Molecular views of viral polyprotein processing revealed by the crystal structure of the hepatitis C virus bifunctional protease-helicase

Background: Hepatitis C virus (HCV) currently infects approximately 3% of t he world's population. HCV RNA is translated into a polyprotein that during maturation is cleaved into functional components. One component, nonstruct ural protein 3 (NS3), is a 631-residue bifunctional enzyme with protease an d helicase activities. The NS3 serine protease processes the HCV polyprotei n by both cis and trans mechanisms. The structural aspects of cis processin g, the autoproteolysis step whereby the protease releases itself from the p olyprotein, have not been characterized. The structural basis for inclusion of protease and helicase activities in a single polypeptide is also unknow n. Results: We report here the 2.5 Angstrom resolution structure of an enginee red molecule containing the complete NS3 sequence and the protease activati on domain of nonstructural protein 4A (NS I A) in a single polypeptide chai n (single chain or scNS3-NS4A). In the molecule, the helicase and protease domains are segregated and connected by a single strand. The helicase nucle oside triphosphate and RNA interaction sites are exposed to solvent. The pr otease active site of scNS3-NS4A is occupied by the NS3 C terminus, which i s part of the helicase domain. Thus, the intramolecular complex shows one p roduct of NS3-mediated cleavage at the NS3-NS4A junction of the HCV polypro tein bound at the protease active site. Conclusions: The scNS3-NS4A structure provides the first atomic view of pol yprotein cis processing. Both local and global structural rearrangements fo llow the cis cleavage reaction, and large segments of the polyprotein can b e folded prior to proteolytic processing. That the product complex of the c is cleavage reaction exists in a stable molecular conformation suggests aut oinhibition and substrate-induced activation mechanisms for regulation of N S3 protease activity.