Key importance of small RNA binding for the activity of a GW motif-containing RNA silencing suppressor

Abstract

Background: GW/WG motif-containing viral suppressors of RNA silencing (VSRs) have been proposed to act through interaction with Argonaute (AGO) proteins. Results: The activity of a GW motif-containing VSR was found to rely on small RNA binding capability rather than on AGO interaction. Conclusion: Overlapping signals in VSRs may lead to misinterpretation of relevant molecular traits. Significance: Knowing primary target(s) of VSRs is critical for better understanding of the host-virus arms race.

Viruses express viral suppressors of RNA silencing (VSRs) to counteract RNA silencing-based host defenses. Although virtually all stages of the antiviral silencing pathway can be inhibited by VSRs, small RNAs (sRNAs) and Argonaute (AGO) proteins seem to be the most frequent targets. Recently, GW/WG motifs of some VSRs have been proposed to dictate their suppressor function by mediating interaction with AGO(s). Here we have studied the VSR encoded by Pelargonium line pattern virus (family Tombusviridae). The results show that p37, the viral coat protein, blocks RNA silencing. Site-directed mutagenesis of some p37 sequence traits, including a conserved GW motif, allowed generation of suppressor-competent and -incompetent molecules and uncoupling of the VSR and particle assembly capacities. The engineered mutants were used to assess the importance of p37 functions for viral infection and the relative contribution of diverse molecular interactions to suppressor activity. Two main conclusions can be drawn: (i) the silencing suppression and encapsidation functions of p37 are both required for systemic Pelargonium line pattern virus infection, and (ii) the suppressor activity of p37 relies on the ability to bind sRNAs rather than on interaction with AGOs. The data also caution against potential misinterpretations of results due to overlap of sequence signals related to distinct protein properties. This is well illustrated by mutation of the GW motif in p37 that concurrently affects nucleolar localization, efficient interaction with AGO1, and sRNA binding capability. These concomitant effects could have been overlooked in other GW motif-containing suppressors, as we exemplify with the orthologous p38 of turnip crinkle virus.