title: The Casein Kinase 2-Dependent Phosphorylation of NS5A Domain 3 from Hepatitis C Virus Followed by Time-Resolved NMR Spectroscopy.
abstract: Hepatitis C virus (HCV) chronically affects millions of individuals worldwide. The HCV nonstructural protein 5A (NS5A) plays a critical role in the viral assembly pathway. Domain 3 (D3) of NS5A is an unstructured polypeptide responsible for the interaction with the core particle assembly structure. Casein kinase 2 (CK2) phosphorylates NS5A-D3 at multiple sites that have mostly been predicted and only observed indirectly. In order to identify the CK2-dependent phosphorylation sites, we monitored the reaction between NS5A-D3 and CK2 in vitro by time-resolved NMR. We unambiguously identified four serine residues as substrates of CK2. The apparent rate constant for each site was determined from the reaction curves. Ser408 was quickly phosphorylated, whereas the three other serine residues reacted more slowly. These results provide a starting point from which to elucidate the role of phosphorylation in the mechanisms of viral assembly-and in the modulation of the viral activity-at the molecular level.
authors.affiliation: Magnetic Resonance Center-CERM, University of Florence, via Luigi Sacconi 6, 50019 Sesto Fiorentino, Florence, Italy.
authors.forename: Erica
authors.identifier: http://orcid.org/0000-0002-2839-6858
authors.initials: E
authors.lastname: Secci
authors.affiliation: Magnetic Resonance Center-CERM, University of Florence, via Luigi Sacconi 6, 50019 Sesto Fiorentino, Florence, Italy.|Department of Biomedical, Experimental and Clinical Sciences, University of Florence, viale Morgagni 50, 50134, Florence, Italy.
authors.forename: Enrico
authors.identifier: http://orcid.org/0000-0003-4183-4311
authors.initials: E
authors.lastname: Luchinat
authors.affiliation: Magnetic Resonance Center-CERM, University of Florence, via Luigi Sacconi 6, 50019 Sesto Fiorentino, Florence, Italy. banci@cerm.unifi.it.|Department of Chemistry, University of Florence, via della Lastruccia 3, 50019 Sesto Fiorentino, Florence, Italy. banci@cerm.unifi.it.
authors.forename: Lucia
authors.identifier: http://orcid.org/0000-0003-0562-5774
authors.initials: L
authors.lastname: Banci
mesh_terms: D000595:Amino Acid Sequence; D047390:Casein Kinase II / Q000378:metabolism*; D016174:Hepacivirus / Q000737:chemistry / Q000378:metabolism*; D006526:Hepatitis C / Q000378:metabolism / Q000821:virology; D006801:Humans; D008969:Molecular Sequence Data; D019906:Nuclear Magnetic Resonance, Biomolecular; D010766:Phosphorylation; D017434:Protein Structure, Tertiary; D012694:Serine / Q000032:analysis / Q000378:metabolism; D017361:Viral Nonstructural Proteins / Q000737:chemistry / Q000378:metabolism*; D012324:RNA-Dependent RNA Polymerase
chemical_list: D047390:Casein Kinase II; D012694:Serine; D017361:Viral Nonstructural Proteins; C087036:NS-5 protein, hepatitis C virus; D012324:RNA-Dependent RNA Polymerase
keywords: NMR spectroscopy; hepatitis C virus (hcv); intrinsically disordered proteins (IDPs); phosphorylation; time-resolved NMR