The 2b protein encoded by Cucumber mosaic virus (CMV) and other cucumoviruses is multifunctional, having roles in local and systemic virus movement, symptom determination, evasion of defense mediated by salicylic acid, and in suppression of antiviral RNA silencing. It also perturbs silencing-mediated regulation of host transcripts, suggesting that another function of 2b protein is to manipulate host gene expression and physiology in a way that may aid the virus. The 2b proteins encoded by the various cucumoviruses (CMV strains, as well as Tomato aspermy virus and Peanut stunt virus) share conserved amino acid sequence domains, suggesting that these might determine specific functions of the protein. We analyzed the effect of mutations in these domains on functions of the 2b protein during viral infection. This revealed that binding of short RNAs, the key determinants of RNA silencing specificity, correlates with RNA silencing suppression activity. Two putative phosphorylation sites were found to be required for virus symptom induction, despite having no influence on RNA silencing suppression. This indicates that the ability to suppress silencing is not the only factor affecting symptom induction by the 2b protein. In accordance with this, our studies also revealed that the 2b protein acts synergistically with some other CMV product(s) to induce symptoms, and that the role of the 2b protein in symptom determination is host species specific.