RESUMO
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.
Assuntos
Nucleotídeos , Integração Viral , Integração Viral/genética , Motivos de NucleotídeosRESUMO
Genetic editing of the germline using CRISPR/Cas9 technology has made it possible to alter livestock traits, including the creation of resistance to viral diseases. However, virus adaptability could present a major obstacle in this effort. Recently, chickens resistant to avian leukosis virus subgroup J (ALV-J) were developed by deleting a single amino acid, W38, within the ALV-J receptor NHE1 using CRISPR/Cas9 genome editing. This resistance was confirmed both in vitro and in vivo. In vitro resistance of W38-/- chicken embryonic fibroblasts to all tested ALV-J strains was shown. To investigate the capacity of ALV-J for further adaptation, we used a retrovirus reporter-based assay to select adapted ALV-J variants. We assumed that adaptive mutations overcoming the cellular resistance would occur within the envelope protein. In accordance with this assumption, we isolated and sequenced numerous adapted virus variants and found within their envelope genes eight independent single nucleotide substitutions. To confirm the adaptive capacity of these substitutions, we introduced them into the original retrovirus reporter. All eight variants replicated effectively in W38-/- chicken embryonic fibroblasts in vitro while in vivo, W38-/- chickens were sensitive to tumor induction by two of the variants. Importantly, receptor alleles with more extensive modifications have remained resistant to the virus. These results demonstrate an important strategy in livestock genome engineering towards antivirus resistance and illustrate that cellular resistance induced by minor receptor modifications can be overcome by adapted virus variants. We conclude that more complex editing will be necessary to attain robust resistance.
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IMPORTANCE: Birds represent important hosts for numerous viruses, including zoonotic viruses and pathogens with the potential to cause major economic losses to the poultry industry. Viral replication and transmission can be inhibited or blocked by the action of antiviral restriction factors (RFs) encoded by the host. One well-characterized RF is tetherin, a protein that directly blocks the release of newly formed viral particles from infected cells. Here, we describe the evolutionary loss of a functional tetherin gene in two galliform birds, turkey (Meleagris gallopavo) and Mikado pheasant (Syrmaticus mikado). Moreover, we demonstrate that the structurally related protein TMCC(aT) exerts antiviral activity in several birds, albeit by a mechanism different from that of tetherin. The evolutionary scenario described here represents the first documented loss-of-tetherin cases in vertebrates.
Assuntos
Proteínas Ligadas por GPI , Galliformes , Animais , Antígenos CD/genética , Antígenos CD/metabolismo , Evolução Biológica , Antígeno 2 do Estroma da Médula Óssea/genética , Proteínas Ligadas por GPI/genética , Proteínas Ligadas por GPI/metabolismo , Galliformes/genética , Evolução Molecular , Proteínas Aviárias/genética , Proteínas Aviárias/metabolismoRESUMO
Avian leukosis virus subgroup J (ALV-J) is an important concern for the poultry industry. Replication of ALV-J depends on a functional cellular receptor, the chicken Na+/H+ exchanger type 1 (chNHE1). Tryptophan residue number 38 of chNHE1 (W38) in the extracellular portion of this molecule is a critical amino acid for virus entry. We describe a CRISPR/Cas9-mediated deletion of W38 in chicken primordial germ cells and the successful production of the gene-edited birds. The resistance to ALV-J was examined both in vitro and in vivo, and the ΔW38 homozygous chickens tested ALV-J-resistant, in contrast to ΔW38 heterozygotes and wild-type birds, which were ALV-J-susceptible. Deletion of W38 did not manifest any visible side effect. Our data clearly demonstrate the antiviral resistance conferred by precise CRISPR/Cas9 gene editing in the chicken. Furthermore, our highly efficient CRISPR/Cas9 gene editing in primordial germ cells represents a substantial addition to genotechnology in the chicken, an important food source and research model.
Assuntos
Vírus da Leucose Aviária/genética , Leucose Aviária/imunologia , Proteínas Aviárias/genética , Doenças das Aves Domésticas/imunologia , Trocador 1 de Sódio-Hidrogênio/genética , Animais , Animais Geneticamente Modificados/genética , Animais Geneticamente Modificados/imunologia , Animais Geneticamente Modificados/virologia , Leucose Aviária/genética , Leucose Aviária/virologia , Vírus da Leucose Aviária/classificação , Vírus da Leucose Aviária/fisiologia , Proteínas Aviárias/imunologia , Sistemas CRISPR-Cas , Galinhas , Resistência à Doença , Feminino , Edição de Genes , Masculino , Doenças das Aves Domésticas/genética , Doenças das Aves Domésticas/virologia , Trocador 1 de Sódio-Hidrogênio/imunologiaRESUMO
The Avian sarcoma and leukosis viruses (ASLVs) are important chicken pathogens. Some of the virus subgroups, including ASLV-A and K, utilize the Tva receptor for cell entrance. Though Tva was identified three decades ago, its physiological function remains unknown. Previously, we have noted an intriguing resemblance and orthology between the chicken gene coding for Tva and the human gene coding for CD320, a receptor involved in cellular uptake of transcobalamin (TC) in complex with vitamin B12/cobalamin (Cbl).Here we show that both the transmembrane and the glycosylphosphatidylinositol (GPI)-anchored form of Tva in the chicken cell line DF-1 promotes the uptake of Cbl with help of expressed and purified chicken TC. The uptake of TC-Cbl complex was monitored using an isotope- or fluorophore-labeled Cbl. We show that (i) TC-Cbl is internalized in chicken cells; and (ii) the uptake is lower in the Tva-knockout cells and higher in Tva-overexpressing cells when compared with wild type chicken cells. The relation between physiological function of Tva and its role in infection was elaborated by showing that infection with ASLV subgroups (targeting Tva) impairs the uptake of TC-Cbl, while this is not the case for cells infected with ASLV-B (not recognized by Tva). In addition, exposure of the cells to a high concentration of TC-Cbl alleviates the infection with Tva-dependent ASLV.IMPORTANCE: We demonstrate that the ASLV receptor Tva participates in the physiological uptake of TC-Cbl, because the viral infection suppresses the uptake of Cbl and vice versa. Our results pave the road for future studies addressing the issues: (i) whether a virus infection can be inhibited by TC-Cbl complexes in vivo; and (ii) whether any human virus employs the human TC-Cbl receptor CD320. In broader terms, our study sheds light on the intricate interplay between physiological roles of cellular receptors and their involvement in virus infection.
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BACKGROUND: Longevity-related genes have been found in several animal species as well as in humans. The goal of this study was to perform genetic analysis of long-lived Cane corso dogs with the aim to find genes that are associated with longevity. RESULTS: SNPs with particular nucleotides were significantly overrepresented in long-lived dogs in four genes, TDRP, MC2R, FBXO25 and FBXL21. In FBXL21, the longevity-associated SNP localises to the exon. In the FBXL21 protein, tryptophan in long-lived dogs replaced arginine present in reference dogs. CONCLUSIONS: Four SNPs associated with longevity in dogs were identified using GWAS and validated by DNA sequencing. We conclude that genes TDRP, MC2R, FBXO25 and FBXL21 are associated with longevity in Cane corso dogs.
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Bengala , Longevidade , Animais , Cães , Longevidade/genética , Polimorfismo de Nucleotídeo ÚnicoRESUMO
BACKGROUND: Human Syncytin-1 is a placentally-expressed cell surface glycoprotein of retroviral origin. After interaction with ASCT2, its cellular receptor, Syncytin-1 triggers cell-cell fusion and formation of a multinuclear syncytiotrophoblast layer of the placenta. The ASCT2 receptor is a multi-spanning membrane protein containing a protruding extracellular part called region C, which has been suggested to be a retrovirus docking site. Precise identification of the interaction site between ASCT2 and Syncytin-1 is challenging due to the complex structure of ASCT2 protein and the background of endogenous ASCT2 gene in the mammalian genome. Chicken cells lack the endogenous background and, therefore, can be used to set up a system with surrogate expression of the ASCT2 receptor. RESULTS: We have established a retroviral heterologous chicken system for rapid and reliable assessment of ectopic human ASCT2 protein expression. Our dual-fluorescence system proved successful for large-scale screening of mutant ASCT2 proteins. Using this system, we demonstrated that progressive deletion of region C substantially decreased the amount of ASCT2 protein. In addition, we implemented quantitative assays to determine the interaction of ASCT2 with Syncytin-1 at multiple levels, which included binding of the soluble form of Syncytin-1 to ASCT2 on the cell surface and a luciferase-based assay to evaluate cell-cell fusions that were triggered by Syncytin-1. Finally, we restored the envelope function of Syncytin-1 in a replication-competent retrovirus and assessed the infection of chicken cells expressing human ASCT2 by chimeric Syncytin-1-enveloped virus. The results of the quantitative assays showed that deletion of the protruding region C did not abolish the interaction of ASCT2 with Syncytin-1. CONCLUSIONS: We present here a heterologous chicken system for effective assessment of the expression of transmembrane ASCT2 protein and its interaction with Syncytin-1. The system profits from the absence of endogenous ASCT2 background and implements the quantitative assays to determine the ASCT2-Syncytin-1 interaction at several levels. Using this system, we demonstrated that the protruding region C was essential for ASCT2 protein expression, but surprisingly, not for the interaction with Syncytin-1 glycoprotein.
Assuntos
Sistema ASC de Transporte de Aminoácidos/genética , Sistema ASC de Transporte de Aminoácidos/metabolismo , Produtos do Gene env/metabolismo , Antígenos de Histocompatibilidade Menor/genética , Antígenos de Histocompatibilidade Menor/metabolismo , Proteínas da Gravidez/metabolismo , Animais , Linhagem Celular , Galinhas , Feminino , Fibroblastos/virologia , Fluorescência , Produtos do Gene env/genética , Humanos , Microscopia Confocal , Placenta/virologia , Gravidez , Proteínas da Gravidez/genéticaRESUMO
Tetherin/BST-2 is an antiviral protein that blocks the release of enveloped viral particles by linking them to the membrane of producing cells. At first, BST-2 genes were described only in humans and other mammals. Recent work identified BST-2 orthologs in nonmammalian vertebrates, including birds. Here, we identify the BST-2 sequence in domestic chicken (Gallus gallus) for the first time and demonstrate its activity against avian sarcoma and leukosis virus (ASLV). We generated a BST-2 knockout in chicken cells and showed that BST-2 is a major determinant of an interferon-induced block of ASLV release. Ectopic expression of chicken BST-2 blocks the release of ASLV in chicken cells and of human immunodeficiency virus type 1 (HIV-1) in human cells. Using metabolic labeling and pulse-chase analysis of HIV-1 Gag proteins, we verified that chicken BST-2 blocks the virus at the release stage. Furthermore, we describe BST-2 orthologs in multiple avian species from 12 avian orders. Previously, some of these species were reported to lack BST-2, highlighting the difficulty of identifying sequences of this extremely variable gene. We analyzed BST-2 genes in the avian orders Galliformes and Passeriformes and showed that they evolve under positive selection. This indicates that avian BST-2 is involved in host-virus evolutionary arms races and suggests that BST-2 antagonists exist in some avian viruses. In summary, we show that chicken BST-2 has the potential to act as a restriction factor against ASLV. Characterizing the interaction of avian BST-2 with avian viruses is important in understanding innate antiviral defenses in birds.IMPORTANCE Birds are important hosts of viruses that have the potential to cause zoonotic infections in humans. However, only a few antiviral genes (called viral restriction factors) have been described in birds, mostly because birds lack counterparts of highly studied mammalian restriction factors. Tetherin/BST-2 is a restriction factor, originally described in humans, that blocks the release of newly formed virus particles from infected cells. Recent work identified BST-2 in nonmammalian vertebrate species, including birds. Here, we report the BST-2 sequence in domestic chicken and describe its antiviral activity against a prototypical avian retrovirus, avian sarcoma and leukosis virus (ASLV). We also identify BST-2 genes in multiple avian species and show that they evolve rapidly in birds, which is an important indication of their relevance for antiviral defense. Analysis of avian BST-2 genes will shed light on defense mechanisms against avian viral pathogens.
Assuntos
Proteínas Aviárias/imunologia , Vírus do Sarcoma Aviário/imunologia , Antígeno 2 do Estroma da Médula Óssea/imunologia , Evolução Molecular , Galliformes/imunologia , Sarcoma Aviário/imunologia , Sequência de Aminoácidos , Animais , Proteínas Aviárias/genética , Vírus do Sarcoma Aviário/genética , Vírus do Sarcoma Aviário/patogenicidade , Antígeno 2 do Estroma da Médula Óssea/genética , Linhagem Celular , Fibroblastos/imunologia , Fibroblastos/virologia , Galliformes/genética , Galliformes/virologia , Regulação da Expressão Gênica , Células HEK293 , HIV-1/genética , HIV-1/imunologia , Interações Hospedeiro-Patógeno/genética , Interações Hospedeiro-Patógeno/imunologia , Humanos , Passeriformes/genética , Passeriformes/imunologia , Passeriformes/virologia , Sarcoma Aviário/genética , Sarcoma Aviário/virologia , Seleção Genética , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Transdução de Sinais , Liberação de Vírus , Replicação Viral , Produtos do Gene gag do Vírus da Imunodeficiência Humana/genética , Produtos do Gene gag do Vírus da Imunodeficiência Humana/imunologiaRESUMO
Avian leukosis virus subgroup K (ALV-K) is composed of newly emerging isolates, which, in sequence analyses, cluster separately from the well-characterized subgroups A, B, C, D, E, and J. However, it remains unclear whether ALV-K represents an independent ALV subgroup with regard to receptor usage, host range, and superinfection interference. In the present study, we examined the host range of the Chinese infectious isolate JS11C1, an ALV-K prototype, and we found substantial overlap of species that were either resistant or susceptible to ALV-A and JS11C1. Ectopic expression of the chicken tva gene in mammalian cells conferred susceptibility to JS11C1, while genetic ablation of the tva gene rendered chicken DF-1 cells resistant to infection by JS11C1. Thus, tva expression is both sufficient and necessary for JS11C1 entry. Receptor sharing was also manifested in superinfection interference, with preinfection of cells with ALV-A, but not ALV-B or ALV-J, blocking subsequent JS11C1 infection. Finally, direct binding of JS11C1 and Tva was demonstrated by preincubation of the virus with soluble Tva, which substantially decreased viral infectivity in susceptible chicken cells. Collectively, these findings indicate that JS11C1 represents a new and bona fide ALV subgroup that utilizes Tva for cell entry and binds to a site other than that for ALV-A.IMPORTANCE ALV consists of several subgroups that are particularly characterized by their receptor usage, which subsequently dictates the host range and tropism of the virus. A few newly emerging and highly pathogenic Chinese ALV strains have recently been suggested to be an independent subgroup, ALV-K, based solely on their genomic sequences. Here, we performed a series of experiments with the ALV-K strain JS11C1, which showed its dependence on the Tva cell surface receptor. Due to the sharing of this receptor with ALV-A, both subgroups were able to interfere with superinfection. Because ALV-K could become an important pathogen and a significant threat to the poultry industry in Asia, the identification of a specific receptor could help in the breeding of resistant chicken lines with receptor variants with decreased susceptibility to the virus.
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Vírus da Leucose Aviária/patogenicidade , Leucose Aviária/genética , Proteínas Aviárias/genética , Proteínas Aviárias/metabolismo , Receptores Virais/genética , Receptores Virais/metabolismo , Animais , Leucose Aviária/metabolismo , Leucose Aviária/virologia , Vírus da Leucose Aviária/classificação , Vírus da Leucose Aviária/fisiologia , Linhagem Celular , Galinhas , Suscetibilidade a Doenças , Fibroblastos/citologia , Fibroblastos/metabolismo , Fibroblastos/virologia , Mesocricetus , Especificidade da Espécie , Internalização do VírusRESUMO
Successful derivation and cultivation of primordial germ cells (PGCs) opened the way to efficient transgenesis and genome editing in the chicken. Furthermore, implantation of male PGCs from non-chicken galliform species into the chicken embryos resulted in cross-species germline chimeras and viable offspring. We have recently improved the PGC technology by demonstrating that chicken male PGCs transplanted into the testes of adult cockerel recipients mature into functional sperms. However, the availability of this orthotopic transplantation for cross-species transfer remains to be explored. Here we tested the capacity of genetically distant male PGCs to mature in the microenvironment of adult testes. We derived PGCs from the Chinese black-bone Silkie and transplanted them into infertile White Leghorn cockerels. Within 15-18 weeks after transplantation, we observed restoration of spermatogenesis in recipient cockerels and production of healthy progeny derived from the transplanted PGCs. Our findings also indicate the possibility of cross-species orthotopic transplantation of PGCs. Thus, our results might contribute to the preservation of endangered avian species and maintaining the genetic variability of the domestic chicken.
Assuntos
Galinhas , Quimera/genética , Conservação dos Recursos Naturais , Células Germinativas/transplante , Espermatozoides/citologia , Animais , Cruzamento/métodos , Células Cultivadas , Embrião de Galinha , Galinhas/classificação , Galinhas/genética , Conservação dos Recursos Naturais/métodos , Cruzamentos Genéticos , Espécies em Perigo de Extinção , Preservação da Fertilidade/métodos , Preservação da Fertilidade/veterinária , Masculino , Espermatogênese/fisiologia , Espermatozoides/transplante , Testículo/citologia , Transplante Heterólogo/veterináriaRESUMO
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 â¼3% 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.
Assuntos
Alpharetrovirus/genética , Elementos Facilitadores Genéticos/genética , Regiões Promotoras Genéticas/genética , Transcrição Gênica , Animais , Linhagem Celular , Ilhas de CpG/genética , Epigênese Genética , Inativação Gênica , Vetores Genéticos/genética , Humanos , Células K562 , Provírus/genética , Integração Viral/genéticaRESUMO
The J subgroup of avian leukosis virus (ALV-J) infects domestic chickens, jungle fowl, and turkeys. This virus enters the host cell through a receptor encoded by the tvj locus and identified as Na+/H+ exchanger 1. The resistance to avian leukosis virus subgroup J in a great majority of galliform species has been explained by deletions or substitutions of the critical tryptophan 38 in the first extracellular loop of Na+/H+ exchanger 1. Because there are concerns of transspecies virus transmission, we studied natural polymorphisms and susceptibility/resistance in wild galliforms and found the presence of tryptophan 38 in four species of New World quails. The embryo fibroblasts of New World quails are susceptible to infection with avian leukosis virus subgroup J, and the cloned Na+/H+ exchanger 1 confers susceptibility on the otherwise resistant host. New World quails are also susceptible to new avian leukosis virus subgroup J variants but resistant to subgroups A and B and weakly susceptible to subgroups C and D of avian sarcoma/leukosis virus due to obvious defects of the respective receptors. Our results suggest that the avian leukosis virus subgroup J could be transmitted to New World quails and establish a natural reservoir of circulating virus with a potential for further evolution. IMPORTANCE: Since its spread in broiler chickens in China and Southeast Asia in 2000, ALV-J remains a major enzootic challenge for the poultry industry. Although the virus diversifies rapidly in the poultry, its spillover and circulation in wild bird species has been prevented by the resistance of most species to ALV-J. It is, nevertheless, important to understand the evolution of the virus and its potential host range in wild birds. Because resistance to avian retroviruses is due particularly to receptor incompatibility, we studied Na+/H+ exchanger 1, the receptor for ALV-J. In New World quails, we found a receptor compatible with virus entry, and we confirmed the susceptibilities of four New World quail species in vitro We propose that a prospective molecular epidemiology study be conducted to identify species with the potential to become reservoirs for ALV-J.
Assuntos
Vírus da Leucose Aviária/fisiologia , Leucose Aviária/genética , Leucose Aviária/virologia , Suscetibilidade a Doenças , Codorniz , Sequência de Aminoácidos , Aminoácidos , Animais , Leucose Aviária/metabolismo , Vírus da Leucose Aviária/classificação , Células Cultivadas , Resistência à Doença/genética , Evolução Molecular , Expressão Gênica , Loci Gênicos , Especificidade de Hospedeiro , Interações Hospedeiro-Patógeno , Filogenia , Polimorfismo Genético , Domínios e Motivos de Interação entre Proteínas , Trocadores de Sódio-Hidrogênio/química , Trocadores de Sódio-Hidrogênio/genética , Trocadores de Sódio-Hidrogênio/metabolismo , Replicação ViralRESUMO
BACKGROUND: Syncytin-1 and 2, human fusogenic glycoproteins encoded by the env genes of the endogenous retroviral loci ERVWE1 and ERVFRDE1, respectively, contribute to the differentiation of multinucleated syncytiotrophoblast in chorionic villi. In non-trophoblastic cells, however, the expression of syncytins has to be suppressed to avoid potential pathogenic effects. Previously, we have shown that the transcriptional suppression of ERVWE1 promoter is controlled epigenetically by DNA methylation and chromatin modifications. In this study, we describe the aberrant expression of syncytin-1 in biopsies of testicular germ cell tumors. RESULTS: We found efficient expression and splicing of syncytin-1 in seminomas and mixed germ cell tumors with seminoma component. Although another fusogenic gene, syncytin-2 was also derepressed in seminomas, its expression was significantly lower than that of syncytin-1. Neither the transcription factor GCM1 nor the increased copy number of ERVWE1 were sufficient for this aberrant expression of syncytin-1 in seminomas. In accordance with our recent finding of the highly increased expression of TET1 dioxygenase in most seminomas, the ERVWE1 promoter was significantly hypomethylated in comparison with the matched controls. In contrast, 5-hydroxymethylcytosine levels were not detectable at the ERVWE1 promoter. We further describe that another endogenous retroviral element adjacent to ERVWE1 remains transcriptionally suppressed and two additional HERV-W family members are only slightly upregulated in seminomas. CONCLUSIONS: We conclude that DNA demethylation of the ERVWE1 promoter in seminomas is a prerequisite for syncytin-1 derepression. We propose the spliced syncytin-1 expression as a marker of seminoma and suggest that aberrant expression of endogenous retroviruses might be a correlate of the hypomethylated genome of seminomas.
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DNA Viral/metabolismo , Retrovirus Endógenos/genética , Regulação da Expressão Gênica , Produtos do Gene env/biossíntese , Proteínas da Gravidez/biossíntese , Seminoma/patologia , Neoplasias Testiculares/patologia , Metilação de DNA , Epigênese Genética , Humanos , Masculino , Seminoma/virologia , Neoplasias Testiculares/virologiaRESUMO
Germ cell tumors and particularly seminomas reflect the epigenomic features of their parental primordial germ cells (PGCs), including genomic DNA hypomethylation and expression of pluripotent cell markers. Because the DNA hypomethylation might be a result of TET dioxygenase activity, we examined expression of TET1-3 enzymes and the level of their product, 5-hydroxymethylcytosine (5hmC), in a panel of histologically characterized seminomas and non-seminomatous germ cell tumors. Expression of TET dioxygenase mRNAs was quantified by real-time PCR. TET1 expression and the level of 5hmC were examined immunohistochemically. Quantitative assessment of 5-methylcytosine (5mC) and 5hmC levels was done by the liquid chromatography-mass spectroscopy technique. We found highly increased expression of TET1 dioxygenase in most seminomas and strong TET1 staining in seminoma cells. Isocitrate dehydrogenase 1 and 2 mutations were not detected, suggesting the enzymatic activity of TET1. The levels of 5mC and 5hmC in seminomas were found decreased in comparison to non-seminomatous germ cell tumors and healthy testicular tissue. We propose that TET1 expression should be studied as a potential marker of seminomas and mixed germ cell tumors and we suggest that elevated expression of TET dioxygenase enzymes is associated with the maintenance of low DNA methylation levels in seminomas. This "anti-methylator" phenotype of seminomas is in contrast to the CpG island methylator phenotype (CIMP) observed in a fraction of tumors of various types.
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Metilação de DNA , Proteínas de Ligação a DNA/genética , Dioxigenases/genética , Oxigenases de Função Mista/genética , Proteínas Proto-Oncogênicas/genética , Seminoma/genética , Neoplasias Testiculares/genética , Testículo/patologia , 5-Metilcitosina/análogos & derivados , 5-Metilcitosina/análise , Adulto , Proteínas de Ligação a DNA/análise , Dioxigenases/análise , Regulação Neoplásica da Expressão Gênica , Humanos , Masculino , Oxigenases de Função Mista/análise , Proteínas Proto-Oncogênicas/análise , Seminoma/patologia , Neoplasias Testiculares/patologia , Testículo/metabolismo , Regulação para CimaRESUMO
Systems of antigen delivery into antigen-presenting cells represent an important novel strategy in chicken vaccine development. In this study, we verified the ability of Rous sarcoma virus (RSV) antigens fused with streptavidin to be targeted by specific biotinylated monoclonal antibody (anti-CD205) into dendritic cells and induce virus-specific protective immunity. The method was tested in four congenic lines of chickens that are either resistant or susceptible to the progressive growth of RSV-induced tumors. Our analyses confirmed that the biot-anti-CD205-SA-FITC complex was internalized by chicken splenocytes. In the cytokine expression profile, several significant differences were evident between RSV-challenged progressor and regressor chicken lines. A significant up-regulation of IL-2, IL-12, IL-15, and IL-18 expression was detected in immunized chickens of both regressor and progressor groups. Of these cytokines, IL-2 and IL-12 were most up-regulated 14 days post-challenge (dpc), while IL-15 and IL-18 were most up-regulated at 28 dpc. On the contrary, IL-10 expression was significantly down-regulated in all immunized groups of progressor chickens at 14 dpc. We detected significant up-regulation of IL-17 in the group of immunized progressors. LITAF down-regulation with iNOS up-regulation was especially observed in the progressor group of immunized chickens that developed large tumors. Based on the increased expression of cytokines specific for activated dendritic cells, we conclude that our system is able to induce partial stimulation of specific cell types involved in cell-mediated immunity.
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Antígenos Virais/imunologia , Galinhas/virologia , Citocinas/fisiologia , Células Dendríticas/imunologia , Vírus do Sarcoma de Rous/imunologia , Sarcoma Aviário/prevenção & controle , Vacinas Virais/imunologia , Animais , Animais Congênicos/imunologia , Animais Congênicos/virologia , Anticorpos Biespecíficos/imunologia , Antígenos CD/imunologia , Galinhas/imunologia , Células Dendríticas/virologia , Imunidade Celular/imunologia , Lectinas Tipo C/imunologia , Antígenos de Histocompatibilidade Menor/imunologia , Receptores de Superfície Celular/imunologia , Sarcoma Aviário/imunologiaRESUMO
UNLABELLED: Transformation of rodent cells with avian Rous sarcoma virus (RSV) opened new ways to studying virus integration and expression in nonpermissive cells. We were interested in (i) the molecular changes accompanying fusion of RSV-transformed mammalian cells with avian cells leading to virus rescue and (ii) enhancement of this process by retroviral gene products. The RSV-transformed hamster RSCh cell line was characterized as producing only a marginal amount of env mRNA, no envelope glycoprotein, and a small amount of unprocessed Gag protein. Egress of viral unspliced genomic RNA from the nucleus was hampered, and its stability decreased. Cell fusion of the chicken DF-1 cell line with RSCh cells led to production of env mRNA, envelope glycoprotein, and processed Gag and virus-like particle formation. Proteosynthesis inhibition in DF-1 cells suppressed steps leading to virus rescue. Furthermore, new aberrantly spliced env mRNA species were found in the RSCh cells. Finally, we demonstrated that virus rescue efficiency can be significantly increased by complementation with the env gene and the highly expressed gag gene and can be increased the most by a helper virus infection. In summary, Env and Gag synthesis is increased after RSV-transformed hamster cell fusion with chicken fibroblasts, and both proteins provided in trans enhance RSV rescue. We conclude that the chicken fibroblast yields some factor(s) needed for RSV replication, particularly Env and Gag synthesis, in nonpermissive rodent cells. IMPORTANCE: One of the important issues in retrovirus heterotransmission is related to cellular factors that prevent virus replication. Rous sarcoma virus (RSV), a member of the avian sarcoma and leukosis family of retroviruses, is able to infect and transform mammalian cells; however, such transformed cells do not produce infectious virus particles. Using the well-defined model of RSV-transformed rodent cells, we established that the lack of virus replication is due to the absence of chicken factor(s), which can be supplemented by cell fusion. Cell fusion with permissive chicken cells led to an increase in RNA splicing and nuclear export of specific viral mRNAs, as well as synthesis of respective viral proteins and production of virus-like particles. RSV rescue by cell fusion can be potentiated by in trans expression of viral genes in chicken cells. We conclude that rodent cells lack some chicken factor(s) required for proper viral RNA processing and viral protein synthesis.
Assuntos
Doenças das Aves Domésticas/virologia , Vírus do Sarcoma de Rous/genética , Sarcoma Aviário/virologia , Animais , Fusão Celular , Linhagem Celular Transformada , Transformação Celular Viral , Galinhas , Cricetinae , Produtos do Gene env/genética , Produtos do Gene env/metabolismo , Produtos do Gene gag/genética , Produtos do Gene gag/metabolismo , Teste de Complementação Genética , Vírus do Sarcoma de Rous/fisiologiaRESUMO
Acellular materials of xenogenic origin are used worldwide as xenografts, and phase I trials of viable pig pancreatic islets are currently being performed. However, limited information is available on transmission of porcine endogenous retrovirus (PERV) after xenotransplantation and on the long-term immune response of recipients to xenoantigens. We analyzed the blood of burn patients who had received living pig-skin dressings for up to 8 wk for the presence of PERV as well as for the level and nature of their long term (maximum, 34 y) immune response against pig Ags. Although no evidence of PERV genomic material or anti-PERV Ab response was found, we observed a moderate increase in anti-αGal Abs and a high and sustained anti-non-αGal IgG response in those patients. Abs against the nonhuman sialic acid Neu5Gc constituted the anti-non-αGal response with the recognition pattern on a sialoglycan array differing from that of burn patients treated without pig skin. These data suggest that anti-Neu5Gc Abs represent a barrier for long-term acceptance of porcine xenografts. Because anti-Neu5Gc Abs can promote chronic inflammation, the long-term safety of living and acellular pig tissue implants in recipients warrants further evaluation.
Assuntos
Antígenos Heterófilos/imunologia , Queimaduras/cirurgia , Ácidos Siálicos/imunologia , Transplante de Pele/efeitos adversos , Transplante Heterólogo/efeitos adversos , Adolescente , Adulto , Idoso , Animais , Antígenos Heterófilos/análise , Criança , Retrovirus Endógenos/imunologia , Ensaio de Imunoadsorção Enzimática , Feminino , Humanos , Imunoglobulina G , Lactente , Masculino , Pessoa de Meia-Idade , RNA Viral/análise , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transplante de Pele/métodos , SuínosRESUMO
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.
Assuntos
Metilação de DNA , Retrovirus Endógenos/genética , Epigênese Genética , Doenças dos Suínos/transmissão , Porco Miniatura/virologia , Replicação Viral , Animais , Células Cultivadas , DNA Viral/genética , Humanos , Rim/metabolismo , Rim/virologia , Provírus/genética , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Suínos , Doenças dos Suínos/genética , Doenças dos Suínos/virologia , Porco Miniatura/genética , Sequências Repetidas Terminais/genéticaRESUMO
Subgroup J avian leukosis virus (ALV-J) is unique among the avian sarcoma and leukosis viruses in using the multimembrane-spanning cell surface protein Na(+)/H(+) exchanger type 1 (NHE1) as a receptor. The precise localization of amino acids critical for NHE1 receptor activity is key in understanding the virus-receptor interaction and potential interference with virus entry. Because no resistant chicken lines have been described until now, we compared the NHE1 amino acid sequences from permissive and resistant galliform species. In all resistant species, the deletion or substitution of W38 within the first extracellular loop was observed either alone or in the presence of other incidental amino acid changes. Using the ectopic expression of wild-type or mutated chicken NHE1 in resistant cells and infection with a reporter recombinant retrovirus of subgroup J specificity, we studied the effect of individual mutations on the NHE1 receptor capacity. We suggest that the absence of W38 abrogates binding of the subgroup J envelope glycoprotein to ALV-J-resistant cells. Altogether, we describe the functional importance of W38 for virus entry and conclude that natural polymorphisms in NHE1 can be a source of host resistance to ALV-J.
Assuntos
Vírus da Leucose Aviária/fisiologia , Receptores Virais/genética , Receptores Virais/metabolismo , Trocadores de Sódio-Hidrogênio/genética , Trocadores de Sódio-Hidrogênio/metabolismo , Tropismo Viral , Internalização do Vírus , Animais , Aves , Análise Mutacional de DNA , Triptofano/genética , Triptofano/metabolismoRESUMO
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.