| Peste des petits ruminants(PPR) is an economic, acute and highly contagious viral disease of small ruminants, which is a notifiable terrestrial animal disease by Office International Des Epizooties(OIE). Peste des petits ruminants virus(PPRV), which belongs to the genus Morbillivirus of the family Paramyxoviridae, is an enveloped virus.Virus enter into the host cells by recognizing and binding the receptor. For paramyxovirus, viral entry involves two viral envelope glycoproteins, the hemagglutinin(H) and fusion(F) proteins, and the envelope proteins mediate the specific membrane fusion of viral envelope and cell membrane. The H proteins of morbillivirus are responsible for viral receptor binding and subsequently viral entry. It is the key factor determining virus infection. F protein is a major factor that determines the virulence of the virus. It is not only directly involved in the fusion of the viral envelope and host cell membrane, but also can mediate the cell-cell fusion. HR1 and HR2 are two conserved heptad repeat regions of F gene, which play important role in fusion. Two natural receptors for PPRV has been identified, signaling lymphocyte activation molecule(CD150/SLAM) and poliovirus-receptor-like-4(nectin 4/PVRL4).The research mainly includes the following three parts:1. Eukaryotic expression of PPRV envelope and receptor protein.Eukaryotic expression vectors p CMV-Myc-SLAM, p CMV-HA-HR1 and p CMV-HA-HR2 were successfully constructed. Expression of recombinant proteins was analyzed by indirect immunofluorescence(IF), flow cytometry(FCM) and Western-blot. The results showed that all recombiant plasmids can express the expected fusion protein in CHO cells.2. Interaction analysis of PPRV H, F and receptor proteins.The recombinant plasmids of receptors and PPRV Hv, Hw, HR1 and HR2 were co-transfected into CHO cells. Protein-protein interactions were analyzed by IF, co-immunoprecipitation(Co-IP) and surface plasmon resonance(SPR). The results showed that receptors and Hv, Hw, HR1 and HR2 were co-expressed and colocalized in the cytoplasm, and the receptors interacted with Hv, Hw, HR1 and HR2, respectively. The immobilization buffer selected was 10 m M sodium acetate p H 5.0 provided the best electrostatic interaction of Hv and Hw with the chip surface, the expected immobilization amount of 6438.7 and 7881.7 RU were detected, respectively. Hv/Hw interacted with SLAM, Nectin4, F, HR1, HR2. Hv has high affinity binding to the analyte except HR1, the equilibrium dissociation constant(KD)values were from 1.3×10-10 to 3.87×10-8 M. The KD value of Hw binding to SLAM was 1.21×10-5 M, while binding to the other analytes were from 7.1×10-12 to 5.97×10-8 M. Recombinant plasmids containing Hw, F and SLAM/Nectin4 induced cell fusion.3. Analysis of promoting cell fusion of PPRV H and F proteins.The recombinant plasmids were co-transfected into CHO cells. The effect of interaction between receptors and viral envelope proteins on the cell fusion and the localization were analyzed by optical microscopy and confocal microscopy at 36 h post-transfected. It was found that co-expression of receptors with PPRV H and F can induce cell fusion, receptor and F protein can also mediate cell fusion in the absence of H protein. When the receptor was co-expressed with H and F, of receptors with H and F, the distribution of the proteins appeared polarization and colocalization.This study of interaction between PPRV envelope proteins and viral receptors laid a foundation for further research on the key amino acid sites of these proteins as well as the invasion mechanism of PPRV. |
