Structure and assembly of Borna disease virus 1 nucleoprotein-RNA complexes

Structures of nucleoprotein (N)-RNA complexes of the Bornaviridae, a virus family in the order Mononegavirales, have not been reported. Here, using cryo-electron microscopy (cryo-EM), we report high-resolution structures of Borna disease virus 1 (BoDV-1) N-RNA complex assemblies, including a dominant hexameric ring-like complex and less populated heptameric and octameric forms, the first RNA-bound N structures reported from this family. These structures reveal key features of N-RNA engagement and a BoDV-1-specific stoichiometry of eight nucleotides per N, providing a framework for comparison with related negative-strand RNA viruses. In addition to these RNA-bound complexes, we identified multiple RNA-free oligomers, indicating substantial conformational flexibility of N. Mutational analyses identified residues essential for nucleocapsid formation and RNA synthesis. Cryo-EM of mutant complexes captured RNA-free assemblies, suggesting that initial N oligomerization precedes RNA binding. These findings clarify the structural organization of the N-RNA complex and suggest how oligomeric plasticity contributes to nucleocapsid assembly.