Mechanisms Of Inhibition Of Type Ⅲ Interferons In Porcine Intestinal Epithelial Cells By Classical Swine Fever Virus

Classical swine fever(CSF)is a highly infectious disease caused by classical swine fever virus(CSFV),which leads to great economic losses to the pig industry.The virus can break the mucosal barrier to establish its infection.Type Ⅰ and Ⅲ interferons(IFN-Ⅰ and IFN-Ⅲ),important components of host innate immunity,are produced upon recognition of the viral components by partern recognition receptors(PRRs)and play crucial roles in maintaining the antiviral state in epithelial cells.CSFV is known to interfere with production of type Ⅰ interferons by inducing degradation of IRF3 through the proteasomes.Type Ⅲ interferon plays a key role in antiviral innate immunity of mucosal epithelial cells.However,no information is available on whether or how CSFV modulates IFN-λs production.This study was attempted to investigate the effects of CSFV and its Npro protein on expression of IFN-Ⅲ as well as expression and nuclear translocation of IRF1 by means of gene deletion,overexpression and RNA silencing in combination with techniques such as reporter assay,immunoblotting and confocal imaging.In IPEC-J2 cells infected with CSFV,qPCR revealed that the ΔNpro mutant induced significantly higher transcription of type Ⅲ IFNs(IFN-λs:IFN-λ1,IFN-λ3 and IFN-λ4)in IPEC-J2 cells from 24 to 48 h than its parental strain(wtCSFV).By qPCR in combination with promoter reporter assay,we found that wtCSFV infection significantly repressed transcription of type Ⅲ IFNs and IFN-λ3 promoter activity in poly(I:C)-treated IPEC-J2 cells.Npro deletion abolished such inhibitory effects.In IPEC-J2 cells transfected with recombinant vector pCMV-Npro,a marked reduction of mRNA transcription of type Ⅲ IFNs and IFN-λ3 promoter activity was observed as a result of Npro expression.These results demonstrate that CSFV employs its Npro to inhibit type Ⅲ IFNs expression.It is known that expression of type Ⅲ IFNs is regulated by IRF 1 in humans and mouse.The IPEC-J2 cells were transfected either with recombinant vector expression IRF1 or with irf1-silencing plasmid and examined for expression of IFN-λs and IFN-λ3 promoter activity.We found that IRF1 is also a positive regulator of type Ⅲ IFNs in pigs.Both wtCSFV and △Npro showed time-dependent increase of IRF1 expression.However,△Npro exhibited more pronounced effect of induction.Infection with wtCSFV led to significant reduction of IRF1 expression and its promoter activity in Poly(I:C)-stimulated IPEC-J2 cells,while ΔNpro infection did not have such inhibitory effects.By Npro expression in IPEC-J2 cells in response to Poly(I:C)stimulation,we found that CSFV Npro was inhibitory to IRF1 expression.Confocal imaging revealed that wtCSFV or Npro expression inhibited IRF 1 translocation into the nuclear compartment.In summary,this research provides clear evidence that CSFV utilizes the Npro protein to inhibit IRF 1 expression and nuclear translocation,thereby inhibiting the production of downstream antiviral IFN-Ⅲ.This could be one of the mechanisms that CSFV evades the host innate immunity on the mucosal surface.This discovery deepens the understanding of the innate antiviral immune mechanism of CSFV.