Establishment of an inducible HBV stable cell line that expresses cccDNA-dependent epitope-tagged HBeAg for screening of cccDNA modulators.
Establishment of an inducible HBV stable cell line that expresses cccDNA-dependent epitope-tagged HBeAg for screening of cccDNA modulators.
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DOI：
10.1016/j.antiviral.2016.05.005
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发表时间：
2016-08
影响因子：
7.6
通讯作者：
Guo, Haitao
中科院分区：
文献类型：
--
作者：
Cai, Dawei;Wang, Xiaohe;Yan, Ran;Mao, Richeng;Liu, Yuanjie;Ji, Changhua;Cuconati, Andrea;Guo, Haitao
B型肝炎病毒（HBV）共价闭合环状（ccc）DNA是病毒生命周期中必不可少的，在慢性感染期间消除它被认为是持久治疗的关键，但目前的抗病毒药物尚未实现。尽管cccDNA是必需的，但它并不是高通量筛选（HTS）的主要靶标，这主要是因为目前HBV组织培养系统的局限性，包括检测cccDNA本身的不切实际性。为了满足这一需求，我们之前已经开发了一种概念验证的HepDE 19细胞系，其中野生型e抗原（HBeAg）的产生依赖于cccDNA。然而，现有的测定系统对于HTS并不理想，因为HBeAg ELISA与病毒HBeAg同源物交叉反应，所述病毒HBeAg同源物是在HepDE 19细胞中主要以cccDNA非依赖性方式表达的核心抗原（HBcAg）。为了进一步提高测定特异性，我们在本文中报告了“第二代”cccDNA报告细胞系，称为HepBHAe 82。按照HepDE 19细胞系的类似原理，在HepBHAe 82细胞的转基因中，将符合读框的HA表位标签引入HBeAg开放阅读框的前核心结构域，而不破坏任何对HBV复制和HBeAg分泌至关重要的顺式元件。为了消除HBcAg的干扰信号，建立了以HA抗体为捕获抗体，HBeAg抗体为检测抗体的化学发光ELISA检测方法。基于小型化HepBHAe 82细胞的测定系统表现出高水平的cccDNA依赖性HA-HBeAg产生和具有低背景的高特异性读出信号。我们还建立了一个HepHA-HBeAg 4细胞系，表达转基因依赖性HA-HBeAg作为反筛选，以确定HBeAg抑制剂。HepBHAe 82系统适用于抗病毒HTS开发，并且可用于鉴定调节cccDNA代谢和转录的宿主因子。
Hepatitis B virus (HBV) covalently closed circular (ccc) DNA is essential to the virus life cycle, its elimination during chronic infection is considered critical to a durable therapy but has not been achieved by current antivirals. Despite being essential, cccDNA has not been the major target of high throughput screening (HTS), largely because of the limitations of current HBV tissue culture systems, including the impracticality of detecting cccDNA itself. In response to this need, we have previously developed a proof-of-concept HepDE19 cell line in which the production of wildtype e antigen (HBeAg) is dependent upon cccDNA. However, the existing assay system is not ideal for HTS because the HBeAg ELISA cross reacts with a viral HBeAg homologue, which is the core antigen (HBcAg) expressed largely in a cccDNA-independent fashion in HepDE19 cells. To further improve the assay specificity, we report herein a “second-generation” cccDNA reporter cell line, termed HepBHAe82. In the similar principle of HepDE19 line, an in-frame HA epitope tag was introduced into the precore domain of HBeAg open reading frame in the transgene of HepBHAe82 cells without disrupting any cis-element critical for HBV replication and HBeAg secretion. A chemiluminescence ELISA assay (CLIA) for the detection of HA-tagged HBeAg with HA antibody serving as capture antibody and HBeAb serving as detection antibody has been developed to eliminate the confounding signal from HBcAg. The miniaturized HepBHAe82 cell based assay system exhibits high level of cccDNA-dependent HA-HBeAg production and high specific readout signals with low background. We have also established a HepHA-HBe4 cell line expressing transgene-dependent HA-HBeAg as a counter screen to identify HBeAg inhibitors. The HepBHAe82 system is amenable to antiviral HTS development, and can be used to identify host factors that regulate cccDNA metabolism and transcription.
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DOI：
10.1007/978-1-62703-484-5_13
发表时间：
2013
期刊：
Methods in molecular biology (Clifton, N.J.)
影响因子：
--
作者：
Cai, Dawei;Nie, Hui;Yan, Ran;Guo, Ju-Tao;Block, Timothy M;Guo, Haitao
通讯作者：
Guo, Haitao
影响因子：
7.6
作者：
Feld, J. J.;Colledge, D.;Locarnini, S. A.
通讯作者：
Locarnini, S. A.
影响因子：
56.9
作者：
Deres, K;Schröder, CH;Rübsamen-Waigmann, H
通讯作者：
Rübsamen-Waigmann, H
DOI：
10.1002/hep.28025
发表时间：
2015-12
期刊：
Hepatology (Baltimore, Md.)
影响因子：
--
作者：
Liang TJ;Block TM;McMahon BJ;Ghany MG;Urban S;Guo JT;Locarnini S;Zoulim F;Chang KM;Lok AS
通讯作者：
Lok AS
影响因子：
4.8
作者：
Messageot, F;Salhi, S;Rossignol, JM
通讯作者：
Rossignol, JM