The cucumber mosaic virus 1a protein regulates interactions between the 2b protein and ARGONAUTE 1 while maintaining the silencing suppressor activity of the 2b protein

The cucumber mosaic virus (CMV) 2b viral suppressor of RNA silencing (VSR) is a potent counter-defense and pathogenicity factor that inhibits antiviral silencing by titration of short double-stranded RNAs. It also disrupts microRNA-mediated regulation of host gene expression by binding ARGONAUTE 1 (AGO1). But in Arabidopsis thaliana complete inhibition of AGO1 is counterproductive to CMV since this triggers another layer of antiviral silencing mediated by AGO2, de-represses strong resistance against aphids (the insect vectors of CMV), and exacerbates symptoms. Using confocal laser scanning microscopy, bimolecular fluorescence complementation, and co-immunoprecipitation assays we found that the CMV 1a protein, a component of the viral replicase complex, regulates the 2b-AGO1 interaction. By binding 2b protein molecules and sequestering them in P-bodies, the 1a protein limits the proportion of 2b protein molecules available to bind AGO1, which ameliorates 2b-induced disease symptoms, and moderates induction of resistance to CMV and to its aphid vector. However, the 1a protein-2b protein interaction does not inhibit the ability of the 2b protein to inhibit silencing of reporter gene expression in agroinfiltration assays. The interaction between the CMV 1a and 2b proteins represents a novel regulatory system in which specific functions of a VSR are selectively modulated by another viral protein. The finding also provides a mechanism that explains how CMV, and possibly other viruses, modulates symptom induction and manipulates host-vector interactions. Author summary Cucumber mosaic virus (CMV) causes disease in over a thousand plant species including many crops. Aphids, insects with probing mouthparts that introduce virus particles directly into host cells, transmit CMV between plants. The 2b protein is the smallest protein encoded by CMV but has multiple functions. The 2b protein can disrupt the host RNA silencing pathways. These comprise mechanisms that regulate host gene expression (using microRNAs) and that degrade viral RNA molecules (using short-interfering RNAs). The 2b protein can bind to short-interfering RNAs to inhibit host resistance to virus infection and it can also bind the host protein Argonaute 1, disrupting microRNA-regulated host gene expression. However, in Arabidopsis thaliana excessive inhibition of Argonaute 1 activity may trigger excessive damage to the host, enhance CMV resistance, and render plants toxic to aphids, which may be deleterious to virus transmission. We found that another CMV protein, the 1a protein (a component of the CMV replication machinery), binds to 2b protein molecules and limits 2b-Argonaute 1 interactions without causing any detectable impairment of the ability of the 2b protein to counteract antiviral RNA silencing. This allows CMV to inhibit resistance against itself and its vectors, while preventing excessive damage to its host plant.