Quantitative phosphoproteomic analysis of host responses in human lung epithelial (A549) cells during influenza virus infection
Quantitative phosphoproteomic analysis of host responses in human lung epithelial (A549) cells during influenza virus infection
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DOI：
10.1016/j.virusres.2013.11.012
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发表时间：
2014-01-22
期刊：
影响因子：
5
通讯作者：
Horigome, Tsuneyoshi
中科院分区：
文献类型：
--
作者：
Dapat, Clyde;Saito, Reiko;Horigome, Tsuneyoshi
抗病毒药物耐药性流感病毒的出现突出了对替代治疗策略的需求。阐明病毒感染过程中所需的宿主因子不仅提供了有关信号传导途径的信息，而且还提供了识别新药物靶点的信息。本研究利用RNA干扰筛选方法对2009年甲型H1N1流感病毒感染的人肺细胞株A549进行磷酸化蛋白质组学定量分析。使用自动纯化系统上的二氧化钛柱，然后进行iTRAQ标记，从感染和模拟感染细胞的总蛋白的胰蛋白酶解物中富集磷酸肽。使用LC-MS/MS对iTRAQ标记的磷酸肽进行鉴定和定量分析。我们在283个蛋白质上鉴定了366个磷酸化位点。其中，我们检测到43个上调和35个下调蛋白在流感病毒感染。基因本体富集分析表明，大部分鉴定的蛋白质是参与RNA加工、免疫系统过程和对感染的反应的磷蛋白。宿主-病毒相互作用网络分析确定了23个密集连接的子网络。其中，13个子网络包含在流感病毒感染期间磷酸化水平改变的蛋白质。我们的研究结果将有助于确定潜在的药物靶点，可以追求流感抗病毒药物的开发。(C)2013 Elsevier B. V.保留所有权利。
The emergence of antiviral drug-resistant influenza viruses highlights the need for alternative therapeutic strategies. Elucidation of host factors required during virus infection provides information not only on the signaling pathways involved but also on the identification of novel drug targets. RNA interference screening method had been utilized by several studies to determine these host factors; however, proteomics data on influenza host factors are currently limited.In this study, quantitative phosphoproteomic analysis of human lung cell line (A549) infected with 2009 pandemic influenza virus A (H1N1) virus was performed. Phosphopeptides were enriched from tryptic digests of total protein of infected and mock-infected cells using a titania column on an automated purification system followed by iTRAQ labeling. Identification and quantitative analysis of iTRAQ-labeled phosphopeptides were performed using LC-MS/MS.We identified 366 phosphorylation sites on 283 proteins. Of these, we detected 43 upregulated and 35 downregulated proteins during influenza virus infection. Gene ontology enrichment analysis showed that majority of the identified proteins are phosphoproteins involved in RNA processing, immune system process and response to infection. Host-virus interaction network analysis had identified 23 densely connected subnetworks. Of which, 13 subnetworks contained proteins with altered phosphorylation levels during by influenza virus infection. Our results will help to identify potential drug targets that can be pursued for influenza antiviral drug development. (C) 2013 Elsevier B.V. All rights reserved.