Aureusvirus P14 Is an Efficient RNA Silencing Suppressor That Binds Double-Stranded RNAs without Size Specificity

Abstract

RNA silencing is a conserved eukaryotic gene regulatory system in which sequence specificity is determined by small RNAs. Plant RNA silencing also acts as an antiviral mechanism; therefore, viral infection requires expression of a silencing suppressor. The mechanism and the evolution of silencing suppression are still poorly understood. Tombusvirus open reading frame (ORF) 5-encoded P19 is a size-selective double-stranded RNA (dsRNA) binding protein that suppresses silencing by sequestering double-stranded small interfering RNAs (siRNAs), the specificity determinant of the antiviral silencing system. To better understand the evolution of silencing suppression, we characterized the suppressor of the type member of Aureusviruses, the closest relatives of the genus Tombusvirus. We show that the Pothos latent virus (PoLV) ORF 5-encoded P14 is an efficient suppressor of both virus-and transgene-induced silencing. Findings that in vitro P14 binds dsRNAs and double-stranded siRNAs without obvious size selection suggest that P14, unlike P19, can suppress silencing by sequestering both long dsRNA and double-stranded siRNA components of the silencing machinery. Indeed, P14 prevents the accumulation of hairpin transcript-derived siRNAs, indicating that P14 inhibits inverted repeat-induced silencing by binding the long dsRNA precursors of siRNAs. However, viral siRNAs accumulate to high levels in PoLV-infected plants; therefore, P14 might inhibit virus-induced silencing by sequestering double-stranded siRNAs. Finally, sequence analyses suggest that P14 and P19 suppressors diverged from an ancient dsRNA binding suppressor that evolved as a nested protein within the common ancestor of aureusvirus-tombusvirus movement proteins.RNA silencing (also termed posttranscriptional gene silencing in plants and RNA interference in animals) is a conserved eukaryotic gene inactivation system that plays regulatory roles in many biological processes including development, maintenance of genome stability, and antiviral responses (2,6,12,25,54). RNA silencing is induced by accumulation of doublestranded RNAs (dsRNAs). dsRNAs are first processed by an RNase III-like nuclease called DICER (in plants termed DICER-LIKE, or DCL) into short (21 to 25 nucleotide [nt]) RNAs, and then these short RNAs incorporate and guide different silencing effector complexes to homologous nucleic acids for suppression (2,6,12,16,25,54). In plants, RNA silencing acts at both single-cell (cell-autonomous silencing) and at whole-plant (systemic silencing) levels. Cell-autonomous silencing inactivates genes in the cells in which dsRNAs accumulated. Moreover, cell-autonomous silencing generates mobile silencing signals that confer suppression of homologous mRNAs in neighboring cells (short distance) and in distant tissues (long-distance systemic silencing) (29,31,32,56).DICERs can process dsRNAs into two functionally different small RNAs, micro-RNAs (miRNAs) and small interfering RNAs (siRNAs). miRNAs are involved in the control of many endogenous protein-encoding mRNAs...

Suppressors of RNA silencing encoded by plant viruses and their role in viral infections

“…Evidence supporting this conclusion comes from the known affinity of p19 of TBSV for siRNA [32], the strong RNA binding activity of TCV CP [44], and the established dsRNA binding affinity of NS1 of Influenza virus and E3L of vaccinia virus. Extending on this theme, a recent report regarding the p14 silencing suppressor encoded by Pothos latent virus (PoLV), showed that it bound to both long and short dsRNA, including the siRNA duplex [45]. Interestingly, although the genome of PoLV is similar to other tombusviruses, its suppressor (p14) is a smaller protein than p19 with higher affinity to long dsRNAs.…”

Genetic variability and evolutionary analyses of the coat protein gene of Tomato mosaic virus

“…It has also been recently shown that V2 suppressor of Geminivirus competes with SGS3, a key component of the secondary cycle of siRNAs amplification, in binding dsRNAs and thus interferes with transitivity (Fukunaga & Doudna 2009). Finally, the CP of some carmoviruses (Meng et al 2006) and the P14 of Aureusvirus (Mérai et al 2005) can also bind long dsRNAs, resulting in the protection of the intermediaries of replication from DICER activity. The first mathematical models of the RNA silencing pathway focused on aberrant cellular mRNA as triggers of the silencing response (Bergstrom et al 2003;Groenenboom et al, 2005).…”

The Importance of the KR-Rich Region of the Coat Protein of Ourmia melon virus for Host Specificity, Tissue Tropism, and Interference With Antiviral Defense

“…Therefore, less efficient encapsidation would actually increase the RNA molecules available for further replication rounds. Taking this into consideration, it is likely that the suppression of antiviral defense in OuMV does not depend on encapsidation efficiency but, instead, relies on other specific protein-RNA interactions, as shown for TCV (Merai et al 2005). Furthermore, the fact that a lower encapsidation efficiency of genomic RNA is complemented functionally in Dicer mutants supports the action of the CP as a silencing suppressor that acts independently of encapsidation.…”

“…Interactions between CP and viral genomic RNA are necessary for encapsidation of the viral genetic material but, in some cases, such as Turnip crinkle virus (TCV) p38 CP, the RNA-binding motif is crucial for silencing-suppression activity (Cao et al 2010). In particular, it has been shown that the N-terminal region of p38 binds double-stranded RNA molecules independently of their size, and that these binding activities also include viral siRNA duplexes (Merai et al 2005). This property is linked with the inhibition of the silencing machinery either by siRNA sequestration or by interference with Dicer activities.…”