Hepatitis C Virus Replicons Volume 3 and 4

Abstract

See “Replication of hepatitis C virus genotype 3a in cultured cells” by Saeed M, Gondeau C, Hmwe S, et al, on page 56 and “Development of robust hepatitis C virus genotype 4 subgenomic replicons” by Peng B, Yu M, Xu S, et al, on page 59. See “Replication of hepatitis C virus genotype 3a in cultured cells” by Saeed M, Gondeau C, Hmwe S, et al, on page 56 and “Development of robust hepatitis C virus genotype 4 subgenomic replicons” by Peng B, Yu M, Xu S, et al, on page 59. Infections with the hepatitis C virus (HCV) are a main cause of acute and chronic liver disease.1Jacobson I.M. Davis G.L. El Serag H. et al.Prevalence and challenges of liver diseases in patients with chronic hepatitis C virus infection.Clin Gastroenterol Hepatol. 2010; 8: 924-933Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar Whereas primary infections are predominantly asymptomatic, in most cases they persist and persistently infected individuals have a high risk to develop serious liver damage, including cirrhosis and hepatocellular carcinoma. HCV is a positive-strand RNA virus that replicates its genome with the help of an RNA-dependent RNA polymerase.2Poenisch M. Bartenschlager R. New insights into structure and replication of the hepatitis C virus and clinical implications.Semin Liver Dis. 2010; 30: 333-347Crossref PubMed Scopus (69) Google Scholar Owing to its high error rate, the virus is highly variable, challenging development of vaccines and treatment with directly acting antivirals (DAAs). Moreover, 7 genotypes can be classified differing in their nucleotide sequence by around 35%. Some of the genotypes are found in rather localized areas, whereas others such as genotype 1 are distributed throughout the world. With the advent of cell culture models recapitulating parts or the complete viral replication cycle in cultured human hepatoma cells, studies on HCV–host cell interaction, viral RNA replication as well as development of antiviral therapies has become possible (reviewed by Murray and Rice3Murray C.L. Rice C.M. Turning hepatitis C into a real virus.Annu Rev Microbiol. 2011; 65: 307-327Crossref PubMed Scopus (50) Google Scholar). However, a serious limitation is the availability of only a few HCV isolates that are capable of replicating in cell culture; these isolates correspond primarily with genotype 1. In fact, the first efficient HCV cell culture system was based on genotype 1b-derived subgenomic replicons, a term referring to the fact that part of the HCV genome had been deleted and that these RNAs can replicate autonomously in cells4Lohmann V. Korner F. Koch J. et al.Replication of subgenomic hepatitis C virus RNAs in a hepatoma cell line.Science. 1999; 285: 110-113Crossref PubMed Scopus (2474) Google Scholar (Figure 1). Upon transfection into cells of the human hepatoma cell line Huh7 and selection with the cytotoxic drug G418, only cells supporting efficient RNA replication could survive, because only those cells contained sufficient amounts of neomycin phosphotransferase that inactivates G418. With this approach, cell lines could be established containing high amounts of self-replicating HCV RNA and proteins.4Lohmann V. Korner F. Koch J. et al.Replication of subgenomic hepatitis C virus RNAs in a hepatoma cell line.Science. 1999; 285: 110-113Crossref PubMed Scopus (2474) Google Scholar However, despite efficient HCV replication, this cell culture model did not support the production of HCV particles, even when using genomic replicons.3Murray C.L. Rice C.M. Turning hepatitis C into a real virus.Annu Rev Microbiol. 2011; 65: 307-327Crossref PubMed Scopus (50) Google Scholar This was because, during replication and G418 selection, replicon RNAs had accumulated mutations that enhanced replication by several orders of magnitude, but at the same time these mutations interfered with assembly.5Pietschmann T. Zayas M. Meuleman P. et al.Production of infectious genotype 1b virus particles in cell culture and impairment by replication enhancing mutations.PLoS Pathog. 2009; 5 (e1000475)Crossref Scopus (117) Google Scholar For instance, the most efficient increase of replication was achieved by mutations residing in NS5A or NS5B, either alone or in combination with mutations residing in the NS3 helicase domain. However, especially the latter blocked assembly of infectious HCV particles. Nevertheless, these replicons provided the first system to study HCV replication in cells and were instrumental for the development of DAAs. In addition, the replicon system allowed selection for highly HCV-permissive Huh7-derived subclones obtained by removal of replicon RNA upon treatment with interferon (IFN) or selective antiviral drugs. Some of these ‘cured’ cell clones such as Huh7.5, Huh7.5.1, or Huh7-Lunet cells turned out to be much more permissive compared with the parental cell line and thus are now widely used.6Blight K.J. McKeating J.A. Rice C.M. Highly permissive cell lines for subgenomic and genomic hepatitis C virus RNA replication.J Virol. 2002; 76: 13001-13014Crossref PubMed Scopus (953) Google Scholar, 7Friebe P. Boudet J. Simorre J.P. et al.Kissing-loop interaction in the 3' end of the hepatitis C virus genome essential for RNA replication.J Virol. 2005; 79: 380-392Crossref PubMed Scopus (295) Google Scholar With the advent of highly permissive cell clones and cell culture–adapted HCV replicons, transient replication systems became possible that were most often based on replicons in which the neo gene had been replaced by a gene encoding for an easy to measure reporter such as luciferase. However, for reasons that are still not well understood, HCV replicons could be established for only a few viral isolates and with one exception they all belonged to genotype 1.3Murray C.L. Rice C.M. Turning hepatitis C into a real virus.Annu Rev Microbiol. 2011; 65: 307-327Crossref PubMed Scopus (50) Google Scholar, 8Bartenschlager R. Sparacio S. Hepatitis C virus molecular clones and their replication capacity in vivo and in cell culture.Virus Res. 2007; 127: 195-207Crossref PubMed Scopus (93) Google Scholar The exception is a particular genotype 2a isolate, designated JFH-1, because it was derived from a clone that had been isolated from a Japanese patient with fulminant hepatitis.9Kato T. Date T. Miyamoto M. et al.Efficient replication of the genotype 2a hepatitis C virus subgenomic replicon.Gastroenterology. 2003; 125: 1808-1817Abstract Full Text Full Text PDF PubMed Scopus (494) Google Scholar This isolate replicates to very high levels without requiring adaptive mutations and because it supports production of infectious HCV particles it recapitulates the complete viral replication cycle.10Wakita T. Pietschmann T. Kato T. et al.Production of infectious hepatitis C virus in tissue culture from a cloned viral genome.Nat Med. 2005; 11: 791-796Crossref PubMed Scopus (2386) Google Scholar Despite this important progress, establishment of cell culture systems derived from HCV genotypes other than 1 and 2 were of limited success. Now, in this issue of Gastroenterology, Saeed et al11Saeed M. Gondeau C. Hmwe S. et al.Replication of hepatitis C virus genotype 3a in cultured cells.Gastroenterology. 2013; 144: 56-58Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar as well as Peng et al12Peng B. Yu M. Xu S. et al.Development of robust hepatitis C virus genotype 4 subgenomic replicons.Gastroenterology. 2013; 144: 59-61Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar were able to establish replicons derived from genotype 3 and 4 HCV isolates, respectively. The starting point of the study by Saeed et al was a consensus HCV isolate termed S310 that had been cloned from a high-titer serum of a patient with recurrent hepatitis after liver transplantation.11Saeed M. Gondeau C. Hmwe S. et al.Replication of hepatitis C virus genotype 3a in cultured cells.Gastroenterology. 2013; 144: 56-58Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar This isolate was used for construction of a selectable subgenomic replicon. Upon transfection of Huh7.5.1 cells and selection with G418, the authors obtained stable cell clones (Figure 1). Importantly, replicons in these cells had accumulated adaptive, replication enhancing mutations in NS3, NS5A, and NS5B at similar or even identical positions as previously reported for genotype 1b isolates.13Lohmann V. Korner F. Dobierzewska A. et al.Mutations in hepatitis C virus RNAs conferring cell culture adaptation.J Virol. 2001; 75: 1437-1449Crossref PubMed Scopus (398) Google Scholar, 14Lohmann V. Hoffmann S. Herian U. et al.Viral and cellular determinants of hepatitis C virus RNA replication in cell culture.J Virol. 2003; 77: 3007-3019Crossref PubMed Scopus (343) Google Scholar In the case of the newly established genotype 4a replicon, Peng et al12Peng B. Yu M. Xu S. et al.Development of robust hepatitis C virus genotype 4 subgenomic replicons.Gastroenterology. 2013; 144: 59-61Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar used the previously cloned HCV consensus isolate ED43, which is infectious in chimpanzees.15Gottwein J.M. Scheel T.K. Callendret B. 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.J Virol. 2010; 84: 5277-5293Crossref PubMed Scopus (111) Google Scholar However, selectable replicons based on this isolate only gave rise to stable cell clones after introducing a replication enhancing mutation at a position in NS5A known to enhance genotype 1 replication (S232I/S2204I16Blight K.J. Kolykhalov A.A. Rice C.M. Efficient initiation of HCV RNA replication in cell culture.Science. 2000; 290: 1972-1974Crossref PubMed Scopus (1268) Google Scholar) and by using a novel highly permissive Huh-7 cell clone (designated 1C12Peng B. Yu M. Xu S. et al.Development of robust hepatitis C virus genotype 4 subgenomic replicons.Gastroenterology. 2013; 144: 59-61Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar). Further adaptive mutations located in NS3 and NS4A were identified in selected replicon clones, increasing replication of this genotype 4a RNA to the level of genotype 1 replicons.12Peng B. Yu M. Xu S. et al.Development of robust hepatitis C virus genotype 4 subgenomic replicons.Gastroenterology. 2013; 144: 59-61Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar Thus, these new genotype 3 and 4 replicons followed the genotype 1 paradigm with wild-type isolates replicating poorly in cell culture and requiring adaptive mutations enhancing RNA replication, which is in contrast with the genotype 2a isolate JFH-1. Nevertheless, independent from the used genotype, steady-state viral RNA levels in selected replicon cell clones are comparable. However, transient replication levels that reflect much better the true dynamics of RNA synthesis vary widely among different isolates. In this respect, the order is JFH-1 ≫ genotype 1 isolates = ED43 > S310. However, low replication of the latter might be improved by using other combinations of adaptive mutations. A recent study also reported the establishment of genotype 3a and 4a replicons17Saeed M. Scheel T.K. Gottwein J.M. et al.Efficient replication of genotype 3a and 4a hepatitis C virus replicons in human hepatoma cells.Antimicrob Agents Chemother. 2012; 56: 5365-5373Crossref PubMed Scopus (106) Google Scholar that were derived from the HCV isolate S52 and also the ED43, respectively.15Gottwein J.M. Scheel T.K. Callendret B. 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.J Virol. 2010; 84: 5277-5293Crossref PubMed Scopus (111) Google Scholar Interestingly, despite the huge genetic difference among HCV genotypes, identical positions of cell culture adaptive mutations were identified. For instance, a mutation residing in NS3 at the junction of the protease and the helicase domains (T1280I in genotype 1b18Krieger N. Lohmann V. Bartenschlager R. Enhancement of hepatitis C virus RNA replication by cell culture-adaptive mutations.J Virol. 2001; 75: 4614-4624Crossref PubMed Scopus (461) Google Scholar and T1286I in S31011Saeed M. Gondeau C. Hmwe S. et al.Replication of hepatitis C virus genotype 3a in cultured cells.Gastroenterology. 2013; 144: 56-58Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar) as well as a mutation at the carboxyterminus of the NS5B RNA-dependent RNA polymerase (R2884G in genotype 1b13Lohmann V. Korner F. Dobierzewska A. et al.Mutations in hepatitis C virus RNAs conferring cell culture adaptation.J Virol. 2001; 75: 1437-1449Crossref PubMed Scopus (398) Google Scholar and R2895G in S31011Saeed M. Gondeau C. Hmwe S. et al.Replication of hepatitis C virus genotype 3a in cultured cells.Gastroenterology. 2013; 144: 56-58Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar) were selected. Moreover, these mutations also increased replication of genotype 4a replicons.17Saeed M. Scheel T.K. Gottwein J.M. et al.Efficient replication of genotype 3a and 4a hepatitis C virus replicons in human hepatoma cells.Antimicrob Agents Chemother. 2012; 56: 5365-5373Crossref PubMed Scopus (106) Google Scholar Likewise, a mutation in NS5A (S2204I14Lohmann V. Hoffmann S. Herian U. et al.Viral and cellular determinants of hepatitis C virus RNA replication in cell culture.J Virol. 2003; 77: 3007-3019Crossref PubMed Scopus (343) Google Scholar, 16Blight K.J. Kolykhalov A.A. Rice C.M. Efficient initiation of HCV RNA replication in cell culture.Science. 2000; 290: 1972-1974Crossref PubMed Scopus (1268) Google Scholar corresponding with S232I12Peng B. Yu M. Xu S. et al.Development of robust hepatitis C virus genotype 4 subgenomic replicons.Gastroenterology. 2013; 144: 59-61Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar or S2210I11Saeed M. Gondeau C. Hmwe S. et al.Replication of hepatitis C virus genotype 3a in cultured cells.Gastroenterology. 2013; 144: 56-58Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar) enhances RNA replication of genotypes 1a, 1b, 3a, and 4a replicons. This high conservation of certain cell culture adaptive mutations will likely help to generate cell culture models for the remaining genotypes 5–7. Nevertheless, the mechanism of replication enhancement remains ill-defined, and it will be important to know whether adaptive mutations interfere with virus production also for these genotype 3 and 4 isolates, as is the case for almost all genotype 1 isolates. The availability of functional genotype 3 and 4 replicons is an important extension of our tool box and thus will foster HCV research and eventually also therapy development in several respects. First, it will now be possible to test DAAs for their inhibitory activity in cell culture. Up to now, this was limited to replication-competent chimeric replicons and genomes into which the NS3 protease or NS5A of a given genotype had been inserted19Imhof I. Simmonds P. Genotype differences in susceptibility and resistance development of hepatitis C virus to protease inhibitors telaprevir (VX-950) and danoprevir (ITMN-191).Hepatology. 2011; 53: 1090-1099Crossref PubMed Scopus (64) Google Scholar, 20Scheel T.K. Gottwein J.M. Mikkelsen L.S. et al.Recombinant HCV variants with NS5A from genotypes 1-7 have different sensitivities to an NS5A inhibitor but not interferon-alpha.Gastroenterology. 2011; 140: 1032-1042Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar; however, such chimeras are not available for the NS5B polymerase, a prime targets for DAAs. Initial results presented in the 2 studies of this issue of Gastroenterology clearly confirm genotype-specific differences in potency of protease inhibitors11Saeed M. Gondeau C. Hmwe S. et al.Replication of hepatitis C virus genotype 3a in cultured cells.Gastroenterology. 2013; 144: 56-58Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar and non-nucleosidic polymerase inhibitors,12Peng B. Yu M. Xu S. et al.Development of robust hepatitis C virus genotype 4 subgenomic replicons.Gastroenterology. 2013; 144: 59-61Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar as expected from in vitro assays and clinical trials. In this respect, the novel replicons will be helpful to tailor effective combination therapies for genotypes 3 and 4. Second, the bigger portfolio of non-genotype 1 or 2 HCV isolates replicating in cell culture might help to understand the molecular basis of genotype-specific differences in IFN treatment response. It is still not clear why patients infected with HCV genotype 2 and 3 viruses clear the virus more frequently compared with infections with other genotype viruses. Interestingly, the newly developed genotype 3 and 4 replicons are as sensitive to IFN-alpha as the replication competent genotype 1 and 2 isolates,11Saeed M. Gondeau C. Hmwe S. et al.Replication of hepatitis C virus genotype 3a in cultured cells.Gastroenterology. 2013; 144: 56-58Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar, 12Peng B. Yu M. Xu S. et al.Development of robust hepatitis C virus genotype 4 subgenomic replicons.Gastroenterology. 2013; 144: 59-61Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar arguing that IFN resistance is not a genotype-intrinsic feature of the HCV nonstructural proteins. Third, comparative analyses of host responses induced in cell culture by different HCV genotypes offers new possibilities to study the molecular determinants of HCV pathogenesis. One obvious example is the hepatic steatosis frequently observed with genotype 3 infections. Last, molecular studies on HCV replication will greatly benefit from the possibility to validate obtained results for several genotypes, thus better reflecting the biological diversity of HCV. This is especially important for the growing number of host cell factors reported to play a role in HCV RNA replication, but at the same time will also broaden our view on interactions among viral nonstructural proteins. In all these respects, the establishment of genotype 3 and 4 replicon models is an important step toward a broader coverage of the biological diversity of HCV that will clearly foster basic research and help to understand the genotype specificity of DAAs. Given this success, we can be optimistic that cell culture models of the remaining genotypes 5–7 will become available soon. Development of Robust Hepatitis C Virus Genotype 4 Subgenomic RepliconsGastroenterologyVol. 144Issue 1PreviewDespite the prevalence of hepatitis C virus genotype 4, no replicon system is available for study of the genotype. To facilitate discovery and development of reagents against this virus, we synthesized and transcribed a genotype 4a subgenomic replicon and transfected Huh7-Lunet cells with it, which yielded very few colonies. However, when we used a new Huh-7–derived cell line, colony formation increased ∼70-fold. We identified multiple adaptive mutations in the virus's nonstructural 3 or 4A proteins that allowed the cells to maintain stable, genotype 4a luciferase-encoding replicons. Full-Text PDF Replication of Hepatitis C Virus Genotype 3a in Cultured CellsGastroenterologyVol. 144Issue 1PreviewHepatitis C virus (HCV) genotype 3a is widespread worldwide, but no replication system exists for its study. We describe a subgenomic replicon system for HCV genotype 3a. We determined the consensus sequence of an HCV genome isolated from a patient, and constructed a subgenomic replicon using this clone. The replicon was transfected into HuH-7 cells and RNA replication was confirmed. We identified cell culture-adaptive mutations that increased colony formation multiple-fold. We have therefore established a genotype 3a replicon system that can be used to study this HCV genotype. Full-Text PDF Covering the CoverGastroenterologyVol. 144Issue 1PreviewUnderstanding of the hepatitis C virus (HCV) and the development of antiviral agents to treat chronic infection was hampered for many years by the inability to achieve viral replication in vitro. The significant advances that have occurred recently in the treatment of chronic hepatitis C infection are, in large part, the result of the development of HCV replicons, self-replicating HCV RNA sequences, for genotypes 1a, 1b, and 2a, and complete cell culture systems. However, no replication systems have been developed for other HCV genotypes, which may be more prevalent in certain areas of the world than HCV genotype 1. Full-Text PDF