Plautia stali intestine virus (PSIV) is a member of the family Dicistroviridae. Dicistroviruses, like picornaviruses, are thought to encode a 3C-like protease; however, the cleavage sites of dicistroviral nonstructural polyprotein precursors are unknown except for those in genome-linked virus protein (VPg) regions. We expressed part of the PSIV polyprotein in Escherichia coli and detected autoprocessed viral proteins. N-terminal sequence analysis of the autoprocessed proteins showed that Q/GWLSW and Q/NGVFD in the PSIV sequence correspond to 2C/3A and 3C/3D cleavage sites in picornaviruses. Alignments of deduced amino acid sequences of the "P3 region" suggest that these cleavage sites can be predicted.