The average number of molecules of Epstein-Barr nuclear antigen 1 per cell does not correlate with the average number of Epstein-Barr virus (EBV) DNA molecules per cell among different clones of EBV-immortalized cells

Sternås, Lars; Middleton, Tim; Sugden, Bill

doi:10.1128/jvi.64.5.2407-2410.1990

Cited by 55 publications

(21 citation statements)

References 26 publications

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“…Moreover, since the EBNA1 and EBNA3 mRNAs are derived from the same transcription unit, in which the EBNA1 coding exon is downstream of those for the EBNA3s, the presence of Sal EBV EBNA1 in the apparent absence of detectable EBNA3A and EBNA3B from the same genomes (EBNA3Cs encoded by the Sal and Akata EBV isolates are indistinguishable by immunoblotting) may be further evidence of a posttranscriptional mechanism of action, possibly one that regulates alternative splicing. Interestingly, this apparent maintenance of specific EBNA1 levels is consistent with an earlier report that the number of EBNA1 molecules per cell is relatively constant (a less than 2-fold variance) within B-cell lines supporting latency III, despite the fact that the EBV genome copy number among these lines varied widely and up to 40-fold (62).…”

Section: Discussionsupporting

confidence: 90%

trans -Repression of Protein Expression Dependent on the Epstein-Barr Virus Promoter Wp during Latency

Hughes

Dickerson

Shaner

et al. 2011

J Virol

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An ordered silencing of Epstein-Barr virus (EBV) latency gene transcription is critical for establishment of persistent infection within B lymphocytes, yet the mechanisms responsible and the role that the virus itself may play are unclear. Here we describe two B-cell superinfection models with which to address these problems. In the first, Burkitt lymphoma (BL) cells that maintain latency I, when superinfected, initially supported transcription from the common EBNA promoters Wp and Cp (latency III) but ultimately transitioned to latency I (Cp/Wp silent), an essential requirement for establishment of EBV latency in vivo. We used this model to test whether the early lytic-cycle gene BHLF1, implicated in silencing of the Cp/Wp locus, is required to establish latency I. Upon superinfection with EBV deleted for the BHLF1 locus, however, we have demonstrated that BHLF1 is not essential for this aspect of EBV latency. In the second model, BL cells that maintain Wp-restricted latency, a variant program in which Cp is silent but Wp remains active, sustained the latency III program of transcription from the superinfecting-virus genomes, failing to transition to latency I. Importantly, there was substantial reduction in Wp-mediated protein expression from endogenous EBV genomes, in the absence of Cp reactivation, that could occur independent of a parallel decrease in mRNA. Thus, our data provide evidence of a novel, potentially posttranscriptional mechanism for trans-repression of Wp-dependent gene expression. We suggest that this may ensure against overexpression of the EBV nuclear antigens (EBNAs) prior to the transcriptional repression of Wp in cis that occurs upon activation of Cp.

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Section: Discussionsupporting

confidence: 90%

trans -Repression of Protein Expression Dependent on the Epstein-Barr Virus Promoter Wp during Latency

Hughes

Dickerson

Shaner

et al. 2011

J Virol

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“…By transfecting LCL expressing wild‐type EBNA1 with an EBNA1ΔGA expression construct and vice versa, the amount of EBNA1 protein derived from the expression construct could be directly correlated with endogenous EBNA1. Endogenous EBNA1 is known to be expressed in a very narrow range at 25,000–44,000 molecules per cell in LCL 27. These experiments showed that NGF‐R‐sorted cells expressed at least 60‐fold more plasmid‐encoded than endogenous EBNA1 protein (Fig.…”

Section: Resultsmentioning

confidence: 79%

Epstein‐Barr virus nuclear antigen 1 evades direct immune recognition by CD4⁺ T helper cells

Mautner

Pich²,

Nimmerjahn³

et al. 2004

Eur J Immunol

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The Epstein-Barr virus (EBV) nuclear antigen 1 (EBNA1) is the only viral protein regularly expressed in EBV-associated malignancies. Immune recognition of EBNA1 by CD8 + T cells is prevented by an internal glycine-alanine repeat (GAr) which blocks proteasomal degradation.To test whether EBV-infected cells could be recognized by T helper cells, human CD4 + T cell clones specific for EBNA1 were isolated from latently EBV-infected individuals. These T cells, however, failed to recognize EBV-positive target cells. To investigate whether endogenous presentation of EBNA1 epitopes on MHC class II was prevented by the GAr domain, a mutant EBV strain with an EBNA1 lacking the GAr (EBNA1DGA) was generated and used to establish an Epstein-Barr virus-immortalized lymphoblastoid B cell line (LCL). The EBNA1DGA LCL were not recognized by the EBNA1-specific T cell clones either, indicating that the GAr domain does not mediate this effect. Immune recognition could be restored by overexpression of EBNA1, for which at least 60-fold higher levels of both EBNA1 or EBNA1DGAr protein were required. These results demonstrate that EBNA1 evades direct recognition by CD4 + T helper cells, since its steady state level is below the threshold required for efficient presentation on MHC class II. These findings have important implications for the design of immunotherapeutic approaches to target EBV-positive malignancies.

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“…Antibodies against EBNA-1. Affinity-purified rabbit antibodies specific for the carboxyl-terminal domain of EBNA-1 were prepared as described previously (37).…”

Section: Methodsmentioning

confidence: 99%

Genetic Evidence that EBNA-1 Is Needed for Efficient, Stable Latent Infection by Epstein-Barr Virus

Lee

Diamond

Yates

1999

J Virol

114

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Replication and maintenance of the 170-kb circular chromosome of Epstein-Barr virus (EBV) during latent infection are generally believed to depend upon a single viral gene product, the nuclear protein EBNA-1. EBNA-1 binds to two clusters of sites at oriP, an 1,800-bp sequence on the EBV genome which can support replication and maintenance of artificial plasmids introduced into cell lines that contain EBNA-1. To investigate the importance of EBNA-1 to latent infection by EBV, we introduced a frameshift mutation into the EBNA-1 gene of EBV by recombination along with a flanking selectable marker. EBV genomes carrying the frameshift mutation could be isolated readily after superinfecting EBV-positive cell lines, but not if recombinant virus was used to infect EBV-negative B-cell lines or to immortalize peripheral blood B cells. EBV mutants lacking almost all of internal repeat 3, which encode a repetitive glycine and alanine domain of EBNA-1, were generated in the same way and found to immortalize B cells normally. An EBNA-1-deficient mutant of EBV was isolated and found to be incapable of establishing a latent infection of the cell line BL30 at a detectable frequency, indicating that the mutant was less than 1% as efficient as an isogenic, EBNA-1-positive strain in this assay. The data indicate that EBNA-1 is required for efficient and stable latent infection by EBV under the conditions tested. Evidence from other studies now indicates that autonomous maintenance of the EBV chromosome during latent infection does not depend on the replication initiation function of oriP. It is therefore likely that the viral chromosome maintenance (segregation) function of oriP and EBNA-1 is what is required.

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The average number of molecules of Epstein-Barr nuclear antigen 1 per cell does not correlate with the average number of Epstein-Barr virus (EBV) DNA molecules per cell among different clones of EBV-immortalized cells

Cited by 55 publications

References 26 publications

trans -Repression of Protein Expression Dependent on the Epstein-Barr Virus Promoter Wp during Latency

trans -Repression of Protein Expression Dependent on the Epstein-Barr Virus Promoter Wp during Latency

Epstein‐Barr virus nuclear antigen 1 evades direct immune recognition by CD4⁺ T helper cells

Genetic Evidence that EBNA-1 Is Needed for Efficient, Stable Latent Infection by Epstein-Barr Virus

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The average number of molecules of Epstein-Barr nuclear antigen 1 per cell does not correlate with the average number of Epstein-Barr virus (EBV) DNA molecules per cell among different clones of EBV-immortalized cells

Cited by 55 publications

References 26 publications

trans -Repression of Protein Expression Dependent on the Epstein-Barr Virus Promoter Wp during Latency

trans -Repression of Protein Expression Dependent on the Epstein-Barr Virus Promoter Wp during Latency

Epstein‐Barr virus nuclear antigen 1 evades direct immune recognition by CD4+ T helper cells

Genetic Evidence that EBNA-1 Is Needed for Efficient, Stable Latent Infection by Epstein-Barr Virus

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Epstein‐Barr virus nuclear antigen 1 evades direct immune recognition by CD4⁺ T helper cells