Abstract
BackgroundTombusvirus P19 is a protein encoded by tomato bushy stunt virus and related tombusviruses. Earlier studies have demonstrated that P19 is an RNA silencing suppressor (RSS) in plant cells. However, it has not been systematically investigated how P19 suppresses RNA interference in various mammalian cell settings.ResultsWe 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).ConclusionsOur 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.
Highlights
Tombusvirus P19 is a protein encoded by tomato bushy stunt virus and related tombusviruses
P19 suppresses shRNA- and siRNA- mediated RNA interference (RNAi) silencing in mammalian cells To investigate systematically tomato bushy stunt virus (TBSV) P19 suppression of RNAi-silencing in mammalian cells, we first studied its activity in HEK293T cells, where its RNA silencing suppressor (RSS) activity, using a V5-epitope tagged P19 expression vector, was previously reported as inactive [31]
We individually co-introduced into the transfected cells expression vectors for FLAG-tagged P19, HIV-1 Tat protein, VP35 Ebola virus protein, or a CMV-immediate early promoter driven expression vector that expresses a polypeptide of 45 repeated arginines
Summary
P19 suppresses shRNA- and siRNA- mediated RNAi silencing in mammalian cells To investigate systematically TBSV P19 suppression of RNAi-silencing in mammalian cells, we first studied its activity in HEK293T cells, where its RSS activity, using a V5-epitope tagged P19 expression vector, was previously reported as inactive [31]. We investigated the latter cells because the PACT protein has been reported to be an important component of the mammalian RNAi machinery [38,39] In both wild type and PACT−/− MEFs, P19 was effective in suppressing both sh- and si- RNA-mediated silencing of Fluc (Figure 7). These findings suggested that there is no cell type or species-specific differences between 293T cells and primary MEFs for P19’s RSS function and that this P19activity does not require PACT as a co-factor. Signals were visualized using chemiluminescence following the manufacturer’s protocol (Chemicon)
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