Hepatitis B virus genome recycling and de novo secondary infection events maintain stable cccDNA levels

Published:August 22, 2018DOI:


      • Studying HBV has been limited by the availability of in vitro and in vivo models.
      • A selected HepG2 cell clone expressing NTCP supports long-term HBV infection.
      • HBV has slow infection kinetics requiring 3 days for full establishment of infection.
      • HBV establishes 1–9 copies of cccDNA per cell which have an estimated half-life of 40 days.
      • cccDNA levels remain stable by intracellular genome recycling and secondary infection.

      Background & Aims

      Several steps in the HBV life cycle remain obscure because of a lack of robust in vitro infection models. These steps include particle entry, formation and maintenance of covalently closed circular (ccc) DNA, kinetics of gene expression and viral transmission routes. This study aimed to investigate infection kinetics and cccDNA dynamics during long-term culture.


      We selected a highly permissive HepG2-NTCP-K7 cell clone engineered to express sodium taurocholate co-transporting polypeptide (NTCP) that supports the full HBV life cycle. We characterized the replication kinetics and dynamics of HBV over six weeks of infection.


      HBV infection kinetics showed a slow infection process. Nuclear cccDNA was only detected 24 h post-infection and increased until 3 days post-infection (dpi). Viral RNAs increased from 3 dpi reaching a plateau at 6 dpi. HBV protein levels followed similar kinetics with HBx levels reaching a plateau first. cccDNA levels modestly increased throughout the 45-day study period with 5–12 copies per infected cell. Newly produced relaxed circular DNA within capsids was reimported into the nucleus and replenished the cccDNA pool. In addition to intracellular recycling of HBV genomes, secondary de novo infection events resulted in cccDNA formation. Inhibition of relaxed circular DNA formation by nucleoside analogue treatment of infected cells enabled us to measure cccDNA dynamics. HBV cccDNA decayed slowly with a half-life of about 40 days.


      After a slow infection process, HBV maintains a stable cccDNA pool by intracellular recycling of HBV genomes and via secondary infection. Our results provide important insights into the dynamics of HBV infection and support the future design and evaluation of new antiviral agents.

      Lay summary

      Using a unique hepatocellular model system designed to support viral growth, we demonstrate that hepatitis B virus (HBV) has remarkably slow infection kinetics. Establishment of the episomal transcription template and the persistent form of the virus, so called covalently closed circular DNA, as well as viral transcription and protein expression all take a long time. Once established, HBV maintains a stable pool of covalently closed circular DNA via intracellular recycling of HBV genomes and through infection of naïve cells by newly formed virions.

      Graphical abstract


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      Author names in bold designate shared co-first authorship

        • Seeger C.
        • Mason W.S.
        Molecular biology of hepatitis B virus infection.
        Virology. 2015; 479–480C: 672-686
        • Schulze A.
        • Gripon P.
        • Urban S.
        Hepatitis B virus infection initiates with a large surface protein-dependent binding to heparan sulfate proteoglycans.
        Hepatology. 2007; 46: 1759-1768
        • Verrier E.R.
        • Colpitts C.C.
        • Bach C.
        • Heydmann L.
        • Weiss A.
        • Renaud M.
        • et al.
        A targeted functional RNA interference screen uncovers glypican 5 as an entry factor for hepatitis B and D viruses.
        Hepatology. 2016; 63: 35-48
        • Yan H.
        • Zhong G.
        • Xu G.
        • He W.
        • Jing Z.
        • Gao Z.
        • et al.
        Sodium taurocholate cotransporting polypeptide is a functional receptor for human hepatitis B and D virus.
        eLife. 2012; 1: e00049
        • Ni Y.
        • Lempp F.A.
        • Mehrle S.
        • Nkongolo S.
        • Kaufman C.
        • Falth M.
        • et al.
        Hepatitis B and D viruses exploit sodium taurocholate co-transporting polypeptide for species-specific entry into hepatocytes.
        Gastroenterology. 2014; 146: 1070-1083
        • Nassal M.
        HBV cccDNA: viral persistence reservoir and key obstacle for a cure of chronic hepatitis B.
        Gut. 2015; 64: 1972-1984
        • Watanabe T.
        • Sorensen E.M.
        • Naito A.
        • Schott M.
        • Kim S.
        • Ahlquist P.
        Involvement of host cellular multivesicular body functions in hepatitis B virus budding.
        Proc Natl Acad Sci USA. 2007; 104: 10205-10210
        • Tuttleman J.S.
        • Pourcel C.
        • Summers J.
        Formation of the pool of covalently closed circular viral DNA in hepadnavirus-infected cells.
        Cell. 1986; 47: 451-460
        • Kock J.
        • Rosler C.
        • Zhang J.J.
        • Blum H.E.
        • Nassal M.
        • Thoma C.
        Generation of covalently closed circular DNA of hepatitis B viruses via intracellular recycling is regulated in a virus specific manner.
        PLoS Pathog. 2010; 6: e1001082
        • Guo F.
        • Zhao Q.
        • Sheraz M.
        • Cheng J.
        • Qi Y.
        • Su Q.
        • et al.
        HBV core protein allosteric modulators differentially alter cccDNA biosynthesis from de novo infection and intracellular amplification pathways.
        PLoS Pathog. 2017; 13: e1006658
        • Sattentau Q.
        Avoiding the void: cell-to-cell spread of human viruses.
        Nat Rev Microbiol. 2008; 6: 815-826
        • Timpe J.M.
        • Stamataki Z.
        • Jennings A.
        • Hu K.
        • Farquhar M.J.
        • Harris H.J.
        • et al.
        Hepatitis C virus cell-cell transmission in hepatoma cells in the presence of neutralizing antibodies.
        Hepatology. 2008; 47: 17-24
        • Xia Y.
        • Stadler D.
        • Ko C.
        • Protzer U.
        Analyses of HBV cccDNA quantification and modification.
        Methods Mol Biol. 2017; 1540: 59-72
        • Liu C.
        • Cai D.
        • Zhang L.
        • Tang W.
        • Yan R.
        • Guo H.
        • et al.
        Identification of hydrolyzable tannins (punicalagin, punicalin and geraniin) as novel inhibitors of hepatitis B virus covalently closed circular DNA.
        Antiviral Res. 2016; 134: 97-107
        • Ko C.
        • Shin Y.C.
        • Park W.J.
        • Kim S.
        • Kim J.
        • Ryu W.S.
        Residues Arg703, Asp777, and Arg781 of the RNase H domain of hepatitis B virus polymerase are critical for viral DNA synthesis.
        J Virol. 2014; 88: 154-163
        • Appelman M.D.
        • Chakraborty A.
        • Protzer U.
        • McKeating J.A.
        • van de Graaf S.F.
        N-Glycosylation of the Na+-taurocholate cotransporting polypeptide (NTCP) determines its trafficking and stability and is required for hepatitis b virus infection.
        PLoS ONE. 2017; 12: e0170419
        • Gripon P.
        • Diot C.
        • Guguen-Guillouzo C.
        Reproducible high level infection of cultured adult human hepatocytes by hepatitis B virus: effect of polyethylene glycol on adsorption and penetration.
        Virology. 1993; 192: 534-540
        • Guo H.
        • Jiang D.
        • Zhou T.
        • Cuconati A.
        • Block T.M.
        • Guo J.T.
        Characterization of the intracellular deproteinized relaxed circular DNA of hepatitis B virus: an intermediate of covalently closed circular DNA formation.
        J Virol. 2007; 81: 12472-12484
        • Gao W.
        • Hu J.
        Formation of hepatitis B virus covalently closed circular DNA: removal of genome-linked protein.
        J Virol. 2007; 81: 6164-6174
        • Nikolaou N.
        • Green C.J.
        • Gunn P.J.
        • Hodson L.
        • Tomlinson J.W.
        Optimizing human hepatocyte models for metabolic phenotype and function: effects of treatment with dimethyl sulfoxide (DMSO).
        Physiol Rep. 2016; 4
        • Allweiss L.
        • Volz T.
        • Giersch K.
        • Kah J.
        • Raffa G.
        • Petersen J.
        • et al.
        Proliferation of primary human hepatocytes and prevention of hepatitis B virus reinfection efficiently deplete nuclear cccDNA in vivo.
        Gut. 2018; 67: 542-552
        • Michailidis E.
        • Pabon J.
        • Xiang K.
        • Park P.
        • Ramanan V.
        • Hoffmann H.H.
        • et al.
        A robust cell culture system supporting the complete life cycle of hepatitis B virus.
        Sci Rep. 2017; 7: 16616
        • Sprinzl M.F.
        • Oberwinkler H.
        • Schaller H.
        • Protzer U.
        Transfer of hepatitis B virus genome by adenovirus vectors into cultured cells and mice: crossing the species barrier.
        J Virol. 2001; 75: 5108-5118
        • Xia Y.
        • Carpentier A.
        • Cheng X.
        • Block P.D.
        • Zhao Y.
        • Zhang Z.
        • et al.
        Human stem cell-derived hepatocytes as a model for hepatitis B virus infection, spreading and virus-host interactions.
        J Hepatol. 2017; 66: 494-503
        • Zhu Y.
        • Yamamoto T.
        • Cullen J.
        • Saputelli J.
        • Aldrich C.E.
        • Miller D.S.
        • et al.
        Kinetics of hepadnavirus loss from the liver during inhibition of viral DNA synthesis.
        J Virol. 2001; 75: 311-322
        • Addison W.R.
        • Walters K.A.
        • Wong W.W.
        • Wilson J.S.
        • Madej D.
        • Jewell L.D.
        • et al.
        Half-life of the duck hepatitis B virus covalently closed circular DNA pool in vivo following inhibition of viral replication.
        J Virol. 2002; 76: 6356-6363
        • Thimme R.
        • Wieland S.
        • Steiger C.
        • Ghrayeb J.
        • Reimann K.A.
        • Purcell R.H.
        • et al.
        CD8(+) T cells mediate viral clearance and disease pathogenesis during acute hepatitis B virus infection.
        J Virol. 2003; 77: 68-76
        • Volz T.
        • Allweiss L.
        • Ben M.M.
        • Warlich M.
        • Lohse A.W.
        • Pollok J.M.
        • et al.
        The entry inhibitor Myrcludex-B efficiently blocks intrahepatic virus spreading in humanized mice previously infected with hepatitis B virus.
        J Hepatol. 2013; 58: 861-867
        • Lempp F.A.
        • Mutz P.
        • Lipps C.
        • Wirth D.
        • Bartenschlager R.
        • Urban S.
        Evidence that hepatitis B virus replication in mouse cells is limited by the lack of a host cell dependency factor.
        J Hepatol. 2016; 64: 556-564
        • Summers J.
        • Smith P.M.
        • Huang M.J.
        • Yu M.S.
        Morphogenetic and regulatory effects of mutations in the envelope proteins of an avian hepadnavirus.
        J Virol. 1991; 65: 1310-1317