Here, we report that the RSS activity of FLAG-tagged TBSV P19 is conserved in human HEK293T cells and mouse embryonic fibroblasts (MEF). We also found that FLAG-P19 has similar RSS activity in HeLa cells (Additional file 1: Figure S1) as in HEK293T cells. Our work revisits earlier reports that untagged P19 exhibited RSS activity in HeLa cells  while epitope-tagged P19 showed little to no RSS activity in 293T cells , suggesting that neither epitope-tagging nor cell type-specific factors influence inherent P19 RSS activity in mammalian cells. We should, however, caution that our assay approaches are similar to, but not identical with, the previous studies; hence, we cannot exclude that small non-identical experimental details may account for the dissimilar findings.
In trying to characterize P19’s RSS activity in animal cells, we point mutated 18 positively charged lysine or arginine amino acids to neutral amino acid counterparts. In these analyses, we discovered 6 positively charged residues that are important for P19-mediated RSS effect. Mutation of these residues also abrogated the ability of the respective protein to bind siRNA. Our mutagenesis results on P19 in animal cells can be compared to parallel point mutation studies of P19 in plant cells. Thus, Chu et al. had shown that mutations upstream from residue K71 or downstream from residue R85 did not noticeably affect the ability of TBSV to systematically invade spinach plants . Mutation of R72, R75 or R85 displayed a reduced lethal necrosis phenotype in three different plants (N. benthamiana, N. clevelandii and spinach) , and the mutation of R43 was shown to decrease the stability of interaction between P19 (R43) protein and siRNA in N. benthamiana. Crystal structure of P19 revealed that K71 and R115 form direct contacts with phosphate groups in the siRNA . Viewed in the above context, our results in mammalian cells show that K71 is important for RSS activity of P19, but mutation of R115 did not affect this activity. Previously, mutation of K60 in infected plants showed necrotic lower leaves, but not systemic collapse ; and our results also showed that mutation of K60A greatly reduced the RSS effect and RNA binding activity of P19. Therefore, for the most part, those positively charged residues that contribute to RSS activity of P19 maintain similar functional roles in mammalian and plant cells, further supporting the notion that the P19 RSS effect in plants and animals arises from co-factor independent direct RNA-binding.
An unexpected observation from our work is that sh-/si-Fluc-mediated silencing was more efficient in PACT−/− MEFs (Figure 7) than TRBP−/− MEFs (Figure 8). These results suggest a role for TRBP in siRNA loading into RISC that may not be equivalently substituted by PACT. Although both TRBP and PACT are found in the 500 kDa complex with Dicer and Ago2 and contribute to the processing of miRNA and shRNA, increasingly nuanced studies had indicated that TRBP appears to have a more critical role than PACT [44, 45] in the cellular RNAi process. Indeed, a recent study showed that TRBP, but not PACT, can directly influence the specificity of Dicer cleavage of pre-miRNA . Relevant to P19, our results suggest that neither PACT nor TRBP plays an essential co-factor role for P19’s RSS activity.
Our results here reinforce the earlier notion that many RNA-binding proteins can function as RNAi-suppressors [5, 19, 20, 48, 49] . Indeed, we have previously shown , and reaffirmed in Figure 1, that simple RNA-binding polypeptides like poly-arginine can exhibit RNAi-suppressing activity. Viral RNA-binding proteins like HIV-1 Tat and HTLV-1 Rex have evolved to serve virus-specific roles, but consistent with our current findings on P19, they also show RSS activity [20, 50], suggesting that they participate in aspects of virus-cellular RNAi engagement . RNAi activity contributes wide-ranging and diverse roles in cellular proliferation, gene regulation, development, metabolism, immune response, infection, and pathogenesis [51–54]. Physiologically, a reasonable notion is that organisms should have evolved biological means that either enhance or repress RNAi activities. We hypothesize that many cellular RNA-binding proteins  may possess suppressive RSS activities while others like TRBP may positively enhance RNAi function. Recently, a computational strategy was used to screen for small molecules with the potential to inhibit miRNA functions . Going forward, further work on the discovery of small molecule inhibitors may help us develop tools to understand better how cellular RNA-binding proteins influence RNAi functions in cells.