Separation of Epstein-Barr Virus DNA from Large Chromosomal DNA in Non-virus-producing Cells

Nonoyama, Meihan; Pagano, Joseph S.

doi:10.1038/newbio238169a0

Cited by 173 publications

(108 citation statements)

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“…In agreement with the previous studies of Nonoyama and Pagano (10), no alkali-resistant association between EBV DNA and Raji cell DNA was observed. It would therefore appear that the mechanism of integration of EBV DNA is different from that found for SV40 (9) or adenoviruses (22), in that it does not involve DNA-DNA covalent linkage.…”

Section: >1supporting

confidence: 82%

“…The Raji ['4C]DNA in fraction III, which had a molecular weight of 110 X 106 in neutral solution, varied in size in alkaline solution from 10 X 106 to 50 X 106. Thus, some intact single strands were present, and most strands only con- (10), and in situ hybridization experiments suggest that virus DNA is associated with several different chromosomes (21). Here, the density distribution of the intrinsic EBV DNA component in Raji cells shows that a large proportion of the virus DNA molecules is directly associated with cellular DNA sequences.…”

Section: >1mentioning

confidence: 99%

“…By alkaline sucrose gradient centrifugation of very-high-molecular-weight cellular DNA, followed by localization of viral sequences by nucleicacid hybridization, it was demonstrated that simian virus 40 (SV40) DNA is covalently integrated into host DNA in SV40-transformed cells (9). In applying this technique to Raji cells, however, Nonoyama and Pagano (10) found no detectable EBV DNA associated with the cellular DNA. Instead, the intracellular EBV DNA had the same sedimentation properties as alkali-denatured DNA isolated from virus particles.…”

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confidence: 99%

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Linear Association Between Cellular DNA and Epstein-Barr Virus DNA in a Human Lymphoblastoid Cell Line

Adams

Lindahl

Klein

1973

Proc. Natl. Acad. Sci. U.S.A.

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Section: >1supporting

confidence: 82%

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confidence: 99%

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confidence: 99%

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Linear Association Between Cellular DNA and Epstein-Barr Virus DNA in a Human Lymphoblastoid Cell Line

Adams

Lindahl

Klein

1973

Proc. Natl. Acad. Sci. U.S.A.

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“…2 During latency, EBV DNA persists as an episome in the nuclei of host cells, or can be integrated into the cellular genome via the TR sequences. [3][4][5][6][7] Integration may initiate transformation events by causing chromosomal instability, [8][9][10] or may provide a cis-acting mechanism for altering gene regulation or structure. 11,12 Hence, if integration has an effect on viral or cellular gene expression, 5,13 it could be directly linked to the oncogenic properties of EBV, 14 mediating continuous cell proliferation.…”

mentioning

confidence: 99%

Visualization of episomal and integrated Epstein‐Barr virus DNA by fiber fluorescence in situ hybridization

Reisinger

Rumpler

Lion

et al. 2005

Intl Journal of Cancer

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For many Epstein-Barr virus (EBV)-associated malignancies, it is still a matter of controversy whether infected cells harbor episomal or chromosomally integrated EBV genomes or both. It is well established that the expression of EBV genes per se carries oncogenic potential, but the discrimination between episomal and integrated forms is of great relevance because integration events can contribute to the oncogenic properties of EBV, whereas host cells that exclusively harbor viral episomes may not carry the risks mediated by chromosomal integration. This notion prompted us to establish a reliable technique that not only allows to unequivocally discriminate episomal from integrated EBV DNA, but also provides detailed insights into the genomic organization of the virus. Here, we show that dynamic molecular combing of host cell DNA combined with fluorescence in situ hybridization (FISH) using EBV-specific DNA probes facilitate unambiguous discrimination of episomal from integrated viral DNA. Furthermore, the detection of highly elongated internal repeat 1 (IR1) sequences provides evidence that this method permits detection of major genomic alterations within the EBV genome. Thus, fiber FISH may also provide valuable insights into the genomic organization of viral genomes other than EBV. ' 2005 Wiley-Liss, Inc.Key words: EBV; fiber FISH; episomal; integration Epstein-Barr virus (EBV, human herpes virus 4) is associated with a number of benign and malignant diseases, including infectious mononucleosis, Burkitt's lymphoma, Hodgkin's disease, immunoblastic and transplantation-associated lymphomas. The virus preferentially infects B lymphocytes. 1 Upon infection, the linear viral DNA is covalently closed by its terminal repeat (TR) sequences and circularizes. 2 During latency, EBV DNA persists as an episome in the nuclei of host cells, or can be integrated into the cellular genome via the TR sequences. [3][4][5][6][7] Integration may initiate transformation events by causing chromosomal instability, [8][9][10] or may provide a cis-acting mechanism for altering gene regulation or structure. 11,12 Hence, if integration has an effect on viral or cellular gene expression, 5,13 it could be directly linked to the oncogenic properties of EBV, 14 mediating continuous cell proliferation. It has recently been shown that loss of expression of the putative tumor suppressor gene BACH2 in a Burkitt's lymphoma cell line is attributable to EBV integration within the first intron of the gene. 13 Another Burkitt's lymphoma cell line has been reported to contain integrated EBV DNA between the oncogenes REL and BCL11A associated with an increased expression of REL. 15 The notion that host cells, which exclusively contain episomal viral DNA, are not susceptible to the risks linked to chromosomal integration emphasizes the necessity of having a reliable method permitting distinction between episomal and integrated EBV DNA. We have therefore established a technique based on DNA fiber analysis, facilitating detailed insights into the genomic or...

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“…1). The double-stranded EBV DNA genome usually persists in latently infected cell nuclei as a multicopy, circular episome (2,3) that associates with chromosomes during mitosis (4). EBV episomes are replicated by cell DNA polymerase during S phase (5).…”

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confidence: 99%

Maintenance of Epstein-Barr virus (EBV) oriP-based episomes requires EBV-encoded nuclear antigen-1 chromosome-binding domains, which can be replaced by high-mobility group-I or histone H1

Hung¹

2001

Proceedings of the National Academy of Sciences

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EBV-encoded nuclear antigen-1 (EBNA-1) binding to a cis-acting viral DNA element, oriP, enables plasmids to persist in dividing human cells as multicopy episomes that attach to chromosomes during mitosis. In investigating the significance of EBNA-1 binding to mitotic chromosomes, we identified the basic domains of EBNA-1 within amino acids 1-89 and 323-386 as critical for chromosome binding. In contrast, the EBNA-1 C terminus (amino acids 379 -641), which includes the nuclear localization signal and DNAbinding domain, does not associate with mitotic chromosomes or retain oriP plasmid DNA in dividing cell nuclei, but does enable the accumulation of replicated oriP-containing plasmid DNA in transient replication assays. The importance of chromosome association in episome maintenance was evaluated by replacing EBNA-1 amino acids 1-378 with cell proteins that have similar chromosome binding characteristics. High-mobility group-I amino acids 1-90 or histone H1-2 could substitute for EBNA-1 amino acids 1-378 in mediating more efficient accumulation of replicated oriP plasmid, association with mitotic chromosomes, nuclear retention, and long-term episome persistence. These data strongly support the hypothesis that mitotic chromosome association is a critical factor for episome maintenance. The replacement of 60% of EBNA-1 with cell protein is a significant step toward eliminating the need for noncellular protein sequences in the maintenance of episomal DNA in human cells.

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Separation of Epstein-Barr Virus DNA from Large Chromosomal DNA in Non-virus-producing Cells

Cited by 173 publications

References 10 publications

Linear Association Between Cellular DNA and Epstein-Barr Virus DNA in a Human Lymphoblastoid Cell Line

Linear Association Between Cellular DNA and Epstein-Barr Virus DNA in a Human Lymphoblastoid Cell Line

Visualization of episomal and integrated Epstein‐Barr virus DNA by fiber fluorescence in situ hybridization

Maintenance of Epstein-Barr virus (EBV) oriP-based episomes requires EBV-encoded nuclear antigen-1 chromosome-binding domains, which can be replaced by high-mobility group-I or histone H1

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