摘要
二肽基肽酶4(DPP4)是猪血凝性脑脊髓炎病毒(PHEV)刺突(S)蛋白的结合受体之一。为鉴定DPP4蛋白与PHEV S蛋白结合界面的关键氨基酸位点,并探究其突变对病毒感染的影响,本研究构建了猪源、鼠源、人源DPP4(pDPP4、mDPP4、hDPP4)与S蛋白截短体(S311-608、S13-298)重组质粒,将两者共转染HEK293T细胞,通过免疫共沉淀技术(CoIP)检测蛋白间的互作;同时,在PHEV非易感的HeLa细胞中过表达DPP4同源物并接种病毒,通过Western blot与RT-qPCR检测PHEV蛋白和基因表达。随后,利用点突变试剂盒对互作界面中重要的pDPP4氨基酸位点进行逐一突变构建突变型过表达质粒,并将突变型、野生型pDPP4分别与病毒S蛋白截短体共转染HEK293T细胞后接种PHEV,利用Western blot和RT-qPCR检测PHEV复制水平。结果显示,pDPP4、mDPP4与PHEV S蛋白结合作用显著强于hDPP4,且与hDPP4相比,pDPP4、mDPP4可显著促进PHEV复制。同时发现pDPP4糖基化位点突变可显著增强与PHEV S蛋白的相互作用,糖基化位点(N229、N321)突变能够显著促进PHEV感染。结果表明,DPP4糖基化修饰在S蛋白介导PHEV侵入靶细胞过程中发挥着重要的屏蔽作用。
Dipeptidyl peptidase 4(DPP4)is one of the binding receptors for the spike(S)protein of porcine hemagglutinating encephalomyelitis virus(PHEV).To identify the key amino acid binding sites at the interface of DPP4 protein and PHEV spike protein and explore the impact of their mu-tations on viral infection,recombinant plasmids of porcine DPP4,murine DPP4 and human DPP4(pDPP4,mDPP4,hDPP4)and spike protein truncations(S311-608,S13-298)were constructed and co-transfected into HEK293T cells to detect the protein binding by co-immunoprecipitation(CoIP).Simultaneously,the expression of PHEV proteins and genes was detected in DPP4-over-expressing HeLa cells infected by PHEV using Western blot and RT-qPCR.homologues were overexpressed in HeLa cells which were not susceptible to and then inoculated with the virus,and.Subsequently,the important pDPP4 amino acid sites at the interaction interface were mutated one by one using a point mutation kit to construct mutant overexpression plasmids.The mutant and wild-type pDPP4 were co-transfected into HEK293T cells with the spike protein truncations re-spectively to assay the protein interaction ability by co-immunoprecipitation.After HeLa cells over-expressing the mutant and wild-type pDPP4 were infected by PHEV,the replication level of PHEV was detected by Western blot and RT-qPCR.Compared with hDPP4,the pDPP4 and mDPP4 had significantly stronger binding to PHEV spike protein,which significantly promote PHEV infection.Moreover,mutation of pDPP4 glycosylation sites significantly enhanced the inter-action with PHEV spike protein,and the mutation of glycosylation sites(N229,N321)dramatical-ly promoted PHEV infection.The above results indicated that DPP4 glycosylation modification plays an important shielding role in the process of PHEV invading target cells mediated by spike protein.
作者
张乐
姜含月
韦寒露
李姿
贺文琦
ZHANG Le;JANG Hanyue;WEI Hanlu;LI Zi;HE Wenqi(Key Laboratory forZoonosis Research of the Ministry of Education,College of Veterinary Medicine,Jilin University,Changchun 130062,China)
出处
《中国兽医学报》
北大核心
2025年第6期1103-1108,共6页
Chinese Journal of Veterinary Science
基金
国家自然科学基金资助项目(3217190328)。
