A Viral Long Non-Coding RNA Modulates Viral RNA Silencing Suppressor and DCL4-Associated DRB4 Protein Interaction

The expression of the BNYVV silencing suppressor (VSR) p14 protein, together with the production of a viral non-coding RNA (ncRNA3), affects the long-distance movement on natural host Beta species. Through immunoprecipitation of Flag-p14 and expression of TurboID-p14 coupled to mass spectrometry, we identified several potential p14 cellular partners. These include proteins related to RNA metabolism, proteasome activation, and notably, SGS3-a key player in the siRNA transitivity pathway-and DRB4, the DCL4 cofactor described in A. thaliana silencing pathways. The interaction between p14 and DRB4 was specifically retrieved with the hypomorphic p14BA2 VSR mutant in nucleoli and was disrupted in the presence of ncRNA3, a condition that allows the viral mutant to move long distances. Subcellular fractionation of infected tissues revealed that ncRNA3 accumulates in the nucleus through its interaction with p14, while genomic RNA3 remains cytoplasmic. Our results suggest that a specific viral RNA is targeted to the nucleus to participate in silencing suppression function by destabilizing the p14-DRB4 complex. Moreover, the interaction between p14 and SGS3 provides a mechanistic explanation of the role of this VSR in silencing transitivity.