鸭肝炎 A 病毒的 VP3 蛋白介导宿主细胞吸附和凋亡。
The VP3 protein of duck hepatitis A virus mediates host cell adsorption and apoptosis.
机构信息
Institute of Preventive Veterinary Medicine, Sichuan Agricultural University, Wenjiang, Chengdu City, 611130, Sichuan, P.R. China.
Key Laboratory of Animal Disease and Human Health of Sichuan Province, Sichuan Agricultural University, Wenjiang, Chengdu City, 611130, Sichuan, P.R. China.
出版信息
Sci Rep. 2019 Nov 14;9(1):16783. doi: 10.1038/s41598-019-53285-0.
Duck hepatitis A virus (DHAV) causes an infectious disease that mainly affects 1- to 4-week-old ducklings, resulting in considerable loss to the duck industry. Although there have been many studies on DHAV in recent years, the effects on host infection and pathogenesis of DHAV-1 remain largely unknown. This study investigated the effects of the DHAV-1 structural protein VP3 on DHAV-1 virus adsorption and apoptosis to explore the role of VP3 in the viral life cycle. The effects of DHAV-1 VP3 and an antibody against the protein on virion adsorption was analyzed by qRT-PCR. The results showed that the virus copy number for the rabbit anti-VP3 IgG-treated group was significantly lower than that for the negative control group but higher than that for the rabbit anti-DHAV-1 IgG-treated group. This result indicates that VP3 mediates DHAV-1 virus adsorption but that it is not the only protein that involved in this process. In addition, a eukaryotic recombinant plasmid, pCAGGS/VP3, was transfected into duck embryo fibroblasts (DEFs), and the apoptotic rate was determined by DAPI staining, the TUNEL assay and flow cytometry. DAPI staining showed nucleus fragmentation and nuclear edge shifting. TUNEL assay results revealed yellow nuclei, and flow cytometry indicated a significant increase in the apoptotic rate. In addition, qRT-PCR revealed increased in the transcriptional levels of the apoptotic caspase-3, -8 and -9, with the largest increase for caspase-3, followed by caspase-9 and caspase-8. Enzyme activity analysis confirmed these results. Furthermore, the VP3 protein decreased the mitochondrial membrane potential, and the transcriptional levels of the proapoptotic factors Bak, Cyt c and Apaf-1 in the mitochondrial apoptotic pathway were significantly upregulated. These data suggest that expression of VP3 in DEFs induces apoptosis and may primarily activate caspase-3-induced apoptosis through mitochondrion-mediated intrinsic pathways. The findings provide scientific data to clarify DHAV-1 infection and pathogenesis.
鸭甲型肝炎病毒 (DHAV) 引起的传染病主要影响 1-4 周龄的雏鸭，给养鸭业造成了巨大的损失。尽管近年来对 DHAV 进行了许多研究，但 DHAV-1 对宿主感染和发病机制的影响在很大程度上仍不清楚。本研究探讨了 DHAV-1 结构蛋白 VP3 对 DHAV-1 病毒吸附和凋亡的影响，以探索 VP3 在病毒生命周期中的作用。通过 qRT-PCR 分析 DHAV-1 VP3 和针对该蛋白的抗体对病毒粒子吸附的影响。结果表明，兔抗-VP3 IgG 处理组的病毒拷贝数明显低于阴性对照组，但高于兔抗-DHAV-1 IgG 处理组。这一结果表明，VP3 介导 DHAV-1 病毒吸附，但它不是参与该过程的唯一蛋白。此外，将真核重组质粒 pCAGGS/VP3 转染入鸭胚成纤维细胞（DEF），通过 DAPI 染色、TUNEL 检测和流式细胞术测定细胞凋亡率。DAPI 染色显示细胞核碎裂和核边缘移位。TUNEL 检测结果显示黄色核，流式细胞术表明凋亡率显著增加。此外，qRT-PCR 显示凋亡 Caspase-3、-8 和 -9 的转录水平升高，其中 Caspase-3 增加最明显，其次是 Caspase-9 和 Caspase-8。酶活性分析证实了这些结果。此外，VP3 蛋白降低了线粒体膜电位，线粒体凋亡途径中促凋亡因子 Bak、Cyt c 和 Apaf-1 的转录水平显著上调。这些数据表明，VP3 在 DEFs 中的表达诱导了细胞凋亡，并且可能主要通过线粒体介导的内在途径激活 caspase-3 诱导的凋亡。这些发现为阐明 DHAV-1 感染和发病机制提供了科学数据。
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