apolipoprotein e mediates evasion from hepatitis c virus … · 2018-04-05 · immune system lags...

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Gastroenterology 2015;-:1–13

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Apolipoprotein E Mediates Evasion From HepatitisC VirusLNeutralizing Antibodies

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Catherine Fauvelle,1,2,* Daniel J. Felmlee,1,2,3,* Emilie Crouchet,1,2 JiYoung Lee,4

Laura Heydmann,1,2 Mathieu Lefèvre,1,2 Andrea Magri,5 Marie-Sophie Hiet,4 Isabel Fofana,1,2

François Habersetzer,1,2,6 Steven K. H. Foung,7 Ross Milne,8 Arvind H. Patel,5

Koen Vercauteren,9 Philip Meuleman,9 Mirjam B. Zeisel,1,2 Ralf Bartenschlager,4

Catherine Schuster,1,2 and Thomas F. Baumert1,2,6

1Inserm, U1110, Institut de Recherche sur les Maladies Virales et Hépatiques, Strasbourg, France; 2Université de Strasbourg,Strasbourg, France; 3University of Plymouth, Centre for Biomedical Research, Plymouth, UK; 4Department of InfectiousDiseases, Molecular Virology, Heidelberg University, Heidelberg, Germany; 5MRC, University of Glasgow, Centre for VirusResearch, Glasgow, UK; 6Institut Hospitalo-Universitaire, Pôle Hépato-digestif, Hôpitaux Universitaires de Strasbourg,Strasbourg, France; 7Department of Pathology, Stanford University School of Medicine, Stanford, California;8Department of Pathology and Laboratory Medicine, University of Ottawa Heart Institute, Ottawa, Ontario, Canada; and9Center for Vaccinology, Ghent University, Ghent University Hospital, Ghent, Belgium

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BACKGROUND & AIMS: Efforts to develop an effective vaccineagainst hepatitis C virus (HCV) have been hindered by the pro-pensity of the virus to evade host immune responses. HCV parti-cles in serumand in cell culture associatewith lipoproteins, whichcontribute to viral entry. Lipoprotein association has also beenproposed to mediate viral evasion of the humoral immuneresponse, though themechanisms are poorly defined.METHODS:Weused small interfering RNAs to reduce levels of apolipoproteinE (apoE) in cell culture�derivedHCV�producingHuh7.5-derivedhepatoma cells and confirmed its depletion by immunoblot ana-lyses of purified viral particles. Before infection of naïvehepatomacells, we exposed cell culture�derived HCV strains of differentgenotypes, subtypes, and variants to serum and polyclonal andmonoclonal antibodies isolated from patients with chronic HCVinfection.We analyzed the interaction of apoEwith viral envelopeglycoprotein 2 and HCV virions by immunoprecipitation.RESULTS: Through loss-of-function studies on patient-derivedHCV variants of several genotypes and subtypes, we found thatthe HCV particle apoE allows the virus to avoid neutralization bypatient-derived antibodies. Functional studies with humanmonoclonal antiviral antibodies showed that conformationalepitopes of envelope glycoprotein 2 domains B and C wereexposed after depletion of apoE. The level and conformation ofvirion-associated apoE affected the ability of the virus to escapeneutralization by antibodies. CONCLUSIONS: In cell-infectionstudies, we found that HCV-associated apoE helps the virusavoid neutralization by antibodies against HCV isolated fromchronically infected patients. This method of immune evasionposes a challenge for the development of HCV vaccines.

*Authors share co-first authorship.

Abbreviations used in this paper: apo, apolipoprotein; E2, envelopeglycoprotein 2; HCV, hepatitis C virus; HCVcc, cell cultureLderived HCV;Jc1E2(FLAG), J6-JFH1 chimera with FLAG epitope in E2; JFH1, Japanesefulminant hepatitis virus; LDL, low-density lipoprotein; Luc-Jc1, J6-JFH1chimera with luciferase reporter; mAb, monoclonal antibody; mRNA,

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Keywords: Lipoviral Particle; Vaccination; Viral Escape; Lipid.

epatitis C virus (HCV) is a major health problem

messenger RNA; nAb, neutralizing antibody; PCR, polymerase chain re-action; siRNA, silencing RNA; TRL, triglyceride-rich lipoprotein Q5.

© 2015 by the AGA Institute0016-5085/$36.00

http://dx.doi.org/10.1053/j.gastro.2015.09.014

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Hinfecting approximately 130 million individualsworldwide. HCV infection typically results in a chronicinfection that can lead to liver cirrhosis and hepatocellularcarcinoma.1 Direct-acting antivirals have markedlyimproved the treatment efficacy, but limitations due to

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access to screening and therapy persist, highlighting theneed for an effective vaccine for global control and eradi-cation of HCV infection. A consistent hallmark of vaccinesagainst pathogens is their reliance on immunogens thatelicit neutralizing antibodies (nAbs).2 HCV vaccine devel-opment has been impeded by the viral adaptations to hostimmunity that enable chronic infection. Indeed, the hostimmune system lags behind the continuous evolution ofHCV, allowing the virus to evade humoral immunity.3,4

However, the escape mechanisms from nAbs duringchronic HCV infection are only partially understood. Clearly,the development of an effective vaccine requires a detailedunderstanding of viral evasion from host immuneresponses, including nAbs. Previous studies investigatingthe molecular mechanisms of HCV liver graft infection,identified a viral variant termed VL5,6 with efficient escapefrom patient nAbs.5,6 Functional genetics had identifiedphenylalanine at HCV polyprotein residue 447 as beingimportant for neutralization escape.6 This amino acid iswidely conserved among HCV isolates, as shown by itsprevalence of 98.4% in all genotypes (including Jc1, Japa-nese fulminant hepatitis virus [JFH1], and H77) and 96.2%in genotype 1b strains (including VL).6 In addition, previousstudies had shown that replacement of phenylalanine byleucine (F447L) rendered HCV highly susceptible toneutralization.6

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2 Fauvelle et al Gastroenterology Vol. -, No. -

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An interesting characteristic of HCV particles is theirassociation with triglyceride-rich lipoproteins (TRL) andlow-density lipoproteins (LDL) forming hybrid lipoviralparticles, which results in a population of virions that areheterogenous in buoyant density.7–9 Apolipoprotein (apo) B,E, AI, and CI have been described as lipoviral particlescomponents.10 ApoE is important for both HCV infection ofhepatocytes and hepatic uptake of TRL remnants, while therole of apoB, the structural protein of TRL and LDL, in theHCV life cycle is less clear. ApoCI may be involved in viralfusion,11 and apoAI can affect HCV replication and produc-tion. Although association with TRL and LDL has been hy-pothesized to contribute to viral evasion,7,12 the role ofspecific apolipoproteins in HCV persistence is unknown.Given that apoE is a host protein incorporated into the HCVparticle and is required for virion production, we sought to


determine its functional role in viral evasion from hostnAbs. Our findings reveal a previously undiscovered mech-anism of viral escape specific to apoE, independent of TRLbinding, and identify a residue in envelope glycoprotein 2(E2) that contributes to this phenotype. These results definea novel challenge for the development of vaccines andimmunopreventive approaches.

Materials and MethodsPatient Samples

Serum samples from patients with chronic HCV infectionwere obtained with informed consent and approval from theStrasbourg University Hospital’s Institutional Review Board(CPP 10-17). Sera termed 1, 2, and 4 came from patients

Figure 1. Depletionof apoEin HCV producer cells effi-ciently sensitizes HCV vi-rions to antibody-mediatedneutralization. Huh7.5.1cells were either electro-porated with VL:JFH1 (A),H77R2a (B) or Luc-Jc1RNA(C) alone, or co-electroporated with eitherscrambled siRNA (siCtrl) orsiRNA targeting apoEmRNA (siApoE). Neutrali-zation experiments usingviruses produced fromthese cells were performedusing sera from 4 differentHCV-infected patients (1, 2,3, or 4) at the indicated di-lutions. Infection wasdetermined by end-pointdilution assay (A) or lucif-erase activity (B,C).Mean±SEM from7experiments (A)or 3 experiments performedin duplicate (B, C) areshown. Results areexpressed as fold increaseof neutralization relative tocontrol serum. ApoEdepletion in producer cellswas confirmed 72 hourspost electroporation byimmunoblotting of cell ly-sates; actin was detectedas loading control. *P< .05. Q19

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Figure 2. (A) Trans-complementation of apoE expression in producer cells restores viral evasion from nAbs. Huh7.5.1 cells wereelectroporatedwith Luc-Jc1RNAalone, or co-electroporatedwith either scrambled siRNA (siCtrl) or siRNA targeting apoEmRNA(siApoE). ApoE expression was then rescued using adenoviral apoE expression vectors transducing apoE knockdown cells.Neutralization experiments of viruses were performed using HCV-infected patient serum 1 at dilution 1/200. Infection wasdetermined by quantification of luciferase activity. Results are expressed as fold increase of neutralization relative to controlserum. ApoE knockdown was confirmed 72 hours post electroporation by immunoblotting (lower panel); actin was detected asloading control.Mean±SEM from3experiments are shown. *P< .01. (B,C) ApoE-depletion results in enhanced neutralization bypurified IgG frompatient sera and humanmonoclonal antibodies. Huh7.5.1 cells were electroporatedwith Luc-Jc1RNA alone, orco-electroporated with either scrambled siRNA (siCtrl) or siRNA targeting apoE mRNA (siApoE). Viruses produced from thesecells were incubated with (B) purified IgG (25 mg/mL) from HCV-infected patient serum 2 and 3 or (C) human monoclonal anti-E2antibodies CBH-23 andHC-1 (10 mg/mL). Infection was determined as described in (A). Results are expressed as fold increase ofneutralization relative to control IgG. Mean ± SEM from 3 experiments are shown. *P < .01.

- 2015 ApoE protects HCV From nAbs 3

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infected by HCV genotype 1 and serum 3 from a genotype 2patient.

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Cells and ReagentsHuh7.5.1 and Huh7.5 green fluorescent protein cells were

cultured as described previously.13 Silencing RNAs (siRNAs)targeting apoE 30 untranslated region (siApoE)(50CUGCAGCGGGAGACCCUGU 30) or apoB and control siRNAswere from Dharmacon (Lafayette, CO). Mouse anti-apoE(ab8226) monoclonal antibody (mAb) and anti�b-actin(ab1906) for immunoblot were obtained from Abcam (Cam-bridge, MA). Mouse anti-apoE (1D7, 3H1, 6C5), rat anti-CD81(QV-6A8-F2-C4), mouse anti-E2 (AP33),14–16 and human


anti-E2 (CBH-23 and HC-1)17 mAbs have been described. Sheepanti-NS5A serum was a kind gift from M. Harris.18 Humananti-E2 antibody AR3B was a kind gift from M. Law.19 Anti-bodies and peptides for FLAG purification and detection wereobtained from Sigma-Aldrich (St Louis, MO). Purification of IgGfrom patient serum was performed using MAbTrap kit fromAmersham (Little Chalfont, UK).

Cell Culture�Derived Hepatitis C VirusProduction, Infection, and Neutralization

Plasmids for cell culture�derived HCV (HCVcc) productionof Jc1, J6-JFH1 chimera with luciferase reporter (Luc-Jc1),Jc1E2FLAG (all genotype 2a/2a), H77R2a (genotype 1a/2a),

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Figure 3. Silencing ofapoE expression in HCVproducer cells alters apoEcontent in immunopurifiedvirions. Huh7.5.1 cellswere electroporated withJc1E2(FLAG) orVL:JFH1E2(FLAG) RNAalone, or co-electroporated with eitherscrambled siRNA (siCtrl) orsiRNA targeting apoEmRNA (siApoE) and FLAG-tagged viruses wereimmunopurified using ananti-FLAG antibody. (A, C)Purified FLAG-tagged vi-rions were subjected toimmunoblot using FLAG-and apoE-specific anti-bodies (upper panels).ApoE knockdown in HCVproducer cells wasconfirmed 72 hours post-electroporation by immu-noblotting; actin wasdetected as loading con-trol (lower panels). (B, D)relative quantities of apoE,E2(FLAG)-tagged virionand actin were determinedas described in Materialsand Methods. Results areexpressed as percentageof apoE:E2 ratio relative tocontrol virus. Mean ± SEMfrom 3 experiments areshown.


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VL:JFH1, and VL:JFH1FLAG (genotype 1b/2a) have beendescribed previously.6,9,20–22 HCVcc were produced inHuh7.5.1 as described previously.20 Infectivity was quantifiedby luciferase activity or tissue culture infectious dose 50%using anti-NS5A antibody.23 HCVcc neutralization using pa-tient serum, IgG, and mAbs was analyzed as describedpreviously.6


Silencing of Apolipoproteins in HepatitisC Virus Producer Cells

Silencing of apoE and apoB expression and immunoblottingin Huh7.5.1 cells were performed as described previously.24

Huh7.5.1 cells were either electroporated with HCV RNA co-electroporated with 200 pmol of either scrambled siRNA

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Figure 4. Depletion of apoB in HCV producer cells has no effect on HCV neutralization. Huh7.5.1 cells were either electroporatedwith VL:JFH1 or Luc-Jc1 RNA alone, or co-electroporated with either scrambled siRNA (siCtrl) or siRNA targeting apoB mRNA(siApoB). Neutralization experiments on viruses produced from these cells were performed using patient serum 1 at the indicateddilutions. Infection was determined by (A) end-point dilution assay, and (B) quantification of luciferase activity. Results areexpressed as fold increase of neutralization relative to control serum. ApoB knockdown was confirmed 72 hours post electropo-ration by immunoblotting (lower panel); actin was detected as loading control. Means ± SEM from 3 experiments are shown.


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(siCtrl) or siRNA targeting apoE messenger RNA (mRNA)(siApoE) or apoB mRNA (siApoB) according to the manufac-turer’s instructions. Supernatants and cells were harvested 72hours post electroporation. Protein expression was analyzed byimmunoblotting as described previously.24

Recombinant Adenoviruses and Rescueof Gene Silencing

The recombinant adenoviral genomes were generated asdescribed previously.25 Recombinant adenoviruses Ad-apoEwere generated by transfection of these plasmids into the293T packaging cell line after PacI digestion.25 Huh7.5.1 cellswere electroporated with HCV RNA and siApoE or a scramblesiRNA (siCtrl). Twenty-four hours post electroporation, cellswere transduced with adenoviruses expressing apoE. After 72hours, viruses were harvested and tested in neutralization ex-periments and both silencing and rescue of protein expressionwas confirmed by immunoblotting.

Generation and Purification of E2-FLAGTagged Viruses

The VL:JFH1 derivatives encoding a E2(FLAG) fusionprotein were generated using overlap polymerase chainreaction (PCR) with 2 sets of primers: S_E1_AgeI_Con1(50ATAGTGGTCTGCGGAACCGGT30) and A_E1_FLAG (50CCCTTGTCATCGTCGTCCTTGTAGTCCCCGTCAACGCCGGCAAAA30); S_E1_FLAG (50GGACGACGATGACAAGGGATCAGGAGCATCCACCTACA


CGACGGGGGG30); and A_E2_AleI (50TGTATGGATAGTCAACCAT30).Amplicons were amplified by PCR from VL:JFH1 derivates(VL:JFH1 or F447L or F447A), then combined by overlap PCRusing S_E1_AgeI_Con1 and A_E2_AleI before insertion of theresulting fragments into the VL:JFH1 construct. E2(FLAG)encoding HCV viruses were purified using anti-FLAG M2affinity gel, as described previously.9 Virion-associated apoEwas detected by immunoblotting using mouse anti-apoE mAb(ab8226; Abcam), while virion E2(FLAG) was detected byanti-FLAG M2 mAb (Sigma-Aldrich). Quantification of apoE,E2(FLAG), and actin protein expression was analyzedusing ImageJ software (National Institutes of Health, Bethesda,MD).

Quantification of Hepatitis C Virus ParticleBuoyant Density Distribution

VL:JFH1 HCVcc virus was concentrated 10-fold using aVivaspin column (GE Healthcare, Little Chalfont, UK). Densitydistributions of infectious HCVcc were determined by over-laying 0.5 mL culture media on a 5-mL, 4%�40% iodixanolstep gradient, and ultracentrifuging samples for 16 hours at40,000 rpm on a SW-55 rotor (Beckman Coulter, Brea, CA). Sixhundred and twenty-five microliter fractions were carefullyharvested from the top of each tube, and density was deter-mined by weighing 0.5 mL of each fraction. Infectivity of eachfraction was quantified by luciferase activity or by tissue cul-ture infectious dose 50%.

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Figure 5. ApoE depletion in HCV producer cells does not modulate the buoyant density profile of virions. Huh7.5.1 cells wereeither electroporated with VL:JFH1 RNA or Luc-Jc1 alone (black squares and black circles, respectively), or co-electroporatedwith either scrambled siRNA (siCtrl) (dark gray squares and dark gray circles) or siRNA targeting apoE mRNA (siApoE) (light graysquares and light gray circles). HCVcc produced from these cells were fractionated using 4%�40% iodixanol density gradientultracentrifugation. Each fraction was assayed for infectivity by (A) end-point dilution assay (log10 tissue culture infectious dose50%/mL) for VL:JFH1 or (B) quantification of luciferase activity (log10 RLU) for Luc-Jc1. Mean ± SEM from 3 experiments areshown. The light gray circles on the density plot show the mean density for each fraction and the error bars indicate the SD ofthe density of the respective fraction from 6 independent experiments. ApoE knockdown was confirmed 72 hours post-electroporation by immunoblotting (lower panels); actin was detected as loading control.


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Hepatitis C Virus Cell-to-Cell TransmissionHCV cell-to-cell transmission was assessed as described

previously.13 Producer Huh7.5.1 cells were electroporated withJc1 RNA or VL:JFH1 RNA. Medium was exchanged 4 hours andagain 24 hours post electroporation, and naïve target Huh7.5green fluorescent protein cells were added concomitantly withmouse nAb AP33 (10 mg/mL) to block cell-free transmission.For analysis of the role of virus and host cell factors, cells wereincubated with 10 mg/mL of either control IgG, anti-CD81 (QV-6A8-F2-C4),14 or anti-apoE (1D7)16 antibodies. Seventy-twohours post electroporation, cells were fixed with para-formaldehyde, stained with a human anti-E2 (AR3B)19 antibodyand analyzed via flow cytometry.

Immunoprecipitation, RNA Extraction, andReverse Transcription Quantitative PolymeraseChain Reaction

VL:JFH1 HCVcc virus was concentrated 10-fold from cellsupernatants using a Vivaspin column (GE Healthcare) andimmunoprecipitations were performed using Protein A/GPLUS-agarose beads according to the manufacturer instructions(Santa Cruz Biotechnology, Santa Cruz, CA). HCV RNA fromimmunoprecipitated viruses was isolated using RNeasy


extraction (Qiagen, Valencia, CA) and quantified using reversetranscription quantitative PCR as described previously.13,24

E2-apoE co-immunoprecipitation using lysates of Huh7/LunetCD81H cells stably expressing ApoEWT or ApoEHA andtransfected with VL:JFH1 or the 447 mutant were performed asdescribed elsewhere.26 Immunoprecipitation of apoE fromHuh7.5.1 cells was performed as described previously.26

StatisticsDatasets were analyzed using the Mann�Whitney test.

ResultsBecause apoE has been shown to be part of the HCV in-

fectious virion mediating viral attachment, we investigatedwhether particle-associated apoE influences viral evasionfrom host nAbs. To experimentally probe this question, weused3differentHCV strains comprising genotypes 1a, 1b, and2a. We first studied HCV strain JFH1 carrying the structuralgenes of a patient-derived viral variant termed VL. We hadpreviously shown that this genotype 1b variant is selectedduring liver graft infection due to envelope-mediatedenhanced viral entry and broad escape from patient-derived

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Figure 6. ApoE and cell-to-cell transmission of HCV. (A) Interference of cell-to-cell transmission of Jc1 and VL:JFH1 by anti-apoE antibody 1D7, nonspecific IgG as a negative control (Ctrl), or anti-CD81 antibody as a positive control was tested. Forcell-to-cell transmission, green fluorescent protein�expressing naïve target cells were co-cultured with cells containing HCVwith neutralizing mouse anti-E2 antibody AP33. Representative results of fluorescence-activated cell sorting analysis areshown. The quantity of positively stained cells for HCV E2 (y-axis), and green fluorescent protein�labeled target cells (x-axis)are represented. (B) Histograms of cell-to-cell transmission for Jc1 and VL:JFH1 summarized from 3 independent experimentsperformed in duplicate are shown. Values represent percentage of cell-to-cell transmission relative to control. (C) The capacityof antibodies to inhibit infection was tested by inoculation of naïve Huh7.5.1 cells with HCV in the presence of antibodiesspecified below the plot. Values were normalized to those obtained with nonspecific IgG (Ctrl). Mean ± SD from 3 experimentsare shown. *P < .05.

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autologous and heterologous nAbs.5,6 Thus, it represents aprototype variant that may be largely refractory to immu-nopreventive approaches or vaccines. We first generatedVL:JFH1 HCVcc by cotransfecting HCV RNA alone, with ascrambled nucleotide control (siCtrl), or with siRNA thattargets apoE expression (siApoE). Similar to the range re-ported previously,24,27,28 silencing of apoE expression inHuh7.5.1 producer cells resulted in a marked (50%�70%)


and significant decrease (P < .001) in infectivity of virusesrelative to viruses with unchanged apoE expression. Expo-sure of VL:JFH1 HCVcc produced from apoE-depleted cells tomultiple patient sera with chronic HCV infection modifiedthese HCVcc to be highly sensitive to nAbs (Figure 1A).Depending on the patient serumand thedilutionused, virusesproduced from apoE-depleted cells were 4–17 times moresensitive to neutralization by patient sera than viruses from

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control cells with unchanged apoE expression (Figure 1A). Tocorroborate these findings, we studied the phenotype of 2additional variants, H77R2a21 expressing the structuralgenes of the HCV strain H77 (genotype 1a) and J6:JFH1


prototype Luc-Jc129 expressing the structural genes of thegenotype 2 HCV strain J6. Similar to findings obtained forVL:JFH1 viruses, apoE depletion resulted in enhanced sensi-tivity to neutralization by patient sera (Figure 1B and C).

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H77R2a and Luc-Jc1 viruses produced from apoE-depletedcells were 2 to 10 times and 1.5 to 2 times more sensitive toneutralization than control viruses, respectively (Figure 1Band C). Luc-Jc1 derived from apoE-deficient cells were moresensitive to nAbs regardless of their viral infectivity titers(Supplementary Figure 1). In addition, similar neutralizationresults were obtained upon adjustment of input virus titersbetween viruses produced from control and apoE-depletedcells (Supplementary Figure 2), ruling out that alteredsensitivity was due to a difference in molar ratios betweennAbs and HCVcc.

To further explore the role of apoE in viral evasion, wenext performed trans-complementation assays usingadenoviral apoE expression vectors transducing apoEknockdown cells. Ectopic apoE expression restored the HCVnAb escape to the same level as HCV produced in controlcells (Figure 2A), excluding off-target effects caused by thesiRNA. In addition, to determine that this observation wasantibody mediated, we confirmed our results using IgGpurified from sera of patients with chronic HCV infection(Figure 2B). Similar to the experiments with patient sera, weobserved that Luc-Jc1 viruses produced from apoE-depletedcells were 2 times more sensitive to neutralization by pu-rified IgG than control viruses. These findings specificallypoint to serum IgG and exclude other serum factors indetermining the sensitivity of HCV from apoE-silenced cells(Figure 2B). In addition, we used 2 human mAbs targetingHCV E2 domain B (HC-1) or domain C (CBH-23) and testedtheir efficacy to neutralize viruses produced from controland apoE-silenced cells (Figure 2C). Consistent with theresults obtained with patient sera and anti-HCV polyclonalIgG, we found that apoE silencing markedly (3- to 4-fold)and significantly (P < .01) increased sensitivity to neutral-ization compared with controls. These results clearly indi-cate that apoE plays a key role in mediating viral escapefrom nAbs and suggest that conformational epitopes of E2domain B and domain C are exposed to nAbs after apoEdepletion (Figure 2C).

To confirm the hypothesis that apoE modulation affectedvirion composition, we utilized J6-JFH1 chimera with FLAG

=Figure 7. Envelope glycoprotein E2 residue 447 and apoE-HCVinfection by anti-apoE antibodies. VL:JFH1 and 447 variant HCVto infection of Huh7.5.1 cells. Infectivity was assessed by tispercentage infectivity relative to Ctrl. Mean ± SEM from 4 eximmunoprecipitation of virions by anti-apoE antibodies. VL:JFHnonspecific mouse IgG (Ctrl), or 3 different anti-apoE antibodiesand quantified by reverse transcription quantitative polymerasecrease of HCV RNA immunoprecipitated relative to control IgG.was quantified by immunoblot evaluating the amount of core prHCV producer cells impairs immunoprecipitation of virions by anproducer cells using control IgG or anti-apoE antibody (1D7), HCnormalized HCV RNA. Mean ± SEM from three experiments areHuh7-derived cells stably expressing ApoEWT or ApoEHA weremutant and lysed 72 hours later. Immunoprecipitation was percomplexes were analyzed by immunoblotting using anti-E2 anlysates used for immunoprecipiation were analyzed in parallel (inE2 associations in purified virions of wild-type and mutant viruproducer cells using an anti-FLAG antibody. Virion-associated pspecific antibodies. Results of 2 independent purifications and


epitope in E2 (Jc1E2[FLAG])9 and VL:JFH1E2(FLAG) HCVccpurified from nonassociated serum component contami-nants. Immune-purification of Jc1E2(FLAG) andVL:JFH1E2(FLAG) using an antibody targeting the FLAG-tagof the virion E2 revealed that silencing of cellular apoEresulted in an approximate 50% and 80% decrease ofvirion-E2 associated apoE for Jc1E2(FLAG) andVL:JFH1E2(FLAG) compared with controls, respectively(Figure 3). Intracellular levels of apoE were decreased by75% and 85%, respectively (Figure 3). Furthermore, virionsproduced from apoE-depleted cells appeared to have adecreased inhibition of infection by anti-apoE antibodies(Supplementary Figure 3). These findings highlight virion-associated apoE’s critical role in mediating escape fromnAbs.

To test if apoB, another component of HCV, plays a rolesimilar to apoE’s in mediating escape, we silenced apoBexpression in HCV-producing cells. Silencing of eitherapolipoprotein had no detectable effect on the level of theother apoliprotein (Supplementary Figure 4) ensuringabsence of the interfering effects of apoE/B silencing.Interestingly, while apoB knockdown was effective(Figure 4A and B), it did not alter the production of VL:JFH1and Luc-Jc1. Furthermore, neither VL:JFH1 nor Luc-Jc1viruses produced from apoB-silenced cells were altered insensitivity to nAbs (Figure 4A and B). These results pointdirectly to apoE as a key apolipoprotein mediating nAbescape and do not support a major functional role of apoB innAb evasion.

ApoE expression is an important regulator of HCV pro-duction, and a key component of TRLs. TRL association,measured by buoyant density distribution, could shield HCVglycoproteins from nAbs.12 Two distinct models have beenproposed regarding HCV�apoE interaction and lipoviralparticles formation: a 1-particle model presenting the virioninextricably fused to the lipoprotein with apoE on its sur-face, and a 2-particle model where HCV glycoproteinsinteract with apoE directly, which may mediate lipoproteinattachment.30 In addition, the envelope glycoprotein, andthe transmembrane domain of E2 specifically is critical for

interactions. (A) Residue 447 is relevant for inhibition of HCVcc were incubated with control IgG (Ctrl) or 1D7 antibody priorsue culture infectious dose 50%. Results are expressed asperiments are shown. *P < .05. (B) Residue 447 modulates1 and 447 mutants HCVcc were immunoprecipitated using(1D7, 3H1, 6C5). HCV RNA was extracted from precipitateschain reaction (RT-qPCR). Results are expressed as fold in-Means ± SEMs from 2 experiments are shown. The viral inputotein in the culture media (lower panel). (C) ApoE depletion inti-apoE antibody. After immunoprecipitation of virions in HCVV RNA was quantified by RT-qPCR. Results are expressed asshown. *P < .01. (D) Intracellular interaction of apoE and E2.electroporated with RNA genomes of VL:JFH1 or the F447Lformed with a hemagglutinin Q20-specific antibody and capturedtibodies. To determine capture efficiency, 0.5% of total cellput). One representative immunoblot is shown. (E) ApoE andses. Virions were immunopurified from supernatants of HCVroteins were analyzed by immunoblots using FLAG and apoE-immunoblots are shown.

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apoE association.26,31 If apoE protects HCV glycoproteinsfrom nAbs directly, then our observations regarding thenAb-sensitivity of HCV generated from apoE knockdowncells will not be mediated by a marked difference in buoyantdensity distribution. To test this hypothesis, we analyzed thebuoyant density distribution of infectious particles usingisopycnic density gradient ultracentrifugation. Interestingly,the density distribution of VL:JFH1 and Luc-Jc1 was unal-tered after apoE knockdown, though infectivity was dimin-ished approximately equally in each density fraction in bothVL:JFH1 and Luc-Jc1 viruses (Figure 5A and B). These datasuggest that apoE knockdown does not modulate HCV-TRLassociation, and apoE directly associated with the virionaccounts for nAb evasion.

Cell-to-cell transmission is another mechanism to avoidnAbs.32 To investigate whether apoE association is relevantin cell-to-cell transmission, we used an anti-apoE antibody(1D7) that binds the low-density lipoprotein recep-tor�binding domain of apoE16 and inhibits HCV infection(Figure 7A). Jc1 demonstrated an approximately 3-foldhigher capacity to spread by direct cell-to-cell trans-mission than VL:JFH1 in this assay (Figure 6A). While 1D7did not affect cell-to-cell spread, despite its capacity tostrongly inhibit extracellular transmission, antibodiesrecognizing HCV entry factor CD8114 inhibited 70%�80%of cell-to-cell transmission of both Jc1 and VL:JFH1(Figure 6A, B, and C). These data indicate that cell-celltransmission is not mediated by apoE domains targeted byanti-apoE antibody 1D7.

Recently, 2 studies demonstrated that apoE interactswith HCV E2.26,31 To define E2 domains relevant for asso-ciation with apoE and neutralization escape, we tookadvantage of a mutation identified in variant A (VA), a pre-transplant variant that was selected against in the sametransplant recipient that contained the predominant VLvariant.6 Indeed, replacing phenylalanine with the leucineencoded in VA rendered the virus both sensitive toneutralization and less dependent on CD81 for cell entry.6

To determine whether this residue is involved in the dif-ferential utilization of apoE, we used the previouslydescribed mutant restoring neutralization escape (F447L),as well as an additional mutant where phenylalanine isreplaced by alanine (F447A), a smaller amino acid residuethan leucine. Interestingly, the wild-type VL:JFH1 escapevariant was efficiently neutralized by anti-apoE 1D7, whilethe 447L and 447A mutants were at least 3-fold less sen-sitive to neutralization by this antibody, indicating that thewild-type virus is more dependent on apoE for infection(Figure 7A). In contrast, 2 other apoE-specific antibodies(3H1 and 6C5) that bind the N- and C-terminal regions ofapoE, respectively,16 did not inhibit HCV infection to thesame degree (data not shown) despite their capacity toimmunoprecipitate apoE from cell lysates (SupplementaryFigure 5). To determine if this modulation might be due toaltered apoE association with virus particles, we immuno-precipitated VL:JFH1 with either nonspecific mouse IgG orwith each of the three apoE-specific antibodies 1D7, 3H1, or6C5.16 Interestingly, all 3 anti-apoE antibodies were at least2.5-fold more efficient at precipitating the VL:JFH1 than the


447 mutants (Figure 7B), suggesting a more robust associ-ation of the escape variant with apoE or a different E2-apoEconformation with different exposure of these epitopes onthis variant. The functional relevance of apoE for immuno-precipitation of virions was confirmed by less efficientimmunoprecipitation of HCV RNA by anti-apoE usingVL:JFH1 and Jc1 viruses produced in apoE-depleted cells(Figure 7C and data not shown).

To investigate whether the difference in sensitivity andpull-down of VL:JFH1 447 mutants by anti-apoE antibodieswas due to an altered association of apoE and virion E2 ormediated by a different conformation of the apoE-E2 com-plex, we performed co-immunoprecipitation analyses in celllysates of HCV producer cells. We observed similar intra-cellular apoE-E2 binding in wild-type and mutant viruses(Figure 7D). Next, we determined the amount of apoEincorporated into virions by using immunopurified E2FLAG-tagged viruses. We found that FLAG-tagged wild-type andF447L mutant viruses contained similar levels of apoE(Figure 7E). These data indicate that residue 447 alters theconformation of the virion E2-apoE complex withoutchanging apoE content of the particles. Collectively, our dataindicate that both the level (Figures 1–3), as well as theconformation (Figure 7) of virion apoE are relevant for theevasion from nAbs.

DiscussionHere we present experimental evidence indicating that

apoE levels in HCV-producing cells determine HCV’s ca-pacity to avoid the effect of nAbs through modifying incor-poration of apoE into the viral particle. These results wereobtained using structural genes from 3 different viral strainsfrom genotypes 1a, 1b, and 2a, sera from multiple patientsas well as patient-derived purified IgG and mAbs, indicatingthat utilization of the host factor apoE as a mechanism toescape from nAbs is pan-genotypic. While the utilization ofapoE is consistently employed, it was most prominentlyobserved in both genotype 1 variants H77 and VL, a clini-cally derived variant previously characterized as effective atnAb escape, highlighting that apoE “shielding” may beemployed by the most difficult to neutralize variants. Thesedifferences of neutralization profiles between H77 orVL:JFH1 and Jc1 may be partially explained by a more effi-cient depletion of apoE in VL:JFH1 compared to Jc1 asshown in Figure 3. Alternatively, these results may point togenotype- or isolate-dependent differences in utilization ofapoE and neutralization escape. Finally, the differences maybe due to different cross-reactivity of the patient sera used.

We further confirmed the role of apoE throughobserving altered apoE:E2 ratios on purified virions aftersilencing (Figure 3) and trans-complementing apoEexpression after knockdown, which rescued HCV’s capacityto avoid inhibition of HCV entry by nAbs present in serum ofchronically infected patients (Figure 2A). However, down-regulation of apoE expression did not affect HCV-TRL as-sociation (Figure 5), indicating that the apoE-mediatedmechanism of nAb escape is distinct from the previouslydescribed role of TRL in escape.12

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Using an anti-apoE antibody that blocks extracellulartransmission, we did not detect a role of apoE in cell-to-celltransmission of Jc1, VL:JFH1, as well as the 2 VL:JFH1 var-iants (Supplementary Figure 6). This is consistent with thereport from Barretto et al.33 Engineering nonhepatic cells toproduce HCV through ectopic apoE expression can confercell-to-cell transmission capacity, albeit to a limited degreerelative to the levels observed in the current study.34 Wethus cannot exclude that epitopes not recognized by theutilized anti-apoE antibody play a role in cell-to-cell trans-mission or that this antibody may have limited access to thevirus during this process.

In variants that were selected during liver trans-plantation, replacement of phenylalanine at E2 residue 447appeared to alter the immunoprecipitation of virions byanti-apoE antibodies (Figure 7B). Given the comparablelevel of intracellular E2-apoE association (Figure 7D) andapoE incorporation into virions (Figure 7E) of the wild-typeand the mutant, mutations of E2 residue 447 appear toinduce conformational changes in the E2-apoE complexaffecting the sensitivity to neutralization by both apoE- andE2-specific nAbs (Figures 1, 2, and 7). Collectively our dataindicate that virion apoE levels (Figures 1–3) and confor-mation (Figure 7) are relevant for the evasion from nAbs.

ApoE plays a key role in viral attachment to heparansulfate proteoglycans,25,35 whereas subsequent steps ofviral entry require E2-SR-BI and E2-CD81 interactions(reviewed in Zeisel et al.36). Thus, dissociation of apoE and/or alteration of the apoE-E2 complex might be required forenvelope-SR-BI and CD81 interactions occurring postbinding. Supporting this model, our results demonstratethat conformational human mAbs CBH-23 and HC-1, whichimpair HCV entry by interfering with E2�CD81 interactionsand act at a post-binding step,6 more effectively neutralizeapoE-depleted HCV.

HCV association with very-low-density lipoprotein dur-ing viral production was hypothesized to block nAbs-envelope glycoprotein binding and confer viral resistanceto nAbs. Our findings point to apoE shielding of E2 fromneutralization, independently from association with lipo-proteins as measured by density distribution. This new roleof apoE is consistent with our previous observationsshowing that nonhepatic cell lines lacking very-low-densitylipoprotein�producing components and engineered to ex-press apoE can sustain the entire HCV life cycle and produceviruses with buoyant density profiles similar to virus pro-duced in hepatoma cells.37

Collectively, we demonstrate that apart from lipoproteinassociation, apoE mediates escape from nAbs. This findingreveals a novel strategy contributing to HCV’s remarkablecapacity to establish chronic infection. According to the corecrystal structure of E2,38,39 residue 447 that appears to alterthe conformation of the E2-apoE complex resides on theperiphery of a nonstructured E2 region and, thus, would bean ideal candidate for association with exposed apoE re-gions. This finding might be relevant for vaccine design andwe assume that immunogens that mimic epitopes at the E2/apoE interface might help to achieve a broadly neutralizinghumoral immune response.


Supplementary MaterialNote: To access the supplementary material accompanyingthis article, visit the online version of Gastroenterology atwww.gastrojournal.org, and at http://dx.doi.org/10.1053/j.gastro.2015.09.014.

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References
1. Yamane D, McGivern DR, Masaki T, et al. Liver injury and
disease pathogenesis in chronic hepatitis C. Curr TopMicrobiol Immunol 2013;369:263–288.

2. Nabel GJ. Designing tomorrow’s vaccines. N Engl J Med2013;368:551–560.

3. von Hahn T, Yoon JC, Alter H, et al. Hepatitis C viruscontinuously escapes from neutralizing antibody andT-cell responses during chronic infection in vivo.Gastroenterology 2007;132:667–678.

4. Osburn WO, Fisher BE, Dowd KA, et al. Spontaneouscontrol of primary hepatitis C virus infection and immu-nity against persistent reinfection. Gastroenterology2010;138:315–324.

5. Fafi-Kremer S, Fofana I, Soulier E, et al. Viral entry andescape from antibody-mediated neutralization influencehepatitis C virus reinfection in liver transplantation. J ExpMed 2010;207:2019–2031.

6. Fofana I, Fafi-Kremer S, Carolla P, et al. Mutations thatalter use of hepatitis C virus cell entry factors mediateescape from neutralizing antibodies. Gastroenterology2012;143:223–233 e9. Q

7. Andre P, Komurian-Pradel F, Deforges S, et al. Charac-terization of low- and very-low-density hepatitis C virusRNA-containing particles. J Virol 2002;76:6919–6928.

8. Bartenschlager R, Penin F, Lohmann V, et al. Assemblyof infectious hepatitis C virus particles. Trends Microbiol2011;19:95–103.

9. Merz A, Long G, Hiet MS, et al. Biochemical andmorphological properties of hepatitis C virus particlesand determination of their lipidome. J Biol Chem 2011;286:3018–3032. Q

10. Catanese MT, Uryu K, Kopp M, et al. Ultrastructuralanalysis of hepatitis C virus particles. Proc Natl Acad SciU S A 2013;110:9505–9510.

11. Dreux M, Boson B, Ricard-Blum S, et al. Theexchangeable apolipoprotein ApoC-I promotes mem-brane fusion of hepatitis C virus. J Biol Chem 2007;282:32357–32369. Q

12. Grove J, Nielsen S, Zhong J, et al. Identification of aresidue in hepatitis C virus E2 glycoprotein that de-termines scavenger receptor BI and CD81 receptor de-pendency and sensitivity to neutralizing antibodies.J Virol 2008;82:12020–12029.

13. Lupberger J, Zeisel MB, Xiao F, et al. EGFR andEphA2 are host factors for hepatitis C virus entry andpossible targets for antiviral therapy. Nat Med 2011;17:589–595. Q

14. Fofana I, Xiao F, Thumann C, et al. A novel monoclonalanti-CD81 antibody produced by genetic immunizationefficiently inhibits hepatitis C virus cell-cell transmission.PLoS One 2013;8:e64221.

ovember 2015 � 1:29 am � ce

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http://www.gastrojournal.org



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15. Owsianka A, Tarr AW, Juttla VS, et al. Monoclonalantibody AP33 defines a broadly neutralizing epitope onthe hepatitis C virus E2 envelope glycoprotein. J Virol2005;79:11095–11104.

16. Weisgraber KH, Rall SC Jr, Mahley RW, et al. Humanapolipoprotein E. Determination of the heparin bindingsites of apolipoprotein E3. J Biol Chem 1986;261:2068–2076.

17. Keck ZY, Xia J, Wang Y, et al. Human monoclonalantibodies to a novel cluster of conformational epi-topes on HCV E2 with resistance to neutralizationescape in a genotype 2a isolate. PLoS Pathog 2012;8:e1002653.

18. Macdonald A, Crowder K, Street A, et al. The hepatitis Cvirus non-structural NS5A protein inhibits activatingprotein-1 function by perturbing ras-ERK pathwaysignaling. J Biol Chem 2003;278:17775–17784.

19. Law M, Maruyama T, Lewis J, et al. Broadly neutralizingantibodies protect against hepatitis C virus quasispecieschallenge. Nat Med 2008;14:25–27.

20. Wakita T, Pietschmann T, Kato T, et al. Production ofinfectious hepatitis C virus in tissue culture from a clonedviral genome. Nat Med 2005;11:791–796.

21. Reiss S, Rebhan I, Backes P, et al. Recruitment andactivation of a lipid kinase by hepatitis C virus NS5A isessential for integrity of the membranous replicationcompartment. Cell Host Microbe 2011;9:32–45.

22. Pietschmann T, Kaul A, Koutsoudakis G, et al. Con-struction and characterization of infectious intra-genotypic and intergenotypic hepatitis C virus chimeras.Proc Natl Acad Sci U S A 2006;103:7408–7413.

23. Lindenbach BD, Evans MJ, Syder AJ, et al. Completereplication of hepatitis C virus in cell culture. Science2005;309:623–626.

24. Benga WJ, Krieger SE, Dimitrova M, et al. Apolipo-protein E interacts with hepatitis C virus nonstructuralprotein 5A and determines assembly of infectious parti-cles. Hepatology 2010;51:43–53.14

25. Lefevre M, Felmlee DJ, Parnot M, et al. Syndecan 4 isinvolved in mediating HCV entry through interaction withlipoviral particle-associated apolipoprotein E. PLoS One2014;9:e95550.

26. Lee JY, Acosta EG, Stoeck IK, et al. Apolipoprotein Elikely contributes to a maturation step of infectioushepatitis C virus particles and interacts with viral enve-lope glycoproteins. J Virol 2014;88:12422–12437.

27. Chang KS, Jiang J, Cai Z, Luo G, et al. Human apoli-poprotein e is required for infectivity and production ofhepatitis C virus in cell culture. J Virol 2007;81:13783–13793.

28. Jiang J, Luo G. Apolipoprotein E but not B is required forthe formation of infectious hepatitis C virus particles.J Virol 2009;83:12680–12691.

29. Koutsoudakis G, Kaul A, Steinmann E, et al. Character-ization of the early steps of hepatitis C virus infection byusing luciferase reporter viruses. J Virol 2006;80:5308–5320.

30. Lindenbach BD, Rice CM. The ins and outs of hepatitis Cvirus entry and assembly. Nat Rev Microbiol 2013;11:688–700.


31. Boyer A, Dumans A, Beaumont E, et al. The associationof hepatitis C virus glycoproteins with apolipoproteins Eand B early in assembly is conserved in lipoviral particles.J Biol Chem 2014;289:18904–18913.

32. Brimacombe CL, Grove J, Meredith LW, et al. Neutral-izing antibody-resistant hepatitis C virus cell-to-celltransmission. J Virol 2011;85:596–605. Q

33. Barretto N, Sainz B Jr, Hussain S, et al. Determining theinvolvement and therapeutic implications of host cellularfactors in hepatitis C virus cell-to-cell spread. J Virol2014;88:5050–5061. Q

34. Hueging K, Doepke M, Vieyres G, et al. Apolipoprotein Ecodetermines tissue tropism of hepatitis C virus and iscrucial for viral cell-to-cell transmission by contributing toa postenvelopment step of assembly. J Virol 2014;88:1433–1446.

35. Jiang J, Cun W, Wu X, et al. Hepatitis C virus attachmentmediated by apolipoprotein E binding to cell surfaceheparan sulfate. J Virol 2012;86:7256–7267.

36. Zeisel MB, Felmlee DJ, Baumert TF. Hepatitis C virusentry. Curr Top Microbiol Immunol 2013;369:87–112.

37. Da Costa D, Turek M, Felmlee DJ, et al. Reconstitutionof the entire hepatitis C virus life cycle in nonhepaticcells. J Virol 2012;86:11919–11925. Q

38. Kong L, Giang E, Nieusma T, et al. Hepatitis C virus E2envelope glycoprotein core structure. Science 2013;342:1090–1094.

39. Khan AG, Whidby J, Miller MT, et al. Structure of the coreectodomain of the hepatitis C virus envelope glycopro-tein 2. Nature 2014;509:381–384.

Author names in bold designate shared co-first authorship.

Received September 24, 2014. Accepted September 11, 2015.

Reprint requestsAddress requests for reprints to: Thomas F. Baumert, MD, Inserm Unit 1110,Institut de Recherche sur les Maladies Virales et Hépatiques, Université deStrasbourg, 3 rue Koeberlé, F-67000 Strasbourg, France. e-mail:[email protected]; fax: (33) 3 68 85 37 50. Q

AcknowledgmentsThe authors thank F. Chisari (The Scripps Research Institute, La Jolla, CA) forthe gift of Huh7.5.1 cells, T. Wakita (Department of Virology II, NationalInstitutes of Health, Tokyo, Japan) and C. M. Rice (Rockefeller University,New York, NY) for providing HCV strain JFH1, M. Harris (University of Leeds,Leeds, UK), for the gift of the sheep anti-NS5A serum, C. M. Rice(Rockefeller University) for Huh7.5-derived cells, and M. Law (The ScrippsResearch Institute, La Jolla, CA) for anti-E2 AR3B antibody. The authorsthank S. Fafi-Kremer and F. Stoll-Keller (Laboratoire de Virologie, HôpitauxUniversitaires Strasbourg) for support and discussions during the early phaseof the study and J. Boileau (Hôpitaux Universitaires Strasbourg) for providingserum samples.Author contributions: C.F., D.J.F., M.B.Z., and T.F.B. wrote the manuscript.

C.F., D.J.F., M.B.Z., R.B., C.S., and T.F.B. designed experiments. C.F.,D.J.F., E.C., JY.L., L.H., M.L., A.M. K.V, I.F. performed experiments. C.F.,D.J.F., E.C., JY.L., P.M., I.F., M.B.Z., R.B., C.S., and T.F.B. analyzed data.M-S.H., R.M., R.B., A.H.P., S.K.H.F., and F.H. contributed essential reagents.T.F.B. initiated and directed the project.

Conflicts of interestThe authors disclose no conflicts. Q

FundingThis study was supported through funding by the European Union (ERC-2008-AdG-233130-HEPCENT, FP7 HepaMAb and Interreg-IV-Rhin Supérieur-FEDER-Hepato-Regio-Net 2012), the Agence Nationale de Recherches sur leSIDA (ANRS) (2012/239, 2012/318, 2013/108), the Direction Générale del’Offre de Soins (A12027MS) and the University of Strasbourg Foundation. Thiswork has been published under the framework of the LABEX ANR-10-LABX-

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0028_HEPSYS and benefits from funding from the state managed by the FrenchNational Research Agency aspart of the Investments for the future program.D. J.Felmlee acknowledges fellowships from European Association for Study of theLiver (EASL) and ANRS, E. Crouchet a PhD student acknowledges a MRTfellowship from the French Ministry for Education. R. Bartenschlager was


supported by the Deutsche Forschungsgemeinschaft, FOR1202, TP1. P.Meuleman was supported by the Research Foundation Flanders (FWO project#3G052112), the Ghent University (GOA #01G01712) and the Belgian state(IUAP P7/47-HEPRO2). C. Schuster acknowledges funding by ANRS (2010-307/2011-415) and IdEx University of Strasbourg. Q

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Supplementary Figure 1. Increased nAb sensitivity of apoE-depleted HCV is not due to decreased virus quantity or theratio of infectious virions and nAbs. Huh7.5.1 cells wereelectroporated with Luc-Jc1 RNA alone, or co-electroporatedwith either scrambled siRNA (siCtrl) or siRNA targeting apoEmRNA (siApoE). Two experiments using different HCV viraltiters (high viral titer vs low viral titer) were performed to studythe impact of virus quantity on the sensitivity to patientserum. Results of neutralization assay of HCV produced fromthese cells using serum from a patient without HCV infection(Ctrl) or indicated dilution of serum from a patient with ge-notype 1 HCV infection was determined by quantification ofluciferase activity (log10 RLU). Mean ± SEM of both experi-ments performed in triplicate are shown.

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Supplementary Figure 2. Adjustment of input virus titers of viruses produced from control and apoE-depleted cells confirmsassociation of virion apoE content and sensitivity to antibody-mediated neutralization. Huh7.5.1 cells were electroporated withH77R2a (A–C) or Luc-Jc1 (D–F) RNA alone, or co-electroporated with either scrambled siRNA (siCtrl) or siRNA targeting apoEmRNA (siApoE). (A, D). ApoE depletion in producer cells was confirmed 72 hours post electroporation by immunoblotting ofcell lysates; actin was detected as loading control (right panels). (B, E) Titers of viruses produced in supernatants of transfectedHuh7.5.1 cells (72 hours post electroporation) were determined by tissue culture infectious dose 50% (TCID50) using infectionof naïve Huh7.5.1 for 72 hours as described in the Materials and Methods. After quantification of viral titers, titers wereadjusted by dilution in cell culture medium. Titers of viruses from apoE-depleted cells served as the reference. Viral titersbefore and after dilution are shown as TCID50 of a representative titration analysis. (C, F) Neutralization experiments usingviruses with adjusted titers shown in (B, E) were performed using sera from 4 different HCV-infected patients (1, 2, 3 or 4) asdescribed in Figure 1. Means ± SDs from one representative experiment performed in duplicate are shown. Results areexpressed as fold increase of neutralization relative to control serum.

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Supplementary Figure 3. Inhibition of viruses produced fromapoE silenced producer cells by anti-apoE or anti-CD81 an-tibodies. Huh7.5.1 cells were electroporated with VL:JFH1alone, or co-electroporated with either scrambled siRNA(siCtrl) or siRNA targeting apoE mRNA (siApoE). To assessthe role of CD81, cells were pretreated with 10 mg/mL anti-CD81 antibody for 30 minutes before HCVcc infection ofHuh7.5.1 cells. To assess the role of apoE, HCVcc werepreincubated with 10 mg/mL anti-apoE antibody (1D7) for 1hour before infection of Huh7.5.1 cells. Infection was deter-mined by end-point dilution assay and expressed as per-centage of infection relative to control antibodies. Mean ±SEM from 4 independent experiments are shown. P < .05.

Supplementary Figure 4. Silencing of apoE expression inHCV producer cells has no effect on apoB expression andvice versa. Huh7.5.1 cells were electroporated with Luc-Jc1RNA alone, or co-electroporated with either scrambledsiRNA (siCtrl) or siRNA targeting either apoE mRNA (siApoE)or apoB mRNA (siApoB). After 72 hours, apoB and apoEexpression was quantified using immunoblots of cell lysatesand anti-apoE and apoB specific antibodies; actin expressionwas analyzed as control. A representative immunoblot isshown.

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Supplementary Figure 6. Absent effect of anti-apoE anti-body 1D7 on the cell-to-cell transmission of VL:JFH1 F447Land F447A mutants. Interference of cell-to-cell transmissionof VL:JFH1 F447L and F447A by anti-apoE antibody 1D7,nonspecific IgG as a negative control (Ctrl), or anti-CD81 as apositive control was tested. For cell-to-cell transmission,green fluorescent protein�expressing naïve target cells wereco-cultured with cells containing HCV with neutralizing anti-body AP33. Histograms of cell-to-cell transmission summa-rized from 2 independent experiments performed in duplicateare shown. Values represent percentage of cell-to-celltransmission relative to control. P < .05.

Supplementary Figure 5. Immunoprecipitation of apoE usinganti-apoE antibodies 1D7, 3H1 and 6C5 in Huh7.5.1 cell ly-sates. ApoE was immunoprecipitated from cell lysates asdescribed in Materials and Methods and apoE on immuno-precipitated beads was subjected to immunoblot using anti-apoE antibody (Abcam). Isotype IgG served as negativecontrol for immunoprecipitation. Two independent experi-ments have been performed. A representative immunoblot isshown.

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apolipoprotein e mediates evasion from hepatitis c virus … · 2018-04-05 · immune system lags...

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