A 2ND HEPATITIS-C VIRUS-ENCODED PROTEINASE

Host and viral proteinases are believed to be required for the product ion of at least nine hepatitis C virus (HCV)-specific polyprotein clea vage products. Although several cleavages appear to be catalyzed by ho st signal peptidase or the HCV NS3 serine proteinase, the enzyme respo nsible for cleavage at the 2/3 site has not been identified. In this r eport, we have defined the 2/3 cleavage site and obtained evidence whi ch suggests that this cleavage is mediated by a second HCV-encoded pro teinase, located between aa 827 and 1207. This region encompasses the C-terminal portion of the 23-kDa NS2 protein, the 2/3 cleavage site, a nd the serine proteinase domain of NS3. Efficient processing at the 2/ 3 site was observed in mammalian cells, Escherichia coli, and in plant or animal cell-free translation systems in the absence of microsomal membranes. Cleavage at the 2/3 site was abolished by alanine substitut ions for NS2 residues His-952 or Cys-993 but was unaffected by several other substitution mutations, including those that inactivate NS3 ser ine proteinase function. Mutations abolishing cleavage at the 2/3 site did not block cleavage at other sites in the HCV polyprotein. Cotrans fection experiments indicate that the 2/3 site can be cleaved in trans , which should facilitate purification and further characterization of this enzyme.