Nicotiana benthamiana Raw sequence reads. Nicotiana benthamiana

Potyvirus HCPro is a much studied suppressor of RNA silencing in plants that in the case of Turnip mosaic virus was recently shown to bind to small RNAs (sRNAs) to viral sequences (vsRNAs) in vivo, preventing their loading into the argonaute (AGO) effector components of the antiviral RNA-induced silencing complexes (RISCs). In this work, we have performed deep sequencing of the short RNAs of less than 500 nucleotides in length that could be bound to HCPro from Potato virus Y (PVY), expressed from a virus vector and purified from infected Nicotiana benthamiana plants. We found that PVY HCPro bound in vivo mainly sRNAs of 21- and 22- nucleotides (nts) in length and to a much lesser extent also some larger RNAs. Furthermore, we found a binding preference for viral over plant sequences in both cases, and within the HCPro-bound vsRNAs fraction a bias towards those containing 5’ terminal adenines, not observed in the bound fraction of sRNAs to plant sequences. Our data support vsRNA-sequestering as a mode of action of HCPro in silencing suppression, and suggest also a potential targeting of vsRNAs that could load into AGO2, a RISC effector whose antiviral role has been highlighted by recent studies.