High-Throughput Minigenome System for Identifying Small-Molecule Inhibitors of Ebola Virus Replication

Abstract

Ebola virus (EBOV), a member of the family Filoviridae, is a nonsegmented negative-sense RNA virus that causes severe, often lethal, disease in humans. EBOV RNA synthesis is carried out by a complex that includes several viral proteins. The function of this machinery is essential for viral gene expression and viral replication and is therefore a potential target for antivirals. We developed and optimized a high-throughput screening (HTS) assay based on an EBOV minigenome assay, which assesses the function of the polymerase complex. The assay is robust in 384-well format and displays a large signal to background ratio and high Z-factor values. We performed a pilot screen of 2080 bioactive compounds, identifying 31 hits (1.5% of the library) with >70% inhibition of EBOV minigenome activity. We further identified eight compounds with 50% inhibitory concentrations below their 50% cytotoxic concentrations, five of which had selectivity index (SI) values >10, suggesting specificity against the EBOV polymerase complex. These included an inhibitor of inosine monophosphate dehydrogenase, a target known to modulate the EBOV replication complex. They also included novel classes of inhibitors, including inhibitors of protein synthesis and hypoxia inducible factor-1. Five compounds were tested for their ability to inhibit replication of a recombinant EBOV that expresses GFP (EBOV-GFP), and four inhibited EBOV-GFP growth at sub-cytotoxic concentrations. These data demonstrate the utility of the HTS minigenome assay for drug discovery and suggest potential directions for antifiloviral drug development.

An RNA polymerase II-driven Ebola virus minigenome system as an advanced tool for antiviral drug screening

“…Due to the large-scale transfections and subsequent re-seeding of the cells, this system assures consistent well-to-well transfection efficiency, as also observed for other minigenome-based antiviral drug screening platforms (Edwards et al, 2015; Jasenosky et al, 2010; Uebelhoer et al, 2014). Our data therefore indicate that the newly established pol II-based EBOV minigenome system is improved and reliable, making it an ideal tool for antiviral drug screening.…”

“…Use of the pol II minigenome 96-well format resulted in a robust assay with a Z -factor of 0.74, regardless of the negative control used (Figure 5B), similar to or better than established EBOV minigenome systems (Edwards et al, 2015; Jasenosky et al, 2010; Uebelhoer et al, 2014). Due to the large-scale transfections and subsequent re-seeding of the cells, this system assures consistent well-to-well transfection efficiency, as also observed for other minigenome-based antiviral drug screening platforms (Edwards et al, 2015; Jasenosky et al, 2010; Uebelhoer et al, 2014).…”

“…High-throughput assays using filovirus minigenome systems have been used with a variety of negative controls, including not expressing VP35 (Edwards et al, 2015) or not expressing L (Uebelhoer et al, 2014). To examine if the choice of the negative control affects the efficiency and reliability of the pol II minigenome system in a 96-well format, we compared different negative controls, including minus L, minus VP35 and L synth− .…”

“…DNA encoding the minigenome is cloned into an expression plasmid and co-transfected into cells along with plasmids encoding the viral proteins required for EBOV genome replication and transcription. Importantly, EBOV minigenome systems have also been used extensively for antiviral drug screening (Edwards et al, 2015; Enterlein et al, 2006; Filone et al, 2013; Jasenosky et al, 2010; Olsen et al, 2016; Uebelhoer et al, 2014). Most of the currently existing EBOV minigenomes are under the control of the T7 RNA polymerase (T7) promoter and thus require T7 expression in the transfected cells (Garcia-Dorival et al, 2016; Mühlberger et al, 1999; Uebelhoer et al, 2014).…”

Electron Cryo-microscopy Structure of Ebola Virus Nucleoprotein Reveals a Mechanism for Nucleocapsid-like Assembly

“…HEK293T cells were transfected in 96 well format with expression plasmids encoding eL (125 ng), eVP30 (25 ng), eVP35 (31.25 ng), T7 polymerase (50 ng) and firefly luciferase (1 ng), eNP or eNP mutant constructs (62.5ng or as indicated in the figure legend) along with minigenome plasmid construct expressing Renilla luciferase (50ng) using Lipofectamine 2000 (Invitrogen) (Edwards et al, 2015; Xu et al, 2017). Forty-eight hours later the luciferase activity was determined using Dual-Glo luciferase kit (Promega).…”