Two chimeras of cucumber mosaic virus (CMV), FCb7(2b)-CMV and FRad35(2b)-CMV, with the 2b genes of strains Cb7-CMV and Rad35-CMV, respectively, in an Fny-CMV background, gave different responses on Nicotiana glutinosa: FCb7(2b)-CMV induced systemic necrosis while FRad35(2b)-CMV caused only mild mosaic. This differential virulence was attributable to the nature of amino acid 55 of their 2b proteins. However, sequence analysis revealed that Leu(55) of the 2b protein was necessary but not sufficient for FCb7(2b)-CMV to induce systemic necrosis. Surprisingly, inhibition of translation of the 2a/2b overlapping region of the 2a protein in FCb7(2b)-CMV led to a loss of systemic necrosis and a reduction in accumulation of viral progeny RNAs. The 2a/2b overlapping region of Fny-CMV had a similar effect on virulence and viral accumulation. Thus, the 2a protein C-terminus of subgroup I strains, as well as the 2b protein, play a role in symptom induction and accumulation of viral RNAs.