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Originally published In Press as doi:10.1074/jbc.M709413200 on January 15, 2008

J. Biol. Chem., Vol. 283, Issue 13, 8089-8101, March 28, 2008
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Identification of the Membrane-active Regions of Hepatitis C Virus p7 Protein

BIOPHYSICAL CHARACTERIZATION OF THE LOOP REGION*

Ana J. Pérez-Berná{ddagger}1, Jaime Guillén{ddagger}1, Miguel R. Moreno{ddagger}, Angela Bernabeu{ddagger}, Georg Pabst§, Peter Laggner2, and José Villalaín{ddagger}2

From the {ddagger}Instituto de Biología Molecular y Celular, Universidad "Miguel Hernández," E-03202 Alicante, Spain and the §Institute of Biophysics and Nanosystems Research, Austrian Academy of Sciences, Graz A-8042, Austria

Received for publication, November 16, 2007 , and in revised form, January 14, 2008.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
We have identified the membrane-active regions of the hepatitis C virus p7 protein by performing an exhaustive study of membrane rupture, hemifusion, and fusion induced by a p7-derived peptide library on model membranes having different phospholipid compositions. We report the identification in p7 of a highly membranotropic region located at the loop domain of the protein. Here, we have investigated the interaction of a peptide patterned after the p7 loop (peptide p7L), studying its binding and interaction with the lipid bilayer, and evaluated the binding-induced structural changes of the peptide and the phospholipids. We show that positively rich p7L strongly binds to negatively charged phospholipids and it is localized in a shallow position in the bilayer. Furthermore, peptide p7L exhibits a high tendency to oligomerize in the presence of phospholipids, which could be the driving force for the formation of the active ion channel. Therefore, our findings suggest that the p7 loop could be an attractive candidate for antiviral drug development, because it could be a target for antiviral compounds that may lead to new vaccine strategies.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Hepatitis C virus (HCV)3 is an enveloped positive single-stranded RNA virus included in the genus Hepacivirus that belongs to the Flaviviridae family. HCV is an important public health problem because it is the leading cause of acute and chronic liver disease in humans, including chronic hepatitis, cirrhosis, and hepatocellular carcinoma (1–3). Currently, there are no vaccines to prevent HCV infection and the available therapeutic agents have very limited efficacy against the virus (4). The HCV genome consists of one translational open reading frame encoding a polyprotein precursor of ~3010 amino acids in length, including structural and non-structural proteins, which is cleaved by host and viral proteases (Fig. 1A). The HCV genome is widely heterogeneous; replication errors cause a high rate of mutations (5). HCV entry into the host cell is achieved by fusion of viral and cellular membranes, and the morphogenesis and virion budding has been suggested to take place in the endoplasmic reticulum (6). Therefore, the viral region implicated in fusion to and/or budding from the cells must interact with the membrane and should be a conserved sequence. The variability of the HCV proteins gives the virus the ability to escape the host immune surveillance system and notably hampers the development of an efficient vaccine. Thus, finding inhibitors of protein-membrane and protein-protein interactions involved in virus fusion and/or budding could be an alternative and valuable strategy against HCV infection because they could be potential therapeutic agents.

Protein p7 gene is located between the structural and the non-structural regions of the HCV polyprotein precursor, specifically between the E2 and NS2 genes. Cleavage of p7 is mediated by the endoplasmic reticulum (ER) signal peptidases of the host cell (7, 8). The protein p7 is classified neither as a structural protein nor as non-structural (9) and locates in the cell ER. The role of p7 in the virus life cycle has been elusive. It has been shown that p7 is not critical for RNA replication (10), although homologous proteins from other members of the Flaviviridae family are critical for cell culture infectivity (11). A recent report, however, demonstrated that p7 is essential for efficient assembly and release of infectious virions indicating that p7 is primarily involved in the late phase of the virus replication cycle (12).

At a molecular level, p7 is a small transmembrane protein of 63 amino acids with two transmembrane helical domains, TM1 and TM2, connected by a loop. Whereas the loop is oriented toward the cytoplasm, the amino- and carboxyl-terminal tails are oriented toward the ER lumen (13, 14). Mutations in the loop region abrogate the channel activity of p7, which has been described to be a viroporin-like protein (12, 15, 16). These functional groups of proteins form ion channels that might be important for virus assembly and/or release; p7 is capable of forming cation-selective ion channels in artificial lipid membranes at physiological pH. Akin to other viral proteins such as M2, NB, and Vpu, secondary structure predictions suggest that p7 may form a hexameric bundle of helical dimers with a pore diameter of 3–5 nm (13, 16). One possible role for this protein could be the transport of ions from the ER to the cytoplasm of HCV-infected host cells. Amantadine, hexamethylene amiloride, and long alkyl-chain amino sugar derivatives are ion channel inhibitors that block ion transport mediated by p7 in lipid membranes (16–18), suggesting that p7 could be a attractive candidate for antiviral drug development.

We have recently identified the membrane-active regions of the HIV gp41, SARS-CoV spike, and HCV E1 and E2 glycoproteins by observing the effect of glycoprotein-derived peptide libraries on model membrane integrity (19–21). These results allowed us to propose the location of different segments in these proteins that are implicated in protein-lipid and protein-protein interactions. These studies have helped us to understand the mechanisms underlying the interaction between viral proteins and membranes. Using a similar approach, we have carried the analysis of the membrane-active regions of p7 by investigating the effect of a p7-derived peptide library from the HCV strain HCV_1B4J, on the integrity of different membrane model systems. Here, we report the identification of a membranotropic region in p7 coincidental with the loop domain of the protein, which exhibits membrane-interacting properties akin to those found for the loop domain of HIV gp41 (21–23). Furthermore, we have focused on the possible functional roles of the p7 loop domain by an in-depth study of a peptide patterned after this domain, peptide p7L. We have studied the binding and interaction of p7L with the lipid bilayer, as well as the structural changes induced in both the peptide and phospholipid molecules upon membrane binding. We show that p7L strongly partitions into phospholipid membranes, interacts with negatively charged phospholipids, locates in a shallow position in the membrane, and oligomerizes in the membrane.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Materials and Reagents—Three sets of 11 peptides derived from HCV_1B4J protein p7 (strictly speaking, p7 spans from residue 746 to residue 809 of the HCV polyprotein precursor) were obtained through the National Institutes of Health AIDS Research and Reference Reagent Program (Division of AIDS, NIAID, NIH, Bethesda, MD). The peptide p7L corresponding to the sequence 771FFCAAWYIKGRLAPGAAY788 (with NH2-terminal acetylation and COOH-terminal amidation) was obtained from Genemed Synthesis, San Francisco, CA. The peptide p7L was purified by reverse-phase high pressure liquid chromatography (Vydac C-8 column, 250 x 4.6 mm, flow rate 1 ml/min, solvent A, 0.1% trifluoroacetic acid, solvent B, 99.9 acetonitrile and 0.1% trifluoroacetic acid) to better than 95% purity, and its composition and molecular mass were confirmed by amino acid analysis and mass spectroscopy. Because trifluoroacetate has a strong infrared absorbance at ~1673 cm–1, which interferes with the characterization of the peptide Amide I band (24), residual trifluoroacetic acid, used both in peptide synthesis and in the high-performance liquid chromatography mobile phase, was removed by several lyophilization/solubilization cycles in 10 mM HCl (25). Egg L-{alpha}-phosphatidylcholine (EPC), egg L-{alpha}-phosphatidic acid (EPA), egg sphingomyelin (SM), bovine brain L-{alpha}-phosphatidylinositol; bovine brain L-{alpha}-phosphatidylserine (BPS), 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), 1,2-dimyristoyl-sn-glycero-3-(phospho-rac-(1-glycerol)) (DMPG), 1,2-dimyristoyl-sn-glycero-3-(phospho-L-serine), 1,2-dimyristoyl-sn-glycero-3-phosphate (DMPA), cholesterol (Chol), liver lipid extract, Lissamine rhodamine B 1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine (N-Rh-PE), and N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)-1,2-dihexadecanoyl-sn-glycero-3-phosphatidylethanolamine (NBD-PE) were obtained from Avanti Polar Lipids (Alabaster, AL). 5-Carboxyfluorescein (CF) (>95% by high pressure liquid chromatography), fluorescein isothiocyanate-labeled dextran FD10 (average molecular weight 10,000), 5-doxyl-stearic acid (5-NS), 16-doxyl-stearic acid (16-NS), dehydroergosterol (ergosta-5,7,9(11),22-tetraen-3β-ol, DHE), sodium dithionite, deuterium oxide (99.9% by atom), Triton X-100, EDTA, and HEPES were purchased from Sigma. 1,6-Diphenyl-1,3,5-hexatriene (DPH), 3-(4-(6-phenyl)-1,3,5-hexatrienyl)-phenylpropionic acid (PA-DPH), 1-(4-trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene (TMA-DPH), 4-(2-(6-(dioctylamino)-2-naphthalenyl)(ethenyl)-1-(3-sulfopropyl)-pyridinium inner salt (di-8-ANEPPS) were obtained from Molecular Probes (Eugene, OR). The lipid composition of the synthetic endoplasmic reticulum was EPC/BPS/bovine brain L-{alpha}-phosphatidylinositol/SM/Chol at a molar ratio of 51:2.4:5.3:7.48:33.42 (26). All other reagents used were of analytical grade from Sigma. Water was deionized, twice distilled, and passed through Milli-Q equipment (Millipore Ibérica, Madrid, Spain) to a resistivity better than 18 M{Omega} cm.

Vesicle Preparation—Aliquots containing the appropriate amount of lipid in chloroform/methanol (2:1, v/v) were placed in a test tube, the solvents were removed by evaporation under a stream of O2-free nitrogen, and finally, traces of solvents were eliminated under vacuum in the dark for more than 3 h. The lipid films were resuspended in an appropriate buffer and incubated either at 25 or 10 °C above the phase transition temperature (Tm) with intermittent vortexing for 30 min to hydrate the samples and obtain multilamellar vesicles (MLV). The samples were frozen and thawed five times to ensure complete homogenization and maximization of peptide/lipid contacts with occasional vortexing. Large unilamellar vesicles (LUV) with a mean diameter of 0.1 and 0.2 µm for either leakage or hemifusion and fusion experiments were prepared from multilamellar vesicles by the extrusion method (27) using polycarbonate filters with a pore size of 0.1 and 0.2 µm (Nuclepore Corp., Cambridge, CA). The phospholipid and peptide concentration were measured by methods described previously (28, 29).

Membrane Leakage Measurement—LUVs with a mean diameter of 0.1 µm were prepared as indicated above in buffer containing 10 mM Tris, 20 mM NaCl, pH 7.4, and either CF at a concentration of 40 mM or FD10 at a concentration of 100 mg/ml. Non-encapsulated CF or FD10 were separated from the vesicle suspension through a filtration column containing Sephadex G-75 or Sephadex S500HR Sephacryl, respectively (GE Healthcare), eluted with buffer containing 10 mM Tris, 100 mM NaCl, 0.1 mM EDTA, pH 7.4. Membrane rupture (leakage) of intraliposomal CF was assayed by treating the probe-loaded liposomes (final lipid concentration, 0.125 mM) with the appropriate amounts of peptide on microtiter plates using a microplate reader (FLUOstar; BMG Labtech, Offenburg, Germany), stabilized at 25 °C, with the appropriate amounts of peptide, each well containing a final volume of 170 µl. The medium in the microtiter plates was continuously stirred to allow the rapid mixing of peptide and vesicles. Membrane leakage of intraliposomal FD10 was carried out using 5 x 5-mm quartz cuvettes stabilized at 25 °C in a final volume of 400 µl (100 µM lipid concentration). Leakage was assayed until no more change in fluorescence was obtained. The fluorescence was measured using a Varian Cary Eclipse spectrofluorimeter. Changes in fluorescence intensity were recorded with excitation and emission wavelengths set at 492 and 517 nm, respectively. Excitation and emission slits were set at 5 nm. One hundred percent release was achieved by adding Triton X-100 to either the microtiter plate or to the cuvette to a final concentration of 0.5% (w/w). For details see Refs. 30 and 31.

Phospholipid Mixing Measurement—Peptide-induced vesicle lipid mixing was measured by resonance energy transfer (32). This assay is based on the decrease in resonance energy transfer between two probes (NBD-PE and RhB-PE) when the lipids of the probe-containing vesicles are allowed to mix with lipids from vesicles lacking the probes. The concentration of each of the fluorescent probes within the liposome membrane was 0.6% mol. LUVs with a mean diameter of 0.2 µm were prepared as described above. Labeled and unlabeled vesicles at a proportion of 1:4 were placed in a 5 x 5-mm fluorescence cuvette at a final lipid concentration of 100 µM in a final volume of 400 µl, stabilized at 25 °C under constant stirring. The fluorescence was measured using a Varian Cary Eclipse fluorescence spectrometer using 467 and 530 nm for excitation and emission, respectively. Excitation and emission slits were set at 10 nm. The proportion of labeled and unlabeled vesicles, lipid concentration, and other experimental and measurement conditions were the same as indicated previously (31).

Inner Monolayer Phospholipid Mixing (Fusion) Measurement—Peptide-induced phospholipid mixing of the inner monolayer was measured by a modification of the phospholipid mixing measurement stated above (33). This assay is based on the decrease in resonance energy transfer between two probes (NBD-PE and RhB-PE) when the lipids of the probe-containing vesicles are allowed to mix with lipids from vesicles lacking the probes. The concentration of each of the fluorescent probes within the liposome membrane was 0.6% mol. LUVs with a mean diameter of 0.2 µm were prepared as described above. LUVs were treated with sodium dithionite to completely reduce the NBD-labeled phospholipid located at the outer monolayer of the membrane. Final concentration of sodium dithionite was 100 mM (from a stock solution of 1 M dithionite in 1 M Tris, pH 10.0) and incubated for ~1 h on ice in the dark. Sodium dithionite was then removed by size exclusion chromatography through a Sephadex G-75 filtration column (GE Healthcare) eluted with buffer containing 10 mM Tris, 100 mM NaCl, 1 mM EDTA, pH 7.4. The proportion of labeled and unlabeled vesicles, lipid concentration, and other experimental and measurement conditions were the same as indicated above for the phospholipid mixing assay.

Peptide Binding to Vesicles—The partitioning of the peptide into the phospholipid bilayer was monitored by the fluorescence enhancement of tryptophan. Fluorescence spectra were recorded in a SLM Aminco 8000C spectrofluorometer with excitation and emission wavelengths of 290 and 348 nm, respectively, and 4-nm spectral bandwidths. Measurements were carried out in 20 mM HEPES, 50 mM NaCl, 0.1 mM EDTA, pH 7.4. Intensity values were corrected for dilution, and the scatter contribution was derived from lipid titration of a vesicle blank. The data were analyzed as previously described (31).

Steady-state Fluorescence Anisotropy—DPH and its derivatives represent popular membrane fluorescent probes for monitoring the organization and dynamics of membranes; whereas DPH is known to partition mainly into the hydrophobic core of the membrane, PA-DPH and TMA-DPH probes are oriented at the membrane bilayer with their charge localized at the lipid-water interface, TMA-DPH nearer the membrane surface than PA-DPH (34). MLVs were formed in 100 mM NaCl, 0.05 mM EDTA, and 25 mM HEPES, pH 7.4. Aliquots of PA-DPH, TMA-DPH, or DPH in N,N'-dimethylformamide (2 x 10–4 M) were directly added into the lipid dispersion to obtain a probe/lipid molar ratio of 1:500. Samples were incubated for 15, 45, or 60 min when TMA-DPH, PA-DPH, or DPH were used, respectively, 10 °C above the gel to liquid-crystalline phase transition temperature Tm of the phospholipid mixture. Afterward, the peptides were added to obtain a peptide/lipid molar ratio of 1:15 and incubated 10 °C above the Tm of each lipid for 1 h, with occasional vortexing. All fluorescence studies were carried using 5 x 5-mm quartz cuvettes in a final volume of 400 µl (315 µM lipid concentration). All the data were corrected for background intensities and progressive dilution. The steady-state fluorescence anisotropy, <r>, was measured with an automated polarization accessory using a Varian Cary Eclipse fluorescence spectrometer, coupled to a Peltier device (Varian) for automatic temperature change. The data were analyzed as previously described (31).

Fluorescence Quenching of Trp Emission by Water-soluble and Lipophilic Probes—For acrylamide quenching assays, aliquots from a 4 M solution of the water-soluble quencher were added to the solution-containing peptide in the presence and absence of liposomes at a peptide/lipid molar ratio of 1:100. The results obtained were corrected for dilution and the scatter contribution was derived from acrylamide titration of a vesicle blank. The data were analyzed according to the Stern-Volmer equation (35), I0/I = 1 + KSV(Q), where I0 and I represent the fluorescence intensities in the absence and the presence of the quencher (Q), respectively, and Ksv is the Stern-Volmer quenching constant, which is a measure of the accessibility of Trp to acrylamide. Quenching studies with lipophilic probes were performed by successive addition of small amounts of 5-NS or 16-NS in ethanol to the samples of the peptide incubated with LUV. The final concentration of ethanol was kept below 2.5% (v/v) to avoid any significant bilayer alterations. After each addition an incubation period of 15 min was kept before the measurement. The data were analyzed as previously described (31).

Fluorescence Measurements Using FPE-labeled Membranes—LUVs with a mean diameter of 0.1 µm were prepared in buffer containing 10 mM Tris-HCl, pH 7.4. The vesicles were labeled exclusively in the outer bilayer leaflet with FPE as described previously (36). Briefly, LUVs were incubated with 0.1 mol % FPE dissolved in ethanol (never more than 0.1% of the total aqueous volume) at 37 °C for 1 h in the dark. Any remaining unincorporated FPE was removed by gel filtration on a Sephadex G-25 column equilibrated with the appropriate buffer. FPE vesicles were stored at 4 °C until use in an oxygen-free atmosphere. Fluorescence time courses of FPE-labeled vesicles were measured after the desired amount of peptide was added into 400 µl of lipid suspensions (200 µM lipid) using a Varian Cary Eclipse fluorescence spectrometer. Excitation and emission wavelengths were set at 490 and 520 nm, respectively, using excitation and emission slits set at 5 nm. Temperature was controlled with a thermostatic bath at 25 °C. The contribution of light scattering to the fluorescence signals was measured in experiments without the dye and was subtracted from the fluorescence traces. Data were fitted either to a hyperbolic binding model (37) using the following equation, F = (Fmax [P]/Kd + [P], where F is the fluorescence variation, Fmax the maximum fluorescence variation, [P] the peptide concentration, and Kd the dissociation constant of the membrane binding process. The experimental points shown in the figures are the mean values of at least three measurements.

Thioflavin T Assay for Peptide Aggregation—Peptide aggregation was assayed by using thioflavin T (ThT). Thioflavin T associates rapidly with aggregated peptides giving rise to a new excitation maximum at 450 nm and a enhanced emission at 482 nm (38). Buffer contained 100 mM NaCl, 10 mM Tris-HCl, 25 µM ThT, pH 7.4, LUVs (final phospholipid concentration of 0.5 mM), and a peptide concentration of 5 µM. Fluorescence was measured before and after the desired amount of peptide was added into the cuvette using a Varian Cary Eclipse fluorescence spectrometer. Temperature was controlled with a thermostatic bath at 25 °C under constant stirring. Samples were excited at 450 nm (slit width, 5 nm) and fluorescence emission was recorded at 482 nm (slit width, 5 nm). Aggregation was quantified on a percentage basis according to equation: %A = [(Ff – F0) x 100] (Fmax – F0), where Ff is the value of fluorescence after peptide addition, F0 the initial fluorescence in the absence of peptide, and Fmax is the fluorescence maximum obtained after peptide addition.

Measurement of the Membrane Dipole Potential Using Di-8-ANEPPS-labeled Membranes—Aliquots containing the appropriate amount of lipid in chloroform/methanol (2:1, v/v) and di-8-ANEPPS were placed in a test tube to obtain a probe/lipid molar ratio of 1:100 and LUVs, with a mean diameter of 90 nm, were prepared as described previously. Steady-state fluorescence measurements were recorded with a Varian Cary Eclipse spectrofluorimeter. Dual wavelength recordings with the dye di-8-ANEPPS were obtained by exciting the samples at two different wavelengths (450 and 520 nm) and measuring their intensity ratio, R(450:520), at an emission wavelength of 620 nm (39, 40). Changes in the total membrane dipole moment cause a shift in the excitation spectrum maximum of di-8-ANEPPS. By exciting the membrane suspensions at two different wavelengths corresponding to the maximum and the minimum of the difference spectrum, a fluorescence intensity ratio R can be calculated, which can be used as a measure of the relative changes in the magnitude of the dipole potential. The fluorescence ratio R is defined as the ratio of the fluorescence intensity at an excitation wavelength of 450 nm divided by that at 520 nm. The lipid concentration was 200 µM, and all experiments were performed at 25 °C.

Infrared Spectroscopy—For Fourier transfer infrared spectroscopy, the samples were prepared as above but in D2O buffer. Approximately 25 µl of a pelleted sample in D2O were placed between two CaF2 windows separated by 50-µm thick Teflon spacers in a liquid demountable cell (Harrick, Ossining, NY). The spectra were obtained in a Bruker IFS55 spectrometer using a deuterated triglycine sulfate detector. Each spectrum was obtained by collecting 200 interferograms with a nominal resolution of 2 cm–1, transformed using triangular apodization and, to average background spectra between sample spectra over the same time period, a sample shuttle accessory was used to obtain sample and background spectra. The spectrometer was continuously purged with dry air at a dew point of –40 °C to remove atmospheric water vapor from the bands of interest. All samples were equilibrated at the lowest temperature for 20 min before acquisition. An external bath circulator, connected to the infrared spectrometer, controlled the sample temperature. For temperature studies, samples were scanned using 2 °C intervals and a 2-min delay between each consecutive scan. The data were analyzed as previously described (30, 31).

Magic Angle Spinning (MAS) 31P NMR—Samples were prepared as described above and concentrated by centrifugation (14,000 x g for 15 min). MAS 31P NMR spectra were acquired on a Bruker 500 MHz Avance spectrometer (Bruker BioSpin, Rheinstetten, Germany) using a Bruker 4-mm broad band MAS probe under both static and MAS conditions. The samples were packed into 4-mm zirconia rotors and placed in the spinning module of the MAS probe; no cross-polarization was used. The spinning speed was 9 kHz, regulated to ±3 Hz by a Bruker pneumatic unit and the temperature was 25 °C. A single 31P 90° pulse (typically 5 µs) was used for excitation, a gated broadband decoupling of 10 W, 32k data points, 1600 transients, and 5-s delay time between acquisitions. Under static conditions, the samples showed a broad asymmetrical signal with a low-frequency peak and a high-frequency shoulder characteristic of bilayer structures (data not shown).

Small-angle X-ray Scattering Experiments—MLVs at a concentration of 5% (w/w) prepared without or with the peptide at a lipid/peptide molar ratio of 50:1 were prepared as stated above and submitted to 15 temperature cycles (heating at 45 °C and cooling at –20 °C). Small angle x-ray scattering (SAXD) measurements were carried out using a Hecus SWAX-camera (Hecus x-ray Systems, Graz, Austria) as described previously (41) using nickel-filtered Cu-K{alpha} radiation ({lambda} = 1,542 Å) originating from a sealed tube x-ray generator (Seifert, Alvenburg, Germany) operating at a power of 2 kW (50 kV, 4 mA). Sample-to-detector distance was 27.8 cm. A linear position-sensitive detector was used with 1024-channel resolution. SAXD angle calibration was done with silver stearate. The measurements were performed with the sample placed in a thin-walled 1-mm diameter quartz capillary held in a steel cuvette holder at different temperatures with an exposure time of 1 h. The SAXD curves were analyzed after background subtraction and normalization in terms of a full q-range model using the program GAP (42).


Figure 1
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  		FIGURE 1.
A, scheme of the HCV structural and non-structural proteins according to literature consensus. The sequence and relative location of the 11 peptides derived from the HCV p7 protein are shown with respect to the sequence of the protein. Maximum overlap between adjacent peptides is 11 amino acids. B, analysis of the hydrophobicity and interfaciality distribution according to the scales of Wimley and White (43, 44) using a window of 9 amino acids along the HCV p7 sequence without any assumption about secondary structure.

 

   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
The peptide library used in this study and their correlation with the HCV p7 protein sequence is depicted in Fig. 1A. Note that the p7-derived peptides include the whole HCV p7 protein sequence (residues 746 to 809 of the HCV polyprotein precursor). Two and three consecutive peptides in the library have an overlap of 11 and 4 amino acids, respectively. Two peptides were 7 and 8 amino acid residues in length due to synthetic problems (Fig. 1A). The analysis of the hydrophobicity and interfaciality distribution along the p7 sequence of the HCV_1B4J strain, without any assumption on the secondary structure, is shown in Fig. 1B (19, 43, 44). The three-dimensional structure of the p7 protein is not known (13), but this analysis renders to us the potential surface zones potentially implicated in the modulation of membrane binding. As depicted in Fig. 1B, it is evident the existence of different regions with large hydrophobic and interfacial values in the p7 sequence. These regions could mediate the interaction with similar domains of other p7 proteins, other proteins, or with the surface of the membrane.

We have studied the effect of the p7-derived peptide library on membrane rupture by monitoring the CF leakage from six different liposome compositions (Fig. 2). The lipidic composition of the model membranes was EPC/Chol at a phospholipid molar ratio of 5:1, EPC/SM at a phospholipid molar ratio of 5:1, EPC/SM/Chol at a phospholipid molar ratio of 5:1:1, EPC/SM/Chol at a phospholipid molar ratio of 26:9:15, a complex lipid composition resembling the ER membrane (containing 51% EPC, 2.4% BPS, 5.3% bovine brain L-{alpha}-phosphatidylinositol, 7.48% SM, and 33.42% Chol (26)), and a lipid extract of liver membranes (containing 42%, PC, 22% PE, 7% Chol, 8% PI, 1% lyso-{alpha}-phosphatidylcholine, and 21% neutral lipids as stated by the manufacturer). The presence of both SM and Chol has been related to the occurrence of laterally segregated membrane microdomains or "lipid rafts," and, for several viruses, a strong relationship between viral interaction with membranes and their content of Chol and SM was found (45). When the p7 peptides were assayed on liposomes, some exerted an important leakage effect (Fig. 2). The most remarkable effect was observed for peptide 771–788, which produced leakage values between 80 and 98%, depending on the specific lipid composition of the liposomes (Fig. 2). Other peptides that elicited significant leakage were those comprising residues 764–781 and 785–802, which displayed values between 15 and 65% depending on the lipid composition (Fig. 2). It is interesting to note that for liposomes composed of a natural lipid extract of liver membranes, the induced leakage was, in general, higher than for synthetic liposomes. Nevertheless, peptide 771–788 displayed the highest effect, followed by peptides 764–781 and 785–80.


Figure 2
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  		FIGURE 2.
Effect of the peptides derived from the HCV p7 protein of the HCV_1B4J strain on the release (membrane rupture) of LUV contents for different lipid compositions. Leakage data for LUVs composed of EPC/Chol at a phospholipid molar ratio of 5:1 (A), EPC/SM at a phospholipid molar ratio of 5:1 (B), EPC/SM/Chol at a phospholipid molar ratio of 5:1:1 (C), EPC/SM/Chol at a phospholipid molar ratio of 26:9:15 (D), synthetic ER membranes (E), and lipid extract of liver membranes (F). Vertical bars indicate S.D. of the mean of triplicate samples.

 
The global average of membrane leakage obtained for the p7 peptides is illustrated in Fig. 3A. The segment corresponding to peptide 771–788 displays the largest leakage efficacy. Based on previous work (13), a model of p7 in the monomeric form is displayed in Fig. 3B. The model shows the specific residues that display the highest leakage effect. This sequence corresponds to the putative extracellular loop (the p7 loop domain) of the protein and it is the best candidate to interact with the phospholipid head groups. Taking into account these results, we performed an in-depth study of a peptide mimicking this domain, p7L (Fig. 3B, inset). We investigated its binding and interaction with different membrane model systems, as well as characterized the structural changes taking place in both the peptide and phospholipid molecules.

The ability of the p7L peptide to interact with lipid bilayers was determined from the increment in the intensity of the fluorescence emission maximum along with the shift toward shorter wavelengths (46) of the single p7L Trp residue in the presence of phospholipid model membranes at different lipid/peptide ratios (Fig. 4A). The change in the Trp fluorescent spectral properties in the presence of phospholipids indicates that the p7L peptide interacts with these model membranes. This approach has allowed us to obtain the peptide partition coefficient, Kp, which gave values in the 105 range for the different phospholipid compositions studied (Table 1). These Kp values are consistent with the tenet that the peptide was bound to the membrane surface with high affinity. Similar Kp values have been found for other peptides in the presence of model membranes (22, 23, 46, 47). Analysis of these data indicated that the peptide interacted stronger with negatively charged phospholipid-containing bilayers. These results were further corroborated by the larger displacement of the Trp emission frequency maximum of Trp in the presence of LUVs. In solution the peptide had an emission maximum centered at 344 nm, whereas in the presence of increasing concentrations of liposomes the emission maximum of the Trp presented a shift of about 9–11 nm to lower wavelengths depending on liposome composition. This finding implies that Trp sensed a low-polarity environment (entered in a hydrophobic environment) upon interaction with the membrane. We have also used the electrostatic surface potential probe FPE (48) to monitor the binding of the p7L peptide to model membranes composed of different lipid compositions at different lipid/peptide ratios (Fig. 4B). As observed in the figure, p7L had a higher affinity for model membranes containing negatively charged phospholipids than for the other compositions, including the liver lipid extract (Table 1).


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  		TABLE 1
Partition coefficients, Stern-Volmer quenching constants, and maximal leakage, hemifusion, and fusion values at a phospholipid/peptide ratio of 5:1 for the p7L peptide incorporated in LUVs of different compositions

 
Changes in the magnitude of the membrane dipole potential elicited by p7L were monitored by means of the spectral shift of the fluorescence probe di-8-ANEPPS (39, 49, 50). The variation of the fluorescence intensity ratio R450/520 normalized as a function of the peptide concentration for different membrane compositions is shown in Fig. 4C. In the presence of the peptide, the greater decrease in the R450/520 value was measured in bilayers with negatively charged lipids, demonstrating that the peptide was capable of inserting into the lipid bilayer and modifying the dipole potential. Because the p7L peptide has a positive net formal charge of +2, it is reasonable to assume that an electrostatic interaction underlies the high Kp values obtained with negatively charged phospholipids.


Figure 3
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  		FIGURE 3.
A, summary of the normalized global experimental membrane rupture data (leakage) corresponding to the peptide library derived from HCV_1B4J protein p7; and B, model of a monomer of the p7 protein showing the transmembrane {alpha}-helices TM1 and TM2 (13), the relative leakage effect for each p7 amino acid, key residues along the loop domain, as well as the sequence of the peptide p7L studied in this work. See text for details.

 


Figure 4
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  		FIGURE 4.
Determination of the partition constant (Kp) of p7L through the change of the intrinsic tryptophan fluorescence in the presence of increasing lipid concentrations (A), determination of the dissociation constant (Kd) of p7L through the change of the fluorescence signal amplitude of FPE (B), and effect of p7L on the membrane dipole potential monitored through the fluorescence ratio (R) of di-8-ANEPPS in the presence of LUVs containing different lipid compositions at different lipid-to-peptide molar ratios. The lipid compositions were EPC/Chol at a molar ratio of 5:1 (•), BPS/Chol at a molar ratio of 5:1 ({blacksquare}), EPG/Chol at a molar ratio of 5:1 ({square}), EPA/Chol at a molar ratio of 5:1 ({blacktriangleup}), EPC/SM/CHOL at a molar ratio of 5:1:1 ({diamondsuit}), and lipidic liver extract ({blacktriangledown}). In B and C the lipid concentration was 200 µM. Vertical bars indicate S.D. of the mean of triplicate samples.

 
To investigate the accessibility of the Trp residues of the p7L peptide to the aqueous phase in the presence of model membranes, we used acrylamide, a neutral, water-soluble, highly efficient quenching probe. Stern-Volmer plots for the quenching of Trp by acrylamide, recorded in the absence and presence of lipid vesicles, are shown in Fig. 5A. Linear Stern-Volmer plots indicate that the Trp residue is fairly accessible to acrylamide, and in all cases, the quenching of the peptide Trp residue showed an acrylamide-dependent concentration behavior. In aqueous solution the Trp residue was highly exposed to the solvent allowing for a more efficient quenching. However, in the presence of the phospholipid membranes, the extent of quenching was significantly reduced, indicating a poor accessibility of the Trp to the aqueous phase, consistent with its incorporation into the lipid bilayer. This notion is substantiated by the lower Ksv values obtained from the Stern-Volmer plots (Table 1). As expected, Ksv values were lower in the presence of negatively charged phospholipids than in the presence of zwitterionic ones, consistent with a deeper location in negative bilayers.

The transverse location of the p7L peptide into the lipid bilayer was further investigated by monitoring the relative quenching of the Trp fluorescence by the lipophylic spin probes 5-NS and 16-NS when the peptide was incorporated in the fluid phase of the bilayers. These two derivatized fatty acids differ in the position of the quencher moiety along the hydrocarbon chain, thus allowing to determine the relative deepness of the peptide in the membrane. The 5-NS probe is a better quencher for molecules near or at the membrane interface, whereas the 16-NS probe is a better probe for molecules buried deeply in the bilayer. The variation of the fluorescence intensity as a function of the effective concentration of both 5-NS and 16-NS probes is shown in Fig. 5B, whereas the KSV values for both probes are presented in Table 1. In general, 16-NS quenches the p7L peptide fluorescence less efficiently than 5-NS, which is consistent with the location of the Trp residue in a shallow position in the membrane. The quenching depends on phospholipid composition, because the peptide is quenched by 5-NS with higher efficacy in model membranes composed of negatively charged phospholipids.


Figure 5
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  		FIGURE 5.
A, Stern-Volmer plots of the quenching of the Trp fluorescence emission of p7L by acrylamide in aqueous buffer (Figure 5), and in the presence of LUVs composed of EPC/Chol at a molar ratio of 5:1 ({circ}), EPG/Chol at a molar ratio of 5:1 ({square}), EPA/Chol at a molar ratio of 5:1 ({Delta}), BPS/Chol at a molar ratio of 5:1 ({star}), EPC/SM/Chol at a molar ratio of 5:1:1 (224) and lipidic liver extract ({triangledown}). B, depth-dependent quenching of the Trp fluorescence emission of p7L by 5-NS (filled symbols) and 16-NS (empty symbols) in LUVs composed of EPC/Chol at a molar ratio of 5:1 (• and {circ}), EPG/Chol at a molar ratio of 5:1 ({blacksquare} and {square}), EPA/Chol at a molar ratio of 5:1 ({blacktriangleup} and {Delta}), BPS/Chol at a molar ratio of 5:1 (* and {star}), EPC/SM/Chol at a molar ratio of 5:1:1 ({diamondsuit} and 224), and lipidic liver extract ({blacktriangledown} and {triangledown}). The lipid to peptide ratio was 50:1.

 
To explore the effect of the p7L peptide in the destabilization of membrane vesicles, we studied its effect on the release of the encapsulated fluorophores CF (Stokes radius around 6 Å) and FD10 (Stokes radius around 23 Å) in model membranes (51, 52). The extent of CF leakage observed at different peptide to lipid molar ratios and the effect on different phospholipid compositions is shown in Fig. 6A and Table 1. It is interesting to note that the p7L peptide induced a high percentage of leakage (90–100%), even at lipid/peptide ratios as high as 30:1, for liposomes composed of BPS/Chol, EPG/Chol, and EPA/Chol (Fig. 6A). Lower, but significant, leakage values were obtained for liposomes composed of EPC/Chol, EPC/SM/Chol, and the liver extract (at the highest peptide to lipid ratio studied, i.e. 1:5, leakage values were about 80–90%). Next, we carried out a series of experiments using FD10 to characterize the size of the possible pores formed by p7L. However, p7L did not induce any leakage of FD10 entrapped in liposomes having different compositions (data not shown). Because p7L promoted the leak of CF but not FD10, the pore size that would be formed by the peptide should be comprised between 6 and 23 Å.

The induction of outer and inner monolayer lipid mixing (hemifusion and true fusion, respectively) by the p7L peptide was tested with several types of vesicles utilizing a probe dilution assay (32, 33). As shown in Fig. 6, B and C, and Table 1, the higher hemifusion and fusion values were found for liposomes containing negatively charged phospholipids. Liposomes containing BPS/Chol, EPG/Chol, and EPA/Chol showed hemifusion and fusion values of about 30–45 and 12–27%, respectively. In all cases the highest values were found when liposomes containing BPS were used. It is worth noting that ER and the liver extract lipids contain negatively charged phospholipids but at lower relative quantities, consistent with their smaller hemifusion and fusion efficacies. The p7L peptide exhibits a CRAC motif, characterized by the presence of the consensus sequence –L/V-(X)(1–5)-Y-(X)(1–5)-R/K-, where (X)(1–5) represents 1–5 residues of any amino acid (53). The CRAC motif has been suggested to induce formation of cholesterolrich domains. Therefore, we studied the possibility that p7L might interact specifically with cholesterol by a fluorescence resonance energy transfer strategy using DHE as an acceptor (54). We did not find any specificity of the peptide for cholesterol-rich domains (data not shown).

The p7L peptide aggregation state in the presence of membranes was assayed using ThT (38). As observed in Fig. 6D, the peptide remained slightly aggregated in an aqueous medium, as the fluorescence increased after adding the peptide. However, the presence of liposomes composed of EPC/Chol, EPA/Chol, EPG/Chol, BPS/Chol, or a lipidic liver extract provoked a significant and fast peptide aggregation (Fig. 6D). These data suggest that the membrane insertion of the peptide concomitantly induces the peptide oligomerization.

The effect of the p7L peptide on the structural and thermotropic properties of phospholipid membranes was also investigated by measuring the steady-state fluorescence anisotropy of the fluorescent probes DPH, PA-DPH, and TMA-DPH incorporated into DMPC, DMPA, and DMPG membranes as a function of the temperature (Fig. 7). DPH and its derivatives are very useful fluorescent probes for monitoring the organization and dynamics of membranes, because fluorescence polarization correlates with the rotational diffusion of membrane-embedded probes, which are highly sensitive to the packing of the fatty acyl chains (34). DPH is known to partition mainly into the hydrophobic core of the membrane, whereas TMA-DPH is oriented at the membrane bilayer with its charge localized at the lipid-water interface (34, 55, 56). Their different location and orientation in the membrane allows to analyze the effect of the p7L peptide on the structural and thermotropic properties along the full-length of the membrane. For DMPC bilayers, the presence of the p7L peptide decreased the cooperativity of the thermal transition as well as induced a decrease of the anisotropy of all types of probes. These data suggest that the peptide was able to increase the mobility of the phospholipid acyl chains when compared with the pure phospholipid (Fig. 7, A–C). In contrast, for DMPA we did not observe a significant decrease in cooperativity, although a slight shift of Tm to lower temperatures is evident, more apparent for TMA-DPH and PA-DPH than for DPH. Similarly, a small decrease in anisotropy for PA-DPH and DPH was also seen (Fig. 7, D–F). For vesicles composed of DMPG, the p7L peptide decreased the cooperativity of the thermal transition, as well as it decreased the anisotropy below the Tm of the phospholipid (Fig. 7, G–I). In this case, the presence of the peptide decreases the anisotropy values compared with the pure phospholipids, suggesting that the peptide was able to increase the mobility of the phospholipid acyl chains below but not above the Tm. These differences could suggest that the difference in charge between DMPC, DMPA, and DMPG could slightly affect the peptide incorporation into the lipid bilayer. Nevertheless, these data demonstrate that the p7L peptide influences the fluidity of these phospholipids. Taking together all these results, it could be suggested that p7L, although interacting with the membrane, should be primarily located at the lipid-water interface (23). It should be stressed that we did not observe quenching of the probes by the peptide in the concentration range used. Thus, this change of anisotropy cannot be ascribed to a shorter probe lifetime.


Figure 6
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  		FIGURE 6.
Effect of the p7L peptide on (A) membrane rupture, i.e. leakage, (B) membrane phospholipid mixing of the outer monolayer, i.e. hemifusion, and (C) membrane phospholipid mixing of the inner monolayer, i.e. fusion, of fluorescent probes encapsulated in LUVs containing different lipid compositions at different lipid-to-peptide molar ratios. The lipid compositions used were EPC/Chol at a molar ratio of 5:1 ({circ}), EPG/Chol at a molar ratio of 5:1 ({square}), EPA/Chol at a molar ratio of 5:1 ({Delta}), BPS/Chol at a molar ratio of 5:1 ({star}), EPC/SM/Chol at a molar ratio of 5:1:1 (224), and lipidic liver extract ({triangledown}). The lines connecting the experimental data are merely guides to the eye. D, fluorescence variation of ThT after addition of the p7L peptide to an aqueous solution (1) and in the presence of LUVs of EPC/Chol at a molar ratio of 5:1 (2), EPA/Chol at a molar ratio of 5:1 (3), EPG/Chol at a molar ratio of 5:1 (4), BPS/Chol at a molar ratio of 5:1 (5), and lipidic liver extract (6).

 
The infrared spectrum of the Amide I' region of the fully hydrated p7L peptide in D2O buffer at 25 °C and pH 7.4 is shown in Fig. 8A. The spectrum is formed by different underlying components that give place to a broad and asymmetric band with a maximum at about 1642 cm–1. The maximum of the band did not change significantly upon increasing the temperature (data not shown), suggesting a high degree of conformational stability of the peptide in solution. The assignment of the Amide I' component bands to specific structural features has been described previously (46). The intensity maxima at about 1642 cm–1 implies that the most significant structure in aqueous solution corresponds to a mixture of mainly unordered but also helical structures (57). The band envelope of the Amide I' region of the peptide bound to DMPC, DMPG, and DMPA model membranes at a phospholipid/peptide molar ratio of 15:1 was significantly different from that seen for the pure peptide in solution. The frequencies at the maximum of the band appeared at about 1624–1626 cm–1 in all cases (Fig. 8B). As it was found for the peptide in solution, the maximum of the band did not change significantly upon increasing the temperature in the presence of the model membranes (data not shown), indicating also a high degree of conformational stability. The narrow band at about 1624–1626 cm–1 would correspond to either β-sheet structures or self-aggregated peptides forming a intermolecular network of hydrogen-bonded β-structures or both (58). In addition, we tested an increased phospholipid/peptide molar ratio of 200:1 for checking the influence of the lipid to peptide ratio on its secondary structure (Fig. 8C), being very similar to that found at higher molar ratios (Fig. 8B).


Figure 7
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  		FIGURE 7.
Steady-state anisotropy, <r>, of TMA-DPH, PA-DPH, and DPH (left, middle, and right columns, respectively) incorporated into MLVs composed of DMPC, DMPA, and DMPA model membranes (from upper to low rows, respectively) as a function of temperature. Data correspond to vesicles in the absence (•) and presence of the p7L peptide ({circ}). The peptide to phospholipid molar ratio was 1:15.

 
We have used MAS 31P NMR to observe the POPC/SM/Chol mixture at a molar ratio of 5:1:1 because the 31P NMR isotropic chemical shifts of both SM and POPC head groups are resolvable under MAS conditions. Furthermore, their spectral intensities reflect the molar ratio of each lipid in the mixture. This approach allows the observation of the line widths of each phospholipid component in the mixture. As observed in Fig. 9A, the chemical shift for the POPC and SM resonances were not different neither in the absence nor in the presence of the p7L peptide, but the line widths of the 31P resonances of POPC and SM were dissimilar. In the absence of p7L, the 31P line width at half-height of POPC was 51 Hz and that of SM was 55 Hz. These values shifted to 118 and 101 Hz, respectively, when the peptide was present. These results show that both phospholipids, POPC and SM, exhibit a lower degree of mobility and/or an increased heterogeneity of head group environments in the presence of p7L (59). We have also used SAXD to get information on the structural organization of the mixture containing POPC/SM/CHOL at a molar ratio of 5:1:1 in the absence and presence of the peptide (Fig. 9B). In both cases, the diffraction pattern corresponds to the liquid-crystalline L{alpha} phase, showing an interlamellar repeat distance of 67.9 Å for the pure lipid mixture and 68.3 Å in the presence of peptide. The structural results from the global data analysis, shown in the inset of Fig. 9B, showed that the membrane thickness decreased from 55 Å in the absence of the peptide to 53.8 Å in its presence. However, the thickness of the water layer increased from 12.9 to 14.5 Å under the same conditions. The most significant effect was, however, the increase of the diffuse scattering in the presence of the peptide indicating that several bilayers have become positionally uncorrelated due to the influence of the peptide.


Figure 8
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  		FIGURE 8.
Amide I' band of the p7L peptide in buffer (A) and in the presence of DMPC (solid line), DMPG (dashed line), and DMPA (dotted line) at a lipid/peptide molar ratio of 15:1 (B) and 200:1 (C). The spectra were taken at Tm + 10 °C.

 


Figure 9
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  		FIGURE 9.
A, MAS 31P NMR at 25 °C and 9 kHz spinning speed (500 MHz proton frequency) spectra for POPC/SM/Chol (5:1:1 molar ratio) MLV suspension in the absence (—-) and presence of p7L at a lipid/molar ratio of 50:1 (solid line). B, small angle x-ray scattering of POPC/SM/Chol (5:1:1 molar ratio) MLV suspension in the absence ({circ}) and presence of p7L (•) at 25 °C in the L{alpha} phase. Solid lines represent the best fit to the SAXD data applying a global analysis technique. The inset displays the one-dimensional electron density profiles along the bilayer normal calculated from the SAXD diffraction patterns in the absence (dotted line) and presence of p7L (solid line).

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Viral morphogenesis, although one of the most important steps in the viral cycle involving lipid membranes, is not as well characterized as viral-mediated membrane fusion. The assembly of enveloped viruses takes place in the host cell membrane and in the case of HCV it has been suggested that the virus particles assembly occurs in the ER membranes (60) where different proteins, including p7, play a central role in viral particle formation and budding. HCV protein p7 is a small transmembrane protein with two TM helical domains connected by a loop, which is essential for the efficient assembly and release of infectious virions but not critical for RNA replication (10). p7 is capable of forming ion channels and mutations in the loop region abolish the channel activity of the protein, which has been described to be a viroporin-like protein (12, 15, 16). Because the biological roles of p7 can be modulated by membranes, using an approach similar to that published recently (19–21), we have carried the analysis of the membrane-active regions of p7 by observing the effect of a p7-derived peptide library from HCV (strain HCV_1B4J) on the integrity of different membrane model systems. We have identified a membranotropic region in p7 coincidental with the loop domain of the protein. Consequently, we have made a comprehensive study of a peptide patterned after the p7 loop domain, p7L, characterizing its binding and interaction with model membrane systems through a series of complementary experiments. Our findings identify a key region in the viral protein that might be implicated in the HCV life cycle, which could be used as a new target for searching inhibitors of viral assembly, thus leading to new vaccine strategies.

We have been able to discern different regions along the p7 sequence that display distinct membranotropic properties using a peptide library derived from the p7 protein. Although the use of peptide fragments might not fully mimic the properties of the intact protein, our results give an indication of the relative propensity of the different domains to bind, interact, and affect different model membranes. When all the leakage values were taken into account for all lipid compositions assayed, one peptide displayed significant membrane rupture activity, namely the region encompassing amino acids 771–788 (Fig. 3A). Region 771–788 coincides with the p7 loop linking the two TM helices of the protein. Notably, this region displayed a high interfacial value along its sequence. This highly conserved loop seems to be necessary for homodimerization and channel forming (61). Indeed, our findings are consistent with the tenet that the interaction with lipid bilayers induces its oligomerization, as evidenced by aggregation of the p7L peptide in the presence of model membranes. One model of p7 predicted that the side chains of residues Lys779 and Arg781 would project into the channel lumen, perhaps forming a gate controlling the flow of ions (16).

Peptide p7L binds with high affinity to phospholipid model membranes containing negatively charged phospholipids. We and others have previously found similar binding affinities for other peptides pertaining to the loop and NHR regions of the gp41 protein (22, 23, 46, 62, 63). The p7L peptide has a positive net formal charge of +2, implying that an electrostatic force may be responsible for the high Kp values observed for compositions containing negatively charged phospholipids. However, the peptide was also capable to bind significantly to other liposome systems composed of zwitterionic phospholipids. The peptide decreased the dipole potential of the membrane as well. Binding of p7L to liposomes was further demonstrated by hydrophilic and lipophilic quenching probes. p7L was less accessible for quenching by acrylamide in the presence of negatively charged phospholipids implying a buried location. A higher quenching efficiency was observed for model membranes containing negatively charged phospholipids in the presence of NS probes, suggesting that the peptide was more buried in the membrane when negatively charged phospholipids were present, but near the membrane lipid/water interface in a shallow position.

The p7L peptide was also capable of altering the membrane stability causing the release of fluorescent probes of small size, being dependent on lipid composition and on the lipid/peptide molar ratio. The highest CF release was observed for liposomes containing negatively charged phospholipids, although significant leakage values were also observed for liposomes composed of zwitterionic phospholipids. The effect on zwitterionic vesicles should be due primarily to hydrophobic interactions within the bilayer but not to the specific charge of the phospholipid head groups. Notably, p7L released CF but not FD10, implying that the peptide formed pores of diameter between 6 and 23 Å. The induction of hemifusion and fusion by the p7L peptide were also studied and similar results were obtained, because specific and large membrane hemifusion and fusion values were characteristic of liposomes composed of negatively charged phospholipids. It is interesting to note that p7L presents a CRAC motif, although we have not seen specificity of the peptide for cholesterol-rich domains.

We have also shown that the p7L peptide is capable of affecting the steady-state fluorescence anisotropy of DPH fluorescent probes located into the palisade structure of the membrane, because the peptide increased the mobility of the phospholipid acyl chains below but not above the Tm when compared with the pure phospholipids. Additionally, by using MAS NMR, we demonstrated that the phosphate groups of the phospholipid molecules display a lower degree of mobility and/or an increased heterogeneity of head group environments in the presence of p7L. This result strengthens that the location of the peptide is at or near the membrane interface. Moreover, the presence of p7L slightly decreased the membrane thickness and increased its hydration layer. At the same time we found an increase of positionally uncorrelated bilayers. These data reveal that p7L affects the elastic behavior of the membrane and further substantiates that p7L should be located at the lipid-water interface (23). All this information suggests that negatively charged phospholipids could play an important role in the biological function of p7. As observed by infrared, the Amide I' region of the fully hydrated peptide did not change with temperature, indicating a high stability of its conformation. In buffer, p7L presents a high content of random structure. In marked contrast, the overall structure of the peptide in the presence of model membranes was significantly different, because it displayed a high percentage of β-sheet structures and/or self-aggregated peptides. These components were independent of the lipid-to-peptide ratio. The ThT aggregation data supports that p7L has a tendency to oligomerize at the membrane surface.

It is interesting to note that replacement of the two conserved basic amino acids, Lys779 and Arg781, pertaining to the p7 loop suppress dramatically the production of infectious viruses (12), highlighting the importance of this conserved motif as well as the relevance of the loop. Significantly, two other amino acids pertaining to the p7L peptide, Trp776 and Tyr788, are also essential for the p7 function (12). It seems reasonable to hypothesize that the basic residues interact specifically with the phospholipid head groups of the ER. Although it could not be discarded the possibility that p7 may also interact with other proteins from HCV as it has been suggested (9). It is already known that p7 forms ionic channels in the membrane, probably forming an hexameric bundle (16). The folding of p7 could begin by forming two TM helices across the bilayer, followed by stabilization of the monomeric protein and finally the oligomerization of the monomer to form the hexameric assembly through interaction of the helices (13). The conserved positively charged loop, its location on the surface of the membrane, its tendency to oligomerize in the presence of phospholipids, as well as its specific interaction with phospholipid head groups, could be the driving force of the protein oligomerization. Therefore this protein domain may be essential for the formation of the active ion channel. Accordingly, the p7 loop appears as an attractive candidate for antiviral drug development leading to new vaccine strategies.


   FOOTNOTES
 
* This work was supported in part by Grant BFU2005-00186-BMC from the Ministerio de Ciencia y Tecnología, Spain (to J. V.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

1 Recipients of pre-doctoral fellowships from the Autonomous Government of the Comunidad Valenciana, Spain. Back

2 To whom correspondence should be addressed: IBMC, Universidad "Miguel Hernández," E-03202 Alicante, Spain. Tel.: 34-966-658-762; Fax: 34-966-658-758; E-mail: jvillalain{at}umh.es.

3 The abbreviations used are: HCV, hepatitis C virus; BPS, bovine brain L-{alpha}-phosphatidylserine; CF, 5-carboxyfluorescein; Chol, cholesterol; DHE, ergosta-5,7,9(11),22-tetraen-3β-ol; di-8-ANEPPS, 4-(2-(6-(dioctylamino)-2-naphthalenyl)-(ethenyl)-1-(3-sulfopropyl)-pyridinium inner salt; DMPA, 1,2-dimyristoyl-sn-glycero-3-phosphate; DMPC, 1,2-dimyristoyl-sn-glycero-3-phosphocholine; DMPG, 1,2-dimyristoyl-sn-glycero-3-[phospho-rac-(1-glycerol)];DPH,1,6-diphenyl-1,3,5-hexatriene;EPA,eggL-{alpha}-phosphatidicacid; EPC, egg L-{alpha}-phosphatidylcholine; ER, endoplasmic reticulum; FD10, fluorescein isothiocyanate dextran with an average molecular weight of 10,000; LUV, largeunilamellarvesicles;MLV,multilamellarvesicles;NBD-PE,N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)-1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine; N-RhB-PE, Lissamine rhodamine B 1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine; NS, doxylstearic acid; PA-DPH, 3-(4-(6-phenyl)-1,3, 5-hexatrienyl)-phenylpropionic acid; SAXD, small-angle X-ray diffraction; SM, sphingomyelin; Tm, temperature of the gel to liquid crystalline phase transition; TM, transmembrane domain; TMA-DPH, 1-(4-trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene; FPE, fluorescein phosphatidylethanolamine; ThT, thioflavin T; MAS, magic angle spinning; POPC, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine. Back


   ACKNOWLEDGMENTS
 
We are especially grateful to the National Institutes of Health AIDS Research and Reference Reagent Program, Division of AIDS, NIAID, National Institutes of Health, for the peptides used in this work; Ana I. Gómez-Sanchez for outstanding technical assistance; as well as Prof. Antonio Ferrer, IBMC-UMH, for the critical reading of the manuscript and excellent aid in correcting the English language.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
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