The poxviruses comprise a large family of enveloped double-stranded DNA viruses that infect many vertebrates and invertebrates and are responsible for human diseases including smallpox and molluscum contagiosum as well as mpox and other zoonoses.1 In particular, the increasing incidence of mpox in Africa and the global spread due to human-to-human transmission have motivated efforts to better understand the biology of poxviruses and develop more effective vaccines and therapeutics.2 Unlike other DNA viruses, poxviruses replicate entirely in the cytoplasm and encode their own unique transcription and replication systems.3 Another key difference from other viruses is the poxvirus mode of cell entry, which can occur by fusion with the plasma or endosomal membrane. Whereas most enveloped viruses have one or two proteins devoted to membrane fusion, poxviruses have 11 small proteins that form the entry-fusion complex (EFC) and are each required for infection.4 Orthologs of each EFC protein are highly conserved and present in nearly all poxviruses including those that infect vertebrates and invertebrates. Moreover, some EFC homologs are also present in African Swine Fever Virus, a member of the Asfarviridae family5 and giant viruses belonging to the Nucleocytoviricota.6 However, only the entry-fusion proteins expressed by vaccinia virus (VACV), the prototype poxvirus, have been characterized experimentally and demonstrated to exist in a complex. VACV EFC proteins A16, G9, and J5 have C-terminal transmembrane (TM) domains, conserved intramolecular disulfide bonds and partial sequence identity indicating an origin from a common ancestral gene.7, 8, 9 L1 and F9 have C-terminal TM domains, partial sequence identity and likely also arose by gene duplication.10, 11 The other EFC proteins are unrelated: L5 has a C-terminal TM domain,12 whereas A21, A28, G3, H2, and O3 have N-terminal TM domains.13, 14, 15 None of the EFC proteins have cleavable signal peptides or are glycosylated as they apparently bypass the endoplasmic reticulum to insert directly in the cytoplasmic viral membrane.16 Another unique feature of the EFC proteins is the presence of intramolecular disulfide bonds that are formed by a highly conserved poxvirus cytoplasmic redox system.17 Entry entails a hemifusion step, in which lipid mixing of viral and cellular membranes occurs, followed by pore formation and penetration of the core into the cytoplasm.18 However, neither a putative cell activation receptor nor the mechanism of fusion has been determined to date.

Elucidation of the structure of the EFC may help to understand the fusion process and point to improved therapeutics and vaccines. Although the overall structure of the EFC has not been determined, several protein–protein interactions have been found including A28:H2,19 A16:G9,20 and G3:L5.21 At present, structures have been reported for the ectodomains of nine out of the eleven proteins forming the EFC: L1,22 F9,23 the heterodimers of G3:L524 and A16:G9,25 A28,26 H2,27 and J5.28

The present study examines the structure of VACV A21 (OPG147), one of the two remaining uncharacterized EFC proteins. A21 synthesis occurs at a late stage of virus infection and the protein localizes on the lipoprotein membrane of intracellular mature virions (MVs).29 Studies with a conditional lethal mutant in which the A21 gene was downregulated by the lac repressor demonstrated that virions lacking A21 were able to bind to cells, but were unable to mediate cell fusion.29 The association of A21 with the EFC was demonstrated by affinity chromatography and mass spectrometry.9 Here we report on the experimentally determined structure of the ectodomain of A21, its conservation in other poxviruses, and predicted interactions with the other proteins of the poxvirus EFC.