Picornavirus genomes are translated into a single large polyprotein, which is processed by virus-encoded proteases into individual functional proteins. 3C of all picornaviruses is a protease, and the leader (L) and 2A proteins of some picornaviruses are also involved in polyprotein processing. Aichi virus (AiV), which is associated with acute gastroenteritis in humans, is a member of the genus Kobuvirus of the family Picornaviridae. The AiV L and 2A proteins have already been shown to exhibit no protease activity. In this study, we investigated AiV polyprotein processing by 3C and 3CD using a cell-free translation system. 3C and 3CD were capable of processing the polyprotein in trans; 3C, however, cleaved the VP1/2A site inefficiently, while 3CD cleaved this site almost completely. Mammalian two-hybrid and coimmunoprecipitation assays showed an interaction between 2A and 3CD. Using a 3CD mutant and various 2A mutants of substrate proteins, we showed a clear correlation between the 2A-3CD interaction and the VP1/2A cleavage by 3CD. Thus, this study suggests that tight interaction of 3CD with the 2A region of a precursor protein is required for efficient cleavage at the VP1/2A site. (C) 2011 Elsevier B.V. All rights reserved.