Aichi virus, which is associated with human acute gastroenteritis (41), is a member of the genus Kobuvirus of the family Picornaviridae (31, 42). This virus was first isolated in 1989 from a stool specimen from a patient with oyster-associated nonbacterial gastroenteritis in Aichi, Japan (41), and the complete genome sequence was determined in 1998 (42). The single-stranded, positive-sense RNA genome of Aichi virus consists of 8,280 nucleotides (nt) and a poly(A) tract and encodes a polyprotein of 2,432 amino acids (aa) (34,42). One of the features of the genome organization of Aichi virus is that it encodes a leader (L) polypeptide upstream of the capsid coding region.Among picornaviruses besides Aichi virus, aphtho-, cardio-, erbo-, and teschoviruses (10) and porcine enterovirus 8 (18) encode L proteins. The L protein of foot-and-mouth disease virus (FMDV), an aphthovirus, is produced in two forms, Lab and Lb, through initiation of translation at two in-frame AUG codons located 84 nt apart (3). Both forms of the FMDV L protein are papain-like thiol proteinases that cleave at their own C termini (23,30,36). In addition, the FMDV L protein cleaves a eukaryotic translation initiation factor, eIF4G (9), contributing to the shutoff of host cap-dependent protein synthesis. The shutoff of host protein synthesis leads to inhibition of alpha/beta interferon production. The L deletion mutant of FMDV can replicate and spread in non-interferon-responsive baby hamster kidney (BHK) cells (29). However, it can replicate but cannot spread in secondary cells from susceptible animals, such as bovine, ovine, and porcine cells (8). This is associated with the inability of the L deletion mutant to inhibit cap-dependent alpha/beta interferon mRNA translation (7,8). The erbovirus L protein also has autocatalytic activity but does not cleave eIF4GI (15).The cardiovirus L proteins have neither the amino acid sequence motif characteristic of picornavirus proteases nor autocatalytic activity. They are released from a polyprotein by viral protease 3C (27, 33). The L protein of the GDVII strain of Theiler's murine encephalomyelitis virus (TMEV) has been shown to be essential for neurovirulence in mice (6). In addition, it has been reported that L mutants of TMEV and mengovirus exhibit distinct growth properties depending on the host cells: certain L mutants of TMEV and mengovirus can spread in BHK cells but not in mouse L929 cells (1,40,43). For the GDVII strain of TMEV, it has been shown that the L deletion mutant has a defect in the assembly of virions in L929 cells (1), which mainly causes the inability of the mutant to spread in L929 cells. On the other hand, for the DA1 strain of TMEV and mengovirus, the L proteins have been reported to be involved in the inhibition of alpha/beta interferon production by L929 cells (40,44). Furthermore, in the case of encephalomyocarditis virus, it has been suggested that the L protein is involved in internal ribosome entry site-mediated translation of viral RNA (11,17).The Aichi virus L protein does...