Background
Epstein–Barr virus (EBV) is a human
Herpesviridae that infects about 95% of adults worldwide. Most genes encoded by the viral genome are expressed during the lytic cycle and contribute to the production of viral particles. By contrast, only a restricted repertoire of viral genes is expressed during latency, to allow a lifelong persistence of the virus in the organism. EBV’s lytic cycle takes place in the oropharyngeal epithelium whereas the latent cycle is established in the B lymphocytes from the underlying lymphoid tissues [
1]. Chronic infection in immunocompetent individuals is generally asymptomatic with the virus being maintained in a latent state [
2]. However, inefficient control of viral latency contributes to the development of malignancies such as Burkitt's lymphoma, Hodgkin’s lymphoma and Nasopharyngeal carcinoma.
Several primary immune deficiencies (PID) are associated with poor EBV responses and are also at high risk for EBV-related malignancies [
3]. Ataxia telangiectasia (AT) is a rare PID caused by mutations in the
Ataxia Telangiectasia Mutated (
ATM) gene, involved in the DNA damage response (DDR). AT patients have an increased risk of cancer, mostly B-cell lymphoid malignancies, many of which are related to EBV [
4]. The prevailing hypothesis to explain the increased incidence of malignancies in patients with AT is based on the role of the ATM kinase in the DDR [
5]. However, the strong association of lymphomas with EBV also suggests an oncogenic role of the latter. AT patients often present with antibody deficiency and T-cell lymphopenia but rarely overt immunodeficiency [
6]. A number of PIDs exhibit a selective susceptibility to EBV-related malignancies, while displaying a more restricted susceptibility to other opportunistic infections. In such cases, specific mechanisms may include pathways important for T, NK and iNKT cytotoxicity aimed at EBV-infected B-cells, and pathways involved in expansion of EBV-specific T-cells, leading to an inability to cope with intense EBV induced proliferative stress like in XMEN or CTPS1 mutated patients [
7]. We raised the hypothesis that the lack of ATM function in AT patients may be associated with a less stringent control of EBV latency in ATM-deficient B cells, thereby promoting the oncogenic properties of the virus.
Indeed, beside DNA repair, ATM is also involved in a multitude of signaling pathways such as cell cycle checkpoint, apoptosis, mitochondrial metabolism and telomere maintenance [
8]. In addition, ATM is involved both in transcription induction [
9], and in transcription inhibition in the vicinity of a double-strand break, in nuclear [
10] or ribosomal DNA [
11]. ATM has a role in the control of the latent cycle of Kaposi’s sarcoma herpesvirus (KSHV) and Murine γ-herpesvirus 68 (MHV68), both related to EBV [
12,
13].
We performed RNA sequencing (RNA-seq) on lymphoblastoid cell lines (LCL) generated from AT patients and healthy donors, to explore the specific expression pattern of both the cellular and viral genomes and investigate a possible role of ATM in the regulation of EBV latent cycle.
Discussion
AT patients have a high risk of developing lymphoid malignancies with a high rate of association with EBV. Mechanisms associated with this specific susceptibility may be due cellular immune deficiency in a number of AT patients, or to other specific immune dysfunctions that remain to be explored. We raised the hypothesis that ATM defect in EBV-infected cells could play a role per se in the control of EBV latency, favoring a latent program more prone to lymphomagenesis [
19]. In the present study, we used strand-specific RNA seq strategy to profile the RNA expression landscape of ATM deficient LCL versus control, in order to assess the involvement of ATM in EBV latent cycle regulation.
Our data suggests a previously unsuspected ribosomal defect in LCL-AT. In addition, we found that LCL-AT display a distinct pattern of cancer associated gene expression, most notably by overexpressing certain oncogenes and downregulation of tumor suppressors, and also exhibit features of immune dysfunction. We also confirmed that latent gene expression can be studied regardless of lytic gene expression. Our data, based on the single technique of RNA-seq analysis, will require validation by additional studies.
Transcription, translation and mitochondria
Our results suggest that LCL-AT may have a transcriptional and translational defect. Indeed LCL-AT express less 28S RNA and have a lower translational rate than LCL-WT, but transcription capacities did not differ significantly from LCL-WT. The possible transcriptional defect does not appear to affect EBV latency genes, as observed by the absence of DE latency genes between LCL-AT and LCL-WT. Housekeeping genes were not DE, suggesting that transcriptional alterations may affect specific genes such as ribosomal genes. Interestingly, the signaling pathways for transcription and translation were not found in the EGSEA results but stand out significantly with a lower log.fold.dir. This underscores the need for several complementary methods to study RNA-seq data.
The alteration of 28S RNA and translational rate in LCL-AT suggest that the pathophysiology of ataxia-telangiectasia may also include aspects of ribosomal disease. ATM participates in the modification of the nucleolus architecture in case of double-strand break within rDNA [
20] and It has been suggested that ATM participates in basal nucleolar transcription [
21]. Other immunodeficiencies with specific susceptibility to EBV—such as
CTPS1 deficiency—are characterized by altered nucleic acid metabolism leading to rapid T-cell exhaustion upon massive proliferation induced by EBV infection [
22]. We hypothesize that the massive protein synthesis rate in cytotoxic T-cells during EBV-driven proliferative stress is inefficiently sustained in ataxia-telangiectasia, resulting in a defective control of EBV. Further studies are needed to address this hypothesis. Transcription of many mitochondrial genes were decreased in LCL-AT including several genes involved in the respiratory chain and in ribosomal protein synthesis. Inhibition of ATM leads to mitochondrial dysfunction and ROS production [
23]. The latter could be involved in the increased incidence of cancers in patients with ataxia-telangiectasia by increasing genotoxic stress.
Oncogenesis and immune dysfunction
The EGSEA results show enrichment in pro-tumorigenic GS particularly oncogenes, growth factors and downregulation of tumor suppressors in keeping with the increased cancer risk in AT [
24]. Among the main oncogenes induced in LCL-AT, we find BCL11A (log2FC 4.20), a modulator of transcriptional repression frequently upregulated in B-cell malignancies [
25,
26] or TCL1A (log2FC 3.41), a survival promoting factor strongly associated with Burkitt lymphoma and related to other malignancies [
27,
28]. The main tumor suppressors downregulated in AT are PCDH10 (log2FC − 4.76), a protocadherin whose promoter is methylated in diffuse large B-cell lymphomas [
29] or PTPN13 (log FC: − 2.84) an inhibitor of FAS-induced apoptosis associated with aggressive breast cancer [
30].
Telomere maintenance pathway, including
TERT, was downregulated in LCL-AT, (log2FC − 4.78). Inhibition of TERT in LCL decreases cell proliferation and induces apoptosis in an ATM dependent manner [
31] as well as induces the EBV lytic cycle, which was not the case in LCL-AT (data not shown). LCL-AT may use an alternative lengthening of telomeres pathway [
32]. Whether TERT related induction of the EBV lytic cycle is also ATM dependent should be further explored.
A modulation of innate immunity in LCL-AT is suggested by several DE-GS. The gene expression of HLA-C, a major NK cell inhibitory molecule, is upregulated in LCL-AT (log2FC 5.63). Similarly, CD200R1, CD276, SLAMF7, LILRB1 were overexpressed, suggesting that AT patients may have a disrupted NK cell function. On the other hand, LAIR1, another inhibitory molecule, was downregulated (log2FC − 4.99).
IL4 and IL10 were also upregulated in LCL-AT (log2FC 2.23 and 3.58, respectively). These two cytokines participate in the proliferation, plasma cell differentiation and antibody production of B lymphocytes [
33,
34]. IL10 also inhibits CD8 cytotoxic T-cells [
35]. The cGAS, STING (TMEM173) [
36] and interferon β1 (IFNB1) transcripts were downregulated (log2FC − 0.70; − 2.39 and − 2.51 respectively), suggesting a possible defect in antiviral response in LCL-AT.
EBV regulation
We found no significant difference in EBV latent gene expression between LCL-AT and LCL-WT. Several deregulated cellular genes in LCL-AT interact with EBNA-3A, EBNA-3C and LMP1 suggesting an overall differential impact on cellular homeostasis.
ATM participates in the regulation of EBV’s lytic cycle and is necessary for a proper viral replication in epithelial cells [
37]. In LCL however, ATM inhibition through caffeine treatment [
38] or the lack of ATM in our LCL-AT did not affect viral replication. On the other hand, LCL treated by the pan-PIKK inhibitor (to which the ATM kinase belongs) LY294002 were shown to inhibit viral replication. It is thus possible that another kinase compensates for ATM deficiency in LCL, to promote viral replication. ATR is a good candidate as it activates the same downstream targets as ATM.
However, the impact of ATM on the control of EBV latency may not be manifested in a highly artificial system such as LCL, but appear in the context of natural EBV-B cell infection.
Conclusion
In summary, we show that LCL-AT display a gene expression pattern consistent with the observed increased incidence of EBV-related malignancies in patients with Ataxia-Telangiectasia. The dysregulated pathways uncovered by this approach need to be further explored to better understand the biological mechanisms involved in the regulation of EBV latency and lymphomagenesis. Elucidation of these pathways may contribute to the development of novel approaches to treat or prevent EBV associated lymphoproliferations in AT patients where conventional chemotherapy is very toxic because of the DDR defect, and also in the general population.
Methods
Cell lines and culture
LCL were generated according to standard protocols at the Genethon Laboratory and at the Imagine Institute Biological Resource Center. LCL were cultured in RPMI containing 10% FBS at 37 °C in a 5% CO2 incubator and passed every 3 days replacing half of the culture medium with fresh medium.
RNA extraction and sequencing
LCL-WT and LCL-AT were harvested in an exponential growth phase. Total RNA was extracted using the RNeasy Plus Mini Kit (Qiagen). The concentration of total RNA was measured spectrometrically using Xpose (Trinean). The RNA integrity was analyzed by capillary electrophoresis using a Tape-Station (Agilent). The NUGEN Ovation Universal RNA-Seq system was used to construct the RNA-seq libraries from 100 ng of total RNA according to the manufacturer protocol. RNA sequencing was performed by the genomics platform of Imagine Institute on HiSeq 2500 (Illumina), by multiplexing 12 libraries per line to obtain a sequencing depth of 70 million pair-end reads per library, with a read length of approximately 130 nucleotides.
Reads quantification and differential analysis
Salmon v0.8.2 [
39] was used to pseudo-align raw RNA-seq reads to both human and viral genomes and get quantification estimates at the transcript level. The human reference genome GRCh38 was downloaded from the ENSEMBL website [
40] (
http://ftp.ensembl.org/pub/release-92/fasta/homo_sapiens/cdna), and the EBV genome B95-8/Raji from Flemington Lab (
http://www.flemingtonlab.com/rnaseq.html). Differential analysis was performed using R software, version 3.5.0 [
41] and the negative binomial generalized linear modelling implemented in DESeq2 package version 1.20.0 [
42]. A Wald test was applied on viral transcripts to perform comparisons among conditions.
p Values were corrected for multiple testing using Benjamini–Hochberg correction [
43]. The BioMart R package [
44] was used to annotate human differentially expressed genes.
Genome visualization
Reads were mapped on the human genome (hg38) using STAR v2.5.0a [
45] and the unmapped reads were aligned to the EBV genome using bowtie2 v2.3.4.2 [
46]. The format conversions, sorting, and indexing intermediate operations on the data were performed using samtools v1.7 [
47] and bedtools v2.26 [
48]. The snapshots obtained in Fig.
4a and Additional file
7: Fig S4 were obtained with IGV v2.4.15 [
49].
Gene ontology and gene sets analysis
Differentially expressed genes were subjected to GO-Analysis, using the over-representation test within the PANTHER classification system [
50] (version 13.1). Statistically over-represented GO-categories (FDR < 0.05) containing less than 25% of the input were selected for visualization. Functional analysis was carried out using the Ensemble of Gene Set Enrichment Analyses (EGSEA) R package (version 1.8.0). EGSEA uses 12 algorithms and combine the results by calculating a Wilkinson adjusted p-value. The EGSEA database consists of approximately 25.000 genes-sets classified into 16 collections from the MSigDB [
51,
52], GeneSetDB [
53] and KEGG [
54] databases.
Transcription and translation assay
Cells were incubated with 5-ethynyl uridine (EU) on 1 h or with O-Propargyl-puromycin (OPP) on 30 min. Transcription and translation rate were assessed using the Click-it assay Kit (Invitrogen), followed by flow-cytometer analysis. Statistical comparison between LCL-AT and LCL-WT were performed using a Mann–Whitney–Wilcoxon non-parametric test, using Prism (version 7.00, GraphPad Software).
1000 Genome project and EBV interactome data
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