Strain-specific differences in the interactions of the cucumber mosaic virus 2b protein with the viral 1a and host Argonaute 1 proteins

Abstract

Abstract/SummaryThe cucumber mosaic virus (CMV) 2b protein is a potent counter-defense protein and symptom determinant that inhibits antiviral silencing by titration of short double-stranded RNAs. Expression of the 2b protein from the CMV Subgroup IA strain Fny-CMV in transgenic Arabidopsis thaliana plants disrupts microRNA-mediated cleavage of host mRNAs by binding ARGONAUTE 1 (AGO1), leading to symptom-like phenotypes. This also triggers AGO2-mediated resistance against CMV and strong resistance to CMVs aphid vectors, which would be deleterious to viral fitness. However, in authentic viral infections the Fny-CMV 1a protein modulates 2b-AGO1 interactions, which inhibits induction of AGO2-mediated virus resistance and resistance to aphid vectors. Contrastingly, the 2b proteins encoded by the Subgroup II LS-CMV strain or the recently discovered Subgroup IA strain Ho-CMV induce no apparent symptoms. Confocal laser scanning microscopy, bimolecular fluorescence complementation and co-immunoprecipitation showed that the Fny-CMV and Ho-CMV 2b proteins interact with the Fny-CMV and LS-CMV 1a proteins whilst the CMV-LS 2b protein does not. However, the Fny-CMV, Ho-CMV and LS-CMV 2b proteins all interacted with AGO1, but while AGO1-Fny2b complexes occurred in the host cell nucleus and cytoplasm, the corresponding AGO1-2b complexes for LS-CMV and Ho-CMV accumulated almost exclusively in nuclei. AGO2 transcript accumulation was used to assess the inhibition of AGO1-mediated miRNA-regulated mRNA cleavage. While Fny-CMV 2b induced a five-fold increase in AGO2 accumulation, the LS-CMV and Ho-CMV 2b proteins induced only two-fold increases. Thus, these 2b proteins bind AGO1 but are less effective at inhibiting AGO1 activity. We conclude that the intracellular localization sites of 2b-AGO1 complexes influences the degree to which a 2b protein can inhibit microRNA-mediated host mRNA degradation and that cytoplasmic AGO1 has the strongest influence on miRNA-mediated cellular mRNA turnover.