Novel recognition sequence of coxsackievirus 2A proteinase

Coxsackievirus B1 (CVB1) 2A proteinase (2Apro) is a cysteine proteinase that cleaves the viral monocistronic polyprotein between the C-terminus of the VP1 region and the N-terminus of the 2A region, and also shuts off translational initiation in host cells by cleavage of eukaryotic initiation factor 4G (eIF4G) isoforms. We expressed in Escherichia coli a series of fusions in which various C-terminal fragments of VP1 were linked to the N-terminus of 2Apro, and we also employed site-directed mutagenesis to introduce mutations of several amino acid residues. Our results showed that the presence of the C-terminal three amino acid residues of VP1 at the N-terminus of 2Apro is sufficient for specific self-cleavage between VP1 and 2Apro to generate mature 2Apro, but the P4 amino acid also plays an important role. We further found that 2Apro cleaves the amino acid sequence Leu-Val-Pro-Arg-∗Gly-Ser (LVPRGS motif), which is the target sequence of thrombin.