Screening for inhibitor of episomal DNA identified dicumarol as a hepatitis B virus inhibitor.

Fumihiko Takeuchi,Koh Takeuchi,Wan-Ling Yao,Takashi Fujita,Kazuaki Chayama,Yukie Otakaki,Ouda Ryota,Chen Qihao,Ryo Narita,Hijikata Makoto,Hiroki Kato,Yuta Tsukamoto,Koichi Watashi,Sotaro Ikeda,Yoshikazu Iwasawa,Amane Kogure,Takaji Wakita,Wang-Shick Ryu

doi:10.1371/journal.pone.0212233

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.

Highlights

More than 240 million people are infected with hepatitis B virus (HBV)[1]
The assay had little to do with the HBV life cycle, dicumarol inhibited HBV replication (Figs 3–5)
It is important to determine which stage of the HBV life cycle is affected by dicumarol because blockade of any step of the HBV life cycle may reduce HBV closed circular DNA (cccDNA)

Summary

Introduction

More than 240 million people are infected with hepatitis B virus (HBV)[1]. Most infections in adulthood are transient, approximately 5%–10% of infected adults and over 90% of infected neonates fail to mount a sufficient immune response to clear the virus, and develop a lifelong chronic infection[2]. 0.6–1 million die from chronic hepatitis B (CHB). Dicumarol inhibits HBV replication study design, data collection and analysis, decision to publish, or preparation of the manuscript

Methods

Results

Conclusion