Key Laboratory of Animal Virology of Ministry of Agriculture, Zhejiang University, Hangzhou, P. R. China; State Key Laboratory for Diagnosis and Treatment of Infectious Diseases, First Affiliated Hospital, Zhejiang University, Hangzhou, P. R. China.
Proteomics. 2013 Nov;13(22):3309-26. doi: 10.1002/pmic.201300180. Epub 2013 Oct 25.
Cross-species transmissions of swine influenza viruses (SIVs) raise great public health concerns. In this study, subcellular proteomic profiles of human A549 cells inoculated with H3N2 subtype SIV were used to characterize dynamic cellular responses to infection. By 2DE and MS, 27 differentially expressed (13 upregulated, 14 downregulated) cytoplasmic proteins and 20 differentially expressed (13 upregulated, 7 downregulated) nuclear proteins were identified. Gene ontology analysis suggested that these differentially expressed proteins were mainly involved in cell death, stress response, lipid metabolism, cell signaling, and RNA PTMs. Moreover, 25 corresponding genes of the differentially expressed proteins were quantitated by real time RT-PCR to examine the transcriptional profiles between mock- and virus-infected A549 cells. Western blot analysis confirmed that changes in abundance of identified cellular proteins heterogeneous nuclear ribonucleoprotein (hnRNP) U, hnRNP C, ALDH1A1, tryptophanyl-tRNA synthetase, IFI35, and HSPB1 in H3N2 SIV-infected cells were consistent with results of 2DE analysis. By confocal microscopy, nucleus-to-cytoplasm translocation of hnRNP C and colocalization between the viral nonstructural protein 1 and hnRNP C as well as N-myc (and STAT) interactor were observed upon infection. Ingenuity Pathway Analysis revealed that cellular proteins altered during infection were grouped mainly into NFκB and interferon signaling networks. Collectively, these identified subcellular constituents provide an important framework for understanding host/SIV interactions and underlying mechanisms of SIV cross-species infection and pathogenesis.
跨物种传播的猪流感病毒 (SIV) 引起了极大的公共卫生关注。在这项研究中，用人 A549 细胞接种 H3N2 亚型 SIV 的亚细胞蛋白质组学图谱来描述感染后细胞的动态反应。通过 2-DE 和 MS，鉴定出 27 个差异表达的 (13 个上调，14 个下调) 细胞质蛋白和 20 个差异表达的 (13 个上调，7 个下调) 核蛋白。GO 分析表明，这些差异表达的蛋白主要参与细胞死亡、应激反应、脂质代谢、细胞信号转导和 RNA PTMs。此外，通过实时 RT-PCR 对差异表达蛋白的 25 个相应基因进行定量，以检测模拟和病毒感染 A549 细胞之间的转录谱。Western blot 分析证实，在 H3N2 SIV 感染细胞中，异质核核糖核蛋白 (hnRNP) U、hnRNP C、ALDH1A1、色氨酰-tRNA 合成酶、IFI35 和 HSPB1 等鉴定出的细胞蛋白的丰度变化与 2-DE 分析结果一致。通过共聚焦显微镜观察到，hnRNP C 的核质易位以及病毒非结构蛋白 1 与 hnRNP C 以及 N-myc(和 STAT)相互作用蛋白之间的共定位。IPA 分析表明，感染过程中改变的细胞蛋白主要分为 NFκB 和干扰素信号网络。总之，这些鉴定出的亚细胞成分为理解宿主/SIV 相互作用以及 SIV 跨种感染和发病机制的潜在机制提供了重要框架。
