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VP1-binding protein glucose-regulated protein 78 as an important mediator for enterovirus 71 infecting human brain
Wenying Luo1, Peng Liang2, Santhosh Puthiyakunnon3
1Department of Clinical Laboratory, Affiliated Hospital of Guangdong Medical University, Zhanjiang; Department of Laboratory Medicine, School of Laboratory Medicine, Guangdong Medical University, Guangdong, China.
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.
Insights
Enterovirus 71 (EV71) infection involves glucose-regulated protein 78 (GRP78) binding to viral protein 1 (VP1). GRP78 mediates EV71 invasion and replication in brain endothelial cells, impacting disease progression.
Area of Science:
- Virology
- Cell Biology
- Neuroscience
Background:
- Enterovirus 71 (EV71) causes hand, foot, and mouth disease, potentially leading to severe neurological complications.
- The precise mechanisms of EV71 invasion and replication in the central nervous system remain incompletely understood.
Purpose of the Study:
- To identify host cell proteins involved in EV71 invasion of human brain microvascular endothelial cells (HBMECs).
- To elucidate the role of identified proteins in the EV71 infection pathway and pathogenesis.
Main Methods:
- Proteomic analysis (GST pull-down coupled with LC-ESI-MS/MS) to identify EV71 VP1-binding proteins in HBMECs.
- Validation using co-immunoprecipitation, immunofluorescence, and Western blot.
- Functional assays including gene knockdown/overexpression of GRP78 and TCID50 to assess viral replication.
Main Results:
- Ninety-one candidate proteins binding to EV71 VP1 were identified; 78 kDa glucose-regulated protein 78 (GRP78) was validated as a key interactor.
- GRP78 co-localized with VP1 in the cytoplasm of infected HBMECs.
- Knockdown of GRP78 reduced EV71 replication, while its overexpression enhanced viral load.
Conclusions:
- GRP78 is a crucial host factor mediating EV71 infection in HBMECs.
- GRP78 plays a significant role in the replication capacity of EV71.
- Targeting GRP78 may offer a therapeutic strategy against EV71-induced neuropathogenesis.
