title: Recombinant covalently closed circular hepatitis B virus DNA induces prolonged viral persistence in immunocompetent mice.
abstract: It remains crucial to develop a laboratory model for studying hepatitis B virus (HBV) chronic infection. We hereby produced a recombinant covalently closed circular DNA (rcccDNA) in view of the key role of cccDNA in HBV persistence. A loxP-chimeric intron was engineered into a monomeric HBV genome in a precursor plasmid (prcccDNA), which was excised using Cre/loxP-mediated DNA recombination into a 3.3-kb rcccDNA in the nuclei of hepatocytes. The chimeric intron was spliced from RNA transcripts without interrupting the HBV life cycle. In cultured hepatoma cells, cotransfection of prcccDNA and pCMV-Cre (encoding Cre recombinase) resulted in accumulation of nuclear rcccDNA that was heat stable and epigenetically organized as a minichromosome. A mouse model of HBV infection was developed by hydrodynamic injection of prcccDNA. In the presence of Cre recombinase, rcccDNA was induced in the mouse liver with effective viral replication and expression, triggering a compromised T-cell response against HBV. Significant T-cell hyporesponsiveness occurred in mice receiving 4 μg prcccDNA, resulting in prolonged HBV antigenemia for up to 9 weeks. Persistent liver injury was observed as elevated alanine transaminase activity in serum and sustained inflammatory infiltration in the liver. Although a T-cell dysfunction was induced similarly, mice injected with a plasmid containing a linear HBV replicon showed rapid viral clearance within 2 weeks. Collectively, our study provides an innovative approach for producing a cccDNA surrogate that established HBV persistence in immunocompetent mice. It also represents a useful model system in vitro and in vivo for evaluating antiviral treatments against HBV cccDNA. Importance: (i) Unlike plasmids that contain a linear HBV replicon, rcccDNA established HBV persistence with sustained liver injury in immunocompetent mice. This method could be a prototype for developing a mouse model of chronic HBV infection. (ii) An exogenous intron was engineered into the HBV genome for functionally seamless DNA recombination. This original approach could be also extended to other viral studies. (iii) rcccDNA was substantially induced in the nuclei of hepatocytes and could be easily distinguished by its exogenous intron using PCR. This convenient model system affords the opportunity to test antivirals directly targeting HBV cccDNA.
authors.affiliation: Key Laboratory of Molecular Virology and Immunology, Institut Pasteur of Shanghai, Chinese Academy of Sciences, Shanghai, China.
authors.forename: Zhihua
authors.initials: Z
authors.lastname: Qi
authors.affiliation: Key Laboratory of Molecular Virology and Immunology, Institut Pasteur of Shanghai, Chinese Academy of Sciences, Shanghai, China.
authors.forename: Gaiyun
authors.initials: G
authors.lastname: Li
authors.affiliation: Key Laboratory of Molecular Virology and Immunology, Institut Pasteur of Shanghai, Chinese Academy of Sciences, Shanghai, China.
authors.forename: Hao
authors.initials: H
authors.lastname: Hu
authors.affiliation: Key Laboratory of Molecular Virology and Immunology, Institut Pasteur of Shanghai, Chinese Academy of Sciences, Shanghai, China.
authors.forename: Chunhui
authors.initials: C
authors.lastname: Yang
authors.affiliation: Key Laboratory of Molecular Virology and Immunology, Institut Pasteur of Shanghai, Chinese Academy of Sciences, Shanghai, China.
authors.forename: Xiaoming
authors.initials: X
authors.lastname: Zhang
authors.affiliation: Key Laboratory of Molecular Virology and Immunology, Institut Pasteur of Shanghai, Chinese Academy of Sciences, Shanghai, China.
authors.forename: Qibin
authors.initials: Q
authors.lastname: Leng
authors.affiliation: Key Laboratory of Medical Molecular Virology, Shanghai Medical College, Fudan University, Shanghai, China.
authors.forename: Youhua
authors.initials: Y
authors.lastname: Xie
authors.affiliation: Department of Infectious Diseases, Ruijin Hospital, Shanghai Jiaotong University School of Medicine, Shanghai, China.
authors.forename: Demin
authors.initials: D
authors.lastname: Yu
authors.affiliation: Department of Infectious Diseases, Ruijin Hospital, Shanghai Jiaotong University School of Medicine, Shanghai, China.
authors.forename: Xinxin
authors.initials: X
authors.lastname: Zhang
authors.affiliation: Department of Hepatopathy, Shuguang Hospital, Shanghai University of Traditional Chinese Medicine, Shanghai, China.
authors.forename: Yueqiu
authors.initials: Y
authors.lastname: Gao
authors.affiliation: Key Laboratory of Molecular Virology and Immunology, Institut Pasteur of Shanghai, Chinese Academy of Sciences, Shanghai, China lanke@sibs.ac.cn dengqiang@sibs.ac.cn.
authors.forename: Ke
authors.initials: K
authors.lastname: Lan
authors.affiliation: Key Laboratory of Molecular Virology and Immunology, Institut Pasteur of Shanghai, Chinese Academy of Sciences, Shanghai, China lanke@sibs.ac.cn dengqiang@sibs.ac.cn.
authors.forename: Qiang
authors.initials: Q
authors.lastname: Deng
mesh_terms: D000410:Alanine Transaminase / Q000097:blood; D000818:Animals; D002467:Cell Nucleus / Q000821:virology; D002478:Cells, Cultured; D004270:DNA, Circular / Q000235:genetics*; D004279:DNA, Viral / Q000235:genetics*; D004195:Disease Models, Animal; D006515:Hepatitis B virus / Q000235:genetics* / Q000254:growth & development*; D019694:Hepatitis B, Chronic / Q000276:immunology / Q000473:pathology / Q000821:virology; D022781:Hepatocytes / Q000821:virology; D006801:Humans; D008099:Liver / Q000473:pathology; D051379:Mice; D008822:Mice, Transgenic
chemical_list: D004270:DNA, Circular; D004279:DNA, Viral; D000410:Alanine Transaminase