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

ABSTRACT

The cucumber mosaic virus (CMV) 2b protein is a potent counter-defense factor and symptom determinant that inhibits antiviral silencing by titrating short double-stranded RNAs. Expression of the CMV subgroup IA strain Fny-CMV 2b protein 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 antiviral resistance and resistance to CMV's aphid vectors. However, in authentic viral infections, the Fny-CMV 1a protein modulates 2b-AGO1 interactions, inhibiting induction of AGO2-mediated virus resistance and aphid resistance. Contrastingly, 2b proteins encoded by the subgroup II strain LS-CMV and the recently discovered subgroup IA strain Ho-CMV induce no symptoms. Confocal laser scanning microscopy, bimolecular fluorescence complementation, and co-immunoprecipitation showed that Fny-CMV and Ho-CMV 2b proteins interact with Fny-CMV and LS-CMV 1a proteins, while the CMV-LS 2b protein cannot. However, Fny-CMV, Ho-CMV, and LS-CMV 2b proteins, all interacted with AGO1, but while AGO1-Fny2b complexes occurred in the nucleus and cytoplasm, 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 mRNA degradation. Fny-CMV 2b induced a fivefold increase in AGO2 accumulation, but LS-CMV and Ho-CMV 2b proteins induced only twofold increases. Thus, these 2b proteins bind AGO1 but are less effective at inhibiting AGO1 activity. We conclude that the intracellular localization of 2b-AGO1 complexes influences the degree to which a 2b protein inhibits microRNA-mediated host mRNA degradation and that cytoplasmic AGO1 has the strongest influence on miRNA-mediated cellular mRNA turnover. IMPORTANCE The cucumber mosaic virus (CMV) 2b protein was among the first discovered viral suppressors of RNA silencing. It has additional pro-viral functions through effects on plant defensive signaling pathways mediated by salicylic acid and jasmonic acid, the abscisic acid pathway and virus-induced drought resistance, and on host plant interactions with insect vectors. Many of these effects occur due to interaction with the important host RNA silencing component Argonaute 1 (AGO1). It was thought that only 2b proteins of "severe" CMV strains interacted with AGO1 and inhibited its microRNA-mediated "slicing" of cellular mRNAs and that the lack of interaction with AGO1 explained the moderate symptoms typically seen in plants infected with mild CMV strains. Our work overthrows this paradigm by showing that mild strain CMV 2b proteins can interact with AGO1, but their in vivo localization prevents them from interacting with AGO1 molecules present in the infected cell cytoplasm.