Studies On The Molecular Mechanism That Porcine Deltacoronavirus Nsp10 Inhibits IFN-β Production

Porcine deltacoronavirus(PDCoV)is a newly discovered pig intestine coronavirus that causes watery diarrhoea,vomiting,and dehydration in suckling piglets.In 2014,a large-scale outbreak of PDCoV in the United States was reported.Subsequently,Canada,South Korea,China,Thailand,Vietnam,Laos,and other countries or regions had also reported the occurrence and prevalence of PDCoV,which had aroused great concerns in the global pig industry.Previous studies had confirmed that PDCoV induces very low levels of Interferon(IFN)and inhibits Sendai virus(SeV)-induced type I IFN production.However,it is not clear which protein of PDCoV has the property of inhibiting type I IFN production.In this study,we found that PDCoV encoded non-structural protein nsp10 inhibits SeV-induced type I IFN production,and explored its mechanism.The main findings are as follows: 1.PDCoV nsp10 inhibits SeV-induced IFN-β productionThe eukaryotic expression plasmid of PDCoV nsp10 was constructed and transfected into HEK-293 T or LLC-PK1 cells.By detecting the dual luciferase activity and mRNA expression of IFN-β promoter,it was found that overexpression of nsp10 significantly inhibits SeV-induced IFN-β promoter activation and mRNA expression.It was confirmed that PDCoV nsp10 has the property of inhibiting the production of IFN-β.2.PDCoV nsp10 inhibits SeV-induced activation of IRF3 and NF-κBNF-κB and IRF3 are two important transcription factors that cooperatively activate IFN-β production.To investigate whether PDCoV nsp10 inhibits the activation of NF-κB and IRF3,nsp10 eukaryotic expression plasmid was transfected into HEK-293 T or LLC-PK1 cells,NF-κB and IRF3 promoter activity,phosphorylation,and nuclear transport was detected by dual luciferase activity assay,western blot,and indirect immunofluorescence respectively.Dual luciferase activity analysis revealed that the expression of nsp10 significantly inhibits SeV-induced NF-κB and IRF3 activation;Western blot demonstrated that nsp10 has no effect on SeV-induced p65(NF-κB subunit)and IRF3 total protein levels,but significantly decreases phosphorylation of p65 and IRF3;Indirect immunofluorescence also confirmed that nsp10 inhibits SeV-induced nuclear import of p65 and IRF3.The above results indicate that PDCoV nsp10 can inhibit IFN-β production by inhibiting the activation of NF-κB and IRF3.3.PDCoV nsp10 inhibits RIG-I/MAD5-mediated IFN signalingCoronaviruses mediate innate immune responses primarily through RLRs(RIG-I-like receptors).To investigate whether PDCoV nsp10 inhibits the production of IFN-β mediated by the RLR pathway,the nsp10 eukaryotic expression plasmid and signal molecules RIG-I/RIG-IN,MDA5,IPS-1,TBK1/IKKε,IRF3/IRF3-5D in the RLR signaling pathway co-transfected HEK-293 T cells and we found that nsp10 inhibits RIG-I/RIG-IN,MDA5-induced IFN-β promoter activation,but not IPS-1,TBK1/IKKε,IRF3/IRF3-5D.It was speculated that nsp10 may target the signalling molecule IPS-1 or its upstream molecules.However,co-immunoprecipitation(CO-IP)experiments showed that there was no interaction between nsp10 and RIG-I,MDA5,TRIM25,and TRIM65.Therefore,it remains to be further studied which molecule of the RLR pathway is actually targeted by PDCoV nsp10.4.Analysis of the key regions for the inhibition of IFN-β production by PDCoV nsp10 proteinIn order to further determine the functional domains of PDCoV nsp10 in inhibiting the production of IFN-β,the amino acid sequences of different coronavirus(SARS-CoV,PDCoV,PEDV,TGEV)nsp10 were compared,and it was found that the amino acid sequence of different coronavirus nsp10 is relatively conserved.According to the crystal structure of SARS-CoV nsp10,the homology modeling of PDCoV nsp10 was performed.It was found that the N-terminus of PDCoV nsp10 contains a pair of anti-parallel α-helices.The C-terminus contains a loop structure and the middle contains an irregular β-sheet.Based on the homology modeling structure,truncated mutants(1-96 aa,34-96 aa,34-133aa)in different regions of PDCoV nsp10 were constructed,and it was found that 34-96 aa is the main region that inhibits the production of IFN-β.Further analysis found that this region contains a zinc finger structure(C74-C77-H83-C90).Thus,four single-point mutant mutants(ΔN33ΔC37-C74 A,ΔN33ΔC37-C77 A,ΔN33ΔC37-H83 A,ΔN33ΔC37-C90A)and a quadruple-mutant mutant ΔN33ΔC37-4A were constructed against the zinc finger domain.The above mutants and wild-type nsp10 were transfected into HEK-293 T cells or LLC-PK1 cells respectively and their effects on SeV-induced IFN-β production were examined.It was found that the zinc finger structure mutants do not affect SeV-induced IFN-β production,suggesting that PDCoV nsp10 inhibits the production of IFN-β is independent of its zinc finger structure.