Assessment of the RNA Silencing Suppressor Activity of Protein P0 of Pepper Vein Yellows Virus 5: Uncovering Natural Variability, Relevant Motifs and Underlying Mechanism

SIMPLE SUMMARY: Viruses must face their host’s antiviral defenses during the infection process. In plants, one of the main antiviral mechanisms is based on RNA silencing, which ultimately leads to the degradation of viral RNA. To cope with this host’s strategy, plant viruses encode proteins that act as suppressors of RNA silencing (VSRs). These proteins are structurally diverse and may differ in their mode of action. Due to their essential role for viral fitness, VSRs are appealing targets for virus control. In this study, we provide significant information on the structural and functional properties of the VSR encoded by Pepper vein yellows virus 5, a pathogen belonging to a group of emergent and closely related poleroviruses that threaten pepper cultivation worldwide. This virus caused an outbreak nine years ago in Spain and we also present data showing that the virus is still present in the country. ABSTRACT: Pepper vein yellows virus 5 (PeVYV-5) belongs to a group of emerging poleroviruses (family Solemoviridae) which pose a risk to pepper cultivation worldwide. Since its first detection in Spain in 2013 and the determination of the complete genome sequence of an isolate in 2018, little is known on the presence, genomic variation and molecular properties of this pathogen. As other members of genus Polerovirus, PeVYV-5 encodes a P0 protein that was predicted to act as viral suppressor of RNA silencing (VSR), one of the major antiviral defense mechanisms in plants. The results of the present work have indicated that PeVYV-5 P0 is a potent VSR, which is able to induce the degradation of Argonaute (AGO) endonucleases, the main effectors of RNA silencing. New viral isolates have been identified in samples collected in 2020–2021 and sequencing of their P0 gene has revealed limited heterogeneity, suggesting that the protein is under negative selection. Analysis of natural and engineered P0 variants has pinpointed distinct protein motifs as critical for the VSR role. Moreover, a positive correlation between the VSR activity of the protein and its capability to promote AGO degradation could be established, supporting that such activity essentially relies on the clearance of core components of the RNA silencing machinery.