Biology subjects

Chiu, H.-J.

Publications and source records attributed to Chiu, H.-J..

3 recordsLinked to original sources

bioRxiv2026

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 P38YYCWY43 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. ImportanceVaccinia 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 P38YYCWY43 motif and residues 90-110, as key determinants of EFC function during vaccinia virus entry.

microbiology↗

bioRxiv2025

Structure and functional analyses of Vaccinia virus entry- fusion complex component J5 protein

Vaccinia virus enters host cells through an 11-protein entry fusion complex (EFC) that operates by a mechanism distinct from those of canonical viral fusion systems. Understanding how this multiprotein complex mediates membrane fusion is crucial for elucidating poxvirus entry and identifying potential antiviral targets. Here, we determined the NMR structure of a truncated J5 protein, (J5 2-68). To further dissect the functional determinants of J5, we generated recombinant vaccinia viruses expressing various J5 mutants, including substitutions in conserved residues, alterations of exposed charged residues, and chimeric constructs between vaccinia J5 and its orthologous AMV232 gene from an entomopoxvirus. Functional analyses revealed that residues 90-110 and the conserved P38YYCWY43 motif are indispensable for maintaining EFC integrity and promoting membrane fusion. Together, we define the structural and functional elements of J5 that are essential for poxvirus entry and advance our understanding of the unique membrane fusion mechanism employed by poxviruses. ImportanceVaccinia virus enters host cells through an eleven-protein entry fusion complex (EFC) that is mechanistically distinct from canonical viral fusion systems. Understanding how this multiprotein machinery mediates membrane fusion is essential for elucidating poxvirus entry mechanisms and for developing antiviral strategies. Here, we determined the NMR structure of the J5 ectodomain and generated a series of recombinant vaccinia viruses carrying J5 mutations. Functional analyses identified two regions, residues 90-110 and the conserved P38YYCWY43 motif, as essential for EFC stability and membrane fusion. These findings provide the first structure-function framework for J5, a core component of the EFC, and reveal key determinants required for complex integrity and viral entry.

microbiology↗

bioRxiv2025

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.

microbiology↗