IRF2a Cooperates With Phosphoprotein Of Spring Viremia Of Carp Virus To Suppress Antiviral Response In Zebrafish

IFN regulatory factor（IRF）2 belongs to the IRF1 subfamily and its functions have not fully been understood.The studies that fish IRF2 can inhibit the IFN response have been reported,but the mechanism has not been studied in detail.We focused on molecular biology of zebrafish IRF2a,the protein interaction between host antiviral factors and SVCV structure proteins.Therefore,advances in the knowledge of the immune response of IRF2a in zebrafish could help to gain a better understanding of the defense mechanisms against rhabdoviruses or viruses in general in other species.The key results are as follows:1.IRF2a negatively regulates IFN response and promotes viral replicationwe overexpressed IRF2a in the WT cells and examined the expression of antiviral genes after SVCV infection.We found that overexpression of IRF2a repressed the expression of ifnφ1 and mx1 genes at 24 h after infection.Consistently,the expression levels of SVCV-N and SVCV-G genes were elevated and virus titer of IRF2a-overexpressed cells was significantly higher than that of the control cells.Next,we analyzed the promoter activities of genes involved in the IFN-mediated pathway in the EPC cells following infection with SVCV or stimulation with poly（I:C）.It was shown that overexpression of IRF2a consistently inhibited the activities of ifnφ1,ifnφ3 and ISRE promoters depends on DBD domain.Interestingly,overexpression of IRF2a and IRF1 or IRF7 also decreased activities of ifnφ1 and ISRE promoters,suggesting that IRF2a acts as a suppressor for IRF1/IRF7 activated IFN production.2.IRF2a knockout cells exhibit enhanced resistance to virus infectionHaving shown that IRF2a negatively regulates IFN response,we generated irf2a knockout zebrafish for to further investigation on the roles of IRF2a in immune response to viral infection.We found that irf2a^-/-zebrafish larvae had higher survival rate than the irf2a^+/+larvae after infection with SVCV infection.Antiviral genes such as ifnφ1 were induced.Next,we prepared the cell cultures from the caudal fin of irf2a^+/+and irf2a^-/-fish and evaluated their susceptibility to virus.We observed that compared to that in the irf2a^+/+cells,the irf2a^-/-cells exhibited sharp decreases of viruses release to the medium and expression levels of svcv-n and svcv-g genes.Further,the expression levels of genes involved in the IFN pathway such as ifnφ1,mx,isg15 were relatively higher,indicating that the IFN response was more pronounced in the irf2a^-/-cells than irf2a^+/+cells.To address why the irf2a^-/-cells showed enhanced resistance to SVCV infection,we performed RNA-seq analysis to identify differentially expressed genes（DEGs）and found a total of 11,623 differentially expressed genes（DEGs）were identified.To visualize the variation in expression between samples,principal component analysis（PCA）were conducted,they were segregated by irf2a^-/-or WT phenotype（PC1）and then by SVCV（PC2）.Gene set enrichment analysis（GSEA）was performed,the top pathways were mainly upgraded in MAPK signaling pathway,cytokine-cytokine receptor interaction and TGF-beta signaling pathway when in irf2a^-/-group.The ISGs genes are antiviral effectors involved in limiting virus replication,packaging and release,their expression levels were relatively higher in the irf2a^-/-group.Such as mx genes family.RNA-seq analysis revealed that most of STPK genes,including pim,mknk2b,stk and cdk genes,were down-regulated in the irf2a^-/-cells.Therefore,the significantly upregulation of PIM family expression in WT cells infected with SVCV,but those pim genes were impaired in irf2a^-/-cells attracted our attention.We selected pim1,pim2 and pim3 to verify the alteration of expression in the WT and irf2a^-/-cells after SVCV infection.It was apparent that the expression of pim genes was decreased in the irf2a^-/-cells.Relative expression of pim2 was upregulated to 26-fold in WT cells,but decreased to 6-fold in irf2a^-/-cells infected by SVCV.pim1 expression was from 1.3-fold decreased to 0.8-fold,pim3 expression was from 2.2-fold decreased to 1.4-fold.Next,we overexpressed pim genes in the EPC cells and found that overexpression of pim2 and pim3 but not pim1 resulted in enhancement of SVCV replication.3.SVCV-P promotes nuclear translocation and stability of IRF2aGiven that overexpression of IRF2a enhanced viral replication in the irf2a^+/+cells,we wonder whether it is exploited by viruses to favor infection.To test this hypothesis,we sought to determine the interaction between IRF2a and SVCV viral proteins in the HEK293T cells.The results showed that SVCV-P protein but not N protein could be co-precipitated with IRF2a depends on IAD domain interacted with C terminal of SVCV-P.The colocalization of IRF2a and SVCV-P is shown that the SVCV-P protein appeared to be limited in the cytoplasm and was largely undetectable in the nucleus.We also observed that the cellular IRF2a protein levels steadily rose in the EPC cells with the increases of SVCV-P protein depends on SVCV-P significantly reduced the K48-linked ubiquitination of IRF2a.The IRF2a protein levels in the cytoplasm and nucleus were markedly increased by SVCV-P after virus infection or poly（I:C）stimulation.4.IRF2a promotes degradation of STAT1a and blocks its nuclear translocationSTAT1 is the central transcription factor required for IRF2 to regulate gene expression.GFP-IRF2a bound to FLAG-STAT1a which involved the IAD domain of IRF2a.Moreover,formation of IRF2a/STAT1a complex resulted in decreases of STAT1a protein in the EPC cells relayed on IRF2a significantly decreased the K63-linked ubiquitination.After stimulation with zebrafish IFNφ1,IRF2a was still able to decrease the levels of STAT1a and phospho-STAT1a protein.Furthermore,SVCV-infection enhanced IRF2a translocation from the cytoplasm into the nucleus,although the majority of STAT1a remained in the cytoplasm when co-expressed with IRF2a.We found that IRF2a abrogated nuclear accumulation of STAT1a not phospho-STAT1a upon response to SVCV infection and poly（I:C）stimulation.5.SVCV-P promotes degradation of STAT1a and blocks its nuclear translocationViruses target STAT1 to antagonize antiviral interferon signaling^[1,2].We reasoned that SVCV-P protein may inhibit STAT1 mediated signaling.In the EPC cells transfected with FLAG-STAT1a,which was localized in the nucleus after SVCV infection compared to the cytoplasmic presence prior to infection.However,when co-transfected with FLAG-STAT1a and SVCV-P,FLAG-STAT1a was seen in the cytoplasm with or without SVCV infection.We showed that SVCV-P co-immunoprecipitated with STAT1a depends on the C-terminal domain of SVCV-P.As consequence,binding of SVCV-P with STAT1a resulted in decreased protein levels of STAT1a and phospho-STAT1a regardless of activation by IFNφ1.Our data showed that SVCV-P protein could effectively block STAT1a nuclear translocation process.