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

Abstract Hepatitis C virus (HCV) nonstructural protein 5A (NS5A) and its interaction with the human chaperone cyclophilin A (CypA) are both targets for highly potent and promising antiviral drugs that are in late stage of clinical development. Despite its high interest in the development of drugs to counteract the worldwide HCV burden, NS5A is still an enigmatic multifunctional protein poorly characterized at the molecular level. NS5A is required for HCV RNA replication and is involved in viral particles formation and regulation of host pathways. Thus far, no enzymatic activity or precise molecular function has been ascribed to NS5A that is composed of a highly structured domain 1 (-D1) as well as two intrinsically disordered domains 2 (-D2) and 3 (-D3), representing half of the protein. Here we identify a short structural motif in the disordered NS5A-D2 and report its NMR structure. We show that this structural motif, a minimal Pro314-Trp316 turn, is essential for HCV RNA replication and its disruption alters the subcellular distribution of NS5A. We demonstrate that this Pro-Trp (PW) turn is required for proper interaction with the host CypA and influences its peptidyl-prolyl cis/trans isomerase (PPIase) activity on residue P314 of NS5A-D2. This work provides a molecular basis for further understanding of the function of the intrinsically disordered domain 2 of HCV NS5A protein. In addition, our work highlights how very small structural motifs present in intrinsically disordered proteins (IDPs) can exert a specific function.