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.
Highlights
RNA interference (RNAi) is an important defence-response system of eukaryotic cells that results in the silencing of viral gene transcripts (Ratcliff et al, 1997; Fire et al, 1998)
It has been proved that the synergistic interaction was mediated by a class 1 RNase III protein encoded by the RNA1 genome segment of sweet potato chlorotic stunt virus (SPCSV), and its endonuclease function was necessary for its role as an RNA silencing suppressor in inducing synergistic disease (Kreuze et al, 2005; Cuellar et al, 2009; Weinheimer et al, 2015)
Several strategies could be applied in plant epidemiology to control plant viruses by taking into consideration virus–vector and virus– host interactions
Summary
RNA interference (RNAi) is an important defence-response system of eukaryotic cells that results in the silencing of viral gene transcripts (Ratcliff et al, 1997; Fire et al, 1998). Our HTS assay was based on FRET, in which CSR3 cleaves a labelled siRNA and generates a fluorescent signal (Figure 2a,b). For the positive control (or blank control), the reaction was carried out either with the catalytically inactive CSR3-A or in the absence of any added enzyme, so that labelled siRNA integrity was maintained and fluorescence emissions from the reporter remained quenched by HBQ1 (Figure 2b). A very high kinetic rate was observed with 288 and 575 nM CSR3 concentrations during the first cycle (shown for 288 nM in Figure 3b), making it impossible to monitor the reaction at high enzyme concentrations, that is, the labelled siRNA was cleaved before fluorescence could be monitored. The slope of the raw fluorescence between all neighbouring detection cycles was calculated for all cycle numbers These data revealed that the maximal slope (2.2–17.1) increased with both FRET-siRNA concentration (50–800 nM) and reaction cycles (2–10) (Figure 4b). The lone difference is D114 of EcR3, which corresponds to N126 of CSR3, and previous studies have shown that, at this position, aspartic acid (D) is prevalent in most RNase III family enzymes (Nicholson, 2014)
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