Domain 3 of NS5A Protein from the Hepatitis C Virus Has Intrinsic α-Helical Propensity and Is a Substrate of Cyclophilin A

Abstract

Nonstructural protein 5A (NS5A) is essential for hepatitis C virus (HCV) replication and constitutes an attractive target for antiviral drug development. Although structural data for its in-plane membrane anchor and domain D1 are available, the structure of domains 2 (D2) and 3 (D3) remain poorly defined. We report here a comparative molecular characterization of the NS5A-D3 domains of the HCV JFH-1 (genotype 2a) and Con1 (genotype 1b) strains. Combining gel filtration, CD, and NMR spectroscopy analyses, we show that NS5A-D3 is natively unfolded. However, NS5A-D3 domains from both JFH-1 and Con1 strains exhibit a propensity to partially fold into an ␣-helix. NMR analysis identifies two putative ␣-helices, for which a molecular model could be obtained. The amphipathic nature of the first helix and its conservation in all genotypes suggest that it might correspond to a molecular recognition element and, as such, promote the interaction with relevant biological partner(s). Because mutations conferring resistance to cyclophilin inhibitors have been mapped into NS5A-D3, we also investigated the functional interaction between NS5A-D3 and cyclophilin A (CypA). CypA indeed interacts with NS5A-D3, and this interaction is completely abolished by cyclosporin A. NMR heteronuclear exchange experiments demonstrate that CypA has in vitro peptidyl-prolyl cis/trans-isomerase activity toward some, but not all, of the peptidyl-prolyl bonds in NS5A-D3. These studies lead to novel insights into the structural features of NS5A-D3 and its relationships with CypA. Hepatitis C virus (HCV),3 a small enveloped virus from the Flaviviridae family, is a major cause of chronic liver disease that may lead to steatosis, liver cirrhosis, and hepatocellular carcinoma. Given about 180 million chronically infected individuals worldwide, HCV is an important health challenge (1). Current therapy is based on a combination of pegylated interferon-␣ and ribavirin but is not fully satisfying because numerous patients are not responding or suffer from serious side effects caused by this treatment. The development of new drugs to treat HCV infections requires a better understanding of the structural and functional features of the viral proteins and their relationships with host cell factors. The HCV genome (ϳ9.6 kb) encodes for a single polyprotein precursor (ϳ3,000 aa) that is co-and post-translationally processed by cellular and viral proteases to yield 10 mature proteins (2, 3). They are classified into structural proteins (Core, E1, and E2), which constitute the viral particle, and nonstructural proteins, of which two, p7 and nonstructural protein 2 (NS2), are required for virus assembly. The remainder of the nonstructural proteins (NS3, NS4A, NS4B, NS5A, and NS5B) are involved in HCV RNA replication (reviewed in Refs. 2 and 3).Cyclophilins are host cell factors that in addition to viral proteins, are equally essential for HCV replication. The human genome encodes up to 16 different cyclophilins (4) that, despite differences in their tissue distributio...

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

“…We previously assigned the spectra of the D2 and D3 domains of HCV NS5A when isolated in solution (53,56,57,67). To monitor by NMR spectroscopy the effects induced by NS5B ⌬21 on the spectrum of NS5A-D2 (or NS5A-D3), we mixed at equimolar concentrations (i.e.…”

“…Assignment of the NS5A-D2, NS5A-D3, and CypA spectra was taken from our previous studies (53,57). Surface Plasmon Resonance Analyses of the NS5B ⌬21 -NS5A-D2, CypA-NS5B ⌬21 , and CypA-NS5A-D2 Interactions-All materials and chemicals came from Biacore (GE Healthcare).…”

“…It has been shown that NS5A-D1 may adopt two different heterodimeric structures, at least in the context of a crystal (51,52). NS5A-D2 (250 -342), which is essential for RNA replication (47), and NS5A-D3 (356 -447), which is involved in the production and assembly of viral particles (45), have been shown to be natively unfolded (53)(54)(55)(56)(57). In previous studies we have shown that both NS5A-D2 (53) and NS5A-D3 (57) establish direct interactions with CypA in vitro and that these domains are substrates for the peptidyl-prolyl cis-trans isomerase activity of CypA.…”

A Proline-Tryptophan Turn in the Intrinsically Disordered Domain 2 of NS5A Protein Is Essential for Hepatitis C Virus RNA Replication

“…(D1), two additional domains (D2 and D3) that are mainly intrinsically disordered (23)(24)(25) and that we previously characterized by NMR spectroscopy (27,28). IDPs/IDRs exist as highly dynamic and interconverting conformers and thus escape to the rule "one three-dimensional structure, one function."…”

“…In this respect, NS5A is the HCV protein with the highest percentage of its primary sequence predicted to be disordered (26). In vitro, both NS5A-D2 and -D3 have been shown to directly interact with the host CypA and to be substrates of its PPIase enzymatic activity (27,28).…”