Hepatitis B virus (HBV) covalently closed circular (CCC) DNA is the source of HBV transcripts and persistence in chronically infected patients. The novel aspect of this study was to determine the effect of RNA interference (RNAi) on HBV CCC DNA when administered prior to establishment of HBV replication or during chronic HBV infection. HBV replication was initiated in HepG2 cells by transduction with HBV baculovirus. Subculture of HBV-expressing HepG2 cells at 10 days post-transduction generates a system in which HBV replication is ongoing and HBV is expressed largely from CCC DNA, thus simulating chronic HBV infection. HepG2 cells were transduced with short hairpin RNA (shRNA)-expressing baculovirus prior to initiation of HBV replication or during chronic HBV replication, and the levels of HBV RNA, HBV surface antigens (HBsAg) and replicative intermediates (RI), extracellular (EC) and CCC DNA species were measured. HBsAg, HBV RNA and DNA levels were markedly reduced until day 8 whether cells were transduced with shRNA prior to or during a chronic infection; however, the CCC DNA species were only affected when shRNA was administered prior to initiation of infection. We conclude that RNAi may have a therapeutic value for controlling HBV replication at the level of RI and EC DNA and for reducing establishment of CCC DNA during HBV infection. Our data support previous findings demonstrating the stability of HBV CCC DNA following antiviral therapy. This study also reports the development of a novel HBV baculovirus subculture system that can be used to evaluate antiviral effects on chronic HBV replication.


Article metrics loading...

Loading full text...

Full text loading...



  1. Abdelhamed, A. M., Kelley, C. M., Miller, T. G., Furman, P. A. & Isom, H. C.(2002). Rebound of hepatitis B virus replication in HepG2 cells after cessation of antiviral treatment. J Virol 76, 8148–8160.[CrossRef] [Google Scholar]
  2. Abdelhamed, A. M., Kelley, C. M., Miller, T. G., Furman, P. A., Cable, E. E. & Isom, H. C.(2003). Comparison of anti-hepatitis B virus activities of lamivudine and clevudine by a quantitative assay. Antimicrob Agents Chemother 47, 324–336.[CrossRef] [Google Scholar]
  3. Abe, T., Takahashi, H., Hamazaki, H., Miyano-Kurosaki, N., Matsuura, Y. & Takaku, H.(2003). Baculovirus induces an innate immune response and confers protection from lethal influenza virus infection in mice. J Immunol 171, 1133–1139.[CrossRef] [Google Scholar]
  4. Beasley, R. P.(1988). Hepatitis B virus. The major etiology of hepatocellular carcinoma. Cancer 61, 1942–1956.[CrossRef] [Google Scholar]
  5. Boyce, F. M. & Bucher, N. L.(1996). Baculovirus-mediated gene transfer into mammalian cells. Proc Natl Acad Sci U S A 93, 2348–2352.[CrossRef] [Google Scholar]
  6. Chen, C. C., Ko, T. M., Ma, H. I., Wu, H. L., Xiao, X., Li, J., Chang, C. M., Wu, P. Y., Chen, C. H. & other authors(2007). Long-term inhibition of hepatitis B virus in transgenic mice by double-stranded adeno-associated virus 8-delivered short hairpin RNA. Gene Ther 14, 11–19.[CrossRef] [Google Scholar]
  7. Cheng, T. L., Chang, W. W., Su, I. J., Lai, M. D., Huang, W., Lei, H. Y. & Chang, W. T.(2005). Therapeutic inhibition of hepatitis B virus surface antigen expression by RNA interference. Biochem Biophys Res Commun 336, 820–830.[CrossRef] [Google Scholar]
  8. Chomczynski, P. & Sacchi, N.(1987). Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem 162, 156–159. [Google Scholar]
  9. 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]
  10. 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]
  11. Fire, A., Xu, S., Montgomery, M. K., Kostas, S. A., Driver, S. E. & Mello, C. C.(1998). Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. Nature 391, 806–811.[CrossRef] [Google Scholar]
  12. Ganem, D. & Prince, A. M.(2004). Hepatitis B virus infection – natural history and clinical consequences. N Engl J Med 350, 1118–1129.[CrossRef] [Google Scholar]
  13. Ganem, D. & Varmus, H. E.(1987). The molecular biology of the hepatitis B viruses. Annu Rev Biochem 56, 651–693.[CrossRef] [Google Scholar]
  14. Hammond, S. M., Bernstein, E., Beach, D. & Hannon, G. J.(2000). An RNA-directed nuclease mediates post-transcriptional gene silencing in Drosophila cells. Nature 404, 293–296.[CrossRef] [Google Scholar]
  15. Hammond, S. M., Boettcher, S., Caudy, A. A., Kobayashi, R. & Hannon, G. J.(2001). Argonaute2, a link between genetic and biochemical analyses of RNAi. Science 293, 1146–1150.[CrossRef] [Google Scholar]
  16. Han, S. H., Ofman, J., Holt, C., King, K., Kunder, G., Chen, P., Dawson, S., Goldstein, L., Yersiz, H. & other authors(2000). An efficacy and cost-effectiveness analysis of combination hepatitis B immune globulin and lamivudine to prevent recurrent hepatitis B after orthotopic liver transplantation compared with hepatitis B immune globulin monotherapy. Liver Transpl 6, 741–748.[CrossRef] [Google Scholar]
  17. Heipertz, R. A., Jr, Miller, T. G., Kelley, C. M., Delaney, W. E., IV, Locarnini, S. A. & Isom, H. C.(2007). In vitro study of the effects of precore and lamivudine-resistant mutations on hepatitis B virus replication. J Virol 81, 3068–3076.[CrossRef] [Google Scholar]
  18. Hofmann, C., Sandig, V., Jennings, G., Rudolph, M., Schlag, P. & Strauss, M.(1995). Efficient gene transfer into human hepatocytes by baculovirus vectors. Proc Natl Acad Sci U S A 92, 10099–10103.[CrossRef] [Google Scholar]
  19. Jia, F., Zhang, Y. Z. & Liu, C. M.(2006). A retrovirus-based system to stably silence hepatitis B virus genes by RNA interference. Biotechnol Lett 28, 1679–1685.[CrossRef] [Google Scholar]
  20. Kane, M.(1995). Global programme for control of hepatitis B infection. Vaccine 13 (Suppl 1), S47–S49.[CrossRef] [Google Scholar]
  21. Kim, C. H., Yoon, J. S., Sohn, H. J., Kim, C. K., Paik, S. Y., Hong, Y. K. & Kim, T. G.(2007). Direct vaccination with pseudotype baculovirus expressing murine telomerase induces anti-tumor immunity comparable with RNA-electroporated dendritic cells in a murine glioma model. Cancer Lett 250, 276–283.[CrossRef] [Google Scholar]
  22. Knowles, B. B., Howe, C. C. & Aden, D. P.(1980). Human hepatocellular carcinoma cell lines secrete the major plasma proteins and hepatitis B surface antigen. Science 209, 497–499.[CrossRef] [Google Scholar]
  23. Kost, T. A. & Condreay, J. P.(2002). Recombinant baculoviruses as mammalian cell gene-delivery vectors. Trends Biotechnol 20, 173–180.[CrossRef] [Google Scholar]
  24. Kost, T. A., Condreay, J. P. & Jarvis, D. L.(2005). Baculovirus as versatile vectors for protein expression in insect and mammalian cells. Nat Biotechnol 23, 567–575.[CrossRef] [Google Scholar]
  25. Lau, G. K., Tsiang, M., Hou, J., Yuen, S., Carman, W. F., Zhang, L., Gibbs, C. S. & Lam, S.(2000). Combination therapy with lamivudine and famciclovir for chronic hepatitis B-infected Chinese patients: a viral dynamics study. Hepatology 32, 394–399.[CrossRef] [Google Scholar]
  26. Lau, N. C., Lim, L. P., Weinstein, E. G. & Bartel, D. P.(2001). An abundant class of tiny RNAs with probable regulatory roles in Caenorhabditis elegans. Science 294, 858–862.[CrossRef] [Google Scholar]
  27. Li, G. Q., Gu, H. X., Li, D. & Xu, W. Z.(2007). Inhibition of Hepatitis B virus cccDNA replication by siRNA. Biochem Biophys Res Commun 355, 404–408.[CrossRef] [Google Scholar]
  28. Margolis, H. S.(1998). Hepatitis B virus infection. Bull World Health Organ 76 (Suppl. 2), 152–153. [Google Scholar]
  29. Markowitz, J. S., Martin, P., Conrad, A. J., Markmann, J. F., Seu, P., Yersiz, H., Goss, J. A., Schmidt, P., Pakrasi, A. & other authors(1998). Prophylaxis against hepatitis B recurrence following liver transplantation using combination lamivudine and hepatitis B immune globulin. Hepatology 28, 585–589.[CrossRef] [Google Scholar]
  30. McCaffrey, A. P., Nakai, H., Pandey, K., Huang, Z., Salazar, F. H., Xu, H., Wieland, S. F., Marion, P. L. & Kay, M. A.(2003). Inhibition of hepatitis B virus in mice by RNA interference. Nat Biotechnol 21, 639–644.[CrossRef] [Google Scholar]
  31. Melegari, M., Scaglioni, P. P. & Wands, J. R.(1998). Hepatitis B virus mutants associated with 3TC and famciclovir administration are replication defective. Hepatology 27, 628–633.[CrossRef] [Google Scholar]
  32. Moore, M. D., McGarvey, M. J., Russell, R. A., Cullen, B. R. & McClure, M. O.(2005). Stable inhibition of hepatitis B virus proteins by small interfering RNA expressed from viral vectors. J Gene Med 7, 918–925.[CrossRef] [Google Scholar]
  33. Nicholson, L. J., Philippe, M., Paine, A. J., Mann, D. A. & Dolphin, C. T.(2005). RNA interference mediated in human primary cells via recombinant baculoviral vectors. Mol Ther 11, 638–644.[CrossRef] [Google Scholar]
  34. O'Grady, J. G., Smith, H. M., Davies, S. E., Daniels, H. M., Donaldson, P. T., Tan, K. C., Portmann, B., Alexander, G. J. & Williams, R.(1992). Hepatitis B virus reinfection after orthotopic liver transplantation. Serological and clinical implications. J Hepatol 14, 104–111.[CrossRef] [Google Scholar]
  35. O'Reilly, D. R.(1997). Use of baculovirus expression vectors. Methods Mol Biol 62, 235–246. [Google Scholar]
  36. Papatheodoridis, G. V., Dimou, E. & Papadimitropoulos, V.(2002). Nucleoside analogues for chronic hepatitis B: antiviral efficacy and viral resistance. Am J Gastroenterol 97, 1618–1628.[CrossRef] [Google Scholar]
  37. Sambrook, J., Fritsch, E. F. & Maniatis, T.(1989). Analysis and cloning of eukaryotic genomic DNA. In Molecular Cloning, 2nd edn, pp. 9.1–9.62. Edited by C. Nolan. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.
  38. Shlomai, A. & Shaul, Y.(2003). Inhibition of hepatitis B virus expression and replication by RNA interference. Hepatology 37, 764–770.[CrossRef] [Google Scholar]
  39. Strauss, R., Huser, A., Ni, S., Tuve, S., Kiviat, N., Sow, P. S., Hofmann, C. & Lieber, A.(2007). Baculovirus-based vaccination vectors allow for efficient induction of immune responses against Plasmodium falciparum circumsporozoite protein. Mol Ther 15, 193–202.[CrossRef] [Google Scholar]
  40. Todo, S., Demetris, A. J., Van Thiel, D., Teperman, L., Fung, J. J. & Starzl, T. E.(1991). Orthotopic liver transplantation for patients with hepatitis B virus-related liver disease. Hepatology 13, 619–626. [Google Scholar]
  41. Uprichard, S. L., Boyd, B., Althage, A. & Chisari, F. V.(2005). Clearance of hepatitis B virus from the liver of transgenic mice by short hairpin RNAs. Proc Natl Acad Sci U S A 102, 773–778.[CrossRef] [Google Scholar]
  42. Wang, C. Y., Li, F., Yang, Y., Guo, H. Y., Wu, C. X. & Wang, S.(2006). Recombinant baculovirus containing the diphtheria toxin A gene for malignant glioma therapy. Cancer Res 66, 5798–5806.[CrossRef] [Google Scholar]
  43. Wu, H. L., Huang, L. R., Huang, C. C., Lai, H. L., Liu, C. J., Huang, Y. T., Hsu, Y. W., Lu, C. Y., Chen, D. S. & Chen, P. J.(2005). RNA interference-mediated control of hepatitis B virus and emergence of resistant mutant. Gastroenterology 128, 708–716.[CrossRef] [Google Scholar]
  44. Xin, X. M., Li, G. Q., Jin, Y. Y., Zhuang, M. & Li, D.(2008). Combination of small interfering RNAs mediates greater suppression on hepatitis B virus cccDNA in HepG2.2.15 cells. World J Gastroenterol 14, 3849–3854.[CrossRef] [Google Scholar]
  45. Zhang, Y. Y. & Summers, J.(2000). Low dynamic state of viral competition in a chronic avian hepadnavirus infection. J Virol 74, 5257–5265.[CrossRef] [Google Scholar]
  46. Zhang, X. N., Xiong, W., Wang, J. D., Hu, Y. W., Xiang, L. & Yuan, Z. H.(2004). siRNA-mediated inhibition of HBV replication and expression. World J Gastroenterol 10, 2967–2971. [Google Scholar]
  47. Zheng, S., Chen, Y., Liang, T., Lu, A., Wang, W., Shen, Y. & Zhang, M.(2006). Prevention of hepatitis B recurrence after liver transplantation using lamivudine or lamivudine combined with hepatitis B immunoglobulin prophylaxis. Liver Transpl 12, 253–258.[CrossRef] [Google Scholar]

Data & Media loading...

This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error