Flow Cytometric Analysis of Epstein-Barr Virus (EBV) Latent Membrane Protein Expression in EBV-infected Raji cells

Boos, Harald; Stoehr, Michael; Sauter, Marlies; Mueller‐Lantzsch, Nikolaus

doi:10.1099/0022-1317-71-8-1811

Cited by 12 publications

(4 citation statements)

References 17 publications

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“…By using a cell line that expressed EBNA-3C in an inducible fashion, we were able to determine that the increase in LMP-1 protein occurred only hours after EBNA-3C expression, suggesting that this is a specific effect of EBNA-3C and demonstrating that this occurred not only in growth-arrested cells, as previously described, but also in proliferating cells. As previously reported, LMP-1 levels are increased by serum and fall over time in culture (5,6). The increases seen with EBNA-3C are largely due to sustained levels of LMP-1 at later time points.…”

Section: Discussionsupporting

confidence: 58%

“…The first demonstration that EBNA-3C might regulate LMP-1 came from a study in which EBNA-3C expression was restored in the EBV-positive Burkitt lymphoma cell line Raji, whose EBV genomes are deleted for most of the EBNA-3C open reading frame (1,2). Specifically, in parental Raji cells, levels of LMP-1 fall as cells progress to G 1 (5,6), whereas upon stable expression of EBNA-3C, levels of LMP-1 remain high (2). In reporter gene assays, however, EBNA-3C can mediate either repression or activation of the LMP-1 promoter through J and PU.1 sites, respectively (43,53,67,76,83,84).…”

mentioning

confidence: 99%

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Epstein-Barr Virus EBNA-3C Is Targeted to and Regulates Expression from the Bidirectional LMP-1/2B Promoter

Jiménez-Ramírez

Brooks

Forshell

et al. 2006

J Virol

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Epstein-Barr virus (EBV) nuclear antigen 3C (EBNA-3C) is essential for EBV-mediated immortalization of human B lymphocytes and regulates both the cell cycle and transcription. Transient reporter gene assays have implicated a pivotal role for EBNA-3C in the regulation of transcription of the majority of latency-associated genes expressed during the EBV growth program, including the viral oncoprotein LMP-1. To examine the regulation of latency gene expression by EBNA-3C, we generated an EBV-positive cell line that inducibly expresses EBNA-3C. This cell line allowed us to examine expression from the endogenous latency gene promoters in the context of an actual latent infection and the presence of other EBNA proteins, in particular EBNA-2, which is presumed to coregulate transcription with EBNA-3C. EBNA-3C induced the expression of both LMP-1 and LMP-2B mRNAs from the bidirectional LMP-1/LMP-2B promoter. In contrast, no effect was seen on expression from the common EBNA promoter Cp, which is responsive to EBNA-3C in reporter assays. Activation of LMP expression was not the consequence of increases in EBNA-2, PU.1 or Spi-B transcription factors, all of which are believed to be critical for activation of LMP-1. Chromatin immunoprecipitation assays furthermore indicated that EBNA-3C is present at the bidirectional LMP-1/LMP-2B promoter. These results indicate that EBNA-3C directly activates the expression of LMP-1 and LMP-2B but is unlikely to significantly regulate EBNA expression via Cp under normal growth conditions. Epstein-Barr virus (EBV) nuclear antigen 3C (EBNA-3C) is one of only six viral proteins known to be essential for EBV to immortalize primary human B lymphocytes (9,22,33,42,52,72). Although the EBNA-3 genes (3A, 3B, and 3C) probably arose through gene duplication, the sequences of the EBNA-3 proteins are highly divergent, and the fact that both EBNA-3A and EBNA-3C (but not EBNA-3B) are essential for EBVmediated immortalization suggests that their functions have also diverged. These proteins do have related functions, however, regulating both cell cycle progression and transcription.

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Section: Discussionsupporting

confidence: 58%

mentioning

confidence: 99%

Epstein-Barr Virus EBNA-3C Is Targeted to and Regulates Expression from the Bidirectional LMP-1/2B Promoter

Jiménez-Ramírez

Brooks

Forshell

et al. 2006

J Virol

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“…originally observed that LMP1 is induced in Raji after the dilution of a dense culture with fresh medium, it was concluded that this induction and the accompanying cell cycle progression were in response to the serum included in the medium (10,11).…”

Section: Resultsmentioning

confidence: 99%

Epstein-Barr virus nuclear antigen EBNA3C/6 expression maintains the level of latent membrane protein 1 in G1-arrested cells

Allday

Farrell

1994

J Virol

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The Epstein-Barr virus in the Burkitt lymphoma-derived cell line Raji has a deletion in the EBNA3C gene. When Raji cells are allowed to grow to high density and most of the cells become growth arrested in the G, phase of the cell cycle, the level of detectable latent membrane protein 1 (LMP1) is substantially reduced. After dilution of the cells with fresh growth medium, within 8 h, there is a large increase in LMP1 mRNA, and by 12 h, LMP1 is expressed to a high level (H.

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“…Trypan blue (TB) quenches the fluorescence of bacteria on the surface but not those inside leucocytes [20]. The quenching capacity of TB was determined by using Raji-cells [21] and neutrophils in the presence of 10 nM cytochalasin D [22]; both cause adherence of FITClabelled opsonised target particles on the cell surface without phagocytosis. The maximum quenching capacity was achieved with TB at 5 mg/ml.…”

mentioning

confidence: 99%

Colostral proteins from cows immunised with Streptococcus mutans/S. sobrinus support the phagocytosis and killing of mutans streptococci by human leucocytes

Loimaranta¹,

Nuutila

Marnila

et al. 1999

Journal of Medical Microbiology

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Passive immunisation, based on bovine colostral preparations, is an area of active research. Specific bovine antibodies inhibit the virulence factors of target pathogens but the interactions between whey preparations and human immune defence cells are not well known. Bovine colostrum inhibits the phagocytic activity of bovine leucocytes and this may reflect the biological activity of immunoglobulins in it. Therefore, this study aimed to examine the effects of bovine whey protein preparations from the colostrum of Streptococcus mutuns/S. sobrinus-immunised and sham-immunised cows on binding, ingestion and killing of these bacteria by human leucocytes. Binding and ingestion of FITC-labelled bacteria were estimated by flow cytometry and leukocyte activation was measured as chemiluminescence. Killing rate was estimated by plate counting and by measuring bioluminescence from S. mutans-containing the insect luciferase gene. Colostral whey protein preparation from hyperimmunised cows activated human leucocytes by opsonising specific bacteria. Neutrophils, eosinophils and monocytes weakly phagocytosed non-opsonised bacteria and bacteria opsonised with control product. On the contrary, binding and ingestion were efficient in the presence of the preparation from immunised cows. Thus, these results show that bovine colostral whey proteins are able to support the activation of human phagocytes against pathogenic microbes and that this property is related to specific antibodies in whey preparations. These whey proteins may also be clinically useful, especially in preventing the colonisation of newly erupted teeth by mutans streptococci.

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Flow Cytometric Analysis of Epstein-Barr Virus (EBV) Latent Membrane Protein Expression in EBV-infected Raji cells

Cited by 12 publications

References 17 publications

Epstein-Barr Virus EBNA-3C Is Targeted to and Regulates Expression from the Bidirectional LMP-1/2B Promoter

Epstein-Barr Virus EBNA-3C Is Targeted to and Regulates Expression from the Bidirectional LMP-1/2B Promoter

Epstein-Barr virus nuclear antigen EBNA3C/6 expression maintains the level of latent membrane protein 1 in G1-arrested cells

Colostral proteins from cows immunised with Streptococcus mutans/S. sobrinus support the phagocytosis and killing of mutans streptococci by human leucocytes

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