NMR reveals the intrinsically disordered domain 2 of NS5A protein as an allosteric regulator of the hepatitis C virus RNA polymerase NS5B

Abstract

Non-structural protein 5B (NS5B) is the RNA-dependent RNA polymerase that catalyzes replication of the hepatitis C virus (HCV) RNA genome and therefore is central for its life cycle. NS5B interacts with the intrinsically disordered domain 2 of NS5A (NS5A-D2), another essential multifunctional HCV protein that is required for RNA replication. As a result, these two proteins represent important targets for anti-HCV chemotherapies. Despite this importance and the existence of NS5B crystal structures, our understanding of the conformational and dynamic behavior of NS5B in solution and its relationship with NS5A-D2 remains incomplete. To address these points, we report the first detailed NMR spectroscopic study of HCV NS5B lacking its membrane anchor (NS5B). Analysis of constructs with selective isotope labeling of the δ1 methyl groups of isoleucine side chains demonstrates that, in solution, NS5B is highly dynamic but predominantly adopts a closed conformation. The addition of NS5A-D2 leads to spectral changes indicative of binding to both allosteric thumb sites I and II of NS5B and induces long-range perturbations that affect the RNA-binding properties of the polymerase. We compared these modifications with the short- and long-range effects triggered in NS5B upon binding of filibuvir, an allosteric inhibitor. We demonstrate that filibuvir-bound NS5B is strongly impaired in the binding of both NS5A-D2 and RNA. NS5A-D2 induces conformational and functional perturbations in NS5B similar to those triggered by filibuvir. Thus, our work highlights NS5A-D2 as an allosteric regulator of the HCV polymerase and provides new insight into the dynamics of NS5B in solution.

Cyclophilin A allows the allosteric regulation of a structural motif in the disordered domain 2 of NS5A and thereby fine-tunes HCV RNA replication

“…1e). 15 N spin relaxation data on the full-length domain confirmed this, with higher R 2 values for the 304 -321 region of NS5A-D2. Heteronuclear NOE values in this region are also clearly positive, whereas they are negative or close to zero for most residues in the N-terminal half of the fragment.…”

“…The 1 H, 15 N-HSQC NMR spectrum of NS5A-D2 of the HCV JFH1 strain (genotype 2a) ( Fig. 1b) displays a narrow 1 H chemical shift dispersion that confirms the high level of intrinsic disorder in this domain (Fig.…”

“…The plot of the 1 H, 15 N-HSQC peak intensity along the NS5A-D2 sequence reveals that the resonances in the N-terminal moiety are narrower than those in the C-terminal half (Fig. 1e).…”

“…S3). All the NMR data ( 1 H, 15 N, 13 C chemical shifts, NOE contacts, as well 3 J HN-H␣ couplings) were used to calculate a NMR structural model of this PW-turn ( Fig. 2 and Fig.…”

“…NS5A is a key multifunctional phosphoprotein (49 kDa) that is essential for HCV genome replication (10,11) and is also involved in virion production (12). NS5A is known to interact with numerous viral and host proteins (4,(13)(14)(15). NS5A, which is bound to the ER membrane via an N-terminal amphipathic helix (16), is composed by a folded cytoplasmic domain (NS5A-D1) (11) and two intrinsically disordered domains (NS5A-D2 and -D3) (17)(18)(19) (Fig.…”