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

Lourdes Fernández-Calvino, Llúcia Martínez-Priego, Edit Z. Szabo, Irene Guzmán-Benito, Inmaculada González, Tomás Canto, L. Lakatos, Cásar Llave

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Abstract

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.

Original languageEnglish
Pages (from-to)246-257
Number of pages12
JournalJournal of General Virology
Volume97
Issue number1
DOIs
Publication statusPublished - Jan 1 2016

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ASJC Scopus subject areas

  • Virology

Cite this

Fernández-Calvino, L., Martínez-Priego, L., Szabo, E. Z., Guzmán-Benito, I., González, I., Canto, T., Lakatos, L., & Llave, C. (2016). Tobacco rattle virus 16K silencing suppressor binds ARGONAUTE 4 and inhibits formation of RNA silencing complexes. Journal of General Virology, 97(1), 246-257. https://doi.org/10.1099/jgv.0.000323