SUBSTRATE REQUIREMENTS OF HEPATITIS-C VIRUS SERINE PROTEINASE FOR INTERMOLECULAR POLYPEPTIDE CLEAVAGE IN ESCHERICHIA-COLI

Using as substrates a series of chimeric proteins containing various f ragments of the hepatitis C virus precursor polyprotein between Escher ichia coli maltose binding protein and dihydrofolate reductase, we ana lyzed the substrate requirements of hepatitis C viral serine proteinas e (Cpro-2) for intermolecular polypeptide cleavage in E. coli. Cpro-2- dependent substrate cleavage was observed in E. coli cells simultaneou sly transformed with expression plasmids for the Cpro-2 molecule and s ubstrate protein. The cleavage sites were estimated by determining the amino (N)-terminal aminoacid sequences of dihydrofolate reductase-fus ed processed products purified partially by affinity chromatography fr om the lysates, indicating that cleavage occurred at sites identical t o those observed in eukaryotic cells. Mutation analysis using the chim eric substrate indicated that the presence of cysteine and small uncha rged residues at positions P1 and P1', respectively, of the putative c leavage site is necessary for cleavage and that acidic residues in the region upstream of the cleavage site are required for efficient cleav age.