title: Characterization of the interactions between the nucleoprotein and the phosphoprotein of Henipavirus.
abstract: The Henipavirus genome is encapsidated by the nucleoprotein (N) within a helical nucleocapsid that recruits the polymerase complex via the phosphoprotein (P). In a previous study, we reported that in henipaviruses, the N-terminal domain of the phosphoprotein and the C-terminal domain of the nucleoprotein (N(TAIL)) are both intrinsically disordered. Here we show that Henipavirus N(TAIL) domains are also disordered in the context of full-length nucleoproteins. We also report the cloning, purification, and characterization of the C-terminal X domains (P(XD)) of Henipavirus phosphoproteins. Using isothermal titration calorimetry, we show that N(TAIL) and P(XD) form a 1:1 stoichiometric complex that is stable under NaCl concentrations as high as 1 M and has a K(D) in the μM range. Using far-UV circular dichroism and nuclear magnetic resonance, we show that P(XD) triggers an increase in the α-helical content of N(TAIL). Using fluorescence spectroscopy, we show that P(XD) has no impact on the chemical environment of a Trp residue introduced at position 527 of the Henipavirus N(TAIL) domain, thus arguing for the lack of stable contacts between the C termini of N(TAIL) and P(XD). Finally, we present a tentative structural model of the N(TAIL)-P(XD) interaction in which a short, order-prone region of N(TAIL) (α-MoRE; amino acids 473-493) adopts an α-helical conformation and is embedded between helices α2 and α3 of P(XD), leading to a relatively small interface dominated by hydrophobic contacts. The present results provide the first detailed experimental characterization of the N-P interaction in henipaviruses and designate the N(TAIL)-P(XD) interaction as a valuable target for rational antiviral approaches.
authors.affiliation: Laboratoire d' Architecture et Fonction des Macromolécules Biologiques, UMR 6098 CNRS, Aix-Marseille University, Campus de Luminy, 13288 Marseille Cedex 9, France.
authors.forename: Johnny
authors.initials: J
authors.lastname: Habchi
authors.forename: Stéphanie
authors.initials: S
authors.lastname: Blangy
authors.forename: Laurent
authors.initials: L
authors.lastname: Mamelli
authors.forename: Malene Ringkjøbing
authors.initials: MR
authors.lastname: Jensen
authors.forename: Martin
authors.initials: M
authors.lastname: Blackledge
authors.forename: Hervé
authors.initials: H
authors.lastname: Darbon
authors.forename: Michael
authors.initials: M
authors.lastname: Oglesbee
authors.forename: Yaoling
authors.initials: Y
authors.lastname: Shu
authors.forename: Sonia
authors.initials: S
authors.lastname: Longhi
mesh_terms: D045403:Henipavirus / Q000737:chemistry* / Q000235:genetics; D008958:Models, Molecular; D009698:Nucleoproteins / Q000737:chemistry* / Q000235:genetics; D010750:Phosphoproteins / Q000737:chemistry* / Q000235:genetics; D020836:Protein Structure, Quaternary; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics; D014764:Viral Proteins / Q000737:chemistry* / Q000235:genetics
chemical_list: D009698:Nucleoproteins; D010750:Phosphoproteins; D011994:Recombinant Proteins; D014764:Viral Proteins