Tobacco rattle virus 16K silencing suppressor binds ARGONAUTE 4 and inhibits formation of RNA silencing complexes.

The cysteine-rich 16K protein of tobacco rattle virus (TRV), the type member of the genus Tobravirus, is known to suppress RNA silencing. However, the mechanism of action of the 16K suppressor is not well understood. In this study, we used a GFP-based sensor strategy and an Agrobacterium-mediated transient assay in Nicotiana benthamiana to show that 16K was unable to inhibit the activity of existing small interfering RNA (siRNA)- and microRNA (miRNA)-programmed RNA-induced silencing effector complexes (RISCs). In contrast, 16K efficiently interfered with de novo formation of miRNA- and siRNA-guided RISCs, thus preventing cleavage of target RNA. Interestingly, we found that transiently expressed endogenous miR399 and miR172 directed sequence-specific silencing of complementary sequences of viral origin. 16K failed to bind small RNAs, although it interacted with ARGONAUTE 4, as revealed by bimolecular fluorescence complementation and immunoprecipitation assays. Site-directed mutagenesis demonstrated that highly conserved cysteine residues within the N-terminal and central regions of the 16K protein are required for protein stability and/or RNA silencing suppression.

Switching on RNA silencing suppressor activity by restoring argonaute binding to a viral protein.

We found that Sweet potato feathery mottle virus (SPFMV) P1, a close homologue of Sweet potato mild mottle virus P1, did not have any silencing suppressor activity. Remodeling the Argonaute (AGO) binding domain of SPFMV P1 by the introduction of two additional WG/GW motifs converted it to a silencing suppressor with AGO binding capacity. To our knowledge, this is the first instance of the transformation of a viral protein of unknown function to a functional silencing suppressor.