Hepatitis C Virus NS5B and Host Cyclophilin A Share a Common Binding Site on NS5A

Rosnoblet, Claire; Fritzinger, Bernd; Legrand, Dominique; Launay, Hélène; Wieruszeski, Jean‐Michel; Lippens, Guy; Hanoulle, Xavier

doi:10.1074/jbc.m112.392209

Cited by 36 publications

(49 citation statements)

References 76 publications

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“…A 2012 study does provide some molecular details on the interaction of NS5A with NS5B, the viral RNA polymerase, and the host factor cyclophilin A (CypA), both of which are crucial for viral RNA replication. The NS5A binding sites for both NS5B and CypA were localized in D2 (29). However, no structural details about the complexes are available as of this writing, and no information whether the binding requires preformed molecular recognition elements in this highly disordered D2 domain.…”

Section: Introductionmentioning

confidence: 91%

The Disordered Region of the HCV Protein NS5A: Conformational Dynamics, SH3 Binding, and Phosphorylation

Solyom

Schwarten

et al. 2015

Biophysical Journal

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Intrinsically disordered proteins (IDPs) perform their physiological role without possessing a well-defined three-dimensional structure. Still, residual structure and conformational dynamics of IDPs are crucial for the mechanisms underlying their functions. For example, regions of transient secondary structure are often involved in molecular recognition, with the structure being stabilized (or not) upon binding. Long-range interactions, on the other hand, determine the hydrodynamic radius of the IDP, and thus the distance over which the protein can catch binding partners via so-called fly-casting mechanisms. The modulation of long-range interactions also presents a convenient way of fine-tuning the protein's interaction network, by making binding sites more or less accessible. Here we studied, mainly by nuclear magnetic resonance spectroscopy, residual secondary structure and long-range interactions in nonstructural protein 5A (NS5A) from hepatitis C virus (HCV), a typical viral IDP with multiple functions during the viral life cycle. NS5A comprises an N-terminal folded domain, followed by a large (∼250-residue) disordered C-terminal part. Comparing nuclear magnetic resonance spectra of full-length NS5A with those of a protein construct composed of only the C-terminal residues 191-447 (NS5A-D2D3) allowed us to conclude that there is no significant interaction between the globular and disordered parts of NS5A. NS5A-D2D3, despite its overall high flexibility, shows a large extent of local residual (α-helical and β-turn) structure, as well as a network of electrostatic long-range interactions. Furthermore, we could demonstrate that these long-range interactions become modulated upon binding to the host protein Bin1, as well as after NS5A phosphorylation by CK2. As the charged peptide regions involved in these interactions are well conserved among the different HCV genotypes, these transient long-range interactions may be important for some of the functions of NS5A over the course of the HCV life cycle.

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Section: Introductionmentioning

confidence: 91%

The Disordered Region of the HCV Protein NS5A: Conformational Dynamics, SH3 Binding, and Phosphorylation

Solyom

Schwarten

et al. 2015

Biophysical Journal

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“…Fourth, NS5A proteins derived from all genotypes tested so far (1a, 1b, 2a, 2b, and 3) bind CypA directly, suggesting that CypA-NS5A interactions are conserved among HCV genotypes , which correlates well with the fact that CypI possess a pan-genotypic anti-HCV activity in HCV-infected patients (Flisiak et al 2007;Gallay 2012;Fischer et al 2010;Pockros 2010;von Hahn et al 2011;Vermehren and Sarrazin 2011;Pawlotsky et al 2012). Fifth, nuclear magnetic resonance (NMR) studies showed that CypA, via its enzymatic pocket, interacts with proline residues located within domains II and III of NS5A (Hanoulle et al 2009;Coelmont et al 2010;Verdegem et al 2011;Rosnoblet et al 2012). This is in accordance with the fact that CypA possesses the ability to catalyze the cis to trans isomerization of proline-containing peptides (Fischer et al 1984).…”

Section: Introductionmentioning

confidence: 75%

“…Handbook of Antimicrobial Resistance DOI 10.1007/978-1-4939-0667-3_3-1 # Springer Science+Business Media New York 2014 (Shirota et al 2002) and that CypA and NS5B share a binding region in the domain II of NS5A (Rosnoblet et al 2012). One thus can envision that CypA modulates an NS5A and/or NS5B function.…”

Section: Introductionmentioning

confidence: 98%

“…In this scenario, the elimination of CypA-NS5A contacts by CypI blocks the NS5A-mediated activation of the NS5B polymerase activity, resulting in defective RNA replication. The action of CypA upon NS5B should be indirect since CypA does not bind to NS5B (Rosnoblet et al 2012). Further work is required to determine which, if any, of these interesting findings represent the biologically relevant functionality of NS5A-CypA interactions.…”

Section: Introductionmentioning

confidence: 98%

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Resistance to Cyclophilin Inhibitors

Gallay

2014

Handbook of Antimicrobial Resistance

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The best approach to avoid hepatitis C virus (HCV) resistance to a specific therapy is rapid and massive suppression of viral replication. This is best accomplished by combining several drugs with potent antiviral activity across multiple genotypes, with each possessing a high barrier to resistance, different mechanisms of action, and no cross-resistance. A novel class of anti-HCV agents that have shown great promise in HCV patients -the cyclophilin inhibitors (CypI) -possess such properties. CypI are host-targeting antivirals (HTAs) with a mechanism of action that differs from those of all existing direct-acting antivirals (DAAs). CypI are pan-genotypic due to their distinct mechanism of action that targets the host protein cyclophilin A (CypA), which is required for HCV replication. HCV has to develop a lengthy mutational strategy to efficiently replicate in vitro independently of the host factor CypA leading to a high genetic barrier that the virus has to cross to develop resistance to CypI. CypI mediate rapid and profound viral load suppression in patients. Very low viral breakthrough rates are associated with the CypI treatment, which result mostly from suboptimal drug exposure rather than viral resistance. The high genetic barrier and the lack of cross-resistance to DAAs make CypI attractive drug candidates to be part of a regimen with one or two DAAs that may constitute the backbone of a new, safe, and effective IFN-free therapy. The characteristic resistance profile of CypI offers an exceptional opportunity to cure HCV. KeywordsHepatitic; cyclophilin; cyclophilia inhibitas; NSSA; resistance Abbreviations BIDTaken twice a day cEVR Complete early virological response = no virus detected after 12 weeks eRVR Extended rapid virological response = no virus detected at week 4 and week 12 EVR Early virological response = 2 log drop of HCV RNA after 12 weeks QD Taken once a day RVR Rapid virological response = no virus detected at week 4 SVR12 Sustained virological response = no virus detected at 12 weeks after completion of treatment SVR24 No virus detected at 24 weeks after completion of treatment

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“…NS5B has been shown to interact with cyclophilin and share its cyclophilin-binding region with that NS5A. It was indirectly inferred from NMR binding studies that NS5B thumb site region could be involved in its interaction with NS5A, and ligand binding at thumb site prevented the NS5B and NS5A interactions [13]. A recent study exemplified that ligand binding at the thumb site II induced the conformational changes, re-orient the C-terminal tail, β-loop structural elements into the palm site of NS5B polymerase.…”

Section: Introductionmentioning

confidence: 97%

Combination of pharmacophore hypothesis and molecular docking to identify novel inhibitors of HCV NS5B polymerase

Harikishore

Lee

et al. 2015

Mol Divers

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Hepatitis C virus (HCV) infection or HCV-related liver diseases are now shown to cause more than 350,000 deaths every year. Adaptability of HCV genome to vary its composition and the existence of multiple strains makes it more difficult to combat the emergence of drug-resistant HCV infections. Among the HCV polyprotein which has both the structural and non-structural regions, the non-structural protein NS5B RNA-dependent RNA polymerase (RdRP) mainly mediates the catalytic role of RNA replication in conjunction with its viral protein machinery as well as host chaperone proteins. Lack of such RNA-dependent RNA polymerase enzyme in host had made it an attractive and hotly pursued target for drug discovery efforts. Recent drug discovery efforts targeting HCV RdRP have seen success with FDA approval for sofosbuvir as a direct-acting antiviral against HCV infection. However, variations in drug-binding sites induce drug resistance, and therefore targeting allosteric sites could delay the emergence of drug resistance. In this study, we focussed on allosteric thumb site II of the non-structural protein NS5B RNA-dependent RNA polymerase and developed a five-feature pharmacophore hypothesis/model which estimated the experimental activity with a strong correlation of 0.971 & 0.944 for training and test sets, respectively. Further, the Güner-Henry score of 0.6 suggests that the model was able to discern the active and inactive compounds and enrich the true positives during a database search. In this study, database search and molecular docking results supported by experimental HCV viral replication inhibition assays suggested ligands with best fitness to the pharmacophore model dock to the key residues involved in thumbs site II, which inhibited the HCV 1b viral replication in sub-micro-molar range.

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Hepatitis C Virus NS5B and Host Cyclophilin A Share a Common Binding Site on NS5A

Cited by 36 publications

References 76 publications

The Disordered Region of the HCV Protein NS5A: Conformational Dynamics, SH3 Binding, and Phosphorylation

The Disordered Region of the HCV Protein NS5A: Conformational Dynamics, SH3 Binding, and Phosphorylation

Resistance to Cyclophilin Inhibitors

Combination of pharmacophore hypothesis and molecular docking to identify novel inhibitors of HCV NS5B polymerase

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