Atomic Resolution Description of the Interaction between the Nucleoprotein and Phosphoprotein of Hendra Virus

Hendra virus (HeV) is a recently emerged severe human pathogen that belongs to the Henipavirus genus within the Paramyxoviridae family. The HeV genome is encapsidated by the nucleoprotein (N) within a helical nucleocapsid. Recruitment of the viral polymerase onto the nucleocapsid template relies on the interaction between the C-terminal domain, N-TAIL, of N and the C-terminal X domain, XD, of the polymerase co-factor phosphoprotein (P). Here, we provide an atomic resolution description of the intrinsically disordered N-TAIL domain in its isolated state and in intact nucleocapsids using nuclear magnetic resonance (NMR) spectroscopy. Using electron microscopy, we show that HeV nucleocapsids form herringbone-like structures typical of paramyxoviruses. We also report the crystal structure of XD of P that consists of a three-helix bundle. We study the interaction between N-TAIL and XD using NMR titration experiments and provide a detailed mapping of the reciprocal binding sites. We show that the interaction is accompanied by alpha-helical folding of the molecular recognition element of N-TAIL upon binding to a hydrophobic patch on the surface of XD. Finally, using solution NMR, we investigate the interaction between intact nucleocapsids and XD. Our results indicate that monomeric XD binds to N-TAIL without triggering an additional unwinding of the nucleocapsid template. The present results provide a structural description at the atomic level of the protein-protein interactions required for transcription and replication of HeV, and the first direct observation of the interaction between the X domain of P and intact nucleocapsids in Paramyxoviridae.