Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2010 Mar;84(5):2236-44.
doi: 10.1128/JVI.01972-09. Epub 2009 Dec 16.

Augmented latent membrane protein 1 expression from Epstein-Barr virus episomes with minimal terminal repeats

Allison M Repic  1 , Mingxia Shi, Rona S Scott, John W Sixbey
Affiliations

Augmented latent membrane protein 1 expression from Epstein-Barr virus episomes with minimal terminal repeats

Allison M Repic et al. J Virol. 2010 Mar.

Abstract

The major oncogene of the Epstein-Barr virus (EBV), latent membrane protein 1 (LMP1), can be expressed from either of two promoters, ED-L1 or L1-TR, producing mRNAs of 2.8 kb or 3.5 kb, respectively. L1-TR, active in nasopharyngeal carcinoma and Hodgkin's lymphoma, is located within the first of a highly variable reiteration of terminal repeat (TR) sequences that are joined by random recombination upon circularization of the linear genome at entry into cells. To determine whether the resultant TR number affects LMP1 promoter activity, we isolated single-cell clones bearing episomes of distinct TR numbers (6TR to 12TR) from epithelial cells newly infected with EBV. LMP1 mRNA levels correlated directly with the quantity of LMP1 protein expressed but varied inversely to TR number. Unexpectedly, the 3.5-kb transcript predominated only at lower TR reiterations. Diminished L1-TR activity in the context of a higher TR count was confirmed with a green fluorescent protein (GFP) reporter construct driven by L1-TR. Various levels of LMP1, expressed from virus isogenic in all but TR number, produced divergent morphological and growth phenotypes in each cell clone. Abundant LMP1 in 6TR cells yielded a relatively cytostatic state compared to the proliferative one produced by intermediate and smaller amounts in 8TR and 12TR clones. These findings suggest that the diversification of TR number, inherent in a round of EBV reactivation and reinfection, may itself be a component of the oncogenic process. The replicative burst preceding onset of many EBV-linked cancers may increase the likelihood that LMP1 levels compatible with clonal outgrowth are achieved in a subset of infected cells.

PubMed Disclaimer

Figures

FIG. 1.
FIG. 1.
Effect of EBV terminal repeat number on levels of LMP1 expression. (A) Schematic of fused TRs (vertical lines) after circularization of the EBV genome in relation to LMP1 regulatory elements. Numerals are EBV coordinates for B95-8 (2) (NCBI GenBank accession number VO1555). Bent arrows indicate LMP1 promoters; the black box is the EBNA1-dependent enhancer (FR, family of repeats) in the origin of plasmid replication (oriP). Gray boxes represent exons 1 to 3 of LMP1. Arrowheads indicate location of PCR primers used to distinguish 3.5-kb LMP1 transcript from total LMP1 mRNA. Insertion sites of antibiotic-resistant cassettes in EBV strains Akata and B95-8 are located in BDLF3 and LMP2 exons 2 to 4, respectively. (B) Southern blot of BamHI-digested cellular DNA from three CCL20.2 epithelial cell clones infected with B95-8 recombinant EBfaV-GFP that was hybridized to a NJhet (TR-specific) probe. Terminal repeat number (indicated at top) was calculated from molecular weight of band minus size of unique sequences at either end of TRs encompassed by BamHI restriction sites divided by size of one TR unit. Note that the EGFP-Neor cassette in recombinant B95-8 introduced a novel BamHI restriction site, thereby reducing the quantity of unique EBV DNA normally contained in the like restriction fragment of wild-type EBV from ∼7.5 kb to 4.1 kb (45, 51). Molecular size markers are indicated on the left. (C) Relative fold difference in LMP1 mRNA as determined by RQ RT-PCR. Each cell clone is designated by its distinct episomal TR number. Shown are values from four independent experiments, with error bars representing the standard error of the mean (SEM). (D) Immunoblot of LMP1 protein in infected cell clones. Numerical values represent fold difference in LMP1 expression relative to cell clone indicated after normalization to tubulin loading control, as determined by densitometry.
FIG. 2.
FIG. 2.
Differential LMP1 promoter usage in epithelial cell culture linked to TR number. (A) Inverse correlation of LMP1 expression to terminal repeat number in CCL185/Akata. Southern blot (left) of BamHI-digested DNA from four infected epithelial cell clones probed for EBV terminal repeat sequences, with the TR number being calculated as indicated in the legend for Fig. 1. Molecular size markers are indicated on the left. Immunoblot (right) of LMP1 protein in each of four CCL185 clones. (B) Northern blot for LMP1 mRNA in CCL20.2/B95-8 clones. The 3.5-kb mRNA initiating from L1-TR promoter predominates in 6TR clone only. The sizes of the molecular weight markers (in thousands) are indicated on the left. (C) Increased L1-TR promoter activity at lower TR reiterations as determined by RT-PCR in two cell lines. PCR primers for amplification of cDNA positioned as shown in Fig. 1A. Top panels show amplification product specific for 3.5-kb transcript only, whereas bottom panels indicate PCR product derived from sequences common to both 2.8-kb and 3.5-kb transcripts.
FIG. 3.
FIG. 3.
TR reiteration influences GFP production from a L1-TR promoter-driven reporter construct in CCL20.2 epithelial cells. (A) Schematic of GFP reporters of equivalent size, one with 2TRs and a 7.6-kb λ phage DNA stuffer and the second with 17TRs. (B) Fluorescence microscopy of epithelial cells 24 h posttransfection with GFP reporter constructs (top); cell density as shown by phase-contrast microscopy (middle); GFP expression in respective cell populations by flow cytometric analysis (bottom). (C) GFP mRNA relative to transfected plasmid copy number as determined by RQ RT-PCR. Values shown are from three independent experiments, with error bars representing the SEM.
FIG. 4.
FIG. 4.
Morphological and growth phenotypes of TR-defined epithelial cell clones. (A) CCL20.2/B95-8 clones examined 24 h post seeding at 104 cells/ml. From left to right, uninfected cells (UN) followed by infected cells with least (12TR) to most (6TR) LMP1 expression. Arrowheads indicate vacuolization. (B) Population doublings in short-term growth assay of CCL20.2/B95-8 clones. Cells were seeded at 104 cells/ml and then counted at indicated times prior to reaching confluence. Each time point represents the mean of 4 independent experiments, the error bars indicating SEM. (C) Proliferation of CCL20.2/B95-8 clones as indicated by metabolic conversion of dye in MTS assay. Colorimetric values plotted were readings minus values obtained from blanks. Error bars indicate the SEM of values from 3 independent experiments. (D) Relative rates of DNA synthesis in CCL20.2/B95-8 cell clones as quantified by BrdU incorporation. At left, BrdU uptake in 1 h by cells seeded 24 h earlier, as visualized by fluorescent microscopy after staining with anti-BrdU FLUOS-conjugated antibody. At right, percent of BrdU-positive cells as measured by flow cytometry. Cells were treated for 1 h with BrdU 24 h after seeding and stained as described above. Error bars denote SEM from 3 independent experiments.
FIG. 5.
FIG. 5.
Cytostatic versus proliferative phenotypes of the three CCL20.2/B95-8 epithelial cell clones in mixed culture, with emergence of 8TR clone as predominant. (A) Southern blot hybridized to EBV terminus-specific probe, with each lane representing a sequential passage as indicated by number. 12TR, 8TR, and 6TR clones were mixed disproportionately for seeding at time zero to favor slower-growing 6TR clone, and then passaged every 48 h × 15. Results shown are representative of 3 independent experiments. (B) Proportion of total cell population represented by each clone at indicated passage, as determined by densitometry of bands within single lane.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Ali, A. K. M., S. Saito, S. Shibata, K. Takada, and T. Kanda. 1 July 2009. Distinctive effects of the Epstein-Barr virus family of repeats on viral latent gene promoter activity and B-lymphocyte transformation. J. Virol. doi:10.1128/JVI.01979-08. - DOI - PMC - PubMed
    1. Baer, R. J., A. T. Bankier, M. D. Biggin, P. L. Deininger, P. J. Farrell, T. J. Gibson, G. F. Hatfill, G. S. Hudson, S. C. Satchwell, C. Seguin, P. S. Tuffnell, and B. C. Barrell. 1984. DNA sequences and expression of the B95-8 Epstein-Barr virus genome. Nature 310:207-211. - PubMed
    1. Blackwood, E. M., and J. T. Kadonaga. 1998. Going the distance: a current review of enhancer action. Science 281:60-63. - PubMed
    1. Borza, C. M., and L. M. Hutt-Fletcher. 1998. Epstein-Barr virus recombinant lacking expression of glycoprotein gp150 infects B cells normally but is enhanced for infection of epithelial cells. J. Virol. 72:7577-7582. - PMC - PubMed
    1. Brown, N. A., C.-R Liu, Y.-F. Wang, and C. R. Garcia. 1988. B-cell lymphoproliferation and lymphomagenesis are associated with clonotypic intracellular terminal regions of the Epstein-Barr virus. J. Virol. 62:962-969. - PMC - PubMed

Publication types

Substances