Department of Microbiology and Immunology, College of Medicine, National Cheng Kung University, 1, University Road, Tainan, 701401, Taiwan.
Ditmanson Medical Foundation Chia-Yi Christian Hospital, Chiayi, Taiwan.
J Biomed Sci. 2023 Feb 23;30(1):14. doi: 10.1186/s12929-023-00901-x.
Influenza is one of the most important viral infections globally. Viral RNA-dependent RNA polymerase (RdRp) consists of the PA, PB1, and PB2 subunits, and the amino acid residues of each subunit are highly conserved among influenza A virus (IAV) strains. Due to the high mutation rate and emergence of drug resistance, new antiviral strategies are needed. Host cell factors are involved in the transcription and replication of influenza virus. Here, we investigated the role of galectin-3, a member of the β-galactoside-binding animal lectin family, in the life cycle of IAV infection in vitro and in mice.
We used galectin-3 knockout and wild-type mice and cells to study the intracellular role of galectin-3 in influenza pathogenesis. Body weight and survival time of IAV-infected mice were analyzed, and viral production in mouse macrophages and lung fibroblasts was examined. Overexpression and knockdown of galectin-3 in A549 human lung epithelial cells were exploited to assess viral entry, viral ribonucleoprotein (vRNP) import/export, transcription, replication, virion production, as well as interactions between galectin-3 and viral proteins by immunoblotting, immunofluorescence, co-immunoprecipitation, RT-qPCR, minireplicon, and plaque assays. We also employed recombinant galectin-3 proteins to identify specific step(s) of the viral life cycle that was affected by exogenously added galectin-3 in A549 cells.
Galectin-3 levels were increased in the bronchoalveolar lavage fluid and lungs of IAV-infected mice. There was a positive correlation between galectin-3 levels and viral loads. Notably, galectin-3 knockout mice were resistant to IAV infection. Knockdown of galectin-3 significantly reduced the production of viral proteins and virions in A549 cells. While intracellular galectin-3 did not affect viral entry, it increased vRNP nuclear import, RdRp activity, and viral transcription and replication, which were associated with the interaction of galectin-3 with viral PA subunit. Galectin-3 enhanced the interaction between viral PA and PB1 proteins. Moreover, exogenously added recombinant galectin-3 proteins also enhanced viral adsorption and promoted IAV infection in A549 cells.
We demonstrate that galectin-3 enhances viral infection through increases in vRNP nuclear import and RdRp activity, thereby facilitating viral transcription and replication. Our findings also identify galectin-3 as a potential therapeutic target for influenza.
流感是全球最重要的病毒性传染病之一。病毒 RNA 依赖性 RNA 聚合酶（RdRp）由 PA、PB1 和 PB2 亚基组成，每个亚基的氨基酸残基在流感病毒（IAV）株之间高度保守。由于高突变率和耐药性的出现，需要新的抗病毒策略。宿主细胞因子参与流感病毒的转录和复制。在这里，我们研究了半乳糖凝集素-3（一种动物凝集素家族的β-半乳糖苷结合蛋白）在体外和小鼠体内 IAV 感染生命周期中的作用。
我们使用半乳糖凝集素-3 敲除和野生型小鼠和细胞来研究流感发病机制中半乳糖凝集素-3 的细胞内作用。分析 IAV 感染小鼠的体重和存活时间，并检测小鼠巨噬细胞和肺成纤维细胞中的病毒产生情况。利用 A549 人肺上皮细胞中转染半乳糖凝集素-3 过表达和敲低载体，通过免疫印迹、免疫荧光、共免疫沉淀、RT-qPCR、小复制子和噬斑分析来评估病毒进入、病毒核糖核蛋白（vRNP）输入/输出、转录、复制、病毒粒子产生以及半乳糖凝集素-3 与病毒蛋白之间的相互作用。我们还使用重组半乳糖凝集素-3 蛋白来鉴定在 A549 细胞中外源添加半乳糖凝集素-3 影响病毒生命周期的特定步骤。
IAV 感染小鼠的支气管肺泡灌洗液和肺组织中半乳糖凝集素-3 水平增加。半乳糖凝集素-3 水平与病毒载量呈正相关。值得注意的是，半乳糖凝集素-3 敲除小鼠对 IAV 感染具有抗性。A549 细胞中半乳糖凝集素-3 敲低显著降低了病毒蛋白和病毒粒子的产生。虽然细胞内半乳糖凝集素-3 不影响病毒进入，但它增加了 vRNP 核输入、RdRp 活性以及病毒转录和复制，这与半乳糖凝集素-3 与病毒 PA 亚基的相互作用有关。半乳糖凝集素-3 增强了病毒 PA 和 PB1 蛋白之间的相互作用。此外，外源性添加的重组半乳糖凝集素-3 蛋白也增强了病毒吸附并促进了 A549 细胞中的 IAV 感染。
我们证明半乳糖凝集素-3 通过增加 vRNP 核输入和 RdRp 活性来增强病毒感染，从而促进病毒转录和复制。我们的研究结果还表明半乳糖凝集素-3 是流感的潜在治疗靶点。
