RNA2, the short segment of the genome of Dicenthrarchus labrax encephalitis virus (DIEV), a fish nodavirus causing seabass encephalitis, was cloned. Sequence analysis revealed that DIEV RNA2 contains a single open reading frame (ORF), which carries the catalytic D-75 residue but lacks the site for autocatalytic proteolysis, the process yielding the two capsid proteins of insect nodaviruses. Nevertheless, SDS-PAGE analysis of mature virions revealed a 43-45 kDa protein doublet. In order to determine the mechanism of synthesis of the two capsid proteins in DIEV, wild type and mutagenized forms of RNA2 were expressed in cell-free translation extracts and in transfected cells. Results showed that, despite the presence of the catalytic D-75 residue, the DIEV capsid protein doublet did not result from the assembly-dependent autocatalytic cleavage of a protein precursor. Moreover, our data show that, although suggested by sequence analysis, the DIEV capsid protein doublet results from neither an alternative initiation codon usage nor from a--1 ribosomal frameshift. Results of cell-free translation experiments demonstrate that the capsid protein doublet neither results of the proteolytic cleavage of a precursor nor of a degradation process. Kinetics of capsid protein synthesis in cell-free translation programmed with RNA2 revealed, instead, that the two capsid proteins are cosynthesized. Together these data strongly suggest that the DIEV capsid protein doublet results from cotranslational modification(s) of the ORF-encoded protein.