RNAi-associated ssRNA-specific ribonucleases in Tombusvirus P19 mutant-infected plants and evidence for a discrete siRNA-containing effector complex

Abstract

Tomato bushy stunt virus (TBSV) and other tombusviruses encode a p19 protein (P19), which is a suppressor of RNAi. Wild-type TBSV or p19-defective mutants initially show a similar infection course in Nicotiana benthamiana, but the absence of an active P19 results in viral RNA degradation followed by recovery from infection. P19 homodimers sequester 21-nt virus-derived duplex siRNAs, and it is thought that this prevents the programming of an antiviral RNAinduced silencing complex to avoid viral RNA degradation. Here we report on chromatographic fractionation (gel filtration, ion exchange, and hydroxyapatite) of extracts from healthy or infected Nicotiana benthamiana plants in combination with in vitro assays for ribonuclease activity and detection of TBSV-derived siRNAs. Only extracts of plants infected with p19 mutants provided a source of sequence-nonspecific but ssRNA-targeted in vitro ribonuclease activity that coeluted with components of a wide molecular weight range. In addition, we isolated a discrete Ϸ500-kDa protein complex that contained Ϸ21-nt TBSV-derived siRNAs and that exhibited ribonuclease activity that was TBSV sequencepreferential, ssRNA-specific, divalent cation-dependent, and insensitive to a ribonuclease inhibitor. We believe that this study provides biochemical evidence for a virus-host system that infection in the absence of a fully active RNAi suppressor induces ssRNA-specific ribonuclease activity, including that conferred by a RNA-induced silencing complex, which is likely the cause for the recovery of plants from infection.suppression ͉ silencing ͉ RNA-induced silencing complex

Arabidopsis DRB4, AGO1, AGO7, and RDR6 participate in a DCL4-initiated antiviral RNA silencing pathway negatively regulated by DCL1

“…S1, compare lanes 2 and 3), suggesting that DCL1 does not influence TCV replication (19). It has been established that P19 suppresses silencing by binding to siRNAs and inhibiting the formation of RISC complex (20,21). TCV CP, however, functions to prevent DCL4-mediated processing of viral dsRNA, a process thought to not only feed RISC with siRNAs, but also destroy the viral RNA itself (6,22).…”

The 21-Nucleotide, but Not 22-Nucleotide, Viral Secondary Small Interfering RNAs Direct Potent Antiviral Defense by Two Cooperative Argonautes inArabidopsis thaliana

“…Viral secondary siRNAs may guide specific silencing of viral RNAs in an AGO-containing effector complex as shown in previous studies that did not distinguish between viral primary and secondary siRNAs (Omarov et al, 2007;Pantaleo et al, 2007;Azevedo et al, 2010). However, the step of dicing to produce viral secondary siRNAs may be sufficient to confer virus resistance because it would destroy the viral RNAs targeted for dsRNA synthesis by the RDR6 pathway.…”

HIV-1 Tat RNA silencing suppressor activity is conserved across kingdoms and counteracts translational repression of HIV-1

“…Benasser, et al (16) determined that Tat RSS activity was genetically separable from Tat transcriptional activity but segregated with the arginine-rich double-stranded RNA-binding domain (16). A similar RNA binding domain is conserved in plant virus RSS and confers interaction with miRNA duplexes in a sequence nonspecific manner that blocks programming of RISC for RNA silencing (19)(20)(21). For Tat, tomato bushy stunt virus (TBSV) P19 and other Tombusvirus RSS, mutation of this domain eliminates RNA silencing suppression (16,22), which posits a common mechanism of activity.…”

“…For Tat, tomato bushy stunt virus (TBSV) P19 and other Tombusvirus RSS, mutation of this domain eliminates RNA silencing suppression (16,22), which posits a common mechanism of activity. The outcome of P19 mutation is reduced TBSV RNA, which culminates in impaired virus propagation and attenuation of disease pathogenesis (19)(20)(21). To investigate whether HIV-1 Tat RSS protects against viral RNA degradation, we compared the activity of Tat and P19 in plant protoplasts and animal cells.…”