Porcine Reproductive And Respiratory Syndrome Virus Enhanced CD83 Expression In Porcine Dendritic Cell And Its Molecular Mechanism

Porcine reproductive and respiratory syndrome virus(PRRSV)is one of the most important pathogenes that caused a panzootic disease of the most economically costly to the swine industry.A key aspect of PRRSV virulence is that the virus suppresses the innate immune response and induces persistent infection.The underlying mechanisms are not well understood.The dendritic cells(DCs)marker CD83 belongs to the immunoglobulin superfamily and is associated with DCs activation and immunosuppression of T cell proliferation when expressed as soluble CD83(sCD83).The relationship between PRRSV and CD83 and its mechanism has not been reported.The main contents of this study are as follows:1.CD83 is strongly induced in MoDCs by PRRSV infection and exploration of its key functional ProteinsMoDCs were incubated with strains differing in virulence(HP-PRRSV BB0907,C-PRRSV S1,and LP-PRRSV NT0801).mCD83,sCD83,and CD83 mRNA expression increased significantly as determined by FACS analysis,ELISA,and qRT-PCR.mRNA expression also increased significantly in the TNF-? positive control.MoDCs were inoculated with live or UV-inactivated HP-PRRSV BB0907 at an MOI of 1,and then harvested for CD83 analysis at differ hpi.The results showed that mCD83 and CD83 mRNA expression increased strongly as a result of TNF-? treatment and PRRSV infection over time.sCD83 levels increased significantly only in cells infected with PRRSV,and CD83 induction is dependent on PRRSV replication.Fothermore,mCD83 and CD83 mRNA expression increases in PRRSV infected-cells in a dose dependent manner.In this study,we obtained the 850bp CD83 gene promoter sequence by genome walking kit,cloned into pGL3-Basic plasmid and obtained pCD83 to establish corresponding dual luciferase reporter gene detection system.Meanwhile,in order to identify viral proteins with the ability to stimulate the CD83 promoter,we constructed a series of recombinant plasmids using the pVAX vector.HEK293T cells were co-transfected with pCD83-luc and pRL-TK,together with a plasmid carrying the nsps,N,or GP5 genes.The results showed that N,nsp1,and nsp10 strongly stimulate the porcine CD83 promoter.2.Molecular mechanism of CD83 induced by PRRSV N protein in dendritic cellsTo confirm that N protein is involved in CD83 promoter activation,HEK293T cells were co-transfected with pCD83-luc and pRL-TK,together with the plasmid pVAX-NP.The results showed that CD83 promoter activity varies directly with N protein.In addition,promoter activity increases from 24 to 48 hpi,and is maintained as long as 72 hpi.Ten single or multi-nucleotide mutations were introduced into the putative functional region of the N protein,in which amino acid residues C23,C75,C90,K10-4A,Q33-5A,K43-5A,P56-4A,E60-5A,H65-4A,and V112-4A were substituted with alanine.HEK293T or Marc-145 cells were then transfected with pCD83-luc and pRL-TK,together with pVAX-NP or the N-terminal constructs.The luciferase assays show that the construct ORF7t containing nucleotides 1-112,and mutants C23,C75 and C90,K10-4A,P56-4A,E60-5 A,H65-4A,and V112-4A are indistinguishable from wild type N protein.In contrast,promoter activity is significantly reduced in the N protein mutant Q33-5A,K43-5A.This result suggests that these amino acids encode two domains that can stimulate porcine CD83 promoter activity.To identify individual amino acid residues within the functional domains that are necessary for inducing CD83 promoter activity,point mutations were constructed in the regions spanning amino acid residues 33-35 and 43-45.Mutations at Ser36,Arg43,and Lys44 significantly depressed CD83 promoter activity compared to the wild type protein.In addition,we found that NF-?B inhibitor and Sp1 siRNA reduce the PRRSV-mediated activation of the CD83 promoter,and that PRRSV infection significantly stimulates expression of Spl and NF-?B mRNA in MoDCs.This suggests that Spl and NF-?B signaling pathways are activated in MoDCs by PRRSV infection.Forthermore,the rR43A,rK44A mutant viruses exhibit altered Spl-dependent transcriptional activity in transient transfection assays.Sp1 and NF-??B mRNA levels in cells infected by these mutants did not recover to wild type levels.CD83 activation by these mutants did not decrease in response to treatment with siRNA targeted to Spl or NF-kB inhibitor.3.Molecular mechanism of CD83 up-regulation by PRRSV Nsp10 in dendritic cellsTo confirm that nsp10 protein is involved in CD83 promoter activation,HEK293T cells were co-transfected with pCD83-luc and pRL-TK,together with the plasmid pVAX-nsp10.The results show that CD83 promoter activity varies directly with nsp10 protein in a time and does dependent manner.HEK293T or Marc-145 cells were then transfected with pCD83-luc and pRL-TK,together with pVAX-Nsp10 or the truncated constructs.The luciferase assays show that amino acids 182 through 221 in nsp10 encode a domain that can stimulate porcine CD83 promoter activity.To refine these results,we constructed 8 multi-point mutations in the putative functional region of nsp10,in which residues R182-5A,C187-5A,P192-5A,L197-5A,P202-5A,P207-5A,L212-5A and C217-5A were substituted with alanine.P192-5A(Pro 192-5A)and L212-5A(Leu212-5A)do not activate the CD83 promoter as well as wild type nsp10 protein or any of the other multi-point mutants.To identify individual amino acid residues within the functional domain that are necessary for inducing CD83 promoter activity,point mutations were introduced to the regions spanning amino acid residues 192-196 and 212-216.Mutations at Prol92,Gly194,Thrl95,Thr196,Gly214,Thr215 and Thr216 in nsp10 obviously depressed CD83 promoter activity compared to the wild type protein nsp10.In addition,we found that Spl and NF-?B therefore appear to be positive modulators of the PRRSV-triggered CD83 expression signaling pathway,probably mediated by binding via domains defined by amino acids P192-5 A and G214-3A of nsp10.4 PRRSV inhibits the immune regulation of MoDC by sCD83 expression.In this section,soluble CD83 gene was cloned from porcine PBMCs by RT-PCR,and a recombinant plasmid expressing soluble CD83 gene were constructed and identified.Both SDS-PAGE and Sandwich ELISA analysis showed soluble CD83 gene was expressed.The sCD83 protein was purified by GST affinity chromatography.We next confirmed that the sCD83 protein has the ability to inhibit TAP1 and ERp57 expression or MoDC-mediated allogeneic T cell proliferation.PRRSV-infected MoDCs were then co-cultured with T cells,which were isolated from PBMC.It was found that PRRSV infection inhibited MoDCs-mediated T cell proliferation in a dose-dependent manner.Finally,by using antibody against CD83,our study demonstrated that PRRSV inhibit the capability of antigen presentation of MoDCs and MoDC-mediated T cell proliferation via sCD83 expression.It provides a new idea for the mechanism of immunosuppression in PRRSV.5.Key amino acid of nspl of PRRSV inhibiting the immune regulation of MoDC by sCD83 expression.To investigate whether nspl protein was involved in CD83 upregulation,a series of recombinant plasmids using the pCI vector containing nsp1? from PRRSV BB0907,S1 and FJ1402 strain were constructed.HEK293T cells were co-transfected with pCD83-luc and pRL-TK,together with the plasmids.The results showed that CD83 promoter activity were upregulated by nsp1? consistently.Then we confirmed that nsp1? protein is involved in CD83 promoter activation.In addition,HEK293T or Marc-145 cells were then transfected with pCD83-luc and pRL-TK,together with pCI-nspla or the truncated constructs.The luciferase assays show that amino acids 1 through 66 can stimulate porcine CD83 promoter activity.Then,12 multi-point mutations in the putative functional region of nsp1,L2-5A,G45-10 and L61-6A do not activate the CD83 promoter as well as wild type nsp1? protein or any of the other multi-point mutants.To identify individual amino acid residues within the functional domain that are necessary for inducing CD83 promoter activity,we finally find mutations at L5-2A,G45,G48 and L61-6A in nspla obviously depressed CD83 promoter activity compared to the wild type protein nspla.By comparison the immune regulation of wild type virus-infected with those of the mutant viruses(rL5-2A,rG45A/G48A,rL61-6A,rNspla-2m,rNspla-3m,rL5-2A(R),rL61-6A(R),rG45A/G48A(R),rNspla-2m(R)and rNspla-3m(R))in MoDCs,our results showed that the wild type and recovery viruses infection induced substantial higher down-regulation of expression of TAP1 and ERp57 than the mutant viruses(rL5-2A,rG45A/G48A,rL61-6A,rNsp1?-2m,rNsp1?-3m).It highlightes the novel mechanism of innate escape responses to viral infections and revealed the suppression of soluble CD83 in the adaptive immune responses during viral infection.