H epatitis C virus (HCV) is a major cause of chronic liver disease (CLD). Approximately 160 million individuals suffer from chronic hepatitis C, putting them at risk to develop cirrhosis and hepatocellular carcinoma. 1 Recent improvements in the standard-of-care therapy, currently a combination of pegylated interferon (IFN) alpha (Peg-IFN-a), ribavirin (RBV), and one of two drugs interfering with the virally encoded NS3/4A protease, have raised the hope that HCV infection can be managed efficiently in countries with adequate medical infrastructure. 2 However, the current mode of treatment is not equally effective for all HCV genotypes and significant side effects are still observed. Efforts are currently being made to develop a combination of new direct-acting antivirals (DAAs) not leading to the emergence of escape mutations and, if possible, free of IFN. First proofs of concept recently emerged from clinical trials demonstrating that combinations of DAAs can result in the cure of chronic HCV infection. 3, 4 However, combinations of drugs targeting different steps of the viral life cycle, including virus entry, will likely improve viral response rates and therapeutic success.
HCV is a small enveloped virus with a positive stranded RNA genome belonging to the Hepacivirus genus in the Flaviviridae family. 5 Its genome encodes two envelope glycoproteins (E1 and E2), which play a key role in virus entry into the hepatocyte. However, as a result of its association with low-or verylow-density lipoproteins, 6 the lipoprotein moiety can also play a role in the entry process of HCV particle. HCV entry is currently viewed as a complex multistep process, because a series of specific cellular entry factors have been shown to be essential in the early steps of the HCV life cycle. 7 These molecules include the scavenger receptor class B type 1 (SRB1), the tetraspanin CD81, tight-junction proteins claudin 1 (CLDN1) and occludin (OCLN), and receptor tyrosine kinase-like epidermal growth factor receptor. After its interaction with entry factors at the cell surface, HCV particle is internalized by clathrinmediated endocytosis. 8 Importantly, as for several other viruses, HCV can also spread by direct cell-tocell transfer. 9, 10 Ferroquine (FQ; SSR97193) is a ferrocenic analog of chloroquine (CQ) that has been developed as a new antimalarial drug (Fig. 1A) . 11 This bioorganometallic compound, which has a mechanism of action different from CQ, 12 is currently one of the most promising new candidate drugs in the antimalarial pipeline, and it is about to complete phase II clinical trials as a treatment for uncomplicated malaria. 13 In addition to its antimalarial activity, FQ also shows an antiviral effect against SARS coronavirus infection. 14 This prompted us to test whether FQ exhibits an antiviral activity against HCV. Our data show that FQ inhibits HCV infection at a half-maximal effective concentration below 1 lM by blocking virus entry at the fusion step.
Chemicals. FQ was synthesized as previously described. 11 Antibodies. Monoclonal antibodies (mAbs) anti-HCV E1 glycoprotein (A4), 16 anti-HCV E2 glycoprotein (3/11; kindly provided by J. McKeating, University of Birmingham, UK)(17), 17 anti-yellow fever virus (YFV) envelope protein (2D12) (ATCC CRL-1689), anti-bovine viral diarrhea virus (BVDV) NS3 protein (Osc-23), 18 anti-NS5A antibody (Ab) (AUSTRAL Biologicals, San Ramon, CA), and anti-CD81 mAb JS-81 CD81 (BD Pharmingen, San Diego, CA) were used in this study. Cy3-, Alexa 488-and phycoerythrin (PE)conjugated secondary Abs were from Jackson Immu-noResearch (West Grove, PA), Invitrogen, and BD Pharmingen, respectively.
Viruses. To produce cell-cultured HCV (HCVcc), we used a modified version of the plasmid encoding Japanese fulminant hepatitis type 1 (JFH-1) genome (genotype 2a; GenBank access no.: AB237837), kindly provided by T. Wakita (National Institute of Infectious Diseases, Tokyo, Japan). 19 Briefly, HCVcc was produced in Huh-7 cells electroporated with in vitrotranscribed RNA of JFH-1 containing (JFH-1/Luc) or not, the Renilla luciferase reporter gene, and engineered to express A4 epitope 20 and titer-enhancing mutations. 21 JFH-1 stocks were produced by further amplification in Huh-7 cells. In the JFH-1/Luc construct, the Renilla luciferase gene is fused with the viral open reading frame in a monocistronic configuration. With this virus, we verified that our luciferase data were in the linear range of the assay. The JFH-1/DE1E2/Luc plasmid, containing an in-frame deletion in the E1/E2 region, and the JFH-1/GND/Luc replication-defective mutant have been described previously. 20, 22 The inhibitory effect of the drugs was determined by quantifying infectivity by indirect immunofluorescence (IF) with the anti-E1 mAb A4 16 or by measuring viral titers with the same Ab. For quantitative binding experiments, purified virus was obtained by precipitation of HCVcc-infected Huh-7 cells supernatants with 8% polyethylene glycol 6000. Pelleted virus was then loaded onto a continuous 10%-40% iodixanol gradient. One-milliter fractions were collected and the most infectious fractions were pooled. The titer of the stock was 5 Â 10 6 focus forming units (ffu)/mL. HCV pseudotyped retroviral particles (HCVpp) expressing the Firefly luciferase reporter gene were produced in HEK-293T as previously described. 23 The intergenotypic HCV chimera GT3a(452)/JFH-1 24 was also used in some experiments. Furthermore, BVDV strain NADL and YFV strain 17D were also used to test the effect of the compounds on other viruses.
Cell-to-Cell Inhibition Assay. HCV cell-to-cell transmission was measured by two different approaches, as previously described. 25, 26 Indirect IF. Infected cells grown on glass coverslips were processed for IF detection of viral proteins as previously described. 27 Nuclei were stained with 1 lg/mL of 4',6'-diamidino-2-phenylindole. Coverslips were observed with a Zeiss Axiophot microscope (Carl Zeiss, Oberkochen, Germany), and fluorescent signals were collected with a Coolsnap ES camera (Photometrix, Kew, Australia). For quantification of antigen-positive cells, images of randomly picked areas from each coverslip were recorded.
Quantification of HCV Core Protein. Huh-7 cells were inoculated for 2 hours with HCVcc in six-well plates. At the indicated time, HCV core antigen expressed within cells or secreted into the supernatant was quantified using chemiluminescent microparticle technology (Architect HCV Ag Test; Abbott Diagnostics, Rungis, France), as previously described. 28 Quantitative Binding and Virus Internalization Assays. Virions bound to Huh-7 cells were determined by quantitative real-time reverse-transcription polymerase chain reaction (qRT-PCR) assay as described previously. 29 Internalization was measured as previously described. 30 Detection of HCV Receptor Expression by Flow Cytometry and Western Blotting. Huh-7 cells were treated with FQ for 48 hours. After incubation with FQ, cells were stained with Abs for flow cytometry and/or western blotting, as previously reported. 31 Cell-Cell Fusion Assay. Cell-cell fusion assay was performed as previously reported. 32 Selection of a FQ-Resistant Virus. Supernatant of HCV-infected cells were serially passaged under increasing concentrations of FQ. The structural region of HCV genome was amplified by RT-PCR and sequenced. Amino acid changes that arose during inhibitor selection were identified by analysis of the DNA sequence, compared to the initial and control passages, in the presence of solvent alone. Identified mutations were reintroduced in JFH-1 plasmid by PCR mutagenesis, and the plasmids were sequenced.
Combination Assays. Antiviral activity of a range of FQ concentrations alone or combined to IFN-a or boceprevir was determined by measuring half-maximal inhibitory concentration (IC 50 ) values. Then, combination index was calculated according to a previously reported method. 33 Prichard and Shipman's method 34 was also used to analyze interaction effects using a three-dimensional (3D) approach. This method presents complete drug interactions.
Statistical Analysis. Prism v5.0c software (Graph-Pad Software, Inc., La Jolla, CA) was used to prepare graphs, calculate IC 50 values, and determine statistical significance of differences between data sets.
FQ Inhibits HCV Infection. Chemical structures of FQ and CQ are presented in Fig. 1A . To test the effect of FQ on the HCV life cycle, the compound was added to Huh-7 target cells before, as well as during, infection. FQ exhibited a dose-dependent inhibition of HCV, indicating that FQ specifically affects the HCV life cycle with an estimated IC 50 value of 0.8 lM (6 0.26) and an 90% inhibitory concentration (IC 90 ) of 1.86 lM (6 0.08) (Fig. 1B,D) . Similar results were obtained using the HepG2 cell line expressing CD81 (data not shown). The inhibitory effect was not the result of cytotoxicity, because parallel experiments did not show any toxic effect of the drug at the concentrations tested (Supporting Fig. 1 ). Furthermore, no cytotoxicity was observed in primary human hepatocytes at the concentrations used in our experiments (Supporting Fig. 1 ). FQ showed a 50% cytotoxic concentration (CC 50 ) of 5.34 lM and a therapeutic index of 6.7. Similar inhibitory effects were observed with FQ-treated cells infected with a chimeric virus containing the structural proteins of a genotype 3a isolate (Supporting Fig. 2) , indicating that the antiviral effect is not specific to JFH-1 isolate. Parallel control experiments with well-characterized HCV inhibitors are presented in Supporting Fig. 3 . CQ was less effective against HCV (Fig. 1C,E) . Indeed, IC 50 and IC 90 values for CQ were 3.93 (6 1.87) and 4.33 lM (6 0.53), respectively. Furthermore, CQ had a CC 50 of 19.58 lM and a therapeutic index of 5. To determine whether HCV is the only member of the Flaviviridae family to be affected by FQ, we tested this compound on two other members of this viral family (BVDV and YFV). BVDV and YFV infections were performed on MDBK and Huh-7 cells, respectively. FQ showed some antiviral effect on these two viruses, albeit at a much higher concentration. Indeed, IC 50 values were 6.74 (6 0.48) and 3.63 lM (6 0.64) for BVDV and YFV, respectively. Altogether, these results indicate that FQ has a potent antiviral activity against HCV.
FQ Inhibits HCV Entry. To determine whether FQ has any effect on HCV entry, the compound was added or removed at different time points before, during, and after inoculation of Huh-7 cells with JFH-1 (Fig. 2) . The highest decrease in HCVcc infection was observed when FQ was present during viral infection, and only a weak antiviral effect was detected when FQ was added postinfection ( Fig. 2A,B) . Similar results were obtained with CQ ( Fig. 2C,D) , and parallel control experiments with well-characterized HCV inhibitors are presented in Supporting Figs. 4 and 5. These results suggest that FQ inhibits an early step of the HCV life cycle, which is most likely the entry step. To investigate the effect of FQ on HCV entry, we used the HCVpp system. These are retroviral cores carrying HCV glycoproteins in their envelope. In this context, only the early steps of the viral life cycle (i.e., virus interaction with receptors, uptake, and fusion) are HCV specific, whereas all later steps are dependent on retroviral nucleocapsid elements. Using this approach, FQ inhibited HCVpp entry in a dose-dependent manner (Fig. 2F) . Furthermore, this effect was specific of HCV envelope glycoproteins because FQ did not affect the entry of control pseudoparticles containing the envelope glycoprotein of the feline endogenous retrovirus, RD114. Together, these data indicate that FQ is an inhibitor of HCV entry.
Because FQ specifically affects HCV entry, this prompted us to test its antiviral effect on different HCV genotypes and subtypes in the context of the HCVpp system. FQ inhibited the infectivity of HCVpp from the different genotypes and subtypes tested, indicating that FQ inhibits HCV entry in a genotype-independent manner ( Table 1) .
Effect of FQ on HCV Replication and Assembly. Although the above data indicate that FQ has a strong effect on HCV entry, we cannot exclude additional effects on other steps of the HCV life cycle. To analyze the effect of FQ on HCV genome replication, Huh-7 cells were electroporated with in vitro-transcribed, assembly-defective JFH-1/DE1E2/Luc RNA, to bypass the entry step, and avoid any interference with late steps of the HCV life cycle. Furthermore, boceprevir was used in parallel as a control of inhibition of viral replication. FQ had also some effect on HCV replication (Fig. 3A) , albeit at higher concentrations (see Figs. 1B and 3A ). This observation is in agreement with the additional weak antiviral effect detected when FQ was added postinfection (Fig. 2B) .
To determine whether FQ could have any effect on HCV assembly or secretion, intra-and extracellular core protein was quantified in infected cells treated postinfection with 1 lM of FQ. The amount of core in the culture supernatant reflects the quantity of secreted viral particles. The amount of core released in the supernatant of infected cells was not significantly reduced in the presence FQ treatment (Fig. 3C) . In contrast, brefeldin A, an inhibitor of HCV release, reduced core secretion by more than 1 log 10 . These data show that FQ does not inhibit virion assembly and egress.
FQ Inhibits a Late
Step of HCV Entry. Because our data show that FQ has an antiviral activity at an early step of the HCV life cycle, we further investigated its mode of action on HCV entry. To determine whether FQ impairs directly the binding of particles to the cell surface, we analyzed virus binding in the presence of FQ. Cells were inoculated with purified HCVcc at 4 C in the presence of FQ, and the amount of bound virions was determined by quantifying HCV genomic RNA (gRNA). Heparin was used as a control of inhibition of HCV binding. As expected, heparin strongly reduced HCV attachment to the cell surface (Fig. 4A ). In the presence of FQ, no effect on virus binding was observed (Fig. 4A) , indicating that FQ does not inhibit HCV entry by impairing virus binding to the cell surface.
To further analyze the mechanism by which FQ inhibits HCV entry, we assessed the expression of known essential HCV entry factors CD81, SRB1, CLDN1, and OCLN. Huh-7 cells were treated with FQ at 1 lM for 48 hours. Then, CD81, SRB1, CLDN1, and OCLN expression was assessed by western blotting and/or flow cytometry. Expression levels of all four entry factors were unaltered, indicating that FQ does not act through their down-regulation (Fig. 4B,C) .
Because FQ does not inhibit the binding of HCV particles to the cell surface and because it has no effect on the expression of HCV receptors, we also analyzed the effect of this molecule on the internalization of the viral particle. HCV internalization was not affected by FQ treatment, indicating that this molecule blocks a postinternalization step (Fig. 4D) . It is also worth noting that FQ has no effect on IFN induction (Supporting Fig. 6) .
To determine the effect of FQ on the fusion process, we used a cell-cell fusion assay that has been previously described. 32 FQ induced a dose-dependent decrease of fusion activity of HCV envelope glycoproteins, whereas no effect was observed on control Chikungunya virus envelope glycoproteins (Fig. 4E) . Together, these results indicate that FQ inhibits the fusion step during the HCV entry process.
FQ Resistance Maps to E1 Glycoprotein. To further investigate the mechanism of action of FQ, we selected a partially resistant mutant by propagation for several passages in the presence of increasing concentrations of drug. After 16 passages, we did not observe any amino acid change in E2, whereas two mutations were identified in E1 glycoprotein (Y297H and S327A). Interestingly, reverse genetics experiments indicate that the S327A mutation is able, by itself, to confer some resistance to FQ (Fig. 5) . It is worth noting that serine 327 is well conserved in genotypes 1-6.
FQ Inhibits Cell-To-Cell Transmission. Subsequent to infection of Huh-7 cells with HCVcc, progeny viruses are transmitted to adjacent cells, resulting in focal areas of spreading infection (foci). This mode of transmission is refractory to neutralization by anti-E2 Abs. 9 To determine whether FQ can block cell-to-cell spread, HCVinfected RFP-NLS-IPS-Huh-7 cells were cocultured with naïve Huh-7 cells in the presence or absence of FQ, as previously described 26 (Fig. 6A) . In a second approach, HCV-infected Huh-7 cells were labeled with CMFDA and cocultured with naïve target cells in the presence or absence of FQ, as previously described 25 (Fig. 6B) . A strong decrease in cell-to-cell transmission was clearly observed in both approaches (Fig. 6) .
Antiviral Effect of FQ in Combination With IFNa or Boceprevir. We tested whether FQ could be combined with other anti-HCV compounds currently (C) Huh-7 cells were inoculated with HCVcc for 2 hours and cultured in the presence or absence of FQ for 48 hours. In parallel, HCV-infected Huh-7 cells were treated with brefeldin A for 8 hours before harvest. Supernatants were collected, cells were lysed, and intracellular and extracellular core protein was quantified. Furthermore, intracellular RNA was measured and the data were normalized by HCV replication level to take into account the potential effect of FQ on HCV replication. No significant (ns) difference was observed between FQ-treated and untreated cells. To determine the effect of FQ on HCV binding, Huh-7 cells were inoculated for 1hour at 4 C with purified HCVcc at a MOI of 10 in the presence of solvent (DMSO), FQ, or 500 lg/mL of heparin. Cells were washed thrice with ice-cold phosphate-buffered saline, and total RNA was extracted. Bound HCV virions were detected by quantification of HCV gRNA by qRT-PCR. Mean values 6 standard deviation (error bars) of three different experiments are presented. Turkey's multiple comparison test was used for statistical analysis. No significant (ns) difference was observed between treated and untreated cells. Receptors expression was assessed in Huh-7 cells, exposed (FQ), or not (Ctrl) for 48 hours to 1 lM of FQ, by flow cytometry for CD81 and SRB1 (B), and by western blotting for CD81, SRB1, CLDN1, and OCLN (C). (D) To determine the effect of FQ on HCV internalization, purified HCV particles were bound to Huh-7 cells, as described in (A), followed by 30-minute incubation at 37 C. Cells were then chilled on ice, and noninternalized virions were removed by trypsinization for 1 hour at 4 C, and internalized viral particles were determined by quantification of HCV gRNA by qRT-PCR. To verify efficacy of trypsin treatment, virus was bound to cells at 4 C and then removed by trypsinization (data not shown). (E) Effect of FQ on fusion function of HCV envelope glycoproteins. 293T ''donor'' cells coexpressing the HCV E1E2 or Chikungunya envelope glycoproteins and a luciferase marker gene under the control of the HIV-1 promoter were cocultured with Huh-7-Tat ''indicator'' cells expressing the HIV-1 Tat protein. After 24 hours, cells incubated for 1 hour in the presence or absence of FQ were treated (at pH 5) for 3 minutes, and luciferase activity induced by fusion between donor and indicator cells was measured 72 hours later. Fusion in the absence of drug was taken as 100%. Graphs represent the averages of three independent experiments (**P < 0.01). A control experiment with pseudoparticles containing the envelope glycoproteins of Chikungunya confirmed that this virus is not sensitive to FQ treatment (data not shown). HIV, human immunodeficiency virus.
used in hepatitis C treatment. We used boceprevir, a drug targeting directly HCV protease NS3/4A, and IFN-a, which has been used for several decades in anti-HCV treatment. These drug combinations were analyzed using the combination index (CI) as well as Prichard and Shipman's method. 34 Each drug was combined with FQ at different fractions of their IC 50 value. Combination of FQ with boceprevir and IFN-a resulted in an additive effect, as reflected by a CI of 0.97 (6 0.03) and 1.08 (6 0.18), respectively. Furthermore, synergy was also observed for some higher concentrations, as measured by Prichard and Shipman's method 34 (Fig. 7 ).
HCV entry represents an attractive target for antiviral intervention, with opportunities to prevent multiple virus-receptor interactions and interfere with virus-cell membrane fusion. 35 In this study, we showed that FQ exhibits a genotype-independent antiviral activity against HCV by inhibiting a postbinding and postinternalization step of HCV entry into target cells and by blocking cell-to-cell spread between neighboring cells.
Although FQ is an analog of CQ, its mechanism of action is potentially different. 12 The mechanism of inhibition by CQ involves impaired endosomal-mediated virus entry, most likely through the prevention of endocytosis and/or endosomal acidification. In contrast, FQ has weaker base properties, compared to CQ. 36 This difference could potentially explain the lack of antiviral activity of FQ against viruses such as vesicular stomatitis virus, influenza virus, or Sindbis virus, 14 whereas CQ blocks cell entry of these viruses as Acceptor cells are naïve Huh-7 cells. Coculture of donor and acceptor cells with or without neutralizing mAb 3/11 allowed for monitoring cell-to-cell or total transmission of HCV. Percentage of cell-free transmission was obtained by subtracting the percentage of cells infected in the presence of mAb 3/11 from the percentage of cells infected in the absence of Ab. After 48 hours, cells were labeled with anti-NS5A mAb, followed by PE-conjugated secondary Ab and analyzed by flow cytometry. In these conditions, newly infected cells are negative for CMFDA staining and positive for PE staining. Results are reported as the percentage of HCV-infected cells from two independent experiments. **P < 0.01; ***P < 0.001. well as other pH-dependent viruses. [37] [38] [39] Other interesting features of FQ are its higher lipophilicity (at pH 7.4) and the peculiar conformation provided by an intramolecular hydrogen bond present in nonpolar conditions, which result in a better potency for FQ to cross membranes. 40 In addition, FQ has also been shown to specifically generate reactive oxygen species and induce lipid peroxidation. 40, 41 Recently, it has been shown that HCV envelope proteins form large molecular complexes stabilized by intermolecular disulfide bonds. 42 This observation strongly suggests that the entry process necessitates a rearrangement of these disulfide bonds for the fusion process to take place. Therefore, it is possible that the oxidative properties of FQ in acidic conditions could inhibit the fusion process by affecting reorganization of the disulfide bonds within endosomes.
FQ inhibits a postbinding and postinternalization step of HCV entry into target cells. Indeed, FQ does not affect binding of HCVcc to Huh-7 cells or the expression of specific cellular entry factors. Furthermore, the effect of FQ on HCVpp strongly suggests that FQ affects the entry function of HCV envelope proteins and not the lipoprotein moiety associated with the virion. Our data also show that FQ blocks HCV entry by inhibiting the fusion process. Finally, the S327A-resistant mutation identified in this work suggests that E1 could be the target of FQ.
In addition to its effect on HCV entry, FQ can also affect HCV RNA replication, albeit at higher concentrations. This inhibitory effect is similar to the inhibition of an HCV subgenomic replicon by CQ. 43 HCV is known to exploit autophagy for its replication, 44 and inhibition of replication by CQ targets virus-associated autophagy. 43 However, it remains to be determined whether inhibition of HCV RNA replication by FQ depends on the same mechanism.
In clinical studies with healthy human volunteers, it has been shown that FQ is safe and very well tolerated with oral doses of 400-1,600 mg FQ, and the mean estimate for blood apparent terminal half-life of FQ was 16 days. 45 In addition, a maximum blood concentration of 487 ng/mL (or 1.1 lM) was observed for the highest dose of FQ, which is slightly above the IC 50 value calculated for HCV in cell culture. Although such a concentration would probably not be high enough to eliminate HCV completely, it would likely be sufficient in combination therapies with other drugs showing a synergistic or additive effect. Further studies will be necessary to determine the in vivo potency of FQ against HCV.
FQ may provide a new approach to prevent HCV infection, especially in the setting of liver transplantation (LT) of chronically infected HCV patients. Indeed, a major problem for LT resulting from HCV is the reinfection of the graft, which is always observed with an accelerated progression of liver disease. 46 Thus, the ability of FQ at inhibiting HCV cell-to-cell transmission is a major asset for an entry inhibitor. Furthermore, FQ exhibits an antiviral activity against 34 Combination studies for each pair of compounds were performed in triplicate. The theoretical additive effect is calculated from the dose-response curves of individual compounds by the equation, Z ¼ X þ Y(1-X), where X and Y represent the inhibition produced by the individual compounds and Z represents the effect produced by the combination of compounds. The theoretical additive surface is subtracted from the actual experimental surface, resulting in a horizontal surface that equals the zero plane when the combination is additive. A surface raising more than 20% above the zero plane indicates a synergistic effect of the combination, and a surface dropping lower than 20% below the zero plane indicates antagonism.
all HCV genotypes, tested in the HCVpp system, increasing its potential interest as a general anti-HCV agent. The combination of entry, replication, and polyprotein-processing inhibitors in a context of a multidrug therapy might be the way to reduce the risk of emergence of resistant viruses. FQ might thus be a valuable option to be tested in low-cost anti-HCV combinations. Finally, these findings highlight the potential interest of FQ use in countries where malaria coinfection with HCV can occur.