High-Throughput Screening Using a Whole-Cell Virus Replication Reporter Gene Assay to Identify Inhibitory Compounds against Rift Valley Fever Virus Infection

Abstract

Rift Valley fever virus (RVFV) is an emerging virus that causes serious illness in humans and livestock. There are no approved vaccines or treatments for humans. The purpose of the study was to identify inhibitory compounds of RVFV infection without any preconceived idea of the mechanism of action. A whole-cell-based high-throughput drug screening assay was developed to screen 28,437 small chemical compounds targeting RVFV infection. To accomplish both speed and robustness, a replication-competent NSs-deleted RVFV expressing a fluorescent reporter gene was developed. Inhibition of fluorescence intensity was quantified by spectrophotometry and related to virus infection in human lung epithelial cells (A549). Cell toxicity was assessed by the Resazurin cell viability assay. After primary screening, 641 compounds were identified that inhibited RVFV infection by ≥80%, with ≥50% cell viability at 50 µM concentration. These compounds were subjected to a second screening regarding dose-response profiles, and 63 compounds with ≥60% inhibition of RVFV infection at 3.12 µM compound concentration and ≥50% cell viability at 25 µM were considered hits. Of these, six compounds with high inhibitory activity were identified. In conclusion, the high-throughput assay could efficiently and safely identify several promising compounds that inhibited RVFV infection.

A Genome-Wide RNA Interference Screen Identifies a Role for Wnt/β-Catenin Signaling during Rift Valley Fever Virus Infection

“…Quantitative real-time RT-PCR (qRT-PCR) analysis was used to measure the mRNA expression levels of β-catenin, cyclin D1, Dvl, LRP5, or matrix metalloproteinase-7 (MMP7) after infection with RVFV MP12 or RVFV MP12-GFP (MOI of 1) or treatment with Wnt3A in A549 cells. The GFP reporter signal from RVFV MP12-GFP was readily observed between 5 and 6 hpi in this cell type, ensuring that viral mRNA transcription and translation occurred within this measurement time frame ( 42 , 43 ). RVFV MP12 and RVFV MP12-GFP infection led to significant upregulation of expression of all five genes compared to that in mock-infected cells ( Fig.…”

Anti-Rift Valley fever virus activity in vitro, pre-clinical pharmacokinetics and oral bioavailability of benzavir-2, a broad-acting antiviral compound

“…Recombinant RVFV expressing the far-red fluorescent protein Katushka instead of the deleted NSs protein (rRVFVΔNSs::Katushka) was used in this study. Generation of rRVFVΔNSs::Katushka has previously been described 27 .…”

Recent Advances in the Development of Antiviral Therapeutics for Rift Valley Fever Virus Infection

“…Using a replication-competent recombinant RVFV (MOI = 3 for the initial screen) encoding red fluorescent protein instead of the NSs ORF, Islam and colleagues identified a subset of six compounds out of a library of 28,437 compounds total to be effective at reducing red fluorescent protein expression in a dose-dependent manner with EC 50 values in the low micromolar range (ranging from 2.1 to 7.0 μM). Interestingly, each identified molecule belonged to a separate compound class, although all contained many cyclic motifs, which may present similar binding profiles to their target protein [115]. Future studies will be necessary to further characterize the mechanism of action, as well as in vivo efficacy.…”

“…6051, 7007 NP oligomerization inhibition Ͻ0.4 μM (Vero E6) [109] Suramin vRNA/N Interaction 22.3 μM (HEK 293T) [110] BCX4430 Adenosine analog inhibiting RNA polymerase 41.6 μM (HeLa) C57/BL6 mice [113,114] Six related compounds Not known 2.1-7.0 μM (A549) [115] LJ001, JL-compound series Membrane disruption Ͻ0.5 μM (BHK-S) BALB/c mice [93,118] Sorafanib RNA synthesis, egress 6.4 μM (Vero) BALB/c mice [119] Bortezomib Ubiquitin proteasome inhibitor Ͻ0.01 μM (HSAEC) [122] FGI-106 Unknown host protein 800 nM (Vero E6) [123] Rapamycin mTOR Inhibition 11 μM (H2. 35) BALB/c mice [129] 17AAG, BAPT A-AM HSP90 Inhibition Ͻ10 μM (Vero) [130] 5,6-dimethoxyindan-1-one Undefined 0.73 μg/ml [131] 1E7-03 PP1 inhibition 3.5 μM (HSAEC03) [136] 25HC Viral-cell membrane fusion block Ͻ1 μM (HeLa) [137] GYY4137 (H2S-donor) Reactive species scavenger Ͻ5 mM (Vero) [143] Four signal transduction inhibitors Block early stages of infection 4.7-36.4 μM (HeLa) [145] Four protease inhibitors Proteasome system inhibition 13.6-61.5 μM (HeLa) [146] RNASEK Internalization block siRNA knockdown [147] SMER28 Autohagy inhibition 6.9 μM (U2OS) [124] NSC 62914 Reactive species scavenger Ͻ10 μM (A549) C57/BL6 mice [151] RNA aptamers RNA binding inhibition Not determined [112] GC stem fragment Peptide fusion inhibition Not determined [159] † In vitro cell culture system used to evaluate antiviral activity against Rift Valley fever virus.…”