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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.
Kryštof Štafl; Martin Trávníček; Dana Kučerová; Ľubomíra Pecnová; Veronika Krchlíková; Eliška Gáliková; Volodymyr Stepanets; Jiří Hejnar; Kateřina Trejbalová. Heterologous avian system for quantitative analysis of Syncytin-1 interaction with ASCT2 receptor. Retrovirology 2021, 18, 1 -18.
AMA StyleKryštof Štafl, Martin Trávníček, Dana Kučerová, Ľubomíra Pecnová, Veronika Krchlíková, Eliška Gáliková, Volodymyr Stepanets, Jiří Hejnar, Kateřina Trejbalová. Heterologous avian system for quantitative analysis of Syncytin-1 interaction with ASCT2 receptor. Retrovirology. 2021; 18 (1):1-18.
Chicago/Turabian StyleKryštof Štafl; Martin Trávníček; Dana Kučerová; Ľubomíra Pecnová; Veronika Krchlíková; Eliška Gáliková; Volodymyr Stepanets; Jiří Hejnar; Kateřina Trejbalová. 2021. "Heterologous avian system for quantitative analysis of Syncytin-1 interaction with ASCT2 receptor." Retrovirology 18, no. 1: 1-18.
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.
Anna Koslová; Pavel Trefil; Jitka Mucksová; Markéta Reinišová; Jiří Plachý; Jiří Kalina; Dana Kučerová; Josef Geryk; Veronika Krchlíková; Barbora Lejčková; Jiří Hejnar. Precise CRISPR/Cas9 editing of the NHE1 gene renders chickens resistant to the J subgroup of avian leukosis virus. Proceedings of the National Academy of Sciences 2020, 117, 2108 -2112.
AMA StyleAnna Koslová, Pavel Trefil, Jitka Mucksová, Markéta Reinišová, Jiří Plachý, Jiří Kalina, Dana Kučerová, Josef Geryk, Veronika Krchlíková, Barbora Lejčková, Jiří Hejnar. Precise CRISPR/Cas9 editing of the NHE1 gene renders chickens resistant to the J subgroup of avian leukosis virus. Proceedings of the National Academy of Sciences. 2020; 117 (4):2108-2112.
Chicago/Turabian StyleAnna Koslová; Pavel Trefil; Jitka Mucksová; Markéta Reinišová; Jiří Plachý; Jiří Kalina; Dana Kučerová; Josef Geryk; Veronika Krchlíková; Barbora Lejčková; Jiří Hejnar. 2020. "Precise CRISPR/Cas9 editing of the NHE1 gene renders chickens resistant to the J subgroup of avian leukosis virus." Proceedings of the National Academy of Sciences 117, no. 4: 2108-2112.
Avian sarcoma and leukosis virus (ASLV), diversified into seven phylogenetically relativesubgroups (A, B, C, D, E, J, and K), present as either exogenous or endogenous viruses in domesticchicken.
Jiří Hejnar; Anna Koslová; Pavel Trefil; Jiří Plachý; Markéta Reinišová; Dana Kučerová; Jitka Mucksová; Jiří Kalina. CRISPR/Cas9 Editing of Viral Receptors and Biotechnological Approach to Host Resistance. Proceedings 2020, 50, 22 .
AMA StyleJiří Hejnar, Anna Koslová, Pavel Trefil, Jiří Plachý, Markéta Reinišová, Dana Kučerová, Jitka Mucksová, Jiří Kalina. CRISPR/Cas9 Editing of Viral Receptors and Biotechnological Approach to Host Resistance. Proceedings. 2020; 50 (1):22.
Chicago/Turabian StyleJiří Hejnar; Anna Koslová; Pavel Trefil; Jiří Plachý; Markéta Reinišová; Dana Kučerová; Jitka Mucksová; Jiří Kalina. 2020. "CRISPR/Cas9 Editing of Viral Receptors and Biotechnological Approach to Host Resistance." Proceedings 50, no. 1: 22.
It has now been more than two years since we said our last goodbye to Jan Svoboda (14 [...].
Jiří Hejnar; Tomáš Ruml. The Current View of Retroviruses as Seen from the Shoulders of a Giant. Viruses 2019, 11, 828 .
AMA StyleJiří Hejnar, Tomáš Ruml. The Current View of Retroviruses as Seen from the Shoulders of a Giant. Viruses. 2019; 11 (9):828.
Chicago/Turabian StyleJiří Hejnar; Tomáš Ruml. 2019. "The Current View of Retroviruses as Seen from the Shoulders of a Giant." Viruses 11, no. 9: 828.
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.
David Přikryl; Jiří Plachý; Dana Kučerová; Anna Koslová; Markéta Reinišová; Filip Šenigl; Jiří Hejnar. The Novel Avian Leukosis Virus Subgroup K Shares Its Cellular Receptor with Subgroup A. Journal of Virology 2019, 93, 1 .
AMA StyleDavid Přikryl, Jiří Plachý, Dana Kučerová, Anna Koslová, Markéta Reinišová, Filip Šenigl, Jiří Hejnar. The Novel Avian Leukosis Virus Subgroup K Shares Its Cellular Receptor with Subgroup A. Journal of Virology. 2019; 93 (17):1.
Chicago/Turabian StyleDavid Přikryl; Jiří Plachý; Dana Kučerová; Anna Koslová; Markéta Reinišová; Filip Šenigl; Jiří Hejnar. 2019. "The Novel Avian Leukosis Virus Subgroup K Shares Its Cellular Receptor with Subgroup A." Journal of Virology 93, no. 17: 1.
Avian leukosis viruses (ALVs), which are pathogens of concern in domestic poultry, utilize specific receptor proteins for cell entry that are both necessary and sufficient for host susceptibility to a given ALV subgroup. This unequivocal relationship offers receptors as suitable targets of selection and biotechnological manipulation with the aim of obtaining virus-resistant poultry. This approach is further supported by the existence of natural knock-outs of receptor genes that segregate in inbred lines of chickens. We used CRISPR/Cas9 genome editing tools to introduce frame-shifting indel mutations into tva, tvc, and tvj loci encoding receptors for the A, C, and J ALV subgroups, respectively. For all three loci, the homozygous frame-shifting indels generating premature stop codons induced phenotypes which were fully resistant to the virus of respective subgroup. In the tvj locus, we also obtained in-frame deletions corroborating the importance of W38 and the four amino-acids preceding it. We demonstrate that CRISPR/Cas9-mediated knock-out or the fine editing of ALV receptor genes might be the first step in the development of virus-resistant chickens.
Anna Koslová; Dana Kučerová; Markéta Reinišová; Josef Geryk; Pavel Trefil; Jiří Hejnar. Genetic Resistance to Avian Leukosis Viruses Induced by CRISPR/Cas9 Editing of Specific Receptor Genes in Chicken Cells. Viruses 2018, 10, 605 .
AMA StyleAnna Koslová, Dana Kučerová, Markéta Reinišová, Josef Geryk, Pavel Trefil, Jiří Hejnar. Genetic Resistance to Avian Leukosis Viruses Induced by CRISPR/Cas9 Editing of Specific Receptor Genes in Chicken Cells. Viruses. 2018; 10 (11):605.
Chicago/Turabian StyleAnna Koslová; Dana Kučerová; Markéta Reinišová; Josef Geryk; Pavel Trefil; Jiří Hejnar. 2018. "Genetic Resistance to Avian Leukosis Viruses Induced by CRISPR/Cas9 Editing of Specific Receptor Genes in Chicken Cells." Viruses 10, no. 11: 605.
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.
Dalibor Miklik; Filip Senigl; Jirí Hejnar. Proviruses with Long-Term Stable Expression Accumulate in Transcriptionally Active Chromatin Close to the Gene Regulatory Elements: Comparison of ASLV-, HIV- and MLV-Derived Vectors. Viruses 2018, 10, 116 .
AMA StyleDalibor Miklik, Filip Senigl, Jirí Hejnar. Proviruses with Long-Term Stable Expression Accumulate in Transcriptionally Active Chromatin Close to the Gene Regulatory Elements: Comparison of ASLV-, HIV- and MLV-Derived Vectors. Viruses. 2018; 10 (3):116.
Chicago/Turabian StyleDalibor Miklik; Filip Senigl; Jirí Hejnar. 2018. "Proviruses with Long-Term Stable Expression Accumulate in Transcriptionally Active Chromatin Close to the Gene Regulatory Elements: Comparison of ASLV-, HIV- and MLV-Derived Vectors." Viruses 10, no. 3: 116.
The ongoing progress in primordial germ cell derivation and cultivation is opening new ways in reproductive biotechnology. This study tested whether functional sperm cells can be matured from genetically manipulated primordial germ cells after transplantation in adult testes and used to restore fertility. We show that spermatogenesis can be restored after mCherry-expressing or GFP-expressing primordial germ cells are transplantated into the testes of sterilized G0 roosters and that mCherry-positive or GFP-positive non-chimeric transgenic G1 offspring can be efficiently produced. Compared with the existing approaches to primordial germ cell replacement, this new technique eliminates the germ line chimerism of G0 roosters and is, therefore, faster, more efficient and requires fewer animals. Furthermore, this is the only animal model, where the fate of primordial germ cells in infertile recipients can be studied.
Pavel Trefil; Dorothea Aumann; Anna Koslová; Jitka Mucksová; Barbora Benešová; Jiří Kalina; Christine Wurmser; Ruedi Fries; Daniel Elleder; Benjamin Schusser; Jiří Hejnar. Male fertility restored by transplanting primordial germ cells into testes: a new way towards efficient transgenesis in chicken. Scientific Reports 2017, 7, 1 -9.
AMA StylePavel Trefil, Dorothea Aumann, Anna Koslová, Jitka Mucksová, Barbora Benešová, Jiří Kalina, Christine Wurmser, Ruedi Fries, Daniel Elleder, Benjamin Schusser, Jiří Hejnar. Male fertility restored by transplanting primordial germ cells into testes: a new way towards efficient transgenesis in chicken. Scientific Reports. 2017; 7 (1):1-9.
Chicago/Turabian StylePavel Trefil; Dorothea Aumann; Anna Koslová; Jitka Mucksová; Barbora Benešová; Jiří Kalina; Christine Wurmser; Ruedi Fries; Daniel Elleder; Benjamin Schusser; Jiří Hejnar. 2017. "Male fertility restored by transplanting primordial germ cells into testes: a new way towards efficient transgenesis in chicken." Scientific Reports 7, no. 1: 1-9.
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.
Filip Šenigl; Dalibor Miklík; Miroslav Auxt; Jiří Hejnar. Accumulation of long-term transcriptionally active integrated retroviral vectors in active promoters and enhancers. Nucleic Acids Research 2017, 45, 12752 -12765.
AMA StyleFilip Šenigl, Dalibor Miklík, Miroslav Auxt, Jiří Hejnar. Accumulation of long-term transcriptionally active integrated retroviral vectors in active promoters and enhancers. Nucleic Acids Research. 2017; 45 (22):12752-12765.
Chicago/Turabian StyleFilip Šenigl; Dalibor Miklík; Miroslav Auxt; Jiří Hejnar. 2017. "Accumulation of long-term transcriptionally active integrated retroviral vectors in active promoters and enhancers." Nucleic Acids Research 45, no. 22: 12752-12765.
Jiří Hejnar. Jan Svoboda (1934-2017): sixty years with retroviruses. Retrovirology 2017, 14, 32 .
AMA StyleJiří Hejnar. Jan Svoboda (1934-2017): sixty years with retroviruses. Retrovirology. 2017; 14 (1):32.
Chicago/Turabian StyleJiří Hejnar. 2017. "Jan Svoboda (1934-2017): sixty years with retroviruses." Retrovirology 14, no. 1: 32.
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.
Jitka Mucksová; Jiří Plachý; Ondřej Staněk; Jiří Hejnar; Jiří Kalina; Barbora Benešová; Pavel Trefil. Cytokine response to the RSV antigen delivered by dendritic cell-directed vaccination in congenic chicken lines. Veterinary Research 2017, 48, 1 -14.
AMA StyleJitka Mucksová, Jiří Plachý, Ondřej Staněk, Jiří Hejnar, Jiří Kalina, Barbora Benešová, Pavel Trefil. Cytokine response to the RSV antigen delivered by dendritic cell-directed vaccination in congenic chicken lines. Veterinary Research. 2017; 48 (1):1-14.
Chicago/Turabian StyleJitka Mucksová; Jiří Plachý; Ondřej Staněk; Jiří Hejnar; Jiří Kalina; Barbora Benešová; Pavel Trefil. 2017. "Cytokine response to the RSV antigen delivered by dendritic cell-directed vaccination in congenic chicken lines." Veterinary Research 48, no. 1: 1-14.
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. 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. 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.
Martina Benešová; Kateřina Trejbalová; Denisa Kovářová; Zdenka Vernerová; Tomáš Hron; Dana Kučerová; Jiří Hejnar. DNA hypomethylation and aberrant expression of the human endogenous retrovirus ERVWE1/syncytin-1 in seminomas. Retrovirology 2017, 14, 1 -17.
AMA StyleMartina Benešová, Kateřina Trejbalová, Denisa Kovářová, Zdenka Vernerová, Tomáš Hron, Dana Kučerová, Jiří Hejnar. DNA hypomethylation and aberrant expression of the human endogenous retrovirus ERVWE1/syncytin-1 in seminomas. Retrovirology. 2017; 14 (1):1-17.
Chicago/Turabian StyleMartina Benešová; Kateřina Trejbalová; Denisa Kovářová; Zdenka Vernerová; Tomáš Hron; Dana Kučerová; Jiří Hejnar. 2017. "DNA hypomethylation and aberrant expression of the human endogenous retrovirus ERVWE1/syncytin-1 in seminomas." Retrovirology 14, no. 1: 1-17.
Human immunodeficiency virus type 1 (HIV-1) latency represents the major barrier to virus eradication in infected individuals because cells harboring latent HIV-1 provirus are not affected by current antiretroviral therapy (ART). We previously demonstrated that DNA methylation of HIV-1 long terminal repeat (5′ LTR) restricts HIV-1 reactivation and, together with chromatin conformation, represents an important mechanism of HIV-1 latency maintenance. Here, we explored the new issue of temporal development of DNA methylation in latent HIV-1 5′ LTR. In the Jurkat CD4(+) T cell model of latency, we showed that the stimulation of host cells contributed to de novo DNA methylation of the latent HIV-1 5′ LTR sequences. Consecutive stimulations of model CD4(+) T cell line with TNF-α and PMA or with SAHA contributed to the progressive accumulation of 5′ LTR DNA methylation. Further, we showed that once established, the high DNA methylation level of the latent 5′ LTR in the cell line model was a stable epigenetic mark. Finally, we explored the development of 5′ LTR DNA methylation in the latent reservoir of HIV-1- infected individuals who were treated with ART. We detected low levels of 5′ LTR DNA methylation in the resting CD4(+) T cells of the group of patients who were treated for up to 3 years. However, after long-term ART, we observed an accumulation of 5′ LTR DNA methylation in the latent reservoir. Importantly, within the latent reservoir of some long-term- treated individuals, we uncovered populations of proviral molecules with a high density of 5′ LTR CpG methylation. Our data showed the presence of 5′ LTR DNA methylation in the long-term reservoir of HIV-1-infected individuals and implied that the transient stimulation of cells harboring latent proviruses may contribute, at least in part, to the methylation of the HIV-1 promoter.
Katerina Trejbalova; Denisa Kovarova; Jana Blazkova; Ladislav Machala; David Jilich; Jan Weber; Dana Kucerova; Ondrej Vencalek; Ivan Hirsch; Jiri Hejnar. P-D1 Transient stimulation of cells harboring latent proviruses contributes to the methylation of the HIV-1 promoter. JAIDS Journal of Acquired Immune Deficiency Syndromes 2017, 74, 89 .
AMA StyleKaterina Trejbalova, Denisa Kovarova, Jana Blazkova, Ladislav Machala, David Jilich, Jan Weber, Dana Kucerova, Ondrej Vencalek, Ivan Hirsch, Jiri Hejnar. P-D1 Transient stimulation of cells harboring latent proviruses contributes to the methylation of the HIV-1 promoter. JAIDS Journal of Acquired Immune Deficiency Syndromes. 2017; 74 ():89.
Chicago/Turabian StyleKaterina Trejbalova; Denisa Kovarova; Jana Blazkova; Ladislav Machala; David Jilich; Jan Weber; Dana Kucerova; Ondrej Vencalek; Ivan Hirsch; Jiri Hejnar. 2017. "P-D1 Transient stimulation of cells harboring latent proviruses contributes to the methylation of the HIV-1 promoter." JAIDS Journal of Acquired Immune Deficiency Syndromes 74, no. : 89.
Crosslinking of regulatory immunoreceptors, such as BDCA-2 (CD303) or ILT7 (CD85g), of plasmacytoid dendritic cells (pDCs) efficiently suppresses production of type-I interferon (IFN)-α/β and other cytokines in response to Toll-like receptor (TLR) 7/9 ligands. Viral surface glycoproteins, HIV-1 gp120, hepatitis C virus (HCV) E2 and hepatitis B virus (HBV) HBsAg were shown to be ligands of pDC regulatory immunoreceptors. Cytokine-inhibitory pathway triggered by ligation of regulatory immunoreceptors is mediated by spleen tyrosine kinase (Syk) associated with the ITAM-containing adapter of regulatory immunoreceptors. Here we demonstrate by pharmacological targeting of Syk that in addition to the negative regulation of TLR7/9 signaling via regulatory immunoreceptors, Syk also positively regulates the TLR7/9 pathway in human pDCs. Novel highly specific Syk inhibitor AB8779 suppressed IFN-α, TNF-α and IL-6 production induced by TLR7/9 agonists in primary pDCs and in the pDC cell line GEN2.2. Triggering of TLR9 or regulatory immunoreceptors signaling induced a differential kinetics of phosphorylation at Y352 and Y525/526 of Syk and a differential sensitivity to AB8779. Consistent with the different roles of Syk in TLR7/9 and regulatory immunoreceptors signaling, a concentration of AB8779 insufficient to block TLR7/9 signaling still released the block of IFN-α production triggered via the regulatory immunoreceptor pathway, including that induced by HIV, HBV and HCV. Thus, pharmacological targeting of Syk partially restored the main pDC function—IFN-α production. Opposing roles of Syk in TLR7/9 and regulatory immunoreceptor pathways may regulate the innate immune response to weaken inflammation reaction.
Besma Aouar; Denisa Kovarova; Albert Font-Haro; Jan Weber; David Durantel; Katerina Trejbalova; Jiri Hejnar; Jacques Nunes; Daniel Olive; Patrice Dubreuil; Ivan Hirsch; Ruzena Stranska. P-C23 Spleen tyrosine kinase (Syk) controls IFN-α production inhibited in plasmacytoid dendritic cells by surface glycoproteins of HIV, HBV and HCV. JAIDS Journal of Acquired Immune Deficiency Syndromes 2017, 74, 1 .
AMA StyleBesma Aouar, Denisa Kovarova, Albert Font-Haro, Jan Weber, David Durantel, Katerina Trejbalova, Jiri Hejnar, Jacques Nunes, Daniel Olive, Patrice Dubreuil, Ivan Hirsch, Ruzena Stranska. P-C23 Spleen tyrosine kinase (Syk) controls IFN-α production inhibited in plasmacytoid dendritic cells by surface glycoproteins of HIV, HBV and HCV. JAIDS Journal of Acquired Immune Deficiency Syndromes. 2017; 74 ():1.
Chicago/Turabian StyleBesma Aouar; Denisa Kovarova; Albert Font-Haro; Jan Weber; David Durantel; Katerina Trejbalova; Jiri Hejnar; Jacques Nunes; Daniel Olive; Patrice Dubreuil; Ivan Hirsch; Ruzena Stranska. 2017. "P-C23 Spleen tyrosine kinase (Syk) controls IFN-α production inhibited in plasmacytoid dendritic cells by surface glycoproteins of HIV, HBV and HCV." JAIDS Journal of Acquired Immune Deficiency Syndromes 74, no. : 1.
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.
Martina Benešová; Kateřina Trejbalová; Dana Kučerová; Zdenka Vernerová; Tomáš Hron; Arpád Szabó; Rachel Amouroux; Petr Klézl; Petra Hajkova; Jiří Hejnar. Overexpression of TET dioxygenases in seminomas associates with low levels of DNA methylation and hydroxymethylation. Molecular Carcinogenesis 2017, 56, 1837 -1850.
AMA StyleMartina Benešová, Kateřina Trejbalová, Dana Kučerová, Zdenka Vernerová, Tomáš Hron, Arpád Szabó, Rachel Amouroux, Petr Klézl, Petra Hajkova, Jiří Hejnar. Overexpression of TET dioxygenases in seminomas associates with low levels of DNA methylation and hydroxymethylation. Molecular Carcinogenesis. 2017; 56 (8):1837-1850.
Chicago/Turabian StyleMartina Benešová; Kateřina Trejbalová; Dana Kučerová; Zdenka Vernerová; Tomáš Hron; Arpád Szabó; Rachel Amouroux; Petr Klézl; Petra Hajkova; Jiří Hejnar. 2017. "Overexpression of TET dioxygenases in seminomas associates with low levels of DNA methylation and hydroxymethylation." Molecular Carcinogenesis 56, no. 8: 1837-1850.
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.
Jiří Plachý; Markéta Reinišová; Dana Kučerová; Filip Šenigl; Volodymyr Stepanets; Tomas Hron; Kateřina Trejbalová; Daniel Elleder; Jiří Hejnar. Identification of New World Quails Susceptible to Infection with Avian Leukosis Virus Subgroup J. Journal of Virology 2017, 91, 1 .
AMA StyleJiří Plachý, Markéta Reinišová, Dana Kučerová, Filip Šenigl, Volodymyr Stepanets, Tomas Hron, Kateřina Trejbalová, Daniel Elleder, Jiří Hejnar. Identification of New World Quails Susceptible to Infection with Avian Leukosis Virus Subgroup J. Journal of Virology. 2017; 91 (3):1.
Chicago/Turabian StyleJiří Plachý; Markéta Reinišová; Dana Kučerová; Filip Šenigl; Volodymyr Stepanets; Tomas Hron; Kateřina Trejbalová; Daniel Elleder; Jiří Hejnar. 2017. "Identification of New World Quails Susceptible to Infection with Avian Leukosis Virus Subgroup J." Journal of Virology 91, no. 3: 1.
J subgroup avian leukosis virus (ALV-J) infects domestic chicken, jungle fowl, and turkey and enters the host cell through a receptor encoded by tvj locus and identified as Na+/H+ exchanger 1 (NHE1). The resistance to ALV-J in a great majority of examined galliform species was explained by deletions or substitutions of the critical tryptophan 38 in the first extracellular loop of NHE1, and genetic polymorphisms around this site predict the susceptibility or resistance of a given species or individual. In this study, we examined the NHE1 polymorphism in domestic chicken breeds and documented quantitative differences in their susceptibility to ALV-J in vitro. In a panel of chicken breeds assembled with the aim to cover the maximum variability encountered in domestic chickens, we found a completely uniform sequence of NHE1 extracellular loop 1 (ECL1) without any source of genetic variation for the selection of ALV-J-resistant poultry. In parallel, we studied the natural polymorphisms of NHE1 in wild ducks and geese because of recent reports on ALV-J positivity in feral Asian species. In anseriform species, we demonstrate a specific and highly conserved critical ECL1 sequence without any homologue of tryptophan 38 in accordance with the resistance of duck cells to prototype ALV-J. Last, we demonstrated that the new Asian strains of ALV-J have not evolved their envelope glycoprotein to the entry the duck cells. Our results contribute substantially to the current discussion of possible heterotransmission of ALV-J and its spill-over into the wild ducks and geese.
Markéta Reinišová; Jiří Plachý; Dana Kučerová; Filip Šenigl; Michal Vinkler; Jiří Hejnar. Genetic Diversity of NHE1, Receptor for Subgroup J Avian Leukosis Virus, in Domestic Chicken and Wild Anseriform Species. PLOS ONE 2016, 11, e0150589 .
AMA StyleMarkéta Reinišová, Jiří Plachý, Dana Kučerová, Filip Šenigl, Michal Vinkler, Jiří Hejnar. Genetic Diversity of NHE1, Receptor for Subgroup J Avian Leukosis Virus, in Domestic Chicken and Wild Anseriform Species. PLOS ONE. 2016; 11 (3):e0150589.
Chicago/Turabian StyleMarkéta Reinišová; Jiří Plachý; Dana Kučerová; Filip Šenigl; Michal Vinkler; Jiří Hejnar. 2016. "Genetic Diversity of NHE1, Receptor for Subgroup J Avian Leukosis Virus, in Domestic Chicken and Wild Anseriform Species." PLOS ONE 11, no. 3: e0150589.
Human immunodeficiency virus type 1 (HIV-1) latency represents the major barrier to virus eradication in infected individuals because cells harboring latent HIV-1 provirus are not affected by current antiretroviral therapy (ART). We previously demonstrated that DNA methylation of HIV-1 long terminal repeat (5' LTR) restricts HIV-1 reactivation and, together with chromatin conformation, represents an important mechanism of HIV-1 latency maintenance. Here, we explored the new issue of temporal development of DNA methylation in latent HIV-1 5' LTR. In the Jurkat CD4(+) T cell model of latency, we showed that the stimulation of host cells contributed to de novo DNA methylation of the latent HIV-1 5' LTR sequences. Consecutive stimulations of model CD4(+) T cell line with TNF-α and PMA or with SAHA contributed to the progressive accumulation of 5' LTR DNA methylation. Further, we showed that once established, the high DNA methylation level of the latent 5' LTR in the cell line model was a stable epigenetic mark. Finally, we explored the development of 5' LTR DNA methylation in the latent reservoir of HIV-1-infected individuals who were treated with ART. We detected low levels of 5' LTR DNA methylation in the resting CD4(+) T cells of the group of patients who were treated for up to 3 years. However, after long-term ART, we observed an accumulation of 5' LTR DNA methylation in the latent reservoir. Importantly, within the latent reservoir of some long-term-treated individuals, we uncovered populations of proviral molecules with a high density of 5' LTR CpG methylation. Our data showed the presence of 5' LTR DNA methylation in the long-term reservoir of HIV-1-infected individuals and implied that the transient stimulation of cells harboring latent proviruses may contribute, at least in part, to the methylation of the HIV-1 promoter.
Kateřina Trejbalová; Denisa Kovarova; Jana Blazkova; Ladislav Machala; David Jilich; Jan Weber; Dana Kučerová; Ondřej Vencálek; Ivan Hirsch; Jiří Hejnar. Development of 5' LTR DNA methylation of latent HIV-1 provirus in cell line models and in long-term-infected individuals. Clinical Epigenetics 2016, 8, 19 .
AMA StyleKateřina Trejbalová, Denisa Kovarova, Jana Blazkova, Ladislav Machala, David Jilich, Jan Weber, Dana Kučerová, Ondřej Vencálek, Ivan Hirsch, Jiří Hejnar. Development of 5' LTR DNA methylation of latent HIV-1 provirus in cell line models and in long-term-infected individuals. Clinical Epigenetics. 2016; 8 (1):19.
Chicago/Turabian StyleKateřina Trejbalová; Denisa Kovarova; Jana Blazkova; Ladislav Machala; David Jilich; Jan Weber; Dana Kučerová; Ondřej Vencálek; Ivan Hirsch; Jiří Hejnar. 2016. "Development of 5' LTR DNA methylation of latent HIV-1 provirus in cell line models and in long-term-infected individuals." Clinical Epigenetics 8, no. 1: 19.
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.
Magda Matouskova; Pavel Vesely; Petr Daniel; Giada Mattiuzzo; Ralph D. Hector; Linda Scobie; Yasuhiro Takeuchi; Jiří Hejnar. Role of DNA Methylation in Expression and Transmission of Porcine Endogenous Retroviruses. Journal of Virology 2013, 87, 12110 -12120.
AMA StyleMagda Matouskova, Pavel Vesely, Petr Daniel, Giada Mattiuzzo, Ralph D. Hector, Linda Scobie, Yasuhiro Takeuchi, Jiří Hejnar. Role of DNA Methylation in Expression and Transmission of Porcine Endogenous Retroviruses. Journal of Virology. 2013; 87 (22):12110-12120.
Chicago/Turabian StyleMagda Matouskova; Pavel Vesely; Petr Daniel; Giada Mattiuzzo; Ralph D. Hector; Linda Scobie; Yasuhiro Takeuchi; Jiří Hejnar. 2013. "Role of DNA Methylation in Expression and Transmission of Porcine Endogenous Retroviruses." Journal of Virology 87, no. 22: 12110-12120.
Comparing the gene expression profiles of metastatic and nonmetastatic cells has the power to reveal candidate metastasis-associated genes, whose involvement in metastasis can be experimentally tested. In this study, differentially expressed genes were explored in the v-src-transformed metastatic cell line PR9692 and its nonmetastatic subclone PR9692-E9. First, the contribution of homeodomain only protein X (HOPX) in metastasis formation and development was assessed. HOPX-specific knockdown decreased HOPX expression in the nonmetastatic subclone and displayed reduced cell motility in vitro. Critically, HOPX knockdown decreased the in vivo metastatic capacity in a syngeneic animal model system. Genomic analyses identified a cadre of genes affected by HOPX knockdown that intersected significantly with genes previously found to be differentially expressed in metastatic versus nonmetastatic cells. Furthermore, 232 genes were found in both screens with at least a two-fold change in gene expression, and a number of high-confidence targets were validated for differential expression. Importantly, significant changes were demonstrated in the protein expression level of three metastatic-associated genes (NCAM, FOXG1, and ITGA4), and knockdown of one of the identified HOPX-regulated metastatic genes, ITGA4, showed marked inhibition of cell motility and metastasis formation. These data demonstrate that HOPX is a metastasis-associated gene and that its knockdown decreases the metastatic activity of v-src-transformed cells through altered gene expression patterns. Implications: This study provides new mechanistic insight into a HOPX-regulated metastatic dissemination signature. Mol Cancer Res; 11(10); 1235–47. ©2013 AACR.
Denisa Kovářová; Jiří Plachý; Jan Kosla; Kateřina Trejbalová; Vladimír Čermák; Jiří Hejnar. Downregulation of HOPX Controls Metastatic Behavior in Sarcoma Cells and Identifies Genes Associated with Metastasis. Molecular Cancer Research 2013, 11, 1235 -1247.
AMA StyleDenisa Kovářová, Jiří Plachý, Jan Kosla, Kateřina Trejbalová, Vladimír Čermák, Jiří Hejnar. Downregulation of HOPX Controls Metastatic Behavior in Sarcoma Cells and Identifies Genes Associated with Metastasis. Molecular Cancer Research. 2013; 11 (10):1235-1247.
Chicago/Turabian StyleDenisa Kovářová; Jiří Plachý; Jan Kosla; Kateřina Trejbalová; Vladimír Čermák; Jiří Hejnar. 2013. "Downregulation of HOPX Controls Metastatic Behavior in Sarcoma Cells and Identifies Genes Associated with Metastasis." Molecular Cancer Research 11, no. 10: 1235-1247.