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.

• Klíčová slova
• epigenetics, expression, integration site, retrovirus, silencing, vectors,
• MeSH
• buněčné linie MeSH
• Gammaretrovirus * genetika   MeSH
• genetické vektory genetika   MeSH
• integrace viru * MeSH
• koncové repetice * genetika   MeSH
• lidé MeSH
• promotorové oblasti (genetika) MeSH
• proviry * genetika   MeSH
• regulace exprese virových genů MeSH
• transgeny MeSH
• umlčování genů MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH

It has now been more than two years since we said our last goodbye to Jan Svoboda (14 [...].

• MeSH
• lidé MeSH
• Retroviridae klasifikace   genetika   izolace a purifikace   fyziologie   MeSH
• retrovirové infekce virologie   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• práce podpořená grantem MeSH
• úvodní články MeSH
• úvodníky 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.

• Klíčová slova
• gene regulatory elements, genome-wide provirus distribution, provirus silencing, retrovirus integration,
• MeSH
• aktivace transkripce * MeSH
• Alpharetrovirus genetika   MeSH
• buněčné linie MeSH
• chromatin genetika   MeSH
• epigeneze genetická MeSH
• genetické vektory genetika   MeSH
• genový targeting MeSH
• HIV-1 genetika   MeSH
• integrace viru MeSH
• lidé MeSH
• myši MeSH
• plazmidy genetika   MeSH
• počátek transkripce MeSH
• proviry genetika   MeSH
• regulace exprese virových genů MeSH
• regulační oblasti nukleových kyselin * MeSH
• stabilita RNA MeSH
• umlčování genů MeSH
• virus myší leukemie genetika   MeSH
• zesilovače transkripce MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• 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 genetika   MeSH
• buněčné linie MeSH
• buňky K562 MeSH
• CpG ostrůvky genetika   MeSH
• epigeneze genetická MeSH
• genetická transkripce * MeSH
• genetické vektory genetika   MeSH
• integrace viru genetika   MeSH
• lidé MeSH
• promotorové oblasti (genetika) genetika   MeSH
• proviry genetika   MeSH
• umlčování genů MeSH
• zesilovače transkripce genetika   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH

This article summarizes the essential steps in understanding the chicken Rous sarcoma virus (RSV) genome association with a nonpermissive rodent host cell genome. This insight was made possible by in-depth study of RSV-transformed rat XC cells, which were called virogenic because they indefinitely carry virus genetic information in the absence of any infectious virus production. However, the virus was rescued by association of XC cells with chicken fibroblasts, allowing cell fusion between both partners. This and additional studies led to the interpretation that the RSV genome gets integrated into the host cell genome as a provirus. Study of additional rodent virogenic cell lines provided evidence that the transcript of oncogene v-src can be transmitted to other retroviruses and produce cell transformation by itself. As discussed in the text, two main questions related to nonpermissiveness to retrovirus infection remain to be solved. The first is changes in the retrovirus envelope gene allowing virus entry into a nonpermissive cell. The second is the nature of the permissive cell functions required by the nonpermissive cell to ensure infectious virus production. Both lines of investigation are being pursued.

• Klíčová slova
• cell transformation, nonpermissiveness to virus infection, virus integration, virus rescue,
• MeSH
• buněčné linie MeSH
• fúze buněk * MeSH
• genom virový genetika   MeSH
• genové produkty env genetika   MeSH
• krysa rodu Rattus MeSH
• kur domácí virologie   MeSH
• onkogenní protein pp60(v-src) genetika   MeSH
• proviry genetika   růst a vývoj   MeSH
• virová transformace buněk MeSH
• virus Rousova sarkomu genetika   růst a vývoj   MeSH
• zvířata MeSH
• Check Tag
• krysa rodu Rattus MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• genové produkty env MeSH
• onkogenní protein pp60(v-src) 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 genetika   MeSH
• DNA methyltransferasa 3A MeSH
• DNA-(cytosin-5-)methyltransferasa genetika   metabolismus   MeSH
• DNA-methyltransferasa 3B MeSH
• epigeneze genetická * MeSH
• genetická transkripce MeSH
• integrace viru * MeSH
• lidé MeSH
• metylace DNA * MeSH
• nádorové buněčné linie MeSH
• proviry genetika   MeSH
• umlčování genů * MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• DNA methyltransferasa 3A MeSH
• DNA-(cytosin-5-)methyltransferasa MeSH
• DNMT3A protein, human MeSH Prohlížeč

Retroviruses and retrovirus-derived vectors integrate nonrandomly into the genomes of host cells with specific preferences for transcribed genes, gene-rich regions, and CpG islands. However, the genomic features that influence the transcriptional activities of integrated retroviruses or retroviral vectors are poorly understood. We report here the cloning and characterization of avian sarcoma virus integration sites from chicken tumors. Growing progressively, dependent on high and stable expression of the transduced v-src oncogene, these tumors represent clonal expansions of cells bearing transcriptionally active replication-defective proviruses. Therefore, integration sites in our study distinguished genomic loci favorable for the expression of integrated retroviruses and gene transfer vectors. Analysis of integration sites from avian sarcoma virus-induced tumors showed strikingly nonrandom distribution, with proviruses found prevalently within or close to transcription units, particularly in genes broadly expressed in multiple tissues but not in tissue-specifically expressed genes. We infer that proviruses integrated in these genomic areas efficiently avoid transcriptional silencing and remain active for a long time during the growth of tumors. Defining the differences between unselected retroviral integration sites and sites selected for long-terminal-repeat-driven gene expression is relevant for retrovirus-mediated gene transfer and has ramifications for gene therapy.

• MeSH
• chromozomy virologie   MeSH
• exprese genu MeSH
• genetická terapie metody   MeSH
• genetické vektory MeSH
• integrace viru * MeSH
• kur domácí MeSH
• proviry genetika   fyziologie   MeSH
• ptačí sarkom virologie   MeSH
• viry ptačího sarkomu genetika   fyziologie   MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Unmethylated CpG islands are known to keep adjacent promoters transcriptionally active. In the CpG island adjacent to the adenosine phosphoribosyltransferase gene, the protection against transcriptional silencing can be attributed to the short CpG-rich core element containing Sp1 binding sites. We report here the insertion of this CpG island core element, IE, into the long terminal repeat of a retroviral vector derived from Rous sarcoma virus, which normally suffers from progressive transcriptional silencing in mammalian cells. IE insertion into a specific position between enhancer and promoter sequences led to efficient protection of the integrated vector from silencing and gradual CpG methylation in rodent and human cells. Individual cell clones with IE-modified reporter vectors display high levels of reporter expression for a sustained period and without substantial variegation in the cell culture. The presence of Sp1 binding sites is important for the protective effect of IE, but at least some part of the entire antisilencing capacity is maintained in IE with mutated Sp1 sites. We suggest that this strategy of antisilencing protection by the CpG island core element may prove generally useful in retroviral vectors.

• MeSH
• biologické modely MeSH
• CpG ostrůvky * MeSH
• genetická transkripce * MeSH
• koncové repetice MeSH
• lidé MeSH
• mutace MeSH
• průtoková cytometrie MeSH
• ptačí sarkom genetika   virologie   MeSH
• ptáci MeSH
• reportérové geny MeSH
• transkripční faktor Sp1 metabolismus   MeSH
• umlčování genů * MeSH
• vazebná místa MeSH
• virus ptačí leukózy metabolismus   MeSH
• virus Rousova sarkomu metabolismus   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• transkripční faktor Sp1 MeSH

CpG islands are important in the protection of adjacent housekeeping genes from de novo DNA methylation and for keeping them in a transcriptionally active state. However, little is known about their capacity to protect heterologous genes and assure position-independent transcription of adjacent transgenes or retroviral vectors. To tackle this question, we have used the mouse aprt CpG island to flank a Rous sarcoma virus (RSV)-derived reporter vector and followed the transcriptional activity of integrated vectors. RSV is an avian retrovirus which does not replicate in mammalian cells because of several blocks at all levels of the replication cycle. Here we show that our RSV-derived reporter proviruses linked to the mouse aprt gene CpG island remain undermethylated and keep their transcriptional activity after stable transfection into both avian and nonpermissive mammalian cells. This effect is most likely caused by the protection from de novo methylation provided by the CpG island and not by enhancement of the promoter strength. Our results are consistent with previous finding of CpG islands in proximity to active but not inactive proviruses and support further investigation of the protection of the gene transfer vectors from DNA methylation.

• MeSH
• adeninfosforibosyltransferasa genetika   MeSH
• buněčné linie virologie   MeSH
• CpG ostrůvky * MeSH
• defektní viry genetika   MeSH
• DNA virů chemie   genetika   MeSH
• DNA-(cytosin-5-)methyltransferasa metabolismus   MeSH
• experimentální sarkom genetika   virologie   MeSH
• fibroblasty virologie   MeSH
• genetická transkripce * MeSH
• genetické vektory genetika   fyziologie   MeSH
• integrace viru MeSH
• koncové repetice MeSH
• křečci praví MeSH
• křeček rodu Mesocricetus MeSH
• kuřecí embryo MeSH
• metylace DNA MeSH
• myši MeSH
• proviry genetika   MeSH
• regulace exprese virových genů * MeSH
• replikace viru MeSH
• reportérové geny MeSH
• umlčování genů * MeSH
• viry ptačího sarkomu genetika   fyziologie   MeSH
• zvířata MeSH
• Check Tag
• křečci praví MeSH
• kuřecí embryo MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• srovnávací studie MeSH
• Názvy látek
• adeninfosforibosyltransferasa MeSH
• DNA virů MeSH
• DNA-(cytosin-5-)methyltransferasa MeSH