Function And Molecular Mechanism Of Endogenous Retrovirus ALVE1 In Innate Immunity

DNA methylation inhibitor 5-Aza-2'-Deoxycytidine(5-Aza-dC),one of the first epigenetic modified drugs approved by the FDA in USA,which has been used for the treatment of human acute myeloid leukemia.By suppressing the activity of DNA methyltransferase(DNMT),5-Aza-dC leads to decreased DNA methylation of the tumor suppressor gene,which is considered to be a broad-spectrum anti-tumor drug.Recent studies have shown that endogenous retroviruses that were reactivated by 5-Aza-dC that was used for treatment of colon and ovarian cancer cells,which can induce type I interferon response.Avian leukemia,a chronic infectious tumor disease caused by avian leukosis virus(ALV),which is extremely harmful to the poultry industry.The prevention of avian leukemia has always been a worldwide problem that plagues the poultry industry.Our results revealed that DNA methylation inhibitor 5-Aza-dC functions in a manner similar to anti-tumor in humans,when it was used for anti-ALVJ.Since ERV is an important source of long non-coding RNA in vivo due to mutations and deletions,therefore,whether 5-Aza-dC exert anti-tumor and anti-viral functions by long non-coding RNAs derived from ERV?Based on the above scientific questions,the study was carried out as follows:(1)DNA methylation inhibitor 5-Aza-dC play a key role in inhibiting avian leukosis virus ALVJ.To test the antiviral function of 5-Aza-dC,using a virus-free,chicken embryo fibroblast(CEF)cell infected with ALVJ for 24 h as the virus infected model,we treated the ALVJ-infected CEFs with a 5 ?M of 5-Aza-dC for 96 h.The results showed that treatment of ALVJ-infected CEFs with 5 ?M of 5-Aza-dC had no effect on the proliferation and activity of CEFs,but which significantly inhibited the envelope protein secreted by ALVJ,which means 5-Aza-dC inhibited the proliferation of ALVJ.And we also further confirmed the conclusion that treatment of ALVJ-infected CEFs with 5-Aza-dC significantly inhibited the proliferation of ALVJ in CEF by confocal immunofluorescence technology.To further reveal the underlying molecular mechanism of antivirus of 5-Aza-dC,we employed the high-throughput RNA sequencing(RNA-seq)to analyze and identify the lncRNA derived from chicken endogenous retroviruses(ERV-lncRNA).Transcriptome sequencing revealed that 5-Aza-dC not only activates the expression of innate immune-related genes,but also induces the abnormal expression of IncRNA in 5-Aza-dC treatment group.Moreover,sequence alignment analysis revealed that a total of 59 of the 405 differentially expressed lncRNAs were derived from chicken endogenous retroviruses.We also found that 51 of differentially expressed lncRNA derived from CERV were significantly up-regulated,but only 8 of which were significantly down-regulated,which suggests that the molecular mechanism by which 5-Aza-dC inhibits the proliferation of ALVJ may be not only related to its activated innate immune-related genes,but also related to abnormally activated ERV-lncRNA.(2)Identification of long noncoding RNA derived from chicken endogenous retroviruses.To test our hypothesis that IncRNAs derived from endogenous retrovirus play a role in reactivation of imiate immunity response,a most upregulated ERV-lncRNA ALDBGALT0000000876 after treatment of 5-Aza-dC in CEFs was chose to study its function on reactivation of innate immunity response.It not only was more highly expressed after treatment of 5-Aza-dC in CEFs,but also derived from the chicken endogenous retrovirus ALVEI that has high homology with ALVJ,which is why we chose ALDBGALT0000000876 as the research object.Therefore,we identified the full length of ALDBGALT0000000876 firstly,and two-end sequences of ALDBGALT0000000876 were amplified by 3' and 5' RACE techniques,separately following amplification of the full length of the transcript ALDBGALT0000000876 by PCR.Finally,we get the full-length sequence with a length of 2136 nt.Since ALDBGALT0000000876 is derived from the antisense transcript of ALVEI,we named the new full-length sequence as lnc-ALVE1-AS1.Next,we hybridized lnc-ALVE1-AS1 with a specific probe labeled with digoxigenin,and the results showed that lnc-ALVE1-AS1 was present as full length isoform.Furthermore,we analyzed the protein coding potential and secondary structure by bioinformatics.The results showed that it does not have the ability to encode proteins.And its secondary structure is complex,which means it may play important biological role in CEFs.A HA fusion protein system covering three different coding potential of lnc-ALVE1-AS1 was designed to further confirm the protein coding potential of lnc-ALVE1-AS1.Western Blot analysis showed that lnc-ALVE1-AS1 does not have the ability to translate proteins,which indicates that lnc-ALVE1-AS 1 is a long non-coding RNA.In addition,we determined the subcellular localization of lnc-ALVE1-AS1 by RNA FISH.The results showed that lnc-ALVE1-AS1 localizes to the cytoplasm and nucleus,and is mainly distributed in the cytoplasm.(3)Long noncoding RNA lnc-ALVE1-AS1 exert innate antiviral immune functions in CEF.To clarify the biological function of lnc-ALVE1-AS1,especially for antiviral innate immunity,we analyzed the expression of innate immune-related genes after gain or loss of lnc-ALVE1-AS1 function.Gain of function was achieved by overexpression of lnc-ALVE1-AS1 via the pcDNA3.1 expression vector and two positive controls including transfection of GFP and antisense of Inc-ALVEI-AS 1 were designed.The results showed that both IRF7 and STAT1,which are related to the activation of type I interferon,were significantly up-regulated in CEFs transfected with lnc-ALVE1-AS1 compared with GFP and antisense of lnc-ALVE1-AS1,indicating that lnc-ALVE1-AS1 may be play a vital role in inducing type I interferon.At the same time,we also found that the expression of IFN-? was significantly up-regulated by nearly 4-fold in the group of transfection with lnc-ALVE1-AS1 compared with the control group.In conclusion,overexpression of lnc-ALVE1-AS1 in CEFs resulted in activation of interferon signaling pathway,inducing type I interferon-IFN-?.In addition,six interferon stimulated genes(ISGs)including IFI27-L2,IFIT5,MX1,OASL,RASD2 and ISG12-2 in chicken cells six genes were selected for further evaluation of the role of lnc-ALVE1-AS1 in inducing type I interferon by qRT-PCR.We found that the expression of IFI27-L2,IFIT5,MX1,OASL,RASD2 and ISG12-2 were significantly up-regulated in the group of transfection with lnc-ALVE1-ASl compared with the control group,and the expression level of the interferon-stimulated gene RASD2 was up-regulated by more than 40 times than that of the control group.Loss of function was achieved by interfere with the expression of lnc-ALVE1-AS1 via RNAi.The results showed that the expression of transcription factors IRF7 and STAT1 were also decreased and the expression of interferon-? was significantly decreased after knockdown of lnc-ALVE1-AS1 expression;At the same time,the expression of ISGs were significantly lower in the group of knockdown of lnc-ALVE1-AS1 than that of the RNAi control group.In conclusion,Gain and loss of function of lnc-ALVE1-AS1 affect the expression of type I interferon-related transcription factors in CEFs,which in turn affects the expression of ISGs,and lnc-ALVEI-AS1 appears to play an important role in activation of type I interferon.To verify whether lnc-ALVE1-AS also inhibit ALVJ in a viral infected model of CEFs infected with ALVJ,an enzyme-linked imunosorbent assay(ELISA)was conducted to evaluate the proliferation of ALVJ in ALVJ-infected CEFs after overexpression of lnc-ALVE1-AS1.We found that p27 protein secreted by ALVJ was significantly decreased after overexpression of lnc-ALVE1-AS1 in ALVJ-infected CEFs compared with GFP and its antisense sequence;following ALVJ virus titer in supernatant was significantly decreased after overexpression of lnc-ALVE1-AS1 compared with the control group.Then,the point that lnc-ALVE1-AS1 can significantly inhibited the proliferation of exogenous avian leukosis virus ALVJ was confirmed by indirect immunofluorescence assay.In conclusion,lnc-ALVE1-AS1,which is derived from chicken endogenous retrovirus,has the ability to inhibit its highly homologous exogenous retrovirus ALVJ.The results also indirectly reveal that lnc-ALVE1-AS1 that derived from chicken endogenous retrovirus may play an important role in the inhibition of exogenous retrovirus ALVJ by 5-Aza-dC.(4)The molecular mechanism by which long noncoding RNA Inc-ALVE1-AS1 induces type I interferon and exerts innate antiviral immunity.To elucidate how lnc-ALVE1-AS1 induces type I-interferon response,we further investigated the effect on the expression of cytosolic nucleic acids sensor by lnc-ALVE1-AS1 in CEFs.Our results indicate that overexpression of Inc-ALVE1-AS1 significantly induced the expression of long dsDNA sensor IFIH1 and short dsDNA sensor TLR3,and the expression of IFIH1,TLR3 and ds DNA sensor MB21D1 were significantly downregulated following knockdown the expression of lnc-ALVE1-AS1 by RNAi.Thus,it helps us to infer molecular mechanisms by which overexpression of lnc-ALVE1-AS1 induced an innate immune response may be related to cytosolic nucleic acids sensors including dsRNA sensors TLR3 and IFIH1.Further,we detected the lnc-ALVE1-AS1 binding protein by RNA pulldown.Mass spectrometry analysis indicated that TLR3 specifically binds or recognizes lnc-ALVE1-AS1 in vitro.To demonstrate that TLR3 also recognizes lnc-ALVE1-AS1 in vivo,immunofluorescence and RNA FISH were performed.And we observed the co-localization of TLR3 and lnc-ALVE1-AS1 in CEFs by confocal microscope.Thus,the dsRNA nucleic acid sensor of TLR3 was a binding protein of Inc-ALVE1-AS1,which induces the innate immune response in CEFs.Based on the above results,it came to the conclusion that lnc-ALVE1-AS1 derived from endogenous retrovirus ALVE1 can effectively inhibit the proliferation of exogenous retrovirus-ALVJ,indicating that endogenous retrovirus ALVE1 is not a "Junk DNA" or "transcriptional noise",but functions in important biological,especially in innate immunity,in CEFs.Therefore,the function of innate immunity of ALVE1 may be achieved by employing its long noncoding RNA lnc-ALVE1-AS1.This study will help to understand the antiviral innate immunity and its mechanisms in animals,and also provide new ideas for more effective prevention of ALVJ infection in chicken.