Effect Of P53 On Antiviral Actions Of Recombinant Columba Livia Interferon-?

Interferon-alpha(IFN-a)is first line of cellular innate defense against viral infection.IFN-a has a wide variety of effects on various cell types and functions.Recently,the studies of animal interferon focused on economic animals,and have made great progress,like chickens,dogs,pigs recombinant interferon product were applied on the market.However,information regarding antiviral response and the specific molecular mechanism of Columba livia interferon-a(CoIFN-a)has not been reported to date.During the last decades,pigeon populations have increased exponentially in different countries,reaching densities higher than 2000 birds/km2 in many European cites.Because most virus replicates in the respiratory and intestinal tracts of pigeon,infection typically follows the fecal-oral or aerosol route of transmission.To date,the use of vaccines to control some diseases has not been successful.This is at least partially due to the immunological tolerance of pigeons,the high virus mutation rate and host factors involved in viral infection.Interest in IFNs of birds has recently sparked from increasing problems with viral diseases in the poultry industry.Therefore,definite characterization and signaling pathway of Columba livia IFN-a(CoIFN-a)can bring a large economic interest.In this study,we cloned a 723bp complete ORF of Columba livia interferon-a(CoIFN-a)gene,which encoding 240 amino acids,sharing less than 40%identity with mammalian IFN-a and 54 to 80%identity with avian IFN-a,has a closer relationship with the parrot(sharing 79.49%identity with the Grus japonensis).The CoIFN-a gene encoding the mature peptide was cloned into the expression vector pET32a(+)to construct the recombinant expression plasmid pET32a-IFN-a,which was transformed into E.coli BL21 to express the recombinant protein.The results of SDS-PAGE showed that the protein was mainly expressed in inclusion bodies,approximately 43KDa.The specific antiviral activity of CoIFN-a in VSV(TCID50 =10-5.87/100?L)-infected CEFs reached 5.5 × 105U/mg.CoIFN-a also showed high sensitivity to trypsin and relatively stable after acid,alkali or heat treatment.Moreover,CoIFN-a activated STAT/Jak signaling and PI3K-AKT-mTORC pathway to inhibit VSV-induced apoptosis.Although the expression of p53 was further increased,apoptosis was not happened under CoIFN-a against VSV.Notably,although STAT signaling was efficiently activated,knockdown p53 dose inhibit the antiviral activity of the CoIFN-a via decreasing the expression of Mx1 but not weakened Jak phosphorylation.Moreover,VSV.aggravated the apoptosis and the expression of cleaved Mdm2 in knockdown p53 under preincubated CoIFN-a.Taken together,p53 might as a highly interconnected regulator in IFN-a antiviral response and cleaved Mdm2 might as a dominant-negative regulator by competing with full length Mdm2 for p53 binding in virus infection.Overall,our research not only enriches CoIFN-? antiviral features but also helps explain that p53 enhance the CoIFN-a antiviral response against pigeon viral diseases.Through this study,we cloned the CoIFN-a gene,expressed the CoIFN-a protein in Escherichia coli.Moreover,CoIFN-a exerted only more VSV than chicken IFN-a We focused on the response of primary fibroblasts to autophagy and apoptosis to best understand how p53 behaves in IFN-a.Our data presented in this study illustrated that(a)CoIFN-a project protective effects on virus-induced apoptosis,(b)CoIFN-a triggered autophagy via PI3K/AKT/mTORC pathway(c)knockdown p53 projected suppressed effects on CoIFN-a-induced antiviral response(d)Mdm2 might positive effects on the protection of CoIFN-a under virus exposure to targeting p53 for degradation.