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

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.

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References

  1. Alter H J, and Seeff L B. 2000. Recovery, persistence, and sequelae in hepatitis C virus infection: a perspective on long-term outcome. Semin Liver Dis, 20: 17–35.

    CAS  PubMed  Article  Google Scholar 

  2. Beran R K, and Pyle A M. 2008. Hepatitis C viral NS3-4A protease activity is enhanced by the NS3 helicase. J Biol Chem, 283: 29929–29937.

    CAS  PubMed  Article  Google Scholar 

  3. Beran R K, Serebrov V, and Pyle A M. 2007. The serine protease domain of hepatitis C viral NS3 activates RNA helicase activity by promoting the binding of RNA substrate. J Biol Chem, 282: 34913–34920.

    CAS  PubMed  Article  Google Scholar 

  4. Buck M. 2008. Direct infection and replication of naturally occurring hepatitis C virus genotypes 1, 2, 3 and 4 in normal human hepatocyte cultures. PLoS ONE, 3: e2660.

    PubMed Central  PubMed  Article  Google Scholar 

  5. Bukh J, Pietschmann T, Lohmann V, Krieger N, Faulk K, Engle R E, Govindarajan S, Shapiro M, St Claire M, and Bartenschlager R. 2002. Mutations that permit efficient replication of hepatitis C virus RNA in Huh-7 cells prevent productive replication in chimpanzees. Proc Natl Acad Sci USA, 99: 14416–14421.

    CAS  PubMed  Article  Google Scholar 

  6. Chinnaswamy S, Murali A, Cai H, Yi G, Palaninathan S, and Kao C. 2010. Conformations of the monomeric hepatitis C virus RNA-dependent RNA polymerase. Virus Treatments and Adaptation, 2: 21–39.

    CAS  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, and Wakita T. 2012. Novel cell culture-adapted genotype 2a hepatitis C virus infectious clone. J Virol, 86: 10805–10820.

    CAS  PubMed Central  PubMed  Article  Google Scholar 

  8. Dimitrova M, Imbert I, Kieny M P, and Schuster C. 2003. Protein-protein interactions between hepatitis C virus nonstructural proteins. J Virol, 77: 5401–5414.

    CAS  PubMed Central  PubMed  Article  Google Scholar 

  9. Frick D N. 2007. The hepatitis C virus NS3 protein: a model RNA helicase and potential drug target. Curr Issues Mol Biol, 9: 1–20.

    CAS  PubMed Central  PubMed  Google Scholar 

  10. Gottwein J M, Scheel T K, Hoegh A M, Lademann J B, Eugen-Olsen J, Lisby G, and Bukh J. 2007. Robust hepatitis C genotype 3a cell culture releasing adapted intergenotypic 3a/2a (S52/JFH1) viruses. Gastroenterology, 133: 1614–1626.

    CAS  PubMed  Article  Google Scholar 

  11. Gottwein J M, Scheel T K, Jensen T B, Lademann J B, Prentoe J C, Knudsen M L, Hoegh A M, and Bukh J. 2009. 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, 49: 364–377.

    CAS  PubMed  Article  Google Scholar 

  12. Grakoui A, Wychowski C, Lin C, Feinstone S M, and Rice C M. 1993. Expression and identification of hepatitis C virus polyprotein cleavage products. J Virol, 67: 1385–1395.

    CAS  PubMed Central  PubMed  Google Scholar 

  13. Han Q, Xu C, Wu C, Zhu W, Yang R, and Chen X. 2009. Compensatory mutations in NS3 and NS5A proteins enhance the virus production capability of hepatitis C reporter virus. Virus Res, 145: 63–73.

    CAS  PubMed  Article  Google Scholar 

  14. Hijikata M, Mizushima H, Tanji Y, Komoda Y, Hirowatari Y, Akagi T, Kato N, Kimura K, and Shimotohno K. 1993. Proteolytic processing and membrane association of putative nonstructural proteins of hepatitis C virus. Proc Natl Acad Sci U S A, 90: 10773–10777.

    CAS  PubMed Central  PubMed  Article  Google Scholar 

  15. Jiang J, and Luo G. 2012. Cell culture-adaptive mutations promote viral protein-protein interactions and morphogenesis of infectious hepatitis C virus. J Virol, 86: 8987–8997.

    CAS  PubMed Central  PubMed  Article  Google Scholar 

  16. Kato T, Date T, Miyamoto M, Furusaka A, Tokushige K, Mizokami M, and Wakita T. 2003. Efficient replication of the genotype 2a hepatitis C virus subgenomic replicon. Gastroenterology, 125: 1808–1817.

    CAS  PubMed  Article  Google Scholar 

  17. Kuiken C, Yusim K, Boykin L, and Richardson R. 2005. The Los Alamos hepatitis C sequence database. Bioinformatics, 21: 379–384.

    CAS  PubMed  Article  Google Scholar 

  18. Li Y P, Ramirez S, Gottwein J M, Scheel T K, Mikkelsen L, Purcell R H, and Bukh J. 2012. 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: E1101–1110.

    CAS  PubMed  Article  Google Scholar 

  19. Lindenbach B D, and Rice C M. 2005. Unravelling hepatitis C virus replication from genome to function. Nature, 436: 933–938.

    CAS  PubMed  Article  Google Scholar 

  20. Lohmann V, Korner F, Koch J, Herian U, Theilmann L, and Bartenschlager R. 1999. Replication of subgenomic hepatitis C virus RNAs in a hepatoma cell line. Science, 285: 110–113.

    CAS  PubMed  Article  Google Scholar 

  21. Lu J, Tao W, Li R, Xiang Y, Zhang N, Xiang X, Xie Q, and Zhong J. 2013. Construction and characterization of infectious hepatitis C virus chimera containing structural proteins directly from genotype 1b clinical isolates. Virology, 443: 80–88.

    CAS  PubMed  Article  Google Scholar 

  22. Lu J, Xiang Y, Tao W, Li Q, Wang N, Gao Y, Xiang X, Xie Q, and Zhong J. 2014. A novel strategy to develop robust infectious hepatitis C virus cell culture system directly from a clinical isolate. J Virol, 88: 1484–1491.

    PubMed  Article  Google Scholar 

  23. Ma Y, Yates J, Liang Y, Lemon S M, and Yi M. 2008. NS3 helicase domains involved in infectious intracellular hepatitis C virus particle assembly. J Virol, 82: 7624–7639.

    CAS  PubMed Central  PubMed  Article  Google Scholar 

  24. Murayama A, Date T, Morikawa K, Akazawa D, Miyamoto M, Kaga M, Ishii K, Suzuki T, Kato T, Mizokami M, and Wakita T. 2007. 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, 81: 8030–8040.

    CAS  PubMed Central  PubMed  Article  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, and Wakita T. 2012. Production of infectious chimeric hepatitis C virus genotype 2b harboring minimal regions of JFH-1. J Virol, 86: 2143–2152.

    CAS  PubMed Central  PubMed  Article  Google Scholar 

  26. Otsuka M, Kato N, Moriyama M, Taniguchi H, Wang Y, Dharel N, Kawabe T, and Omata M. 2005. Interaction between the HCV NS3 protein and the host TBK1 protein leads to inhibition of cellular antiviral responses. Hepatology, 41: 1004–1012.

    CAS  PubMed  Article  Google Scholar 

  27. Pietschmann T, Zayas M, Meuleman P, Long G, Appel N, Koutsoudakis G, Kallis S, Leroux-Roels G, Lohmann V, and Bartenschlager R. 2009. Production of infectious genotype 1b virus particles in cell culture and impairment by replication enhancing mutations. PLoS Pathog, 5: e1000475.

    PubMed Central  PubMed  Article  Google Scholar 

  28. Pietschmann T, Kaul A, Koutsoudakis G, Shavinskaya A, Kallis S, Steinmann E, Abid K, Negro F, Dreux M, Cosset F L, and Bartenschlager R. 2006. Construction and characterization of infectious intragenotypic and intergenotypic hepatitis C virus chimeras. Proc Natl Acad Sci USA, 103: 7408–7413.

    CAS  PubMed  Article  Google Scholar 

  29. Ramirez S, Li Y P, Jensen S B, Pedersen J, Gottwein J M, and Bukh J. 2013. Highly efficient infectious cell culture of three HCV genotype 2b strains and sensitivity to lead protease, NS5A, and polymerase inhibitors. Hepatology, DOI: 10.1002/hep.26660.

    Google Scholar 

  30. Ray U, and Das S. 2011. Interplay between NS3 protease and human La protein regulates translation-replication switch of Hepatitis C virus. Sci Rep, 1: 1.

    PubMed Central  PubMed  Article  Google Scholar 

  31. Scheel T K, Gottwein J M, Jensen T B, Prentoe J C, Hoegh A M, Alter H J, Eugen-Olsen J, and Bukh J. 2008. 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, 105: 997–1002.

    CAS  PubMed Central  PubMed  Article  Google Scholar 

  32. Scheel T K, Gottwein J M, Carlsen T H, Li Y P, Jensen T B, Spengler U, Weis N, and Bukh J. 2011. Efficient culture adaptation of hepatitis C virus recombinants with genotype-specific core-NS2 by using previously identified mutations. J Virol, 85: 2891–2906.

    CAS  PubMed Central  PubMed  Article  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, and Bressanelli S. 2011. 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, 85: 2565–2581.

    CAS  PubMed Central  PubMed  Article  Google Scholar 

  34. Scrima N, Caillet-Saguy C, Ventura M, Harrus D, Astier-Gin T, and Bressanelli S. 2012. Two crucial early steps in RNA synthesis by the hepatitis C virus polymerase involve a dual role of residue 405. J Virol, 86: 7107–7117.

    CAS  PubMed Central  PubMed  Article  Google Scholar 

  35. Shimotohno K. 2000. Hepatitis C virus and its pathogenesis. Semin Cancer Biol, 10: 233–240.

    CAS  PubMed  Article  Google Scholar 

  36. Simister P, Schmitt M, Geitmann M, Wicht O, Danielson U H, Klein R, Bressanelli S, and Lohmann V. 2009. Structural and functional analysis of hepatitis C virus strain JFH1 polymerase. J Virol, 83: 11926–11939.

    CAS  PubMed Central  PubMed  Article  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, and Liang T J. 2005. Production of infectious hepatitis C virus in tissue culture from a cloned viral genome. Nat Med, 11: 791–796.

    CAS  PubMed Central  PubMed  Article  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, and Chen Z. 1998. 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, 7: 837–847.

    CAS  PubMed  Article  Google Scholar 

  39. Yanagi M, Purcell R H, Emerson S U, and Bukh J. 1999. Hepatitis C virus: an infectious molecular clone of a second major genotype (2a) and lack of viability of intertypic 1a and 2a chimeras. Virology, 262: 250–263.

    CAS  PubMed  Article  Google Scholar 

  40. Yi M, Villanueva R A, Thomas D L, Wakita T, and Lemon S M. 2006. Production of infectious genotype 1a hepatitis C virus (Hutchinson strain) in cultured human hepatoma cells. Proc Natl Acad Sci USA, 103: 2310–2315.

    CAS  PubMed  Article  Google Scholar 

  41. Zhong J, Gastaminza P, Cheng G, Kapadia S, Kato T, Burton D R, Wieland S F, Uprichard S L, Wakita T, and Chisari F V. 2005. Robust hepatitis C virus infection in vitro. Proc Natl Acad Sci U S A, 102: 9294–9299.

    CAS  PubMed Central  PubMed  Article  Google Scholar 

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Correspondence to Xinwen Chen.

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Cao, H., Zhu, W., Han, Q. et al. Construction of a chimeric hepatitis C virus replicon based on a strain isolated from a chronic hepatitis C patient. Virol. Sin. 29, 61–70 (2014). https://doi.org/10.1007/s12250-014-3408-z

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Keywords

  • hepatitis C virus
  • subgenomic replicon
  • mutation