Cryo-electron microscopy structure of the bovine ephemeral fever virus RNA-nucleoprotein assembly

Bovine ephemeral fever virus (BEFV), a member of the Rhabdoviridae family, is an arthropod-borne pathogen that causes acute febrile disease in cattle. The structural basis of its genome encapsidation and virion assembly remains unexplored, with the current knowledge largely limited to predictions derived from bioinformatic comparisons with other rhabdoviruses. Furthermore, the structural principles that permit the formation of variable-diameter nucleocapsids resulting in the distinctive bullet-shaped morphology of rhabdoviruses remain poorly understood. Here, we report the cryo-electron microscopy structure of the BEFV nucleoprotein (N) in complex with RNA, in the absence of other viral components. The complex predominantly forms circular decameric oligomers that we propose to act as nucleation intermediates during assembly of the bullet-shaped nucleocapsids. Direct subunit interactions are limited to a small polar surface area, with additional intersubunit links mediated by flexible N- and C-terminal loops. These interfaces generate a structurally plastic oligomeric lattice in which neighbouring N subunits can undergo substantial rigid-body rotations and positional rearrangements while preserving conserved local contacts and continuous RNA encapsidation. Such quasi-equivalent interactions provide a plausible mechanism for accommodating the progressive changes in helical diameter required for the transition from the highly curved bullet tip to the wider cylindrical trunk of rhabdovirus nucleocapsids. The assembly is stabilised by the bound RNA molecule, where nine RNA bases are accommodated by each N subunit. The RNA-binding mechanism is consistent with that of VSV, the closest BEFV homologue characterised structurally, but differs at about half of the RNA-binding residues, demonstrating the versatility of the nucleoprotein scaffold in interacting with ssRNA. Comparative analysis with other rhabdoviruses, as well as negative-sense RNA viruses with constant-diameter nucleocapsids, such as Ebola, further confirms the structural features that enable bullet-shaped versus cylindrical nucleocapsid assembly.