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Abstract Background There are two major genetic types of Epstein-Barr Virus (EBV): type 1 (EBV-1) and type 2 (EBV-2). EBV functions by manipulating gene expression in host B cells, using virus-encoded gene regulatory proteins including Epstein-Barr Nuclear Antigen 2 (EBNA2). While type 1 EBNA2 is known to interact with human transcription factors (hTFs) such as RBPJ, EBF1, and SPI1 (PU.1), type 2 EBNA2 shares only ~ 50% amino acid identity with type 1 and thus may have distinct binding partners, human genome binding locations, and functions. Results In this study, we examined genome-wide EBNA2 binding in EBV-1 and EBV-2 transformed human B cells to identify shared and unique EBNA2 interactions with the human genome, revealing thousands of type-specific EBNA2 ChIP-seq peaks. Computational predictions based on hTF motifs and subsequent ChIP-seq experiments revealed that both type 1 and 2 EBNA2 co-occupy the genome with SPI1 and AP-1 (BATF and JUNB) hTFs. However, type 1 EBNA2 showed preferential co-occupancy with EBF1, and type 2 EBNA2 preferred RBPJ. These differences in hTF co-occupancy revealed possible mechanisms underlying type-specific gene expression of known EBNA2 human target genes: MYC (shared), CXCR7 (type 1 specific), and CD21 (type 2 specific). Both type 1 and 2 EBNA2 binding events were enriched at systemic lupus erythematosus (SLE) and multiple sclerosis (MS) risk loci, while primary biliary cholangitis (PBC) risk loci were specifically enriched for type 2 peaks. Conclusions This study reveals extensive type-specific EBNA2 interactions with the human genome, possible differences in EBNA2 interaction partners, and a possible new role for type 2 EBNA2 in autoimmune disorders. Our results highlight the importance of considering EBV type in the control of human gene expression and disease-related investigations.

Background: There are two major genetic types of Epstein-Barr Virus (EBV): type 1 (EBV-1) and type 2 (EBV-2). EBV functions by manipulating gene expression in host B cells, using virus-encoded gene regulatory proteins including Epstein-Barr Nuclear Antigen 2 (EBNA2). While type 1 EBNA2 is known to interact with human transcription factors (hTFs) such as RBPJ, EBF1, and SPI1 (PU.1), type 2 EBNA2 shares only ~ 50% amino acid identity with type 1 and thus may have distinct binding partners, human genome binding locations, and functions. Results: In this study, we examined genome-wide EBNA2 binding in EBV-1 and EBV-2 transformed human B cells to identify shared and unique EBNA2 interactions with the human genome, revealing thousands of type-specific EBNA2 ChIP-seq peaks. Computational predictions based on hTF motifs and subsequent ChIP-seq experiments revealed that both type 1 and 2 EBNA2 co-occupy the genome with SPI1 and AP-1 (BATF and JUNB) hTFs. However, type 1 EBNA2 showed preferential co-occupancy with EBF1, and type 2 EBNA2 preferred RBPJ. These differences in hTF co-occupancy revealed possible mechanisms underlying type-specific gene expression of known EBNA2 human target genes: MYC (shared), CXCR7 (type 1 specific), and CD21 (type 2 specific). Both type 1 and 2 EBNA2 binding events were enriched at systemic lupus erythematosus (SLE) and multiple sclerosis (MS) risk loci, while primary biliary cholangitis (PBC) risk loci were specifically enriched for type 2 peaks. Conclusions: This study reveals extensive type-specific EBNA2 interactions with the human genome, possible differences in EBNA2 interaction partners, and a possible new role for type 2 EBNA2 in autoimmune disorders. Our results highlight the importance of considering EBV type in the control of human gene expression and disease-related investigations. [ABSTRACT FROM AUTHOR]

Epstein-Barr virus (EBV) contributes to the development of a significant subset of human lymphomas. As a herpes virus, EBV can transition between a lytic state which is required to establish infection and a latent state where a limited number of viral antigens are expressed which allows infected cells to escape immune surveillance. Three broad latency programs have been described which are defined by the expression of viral proteins RNA, with latency I being the most restrictive expressing only EBV nuclear antigen 1 (EBNA1) and EBV-encoded small RNAs (EBERs) and latency III expressing the full panel of latent viral genes including the latent membrane proteins 1 and 2 (LMP1/2), and EBNA 2, 3, and leader protein (LP) which induce a robust T-cell response. The therapeutic use of EBV-specific T-cells has advanced the treatment of EBV-associated lymphoma, however this approach is only effective against EBV-associated lymphomas that express the latency II or III program. Latency I tumors such as Burkitt lymphoma (BL) and a subset of diffuse large B-cell lymphomas (DLBCL) evade the host immune response to EBV and are resistant to EBV-specific T-cell therapies. Thus, strategies for inducing a switch from the latency I to the latency II or III program in EBV+ tumors are being investigated as mechanisms to sensitize tumors to Tcell mediated killing. Here, we review what is known about the establishment and regulation of latency in EBV infected B-cells, the role of EBV-specific T-cells in lymphoma, and strategies to convert latency I tumors to latency II/III. [ABSTRACT FROM AUTHOR]

Background: Epstein–Barr virus (EBV) is a human lymphotropic herpesvirus with a causative agent in cancer. There are two genotypes of EBV (EBV genotype 1 and EBV genotype 2) that have been shown to infect humans. This study aimed to characterize the EBV genotype among people with human immunodeficiency virus (PWH) and HIV-negative individuals in Ethiopia. Methods: DNA was extracted from peripheral blood mononuclear cells (PBMCs). Conventional polymerase chain reaction (cPCR) targeting EBNA3C genes was performed for genotyping. A quantitative real-time PCR (q-PCR) assay for EBV DNA (EBNA1 ORF) detection and viral load quantification was performed. Statistical significance was determined at a value of p  <  0.05. Result: In this study, 155 EBV-seropositive individuals were enrolled, including 128 PWH and 27 HIV-negative individuals. Among PWH, EBV genotype 1 was the most prevalent (105/128, 82.0%) genotype, followed by EBV genotype 2 (17/128, 13.3%), and mixed infection (6/128, 4.7%). In PWH, the median log10 of EBV viral load was 4.23 copies/ml [interquartile range (IQR): 3.76–4.46], whereas it was 3.84 copies/ ml (IQR: 3.74–4.02) in the HIV-negative group. The EBV viral load in PWH was significantly higher than that in HIV-negative individuals (value of p  =  0.004). In PWH, the median log10 of EBV viral load was 4.25 copies/ml (IQR: 3.83–4.47) in EBV genotype 1 and higher than EBV genotype 2 and mixed infection (p  =  0.032). Conclusion: In Ethiopia, EBV genotype 1 was found to be the most predominant genotype, followed by EBV genotype 2. Understanding the genotype characterization of EBV in PWH is essential for developing new and innovative strategies for preventing and treating EBV-related complications in this population. [ABSTRACT FROM AUTHOR]

Aim: To investigate the correlation between Epstein–Barr virus (EBV) variants and EBV-associated gastric cancer (EBVaGC) in Iran. Materials & methods: Biopsies from 21 gastric cancer (4 positive EBER-ISH [EBVaGC] and 17 negative EBER-ISH) and 11 gastritis patients were analyzed using nested PCR and sequencing. Results: Genotype 1 (75%) was the predominant EBV genotype. The P-thr variant was the most common in all groups (positive EBER-ISH 75%, negative EBER-ISH 76.4%, and gastritis 81.8%), followed by P-ala. Additionally, the predominant LMP1 strain in all groups was Med. Sequencing analysis revealed a strong correlation between the P-thr and type 1. Conclusion: Since no preferential correlations were observed between distinct EBV variants and the study groups, these results might indicate geographic polymorphisms. [ABSTRACT FROM AUTHOR]

Epstein-Barr virus (EBV) contributes to the development of a significant subset of human lymphomas. As a herpes virus, EBV can transition between a lytic state which is required to establish infection and a latent state where a limited number of viral antigens are expressed which allows infected cells to escape immune surveillance. Three broad latency programs have been described which are defined by the expression of viral proteins RNA, with latency I being the most restrictive expressing only EBV nuclear antigen 1 (EBNA1) and EBV-encoded small RNAs (EBERs) and latency III expressing the full panel of latent viral genes including the latent membrane proteins 1 and 2 (LMP1/2), and EBNA 2, 3, and leader protein (LP) which induce a robust T-cell response. The therapeutic use of EBV-specific T-cells has advanced the treatment of EBV-associated lymphoma, however this approach is only effective against EBV-associated lymphomas that express the latency II or III program. Latency I tumors such as Burkitt lymphoma (BL) and a subset of diffuse large B-cell lymphomas (DLBCL) evade the host immune response to EBV and are resistant to EBV-specific T-cell therapies. Thus, strategies for inducing a switch from the latency I to the latency II or III program in EBV+ tumors are being investigated as mechanisms to sensitize tumors to T-cell mediated killing. Here, we review what is known about the establishment and regulation of latency in EBV infected B-cells, the role of EBV-specific T-cells in lymphoma, and strategies to convert latency I tumors to latency II/III.

BackgroundEpstein–Barr virus (EBV) is a human lymphotropic herpesvirus with a causative agent in cancer. There are two genotypes of EBV (EBV genotype 1 and EBV genotype 2) that have been shown to infect humans. This study aimed to characterize the EBV genotype among people with human immunodeficiency virus (PWH) and HIV-negative individuals in Ethiopia.MethodsDNA was extracted from peripheral blood mononuclear cells (PBMCs). Conventional polymerase chain reaction (cPCR) targeting EBNA3C genes was performed for genotyping. A quantitative real-time PCR (q-PCR) assay for EBV DNA (EBNA1 ORF) detection and viral load quantification was performed. Statistical significance was determined at a value of p

Multiple sclerosis (MS) is a chronic neurodegenerative autoimmune disease, characterised by the demyelination of neurons in the central nervous system. Whilst it is unclear what precisely leads to MS, it is believed that genetic predisposition combined with environmental factors plays a pivotal role. It is estimated that close to half the disease risk is determined by genetic factors. However, the risk of developing MS cannot be attributed to genetic factors alone, and environmental factors are likely to play a significant role by themselves or in concert with host genetics. Epstein–Barr virus (EBV) infection is the strongest known environmental risk factor for MS. There has been increasing evidence that leaves little doubt that EBV is necessary, but not sufficient, for developing MS. One plausible explanation is EBV may alter the host immune response in the presence of MS risk alleles and this contributes to the pathogenesis of MS. In this review, we discuss recent findings regarding how EBV infection may contribute to MS pathogenesis via interactions with genetic risk loci and discuss possible therapeutic interventions. [ABSTRACT FROM AUTHOR]

Abstract Multiple sclerosis (MS) is a chronic neurodegenerative autoimmune disease, characterised by the demyelination of neurons in the central nervous system. Whilst it is unclear what precisely leads to MS, it is believed that genetic predisposition combined with environmental factors plays a pivotal role. It is estimated that close to half the disease risk is determined by genetic factors. However, the risk of developing MS cannot be attributed to genetic factors alone, and environmental factors are likely to play a significant role by themselves or in concert with host genetics. Epstein–Barr virus (EBV) infection is the strongest known environmental risk factor for MS. There has been increasing evidence that leaves little doubt that EBV is necessary, but not sufficient, for developing MS. One plausible explanation is EBV may alter the host immune response in the presence of MS risk alleles and this contributes to the pathogenesis of MS. In this review, we discuss recent findings regarding how EBV infection may contribute to MS pathogenesis via interactions with genetic risk loci and discuss possible therapeutic interventions.

Approximately 90% of the world's population is infected with the Epstein–Barr virus (EBV). Damage to health and high economical costs require the development of vaccines against EBV. The variability of individual genes may affect the success of the vaccination campaign and will necessitate dynamic update of antigenic composition of vaccines. The aim of this study was to assess the variability of EBV gp350 and EBNA2 genes isolated from saliva of dental clinic personnel in Moscow oblast. Biosamples were obtained from 105 employees at four dental clinics. For EBV DNA-positive samples, the EBNA2 and gp350 genes were amplified using nested PCR. The nucleotide sequence of gp350 gene was determined during sequencing. A maximum likelihood phylogenetic tree was constructed from the N-terminal fragment of gp350 protein, using B95-8 strain as a reference genome. Phylogenetic analysis also included 222 EBV samples from the NCBI database. The EBNA2 genotype and the whole gp350 gene sequence were determined for 31 DNA samples. Based on the phylogenetic analysis of gp350 protein, the Russian virus population was uniformly distributed along the tree. Meanwhile, 30 samples fell into the Aa clade (A, genotype A for EBNA2 gene; a, similarity of the gp350 sequence with B95-8 [NC_007605.1]) and one sample belonged to the Bb clade (B, genotype B for EBNA2; b , similarity of gp350 with Jijoye [LN827800.1]). For 30 Russian samples of Aa genotype, 22 individual profiles and 16 unique mutations were found. Identical gp350 profiles were found amongst staff working in close professional contact. The identified features indicated the need for further phylogenetic studies of samples collected in Russia to develop and introduce vaccines and monitor their efficiency. [ABSTRACT FROM AUTHOR]

Abstract Introduction N6‐methyladenosine (m6A) is the most prevalent modification that occurs in messenger RNA (mRNA), affecting mRNA splicing, translation, and stability. This modification is reversible, and its related biological functions are mediated by “writers,” “erasers,” and “readers.” The field of viral epitranscriptomics and the role of m6A modification in virus–host interaction have attracted much attention recently. When Epstein–Barr virus (EBV) infects a human B lymphocyte, it goes through three phases: the pre‐latent phase, latent phase, and lytic phase. Little is known about the viral and cellular m6A epitranscriptomes in EBV infection, especially in the pre‐latent phase during de novo infection. Methods Methylated RNA immunoprecipitation sequencing (MeRIP‐seq) and MeRIP‐RT‐qPCR were used to determine the m6A‐modified transcripts during de novo EBV infection. RIP assay was used to confirm the binding of EBNA2 and m6A readers. Quantitative reverse‐transcription polymerase chain reaction (RT‐qPCR) and Western blot analysis were performed to test the effect of m6A on the host and viral gene expression. Results Here, we provided mechanistic insights by examining the viral and cellular m6A epitranscriptomes during de novo EBV infection, which is in the pre‐latent phase. EBV EBNA2 and BHRF1 were highly m6A‐modified upon EBV infection. Knockdown of METTL3 (a “writer”) decreased EBNA2 expression levels. The emergent m6A modifications induced by EBV infection preferentially distributed in 3ʹ untranslated regions of cellular transcripts, while the lost m6A modifications induced by EBV infection preferentially distributed in coding sequence regions of mRNAs. EBV infection could influence the host cellular m6A epitranscriptome. Conclusions These results reveal the critical role of m6A modification in the process of de novo EBV infection.

Epstein-Barr virus (EBV) is an etiologic agent of infectious mononucleosis and several malignancies. Here, we found that reactivation of EBV resulted in increased programmed cell death-ligand 1 (PD-L1) expression in a cell type-dependent manner. Lytic induction in EBV-positive Akata, AGS, MutuI, and Jijoye cell lines increased PD-L1 levels, but cells such as EBV-negative Akata, MutuIII, and P3HR1 did not have increased PD-L1. EBV in the P3HR1 cell line has a deletion in the EBNA2 gene, while EBV in its parental cell line, Jijoye, has the complete EBNA2 gene. PD-L1 expression by lytic induction was reduced when EBNA2 was knocked down. In addition, pharmacological inhibition indicated involvement of nuclear factor kappa B, mitogen-activated protein kinase, and AKT signaling. These results suggest that EBV likely evades immunity by inducing PD-L1 upon reactivation, through the increased expression of EBNA2 and activation of signaling pathways. [Display omitted] • PD-L1 was upregulated by lytic induction of EBV in Akata(+) and some other EBV-positive cell lines. • However, PD-L1 was not increased by lytic induction in some other cell lines. • EBNA2, induced by lytic reactivation, was suggested to play a role in the PD-L1 upregulation. [ABSTRACT FROM AUTHOR]

While Epstein–Barr virus (EBV) establishes a life‐long latent infection in apparently healthy human immunocompetent hosts, immunodeficient individuals are at particular risk to develop lymphoproliferative B‐cell malignancies caused by EBV. A key EBV protein is the transcription factor EBV nuclear antigen 2 (EBNA2), which initiates B‐cell proliferation. Here, we combine biochemical, cellular, and in vivo experiments demonstrating that the mitotic polo‐like kinase 1 (PLK1) binds to EBNA2, phosphorylates its transactivation domain, and thereby inhibits its biological activity. EBNA2 mutants that impair PLK1 binding or prevent EBNA2 phosphorylation are gain‐of‐function mutants. They exhibit enhanced transactivation capacities, accelerate the proliferation of infected B cells, and promote the development of monoclonal B‐cell lymphomas in infected mice. Thus, PLK1 coordinates the activity of EBNA2 to attenuate the risk of tumor incidences in favor of the establishment of latency in the infected but healthy host. SYNOPSIS: EBNA2 is a key transactivator that initiates B cell immortalization. PLK1 phosphorylates the C‐ terminal transactivation domain of EBNA2 to attenuate its oncogenic activities and promote the establishment of long term latency in the infected host. CDK1 phosphorylation of EBNA2 (S379) primes PLK1 binding via its polo box domain (PBD).The PLK1 kinase domain (KD) phosphorylates two residues in the EBNA2 transactivation domain (S457/T465).A second PLK1 binding region within the C‐terminus of EBNA2 confers weak CDK1 phosphorylation independent binding.Monoclonal B cell lymphomas more frequently arise in mice infected with EBV mutants expressing either EBNA2 (S379A) or EBNA2 (S457A/T465V) in comparison to EBV wild‐type infected mice. [ABSTRACT FROM AUTHOR]

Background: Epstein-Barr virus (EBV) infection may be necessary for the development of Multiple sclerosis (MS). Earlier we had identified six MS risk loci that are co-located with binding sites for the EBV transcription factor Epstein-Barr Nuclear Antigen 2 (EBNA2) in EBV-infected B cells (lymphoblastoid cell lines – LCLs). Methods: We used an allele-specific chromatin immunoprecipitation PCR assay to assess EBNA2 allelic preference. We treated LCLs with a peptide inhibitor of EBNA2 (EBNA2-TAT), reasoning that inhibiting EBNA2 function would alter gene expression at these loci if it was mediated by EBNA2. Findings: We found that EBNA2 binding was dependent on the risk allele for five of these six MS risk loci (p < 0·05). Treatment with EBNA2-TAT significantly altered the expression of TRAF3 (p < 0·05), CD40 (p < 0·001), CLECL1 (p

Introduction: N6‐methyladenosine (m6A) is the most prevalent modification that occurs in messenger RNA (mRNA), affecting mRNA splicing, translation, and stability. This modification is reversible, and its related biological functions are mediated by "writers," "erasers," and "readers." The field of viral epitranscriptomics and the role of m6A modification in virus–host interaction have attracted much attention recently. When Epstein–Barr virus (EBV) infects a human B lymphocyte, it goes through three phases: the pre‐latent phase, latent phase, and lytic phase. Little is known about the viral and cellular m6A epitranscriptomes in EBV infection, especially in the pre‐latent phase during de novo infection. Methods: Methylated RNA immunoprecipitation sequencing (MeRIP‐seq) and MeRIP‐RT‐qPCR were used to determine the m6A‐modified transcripts during de novo EBV infection. RIP assay was used to confirm the binding of EBNA2 and m6A readers. Quantitative reverse‐transcription polymerase chain reaction (RT‐qPCR) and Western blot analysis were performed to test the effect of m6A on the host and viral gene expression. Results: Here, we provided mechanistic insights by examining the viral and cellular m6A epitranscriptomes during de novo EBV infection, which is in the pre‐latent phase. EBV EBNA2 and BHRF1 were highly m6A‐modified upon EBV infection. Knockdown of METTL3 (a "writer") decreased EBNA2 expression levels. The emergent m6A modifications induced by EBV infection preferentially distributed in 3ʹ untranslated regions of cellular transcripts, while the lost m6A modifications induced by EBV infection preferentially distributed in coding sequence regions of mRNAs. EBV infection could influence the host cellular m6A epitranscriptome. Conclusions: These results reveal the critical role of m6A modification in the process of de novo EBV infection. [ABSTRACT FROM AUTHOR]

Epstein-Barr virus (EBV) is a causative agent of infectious mononucleosis and several types of malignancy. RNAseq of peripheral blood primary B cell samples infected with wild-type EBV revealed that expression of programmed cell death ligand-1 (PD-L1) is markedly induced by infection. This induction of PD-L1 was alleviated by knockout of the EBNA2 gene, but knockout of LMP1 had little effect. ChIPseq, ChIA-PET, and reporter assays further confirmed that EBNA2-binding sites in the promoter region and at 130 kb downstream of the PD-L1 gene played important roles in PD-L1 induction. Our results indicate that EBV mainly utilizes the EBNA2 gene for induction of PD-L1 and to evade host immunity on infection of primary B cells. Furthermore, pathway analysis revealed that genes involved in the cell cycle, metabolic processes, membrane morphogenesis, and vesicle regulation were induced by EBNA2, and that EBNA2 suppressed genes related to immune signaling. • EBV infection to primary B cells causes induction of PD-L1. • Disruption of EBNA2/LMP1 decreases the PD-L1 induction. • EBNA2 binds to enhancer sites of PD-L1 and increases PD-L1 transcription. • In addition, knockout of EBNA2/LMP1 clearly shows the roles of these genes in transcriptome. [ABSTRACT FROM AUTHOR]

Biological macromolecule condensates formed by liquid-liquid phase separation (LLPS) have been discovered in recent years to be prevalent in biology. These condensates are involved in diverse processes, including the regulation of gene expression. LLPS of proteins have been found in animal, plant, and bacterial species but have scarcely been identified in viral proteins. Here, we discovered that Epstein-Barr virus (EBV) EBNA2 and EBNALP form nuclear puncta that exhibit properties of liquid-like condensates (or droplets), which are enriched in superenhancers of MYC and Runx3. EBNA2 and EBNALP are transcription factors, and the expression of their target genes is suppressed by chemicals that perturb LLPS. Intrinsically disordered regions (IDRs) of EBNA2 and EBNALP can form phase-separated droplets, and specific proline residues of EBNA2 and EBNALP contribute to droplet formation. These findings offer a foundation for understanding the mechanism by which LLPS, previously determined to be related to the organization of P bodies, membraneless organelles, nucleolus homeostasis, and cell signaling, plays a key role in EBV-host interactions and is involved in regulating host gene expression. This work suggests a novel anti-EBV strategy where developing appropriate drugs of interfering LLPS can be used to destroy the function of the EBV's transcription factors. [ABSTRACT FROM AUTHOR]

Epstein–Barr virus (EBV) is an usually harmless virus whose oncogenic properties in vitro are related to its ability to transform lymphoid cells, and, in consequence, it can be associated with lymphomas. Since a few studies detected EBV presence in supposedly EBV-negative lymphomas, our aim was to evaluate EBV presence by sensitive gene expression assays in the tonsils from healthy pediatric donors from a region with high incidence of EBV-associated lymphomas. EBERs transcripts were detected by View RNA ISH in all cases, even in cases assessed negative by widely used in situ hybridization. The presence of LMP1 transcripts was proved in 93% of cases, co-expressed with EBNA2 in 30%. In this study, evidence for the expression of different latent and lytic viral genes in a population of young age of primary infection, detected with more sensitive methods, in particular at the germinal center, where most EBV-associated lymphomas originate, was provided. • EBV has been detected in viral-associated lymphomas with sensitive methods. • EBV and its viral genes were detected in tonsils from pediatric patients at the germinal center and interfollicular regions. • The expression of latent and lytic genes was confirmed by laser capture microdisection • More studies are required to further explore the involvement of EBV detected by this methods in EBV mediated lymphomagenesis. [ABSTRACT FROM AUTHOR]

Although the causes of Multiple Sclerosis (MS) still remain largely unknown, multiple lines of evidence suggest that Epstein–Barr virus (EBV) infection may contribute to the development of MS. Here, we aimed to identify the potential contribution of EBV-encoded and host cellular miRNAs to MS pathogenesis. We identified differentially expressed host miRNAs in EBV infected B cells (LCLs) and putative host/EBV miRNA interactions with MS risk loci. We estimated the genotype effect of MS risk loci on the identified putative miRNA:mRNA interactions in silico. We found that the protective allele of MS risk SNP rs4808760 reduces the expression of hsa-mir-3188-3p. In addition, our analysis suggests that hsa-let-7b-5p may interact with ZC3HAV1 differently in LCLs compared to B cells. In vitro assays indicated that the protective allele of MS risk SNP rs10271373 increases ZC3HAV1 expression in LCLs, but not in B cells. The higher expression for the protective allele in LCLs is consistent with increased IFN response via ZC3HAV1 and so decreased immune evasion by EBV. Taken together, this provides evidence that EBV infection dysregulates the B cell miRNA machinery, including MS risk miRNAs, which may contribute to MS pathogenesis via interaction with MS risk genes either directly or indirectly.

The oncogenic microRNA (miRNA) miR-155 is the most frequently up-regulated miRNA in Epstein-Barr virus (EBV)-positive B cell malignancies and is up-regulated in other nonviral lymphomas. Both EBV nuclear antigen 2 (EBNA2) and the B cell transcription factor interferon regulatory factor 4 (IRF4) are known to activate transcription of the host cell gene from which miR-155 is processed (miR-155HG; BIC). EBNA2 also activates IRF4 transcription, indicating that EBV may upregulate miR-155 through direct and indirect mechanisms. The mechanism of transcriptional regulation of IRF4 and miR-155HG by EBNA2, however, has not been defined. We demonstrate that EBNA2 can activate IRF4 and miR-155HG expression through specific upstream enhancers that are dependent on the Notch signaling transcription factor RBPJ, a known binding partner of EBNA2. We demonstrate that in addition to the activation of the miR-155HG promoter, IRF4 can also activate miR-155HG via the upstream enhancer also targeted by EBNA2. Gene editing to remove the EBNA2- and IRF4-responsive miR-155HG enhancer located 60 kb upstream of miR-155HG led to reduced miR-155HG expression in EBV-infected cells. Our data therefore demonstrate that specific RBPJ-dependent enhancers regulate the IRF4-miR-155 expression network and play a key role in the maintenance of miR-155 expression in EBV-infected B cells. These findings provide important insights that will improve our understanding of miR-155 control in B cell malignancies. [ABSTRACT FROM AUTHOR]

Background: Epstein-Barr virus is a worldwide disease that can cause a wide range of human diseases, the person will become a lifelong carrier of the virus once infected. To investigate the mechanism of Epstein-Barr virus nuclear antigen 2 (EBNA2) on B-lymphocytic activity, sera from 35 patients with infectious mononucleosis and from 34 cases without Epstein-Barr virus infection are collected for experimental analysis. Methods: Quantitative real-time PCR, western blot, and MTT assays were used to evaluate the relative expression level of EBNA2 and to examine its impact on cell vitality. Results: Research found that the average EBNA2 mRNA and protein levels in 35 patients with infectious mononucleosis were higher than that 34 cases without Epstein-Barr virus infection. The MTT assay indicates that EBNA2 can promote the growth and proliferation of B lymphocytes. Conclusions: Combining the above implies that EBNA2 plays an important role in diseases that are induced by the Epstein-Barr virus. Other experiments reveal that ATO promotes the degradation of EBNA2 protein and induces the apoptosis of B lymphocytes which are EBNA2-positive. [ABSTRACT FROM AUTHOR]

Epstein-Barr virus (EBV) latency and its associated carcinogenesis are regulated by dynamic changes in DNA methylation of both virus and host genomes. We show here that the ten-eleven translocation 2 (TET2) gene, implicated in hydroxymethylation and active DNA demethylation, is a key regulator of EBV latency type DNA methylation patterning. EBV latency types are defined by DNA methylation patterns that restrict expression of viral latency genes. We show that TET2 mRNA and protein expression correlate with the highly demethylated EBV type III latency program permissive for expression of EBNA2, EBNA3s, and LMP transcripts. We show that short hairpin RNA (shRNA) depletion of TET2 results in a decrease in latency gene expression but can also trigger a switch to lytic gene expression. TET2 depletion results in the loss of hydroxymethylated cytosine and a corresponding increase in cytosine methylation at key regulatory regions on the viral and host genomes. This also corresponded to a loss of RBP-jκ binding and decreased histone H3K4 trimethylation at these sites. Furthermore, we show that the TET2 gene itself is regulated in a fashion similar to that of the EBV genome. Chromatin immunoprecipitation high-throughput sequencing (ChIP-seq) revealed that the TET2 gene contains EBNA2-dependent RBP-jκ and EBF1 binding sites and is subject to DNA methylationassociated transcriptional silencing similar to what is seen in EBV latency type III genomes. Finally, we provide evidence that TET2 colocalizes with EBNA2-EBF1-RBP-jκ binding sites and can interact with EBNA2 by coimmunoprecipitation. Taken together, these findings indicate that TET2 gene transcripts are regulated similarly to EBV type III latency genes and that TET2 protein is a cofactor of EBNA2 and coregulator of the EBV type III latency program and DNA methylation state. [ABSTRACT FROM AUTHOR]

Epstein-Barr virus (EBV) latently infects normal B cells and contributes to the development of certain human lymphomas. Newly infected B cells support a highly transforming form (type III) of viral latency; however, long-term EBV infection in immunocompetent hosts is limited to B cells with a more restricted form of latency (type I) in which most viral gene expression is silenced by promoter DNA methylation. How EBV converts latency type is unclear, although it is known that type I latency is associated with a germinal center (GC) B cell phenotype, and type III latency with an activated B cell (ABC) phenotype. In this study, we have examined whether expression of TET2, a cellular enzyme that initiates DNA demethylation by converting 5-methylcytosine (5mC) into 5-hydroxymethylcytosine (5hmC), regulates EBV latency type in B cells. We found that TET2 expression is inhibited in normal GC cells and GC type lymphomas. In contrast, TET2 is expressed in normal naive B cells and ABC type lymphomas. We also demonstrate that GC type cell lines have increased 5mC levels and reduced 5hmC levels in comparison to those of ABC type lines. Finally, we show that TET2 promotes the ability of the EBV transcription factor EBNA2 to convert EBV-infected cells from type I to type III latency. These findings demonstrate that TET2 expression is repressed in GC cells independent of EBV infection and suggest that TET2 promotes type III EBV latency in B cells with an ABC or naive phenotype by enhancing EBNA2 activation of methylated EBV promoters. IMPORTANCE EBV establishes several different types of viral latency in B cells. However, cellular factors that determine whether EBV enters the highly transforming type III latency, versus the more restricted type I latency, have not been well characterized. Here we show that TET2, a cellular enzyme that initiates DNA demethylation by converting 5-methylcytosine (5mC) into 5-hydroxymethylcytosine (5hmC), regulates EBV latency type in B cells by enhancing the ability of the viral transcription factor EBNA2 to activate methylated viral promoters that are expressed in type III (but not type I) latency. Furthermore, we demonstrate that (independent of EBV) TET2 is turned off in normal and malignant germinal center (GC) B cells but expressed in other B cell types. Thus, restricted TET2 expression in GC cells may promote type I EBV latency. [ABSTRACT FROM AUTHOR]

Epstein-Barr virus (EBV) can infect the majority of the human population with no obvious symptoms and is associated with tumor development, although the mechanism is still largely unknown. In this study, we investigated the role and the underlying mechanism of EBV nuclear antigen 2 (EBNA2) in tumorigenesis. We found that the infection of EBNA2 in human B lymphocytes (HBL) upregulated the expression of activating transcription factor 4 (ATF4). Furthermore, we used gene expression or knockdown approach to demonstrate the effect of EBNA2 on redox balance, mitochondrial function, lipid metabolism, and cell proliferation in both HBL and EBV-transformed lymphocyte cell line (LCL). More importantly, we applied in vivo xenograft tumor mouse model to explore the contribution of EBNA2 and ATF4 in tumor growth and mouse survival. Mechanistically, we revealed that EBNA2 exposure caused persistent expression of ATF4 via EBNA2-mediated epigenetic changes, which increased the binding ability of upstream stimulating factor 1 (USF1) on the ATF4 promoter. ATF4 activation in HBL cells modulated the expression of lipid metabolism-related genes and potentiated fatty acid oxidation and lipogenesis. Conversely, knockdown of either EBNA2 or ATF4 in LCL suppressed lipid metabolism, modulated redox balance and mitochondrial function, as well as inhibited tumor cell proliferation. In consistent with these findings from in vitro study, an in vivo xenograft model confirmed that knockdown of either EBNA2 or ATF4 inhibited the gene expression of SREBP1, ChREBP, and FAS, as well as suppressed tumor growth and prolonged animal survival. Collectively, this study demonstrates that EBNA2 mediates tumorigenesis through ATF4 activation and the modulation of lipid metabolism; therefore, our findings provide a novel avenue for the clinical treatment of EBV-mediated cancer., Competing Interests: None., (AJCR Copyright © 2023.)

Lymphomagenesis in the presence of deregulated MYC requires suppression of MYC-driven apoptosis, often through downregulation of the pro-apoptotic BCL2L11 gene (Bim). Transcription factors (EBNAs) encoded by the lymphoma-associated Epstein-Barr virus (EBV) activate MYC and silence BCL2L11. We show that the EBNA2 transactivator activates multiple MYC enhancers and reconfigures the MYC locus to increase upstream and decrease downstream enhancer-promoter interactions. EBNA2 recruits the BRG1 ATPase of the SWI/SNF remodeller to MYC enhancers and BRG1 is required for enhancer-promoter interactions in EBV-infected cells. At BCL2L11, we identify a haematopoietic enhancer hub that is inactivated by the EBV repressors EBNA3A and EBNA3C through recruitment of the H3K27 methyltransferase EZH2. Reversal of enhancer inactivation using an EZH2 inhibitor upregulates BCL2L11 and induces apoptosis. EBV therefore drives lymphomagenesis by hijacking long-range enhancer hubs and specific cellular co-factors. EBV-driven MYC enhancer activation may contribute to the genesis and localisation of MYC-Immunoglobulin translocation breakpoints in Burkitt's lymphoma.

To investigate the molecular mechanisms through which Epstein-Barr virus (EBV) may contribute to Systemic Lupus Erythematosus (SLE) pathogenesis, we interrogated SLE genetic risk loci for signatures of EBV infection. We first compared the gene expression profile of SLE risk genes across 459 different cell/tissue types. EBV-infected B cells (LCLs) had the strongest representation of highly expressed SLE risk genes. By determining an SLE risk allele effect on gene expression (expression quantitative trait loci, eQTL) in LCLs and 16 other immune cell types, we identified 79 SLE risk locus:gene pairs putatively interacting with EBV infection. A total of 10 SLE risk genes from this list (CD40 , LYST , JAZF1 , IRF5 , BLK , IKZF2 , IL12RB2 , FAM167A , PTPRC and SLC15A) were targeted by the EBV transcription factor, EBNA2 , differentially expressed between LCLs and B cells, and the majority were also associated with EBV DNA copy number, and expression level of EBV encoded genes. Our final gene network model based on these genes is suggestive of a nexus involving SLE risk loci and EBV latency III and B cell proliferation signalling pathways. Collectively, our findings provide further evidence to support the interaction between SLE risk loci and EBV infection that is in part mediated by EBNA2. This interplay may increase the tendency towards EBV lytic switching dependent on the presence of SLE risk alleles. These results support further investigation into targeting EBV as a therapeutic strategy for SLE. • Approximately 20% of SLE risk loci putatively involved in EBV infection processes. • SLE risk genes show highest enrichment of expression in EBV infected B cells (LCLs). • Interactions between SLE risk loci and EBNA2 may increase EBV lytic switching. [ABSTRACT FROM AUTHOR]

A role for Epstein Barr virus (EBV) in Hodgkin lymphoma (HL) pathogenesis is supported by the detection of EBV genome in about one-third of HL cases, but is not well defined. We previously reported that an elevated prediagnosis antibody titer against EBV nuclear antigens (EBNA) was the strongest serologic predictor of subsequent HL. For the present analysis, we measured antibody levels against EBNA components EBNA1 and EBNA2 and computed their titer ratio (anti-EBNA1:2) in serum samples from HL cases and healthy siblings. We undertook this analysis to examine whether titer patterns atypical of well-resolved EBV infection, such as an anti-EBNA1:2 ratio ≤1.0, simply reflect history of infectious mononucleosis (IM), an HL risk factor, or independently predict HL risk. Participants were selected from a previous population-based case-control study according to their history of IM. We identified 55 EBV-seropositive persons with a history of IM (IM+; 33 HL cases, 22 siblings) and frequency-matched a comparison series of 173 IM history-negative, EBV-seropositive subjects on HL status, gender, age and year of blood draw (IM−; 105 cases, 58 siblings). In multivariate logistic regression models, an anti-EBNA1:2 ratio ≤1.0 was significantly more prevalent in HL cases than siblings (odds ratio, 95% confidence interval = 2.43, 1.05-5.65); similar associations were apparent within the IM+ and IM− groups. EBNA antibodies were not significantly associated with IM history in HL cases or siblings. These associations suggest that chronic or more severe EBV infection is a risk factor for HL, independent of IM history. [ABSTRACT FROM AUTHOR]

Abstract: Association of EBV nuclear proteins EBNA2, EBNA3A and EBNA3C with RBP/CSL, is essential for lymphoblastoid cell line (LCL) proliferation. Conserved residues in the EBNA3 homology domain, required for RBP/CSL interaction, lack the WΦP motif that mediates EBNA2 and Notch binding to the RBP/CSL beta-trefoil domain (BTD). We map RBP/CSL interacting residues within EBNA3A(aa128–204) and EBNA3C(aa211–233). The EBNA3A results are consistent with an earlier report (aa125–222), but the EBNA3C domain is unexpectedly small and includes a “WTP” sequence. This EBNA3C WTP motif confers RBP/CSL binding in vitro, in yeast, and in mammalian cells. Further, an EBNA3C WTP→STP(W227S) mutation impaired BTD binding whereas EBNA3 homology domain mutations disrupted RBP/CSL N-terminal domain (NTD) binding. WTP was not essential for EBNA3C repression of EBNA2 in reporter assays or for maintenance of LCL growth. Our results indicate that EBNA3 proteins interact with multiple RBP/CSL domains, but only NTD interactions are required for LCL growth. [Copyright &y& Elsevier]

Abstract: The Epstein–Barr virus (EBV) growth-transforms B-lymphocytes. The virus-encoded nuclear antigen 2 (EBNA2) is essential for transformation and activates gene expression by association with DNA-bound transcription factors such as RBPJκ (CSL/CBF1). We have previously shown that EBNA2 contains symmetrically dimethylated Arginine (sDMA) residues. Deletion of the RG-repeat results in a reduced ability of the virus to immortalise B-cells. We now show that the RG repeat also contains asymmetrically dimethylated Arginines (aDMA) but neither non-methylated (NMA) Arginines nor citrulline residues. We demonstrate that only aDMA-containing EBNA2 is found in a complex with DNA-bound RBPJκ in vitro and preferentially associates with the EBNA2-responsive EBV C, LMP1 and LMP2A promoters in vivo. Inhibition of methylation in EBV-infected cells results in reduced expression of the EBNA2-regulated viral gene LMP1, providing additional evidence that methylation is a prerequisite for DNA-binding by EBNA2 via association with the transcription factor RBPJκ. [Copyright &y& Elsevier]

Abstract: The Epstein–Barr virus (EBV)-encoded latency protein EBNA2 is a nuclear transcriptional activator that is essential for EBV-induced cellular transformation. Here, we show that EBNA2 interacts with STAT3, a signal transducer for an interleukin-6 family cytokine, and enhances the transcriptional activity of STAT3 by influencing its DNA-binding activity. Furthermore, EBNA2 cooperatively acts on STAT3 activation with LMP1. These data demonstrate that EBNA2 acts as a transcriptional coactivator of STAT3. [Copyright &y& Elsevier]

Abstract: The epigenetic regulation of viral genes may be important for the life cycle of EBV. We determined the methylation status of three viral promoters (Wp, Cp, Qp) from EBV B-lymphoblastoid cell lines (LCLs) by pyrosequencing. Our pyrosequencing data showed that the CpG region of Wp was methylated, but the others were not. Interestingly, Wp methylation was increased with proliferation of LCLs. Wp methylation was as high as 74.9% in late-passage LCLs, but 25.6% in early-passage LCLs. From two Burkitt’s lymphoma cell lines, Wp specific hypermethylation was also found (>80%). Interestingly, the expression of EBNA2 gene which located directly next to Wp was associated with its methylation. Our data suggested that Wp specific methylation may be important for the indicator of the proliferation status of LCLs, and the epigenetic viral gene regulation of EBNA2 gene by Wp should be further defined possibly with other biological processes. [Copyright &y& Elsevier]

Abstract: Latent Epstein–Barr virus (EBV) infection is strongly associated with B-cell proliferative diseases such as Burkitt''s lymphoma. Here we show that the oncogenic serine/threonine kinases Pim-1 and Pim-2 enhance the activity of the viral transcriptional activator EBNA2. During EBV infection of primary B-lymphocytes, the mRNA expression levels of pim genes, especially of pim-2, are upregulated and remain elevated in latently infected B-cell lines. Thus, EBV-induced upregulation of Pim kinases and Pim-stimulated EBNA2 transcriptional activity may contribute to the ability of EBV to immortalize B-cells and predispose them to malignant growth. [Copyright &y& Elsevier]

In a screen for binding partners of the Epstein–Barr virus transformation-related protein EBNA2, we cloned a novel, evolutionarily conserved protein showing similarity to the Drosophila Parallel Sister Chromatids Protein (PASC). We have named this protein “Friend of EBNA2” (FOE). Human FOE encodes a protein of 1227 amino acids with a functional bipartite nuclear localization signal, an arginine-rich motif, a putative nuclear export signal as well as with three highly acidic regions and a predicted coiled-coil domain. FOE and EBNA2 coimmunoprecipitate from lymphocyte nuclear extracts. RNA and protein blots show that FOE is expressed in all human tissues. FOE is a nuclear protein with the bulk of the protein associated with the insoluble nuclear fraction biochemically defined as the nuclear matrix. Indirect immunofluorescence and dynamic imaging studies suggest that FOE associates with transcriptionally active nuclear subregions in interphase cells and concentrates at the ends of formed chromosomes during mitosis. [Copyright &y& Elsevier]

In the human B cell line P493-6 two mitogenic signals, the Epstein-Barr virus nuclear antigen 2 (EBNA2) and myc, can be independently regulated by means of an estrogen receptor fusion construct or an inducible expression vector, respectively. Shut off of EBNA2, either in the presence or absence of myc, leads to a significant increase in enzymatic activity and surface expression of ecto-5'-nucleotidase (CD73) as well as an increased adenosine receptor response in cyclic AMP formation. Shut off of myc expression has a small additional positive effect on CD73 activity. Among the four different subtypes of adenosine receptors, the A2a receptor exclusively is subject to regulation in this system, which is substantiated by pharmacologic data (specific agonists and inhibitors), as well as on the mRNA level. With up-regulated CD73 and A2a, cells also respond to 5'-AMP with increased cyclic AMP formation. Turn on of EBNA2 has the reverse effect of repression of CD73 and A2a expression. The time course of both induction and repression of CD73 and A2a is rather slow. [ABSTRACT FROM AUTHOR]

We showed previously that Epstein–Barr virus (EBV) latency is disrupted and the virus-replicative cycle is activated after expression of EBNA2 in the Burkitt's lymphoma-derived Akata cells. Here, an EBNA2 deletion mutant lacking the amino acid residues 248–382, including the region responsible for association with RBP-J κ, was generated and tested for its ability to activate EBV replication in Akata cells. This mutant was shown clearly deficient in inducing the EBV-replicative cycle, suggesting that association with RBP-J κ is necessary for the EBV activating function of EBNA2. It is thus likely that EBV activation by EBNA2, seemingly in conflict with its involvement in lymphocyte immortalization, is nevertheless based on the standard mechanism of transactivation by the protein. [ABSTRACT FROM AUTHOR]

Epstein–Barr virus nuclear antigen 2 (EBNA2) and the Notch protein both function within the nucleus as transcriptional adaptor proteins. EBNA2 plays a key role during the immortalization of primary B-cells by Epstein–Barr virus (EBV). Notch proteins are involved in lymphomagenesis as well as in multiple cell fate decisions during tissue differentiation and development. Both, EBNA2 and Notch interact with the DNA binding protein RBP-J and thereby gain access to the promoter of their target genes. In order to identify regions within the J recombination signal sequence binding protein (RBP-J), that are relevant for either the Notch or the EBNA2 interaction, we have performed a mutational analysis of RBP-J. A library of RBP-J mutants was screened by a reverse two-hybrid system for alleles that fail to bind to either EBNA2 or Notch. The sequence analysis of these alleles reveals that a limited and particularly distinct number of amino-acid positions are relevant for either interaction only. Given the important role of RBP-J in B-cell immortalization, the EBNA2/RBP-J protein–protein interaction could be a candidate target for therapeutic intervention in EBV related diseases. [ABSTRACT FROM AUTHOR]

To reveal sequence variations in the Epstein-Barr virus nuclear antigen 2 (EBNA2) genes of Epstein-Barr virus (EBV) strains circulating in the Korean population, the EBNA2 divergent region was amplified and sequenced from 13 EBV-l isolates, 2 EBNA2 type 1 intertypic EBV isolates, and two EBV-2 isolates, all derived from Korean cancer patients. Comparative sequence analysis revealed that type 1 and type 2 EBNA2 divergent regions of Korean EBV isolates were almost identical to the respective regions of the B95-8 EBV-l and AG876 EBV-2 strains. Two of the nucleotide changes found in the type 1 divergent regions of all Korean isolates, G-toT and C-to-A at B95-8 EBV positions 48,991 and 48,998, respectively, are also present in all EBV-l strains of non-Asian origins, indicating that these two sites might be mutational hot sites. Besides these two mutations, EBV-l strains from Papua New Guinea or European Caucasians and Africa show unique patterns of identical sequence variations from B95-8, which were not found in type 1 isolates from Korea, suggesting that Korean type 1 are evolving as a lineage distinct from isolates from Papua New Guinea or European Caucasian and Africa. [ABSTRACT FROM AUTHOR]

Background: Epstein-Barr virus (EBV) infection may be necessary for the development of Multiple sclerosis (MS). Earlier we had identified six MS risk loci that are co-located with binding sites for the EBV transcription factor Epstein-Barr Nuclear Antigen 2 (EBNA2) in EBV-infected B cells (lymphoblastoid cell lines - LCLs)., Methods: We used an allele-specific chromatin immunoprecipitation PCR assay to assess EBNA2 allelic preference. We treated LCLs with a peptide inhibitor of EBNA2 (EBNA2-TAT), reasoning that inhibiting EBNA2 function would alter gene expression at these loci if it was mediated by EBNA2., Findings: We found that EBNA2 binding was dependent on the risk allele for five of these six MS risk loci (p < 0·05). Treatment with EBNA2-TAT significantly altered the expression of TRAF3 (p < 0·05), CD40 (p < 0·001), CLECL1 (p <0·0001), TNFAIP8 (p < 0·001) and TNFRSF1A (p < 0·001)., Interpretation: These data suggest that EBNA2 can enhance or reduce expression of the gene depending on the risk allele, likely promoting EBV infection. This is consistent with the concept that these MS risk loci affect MS risk through altering the response to EBNA2. Together with the extensive data indicating a pathogenic role for EBV in MS, this study supports targeting EBV and EBNA2 to reduce their effect on MS pathogenesis., Funding: Funding was provided by grants from MS Research Australia, National Health and Medical Research Council of Australia, Australian Government Research Training Program, Multiple Sclerosis International Federation, Trish Multiple Sclerosis Research Foundation., Competing Interests: Declaration of Competing Interest Funding for materials for the current manuscript were provided by MS Research Australia Project and Incubator grants. Australian Government financial support was provided to JTK and AA through the Research Training Program, and to DRB by a National Health and Medical Research Council of Australia fellowship. GPP was supported by an MS Research Australia Postdoctoral Fellowship and a Trish Multiple Sclerosis Research Foundation Project Grant. The authors declare no competing financial and non-financial interests., (Copyright © 2021 The Authors. Published by Elsevier B.V. All rights reserved.)

Although the causes of Multiple Sclerosis (MS) still remain largely unknown, multiple lines of evidence suggest that Epstein–Barr virus (EBV) infection may contribute to the development of MS. Here, we aimed to identify the potential contribution of EBV-encoded and host cellular miRNAs to MS pathogenesis. We identified differentially expressed host miRNAs in EBV infected B cells (LCLs) and putative host/EBV miRNA interactions with MS risk loci. We estimated the genotype effect of MS risk loci on the identified putative miRNA:mRNA interactions in silico. We found that the protective allele of MS risk SNP rs4808760 reduces the expression of hsa-mir-3188-3p. In addition, our analysis suggests that hsa-let-7b-5p may interact with ZC3HAV1 differently in LCLs compared to B cells. In vitro assays indicated that the protective allele of MS risk SNP rs10271373 increases ZC3HAV1 expression in LCLs, but not in B cells. The higher expression for the protective allele in LCLs is consistent with increased IFN response via ZC3HAV1 and so decreased immune evasion by EBV. Taken together, this provides evidence that EBV infection dysregulates the B cell miRNA machinery, including MS risk miRNAs, which may contribute to MS pathogenesis via interaction with MS risk genes either directly or indirectly. [ABSTRACT FROM AUTHOR]

Introduction: N 6 -methyladenosine (m 6 A) is the most prevalent modification that occurs in messenger RNA (mRNA), affecting mRNA splicing, translation, and stability. This modification is reversible, and its related biological functions are mediated by "writers," "erasers," and "readers." The field of viral epitranscriptomics and the role of m 6 A modification in virus-host interaction have attracted much attention recently. When Epstein-Barr virus (EBV) infects a human B lymphocyte, it goes through three phases: the pre-latent phase, latent phase, and lytic phase. Little is known about the viral and cellular m 6 A epitranscriptomes in EBV infection, especially in the pre-latent phase during de novo infection., Methods: Methylated RNA immunoprecipitation sequencing (MeRIP-seq) and MeRIP-RT-qPCR were used to determine the m 6 A-modified transcripts during de novo EBV infection. RIP assay was used to confirm the binding of EBNA2 and m 6 A readers. Quantitative reverse-transcription polymerase chain reaction (RT-qPCR) and Western blot analysis were performed to test the effect of m 6 A on the host and viral gene expression., Results: Here, we provided mechanistic insights by examining the viral and cellular m 6 A epitranscriptomes during de novo EBV infection, which is in the pre-latent phase. EBV EBNA2 and BHRF1 were highly m 6 A-modified upon EBV infection. Knockdown of METTL3 (a "writer") decreased EBNA2 expression levels. The emergent m 6 A modifications induced by EBV infection preferentially distributed in 3' untranslated regions of cellular transcripts, while the lost m 6 A modifications induced by EBV infection preferentially distributed in coding sequence regions of mRNAs. EBV infection could influence the host cellular m 6 A epitranscriptome., Conclusions: These results reveal the critical role of m 6 A modification in the process of de novo EBV infection., (© 2021 The Authors. Immunity, Inflammation and Disease published by John Wiley & Sons Ltd.)