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. 2013 May 1;132(9):2076-86.
doi: 10.1002/ijc.27893. Epub 2012 Nov 2.

Epstein-Barr virus-induced epigenetic alterations following transient infection

Krista J Queen  1 , Mingxia Shi, Fangfang Zhang, Urska Cvek, Rona S Scott
Affiliations

Epstein-Barr virus-induced epigenetic alterations following transient infection

Krista J Queen et al. Int J Cancer. .

Abstract

Epstein-Barr virus (EBV) is a known tumor virus associated with an increasing array of malignancies; however, the association of the virus with certain malignancies is often erratic. To determine EBV's contributions to tumorigenesis in a setting of incomplete association, a transient model of infection was established where a clonal CCL185 carcinoma cell line infected with recombinant EBV was allowed to lose viral genomes by withdrawal of selection pressure. Global gene expression comparing EBV-negative, transiently infected clones to uninfected controls identified expression changes in more than 1,000 genes. Among downregulated genes, several genes known to be deoxyribonucleic acid (DNA) methylated in cancer were identified including E-cadherin and PYCARD. A cadherin switch, increased motility and enhanced cellular invasiveness present in EBV-positive cells were retained after viral loss, indicating an epigenetic effect. Repression of PYCARD expression was a result of increased promoter CpG methylation, whereas loss of E-cadherin expression after transient EBV infection did not correlate with increased DNA methylation of the E-cadherin promoter. Rather, repression of E-cadherin was consistent with the formation of a repressive chromatin state. Decreased histone 3 or 4 acetylation at the promoter and 5' end of the E-cadherin gene was observed in an EBV-negative, transiently infected clone relative to the uninfected controls. These results suggest that EBV can stably alter gene expression in a heritable fashion in formerly infected cells, whereas its own contribution to the oncogenic process is masked.

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Figures

Figure 1
Figure 1. Loss of EBV DNA following transient infection
A) Schematic representation for transient EBV infection. The CCL185 (lung carcinoma) cell line was infected with recombinant Akata BDLF3 carrying a neomycin resistance (neor) cassette. Following ten passages on selection, selective pressure was removed for an additional ten passages to allow loss of the virus. Cells were single-cell cloned and screened for the absence of EBV. B) EBER in situ hybridization of clonally-derived CCL185 cells transfected with vector, after infection with EBV, and three transiently infected, EBV-negative clones (10-9, 10-10, and 10–14). C) RT-PCR depicting EBV transcriptional latency program. Shown are the ethidium bromide stained (white bands) or Southern hybridized (black bands) PCR amplicons for Qp-initiated EBNA1, EBNA2, LMP1, LMP2, and GAPDH in uninfected (UN) or infected (EBV+) CCL185 clone. The EBV-positive B958 cell line, expressing a type III latency program, served as a positive control. D) PCR analysis using primers that amplify various regions across the EBV genome. Serial dilutions of DNA (100ng, 10ng, and 1ng) from the EBV-positive Namalwa BL cell line carrying two integrated copies of EBV in a total background of 100ng of DNA reconstituted from the EBV-negative BL2 cell line was used to determine the limit of detection in each PCR assay. Shown are uninfected (UN), vector control (V), three transiently infected, EBV-negative clones (−), and an EBV-positive clone (+) derived from a CCL185 clonal population.
Figure 2
Figure 2. Alterations to cellular gene expression following transient EBV infection
qRT-PCR was performed for various cellular genes identified as targets of repression [CDH1 (E-cadherin), CLDN3 (claudin 3), INHBB (inhibin-beta), AR (androgen receptor), and PYCARD] or activation [CDH2, (N-cadherin)] and normalized to GAPDH mRNA levels. The ratio of GAPDH to rRNA (ribosomal RNA) was also shown as validation for use of GAPDH as a normalization control. Shown is the mean and standard error of the mean from three independent experiments where the uninfected clone (UN) was arbitrarily set to 1. Samples shown are uninfected control (UN), Vector control (V), an EBV-positive clone, and three EBV-negative, transiently infected clones (cl10-9, cl10-10 and cl10–14). *p<0.05 relative to UN
Figure 3
Figure 3. Retention of an EMT-like phenotype following transient EBV infection
A) Immunoblot analysis for E-cadherin, N-cadherin, PYCARD, GAPDH and tubulin. B) Analysis of cell motility. C) Analysis of cell invasion. Shown is the average number of cells per field of view in four independent experiments with error bars representing the standard error of the mean. Samples shown are uninfected parental cells (UN), vector control (V), an EBV-positive clone, and three transiently infected EBV-negative clones (cl10-9, cl10-10, and cl10–14). * p<0.05 relative to UN
Figure 4
Figure 4. Reversal of E-cadherin and PYCARD expression after transient EBV infection with DNA methyltransferases and/or histone deacetylase inhibitors
CCL185 uninfected parental clonal (UN), vector control (V), and transiently infected, EBV-negative clones (cls 10-9, 10-10, 10–14) were treated with vehicle control (mock), 5 µM 5aza2DC, 300 nM TSA, or in combination (5aza2DC+TSA). A) E-cadherin transcript levels or B) PYCARD transcript levels were measured by qRT-PCR and normalized to GAPDH mRNA levels. UN mock-treated was arbitrarily set to 1. Shown is the mean and standard error of the mean from 3 independent experiments.
Figure 5
Figure 5. DNA methylation status of CpG islands at the E-cadherin and PYCARD gene loci after transient EBV infection
Bisulfite sequencing was performed to map the methylation status of 44 CpG residues within A) the PYCARD promoter and 21 CpG residues within B) the E-cadherin (CDH1) promoter. Each row represents the sequence analysis of a cloned PCR product after bisulfite treatment. Closed circles illustrate methylated CpGs and open circles represent unmethylated CpGs. Samples shown are uninfected (UN) clone, vector control (V), EBV-positive clone, EBV-negative clones 10-9, 10-10, and 10–14, methylated DNA control (Me) and unmethylated DNA control (UNMe).
Figure 6
Figure 6. Formation of repressive chromatin structure at the E-cadherin promoter after transient EBV infection
Chromatin immunoprecipitation (ChIP) was performed to determine the abundance of histone acetylation at the promoter, transcriptional start site (TSS) and intron regions of the E-cadherin gene using qPCR. The data shown are the mean and standard error of the mean normalized to input from three independent ChIPs. For the E-cadherin analysis, the uninfected (UN) control was arbitrarily set to 1. A) Abundance of histone 3 (H3) acetylation at the E-cadherin gene. B) Abundance of histone 4 (H4) acetylation at the E-cadherin gene. C) Abundance of total H3 at the E-cadherin gene. D) Abundance of histone acetylation within the GAPDH gene. For the GAPDH analysis, the vector control was arbitrarily set to 1. Samples are uninfected clone (UN), vector control (V), and the transiently infected, EBV-negative clone (cl10–14). *p<0.05 relative to UN.
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