Tandem mass tag-based quantitative proteomic analysis of lycorine treatment in highly pathogenic avian influenza H5N1 virus infection
Tandem mass tag-based quantitative proteomic analysis of lycorine treatment in highly pathogenic avian influenza H5N1 virus infection
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基于串联质量标签的石蒜碱治疗高致病性禽流感 H5N1 病毒感染的定量蛋白质组学分析
作者：
Yang, Li;Zhang, Jia Hao;He, Jun
高致病性H5 N1流感病毒（HPAIV）在易感动物中引起快速全身性疾病和死亡，导致具有高发病率和死亡率的疾病。虽然疫苗和药物是预防这种威胁的最佳解决方案，但尚未开发出更有效的H5流感病毒株治疗方法。因此，对抗H5 N1流感病毒感染的治疗剂/药物的开发变得越来越重要。石蒜碱是石蒜科植物生物碱的主要成分，在我们前期的研究中，石蒜碱对A/CK/GD/178/04（H5 N1）（GD 178）病毒的保护作用优于商业化的神经氨酸酶（NA）抑制剂奥司他韦。石蒜碱表现出突出的抗病毒活性，因为其抑制病毒核糖核蛋白复合物（vRNP）从细胞核输出的活性。然而石蒜碱如何影响AIV感染细胞的蛋白质组尚不清楚。因此，我们进行了比较蛋白质组学分析，以确定蛋白质表达的变化，在AIV感染的Madin-Darby犬肾细胞石蒜碱处理。设计了三组：假感染组（M）、病毒感染组（V）、病毒感染后石蒜碱处理组（L）。本研究采用多重串联质量标签（TMT）方法分析蛋白质水平。通过使用TMT蛋白质组学分析，总共从三组细胞中鉴定了5，786种蛋白质。在V/M组中，鉴定了1，101个蛋白质，其中340个差异表达蛋白（DEP）在HPAIV感染期间被确定;在石蒜碱处理组鉴定的1，059个蛋白质中，258个蛋白质呈现显著变化。在此，71种蛋白质在病毒感染/模拟和病毒感染/石蒜碱处理的比较中显示出显著的表达上调或下调，并且每个级分中的蛋白质在功能上进一步分类。有趣的是，石蒜碱处理降低了核孔复合物蛋白93（Nup 93，E2 RSV 7）的水平，这是与核质转运。此外，Western blot实验证实，在石蒜碱处理中Nup 93的表达显著下调，但在病毒感染后被诱导。我们的研究结果可能为石蒜碱如何在细胞核中捕获vRNP提供新的见解，并为流感病毒提供新的潜在治疗靶点。
Highly pathogenic H5N1 influenza viruses (HPAIV) cause rapid systemic illness and death in susceptible animals, leading to a disease with high morbidity and mortality rates. Although vaccines and drugs are the best solution to prevent this threat, a more effective treatment for H5 strains of influenza has yet to be developed. Therefore, the development of therapeutics/drugs that combat H5N1 influenza virus infection is becoming increasingly important. Lycorine, the major component of Amaryllidaceae alkaloids, exhibits better protective effects against A/CK/GD/178/04 (H5N1) (GD178) viruses than the commercial neuraminidase (NA) inhibitor oseltamivir in our prior study. Lycorine demonstrates outstanding antiviral activity because of its inhibitory activity against the export of viral ribonucleoprotein complexes (vRNPs) from the nucleus. However, how lycorine affects the proteome of AIV infected cells is unknown. Therefore, we performed a comparative proteomic analysis to identify changes in protein expression in AIV-infected Madin-Darby Canine Kidney cells treated with lycorine. Three groups were designed: mock infection group (M), virus infection group (V), and virus infection and lycorine-treated after virus infection group (L). The multiplexed tandem mass tag (TMT) approach was employed to analyze protein level in this study. In total, 5,786 proteins were identified from the three groups of cells by using TMT proteomic analysis. In the V/M group, 1,101 proteins were identified, of which 340 differentially expressed proteins (DEPs) were determined during HPAIV infection; among the 1,059 proteins identified from the lycorine-treated group, 258 proteins presented significant change. Here, 71 proteins showed significant upregulation or downregulation of expression in the virus-infected/mock and virus-infected/lycorine-treated comparisons, and the proteins in each fraction were functionally classified further. Interestingly, lycorine treatment decreased the levels of the nuclear pore complex protein 93 (Nup93, E2RSV7), which is associated with nuclear-cytoplasmic transport. In addition, Western blot experiments confirmed that the expression of Nup93 was significantly downregulated in lycorine treatment but induced after viral infection. Our results may provide new insights into how lycorine may trap vRNPs in the nucleus and suggest new potential therapeutic targets for influenza virus.