Grass Carp (Ctenopharyngodon Idellus) PIAS1 Negatively Regulates IFN1 Expression By Inhabiting The Phosphorylation Of STAT1

Innate immunity,as an important line of defense,plays a crucial role in antiviral response.Negative regulators which are the key regulators in immune signaling pathways have significant biological functions in a variety of signaling pathways to maintain immune balance of the body.IFN is a vital member of the innate immune system and plays a crucial role in both innate and acquired immunity.Among them,PIAS1 protein family,as one of the typical negative regulators,also participates in the innate immune response of JAK/STAT signal pathway and virus-induced typeⅠIFN signal pathway.Although the specific inhibitory mechanism of PIAS as a negative regulator has been well studied in mammals,its mechanism in fish innate immunity has not been clearly studied so far.In this study,we explored for the first time how grass carp PIAS1 regulated typeⅠIFN expression by participating in JAK/STAT signal pathway,and then responded to viruses,virus analogues,and bacteria stimulation.We obtained the gene sequence of grass carp PIAS1 by homologous cloning method,predicted grass carp PIAS1 domain using online structure prediction websites such as SMART,constructed its phylogenetic tree,and explored the evolutionary relation of PIAS1.The results showed that there are two isoforms of PIAS1 a and PIAS1 b in grass carp that are homologous to mammalian PIAS1.Their ORF sizes are 1953 bp and 1974 bp,respectively.Grass carp PIAS1 a,b have high homology with zebrafish PIAS1 a,b,respectively.They also have five conserved domains similar to mammals,including SAP,PINIT,RLD,AD and S/T enriched domains from N-terminal to C-terminal,respectively.The expression of PIAS1 m RNA in grass carp was detected by q-PCR at cell and individual level.After stimulation with GCRV(a ds RNA virus)or viral analogue Poly I:C and lipopolysaccharide LPS,the m RNA expression levels of grass carp PIAS1 in different tissues(brain,eyes,intestines,gills,skin,spleen,liver and kidney)and cultured cells during the gradient time(0h,6h,12 h,24h,48 h and 72h)were detected.We found that after the stimulation the m RNA expression level of PIAS1 a,b is higher than those in PBS control group,respectively.Subsequently,relevant eukaryotic expression vectors of grass carp PIAS1 were constructed to further explore how grass carp PIAS1 regulates innate immunity in cells.We first explored whether PIAS1 could interact with STAT1 in grass carp.The distribution of grass carp PIAS1 in CIK cells was investigated by immunofluorescence localization and cytoplasmic extraction methods.Meanwhile,we found that the grass carp of PIAS1 a,b are mainly distributed in the nucleus,while STAT1 was mainly distributed in the cytoplasm.Under the stimulation of Poly I:C,STAT1 gradually enters into the nucleus,and co-distribution with PIAS1 a,b.In addition,we also used Co-IP and WB experimental methods to find whether grass carp PIAS1 a,b could interact with STAT1 and regulates the phosphorylation of STAT1,respectively.Finally,in order to explore how grass carp PIAS1 regulates STAT1 and IFN1,we respectively overexpressed grass carp PIAS1 a,b,and found that they could inhibit the expression of IFN1.On the contrary,the expression of IFN1 is up-regulated after knocking down PIAS1 a,b.Moreover,regardless of overexpression or interference PIAS1 a,b do not affect the expression of STAT1,respectively,but could regulate STAT1 phosphorylation.In summary,grass carp PIAS1 a,b could respond to viral stimulation and interact with STAT1 to inhibit STAT1 phosphorylation,and finally they negatively regulates IFN1 expression through the JAK/STAT signaling pathway.