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

Avian leukosis virus subgroup K (ALV-K) is composed of newly emerging isolates, which, in sequence analyses, cluster separately from the well-characterized subgroups A, B, C, D, E, and J. However, it remains unclear whether ALV-K represents an independent ALV subgroup with regard to receptor usage, host range, and superinfection interference. In the present study, we examined the host range of the Chinese infectious isolate JS11C1, an ALV-K prototype, and we found substantial overlap of species that were either resistant or susceptible to ALV-A and JS11C1. Ectopic expression of the chicken tva gene in mammalian cells conferred susceptibility to JS11C1, while genetic ablation of the tva gene rendered chicken DF-1 cells resistant to infection by JS11C1. Thus, tva expression is both sufficient and necessary for JS11C1 entry. Receptor sharing was also manifested in superinfection interference, with preinfection of cells with ALV-A, but not ALV-B or ALV-J, blocking subsequent JS11C1 infection. Finally, direct binding of JS11C1 and Tva was demonstrated by preincubation of the virus with soluble Tva, which substantially decreased viral infectivity in susceptible chicken cells. Collectively, these findings indicate that JS11C1 represents a new and bona fide ALV subgroup that utilizes Tva for cell entry and binds to a site other than that for ALV-A.IMPORTANCE ALV consists of several subgroups that are particularly characterized by their receptor usage, which subsequently dictates the host range and tropism of the virus. A few newly emerging and highly pathogenic Chinese ALV strains have recently been suggested to be an independent subgroup, ALV-K, based solely on their genomic sequences. Here, we performed a series of experiments with the ALV-K strain JS11C1, which showed its dependence on the Tva cell surface receptor. Due to the sharing of this receptor with ALV-A, both subgroups were able to interfere with superinfection. Because ALV-K could become an important pathogen and a significant threat to the poultry industry in Asia, the identification of a specific receptor could help in the breeding of resistant chicken lines with receptor variants with decreased susceptibility to the virus.

• Klíčová slova
• Tva, avian leukosis virus K, host range, resistance/susceptibility to retrovirus, retrovirus receptor, superinfection interference,
• MeSH
• buněčné linie MeSH
• druhová specificita MeSH
• fibroblasty cytologie   metabolismus   virologie   MeSH
• internalizace viru MeSH
• křeček rodu Mesocricetus MeSH
• kur domácí MeSH
• náchylnost k nemoci MeSH
• ptačí leukóza genetika   metabolismus   virologie   MeSH
• ptačí proteiny genetika   metabolismus   MeSH
• virové receptory genetika   metabolismus   MeSH
• virus ptačí leukózy klasifikace   patogenita   fyziologie   MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• ptačí proteiny MeSH
• Tva receptor MeSH Prohlížeč
• virové receptory 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.

• Klíčová slova
• 5-Hydroxymethylcytosine, ERVWE1, Germ cell tumor, Human endogenous retrovirus, Promoter DNA methylation, RNA splicing, Seminoma, Transcription,
• MeSH
• DNA virů metabolismus   MeSH
• endogenní retroviry genetika   MeSH
• epigeneze genetická MeSH
• genové produkty env biosyntéza   MeSH
• lidé MeSH
• metylace DNA MeSH
• regulace genové exprese * MeSH
• seminom patologie   virologie   MeSH
• těhotenské proteiny biosyntéza   MeSH
• testikulární nádory patologie   virologie   MeSH
• Check Tag
• lidé MeSH
• mužské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• DNA virů MeSH
• genové produkty env MeSH
• syncytin MeSH Prohlížeč
• těhotenské proteiny 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
• Názvy látek
• ERVFRD-1 protein, human MeSH Prohlížeč
• genové produkty env MeSH
• glykoproteiny MeSH
• histony MeSH
• messenger RNA MeSH
• syncytin MeSH Prohlížeč
• těhotenské proteiny MeSH