RESUMO
Acellular materials of xenogenic origin are used worldwide as xenografts, and phase I trials of viable pig pancreatic islets are currently being performed. However, limited information is available on transmission of porcine endogenous retrovirus (PERV) after xenotransplantation and on the long-term immune response of recipients to xenoantigens. We analyzed the blood of burn patients who had received living pig-skin dressings for up to 8 wk for the presence of PERV as well as for the level and nature of their long term (maximum, 34 y) immune response against pig Ags. Although no evidence of PERV genomic material or anti-PERV Ab response was found, we observed a moderate increase in anti-αGal Abs and a high and sustained anti-non-αGal IgG response in those patients. Abs against the nonhuman sialic acid Neu5Gc constituted the anti-non-αGal response with the recognition pattern on a sialoglycan array differing from that of burn patients treated without pig skin. These data suggest that anti-Neu5Gc Abs represent a barrier for long-term acceptance of porcine xenografts. Because anti-Neu5Gc Abs can promote chronic inflammation, the long-term safety of living and acellular pig tissue implants in recipients warrants further evaluation.
Assuntos
Antígenos Heterófilos/imunologia , Queimaduras/cirurgia , Ácidos Siálicos/imunologia , Transplante de Pele/efeitos adversos , Transplante Heterólogo/efeitos adversos , Adolescente , Adulto , Idoso , Animais , Antígenos Heterófilos/análise , Criança , Retrovirus Endógenos/imunologia , Ensaio de Imunoadsorção Enzimática , Feminino , Humanos , Imunoglobulina G , Lactente , Masculino , Pessoa de Meia-Idade , RNA Viral/análise , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transplante de Pele/métodos , SuínosRESUMO
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.
Assuntos
Metilação de DNA , Retrovirus Endógenos/genética , Epigênese Genética , Doenças dos Suínos/transmissão , Porco Miniatura/virologia , Replicação Viral , Animais , Células Cultivadas , DNA Viral/genética , Humanos , Rim/metabolismo , Rim/virologia , Provírus/genética , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Suínos , Doenças dos Suínos/genética , Doenças dos Suínos/virologia , Porco Miniatura/genética , Sequências Repetidas Terminais/genéticaRESUMO
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.
Assuntos
Retrovirus Endógenos , Epigênese Genética , Produtos do Gene env/genética , Proteínas da Gravidez/genética , Splicing de RNA , Transcrição Gênica , Linhagem Celular , Produtos do Gene env/metabolismo , Inativação Gênica , Glicoproteínas/genética , Glicoproteínas/metabolismo , Células HeLa , Histonas/metabolismo , Humanos , Masculino , Neoplasias Embrionárias de Células Germinativas/genética , Neoplasias Embrionárias de Células Germinativas/metabolismo , Proteínas da Gravidez/metabolismo , Provírus/genética , Provírus/metabolismo , RNA Mensageiro/metabolismo , Testículo/metabolismoRESUMO
BACKGROUND: The risk of zoonotic infection by porcine endogenous retroviruses (PERV) has been highlighted in the context of pig-to-human xenotransplantation. The use of receptors for cell entry often determines the host range of retroviruses. A human-tropic PERV subgroup, PERV-A, can enter human cells through either of two homologous multitransmembrane proteins, huPAR-1 and huPAR-2. Here, we characterised human PARs and their homologues in the PERV-A resistant rodent species, mouse and rat (muPAR and ratPAR, respectively). RESULTS: Upon exogenous expression in PERV-A resistant cells, human and rat PARs, but not muPAR, conferred PERV-A sensitivity. Exogenously expressed ratPAR binds PERV-A Env and allows PERV-A infection with equivalent efficiency to that of huPAR-1. Endogenous ratPAR expression in rat cell lines appeared to be too low for PERV-A infection. In contrast, the presence of Pro at position 109 in muPAR was identified to be the determinant for PERV-A resistance. Pro109. was shown to be located in the second extracellular loop (ECL2) and affected PERV-A Env binding to PAR molecules. CONCLUSION: The basis of resistance to PERV-A infection in two rodent species is different. Identification of a single a.a. mutation in muPAR, which is responsible for mouse cell resistance to PERV-A highlighted the importance of ECL-2 for the viral receptor function.
Assuntos
Retrovirus Endógenos/fisiologia , Receptores Acoplados a Proteínas G/metabolismo , Receptores Virais/metabolismo , Ligação Viral , Sequência de Aminoácidos , Animais , Linhagem Celular , Clonagem Molecular , Retrovirus Endógenos/genética , Produtos do Gene env/metabolismo , Interações Hospedeiro-Patógeno , Humanos , Camundongos , Dados de Sequência Molecular , Prolina/fisiologia , Ligação Proteica , Estrutura Terciária de Proteína , Codorniz , Ratos , Receptores Acoplados a Proteínas G/química , Receptores Acoplados a Proteínas G/genética , Receptores Virais/química , Receptores Virais/genética , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Infecções por Retroviridae , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , SuínosRESUMO
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.