RNA silencing (termed RNA interference [RNAi] in animals) is an RNA-based eukaryotic gene regulatory system that plays essential roles in many biological processes. RNA silencing is induced by accumulation of double-stranded RNAs (dsRNAs). dsRNAs are first processed by an RNase III-like nuclease called DICER (in plants it is termed DICER-LIKE [DCL]) into (21- to 25-nucleotide [nt]) small dsRNAs (ds-sRNAs) having 2-nt 3′ overhangs, and then these sRNAs incorporate into different silencing effector complexes. In the active effector complexes sRNAs are present as single-stranded molecules, which guide these complexes to the complementary nucleic acids for suppression (2
In plants, different dsRNA precursors are processed by distinct DCLs into functionally different short (21- to 22-nt) and long (23- to 25-nt) sRNAs (24
). Short sRNAs guide a multicomponent nuclease (RNA-induced silencing complex [RISC]) to homologous mRNAs for suppression. RISC cleaves targeted mRNA in the case of (near) perfect base pairing between mRNA and guide RNA. When the guide RNA is only partially complementary to the mRNA, RISC mediates translational repression. Short sRNAs could also provide (directly or indirectly) sequence specificity for plant-encoded RNA-dependent RNA polymerase (RdRP) which transforms homologous mRNAs into dsRNAs, thus amplifying silencing (3
). Long sRNAs play a role in transcriptional silencing by directing the histone and DNA methylation of homologous DNA (66
RNA silencing plays important antiviral roles in plants and animals (15
). In plants, double-stranded replicative intermediates of RNA viruses or strongly structured segments of viral mRNAs (40
) are processed by DCL2 and perhaps by other DCL enzymes into viral sRNAs (18
), short 21-nt dsRNAs having 2-nt 3′ overhangs (also called viral short interfering RNAs [siRNAs]). Viral sRNAs incorporate and target RISC to viral mRNAs for suppression, and thereby silencing could reduce pathogen levels in the infected cells. Virus-induced silencing can also act as a systemic defense system. Viral sRNAs are supposed to spread 10 to 15 cell layers and activate silencing in still noninvaded neighboring cells, limiting the extent of virus invasion (27
To counteract RNA silencing most plant viruses express silencing suppressor proteins (44
). Since silencing suppressors operate differently in widely used silencing inhibition assays it is believed that viral suppressors target different steps of the silencing response (64
). However, as dsRNAs play key roles in silencing, it was speculated that binding of a dsRNA component of the silencing machinery could be a frequently used viral suppression strategy (53
). In theory, dsRNA-binding silencing suppressors could target silencing response at two different steps, by binding and sequestering the silencing inducer long viral dsRNAs and/or by binding and sequestering viral ds-sRNAs.
Recent results suggest that two unrelated viral suppressors (tombusvirus P19 and closterovirus P21) inhibit silencing by binding viral ds-sRNAs (11
). It has been shown that P19 sequesters viral sRNAs in infected plants, and thus it depletes the specificity determinant of antiviral silencing effector complexes (31
). As P21 also binds ds-sRNAs in plant cells (11
), it is likely that sequestering of ds-sRNAs is a silencing suppression strategy that has evolved at least twice independently. Moreover, the P19-related aureusvirus P14 silencing suppressor is also a dsRNA-binding protein (39
Interestingly, P19 and P14 bind dsRNAs differently. P19 binds dsRNAs size-selectively (62
) and it binds efficiently only short, 21-nt ds-sRNAs, whereas P14 efficiently forms complexes with long dsRNAs and with 21-nt ds-sRNAs (39
). These differences could be functionally relevant. P14 might suppress silencing by sequestering both long viral dsRNAs and viral ds-sRNAs, whereas P19 sequesters only viral ds-sRNAs. Moreover, flock house virus B2 is also a dsRNA-binding protein that could suppress antiviral silencing in Caenorhabditis elegans
by targeting the dsRNA precursor of viral sRNAs (38
In this study we wanted to test the hypothesis that dsRNA binding is a general silencing suppression strategy of plant RNA viruses. Here we show that many viral suppressors can bind dsRNAs. CP of Turnip crinkle virus (TCV), a carmovirus, like P14, binds dsRNA without obvious size selection. By contrast, P15 of Peanut clump virus (PCV), a pecluvirus, γB of Barley stripe mosaic virus (BSMV), a hordeivirus, P21 of Beet yellows virus (BYV), a closterovirus, and HC-Pro of Tobacco etch virus (TEV), a potyvirus, like P19, are size-specific ds-sRNA-binding silencing suppressors. These proteins efficiently form complexes with 21-nt ds-sRNA but fail to bind long dsRNA. We have also demonstrated that the structural basis of size-selective ds-sRNA binding is different for P19 and for PCV P15, BSMV γB, and TEV HC-Pro size-specific dsRNA-binding suppressors.
Our data strongly suggest that dsRNA binding is a general plant viral silencing suppression strategy which has evolved independently many times.