Hepatitis B virus (HBV) covalently closed circular DNA (cccDNA), existing in hepatocyte nuclei as a stable minichromosome, plays a central role in the life cycle of the virus and permits the persistence of infection. Despite being essential for HBV infection, little is known about the molecular mechanisms of cccDNA formation, regulation and degradation, and there is no therapeutic agents directly targeting cccDNA, fore mostly due to the lack of robust, reliable and quantifiable HBV cccDNA models. In this study, combined the Cre/loxP and sleeping beauty transposons system, we established HepG2-derived cell lines integrated with 2–60 copies of monomeric HBV genome flanked by loxP sites (HepG2-HBV/loxP). After Cre expression via adenoviral transduction, 3.3-kb recombinant cccDNA (rcccDNA) bearing a chimeric intron can be produced in the nuclei of these HepG2-HBV/loxP cells. The rcccDNA could be accurately quantified by quantitative PCR using specific primers and cccDNA pool generated in this model could be easily detected by Southern blotting using the digoxigenin probe system. We demonstrated that the rcccDNA was epigenetically organized as the natural minichromosome and served as the template supporting pgRNA transcription and viral replication. As the expression of HBV S antigen (HBsAg) is dependent on the newly generated cccDNA, HBsAg is the surrogate marker of cccDNA. Additionally, the efficacies of 3 classes of anti-HBV agents were evaluated in HepG2-HBV/loxP cells and antiviral activities with different mechanisms were confirmed. These data collectively suggested that HepG2-HBV/loxP cell system will be powerful platform for studying cccDNA related biological mechanisms and developing novel cccDNA targeting drugs.

乙型肝炎病毒（HBV）共价闭合的环状DNA（cccDNA）作为稳定的微染色体存在于肝细胞核中，在病毒的生命周期中起着核心作用，并允许感染持续存在。尽管对于HBV感染至关重要，但对cccDNA形成，调控和降解的分子机制知之甚少，并且没有直接针对cccDNA的治疗剂，主要是由于缺乏健壮，可靠且可量化的HBV cccDNA模型。在这项研究中，结合了Cre / loxP和睡眠美容转座子系统，我们建立了HepG2衍生的细胞系，该细胞系整合了2-60拷贝的单体HBV基因组，两侧是loxP位点（HepG2-HBV / loxP）。通过腺病毒转导表达Cre后，可以在这些HepG2-HBV / loxP细胞的细胞核中产生带有嵌合内含子的3.3 kb重组cccDNA（rcccDNA）。可以使用特异性引物通过定量PCR准确定量rcccDNA，并且使用洋地黄毒苷探针系统通过Southern印迹很容易检测该模型中生成的cccDNA池。我们证明rcccDNA被表观遗传组织为天然的微型染色体，并用作支持pgRNA转录和病毒复制的模板。由于HBV S抗原（HBsAg）的表达依赖于新产生的cccDNA，因此HBsAg是cccDNA的替代标记。此外，在HepG2-HBV / loxP中评估了3类抗HBV药物的疗效细胞和抗病毒活性具有不同的机制被证实。这些数据共同表明，HepG2-HBV / loxP细胞系统将成为研究cccDNA相关生物学机制和开发新型cccDNA靶向药物的强大平台。