Screening for inhibitor of episomal DNA identified dicumarol as a hepatitis B virus inhibitor
Screening for inhibitor of episomal DNA identified dicumarol as a hepatitis B virus inhibitor
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DOI：
10.1371/journal.pone.0212233
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发表时间：
2019-02-19
期刊：
影响因子：
3.7
通讯作者：
Fujita, Takashi
中科院分区：
文献类型：
--
作者：
Takeuchi, Fumihiko;Ikeda, Sotaro;Fujita, Takashi
目前，没有可用的疗法来根除慢性感染个体中的B型肝炎病毒（HBV）。这是由于消除病毒共价闭合环状（ccc）DNA的困难，这是HBV基因表达和复制的核心。我们开发了一个检测系统，使用整合缺陷型慢病毒载体的核环状DNA。该载体在感染细胞的细胞核中产生非整合的环状DNA。我们设计了这个载体来编码萤火虫荧光素酶以监测慢病毒附加体DNA。我们通过这种测定筛选了3，840种化学物质的还原酶活性，并鉴定了双香豆素，这是已知的具有抗凝活性。我们证实双香豆素减少慢病毒附加体DNA。此外，双香豆素在使用表达NTCP的HepG 2和原代人肝细胞的细胞培养物中抑制HBV复制。双香豆素减少细胞内HBV RNA、DNA、上清液HBV抗原和DNA。我们还发现双香豆素降低了HBV感染细胞中的cccDNA水平，但不影响HBV吸附/进入。这是一种用于筛选靶向核cccDNA的抑制剂的新型测定系统，可用于寻找新的HBV抗病毒物质。
Currently, there is no available therapy to eradicate hepatitis B virus (HBV) in chronically infected individuals. This is due to the difficulty in eliminating viral covalently closed circular (ccc) DNA, which is central to the gene expression and replication of HBV. We developed an assay system for nuclear circular DNA using an integration-deficient lentiviral vector. This vector produced non-integrated circular DNA in nuclei of infected cells. We engineered this vector to encode firefly luciferase to monitor the lentiviral episome DNA. We screened 3,840 chemicals by this assay for luciferase-reducing activity and identified dicumarol, which is known to have anticoagulation activity. We confirmed that dicumarol reduced lentiviral episome DNA. Furthermore, dicumarol inhibited HBV replication in cell culture using NTCP-expressing HepG2 and primary human hepatocytes. Dicumarol reduced intracellular HBV RNA, DNA, supernatant HBV antigens and DNA. We also found that dicumarol reduced the cccDNA level in HBV infected cells, but did not affect HBV adsorption/entry. This is a novel assay system for screening inhibitors targeting nuclear cccDNA and is useful for finding new antiviral substances for HBV.