). Lipid Mixing Assay. Lipid mixing of LUVs of POPG was measured using the resonance energy transfer assay of Struck et al.15 Lipid vesicles containing 0.6 mol % each of NBD-PE (energy donor) and Rho-PE (energy acceptor) and unlabeled vesicles were prepared at a 1:5 mixture of labeled and unlabeled vesicles (100 µM total phospholipid concentration) and were suspended in 500 µL of 10 mM Tris buffer, pH 7.4. Accordingly to ref 16, a relationship of 1/15 HIV-1 FP/liposome was chosen to study the capacity of GBV-C/HGV peptides to inhibit the lipid mixing induced by the HIV-1 FP. The HIV-1 FP was incubated for 30 min in DMSO with GBV-C/HGV peptides at different molar ratios ranging from 1/1 to 1/40 and subsequently was added to the liposome suspension. The increase at 540 nm was monitored, the excitation being at 467 nm. The fluorescence intensity of the lipid vesicles without peptide was the 0 % of lipid mixing, and the fluorescence upon the addition of Triton X-100 (0.1% v/v) was referred to as 100% of lipid mixing. Infrared Spectroscopy. The infrared measurements were performed on a Nicolet Avatar 360 Fourier-transform infrared spectrometer equipped with a deuterated triglyceride sulfate detector. Each spectrum was obtained by collecting 50 interferograms with a nominal resolution of 4 cm-1. A Ca2F flow cell with a window of a 100 µm path length spacer was used. Spectra of lipid-peptide mixtures were obtained, and reference spectra of solvents were recorded in the same microcells and under identical instrument conditions as those of the samples, which contained a peptide concentration of 2 mg/mL. Difference spectra were obtained by digitally subtracting the solvent spectrum from the sample spectra. To resolve overlapping bands, the spectra were processed using Peakfit software. Second derivative spectra were calculated to identify the positions of the component bands in the spectra. The deconvoluted spectrum was fitted with Gaussian band shapes by an iterative curve fitting procedure until good agreements were achieved between experimental and simulated spectra. Circular Dichroism. CD spectra were recorded on a Jasco J810 spectropolarimeter (Hachioji, Tokyo, Japan) equipped with a Peltier type temperature controller at room temperature flushed with a nitrogen flux of 10 L min-1 in a quartz cell with a 0.1 mm path length. CD spectra were acquired between 190 and 260 nm using a spectral bandwidth of 0.2 mm at a scan speed of 10 nm/min. Fifteen scans were averaged for each sample, and the respective buffer baselines were subtracted from the sample. CD data and the curves were smoothed using the program supplied by Jasco. Peptide conformation experiments were performed at 50 µM in aqueous buffer (phosphate 10 mM, pH 7.4) and in the presence of structure-promoting solvents such as trifluoroethanol (TFE) at different percentages. Isothermal Titration Calorimetry. Isothermal titration calorimetric (ITC) experiments were recorded on VP-ITC

Figure 1. Inhibitory effect of E2(279-284) and E2(264-287) GBVC/HGV peptides in induced leakage of POPG LUVs by HIV-1 FP.

microcalorimeter (MicroCal, LLC, Northampton, MA). Both purified peptides were dissolved in DMSO and then degassed for 5 min prior to sample loading. Briefly, a solution of 2 mM E2(269-286) in DMSO was injected into the chamber containing 100 µM HIV-1 FP. The calorimeter was first equilibrated at 20 °C, and the baseline was monitored during equilibration. The time between injections was 10 min, and the stirring speed was 300 rpm. The heats of dilution were determined in control experiments by injecting E2(269-286) into DMSO and subtracted from the heats produced in the corresponding peptidepeptide binding experiments. Control experiments were also performed by titrating DMSO into HIV-1 FP. The total observed heat effects were corrected for these small contributions. All titration data were subsequently analyzed using the Origin 7 software (MicroCal, LLC). 1 H NMR Spectroscopy. The NMR spectra were recorded with a Bruker AV-600 spectrometer, operating at a frequency of 600.10 MHz, equipped with a 5 mm inverse probe TXI and z-axis gradients. NMR samples were prepared by dissolving 1 mg of E2 and gp41 in 50 µL of DMSO and adding 0.50 mL of a H2O/CF3CD2OH (40:60 v/v) mixture, pH 3.5. Solutions were immediately transferred into 5 mm NMR tubes. 1D spectra were measured at temperatures of 25 °C. 1H spectra were referenced on external DSS, set at 0 ppm. Titration experiments were performed by adding of increasing amounts of gp41 dissolved in DMSO to a solution of E2 in H2O/CF3CD2OH (40:60 v/v) until R ) [gp41]/[E2] is equal to 1.0 with and without roomtemperature incubation. The 1H assignments for gp41 have been taken from ref 17, while the 1H assignments for E2 are reported in Table 1 in the Supporting Information. Two-dimensional homonuclear correlation spectra NOESY18 and TOCSY19 were acquired using standard pulse sequences in the phase-sensitive mode. 2D-NOESY spectra were obtained at 25 °C consecutively with different mixing times (tmix ) 50, 100, 200, and 300 ms). TOCSY spectra were obtained with a spin-lock total duration of 60 ms. Typically, 800 × 4096 spectra were acquired using TPPI and transformed to a final 2K × 2K real data matrix after apodization with a 90°-shifted sine-bell squared function. Baseline correction was achieved by a fifthdegree polynomial function. Solvent suppression was achieved by gradient-based pulse sequences as excitation sculpting for H2O/CF3CD2OH. Data processing was performed using TOPSPIN software (v. 1.5, Bruker Spectrospin, Germany). Pseudotwo-dimensional DOSY experiments20 were acquired using a diffusion delay, 0.12-0.45s, gradient pulse, 1.5 ms, and number of increments, 64. Raw data were processed using the standard DOSY software present in the Bruker library (TOPSPIN v. 1.3).

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Figure 2. Inhibitory effect on HIV-1 FP induced leakage of E2(259-287) GBV-C/HGV overlapped peptides.

Figure 3. Inhibitory effects on both HIV-1 FP induced leakage and lipid mixing of the E2(269-286) GBV-C/HGV peptide. The extent of both leakage and lipid mixing inhibition was plotted as a function of the E2(269-286)/HIV-1 FP molar ratio.

Results and Discussion Previous literature has proven useful for the application of a biophysical assay that included isolated synthetic fusion peptide derivatives and model membranes in the screening and identification of compounds with specific anti-gp41 activity.13 Taking into account this approach, the initial proposed work consists of studying the inhibitory capacity of the interaction and destabilization process of membranes induced by the fusion peptide of the HIV glycoprotein, gp41, by two previously described overlapping E2 peptides,5,7 E2(267-284) and E2(279-298), using the vesicular content leakage assay. These peptides were evaluated in regard to their capacity to inhibit the destabilization process of lipid vesicles induced by the HIV-I fusion peptide. In contrast to the E2(279-298) peptide, the sequence E2(267-284) was able to inhibit the leakage induced by the HIV-1 FP to an extent around 40% (Figure 1). As the (279-298) peptide did not shown any leakage effect on the interaction with the FP, we studied the amino acid requirements regarding the elongation of the sequence E2(267-284) at the N-terminus to inhibit vesicular content leakage induced by the HIV FP. To this end, a multiple synthesis scanning the (259-287) region of the E2 protein was carried out. The first 10 peptide sequences (P1-P10) are 18-mer overlapped peptides by 17 amino acid residues that scan the E2(261-287) region. To test the effect of the synthetic peptide length on the vesicular leakage inhibition assay, peptide sequences ranging from 19 to 25 amino acid residues were also synthesized (P11-P17). Finally, two sets of three peptides of 19-mer (P18-P20) and 20-mer (P21-P23) overlapped by 18 amino acids were synthesized to scan the region E2(259-283). The peptides were correctly characterized by analytical HPLC and MS-ES spectrometry (Table 1). As shown in Figure 2, the peptides P1 and P2 inhibit the leakage induced by the HIV-1 FP to an extent around 50%.

Figure 4. Isothermal titration calorimetry results of the E2(269- 286)/ HIV-1 FP interaction.

Taking into account the primary sequence of the peptides, it seems that the carboxi-terminal region of the scanned E2(259-287) sequence is important in the leakage inhibition induced by the HIV-1 FP, the amino acid residues 283-286 being particularly involved in the interaction with the HIV FP. P2 corresponding to the region 269-286 of the GBV-C/HGV E2 protein was capable of inhibiting the activity of the HIV-1 fusion peptide in an extend higher than 50%. The intrinsic lytic effect of the GBV-C/HGV peptide alone was null or negligible. Several ratios of the HIV-1 fusion peptide and the E2(269286) peptide (1:1,1:2, 1:5, 1:10, 1:20, and 1:40) were tested both in lipid mixing and in leakage assays. As shown in Figure 3, E2(269-286) inhibit the permeabilization vesicular process as well as the intervesicular lipid mixing induced by the HIV-1 FP; 50% inhibition was observed for a HIV-1 FP/E2(269-286) ratio of 1/10. Leakage inhibition correlated with inhibition of HIV-1 FP induced intervesicle mixing of lipids. In order to test the specificity of the interaction between the E2(269-286) and HIV FP, we used melittin as a control peptide. Melittin, the main proteinaceous component of honeybee venom, has been extensively studied as a model peptide for lytic peptide-lipid interactions. 21,22 Melittin

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Figure 5. CD spectra of (A) 50 µM HIV-1 FP in aqueous buffer (phosphate 10 mM, pH 7.4); (B) TFE titration of 50 µM HIV-1 FP in aqueous buffer; (C) 50 µM E2(269-286) GBV-C/HGV peptide; (D) TFE titration of 50 µM E2(269-286) GBV-C/HGV peptide in aqueous buffer; (E) equimolecular mixture of HIV-1 FP/E2(269-286) peptides in aqueous buffer.

induced ANTS/DPX leakage from POPG LUVs at peptideto-lipid mole ratios higher than 1/50. The 50% POPG vesicular content leakage induced by melittin was establish at a peptide-to-lipid mole ratio of 1/10. Performing the assay in the same conditions as those using the HIV-1 FP, several

ratios of melittin and E2(269-286) (1:1; 1:2, 1:5, 1:10, 1:20, 1:40) were premixed in DMSO and tested in the leakage assay. The results showed that the E2(269-286) peptide was unable to inhibit the membrane lytic activity of melittin at any relationship studied, thus indicating the specificity of the

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Figure 6. FTIR spectra of (A) HIV-1 FP; (B) E2(269-286) peptide; and (C) an equimolecular mixture of HIV-1 FP/E2(269-286) peptides in deuterated DMSO.

interaction between GBV-C/HGV E2(269-286) peptide and the HIV-1 FP. To get further into this interaction, thermodynamic parameters associated with the binding of E2(269-286) to HIV-1 FP were determined by isothermal titration calorimetry (ITC). This information can provide insight into the dominant forces associated with binding. Regarding the insolubility of the HIV-1 FP, a microcalorimetry titration experiment was performed using DMSO as a solvent. The binding of the E2(269-286) to HIV-1 FP was exothermic, as indicated by negative peaks (Figure 4 (top)). The hyperbolic titration curve demonstrates that the binding site of HIV-1 FP was saturated with the corresponding peptide. The cumulative reaction enthalpy as a function of the molar ratio is shown in Figure 4 (bottom). The solid line corresponds to the best theoretical fit to the experimental data. The binding enthalpy (∆H) of the GBV-C/HGV peptide to the HIV-1 FP was -4.9 ( 0.37 kcal/mol, and the entropy change -T∆S was -1.3 kcal/mol. This information provide insight into the

Figure 7. FTIR spectra of (A) HIV-1 FP; (B) E2(269-286) peptide; and (C) an equimolecular mixture of HIV-1 FP/E2(269-286) peptides in 50% TFE/D2O.

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Figure 8. 600 MHz 1H NMR spectrum of (a) gp41, (b) E2, and (c) E2 + gp41 at R ) [gp41]/[E2] ) 1 at 25 °C in H2O/CF3CD2OH (40:60 v/v).

dominant forces driving association between E2(269-286) and HIV-1 FP. This association is primarily driven by enthalpy, with an unfavorable entropic contribution. The dominant negative enthalpy suggest that there are a large number of favorable hydrogen bond contacts or van der Waals interactions between E2(269-286) and HIV-1 FP. The negative Gibbs energy (∆G) indicates that the binding of these two peptides is spontaneous at all temperatures.23 The fit yielded a binding affinity (Ka) of 4.83 ( 0.37 × 104 -1 M (Kd ) 20.7 µM) and a binding stoichiometry of about 1, indicating that this peptide binds to a monomer of HIV-1 FP. Besides, we used CD to analyze if the ability of the E2(269-286) peptide to bind HIV-1 FP induces a conformational change in its secondary structure. In this sense, CD spectra of the isolated HIV-1 FP in aqueous buffer showed a negative band with a peak between 215 and 220 nm which could be attributed to a β-structure contribution probably due to peptide aggregation (Figure 5A). As reported by Gordon et al.,24 the addition of TFE for stabilizing the secondary conformation promoted a characteristic double minimum at 208 and 222 nm, suggesting R-helical content for HIV-1 FP in this environment. In addition, a structural transition with an isodichroic point at 200 nm provides evidence that simple two-

state equilibrium (β-sheet T R-helix) has occurred for this peptide at 50 µM (Figure 5B). On the other hand, isolated E2(269-286) peptide was also analyzed by CD in different environments. When studying the conformational behavior of the peptide in aqueous buffer, the CD spectrum showed a minimum around 200 nm that was assigned to the random coil conformation (Figure 5C). When TFE titration was affected, the characteristic bands located at 207 and 222 nm appeared, and as a result, the spectra (Figure 5D) looked more like those of an R-helix structure. As shown in HIV-1 FP, a structural transition with an isodichroic point at 200 nm proves that simple two-state equilibrium (random coil T R-helix) has occurred for this peptide at 50 µM. The experimental CD spectrum of the mixture of E2(269-286) peptide and HIV-1 FP at equimolecular concentration in aqueous buffer was different from the theoretical spectrum obtained by summing experimental spectra of equivalent amounts of the peptides alone (Figure 5E). The results suggested that the E2(269-286) peptide interacts with HIV-1 FP. The equimolecular mixture demonstrated that the addition of E2(269-286) to the HIV-1 FP reduced significantly the mean residue ellipticity

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Figure 9. 600 MHz 1H NMR spectrum of (a) E2 and E2 + gp41 at R ) [gp41]/[E2] equal to (b) 0.1, (c) after 20 min, (d) R ) 0.2, (e) after 20 min, (f) R ) 0.3 after 30 min, (g and h) R ) 1.0 after 15 and 45 min, respectively, at 25 °C in H2O/CF3CD2OH (40:60 v/v).

at 220 nm, thus avoiding HIV-1 FP aggregation in aqueous buffer (Figure 5E). To gain insight into the conformational changes taking place on the peptide-peptide interaction, the secondary structure variations of an equimolecular mixture of HIV FP and E2(269-286) in different environments were monitored by Fourier-transform infrared spectroscopy (FTIR). Regarding that the peptide-peptide interaction was first characterized by ITC in DMSO and due to the impossibility of using this solvent in circular dichroism spectroscopy, we analyzed the structural changes upon peptide-peptide interaction in DMSO, which is the solvent more commonly used to stock the fusion peptides. The experimental FTIR spectrum of FP HIV-1 in deuterated DMSO showed the maximum of the amide I band centered at 1661 cm-1 (Figure 6A). Such an amide I maximum has been assigned, for proteins and peptides dissolved in DMSO, to the stretching vibration of amide carbonyls which do not form hydrogen bonds with the amide NH groups.25 According to V. Buzon et al.,26 the HIV FPs in pure DMSO are, therefore, monomeric and unestructured. The infrared transmission spectrum of the E2(269-286) peptide in DMSO shows a broad band centered at 1648 cm-1, which could be interpreted as being the result of a mixture of spectral components assignable to helical and unordered structures (Figure 6B). When analyzing an equimolecular mixture of HIV-1 FP and the E2(269-287) peptide in DMSO, there is a conformational

change which is also observed by FTIR measurements (Figure 6C). In this sense, the band located at around 1655 cm-1 could be attributed to an R-helix conformation. Moreover, the band located at 1670 cm-1 could be assigned to a turn conformation. Besides, in order to characterize the conformational changes upon peptide-peptide interaction in membrane-mimic solvents, 50% TFE in water was used. As it has been described by Gordon et al.,24 a principal band occurs at 1655 cm-1 for the TFE spectra, consistent with high R-helical content for the HIV-1 FP in this membrane-mimic environment (Figure 7A). The spectrum corresponding to the E2(269-286) peptide when added into the TFE/water solvent also reveals a dominant band at 1653 cm-1, which indicates that the predominant component is an R-helix (Figure 7B). As compared with the spectra of each peptide at the same peptide concentration, the experimental spectrum of the mixture of these two peptides showed contributions of β-turn (1660 cm-1 band) and random (1645 cm-1 band) structures (Figure 7C). Similar experimental conditions were used for 1H NMR experiments. 1H NMR spectra of E2 and gp41 peptides were performed in water/TFE (40:60 v/v) solution, pH 3.5. The addition of TFE was important for the acquisition of good-quality spectra. Characteristic amino acid spin systems and the presence of the helical structure were identified from the analysis of 2D-TOCSY and 2DNOESY experiments and from the observation of the NH(i)-NH(i+1) interactions for both peptides observed in 2D-

Synthetic Peptides of Hepatitis G Virus NOESY spectra, respectively. Titration experiments were performed by adding of increasing amounts of gp41 dissolved in DMSO to a solution of E2 until R ) [gp41]/[E2] was equal to 1.0 with and without room-temperature incubation (Figure 8). The spectrum of the mixture was characterized by a generalized line broadening, suggesting an increase of the molecular correlation time and the presence of a high-molecular-weight aggregates. The spectra showed, even at low ratio (R ) 0.1), an upfield chemical shift variation (0.2 ppm) of the gp41 amide resonances (Figure 9) and the change of the 1H line widths starting from R ) 0.2. NMR DOSY experiments, performed at room temperature, allowed one to obtain a single diffusion coefficient (D) for the mixture at the ratio R ) [gp41]/[E2] ) 1.0, and this reflects, as a first consideration, that E2 and gp41 move together in solution, forming stable interactions. The value of D (10-10.04 m2 s-1) is in agreement with a high molecular weight corresponding to an aggregate species. Thus, it seems that there is a conformational change upon mixing HIV FP and E2(269-286) which could be involved in the inhibition of the membrane destabilization process induced by the HIV-1 FP. It has been described that obliquely inserted R-helices and aggregated β-structures are the two secondary structures of the HIV-1 FP responsible for destabilizing the membrane and triggering the fusion process.27,28 In addition, it has been recently stated by the group of Cladera that fusion is preceded by a conformational change which consists of the transformation of helical and unordered structures into β-aggregated ones, and such a conformational change takes place upon binding of the peptide to the membrane.26 In this sense, it seems that the interaction between HIV-1 FP and E2(269-286) characterized in this work avoids oligomerization of the HIV-1 FP upon membrane binding, thus inhibiting the membrane destabilization effect concomitant to membrane fusion. Conclusions We have found a GBV-C/HGV-related peptide, E2(269-286), with the ability to interact and inhibit the activity of the HIV-1 fusion peptide in model membranes. The results show that E2(269-286) peptide interacts with target HIV-1 FP and modifies its conformation, thus indicating that E2(269-286) peptide could be able to alter the HIV-1 FP interaction with membranes. Thus, this peptide could be involved in the prevention of HIV-1 entry by its binding to the HIV-1 FP, which avoids the triggering of the HIV-cell fusion process. In order to test the putative use of this peptide as an antiviral therapeutic agent, cell-cell fusion assays as well as HIV-1 replication assays will be performed. Acknowledgment. This work was funded by Grant CTQ200615396-CO2-01/BQU from the Ministerio de Ciencia e Innovacio´n, Spain.

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