Studies On The Mechanism Of Porcine Deltacoronavirus Non-structural Protein Nsp15 Inhibits IFN-? Production

Porcine deltacoronavirus(PDCoV)is an emerging swine coronavirus causing diarrhea and intestinal damage in nursing piglets.PDCoV is single-stranded,positive-sense RNA virus with envelop,and its full-length genome,approximately 25.4kb,the typical genome of PDCoV contains successively 5?UTR-ORF1a-ORF1b-S-E-M-NS6-N-NS7-NS7a-3?UTR,which encodes two viral replicase polyproteins that are predicted to be proteolytically processed to yield replication and transcription-associated 15 mature nonstructural proteins(nsp2-nsp16),four structural proteins and three accessory proteins,respectively.Based on studies of other known CoVs,the non-structural protein 15 of the order Nidovirales(families Arteriviridae and Coronaviridae)infected with vertebrates has a highly conservative endoribonuclease,termed NendoU,which exerts a critical role in viral replication and transcription.And the nsp15 of some CoVs and its orthologs nsp11 in arteriviruses have been demonstrated to antagonize IFN production.Our previous studies have shown that PDCoV infection suppresses RIG-I-mediated the production of IFN-?.Whether PDCoV nsp15 antagonizes IFN-? production remains unclear.In this study,PDCoV nsp15 was taked as the research object to investigate its role in the production of IFN-? and further explained the molecular mechanism.The main research contents are as follows: 1.PDCoV nsp15 dose-dependently inhibit IFN-? production.LLC-PK1 cells or HEK-293 T cells were co-transfected with increasing amounts of PDCoV nsp15 expression plasmid and IFN-?-Luc reporter plasmid,along with pRL-TK(an internal control plasmid).The cell lysates were harvested and the IFN-? promoter-driven luciferase activity and IFN-? mRNA expression levels were measured by dual-luciferase reporter assay and real-time RT-PCR analysis,respectively.It showed that PDCoV nsp15 significantly inhibited SeV-induced IFN-? promoter activity and mRNA expression levels in a dose-dependent manner,these results suggested that PDCoV nsp15 is an IFN-? antagonist.2.PDCoV nsp15 mainly inhibits activation of NF-?B.It is well known that the induction of IFN-? requires the coordinated and cooperative actions of the transcription factors interferon regulatory factor 3(IRF3)and nuclear factor-?B(NF-?B).Therefore,it is essential to investigate whether PDCoV nsp15 is involved in the SeV-induced activation of IRF3 and NF-?B in both LLC-PK1 and HEK-293 T cells.The results of luciferase reporter assay showed that PDCoV nsp15 blocked the SeV-induced promoter activity of NF-?B in a dose-dependent manner,but it had no effect on SeV-induced promoter activity of IRF3.The phosphorylation and nuclear translocation of IRF3 and p65 subunit are the hallmarks for activation of IRF3 and NF-?B,respectively.In line with our foregoing results of luciferase reporter assays,SeV-induced p65 phosphorylation and nuclear translocation was notably inhibited in nsp15-expressing cells,but nsp15 failed to impede the IRF3 phosphorylation and nuclear translocation by western blot and IFA analysis.Overall,these results demonstrated that PDCoV nsp15 mainly inhibits SeV-induced activation of NF-?B to antagonize IFN-? production.3.His219,His234 and Lys269 are the key endoribonuclease activity sites of PDCoVnsp15.Amino acid residues of PDCoV nsp15 His219,His234 and Lys269 were predicted to be the NendoU active catalytic amino acid sites via amino acid homologous sequence alignment and structural homology analysis of nsp15 orthologs from PDCoV and others typical coronavirus.Therefore,Alanine(A)substitutions at residues H219,H234,and K269 of PDCoV nsp15 were conducted to generate three mutants H219 A,H234A,and K269 A,respectively,and then cloned into expression vector to express the recombinant proteins of wild type(WT)or mutants of PDCoV nsp15.Their endoribonuclease activities were analyzed under identical conditions by using fluorescence resonance energy transfer(FRET)as described previously.The endoribonuclease activities of three mutants were significantly decreased compared with the WT nsp15,confirming that His219,His234 and Lys269 are the key endoribonuclease activity sites of PDCoV nsp15.4.PDCoV nsp15 inhibits IFN-? production independently of its endoribonucleaseactivityThe endoribonuclease activity of CoV nsp15 s or arterivirus nsp11 s are essential for antagonizing type I IFN.In addition,some studies reported that arterivirus nsp11 s are extremely toxic to effect on the production of type I IFN.To explore whether PDCoV nsp15 inhibits IFN-? production independently of its endoribonuclease activity or cytotoxicity,the cytotoxicity of the WT and mutants of nsp15 were first detected,and it was found that overexpression of nsp15 had a certain cytotoxicity,while no cytotoxicity could be observed in cells expressing three mutants.Bases on this,we showed by the results of dual-luciferase reporter assay that,similar to the WT nsp15,all three mutants significantly inhibited SeV-induced the IFN-? promoter activity,and no significant difference between each other.We further found that all three mutants inhibited NF-?B activation by markedly decreased p65 phosphorylation,while not affected the phosphorylation level of IRF3.By using the mutant H234 A as a representative,we analyzed the nuclear translocation of endogenous IRF3 and p65 after overexpression of nsp15 mutants.Similar to the WT nsp15,ectopic expression of mutant H234 A inhibited SeV-induced nuclear translocation of p65,while not affected IRF3.Based on these results,it was suggested that PDCoV nsp15 inhibits the production of IFN-? and the activation of NF-?B independently of its endoribonuclease activity and cytotoxicity.