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

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

Author information

1
Department of Microbiology, Molecular Genetics, and Immunology, University of Kansas Medical Center, Kansas City, Kansas, USA.
2
ViraCor Eurofins Laboratories, Lee's Summit, Missouri, USA.
3
Department of Molecular and Cellular Biology, University of Arizona, Tucson, Arizona, USA.
4
Center for World Health and Medicine, Saint Louis University, St. Louis, Missouri, USA.
5
Department of Chemistry, Saint Louis University, St. Louis, Missouri, USA.
6
Department of Molecular Microbiology and Immunology, Saint Louis University, St. Louis, Missouri, USA.
7
Department of Microbiology, Molecular Genetics, and Immunology, University of Kansas Medical Center, Kansas City, Kansas, USA jqiu@kumc.edu.

Abstract

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

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