Characterization of the interaction domains between the phosphoprotein and the nucleocapsid of human Metapneumovirus

Human metapneumovirus (HMPV) causes severe respiratory diseases in young children. The HMPV RNA genome is encapsidated by the viral nucleoprotein, forming an RNA-N complex (NNuc), which serves as template for genome replication and mRNA transcription by the RNA-dependent RNA polymerase (RdRp). The RdRp is formed by the association of the large polymerase subunit (L), which has RNA polymerase, capping and methyltransferase activities, and the tetrameric phosphoprotein (P). P plays a central role in the RdRp complex by binding to NNuc and L, allowing the attachment of the L polymerase to the nucleocapsid template. During infection these proteins concentrate in cytoplasmic inclusion bodies (IBs) where viral RNA synthesis occurs. By analogy to the closely related pneumovirus respiratory syncytial virus (RSV), it is likely that the formation of IBs depends on the interaction between P and NNuc. However, the HMPV P-NNuc interaction still remains to characterize. Here, we finely characterized the binding domains involved in HMPV P and NNuc interaction by studying binding between recombinant proteins, combined with the use of a functional assay of the polymerase complex activity and the study of the recruitment of these proteins to IBs by immunofluorescence. We show that the last 6 C-terminal residues of HMPV P are necessary and sufficient for binding to NNuc, that P binds the N-terminal domain of N (NNTD), and identified conserved N residues critical for the interaction. Our results allowed to propose a structural model of the HMPV P-NNuc interaction.