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Cucumber Mosaic Virus 2b Protein Subcellular Targets and Interactions: Their Significance to RNA Silencing Suppressor Activity

¹Centro de Investigaciones Biológicas, CIB, CSIC. Ramiro de Maeztu 9, 28040 Madrid, Spain; ²A. N. Belozersky Institute of Physisco-Chemical Biology, Moscow State University. Leninskie Gory 119991, Russia; ³Department of Plant Sciences, University of Cambridge, Cambridge, CB2 3EA, United Kingdom; ⁴Department of Plant Pathology, Scottish Crop Research Institute, Dundee DD2 5DA, United Kingdom

The RNA silencing suppressor activity of the 2b protein of Cucumber mosaic virus (CMV) has been variously attributed to its nuclear targeting, its interaction with and inhibition of Argonaute 1 (AGO1), or its ability to bind small RNAs in vitro. In addition, the 2b ortholog of Tomato aspermy virus forms aggregates and binds RNAs in vitro. We have further studied the relationships between CMV 2b protein silencing suppressor activity and its subcellular distribution, protein-protein interactions in vivo, and interactions with small interfering RNAs in vitro. To do this, we tagged the protein with fluorescent markers and showed that it retained suppressor activity. We showed that the 2b protein is present in the nucleolus and that it self-interacts and interacts with AGO1 and AGO4 in vivo. Using a battery of mutants, we showed that the putative nuclear localization signals and phosphorylation motif of the 2b protein are not required for self-interaction or for interaction with AGO proteins. The occurrence of neither of these interactions or of nucleolar targeting was sufficient to provide local silencing-suppression activity. In contrast, the ability of the 2b protein to bind small RNAs appears to be indispensable for silencing suppressor function.