Journal articles on the topic 'Episomal'
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1
Habel, Marie-Eve, Mathieu Drouin, and Daniel Jung. "Maintenance of Epstein–Barr virus-derived episomal vectors in the murine Sp2/0 myeloma cell line is dependent upon exogenous expression of human EBP2." Biochemistry and Cell Biology 82, no. 3 (June 1, 2004): 375–80. http://dx.doi.org/10.1139/o04-037.
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Vectors carrying the origin of replication (oriP) and driving expression of the EBNA-1 protein from Epstein–Barr virus (EBV) replicate as extrachromosomal episomes in human cells. Whether these vectors can be maintained as episomes in murine cells is still controversial. Here we demonstrate that EBNA-1 expression alone was unable to maintain episomal expression of an EBV-based vector in the murine Sp2/0 cell line. However, we were able to obtain long-term episome maintenance in Sp2/0 cells after exogenously expressing human EBP2 by genetic engineering. Our results provide further evidence for the fundamental role of human EBP2 in episomal maintenance of EBV-based vectors. Moreover, we demonstrate that EBV-based vectors can be successfully used in cells presumably incompetent for episomal maintenance.Key words: EBV vector, EBNA-1, EBP2, episomal maintenance, mouse cell, Sp2/0 myeloma cell line.
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Griffiths, Rhoswyn, Sally M. Harrison, Stuart Macnab, and Adrian Whitehouse. "Mapping the minimal regions within the ORF73 protein required for herpesvirus saimiri episomal persistence." Journal of General Virology 89, no. 11 (November 1, 2008): 2843–50. http://dx.doi.org/10.1099/vir.0.2008/002725-0.
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Herpesvirus saimiri (HVS) establishes a persistent infection in which the viral genome persists as a circular non-integrated episome. ORF73 tethers HVS episomes to host mitotic chromosomes, allowing episomal persistence via an interaction with the chromosome-associated protein, MeCP2. Here we demonstrate that ORF73 also interacts with the linker histone H1 via its C terminus, suggesting it associates with multiple chromosome-associated proteins. In addition, we show that the C terminus is also required for the ability of ORF73 to bind the terminal repeat region of the HVS genome. These results suggest that the ORF73 C terminus contains all the necessary elements required for HVS episomal persistence. Using a range of ORF73 C terminus deletions to rescue the episomal maintenance properties of a HVSΔ73 recombinant virus, we show that a C terminus region comprising residues 285–407 is sufficient to maintain the HVS episome in a dividing cell population.
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Angeletti, Peter C., Kitai Kim, Fiona J. Fernandes, and Paul F. Lambert. "Stable Replication of Papillomavirus Genomes in Saccharomyces cerevisiae." Journal of Virology 76, no. 7 (April 1, 2002): 3350–58. http://dx.doi.org/10.1128/jvi.76.7.3350-3358.2002.
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ABSTRACT Papillomaviruses normally replicate in stratified squamous epithelial tissues of their mammalian hosts, in which the viral genome is found as a nuclear plasmid. Two viral proteins, E1, a helicase, and E2, a transcriptional activator and plasmid maintenance factor, are known to contribute to the episomal replication of the viral genome. Recently, our laboratory discovered that papillomaviruses can also replicate in an E1-independent manner in mammalian cells (K. Kim and P. F. Lambert, Virology, in press; K. Kim and P. F. Lambert, submitted for publication). In this study, we describe experiments investigating the capacity of the human papillomavirus type 16 (HPV16) genome to replicate in yeast (Saccharomyces cerevisiae). The full-length HPV16 genome, when linked in cis to a selectable yeast marker gene, either TRP1 or URA3, could replicate stably as an episome in yeast. The replication of papillomavirus genomes in yeast is not limited to HPV16. Bovine papillomavirus type 1 and HPV6b, -11, -16, -18, and -31 were all capable of replicating in short-term assays over a period of 20 cell doublings. The long-term persistence of viral episomes did not require any one viral gene, as mutant genomes defective in single genes also replicated episomally. These results indicate that the viral episome can replicate in the absence of the E1 DNA helicase. Similarly, E2 was also not required for replication in yeast, and E2 mutant viral genomes were stably maintained in the absence of selection, indicating the existence of an E2-independent mechanism for plasmid maintenance. The episomal replication of papillomavirus genomes in yeast provides a genetically manipulatable system in which to investigate cellular factors required for episomal replication and may provide a novel means for generating infectious papillomavirus.
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Sharkey, Mark, Karine Triques, Daniel R. Kuritzkes, and Mario Stevenson. "In Vivo Evidence for Instability of Episomal Human Immunodeficiency Virus Type 1 cDNA." Journal of Virology 79, no. 8 (April 15, 2005): 5203–10. http://dx.doi.org/10.1128/jvi.79.8.5203-5210.2005.
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ABSTRACT Current regimens for the management of human immunodeficiency virus type 1 (HIV-1) infection suppress plasma viremia to below detectable levels for prolonged intervals. Nevertheless, there is a rapid resumption in plasma viremia if therapy is interrupted. Attempts to characterize the extent of viral replication under conditions of potent suppression and undetectable plasma viremia have been hampered by a lack of convenient assays that can distinguish latent from ongoing viral replication. Using episomal viral cDNA as a surrogate for ongoing replication, we previously presented evidence that viral replication persists in the majority of infected individuals with a sustained aviremic status. The labile nature of viral episomes and hence their validity as surrogate markers of ongoing replication in individuals with long-term-suppressed HIV-1 infection have been analyzed in short-term in vitro experiments with conflicting results. Since these in vitro experiments do not shed light on the long-term in vivo dynamics of episomal cDNA or recapitulate the natural targets of infection in vivo, we have analyzed the dynamics of episomal cDNA turnover in vivo by following the emergence of an M184V polymorphism in plasma viral RNA, in episomal cDNA, and in proviral DNA in patients on suboptimal therapies. We demonstrate that during acquisition of drug resistance, wild-type episomal cDNAs are replaced by M184V-harboring episomes. Importantly, a complete replacement of wild-type episomes with M184V-containing episomes occurred while proviruses remained wild type. This indicates that episomal cDNAs are turned over by degradation rather than through death or tissue redistribution of the infected cell itself. Therefore, evolution of episomal viral cDNAs is a valid surrogate of ongoing viral replication in HIV-1-infected individuals.
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Wang, Xiao-Yin, Xi Zhang, Tian-Yun Wang, Yan-Long Jia, Dan-Hua Xu, and Dan-Dan Yi. "Shortened nuclear matrix attachment regions are sufficient for replication and maintenance of episomes in mammalian cells." Molecular Biology of the Cell 30, no. 22 (October 15, 2019): 2761–70. http://dx.doi.org/10.1091/mbc.e19-02-0108.
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Matrix attachment regions (MARs) can mediate the replication of vector episomes in mammalian cells; however, the molecular mode of action remains unclear. Here, we assessed the characteristics of MARs and the mechanism that mediates episomal vector replication in mammalian cells. Five shortened subfragments of β-interferon MAR fragments were cloned and transferred into CHO cells, and transgene expression levels, presence of the gene, and the episomal maintenance mechanism were determined. Three shortened MAR derivatives (position 781–1320, 1201–1740, and 1621–2201) retained full MAR activity and mediated episomal vector replication. Moreover, the three shortened MARs showed higher transgene expression levels, greater efficiency in colony formation, and more persistent transgene expression compared with those of the original pEPI-1 plasmid, and three functional truncated MARs can bind to SAF-A MAR-binding protein. These results suggest that shortened MARs are sufficient for replication and maintenance of episomes in CHO cells.
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Madireddy, Advaitha, Pravinkumar Purushothaman, Christopher P. Loosbroock, Erle S. Robertson, Carl L. Schildkraut, and Subhash C. Verma. "G-quadruplex-interacting compounds alter latent DNA replication and episomal persistence of KSHV." Nucleic Acids Research 44, no. 8 (February 2, 2016): 3675–94. http://dx.doi.org/10.1093/nar/gkw038.
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Abstract Kaposi's sarcoma associated herpesvirus (KSHV) establishes life-long latent infection by persisting as an extra-chromosomal episome in the infected cells and by maintaining its genome in dividing cells. KSHV achieves this by tethering its epigenome to the host chromosome by latency associated nuclear antigen (LANA), which binds in the terminal repeat (TR) region of the viral genome. Sequence analysis of the TR, a GC-rich DNA element, identified several potential Quadruplex G-Rich Sequences (QGRS). Since quadruplexes have the tendency to obstruct DNA replication, we used G-quadruplex stabilizing compounds to examine their effect on latent DNA replication and the persistence of viral episomes. Our results showed that these G-quadruplex stabilizing compounds led to the activation of dormant origins of DNA replication, with preferential bi-directional pausing of replications forks moving out of the TR region, implicating the role of the G-rich TR in the perturbation of episomal DNA replication. Over time, treatment with PhenDC3 showed a loss of viral episomes in the infected cells. Overall, these data show that G-quadruplex stabilizing compounds retard the progression of replication forks leading to a reduction in DNA replication and episomal maintenance. These results suggest a potential role for G-quadruplex stabilizers in the treatment of KSHV-associated diseases.
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Sears, John, John Kolman, Geoffrey M. Wahl, and Ashok Aiyar. "Metaphase Chromosome Tethering Is Necessary for the DNA Synthesis and Maintenance of oriP Plasmids but Is Insufficient for Transcription Activation by Epstein-Barr Nuclear Antigen 1." Journal of Virology 77, no. 21 (November 1, 2003): 11767–80. http://dx.doi.org/10.1128/jvi.77.21.11767-11780.2003.
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ABSTRACT Epstein-Barr Virus (EBV) infects resting B cells, within which it establishes latency as a stable, circular episome with only two EBV components, the cis element oriP and the latently expressed protein EBNA1. It is believed that EBNA1's ability to tether oriP episomes to metaphase chromosomes is required for its stable replication. We created fusions between the DNA-binding domain (DBD) of EBNA1 and the cellular chromatin-binding proteins HMGA1a and HMG1 to determine the minimal requirements for stable maintenance of an oriP-based episome. These two proteins differ in that HMGA1a can associate with metaphase chromosomes but HMG1 cannot. Interestingly, coinciding with metaphase chromosome association, HMGA1a-DBD but not HMG1-DBD supported both the transient replication and stable maintenance of oriP plasmids, with efficiencies quantitatively similar to that of EBNA1. However, HMGA1a-DBD activated transcription from EBNA1-dependent episomal reporter to only 20% of the level of EBNA1. Furthermore, EBNA1 but not HMGA1a-DBD activated transcription from a chromosomally integrated EBNA1-dependent transcription reporter. This indicates that EBNA1 possesses functional domains that support transcription activation independent of its ability to tether episomal oriP plasmids to cellular chromosomes. We provide evidence that metaphase chromosome tethering is a fundamental requirement for maintenance of an oriP plasmid but is insufficient for EBNA1 to activate transcription.
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Calderwood, Michael, Robert E. White, Rhoswyn A. Griffiths, and Adrian Whitehouse. "Open reading frame 73 is required for herpesvirus saimiri A11-S4 episomal persistence." Journal of General Virology 86, no. 10 (October 1, 2005): 2703–8. http://dx.doi.org/10.1099/vir.0.81230-0.
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Herpesvirus saimiri (HVS) establishes a latent infection in which the viral genome persists as a non-integrated episome. Analysis has shown that only open reading frames (ORFs) 71–73 are transcribed in an in vitro model of HVS latency. ORF73 also colocalizes with HVS genomic DNA on host mitotic chromosomes and maintains the stability of HVS terminal-repeat-containing plasmids. However, it is not known whether ORF73 is the only HVS-encoded protein required for episomal maintenance. In this study, the elements required for episomal maintenance in the context of a full-length HVS genome were examined by mutational analysis. A recombinant virus, HVS-BACΔ71-73, lacking the latency-associated genes was unable to persist in a dividing cell population. However, retrofitting an ORF73 expression cassette into the recombinant virus rescued episomal maintenance. This indicates that ORF73 is the key trans-acting factor for episomal persistence and efficient establishment of a latent infection.
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Duensing, Stefan, Anette Duensing, Elsa R. Flores, Anh Do, Paul F. Lambert, and Karl Münger. "Centrosome Abnormalities and Genomic Instability by Episomal Expression of Human Papillomavirus Type 16 in Raft Cultures of Human Keratinocytes." Journal of Virology 75, no. 16 (August 15, 2001): 7712–16. http://dx.doi.org/10.1128/jvi.75.16.7712-7716.2001.
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ABSTRACT Primary human keratinocytes with ectopic expression of high-risk human papillomavirus (HPV) E6 and E7 oncoproteins display abnormal centrosome numbers, multipolar mitoses, and aneusomy. However, it has not been explored whether these abnormalities can occur in cells containing HPV episomes where E6 and E7 expression is under viral transcriptional control. Here, we demonstrate that centrosome abnormalities and genomic instability occur in organotypic raft cultures of human keratinocytes with episomal HPV-16 even at low copy numbers. We conclude that HPV-16 DNA, when maintained as an episome, can disturb centrosome homeostasis and subvert genomic integrity of the host cell during early stages of the viral infection.
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Oh, Stephen T., Michelle S. Longworth, and Laimonis A. Laimins. "Roles of the E6 and E7 Proteins in the Life Cycle of Low-Risk Human Papillomavirus Type 11." Journal of Virology 78, no. 5 (March 1, 2004): 2620–26. http://dx.doi.org/10.1128/jvi.78.5.2620-2626.2004.
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ABSTRACT Many important functions have been attributed to the high-risk human papillomavirus (HPV) E6 and E7 proteins, including binding and degradation of p53 as well as interacting with Rb proteins. In contrast, the physiological roles of the low-risk E6 and E7 proteins remain unclear. Previous studies demonstrated that the high-risk E6 and E7 proteins also play roles in the productive life cycle by facilitating the maintenance of viral episomes (J. T. Thomas, W. G. Hubert, M. N. Ruesch, and L. A. Laimins, Proc. Natl. Acad. Sci. USA 96:8449-8454, 1999). In order to determine whether low-risk E6 or E7 is similarly necessary for the stable maintenance of episomes, HPV type 11 (HPV-11) genomes that contained translation termination mutations in E6 or E7 were constructed. Upon transfection into normal human keratinocytes, genomes in which E6 function was abolished were unable to be maintained episomally. Transfection of genomes containing substitution mutations in amino acids conserved in high- and low-risk HPV types suggested that multiple protein domains are involved in this process. Examination of cells transfected with HPV-11 genomes in which E7 function was inhibited were found to exhibit a more complex phenotype. At the second passage following transfection, mutant genomes were maintained as episomes but at significantly reduced levels than in cells transfected with the wild-type HPV-11 genome. Upon further passage in culture, however, the episomal forms of these E7 mutant genomes quickly disappeared. These findings identify important new functions for the low-risk E6 and E7 proteins in the episomal maintenance of low-risk HPV-11 genomes and suggest that they may act in a manner similar to that observed for the high-risk proteins.
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Meneses, Patricio, Kenneth I. Berns, and Ernest Winocour. "DNA Sequence Motifs Which Direct Adeno-Associated Virus Site-Specific Integration in a Model System." Journal of Virology 74, no. 13 (July 1, 2000): 6213–16. http://dx.doi.org/10.1128/jvi.74.13.6213-6216.2000.
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ABSTRACT The DNA sequence motifs which direct adeno-associated virus type 2 site-specific integration are being investigated using a shuttle vector, propagated as a stable episome in cultured cell lines, as the target for integration. Previously, we reported that the minimum episomal targeting elements comprise a 16-bp binding motif (Rep binding site [RBS]) for a viral regulatory protein (Rep) separated by a short DNA spacer from a sequence (terminal resolution site [TRS]) that can serve as a substrate for Rep-mediated nicking activity (R. M. Linden, P. Ward, C. Giraud, E. Winocour, and K. I. Berns, Proc. Natl. Acad. Sci. USA 93:11288–11294, 1996; R. M. Linden, E. Winocour, and K. I. Berns, Proc. Natl. Acad. Sci. USA 93:7966–7972, 1996). We now report that episomal integration depends upon both the sequence and the position of the spacer DNA separating the RBS and TRS motifs. The spacer thus constitutes a third element required for site-specific episomal integration.
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Park, Regina B., and Elliot J. Androphy. "Genetic Analysis of High-Risk E6 in Episomal Maintenance of Human Papillomavirus Genomes in Primary Human Keratinocytes." Journal of Virology 76, no. 22 (November 15, 2002): 11359–64. http://dx.doi.org/10.1128/jvi.76.22.11359-11364.2002.
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ABSTRACT Papillomaviruses possess small DNA genomes that encode five early (E) proteins. Transient DNA replication requires activities of the E1 and E2 proteins and a DNA segment containing their binding sites. The E6 and E7 proteins of cancer-associated human papillomavirus (HPV) transform cells in culture. Recent reports have shown that E6 and E7 are necessary for episomal maintenance of HPV in primary keratinocytes. The functions of E6 necessary for viral replication have not been determined, and to address this question we used a recently developed transfection system based on HPV31. To utilize a series of HPV16 E6 mutations, HPV31 E6 was replaced by its HPV16 counterpart. This chimeric genome was competent for both transient and stable replication in keratinocytes. Four HPV16 E6 mutations that do not stimulate p53 degradation were unable to support stable viral replication, suggesting this activity may be necessary for episomal maintenance. E7 has also been shown to be essential for episomal maintenance of the HPV31 genome. A point mutation in the Rb binding motif of HPV E7 has been reported to render HPV31 unable to stably replicate. Interestingly, HPV31 genomes harboring two of the three p53 degradation-defective E6 mutations combined with this E7 mutation were maintained as replicating episomes. These findings imply that the balance between E6 and E7 functions in infected cells is critical for episomal maintenance of high-risk HPV genomes. This model will be useful to dissect the activities of E6 and E7 necessary for viral DNA replication.
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Graux, Carlos, Marina Lafage, Nicole Dastugue, Francine Mugneret, Roland Berger, Stéphanie Struski, Marie-José Gregoire, et al. "Characterization of 14 NUP214-ABL1 Fusions in T-Cell Acute Lymphoblastic Leukaemia (T-ALL) Exhibits a Genomic Heterogeneity." Blood 108, no. 11 (November 1, 2006): 2075. http://dx.doi.org/10.1182/blood.v108.11.2075.2075.
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Abstract The recently described episomal amplification of the NUP214-ABL1 fusion in 6% of T-ALL leads to the expression of a constitutively activated chimeric tyrosine kinase sensitive to imatinib. We collected additional cases in order to better characterize this new entity with respect to genetic presentations and clinical course. We collected 14 new NUP214-ABL1 positive cases by FISH (LSI-BCR/ABL1 ES, Vysis and ABL1 break-apart home-made probes) or RT-PCR (fusion transcript) screening. FISH analysis detected episomal amplification of NUP214-ABL1 in 11 patients with a highly variable number of nuclei with amplification (<1% to 90%). Interestingly, one case showed a higher percentage of nuclei with amplification when using frozen non cultured cells rather than cultured fixed cells (10% vs 1%) suggesting loss of episomes during culture. FISH showed also intrachromosomal amplification of NUP214-ABL1 in two cases: one presented a HSR at the original 9q34 site without detectable episomes, the other associated HSR (probably on a chromosome 10), episomes (<1% of nuclei) and 9q34 chromosomal insertions including NUP214 and ABL3′ that encode the tyrosine kinase domain but not ABL5′, on variable chromosomes including 14p (33%). One NUP214-ABL1 RT-PCR positive case did not show any FISH aberration. Median age: 16 y (3–45) with a male predominance (10:4). There were no T-cell lymphoblastic lymphoma. Immunophenotype (EGIL): mature (n=2), cortical (n=6) or pre-T (n=4). Karyotype: structural chromosomal alterations in 8 patients (including 4 with 10q24/HOX11 rearrangements), only numerical chromosomal alterations in 4 (including 2 with + 8), normal in 1, failed in 1. All samples with available information (10/14) showed a HOX11 or HOX11L2 abnormality. Among the 13 cases with available outcome data, we observed 5 early relapses, including both patients with NUP214-ABL1 HSR, and 1 refractory ALL. These observations emphasize the interest of combining both (quantitative) RT-PCR and FISH for the screening and characterisation of NUP214-ABL1 fusion and amplification, demonstrate the coexistence of different NUP214-ABL1 genomic presentations in one patient (episomal amplification, 9q34 insertions, HSR) compatible with the model in which genomic amplification start with episome formation in order to create the NUP214-ABL1 fusion followed by their amplification and optional secondary reintegration, confirm occurrence of NUP214-ABL1 in T-ALL with HOX11 and HOX11L2 involvement, raise the question of the rather worse prognosis for cases with intrachromosomal amplification as previously suggested, raise the signification of minor NUP214-ABL1 clones and variable genomic presentations in the leukemogenesis of this subgroup of T-ALL that could potentially benefit from imatimib. ° on behalf of the GFCH (Groupe Francophone de Cytogénétique Hématologique) and the BCGHO (Belgian Cytogenetic Group for Hematology and Oncology).
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Meyer, J. B., G. G. F. Kasdorf, L. H. Nel, and G. Pietersen. "Transmission of Activated-Episomal Banana streak OL (badna)virus (BSOLV) to cv. Williams Banana (Musa sp.) by Three Mealybug Species." Plant Disease 92, no. 8 (August 2008): 1158–63. http://dx.doi.org/10.1094/pdis-92-8-1158.
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Four different mealybug species (Dysmicoccus brevipes, Planococcus citri, P. ficus, and Pseudococcus longispinus) were evaluated for their ability to transmit putative activated-episomal Banana streak OL (badna)virus (BSOLV) to banana cv. Williams (Cavendish subgroup, AAA). Expressible endogenous sequences of banana streak viruses (BSVs) have been reported to be present in the DNA of various Musa hybrids, including FHIA-21 (AAAB). To obtain activated episomal BSOLV for this experimental transmission study, intentional stress by tissue culture propagation was applied to indexed FHIA-21 which, while free of other viruses, can contain activated episomal BSOLV. Immunocapture polymerase chain reaction and triple-antibody sandwich enzyme-linked immunosorbent assay results revealed that 13.4% of the derived progeny of the mother plants were infected with episomal BSOLV. Four of these BSOLV-infected progeny were used as sources of episomal virus for transmission studies. D. brevipes, Planococcus citri, and P. ficus mealybugs were able to transmit the putative activated episomal BSOLV. Control plants for the transmission experiments included FHIA-21 corms with no background history of tissue culture, as well as virus-free Williams plants. Episomal Banana streak GF (badna)virus (BSGFV) was transmitted from asymptomatic corm-derived FHIA-21 plants by P. citri and P. ficus. This is the first report of P. ficus as a vector of BSVs.
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Dorigo, Oliver, Jose S. Gil, Sean D. Gallaher, Brenton T. Tan, Maria G. Castro, Pedro R. Lowenstein, Michele P. Calos, and Arnold J. Berk. "Development of a Novel Helper-Dependent Adenovirus-Epstein-Barr Virus Hybrid System for the Stable Transformation of Mammalian Cells." Journal of Virology 78, no. 12 (June 15, 2004): 6556–66. http://dx.doi.org/10.1128/jvi.78.12.6556-6566.2004.
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ABSTRACT Epstein-Barr virus (EBV) episomes are stably maintained in permissive proliferating cell lines due to EBV nuclear antigen 1 (EBNA-1) protein-mediated replication and segregation. Previous studies showed the ability of EBV episomes to confer long-term transgene expression and correct genetic defects in deficient cells. To achieve quantitative delivery of EBV episomes in vitro and in vivo, we developed a binary helper-dependent adenovirus (HDA)-EBV hybrid system that consists of one HDA vector for the expression of Cre recombinase and a second HDA vector that contains all of the sequences for the EBV episome flanked by loxP sites. Upon coinfection of cells, Cre expressed from the first vector recombined loxP sites on the second vector. The resulting circular EBV episomes expressed a transgene and contained the EBV-derived family of repeats, an EBNA-1 expression cassette, and 19 kb of human DNA that functions as a replication origin in mammalian cells. This HDA-EBV hybrid system transformed 40% of cultured cells. Transgene expression in proliferating cells was observed for over 20 weeks under conditions that selected for the expression of the transgene. In the absence of selection, EBV episomes were lost at a rate of 8 to 10% per cell division. Successful delivery of EBV episomes in vivo was demonstrated in the liver of transgenic mice expressing Cre from the albumin promoter. This novel gene transfer system has the potential to confer long-term episomal transgene expression and therefore to correct genetic defects with reduced vector-related toxicity and without insertional mutagenesis.
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Sharkey, Mark. "Tracking episomal HIV DNA." Current Opinion in HIV and AIDS 8, no. 2 (March 2013): 93–99. http://dx.doi.org/10.1097/coh.0b013e32835d08c2.
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Griffiths, Rhoswyn, and Adrian Whitehouse. "Herpesvirus Saimiri Episomal Persistence Is Maintained via Interaction between Open Reading Frame 73 and the Cellular Chromosome-Associated Protein MeCP2." Journal of Virology 81, no. 8 (January 31, 2007): 4021–32. http://dx.doi.org/10.1128/jvi.02171-06.
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ABSTRACT Herpesvirus saimiri (HVS) is the prototype gamma-2 herpesvirus, which naturally infects the squirrel monkey Saimiri sciureus, causing an asymptomatic but persistent infection. The latent phase of gamma-2 herpesviruses is characterized by their ability to persist in a dividing cell population while expressing a limited subset of latency-associated genes. In HVS only three genes, open reading frame 71 (ORF71), ORF72, and ORF73, are expressed from a polycistronic mRNA. ORF73 has been shown to be the only gene essential for HVS episomal maintenance and can therefore be functionally compared to the human gammaherpesvirus latency-associated proteins, EBNA-1 and Kaposi's sarcoma-associated herpesvirus (KSHV) latency-associated nuclear antigen (LANA). HVS ORF73 is the positional homologue of KSHV LANA and, although it shares limited sequence homology, has significant structural and functional similarities. Investigation of KSHV LANA has demonstrated that it is able to mediate KSHV episomal persistence by tethering the KSHV episome to host mitotic chromosomes via interactions with cellular chromosome-associated proteins. These include associations with core and linker histones, several bromodomain proteins, and the chromosome-associated proteins methyl CpG binding protein 2 (MeCP2) and DEK. Here we show that HVS ORF73 associates with MeCP2 via a 72-amino-acid domain within the ORF73 C terminus. Furthermore, we have assessed the functional significance of this interaction, using a variety of techniques including small hairpin RNA knockdown, and show that association between ORF73 and MeCP2 is essential for HVS chromosomal attachment and episomal persistence.
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Deyle, David R., Yi Li, Erik M. Olson, and David W. Russell. "Nonintegrating Foamy Virus Vectors." Journal of Virology 84, no. 18 (June 30, 2010): 9341–49. http://dx.doi.org/10.1128/jvi.00394-10.
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ABSTRACT Foamy viruses (FVs), or spumaviruses, are integrating retroviruses that have been developed as vectors. Here we generated nonintegrating foamy virus (NIFV) vectors by introducing point mutations into the highly conserved DD35E catalytic core motif of the foamy virus integrase sequence. NIFV vectors produced high-titer stocks, transduced dividing cells, and did not integrate. Cells infected with NIFV vectors contained episomal vector genomes that consisted of linear, 1-long-terminal-repeat (1-LTR), and 2-LTR circular DNAs. These episomes expressed transgenes, were stable, and became progressively diluted in the dividing cell population. 1-LTR circles but not 2-LTR circles were found in all vector stocks prior to infection. Residual integration of NIFV vectors occurred at a frequency 4 logs lower than that of integrase-proficient FV vectors. Cre recombinase expressed from a NIFV vector mediated excision of both an integrated, floxed FV vector and a gene-targeted neo expression cassette, demonstrating the utility of these episomal vectors. The broad host range and large packaging capacity of NIFV vectors should make them useful for a variety of applications requiring transient gene expression.
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Simpson, K., A. McGuigan, and C. Huxley. "Stable episomal maintenance of yeast artificial chromosomes in human cells." Molecular and Cellular Biology 16, no. 9 (September 1996): 5117–26. http://dx.doi.org/10.1128/mcb.16.9.5117.
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Plasmids carrying the Epstein-Barr virus origin of plasmid replication (oriP) have been shown to replicate autonomously in latently infected human cells (J. Yates, N. Warren, D. Reisman, and B. Sugden, Proc. Natl. Acad. Sci. USA 81:3806-3810, 1984). We demonstrate that addition of this domain is sufficient for stable episomal maintenance of yeast artificial chromosomes (YACs), up to at least 660 kb, in human cells expressing the viral protein EBNA-1. To better approximate the latent viral genome, YACs were circularized before addition of the oriP domain by homologous recombination in yeast cells. The resulting OriPYACs were maintained as extrachromosomal molecules over long periods in selection; a 90-kb OriPYAC was unrearranged in all cell lines analyzed, whereas the intact form of a 660-kb molecule was present in two of three cell lines. The molecules were also relatively stable in the absence of selection. This finding indicates that the oriP-EBNA-1 interaction is sufficient to stabilize episomal molecules of at least 660 kb and that such elements do not undergo rearrangements over time. Fluorescence in situ hybridization analysis demonstrated a close association of OriPYACs, some of which were visible as pairs, with host cell chromosomes, suggesting that the episomes replicate once per cell cycle and that stability is achieved by attachment to host chromosomes, as suggested for the viral genome. The wide availability of YAC libraries, the ease of manipulation of cloned sequences in yeast cells, and the episomal stability make OriPYACs ideal for studying gene function and control of gene expression.
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Goffinet, Christine, Ina Allespach, Lena Oberbremer, Pamela L. Golden, Scott A. Foster, Brian A. Johns, Jason G. Weatherhead, et al. "Pharmacovirological Impact of an Integrase Inhibitor on Human Immunodeficiency Virus Type 1 cDNA Species In Vivo." Journal of Virology 83, no. 15 (May 20, 2009): 7706–17. http://dx.doi.org/10.1128/jvi.00683-09.
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ABSTRACT Clinical trials of the first approved integrase inhibitor (INI), raltegravir, have demonstrated a drop in the human immunodeficiency virus type 1 (HIV-1) RNA loads of infected patients that was unexpectedly more rapid than that with a potent reverse transcriptase inhibitor, and apparently dose independent. These clinical outcomes are not understood. In tissue culture, although their inhibition of integration is well documented, the effects of INIs on levels of unintegrated HIV-1 cDNAs have been variable. Furthermore, there has been no report to date on an INI's effect on these episomal species in vivo. Here, we show that prophylactic treatment of transgenic rats with the strand transfer INI GSK501015 reduced levels of viral integrants in the spleen by up to 99.7%. Episomal two-long-terminal-repeat (LTR) circles accumulated up to sevenfold in this secondary lymphoid organ, and this inversely correlated with the impact on the proviral burden. Contrasting raltegravir's dose-ranging study with HIV patients, titration of GSK501015 in HIV-infected animals demonstrated dependence of the INI's antiviral effect on its serum concentration. Furthermore, the in vivo 50% effective concentration calculated from these data best matched GSK501015's in vitro potency when serum protein binding was accounted for. Collectively, this study demonstrates a titratable, antipodal impact of an INI on integrated and episomal HIV-1 cDNAs in vivo. Based on these findings and known biological characteristics of viral episomes, we discuss how integrase inhibition may result in additional indirect antiviral effects that contribute to more rapid HIV-1 decay in HIV/AIDS patients.
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Collins, Christopher M., Maria M. Medveczky, Troy Lund, and Peter G. Medveczky. "The terminal repeats and latency-associated nuclear antigen of herpesvirus saimiri are essential for episomal persistence of the viral genome." Journal of General Virology 83, no. 9 (September 1, 2002): 2269–78. http://dx.doi.org/10.1099/0022-1317-83-9-2269.
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The simian herpesvirus saimiri (HVS) induces malignant T cell lymphomas and is closely related to Kaposi’s sarcoma-associated herpesvirus (KSHV or HHV-8). Both belong to the gamma-2 herpesvirus subgroup. The viral genome of HVS consists of a unique region (L-DNA) that contains all of the viral genes flanked by non-coding terminal repeats (H-DNA). Here we describe the cloning of a 113 kb restriction fragment containing the L-DNA of an oncogenic HVS strain in an F′ replicon-based E. coli vector. Cloned DNA was infectious and the ends of the progeny viral genome consisted of amplified tandem alternating repeats of vector and a single H-DNA unit. T cells infected with these viruses contained the linear DNA typically found a few weeks after infection, but were unable to form episomal circular viral DNA, which is the latent form of the viral genome. Recombinant viruses with reconstructed H-DNA were generated and T cells infected with these rescued viruses contained high copy numbers of episomal DNA. Plasmids expressing the latency-associated nuclear antigen (LANA) and containing various numbers of H-DNA repeats stably replicated as episomes, but constructs containing three repeat units produced the highest copy numbers. These data show that intact and multiple terminal repeats are essential components for episomal replication in latently infected T cells. Moreover, LANA and terminal repeats are sufficient for stable plasmid persistence. Cloned HVS can also be utilized for mutagenesis of HVS and for the expression of foreign genes through efficient manipulation of plasmids in E. coli.
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Hsieh, Chih-Lin. "In Vivo Activity of Murine De Novo Methyltransferases, Dnmt3a and Dnmt3b." Molecular and Cellular Biology 19, no. 12 (December 1, 1999): 8211–18. http://dx.doi.org/10.1128/mcb.19.12.8211.
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ABSTRACT The putative de novo methyltransferases, Dnmt3a and Dnmt3b, were reported to have weak methyltransferase activity in methylating the 3′ long terminal repeat of Moloney murine leukemia virus in vitro. The activity of these enzymes was evaluated in vivo, using a stable episomal system that employs plasmids as targets for DNA methylation in human cells. De novo methylation of a subset of the CpG sites on the stable episomes is detected in human cells overexpressing the murine Dnmt3a or Dnmt3b1 protein. This de novo methylation activity is abolished when the cysteine in the P-C motif, which is the catalytic site of cytosine methyltransferases, is replaced by a serine. The pattern of methylation on the episome is nonrandom, and different regions of the episome are methylated to different extents. Furthermore, Dnmt3a also methylates the sequence methylated by Dnmt3a on the stable episome in the corresponding chromosomal target. Overexpression of human DNMT1 or murine Dnmt3b does not lead to the same pattern or degree of de novo methylation on the episome as overexpression of murine Dnmt3a. This finding suggests that these three enzymes may have different targets or requirements, despite the fact that weak de novo methyltransferase activity has been demonstrated in vitro for all three enzymes. It is also noteworthy that both Dnmt3a and Dnmt3b proteins coat the metaphase chromosomes while displaying a more uniform pattern in the nucleus. This is the first evidence that Dnmt3a and Dnmt3b have de novo methyltransferase function in vivo and the first indication that the Dnmt3a and Dnmt3b proteins may have preferred target sites.
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Verghese, Santhosh Chakkaramakkil, Amy M. Skinner, Karen L. Huan, Hans J. Lipps, and Peter Kurre. "Episomal Anchorage Maintains Non-Integrating Lentiviral Vectors in Dividing Cells." Blood 120, no. 21 (November 16, 2012): 4224. http://dx.doi.org/10.1182/blood.v120.21.4224.4224.
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Abstract Abstract 4224 Insertional mutagenesis has been a major setback in retroviral stem cell gene therapy. Vector integration events can induce alternative splicing, aberrant transcripts and read-through transcription of endogenous genes. Non-integrating vectors provide a potentially safer approach in some tissue targets. However, due to the lack of nuclear retention, the vector genome is rapidly lost during cell division and differentiation. Genomic Scaffold/Matrix Associated Regions (S/MARs) efficiently anchor the DNA to nuclear matrix proteins to generate chromosome domains in the nucleus and have been successfully used for non-viral approaches to genetic modification. Here we incorporated human ß-interferon derived S/MAR sequence between GFP reporter reading frame and 3'LTR of a non-integrating 3rd generation lentiviral transfer vector, in an effort to ‘anchor’ the vector (termed ‘aLV’) to the host cell genome in dividing cells. Our approach exploits two key properties of lentiviral life cycle, i.e. nuclear translocation of the viral genomic DNA and circularization to generate episomal genomes comprised of 1-LTR or 2-LTR circles. Integrating vector (iLV) and aLV (integrase-defective gag packaging plasmid) were VSV-G pseudotyped and concentrated, yielding stocks with titers of 2.3 x108 and 3×106 TU/ml, respectively. Transduction of 293T cells with aLV showed dose-dependent transduction and GFP expression similar to iLV. Transduced 293T cells were clonally expanded and maintained in vitro up to 10 weeks. A subset of 22 aLV clones and 9 iLV clones were followed over time and analyzed for transgene expression and molecular persistence of LTR circles. After >70 rounds of cell division, GFP fluorescence intensity was consistently lower, but persisted in aLV-293T clones at MFI 406.5±34.9 versus iLV at MFI 786.3±58.5. As predicted, 1-LTR and 2-LTR vector episomes were detected by PCR in genomic DNA from aLV transduced cells at the 10-week time point. Additional sequencing confirmed the presence of episomal junctions indicating the persistence of completely circularized episomes. With evidence of molecular persistence and GFP expression of S/MAR-anchored, episomal vector genomes in rapidly dividing cell lines, we next tested the performance of aLV in lineage-depleted murine hematopoietic stem and progenitor cells (mHSPCs). Overnight transduction of lin- progenitors at matched MOI yielded 96.3%±22 GFP-positive clonogenic methylcellulose colonies after iLV exposure versus 39.4%±21 after aLV exposure. Transduced mHSPCs were also transplanted into sublethally irradiated C57B/6 mice and peripheral blood leukocytes were analyzed for gene expression. Here GFP expression in the donor compartment averaged 62% for iLV compared with 10% for aLV. We are evaluating leukocyte donor subset GFP expression and will undertake secondary transplantation to formally demonstrate transduction in the stem cell compartment. In summary, lentivector mediated nuclear localization followed by beta-interferon S/MAR anchorage of the vector genome leads to episomal transgene expression in dividing target cells. Our studies suggest that S/MAR elements can serve as molecular anchors for non-integrating lentiviral episomes to provide sustained gene expression through successive rounds of cell division and progenitor differentiation in vitro and in vivo. We propose further study of aLV as a candidate vector for gene delivery to HSPCs while avoiding proviral integration and its potentially deleterious consequences. Disclosures: No relevant conflicts of interest to declare.
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Penaud-Budloo, Magalie, Caroline Le Guiner, Ali Nowrouzi, Alice Toromanoff, Yan Chérel, Pierre Chenuaud, Manfred Schmidt, et al. "Adeno-Associated Virus Vector Genomes Persist as Episomal Chromatin in Primate Muscle." Journal of Virology 82, no. 16 (June 4, 2008): 7875–85. http://dx.doi.org/10.1128/jvi.00649-08.
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ABSTRACT Recombinant adeno-associated virus (rAAV) vectors are capable of mediating long-term gene expression following administration to skeletal muscle. In rodent muscle, the vector genomes persist in the nucleus in concatemeric episomal forms. Here, we demonstrate with nonhuman primates that rAAV vectors integrate inefficiently into the chromosomes of myocytes and reside predominantly as episomal monomeric and concatemeric circles. The episomal rAAV genomes assimilate into chromatin with a typical nucleosomal pattern. The persistence of the vector genomes and gene expression for years in quiescent tissues suggests that a bona fide chromatin structure is important for episomal maintenance and transgene expression. These findings were obtained from primate muscles transduced with rAAV1 and rAAV8 vectors for up to 22 months after intramuscular delivery of 5 × 1012 viral genomes/kg. Because of this unique context, our data, which provide important insight into in situ vector biology, are highly relevant from a clinical standpoint.
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Jenkins, Peter J., Ulrich K. Binné, and Paul J. Farrell. "Histone Acetylation and Reactivation of Epstein-Barr Virus from Latency." Journal of Virology 74, no. 2 (January 15, 2000): 710–20. http://dx.doi.org/10.1128/jvi.74.2.710-720.2000.
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ABSTRACT Induction of the viral BZLF1 gene has previously been shown to be one of the first steps in the reactivation of Epstein-Barr virus (EBV). Using an EBV oriP episomal vector system, we have reconstituted the regulation of the promoter for BZLF1 on stably transfected episomes, mapped promoter elements required for that regulation, and investigated mechanisms that may control the switch between latency and the lytic cycle. Changes in histone acetylation at the promoter for the BZLF1 gene appear to be a key part of the reactivation mechanism of this herpesvirus.
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Ehrhardt, Anja, Rudolf Haase, Aloys Schepers, Manuel Deutsch, Hans Lipps, and Armin Baiker. "Episomal Vectors for Gene Therapy." Current Gene Therapy 8, no. 3 (June 1, 2008): 147–61. http://dx.doi.org/10.2174/156652308784746440.
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O'Brochta, David A., Christina D. Stosic, Kristina Pilitt, Ramanand A. Subramanian, Robert H. Hice, and Peter W. Atkinson. "Transpositionally active episomal hAT elements." BMC Molecular Biology 10, no. 1 (2009): 108. http://dx.doi.org/10.1186/1471-2199-10-108.
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Spanos, William C., Mark El-Deiry, and John H. Lee. "Cidofovir Incorporation into Human Keratinocytes with Episomal HPV 16 Results in Nonselective Cytotoxicity." Annals of Otology, Rhinology & Laryngology 114, no. 11 (November 2005): 840–46. http://dx.doi.org/10.1177/000348940511401106.
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Objectives: Recurrent respiratory papillomatosis (RRP) is caused by human papillomavirus (HPV). Surgical excision is the mainstay of treatment; however, medical therapy including cidofovir, a cytosine analog, has been investigated. Human papillomavirus does not encode a viral DNA polymerase, which is the known target of cidofovir in cytomegalovirus infections. Methods: In an effort to better understand the usefulness of cidofovir in the treatment of HPV-related disease, we tested cidofovir's ability to inhibit growth, alter gene expression, and inhibit genome replication. Results: With the use of carbon 14-labeled cidofovir in episomal HPV 16-containing keratinocytes, there was a minimal increase in cidofovir incorporation into episomal DNA versus genomic DNA. Cidofovir decreased the copies of episomal HPV 16 in keratinocytes; however, the copies per cell returned to baseline levels once cidofovir was removed. Expression of a viral oncogene (HPV 16 E6) in transformed keratinocytes with episomal HPV 16 was not decreased by cidofovir. Cytotoxicity in head and neck squamous cell carcinoma lines exposed to cidofovir correlated with cell doubling time, and not with HPV status. Also, tonsil keratinocytes transformed with episomal HPV 16 did not exhibit greater cidofovir-mediated toxicity than did telomerase-transformed keratinocytes. Conclusions: These findings suggest that any potential in vivo benefit of cidofovir therapy results from non–viral-specific cell toxicity at the site of application.
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Janjetovic, Snjezana, Juliane Hinke, Saranya Balachandran, Nuray Akyüz, Petra Behrmann, Carsten Bokemeyer, Judith Dierlamm, and Eva Maria Murga Penas. "Non-Random Pattern of Integration for Epstein-Barr Virus with Preference for Gene-Poor Genomic Chromosomal Regions into the Genome of Burkitt Lymphoma Cell Lines." Viruses 14, no. 1 (January 4, 2022): 86. http://dx.doi.org/10.3390/v14010086.
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Background: Epstein-Barr virus (EBV) is an oncogenic virus found in about 95% of endemic Burkitt lymphoma (BL) cases. In latently infected cells, EBV DNA is mostly maintained in episomal form, but it can also be integrated into the host genome, or both forms can coexist in the infected cells. Methods: In this study, we mapped the chromosomal integration sites of EBV (EBV-IS) into the genome of 21 EBV+ BL cell lines (BL-CL) using metaphase fluorescence in situ hybridization (FISH). The data were used to investigate the EBV-IS distribution pattern in BL-CL, its relation to the genome instability, and to assess its association to common fragile sites and episomes. Results: We detected a total of 459 EBV-IS integrated into multiple genome localizations with a preference for gene-poor chromosomes. We did not observe any preferential affinity of EBV to integrate into common and rare fragile sites or enrichment of EBV-IS at the chromosomal breakpoints of the BL-CL analyzed here, as other DNA viruses do. Conclusions: We identified a non-random integration pattern into 13 cytobands, of which eight overlap with the EBV-IS in EBV-transformed lymphoblastoid cell lines and with a preference for gene- and CpGs-poor G-positive cytobands. Moreover, it has been demonstrated that the episomal form of EBV interacts in a non-random manner with gene-poor and AT-rich regions in EBV+ cell lines, which may explain the observed affinity for G-positive cytobands in the EBV integration process. Our results provide new insights into the patterns of EBV integration in BL-CL at the chromosomal level, revealing an unexpected connection between the episomal and integrated forms of EBV.
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Ajay Kumar, R., Samisubbu R. Naidu, Xiaoyu Wang, Anthony N. Imbalzano, and Elliot J. Androphy. "Interaction of Papillomavirus E2 Protein with the Brm Chromatin Remodeling Complex Leads to Enhanced Transcriptional Activation." Journal of Virology 81, no. 5 (December 6, 2006): 2213–20. http://dx.doi.org/10.1128/jvi.01746-06.
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ABSTRACT Papillomavirus E2 is a sequence-specific DNA binding protein that regulates transcription and replication of the viral genome. The transcriptional activities of E2 are typically evaluated by transient transfection of nonreplicating E2-dependent reporters. We sought to address whether E2 activates transcription in an episomal context and its potential interaction with the chromatin remodeling proteins. Using an Epstein-Barr virus-based episomal reporter, we demonstrate that E2 stimulates transcription from an E2-dependent promoter in a chromatin context. This activation is enhanced by the presence of proteins associated with SWI/SNF complexes, which are ATP-dependent chromatin remodeling enzymes. We show that exogenous expression of the Brm ATPase enhances E2 activity in SWI/SNF-deficient cell lines and that the amino-terminal transactivation domain of E2 mediates association with the Brm complex in vivo. Using chromatin immunoprecipitation assays, we demonstrate that Brm enhances promoter occupancy by E2 in an episomal context. Our results demonstrate that E2 activates transcription from an episomal reporter system and reveal a novel property of E2 in collaborating with the Brm chromatin remodeling complex in enhancing transcriptional activation.
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James, A. P., R. J. Geijskes, J. L. Dale, and R. M. Harding. "Development of a Novel Rolling-Circle Amplification Technique to Detect Banana streak virus that also Discriminates Between Integrated and Episomal Virus Sequences." Plant Disease 95, no. 1 (January 2011): 57–62. http://dx.doi.org/10.1094/pdis-07-10-0519.
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Banana plants are hosts to a large number of Banana streak virus (BSV) species. However, diagnostic methods for BSV are inadequate because of the considerable genetic and serological diversity among BSV isolates and the presence of integrated BSV sequences in some banana cultivars which leads to false positives. In this study, a sequence-nonspecific, rolling-circle amplification (RCA) technique was developed and shown to overcome these limitations for the detection and subsequent characterization of BSV isolates infecting banana. This technique was shown to discriminate between integrated and episomal BSV DNA, specifically detecting the latter in several banana cultivars known to contain episomal or integrated sequences of Banana streak Mysore virus (BSMyV), Banana streak OL virus (BSOLV), and Banana streak GF virus (BSGFV). Using RCA, the presence of BSMyV and BSOLV was confirmed in Australia, while BSOLV, BSGFV, Banana streak Uganda I virus (BSUgIV), Banana streak Uganda L virus (BSUgLV), and Banana streak Uganda M virus (BSUgMV) were detected in Uganda. This is the first confirmed report of episomally-derived BSUglV, BSUgLV, and BSUgMV in Uganda. As well as its ability to detect BSV, RCA was shown to detect two other pararetroviruses, Sugarcane bacilliform virus in sugarcane and Cauliflower mosaic virus in turnip.
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Coleman, Heather M., Stacey Efstathiou, and Philip G. Stevenson. "Transcription of the murine gammaherpesvirus 68 ORF73 from promoters in the viral terminal repeats." Journal of General Virology 86, no. 3 (March 1, 2005): 561–74. http://dx.doi.org/10.1099/vir.0.80565-0.
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Gammaherpesviruses persist as latent episomes in a dynamic lymphocyte pool. The regulated production of an episome maintenance protein is therefore crucial to their survival. The transcription initiation site of the murine gammaherpesvirus 68 episome maintenance protein, ORF73, was mapped to the viral terminal repeats, more than 10 kb distant from the open reading frame (ORF) itself. A 5′ non-coding exon in the terminal repeats was spliced to the right end of the viral unique sequence, and then across ORFs 75a, 75b, 75c and 74 to ORF73. The right-hand portion of a single repeat unit was sufficient for constitutive promoter activity. The unique left end of the viral genome further enhanced ORF73 transcription. This, together with the large size of the predominant ORF73 mRNA, suggested that transcription initiates in distal repeat units and then splices between repeats to generate an extensive 5′ untranslated region. A second promoter in the left-hand portion of the proximal terminal repeat unit generated a transcript which overlapped that of ORF73, but failed to splice to the ORF73 coding exon and so transcribed ORF75a. In distal repeat copies, however, transcription from this promoter would enter the next repeat unit to become an ORF73 mRNA. There was a third promoter just upstream of ORF73 itself. These data indicate that ORF73 transcription is highly complex, and support the idea that the terminal repeats of gamma-2-herpesviruses constitute a vital component of episomal persistence.
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Papapetrou, Eirini, Panos Ziros, Ilina Mitcheva, Aglaia Athanassiadou, and Nicholas Zoumbos. "Gene Transfer in Hematopoietic Progenitor Cells Mediated by an SV40 Based Episomal Vector Carrying A S/MAR Element." Blood 104, no. 11 (November 16, 2004): 5264. http://dx.doi.org/10.1182/blood.v104.11.5264.5264.
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Abstract Background/Aims: Replicating episomal vectors present a potential alternative to currently used oncoretroviral vectors for gene transfer in hematopoietic progenitor/stem cells. Their main advantage is that they can persist in the recipient nucleus as independent units, without integrating into the host’s genome, eliminating thus the risk of insertional mutagenesis. In the present study we explored the capacity of a recently developed SV40-based episomal vector, pEPI-eGFP, to stably transfect hematopoietic progenitor cell lines and primary cells, in order to evaluate its potential for therapeutic applications. pEPI-eGFP contains the enhanced green fluorescent protein (eGFP) cDNA and a Scaffold/Matrix Attachment Region (S/MAR) and it does not code for any proteins of viral origin. These unique properties qualify pEPI-eGFP as an attractive vehicle for gene therapy applications. Results: The vector was maintained as a stable episome in K562 cells for at least 100 generations and supported long-term EGFP expression, even in cells cultured in non-selective medium. Methylation-dependent cleavage assays demonstrated the vector’s ability to self-replicate in K562 cells and MEL cells, whereas its episomal status was confirmed by Southern blotting and plasmid rescue in E. coli. The vector was also maintained in primary human fibroblasts for at least 30 passages with and without selection pressure. Transfection of CD34+ cells from umbilical cord blood with pEPI-eGFP was feasible by electroporation with an efficiency of up to 30%, as estimated by flow cytometric evaluation of eGFP expression 24–48 h post-transfection. Cytokine prestimulation of CD34+ cells did not enhance transfection efficiency, whereas transfection of post-mitotic cells, such as dendritic cells was also feasible. This suggests that efficient transfection with the vector does not require cell cycling. PCR with eGFP-specific primers in single CFU colonies derived from CD34+/eGFP+/PI− FACS-sorted cells demonstrated the presence of the vector in ~20% of the colonies. Conclusion: Our results demonstrate that pEPI-2 exhibits a considerable potential as a gene transfer vector for hematopoietic cells, as it confers long-term transgene expression in hematopoietic progenitor cell lines as a stable episome and can efficiently transfect unstimulated CD34+ cells. Further studies, both in vitro and in vivo, are required to assess the long-term maintenance in hematopoietic progenitor/stem cells and their progeny as well as other features of the vector, in order to evaluate its overall efficacy and possibly improve its performance.
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Viejo-Borbolla, Abel, Matthias Ottinger, Eva Brüning, Antje Bürger, Regina König, Emrah Kati, Julie A. Sheldon, and Thomas F. Schulz. "Brd2/RING3 Interacts with a Chromatin-Binding Domain in the Kaposi's Sarcoma-Associated Herpesvirus Latency-Associated Nuclear Antigen 1 (LANA-1) That Is Required for Multiple Functions of LANA-1." Journal of Virology 79, no. 21 (November 1, 2005): 13618–29. http://dx.doi.org/10.1128/jvi.79.21.13618-13629.2005.
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ABSTRACT Latency-associated nuclear antigen 1 (LANA-1) of Kaposi's sarcoma-associated herpesvirus (KSHV) mediates the episomal replication of the KSHV genome, as well as transcriptional regulation, in latently infected cells. Interaction of LANA-1 with cellular chromatin is required for both these functions. An N-terminal heterochromatin-binding site in LANA-1 is essential for the replication and maintenance of latent episomes, as well as transcriptional regulation. We have recently described a C-terminal domain in LANA-1 that modulates the interaction with cellular interphase chromatin or elements of the nuclear matrix. Here, we used a series of LANA-1 deletion mutants to investigate the relationship between the different functions of LANA-1 and its interaction with the host chromatin-binding protein Brd2/RING3. Our findings suggest that the C-terminal chromatin-binding domain in LANA-1 is required for multiple LANA-1 functions, including the ability to bind to and replicate viral episomal DNA, to modulate transcription, and to interact with Brd2/RING3. Similar to the recently described tethering of bovine papillomavirus E2 protein to host chromatin via Brd4/MCAP, Brd2/RING3, another member of the Brd family of chromatin-binding proteins, therefore interacts with a chromatin-binding region of another viral latent nuclear protein and could play a role in its multiple functions.
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Wang, Aline Yen Ling, and Charles Yuen Yung Loh. "Episomal Induced Pluripotent Stem Cells: Functional and Potential Therapeutic Applications." Cell Transplantation 28, no. 1_suppl (November 14, 2019): 112S—131S. http://dx.doi.org/10.1177/0963689719886534.
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The term episomal induced pluripotent stem cells (EiPSCs) refers to somatic cells that are reprogrammed into induced pluripotent stem cells (iPSCs) using non-integrative episomal vector methods. This reprogramming process has a better safety profile compared with integrative methods using viruses. There is a current trend toward using episomal plasmid reprogramming to generate iPSCs because of the improved safety profile. Clinical reports of potential human cell sources that have been successfully reprogrammed into EiPSCs are increasing, but no review or summary has been published. The functional applications of EiPSCs and their potential uses in various conditions have been described, and these may be applicable to clinical scenarios. This review summarizes the current direction of EiPSC research and the properties of these cells with the aim of explaining their potential role in clinical applications and functional restoration.
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Vines, Richard R., Jay E. Purdy, Brian D. Ragland, John Samuelson, Barbara J. Mann, and William A. Petri. "Stable episomal transfection of Entamoeba histolytica." Molecular and Biochemical Parasitology 71, no. 2 (May 1995): 265–67. http://dx.doi.org/10.1016/0166-6851(95)00057-8.
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Ndunguru, Joseph, Leandro De León, Catherine D. Doyle, Peter Sseruwagi, German Plata, James P. Legg, Graham Thompson, et al. "Two Novel DNAs That Enhance Symptoms and Overcome CMD2 Resistance to Cassava Mosaic Disease." Journal of Virology 90, no. 8 (February 10, 2016): 4160–73. http://dx.doi.org/10.1128/jvi.02834-15.
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ABSTRACTCassava mosaic begomoviruses (CMBs) cause cassava mosaic disease (CMD) across Africa and the Indian subcontinent. Like all members of the geminivirus family, CMBs have small, circular single-stranded DNA genomes. We report here the discovery of two novel DNA sequences, designated SEGS-1 and SEGS-2 (forsequencesenhancinggeminivirussymptoms), that enhance symptoms and break resistance to CMD. The SEGS are characterized by GC-rich regions and the absence of long open reading frames. Both SEGS enhanced CMD symptoms in cassava (Manihot esculentaCrantz) when coinoculated withAfrican cassava mosaic virus(ACMV),East African cassava mosaic Cameroon virus(EACMCV), orEast African cassava mosaic virus-Uganda(EACMV-UG). SEGS-1 also overcame resistance of a cassava landrace carrying the CMD2 resistance locus when coinoculated with EACMV-UG. Episomal forms of both SEGS were detected in CMB-infected cassava but not in healthy cassava. SEGS-2 episomes were also found in virions and whiteflies. SEGS-1 has no homology to geminiviruses or their associated satellites, but the cassava genome contains a sequence that is 99% identical to full-length SEGS-1. The cassava genome also includes three sequences with 84 to 89% identity to SEGS-2 that together encompass all of SEGS-2 except for a 52-bp region, which includes the episomal junction and a 26-bp sequence related to alphasatellite replication origins. These results suggest that SEGS-1 is derived from the cassava genome and facilitates CMB infection as an integrated copy and/or an episome, while SEGS-2 was originally from the cassava genome but now is encapsidated into virions and transmitted as an episome by whiteflies.IMPORTANCECassava is a major crop in the developing world, with its production in Africa being second only to maize. CMD is one of the most important diseases of cassava and a serious constraint to production across Africa. CMD2 is a major CMD resistance locus that has been deployed in many cassava cultivars through large-scale breeding programs. In recent years, severe, atypical CMD symptoms have been observed occasionally on resistant cultivars, some of which carry the CMD2 locus, in African fields. In this report, we identified and characterized two DNA sequences, SEGS-1 and SEGS-2, which produce similar symptoms when coinoculated with cassava mosaic begomoviruses onto a susceptible cultivar or a CMD2-resistant landrace. The ability of SEGS-1 to overcome CMD2 resistance and the transmission of SEGS-2 by whiteflies has major implications for the long-term durability of CMD2 resistance and underscore the need for alternative sources of resistance in cassava.
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Godoy, V. G., F. S. Gizatullin, and Maurice S. Fox. "Some Features of the Mutability of Bacteria During Nonlethal Selection." Genetics 154, no. 1 (January 1, 2000): 49–59. http://dx.doi.org/10.1093/genetics/154.1.49.
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Abstract We describe the mutability of the Trp− chromosomal +1 frameshift mutation trpE7999 during nonlethal selection, finding that the appearance of Trp+ revertants behaves similarly to that of episomal Lac+ revertants. In addition, we show that a feature of the Lac+ and Trp+ mutability is the accumulation of Trp+ and Lac+ revertants with additional unselected mutations, most of which are not due to heritable mutators. The cells undergoing nonlethal selection apparently experience an epigenetic change resulting in a subset of bacteria with elevated mutability that often remain hypermutable for the duration of selection. The epigenetic change provoked by nonlethal selection appears to be mediated by a unique function provided by the F'128 episome.
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Wada, Akihito, and Haruo Watanabe. "Penicillin-Binding Protein 1 ofStaphylococcus aureus Is Essential for Growth." Journal of Bacteriology 180, no. 10 (May 15, 1998): 2759–65. http://dx.doi.org/10.1128/jb.180.10.2759-2765.1998.
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ABSTRACT pbpA, a gene encoding penicillin-binding protein (PBP) 1 of Staphylococcus aureus, was cloned in anEscherichia coli MC1061 transformant which grew on a plate containing 512 μg of vancomycin per ml. This gene encodes a 744-amino-acid sequence which conserves three motifs of PBPs, SXXK, SXN, and KTG. The chromosomal copy of pbpA could be disrupted only when RN4220, a methicillin-sensitive S. aureus strain, had additional copies of pbpA in its episome. Furthermore, these episomal copies of pbpA could not be eliminated by an incompatible plasmid when the chromosomal copy of pbpA was disrupted beforehand. Based on these observations, we concluded that pbpA is essential for the growth of methicillin-sensitive S. aureus.
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Brezgin, Sergey, Anastasiya Kostyusheva, Natalia Ponomareva, Viktoriia Volia, Irina Goptar, Anastasiya Nikiforova, Igor Shilovskiy, Valery Smirnov, Dmitry Kostyushev, and Vladimir Chulanov. "Clearing of Foreign Episomal DNA from Human Cells by CRISPRa-Mediated Activation of Cytidine Deaminases." International Journal of Molecular Sciences 21, no. 18 (September 18, 2020): 6865. http://dx.doi.org/10.3390/ijms21186865.
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Restriction of foreign DNA is a fundamental defense mechanism required for maintaining genomic stability and proper function of mammalian cells. APOBEC cytidine deaminases are crucial effector molecules involved in clearing pathogenic DNA of viruses and other microorganisms and improperly localized self-DNA (DNA leakages). Mastering the expression of APOBEC provides the crucial means both for developing novel therapeutic approaches for combating infectious and non-infectious diseases and for numerous research purposes. In this study, we report successful application of a CRISPRa approach to effectively and specifically overexpress APOBEC3A and APOBEC3B deaminases and describe their effects on episomal and integrated foreign DNA. This method increased target gene transcription by >6–50-fold in HEK293T cells. Furthermore, CRISPRa-mediated activation of APOBEC3A/APOBEC3B suppressed episomal but not integrated foreign DNA. Episomal GC-rich DNA was rapidly destabilized and destroyed by CRISPRa-induced APOBEC3A/APOBEC3B, while the remaining DNA templates harbored frequent deaminated nucleotides. To conclude, the CRISPRa approach could be readily utilized for manipulating innate immunity and investigating the effects of the key effector molecules on foreign nucleic acids.
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Hübscher, Daniela, Sabine Rebs, Luis Haupt, Thomas Borchert, Celina Isabell Guessoum, Franziska Treu, Steffen Köhne, et al. "A High-Throughput Method as a Diagnostic Tool for HIV Detection in Patient-Specific Induced Pluripotent Stem Cells Generated by Different Reprogramming Methods." Stem Cells International 2019 (August 5, 2019): 1–11. http://dx.doi.org/10.1155/2019/2181437.
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Induced pluripotent stem cells (iPSCs) provide a unique opportunity for generation of patient-specific cells for use in translational purposes. We aimed to compare iPSCs generated by different reprogramming methods regarding their reprogramming efficiency, pluripotency capacity, and the possibility to use high-throughput PCR-based methods for detection of human pathogenic viruses. iPSCs from skin fibroblasts (FB), peripheral blood mononuclear cells (PBMCs), or mesenchymal stem cells (MSCs) were generated by using three different reprogramming systems including chromosomal integrating and nonintegrating methods. Reprogramming efficiencies were in accordance with the literature, indicating that the parental cell type and the reprogramming method play a major role for the reprogramming efficiencies (FB: STEMCCA: 1.30±0.18, Sendai virus: 1.37±0.01, and episomal plasmids: 0.04±0.02; PBMCs: Sendai virus: 0.002±0.001, episomal plasmids: 0) but result in the same characteristics of pluripotency. We found the highest reprogramming efficiencies for MSC with 3.32±1.2 by using episomal plasmids. Since GMP standard working procedures and screening units need virus contamination-free cell lines, we studied HIV-1 contamination in the generated iPSCs. We used the high-throughput cobas® 6800/8800 system, which is normally used for detection of HIV-1 in plasma of patients, and found that footprint-free reprogramming methods as episomal plasmids and Sendai virus are useful for the described virus detection method. This fast, cost-effective, robust, and reliable assay demonstrates the feasibility to use high-throughput PCR-based methods for detection of human pathogenic viruses in ps-iPSC lines that were generated with nongenome integrating reprogramming methods.
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Sen, Shrinka, Paramita Mandal, Amrapali Bhattacharya, Sudip Kundu, Rahul Roy Chowdhury, Nidhu Ranjan Mondal, Tanmay Chatterjee, Biman Chakravarty, Sudipta Roy, and Sharmila Sengupta. "Impact of viral and host DNA methylations on HPV16-related cervical cancer pathogenesis." Tumor Biology 39, no. 5 (May 2017): 101042831769979. http://dx.doi.org/10.1177/1010428317699799.
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Epigenetic alterations within human papillomavirus (HPV) and host cellular genomes are known to occur during cervical carcinogenesis. Our objective was to analyse the influence of (1) methylation within two immunostimulatory CpG motifs within HPV16 E6 and E7 genes around the viral late promoter and their correlation, if any, with expression deregulation of host receptor (TLR9) and DNA methyltransferases (DNMT1, DNMT3A and DNMT3B) and (2) global DNA methylation levels within CpGs of the repetitive Alu sequences, on cervical cancer (CaCx) pathogenesis. Significantly higher proportions of CaCx samples portrayed methylation in immunostimulatory CpG motifs, compared to HPV16-positive non-malignant samples, with cases harbouring episomal HPV16 showing decreased methylation compared to those with viral integration. A significant linear trend of TLR9 upregulation was recorded in the order of HPV–negative controls < HPV16-positive non-malignant samples < HPV16-positive CaCx cases. TLR9 upregulation in cases with episomal HPV16 was again higher among those with non-methylated immunostimulatory CpG motifs. Comparison of cases with HPV–negative controls revealed that DNMT3A was significantly downregulated only among integrated cases, DNMT3B was significantly overexpressed among both categories of cases, although at variable levels, while DNMT1 failed to show any deregulated expression among the cases. Global host DNA hypomethylation, also showed a significant linear increasing trend through the progressive CaCx development stages mentioned above and was most prominently higher among cases with episomal HPV16 as opposed to viral integration. Thus, HPV16 and host methylations appear to influence CaCx pathogenesis, with differential molecular signatures among CaCx cases with episomal and integrated HPV16.
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Young, Won-Bin, E. Jeffery Beecham, Gary L. Lindberg, and Charles J. Link. "Restriction Mapping of Retroviral Vector Episomal DNA." BioTechniques 28, no. 3 (March 2000): 562–65. http://dx.doi.org/10.2144/00283cr01.
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Horlick, Robert A., Adriane E. Schilling, Philippe Samama, Robert N. Swanson, V. Danial Fitzpatrick, Alan K. Robbins, and Bassam Damaj. "Combinatorial gene expression using multiple episomal vectors." Gene 243, no. 1-2 (February 2000): 187–94. http://dx.doi.org/10.1016/s0378-1119(99)00561-2.
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D'Aiuto, L., J. I. delasHeras, A. Ross, M. H. Shen, and H. Cooke. "Generation of a Telomere-Based Episomal Vector." Biotechnology Progress 19, no. 6 (December 5, 2003): 1775–80. http://dx.doi.org/10.1021/bp0341500.
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Silla, Toomas, Ingrid Hääl, Jelizaveta Geimanen, Kadri Janikson, Aare Abroi, Ene Ustav, and Mart Ustav. "Episomal Maintenance of Plasmids with Hybrid Origins in Mouse Cells." Journal of Virology 79, no. 24 (December 15, 2005): 15277–88. http://dx.doi.org/10.1128/jvi.79.24.15277-15288.2005.
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ABSTRACT Bovine papillomavirus type 1 (BPV1), Epstein-Barr virus (EBV), and human herpesvirus 8 genomes are stably maintained as episomes in dividing host cells during latent infection. The mitotic segregation/partitioning function of these episomes is dependent on single viral protein with specific DNA-binding activity and its multimeric binding sites in the viral genome. In this study we show that, in the presence of all essential viral trans factors, the segregation/partitioning elements from both BPV1 and EBV can provide the stable maintenance function to the mouse polyomavirus (PyV) core origin plasmids but fail to do so in the case of complete PyV origin. Our study is the first which follows BPV1 E2- and minichromosome maintenance element (MME)-dependent stable maintenance function with heterologous replication origins. In mouse fibroblast cell lines expressing PyV large T antigen (LT) and either BPV1 E2 or EBV EBNA1, the long-term episomal replication of plasmids carrying the PyV minimal origin together with the MME or family of repeats (FR) element can be monitored easily for 1 month under nonselective conditions. Our data demonstrate clearly that the PyV LT-dependent replication function and the segregation/partitioning function of the BPV1 or EBV are compatible in certain, but not all, configurations. The quantitative analysis indicates a loss rate of 6% per cell, doubling in the case of MME-dependent plasmids, and 13% in the case of FR-dependent plasmids in nonselective conditions. Our data clearly indicate that maintenance functions from different viruses are principally interexchangeable and can provide a segregation/partitioning function to different heterologous origins in a variety of cells.
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Grant, Margaret J., Matthew S. Loftus, Aiola P. Stoja, Dean H. Kedes, and M. Mitchell Smith. "Superresolution microscopy reveals structural mechanisms driving the nanoarchitecture of a viral chromatin tether." Proceedings of the National Academy of Sciences 115, no. 19 (April 2, 2018): 4992–97. http://dx.doi.org/10.1073/pnas.1721638115.
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By tethering their circular genomes (episomes) to host chromatin, DNA tumor viruses ensure retention and segregation of their genetic material during cell divisions. Despite functional genetic and crystallographic studies, there is little information addressing the 3D structure of these tethers in cells, issues critical for understanding persistent infection by these viruses. Here, we have applied direct stochastic optical reconstruction microscopy (dSTORM) to establish the nanoarchitecture of tethers within cells latently infected with the oncogenic human pathogen, Kaposi’s sarcoma-associated herpesvirus (KSHV). Each KSHV tether comprises a series of homodimers of the latency-associated nuclear antigen (LANA) that bind with their C termini to the tandem array of episomal terminal repeats (TRs) and with their N termini to host chromatin. Superresolution imaging revealed that individual KSHV tethers possess similar overall dimensions and, in aggregate, fold to occupy the volume of a prolate ellipsoid. Using plasmids with increasing numbers of TRs, we found that tethers display polymer power law scaling behavior with a scaling exponent characteristic of active chromatin. For plasmids containing a two-TR tether, we determined the size, separation, and relative orientation of two distinct clusters of bound LANA, each corresponding to a single TR. From these data, we have generated a 3D model of the episomal half of the tether that integrates and extends previously established findings from epifluorescent, crystallographic, and epigenetic approaches. Our findings also validate the use of dSTORM in establishing novel structural insights into the physical basis of molecular connections linking host and pathogen genomes.
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Uphoff, Cord C., Sabine A. Denkmann, and Hans G. Drexler. "Analysis of Epstein-Barr Virus Infections in Lymphoma Cell Lines." Blood 106, no. 11 (November 16, 2005): 3906. http://dx.doi.org/10.1182/blood.v106.11.3906.3906.
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Abstract Epstein-Barr virus (EBV, human herpesvirus type 4) is ubiquitously distributed in all human populations, reaching infection rates of more than 90%. EBV is known to infect B- lymphocytes and mucosal epithelium cells and to establish latent or productive infections. The virus is the causative agent of infectious mononucleosis and closely associated with the endemic form of Burkitt lymphoma (BL). EBV has also been associated with various lymphoid and epithelial malignancies, such as Hodgkin, T-cell, and AIDS-related lymphomas, and lymphoepithelioma-like carcinomas of several organs. In vitro, B- lymphocytes are transformed by EBV into permanent lymphoblastoid cell lines (B-LCL). We investigated the EBV infection status of primate cell lines by PCR (406 human, 4 monkey). This method detects EBV genomes integrated into the eukaryotic chromosomes, non-integrated EBV episomes, and linear genomes of active EBV particles. The analyses revealed that 38/410 cell lines contain the EBV genome. All EBV+ cell lines were established from B- lineage leukemia/lymphoma cells (13/52 B-non-Hodgkin cell lines, 10/13 BL cell lines, 2/2 hairy cell leukemia cell lines, 1/6 plasma cell leukemia/myeloma cell lines) or are B-LCLs (9/9), natural killer cells (2/2), and one monkey cell line. No cell lines from other tissues were found to be EBV+. To further examine the production of EBV particles in the PCR+ cell lines, we analyzed the expression of the BZLF1 protein by Western blotting applying a ZEBRA monoclonal antibody. The cell lines were analyzed untreated as well as treated with the phorbol ester TPA for 3 days to induce the lytic phase of the EBV infection. Four cell lines exhibited a BZLF1 specific band a priori; after stimulation with TPA, 4 further cell lines expressed BZLF1 protein to various extents. To distinguish between linear DNA of herpesviruses (DNA form of active viruses) and covalently closed circles of episomal DNA, we performed Gardella gels applying crude lysates from cell cultures. Except for cell line NAMALWA and its subclones, DG-75, DOHH-2, and OCI-LY19 (all EBV-PCR+ cell lines) showed at least one band of episomal genomes. Some cell lines showed two episomal bands pointing to a double infection or to mutated episomes. The amount of linear DNA does not correlate with the number of episomes. Southern blots of genomic DNA revealed different genotypes of EBV, except for those cell lines which were established with B95-8 virus particles. To determine distribution of EBV genomes in single cells, we established a fluorescence in situ hybridization (FISH) method with a Cy3-labeled cosmid clone containing a genomic EBV fragment. The method showed for various cell lines that only a few cells contain high amounts of EBV genomes (several hundred) whereas the vast majority harbors only a few genomes in the nuclei. FISH appears to be superior to other methods, allowing for EBV analysis at the single cell level to determine the cellular permissiveness. In summary, we could show that EBV is constitutively produced in a few B-lymphoma derived cell lines and can be induced in several other cell lines. These cell lines represent valuable tools for further investigation into the biology of EBV infection.
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Xia, Wei, Mengkai Hu, Yang Pan, Dan Wu, and Jing Wu. "Improved Production of Streptomyces sp. FA1 Xylanase in a Dual-Plasmid Pichia pastoris System." Current Issues in Molecular Biology 43, no. 3 (December 18, 2021): 2289–304. http://dx.doi.org/10.3390/cimb43030161.
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Methanol is considered as a potential hazard in the methanol-induced yeast expression of food-related enzymes. To increase the production efficiency of recombinant proteins in Pichia pastoris without methanol induction, a novel dual-plasmid system was constructed, for the first time, by a combining the strategies of genomic integration and episomal expression. To obtain a high copy number of the target gene, the autonomously replicating sequence derived from Kluyveromyces lactis (PARS) was used to construct episomal vectors carrying the constitutive promoters PGAP and PGCW14. In addition, an integrative vector carrying the PGCW14 promoter was constructed by replacing the PGAP promoter sequence with a partial PGCW14 promoter. Next, using xylanase XynA from Streptomyces sp. FA1 as the model enzyme, recombination strains were transformed with different combinations of integrating and episomal vectors that were constructed to investigate the changes in the protein yield. Results in shake flasks indicated that the highest enzyme yield was achieved when integrated PGAP and episomal PGCW14 were simultaneously transformed into the host strain. Meanwhile, the copy number of xynA increased from 1.14 ± 0.46 to 3.06 ± 0.35. The yield of XynA was successfully increased to 3925 U·mL−1 after 102 h of fermentation in a 3.6 L fermenter, which was 16.7-fold and 2.86-fold of the yields that were previously reported for the constitutive expression and methanol-induced expression of the identical protein, respectively. Furthermore, the high-cell-density fermentation period was shortened from 132 h to 102 h compared to that of methanol-induced system. Since the risk of methanol toxicity is removed, this novel expression system would be suitable for the production of proteins related to the food and pharmaceutical industries.
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Duan, Dongsheng, Prerna Sharma, Jusan Yang, Yongping Yue, Lorita Dudus, Yulong Zhang, Krishna J. Fisher, and John F. Engelhardt. "Circular Intermediates of Recombinant Adeno-Associated Virus Have Defined Structural Characteristics Responsible for Long-Term Episomal Persistence in Muscle Tissue." Journal of Virology 72, no. 11 (November 1, 1998): 8568–77. http://dx.doi.org/10.1128/jvi.72.11.8568-8577.1998.
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ABSTRACT Adeno-associated viral (AAV) vectors have demonstrated great utility for long-term gene expression in muscle tissue. However, the mechanisms by which recombinant AAV (rAAV) genomes persist in muscle tissue remain unclear. Using a recombinant shuttle vector, we have demonstrated that circularized rAAV intermediates impart episomal persistence to rAAV genomes in muscle tissue. The majority of circular intermediates had a consistent head-to-tail configuration consisting of monomer genomes which slowly converted to large multimers of >12 kbp by 80 days postinfection. Importantly, long-term transgene expression was associated with prolonged (80-day) episomal persistence of these circular intermediates. Structural features of these circular intermediates responsible for increased persistence included a DNA element encompassing two viral inverted terminal repeats (ITRs) in a head-to-tail orientation, which confers a 10-fold increase in the stability of DNA following incorporation into plasmid-based vectors and transfection into HeLa cells. These studies suggest that certain structural characteristics of AAV circular intermediates may explain long-term episomal persistence with this vector. Such information may also aid in the development of nonviral gene delivery systems with increased efficiency.