Ezrin is essential for the entry of Japanese encephalitis virus into the human brain microvascular endothelial cells

Liu, Yangang; Yang, Chen; Wang, Xiaohang; Zhao, Ping; Zhu, Yu; Qi, Zhongtian

doi:10.1080/22221751.2020.1757388

Cited by 20 publications

(9 citation statements)

References 50 publications

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“…In vitro experiments have confirmed that some flaviviruses can infect and replicate in BMECs in vertebrates. 36 , 37 However, the infection and replication of flavivirus in BMECs of human patients still lack sufficient direct evidence and require histological confirmation. ZIKV persistently infects and continuously replicates in primary hBMECs in vitro , without obvious cytopathology or increased endothelial cell permeability.…”

Section: How Do Endothelial Cells Of the Bbb Respond To Viral Infectimentioning

confidence: 99%

Immune response and blood–brain barrier dysfunction during viral neuroinvasion

Chen

2020

Innate Immun

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The blood-brain barrier (BBB), which protects the CNS from pathogens, is composed of specialized brain microvascular endothelial cells (BMECs) joined by tight junctions and ensheathed by pericytes and astrocyte endfeet. The stability of the BBB structure and function is of great significance for the maintenance of brain homeostasis. When a neurotropic virus invades the CNS via a hematogenous or non-hematogenous route, it may cause structural and functional disorders of the BBB, and also activate the BBB anti-inflammatory or pro-inflammatory innate immune response. This article focuses on the structural and functional changes that occur in the three main components of the BBB (endothelial cells, astrocytes, and pericytes) in response to infection with neurotropic viruses transmitted by hematogenous routes, and also briefly describes the supportive effect of three cells on the BBB under normal physiological conditions. For example, all three types of cells express several PRRs, which can quickly sense the virus and make corresponding immune responses. The pro-inflammatory immune response will exacerbate the destruction of the BBB, while the anti-inflammatory immune response, based on type I IFN, consolidates the stability of the BBB. Exploring the details of the interaction between the host and the pathogen at the BBB during neurotropic virus infection will help to propose new treatments for viral encephalitis. Enhancing the defense function of the BBB, maintaining the integrity of the BBB, and suppressing the pro-inflammatory immune response of the BBB provide more ideas for limiting the neuroinvasion of neurotropic viruses. In the future, these new treatments are expected to cooperate with traditional antiviral methods to improve the therapeutic effect of viral encephalitis.

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Section: How Do Endothelial Cells Of the Bbb Respond To Viral Infectimentioning

confidence: 99%

Immune response and blood–brain barrier dysfunction during viral neuroinvasion

Chen

2020

Innate Immun

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“…ATP6V1A directly interacts with rabies viral matrix protein facilitating uncoating [52]. EZR is an essential host factor required for the entry of [21] Japanese encephalitis virus into human brain microvascular endothelial cells [53]. HSPA9 has been identified as a putative receptor for Tembusu virus [54].…”

Section: Discussionmentioning

confidence: 99%

Proteome-wide cross-linking mass spectrometry to identify specific virus capsid-host interactions between tick-borne encephalitis virus and neuroblastoma cells

Barrass

Pulkkinen

Vapalahti

et al. 2021

Preprint

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Virus-host protein-protein interactions are central to viral infection, but are challenging to identify and characterise, especially in complex systems involving intact viruses and cells. In this work, we demonstrate a proteome-wide approach to identify virus-host interactions using chemical cross-linking coupled with mass spectrometry. We adsorbed tick-borne encephalitis virus onto metabolically-stalled neuroblastoma cells, covalently cross-linked interacting virus-host proteins, and performed limited proteolysis to release primarily the surface-exposed proteins for identification by mass spectrometry. Using the intraviral protein cross-links as an internal control to assess cross-link confidence levels, we identified 22 high confidence unique intraviral cross-links and 59 high confidence unique virus-host protein-protein interactions. The identified host proteins were shown to interact with eight distinct sites on the outer surface of the virus. Notably, we identified an interaction between the substrate-binding domain of heat shock protein family A member 5, an entry receptor for four related flaviviruses, and the hinge region of the viral envelope protein. We also identified host proteins involved in endocytosis, cytoskeletal rearrangement, or located in the cytoskeleton, suggesting that entry mechanisms for tick-borne encephalitis virus could include both clathrin-mediated endocytosis and macropinocytosis. Additionally, cross-linking of the viral proteins showed that the capsid protein forms dimers within tick-borne encephalitis virus, as previously observed with purified C proteins for other flaviviruses. This method enables the identification and mapping of transient virus-host interactions, under near-physiological conditions, without the need for genetic manipulation.Author summaryTick-borne encephalitis virus is an important human pathogen that can cause severe infection often resulting in life-long neurological complications or even death. As with other viruses, it fully relies on the host cells, and any successful infection starts with interactions between the viral structural proteins and cellular surface proteins. Mapping these interactions is essential both for the fundamental understanding of viral entry mechanisms, and for guiding the design of new antiviral drugs and vaccines. Here, we stabilise the interactions between tick-borne encephalitis virus and human proteins by chemical cross-linking. We then detect the interactions using mass spectrometry and analyse the data to identify protein-protein complexes. We demonstrate that we can visualise the protein interaction interfaces by mapping the cross-linked sites onto the host and viral protein structures. We reveal that there are eight distinct sites on the outer surface of the viral envelope protein that interact with host. Using this approach, we mapped interactions between the tick-borne encephalitis virus envelope protein, and 59 host proteins, identifying a possible new virus receptor. These results highlight the potential of chemical cross-linking coupled with mass spectrometry to identify and map interactions between viral and host proteins.

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“…As described in previous studies, 12 HBMECs (2 × 10 4 cells/well) were seeded on a collagen‐coated 96‐well black optical base plate (Nunc; Thermo Fisher Scientific). The cells (70% confluence) were then transfected with 75 nM siRNA and incubated for 60 h. After 48 h of infection with EV71 at a multiplicity of infection (MOI) of five, the cells were fixed and immunofluorescence was then used to analyze the expression of EV71 capsid VP1.…”

Section: Methodsmentioning

confidence: 99%

Enterovirus 71 enters human brain microvascular endothelial cells through an ARF6‐mediated endocytic pathway

Zhu¹,

Wang²,

He³

et al. 2023

Journal of Medical Virology

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Infection of the central nervous system caused by enterovirus 71 (EV71) remains the main cause of death in hand‐foot‐and‐mouth disease. However, the mechanism responsible for how EV71 breaks through the blood‐brain barrier to infect brain cells has yet to be elucidated. By performing a high‐throughput small interfering RNA (siRNA) screening and validation, we found that the infection of human brain microvascular endothelial cells (HBMECs) by EV71 was independent of the endocytosis pathways mediated by caveolin, clathrin, and macropinocytosis but dependent on ADP‐ribosylation factor 6 (ARF6), a small guanosinetriphosphate (GTP)‐binding protein of the Ras superfamily. The specific siRNA targeting ARF6 markedly inhibited HBMECs susceptibility to EV71. EV71 infectivity was inhibited by NAV‐2729, a specific inhibitor of ARF6, in a dose‐dependent manner. The subcellular analysis demonstrated the co‐localization of the endocytosed EV71 and ARF6, while knockdown of ARF6 with siRNA remarkably influenced EV71 endocytosis. By immunoprecipitation assays, we found a direct interaction of ARF6 with EV71 viral protein. Furthermore, ARF1, another small GTP‐binding protein, was also found to participate in ARF6‐mediated EV71 endocytosis. Murine experiments demonstrated that NAV‐2729 significantly alleviated mortality caused by EV71 infection. Our study revealed a new pathway by which EV71 enters the HBMECs and provides new targets for drug development.

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Ezrin is essential for the entry of Japanese encephalitis virus into the human brain microvascular endothelial cells

Cited by 20 publications

References 50 publications

Immune response and blood–brain barrier dysfunction during viral neuroinvasion

Immune response and blood–brain barrier dysfunction during viral neuroinvasion

Proteome-wide cross-linking mass spectrometry to identify specific virus capsid-host interactions between tick-borne encephalitis virus and neuroblastoma cells

Enterovirus 71 enters human brain microvascular endothelial cells through an ARF6‐mediated endocytic pathway

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