1887

Abstract

A -packaging system for hepatitis C virus (HCV) replicons lacking envelope glycoproteins was developed. The replicons were efficiently encapsidated into infectious particles after expression of homologous HCV envelope proteins under the control of an adenoviral vector. Interestingly, expression of core or core, p7 and NS2 with envelope proteins did not enhance encapsidation. Expression of heterologous envelope proteins, in the presence or absence of heterologous core, p7 and NS2, did not rescue single-round infectious particle production. To increase the titre of homologous, single-round infectious particles in our system, successive cycles of encapsidation and infection were performed. Four cycles resulted in a 100-fold increase in the yield of particles. Sequence analysis revealed a total of 16 potential adaptive mutations in two independent experiments. Except for a core mutation in one experiment, all the mutations were located in non-structural regions mainly in NS5A (four in domain III and two near the junction with the NS5B gene). Reverse genetics studies suggested that D2437A and S2443T adaptive mutations, which are located at the NS5A-B cleavage site did not affect viral replication, but enhanced the single-round infectious particles assembly only -encapsidation model. In conclusion, our encapsidation system enables the production of HCV single-round infectious particles. This system is adaptable and can positively select variants. The adapted variants promote encapsidation and should constitute a valuable tool in the development of replicon-based HCV vaccines.

Funding
This study was supported by the:
  • Conseil Régional de Picardie (Award AAP08-12)
  • Université de Picardie–Jules Verne
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2013-05-01
2022-01-27
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References

  1. Adair R., Patel A. H., Corless L., Griffin S., Rowlands D. J., McCormick C. J. 2009; Expression of hepatitis C virus (HCV) structural proteins in trans facilitates encapsidation and transmission of HCV subgenomic RNA. J Gen Virol 90:833–842 [View Article][PubMed]
    [Google Scholar]
  2. Appel N., Herian U., Bartenschlager R. 2005; Efficient rescue of hepatitis C virus RNA replication by trans-complementation with nonstructural protein 5A. J Virol 79:896–909 [View Article][PubMed]
    [Google Scholar]
  3. Appel N., Zayas M., Miller S., Krijnse-Locker J., Schaller T., Friebe P., Kallis S., Engel U., Bartenschlager R. 2008; Essential role of domain III of nonstructural protein 5A for hepatitis C virus infectious particle assembly. PLoS Pathog 4:e1000035 [View Article][PubMed]
    [Google Scholar]
  4. Brohm C., Steinmann E., Friesland M., Lorenz I. C., Patel A., Penin F., Bartenschlager R., Pietschmann T. 2009; Characterization of determinants important for hepatitis C virus p7 function in morphogenesis by using trans-complementation. J Virol 83:11682–11693 [View Article][PubMed]
    [Google Scholar]
  5. Choo Q. L., Richman K. H., Han J. H., Berger K., Lee C., Dong C., Gallegos C., Coit D., Medina-Selby R., Barr P. J. 1991; Genetic organization and diversity of the hepatitis C virus. Proc Natl Acad Sci U S A 88:2451–2455 [View Article][PubMed]
    [Google Scholar]
  6. Delgrange D., Pillez A., Castelain S., Cocquerel L., Rouillé Y., Dubuisson J., Wakita T., Duverlie G., Wychowski C. 2007; Robust production of infectious viral particles in Huh-7 cells by introducing mutations in hepatitis C virus structural proteins. J Gen Virol 88:2495–2503 [View Article][PubMed]
    [Google Scholar]
  7. Dimitrova M., Imbert I., Kieny M. P., Schuster C. 2003; Protein-protein interactions between hepatitis C virus nonstructural proteins. J Virol 77:5401–5414 [View Article][PubMed]
    [Google Scholar]
  8. Eyre N. S., Phillips R. J., Bowden S., Yip E., Dewar B., Locarnini S. A., Beard M. R. 2009; Hepatitis B virus and hepatitis C virus interaction in Huh-7 cells. J Hepatol 51:446–457 [View Article][PubMed]
    [Google Scholar]
  9. Gottwein J. M., Bukh J. 2008; Cutting the gordian knot-development and biological relevance of hepatitis C virus cell culture systems. Adv Virus Res 71:51–133 [View Article][PubMed]
    [Google Scholar]
  10. Gottwein J. M., Scheel T. K., Jensen T. B., Lademann J. B., Prentoe J. C., Knudsen M. L., Hoegh A. M., 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 [View Article][PubMed]
    [Google Scholar]
  11. Han Q., Xu C., Wu C., Zhu W., Yang R., 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 [View Article][PubMed]
    [Google Scholar]
  12. Helle F., Vieyres G., Elkrief L., Popescu C. I., Wychowski C., Descamps V., Castelain S., Roingeard P., Duverlie G., Dubuisson J. 2010; Role of N-linked glycans in the functions of hepatitis C virus envelope proteins incorporated into infectious virions. J Virol 84:11905–11915 [View Article][PubMed]
    [Google Scholar]
  13. Ishii K., Murakami K., Hmwe S. S., Zhang B., Li J., Shirakura M., Morikawa K., Suzuki R., Miyamura T. & other authors ( 2008; Trans-encapsidation of hepatitis C virus subgenomic replicon RNA with viral structure proteins. Biochem Biophys Res Commun 371:446–450 [View Article][PubMed]
    [Google Scholar]
  14. Jiang J., Luo G. 2012; Cell culture-adaptive mutations promote viral protein-protein interactions and morphogenesis of infectious hepatitis C virus. J Virol 86:8987–8997 [View Article][PubMed]
    [Google Scholar]
  15. Jirasko V., Montserret R., Appel N., Janvier A., Eustachi L., Brohm C., Steinmann E., Pietschmann T., Penin F., Bartenschlager R. 2008; Structural and functional characterization of nonstructural protein 2 for its role in hepatitis C virus assembly. J Biol Chem 283:28546–28562 [View Article][PubMed]
    [Google Scholar]
  16. Jones D. M., Patel A. H., Targett-Adams P., McLauchlan J. 2009; The hepatitis C virus NS4B protein can trans-complement viral RNA replication and modulates production of infectious virus. J Virol 83:2163–2177 [View Article][PubMed]
    [Google Scholar]
  17. Kang J. I., Kim J. P., Wakita T., Ahn B. Y. 2009; Cell culture-adaptive mutations in the NS5B gene of hepatitis C virus with delayed replication and reduced cytotoxicity. Virus Res 144:107–116 [View Article][PubMed]
    [Google Scholar]
  18. Kato N., Hijikata M., Ootsuyama Y., Nakagawa M., Ohkoshi S., Sugimura T., Shimotohno K. 1990; Molecular cloning of the human hepatitis C virus genome from Japanese patients with non-A, non-B hepatitis. Proc Natl Acad Sci U S A 87:9524–9528 [View Article][PubMed]
    [Google Scholar]
  19. Kato T., Matsumura T., Heller T., Saito S., Sapp R. K., Murthy K., Wakita T., Liang T. J. 2007; Production of infectious hepatitis C virus of various genotypes in cell cultures. J Virol 81:4405–4411 [View Article][PubMed]
    [Google Scholar]
  20. Kaul A., Woerz I., Meuleman P., Leroux-Roels G., Bartenschlager R. 2007; Cell culture adaptation of hepatitis C virus and in vivo viability of an adapted variant. J Virol 81:13168–13179 [View Article][PubMed]
    [Google Scholar]
  21. Lavillette D., Tarr A. W., Voisset C., Donot P., Bartosch B., Bain C., Patel A. H., Dubuisson J., Ball J. K., Cosset F. L. 2005; Characterization of host-range and cell entry properties of the major genotypes and subtypes of hepatitis C virus. Hepatology 41:265–274 [View Article][PubMed]
    [Google Scholar]
  22. Li R., Qin Y., He Y., Tao W., Zhang N., Tsai C., Zhou P., Zhong J. 2011; Production of hepatitis C virus lacking the envelope-encoding genes for single-cycle infection by providing homologous envelope proteins or vesicular stomatitis virus glycoproteins in trans. J Virol 85:2138–2147 [View Article][PubMed]
    [Google Scholar]
  23. Masaki T., Suzuki R., Murakami K., Aizaki H., Ishii K., Murayama A., Date T., Matsuura Y., Miyamura T. & other authors ( 2008; 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 [View Article][PubMed]
    [Google Scholar]
  24. Miyanari Y., Atsuzawa K., Usuda N., Watashi K., Hishiki T., Zayas M., Bartenschlager R., Wakita T., Hijikata M., Shimotohno K. 2007; The lipid droplet is an important organelle for hepatitis C virus production. Nat Cell Biol 9:1089–1097 [View Article][PubMed]
    [Google Scholar]
  25. Morel V., Fournier C., François C., Brochot E., Helle F., Duverlie G., Castelain S. 2011; Genetic recombination of the hepatitis C virus: clinical implications. J Viral Hepat 18:77–83 [View Article][PubMed]
    [Google Scholar]
  26. Pacini L., Graziani R., Bartholomew L., De Francesco R., Paonessa G. 2009; Naturally occurring hepatitis C virus subgenomic deletion mutants replicate efficiently in Huh-7 cells and are trans-packaged in vitro to generate infectious defective particles. J Virol 83:9079–9093 [View Article][PubMed]
    [Google Scholar]
  27. Pietschmann T., Kaul A., Koutsoudakis G., Shavinskaya A., Kallis S., Steinmann E., Abid K., Negro F., Dreux M. & other authors ( 2006; Construction and characterization of infectious intragenotypic and intergenotypic hepatitis C virus chimeras. Proc Natl Acad Sci U S A 103:7408–7413 [View Article][PubMed]
    [Google Scholar]
  28. Russell R. S., Meunier J. C., Takikawa S., Faulk K., Engle R. E., Bukh J., Purcell R. H., Emerson S. U. 2008; Advantages of a single-cycle production assay to study cell culture-adaptive mutations of hepatitis C virus. Proc Natl Acad Sci U S A 105:4370–4375 [View Article][PubMed]
    [Google Scholar]
  29. Scheel T. K., Gottwein J. M., Jensen T. B., Prentoe J. C., Hoegh A. M., Alter H. J., Eugen-Olsen J., 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 [View Article][PubMed]
    [Google Scholar]
  30. Steinmann E., Brohm C., Kallis S., Bartenschlager R., Pietschmann T. 2008; Efficient trans-encapsidation of hepatitis C virus RNAs into infectious virus-like particles. J Virol 82:7034–7046 [View Article][PubMed]
    [Google Scholar]
  31. Sugiyama K., Suzuki K., Nakazawa T., Funami K., Hishiki T., Ogawa K., Saito S., Shimotohno K. W., Suzuki T. & other authors ( 2009; Genetic analysis of hepatitis C virus with defective genome and its infectivity in vitro. J Virol 83:6922–6928 [View Article][PubMed]
    [Google Scholar]
  32. Takeda M., Ikeda M., Ariumi Y., Wakita T., Kato N. 2012; Development of hepatitis C virus production reporter-assay systems using two different hepatoma cell lines. J Gen Virol 93:1422–1431 [View Article][PubMed]
    [Google Scholar]
  33. Tang H., Grisé H. 2009; Cellular and molecular biology of HCV infection and hepatitis. Clin Sci (Lond) 117:49–65 [View Article][PubMed]
    [Google Scholar]
  34. Tellinghuisen T. L., Foss K. L., Treadaway J. 2008; Regulation of hepatitis C virion production via phosphorylation of the NS5A protein. PLoS Pathog 4:e1000032 [View Article][PubMed]
    [Google Scholar]
  35. Tong X., Malcolm B. A. 2006; Trans-complementation of HCV replication by non-structural protein 5A. Virus Res 115:122–130 [View Article][PubMed]
    [Google Scholar]
  36. Wakita T., Pietschmann T., Kato T., Date T., Miyamoto M., Zhao Z., Murthy K., Habermann A., Kräusslich H. G. & other authors ( 2005; Production of infectious hepatitis C virus in tissue culture from a cloned viral genome. Nat Med 11:791–796 [View Article][PubMed]
    [Google Scholar]
  37. Widjojoatmodjo M. N., van Gennip H. G., Bouma A., van Rijn P. A., Moormann R. J. 2000; Classical swine fever virus Erns deletion mutants: trans-complementation and potential use as nontransmissible, modified, live-attenuated marker vaccines. J Virol 74:2973–2980 [View Article][PubMed]
    [Google Scholar]
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