建立Cre介导的HBV重组cccDNA（rcccDNA）细胞系，用于cccDNA生物学

StephenW

Antiviral Res. 2018 Feb 9. pii: S0166-3542(17)30590-9. doi: 10.1016/j.antiviral.2018.02.007. [Epub ahead of print]
Establishment of Cre-mediated HBV recombinant cccDNA (rcccDNA) cell line for cccDNA biology and antiviral screening assays.Wu M1, Li J1, Yue L1, Bai L1, Li Y1, Chen J1, Zhang X2, Yuan Z3.
Author information
1Research Unit, Shanghai Public Health Clinical Center, Key Laboratory of Medical Molecular Virology, School of Basic Medical Sciences of Shanghai Medical College, Fudan University, Shanghai, China.2Research Unit, Shanghai Public Health Clinical Center, Key Laboratory of Medical Molecular Virology, School of Basic Medical Sciences of Shanghai Medical College, Fudan University, Shanghai, China. Electronic address: [email protected].3Research Unit, Shanghai Public Health Clinical Center, Key Laboratory of Medical Molecular Virology, School of Basic Medical Sciences of Shanghai Medical College, Fudan University, Shanghai, China. Electronic address: [email protected].

AbstractHepatitis 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.


KEYWORDS: Cre/loxP; Hepatitis B virus (HBV); Recombinant covalently closed circular DNA (rcccDNA); Stable cell line

PMID:29432776DOI:10.1016/j.antiviral.2018.02.007

StephenW

抗病毒研究。 2018年2月9日。pii：S0166-3542（17）30590-9。 doi：10.1016 / j.antiviral.2018.02.007。 [电子版提前打印]
建立Cre介导的HBV重组cccDNA（rcccDNA）细胞系，用于cccDNA生物学和抗病毒筛选试验。
Wu M1，Li J1，Yue L1，Bai L1，Li Y1，Chen J1，Zhang X2，Yuan Z3。
作者信息

1
    上海市公共卫生临床中心研究员，复旦大学上海医学院基础医学院医学分子病毒学重点实验室，中国上海。
2
    上海市公共卫生临床中心研究员，复旦大学上海医学院基础医学院医学分子病毒学重点实验室，中国上海。电子地址：[email protected]。
3
    上海市公共卫生临床中心研究员，复旦大学上海医学院基础医学院医学分子病毒学重点实验室，中国上海。电子地址：[email protected]。

抽象

乙型肝炎病毒（HBV）共价闭合环状DNA（cccDNA）作为稳定的微型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的持续存在。尽管对于HBV感染至关重要，但对cccDNA形成，调节和降解的分子机制知之甚少，并且没有直接靶向cccDNA的治疗剂，主要是由于缺乏稳健，可靠和可量化的HBV cccDNA模型。在本研究中，将Cre / loxP和睡美转座子系统相结合，我们建立了HepG2衍生的细胞系，整合了2-60个拷贝的单体HBV基因组，位于loxP位点（HepG2-HBV / loxP）侧翼。通过腺病毒转导Cre表达后，可在这些HepG2-HBV / loxP细胞的细胞核中产生携带嵌合内含子的3.3-kb重组cccDNA（rcccDNA）。通过使用特异性引物的定量PCR可以准确定量rcccDNA，并且使用地高辛探针系统通过Southern印迹可以容易地检测在该模型中产生的cccDNA库。我们证明了rcccDNA在表观遗传学上被组织为天然微染色体并且充当支持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，所以HBsAg是cccDNA的替代标记。此外，在HepG2-HBV / loxP细胞中评估了3类抗HBV药物的功效，并证实了具有不同机制的抗病毒活性。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。
关键词：

酶Cre / loxP位;乙型肝炎病毒（HBV）;重组共价闭环DNA（rcccDNA）;稳定的细胞系

结论：
    29432776
DOI：
    10.1016 / j.antiviral.2018.02.007