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 ORIGINAL ARTICLE
Year : 2019  |  Volume : 37  |  Issue : 3  |  Page : 406-414

VP1-binding protein glucose-regulated protein 78 as an important mediator for enterovirus 71 infecting human brain microvascular endothelial cells


1 Department of Clinical Laboratory, Affiliated Hospital of Guangdong Medical University, Zhanjiang; Department of Laboratory Medicine, School of Laboratory Medicine, Guangdong Medical University, Guangdong, China
2 Department of Clinic Laboratory, The Fifth Affiliated Hospital of Guangzhou Medical University, Guangzhou, China
3 Department of Microbiology, Guangdong Provincial Key Laboratory of Tropical Disease Research, School of Public Health, Southern Medical University, Guangzhou, China
4 Department of Clinical Laboratory, Affiliated Hospital of Guangdong Medical University, Zhanjiang, China

Correspondence Address:
Prof. Cao Hong
Department of Microbiology, Guangdong Provincial Key Laboratory of Tropical Disease Research, School of Public Health, Southern Medical University, Guangzhou 510515
China
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/ijmm.IJMM_19_194

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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.






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