Abstract: 【Objective】 The aim of the experiment was to explore the role of chicken derived ring finger protein 165 (RNF165) in Avian leukosis virus subgroup A (ALV-A) replication,and provide reference for further research on the impact of chicken derived RNF165 on ALV-A pathogenicity.【Method】 ALV-A was inoculated into DF-1 cells and 1-day-old chicks.Western blotting and Real-time PCR were used to verify the effect of ALV-A on the expression of host RNF165 in vitro and in vivo.The expression primer was designed according to the published sequence of RNF165 gene.The RNF165 gene was amplified by PCR and cloned into the eukaryotic expression vector pcDNA3.1.The recombinant plasmid was transfected into DF-1 cells and inoculated with ALV-A one day later.The effect of RNF165 expression on virus replication was detected by Western blotting.Finally,the mutation primer was designed to construct the RNF165 functional domain mutation plasmid.The overexpression plasmid and the mutant plasmid were transfected into DF-1 cells respectively.One day later,they were inoculated with ALV-A.Western blotting was used to determine the difference of gp85 protein expression.【Result】 The endogenous RNF165 protein could not be detected after ALV-A infected DF-1 cells,but the gene transcription level was significantly lower than that of control group(P＜0.01).In vivo experiments,the gene and protein expression level of RNF165 in chicken liver was lower than that of control group(P＜0.01).The 1 068 bp target fragment was successfully amplified by PCR and cloned into pcDNA3.1.Western blotting results showed that the target band appeared at 39 ku,and the over-expression of RNF165 promoted virus replication.The sequencing results showed that the functional domain mutant plasmid was successfully constructed,and the replication level of the RNF165 mutant plasmid group was significantly inhibited compared with the overexpression plasmid group.【Conclusion】 The infection of ALV-A inhibited the expression of RNF165 in DF-1 cells and chicken liver.In vitro,overexpression of RNF165 would promote the replication of ALV-A,and this effect was related to its conservative domain.

Key words: ring finger protein 165; Avian leukosis virus subgroup A (ALV-A); virus replication; functional domain mutation