Role Of Chicken 14-3-3σ On Acute Fibrous Tissue Hyperplasia Caused By Subgroup J Avian Leukosis Virus Strain

Avian leukosis virus(ALV)is a tumorigenic retrovirus,divided into 11 subtypes A-K,of which subgroup J avian leukosis virus(ALV-J)has the highest infection rate,the strongest pathogenicity and the most serious damage among all subgroups of ALV,causing severe immunosuppression and multi-tissue tumorigenesis in chickens after infection.In recent years,an acute fibrosarcoma-causing ALV-J strain(ALV-J-SD1005)carrying the v-fps tumor gene was found in local chickens in Shandong,which could induce fibrosarcoma visible to the naked eye in infected chickens at about 10 d.The chickens died at 21 d due to tumor overgrowth,and its tumorigenic mechanism is still unclear.14-3-3σ protein is a cell cycle inhibitor that negatively regulates cell cycle progression;it was found that 14-3-3σ is dysregulated or absent during the development of various tumors and loses its inhibitory effect on tumor cell proliferation,while its role in acute fibrous tissue proliferation and cell proliferation caused by ALV-J has not been reported.In this study,we investigated the role of chicken 14-3-3σ in the acute fibrosarcoma-causing process of ALV-J strain using animal and cellular assays to provide a new scientific basis for analyzing the tumorigenic mechanism of ALV-J.In this study,we first designed primers to amplify the chicken 14-3-3σ gene based on the published chicken 14-3-3σ gene sequence and performed sequence analysis,and constructed a recombinant prokaryotic expression vector to prepare recombinant protein,and immunized rabbits to prepare polyclonal antibodies to detect the expression of chicken 14-3-3σ.Secondly,the ALV-J-SD1005 strain was subcutaneously injected into infected chicks at the neck to analyze the viral replication changes,fibrous tissue proliferation lesions and 14-3-3σexpression changes in different tissues of infected chickens.Third,the ALV-J-SD1005 strain infected DF-1 cells were analyzed for changes in viral replication,cell proliferation and14-3-3σ expression.Fourth,chicken 14-3-3σ overexpression plasmids with small interfering RNA were prepared and transfected into ALV-J-SD1005-infected DF-1 cells,respectively,to analyze the effects of 14-3-3σ on cell proliferation,cell cycle,and cell cycle regulators(p53,CDC2,and CDK2).The results showed that the chicken 14-3-3σ gene with a size of 741 bp was amplified,with 79% homology to human;the prepared polyclonal antibody could specifically bind the14-3-3σ protein and induced expression of the protein in chicken tissue cells.The virus replicated rapidly in chicks infected with strain ALV-J-SD1005;the incidence of subcutaneous fibrosarcoma in the neck was 100% at 12 d;the sarcoma index increased with increasing age of infection and could reach 40.4% at 21 d;14-3-3σ expression decreased significantly.DF-1cells infected with the virus showed a significant increase in cell proliferation and a significant decrease in 14-3-3σ protein expression.The results of cell transfection assay showed that chicken 14-3-3σ overexpression significantly decreased the cell proliferation rate and S-phase cell ratio,but increased the G2/M-phase cell ratio in ALV-J-SD1005-infected DF-1 cells;on the contrary,chicken 14-3-3σ knock-down expression significantly increased the cell proliferation rate and S-phase cell ratio,but decreased the G2/M-phase cell ratio in ALV-J-SD1005-infected DF-1 cells.Also,chicken 14-3-3σ overexpression significantly downregulated the expression of CDK2 and CDC2 and upregulated the expression of p53 in DF-1 cells;knockdown expression significantly increased the expression of CDK2 and CDC2 and decreased the expression of p53 in DF-1 cells.The above results show that the successful amplification of chicken 14-3-3σ gene and the preparation of polyclonal antibodies to detect its expression provide the necessary material for functional studies;chicken 14-3-3σ protein can regulate the expression of CDK2,CDC2 and p53 in cells causing G2/M phase cell block and inhibiting cell proliferation;ALV-J-SD1005 strain can release the G2/M phase cell block and promote cell proliferation by inhibiting the expression of 14-3-3σ protein.