人血管内皮细胞上与2型登革病毒相互作用蛋白的筛选与鉴定

Screening and identification of DENV-2 interacting proteins on human vascular endothelial cells

目的建立高效病毒受体筛选方法,筛选并鉴定人血管内皮细胞膜上与2型登革病毒(DENV-2)相互作用的蛋白。方法用纯化的DENV-2 E蛋白DⅢ区(EDⅢ)分子代替全病毒颗粒与转印至PVDF膜的人血管内皮ECV304细胞膜蛋白进行孵育,建立改良的VOPBA和2D-VOPBA技术筛选相互作用蛋白,筛选出的候选蛋白经质谱分析、Western blot和免疫共沉淀(Co-IP)实验鉴定;利用抗体阻断实验和激光共聚焦显微技术观察和验证候选蛋白与DENV-2感染的关系;经生物信息学分析,预测候选蛋白与DENV-2 EDⅢ蛋白的对接复合物模型及其结合热点残基。结果筛选出相对分子质量34 000、43 000和55 000这3个候选蛋白,其中相对分子质量43 000和55 000蛋白经鉴定分别为细胞骨架微丝蛋白(fibrous-actin,F-actin)和波形蛋白(vimentin),均可与DENV-2 EDⅢ蛋白在体外发生特异性结合,并且vimentin蛋白可表达于人血管内皮细胞表面,其抗体可部分阻断DENV-2感染;激光共聚焦显微镜观察到在DENV-2感染的前5 min就可引起ECV304细胞中actin的重排,并且actin与病毒E蛋白有明显的随感染时间增强的共存现象。最后,利用生物信息学分别预测了actin和vimentin与DENV-2 EDⅢ蛋白的结合模型及结合残基。结论成功建立了高效的病毒受体筛选技术,并证实筛选到的actin和vimentin蛋白可作为辅助受体或相互作用蛋白,通过与DENV-2 EDⅢ蛋白结合参与DENV-2感染人血管内皮细胞。

This study was designed to establish a method to screen DENV interacting proteins on human vascular endothelial cells by using improved VOPBA and 2D-VOPBA techniques.Instead of the whole virus particles,the purified DENV-2 E protein domainⅢ(EDⅢ)was used to incubate with the transferred membrane of the ECV304 cell plasma membrane proteins,and the DENV-2-binding proteins were verified by mass spectrometry and Western blot analysis.Co-immunoprecipitation(Co-IP)was performed to confirm the interaction between DENV-2-binding proteins and DENV-2 EDⅢprotein;antibody blocking assay and indirect immunofluorescence staining were also conducted to determine the role of DENV-2-binding proteins involved in the DENV-2 infection.Then the binding models and hot binding residues of DENV-2-binding proteins and DENV-2 EDⅢprotein were simulated by bioinformatics software.There were three putative proteins with molecular weights of 34,43,and 55 kDa.Mass spectrometry identified 43 kDa protein matched to actin,and 55 kDa protein matched to vimentin,both of which are the cytoskeleton proteins in eukaryotic cells.The interaction of 43 kDa actin or 55 kDa vimentin with DENV-2 EDⅢwere further confirmed by Co-IP assay.Vimentin protein could be expressed on the surface of human vascular endothelial cells,and its antibodies can partially block DENV-2 infection.Confocal immunofluorescent assay confirmed actin cytoskeleton rearrangement in DENV-2-infected ECV304 cells,and the coexistence of actin filaments and DENV-2 E protein gradually increased with infection times.Furthermore,two docking models and their binding residues of actin-DENV-2 EDⅢmodel and vimentin-DENV-2 EDⅢmodel were simulated by bioinformatics software.In conclusion,efficient virus receptor screening techniques have constructed successfully,which demonstrate that the actin and vimentin proteins involved in DENV-2 infection of human vascular endothelial cells.