HEPATITIS-C VIRUS NS3 SERINE PROTEINASE - TRANS-CLEAVAGE REQUIREMENTSAND PROCESSING KINETICS

The hepatitis C virus H strain (HCV H) polyprotein is cleaved to produ ce at least 10 distinct products, in the order of NH2-C-E1-E2-p7-NS2-N S3-NS4A-NS4B-NS5A-NS5B-COOH. An HCV-encoded serine proteinase activity in NS3 is required for cleavage at four sites in the nonstructural re gion (3/4A, 4A/4B, 4B/5A, and 5A/5B). In this report, the HCV-H serine proteinase domain (the N-terminal 181 residues of NS3) was tested for its ability to mediate trans-processing at these four sites. By using an NS3-5B substrate with an inactivated serine proteinase domain, tra ns-cleavage was observed at all sites except for the 3/4A site. Deleti on of the inactive proteinase domain led to efficient trans-processing at the 3/4A site. Smaller NS4A-4B and NS5A-5B substrates were process ed efficiently in trans; however, cleavage of an NS4B-5A substrate occ urred only when the serine proteinase domain was coexpressed with NS4A . Only the N-terminal 35 amino acids of NS4A required for this activit y. Thus, while NS4A appears to be absolutely required for trans-cleava ge at the 4B/5A site, it is not an essential cofactor for serine prote inase activity. To begin to examine the conservation (or divergence) o f serine proteinase-substrate interactions during HCV evolution, we de monstrated that similar trans-processing occurred when the proteinase domains and substrates were derived from two different HCV subtypes. T hese results are encouraging for the development of broadly effective HCV serine proteinase inhibitors as antiviral agents. Finally, the kin etics of processing in the nonstructural region was examined by pulse- chase analysis. NS3-containing precursors were absent, indicating that the 2/3 and 3/4A cleavages occur rapidly. In contrast, processing of the NS4A-5B region appeared to involve multiple pathways, and signific ant quantities of various polyprotein intermediates were observed. NS5 B, the putative RNA polymerase, was found to be significantly less sta ble than the other mature cleavage products. This instability appeared to be an inherent property of NS5B and did not depend on expression o f other viral polypeptides, including the HCV-encoded proteinases.