E3 Ubiquitin Ligase RNF114 Inhibits Classical Swine Fever Virus Replication Via Ubiquitination And Degradation Of Viral NS4B Protein

Classical swine fever(CSF)poses a serious threat to the pig industry in many countries.Classical swine fever virus(CSFV),the causative agent of CSF,belongs to the genus Pestivirus of the family Flaviviridae.Ubiquitination is an important and extensive post-translational modification,which is involved in various cellular processes.Viruses employ ubiquitination to facilitate their replication and the hosts use ubiquitination to limit viral replication.Recently,increasing studies have reported that ubiquitination is involved in the viral replication of family Flaviviridae.Previously,we have screened the porcine RING finger protein 114(pRNF114)as a potential anti-CSFV protein using a reporter CSFV in ISGs-overexpressing PK-15 cells.However,the anti-CSFV activity mechanism of pRNF114 remains unknown.This study was aimed to clarify the mechanism of pRNF114 against CSFV replication.First,we detected the effects of CSFV infection on the expression level of pRNF114.The results showed that the mRNA level of pRNF114 was increased significantly in the peripheral blood mononuclear cells(PBMCs)of the CSFV-infected pigs(in vivo)and in PK-15,PAM or PBMCs infected with CSFV(in vitro)at an increasing multiplicity of infection(MOI)and time points postinfection.These data suggested that CSFV infection can upregulate the expression of pRNF114,indicating a potential role of pRNF114 in CSFV infection.Next,we found that CSFV replication was suppressed in PK-pRNF114 cells stably expressing pRNF114,whereas CSFV growth was enhanced in PK-RNF114-KO cells with pRNF114 knockout.These indicated that pRNF114 is an anti-CSFV protein.Considering that pRNF114 is an E3 ubiquitin ligase,we generated a pRNF114 RING domain mutant-expressing plasmid pRNF114(C64/67A),which lacks ubiquitin ligase activity.The results demonstrated that pRNF114(C64/67A)failed to display an anti-CSFV action,suggesting that anti-CSFV activity of pRNF114 is dependent on its E3 ligase activity.Moreover,we showed that pRNF114 interacts with NS4 B by coimmunoprecipitation and pRNF114 degrades NS4 B through the proteasome pathway.Since protein ubiquitination is a critical step in the proteasome pathway,we generated a panel of ubiquitin mutants and found that pRNF114 mediates the K27-linked polyubiquitination of the NS4 B.Taken together,pRNF114 is an anti-CSFV protein and exerts antiviral activity through mediating the K27-linked polyubiquitination of the NS4 B and promoting its degradation by the proteasome pathway.