An in vitro reprogrammable antiviral RISC with size-preferential ribonuclease activity

Infection of Nicotiana benthamiana plants with Tomato bushy stunt virus (TBSV) mutants compromised for silencing suppression induces formation of an antiviral RISC (vRISC) that can be isolated using chromatography procedures. The isolated vRISC sequence-specifically degrades TBSV RNA in vitro, its activity can be down-regulated by removing siRNAs, and re-stimulated by exogenous supply of siRNAs. vRISC is most effective at hydrolyzing the ~4.8 kb genomic RNA, but less so for a ~2.2 kb TBSV subgenomic mRNA (sgRNA1), while the 3′ co-terminal sgRNA2 of ~0.9 kb appears insensitive to vRISC cleavage. Moreover, experiments with in vitro generated 5′ co-terminal viral transcripts show that RNAs of ~2.7 kb are efficiently cleaved while those of ~1.1 kb or shorter are unaffected. The isolated antiviral ribonuclease complex fails to degrade ~0.4 kb defective interfering RNAs (DIs) in vitro, agreeing with findings that in plants DIs are not targeted by silencing.