Structural and functional relationships between the NS5A and NS5B Hepatitis C Virus proteins and the host Cyclophilin A. – StruFunc5A5B

To study the relationship between NS5A and NS5B proteins from HCV and human Cyclophiline A (CypA) we use nuclear magnetic resonance spectroscopy (NMR). This technique allows performing, at atomic level and in solution, structural and/or functional studies. This method is particularly suited to the study of intrinsically disordered proteins that do not have stable 3D structure but rather correspond to dynamic ensemble of conformers. Domains 2 and 3 of NS5A are disordered. We will continue their molecular characterization also in the presence of point mutations that confer to HCV resistance to CsA derivatives. Some of these mutations are located in a conserved sequence of NS5A that interacts with the human CypA. Moreover, NMR spectroscopy allows studying protein/protein interactions processes or even enzymatic activities as the peptidyl-prolyl cis-trans isomerase (PPIase) one of CypA. Whereas the 3D structure of NS5B, the HCV RNA polymerase, has been solved by XRay, no structure of this protein in complex with another protein is available. A study of NS5B by NMR spectroscopy would allow characterizing its interactions with other viral (NS5A) or human (CypA) proteins. To reach this goal we will have to overcome several scientific and technical challenges. The first one will be to produce (in a recombinant way in E. coli) and purify all of these proteins enriched with stable isotopes (15N, 13C, 2H). These samples must meet high level criteria in terms of purity and quantity. We will have to study high molecular weight proteins or complexes for which conventional liquid NMR techniques are not suitable. So we will use and adapt selective isotope labeling strategies.

Up to now we managed to express and then purify NS5B protein, the HCV RNA polymerase, with an isotopic labeling that is consistent with a study by NMR spectroscopy. With to the use of a very high field NMR spectrometer at 900 MHz, we obtained a good quality spectrum on this protein, which has a high molecular weight (65kDa). This spectrum constitutes a powerful tool for studying the interactions of NS5B, a therapeutic target, with all kinds of molecules (proteins, RNA, inhibitors...)

With this research project we intend to get a better understanding of the biological roles that are played by intrinsically disordered viral proteins, which are involved in the pathogenicity of HCV

Rosnoblet C, Fritzinger B, Legrand D, Launay H, Wieruszeski JM, Lippens G, Hanoulle X.
Hepatitis C virus NS5B and host cyclophilin A share a common binding site on NS5A.
J Biol Chem. 2012 Dec 28;287(53):44249-60.

Hepatitis C virus (HCV) is a small RNA virus that infects chronically about 180 millions individuals worldwide. Current therapies at this time are not fully satisfying as numerous patients are not responding or suffer the side effects of treatment. To develop new drugs against this viral infection, which is a serious health challenge, it is necessary to have a better understanding of the HCV virus itself and also of its relationships with cellular host factors. There is a worldwide interest for deciphering the interplay between HCV and host cell factors, as each interaction is a potential target for the development of a new antiviral. Moreover, targeting specific host factors that are required for HCV life cycle is preferred because the very high mutation rate in viruses often leads to rapid drug resistance when the target is a viral protein. Also, for virologists, there is an ongoing need for structural data that can help to discover or understand the molecular mechanisms of the essential HCV life cycle steps (RNA replication, particles production). Currently, structural data are available for at least half of HCV proteins and most of them have been obtained by X-ray crystallography. Two categories of unknown structures remain. The first one concerns some membrane embedded proteins and the second one corresponds to intrinsically unstructured domains or proteins (IUP). These latter proteins or protein domains, when studied in an isolated form by the classical biophysical techniques, seem not to adopt a stable tertiary structure, or not even the characteristic secondary structure elements. To gain molecular information of IUP, at the atomic level, NMR spectroscopy is a method of choice. Furthermore, until know all the small anti-HCV molecules that target viral components have well-structured proteins as molecular targets (i.e. NS5B, NS3), so a better understanding of the intrinsically disordered viral proteins may constitute an additional set of antiviral targets.
The main purpose of this project is to better understand the relationships at the structural and/or functional level between two HCV proteins, NS5A and NS5B, and a human host protein, the Cyclophilin A (CypA). NS5A and NS5B are essential for viral RNA replication and for the production of infectious particles. Whereas the structure and function of NS5B, the viral RNA-dependant RNA-polymerase, are known, very little is known for NS5A. A multi-domain protein that is anchored to the cytoplasmic leaflet of endoplasmic reticulum (ER) membrane via an amphipatic helix, its domain 1 (NS5A-D1) is a zinc-binding domain and its three dimensional crystal structure has been solved. For domains 2 and 3 (NS5A-D2 and -D3) no crystallographic data are available. Very recently, we performed structural and functional characterizations of these domains by liquid NMR spectroscopy, and demonstrated they are intrinsically disordered. As an anti-HCV activity has been demonstrated for Cyslosporin A (CsA), a well-known inhibitor of humans Cyclophilins, and mutations that confer CsA resistance for HCV have been identified in NS5A and NS5B, this suggests that these viral proteins may interact with host Cyclophilins. However, the underlying molecular mechanisms are still to be elucidated. To achieve this, a detailed molecular characterization of each protein is required, followed by the understanding of the structural and/or functional relationships that may exist between them.
The members involved in this project bring together all the competences that are required to achieve it successfully: molecular biology, protein biochemistry, NMR spectroscopy and methodology development. They form a new HCV_NMR team into the NMR laboratory in Lille where there is both a strong scientific and technologic background in the structural and functional biochemistry, the NMR study of unstructured proteins and a high level equipment with notably 600MHZ, 800MH and a recent 900MHz NMR spectrometers.