Identification and analysis of key binding sites between porcine haemagglutinating encephalomyelitis virus spike protein and DPP4 receptor
10.16303/j.cnki.1005-4545.2025.06.01
- VernacularTitle:猪血凝性脑脊髓炎病毒S蛋白与DPP4受体结合的关键位点鉴定与分析
- Author:
Le ZHANG
1
;
Hanyue JANG
1
;
Hanlu WEI
1
;
Zi LI
1
;
Wenqi HE
1
Author Information
1. 吉林大学动物医学学院人兽共患病研究教育部重点实验室,吉林长春 130062
- Publication Type:Journal Article
- Keywords:
PHEV;
cellular receptor;
dipeptidyl peptidase-4;
spike protein
- From:
Chinese Journal of Veterinary Science
2025;45(6):1103-1108
- CountryChina
- Language:Chinese
-
Abstract:
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.
