Structures of in vitro assembly products of poxvirus scaffolding protein reveal transition from pre-assembly state to fully assembled scaffold

During poxvirus morphogenesis, the external scaffold plays a key role in defining the shape and size of immature virions. However, its structural characterization has been hindered by the pleomorphic nature of authentic virus particles. Here, we present single-particle cryo-electron microscopy and cryo-electron tomography structures of two distinct in vitro assemblies of the vaccinia virus scaffold protein D13 that recapitulate oligomeric states previously observed in situ . These structures reveal a dramatic transition from rod-like oligomers, representing a pre-assembly state, to a fully formed scaffold, triggered by the docking of N-terminal peptide of the membrane protein A17 into the base cavity of D13. We propose that this interaction destabilizes the pre-assembly conformation and initiates scaffold formation upon viral membrane recruitment, ultimately leading to the immature virion. Our findings provide novel structural insights into the early stages of poxvirus morphogenesis and establish a mechanistic link between conformational changes in D13 and scaffold assembly.