Retroviruses integrate into the genomes of infected host cells to form proviruses, a genetic platform for stable viral gene expression. Epigenetic silencing can, however, hamper proviral transcriptional activity. As gammaretroviruses (γRVs) preferentially integrate into active promoter and enhancer sites, the high transcriptional activity of γRVs can be attributed to this integration preference. In addition, long terminal repeats (LTRs) of some γRVs were shown to act as potent promoters by themselves. Here, we investigate the capacity of different γRV LTRs to drive stable expression within a non-preferred epigenomic environment in the context of diverse retroviral vectors. We demonstrate that different γRV LTRs are either rapidly silenced or remain active for long periods of time with a predominantly active proviral population under normal and retargeted integration. As an alternative to the established γRV systems, the feline leukemia virus and koala retrovirus LTRs are able to drive stable, albeit intensity-diverse, transgene expression. Overall, we show that despite the occurrence of rapid silencing events, most γRV LTRs can drive stable expression outside of their preferred chromatin landscape after retrovirus integrations.

• Keywords
• epigenetics, expression, integration site, retrovirus, silencing, vectors,
• MeSH
• Cell Line MeSH
• Gammaretrovirus * genetics   MeSH
• Genetic Vectors genetics   MeSH
• Virus Integration * MeSH
• Terminal Repeat Sequences * genetics   MeSH
• Humans MeSH
• Promoter Regions, Genetic MeSH
• Proviruses * genetics   MeSH
• Gene Expression Regulation, Viral MeSH
• Transgenes MeSH
• Gene Silencing MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Animals MeSH
• Publication type
• Journal Article 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

The ongoing progress in primordial germ cell derivation and cultivation is opening new ways in reproductive biotechnology. This study tested whether functional sperm cells can be matured from genetically manipulated primordial germ cells after transplantation in adult testes and used to restore fertility. We show that spermatogenesis can be restored after mCherry-expressing or GFP-expressing primordial germ cells are transplantated into the testes of sterilized G0 roosters and that mCherry-positive or GFP-positive non-chimeric transgenic G1 offspring can be efficiently produced. Compared with the existing approaches to primordial germ cell replacement, this new technique eliminates the germ line chimerism of G0 roosters and is, therefore, faster, more efficient and requires fewer animals. Furthermore, this is the only animal model, where the fate of primordial germ cells in infertile recipients can be studied.

• MeSH
• Phenotype MeSH
• Fertility * MeSH
• Chickens genetics   physiology   MeSH
• Spermatogenesis genetics   MeSH
• Spermatozoa cytology   MeSH
• Gene Transfer Techniques * MeSH
• Testis cytology   physiology   MeSH
• Transduction, Genetic MeSH
• Cell Transplantation * MeSH
• Animals MeSH
• Check Tag
• Male MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

Porcine endogenous retroviruses (PERV) represent a major safety concern in pig-to-human xenotransplantation. To date, no PERV infection of a xenograft recipient has been recorded; however, PERVs are transmissible to human cells in vitro. Some recombinants of the A and C PERV subgroups are particularly efficient in infection and replication in human cells. Transcription of PERVs has been described in most pig cells, but their sequence and insertion polymorphism in the pig genome impede identification of transcriptionally active or silenced proviral copies. Furthermore, little is known about the epigenetic regulation of PERV transcription. Here, we report on the transcriptional suppression of PERV by DNA methylation in vitro and describe heavy methylation in the majority of PERV 5' long terminal repeats (LTR) in porcine tissues. In contrast, we have detected sparsely methylated or nonmethylated proviruses in the porcine PK15 cells, which express human cell-tropic PERVs. We also demonstrate the resistance of PERV DNA methylation to inhibitors of methylation and deacetylation. Finally, we show that the high permissiveness of various human cell lines to PERV infection coincides with the inability to efficiently silence the PERV proviruses by 5'LTR methylation. In conclusion, we suggest that DNA methylation is involved in PERV regulation, and that only a minor fraction of proviruses are responsible for the PERV RNA expression and porcine cell infectivity.

• MeSH
• DNA, Viral genetics   MeSH
• Endogenous Retroviruses genetics   MeSH
• Epigenesis, Genetic * MeSH
• Terminal Repeat Sequences genetics   MeSH
• Cells, Cultured MeSH
• Real-Time Polymerase Chain Reaction MeSH
• Kidney metabolism   virology   MeSH
• Humans MeSH
• RNA, Messenger genetics   MeSH
• DNA Methylation * MeSH
• Swine, Miniature genetics   virology   MeSH
• Swine Diseases genetics   transmission   virology   MeSH
• Reverse Transcriptase Polymerase Chain Reaction MeSH
• Swine MeSH
• Proviruses genetics   MeSH
• Virus Replication * MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• DNA, Viral MeSH
• RNA, Messenger MeSH