Mechanisms Of Endocytosis Pathway Used By Japanese Encephalitis Virus To Enter Nerve Cells

Japanese encephalitis virus (JEV) is a mosquito-borne flavivirus and one of themost common agents of viral encephalitis. In humans, JEV infection can cause severecentral nerve system disease which result in high mortality or developing permanentneurological sequelae. Despite the existence of effective vaccines, JE cases appear to beincreasing throughout Southeast Asia, probably as a result of increases in populationdensity, deforestation, and the expanding irrigation of agricultural areas. Therefore, JEVinfections remain a leading cause of encephalitis.Initiation of a viral infection requires entry of the virus into the host cell，which is anearly event and critical to the replication of virus．Viruses can utilize several endocyticpathways to enter host cells: macropinocytosis, clathrin-mediated endocytosis,caveolae/cholesterol-dependent uptake, and clathrin-and caveolae-independentendocytosis. The flavivirus internalization to mammalian cell is a complex phenomenonwherein a virus of diverse strains may use different mechanisms to enter different celltype. However, the infectious entry process of JEV into host cells remains largelyunknown.The aim of this study was to elucidate mechanism of JEV internalization into B104rat neuroblastoma cells. The chemical inhibitors, RNA interference (RNAi) silencing,and the expression of dominant negative (DN) constructs were used to examine the keycellular molecules involved in JEV entry. Firstly, we set up the infectious cell model ofJEV, and determined the kinetics and rate of virus penetration. Secondly, pharmacologicinhibitors were used to block corresponding endocytic pathways, the effects of which onviral entry were analyzed by the infectious centre assay. In addition, B104cells weretransfected with dominant-negative construct and siRNA targeting the key elements ofdifferent endocytic pathways, to confirm the endocytic pathways involved in the JEVentry process. To determine whether the endocytotic pathways utilized by JEV to enterB104cells are virus strain specific, the effect of the pharmacological inhibitors on theinternalization of the street virus SA14belonging to genotype III and the isolated JEVstrain SH-53belonging to genotype I were assessed. Finally, the effect of thepharmacological inhibitors on the internalization of JEV into B104, Vero, CHO, Huh7 cells were assessed to determine whether the endocytotic pathways utilized by JEV toinfect different cell lines were different.Results:1. Clathrin is not required for JEV entry into rat neuroblastoma cells.It was observed that chlorpromazine, the expression of EPS15dominant negativeconstructs, and siRNA silencing of clathrin all allowed efficient JEV internalization,suggesting JEV enters B104cells via clathrin-independent endocytosis.2. JEV endocytosis to rat neuroblastoma cells depends on dynamin.It was found that Dynasore, a specific inhibitor of the dynamin GTPase activitysignificantly inhibited JEV entry and blocked viral internalization. The expression ofdynamin II dominant negative mutant K44A and siRNA silencing of dynamin II also ledto a significant decrease in JEV internalization and entry. These data demonstrate thatJEV entry occurs through a dynamin-dependent endocytosis.3. JEV enters rat neuroblastoma cells via a caveolae-mediated endocytosispathway.It was observed that disruption of membrane rafts by MβCD orcholesterol-sequester filipin efficiently inhibited viral entry into B104cells, suggestingthe involvement of cholesterol-enriched membrane microdomains in virus internalization.It was also found that inactivation of caveolin-1by siRNA or dominant negative mutantssignificantly decreased the JEV internalization, suggesting the involvement of caveolin-1during viral entry. Immunofluorescence staining showed that colocalization of virus withCholera toxin B (CTB) and caveolin-1. These data demonstrate that the infectious entryof JEV into rat neuroblastoma cells is dependent on caveola-mediated endocytosis.4. JEV entry into rat neuroblastoma cells is independent of macropinocytosis.Pretreatment of B104cells with EIPA did not lead to a decrease in JEV entry and noinhibition was observed on JEV entry after treatment with the PI3K inhibitor-wortmannin at the same time. Consistent with the results obtained with wortmannin, JEVinternalization was not inhibited in B104cells transfected with siRNA against PI3K;instead, depletion of this macropinocytosis regulatory molecule seemed to enhance JEVentry. These results suggest that the endocytosis of JEV to rat neuroblastoma cells is notdependent on macropinocytosis. 5. JEV entry into rat neuroblastoma cells is dependent on acidic pH.JEV internalization was inhibited by chloroquine or ammonium chloride in adose-dependent manner, supporting the notion that JEV cell entry is sensitive to elevationof the pH in intracellular acidic compartments.6. The endocytonic pathways utilized by JEV to enter rat neuroblastoma cellsare not virus strain specific.It was observed that no inhibitory effect on the internalization of JEV strain SA14and SH-53was detected by chlorpromazine or EIPA treatment. However, MβCD,chloroquine, and dynasore all resulted in a significant reduction in the internalization ofdifferent JEV genotypes. These results suggest that the atypical dynamin-dependentcaveola-mediated pathway for JEV entry into B104cells is virus strain independent.7. JEV entry into rat neuroblastoma cells differs from non-neurocyte cell lines.JEV infection of Vero, CHO, Huh7cells occured through a clathrin-mediatedendocytic pathway, while infection of B104cell line through a caveolae-mediatedendocytosis. These results suggest that JEV entry into rat neuroblastoma cells differsfrom non-neurocyte cell lines.In conclusion, we conducted a systematic study to identify the internalizationmechanism of JEV in rat neuroblastoma cells. The evidence presented here indicates thatJEV entry into B104cells occurs by a pH-dependent, dynamin-dependent andcaveolae-mediated endocytosis. Understanding the viral and cellular components involvedin JEV invasion into the host cell, combined with a comprehension of the mechanisms thatgovern this process, should therefore open the possibility of developing new therapeuticapproaches.