Studies On The Mechanism Of Japanese Encephalitis Virus Entry Into Brain Microvascular Endothelial Cells

Background and ObjectiveJapanese encephalitis is a serious neurological disease caused by Japanese encephalitis virus(JEV)infection.It is currently the most common viral encephalitis in humans.Although the use of vaccines has greatly reduced its incidence,nearly 70,000 clinical cases are currently reported worldwide each year.Most of these cases are distributed in the Asia-Pacific region,of which more than 50%are in china’s Henan,Yunnan,Guizhou and Sichuan provinces.In recent years,with climate change,population migration and virus mutation,the incidence is increasing.As there is no available specific drug for Japanese encephalitis in clinical practice,its mortality remains high,and 30%-50%of survivors have neurological or mental sequelae,which has brought a heavy burden on public health and society.Therefore,there is an urgent need to explore the pathogenic mechanism of JEV in order to provide a solid theoretical basis for the development of effective drugs for the treatment of Japanese encephalitis.JEV is a mosquito-borne flavivirus that initiates local infection by mosquito biting,then the virus is released into circulation and crossed the blood-brain barrier(BBB)to infect neurons,causing widespread inflammation in the central nervous system.Disruption of the BBB is a key and prerequisite step for JEV pathogenesis,but currently little is known about its mechanism.Brain microvascular endothelial cells(BMEC)are the main components of the BBB,which determine the structural and functional integrity of the BBB.It is also the first line of defensing against JEV crossing the BBB.JEV infection of BMEC is the initial event of crossing the BBB.The first step of viral infection is the entry of cells by hijacking the endocytic pathway involving multiple cellular processes and host proteins.Although studies have characterized the entry routes of JEV in many types of host cells,the mechanism of its entry in BMEC is still unclear.In this study,through the systematical si RNA library screening,we identified the key roles of host factors such as endocytic-related caveolin-1 and ezrin in JEV infection of BMEC.Based on this,we will systematically study the endocytic pathways and specific molecular mechanisms involved in JEV entry in BMEC.This will not only deepen the understanding of the mechanism of JEV crossing the BBB,but also provide important targets for the development of anti-JEV drugs.Part Ⅰ:Screening of host factors involved in Japanese encephalitis virus infecting brain microvascular endothelial cells and study of endocytic pathwayMethods1.A si RNA library targeting human membrane trafficking-related protein was utilized to screen for key host factors involved in JEV infection of human brain microvascular endothelial cell(HBMEC)monolayer model.2.The expression or function of clathrin was inhibited by si RNA or chemical inhibitor chlorpromazine,and then the infection and entry of JEV were detected by indirect immunofluorescence and endocytic(internalization)assays,respectively.3.Caveolin-dependent endocytosis was inhibited by si RNA,chemical inhibitor filipin III or expression of dominant negative mutants of caveolin-1 and then JEV infectivity and entry were determined.4.JEV and markers of endocytic pathway were double-stained,and their localization relationship was analyzed by laser confocal technology.Results1.After 48 h of contact culture,HBMEC grew as a polarized monolayer,microvilli structure was formed on the apical surface,endothelial marker von Willebrand factor and tight junction protein Claudin-5 are highly expressed.2.Through screening,it was identified that the down-regulation of 18 factors inhibited JEV infection in HBMEC by more than 50%.Including:CAV1(caveolin-1),a marker of endocytic pathway;HGS and VPS4A,components of endosomal sorting complex required for transport;NSF and SYT1,key molecules in the membrane fusion process;ARPC4,RAC1,EZR and WAS,actin reorganization related proteins;COPA,GAF1,RAB4B,RAB5A,RAB5B,and RAB11B,endosome and vesicle transport related proteins.3.Corrected viral entry kinetics curve was drawn.It shows that JEV internalization(endocytosis)increases continuously from 0 to 6 h,reaching a maximum at 6 h.4.Knocking down or inhibition of clathrin all did not affect JEV infectivity and endocytosis in HBMEC.5.After knocking down or inhibition of caveolin-1,JEV infectivity and endocytosis in HBMEC were significantly reduced.6.Two hours after infection,JEV particles were extensively colocalized with caveolin-1 in the membrane ruffles of HBMEC,but not with clathrin in both membrane and cytoplasm.ConclusionJEV entry into BMEC by caveolin-mediated endocytosis,without the participation of clathrin-mediated endocytosis.At the same time,processes such as actin cytoskeleton reorganization,endosome and vesicle transport,protein sorting,and membrane fusion may also play important roles in JEV infection in BMEC.Part Ⅱ:The role of ezrin in Japanese encephalitis virus entry into brain microvascular endothelial cellsMethods1.The ERM member proteins moesin,ezrin,and radixin in HBMEC were independently knocked down by si RNA,and its effects on JEV binding,entry and infection were tested by indirect immunofluorescence,binding assay,and endocytic assay.2.To determine the stage ezrin involved in JEV infection,viral infection and internalization or CT-B endocytosis were tested,with the treatment of ezrin-specific inhibitor NSC668394.3.Phalloidin staining was used to detect actin morphology during JEV invasion.To analyze the role of actin reorganization in JEV infection and entry,HBMEC were treated by chemical inhibitors for actin reorganization,and JEV infection and entry were detected.4.To analyze the effect of JEV on actin reorganization and the role of ezrin in it,the G/F-actin ratio determination assay combined with colocalization analysis of virus and cytoskeleton were performed,with the treatment of NSC668394.5.The common interaction proteins of ezrin and caveolin-1 was predicted based on STRING database,and the proteins involved in JEV entry in HBMEC were verified by si RNA and specific inhibitors.6.To analyze the activity status and activation relationship of Src,ezrin,and caveolin-1 in JEV entry into HBMEC,inhibitors of Src(PP2)and ezrin,or si RNA of cavelin-1 were utilized combined with immunoblotting.7.Co-immunoprecipitation combined with recombinant protein in vitro kinase assay were used to analyze the interaction between Src,ezrin and caveolin-1 during JEV entry.8.To verify the interaction of Src-caveolin-1 and the role of ezrin in it,colocalization analysis of activated Src(p-Src)and caveolin-1(p-caveolin-1)were performed,with the treatment of NSC668394.Results1.Of the three ERM proteins,only the down-regulation of ezrin showed a significant inhibitory effect on JEV internalization.2.NSC668394 inhibited JEV infection in a dose-dependent manner,and its pretreatment also inhibited JEV endocytosis by more than 96%.After pretreated HBMEC with NSC668394,the endocytosis of CT-B was also significantly reduced.3.Actin was widely reorganized in JEV-infected HBMEC.JEV infectivity and endocytosis was markedly reduced in HBMEC treated with inhibitors of actin reorganization.4.After treated with NSC668394,the polymerization of actin induced by JEV and the colocalization of virus and actin cytoskeleton was markedly reduced.5.STRING database prediction showed three proteins that may be directly related to ezrin and caveolin-1,among which only the knocking down or inhibition of Src showed significantly decreasing of JEV infectivity and endocytosis.6.JEV-induced ezrin and caveolin-1 activation(phosphorylation)were reduced after Src inhibition.After inhibiting ezrin,caveolin-1 activation was reduced while Src activation was not affected.After knocking down caveolin-1,the activation level of ezrin and Src increased.7.Antibodies against Src,ezrin,and caveolin-1 all precipitated p-Src,p-ezrin,and p-caveolin-1 from JEV-infected HBMEC.In vitro,Src phosphorylated ezrin and caveolin-1directly.8.Two hours after JEV infection,extensively colocalization of activated Src and caveolin-1 on membrane ruffles was observed.P-caveolin-1 and its co-localization with Src were significantly reduced after NSC668394 treatment.ConclusionJEV induced the activation of Src/ezrin/caveolin-1 pathway and organization of p-Src/p-ezrin/p-caveolin-1 complex,and established entry into HBMEC.Ezrin is a key factor in Src-mediated caveolin-1 phosphorylation and promotes JEV entry into HBMEC through caveolin-dependent endocytosis.Ezrin is also essential for actin reorganization.Part Ⅲ:Src/ezrin-targeted inhibitors are potential anti-JEV drugsMethods1.Suckling ICR mice was infected subcutaneously to determine the experimental conditions for establishing animal model of JEV infection,by observing symptoms,survival analysis and hematoxylin-eosin(HE)staining analysis.2.To determine whether NSC668394 and PP2 have protective effects on the lethality of JEV,drugs were injected subcutaneously repeatedly after infection,and survival analysis was conducted.3.Repeated high-dose of NSC668394(6 mg/kg)and PP2(7.5 mg/kg)were administered subcutaneously after virus inoculation.RT-PCR,indirect immunofluorescence,and HE staining were used to detect JEV load and pathological damage in mouse brain.Results1.After subcutaneous injection of 4×10~3 PFU/mouse JEV,the mortality of the mice was73.33%,and typical neurological symptoms and pathological changes of Japanese encephalitis was observed.2.Continuous injection of high-dose PP2(7.5 mg/kg)or NSC668394(6 mg/kg)for 6days elevated the survival rate of infected mice to 54.55%or 61.91%,respectively.3.After repeated administration of high doses of PP2 and NSC668394,the viral load in the brain of JEV-infected mice was reduced to about 1%of the model group,and the pathological damage of the brain was also significantly improved.ConclusionRepeated administration of PP2 and NSC668394 after JEV infection all can reduce viral load and improve pathological damage in brain,and rescue animals from JEV-caused death.