Genome-Wide Profiling of Epstein-Barr Virus (EBV) Isolated from EBV-Related Malignancies

*Ying Liu, Zheming Lu and Hongying Huang*

## **Abstract**

Epstein–Barr virus (EBV) is the cause of certain cancers, such as Burkitt lymphoma, Hodgkin lymphoma, NK/T cell lymphoma, nasopharyngeal carcinoma, and a subset of gastric carcinomas. The genome-wide characteristics of EBV are essential to understand the diversity of strains isolated from EBV-related malignancies, provide the first opportunity to test the general validity of the EBV genetic map and explore recombination, geographic variation, and the major features of variation in this virus. Moreover, understanding more about EBV sequence variations isolated from EBV-related malignancies might give important implications for the development of effective prophylactic and therapeutic vaccine approaches targeting the personalized or geographic-specific EBV antigens in these aggressive diseases. In this chapter, we will mainly focus on the EBV genome-wide profiling in three common EBV-related cancers in Asia, including nasopharyngeal carcinoma, EBV-associated gastric carcinoma, and NK/T-cell lymphoma.

**Keywords:** Epstein–Barr virus (EBV), next-generation sequencing (NGS), nasopharyngeal carcinoma (NPC), EBV-associated gastric carcinoma (EBVaGC), NK/T-cell lymphoma (NKTCL)

## **1. Introduction**

Epstein–Barr virus (EBV), a ubiquitous human herpesvirus discovered in 1964 is classified as a group I carcinogen by the International Agency for Research on Cancer (IARC), since the latent infection by EBV has been estimated to be responsible for 200,000 cancer cases worldwide [1], including Burkitt lymphoma, Hodgkin lymphoma, NK/T cell lymphoma (NKTCL), nasopharyngeal carcinoma (NPC), and a subset of gastric carcinomas. It has been shown that viruses can contribute to the biology of multistep oncogenesis and are implicated in many of the hallmarks of cancer [2]. Notably, the discovery of links between viral infection and cancer types has provided actionable opportunities, such as the use of human papilloma virus (HPV) vaccines as a preventive measure, to reduce the global impact of cancer. However, until now, approved vaccines for EBV have not been available.

EBV has a double stranded DNA genome comprised of approximately 172 kilobases. The expression products cover at least 86 proteins and 46 functional small-untranslated RNAs [3–5]. EBV has two distinct life cycles: latency and lytic replication. During latency, viral genomes only express a limited number of latent proteins (EBV-determined nuclear antigen 1 (EBNA1), 2, 3A, 3B, and 3C and EBNA leader protein (EBNA-LP); latent membrane protein 1 (LMP1) and LMP2 (which encodes two isoforms, LMP2A and LMP2B)), noncoding EBV-encoded RNAs (EBER1 and EBER2), and viral miRNAs (BHRF1-miRNA and BART-miRNA). EBV latency is categorized as three latency types (latency I–III). EBV genomes in type-I latency are known to express EBNA1 and EBER. EBV genomes in type-II latency are known to express more genes such as *EBNA-LP*, *LMP1*, *LMP2A*, and *LMP2B*. EBV genomes in type-III latency are known to express most restricted latent genes including *EBNA2*, *EBNA3A*, *EBNA3B*, and *EBNA3C*. Lytic genes encode viral transcription factors (e.g., BZLF1), a viral DNA polymerase (BALF5) and associated factors, and viral glycoproteins (e.g., gp350/220 and gp110) and structural proteins (capsid and tegument proteins).

Southern blot of restriction fragment length polymorphisms was first used to detect EBV strain variation, and Sanger sequencing of certain specific viral genes (e.g., EBNA1 and LMP1) was later developed to detect sequence diversity. Now, on the basis of high-throughput sequencing, genome-wide analysis is becoming possible.

Prior to 2013, EBV whole genome sequences available from GenBank were limited to less than 10 strains (B95-8, EBV-WT, GD1, AG876, GD2, HKNPC1, Akata, and Mutu). The prototypic type 1 EBV strain B95-8 was the first complete genome sequenced from an individual with infectious mononucleosis using a conventional strategy (i.e., subcloning followed by Sanger sequencing) [6]. Subsequently, a more representative type 1 EBV reference genome, human herpesvirus 4 complete wild type genome, was constructed by using B95-8 as the backbone with an 11-kb deletion segment provided by the Raji sequences (named EBV-WT) [7]. AG876 was the unique complete type 2 EBV sequence from a Ghanaian case of Burkitt lymphoma [8]. Akata and Mutu were sequenced from Burkitt lymphoma cell lines from a Japanese patient and a Kenyan patient, respectively [9]. GD1 [10], GD2 [11] and HKNPC1 [12] were isolated from NPC patients.

Since 2014, a new technology named Hybrid Capture (**Figure 1**), has marked a new era of EBV genome sequencing. Using the method of target enrichment of EBV DNA by hybridization, followed by next-generation sequencing, de novo assembly, and joining of contigs can yield complete EBV genomes. The development of highthroughput sequencing technologies enabled sequencing of EBV genomes derived from a wide variety of clinical samples, such as tumor biopsy samples [13]. The number of available EBV sequences is increasing exponentially and up to now, more than 500 EBV genomes have been sequenced from a variety of human malignancies, including NPC, lymphoma, gastric cancer, and lung cancer, as well as from healthy carriers [14–25]. Progress has made it possible that the population-based case–control studies of EBV strain variation in EBV-related cancer patients as compared with the healthy population and a comprehensive survey of EBV integration in a variety of human malignancies can be effectively conducted [20, 25–27]. These developments have revealed that various EBV strains are differentially distributed throughout the world, and that the behavior of cancer-derived EBV strains is different from that of the prototype EBV strain of noncancerous origin.

Hence, the genome-wide characteristics of EBV are essential to assess the diversity of strains isolated from EBV-related malignancies. Meanwhile, understanding the pattern of EBV sequence variation is important for knowing whether there is a diseaserelated strain-specific or geographic regional variation of EBV strain, and might provide important implications for the development of effective prophylactic and therapeutic vaccine approaches targeting the personalized or geographic-specific EBV antigens in these aggressive diseases.

*Genome-Wide Profiling of Epstein-Barr Virus (EBV) Isolated from EBV-Related Malignancies DOI: http://dx.doi.org/10.5772/intechopen.93244*

#### **Figure 1.**

*Complete workflow for EBV DNA capture and sequencing.*


#### **Table 1.**

*EBV genomes reviewed in this chapter*

In this chapter, EBV genomes reviewed are from three common EBV-related cancers in Asia, including NPC, EBV-associated gastric carcinoma (EBVaGC), and NKTCL. The EBV strains include GD1 [10], GD2 [11], HKNPC1 [12],

HKNPC2-HKNPC9 [14], EBVaGC1-EBVaGC9 [17], GDGC1-GDGC2 [21], NKTCL-EBV1-NKTCL-EBV8 [23], NKTCL-SC01-NKTCL-SC15 and NKTCL-SG01- NKTCL-SG12 [24] (**Table 1**).

## **2. Genomic diversity of EBV-related malignances**

### **2.1 NPC**

NPC, an EBV-associated epithelial carcinoma, has a unique geographical distribution [28]. A recent World Health Organization (WHO) report estimated that there were around 130,000 new NPC cases worldwide in 2018 [29]. Rare in most of the world, NPC is particularly prevalent in South China and Southeast Asia [30]. In Hong Kong and Guangdong in South China, NPC incidence is as high as 12.8–25.0/100,000 per year [28, 29]. The cause of NPC endemicity remains unknown.

Many studies have shown that EBV genome is present in almost all endemic NPC tumors with a unique pattern of virus latent gene expression, suggesting that EBV plays an important role in the tumorigenesis of NPC [31]. Whole genome sequencing is useful for us to understand genomic characterization and divergence. Here, we mainly focus on 11 mostly available full-length genomes of NPC.

#### *2.1.1 GD1*

GD1 (Guangdong strain 1), the first NPC-derived EBV strain with full-length sequences determined using PCR amplification and sub-cloning followed by conventional Sanger sequencing technology, was analyzed from established a lymphoblastoid cell line (LCL) from umbilical cord blood mononuclear cells transformed by saliva virus from a Cantonese NPC patient in 2005 [10]. The entire GD1 sequence is 171,656 bp in length and GD1 belongs to type 1 strain. Many sequence variations in GD1 compared to prototypical strain B95-8 were detected, including 43 deletion sites, 44 insertion sites, and 1413 point mutations. Furthermore, the frequency of some GD1 mutations in Cantonese NPC patients was evaluated, such as a 30-bp deletion in the C terminus of LMP1, and the V-Val subtypes of EBNA1. The results suggested that GD1 is highly representative of the EBV strains isolated from NPC patients in Guangdong, China, an area with the highest incidence of NPC in the world.

#### *2.1.2 GD2*

With the invention of next-generation sequencing (NGS) systems, it is possible to determine genome-wide sequences and the viral clonality of EBV strains by direct sequencing of EBV genomes in clinical tumors in a time- and cost-effective manner. GD2 with 164,701 bp long was directly sequenced using the Illumina (Solexa) platform, and successfully assembled from an NPC tumor of a patient in Guangdong province, a region in China by the same group who determined GD1 [11]. GD2 was closely related to GD1 by sequence and phylogenetic analyses. The sequence similarities between GD2 and GD1 were 98.76%. GD2 and GD1 shared 505 common single-nucleotide variations (SNVs), including most SNVs in the coding regions (348 [68.91%] SNVs) and seven insertion and deletions (indels). From a comparison with the EBV-WT reference genome, a total of 927 SNVs and 160 indels with genome-wide distribution were found in the GD2 genome. The results

revealed that NGS allows the characterization of genome-wide variations of EBV in clinical tumors and provides evidence of monoclonal expansion of EBV *in vivo*.

## *2.1.3 HKNPC1*

Because of the relatively small quantity of viral DNA present in the tumor sample, next-generation sequencing total cellular and viral DNA in a sample is costly and inefficient, and may limit the generation of the high read depth necessary to make high confident base calls of the viral genome. Using target enrichment technology could increase the relative amount of viral DNA. Kwok et al. reported an approach of PCR enrichment (Amplicon Sequencing) followed by sequencing the amplified products on the Illumina Genome Analyzer IIx platform to determine the genome sequence of an EBV isolate from NPC tumor of a Chinese patient in Hong Kong, designated as HKNPC1 [12]. HKNPC1 is approximately 171,549 bp, and contains 1589 SNVs and 132 indels in comparison to the reference EBV-WT sequence. Nonsynonymous SNVs were mainly found in the latent, tegument and glycoprotein genes. The same point mutations were found in glycoprotein (BLLF1 and BALF4) genes of GD1, GD2 and HKNPC1 strains and might affect cell type specific binding. The results showed that whole genome sequencing of EBV in NPC may facilitate discovery of previously unknown variations of pathogenic significance.

## *2.1.4 HKNPC2-9*

The group of Kwok and colleagues established a complete sequencing workflow comprising target enrichment of EBV DNA by hybridization, followed by nextgeneration sequencing, *de novo* assembly, and joining of contigs by Sanger sequencing to yield whole EBV genomes. The sequences of eight NPC biopsy specimen-derived EBV (NPC-EBV) genomes, designated HKNPC2 to HKNPC9, were then determined in the same geographic location in order to reveal their sequence diversity [14]. The eight NPC-EBV genome sizes estimated based on the reference EBV-WT sequence ranged from 170,062 bp (HKNPC2) to 171,556 bp (HKNPC3 and -6). A total of 1736 variations were found, including 1601 substitutions, 64 insertions, and 71 deletions, compared to the reference EBV-WT genome. Furthermore, genes encoding latent, early lytic, and tegument proteins and glycoproteins were found to contain nonsynonymous mutations of potential biological significance. Thus, much greater sequence diversity among EBV isolates derived from NPC biopsy specimens is demonstrated on a whole-genome level through a complete sequencing workflow.

Obtaining whole-genome sequence information for more clinical EBV isolates, with good representation of the EBV repertoire in tumors, could help to address that hypothesis and uncover the pathogenic subtypes of EBV in NPC tumorigenesis. A case–control (62 NPC patients and 142 population carriers) study of NPC in Hong Kong has identified high-risk EBV subtypes with polymorphisms in the EBVencoded small RNA (EBER) locus [26]. A recent study published in Nature Genetics entitled 'Genome sequencing analysis identifies high-risk Epstein–Barr virus subtypes for nasopharyngeal carcinoma' by Xu et al. used large-scale EBV whole-genome sequencing to examine EBV subtypes in an attempt to explain the unique NPC endemicity in South China [25]. Through EBV genomes from 156 NPC cases and 47 controls and two-stage association study, they identified two non-synonymous EBV variants within the BALF2 gene (BamHIA leftward reading frame 2 encoding a single strand DNA binding protein associated with EBV replication) strongly associated with the risk of NPC (odds ratio [OR] = 8.69 for SNP162476\_C and OR = 6.14 for SNP163364\_T). The cumulative effects of these variants contribute to 83% of

the overall risk of NPC in southern China. These studies confirmed the critical role of EBV infection in the pathogenesis of NPC and provided an explanation for the striking epidemiological distribution of this tumor in South China.

#### **2.2 EBVaGC**

EBVaGC has been recognized as a distinct subset of gastric carcinoma, accounting for about 10% of total gastric carcinomas [32–35]. The monoclonal presence of the virus was uniformly distributed in malignant cells of EBV-positive tumors but not observed in the surrounding normal epithelial cells, providing strong evidence to support the role of EBV as an etiologic agent [32, 33]. However, the exact role of EBV in the development and progression of this specific type of gastric carcinoma is not yet clear.

Progress has been made in understanding the full spectrum of diversity existent within the EBV genome from EBVaGC clinical tumor samples, since the NGS technology has been developed. Here, 11 EBV strains from primary EBVaGC biopsy samples were included.

#### *2.2.1 EBVaGC1-EBVaGC9*

Our group reported the first genome-wide view of sequence variation of EBV isolated from primary EBVaGC biopsy specimens in 2016 [17]. We used the method of target enrichment of EBV DNA by hybridization, followed by next-generation sequencing. EBV probes were designed according to full-length genome of six available EBV strains, including EBV-WT, B95-8, AG876, GD1, GD2, and HKNPC1. According to the value of coverage of the target region, all DNA sequence generated from GC-EBV strains most resembled GD1. Thus, GD1 was used as the reference EBV genome in our study. *De novo* assembly was performed for nine sequenced GC-EBV strains. Finally, nine EBVaGC genomes were successfully sequenced, designated EBVaGC1 to EBVaGC9. The genome sizes, estimated based on the reference GD1 sequence, ranged from 171,612 bp (EBVaGC6) to 171,957 bp (EBVaGC1).

Whole-genome sequencing of EBV enabled the comparison and thus the determination of EBV variations at the genome level. In our study, 961 variations were observed in the EBVaGC1 to 9 genomes in comparison to the reference GD1, including 919 substitutions, 23 insertions, and 19 deletions. Both latent genes and genes encoding tegument proteins in nine GC-EBV genomes were found to harbor the majority of nonsynonymous mutations, accounting for 58.4% (EBVaGC8) to 84.3% (EBVaGC3) of all nonsynonymous mutations detected for each genome.

EBNA1 is essential for maintenance of the EBV episome in latently infected cells and is the only EBV antigen that is consistently expressed in all EBV associated malignancies [36]. Based on the amino acid changes at position 487 in the COOH-terminal region in EBNA1 relative to B95-8 (P-ala), V-val was the most common subtype, accounting for 77.7% of nine GC-EBV strains, followed by P-thrV, accounting for 22.3%. Multiple results showed that V-val is the dominant subtype in Asian regions studies, not only in EBVaGC but also in NPC and healthy donors, while V-val subtype was rarely found in Africa, Europe, and America irrespective of source (lymphoma, NPC, EBVaGC, or healthy donors) [37–39], indicating that polymorphism of EBNA1 subtypes has geographic differences but is not tumor-specific. Apart from changes in the C-terminus, EBNA1 has variations in the N-terminus. Interestingly, we identified two interstrain recombinants at the EBNA1 locus, which provided a further mechanism for the generation of diversity. EBNA1 N-terminus changes have revealed additional variants that were not simply

*Genome-Wide Profiling of Epstein-Barr Virus (EBV) Isolated from EBV-Related Malignancies DOI: http://dx.doi.org/10.5772/intechopen.93244*

classified based on the signature amino acid residue 487 in the C-terminus as widely used previously. The N-terminus changes reinforce the need to evaluate the EBV genome more comprehensively in order to characterize the full extent of EBV genetic diversity. A comprehensive investigation into the functional and immunological impact of the naturally occurred *EBNA1* sequence variations and interstrain recombinants is required to evaluate their possible significance, which may also be helpful for clarifying the association of EBNA1 subtypes and EBVaGC.

### *2.2.2 GDGC1 and GDGC2*

In 2018, NGS was employed to determine the EBV genomes from two EBVaGC specimens, designated as GDGC1 and GDGC2, from Guangdong, China, an endemic area of NPC [21]. Due to the presence of the much more abundant cellular genomic DNA in the DNA preparations, the number of reads belonging to EBV was low, accounting for only 0.02–0.23% of the total reads. However, since the original data were sufficient, the average sequencing depth for genomes GDGC1 and GDGC2 was ~73x and ~24x, respectively, which was sufficient for further analysis. The genome sizes, estimated based on the reference EBV-WT genome sequence, were as follows: GDGC1 (169,611 bp) and GDGC2 (171,299 bp).

The authors reported that a total of 1815 SNPs (146 indels) and 1519 SNPs (106 indels) were found in GDGC1 and GDGC2, respectively, compared with the reference EBV-WT genome. Among these, 1229 SNPs (66 indels) and 1076 SNPs (54 indels) were located in the coding regions for GDGC1 and GDGC2, respectively, while the remaining variations were found in the non-coding regions. Consistent with previous reports [17], there is clear evidence for a higher frequency of SNPs in latent genes, followed by the genes encoding tegument and membrane glycoproteins. In contrast to the frequent mutations that occurred in latent genes, the sequences of promoters and ncRNAs were investigated to be strictly conserved. A few point mutations were found in the sequences of Cp, Qp, Fp and LMP2Ap, and only scattered mutations could be identified in certain ncRNA sequences. Promoters and EBV-generated ncRNAs play important roles in regulating viral processes and in mediating host-virus interactions. Thus, a detailed EBV genomewide analysis of EBVaGC from Guangdong was performed, which would be helpful for further understanding of the relationship between EBV genomic variation and EBVaGC carcinogenesis.

The features of the disease and geographically associated EBV genetic variation as well as the roles that the variation plays in carcinogenesis and evolution remain unclear. A recent study sequenced 95 geographically distinct EBV isolates from EBVaGC biopsies (n = 41) and saliva of healthy donors (n = 54) to detect variants and genes associated with gastric carcinoma from a genome-wide spectrum [20]. BRLF1, BBRF3, and BBLF2/BBLF3 genes had significant associations with gastric carcinoma. LMP1 and BNLF2a genes were strongly geographically associated genes in EBV. The results provided insights into the genetic basis of oncogenic EBV for gastric carcinoma, and the genetic variants associated with gastric carcinoma could serve as biomarkers for oncogenic EBV.

#### **2.3 NKTCL**

Extranodal NKTCL, a rare type of non-Hodgkin lymphoma, is characterized by the presence of EBV in virtually all cases, irrespective of their ethnicity or geographical origin. NKTCL is an aggressive malignancy, predominantly occurs in the nasal, paranasal, and oropharyngeal sites, and is much more prevalent in East Asia and Latin America than in Western countries [40].

Although the association of this B lymphotropic virus with malignancies of T and NK cell origin was quite unexpected, both the presence of virus sequences in tumor cells and the virus's oncogenic potency have led to the hypothesis that whether particular EBV strains are preferentially selected in NKTCL. Pathogenesis and genotype analyses of NKTCL have mainly focused on genetic variations in a small fraction of EBV genes before, which is limited to define the spectrum of diversity within the whole genome of EBV. The genome-wide characteristics of EBV are essential to understand the diversity of strains isolated from NKTCL. In 2019, for the first time, 35 NKTCL-derived EBV genomic landscapes at genome-wide level were simultaneously systematically characterized by two groups.

### *2.3.1 NKTCL-EBV1-NKTCL-EBV8*

Our group directly sequenced EBV-captured DNA from eight primary NKTCL biopsy samples from China using Illumina HiSeq 2500 sequencer platform and presented the eight EBV sequences, designated NKTCL-EBV1-NKTCL-EBV8 [23]. Aiming at knowing the detail of subtype, the obtained DNA sequences were compared with six mostly referenced sequences, including AG876, B95-8, EBV WT, GD1, GD2, and HKNPC1. The GD1 coverage percentages are higher than the rest. The genome sizes, estimated based on the reference GD1 sequence, ranged from 171,590 bp (NKTCL-EBV8) to 172,059 bp (NKTCL-EBV1).

Whole-genome sequence alignments revealed extensive nucleotide variation in the eight NKTCL-EBV genomes. In comparison with the most similar GD1 strain, the NKTCL-EBV1 to NKTCL-EBV8 harbored 2072 variations in total, including 1938 substitutions, 58 insertions, and 76 deletions. Among them, 1218 substitutions, 15 insertions, and 26 deletions were located in the coding regions. The number of the nonsynonymous mutations is highest in the gene regions encoding latent proteins in each of the NKTCL-EBV genomes, followed by genes encoding the tegument protein and membrane glycoproteins.

EBNA1 and LMP1 are the most frequently studied regions to date. Based on the amino acid changes in certain residues of LMP1 and EBNA1, eight NKTCL-EBVs were sorted to China 1 and V-val subtype, respectively. Of interest, EBNA1 of NKTCL-EBV3 sequence showed clustered away from the other seven NKTCL-EBV strains. Analysis of amino acid sequences of EBNA1 supported that EBNA1 of NKTCL-EBV3 may arise from recombination of GD1 and B95-8. Other two commonly classification systems for LMP-1 gene polymorphisms include a 30-bp deletion in the C terminus and the loss of restriction site Xho I in the N terminus of the gene. LMP1 is a key latent protein with abilities to promote cell proliferation and inhibit cell apoptosis in NKTCL. In our study, the LMP1 strain in NKTCL-EBV1-NKTCL-EBV7, but not NKTCL-EBV8, harbored the 30-bp deletion. The variant of 30-bp deletion of LMP1 has been demonstrated that it is associated with poor prognosis of patients with NKTCL, which might serve as a potential marker to monitor treatment [41]. In addition, eight NKTCL-EBV strains had Xho I restriction site loss at exon 1 of the LMP1 gene.

#### *2.3.2 NKTCL-SC01-NKTCL-SC15 and NKTCL-SG01-NKTCL-SG12*

The other group assembled 27 NKTCL-derived EBV genome sequences retrieved from whole-genome sequencing data using the Hiseq sequencer (Illumina), including 15 EBV-positive NKTCL tumor samples from Southern China and 12 samples from Singapore [24]. The average percentage of EBV sequences in WGS data is 0.45% (0.03–1.06%), and the coverage depth is 222.2X in average (26.7X–612.8X). As ~34 kb of 172 kb of EBV genome are repeat regions, which could not be properly

*Genome-Wide Profiling of Epstein-Barr Virus (EBV) Isolated from EBV-Related Malignancies DOI: http://dx.doi.org/10.5772/intechopen.93244*

assembled with short-reads sequencing technology, the groups assigned "N" for these regions and subsequently joined the scaffolds, resulting in EBV genomes with ~172 kb in length.

The authors reported that among the 27 NKTCL samples, in average 1152 EBV SNVs for each sample were determined by aligning the viral reads against the reference EBV-WT genome. The most frequent tumor-specific non-synonymous mutations in NKTCL-derived EBV were located at BPLF1 gene (position 49,790–59,239 bp). An average of 44.8 small indels (<50 bp) of EBV were found in each NKTCL sample, and the 30-bp deletion of LMP1 was commonly found in the samples (21/27), with a frequency consistent with the previous study revealed by using Sanger sequencing [42]. Large deletions of EBV (>1 kb) were found in 10 of 27 NKTCL samples, without any sequencing coverage in the deleted regions. The findings provided insights into the understanding of EBV's role the etiology of NKTCL.

A genome-wide association study of 189 patients with extranodal NKTCL, nasal type and 957 controls from Guangdong province, Southern China was performed to identify common genetic variants affecting individual risk of NKTCL [43]. All cases were genotyped with Illumina Human OmniExpress ZhongHua-8 BeadChip, and population controls were scanned by Illumina OmniHumanExpress-24 V1.0 (both Illumina, San Diego, CA, USA). The findings were validated in four independent case–control series. The SNP with the strongest association was rs9277378 (OR 2.65 [95% CI 2.08–3.37]) located with HLA-DPB1, indicating the importance of HLA-DP antigen presentation in the pathogenesis of NKTCL. The pathogenic subtypes of EBV in NKTCL tumorigenesis should be further explored.

## **3. Phylogenetic analysis of the EBV genomes**

Phylogenetic analysis of EBV genomes could demonstrate detailed overall genomic differences in EBV genome within or beyond subtypes of EBV-associated diseases, thus, EBV genomic similarity is likely to better infer the phylogenetic relatedness among EBV genomes.

Traditionally, EBV has two distinct subtypes, type 1 and type 2. Type 1 EBV (e.g., B95-8, GD1 and Akata) is the main EBV strain prevalent worldwide, while type 2 EBV (e.g., AG876) is abundant only in parts of Africa and New Guinea. Type 1 and type 2 EBV encode different *EBNA2* genes, with only 54% amino acid sequence identity. A recent whole genome sequencing study confirmed that *EBNA2* and *EBNA3* are the only genes that can distinguish type 1 and type 2 EBV strains [16]. Technologies for genome sequencing were currently developed with tools for genome analysis. High-throughput sequencing technology such as illumine dye sequencing was introduced to successfully sequence viral genomes. As exemplary tools for genome analysis, Molecular Evolutionary Genetics Analysis (MEGA) is used for both conducting statistical analysis of molecular evolution and constructing phylogenetic trees [44].

The NPC genomes from Asian EBV strains, including GD1, GD2, and HKNPC1- HKNPC9, are type 1 viruses and were clustered in a branch distant to the non-Asian strains AG876, B95-8 [14]. Analysis of LMP1 and -2 showed a phylogenetic relationship corresponding to the geographical origin of the viral genomes instead of the type 1 and 2 dichotomy, indicating that LMP1 and -2 genes can serve as geographical markers. GD1 seemed to harbor many mutations that were not present in the other Chinese strains. HKNPC6 and -7 genomes, which were isolated from tumor biopsy specimens of advanced metastatic NPC cases, were distinct from the other NPC-EBV genomes. Future work should investigate the relationship between the distinct lineage of EBV and the clinical stages of NPC.

GC-EBV strains, EBVaGC1-EBVaGC9 and GDGC1-GDGC2 involved here, were closely related to all Asian-derived EBV strains, distant to the non-Asian strains, and also showed that the EBV sequences generally clustered in a manner consistent with geographical location [17, 21]. Neighbor-joining trees derived from the sequences of *EBNA2* gene showed that all the GC-EBV genomes are type 1 viruses, clustered in a branch with other type 1 EBV strains, distant to the only type 2 EBV strain, AG876. Phylogenetic trees based on the LMP1 gene and whole EBV genomes indicated that the nine EBVaGC strains were closely related to all Asian-derived EBV strains and distant to the non-Asian strains, suggesting that the LMP1 gene can serve as a geographical marker [17]. This is in line with the previous results from the NPC-EBV genomes [14]. In addition, phylogenetic analyses on GDGC1 and GDGC2 derived from specific EBV-encoded gene suggested the presence of at least two parental lineages of EBV, as GDGC1 and GD2 clustered closely, while GDGC2 and GD1 clustered closely [21].

In our recent study, the phylogenetic trees were conducted based on alignment of eight full-length NKTCL-EBVs and previously published 28 strains [23]. Of note, eight NKTCL-EBVs genomes clearly sort into type 1, based on differences in whole genome and especially EBNA2. Eight NKTCL-EBVs were related to other Asian EBV strains, including EBVaGC1–9, HKNPC1–9, GD1, and GD2 obtained from China, and Akata from Japan, whereas none of the specimens was clustered

#### **Figure 2.**

*Phylogenetic trees of EBV genomes. Phylogenetic analyses were conducted using the neighbor-joining (NJ) algorithm implemented in MEGA software (version 6). Bootstrap analysis of 1000 replicates was performed to determine the confidence.*

*Genome-Wide Profiling of Epstein-Barr Virus (EBV) Isolated from EBV-Related Malignancies DOI: http://dx.doi.org/10.5772/intechopen.93244*

in a branch of non-Asian strains AG876, B95-8, and Mutu. Other group compared the sequences between 27 NKTCL-derived EBV and 164 EBV genome sequences from public database to determine the sequence diversity of EBV [24]. Phylogenetic analysis revealed clear clustering of EBV isolates firstly according to their respective geographic origin; moreover, EBV isolates derived from NKTCL samples tend to cluster closely, apart from clusters by other diseases, supporting the hypothesis of the existence of disease-specific EBV. However, whether the unique EBV has been driving the development of NKTCL or simply adapted to the niche of NKTCL as bystander await further investigations.

In this chapter, phylogenetic analysis was conducted on full-length EBV genomes, including 11 NPC-EBV strains (GD1, GD2, HKNPC1-HKNPC9), 11 GC-EBV strains (EBVaGC1-EBVaGC9, GDGC1-GDGC2), 35 NKTCL-EBV strains (NKTCL-EBV1-NKTCL-EBV8, NKTCL-SC01-NKTCL-SC15, NKTCL-SG01- NKTCL-SG12), B95-8, EBV-WT, Mutu, Akata, and AG876 (**Figure 2**). The result of phylogenetic tree supports the conclusion that EBV infections are more likely affected by different geographic regions rather than particular EBV-associated malignancies.

#### **4. Amino acid changes in CD4<sup>+</sup> and CD8+ T-cell epitopes**

Sequence variations of EBV genes also result in amino acid epitope exchanges, which should have a significant impact on EBV-specific T-cell immunity.

Among the shared non-synonymous SNVs of the Chinese derived GD1, GD2 and HKNPC1 isolates, 34 are associated with known EBV-specific epitopes; 19 and 15 are found in CD8+ and CD4+ epitopes, respectively [12]. HKNPC2-9 genomes harbored nonsynonymous mutations in epitopes specific for both CD4+ and CD8+ T cells [14]. Amino acid changes were found in seven CD8+ epitopes of LMP2, five epitopes of EBNA3A, and three or fewer in other proteins. Thirteen CD4<sup>+</sup> epitopes of EBNA1, six in LMP1, six in LMP2, five in EBNA2, and three or fewer in other proteins contained amino acid changes. Some of the nonsynonymous mutations were affecting multiple epitopes.

EBVaGC shows EBV type I latency neoplasm, in which EBNA1 is expressed in 100% and LMP2A in about half of EBVaGC cases, respectively [45]. Recent studies show that EBNA1, as well as LMP2A, can be presented to both CD4<sup>+</sup> and CD8+ T cells, highlighting its potential importance in the development of therapeutic strategies against EBV-associated malignancies [46, 47]. There is some clear evidence for sequence variation affecting immune recognition of EBNA1 and potential epitope selection for vaccine development [46]. So far, most research on the EBNA1 protein has been focused exclusively on the B95-8 strain alone [46, 47]. Sequence analysis of the gene encoding EBNA1 in EBV isolates from nine EBVaGC specimens has revealed considerable *EBNA1* sequence divergence from the B95-8 strain [17]. Importantly, T cell recognition of EBNA1 epitope might be greatly influenced by this sequence polymorphism as adoptive transfer of EBNA1-targeted T cells has a potential use in immunotherapy of EBV associated carcinomas.

NKTCL is associated with type II EBV latency, in which only restricted EBV antigens, namely EBNA1, and LMP1 and 2, are expressed [48]. These EBV encoded proteins might be the targets of immune recognition during its persistent infection, and their nonsynonymous variations in CD4<sup>+</sup> and CD8<sup>+</sup> T-cell epitopes may affect the efficacy for a cytotoxic T lymphocyte (CTL)-based therapy. Many epitopes were defined and were mapped in EBV antigens and correlated with major histocompatibility complex type in previous studies. In our study, we mainly investigated the amino acid changes in CD4<sup>+</sup> and CD8<sup>+</sup> T-cell epitopes of

EBNA1, LMP1, and LMP2A. Compared with B95-8, amino acids changes were found in 3 CD8<sup>+</sup> epitopes of EBNA1, 8 epitopes of LMP1, and 12 epitopes of LMP2A. Eleven CD4<sup>+</sup> epitopes of EBNA1, 13 in LMP1, and 9 in LMP2A contained amino acids. Some of the nonsynonymous mutations were affecting multiple epitopes [23]. In another study, alterations of the known T-cell epitopes were examined in EBV sequences derived from NKTCL [24]. Alterations of T-cell epitopes were detected in EBV derived from NKTCL samples. Notably, 21 of these epitopes with significant enrichment in NKTCL samples were restricted to six EBV genes, including EBNA3A (G373D, F325L, I333K, L406P, S412R, H464R, M466R, T585I, and A588P), EBNA3B (A399S, V400L, V417L, K424T, Y662D, and K663E), EBNA3C (P916S), BARF1 (V29A), BCRF1 (V6M), and BNRF1 (G456R, S497G, and A1289T).

Therefore, these data have implications for the development of effective prophylactic and therapeutic vaccine approaches targeting the personalized EBV antigens in these aggressive diseases. Adoptive transfer of cytotoxic T cells (CTLs) specific for EBV antigens has proved safe and effective as prophylaxis and treatment for EBV-associated lymphoproliferative disease. Some patients with advanced stage or relapsed EBV-associated malignancies achieved complete remission after treatment with autologous LMP1/2- and EBNA1-specific CTLs or activated by peptides derived from LMP1/2 [49, 50]. Nonetheless, some cases still did not respond to LMP-CTL therapy, and this failure was usually attributed to immune escapes by antigen loss. It is worth noting that all these previous studies used prototype EBV sequence, B95-8, to design full-length LMP epitopes. Therefore, recent work gives an alternative explanation for the lack of tumor response. Whether changes in such epitopes confer immune evasion of the tumor cells may constitute another hypothesis for future testing.

## **5. Genomic integration of viral sequences**

Viral integration into the host genome has been shown to be a causal mechanism that can lead to the development of cancer [51]. Not surprisingly, known tumorassociated viruses, such as EBV, HBV, HPV16 and HPV18, were among the most frequently detected targets [52]. Notably, the approach of WGS is sensitive to detect viruses. This is particularly true for the common integration verified for HBV, HPV16 and HPV18 in a variety of studies [53–55]. The known causal role of HPV16 and HPV18 in several tumor entities, which triggered one of the largest measures in cancer prevention, has been the motivation for extensive elucidation of the pathogenetic processes involved. Integration events with high confidence were demonstrated for HBV (liver cancer), HPV16 and HPV18 (in both cervical and head-and neck carcinoma), however, low-confidence integration events were detected for EBV (gastric cancer and malignant lymphoma) [56].

Comprehensive analyses of WGS datasets may reveal some novel findings on EBV integration. Recently, a comprehensive survey of EBV integration in a variety of human malignancies, including NPC, EBVaGC, and NKTCL was conducted, using EBV genome capture combined with ultra-deep sequencing, which could efficiently detect integrated EBV sequences from background "noise" introduced by nuclear EBV episomes [27]. The EBV integration rates were 25.6% (10/39), 16.0% (4/25), 9.6% (17/177) in the EBVaGC, NKTCL, and NPC tumors, respectively, which were lower than HPV integration in cervical cancer (76.3%) and head and neck squamous cell carcinoma (60.7%), and HBV in hepatocellular carcinoma (92.6%) [54, 57–59]. They found that EBV

*Genome-Wide Profiling of Epstein-Barr Virus (EBV) Isolated from EBV-Related Malignancies DOI: http://dx.doi.org/10.5772/intechopen.93244*

integrations into the introns could decrease the expression of the inflammationrelated genes, TNFAIP3, PARK2, and CDK15, in NPC tumors [27]. The EBV integration breakpoints were frequently at oriP or terminal repeats, and were surrounded by microhomology sequences, consistent with a mechanism for integration involving viral genome replication and microhomology-mediated recombination, which has an important role in the integration of other tumorigenic viruses, HBV and HPV [54, 59]. Meanwhile, researchers also observed integrations of short EBV fragments into human chromosomes, coincident with episomal EBV genomes in NKTCL, and showed that 31 EBV-host integration sites were detected from eight NKTCL samples, and enriched in the repeat regions of human genome, such as SINE, LINE, and satellite [24].

However, there are still few studies on EBV integration based on WGS technology. In addition, authors only selected some potential breakpoints to perform PCR and Sanger sequencing for validating. For example, Xu et al. randomly select 12 integrations from 197 breakpoints identified from NPC and other EBVassociated malignancies, and only 10 breakpoints were successfully validated [27]. As integration of EBV sequence into the host genome and the consequent disruption of the important host genes might represent a novel tumorigenesis mechanism in EBV associated malignancies, all the potential EBV integration breakpoints should be validated and biological function of host genes involved should be further conducted.

### **6. Summary**

In conclusion, full-length EBV genomes isolated from primary NPC, EBVaGC, and NKTCL biopsy specimens have been successfully sequenced and the sequence diversity on a whole-genome level has been analyzed, although their pathogenesis remains to be clarified. Phylogenetic analysis has shown that all aforementioned NPC, GC, and NKTCL-EBV strains are type 1 EBV and close to other Asian subtypes, leading to the conclusion that EBV infections are more likely affected by different geographic regions rather than particular EBV-associated malignancies. In addition, sequence variations of EBV genes also result in amino acid epitope exchanges, which should have a significant impact on EBV-specific T-cell immunity. Recent data have provided optimization proposal for selecting EBV genome for treatment from individual patients or at least predominant strains prevalent in geographical regions instead of commonly used B95-8 genome. We acknowledge that further characterizations of the molecular events would provide more information on the exact mechanisms underlying their pathogenic potentials and clinical significance.

### **Acknowledgements**

This work was supported by grants from National Natural Science Foundation of China (81903155 to Y.L.), and Beijing Municipal Natural Science Foundation (7202023 to Y.L.), and Beijing Hospitals Authority Youth Program (QML20181106 to Y.L.)

#### **Conflict of interest**

The authors declare no conflict of interest.

*Epstein-Barr Virus - New Trends*

## **Author details**

Ying Liu1 , Zheming Lu2 \* and Hongying Huang3 \*

1 Laboratory of Genetics, Key Laboratory of Carcinogenesis and Translational Research (Ministry of Education), Peking University Cancer Hospital and Institute, Beijing, People's Republic of China

2 Laboratory of Biochemistry and Molecular Biology, Key Laboratory of Carcinogenesis and Translational Research (Ministry of Education), Peking University Cancer Hospital and Institute, Beijing, People's Republic of China

3 Department of Pathology, New York University Langone Medical Center, New York, USA

\*Address all correspondence to: luzheming@bjmu.edu.cn; hongying.huang@nyumc.org

© 2020 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

*Genome-Wide Profiling of Epstein-Barr Virus (EBV) Isolated from EBV-Related Malignancies DOI: http://dx.doi.org/10.5772/intechopen.93244*

## **References**

[1] Parkin DM. The global health burden of infection-associated cancers in the year 2002. International Journal of Cancer. 2006;**118**(12):3030-3044

[2] Mesri EA, Feitelson MA, Munger K. Human viral oncogenesis: A cancer hallmarks analysis. Cell Host & Microbe. 2014;**15**(3):266-282

[3] Tarbouriech N, Buisson M, Geoui T, Daenke S, Cusack S, Burmeister WP. Structural genomics of the Epstein-Barr virus. Acta Crystallographica. Section D, Biological Crystallography. 2006;**62**(Pt 10):1276-1285

[4] Swaminathan S. Noncoding RNAs produced by oncogenic human herpesviruses. Journal of Cellular Physiology. 2008;**216**(2):321-326

[5] Chen SJ, Chen GH, Chen YH, Liu CY, Chang KP, Chang YS, et al. Characterization of Epstein-Barr virus miRNAome in nasopharyngeal carcinoma by deep sequencing. PLoS One. 2010;**5**(9):e12745

[6] Farrell PJ. Epstein-Barr virus. The B95-8 strain map. Methods in Molecular Biology. 2001;**174**:3-12

[7] de Jesus O, Smith PR, Spender LC, Elgueta Karstegl C, Niller HH, Huang D, et al. Updated Epstein-Barr virus (EBV) DNA sequence and analysis of a promoter for the BART (CST, BARF0) RNAs of EBV. The Journal of General Virology. 2003;**84**(Pt 6):1443-1450

[8] Dolan A, Addison C, Gatherer D, Davison AJ, McGeoch DJ. The genome of Epstein-Barr virus type 2 strain AG876. Virology. 2006;**350**(1):164-170

[9] Lin Z, Wang X, Strong MJ, Concha M, Baddoo M, Xu G, et al. Whole-genome sequencing of the Akata and Mutu Epstein-Barr virus strains. Journal of Virology. 2013;**87**(2):1172-1182

[10] Zeng MS, Li DJ, Liu QL, Song LB, Li MZ, Zhang RH, et al. Genomic sequence analysis of Epstein-Barr virus strain GD1 from a nasopharyngeal carcinoma patient. Journal of Virology. 2005;**79**(24):15323-15330

[11] Liu P, Fang X, Feng Z, Guo YM, Peng RJ, Liu T, et al. Direct sequencing and characterization of a clinical isolate of Epstein-Barr virus from nasopharyngeal carcinoma tissue by using next-generation sequencing technology. Journal of Virology. 2011;**85**(21):11291-11299

[12] Kwok H, Tong AH, Lin CH, Lok S, Farrell PJ, Kwong DL, et al. Genomic sequencing and comparative analysis of Epstein-Barr virus genome isolated from primary nasopharyngeal carcinoma biopsy. PLoS One. 2012;**7**(5):e36939

[13] Kwok H, Chiang AK. From conventional to next generation sequencing of Epstein-Barr virus genomes. Viruses. 2016;**8**(3):60

[14] Kwok H, Wu CW, Palser AL, Kellam P, Sham PC, Kwong DL, et al. Genomic diversity of Epstein-Barr virus genomes isolated from primary nasopharyngeal carcinoma biopsy samples. Journal of Virology. 2014;**88**(18):10662-10672

[15] Santpere G, Darre F, Blanco S, Alcami A, Villoslada P, Mar Alba M, et al. Genome-wide analysis of wildtype Epstein-Barr virus genomes derived from healthy individuals of the 1,000 genomes project. Genome Biology and Evolution. 2014;**6**(4):846-860

[16] Palser AL, Grayson NE, White RE, Corton C, Correia S, Ba Abdullah MM, et al. Genome diversity of Epstein-Barr virus from multiple tumor types and

normal infection. Journal of Virology. 2015;**89**(10):5222-5237

[17] Liu Y, Yang W, Pan Y, Ji J, Lu Z, Ke Y. Genome-wide analysis of Epstein-Barr virus (EBV) isolated from EBVassociated gastric carcinoma (EBVaGC). Oncotarget. 2016;**7**(4):4903-4914

[18] Wang S, Xiong H, Yan S, Wu N, Lu Z. Identification and characterization of Epstein-Barr virus genomes in lung carcinoma biopsy samples by next-generation sequencing technology. Scientific Reports. 2016;**6**:26156

[19] Tu C, Zeng Z, Qi P, Li X, Yu Z, Guo C, et al. Genome-wide analysis of 18 Epstein-Barr viruses isolated from primary nasopharyngeal carcinoma biopsy specimens. Journal of Virology. 2017;**91**(17):e00301-17

[20] Yao Y, Xu M, Liang L, Zhang H, Xu R, Feng Q, et al. Genome-wide analysis of Epstein-Barr virus identifies variants and genes associated with gastric carcinoma and population structure. Tumour Biology. 2017;**39**(10):1010428317714195

[21] Chen JN, Zhou L, Qiu XM, Yang RH, Liang J, Pan YH, et al. Determination and genome-wide analysis of Epstein-Barr virus (EBV) sequences in EBV-associated gastric carcinoma from Guangdong, an endemic area of nasopharyngeal carcinoma. Journal of Medical Microbiology. 2018;**67**(11):1614-1627

[22] Hong S, Liu D, Luo S, Fang W, Zhan J, Fu S, et al. The genomic landscape of Epstein-Barr virus-associated pulmonary lymphoepithelioma-like carcinoma. Nature Communications. 2019;**10**(1):3108

[23] Lin N, Ku W, Song Y, Zhu J, Lu Z. Genome-wide analysis of Epstein-Barr virus isolated from extranodal

NK/T-cell lymphoma, nasal type. The Oncologist. 2019;**24**(9):e905–e913

[24] Peng RJ, Han BW, Cai QQ, Zuo XY, Xia T, Chen JR, et al. Genomic and transcriptomic landscapes of Epstein-Barr virus in extranodal natural killer T-cell lymphoma. Leukemia. 2019;**33**(6):1451-1462

[25] Xu M, Yao Y, Chen H, Zhang S, Cao SM, Zhang Z, et al. Genome sequencing analysis identifies Epstein-Barr virus subtypes associated with high risk of nasopharyngeal carcinoma. Nature Genetics. 2019;**51**(7):1131-1136

[26] Hui KF, Chan TF, Yang W, Shen JJ, Lam KP, Kwok H, et al. High risk Epstein-Barr virus variants characterized by distinct polymorphisms in the EBER locus are strongly associated with nasopharyngeal carcinoma. International Journal of Cancer. 2019;**144**(12):3031-3042

[27] Xu M, Zhang WL, Zhu Q, Zhang S, Yao YY, Xiang T, et al. Genome-wide profiling of Epstein-Barr virus integration by targeted sequencing in Epstein-Barr virus associated malignancies. Theranostics. 2019;**9**(4):1115-1124

[28] Chen YP, Chan ATC, Le QT, Blanchard P, Sun Y, Ma J. Nasopharyngeal carcinoma. Lancet. 2019;**394**(10192):64-80

[29] de Martel C, Georges D, Bray F, Ferlay J, Clifford GM. Global burden of cancer attributable to infections in 2018: A worldwide incidence analysis. The Lancet Global Health. 2020;**8**(2):e180–e190

[30] Mahdavifar N, Ghoncheh M, Mohammadian-Hafshejani A, Khosravi B, Salehiniya H. Epidemiology and inequality in the incidence and mortality of nasopharynx cancer in Asia. Osong Public Health and Research Perspectives. 2016;**7**(6):360-372

*Genome-Wide Profiling of Epstein-Barr Virus (EBV) Isolated from EBV-Related Malignancies DOI: http://dx.doi.org/10.5772/intechopen.93244*

[31] Young LS, Yap LF, Murray PG. Epstein-Barr virus: More than 50 years old and still providing surprises. Nature Reviews Cancer. 2016;**16**(12):789-802

[32] Shibata D, Weiss LM. Epstein-Barr virus-associated gastric adenocarcinoma. The American Journal of Pathology. 1992;**140**(4):769-774

[33] Tokunaga M, Land CE, Uemura Y, Tokudome T, Tanaka S, Sato E. Epstein-Barr virus in gastric carcinoma. The American Journal of Pathology. 1993;**143**(5):1250-1254

[34] Takada K. Epstein-Barr virus and gastric carcinoma. Molecular Pathology. 2000;**53**(5):255-261

[35] Murphy G, Pfeiffer R, Camargo MC, Rabkin CS. Metaanalysis shows that prevalence of Epstein-Barr virus-positive gastric cancer differs based on sex and anatomic location. Gastroenterology. 2009;**137**(3):824-833

[36] Fukayama M, Ushiku T. Epstein-Barr virus-associated gastric carcinoma. Pathology, Research and Practice. 2011;**207**(9):529-537

[37] Gutierrez MI, Raj A, Spangler G, Sharma A, Hussain A, Judde JG, et al. Sequence variations in EBNA-1 may dictate restriction of tissue distribution of Epstein-Barr virus in normal and tumour cells. The Journal of General Virology. 1997;**78**(Pt 7):1663-1670

[38] Chang KL, Chen YY, Chen WG, Hayashi K, Bacchi C, Bacchi M, et al. EBNA-1 gene sequences in Brazilian and American patients with Hodgkin's disease. Blood. 1999;**94**(1):244-250

[39] Chen YY, Chang KL, Chen WG, Shibata D, Hayashi K, Weiss LM. Epstein-Barr virus-associated nuclear antigen-1 carboxy-terminal gene sequences in Japanese and American patients with gastric

carcinoma. Laboratory Investigation. 1998;**78**(7):877-882

[40] Xiong J, Zhao W. What we should know about natural killer/T-cell lymphomas. Hematological Oncology. 2019;**37**(Suppl 1):75-81

[41] Halabi MA, Jaccard A, Moulinas R, Bahri R, Al Mouhammad H, Mammari N, et al. Clonal deleted latent membrane protein 1 variants of Epstein-Barr virus are predominant in European extranodal NK/T lymphomas and disappear during successful treatment. International Journal of Cancer. 2016;**139**(4):793-802

[42] Nagamine M, Takahara M, Kishibe K, Nagato T, Ishii H, Bandoh N, et al. Sequence variations of Epstein-Barr virus LMP1 gene in nasal NK/T-cell lymphoma. Virus Genes. 2007;**34**(1):47-54

[43] Li Z, Xia Y, Feng LN, Chen JR, Li HM, Cui J, et al. Genetic risk of extranodal natural killer T-cell lymphoma: A genome-wide association study. The Lancet Oncology. 2016;**17**(9):1240-1247

[44] Tamura K, Peterson D, Peterson N, Stecher G, Nei M, Kumar S. MEGA5: Molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Molecular Biology and Evolution. 2011;**28**(10):2731-2739

[45] Chen JN, Ding YG, Feng ZY, Li HG, He D, Du H, et al. Association of distinctive Epstein-Barr virus variants with gastric carcinoma in Guangzhou, southern China. Journal of Medical Virology. 2010;**82**(4):658-667

[46] Bell MJ, Brennan R, Miles JJ, Moss DJ, Burrows JM, Burrows SR. Widespread sequence variation in Epstein-Barr virus nuclear antigen 1 influences the antiviral T cell response. The Journal of Infectious Diseases. 2008;**197**(11):1594-1597

[47] Icheva V, Kayser S, Wolff D, Tuve S, Kyzirakos C, Bethge W, et al. Adoptive transfer of epstein-barr virus (EBV) nuclear antigen 1-specific t cells as treatment for EBV reactivation and lymphoproliferative disorders after allogeneic stem-cell transplantation. Journal of Clinical Oncology. 2013;**31**(1):39-48

[48] Fox CP, Haigh TA, Taylor GS, Long HM, Lee SP, Shannon-Lowe C, et al. A novel latent membrane 2 transcript expressed in Epstein-Barr virus-positive NK- and T-cell lymphoproliferative disease encodes a target for cellular immunotherapy. Blood. 2010;**116**(19):3695-3704

[49] Bollard CM, Gottschalk S, Torrano V, Diouf O, Ku S, Hazrat Y, et al. Sustained complete responses in patients with lymphoma receiving autologous cytotoxic T lymphocytes targeting Epstein-Barr virus latent membrane proteins. Journal of Clinical Oncology. 2014;**32**(8):798-808

[50] Cho SG, Kim N, Sohn HJ, Lee SK, Oh ST, Lee HJ, et al. Long-term outcome of Extranodal NK/T cell lymphoma patients treated with postremission therapy using EBV LMP1 and LMP2aspecific CTLs. Molecular Therapy. 2015;**23**(8):1401-1409

[51] Tang KW, Larsson E. Tumour virology in the era of high-throughput genomics. Philosophical Transactions of the Royal Society of London. Series B, Biological Sciences. 2017;**372**(1732):20160265

[52] Plummer M, de Martel C, Vignat J, Ferlay J, Bray F, Franceschi S. Global burden of cancers attributable to infections in 2012: A synthetic analysis. The Lancet Global Health. 2016;**4**(9):e609-e616

[53] Jiang Z, Jhunjhunwala S, Liu J, Haverty PM, Kennemer MI, Guan Y, et al. The effects of hepatitis B virus integration into the genomes of hepatocellular carcinoma patients. Genome Research. 2012;**22**(4):593-601

[54] Hu Z, Zhu D, Wang W, Li W, Jia W, Zeng X, et al. Genome-wide profiling of HPV integration in cervical cancer identifies clustered genomic hot spots and a potential microhomologymediated integration mechanism. Nature Genetics. 2015;**47**(2):158-163

[55] Liu Y, Lu Z, Xu R, Ke Y. Comprehensive mapping of the human papillomavirus (HPV) DNA integration sites in cervical carcinomas by HPV capture technology. Oncotarget. 2016;**7**(5):5852-5864

[56] Zapatka M, Borozan I, Brewer DS, Iskar M, Grundhoff A, Alawi M, et al. The landscape of viral associations in human cancers. Nature Genetics. 2020;**52**(3):320-330

[57] Koneva LA, Zhang Y, Virani S, Hall PB, McHugh JB, Chepeha DB, et al. HPV integration in HNSCC correlates with survival outcomes, immune response signatures, and candidate drivers. Molecular Cancer Research. 2018;**16**(1):90-102

[58] Sung WK, Zheng H, Li S, Chen R, Liu X, Li Y, et al. Genome-wide survey of recurrent HBV integration in hepatocellular carcinoma. Nature Genetics. 2012;**44**(7):765-769

[59] Zhao LH, Liu X, Yan HX, Li WY, Zeng X, Yang Y, et al. Genomic and oncogenic preference of HBV integration in hepatocellular carcinoma. Nature Communications. 2016;**7**:12992

## **Chapter 2**

## EBV Genome Mutations and Malignant Proliferations

*Sylvie Ranger-Rogez*

## **Abstract**

The Epstein-Barr virus (EBV) is a DNA virus with a relatively stable genome. Indeed, genomic variability is reported to be around 0.002%. However, some regions are more variable such as those carrying latency genes and specially *EBNA1*, *-2*, *-LP*, and *LMP1*. Tegument genes, particularly *BNRF1*, *BPLF1*, and *BKRF3*, are also quite mutated. For a long time, it has been considered for this ubiquitous virus, which infects a very large part of the population, that particular strains could be the cause of certain diseases. However, the mutations found, in some cases, are more geographically restricted rather than associated with proliferation. In other cases, they appear to be involved in oncogenesis. The objective of this chapter is to provide an update on changes in viral genome sequences in malignancies associated with EBV. We focused on describing the structure and function of the proteins corresponding to the genes mentioned above in order to understand how certain mutations of these proteins could increase the tumorigenic character of this virus. Mutations described in the literature for these proteins were identified by reporting viral and/or cellular functional changes as they were described.

**Keywords:** Epstein-Barr virus, lymphoma, carcinoma, mutation, sequence, next generation sequencing

### **1. Introduction**

Epstein-Barr virus (EBV), a ubiquitous gamma-herpesvirus, infects the vast majority of the worldwide human population. This virus was initially discovered in cultured lymphoma cells from patients with Burkitt's lymphoma (BL) in 1964 [1]. During the primary infection, EBV infects epithelial cells of the oropharynx where it actively replicates and also infects B cells where it establishes a life-long latency in the form of an episome located in the host cell nucleus. During latency, EBV may produce nine viral latency proteins, including six so-called "Epstein-Barr Nuclear Antigens" (EBNA1, -2, -3A, -3B, -3C, and -LP), involved in transcriptional regulation, and three "Latent Membrane Proteins" (LMP1, -2A, and -2B), mimicking signals needed for B cell maturation, as well as two small noncoding RNAs (EBER-1 and EBER-2), BamHI-A rightward transcripts (BARTs), and miRNAs. Four different latency programs can be identified, based on the proteins that are expressed (**Table 1**). EBV primary infection, which occurs more often in childhood, is usually asymptomatic in children, whereas it may be responsible for infectious mononucleosis (IM) in teenagers or young adults in western countries. In addition to this nonmalignant disease, EBV can also be associated with diverse malignant pathologies. In particular, EBV is involved in the development of several malignancies of lymphoid


#### **Table 1.**

*Proteins expressed during the different latency programs.*

origin including endemic Burkitt's lymphoma [2], nasal NK/T lymphoma [3], some Hodgkin's lymphoma [4], and B- or T-cell lymphoproliferations in immunocompromised patients [5]. It is also implicated in epithelial malignancies such as undifferentiated nasopharyngeal carcinoma (NPC) [6] and 10% of cases of gastric carcinoma [7]. Although populations from all geographic areas are infected by the virus, the incidence of the pathologies in which it occurs varies significantly depending on the region [8]. For example, BL occurs mainly in children living in sub-Saharan Africa [9], and the prevalence of NPC is particularly high in adults living in Southern China, Southeast Asia, and Northern Africa [10]. The differences observed in the geographic distribution of these pathologies suggest that there could be various genetic variants of EBV, of different global distributions, and with different levels of transforming capacity. This question of a specific disease variant is raised by many authors and is still being debated. In this chapter, we wish to take inventory of the state of knowledge concerning the variability observed on the most mutated genes among all EBV genes and the possible implications in human pathology.

### **2. Evolving knowledge of the EBV genome**

The fact that the viral genome is relatively large (175 kb), that it is made up of DNA, therefore less variable than if it was an RNA genome, and that it carries repetitive regions, limited its sequencing for a long time. The first published sequences were small fragments of the B95-8 genome; then, the entire B95-8 genome was sequenced in 1984 [11]. The B95-8 strain was the first cultured EBV cell line able to secrete large amounts of viral particles into the culture medium. It was originally obtained from a spontaneous human lymphoblastoid cell line (LCL) established from a North American case of infectious mononucleosis, the 883L cell line, whose virus was used to transform lymphocytes from a cotton top marmoset. Since it was the first strain with a fully published genome, B95-8 has been extensively studied and mapped for transcripts, promoters, and open reading frames.

This first EBV whole genome sequencing was followed by others, and complete viral genome sequences of the cell lines AG876, originating from a Ghanaian case of African BL [12] and GD1, obtained from cord B cells infected with EBV from saliva of an NPC patient in Guangzhou, China [13] were published. Sequences of some genes, mainly latency genes, were also studied, especially in lines established from patients [14, 15]. B95-8, GD1, and AG876 were sequenced by conventional

#### *EBV Genome Mutations and Malignant Proliferations DOI: http://dx.doi.org/10.5772/intechopen.93194*

shotgun sequencing (Sanger's method). The comparison of sequences obtained for various cell lines revealed the existence of two types of EBV: type 1 or A, of which B95-8 can be considered as the prototype, and type 2 or B, exemplified by AG876. The main difference between the two types concerns the *EBNA2* gene, with only 70% identity at the nucleotide level and 54% identity in the protein sequence [16]. Additional variations have also been observed in the *EBNA3* genes, but to a lesser extent: 10, 12, and 19% of base pair differences for *EBNA3A, 3B,* and *3C,* respectively [17]. The comparison of viral sequences also highlighted that the B95-8 cell line has a significant 11.8 kb deletion (positions 139,724–151,554) corresponding to some of the *BART* miRNA genes, one of the origins of lytic replication [11], the *LF2* and *LF3* genes, and a part of the *LF1* gene. More complete sequence comprising the B95-8 sequence supplemented with a Raji fragment at the level of deletion has been constructed. It was annotated in 2010 as RefSeq HHV4 (EBV) sequence NC\_007605 and is now used as a wild-type strain reference [18].

As adaptation of the virus to *in vitro* culture is possible, thus generating a bias in the results, some authors have preferred to sequence the viral genome directly in samples from patients. Therefore, the sequences GD2, from a Guangzhou NPC biopsy, and HKNPC1, from a Hong Kong NPC biopsy, were published [19, 20], both using a more recent sequencing technique, "next generation sequencing" (NGS). This technology can be used directly on samples or after enrichment, which avoids artifacts due to cellular DNA. Enrichment can be achieved by PCR or cloning into F-factor plasmids, but most frequently, it is carried out using target DNA capture by hybridization. NGS delivers a wealth of information and requires extensive bioinformatic analysis. This technology has made it possible to rapidly increase the number of fully sequenced viral genomes originating from healthy subjects or patients and thus obtain more information.

## **3. The most variable regions of the genome**

Authors who sequenced the entire viral genome and analyzed the genomic variations came to the conclusion that the latent genes harbored the highest numbers of nonsynonymous mutations [20–24]. For example, Liu et al. [25] compared the sequences of nine strains of EBV to GD1, of which they were most closely related, and showed that latency genes were the most mutated. In this study, latent and tegument genes were found to harbor 58.4 to 84.3% of all nonsynonymous mutations detected for each genome. Santpere et al. [26] found that latent genes were twice as mutated as lytic genes. The observation that the latent genes harbor more nucleotide diversity than lytic genes was made regardless of the type of pathology: nasopharyngeal carcinoma [20, 21], NK/T lymphoma [27], endemic Burkitt's lymphoma [22], Hodgkin's lymphoma [22], posttransplant lymphoproliferative disease [22], gastric carcinoma [25], lung carcinoma [23], and also strains originating from infectious mononucleosis [22] or healthy subjects [26]. Why latent genes are the most variable is not clear today. By analyzing their data according to the Yang model [28], Santpere et al. [26] showed that the lytic genes had an evolutionary constraint close to that of the host: a strong purifying selection was objectified for 11 lytic genes. However, signatures of accelerated protein evolution rates were found in coding regions related to virus attachment and entry into host cells. The latency genes, on the other hand, show a positive selection, perhaps in relation to the MHC, which can be the cause of their large diversity. Changes in amino acids (aa) often occur in immune epitopes. Amino acid changes in CD8+ epitopes were described in all latent proteins, while changes in CD4+ epitopes were shown only for EBNA1 and -2 and LMP1 and -2 [20]. However, most codons of the *EBNA3* gene

#### *Epstein-Barr Virus - New Trends*

under positive selection are not cytotoxic T-lymphocyte epitopes: either there are epitopes not described to date or the selection relates to other functionalities. The selection of mutants may depend on a difference in immunity in relation to the geography and/or capacity of a strain to infect and persist.

## **4. Variability of main latency proteins**

After the virus enters a host cell, the genome circularizes through recombination of the terminal repeats (TRs) located at each end of the genome to form an episome that will be chromatinized and methylated in the same way as the human genome. Latent transcription programs in B cells are due to the differential activity of epigenetically regulated promoters and take place in three successive waves. The EBNA2 and EBNA-LP, as well as BHRF1, a bcl2 homolog, are the first viral proteins to be expressed, under the dependence of Wp promoter. The two expressed EBNAs and the cellular factor recombination signal-binding protein for immunoglobulin Kappa J region (RBP-Jk) activate then the Cp promoter, which drives the expression of all of the EBNA proteins, while Wp becomes progressively hypermethylated; the transcription will gradually be under Cp control. Subsequently, LMP1, LMP2A, and LMP2B proteins are expressed due to activation of their respective promoters. During latency I or II, Qp promoter controls EBNA1 expression, and Cp methylation is responsible for the five other EBNA silencing. Methylation does not control the Qp promoter, which is switched off by binding to a repressor protein.

As previously developed, latency proteins show the most sequence variations, and among them, EBNA1, EBNA2, EBNA-LP, and LMP1 are the most mutated. The main properties of these proteins are reported in **Table 2**.

#### **4.1 EBNA1**

EBNA1, expressed in both latent and lytic EBV infections, was the first EBV protein detected. EBNA1, whose structure (**Figure 1**) and functions have largely been studied [29, 30], is a 641 aa protein. However, EBNA1 proteins frequently exhibit size variations due to differing numbers of gly-ala repeats (aa 89–325). During latency, EBNA1 is the only protein expressed in all forms of latency in proliferating cells and also in all EBV associated malignancies. EBNA1, which acts as a homodimer, is essential for initiating EBV episome replication before mitosis, once per cell cycle, and mitotic segregation of EBV episomes, thus for the maintenance of EBV episome in latently infected cells [31]. The EBNA1 DNA-binding domain is essential but not sufficient for the replication function, and the N-terminal half of EBNA1 is also required. Two EBNA1 regions (aa 8–67 and aa 325–376) are particularly important for this activity, and the point mutations G81 or G425 enhance EBNA1 dependent DNA replication. Inversely, the EBNA1 aa 395–450 region mediates an interaction with the human ubiquitin-specific protease, USP7, which may negatively regulate replication. The partitioning of EBV episomes in two dividing cells requires two viral components: the *ori P* FR element and EBNA1, mainly the central Gly-Arg region aa 325–376 and secondarily the aa 8–67 sequence. EBNA1 also activates the expression of other latency genes participating in immortalization: the regions involved are the central Gly-Arg sequence and the 61–89 region. Interaction with the recognition sites located on FR, DS of *ori P*, and Bam-HI-Q takes place through binding sites located in the C-terminal of EBNA1 (aa 459-607), sequence which also mediates the dimerization of EBNA1 (aa 504–604). Through its interaction with both human casein kinase CK2 (aa 383–395) and cellular ubiquitin-specific protease USP7 (aa 442–448), EBNA1 is also able to disrupt promyelocytic leukemia protein


#### *EBV Genome Mutations and Malignant Proliferations DOI: http://dx.doi.org/10.5772/intechopen.93194*

#### **Table 2.**

*Main properties of proteins developed in this chapter.*

#### **Figure 1.**

*Schematic representation of basic structure of EBNA1 protein with the different motifs and their position. Gly-Arg: region rich in Gly-Arg; Gly-Ala: Gly-Ala repeats; CK2: interaction with human casein kinase, CK2; USP7: interaction with the human ubiquitin specific protease, USP7; DNA binding: DNA-binding domain; Dimerization: region that mediates the dimerization of EBNA1. The different mutations discussed are noted.*

(PML) bodies and degrade PML. In addition to its role in latent infection, EBNA1 can therefore participate in lytic infection by overcoming suppression by PML proteins [32]. Indeed, PML proteins and nuclear bodies were found to suppress lytic infection by EBV. Recently [33], organization in an oligomeric hexameric ring form was described for the EBNA1 DNA-binding domain, the oligomeric interface pivoting around residue T585. Mutations occurring on this residue had both positive and negative effects on EBNA1-dependent DNA replication and episome maintenance.

Based on polymorphisms observed at 15 codons, Bhatia et al. [14] reported two strains named P (prototype) and V (variant), each having two subtypes defined by the aa at position 487 (P-ala, P-thr, V-pro, and V-leu). They detected mostly the P-thr and the V-leu variants, respectively, in African and American BL tumors,

but these findings were not confirmed by another group who reported different spectra of EBNA1 subtypes according to different geographical areas in both healthy patients and BL tumors [34]. A fifth subtype, V-val, was later recognized in South-East Asia and was found to be prevalent in NPC samples by numerous authors [20, 35–37]. These findings suggest that the V-val variant might adapt particularly well to the nasopharyngeal epithelium or that this strain possesses an increased oncogenic potential. Indeed, most of the variant codons, localized in the DNA-binding domain, may have an impact on the EBV phenotype resulting in impaired ability to transform B-lymphocytes [30]. However, other reports observed that this subtype had no tumor-specific expression [38], and it is likely that it probably represents a dominant EBNA1 subtype in Asian regions, not found in other areas of the world [8, 23, 25]. The P-thr subtype is the most commonly observed in peripheral blood of American and African subjects as well as in African tumors. In our experience, P-thr is also the most prevalent in France and particularly in the course of lymphoproliferative diseases.

Apart from these mutations, others have been reported. For example, Borozan et al. [39] looked at gastric carcinomas and mainly found two mutations already described in NPC, H418L and A439T, located outside the DNA-binding domain and common in both NPC and GC but uncommon in other EBV isolates, from lymphomas or healthy subjects. They also described a new mutation, T85A, positioned in the region required for transcriptional activation of other latency genes and thus able to modify this function. Wang et al. [23] described the substitution T585I. T585 is subject to substitutions, and T585 polymorphism is found frequently in NPC tumors and Burkitt's lymphoma. T585I was previously found, and this strain was defective in replication and maintenance of the viral episome [40], as well as deficient in suppressing lytic cycle gene transcription and lytic DNA replication.

In summary, EBNA1 V-val variant seems to be a geographic variant almost exclusively present in South-East Asia. Conversely, mutations T85 and T585, which occur in functional regions of the protein, could have biological consequences and especially the substitution T585I, which promotes lytic replication and is found in NPC.

#### **4.2 EBNA2**

EBNA2, a 487 aa protein, is expressed *in vivo* during latency III shortly after infection of B cells or in lymphomas occurring in immunocompromised patients and in LCL. As mentioned above, the variations in EBNA2 make it possible to classify EBV as types 1 and 2 (or A and B) since only 70% identity at the nucleotide level and 54% homology in the protein sequence were observed. The overall structure of the EBNA2 protein (**Figure 2**) is characterized by poly-P and poly-RG areas, this last one being a protein-protein and protein-nucleic acid interaction domain important for efficient cell growth transformation, and nine regions conserved throughout the gene [41]. EBNA2 acts principally as a transcription factor and contains three categories of domains critical for its transcription regulation function: transactivation domains (TAD), self-association domains (SAD), and nuclear localization signals (NLS). EBNA2 does not bind directly to DNA. It uses cell proteins as adapters to access viral or cellular enhancer and promoter sites. The C-terminal TAD (aa 448–471) is able to recruit components of basic transcriptional machinery as well as chromatin modifiers and can bind to the viral coactivator EBNA-LP, while the N-terminal TAD (aa 1–58) cannot bind EBNA-LP, although its activity can be enhanced by this protein. Two SADs (aa 1–58 and 97–121), separated by the polyproline stretch, were identified in the N-terminal region [42]. An additional third one has been reported, localized in a nonconserved region, and flanked by the second SAD and the adapter region [43]. EBNA2 contributes to B-cell immortalization, and

*EBV Genome Mutations and Malignant Proliferations DOI: http://dx.doi.org/10.5772/intechopen.93194*

#### **Figure 2.**

*Schematic representation of basic structure of EBNA2 protein with the different motifs and their position. The two transactivation domains (TADs), the three self-association domains (SADs), and the two nuclear localization signals (NLSs) are mentioned. Poly P: area rich in P; PolyRG: area rich in RG; CR5: conserved region 5, which interacts with SKIP (Ski-interacting protein); CR6: conserved region 6, which interacts with CBF1 (C promoter-binding factor 1). The different mutations discussed are mentioned.*

it has been demonstrated that type 1 EBV, which is predominantly found in EBVassociated diseases, immortalizes B cells *in vitro* much more efficiently than type 2 [44], which is predominantly determined by sequence variation in the C-terminus of EBNA2 [45]. During the early events of EBV infection in resting B cells, EBNA2 initiates the transcription of a cascade of primary and secondary viral and cellular target genes and therefore is responsible for the initiation of immortalization by reprogramming the resting state into a proliferative state. For this, EBNA2 interacts with chromatin remodelers and as a transcription factor cofactor [46]. Mühe et al. [47] demonstrated that the first 150 N-terminal aa of EBNA2 are important for the initiation of immortalization. EBNA2 is also involved in immortalization maintenance; the region implicated here (aa 295–378) includes the conserved regions CR5 (aa 295–307) and CR6 (aa 320–326), particularly important for this function. CR5 mediates the contact between EBNA2 and SKIP (Ski-interacting protein), and CR6 is the CBF1 (C promoter-binding factor 1) or RBP-Jk targeting domain. Mechanisms to initiate and maintain B cell immortalization are not completely understood today.

Wang et al. [41], working on 25 EBV-associated GCs, 56 NPCs, and 32 throat washings from healthy donors in Northern China, described 4 EBNA2 subtypes according to the presence of a deletion, namely subtypes E2-A (no aa deletion), E2-B (aa 294Q deletion), E2-C (aa 357K and 358G deletion), and E2-D (aa 357K, 358G, and 294Q deletion). The E2-A subtype exhibited six nonsilent mutations, P291T, R413G, I438L, E476G, P484H, and I486T; the substitution P291T was present in six NPC E2-D and six NPC E2-C. The substitution R413G was detected in E2-C for one patient. They found that E2-A and E2-C were dominant in the samples they analyzed and that the E2-D pattern was detected only in the NPC specimens. The mutation R163M was detected in all samples. This mutation has previously been described worldwide and in different diseases.

Mutations 357 and 358 occurred in the RG domain (aa 335–362), a downregulator of EBNA2 activation of the LMP1 promoter [48]. Moreover, aa 357–363 (KGKSRDK) constitutes the PKC phosphorylation site, which can reduce the amounts of EBNA2/CBF1 complex formed. EBNA2 is suspected to be involved in the development of malignancies as a result of sequence variations most frequently affecting its regulation function.

Interestingly, *EBNA2* entire-gene deletion has been shown in some endemic BL cell lines such as P3HR1, Daudi, Sav, Oku, and Ava [49]; it remains to determine if this deletion occurs classically *in vivo* in African BL.

In short, geographic variants were not formally demonstrated for EBNA2. Among the described mutations, the most interesting are those occurring in the PKC phosphorylation site because they can activate the Cp and/or LMP1p and thus increase the production of latency proteins.

#### **4.3 EBNA-LP (EBNA-leader protein)**

EBNA-LP, like EBNA2 and concomitantly with EBNA2, is expressed shortly after the infection of B cells in healthy individuals as well as in EBV-related malignant diseases in immunodeficient patients and LCLs. EBNA-LP acts mostly as a coactivator of the transcriptional activator EBNA2, thus inducing the expression of some cellular genes, including *cyclin D2* [50], or viral genes, that is, *LMP1* [51], *LMP2b,* and *Cp* and therefore having an important role in B cell immortalization. EBNA-LP also can directly interact with several cell proteins such as tumor suppressors or proteins involved in apoptosis or cell cycle regulation.

EBNA-LP is comprised of a variable number of 66 aa repetitive units, corresponding to the variable number of W1 and W2 exons located in the EBV internal repeat IR1, followed by a unique 45 aa domain, encoded by two unique 3′ exons Y1 and Y2 (**Figure 3**). Therefore, EBNA-LP protein may vary in size according to the number of W1–W2 repeats contained in each EBV isolate. By convention, the protein annotation is based on a single W repeat isoform (**Figure 4**). In this configuration, the protein has 110 aa. Conserved regions were identified in the N extremity of the protein (CR1 to CR3, respectively, aa 11–33, 45–52, and 55–62, implicated in EBNA2 binding), and in the C-terminal region (CR4 and CR5, respectively, aa 76–82 and 101–110). CR3 and a serine within W2 (S35) were demonstrated to be important for EBNA2 coactivation. EBV-mediated B cell immortalization maps to the W1W2 repeated domains and requires at least two IR1 repetitions to be effective, but a number greater than or equal to 5 is optimal [53]. Some interactions with cell proteins are mediated by the repeated W1W2 N-terminus [54]. *EBNA-LP* gene transcription initiates from the W promoter (Wp) residing in each IR1 repeat during the early stages of infection, and multiple EBNA-LP protein isoforms are produced. During the later stages of infection and in LCLs, transcription initiates from the C promotor (Cp) [55]. The level of transcription initiated by Cp compared to Wp varies according to different circumstances [56].

About 15% of BL tumors host a virus, which uses exclusively the W promoter, expressing an EBV atypical latency program [49], harboring EBNA1, EBNA3A, 3B, 3C, and a truncated form of EBNA-LP. In these cases, EBV genome lacks the *EBNA2*

#### **Figure 3.**

*Schematic representation of the IR1 region of EBV genome (according to Ref. [52]). The promoters Wp, Cp, and Qp are represented, as well as the different proteins expressed according to the stage of infection.*

gene and the unique Y1Y2 exons of *EBNA-LP*. This was firstly described in P3HR1 and Daudi BL cell lines [57]. Subsequently, these cells were shown to be more resistant to apoptosis than cells infected by wild-type virus, what would be related to the truncated shape of EBNA-LP.

Given the difficulty of sequencing repetitive regions, only few authors have sequenced the IR1 region, including the EBNA-LP coding region. Previous studies identified two EBNA-LP distinct isoforms, type 1 and type 2 variants, based on the presence of G8/T12 or V8/A12 in exon W1 [58]. The Q54R substitution was also described in exon W2 from an African type 2 spontaneous lymphoblastoid cell line LCL [59]. Despite this, a high degree of conservation was reported for the Wp promoter and the W1-W2 intron, while the most diversity was observed for the BWRF1 ORF, which only shows 80% homology between various strains, and for Y exons [60]. The sequence variations in the Y exons, and especially the Y2 exon, made it possible to define four main subgroups, called A, B, C, and Z. The Akata strain belongs to subgroup A and B95-8 to subgroup B. Subgroup Z is found in type 2 EBVs, and the C subtype is characterized by V95E and V102I. Finally, it has been reported that tumor-derived strains are more prone to interstrain genetic exchange in IR1 [60].

## **4.4 LMP1**

LMP1 is considered to be the main oncogenic protein in EBV. LMP1 is a multifunctional self-aggregating protein essential for the transformation of human B cells and rodent fibroblasts [61]. It is a 386 aa protein comprising a 24 aa cytosolic N-terminal (NT) segment, a 162 aa portion consisting of six transmembrane (TM) domains, and a 200 aa cytosolic C-terminal (CT) domain (**Figure 5**) [62]. The NT domain plays an important role in the orientation and anchoring of LMP1 to the membrane and its constitutive aggregation, thus contributing to the transforming function of LMP1 [63]. The TM region is involved in the localization of LMP1 at the level of lipid rafts in the membrane, thus inducing its clustering to activate signaling from the CT tail. It is remarkable that the F38LWY41 pattern in the first

**Figure 4.**

*Sequence of EBNA-LP protein, with the position of the corresponding exons opposite. Conserved regions are represented as well as the key positions. Phosphorylated serins are mentioned by an asterisk.*

#### **Figure 5.**

*Schematic representation of basic structure of LMP1 protein with the different motifs and their position. TM1–6: transmembrane domains 1–6. The FWLY pattern in TM1 and W98 in TM3 are essential for the association of TM1–2 with TM3–6 and oligomerization signaling. CTAR1–3: carboxyl-terminal activating regions 1–3. PQQAT pattern is necessary for the attachment of TRAF adapters. YYD pattern is essential for binding the TNF receptor-associated death domain (TRADD) adapter.*

transmembrane fragment (TM1) and a second pattern consisting of aa W98 in TM3 are essential for the association of TM domains (1–2) with TM domains (3–6) as well as for the oligomerization and signaling of LMP1 [64]. The CT part is involved in the activation of LMP1-induced cell signaling pathways, including two important regions, CTAR1/TES1 and CTAR2/TES2 (Carboxyl-Terminal Activating Region/Transformation Effector Site) critical for EBV-mediated B-cell growth transformation [65]. Together, these regions mimic CD40, a member of the tumor necrosis factor (TNF) receptor family and key B-cell costimulatory receptor, thus enabling the recruitment of cell adapters associated with the TNF receptor family, TNF receptor-associated factors (TRAFs). The CTAR1 region includes the P204- X-Q206-X-T208 consensus pattern necessary for the attachment of TRAF adapters, specifically TRAF1, TRAF2, TRAF3, and TRAF5 [66]. Within the CTAR2 region, the Y384-Y385-D386 pattern is essential for binding the TNF receptor-associated death domain (TRADD) adapter. There is a third region, CTAR3 (aa 232–350), that is not essential for *in vitro* B cell immortalization and is less well known [67]. In this region located between CTAR1 and CTAR2 (aa 253–302), a variable number of repeat 11 aa elements (4 repeats for B95-8) exist.

LMP1 acts principally as a viral pseudoreceptor, which regulates host cell signal transduction by constitutive activation of cell pathways as mitogen-activated protein kinase (MAPK) pathways and principally the extracellular regulated kinases 1 and 2 (ERK1/2), c-Jun amino-terminal kinases 1–3 (JNK1–3), and p38 isoform pathways. LMP1 also induces the phosphatidylinositol 3-kinase (PI3K) pathway, which contributes to survival signals [68] and transcription of activator protein 1 (AP1) [69], PI3K, and AP1 pathways, therefore playing a major role in proliferation and cell cycle control. LMP1 is also responsible for the activation of JAK/STAT and interferon regulatory factor 7 (IRF7) pathways and for aberrant constitutive NF-kB activation. Indeed, the CTAR1 PXQXT pattern is able to engage TRAFs, leading finally to the activation of noncanonical NF-kB pathway that controls processing of the NF-kB2/p100 precursor [70]. The CTAR2 YYD pattern is in turn implicated in the activation of the canonical NF-kB pathway [71] after binding of tumor necrosis factor receptor superfamily member 1A (TNFRSF1A)-associated via TRADD and receptor-inter-acting protein 1 (RIP1). A wider region of LMP1 seems to be responsible for binding RIP1 (aa 351–386), compared to TRADD (aa 375–386) [72]. NF-kB is considered to be the principal factor by which LMP1 regulates gene expression and modifies cell behavior [73]. Activation of NF-kB is associated with upregulation of anti-apoptotic genes [32, 74] and downregulation of pro-apoptotic factors, as well as induction of tumorigenesis-associated B-cell activation markers [75, 76]. CTAR3, less well defined, seems to activate SUMOylation pathways and participate in the maintenance of EBV latency and control of cell migration, a hallmark of oncogenesis [77, 78].

Besides its ability to transform B cells, during the latency state, LMP1 seems also to be able to facilitate the release of virions from B cells during lytic replication [32].

Variations in the LMP1 sequence have been widely studied, particularly in the context of its impact on clinical occurrence or evolution. A 30 bp deletion (del30), resulting in a 10 aa loss in the C-terminal (aa 343–352), was first described in the Cao cell isolate from a Chinese NPC [79]. In addition, this isolate harbored numerous substitutions. A high prevalence of the same deletion, as reviewed by Chang et al. [8], was found in Asian NPC biopsy tissues [80, 81], in lymphomas and EBV-related gastric cancers from Eastern Asia [82] and in Asian nasal NK/T-cell lymphomas [83, 84]. Del30 was shown to be often associated with the G335D mutation in NPC, and such strains were reported to have a greater transforming activity *in vitro* than the reference LMP1 [85, 86]. If the 30 bp deletion is partly localized to CTAR2, it does not alter NF-kB activation [87] and finally does not modify signaling



**Table 3.** *LMP1 mutations described in the literature.*

#### *EBV Genome Mutations and Malignant Proliferations DOI: http://dx.doi.org/10.5772/intechopen.93194*

properties [88]. However, it is clear that strains bearing del30 are selected over the wt-LMP1 variants in NK/T-cell lymphomas [83] and NPC tumors [89]. Given that del30 strains have been currently detected in normal carriers [90] or in various EBVassociated diseases [91], and, because of a low prevalence of del30 strains in samples from Africa, North America, and Europe [8, 92], it is generally admitted that LMP1 del30 may represent a geographic polymorphism rather than a disease-associated polymorphism [93]. In a study, we carried out in France in patients with NK/T lymphoma, we found a del30 EBV in 4/4 biopsies studied and in 46.1% of total blood samples analyzed, while in a control population, the deletion was present in 4.8% of cases [94]. Other deletions were also described, such as the rare C terminal 69 bp deletion reported to weakly activate the AP1 transcription factor [95], or the 15 bp deletion (aa 275–279) frequently encountered in Western Europe [94].

Otherwise, numerous substitutions have been described in LMP1 (**Table 3**), particularly in the N-terminal extremity. Some authors have made attempts to classify viral strains by taking into account these substitutions with the aim of highlighting a viral implication in certain pathologies [99]. Thus, Mainou and Raab-Traub [88] classified EBV into seven variants, namely Alaskan, China 1, China 2, Med+, Med-, NC, and B95-8, all having the same *in vitro* transforming potential and signaling properties. Zuercher et al. [98] mentioned two polymorphisms, I124V/I152L and F144I/D150A/L151I, which seem to be markers of increased NF-kB activation *in vitro*. Lei et al. [96] distinguished four models according to the substitutions occurring in both the *LMP1* gene and its promoter. The patients suffering from NPC that they studied all carried a strain belonging to pattern B, while the BLs were distributed among the four patterns. Many authors recognize two evolutionarily distinct clusters, Asian-derived EBV strains including GD2, HKNPC1, and Akata strains and non-Asian and African/American strains including AG876, B95-8, and Mutu strains, suggesting that the *LMP1* gene could be used as a geographic marker [25, 97].

Finally, it should be noted that LMP1 carries a molecular signature of accelerated evolution rate probably due to positive selection as deduced from a significant proportion of nonsignificant variations [26].

So, regarding LMP1, which is the most oncogenic latency protein, two geographic clusters appear to exist corresponding to an Asian variant and a non-Asiatic variant. The described 30 bp deletion is mainly present on Asian strains, and it shows an obvious tropism for nasopharynx. Although many substitutions have been described, little work is done to analyze changes in LMP1 properties based on these substitutions. NPC could be associated with a particular strain, but this remains to be confirmed.

## **5. Variability of tegument proteins**

After the latency proteins, the tegument proteins carry the most changes, and among them, the most mutated are BNRF1, BPLF1, and BKRF3, which will be detailed, as well as BBRF2. This latter protein appears to play an important role in viral infectivity [100], but its structure and function are poorly known today. For this reason, BBRF2 will not be developed here.

## **5.1 BNRF1**

EBV major tegument protein BNRF1 contains 1318 aa, and its structure is shown schematically in **Figure 6**. BNRF1 is a member of a protein family with homology to the cellular purine biosynthesis enzyme FGARAT. BNRF1 is involved in the

#### **Figure 6.**

*Schematic representation of basic structure of BNRF1 protein with the different motifs and their position. H3.3 and H4 regions, respectively, involved in binding to H3.3 and H4. DID: DAXX-interaction domain, domain implicated in binding to DAXX (death-domain associated protein-6) histone chaperone. PurM-like domain and GATase domain were noted, as well as the different mutations discussed.*

establishment of latency and cell immortalization by hijacking the antiviral DAXX (death domain-associated protein-6) histone chaperone [101]. BNRF1 seems to have lost conventional purine biosynthesis activity. It forms a stable quaternary complex with DAXX histone-binding domain (HBD), H3.3 and H4 [102], responsible for BNRF1 localization to PML nuclear bodies involved in antiviral intrinsic resistance and transcriptional repression of host cells. In the presence of BNFR1, DAXX can no longer collaborate with ATRX to assemble histone variant H3.3 into repressive chromatin at GC-rich repetitive DNA. Binding to DAXX, histone H3.3 and histone H4 occur, respectively, via the BNRF1 DAXX interaction domain (DID) (aa 360–600) and BNRF1 residues 40–52 and 99–102. Huang et al. [102] demonstrated that the quaternary complex formation is abrogated when dual mutations V546D/L548D and D568A/D569A occurred on BRNF1 DID and is partially diminished *in vitro* in case of dual mutations Y390A/K461A and V546S/L548S on BNRF1 DID. BNRF1 mutations at K461A, Y390A/K461A, V546S/L548S or Y390A, V546A/ L548A, and D568A/569A moderately or severely reduced BNRF1 colocalization at PML nuclear bodies, respectively. A PurM-like domain (610–976) and a GATase domain (1037–1318) were defined. It has also recently been shown that BNRF1 can cause an abnormal increase in the number of cellular centrioles [103]. This phenomenon can lead to aneuploidy or structural chromosome abnormalities and, possibly, to carcinogenesis. The gene regions concerned have not been described.

BNRF1 is reported to be one of the most frequently mutated tegument proteins. It is interesting to note that a nonsense mutation was described in C666–1, an EBVpositive NPC cell line, with no major structural alterations in the BNRF1-deleted virus [92].

So, the mutations described for BNRF1 do not appear to correspond to a particular geographical distribution. On the other hand, some mutations seem to be able to modify DNA chromatinization, thus affecting the transcription, and therefore have important consequences on cell functioning.

### **5.2 BPLF1**

BPLF1, the largest EBV protein (3149 aa), is a late lytic tegument protein. BPLF1 possesses a deubiquitinating (DUB) activity. BPLF1 is able to downregulate viral ribonucleotide reductase (RR) activity, by deubiquitination of the large subunit RR1 [104], and to specifically deubiquitinate proliferating cell nuclear antigen (PCNA), a DNA polymerase processivity factor, thus disrupting the repair of damaged DNA [105]. By triggering activation of repair pathways and co-opting DNA repair and replication factors, the virus could create genomic instability. The DUB activity is carried by the first 246 aa of the N-terminal region, and the C61 residue of the catalytic triad (Cys-His-Asp) is essential for activity [104]. BPLF1 relocalizes

#### *EBV Genome Mutations and Malignant Proliferations DOI: http://dx.doi.org/10.5772/intechopen.93194*

Pol to nuclear sites of viral DNA production, thereby bypassing DNA damage [106]. This mechanism contributes to efficient production of infectious virus.

BPLF1 is also able to deubiquinate cell factors, such as TRAF6, NEMO, and IkBα, leading to TLR signaling inhibition through both MyD88- and TRIF-dependent pathways, thus decreasing innate immune responses by reduced NF-kB activation and proinflammatory cytokine production [107]. It is noteworthy that the same catalytic active site also carries a deneddylating activity shown to target cullin ring ligases, potentially affecting viral replication and infectivity [108]. The role of BPLF1 to help drive human B-cell immortalization and lymphoma formation has also been discussed [109].

Sequencing of various viral strains has shown that BPLF1 is one of the proteins with the greatest number of changes [20, 24, 110]. Most of these mutations are not analyzed in detail, but Kwok et al. [21], working on the sequences of eight NPC biopsy specimens, reported two nonsynonymous mutations in the N-terminal region of the protein that exhibit deubiquitinating activity. The same finding was reported by Simbiri et al. [110], who also described 3 C-terminal mutations (L2935P, P2987L, and R3005Q ). A single-nucleotide deletion coupled with a singlenucleotide insertion three nucleotides away was reported by Zeng et al. [13] in a NPC strain. As a result, two aa substitutions (GA/EG) were predicted to occur. Tu et al. [24] undertook phylogenetic analysis based on several reported EBV genome sequences and some major genes as *BPLF1*. They observed that EBV Asian subtypes clustered as a separate branch from the non-Asian ones.

So, as with other proteins, it seems that the Asian strains carry a protein different from the other strains. Substitutions occurring in the region carrying the deubiquitinase activity could have biological consequences.

#### **5.3 BKRF3**

BKRF3 is a small protein (255 aa), which belongs to the early lytic gene family, and encodes an uracil-DNA glycosylase (UDG), which removes inappropriate uracil residues from DNA. BKRF3 excises uracil bases incorporated in double-stranded DNA due to uracil misincorporation or more often cytosine deamination [111, 112]. BKRF3 participates in DNA replication and repair and prevents viral DNA mutagenesis. BKRF3 shares substantial similarity in overall structure with the one UDG family. Four of the five catalytic motifs are completely conserved (aa 90–94, 110–114, 146–149, 191–192), whereas the fifth domain (aa 213–229) carries a sevenresidue insertion in the leucine loop [113]. In addition, the 29 N-terminal aa carry a nuclear localization signal (sequence KRKQ ). Only changes in BKRF3 that do not severely affect viral replication can be retained, but it may be considered that these mutations cause a change in virus-cell interrelations.

## **6. Conclusion**

The aim of this chapter was to take stock of the most frequently observed variations in the EBV genome and more particularly to see if some of these variations are considered to be involved in tumor pathology. The candidate viral genes concerned are numerous; those developed here are the most affected, and the mutations reported in the literature have been identified. Some mutations have been well studied, in particular as regards their impact on the structure or functionality of the protein or the cellular consequences of these modifications. However, most mutations have only been described. If a tumorigenic impact of viral mutations is not yet

certain, many authors agree that geographic variants exist, and it seems clear that Asian strains have different characteristics from non-Asian strains. Further work is necessary to complete the mass of information and analysis, not at the level of one or several genes, but at the level of the entire genome.

## **Author details**

Sylvie Ranger-Rogez1,2

1 Department of Virology, University Hospital Dupuytren, Limoges, France

2 Faculty of Pharmacy, UMR CNRS 7276, UMR INSERM 1262, Limoges, France

\*Address all correspondence to: sylvie.rogez@unilim.fr

© 2020 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

*EBV Genome Mutations and Malignant Proliferations DOI: http://dx.doi.org/10.5772/intechopen.93194*

## **References**

[1] Epstein MA, Achong BG, Barr YM. Virus particles in cultured lymphoblasts from BURKITT'S lymphoma. Lancet. 1964;**1**:702-703. DOI: 10.1016/ s0140-6736(64)91524-7

[2] Zur Hausen H, Schulte-Holthausen H. Presence of EB virus nucleic acid homology in a "virusfree" line of Burkitt tumour cells. Nature. 1970;**227**:245-248. DOI: 10.1038/227245a0

[3] Aozasa K, Takakuwa T, Hongyo T, Yang W-I. Nasal NK/T-cell lymphoma: Epidemiology and pathogenesis. International Journal of Hematology. 2008;**87**:110-117. DOI: 10.1007/ s12185-008-0021-7

[4] Weiss LM, Movahed LA, Warnke RA, Sklar J. Detection of Epstein-Barr viral genomes in reed-Sternberg cells of Hodgkin's disease. The New England Journal of Medicine. 1989;**320**:502-506. DOI: 10.1056/ NEJM198902233200806

[5] Hamilton-Dutoit SJ, Pallesen G, Franzmann MB, Karkov J, Black F, Skinhøj P, et al. AIDS-related lymphoma. Histopathology, immunophenotype, and association with Epstein-Barr virus as demonstrated by in situ nucleic acid hybridization. The American Journal of Pathology. 1991;**138**:149-163

[6] Klein G, Giovanella BC, Lindahl T, Fialkow PJ, Singh S, Stehlin JS. Direct evidence for the presence of Epstein-Barr virus DNA and nuclear antigen in malignant epithelial cells from patients with poorly differentiated carcinoma of the nasopharynx. Proceedings of the National Academy of Sciences of the United States of America. 1974;**71**:4737- 4741. DOI: 10.1073/pnas.71.12.4737

[7] Imai S, Koizumi S, Sugiura M, Tokunaga M, Uemura Y, Yamamoto N, et al. Gastric carcinoma: Monoclonal epithelial malignant cells expressing Epstein-Barr virus latent infection protein. Proceedings of the National Academy of Sciences of the United States of America. 1994;**91**:9131-9135. DOI: 10.1073/pnas.91.19.9131

[8] Chang CM, Yu KJ, Mbulaiteye SM, Hildesheim A, Bhatia K. The extent of genetic diversity of Epstein-Barr virus and its geographic and disease patterns: A need for reappraisal. Virus Research. 2009;**143**:209-221. DOI: 10.1016/j. virusres.2009.07.005

[9] Ziegler JL. Burkitt's lymphoma. The New England Journal of Medicine. 1981;**305**:735-745. DOI: 10.1056/ NEJM198109243051305

[10] Kumar S, Mahanta J. Aetiology of nasopharyngeal carcinoma. A review. Indian Journal of Cancer. 1998;**35**:47-56

[11] Baer R, Bankier AT, Biggin MD, Deininger PL, Farrell PJ, Gibson TJ, et al. DNA sequence and expression of the B95-8 Epstein-Barr virus genome. Nature. 1984;**310**:207-211. DOI: 10.1038/310207a0

[12] Dolan A, Addison C, Gatherer D, Davison AJ, McGeoch DJ. The genome of Epstein-Barr virus type 2 strain AG876. Virology. 2006;**350**:164-170. DOI: 10.1016/j.virol.2006.01.015

[13] Zeng M-S, Li D-J, Liu Q-L, Song L-B, Li M-Z, Zhang R-H, et al. Genomic sequence analysis of Epstein-Barr virus strain GD1 from a nasopharyngeal carcinoma patient. Journal of Virology. 2005;**79**:15323-15330. DOI: 10.1128/ JVI.79.24.15323-15330.2005

[14] Bhatia K, Raj A, Guitierrez MI, Judde JG, Spangler G, Venkatesh H, et al. Variation in the sequence of Epstein Barr virus nuclear antigen 1 in normal peripheral blood lymphocytes and in Burkitt's lymphomas. Oncogene. 1996;**13**:177-181

[15] Walling DM, Shebib N, Weaver SC, Nichols CM, Flaitz CM, Webster-Cyriaque J. The molecular epidemiology and evolution of Epstein-Barr virus: Sequence variation and genetic recombination in the latent membrane protein-1 gene. The Journal of Infectious Diseases. 1999;**179**:763-774. DOI: 10.1086/314672

[16] Adldinger HK, Delius H, Freese UK, Clarke J, Bornkamm GW. A putative transforming gene of Jijoye virus differs from that of Epstein-Barr virus prototypes. Virology. 1985;**141**:221-234. DOI: 10.1016/0042-6822(85)90253-3

[17] Sample J, Young L, Martin B, Chatman T, Kieff E, Rickinson A, et al. Epstein-Barr virus types 1 and 2 differ in their EBNA-3A, EBNA-3B, and EBNA-3C genes. Journal of Virology. 1990;**64**:4084-4092

[18] Tzellos S, Farrell PJ. Epstein-Barr virus sequence variation-biology and disease. Pathogens. 2012;**1**:156-174. DOI: 10.3390/pathogens1020156

[19] Liu P, Fang X, Feng Z, Guo Y-M, Peng R-J, Liu T, et al. Direct sequencing and characterization of a clinical isolate of Epstein-Barr virus from nasopharyngeal carcinoma tissue by using next-generation sequencing technology. Journal of Virology. 2011;**85**:11291-11299. DOI: 10.1128/ JVI.00823-11

[20] Kwok H, Tong AHY, Lin CH, Lok S, Farrell PJ, Kwong DLW, et al. Genomic sequencing and comparative analysis of Epstein-Barr virus genome isolated from primary nasopharyngeal carcinoma biopsy. PLoS One. 2012;**7**:e36939. DOI: 10.1371/journal.pone.0036939

[21] Kwok H, Wu CW, Palser AL, Kellam P, Sham PC, Kwong DLW, et al. Genomic diversity of Epstein-Barr

virus genomes isolated from primary nasopharyngeal carcinoma biopsy samples. Journal of Virology. 2014;**88**:10662-10672. DOI: 10.1128/ JVI.01665-14

[22] Palser AL, Grayson NE, White RE, Corton C, Correia S, Ba Abdullah MM, et al. Genome diversity of Epstein-Barr virus from multiple tumor types and normal infection. Journal of Virology. 2015;**89**:5222-5237. DOI: 10.1128/ JVI.03614-14

[23] Wang S, Xiong H, Yan S, Wu N, Lu Z. Identification and characterization of Epstein-Barr virus genomes in lung carcinoma biopsy samples by nextgeneration sequencing technology. Scientific Reports. 2016;**6**:26156. DOI: 10.1038/srep26156

[24] Tu C, Zeng Z, Qi P, Li X, Guo C, Xiong F, et al. Identification of genomic alterations in nasopharyngeal carcinoma and nasopharyngeal carcinomaderived Epstein-Barr virus by wholegenome sequencing. Carcinogenesis. 2018;**39**:1517-1528. DOI: 10.1093/carcin/ bgy108

[25] Liu Y, Yang W, Pan Y, Ji J, Lu Z, Ke Y. Genome-wide analysis of Epstein-Barr virus (EBV) isolated from EBVassociated gastric carcinoma (EBVaGC). Oncotarget. 2016;**7**:4903-4914. DOI: 10.18632/oncotarget.6751

[26] Santpere G, Darre F, Blanco S, Alcami A, Villoslada P, Mar Albà M, et al. Genome-wide analysis of wildtype Epstein-Barr virus genomes derived from healthy individuals of the 1,000 genomes project. Genome Biology and Evolution. 2014;**6**:846-860. DOI: 10.1093/gbe/evu054

[27] Peng R-J, Han B-W, Cai Q-Q, Zuo X-Y, Xia T, Chen J-R, et al. Genomic and transcriptomic landscapes of Epstein-Barr virus in extranodal natural killer T-cell lymphoma. Leukemia.

*EBV Genome Mutations and Malignant Proliferations DOI: http://dx.doi.org/10.5772/intechopen.93194*

2019;**33**:1451-1462. DOI: 10.1038/ s41375-018-0324-5

[28] Yang Z. PAML 4: Phylogenetic analysis by maximum likelihood. Molecular Biology and Evolution. 2007;**24**:1586-1591. DOI: 10.1093/ molbev/msm088

[29] Frappier L. The Epstein-Barr virus EBNA1 protein. Scientifica (Cairo). 2012;**2012**:438204. DOI: 10.6064/2012/438204

[30] Wilson JB, Manet E, Gruffat H, Busson P, Blondel M, Fahraeus R. EBNA1: Oncogenic activity, immune evasion and biochemical functions provide targets for novel therapeutic strategies against Epstein-Barr virusassociated cancers. Cancers (Basel). 2018;**10**(4):109. 130 pages. DOI: 10.3390/cancers10040109

[31] Young LS, Murray PG. Epstein-Barr virus and oncogenesis: From latent genes to tumours. Oncogene. 2003;**22**:5108-5121. DOI: 10.1038/ sj.onc.1206556

[32] Li C-W, Jheng B-R, Chen B-S. Investigating genetic-and-epigenetic networks, and the cellular mechanisms occurring in Epstein-Barr virus-infected human B lymphocytes via big data mining and genome-wide two-sided NGS data identification. PLoS One. 2018;**13**:e0202537. DOI: 10.1371/journal. pone.0202537

[33] Deakyne JS, Malecka KA, Messick TE, Lieberman PM. Structural and functional basis for an EBNA1 hexameric ring in Epstein-Barr virus episome maintenance. Journal of Virology. 2017;**91**(19):17 pages. DOI: 10.1128/JVI.01046-17

[34] Habeshaw G, Yao QY, Bell AI, Morton D, Rickinson AB. Epstein-Barr virus nuclear antigen 1 sequences in endemic and sporadic Burkitt's lymphoma reflect virus strains prevalent in different geographic areas. Journal of Virology. 1999;**73**:965-975

[35] Gutiérrez MI, Raj A, Spangler G, Sharma A, Hussain A, Judde JG, et al. Sequence variations in EBNA-1 may dictate restriction of tissue distribution of Epstein-Barr virus in normal and tumour cells. The Journal of General Virology. 1997;**78**(Pt 7):1663-1670. DOI: 10.1099/0022-1317-78-7-1663

[36] Wang W-Y, Chien Y-C, Jan J-S, Chueh C-M, Lin J-C. Consistent sequence variation of Epstein-Barr virus nuclear antigen 1 in primary tumor and peripheral blood cells of patients with nasopharyngeal carcinoma. Clinical Cancer Research. 2002;**8**:2586-2590

[37] Zhang X-S, Wang H-H, Hu L-F, Li A, Zhang R-H, Mai H-Q, et al. V-val subtype of Epstein-Barr virus nuclear antigen 1 preferentially exists in biopsies of nasopharyngeal carcinoma. Cancer Letters. 2004;**211**:11-18. DOI: 10.1016/j. canlet.2004.01.035

[38] Sandvej K, Zhou XG, Hamilton-Dutoit S. EBNA-1 sequence variation in Danish and Chinese EBV-associated tumours: Evidence for geographical polymorphism but not for tumourspecific subtype restriction. The Journal of Pathology. 2000;**191**:127- 131. DOI: 10.1002/(SICI)1096- 9896(200006)191:2<127::AID-PATH614>3.0.CO;2-E

[39] Borozan I, Zapatka M, Frappier L, Ferretti V. Analysis of Epstein-Barr virus genomes and expression profiles in gastric adenocarcinoma. Journal of Virology. 2018;**92**(2):17 pages. DOI: 10.1128/JVI.01239-17

[40] Dheekollu J, Malecka K, Wiedmer A, Delecluse H-J, Chiang AKS, Altieri DC, et al. Carcinoma-risk variant of EBNA1 deregulates Epstein-Barr virus episomal latency. Oncotarget. 2017;**8**:7248-7264. DOI: 10.18632/ oncotarget.14540

[41] Wang X, Wang Y, Wu G, Chao Y, Sun Z, Luo B. Sequence analysis of Epstein-Barr virus EBNA-2 gene coding amino acid 148-487 in nasopharyngeal and gastric carcinomas. Virology Journal. 2012;**9**:49. DOI: 10.1186/1743-422X-9-49

[42] Friberg A, Thumann S, Hennig J, Zou P, Nössner E, Ling PD, et al. The EBNA-2 N-terminal transactivation domain folds into a dimeric structure required for target gene activation. PLoS Pathogens. 2015;**11**:e1004910. DOI: 10.1371/journal.ppat.1004910

[43] Tsui S, Schubach WH. Epstein-Barr virus nuclear protein 2A forms oligomers in vitro and in vivo through a region required for B-cell transformation. Journal of Virology. 1994;**68**:4287-4294

[44] Rickinson AB, Young LS, Rowe M. Influence of the Epstein-Barr virus nuclear antigen EBNA 2 on the growth phenotype of virus-transformed B cells. Journal of Virology. 1987;**61**:1310-1317

[45] Tzellos S, Correia PB, Karstegl CE, Cancian L, Cano-Flanagan J, McClellan MJ, et al. A single amino acid in EBNA-2 determines superior B lymphoblastoid cell line growth maintenance by Epstein-Barr virus type 1 EBNA-2. Journal of Virology. 2014;**88**:8743-8753. DOI: 10.1128/ JVI.01000-14

[46] Lu F, Chen H-S, Kossenkov AV, DeWispeleare K, Won K-J, Lieberman PM. EBNA2 drives formation of new chromosome binding sites and target genes for B-cell master regulatory transcription factors RBP-jκ and EBF1. PLoS Pathogens. 2016;**12**:e1005339. DOI: 10.1371/journal.ppat.1005339

[47] Mühe J, Wang F. Species-specific functions of Epstein-Barr virus nuclear antigen 2 (EBNA2) reveal dual roles for initiation and maintenance of B cell immortalization. PLoS Pathogens.

2017;**13**:e1006772. DOI: 10.1371/journal. ppat.1006772

[48] Peng C-W, Zhao B, Kieff E. Four EBNA2 domains are important for EBNALP coactivation. Journal of Virology. 2004;**78**:11439-11442. DOI: 10.1128/JVI.78.20.11439-11442.2004

[49] Kelly G, Bell A, Rickinson A. Epstein-Barr virus-associated Burkitt lymphomagenesis selects for downregulation of the nuclear antigen EBNA2. Nature Medicine. 2002;**8**:1098- 1104. DOI: 10.1038/nm758

[50] Sinclair AJ, Palmero I, Peters G, Farrell PJ. EBNA-2 and EBNA-LP cooperate to cause G0 to G1 transition during immortalization of resting human B lymphocytes by Epstein-Barr virus. The EMBO Journal. 1994;**13**:3321-3328

[51] Nitsche F, Bell A, Rickinson A. Epstein-Barr virus leader protein enhances EBNA-2-mediated transactivation of latent membrane protein 1 expression: A role for the W1W2 repeat domain. Journal of Virology. 1997;**71**:6619-6628

[52] Kempkes B et al. EBNA2 and its coactivator EBNA-LP. In: Münz C, editor. Epstein Barr Virus. Vol. 2. One Herpes Virus: Many Diseases. Heidelberg, Berlin: Springer; 2015. pp. 35-59

[53] Tierney RJ, Kao K-Y, Nagra JK, Rickinson AB. Epstein-Barr virus BamHI W repeat number limits EBNA2/ EBNA-LP coexpression in newly infected B cells and the efficiency of B-cell transformation: A rationale for the multiple W repeats in wild-type virus strains. Journal of Virology. 2011;**85**:12362-12375. DOI: 10.1128/ JVI.06059-11

[54] Han I, Harada S, Weaver D, Xue Y, Lane W, Orstavik S, et al. EBNA-LP associates with cellular proteins

*EBV Genome Mutations and Malignant Proliferations DOI: http://dx.doi.org/10.5772/intechopen.93194*

including DNA-PK and HA95. Journal of Virology. 2001;**75**:2475-2481. DOI: 10.1128/JVI.75.5.2475-2481.2001

[55] Woisetschlaeger M, Yandava CN, Furmanski LA, Strominger JL, Speck SH. Promoter switching in Epstein-Barr virus during the initial stages of infection of B lymphocytes. Proceedings of the National Academy of Sciences of the United States of America. 1990;**87**:1725-1729. DOI: 10.1073/pnas.87.5.1725

[56] Chelouah S, Cochet E, Couvé S, Balkaran S, Robert A, May E, et al. New interactors of the truncated EBNA-LP protein identified by mass spectrometry in P3HR1 Burkitt's lymphoma cells. Cancers (Basel). 2018;**10**(12):14 pages. DOI: 10.3390/cancers10010012

[57] Jones MD, Foster L, Sheedy T, Griffin BE. The EB virus genome in Daudi Burkitt's lymphoma cells has a deletion similar to that observed in a non-transforming strain (P3HR-1) of the virus. The EMBO Journal. 1984;**3**:813-821

[58] Finke J, Rowe M, Kallin B, Ernberg I, Rosén A, Dillner J, et al. Monoclonal and polyclonal antibodies against Epstein-Barr virus nuclear antigen 5 (EBNA-5) detect multiple protein species in Burkitt's lymphoma and lymphoblastoid cell lines. Journal of Virology. 1987;**61**:3870-3878

[59] McCann EM, Kelly GL, Rickinson AB, Bell AI. Genetic analysis of the Epstein-Barr virus-coded leader protein EBNA-LP as a co-activator of EBNA2 function. The Journal of General Virology. 2001;**82**:3067-3079. DOI: 10.1099/0022-1317-82-12-3067

[60] Ba Abdullah MM, Palermo RD, Palser AL, Grayson NE, Kellam P, Correia S, et al. Heterogeneity of the Epstein-Barr virus (EBV) major internal repeat reveals evolutionary mechanisms of EBV and a functional defect in the

prototype EBV strain B95-8. Journal of Virology. 2017;**91**(23):25 pages. DOI: 10.1128/JVI.00920-17

[61] Dirmeier U, Neuhierl B, Kilger E, Reisbach G, Sandberg ML, Hammerschmidt W. Latent membrane protein 1 is critical for efficient growth transformation of human B cells by Epstein-Barr virus. Cancer Research. 2003;**63**:2982-2989

[62] Li H-P, Chang Y-S. Epstein-Barr virus latent membrane protein 1: Structure and functions. Journal of Biomedical Science. 2003;**10**:490-504. DOI: 10.1007/bf02256110

[63] Coffin WF, Erickson KD, Hoedt-Miller M, Martin JM. The cytoplasmic amino-terminus of the latent membrane protein-1 of Epstein-Barr virus: Relationship between transmembrane orientation and effector functions of the carboxy-terminus and transmembrane domain. Oncogene. 2001;**20**:5313-5330. DOI: 10.1038/ sj.onc.1204689

[64] Yasui T, Luftig M, Soni V, Kieff E. Latent infection membrane protein transmembrane FWLY is critical for intermolecular interaction, raft localization, and signaling. Proceedings of the National Academy of Sciences of the United States of America. 2004;**101**:278-283. DOI: 10.1073/ pnas.2237224100

[65] Izumi KM, Kaye KM, Kieff ED. The Epstein-Barr virus LMP1 amino acid sequence that engages tumor necrosis factor receptor associated factors is critical for primary B lymphocyte growth transformation. Proceedings of the National Academy of Sciences of the United States of America. 1997;**94**:1447-1452. DOI: 10.1073/ pnas.94.4.1447

[66] Mosialos G, Birkenbach M, Yalamanchili R, VanArsdale T, Ware C, Kieff E. The Epstein-Barr virus transforming protein LMP1 engages signaling proteins for the tumor necrosis factor receptor family. Cell. 1995;**80**:389-399. DOI: 10.1016/0092-8674(95)90489-1

[67] Izumi KM, Cahir McFarland ED, Riley EA, Rizzo D, Chen Y, Kieff E. The residues between the two transformation effector sites of Epstein-Barr virus latent membrane protein 1 are not critical for B-lymphocyte growth transformation. Journal of Virology. 1999;**73**:9908-9916

[68] Dawson CW, Tramountanis G, Eliopoulos AG, Young LS. Epstein-Barr virus latent membrane protein 1 (LMP1) activates the phosphatidylinositol 3-kinase/Akt pathway to promote cell survival and induce actin filament remodeling. The Journal of Biological Chemistry. 2003;**278**:3694-3704. DOI: 10.1074/jbc.M209840200

[69] Kieser A, Kilger E, Gires O, Ueffing M, Kolch W, Hammerschmidt W. Epstein-Barr virus latent membrane protein-1 triggers AP-1 activity via the c-Jun N-terminal kinase cascade. The EMBO Journal. 1997;**16**:6478-6485. DOI: 10.1093/emboj/16.21.6478

[70] Lavorgna A, Harhaj EW. EBV LMP1: New and shared pathways to NF-κB activation. Proceedings of the National Academy of Sciences of the United States of America. 2012;**109**:2188-2189. DOI: 10.1073/pnas.1121357109

[71] Gewurz BE, Mar JC, Padi M, Zhao B, Shinners NP, Takasaki K, et al. Canonical NF-kappaB activation is essential for Epstein-Barr virus latent membrane protein 1 TES2/CTAR2 gene regulation. Journal of Virology. 2011;**85**:6764-6773. DOI: 10.1128/ JVI.00422-11

[72] Eliopoulos AG, Young LS. LMP1 structure and signal transduction. Seminars in Cancer Biology. 2001;**11**:435-444. DOI: 10.1006/ scbi.2001.0410

[73] Cahir-McFarland ED, Carter K, Rosenwald A, Giltnane JM, Henrickson SE, Staudt LM, et al. Role of NF-kappa B in cell survival and transcription of latent membrane protein 1-expressing or Epstein-Barr virus latency III-infected cells. Journal of Virology. 2004;**78**:4108-4119. DOI: 10.1128/jvi.78.8.4108-4119.2004

[74] Calderwood MA, Venkatesan K, Xing L, Chase MR, Vazquez A, Holthaus AM, et al. Epstein-Barr virus and virus human protein interaction maps. Proceedings of the National Academy of Sciences of the United States of America. 2007;**104**:7606-7611. DOI: 10.1073/pnas.0702332104

[75] Liu M-T, Chang Y-T, Chen S-C, Chuang Y-C, Chen Y-R, Lin C-S, et al. Epstein-Barr virus latent membrane protein 1 represses p53-mediated DNA repair and transcriptional activity. Oncogene. 2005;**24**:2635-2646. DOI: 10.1038/sj.onc.1208319

[76] Wang LW, Jiang S, Gewurz BE. Epstein-Barr virus LMP1-mediated oncogenicity. Journal of Virology. 2017;**91**(21):11 pages. DOI: 10.1128/ JVI.01718-16

[77] Bentz GL, Whitehurst CB, Pagano JS. Epstein-Barr virus latent membrane protein 1 (LMP1) C-terminal-activating region 3 contributes to LMP1-mediated cellular migration via its interaction with Ubc9. Journal of Virology. 2011;**85**:10144- 10153. DOI: 10.1128/JVI.05035-11

[78] Bentz GL, Moss CR, Whitehurst CB, Moody CA, Pagano JS. LMP1-induced sumoylation influences the maintenance of Epstein-Barr virus latency through KAP1. Journal of Virology. 2015;**89**:7465-7477. DOI: 10.1128/ JVI.00711-15

[79] Hu LF, Zabarovsky ER, Chen F, Cao SL, Ernberg I, Klein G, et al. Isolation and sequencing of the *EBV Genome Mutations and Malignant Proliferations DOI: http://dx.doi.org/10.5772/intechopen.93194*

Epstein-Barr virus BNLF-1 gene (LMP1) from a Chinese nasopharyngeal carcinoma. The Journal of General Virology. 1991;**72**(Pt 10):2399-2409. DOI: 10.1099/0022-1317-72-10-2399

[80] Miller WE, Edwards RH, Walling DM, Raab-Traub N. Sequence variation in the Epstein-Barr virus latent membrane protein 1. The Journal of General Virology. 1994;**75**(Pt 10):2729-2740. DOI: 10.1099/0022-1317-75-10-2729

[81] Tan E-L, Peh S-C, Sam C-K. Analyses of Epstein-Barr virus latent membrane protein-1 in Malaysian nasopharyngeal carcinoma: High prevalence of 30-bp deletion, Xho1 polymorphism and evidence of dual infections. Journal of Medical Virology. 2003;**69**:251-257. DOI: 10.1002/ jmv.10282

[82] Mori S, Itoh T, Tokunaga M, Eizuru Y. Deletions and single-base mutations within the carboxy-terminal region of the latent membrane protein 1 oncogene in Epstein-Barr virusrelated gastric cancers of southern Japan. Journal of Medical Virology. 1999;**57**:152-158. DOI: 10.1002/ (sici)1096-9071(199902)57:2<152::aidjmv11>3.0.co;2-k

[83] Chiang AK, Wong KY, Liang AC, Srivastava G. Comparative analysis of Epstein-Barr virus gene polymorphisms in nasal T/NK-cell lymphomas and normal nasal tissues: Implications on virus strain selection in malignancy. International Journal of Cancer. 1999;**80**:356-364. DOI: 10.1002/ (sici)1097-0215(19990129)80:3<356::aidijc4>3.0.co;2-d

[84] Nagamine M, Takahara M, Kishibe K, Nagato T, Ishii H, Bandoh N, et al. Sequence variations of Epstein-Barr virus LMP1 gene in nasal NK/T-cell lymphoma. Virus Genes. 2007;**34**:47-54. DOI: 10.1007/ s11262-006-0008-5

[85] Li SN, Chang YS, Liu ST. Effect of a 10-amino acid deletion on the oncogenic activity of latent membrane protein 1 of Epstein-Barr virus. Oncogene. 1996;**12**:2129-2135

[86] Cheung ST, Leung SF, Lo KW, Chiu KW, Tam JS, Fok TF, et al. Specific latent membrane protein 1 gene sequences in type 1 and type 2 Epstein-Barr virus from nasopharyngeal carcinoma in Hong Kong. International Journal of Cancer. 1998;**76**:399- 406. DOI: 10.1002/(sici)1097- 0215(19980504)76:3<399::aidijc18>3.0.co;2-6

[87] Farrell PJ. Signal transduction from the Epstein-Barr virus LMP-1 transforming protein. Trends in Microbiology. 1998;**6**:175-177; discussion 177-178. DOI: 10.1016/ s0966-842x(98)01262-1

[88] Mainou BA, Raab-Traub N. LMP1 strain variants: Biological and molecular properties. Journal of Virology. 2006;**80**:6458-6468. DOI: 10.1128/ JVI.00135-06

[89] Edwards RH, Sitki-Green D, Moore DT, Raab-Traub N. Potential selection of LMP1 variants in nasopharyngeal carcinoma. Journal of Virology. 2004;**78**:868-881. DOI: 10.1128/jvi.78.2.868-881.2004

[90] Correa RM, Fellner MD, Alonio LV, Durand K, Teyssié AR, Picconi MA. Epstein-Barr virus (EBV) in healthy carriers: Distribution of genotypes and 30 bp deletion in latent membrane protein-1 (LMP-1) oncogene. Journal of Medical Virology. 2004;**73**:583-588. DOI: 10.1002/jmv.20129

[91] Lorenzetti MA, Gantuz M, Altcheh J, De Matteo E, Chabay PA, Preciado MV. Distinctive Epstein-Barr virus variants associated with benign and malignant pediatric pathologies: LMP1 sequence characterization and linkage with other viral gene

polymorphisms. Journal of Clinical Microbiology. 2012;**50**:609-618. DOI: 10.1128/JCM.05778-11

[92] Tso KK-Y, Yip KY-L, Mak CK-Y, Chung GT-Y, Lee S-D, Cheung S-T, et al. Complete genomic sequence of Epstein-Barr virus in nasopharyngeal carcinoma cell line C666-1. Infectious Agents and Cancer. 2013;**8**:29. DOI: 10.1186/1750-9378-8-29

[93] Zhang X-S, Song K-H, Mai H-Q, Jia W-H, Feng B-J, Xia J-C, et al. The 30-bp deletion variant: A polymorphism of latent membrane protein 1 prevalent in endemic and non-endemic areas of nasopharyngeal carcinomas in China. Cancer Letters. 2002;**176**:65-73. DOI: 10.1016/s0304-3835(01)00733-9

[94] Halabi MA, Jaccard A, Moulinas R, Bahri R, Al Mouhammad H, Mammari N, et al. Clonal deleted latent membrane protein 1 variants of Epstein-Barr virus are predominant in European extranodal NK/T lymphomas and disappear during successful treatment. International Journal of Cancer. 2016;**139**:793-802. DOI: 10.1002/ijc.30128

[95] Larcher C, Bernhard D, Schaadt E, Adler B, Ausserlechner MJ, Mitterer M, et al. Functional analysis of the mutated Epstein-Barr virus oncoprotein LMP1(69del): Implications for a new role of naturally occurring LMP1 variants. Haematologica. 2003;**88**:1324-1335

[96] Lei H, Li T, Li B, Tsai S, Biggar RJ, Nkrumah F, et al. Epstein-Barr virus from Burkitt lymphoma biopsies from Africa and South America share novel LMP-1 promoter and gene variations. Scientific Reports. 2015;**5**:16706. DOI: 10.1038/srep16706

[97] Lin Z, Wang X, Strong MJ, Concha M, Baddoo M, Xu G, et al. Whole-genome sequencing of the Akata and Mutu Epstein-Barr virus strains.

Journal of Virology. 2013;**87**:1172-1182. DOI: 10.1128/JVI.02517-12

[98] Zuercher E, Butticaz C, Wyniger J, Martinez R, Battegay M, Boffi El Amari E, et al. Genetic diversity of EBV-encoded LMP1 in the Swiss HIV Cohort Study and implication for NF-κb activation. PLoS One. 2012;**7**:e32168. DOI: 10.1371/journal.pone.0032168

[99] Edwards RH, Seillier-Moiseiwitsch F, Raab-Traub N. Signature amino acid changes in latent membrane protein 1 distinguish Epstein-Barr virus strains. Virology. 1999;**261**:79-95. DOI: 10.1006/ viro.1999.9855

[100] Masud HMAA, Yanagi Y, Watanabe T, Sato Y, Kimura H, Murata T. Epstein-Barr virus BBRF2 is required for maximum infectivity. Microorganisms. 2019;**7**(705):14 pages. DOI: 10.3390/microorganisms7120705

[101] Tsai K, Thikmyanova N, Wojcechowskyj JA, Delecluse H-J, Lieberman PM. EBV tegument protein BNRF1 disrupts DAXX-ATRX to activate viral early gene transcription. PLoS Pathogens. 2011;**7**:e1002376. DOI: 10.1371/journal.ppat.1002376

[102] Huang H, Deng Z, Vladimirova O, Wiedmer A, Lu F, Lieberman PM, et al. Structural basis underlying viral hijacking of a histone chaperone complex. Nature Communications. 2016;**7**:12707. DOI: 10.1038/ ncomms12707

[103] Shumilov A, Tsai M-H, Schlosser YT, Kratz A-S, Bernhardt K, Fink S, et al. Epstein-Barr virus particles induce centrosome amplification and chromosomal instability. Nature Communications. 2017;**8**:14257. DOI: 10.1038/ncomms14257

[104] Whitehurst CB, Ning S, Bentz GL, Dufour F, Gershburg E, Shackelford J, et al. The Epstein-Barr virus (EBV)

*EBV Genome Mutations and Malignant Proliferations DOI: http://dx.doi.org/10.5772/intechopen.93194*

deubiquitinating enzyme BPLF1 reduces EBV ribonucleotide reductase activity. Journal of Virology. 2009;**83**:4345-4353. DOI: 10.1128/JVI.02195-08

[105] Whitehurst CB, Vaziri C, Shackelford J, Pagano JS. Epstein-Barr virus BPLF1 deubiquitinates PCNA and attenuates polymerase η recruitment to DNA damage sites. Journal of Virology. 2012;**86**:8097-8106. DOI: 10.1128/ JVI.00588-12

[106] Dyson OF, Pagano JS, Whitehurst CB. The translesion polymerase pol η is required for efficient Epstein-Barr virus infectivity and is regulated by the viral deubiquitinating enzyme BPLF1. Journal of Virology. 2017;**91**(19):14 pages. DOI: 10.1128/JVI.00600-17

[107] van Gent M, Braem SGE, de Jong A, Delagic N, Peeters JGC, Boer IGJ, et al. Epstein-Barr virus large tegument protein BPLF1 contributes to innate immune evasion through interference with toll-like receptor signaling. PLoS Pathogens. 2014;**10**:e1003960. DOI: 10.1371/ journal.ppat.1003960

[108] Gastaldello S, Hildebrand S, Faridani O, Callegari S, Palmkvist M, Guglielmo CD, et al. A deneddylase encoded by Epstein–Barr virus promotes viral DNA replication by regulating the activity of cullin-RING ligases. Nature Cell Biology. 2010;**12**:351-361. DOI: 10.1038/ncb2035

[109] Whitehurst CB, Li G, Montgomery SA, Montgomery ND, Su L, Pagano JS. Knockout of Epstein-Barr virus BPLF1 retards B-cell transformation and lymphoma formation in humanized mice. MBio. 2015;**6**(5):11 pages. DOI: 10.1128/ mBio.01574-15

[110] Simbiri KO, Smith NA, Otieno R, Wohlford EEM, Daud II, Odada SP, et al. Epstein-Barr virus genetic variation in

lymphoblastoid cell lines derived from Kenyan pediatric population. PLoS One. 2015;**10**:e0125420. DOI: 10.1371/journal. pone.0125420

[111] Lu C-C, Huang H-T, Wang J-T, Slupphaug G, Li T-K, Wu M-C, et al. Characterization of the uracil-DNA glycosylase activity of Epstein-Barr virus BKRF3 and its role in lytic viral DNA replication. Journal of Virology. 2007;**81**:1195-1208. DOI: 10.1128/ JVI.01518-06

[112] Su M-T, Liu I-H, Wu C-W, Chang S-M, Tsai C-H, Yang P-W, et al. Uracil DNA glycosylase BKRF3 contributes to Epstein-Barr virus DNA replication through physical interactions with proteins in viral DNA replication complex. Journal of Virology. 2014;**88**:8883-8899. DOI: 10.1128/ JVI.00950-14

[113] Géoui T, Buisson M, Tarbouriech N, Burmeister WP. New insights on the role of the gammaherpesvirus uracil-DNA glycosylase leucine loop revealed by the structure of the Epstein-Barr virus enzyme in complex with an inhibitor protein. Journal of Molecular Biology. 2007;**366**:117-131. DOI: 10.1016/j. jmb.2006.11.007

## **Chapter 3**
