The intracellular inhibition of HCV replication represents a novel mechanism of action by the innate immune Lactoferrin protein.

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The intracellular inhibition of HCV replication represents a novel mechanism of action by the innate immune Lactoferrin protein

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Frédéric Picard-Jean, Sabrina Bouchard, Geneviève Larivée, Martin Bisaillon ⇑ Département de Biochimie, Faculté de médecine et des sciences de la santé, Université de Sherbrooke, Sherbrooke, Québec J1E 4K8, Canada

a r t i c l e

i n f o

Article history: Received 5 July 2013 Revised 15 August 2014 Accepted 25 August 2014 Available online xxxx Keywords: Hepatitis C virus (HCV) Human Lactoferrin (hLF) Inhibitor Viral replication NS3 ATPase/Helicase

a b s t r a c t Hepatitis C virus (HCV) is a major public-health problem with 130–170 million individuals chronically infected worldwide. In order to halt the epidemic, therapy against HCV will need to be both effective and widely available. Studies focusing on safe and affordable natural product active against HCV have revealed the antiviral activity of the human Lactoferrin (hLF) protein which binds and neutralizes the circulating virion. In the current study, investigation of hLF activity on the HCV subgenomic replicon system, which is independent from viral entry and shedding, revealed a distinct antireplicative activity of hLF against HCV. Hepatocellular uptake of hLF was confirmed and correlated with qualitative HCV staining reduction. Quantitative dose–response inhibition assays confirmed an hLF-mediated and dose-dependent HCV replication reduction reaching up to 60%. The in cellulo anti-HCV activity of hLF was additive to both Ribavirin and Interferon-a-2b. Further investigation of hLF activity against the essential viral proteins involved in HCV genome replication revealed an inhibitory activity against the HCV ATPase/Helicase NS3 protein but not against the HCV RNA-dependent RNA polymerase (NS5B protein). NS3 inhibition was mediated by a direct and specific interaction between hLF and an allosteric binding site on NS3. Taken together, our findings reveal a new antiviral mechanism of action by which hLF inhibits intracellular HCV replication. Ó 2014 Published by Elsevier B.V.

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1. Introduction

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Hepatitis C virus (HCV) is a major public-health problem; 130–170 million individuals are chronically infected by HCV and more than 350,000 die each year due to HCV-related liver diseases (Lavanchy, 2009; Perz et al., 2006). Ever since its discovery in 1989, the transmission of this blood–borne virus has been greatly reduced in developed countries. The reality is discordant in developing countries, where the lack of screening for blood donations and the common reuse of medical and dental supplies are fuelling viral

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Abbreviations: HCV, Hepatitis C virus; hLF, human Lactoferrin; LF, Lactoferrin; HIV, human immunodeficiency virus; HSV1, herpes simplex virus type 1; CMV, cytomegalovirus; RDRP, RNA-dependent RNA polymerase; NGS, normal goat serum; WGA, wheat germ agglutinin; IPTG, isopropyl b-D-thiogalactopyranoside; Ni–NTA, Nickel–nitrilotriacetic acid; DTT, dithiothreitol; Pi, inorganic phosphate; DIP, 2,20 -Bipyridyl; RBV, Ribavirin; IFN-a-2b, Interferon-a-2b; CI, combination index; GRAS, Generally Recognized As Safe; FDA, U.S. Food and Drug Administration; SVR, sustained virological response; SNPs, single-nucleotide polymorphisms; IL-28B, Interleukin-28B. ⇑ Corresponding author. Address: Département de Biochimie, Faculté de médecine et des sciences de la santé, Université de Sherbrooke, 3201 rue Jean-Mignault, Sherbrooke, Québec J1E 4K8, Canada. Tel.: +1 (819) 821 8000x75287. E-mail address: [email protected] (M. Bisaillon).

spread (Gravitz, 2011). As a consequence, the prevalence of HCV infection is unequally distributed around the world and developing countries are much more affected (Gravitz, 2011). In the absence of a vaccine, a low-cost widely distributed therapeutic regimen would be required in order to halt and treat the HCV epidemic. This therapy could be a combination of novel drugs benefiting from a price reduction agreement, patent-expired generics, and active natural products. In that sense, several groups have investigated the antiviral activity of various human circulating proteins. Among those, one interesting hit is the Lactoferrin (LF) protein, a ubiquitous protein present in body fluid such as plasma, tears and milk (Gonzalez-Chavez et al., 2009). LF is a 80 kDa iron-binding protein component of the innate immune system with demonstrated antibiotic, antifungal and antiviral activities, notably against human immunodeficiency virus (HIV), herpes simplex virus type 1 (HSV1), cytomegalovirus (CMV), rotavirus, and HCV (Defer et al., 1995; Fujihara and Hayashi, 1995; Harmsen et al., 1995; Superti et al., 1997; Yi et al., 1997). HCV is an enveloped positive single-stranded RNA virus encoding a single polyprotein which is further processed into seven non-structural proteins (p7, NS2, NS3, NS4A, NS4B, NS5A, NS5B), one capsid protein (core) and two envelope glycoproteins (E1 and E2). It has been shown that both E1 and E2, which are at the surface

http://dx.doi.org/10.1016/j.antiviral.2014.08.012 0166-3542/Ó 2014 Published by Elsevier B.V.

Please cite this article in press as: Picard-Jean, F., et al. The intracellular inhibition of HCV replication represents a novel mechanism of action by the innate immune Lactoferrin protein. Antiviral Res. (2014), http://dx.doi.org/10.1016/j.antiviral.2014.08.012

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of the virion, can be bound by LF which ultimately blocks viral entry (Yi et al., 1997). Limited clinical trials, although not all conclusive, have demonstrated a potential anti-HCV activity of orally administrated LF on viral load and hepatic markers from patients (Hirashima et al., 2004; Ishibashi et al., 2005; Iwasa et al., 2002; Kaito et al., 2007; Okada et al., 2002; Tanaka et al., 1999; Ueno et al., 2006). The LF anti-HCV activity is currently believed to behave as a viral entry inhibitor, binding circulating virions and preventing them from entering into their hepatocellular target. Interestingly, biopsies of HCV patients show immunohistochemical evidence of intrahepatic Lactoferrin accumulation (Tuccari et al., 2002), which raises the question of the impact of LF on the intracellular phase of the HCV viral cycle. In order to maintain viral load, with virions half-life being 2.7 h, HCV relies on a very active viral replication, generating up to 1012 virions per day per patient (Neumann et al., 1998). The HCV genome is replicated in the hepatocellular cytoplasm by the membrane-associated viral replication complex composed of, but not restricted to, the ATPase/Helicase C-terminal domain of NS3 and the RNAdependent RNA polymerase (RDRP) NS5B. Could the intrahepatic hLF accumulation interfere with HCV replication through a novel and distinct mechanism of inhibition independent from virion neutralization? This is the question addressed in the present study.

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2. Material and methods

germ agglutinin Alexa Fluor 488 conjugate (WGA, 5 lg/ml, Invitrogen W11261) for 15 min at 4 °C. After two additional washes with cold PSB, cells were fixed with 4% paraformaldehyde and 4% sucrose for 20 min at 22 °C. Cells were permeabilized with 0.15% triton X100 in PBS for 5 min at 22 °C and blocked in 10% normal goat serum (NGS, Wisent 053-150). Primary mouse anti-hLF (Abcam ab10110) antibody diluted 1:1000 in 10% NGS was added for 4 h at 22 °C. Cells were washed and incubated in the dark for 1 h at 22 °C with an Alexa Fluor 633-labeled anti-mouse (Invitrogen A21053) secondary antibody (1:1000 in 10% NGS). Lastly, the nuclei were stained with Hoechst (1 lg/ml) for 15 min at 22 °C in the dark. Cover glasses were mounted on slides with SlowFade mounting medium. Confocal microscopy was conducted using an Olympus FV1000 inverted microscope (60 oil immersion objective, Olympus). Cells with similar size from each condition were sequentially laser-excited at 405 nm with a diode laser (emission D450/50, Hoechst), 488 nm with a 40 mW Argon laser (emission D515/30, Alexa Fluor 488 conjugate of WGA) and 633 nm with a 10 mW Helium Neon laser (emission D705/100, Alexa Fluor 568 Phalloidin). Serial horizontal optical cross-sections of 1024 1024 pixels were acquired throughout the entire thickness of the cells at interval of 0.5 lm. Identical instrumental settings were used for each condition. Images were pseudocolored to match their fluorochrome color and merged using FluoView software and subsequently assembled using Adobe Photoshop.

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2.1. Reagent

2.3. In cellulo inhibition of HCV replicon

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2.2. Immunofluorescence

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Huh-7 cells supporting HCV subgenomic replicon (expressing HCV NS3, NS4A, NS4B, NS5A, NS5B proteins) were seeded in 24 well plates to a density of 5 104 cells/well. They were treated the next day with 3 lM of hLF purified from human milk. After 0 h, 2 h or 24 h, cells were washed twice with PBS and fixed with 4% paraformaldehyde and 4% sucrose for 20 min at 22 °C. Cells were permeabilized with 0.15% triton X-100 in PBS for 5 min at 22 °C and blocked in 10% normal goat serum (NGS, Wisent 053150). The hLF was stained with primary mouse anti-hLF (Abcam ab10110) antibody diluted 1:1000 in 10% NGS and viral NS5A protein was stained with a primary rabbit anti-NS5A (Abcam ab2594) antibody diluted 1:50 in 10% NGS. After 4 h at 22 °C cells were washed and incubated in the dark for 1 h at 22 °C with an Alexa Fluor 488-labeled anti-mouse (Invitrogen A11017) secondary antibody (1:1000 in 10% NGS) and Alexa Fluor 568-labeled anti-rabbit (Invitrogen A21069) secondary antibody (1:1000 in 10% NGS). Nuclei were stained with Hoechst (1 lg/ml) for 15 min at 22 °C. Cover glasses were mounted on slides with SlowFade mounting medium (Invitrogen S36937). Epifluorescence microscopy was conducted using a Nikon Eclipse TE2000-E visible/epifluorescence inverted microscope using bandpass filters for Hoechst, Alexa Fluor 488 and Alexa Fluor 568. Photomicrographs were acquired at a 60 magnification using an oil immersion objective. Images were processed using the Nikon NIS Elements AR software and assembled using Adobe Photoshop. Alternatively, Huh-7 cells supporting the HCV subgenomic replicon were seeded in 24 well plates to a density of 5 104 cells/ well. They were treated the next day with 3 lM of hLF. Cellular uptake was allowed for 24 h and followed by 5 min of trypsinization. Cells were allowed to re-adhere for 18 h, washed twice with cold PBS and cellular membranes were labeled with wheat

Huh-7 cells containing the HCV subgenomic biscistronic replicon Luc-ubi-neo (kindly provided by R. Bartenschlager) were cultured in Dulbecco’s Modified Eagle Medium (DMEM, Wisent) supplemented with 10% FBS (Wisent), 2 mM L-glutamine and 1 mM sodium pyruvate. In order to maintain the replicon, 250 mg/mL of G-418 (Wisent) was added to cultured cells. Prior to hLF treatment, 5 104 cells/well were seeded in 24 well plates and the G418 selection was removed. Cells were treated for 48 h with various concentrations of hLF or hTF dissolved in PBS, or PBS alone. The luciferase protein encoded by the replicon was quantified using the luciferase reporter system according to the instructions of the manufacturer (Promega). Briefly, cells were washed with PBS and were incubated for 10 min at 22 °C with 150 ll of 1 cell culture lysis buffer (Promega). Cells were next harvested, vortexed for 10 s and centrifuged for 15 s at 12,000g. Supernatants were collected and protein concentration was determined using the Bradford protein assay (Bio-Rad). In order to quantify the luciferase activity, a 20 ll aliquot (22 °C) was mixed with 100 ll of luciferase assay reagent (22 °C), and analyzed with a Glomax 20/20 luminometer (Promega). Huh-7 cells containing the Luc-ubi-neo replicon (5 104 cells/ well) were treated for 24 h in conjunction with hLF (0 or 2 lM) and either Ribavirin or Interferon-a-2b (various concentrations). The combination index were calculated according to formula (1) based on the individual drug concentration (ICx,A and ICx,B) and the combined drug concentrations (CA,x and CB,x) required to obtain 50% of the effect. Alternatively, Huh-7 cells containing the Luc-ubineo replicon (5 104 cells/well) were treated for 48 h with a mono-, bi- or tri-therapy consisting of a Ribavirin (10 lg/ml), Interferon-a-2b (0.3 U/ml), hLF (5 lM) alone or in combination:

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The human Lactoferrin (hLF) and human Transferrin (hTF) were purchased from Sigma–Aldrich (L0520, chromatographically purified, purity >90% and T0665, chromatographically purified, purity >99%).

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CI ¼

C A;x C B;x þ ICx;A ICx;B

ð1Þ

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2.4. Fluorescence-activated cell sorting (FACS)

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Huh-7 cells containing the Luc-ubi-neo replicon were seeded into 6 well plates (2 105 cells/well) 24 h prior treatment. Various

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concentrations of exogenous hLF (0–50 lM) were added to culture media for 48 h. Alternatively, 3 lM hLF was added for 0–48 h. Cells were treated with trypsin for 5 min at 37 °C, washed twice with PBS and fixed with 1.2% paraformaldehyde for 30 min at 4 °C. Cells were permeabilized with 0.1% triton X-100 in PBS for 5 min at 22 °C, washed and incubated for 3 h with primary mouse antihLF (Abcam ab10110) antibody diluted 1:1000 PBS-2% FBS. Cells were then washed and incubated in the dark for 30 min with an Alexa Fluor 488-labeled anti-mouse antibody (Invitrogen A11017) and propidium iodine (5 lg/ml). After two washes, cells were analyzed by a Flow cytometry (Becton Dickinson, Mountain View, CA) with a 15 mW argon ion laser tuned at 488 nm, the emitted fluorescence was detected at 530 ± 15 nm (FL1). Negative staining levels were set by comparison with an isotype-matched control sample.

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2.5. Expression and purification of recombinant HCV NS3h protein

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An expression plasmid containing the ATPase/Helicase domain of HCV NS3 protein (amino acids 165–631, NS3h) was generated by inserting the corresponding cDNA between the EcoRI and HindIII cloning sites of the pET21b plasmid (Novagen). In this context, the NS3h protein is fused in frame with a C-terminal 6-His tag, and expression of the protein is driven by a T7 RNA polymerase promoter. A plasmid for the expression of HCV RDRP NS5B lacking the 21 C-terminal hydrophobic amino acids (NS5BD21) in fusion with a hexa-histidine tag was also generated. Upon transformation of the pET-NS3h or pET-NS5BD21 plasmids into Escherichia coli BL21(DE3), cultures were grown at 37 °C in Luria–Bertani medium containing 30 lg/mL ampicillin until the OD600 reached 0.5. Induction was initiated with 400 lM isopropyl b-D-thiogalactopyranoside (IPTG) and 2% ethanol, protein expression was allowed for 20 h at 18 °C. All subsequent procedures were performed at 4 °C. The bacteria were harvested by centrifugation at 5000 rpm and resuspended in 50 mL of lysis buffer [50 mM Tris–HCl (pH 7.5), 150 mM NaCl, and 10% sucrose]. 50 lg/mL lysozyme and 0.1% Triton were added prior to sonication. After removal of insoluble material by centrifugation at 13,000 rpm, the soluble extract was applied to a nickel–nitrilotriacetic acid agarose (Ni–NTA Agarose, Qiagen) column. The protein was eluted stepwise with elution buffer [50 mM Tris–HCl (pH 8.0), 100 mM NaCl, and 10% glycerol] containing 50, 100, 200, 500, and 1000 mM imidazole. The polypeptide composition of the fractions was monitored by SDS–PAGE. The recombinant NS3h protein was recovered in the 200 mM imidazole eluate. This fraction was dialyzed against 50 mM Tris–HCl (pH 8.0), 50 mM NaCl, 2 mM dithiothreitol (DTT) and 10% glycerol. The protein concentration was determined by the Bio-Rad dye binding method and stored at 80 °C.

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2.6. In vitro HVC NS3h and NS5B inhibition by hLF

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NS3h ATPase assays were carried out in 20 ll reactions containing 150 nM NS3h, 100 lM ATP, 50 mM Tris–HCl (pH 7.5), 5 mM DTT and 1 mM MgCl2 in the absence or in the presence of 10 lM hLF for 10 min at 37 °C. The reaction was stopped with the addition of 400 ll of Biomol Green Reagent (Enzo Life Science). Released inorganic phosphate (Pi) product was detected by measuring the OD620 after 20 min of incubation. NS5B polymerase assays were conducted in 20 ll reactions containing 1 lM NS5B, 1 mM rNTP, 10 lM of partially double-stranded RNA template (50 -GGGCACACACAGTCGACCACACAAAACCACCC-30 hybridized with 50 -GGGTGGTTTTGTGTGGTCGA-30 ), 20 mM Tris–HCl, pH 7.5, 1 mM DTT, 5 mM MgCl2, 20 U RNaseOUT and 1 lg/ml pyrophosphatase in presence or in absence of 10 lM hLF for 10 min at 37 °C. Pi, hydrolyzed from pyrophosphate product, was detected using with the addition of 400 ll of Biomol Green Reagent by measuring the OD620 after

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20 min of incubation. Viral enzyme activity was relativized to the untreated (no hLF) condition.

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2.7. Characterization of HVC NS3h inhibition by hLF

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HCV NS3h ATPase activity was subjected to hLF inhibition. A 20 ll reaction containing 50 mM Tris–HCl, pH 7.5, 5 mM DTT, 1 mM MgCl2, 100 lM ATP and increasing concentrations of either native hLF, heat inactivated hLF of hTF were incubated in the presence of 150 nM NS3h for 10 min at 37 °C. The reaction was stopped with the addition of 400 ll of Biomol Green Reagent (Enzo Life Science). Released inorganic phosphate (Pi) product was detected by measuring the OD620 after 20 min of incubation. Relative ATP hydrolysis was plotted against the milk protein concentration. Alternatively, the assays were conducted as a function of the ATP substrate concentration in the presence of various fixed concentrations of hLF.

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2.8. Ion dependency of the hLF inhibitory activity

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Reactions (20 ll) containing 50 mM Tris–HCl, pH 7.5, 5 mM DTT, 1 mM MgCl2, 100 lM ATP, 0 or 10 lM hLF and 150 nM NS3h were incubated for 10 min at 37 °C in the presence of 1 mM 2,20 -Bipyridyl (DIP), 100 lM FeCl3, 50 mM EDTA or an additional 19 mM MgCl2 (20 mM final). The reactions were stopped by the addition of 400 ll of Biomol Green Reagent (Enzo Life Science). Released Pi product was detected by measuring the OD620 after 20 min of incubation.

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2.9. HCV NS3h and hLF interaction

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The hLF protein (3 lM) was incubated in the presence or absence of 15 lM of histidine-tagged NS3h protein for 5 min at 22 °C in a buffer containing 50 mM Tris–HCl, pH 7.5, 5 mM DTT, 1 mM MgCl2 and 1 mM ATP. The reaction was then applied to a Ni–NTA Agarose (Qiagen) column and washed with 18 volumes of washing buffer [10 mM imidazole, 50 mM Tris–HCl (pH 8.0), 100 mM NaCl, and 10% glycerol]. The retained proteins were eluted with 2 volumes of washing buffer containing 1000 mM imidazole. The polypeptide composition of the eluate was resolved by SDS– PAGE and revealed by Coomassie staining.

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3. Results

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3.1. HCV replication appears to be reduced by hLF treatment

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We observed that HCV subgenomic replicon staining (as measured by NS5A immunofluorescence) was reduced in cells treated with human Lactoferrin (hLF). Huh-7 cells supporting HCV subgenomic replicon replication were treated with 3 lM hLF for 0 h, 2 h or 24 h. The double-immunofluorescence staining for hLF and HCV NS5A proteins was analyzed by epifluorescence microscopy. Fig. 1 reveals a qualitative reduction of approximately 50% in HCV staining intensity after 24 h of hLF treatment. Concomitant with the fading of HCV staining, hLF accumulation began to be detected after 2 h, and strong hLF accumulation was observed after 24 h. The hLF effect on HCV replication is not due to hLF cytotoxicity as measured by XTT assay in Huh-7 cells (Supp. Fig. S1). These results, although qualitative, suggest an inverse correlation between hLF exposition and HCV intracellular replication fitness. These data suggest that the hLf-mediated reduction in HCV replicon replication is not mediated through extracellular virion neutralization.

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Fig. 1. HCV staining is reduced upon hLF treatment. Huh-7 cells supporting HCV subgenomic replicon replication were treated with 3 lM hLF for the indicated period of time and subjected to immunofluorescence. Nuclei were stained with Hoechst (blue channel), HCV was revealed by an anti-NS5A antibody (red channel) and hLF was stained with an anti-hLF antibody (green channel). Images were acquired by epifluorescence microscopy at an original magnification of 60. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

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3.2. Cellular uptake of hLF

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We next sought to confirm that HCV-infected hepatic cells could internalize exogenously administrated hLF. Huh-7 supporting HCV replication cells were seeded in a 24-well plate and 0 lM or 3 lM of hLF was added to the culture media. The hLF internalization was allowed for 24 h, after which, extracellular hLF was washed with PBS and residual membrane bound hLF was degraded with a 5 min trypsin treatment. Cells were reseeded and allowed to readhere for 18 h before immunofluorescence staining. In order to outline the cytoplasmic limits, cells membranes were stained with WGA-Alexa Fluor 488 conjugate. The hLF staining was achieved using an anti-hLF primary antibody and an Alexa Fluor 633 secondary antibody. In order to determine the precise localization of hLF, cells were imaged following sequential 0.5 lm horizontal crosssection by confocal microscopy. Fig. 2 presents an optical cross-section corresponding to mid-cell thickness. Following the addition of exogenous hLF, a significant amount of hLF was detected inside the cytoplasm of hepatocytes. No hLF was observed inside the untreated cells when using identical instrumental settings. Together, these data confirm that hepatocytes infected by HCV have the capacity to acquire hLF from their extracellular environment. The hLF uptake dynamic was addressed using flow cytometry in order to quantify hLF internalization into HCV-infected hepatic cells. Huh-7 cells supporting HCV replicon were treated with 0– 50 lM hLF. After 48 h, cells were treated with trypsin, washed, then hLF was stained using anti-hLF antibody. Intracellular hLF staining was quantified using flow cytometry (Fig. 3). Concurrently, cells were treated with 3 lM hLF for 0–48 h. The intracellular concentration of hLF rises as a function of extracellular hLF concentration up to 1 lM before reaching a plateau, which is coherent with the active uptake model previously described

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(Fig. 3C) (Jiang et al., 2011). Furthermore, hepatic cells rapidly acquire hLF, the maximal intracellular concentration is reached within 4 h and remain constant over 48 h. Together, these data reveal that intracellular hLF concentration is stable overtime and maximum concentration is reached at extracellular hLF concentration equal or greater to 1 lM.

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3.3. In cellulo HCV replication inhibition by hLF

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We next set out to quantitatively investigate the inhibitory potential of hLF on intracellular HCV replication. The use of a firefly luciferase reporter HCV subgenomic replicon system allowed for the quantification of HCV replication. Huh-7 cells carrying the Luc-ubi-neo HCV subgenomic replicon were treated with various exogenous concentrations of hLF or hTF and viral replication was allowed for 48 h. Cells were harvested and luciferase assays were conducted and adjusted to protein levels, which were constant in these assays. Only the cells treated with hLF showed a dose-dependent reduction of up to 60% of the reporter activity (Fig. 3A). Half of the maximal hLF-mediated replicon inhibition was attained with the addition of 2 lM of exogenous hLF. These quantitative data are consistent with our previous qualitative observation where hLF reduced but did not completely abolish HCV replication. Nevertheless, these results demonstrate that hLF can impair intracellular HCV replication in a cellular model. The inhibitory potency of hLF was next tested in combination with two established HCV replication inhibitors: Ribavirin (RBV) and Interferon-a-2b (IFN-a-2b). Huh-7 cells carrying the HCV subgenomic replicon were treated for 24 h with 2 lM hLF and various concentrations of either RBV or IFN-a-2b. These two inhibitors displayed a stronger potency against HCV replication when combined with hLF (Fig. 4A and B). The nature of the interaction between

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Fig. 2. Hepatocellular uptake of exogenous human Lactoferrin detected by confocal microscopy. Huh-7 cells were treated with 0 lM or 3 lM hLF for 24 h, washed twice, submitted to a 5 min trypsin treatment, allowed to re-adhere for 18 h and subjected to immunofluorescence. Nuclei were stained with Hoechst (blue channel), plasma membranes were labeled with WGA (green channel) and hLF was stained with an anti-hLF antibody (red channel). The horizontal optical cross-sections represent the midthickness of the cells. Images were acquired by confocal microscopy at an original magnification of 60. Scale bar, 10 lm. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 3. In cellulo hLF internalization and antireplicative activity. (A) The HCV subgenomic replicon containing the firefly luciferase reporter was transfected in order to stably replicate in Huh-7 cells, allowing for the direct quantification of HCV replication. Replicon infected cells were treated with increasing concentrations of exogenous hLF or human Transferrin for 48 h and HCV quantification was performed by standard luciferase assays. (B) Huh-7 cells supporting HCV replicon were treated with 0 lM (light gray) or 10 lM (dark gray) hLF for 48 h. Upon trypsin treatment and fixation, internalized hLF was stained using anti-hLF antibody and quantified with a FACScan cytometer (FL1-H channel). (C) The hLF uptake was monitored after 48 h of treatment for hLF concentrations ranging from 0.1 to 50 lM. (D) Cells were treated with 3 lM hLF for 0–48 h upon which internalized hLF was quantified using FACScan cytometer.

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these drugs was determined based on the Loewe additivity model and the combination index (CI) (Loewe, 1953). The CI are similar for both combination regiments, and are near the additivity value of 1 at 1.03 and 0.95 for the RBV and IFN-a-2b, respectively. The Loewe’s isobologram representations (embedded in panel A and

B of Fig. 4) also indicate that the coordinates of the combinatory treatment are located within the line of additivity. Furthermore, the inclusion of hLF as part of a tritherapy together with RBV and IFN, displayed a significatively higher potency to reduce cellular HCV replication when compared to mono- or bi-therapy (Fig. 4C).


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Fig. 4. Effect on HCV replication of the hLF inclusion as part of a multidrug therapy. HCV subgenomic replicon containing the firefly luciferase reporter was transfected to stably replicate in Huh-7 cells, allowing for the direct quantification of HCV replication. (A and B) Hepatic cells were treated for 24 h with a mono- or bi-therapy consisting of increasing concentrations of either Ribavirin (A) or Interferon-a-2b (B) and 0 or 2 lM hLF. Loewe’s isobologram of these drug combination interactions is embedded in panel A and B. (C) Huh-7 cells were treated for 48 h with single concentration of Ribavirin (10 lg/ml), Interferon-a-2b (0.3 U/ml) or hLF (5 lM) (all below toxicity level) for single therapy or in combination as bi- or tri-therapy. Data represent the mean ± SD of two independent experiments. (⁄P < 0.05, ⁄⁄P < 0.01, ⁄⁄⁄P < 0.001).

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Together, these results demonstrate that the inhibitory potential of hLF is additive to currently established RBV and IFN-a-2b therapies to reduce in cellulo viral replication.

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3.4. In vitro inhibition of HCV enzymes by hLF

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In an effort to shed light on the intracellular mechanism of hLFmediated HCV inhibition, the potency of hLF to directly inhibit viral enzymes involved in HCV genome replication was addressed in vitro. The ATPase/Helicase NS3 (amino acid 168–631; NS3h) and the RNA-dependant RNA polymerase (RDRP) NS5B (amino acid 1–570) were cloned in fusion with a hexa-histidine tag, expressed in E. coli and purified by nickel-agarose affinity chromatography. The peptides corresponding to NS3h and NS5B protein were the predominant polypeptides (>95%) in the purified fraction (Fig. 5A). NS3h was incubated with ATP and Mg2+ cofactor in the absence or in the presence of hLF. NS3h ATPase activity was strongly inhibited by hLF (Fig. 5B). NS5B was incubated with a partially double-stranded RNA template, rNTP and Mg2+ cofactor in the absence or in the presence of hLF. The NS5B RNA polymerase activity was not affected by the presence of hLF (Fig. 5B). Next, NS3 ATPase activity was assayed in the presence of increasing concentration of hLF or hTF protein. As shown in Fig. 5C, hLF inhibits NS3 ATPase activity in a concentration-dependent manner, with an IC50 of 2 lM. Since hLF is a structured protein, we next investigated if the native conformation of the hLF protein was important for its inhibitory activity. The ATPase assay was performed in the presence of a heat-inactivated (94 °C, 5 min) hLF protein. Unlike

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the native hLF, no significant inhibitory effect against the NS3h ATPase activity was observed with concentrations up to 100 lM of unfolded hLF (Fig. 5C). Thus the hLF protein can inhibit the viral ATPase/Helicase but not the viral RNA polymerase.

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3.5. Cation dependency of the hLF NS3 ATPase inhibition

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Human Lactoferrin is an iron-binding protein. We next investigated if the hLF inhibitory activity was dependent on the cation status of the protein. The potency of hLF to inhibit NS3h was evaluated in depleted (1 mM DIP) or saturated (100 lM FeCl3) iron conditions. As shown in Fig. 6A the sequestration or the addition of Fe3+ did not influence the potency of hLF to inhibit the viral ATPase. Likewise, normal (1 mM) or high (20 mM) Mg2+ concentrations did not impact the hLF-mediated ATPase inhibition. In the absence of hLF, high Mg2+ concentrations created suboptimal ATPase conditions resulting in a slight reduction of the NS3h activity (Fig. 6A). The chelation of the NS3h Mg2+ cofactor resulted in a complete inhibition of the ATPase activity, independent of the presence or absence of hLF. Thus, hLF inhibition of NS3h is independent from its Fe3+ status and does not rely on the sequestration of the Mg2+ cofactor.

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The NS3h ATPase velocity was monitored as a function of ATP substrate concentration in the presence of various concentrations of hLF. As shown in Fig. 6B, the maximal ATPase velocity was

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Fig. 5. The hLF inhibits HCV NS3 ATPase activity. An aliquot of the purified HCV His-tagged NS3h (lane 2) and His-tagged NS5B (lane 3) proteins was analyzed by 12% SDS–PAGE and visualized by Coomassie staining. The sizes (in kDa) of the molecular weight markers (lane 1) are indicated to the left. (B) HCV NS3h (150 nM) was incubated with 100 lM ATP, 50 mM Tris–HCl, pH 7.5, 5 mM DTT and 1 mM MgCl2, in the presence or in the absence of 10 lM hLF. Released inorganic phosphate (Pi) product was quantified with Biomol Green Reagent. HCV NS5B (1 lM) was incubated in the presence of 10 lM of partially double-stranded RNA template, 1 mM rNTP, 20 mM Tris–HCl, pH 7.5, 1 mM DTT, 5 mM MgCl2, 20 U RNaseOUT and 1 lg/ml pyrophosphatase, in the presence or in the absence of 10 lM hLF. Pi hydrolyzed from pyrophosphate product was quantified with Biomol Green Reagent. Viral enzyme activity is presented relative to the untreated condition. (C) A 20 ll reaction containing 100 lM ATP, 50 mM Tris–HCl, pH 7.5, 5 mM DTT, 1 mM MgCl2 and increasing concentrations of either native hLF, heat-inactivated hLF (94 °C, 5 min) or human transferrin was incubated in the presence of 150 nM NS3h. Data represent the mean ± SD of two independent experiments. (⁄P < 0.05, ⁄⁄P < 0.01, ⁄⁄⁄P < 0.001).

protein was retained in the Ni–NTA Agarose resin only in the presence of His-tagged NS3h, demonstrating the interaction between both proteins (Fig. 7, lane 4). In the absence of His-tagged NS3h, hLF was not retained in the Ni–NTA Agarose resin (Fig. 7, lane 3). Since hLF could not be co-purified in the presence of His-tagged NS5B, the hLF–NS3 interaction appears to be specific (Supp. Fig. S4). These in vitro results indicate a direct physical interaction between hLF and NS3h proteins.

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reduced in the presence of varying concentrations of hLF while the KM behaved independently of inhibitor concentration as determined by a Lineweaver–Burk plot (Fig. 6C). Such enzymatic parameters are characteristic of a non-competitive mechanism of inhibition. The latter would suggest that hLF does not bind directly to the NS3h active site but rather to an allosteric binding site on the NS3h protein. Coherent with this allosteric mechanism of action, hLF was unable to inhibit the Paramecium bursaria Chlorella virus 1 ATPase/RNA triphosphatase A449R or the human ATPase/Helicase eIF4A1 (Supp. Fig. S3).

4. Discussion

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The inhibition of NS3h by hLF suggested a physical interaction between both proteins, which had yet to be demonstrated. Purified His-tagged NS3h and untagged hLF were both available in high concentrations, which allowed for the direct measurement of the interaction through Ni–NTA Agarose retention. Micromolar concentrations of both proteins were incubated together for 5 min at room temperature (Fig. 7, lane 1–2) and then applied to an equilibrated Ni–NTA Agarose resin. Unbound proteins were extensively washed with 18 volumes of wash buffer. The hexahistidine-bound proteins were recovered upon the addition of 2 volumes of imidazole containing elution buffer. The entire procedure was completed within 10 min in a continuous flow column. The eluate composition was resolved by SDS–PAGE and revealed by Coomassie staining as the protein concentration was sufficiently high. The hLF

The HCV epidemic remains a global threat, with 80% of newly infected patients developing a chronic infection, putting them at risk of cirrhosis, liver failure or hepatocellular carcinoma. Although chronic, the persistency of HCV infection is not due to latency, but rather to the dynamic equilibrium between the daily production and clearance of 1012 virions per infected individual (Neumann et al., 1998). In this context, the efficiency of HCV replication is primordial to the persistence of infection. Hence, direct-acting antiviral agents targeting HCV replication and maturation represent prime anti-HCV agents, as recently demonstrated by the regulatory approval of two NS3 N-terminus protease inhibitors (Boceprevir and Telaprevir). Our research on the human innate immune protein Lactoferrin presents a novel intracellular anti-HCV mechanism of action (distinct from extracellular virion neutralization), which could potentially be directly acting against the HCV essential protein NS3.

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Fig. 7. Direct physical NS3h–hLF interaction. The untagged hLF protein was incubated in the absence (lane 1) or in the presence of His-tagged NS3h (lane 2) and applied to Ni–NTA Agarose immobilized metal affinity chromatography resin. The resin was extensively washed and bound proteins were further eluted with the addition of 1000 mM imidazole. No peptides were detected in absence of Histagged NS3h (lane 3) and both hLF and His-tagged NS3h proteins were detected in presence of His-tagged NS3h (lane 4). The protein composition was resolved by SDS–PAGE and revealed by Coomassie staining.

Fig. 6. Characterization of the hLF inhibitory activity on HCV NS3. (A) HCV NS3h ATPase assays were performed in the absence (black column) or in the presence (white column) of 10 lM hLF under standard conditions [50 mM Tris–HCl, pH 7.5, 5 mM DTT, 1 mM MgCl2 and 100 lM ATP] and in the presence of either 1 mM DIP (iron chelator), 100 lM FeCl3, 50 mM EDTA or an additional 19 mM MgCl2 (20 mM final). (B) The NS3h ATPase velocity is presented as a function of ATP substrate concentration in the presence of various concentrations of hLF inhibitor. (C) Doublereciprocal representations (Lineweaver–Burk) of NS3h kinetics in the presence of various concentration of hLF. The y-intercept is equivalent to the V 1 max and the xintercept represents the K 1 m . Data represent the mean ± SD of two independent experiments. (⁄P < 0.05, ⁄⁄P < 0.01, ⁄⁄⁄P < 0.001). 489 490 491 492 493 494 495 496 497 498 499 500 501 502

Immunofluorescence of hepatic cells supporting an HCV subgenomic replicon revealed a qualitative reduction in the HCV staining intensity upon treatment with hLF. This is an interesting observation as this subgenomic replicon system is lacking HCV E1 and E2 envelope glycoproteins, which are the commonly accepted pharmacological targets of hLF. The reduction of viral replication was not due to hLF-induced cellular toxicity, which is coherent with the established Lactoferrin safety profile: 7.2 g body-1 day-1 being well tolerated and Lactoferrin is classified as Generally Recognized As Safe (GRAS) by the FDA (Okada et al., 2002). From these results arise the possibility that hLF might impair intracellular HCV replication. Unlike the extracellular virion neutralization activity, hLF would need to be internalized by HCV-infected hepatocytes in order to

impair viral replication. Such a possibility was evaluated by confocal microscopy. Upon pre-incubation on HCV-infected hepatocytes, exogenous hLF was successfully internalized, and shown to impair intracellular viral replication. This cellular uptake is coherent with clinical data revealing that hepatic biopsies from chronically infected HCV patients showed intracellular accumulation of hLF (Tuccari et al., 2002). A reduction in HCV replication could be visualized by immunofluorescence in cells treated with hLF. This result was further confirmed and quantified with the use of a firefly reporter HCV replicon and luciferase assays. Treatment with increasing concentrations of exogenous hLF, but not its related hTF counterpart, led to the reduction of HCV replication by up to 60% in an hLF-dependent manner (EC50 = 2 lM). Interestingly, the antiviral potency plateau observed upon hLF treatment seems to correlate with the drug’s uptake profile, which suggests that hLF efficacy could be limited by its internalization rate. The exact clinical transposition of this moderate in cellulo efficacy is not yet known, but the good potency together with the oral administration possibility and the safety profile of hLF offers reasonable hope for addition of hLF-based treatment to standard of care therapy. Combinations of moderate doses of hLF with known anti-HCV agents could lead to improved effects. In that sense, the hLF inhibitory potential was addressed in combination with RBV or IFN-a-2b, the two most widely used anti-HCV drugs. Both inhibitor regimens showed improved potency compared to the monotherapies. The hLF, as part of these combination therapies, displayed additive interactions with standard of care therapies (CI RBV = 1.03 and IFN-a-2b = 0.95). Furthermore, a tritherapy compose of low concentrations of RBV, IFN and hLF displayed higher in cellulo HCV inhibition compared to mono- or bi-therapy alone, which raises the possibility of adding hLF to the currently established RBV and IFN-a-2b therapies in order to reduce intracellular viral replication. Together, these results confirm the capability of hLF to impair in cellulo HCV replication and reveal an additive effect with the current HCV standard of care. The hLF intracellular activity suggests a novel mechanism of action for the hLF that is distinct from virion neutralization. Despite the previously describe RNAse activity for some isoforms, hLF does not affect intracellular RNA level in Huh-7 (Supp. Fig. S2), which raises the need to unveil a yet unknown mechanism of action for this immune protein (Furmanski et al., 1989). In this context, we investigated the attractive possibility of a direct acting mechanism of action to explain the hLF anti-HCV activity. The core components of the HCV replication complex are the RDRP NS5B protein and the ATPase/Helicase NS3. NS5B replicates the HCV


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RNA genome and synthesizes viral mRNAs while NS3 is believed to unwind RNA structures, dislocate RNA-bound proteins and/or separate HCV positive- and negative-RNA strands (Appel et al., 2006). The abolition of either of these enzymatic activities is detrimental to HCV, thus representing potent pharmacological targets (Lam and Frick, 2006; Lohmann et al., 1997). The potency of hLF to target these viral enzymes was addressed in vitro. While insensitive to hTF, NS3h ATPase activity was inhibited by hLF with an IC50 of 2 lM while NS5B polymerase activity remained inert to hLF. NS3h inhibition is independent of multivalent cation concentrations and is not due to ATP substrate sequestration. This result is supported by hLF inhibition kinetic parameters, which revealed no decrease in the relative ATP affinity of NS3h in the presence of hLF (KM is independent of hLF concentration). The maximal velocity of NS3h ATPase is, however, impaired in the presence of increasing concentrations of hLF. This kinetic profile is reminiscent of a non-competitive mechanism of inhibition and points toward an interaction between hLF and an allosteric site on NS3h. This finding is coherent with the large size of this protein inhibitor and is further supported by the specificity of hLF inhibition toward NS3 ATPase/Helicase but not against other ATPase/Helicase (eIF4A1), ATPase/RNA triphosphatase (A449R) or RDRP (NS5B). Furthermore, hLF directly interacts with NS3h as confirmed by co-affinity purification of untagged hLF in the presence His-tagged NS3h. The finding that hLF mediates NS3h inhibition through a direct and specific protein–protein interaction between hLF and the viral enzyme is coherent with the inability of the unstructured (heat-inactivated) hLF to inhibit NS3h ATPase activity. Together, these data suggest a suitable and novel mechanism of action in which hLF intracellular viral replication reduction could potentially be mediated through a direct HCV NS3 ATPase/Helicase inhibition. Over the years, multiple studies have revealed the antibiotic, antifungal and antiviral activities of hLF against a large spectrum of human pathogens. Such a wide diversity of targets was associated with an array of mechanisms of action including iron chelation and membrane permeabilization against bacteria and fungi (Ellison et al., 1988; Kirkpatrick et al., 1971; Kondori et al., 2011; Oram and Reiter, 1968; Viejo-Diaz et al., 2004; Yamauchi et al., 1993). Antiviral activity was promoted by direct virion neutralization and viral receptor inhibition (Andersen et al., 2004; Beljaars et al., 2004; Ikeda et al., 2000; Legrand et al., 2004; Yi et al., 1997). Against HCV, hLF is currently known to bind the viral envelope proteins (E1 and E2) and neutralize the circulating virions (Ikeda et al., 2000; Yi et al., 1997). The present study reveals a novel and complementary mechanism of action employed by hLF in order to restrict HCV infection: the inhibition of the viral genome replication. This demonstration was accomplished both in cellulo in an HCV replication based system and in vitro on a purified HCV protein. The antiviral activity of hLF was detected in two systems lacking E1 and E2 proteins, further supporting the evidence of a distinct mechanism of HCV inhibition (independent from virion neutralization). The relative importance of each mechanism of action to the global anti-HCV activity of hLF remains to be addressed. Nevertheless, numerous advantages could arise from this novel intracellular mode of inhibition. Through intracellular viral replication inhibition, hLF could maintain its antiviral activity even on persisting viruses evading neutralizing antibodies (and extracellular hLF) through cell-to-cell transmission (without cellfree particles) (Sattentau, 2008; Timpe et al., 2008). The intracellular anti-HCV activity of hLF would be coherent with the finding that viral hepatitis are associated with intra-hepatic hLF accumulation, which could represent a physiological response aimed to fight the virus (Tuccari et al., 2002). The understanding of this novel hLF antiviral activity constitutes a step forward in the comprehension of the global (likely

9

pleiotropic) mechanism of inhibition of this innate immunity protein. Clinical studies suggest a reduction in HCV RNA titer following oral hLF monotherapy and a higher sustained virological response (SVR) to RBV, IFN-a-2b and hLF combination therapy for hLF responder (Kaito et al., 2007). Together, these data raises questions about the role of hLF-mediated innate immunity in HCV spontaneous clearance and prognostic. Numerous studies have correlated various hLF single-nucleotide polymorphisms (SNPs) with increased susceptibility to viral and bacterial infection (Janssen et al., 2007; Keijser et al., 2008; Mohamed et al., 2007; Velliyagounder et al., 2003; Wu et al., 2009). It would be interesting to investigate if various hLF SNPs could play a role (similar to IL-28B SNP (Ge et al., 2009; Thomas et al., 2009)) in HCV spontaneous clearance and therapeutic prognostics. Orally-administrated hLF could be a safe addition to the standard of care for HCV treatment in developed countries. It would also be very interesting to investigate if the hLF-induced viral titer reduction could correlate with reduced morbidity and delayed mortality. In such a case, hLF monotherapy could benefit HCV positive individuals in developing countries. Hopefully, the new insights about the hLF antiviral mechanism of action unveiled here will stimulate further studies aimed at answering these questions.

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We thank Dr. Ralf Bartenschlager for the generous gift of the HCV replicon system. We also thank Dr. Charles Rice and Dr. Daniel Lamarre for kindly providing the hepatic cell line. We also want to thank Guillaume Tremblay for technical assistance as well as Dr. Martin Richter and Dr. Xavier Roucou for suggestion and comments on the manuscript. M.B. is a Chercheur Boursier Senior from the Fonds de Recherche en Santé du Québec and also a member of the Centre de Recherche Clinique Étienne-Lebel. This work was Q2 funded by both the Canadian Institutes of Health Research and Q3 Natural Sciences and the Engineering Research Council of Canada.

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Appendix A. Supplementary data

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Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.antiviral.2014.08. 012.

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Assay Development for Image-Based Quantification of Intracellular Bacterial Replication and Analysis of the Innate Immune Response to Infection.

Mechanism of inhibition of human cytomegalovirus replication by oxetanocin G.

S-palmitoylation represents a novel mechanism regulating the mitochondrial targeting of BAX and initiation of apoptosis.

The Novel Cyclophilin Inhibitor CPI-431-32 Concurrently Blocks HCV and HIV-1 Infections via a Similar Mechanism of Action.

The Adenovirus E4orf4 Protein Provides a Novel Mechanism for Inhibition of the DNA Damage Response.

Innate and Adaptive Immune Responses in Chronic HCV Infection.

Innate and adaptive immune responses in HCV infections.

Differential molecular mechanism of the estrogen action that regulates lactoferrin gene in human and mouse.

Unraveling the mechanisms of action of lactoferrin-derived antihypertensive peptides: ACE inhibition and beyond.

The innate immune roles of host factors TRIM5α and Cyclophilin A on HIV-1 replication.

A novel mechanism for the control of translation of specific mRNAs by tumor suppressor protein Pdcd4: inhibition of translation elongation.

Inhibition of influenza virus replication in tissue culture by 2-deoxy-2,3-dehydro-N-trifluoroacetylneuraminic acid (FANA): mechanism of action.

The circumsporozoite protein of Plasmodium: a mechanism of immune evasion by the malaria parasite?

Suppression of autophagy by mycophenolic acid contributes to inhibition of HCV replication in human hepatoma cells.

Ubiquitination of Innate Immune Regulator TRAF3 Orchestrates Expulsion of Intracellular Bacteria by Exocyst Complex.

Outrunning the Red Queen: bystander activation as a means of outpacing innate immune subversion by intracellular pathogens.

The mechanism of action of novel antipsychotic drugs.

Control of the innate immune response by the mevalonate pathway.