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Plachý, Jiří, Jan Kotáb, Petr Divina, Markéta Reinišová, Filip Šenigl et Jiří Hejnar. « Proviruses Selected for High and Stable Expression of Transduced Genes Accumulate in Broadly Transcribed Genome Areas ». Journal of Virology 84, no 9 (10 février 2010) : 4204–11. http://dx.doi.org/10.1128/jvi.02511-09.
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ABSTRACT Retroviruses and retrovirus-derived vectors integrate nonrandomly into the genomes of host cells with specific preferences for transcribed genes, gene-rich regions, and CpG islands. However, the genomic features that influence the transcriptional activities of integrated retroviruses or retroviral vectors are poorly understood. We report here the cloning and characterization of avian sarcoma virus integration sites from chicken tumors. Growing progressively, dependent on high and stable expression of the transduced v-src oncogene, these tumors represent clonal expansions of cells bearing transcriptionally active replication-defective proviruses. Therefore, integration sites in our study distinguished genomic loci favorable for the expression of integrated retroviruses and gene transfer vectors. Analysis of integration sites from avian sarcoma virus-induced tumors showed strikingly nonrandom distribution, with proviruses found prevalently within or close to transcription units, particularly in genes broadly expressed in multiple tissues but not in tissue-specifically expressed genes. We infer that proviruses integrated in these genomic areas efficiently avoid transcriptional silencing and remain active for a long time during the growth of tumors. Defining the differences between unselected retroviral integration sites and sites selected for long-terminal-repeat-driven gene expression is relevant for retrovirus-mediated gene transfer and has ramifications for gene therapy.
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Schlesinger, Sharon, et Stephen P. Goff. « Retroviral Transcriptional Regulation and Embryonic Stem Cells : War and Peace ». Molecular and Cellular Biology 35, no 5 (29 décembre 2014) : 770–77. http://dx.doi.org/10.1128/mcb.01293-14.
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Retroviruses have evolved complex transcriptional enhancers and promoters that allow their replication in a wide range of tissue and cell types. Embryonic stem (ES) cells, however, characteristically suppress transcription of proviruses formed after infection by exogenous retroviruses and also of most members of the vast array of endogenous retroviruses in the genome. These cells have unusual profiles of transcribed genes and are poised to make rapid changes in those profiles upon induction of differentiation. Many of the transcription factors in ES cells control both host and retroviral genes coordinately, such that retroviral expression patterns can serve as markers of ES cell pluripotency. This overlap is not coincidental; retrovirus-derived regulatory sequences are often used to control cellular genes important for pluripotency. These sequences specify the temporal control and perhaps “noisy” control of cellular genes that direct proper cell gene expression in primitive cells and their differentiating progeny. The evidence suggests that the viral elements have been domesticated for host needs, reflecting the wide-ranging exploitation of any and all available DNA sequences in assembling regulatory networks.
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Evans, Leonard H., A. S. M. Alamgir, Nick Owens, Nick Weber, Kimmo Virtaneva, Kent Barbian, Amenah Babar, Frank Malik et Kyle Rosenke. « Mobilization of Endogenous Retroviruses in Mice after Infection with an Exogenous Retrovirus ». Journal of Virology 83, no 6 (30 décembre 2008) : 2429–35. http://dx.doi.org/10.1128/jvi.01926-08.
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ABSTRACT Mammalian genomes harbor a large number of retroviral elements acquired as germ line insertions during evolution. Although many of the endogenous retroviruses are defective, several contain one or more intact viral genes that are expressed under certain physiological or pathological conditions. This is true of the endogenous polytropic retroviruses that generate recombinant polytropic murine leukemia viruses (MuLVs). In these recombinants the env gene sequences of exogenous ecotropic MuLVs are replaced with env gene sequences from an endogenous polytropic retrovirus. Although replication-competent endogenous polytropic retroviruses have not been observed, the recombinant polytropic viruses are capable of replicating in numerous species. Recombination occurs during reverse transcription of a virion RNA heterodimer comprised of an RNA transcript from an endogenous polytropic virus and an RNA transcript from an exogenous ecotropic MuLV RNA. It is possible that homodimers corresponding to two full-length endogenous RNA genomes are also packaged. Thus, infection by an exogenous virus may result not only in recombination with endogenous sequences, but also in the mobilization of complete endogenous retrovirus genomes via pseudotyping within exogenous retroviral virions. We report that the infection of mice with an ecotropic virus results in pseudotyping of intact endogenous viruses that have not undergone recombination. The endogenous retroviruses infect and are integrated into target cell genomes and subsequently replicate and spread as pseudotyped viruses. The mobilization of endogenous retroviruses upon infection with an exogenous retrovirus may represent a major interaction of exogenous retroviruses with endogenous retroviruses and may have profound effects on the pathogenicity of retroviral infections.
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Pluta, Aneta, Juan P. Jaworski et César N. Cortés-Rubio. « Balance between Retroviral Latency and Transcription : Based on HIV Model ». Pathogens 10, no 1 (29 décembre 2020) : 16. http://dx.doi.org/10.3390/pathogens10010016.
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The representative of the Lentivirus genus is the human immunodeficiency virus type 1 (HIV-1), the causative agent of acquired immunodeficiency syndrome (AIDS). To date, there is no cure for AIDS because of the existence of the HIV-1 reservoir. HIV-1 infection can persist for decades despite effective antiretroviral therapy (ART), due to the persistence of infectious latent viruses in long-lived resting memory CD4+ T cells, macrophages, monocytes, microglial cells, and other cell types. However, the biology of HIV-1 latency remains incompletely understood. Retroviral long terminal repeat region (LTR) plays an indispensable role in controlling viral gene expression. Regulation of the transcription initiation plays a crucial role in establishing and maintaining a retrovirus latency. Whether and how retroviruses establish latency and reactivate remains unclear. In this article, we describe what is known about the regulation of LTR-driven transcription in HIV-1, that is, the cis-elements present in the LTR, the role of LTR transcription factor binding sites in LTR-driven transcription, the role of HIV-1-encoded transactivator protein, hormonal effects on virus transcription, impact of LTR variability on transcription, and epigenetic control of retrovirus LTR. Finally, we focus on a novel clustered regularly interspaced short palindromic repeats-associated protein 9 (CRISPR/dCas9)-based strategy for HIV-1 reservoir purging.
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Dimcheff, Derek E., Mallika Krishnan et David P. Mindell. « Evolution and Characterization of Tetraonine Endogenous Retrovirus : a New Virus Related to Avian Sarcoma and Leukosis Viruses ». Journal of Virology 75, no 4 (15 février 2001) : 2002–9. http://dx.doi.org/10.1128/jvi.75.4.2002-2009.2001.
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ABSTRACT In a previous study, we found avian sarcoma and leukosis virus (ASLV) gag genes in 19 species of birds in the order Galliformes including all grouse and ptarmigan (Tetraoninae) surveyed. Our data suggested that retroviruses had been transmitted horizontally among some host species. To further investigate these elements, we sequenced a replication-defective retrovirus, here named tetraonine endogenous retrovirus (TERV), from Bonasa umbellus (ruffed grouse). This is the first report of a complete, replication-defective ASLV provirus sequence from any bird other than the domestic chicken. We found a replication-defective proviral sequence consisting of putative Gag and Env proteins flanked by long terminal repeats. Reverse transcription-PCR analysis showed that retroviral gagsequences closely related to TERV are transcribed, supporting the hypothesis that TERV is an active endogenous retrovirus. Phylogenetic analyses suggest that TERV may have arisen via recombination between different retroviral lineages infecting birds. Southern blotting usinggag probes showed that TERV occurs in tetraonines but not in chickens or ducks, suggesting that integration occurred after the earliest phasianid divergences but prior to the radiation of tetraonine birds.
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Seifarth, Wolfgang, Oliver Frank, Udo Zeilfelder, Birgit Spiess, Alex D. Greenwood, Rüdiger Hehlmann et Christine Leib-Mösch. « Comprehensive Analysis of Human Endogenous Retrovirus Transcriptional Activity in Human Tissues with a Retrovirus-Specific Microarray ». Journal of Virology 79, no 1 (1 janvier 2005) : 341–52. http://dx.doi.org/10.1128/jvi.79.1.341-352.2005.
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ABSTRACT Retrovirus-like sequences account for 8 to 9% of the human genome. Among these sequences, about 8,000 pol-containing proviral elements have been identified to date. As part of our ongoing search for active and possibly disease-relevant human endogenous retroviruses (HERVs), we have recently developed an oligonucleotide-based microarray. The assay allows for both the detection and the identification of most known retroviral reverse transcriptase (RT)-related nucleic acids in biological samples. In the present study, we have investigated the transcriptional activity of representative members of 20 HERV families in 19 different normal human tissues. Qualitative evaluation of chip hybridization signals and quantitative analysis by real-time RT-PCR revealed distinct HERV activity in the human tissues under investigation, suggesting that HERV elements are active in human cells in a tissue-specific manner. Most active members of HERV families were found in mRNA prepared from skin, thyroid gland, placenta, and tissues of reproductive organs. In contrast, only few active HERVs were detectable in muscle cells. Human tissues that lack HERV transcription could not be found, confirming that human endogenous retroviruses are permanent components of the human transcriptome. Distinct activity patterns may reflect the characteristics of the regulatory machinery in these cells, e.g., cell type-dependent occurrence of transcriptional regulatory factors.
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Wolgamot, Greg, et A. Dusty Miller. « Replication of Mus dunni Endogenous Retrovirus Depends on Promoter Activation Followed by Enhancer Multimerization ». Journal of Virology 73, no 12 (1 décembre 1999) : 9803–9. http://dx.doi.org/10.1128/jvi.73.12.9803-9809.1999.
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ABSTRACT Mus dunni endogenous virus (MDEV) is an apparently intact retrovirus that normally lies transcriptionally silent in cultured M. dunni cells, but the provirus can be activated by treatment of the cells with hydrocortisone or 5-iodo-2′-deoxyuridine. Sequence analysis of a molecular clone of the replicating virus revealed a simple retrovirus with a chimeric VL30/GALV-like structure. Interestingly, in the region of the long terminal repeat (LTR) that typically contains the retroviral transcription enhancers, we found over six 80-bp repeats with only a single mismatch, indicating that acquisition of the repeats was a recent event. Here we provide evidence for the following model of MDEV activation and replication. The MDEV provirus in M. dunnicells has a chimeric structure similar to that of the molecular clone but has only 1.15 copies of the 80-bp repeat sequence found in the molecular clone. Activating chemicals directly stimulate transcription from the LTR, allowing a low level of virus replication. Copying errors made during reverse transcription allow multimerization of the 80-bp enhancer region, resulting in viruses with higher transcriptional rates and improved fitness, but increased enhancer copy number is likely balanced by the natural instability of retroviral repeats and constraints imposed by virion packaging limits. The resultant population of replicating MDEV is widely heterogeneous, having from 2.15 to 13.15 enhancer repeats in the LTR. These results reveal a novel mechanism for regulation of transcription and replication of an endogenous retrovirus, in terms of both activation of the virus by the steroid hydrocortisone and the large number and variation in enhancer repeats observed.
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Kogan, Scott C., Mei Lin Maunakea, Karen L. Himmel, Bin Yin, Michelle M. Le Beau et David A. Largaespada. « Cooperative Pathways to Acute Myeloid Leukemia Include the Combining of Transcription Factor Alterations : PML-RARα Cooperates with SOX4. » Blood 104, no 11 (16 novembre 2004) : 3385. http://dx.doi.org/10.1182/blood.v104.11.3385.3385.
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Abstract One paradigm for transformation of normal myeloid cells into acute myeloid leukemia (AML) is the combining of transcription factor alteration with activation of signaling pathways, including cytokine receptor activation. To identify possible alternative pathways to leukemogenesis we crossed MRP8-PML/RARA transgenic mice with BXH-2 mice, which harbor an endogenous retrovirus that causes AML. Approximately half of the leukemias that arose in this cross showed features of acute promyelocytic leukemia (APL). We identified 22 insertions sites in 8 APL-like leukemias. Of these, 7 represented common insertion sites in the Mouse Retroviral Tagged Cancer Gene Database (RTCGD, http://rtcgd.ncifcrf.gov/mm4/index.html). We introduced into a retroviral vector cDNAs encoding 6 genes located at retroviral insertion sites identified in PML/RARA leukemias (Sox4, Meis1, Lck, Sfpi1, Nfil3, Cblb). PML/RARA transgenic bone marrow was transduced with these retroviruses and the marrow was used to reconstitute lethally irradiated recipient animals. Recipients of PML/RARα transgenic marrow transduced with a SOX4 retrovirus developed APL-like leukemias in 3 months. SOX4 is an HMG box transcription factor that exhibits sequence specific DNA binding and transactivation. It is the most common insertion site in RTCGD, and has been found to be overexpressed in small cell lung cancer and medulloblastoma, as well as other tumors. In light of other data indicating that combining transcription factor abnormalities can rapidly induce leukemia (e.g. Hoxa9 + Meis1), our finding that SOX4 + PML/RARα is potently leukemogenic supports the hypothesis that such cooperativity represents another important paradigm in myeloid leukemogenesis.
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Pietrantoni, Gianfranco, Rodrigo Ibarra-Karmy et Gloria Arriagada. « Microtubule Retrograde Motors and Their Role in Retroviral Transport ». Viruses 12, no 4 (24 avril 2020) : 483. http://dx.doi.org/10.3390/v12040483.
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Following entry into the host cell, retroviruses generate a dsDNA copy of their genomes via reverse transcription, and this viral DNA is subsequently integrated into the chromosomal DNA of the host cell. Before integration can occur, however, retroviral DNA must be transported to the nucleus as part of a ‘preintegration complex’ (PIC). Transporting the PIC through the crowded environment of the cytoplasm is challenging, and retroviruses have evolved different mechanisms to accomplish this feat. Within a eukaryotic cell, microtubules act as the roads, while the microtubule-associated proteins dynein and kinesin are the vehicles that viruses exploit to achieve retrograde and anterograde trafficking. This review will examine the various mechanisms retroviruses have evolved in order to achieve retrograde trafficking, confirming that each retrovirus has its own strategy to functionally subvert microtubule associated proteins.
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Hansen, Regine, Stefanie Czub, Evi Werder, Jens Herold, Georg Gosztonyi, Hans Gelderblom, Simone Schimmer, Stefan Mazgareanu, Volker ter Meulen et Markus Czub. « Abundant Defective Viral Particles Budding from Microglia in the Course of Retroviral Spongiform Encephalopathy ». Journal of Virology 74, no 4 (15 février 2000) : 1775–80. http://dx.doi.org/10.1128/jvi.74.4.1775-1780.2000.
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ABSTRACT A pathogenetic hallmark of retroviral neurodegeneration is the affinity of neurovirulent retroviruses for microglia cells, while degenerating neurons are excluded from retroviral infections. Microglia isolated ex vivo from rats peripherally infected with a neurovirulent retrovirus released abundant mature type C virions; however, infectivity associated with microglia was very low. In microglia, viral transcription was unaffected but envelope proteins were insufficiently cleaved into mature viral proteins and were not detected on the microglia cell surface. These microglia-specific defects in envelope protein translocation and processing not only may have prevented formation of infectious virus particles but also may have caused further cellular defects in microglia with the consequence of indirect neuronal damage. It is conceivable that similar events play a role in neuro-AIDS.
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Zhang, Jiayou. « Host RNA polymerase II makes minimal contributions to retroviral frame-shift mutations ». Journal of General Virology 85, no 8 (1 août 2004) : 2389–95. http://dx.doi.org/10.1099/vir.0.80081-0.
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The rate of mutation during retrovirus replication is high. Mutations can occur during transcription of the viral genomic RNA from the integrated provirus or during reverse transcription from viral RNA to form viral DNA or during replication of the proviral DNA as the host cell is dividing. Therefore, three polymerases may all contribute to retroviral evolution: host RNA polymerase II, viral reverse transcriptases and host DNA polymerases, respectively. Since the rate of mutation for host DNA polymerase is very low, mutations are more likely to be caused by the host RNA polymerase II and/or the viral reverse transcriptase. A system was established to detect the frequency of frame-shift mutations caused by cellular RNA polymerase II, as well as the rate of retroviral mutation during a single cycle of replication in vivo. In this study, it was determined that RNA polymerase II contributes less than 3 % to frame-shift mutations that occur during retrovirus replication. Therefore, the majority of frame-shift mutations detected within the viral genome are the result of errors during reverse transcription.
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Ristevski, Sika, Damian F. J. Purcell, John Marshall, Daniella Campagna, Sara Nouri, Simon P. Fenton, Dale A. McPhee et George Kannourakis. « Novel Endogenous Type D Retroviral Particles Expressed at High Levels in a SCID Mouse Thymic Lymphoma ». Journal of Virology 73, no 6 (1 juin 1999) : 4662–69. http://dx.doi.org/10.1128/jvi.73.6.4662-4669.1999.
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ABSTRACT A xenograft model of the human disease Langerhans cell histiocytosis (LCH) was investigated with severe combined immunodeficiency (SCID) mice. Transplantation of human LCH biopsy material into SCID mice resulted in the generation of mouse tumors resembling lymphomas. A thymoma cell line (ThyE1M6) was generated from one of these mice and found to display significant levels of Mg2+-dependent reverse transcriptase activity. Electron microscopy revealed particles with type D retroviral morphology budding from ThyE1M6 cells at a high frequency, whereas control cultures were negative. Reverse transcription-PCR of virion RNA with degenerate primers for conserved regions of various mouse, human, and primate retroviruses amplified novel sequences related to primate type D retroviruses, murine intracisternal A particles, Jaagsiekte sheep retrovirus, and murine long interspersed nuclear elements but not other retroviral classes. We demonstrate that these sequences represent a novel group of endogenous retroviruses expressed at low levels in mice but expressed at high levels in the ThyE1M6 cell line. Furthermore, we propose that the activation of endogenous retroviral elements may be associated with a high incidence of thymomas in SCID mice.
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Galla, Melanie, Axel Schambach, Greg J. Towers et Christopher Baum. « Cellular Restriction of Retrovirus Particle-Mediated mRNA Transfer ». Journal of Virology 82, no 6 (16 janvier 2008) : 3069–77. http://dx.doi.org/10.1128/jvi.01880-07.
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ABSTRACT Analyzing cellular restriction mechanisms provides insight into viral replication strategies, identifies targets for antiviral drug design, and is crucial for the development of novel tools for experimental or therapeutic delivery of genetic information. We have previously shown that retroviral vector mutants that are unable to initiate reverse transcription mediate a transient expression of any sequence which replaces the gag-pol transcription unit, a process we call retrovirus particle-mediated mRNA transfer (RMT). Here, we further examined the mechanism of RMT by testing its sensitivity to cellular restriction factors and short hairpin RNAs (shRNAs). We found that both human TRIM5α and, to a lesser extent, Fv1 effectively restrict RMT if the RNA is delivered by a restriction-sensitive capsid. While TRIM5α restriction of RMT led to reduced levels of retroviral mRNA in target cells, restriction by Fv1 did not. Treatment with the proteasome inhibitor MG132 partially relieved TRIM5α-mediated restriction of RMT. Finally, cells expressing shRNAs specifically targeting the retroviral mRNA inhibited RMT particles, but not reverse-transcribing particles. Retroviral mRNA may thus serve as a translation template if not used as a template for reverse transcription. Our data imply that retroviral nucleic acids become accessible to host factors, including ribosomes, as a result of particle remodeling during cytoplasmic trafficking.
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Peters, C., W. Rommerskirch, S. Modaressi et K. von Figura. « Restoration of arylsulphatase B activity in human mucopolysaccharidosis-type-VI fibroblasts by retroviral-vector-mediated gene transfer ». Biochemical Journal 276, no 2 (1 juin 1991) : 499–504. http://dx.doi.org/10.1042/bj2760499.
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The Maroteaux-Lamy syndrome (mucopolysaccharidosis type VI; MPS VI) is a lysosomal storage disease caused by deficiency of the enzyme arylsulphatase B (ASB). A human ASB cDNA has been subcloned into the retroviral vector pXT1 containing the bacterial neomycin-resistance gene and an internal thymidine kinase promoter for transcription of the inserted gene. Replication defective retrovirus was generated by transfecting the construct into the amphotropic packaging cell line PA317. Human MPS VI fibroblasts infected with recombinant retrovirus integrated the provirus into their genome and expressed retrovirus-encoded ASB mRNAs. In infected fibroblasts the level of ASB was up to 36-fold higher than in normal fibroblasts. Biosynthesis and processing of ASB in infected MPS VI fibroblasts was accomplished as in normal fibroblasts, and mature, enzymically active, ASB accumulated in dense lysosomes, indicating that the ASB deficiency in MPS VI fibroblasts was corrected by the retroviral gene transfer.
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Temin, H. M. « Retrovirus variation and reverse transcription : abnormal strand transfers result in retrovirus genetic variation. » Proceedings of the National Academy of Sciences 90, no 15 (1 août 1993) : 6900–6903. http://dx.doi.org/10.1073/pnas.90.15.6900.
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LaMere, Sarah A., Judy A. St. Leger, Mark D. Schrenzel, Simon J. Anthony, Bruce A. Rideout et Daniel R. Salomon. « Molecular Characterization of a Novel Gammaretrovirus in Killer Whales (Orcinus orca) ». Journal of Virology 83, no 24 (7 octobre 2009) : 12956–67. http://dx.doi.org/10.1128/jvi.01354-09.
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ABSTRACT There are currently no published data documenting the presence of retroviruses in cetaceans, though the occurrences of cancers and immunodeficiency states suggest the potential. We examined tissues from adult killer whales and detected a novel gammaretrovirus by degenerate PCR. Reverse transcription-PCR also demonstrated tissue and serum expression of retroviral mRNA. The full-length sequence of the provirus was obtained by PCR, and a TaqMan-based copy number assay did not demonstrate evidence of productive infection. PCR on blood samples from 11 healthy captive killer whales and tissues from 3 free-ranging animals detected the proviral DNA in all tissues examined from all animals. A survey of multiple cetacean species by PCR for gag, pol, and env sequences showed homologs of this virus in the DNA of eight species of delphinids, pygmy and dwarf sperm whales, and harbor porpoises, but not in beluga or fin whales. Analysis of the bottlenose dolphin genome revealed two full-length proviral sequences with 97.4% and 96.9% nucleotide identity to the killer whale gammaretrovirus. The results of single-cell PCR on killer whale sperm and Southern blotting are also consistent with the conclusion that the provirus is endogenous. We suggest that this gammaretrovirus entered the delphinoid ancestor's genome before the divergence of modern dolphins or that an exogenous variant existed following divergence that was ultimately endogenized. However, the transcriptional activity demonstrated in tissues and the nearly intact viral genome suggest a more recent integration into the killer whale genome, favoring the latter hypothesis. The proposed name for this retrovirus is killer whale endogenous retrovirus.
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Kim, Rachel, Alla Trubetskoy, Takeshi Suzuki, Nancy A. Jenkins, Neal G. Copeland et Jack Lenz. « Genome-Based Identification of Cancer Genes by Proviral Tagging in Mouse Retrovirus-Induced T-Cell Lymphomas ». Journal of Virology 77, no 3 (1 février 2003) : 2056–62. http://dx.doi.org/10.1128/jvi.77.3.2056-2062.2003.
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ABSTRACT The identification of tumor-inducing genes is a driving force for elucidating the molecular mechanisms underlying cancer. Many retroviruses induce tumors by insertion of viral DNA adjacent to cellular oncogenes, resulting in altered expression and/or structure of the encoded proteins. The availability of the mouse genome sequence now allows analysis of retroviral common integration sites in murine tumors to be used as a genetic screen for identification of large numbers of candidate cancer genes. By positioning the sequences of inverse PCR-amplified, virus-host junction fragments within the mouse genome, 19 target genes were identified in T-cell lymphomas induced by the retrovirus SL3-3. The candidate cancer genes included transcription factors (Fos, Gfi1, Lef1, Myb, Myc, Runx3, and Sox3), all three D cyclins, Ras signaling pathway components (Rras2/TC21 and Rasgrp1), and Cmkbr7/CCR7. The most frequent target was Rras2. Insertions as far as 57 kb away from the transcribed portion were associated with substantially increased transcription of Rras2, and no coding sequence mutations, including those typically involved in Ras activation, were detected. These studies demonstrate the power of genome-based analysis of retroviral insertion sites for cancer gene discovery, identify several new genes worth examining for a role in human cancer, and implicate the pathways in which those genes act in lymphomagenesis. They also provide strong genetic evidence that overexpression of unmutated Rras2 contributes to tumorigenesis, thus suggesting that it may also do so if it is inappropriately expressed in human tumors.
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Haedicke, Juliane, Kenia de los Santos, Stephen P. Goff et Mojgan H. Naghavi. « The Ezrin-Radixin-Moesin Family Member Ezrin Regulates Stable Microtubule Formation and Retroviral Infection ». Journal of Virology 82, no 9 (27 février 2008) : 4665–70. http://dx.doi.org/10.1128/jvi.02403-07.
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ABSTRACT We recently identified the cytoskeletal regulatory protein moesin as a novel gene that inhibits retroviral replication prior to reverse transcription by downregulation of stable microtubule formation. Here, we provide evidence that overexpression of ezrin, another closely related ezrin-radixin-moesin (ERM) family member, also blocks replication of both murine leukemia viruses and human immunodeficiency virus type 1 (HIV-1) in Rat2 fibroblasts before reverse transcription, while knockdown of endogenous ezrin increases the susceptibility of human cells to HIV-1 infection. Together, these results suggest that ERM proteins may be important determinants of retrovirus susceptibility through negative regulation of stable microtubule networks.
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Pavlicev, Mihaela, Kaori Hiratsuka, Kayleigh A. Swaggart, Caitlin Dunn et Louis Muglia. « Detecting Endogenous Retrovirus-Driven Tissue-Specific Gene Transcription ». Genome Biology and Evolution 7, no 4 (11 mars 2015) : 1082–97. http://dx.doi.org/10.1093/gbe/evv049.
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Cone, R. D., A. Weber-Benarous, D. Baorto et R. C. Mulligan. « Regulated expression of a complete human beta-globin gene encoded by a transmissible retrovirus vector ». Molecular and Cellular Biology 7, no 2 (février 1987) : 887–97. http://dx.doi.org/10.1128/mcb.7.2.887-897.1987.
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We introduced a human beta-globin gene into murine erythroleukemia (MEL) cells by infection with recombinant retroviruses containing the complete genomic globin sequence. The beta-globin gene was correctly regulated during differentiation, steady-state mRNA levels being induced 5- to 30-fold after treatment of the cells with the chemical inducer dimethyl sulfoxide. Studies using vectors which yield integrated proviruses lacking transcriptional enhancer sequences indicated that neither retroviral transcription nor the retroviral enhancer sequences themselves had any obvious effect on expression of the globin gene. Viral RNA expression also appeared inducible, being considerably depressed in uninduced MEL cells but approaching normal wild-type levels after dimethyl sulfoxide treatment. We provide data which suggest that the control point for both repression and subsequent activation of virus expression in MEL cells lies in the viral enhancer element.
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Cone, R. D., A. Weber-Benarous, D. Baorto et R. C. Mulligan. « Regulated expression of a complete human beta-globin gene encoded by a transmissible retrovirus vector. » Molecular and Cellular Biology 7, no 2 (février 1987) : 887–97. http://dx.doi.org/10.1128/mcb.7.2.887.
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We introduced a human beta-globin gene into murine erythroleukemia (MEL) cells by infection with recombinant retroviruses containing the complete genomic globin sequence. The beta-globin gene was correctly regulated during differentiation, steady-state mRNA levels being induced 5- to 30-fold after treatment of the cells with the chemical inducer dimethyl sulfoxide. Studies using vectors which yield integrated proviruses lacking transcriptional enhancer sequences indicated that neither retroviral transcription nor the retroviral enhancer sequences themselves had any obvious effect on expression of the globin gene. Viral RNA expression also appeared inducible, being considerably depressed in uninduced MEL cells but approaching normal wild-type levels after dimethyl sulfoxide treatment. We provide data which suggest that the control point for both repression and subsequent activation of virus expression in MEL cells lies in the viral enhancer element.
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Sampey, Gavin C., Sergey Iordanskiy, Michelle L. Pleet, Catherine DeMarino, Fabio Romerio, Renaud Mahieux et Fatah Kashanchi. « Identification of Modulators of HIV-1 Proviral Transcription from a Library of FDA-Approved Pharmaceuticals ». Viruses 12, no 10 (23 septembre 2020) : 1067. http://dx.doi.org/10.3390/v12101067.
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Human immunodeficiency virus 1 (HIV-1) is the most prevalent human retrovirus. Recent data show that 34 million people are living with HIV-1 worldwide. HIV-1 infections can lead to AIDS which still causes nearly 20,000 deaths annually in the USA alone. As this retrovirus leads to high morbidity and mortality conditions, more effective therapeutic regimens must be developed to treat these viral infections. A key target for intervention for which there are no current FDA-approved modulators is at the point of proviral transcription. One successful method for identifying novel therapeutics for treating infectious diseases is the repurposing of pharmaceuticals that are approved by the FDA for alternate indications. Major benefits of using FDA-approved drugs include the fact that the compounds have well established toxicity profiles, approved manufacturing processes, and immediate commercial availability to the patients. Here, we demonstrate that pharmaceuticals previously approved for other indications can be utilized to either activate or inhibit HIV-1 proviral transcription. Specifically, we found febuxostat, eltrombopag, and resveratrol to be activators of HIV-1 transcription, while mycophenolate was our lead inhibitor of HIV-1 transcription. Additionally, we observed that the infected cells of lymphoid and myeloid lineage responded differently to our lead transcriptional modulators. Finally, we demonstrated that the use of a multi-dose regimen allowed for enhanced activation with our transcriptional activators.
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Lascano, Josefina, Pradeep D. Uchil, Walther Mothes et Jeremy Luban. « TRIM5 Retroviral Restriction Activity Correlates with the Ability To Induce Innate Immune Signaling ». Journal of Virology 90, no 1 (14 octobre 2015) : 308–16. http://dx.doi.org/10.1128/jvi.02496-15.
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ABSTRACTHost restriction factor TRIM5 inhibits retroviral transduction in a species-specific manner by binding to and destabilizing the retroviral capsid lattice before reverse transcription is completed. However, the restriction mechanism may not be that simple since TRIM5 E3 ubiquitin ligase activity, the proteasome, autophagy, and TAK1-dependent AP-1 signaling have been suggested to contribute to restriction. Here, we show that, among a panel of seven primate and Carnivora TRIM5 orthologues, each of which has potential for potent retroviral restriction activity, all activated AP-1 signaling. In contrast, TRIM family paralogues most closely related to TRIM5 did not. While each primate species has a single TRIM5 gene, mice have at least seven TRIM5 homologues that cluster into two groups, Trim12a, -b, and -c and Trim30a, -b, -c, and -d. The three Trim12 proteins activated innate immune signaling, while the Trim30 proteins did not, though none of the murine Trim5 homologues restricted any of a panel of cloned retroviruses. To determine if any mouse TRIM5 homologues had potential for restriction activity, each was fused to the human immunodeficiency virus type 1 (HIV-1) CA binding protein cyclophilin A (CypA). The three Trim12-CypA fusions all activated AP-1 and restricted HIV-1 transduction, whereas the Trim30-CypA fusions did neither. AP-1 activation and HIV-1 restriction by the Trim12-CypA fusions were inhibited by disruption of TAK1. Overall then, these experiments demonstrate that there is a strong correlation between TRIM5 retroviral restriction activity and the ability to activate TAK1-dependent innate immune signaling.IMPORTANCEThe importance of retroviruses for the evolution of susceptible host organisms cannot be overestimated. Eight percent of the human genome is retrovirus sequence, fixed in the germ line during past infection. Understanding how metazoa protect their genomes from mutagenic retrovirus infection is therefore of fundamental importance to biology. TRIM5 is a cellular protein that protects host genome integrity by disrupting the retroviral capsid as it transports viral nucleic acid to the host cell nucleus. Previous data suggest that innate immune signaling contributes to TRIM5-mediated restriction. Here, we show that activation of innate immune signaling is conserved among primate and carnivore TRIM5 orthologues and among 3 of the 7 mouse Trim5 homologues and that such activity is required for TRIM5-mediated restriction activity.
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Wang, Cheng, et Stephen P. Goff. « Differential control of retrovirus silencing in embryonic cells by proteasomal regulation of the ZFP809 retroviral repressor ». Proceedings of the National Academy of Sciences 114, no 6 (23 janvier 2017) : E922—E930. http://dx.doi.org/10.1073/pnas.1620879114.
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Replication of the murine leukemia viruses is strongly suppressed in mouse embryonic stem (ES) cells. Proviral DNAs are formed normally but are then silenced by a large complex bound to DNA by the ES cell-specific zinc-finger protein ZFP809. We show here that ZFP809 expression is not regulated by transcription but rather by protein turnover: ZFP809 protein is stable in embryonic cells but highly unstable in differentiated cells. The protein is heavily modified by the accumulation of polyubiquitin chains in differentiated cells and stabilized by the proteasome inhibitor MG132. A short sequence of amino acids at the C terminus of ZFP809, including a single lysine residue (K391), is required for the rapid turnover of the protein. The silencing cofactor TRIM28 was found to promote the degradation of ZFP809 in differentiated cells. These findings suggest that the stem cell state is established not only by an unusual transcriptional profile but also by unusual regulation of protein levels through the proteasomal degradation pathway.
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Stuhlmann, H., et P. Berg. « Homologous recombination of copackaged retrovirus RNAs during reverse transcription. » Journal of Virology 66, no 4 (1992) : 2378–88. http://dx.doi.org/10.1128/jvi.66.4.2378-2388.1992.
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Tillmann, Maribeth, Fred C. Krebs, Renee Wessner, Steven M. Pomeroy, Maureen M. Goodenow et Brian Wigdahl. « Neuroglial-specific factors and the regulation of retrovirus transcription ». Advances in Neuroimmunology 4, no 3 (janvier 1994) : 305–18. http://dx.doi.org/10.1016/s0960-5428(06)80271-8.
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Assinger, Alice, Koon-Chu Yaiw, Ingmar Göttesdorfer, Christine Leib-Mösch et Cecilia Söderberg-Nauclér. « Human Cytomegalovirus (HCMV) induces Human Endogenous Retrovirus (HERV) transcription ». Retrovirology 10, no 1 (2013) : 132. http://dx.doi.org/10.1186/1742-4690-10-132.
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Rovnak, Joel, et Sandra L. Quackenbush. « Walleye Dermal Sarcoma Virus Retroviral Cyclin Directly Contacts TAF9 ». Journal of Virology 80, no 24 (11 octobre 2006) : 12041–48. http://dx.doi.org/10.1128/jvi.01425-06.
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ABSTRACT Walleye dermal sarcoma virus (WDSV) is a complex retrovirus associated with dermal sarcomas in walleye fish. A WDSV accessory gene encodes a cyclin homolog or retroviral cyclin (rv-cyclin). WDSV rv-cyclin was found to be associated with transcription complexes and to affect transcription in a cell-type and promoter-dependent manner. It inhibited the WDSV promoter in walleye fibroblasts and activated transcription from GAL4 promoters when fused to the GAL4 DNA binding domain, and an activation domain (AD) has been localized to 30 amino acids in the carboxyl region. rv-cyclin can block the pulldown of transcription coactivators by the AD of VP16, and the isolated rv-cyclin AD interferes specifically with the interaction between the carboxyl halves of the VP16 AD, VP16C, and TATA-binding protein-associated factor 9 (TAF9). The carboxyl region and isolated AD can bind TAF9 directly in assays of protein-protein interaction in vitro. Furthermore, rv-cyclin and the isolated rv-cyclin AD interfere specifically with the function of VP16C in transcription assays. A previously identified motif within the VP16C sequence mediates TAF9 binding, and this motif is present in the activation domains of a variety of TAF9-binding transcriptional activators. A similar motif is present in the rv-cyclin AD, and point mutations within this motif affect rv-cyclin function and protein-protein interactions. The results support a model of transcription regulation by direct interaction with TAF9.
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Bartholomew, C., et J. N. Ihle. « Retroviral insertions 90 kilobases proximal to the Evi-1 myeloid transforming gene activate transcription from the normal promoter ». Molecular and Cellular Biology 11, no 4 (avril 1991) : 1820–28. http://dx.doi.org/10.1128/mcb.11.4.1820-1828.1991.
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The inappropriate production of the Evi-1 zinc finger protein occurs in retrovirus-induced murine myeloid leukemias and human acute myelogenous leukemias. In murine leukemias, expression of the Evi-1 gene is associated with retroviral insertions either in the Evi-1 locus, which is immediately 5' of the coding region of the gene, or in the genetically linked Cb-1/fim-3 locus. In these studies, we demonstrate by chromosomal walking and pulse field electrophoresis that the Cb-1/fim-3 locus is located 90 kb 5' of the Evi-1 locus. Primary structure analysis of Evi-1 cDNA clones from a Cb-1/fim-3 rearranged cell line (DA-3) demonstrates that transcription initiates 5' of the Evi-1 locus and that the first noncoding exon of the gene is 681 bp larger than previously defined. S1 nuclease protection studies reveal multiple transcription initiation sites within this region. Comparable transcriptional initiation sites were identified in RNA from kidney and ovary, in which the gene is normally expressed, suggesting that retroviral insertions in the Cb-1/fim-3 locus activate transcription from the normal promoter. In one myeloid cell line (DA-3), a single long terminal repeat (LTR) is present in the Cb-1/fim-3 locus. No stable transcripts were detectable from this LTR. In cells with retroviral insertions in the Cb-1/fim-3 locus, one allele of the Evi-1 locus becomes hypermethylated in the 5' region of the gene. Together, these results are most consistent with an LTR-mediated, long-range cis activation of Evi-1 gene expression.
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Bartholomew, C., et J. N. Ihle. « Retroviral insertions 90 kilobases proximal to the Evi-1 myeloid transforming gene activate transcription from the normal promoter. » Molecular and Cellular Biology 11, no 4 (avril 1991) : 1820–28. http://dx.doi.org/10.1128/mcb.11.4.1820.
Texte intégralRésumé :
The inappropriate production of the Evi-1 zinc finger protein occurs in retrovirus-induced murine myeloid leukemias and human acute myelogenous leukemias. In murine leukemias, expression of the Evi-1 gene is associated with retroviral insertions either in the Evi-1 locus, which is immediately 5' of the coding region of the gene, or in the genetically linked Cb-1/fim-3 locus. In these studies, we demonstrate by chromosomal walking and pulse field electrophoresis that the Cb-1/fim-3 locus is located 90 kb 5' of the Evi-1 locus. Primary structure analysis of Evi-1 cDNA clones from a Cb-1/fim-3 rearranged cell line (DA-3) demonstrates that transcription initiates 5' of the Evi-1 locus and that the first noncoding exon of the gene is 681 bp larger than previously defined. S1 nuclease protection studies reveal multiple transcription initiation sites within this region. Comparable transcriptional initiation sites were identified in RNA from kidney and ovary, in which the gene is normally expressed, suggesting that retroviral insertions in the Cb-1/fim-3 locus activate transcription from the normal promoter. In one myeloid cell line (DA-3), a single long terminal repeat (LTR) is present in the Cb-1/fim-3 locus. No stable transcripts were detectable from this LTR. In cells with retroviral insertions in the Cb-1/fim-3 locus, one allele of the Evi-1 locus becomes hypermethylated in the 5' region of the gene. Together, these results are most consistent with an LTR-mediated, long-range cis activation of Evi-1 gene expression.
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Kim, Eui Tae, Tommy E. White, Alberto Brandariz-Núñez, Felipe Diaz-Griffero et Matthew D. Weitzman. « SAMHD1 Restricts Herpes Simplex Virus 1 in Macrophages by Limiting DNA Replication ». Journal of Virology 87, no 23 (25 septembre 2013) : 12949–56. http://dx.doi.org/10.1128/jvi.02291-13.
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Macrophages play important roles in host immune defense against virus infection. During infection by herpes simplex virus 1 (HSV-1), macrophages acquire enhanced antiviral potential. Restriction of HSV-1 replication and progeny production is important to prevent viral spread, but the cellular mechanisms that inhibit the DNA virus in macrophages are unknown. SAMHD1 was recently identified as a retrovirus restriction factor highly expressed in macrophages. The SAMHD1 protein is expressed in both undifferentiated monocytes and differentiated macrophages, but retroviral restriction is limited to differentiated cells by modulation of SAMHD1 phosphorylation. It is proposed to block reverse transcription of retroviral RNA into DNA by depleting cellular deoxynucleotide triphosphates (dNTPs). Viruses with DNA genomes do not employ reverse transcription during infection, but replication of their viral genomes is also dependent on intracellular dNTP concentrations. Here, we demonstrate that SAMHD1 restricts replication of the HSV-1 DNA genome in differentiated macrophage cell lines. Depleting SAMHD1 in THP-1 cells enhanced HSV-1 replication, while ectopic overexpression of SAMHD1 in U937 cells repressed HSV-1 replication. SAMHD1 did not impact viral gene expression from incoming HSV-1 viral genomes. HSV-1 restriction involved the dNTP triphosphohydrolase activity of SAMHD1 and was partially overcome by addition of exogenous deoxynucleosides. Unlike retroviruses, restriction of HSV-1 was not affected by SAMHD1 phosphorylation status. Our results suggest that SAMHD1 functions broadly to inhibit replication of DNA viruses in nondividing macrophages.
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Mizuno, Shin-ichi, Hidetoshi Ozawa, Hirokazu Shigematsu, Yojiro Arinobu, Chong Yong, Tadafumi Iino et Koichi Akashi. « Notch1 Expression Is Regulated at Translational Level by 3′UTR Region in Hematopoietic Stem Cell Development. » Blood 108, no 11 (16 novembre 2006) : 782. http://dx.doi.org/10.1182/blood.v108.11.782.782.
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Abstract In hematopoietic stem cell development, the expression of critical genes is precisely regulated in a stage specific manner, which supports normal hematopoietic development through adequately regulating timing of cell division, self-renewal, and lineage commitment of hematopoietic stem cells. Regulation of gene expression is known to take place at transcriptional level. In addition to the transcriptional regulation, there are growing evidences that translational control of critical genes may play a role, especially in embryogenic development, suggesting an interesting possibility that translational control may also play a role in hematopoiesis. Here, we provide the evidence that the expression of a key transcription factor in lymphoid development, Notch1, is controlled at translational level in hematopoietic stem cell. To examine whether translation of mRNAs of hematopoietic major factors can be regulated at each stage of hematopoietic development, we established a retrovirus sensor vector, in which 3′UTR (untranslated region) of target genes is placed between the GFP coding region and the retrovirus 3′LTR. Since the transcribed mRNA from this vector is a fusion mRNA of GFP and 3′UTR from the gene of interest, this vector allows us to visualize the effect of 3′UTR on translational control of particular genes by GFP as a reporter. We cloned 3′UTR fragments from PU.1, GATA-1, GATA-2, C/EBPα and Notch1 genes into the sensor vector. Then, we introduced these retrovirus vectors into prospectively-purified hematopoietic stem and progenitor cells, including HSC, CMP and GMP, and their GFP intensities were monitored by a flowcytometer. We found that induction of the sensor vector with the 3′UTR sequence from Notch1 showed marked suppression of the GFP intensity at the HSC stage. The 3′UTR sequences from the other 4 transcription factors did not show such intensive inhibitory effect. Suppression of Notch1 transcription by its 3′UTR was further confirmed by using a retrovirus vector which has two distinct markers of YFP and GFP-3′UTR fusion genes under bi-directional EF1α promoter. These data suggest that the expression of Notch1 should be regulated at translational level by its 3′UTR at the HSC stage as well as at transcriptional level. Our data provide the first evidence that the stage-specific translational regulation can play an important role in organization of hematopoietic development.
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Lavie, Laurence, Milena Kitova, Esther Maldener, Eckart Meese et Jens Mayer. « CpG Methylation Directly Regulates Transcriptional Activity of the Human Endogenous Retrovirus Family HERV-K(HML-2) ». Journal of Virology 79, no 2 (15 janvier 2005) : 876–83. http://dx.doi.org/10.1128/jvi.79.2.876-883.2005.
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ABSTRACT A significant proportion of the human genome consists of stably inherited retroviral sequences. Most human endogenous retroviruses (HERVs) became defective over time. The HERV-K(HML-2) family is exceptional because of its coding capacity and the possible involvement in germ cell tumor (GCT) development. HERV-K(HML-2) transcription is strongly upregulated in GCTs. However, regulation of HERV-K(HML-2) transcription remains poorly understood. We investigated in detail the role of CpG methylation on the transcriptional activity of HERV-K(HML-2) long terminal repeats (LTRs). We find that CpG sites in various HERV-K(HML-2) proviral 5′ LTRs are methylated at different levels in the cell line Tera-1. Methylation levels correlate with previously observed transcriptional activities of these proviruses. CpG-mediated silencing of HERV-K(HML-2) LTRs is further corroborated by transcriptional inactivity of in vitro-methylated 5′ LTR reporter plasmids. However, CpG methylation levels do not solely regulate HERV-K(HML-2) 5′ LTR activity, as evidenced by different LTR activities in the cell line T47D. A significant number of mutated CpG sites in evolutionary old HERV-K(HML-2) 5′ LTRs suggests that CpG methylation had already silenced HERV-K(HML-2) proviruses millions of years ago. Direct silencing of HERV-K(HML-2) expression by CpG methylation enlightens upregulated HERV-K(HML-2) expression in usually hypomethylated GCT tissue.
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Gao, Tingting, Fuquan Chen, Wenying Zhang, Xuan Zhao, Xiao Hu et Xinyi Lu. « Nsd2 Represses Endogenous Retrovirus MERVL in Embryonic Stem Cells ». Stem Cells International 2021 (16 janvier 2021) : 1–8. http://dx.doi.org/10.1155/2021/6663960.
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The facilitates chromatin transcription (FACT) complex is a histone H2A/H2B chaperone, which represses endogenous retroviruses (ERVs) and transcription of ERV-chimeric transcripts. It binds to both transcription start site and gene body region. Here, we investigated the downstream targets of FACT complex to identify the potential regulators of MERVL, which is a key 2-cell marker gene. H3K36me2 profile was positively correlated with that of FACT component Ssrp1. Among H3K36me2 deposition enzymes, Nsd2 was downregulated after the loss of Ssrp1. Furthermore, we demonstrated that Nsd2 repressed the expression of ERVs without affecting the expression of pluripotency genes. The expression of MERVL and 2-cell genes was partially rescued by Nsd2 overexpression. The enrichment of H3K36me2 decreased on MERVL-chimeric gene in ESCs without Ssrp1. Our study discovers that Nsd2 is a repressor of MERVL, and FACT partially represses MERVL expression by regulating the expression of Nsd2 and its downstream H3K36me2.
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Bodem, Jochen, Hans-Georg Kräusslich et Axel Rethwilm. « Acetylation of the foamy virus transactivator Tas by PCAF augments promoter-binding affinity and virus transcription ». Journal of General Virology 88, no 1 (1 janvier 2007) : 259–63. http://dx.doi.org/10.1099/vir.0.82169-0.
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It was shown recently that retrovirus transactivators interact with transcriptional coactivators, such as histone acetyltransferases (HATs). Foamy viruses (FVs) direct gene expression from the long terminal repeat and from an internal promoter. The activity of both promoters is strictly dependent on the DNA-binding transactivator Tas. Recently, it was shown that Tas interacts with the HATs p300 and PCAF. Based on these findings, it is demonstrated here that PCAF has the ability to acetylate Tas in vitro and in vivo. Tas acetylation resulted in enhanced DNA binding to the virus promoters. In vitro transcription reactions on non-chromatinized template showed that only acetylated Tas enhanced transcription significantly. These results demonstrate that acetylation of the FV transactivator Tas may be an effective means to regulate virus transcription.
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Perkins, Archibald S., Jacob J. del Campo, Ying-Yi Xiao, Yi Zhang, Sharon J. Lin, Jonathan Dudley, David Tuck et Bogdan Yatsula. « EVI1 Blocks Apoptosis in DA-1 Myeloid Leukemia Cells Via Enhanced Transcription of the Prosurvival Gene Bcl2a1 (A1) ». Blood 112, no 11 (16 novembre 2008) : 3805. http://dx.doi.org/10.1182/blood.v112.11.3805.3805.
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Abstract Retroviral insertion at the Evi1 locus causes high level of expression of the gene in myeloid neoplasms in mice and in nonmalignant expansions of myeloid cells in humans and monkeys. In these settings, it is suggested that EVI1 confers a survival advantage on myeloid cells. Here, we investigate the survival phenotype in DA-1 cells, a leukemic cell line with provirally activated Evi1. We report that short hairpin-mediated suppression of Evi1 in DA-1 cells induces apoptosis via the intrinsic/mitochondrial pathway: DNA fragmentation and histone release are both induced, as is reduction in mitochondrial membrane potential. In addition, procasapses 3 and 9, but not caspase 8 or Bid, are cleaved following Evi1 knockdown; phosphoAKT remains unchanged. Furthermore, mRNA expression profiling following Evi1 suppression show transcriptional changes in several apoptotic regulators, including a 3.5-fold decrease in Bcl2a1 (bfl/A1), a prosurvival member of the Bcl-2 family. To assess whether EVI1 regulates Bcl2a1 expression in a cell type other than DA-1 cells, we transduced primary murine Lin-/Sca-1+/c-Kit+ cells with EVI1 via retroviral vector, and then assessed the expression of Bcl2a1. This revealed that EVI1 induced a more than five-fold increase in Bcl2a1 mRNA expression, as measured by quantitative PCR. Furthermore, transduction of primary murine bone marrow cells with retroviruses bearing either Bcl2a1 or Evi1 resulted in a significant decrease in spontaneous apoptosis, as assessed by the activity of caspases 3 and 7. To further test if Bcl2a1 is necessary for leukemic transformation by Evi1, we assessed the ability of Evi1 to confer serial replating ability on primary bone marrow cells from Bcl2a1−/− mice. Bone marrow was harvested from Bcl2a1−/− and C57BL6 mice and transduced with retrovirus containing either no gene or Evi1. While in C57BL6 mice, Evi1 induced a significant increase in colonies, bone marrow from Bcl2a1−/− mice were resistant to transformation by Evi1. To show that this was due to the lack of Bcl2a1, the gene was added back via retrovirus. While Bcl2a1 by itself did not induce significant number of colonies over vector, when introduced into Bcl2a1−/− cells together with Evi1, there was a significant increase in colony formation. These data indicate that transformation of bone marrow cells by Evi1 depends on the presence of the Bcl2a1 gene. We further show EVI1 can transcriptionally upregulate a BCL2A1::luc reporter that harbors 1.37 kb of the human BCL2A1 upstream sequence. Our analysis of the Bcl2a1 promoter indicates that the effect of EVI1 is likely indirect. Based on our findings, we propose that EVI1 acts to block apoptosis in DA-1 cells by transcriptionally activating Bcl2a1.
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Chen, P. J., A. Cywinski et J. M. Taylor. « Reverse transcription of 7S L RNA by an avian retrovirus. » Journal of Virology 54, no 2 (1985) : 278–84. http://dx.doi.org/10.1128/jvi.54.2.278-284.1985.
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Seifarth, Wolfgang, Corinna Baust, Andreas Murr, Heyko Skladny, Frank Krieg-Schneider, Jürgen Blusch, Thomas Werner, Rüdiger Hehlmann et Christine Leib-Mösch. « Proviral Structure, Chromosomal Location, and Expression of HERV-K-T47D, a Novel Human Endogenous Retrovirus Derived from T47D Particles ». Journal of Virology 72, no 10 (1 octobre 1998) : 8384–91. http://dx.doi.org/10.1128/jvi.72.10.8384-8391.1998.
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ABSTRACT We previously described that type B retrovirus-like particles released from the human mammary carcinoma cell line T47D are pseudotypes and package retroviral RNA of different origins (W. Seifarth, H. Skladny, F. Krieg-Schneider, A. Reichert, R. Hehlmann, and C. Leib-Mösch, J. Virol. 69:6408–6416, 1995). One preferentially packaged retroviral sequence, ERV-MLN, has now been used to isolate the corresponding full-length provirus from a human genomic library. The 9,315-bp proviral genome comprises a complete retroviral structure except for a 3′ long terminal repeat (LTR) truncation. A lysine tRNA primer-binding site and phylogenetic analyses assign this human endogenous retroviral element, now called HERV-K-T47D, to the HML-4 subgroup of the HERV-K superfamily. The gag,prt, pol, and env genes exhibit 40 to 60% amino acid identity to HERV-K10. HERV-K-T47D is located on human chromosome 10, with five closely related elements on chromosomes 8, 9, 15, 16, and 19 and several hundred HERV-K-T47D-related solitary LTRs dispersed over the human genome. HERV-K-T47D-related sequences are detected in the genomes of higher primates and Old World monkeys but not in those of New World monkeys. High HERV-K-T47D transcription levels were observed in human placenta tissue, whereas transcription in T47D cells was strictly steroid dependent.
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Stubbs, Matthew C., InSung Min, Marc W. Izzo, Ravikumar Rallapalli, Assia Derfoul et David J. Hall. « The ZF87/MAZ transcription factor functions as a growth suppressor in fibroblasts ». Biochemistry and Cell Biology 78, no 4 (3 avril 2000) : 477–85. http://dx.doi.org/10.1139/o00-053.
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ZF87/MAZ is a zinc finger transcription factor that activates expression of tissue-specific genes and represses expression of the c-myc proto-oncogene. Infection of NIH3T3 fibroblasts with a retrovirus expressing ZF87/MAZ leads to a significant reduction in G418-resistant colonies, compared to cells infected with a retroviral control. Further, only a small fraction of the G418-resistant colonies express ZF87/MAZ. When the ZF87/MAZ-expressing colonies are expanded, they demonstrate a slow growth phenotype, a delayed transit through G1 phase and a decrease in endogenous c-myc gene expression and cyclin A and cyclin E protein levels. Consistent with a partial G1 arrest, the ZF87/MAZ-expressing cells show a reduced sensitivity to the S phase specific chemotherapeutic agent camptothecin. These data indicate that ZF87/MAZ is a growth suppressor protein in nontransformed cells, in part, by affecting the levels of key cell cycle regulatory proteins.Key words: cell cycle, ZF87/MAZ, cancer.
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Feng, Ru, Lixia Hao, Hua Jin, Xiaolei Wei, Yongqiang Wei, Fen Huang et Changxin Yin. « The Initial Research on Relationship of Transcription Factor C/EBPα and Raji Cell Line ». Blood 118, no 21 (18 novembre 2011) : 4650. http://dx.doi.org/10.1182/blood.v118.21.4650.4650.
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Abstract Abstract 4650 Introduction: Leukemia is the common term for a diverse group of malignancies, defined by accumulation of abnormal hematopoietic progenitor cells, which fail to undergo terminal differentiation. The CCATT/enhancer binding protein alpha, C/EBPα, is a key transcription factor involved in normal hematopoietic system and leukemia. C/EBPα acts as an inhibitor of growth and apoptosis, and induces myeloid progenitors to differentiate. It is also reported that enforced expression of C/EBPα in B cells leads to their rapid and efficient reprogramming into macrophages. According to the phenomenon of cell-type transformation in clinical practice, we explore the role of C/EBPα in transformation of Raji cell line. Materials and Methods We detected expression of C/EBPα gene in Raji cell line by RT-PCR.The retroviral vector was inducted into the packaging cell Phoenix 293 by Lipofectamine™ 2000 (Invitrogen). Retroviruses encoding full-lengh cDNAs of murine C/EBPα were cloned by PCR into the BglII/XhoI sites of the pMIG retrovirus vector by creating a BglII site on the 5 end and a XhoI site at the 3 end. The supernatant of retrovirus was used to transfect Raji cells. To study the biological differences in C/EBPα positive-Raji cells, we used multiple methods such as cytomorphology, flow cytometry, MTT and RT-PCR. Results 1. Raji, 6T-CEM, Molt-4 and K562 cell line were negative for C/EBPα gene.However, HL60 and NB4 cells were positive for C/EBPα gene. Sequencing results showed that there is no genetic mutation. 2. The retrovirus pMIG and pMIG-C/EBPα were used to transfect Raji cell. The 72h transfection rates were 28.5% and 8.4% respectively. We used FACS to sort the GFP+ cells and amplification these cells. The GFP+ cells were taken account above 80%. By RT-PCR, we confirmed that C/EBPα gene was stably expressed. 3. Cytomorphology showed that the nuclein of Raji+pMIG-C/EBPα cells was loose compared to the other two cells. All the three cells were PAS and POX negative. FACS results showed that CD19 positive rate was (97.76±1.48)% A(97.93±0.64)% A(96.98±1.80)% in Raji cells, Raji+pMIG cells and Raji+pMIG-C/EBPα cells separately(P=0.688), and their mean value was 7003.5±276.48, 5803.5±159.10, 4808.5±327.39 (P=0.008). However, they were all negative for CD33 and CD14.RT-QPCR results showed that Raji cells, Raji+pMIG cells and Raji+pMIG-C/EBPα cells were Pax5 and PU.1 positive(P value was 0.450 and 0.186), and negative for MPO, G-CSFR and GM-CSFR gene. 4. We knew that C/EBPα gene may decrease proliferation of the cell. And this was maybe the reason for why we didn’t find a cell-type tansformation we expected. We did FACS 72h after transfection and found that CD19 positive rate of the Raji+pMIG-C/EBPα cell was decreased, but CD14 and CD33 were still nagetive. By sorting the CD19 negative cells which including all the GFP positive cells, we detected that apart from β-actin and C/EBPα gene were positive, the other five genes were all nagetive including Pax5 and PU.1. Conclusion C/EBPα acts as an inhibitor of growth in Raji cell line. B lymphoid cell related antigen CD19 and Pax5 and PU.1 genes were decreased after C/EBPα gene transfection.We conclude that more than one transcription factor were involved in this complex progress, and PU.1 may act an equal important role in it. Disclosures: No relevant conflicts of interest to declare.
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Menees, Thomas M. « RNA Lariat Debranching Enzyme as a Retroviral and Long-Terminal-Repeat Retrotransposon Host Factor ». Annual Review of Virology 7, no 1 (29 septembre 2020) : 189–202. http://dx.doi.org/10.1146/annurev-virology-012720-094902.
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Host cell factors are integral to viral replication. Human immunodeficiency virus 1 (HIV-1), the retroviral agent of acquired immune deficiency syndrome, requires several host factors for reverse transcription of the viral genomic RNA (gRNA) into DNA shortly after viral entry. One of these host factors is the RNA lariat debranching enzyme (Dbr1), which cleaves the 2′–5′ bond of branched and lariat RNAs. A recent study has revealed that Dbr1 cleaves HIV-1 gRNA lariats that form early after viral entry. Without Dbr1 activity, HIV-1 reverse transcription stalls, consistent with blockage of viral reverse transcriptase at gRNA branch points. These findings echo an earlier study with the long-terminal-repeat retrotransposon of Saccharomyces cerevisiae, Ty1, which is a retrovirus model. Currently, branching and debranching of viral gRNA are not widely recognized as features of HIV-1 replication, and the role of a gRNA lariat is not known. Future studies will determine whether these gRNA dynamics represent fundamental features of retroviral biology and whether they occur for other positive-sense RNA viruses.
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Crise, Bruce, Yuan Li, Chiuchin Yuan, David R. Morcock, Denise Whitby, David J. Munroe, Larry O. Arthur et Xiaolin Wu. « Simian Immunodeficiency Virus Integration Preference Is Similar to That of Human Immunodeficiency Virus Type 1 ». Journal of Virology 79, no 19 (1 octobre 2005) : 12199–204. http://dx.doi.org/10.1128/jvi.79.19.12199-12204.2005.
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ABSTRACT Simian immunodeficiency virus (SIV) is a useful model for studying human immunodeficiency virus (HIV) pathogenesis and vaccine efficacy. As with all other retroviruses, integration is a necessary step in the replication cycle of SIV. The location of the retrovirus integration site is known to impact on viral gene expression, establishment of viral latency, and other aspects of the replication cycle of a retrovirus. In this study, 148 SIV provirus integration sites were sequenced and mapped in the human genome. Our analysis showed that SIV integration, like that of HIV type 1 (HIV-1), exhibited a strong preference for actively transcribed regions in the genome (A. R. Schroder et al., Cell 110:521-529, 2002) and no preference for the CpG islands or transcription start sites, in contrast to observations for murine leukemia virus (X. Wu et al., Science 300:1749-1751, 2003). The parallel integration target site preferences of SIV and HIV-1 suggest that these lentiviruses may share similar mechanisms for target site selection and that SIV serves as an accurate model of HIV-1 with respect to integration.
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Klymiuk, Nikolai, Mathias Müller, Gottfried Brem et Bernhard Aigner. « Phylogeny, recombination and expression of porcine endogenous retrovirus γ2 nucleotide sequences ». Journal of General Virology 87, no 4 (1 avril 2006) : 977–86. http://dx.doi.org/10.1099/vir.0.81552-0.
Texte intégralRésumé :
Endogenous retroviral sequences in the pig genome represent a potential infectious risk in xenotransplantation. Porcine endogenous retrovirus (PERV) γ sequences described to date have been classified into several families. The known infectious, human-tropic PERVs have been assigned to the PERV γ1 subfamilies A, B and C. High copy numbers and full-length clones have also been observed for an additional family, designated PERV γ2. The aim of this study was to examine the PERV γ2 family by analysis of retroviral pro/pol gene sequences. The proviral load was observed to be similar among various pig breeds. Although clones harbouring an open reading frame in the examined region were found, analysis of published large PERV γ2 clones revealed multiple deleterious mutations in each of the retroviral genes. Various recombination events between γ2 genomes were revealed. In contrast to PERV γ1, phylogenetic analyses did not distinguish defined subfamilies, but indicated the independent evolution of the proviruses after a single event of retroviral amplification. Expression analysis showed large PERV γ2 transcripts and variable transcription in several tissues. Analysis of the two published γ2 env gene sequences observed the partial lack of the receptor-binding domain. Overall, this study indicated the low infectious potential for PERV γ2.
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Schön, Ulrike, Olivia Diem, Laura Leitner, Walter H. Günzburg, Dixie L. Mager, Brian Salmons et Christine Leib-Mösch. « Human Endogenous Retroviral Long Terminal Repeat Sequences as Cell Type-Specific Promoters in Retroviral Vectors ». Journal of Virology 83, no 23 (9 septembre 2009) : 12643–50. http://dx.doi.org/10.1128/jvi.00858-09.
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ABSTRACT The human genome contains more than half a million human endogenous retrovirus (HERV) long terminal repeats (LTRs) that can be regarded as mobile regulatory modules. Many of these HERV LTRs have been recruited during evolution as transcriptional control elements for cellular gene expression. We have cloned LTR sequences from two HERV families, HERV-H and HERV-L, differing widely in their activity and tissue specificity into a murine leukemia virus (MLV)-based promoter conversion vector (ProCon). Various human cell lines were infected with the HERV-MLV hybrid vectors, and cell type-specific expression of the reporter gene was compared with the promoter specificity of the corresponding HERV LTRs in transient-transfection assays. Transcription start site analysis of HERV-MLV hybrid vectors revealed preferential use of the HERV promoter initiation site. Our data show that HERV LTRs function in the context of retroviral vectors in certain cell types and have the potential to be useful as cell type-specific promoters in vector construction.
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Mendis, Shehara Ramyalini, James Thomas Topham, Emma Titmuss, Laura Williamson, Erin D. Pleasance, Luka Culibrk, Joanna Karasinska et al. « Comprehensive transcriptome analysis reveals link between epigenetic dysregulation, endogenous retrovirus expression and immunogenicity in metastatic colorectal carcinoma. » Journal of Clinical Oncology 37, no 15_suppl (20 mai 2019) : 3535. http://dx.doi.org/10.1200/jco.2019.37.15_suppl.3535.
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3535 Background: Endogenous retrovirus (ERV) elements represent genomic footprints of ancestral retroviral infections within the human genome. Previous studies have demonstrated increases in ERV mRNA as a result of DNA hypomethylation, and ERV transcription has been associated with increased immunogenicity in metastatic renal cell carcinoma. We performed comprehensive bioinformatics analysis of ERV transcription in metastatic colorectal carcinoma (mCRC), to identify novel links between ERV transcription, epigenetic dysregulation and immunogenicity in metastatic colorectal carcinoma (mCRC). Methods: Tumour samples from 63 patients with mCRC were subjected to RNA sequencing as part of the Personalized OncoGenomics program (POG; NCT02155621) at BC Cancer. Patients were enrolled between 07/2012-07/2017. ERV transcription was quantified across 702,533 distinct loci. Tumors were classified ERV-hi if their total ERV expression (RPKM) was greater than the mean across all samples. High antiviral gene expression tumors (AVG-hi) were designated as having a mean expression of IFIH1, DDX58, TLR3, TANK, TBKBP1, TBK1, IRF3 and IRF7 that was greater than the mean across all samples. All pairwise comparisons of gene expression were subjected to multiple hypothesis correction. Results: Median age was 59 years, with 34 (54%) male and 1 tumor microsatellite unstable. ERV-hi tumors showed increased expression of DNA demethylators TET2 ( q=0.0045) and TET3 ( q<0.0001). Significant overlap existed between ERV-hi and AVG-hi tumors (18/27, p=0.016). Tumors both ERV-hi and AVG-hi trended towards increased PD-L1 expression (p=0.055) and showed a significant increase in survival compared to tumors with high antiviral expression in the absence of high ERV transcription (p=0.0043). Conclusions: Our results suggest DNA demethylation drives increased ERV transcription and ERV-associated immunogenicity in mCRC. Moreover, we provide novel insight into the impact of ERV transcription on the biology of mCRC, highlighting ERV transcription as a potential biomarker and target for precision immunotherapy. Clinical trial information: NCT02155621.
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Laverdure, Sylvain, Nicholas Polakowski, Kimson Hoang et Isabelle Lemasson. « Permissive Sense and Antisense Transcription from the 5′ and 3′ Long Terminal Repeats of Human T-Cell Leukemia Virus Type 1 ». Journal of Virology 90, no 7 (20 janvier 2016) : 3600–3610. http://dx.doi.org/10.1128/jvi.02634-15.
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ABSTRACTHuman T-cell leukemia virus type 1 (HTLV-1) is a retrovirus, and, as such, its genome becomes chromosomally integrated following infection. The resulting provirus contains identical 5′ and 3′ peripheral long terminal repeats (LTRs) containing bidirectional promoters. Antisense transcription from the 3′ LTR regulates expression of a single gene,hbz, while sense transcription from the 5′ LTR controls expression of all other viral genes, includingtax. Both the HBZ and Tax proteins are implicated in the development of adult T-cell leukemia (ATL), a T-cell malignancy caused by HTLV-1 infection. However, these proteins appear to harbor opposing molecular functions, indicating that they may act independently and at different time points prior to leukemogenesis. Here, we used bidirectional reporter constructs to test whether transcriptional interference serves as a mechanism that inhibits simultaneous expression of Tax and HBZ. We found that sense transcription did not interfere with antisense transcription from the 3′ LTR and vice versa, even with strong transcription emanating from the opposing direction. Therefore, bidirectional transcription across the provirus might not restricthbzortaxexpression. Single-cell analyses revealed that antisense transcription predominates in the absence of Tax, which transactivates viral sense transcription. Interestingly, a population of Tax-expressing cells exhibited antisense but not activated sense transcription. Consistent with the ability of Tax to induce cell cycle arrest, this population was arrested in G0/G1phase. These results imply that cell cycle arrest inhibits Tax-mediated activation of sense transcription without affecting antisense transcription, which may be important for long-term viral latency.IMPORTANCEThe chromosomally integrated form of the retrovirus human T-cell leukemia virus type 1 (HTLV-1) contains identical DNA sequences, known as long terminal repeats (LTRs), at its 5′ and 3′ ends. The LTRs modulate transcription in both forward (sense) and reverse (antisense) directions. We found that sense transcription from the 5′ LTR does not interfere with antisense transcription from the 3′ LTR, allowing viral genes encoded on opposite DNA strands to be simultaneously transcribed. Two such genes aretaxandhbz, and while they are thought to function at different times during the course of infection to promote leukemogenesis of infected T cells, our results indicate that they can be simultaneously transcribed. We also found that the ability of Tax to induce cell cycle arrest inhibits its fundamental function of activating viral sense transcription but does not affect antisense transcription. This regulatory mechanism may be important for long-term HTLV-1 infection.
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Schuetze, S., P. E. Stenberg et D. Kabat. « The Ets-related transcription factor PU.1 immortalizes erythroblasts ». Molecular and Cellular Biology 13, no 9 (septembre 1993) : 5670–78. http://dx.doi.org/10.1128/mcb.13.9.5670-5678.1993.
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In vivo studies of Friend virus erythroleukemia have implied that proviral integrations adjacent to the gene for the Ets-related transcription factor PU.1 may inhibit the commitment of erythroblasts to differentiate and cause their capability for indefinite transplantation (C. Spiro, B. Gliniak, and D. Kabat, J. Virol. 62:4129-4135, 1988; R. Paul, S. Schuetze, S. L. Kozak, C. Kozak, and D. Kabat, J. Virol. 65:464-467, 1991). To test this hypothesis, we ligated PU.1 cDNA into a retroviral vector and studied its effects on cultured cells. Infection of fibroblasts with PU.1-encoding retrovirus resulted in PU.1 synthesis followed by nuclear pyknosis, cell rounding, and degeneration. In contrast, in long-term bone marrow cultures, erythroblasts were efficiently and rapidly immortalized. The resulting cell lines were polyclonal populations that contained PU.1, were morphologically blast-like, required erythropoietin and bone marrow stromal cells for survival and proliferation, and spontaneously differentiated at low frequency to synthesize hemoglobin. After 9 months in culture, erythroblasts became stroma independent, and they then grew as clonal cell lines. We conclude that PU.1 perturbs the pathway(s) that controls potential for indefinite proliferation and that it can be used to generate permanent erythroblast cell lines.
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Schuetze, S., P. E. Stenberg et D. Kabat. « The Ets-related transcription factor PU.1 immortalizes erythroblasts. » Molecular and Cellular Biology 13, no 9 (septembre 1993) : 5670–78. http://dx.doi.org/10.1128/mcb.13.9.5670.
Texte intégralRésumé :
In vivo studies of Friend virus erythroleukemia have implied that proviral integrations adjacent to the gene for the Ets-related transcription factor PU.1 may inhibit the commitment of erythroblasts to differentiate and cause their capability for indefinite transplantation (C. Spiro, B. Gliniak, and D. Kabat, J. Virol. 62:4129-4135, 1988; R. Paul, S. Schuetze, S. L. Kozak, C. Kozak, and D. Kabat, J. Virol. 65:464-467, 1991). To test this hypothesis, we ligated PU.1 cDNA into a retroviral vector and studied its effects on cultured cells. Infection of fibroblasts with PU.1-encoding retrovirus resulted in PU.1 synthesis followed by nuclear pyknosis, cell rounding, and degeneration. In contrast, in long-term bone marrow cultures, erythroblasts were efficiently and rapidly immortalized. The resulting cell lines were polyclonal populations that contained PU.1, were morphologically blast-like, required erythropoietin and bone marrow stromal cells for survival and proliferation, and spontaneously differentiated at low frequency to synthesize hemoglobin. After 9 months in culture, erythroblasts became stroma independent, and they then grew as clonal cell lines. We conclude that PU.1 perturbs the pathway(s) that controls potential for indefinite proliferation and that it can be used to generate permanent erythroblast cell lines.
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Haupt, Sonja, Michele Tisdale, Michelle Vincendeau, Mary Anne Clements, David T. Gauthier, Raymond Lance, O. John Semmes et al. « Human endogenous retrovirus transcription profiles of the kidney and kidney-derived cell lines ». Journal of General Virology 92, no 10 (1 octobre 2011) : 2356–66. http://dx.doi.org/10.1099/vir.0.031518-0.
Texte intégralRésumé :
The human genome comprises approximately 8–9 % of human endogenous retroviruses (HERVs) that are transcribed with tissue specificity. However, relatively few organs have been examined in detail for individual differences in HERV transcription pattern, nor have tissue-to-cell culture comparisons been frequently performed. Using an HERV-specific DNA microarray, a core HERV transcription profile was established for the human kidney comparing 10 tissue samples. This core represents HERV groups expressed uniformly or nearly so in non-tumour kidney tissue. The profiles obtained from non-tumour tissues were compared to 10 renal tumour tissues (renal cell carcinoma, RCC) derived from the same individuals and additionally, to 22 RCC cell lines. No RCC cell line or tumour-specific differences were observed, suggesting that HERV transcription is not altered in RCC. However, when comparing tissue transcription to cell line transcription, there were consistent differences. The differences were irrespective of cancer state and included cell lines derived from non-tumour kidney tissue, suggesting that a specific alteration of HERV transcription occurs when establishing cell lines. In contrast to previous publications, all known HERV-derived tumour antigens, including those identified in RCC, were expressed both in multiple RCC cell lines and several non-tumour tissue-derived cell lines, a result that contrasts with findings from patient samples. The results establish the core kidney transcription pattern of HERVs and reveal differences between cell culture lines and tissue samples.
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Chan, H., S. Hartung et M. Breindl. « Retrovirus-induced interference with collagen I gene expression in Mov13 fibroblasts is maintained in the absence of DNA methylation ». Molecular and Cellular Biology 11, no 1 (janvier 1991) : 47–54. http://dx.doi.org/10.1128/mcb.11.1.47-54.1991.
Texte intégralRésumé :
We have studied the role of DNA methylation in repression of the murine alpha 1 type I collagen (COL1A1) gene in Mov13 fibroblasts. In Mov13 mice, a retroviral provirus has inserted into the first intron of the COL1A1 gene and blocks its expression at the level of transcriptional initiation. We found that regulatory sequences in the COL1A1 promoter region that are involved in the tissue-specific regulation of the gene are unmethylated in collagen-expressing wild-type fibroblasts and methylated in Mov13 fibroblasts, confirming and extending earlier observations. To directly assess the role of DNA methylation in the repression of COL1A1 gene transcription, we treated Mov13 fibroblasts with the demethylating agent 5-azacytidine. This treatment resulted in a demethylation of the COL1A1 regulatory sequences but failed to activate transcription of the COL1A1 gene. Moreover, the 5-azacytidine treatment induced a transcription-competent chromatin structure in the retroviral sequences but not in the COL1A1 promoter. In DNA transfection and microinjection experiments, we found that the provirus interfered with transcriptional activity of the COL1A1 promoter in Mov13 fibroblasts but not in Xenopus laevis oocytes. In contrast, the wild-type COL1A1 promoter was transcriptionally active in Mov13 fibroblasts. These experiments showed that the COL1A1 promoter is potentially transcriptionally active in the presence of proviral sequences and that Mov13 fibroblasts contain the trans-acting factors required for efficient COL1A1 gene expression. Our results indicate that the provirus insertion in Mov13 can inactivate COL1A1 gene expression at several levels. It prevents the developmentally regulated establishment of a transcription-competent methylation pattern and chromatin structure of the COL1A1 domain and, in the absence of DNA methylation, appears to suppress the COL1A1 promoter in a cell-specific manner, presumably by assuming a dominant chromatin structure that may be incompatible with transcriptional activity of flanking cellular sequences.