Characterization Of Chicken APOBEC4 And Its Antiviral Effect On Newcastle Disease Virus

The apolipoprotein B mRNA editing enzyme family play an important role in both innate and adaptive immunity.Due to the conserved cytidine deaminase motif,APOBEC proteins can induce a wide range of cytosine deamination mutations and participate in immune responese.APOBEC4 is a newly discovered member of this family,and very few studies have been reported on the function of APOBEC4 protein.In order to characterize chicken APOBEC4,we conducted experiments as follow:A prokaryotic expression vector of APOBEC4 gene was constructed,and then,a large amount of APOBEC4 protein was induced to high expression levels in E.coli with IPTG.Then purified chicken APOBEC4 protein was used as an antigen to immunize mice for polyclonal antibodies.It laid the foundation for the following study of the distribution and expression of APOBEC4 protein in chicken.According to the phylogenetic analysis,chicken APOBEC4 was found to be highly homologous to mammals and reptiles APOBEC4,along with a certain genetic distance from fish APOBEC4.In this study,the expression of APOBEC4 in chicken cell lines and the endogenous APOBEC4 distribution in chickens were detected by absolute Real time Quantitative PCR.It was found that except the peripheral blood lymphocytes,the background expression of APOBEC4 was not high in the other cell lines;in vivo,APOBEC4 is highly expressed in the Bursa.Based on the structure of human APOBEC3,3D structures of chicken APOBEC4 and human APOBEC4 catalytic domains were established.The structural similarity indicates that chicken APOBEC4,human APOBEC4 and human APOBEC3G contain the same Zn²⁺coordination motif,carry the same folding factor and may have similar catalytic activities.During the long evolution process,APOBEC4 remains highly conserved in sequence and structure in its ZDD domain,which indicates that it is functional and indispensable for organisms.The high expression of APOBEC4 in vitro has an inhibitory effect on the NDV replication.This antiviral effect is dose dependent and have nothing to do with RNA editing function.By co-IP and IFA identification,the antiviral effect of APOBEC4 may be played by binding with M protein of NDV.At the same time,the overexpression of APOBEC4 protein in chickens could induce caspase-3 activation,suggesting that APOBEC4 in chickens may play a part of antiviral effect by inducing apoptosis.After viral infection,APOBEC4 expression increased significantly in lymphocytes and thymus,similar to that of homolog AID,another member of the APOBEC family.Cytokine tests on virus-infected cells revealed significantly elevated IFN-?mRNA levels.Moreover,when different drugs were used to stimulate cells,T cell activated stimulators could significantly increase the expression of APOBEC4 in cells,indicating that the high expression of APOBEC4 in chickens after viral infection might be related to cellular immunity.While overexpression of APOBEC4 protein in MSB1 cells could not cause the change of IFN-?mRNA expression level,the expression level of APOBEC4 mRNA was significantly increased when treated with IFN-?,indicating that APOBEC4 might be the downstream effector of IFN-?.To sum up,APOBEC4 is a host limiting factor that not only plays an antiviral role which is not RNA editing dependent,but also inhibits viral replication by regulating adaptive immunity.These results indicated that APOBEC4 played an important role in both innate and adaptive immune responses in chickens.