A single methylation site regulates HRSV nucleocapsid architecture and replication

ABSTRACT The nucleoprotein N of the human respiratory syncytial virus (HRSV) encases the viral genome, forming a flexible N–RNA nucleocapsid helix that serves as template for the viral polymerase L. Recent structural analysis revealed a non-canonical helical nucleocapsid arrangement that modulates RNA accessibility, yet its impact on polymerase function remains unknown. Here, we identified symmetric dimethylation of residue R27 of N as a critical modulator of nucleocapsid architecture and viral replication. We also showed that the methylase PRMT5 interacts with N and likely catalyses R27 methylation. Molecular dynamics simulations of RNA-free N dimers indicate that R27 methylation enhances opening and closing of the RNA-binding cavity, whereas a methylation-mimicking R27M substitution favours a closed state. Cryo-electron microscopy reveals a canonical, markedly straight R27M helix with increased rise and pitch. These findings demonstrate that post-translational modifications fine-tune interactions between N protomers, shaping nucleocapsid assembly, structure and dynamics, and thereby controlling HRSV replication.