Recombinant baculoviruses have been constructed which express the hepatitis C virus (HCV) NS3 proteinase and its substrates in insect cells. The expressed proteinase has been shown to carry out -cleavage at the NS3/4A, NS4A/4B, NS4B/5A and NS5A/5B junctions in a cell-based assay. When assayed in a cell-free system using translated substrates, the proteinase could perform -processing of the NS4A/4B and NS5A/5B junctions, but only when coexpressed with NS4A, either as an NS3-4A precursor or by co-infection of cells with NS3- and NS4A-expressing recombinant baculoviruses. Possible reasons for the absolute requirement of the NS3 proteinase for NS4A are discussed.


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