A Southern Blot Assay for Detection of Hepatitis B Virus Covalently Closed Circular DNA from Cell Cultures

  • Dawei Cai
  • Hui Nie
  • Ran Yan
  • Ju-Tao Guo
  • Timothy M. Block
  • Haitao Guo
Part of the Methods in Molecular Biology book series (MIMB, volume 1030)


Chronic hepatitis B remains a substantial public health burden affecting approximately 350 million people worldwide, causing cirrhosis and liver cancer, and about 1 million people die each year from hepatitis B and its complications. Hepatitis B is caused by hepatitis B virus (HBV) infection. As an essential component of the viral life cycle, HBV covalently closed circular DNA (cccDNA) is synthesized and maintained at low copy numbers in the nucleus of infected hepatocytes, and serves as the transcription template for all viral RNAs. Therefore, cccDNA is responsible for the establishment of viral infection and persistence. The presence and longevity of cccDNA may also explain the limitations of current antiviral therapy for hepatitis B. Thus, understanding the mechanisms underlying cccDNA formation and regulation is critical in understanding the HBV pathogenesis and finding a cure for hepatitis B. Here we describe a protocol for HBV cccDNA extraction and detection in detail. The procedure includes two major steps: (1) HBV cccDNA extraction by Hirt protein-free DNA extraction method and (2) HBV cccDNA detection by Southern blot analysis. The method is straightforward and reliable for cccDNA assay with cell culture samples, and it is useful for both HBV molecular biology and antiviral research.

Key words

HBV cccDNA Hirt extraction Southern blot 



We thank Dr. Andrea Cuconati (Institute for Hepatitis and Virus Research) for critical reading of the manuscript. This work was supported by the NIH grants (R01AI094474, R21AI088424) and Hepatitis B Foundation through an appropriation of the Commonwealth of Pennsylvania. Haitao Guo is the Bruce Witte fellow of the Hepatitis B Foundation.


  1. 1.
    Lok AS, McMahon BJ (2009) Chronic hepatitis B: update 2009. Hepatology 50:661–662PubMedCrossRefGoogle Scholar
  2. 2.
    Seeger C, Mason WS (2000) Hepatitis B virus biology. Microbiol Mol Biol Rev 64:51–68PubMedCrossRefGoogle Scholar
  3. 3.
    Block TM, Guo H, Guo JT (2007) Molecular virology of hepatitis B virus for clinicians. Clin Liver Dis 11(4):685–706, viiPubMedCrossRefGoogle Scholar
  4. 4.
    Dejean A, Lugassy C, Zafrani S et al (1984) Detection of hepatitis B virus DNA in pancreas, kidney and skin of two human carriers of the virus. J Gen Virol 65:651–655PubMedCrossRefGoogle Scholar
  5. 5.
    Naumova AK, Favorov MO, Keteladze ES et al (1985) Nucleotide sequences in human chromosomal DNA from nonhepatic tissues homologous to the hepatitis B virus genome. Gene 35:19–25PubMedCrossRefGoogle Scholar
  6. 6.
    Gripon P, Rumin S, Urban S et al (2002) Infection of a human hepatoma cell line by hepatitis B virus. Proc Natl Acad Sci U S A 99:15655–15660PubMedCrossRefGoogle Scholar
  7. 7.
    Schulze-Bergkamen H, Untergasser A, Dax A et al (2003) Primary human hepatocytes–a valuable tool for investigation of apoptosis and hepatitis B virus infection. J Hepatol 38: 736–744PubMedCrossRefGoogle Scholar
  8. 8.
    Huang HC, Chen CC, Chang WC et al (2012) Entry of hepatitis B virus into immortalized human primary hepatocytes by clathrin-dependent endocytosis. J Virol 86:9443–9453PubMedCrossRefGoogle Scholar
  9. 9.
    Guo H, Mao R, Block TM, Guo JT (2010) Production and function of the cytoplasmic deproteinized relaxed circular DNA of hepadnaviruses. J Virol 84:387–396PubMedCrossRefGoogle Scholar
  10. 10.
    Rabe B, Vlachou A, Pante N et al (2003) Nuclear import of hepatitis B virus capsids and release of the viral genome. Proc Natl Acad Sci U S A 100:9849–9854PubMedCrossRefGoogle Scholar
  11. 11.
    Zoulim F (2005) New insight on hepatitis B virus persistence from the study of intrahepatic viral cccDNA. J Hepatol 42:302–308PubMedCrossRefGoogle Scholar
  12. 12.
    Zoulim F, Mason WS (2012) Reasons to consider earlier treatment of chronic HBV infections. Gut 61:333–336PubMedCrossRefGoogle Scholar
  13. 13.
    Werle-Lapostolle B, Bowden S, Locarnini S et al (2004) Persistence of cccDNA during the natural history of chronic hepatitis B and decline during adefovir dipivoxil therapy. Gastroenterology 126:1750–1758PubMedCrossRefGoogle Scholar
  14. 14.
    Guo H, Jiang D, Zhou T, Cuconati A, Block TM, Guo JT (2007) Characterization of the intracellular deproteinized relaxed circular DNA of hepatitis B virus: an intermediate of covalently closed circular DNA formation. J Virol 81:12472–12484PubMedCrossRefGoogle Scholar
  15. 15.
    Zhou T, Guo H, Guo JT, Cuconati A, Mehta A, Block TM (2006) Hepatitis B virus e antigen production is dependent upon covalently closed circular (ccc) DNA in HepAD38 cell cultures and may serve as a cccDNA surrogate in antiviral screening assays. Antiviral Res 72:116–124PubMedCrossRefGoogle Scholar
  16. 16.
    Cai D, Mills C, Yu W, Yan R, Aldrich CE, Saputelli JR, Mason WS, Xu X, Guo JT, Block TM, Cuconati A, Guo H (2012) Identification of disubstituted sulfonamide compounds as specific inhibitors of hepatitis B virus covalently closed circular DNA formation. Antimicrob Agents Chemother 56:4277–4288PubMedCrossRefGoogle Scholar
  17. 17.
    Tuttleman JS, Pourcel C, Summers J (1986) Formation of the pool of covalently closed circular viral DNA in hepadnavirus-infected cells. Cell 47:451–460PubMedCrossRefGoogle Scholar
  18. 18.
    Guo H, Mason WS, Aldrich CE et al (2005) Identification and characterization of avihepadnaviruses isolated from exotic anseriformes maintained in captivity. J Virol 79:2729–2742PubMedCrossRefGoogle Scholar
  19. 19.
    Hirt B (1967) Selective extraction of polyoma DNA from infected mouse cell cultures. J Mol Biol 26:365–369PubMedCrossRefGoogle Scholar
  20. 20.
    Gao W, Hu J (2007) Formation of hepatitis B virus covalently closed circular DNA: removal of genome-linked protein. J Virol 81:6164–6174PubMedCrossRefGoogle Scholar
  21. 21.
    Margeridon S, Carrouee-Durantel S, Chemin I et al (2008) Rolling circle amplification, a powerful tool for genetic and functional studies of complete hepatitis B virus genomes from low-level infections and for directly probing covalently closed circular DNA. Antimicrob Agents Chemother 52:3068–3073PubMedCrossRefGoogle Scholar
  22. 22.
    Mazet-Wagner AA, Baclet MC, Loustaud-Ratti V et al (2006) Real-time PCR quantitation of hepatitis B virus total DNA and covalently closed circular DNA in peripheral blood mononuclear cells from hepatitis B virus-infected patients. J Virol Methods 138: 70–79PubMedCrossRefGoogle Scholar
  23. 23.
    Sells MA, Chen M, Acs G (1987) Production of hepatitis B virus particles in hepG2 cells transfected with cloned hepatitis B virus DNA. Proc Natl Acad Sci U S A 84:1005–1009PubMedCrossRefGoogle Scholar
  24. 24.
    Ladner SK, Otto MJ, Barker CS et al (1997) Inducible expression of human hepatitis B virus (HBV) in stably transfected hepatoblastoma cells: a novel system for screening potential inhibitors of HBV replication. Antimicrob Agents Chemother 41:1715–1720PubMedGoogle Scholar
  25. 25.
    Wang T, Zhao R, Wu Y et al (2011) Hepatitis B virus induces G1 phase arrest by regulating cell cycle genes in HepG2.2.15 cells. Virol J 8:231PubMedCrossRefGoogle Scholar
  26. 26.
    Gearhart TL, Bouchard MJ (2011) The hepatitis B virus HBx protein modulates cell cycle regulatory proteins in cultured primary human hepatocytes. Virus Res 155:363–367PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2013

Authors and Affiliations

  • Dawei Cai
    • 1
  • Hui Nie
    • 1
  • Ran Yan
    • 1
  • Ju-Tao Guo
    • 1
  • Timothy M. Block
    • 1
  • Haitao Guo
    • 1
  1. 1.Institute for Biotechnology and Virology ResearchDrexel University College of MedicineDoylestownUSA

Personalised recommendations