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.

• Klíčová slova
• Syncytin-1, cell-to-cell fusion, endogenous retrovirus, placenta, viral receptor,
• MeSH
• antigeny CD98 - těžký řetězec MeSH
• fúze buněk * MeSH
• genové produkty env * metabolismus   genetika   MeSH
• lidé MeSH
• placenta * metabolismus   MeSH
• těhotenské proteiny * metabolismus   genetika   MeSH
• těhotenství MeSH
• transportní systém ASC pro aminokyseliny * metabolismus   genetika   MeSH
• transportní systémy pro neutrální aminokyseliny metabolismus   genetika   MeSH
• trofoblasty metabolismus   cytologie   MeSH
• vedlejší histokompatibilní antigeny metabolismus   genetika   MeSH
• Check Tag
• lidé MeSH
• těhotenství MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• antigeny CD98 - těžký řetězec MeSH
• genové produkty env * MeSH
• SLC1A5 protein, human MeSH Prohlížeč
• SLC3A2 protein, human MeSH Prohlížeč
• syncytin MeSH Prohlížeč
• těhotenské proteiny * MeSH
• transportní systém ASC pro aminokyseliny * MeSH
• transportní systémy pro neutrální aminokyseliny MeSH
• vedlejší histokompatibilní antigeny 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.

• Klíčová slova
• ASCT2 (SLC1A5), Cell–cell fusion, Envelope glycoprotein, Envelope-receptor interaction, NanoLuc luciferase, Retroviral receptor, Syncytin-1,
• MeSH
• buněčné linie MeSH
• fibroblasty virologie   MeSH
• fluorescence MeSH
• genové produkty env genetika   metabolismus   MeSH
• konfokální mikroskopie MeSH
• kur domácí MeSH
• lidé MeSH
• placenta virologie   MeSH
• těhotenské proteiny genetika   metabolismus   MeSH
• těhotenství MeSH
• transportní systém ASC pro aminokyseliny genetika   metabolismus   MeSH
• vedlejší histokompatibilní antigeny genetika   metabolismus   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• těhotenství MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• genové produkty env MeSH
• SLC1A5 protein, human MeSH Prohlížeč
• syncytin MeSH Prohlížeč
• těhotenské proteiny MeSH
• transportní systém ASC pro aminokyseliny MeSH
• vedlejší histokompatibilní antigeny MeSH