Department of Medical Microbiology, Faculty of Medicine, University of Manitoba, Winnipeg, Manitoba R3E 0J6, Canada.
J Virol. 2010 Oct;84(20):10888-906. doi: 10.1128/JVI.00431-10. Epub 2010 Aug 11.
Because they are obligate intracellular parasites, all viruses are exclusively and intimately dependent upon host cells for replication. Viruses, in turn, induce profound changes within cells, including apoptosis, morphological changes, and activation of signaling pathways. Many of these alterations have been analyzed by gene arrays, which measure the cellular "transcriptome." Until recently, it has not been possible to extend comparable types of studies to globally examine all the host cellular proteins, which are the actual effector molecules. We have used stable isotope labeling by amino acids in cell culture (SILAC), combined with high-throughput two-dimensional (2-D) high-performance liquid chromatography (HPLC)/mass spectrometry, to determine quantitative differences in host proteins after infection of human lung A549 cells with human influenza virus A/PR/8/34 (H1N1) for 24 h. Of the 4,689 identified and measured cytosolic protein pairs, 127 were significantly upregulated at >95% confidence, 153 were significantly downregulated at >95% confidence, and a total of 87 proteins were upregulated or downregulated more than 5-fold at >99% confidence. Gene ontology and pathway analyses indicated differentially regulated proteins and included those involved in host cell immunity and antigen presentation, cell adhesion, metabolism, protein function, signal transduction, and transcription pathways.
由于病毒是专性细胞内寄生虫，因此它们完全且紧密地依赖宿主细胞进行复制。反过来，病毒会在细胞内引起深刻的变化，包括细胞凋亡、形态变化和信号通路的激活。许多这些变化已经通过基因芯片进行了分析，基因芯片测量细胞的“转录组”。直到最近，还不可能进行类似的研究来全面检查所有作为实际效应分子的宿主细胞蛋白。我们使用稳定同位素标记的氨基酸在细胞培养物中进行了研究（SILAC），并结合二维（2-D）高效液相色谱（HPLC）/质谱的高通量技术，以确定在感染人类流感病毒 A/PR/8/34（H1N1）24 小时后，人类肺 A549 细胞中宿主蛋白的定量差异。在所鉴定和测量的 4689 个细胞质蛋白对中，有 127 个以>95%的置信度显著上调，有 153 个以>95%的置信度显著下调，共有 87 个蛋白的上调或下调超过 5 倍，置信度>99%。基因本体论和途径分析表明，差异调节的蛋白包括参与宿主细胞免疫和抗原呈递、细胞黏附、代谢、蛋白功能、信号转导和转录途径的蛋白。
