Structural Dissection of Vaccinia G9 Identifies Residues Essential for Membrane Fusion and Complex Assembly

Vaccinia virus, a prototypical poxvirus, utilizes a unique multi-protein Entry Fusion Complex (EFC), comprising 11 components, to mediate membrane fusion during host cell entry. Although the crystal structure of a truncated form of the G9 protein has been determined, the functional relevance of its structural features remains poorly understood. In this study, we systematically analyzed 47 G9 mutants to identify critical functional residues. Using trans-complementation assays, co-immunoprecipitation, membrane fusion assays, and structural analysis, we identified nine key mutants, which were categorized into three functional groups. Group 1 mutants failed to interact with A16 and other EFC components, highlighting their essential roles in G9-A16 subcomplex formation. Group 2 and Group 3 mutants retained A16 binding but disrupted interactions with other EFC proteins, suggesting their roles in broader complex assembly. Notably, Group 3 mutants targeted a conserved P(R/Y)XCW motif and a loop structure shared among vaccinia G9, A16, and J5 proteins. A similar motif was also identified in G9 homologs from Nucleocytoviricota, suggesting an evolutionarily conserved fusion mechanism. Collectively, our findings demonstrated that G9 function requires multiple domains, including A16-binding interfaces and conserved motifs not resolved in previous protein structure. These results establish G9 as a central EFC component and underscore its potential as a target for antiviral development. ImportanceUnderstanding how viruses enter host cells is critical for developing antiviral strategies. Vaccinia virus, a model poxvirus, uses a unique 11-protein entry fusion complex (EFC) to mediate membrane fusion--unlike other viruses that rely on a single fusion protein. In this study we identified specific residues in the G9 protein that are critical for maintaining EFC function. Notably, we discovered a conserved P(R/Y)XCW motif within G9 that is also present in orthologs from both poxviruses and members of the Nucleocytoviricota phylum, suggesting an evolutionarily conserved mechanism of membrane fusion. These conserved structural elements can serve as potential targets for antiviral intervention against pathogenic poxvirus infections in humans.