Investigating the Ability of a Novel Compound to Inhibit the Cleavage Activity of Human Parvovirus B19’s NS1 Nuclease Domain

Abstract

The Human Parvovirus B19 is a medically relevant single-stranded DNA virus implicated in the development of severe, sometimes fatal illnesses in human populations. Specifically, infection with B19V is associated with the development of erythema infectiosum (fifths disease), arthropathy such as inflammatory arthritis, myocarditis, pure red-cell aplasia, and sometimes transient aplastic crisis. Despite these apparent effects on human health, no antiviral treatments are currently specific for B19 infection. Our study focuses on the N-terminal domain of B19s main replicative protein, nonstructured protein 1 (NS1). The NS1 nuclease domain (NS1-Nuc) cleaves viral DNA at the terminal resolution site (TRS), prompting endless viral replication via the rolling hairpin mechanism. Because of its critical role in facilitating long-term infection, NS1-Nuc has become a favorable target for developing antiviral therapeutics. This work describes attempts to characterize the interactions of the NS1 nuclease domain with the novel synthetic compound SS4 designed to inhibit its cleavage activity. Using isothermal titration calorimetry, we have identified a Kd of 1.7 ± 0.5 µM for the binding of SS4 to a solution of purified NS1-Nuc. Additionally, we present a crystal structure of a new NS1-Nuc construct (His-nuc) solved at 3.0 Å and various attempts at co-crystallization with both the SS4 compound and the viral DNA in which NS1-Nuc cleaves. Finally, we describe the work to optimize an in-vitro cleavage assay to validate the activity of purified NS1-Nuc on 32P radiolabeled TRS DNA assessed by denaturing PAGE. We will ultimately use this test to assess the inhibition of His-nuc cleavage on TRS DNA in the presence of SS4.