NS3 IS A SERINE-PROTEASE REQUIRED FOR PROCESSING OF HEPATITIS-C VIRUSPOLYPROTEIN

Hepatitis C virus (HCV) possesses a positive-sense RNA genome which en codes a large polyprotein of 3,010 amino acids. Previous data and sequ ence analysis have indicated that this polyprotein is processed by cel lular proteases and possibly by a virally encoded serine protease loca lized in the N-terminal domain of nonstructural protein NS3. To charac terize the molecular aspects of HCV protein biogenesis and to clearly identify the protein products derived from the HCV genome, we have exa mined HCV polyprotein expression by using the vaccinia virus T7 transi ent expression system in transfected cells and by cell-free translatio n studies. HCV proteins were identified by immunoprecipitation with re gion-specific antisera. Here we show that the amino-terminal region of the HCV polyprotein is processed in vitro by cellular proteases relea sing three structural proteins: p21 (core), gp37 (El), and gp61 (E2). Processing of the nonstructural region of HCV was evident in transfect ed cells. Two proteins of 24 and 68 kDa were immunoprecipitated with a nti-NS2 and NS3 antisera, respectively. Antiserum against NS4 recogniz ed three proteins of 6, 26, and 31 kDa, while antisera specific for NS 5 immunoprecipitated two polypeptides of 56 and 65 kDa, indicating tha t each of these two genes encodes at least two different proteins. Whe n the NS3 protease domain was inactivated by replacing the proposed ca talytic Ser-1165 with Ala, processing at several sites was abolished. When Ser-1164 was mutated to Ala, no effect on the processing was obse rved. Cleavage activities at three of the four sites affected by NS3 w ere shown to occur in trans, while processing at the carboxy terminus of NS3 could not be mediated in trans. These results provide a detaile d description of the protein products obtained from the processing of the HCV polyprotein. Furthermore, the data obtained implicate NS3 as a serine protease and demonstrate that a catalytically active NS3 is ne cessary for cleavage of the nonstructural region of HCV.