Molecular Characterization Of Env Protein Of J Subgroup Avian Leukosis Virus

Subgroup J avian leukosis virus (ALV-J) was first isolated in 1989 and identified as a new subgroup of avian leukosis virus. The env gene of ALV-J is very unique, has very low homology to other ALV subgroups, however, exhibits 97% identity to EAV-HP sequence, which is existed in the normal chicken's genome. ALV-J most causes myeloid leukosis, while other exogenous ALVs mainly induce lymphoid leukosis. The previous research data showed that env gene is closely related to the ALV oncogenicity, however, the function of the envlope protein is still unclear. This paper is to further investigate the uniqueness of ALV-J env gene and to explore the function of env gene in the ALV-J pathogenicity and oncogenicity.1 Development of Chicken Embryo Fibroblast Cell Line Expressing env Gene of Subgroup J Avian Leukosis VirusThe recombinant of pcDNA-env was constructed by inserting the env gene of ALV-J 4817 strain into the vector pcDNA3.1. Chicken embryo fibroblast cell line transfected with the recombinant pcDNA-env was selected for the resistant cells to Zeocin. The resistant cells were passed for 30 generations and frozen. After 13 months, these cells were refreshed and cultured in the medium free of Zeocin. After another 40 passages, env gene and Env protein in the cells were detected by PCR, Southern-blot, IFA and Western-blot. The results showed that the cells transfected with the recombinant, designated as pcDNA-env-DF1 cells, could stably express the env gene of ALV-J. And pcDNA-env-DF1 cells had the characterization of resistance to ALV-J with a dosage of 104 TCID50. The pcDNA-env-DF1 cells developed in this paper had a vital significance for further exploring the function of ALV-J env gene.2 Function of Envelope Protein of Subgroup J Avian Leukosis Virus Associating with PI3KThe observation in pcDNA-env-DFl cells expressing Env protein of ALV-J 4817 strain showed that the cells appeared large size and became round in morphology comparing with normal cell. The cells look like transformed. Analysis results with 3D-PSSM software showed that there is signal transduction in Env protein of 4817. The further analysis for signal transduction motif released that there is a YxxM motif in cytoplasmic tail of the protein, which could specifically activate PI3K signal pathway. And the changed morphology of the cells could be reversed to normal by inhibitor LY294002 specific to PI3K. These results proved the phenomenon of the cells was directly related to the PI3K and indicated the envelope protein had potential oncogenicity and could activate PI3K through YxxM motif to result in the phenomenon. Some experiments associated with the phenomenon are being undertaken.3 Classification and Characterization of Envelope Protein of Subgroup J Avian Leukosis VirusFurther analysis showed that not only YxxM motif, but also ITIM and ITAM-like were existed in cytoplasmic tails of different ALV-J envelope proteins. Based on significance of YxxM and ITAM motifs in conducting active signal to induce cell proliferation and tumorogenisis, significance of ITIM in conducting inhibitory signal to control or inhibit cell proliferation and tumorogenisis and the distribution of these motifs in ALV-J envelope proteins, ALV-J envelope proteins were classified as three types, designated as inhibitive envelope protein, bi-functional envelope protein and active envelope protein. According to this classification, 28 of 33 ALV-J envelope proteins (Download from Genbank) were bi-functional envelope protein, 3 ones were inhibitive envelope protein and only 4817 and UD3 strain's envelope proteins were active envelope proteins, while all EAV-HPs (Download from Genbank) were classified as inhibitive envelope protein. Based on mechanisms in signal transduction of these signal motifs, three models of signal transduction of the three types of envelope protein were figured out.Antigenic analysis of envelope suggests that the antigenicity of cytoplasmic tail of inhibitive, bi-functional and active envelope proteins be not only directly related to these motifs, but also decreas...