HIV-1 persists lifelong in memory cells of the immune system as latent provirus that rebounds upon treatment interruption. Therefore, the latent reservoir is the main target for an HIV cure. Here, we studied the direct link between integration site and transcription using LEDGINs and Barcoded HIV-ensembles (B-HIVE). LEDGINs are antivirals that inhibit the interaction between HIV-1 integrase and the chromatin-tethering factor LEDGF/p75. They were used as a tool to retarget integration, while the effect on HIV expression was measured with B-HIVE. B-HIVE tracks insert-specific HIV expression by tagging a unique barcode in the HIV genome. We confirmed that LEDGINs retarget integration out of gene-dense and actively transcribed regions. The distance to H3K36me3, the marker recognized by LEDGF/p75, clearly increased. LEDGIN treatment reduced viral RNA expression and increased the proportion of silent provirus. Finally, silent proviruses obtained after LEDGIN treatment were located further away from epigenetic marks associated with active transcription. Interestingly, proximity to enhancers stimulated transcription irrespective of LEDGIN treatment, while the distance to H3K36me3 only changed after treatment with LEDGINs. The fact that proximity to these markers are associated with RNA expression support the direct link between provirus integration site and viral expression.
- MeSH
- buněčné linie MeSH
- chromatin metabolismus MeSH
- histony metabolismus MeSH
- HIV-1 účinky léků genetika metabolismus MeSH
- inhibitory HIV-integrasy farmakologie MeSH
- integrace viru * účinky léků MeSH
- lidé MeSH
- mezibuněčné signální peptidy a proteiny MeSH
- proviry genetika MeSH
- regulace exprese virových genů * účinky léků MeSH
- RNA virová metabolismus MeSH
- umlčování genů * MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Retrovirus assembly is driven mostly by Gag polyprotein oligomerization, which is mediated by inter and intra protein-protein interactions among its capsid (CA) domains. Mason-Pfizer monkey virus (M-PMV) CA contains three cysteines (C82, C193 and C213), where the latter two are highly conserved among most retroviruses. To determine the importance of these cysteines, we introduced mutations of these residues in both bacterial and proviral vectors and studied their impact on the M-PMV life cycle. These studies revealed that the presence of both conserved cysteines of M-PMV CA is necessary for both proper assembly and virus infectivity. Our findings suggest a crucial role of these cysteines in the formation of infectious mature particles.
- MeSH
- buněčné linie MeSH
- cystein genetika MeSH
- genetické vektory MeSH
- HEK293 buňky MeSH
- lidé MeSH
- Masonův-Pfizerův opičí virus genetika fyziologie MeSH
- mutace MeSH
- proviry genetika MeSH
- sestavení viru * MeSH
- virion fyziologie MeSH
- virové plášťové proteiny chemie genetika MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Research Support, N.I.H., Extramural 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.
- 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
Myeloblastosis-associated virus 2 (MAV-2) is a highly tumorigenic simple avian retrovirus. Chickens infected in ovo with MAV-2 develop tumors in the kidneys, lungs, and liver with a short latency, less than 8 weeks. Here we report the results of molecular analyses of MAV-2-induced liver tumors that fall into three classes: hepatic hemangiosarcomas (HHSs), intrahepatic cholangiocarcinomas (ICCs), and hepatocellular carcinomas (HCCs). Comprehensive inverse PCR-based screening of 92 chicken liver tumors revealed that in ca. 86% of these tumors, MAV-2 provirus had integrated into one of four gene loci: HRAS, EGFR, MET, and RON Insertionally mutated genes correlated with tumor type: HRAS was hit in HHSs, MET in ICCs, RON mostly in ICCs, and EGFR mostly in HCCs. The provirus insertions led to the overexpression of the affected genes and, in the case of EGFR and RON, also to the truncation of exons encoding the extracellular ligand-binding domains of these transmembrane receptors. The structures of truncated EGFR and RON closely mimic the structures of oncogenic variants of these genes frequently found in human tumors (EGFRvIII and sfRON).IMPORTANCE These data describe the mechanisms of oncogenesis induced in chickens by the MAV-2 retrovirus. They also show that molecular processes converting cellular regulatory genes to cancer genes may be remarkably similar in chickens and humans. We suggest that the MAV-2 retrovirus-based model can complement experiments performed using mouse models and provide data that could translate to human medicine.
- MeSH
- cholangiokarcinom genetika virologie MeSH
- geny erbB-1 * MeSH
- hemangiosarkom genetika virologie MeSH
- hepatocelulární karcinom genetika virologie MeSH
- integrace viru MeSH
- inzerční mutageneze * MeSH
- karcinogeneze * MeSH
- kur domácí genetika MeSH
- lidé MeSH
- nádory jater genetika virologie MeSH
- onkogeny MeSH
- protoonkogenní proteiny c-met genetika MeSH
- proviry genetika fyziologie MeSH
- ptačí proteiny genetika MeSH
- tyrosinkinasové receptory genetika MeSH
- virus ptačí myeloblastózy genetika fyziologie MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- MeSH
- dějiny 20. století MeSH
- experimentální nádory MeSH
- infekce onkogenními viry genetika přenos MeSH
- kur domácí MeSH
- leukemie * veterinární virologie MeSH
- nádorová transformace buněk MeSH
- nádory dějiny etiologie klasifikace virologie MeSH
- onkogenní viry klasifikace patogenita MeSH
- onkogeny * MeSH
- proviry * enzymologie genetika MeSH
- pseudogeny MeSH
- ptačí sarkom * etiologie virologie MeSH
- ptáci MeSH
- replikace viru MeSH
- retroelementy MeSH
- Retroviridae účinky léků MeSH
- reverzní transkripce MeSH
- reverzní transkriptasa dějiny MeSH
- savci MeSH
- zvířata MeSH
- Check Tag
- dějiny 20. století MeSH
- zvířata MeSH
- Klíčová slova
- probuzení proviru,
- MeSH
- CD4-pozitivní T-lymfocyty MeSH
- chronická nemoc MeSH
- HIV infekce farmakoterapie genetika patologie terapie MeSH
- HIV * genetika účinky léků MeSH
- latence viru * účinky léků MeSH
- lidé MeSH
- novorozenec MeSH
- předškolní dítě MeSH
- proviry * účinky léků MeSH
- vaskularizovaná kompozitní alotransplantace MeSH
- výsledek terapie MeSH
- vysoce aktivní antiretrovirová terapie MeSH
- Check Tag
- lidé MeSH
- novorozenec MeSH
- předškolní dítě MeSH
- Publikační typ
- kazuistiky 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.
- 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
In my article I tried to present the results of early experiments suggesting a significant role for cell association in Rous sarcoma virus transformation of non-permissive cells and revealing that infectious virus can be efficiently rescued from such cells by their fusion with permissive chicken fibroblasts.
- MeSH
- krysa rodu rattus MeSH
- kur domácí virologie MeSH
- proviry patogenita fyziologie MeSH
- ptačí sarkom virologie MeSH
- replikace viru MeSH
- virová transformace buněk MeSH
- virus Rousova sarkomu patogenita fyziologie MeSH
- zvířata MeSH
- Check Tag
- krysa rodu rattus MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- přehledy MeSH
