2 DISTINCT PROTEINASE ACTIVITIES REQUIRED FOR THE PROCESSING OF A PUTATIVE NONSTRUCTURAL PRECURSOR PROTEIN OF HEPATITIS-C VIRUS

Gene products of hepatitis C virus (HCV), a possible major causative a gent of posttransfusion non-A, non-B hepatitis, are considered to be p roduced from a precursor polyprotein via proteolytic processing mediat ed by either host cell or viral proteinases. The presence of HCV serin e proteinase has been proposed from analyses of amino acid sequence ho mology. To examine the processing mechanism of the HCV precursor polyp rotein, the amino-terminal region of the putative nonstructural protei n region of the HCV genome, containing the serine proteinase motif, wa s expressed and analyzed by using an in vitro transcription/translatio n system and a transient expression system in cultured cells. Two dist inct proteinase activities which function in the production of a 70-kD a protein (p70) from the precursor polyprotein were detected. One of t hese proteinase activities, which cleaved the carboxyl (C)-terminal si de of p70, required the presence of the serine proteinase motif, which is located in the amino (N)-terminal region of p70. That suggested th at the predicted HCV serine proteinase was functional. The other activ ity, which was responsible for the cleavage of the N-terminal side of p70, required the expression of the region upstream and downstream of that cleavage site, including the p70 serine proteinase domain. From t he results of pulse-chase analysis, using proteinase inhibitors couple d with a point mutation analysis, the latter activity was proposed to be a novel zinc-dependent metalloproteinase.