A tetracycline-regulated gene expression system and a panel of novel monoclonal antibodies were used to examine the subcellular localization, stability, and trans-cleavage competence of the hepatitis C virus (HCV) NS3-NS4A complex in inducible cell lines. The NS3 serine protease domain and the full-length NS3 protein expressed in the absence of the NS4A cofactor were diffusely distributed in the cytoplasm and nucleus. Coexpression of NS4A, however, directed NS3 to the endoplasmic reticulum (ER) or an ER-like modified compartment, as demonstrated by colocalization with 3,3-dihexyloxacarbocyanine iodide, protein disulfide isomerase, and calnexin, as well as subcellular fractionation analyses. In addition, coexpression with NS4A dramatically increased the intracellular stability of NS3 (mean protein half-life of 26 versus 3 h) and allowed for NS4A-dependent trans-cleavage at the NS4B-NS5A junction. Deletion analyses revealed that the hydrophobic aminoterminal domain of NS4A was required for ER targeting of NS3. These results demonstrate the importance of studying HCV proteins in their biological context and define a well-characterized cell culture system for further analyses of the NS3-NS4A complex and the evaluation of novel antiviral strategies against hepatitis C.Hepatitis C virus (HCV) is the most common etiologic agent of posttransfusion and sporadic non-A, non-B hepatitis (6, 25). The majority of HCV-infected individuals develop chronic infection which may progress to liver cirrhosis and eventually hepatocellular carcinoma (19). HCV contains a single-stranded, positive-sense RNA genome of approximately 9,600 nucleotides (nt) that encodes a polyprotein precursor of 3,010 to 3,033 amino acids (aa). The polyprotein precursor is co-and posttranslationally processed by cellular and viral proteases to yield the mature structural and nonstructural proteins (39). The structural proteins are believed to be processed by the endoplasmic reticulum (ER) signal peptidase (17,26,43). Cleavage at the NS2-NS3 site is mediated by a viral protease composed of NS2 and the amino-terminal one-third of NS3 (13,18,38). A distinct serine protease located in the amino-terminal one-third of NS3 is responsible for the downstream cleavages in the nonstructural region (1, 12, 49). The proteolytic events mediated by the NS3 serine protease have been shown to be essential for viral replication in vivo in the related yellow fever (4) and bovine viral diarrhea viruses (50), as well as very recently also in HCV (23a). This viral enzyme, therefore, has emerged as a major target in the design of novel antiviral agents against hepatitis C. The biochemical features of the NS3 serine protease have been well characterized in cell-free translation and transient cellular expression systems (39). In addition, the crystal structure of the NS3 se-rine protease (23, 31, 51) and RNA helicase (5, 22, 52) domains, the latter located in the carboxy-terminal region of NS3, have recently been elucidated. The 54-aa NS4A polypeptide functions as a cofactor for th...
