The Preliminary Study On Putative Japanese Encephalitis Virus Receptors

Japanese encephalitis virus (JEV) is a member of genus Flavivirus, family Flaviviridae and is an enveloped positive sense single stranded RNA virus. The major envelope glycoprotein (E), associated with neurovirulence and neuroinvasiveness, is the dominant antigen in inducing protective immune response in the infected host. Since E protein is known as the viral attachment protein (VAP), it contains structural and functional elements that participate in the virus-host cell receptor interaction and membrane fusion. The virus has a zoonotic transmission cycle between birds and mosquitoes, with swine serving as an intermediate amplifier hosts from which infected mosquitoes transmit the virus to humans. Japanese encephalitis caused by JEV is an acute and severe disease of central nervous system, and is epidemic nearly throughout of China, and virtually all of the southeastern Asia.JEV pathogenesis remains elusive. At a variable time after inoculation by an infected mosquito, JEV primaryly replicates in the lymph cells, myeloid cells or vascular endothelial cells. There is a temporal viraemia before JEV breaches the blood-brain barrier. After interaction with membrane of cerebral blood vessels endothelial cells and pericytes, JEV fuses with cytoplasmic endocytic vesicles by receptor-mediated endocytosis. Then taken up by the endocytic vesicles,JEV breaches the blood-brain barrier, and infects encephalic neurons rather than glial cells. Macrophage migration inhibitory factor (MIF) is largely expressed not only in neurons but also in glial cells, and involved in broad JEV-induced immune inflammation. JEV infection could also trigger neuronal apoptosis.Aim:The first step in virus infection requires interaction between the virus and the cellular receptors. The interaction of VAP and its cellular receptors is known to contribute to host range, tissue tropism and viral pathogenesis. To date, the cellular receptorfor JEV remains unknown. To identify and characterize the JEV cellular receptors, this study on putative JEV receptors was performed from two strategies as the followings, respectively.METHODS AND RESULTS:Ⅰ. Selection, identification and characterization of a cell mutant lacking the function of JEV receptor.ICR-191, a DNA alkylating agent, was used to introduce random mutations in JEV susceptible baby hamster kidney cells (BHK-21). After challenged with JEV, the surviving cells were cloned by limiting dilution. Individual clones were detected by RT-PCR for JEV RNA. A JEV RNA-negative mutant cell line, 3A10-3F, was selected and further analyzed lacking the function of JEV receptor.1. Indirect immunofluorescence assay (IFA): The fluorescence signals in the infected 3A10-3F cells was significantly weaker than in the infected BHK-21 cells. This suggests that 3A10-3F cells are considerably resistant to infection of JEV. 2. Plaque assay: At MOI of 1 and 10, the highest titer for JEV from 3A10-3F cells was reduced by 2 logs and 1 log compared to the highest titer for JEV from BHK-21 cells, respectively. This shows that the susceptibility to JEV in 3A10-3F cells significant declined.3. Flow cytometry: The binding affinity was only 2.10% between 3A10-3F cells and JEV, while the binding affinity was 48.84% between BHK-21 cells and JEV. Apparently, the ability to bind JEV by 3A10-3F cells was significantly impaired compared with its parental BHK-21 cells, suggesting that 3A10-3F cells lack the function of the JEV receptor.4. The susceptibility to herpes simplex virus 1(HSV-1): 3A10-3F cells were similar to the normal BHK-21 cells in cell morphology, and were still susceptible to HSV-1. After infected with HSV-1, 3A10-3F cells appeared CPE as rapidly as BHK-21 cells did: cells shrinking, turning round and fusion. Thus, resistance of 3A10-3F cells appears to be specific for JEV.5. A proteomic approach, two-dimensional electrophoresis (2-DE) coupled with LC-MS/MS was used to identify differentially expressed proteins in 3A10-3F and BHK-21 cells. For the first time four differentially expressed membrane proteins of 3A10-3F cells, annexin 1 and annexin 2, voltage-dependent anion channel (VDAC) 1 and VDAC 2, were identified.6. The expression of annexin 1 and annexin 2 were significantly reduced on 3A10-3F cells. It is possible that annexin 1 and annexin 2 are responsible for JEV binding by an interaction with phospholipid membrane. Therefore, the decreased expression of annexin 1 and annexin 2 significantly affected the binding between JEV and 3A10-3F cells.7. VDAC 1 was only expressed in 3A10-3F cells, and VDAC 2 showed an increased expression in 3A10-3F cells. It is possible these two VDAC proteins represent roles involving ion transport and other cell functions including signal transduction in JEV entry into 3A10-3F cells.Ⅱ. Isolation and identification of the putative JEV receptors from susceptible cells.1. By using Co-immunoprecipation (Co-IP) approach, several molecules binding with JEV from Aedes albopictus cells (C6/36), African green monkey kidney cells (Vero) and BHK-21 cells membranes were isolated.2. For the first time 74kD molecular from C6/36 cells membrane binding with JEV was identified as heat shock cognate 70 (HSC70) by LC-MS/MS.3. Antibody against HSC70 was able to detect a 74kD protein isolated by Co-IP from C6/36 cells membrane in Western blot assays.4. Polyclonal antibodies against 74kD and 97kD proteins were obtained after immunization of mice with the electro-eluted proteins isolated by Co-IP from C6/36 cells membrane. Then antibodies-mediated blocking JEV binding assay was performed. The results showed that antibodies against 74kD and 97kD proteins as well as antibody against HSC70 were able to block JEV binding partially to C6/36 cells in dose-dependent manner. At JEV MOI of 100 and antibodies concentration of 200μg, the binding were inhibited by 38%, 35% and 20% with antibodies against 97 kD, 74 kD proteins and antibody against HSC70.5. IFA: It was observed that the 74kD, 97kD and HSC70 proteins are located on the surface of C6/36 cells by confocal mocroscopy. Double-labelled IFA showed JEV and 74kD, 97kD or HSC70 proteins were co-localization on the surface of C6/36 cells. These results suggested that 74kD, 97kD or HSC70 proteins involved JEV attachment on the surface of C6/36 cells.CONCLUSIONS: 1. The mutant cell line, 3A10-3F, may be deficient in binding receptor function, but some other putative co-receptors may still exist to mediate JEV entry into cells.2. The 74kD and 97kD proteins from C6/36 cells membrane may be components of JEV receptor complex, and the 74kD protein may be HSC70. Taken together, these results provide important clues to facilitate characterization of JEV receptor molecule(s), and to further elucidate the pathogenesis of JEV infection.