Hong, Y.; Huang, B.; Zhang, J.; Peng, C.; Kong, W.; Tan, W.; Li, S.

The mpox outbreak since 2022 has caused over 97,208 infected cases with 186 deaths across 117 countries/territories/areas and was declared a public health emergency of international concern by the WHO. The causative pathogen, monkeypox virus (MPXV), is a zoonotic double-stranded DNA orthopoxvirus. To investigate the structures and replication mechanism of MPXV, the MPXV recombinant proteins and vaccinia virus (VACV) particles have been studied by cryo-EM, however, in situ structural insights into major structural proteins and assembly of the authentic MPXV particles are missing. Here, we optimized an MPXV propagation and purification workflow. Combining cryo-electron tomography (cryo-ET) and sub-tomogram averaging (STA), we determined the structures and assembly of palisade proteins and portal complexes to unveil the architecture of intact MPXV MVs. We also discovered irregularly-shaped MV particles with distorted core walls, which have not been reported before.