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首页> 外文期刊>Indian Journal of Medical Microbiology >VP1-binding protein glucose-regulated protein 78 as an important mediator for enterovirus 71 infecting human brain microvascular endothelial cells
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VP1-binding protein glucose-regulated protein 78 as an important mediator for enterovirus 71 infecting human brain microvascular endothelial cells

机译：VP1结合蛋白葡萄糖调节蛋白78作为肠道病毒71感染人脑微血管内皮细胞的重要介体

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Purpose: Enterovirus 71 (EV71) is one of the main pathogens causing hand, foot and mouth disease, which could even induce severe brain damage in some patients. As the underlying mechanism of the invasion and replication process still remains largely unknown, we investigated the role of candidate proteins expressed during EV71 invasion in human brain microvascular endothelial cells (HBMECs) to delineate the pathophysiological mechanism of EV-71 infection. Materials and Methods: Ninety-one candidate EV71-associated proteins which could bind the major capsid protein (viral protein 1 [VP1]) of EV71 on the HBMEC were identified by applying an analysis of glutathione-S-transferase pull-down coupling with liquid chromatography-electrospray ionisation-tandem mass spectrometry (LC-ESI-MS/MS). Seventy-eight kDa glucose-regulated protein 78 (GRP78) binding to the VP1 protein was further validated by co-immunoprecipitation, immunofluorescence and western blot analysis. To explore the role of GRP78 in EV71 infection, GRP78 was knocked down and overexpressed in HBMEC and was verified by TCID50 assay. Results: LC-ESI-MS/MS-identified 91 proteins were subjected to gene ontology analysis, and on molecular and biological function analysis revealed GRP78 act as an important binding protein in mediating EV71 infection. In addition, immunofluorescence demonstrated the co-localisation of GRP78 and VP1 in cytoplasm of the infected HBMEC. The TCID50 assay showed that knockdown of GRP78 could attenuate the replication capacity of EV71 in HBMEC, and the overexpression could increase the virus titre in HBEMC at 24 h post-infection suggesting that GRP78 was associated with the replication capacity of EV71 in HBMEC. Conclusion: These findings provided evidence that GRP78 plays an important role during the progression of EV71 infection as a mediator in HBMEC.
机译：目的：肠病毒71（EV71）是引起手足口病的主要病原体之一，在某些患者中甚至可能引起严重的脑损伤。由于入侵和复制过程的潜在机制仍在很大程度上未知，我们调查了人脑微血管内皮细胞（HBMEC）在EV71入侵过程中表达的候选蛋白的作用，以描述EV-71感染的病理生理机制。材料与方法：通过分析与液体结合的谷胱甘肽-S-转移酶下拉结合分析，鉴定了九十一种与EV71的主要衣壳蛋白（病毒蛋白1 [VP1]）结合的候选蛋白。色谱-电喷雾串联质谱法（LC-ESI-MS / MS）。通过共免疫沉淀，免疫荧光和蛋白质印迹分析进一步验证了与VP1蛋白结合的78 kDa葡萄糖调节蛋白78（GRP78）。为了探索GRP78在EV71感染中的作用，将GRP78敲低并在HBMEC中过表达，并通过TCID50分析进行了验证。结果：对LC-ESI-MS / MS鉴定出的91种蛋白质进行了基因本体分析，分子和生物学功能分析表明，GRP78是介导EV71感染的重要结合蛋白。另外，免疫荧光证明了GRP78和VP1在被感染的HBMEC的细胞质中共定位。 TCID50分析表明，敲除GRP78可以减弱HBVEC中EV71的复制能力，并且过表达可以增加感染后24 h HBEMC中的病毒效价，表明GRP78与EV71在HBMEC中的复制能力有关。结论：这些发现提供了证据，表明GRP78在EV71感染的过程中作为HBMEC的介体起着重要作用。

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