Double-stranded RNA binding may be a general plant RNA viral strategy to suppress RNA silencing - PubMed (original) (raw)

Double-stranded RNA binding may be a general plant RNA viral strategy to suppress RNA silencing

Zsuzsanna Mérai et al. J Virol. 2006 Jun.

Abstract

In plants, RNA silencing (RNA interference) is an efficient antiviral system, and therefore successful virus infection requires suppression of silencing. Although many viral silencing suppressors have been identified, the molecular basis of silencing suppression is poorly understood. It is proposed that various suppressors inhibit RNA silencing by targeting different steps. However, as double-stranded RNAs (dsRNAs) play key roles in silencing, it was speculated that dsRNA binding might be a general silencing suppression strategy. Indeed, it was shown that the related aureusvirus P14 and tombusvirus P19 suppressors are dsRNA-binding proteins. Interestingly, P14 is a size-independent dsRNA-binding protein, while P19 binds only 21-nucleotide ds-sRNAs (small dsRNAs having 2-nucleotide 3' overhangs), the specificity determinant of the silencing system. Much evidence supports the idea that P19 inhibits silencing by sequestering silencing-generated viral ds-sRNAs. In this study we wanted to test the hypothesis that dsRNA binding is a general silencing suppression strategy. Here we show that many plant viral silencing suppressors bind dsRNAs. Beet yellows virus Peanut P21, clump virus P15, Barley stripe mosaic virus gammaB, and Tobacco etch virus HC-Pro, like P19, bind ds-sRNAs size-selectively, while Turnip crinkle virus CP is a size-independent dsRNA-binding protein, which binds long dsRNAs as well as ds-sRNAs. We propose that size-selective ds-sRNA-binding suppressors inhibit silencing by sequestering viral ds-sRNAs, whereas size-independent dsRNA-binding suppressors inactivate silencing by sequestering long dsRNA precursors of viral sRNAs and/or by binding ds-sRNAs. The findings that many unrelated silencing suppressors bind dsRNA suggest that dsRNA binding is a general silencing suppression strategy which has evolved independently many times.

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Figures

FIG. 1.

TCV CP binds long dsRNAs. (A) Long-dsRNA-binding activity of plant viral proteins was studied in gel mobility shift assays. Long dsRNAs were incubated with crude viral extracts prepared from symptomatic N. benthamiana (PoLV, CymRSV, BYV, PCV, PVY, PVX, CMV, TMV, and TAV), Arabidopsis (TCV), tobacco (TEV), or wheat leaves (BSMV). Extracts prepared from mock-inoculated plants (−) and extracts isolated from plants that were infected with PΔ14, a mutant PoLV that was unable to express P14 suppressor protein, were used as negative controls. (B) Plant-expressed TCV CP binds long ds-sRNA. Extracts were prepared from nontreated (−) N. benthamiana leaves and from leaves which were infiltrated with PoLV P14 (P14), TCV CP (CP), BYV P21 (P21), PCV P15 (P15), TEV HC-Pro (Hc-TEV), BSMV γB (γB), or reovirus Sigma3 (Reo). P14 and Reo were used as positive controls. Reo strongly binds long dsRNA but does not bind ds-sRNA. (C) High- and low-molecular-weight RNA gel blot analyses were carried out to study the effect of TCV CP and BYV P21 on accumulation of hairpin transcripts, GFP mRNAs, and sRNAs. N. benthamiana leaves were infiltrated (GFP-IR+35SGFP) with 35SGFP and with agrobacteria expressing hairpin GFP transcripts (−), or the leaves were coinfiltrated with GFP-IR+35SGFP and with TCV CP (CP) or with BYV P21 (P21). RNA samples were isolated at 3 d.p.i. GFP-ir indicates hairpin transcripts derived from GFP-IR, while GFP refers to GFP mRNA transcribed from 35SGFP (upper panel). Note that the probe we used for sRNA hybridization (bottom panel) detected both hairpin transcript- and GFP mRNA-derived sRNAs (GFP sRNA). (D) Effect of TCV CP and BYV P21 on accumulation of hairpin transcripts and hairpin-derived sRNAs. N. benthamiana leaves were infiltrated with GFP-IR (−) or coinfiltrated with GFP-IR and with TCV CP (CP) or with P21 (P21). Samples were taken at 3 d.p.i.

FIG. 2.

Many plant viral silencing suppressors can bind ds-sRNA. (A) ds-sRNA-binding activity of plant viral proteins was studied in gel mobility shift assays. Labeled synthetic 21-nt RNAs were annealed to form 21-nt ds-sRNAs (having a 19-nt duplex region and 2-nt 3′ overhangs) and used as probes in binding studies. The 21-nt ds-sRNAs were incubated with crude viral extract. Extracts prepared from mock-inoculated (−) plants and extracts isolated from plants that were infected with PΔ14, a mutant PoLV that was unable to express P14, were used as negative controls. The ds-sRNA-binding activity of extracts prepared from PVX P14 and PVX P19 recombinant PVXs, which express the P14 and P19 suppressor, respectively, shows that ds-sRNA-binding proteins are also active if they are expressed by heterologous viruses. * indicates a host-encoded nonspecific ds-sRNA-binding activity that can be frequently detected in extracts prepared from wild-type, mock-inoculated, agroinfiltrated, or virus-inoculated plants. (B) Plant-expressed viral silencing suppressors bind ds-sRNA. At 3 d.p.i. extracts were prepared from nontreated (−) N. benthamiana leaves and from leaves which were infiltrated with CymRSV P19 (P19), PoLV P14 (P14), PCV P15 (P15), TCV CP (CP), BYV P21 (P21), BSMV γB (γB), PVY HC-Pro (Hc-PVY), TEV HC-Pro (Hc-TEV), TAV 2b, or PVX P25 (P25). Suppressor extracts were probed with labeled 21-nt ds-sRNAs. (C and D) The suppressor proteins are the 21-nt ds-sRNA-binding factors in the suppressor extracts. Epitope-tagged suppressors were expressed in N. benthamiana leaves. To carry out antibody supershift assays, suppressor extracts were incubated with labeled 21-nt ds-sRNAs in the absence of antibody or in the presence of antibody specific against the respective epitope or in the presence of heterologous antibody. Note that Myc antibody-supershifted protein-ds-sRNA complexes stayed in the wells.

FIG. 3.

P21 and P15 are size-selective ds-sRNA-binding proteins, whereas TCV CP binds ds-sRNA size independently. (A to C) P21, P15, and TCV CP suppressor extracts were obtained from agroinfiltrated N. benthamiana leaves at 3 d.p.i. Direct competition assays were carried out with labeled 26-nt ds-sRNAs (upper panels) or with labeled 21-nt ds-sRNAs (bottom panels) and with 26-nt or 21-nt ds-sRNAs as unlabeled competitors. Competitors were added in increasing molar excess (25-, 50-, 100-, 200-, 400-, 800-, and 1,600-fold). Lanes −, no competitor. Note that P15 binds 26-nt ds-sRNAs very weakly, and thus a long exposure was required for a detectable shift.

FIG. 4.

Suppressors show different structural requirements for size-specific ds-sRNA binding. (A) The 19-nt dsRNA-binding activity of suppressor extracts. Labeled 19-nt blunt-ended dsRNA duplexes (19 dsRNA*) were incubated with the same suppressor extracts which were used for the binding studies shown at Fig. 2B. (B to G) Direct competition assays were carried out with 19 dsRNAs* as probes and with 19 dsRNAs or 21-nt ds-sRNAs as unlabeled competitors to test if the CymRSV P19 (B), TEV HC-Pro (C), PoLV P14 (D), and TCV CP (E) suppressors require 2-nt 3′ overhangs for ds-sRNA binding. As PCV P15 and BSMV γB barely bind 19 dsRNA, for the P15 (F) and γB (G) competition assays 21-nt ds-sRNAs* were used as the labeled probes. Unlabeled competitors were added in increasing molar excess (25-, 50-, 100-, 200-, 400-, 800-, and 1,600-fold). Lanes −, no competitor.

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Double-stranded RNA binding may be a general plant RNA viral strategy to suppress RNA silencing - PubMed (original) (raw)