The Molecular Mechanism Of E3 Ubiquitin Ligase TRAIP-mediated Regulation Of Host Innate Immunity

Ubiquitination and small ubiquitin-like modifier are one of the highly conservative post-translational modifications that affect various life activities of viral infection.Tumor necrosis factor receptor-associated factor(TRAF)interacting protein(TRAIP)is a RING E3 ubiquitin ligase that exerts multiple functions in DNA damage response,DNA repair and innate immune pathways.In this study,we used porcine reproductive and respiratory syndrome virus(PRRSV)infected cells as a model,and explored the mechanism by which the virus regulates host protein post-translational modification and inhibits innate immune signaling pathways to promote PRRSV proliferation.In addition,the TRAIP ubiquitinated substrate protein DEx D-Box Helicase 39A(DDX39A)was excavated by mass spectrometry.Furthermore,the type of ubiquitination modification of DDX39A by TRAIP and the new immunobiology mechanism of TRAIP regulating DDX39A to inhibit host innate immunity were further clarified.The main results are as follows:(1)The effect of TRAIP on the proliferation of PRRSV and screening of interacting viral proteinsThe E3 ubiquitin ligase TRAIP,which plays a potentially important role in PPRSV infection,was screened from the 3D4/21 cells transcriptome data of PRRSV in vitro infection.Our experiments showed that PRRSV infection promoted the expression of TRAIP.Analysis of immunofluorescence and Co-IP results revealed TRAIP interacted with the PRRSV nsp1α subunit,and the LZ domain of TRAIP and the PCPα domain of nsp1α were the key interacting domains.Base mutation and functional analysis showed that the LZ domain K205 of TRAIP was the key site for interaction between nsp1α and TRAIP.(2)PRRSV nsp1α affects post-translational modification and nucleocytoplasmic distribution of TRAIP.PRRSV nsp1α inhibited the K48 polyubiquitination and SUMOylation of TRAIP.Nsp1α inhibited polyubiquitination of TRAIP(WT),but it had no effect on TRAIP mutant(K205R),suggesting that the inhibitory effect of nsp1α depended on its interaction.The interaction experiments of site mutations showed that the inhibitory effect of nsp1α on the SUMO modification of TRAIP was not affected by the mutation of nsp1α and TRAIP binding site.The dual protein post-translational modifications regulation of TRAIP led to TRAIP cytoplasmic enrichment.PRRSV nsp1α affected the nuclear localization of TRAIP by inhibiting the SUMOylation of TRAIP.In addition,PRRSV nsp1α inhibited TRAIP K48 polyubiquitination modification and its ubiquitin proteasome degradation pathway,thus maintained the abundance and stability of TRAIP.(3)PRRSV nsp1α cooperates with TRAIP to further suppress the production of type I interferon.Previous studies have shown that TRAIP can promote K48 polyubiquitination of TBK1 and negatively regulate type I interferon production.In this study,immunofluorescence and co-immunoprecipitation assays showed that nsp1α mediates the enrichment of TRAIP in cytoplasm,and forms ternary complexes with TRAIP and TBK1,further promoting the K48 polyubiquitination and degradation of TBK1,thus antagonizing TBK1-IRF3-IFN signaling pathway.The co-expression of nsp1α and TRAIP significantly inhibited the production of IFN-β rather than the single expression of nsp1α and TRAIP.(4)TRAIP-DDX39A negatively regulates RNA virus-induced type I interferon response.TRAIP interacting protein DDX39A,a K63 ubiquitination modified substrate protein of TRAIP,was discovered by mass spectrometry.DDX39A can bind to mRNAs of specific innate immune-related factors(TRAF3,TRAF6,MAVS),promote the nuclear retention of these antiviral transcripts,and down-regulate its protein expression,thus negatively regulating the production of type I interferon.It was further discovered through the cell infection models of Sendai virus(SeV)and vesicular stomatitis virus(VSV)that these RNA viruses can induce TRAIP protein expression,promote the K63 polyubiquitination of TRAIP targeting DDX39A,and promote DDX39A binding to TRAF3,TRAF6,MAVS mRNA,suppresses its expression and further inhibits the IFN production.In summary,modulation of the dual modification of TRAIP by PRRSV nsp1αresults in over-enrichment of TRAIP in the cytoplasm,leading to excessive K48 ubiquitination and degradation of TBK1,thus antagonizing TBK1-IRF3-IFN signaling pathway.Our research proposes a new model of PRRSV escaping IFN signaling pathway to promote PPRSV proliferation from the perspective of the interaction between viral protein and host E3 ubiquitin ligase TRAIP.This deepens our understanding of the pathogenesis of PRRSV escaping host innate immunity.Additionally,TRAIP entering the nucleus mediates K63 polyubiquitination of DDX39A,promotes the binding of DDX39A to antiviral transcripts TRAF3,TRAF6 and MAVS,restricts nuclear export of related mRNAs and protein expression,and inhibit the production of IFN-β.This further enriched a new way for RNA viruses to escape from host innate immunity and promote virus proliferation through regulating the expression of E3 ubiquitin ligase TRAIP.