A Conserved Tandem Cyclophilin-Binding Site in Hepatitis C Virus Nonstructural Protein 5A Regulates Alisporivir Susceptibility

Abstract

Cyclophilin A (CyPA) and its peptidyl-prolyl isomerase (PPIase) activity play an essential role in hepatitis C virus (HCV) replication, and mounting evidence indicates that nonstructural protein 5A (NS5A) is the major target of CyPA. However, neither a consensus CyPA-binding motif nor specific proline substrates that regulate CyPA dependence and sensitivity to cyclophilin inhibitors (CPIs) have been defined to date. We systematically characterized all proline residues in NS5A domain II, low-complexity sequence II (LCS-II), and domain III with both biochemical binding and functional replication assays. A tandem cyclophilinbinding site spanning domain II and LCS-II was identified. The first site contains a consensus sequence motif of AØPXW (where Ø is a hydrophobic residue) that is highly conserved in the majority of the genotypes of HCV (six of seven; the remaining genotype has VØPXW). The second tandem site contains a similar motif, and the ØP sequence is again conserved in six of the seven genotypes. Consistent with the similarity of their sequences, peptides representing the two binding motifs competed for CyPA binding in a spot-binding assay and induced similar chemical shifts when bound to the active site of CyPA. The two prolines (P310 and P341 of Japanese fulminant hepatitis 1 [JFH-1]) contained in these motifs, as well as a conserved tryptophan in the spacer region, were required for CyPA binding, HCV replication, and CPI resistance. Together, these data provide a high-resolution mapping of proline residues important for CyPA binding and identify critical amino acids modulating HCV susceptibility to the clinical CPI Alisporivir. Hepatitis C virus (HCV) is a member of the plus-strand RNA virus family Flaviviridae, which includes several additional significant human pathogens, such as West Nile virus, dengue virus, and yellow fever virus. HCV infection alone affects more than 130 million people worldwide and imposes a significant toll on the global health care system, because the majority of patients are chronically infected and in need of a more effective therapy.Replication of the HCV genome occurs on intracellular membranes and requires the participation of multiple nonstructural proteins, including the nonstructural protein 3 (NS3)/NS4A protease, the NS3 helicase, membranous web-forming protein NS4B, the RNA-dependent RNA polymerase NS5B, and the replicase cofactor NS5A. In addition, the viral replication complex contains cellular proteins that are also essential for HCV replication (46). One of these cellular cofactors is human cyclophilin A (CyPA) (64), a cytosolic protein originally identified as the protein target of the immunosuppressive drug cyclosporine (CsA) (20). The targets of CsA and another immunosuppressive compound, FK506, are CyPs and FK506-binding proteins (FKBPs), respectively. The CsA-CyPA and the FK506-FKBP complexes bind to a common target, calcineurin, and block its phosphatase activity and T-cell activation (32). FK506 does not inhibit HCV in vitro (60), whereas derivatives of CsA,...

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

“…We previously identified the 308 -327 region of NS5A-D2 as the main binding site for the human CypA, which is a mandatory host factor for viral RNA replication (27). Several Pro to Ala mutations in this NS5A region have been evaluated in terms of CypA binding (51). The reduced CypA binding observed for P310A in the JFH-1 strain (equivalent to Pro 314 in Con1) might be due to the loss of the Pro as anchoring residue but also to the disruption of the structural PW turn.…”

“…, which have been previously identified as crucial for replication (12,51), are unaffected. Hence, the short PW turn structural motif in the mainly disordered NS5A-D2 domain plays an essential role for HCV RNA replication.…”

“…CypA-Because the structural motif is located in the CypA binding site (8,27,51) and CypA is a mandatory host factor for HCV replication, we investigated the possibility that the structural PW turn element was involved in the binding of CypA. (Fig.…”

Intragenic Complementation of Hepatitis C Virus NS5A RNA Replication-Defective Alleles

“…7). G337 is located near the C terminus of NS5A domain II within the second of two cyclophilin A (CypA) binding sites identified within JFH1 NS5A (46). It will be interesting to determine whether the G337A amino acid substitution affects CypA binding and whether complementation group C is functionally defined by the interaction between NS5A and CypA.…”

Characterization of the Anti-HCV Activities of the New Cyclophilin Inhibitor STG-175

“…Two mutations-D320E and Y321N -were identified in the NS5A region of the STG-175-resistant replicon colonies. The D320E and Y321N mutations have been previously identified in HCV variants resistant to other CypI including CsA, ALV, SCY-635 and NIM811 [43][44][45][46][47][48][49]. Thus, it is likely that the double E320/N321 substitutions represent a common motif of partial resistance to CypI including STG-175.…”