Structure and functional analyses of vaccinia virus J5 protein reveal distinct determinants for entry-fusion complex assembly and activation

Vaccinia virus enters host cells through a multi-component entry-fusion complex (EFC) that is structurally distinct from canonical viral fusion systems. Understanding how vaccinia virus EFC mediates membrane fusion is crucial for elucidating poxvirus entry and identifying potential antiviral targets. Here, we report the solution NMR structure of a truncated ectodomain of vaccinia J5 protein, residues 2–68. Using recombinant vaccinia viruses expressing J5 mutants, we analyzed substitutions in conserved and surface-exposed residues, as well as chimeric constructs between vaccinia J5 and its entomopoxvirus ortholog AMV232. Functional analyses revealed that the conserved P 38 YYCWY 43 motif is dispensable for EFC assembly but required for membrane fusion activity, whereas the flexible region spanning residues 90–110 mediates interactions required for stable incorporation of J5 into the EFC. IMPORTANCE Vaccinia virus enters host cells through membrane fusion mediated by a unique multi-component entry-fusion complex (EFC) that is distinct from classical viral fusion proteins. Although J5 has been identified as a central component of the pre-fusion EFC, the structural regions of J5 required for membrane fusion remain unclear. Here, we determined the solution NMR structure of the J5 ectodomain and identified two regions, the conserved P 38 YYCWY 43 motif and residues 90–110, as key determinants of EFC function during vaccinia virus entry.