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. 2006 Oct 3;103(40):14935-40.
doi: 10.1073/pnas.0509988103. Epub 2006 Sep 25.

Three restricted forms of Epstein-Barr virus latency counteracting apoptosis in c-myc-expressing Burkitt lymphoma cells

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Three restricted forms of Epstein-Barr virus latency counteracting apoptosis in c-myc-expressing Burkitt lymphoma cells

Gemma L Kelly et al. Proc Natl Acad Sci U S A. .

Abstract

Epstein-Barr virus (EBV), a human herpesvirus, transforms B cell growth in vitro through expressing six virus-coded Epstein-Barr nuclear antigens (EBNAs) and two latent membrane proteins (LMPs). In many EBV-associated tumors, however, viral antigen expression is more restricted, and the aetiological role of the virus is unclear. For example, endemic Burkitt lymphoma (BL) classically presents as a monoclonal, c-myc-translocation-positive tumor in which every cell carries EBV as an EBNA1-only (Latency I) infection; such homogeneity among EBV-positive cells, and the lack of EBV-negative comparators, hampers attempts to understand EBV's role in BL pathogenesis. Here, we describe an endemic BL that was unusually heterogeneous at the single-cell level and, in early passage culture, yielded a range of cellular clones, all with the same c-myc translocation but differing in EBV status. Rare EBV-negative cells were isolated alongside EBV-positive cells displaying one of three forms of restricted latency: (i) conventional Latency I expressing EBNA1 only from a WT virus genome, (ii) Wp-restricted latency expressing EBNAs 1, 3A, 3B, 3C, and -LP only from an EBNA2-deleted genome, and (iii) a previously undescribed EBNA2(+)/LMP1(-) latency in which all six EBNAs are expressed again in the absence of the LMPs. Interclonal comparisons showed that each form of EBV infection was associated with a specific degree of protection from apoptosis. Our work suggests that EBV acts as an antiapoptotic rather than a growth-promoting agent in BL by selecting among three transcriptional programs, all of which, unlike the full virus growth-transforming program, remain compatible with high c-myc expression.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
EBV genome content of Awia-BL. (A Left) PCR analysis of Awia-BL and the Awia-tr-LCL by using primer/probe combinations specific for the EBNA2 sequence or for a BamHI W-H fusion fragment (EBNA2 gene deletion). BJAB is an EBV-negative control line. Rael-BL is a standard Latency I BL line. (A Right) Parallel data from a representative range of Awia-BL clones. The boxed numbers refer to the total EBV genome load for each clone as determined by quantitative PCR assay for the EBV pol gene. (B) Location of the EBNA2 gene deletion in Awia-BL shown below a linearized map of the EBV genome with BamHI restriction fragments. Deletion coordinates (numbered according to the B95.8 prototype strain sequence) are shown relative to those found in the Wp-restricted Sal-BL, Oku-BL, and Ava-BL lines (13). (C) Metaphase spreads of Awia-BL clone e (carrying a single EBNA2-positive genome only) and clone m (carrying a single EBNA2-deleted genome only) after FISH staining with a fluorescein-labeled EBV probe (green) and a spectrum-orange-labeled probe specific either for a chromosome 4 centromeric region or for a chromosome 5 telomeric region (red). Clone e shows integration on chromosome 5; clone m shows integration on chromosome 4 (see white arrows).
Fig. 2.
Fig. 2.
Analysis of EBV latent transcription in the parental Awia-BL line, the Awia tr-LCL, and representative EBV genome-negative, Latency I, Wp-restricted, and EBNA2+/LMP1 Awia-BL clones. Transcript levels (with transcript-specific primer-probe combinations as shown) relative to an appropriate positive control line (assigned a value of 1). Note that the BamHI Q-U-K-spliced transcript levels indeed reflected Qp usage because there was no detectable F-Q-U-K-spliced transcripts from the lytic Fp promoter (data not shown).
Fig. 3.
Fig. 3.
Analysis of EBV latent protein expression in the parental Awia-BL line, the Awia tr-LCL, and representative Awia-BL clones as in Fig. 2. Results are presented as immunoblots probed with antibodies specific for EBNA1, EBNA2, EBNA3A, and LMP1. Positive control lines were the type 1 virus-transformed X50–7 LCL and the type 2 virus-transformed C2+BL16 LCL; BJAB is the EBV-negative control line. Immunoblots of the same samples were probed with antibodies against c-myc and, as a loading control, against β-actin. Note that by immunoblotting levels of EBNA2 in the EBNA2+/LMP1 clones was estimated to be 20–60% of that seen in the C2+BL16 LCL.
Fig. 4.
Fig. 4.
EBNA2 and LMP1 staining of Awia-BL (at the first in vitro passage within 7 days of tumor cell culture), two EBNA2+/LMP1 Awia-BL clones (e and j), and the Awia tr-LCL. Immunofluorescence results are shown alongside bright field photomicrographs of the same area.
Fig. 5.
Fig. 5.
Relationship among EBV status, form of EBV latency, and susceptibility to apoptosis in Awia-BL cells. (A and B) Mean results from multiple apoptosis assays in which Awia-BL clones were exposed to anti-IgM at 10 μg/ml for 72 h (A) or ionomycin at 1 μg/ml for 48 h (B) and then stained with propidium iodide and Syto 16. Results are expressed as the percentage of cells induced to die. Extending the assay period up to 96 h in each case did not affect the pattern of results (data not shown). (C) Mean results from additional assays in which EBV-negative and Latency I clones were tested at lower doses of 2.5 μg/ml anti-IgM and 0.25 μg/ml ionomycin. Note that in all experiments, essentially similar results were obtained by using caspase cleavage as a marker of apoptosis in single cells.
Fig. 6.
Fig. 6.
Diagrammatic representation of the three different forms of restricted EBV latency found in the Awia-BL tumor compared with the standard Latency III infection typical of all LCLs. Note that the Latency I and EBNA2+/LMP1 forms of latency involve different patterns of transcription from an EBNA2 gene-positive WT EBV genome, whereas the Wp-restricted form of latency involves transcription from an EBNA2 gene-deleted genome. In Awia-BL, the Wp-restricted and EBNA2+/LMP1 latent infections have involved the integration of single EBV genomes into chromosomes 4 and 5, respectively.

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