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

  • Research Article
  • Published:
Virologica Sinica

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

  • 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.

    Article  CAS  PubMed  Google Scholar 

  • 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.

    Article  CAS  PubMed  Google Scholar 

  • 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.

    Article  CAS  PubMed  Google Scholar 

  • 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.

    Article  PubMed Central  PubMed  Google Scholar 

  • 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.

    Article  CAS  PubMed  Google Scholar 

  • 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 

  • 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.

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • 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.

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • 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 

  • 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.

    Article  CAS  PubMed  Google Scholar 

  • 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.

    Article  CAS  PubMed  Google Scholar 

  • 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 

  • 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.

    Article  CAS  PubMed  Google Scholar 

  • 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.

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • 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.

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • 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.

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  • 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.

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  • 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.

    Article  CAS  PubMed  Google Scholar 

  • 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.

    Article  CAS  PubMed  Google Scholar 

  • 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.

    Article  PubMed  Google Scholar 

  • 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.

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • 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.

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • 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.

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • 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.

    Article  CAS  PubMed  Google Scholar 

  • 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.

    Article  PubMed Central  PubMed  Google Scholar 

  • 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.

    Article  CAS  PubMed  Google Scholar 

  • 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 

  • 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.

    Article  PubMed Central  PubMed  Google Scholar 

  • 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.

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • 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.

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • 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.

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • 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.

    Article  CAS  PubMed Central  PubMed  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  • 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.

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • 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.

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • 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.

    Article  CAS  PubMed  Google Scholar 

  • 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.

    Article  CAS  PubMed  Google Scholar 

  • 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.

    Article  CAS  PubMed  Google Scholar 

  • 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.

    Article  CAS  PubMed Central  PubMed  Google Scholar 

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Authors and Affiliations

  1. State Key Laboratory of Virology, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan, 430071, China

    Huang Cao, Wandi Zhu, Qingxia Han, Rongjuan Pei & Xinwen Chen

  2. University of the Chinese Academy of Sciences, Beijing, 100049, China

    Huang Cao & Wandi Zhu

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  1. Huang Cao
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  2. Wandi Zhu
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  4. Rongjuan Pei
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  5. Xinwen Chen
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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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  • DOI: https://doi.org/10.1007/s12250-014-3408-z

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