Chiu Hsiao-Jung, Wang Hao-Ching, Chang Wen
Molecular and Cell Biology, Taiwan International Graduate Program, Academia Sinica and Graduate Institute of Life Sciences, National Defense Medical Center, Taipei, Taiwan.
Institute of Molecular Biology, Taipei, Taiwan.
J Virol. 2025 Aug 18:e0072325. doi: 10.1128/jvi.00723-25.
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 , 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 structures. 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 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.
痘苗病毒是一种典型的痘病毒，它利用一种独特的多蛋白进入融合复合体（EFC）（由11种成分组成）在宿主细胞进入过程中介导膜融合。尽管已确定了截短形式的G9蛋白的晶体结构，但其结构特征的功能相关性仍知之甚少。在本研究中，我们系统分析了47个G9突变体以鉴定关键功能残基。通过反式互补分析、免疫共沉淀、膜融合分析和结构分析，我们鉴定出9个关键突变体，它们被分为三个功能组。第1组突变体无法与A16及其他EFC成分相互作用，突出了它们在G9 - A16亚复合体形成中的重要作用。第2组和第3组突变体保留了与A16的结合，但破坏了与其他EFC蛋白的相互作用，表明它们在更广泛的复合体组装中的作用。值得注意的是，第3组突变体靶向一个保守的P(R/Y)XCW基序以及痘苗病毒G9、A16和J5蛋白共有的一个环结构。在来自[未提及的来源]的G9同源物中也鉴定出了类似的基序，表明存在一种进化上保守的融合机制。总体而言，我们的研究结果表明，G9功能需要多个结构域，包括A16结合界面和先前蛋白质结构中未解析的保守基序。这些结果确立了G9作为EFC的核心成分，并强调了其作为抗病毒开发靶点的潜力。
了解病毒如何进入宿主细胞对于制定抗病毒策略至关重要。痘苗病毒作为一种模型痘病毒，与其他依赖单一融合蛋白的病毒不同，它使用一种独特的由11种蛋白组成的进入融合复合体（EFC）来介导膜融合。在本研究中，我们鉴定出了G9蛋白中对于维持EFC功能至关重要的特定残基。值得注意的是，我们在G9中发现了一个保守的P(R/Y)XCW基序，该基序在痘病毒和[未提及的门类]成员的直系同源物中也存在，这表明存在一种进化上保守的膜融合机制。这些保守的结构元件可作为针对人类致病性痘病毒感染的抗病毒干预的潜在靶点。
