Poxviruses are a family of large, complex double-stranded DNA viruses that includes human pathogens such as variola—the cause of smallpox—and monkeypox. Recent outbreaks of mpox underscore the need for a better understanding of poxvirus biology1,2. Poxvirus assembly is a conserved process that involves the formation of a biconcave core inside the membrane of the maturing virus3,4. Here we use cryo-electron tomography combined with subtomogram averaging and structure prediction to determine the structure and composition of the portal complex—a pore that spans the core wall—in vaccinia virus, the prototypical poxvirus. The hexameric complex consists of the E8, E6 and L3 proteins, which are conserved across poxviruses and essential for mRNA release during the establishment of infection5–7. E6, which is also required for virus assembly8–10, forms the central chamber of the portal and interacts with the surrounding core wall. A hexamer of E8 attaches to the exterior side of E6. L3, a target of TRIM5α-mediated restriction11, binds as a hexamer of dimers to the interior side. Furthermore, the viral helicase D5, which is required for genome release from cores12, associates with cytoplasmic cores during infection by docking onto the exterior E8 rim of the portal complex. We propose that the portal complex represents an attractive target for the development of anti-poxvirus therapeutics.
