The Mechanism Of Porcine Deltacoronavirus Nsp5 Mediated Inhibition In Interferon-I Production And Signal Transduction

Porcine deltacoronavirus?PDCoV?belongs to the genus of Deltacoronavirus and Coronavirus subfamily.Clinical research confirmed that PDCoV infection causes diarrhea and mortality in seronegative neonatal piglets,which is similar to that of transmissible gastroenteritis virus?TGEV?,porcine epidemic diarrhea virus?PEDV?.The first outbreak of PDCoV was in the United States in 2014,followed by many countries in Asia,which caused considerable attention.As an emerging swine enteropathogenic coronavirus?CoV?,the pathogenesis and infection mechanism for deltacoronavirus is still unclear.As we known,interferon?IFN?and IFN-induced antiviral responses are important to defend against early viral infection.Our previous research showed that PDCoV infection impaired Sendai virus?SeV?-induced IFN-?production,while the role of PDCoV encoded proteins in IFN signaling is quite unclear.CoV nsp5,the 3C-like protease,mediated the cleavage of polyprotein precursors to produce most of the mature nonstructural proteins and is widely considered to be an attractive target for broad anti-coronavirus drugs.Previous research showed that many viral 3C or 3C-like proteases play the vital function in viral replication and immunoregulation.While the role of PDCoV nsp5 in host immune response is unclear.Here,our work revealed the antagonistic property of PDCoV nsp5 in IFN signaling.1.The mechanism of PDCoV nsp5 antagonizing IFN-I productionThrough the dual luciferase reporter system,PDCoV nsp5,the 3C-like protease,inhibited both IFN-?production and activation of IRF3 and NF-?B induced by SeV through its protease activity.Western blot showed that PDCoV nsp5 targeted the NF-?B essential modulator?NEMO?for cleavage to impair RIG-I/MDA5 signaling pathway and PDCoV infection induced the degradation of endogenous NEMO.By a series of point mutations and truncated mutants,PDCoV nsp5 cleaved glutamine?Q?231 of NEMO and the cleavage products lost the ability to activate the downstream signaling.Our results revealed the new character of PDCoV nsp5 to antagonize IFN-?production.2.The mechanism of PDCoV nsp5 antagonizing IFN-I signal transductionTo analyze the effect of PDCoV nsp5 on IFN-I signal transduction,the IFN-?-mediated response element?ISRE?promoter activity induced by IFN-?was tested under the overexpression of PDCoV nsp5 in both HEK-293T cells and PK-15 cells through dual luciferase assay.The results presented that PDCoV nsp5 overexpression significantly inhibited ISRE promoter activity induced by IFN-?.Quantitative real-time PCR showed that IFN-?induced transcription of IFN-stimulated genes?ISGs?was also impaired in the presence of PDCoV nsp5.Further Western blot assay showed that PDCoV nsp5 significantly inhibited endogenous signal transducer and activator of transcription 2?STAT2?expression and phosphorylation.Interestingly,PDCoV nsp5 could cleave STAT2in a dose-dependent manner and endogenous STAT2 cleavage was also detected in PDCoV infected LLC-PK1 cells.Through a series of point mutations in STAT2,Q685and Q758 residues were identified to be the two sites cleaved by PDCoV nsp5.Compared with full-length STAT2,the ability of the cleavage products to induce ISGs was impaired,indicating that PDCoV nsp5 mediated STAT2 cleavage is an effective mechanism to antagonize IFN-I signal transduction.3.The mechanism of PDCoV nsp5 antagonizing the antiviral effect of DCP1ADue to the important role of PDCoV nsp5 protease activity in IFN-I signaling regulation,we analyzed the possible cleavage of ISGs by PDCoV nsp5.To this end,14classical ISGs were screened in HEK-293T cells.Western blot results showed that PDCoV nsp5 cleaved the porcine mRNA-decapping enzyme 1a?pDCP1A?at Q343 in a dose-dependent manner.The cleavage of endogenous pDCP1A was also confirmed in PDCoV-infected IPI-2I cells.Compared with full-length pDCP1A,the cleavage products,pDCP1A_1–343 and pDCP1A_344–580 both lost the antiviral ability.Remarkably,Mutant pDCP1A-Q343A showed a stronger capacity to inhibit PDCoV infection compared with wild-type pDCP1A.We further test seven mammalian CoVs encoded nsp5 and western blot showed all nsp5 could cleaved both pDCP1A and human DCP1A at common site,revealing the possible common mechanism for CoVs.