HEV与细胞结合的氨基酸位点分析

Analysis of Amino Acid Sites for HEV Binding to Cells

目的:构建模型用于评估戊型肝炎病毒(hepatitis E virus, HEV)突变衣壳蛋白与细胞的结合作用以及各氨基酸位点在细胞结合过程中的作用,探讨HEV的入侵机制,为研究HEV的受体奠定基础。方法:以D66的基因四型重组类病毒颗粒样抗原为研究对象,对其关键结构域位点的氨基酸进行定点突变,利用聚丙烯酰胺凝胶电泳(sodium dodecyl sulfate polyacrylamide gel electrophoresis, SDS-PAGE)和酶联免疫吸附测定(enzyme linked immunosorbent assay, ELISA)方法确认突变蛋白的正常构象,并通过流式细胞仪分析突变蛋白对吸附的影响,探讨影响病毒吸附入胞的关键位点。结果:通过聚合酶链式反应定点诱变技术成功制备并鉴定了45个正确折叠、构象正常的突变蛋白;ELISA检测结果显示,重组类病毒颗粒样抗原D66的单点突变并不会影响蛋白构象;应用模型模拟突变蛋白与细胞的结合过程,T484A、S488A、T489A、P491A、R512A、Y561A、N562A和T585A突变蛋白显著影响了对C3A的吸附。结论:成功建立了一种评估HEV衣壳蛋白与细胞结合性的模型,T484A、S488A、T489A、P491A、R512A等位点的丙氨酸突变显著减弱了HEV衣壳蛋白与细胞的结合,这些位点可能是影响HEV与细胞结合的关键位点。

Objective:In this study,we constructed a model for evaluating the binding of hepatitis E virus(HEV)mutated capsid proteins to cells in a way that allowed us to evaluate the role of various amino acid sites in the cell binding process.This method helps us understand the invasion mechanism of HEV and lays a foundation for studying the receptor of HEV.Methods:We used a genotype IV recombinant virus-like particle-like antigen called D66,which contains the key domain of a.a.459-606.We performed site-specific mutations of amino acids at key D66 domain sites,and then confirmed the normal conformation of all mutated proteins by SDS-PAGE and ELISA methods.After confirming the conformation of the mutated proteins,we analyzed the effect of these mutated proteins on adsorption by flow cytometry to find out the key sites that affect the absorption of virus into the cell.Results:We successfully prepared and identified 45 correctly folded and conformationally normal mutant proteins by polymerase chain reaction site-specific mutagenesis.ELISA results showed that the single point mutation of recombinant viral particle-like antigen D66 did not affect the conformation of the protein.In the application of the model to simulate the binding process of mutant proteins to cells,we found that the mutant proteins of T484A,S488A,T489A,P491A,R512A,Y561A,N562A and T585A significantly affected their adsorption of C3A.Conclusion:In this study,a model was established to evaluate the cell binding of HEV capsid protein,and it was found that alanine mutations at T484A,S488A,T489A,P491A and R512A significantly weakened the cell binding of HEV capsid proteins.These sites may be the key sites that affect HEV binding to cells.