Structure of the Nucleoprotein Binding Domain of Mokola Virus Phosphoprotein
AbstractStructure of the Nucleoprotein Binding Domain of Mokola Virus Phosphoprotein ▿ René Assenberg 1 , Olivier Delmas 2 , Jingshan Ren 1 , Pierre-Olivier Vidalain 3 , Anil Verma 1 , Florence Larrous 2 , Stephen C. Graham 1 , Frédéric Tangy 3 , Jonathan M. Grimes 1 and Hervé Bourhy 2 , * 1 Division of Structural Biology and Oxford Protein Production Facility, Wellcome Trust Centre for Human Genetics, University of Oxford, Roosevelt Drive, Oxford OX3 7BN, United Kingdom 2 UPRE Lyssavirus Dynamics and Host Adaptation, WHO Collaborating Centre for Reference and Research on Rabies, Institut Pasteur, 75724 Paris Cedex 15, France 3 Laboratoire de Génomique Virale et Vaccination, CNRS URA 3015, Institut Pasteur, 28 rue du Dr. Roux, 75724 Paris Cedex 15, France ABSTRACT Mokola virus (MOKV) is a nonsegmented, negative-sense RNA virus that belongs to the Lyssavirus genus and Rhabdoviridae family. MOKV phosphoprotein P is an essential component of the replication and transcription complex and acts as a cofactor for the viral RNA-dependent RNA polymerase. P recruits the viral polymerase to the nucleoprotein-bound viral RNA (N-RNA) via an interaction between its C-terminal domain and the N-RNA complex. Here we present a structure for this domain of MOKV P, obtained by expression of full-length P in Escherichia coli , which was subsequently truncated during crystallization. The structure has a high degree of homology with P of rabies virus, another member of Lyssavirus genus, and to a lesser degree with P of vesicular stomatitis virus (VSV), a member of the related Vesiculovirus genus. In addition, analysis of the crystal packing of this domain reveals a potential binding site for the nucleoprotein N. Using both site-directed mutagenesis and yeast two-hybrid experiments to measure P-N interaction, we have determined the relative roles of key amino acids involved in this interaction to map the region of P that binds N. This analysis also reveals a structural relationship between the N-RNA binding domain of the P proteins of the Rhabdoviridae and the Paramyxoviridae .