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Hepatitis C virus entry into hepatocytes: Molecular mechanisms and targets for antiviral therapies

Open AccessPublished:November 29, 2010DOI:https://doi.org/10.1016/j.jhep.2010.10.014
      Hepatitis C virus (HCV) is a major cause of liver cirrhosis and hepatocellular carcinoma. Preventive modalities are absent and the current antiviral treatment is limited by resistance, toxicity, and high costs. Viral entry is required for initiation, spread, and maintenance of infection, and thus is a promising target for antiviral therapy. HCV entry is a highly orchestrated process involving viral and host cell factors. These include the viral envelope glycoproteins E1 and E2, CD81, scavenger receptor BI, and tight junction proteins claudin-1 and occludin. Recent studies in preclinical models and HCV-infected patients have demonstrated that the virus has developed multiple strategies to escape host immune responses during viral entry. These include evasion from neutralizing antibodies and viral spread by cell–cell transmission. These challenges have to be taken into account for the design of efficient antiviral strategies. Thus, a detailed understanding of the mechanisms of viral entry and escape is a prerequisite to define viral and cellular targets and develop novel preventive and therapeutic antivirals. This review summarizes the current knowledge about the molecular mechanisms of HCV entry into hepatocytes, highlights novel targets and reviews the current preclinical and clinical development of compounds targeting entry. Proof-of-concept studies suggest that HCV entry inhibitors are a novel and promising class of antivirals widening the preventive and therapeutic arsenal against HCV infection.

      Abbreviations:

      CLDN1 (claudin-1), HCV (hepatitis C virus), HCVcc (cell culture-derived HCV), HCVpp (HCV pseudo - particle), HCV-LP (HCV-like particle), JFH1 (Japanese fulminant hepatitis 1), OCLN (occludin), SR-BI (scavenger receptor class B type I)

      Keywords

      Introduction

      Hepatitis C virus (HCV) is a major cause of chronic hepatitis worldwide. The current therapy against HCV infection, consisting of an association of pegylated interferon alpha (PEG-IFN) and ribavirin, is limited by resistance, adverse effects, and high costs. Although the clinical development of novel antivirals targeting HCV protein processing has been shown to improve sustained virological response, toxicity of the individual compounds and development of viral resistance remain major challenges [
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      ]. To date, a vaccine is not available. The absence of preventive strategies is a major limitation for patients undergoing liver transplantation (LT) for HCV-related end-stage liver disease. Re-infection of the graft is universal and characterized by accelerated progression of liver disease [
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      ]. Recurrent HCV liver disease in the graft with poor outcome has become an increasing problem faced by hepatologists and transplant surgeons. Thus, novel antiviral preventive and therapeutic strategies are urgently needed.
      Viral entry is the first step of virus–host cell interactions leading to productive infection and thus represents an interesting target for antiviral therapy. HCV entry is believed to be a highly orchestrated process involving several viral and host cell factors, thereby offering multiple novel targets for antiviral therapy. However, multiple strategies evolved by the virus in order to escape the host immune system, such as escape from neutralizing antibodies and direct cell–cell transmission, have to be taken into account for the design of efficient novel antiviral strategies. Understanding the mechanisms of viral entry and escape is thus a prerequisite to define the viral and cellular targets that will give broad protection against HCV infection.
      HCV is an enveloped single-strand RNA virus that mainly targets hepatocytes. Due to the difficulty to grow HCV in vitro and the species specificity of this virus, surrogate model systems have been developed to study HCV entry into hepatocytes: recombinant envelope glycoproteins [
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      ] have been used to study the interactions of the viral envelope with human hepatoma cells or primary human hepatocytes. Moreover, the use of transgenic immunodeficient mice with hepatocyte-lethal phenotype (Alb-uPA/SCID [
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      ]), that can be successfully transplanted with primary human hepatocytes, allowed to establish a small animal model to study certain aspects of HCV infection in vivo [
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      Using the above described model systems, tremendous progress has been made over the past years in deciphering the mechanisms of HCV-host interactions leading to viral entry. The understanding of these mechanisms has allowed researchers to identify novel targets for antivirals, and several compounds are reaching early clinical development. The aim of this review is to summarize the current knowledge on the complex mechanisms of HCV entry into host cells, as well as to highlight the antiviral targets and to review the current development of HCV entry inhibitors that represent a novel important class of antivirals. Developing efficient HCV entry inhibitors may hold great promises to improve the sustained virological response in chronic HCV-infected patients and thus prevent HCV re-infection during LT.

      Hepatitis C virus evades host immune responses to enter the hepatocyte

      Viral entry is the first step of HCV infection that requires interaction of the HCV envelope glycoproteins E1 and E2 and the host cell membrane. E1 and E2 are type I transmembrane proteins with an N-terminal ectodomain and a short C-terminal transmembrane domain (TMD). Functional virion-associated E1E2 envelope glycoproteins mediating viral entry form large covalent complexes stabilized by disulfide bridges [
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      ]. The N-terminal ectodomains of E1 and E2 are heavily glycosylated. The glycans play a major role in E1E2 folding as well as HCV entry [
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      ] and are of crucial importance for the evasion from the host immune responses by masking immunogenic envelope epitopes [
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      ]. Moreover, HCV exists in heterogenous forms in human serum and may be associated with VLDL, LDL, and HDL [
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      ] also shielding the virus from neutralizing antibodies targeting the HCV envelope glycoproteins.
      Figure thumbnail fx2
      Both E1 and E2 contain putative fusion domains [
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      Functional analysis of hepatitis C virus envelope proteins, using a cell–cell fusion assay.
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      Characterization of fusion determinants points to the involvement of three discrete regions of both E1 and E2 glycoproteins in the membrane fusion process of hepatitis C virus.
      ]. While the role of E1 in HCV entry is not completely understood, several E2 domains play pivotal roles in viral entry, i.e. putative domain binding to two HCV entry factors, CD81 and scavenger receptor class B type I (SR-BI), and escape from host immune responses. Hypervariable regions (HVR) have been identified in E2. The first 27 amino acids of E2 called hypervariable region 1 (HVR1), are the most divergent among HCV isolates. HVR1 plays an important role in viral fitness, likely due to an involvement in SR-BI-mediated entry [
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      Cell entry of hepatitis C virus requires a set of co-receptors that include the CD81 tetraspanin and the SR-B1 scavenger receptor.
      ], assembly and release of virus particles [
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      Hepatitis C virus hypervariable region 1 modulates receptor interactions, conceals the CD81 binding site, and protects conserved neutralizing epitopes.
      ] as well as HCV membrane fusion process [
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      ]. HVR1 is a target for neutralizing antibodies. However, due to its high variability, antibodies targeting HVR1 exhibit poor cross-neutralization potency across different HCV isolates [
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      ]. Broadly neutralizing antibodies are directed against conserved conformational epitopes within E2 [
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      ] and mostly inhibit E2–CD81 interaction [
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      Analysis of antigenicity and topology of E2 glycoprotein present on recombinant hepatitis C virus-like particles.
      ]. The region located immediately downstream of HVR1 contains a potent and highly conserved epitope. This epitope defined by the mouse monoclonal antibody (mAb) AP33 and a rat mAb 3/11, is involved in E2–CD81 [
      • Owsianka A.
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      ] and E2-heparan sulfate interaction [
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      Viral and cellular determinants of the hepatitis C virus envelope–heparan sulfate interaction.
      ]. Importantly, mutated variants that escape from AP33 neutralization show very low infectivity [
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      Mutations in HCV E2 located outside the CD81 binding sites lead to escape from broadly neutralizing antibodies but compromise virus infectivity.
      ]. Recently, new conformational and conserved epitopes were identified in the N-terminal part of E2. Antibodies targeting these epitopes neutralize genetically diverse HCV isolates and protect against heterologous HCV quasispecies challenge in the human liver-chimeric Alb-uPA/SCID mouse model [
      • Law M.
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      Broadly neutralizing antibodies protect against hepatitis C virus quasispecies challenge.
      ]. Since these epitopes are thought to be involved in HCV entry, viral mutation could induce escape from broadly neutralizing antibodies but at a substantial cost in viral fitness [
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      Mutations in HCV E2 located outside the CD81 binding sites lead to escape from broadly neutralizing antibodies but compromise virus infectivity.
      ]. The conserved nature of these epitopes makes them of interest for vaccine and immunotherapeutic development.
      In vivo, humoral responses are thought to play an important role in controlling HCV infection. Indeed, spontaneous resolvers tend to have an early induction of neutralizing antibody responses, whereas chronically evolving subjects have a delayed initiation of neutralizing antibody responses [
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      Rapid induction of virus-neutralizing antibodies and viral clearance in a single-source outbreak of hepatitis C.
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      Human serum facilitates hepatitis C virus infection, and neutralizing responses inversely correlate with viral replication kinetics at the acute phase of hepatitis C virus infection.
      ]. Furthermore, the generation of cross-reactive humoral responses is associated with protection against HCV re-infection [
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      • Urban G.
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      Spontaneous control of primary hepatitis C virus infection and immunity against persistent reinfection.
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      • Urban G.
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      Spontaneous control of primary hepatitis C virus infection and immunity against persistent reinfection.
      ]. However, the accelerated evolution [
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      ] and the diversity of HCV, as well as the variety of strategies the virus evolved to escape antibody-mediated neutralization (reviewed in [
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      ]), are a major challenge. Indeed, due to its very high replication rate and the highly error prone viral polymerase, HCV circulates as a pool of genetically distinct but closely related variants known as viral quasi-species. The capacity of HCV to mutate continuously allows a high plasticity, an ability of the virus to adapt to variable environmental conditions and escape the host’s immune responses leading to HCV persistence [
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      ]. Noteworthy, a recent longitudinal analysis of six HCV-infected patients undergoing LT suggests that efficient entry and escape from host neutralizing antibodies represent important mechanisms for the selection of HCV during LT [
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      ]. As strains selected during LT could be neutralized by broadly neutralizing antibodies, the major challenge for developing efficient antiviral strategies targeting the HCV envelope glycoproteins will be to identify epitopes largely conserved among genotypes and selected isolates.

      Hepatitis C virus uses multiple host factors to enter its target cell

      HCV attachment and entry into host cells is a complex and multistep process. Using various model systems, several cell surface molecules have been identified to interact with HCV. These include CD81 [
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      Binding of hepatitis C virus to CD81.
      ], the LDL receptor [
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      Hepatitis C virus and other flaviviridae viruses enter cells via low density lipoprotein receptor.
      ], highly sulfated heparan sulfate (HS) [
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      Cellular binding of hepatitis C virus envelope glycoprotein E2 requires cell surface heparan sulfate.
      ], SR-BI [
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      The human scavenger receptor class B type I is a novel candidate receptor for the hepatitis C virus.
      ], DC-SIGN (dendritic cell-specific intercellular adhesion molecule three grabbing non integrin)/L-SIGN (DC-SIGNr, liver and lymph node specific) [
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      Hepatitis C virus glycoproteins interact with DC-SIGN and DC-SIGNR.
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      C-type lectins L-SIGN and DC-SIGN capture and transmit infectious hepatitis C virus pseudotype particles.
      ], claudin-1 (CLDN1) [
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      Claudin-1 is a hepatitis C virus co-receptor required for a late step in entry.
      ], and occludin (OCLN) [
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      Human occludin is a hepatitis C virus entry factor required for infection of mouse cells.
      ,
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      Tight junction proteins claudin-1 and occludin control hepatitis C virus entry and are downregulated during infection to prevent superinfection.
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      Correlation of the tight junction-like distribution of Claudin-1 to the cellular tropism of hepatitis C virus.
      ].
      In vivo, HCV enters the liver through the sinusoidal blood. Capture of circulating HCV particles by liver sinusoidal cells may thus facilitate the viral infection of neighbouring hepatocytes which are not in direct contact with circulating blood. This process may be mediated by DC-SIGN, which is expressed in Kupffer cells that localize close to liver sinusoidal endothelial cells (LSEC) and hepatocytes [
      • van Kooyk Y.
      • Geijtenbeek T.B.
      DC-SIGN: escape mechanism for pathogens.
      ], and L-SIGN that is highly expressed in LSEC. DC-SIGN and L-SIGN have been shown to bind envelope glycoprotein E2 with high affinity [
      • Pohlmann S.
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      • Chen Z.
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      Hepatitis C virus glycoproteins interact with DC-SIGN and DC-SIGNR.
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      L-SIGN (CD 209L) is a liver-specific capture receptor for hepatitis C virus.
      ]. On hepatocytes, HS glycosaminoglycans represent first attachment sites [
      • Barth H.
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      • Zhang F.
      • Linhardt R.J.
      • Depla E.
      • Boson B.
      • et al.
      Viral and cellular determinants of the hepatitis C virus envelope–heparan sulfate interaction.
      ,
      • Barth H.
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      • Zhang F.
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      • Toyoda H.
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      Cellular binding of hepatitis C virus envelope glycoprotein E2 requires cell surface heparan sulfate.
      ,
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      Characterization of the early steps of hepatitis C virus infection by using luciferase reporter viruses.
      ] that may help to concentrate the virus on the target cell surface and allow further interactions with other host factors triggering viral entry.
      CD81 is a ubiquitously expressed 25 kDa tetraspanin, containing a small extracellular and a large extracellular loop (LEL). CD81 has been the first molecule described to interact with a soluble truncated form of HCV E2 and to be a critical host cell factor for viral entry [
      • Lindenbach B.D.
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      ]. The LEL seems to play an important role in this process, as soluble recombinants forms of CD81 LEL have been shown to inhibit HCVpp and HCVcc infections [
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      ]. Several amino acid residues critical for E2–CD81 binding have been identified throughout the CD81 LEL and HCV E2 [
      • Owsianka A.
      • Clayton R.F.
      • Loomis-Price L.D.
      • McKeating J.A.
      • Patel A.H.
      Functional analysis of hepatitis C virus E2 glycoproteins and virus-like particles reveals structural dissimilarities between different forms of E2.
      ,
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      • Uematsu Y.
      • Campagnoli S.
      • Galli G.
      • Falugi F.
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      Binding of hepatitis C virus to CD81.
      ,
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      Characterization of hepatitis C virus E2 glycoprotein interaction with a putative cellular receptor CD81.
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      • Dragic T.
      Different domains of CD81 mediate distinct stages of hepatitis C virus pseudoparticle entry.
      ]. In recent years, studies using HCVpp and HCVcc have provided additional valuable information about E2–CD81 interactions and highlighted the importance of E2 residues at positions 415, 420, 527, 529, 530, and 535 [
      • Owsianka A.M.
      • Timms J.M.
      • Tarr A.W.
      • Brown R.J.
      • Hickling T.P.
      • Szwejk A.
      • et al.
      Identification of conserved residues in the E2 envelope glycoprotein of the hepatitis C virus that are critical for CD81 binding.
      ,
      • Dhillon S.
      • Witteveldt J.
      • Gatherer D.
      • Owsianka A.M.
      • Zeisel M.B.
      • Zahid M.N.
      • et al.
      Mutations within a conserved region of the hepatitis C virus E2 glycoprotein that influence virus–receptor interactions and sensitivity to neutralizing antibodies.
      ] for virus particle–CD81 interaction.
      Human SR-BI or CLA-1 (CD36 and LIMPII Analogous-1) is an 82 kDa glycoprotein with a large extracellular loop highly expressed in the liver and steroidogenic tissues [
      • Krieger M.
      Scavenger receptor class B type I is a multiligand HDL receptor that influences diverse physiologic systems.
      ]. SR-BI binds a variety of lipoproteins (HDL, LDL, oxLDL) and is involved in bi-directional cholesterol transport at the cell membrane. The SR-BI extracellular loop has been demonstrated to interact with E2 HVR1 [
      • Scarselli E.
      • Ansuini H.
      • Cerino R.
      • Roccasecca R.M.
      • Acali S.
      • Filocamo G.
      • et al.
      The human scavenger receptor class B type I is a novel candidate receptor for the hepatitis C virus.
      ]. Recent evidence suggests that amino acids 70–87 and the single residue E210 of SR-BI are required for E2 recognition [
      • Catanese M.T.
      • Ansuini H.
      • Graziani R.
      • Huby T.
      • Moreau M.
      • Ball J.K.
      • et al.
      Role of scavenger receptor class B type I in hepatitis C virus entry: kinetics and molecular determinants.
      ]. SR-BI may play a dual role during the HCV entry process, during both binding and post-binding steps [
      • Catanese M.T.
      • Ansuini H.
      • Graziani R.
      • Huby T.
      • Moreau M.
      • Ball J.K.
      • et al.
      Role of scavenger receptor class B type I in hepatitis C virus entry: kinetics and molecular determinants.
      ,
      • Zeisel M.B.
      • Koutsoudakis G.
      • Schnober E.K.
      • Haberstroh A.
      • Blum H.E.
      • Cosset F.-L.
      • et al.
      Scavenger receptor BI is a key host factor for hepatitis C virus infection required for an entry step closely linked to CD81.
      ]. Physiological SR-BI ligands have been shown to modulate HCV infection: HDL is able to enhance HCVpp and HCVcc infections [
      • Bartosch B.
      • Verney G.
      • Dreux M.
      • Donot P.
      • Morice Y.
      • Penin F.
      • et al.
      An interplay between hypervariable region 1 of the hepatitis C virus E2 glycoprotein, the scavenger receptor BI, and high-density lipoprotein promotes both enhancement of infection and protection against neutralizing antibodies.
      ,
      • Voisset C.
      • Callens N.
      • Blanchard E.
      • Op De Beeck A.
      • Dubuisson J.
      • Vu-Dac N.
      High density lipoproteins facilitate hepatitis C virus entry through the scavenger receptor class B type I.
      ] whereas oxidized LDL inhibits HCVpp and HCVcc infections [
      • von Hahn T.
      • Lindenbach B.D.
      • Boullier A.
      • Quehenberger O.
      • Paulson M.
      • Rice C.M.
      • et al.
      Oxidized low-density lipoprotein inhibits hepatitis C virus cell entry in human hepatoma cells.
      ]. Interestingly, high concentrations of HDL and LDL inhibited HCV replication in human hepatocytes infected with serum-derived HCV [
      • Molina S.
      • Castet V.
      • Fournier-Wirth C.
      • Pichard-Garcia L.
      • Avner R.
      • Harats D.
      • et al.
      The low-density lipoprotein receptor plays a role in the infection of primary human hepatocytes by hepatitis C virus.
      ]. Moreover, using serum-derived HCV, it has been suggested that the virus-associated lipoproteins rather than the E2 protein interact with SR-BI in transfected CHO cells [
      • Maillard P.
      • Huby T.
      • Andreo U.
      • Moreau M.
      • Chapman J.
      • Budkowska A.
      The interaction of natural hepatitis C virus with human scavenger receptor SR-BI/Cla1 is mediated by ApoB-containing lipoproteins.
      ]. A recent mapping study reported that HCV and HDL binding to SR-BI as well as the lipid transfer properties of SR-BI are required for SR-BI function as an HCV entry factor [
      • Dreux M.
      • Dao Thi V.L.
      • Fresquet J.
      • Guerin M.
      • Julia Z.
      • Verney G.
      • et al.
      Receptor complementation and mutagenesis reveal SR-BI as an essential HCV entry factor and functionally imply its intra- and extra-cellular domains.
      ]. This study also suggests that the C-terminal cytoplasmic tail of SR-BI modulates the basal HCV entry process, but does not seem to influence HDL-mediated infection-enhancement whereas the extracellular domain is required for E2 binding and lipid transfer function [
      • Dreux M.
      • Dao Thi V.L.
      • Fresquet J.
      • Guerin M.
      • Julia Z.
      • Verney G.
      • et al.
      Receptor complementation and mutagenesis reveal SR-BI as an essential HCV entry factor and functionally imply its intra- and extra-cellular domains.
      ]. Taken together, these results suggest that HCV entry requires the existence of a complex interplay between lipoproteins, SR-BI, and HCV envelope glycoproteins that all need to be taken into account for the development of antivirals targeting SR-BI.
      CLDN1, a 23 kDa four transmembrane protein, has been identified as a critical HCV hepatocyte entry factor by expression cloning [
      • Evans M.J.
      • von Hahn T.
      • Tscherne D.M.
      • Syder A.J.
      • Panis M.
      • Wolk B.
      • et al.
      Claudin-1 is a hepatitis C virus co-receptor required for a late step in entry.
      ]. Interestingly, CLDN6 and CLDN9 are also able to mediate HCV entry in hepatoma cells [
      • Zheng A.
      • Yuan F.
      • Li Y.
      • Zhu F.
      • Hou P.
      • Li J.
      • et al.
      Claudin-6 and claudin-9 function as additional coreceptors for hepatitis C virus.
      ,
      • Meertens L.
      • Bertaux C.
      • Cukierman L.
      • Cormier E.
      • Lavillette D.
      • Cosset F.L.
      • et al.
      The tight junction proteins claudin-1, -6, and -9 are entry cofactors for hepatitis C virus.
      ]. CLDNs are critical components of tight junctions (TJ) regulating paracellular permeability and polarity. CLDN1 is expressed in all epithelial tissues but predominantly in the liver [
      • Furuse M.
      • Fujita K.
      • Hiiragi T.
      • Fujimoto K.
      • Tsukita S.
      Claudin-1 and -2: novel integral membrane proteins localizing at tight junctions with no sequence similarity to occludin.
      ]. Of note, CLDN1 may localize to TJ of hepatocytes but also to the basolateral surfaces of these cells [
      • Reynolds G.M.
      • Harris H.J.
      • Jennings A.
      • Hu K.
      • Grove J.
      • Lalor P.F.
      • et al.
      Hepatitis C virus receptor expression in normal and diseased liver tissue.
      ]. Recent studies suggest that non-junctional CLDN1 may be involved in HCV entry [
      • Evans M.J.
      • von Hahn T.
      • Tscherne D.M.
      • Syder A.J.
      • Panis M.
      • Wolk B.
      • et al.
      Claudin-1 is a hepatitis C virus co-receptor required for a late step in entry.
      ,
      • Cukierman L.
      • Meertens L.
      • Bertaux C.
      • Kajumo F.
      • Dragic T.
      Residues in a highly conserved claudin-1 motif are required for hepatitis C virus entry and mediate the formation of cell–cell contacts.
      ] probably during a post-binding step [
      • Evans M.J.
      • von Hahn T.
      • Tscherne D.M.
      • Syder A.J.
      • Panis M.
      • Wolk B.
      • et al.
      Claudin-1 is a hepatitis C virus co-receptor required for a late step in entry.
      ,
      • Krieger S.E.
      • Zeisel M.B.
      • Davis C.
      • Thumann C.
      • Harris H.J.
      • Schnober E.K.
      • et al.
      Inhibition of hepatitis C virus infection by anti-claudin-1 antibodies is mediated by neutralization of E2–CD81–claudin-1 associations.
      ]. So far, no direct HCV–CLDN1 interaction has been demonstrated [
      • Evans M.J.
      • von Hahn T.
      • Tscherne D.M.
      • Syder A.J.
      • Panis M.
      • Wolk B.
      • et al.
      Claudin-1 is a hepatitis C virus co-receptor required for a late step in entry.
      ,
      • Krieger S.E.
      • Zeisel M.B.
      • Davis C.
      • Thumann C.
      • Harris H.J.
      • Schnober E.K.
      • et al.
      Inhibition of hepatitis C virus infection by anti-claudin-1 antibodies is mediated by neutralization of E2–CD81–claudin-1 associations.
      ]. Mapping studies suggest that the first extracellular loop (ECL1), and more particularly residues in the highly conserved claudin motif W(30)–GLW(51)–C(54)–C(64), are critical for HCV entry [
      • Evans M.J.
      • von Hahn T.
      • Tscherne D.M.
      • Syder A.J.
      • Panis M.
      • Wolk B.
      • et al.
      Claudin-1 is a hepatitis C virus co-receptor required for a late step in entry.
      ,
      • Cukierman L.
      • Meertens L.
      • Bertaux C.
      • Kajumo F.
      • Dragic T.
      Residues in a highly conserved claudin-1 motif are required for hepatitis C virus entry and mediate the formation of cell–cell contacts.
      ]. CLDN1 associates to CD81 in a variety of cell types and the formation of CLDN1–CD81 complexes is essential for HCV infection [
      • Harris H.J.
      • Farquhar M.J.
      • Mee C.J.
      • Davis C.
      • Reynolds G.M.
      • Jennings A.
      • et al.
      CD81 and claudin 1 coreceptor association: role in hepatitis C virus entry.
      ,
      • Harris H.J.
      • Davis C.
      • Mullins J.G.
      • Hu K.
      • Goodall M.
      • Farquhar M.J.
      • et al.
      Claudin association with CD81 defines hepatitis C virus entry.
      ]. Mutations at residues 32 and 48 in CLDN1 ECL1 ablate the association with CD81 and the viral receptor activity [
      • Harris H.J.
      • Davis C.
      • Mullins J.G.
      • Hu K.
      • Goodall M.
      • Farquhar M.J.
      • et al.
      Claudin association with CD81 defines hepatitis C virus entry.
      ].
      OCLN has been identified as another host cell factor critical for HCV entry, probably at a late post-binding event [
      • Ploss A.
      • Evans M.J.
      • Gaysinskaya V.A.
      • Panis M.
      • You H.
      • de Jong Y.P.
      • et al.
      Human occludin is a hepatitis C virus entry factor required for infection of mouse cells.
      ,
      • Liu S.
      • Yang W.
      • Shen L.
      • Turner J.R.
      • Coyne C.B.
      • Wang T.
      Tight junction proteins claudin-1 and occludin control hepatitis C virus entry and are downregulated during infection to prevent superinfection.
      ,
      • Benedicto I.
      • Molina-Jimenez F.
      • Bartosch B.
      • Cosset F.L.
      • Lavillette D.
      • Prieto J.
      • et al.
      The tight junction-associated protein occludin is required for a postbinding step in hepatitis C virus entry and infection.
      ]. OCLN is a 65 kDa four transmembrane protein expressed in TJ of polarized cells. To date, there is no evidence of a direct interaction with HCV. It is worth noting that OCLN has been reported to be one of the two HCV host entry factors responsible for the species specificity of HCV: expression of human OCLN and human CD81 may confer HCV permissivity to mouse cell lines [
      • Ploss A.
      • Evans M.J.
      • Gaysinskaya V.A.
      • Panis M.
      • You H.
      • de Jong Y.P.
      • et al.
      Human occludin is a hepatitis C virus entry factor required for infection of mouse cells.
      ]. The species-specific determinants of this protein have been mapped to the second extracellular loop [
      • Ploss A.
      • Evans M.J.
      • Gaysinskaya V.A.
      • Panis M.
      • You H.
      • de Jong Y.P.
      • et al.
      Human occludin is a hepatitis C virus entry factor required for infection of mouse cells.
      ]. Interestingly, OCLN expression on hepatocytes as well as HCV entry is increased upon glucocorticoid treatment [
      • Ciesek S.
      • Steinmann E.
      • Iken M.
      • Ott M.
      • Helfritz F.A.
      • Wappler I.
      • et al.
      Glucocorticosteroids increase cell entry by hepatitis C virus.
      ] while OCLN expression is down-regulated upon HCV infection to prevent super-infection [
      • Liu S.
      • Yang W.
      • Shen L.
      • Turner J.R.
      • Coyne C.B.
      • Wang T.
      Tight junction proteins claudin-1 and occludin control hepatitis C virus entry and are downregulated during infection to prevent superinfection.
      ]. Further studies are needed to decipher the interplay between HCV, OCLN, and the other known host factors.
      As HCV circulates in the blood in association with LDL and VLDL, the LDL receptor has also been proposed as an attachment and/or entry factor for HCV [
      • Agnello V.
      • Abel G.
      • Elfahal M.
      • Knight G.B.
      • Zhang Q.X.
      Hepatitis C virus and other flaviviridae viruses enter cells via low density lipoprotein receptor.
      ,
      • Wunschmann S.
      • Medh J.D.
      • Klinzmann D.
      • Schmidt W.N.
      • Stapleton J.T.
      Characterization of hepatitis C virus (HCV) and HCV E2 interactions with CD81 and the low-density lipoprotein receptor.
      ]. As HCVpp are not associated with lipoproteins, studies investigating the role of LDLR in HCVpp entry did not show a major role for LDLR [
      • Bartosch B.
      • Dubuisson J.
      • Cosset F.L.
      Infectious hepatitis C virus pseudo-particles containing functional E1–E2 envelope protein complexes.
      ]. Moreover, no direct interaction between envelope glycoprotein E2 and LDL or LDLR was demonstrated [
      • Wunschmann S.
      • Medh J.D.
      • Klinzmann D.
      • Schmidt W.N.
      • Stapleton J.T.
      Characterization of hepatitis C virus (HCV) and HCV E2 interactions with CD81 and the low-density lipoprotein receptor.
      ]. However, the LDLR has been shown to mediate the internalization of serum-derived HCV into CD81-deficient HepG2 cells by binding virus-LDL particles [
      • Agnello V.
      • Abel G.
      • Elfahal M.
      • Knight G.B.
      • Zhang Q.X.
      Hepatitis C virus and other flaviviridae viruses enter cells via low density lipoprotein receptor.
      ]. This will have to be taken into account for the development of antiviral therapies targeting HCV host factors.

      HCV entry is a multistep process

      In vivo, HCV most likely first interacts with the basolateral surfaces of hepatocytes. HS glycosaminoglycans represent first attachment sites [
      • Barth H.
      • Schnober E.K.
      • Zhang F.
      • Linhardt R.J.
      • Depla E.
      • Boson B.
      • et al.
      Viral and cellular determinants of the hepatitis C virus envelope–heparan sulfate interaction.
      ,
      • Barth H.
      • Schäfer C.
      • Adah M.I.
      • Zhang F.
      • Linhardt R.J.
      • Toyoda H.
      • et al.
      Cellular binding of hepatitis C virus envelope glycoprotein E2 requires cell surface heparan sulfate.
      ,
      • Koutsoudakis G.
      • Kaul A.
      • Steinmann E.
      • Kallis S.
      • Lohmann V.
      • Pietschmann T.
      • et al.
      Characterization of the early steps of hepatitis C virus infection by using luciferase reporter viruses.
      ] before the virus interacts with several entry factors, SR-BI [
      • Bartosch B.
      • Vitelli A.
      • Granier C.
      • Goujon C.
      • Dubuisson J.
      • Pascale S.
      • et al.
      Cell entry of hepatitis C virus requires a set of co-receptors that include the CD81 tetraspanin and the SR-B1 scavenger receptor.
      ,
      • Scarselli E.
      • Ansuini H.
      • Cerino R.
      • Roccasecca R.M.
      • Acali S.
      • Filocamo G.
      • et al.
      The human scavenger receptor class B type I is a novel candidate receptor for the hepatitis C virus.
      ,
      • Zeisel M.B.
      • Koutsoudakis G.
      • Schnober E.K.
      • Haberstroh A.
      • Blum H.E.
      • Cosset F.-L.
      • et al.
      Scavenger receptor BI is a key host factor for hepatitis C virus infection required for an entry step closely linked to CD81.
      ,
      • Voisset C.
      • Callens N.
      • Blanchard E.
      • Op De Beeck A.
      • Dubuisson J.
      • Vu-Dac N.
      High density lipoproteins facilitate hepatitis C virus entry through the scavenger receptor class B type I.
      ,
      • Barth H.
      • Schnober E.K.
      • Neumann-Haefelin C.
      • Thumann C.
      • Zeisel M.B.
      • Diepolder H.M.
      • et al.
      Scavenger receptor class B is required for hepatitis C virus uptake and cross-presentation by human dendritic cells.
      ], CD81 [
      • Pileri P.
      • Uematsu Y.
      • Campagnoli S.
      • Galli G.
      • Falugi F.
      • Petracca R.
      • et al.
      Binding of hepatitis C virus to CD81.
      ,
      • Koutsoudakis G.
      • Kaul A.
      • Steinmann E.
      • Kallis S.
      • Lohmann V.
      • Pietschmann T.
      • et al.
      Characterization of the early steps of hepatitis C virus infection by using luciferase reporter viruses.
      ], CLDN1 [
      • Evans M.J.
      • von Hahn T.
      • Tscherne D.M.
      • Syder A.J.
      • Panis M.
      • Wolk B.
      • et al.
      Claudin-1 is a hepatitis C virus co-receptor required for a late step in entry.
      ,
      • Krieger S.E.
      • Zeisel M.B.
      • Davis C.
      • Thumann C.
      • Harris H.J.
      • Schnober E.K.
      • et al.
      Inhibition of hepatitis C virus infection by anti-claudin-1 antibodies is mediated by neutralization of E2–CD81–claudin-1 associations.
      ], and OCLN [
      • Ploss A.
      • Evans M.J.
      • Gaysinskaya V.A.
      • Panis M.
      • You H.
      • de Jong Y.P.
      • et al.
      Human occludin is a hepatitis C virus entry factor required for infection of mouse cells.
      ,
      • Liu S.
      • Yang W.
      • Shen L.
      • Turner J.R.
      • Coyne C.B.
      • Wang T.
      Tight junction proteins claudin-1 and occludin control hepatitis C virus entry and are downregulated during infection to prevent superinfection.
      ,
      • Yang W.
      • Qiu C.
      • Biswas N.
      • Jin J.
      • Watkins S.C.
      • Montelaro R.C.
      • et al.
      Correlation of the tight junction-like distribution of Claudin-1 to the cellular tropism of hepatitis C virus.
      ] (Fig. 1). It is worth noting that all these entry factors are required for productive HCV infection. This suggests that HCV entry may be mediated through the formation of a tightly orchestrated HCV-entry factor complex at the plasma membrane [
      • Zeisel M.B.
      • Koutsoudakis G.
      • Schnober E.K.
      • Haberstroh A.
      • Blum H.E.
      • Cosset F.-L.
      • et al.
      Scavenger receptor BI is a key host factor for hepatitis C virus infection required for an entry step closely linked to CD81.
      ,
      • Krieger S.E.
      • Zeisel M.B.
      • Davis C.
      • Thumann C.
      • Harris H.J.
      • Schnober E.K.
      • et al.
      Inhibition of hepatitis C virus infection by anti-claudin-1 antibodies is mediated by neutralization of E2–CD81–claudin-1 associations.
      ]. First evidence for such co-entry factor complexes has been provided by fluorescence resonance energy transfer (FRET) studies demonstrating the role of CLDN1–CD81 complexes in HCV infection [
      • Harris H.J.
      • Farquhar M.J.
      • Mee C.J.
      • Davis C.
      • Reynolds G.M.
      • Jennings A.
      • et al.
      CD81 and claudin 1 coreceptor association: role in hepatitis C virus entry.
      ,
      • Harris H.J.
      • Davis C.
      • Mullins J.G.
      • Hu K.
      • Goodall M.
      • Farquhar M.J.
      • et al.
      Claudin association with CD81 defines hepatitis C virus entry.
      ]. The fact that only members of the CLDN family supporting HCV entry, i.e. CLDN1, CLDN6, and CLDN9, were able to form complexes with CD81 suggests that CLDN–CD81 complex formation is essential for HCV entry [
      • Krieger S.E.
      • Zeisel M.B.
      • Davis C.
      • Thumann C.
      • Harris H.J.
      • Schnober E.K.
      • et al.
      Inhibition of hepatitis C virus infection by anti-claudin-1 antibodies is mediated by neutralization of E2–CD81–claudin-1 associations.
      ,
      • Harris H.J.
      • Davis C.
      • Mullins J.G.
      • Hu K.
      • Goodall M.
      • Farquhar M.J.
      • et al.
      Claudin association with CD81 defines hepatitis C virus entry.
      ]. To date, our knowledge about the molecular mechanisms of potential other co-factor association(s) is still rudimental. First studies showed that the majority of CLDN1 proteins at the plasma membrane interact with OCLN but did not show any relationship between CLDN1–OCLN association and HCV infection [
      • Harris H.J.
      • Davis C.
      • Mullins J.G.
      • Hu K.
      • Goodall M.
      • Farquhar M.J.
      • et al.
      Claudin association with CD81 defines hepatitis C virus entry.
      ]. In addition, it has been demonstrated that cell contacts modulate SR-BI and CLDN1 expression levels and favour HCV internalization through facilitation of entry factor complexes [
      • Schwarz A.K.
      • Grove J.
      • Hu K.
      • Mee C.J.
      • Balfe P.
      • McKeating J.A.
      Hepatoma cell density promotes claudin-1 and scavenger receptor BI expression and hepatitis C virus internalization.
      ]. Further studies are thus necessary to assess which set of host factors are present with HCV in these complexes.
      Figure thumbnail gr1
      Fig. 1Hepatitis C virus entry into hepatocytes: molecular mechanisms and targets for antiviral therapies. A model of HCV life cycle, with potential targets for virus neutralizing antibodies and other entry inhibitors, is shown. HCV is believed to first interact with HS and LDLR on the basolateral membrane surface of hepatocytes to allow concentration of the virion. Subsequently, interaction with other host factors such as SR-BI, CD81, CLDN1, and OCLN ultimately leads to viral internalization via clathrin-mediated endocytosis. For CLDN1 both junctional and non-junctional forms have been described (for review see
      [
      • Zeisel M.B.
      • Turek M.
      • Baumert T.F.
      Tight junctions and viral entry.
      ]
      ). Fusion between viral and endosomal membranes is followed by release of the viral genome into the cytosol where translation and replication take place. HCV particles are then assembled and released from the host cell. An alternative route of viral entry is direct cell–cell transmission which is resistant to neutralizing antibodies. Entry inhibitors can potentially interfere with the viral life cycle at different steps, i.e. viral binding, post-binding and fusion. Ab, antibody; apo, apolipoprotein; BC, bile canaliculi; CLDN1, claudin 1; HCV, hepatitis C virus; HS, heparan sulfate; JAM, junction-associated adhesion molecule; LDLR, low-density lipoprotein receptor; nAb, neutralizing antibody; OCLN, occludin; PS-ON, phosphorothioate oligonucleotides; SR-BI, scavenger receptor class B type I; ZO, zona occludens.
      To date, the sequence of events leading from HCV-interaction with host factors on the plasma membrane to internalization, viral fusion, and replication still remains elusive. Studies using HCVpp and HCVcc have demonstrated that HCV entry into both hepatoma cells and primary human hepatocytes depends on clathrin-mediated endocytosis [
      • Blanchard E.
      • Belouzard S.
      • Goueslain L.
      • Wakita T.
      • Dubuisson J.
      • Wychowski C.
      • et al.
      Hepatitis C virus entry depends on clathrin-mediated endocytosis.
      ,
      • Meertens L.
      • Bertaux C.
      • Dragic T.
      Hepatitis C virus entry requires a critical postinternalization step and delivery to early endosomes via clathrin-coated vesicles.
      ,
      • Codran A.
      • Royer C.
      • Jaeck D.
      • Bastien-Valle M.
      • Baumert T.F.
      • Kieny M.P.
      • et al.
      Entry of hepatitis C virus pseudotypes into primary human hepatocytes by clathrin-dependent endocytosis.
      ,
      • Trotard M.
      • Lepere-Douard C.
      • Regeard M.
      • Piquet-Pellorce C.
      • Lavillette D.
      • Cosset F.L.
      • et al.
      Kinases required in hepatitis C virus entry and replication highlighted by small interference RNA screening.
      ,
      • Coller K.E.
      • Berger K.L.
      • Heaton N.S.
      • Cooper J.D.
      • Yoon R.
      • Randall G.
      RNA interference and single particle tracking analysis of hepatitis C virus endocytosis.
      ], the most common route of endocytosis for viruses that require internalization. Moreover, actin and clathrin–actin associations have also been shown to be involved in efficient HCV endocytosis [
      • Coller K.E.
      • Berger K.L.
      • Heaton N.S.
      • Cooper J.D.
      • Yoon R.
      • Randall G.
      RNA interference and single particle tracking analysis of hepatitis C virus endocytosis.
      ]. The question whether all or part of the plasma membrane expressed HCV host factors internalize together with HCV still remains unanswered. A recent study suggests that during internalization, HCV associates with CD81 and CLDN1 [
      • Coller K.E.
      • Berger K.L.
      • Heaton N.S.
      • Cooper J.D.
      • Yoon R.
      • Randall G.
      RNA interference and single particle tracking analysis of hepatitis C virus endocytosis.
      ]. Moreover, PKA has been suggested to play a role during this process as inhibition of PKA lead to the reorganization of CLDN1 from the plasma membrane to intracellular vesicular location(s) and disrupted CD81–CLDN1 co-receptor association [
      • Farquhar M.J.
      • Harris H.J.
      • Diskar M.
      • Jones S.
      • Mee C.J.
      • Nielsen S.U.
      • et al.
      Protein kinase A-dependent step(s) in hepatitis C virus entry and infectivity.
      ]. Interestingly, in line with the fact that polarization restricts HCV entry [
      • Mee C.J.
      • Harris H.J.
      • Farquhar M.J.
      • Wilson G.
      • Reynolds G.
      • Davis C.
      • et al.
      Polarization restricts hepatitis C virus entry into HepG2 hepatoma cells.
      ] and that HCV co-entry factors are co-expressed on basolateral sites of hepatocytes but not at TJ [
      • Reynolds G.M.
      • Harris H.J.
      • Jennings A.
      • Hu K.
      • Grove J.
      • Lalor P.F.
      • et al.
      Hepatitis C virus receptor expression in normal and diseased liver tissue.
      ], imaging studies suggest that HCV internalization does not preferentially take place at sites of cell–cell contacts [
      • Coller K.E.
      • Berger K.L.
      • Heaton N.S.
      • Cooper J.D.
      • Yoon R.
      • Randall G.
      RNA interference and single particle tracking analysis of hepatitis C virus endocytosis.
      ].
      Clathrin-mediated endocytosis transports incoming viruses together with their receptors into early and late endosomes [
      • Marsh M.
      • Helenius A.
      Virus entry: open sesame.
      ]. HCVpp have been suggested to be delivered to early but not late endosomes [
      • Meertens L.
      • Bertaux C.
      • Dragic T.
      Hepatitis C virus entry requires a critical postinternalization step and delivery to early endosomes via clathrin-coated vesicles.
      ]. This is in line with recent imaging data showing colocalization between HCV and Rab5a, an early endosome marker [
      • Coller K.E.
      • Berger K.L.
      • Heaton N.S.
      • Cooper J.D.
      • Yoon R.
      • Randall G.
      RNA interference and single particle tracking analysis of hepatitis C virus endocytosis.
      ]. The acidic pH in endosomes provides an essential cue that triggers penetration and uncoating. Penetration of enveloped virus occurs by membrane fusion catalyzed by fusion peptides embedded in the viral envelope glycoproteins [
      • Smith A.E.
      • Helenius A.
      How viruses enter animal cells.
      ]. To date, the mechanisms of HCV fusion have not been completely elucidated but it has been suggested that similar fusion mechanisms as described for other flaviviridae may apply to HCV [
      • Penin F.
      • Dubuisson J.
      • Rey F.A.
      • Moradpour D.
      • Pawlotsky J.M.
      Structural biology of hepatitis C virus.
      ,
      • Yagnik A.T.
      • Lahm A.
      • Meola A.
      • Roccasecca R.M.
      • Ercole B.B.
      • Nicosia A.
      • et al.
      A model for the hepatitis C virus envelope glycoprotein E2.
      ,
      • Heinz F.X.
      • Stiasny K.
      • Allison S.L.
      The entry machinery of flaviviruses.
      ,
      • Voisset C.
      • Dubuisson J.
      Functional hepatitis C virus envelope glycoproteins.
      ]. This hypothesis is supported by the observation that HCVpp entry [
      • Bartosch B.
      • Dubuisson J.
      • Cosset F.L.
      Infectious hepatitis C virus pseudo-particles containing functional E1–E2 envelope protein complexes.
      ,
      • Hsu M.
      • Zhang J.
      • Flint M.
      • Logvinoff C.
      • Cheng-Mayer C.
      • Rice C.M.
      • et al.
      Hepatitis C virus glycoproteins mediate pH-dependent cell entry of pseudotyped retroviral particles.
      ,
      • Lavillette D.
      • Bartosch B.
      • Nourrisson D.
      • Verney G.
      • Cosset F.L.
      • Penin F.
      • et al.
      Hepatitis C virus glycoproteins mediate low pH-dependent membrane fusion with liposomes.
      ] and HCVcc infection [
      • Blanchard E.
      • Belouzard S.
      • Goueslain L.
      • Wakita T.
      • Dubuisson J.
      • Wychowski C.
      • et al.
      Hepatitis C virus entry depends on clathrin-mediated endocytosis.
      ,
      • Tscherne D.M.
      • Jones C.T.
      • Evans M.J.
      • Lindenbach B.D.
      • McKeating J.A.
      • Rice C.M.
      Time- and temperature-dependent activation of hepatitis C virus for low-pH-triggered entry.
      ] are pH-dependent, suggesting that a pH-dependent membrane fusion process may be required for delivery of the HCV genome into the host cell cytosol. It is worth noting that although HCV entry requires an acidification step, extracellular HCV is resistant to low pH treatment [
      • Meertens L.
      • Bertaux C.
      • Dragic T.
      Hepatitis C virus entry requires a critical postinternalization step and delivery to early endosomes via clathrin-coated vesicles.
      ,
      • Tscherne D.M.
      • Jones C.T.
      • Evans M.J.
      • Lindenbach B.D.
      • McKeating J.A.
      • Rice C.M.
      Time- and temperature-dependent activation of hepatitis C virus for low-pH-triggered entry.
      ]. As HCV fusion kinetics are delayed as compared to other viruses, it has been suggested that after internalization, HCVpp entry necessitates additional, low-pH-dependent interactions, modifications, or trafficking [
      • Meertens L.
      • Bertaux C.
      • Dragic T.
      Hepatitis C virus entry requires a critical postinternalization step and delivery to early endosomes via clathrin-coated vesicles.
      ]. However, neither HCVpp nor HCVcc require cleavage by endosomal proteases for fusion [
      • Meertens L.
      • Bertaux C.
      • Dragic T.
      Hepatitis C virus entry requires a critical postinternalization step and delivery to early endosomes via clathrin-coated vesicles.
      ,
      • Tscherne D.M.
      • Jones C.T.
      • Evans M.J.
      • Lindenbach B.D.
      • McKeating J.A.
      • Rice C.M.
      Time- and temperature-dependent activation of hepatitis C virus for low-pH-triggered entry.
      ]. Several in vitro fusion assays have been set up in the last years [
      • Kobayashi M.
      • Bennett M.C.
      • Bercot T.
      • Singh I.R.
      Functional analysis of hepatitis C virus envelope proteins, using a cell–cell fusion assay.
      ,
      • Lavillette D.
      • Pecheur E.I.
      • Donot P.
      • Fresquet J.
      • Molle J.
      • Corbau R.
      • et al.
      Characterization of fusion determinants points to the involvement of three discrete regions of both E1 and E2 glycoproteins in the membrane fusion process of hepatitis C virus.
      ,
      • Evans M.J.
      • von Hahn T.
      • Tscherne D.M.
      • Syder A.J.
      • Panis M.
      • Wolk B.
      • et al.
      Claudin-1 is a hepatitis C virus co-receptor required for a late step in entry.
      ,
      • Lavillette D.
      • Bartosch B.
      • Nourrisson D.
      • Verney G.
      • Cosset F.L.
      • Penin F.
      • et al.
      Hepatitis C virus glycoproteins mediate low pH-dependent membrane fusion with liposomes.
      ]. Liposome/HCVpp fusion assays suggest that HCVpp-induced fusion was low pH and temperature-dependent, and facilitated by cholesterol [
      • Lavillette D.
      • Bartosch B.
      • Nourrisson D.
      • Verney G.
      • Cosset F.L.
      • Penin F.
      • et al.
      Hepatitis C virus glycoproteins mediate low pH-dependent membrane fusion with liposomes.
      ]. Interestingly, patient-derived anti-HCV antibodies were able to inhibit liposome/HCVpp fusion [
      • Haberstroh A.
      • Schnober E.K.
      • Zeisel M.B.
      • Carolla P.
      • Barth H.
      • Blum H.E.
      • et al.
      Neutralizing host responses in hepatitis C virus infection target viral entry at postbinding steps and membrane fusion.
      ] thus highlighting the importance of HCV envelope glycoproteins in this process. These data have been recently confirmed in a novel liposome/HCVcc fusion assay showing that HCVcc fusion was dependent on pH, lipid composition of both viral and target membranes, and HCV E2 [
      • Haid S.
      • Pietschmann T.
      • Pecheur E.I.
      Low pH-dependent hepatitis C virus membrane fusion depends on E2 integrity, target lipid composition, and density of virus particles.
      ]. However, in this kind of assay no host cell factor is necessary to allow fusion to occur. To study the role of both viral and host factors in HCV fusion, cell–cell fusion assays have been used where HCV envelope glycoproteins are expressed on one cell type and host entry factors on another cell type [
      • Kobayashi M.
      • Bennett M.C.
      • Bercot T.
      • Singh I.R.
      Functional analysis of hepatitis C virus envelope proteins, using a cell–cell fusion assay.
      ]. Cell–cell fusion assays are also pH-dependent and most interestingly, these assays highlighted the importance of CD81 and CLDN1 in this process [
      • Kobayashi M.
      • Bennett M.C.
      • Bercot T.
      • Singh I.R.
      Functional analysis of hepatitis C virus envelope proteins, using a cell–cell fusion assay.
      ,
      • Evans M.J.
      • von Hahn T.
      • Tscherne D.M.
      • Syder A.J.
      • Panis M.
      • Wolk B.
      • et al.
      Claudin-1 is a hepatitis C virus co-receptor required for a late step in entry.
      ]. To date, it still remains unclear whether these host factors directly participate in the HCV fusion process or whether they play a role in an earlier entry step required to enable efficient subsequent fusion. Taken together, these data suggest that HCV internalization and fusion offer multiple targets for the development of HCV entry inhibitors.

      An alternative route of entry and spread by cell–cell transmission

      The above described entry mechanisms have been unravelled using cell-free HCV, i.e. the virus infects surrounding cells after the formation of viral particles that are released from infected cells and enter naïve cells by a host factor-dependent mechanism. In addition, viruses may also use direct cell–cell transfer to infect neighbouring cells [
      • Marsh M.
      • Helenius A.
      Virus entry: open sesame.
      ] thereby escaping potential interactions with neutralizing antibodies in the extracellular milieu.
      Direct cell–cell transfer or neutralizing antibody-resistant transmission has been described for HCV [
      • Timpe J.M.
      • Stamataki Z.
      • Jennings A.
      • Hu K.
      • Farquhar M.J.
      • Harris H.J.
      • et al.
      Hepatitis C virus cell–cell transmission in hepatoma cells in the presence of neutralizing antibodies.
      ]. CLDN1, CD81, and probably SR-BI are involved in this process [
      • Schwarz A.K.
      • Grove J.
      • Hu K.
      • Mee C.J.
      • Balfe P.
      • McKeating J.A.
      Hepatoma cell density promotes claudin-1 and scavenger receptor BI expression and hepatitis C virus internalization.
      ,
      • Timpe J.M.
      • Stamataki Z.
      • Jennings A.
      • Hu K.
      • Farquhar M.J.
      • Harris H.J.
      • et al.
      Hepatitis C virus cell–cell transmission in hepatoma cells in the presence of neutralizing antibodies.
      ]. Interestingly, CD81-independent routes of cell–cell transport have also been described [
      • Timpe J.M.
      • Stamataki Z.
      • Jennings A.
      • Hu K.
      • Farquhar M.J.
      • Harris H.J.
      • et al.
      Hepatitis C virus cell–cell transmission in hepatoma cells in the presence of neutralizing antibodies.
      ,
      • Witteveldt J.
      • Evans M.J.
      • Bitzegeio J.
      • Koutsoudakis G.
      • Owsianka A.M.
      • Angus A.G.
      • et al.
      CD81 is dispensable for hepatitis C virus cell-to-cell transmission in hepatoma cells.
      ]. Direct cell–cell transfer has an important impact for the development of antivirals as this process allows viral spreading by escaping extracellular neutralizing antibodies as well as defined antibodies interfering with host cell entry factors. It will be challenging to develop novel anti-HCV therapeutics interfering with this process.

      Viral entry offers promising targets for antiviral therapy

      In contrast to the current standard of care therapy for HCV infection, new therapeutic approaches aim at the development of more specific compounds targeting the virus and/or host cell factors. This represents the concept of specifically targeted antiviral therapy for HCV (STAT-C). This concept consists in developing more efficient and better tolerated combination therapies that need shorter treatment periods. To date, several small molecular compounds targeting the HCV non-structural proteins including protease, polymerase, and NS5A have been developed and are at various stages of clinical development [
      • Hezode C.
      • Forestier N.
      • Dusheiko G.
      • Ferenci P.
      • Pol S.
      • Goeser T.
      • et al.
      Telaprevir and peginterferon with or without ribavirin for chronic HCV infection.
      ,
      • Pereira A.A.
      • Jacobson I.M.
      New and experimental therapies for HCV.
      ,
      • Sarrazin C.
      • Zeuzem S.
      Resistance to direct antiviral agents in patients with hepatitis C virus infection.
      ,
      • Gao M.
      • Nettles R.E.
      • Belema M.
      • Snyder L.B.
      • Nguyen V.N.
      • Fridell R.A.
      • et al.
      Chemical genetics strategy identifies an HCV NS5A inhibitor with a potent clinical effect.
      ]. First clinical trial data are promising but toxicity of the individual compounds and the emergence of resistance against these drugs limit their use in monotherapy. This suggests that additional drugs, ideally targeting different steps of the viral life cycle, are needed for efficient anti-HCV therapy.
      Figure thumbnail fx3
      HCV entry into target cells is a promising target for preventive and therapeutic antiviral strategies since it is essential for the initiation, spread, and maintenance of infection. Interfering with HCV entry holds great promises for drug design and offers several targets: (i) blocking virus–target cell interaction during attachment and binding, (ii) interfering with post-binding events, and (iii) interfering with viral fusion (Fig. 1). Various modalities may be developed as HCV entry inhibitors: these include neutralizing antibodies targeting the viral envelope and inhibitory/blocking antibodies targeting host cell surface factors as well as small molecule compounds or siRNAs against host cell factors or viral proteins [
      • Zeisel M.B.
      • Barth H.
      • Schuster C.
      • Baumert T.F.
      Hepatitis C virus entry: molecular mechanisms and targets for antiviral therapy.
      ,
      • Khaliq S.
      • Khaliq S.A.
      • Zahur M.
      • Ijaz B.
      • Jahan S.
      • Ansar M.
      • et al.
      RNAi as a new therapeutic strategy against HCV.
      ].
      Several non-HCV specific molecules interfering with HCV envelope glycoproteins and abrogating viral attachment have been described. As HCV envelope proteins are highly glycosylated, molecules interfering with glycoproteins may possess antiviral activity against HCV. As shown for HIV, targeting the glycans may represent a new therapeutic concept for controlling HCV infection [
      • Balzarini J.
      Carbohydrate-binding agents: a potential future cornerstone for the chemotherapy of enveloped viruses?.
      ]. Carbohydrate-binding agents that interact with the viral-envelope glycans may compromise the efficient entry of the virus into its susceptible target cells and induce a progressive creation of deletions in the envelope glycan shield, thereby triggering the immune system to act against previously hidden immunogenic epitopes of the viral envelope [
      • Balzarini J.
      Carbohydrate-binding agents: a potential future cornerstone for the chemotherapy of enveloped viruses?.
      ]. The lectin cyanovirin-N (CV-N) interacts with high-mannose oligosaccharides on viral envelope glycoproteins and has been demonstrated to possess antiviral activity against several enveloped viruses [
      • Boyd M.R.
      • Gustafson K.R.
      • McMahon J.B.
      • Shoemaker R.H.
      • O’Keefe B.R.
      • Mori T.
      • et al.
      Discovery of cyanovirin-N, a novel human immunodeficiency virus-inactivating protein that binds viral surface envelope glycoprotein gp120: potential applications to microbicide development.
      ,
      • O’Keefe B.R.
      • Smee D.F.
      • Turpin J.A.
      • Saucedo C.J.
      • Gustafson K.R.
      • Mori T.
      • et al.
      Potent anti-influenza activity of cyanovirin-N and interactions with viral hemagglutinin.
      ,
      • Shenoy S.R.
      • O’Keefe B.R.
      • Bolmstedt A.J.
      • Cartner L.K.
      • Boyd M.R.
      Selective interactions of the human immunodeficiency virus-inactivating protein cyanovirin-N with high-mannose oligosaccharides on gp120 and other glycoproteins.
      ]. It has been shown that oligomannose glycans within the HCV envelope glycoproteins interact with CV-N resulting in HCV antiviral activity by blocking HCV entry into target cells [
      • Helle F.
      • Wychowski C.
      • Vu-Dac N.
      • Gustafson K.R.
      • Voisset C.
      • Dubuisson J.
      Cyanovirin-N inhibits hepatitis C virus entry by binding to envelope protein glycans.
      ]. As most of the HCV glycosylation sites are highly conserved, drugs that target glycans on HCV glycoproteins may not lead so rapidly to viral escape/resistance as it is the case for HIV [
      • Wei X.
      • Decker J.M.
      • Wang S.
      • Hui H.
      • Kappes J.C.
      • Wu X.
      • et al.
      Antibody neutralization and escape by HIV-1.
      ]. Other carbohydrate-binding agents that have been shown to prevent HIV infectivity [
      • Balzarini J.
      Carbohydrate-binding agents: a potential future cornerstone for the chemotherapy of enveloped viruses?.
      ] might also be efficient against other viruses that require a glycosylated envelope for entry into target cells. Interfering with the interaction of viral envelope proteins and glycosaminoglycans on the cell surface is another way to abrogate viral attachment. HS glycosaminoglycans mediate HCV and dengue virus binding to host cells and heparin, a structural analogue of HS, has been demonstrated to inhibit dengue virus infection as wells as HCV E2, HCVpp, HCV-LP, and HCVcc binding to hepatoma cells [
      • Barth H.
      • Schnober E.K.
      • Zhang F.
      • Linhardt R.J.
      • Depla E.
      • Boson B.
      • et al.
      Viral and cellular determinants of the hepatitis C virus envelope–heparan sulfate interaction.
      ,
      • Barth H.
      • Schäfer C.
      • Adah M.I.
      • Zhang F.
      • Linhardt R.J.
      • Toyoda H.
      • et al.
      Cellular binding of hepatitis C virus envelope glycoprotein E2 requires cell surface heparan sulfate.
      ,
      • Koutsoudakis G.
      • Kaul A.
      • Steinmann E.
      • Kallis S.
      • Lohmann V.
      • Pietschmann T.
      • et al.
      Characterization of the early steps of hepatitis C virus infection by using luciferase reporter viruses.
      ,
      • Cormier E.G.
      • Tsamis F.
      • Kajumo F.
      • Durso R.J.
      • Gardner J.P.
      • Dragic T.
      CD81 is an entry coreceptor for hepatitis C virus.
      ]. HS-like molecules and semisynthetic derivatives are already explored as an antiviral approach against dengue virus infection [
      • Lee E.
      • Pavy M.
      • Young N.
      • Freeman C.
      • Lobigs M.
      Antiviral effect of the heparan sulfate mimetic, PI-88, against dengue and encephalitic flaviviruses.
      ]. Such molecules may also have antiviral activity against HCV.
      Neutralization of the viral particle may be achieved by targeting the HCV envelope or host derived factors associated with the mature viral particle. The molecular mechanisms of viral assembly and the exact composition of released HCV particles still remain elusive but recent studies suggest that HCV and VLDL assembly are closely linked [
      • Huang H.
      • Sun F.
      • Owen D.M.
      • Li W.
      • Chen Y.
      • Gale Jr., M.
      • et al.
      Hepatitis C virus production by human hepatocytes dependent on assembly and secretion of very low-density lipoproteins.
      ,
      • Gastaminza P.
      • Cheng G.
      • Wieland S.
      • Zhong J.
      • Liao W.
      • Chisari F.V.
      Cellular determinants of hepatitis C virus assembly, maturation, degradation, and secretion.
      ]. Noteworthy, apolipoprotein E (apoE) is required for HCV assembly [
      • Chang K.S.
      • Jiang J.
      • Cai Z.
      • Luo G.
      Human apolipoprotein e is required for infectivity and production of hepatitis C virus in cell culture.
      ,
      • Benga W.J.
      • Krieger S.E.
      • Dimitrova M.
      • Zeisel M.B.
      • Parnot M.
      • Lupberger J.
      • et al.
      Apolipoprotein E interacts with hepatitis C virus nonstructural protein 5A and determines assembly of infectious particles.
      ] and is also part of infectious HCV particles [
      • Chang K.S.
      • Jiang J.
      • Cai Z.
      • Luo G.
      Human apolipoprotein e is required for infectivity and production of hepatitis C virus in cell culture.
      ]. Interestingly, anti-apoE antibodies are able to inhibit HCV entry [
      • Agnello V.
      • Abel G.
      • Elfahal M.
      • Knight G.B.
      • Zhang Q.X.
      Hepatitis C virus and other flaviviridae viruses enter cells via low density lipoprotein receptor.
      ,
      • Chang K.S.
      • Jiang J.
      • Cai Z.
      • Luo G.
      Human apolipoprotein e is required for infectivity and production of hepatitis C virus in cell culture.
      ] suggesting that HCV may be neutralized using compounds directed against the lipoprotein moiety of the viral particle (Table 1).
      Table 1Examples of compounds targeting viral entry. Targets within the HCV entry process are depicted, followed by examples of compounds targeting the respective entry step. Stages of development and references are indicated. CLDN, claudin; mAb, monoclonal antibody; pAb, polyclonal antibodies; SR-BI, scavenger receptor class B type I; PS-ON: phosphorothioate oligonucleotides. (
      • Davis G.L.
      • Nelson D.R.
      • Terrault N.
      • Pruett T.L.
      • Schiano T.D.
      • Fletcher C.V.
      • et al.
      A randomized, open-label study to evaluate the safety and pharmacokinetics of human hepatitis C immune globulin (Civacir) in liver transplant recipients.
      ,
      • Borgia G.
      HepeX-C (XTL Biopharmaceuticals).
      ,
      • Schiano T.D.
      • Charlton M.
      • Younossi Z.
      • Galun E.
      • Pruett T.
      • Tur-Kaspa R.
      • et al.
      Monoclonal antibody HCV-AbXTL68 in patients undergoing liver transplantation for HCV: results of a phase 2 randomized study.
      ,
      • Eren R.
      • Landstein D.
      • Terkieltaub D.
      • Nussbaum O.
      • Zauberman A.
      • Ben-Porath J.
      • et al.
      Preclinical evaluation of two neutralizing human monoclonal antibodies against hepatitis C virus (HCV): a potential treatment to prevent HCV reinfection in liver transplant patients.
      ,
      • Keck Z.Y.
      • Li T.K.
      • Xia J.
      • Bartosch B.
      • Cosset F.L.
      • Dubuisson J.
      • et al.
      Analysis of a highly flexible conformational immunogenic domain a in hepatitis C virus E2.
      ,
      • Owsianka A.
      • Tarr A.W.
      • Juttla V.S.
      • Lavillette D.
      • Bartosch B.
      • Cosset F.L.
      • et al.
      Monoclonal antibody AP33 defines a broadly neutralizing epitope on the hepatitis C virus E2 envelope glycoprotein.
      ,
      • Grove J.
      • Nielsen S.
      • Zhong J.
      • Bassendine M.F.
      • Drummer H.E.
      • Balfe P.
      • et al.
      Identification of a residue in hepatitis C virus E2 glycoprotein that determines scavenger receptor BI and CD81 receptor dependency and sensitivity to neutralizing antibodies.
      ,
      • Broering T.J.
      • Garrity K.A.
      • Boatright N.K.
      • Sloan S.E.
      • Sandor F.
      • Thomas Jr., W.D.
      • et al.
      Identification and characterization of broadly neutralizing human monoclonal antibodies directed against the E2 envelope glycoprotein of hepatitis C virus.
      ,
      • Biermer M.
      • Berg T.
      Rapid suppression of hepatitis C viremia induced by intravenous silibinin plus ribavirin.
      )
      Viral attachment and entry is a major target of adaptive host cell defenses and anti-HCV antibodies represent unique tools to interfere with the HCV entry process. Virus-specific neutralizing antibodies are defined by their antiviral activity enabling them to block viral entry and control viral spread. Neutralizing antibodies may interfere with different steps of the viral entry process, such as attachment, post-binding steps, and fusion [
      • Zeisel M.B.
      • Fafi-Kremer S.
      • Fofana I.
      • Barth H.
      • Stoll-Keller F.
      • Doffoel M.
      • et al.
      Neutralizing antibodies in hepatitis C virus infection.
      ]. Two studies of large-scale accidental HCV infections demonstrated that rapid induction of neutralizing antibodies in the early phase of infection correlates with viral clearance or control of infection [
      • Pestka J.M.
      • Zeisel M.B.
      • Blaser E.
      • Schurmann P.
      • Bartosch B.
      • Cosset F.L.
      • et al.
      Rapid induction of virus-neutralizing antibodies and viral clearance in a single-source outbreak of hepatitis C.
      ,
      • Lavillette D.
      • Morice Y.
      • Germanidis G.
      • Donot P.
      • Soulier A.
      • Pagkalos E.
      • et al.
      Human serum facilitates hepatitis C virus infection, and neutralizing responses inversely correlate with viral replication kinetics at the acute phase of hepatitis C virus infection.
      ]. These studies suggest that neutralizing antibodies represent an interesting approach for the development of novel preventive and therapeutic antiviral strategies (Table 1). In line with this concept, it has been shown that immunoglobulins prepared from unscreened donors or from selected patients with chronic HCV infection have prevented HCV infection in recipients when administered before exposure to the virus [
      • Rosa D.
      • Campagnoli S.
      • Moretto C.
      • Guenzi E.
      • Cousens L.
      • Chin M.
      • et al.
      A quantitative test to estimate neutralizing antibodies to the hepatitis C virus: cytofluorimetric assessment of envelope glycoprotein 2 binding to target cells.
      ,
      • Zibert A.
      • Schreier E.
      • Roggendorf M.
      Antibodies in human sera specific to hypervariable region 1 of hepatitis C virus can block viral attachment.
      ]. Moreover, administration of polyclonal immunoglobulins from a chronically infected patient conveyed sterilizing immunity toward a homologous strain in human liver-chimeric Alb-uPA/SCID mouse model [
      • Vanwolleghem T.
      • Bukh J.
      • Meuleman P.
      • Desombere I.
      • Meunier J.C.
      • Alter H.
      • et al.
      Polyclonal immunoglobulins from a chronic hepatitis C virus patient protect human liver-chimeric mice from infection with a homologous hepatitis C virus strain.
      ]. Human mAbs provide an attractive alternative to polyclonal immune globulin for immunotherapy, since mAbs can be more readily standardized. The recent production of human mAbs efficiently cross-neutralizing HCV may represent an important step for the development of immunopreventive strategies against HCV infection [
      • Law M.
      • Maruyama T.
      • Lewis J.
      • Giang E.
      • Tarr A.W.
      • Stamataki Z.
      • et al.
      Broadly neutralizing antibodies protect against hepatitis C virus quasispecies challenge.
      ,
      • Perotti M.
      • Mancini N.
      • Diotti R.A.
      • Tarr A.W.
      • Ball J.K.
      • Owsianka A.
      • et al.
      Identification of a broadly cross-reacting and neutralizing human monoclonal antibody directed against the hepatitis C virus E2 protein.
      ,
      • Johansson D.X.
      • Voisset C.
      • Tarr A.W.
      • Aung M.
      • Ball J.K.
      • Dubuisson J.
      • et al.
      Human combinatorial libraries yield rare antibodies that broadly neutralize hepatitis C virus.
      ,
      • Meunier J.C.
      • Russell R.S.
      • Goossens V.
      • Priem S.
      • Walter H.
      • Depla E.
      • et al.
      Isolation and characterization of broadly neutralizing human monoclonal antibodies to the e1 glycoprotein of hepatitis C virus.
      ] as such antibodies have been demonstrated to protect against HCV quasi-species challenges in vivo in the human liver-chimeric Alb-uPA/SCID mouse model [
      • Law M.
      • Maruyama T.
      • Lewis J.
      • Giang E.
      • Tarr A.W.
      • Stamataki Z.
      • et al.
      Broadly neutralizing antibodies protect against hepatitis C virus quasispecies challenge.
      ] (Table 1). However, due to the high variability of HCV, it will be a major challenge to develop efficient cross-neutralizing antibodies able to target conserved epitopes across all genotypes to avoid escape. Examples for neutralizing antibodies in preclinical or clinical development are provided in Table 1.
      Targeting the host entry factors, which are indispensable for the propagation of the virus, represents an additional approach for the development of antivirals because they may impose a higher genetic barrier for resistance. HCV interaction with host entry factors offers multiple targets for the development of specific entry inhibitors (Table 1).
      CD81 is one of these potential targets. Imidazole based compounds mimicking an alpha helix in the LEL of CD81 compete for HCV E2–CD81 binding. These drugs bind E2 in a reversible manner and block E2–CD81 interaction while having no effect on CD81 expression nor on CD81 interaction with physiological partner molecules [
      • VanCompernolle S.E.
      • Wiznycia A.V.
      • Rush J.R.
      • Dhanasekaran M.
      • Baures P.W.
      • Todd S.C.
      Small molecule inhibition of hepatitis C virus E2 binding to CD81.
      ]. Interestingly, anti-CD81 antibodies inhibiting HCV infection in vitro have also been demonstrated to prevent HCV infection in the human liver-chimeric Alb-uPA/SCID mouse model [
      • Meuleman P.
      • Hesselgesser J.
      • Paulson M.
      • Vanwolleghem T.
      • Desombere I.
      • Reiser H.
      • et al.
      Anti-CD81 antibodies can prevent a hepatitis C virus infection in vivo.
      ]. This study suggests that targeting CD81 may be an efficient strategy to prevent HCV infection in vivo and demonstrates the proof-of-concept that anti-receptor antibodies prevent HCV infection in a clinically relevant animal model.
      SR-BI binds a wide variety of molecules and is thus another interesting target for anti-HCV drugs. SR-BI binds and internalizes serum amyloid A (SAA), an acute phase protein produced in the liver [
      • Baranova I.N.
      • Vishnyakova T.G.
      • Bocharov A.V.
      • Kurlander R.
      • Chen Z.
      • Kimelman M.L.
      • et al.
      Serum amyloid A binding to CLA-1 (CD36 and LIMPII analogous-1) mediates serum amyloid A protein-induced activation of ERK1/2 and p38 mitogen-activated protein kinases.
      ,
      • Cai L.
      • de Beer M.C.
      • de Beer F.C.
      • van der Westhuyzen D.R.
      Serum amyloid A is a ligand for scavenger receptor class B type I and inhibits high density lipoprotein binding and selective lipid uptake.
      ]. SAA inhibits HCV entry by interacting with the virus thereby reducing its infectivity [
      • Lavie M.
      • Voisset C.
      • Vu-Dac N.
      • Zurawski V.
      • Duverlie G.
      • Wychowski C.
      • et al.
      Serum amyloid A has antiviral activity against hepatitis C virus by inhibiting virus entry in a cell culture system.
      ]. Anti-SR-BI antibodies blocking interaction with HCV are another interesting strategy to prevent HCV entry. Anti-SR-BI antibodies have been demonstrated to inhibit HCVcc infection in vitro [
      • Zeisel M.B.
      • Koutsoudakis G.
      • Schnober E.K.
      • Haberstroh A.
      • Blum H.E.
      • Cosset F.-L.
      • et al.
      Scavenger receptor BI is a key host factor for hepatitis C virus infection required for an entry step closely linked to CD81.
      ,
      • Bartosch B.
      • Verney G.
      • Dreux M.
      • Donot P.
      • Morice Y.
      • Penin F.
      • et al.
      An interplay between hypervariable region 1 of the hepatitis C virus E2 glycoprotein, the scavenger receptor BI, and high-density lipoprotein promotes both enhancement of infection and protection against neutralizing antibodies.
      ,
      • Catanese M.T.
      • Graziani R.
      • von Hahn T.
      • Moreau M.
      • Huby T.
      • Paonessa G.
      • et al.
      High-avidity monoclonal antibodies against the human scavenger class B type I receptor efficiently block hepatitis C virus infection in the presence of high-density lipoprotein.
      ]. Finally, small molecule inhibitors of SR-BI have recently been developed. ITX5061 is a compound that inhibits entry of HCVpp from all major genotypes and HCVcc infection without affecting viral replication [
      • Syder A.J.
      • Haekyung L.
      • Zeisel M.B.
      • Grove J.
      • Soulier E.
      • MacDonald J.
      • et al.
      Small molecule scavenger receptor bi antagonists are potent HCV entry inhibitors.
      ]. Kinetic studies suggest that this small molecule inhibitor targets HCV entry during an early post-binding stage [
      • Syder A.J.
      • Haekyung L.
      • Zeisel M.B.
      • Grove J.
      • Soulier E.
      • MacDonald J.
      • et al.
      Small molecule scavenger receptor bi antagonists are potent HCV entry inhibitors.
      ]. The safety of this compound has been evaluated in patients for another clinical indication [
      • Syder A.J.
      • Haekyung L.
      • Zeisel M.B.
      • Grove J.
      • Soulier E.
      • MacDonald J.
      • et al.
      Small molecule scavenger receptor bi antagonists are potent HCV entry inhibitors.
      ] allowing future clinical trials in HCV infected patients.
      CLDN1 is a promising antiviral target since it is essential for HCV entry and to date there is no evidence for CLDN1-independent HCV entry. Furthermore, CLDN1 has been suggested to play an important role in cell–cell transmission. In contrast to other HCV entry factors such as CD81 or SR-BI, CLDN1 is predominantly expressed in the liver. Recently, anti-CLDN1 antibodies inhibiting HCV infection in vitro have been developed [
      • Krieger S.E.
      • Zeisel M.B.
      • Davis C.
      • Thumann C.
      • Harris H.J.
      • Schnober E.K.
      • et al.
      Inhibition of hepatitis C virus infection by anti-claudin-1 antibodies is mediated by neutralization of E2–CD81–claudin-1 associations.
      ,
      • Fofana I.
      • Krieger S.E.
      • Grunert F.
      • Glauben S.
      • Xiao F.
      • Fafi-Kremer S.
      • et al.
      Monoclonal anti-claudin 1 antibodies prevent hepatitis C virus infection of primary human hepatocytes.
      ]. Anti-CLDN1 antibodies inhibit HCV infectivity by reducing HCV E2 association with the cell surface and disrupting CLDN1–CD81 interactions [
      • Krieger S.E.
      • Zeisel M.B.
      • Davis C.
      • Thumann C.
      • Harris H.J.
      • Schnober E.K.
      • et al.
      Inhibition of hepatitis C virus infection by anti-claudin-1 antibodies is mediated by neutralization of E2–CD81–claudin-1 associations.
      ]. Interestingly, monoclonal anti-CLDN1 antibodies efficiently block cell entry of highly infectious escape variants of HCV that are resistant to host neutralizing antibodies [
      • Fofana I.
      • Krieger S.E.
      • Grunert F.
      • Glauben S.
      • Xiao F.
      • Fafi-Kremer S.
      • et al.
      Monoclonal anti-claudin 1 antibodies prevent hepatitis C virus infection of primary human hepatocytes.
      ]. These data suggest that anti-CLDN1 antibodies might be used to prevent HCV infection, such as after LT, and might also restrain virus spread in chronically infected patients [
      • Fofana I.
      • Krieger S.E.
      • Grunert F.
      • Glauben S.
      • Xiao F.
      • Fafi-Kremer S.
      • et al.
      Monoclonal anti-claudin 1 antibodies prevent hepatitis C virus infection of primary human hepatocytes.
      ].
      Finally, OCLN may also be considered as a potential target for interfering with HCV entry. To date, no anti-OCLN antibodies inhibiting HCV infection have been described. Further characterization of the role of this host cell factor in the HCV entry process may lead to designing compounds interfering with OCLN and inhibiting HCV entry.
      In addition to cell surface expressed host factors, HCV internalization and fusion are complex processes that also offer several targets for antivirals. Long phosphorothioate oligonucleotides (PS-ON) are a promising new class of antiviral compounds. These amphipathic DNA polymers display a sequence-independent antiviral activity against HIV by blocking virus–cell fusion [
      • Vaillant A.
      • Juteau J.M.
      • Lu H.
      • Liu S.
      • Lackman-Smith C.
      • Ptak R.
      • et al.
      Phosphorothioate oligonucleotides inhibit human immunodeficiency virus type 1 fusion by blocking gp41 core formation.
      ]. A recent study demonstrated that PS-ON inhibited HCV internalization without affecting viral binding and replication [
      • Matsumura T.
      • Hu Z.
      • Kato T.
      • Dreux M.
      • Zhang Y.Y.
      • Imamura M.
      • et al.
      Amphipathic DNA polymers inhibit hepatitis C virus infection by blocking viral entry.
      ]. A noteworthy observation is that PS-ON block de novo HCV infection in the human liver-chimeric Alb-uPA/SCID mouse model [
      • Matsumura T.
      • Hu Z.
      • Kato T.
      • Dreux M.
      • Zhang Y.Y.
      • Imamura M.
      • et al.
      Amphipathic DNA polymers inhibit hepatitis C virus infection by blocking viral entry.
      ] highlighting the promise of PS-ON as future clinical HCV entry inhibitors (Table 1). A peptide-based HIV fusion inhibitor (Enfuvirtide) has already been approved for treatment of HIV infected patients [
      • Poveda E.
      • Briz V.
      • Soriano V.
      Enfuvirtide, the first fusion inhibitor to treat HIV infection.
      ]. As HCV fusion requires acidification of the endosome, molecules able to prevent acidification of endosomes, such as chloroquine, prevent HCV fusion in vitro [
      • Blanchard E.
      • Belouzard S.
      • Goueslain L.
      • Wakita T.
      • Dubuisson J.
      • Wychowski C.
      • et al.
      Hepatitis C virus entry depends on clathrin-mediated endocytosis.
      ,
      • Tscherne D.M.
      • Jones C.T.
      • Evans M.J.
      • Lindenbach B.D.
      • McKeating J.A.
      • Rice C.M.
      Time- and temperature-dependent activation of hepatitis C virus for low-pH-triggered entry.
      ]. In the last few years, other compounds interfering with HCV fusion have been described (Table 1). Arbidol is a broad-spectrum antiviral that has already been evaluated in humans indicating good safety and tolerability (for review see [
      • Boriskin Y.S.
      • Leneva I.A.
      • Pecheur E.I.
      • Polyak S.J.
      Arbidol: a broad-spectrum antiviral compound that blocks viral fusion.
      ]). Arbidol inhibits HCV fusion in the HCVpp-liposome assay and prevents HCVpp and HCVcc infection in vitro [
      • Boriskin Y.S.
      • Leneva I.A.
      • Pecheur E.I.
      • Polyak S.J.
      Arbidol: a broad-spectrum antiviral compound that blocks viral fusion.
      ]. In addition, this molecule also targets other steps of the viral life cycle such as replication [
      • Boriskin Y.S.
      • Pecheur E.I.
      • Polyak S.J.
      Arbidol: a broad-spectrum antiviral that inhibits acute and chronic HCV infection.
      ]. Silymarin is another compound inhibiting HCVpp-liposome fusion as well as other steps of the HCV life cycle, such as replication, protein expression, and infectious virus production without affecting viral assembly [
      • Polyak S.J.
      • Morishima C.
      • Shuhart M.C.
      • Wang C.C.
      • Liu Y.
      • Lee D.Y.
      Inhibition of T-cell inflammatory cytokines, hepatocyte NF-κB signaling, and HCV infection by standardized silymarin.
      ,
      • Wagoner J.
      • Negash A.
      • Kane O.J.
      • Martinez L.E.
      • Nahmias Y.
      • Bourne N.
      • et al.
      Multiple effects of silymarin on the hepatitis C virus lifecycle.
      ]. Interestingly, silymarin inhibited HCV infection in vitro irrespective of the entry route, i.e. cell-free and cell–cell transmission [
      • Wagoner J.
      • Negash A.
      • Kane O.J.
      • Martinez L.E.
      • Nahmias Y.
      • Bourne N.
      • et al.
      Multiple effects of silymarin on the hepatitis C virus lifecycle.
      ], highlighting the potential of such drugs for in vivo use [
      • Zeisel M.B.
      • Turek M.
      • Baumert T.F.
      Tight junctions and viral entry.
      ].

      Conclusions and perspectives

      In recent years, substantial progress unravelling the molecular mechanisms of HCV entry has been made and revealed a multitude of novel targets for antivirals. Several compounds interfering with HCV entry have been demonstrated to efficiently inhibit HCV infection using in vitro assays or state of the art animal models and may thus be valuable for future anti-HCV therapy or prevention of HCV infection during LT. As for other chronic viral infections such as HIV, the future therapeutic and preventive approach for HCV infection will probably be based on the combination of several drugs [
      • Pereira A.A.
      • Jacobson I.M.
      New and experimental therapies for HCV.
      ,
      • Sarrazin C.
      • Zeuzem S.
      Resistance to direct antiviral agents in patients with hepatitis C virus infection.
      ]. HCV entry inhibitors represent a promising class of novel antivirals since they are complementary to current approaches and target an essential step of the viral life cycle. Indeed, the first compounds have reached the early stage of clinical development. Moreover, recent data suggest that combination of antivirals targeting the virus and host factors such as CLDN1 act in an additive manner in suppressing HCV infection [
      • Fofana I.
      • Krieger S.E.
      • Grunert F.
      • Glauben S.
      • Xiao F.
      • Fafi-Kremer S.
      • et al.
      Monoclonal anti-claudin 1 antibodies prevent hepatitis C virus infection of primary human hepatocytes.
      ]. Thus, combining compounds targeting viral and host cell factors and complementary steps of the viral life cycle such as entry and replication is a promising approach for the prevention of infection in LT and for cure of chronic HCV infection.

      Conflict of interest

      The authors who have taken part in this study declare that they do not have anything to disclose regarding funding or conflict of interest with respect to this manuscript. Inserm, the University of Strasbourg and Genovac have filed a patent application on monoclonal anti-claudin 1 antibodies for the inhibition of hepatitis C virus infection.

      Acknowledgements

      The authors acknowledge financial support of their work by the European Union (ERC-2008-AdG-233130-HEPCENT and INTERREG-IV-2009-FEDER-Hepato-Regio-Net), ANRS (2007/306 and 2008/354), the Région Alsace (2007/09), the Else Kröner-Fresenius Foundation (EKFS P17//07//A83/06), the Ligue Contre le Cancer (CA 06/12), Inserm, University of Strasbourg, and the Strasbourg University Hospitals, France. We apologize to all authors whose work could not be cited due to space restrictions.

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