A weak palindromic nucleotide motif is the hallmark of retroviral integration site alignments. Given that the majority of target sequences are not palindromic, the current model explains the symmetry by an overlap of the nonpalindromic motif present on one of the half-sites of the sequences. Here, we show that the implementation of multicomponent mixture models allows for different interpretations consistent with the existence of both palindromic and nonpalindromic submotifs in the sets of integration site sequences. We further show that the weak palindromic motifs result from freely combined site-specific submotifs restricted to only a few positions proximal to the site of integration. The submotifs are formed by either palindrome-forming nucleotide preference or nucleotide exclusion. Using the mixture models, we also identify HIV-1-favored palindromic sequences in Alu repeats serving as local hotspots for integration. The application of the novel statistical approach provides deeper insight into the selection of retroviral integration sites and may prove to be a valuable tool in the analysis of any type of DNA motifs.

• MeSH
• integrace viru * genetika   MeSH
• nukleotidové motivy MeSH
• nukleotidy * MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• nukleotidy * MeSH

Both temperate and obligately lytic phages have crucial roles in the biology of staphylococci. While superinfection exclusion among closely related temperate phages is a well-characterized phenomenon, the interactions between temperate and lytic phages in staphylococci are not understood. Here, we present a resistance mechanism toward lytic phages of the genus Kayvirus, mediated by the membrane-anchored protein designated PdpSau encoded by Staphylococcus aureus prophages, mostly of the Sa2 integrase type. The prophage accessory gene pdpSau is strongly linked to the lytic genes for holin and ami2-type amidase and typically replaces genes for the toxin Panton-Valentine leukocidin (PVL). The predicted PdpSau protein structure shows the presence of a membrane-binding α-helix in its N-terminal part and a cytoplasmic positively charged C terminus. We demonstrated that the mechanism of action of PdpSau does not prevent the infecting kayvirus from adsorbing onto the host cell and delivering its genome into the cell, but phage DNA replication is halted. Changes in the cell membrane polarity and permeability were observed from 10 min after the infection, which led to prophage-activated cell death. Furthermore, we describe a mechanism of overcoming this resistance in a host-range Kayvirus mutant, which was selected on an S. aureus strain harboring prophage 53 encoding PdpSau, and in which a chimeric gene product emerged via adaptive laboratory evolution. This first case of staphylococcal interfamily phage-phage competition is analogous to some other abortive infection defense systems and to systems based on membrane-destructive proteins. IMPORTANCE Prophages play an important role in virulence, pathogenesis, and host preference, as well as in horizontal gene transfer in staphylococci. In contrast, broad-host-range lytic staphylococcal kayviruses lyse most S. aureus strains, and scientists worldwide have come to believe that the use of such phages will be successful for treating and preventing bacterial diseases. The effectiveness of phage therapy is complicated by bacterial resistance, whose mechanisms related to therapeutic staphylococcal phages are not understood in detail. In this work, we describe a resistance mechanism targeting kayviruses that is encoded by a prophage. We conclude that the defense mechanism belongs to a broader group of abortive infections, which is characterized by suicidal behavior of infected cells that are unable to produce phage progeny, thus ensuring the survival of the host population. Since the majority of staphylococcal strains are lysogenic, our findings are relevant for the advancement of phage therapy.

• Klíčová slova
• Kayvirus, Staphylococcus aureus, abortive infection, bacteriophage evolution, bacteriophage therapy, bacteriophages, cell death, lysogeny, phage resistance, phage therapy,
• MeSH
• lidé MeSH
• lyzogenie MeSH
• membránové proteiny genetika   MeSH
• profágy * genetika   MeSH
• stafylokokové bakteriofágy genetika   MeSH
• stafylokokové infekce * mikrobiologie   MeSH
• Staphylococcus aureus genetika   MeSH
• Staphylococcus MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• membránové proteiny MeSH

Widely distributed in water environments and in soil, cyanobacteria are hosts of lysogenic or lytic bacterioviruses. A novel, probably lysogenic virus (phage) for which the name Arthronema africanum virus TR020 (Aa-TR020) is proposed, has been isolated from filamentous freshwater cyanobacterium Arthronema africanum. The virus formed turbid plaques on plate culture of A. africanum strain 1980/01 but not on other Arthronema strain and other bacterial species. The genome of Aa-TR020 is linear molecule of dsDNA, 44,805 bp in length with 216 bp long terminal repeats and with G + C content of 46%. Fifty-five genes organized on plus and minus strands were predicted there. The genome size, gene arrangement, and selected protein sequences showed relatedness to Phormidium virus Pf-WMP3 and other viruses known to infect cyanobacteria and classified in the family Podoviridae.

• Klíčová slova
• Complete sequence, Podophage, Podovirus,
• MeSH
• bakteriofágy klasifikace   genetika   fyziologie   MeSH
• délka genomu MeSH
• DNA virů genetika   MeSH
• fylogeneze MeSH
• genom virový genetika   MeSH
• hostitelská specificita MeSH
• lyzogenie MeSH
• Podoviridae klasifikace   genetika   fyziologie   MeSH
• sekvence aminokyselin MeSH
• sekvenční analýza DNA MeSH
• sinice virologie   MeSH
• sladká voda mikrobiologie   MeSH
• virové proteiny genetika   MeSH
• zastoupení bazí MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• DNA virů MeSH
• virové proteiny MeSH

During the last two decades, progresses in bioimaging and the development of various strategies to fluorescently label the viral components opened a wide range of possibilities to visualize the early phase of Human Immunodeficiency Virus 1 (HIV-1) life cycle directly in infected cells. After fusion of the viral envelope with the cell membrane, the viral core is released into the cytoplasm and the viral RNA (vRNA) is retro-transcribed into DNA by the reverse transcriptase. During this process, the RNA-based viral complex transforms into a pre-integration complex (PIC), composed of the viral genomic DNA (vDNA) coated with viral and host cellular proteins. The protective capsid shell disassembles during a process called uncoating. The viral genome is transported into the cell nucleus and integrates into the host cell chromatin. Unlike biochemical approaches that provide global data about the whole population of viral particles, imaging techniques enable following individual viruses on a single particle level. In this context, quantitative microscopy has brought original data shedding light on the dynamics of the viral entry into the host cell, the cytoplasmic transport, the nuclear import, and the selection of the integration site. In parallel, multi-color imaging studies have elucidated the mechanism of action of host cell factors implicated in HIV-1 viral cycle progression. In this review, we describe the labeling strategies used for HIV-1 fluorescence imaging and report on the main advancements that imaging studies have brought in the understanding of the infection mechanisms from the viral entry into the host cell until the provirus integration step.

• Klíčová slova
• HIV-1, fluorescence microscopy, virus labeling,
• MeSH
• buněčné jádro virologie   MeSH
• fluorescenční mikroskopie MeSH
• HIV infekce virologie   MeSH
• HIV-1 chemie   genetika   fyziologie   MeSH
• integrace viru MeSH
• internalizace viru * MeSH
• lidé MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• přehledy MeSH

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

Endogenous retroviruses (ERVs) constitute a significant part of vertebrate genomes. They originated from past retroviral infections and some of them retain transcriptional activity. The key mechanism avoiding uncontrolled ERV transcription is DNA methylation-mediated epigenetic silencing. Despite numerous studies describing the involvement of ERV activity in cellular processes, epigenetic regulation of ERVs is still poorly understood. We previously described a cervid endogenous retrovirus (CrERV) in the mule deer genome. This virus exhibits massive insertional polymorphism, suggesting recent activity. Here we employed NGS-based strategy to determine the methylation pattern of CrERV integrations in four mule deer. Besides the vast majority of methylated integrations, we identified a tiny fraction of demethylated proviral copies. These copies represent evolutionary older integrations located near gene promoters. In general, our work is a first attempt to characterize the epigenetic landscape of insertionally polymorphic ERV on a whole-genome scale and offers insight into its interactions with a host.

• Klíčová slova
• DNA methylation, Endogenous retrovirus, Epigenetics, Mule deer, Retrovirus,
• MeSH
• endogenní retroviry * MeSH
• epigeneze genetická * MeSH
• Gammaretrovirus genetika   MeSH
• integrace viru MeSH
• koncové repetice MeSH
• metylace DNA MeSH
• vysoká zvěř genetika   MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

The human papillomavirus (HPV) integration, the critical step in viral carcinogenesis, most frequently occurs in the E2 gene, which results in its inactivation and in an increase of E6/E7 transcription. However, in a substantial number of tumors, the virus is present in an extrachromosomal form. For those tumors, the transformation mechanisms are not fully elucidated. Here we evaluated the possible mechanism of inactivating the E2 without interruption of the gene, methylation or mutation of the E2 binding sites (E2BSs) in HPV16-positive tonsillar tumors by next-generation and Sanger sequencing. Viral genome status was analyzed by the amplification of papillomavirus oncogene transcripts assay (APOT) and mRNA mapping, and expression of viral oncogenes was performed by qPCR. The methylation of E2BSs was significantly higher in tumors with an integrated, in comparison to extrachromosomal, form of the viral genome. No mutations were detected in the E2BSs. The viral oncogenes were equally expressed in samples with an integrated and extrachromosomal form of the virus. Only the nucleotide variants were identified in the E2 gene. No proposed mechanism of E2 inactivation was confirmed in tonsillar tumors with an extrachromosomal form of the HPV genome. The expression of E6/E7 genes seems to be sufficient to initiate and maintain the carcinogenic process.

• Klíčová slova
• E2 binding sites, genome status, human papillomavirus, methylation,
• MeSH
• DNA vazebné proteiny genetika   MeSH
• genom virový * MeSH
• infekce papilomavirem virologie   MeSH
• integrace viru * MeSH
• lidé MeSH
• lidský papilomavirus 16 genetika   MeSH
• metylace DNA MeSH
• mutace MeSH
• onkogenní proteiny virové genetika   MeSH
• tonzilární nádory virologie   MeSH
• vazebná místa MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• DNA vazebné proteiny MeSH
• E2 protein, Human papillomavirus type 16 MeSH Prohlížeč
• onkogenní proteiny virové 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

Critical behaviour pervades scientific disciplines as diverse as geology, economy or sociology. The critical behaviour of cell control systems is an open issue whose role has not yet been fully explored. The control of the expression of lambda phage DNA in the host cell can be classified as a system with critical behaviour. Lambda phage is a virus that infects Escherichia coli. Its core genes maintain one of two states; lysogeny or lysis. Current knowledge of the lambda phage genetic network allows to build a computational model of transcriptional control of the genes involved in the lytic-lysogenic switch and to simulate the temporal changes of their expression. Here, we focused on the computational simulation of these gene expressions to demonstrate critical behaviour of the system.

• Klíčová slova
• Computational simulation, Critical behaviour, Genetic networks, Phage lambda,
• MeSH
• bakteriofág lambda genetika   MeSH
• Escherichia coli virologie   MeSH
• genové regulační sítě MeSH
• lyzogenie genetika   MeSH
• modely genetické * MeSH
• regulace exprese virových genů MeSH
• virové geny MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH