1887

Abstract

One of the major problems in gaining further insight into hepatitis B virus (HBV)/host-cell interactions is to improve the existing cellular models for the study of HBV replication. The first objective of this study was to improve the system based on transduction of HepG2 cells with a recombinant baculovirus to study HBV replication. A new HBV recombinant baculovirus, Bac-HBV-1.1, in which the synthesis of pre-genomic RNA is driven by a strong mammalian promoter, was generated. Transduction with this new recombinant baculovirus led to higher levels of HBV replication in HepG2 cells compared with levels obtained with previously described baculovirus vectors. The initiation of a complete HBV DNA replication cycle in Bac-HBV-1.1-transduced HepG2 cells was shown by the presence of HBV replicative intermediates, including covalently closed circular DNA (cccDNA). Only low levels of cccDNA were detected in the nucleus of infected cells. Data showed that cccDNA resulted from the recycling of newly synthesized nucleocapsids and was bound to acetylated histones in a chromatin-like structure. HBV particles released into the supernatant of transduced HepG2 cells were infectious in differentiated HepaRG cells. Several Bac-HBV-1.1 baculoviruses containing HBV strains carrying mutations conferring resistance to lamivudine and/or adefovir were constructed. Phenotypic analysis of these mutants confirmed the results obtained with the transfection procedures. In conclusion, an improved cell-culture system was established for the transduction of replication-competent HBV genomes. This will be useful for future studies of the fitness of HBV mutants.

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2008-08-01
2020-01-27
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References

  1. Allen, M. I., Deslauriers, M., Andrews, C. W., Tipples, G. A., Walters, K. A., Tyrrell, D. L., Brown, N. & Condreay, L. D. ( 1998; ). Identification and characterization of mutations in hepatitis B virus resistant to lamivudine. Lamivudine Clinical Investigation Group. Hepatology 27, 1670–1677.[CrossRef]
    [Google Scholar]
  2. Bock, C. T., Schwinn, S., Locarnini, S., Fyfe, J., Manns, M. P., Trautwein, C. & Zentgraf, H. ( 2001; ). Structural organization of the hepatitis B virus minichromosome. J Mol Biol 307, 183–196.[CrossRef]
    [Google Scholar]
  3. Brunelle, M. N., Jacquard, A. C., Pichoud, C., Durantel, D., Carrouee-Durantel, S., Villeneuve, J. P., Trepo, C. & Zoulim, F. ( 2005; ). Susceptibility to antivirals of a human HBV strain with mutations conferring resistance to both lamivudine and adefovir. Hepatology 41, 1391–1398.[CrossRef]
    [Google Scholar]
  4. Delaney, W. E., IV & Isom, H. C. ( 1998; ). Hepatitis B virus replication in human HepG2 cells mediated by hepatitis B virus recombinant baculovirus. Hepatology 28, 1134–1146.[CrossRef]
    [Google Scholar]
  5. Delaney, W. E., IV, Miller, T. G. & Isom, H. C. ( 1999; ). Use of the hepatitis B virus recombinant baculovirus-HepG2 system to study the effects of (−)-β-2′,3′-dideoxy-3′-thiacytidine on replication of hepatitis B virus and accumulation of covalently closed circular DNA. Antimicrob Agents Chemother 43, 2017–2026.
    [Google Scholar]
  6. Delaney, W. E., IV, Yang, H., Westland, C. E., Das, K., Arnold, E., Gibbs, C. S., Miller, M. D. & Xiong, S. ( 2003; ). The hepatitis B virus polymerase mutation rtV173L is selected during lamivudine therapy and enhances viral replication in vitro. J Virol 77, 11833–11841.[CrossRef]
    [Google Scholar]
  7. Durantel, D., Carrouee-Durantel, S., Werle-Lapostolle, B., Brunelle, M. N., Pichoud, C., Trepo, C. & Zoulim, F. ( 2004; ). A new strategy for studying in vitro the drug susceptibility of clinical isolates of human hepatitis B virus. Hepatology 40, 855–864.[CrossRef]
    [Google Scholar]
  8. Durantel, D., Brunelle, M. N., Gros, E., Carrouee-Durantel, S., Pichoud, C., Villet, S., Trepo, C. & Zoulim, F. ( 2005; ). Resistance of human hepatitis B virus to reverse transcriptase inhibitors: from genotypic to phenotypic testing. J Clin Virol 34 (Suppl. 1), S34–S43.[CrossRef]
    [Google Scholar]
  9. Ganem, D. ( 1991; ). Assembly of hepadnaviral virions and subviral particles. Curr Top Microbiol Immunol 168, 61–83.
    [Google Scholar]
  10. Gripon, P., Diot, C., Theze, N., Fourel, I., Loreal, O., Brechot, C. & Guguen-Guillouzo, C. ( 1988; ). Hepatitis B virus infection of adult human hepatocytes cultured in the presence of dimethyl sulfoxide. J Virol 62, 4136–4143.
    [Google Scholar]
  11. Gripon, P., Rumin, S., Urban, S., Le Seyec, J., Glaise, D., Cannie, I., Guyomard, C., Lucas, J., Trepo, C. & Guguen-Guillouzo, C. ( 2002; ). Infection of a human hepatoma cell line by hepatitis B virus. Proc Natl Acad Sci U S A 99, 15655–15660.[CrossRef]
    [Google Scholar]
  12. Karayiannis, P. ( 2003; ). Hepatitis B virus: old, new and future approaches to antiviral treatment. J Antimicrob Chemother 51, 761–785.[CrossRef]
    [Google Scholar]
  13. King, L. A. & Possee, R. D. ( 1992; ). The Baculovirus Expression System: a Laboratory Guide. London: Chapman & Hall.
  14. Lasken, R. S. & Egholm, M. ( 2003; ). Whole genome amplification: abundant supplies of DNA from precious samples or clinical specimens. Trends Biotechnol 21, 531–535.[CrossRef]
    [Google Scholar]
  15. Lau, D. T., Khokhar, M. F., Doo, E., Ghany, M. G., Herion, D., Park, Y., Kleiner, D. E., Schmid, P., Condreay, L. D. & other authors ( 2000; ). Long-term therapy of chronic hepatitis B with lamivudine. Hepatology 32, 828–834.[CrossRef]
    [Google Scholar]
  16. Marcellin, P., Chang, T. T., Lim, S. G., Tong, M. J., Sievert, W., Shiffman, M. L., Jeffers, L., Goodman, Z., Wulfsohn, M. S. & other authors ( 2003; ). Adefovir dipivoxil for the treatment of hepatitis B e antigen-positive chronic hepatitis B. N Engl J Med 348, 808–816.[CrossRef]
    [Google Scholar]
  17. Margeridon, S., Durantel, S., Chemin, I., Barraud, L., Zoulim, F., Trépo, C. & Kay, A. ( 2006; ). Rolling circle amplification technology for the study of HBV variants. J Clin Virol 36, S51–S52.
    [Google Scholar]
  18. Newbold, J. E., Xin, H., Tencza, M., Sherman, G., Dean, J., Bowden, S. & Locarnini, S. ( 1995; ). The covalently closed duplex form of the hepadnavirus genome exists in situ as a heterogeneous population of viral minichromosomes. J Virol 69, 3350–3357.
    [Google Scholar]
  19. Peters, M. G., Hann Hw, H., Martin, P., Heathcote, E. J., Buggisch, P., Rubin, R., Bourliere, M., Kowdley, K., Trepo, C. & other authors ( 2004; ). Adefovir dipivoxil alone or in combination with lamivudine in patients with lamivudine-resistant chronic hepatitis B. Gastroenterology 126, 91–101.[CrossRef]
    [Google Scholar]
  20. Pollicino, T., Belloni, L., Raffa, G., Pediconi, N., Squadrito, G., Raimondo, G. & Levrero, M. ( 2006; ). Hepatitis B virus replication is regulated by the acetylation status of hepatitis B virus cccDNA-bound H3 and H4 histones. Gastroenterology 130, 823–837.[CrossRef]
    [Google Scholar]
  21. Qi, X., Xiong, S., Yang, H., Miller, M. & Delaney, W. E., 4th ( 2007; ). In vitro susceptibility of adefovir-associated hepatitis B virus polymerase mutations to other antiviral agents. Antivir Ther 12, 355–362.
    [Google Scholar]
  22. Ren, S. & Nassal, M. ( 2001; ). Hepatitis B virus (HBV) virion and covalently closed circular DNA formation in primary tupaia hepatocytes and human hepatoma cell lines upon HBV genome transduction with replication-defective adenovirus vectors. J Virol 75, 1104–1116.[CrossRef]
    [Google Scholar]
  23. Seeger, C. & Mason, W. S. ( 2000; ). Hepatitis B virus biology. Microbiol Mol Biol Rev 64, 51–68.[CrossRef]
    [Google Scholar]
  24. Seigneres, B., Pichoud, C., Martin, P., Furman, P., Trepo, C. & Zoulim, F. ( 2002; ). Inhibitory activity of dioxolane purine analogs on wild-type and lamivudine-resistant mutants of hepadnaviruses. Hepatology 36, 710–722.
    [Google Scholar]
  25. Sells, M. A., Chen, M. L. & Acs, G. ( 1987; ). Production of hepatitis B virus particles in Hep G2 cells transfected with cloned hepatitis B virus DNA. Proc Natl Acad Sci U S A 84, 1005–1009.[CrossRef]
    [Google Scholar]
  26. Summers, J., Smith, P. M. & Horwich, A. L. ( 1990; ). Hepadnavirus envelope proteins regulate covalently closed circular DNA amplification. J Virol 64, 2819–2824.
    [Google Scholar]
  27. Sun, D. & Nassal, M. ( 2006; ). Stable HepG2- and Huh7-based human hepatoma cell lines for efficient regulated expression of infectious hepatitis B virus. J Hepatol 45, 636–645.[CrossRef]
    [Google Scholar]
  28. Tuttleman, J. S., Pourcel, C. & Summers, J. ( 1986; ). Formation of the pool of covalently closed circular viral DNA in hepadnavirus-infected cells. Cell 47, 451–460.[CrossRef]
    [Google Scholar]
  29. Villeneuve, J. P., Durantel, D., Durantel, S., Westland, C., Xiong, S., Brosgart, C. L., Gibbs, C. S., Parvaz, P., Werle, B. & other authors ( 2003; ). Selection of a hepatitis B virus strain resistant to adefovir in a liver transplantation patient. J Hepatol 39, 1085–1089.[CrossRef]
    [Google Scholar]
  30. Villet, S., Pichoud, C., Villeneuve, J. P., Trepo, C. & Zoulim, F. ( 2006; ). Selection of a multiple drug-resistant hepatitis B virus strain in a liver-transplanted patient. Gastroenterology 131, 1253–1261.[CrossRef]
    [Google Scholar]
  31. Villet, S., Ollivet, A., Pichoud, C., Barraud, L., Villeneuve, J. P., Trepo, C. & Zoulim, F. ( 2007; ). Stepwise process for the development of entecavir resistance in a chronic hepatitis B virus infected patient. J Hepatol 46, 531–538.[CrossRef]
    [Google Scholar]
  32. Werle-Lapostolle, B., Bowden, S., Locarnini, S., Wursthorn, K., Petersen, J., Lau, G., Trepo, C., Marcellin, P., Goodman, Z. & other authors ( 2004; ). Persistence of cccDNA during the natural history of chronic hepatitis B and decline during adefovir dipivoxil therapy. Gastroenterology 126, 1750–1758.[CrossRef]
    [Google Scholar]
  33. Zhu, Y., Yamamoto, T., Cullen, J., Saputelli, J., Aldrich, C. E., Miller, D. S., Litwin, S., Furman, P. A., Jilbert, A. R. & Mason, W. S. ( 2001; ). Kinetics of hepadnavirus loss from the liver during inhibition of viral DNA synthesis. J Virol 75, 311–322.[CrossRef]
    [Google Scholar]
  34. Zoulim, F. ( 2004; ). Mechanism of viral persistence and resistance to nucleoside and nucleotide analogs in chronic hepatitis B virus infection. Antiviral Res 64, 1–15.[CrossRef]
    [Google Scholar]
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