Journal of Medical Microbiology (2016), 65, 1243-1252 DOI 10.1099/jmm.0.000352

Analysis of Epstein-Barr virus and cellular gene expression during the early phases of Epstein-Barr virus lytic induction.

Helen Auburn, Mark Zuckerman and Melvyn Smith

Department of Virology, South London Specialist Virology Centre, King’s College NHS Foundation

Trust, Denmark Hill, London SE5 9RS, UK



In order to develop novel host/pathogen real-time PCR assays for routine diagnostic use, early gene expression patterns from both Epstein-Barr virus (EBV) and Raji cells were examined after inducing the lytic life cycle using 12-O-tetradecanoyl-13-phorbol ester and sodium butyrate. Real-time PCR identified several highly induced (>90-fold) EBV lytic genes over a 48hr time course. All latent genes were also expressed, although at lower levels. Human genome microarray analysis identified 113 cellular genes regulated by EBV, including 63 upregulated and 46 downregulated genes over a 24hr time course post-induction. Gene Ontology enrichment analysis revealed cell cycle (core), cell cycle (role of anaphase-promoting complex in cell cycle regulation) and lymphatic diseases as the most significantly represented network processes, canonical pathways and disease biomarkers, respectively. Chemotaxis, DNA damage and inflammation (IL-4 signalling) together with lymphoproliferative disorders and non-Hodgkin’s lymphoma were significantly represented biological processes and disease biomarkers. Copyright © 2016 The Authors


EBV natural history:

EBV infection is widespread, with over 90% of the adult population affected worldwide. Primary EBV infection occurs during childhood when the virus can establish a lifelong, latent, infection in the memory B-cell pool [1]. Reactivation of the latent virus to a lytic infection occurs via a cascade of activation events involving immediate early, early and lytic gene expression. The lytic life cycle is associated with a number of malignancies, including Burkitt’s lymphoma, nasopharyngeal carcinoma, Hodgkin’s lymphoma, gastric carcinoma, and in immunosuppressed individuals, AIDS-associated lymphomas and post-transplant lymphoproliferative disease (PTLD) [2].


EBV can transform normal resting B cells in-vitro, driving them into immortalized lymphoblastoid cell lines (LCLs) [3]. In LCLs, a limited set of EBV latent transcripts are expressed, including six EBV nuclear antigens (EBNA-1, -2, -3A, -3B, -3C and -LP), three latent membrane proteins (LMP-1, LMP-2A and LMP-2B), two small non-coding RNAs (EBER-1 and -2) and 25 pre-miRNAs [4, 5]. This expression profile is defined as latency III and drives the indefinite proliferation of primary B cells and maintains the viral genome [5]. EBV reactivation in LCLs can be induced using various reagents, including phorbol esters such as 12-O-tetradecanoyl-13-phorbol ester (TPA) and histone deacetylases, such as sodium butyrate (NaB).


Methods: Raji cells maintain a stable copy number of EBV episomes and due to the deletion of three genes (BALF1, BARF1 and BZLF2) and two truncations (BALF2 and EBNA-3C), prevent lytic replication. This minimises the potential effects of late gene expression during the induction experiments. Additionally, Raji cells exhibit latency type III, similar to that seen in PTLD. The EBV-negative cell line Ramos was used as a control. SBYR Green-based real-time RT-PCRs using were used to analyse EBV gene expression at 0, 2, 6, 24 and 48hrs post induction (PI) on RNA isolated from duplicate cultures of TPA/NaB-induced and uninduced Raji and Ramos cell lines. Cellular gene expression patterns were analysed using Affymetrix Human Genome U133 Plus 2.0 GeneChips for the 24hr time point only.



EBV lytic and latent gene expression: Induction levels at the early time points were less than 2-fold; therefore, analyses were carried out only on samples from the 24 and 48hr inductions.


Both the immediate early lytic genes, BRLF1 (60.3-fold) and BZLF1 (156.0-fold), showed increased expression at 48hrs post induction (PI). A similar increase at 48hrs was also found for the early lytic genes, where 67% (22/33) showed higher fold changes at 48hrs and for the late lytic genes, where 93% (39/42) showed higher expression at 48hrs PI. The latent genes were all expressed, with the exception of the deleted EBNA-3c, although generally at lower levels than the lytic genes (ranging from 0.2-fold for EBNA-LP and 15.3-fold for EBNA-3A at 48hrs PI), with 60% (6/10) expressed more highly 48hrs PI.


Cellular gene expression in the lytic induction phase: A total of 3884 differentially expressed probe sets were identified from the two Raji cell replicates at 24hrs PI. Of these 191 were commonly modulated in both biological replicates. To identify which of the 191 probe sets were specific to EBV replication and not in response to the inducing reagents, the overlap of the 191 probe sets with the 199 most highly differentially expressed probe sets from induced EBV-negative Ramos cells was evaluated. From this, 58 probe sets were identified as commonly modulated in both Raji and Ramos cells, leaving 133 representing 113 genes specifically regulated in Raji cells. Of the 113 genes regulated specifically by EBV lytic infection, 64 genes were upregulated (up to 18.1-fold), the remaining 49 genes were downregulated (down to -7.6-fold) at 24hrs PI. The most upregulated cellular gene was CHI3L1, a chitinase (18.1-fold) and the most downregulated was TYMS, a thymidylate synthetase (-7.6-fold). The median fold changes of the upregulated and downregulated genes were 5.0 (range, 2.1-18.1) and -3.6 (range, -2.1 to -7.6), respectively. 



Latent genes predominate in EBV-related cancers. Although reactivation from latent EBV infection during immunosuppression is thought to contribute to the development of PTLD, through an increase in viral replication, the exact role of the EBV lytic genes in oncogenesis is unclear [6].


Eight early EBV genes were more highly induced at 24hrs PI compared with the 48hrs, six of which are involved directly in viral DNA replication, with four (BALF5, BBLF2, BBLF3 and BBLF4) forming part of the replication initiation complex [7]. Six genes (BALF2, BALF3, BALF5, BBLF2, BBLF3 and BBLF4) also contain methylated sites in their promoters [8]. The EBV BZLF1 IE protein, Zta, binds preferentially to methylated sites within promoter regions to activate the lytic cycle cascade ([8] and the early, high-level expression of these six genes accords with this function. Two genes (BcRF1 and BGLF4) were induced over 100-fold. BcRF1 encodes a TATA box-binding like protein essential for activation of late viral gene expression [9]. BGLF4, a serine/threonine protein kinase, is localized in the virus replication compartment, acting by phosphorylating BMRF1, a DNA polymerase processivity factor, essential for lytic replication. In addition, BFRF1, BFRF2, BMRF1 and BXLF1 were also highly induced. Although not directly involved in lytic replication, BFRF1 plays a role in transporting mature virions across the nuclear membrane [10]. BFRF2 encodes a viral tegument protein but appears to be present only at a transcript level [11], while BXLF1 encodes a viral thymidine kinase; however, their exact roles are not fully understood.


Whole-genome microarray analysis revealed the altered expression of 113 cellular genes more than twofold PI. The most upregulated was CHI3L1 (18.1-fold), a chitinase-3-like 1 protein, implicated in several cancers. The most downregulated cellular gene was TYMS (-7.6-fold), a thymidylate synthetase and a key enzyme for pyrimidine biosynthesis and essential for DNA synthesis. Low levels of its expression may cause an imbalance of deoxynucleotides, leading to increased levels of deoxyuridine monophosphate, resulting in DNA damage. A number of genes were upregulated, with functions relevant to viral infections, such as apoptosis, inactivation of MAPK activity and the type 1 interferon signalling in antiviral response. These include SQSTM1 (12.4-fold); OAS1 (5.6-fold); OASL (9.5-fold) and PHLDA (13.1-fold). Of interest were two of the downregulated genes, (IGJ; -2.6-fold & RGS13; -5.7-fold) that have direct functions relating to immune response and cell signalling. A series of chemokine genes previously shown to be regulated by the latent genes LMP-1, EBNA-2 and EBNA-3A were found, these include CCL3 (3.7-fold), CCL5 (9.6-fold), CCL22 (4.4-fold), CXCL9 (10.2-fold) and CXCL10 (4.0-fold).


A number of differentially expressed genes, previously shown to be regulated more than twofold in microarray analyses of monomorphic PTLD tumour biopsies were found. These include ANP32E, CDC2, CD52, HDGF1, HMGB1, LRMP, MAD2L1, PCNA and TIMP1. The products of these genes are involved in processes relevant to PTLD progression such as anti-apoptosis; (ANP32E) [14]; cell growth and apoptosis; (CDC2, CD52, MAD2L1 and LRMP) [15-19]; apoptosis, cell growth, cell invasion and apoptosis; (HDGF1) [19]; regulation of transcription/antitumour immunity; (HMGB1) [20]; cell proliferation; (PCNA) [21] and cell proliferation, apoptosis, angiogenesis and cellular signalling; (TIMP1) [22].


Gene ontology (GO) analysis showed the functions of the differentially expressed genes ranged from cell cycle, chemotaxis, DNA damage and inflammation (IL-4 signalling), with the cell cycle as the most significantly represented canonical pathway. Although EBV is known to use host cell genes to control pathways involved in cycle checkpoints and DNA repair, their regulation during EBV lytic replication is not well understood [23]. It is likely that interleukin signalling, chemotaxis and inflammation are regulated in response to virally induced chemokine genes. We found BZLF1 was upregulated 19.4-fold, and BGLF4 was upregulated 114.2-fold at 24hrs PI. The BZLF1 product, Zta, is postulated to cause cell growth arrest at the G1/S boundary, similar to that seen in both cytomegalovirus infection [24] and herpes simplex virus 1 infection [25]. The BGLF4 product, a proline-dependent serine/threonine protein kinase also interferes with DNA replication and S-phase progression by mimicking cellular Cdk activities [26]. It was shown that BGLF4 prevents cells from entering G2/M phase by inducing highly condensed chromosomal structures, preventing cell cycle progression and facilitating viral DNA replication [26]. In fact, chromosome separation and chromosome condensation were highly represented in cell cycle canonical pathways.


The DNA damage response is also elicited during EBV lytic replication by activation of the ataxia telangiectasia-mutated (ATM) checkpoint signalling pathway and shown to be necessary for virus replication [27]. Genes enriched for disease biomarkers were analysed to identify altered expression patterns during lytic reactivation. Despite the uncertain role of lytic gene expression in oncogenesis, lymphatic diseases were the most highly represented. Lymphoproliferative disorders (LPDs) and non-Hodgkin lymphomas, both EBV-associated, were significantly represented. Levels of BZLF1 and BRLF1 were both upregulated in our experiments, 19.4-fold and 156.0-fold, respectively at 24 h and 3.7-fold and 60.3-fold, respectively at 48hrs PI. These results suggest that lytic gene expression may contribute to EBV-associated lymphoproliferative disease, potentially through induction of paracrine B-cell growth factors.


In summary, real-time PCR analysis of EBV gene expression identified several highly induced EBV early lytic genes. Microarray analysis revealed 113 cellular genes regulated by EBV infection. These altered patterns of expression during the lytic induction phase may also be reflected in vivo and, therefore, play a role in the development of EBV-associated malignancies and could be exploited as potential diagnostic markers.


Based on fold change data, we are actively assessing a range of host and EBV targets derived from this work using sequential samples from post-transplant recipients collected over the past 12 months. By using real-time RT-PCR assays to analyse both host and EBV gene expression levels we hope to further investigate the development and develop diagnostic assays capable of the early prediction of EBV-related disease in this setting.



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