Division of International Health (Public Health), Graduate School of Medical, Dental Sciences, Niigata University, Japan.
Division of International Health (Public Health), Graduate School of Medical, Dental Sciences, Niigata University, Japan.
Virus Res. 2014 Jan 22;179:53-63. doi: 10.1016/j.virusres.2013.11.012. Epub 2013 Nov 27.
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.
抗病毒药物耐药流感病毒的出现凸显了需要替代治疗策略。阐明病毒感染过程中所需的宿主因素不仅提供了有关涉及的信号通路的信息，还提供了鉴定新的药物靶标的信息。一些研究已经利用 RNA 干扰筛选方法来确定这些宿主因素；然而，目前流感宿主因素的蛋白质组学数据有限。在这项研究中，对感染 2009 年大流行性流感病毒 A（H1N1）的人肺细胞系（A549）进行了定量磷酸化蛋白质组学分析。使用自动纯化系统上的 TiO2 柱从感染和模拟感染细胞的总蛋白的胰蛋白酶消化物中富集磷酸肽，然后进行 iTRAQ 标记。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 种蛋白质上鉴定了 366 个磷酸化位点。其中，我们在流感病毒感染期间检测到 43 个上调和 35 个下调蛋白。GO 富集分析表明，大多数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酸化蛋白。宿主-病毒相互作用网络分析确定了 23 个密集连接的子网。其中，13 个子网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鉴定可用于流感抗病毒药物开发的潜在药物靶标。
