RNA silencing regulates plant gene expression and antiviral defenses and functions by cleaving target RNAs or repressing translation. As a counter defense, many plant viruses encode suppressor proteins that sequester small RNAs or inactivate Argonaute (AGO) proteins. All known plant virus silencing suppressor activities eventually inhibit the degradation of target mRNAs. Using a transiently expressed green fluorescent protein (GFP) reporter gene, we show that Tomato ringspot virus (ToRSV) coat protein (CP) is a suppressor of RNA silencing that enhances GFP expression but does not prevent the degradation of the GFP mRNA or the accumulation of GFP small interfering RNAs (siRNAs). Coexpression of the CP with GFP resulted in increased association of residual GFP mRNAs with polysome fractions and reduced association of GFP siRNAs with monosome fractions. AGO1 was co-immunoprecipitated with the CP and CP expression destabilized AGO1. A WG motif within the CP was critical for the enhanced GFP expression, AGO1 interaction, and AGO1 destabilization, suggesting that the ToRSV CP acts as an AGO-hook protein and competes for AGO binding with a plant cellular GW/WG protein involved in translation repression.

Pokeweed antiviral protein (PAP) is a glycosidase of plant origin that has been shown to depurinate some viral RNAs in vitro. We have demonstrated previously that treatment of Brome mosaic virus (BMV) RNAs with PAP inhibited their translation in a cell-free system and decreased their accumulation in barley protoplasts. In the current study, we map the depurination sites on BMV RNA3 and describe the mechanism by which replication of the viral RNA is inhibited by depurination. Specifically, we demonstrate that the viral replicase exhibited reduced affinity for depurinated positive-strand RNA3 compared with intact RNA3, resulting in less negative-strand product. This decrease was due to depurination within the intergenic region of RNA3, between ORF3 and 4, and distant from the 3′ terminal core promoter required for initiation of negative-strand RNA synthesis. Depurination within the intergenic region alone inhibited the binding of the replicase to full-length RNA3, whereas depurination outside the intergenic region permitted the replicase to initiate negative-strand synthesis; however, elongation of the RNA product was stalled at the abasic nucleotide. These results support a role of the intergenic region in controlling negative-strand RNA synthesis and contribute new insight into the effect of depurination by PAP on BMV replication.

Ribosome inactivating proteins are glycosidases synthesized by many plants and have been hypothesized to serve in defence against pathogens. These enzymes catalytically remove a conserved purine from the sarcin/ricin loop of the large ribosomal RNA, which has been shown in vitro to limit protein synthesis. The resulting toxicity suggests that plants may possess a mechanism to protect their ribosomes from depurination during the synthesis of these enzymes. For example, pokeweed antiviral protein (PAP) is cotranslationally inserted into the lumen of the endoplasmic reticulum and travels via the endomembrane system to be stored in the cell wall. However, some PAP may retrotranslocate across the endoplasmic reticulum membrane to be released back into the cytosol, thereby exposing ribosomes to depurination. In this work, we isolated and characterized a complexed form of the enzyme that exhibits substantially reduced activity. We showed that this complex is a homodimer of PAP and that dimerization involves a peptide that contains a conserved aromatic amino acid, tyrosine 123, located in the active site of the enzyme. Bimolecular fluorescence complementation demonstrated that the homodimer may form in vivo and that dimerization is prevented by the substitution of tyrosine 123 for alanine. The homodimer is a minor form of PAP, observed only in the cytosol of cells and not in the apoplast. Taken together, these data support a novel mechanism for the limitation of depurination of autologous ribosomes by molecules of the protein that escape transport to the cell wall by the endomembrane system.

RNA toxins are a group of enzymes primarily synthesized by bacteria, fungi, and plants that either cleave or depurinate RNA molecules. These proteins may be divided according to their RNA substrates: ribotoxins are nucleases that cleave ribosomal RNA (rRNA), ribosome inactivating proteins are glycosidases that remove a base from rRNA, messenger RNA (mRNA) interferases are nucleases that cleave mRNAs, and anticodon nucleases cleave transfer RNAs (tRNAs). These modifications to the RNAs may substantially alter gene expression and translation rates. Given that some of these enzymes cause cell death, it has been suggested that they function mainly in defense, either to kill competing cells or to elicit suicide and thereby limit pathogen spread from infected cells. Although good correlations have been drawn between their enzymatic functions and toxicity, recent work has shown that some RNA toxins cause apoptosis in the absence of damage to RNA and that defense against pathogens can be achieved without host cell death. Moreover, a decrease in cellular translation rate, insufficient to cause cell death, allows some organisms to adapt to stress and environmental change. Although ascribing effects observed in vitro to the roles of these toxins in nature has been challenging, recent results have expanded our understanding of their modes of action, and emphasized the importance of these toxins in development, adaptation to stress and defense against pathogens.

Packaging of the segmented RNA genome of Brome mosaic virus (BMV) into discrete particles is an essential step in the virus life cycle; however, questions remain regarding the mechanism of RNA packaging and the degree to which the viral coat protein controls the process. In this study, we used a plant-derived glycosidase, Pokeweed antiviral protein, to remove 14 specific bases from BMV RNA3 to examine the effect of depurination on virus assembly. Depurination of A771 within ORF3 and A1006 in the intergenic region inhibited coat protein binding and prevented RNA3 incorporation into particles. The disruption of interaction was not based on sequence identity, as mutation of these two purines to pyrimidines did not decrease coat protein-binding affinity. Rather, we suggest that base removal results in decreased thermodynamic stability of local RNA structures required for packaging, and that this instability is detected by coat protein. These results describe a new level of discrimination by coat protein, whereby it recognizes damage to specific viral RNA elements in the form of base removal and selects against incorporating the RNA into particles.