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.
J Virol Methods. 2021 Jun;292:114127. doi: 10.1016/j.jviromet.2021.114127. Epub 2021 Mar 22.
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 RNA:DNA 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 total 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.
乙型肝炎病毒（HBV）核糖核酸酶 H（RNaseH）是一个有前景但尚未开发的药物靶点。抑制 RNaseH 通过截断负链 DNA 来阻断病毒的逆转录，导致 RNA:DNA 杂种的积累，并终止正链 DNA 的合成。由于即使复制完全被抑制，负链 DNA 仍然存在，因此筛选 RNaseH 抑制剂变得复杂，因为这些残留的 DNA 可以通过标准的筛选检测方法检测到，这些方法通过检测 HBV 总 DNA 积累的减少来测量。我们之前开发了一种链偏好 qPCR 检测法，通过测量对病毒正链 DNA 链的优先抑制来检测 RNaseH 复制抑制剂。然而，该检测法使用在 6 孔或 12 孔板中生长的细胞，因此通量非常低。在此，我们将该检测法适配至 96 孔板格式，并对 727 种化合物进行了原理验证筛选。新开发的检测法是抗 HBV 药物发现的有用工具，特别是在筛选 RNaseH 抑制剂时。
