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Interferon-stimulated genes and their role in controlling hepatitis C virus

  • Author Footnotes
    † These authors contributed equally to this work.
    Philippe Metz
    Footnotes
    † These authors contributed equally to this work.
    Affiliations
    Department of Infectious Diseases, Molecular Virology, Heidelberg University, Heidelberg, Germany
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  • Author Footnotes
    † These authors contributed equally to this work.
    Antje Reuter
    Footnotes
    † These authors contributed equally to this work.
    Affiliations
    Department of Infectious Diseases, Molecular Virology, Heidelberg University, Heidelberg, Germany
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  • Author Footnotes
    † These authors contributed equally to this work.
    Silke Bender
    Footnotes
    † These authors contributed equally to this work.
    Affiliations
    Department of Infectious Diseases, Molecular Virology, Heidelberg University, Heidelberg, Germany
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  • Ralf Bartenschlager
    Correspondence
    Corresponding author. Address: Department of Infectious Diseases, Molecular Virology, Heidelberg University, Im Neuenheimer Feld 345, Heidelberg, Germany. Tel.: +49 6221 564225; fax: +49 6221 564570.
    Affiliations
    Department of Infectious Diseases, Molecular Virology, Heidelberg University, Heidelberg, Germany
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  • Author Footnotes
    † These authors contributed equally to this work.
Open AccessPublished:August 09, 2013DOI:https://doi.org/10.1016/j.jhep.2013.07.033

      Summary

      Infections with the hepatitis C virus (HCV) are a major cause of chronic liver disease. While the acute phase of infection is mostly asymptomatic, this virus has the high propensity to establish persistence and in the course of one to several decades liver disease can develop. HCV is a paradigm for the complex interplay between the interferon (IFN) system and viral countermeasures. The virus induces an IFN response within the infected cell and is rather sensitive against the antiviral state triggered by IFNs, yet in most cases HCV persists. Numerous IFN-stimulated genes (ISGs) have been reported to suppress HCV replication, but in only a few cases we begin to understand the molecular mechanisms underlying antiviral activity. It is becoming increasingly clear that blockage of viral replication is mediated by the concerted action of multiple ISGs that target different steps of the HCV replication cycle. This review briefly summarizes the activation of the IFN system by HCV and then focuses on ISGs targeting the HCV replication cycle and their possible mode of action.

      Keywords

      Introduction

      The hepatitis C virus (HCV) is a member of the Flaviviridae family where it forms the genus hepacivirus. These members have in common a single stranded RNA genome of positive polarity and an enveloped virus particle. Seven HCV genotypes (1–7), in most cases with numerous subtypes (a, b etc.), are distinguished, differing in their nucleotide sequence by ∼33% and 25%, respectively [
      • Simmonds P.
      The origin of hepatitis C virus.
      ]. HCV infects only humans and chimpanzees. This narrow host range is due to species-specific host cell factors promoting or restricting HCV replication [
      • Ploss A.
      • Rice C.M.
      Towards a small animal model for hepatitis C.
      ].
      Around 2% of the world population is chronically infected with HCV [

      WHO. Hepatitis C, fact sheet N°164; 2012 [cited; Available from: <http://www.who.int/mediacentre/factsheets/fs164/en/>.

      ] and these people are at high risk to develop liver diseases. Treatment of hepatitis C to the most part depends on pegylated interferon-alpha (pegIFN-α) and ribavirin. A successful therapy is determined by multiple parameters such as age and sex of the patient, duration and degree of liver damage, co-infection with other viruses (e.g., HIV) and alcohol consumption. However, the most important predictors of treatment outcome are distinct genetic polymorphisms in the interleukin (IL)28B gene locus [
      • Prokunina-Olsson L.
      • Muchmore B.
      • Tang W.
      • Pfeiffer R.M.
      • Park H.
      • Dickensheets H.
      • et al.
      A variant upstream of IFNL3 (IL28B) creating a new interferon gene IFNL4 is associated with impaired clearance of hepatitis C virus.
      ,
      • Heim M.H.
      Innate immunity and HCV.
      ] and the genotype of the infecting virus. More than 80% of the individuals infected with genotype 2 or 3 viruses, but only ∼45% of genotype 1-infected individuals, achieve sustained viral response with this treatment regimen. In the latter case, this number has increased to ∼75% with the recent implementation of a triple combination therapy, composed of pegIFN-α, ribavirin, and a direct-acting antiviral (DAAs) targeting the HCV protease that resides in non-structural protein 3 (NS3) [
      • EASL Clinical Practice Guidelines
      Management of hepatitis C virus infection.
      ,
      • Ghany M.G.
      • Nelson D.R.
      • Strader D.B.
      • Thomas D.L.
      • Seeff L.B.
      An update on treatment of genotype 1 chronic hepatitis C virus infection: 2011 practice guideline by the American Association for the Study of liver diseases.
      ]. However, this therapy has serious side effects, is very costly and still depends on the individual patient response to pegIFN-α and ribavirin as deduced from the fact that in treatment-experienced patients, response rates are higher among previous relapsers as compared to patients with a previous breakthrough or non-response [
      • Cunningham M.
      • Foster G.R.
      Efficacy and safety of telaprevir in patients with genotype 1 hepatitis C infection.
      ].
      The possibility to propagate HCV in cultured hepatoma cells and the recent availability of cell systems based on immortalized and primary human hepatocytes (PHHs) provided important insights into induction of innate antiviral defense by HCV and viral countermeasures [
      • Steinmann E.
      • Pietschmann T.
      Cell culture systems for hepatitis C virus.
      ]. Moreover, complementing studies with human liver samples have become available [
      • Sarasin-Filipowicz M.
      • Oakeley E.J.
      • Duong F.H.
      • Christen V.
      • Terracciano L.
      • Filipowicz W.
      • et al.
      Interferon signaling and treatment outcome in chronic hepatitis C.
      ,
      • Thomas E.
      • Gonzalez V.D.
      • Li Q.
      • Modi A.A.
      • Chen W.
      • Noureddin M.
      • et al.
      HCV infection induces a unique hepatic innate immune response associated with robust production of type III interferons.
      ,
      • Mihm S.
      • Frese M.
      • Meier V.
      • Wietzke-Braun P.
      • Scharf J.G.
      • Bartenschlager R.
      • et al.
      Interferon type I gene expression in chronic hepatitis C.
      ]. The common denominator is that HCV induces an innate antiviral response that suppresses virus replication. However, the mechanisms by which HCV replication is suppressed are poorly understood. In this review, we will briefly summarize how HCV is sensed in the infected cell and how this leads to the activation of the IFN system. Further, we will focus on ISGs and their role in controlling HCV replication, emphasizing those ISGs for which we begin to understand the molecular mechanisms responsible for replication suppression. We will not cover the clinical implications of the IFN response with respect to antiviral therapy or pathogenesis of liver disease as these topics have been covered in excellent recent reviews [
      • Heim M.H.
      Innate immunity and HCV.
      ,
      • Thimme R.
      • Binder M.
      • Bartenschlager R.
      Failure of innate and adaptive immune responses in controlling hepatitis C virus infection.
      ,
      • Lauer G.M.
      Immune responses to hepatitis C virus (HCV) infection and the prospects for an effective HCV vaccine or immunotherapies.
      ].

      Detection of invading hepatitis C virus

      Hepatitis C virus detection by RIG-I-like receptors

      The innate immune system comprises several pathogen recognition receptors (PRRs) (Fig. 1). In case of viral infections, major key players are the Toll-like receptor (TLR) family and the RIG-I-like receptors (RLRs). Three proteins belong to the latter: retinoic acid inducible gene-I (RIG-I), melanoma differentiation associated gene 5 (MDA5, IFIH1) and laboratory of genetics and physiology 2 (LGP2, DHX58). Upon stimulation, RIG-I as well as MDA5 activate mitochondrial antiviral-signaling protein (MAVS, IPS-1, Cardif, VISA) located at mitochondria, peroxisomes and mitochondria-associated membranes (MAMs) [
      • Dixit E.
      • Boulant S.
      • Zhang Y.
      • Lee A.S.
      • Odendall C.
      • Shum B.
      • et al.
      Peroxisomes are signaling platforms for antiviral innate immunity.
      ,
      • Horner S.M.
      • Liu H.M.
      • Park H.S.
      • Briley J.
      • Gale Jr., M.
      Mitochondrial-associated endoplasmic reticulum membranes (MAM) form innate immune synapses and are targeted by hepatitis C virus.
      ] (Fig. 1). Activation of MAVS leads to phosphorylation of IFN regulatory factor 3 (IRF3) and IRF7. They are translocated into the nucleus to stimulate transcription of IFN-β and several ISGs.
      Figure thumbnail gr1
      Fig. 1IFN signaling pathways. Simplified schematic representation of major sensor pathways leading to the expression of antiviral effector genes upon virus infection. Viral RNA is detected by PRRs, such as TLR3 residing in the endosome (ES), or by the cytoplasmic sensors RIG-I or MDA5. Activation of these PRRs leads to the phosphorylation and dimerization of IRF3 that translocates into the nucleus to stimulate transcription of IFN genes as well as genes encoding ISGs and proinflammatory cytokines. Secreted IFNs bind to their cognate receptor, thus activating the JAK/STAT pathway, which leads to the formation of Stat1/2/IRF9 heterotrimers that translocate into the nucleus to induce expression of further ISGs and IFN genes. A subset of these ISGs suppresses HCV replication. Several HCV proteins counteract signaling molecules, most notably MAVS and TRIF that are cleaved by the NS3/4A protease. Additionally, core and NS5SA may interfere with the JAK/STAT signaling pathway. ER, endoplasmatic reticulum; MAM, mitochondrion-associated membrane.
      RIG-I preferentially senses short dsRNA molecules with 5′-triphosphorylated RNA [
      • Kato H.
      • Takeuchi O.
      • Mikamo-Satoh E.
      • Hirai R.
      • Kawai T.
      • Matsushita K.
      • et al.
      Length-dependent recognition of double-stranded ribonucleic acids by retinoic acid-inducible gene-I and melanoma differentiation-associated gene 5.
      ,
      • Takahasi K.
      • Yoneyama M.
      • Nishihori T.
      • Hirai R.
      • Kumeta H.
      • Narita R.
      • et al.
      Nonself RNA-sensing mechanism of RIG-I helicase and activation of antiviral immune responses.
      ,
      • Cui S.
      • Eisenacher K.
      • Kirchhofer A.
      • Brzozka K.
      • Lammens A.
      • Lammens K.
      • et al.
      The C-terminal regulatory domain is the RNA 5′-triphosphate sensor of RIG-I.
      ,
      • Binder M.
      • Eberle F.
      • Seitz S.
      • Mucke N.
      • Huber C.M.
      • Kiani N.
      • et al.
      Molecular mechanism of signal perception and integration by the innate immune sensor retinoic acid-inducible gene-I (RIG-I).
      ] whereas MDA5 appears to sense long dsRNA independent of a 5′-triphosphorylated end [
      • Kato H.
      • Takeuchi O.
      • Mikamo-Satoh E.
      • Hirai R.
      • Kawai T.
      • Matsushita K.
      • et al.
      Length-dependent recognition of double-stranded ribonucleic acids by retinoic acid-inducible gene-I and melanoma differentiation-associated gene 5.
      ]. In agreement with this specificity, RIG-I seems to be the key sensor of HCV RNA, with the poly(U/UC) tract in the 3′ non-translated region of the HCV genome playing an important role [
      • Saito T.
      • Owen D.M.
      • Jiang F.
      • Marcotrigiano J.
      • Gale Jr., M.
      Innate immunity induced by composition-dependent RIG-I recognition of hepatitis C virus RNA.
      ,
      • Uzri D.
      • Gehrke L.
      Nucleotide sequences and modifications that determine RIG-I/RNA binding and signaling activities.
      ]. In fact, mouse embryonic fibroblasts (MEFs) from RIG-I−/− mice stimulated with HCV RNA do not produce IFN [
      • Saito T.
      • Owen D.M.
      • Jiang F.
      • Marcotrigiano J.
      • Gale Jr., M.
      Innate immunity induced by composition-dependent RIG-I recognition of hepatitis C virus RNA.
      ] and analogous results were obtained with HuH7-derived human hepatoma cell lines as well as PHHs with attenuated RIG-I expression [
      • Eksioglu E.A.
      • Kielbasa J.
      • Eisen S.
      • Reddy V.
      Granulocyte-macrophage colony-stimulating factor increases the proportion of circulating dendritic cells after autologous but not after allogeneic hematopoietic stem cell transplantation.
      ,
      • Yang D.
      • Liu N.
      • Zuo C.
      • Lei S.
      • Wu X.
      • Zhou F.
      • et al.
      Innate host response in primary human hepatocytes with hepatitis C virus infection.
      ].
      Whether MDA5 can detect invading HCV remains to be clarified. One study reported suppression of HCV in vitro upon overexpression of MDA5 [
      • Schoggins J.W.
      • Wilson S.J.
      • Panis M.
      • Murphy M.Y.
      • Jones C.T.
      • Bieniasz P.
      • et al.
      A diverse range of gene products are effectors of the type I interferon antiviral response.
      ]. Moreover, HCV replication is enhanced in cells expressing the V protein of paramyxovirus, which is a known inhibitor of MDA5 [
      • Andrus L.
      • Marukian S.
      • Jones C.T.
      • Catanese M.T.
      • Sheahan T.P.
      • Schoggins J.W.
      • et al.
      Expression of paramyxovirus V proteins promotes replication and spread of hepatitis C virus in cultures of primary human fetal liver cells.
      ,
      • Ramachandran A.
      • Horvath C.M.
      Dissociation of paramyxovirus interferon evasion activities: universal and virus-specific requirements for conserved V protein amino acids in MDA5 interference.
      ]. However, inhibition of STAT1 (signal transducer and activator of transcription1), rather than MDA5, by the V protein might account for increased HCV replication [
      • Andrejeva J.
      • Poole E.
      • Young D.F.
      • Goodbourn S.
      • Randall R.E.
      The p127 subunit (DDB1) of the UV-DNA damage repair binding protein is essential for the targeted degradation of STAT1 by the V protein of the paramyxovirus simian virus 5.
      ,
      • Rodriguez J.J.
      • Parisien J.P.
      • Horvath C.M.
      Nipah virus V protein evades alpha and gamma interferons by preventing STAT1 and STAT2 activation and nuclear accumulation.
      ]. While other members of the Flaviviridae family are detected by both RIG-I and MDA5 [
      • Loo Y.M.
      • Gale Jr., M.
      Immune signaling by RIG-I-like receptors.
      ], the observation that MDA5−/− MEFs still produce IFN-β upon stimulation with HCV RNA [
      • Saito T.
      • Owen D.M.
      • Jiang F.
      • Marcotrigiano J.
      • Gale Jr., M.
      Innate immunity induced by composition-dependent RIG-I recognition of hepatitis C virus RNA.
      ] suggests that RIG-I is the key player in detecting HCV.

      Hepatitis C virus detection by Toll-like receptors

      TLRs are membrane-bound receptors that detect invading pathogens either on the plasma membrane or in endosomes (Fig. 1). In case of HCV, TLR3 and TLR7 that recognize dsRNA and ssRNA, respectively, appear to be the main sensors. They both reside in endosomes and signal either via TRIF (TIR-domain-containing adapter-inducing interferon-β) or MyD88 to activate IRF3/7 and NFκB (Fig. 1). This finally leads to the production of proinflammotory cytokines and chemokines (Fig. 1). Several lines of evidence suggest that HCV is sensed by these TLRs. First, reconstitution of TLR3 expression in HuH7 human hepatoma cells that normally do not express TLR3 induces an antiviral state upon HCV infection [
      • Wang B.
      • Trippler M.
      • Pei R.
      • Lu M.
      • Broering R.
      • Gerken G.
      • et al.
      Toll-like receptor activated human and murine hepatic stellate cells are potent regulators of hepatitis C virus replication.
      ,
      • Li K.
      • Li N.L.
      • Wei D.
      • Pfeffer S.R.
      • Fan M.
      • Pfeffer L.M.
      Activation of chemokine and inflammatory cytokine response in hepatitis C virus-infected hepatocytes depends on Toll-like receptor 3 sensing of hepatitis C virus double-stranded RNA intermediates.
      ], which is likely enhanced by the TLR3-dependent transcriptional upregulation of RIG-I (reviewed in [
      • Schoggins J.W.
      • Rice C.M.
      Innate immune responses to hepatitis C virus.
      ,
      • Manuse M.J.
      • Parks G.D.
      TLR3-dependent upregulation of RIG-I leads to enhanced cytokine production from cells infected with the parainfluenza virus SV5.
      ]). Second, a recent study reported dsRNA replication intermediates inducing TLR3 signaling [
      • Li K.
      • Li N.L.
      • Wei D.
      • Pfeffer S.R.
      • Fan M.
      • Pfeffer L.M.
      Activation of chemokine and inflammatory cytokine response in hepatitis C virus-infected hepatocytes depends on Toll-like receptor 3 sensing of hepatitis C virus double-stranded RNA intermediates.
      ]. Third, by using the macrophage cell line THP1, it was found that HCV RNA is sensed by TLR7 leading to the expression of interleukin-1β (IL-1β), which is proposed to be a central factor of liver inflammation [
      • Negash A.A.
      • Ramos H.J.
      • Crochet N.
      • Lau D.T.
      • Doehle B.
      • Papic N.
      • et al.
      IL-1beta production through the NLRP3 inflammasome by hepatic macrophages links hepatitis C virus infection with liver inflammation and disease.
      ]. Forth, it was shown that HCV-infected cells activate IFN production by plasmacytoid dendritic cells (pDCs) via TLR7 [
      • Takahashi K.
      • Asabe S.
      • Wieland S.
      • Garaigorta U.
      • Gastaminza P.
      • Isogawa M.
      • et al.
      Plasmacytoid dendritic cells sense hepatitis C virus-infected cells, produce interferon, and inhibit infection.
      ]. In summary, HCV is recognized both by TLR3 and by TLR7, at least in cell cultures.

      Interferons and their role in controlling hepatitis C virus

      Activation of the interferon response by hepatitis C virus

      Based on receptor usage, IFNs are divided into three groups. Type I IFNs comprise a multitude of cytokines with IFN-α and IFN-β being the most important ones with respect to viral infections. Type III IFNs comprise IFN-λ1, IFN-λ2, and IFN-λ3 in humans (also called IL-29, IL-28A and IL-28B, respectively). Moreover, a recent study reported an additional open reading frame, which encodes for IFN-λ4 [
      • Prokunina-Olsson L.
      • Muchmore B.
      • Tang W.
      • Pfeiffer R.M.
      • Park H.
      • Dickensheets H.
      • et al.
      A variant upstream of IFNL3 (IL28B) creating a new interferon gene IFNL4 is associated with impaired clearance of hepatitis C virus.
      ,
      • Lupberger J.
      • Felmlee D.J.
      • Baumert T.F.
      Interferon-lambda polymorphisms and hepatitis C virus clearance revisited.
      ]. Type I and type III IFNs signal via the JAK-STAT pathway (Fig. 1). However, they bind to different receptors that in case of type III IFN is expressed mainly in epithelial cells and hepatocytes (reviewed in [
      • Lupberger J.
      • Felmlee D.J.
      • Baumert T.F.
      Interferon-lambda polymorphisms and hepatitis C virus clearance revisited.
      ]) whereas the type I IFN receptor is expressed ubiquitously. Type II IFN comprises only IFN-γ, which is mainly produced by T cells and NK cells and acts on multiple cell types including immune cells.
      Varying experimental systems such as cell culture models [
      • Marukian S.
      • Andrus L.
      • Sheahan T.P.
      • Jones C.T.
      • Charles E.D.
      • Ploss A.
      • et al.
      Hepatitis C virus induces interferon-lambda and interferon-stimulated genes in primary liver cultures.
      ,
      • Stevenson N.J.
      • Murphy A.G.
      • Bourke N.M.
      • Keogh C.A.
      • Hegarty J.E.
      • O’Farrelly C.
      Ribavirin enhances IFN-alpha signalling and MxA expression: a novel immune modulation mechanism during treatment of HCV.
      ,
      • Kanazawa N.
      • Kurosaki M.
      • Sakamoto N.
      • Enomoto N.
      • Itsui Y.
      • Yamashiro T.
      • et al.
      Regulation of hepatitis C virus replication by interferon regulatory factor 1.
      ,
      • Raychoudhuri A.
      • Shrivastava S.
      • Steele R.
      • Kim H.
      • Ray R.
      • Ray R.B.
      ISG56 and IFITM1 proteins inhibit hepatitis C virus replication.
      ,
      • Zhu H.
      • Butera M.
      • Nelson D.R.
      • Liu C.
      Novel type I interferon IL-28A suppresses hepatitis C viral RNA replication.
      ,
      • Wang C.
      • Pflugheber J.
      • Sumpter Jr., R.
      • Sodora D.L.
      • Hui D.
      • Sen G.C.
      • et al.
      Alpha interferon induces distinct translational control programs to suppress hepatitis C virus RNA replication.
      ,
      • Itsui Y.
      • Sakamoto N.
      • Kurosaki M.
      • Kanazawa N.
      • Tanabe Y.
      • Koyama T.
      • et al.
      Expressional screening of interferon-stimulated genes for antiviral activity against hepatitis C virus replication.
      ], transgenic mice with human liver xenografts [
      • Walters K.A.
      • Joyce M.A.
      • Thompson J.C.
      • Smith M.W.
      • Yeh M.M.
      • Proll S.
      • et al.
      Host-specific response to HCV infection in the chimeric SCID-beige/Alb-uPA mouse model: role of the innate antiviral immune response.
      ,
      • Tsuge M.
      • Fujimoto Y.
      • Hiraga N.
      • Zhang Y.
      • Ohnishi M.
      • Kohno T.
      • et al.
      Hepatitis C virus infection suppresses the interferon response in the liver of the human hepatocyte chimeric mouse.
      ], experimentally inoculated chimpanzees [
      • Thomas E.
      • Gonzalez V.D.
      • Li Q.
      • Modi A.A.
      • Chen W.
      • Noureddin M.
      • et al.
      HCV infection induces a unique hepatic innate immune response associated with robust production of type III interferons.
      ,
      • Bigger C.B.
      • Brasky K.M.
      • Lanford R.E.
      DNA microarray analysis of chimpanzee liver during acute resolving hepatitis C virus infection.
      ,
      • Bigger C.B.
      • Guerra B.
      • Brasky K.M.
      • Hubbard G.
      • Beard M.R.
      • Luxon B.A.
      • et al.
      Intrahepatic gene expression during chronic hepatitis C virus infection in chimpanzees.
      ,
      • Park H.
      • Serti E.
      • Eke O.
      • Muchmore B.
      • Prokunina-Olsson L.
      • Capone S.
      • et al.
      IL-29 is the dominant type III interferon produced by hepatocytes during acute hepatitis C virus infection.
      ,
      • Barth H.
      • Rybczynska J.
      • Patient R.
      • Choi Y.
      • Sapp R.K.
      • Baumert T.F.
      • et al.
      Both innate and adaptive immunity mediate protective immunity against hepatitis C virus infection in chimpanzees.
      ,
      • Shin E.C.
      • Seifert U.
      • Kato T.
      • Rice C.M.
      • Feinstone S.M.
      • Kloetzel P.M.
      • et al.
      Virus-induced type I IFN stimulates generation of immunoproteasomes at the site of infection.
      ,
      • Lanford R.E.
      • Guerra B.
      • Bigger C.B.
      • Lee H.
      • Chavez D.
      • Brasky K.M.
      Lack of response to exogenous interferon-alpha in the liver of chimpanzees chronically infected with hepatitis C virus.
      ] and human liver samples from HCV-infected patients [
      • Sarasin-Filipowicz M.
      • Oakeley E.J.
      • Duong F.H.
      • Christen V.
      • Terracciano L.
      • Filipowicz W.
      • et al.
      Interferon signaling and treatment outcome in chronic hepatitis C.
      ,
      • Thomas E.
      • Gonzalez V.D.
      • Li Q.
      • Modi A.A.
      • Chen W.
      • Noureddin M.
      • et al.
      HCV infection induces a unique hepatic innate immune response associated with robust production of type III interferons.
      ,
      • Mihm S.
      • Frese M.
      • Meier V.
      • Wietzke-Braun P.
      • Scharf J.G.
      • Bartenschlager R.
      • et al.
      Interferon type I gene expression in chronic hepatitis C.
      ,
      • Pfeffer L.M.
      • Madey M.A.
      • Riely C.A.
      • Fleckenstein J.F.
      The induction of type I interferon production in hepatitis C-infected patients.
      ] have been used to characterize the IFN response induced by HCV. The common denominator is that HCV induces an upregulation of ISGs, but the source of IFNs in the infected liver remains to be elucidated. One likely source might be infected hepatocytes themselves, as inferred from the IFN production by PHHs infected with HCV in vitro [
      • Thomas E.
      • Gonzalez V.D.
      • Li Q.
      • Modi A.A.
      • Chen W.
      • Noureddin M.
      • et al.
      HCV infection induces a unique hepatic innate immune response associated with robust production of type III interferons.
      ,
      • Metz P.
      • Dazert E.
      • Ruggieri A.
      • Mazur J.
      • Kaderali L.
      • Kaul A.
      • et al.
      Identification of type I and type II interferon-induced effectors controlling hepatitis C virus replication.
      ]. In addition, IFNs and other proinflammatory cytokines can be produced by resident or infiltrating immune cells such as macrophages, Kupffer cells or DCs that can be stimulated, e.g., by HCV RNA-containing exosomes released from infected cells [
      • Lau D.T.
      • Negash A.
      • Chen J.
      • Crochet N.
      • Sinha M.
      • Zhang Y.
      • et al.
      Innate immune tolerance and the role of kupffer cells in differential responses to interferon therapy among patients with HCV genotype 1 infection.
      ,
      • Dreux M.
      • Garaigorta U.
      • Boyd B.
      • Decembre E.
      • Chung J.
      • Whitten-Bauer C.
      • et al.
      Short-range exosomal transfer of viral RNA from infected cells to plasmacytoid dendritic cells triggers innate immunity.
      ].
      The pattern of ISGs detected in patients with chronic hepatitis C clearly corresponds to a type I or III IFN signature [
      • Sarasin-Filipowicz M.
      • Oakeley E.J.
      • Duong F.H.
      • Christen V.
      • Terracciano L.
      • Filipowicz W.
      • et al.
      Interferon signaling and treatment outcome in chronic hepatitis C.
      ,
      • Bigger C.B.
      • Guerra B.
      • Brasky K.M.
      • Hubbard G.
      • Beard M.R.
      • Luxon B.A.
      • et al.
      Intrahepatic gene expression during chronic hepatitis C virus infection in chimpanzees.
      ,
      • Dill M.T.
      • Makowska Z.
      • Duong F.H.
      • Merkofer F.
      • Filipowicz M.
      • Baumert T.F.
      • et al.
      Interferon-gamma-stimulated genes, but not USP18, are expressed in livers of patients with acute hepatitis C.
      ]. Studies based on PHHs infected with cell culture-produced HCV, or liver biopsies from infected patients and chimpanzees show that the ISG expression pattern predominantly reflects a type I and/or type III IFN response [
      • Bigger C.B.
      • Guerra B.
      • Brasky K.M.
      • Hubbard G.
      • Beard M.R.
      • Luxon B.A.
      • et al.
      Intrahepatic gene expression during chronic hepatitis C virus infection in chimpanzees.
      ,
      • Shin E.C.
      • Seifert U.
      • Kato T.
      • Rice C.M.
      • Feinstone S.M.
      • Kloetzel P.M.
      • et al.
      Virus-induced type I IFN stimulates generation of immunoproteasomes at the site of infection.
      ]. Moreover, several groups reported upregulation of type III IFN in PHHs and in liver samples from patients as well as experimentally infected chimpanzees [
      • Thomas E.
      • Gonzalez V.D.
      • Li Q.
      • Modi A.A.
      • Chen W.
      • Noureddin M.
      • et al.
      HCV infection induces a unique hepatic innate immune response associated with robust production of type III interferons.
      ,
      • Marukian S.
      • Andrus L.
      • Sheahan T.P.
      • Jones C.T.
      • Charles E.D.
      • Ploss A.
      • et al.
      Hepatitis C virus induces interferon-lambda and interferon-stimulated genes in primary liver cultures.
      ,
      • Park H.
      • Serti E.
      • Eke O.
      • Muchmore B.
      • Prokunina-Olsson L.
      • Capone S.
      • et al.
      IL-29 is the dominant type III interferon produced by hepatocytes during acute hepatitis C virus infection.
      ]. However, whether type I or type III IFN is the main driver of HCV-induced ISG expression remains to be determined.
      Several single nucleotide polymorphisms (SNPs) have been found in the IL28B gene locus, which seem to influence the response to HCV [
      • Tanaka Y.
      • Nishida N.
      • Sugiyama M.
      • Kurosaki M.
      • Matsuura K.
      • Sakamoto N.
      • et al.
      Genome-wide association of IL28B with response to pegylated interferon-alpha and ribavirin therapy for chronic hepatitis C.
      ,
      • Suppiah V.
      • Moldovan M.
      • Ahlenstiel G.
      • Berg T.
      • Weltman M.
      • Abate M.L.
      • et al.
      IL28B is associated with response to chronic hepatitis C interferon-alpha and ribavirin therapy.
      ,
      • Ge D.
      • Fellay J.
      • Thompson A.J.
      • Simon J.S.
      • Shianna K.V.
      • Urban T.J.
      • et al.
      Genetic variation in IL28B predicts hepatitis C treatment-induced viral clearance.
      ]. The best studied SNPs do not reside in the coding region of the IL28A and IL28B genes, but rather upstream (rs12979860, rs8099917) or downstream (rs12980275) of IL28B (reviewed in [
      • Heim M.H.
      Innate immunity and HCV.
      ]). Of note, the major genotype of SNP rs12979860 (called C/C because of a homozygous cytosine at this position) correlates with better treatment response and viral clearance [
      • Rauch A.
      • Kutalik Z.
      • Descombes P.
      • Cai T.
      • Di Iulio J.
      • Mueller T.
      • et al.
      Genetic variation in IL28B is associated with chronic hepatitis C and treatment failure: a genome-wide association study.
      ,
      • Thomas D.L.
      • Thio C.L.
      • Martin M.P.
      • Qi Y.
      • Ge D.
      • O’Huigin C.
      • et al.
      Genetic variation in IL28B and spontaneous clearance of hepatitis C virus.
      ,
      • Tillmann H.L.
      • Thompson A.J.
      • Patel K.
      • Wiese M.
      • Tenckhoff H.
      • Nischalke H.D.
      • et al.
      A polymorphism near IL28B is associated with spontaneous clearance of acute hepatitis C virus and jaundice.
      ]. However, the underlying mechanism is unknown and appears to be independent of a global upregulation of ISGs [
      • Abe H.
      • Hayes C.N.
      • Ochi H.
      • Maekawa T.
      • Tsuge M.
      • Miki D.
      • et al.
      IL28 variation affects expression of interferon stimulated genes and peg-interferon and ribavirin therapy.
      ]. Moreover, non-responders have elevated ISG levels irrespective of the SNP genotype [
      • Dill M.T.
      • Duong F.H.
      • Vogt J.E.
      • Bibert S.
      • Bochud P.Y.
      • Terracciano L.
      • et al.
      Interferon-induced gene expression is a stronger predictor of treatment response than IL28B genotype in patients with hepatitis C.
      ] arguing that IFN-λ3, encoded by the IL28B gene, does not directly control ISG induction.
      More recently, a fourth SNP (ss469415590 (TT or ΔG)) was found upstream of IL28B [
      • Prokunina-Olsson L.
      • Muchmore B.
      • Tang W.
      • Pfeiffer R.M.
      • Park H.
      • Dickensheets H.
      • et al.
      A variant upstream of IFNL3 (IL28B) creating a new interferon gene IFNL4 is associated with impaired clearance of hepatitis C virus.
      ]. Interestingly, the ΔG variant is a frameshift mutation creating a novel open reading frame, which encodes for a protein that shares 40.8% amino acid sequence similarity to IFN-λ3 and hence has been designated IFN-λ4 [
      • Prokunina-Olsson L.
      • Muchmore B.
      • Tang W.
      • Pfeiffer R.M.
      • Park H.
      • Dickensheets H.
      • et al.
      A variant upstream of IFNL3 (IL28B) creating a new interferon gene IFNL4 is associated with impaired clearance of hepatitis C virus.
      ,
      • Lupberger J.
      • Felmlee D.J.
      • Baumert T.F.
      Interferon-lambda polymorphisms and hepatitis C virus clearance revisited.
      ]. Overexpression of an engineered IFN-λ4 fusion protein in a hepatoma cell line induced phosphorylation of STAT1 and STAT2, expression of several ISGs, and suppression of HCV replication. However, in spite of this antiviral activity, the presence of the ΔG variant is in high disequilibrium with rs12979860 and correlates with reduced response to IFN-α treatment as well as low HCV clearance. Since overexpression of the engineered IFN-λ4 protein was found to weakly pre-activate the IFN signaling pathway, which prevented further activation by IFN-α or -λ, it is tempting to speculate that this refractoriness impairs HCV clearance. Alternatively, methylation of a cysteine residue adjacent to the ΔG variant might be important for HCV clearance [
      • Bibert S.
      • Roger T.
      • Calandra T.
      • Bochud M.
      • Cerny A.
      • Semmo N.
      • et al.
      IL28B expression depends on a novel TT/-G polymorphism which improves HCV clearance prediction.
      ].

      Interferon-stimulated genes and their impact on controlling hepatitis C virus

      More than 300 ISGs can be upregulated by IFNs. For obvious reasons, most transcriptome profiles have been established by using cell culture systems and only a limited number of profiles are available from in vivo studies. In line with the induction of ISGs by HCV infection itself enhanced transcription of ISGs in acutely and chronically HCV-infected chimpanzees as well as chronically infected patients has been observed. Importantly, the subsets of upregulated ISGs detected in vivo overlap to a large degree with those detected in IFN-treated cell cultures in in vitro [
      • Sarasin-Filipowicz M.
      • Oakeley E.J.
      • Duong F.H.
      • Christen V.
      • Terracciano L.
      • Filipowicz W.
      • et al.
      Interferon signaling and treatment outcome in chronic hepatitis C.
      ,
      • Thomas E.
      • Gonzalez V.D.
      • Li Q.
      • Modi A.A.
      • Chen W.
      • Noureddin M.
      • et al.
      HCV infection induces a unique hepatic innate immune response associated with robust production of type III interferons.
      ,
      • Bigger C.B.
      • Brasky K.M.
      • Lanford R.E.
      DNA microarray analysis of chimpanzee liver during acute resolving hepatitis C virus infection.
      ,
      • Bigger C.B.
      • Guerra B.
      • Brasky K.M.
      • Hubbard G.
      • Beard M.R.
      • Luxon B.A.
      • et al.
      Intrahepatic gene expression during chronic hepatitis C virus infection in chimpanzees.
      ,
      • Shin E.C.
      • Seifert U.
      • Kato T.
      • Rice C.M.
      • Feinstone S.M.
      • Kloetzel P.M.
      • et al.
      Virus-induced type I IFN stimulates generation of immunoproteasomes at the site of infection.
      ,
      • Lanford R.E.
      • Guerra B.
      • Lee H.
      • Averett D.R.
      • Pfeiffer B.
      • Chavez D.
      • et al.
      Antiviral effect and virus-host interactions in response to alpha interferon, gamma interferon, poly(i)-poly(c), tumor necrosis factor alpha, and ribavirin in hepatitis C virus subgenomic replicons.
      ,
      • Helbig K.J.
      • Lau D.T.
      • Semendric L.
      • Harley H.A.
      • Beard M.R.
      Analysis of ISG expression in chronic hepatitis C identifies viperin as a potential antiviral effector.
      ]. Of note, the spectrum of ISGs induced in cultured cells in the absence or presence of HCV does not differ, supporting the notion that HCV has little effect on the signaling cascade leading to ISG activation, at least in vitro (M. Binder and R.B., unpublished).
      In the absence of immuno-competent small animal models that are fully permissive for HCV, no mechanistic in vivo evaluation of specific ISGs is possible. Thus, our knowledge about the mechanisms by which ISGs control HCV replication is based on in vitro models. Below, we will first summarize screens conducted to identify ISGs that are involved in the control of HCV and then focus on those ISGs for which insights into the underlying mechanism are available.

      Identification of ISGs targeting hepatitis C virus

      To identify genes responsible for the suppression of HCV replication in IFN-treated cells, several biased and unbiased screens have been conducted in hepatoma cells. Identified ISGs restricting or promoting HCV replication are summarized in Fig. 2 as well as Table 1, Table 2, respectively, that also specify the used ISG abbreviations.
      Figure thumbnail gr2
      Fig. 2Phases of the HCV replication cycle and ISGs targeting these phases. Individual steps of the HCV replication cycle are given in the filled circles. These are virus entry, RNA translation, replication of the plus-strand (+) RNA genome via a minus-strand copy (−), assembly of infectious HCV particles that seems to require cytosolic lipid droplets (cLDs), and release of infectious HCV particles. ISGs targeting these individual steps are given in the respective sector. ISGs written in dark brown letters target only one step; ISGs marked in blue target multiple steps.
      Table 1Interferon-stimulated genes described to restrict the HCV replication cycle.
      1Cell line used for the study.
      22ISGs were validated by using either infection of cells with HCV (inf) or subgenomic replicons (rep) or transfection of genomic in vitro transcripts (genome transf).
      3Antiviral effects were determined by using overexpression of a given ISG (oe) or by knockdown (kd) of the respective ISG in cells treated with IFN and measuring the impact on HCV replication.
      Table 2Interferon-stimulated genes described to promote the HCV replication cycle.
      1Cell line used for the study.
      2ISGs were validated by using either infection of cells with HCV (inf) or subgenomic replicons (rep).
      3Antiviral effects were determined by using overexpression of a given ISG (oe) or by knockdown (kd) of the respective ISG in cells treated with IFN and measuring the impact on HCV replication.
      A FACS-based phenotypic screen was used to determine the antiviral activity of 389 ISGs upon overexpression in HuH7 and HuH7.5 cells [
      • Schoggins J.W.
      • Wilson S.J.
      • Panis M.
      • Murphy M.Y.
      • Jones C.T.
      • Bieniasz P.
      • et al.
      A diverse range of gene products are effectors of the type I interferon antiviral response.
      ]. Lentivirus-based transduction was used to introduce each ISG individually and transduced cells were infected with an HCV reporter virus. A reduction of HCV-specific signal in ISG-expressing cells indicated inhibition of viral replication. In this way, strongest antiviral effects were observed for IRF1, RIG-I, MDA5, IRF2, and IRF7. While this result underscores the important role of these RNA-sensing and key signaling molecules in mounting an antiviral response against HCV, additional ISGs were detected suppressing HCV replication to a much lesser extent. These ISGs included DDIT4, NT5C3, IFI44L, MAP3K14, and OASL (Table 1). It is likely that these factors exert a more direct effect against HCV, e.g., by enhancing turnover of RNA or slowing down RNA translation. The fact that none of these ISGs was sufficient to suppress HCV replication to an extent observed by IFN treatment suggests that virus inhibition is brought about by the concerted action of several ISGs.
      In contrast to this overexpression approach, a whole-genome RNA interference-based screen was performed by Zhao and co-workers [
      • Zhao H.
      • Lin W.
      • Kumthip K.
      • Cheng D.
      • Fusco D.N.
      • Hofmann O.
      • et al.
      A functional genomic screen reveals novel host genes that mediate interferon-alpha’s effects against hepatitis C virus.
      ]. The authors used HuH7 cells containing a stably replicating subgenomic HCV replicon and determined rescue of viral replication by knockdown of a given gene with cells that had been treated with IFN-α. With this approach, 93 genes were identified contributing to suppression of HCV replication. Identified hits were enriched for genes involved in IFN signaling, RNA translation, and mRNA processing [
      • Zhao H.
      • Lin W.
      • Kumthip K.
      • Cheng D.
      • Fusco D.N.
      • Hofmann O.
      • et al.
      A functional genomic screen reveals novel host genes that mediate interferon-alpha’s effects against hepatitis C virus.
      ]. One of these genes, SART1, was characterized in more detail. This factor was not induced by IFN and reported to play a more general role in regulation of ISG expression, which would explain its antiviral effect [
      • Zhao H.
      • Lin W.
      • Kumthip K.
      • Cheng D.
      • Fusco D.N.
      • Hofmann O.
      • et al.
      A functional genomic screen reveals novel host genes that mediate interferon-alpha’s effects against hepatitis C virus.
      ].
      By using an analogous ‘gain-of-function’ RNA interference-based screen, Metz and co-workers identified 7 ISGs that rescue HCV replication in IFN-α or IFN-γ treated cells [
      • Metz P.
      • Dazert E.
      • Ruggieri A.
      • Mazur J.
      • Kaderali L.
      • Kaul A.
      • et al.
      Identification of type I and type II interferon-induced effectors controlling hepatitis C virus replication.
      ]. All of these genes are induced by either IFN, showing a substantial overlap of the ISG spectrum triggered by these cytokines. Nevertheless, some differences exist. For instance, phospholipid scramblase 1 and inducible nitric oxide synthase 2 were identified as main effectors of IFN-γ. Similar to the results by Schoggins and co-workers [
      • Schoggins J.W.
      • Wilson S.J.
      • Panis M.
      • Murphy M.Y.
      • Jones C.T.
      • Bieniasz P.
      • et al.
      A diverse range of gene products are effectors of the type I interferon antiviral response.
      ], it was found that the antiviral state blocking HCV replication requires the combined activity of multiple ISGs [
      • Metz P.
      • Dazert E.
      • Ruggieri A.
      • Mazur J.
      • Kaderali L.
      • Kaul A.
      • et al.
      Identification of type I and type II interferon-induced effectors controlling hepatitis C virus replication.
      ].
      Very recently, an unbiased genome-wide siRNA screen was performed to identify ISGs as well as non-transcriptionally induced genes required for the antiviral effect of IFN-α [
      • Fusco D.N.
      • Brisac C.
      • John S.P.
      • Huang Y.W.
      • Chin C.R.
      • Xie T.
      • et al.
      A genetic screen identifies interferon-alpha effector genes required to suppress hepatitis C virus replication.
      ]. HuH7-derived cells were transfected with siRNAs and after stimulation with IFN-α infected with HCV. By using an image-based analysis, 9 genes were identified to be responsible for IFN-α-mediated suppression of HCV replication (Table 1). Furthermore, the individual contribution of each of these candidates to inhibit distinct steps in the HCV replication cycle was determined. Thereby, effector groups were identified targeting viral entry, replication, RNA production or virus particle production/release. Three of the candidates, MYST1, ALG10B, and PDIP1, were found to target nearly all steps of the HCV life cycle. These findings underscore that both ISGs and non-ISGs are required for efficient suppression of HCV.
      In addition to the high-content screens described above, several more targeted screens have been conducted. Although a direct comparison of obtained results is flawed by differences of used experimental approaches, several ISGs have been identified consistently. These include members of the IFITM family, PKR, and viperin (Table 1).

      Possible anti-hepatitis C virus mechanisms of selected ISGs

      Protein kinase R

      PKR is a central component that links pathogen sensing to stress [
      • Dabo S.
      • Meurs E.F.
      DsRNA-dependent protein kinase PKR and its role in stress, signaling and HCV infection.
      ]. The kinase is a cytosolic sensor of viral dsRNA [
      • Saunders L.R.
      • Barber G.N.
      The dsRNA binding protein family: critical roles, diverse cellular functions.
      ] and PKR activation results in a global suppression of RNA translation and thus, protein synthesis. Binding to dsRNA promotes PKR homodimerization and activates the kinase domain that phosphorylates the alpha subunit of eukaryotic initiation factor 2 (eIF2α). This factor is essential for initiation of cap-dependent mRNA translation and its phosphorylation blocks this process [
      • Chong K.L.
      • Feng L.
      • Schappert K.
      • Meurs E.
      • Donahue T.F.
      • Friesen J.D.
      • et al.
      Human p68 kinase exhibits growth suppression in yeast and homology to the translational regulator GCN2.
      ]. In addition, translation arrest leads to the formation of complexes that are composed of non-translated mRNAs and RNA-binding proteins in so-called stress granules [
      • Anderson P.
      • Kedersha N.
      Visibly stressed: the role of eIF2, TIA-1, and stress granules in protein translation.
      ,
      • Ruggieri A.
      • Dazert E.
      • Metz P.
      • Hofmann S.
      • Bergeest J.P.
      • Mazur J.
      • et al.
      Dynamic oscillation of translation and stress granule formation mark the cellular response to virus infection.
      ]. Apart from that, PKR links pathogen sensing to metabolic homeostasis, virus-induced autophagy [
      • Talloczy Z.
      • Jiang W.
      • Virgin H.W.
      • Leib D.A.
      • Scheuner D.
      • Kaufman R.J.
      • et al.
      Regulation of starvation- and virus-induced autophagy by the eIF2alpha kinase signaling pathway.
      ], NF-κB signaling [
      • Zamanian-Daryoush M.
      • Mogensen T.H.
      • DiDonato J.A.
      • Williams B.R.
      NF-kappaB activation by double-stranded-RNA-activated protein kinase (PKR) is mediated through NF-kappaB-inducing kinase and IkappaB kinase.
      ], and activation of the inflammasome [
      • Lu B.
      • Nakamura T.
      • Inouye K.
      • Li J.
      • Tang Y.
      • Lundback P.
      • et al.
      Novel role of PKR in inflammasome activation and HMGB1 release.
      ].
      In vitro binding studies suggest that after HCV infection, PKR is activated as a result of binding to distinct RNA structures in the IRES residing in the 5′ NTR of the genome [
      • Shimoike T.
      • McKenna S.A.
      • Lindhout D.A.
      • Puglisi J.D.
      Translational insensitivity to potent activation of PKR by HCV IRES RNA.
      ]. However, the benefits and/or disadvantages of this activation for HCV replication are controversial. PKR was initially recognized as an effector ISG that is able to inhibit HCV replication [
      • Wang C.
      • Pflugheber J.
      • Sumpter Jr., R.
      • Sodora D.L.
      • Hui D.
      • Sen G.C.
      • et al.
      Alpha interferon induces distinct translational control programs to suppress hepatitis C virus RNA replication.
      ,
      • Pflugheber J.
      • Fredericksen B.
      • Sumpter Jr., R.
      • Wang C.
      • Ware F.
      • Sodora D.L.
      • et al.
      Regulation of PKR and IRF-1 during hepatitis C virus RNA replication.
      ]. Two viral proteins, NS5A and E2, were reported as PKR inhibitors by direct binding to the kinase. Comparison of genotype 1b full-length virus sequences of IFN-α-responsive and non-responsive patients suggested that HCV variants, unable to escape IFN antiviral effects, accumulated mutations in the C-terminal region of NS5A. This region was designated the IFN sensitivity determining region (ISDR) [
      • Enomoto N.
      • Sakuma I.
      • Asahina Y.
      • Kurosaki M.
      • Murakami T.
      • Yamamoto C.
      • et al.
      Comparison of full-length sequences of interferon-sensitive and resistant hepatitis C virus 1b. Sensitivity to interferon is conferred by amino acid substitutions in the NS5A region.
      ]. Cell culture assays and in vitro studies suggest that the ISDR overlaps with the PKR binding region in NS5A, arguing that ISDR mutations might cause a loss of PKR binding and thereby reduce susceptibility to IFN therapy [
      • Tan S.L.
      • Katze M.G.
      How hepatitis C virus counteracts the interferon response: the jury is still out on NS5A.
      ]. This is an attractive hypothesis, however, recent studies propose that PKR acts as an HCV proviral rather than antiviral factor [
      • Metz P.
      • Dazert E.
      • Ruggieri A.
      • Mazur J.
      • Kaderali L.
      • Kaul A.
      • et al.
      Identification of type I and type II interferon-induced effectors controlling hepatitis C virus replication.
      ,
      • Garaigorta U.
      • Chisari F.V.
      Hepatitis C virus blocks interferon effector function by inducing protein kinase R phosphorylation.
      ,
      • Arnaud N.
      • Dabo S.
      • Maillard P.
      • Budkowska A.
      • Kalliampakou K.I.
      • Mavromara P.
      • et al.
      Hepatitis C virus controls interferon production through PKR activation.
      ]. It has been suggested that by activation of PKR, translation of ISG mRNAs is attenuated without affecting translation of the HCV genome that is mediated by the IRES and thus, eIF2α independent [
      • Garaigorta U.
      • Chisari F.V.
      Hepatitis C virus blocks interferon effector function by inducing protein kinase R phosphorylation.
      ,
      • Terenin I.M.
      • Dmitriev S.E.
      • Andreev D.E.
      • Shatsky I.N.
      Eukaryotic translation initiation machinery can operate in a bacterial-like mode without eIF2.
      ]. While these results appear to be conflicting, it is possible that activation and inhibition of PKR occur in a sequential manner, with activation predominating at the early stage of infection and inhibition of PKR at a later stage when high amounts of NS5A have accumulated. However, how the reported enhancement of PKR autophosphorylation induced by interaction with the HCV core protein fits into this scenario remains to be determined [
      • Delhem N.
      • Sabile A.
      • Gajardo R.
      • Podevin P.
      • Abadie A.
      • Blaton M.A.
      • et al.
      Activation of the interferon-inducible protein kinase PKR by hepatocellular carcinoma derived-hepatitis C virus core protein.
      ].
      NS5A is not the only viral protein assumed to interfere with PKR. One study reported an inhibition by the viral envelope glycoprotein E2 via direct interaction with PKR [
      • Taylor D.R.
      • Shi S.T.
      • Romano P.R.
      • Barber G.N.
      • Lai M.M.
      Inhibition of the interferon-inducible protein kinase PKR by HCV E2 protein.
      ]. Interestingly, the interaction site in E2 was mapped to a highly conserved amino acid sequence with high sequence homology to PKR autophosphorylation and eIF2α phosphorylation sites. In addition, the extent of sequence homology correlated with the IFN-resistance phenotypes of different HCV genotypes [
      • Taylor D.R.
      • Shi S.T.
      • Romano P.R.
      • Barber G.N.
      • Lai M.M.
      Inhibition of the interferon-inducible protein kinase PKR by HCV E2 protein.
      ]. Although this is an elegant way to explain how HCV might block PKR, the physiological relevance is not clear. In the study by Taylor and colleagues, only E2 was used for expression-based interaction assays, even though proper folding and membrane association of E2 require co-expression with E1 [
      • Moradpour D.
      • Penin F.
      Hepatitis C virus proteins: from structure to function.
      ]. Thus, these observations need to be revisited by using more authentic viral proteins and adequate cell culture systems.
      To conclude, studies on inhibition or activation of PKR by HCV are conflicting. This controversy is further emphasized by the discrepant reports related to the effects that PKR silencing, inhibition or overexpression have on HCV replication that range from inhibitory [
      • Arnaud N.
      • Dabo S.
      • Maillard P.
      • Budkowska A.
      • Kalliampakou K.I.
      • Mavromara P.
      • et al.
      Hepatitis C virus controls interferon production through PKR activation.
      ] to non-existent [
      • Schoggins J.W.
      • Wilson S.J.
      • Panis M.
      • Murphy M.Y.
      • Jones C.T.
      • Bieniasz P.
      • et al.
      A diverse range of gene products are effectors of the type I interferon antiviral response.
      ,
      • Metz P.
      • Dazert E.
      • Ruggieri A.
      • Mazur J.
      • Kaderali L.
      • Kaul A.
      • et al.
      Identification of type I and type II interferon-induced effectors controlling hepatitis C virus replication.
      ]. One obvious explanation is the differences of used experimental conditions that are very critical when analyzing rather subtle effects. One other source of discrepancy is the multiple roles that PKR has in controlling cell homeostasis, making experimental outcomes very dependent on cell status. Thus, the role PKR plays in IFN-mediated suppression of HCV replication remains to be determined. We note that in our hands neither silencing of PKR expression in IFN-α-treated cells, nor PKR overexpression has an effect on HCV replication in the commonly used cell line HuH7 [
      • Metz P.
      • Dazert E.
      • Ruggieri A.
      • Mazur J.
      • Kaderali L.
      • Kaul A.
      • et al.
      Identification of type I and type II interferon-induced effectors controlling hepatitis C virus replication.
      ].

      Interferon-inducible transmembrane proteins

      The IFITM family consists of 4 members: IFITM1, IFITM2, IFITM3, and IFITM5. With the exception of the latter, all members are ubiquitously expressed in humans and their expression is upregulated by all types of IFNs. IFITMs contain two anti-parallel transmembrane domains, a leucin-zipper motif, and a short cytoplasmic domain. A membrane-proximal cysteine residue can be modified post-translationally by S-palmitoylation controlling IFITM localisation in membrane compartments [
      • Yount J.S.
      • Karssemeijer R.A.
      • Hang H.C.
      S-palmitoylation and ubiquitination differentially regulate interferon-induced transmembrane protein 3 (IFITM3)-mediated resistance to influenza virus.
      ]. IFITM proteins appear to counteract a wide range of viruses, but the mode of action remains unclear [
      • Raychoudhuri A.
      • Shrivastava S.
      • Steele R.
      • Kim H.
      • Ray R.
      • Ray R.B.
      ISG56 and IFITM1 proteins inhibit hepatitis C virus replication.
      ,
      • Brass A.L.
      • Huang I.C.
      • Benita Y.
      • John S.P.
      • Krishnan M.N.
      • Feeley E.M.
      • et al.
      The IFITM proteins mediate cellular resistance to influenza A H1N1 virus, West Nile virus, and dengue virus.
      ,
      • Lu J.
      • Pan Q.
      • Rong L.
      • He W.
      • Liu S.L.
      • Liang C.
      The IFITM proteins inhibit HIV-1 infection.
      ,
      • Huang I.C.
      • Bailey C.C.
      • Weyer J.L.
      • Radoshitzky S.R.
      • Becker M.M.
      • Chiang J.J.
      • et al.
      Distinct patterns of IFITM-mediated restriction of filoviruses, SARS coronavirus, and influenza A virus.
      ,
      • Yao L.
      • Dong H.
      • Zhu H.
      • Nelson D.
      • Liu C.
      • Lambiase L.
      • et al.
      Identification of the IFITM3 gene as an inhibitor of hepatitis C viral translation in a stable STAT1 cell line.
      ]. In case of HCV, IFITM1, and IFITM3 have been reported to interfere with viral entry and RNA replication, respectively [
      • Raychoudhuri A.
      • Shrivastava S.
      • Steele R.
      • Kim H.
      • Ray R.
      • Ray R.B.
      ISG56 and IFITM1 proteins inhibit hepatitis C virus replication.
      ,
      • Metz P.
      • Dazert E.
      • Ruggieri A.
      • Mazur J.
      • Kaderali L.
      • Kaul A.
      • et al.
      Identification of type I and type II interferon-induced effectors controlling hepatitis C virus replication.
      ,
      • Yao L.
      • Dong H.
      • Zhu H.
      • Nelson D.
      • Liu C.
      • Lambiase L.
      • et al.
      Identification of the IFITM3 gene as an inhibitor of hepatitis C viral translation in a stable STAT1 cell line.
      ,
      • Wilkins C.
      • Woodward J.
      • Lau D.T.
      • Barnes A.
      • Joyce M.
      • McFarlane N.
      • et al.
      IFITM1 is a tight junction protein that inhibits hepatitis C virus entry.
      ]. IFITM1 is able to bind to two of the HCV co-receptors: the tetraspanin CD81 and the tight junction protein occludin [
      • Wilkins C.
      • Woodward J.
      • Lau D.T.
      • Barnes A.
      • Joyce M.
      • McFarlane N.
      • et al.
      IFITM1 is a tight junction protein that inhibits hepatitis C virus entry.
      ,
      • Takahashi S.
      • Doss C.
      • Levy S.
      • Levy R.
      TAPA-1, the target of an antiproliferative antibody, is associated on the cell surface with the Leu-13 antigen.
      ]. These co-receptors have to interact with each other to enable virus particle entry into hepatocytes. It is speculated that IFITM1, and to a minor extent IFITM3, disrupt co-receptor interaction, thus inhibiting the entry process [
      • Wilkins C.
      • Woodward J.
      • Lau D.T.
      • Barnes A.
      • Joyce M.
      • McFarlane N.
      • et al.
      IFITM1 is a tight junction protein that inhibits hepatitis C virus entry.
      ]. However, two alternative hypotheses have been put forward. First, IFITMs might inhibit the fusion of the viral envelope membrane with endosomes or lysosomes by altering lipid components or blocking acidification of virus-containing endocytic vesicles. Second, IFITMs might alter vesicle trafficking in a way that the invading viruses are redirected to a non-fusogenic pathway [
      • Huang I.C.
      • Bailey C.C.
      • Weyer J.L.
      • Radoshitzky S.R.
      • Becker M.M.
      • Chiang J.J.
      • et al.
      Distinct patterns of IFITM-mediated restriction of filoviruses, SARS coronavirus, and influenza A virus.
      ,
      • Feeley E.M.
      • Sims J.S.
      • John S.P.
      • Chin C.R.
      • Pertel T.
      • Chen L.M.
      • et al.
      IFITM3 inhibits influenza A virus infection by preventing cytosolic entry.
      ]. Apart from viral entry inhibition, IFITM proteins also suppress HCV replication as deduced from knockdown as well as overexpression studies [
      • Raychoudhuri A.
      • Shrivastava S.
      • Steele R.
      • Kim H.
      • Ray R.
      • Ray R.B.
      ISG56 and IFITM1 proteins inhibit hepatitis C virus replication.
      ,
      • Metz P.
      • Dazert E.
      • Ruggieri A.
      • Mazur J.
      • Kaderali L.
      • Kaul A.
      • et al.
      Identification of type I and type II interferon-induced effectors controlling hepatitis C virus replication.
      ,
      • Yao L.
      • Dong H.
      • Zhu H.
      • Nelson D.
      • Liu C.
      • Lambiase L.
      • et al.
      Identification of the IFITM3 gene as an inhibitor of hepatitis C viral translation in a stable STAT1 cell line.
      ]. Interestingly, the combined knockdown of IFITM1 and IFITM3 has synergistic effects arguing for a redundant antiviral mode-of-action [
      • Metz P.
      • Dazert E.
      • Ruggieri A.
      • Mazur J.
      • Kaderali L.
      • Kaul A.
      • et al.
      Identification of type I and type II interferon-induced effectors controlling hepatitis C virus replication.
      ].
      The mechanism of replication inhibition by IFITMs remains to be determined. One study describes an interference of IFITM3 with HCV IRES-mediated RNA translation, however, this hypothesis needs further proof as in this study also inhibition of cap-dependent RNA translation was observed and a rather artificial in vitro system based on extracts from HeLa cells has been used [
      • Yao L.
      • Dong H.
      • Zhu H.
      • Nelson D.
      • Liu C.
      • Lambiase L.
      • et al.
      Identification of the IFITM3 gene as an inhibitor of hepatitis C viral translation in a stable STAT1 cell line.
      ]. Given the ‘membrane activity’ of IFITMs, it is much more intuitive to speculate that IFITMs have a negative influence on formation of the membranous web, which is the site of HCV RNA replication. A recent study describes an inhibitory effect of IFITM3 on cholesterol homeostasis [
      • Amini-Bavil-Olyaee S.
      • Choi Y.J.
      • Lee J.H.
      • Shi M.
      • Huang I.C.
      • Farzan M.
      • et al.
      The antiviral effector IFITM3 disrupts intracellular cholesterol homeostasis to block viral entry.
      ]. Amongst others, cholesterol synthesis is regulated by the vesicle-membrane-protein-associated protein A (VAP-A) and oxysterol-binding protein (OSBP). Interestingly, IFITM1, IFITM2, and IFITM3 can bind to VAP-A and inhibit its interaction with OSBP. This results in cholesterol-enriched multivesicular bodies and late endosomes that inhibit release of several viruses into the cytosol. In the context of HCV infection, the impact of IFITMs on cholesterol homeostasis might have consequences on the formation of the membranous web, which is a highly specialized membrane compartment that contains high amounts of cholesterol [
      • Paul D.
      • Hoppe S.
      • Saher G.
      • Krijnse-Locker J.
      • Bartenschlager R.
      Morphological and biochemical characterization of the membranous hepatitis C virus replication compartment.
      ]. Moreover, IFITM-mediated sequestration of VAP-A, which is an important cofactor for efficient HCV RNA replication, might contribute to this inhibitory effect.

      Viperin

      Viperin (RSAD2) localizes to the ER and lipid droplets (LDs), which are both important for HCV replication [
      • Hinson E.R.
      • Cresswell P.
      The N-terminal amphipathic alpha-helix of viperin mediates localization to the cytosolic face of the endoplasmic reticulum and inhibits protein secretion.
      ]. This protein belongs to the radical S-adenosyl-L-methionine (SAM) superfamily [
      • Duschene K.S.
      • Broderick J.B.
      The antiviral protein viperin is a radical SAM enzyme.
      ] that is characterized by a SAM domain, which is responsible for methylation of nucleic acids and proteins. It was speculated that SAM transferase activity is important for the antiviral activity of viperin [
      • Jiang D.
      • Guo H.
      • Xu C.
      • Chang J.
      • Gu B.
      • Wang L.
      • et al.
      Identification of three interferon-inducible cellular enzymes that inhibit the replication of hepatitis C virus.
      ]. However, mutagenesis studies showed that not the SAM domain, but rather the N-terminal amphipathic helix and the C-terminal region of the protein are necessary for suppression of HCV replication [
      • Hinson E.R.
      • Cresswell P.
      The antiviral protein, viperin, localizes to lipid droplets via its N-terminal amphipathic alpha-helix.
      ]. The N-terminal helix of viperin is responsible for its anchoring to the ER and LDs [
      • Hinson E.R.
      • Cresswell P.
      The antiviral protein, viperin, localizes to lipid droplets via its N-terminal amphipathic alpha-helix.
      ]. Importantly, this helix inhibits protein secretion and induces ER membrane curvature [
      • Drin G.
      • Casella J.F.
      • Gautier R.
      • Boehmer T.
      • Schwartz T.U.
      • Antonny B.
      A general amphipathic alpha-helical motif for sensing membrane curvature.
      ]. During HCV infection, viperin resides in small cytoplasmic foci at the ER–LD interface. These foci are thought to correspond to viral replication complexes. It is thought that viperin interacts via its C-terminal region with the core protein and NS5A that also localize to LDs and LD-proximal ER membranes [
      • Hinson E.R.
      • Cresswell P.
      The antiviral protein, viperin, localizes to lipid droplets via its N-terminal amphipathic alpha-helix.
      ,
      • Wang S.
      • Wu X.
      • Pan T.
      • Song W.
      • Wang Y.
      • Zhang F.
      • et al.
      Viperin inhibits hepatitis C virus replication by interfering with binding of NS5A to host protein hVAP-33.
      ,
      • Helbig K.J.
      • Eyre N.S.
      • Yip E.
      • Narayana S.
      • Li K.
      • Fiches G.
      • et al.
      The antiviral protein viperin inhibits hepatitis C virus replication via interaction with nonstructural protein 5A.
      ]. In addition, viperin binds to VAP-A, an important host factor of HCV replication [
      • Wang S.
      • Wu X.
      • Pan T.
      • Song W.
      • Wang Y.
      • Zhang F.
      • et al.
      Viperin inhibits hepatitis C virus replication by interfering with binding of NS5A to host protein hVAP-33.
      ,
      • Helbig K.J.
      • Eyre N.S.
      • Yip E.
      • Narayana S.
      • Li K.
      • Fiches G.
      • et al.
      The antiviral protein viperin inhibits hepatitis C virus replication via interaction with nonstructural protein 5A.
      ] that also regulates trafficking and biogenesis of lipids and sterols [
      • Gao L.
      • Aizaki H.
      • He J.W.
      • Lai M.M.
      Interactions between viral nonstructural proteins and host protein hVAP-33 mediate the formation of hepatitis C virus RNA replication complex on lipid raft.
      ]. Therefore, one proposed mechanism of viperin’s antiviral activity is the disintegration of the membranous HCV replication compartment as a result of altered NS5A – VAP-A interaction. This might cause an alteration of the lipid composition of the membranous web thereby inhibiting HCV replication. Viperin has also been found to bind to and inhibit farnesyl diphosphate synthetase (FPPS) [
      • Wang X.
      • Hinson E.R.
      • Cresswell P.
      The interferon-inducible protein viperin inhibits influenza virus release by perturbing lipid rafts.
      ]. Like VAP-A, FPPS is involved in the cholesterol and isoprenoid biosynthesis at the ER membrane [
      • Szkopinska A.
      • Plochocka D.
      Farnesyl diphosphate synthase; regulation of product specificity.
      ]. It is assumed that the inhibition of FPPS by viperin may change the type or quantity of lipids in the ER membrane and thus affect the composition of the ER-derived HCV replication complex. Given the recent establishment of a method to isolate functional membrane-associated HCV replication complexes, these attractive hypotheses can now be addressed experimentally [
      • Paul D.
      • Hoppe S.
      • Saher G.
      • Krijnse-Locker J.
      • Bartenschlager R.
      Morphological and biochemical characterization of the membranous hepatitis C virus replication compartment.
      ].

      The oligoadenylate synthetase/ribonuclease L system

      RNase L was one of the first identified restriction factors of HCV [
      • Han J.Q.
      • Barton D.J.
      Activation and evasion of the antiviral 2’-5’-oligoadenylate synthetase/ribonuclease L pathway by hepatitis C virus mRNA.
      ]. Subsequent studies could confirm that the components of this pathway are important for the counteraction of HCV infection [
      • Schoggins J.W.
      • Wilson S.J.
      • Panis M.
      • Murphy M.Y.
      • Jones C.T.
      • Bieniasz P.
      • et al.
      A diverse range of gene products are effectors of the type I interferon antiviral response.
      ,
      • Metz P.
      • Dazert E.
      • Ruggieri A.
      • Mazur J.
      • Kaderali L.
      • Kaul A.
      • et al.
      Identification of type I and type II interferon-induced effectors controlling hepatitis C virus replication.
      ,
      • Ishibashi M.
      • Wakita T.
      • Esumi M.
      2’,5’-Oligoadenylate synthetase-like gene highly induced by hepatitis C virus infection in human liver is inhibitory to viral replication in vitro.
      ]. RNase L contains 9 so-called ankyrin repeats serving as protein-protein interaction platforms, several protein kinase-like motifs, and a ribonuclease domain. This domain has homology to IRE1 (inositol-requiring protein 1), an enzyme important for the unfolded protein response (UPR) [
      • Lee K.P.
      • Dey M.
      • Neculai D.
      • Cao C.
      • Dever T.E.
      • Sicheri F.
      Structure of the dual enzyme Ire1 reveals the basis for catalysis and regulation in nonconventional RNA splicing.
      ]. RNase L is constitutively expressed in a wide range of tissues, but requires stimulation of enzymatic activity by a particular oligonucleotide (2′,5′-oligoadenylate) that is generated by the IFN-inducible 2,5-linked oligoadenylate synthetases (OAS1, OAS3, and OASL). Similar to PKR, OAS proteins require activation by dsRNA that is generated during viral replication. Synthesized 2′,5′-oligoadenylate binds to RNase L to induce a conformational change leading to its homodimerization and activation of the endonuclease activity. RNase L cleaves viral as well as cellular RNAs in single-stranded regions, mainly after UU or UA dinucleotides leaving a 5′-OH and a 3′-monophosphate [
      • Floyd-Smith G.
      • Slattery E.
      • Lengyel P.
      Interferon action: RNA cleavage pattern of a (2′–5′)oligoadenylate–dependent endonuclease.
      ]. This unspecific cleavage counteracts several viral pathogens, including HCV. Recent studies showed that all three OAS proteins induce RNase L-dependent antiviral activity against HCV [
      • Schoggins J.W.
      • Wilson S.J.
      • Panis M.
      • Murphy M.Y.
      • Jones C.T.
      • Bieniasz P.
      • et al.
      A diverse range of gene products are effectors of the type I interferon antiviral response.
      ,
      • Ishibashi M.
      • Wakita T.
      • Esumi M.
      2’,5’-Oligoadenylate synthetase-like gene highly induced by hepatitis C virus infection in human liver is inhibitory to viral replication in vitro.
      ,
      • Kwon Y.C.
      • Kang J.I.
      • Hwang S.B.
      • Ahn B.Y.
      The ribonuclease L-dependent antiviral roles of human 2′,5′-oligoadenylate synthetase family members against hepatitis C virus.
      ]. Some of the cleavage products of the HCV genome might serve as ligands for RIG-I and MDA5 [
      • Malathi K.
      • Saito T.
      • Crochet N.
      • Barton D.J.
      • Gale Jr., M.
      • Silverman R.H.
      RNase L releases a small RNA from HCV RNA that refolds into a potent PAMP.
      ] thereby enhancing the induction phase of the IFN pathway. Interestingly, HCV genomes of less IFN-responsive genotypes (1a and 1b) have a lower frequency of UA and UU dinucleotides as compared to genotypes with higher IFN response (genotypes 2a, b and 3a, b) [
      • Han J.Q.
      • Barton D.J.
      Activation and evasion of the antiviral 2’-5’-oligoadenylate synthetase/ribonuclease L pathway by hepatitis C virus mRNA.
      ], indicating that the OAS/RNase L system contributes to the control of HCV replication also in vivo.

      Interferon-stimulated gene 15

      ISG15 is one of the most highly induced ISGs. It is a 15 kDa protein with two ubiquitin-like domains in the C- and N-terminal region. Comparable to ubiquitin, ISG15 can be conjugated to lysine residues of target proteins. This so-called ISGylation occurs through the sequential reaction of an E1-activating, an E2-conjugating, and an E3 ligation enzyme. Mass spectrometry-based studies identified more than 160 host proteins that are ISGylated, including the important dsRNA sensors PKR and RIG-I [
      • Zhao C.
      • Denison C.
      • Huibregtse J.M.
      • Gygi S.
      • Krug R.M.
      Human ISG15 conjugation targets both IFN-induced and constitutively expressed proteins functioning in diverse cellular pathways.
      ]. ISGylation has two effects: first, it alters protein property directly by addition of ISG15; second, it reduces degradation of the target protein by competing with ubiquitin conjugation [
      • Shi H.X.
      • Yang K.
      • Liu X.
      • Liu X.Y.
      • Wei B.
      • Shan Y.F.
      • et al.
      Positive regulation of interferon regulatory factor 3 activation by Herc5 via ISG15 modification.
      ]. In addition, ISG15 can be secreted and acts like a cytokine by modulating immune responses such as activation of T cells and NK cells leading to the production of IFN-γ [
      • D’Cunha J.
      • Knight Jr., E.
      • Haas A.L.
      • Truitt R.L.
      • Borden E.C.
      Immunoregulatory properties of ISG15, an interferon-induced cytokine.
      ].
      Reports on the role of ISG15 in the HCV replication cycle are controversial, but the majority of studies argue for a proviral effect. For instance, ISG15 overexpression has been reported to increase HCV replication while RNAi-mediated silencing of ISG15 expression was found to inhibit HCV replication, albeit to a rather low extent (∼2 to 3-fold) [
      • Broering R.
      • Zhang X.
      • Kottilil S.
      • Trippler M.
      • Jiang M.
      • Lu M.
      • et al.
      The interferon stimulated gene 15 functions as a proviral factor for the hepatitis C virus and as a regulator of the IFN response.
      ,
      • Chen L.
      • Sun J.
      • Meng L.
      • Heathcote J.
      • Edwards A.M.
      • McGilvray I.D.
      ISG15, a ubiquitin-like interferon-stimulated gene, promotes hepatitis C virus production in vitro: implications for chronic infection and response to treatment.
      ]. At the first glance, the proviral role of ISG15 appears counterintuitive, but it was found that ISG15 overexpression inhibited induction of IFN-β by HCV [
      • Arnaud N.
      • Dabo S.
      • Akazawa D.
      • Fukasawa M.
      • Shinkai-Ouchi F.
      • Hugon J.
      • et al.
      Hepatitis C virus reveals a novel early control in acute immune response.
      ]. In line with this report, silencing of USP18 expression, which is a negative regulator of ISGylation, potentiates IFN-α mediated HCV suppression [
      • Randall G.
      • Chen L.
      • Panis M.
      • Fischer A.K.
      • Lindenbach B.D.
      • Sun J.
      • et al.
      Silencing of USP18 potentiates the antiviral activity of interferon against hepatitis C virus infection.
      ]. These results suggest that ISG15 counteracts the IFN-α response by ISGylation, e.g., of important signaling factors, and this negative regulation is counteracted by USP18 that removes ISG15 from target proteins. Although this is a plausible assumption, one report seems to contradict it. Kim and Yoo reported reduced HCV replication upon overexpression of ISG15 or ISG15-conjugating enzymes [
      • Kim M.J.
      • Yoo J.Y.
      Inhibition of hepatitis C virus replication by IFN-mediated ISGylation of HCV-NS5A.
      ]. This destabilization was blocked by a particular arginine for lysine substitution in NS5A, arguing for an ISGylation-dependent antiviral mechanism. However, these results have been obtained in highly selected HCV replicon cell clones and thus, need to be revisited in more authentic culture systems and by using replication-competent HCV isolates from different strains and genotypes. We note that high hepatic ISG15 levels were found to correlate with low antiviral IFN-response during the early phase of antiviral therapy, supporting the notion that ISG15 is a negative regulator of the IFN system [
      • Broering R.
      • Zhang X.
      • Kottilil S.
      • Trippler M.
      • Jiang M.
      • Lu M.
      • et al.
      The interferon stimulated gene 15 functions as a proviral factor for the hepatitis C virus and as a regulator of the IFN response.
      ].

      Conclusions

      With the advent of HCV cell culture models, important insights into the suppression of viral replication by the IFN-induced antiviral state have been gained. Nevertheless, several key questions remain to be clarified.
      • 1.
        High-content screens have identified numerous ISGs that might contribute to IFN-mediated control of HCV. However, for a few of them we begin to understand the underlying mechanism and for most of them, the mechanism is not known.
      • 2.
        A concerted action of multiple ISGs is responsible for the suppression of HCV replication, but the relevant ISGs remain to be identified. Determination of the ‘real’ ISG set will require systems that allow combination of multiple ISGs.
      • 3.
        Given the frequent discrepancies reported for HCV-targeting ISGs, more authentic cell culture systems as well as standardization of used methods are urgently needed. Since many results were obtained by using rather artificial experimental systems, re-evaluations in more appropriate models are required.
      • 4.
        Validation of ISGs identified in cell culture or deduced from analyses of patient samples in adequate in vivo models is necessary. This will require a fully permissive and immuno-competent mouse model, which is not yet available.

      References

        • Simmonds P.
        The origin of hepatitis C virus.
        Curr Top Microbiol Immunol. 2013; 369: 1-15
        • Ploss A.
        • Rice C.M.
        Towards a small animal model for hepatitis C.
        EMBO Rep. 2009; 10: 1220-1227
      1. WHO. Hepatitis C, fact sheet N°164; 2012 [cited; Available from: <http://www.who.int/mediacentre/factsheets/fs164/en/>.

        • Prokunina-Olsson L.
        • Muchmore B.
        • Tang W.
        • Pfeiffer R.M.
        • Park H.
        • Dickensheets H.
        • et al.
        A variant upstream of IFNL3 (IL28B) creating a new interferon gene IFNL4 is associated with impaired clearance of hepatitis C virus.
        Nat Genet. 2013; 45: 164-171
        • Heim M.H.
        Innate immunity and HCV.
        J Hepatol. 2013; 58: 564-574
        • EASL Clinical Practice Guidelines
        Management of hepatitis C virus infection.
        J Hepatol. 2011; 55: 245-264
        • Ghany M.G.
        • Nelson D.R.
        • Strader D.B.
        • Thomas D.L.
        • Seeff L.B.
        An update on treatment of genotype 1 chronic hepatitis C virus infection: 2011 practice guideline by the American Association for the Study of liver diseases.
        Hepatology. 2011; 54: 1433-1444
        • Cunningham M.
        • Foster G.R.
        Efficacy and safety of telaprevir in patients with genotype 1 hepatitis C infection.
        Therap Adv Gastroenterol. 2012; 5: 139-151
        • Steinmann E.
        • Pietschmann T.
        Cell culture systems for hepatitis C virus.
        Curr Top Microbiol Immunol. 2013; 369: 17-48
        • Sarasin-Filipowicz M.
        • Oakeley E.J.
        • Duong F.H.
        • Christen V.
        • Terracciano L.
        • Filipowicz W.
        • et al.
        Interferon signaling and treatment outcome in chronic hepatitis C.
        Proc Natl Acad Sci U S A. 2008; 105: 7034-7039
        • Thomas E.
        • Gonzalez V.D.
        • Li Q.
        • Modi A.A.
        • Chen W.
        • Noureddin M.
        • et al.
        HCV infection induces a unique hepatic innate immune response associated with robust production of type III interferons.
        Gastroenterology. 2012; 142: 978-988
        • Mihm S.
        • Frese M.
        • Meier V.
        • Wietzke-Braun P.
        • Scharf J.G.
        • Bartenschlager R.
        • et al.
        Interferon type I gene expression in chronic hepatitis C.
        Lab Invest. 2004; 84: 1148-1159
        • Thimme R.
        • Binder M.
        • Bartenschlager R.
        Failure of innate and adaptive immune responses in controlling hepatitis C virus infection.
        FEMS Microbiol Rev. 2012; 36: 663-683
        • Lauer G.M.
        Immune responses to hepatitis C virus (HCV) infection and the prospects for an effective HCV vaccine or immunotherapies.
        J Infect Dis. 2013; 207: S7-S12
        • Dixit E.
        • Boulant S.
        • Zhang Y.
        • Lee A.S.
        • Odendall C.
        • Shum B.
        • et al.
        Peroxisomes are signaling platforms for antiviral innate immunity.
        Cell. 2010; 141: 668-681
        • Horner S.M.
        • Liu H.M.
        • Park H.S.
        • Briley J.
        • Gale Jr., M.
        Mitochondrial-associated endoplasmic reticulum membranes (MAM) form innate immune synapses and are targeted by hepatitis C virus.
        Proc Natl Acad Sci U S A. 2011; 108: 14590-14595
        • Kato H.
        • Takeuchi O.
        • Mikamo-Satoh E.
        • Hirai R.
        • Kawai T.
        • Matsushita K.
        • et al.
        Length-dependent recognition of double-stranded ribonucleic acids by retinoic acid-inducible gene-I and melanoma differentiation-associated gene 5.
        J Exp Med. 2008; 205: 1601-1610
        • Takahasi K.
        • Yoneyama M.
        • Nishihori T.
        • Hirai R.
        • Kumeta H.
        • Narita R.
        • et al.
        Nonself RNA-sensing mechanism of RIG-I helicase and activation of antiviral immune responses.
        Mol Cell. 2008; 29: 428-440
        • Cui S.
        • Eisenacher K.
        • Kirchhofer A.
        • Brzozka K.
        • Lammens A.
        • Lammens K.
        • et al.
        The C-terminal regulatory domain is the RNA 5′-triphosphate sensor of RIG-I.
        Mol Cell. 2008; 29: 169-179
        • Binder M.
        • Eberle F.
        • Seitz S.
        • Mucke N.
        • Huber C.M.
        • Kiani N.
        • et al.
        Molecular mechanism of signal perception and integration by the innate immune sensor retinoic acid-inducible gene-I (RIG-I).
        J Biol Chem. 2011; 286: 27278-27287
        • Saito T.
        • Owen D.M.
        • Jiang F.
        • Marcotrigiano J.
        • Gale Jr., M.
        Innate immunity induced by composition-dependent RIG-I recognition of hepatitis C virus RNA.
        Nature. 2008; 454: 523-527
        • Uzri D.
        • Gehrke L.
        Nucleotide sequences and modifications that determine RIG-I/RNA binding and signaling activities.
        J Virol. 2009; 83: 4174-4184
        • Eksioglu E.A.
        • Kielbasa J.
        • Eisen S.
        • Reddy V.
        Granulocyte-macrophage colony-stimulating factor increases the proportion of circulating dendritic cells after autologous but not after allogeneic hematopoietic stem cell transplantation.
        Cytotherapy. 2011; 13: 888-896
        • Yang D.
        • Liu N.
        • Zuo C.
        • Lei S.
        • Wu X.
        • Zhou F.
        • et al.
        Innate host response in primary human hepatocytes with hepatitis C virus infection.
        PLoS One. 2011; 6: e27552
        • Schoggins J.W.
        • Wilson S.J.
        • Panis M.
        • Murphy M.Y.
        • Jones C.T.
        • Bieniasz P.
        • et al.
        A diverse range of gene products are effectors of the type I interferon antiviral response.
        Nature. 2011; 472: 481-485
        • Andrus L.
        • Marukian S.
        • Jones C.T.
        • Catanese M.T.
        • Sheahan T.P.
        • Schoggins J.W.
        • et al.
        Expression of paramyxovirus V proteins promotes replication and spread of hepatitis C virus in cultures of primary human fetal liver cells.
        Hepatology. 2011; 54: 1901-1912
        • Ramachandran A.
        • Horvath C.M.
        Dissociation of paramyxovirus interferon evasion activities: universal and virus-specific requirements for conserved V protein amino acids in MDA5 interference.
        J Virol. 2010; 84: 11152-11163
        • Andrejeva J.
        • Poole E.
        • Young D.F.
        • Goodbourn S.
        • Randall R.E.
        The p127 subunit (DDB1) of the UV-DNA damage repair binding protein is essential for the targeted degradation of STAT1 by the V protein of the paramyxovirus simian virus 5.
        J Virol. 2002; 76: 11379-11386
        • Rodriguez J.J.
        • Parisien J.P.
        • Horvath C.M.
        Nipah virus V protein evades alpha and gamma interferons by preventing STAT1 and STAT2 activation and nuclear accumulation.
        J Virol. 2002; 76: 11476-11483
        • Loo Y.M.
        • Gale Jr., M.
        Immune signaling by RIG-I-like receptors.
        Immunity. 2011; 34: 680-692
        • Wang B.
        • Trippler M.
        • Pei R.
        • Lu M.
        • Broering R.
        • Gerken G.
        • et al.
        Toll-like receptor activated human and murine hepatic stellate cells are potent regulators of hepatitis C virus replication.
        J Hepatol. 2009; 51: 1037-1045
        • Li K.
        • Li N.L.
        • Wei D.
        • Pfeffer S.R.
        • Fan M.
        • Pfeffer L.M.
        Activation of chemokine and inflammatory cytokine response in hepatitis C virus-infected hepatocytes depends on Toll-like receptor 3 sensing of hepatitis C virus double-stranded RNA intermediates.
        Hepatology. 2012; 55: 666-675
        • Schoggins J.W.
        • Rice C.M.
        Innate immune responses to hepatitis C virus.
        Curr Top Microbiol Immunol. 2013; 369: 219-242
        • Manuse M.J.
        • Parks G.D.
        TLR3-dependent upregulation of RIG-I leads to enhanced cytokine production from cells infected with the parainfluenza virus SV5.
        Virology. 2010; 397: 231-241
        • Negash A.A.
        • Ramos H.J.
        • Crochet N.
        • Lau D.T.
        • Doehle B.
        • Papic N.
        • et al.
        IL-1beta production through the NLRP3 inflammasome by hepatic macrophages links hepatitis C virus infection with liver inflammation and disease.
        PLoS Pathog. 2013; 9: e1003330
        • Takahashi K.
        • Asabe S.
        • Wieland S.
        • Garaigorta U.
        • Gastaminza P.
        • Isogawa M.
        • et al.
        Plasmacytoid dendritic cells sense hepatitis C virus-infected cells, produce interferon, and inhibit infection.
        Proc Natl Acad Sci U S A. 2010; 107: 7431-7436
        • Lupberger J.
        • Felmlee D.J.
        • Baumert T.F.
        Interferon-lambda polymorphisms and hepatitis C virus clearance revisited.
        Hepatology. 2013; 58: 439-441
        • Marukian S.
        • Andrus L.
        • Sheahan T.P.
        • Jones C.T.
        • Charles E.D.
        • Ploss A.
        • et al.
        Hepatitis C virus induces interferon-lambda and interferon-stimulated genes in primary liver cultures.
        Hepatology. 2011; 54: 1913-1923
        • Stevenson N.J.
        • Murphy A.G.
        • Bourke N.M.
        • Keogh C.A.
        • Hegarty J.E.
        • O’Farrelly C.
        Ribavirin enhances IFN-alpha signalling and MxA expression: a novel immune modulation mechanism during treatment of HCV.
        PLoS One. 2011; 6: e27866
        • Kanazawa N.
        • Kurosaki M.
        • Sakamoto N.
        • Enomoto N.
        • Itsui Y.
        • Yamashiro T.
        • et al.
        Regulation of hepatitis C virus replication by interferon regulatory factor 1.
        J Virol. 2004; 78: 9713-9720
        • Raychoudhuri A.
        • Shrivastava S.
        • Steele R.
        • Kim H.
        • Ray R.
        • Ray R.B.
        ISG56 and IFITM1 proteins inhibit hepatitis C virus replication.
        J Virol. 2011; 85: 12881-12889
        • Zhu H.
        • Butera M.
        • Nelson D.R.
        • Liu C.
        Novel type I interferon IL-28A suppresses hepatitis C viral RNA replication.
        Virol J. 2005; 2: 80
        • Wang C.
        • Pflugheber J.
        • Sumpter Jr., R.
        • Sodora D.L.
        • Hui D.
        • Sen G.C.
        • et al.
        Alpha interferon induces distinct translational control programs to suppress hepatitis C virus RNA replication.
        J Virol. 2003; 77: 3898-3912
        • Itsui Y.
        • Sakamoto N.
        • Kurosaki M.
        • Kanazawa N.
        • Tanabe Y.
        • Koyama T.
        • et al.
        Expressional screening of interferon-stimulated genes for antiviral activity against hepatitis C virus replication.
        J Viral Hepat. 2006; 13: 690-700
        • Walters K.A.
        • Joyce M.A.
        • Thompson J.C.
        • Smith M.W.
        • Yeh M.M.
        • Proll S.
        • et al.
        Host-specific response to HCV infection in the chimeric SCID-beige/Alb-uPA mouse model: role of the innate antiviral immune response.
        PLoS Pathog. 2006; 2: e59
        • Tsuge M.
        • Fujimoto Y.
        • Hiraga N.
        • Zhang Y.
        • Ohnishi M.
        • Kohno T.
        • et al.
        Hepatitis C virus infection suppresses the interferon response in the liver of the human hepatocyte chimeric mouse.
        PLoS One. 2011; 6: e23856
        • Bigger C.B.
        • Brasky K.M.
        • Lanford R.E.
        DNA microarray analysis of chimpanzee liver during acute resolving hepatitis C virus infection.
        J Virol. 2001; 75: 7059-7066
        • Bigger C.B.
        • Guerra B.
        • Brasky K.M.
        • Hubbard G.
        • Beard M.R.
        • Luxon B.A.
        • et al.
        Intrahepatic gene expression during chronic hepatitis C virus infection in chimpanzees.
        J Virol. 2004; 78: 13779-13792
        • Park H.
        • Serti E.
        • Eke O.
        • Muchmore B.
        • Prokunina-Olsson L.
        • Capone S.
        • et al.
        IL-29 is the dominant type III interferon produced by hepatocytes during acute hepatitis C virus infection.
        Hepatology. 2012; 56: 2060-2070
        • Barth H.
        • Rybczynska J.
        • Patient R.
        • Choi Y.
        • Sapp R.K.
        • Baumert T.F.
        • et al.
        Both innate and adaptive immunity mediate protective immunity against hepatitis C virus infection in chimpanzees.
        Hepatology. 2011; 54: 1135-1148
        • Shin E.C.
        • Seifert U.
        • Kato T.
        • Rice C.M.
        • Feinstone S.M.
        • Kloetzel P.M.
        • et al.
        Virus-induced type I IFN stimulates generation of immunoproteasomes at the site of infection.
        J Clin Invest. 2006; 116: 3006-3014
        • Lanford R.E.
        • Guerra B.
        • Bigger C.B.
        • Lee H.
        • Chavez D.
        • Brasky K.M.
        Lack of response to exogenous interferon-alpha in the liver of chimpanzees chronically infected with hepatitis C virus.
        Hepatology. 2007; 46: 999-1008
        • Pfeffer L.M.
        • Madey M.A.
        • Riely C.A.
        • Fleckenstein J.F.
        The induction of type I interferon production in hepatitis C-infected patients.
        J Interferon Cytokine Res. 2009; 29: 299-306
        • Metz P.
        • Dazert E.
        • Ruggieri A.
        • Mazur J.
        • Kaderali L.
        • Kaul A.
        • et al.
        Identification of type I and type II interferon-induced effectors controlling hepatitis C virus replication.
        Hepatology. 2012; 56: 2082-2093
        • Lau D.T.
        • Negash A.
        • Chen J.
        • Crochet N.
        • Sinha M.
        • Zhang Y.
        • et al.
        Innate immune tolerance and the role of kupffer cells in differential responses to interferon therapy among patients with HCV genotype 1 infection.
        Gastroenterology. 2013; 144 (e12): 402-413
        • Dreux M.
        • Garaigorta U.
        • Boyd B.
        • Decembre E.
        • Chung J.
        • Whitten-Bauer C.
        • et al.
        Short-range exosomal transfer of viral RNA from infected cells to plasmacytoid dendritic cells triggers innate immunity.
        Cell Host Microbe. 2012; 12: 558-570
        • Dill M.T.
        • Makowska Z.
        • Duong F.H.
        • Merkofer F.
        • Filipowicz M.
        • Baumert T.F.
        • et al.
        Interferon-gamma-stimulated genes, but not USP18, are expressed in livers of patients with acute hepatitis C.
        Gastroenterology. 2012; 143 ([e771–776]): 777-786
        • Tanaka Y.
        • Nishida N.
        • Sugiyama M.
        • Kurosaki M.
        • Matsuura K.
        • Sakamoto N.
        • et al.
        Genome-wide association of IL28B with response to pegylated interferon-alpha and ribavirin therapy for chronic hepatitis C.
        Nat Genet. 2009; 41: 1105-1109
        • Suppiah V.
        • Moldovan M.
        • Ahlenstiel G.
        • Berg T.
        • Weltman M.
        • Abate M.L.
        • et al.
        IL28B is associated with response to chronic hepatitis C interferon-alpha and ribavirin therapy.
        Nat Genet. 2009; 41: 1100-1104
        • Ge D.
        • Fellay J.
        • Thompson A.J.
        • Simon J.S.
        • Shianna K.V.
        • Urban T.J.
        • et al.
        Genetic variation in IL28B predicts hepatitis C treatment-induced viral clearance.
        Nature. 2009; 461: 399-401
        • Rauch A.
        • Kutalik Z.
        • Descombes P.
        • Cai T.
        • Di Iulio J.
        • Mueller T.
        • et al.
        Genetic variation in IL28B is associated with chronic hepatitis C and treatment failure: a genome-wide association study.
        Gastroenterology. 2010; 138 ([1345 e1331–1337]): 1338-1345
        • Thomas D.L.
        • Thio C.L.
        • Martin M.P.
        • Qi Y.
        • Ge D.
        • O’Huigin C.
        • et al.
        Genetic variation in IL28B and spontaneous clearance of hepatitis C virus.
        Nature. 2009; 461: 798-801
        • Tillmann H.L.
        • Thompson A.J.
        • Patel K.
        • Wiese M.
        • Tenckhoff H.
        • Nischalke H.D.
        • et al.
        A polymorphism near IL28B is associated with spontaneous clearance of acute hepatitis C virus and jaundice.
        Gastroenterology. 2010; 139 (1592 e1581): 1586-1592
        • Abe H.
        • Hayes C.N.
        • Ochi H.
        • Maekawa T.
        • Tsuge M.
        • Miki D.
        • et al.
        IL28 variation affects expression of interferon stimulated genes and peg-interferon and ribavirin therapy.
        J Hepatol. 2011; 54: 1094-1101
        • Dill M.T.
        • Duong F.H.
        • Vogt J.E.
        • Bibert S.
        • Bochud P.Y.
        • Terracciano L.
        • et al.
        Interferon-induced gene expression is a stronger predictor of treatment response than IL28B genotype in patients with hepatitis C.
        Gastroenterology. 2011; 140: 1021-1031
        • Bibert S.
        • Roger T.
        • Calandra T.
        • Bochud M.
        • Cerny A.
        • Semmo N.
        • et al.
        IL28B expression depends on a novel TT/-G polymorphism which improves HCV clearance prediction.
        J Exp Med. 2013; 210: 1109-1116
        • Lanford R.E.
        • Guerra B.
        • Lee H.
        • Averett D.R.
        • Pfeiffer B.
        • Chavez D.
        • et al.
        Antiviral effect and virus-host interactions in response to alpha interferon, gamma interferon, poly(i)-poly(c), tumor necrosis factor alpha, and ribavirin in hepatitis C virus subgenomic replicons.
        J Virol. 2003; 77: 1092-1104
        • Helbig K.J.
        • Lau D.T.
        • Semendric L.
        • Harley H.A.
        • Beard M.R.
        Analysis of ISG expression in chronic hepatitis C identifies viperin as a potential antiviral effector.
        Hepatology. 2005; 42: 702-710
        • Zhao H.
        • Lin W.
        • Kumthip K.
        • Cheng D.
        • Fusco D.N.
        • Hofmann O.
        • et al.
        A functional genomic screen reveals novel host genes that mediate interferon-alpha’s effects against hepatitis C virus.
        J Hepatol. 2012; 56: 326-333
        • Fusco D.N.
        • Brisac C.
        • John S.P.
        • Huang Y.W.
        • Chin C.R.
        • Xie T.
        • et al.
        A genetic screen identifies interferon-alpha effector genes required to suppress hepatitis C virus replication.
        Gastroenterology. 2013; 144 (e1-9): 1449
        • Dabo S.
        • Meurs E.F.
        DsRNA-dependent protein kinase PKR and its role in stress, signaling and HCV infection.
        Viruses. 2012; 4: 2598-2635
        • Saunders L.R.
        • Barber G.N.
        The dsRNA binding protein family: critical roles, diverse cellular functions.
        FASEB J. 2003; 17: 961-983
        • Chong K.L.
        • Feng L.
        • Schappert K.
        • Meurs E.
        • Donahue T.F.
        • Friesen J.D.
        • et al.
        Human p68 kinase exhibits growth suppression in yeast and homology to the translational regulator GCN2.
        EMBO J. 1992; 11: 1553-1562
        • Anderson P.
        • Kedersha N.
        Visibly stressed: the role of eIF2, TIA-1, and stress granules in protein translation.
        Cell Stress Chaperones. 2002; 7: 213-221
        • Ruggieri A.
        • Dazert E.
        • Metz P.
        • Hofmann S.
        • Bergeest J.P.
        • Mazur J.
        • et al.
        Dynamic oscillation of translation and stress granule formation mark the cellular response to virus infection.
        Cell Host Microbe. 2012; 12: 71-85
        • Talloczy Z.
        • Jiang W.
        • Virgin H.W.
        • Leib D.A.
        • Scheuner D.
        • Kaufman R.J.
        • et al.
        Regulation of starvation- and virus-induced autophagy by the eIF2alpha kinase signaling pathway.
        Proc Natl Acad Sci U S A. 2002; 99: 190-195
        • Zamanian-Daryoush M.
        • Mogensen T.H.
        • DiDonato J.A.
        • Williams B.R.
        NF-kappaB activation by double-stranded-RNA-activated protein kinase (PKR) is mediated through NF-kappaB-inducing kinase and IkappaB kinase.
        Mol Cell Biol. 2000; 20: 1278-1290
        • Lu B.
        • Nakamura T.
        • Inouye K.
        • Li J.
        • Tang Y.
        • Lundback P.
        • et al.
        Novel role of PKR in inflammasome activation and HMGB1 release.
        Nature. 2012; 488: 670-674
        • Shimoike T.
        • McKenna S.A.
        • Lindhout D.A.
        • Puglisi J.D.
        Translational insensitivity to potent activation of PKR by HCV IRES RNA.
        Antiviral Res. 2009; 83: 228-237
        • Pflugheber J.
        • Fredericksen B.
        • Sumpter Jr., R.
        • Wang C.
        • Ware F.
        • Sodora D.L.
        • et al.
        Regulation of PKR and IRF-1 during hepatitis C virus RNA replication.
        Proc Natl Acad Sci U S A. 2002; 99: 4650-4655
        • Enomoto N.
        • Sakuma I.
        • Asahina Y.
        • Kurosaki M.
        • Murakami T.
        • Yamamoto C.
        • et al.
        Comparison of full-length sequences of interferon-sensitive and resistant hepatitis C virus 1b. Sensitivity to interferon is conferred by amino acid substitutions in the NS5A region.
        J Clin Invest. 1995; 96: 224-230
        • Tan S.L.
        • Katze M.G.
        How hepatitis C virus counteracts the interferon response: the jury is still out on NS5A.
        Virology. 2001; 284: 1-12
        • Garaigorta U.
        • Chisari F.V.
        Hepatitis C virus blocks interferon effector function by inducing protein kinase R phosphorylation.
        Cell Host Microbe. 2009; 6: 513-522
        • Arnaud N.
        • Dabo S.
        • Maillard P.
        • Budkowska A.
        • Kalliampakou K.I.
        • Mavromara P.
        • et al.
        Hepatitis C virus controls interferon production through PKR activation.
        PLoS One. 2010; 5: e10575
        • Terenin I.M.
        • Dmitriev S.E.
        • Andreev D.E.
        • Shatsky I.N.
        Eukaryotic translation initiation machinery can operate in a bacterial-like mode without eIF2.
        Nat Struct Mol Biol. 2008; 15: 836-841
        • Delhem N.
        • Sabile A.
        • Gajardo R.
        • Podevin P.
        • Abadie A.
        • Blaton M.A.
        • et al.
        Activation of the interferon-inducible protein kinase PKR by hepatocellular carcinoma derived-hepatitis C virus core protein.
        Oncogene. 2001; 20: 5836-5845
        • Taylor D.R.
        • Shi S.T.
        • Romano P.R.
        • Barber G.N.
        • Lai M.M.
        Inhibition of the interferon-inducible protein kinase PKR by HCV E2 protein.
        Science. 1999; 285: 107-110
        • Moradpour D.
        • Penin F.
        Hepatitis C virus proteins: from structure to function.
        Curr Top Microbiol Immunol. 2013; 369: 113-142
        • Yount J.S.
        • Karssemeijer R.A.
        • Hang H.C.
        S-palmitoylation and ubiquitination differentially regulate interferon-induced transmembrane protein 3 (IFITM3)-mediated resistance to influenza virus.
        J Biol Chem. 2012; 287: 19631-19641
        • Brass A.L.
        • Huang I.C.
        • Benita Y.
        • John S.P.
        • Krishnan M.N.
        • Feeley E.M.
        • et al.
        The IFITM proteins mediate cellular resistance to influenza A H1N1 virus, West Nile virus, and dengue virus.
        Cell. 2009; 139: 1243-1254
        • Lu J.
        • Pan Q.
        • Rong L.
        • He W.
        • Liu S.L.
        • Liang C.
        The IFITM proteins inhibit HIV-1 infection.
        J Virol. 2011; 85: 2126-2137
        • Huang I.C.
        • Bailey C.C.
        • Weyer J.L.
        • Radoshitzky S.R.
        • Becker M.M.
        • Chiang J.J.
        • et al.
        Distinct patterns of IFITM-mediated restriction of filoviruses, SARS coronavirus, and influenza A virus.
        PLoS Pathog. 2011; 7: e1001258
        • Yao L.
        • Dong H.
        • Zhu H.
        • Nelson D.
        • Liu C.
        • Lambiase L.
        • et al.
        Identification of the IFITM3 gene as an inhibitor of hepatitis C viral translation in a stable STAT1 cell line.
        J Viral Hepat. 2011; 18: e523-e529
        • Wilkins C.
        • Woodward J.
        • Lau D.T.
        • Barnes A.
        • Joyce M.
        • McFarlane N.
        • et al.
        IFITM1 is a tight junction protein that inhibits hepatitis C virus entry.
        Hepatology. 2013; 57: 461-469
        • Takahashi S.
        • Doss C.
        • Levy S.
        • Levy R.
        TAPA-1, the target of an antiproliferative antibody, is associated on the cell surface with the Leu-13 antigen.
        J Immunol. 1990; 145: 2207-2213
        • Feeley E.M.
        • Sims J.S.
        • John S.P.
        • Chin C.R.
        • Pertel T.
        • Chen L.M.
        • et al.
        IFITM3 inhibits influenza A virus infection by preventing cytosolic entry.
        PLoS Pathog. 2011; 7: e1002337
        • Amini-Bavil-Olyaee S.
        • Choi Y.J.
        • Lee J.H.
        • Shi M.
        • Huang I.C.
        • Farzan M.
        • et al.
        The antiviral effector IFITM3 disrupts intracellular cholesterol homeostasis to block viral entry.
        Cell Host Microbe. 2013; 13: 452-464
        • Paul D.
        • Hoppe S.
        • Saher G.
        • Krijnse-Locker J.
        • Bartenschlager R.
        Morphological and biochemical characterization of the membranous hepatitis C virus replication compartment.
        J Virol. 2013; 87: 10612-10627
        • Hinson E.R.
        • Cresswell P.
        The N-terminal amphipathic alpha-helix of viperin mediates localization to the cytosolic face of the endoplasmic reticulum and inhibits protein secretion.
        J Biol Chem. 2009; 284: 4705-4712
        • Duschene K.S.
        • Broderick J.B.
        The antiviral protein viperin is a radical SAM enzyme.
        FEBS Lett. 2010; 584: 1263-1267
        • Jiang D.
        • Guo H.
        • Xu C.
        • Chang J.
        • Gu B.
        • Wang L.
        • et al.
        Identification of three interferon-inducible cellular enzymes that inhibit the replication of hepatitis C virus.
        J Virol. 2008; 82: 1665-1678
        • Hinson E.R.
        • Cresswell P.
        The antiviral protein, viperin, localizes to lipid droplets via its N-terminal amphipathic alpha-helix.
        Proc Natl Acad Sci U S A. 2009; 106: 20452-20457
        • Drin G.
        • Casella J.F.
        • Gautier R.
        • Boehmer T.
        • Schwartz T.U.
        • Antonny B.
        A general amphipathic alpha-helical motif for sensing membrane curvature.
        Nat Struct Mol Biol. 2007; 14: 138-146
        • Wang S.
        • Wu X.
        • Pan T.
        • Song W.
        • Wang Y.
        • Zhang F.
        • et al.
        Viperin inhibits hepatitis C virus replication by interfering with binding of NS5A to host protein hVAP-33.
        J Gen Virol. 2012; 93: 83-92
        • Helbig K.J.
        • Eyre N.S.
        • Yip E.
        • Narayana S.
        • Li K.
        • Fiches G.
        • et al.
        The antiviral protein viperin inhibits hepatitis C virus replication via interaction with nonstructural protein 5A.
        Hepatology. 2011; 54: 1506-1517
        • Gao L.
        • Aizaki H.
        • He J.W.
        • Lai M.M.
        Interactions between viral nonstructural proteins and host protein hVAP-33 mediate the formation of hepatitis C virus RNA replication complex on lipid raft.
        J Virol. 2004; 78: 3480-3488
        • Wang X.
        • Hinson E.R.
        • Cresswell P.
        The interferon-inducible protein viperin inhibits influenza virus release by perturbing lipid rafts.
        Cell Host Microbe. 2007; 2: 96-105
        • Szkopinska A.
        • Plochocka D.
        Farnesyl diphosphate synthase; regulation of product specificity.
        Acta Biochim Pol. 2005; 52: 45-55
        • Han J.Q.
        • Barton D.J.
        Activation and evasion of the antiviral 2’-5’-oligoadenylate synthetase/ribonuclease L pathway by hepatitis C virus mRNA.
        RNA. 2002; 8: 512-525
        • Ishibashi M.
        • Wakita T.
        • Esumi M.
        2’,5’-Oligoadenylate synthetase-like gene highly induced by hepatitis C virus infection in human liver is inhibitory to viral replication in vitro.
        Biochem Biophys Res Commun. 2010; 392: 397-402
        • Lee K.P.
        • Dey M.
        • Neculai D.
        • Cao C.
        • Dever T.E.
        • Sicheri F.
        Structure of the dual enzyme Ire1 reveals the basis for catalysis and regulation in nonconventional RNA splicing.
        Cell. 2008; 132: 89-100
        • Floyd-Smith G.
        • Slattery E.
        • Lengyel P.
        Interferon action: RNA cleavage pattern of a (2′–5′)oligoadenylate–dependent endonuclease.
        Science. 1981; 212: 1030-1032
        • Kwon Y.C.
        • Kang J.I.
        • Hwang S.B.
        • Ahn B.Y.
        The ribonuclease L-dependent antiviral roles of human 2′,5′-oligoadenylate synthetase family members against hepatitis C virus.
        FEBS Lett. 2013; 587: 156-164
        • Malathi K.
        • Saito T.
        • Crochet N.
        • Barton D.J.
        • Gale Jr., M.
        • Silverman R.H.
        RNase L releases a small RNA from HCV RNA that refolds into a potent PAMP.
        RNA. 2010; 16: 2108-2119
        • Zhao C.
        • Denison C.
        • Huibregtse J.M.
        • Gygi S.
        • Krug R.M.
        Human ISG15 conjugation targets both IFN-induced and constitutively expressed proteins functioning in diverse cellular pathways.
        Proc Natl Acad Sci U S A. 2005; 102: 10200-10205
        • Shi H.X.
        • Yang K.
        • Liu X.
        • Liu X.Y.
        • Wei B.
        • Shan Y.F.
        • et al.
        Positive regulation of interferon regulatory factor 3 activation by Herc5 via ISG15 modification.
        Mol Cell Biol. 2010; 30: 2424-2436
        • D’Cunha J.
        • Knight Jr., E.
        • Haas A.L.
        • Truitt R.L.
        • Borden E.C.
        Immunoregulatory properties of ISG15, an interferon-induced cytokine.
        Proc Natl Acad Sci U S A. 1996; 93: 211-215
        • Broering R.
        • Zhang X.
        • Kottilil S.
        • Trippler M.
        • Jiang M.
        • Lu M.
        • et al.
        The interferon stimulated gene 15 functions as a proviral factor for the hepatitis C virus and as a regulator of the IFN response.
        Gut. 2010; 59: 1111-1119
        • Chen L.
        • Sun J.
        • Meng L.
        • Heathcote J.
        • Edwards A.M.
        • McGilvray I.D.
        ISG15, a ubiquitin-like interferon-stimulated gene, promotes hepatitis C virus production in vitro: implications for chronic infection and response to treatment.
        J Gen Virol. 2010; 91: 382-388
        • Arnaud N.
        • Dabo S.
        • Akazawa D.
        • Fukasawa M.
        • Shinkai-Ouchi F.
        • Hugon J.
        • et al.
        Hepatitis C virus reveals a novel early control in acute immune response.
        PLoS Pathog. 2011; 7: e1002289
        • Randall G.
        • Chen L.
        • Panis M.
        • Fischer A.K.
        • Lindenbach B.D.
        • Sun J.
        • et al.
        Silencing of USP18 potentiates the antiviral activity of interferon against hepatitis C virus infection.
        Gastroenterology. 2006; 131: 1584-1591
        • Kim M.J.
        • Yoo J.Y.
        Inhibition of hepatitis C virus replication by IFN-mediated ISGylation of HCV-NS5A.
        J Immunol. 2010; 185: 4311-4318
        • Taylor D.R.
        • Puig M.
        • Darnell M.E.
        • Mihalik K.
        • Feinstone S.M.
        New antiviral pathway that mediates hepatitis C virus replicon interferon sensitivity through ADAR1.
        J Virol. 2005; 79: 6291-6298
        • Chang J.H.
        • Kato N.
        • Muroyama R.
        • Taniguchi H.
        • Guleng B.
        • Dharel N.
        • et al.
        Double-stranded RNA-activated protein kinase inhibits hepatitis C virus replication but may be not essential in interferon treatment.
        Liver Int. 2010; 30: 311-318
        • Dafa-Berger A.
        • Kuzmina A.
        • Fassler M.
        • Yitzhak-Asraf H.
        • Shemer-Avni Y.
        • Taube R.
        Modulation of hepatitis C virus release by the interferon-induced protein BST-2/tetherin.
        Virology. 2012; 428: 98-111