Receptor Identification And Binding Mechanism Of PHEV S Protein In Neurons

Porcine hemagglutinating encephalomyelitis virus(PHEV),which is a neurotropic?-coronaviruses,mainly affects piglets within 3 weeks of age,causing porcine hemagglutinating encephalomyelitis(PHE),vomiting and wasting disease.The mortality rate can reach 20%-100%.PHEV generally infects the cells of the respiratory tract,and spreads to the central nervous system,where PHEV replicates and multiplies greatly,through olfactory nerve and trigeminal nerve in host animals.However,the mechanism of PHEV infection of nerve cells remains to be further elucidated.The first step of PHEV infecting the cell is to make the spike protein(S protein)bind cell surface receptors whose types(including protein receptor and carbohydrate receptor)determine the tissue tropism of the virus.At present,the types of receptors and binding mechanism of PHEV are not stepelucidated.In this study,the susceptible mouse's neuroblastoma cells(N2a cells)by PHEV are used as a sample to identify the receptor types of S protein and further elucidate the molecular mechanism of their interaction.Firstly,the paper studies whether carbohydrate receptors on the surface of nerve cells are involved in the infection process of PHEV.Exogenous enzymes such as neuraminidase and heparin sulfate are used to cleave the two sugar chains of neuraminic acid(SA)and heparin sulfate(HS)on the cell surface respectively,and IFA and q RT-PCR are performed after virus inoculation.The study shows that removal of SA and HS on the surface of target cells could effectively inhibit the infection level of PHEV.Co-incubated with N-acetylneuraminic acid,PHEV is used to infect N2a cells,then FCM,Western blot and q RT-PCR are performed,showing that soluble SA could specifically inhibit the attachment of PHEV to target cells.Virions are pretreated with sugar molecules such as mannose,glucose and galactose or soluble glycosaminoglycan to conduct competitive binding tests.It turns out that both the soluble monosaccharides and glycosaminoglycan do not affect the attachment of PHEV to N2a cells.These results indicate that SA and HS are important carbohydrate receptors involved in PHEV infection of N2a cells.HSPG is a class of HS-containing proteoglycan and is widely distributed on the cell surface.To investigate whether HSPG on nerve cell surface is involved in PHEV infection,the amount of HSPG(including SDC and GPC)and the expression levels of HPSE proteins were detected by q RT-PCR after N2a cells inoculating with the virus for 24 hours,48 hours and 72 hours.It is found that the expression levels of SDC3,SDC4 and HPSE are significantly increased,while the expression levels of SDC1protein and m RNA are significantly decreased.RNAi technology is used to inhibit the expression of SDC1 protein in N2a cells,and IFA,Western blot and q RT-PCR are performed after virus inoculation.The expression of SDC1 protein is inhibited and the infection level of PHEV is effectively inhibited,suggesting that SDC1 is involved in PHEV infection of N2a cells.Transfection assay with miRNA mimic and inhibitor and dual-luciferase assay have proved that miR-10a-5p specifically targets SDC1m RNA3'UTR gene sequence and regulates the expression of SDC1.These results suggest that SDC1,SDC3 and SDC4 are proteoglycans involved in PHEV infection,and SDC1 expression is regulated by miR-10a-5p in N2a cells.Secondly,this study investigates whether protein receptors on the surface of nerve cells are involved in the invasion process of PHEV.PHEV is cultured on different cell lines for three consecutive generations,and IFA and Western blot analysis show that PHEV could effectively infect two nerve cells including N2a and HT-22 and four non-nerve cells including PK-15,IPI-2I,PEF and RAW264.7.Membrane proteins of N2a are extracted after infection,and the proteins with different expression levels after PHEV infection are obtained by LC-MS/MS.Combined with the function analysis of known coronavirus receptors,potential receptor proteins including CD81,Ezrin,APN,CEACAM1,ACE2 and DPP4 are obtained.Co-transfected p FUSE-PHEV-S1-FC and p FUSE-PHEV-S_1-523-FC and receptor proteins expression plasmid including APN,DPP4,APN,ACE2 and CD81 respectively into 293T cells,and then immunoprecipitation(CO-IP)and IFA assay are performed,showing that DPP4 protein could specifically bind PHEV S1 and truncated PHEV S_1-523.Cas9 gene editing system and RNAi technology are used to knock out or lower DPP4 protein in N2a cells,and Western blot and q RT-PCR are performed after virus inoculation.The results show that the inhibition of DPP4 protein expression could effectively inhibit the infection level of PHEV.Meanwhile,DPP4 overexpression vector is transfected into N2a cells.After inoculation,q RT-PCR shows that the overexpression of DPP4protein promotes the replication and proliferation of PHEV,which proves the infection efficiency of PHEV on N2a cells is positively correlated with the expression of DPP4 protein.These results indicate that DPP4 protein is a functional PHEV receptorFinally,in order to clarify the binding mechanism between S protein of PHEV and DPP4 protein,N2a cells are treated with Vildagliptin,a bioenzyme inhibitor.After inoculation,q RT-PCR assay shows that DPP4 protease activity does not affect the replication and proliferation of PHEV.p FUSE-PHEV-S1-523-Fc and DPP4 mutant protein expression plasmid are co-transfected into 293T cells.Co-ip assay is used to confirm that DPP4 A288,R289 and glycoylation site N223 are the key amino acid sites of the binding between S proteins of PHEV and receptor proteins.In conclusion,this study successfully identifies the receptor molecules that PHEV invasion depends on,including glucose-assisted receptor HS and SA and protein functional receptor DPP4,and further demonstrates that the key amino acid sites of the binding between S protein RBD and DPP4 are A288,R289 and glycoylation site N223.The above research results systematically clarify the recognition and binding mechanism between PHEV and nerve cell receptor,provide a theoretical reference for a comprehensive understanding of PHEV life cycle and tissue tropism,and also provide a reference for the development of coronavirus receptor blocking drugs and vaccine design.