Protease Activity, Self Interaction, and Small Interfering RNA Binding of the Silencing Suppressor P1b fromCucumber Vein Yellowing Ipomovirus

Abstract

The RNA silencing pathway mediated by small interfering RNAs (siRNAs) plays an important antiviral role in eukaryotes. To counteract this defense barrier, a large number of plant viruses express proteins with RNA silencing suppression activity. Recently, it was reported that the ipomovirus Cucumber vein yellowing virus (CVYV), which lacks the typical silencing suppressor of members of the family Potyviridae, i.e., HCPro, has a duplicated P1 coding sequence and that the downstream P1 copy, named P1b, has silencing suppression activity. In this study, we provide experimental evidence that P1b is a serine protease that self-cleaves at its C terminus but that its proteolytic activity is not essential for silencing suppression. In contrast, a putative zinc finger and a conserved basic motif in the N-terminal region of the protein are required for efficient silencing suppression. In vitro gel filtration-fast protein liquid chromatography and in vivo bimolecular fluorescence complementation assays showed that P1b binds itself to form oligomeric structures and that the zinc finger-like motif is essential for the self interaction. Moreover, we observed that CVYV P1b forms complexes with synthetic siRNAs, and this ability correlated with both silencing suppression activity and enhancement of Potato virus X pathogenicity in a mutational analysis. Together, these results suggest that CVYV P1b resembles potyviral HCPro and other viral proteins in interfering RNA silencing by preventing siRNA loading into the RNAinduced silencing complex.

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

“…The CVYV P1b is able to bind in vitro siRNAs-key elements of the RNA silencing machinery-and it seemed likely that siRNA sequestration plays a major role in the suppression activity of this protein (Valli et al 2008), as has been demonstrated or suggested for other RSSs (Vargason et al 2003;Lakatos et al 2006). In the present study, consistent with that prediction, we show that CVYV P1b binds small RNAs in vivo and that this property is strictly correlated with its silencing suppression activity.…”

“…The P1b protein of CVYV is a RSS that resembles the typical potyviral RSS HCPro in its ability to interact with synthetic siRNA molecules in vitro (Valli et al 2008). To further characterize this siRNA binding activity of CVYV P1b, a NTAP (N-terminal Tandem Affinity Purification)-tagged P1b was expressed by agroinfiltration in Nicotiana benthamiana plants, partially purified by affinity chromatography, and then probed with various single-stranded (ss-) or double-stranded (ds-) forms of 32 P-labeled siRNAs in an electrophoretic mobility shift assay (EMSA).…”

“…To determine whether CVYV P1b binds siRNAs in vivo, we partially purified NTAP-P1b by affinity from N. benthamiana leaves in which three constructs were transiently expressed: p35S:GFP (expressing GFP mRNA as reporter), the strong silencing inducer p35S:GF-IR (expressing an inverted repeat that generates a partial GFP dsRNA) and the expression construct of wildtype NTAP-P1b or its RK68,69AA inactive mutant (Valli et al 2008).…”

Análisis Funcional De Proteínas Codificadas Por El Virus De La Rotura Del Color De La Flor Del Pelargonium

“…También se han etiquetado en amarillo dos residuos de Arg (R127 y R134) conservados en varias CP de carmovirus con actividad supresora e identificados en la CP homóloga del TCV (p38) como esenciales para la unión a dsRNAs [Cao et al, 2010]. (B) Modelo de p37 obtenido con ayuda del programa Rosetta [Rohl et al, 2004], validado empleando las herramientas QMEAN [Benkert et al, 2009] [ Vargason et al, 2003;Ye et al, 2003;Bragg & Jackson, 2004;Lingel et al, 2005;Rashid et al, 2008;Valli et al, 2008;Yang et al, 2011], y que la mutación P323L afecte este supuesto proceso de dimerización. Si bien esta cuestión no ha sido abordada experimentalmente en este trabajo, se ha descrito que las CP de carmovirus dimerizan antes de ensamblarse al RNA y se ha sugerido que el estado dimerizado constituye la forma estable de la proteína en la célula [Sorger et al, 1986].…”

“…Silencing suppressors are commonly involved in enhancement of viral pathogenicity and accumulation of viruses [Voinnet et al, 1999], thus many of them have been shown to accentuate symptoms when expressed by a heterologous virus [Pruss et al, 1997;Valli et al, 2008;Zhou et al, 2006]. To investigate this, the PFBV CP gene was cloned in a PVX vector [pPVX202; Sablowski et al, 1995] and the resulting construct (pPVX-CP) was used to mechanically inoculate N. benthamiana and Nicotiana clevelandii plants.…”