Stanton R J, McSharry B P, Rickards C R, Wang E C Y, Tomasec P, Wilkinson G W G
Department of Medical Microbiology, Tenovus Building, Heath Park, Cardiff CF14 4XX, United Kingdom.
J Virol. 2007 Aug;81(15):7860-72. doi: 10.1128/JVI.02247-06. Epub 2007 May 23.
Human cytomegalovirus (HCMV) systematically manages the expression of cellular functions, rather than exerting the global shutoff of host cell protein synthesis commonly observed with other herpesviruses during the lytic cycle. While microarray technology has provided remarkable insights into viral control of the cellular transcriptome, HCMV is known to encode multiple mechanisms for posttranscriptional and post-translation regulation of cellular gene expression. High-throughput Western blotting (BD Biosciences Powerblot technology) with 1,009 characterized antibodies was therefore used to analyze and compare the effects of infection with attenuated high-passage strain AD169 and virulent low-passage strain Toledo at 72 hpi across gels run in triplicate for each sample. Six hundred ninety-four proteins gave a positive signal in the screen, of which 68 from strain AD169 and 71 from strain Toledo were defined as being either positively or negatively regulated by infection with the highest level of confidence (BD parameters). In follow-up analyses, a subset of proteins was selected on the basis of the magnitude of the observed effect or their potential to contribute to defense against immune recognition. In analyses performed at 24, 72, and 144 hpi, connexin 43 was efficiently downregulated during HCMV infection, implying a breakdown of intercellular communication. Mitosis-associated protein Eg-5 was found to be differentially upregulated in the AD169 and Toledo strains of HCMV. Focal adhesions link the actin cytoskeleton to the extracellular matrix and have key roles in initiating signaling pathways and substrate adhesion and regulating cell migration. HCMV suppressed expression of the focal-adhesion-associated proteins Hic-5, paxillin, and alpha-actinin. Focal adhesions were clearly disrupted in HCMV-infected fibroblasts, with their associated intracellular and extracellular proteins being dispersed. Powerblot shows potential for rapid screening of the cellular proteome during HCMV infection.
人巨细胞病毒（HCMV）系统性地调控细胞功能的表达，而非像其他疱疹病毒在裂解周期中那样导致宿主细胞蛋白质合成的全面关闭。虽然微阵列技术为病毒对细胞转录组的控制提供了显著的见解，但已知HCMV编码多种用于细胞基因表达的转录后和翻译后调控机制。因此，使用具有1009种特征明确抗体的高通量蛋白质印迹法（BD Biosciences Powerblot技术），分析并比较在感染后72小时，减毒高代次菌株AD169和强毒低代次菌株托莱多感染的效果，每个样品的凝胶进行一式三份电泳。在筛选中，694种蛋白质给出了阳性信号，其中来自菌株AD169的68种和来自菌株托莱多的71种被确定为受感染正向或负向调控，置信度最高（BD参数）。在后续分析中，根据观察到的效应大小或其对抵御免疫识别的潜在贡献，选择了一部分蛋白质。在感染后24、72和144小时进行的分析中，连接蛋白43在HCMV感染期间被有效下调，这意味着细胞间通讯的破坏。有丝分裂相关蛋白Eg-5在HCMV的AD169和托莱多菌株中被发现有差异上调。粘着斑将肌动蛋白细胞骨架与细胞外基质连接起来，在启动信号通路、底物粘附和调节细胞迁移中起关键作用。HCMV抑制粘着斑相关蛋白Hic-5、桩蛋白和α-辅肌动蛋白的表达。在HCMV感染的成纤维细胞中，粘着斑明显被破坏，其相关的细胞内和细胞外蛋白分散。Powerblot显示了在HCMV感染期间快速筛选细胞蛋白质组的潜力。
