The specific binding to 21-nt double-stranded RNAs is crucial for the anti-silencing activity of Cucumber vein yellowing virus P1b and perturbs endogenous small RNA populations

Abstract

RNA silencing mediated by siRNAs plays an important role as an anti-viral defense mechanism in plants and other eukaryotic organisms, which is usually counteracted by viral RNA silencing suppressors (RSSs). The ipomovirus Cucumber vein yellowing virus (CVYV) lacks the typical RSS of members of the family Potyviridae, HCPro, which is replaced by an unrelated RSS, P1b. CVYV P1b resembles potyviral HCPro in forming complexes with synthetic siRNAs in vitro. Electrophoretic mobility shift assays showed that P1b, like potyviral HCPro, interacts with double-stranded siRNAs, but is not able to bind single-stranded small RNAs or small DNAs. These assays also showed a preference of CVYV P1b for binding to 21-nt siRNAs, a feature also reported for HCPro. However, these two potyvirid RSSs differ in their requirements of 2-nucleotide (nt) 39 overhangs and 59 terminal phosphoryl groups for siRNA binding. Copurification assays confirmed in vivo P1b-siRNA interactions. We have demonstrated by deep sequencing of small RNA populations interacting in vivo with CVYV P1b that the size preference of P1b for small RNAs of 21 nt also takes place in the plant, and that expression of this RSS causes drastic changes in the endogenous small RNA populations. In addition, a site-directed mutagenesis analysis strongly supported the assumption that P1b-siRNA binding is decisive for the anti-silencing activity of P1b and localized a basic domain involved in the siRNA-binding activity of this protein.

Biochemical and genetic functional dissection of the P38 viral suppressor of RNA silencing

“…These factors have all been characterized as VSRs in planta, and amino acids essential to their anti-silencing functions have been identified in all cases (Kasschau et al 1997;Chu et al 2000;Chen et al 2008;Azevedo et al 2010;Valli et al 2011). The BYL system was applied for the de novo expression of wild-type VSRs and their corresponding point-mutant derivatives.…”

The P1N-PISPO trans -Frame Gene of Sweet Potato Feathery Mottle Potyvirus Is Produced during Virus Infection and Functions as an RNA Silencing Suppressor

“…Ethidium bromide staining was used to verify equal loading of lanes and monitor the transference processes. After UV cross-linking and prehybridization in UltraHyb buffer (Ambion), blots were hybridized in the same solution with probes specific to the GFP sequence, using a 32 Plabeled DNA probe (33) in the case of mRNAs or a 32 P-labeled RNA probe (36) in the case of small RNAs. Hybridization signals were detected with a PhosphorImager.…”

A Novel Role of the Potyviral Helper Component Proteinase Contributes To Enhance the Yield of Viral Particles

“…Several reviews of RNA silencing and its role in antiviral defense have been recently published (22)(23)(24)(25). The mechanism by which HCPro acts as an RNA-silencing suppressor (RSS) appears to rely on its capacity to specifically interact with/ sequester virus-derived 21-nucleotide (nt) small RNAs (26)(27)(28) and to interact with the small RNA methyltransferase HEN1 (29) and other host factors (30,31). Interestingly, since its first characterization, HCPro has also been related to several events during viral infection, such as genome amplification, both cell-to-cell and long-distance movements, pathogenicity, and viral synergism (reviewed in references 32 and 33).…”