Study On The BVDV Npro Non-structural Protein Antagonism Against Type Ⅰ Interferon Producting Mediated By ELF4

BVDV is a member of Flaviviridae and prion genus,and it mainly causes diarrhea,reproductive problems and severe acute and chronic mucosal diseases.The most serious hazard is the formation of persistent infection of cattle and immunosuppression in cattle.The mechanism of immunosuppression cannot be fully explained yet.Type I interferon is a key factor in the body’s establishment of an antiviral state that prevents the replication of the virus in the host cell.In 2013,Funfu et al.first discovered that ELF4 inhibited the replication of the virus by directly regulating type I IFNs.We speculate that BVDV infection in bovine organisms may also inhibit viral replication through ELF4-mediated type I IFNs.It has been reported that NPV-type BVDV-infected macrophages do not produce type I interferons,and BVDV non-structural protein Npro mediates degradation of the ubiquitinated proteasomal targeted to IRF3 and inhibits type I interferon production.We speculate that the Npro protein may antagonize the type I IFNs mediated by BVDV by degrading ELF4.Based on this study,we investigated the molecular mechanism of BoELF4-induced type I interferon production during BVDV infection and the role of Npro in antagonizing the expression of type I interferon.The first overexpression of BoELF4 was found to promote the activation of HuIFNβand BoIFNβ3 promoters;shRNA knock down the expression of ELF4 gene in MDBK cells,and qPCR and reporter gene detection showed that the expression levels of BoIFNβ3 and ISG15were reduced.The study showed that bovine ELF4 was introduced.Guided by the expression of type I interferon.Overexpression of BoELF4 different domain deletion plasmids and the addition of viral stimulation revealed that the NLS domain is a key binding site for the formation of dimers.The ETS domain binds to and activates the BoIFNβ3 promoter to promote type I interferons after the dimer enters the nucleus.It plays a key role in the production process.Second,BVDV was used to infect MDBK cells,mouse iBMDM cells,mouse peritoneal macrophages,and ELF4^–/–mouse models.It was detected that BVDV infection could activate the promoter activity of HuIFNβand BoIFNβ3;after infecting MDBK,qPCR was used to detect ELF4 at different times.The transcription levels of the upstream and downstream transcription factors were changed and it was found that the ELF4 transcription level increased with the amount of virus,and compared with the normal MDBK cells,the titer of the virus that overexpressed the ELF4 cell BVDV infection weakened,knocking down the ELF4 MDBK cell BVDV The titer of the infected virus was higher.The transcription levels of IFNβand ISG15 in the BVDV-infected ELF4^–/–mice group were lower than those in the control group.The results showed that BoELF4 played a positive regulatory role in the expression of type I IFN during BVDV infection.At last,the Npro gene eukaryotic expression plasmid Pcmv7.1-Npro of BVDV was constructed and transfected into 293T and MDBK cells.The results of qPCR and Western blot indicated that Npro could degrade ELF4 through ubiquitination pathway.The intracellular type I interferon IFNβ3 and ISG15 The level of transcription is also reduced.The results indicate that Npro protein inhibits type I interferon expression through the regulation of BoELF4.In summary,this study demonstrated that BoELF4 can form dimer into the nucleus during BVDV infection and directly activate type I IFN promoter to induce type I IFN production.BVDV nonstructural protein Npro degrades BoELF4 via ubiquitination pathway to antagonize type I interferon expression.This study provides a theoretical basis for understanding the antiviral mechanism of BVDV antagonism against innate immunity.