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. 2014 Jan 20;29(1):61–70. doi: 10.1007/s12250-014-3408-z

Construction of a chimeric hepatitis C virus replicon based on a strain isolated from a chronic hepatitis C patient

Huang Cao 1,2, Wandi Zhu 1,2, Qingxia Han 1, Rongjuan Pei 1, Xinwen Chen 1,✉
PMCID: PMC8206306  PMID: 24452538

Abstract

Subgenomic replicons of hepatitis C virus (HCV) have been widely used for studying HCV replication. Here, we report a new subgenomic replicon based on a strain isolated from a chronically infected patient. The coding sequence of HCV was recovered from a Chinese chronic hepatitis C patient displaying high serum HCV copy numbers. A consensus sequence designated as CCH strain was constructed based on the sequences of five clones and this was classified by sequence alignment as belonging to genotype 2a. The subgenomic replicon of CCH was replication-deficient in cell culture, due to dysfunctions in NS3 and NS5B. Various JFH1/CCH chimeric replicons were constructed, and specific mutations were introduced. The introduction of mutations could partially restore the replication of chimeric replicons. A replication-competent chimeric construct was finally obtained by the introduction of NS3 from JFH1 into the backbone of the CCH strain.

Keywords: hepatitis C virus, subgenomic replicon, mutation

References

  1. Alter H J, Seeff L B. Recovery, persistence, and sequelae in hepatitis C virus infection: a perspective on long-term outcome. Semin Liver Dis. 2000;20:17–35. doi: 10.1055/s-2000-9505. [DOI] [PubMed] [Google Scholar]
  2. Beran R K, Pyle A M. Hepatitis C viral NS3-4A protease activity is enhanced by the NS3 helicase. J Biol Chem. 2008;283:29929–29937. doi: 10.1074/jbc.M804065200. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Beran R K, Serebrov V, Pyle A M. The serine protease domain of hepatitis C viral NS3 activates RNA helicase activity by promoting the binding of RNA substrate. J Biol Chem. 2007;282:34913–34920. doi: 10.1074/jbc.M707165200. [DOI] [PubMed] [Google Scholar]
  4. Buck M. Direct infection and replication of naturally occurring hepatitis C virus genotypes 1, 2, 3 and 4 in normal human hepatocyte cultures. PLoS ONE. 2008;3:e2660. doi: 10.1371/journal.pone.0002660. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Bukh J, Pietschmann T, Lohmann V, Krieger N, Faulk K, Engle R E, Govindarajan S, Shapiro M, St Claire M, Bartenschlager R. Mutations that permit efficient replication of hepatitis C virus RNA in Huh-7 cells prevent productive replication in chimpanzees. Proc Natl Acad Sci USA. 2002;99:14416–14421. doi: 10.1073/pnas.212532699. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Chinnaswamy S, Murali A, Cai H, Yi G, Palaninathan S, Kao C. Conformations of the monomeric hepatitis C virus RNA-dependent RNA polymerase. Virus Treatments and Adaptation. 2010;2:21–39. [Google Scholar]
  7. Date T, Kato T, Kato J, Takahashi H, Morikawa K, Akazawa D, Murayama A, Tanaka-Kaneko K, Sata T, Tanaka Y, Mizokami M, Wakita T. Novel cell culture-adapted genotype 2a hepatitis C virus infectious clone. J Virol. 2012;86:10805–10820. doi: 10.1128/JVI.07235-11. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Dimitrova M, Imbert I, Kieny M P, Schuster C. Protein-protein interactions between hepatitis C virus nonstructural proteins. J Virol. 2003;77:5401–5414. doi: 10.1128/JVI.77.9.5401-5414.2003. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Frick D N. The hepatitis C virus NS3 protein: a model RNA helicase and potential drug target. Curr Issues Mol Biol. 2007;9:1–20. [PMC free article] [PubMed] [Google Scholar]
  10. Gottwein J M, Scheel T K, Hoegh A M, Lademann J B, Eugen-Olsen J, Lisby G, Bukh J. Robust hepatitis C genotype 3a cell culture releasing adapted intergenotypic 3a/2a (S52/JFH1) viruses. Gastroenterology. 2007;133:1614–1626. doi: 10.1053/j.gastro.2007.08.005. [DOI] [PubMed] [Google Scholar]
  11. Gottwein J M, Scheel T K, Jensen T B, Lademann J B, Prentoe J C, Knudsen M L, Hoegh A M, Bukh J. 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. Hepatology. 2009;49:364–377. doi: 10.1002/hep.22673. [DOI] [PubMed] [Google Scholar]
  12. Grakoui A, Wychowski C, Lin C, Feinstone S M, Rice C M. Expression and identification of hepatitis C virus polyprotein cleavage products. J Virol. 1993;67:1385–1395. doi: 10.1128/jvi.67.3.1385-1395.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Han Q, Xu C, Wu C, Zhu W, Yang R, Chen X. Compensatory mutations in NS3 and NS5A proteins enhance the virus production capability of hepatitis C reporter virus. Virus Res. 2009;145:63–73. doi: 10.1016/j.virusres.2009.06.005. [DOI] [PubMed] [Google Scholar]
  14. Hijikata M, Mizushima H, Tanji Y, Komoda Y, Hirowatari Y, Akagi T, Kato N, Kimura K, Shimotohno K. Proteolytic processing and membrane association of putative nonstructural proteins of hepatitis C virus. Proc Natl Acad Sci U S A. 1993;90:10773–10777. doi: 10.1073/pnas.90.22.10773. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Jiang J, Luo G. Cell culture-adaptive mutations promote viral protein-protein interactions and morphogenesis of infectious hepatitis C virus. J Virol. 2012;86:8987–8997. doi: 10.1128/JVI.00004-12. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Kato T, Date T, Miyamoto M, Furusaka A, Tokushige K, Mizokami M, Wakita T. Efficient replication of the genotype 2a hepatitis C virus subgenomic replicon. Gastroenterology. 2003;125:1808–1817. doi: 10.1053/j.gastro.2003.09.023. [DOI] [PubMed] [Google Scholar]
  17. Kuiken C, Yusim K, Boykin L, Richardson R. The Los Alamos hepatitis C sequence database. Bioinformatics. 2005;21:379–384. doi: 10.1093/bioinformatics/bth485. [DOI] [PubMed] [Google Scholar]
  18. Li Y P, Ramirez S, Gottwein J M, Scheel T K, Mikkelsen L, Purcell R H, Bukh J. 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. 2012;109:E1101–1110. doi: 10.1073/pnas.1203829109. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Lindenbach B D, Rice C M. Unravelling hepatitis C virus replication from genome to function. Nature. 2005;436:933–938. doi: 10.1038/nature04077. [DOI] [PubMed] [Google Scholar]
  20. Lohmann V, Korner F, Koch J, Herian U, Theilmann L, Bartenschlager R. Replication of subgenomic hepatitis C virus RNAs in a hepatoma cell line. Science. 1999;285:110–113. doi: 10.1126/science.285.5424.110. [DOI] [PubMed] [Google Scholar]
  21. Lu J, Tao W, Li R, Xiang Y, Zhang N, Xiang X, Xie Q, Zhong J. Construction and characterization of infectious hepatitis C virus chimera containing structural proteins directly from genotype 1b clinical isolates. Virology. 2013;443:80–88. doi: 10.1016/j.virol.2013.04.030. [DOI] [PubMed] [Google Scholar]
  22. Lu J, Xiang Y, Tao W, Li Q, Wang N, Gao Y, Xiang X, Xie Q, Zhong J. A novel strategy to develop robust infectious hepatitis C virus cell culture system directly from a clinical isolate. J Virol. 2014;88:1484–1491. doi: 10.1128/JVI.02929-13. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Ma Y, Yates J, Liang Y, Lemon S M, Yi M. NS3 helicase domains involved in infectious intracellular hepatitis C virus particle assembly. J Virol. 2008;82:7624–7639. doi: 10.1128/JVI.00724-08. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Murayama A, Date T, Morikawa K, Akazawa D, Miyamoto M, Kaga M, Ishii K, Suzuki T, Kato T, Mizokami M, Wakita T. The NS3 helicase and NS5B-to-3’X regions are important for efficient hepatitis C virus strain JFH-1 replication in Huh7 cells. J Virol. 2007;81:8030–8040. doi: 10.1128/JVI.02088-06. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Murayama A, Kato T, Akazawa D, Sugiyama N, Date T, Masaki T, Nakamoto S, Tanaka Y, Mizokami M, Yokosuka O, Nomoto A, Wakita T. Production of infectious chimeric hepatitis C virus genotype 2b harboring minimal regions of JFH-1. J Virol. 2012;86:2143–2152. doi: 10.1128/JVI.05386-11. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Otsuka M, Kato N, Moriyama M, Taniguchi H, Wang Y, Dharel N, Kawabe T, Omata M. Interaction between the HCV NS3 protein and the host TBK1 protein leads to inhibition of cellular antiviral responses. Hepatology. 2005;41:1004–1012. doi: 10.1002/hep.20666. [DOI] [PubMed] [Google Scholar]
  27. Pietschmann T, Zayas M, Meuleman P, Long G, Appel N, Koutsoudakis G, Kallis S, Leroux-Roels G, Lohmann V, Bartenschlager R. Production of infectious genotype 1b virus particles in cell culture and impairment by replication enhancing mutations. PLoS Pathog. 2009;5:e1000475. doi: 10.1371/journal.ppat.1000475. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Pietschmann T, Kaul A, Koutsoudakis G, Shavinskaya A, Kallis S, Steinmann E, Abid K, Negro F, Dreux M, Cosset F L, Bartenschlager R. Construction and characterization of infectious intragenotypic and intergenotypic hepatitis C virus chimeras. Proc Natl Acad Sci USA. 2006;103:7408–7413. doi: 10.1073/pnas.0504877103. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Ramirez S, Li Y P, Jensen S B, Pedersen J, Gottwein J M, Bukh J. Hepatology. 2013. Highly efficient infectious cell culture of three HCV genotype 2b strains and sensitivity to lead protease, NS5A, and polymerase inhibitors. [DOI] [PubMed] [Google Scholar]
  30. Ray U, Das S. Interplay between NS3 protease and human La protein regulates translation-replication switch of Hepatitis C virus. Sci Rep. 2011;1:1. doi: 10.1038/srep00001. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Scheel T K, Gottwein J M, Jensen T B, Prentoe J C, Hoegh A M, Alter H J, Eugen-Olsen J, Bukh J. Development of JFH1-based cell culture systems for hepatitis C virus genotype 4a and evidence for cross-genotype neutralization. Proc Natl Acad Sci U S A. 2008;105:997–1002. doi: 10.1073/pnas.0711044105. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Scheel T K, Gottwein J M, Carlsen T H, Li Y P, Jensen T B, Spengler U, Weis N, Bukh J. Efficient culture adaptation of hepatitis C virus recombinants with genotype-specific core-NS2 by using previously identified mutations. J Virol. 2011;85:2891–2906. doi: 10.1128/JVI.01605-10. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Schmitt M, Scrima N, Radujkovic D, Caillet-Saguy C, Simister P C, Friebe P, Wicht O, Klein R, Bartenschlager R, Lohmann V, Bressanelli S. A comprehensive structure-function comparison of hepatitis C virus strain JFH1 and J6 polymerases reveals a key residue stimulating replication in cell culture across genotypes. J Virol. 2011;85:2565–2581. doi: 10.1128/JVI.02177-10. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Scrima N, Caillet-Saguy C, Ventura M, Harrus D, Astier-Gin T, Bressanelli S. Two crucial early steps in RNA synthesis by the hepatitis C virus polymerase involve a dual role of residue 405. J Virol. 2012;86:7107–7117. doi: 10.1128/JVI.00459-12. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Shimotohno K. Hepatitis C virus and its pathogenesis. Semin Cancer Biol. 2000;10:233–240. doi: 10.1006/scbi.2000.0322. [DOI] [PubMed] [Google Scholar]
  36. Simister P, Schmitt M, Geitmann M, Wicht O, Danielson U H, Klein R, Bressanelli S, Lohmann V. Structural and functional analysis of hepatitis C virus strain JFH1 polymerase. J Virol. 2009;83:11926–11939. doi: 10.1128/JVI.01008-09. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Wakita T, Pietschmann T, Kato T, Date T, Miyamoto M, Zhao Z, Murthy K, Habermann A, Krausslich H G, Mizokami M, Bartenschlager R, Liang T J. Production of infectious hepatitis C virus in tissue culture from a cloned viral genome. Nat Med. 2005;11:791–796. doi: 10.1038/nm1268. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Yan Y, Li Y, Munshi S, Sardana V, Cole J L, Sardana M, Steinkuehler C, Tomei L, De Francesco R, Kuo L C, Chen Z. Complex of NS3 protease and NS4A peptide of BK strain hepatitis C virus: a 2.2 A resolution structure in a hexagonal crystal form. Protein Sci. 1998;7:837–847. doi: 10.1002/pro.5560070402. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. 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. Virology. 1999;262:250–263. doi: 10.1006/viro.1999.9889. [DOI] [PubMed] [Google Scholar]
  40. 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. 2006;103:2310–2315. doi: 10.1073/pnas.0510727103. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Zhong J, Gastaminza P, Cheng G, Kapadia S, Kato T, Burton D R, Wieland S F, Uprichard S L, Wakita T, Chisari F V. Robust hepatitis C virus infection in vitro. Proc Natl Acad Sci U S A. 2005;102:9294–9299. doi: 10.1073/pnas.0503596102. [DOI] [PMC free article] [PubMed] [Google Scholar]

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