【作者】 任君萍；

【导师】 马文煜； 丁天兵；

【作者基本信息】 第四军医大学 ， 病原生物学， 2008， 博士

【摘要】 日本脑炎病毒(Japanese encephalitis virus,JEV)属于黄病毒科黄病毒属,是有囊膜的正链RNA病毒,主要的囊膜糖蛋白E与病毒的神经毒性和神经侵袭性相关,能够诱导机体产生保护性免疫反应。作为JEV的病毒吸附蛋白(virus attachment protein,VAP),E蛋白介导JEV与宿主细胞受体结合及膜融合。JEV在鸟与蚊子之间有一个区域性传播循环,猪作为中间扩增宿主,带毒蚊子通过叮咬将感染性病毒传播给人。JEV引起的流行性乙型脑炎(乙脑),是一种严重的急性中枢神经系统传染病,主要流行于我国广大地区及东亚、东南亚和南亚。JEV致病机制尚未完全阐明。人被携带JEV的蚊子叮咬后,病毒先在淋巴细胞、骨髓细胞及血管内皮细胞增殖。在侵袭中枢神经系统前,有一个短暂的病毒血症期。JEV和脑血管内皮细胞及其周围细胞的细胞膜相互作用后,通过受体介导的内吞,JEV与胞浆囊泡膜融合形成内化囊泡,再被转运穿过血脑屏障。在颅内,JEV仅在神经元中复制。神经元和胶质细胞大量表达巨噬细胞移动抑制因子(MIF),介导广谱的免疫炎症反应。JEV的感染也诱导神经元的凋亡。目的:病毒与细胞表面相应受体的结合是病毒感染复制过程中的始动环节,也是影响病毒宿主特异性、组织亲嗜性和致病性的决定因素之一。目前,JEV在易感细胞上的受体分子及其特性与功能仍然不清楚。为了鉴定JEV的受体,本研究从以下两种思路出发,分别进行了JEV受体候选分子的初步研究。方法和结果:一、筛选建立JEV受体功能缺陷的细胞系,并鉴定其功能。应用ICR-191(一种DNA烷化剂,通过DNA小片段缺失和移码突变使细胞随机突变)诱变JEV易感细胞——地鼠肾细胞BHK-21。经过JEV攻击、克隆化和RT-PCR筛选,获得了一株突变细胞系3A10-3F,并鉴定其为JEV受体功能缺陷。1.间接免疫荧光实验:JEV感染的3A10-3F细胞的荧光强度明显弱于JEV感染的BHK-21细胞,说明3A10-3F细胞对JEV的感染有相当的抵抗。2.空斑实验: MOI 1和MOI 10的JEV感染后,3A10-3F细胞比BHK细胞上清中JEV最高滴度分别降低了2个数量级和1个数量级,进一步证实3A10-3F细胞的确对JEV的敏感性降低了许多。3.流式细胞术: 3A10-3F细胞与JEV的结合率仅有2.10 % ,而BHK-21细胞与JEV的结合率可达48.84 %。3A10-3F细胞对JEV的结合率明显降低,进一步说明3A10-3F细胞为JEV受体功能缺陷。4.对单纯疱疹病毒1型(HSV-1)的易感性:在形态上,3A10-3F细胞与BHK-21细胞相似,仍然保留着对HSV-1的易感性。HSV-1感染后,与BHK-21一样出现细胞病变:细胞融合,变圆及融合。所以这株突变细胞系对JEV感染的抵抗是特异的。5.为了鉴定突变细胞3A10-3F与亲代细胞BHK-21膜分子的表达差异,应用了蛋白质组学的方法,即二维电泳(2-DE)组合液质联用质谱(LC-MS/MS)技术,首次鉴定了3A10-3F细胞上有明显差异表达的四种膜蛋白,即钙结合蛋白annexin 1, annexin 2;电压依赖的离子通道(VDAC)蛋白VDAC 1, VDAC 2。6. 3A10-3F细胞上annexin 1, annexin 2的表达明显下降。这两个分子有可能通过与脂膜的相互作用与JEV的结合相关,它们在3A10-3F细胞上表达的下调,导致JEV与3A10-3F细胞膜的结合能力明显下降。7. VDAC 1仅仅表达在3A10-3F细胞上,VDAC 2在3A10-3F细胞上的表达升高。这两个VDAC分子可能在JEV穿入3A10-3F细胞过程中起着离子通道的作用,同时也参与其它包括信号转导的细胞功能。二、从易感细胞上分离JEV受体候选分子,并初步鉴定其功能。1.应用免疫共沉淀方法(Co-immunoprecipitation, Co-IP),从白纹伊蚊细胞C6/36、非洲绿猴肾细胞Vero及BHK-21细胞膜上,分离到多个与JEV结合的分子条带。2.质谱分析首次鉴定出一个蛋白,即C6/36细胞上与JEV结合的74 kD分子为HSC70蛋白。3.免疫印记实验中,用抗HSC70抗体检测到从C6/36细胞膜上免疫共沉淀分离到的74 kD蛋白。4.将主要的JEV受体候选分子,即C6/36细胞膜上免疫共沉淀分离到的74 kD、97 kD分子电洗脱下来后,分别免疫小鼠制备抗体,进行病毒结合的阻断实验。结果显示,抗74 kD、97 kD抗体及抗HSC70抗体均以剂量依赖的方式部分阻断JEV与C6/36细胞的结合。在JEV MOI 100,抗体浓度为200μg时,抗97kD、74 kD抗体和抗HSC70抗体分别使JEV的结合率下降38%、35%、20%。5.间接免疫荧光实验:激光共聚焦显微镜观察到74 kD、97 kD及HSC70分子定位在C6/36细胞膜上。免疫荧光双标记实验结果显示JEV与74 kD、97 kD及HSC70分子共定位在C6/36细胞膜上。这些结果说明74 kD、97 kD及HSC70分子参与JEV吸附到C6/36细胞膜上。结论:1.突变细胞系3A10-3F可能是缺乏结合受体的功能,而辅受体仍然能够介导JEV进入细胞内。2. C6/36细胞上74 kD、97 kD分子可能是JEV受体的复合物组分,而74 kD分子可能是HSC70。综上所述,这些研究结果将为深入研究JEV受体特性与功能,阐明JEV致病机制提供有益的线索。更多还原

【Abstract】 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.更多还原