Biochemical characterization of the respiratory syncytial virus N0-P complex in solution

As all the viruses belonging to the Mononegavirales order, the nonsegmented negative-strand RNA genome of respiratory syncytial virus (RSV) is encapsidated by the viral nucleoprotein N. N protein polymerizes along the genomic and anti-genomic RNAs during replication. This requires the maintenance of the neosynthesized N protein in a monomeric and RNA-free form by the viral phosphoprotein P that plays the role of a chaperone protein, forming a soluble N⁰-P complex. We have previously demonstrated that residues 1–30 of P specifically bind to N⁰. Here, to isolate a stable N⁰-P complex suitable for structural studies, we used the N-terminal peptide of P (P40) to purify truncated forms of the N protein. We show that to purify a stable N⁰-P–like complex, a deletion of the first 30 N-terminal residues of N (N_Δ30) is required to impair N oligomerization, whereas the presence of a full-length C-arm of N is required to inhibit RNA binding. We generated structural models of the RSV N⁰-P with biophysical approaches, including hydrodynamic measurements and small-angle X-ray scattering (SAXS), coupled with biochemical and functional analyses of human RSV (hRSV) N_Δ30mutants. These models suggest a strong structural homology between the hRSV and the human metapneumovirus (hMPV) N⁰-P complexes. In both complexes, the P40-binding sites on N⁰ appear to be similar, and the C-arm of N provides a high flexibility and a propensity to interact with the N RNA groove. These findings reveal two potential sites to target on N⁰-P for the development of RSV antivirals.