Structural Dynamics of the C-terminal X Domain of Nipah and Hendra Viruses Controls the Attachment to the C-terminal Tail of the Nucleocapsid Protein

Abstract

To understand the dynamic interactions between the phosphoprotein (P) and the nucleoprotein (N) within the transcription/replication complex of the Paramyxoviridae and to decipher their roles in regulating viral multiplication, we characterized the structural properties of the C-terminal X domain (P_XD) of Nipah (NiV) and Hendra virus (HeV) P protein. In crystals, isolated NiV P_XD adopted a two-helix dimeric conformation, which was incompetent for binding its partners, but in complex with the C-terminal intrinsically disordered tail of the N protein (N_TAIL), it folded into a canonical 3H bundle conformation. In solution, SEC-MALLS, SAXS and NMR spectroscopy experiments indicated that both NiV and HeV P_XD were larger in size than expected for compact proteins of the same molecular mass and were in conformational exchange between a compact three-helix (3H) bundle and partially unfolded conformations, where helix α₃ is detached from the other two. Some measurements also provided strong evidence for dimerization of NiV P_XD in solution but not for HeV P_XD. Ensemble modeling of experimental SAXS data and statistical-dynamical modeling reconciled all these data, yielding a model where NiV and HeV P_XD exchanged between different conformations, and where NiV but not HeV P_XD formed dimers. Finally, recombinant NiV comprising a chimeric P carrying HeV P_XD was rescued and compared with parental NiV. Experiments carried out in cellula demonstrated that the replacement of P_XD did not significantly affect the replication dynamics while caused a slight virus attenuation, suggesting a possible role of the dimerization of NiV P_XD in viral replication.

Keywords: intrinsically disordered protein; mononegavirales; nipah virus; phosphoprotein; small-angle X-ray scattering.