AN IN-VITRO ASSAY FOR HEPATITIS-C VIRUS NS3 SERINE PROTEINASE

Hepatitis C virus (HCV) encodes a polyprotein of which the majority of nonstructural proteins are matured by the viral serine proteinase loc ated in the N terminus of NS3. Intracellular studies using recombinant vaccinia virus have shown that both NS3 and its cofactor NS4A are req uired to enhance processing at the NS3-dependent cleavage sites. We de veloped an in vitro (cell-free) assay in which the HCV serine proteina se was shown to be enzymatically active, by mixing lysates of cells ex pressing either the serine proteinase or a nonstructural protein subst rate. NS3 cleaved in a highly reproducible manner at the NS5A/5B site in the presence of NS4A, whereas NS3 alone was enzymatically inactive. NS4A could be provided either linked to NS3 or as part of the substra te. In contrast, irrespective of the presence or absence of NS4A, no N S3-mediated processing was observed at the NS3/4A, NS4A/4B, and NS4B/5 A sites in this assay. in vitro cleavage at the NS5A/5B site occurred rapidly, within 1 min at temperatures ranging from 0 to 20 degrees, bu t was incomplete and required detergent-solubilized lysates. General s erine proteinase inhibitors did not decrease processing activity. The in vitro model described in this study is a new tool: (1) to study the structure and the function of HCV serine proteinase and NS5A/5B cleav age site, and (2) to test NS3 serine proteinase inhibitors. (C) 1995 A cademic Press, Inc.