Tombusvirus P19 RNA silencing suppressor (RSS) activity in mammalian cells correlates with charged amino acids that contribute to direct RNA-binding

We have studied the RSS effect of P19 in mammalian cells, HEK293T, HeLa, and mouse embryonic fibroblasts. We have individually mutated 18 positively charged residues in P19 and found that 6 of these charged residues in P19 reduce its ability to suppress RNA interference. In each case, the reduction of silencing of RNA interference correlated with the reduced ability by these P19 mutants to bind siRNAs (small interfering RNAs).Our findings characterize a class of RNA-binding proteins that function as RSS moieties. We find a tight correlation between positively charged residues in P19 accounting for siRNA-binding and their RSS activity. Because P19’s activity is conserved in plant and animal cells, we conclude that its RSS function unlikely requires cell type-specific co-factors and likely arises from direct RNA-binding.RNA interference (RNAi) is a mechanism of gene regulation that is conserved in a wide range of organisms, from plants to animals [1-3]. RNAi is also reported to function as an antiviral defense against viral infections [4-9]. To counteract host cell RNAi-mediated immunity, viruses have evolved a variety of countermeasures, one of which is to encode RNA silencing suppressor (RSS) proteins [10-14]. Many RSS proteins have been reported; they include tomato bushy stunt virus (TBSV) P19 protein, rice hoja blanca virus NS3 protein, vaccinia virus E3L, influenza A virus NS1 protein, the Ebola virus VP35 protein, HIV-1 Tat protein, amongst others [5,15-23]. Currently, it is incompletely understood how each of these RSS proteins works mechanistically.One of the better characterized RSS is the P19 protein [13,16,24] encoded by TBSV and related tombusviruses [25]. An association between P19 and siRNAs has been demonstrated in infected plants [26]. The crystal structure of P19-siRNA complex reveals that a P19 homodimer tightly binds a single 21-nucleotide (nt) siRNA duplex in a positively charged surface cleft, but that this binding is progressively weaker for a s