Unmasking the Active Helicase Conformation of Nonstructural Protein 3 from Hepatitis C Virus

The nonstructural protein 3 (NS3) helicase/protease is an important component of the hepatitis C virus (HCV) replication complex. We hypothesized that a specific beta-strand tethers the C terminus of the helicase domain to the protease domain, thereby maintaining HCV NS3 in a compact conformation that differs from the extended conformations observed for other Flaviviridae NS3 enzymes. To test this hypothesis, we removed the beta-strand and explored the structural and functional attributes of the truncated NS3 protein (NS3 Delta C7). Limited proteolysis, hydrodynamic, and kinetic measurements indicate that NS3 Delta C7 adopts an extended conformation that contrasts with the compact form of the wild-type (WT) protein. The extended conformation of NS3 Delta C7 allows the protein to quickly form functional complexes with RNA unwinding substrates. We also show that the unwinding activity of NS3 Delta C7 is independent of the substrate 3'-overhang length, implying that a monomeric form of the protein promotes efficient unwinding. Our findings indicate that an open, extended conformation of NS3 is required for helicase activity and represents the biologically relevant conformation of the protein during viral replication.