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1 The Scripps Research Institute – Florida, Department of Infectology, 130 Scripps Way, #3C1 Jupiter, FL 33458, USA
2 Institut de Biologie et Chimie des Protéines, UMR5086, CNRS-Université Lyon I, IFR128, 7 Passage du Vercors, 69367 Lyon Cedex 07, France
Correspondence
A. D. Strosberg
strosber{at}scripps.edu
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). Recent efforts have concentrated on designing specific inhibitors that target several of the 10 individual HCV proteins that comprise the virus (Choo et al., 1989; Lindenbach & Rice, 2005), including the non-structural proteins NS3A/NS4A protease (Thibeault et al., 2004) and the NS5B polymerase (De Francesco & Carfí, 2007). One HCV structural protein that has not yet been targeted is core, the 191-residue protein that constitutes the N terminus of the HCV polyprotein and is responsible for assembly and packaging of the HCV plus-strand RNA genome (Lindenbach & Rice, 2005). Core dimers and higher-order complexes associate with the virus RNA to form the nucleocapsid and associate with envelope proteins E1 and E2 to form the infectious particle (Boulant et al., 2005; Klein et al., 2004; Kunkel et al., 2001; McLauchlan, 2000; McLauchlan et al., 2002). Core is cleaved from the polyprotein precursor by a host signal peptidase located in the endoplasmic reticulum (ER) (Boulant et al., 2005; Klein et al., 2005; Kunkel et al., 2001), but remains anchored to the ER through a 22-residue C-terminal hydrophobic region that is removed by a signal peptide peptidase to yield a mature core comprising residues 169–179 (Grakoui et al., 1993; Hijikata et al., 1991; Liu et al., 1997; McLauchlan et al., 2002; Okamoto et al., 2008). Most interactions with intracellular proteins are mediated via the N-terminal two-thirds of core, whereas binding to glycoprotein E1 has been mapped to the C-terminal region (Lo et al., 1996; Ma et al., 2002). Core plays an essential role in the HCV cycle during assembly and release of the infectious virus (Penin et al., 2004) and also during disassembly of virus particles upon entering host cells and binding to lipid droplets (Miyanari et al., 2007; Penin et al., 2004; Shavinskaya et al., 2007). Self-association of HCV core protein may well lead to dimerization of RNA and formation of virus nucleocapsid. Blocking such self-assembly by specific inhibitors would provide useful molecular tools to investigate the sequence of events leading to virus particle assembly.
To improve our understanding of the role of core in HCV assembly, an amplified luminescent proximity homogeneous assay (ALPHA) screen was developed to identify inhibitors of dimerization of a fragment of core comprising residues 1–106 (core106). Peptides containing an 11-residue sequence inhibited core dimerization by up to 68 %. A fluorescence polarization (FP) assay was used to measure direct binding of an inhibitor peptide to core106 (Dandliker et al., 1973) and a surface plasmon resonance (SPR) assay was used to measure binding to core169. Two inhibitor peptides blocked production of infectious HCV in a hepatoma cell-culture system, and one of these was recently confirmed independently to strongly reduce HCV production (Cheng et al., 2008).
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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Cloning, expression and purification of proteins.
The sequence of core covering the first 106 residues was amplified by PCR from HCV H77 genotype 1a plasmid pCV-H77C. The amplified sequence was then digested with restriction enzymes and ligated to expression vector pET42b (Novagen) or pT7Flag (Sigma-Aldrich) to generate constructs that were verified by DNA sequencing (Davis Sequencing).
For expression, the pET42b and pT7Flag plasmids encoding recombinant core106, FLAG–core106 and glutathione S-transferase (GST)–core106 proteins were transformed into Escherichia coli BL21DE3 cells by using standard expression procedures (Boulant et al., 2005). Core106- and FLAG–core106-containing pellets were solubilized by using lysis buffer containing 2 M guanidine. The soluble fraction containing GST–core106 was purified by affinity chromatography on an Ni-NTA column. Recombinant core106 and FLAG–core106 proteins in the clarified lysates were applied to affinity columns (HiTrap Ni-NTA; Amersham Biosciences) pre-equilibrated with 20 mM Tris/HCl (pH 8.0), 0.5 M NaCl. Recombinant proteins were eluted with a linear 0–1 M imidazole gradient. Eluted fractions containing recombinant protein were dialysed overnight at 4 °C against 20 mM Tris (pH 8.0), 0.5 M NaCl. The homogeneity of the purified proteins was determined by SDS-PAGE and confirmed by Western immunoblot analysis using rabbit polyclonal antibodies generated against core106. Core169 was prepared and purified as described by Boulant et al. (2005).
Cross-linking of core106 with dimethyl suberimidate (DMS).
Purified core106 was concentrated by Centricon (Millipore), dialysed against 50 mM phosphate buffer (pH 7.2), 150 mM NaCl (PBS) and stored at –80 °C. Core106 dimerization was confirmed by cross-linking with DMS (Thermo Scientific). A 100 µM core106 solution was freshly made by diluting the protein in buffer A [200 mM phosphate buffer (pH 8.0)]. DMS was added to the protein at 0.2, 0.5, 1.0, 2.5 or 5.0 mM final concentration in a final volume of 100 µl, incubated for 30 min at room temperature, stopped by addition of 20 mM Tris (pH 8.0) (final concentration) and by addition of SDS-PAGE loading buffer (1 : 1) and boiled for 10 min at 95 °C. The cross-linked proteins were analysed by SDS-PAGE (Tris–glycine 4–20 %; Invitrogen) and confirmed by Western blot analysis.
Sandwich ELISA for monitoring core106 dimerization.
Glutathione-coated 96-well plates (Sigma-Aldrich) were blocked with 5 % milk in PBS, then coated with 27 nM GST–core106 and incubated overnight. The uncoated proteins at each step were washed off with PBS. FLAG–core106 was dosed from 0.068 to 68 nM and the plate was incubated at 37 °C for 1 h and then washed with PBS. Mouse anti-FLAG antibody (Sigma-Aldrich) was added at a dilution of 1 : 1000 and incubated for 1 h at room temperature. The plate was washed twice with PBS and twice with 0.01 % Tween–PBS. Anti-mouse IgG conjugated to horseradish peroxidase (HRP) (Jackson ImmunoResearch) was added at a dilution of 1 : 10 000 and the plate was incubated for 1 h at room temperature. The plate was washed twice with PBS and twice with 0.01 % Tween–PBS. Ultra TMB (Pierce), an HRP substrate, was added to the plate. The colour reaction was stopped by adding 0.5 M H2SO4. A450 was measured on a Biotek plate reader.
ALPHA screen for monitoring core106 dimerization and screen for peptide inhibitors.
The ALPHA screen is based on the use of photoactive donor and acceptor beads that recognize specific tags on interacting proteins (Peppard et al., 2003). Core106 dimerization was confirmed by using ALPHA screen technology in which a core106 protein domain was tagged with either a GST or a FLAG peptide tag. The untagged core106 protein domain was used as a model competitor in the assay. The proteins were diluted to working concentrations in protein buffer [100 mM HEPES (pH 7.5), 1 mM EDTA, 5 mM dithiothreitol (DTT), 0.1 % CHAPS, 10 % glycerol]. The donor and acceptor beads were diluted to working concentrations in bead buffer [20 mM HEPES (pH 7.5), 125 mM NaCl, 0.1 % bovine serum albumin, 0.1 % CHAPS]. GST-tagged core106 (150 nM) was incubated with FLAG-tagged core106 (150 nM) for 1 h at room temperature. Anti-FLAG acceptor beads (PerkinElmer Life Sciences) were added to the proteins at a final concentration of 20 µg ml–1 and incubated for 1 h at room temperature. Then, glutathione donor beads (PerkinElmer Life Sciences) were added to the proteins at a final concentration of 20 µg ml–1 and incubated for 1 h. The assays were executed in a white Packard OptiPlate-384 (PerkinElmer Life Sciences) and were read on a PerkinElmer EnVision multilabel plate reader. The core106 dimerization ALPHA screen assay was used to screen a set of 14 core-derived peptides spanning residues 1–109 of the core protein at a final concentration of 20 µM. The free core106 domain was used as a control inhibitor for screening inhibitors of the core–core interaction.
FP.
Alexa Fluor 488 carboxylic acid TFP ester (A488; Invitrogen) was coupled to the primary amine of peptide SL175 in solution, followed by HPLC purification and electrospray ionization liquid chromatography mass spectrometry analysis (as recommended by Invitrogen). Peptide SL175 (50 µg) in 0.1 M sodium bicarbonate buffer (pH 8.3) was added to Alexa Fluor 488 Protein Labelling kit reagents (Invitrogen) for 2 h at room temperature with continuous stirring in the dark. For FP experiments, A488–SL175 and core106 were incubated in a black 384-well Greiner polystyrene plate for 30 min at room temperature. FP measurements (integration time of 100 000 µs) were taken on an Analyst GT Multimode reader using the FP program of the AnalystHost 3.01 software (Molecular Devices). The fluorophore–peptides were studied by using filters (excitation, 485 nm; emission, 530 nm) and a beam splitter (505 nm). The Z height was set at 0 mm and the G factor was determined experimentally to be 1.0. A dose–response analysis was done with a concentration of coupled peptide at 309 nM with various concentrations of core106 (between 0 and 800 nM) in 20 mM Tris buffer (pH 7.7). As a control, the same experiment was done with the fluorophore (309 nM) in the presence of the protein. For specificity controls, unlabelled peptide SL175 (60 µM) was added to the complex. A titration with the free peptide SL175 (0–100 µM) was done to displace the complex A488–SL175–core106 (309 and 600 nM, respectively). Kd was calculated (six points for each concentration) with GraphPad Prism 5 software, using the non-linear regression ‘Binding saturation: total and non-specific binding’ and data from the inhibition by free SL175 (60 µM) as the non-specific binding. IC50 was calculated by using a non-linear regression ‘log(inhibitor) vs response – variable slope’ with nine points for each concentration.
SPR.
Binding of peptides to core169 was measured in a BIACore 3000 Instrument using a Sensor Chip NTA as described previously (Maillard et al., 2004). The running buffer was 20 mM HEPES (pH 7.4), 150 mM NaCl, 50 mM EDTA, 0.1 % n-dodecyl β-D-maltoside and 1 mM DTT. At the end of each cycle, proteins were removed by injecting 10 ml regeneration solution consisting of 0.35 M EDTA, 0.05 % SDS. Sensorgrams were analysed by using BIAevaluation 4.1 software and kinetic constants were obtained by non-linear fitting of the sensorgrams to a 1 : 1 binding model and taking into account the drift of the baseline. The apparent Kd value was determined from the ratio of the kinetic constants (koff/kon). The quality of the fit was assessed by verifying the 
2 values and the random distribution of the residuals. For control experiments, peptides were injected over the surface of the sensor chip previously coated with NiCl2 and the signal was subtracted from the assays.
Cell-culture protocols for first and second transfers of infectious HCV and addition of peptides.
Infectious HCV 2a strain J6/JFH-1 was produced by using a previously described protocol (Lindenbach et al., 2005; Wakita et al., 2005; Zhong et al., 2005). Huh-7.5 cells (100 µl at 8000 cells ml–1 per well) were plated in 96-well plates for 24 h before infection with infectious HCV incubated for 1 min with or without core peptides before addition to naïve Huh-7.5 cells. After 24 h, the supernatant was removed. Fresh medium with the same core peptides as incubated previously was added for 72 h; the supernatant of these cells was harvested as the first transfer of infectious J6/JFH-1 and the cells were fixed by 100 % methanol at –20 °C for 15 min. The cells were then stored in PBS at 4 °C. The first transfer of infectious supernatant was added to naïve Huh-7.5 cells grown for 24 h in 96-well plates (100 µl at 8000 cells ml–1 per well); this constituted the second transfer. After 24 h, the infectious supernatant was removed and fresh medium without peptide was added for 72 h until the cells were fixed with 100 % methanol at –20 °C for 15 min. The cells were then stored in PBS at 4 °C.
Measurement of effects of peptides.
Staining of HCV-infected cells by immunohistochemistry was performed by using an NS5A antibody (Tellinghuisen et al., 2008). Tissue culture infectious doses 50 % (TCID50) were calculated as described by Lindenbach et al. (2005). Real-time RT-PCR protocol, primers and probes were used as described previously (Jones et al., 2007; Tellinghuisen et al., 2008).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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; Kunkel et al., 2001; McLauchlan, 2000). Fromentin et al. (2007) studied a residue 1–82 core construct to follow nucleocapsid formation (Majeau et al., 2004; Fromentin et al., 2007). Based on the discovery of a residue 1–84 self-associating fragment by the yeast two-hybrid method applied to a library of fragments of the HCV polyprotein (Flajolet et al., 2000) and the decision to include the residue 82–106 ‘homotypic’ domain that had been reported to be essential for core dimerization (Murray et al., 2007; Nolandt et al., 1997), we prepared the longer, residue 1–106 N-terminal fragment. To confirm core106 dimerization, we used a sandwich ELISA, and to measure dimerization and its inhibition accurately, we developed a quantitative ALPHA screen. Three core fragments were used in these assays: core106, GST–core106 and FLAG–core106, which collectively could form at least two types of homodimer and one type of heterodimer. Whilst homodimers were seen by electrophoresis, only heterodimers were detectable in our quantitative assays.
Purification of core106 fragments and verification of dimerization
The different HCV core106 fragments tagged with an N-terminal GST or FLAG tag and a C-terminal sequence of six or eight His residues were expressed in E. coli and purified to homogeneity by affinity chromatography on an Ni-NTA agarose column. The composition and mass of core106 were confirmed by mass spectrometry. The size of the tagged core proteins was verified by SDS-PAGE (Fig. 1a). Coomassie blue staining revealed major bands for core106 at 14.5 kDa, for FLAG–core106 at 15 kDa and for GST–core106 at 34 kDa, and weak bands for their corresponding dimers. The identity of the proteins was confirmed by immunoblot analysis (Fig. 1b). The rabbit anti-core106 antibody recognized the bands for core106 at 14.5 kDa, for FLAG–core106 at 15 kDa and for GST–core106 at 34 kDa. Except for the weak bands corresponding to the dimers, no larger-molecular-mass bands were observed, probably because the level of self-association of the core106 fragments is affected by SDS.
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) with only two molar equivalents of DMS over core106. Trimeric and higher polymeric forms become more distinct with higher concentrations of added reagent (Fig. 1c). Surprisingly, only the monomer and the dimer were identified by Western blotting using the anti-core106 antibody, suggesting that the cross-linking that leads to higher oligomerization forms results in loss of the specific epitopes (Fig. 1d).
Heterodimerization of core106 as shown by ELISA and quantified by ALPHA screen
A sandwich ELISA was used for initial confirmation of the GST–core106/FLAG–core106 heterodimerization. GST–core106 was adsorbed onto a microtitre plate coated with glutathione. FLAG–core106 was added and mouse anti-FLAG antibody, anti-mouse IgG–HRP and an HRP substrate were used to visualize core106 heterodimerization. As shown in Fig. 2, heterodimerization of core106 was quantified over a range of concentrations (0.068–68 nM). The background controls in the assay were buffer only, GST–core106 alone and FLAG–core106 alone. An unrelated GST-tagged protein (1 µg) was adsorbed onto a glutathione-coated plate and, when mixed with FLAG–core106, gave a signal similar to the background in the assay, confirming that the GST–core106/FLAG–core106 interaction was specific. The signal for the interaction weakened with a decrease in the dose of FLAG–core106 used in the assay. Free GST and free FLAG peptide added to the assay were able to displace the binding of GST–core106 to FLAG–core106 to background levels (data not shown). The data shown in Fig. 2 were representative of five independent experiments with triplicate data points in each experiment.
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), which thus allowed quantification of core dimerization over a range of concentrations up to 10 µM. The IC50 of core106 inhibition of GST–core106/FLAG–core106 dimerization was calculated to be 89 nM. Core106 was able to reduce the specific signal completely to background levels. The data of the uninhibited control compared with inhibition by the peptides were analysed by using an unpaired Student's t-test (Fig. 3b).
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). To explore this possibility, a library was obtained from NIH (http://www.aidsreagent.org), consisting of 441 overlapping 18-mer peptides spanning the HCV genotype 1a polyprotein sequence. Fourteen 18-residue peptides derived from core (spanning residues 1–109 and overlapping with each other by 11 C-terminal residues) were tested for their inhibitory capacity at a final concentration of 20 µM by ALPHA screen (Fig. 3a). Two successive peptides, SL173 and SL174, showed 68 and 63 % inhibition of core dimerization, respectively (Tables 1 and 2). Peptide SL175, which corresponds to peptide SL174 truncated by three residues at its C terminus, inhibited by 50 %. This shorter peptide, which is much easier to produce than its longer counterparts, was evaluated in a dose–response analysis (0.5–100 µM); the IC50 was calculated to be 21.9 µM (Fig. 4a). Peptide SL571, which contains the reversed sequence of SL175, displayed no effect on core dimerization. Student's t-tests were performed on the uninhibited control compared individually with peptides SL173, SL174, SL175 and SL571 (reverse control) to confirm that the inhibition values were statistically significant compared with the control.
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). As a control, the same experiment was done with the fluorophore alone (309 nM) in the presence and absence of the protein (data not shown). When a titration with the free peptide SL175 (0–100 µM) was done to displace the A488–SL175–core106 complex (at respectively 309 and 600 nM), the IC50 was 18.7 µM, in good agreement with the value obtained by ALPHA screen analysis (21.9 µM) (Fig. 4a, c).
Binding of peptide SL175 to core169, previously characterized by Boulant et al. (2005), was also evaluated by SPR. For this purpose, core169 was immobilized on the surface of a Sensor Chip NTA previously coated with NiCl2, and different concentrations of peptide SL175 were injected (Fig. 4b). The kinetics of the interaction could be fitted to a single-site binding model with a kon value of 1.07x103 M–1 s–1 and a koff value of 7.68x10–3 s–1, giving an apparent Kd value of 7.2 µM with good confidence (2 value of 4.21). This result indicated that peptide SL175 could interact with core169, which encompasses the domain involved in lipid-droplet targeting (Boulant et al., 2005). In contrast, peptide SL571, as expected, did not bind to core169.
Effect of peptides on HCV production
We evaluated the effects of the inhibitor and reverse peptides on production of infectious virus by individual addition to cells infected with HCV J6/JFH-1. Peptides were added to cells before or during the first 24 h of contact with HCV and after replacing medium containing virus with fresh medium. Cell supernatants containing virus released from infected cells were harvested 72 h after addition of fresh medium and tested for infectivity on naïve Huh-7.5 cells.
Virus replication efficiency was quantified (TCID50) by counting infected cells after treatment with peptide, by using immunohistochemical staining with a mouse anti-NS5A antibody. Virus production was analysed during two transfers: transfer 1 (T1) corresponded to naïve cells infected with virus stock, whereas T2 corresponded to naïve cells infected with supernatant of cells from T1. When any of peptides SL173, SL174, SL175 and SL571 were added before the addition of the virus (i.e. before T1), no inhibition of virus infection was observed in either T1 or T2. When peptides were added at the same time as the virus (during T1), no significant effect was observed (shown for SL175 and SL571; Fig. 5a, b). SL175 at 10 µM added during T1 had a strong inhibitory effect in T2 (50 % reduction of NS5A expression); SL571 showed no effect (Fig. 5c, d). SL173 at 20 µM showed also a strong inhibitory effect: 80 % reduction of NS5A expression, compared with 15 % reduction caused reproducibly by SL174 (Table 2).
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) confirmed that SL175 activity was based specifically on its sequence and not on its composition, in contrast to what was observed for another HCV-derived peptide, where a scrambled peptide displayed the same activity as the original NS5A peptide (Cheng et al., 2008). TCID50 calculations (Fig. 5d) confirmed that the activity of SL175 at T2 could be measured by using as little as 0.1 µM, and was easily observed at 1 µM. Quantitative real-time RT-PCR measurements confirmed the effects of the peptides on HCV production in T2, showing a decrease of nearly 7-fold in RNA levels (Fig. 5f). Absence of non-specific toxicity was inferred from the fact that peptides were added on two alternative occasions, i.e. before or during virus infection, but the effect on virus production was seen only during infection. The peptides were stored in 10 % DMSO, but the working stock of the peptides was at a maximum of 0.1 % DMSO, which was not toxic for the cells. To investigate the specificity of the effect of SL175 on HCV, we evaluated its activity on the propagation of human immunodeficiency virus (HIV), another RNA-encoded virus, and found it to have no effect (M. Caputi, personal communication).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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; Roingeard & Hourioux, 2008; Suzuki et al., 2005). Dimerization and interaction with other HCV proteins and with cellular proteins are the basis for these functions (McLauchlan, 2000). Thus, developing inhibitors that act specifically on these protein–protein interactions may help to elucidate the pathways by which core exerts its effects and aid in developing novel anti-HCV drugs. For this purpose, we developed novel protein-interaction assays using the N-terminal half of core, the region essential for self-association, in order to identify key residues involved in homologous core interaction and possibly identify specific dimerization inhibitors. Core-derived peptides SL173–SL175 indeed inhibited core106 dimerization, whereas the reverse-sequence control peptide SL571 had no effect. Furthermore, SL175 binds directly to core106 and to core169, a nearly complete form of core, with comparable affinities. A peptide identical to SL173 was recently (Cheng et al., 2008) reported to inhibit HCV focus formation by >90 % and RNA levels were reduced by 11-fold 72 h after infection.
The three inhibitory peptides all contain the hydrophobic 11-residue sequence located between positions 82 and 102, well within the previously reported homotypic domain (Nolandt et al., 1997), including serine 99, described as essential for core function, as seen by the decrease in HCV viability when this residue was substituted by alanine (Murray et al., 2007). This 11-residue sequence is identical for genotypes 1a, 1b, 2a, 3 and 4, suggesting further that this region is important for the virus cycle. Attempts to block dimerization of core106 with peptide SL200, consisting solely of these 11 residues, gave maximal inhibition of 30–40 %. Data were, however, quite variable from one experiment to another, possibly because of the peptide's high hydrophobic composition and low solubility in our assay format (unpublished results). The 15 residues corresponding to peptide SL175 were identical across genotypes 1a, 1b, 2a, 3 and 4. Genotype 5 differs at position 102 by glycine to serine and at position 106 from serine to asparagine, and genotype 6 differs at position 98 by a leucine to methionine substitution, confirming that core is extremely well conserved across the six major genotypes.
Fromentin et al. (2007) also used core-derived peptides that inhibited complex formation with RNAs of various origins. Surprisingly, these peptides did not originate from the residue 82–106 homotypic region. Unfortunately, the peptides' specificities were not verified with any control, nor was their effect on HCV production studied (Fromentin et al., 2007).
For this report, we chose not to work on nucleocapsid assembly, given the considerable difficulty in selecting the right RNA size and composition, the right core sequence, the reaction conditions and the best method of characterization of the resulting HCV nucleocapsid (Boulant et al., 2005; Fromentin et al., 2007). Instead, we evaluated the influence of the inhibitor peptides on infectious virus production by direct individual addition to cells infected with HCV. No effect was observed at either stage (T1 or T2) when peptides were added before infection. When any of the peptides were added during T1 transfer, no specific inhibition was observed during T1. In contrast, during the T2 transfer, the peptides caused up to 80 % reduction of the number of infected cells, as measured by immunofluorescence, immunohistochemistry (TCID50) or measurement of RNA levels by real-time RT-PCR. Control peptide SL571 had no effect. These data confirm that peptides selected for inhibiting core dimerization indeed affected virus release specifically. SL175 had no effect on the propagation of HIV (M. Caputi, personal communication).
Cheng et al. (2008) also explored the NIH set of HCV 18-residue peptides for anti-HCV activity. One of the active peptides has the same sequence as SL173, and was reported to inhibit HCV focus formation by >90 % and to reduce HCV RNA by 11-fold 72 h after infection, in excellent agreement with our own quantification data.
In summary, an efficient method was established to measure core dimerization in vitro, to identify core-derived peptides that inhibit the protein–protein interaction and to block production of HCV by Huh-7.5 cells. The GWAGWLLSPRG sequence common to these peptides corresponds to a protein-interaction ‘hot spot’, defined by Wells & McClendon (2007) as the small subset of residues involved in protein–protein interaction that contribute most of the free energy of binding. These core-derived peptides will be useful as tools to study the role of core dimerization in the virus cycle and in the interaction of core with cellular proteins such as PKR (Yan et al., 2007) and Dicer (Chen et al., 2008). When modified to improve affinity and serum stability, these peptides may by themselves constitute useful building blocks for peptidomimetic drugs, much along the lines of the NS3/NS4A inhibitor developed previously (Thibeault et al., 2004) or the cyclosporine A analogues now in clinical development as anti-HCV drugs (Paeshuyse et al., 2006).
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ACKNOWLEDGEMENTS |
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Received 5 November 2008;
accepted 4 February 2009.
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