Chicken Endogenous Retrovirus Mediates The Innate Immunity Of Macrophages Against ALV-J Via Marco

According to recent studies some endogenous retroviruses can not only participate in regulating early embryonic development and the stemness of embryonic stem cells,but also may be related to the effects of antiviral and antitumor innate immunity.In order to lay the foundation for it as a standard for screening avian leukemia-resistant chickens in the future,in our research group,the entire sequence of the chicken endogenous retrovirus ALVE1 has been identified.Then the CRISPR/Cas9 system was used to insert a SV40 polyA termination sequence in the LTR region of the ALVE1 promoter for terminating the expression of ALVE1 in chicken macrophages HD11 cells.Still,the relationship between ALVE1 and innate antiviral immunity has not been studied in depthIn first part of the current study,we utilized RNA-seq and bioinformatics analysis methods to identify differentially expressed mRNA and related signal pathways in HD11 cells that had had potential to terminated the expression of chicken endogenous retrovirus ALVE1.Later,the differentially expressed gene Marco related to inflammation was screened out,and investigated whether there is a positive or negative regulatory relationship between env and Marco at the mRNA level.Finally,it was further investigated whether Marco can resist viruses and preliminarily explore whether the chicken endogenous retrovirus env sequence can mediate the innate antiviral immunity of macrophages through the Marco gene.The main research methods and results are as follows:1)Effect of ALVE1 knockout on the gene expression profile of macrophagesFirst,to study the effect of ALVE1 knockout on chicken macrophages gene expression profile,high-throughput RNA-sequencing was performed in HD11ALVE1-/-cells and HD11ALVE1+/+ cells.The results showed that there were a total of 1537 differentially expressed genes(DEG);GO analysis revealed that DEGs were mainly enriched in GO categories such as immune system process,cell development process,catalytic activity and signal transduction activity.The KEGG analysis results indicated that DEGs were mainly enriched in cytokines-Cytokine receptor interaction,transcription disorders in cancer,IL-17 signaling pathway,rheumatoid arthritis and other pathways.On the other hand,GSEA enrichment analysis revealed that Marco may be a potential key gene involved in the process of inflammation caused by chicken endogenous retrovirus ALVE1.2)Positive regulatory relationship between env and Marco.To explore whether there is a positive or negative regulatory relationship between env and Marco,we constructed pcDNA3.1-env1-ALVE 1,pcDNA3.1-env2-ALVE1 and pcDNA3.1-Marco eukaryotic overexpression vetors and pCDH-env1-ALVE1 and pCDH-env2-ALVE1 lenti viral backbone vetors.The pLV-env1-ALVE 1 and pLV-env2-ALVE1 lentiviruses were packaged using HEK-293T cells and siRNA for env was designed and synthesized by the company.CRISPR/Cas9 system was applied to site-directed mutation of the first exon of the Marco gene.And the HD11 cell line without Marco expression was successfully obtained.The intriguing results showed the env overexpression can significantly up-regulate the transcription level of Marco(p<0.05),and env interference can down-regulate the transcription level of Marco(p<0.05)substantially.The Marco overexpression can significantly up-regulate the transcription level of env(p<0.01),Marco knockout can significantly down-regulate the transcription level of env(p<0.01),indicating that there is a positive control between env and Marco.3)env mediates the innate antiviral immunity of macrophages through the Marco gene.To investigate whether env mediates the innate antiviral immunity of HD 11 cells through the Marco gene.The results also showed that the ALV-J env gene’s over-expression would significantly inhibit the Marco gene’s transcription level(p<0.01).When infecting with ALV-J GY3 strain after Marco overexpression,the Marco gene can significantly inhibited the replication of ALV-J in the process of transcription and translation,respectively(p<0.01).The overexpression of Marco can not only significantly up-regulated the mRNA transcription levels of viral dsRNA recognition receptor genes Tlr3 and Ifih1(p<0.01),but also significantly up-regulated the mRNA transcription levels of innate antiviral immune genes Il-1β and inos(p<0.01),indicating that Marco may achieve the antiviral effect by activating the expression of innate immune genes.In summary,this study found a positive regulatory circuit between env and Marco,and that env mediates the innate antiviral immunity of HD11 cells through the Marco gene.These results shed new light on the innate immune mechanism activated by endogenous chicken leukosis virus and lay a foundation for further study on the function of the Marco gene and the treatment of chicken leukosis in the future.