Subcellular localization, stability, and trans-cleavage competence of the hepatitis C virus NS3-NS4A complex expressed in tetracycline-regulated celllines

A tetracycline-regulated gene expression system and a panel of novel monocl onal antibodies were used to examine the subcellular localization, stabilit y, and trans-cleavage competence of the hepatitis C virus (HCV) NS3-NS4A co mplex in inducible cell lines. The NS3 serine protease domain and the full- length NS3 protein expressed in the absence of the NS4A cofactor were diffu sely distributed in the cytoplasm and nucleus. Coexpression of NS4A, howeve r, directed NS3 to the endoplasmic: reticulum (ER) or an ER-like modified c ompartment, as demonstrated by colocalization with 3,3'-dihexyloxacarbocyan ine iodide, protein disulfide isomerase, and calnexin, as well as subcellul ar 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 do main of NS4A was required for ER targeting;of NS3. These results demonstrat e the importance of studying HCV proteins in their biological context and d efine a well-characterized cell culture system for further analyses of the NS3-NS4A complex and the evaluation of novel antiviral strategies against h epatitis C.