Syncytin-1, a human fusogenic protein of retroviral origin, is crucial for placental syncytiotrophoblast formation. To mediate cell-to-cell fusion, Syncytin-1 requires specific interaction with its cognate receptor. Two trimeric transmembrane proteins, Alanine, Serine, Cysteine Transporters 1 and 2 (ASCT1 and ASCT2), were suggested and widely accepted as Syncytin-1 cellular receptors. To quantitatively assess the individual contributions of human ASCT1 and ASCT2 to the fusogenic activity of Syncytin-1, we developed a model system where the ASCT1 and ASCT2 double knockout was rescued by ectopic expression of either ASCT1 or ASCT2. We demonstrated that ASCT2 was required for Syncytin-1 binding, cellular entry, and cell-to-cell fusion, while ASCT1 was not involved in this receptor interaction. We experimentally validated the ASCT1-ASCT2 heterotrimers as a possible explanation for the previous misidentification of ASCT1 as a receptor for Syncytin-1. This redefinition of receptor specificity is important for proper understanding of Syncytin-1 function in normal and pathological pregnancy.

• Keywords
• Syncytin-1, cell-to-cell fusion, endogenous retrovirus, placenta, viral receptor,
• MeSH
• Fusion Regulatory Protein 1, Heavy Chain MeSH
• Cell Fusion * MeSH
• Gene Products, env * metabolism   genetics   MeSH
• Humans MeSH
• Placenta * metabolism   MeSH
• Pregnancy Proteins * metabolism   genetics   MeSH
• Pregnancy MeSH
• Amino Acid Transport System ASC * metabolism   genetics   MeSH
• Amino Acid Transport Systems, Neutral metabolism   genetics   MeSH
• Trophoblasts metabolism   cytology   MeSH
• Minor Histocompatibility Antigens metabolism   genetics   MeSH
• Check Tag
• Humans MeSH
• Pregnancy MeSH
• Female MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Fusion Regulatory Protein 1, Heavy Chain MeSH
• Gene Products, env * MeSH
• SLC1A5 protein, human MeSH Browser
• SLC3A2 protein, human MeSH Browser
• syncytin MeSH Browser
• Pregnancy Proteins * MeSH
• Amino Acid Transport System ASC * MeSH
• Amino Acid Transport Systems, Neutral MeSH
• Minor Histocompatibility Antigens MeSH

BACKGROUND: Human Syncytin-1 is a placentally-expressed cell surface glycoprotein of retroviral origin. After interaction with ASCT2, its cellular receptor, Syncytin-1 triggers cell-cell fusion and formation of a multinuclear syncytiotrophoblast layer of the placenta. The ASCT2 receptor is a multi-spanning membrane protein containing a protruding extracellular part called region C, which has been suggested to be a retrovirus docking site. Precise identification of the interaction site between ASCT2 and Syncytin-1 is challenging due to the complex structure of ASCT2 protein and the background of endogenous ASCT2 gene in the mammalian genome. Chicken cells lack the endogenous background and, therefore, can be used to set up a system with surrogate expression of the ASCT2 receptor. RESULTS: We have established a retroviral heterologous chicken system for rapid and reliable assessment of ectopic human ASCT2 protein expression. Our dual-fluorescence system proved successful for large-scale screening of mutant ASCT2 proteins. Using this system, we demonstrated that progressive deletion of region C substantially decreased the amount of ASCT2 protein. In addition, we implemented quantitative assays to determine the interaction of ASCT2 with Syncytin-1 at multiple levels, which included binding of the soluble form of Syncytin-1 to ASCT2 on the cell surface and a luciferase-based assay to evaluate cell-cell fusions that were triggered by Syncytin-1. Finally, we restored the envelope function of Syncytin-1 in a replication-competent retrovirus and assessed the infection of chicken cells expressing human ASCT2 by chimeric Syncytin-1-enveloped virus. The results of the quantitative assays showed that deletion of the protruding region C did not abolish the interaction of ASCT2 with Syncytin-1. CONCLUSIONS: We present here a heterologous chicken system for effective assessment of the expression of transmembrane ASCT2 protein and its interaction with Syncytin-1. The system profits from the absence of endogenous ASCT2 background and implements the quantitative assays to determine the ASCT2-Syncytin-1 interaction at several levels. Using this system, we demonstrated that the protruding region C was essential for ASCT2 protein expression, but surprisingly, not for the interaction with Syncytin-1 glycoprotein.

• Keywords
• ASCT2 (SLC1A5), Cell–cell fusion, Envelope glycoprotein, Envelope-receptor interaction, NanoLuc luciferase, Retroviral receptor, Syncytin-1,
• MeSH
• Cell Line MeSH
• Fibroblasts virology   MeSH
• Fluorescence MeSH
• Gene Products, env genetics   metabolism   MeSH
• Microscopy, Confocal MeSH
• Chickens MeSH
• Humans MeSH
• Placenta virology   MeSH
• Pregnancy Proteins genetics   metabolism   MeSH
• Pregnancy MeSH
• Amino Acid Transport System ASC genetics   metabolism   MeSH
• Minor Histocompatibility Antigens genetics   metabolism   MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Pregnancy MeSH
• Female MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Gene Products, env MeSH
• SLC1A5 protein, human MeSH Browser
• syncytin MeSH Browser
• Pregnancy Proteins MeSH
• Amino Acid Transport System ASC MeSH
• Minor Histocompatibility Antigens MeSH

BACKGROUND: 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. Previously, we have shown that the transcriptional suppression of ERVWE1 promoter is controlled epigenetically by DNA methylation and chromatin modifications. In this study, we describe the aberrant expression of syncytin-1 in biopsies of testicular germ cell tumors. RESULTS: We found efficient expression and splicing of syncytin-1 in seminomas and mixed germ cell tumors with seminoma component. Although another fusogenic gene, syncytin-2 was also derepressed in seminomas, its expression was significantly lower than that of syncytin-1. Neither the transcription factor GCM1 nor the increased copy number of ERVWE1 were sufficient for this aberrant expression of syncytin-1 in seminomas. In accordance with our recent finding of the highly increased expression of TET1 dioxygenase in most seminomas, the ERVWE1 promoter was significantly hypomethylated in comparison with the matched controls. In contrast, 5-hydroxymethylcytosine levels were not detectable at the ERVWE1 promoter. We further describe that another endogenous retroviral element adjacent to ERVWE1 remains transcriptionally suppressed and two additional HERV-W family members are only slightly upregulated in seminomas. CONCLUSIONS: We conclude that DNA demethylation of the ERVWE1 promoter in seminomas is a prerequisite for syncytin-1 derepression. We propose the spliced syncytin-1 expression as a marker of seminoma and suggest that aberrant expression of endogenous retroviruses might be a correlate of the hypomethylated genome of seminomas.

• Keywords
• 5-Hydroxymethylcytosine, ERVWE1, Germ cell tumor, Human endogenous retrovirus, Promoter DNA methylation, RNA splicing, Seminoma, Transcription,
• MeSH
• DNA, Viral metabolism   MeSH
• Endogenous Retroviruses genetics   MeSH
• Epigenesis, Genetic MeSH
• Gene Products, env biosynthesis   MeSH
• Humans MeSH
• DNA Methylation MeSH
• Gene Expression Regulation * MeSH
• Seminoma pathology   virology   MeSH
• Pregnancy Proteins biosynthesis   MeSH
• Testicular Neoplasms pathology   virology   MeSH
• Check Tag
• Humans MeSH
• Male MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• DNA, Viral MeSH
• Gene Products, env MeSH
• syncytin MeSH Browser
• Pregnancy Proteins MeSH