title: Characterization of a viral phosphoprotein binding site on the surface of the respiratory syncytial nucleoprotein.
abstract: The human respiratory syncytial virus (HRSV) genome is composed of a negative-sense single-stranded RNA that is tightly associated with the nucleoprotein (N). This ribonucleoprotein (RNP) complex is the template for replication and transcription by the viral RNA-dependent RNA polymerase. RNP recognition by the viral polymerase involves a specific interaction between the C-terminal domain of the phosphoprotein (P) (P(CTD)) and N. However, the P binding region on N remains to be identified. In this study, glutathione S-transferase (GST) pulldown assays were used to identify the N-terminal core domain of HRSV N (N(NTD)) as a P binding domain. A biochemical characterization of the P(CTD) and molecular modeling of the N(NTD) allowed us to define four potential candidate pockets on N (pocket I [PI] to PIV) as hydrophobic sites surrounded by positively charged regions, which could constitute sites complementary to the P(CTD) interaction domain. The role of selected amino acids in the recognition of the N-RNA complex by P was first screened for by site-directed mutagenesis using a polymerase activity assay, based on an HRSV minigenome containing a luciferase reporter gene. When changed to Ala, most of the residues of PI were found to be critical for viral RNA synthesis, with the R132A mutant having the strongest effect. These mutations also reduced or abolished in vitro and in vivo P-N interactions, as determined by GST pulldown and immunoprecipitation experiments. The pocket formed by these residues is critical for P binding to the N-RNA complex, is specific for pneumovirus N proteins, and is clearly distinct from the P binding sites identified so far for other nonsegmented negative-strand viruses.
authors.affiliation: INRA, Unité de Virologie Immunologie Moléculaires UR892, Jouy-en-Josas, France.
authors.forename: Marie
authors.initials: M
authors.lastname: Galloux
authors.forename: Bogdan
authors.initials: B
authors.lastname: Tarus
authors.forename: Ilfad
authors.initials: I
authors.lastname: Blazevic
authors.forename: Jenna
authors.initials: J
authors.lastname: Fix
authors.forename: Stéphane
authors.initials: S
authors.lastname: Duquerroy
authors.forename: Jean-François
authors.initials: JF
authors.lastname: Eléouët
mesh_terms: D019943:Amino Acid Substitution; D000818:Animals; D001665:Binding Sites; D002460:Cell Line; D006224:Cricetinae; D047468:Immunoprecipitation; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D050505:Mutant Proteins / Q000235:genetics / Q000378:metabolism; D009698:Nucleoproteins / Q000378:metabolism*; D011485:Protein Binding; D025941:Protein Interaction Mapping; D018113:Respiratory Syncytial Virus, Human / Q000235:genetics / Q000378:metabolism*; D015678:Viral Structural Proteins / Q000378:metabolism*
chemical_list: D050505:Mutant Proteins; D009698:Nucleoproteins; C529603:P protein, human respiratory syncytial virus; D015678:Viral Structural Proteins