RNA binding is more critical to the suppression of silencing function ofCucumber mosaic virus2b protein than nuclear localization

Abstract

Previously, we found that silencing suppression by the 2b protein and six mutants correlated both with their ability to bind to double-stranded (ds) small RNAs (sRNAs) in vitro and with their nuclear/nucleolar localization. To further discern the contribution to suppression activity of sRNA binding and of nuclear localization, we have characterized the kinetics of in vitro binding to a ds sRNA, a single-stranded (ss) sRNA, and a micro RNA (miRNA) of the native 2b protein and eight mutant variants. We have also added a nuclear export signal (NES) to the 2b protein and assessed how it affected subcellular distribution and suppressor activity. We found that in solution native protein bound ds siRNA, miRNA, and ss sRNA with high affinity, at protein:RNA molar ratios~2:1. Of the four mutants that retained suppressor activity, three showed sRNA binding profiles similar to those of the native protein, whereas the remaining one bound ss sRNA at a 2:1 molar ratio, but both ds sRNAs with 1.5-2 times slightly lower affinity. Three of the four mutants lacking suppressor activity failed to bind to any sRNA, whereas the remaining one bound them at far higher ratios. NES-tagged 2b protein became cytoplasmic, but suppression activity in patch assays remained unaffected. These results support binding to sRNAs at molar ratios at or near 2:1 as critical to the suppressor activity of the 2b protein. They also show that cytoplasmically localized 2b protein retained suppressor activity, and that a sustained nuclear localization was not required for this function.

Key Importance of Small RNA Binding for the Activity of a Glycine-Tryptophan (GW) Motif-containing Viral Suppressor of RNA Silencing

“…However, such interactions by themselves seem to be insufficient to sustain the VSR activity of p37 because the interaction with AGO4 was maintained by all the assayed p37 variants, and the interaction with AGO1, although notably diminished regarding p37-W 28 , was retained at WT levels in the case of the loss-of-function form p37-3R 67 . The great importance of the sRNA binding capability over other protein properties is in agreement with recent reports on other VSRs, such as cucumber mosaic virus 2b, which, like p37, exhibits nucleolar localization, self-interaction, and interaction with AGO1 and AGO4 proteins (42,62,75,78,82).…”

Using a Viral Vector to Reveal the Role of MicroRNA159 in Disease Symptom Induction by a Severe Strain of Cucumber mosaic virus

“…1C). This increased accumulation of miR159* and miR159 is likely to be the consequence of miR159/miR159* duplex stabilization by binding with Fny2b (Goto et al, 2007;González et al, 2010González et al, , 2012Duan et al, 2012;Hamera et al, 2012). Duplex stabilization likely prevents recruitment of miR159 strands into RISCs, an idea supported by increased steady-state levels of the miR159 target transcripts MYB33 and MYB65 in Fny2b-transgenic plants (Fig.…”

“…The 3a movement protein is translated directly from RNA3, whereas a second protein encoded by this genomic segment, the coat protein (CP), is translated from the RNA3-derived subgenomic mRNA RNA4. CMV 2b protein, which was one of the first VSRs identified, disrupts RNA silencing predominantly by sequestration of small dsRNA (Brigneti et al, 1998;Goto et al, 2007;González et al, 2010González et al, , 2012Duan et al, 2012). In addition to inhibiting the establishment of antiviral silencing, the 2b protein also interferes with salicylic acid-induced CMV resistance (Brigneti et al, 1998;Ji and Ding, 2001;Lewsey and Carr, 2009), disrupts signaling mediated by jasmonic acid and abscisic acid Westwood et al, 2013a), and modifies plant-aphid interactions (Ziebell et al, 2011;Westwood et al, 2013b).…”

Antiviral Silencing and Suppression of Gene Silencing in Plants

“…2b of CMV (CM95R strain) and TAV exhibit high affinity for long dsRNAs and ds-sRNAs Duan et al 2012;Gonzalez et al 2012;Goto et al 2007). CMV 2b (Fny and SD strains) was also shown to interact with AGO1 through the PAZ-and partly PIWI domains and blocks RISC slicer activity (Duan et al 2012;Zhang et al 2006).…”

“…2b interacts both with AGO4-related siRNAs and with AGO4 protein through PAZ and PIWI domains. The interaction with 2b reduces AGO4 access to endogenous target loci and consequently modulates endogenous transcription to create a favorable niche for CMV infection (Duan et al 2012;Gonzalez et al 2010Gonzalez et al , 2012Hamera et al 2012). P38 of TCV may also suppress silencing at multiple levels.…”