The palisade layer of the poxvirus core is composed of flexible a10-trimers

Although vaccinia virus (VACV) is the best studied poxvirus, the structure of the mature virus (MV) remains poorly understood. Its asymmetric shape, size and compactness poses a major challenge for electron microscopy (EM) analysis, including cryoEM. Sub-viral particles, in particular membrane-free viral cores, may overcome these limitations. We compare cores obtained by detergent-stripping MVs with cores in the cellular cytoplasm, early in infection. By combining cryo-electron tomography (cryoET), subtomogram averaging (STA) and AlphaFold2 (AF2), abundant core-structures are analyzed, focusing on the prominent palisade layer on the core surface. On detergent-stripped cores, the palisade is composed of densely packed trimers of the major core protein A10. On the core surface they display a random order and their classification indicate structural flexibility. On cytoplasmic cores A10 is organized in a similar manner, indicating that the structures obtained in vitro are physiologically relevant. CryoET and STA also uncover unexpected details of the layers beneath the palisade both on in vitro and in situ cores, that are compared to AF2 structure predictions of known VACV core-associated proteins. Altogether, our data identify for the first time the structure and molecular composition of the palisade units. The results are discussed in the context of the VACV replicative cycle, the assembly and disassembly of the infectious MV. ### Competing Interest Statement The authors have declared no competing interest.