RNA Silencing Suppression by a Second Copy of the P1 Serine Protease ofCucumber Vein Yellowing Ipomovirus, a Member of the FamilyPotyviridaeThat Lacks the Cysteine Protease HCPro

Abstract

The P1 protein of viruses of the family Potyviridae is a serine proteinase, which is highly variable in length and sequence, and its role in the virus infection cycle is not clear. One of the proposed activities of P1 is to assist HCPro, the product that viruses of the genus Potyvirus use to counteract antiviral defense mediated by RNA silencing. Indeed, an HCPro-coding region is present in all the genomes of members of the genera Potyvirus, Rymovirus, and Tritimovirus that have been sequenced. However, it was recently reported that a sequence coding for HCPro is lacking in the genome of Cucumber vein yellowing virus (CVYV), a member of the genus Ipomovirus, the fourth monopartite genus of the family. In this study, we provide further evidence that P1 enhances the activity of HCPro in members of the genus Potyvirus and show that it is duplicated in the ipomovirus CVYV. The two CVYV P1 copies are arranged in tandem, and the second copy (P1b) has RNA silencing suppression activity. CVYV P1b suppressed RNA silencing induced either by sense green fluorescent protein (GFP) mRNA or by a GFP inverted repeat RNA, indicating that CVYV P1b acts downstream of the formation of double-stranded RNA. CVYV P1b also suppressed local silencing in agroinfiltrated patches of transgenic Nicotiana benthamiana line 16c and delayed its propagation to the neighboring cells. However, neither the short-distance nor long-distance systemic spread of silencing of the GFP transgene was completely blocked by CVYV P1b. CVYV P1b and P1-HCPro from the potyvirus Plum pox virus showed very similar behaviors in all the assays carried out, suggesting that evolution has found a way to counteract RNA silencing by similar mechanisms using very different proteins in viruses of the same family.The regulatory systems of gene expression mediated by sequence-specific RNA silencing are mechanisms conserved in a wide variety of eukaryotic organisms (50). These pathways are triggered by double-stranded RNA (dsRNA), which is recognized by Dicer-like RNases to produce short dsRNAs of 21 to 26 nucleotides (nt) in length (small interfering RNAs [siRNAs]) (3, 32). One strand of these small RNAs is then incorporated into different silencing effector complexes, guiding them, by sequence complementarity, to degrade mRNA, inhibit RNA translation, or interfere with transcription by chromatin rearrangements (47).Different RNA silencing pathways have been identified, and one of them, active in the cytoplasm, has been shown to play an antiviral role in eukaryotes, where viruses generate dsRNA tracts in replicative intermediates, highly structured mRNAs, or other RNA molecules as a consequence of the action of cellular RNA-dependent RNA polymerases (10, 56). In plants, fungi, and Caenorhabditis elegans at least, this is a non-cellautonomous process, which spreads to remote cells or tissues (57, 59). The systemic silencing signal has still not been identified, although the sequence specificity of this pathway suggests the involvement of a nucleic acid component (19,34).To cou...

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

“…Nicotiana benthamiana plants were infiltrated with A. tumefaciens C58C1 strain carrying the indicated plasmids as previously described (Valli et al 2006). GFP fluorescence was observed under a Leica MZ FLIII fluorescence microscope (Leica) with excitation and barrier filters of 480/40 and 510 nm, respectively.…”

“…p35S-NTAP-P1b (wild-type and RK68,69AA mutant) and p35S-P1HC has been previously described (Valli et al 2006(Valli et al , 2008. Site-directed mutagenesis of P1b was carried out by two PCR steps as described by Herlitze and Koenen (1990).…”

“…Samples of large and small RNAs were prepared from agroinfiltrated leaf tissue as previously described (Valli et al 2006). For the analysis of small RNAs interacting with P1b in vivo, wild-type and mutant NTAP-P1b were purified by affinity chromatography in calmoduline-Sepharose beads (Valli et al 2008).…”

“…However, the sequence encoding this protein is absent from the genome of some members of the Ipomovirus genus, such as Cucumber vein yellowing virus (CVYV), Squash vein yellowing virus (SqVYV), and Cassava brown streak virus (CBSV) (Janssen et al 2005;Li et al 2008;Mbanzibwa et al 2009). Whereas in typical potyviruses the 59 terminal region of the genome codes for a single serine proteinase (P1), the 59 terminal region of the genome of CVYV and SqVYV codes for two similar serine proteinases arranged in tandem (P1a and P1b), and the P1b has been shown to have RNA silencing suppression activity (Valli et al 2006;Li et al 2008). These P1b proteins, together with homologs in the Ipomovirus, Brambyvirus, and Poacevirus (or Susmovirus) genera form a group of P1 proteins that is phylogenetically and functionally distinct from the potyviral P1s, and the P1a proteins of CVYV and SqVYV (Valli et al 2007;Susaimuthu et al 2008;Fellers et al 2009;Tatineni et al 2009).…”

Cassava brown streak disease: a threat to food security in Africa

“…In contrast, the other two ipomoviruses cucumber vein yellowing virus (CVYV) and squash vein yellowing virus (SqVYV) possess two P1 serine proteinases (P1a and P1b) (Valli et al, 2007). The P1 protein of SPMMV is known to suppress RNA silencing whilst HC-Pro contributes to the durability of silencing suppression (Giner et al, 2008), whereas in the case of ipomoviruses lacking HC-Pro, the P1b protein has taken over the function of silencing suppression (Valli et al, 2006). However, in the case of UCBSV/CBSV, the single P1 protein possesses silencing suppression activity due to the presence of the basic LxKA and Zn-finger motifs (Mbanzibwa et al, 2009a).…”