The nanovirus U2 protein suppresses RNA silencing via three conserved cysteine residues

Nanoviruses have multipartite, circular, single-stranded DNA genomes and cause huge production losses in legumes and other crops. No viral suppressor of RNA silencing (VSR) has yet been reported from a member of the genus Nanovirus. Here, we demonstrate that the nanovirus U2 protein is a VSR. The U2 protein of milk vetch dwarf virus (MDV) suppressed the silencing of the green fluorescent protein (GFP) gene induced by single-stranded and double-stranded RNA, and the systemic spread of the GFP silencing signal. An electrophoretic mobility shift assay showed that the U2 protein was able to bind double-stranded 21-nucleotide small interfering RNA (siRNA). The cysteine residues at positions 43, 79 and 82 in the MDV U2 protein are critical to its nuclear localization, self-interaction and siRNA-binding ability, and were essential for its VSR activity. In addition, expression of the U2 protein via a potato virus X vector induced more severe necrosis symptoms in Nicotiana benthamiana leaves. The U2 proteins of other nanoviruses also acted as VSRs, and the three conserved cysteine residues were indispensable for their VSR activity.