NONSTRUCTURAL PROTEIN-3 OF THE HEPATITIS-C VIRUS ENCODES A SERINE-TYPE PROTEINASE REQUIRED FOR CLEAVAGE AT THE NS3 4 AND NS4/5 JUNCTIONS/

We have studied processing of the nonstructural (NS) polyprotein of th e hepatitis C virus. A series of cDNAs corresponding to predicted NS2/ 3/4 or NS3/4 regions were constructed, and processing of the polyprote ins was studied in an in vitro transcription-translation system. We re port that a catalytically active serine-type proteinase is encoded by the NS3 region. Substitution of the serine residue of the putative cat alytic triad (H, D, and S) by alanine blocked cleavage at the NS3/4 ju nction, while processing between NS2 and NS3 was not affected. Thus, c leavage at the NS2/3 junction is mediated either by cellular enzymes o r by an NS-2 inherent proteinase activity. Deletion analysis of an NS3 /4 cDNA construct mapped the amino terminus of the enzymatically activ e proteinase between amino acids 1049 and 1065 of the polyprotein. As internal deletions of variable segments of the presumed helicase domai n prevented processing at the NS314 junction, a continuous NS3 region appears to be required for processing at this site. To analyze hepatit is C virus polyprotein cleavage in vivo, recombinant vaccinia viruses expressing NS2/3/4 or NS3/4/5 proteins were generated. In agreement wi th the in vitro data, cleavage between NS2 and NS3 was independent of a catalytically active NS3 proteinase, whereas substitution of the act ive-site serine residue by the amino acid alanine completely blocked p rocessing at the NS3/4 and NS4/5 junctions. These results demonstrate that NS3 encodes the viral proteinase essential for generating the ami no termini of NS4 and NS5.