Development of FRET-based high-throughput screening for viral RNase III inhibitors

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Wang , L , Saarela , J , Poque , S & Valkonen , J P T 2020 , ' Development of FRET-based high-throughput screening for viral RNase III inhibitors ' , Molecular Plant Pathology , vol. 21 , no. 7 , pp. 961-974 .

Title: Development of FRET-based high-throughput screening for viral RNase III inhibitors
Author: Wang, Linping; Saarela, Jani; Poque, Sylvain; Valkonen, Jari P. T.
Contributor organization: Department of Agricultural Sciences
Institute for Molecular Medicine Finland
Plant Pathology and Virology
Viikki Plant Science Centre (ViPS)
Plant Production Sciences
Plant-Virus Interactions
Date: 2020-07
Language: eng
Number of pages: 14
Belongs to series: Molecular Plant Pathology
ISSN: 1464-6722
Abstract: The class 1 ribonuclease III (RNase III) encoded by Sweet potato chlorotic stunt virus (CSR3) suppresses RNA silencing in plant cells and thereby counters the host antiviral response by cleaving host small interfering RNAs, which are indispensable components of the plant RNA interference (RNAi) pathway. The synergy between sweet potato chlorotic stunt virus and sweet potato feathery mottle virus can reduce crop yields by 90%. Inhibitors of CSR3 might prove efficacious to counter this viral threat, yet no screen has been carried out to identify such inhibitors. Here, we report a novel high-throughput screening (HTS) assay based on fluorescence resonance energy transfer (FRET) for identifying inhibitors of CSR3. For monitoring CSR3 activity via HTS, we used a small interfering RNA substrate that was labelled with a FRET-compatible dye. The optimized HTS assay yielded 109 potential inhibitors of CSR3 out of 6,620 compounds tested from different small-molecule libraries. The three best inhibitor candidates were validated with a dose-response assay. In addition, a parallel screen of the selected candidates was carried out for a similar class 1 RNase III enzyme from Escherichia coli (EcR3), and this screen yielded a different set of inhibitors. Thus, our results show that the CSR3 and EcR3 enzymes were inhibited by distinct types of molecules, indicating that this HTS assay could be widely applied in drug discovery of class 1 RNase III enzymes.
Subject: fluorescence resonance energy transfer
high-throughput screening
RNA silencing suppressor
small interfering RNA
1182 Biochemistry, cell and molecular biology
11832 Microbiology and virology
Peer reviewed: Yes
Rights: cc_by
Usage restriction: openAccess
Self-archived version: publishedVersion

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