Epstein - Barr Virus Transforming Protein LMP-1 Alters B Cells Gene Expression by Promoting Accumulation of the Oncoprotein ΔNp73α

<div><p>Many studies have proved that oncogenic viruses develop redundant mechanisms to alter the functions of the tumor suppressor p53. Here we show that Epstein-Barr virus (EBV), via the oncoprotein LMP-1, induces the expression of ΔNp73α, a strong antagonist of p53. This phenomenon is mediated by the LMP-1 dependent activation of c-Jun NH2-terminal kinase 1 (JNK-1) which in turn favours the recruitment of p73 to ΔNp73α promoter. A specific chemical inhibitor of JNK-1 or silencing JNK-1 expression strongly down-regulated ΔNp73α mRNA levels in LMP-1-containing cells. Accordingly, LMP-1 mutants deficient to activate JNK-1 did not induce ΔNp73α accumulation. The recruitment of p73 to the ΔNp73α promoter correlated with the displacement of the histone-lysine N-methyltransferase EZH2 which is part of the transcriptional repressive polycomb 2 complex. Inhibition of ΔNp73α expression in lymphoblastoid cells (LCLs) led to the stimulation of apoptosis and up-regulation of a large number of cellular genes as determined by whole transcriptome shotgun sequencing (RNA-seq). In particular, the expression of genes encoding products known to play anti-proliferative/pro-apoptotic functions, as well as genes known to be deregulated in different B cells malignancy, was altered by ΔNp73α down-regulation. Together, these findings reveal a novel EBV mechanism that appears to play an important role in the transformation of primary B cells.</p> </div

ΔNp73α inhibit expression of pro-apoptotic genes in EBV-infected cells.

<p>(<b>A</b>) LCLs were transfected with 2 µg S (S-high) and 2 increasing concentration of AS (0.5 µg, AS-low and 2 µg, AS-high), against ΔNp73α. Thirty hours after transfection cells were collected, the total lysates were extracted and analysed by immunoblotting for the indicated proteins. (<b>B</b>) Cells were treated as described in the legend of <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003186#ppat-1003186-g007" target="_blank">Figure 7A</a> and live stained with PI. The percentage of PI stained cells (dead cells) were calculated by flow cytometer as explained in Material and Methods. (<b>C</b>) LCLs treated with S-high (2 µg), AS-low (0.5 µg) and AS-high (2 µg) were used to perform RNAseq. The p53 target genes which were significantly deregulated (p value<0,01 EdgeR software) in S vs. AS were represented in the histogram and expressed as relative RPMK values. (<b>D</b>) SaOS-2 cells were transfected with different pcDNA3 constructs in the indicated combinations. After 36 hours, ChIP was performed using an anti HA-tag antibody and followed by real-time PCR, using primers flanking the p53-RE BS1 within the PLK2 promoter. The percentage of binding of p73 and ΔNp73 to PLK2 promoter was determined as described in the legend of <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003186#ppat-1003186-g004" target="_blank">Figure 4A</a>. (<b>E</b>) LCLs were transfected with 2 µg of ΔNp73α S (S-high) and 3 increasing concentration of ΔNp73α AS (0.5 µg, AS-low; 1 µg, AS-medium; 2 µg, AS-high). Thirty-six hours after transfection, cells were collected and processed for RNA extraction. Pig3 and PLK2 mRNA levels were determined by quantitative RT-PCR. The data are the mean of two independent experiments. The difference of Pig3 or PLK2 mRNA levels in LCLs transfected with S and AS is statistically significant (p values = 0.02 and 0.01 for Pig3 and PLK2 respectively).</p

EBV infection of B cells determines epigenetic changes on the p2 promoter.

<p>(<b>A and B</b>). Primary B cells were infected and immortalized with EBV (LCL). At the day of the extraction and 2-weeks post infection, primary and immortalized B cell respectively, were fixed and processed for quantitative ChIP analysis. (A) ChIP was carried out using EZH2, Histone 3 Trimethylated Lysine 27 (H3K27), Histone 3 acetylated Lysine 9 (H3K9Ac) antibodies and IgG antibody as negative control. After ChIP the eluted DNA was used as template for quantitative PCR with primers spanning the p73RE within the ΔNp73 promoter. Part of the total chromatin fraction (1/10) was processed at the same time and used as input. After subtracting the background of the unspecific binding (ChIP for IgG), the amount of promoter specifically bound by each protein was expressed as a percentage of the total amount of ΔNp73 promoter (% of input). (B) Quantitative-ChIP for p73 was carried out and analyzed as described in the legend of <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003186#ppat-1003186-g006" target="_blank">Figure 6A</a>. The data shown in (A) and (B) are the mean of three independent experiments with primary B cells of three different donors. The differences between the % of binding to the promoter in primary B cells and in LCL are significant (p value = 0.01 for EZH2, p value = 0.01 for H3K27Met, p value = 0.002 for H3K9Ac). (<b>C</b>) Primary B cells or primary B cells infected with wild-type EBV (EBV) or EBV lacking the entire LMP-1 gene (EBVΔLMP-1) were fixed and processed for EZH2 ChIP by using the LowCell ChIP Kit (Diogenode). Quantitative ChIP analysis was performed according to the manufacturers' protocol. The data are the mean of two independent experiments. The difference between the % of EZH2 binding to p2 in primary and LCL is significant (p value = 0.01). (<b>D</b>) RPMI-pLXSN (pLXSN) and RPMI-pLXSN-LMP-1 (LMP-1), RPMI EBV and RPMI EBVΔLMP-1 were fixed and processed for ChIP for EZH2 as described in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003186#ppat-1003186-g006" target="_blank">figure 6C</a>. The difference in the levels of EZH2 recruited to ΔNp73α promoter in the different conditions is significant (pLXSN vesus LMP-1 p value = 0.038, EBVΔLMP-1 versus EBV p value = 0.008). (<b>E</b>) Primary and LCLs non-transfected or transfected with scramble (Scr) or JNK-1 siRNA were fixed and processed for quantitative ChIP analysis using an antibody against the acetylated form of histone H4 following the procedure described in the legend of <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003186#ppat-1003186-g006" target="_blank">Figure 6A</a>. The data are the mean of three independent experiments. The difference in the levels of H4Ac binding to ΔNp73α promoter in the different conditions is significant (primary B cells versus LCL p value = 0.002, scramble siRNA versus JNK-1 siRNA p value = 0.04). (<b>F</b>) LCLs were cultured in absence (DMSO) or presence of 20 µM of JNK-1 inhibitor for 5 hours, fixed and processed for quantitative ChIP analysis using an antibody against the acetylated form of histone H4 or p73 following the procedure described in the legend of <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003186#ppat-1003186-g006" target="_blank">Figure 6A</a>. The data are the mean of three independent experiments. The difference in the percentage of H4Ac or p73 binding to ΔNp73α promoter in the different conditions is significant (DMSO versus JNK-1 inhibitor p value = 0.004 for H4Ac and p value = 0.001 for p73). (<b>G</b>) Primary B cells and EBV-positive B cells were analyzed by immunoblotting for the levels of the indicated proteins. (<b>H</b>) Forty µg of total extract from RPMI- EBVΔLMP-1 pLXSN and pLXSN-LMP-1 cells were analyzed by immunoblotting for the levels of the indicated proteins. (<b>I</b>) Nuclear and cytoplasmic fractions from RPMI- EBVΔLMP-1 pLXSN and pLXSN-LMP-1 were analyzed by immunoblotting for the levels of LMP-1, EZH2, β-actin and PARP. (<b>J</b>) Cellular localization of EZH2 was determined by immunofluorescent in Primary B cells and LCLs. Fluorescent signal was visualized using Axioplan2 microscope from Zeiss laser microscopy. The pictures shown are representative of three independent staining.</p

Expression of LMP-1 in cells infected by EBVΔLMP-1 restores ΔNp73α levels.

<p>(<b>A and B</b>) Two independent infections of RPMI with EBVΔLMP-1 recombinant virus, not infected RPMI and RPMI carrying the wild-type EBV genome were collected and processed to obtain total RNA or whole cell extracts. (A) The levels of ΔNp73, LMP-1, EBER-2 and GAPDH transcripts were determined by RT-PCR. (B) Protein extracts were analyzed by immunoblotting using the indicated antibodies. Please note that lane 1 to 3 and lane 4 have been joined from 2 different areas of the same immunoblot. (<b>C and D</b>) Total RNA and total cell extracts from RPMI- EBVΔLMP-1 were transduced with pLXSN or pLXSN-LMP-1 retroviruses were prepared. (C) Transcript levels of ΔNp73α, LMP-1, EBER-2 and GAPDH were measured by RT-PCR. (D) Protein extracts were analyzed by immunoblotting for the levels of ΔNp73, LMP-1. β-actin was used as loading control.</p

EBV LMP-1 induces up-regulation of ΔNp73α.

<p>(<b>A and B</b>) RPMI-pLXSN (pLXSN) and RPMI-pLXSN-LMP-1 (LMP-1) cells were collected and processed for the preparation of total RNA or protein extract. RNA was retro-trancribed to cDNA and ΔNp73, LMP-1 and GAPDH mRNA levels were measured by RT-PCR (A right panel) or quantitative PCR (A left panel). Protein extracts were analyzed by immunoblotting using the indicated antibodies (B left panel). The ΔNp73α protein signal was quantified by Quantity one (Biorad), normalized on the levels of β-actin, and the values obtained were reported in the histogram (B left panel). The data are the mean of three independent experiments.</p

p73 is recruited to ΔNp73 in promoter LMP-1 expressing cells.

<p>(<b>A</b>) SaOS-2 cells were transfected with different pcDNA3 constructs in the indicated combinations. After 36 hours, ChIP was performed using an anti HA-tag antibody and followed by real-time PCR, using primers flanking the p53-RE within the ΔNp73 promoter. Simultaneously, 1/10 of the total chromatin was processed. The values in the histogram were obtained by dividing for each sample the amount of ΔNp73 promoter, which is bound by p73-HA and p53-HA for the total amount of ΔNp73 promoter present in the input. The reported values are the mean of three independent experiments. (<b>B and C</b>) RPMI-pLXSN (pLXSN) and RPMI-pLXSN-LMP-1 (LMP-1) were fixed and processed for ChIP, or collected to perform DNA-pull down. (B) ChIP was carried out using an anti p73 antibody. The real time PCR and the quantification of the % of p73 binding to the p73-RE within the ΔNp73 promoter, was performed as explained in the legend of <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003186#ppat-1003186-g004" target="_blank">Figure 4A</a>. The data are the mean of three independent experiments. The difference in the p73 levels recruited to ΔNp73α promoter in presence and absence of LMP-1 is statistically significant ((p = 0.000254) (C) After the DNA pull-down assay the levels of p53 and p73 proteins binding to biotinylated DNA probe were analysed by immunoblotting. (<b>D</b>) LCLs were transfected with scrambled RNA and siRNA for p53 (sip53) or transduced with lentivirus carrying two different OmicsLink p73 shRNAs (shp73-1, shp73-2) and scramble shRNA. Thirty-six hours after transfection or one-week post lentivirus transduction cells were collected and processed for RNA extraction and cDNA synthesis. The levels of p53, p73 and ΔNp73α transcript were determined by quantitative RT-PCR. The data are the mean of three independent experiments. The difference in mRNA ΔNp73α levels in scramble or sip53 transfected cells was not significant, while it was statistically significant between scramble and shp73-1 (p value = 0.0022) and scramble and shp73-2 (p value = 0.005).</p

JNK-1 is involved in LMP-1-mediated activation of ΔNp73α expression.

<p>(<b>A</b>) BJAB cells were transfected with JNK-1 expression vector or empty pcDNA3 construct as control. After 36 hours cells were collected and processed for RNA extraction. The levels of ΔNp73, JNK-1 and GAPDH transcripts were determined by quantitative RT-PCR. The data are the mean of three independent experiments. The difference of ΔNp73α mRNA levels in mock cells (BJAB) and BJAB expressing JNK-1 is statistically significant (p value = 0.022). (<b>B</b>) LCLs were treated with JNK-1 inhibitor (SP600125) at 20 µM. After the indicated times cells were collected and processed for RNA extraction. ΔNp73 mRNA levels were determined by quantitative RT-PCR. The data are the mean of three independent experiments. The difference of ΔNp73α mRNA levels in LCLs cultured in presence (7 hours) or absence (DMSO) of JNK-1 inhibitor is statistically significant ((p value = 0.015). (<b>C and D</b>) Scrambled siRNA and siRNA for JNK-1 (siJNK-1) was transfected in LCLs. Thirty-six hours after transfection, cells were collected and processed for RNA or total cells proteins extraction. (C) ΔNp73 and JNK-1 transcript levels were determined by quantitative RT-PCR. The data are the mean of three independent experiments. The difference of JNK-1 or ΔNp73α mRNA levels in LCLs transfected with scramble siRNA or siJNK-1 is statistically significant (p value = 0.008 and p value = 0.0002, respectively). (D) Forty µg of whole cell lysate was analyzed by immunoblotting for the levels of ΔNp73α, JNK-1 or β-actin proteins (left panel). ΔNp73α and JNK-1 protein signal was quantified by Quantity one (Biorad), normalized on the levels of β-actin, and the values obtained were reported in the histogram (right panel). The data are the mean of three independent experiments. The difference of ΔNp73α mRNA levels in LCLs transfected with scramble siRNA or siJNK-1 is statistically significant (p value = 0.01). (<b>E and F</b>) SaOS-2 cells were transfected with different pcDNA3 constructs in the indicated combinations. After 36 hours, cells were collected and fixed for ChIP experiments performed with a HA antibody and followed by quatitative PCR (E) or analysed by immunoblot for the indicated proteins (F). (<b>G</b>) RPMI- EBVΔLMP-1 cells were transduced with pLXSN, pLXSN-LMP-1 wild-type or 378/Stop mutant retroviruses. Cells were selected for neomycin resistance, and then collected for RNA extraction. The levels of ΔNp73α, LMP-1 and EBER2 transcript were determined by RT-PCR.</p

ΔNp73α mRNA levels are high in the skin of HPV38 E6/E7 Tg mice, but are decreased in the UV-induced skin lesions harbouring p53 mutations.

<p>Total RNA was extracted from the skin of WT (<i>n</i> = 4) or K14 HPV38 E6/E7 Tg animals (<i>n</i> = 5) as well as histologically confirmed pre-malignant (pre-m) and SCC from three independent mice and harbouring mutated p53. ΔN73α levels were measured by quantitative RT-PCR. The data shown are the mean of two independent experiments. The differences in ΔN73α mRNA levels between WT and K14 HPV38 E6/E7 Tg animals were statistically significant: * <0.05.</p

Schematic representation of well-known and hypothetical models of virus-associated carcinogenesis.

<p>Schematic representation of well-known and hypothetical models of virus-associated carcinogenesis.</p

Cancer-related genes recurrently mutated in cSCCs of K14 HPV38 E6/E7 Tg mice.

<p>(A) Circos presentation of mutations occurring in the same genes between the different mice. From the centre to the outside, the skin samples (white), the lesion samples (yellow), and the SCC samples (grey) are displayed for <i>n</i> = 3 mice each. Each track (three per colour) corresponds to one animal. Red dots represent C:G > T:A mutations, and black dots represent the other types of mutations. For Circos A, only the mutations that occur in genes present in the Cancer Gene Census list from the COSMIC database are displayed, with the number of recurrent mutations in these genes in parentheses. (B) For the epigenetic drivers/modifiers, only the mutations that occur in the epi-driver or the epi-modifier gene lists are displayed. Blue gene names correspond to genes that are only involved in epigenetic processes, and purple gene names correspond to genes that are involved in epigenetic processes and that are present in the Cancer Gene Census list. The total number of recurrent mutations occurring in each of these genes is also displayed in parentheses.</p