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

Abstract

Human parvovirus B19 (B19V), a member of the genus <i>Erythroparvovirus</i> of the family <i>Parvoviridae</i>, 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 <i>in vitro</i> nicking assay of the replication origin using the origin-binding domain of NS1 and compared it with the radioactive <i>in vitro</i> nicking assay. We used both assays to screen a set of small-molecule compounds (<i>n</i> = 96) that have potential antinuclease activity. We found that the fluorophore-based <i>in vitro</i> 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 <i>in vitro</i> nicking assays. We further tested 3 compounds that have a flavonoid-like structure and an <i>in vitro</i> 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 <i>ex vivo</i>-expanded human erythroid progenitor cells.