Preliminary Study On Immunosuppressive Mechanism Of Capsid Protein P27 Of Avian Leukosis Virus

The Avian leukosis virus(ALV)has caused enormous economic losses in the global po?ltry industry since it was first reported.Chicken infected with ALV were characterized with reduced egg production,secondary and mixed infections,increased death and c?lling rate,immune suppression,and many types of tumors.However,no vaccine against ALV was available so far.Since the p27 protein(ALV-p27),group-specific capsid protein with high content in the virus,has many easily detectable antigenic sites,it was often used as detection antigen in ELISA and IFA.Many researches have shown that p27 influences the expression of several cytokines,suggesting that ALV-p27 may play a critical role in the immunosuppression caused by viral infection.In this study,the full-length and truncated p27 was cloned into pcDNA3.1 for the preliminary study on the immunosuppressive mechanism of p27 protein in DF1 cells.1.ALV-p27 inhibits stimulation of LPS and Poly(I:C)in DF-1 cellsIn order to study the immunosuppression of overexpressed ALV-p27 in DF-1 cells after LPS and Poly(I:C)stimulation,the f?ll-length p27 was truncated into two segments and cloned into pcDNA3.1,which were named as pcDNA3.1-p27A(1-480 bp)and pcDNA3.1-p27B(241-720 bp).Then DF1 cells were transfected with pcDNA3.1-p27 followed by treatment with LPS and Poly(I:C),and RNA was extracted at 1h,6h,12h and 24h respectively.After that,Real-time PCR was carried out to detect the expression of cytokines such as IL-1?,IL-6,IFN-P etc.The res?lts showed that overexpression of p27 significantly inhibited the expression of cytokines in DF1 cells in contrast to negative control.At the same time,DF1 cells transfected with the truncated p27,namely pcDNA3.1-p27B,showed the same res?lts,which means the immunosuppression domain of p27 is located within 480-720bp.As far as concerned,the results above showed that the ALV-p27 protein may induce immunosuppression through inhibiting the expression of cytokines,and the immunosuppression domain is located within 480-720bp,that is C terminal of p27.2.Preliminary study on the mechanism of ALV-p27 for inhibiting the immune response in DF-1 cellsIn order to further investigate the mechanism of ALV-p27 immunosuppression in DF-1 cells,gene expression of some key molecules in LPS and Poly(I:C)-related signaling pathways was detected by Real-time PCR.The results showed that the expression of IRAK4 and IRF-7 was significantly inhibited after LPS treatment,however,the expression of common molecules did not change after Poly(I:C)treatment.Further experiment of dual-luciferase reporter assays showed that p27 protein may inhibit the expression of common molecules in Poly(I:C)-related signaling pathways by inhibiting the initiation of IFN-? expression after Poly(I:C)treatment;and may inhibit the expression of IRF-7 promoter after LPS treatment.Besides,the results of Western blot for detection of phosphorylated proteins in MAPK-associated signaling pathways revealed that the p27 may induce immunosuppression through decreasing the phosphorylation of p38.In conclusion,the ALV-p27 protein may induce immunosuppression through inhibiting the expression of relative genes in certain signaling pathways,inhibiting the initiation of cytokines expression and interfering the phosphorylation of key proteins in signaling pathways.