Mechanisim Of Negative Regulation Of Innate Immunity By Porcine E3 Ubiquitin Ligase ASB8

The immune system recognizes viral nucleic acids through pattern recognition receptors(PRRs)that transmit signals to IκB kinases(IKKs)via different adaptor protein,including canonical IKKα,IKKβ and its essential regulatory subunit NEMO and non-canonical TANK-binding kinase-1(TBK1)and IKKi,which activate the transcription factors NF-κB and IRF3/7 signaling pathways.And proinflammatory cytokines,type I interferons(IFN)and IFN-stimulated genes(ISGs)are further induced to exert an intracellular antiviral effect.Ubiquitination is the major intracellular pathway for degradation and functional modification of cellular proteins.It plays a key role in the regulation of IFN signaling pathway.Porcine reproductive and respiratory syndrome virus(PRRSV)infection causes huge economic losses to the swine industry worldwide.The PRRS clinical signs include respiratory disorders,abortion,stillbirth and severe immunosuppression in pigs.Suppression of type I IFN production is one of the major characteristics of PRRSV.By analyzing the transcriptome of PRRSV-infected porcine alveolar macrophages(3D4/21),we found E3 ubiquitin ligase ankyrin repeat and SOCS protein containing protein-8(ASB8)was obviously upregulated in PRRSV-infected cells.However,the function knowledge of ASB8 was limited.The purpose of this study is to investigate the mechanism of ASB8 in PRRSV infection and its effect on IFN pathway,and to explore the intracellular interaction and modified proteins of ASB8 and clarify the details of their interaction modification,and to understand the role of ASB8 in the regulation of PRRs pathway,revealing a new mechanism of PRRSV evades host antiviral immunity.The main results are listed as follows:(1)ASB8 mediated viral innate immune response: up-regulation of ASB8 expression occurred in RNA virus(PRRSV,Se V,PRV,EMCV)and DNA virus(HSV and PCV2)infection.(2)Effect of ASB8 on IFNβ signaling pathway and proliferation of PRRSV: The luciferase assay showed that ASB8 inhibited Se V-induced IFNβ,ISRE and NF-κB promoter activity in a dose-dependent manner.q PCR analysis indicated that overexpression of ASB8 inhibited the m RNA levels of IFNβ,CCL5 and CXCL10 genes after treated with poly(I:C)and Se V or VSV infection.And the expression of VSV and Se V specific m RNA were enhanced in HEK293 T cells by q PCR in overexpressed-ASB8 cells.Our results indicated that the upregulated ASB8 expression promoted the replication of PRRSV,on the contrary,silencing expression of ASB8 could inhibit the replication of PRRSV.Co-IP and immunofluorescence experiments confirmed the interaction between ASB8 and PRRSV Nsp1α.Moreover,the inhibition of IFNβ and NF-κB were further enhanced after co-transfection ASB8 and Nsp1α.Ubiquitination assay revealed that ASB8 mediated K63-linked ubiquitination of Nsp1α in an SOCS domain-dependent.And ASB8 enhanced the stability of Nsp1α.(3)Interaction regulation between ASB8 and kinase complex: A number of proteins interacted with ASB8,including IKKβ,heat shock protein 70(HSP70)and leucine-rich repeat 10B(LRRC10B)were identified by mass spectrometry.Co-IP and immunofluorescence experiments further confirmed the interaction between ASB8 and IKKβ or NEMO.ASB8 promoted K48-linked ubiquitination and proteasomal degradation of IKKβ,which suppress P65 and IκBα phosphorylation and NF-κB production.In addition,ASB8 could be phosphorylated by the kinases(IKKβ,TBK1 and IKKi)when ASB8 was co-transfected with kinase complex(IKKα/β/NEMO and TBK1/IKKi).Mass spectrometry and transfection experiments confirmed that Ser31 phosphorylation of ASB8 by IKKβ is essential for inhibition of the NF-κB signaling pathway.Further luciferase assay and Co-IP experiments demonstrated ASB8 also interacted with the kinases TBK1 and IKKi and catalyzed the K48-linked ubiquitination and degradation of TBK1 and IKKi kinases,resulting in a marked inhibition of IRF3 phosphorylation and IFNβ production.(4)LRRC10B participated in the interaction between ASB8 and kinase complex: RNA virus(PRRSV,Se V and TGEV)infection induced the expression of LRRC10 B.And LRRC10 B overexpression attenuated Se V and VSV-induced activation of IFN-β and NF-κB.In addition,LRRC10 B was involved in the interaction between ASB8 and kinases(IKKβ,TBK1 and IKKi).Taken together,our results provide evidence that PRRSV Nsp1α hijacks host E3 ubiquitin ligase ASB8 to promote K63–linked ubiquitination and increase stability of Nsp1α,thereby promoting PRRSV proliferation.ASB8 inhibited IFN signaling pathway by interacting with kinases(IKKβ,TBK1 and IKKi)and phosphorylation by kinases,which facilitate K48–linked ubiquitination and degradation of kinase.LRRC10 B was involved in the interaction between ASB8 and kinases and the regulation of innate immune pathway of PRRs.Our findings enrich the theoretical understanding of PRRSV-induced immunosuppression and provide a new mechanism for innate antiviral immunity.