Broad anti-herpesviral activity of α-hydroxytropolones

Abstract

Herpesviruses are ubiquitous in animals and cause economic losses concomitant with many diseases. Most of the domestic animal herpesviruses are within the subfamily Alphaherpesvirinae, which includes human herpes simplex virus 1 (HSV-1). Suppression of HSV-1 replication has been reported with α-hydroxytropolones (αHTs), aromatic ring compounds that have broad bioactivity due to potent chelating activity. It is postulated that αHTs inhibit enzymes within the nucleotidyltransferase superfamily (NTS). These enzymes require divalent cations for nucleic acid cleavage activity. Potential targets include the nuclease component of the herpesvirus terminase (pU_L15C), a highly conserved NTS-like enzyme that cleaves viral DNA into genomic lengths prior to packaging into capsids. Inhibition of pU_L15C activity in biochemical assays by various αHTs previously revealed a spectrum of potencies. Interestingly, the most potent anti-pU_L15C αHT inhibited HSV-1 replication to a limited extent in cell culture. The aim of this study was to evaluate three different αHT molecules with varying biochemical anti-pU_L15C activity for a capacity to inhibit replication of veterinary herpesviruses (BoHV-1, EHV-1, and FHV-1) and HSV-1. Given the known discordant potencies between anti-pU_L15C and HSV-1 replication inhibition, a second objective was to elucidate the mechanism of action of these compounds. The results show that αHTs broadly inhibit herpesviruses, with similar inhibitory effect against HSV-1, BoHV-1, EHV-1, and FHV-1. Based on immunoblotting, Southern blotting, and real-time qPCR, the compounds were found to specifically inhibit viral DNA replication. Thus, αHTs represent a new class of broadly active anti-herpesviral compounds with potential veterinary applications.

Keywords: Anti-herpesvirus; Herpesvirus; Nucleotidyltransferase superfamily; α-hydroxytropolones.

Fig. 1.

The chemical structure of the three synthetic αHTs compounds used in this study.

Fig. 2.

TEM of MDBK cells inoculated with BoHV-1 and treated with DMSO control (A), 50 μM αHT-106 (B), or 8 μM αHT-115 (C). Note the numerous extracellular virions in the control sample (A). No virions or capsids were observed in the αHT-106 treated (B) and αHT −115 treated samples (not pictured). Clusters of round electron dense intranuclear structures were very rarely present in the αHT-115 sample (C).

Fig. 3.

Immunoblotting of α (A) and γ2 (B) protein in mock-infected or αHT-treated HSV-1-infected samples. (A). α protein ICP27 and β-actin, loading control. (B). γ2 protein gC and β-actin, loading control.

Fig. 4.

Real-time qPCR of viral DNA in wild-type HSV-1 control and αHT-treated samples, performed in triplicate. The decrease in HSV-1 DNA copy number is significant, as determined by one-way ANOVA and Tukey’s post-hoc analysis (**, p < 0.05). No significant differences were observed between αHT treated samples (50 μM αHT-106, 30 μM αHT-111, or 8 μM αHT-115).

Fig. 5.

Viral DNA analysis. (A and B) Schematic diagram of the HSV-1 genome indicating the unique long (U_L) and unique short (U_S) components, and positions of the BamHI P, S, or junctional S-P fragments. The P and S are end fragments unique to terminase-cleaved viral DNA (i.e. the monomer shown in A). (C) Fluorographic image of viral DNA probed with radiolabeled terminus of the short component (P fragment). CV1 cells were infected with HSV-1(F) or UL15-null. The HSV-1(F) infected samples were treated with PAA, αHT-106, αHT-111, αHT-115 or DMSO vehicle-control. The positions of the S-P junctional fragments and BamHI P fragments are indicated. Decreased band intensities representing the junctional and end fragments are noted in the αHT-treated samples and are comparable in level to that of the PAA-treated sample.