Porcine endogenous retroviruses (PERV) represent a major safety concern in pig-to-human xenotransplantation. To date, no PERV infection of a xenograft recipient has been recorded; however, PERVs are transmissible to human cells in vitro. Some recombinants of the A and C PERV subgroups are particularly efficient in infection and replication in human cells. Transcription of PERVs has been described in most pig cells, but their sequence and insertion polymorphism in the pig genome impede identification of transcriptionally active or silenced proviral copies. Furthermore, little is known about the epigenetic regulation of PERV transcription. Here, we report on the transcriptional suppression of PERV by DNA methylation in vitro and describe heavy methylation in the majority of PERV 5' long terminal repeats (LTR) in porcine tissues. In contrast, we have detected sparsely methylated or nonmethylated proviruses in the porcine PK15 cells, which express human cell-tropic PERVs. We also demonstrate the resistance of PERV DNA methylation to inhibitors of methylation and deacetylation. Finally, we show that the high permissiveness of various human cell lines to PERV infection coincides with the inability to efficiently silence the PERV proviruses by 5'LTR methylation. In conclusion, we suggest that DNA methylation is involved in PERV regulation, and that only a minor fraction of proviruses are responsible for the PERV RNA expression and porcine cell infectivity.
- MeSH
- DNA virů genetika MeSH
- endogenní retroviry genetika MeSH
- epigeneze genetická * MeSH
- koncové repetice genetika MeSH
- kultivované buňky MeSH
- kvantitativní polymerázová řetězová reakce MeSH
- ledviny metabolismus virologie MeSH
- lidé MeSH
- messenger RNA genetika MeSH
- metylace DNA * MeSH
- miniaturní prasata genetika virologie MeSH
- nemoci prasat genetika přenos virologie MeSH
- polymerázová řetězová reakce s reverzní transkripcí MeSH
- prasata MeSH
- proviry genetika MeSH
- replikace viru * MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Syncytin-1 and -2, human fusogenic glycoproteins encoded by the env genes of the endogenous retroviral loci ERVWE1 and ERVFRDE1, respectively, contribute to the differentiation of multinucleated syncytiotrophoblast in chorionic villi. In non-trophoblastic cells, however, the expression of syncytins has to be suppressed to avoid potential pathogenic effects. We studied the epigenetic suppression of ERVWE1 and ERVFRDE1 5'-long terminal repeats by DNA methylation and chromatin modifications. Immunoprecipitation of the provirus-associated chromatin revealed the H3K9 trimethylation at transcriptionally inactivated syncytins in HeLa cells. qRT-PCR analysis of non-spliced ERVWE1 and ERVFRDE1 mRNAs and respective env mRNAs detected efficient splicing of endogenously expressed RNAs in trophoblastic but not in non-placental cells. Pointing to the pathogenic potential of aberrantly expressed syncytin-1, we have found deregulation of transcription and splicing of the ERVWE1 in biopsies of testicular seminomas. Finally, ectopic expression experiments suggest the importance of proper chromatin context for the ERVWE1 splicing. Our results thus demonstrate that cell-specific retroviral splicing represents an additional epigenetic level controling the expression of endogenous retroviruses.
- MeSH
- buněčné linie MeSH
- endogenní retroviry MeSH
- epigeneze genetická MeSH
- genetická transkripce MeSH
- genové produkty env genetika metabolismus MeSH
- germinální a embryonální nádory genetika metabolismus MeSH
- glykoproteiny genetika metabolismus MeSH
- HeLa buňky MeSH
- histony metabolismus MeSH
- lidé MeSH
- messenger RNA metabolismus MeSH
- proviry genetika metabolismus MeSH
- sestřih RNA MeSH
- těhotenské proteiny genetika metabolismus MeSH
- testis metabolismus MeSH
- umlčování genů MeSH
- Check Tag
- lidé MeSH
- mužské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Syncytin-1 is a captive envelope glycoprotein encoded by one of human endogenous retroviruses W. It is expressed exclusively in the placental trophoblast where it participates in cell-to-cell fusion during differentiation of syncytiotrophobast. In other tissues, however, syncytin-1 expression must be kept in check because inadvertent cell fusion might be dangerous for tissue organization and integrity. We describe here an inverse correlation between CpG methylation of syncytin-1 5' long terminal repeat and its expression. Hypomethylation of the syncytin-1 5' long terminal repeat in the placenta and in the choriocarcinoma-derived cell line BeWo was detected. However, other analyzed primary cells and cell lines non-expressing syncytin-1 contain proviruses heavily methylated in this sequence. CpG methylation of syncytin-1 is resistant to the effect of the demethylating agent 5-azacytidine. The inhibitory role of CpG methylation is further confirmed by transient transfection of in-vitro-methylated syncytin-1 promoter-driven reporter construct. Altogether, we conclude that CpG methylation plays a principal role in the transcriptional suppression of syncytin-1 in non-placental tissues, and, in contrast, demethylation of the syncytin-1 promoter in trophoblast is a prerequisite for its expression and differentiation of multinucleated syncytiotrophoblast.
- MeSH
- buněčné linie MeSH
- CpG ostrůvky fyziologie MeSH
- down regulace imunologie MeSH
- financování organizované MeSH
- genové produkty env antagonisté a inhibitory biosyntéza genetika MeSH
- HeLa buňky MeSH
- koncové repetice MeSH
- lidé MeSH
- metylace DNA MeSH
- placenta metabolismus MeSH
- promotorové oblasti (genetika) MeSH
- těhotenské proteiny antagonisté a inhibitory biosyntéza genetika MeSH
- transkripční faktory antagonisté a inhibitory biosyntéza genetika MeSH
- trofoblasty metabolismus MeSH
- Check Tag
- lidé MeSH
