The transcriptional activity of an integrated retroviral copy strongly depends on the adjacent host-cell DNA at the site of integration. Transcribed DNA loci as well as cis-acting sequences like enhancers or CpG islands usually permit expression of nearby integrated proviruses. In contrast, proviruses residing close to cellular silencers tend to transcriptional silencing and CpG methylation. Little is known, however, about the influence of provirus integration on the target sequence in the host genome. Here, we report interesting features of a simplified Rous sarcoma virus integrated into a non-transcribed hypermethylated DNA sequence in the Syrian hamster genome. After integration, CpG methylation of this sequence has been lost almost completely and hypomethylated DNA permits proviral transcription and hamster cell transformation by the proviral v-src oncogene. This, however, is not a stable state, and non-transformed revertants bearing transcriptionally silenced proviruses segregate with a high rate. The provirus silencing is followed by DNA methylation of both provirus regulatory regions and adjacent cellular sequences. This CpG methylation is very dense and resistant to the demethylation effects of 5-aza-2(')-deoxycytidine and/or trichostatin A. Our description exemplifies the capacity of retroviruses/retroviral vectors to overcome, at least transiently, negative position effects of DNA methylation at the site of integration.

• MeSH
• agar farmakologie   MeSH
• azacytidin analogy a deriváty   farmakologie   MeSH
• buněčné linie MeSH
• CpG ostrůvky MeSH
• decitabin MeSH
• genetická transkripce MeSH
• geny src genetika   MeSH
• histondeacetylasy metabolismus   MeSH
• inhibitory enzymů farmakologie   MeSH
• inhibitory syntézy proteinů farmakologie   MeSH
• integrace viru * MeSH
• koncové repetice MeSH
• křečci praví MeSH
• křeček rodu Mesocricetus MeSH
• kyseliny hydroxamové farmakologie   MeSH
• metylace DNA * MeSH
• modely genetické MeSH
• molekulární sekvence - údaje MeSH
• nádorové buněčné linie MeSH
• Retroviridae genetika   MeSH
• siřičitany farmakologie   MeSH
• zvířata MeSH
• Check Tag
• křečci praví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• agar MeSH
• azacytidin MeSH
• decitabin MeSH
• histondeacetylasy MeSH
• inhibitory enzymů MeSH
• inhibitory syntézy proteinů MeSH
• kyseliny hydroxamové MeSH
• siřičitany MeSH
• trichostatin A MeSH Prohlížeč

Integration, which leads to the disruption of the circular HPV genome, is considered as a critical, albeit not obligatory, step in carcinogenic progression. Although cervical carcinomas with extrachromosomal HPV plasmid genomes have been described, the virus is integrated in 70% of HPV16-positive cervical tumours. Limited information is available about HPV integration in head and neck tumours (HNC). In this study, we have characterised the physical status of HPV in a set of tonsillar tumour samples using different methods--the mapping of E2 integration breakpoint at the mRNA level, the 3' RACE based Amplification of Papillomavirus Oncogene Transcripts (APOT) assay and Southern blot. Furthermore, the impact of HPV integration on patients' prognosis has been evaluated in a larger set of 186 patients with head and neck cancer. Based on the analysis of E2 mRNA, HPV was integrated in the host genome in 43% of the HPV-positive samples. Extrachromosomal or mixed form was present in 57%. In fresh frozen samples, the APOT and E2 mapping results were in agreement. The results were confirmed using Southern blotting. Furthermore, the type and exact site of integration were determined. The survival analysis of 186 patients revealed HPV positivity, tumour size and lymph node positivity as factors that influence disease specific survival. However, no statistically significant difference was found in disease specific survival between patients with HPV-positive integrated vs. extrachromosomal/mixed forms of the virus.

• Klíčová slova
• HPV, head and neck tumours, integration, prognosis, survival,
• MeSH
• dlaždicobuněčné karcinomy hlavy a krku MeSH
• infekce papilomavirem komplikace   virologie   MeSH
• integrace viru genetika   MeSH
• lidé MeSH
• nádory hlavy a krku mortalita   virologie   MeSH
• prognóza MeSH
• proporcionální rizikové modely MeSH
• Southernův blotting MeSH
• spinocelulární karcinom virologie   MeSH
• tonzilární nádory virologie   MeSH
• Check Tag
• lidé MeSH
• mužské pohlaví MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem 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

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
• Názvy látek
• chromatin MeSH
• histony MeSH
• inhibitory HIV-integrasy MeSH
• lens epithelium-derived growth factor MeSH Prohlížeč
• mezibuněčné signální peptidy a proteiny MeSH
• RNA virová MeSH

The genes involved in the transformation of kidney blastema cells were searched for in avian nephroblastomas induced by the MAV2 retrovirus. The twist gene was identified as a common site of provirus integration in tumor cells. Twist was rearranged by the MAV2 provirus in three out of 76 independent nephroblastoma samples. The MAV2 integration sites were localized within 40 nucleotides of the twist 5'UTR region, right upstream from the ATG initiation codon. The integrated proviruses were deleted at their 5'ends. As a consequence, twist transcription became controlled by the retroviral 3'LTR promoter and was strongly upregulated, more than 200 times. In addition, 2-100 times elevated twist transcription was also detected in the majority of other nephroblastoma samples not containing MAV2 in the twist locus. We propose that chicken nephroblastoma originates from a single blastemic cell in which the MAV retrovirus, through its integration, has deregulated specific combinations of genes controlling proliferation and differentiation. The activation of the twist gene expression appears to contribute to tumorigenesis, as there is an in vivo positive selection of tumor cell clones containing the twist gene hyperactivated by MAV2 sequences inserted within the twist promoter.

• MeSH
• 5' nepřekládaná oblast MeSH
• genetická transkripce fyziologie   MeSH
• integrace viru genetika   MeSH
• jaderné proteiny genetika   MeSH
• klonování DNA MeSH
• kur domácí genetika   MeSH
• kuřecí embryo MeSH
• polymerázová řetězová reakce MeSH
• promotorové oblasti (genetika) MeSH
• regulace exprese virových genů fyziologie   MeSH
• regulace genové exprese u nádorů fyziologie   MeSH
• Retroviridae genetika   MeSH
• transkripční faktor Twist MeSH
• transkripční faktory * MeSH
• upregulace MeSH
• Wilmsův nádor genetika   MeSH
• zvířata MeSH
• Check Tag
• kuřecí embryo MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• 5' nepřekládaná oblast MeSH
• jaderné proteiny MeSH
• transkripční faktor Twist MeSH
• transkripční faktory * MeSH

Gene deregulation is a frequent cause of malignant transformation. Alteration of the gene structure and/or expression leading to cellular transformation and tumor growth can be experimentally achieved by insertion of the retroviral genome into the host DNA. Retrovirus-containing host loci found repeatedly in clonal tumors are called common viral integration sites (cVIS). cVIS are located in genes or chromosomal regions whose alterations participate in cellular transformation. Here, we present the chicken model for the identification of oncogenes and tumor suppressor genes in solid tumors by mapping the cVIS. Using the combination of inverse PCR and long terminal repeat-rapid amplification of cDNA ends technique, we have analyzed 93 myeloblastosis-associated virus type 2-induced clonal nephroblastoma tumors in detail, and mapped >500 independent retroviral integration sites. Eighteen genomic loci were hit repeatedly and thus classified as cVIS, five of these genomic loci have previously been shown to be involved in malignant transformation of different human cell types. The expression levels of selected genes and their human orthologues have been assayed in chicken and selected human renal tumor samples, and their possible correlation with tumor development, has been suggested. We have found that genes associated with cVIS are frequently, but not in all cases, deregulated at the mRNA level as a result of proviral integration. Furthermore, the deregulation of their human orthologues has been observed in the samples of human pediatric renal tumors. Thus, the avian nephroblastoma is a valid source of cancer-associated genes. Moreover, the results bring deeper insight into the molecular background of tumorigenesis in distant species.

• MeSH
• DNA vazebné proteiny genetika   MeSH
• geny ras genetika   MeSH
• integrace viru genetika   MeSH
• koncové repetice MeSH
• kur domácí * MeSH
• kuřecí embryo MeSH
• lidé MeSH
• mapování chromozomů MeSH
• nádory ledvin genetika   virologie   MeSH
• nemoci drůbeže genetika   MeSH
• onkogenní proteiny genetika   MeSH
• onkogeny genetika   MeSH
• polymerázová řetězová reakce MeSH
• proviry genetika   MeSH
• ptačí proteiny genetika   MeSH
• transkripční faktor Twist genetika   MeSH
• tumor supresorové geny MeSH
• virus ptačí myeloblastózy genetika   MeSH
• Wilmsův nádor genetika   virologie   MeSH
• zvířata MeSH
• Check Tag
• kuřecí embryo MeSH
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• DNA vazebné proteiny MeSH
• onkogenní proteiny MeSH
• ptačí proteiny MeSH
• transkripční faktor Twist MeSH
• Twist protein, Gallus gallus MeSH Prohlížeč

BACKGROUND: Unicellular green algae of the genus Micrasterias (Desmidiales) have complex cells with multiple lobes and indentations, and therefore, they are considered model organisms for research on plant cell morphogenesis and variation. Micrasterias cells have a typical biradial symmetric arrangement and multiple terminal lobules. They are composed of two semicells that can be further differentiated into three structural components: the polar lobe and two lateral lobes. Experimental studies suggested that these cellular parts have specific evolutionary patterns and develop independently. In this study, different geometric morphometric methods were used to address whether the semicells of Micrasterias compereana are truly not integrated with regard to the covariation of their shape data. In addition, morphological integration within the semicells was studied to ascertain whether individual lobes constitute distinct units that may be considered as separate modules. In parallel, I sought to determine whether the main components of morphological asymmetry could highlight underlying cytomorphogenetic processes that could indicate preferred directions of variation, canalizing evolutionary changes in cellular morphology. RESULTS: Differentiation between opposite semicells constituted the most prominent subset of cellular asymmetry. The second important asymmetric pattern, recovered by the Procrustes ANOVA models, described differentiation between the adjacent lobules within the quadrants. Other asymmetric components proved to be relatively unimportant. Opposite semicells were shown to be completely independent of each other on the basis of the partial least squares analysis analyses. In addition, polar lobes were weakly integrated with adjacent lateral lobes. Conversely, higher covariance levels between the two lateral lobes of the same semicell indicated mutual interconnection and significant integration between these parts. CONCLUSIONS: Micrasterias cells are composed of several successively disintegrated parts. These integration patterns concurred with presumed scenarios of morphological evolution within the lineage. In addition, asymmetric differentiation in the shape of the lobules involves two major patterns: asymmetry across the isthmus axis and among the adjacent lobules. Notably, asymmetry among the adjacent lobules may be related to evolutionary differentiation among species, but it may also point out developmental instability related to environmental factors.

• Klíčová slova
• Desmidiales, Geometric morphometrics, Green algae, Micrasterias, Morphological asymmetry, Morphological integration,
• MeSH
• biologická evoluce MeSH
• Micrasterias anatomie a histologie   MeSH
• morfogeneze MeSH
• Publikační typ
• časopisecké články MeSH

The tumor hamster cell line H-19 harbors a single copy LTR, v-src, LTR provirus that becomes permanently transcriptionally suppressed in morphological revertants segregating at high rate from this cell line. Our previous data document that the provirus suppression is mediated by epigenetic cell-regulatory mechanisms. In this report, we concentrate on cellular sequences neighboring the integration site. The locus is unique for Syrian hamster and is not detectable in DNA of several animal species. No restriction sites that usually hint at the presence of CpG islands were found in the significantly close vicinity of the provirus. Nevertheless, the chromosomal DNA flanking the provirus is rich in GC content (57.8%). We localized a 0.5-kb region downstream from the provirus that remarkably inhibits transcription in the transient expression assay and is effective both on the homologous RSV LTR promoter/enhancer and heterologous SV40 promoter. We propose that a cellular trans-acting factor is involved in the silencing of the reporter gene. Since this activity is comparable both in transformed and revertant cells, we speculate that this down-regulatory region makes the permissive integration locus prone to provirus silencing initiated by other fluctuating stimuli.

• MeSH
• aktivace transkripce * MeSH
• DNA virů genetika   MeSH
• DNA genetika   MeSH
• geny src * MeSH
• integrace viru genetika   MeSH
• křečci praví MeSH
• křeček rodu Mesocricetus MeSH
• molekulární sekvence - údaje MeSH
• nádorové buňky kultivované MeSH
• proviry genetika   MeSH
• repetitivní sekvence nukleových kyselin genetika   MeSH
• sekvenční analýza DNA MeSH
• viry ptačího sarkomu fyziologie   MeSH
• zvířata MeSH
• Check Tag
• křečci praví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, U.S. Gov't, P.H.S. MeSH
• Názvy látek
• DNA virů MeSH
• DNA MeSH

To understand the persistence of latently HIV-1 infected cells in virally suppressed infected patients, a number of in vitro models of HIV latency have been developed. In an attempt to mimic the in vivo situation as closely as possible, several models use primary cells and replication-competent viruses in combination with antiretroviral compounds to prevent ongoing replication. Latency is subsequently measured by HIV RNA and/or protein production after cellular activation. To discriminate between pre- and post-integration latency, integrase inhibitors are routinely used, preventing novel integrations upon cellular activation. Here, we show that this choice of antiretrovirals may still cause a bias of pre-integration latency in these models, as unintegrated HIV DNA can form and directly contribute to the levels of HIV RNA and protein production. We further show that the addition of reverse transcriptase inhibitors effectively suppresses the levels of episomal HIV DNA (as measured by 2-LTR circles) and decreases the levels of HIV transcription. Consequently, we show that latency levels described in models that only use integrase inhibitors may be overestimated. The inclusion of additional control conditions, such as 2-LTR quantification and the addition of reverse transcriptase inhibitors, is crucial to fully elucidate the actual levels of post-integration latency.

• MeSH
• aktivace lymfocytů MeSH
• alkyny MeSH
• benzoxaziny farmakologie   MeSH
• biologické modely MeSH
• CD4-pozitivní T-lymfocyty imunologie   virologie   MeSH
• cyklopropany MeSH
• DNA virů antagonisté a inhibitory   biosyntéza   genetika   MeSH
• HIV infekce farmakoterapie   imunologie   virologie   MeSH
• HIV-1 účinky léků   fyziologie   MeSH
• inhibitory HIV-integrasy farmakologie   MeSH
• inhibitory HIV-proteasy farmakologie   MeSH
• inhibitory reverzní transkriptasy farmakologie   MeSH
• integrace viru účinky léků   MeSH
• latence viru účinky léků   MeSH
• lidé MeSH
• nevirapin farmakologie   MeSH
• primární buněčná kultura MeSH
• raltegravirum kalicum farmakologie   MeSH
• replikace viru účinky léků   MeSH
• reportérové geny MeSH
• ritonavir farmakologie   MeSH
• virové proteiny antagonisté a inhibitory   biosyntéza   genetika   MeSH
• zelené fluorescenční proteiny genetika   metabolismus   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• alkyny MeSH
• benzoxaziny MeSH
• cyklopropany MeSH
• DNA virů MeSH
• efavirenz MeSH Prohlížeč
• enhanced green fluorescent protein MeSH Prohlížeč
• inhibitory HIV-integrasy MeSH
• inhibitory HIV-proteasy MeSH
• inhibitory reverzní transkriptasy MeSH
• nevirapin MeSH
• raltegravirum kalicum MeSH
• ritonavir MeSH
• virové proteiny MeSH
• zelené fluorescenční proteiny MeSH

Two patients with the characteristic high human herpesvirus 6 (HHV-6) DNA loads in peripheral blood caused by chromosomally integrated (CI) virus received a haematopoietic stem cell transplant (HSCT) from a donor without CI HHV-6. Both patients died in consequence of cytomegalovirus (CMV) pneumonitis. At autopsy, high amounts of CMV DNA were detected in lungs but at much lower levels in other organs. In contrast HHV-6 DNA was detected at high levels throughout the organs with the exception of donor-derived haematopoietic tissue. In individuals with chromosomal integration, HHV-6 DNA is found in every tissue of recipient origin indicating inheritance through the germ line.

• MeSH
• akutní lymfatická leukemie mortalita   terapie   virologie   MeSH
• akutní myeloidní leukemie mortalita   terapie   virologie   MeSH
• cytomegalovirové infekce mortalita   MeSH
• Cytomegalovirus izolace a purifikace   MeSH
• dítě MeSH
• DNA virů analýza   MeSH
• dospělí MeSH
• fatální výsledek MeSH
• infekce roseoloviry genetika   virologie   MeSH
• integrace viru genetika   MeSH
• lidé MeSH
• lidské chromozomy virologie   MeSH
• lidský herpesvirus 6 genetika   MeSH
• plíce virologie   MeSH
• pneumonie mortalita   virologie   MeSH
• polymerázová řetězová reakce s reverzní transkripcí MeSH
• transplantace hematopoetických kmenových buněk * MeSH
• Check Tag
• dítě MeSH
• dospělí MeSH
• lidé MeSH
• mužské pohlaví MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• kazuistiky MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH
• Názvy látek
• DNA virů MeSH