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

Human parvovirus B19 (B19V), a member of the genus Erythroparvovirus of the family Parvoviridae, is a small non-enveloped virus that has a single-stranded DNA (ssDNA) genome of 5.6 kilobases 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 non-structural 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 the NS1 and compared it with the radioactive in vitro nicking assay. We used both assays to screen a set of small molecule compounds (96) that have potential anti-nuclease activity. We found that the fluorophore-based in vitro nicking assay demonstrate sensitivity and specificity values as high as 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 flavonoid-like structure for IC₅₀in vitro that falls in the range of 1-3 µM. Importantly, they also exhibited inhibition of B19V DNA replication in UT7/Epo-S1 cells and ex vivo expanded human erythroid progenitor cells.

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