Retention of plasmid DNA in mammalian cells is enhanced by binding of the Epstein-Barr virus replication protein EBNA1

Middleton, Tim; Sugden, Bill

doi:10.1128/jvi.68.6.4067-4071.1994

Cited by 118 publications

(41 citation statements)

References 20 publications

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“…In fact, the catastrophic loss of plasmid DNAs synthesized in the absence of EBNA-1 may reflect their being relegated to the cytoplasm after mitosis, perhaps in a manner akin to the loss of double-minute chromosomes (Von Hoff et al, 1992;Shimizu et al, 1998). This model is consistent with the observations that EBNA-1 (Grogan et al, 1983), EBV DNA (Delecluse et al, 1993) and large oriP plasmids (Simpson et al, 1996) associate with mitotic chromosomes, and that EBNA-1 enhances the retention of FR-containing DNAs in human cells (Middleton and Sugden, 1994). We note, however, that EBV DNA or oriP DNA does not consistently stain symmetrically on pairs of sister chromatids.…”

Section: Discussionsupporting

confidence: 68%

The plasmid replicon of EBV consists of multiplecis-acting elements that facilitate DNA synthesis by the cell and a viral maintenance element

1998

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Plasmids containing oriP, the plasmid origin of Epstein-Barr virus (EBV), are replicated stably in human cells that express a single viral trans-acting factor, EBNA-1. Unlike plasmids of other viruses, but akin to human chromosomes, oriP plasmids are synthesized once per cell cycle, and are partitioned faithfully to daughter cells during mitosis. Although EBNA-1 binds multiple sites within oriP, its role in DNA synthesis and partitioning has been obscure. EBNA-1 lacks enzymatic activities that are present in the origin-binding proteins of other mammalian viruses, and does not interact with human cellular proteins that provide equivalent enzymatic functions. We demonstrate that plasmids with oriP or its constituent elements are synthesized efficiently in human cells in the absence of EBNA-1. Further, we show that human cells rapidly eliminate or destroy newly synthesized plasmids, and that both EBNA-1 and the family of repeats of oriP are required for oriP plasmids to escape this catastrophic loss. These findings indicate that EBV's plasmid replicon consists of genetic elements with distinct functions, multiple cis-acting elements that facilitate DNA synthesis and viral cis/trans elements that permit retention of replicated DNA in daughter cells. They also explain historical failures to identify mammalian origins of DNA synthesis as autonomously replicating sequences.

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Section: Discussionsupporting

confidence: 68%

The plasmid replicon of EBV consists of multiplecis-acting elements that facilitate DNA synthesis by the cell and a viral maintenance element

1998

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“…These results strongly suggest that binding sites for the E2 protein can be responsible for providing MME activity to the BPV-1 origin. MME enhances the frequency of formation of G418-resistant colonies without replication It has been observed previously that, for EBV, multimerized EBNA-1 binding sites (FR) are required for stable replication of oriP-containing plasmids in an EBNA-1dependent fashion (Krysan et al, 1989;Middleton and Sugden, 1994;Kirchmaier and Sugden, 1995). This activity can be measured by increased transformation frequency of the plasmids carrying FR, and is thought to be caused by enhanced nuclear retention of plasmids containing FR.…”

Section: Mme Is Composed Of Redundant Sequencesmentioning

confidence: 99%

Cis and trans requirements for stable episomal maintenance of the BPV-1 replicator.

et al. 1996

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Papillomavirus genomes are maintained as multicopy nuclear plasmids in transformed cells. To address the mechanisms by which the viral DNA is stably propagated in the transformed cells, we have constructed a cell line CH04.15 expressing constitutively the viral proteins E1 and E2, that are required for initiation of viral DNA replication. We show that these viral proteins are necessary and sufficient for stable extrachromosomal replication. Using the cell line CH04.15, we have shown that the bovine papillomavirus‐1 (BPV‐1) minimal origin of replication (MO) is absolutely necessary, but is not sufficient for stable extrachromosomal replication of viral plasmids. By deletion and insertion analysis, we identified an additional element (minichromosome maintenance element, MME) in the upstream regulatory region of BPV‐1 which assures stable replication of the MO‐containing plasmids. This element is composed of multiple binding sites for the transcription activator E2. MME appears to function in the absence of replication but requires E1 and E2 proteins for activity. In contrast to, for example, Epstein‐Barr virus oriP, stably maintained BPV‐1 plasmids are not subject to once‐per‐cell cycle replication as determined by density labelling experiments. These results indicate that papillomavirus episomal replicators replicate independently of the chromosomal DNA of their hosts.

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“…22,23 One of the main drawbacks of TGE is that plasmid DNA is not integrated into the cell genome and thus the gene of interest is lost as cells divide. 24,25 Some experimental approaches have been developed to overcome this problem. One approach is the so-called extended gene expression (EGE) technique.…”

Section: Introductionmentioning

confidence: 99%

Extended gene expression for Gag VLP production achieved at bioreactor scale

Fuenmayor

Cervera

Gòdia

et al. 2018

J of Chemical Tech & Biotech

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BACKGROUND Transient gene expression has been widely used for virus‐like particles (VLP) production in both small‐ and large‐scale systems. The extended gene expression (EGE) technique is based on repeated medium exchanges and retransfections of cell culture to prolong the production phase. The aim of this study was to demonstrate scalability of the approach by operating EGE continuously in a controlled bioreactor to obtain similar results to those achieved with shake flask EGE for VLP production. A standard batch cultivation was also carried out in shake flask. RESULTS For EGE in shake flasks and in a bioreactor, cell viability was comparable; however, the bioreactor enabled much higher cell densities and specific growth rates than the shake flasks. Owing to this increased cell growth in the bioreactor, the percentage of GFP‐positive cells was considerably lower at the end than in the shake flasks. GagGFP VLP titers were similar in both shake flasks and bioreactor. Nanoparticle tracking analysis revealed that the ratio of VLPs/total particles (VLPs and cell vesicles) was higher in the shake flasks than in the bioreactor, possibly due to higher cell densities achieved in the bioreactor. CONCLUSIONS In this study, EGE methodology was carried out in a bioreactor system for the first time while maintaining GagGFP production titers. Overall, our findings call for further optimization and implementation of common downstream processing steps to improve yield and VLP quality. © 2018 Society of Chemical Industry

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Retention of plasmid DNA in mammalian cells is enhanced by binding of the Epstein-Barr virus replication protein EBNA1

Cited by 118 publications

References 20 publications

The plasmid replicon of EBV consists of multiplecis-acting elements that facilitate DNA synthesis by the cell and a viral maintenance element

The plasmid replicon of EBV consists of multiplecis-acting elements that facilitate DNA synthesis by the cell and a viral maintenance element

Cis and trans requirements for stable episomal maintenance of the BPV-1 replicator.

Extended gene expression for Gag VLP production achieved at bioreactor scale

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