Establishment of Cre-mediated HBV recombinant cccDNA (rcccDNA) cell line for cccDNA biology and antiviral screening assays
Establishment of Cre-mediated HBV recombinant cccDNA (rcccDNA) cell line for cccDNA biology and antiviral screening assays
复制标题
Cre 介导的 HBV 重组 cccDNA (rcccDNA) 细胞系的建立，用于 cccDNA 生物学和抗病毒筛选分析
DOI：
10.1016/j.antiviral.2018.02.007
复制
发表时间：
2018
影响因子：
7.6
通讯作者：
Yuan Zhenghong
中科院分区：
文献类型：
--
作者：
Wu Min;Li Jin;Yue Lei;Bai Lu;Li Yaming;Chen Jieliang;Zhang Xiaonan;Yuan Zhenghong
B型肝炎病毒（HBV）共价闭合环状DNA（cccDNA）作为稳定的微小染色体存在于肝细胞核中，在病毒的生命周期中起着中心作用，并允许感染的持续。尽管cccDNA对于HBV感染是必需的，但关于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。本研究将Cre/loxP与睡美人转座子系统相结合，建立了2-60拷贝HBV单体基因组两侧带有loxP位点的HepG 2细胞系（HepG 2-HBV/loxP）。通过腺病毒转导表达Cre后，可在这些HepG 2-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）。使用特异性引物通过定量PCR可以准确地定量rcccDNA，并且在该模型中产生的cccDNA池可以通过使用地高辛探针系统的Southern印迹容易地检测。我们证明，rcccDNA是表观遗传学组织的天然微型染色体，并作为模板支持pgRNA转录和病毒复制。由于HBV S抗原（HBsAg）的表达依赖于新生成的cccDNA，因此HBsAg是cccDNA的替代标志物。此外，在HepG 2-HBV/loxP细胞中评价了3类抗HBV药物的效力，并证实了不同机制的抗病毒活性。这些数据表明HepG 2-HBV/loxPcell系统将成为研究cccDNA相关生物学机制和开发新型cccDNA靶向药物的有力平台。
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/loxPand sleeping beauty transposons system, we established HepG2-derived cell lines integrated with 2–60 copies of monomeric HBV genome flanked byloxPsites (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/loxPcells. 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/loxPcells and antiviral activities with different mechanisms were confirmed. These data collectively suggested that HepG2-HBV/loxPcell system will be powerful platform for studying cccDNA related biological mechanisms and developing novel cccDNA targeting drugs.