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A mid-throughput HBV replication inhibition assay capable of detecting ribonuclease H inhibitors
Qilan Li 1 , Tiffany C Edwards 2 , Nathan L Ponzar 3 , John E Tavis 4
Affiliations
Affiliations
1
Department of Molecular Microbiology and Immunology, Saint Louis University School of Medicine, St. Louis, MO, USA; Saint Louis University Liver Center, Saint Louis University School of Medicine, St. Louis, MO, USA. Electronic address: [email protected].
2
Department of Molecular Microbiology and Immunology, Saint Louis University School of Medicine, St. Louis, MO, USA; Saint Louis University Liver Center, Saint Louis University School of Medicine, St. Louis, MO, USA. Electronic address: [email protected].
3
Department of Molecular Microbiology and Immunology, Saint Louis University School of Medicine, St. Louis, MO, USA; Saint Louis University Liver Center, Saint Louis University School of Medicine, St. Louis, MO, USA. Electronic address: [email protected].
4
Department of Molecular Microbiology and Immunology, Saint Louis University School of Medicine, St. Louis, MO, USA; Saint Louis University Liver Center, Saint Louis University School of Medicine, St. Louis, MO, USA. Electronic address: [email protected].
PMID: 33766659 DOI: 10.1016/j.jviromet.2021.114127
Abstract
The hepatitis B virus (HBV) ribonuclease H (RNaseH) is a promising but unexploited drug target. Inhibiting the RNaseH blocks viral reverse transcription by truncating the minus-polarity DNA strand, causing accumulation of RNANA heteroduplexes, and abrogating plus-polarity DNA synthesis. Screening for RNaseH inhibitors is complicated by the presence of the minus-polarity DNA strand even when replication is fully inhibited because this residual DNA can be detected by standard screening assays that measure reduction in HBV DNA accumulation. We previously developed a strand-preferential qPCR assay that detects RNaseH replication inhibitors by measuring preferential suppression of the viral plus-polarity DNA strand. However, this assay employed cells grown in 6- or 12-well plates and hence was of very low throughput. Here, we adapted the assay to a 96-well format and conducted a proof-of-principle screen of 727 compounds. The newly developed assay is a valuable tool for anti-HBV drug discovery, particularly when screening for RNaseH inhibitors.
Keywords: Hepatitis B virus; assay optimization; replication inhibition assay; ribonuclease H.
Copyright © 2021. Published by Elsevier B.V. |
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