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

Abstract

The nonstructural protein 3 (NS3) helicase/protease is an important component of the hepatitis C virus (HCV) replication complex. We hypothesized that a specific ␤-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 ␤-strand and explored the structural and functional attributes of the truncated NS3 protein (NS3⌬C7). Limited proteolysis, hydrodynamic, and kinetic measurements indicate that NS3⌬C7 adopts an extended conformation that contrasts with the compact form of the wild-type (WT) protein. The extended conformation of NS3⌬C7 allows the protein to quickly form functional complexes with RNA unwinding substrates. We also show that the unwinding activity of NS3⌬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.Nonstructural protein 3 (NS3) is an essential member of the hepatitis C virus (HCV) replication complex (2, 33). It is a bifunctional enzyme that contains a serine protease domain within the N-terminal third of the protein and a nucleic acidstimulated NTPase/helicase domain within the C-terminal twothirds of the protein (40). Both enzymatic activities are critical for HCV replication. In the presence of the viral NS4A cofactor protein, the NS3 protease activity cleaves and releases downstream viral proteins from the precursor polyprotein (13). Furthermore, the NS3 protease represses the host innate immune response by cleaving cellular proteins such as TRIF and MAVS, thereby preventing a signaling cascade that leads to an antiviral cellular response (17,24,32,44,51). In addition to its protease activity, NS3 displays robust helicase activity (46). NS3 helicase activity is essential for replication of the viral RNA genome (21), potentially functioning together with the NS5B polymerase during viral replication (16). NS3 also participates in the intracellular assembly and packaging of infectious virus particles (30). Given its multiple roles throughout the viral life cycle, NS3 is an important target for antiviral drug discovery against HCV (5).The NS3 helicase (NS3hel) domain belongs to the DExH/D subgroup of DNA and RNA helicases within helicase superfamily 2 (37, 52). Members of this family contain a core helicase structure consisting of two RecA-like folds (domains 1 and 2) arranged in tandem. Together with these two RecA-like domains, NS3hel has a third domain (domain 3) that forms a single-stranded DNA/RNA binding groove (Fig. 1A) (20, 29). The NS3hel construct has been studied extensively and displays modest helicase activity in isolation. However, a unique feature of NS3 that distinguishes it from its other family members is the ...

A macrocyclic HCV NS3/4A protease inhibitor interacts with protease and helicase residues in the complex with its full-length target

“…Ding et al (15) have recently reported enzymatic data supporting a fully open form as proposed by Brass et al (4).…”

Protease Inhibitors Block Multiple Functions of the NS3/4A Protease-Helicase during the Hepatitis C Virus Life Cycle

“…This difference may be explained by the conformation-dependent binding of the APHI to NS3, in which the APHI targets a site in the interdomain interface that is present in the compact crystallographic conformation of NS3 but not in the extended conformation, which biochemical studies suggest represents the active conformation during RNA synthesis (30). Partial inhibition of RNA synthesis by AT23708 is consistent with NS3 adopting the extended conformation at least some of the time during RNA synthesis.…”

Hepatitis C Virus RNA Replication and Virus Particle Assembly Require Specific Dimerization of the NS4A Protein Transmembrane Domain

“…This model predicts that the protease domain is anchored tightly against the membrane and is free to rotate only in the plane parallel to the membrane surface. Previous structural and biochemical work on NS3 from HCV, dengue virus, and Murray Valley encephalitis virus indicates that the ϳ10-amino-acid linker region between the protease and helicase domains can accommodate a rotation of up to 180°between domains (93)(94)(95). This flexibility is sufficient for placement of adjacent NS3 helicase domains and would allow both domains to orient themselves above the membrane (Fig.…”