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The history of hepatitis C virus (HCV): Basic research reveals unique features in phylogeny, evolution and the viral life cycle with new perspectives for epidemic control

  • Jens Bukh
    Correspondence
    Corresponding author. Address: Department of Infectious Diseases, Hvidovre Hospital, Kettegaard Alle 30, DK-2650 Hvidovre, Denmark. Tel.: +45 23 41 89 69.
    Affiliations
    Copenhagen Hepatitis C Program (CO-HEP), Department of Infectious Diseases and Clinical Research Centre, Hvidovre Hospital and Department of Immunology and Microbiology, Faculty of Health and Medical Sciences, University of Copenhagen, Denmark
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      Summary

      The discovery of hepatitis C virus (HCV) in 1989 permitted basic research to unravel critical components of a complex life cycle for this important human pathogen. HCV is a highly divergent group of viruses classified in 7 major genotypes and a great number of subtypes, and circulating in infected individuals as a continuously evolving quasispecies destined to escape host immune responses and applied antivirals. Despite the inability to culture patient viruses directly in the laboratory, efforts to define the infectious genome of HCV resulted in development of experimental recombinant in vivo and in vitro systems, including replicons and infectious cultures in human hepatoma cell lines. And HCV has become a model virus defining new paradigms in virology, immunology and biology. For example, HCV research discovered that a virus could be completely dependent on microRNA for its replication since microRNA-122 is critical for the HCV life cycle. A number of other host molecules critical for HCV entry and replication have been identified. Thus, basic HCV research revealed important molecules for development of host targeting agents (HTA). The identification and characterization of HCV encoded proteins and their functional units contributed to the development of highly effective direct acting antivirals (DAA) against the NS3 protease, NS5A and the NS5B polymerase. In combination, these inhibitors have since 2014 permitted interferon-free therapy with cure rates above 90% among patients with chronic HCV infection; however, viral resistance represents a challenge. Worldwide control of HCV will most likely require the development of a prophylactic vaccine, and numerous candidates have been pursued. Research characterizing features critical for antibody-based virus neutralization and T cell based virus elimination from infected cells is essential for this effort. If the world community promotes an ambitious approach by applying current DAA broadly, continues to develop alternative viral- and host- targeted antivirals to combat resistant variants, and invests in the development of a vaccine, it would be possible to eradicate HCV. This would prevent about 500 thousand deaths annually. However, given the nature of HCV, the millions of new infections annually, a high chronicity rate, and with over 150 million individuals with chronic infection (which are frequently unidentified), this effort remains a major challenge for basic researchers, clinicians and communities.

      Abbreviations:

      HCV (hepatitis C virus), HTA (host targeting agents), DAA (direct acting antivirals), ORF (open reading frame), UTR (untranslated region), NS (non-structural), E (envelope), IFN (interferon), GBV-B (GB virus B), miR-122 (microRNA-122), HBV (hepatitis B virus)

      Keywords

      Discovery and basic characterization of an important human pathogen

      Hepatitis C virus (HCV) is a main contributor to chronic liver diseases worldwide. Its existence was first fully recognized in 1975 when Feinstone et al. found that most cases of transfusion-associated hepatitis were not associated with hepatitis A virus or hepatitis B virus (HBV) infections, and thus defined the disease non-A, non-B hepatitis [
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      ]. However, since 2011 their use has been highly restricted and in reality eliminated even in the U.S. Thus, HCV studies in animal models are now dependent on rodent models which all have severe shortcomings [
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      ]. More recently, Ploss and colleagues succeeded in developing genetically humanized mice with limited HCV replication, which might open up new possibilities for studies of HCV, such as studies of protective immunity in vaccine studies [
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      ]. Experimental infection of New World monkeys with GBV-B causes acute hepatitis, and it was suggested as a potential surrogate animal model for HCV [
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      Phylogenetic classification of HCV variants into highly diverse genotypes and subtypes

      A distinctive characteristic of HCV is its extensive genetic heterogeneity, which exist at several levels among viral populations in individual infected patients at any given time and during evolution (quasispecies; see below), and worldwide among isolates from different patients (genotypes, subtypes, and isolates/strains). Thus, in 1993 phylogenetic analysis of partial HCV sequences recovered from a large number of patient isolates from around the world demonstrated that the virus could be classified into 6 major genotypes with important subtypes [
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      ]. The genomes of the HCV isolates belonging to different major genotypes differ by about 30% at the nucleotide and deduced amino acid level; subtypes typically differ by >15%. Even within subtypes, different isolates can vary by up to 10%. Thus, HCV has a high level of genetic heterogeneity throughout the genome with important implications for diagnosis and treatment, as well as the possibility of developing an effective vaccine. Naturally occurring intra- or intergenotypic recombinants are rare, but a particular 1b/2k intergenotypic recombinant has spread to become of epidemiological importance [
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      Hepatitis C virus (HCV) exhibits extensive genetic heterogeneity, and has been classified into 7 major genotypes and over 75 subtypes with important implications for diagnosis and treatment. The major genotypes are found worldwide, but with distinct differences in their geographical distribution. Among individuals with persistent HCV genotypes 1 and 3 account for about 75% of all infections.
      Figure thumbnail gr1
      Fig. 1Classification of hepatitis C virus (HCV) into 7 major genotypes and a large number of subtypes. The tree is based on phylogenetic analysis of the open reading frame (nucleotide) sequences. The overall prevalence and distribution is indicated for each major genotype. In part adapted from Smith et al.
      [
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      ]
      .
      The impact of HCV genotypes on the long-term outcome of HCV infection appears to be minimal. Thus, all genotypes are associated with severe liver diseases; genotype 3 patients might have an increased tendency to develop liver steatosis [
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      ]. It is well established that HCV genotype was associated with response to IFN-based treatments; patients infected with genotype 1 and 4 responded poorly to treatment compared to patients infected with genotypes 2 or 3 [
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      ].
      Although the different major genotypes have all been found worldwide, there are clear differences in their geographical distribution (Fig. 1). A recent global survey found that genotypes 1 and 3 are the most prevalent, accounting for 46% and 30% of all infections, respectively; genotypes 2, 4, 5 and 6 accounted for 9%, 8%, 1% and 6%, respectively [
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      • Quer J.
      The changing epidemiology of hepatitis C virus infection in Europe.
      ]; genotype 1 is the most prevalent in most countries, but genotype 3 has a significant prevalence in many countries and genotype 2 is a prevalent genotype in Italy. Among genotype 1, subtype 1a is the most prevalent in Northern Europe, whereas 1b is most prevalent in Southern Europe. Among genotype 2, subtype 2b is most prevalent in Northern Europe, whereas 2c is prevalent in Southern Europe, in particular in Italy; 2c was originally identified in a patient from Sardinia [
      • Bukh J.
      • Purcell R.H.
      • Miller R.H.
      At least 12 genotypes of hepatitis C virus predicted by sequence analysis of the putative E1 gene of isolates collected worldwide.
      ]. Genotype 3 infections are represented almost exclusively by subtype 3a. Genotypes 4 and 5 have increased presence due to emigration from the Middle East and Africa and spread of specific subtypes in intravenous drug addict populations. Genotype 6 is only found sporadically, mostly in emigrants from Southeast Asia. However, genotypes 3a and 4d for example, have had increasing importance in Europe because of transmission among intravenous drug addicts. Thus, in some Northern European countries genotype 3a constitute nearly half of all infections [
      • Corbet S.
      • Bukh J.
      • Heinsen A.
      • Fomsgaard A.
      Hepatitis C virus subtyping by a core-envelope 1-based reverse transcriptase PCR assay with sequencing and its use in determining subtype distribution among Danish patients.
      ]. Furthermore, although genotype 4 is primarily found in the Middle East and Africa, 4d was originally identified in a Danish patient [
      • Bukh J.
      • Purcell R.H.
      • Miller R.H.
      At least 12 genotypes of hepatitis C virus predicted by sequence analysis of the putative E1 gene of isolates collected worldwide.
      ,
      • Bukh J.
      • Purcell R.H.
      • Miller R.H.
      Sequence analysis of the core gene of 14 hepatitis C virus genotypes.
      ] and is an important genotype among intravenous drug addicts in Europe.
      In most countries of the Americas the majority of infections are genotype 1 (subtypes 1a and 1b); the remaining infections are genotypes 2 (in particular 2a) and 3a [
      • Messina J.P.
      • Humphreys I.
      • Flaxman A.
      • Brown A.
      • Cooke G.S.
      • Pybus O.G.
      • et al.
      Global distribution and prevalence of hepatitis C virus genotypes.
      ]. Genotype 3 is found infrequently in Africa (and nearly exclusively in North and South Africa), where genotypes 1, 2 and 4 are the prevalent genotypes in North and Central Africa and genotypes 1 and 5 in South Africa; genotypes 4 is the most prevalent in North East and Central Africa and genotype 5 in South Africa. There are numerous subtypes of genotype 4, as originally identified among HIV infected individuals from the Democratic Republic of the Congo (Zaire at the time) [
      • Bukh J.
      • Purcell R.H.
      • Miller R.H.
      At least 12 genotypes of hepatitis C virus predicted by sequence analysis of the putative E1 gene of isolates collected worldwide.
      ,
      • Bukh J.
      • Purcell R.H.
      • Miller R.H.
      Sequence analysis of the core gene of 14 hepatitis C virus genotypes.
      ]. In Egypt most infections are subtype 4a, resulting from extensive transmission during a national anti-schistosomiasis injection campaign [
      • Pybus O.G.
      • Drummond A.J.
      • Nakano T.
      • Robertson B.H.
      • Rambaut A.
      The epidemiology and iatrogenic transmission of hepatitis C virus in Egypt: a Bayesian coalescent approach.
      ,
      • Ray S.C.
      • Arthur R.R.
      • Carella A.
      • Bukh J.
      • Thomas D.L.
      Genetic epidemiology of hepatitis C virus throughout Egypt.
      ]. Only a single subtype of genotype 5 was identified [
      • Bukh J.
      • Purcell R.H.
      • Miller R.H.
      At least 12 genotypes of hepatitis C virus predicted by sequence analysis of the putative E1 gene of isolates collected worldwide.
      ,
      • Bukh J.
      • Purcell R.H.
      • Miller R.H.
      Sequence analysis of the core gene of 14 hepatitis C virus genotypes.
      ]. In the Middle East genotypes 1 and 4 predominates. Although genotype 1 is most prevalent in Asia, genotypes 2, 3 and 6 are other important genotypes. Most infections in India, Pakistan, Bangladesh, Myanmar, Nepal, and Thailand are genotype 3, including numerous subtypes as originally evidenced in samples from Nepal [
      • Tokita H.
      • Shrestha S.M.
      • Okamoto H.
      • Sakamoto M.
      • Horikita M.
      • Iizuka H.
      • et al.
      Hepatitis C virus variants from Nepal with novel genotypes and their classification into the third major group.
      ]. In Japan most infections are genotypes 1b and 2a. Genotype 6 is found in 10–20% of the population in many areas in East and South East Asia; the relative high prevalence of subtype 6a, originally identified in a patient from Hong Kong [
      • Bukh J.
      • Purcell R.H.
      • Miller R.H.
      At least 12 genotypes of hepatitis C virus predicted by sequence analysis of the putative E1 gene of isolates collected worldwide.
      ,
      • Bukh J.
      • Purcell R.H.
      • Miller R.H.
      Sequence analysis of the core gene of 14 hepatitis C virus genotypes.
      ,
      • Bukh J.
      • Purcell R.H.
      • Miller R.H.
      Sequence analysis of the 5′ noncoding region of hepatitis C virus.
      ], is the result of spread among intravenous drug addicts in Vietnam and Hong Kong. Numerous subtypes of genotype 6 exist, as originally found in individuals from Vietnam [
      • Tokita H.
      • Okamoto H.
      • Tsuda F.
      • Song P.
      • Nakata S.
      • Chosa T.
      • et al.
      Hepatitis C virus variants from Vietnam are classifiable into the seventh, eighth, and ninth major genetic groups.
      ]. Finally, in Russia and Australia/New Zealand genotypes 1 (subtypes 1a and 1b) are most prevalent, followed by 3a.

      Quasispecies nature of HCV – a moving target for drug and vaccine development

      Within infected individuals, HCV circulates as a quasispecies, which is a mixture of closely related but distinctly different genomes. The viral genomes of a quasispecies typically differ by 1–3%. The quasispecies composition of HCV in an infected individual is the result of mutations that accumulate over time during infection or mutations that are present from the onset of the infection due to simultaneous transmission of multiple viral species. A new dominant HCV sequence can result from accumulation of mutations over time and/or from the selection of a preexisting minor viral species (evolution). Such mutations might enable HCV to replicate more efficiently or might help the virus evade host immune responses or antivirals. Although the genetic heterogeneity defining a quasispecies is found throughout the genome, certain regions are hypervariable, including hypervariable region 1 (HVR1) at the N-terminus of E2. The quasispecies nature of HCV might have implications for the natural history, for the response to antiviral therapy, and for the effectiveness of vaccine candidates [
      • Pawlotsky J.M.
      Hepatitis C virus resistance to direct-acting antiviral drugs in interferon-free regimens.
      ,
      • Farci P.
      • Bukh J.
      • Purcell R.H.
      The quasispecies of hepatitis C virus and the host immune response.
      ,
      • Forns X.
      • Purcell R.H.
      • Bukh J.
      Quasispecies in viral persistence and pathogenesis of hepatitis C virus.
      ].
      In infected individuals, hepatitis C virus (HCV) circulates as a continuously evolving quasispecies destined to escape host immune responses, including neutralizing antibodies and activated T-cells, and antivirals. Evolution has grave implications for the effectiveness of vaccine candidates and contributes to viral escape from DAA, with variants evolving from preexisting resistant associated substitutions or developing de novo during treatment.
      The great potential for HCV to introduce functional genome changes have been experimentally shown to promote escape from neutralizing antibodies and cellular immune responses [
      • Ball J.K.
      • Tarr A.W.
      • McKeating J.A.
      The past, present and future of neutralizing antibodies for hepatitis C virus.
      ,
      • Holz L.
      • Rehermann B.
      T cell responses in hepatitis C virus infection: historical overview and goals for future research.
      ]. It is associated with the outcome of acute HCV infection [
      • Farci P.
      • Shimoda A.
      • Coiana A.
      • Diaz G.
      • Peddis G.
      • Melpolder J.C.
      • et al.
      The outcome of acute hepatitis C predicted by the evolution of the viral quasispecies.
      ]. Further, it affects the viral population following reinfection, for example, after liver transplantations [
      • Ramirez S.
      • Perez-del-Pulgar S.
      • Carrion J.A.
      • Costa J.
      • Gonzalez P.
      • Massaguer A.
      • et al.
      Hepatitis C virus compartmentalization and infection recurrence after liver transplantation.
      ,
      • Fofana I.
      • Fafi-Kremer S.
      • Carolla P.
      • Fauvelle C.
      • Zahid M.N.
      • Turek M.
      • et al.
      Mutations that alter use of hepatitis C virus cell entry factors mediate escape from neutralizing antibodies.
      ,
      • Perez-del-Pulgar S.
      • Gregori J.
      • Rodriguez-Frias F.
      • Gonzalez P.
      • Garcia-Cehic D.
      • Ramirez S.
      • et al.
      Quasispecies dynamics in hepatitis C liver transplant recipients receiving grafts from hepatitis C virus infected donors.
      ]. Importantly, it was evidenced that this HCV heterogeneity found in the individual patient can contribute to viral escape from DAA, with variants evolving from preexisting resistant associated substitutions or developing de novo during treatment [
      • Pawlotsky J.M.
      Hepatitis C virus resistance to direct-acting antiviral drugs in interferon-free regimens.
      ,
      • Susser S.
      • Welsch C.
      • Wang Y.
      • Zettler M.
      • Domingues F.S.
      • Karey U.
      • et al.
      Characterization of resistance to the protease inhibitor boceprevir in hepatitis C virus-infected patients.
      ].

      Recombinant systems to study HCV replication for the different genotypes: an arduous undertaking

      Since clinical isolates of HCV cannot be cultured efficiently in vitro, even in cells now known to be susceptible to functional recombinant viruses, it has been an enormous challenge to develop experimental systems required for true basic studies of the viral life cycle [
      • Lohmann V.
      • Bartenschlager R.
      On the history of hepatitis C virus cell culture systems.
      ]. Infections were originally reported in continuous human T and B cell lines, but these replication systems are very inefficient and results have been difficult to reproduce. In addition, it turned out that the HCV genome originally described were missing the 3′ terminal sequence. Thus, the development of recombinant lab-generated culture systems only became possible after the identification, in pioneering studies in 1995–1996, of a structured sequence following the poly-pyrimidine tract at the 3′ terminus of HCV [
      • Kolykhalov A.A.
      • Feinstone S.M.
      • Rice C.M.
      Identification of a highly conserved sequence element at the 3′ terminus of hepatitis C virus genome RNA.
      ,
      • Tanaka T.
      • Kato N.
      • Cho M.J.
      • Shimotohno K.
      A novel sequence found at the 3′ terminus of hepatitis C virus genome.
      ].

      Infectious cDNA clones – in vivo studies

      A major breakthrough in the development of recombinant systems to study HCV was the generation of infectious molecular cDNA clones in 1997 [
      • Kolykhalov A.A.
      • Agapov E.V.
      • Blight K.J.
      • Mihalik K.
      • Feinstone S.M.
      • Rice C.M.
      Transmission of hepatitis C by intrahepatic inoculation with transcribed RNA.
      ,
      • Yanagi M.
      • Purcell R.H.
      • Emerson S.U.
      • Bukh J.
      Transcripts from a single full-length cDNA clone of hepatitis C virus are infectious when directly transfected into the liver of a chimpanzee.
      ]. This was achieved by determining the consensus sequence of the prototype strain H77 (genotype 1a) of HCV, and thereafter generating molecular clones encompassing this sequence (Fig. 2). In the absence of cell culture systems, infectivity was demonstrated by inoculating RNA transcripts synthesized in vitro directly into the liver of chimpanzees. Subsequently, in vivo infectious cDNA clones have been developed for HCV strains of other genotypes, including 1b, 2a, 3a and 4a [
      • Gottwein J.M.
      • Scheel T.K.
      • Callendret B.
      • Li Y.P.
      • Eccleston H.B.
      • Engle R.E.
      • et al.
      Novel infectious cDNA clones of hepatitis C virus genotype 3a (strain S52) and 4a (strain ED43): genetic analyses and in vivo pathogenesis studies.
      ,
      • Yanagi M.
      • St C.M.
      • Shapiro M.
      • Emerson S.U.
      • Purcell R.H.
      • Bukh J.
      Transcripts of a chimeric cDNA clone of hepatitis C virus genotype 1b are infectious in vivo.
      ,
      • Yanagi M.
      • Purcell R.H.
      • Emerson S.U.
      • Bukh J.
      Hepatitis C virus: an infectious molecular clone of a second major genotype (2a) and lack of viability of intertypic 1a and 2a chimeras.
      ]. This effort was greatly aided by the availability of prototype strains characterized in chimpanzees [
      • Bukh J.
      • Meuleman P.
      • Tellier R.
      • Engle R.E.
      • Feinstone S.M.
      • Eder G.
      • et al.
      Challenge pools of hepatitis C virus genotypes 1–6 prototype strains: replication fitness and pathogenicity in chimpanzees and human liver-chimeric mouse models.
      ]. The resulting monoclonal HCV infection in chimpanzees did not differ significantly from the polyclonal infection observed in animals infected intravenously with wild-type virus [
      • Bukh J.
      A critical role for the chimpanzee model in the study of hepatitis C.
      ,
      • Callendret B.
      • Bukh J.
      • Eccleston H.B.
      • Heksch R.
      • Hasselschwert D.L.
      • Purcell R.H.
      • et al.
      Transmission of clonal hepatitis C virus genomes reveals the dominant but transitory role of CD8 T cells in early viral evolution.
      ,
      • Major M.E.
      • Mihalik K.
      • Fernandez J.
      • Seidman J.
      • Kleiner D.
      • Kolykhalov A.A.
      • et al.
      Long-term follow-up of chimpanzees inoculated with the first infectious clone for hepatitis C virus.
      ,
      • Major M.E.
      • Dahari H.
      • Mihalik K.
      • Puig M.
      • Rice C.M.
      • Neumann A.U.
      • et al.
      Hepatitis C virus kinetics and host responses associated with disease and outcome of infection in chimpanzees.
      ]. Furthermore, these studies formally proved that HCV causes liver disease, since chimpanzees transfected with HCV genomic RNA developed acute hepatitis (Fig. 2). Finally, the availability of infectious HCV clones permitted for the first time true reverse genetics studies of the importance of genetic components, including the 3′UTR, p7 and key viral enzymes, for virus infectivity [
      • Kolykhalov A.A.
      • Mihalik K.
      • Feinstone S.M.
      • Rice C.M.
      Hepatitis C virus-encoded enzymatic activities and conserved RNA elements in the 3′ nontranslated region are essential for virus replication in vivo.
      ,
      • Sakai A.
      • Claire M.S.
      • Faulk K.
      • Govindarajan S.
      • Emerson S.U.
      • Purcell R.H.
      • et al.
      The p7 polypeptide of hepatitis C virus is critical for infectivity and contains functionally important genotype-specific sequences.
      ,
      • Yanagi M.
      • St C.M.
      • Emerson S.U.
      • Purcell R.H.
      • Bukh J.
      In vivo analysis of the 3′ untranslated region of the hepatitis C virus after in vitro mutagenesis of an infectious cDNA clone.
      ].
      Figure thumbnail gr2
      Fig. 2The generation of infectious cDNA clones of HCV. The full-length consensus HCV sequence for selected HCV strains was engineered into plasmids. In the absence of cell culture systems, infectivity was demonstrated by inoculating in vitro generated RNA transcripts directly into the liver of chimpanzees. Such in vivo infectious cDNA clones have been developed for HCV strains of genotypes 1a, 1b, 2a, 3a and 4a. The resulting monoclonal HCV infection in chimpanzees did not differ significantly from the polyclonal infection observed in animals infected intravenously with wild-type virus; here is shown an example of the course of infection observed in a chimpanzee transfected with RNA transcripts from an infectious cDNA clone of HCV (pCV-H77C) from patient H. The chimpanzee developed acute hepatitis with elevated liver enzyme levels and necro-inflammatory changes in liver biopsies. Based on original findings by Kolykhalov et al.
      [
      • Kolykhalov A.A.
      • Agapov E.V.
      • Blight K.J.
      • Mihalik K.
      • Feinstone S.M.
      • Rice C.M.
      Transmission of hepatitis C by intrahepatic inoculation with transcribed RNA.
      ]
      and Yanagi et al.
      [
      • Yanagi M.
      • Purcell R.H.
      • Emerson S.U.
      • Bukh J.
      Transcripts from a single full-length cDNA clone of hepatitis C virus are infectious when directly transfected into the liver of a chimpanzee.
      ]
      .
      The wild-type in vivo infectious HCV sequences turned out not to be replication competent in transfected continuous cell lines, including Huh7 derived hepatoma cell lines [
      • Gottwein J.M.
      • Scheel T.K.
      • Callendret B.
      • Li Y.P.
      • Eccleston H.B.
      • Engle R.E.
      • et al.
      Novel infectious cDNA clones of hepatitis C virus genotype 3a (strain S52) and 4a (strain ED43): genetic analyses and in vivo pathogenesis studies.
      ,
      • Sakai A.
      • Takikawa S.
      • Thimme R.
      • Meunier J.C.
      • Spangenberg H.C.
      • Govindarajan S.
      • et al.
      In vivo study of the HC-TN strain of hepatitis C virus recovered from a patient with fulminant hepatitis: RNA transcripts of a molecular clone (pHC-TN) are infectious in chimpanzees but not in Huh7.5 cells.
      ]. However, they facilitated development of in vitro systems for HCV, since subsequent studies could be performed with genomes known to have all genetic elements required for infection [
      • Lohmann V.
      • Bartenschlager R.
      On the history of hepatitis C virus cell culture systems.
      ,
      • Bartenschlager R.
      Hepatitis C virus molecular clones: from cDNA to infectious virus particles in cell culture.
      ]. This research resulted in the development of HCV replicons in 1999 and in a true infectious culture system in 2005 [
      • Bartenschlager R.
      Hepatitis C virus molecular clones: from cDNA to infectious virus particles in cell culture.
      ,
      • Bartenschlager R.
      • Sparacio S.
      Hepatitis C virus molecular clones and their replication capacity in vivo and in cell culture.
      ].
      Despite the inability to culture patient viruses, detailed characterization of the infectious genome of hepatitis C virus (HCV) resulted in the development of recombinant systems in vivo (infectious clones) and in vitro (replicons, pseudoviruses, and infectious cultures in human hepatoma cell lines). They have made it possible to discover key viral and host elements in the HCV life-cycle.

      HCV replicons – in vitro studies

      In a landmark study by Lohmann et al. that literally changed the perspective of developing drugs targeting HCV replication it was demonstrated that a subgenomic viral sequence consisting of the 5′ UTR, NS3-NS5B and the 3′ UTR from strain Con1 (genotype 1b) in a selectable bicistronic construct can function as a self-replicating autonomous unit in Huh7 hepatoma cell lines [
      • Lohmann V.
      • Korner F.
      • Koch J.
      • Herian U.
      • Theilmann L.
      • Bartenschlager R.
      Replication of subgenomic hepatitis C virus RNAs in a hepatoma cell line.
      ] (Fig. 3). This breakthrough finding permitted for the first time in vitro studies of HCV RNA replication. Subsequent analysis demonstrated that their replication capacity was determined by adaptive mutations of the replicating HCV RNA and by increased host cell permissiveness [
      • Blight K.J.
      • Kolykhalov A.A.
      • Rice C.M.
      Efficient initiation of HCV RNA replication in cell culture.
      ,
      • Blight K.J.
      • McKeating J.A.
      • Rice C.M.
      Highly permissive cell lines for subgenomic and genomic hepatitis C virus RNA replication.
      ,
      • Lohmann V.
      • Korner F.
      • Dobierzewska A.
      • Bartenschlager R.
      Mutations in hepatitis C virus RNAs conferring cell culture adaptation.
      ,
      • Lohmann V.
      • Hoffmann S.
      • Herian U.
      • Penin F.
      • Bartenschlager R.
      Viral and cellular determinants of hepatitis C virus RNA replication in cell culture.
      ]. Numerous studies addressing the function of these adaptive mutations led to highly adapted subgenomic and full-genome length replicons, and by curing Huh7 cell lines with high level RNA replication highly permissive cell lines [
      • Lohmann V.
      HCV replicons: overview and basic protocols.
      ,
      • Bartenschlager R.
      The hepatitis C virus replicon system: from basic research to clinical application.
      ]. The improved permissiveness of Huh7.5 cells appears to be the result of a defect in the IFN signaling pathway of major importance for antiviral immunity. Subsequently, HCV replicons depending on signature adaptive mutations have been developed for other genotype 1 strains [
      • Blight K.J.
      • McKeating J.A.
      • Marcotrigiano J.
      • Rice C.M.
      Efficient replication of hepatitis C virus genotype 1a RNAs in cell culture.
      ,
      • Grobler J.A.
      • Markel E.J.
      • Fay J.F.
      • Graham D.J.
      • Simcoe A.L.
      • Ludmerer S.W.
      • et al.
      Identification of a key determinant of hepatitis C virus cell culture adaptation in domain II of NS3 helicase.
      ,
      • Ikeda M.
      • Yi M.
      • Li K.
      • Lemon S.M.
      Selectable subgenomic and genome-length dicistronic RNAs derived from an infectious molecular clone of the HCV-N strain of hepatitis C virus replicate efficiently in cultured Huh7 cells.
      ,
      • Yi M.
      • Lemon S.M.
      Adaptive mutations producing efficient replication of genotype 1a hepatitis C virus RNA in normal Huh7 cells.
      ], as well as for selected strains of genotypes 2–6 [
      • Saeed M.
      • Scheel T.K.
      • Gottwein J.M.
      • Marukian S.
      • Dustin L.B.
      • Bukh J.
      • et al.
      Efficient replication of genotype 3a and 4a hepatitis C virus replicons in human hepatoma cells.
      ,
      • Saeed M.
      • Gondeau C.
      • Hmwe S.
      • Yokokawa H.
      • Date T.
      • Suzuki T.
      • et al.
      Replication of hepatitis C virus genotype 3a in cultured cells.
      ,
      • Kato T.
      • Date T.
      • Miyamoto M.
      • Furusaka A.
      • Tokushige K.
      • Mizokami M.
      • et al.
      Efficient replication of the genotype 2a hepatitis C virus subgenomic replicon.
      ,
      • Peng B.
      • Yu M.
      • Xu S.
      • Lee Y.J.
      • Tian Y.
      • Yang H.
      • et al.
      Development of robust hepatitis C virus genotype 4 subgenomic replicons.
      ,
      • Wose Kinge C.N.
      • Espiritu C.
      • Prabdial-Sing N.
      • Sithebe N.P.
      • Saeed M.
      • Rice C.M.
      Hepatitis C virus genotype 5a subgenomic replicons for evaluation of direct-acting antiviral agents.
      ,
      • Yu M.
      • Peng B.
      • Chan K.
      • Gong R.
      • Yang H.
      • Delaney W.
      • et al.
      Robust and persistent replication of the genotype 6a hepatitis C virus replicon in cell culture.
      ]. An important finding was that a replicon of JFH1, a genotype 2a strain from a Japanese patient with fulminant hepatitis [
      • Kato T.
      • Furusaka A.
      • Miyamoto M.
      • Date T.
      • Yasui K.
      • Hiramoto J.
      • et al.
      Sequence analysis of hepatitis C virus isolated from a fulminant hepatitis patient.
      ], could replicate in original Huh7 derived cell lines without the requirement for cell culture adaptive mutations [
      • Kato T.
      • Date T.
      • Miyamoto M.
      • Furusaka A.
      • Tokushige K.
      • Mizokami M.
      • et al.
      Efficient replication of the genotype 2a hepatitis C virus subgenomic replicon.
      ], thus leading the way to the development of the first infectious cell culture system for HCV (see below).
      Figure thumbnail gr3
      Fig. 3Principle of the generation of HCV replicons. Diagram showing the composition of a sub-genomic viral replicon sequence consisting of the 5′ UTR, NS3-NS5B and the 3′ UTR from strain Con1 (genotype 1b) in a selectable bicistronic construct. To demonstrate viral replication, RNA transcripts generated from this genome were transfected into human hepatoma derived cells and the cells with replicating RNA was selected following treatment with neomycin. Subsequent analysis demonstrated that their replication capacity was determined by adaptive mutations of the replicating HCV RNA and by increased host cell permissiveness. Based on findings by Lohmann et al.
      [
      • Lohmann V.
      • Korner F.
      • Koch J.
      • Herian U.
      • Theilmann L.
      • Bartenschlager R.
      Replication of subgenomic hepatitis C virus RNAs in a hepatoma cell line.
      ]
      .
      It was unclear why the HCV RNA of most isolates required adaptive mutations to autonomously replicate in Huh7 hepatoma cells. However, in 2015 it was found that the host cells lack a key factor, SEC14L2, which permit replication of HCV sequences of different genotypes without the requirement for adaptive mutations [
      • Saeed M.
      • Andreo U.
      • Chung H.Y.
      • Espiritu C.
      • Branch A.D.
      • Silva J.M.
      • et al.
      SEC14L2 enables pan-genotype HCV replication in cell culture.
      ]. The dramatic effect of SEC14L2 for HCV replication was confirmed for genotypes 1–4 replicons in a recent study that also showed that specific modifications of the non-HCV replicon sequences could enhance HCV replication in this system [
      • Witteveldt J.
      • Martin-Gans M.
      • Simmonds P.
      Enhancement of the replication of hepatitis C virus replicons of genotypes 1 to 4 by manipulation of CpG and UpA dinucleotide frequencies and use of cell lines expressing SECL14L2 for antiviral resistance testing.
      ].
      The development of full-length adapted replication competent HCV genomes provided hope that these systems would eventually yield cell culture derived infectious viruses. However, they turned out to be incapable or restricted in generating infectious HCV particles. Thus, it was originally found that an in vitro optimized combination of highly adaptive mutations into the Con1 full-length genome abrogated its ability to productively infect chimpanzees [
      • Bukh J.
      • Pietschmann T.
      • Lohmann V.
      • Krieger N.
      • Faulk K.
      • Engle R.E.
      • et al.
      Mutations that permit efficient replication of hepatitis C virus RNA in Huh-7 cells prevent productive replication in chimpanzees.
      ] (Fig. 4). Similarly, it was observed that the in vivo infectious Con1 full-length genome without adaptive mutations could produce virions in culture, albeit at very low levels, in contrast to the genome with adaptive mutations which did not [
      • Pietschmann T.
      • Zayas M.
      • Meuleman P.
      • Long G.
      • Appel N.
      • Koutsoudakis G.
      • et al.
      Production of infectious genotype 1b virus particles in cell culture and impairment by replication enhancing mutations.
      ]. Thus, cell culture adaptive mutations in the Con1 strain apparently prevented formation of infectious virions in vitro and in vivo. After the development of infectious JFH1-based culture systems, however, the use of replicon adaptive mutations yielded infectious cultures of isolates of genotypes 1a, 2a and 3a [
      • Date T.
      • Kato T.
      • Kato J.
      • Takahashi H.
      • Morikawa K.
      • Akazawa D.
      • et al.
      Novel cell culture-adapted genotype 2a hepatitis C virus infectious clone.
      ,
      • Kim S.
      • Date T.
      • Yokokawa H.
      • Kono T.
      • Aizaki H.
      • Maurel P.
      • et al.
      Development of hepatitis C virus genotype 3a cell culture system.
      ,
      • Yi M.
      • Villanueva R.A.
      • Thomas D.L.
      • Wakita T.
      • Lemon S.M.
      Production of infectious genotype 1a hepatitis C virus (Hutchinson strain) in cultured human hepatoma cells.
      ]. However, these systems are inefficient with low virus titers and poor capacity to infect naïve cells.
      Figure thumbnail gr4
      Fig. 4Replication enhancing adaptive mutations renders full-length HCV clone non-viable in vivo. The wild-type Con1 genome was viable in vivo, but did not replicate in cell culture. Contrarily, genomes with mutations that permitted replication in cell culture were non-viable in vivo. Based on findings by Bukh et al.
      [
      • Bukh J.
      • Pietschmann T.
      • Lohmann V.
      • Krieger N.
      • Faulk K.
      • Engle R.E.
      • et al.
      Mutations that permit efficient replication of hepatitis C virus RNA in Huh-7 cells prevent productive replication in chimpanzees.
      ]
      .
      The described replicon systems proved extremely valuable for studies of the role of different HCV genome segments and proteins for HCV RNA replication, of the intracellular localization of HCV proteins, of virus-host interactions, and for testing of therapeutic compounds interfering with HCV replication [
      • Lohmann V.
      Hepatitis C virus RNA replication.
      ]. Thus, these systems have had a major impact on identifying and advancing candidate DAA [
      • Bartenschlager R.
      The hepatitis C virus replicon system: from basic research to clinical application.
      ,
      • Bartenschlager R.
      • Lohmann V.
      • Penin F.
      The molecular and structural basis of advanced antiviral therapy for hepatitis C virus infection.
      ].

      Infectious HCV cell culture systems (HCVcc)

      The development of a recombinant cell culture system resulting in production of significant levels of infectious virus particles has accelerated understanding of the complete HCV viral life cycle [
      • Wakita T.
      • Pietschmann T.
      • Kato T.
      • Date T.
      • Miyamoto M.
      • Zhao Z.
      • et al.
      Production of infectious hepatitis C virus in tissue culture from a cloned viral genome.
      ] (Fig. 5). In this breakthrough study, Wakita and Bartenschlager’s research teams demonstrated that RNA transcripts from the full-length JFH1 genome (genotype 2a) could produce viruses in Huh7 cells [
      • Wakita T.
      • Pietschmann T.
      • Kato T.
      • Date T.
      • Miyamoto M.
      • Zhao Z.
      • et al.
      Production of infectious hepatitis C virus in tissue culture from a cloned viral genome.
      ], and subsequently it was found by Chisari and Wakita’s research teams that this system could be adapted to generate relatively high titers of HCV [
      • Zhong J.
      • Gastaminza P.
      • Cheng G.
      • Kapadia S.
      • Kato T.
      • Burton D.R.
      • et al.
      Robust hepatitis C virus infection in vitro.
      ]. In a different approach, Rice’s research team generated a chimeric genome in which the structural genes (Core, E1 and E2), p7 and NS2 from an infectious clone of 2a strain J6 [
      • Yanagi M.
      • Purcell R.H.
      • Emerson S.U.
      • Bukh J.
      Hepatitis C virus: an infectious molecular clone of a second major genotype (2a) and lack of viability of intertypic 1a and 2a chimeras.
      ] were inserted into the subgenomic replicon sequence of the JFH1 strain [
      • Kato T.
      • Date T.
      • Miyamoto M.
      • Furusaka A.
      • Tokushige K.
      • Mizokami M.
      • et al.
      Efficient replication of the genotype 2a hepatitis C virus subgenomic replicon.
      ], and demonstrated that RNA transcripts from this full-length chimeric genotype 2a genome could produce relatively high titers of HCV [
      • Lindenbach B.D.
      • Evans M.J.
      • Syder A.J.
      • Wolk B.
      • Tellinghuisen T.L.
      • Liu C.C.
      • et al.
      Complete replication of hepatitis C virus in cell culture.
      ].
      Figure thumbnail gr5
      Fig. 5Principle of the generation of infectious HCV in cell culture, and demonstration of in vivo infectivity. Diagram showing the composition of full-length JFH1 and J6/JFH1 genomes. To demonstrate infectivity RNA transcripts generated from these genomes were transfected into human hepatoma derived cells and collected supernatant viruses were used to infect naïve cells. The immuno-staining shown was performed with a mouse monoclonal NS5A antibody. When passaged viruses were transmitted to chimpanzees or SCID-uPA mice with a human liver graft (human liver chimeric mice), the animals developed a productive HCV infection. Compared to culture derived viruses, the in vivo derived viruses had a high specific infectivity (ratio of infectivity titer to RNA titer). In part adapted from Bukh and Purcell
      [
      • Bukh J.
      • Purcell R.H.
      A milestone for hepatitis C virus research: a virus generated in cell culture is fully viable in vivo.
      ]
      .
      Whereas the J6/JFH1 genome can function without the requirement for adaptive mutations [
      • Gottwein J.M.
      • Scheel T.K.
      • Hoegh A.M.
      • Lademann J.B.
      • Eugen-Olsen J.
      • Lisby G.
      • et al.
      Robust hepatitis C genotype 3a cell culture releasing adapted intergenotypic 3a/2a (S52/JFH1) viruses.
      ,
      • Scheel T.K.
      • Gottwein J.M.
      • Jensen T.B.
      • Prentoe J.C.
      • Hoegh A.M.
      • Alter H.J.
      • et al.
      Development of JFH1-based cell culture systems for hepatitis C virus genotype 4a and evidence for cross-genotype neutralization.
      ], the original JFH1 genome requires adaptive mutations for efficient virus production [
      • Russell R.S.
      • Meunier J.C.
      • Takikawa S.
      • Faulk K.
      • Engle R.E.
      • Bukh J.
      • et al.
      Advantages of a single-cycle production assay to study cell culture-adaptive mutations of hepatitis C virus.
      ,
      • Kaul A.
      • Woerz I.
      • Meuleman P.
      • Leroux-Roels G.
      • Bartenschlager R.
      Cell culture adaptation of hepatitis C virus and in vivo viability of an adapted variant.
      ]. Both systems can be adapted to grow to high viral titers by continuous passage in culture [
      • Lohmann V.
      • Bartenschlager R.
      On the history of hepatitis C virus cell culture systems.
      ]. In addition, culture derived viruses were viable also in vivo, as tested in chimpanzees and human liver chimeric mice [
      • Kato T.
      • Choi Y.
      • Elmowalid G.
      • Sapp R.K.
      • Barth H.
      • Furusaka A.
      • et al.
      Hepatitis C virus JFH-1 strain infection in chimpanzees is associated with low pathogenicity and emergence of an adaptive mutation.
      ,
      • Lindenbach B.D.
      • Meuleman P.
      • Ploss A.
      • Vanwolleghem T.
      • Syder A.J.
      • McKeating J.A.
      • et al.
      Cell culture-grown hepatitis C virus is infectious in vivo and can be recultured in vitro.
      ,
      • Bukh J.
      • Purcell R.H.
      A milestone for hepatitis C virus research: a virus generated in cell culture is fully viable in vivo.
      ] (Fig. 5). Therefore, these culture systems produce viruses that are biologically relevant, although the viruses recovered from animals had a specific infectivity, determined by comparing the infectivity titer with the HCV genome titer, that was 10–100 times greater than virus recovered from infected Huh7 cells. Overall, an important milestone was achieved in 2005 with the development of JFH1-based true cell culture systems that permits classical virological studies, but it was clear that new developments were required to expand the system beyond one virus strain and one type of cell line.
      Initial research to expand the infectious culture system to other HCV variants and genotypes took advantage of the unique replication capacity of JFH1. Thus, JFH1-based recombinants comprising 5′UTR-NS2, Core-NS2, NS3P/NS4A, NS4A, NS5A, Core-NS2 plus NS5A, 5′UTR-NS3-protease plus NS4A-NS5A or most recently 5′UTR-NS5A of other HCV genotype strains have been developed; most of these systems depend on specific adaptive mutations [
      • Gottwein J.M.
      • Scheel T.K.
      • Hoegh A.M.
      • Lademann J.B.
      • Eugen-Olsen J.
      • Lisby G.
      • et al.
      Robust hepatitis C genotype 3a cell culture releasing adapted intergenotypic 3a/2a (S52/JFH1) viruses.
      ,
      • Scheel T.K.
      • Gottwein J.M.
      • Jensen T.B.
      • Prentoe J.C.
      • Hoegh A.M.
      • Alter H.J.
      • et al.
      Development of JFH1-based cell culture systems for hepatitis C virus genotype 4a and evidence for cross-genotype neutralization.
      ,
      • Gottwein J.M.
      • Scheel T.K.
      • Jensen T.B.
      • Lademann J.B.
      • Prentoe J.C.
      • Knudsen M.L.
      • et al.
      Development and characterization of hepatitis C virus genotype 1–7 cell culture systems: role of CD81 and scavenger receptor class B type I and effect of antiviral drugs.
      ,
      • Gottwein J.M.
      • Scheel T.K.
      • Jensen T.B.
      • Ghanem L.
      • Bukh J.
      Differential efficacy of protease inhibitors against HCV genotypes 2a, 3a, 5a, and 6a NS3/4A protease recombinant viruses.
      ,
      • Gottwein J.M.
      • Jensen S.B.
      • Serre S.B.
      • Ghanem L.
      • Scheel T.K.
      • Jensen T.B.
      • et al.
      Adapted J6/JFH1-based Hepatitis C virus recombinants with genotype-specific NS4A show similar efficacies against lead protease inhibitors, alpha interferon, and a putative NS4A inhibitor.
      ,
      • Gottwein J.M.
      • Jensen S.B.
      • Li Y.P.
      • Ghanem L.
      • Scheel T.K.
      • Serre S.B.
      • et al.
      Combination treatment with hepatitis C virus protease and NS5A inhibitors is effective against recombinant genotype 1a, 2a, and 3a viruses.
      ,
      • Jensen T.B.
      • Gottwein J.M.
      • Scheel T.K.
      • Hoegh A.M.
      • Eugen-Olsen J.
      • Bukh J.
      Highly efficient JFH1-based cell-culture system for hepatitis C virus genotype 5a: Failure of Homologous Neutralizing-Antibody Treatment to Control Infection.
      ,
      • Li Y.P.
      • Gottwein J.M.
      • Scheel T.K.
      • Jensen T.B.
      • Bukh J.
      MicroRNA-122 antagonism against hepatitis C virus genotypes 1–6 and reduced efficacy by host RNA insertion or mutations in the HCV 5′ UTR.
      ,
      • Li Y.P.
      • Ramirez S.
      • Humes D.
      • Jensen S.B.
      • Gottwein J.M.
      • Bukh J.
      Differential sensitivity of 5′UTR-NS5A recombinants of hepatitis C virus genotypes 1–6 to protease and NS5A inhibitors.
      ,
      • Pedersen J.
      • Carlsen T.H.
      • Prentoe J.
      • Ramirez S.
      • Jensen T.B.
      • Forns X.
      • et al.
      Neutralization resistance of hepatitis C virus can be overcome by recombinant human monoclonal antibodies.
      ,
      • Pietschmann T.
      • Kaul A.
      • Koutsoudakis G.
      • Shavinskaya A.
      • Kallis S.
      • Steinmann E.
      • et al.
      Construction and characterization of infectious intragenotypic and intergenotypic hepatitis C virus chimeras.
      ,
      • Scheel T.K.
      • Gottwein J.M.
      • Carlsen T.H.
      • Li Y.P.
      • Jensen T.B.
      • Spengler U.
      • et al.
      Efficient culture adaptation of hepatitis C virus recombinants with genotype-specific core-NS2 by using previously identified mutations.
      ,
      • Scheel T.K.
      • Gottwein J.M.
      • Mikkelsen L.S.
      • Jensen T.B.
      • Bukh J.
      Recombinant HCV variants with NS5A from genotypes 1–7 have different sensitivities to an NS5A inhibitor but not interferon-alpha.
      ,
      • Yi M.
      • Ma Y.
      • Yates J.
      • Lemon S.M.
      Compensatory mutations in E1, p7, NS2, and NS3 enhance yields of cell culture-infectious intergenotypic chimeric hepatitis C virus.
      ,
      • Galli A.
      • Scheel T.K.
      • Prentoe J.C.
      • Mikkelsen L.S.
      • Gottwein J.M.
      • Bukh J.
      Analysis of hepatitis C virus core/NS5A protein co-localization using novel cell culture systems expressing core-NS2 and NS5A of genotypes 1–7.
      ,
      • Murayama A.
      • Kato T.
      • Akazawa D.
      • Sugiyama N.
      • Date T.
      • Masaki T.
      • et al.
      Production of infectious chimeric hepatitis C virus genotype 2b harboring minimal regions of JFH-1.
      ]. The 5′UTR-NS2, Core-NS2, NS5A, Core-NS2 plus NS5A, and 5′UTR-NS5A systems have been developed for HCV strains of genotypes 1–6; the latter system depended on mutations identified in efforts to develop full-length culture systems (see below). Efforts have been made to adapt the cultures to grow to higher titers, in particular for the Core-NS2 systems, which makes them more relevant in particular for attempts to generate inactivated whole virus vaccine candidates [
      • Mathiesen C.K.
      • Prentoe J.
      • Meredith L.W.
      • Jensen T.B.
      • Krarup H.
      • McKeating J.A.
      • et al.
      Adaptive mutations enhance assembly and cell-to-cell transmission of a high-titer hepatitis C virus genotype 5a Core-NS2 JFH1-based recombinant.
      ]. The chimeric systems have permitted genotype-specific studies of novel antivirals, including human monoclonal antibodies (HMAb) and DAA [
      • Gottwein J.M.
      • Scheel T.K.
      • Jensen T.B.
      • Ghanem L.
      • Bukh J.
      Differential efficacy of protease inhibitors against HCV genotypes 2a, 3a, 5a, and 6a NS3/4A protease recombinant viruses.
      ,
      • Gottwein J.M.
      • Jensen S.B.
      • Li Y.P.
      • Ghanem L.
      • Scheel T.K.
      • Serre S.B.
      • et al.
      Combination treatment with hepatitis C virus protease and NS5A inhibitors is effective against recombinant genotype 1a, 2a, and 3a viruses.
      ,
      • Scheel T.K.
      • Gottwein J.M.
      • Mikkelsen L.S.
      • Jensen T.B.
      • Bukh J.
      Recombinant HCV variants with NS5A from genotypes 1–7 have different sensitivities to an NS5A inhibitor but not interferon-alpha.
      ,
      • Giang E.
      • Dorner M.
      • Prentoe J.C.
      • Dreux M.
      • Evans M.J.
      • Bukh J.
      • et al.
      Human broadly neutralizing antibodies to the envelope glycoprotein complex of hepatitis C virus.
      ,
      • Keck Z.
      • Wang W.
      • Wang Y.
      • Lau P.
      • Carlsen T.H.
      • Prentoe J.
      • et al.
      Cooperativity in virus neutralization by human monoclonal antibodies to two adjacent regions located at the amino terminus of hepatitis C virus E2 glycoprotein.
      ,
      • Keck Z.Y.
      • Xia J.
      • Wang Y.
      • Wang W.
      • Krey T.
      • Prentoe J.
      • et al.
      Human monoclonal antibodies to a novel cluster of conformational epitopes on HCV E2 with resistance to neutralization escape in a genotype 2a isolate.
      ]. However, systems for key viral enzymes, in particular the NS5B polymerase, were missing.
      In breakthrough studies in 2012, it finally became possible to robustly culture other HCV strains independent of JFH1 elements. Li et al. identified LSG substitutions F1464L (NS3-helicase), A1672S (NS4A), and D2979G (NS5B) [
      • Li Y.P.
      • Ramirez S.
      • Gottwein J.M.
      • Scheel T.K.
      • Mikkelsen L.
      • Purcell R.H.
      • et al.
      Robust full-length hepatitis C virus genotype 2a and 2b infectious cultures using mutations identified by a systematic approach applicable to patient strains.
      ], which have permitted the development of robust full-length HCV genotype 1a, 2a, and 2b infectious cell cultures thus expanding efficient systems to other genotypes and subtypes [
      • Li Y.P.
      • Ramirez S.
      • Gottwein J.M.
      • Scheel T.K.
      • Mikkelsen L.
      • Purcell R.H.
      • et al.
      Robust full-length hepatitis C virus genotype 2a and 2b infectious cultures using mutations identified by a systematic approach applicable to patient strains.
      ,
      • Li Y.P.
      • Ramirez S.
      • Jensen S.B.
      • Purcell R.H.
      • Gottwein J.M.
      • Bukh J.
      Highly efficient full-length hepatitis C virus genotype 1 (strain TN) infectious culture system.
      ,
      • Ramirez S.
      • Li Y.P.
      • Jensen S.B.
      • Pedersen J.
      • Gottwein J.M.
      • Bukh J.
      Highly efficient infectious cell culture of three hepatitis C virus genotype 2b strains and sensitivity to lead protease, nonstructural protein 5A, and polymerase inhibitors.
      ,
      • Li Y.P.
      • Ramirez S.
      • Mikkelsen L.
      • Bukh J.
      Efficient infectious cell culture systems of the hepatitis C virus (HCV) prototype strains HCV-1 and H77.
      ]. The NS4A substitution might help overcome defects in oligomerization of the NS4A protein in non-replicative genomes [
      • Kohlway A.
      • Pirakitikulr N.
      • Barrera F.N.
      • Potapova O.
      • Engelman D.M.
      • Pyle A.M.
      • et al.
      Hepatitis C virus RNA replication and virus particle assembly require specific dimerization of the NS4A protein transmembrane domain.
      ]. Culture systems were developed for the prototype HCV isolates HCV-1, H77 and TN of genotype 1a, and prototype strains J6 and J8 of genotypes 2a and 2b, respectively. The adaptation and efficient growth in culture of the TN genome correlated with resistance to lipid peroxidation [
      • Yamane D.
      • McGivern D.R.
      • Wauthier E.
      • Yi M.
      • Madden V.J.
      • Welsch C.
      • et al.
      Regulation of the hepatitis C virus RNA replicase by endogenous lipid peroxidation.
      ]. Most recently, Ramirez et al. succeeded in developing highly efficient adapted full-length genotype 3a culture systems [

      Ramirez S, Mikkelsen LS, Gottwein JM, Bukh J. Robust HCV genotype 3a infectious cell culture system permits identification of escape variants with resistance to sofosbuvir. Gastroenterology. In press. http://dx.doi.org/10.1053/j.gastro.2016.07.013.

      ]. Robust infectious HCV cell culture systems for isolates of different genotypes represent valuable tools for studies of the importance of genetic heterogeneity for antiviral therapy and vaccine development. It will therefore be important to also develop robust full-length culture systems of other important subtypes of genotypes 1, 2 and 3, and to succeed in developing such systems for genotypes 4, 5, 6 and 7.
      A limitation of the current robust cell culture systems is their dependence on a single cell line, which is a hepatoma cell line known to have numerous genetic anomalies compared with hepatocytes. Such differences could provide data on virus-host interactions that might not be fully biologically relevant. Thus efforts have been undertaken to develop systems depending on cells more closely resembling the hepatocyte, including primary human hepatocyte cultures and hepatocyte-like cells derived from pluripotent stem cells [
      • Scheel T.K.
      • Rice C.M.
      Understanding the hepatitis C virus life cycle paves the way for highly effective therapies.
      ,
      • Ploss A.
      • Khetani S.R.
      • Jones C.T.
      • Syder A.J.
      • Trehan K.
      • Gaysinskaya V.A.
      • et al.
      Persistent hepatitis C virus infection in microscale primary human hepatocyte cultures.
      ,
      • Roelandt P.
      • Obeid S.
      • Paeshuyse J.
      • Vanhove J.
      • Van L.A.
      • Nahmias Y.
      • et al.
      Human pluripotent stem cell-derived hepatocytes support complete replication of hepatitis C virus.
      ,
      • Helle F.
      • Brochot E.
      • Fournier C.
      • Descamps V.
      • Izquierdo L.
      • Hoffmann T.W.
      • et al.
      Permissivity of primary human hepatocytes and different hepatoma cell lines to cell culture adapted hepatitis C virus.
      ,
      • Yoshida T.
      • Takayama K.
      • Kondoh M.
      • Sakurai F.
      • Tani H.
      • Sakamoto N.
      • et al.
      Use of human hepatocyte-like cells derived from induced pluripotent stem cells as a model for hepatocytes in hepatitis C virus infection.
      ]. It would be advantageous to identify cell lines approved for vaccine production, e.g., for an inactivated whole virus vaccine. Here, a recent report of Vero cell expressing critical HCV host factors with the capacity to complete the entire HCV life cycle has interest, since Vero cells have been used in vaccine production against other viruses [
      • Murayama A.
      • Sugiyama N.
      • Wakita T.
      • Kato T.
      Completion of the entire hepatitis C virus life cycle in vero cells derived from monkey kidney.
      ].

      Discovery of unique features of the viral life cycle of HCV with relevance for development of host targeting agents (HTA)

      A number of host molecules critical for hepatitis C virus (HCV) entry and replication have been identified. Thus, basic HCV research revealed important molecules for development of host targeting agents (HTA), including microRNA-122, viral receptors and cyclophilins.
      An impressive amount of data has been generated on key viral and host elements in the HCV life cycle [
      • Lohmann V.
      Hepatitis C virus RNA replication.
      ,
      • Gottwein J.M.
      • Bukh J.
      Hepatitis C virus host cell interactions uncovered.
      ,
      • Randall G.
      • Panis M.
      • Cooper J.D.
      • Tellinghuisen T.L.
      • Sukhodolets K.E.
      • Pfeffer S.
      • et al.
      Cellular cofactors affecting hepatitis C virus infection and replication.
      ]. Here it is attempted to merely highlight basic research that has revealed unique features in the viral life cycle with new perspectives for epidemic control. Thus, basic research on HCV has revealed a number of potential molecules for HTA. An example is the novel finding, in 2005, demonstrating that a virus could be completely dependent of microRNA for its replication [
      • Jopling C.L.
      • Yi M.
      • Lancaster A.M.
      • Lemon S.M.
      • Sarnow P.
      Modulation of hepatitis C virus RNA abundance by a liver-specific microRNA.
      ]. Thus, the essential role of miR-122 in HCV is a good example of how basic research revealed novel principles in biology, and thus identified drug targets. It is expected that future research on HCV RNA interactions could reveal other RNA-based drug targets.

      Discovery of an essential role of microRNA 122 (miR-122) in HCV replication

      The 5′UTR of HCV is a highly conserved region of about 340 nucleotides forming four major structured domains [
      • Bukh J.
      • Purcell R.H.
      • Miller R.H.
      Sequence analysis of the 5′ noncoding region of hepatitis C virus.
      ,
      • Honda M.
      • Beard M.R.
      • Ping L.H.
      • Lemon S.M.
      A phylogenetically conserved stem-loop structure at the 5′ border of the internal ribosome entry site of hepatitis C virus is required for cap-independent viral translation.
      ]. Three domains, including most of the 5′UTR sequence, create an internal ribosome entry site that controls translation of the HCV polyprotein. The fourth domain at the 5′ termini of the HCV genome contains a stem-loop structure [
      • Honda M.
      • Beard M.R.
      • Ping L.H.
      • Lemon S.M.
      A phylogenetically conserved stem-loop structure at the 5′ border of the internal ribosome entry site of hepatitis C virus is required for cap-independent viral translation.
      ], followed by two miR-122 binding sites named S1 and S2 [
      • Jopling C.L.
      • Yi M.
      • Lancaster A.M.
      • Lemon S.M.
      • Sarnow P.
      Modulation of hepatitis C virus RNA abundance by a liver-specific microRNA.
      ]. The stem-loop structure was found to be essential for the viability of infectious HCV genotype 1–6 5′UTR-NS2 recombinants [
      • Li Y.P.
      • Gottwein J.M.
      • Scheel T.K.
      • Jensen T.B.
      • Bukh J.
      MicroRNA-122 antagonism against hepatitis C virus genotypes 1–6 and reduced efficacy by host RNA insertion or mutations in the HCV 5′ UTR.
      ]. In a landmark study, Jopling et al. discovered that the liver-abundant miR-122 is required for HCV RNA replication [
      • Jopling C.L.
      • Yi M.
      • Lancaster A.M.
      • Lemon S.M.
      • Sarnow P.
      Modulation of hepatitis C virus RNA abundance by a liver-specific microRNA.
      ]. Subsequent studies have confirmed that this microRNA permits viral RNA replication and translation by binding to S1 and S2, as well as to upstream nucleotides at the 5′ end of the HCV genome, and apparently promote RNA production by controlling the relative amount of RNA involved with replication compared to that involved with translation [
      • Henke J.I.
      • Goergen D.
      • Zheng J.
      • Song Y.
      • Schuttler C.G.
      • Fehr C.
      • et al.
      MicroRNA-122 stimulates translation of hepatitis C virus RNA.
      ,
      • Jopling C.L.
      Regulation of hepatitis C virus by microRNA-122.
      ,
      • Machlin E.S.
      • Sarnow P.
      • Sagan S.M.
      Masking the 5′ terminal nucleotides of the hepatitis C virus genome by an unconventional microRNA-target RNA complex.
      ,
      • Roberts A.P.
      • Lewis A.P.
      • Jopling C.L.
      MiR-122 activates hepatitis C virus translation by a specialized mechanism requiring particular RNA components.
      ,
      • Masaki T.
      • Arend K.C.
      • Li Y.
      • Yamane D.
      • McGivern D.R.
      • Kato T.
      • et al.
      MiR-122 stimulates hepatitis C virus RNA synthesis by altering the balance of viral RNAs engaged in replication versus translation.
      ]. miR-122 associates with host Argonaute 2 to bind the HCV RNA, and through this interaction stabilizes the viral RNA [
      • Shimakami T.
      • Yamane D.
      • Jangra R.K.
      • Kempf B.J.
      • Spaniel C.
      • Barton D.J.
      • et al.
      Stabilization of hepatitis C virus RNA by an Ago2-miR-122 complex.
      ] and most likely protects its 5′ end from degradation [
      • Li Y.
      • Masaki T.
      • Yamane D.
      • McGivern D.R.
      • Lemon S.M.
      Competing and noncompeting activities of miR-122 and the 5′ exonuclease Xrn1 in regulation of hepatitis C virus replication.
      ]. In this fashion HCV also has a sponge effect in depleting the host cell for miR-122, which could impact the cell and perhaps even contribute to the oncogenic potential of HCV [
      • Luna J.M.
      • Scheel T.K.
      • Danino T.
      • Shaw K.S.
      • Mele A.
      • Fak J.J.
      • et al.
      Hepatitis C virus RNA functionally sequesters miR-122.
      ]. Overall, basic research demonstrated that miR-122 was essential for replication of the different HCV variants. Since it binds universally conserved HCV sequences, it was considered an exciting novel drug target.
      The drug miravirsen is a locked nucleic acid antisense oligonucleotide that targets and inhibits miR-122 function in liver cells [
      • Elmen J.
      • Lindow M.
      • Schutz S.
      • Lawrence M.
      • Petri A.
      • Obad S.
      • et al.
      LNA-mediated microRNA silencing in non-human primates.
      ]. In infectious cell culture systems miravirsen inhibits HCV genotypes 1–6 [
      • Li Y.P.
      • Gottwein J.M.
      • Scheel T.K.
      • Jensen T.B.
      • Bukh J.
      MicroRNA-122 antagonism against hepatitis C virus genotypes 1–6 and reduced efficacy by host RNA insertion or mutations in the HCV 5′ UTR.
      ], and in vivo it suppresses HCV genotype 1 chronic infection in experimentally infected chimpanzees and in patients with no or limited evidence of virus resistance [
      • Janssen H.L.
      • Reesink H.W.
      • Lawitz E.J.
      • Zeuzem S.
      • Rodriguez-Torres M.
      • Patel K.
      • et al.
      Treatment of HCV infection by targeting microRNA.
      ,
      • Lanford R.E.
      • Hildebrandt-Eriksen E.S.
      • Petri A.
      • Persson R.
      • Lindow M.
      • Munk M.E.
      • et al.
      Therapeutic silencing of microRNA-122 in primates with chronic hepatitis C virus infection.
      ,
      • Ottosen S.
      • Parsley T.B.
      • Yang L.
      • Zeh K.
      • van Doorn L.J.
      • van der Veer E.
      • et al.
      In vitro antiviral activity and preclinical and clinical resistance profile of miravirsen, a novel anti-hepatitis C virus therapeutic targeting the human factor miR-122.
      ], thus showing potential as a host targeting antiviral drug for HCV therapy. However, in recombinant culture systems it is possible to introduce mutations in the miR-122 binding sites or sequences in close proximity that confer virus resistance to miravirsen treatment [
      • Li Y.P.
      • Gottwein J.M.
      • Scheel T.K.
      • Jensen T.B.
      • Bukh J.
      MicroRNA-122 antagonism against hepatitis C virus genotypes 1–6 and reduced efficacy by host RNA insertion or mutations in the HCV 5′ UTR.
      ,
      • Li Y.P.
      • Van Pham L.
      • Uzcategui N.
      • Bukh J.
      Functional analysis of microRNA-122 binding sequences of hepatitis C virus and identification of variants with high resistance against a specific antagomir.
      ,
      • Israelow B.
      • Mullokandov G.
      • Agudo J.
      • Sourisseau M.
      • Bashir A.
      • Maldonado A.Y.
      • et al.
      Hepatitis C virus genetics affects miR-122 requirements and response to miR-122 inhibitors.
      ]. Miravirsen has been tested in clinical trials with promising results, but at present it is unclear whether it will be developed further for HCV therapy. The finding, however, that a host targeting RNA drug can be developed as an effective antiviral, has wide reaching perspectives in medicine.

      HCV co-receptors

      The mechanism by which HCV enters the human hepatocyte to initiate infection is not fully known, but a number of molecules with important roles in a complex multistep entry process have been identified [
      • Douam F.
      • Lavillette D.
      • Cosset F.L.
      The mechanism of HCV entry into host cells.
      ]. The E1 and E2 glycoproteins are involved in binding to receptors and subsequent fusion with the host cell. An essential receptor is CD81, and its discovery in 1998 represented a major breakthrough. By preparing a cDNA expression library from a cell line with a high capacity to bind recombinant HCV E2 protein, Pileri et al. identified the surface expressed CD81 as a binding partner [
      • Pileri P.
      • Uematsu Y.
      • Campagnoli S.
      • Galli G.
      • Falugi F.
      • Petracca R.
      • et al.
      Binding of hepatitis C virus to CD81.
      ]. In vitro studies using HCV pseudo-particles, developed in 2003 [
      • Bartosch B.
      • Dubuisson J.
      • Cosset F.L.
      Infectious hepatitis C virus pseudo-particles containing functional E1–E2 envelope protein complexes.
      ], and infectious HCVcc systems have confirmed an essential role of CD81 for viral entry [
      • Bartosch B.
      • Vitelli A.
      • Granier C.
      • Goujon C.
      • Dubuisson J.
      • Pascale S.
      • et al.
      Cell entry of hepatitis C virus requires a set of co-receptors that include the CD81 tetraspanin and the SR-B1 scavenger receptor.
      ,
      • Akazawa D.
      • Date T.
      • Morikawa K.
      • Murayama A.
      • Miyamoto M.
      • Kaga M.
      • et al.
      CD81 expression is important for the permissiveness of Huh7 cell clones for heterogeneous hepatitis C virus infection.
      ].
      The identification of the HCV-CD81 interaction led to searches for other putative receptors. Both the identified low density lipoprotein receptor (LDLr) [
      • Agnello V.
      • Abel G.
      • Elfahal M.
      • Knight G.B.
      • Zhang Q.X.
      Hepatitis C virus and other flaviviridae viruses enter cells via low density lipoprotein receptor.
      ,
      • Mathiesen C.K.
      • Jensen T.B.
      • Prentoe J.
      • Krarup H.
      • Nicosia A.
      • Law M.
      • et al.
      Production and characterization of high-titer serum-free cell culture grown hepatitis C virus particles of genotype 1–6.
      ,
      • Prentoe J.
      • Serre S.B.
      • Ramirez S.
      • Nicosia A.
      • Gottwein J.M.
      • Bukh J.
      Hypervariable region 1 deletion and required adaptive envelope mutations confer decreased dependency on scavenger receptor class B type I and low-density lipoprotein receptor for hepatitis C virus.
      ] and scavenger receptor class B type I (SR-BI) [
      • Scarselli E.
      • Ansuini H.
      • Cerino R.
      • Roccasecca R.M.
      • Acali S.
      • Filocamo G.
      • et al.
      The human scavenger receptor class B type I is a novel candidate receptor for the hepatitis C virus.
      ] are believed to be involved in early interactions between the host cell and HCV, promoting interactions with late-stage receptor CD81 or identified tight-junction factors Claudin-I and Occludin [
      • Ploss A.
      • Evans M.J.
      • Gaysinskaya V.A.
      • Panis M.
      • You H.
      • de Jong Y.P.
      • et al.
      Human occludin is a hepatitis C virus entry factor required for infection of mouse cells.
      ,
      • Mensa L.
      • Crespo G.
      • Gastinger M.J.
      • Kabat J.
      • Perez-del-Pulgar S.
      • Miquel R.
      • et al.
      Hepatitis C virus receptors claudin-1 and occludin after liver transplantation and influence on early viral kinetics.
      ,
      • Evans M.J.
      • von Hahn T.
      • Tscherne D.M.
      • Syder A.J.
      • Panis M.
      • Wolk B.
      • et al.
      Claudin-1 is a hepatitis C virus co-receptor required for a late step in entry.
      ]. Virus association with apolipoproteins most likely also has important roles in the entry process [
      • Douam F.
      • Lavillette D.
      • Cosset F.L.
      The mechanism of HCV entry into host cells.
      ,
      • Meunier J.C.
      • Engle R.E.
      • Faulk K.
      • Zhao M.
      • Bartosch B.
      • Alter H.
      • et al.
      Evidence for cross-genotype neutralization of hepatitis C virus pseudo-particles and enhancement of infectivity by apolipoprotein C1.
      ]. Several other molecules have been identified as important host HCV entry molecules, including most recently CD36 that apparently is a co-receptor for HCV E1 protein attachment [
      • Sainz Jr., B.
      • Barretto N.
      • Martin D.N.
      • Hiraga N.
      • Imamura M.
      • Hussain S.
      • et al.
      Identification of the Niemann-Pick C1-like 1 cholesterol absorption receptor as a new hepatitis C virus entry factor.
      ,
      • Lupberger J.
      • Zeisel M.B.
      • Xiao F.
      • Thumann C.
      • Fofana I.
      • Zona L.
      • et al.
      EGFR and EphA2 are host factors for hepatitis C virus entry and possible targets for antiviral therapy.
      ,
      • Cheng J.J.
      • Li J.R.
      • Huang M.H.
      • Ma L.L.
      • Wu Z.Y.
      • Jiang C.C.
      • et al.
      CD36 is a co-receptor for hepatitis C virus E1 protein attachment.
      ,
      • Martin D.N.
      • Uprichard S.L.
      Identification of transferrin receptor 1 as a hepatitis C virus entry factor.
      ]. A combination of multiple receptors is involved in both cell free and cell-to-cell transmission. However, despite these advances in the understanding of the HCV entry process it would be important to get a more complete insight including detailed data on the structural changes to the HCV envelope proteins and cell membranes during this multistep process.
      Important proof-of-concept studies were published of the protective potential of antibodies against key host cell HCV receptors [
      • Bukh J.
      Animal models for the study of hepatitis C virus infection and related liver disease.
      ]. Monoclonal antibodies that block CD81 or SR-BI protected against subsequent HCV challenge with different genotypes in human liver chimeric mice [
      • Lacek K.
      • Vercauteren K.
      • Grzyb K.
      • Naddeo M.
      • Verhoye L.
      • Slowikowski M.P.
      • et al.
      Novel human SR-BI antibodies prevent infection and dissemination of HCV in vitro and in humanized mice.
      ,
      • Meuleman P.
      • Hesselgesser J.
      • Paulson M.
      • Vanwolleghem T.
      • Desombere I.
      • Reiser H.
      • et al.
      Anti-CD81 antibodies can prevent a hepatitis C virus infection in vivo.
      ,
      • Meuleman P.
      • Catanese M.T.
      • Verhoye L.
      • Desombere I.
      • Farhoudi A.
      • Jones C.T.
      • et al.
      A Human monoclonal antibody targeting scavenger receptor class B type I precludes hepatitis C virus infection and viral spread in vitro and in vivo.
      ]. The anti-SR-BI entry inhibitor ITX5061 has been tested in the clinic [
      • Rowe I.A.
      • Tully D.C.
      • Armstrong M.J.
      • Parker R.
      • Guo K.
      • Barton D.
      • et al.
      Effect of scavenger receptor class B type I antagonist ITX5061 in patients with hepatitis C virus infection undergoing liver transplantation.
      ,
      • Sulkowski M.S.
      • Kang M.
      • Matining R.
      • Wyles D.
      • Johnson V.A.
      • Morse G.D.
      • et al.
      Safety and antiviral activity of the HCV entry inhibitor ITX5061 in treatment-naive HCV-infected adults: a randomized, double-blind, phase 1b study.
      ], and showed some evidence of reducing HCV RNA titers and viral evolution in patients undergoing liver transplantation [
      • Rowe I.A.
      • Tully D.C.
      • Armstrong M.J.
      • Parker R.
      • Guo K.
      • Barton D.
      • et al.
      Effect of scavenger receptor class B type I antagonist ITX5061 in patients with hepatitis C virus infection undergoing liver transplantation.
      ]. An antibody against Claudin-1 can control HCV in human liver chimeric mice [
      • Mailly L.
      • Xiao F.
      • Lupberger J.
      • Wilson G.K.
      • Aubert P.
      • Duong F.H.
      • et al.
      Clearance of persistent hepatitis C virus infection in humanized mice using a claudin-1-targeting monoclonal antibody.
      ]. It is possible that these entry inhibitors will have a role in future HCV therapy [

      Colpitts CC, Baumert TF. Hepatitis C virus cell entry: a target for novel antiviral strategies to address limitations of direct acting antivirals. Hepatol Int. In press. http://dx.doi.org/10.1007/s12072-016-9724-7.

      ].

      Cyclophilin inhibitors against HCV

      Basic research in replicon and infectious culture models revealed that host cell cyclophilins, which affects protein folding, stimulate HCV replication. In addition, inhibitors of this molecule reduce HCV replication [
      • Hopkins S.
      • Gallay P.
      Cyclophilin inhibitors: an emerging class of therapeutics for the treatment of chronic hepatitis C infection.
      ,
      • Lin K.
      • Gallay P.
      Curing a viral infection by targeting the host: the example of cyclophilin inhibitors.
      ]. Cyclophilins apparently affect several steps of the HCV life cycle including viral assembly; inhibitors thus have multiple modes of action [
      • Watashi K.
      • Ishii N.
      • Hijikata M.
      • Inoue D.
      • Murata T.
      • Miyanari Y.
      • et al.
      Cyclophilin B is a functional regulator of hepatitis C virus RNA polymerase.
      ,
      • Nakagawa M.
      • Sakamoto N.
      • Tanabe Y.
      • Koyama T.
      • Itsui Y.
      • Takeda Y.
      • et al.
      Suppression of hepatitis C virus replication by cyclosporin a is mediated by blockade of cyclophilins.
      ,
      • Ma S.
      • Boerner J.E.
      • TiongYip C.
      • Weidmann B.
      • Ryder N.S.
      • Cooreman M.P.
      • et al.
      NIM811, a cyclophilin inhibitor, exhibits potent in vitro activity against hepatitis C virus alone or in combination with alpha interferon.
      ,
      • Kaul A.
      • Stauffer S.
      • Berger C.
      • Pertel T.
      • Schmitt J.
      • Kallis S.
      • et al.
      Essential role of cyclophilin A for hepatitis C virus replication and virus production and possible link to polyprotein cleavage kinetics.
      ,
      • Wagoner J.
      • Negash A.
      • Kane O.J.
      • Martinez L.E.
      • Nahmias Y.
      • Bourne N.
      • et al.
      Multiple effects of silymarin on the hepatitis C virus lifecycle.
      ]. Thus cyclophilin inhibitors have been advanced for testing in clinical trials, but it is unclear whether they will have a role in future HCV therapy.

      Features of the viral life cycle of HCV, leading to development of direct acting antivirals (DAA)

      The approval of numerous effective DAA for HCV treatment since 2011 represents a huge success for basic and translational research, and the interaction with the pharmaceutical industry. These drugs are directed against the more classical viral targets, the NS3 protease and NS5B polymerase, as well as a novel viral target, the NS5A protein. Used in combination they can eradicate HCV from patients with chronic HCV in 8–24 weeks of oral treatment [
      • Pawlotsky J.M.
      New hepatitis C therapies: the toolbox, strategies, and challenges.
      ]. Many other viral targets in HCV has been pursued, including p7, the NS3 helicase and NS4B, but they have not lead to drugs introduced in the clinic for treatment of HCV [
      • Scheel T.K.
      • Rice C.M.
      Understanding the hepatitis C virus life cycle paves the way for highly effective therapies.
      ].

      Characterization of the NS3 protease and development of the first approved DAA

      The amino-terminal part of the NS3 protein has serine-protease activity and has been shown to cleave NS3/4A, 4A/4B, 4B/5A, and 5A/5B junctions of the polyprotein [
      • Grakoui A.
      • McCourt D.W.
      • Wychowski C.
      • Feinstone S.M.
      • Rice C.M.
      Characterization of the hepatitis C virus-encoded serine proteinase: determination of proteinase-dependent polyprotein cleavage sites.
      ]. The protease forms a stable complex with the NS4A protein, and NS4A functions as an essential cofactor in the processing of NS3/4A and NS4B/5A sites, and enhances cleavage at the other sites. In advances of great importance for development of DAA, the crystal structure of the NS3 protease domain and of the protease domain complexed with a synthetic NS4A cofactor peptide were determined [
      • Kim J.L.
      • Morgenstern K.A.
      • Lin C.
      • Fox T.
      • Dwyer M.D.
      • Landro J.A.
      • et al.
      Crystal structure of the hepatitis C virus NS3 protease domain complexed with a synthetic NS4A cofactor peptide.
      ,
      • Lorenz I.C.
      • Marcotrigiano J.
      • Dentzer T.G.
      • Rice C.M.
      Structure of the catalytic domain of the hepatitis C virus NS2-3 protease.
      ,
      • Love R.A.
      • Parge H.E.
      • Wickersham J.A.
      • Hostomsky Z.
      • Habuka N.
      • Moomaw E.W.
      • et al.
      The crystal structure of hepatitis C virus NS3 proteinase reveals a trypsin-like fold and a structural zinc binding site.
      ,
      • Love R.A.
      • Parge H.E.
      • Wickersham J.A.
      • Hostomsky Z.
      • Habuka N.
      • Moomaw E.W.
      • et al.
      The conformation of hepatitis C virus NS3 proteinase with and without NS4A: a structural basis for the activation of the enzyme by its cofactor.
      ]. Solving the crystal structure gave the possibility of designing specific inhibitors of the enzyme for therapeutic use as inhibitors of viral replication.
      The identification and characterization of HCV encoded proteins and their functional units led to effective antivirals against the NS3 protease, NS5A and the NS5B polymerase. In combination, these inhibitors permitted interferon-free therapy with high cure rates and minimal side effects. However, viral resistance represents a challenge for the continued success of these drugs.
      Several NS3 protease drugs have been developed and approved for HCV treatment, including telaprevir, boceprevir, simeprevir, paritaprevir and grazoprevir [
      • Pawlotsky J.M.
      New hepatitis C therapies: the toolbox, strategies, and challenges.
      ]. Basic studies in infectious cell culture systems have shown great variation in their potency against different HCV variants and genotypes, and in their pattern of resistance [
      • Gottwein J.M.
      • Scheel T.K.
      • Jensen T.B.
      • Ghanem L.
      • Bukh J.
      Differential efficacy of protease inhibitors against HCV genotypes 2a, 3a, 5a, and 6a NS3/4A protease recombinant viruses.
      ,
      • Imhof I.
      • Simmonds P.
      Development of an intergenotypic hepatitis C virus (HCV) cell culture method to assess antiviral susceptibilities and resistance development of HCV NS3 protease genes from HCV genotypes 1 to 6.
      ,
      • Jensen S.B.
      • Serre S.B.
      • Humes D.G.
      • Ramirez S.
      • Li Y.P.
      • Bukh J.
      • et al.
      Substitutions at NS3 residue 155, 156, or 168 of hepatitis C virus genotypes 2 to 6 induce complex patterns of protease inhibitor resistance.
      ,
      • Serre S.B.
      • Jensen S.B.
      • Ghanem L.
      • Humes D.G.
      • Ramirez S.
      • Li Y.P.
      • et al.
      Hepatitis C virus genotype 1 to 6 protease inhibitor escape variants. In vitro selection, fitness, and resistance patterns in the context of the infectious viral life cycle.
      ]; since they all target the protease active site substitutions conferring cross-resistance have been identified [
      • Pawlotsky J.M.
      Hepatitis C virus resistance to direct-acting antiviral drugs in interferon-free regimens.
      ].

      Identification of the HCV NS5A protein as a novel drug target

      From the early time in HCV research NS5A has been of great interest for studies on therapy of HCV. The NS5A protein is phosphorylated; protein phosphorylation can regulate protein-protein interactions as well as protein-nucleic acid interactions. A short region of the NS5A protein has been implicated in the modulation of the host IFN-mediated antiviral response. Mutations in this region, called the IFN-sensitive determining region appeared to correlate with the sensitivity of HCV genotype 1b viruses to IFN treatment [
      • 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.
      ]; NS5A interacts with the IFN-induced cellular protein kinase R (PKR), which could represent mechanisms used by the virus to escape IFNs antiviral activity.
      The NS5A protein, consisting of three defined domains, is an essential component of the viral replication complex [
      • Gosert R.
      • Egger D.
      • Lohmann V.
      • Bartenschlager R.
      • Blum H.E.
      • Bienz K.
      • et al.
      Identification of the hepatitis C virus RNA replication complex in Huh-7 cells harboring subgenomic replicons.
      ,
      • Tellinghuisen T.L.
      • Marcotrigiano J.
      • Gorbalenya A.E.
      • Rice C.M.
      The NS5A protein of hepatitis C virus is a zinc metalloprotein.
      ]. Another major achievement was the identification of NS5A as a regulator of replication and viral assembly. Reverse genetic studies demonstrated that the NS5A N-terminal amphipathic domain, which anchors this protein to ER membranes [
      • Elazar M.
      • Cheong K.H.
      • Liu P.
      • Greenberg H.B.
      • Rice C.M.
      • Glenn J.S.
      Amphipathic helix-dependent localization of NS5A mediates hepatitis C virus RNA replication.
      ,
      • Penin F.
      • Brass V.
      • Appel N.
      • Ramboarina S.
      • Montserret R.
      • Ficheux D.
      • et al.
      Structure and function of the membrane anchor domain of hepatitis C virus nonstructural protein 5A.
      ], as well as four conserved cysteine residues, localizing to the NS5A zinc binding site [
      • Tellinghuisen T.L.
      • Marcotrigiano J.
      • Gorbalenya A.E.
      • Rice C.M.
      The NS5A protein of hepatitis C virus is a zinc metalloprotein.
      ], were critical for replication. In addition, crystal structures were determined for the N-terminal domain I [
      • Love R.A.
      • Brodsky O.
      • Hickey M.J.
      • Wells P.A.
      • Cronin C.N.
      Crystal structure of a novel dimeric form of NS5A domain I protein from hepatitis C virus.
      ,
      • Tellinghuisen T.L.
      • Marcotrigiano J.
      • Rice C.M.
      Structure of the zinc-binding domain of an essential component of the hepatitis C virus replicase.
      ], which has RNA binding capacity [
      • Huang L.
      • Hwang J.
      • Sharma S.D.
      • Hargittai M.R.
      • Chen Y.
      • Arnold J.J.
      • et al.
      Hepatitis C virus nonstructural protein 5A (NS5A) is an RNA-binding protein.
      ]. Overall, it has been found that the amphipathic alpha-helix and domains I and II are essential for HCV RNA replication [
      • Tellinghuisen T.L.
      • Marcotrigiano J.
      • Gorbalenya A.E.
      • Rice C.M.
      The NS5A protein of hepatitis C virus is a zinc metalloprotein.
      ,
      • Elazar M.
      • Cheong K.H.
      • Liu P.
      • Greenberg H.B.
      • Rice C.M.
      • Glenn J.S.
      Amphipathic helix-dependent localization of NS5A mediates hepatitis C virus RNA replication.
      ,
      • Tellinghuisen T.L.
      • Foss K.L.
      • Treadaway J.C.
      • Rice C.M.
      Identification of residues required for RNA replication in domains II and III of the hepatitis C virus NS5A protein.
      ,
      • Appel N.
      • Pietschmann T.
      • Bartenschlager R.
      Mutational analysis of hepatitis C virus nonstructural protein 5A: potential role of differential phosphorylation in RNA replication and identification of a genetically flexible domain.
      ]; a recent study showed a critical role for generation of double-membrane vesicles associated with replication [
      • Romero-Brey I.
      • Berger C.
      • Kallis S.
      • Kolovou A.
      • Paul D.
      • Lohmann V.
      • et al.
      NS5A Domain 1 and Polyprotein Cleavage Kinetics Are Critical for Induction of Double-Membrane Vesicles Associated with Hepatitis C Virus Replication.
      ]. In addition, domain III has a primary role in production of infectious particles with a direct role in coordinating viral assembly [
      • Appel N.
      • Zayas M.
      • Miller S.
      • Krijnse-Locker J.
      • Schaller T.
      • Friebe P.
      • et al.
      Essential role of domain III of nonstructural protein 5A for hepatitis C virus infectious particle assembly.
      ,
      • Scheel T.K.
      • Prentoe J.
      • Carlsen T.H.
      • Mikkelsen L.S.
      • Gottwein J.M.
      • Bukh J.
      Analysis of functional differences between hepatitis C virus NS5A of genotypes 1–7 in infectious cell culture systems.
      ,
      • Zayas M.
      • Long G.
      • Madan V.
      • Bartenschlager R.
      Coordination of hepatitis C virus assembly by distinct regulatory regions in nonstructural protein 5A.
      ].
      Gao et al. developed a highly efficient HCV NS5A inhibitor daclatasvir that was found to have high potency against the different HCV genotypes in vitro although genotype 3 was less sensitive [
      • Scheel T.K.
      • Gottwein J.M.
      • Mikkelsen L.S.
      • Jensen T.B.
      • Bukh J.
      Recombinant HCV variants with NS5A from genotypes 1–7 have different sensitivities to an NS5A inhibitor but not interferon-alpha.
      ,
      • Gao M.
      • Nettles R.E.
      • Belema M.
      • Snyder L.B.
      • Nguyen V.N.
      • Fridell R.A.
      • et al.
      Chemical genetics strategy identifies an HCV NS5A inhibitor with a potent clinical effect.
      ]. In addition, great differences in sensitivity were observed even within a single subtype due to differences at individual amino acid residues [
      • Scheel T.K.
      • Gottwein J.M.
      • Mikkelsen L.S.
      • Jensen T.B.
      • Bukh J.
      Recombinant HCV variants with NS5A from genotypes 1–7 have different sensitivities to an NS5A inhibitor but not interferon-alpha.
      ]. This discovery of NS5A as a DAA target paved the way for the development of several other similar NS5A inhibitors, including clinically approved elbasvir, ledipasvir, obitasvir, and velpatasvir [
      • Pawlotsky J.M.
      New hepatitis C therapies: the toolbox, strategies, and challenges.
      ,

      Ramirez S, Mikkelsen LS, Gottwein JM, Bukh J. Robust HCV genotype 3a infectious cell culture system permits identification of escape variants with resistance to sofosbuvir. Gastroenterology. In press. http://dx.doi.org/10.1053/j.gastro.2016.07.013.

      ]. They all target domain 1 of NS5A and although their exact mechanism of action has not been determined, it appears that their effect goes beyond merely inhibiting viral replication [
      • Berger C.
      • Romero-Brey I.
      • Radujkovic D.
      • Terreux R.
      • Zayas M.
      • Paul D.
      • et al.
      Daclatasvir-like inhibitors of NS5A block early biogenesis of hepatitis C virus-induced membranous replication factories, independent of RNA replication.
      ]. They have become a central part of current DAA therapy combinations, but they have a relatively low barrier of resistance [
      • Pawlotsky J.M.
      Hepatitis C virus resistance to direct-acting antiviral drugs in interferon-free regimens.
      ,
      • Sarrazin C.
      The importance of resistance to direct antiviral drugs in HCV infection in clinical practice.
      ].

      Development of a blockbuster drug against the NS5B polymerase

      The NS5B protein of HCV is an RNA-dependent RNA polymerase that has provided important targets for inhibition of viral replication [
      • Pawlotsky J.M.
      New hepatitis C therapies: the toolbox, strategies, and challenges.
      ,
      • Simmonds P.
      • Becher P.
      • Collett M.S.
      • Gould E.A.
      • Heinz F.X.
      • Meyers G.
      • et al.
      FLAVIVIRIDAE., Virus taxonomy: classification and nomenclature of viruses.
      ]. The solving of the crystal structure of the NS5B protein provided critical data of relevance for DAA development [
      • Love R.A.
      • Parge H.E.
      • Yu X.
      • Hickey M.J.
      • Diehl W.
      • Gao J.
      • et al.
      Crystallographic identification of a noncompetitive inhibitor binding site on the hepatitis C virus NS5B RNA polymerase enzyme.
      ,
      • Lesburg C.A.
      • Cable M.B.
      • Ferrari E.
      • Hong Z.
      • Mannarino A.F.
      • Weber P.C.
      Crystal structure of the RNA-dependent RNA polymerase from hepatitis C virus reveals a fully encircled active site.
      ,
      • Bressanelli S.
      • Tomei L.
      • Roussel A.
      • Incitti I.
      • Vitale R.L.
      • Mathieu M.
      • et al.
      Crystal structure of the RNA-dependent RNA polymerase of hepatitis C virus.
      ,
      • Ago H.
      • Adachi T.
      • Yoshida A.
      • Yamamoto M.
      • Habuka N.
      • Yatsunami K.
      • et al.
      Crystal structure of the RNA-dependent RNA polymerase of hepatitis C virus.
      ]. Basic research on the NS5B polymerase has thus contributed to the development of highly effective nucleos(t)ide analogs against HCV that have pangenotypic activity, and most importantly with a high genetic barrier to resistance [
      • Pawlotsky J.M.
      Hepatitis C virus resistance to direct-acting antiviral drugs in interferon-free regimens.
      ,

      Ramirez S, M