Functional analyses of viral RNA silencing suppressors and a strategy to screen antiviral compounds that target viral RNA silencing suppressors

Abstract

RNA silencing is triggered by double-stranded RNAs (dsRNAs), and the dsRNAs are processed into 21to 24-nulceotide (nt) short interfering RNAs (siRNAs) by a host dsRNA-specific ribonuclease, Dicer. The siRNAs are subsequently incorporated into ARGONAUTE (AGO) proteins and serve as a guide for either sequence-specific cleavage or translational repression of a target RNA. Because viral dsRNAs originating from either replicative intermediates or hairpin structures on viral genomes can become inducers of RNA silencing, the RNA silencing machinery in plants is considered to be a natural antiviral defense mechanism. In contrast, plant viruses have evolved a counter defense strategy, producing RNA silencing suppressors (RSSs) that interfere with the RNA silencing pathway. Among RSSs of plant viruses, HC-Pro of potyviruses, 2b of Cucumber mosaic virus (CMV) and P19 of tombusviruses have been extensively studied. These RSSs are structurally diverse, but many use a common strategy to interfere with the RNA silencing pathway by binding to siRNAs. In this study, we first developed a protoplastbased system for RNA silencing to measure RSS activity more stringently than with the commonly used Agrobacterium-mediated transient expression system. Using the protoplast assay, we performed various functional analyses of the 2b protein (2b) of Cucumovirus. In addition, we established a strategy for screening and assessing chemical compounds that inhibit the interaction of viral RSS and siRNAs, leading to attenuation of viral disease symptoms.