PROCESSING OF HEPATITIS-C VIRAL POLYPROTEIN IN ESCHERICHIA-COLI

Two proteinase activities, encoded by hepatitis C virus (HCV), Cpro-1 and Cpro-2. Cpro-1 and Cpro-2 appear to process the precursor polyprot ein from which they originate. Mutant HCV polypeptides containing the region for these proteinases were produced in Escherichia coli as fusi on proteins. The N- and C-terminal ends of the HCV polypeptides were f used with the E. coli maltose-binding protein (MBP) and E. coli dihydr ofolate reductase (DHFR), respectively. The proteinase activities clea ved the fusion polypeptides by the same processing pathway used in euk aryotic protein production systems. The N-terminal amino acid (aa) seq uences of the processed fusion proteins were determined. A comparison of those N-terminal sequences with the aa sequence of the HCV precurso r polyprotein showed that the N-terminal and C-terminal cleavage sites of p70(NS3), one of the HCV nonstructural (NS) proteins, were the sam e as those identified in other processing studies: cleavages were esti mated to be between aa 1026 and 1027 and between aa 1657 and 1658 of t he HCV precursor protein, which are known to be cleaved by Cpro-1 and Cpro-2, respectively. Cpro-1 and Cpro-2 both functioned in E. coli and possessed authentic characteristic features.