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

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

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

J Biol Chem. 2017 Sep 14;:

Authors: Bessa LM, Launay H, Dujardin M, Cantrelle FX, Lippens G, Landrieu I, Schneider R, Hanoulle X

Abstract
Non-structural protein 5B (NS5B) is the RNAdependent RNA polymerase that catalyses replication of the hepatitis C virus (HCV) RNA genome, and so 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 behaviour of NS5B in solution and its relationship with NS5A-D2 remain incomplete. To address these points, we report the first detailed NMR spectroscopic study of HCV NS5B lacking its membrane anchor (NS5B?21). Analysis of constructs with selective isotope labelling of the ?1 methyl groups of isoleucine side chains demonstrates that, in solution, NS5B?21 is highly dynamic, but predominantly adopts a closed conformation. Addition of NS5A-D2 leads to spectral changes indicative of binding to both allosteric thumb sites I and II of NS5B?21, and induces long range perturbations that affect the RNA-binding properties of the polymerase. We compared these modifications to the short- and long-range effects triggered in NS5B?21 upon binding of filibuvir, an allosteric inhibitor. We demonstrate that filibuvir-bound NS5B?21 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.


PMID: 28912275 [PubMed - as supplied by publisher]



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