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. 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. 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.

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.

Syncytin-1 and Syncytin-2 are envelope glycoproteins encoded by human endogenous retroviruses that have been exapted for the fusion of cytotrophoblast cells into syncytiotrophoblasts during placental development. Pregnancy complications like preeclampsia are associated with altered expression of interferon-stimulated genes, including guanylate-binding protein 5 (GBP5). Here, we show that misdirected antiviral activity of GBP5 impairs processing and activation of Syncytin-1. In contrast, the proteolytic activation of Syncytin-2 is not affected by GBP5, and its fusogenic activity is only modestly reduced. Mechanistic analyses revealed that Syncytin-1 is mainly cleaved by the GBP5 target furin, whereas Syncytin-2 is also efficiently processed by the proprotein convertase subtilisin/kexin type 7 (PCSK7) and thus resistant to GBP5-mediated restriction. Mutational analyses mapped PCSK7 processing of Syncytin-2 to a leucine residue upstream of the polybasic cleavage site. In summary, we identified an innate immune mechanism that impairs the activity of a co-opted endogenous retroviral envelope protein during pregnancy and may potentially contribute to the pathogenesis of pregnancy disorders.

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. Graphical

Depletion and functional impairment of circulating plasmacytoid dendritic cells (pDCs) are characteristic attributes of HIV-1-infection. The mechanism of dysfunction of pDCs is unclear. Here, we studied the development of phenotype of pDCs in a cohort of HIV-1-infected individuals monitored before the initiation and during a 9-month follow up with antiretroviral therapy (ART). Using polychromatic flow cytometry, we detected significantly higher pDC-surface expression of the HIV-1 receptor CD4, regulatory receptor BDCA-2, Fcγ receptor CD32, pDC dysfunction marker TIM-3, and the marker of killer pDC, TRAIL, in treatment-naïve HIV-1-infected individuals before initiation of ART when compared to healthy donors. After 9 months of ART, all of these markers approached but did not reach the expression levels observed in healthy donors. We found that the rate of decline in HIV-1 RNA level over the first 3 months of ART negatively correlated with the expression of TIM-3 on pDCs. We conclude that immunogenic phenotype of pDCs is not significantly restored after sustained suppression of HIV-1 RNA level in ART-treated patients and that the level of the TIM-3 expressed on pDCs in treatment naïve patients could be a predictive marker of the rate of decline in the HIV-1 RNA level during ART.

Avian leukosis virus (ALV), the prototypical alpharetrovirus, causes tumorigenesis, immunosuppression, and wasting disease in poultry. The ALV genus is classified into ten subgroups, which differ in their host range, cell tropism, and receptor usage. The subgroups A, B, K, and J cause significant economic losses worldwide. The most recently discovered subgroup, ALV-K, which is now widespread in China, has been shown to use the tva cell receptor and share it with ALV-A. However, the specific amino acid residues crucial for ALV-K host cell entry remain unknown. Using precise tva expression and chimeric tva receptors, we further elucidated the significance of the cysteine-rich domain in mediating interactions with both ALV-A and ALV-K. Through a comprehensive analysis of mutated tva receptor variants, we pinpointed tryptophan at position 33 (W33) as a pivotal amino acid residue essential for ALV-K virus binding and entry. Of note is the finding that the substitution of W33 induced resistance to ALV-K while preserving sensitivity to ALV-A. This study not only represents an advance in the understanding of the specificity of the tva receptor for ALV-K, but also offers a biotechnological strategy for the prevention of ALV-K infections in poultry.