| Endogenous retrovirus(ERV)is a genomic component and its biological function is being discovered.Recent studies have shown that the expression of endogenous retroviruses in mammals is regulated by epigenetic and nutritional factors,and ERV can involve in innate immune response by regulating the expression of innate immune related genes.However,the study of avian ERV is still very limited.For instance,it is unknown whether the expression of ERV is regulated by epigenetic and nutritional factors in poultry.and whether nutritional factors can affect natural immune response by regulating ERV expression.This study aimed to address these questions in depth,so as to elucidate the effects of nutritional factors on the expression of chicken ERV and the related mechanisms.The main research methods and results are shown as follows:(1)Analysis of sequences and expression of ALVEIThe PCR with gene specific primers was performed to amplify the full length of ALVE1 using genomic DNA templates isolated from HD 11 cells,a macrophage cell line.The sequencing result showed that ALVE1 sequence of HD 11 cells was 99%homologous to the reference sequence of ALVE1 deposited in NCBI database,with nucleic acid polymorphism at multiple sites.Quantitative PCR was also used to determine the expression of ALVEI in both CEF and HD11 cell lines,as well as the expression of ALVEI env gene in the brain,bursa,thymus,spleen,heart,liver,kidneys,lungs,and skin of 2-day-old and 35-day-old SPF chickens.The results indicated that the expression of env gene in immune cell line(HD 11)was significantly higher than that of non-immune cell line(CEF)(P<0.05),the expression of ALVE1 in the spleen(an immune organ)of 2-day-old SPF chickens was significantly higher than that in other tissues(P<0.05),and the env expression in the immune organs(bursa and spleen)of 2-day-old SPF chickens was significantly higher than that in 35-day-old chickens(P<0.05).These results suggest that ALVEI may be involved in early development of immune organs and innate immune response.(2)Effect of nutritional factors on ALVE1 expressionThe HD 11 cells were treated with methionine and vitamin C,respectively,qPCR assays with these cells showed that treatment of methionine activated the expression of ALVEI LTR and significantly increased the expression of innate immune related genes(P<0.05).However,the treatment of Vitamin C had no significant effect on the expression of ALVE1(P>0.05).(3)Analysis of DNA methylation of ALVE1Bioinformatics predicted that there were two CpG methylation islands in the ALVEl gene,and miR-155 targeted to ALVE1 env transcripts.The bisulfite-pyrosequencing analysis demonstrated that the DNA methylation level of ALVEI CpG Island was decreased in the HD11 cells treated with DNA methyltransferase inhibitor AZA compared to the control cells only treated with DMSO.The treatment of AZA also activated the expression of ALVE1 and innate immune related genes.Through overlap PCR and luciferase reporter assay,it was verified that miR-155 indeed targeted to env transcripts.Together,the results showed that the expression of ALVE1 was under epigenetic control,and activation of ALVE1 could play a role in innate immunity of chicken cells.Additionally,however,overexpression of ALVE1 env gene in HD 11 cells did not significantly increase the expression of innate immune related genes.The reason remains to investigate in the future.In conclusion,the env gene of chicken endogenous retrovirus ALVE1 was highly expressed at early life stage.The expression of ALVEI was regulated by nutritient methionine,miR-155 and DNA methylation.Methionine could induce the expression of innate immune related genes by activating ALVE1 and thus play a role in animal immune response. |
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