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

Somatic hypermutation (SHM) introduces point mutations into immunoglobulin (Ig) genes but also causes mutations in other parts of the genome. We have used lentiviral SHM reporter vectors to identify regions of the genome that are susceptible ("hot") and resistant ("cold") to SHM, revealing that SHM susceptibility and resistance are often properties of entire topologically associated domains (TADs). Comparison of hot and cold TADs reveals that while levels of transcription are equivalent, hot TADs are enriched for the cohesin loader NIPBL, super-enhancers, markers of paused/stalled RNA polymerase 2, and multiple important B cell transcription factors. We demonstrate that at least some hot TADs contain enhancers that possess SHM targeting activity and that insertion of a strong Ig SHM-targeting element into a cold TAD renders it hot. Our findings lead to a model for SHM susceptibility involving the cooperative action of cis-acting SHM targeting elements and the dynamic and architectural properties of TADs.

• Keywords
• activation induced deaminase, chromatin loop extrusion, chromatin structure, somatic hypermutation, topologically associated domain, transcription factor,
• MeSH
• Cytidine Deaminase genetics   metabolism   MeSH
• HEK293 Cells MeSH
• Lentivirus MeSH
• Humans MeSH
• Mutation genetics   MeSH
• Cell Line, Tumor MeSH
• Plasmids genetics   MeSH
• RNA Polymerase II genetics   metabolism   MeSH
• Somatic Hypermutation, Immunoglobulin genetics   MeSH
• Enhancer Elements, Genetic genetics   MeSH
• Check Tag
• Humans MeSH
• Male MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Research Support, N.I.H., Extramural MeSH
• Names of Substances
• Cytidine Deaminase MeSH
• RNA Polymerase II MeSH

Individual groups of retroviruses and retroviral vectors differ in their integration site preference and interaction with the host genome. Hence, immediately after infection genome-wide distribution of integrated proviruses is non-random. During long-term in vitro or persistent in vivo infection, the genomic position and chromatin environment of the provirus affects its transcriptional activity. Thus, a selection of long-term stably expressed proviruses and elimination of proviruses, which have been gradually silenced by epigenetic mechanisms, helps in the identification of genomic compartments permissive for proviral transcription. We compare here the extent and time course of provirus silencing in single cell clones of the K562 human myeloid lymphoblastoma cell line that have been infected with retroviral reporter vectors derived from avian sarcoma/leukosis virus (ASLV), human immunodeficiency virus type 1 (HIV) and murine leukaemia virus (MLV). While MLV proviruses remain transcriptionally active, ASLV proviruses are prone to rapid silencing. The HIV provirus displays gradual silencing only after an extended time period in culture. The analysis of integration sites of long-term stably expressed proviruses shows a strong bias for some genomic features-especially integration close to the transcription start sites of active transcription units. Furthermore, complex analysis of histone modifications enriched at the site of integration points to the accumulation of proviruses of all three groups in gene regulatory segments, particularly close to the enhancer loci. We conclude that the proximity to active regulatory chromatin segments correlates with stable provirus expression in various retroviral species.

• Keywords
• gene regulatory elements, genome-wide provirus distribution, provirus silencing, retrovirus integration,
• MeSH
• Transcriptional Activation * MeSH
• Alpharetrovirus genetics   MeSH
• Cell Line MeSH
• Chromatin genetics   MeSH
• Epigenesis, Genetic MeSH
• Genetic Vectors genetics   MeSH
• Gene Targeting MeSH
• HIV-1 genetics   MeSH
• Virus Integration MeSH
• Humans MeSH
• Mice MeSH
• Plasmids genetics   MeSH
• Transcription Initiation Site MeSH
• Proviruses genetics   MeSH
• Gene Expression Regulation, Viral MeSH
• Regulatory Sequences, Nucleic Acid * MeSH
• RNA Stability MeSH
• Gene Silencing MeSH
• Leukemia Virus, Murine genetics   MeSH
• Enhancer Elements, Genetic MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Chromatin MeSH

Most retroviruses preferentially integrate into certain genomic locations and, as a result, their genome-wide integration patterns are non-random. We investigate the epigenetic landscape of integrated retroviral vectors and correlate it with the long-term stability of proviral transcription. Retroviral vectors derived from the avian sarcoma/leukosis virus expressing the GFP reporter were used to transduce the human myeloid lymphoblastoma cell line K562. Because of efficient silencing of avian retrovirus in mammalian cells, only ∼3% of established clones displayed stable proviral expression. We analyzed the vector integration sites in non-selected cells and in clones selected for the GFP expression. This selection led to overrepresentation of proviruses integrated in active transcription units, with particular accumulation in promoter-proximal areas. In parallel, we investigated the integration of vectors equipped with an anti-silencing CpG island core sequence. Such modification increased the frequency of stably expressing proviruses by one order. The modified vectors are also overrepresented in active transcription units, but stably expressed in distal parts of transcriptional units further away from promoters with marked accumulation in enhancers. These results suggest that integrated retroviruses subject to gradual epigenetic silencing during long-term cultivation. Among most genomic compartments, however, active promoters and enhancers protect the adjacent retroviruses from transcriptional silencing.

• MeSH
• Alpharetrovirus genetics   MeSH
• Cell Line MeSH
• K562 Cells MeSH
• CpG Islands genetics   MeSH
• Epigenesis, Genetic MeSH
• Transcription, Genetic * MeSH
• Genetic Vectors genetics   MeSH
• Virus Integration genetics   MeSH
• Humans MeSH
• Promoter Regions, Genetic genetics   MeSH
• Proviruses genetics   MeSH
• Gene Silencing MeSH
• Enhancer Elements, Genetic genetics   MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH

UNLABELLED: Transformation of rodent cells with avian Rous sarcoma virus (RSV) opened new ways to studying virus integration and expression in nonpermissive cells. We were interested in (i) the molecular changes accompanying fusion of RSV-transformed mammalian cells with avian cells leading to virus rescue and (ii) enhancement of this process by retroviral gene products. The RSV-transformed hamster RSCh cell line was characterized as producing only a marginal amount of env mRNA, no envelope glycoprotein, and a small amount of unprocessed Gag protein. Egress of viral unspliced genomic RNA from the nucleus was hampered, and its stability decreased. Cell fusion of the chicken DF-1 cell line with RSCh cells led to production of env mRNA, envelope glycoprotein, and processed Gag and virus-like particle formation. Proteosynthesis inhibition in DF-1 cells suppressed steps leading to virus rescue. Furthermore, new aberrantly spliced env mRNA species were found in the RSCh cells. Finally, we demonstrated that virus rescue efficiency can be significantly increased by complementation with the env gene and the highly expressed gag gene and can be increased the most by a helper virus infection. In summary, Env and Gag synthesis is increased after RSV-transformed hamster cell fusion with chicken fibroblasts, and both proteins provided in trans enhance RSV rescue. We conclude that the chicken fibroblast yields some factor(s) needed for RSV replication, particularly Env and Gag synthesis, in nonpermissive rodent cells. IMPORTANCE: One of the important issues in retrovirus heterotransmission is related to cellular factors that prevent virus replication. Rous sarcoma virus (RSV), a member of the avian sarcoma and leukosis family of retroviruses, is able to infect and transform mammalian cells; however, such transformed cells do not produce infectious virus particles. Using the well-defined model of RSV-transformed rodent cells, we established that the lack of virus replication is due to the absence of chicken factor(s), which can be supplemented by cell fusion. Cell fusion with permissive chicken cells led to an increase in RNA splicing and nuclear export of specific viral mRNAs, as well as synthesis of respective viral proteins and production of virus-like particles. RSV rescue by cell fusion can be potentiated by in trans expression of viral genes in chicken cells. We conclude that rodent cells lack some chicken factor(s) required for proper viral RNA processing and viral protein synthesis.

• MeSH
• Cell Fusion MeSH
• Gene Products, env genetics   metabolism   MeSH
• Gene Products, gag genetics   metabolism   MeSH
• Cricetinae MeSH
• Chickens MeSH
• Poultry Diseases virology   MeSH
• Sarcoma, Avian virology   MeSH
• Genetic Complementation Test MeSH
• Cell Line, Transformed MeSH
• Cell Transformation, Viral MeSH
• Rous sarcoma virus genetics   physiology   MeSH
• Animals MeSH
• Check Tag
• Cricetinae MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Gene Products, env MeSH
• Gene Products, gag MeSH

The autonomous transcription of integrated retroviruses strongly depends on genetic and epigenetic effects of the chromatin at the site of integration. These effects are mostly suppressive and proviral activity can be finally silenced by mechanisms, such as DNA methylation and histone modifications. To address the role of the integration site at the whole-genome-scale, we performed clonal analysis of provirus silencing with an avian leucosis/sarcoma virus-based reporter vector and correlated the transcriptional silencing with the epigenomic landscape of respective integrations. We demonstrate efficient provirus silencing in human HCT116 cell line, which is strongly but not absolutely dependent on the de novo DNA methyltransferase activity, particularly of Dnmt3b. Proviruses integrated close to the transcription start sites of active genes into the regions enriched in H3K4 trimethylation display long-term stability of expression and are resistant to the transcriptional silencing after over-expression of Dnmt3a or Dnmt3b. In contrast, proviruses in the intergenic regions tend to spontaneous transcriptional silencing even in Dnmt3a(-/-) Dnmt3b(-/-) cells. The silencing of proviruses within genes is accompanied with DNA methylation of long terminal repeats, whereas silencing in intergenic regions is DNA methylation-independent. These findings indicate that the epigenomic features of integration sites are crucial for their permissivity to the proviral expression.

• MeSH
• Alpharetrovirus genetics   MeSH
• DNA Methyltransferase 3A MeSH
• DNA (Cytosine-5-)-Methyltransferases genetics   metabolism   MeSH
• DNA Methyltransferase 3B MeSH
• Epigenesis, Genetic * MeSH
• Transcription, Genetic MeSH
• Virus Integration * MeSH
• Humans MeSH
• DNA Methylation * MeSH
• Cell Line, Tumor MeSH
• Proviruses genetics   MeSH
• Gene Silencing * MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• DNA Methyltransferase 3A MeSH
• DNA (Cytosine-5-)-Methyltransferases MeSH
• DNMT3A protein, human MeSH Browser

The past 15 years opened new avenues for retrovirus and retroelement research. Not surprisingly, they stemmed from essential knowledge collected in the past, which remains the ground of the present and therefore should be remembered. However, a short supplement of new break-through discoveries and ideas should be recollected. Using selected examples of recent works, I tried to extend and supplement my original article published in Folia Biologica (1996).

• Publication type
• Journal Article MeSH