PLoS One. 2019 Feb 19;14(2):e0212233. doi: 10.1371/journal.pone.0212233. eCollection 2019.
Screening for inhibitor of episomal DNA identified dicumarol as a hepatitis B virus inhibitor.
Takeuchi F1,2, Ikeda S1,2, Tsukamoto Y2,3, Iwasawa Y1,2, Qihao C1,2, Otakaki Y1,2, Ryota O2,4, Yao WL1,2, Narita R2,5, Makoto H1,2, Watashi K6,7,8, Wakita T6, Takeuchi K9, Chayama K10, Kogure A2, Kato H1,2,3, Fujita T1,2.
Author information
1
Graduate School of Biostudies, Kyoto University, Kyoto, Japan.
2
Department of Virus Research, Institute for Frontier Life and Medical Sciences, Kyoto University, Kyoto, Japan.
3
Institute of Cardiovascular Immunology, University Hospital Bonn, Bonn, Germany.
4
Department of Immunology, Faculty of Medicine and Graduate School of Medicine, Hokkaido University, Hokkaido, Japan.
5
Centre for Structural Biology, Department of Molecular Biology and Genetics, Aarhus University, Aarhus, Denmark.
6
Department of Virology II, National Institute of Infectious Diseases, Tokyo, Japan.
7
Department of Applied Biological Science, Tokyo University of Science, Noda, Japan.
8
CREST, Japan Science and Technology Agency (JST), Saitama, Japan.
9
Molecular Profiling Research Center for Drug Discovery, National Institute of Advanced Industrial Science and Technology (AIST), Tokyo, Japan.
10
Liver Research Project Center, Hiroshima University, Hiroshima, Japan.
Abstract
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.