| Avian influenza(AI)is an avian infectious disease which caused by avian influenza viruses(AIVs).The poultry infected by highly pathogenic avian influenza virus(HPAIV),often appears severe systemic symptoms and even death.Occasionally,HPAIVs can cause human infection and even death.Waterfowl,including ducks and geese,is the natural reservoir of AIVs.The waterfowl is also the source of donor genetic fragments for viral recombination,and always acts as asymptomatic carriers of AIV.China is one of the largest countries in poultry breeding with a variety of breeding modes.The unreasonable breeding mode accelerates the risk of AIV transmission from waterfowl to chickens and even humans.In addition,the replication of AIV requires many cellular proteins.At present,most studies focus on how host proteins regulate AIV replication in mammals.However,there are few studies on how birds,especially proteins of duck regulate AIV replication.In this study,we analysis the biological functions of cellular protein during AIV infection and the mechanism of the cellular protein regulating AIV replication in primary duck embryo fibroblast(DEF)cells.Nucleoprotein(NP)is a conserved structural protein and plays multiple essential functions during AIV infection.To complete these functions,NP must interact with host factors and utilize the host post-translational modification(PTM)machinery to regulate its functions in different phases of viral replication.In this study,we enriched NP from sheldrake DEF cells,which were infected with HPAIV H5N1 strain of DK212 by immunoprecipitation and applied the LC-MS/MS method to identify immunoprecipitated proteins.A total of 590 proteins were identified,in which 351 proteins contained quantitative information.Compared with the control group,77 proteins showed increased expression and 39 proteins showed decreased expression.Most of the identified proteins are proteins involved in PTM,negative regulation of genes,cytoskeleton,RNA processing,and ribosomal structure.Among these proteins,the expression of duck protein inhibitor of activated STAT2(du PIAS2)protein is increased.And PIAS proteins are SUMO E3 ligases that play important roles in regulating gene expression and viral replication in mammals.In order to identify whether du PIAS proteins have the similar functions,we selected du PIAS in the following study.PIAS proteins are important signal transduction modulator family and regulate the innate immune signaling pathway induced by certain transcription factors,including nuclear factor κB(NF-κB),interferon regulatory factor(IRF),and JAK/STAT.The PIAS protein mechanism that regulates innate immune response in mammals has been well described in the literature;however,whether the PIAS gene exists in ducks as well as the role of PIAS in duck IFN-β expression is still unclear.Here,we cloned sheldrake PIAS,finding du PIAS2 could repress IFN-β production.du PIAS2 contains SAP-PINIT-RLD-S/T characteristic domains,and its overexpression could inhibit virus-induced IFN-β promoter activation.Moreover,du PIAS2 interacted with duck IRF7 and inhibited IFN-β promoter activation induced by duck IRF7.Additionally,its inhibitory function does not rely on its SUMO E3 ligase activity but rather its C-terminal portion.The above results demonstrate that du PIAS2 is a repressor of IFN-β production induced by duck IRF7.PIAS2 has been implicated in many cellular processes and can also regulate viral replication in mammals.However,the role of du PIAS2 in HPAIV H5N1 replication in ducks is still unclear.Through liquid chromatography-tandem mass spectrometry(LC-MS/MS)assay,we identified that du PIAS2 was one protein that interacted with the NP of DK212.Through confocal microscopy images and Co-IP assay,we confirmed NP could interact with du PIAS2.Overexpression of du PIAS2 in DEF cells was shown to promote DK212 replication,and knockdown of du PIAS2 could repress DK212 replication.We further found du PIAS2 could promote NP SUMOylation through du SUMO1,and the potential SUMOylation sites of NP were at lysines 7,48,and 87.Furthermore,du PIAS2 promoted the replication of DK212,here relying on the activity of its SUMO E3 ligase.du SENP1,a de SUMOylation enzyme,could repress NP SUMOylation and also inhibit DK212 replication.Together,we identified du PIAS2 could interact with NP,and that du PIAS2 promoted H5N1 HPAIV replication,which might be related to NP SUMOylation.In conclusion,during AIV infection,du PIAS2 could interact with IRF7 to negative regulate the activation of IFN-β production mediated by duck RIG-Ⅰ.And its inhibitory function does not rely on its SUMO E3 ligase activity but its C-terminal portion.In addition,du PIAS2 can interact with NP and promote NP SUMOylation.It could also promote the replication of H5N1 AIV,and this promotion function depends on the activity of its SUMO E3 ligase. |
