Study On Cell Fusion Efficiency Difference Induced By The Interaction Between Canine Distemper Virus H And Different Animal SLAM

Canine distemper virus belongs to the paramyxoviridae and measles virus genus.It is a single-strand RNA virus with capsular.The genome encodes six structural proteins from the 3' to the 5',in which the hemagglutinin(H)and fusion(F)proteins bind and fuse with the host cell membrane,respectively.The expression of virus-specific receptors on the surface of host cells is the primary condition for viral invasion,and it is also an important factor that determines the tropism and pathogenesis of viral tissues.SLAM(CD150)and nectin-4(PVRL-4)identified recently as the cellular receptors of CDV,play critical roles on the invasion of CDV.Signaling lymphocyte activation molecules(SLAM)is a mainly membrane receptor of immune cells in hosts infected with canine distemper virus(CDV),and its recognition with the CDV H protein is the basis for viral cross-host infection.Differences in SLAM variable(V)region amino acids affect the interaction between SLAM and CDV hemagglutinin(H)protein,and contribute to variations in host susceptibility to CDV.In our preliminary study,we demonstrated that the pathogenicity of a single CDV strain was highest in raccoon dogs and weakest in mink.Variations in the size of the syncytia and the titer caused by the same CDV in cell lines expressing raccoon dog SLAM(rSLAM)and mink SLAM(mSLAM)were observed.Further comparison of SLAM amino acid sequences revealed the existence of two mutation sites(I74V and Q129R)in the mSLAM variable(V)region compared with rSLAM.We speculate that the amino acid mutations may alter the interaction between CDV H protein and SLAM,and induce the change of cell membrane fusion efficiency.Here,to study the relationship between SLAM of mink and raccoon dog and cell fusion efficiency,we performed SLAM-V homologous modeling,amino acids site-directed mutagenesis and expression analysis,as wells as CDV-H/fusion(F)protein-SLAM cell fusion experiments to study the interaction between SLAM and CDV-H based on the amino acids 74 and 129 of the SLAM.Our results indicated that amino acids 74 and 129 of the SLAM are both located in the interface region of the 3D structure of SLAM.The SLAM-V74 I mutation in mink down-regulated the average protein expression rate from 95.6% to 91.1%(P < 0.05),but had no impact on its cell fusion efficiency.In contrast,the SLAM-R129 Q mutation in mink significantly improved cell fusion efficiency of CDV,with the mean number of syncytia increased from 23.9 to 32.1(P < 0.001).However,The SLAM-Q129 R mutation in raccoon dog significantly reduced cell fusion efficiency,with the mean number of syncytia decreased from 19.4 to 15.8(P < 0.05).The results verify our hypothesis and confirms the effect of mink,raccoon 74 and 129 amino acid on cell fusion efficiency.In order to establish a cell line that culturing CDV easily,pDisplay-mink SLAM eukaryotic expression plasmid was transfected into BHK-21 cells,and monoclonal cell line named BMS stably expressing mink SLAM was successfully constructed by screening with G418 and limiting dilution assay.Indirect immunofluorescence and PCR experimental methods for initial identification of BMS cell line.Finally,a mink CDV from Zhucheng City,Shandong Province was successfully isolated by the BMS cell line,named ZCM(17)2.