Endonuclease Activity Inhibition of the NS1 Protein of Parvovirus B19 as a Novel Target for Antiviral Drug Development

Antimicrob Agents Chemother. 2019 Feb 26;63(3):e01879-18. doi: 10.1128/AAC.01879-18. Print 2019 Mar.


Human parvovirus B19 (B19V), a member of the genus Erythroparvovirus of the family Parvoviridae, is a small nonenveloped virus that has a single-stranded DNA (ssDNA) genome of 5.6 kb with two inverted terminal repeats (ITRs). B19V infection often results in severe hematological disorders and fetal death in humans. B19V replication follows a model of rolling hairpin-dependent DNA replication, in which the large nonstructural protein NS1 introduces a site-specific single-strand nick in the viral DNA replication origins, which locate at the ITRs. NS1 executes endonuclease activity through the N-terminal origin-binding domain. Nicking of the viral replication origin is a pivotal step in rolling hairpin-dependent viral DNA replication. Here, we developed a fluorophore-based in vitro nicking assay of the replication origin using the origin-binding domain of NS1 and compared it with the radioactive in vitro nicking assay. We used both assays to screen a set of small-molecule compounds (n = 96) that have potential antinuclease activity. We found that the fluorophore-based in vitro nicking assay demonstrates sensitivity and specificity values as high as those of the radioactive assay. Among the 96 compounds, we identified 8 which have an inhibition of >80% at 10 µM in both the fluorophore-based and radioactive in vitro nicking assays. We further tested 3 compounds that have a flavonoid-like structure and an in vitro 50% inhibitory concentration that fell in the range of 1 to 3 µM. Importantly, they also exhibited inhibition of B19V DNA replication in UT7/Epo-S1 cells and ex vivo-expanded human erythroid progenitor cells.

Keywords: antivirals; in vitro nicking assay; parvovirus B19.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Antiviral Agents / pharmacology*
  • Cell Line
  • DNA Replication / drug effects*
  • DNA, Viral / genetics
  • Drug Development
  • Erythroid Precursor Cells
  • Humans
  • Parvoviridae Infections / drug therapy*
  • Parvoviridae Infections / virology
  • Parvovirus B19, Human / drug effects*
  • Viral Nonstructural Proteins / antagonists & inhibitors*
  • Virus Replication / drug effects*
  • Virus Replication / genetics


  • Antiviral Agents
  • DNA, Viral
  • NS1 protein, parvovirus
  • Viral Nonstructural Proteins