Zika virus NS1 affects the junctional integrity of human brain microvascular endothelial cells

doi:10.1016/j.biochi.2020.06.011

Biochimie

Volume 176, September 2020, Pages 52-61

https://doi.org/10.1016/j.biochi.2020.06.011 Get rights and content

Highlights

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The BMVECs exposed to Zika virus (ZIKV) NS1 protein disrupts the endothelial barrier integrity.
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The ZIKV-NS1 exposure to BMVECs, increases the reactive oxygen species (ROS).
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The ROS production affects the tyrosine kinase, PYK2 and phosphatases, SHP2.
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The inhibition of ROS by Diphenyleneiodonium chloride (DPI) rescued the endothelial barrier-integrity.

Abstract

Zika virus (ZIKV) infection leads to microcephaly in newborns. Flaviviruses are known to secrete NS1 protein extracellularly and its concentration in serum directly co-relate to disease severity. The presence of ZIKV-NS1 near the brain microvascular endothelial cells (BMVECs) affects blood-brain-barrier, which is composed of tight junctions (TJs) and adherens junctions (AJs). Viruses utilize different strategies to circumvent this barrier to enter in brain. The present study demonstrated the mechanism of junctional integrity disruption in BMVECs by ZIKV-NS1 protein exposure. The Transendothelial Electrical Resistance and sodium fluorescein migration assays revealed the endothelial barrier disruption in BMVECs exposed to ZIKV-NS1 at different time (12hr and 24hr) and doses (500 ng/mL, 1000 ng/mL and 1500 ng/mL). The exposure of ZIKV-NS1 on BMVECs led to the phosphorylation of AJs and suppression of TJs through secreted ZIKV-NS1 in a bystander fashion. The activation of NADPH dependent reactive oxygen species activity and redox sensitive tyrosine kinase further increased the phosphorylation of AJs. The reduced expression of the phosphatase led to the increased phosphorylation of the AJs. The treatment with Diphenyleneiodonium chloride rescued the phosphatase and TJs expression and suppressed the expression of kinase and AJs in BMVECs exposed to ZIKV-NS1.

Introduction

The Zika virus (ZIKV) is an arbovirus, transmitted through Aedes sp. Mosquitoes. The vertical transmission of ZIKV has been reported from infected mother to child during pregnancy [1]. The virus belongs to the family of Flaviviridae and genetically related to Japanese Encephalitis Virus (JEV), West Nile Virus (WNV), Dengue Virus (DENV), Yellow Fever virus (YFV) and Tick-borne Encephalitis virus (TBEV) [2]. The ZIKV was first isolated from a monkey in the Zika forest of Uganda, East Africa in the year 1947 [3]. The recent outbreaks of ZIKV and ZIKV-mediated neurological manifestations have been declared as Public Health Emergency of International concerns (PHEIC) [4]. ZIKV infection causes microcephaly in infants and Guillain Barre Syndrome in adults [5]. The flavivirus genome consists of a single positive-sense RNA virus which directly encodes into a polyprotein containing 3 structural proteins (C, prM, and E) and 7 non-structural proteins (NS1, NS2AB, NS3, NS4AB, and NS5). Flavivirus NS1 is a multifaceted viral protein present in three isoforms, monomer (cytoplasm), dimer (trans-membrane NS1), and hexamer (secreted NS1). The association of the disease severity with the flavivirus NS1 has been reported previously [6,7]. The concentration of the secreted NS1 (sNS1) has been reported in the range of 1–2 μg/mL in the blood circulation of patients infected with flaviviruses [8,9]. The circulation of NS1 has been reported in the mouse models of flavivirus infections [10]. Tabata et al., 2016 reported the ZIKV dissemination through placental and para-placental routes [11]. The transmigration of ZIKV-NS1 through placenta may affect the fetal brain development [12]. A more recent study reported the tissue-specific vascular endothelial dysfunction by flavivirus NS1, where the endothelial glycocalyx-like layer (EGL) of the endothelial cells gets disrupted due to the activation of endothelial sialidases, cathepsin L and heparinase [13].

The endothelial cells form an interface between the systemic blood circulation and brain tissues. The cell-to-cell junctions, adherens junctions (AJs) and tight junctions (TJs), between the endothelial cells maintain the integrity of the BBB [14]. The VE-cadherin provide a basic structure to endothelial cells by interacting with the catenin protein family (p120-catenin, β-catenin, or plakoglobin) through its cytoplasmic tails [15]. The tight junction proteins, like Claudin-5 (CLDN5) interacts with VE-cadherin, where VE-cadherin is a positive regulator of CLDN5 [16]. We previously reported the role of tyrosine phosphorylation of VE-cadherin (Y731) and β-catenin (Y654) in compromising the endothelial integrity [17] and have demonstrated the regulation of tight junction protein CLDN5 by VE-cadherin during HIV-1 Tat C exposure on BMVECs through miR-101 [18].

The phosphatases like SHP2, VE-PTP, PTP-μ, Csk interact with the VE-cadherin and β-catenin to keep AJs in the dephosphorylated state, but the suppression of different phosphatases results in uncontrolled phosphorylation of AJs [17]. The NADPH mediated reactive oxygen species (ROS) generation has been reported to activate the redox-sensitive protein tyrosine kinase, PYK2 [19]. In addition, the phosphorylation of PYK2 results in the destabilization of VE-cadherin and β-catenin by phosphorylating the proteins [20].

There may be different forms of the inflammatory responses affecting the phosphorylated state of AJs, but we explored the bystander effect of ZIKV-NS1 on the BMVECs. The presence of ZIKV-NS1 in peripheral circulation may affect the permeability of BMVECs, which may lead to the endothelial barrier dysfunctions resulting in vascular leakage. In this study, we exposed BMVECs to ZIKV-NS1 in time and dose-dependent manner. We observed the expression of NADPH oxidases (NOX2, NOX4), redox-sensitive protein tyrosine kinase, PYK2 and tyrosine phosphatase, SHP2 and their implications in the maintenance of barrier integrity through AJs and TJs proteins. This study is focused to understand the molecular mechanism of the barrier dysfunctions in terms of the compromised endothelial barrier integrity upon ZIKV-NS1 exposure to BMVECs.

Section snippets

Cell culture

The Primary Human Brain Microvascular Endothelial Cells (BMVECs) were purchased from the Cell Systems (#ACBRI 376). The BMVECs were obtained by elutriation of dispase-dissociated human brain cortex tissue [21]. The cells were cultured in Endothelial Cell Growth Medium-2 (#CC-3162, Lonza) with the growth factors and supplemented with the Fetal Bovine Serum (FBS, Lonza). The tissue culture plates and flasks were coated with 0.2% bovine type B gelatin (#TCL059, HiMedia) for an hour at 37 °C in a CO

The ZIKV-NS1 protein triggers the endothelial barrier dysfunction

The vertical transmission of ZIKV from mothers to their fetuses and the presence of ZIKV-NS1 in placental tissues have been reported among the ZIKV infected patients [22]. Through this process, the CNS of developing fetuses gets exposed to ZIKV as well as the products of ZIKV infection such as NS1 protein in the bystander fashion. Here, we have exposed ZIKV-NS1 on BMVECs, to study the effect on BBB integrity by trans-endothelial electrical resistance (TEER) and sodium fluorescein migration

Discussion

ZIKV is a flavivirus (positive ssRNA virus) of the family Flaviviridae. The ZIKV infections have been linked with microcephaly in newborns [5]. ZIKV, being a neurotropic virus infects the neural progenitor cells (NPCs) and cortical neurons in the newborns presenting with microcephaly [29]. The animal model based studies further re-enforced the notion of the vertical transmission of ZIKV from infected pregnant mothers to their fetuses which may lead to microcephaly [30]. The flaviviruses are

Conclusion

ZIKV-NS1 protein may affect the integrity of the brain microvascular endothelial cells during the late stages of the ZIKV infection among the patients having higher levels of circulating form of the secreted ZIKV-NS1. The longer the ZIKV-NS1 protein interacted with endothelial cells, the NADPH-dependent ROS production increased. The NADPH-dependent ROS production (NOX2 and NOX4) activated the redox-sensitive tyrosine kinase, PYK2, which in turn phosphorylated the VE-cadherin and β-catenin

Funding

This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

CRediT authorship contribution statement

Meghana Rastogi: Formal analysis, Data curation, Writing - original draft. Sunit K. Singh: Supervision, Data curation, Writing - original draft.

Declaration of competing interest

The authors declare they have no conflict of interest.

Acknowledgment

The Authors highly acknowledge the facilities provided by the Banaras Hindu University for conducting the present study. Meghana Rastogi is a PhD student and a recipient of CSIR-SRF-(Direct) Fellowship.

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Zika virus NS1 affects the junctional integrity of human brain microvascular endothelial cells

Highlights

Abstract

Introduction

Section snippets

Cell culture

The ZIKV-NS1 protein triggers the endothelial barrier dysfunction

Discussion

Conclusion

Funding

CRediT authorship contribution statement

Declaration of competing interest

Acknowledgment

J. Neuroimmunol.

Trans. R. Soc. Trop. Med. Hyg.

Lancet

Cell Host Microbe

Dev. Cell

Prog. Mol. Biol. Transl. Sci.

Microvasc. Res.

Blood

J. Biol. Chem.

J. Biol. Chem.

Cell. Stem Cell.

Neurobiol. Dis.

Blood

Eur. J. Cell Biol.

J. Biol. Chem.

Biochim. Biophys. Acta

J. Biol. Chem.

Fields Virology

Zika virus infection as a cause of congenital brain abnormalities and guillain-barre Syndrome: systematic review

PLoS Med.

Flavivirus NS1: a multifaceted enigmatic viral protein

Virol. J.

Secretion of flaviviral non-structural protein NS1: from diagnosis to pathogenesis

Novartis Found. Symp.

NS1 protein secretion during the acute phase of West Nile virus infection

J. Virol.

An antigen capture enzyme-linked immunosorbent assay reveals high levels of the dengue virus protein NS1 in the sera of infected patients

J. Clin. Microbiol.

Evolutionary enhancement of Zika virus infectivity in Aedes aegypti mosquitoes

Nature

Zika virus infection with prolonged maternal viremia and fetal brain abnormalities

N. Engl. J. Med.