Duck Enteritis Virus VP16 Antagonizes IFN-? Mediated Antiviral Immunity

Duck enteritis virus(DEV)tegument protein VP16 encoded by the UL48 gene locatingin the long unique region(UL)of the viral genome and is an important and highly functionally protein of DEV.In this study,VP16 and its truncated mutants are expressed in duck embryo fibroblast(DEF)cells,the abilityof VP16 in antiviral innate immunity was studied,following results obtained:1.VP16 inhibited the mRNA transcriptional level and activation of IFN-?in DEFs Duck embryo fibroblast cells were inoculated with 1MOI duck enteritis virus.RT-qPCR were used to detectthe transcription level of duck IFN-?mRNA at different time points after infection.Results showed that duck IFN-?mRNA transcription level was significantly down-regulated at 24h after infection,it is 85%lower than that at 12h.Transfecting VP16expressing plasmids in DEF cells,DEV VP16 protein was functionally expressed,we found that it can dose-dependently inhibit IFN-?mRNA transcription.The recombinant expression plasmids which express VP16 protein was transfected in DEF cells at amount of10?g/9.6 cm~2,the inhibitory effect of IFN-?mRNA transcription level was increased by about 3 times compared with the group that transfected 4?g/9.6 cm~2cells.Using dual-luciferase reporter system to detect the effect of VP16 on the promoter activity of IFN-?in DEF cells.Results showed that in the experimental group expressing VP16 protein,the IFN-?promoter activity which up-regulated by poly I:C was down-regulated by 80%,which was significantly different from the control group(p<0.01).Overexpression of cGAS,STING and IRF7 can obviously induce the IFN-?promoter activity in DEF.Infection with DEV or transient expression of VP16 can significantly inhibit the activationof IFN-?promoter that is induced by cGAS,STING and IRF7,meanwhile p CAGGS did not exhibit inhibitory ability by comparing with the control group,the difference was extremely significant(p<0.001 or p<0.005).2.Screening of the key site where VP16 protein utilized to dampen IFN-?activation.VP16 protein can not only inhibit poly I:C induced Mx and OASL mRNA transcription,but also exerted the same function to poly d A:d T induced ISGs transcription.The transcription level of Mx protein mRNA induced by poly I:C down-regulated by VP16 was statistically different,P value<0.05.The transcription level of OASL protein mRNA induced by poly I:C down-regulated by VP16 had significantlystatistical difference,P value<0.01.VP16 down-regulated poly d A:d T-induced Mx protein and OASL protein mRNA transcription levels,both of which had extremely significant statistical differences,P value<0.001.The results being that VP16 protein acted on the common node of innateimmune pathway recognized by DNA virus andRNA virus.The over-expression of heterogeneous DNA sensor-cGAS stimulated the down-stream immunologic pathway.Overexpression of cGAS/STING,TBK-1,IRF7 or IRF1 protein can activate the promoter activity of IFN-?,but when VP16 protein was expressed as well,the activity of IFN-?promoter activated by cGAS/STING,TBK-1 and IRF7 were all down-regulated,and the level of inhibition was significantly or significantly different compared with the control group(P<0.005 or P<0.001).However,VP16 protein cannot inhibit the activity of IFN-?promoter activated by IRF1,and the activity of IFN-?promoter is only reduced by less than 15%.This indicated that the VP16 protein could act at IRF7 level which is downstream of TBK1.3.Preliminary study on the mechanism of VP16 protein acting on IRF7 site.Using the eukaryotic expression cell line HEK293T to co-transfect and express VP16protein and IRF7 protein.Immunofluorescence colocalization and immunoprecipitation experiments were carried out in HEK293T cells.As a result,VP16 and IRF7 could co-colocalized with each other in the cytoplasm,whatsmore,VP16 combined with IRF7 using co-IP method,indicating that VP16 associated with IRF7 in vitro.Further study focused on the different truncated VP16 proteins affecting IFN-?promoter activity in DEF,the results showed that VP16(aa1-200)lacking most of C-terminal down-regulated 70%more poly I:C-induced IFN-?promoter activity than that caused by VP16(aa110-475)which lacks most of the N-terminal.the difference between these two groups was significant(p<0.05).In summary,DEV owns the immune-evasion ability to escape host innate immunity,especially exerts their function on the cGAS recognized pathway.VP16 is an important immune-modulatory protein who helps DEV to be free of elimination from immune system.VP16affected interferon regulatory factor 7 by binding with it and utilizing N-terminal region to inhibit interferon beta-mediated antiviral immunity.