Targeted gene disruption in Epstein-Barr virus

Lee, May-Ann; Kim, Okjin; Yates, John L.

doi:10.1016/0042-6822(92)90701-p

Cited by 15 publications

(17 citation statements)

References 21 publications

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“…Both approaches have demonstrated that noncoding exons and introns of the long EBNA transcript and most of the lytic genes between EBNA1 and LMP1 can be deleted (13,23,25). An analysis of all the deletions made in the EBV genome demonstrates that the residual complexity of the genome required for transformation of a B lymphocyte in vitro is approximately 48 kbp, or 26% of the genome (14,(25)(26)(27)(28)(29)(30)(31). The construction of mini-transforming EBV genomes with large segment(s) of heterologous DNA could be independent of P3HR-1 since direct transformation of primary B lymphocyte with a cosmid DNA should be feasible given the small residual complexity of the transforming EBV genome.…”

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confidence: 99%

“…Progeny virus can then be used to infect primary B lymphocytes or a non-EBV-infected continuous B-cell line such as an EBVnegative Burkitt lymphoma (BL) cell line. Cells infected with the recombinant EBV can then be positively selected by growing the infected cells in the presence of the toxic drug (11,12,(14)(15)(16)). An advantage of this strategy is that mutations can be made in a gene that is essential for primary B-lymphocyte transformation and the mutant progeny genome can be positively identified by infection of BL cells and by plating of the infected cells under selective conditions (11,12,16).…”

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confidence: 99%

“…The progeny virus could then be harvested and plated onto lymphocytes at a low multiplicity, and recombinant infected cells could be identified by genetic selection or marker-specific PCR (11)(12)(13). One strategy is to transfect a cell carrying a wild-type latent EBV genome, with a mutated EBV DNA fragment carrying an expression vector for a gene that encodes an enzyme that inactivates a toxic drug (11,12,(14)(15)(16). Progeny virus can then be used to infect primary B lymphocytes or a non-EBV-infected continuous B-cell line such as an EBVnegative Burkitt lymphoma (BL) cell line.…”

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confidence: 99%

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Epstein-Barr virus vectors for gene delivery to B lymphocytes.

Robertson

Ooka

Kieff

1996

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

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Basic research in Epstein-Barr virus (EBV) molecular genetics has provided means to maintain episomes in human cells, to efficiently deliver episomes with up to 150 kbp of heterologous DNA to human B lymphocytes, and to immortalize human B lymphocytes with EBV recombinants that can maintain up to 120 kbp of heterologous DNA. Episome maintenance requires an EBV nuclear protein, EBNA1, whereas immortalization of cells with EBV recombinants requires EBNA1, EBNA2, EBNA3A, EBNA3C, EB-NALP, and LMP1. EBV-derived vectors are useful for experimental genetic reconstitution in B lymphocytes, a cell type frequently used in establishing repositories of human genetic deficiencies. The ability of EBV-infected cells to establish a balanced state of persistence in normal humans raises the possibility that cells infected with EBV recombinants could be useful for genetic reconstitution, in vivo.The Epstein-Barr virus (EBV) genome has yielded reagents that have been useful in vector design. Genetic engineering of specifically mutated EBV recombinants has also had a major impact on basic EBV research and is useful for experimental genetic reconstitution in human cells. The purpose of this article is to review previous work, indicate potential utilities and liabilities, and describe recent experiments that should enable intensive investigation of an under explored and important part of the EBV genome.EBV is similar in its replication to other herpes viruses and many of the genetic approaches have substantial precedence in alpha herpes virus research. However, EBV also differs from alpha herpes viruses in that it establishes latency in and alters the growth of human B lymphocytes (for reviews, see refs. 1 and 2). These properties derive in large measure from a unique set of genes that encode nuclear and integral membrane proteins that have been given the acronyms, EBNAs (EBV nuclear antigens) and LMPs (latent membrane proteins). In initiation of latent infection in B lymphocytes, EBV first expresses EBNALP and EBNA2. EBNA2 upregulates the EBNA promoter leading to a longer primary transcript from which EBNALP, EBNA2, EBNA3A, EBNA3B, EBNA3C, and EBNA1 transcripts are derived (Fig. 1). EBNA2 also activates the LMP1 and LMP2 promoters. These proteins act in concert to alter B-lymphocyte growth and enable the maintenance of the EBV genome as a multicopy episome in a state of latent infection. Each of these proteins expressed in latently infected cells, with the exception of EBNA1, has epitopes that are presented on the B-cell surface in the context of common class I major histocompatibility complex (MHC) molecules and are recognized by immune human cytotoxic T lymphocytes. This high level of cytotoxic T-cell recognition and the ability of latently infected cells to shift between full latent gene expression with cell proliferation and an EBNA1 only type of latent infection that is immunologically privileged enables latently infected cells to achieve a balanced state of long-term persistence in humans. This state of long-term persis...

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Epstein-Barr virus vectors for gene delivery to B lymphocytes.

Robertson

Ooka

Kieff

1996

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

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“…The sizes of these recombinant fragments, around 24 kb, is close to the sum of the sizes of the 11.3-kb wild-type SacI fragment and pTE3C-Hyg (also 11.3 kb). These data suggest that the entire pTE3C-Hyg construct has been inserted into the 11.3-kb SacI fragment, something that could be expected from earlier experiments in which EBV recombinants were generated by using circular targeting plasmids (Lee et al, 1992; and data not shown). The sizes of these recombinant fragments vary slightly, probably due to amplification of a portion of pTE3C-Hyg (Fujiwara and Ono, 1995).…”

Section: Structures Of the Ebv Recombinantsmentioning

confidence: 66%

Epstein-Barr-virus-infected human T-cell line with a unique pattern of viral-gene expression

et al. 1998

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“…A recombinant virus that carries a deletion of the BARF2 gene that encodes the single strand DNA binding protein was not able to support the lytic cycle, an observation in line with the function of this protein. 12 The analysis of EBV virions in which the BHRF1, BHLF1, LMP2, or EBER genes were disrupted showed no diVerence compared with wild-type EBV in terms of lytic propagation. 45 57 59 65 This observation suggests that the functions of these proteins during viral replication are redundant or are not apparent in vitro.…”

Section: Dissection Of Genes Involved In the Ebvmentioning

confidence: 97%

The genetic approach to the Epstein-Barr virus: from basic virology to gene therapy

Delecluse¹,

Hammerschmidt

2000

Molecular Pathology

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The Epstein-Barr virus (EBV) infects humans and the genome of this infectious agent has been detected in several tumour types, ranging from lymphomas to carcinomas. The analysis of the functions of the numerous viral proteins encoded by EBV has been impeded by the large size of the viral genome, which renders the construction of viral mutants diYcult. To overcome these limitations, several genetic systems have been developed that allow the modification of the viral genome. Two diVerent approaches, depending on the host cell type in which the viral mutants are generated, have been used in the past. Traditionally, mutants were constructed in EBV infected eukaryotic cells, but more recently, approaches that make use of a recombinant EBV cloned in Escherichia coli have been proposed. The phenotype associated with the inactivation or modification of nearly 20 of the 100 EBV viral genes has been reported in the literature. In most of the reported cases, the EBV latent genes that mediate the ability of EBV to immortalise infected cells were the targets of the genetic analysis, but some virus mutants in which genes involved in DNA lytic replication or infection were disrupted have also been reported. The ability to modify the viral genome also opens the way to the construction of viral strains with medical relevance. A cell line infected by a virus that lacks the EBV packaging sequences can be used as a helper cell line for the encapsidation of EBV based viral vectors. This cell line will allow the evaluation of EBV as a gene transfer system with applications in gene therapy. Finally, genetically modified non-pathogenic strains will provide a basis for the design of an attenuated EBV live vaccine.

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Targeted gene disruption in Epstein-Barr virus

Cited by 15 publications

References 21 publications

Epstein-Barr virus vectors for gene delivery to B lymphocytes.

Epstein-Barr virus vectors for gene delivery to B lymphocytes.

Epstein-Barr-virus-infected human T-cell line with a unique pattern of viral-gene expression

The genetic approach to the Epstein-Barr virus: from basic virology to gene therapy

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