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首页> 外文期刊>Molecular & cellular proteomics: MCP >A Comprehensive Proteomic View of Responses of A549 Type II Alveolar Epithelial Cells to Human Respiratory Syncytial Virus Infection
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A Comprehensive Proteomic View of Responses of A549 Type II Alveolar Epithelial Cells to Human Respiratory Syncytial Virus Infection

机译：A549 II型肺泡上皮细胞对人类呼吸道合胞病毒感染的反应的综合蛋白质组学观点。

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Human respiratory syncytial virus is a major respiratory pathogen for which there are no suitable antivirals or vaccines. A better understanding of the host cell response to this virus may redress this problem. The present report concerns analysis of multiple independent biological replicates of control and 24 h infected lysates of A549 cells by two different proteomic workflows. One workflow involved fractionation of lysates by in-solution protein IEF and individual fractions were digested using trypsin prior to capillary HPLC-LTQ-OrbitrapXL-MS/MS. A second workflow involved digestion of whole cell lysates and analysis by nanoUltraHPLC-LTQ-OrbitrapElite-MS/MS. Both workflows resulted in the quantification of viral proteins exclusively in lysates of infected cells in the relative abundances anticipated from previous studies. Unprecedented numbers (3247 - 5010) of host cell protein groups were also quantified and the infection-specific regulation of a large number (191) of these protein groups was evident based on a stringent false discovery rate cut-off (<1%). Bioinformatic analyses revealed that most of the regulated proteins were potentially regulated by type I, II, and III interferon, TNF-alpha and noncanonical NF-kappa B2 mediated antiviral response pathways. Regulation of specific protein groups by infection was validated by quantitative Western blotting and the cytokine-/key regulator-specific nature of their regulation was confirmed by comparable analyses of cytokine treated A549 cells. Overall, it is evident that the workflows described herein have produced the most comprehensive proteomic characterization of host cell responses to human respiratory syncytial virus published to date. These workflows will form the basis for analysis of the impacts of specific genes of human respiratory syncytial virus responses of A549 and other cell lines using a gene-deleted version of the virus. They should also prove valuable for the analysis of the impact of other infectious agents on host cells.
机译：人呼吸道合胞病毒是主要的呼吸道病原体，没有合适的抗病毒药或疫苗。更好地了解宿主细胞对此病毒的反应可能会解决此问题。本报告涉及通过两种不同的蛋白质组学工作流程对对照和24小时感染的A549细胞裂解液进行多个独立生物学复制的分析。一种工作流程涉及通过溶液中蛋白质IEF分离裂解物，并在毛细管HPLC-LTQ-OrbitrapXL-MS / MS之前使用胰蛋白酶消化各个部分。第二个工作流程涉及消化全细胞裂解物并通过nanoUltraHPLC-LTQ-OrbitrapElite-MS / MS进行分析。两种工作流程均导致仅在感染细胞的裂解液中对病毒蛋白进行定量，其定量结果是先前研究预期的。宿主细胞蛋白质组的空前数量（3247-5010）也得到了量化，并且基于严格的错误发现率临界值（<1％），大量（191）这些蛋白质组的感染特异性调节也很明显。生物信息学分析表明，大多数受调节的蛋白都可能受到I，II和III型干扰素，TNF-α和非经典NF-κB2介导的抗病毒应答途径的调节。通过定量蛋白质印迹法验证了感染对特定蛋白基团的调节，并且通过对细胞因子处理过的A549细胞进行了可比分析，证实了其调节的细胞因子/关键调节因子特异性。总体而言，显然，本文所述的工作流程已产生了迄今为止对宿主细胞对人呼吸道合胞病毒的反应的最全面的蛋白质组学表征。这些工作流程将构成使用该基因删除版本的病毒分析A549和其他细胞系的人类呼吸道合胞病毒反应特定基因影响的基础。它们还应被证明对于分析其他传染因子对宿主细胞的影响是有价值的。

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