Hepatitis C virus (HCV) infection is a major cause of chronic hepatitis, liver cirrhosis and hepatocellular carcinoma worldwide.
Cell culture systems for HCV, especially the replicon and cell culture-derived HCV (HCVcc) systems, have been essential for researchers to gain insights into the viral replication cycle and for the development of selective drugs.
Prime targets for direct-acting antiviral agents (DAAs) against HCV are the protease NS3-4A, the replicase factor NS5A and the RNA-dependent RNA polymerase NS5B.
Knowledge of the biochemical and structural properties of NS3-4A, NS5A and NS5B has been a key factor for the development of highly efficient drugs targeting these proteins.
Additional viral proteins, such as the ion channel formed by p7 or the membrane-active protein NS4B, represent alternative targets for antiviral therapy.
Drugs directed against certain host cell factors on which HCV is dependent, such as cyclophilin A or microRNA miR-122, are highly efficient in vitro and in vivo.
New drug regimens based on the combination of DAAs and independent of interferon and, eventually, ribavirin (both of which drugs account for serious side effects) appear to be within reach in the near future.
The availability of the first molecular clone of the hepatitis C virus (HCV) genome allowed the identification and biochemical characterization of two viral enzymes that are targets for antiviral therapy: the protease NS3-4A and the RNA-dependent RNA polymerase NS5B. With the advent of cell culture systems that can recapitulate either the intracellular steps of the viral replication cycle or the complete cycle, additional drug targets have been identified, most notably the phosphoprotein NS5A, but also host cell factors that promote viral replication, such as cyclophilin A. Here, we review insights into the structures of these proteins and the mechanisms by which they contribute to the HCV replication cycle, and discuss how these insights have facilitated the development of new, directly acting antiviral compounds that have started to enter the clinic.
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Ascione, A., Tartaglione, T. & Di Costanzo, G. G. Natural history of chronic hepatitis C virus infection. Dig. Liver Dis. 39 (Suppl. 1), S4–S7 (2007).
Jacobson, I. M., Davis, G. L., El Serag, H., Negro, F. & Trepo, C. Prevalence and challenges of liver diseases in patients with chronic hepatitis C virus infection. Clin. Gastroenterol. Hepatol. 8, 924–933 (2010).
Perz, J. F., Armstrong, G. L., Farrington, L. A., Hutin, Y. J. & Bell, B. P. The contributions of hepatitis B virus and hepatitis C virus infections to cirrhosis and primary liver cancer worldwide. J. Hepatol. 45, 529–538 (2006).
Houghton, M. Prospects for prophylactic and therapeutic vaccines against the hepatitis C viruses. Immunol. Rev. 239, 99–108 (2011).
Simmonds, P. The origin of hepatitis C virus. Curr. Top. Microbiol. Immunol. 369, 1–15 (2013).
Thimme, R., Binder, M. & Bartenschlager, R. Failure of innate and adaptive immune responses in controlling hepatitis C virus infection. FEMS Microbiol. Rev. 36, 663–683 (2012).
Sievert, W. et al. A systematic review of hepatitis C virus epidemiology in Asia, Australia and Egypt. Liver Int. 31 (Suppl. 2), 61–80 (2011).
Cornberg, M. et al. A systematic review of hepatitis C virus epidemiology in Europe, Canada and Israel. Liver Int. 31 (Suppl. 2), 30–60 (2011).
Yahia, M. Global health: a uniquely Egyptian epidemic. Nature 474, S12–S13 (2011).
Schaefer, E. A. & Chung, R. T. The impact of human gene polymorphisms on HCV infection and disease outcome. Semin. Liver Dis. 31, 375–386 (2011).
Alvisi, G., Madan, V. & Bartenschlager, R. Hepatitis C virus and host cell lipids: an intimate connection. RNA Biol. 8, 258–269 (2011).
Murray, C. L. & Rice, C. M. Turning hepatitis C into a real virus. Annu. Rev. Microbiol. 65, 307–327 (2011).
Moradpour, D., Penin, F. & Rice, C. M. Replication of hepatitis C virus. Nature Rev. Microbiol. 5, 453–463 (2007).
Bartenschlager, R., Penin, F., Lohmann, V. & Andre, P. Assembly of infectious hepatitis C virus particles. Trends Microbiol. 19, 95–103 (2011).
Lohmann, V. et al. Replication of subgenomic hepatitis C virus RNAs in a hepatoma cell line. Science 285, 110–113 (1999). The first description of a functional HCV replicon capable of efficient self-amplification in cultured human hepatoma cells.
Lindenbach, B. D. Virion assembly and release. Curr. Top. Microbiol. Immunol. 369, 199–218 (2013).
Andre, P. et al. Characterization of low- and very-low-density hepatitis C virus RNA-containing particles. J. Virol. 76, 6919–6928 (2002).
Chang, K. S., Jiang, J., Cai, Z. & Luo, G. Human apolipoprotein E is required for infectivity and production of hepatitis C virus in cell culture. J. Virol. 81, 13783–13793 (2007).
Lindenbach, B. D. et al. Cell culture-grown hepatitis C virus is infectious in vivo and can be recultured in vitro. Proc. Natl Acad. Sci. USA 103, 3805–3809 (2006).
Zeisel, M. B., Felmlee, D. J. & Baumert, T. F. Hepatitis C virus entry. Curr. Top. Microbiol. Immunol. 369, 87–112 (2013).
Blanchard, E. et al. Hepatitis C virus entry depends on clathrin-mediated endocytosis. J. Virol. 80, 6964–6972 (2006).
Bartenschlager, R., Frese, M. & Pietschmann, T. Novel insights into hepatitis C virus replication and persistence. Adv. Virus Res. 63, 71–180 (2004).
Ferraris, P., Blanchard, E. & Roingeard, P. Ultrastructural and biochemical analyses of hepatitis C virus-associated host cell membranes. J. Gen. Virol. 91, 2230–2237 (2010).
Romero-Brey, I. et al. Three-dimensional architecture and biogenesis of membrane structures associated with hepatitis C virus replication. PLoS. Pathog. 8, e1003056 (2012).
Gosert, R. et al. Identification of the hepatitis C virus RNA replication complex in Huh-7 cells harboring subgenomic replicons. J. Virol. 77, 5487–5492 (2003).
Lohmann, V. Hepatitis C virus RNA replication. Curr. Top. Microbiol. Immunol. 369, 167–198 (2013).
Miyanari, Y. et al. The lipid droplet is an important organelle for hepatitis C virus production. Nature Cell Biol. 9, 1089–1097 (2007). The first report providing a functional link between the accumulation of core protein on lipid droplets and HCV assembly.
Huang, H. et al. Hepatitis C virus production by human hepatocytes dependent on assembly and secretion of very low-density lipoproteins. Proc. Natl Acad. Sci. USA 104, 5848–5853 (2007).
Coller, K. E. et al. Molecular determinants and dynamics of hepatitis C virus secretion. PLoS. Pathog. 8, e1002466 (2012).
Counihan, N. A., Rawlinson, S. M. & Lindenbach, B. D. Trafficking of hepatitis C virus core protein during virus particle assembly. PLoS. Pathog. 7, e1002302 (2011).
Behrens, S. E., Tomei, L. & De Francesco, R. Identification and properties of the RNA-dependent RNA polymerase of hepatitis C virus. EMBO J. 15, 12–22 (1996). The first biochemical characterization of a recombinant NS5B.
Lohmann, V., Korner, F., Herian, U. & Bartenschlager, R. Biochemical properties of hepatitis C virus NS5B RNA-dependent RNA polymerase and identification of amino acid sequence motifs essential for enzymatic activity. J. Virol. 71, 8416–8428 (1997).
Pawlotsky, J. M. Treatment of chronic hepatitis C: current and future. Curr. Top. Microbiol. Immunol. 369, 321–342 (2013).
Morikawa, K. et al. Nonstructural protein 3-4A: the Swiss army knife of hepatitis C virus. J. Viral Hepat. 18, 305–315 (2011).
Phan, T., Kohlway, A., Dimberu, P., Pyle, A. M. & Lindenbach, B. D. The acidic domain of hepatitis C virus NS4A contributes to RNA replication and virus particle assembly. J. Virol. 85, 1193–1204 (2011).
Meylan, E. et al. Cardif is an adaptor protein in the RIG-I antiviral pathway and is targeted by hepatitis C virus. Nature 437, 1167–1172 (2005). A study describing the identification of MAVS (in this report, called CARDIF) and its cleavage by the HCV protease NS3-4A. Subsequent studies confirmed cleavage in hepatocytes of infected patients.
Li, K. et al. Immune evasion by hepatitis C virus NS3/4A protease-mediated cleavage of the Toll-like receptor 3 adaptor protein TRIF. Proc. Natl Acad. Sci. USA 102, 2992–2997 (2005).
Brass, V. et al. Structural determinants for membrane association and dynamic organization of the hepatitis C virus NS3-4A complex. Proc. Natl Acad. Sci. USA 105, 14545–14550 (2008).
Llinas-Brunet, M. et al. Peptide-based inhibitors of the hepatitis C virus serine protease. Bioorg. Med. Chem. Lett. 8, 1713–1718 (1998).
Steinkuhler, C. et al. Product inhibition of the hepatitis C virus NS3 protease. Biochemistry 37, 8899–8905 (1998).
Lamarre, D. et al. An NS3 protease inhibitor with antiviral effects in humans infected with hepatitis C virus. Nature 426, 186–189 (2003). The first paper to describe an HCV-specific DAA and its antiviral potency in individuals infected with HCV.
Delang, L. et al. Hepatitis C virus-specific directly acting antiviral drugs. Curr. Top. Microbiol. Immunol. 369, 289–320 (2013).
Thompson, A. J., Locarnini, S. A. & Beard, M. R. Resistance to anti-HCV protease inhibitors. Curr. Opin. Virol. 1, 599–606 (2011).
Summa, V. et al. MK-5172, a selective inhibitor of hepatitis C virus NS3/4a protease with broad activity across genotypes and resistant variants. Antimicrob. Agents Chemother. 56, 4161–4167 (2012).
Huang, M. et al. ACH-2684: HCV NS3 protease inhibitor with potent activity against multiple genotypes and known resistance variants. Hepatology 52, 1204A (2010).
Romano, K. P. et al. The molecular basis of drug resistance against hepatitis C virus NS3/4A protease inhibitors. PLoS. Pathog. 8, e1002832 (2012).
Tellinghuisen, T. L., Marcotrigiano, J., Gorbalenya, A. E. & Rice, C. M. The NS5A protein of hepatitis C virus is a zinc metalloprotein. J. Biol. Chem. 279, 48576–48587 (2004).
Penin, F. et al. Structure and function of the membrane anchor domain of hepatitis C virus nonstructural protein 5A. J. Biol. Chem. 279, 40835–40843 (2004).
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. J. Virol. 82, 1073–1083 (2008).
Masaki, T. et al. Interaction of hepatitis C virus nonstructural protein 5A with core protein is critical for the production of infectious virus particles. J. Virol. 82, 7964–7976 (2008).
Appel, N. et al. Essential role of domain III of nonstructural protein 5A for hepatitis C virus infectious particle assembly. PLoS. Pathog. 4, e1000035 (2008).
Tellinghuisen, T. L., Marcotrigiano, J. & Rice, C. M. Structure of the zinc-binding domain of an essential component of the hepatitis C virus replicase. Nature 435, 374–379 (2005). The first description of the X-ray crystallograohic structure of NS5A D1.
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. J. Virol. 83, 4395–4403 (2009). A report describing the X-ray crystallographic structure of an alternative dimer of NS5A D1.
Hwang, J. et al. Hepatitis C virus nonstructural protein 5A: biochemical characterization of a novel structural class of RNA-binding proteins. J. Virol. 84, 12480–12491 (2010).
Appel, N., Schaller, T., Penin, F. & Bartenschlager, R. From structure to function: new insights into hepatitis C virus RNA replication. J. Biol. Chem. 281, 9833–9836 (2006).
Hanoulle, X. et al. Hepatitis C virus NS5A protein is a substrate for the peptidyl-prolyl cis/trans isomerase activity of cyclophilins A and B. J. Biol. Chem. 284, 13589–13601 (2009).
Verdegem, D. et al. Domain 3 of NS5A protein from the hepatitis C virus has intrinsic α-helical propensity and is a substrate of cyclophilin A. J. Biol. Chem. 286, 20441–20454 (2011).
de Chassey, B. et al. Hepatitis C virus infection protein network. Mol. Syst. Biol. 4, 230 (2008).
Yang, F. et al. Cyclophilin A is an essential cofactor for hepatitis C virus infection and the principal mediator of cyclosporine resistance in vitro. J. Virol. 82, 5269–5278 (2008).
Reiss, S. et al. Recruitment and activation of a lipid kinase by hepatitis C virus NS5A is essential for integrity of the membranous replication compartment. Cell Host. Microbe 9, 32–45 (2011).
Berger, K. L., Kelly, S. M., Jordan, T. X., Tartell, M. A. & Randall, G. Hepatitis C virus stimulates the phosphatidylinositol 4-kinase III alpha-dependent phosphatidylinositol 4-phosphate production that is essential for its replication. J. Virol. 85, 8870–8883 (2011).
Kaneko, T. et al. Production of two phosphoproteins from the NS5A region of the hepatitis C viral genome. Biochem. Biophys. Res. Commun. 205, 320–326 (1994).
Quintavalle, M. et al. Hepatitis C virus NS5A is a direct substrate of casein kinase I-α, a cellular kinase identified by inhibitor affinity chromatography using specific NS5A hyperphosphorylation inhibitors. J. Biol. Chem. 282, 5536–5544 (2007).
Tellinghuisen, T. L., Foss, K. L. & Treadaway, J. Regulation of hepatitis C virion production via phosphorylation of the NS5A protein. PLoS. Pathog. 4, e1000032 (2008).
Chen, Y. C. et al. Polo-like kinase 1 is involved in hepatitis C virus replication by hyperphosphorylating NS5A. J. Virol. 84, 7983–7993 (2010).
Macdonald, A. & Harris, M. Hepatitis C virus NS5A: tales of a promiscuous protein. J. Gen. Virol. 85, 2485–2502 (2004).
Bianco, A. et al. Metabolism of phosphatidylinositol 4-kinase IIIα-dependent PI4P Is subverted by HCV and is targeted by a 4-anilino quinazoline with antiviral activity. PLoS. Pathog. 8, e1002576 (2012).
Gao, M. et al. Chemical genetics strategy identifies an HCV NS5A inhibitor with a potent clinical effect. Nature 465, 96–100 (2010). The first description of an NS5A inhibitor (daclatasvir) and its antiviral potency in individuals infected with HCV.
Guedj, J. et al. Modeling shows that the NS5A inhibitor daclatasvir has two modes of action and yields a shorter estimate of the hepatitis C virus half-life. Proc. Natl Acad. Sci. USA 110, 3991–3996 (2013).
Targett-Adams, P. et al. Small molecules targeting hepatitis C virus-encoded NS5A cause subcellular redistribution of their target: insights into compound modes of action. J. Virol. 85, 6353–6368 (2011).
Bressanelli, S. et al. Crystal structure of the RNA-dependent RNA polymerase of hepatitis C virus. Proc. Natl Acad. Sci. USA 96, 13034–13039 (1999).
Ago, H. et al. Crystal structure of the RNA-dependent RNA polymerase of hepatitis C virus. Structure 7, 1417–1426 (1999).
Lesburg, C.A. et al. Crystal structure of the RNA-dependent RNA polymerase from hepatitis C virus reveals a fully encircled active site. Nature Struct. Biol. 6, 937–943 (1999). References 71–73 describe the first X-ray crystallographic structures of the NS5B RdRp domain.
Moradpour, D. et al. Membrane association of the RNA-dependent RNA polymerase is essential for hepatitis C virus RNA replication. J. Virol. 78, 13278–13284 (2004).
Harrus, D. et al. Further insights into the roles of GTP and the C terminus of the hepatitis C virus polymerase in the initiation of RNA synthesis. J. Biol. Chem. 285, 32906–32918 (2010).
Simister, P. et al. Structural and functional analysis of hepatitis C virus strain JFH1 polymerase. J. Virol. 83, 11926–11939 (2009).
Mosley, R. T. et al. Structure of hepatitis C virus polymerase in complex with primer-template RNA. J. Virol. 86, 6503–6511 (2012).
Madela, K. & McGuigan, C. Progress in the development of anti-hepatitis C virus nucleoside and nucleotide prodrugs. Future Med. Chem. 4, 625–650 (2012).
Shim, J., Larson, G., Lai, V., Naim, S. & Wu, J. Z. Canonical 3′-deoxyribonucleotides as a chain terminator for HCV NS5B RNA-dependent RNA polymerase. Antiviral Res. 58, 243–251 (2003).
Le Pogam, S. et al. In vitro selected Con1 subgenomic replicons resistant to 2′-C-methyl-cytidine or to R1479 show lack of cross resistance. Virology 351, 349–359 (2006).
Pawlotsky, J. M., Najera, I. & Jacobson, I. Resistance to mericitabine, a nucleoside analogue inhibitor of HCV RNA-dependent RNA polymerase. Antivir. Ther. 17, 411–423 (2012).
Arnold, J. J. et al. Sensitivity of mitochondrial transcription and resistance of RNA polymerase II dependent nuclear transcription to antiviral ribonucleosides. PLoS. Pathog. 8, e1003030 (2012).
Pockros, P. J. et al. JUMP-C: a randomized trial of mericitabine plus peginterferon alfa-2a/ribavirin for 24 weeks in treatment-naive HCV genotype 1/4 patients. Hepatology 28 Jan 2013 (doi:10.1002/hep.26275).
Sofia, M. J. et al. Discovery of a β-d-2′-deoxy-2′-α-fluoro-2′-β-C-methyluridine nucleotide prodrug (PSI-7977) for the treatment of hepatitis C virus. J. Med. Chem. 53, 7202–7218 (2010). A report describing the development of sofosbuvir.
Lawitz, E. et al. Sofosbuvir for previously untreated chronic hepatitis C infection. N. Engl. J. Med. 368, 1878–1887 (2013). A Phase III clinical trial to test sofosbuvir and its efficacy in IFNα-containing and IFNα-free regimens.
Gane, E. J. et al. Nucleotide polymerase inhibitor sofosbuvir plus ribavirin for hepatitis C. N. Engl. J. Med. 368, 34–44 (2013). A study summarizing the first clinical trial with sofosbuvir and its efficacy in IFN-containing and IFN-free regimens.
Jacobson, I. M. et al. Sofosbuvir for hepatitis C genotype 2 or 3 in patients without treatment options. N. Engl. J. Med. 368, 1878–1887 (2013).
Powdrill, M. H., Bernatchez, J. A. & Gotte, M. Inhibitors of the hepatitis C virus RNA-dependent RNA polymerase NS5B. Viruses 2, 2169–2195 (2010).
Sarrazin, C., Hezode, C., Zeuzem, S. & Pawlotsky, J. M. Antiviral strategies in hepatitis C virus infection. J. Hepatol. 56 (Suppl. 1), S88–S100 (2012).
Kukolj, G. et al. Binding site characterization and resistance to a class of non-nucleoside inhibitors of the hepatitis C virus NS5B polymerase. J. Biol. Chem. 280, 39260–39267 (2005).
Biswal, B. K. et al. Crystal structures of the RNA-dependent RNA polymerase genotype 2a of hepatitis C virus reveal two conformations and suggest mechanisms of inhibition by non-nucleoside inhibitors. J. Biol. Chem. 280, 18202–18210 (2005).
Yi, G. et al. Biochemical study of the comparative inhibition of hepatitis C virus RNA polymerase by VX-222 and filibuvir. Antimicrob. Agents Chemother. 56, 830–837 (2012).
Poordad, F. et al. Exploratory study of oral combination antiviral therapy for hepatitis C. N. Engl. J. Med. 368, 45–53 (2013).
Howe, A. Y. et al. Molecular mechanism of hepatitis C virus replicon variants with reduced susceptibility to a benzofuran inhibitor, HCV-796. Antimicrob. Agents Chemother. 52, 3327–3338 (2008).
Shih, I. H. et al. Mechanistic characterization of GS-9190 (Tegobuvir), a novel nonnucleoside inhibitor of hepatitis C virus NS5B polymerase. Antimicrob. Agents Chemother. 55, 4196–4203 (2011).
Hebner, C. M. et al. The HCV non-nucleoside inhibitor tegobuvir utilizes a novel mechanism of action to inhibit NS5B polymerase function. PLoS ONE. 7, e39163 (2012).
Egger, D. et al. Expression of hepatitis C virus proteins induces distinct membrane alterations including a candidate viral replication complex. J. Virol. 76, 5974–5984 (2002).
Yu, G. Y., Lee, K. J., Gao, L. & Lai, M. M. Palmitoylation and polymerization of hepatitis C virus NS4B protein. J. Virol. 80, 6013–6023 (2006).
Gouttenoire, J. et al. Identification of a novel determinant for membrane association in hepatitis C virus nonstructural protein 4B. J. Virol. 83, 6257–6268 (2009).
Lundin, M., Lindstrom, H., Gronwall, C. & Persson, M. A. Dual topology of the processed hepatitis C virus protein NS4B is influenced by the NS5A protein. J. Gen. Virol. 87, 3263–3272 (2006).
Gouttenoire, J., Roingeard, P., Penin, F. & Moradpour, D. Amphipathic α-helix AH2 is a major determinant for the oligomerization of hepatitis C virus nonstructural protein 4B. J. Virol. 84, 12529–12537 (2010).
Paul, D. et al. NS4B self-interaction through conserved C-terminal elements is required for the establishment of functional hepatitis C virus replication complexes. J. Virol. 85, 6963–6976 (2011).
Einav, S. et al. Discovery of a hepatitis C target and its pharmacological inhibitors by microfluidic affinity analysis. Nature Biotech. 26, 1019–1027 (2008).
Thompson, A. A. et al. Biochemical characterization of recombinant hepatitis C virus nonstructural protein 4B: evidence for ATP/GTP hydrolysis and adenylate kinase activity. Biochemistry 48, 906–916 (2009).
Jones, D. M., Patel, A. H., Targett-Adams, P. & McLauchlan, J. The hepatitis C virus NS4B protein can trans-complement viral RNA replication and modulates production of infectious virus. J. Virol. 83, 2163–2177 (2009).
Einav, S. et al. The nucleotide binding motif of hepatitis C virus NS4B can mediate cellular transformation and tumor formation without Ha-ras co-transfection. Hepatology 47, 827–835 (2008).
Cho, N. J. et al. Identification of a class of HCV inhibitors directed against the nonstructural protein NS4B. Sci. Transl. Med. 2, 15ra6 (2010).
Esser-Nobis, K. et al. Analysis of hepatitis C virus resistance to silibinin in vitro and in vivo points to a novel mechanism involving nonstructural protein 4B. Hepatology 57, 953–963 (2013).
Steinmann, E. & Pietschmann, T. Hepatitis C virus p7 — a viroporin crucial for virus assembly and an emerging target for antiviral therapy. Viruses 2, 2078–2095 (2010).
Luik, P. et al. The 3-dimensional structure of a hepatitis C virus p7 ion channel by electron microscopy. Proc. Natl Acad. Sci. USA 106, 12712–12716 (2009).
Chandler, D. E., Penin, F., Schulten, K. & Chipot, C. The p7 protein of hepatitis C virus forms structurally plastic, minimalist ion channels. PLoS. Comput. Biol. 8, e1002702 (2012).
Wozniak, A. L. et al. Intracellular proton conductance of the hepatitis C virus p7 protein and its contribution to infectious virus production. PLoS. Pathog. 6, e1001087 (2010).
Chatel-Chaix, L., Germain, M. A., Gotte, M. & Lamarre, D. Direct-acting and host-targeting HCV inhibitors: current and future directions. Curr. Opin. Virol. 2, 588–598 (2012).
Zhu, H. et al. Evaluation of ITX 5061, a scavenger receptor B1 antagonist: resistance selection and activity in combination with other hepatitis C virus antivirals. J. Infect. Dis. 205, 656–662 (2012).
Watashi, K., Hijikata, M., Hosaka, M., Yamaji, M. & Shimotohno, K. Cyclosporin A suppresses replication of hepatitis C virus genome in cultured hepatocytes. Hepatology 38, 1282–1288 (2003). An investigation that demonstrates the HCV-suppressive activity of CsA in a replicon system and lays the foundation for the clinical development of cyclophilin antagonists.
Kaul, A. et al. Essential role of cyclophilin A for hepatitis C virus replication and virus production and possible link to polyprotein cleavage kinetics. PLoS. Pathog. 5, e1000546 (2009).
Coelmont, L. et al. DEB025 (Alisporivir) inhibits hepatitis C virus replication by preventing a cyclophilin A induced cis-trans isomerisation in domain II of NS5A. PLoS ONE. 5, e13687 (2010).
Liu, Z., Yang, F., Robotham, J. M. & Tang, H. Critical role of cyclophilin A and its prolyl-peptidyl isomerase activity in the structure and function of the hepatitis C virus replication complex. J. Virol. 83, 6554–6565 (2009).
Flisiak, R., Jaroszewicz, J., Flisiak, I. & Lapinski, T. Update on alisporivir in treatment of viral hepatitis C. Expert. Opin. Investig. Drugs 21, 375–382 (2012).
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. Science 309, 1577–1581 (2005). The identification of miR-122 as an important HCV replication-promoting host cell factor, laying the foundation for the clinical development of miR-122-antagonizing compounds.
Jopling, C. L., Schutz, S. & Sarnow, P. Position-dependent function for a tandem microRNA miR-122-binding site located in the hepatitis C virus RNA genome. Cell Host Microbe 4, 77–85 (2008).
Niepmann, M. Hepatitis C virus RNA translation. Curr. Top. Microbiol. Immunol. 369, 143–166 (2013).
Randall, G. et al. Cellular cofactors affecting hepatitis C virus infection and replication. Proc. Natl Acad. Sci. USA 104, 12884–12889 (2007).
Shimakami, T. et al. Stabilization of hepatitis C virus RNA by an Ago2–miR-122 complex. Proc. Natl Acad. Sci. USA 109, 941–946 (2012).
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. Proc. Natl Acad. Sci. USA 110, 1881–1886 (2013).
Henke, J. I. et al. microRNA-122 stimulates translation of hepatitis C virus RNA. EMBO J. 27, 3300–3310 (2008).
Lanford, R. E. et al. Therapeutic silencing of microRNA-122 in primates with chronic hepatitis C virus infection. Science 327, 198–201 (2010). Work assessing the antiviral activity of miR-122-antagonizing compounds in HCV-infected chimpanzees.
Janssen, H. L. et al. Treatment of HCV infection by targeting microRNA. N. Engl. J. Med. 368, 1685–1694 (2013).
Tsai, W. C. et al. MicroRNA-122 plays a critical role in liver homeostasis and hepatocarcinogenesis. J. Clin. Invest. 122, 2884–2897 (2012).
Hsu, S. H. et al. Essential metabolic, anti-inflammatory, and anti-tumorigenic functions of miR-122 in liver. J. Clin. Invest. 122, 2871–2883 (2012).
Choo, Q. L. et al. Isolation of a cDNA clone derived from a blood-borne non-A, non-B viral hepatitis genome. Science 244, 359–362 (1989). A landmark paper that describes the first molecular clone of HCV and paves the way for the development of diagnostic tests, as well as basic and translational HCV research.
Zeuzem, S. et al. Telaprevir for retreatment of HCV infection. N. Engl. J. Med. 364, 2417–2428 (2011). A Phase III clinical trial of telaprevir, providing part of the basis for the approval of this drug.
Jacobson, I. M. et al. Telaprevir for previously untreated chronic hepatitis C virus infection. N. Engl. J. Med. 364, 2405–2416 (2011). Another Phase III clinical trial of telaprevir, providing part of the basis for the approval of this drug.
Poordad, F. et al. Boceprevir for untreated chronic HCV genotype 1 infection. N. Engl. J. Med. 364, 1195–1206 (2011). A Phase III clinical trial of boceprevir, providing part of the basis for the approval of this drug.
Bacon, B. R. et al. Boceprevir for previously treated chronic HCV genotype 1 infection. N. Engl. J. Med. 364, 1207–1217 (2011). Another Phase III clinical trial of boceprevir providing part of the basis for the approval of this drug.
Lok, A. S. et al. Preliminary study of two antiviral agents for hepatitis C genotype 1. N. Engl. J. Med. 366, 216–224 (2012). The first study describing HCV elimination in patients treated with an IFNα-free regimen.
Blight, K. J., Kolykhalov, A. A. & Rice, C. M. Efficient initiation of HCV RNA replication in cell culture. Science 290, 1972–1974 (2000).
Lohmann, V., Körner, F., Dobierzewska, A. & Bartenschlager, R. Mutations in hepatitis C virus RNAs conferring cell culture adaptation. J. Virol. 75, 1437–1449 (2001).
Bartenschlager, R. Hepatitis C virus replicons: potential role for drug development. Nature Rev. Drug Discov. 1, 911–916 (2002).
Saeed, M. et al. Efficient replication of genotype 3a and 4a hepatitis C virus replicons in human hepatoma cells. Antimicrob. Agents Chemother. 56, 5365–5373 (2012).
Saeed, M. et al. Replication of hepatitis C virus genotype 3a in cultured cells. Gastroenterology 144, 56–58 (2013).
Peng, B. et al. Development of robust hepatitis C virus genotype 4 subgenomic replicons. Gastroenterology 144, 59–61 (2013).
Pietschmann, T. et al. Production of infectious genotype 1b virus particles in cell culture and impairment by replication enhancing mutations. PLoS. Pathog. 5, e1000475 (2009).
Bartosch, B., Dubuisson, J. & Cosset, F. L. Infectious hepatitis C virus pseudo-particles containing functional E1–E2 envelope protein complexes. J. Exp. Med. 197, 633–642 (2003).
Hsu, M. et al. Hepatitis C virus glycoproteins mediate pH-dependent cell entry of pseudotyped retroviral particles. Proc. Natl Acad. Sci. USA 100, 7271–7276 (2003). References 144 and 145 describe the HCV pseudoparticle (HCVpp) system for the first time.
Bartosch, B. & Dubuisson, J. Recent advances in hepatitis C virus cell entry. Viruses 2, 692–709 (2010).
Kato, T. et al. Efficient replication of the genotype 2a hepatitis C virus subgenomic replicon. Gastroenterology 125, 1808–1817 (2003).
Wakita, T. et al. Production of infectious hepatitis C virus in tissue culture from a cloned viral genome. Nature Med. 11, 791–796 (2005). A report describing the establishment of the cell culture-derived HCV (HCVcc) system and the in vivo infectivity of cell culture-grown HCV.
Lindenbach, B. D. et al. Complete replication of hepatitis C virus in cell culture. Science 309, 623–626 (2005). A paper that describes the establishment of the HCVcc system by using a JFH-1-derived chimeric HCV genome.
Zhong, J. et al. Robust hepatitis C virus infection in vitro. Proc. Natl Acad. Sci. USA 102, 9294–9299 (2005). A description of the HCVcc system and the increase of viral titres by long-term cell culture passage.
Steinmann, E. & Pietschmann, T. Cell culture systems for hepatitis C virus. Curr. Top. Microbiol. Immunol. 369, 17–48 (2013).
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. Proc. Natl Acad. Sci. USA 103, 2310–2315 (2006).
Li, Y. P. et al. Highly efficient full-length hepatitis C virus genotype 1 (strain TN) infectious culture system. Proc. Natl Acad. Sci. USA 109, 19757–19762 (2012).
Li, Y. P. 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. Proc. Natl Acad. Sci. USA 109, E1101–E1110 (2012).
Ahmad, J., Eng, F. J. & Branch, A. D. HCV and HCC: clinical update and a review of HCC-associated viral mutations in the core gene. Semin. Liver Dis. 31, 347–355 (2011).
Fridell, R. A., Qiu, D., Wang, C., Valera, L. & Gao, M. Resistance analysis of the hepatitis C virus NS5A inhibitor BMS-790052 in an in vitro replicon system. Antimicrob. Agents Chemother. 54, 3641–3650 (2010).
Gouttenoire, J., Penin, F. & Moradpour, D. Hepatitis C virus nonstructural protein 4B: a journey into unexplored territory. Rev. Med. Virol. 20, 117–129 (2010).
Rai, R. & Deval, J. New opportunities in anti-hepatitis C virus drug discovery: targeting NS4B. Antiviral Res. 90, 93–101 (2011).
The authors apologize to all colleagues whose work could not be cited owing to space limitations. Research in the R.B. and V.L. laboratories is supported by the Deutsche Forschungsgemeinschaft (grants SFB/TRR 83 (TP 13), SFB 638 (TP A1), SFB/TRR77 (TP A1) and FOR1202 (TP1) to R.B.; and grants LO1556/1-2, SFB/TRR77 (TP A1) and FOR1202 (TP3) to V.L.). F.P. is supported by the French National Agency for Research on AIDS and Viral Hepatitis (ANRS).
R.B. and V.L. are co-founders of ReBLikon GmbH, which holds commercial rights to hepatitis C virus replicon technology. F.P. declares no competing financial interests.
- Internal ribosome entry site
An RNA sequence that allows ribosomes to bind an mRNA internally, independently of a cap structure, and that thus mediates translation of a downstream ORF.
- Low-density and very-low-density lipoproteins
Lipoproteins that are made in the liver from triglycerides, cholesterol and apolipoproteins and are used to transport lipids in the blood.
- Membranous web
Originally, a term describing a discrete accumulation of the membranous vesicles that have been detected in cells containingreplicating hepatitis C virus (HCV) RNA. More recent studies have shown that this web is composed of single-, double- and multi-membraned vesicles, complex ER membrane rearrangements and lipid droplets. However, in most reports, the term is used as a synonym for the membranous HCV replication compartment, although firm proof of exactly where HCV RNA replication takes place is not available.
- Cyclophilin A
A highly abundant protein that catalyses the cis–trans isomerization of peptide bonds at Pro residues and thus facilitates protein folding. Cyclophilin A binds to the immunosuppressive drug cyclosporin A and is involved in numerous biological processes.
A short, non-coding RNA that is highly expressed in hepatocytes, where it regulates the translation and turnover of mRNAs involved in numerous activities, such as iron and cholesterol homeostasis. In addition, miR-122 was found to act as a tumour suppressor.
(Mitochondrial antiviral-signalling protein). An important factor involved in the activation of a rapid interferon response following the triggering of intracellular RNA sensors such as RIG-I or MDA5.
(TIR domain-containing adaptor inducing interferon-β). A protein involved in the induction of an interferon response following the activation of certain Toll-like receptors, for example Toll-like receptor 3.
- Phosphatidylinositol 4-kinase IIIα
(PtdIns4KIIIα). A lipid kinase that generates PtdIns 4-phosphate by adding a phosphate group to the 4-hydroxy group of PtdIns. PtdIns phosphates are membrane-localized metabolites that have important roles in intracellular signalling and membrane trafficking.
- Sustained virological response rates
The percentages of patients with undetectable hepatitis C virus RNA in the blood at least 6 months after the completion of antiviral therapy.
A virally encoded membrane protein that localizes mainly to the ER or the cell membrane of the host cell and forms an ion channel or pore.
- RNA-induced silencing complex
A multiprotein complex that incorporates a microRNA to recognize complementary sequences in mRNAs and block protein expression.
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Bartenschlager, R., Lohmann, V. & Penin, F. The molecular and structural basis of advanced antiviral therapy for hepatitis C virus infection. Nat Rev Microbiol 11, 482–496 (2013). https://doi.org/10.1038/nrmicro3046
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