1. IntroductionEpstein–Barr virus (EBV) is a ubiquitous human tumor virus that resides as a lifelong infection in the oral epithelium and B lymphocytes that involves latency and productive viral replication (Figure 1). B lymphocytes support the latent phase of the viral lifecycle exemplified by a restricted viral gene expression program that supports viral evasion of immune surveillance and long-term persistence [1]. Latently infected EBV-positive B lymphocytes can be reactivated into the productive lifecycle, referred to as viral reactivation.Epithelial cells also support the productive phase of the viral lifecycle following a coordinated viral gene expression cascade resulting in amplification of the viral genome and production of progeny virions [2]. Latency in epithelial cells is observed in carcinomas and in basal tonsillar epithelial cells [3,4].Infection with EBV is typically acquired asymptomatically in childhood. However, delayed infection until adolescence often causes infectious mononucleosis, a self-limiting lymphoproliferative response to primary EBV infection [5]. Although long-term carriage of the virus is generally benign, EBV is a tumor virus associated with several epithelial and lymphoid malignancies that reflect the tissue tropism of the virus. EBV linked malignancies occur more frequently in immunosuppressed individuals but also develop in immunocompetent persons. EBV-associated cancers include nasopharyngeal carcinoma (NPC), gastric carcinoma (GC), oral squamous carcinoma, Burkitt’s lymphoma, Hodgkin’s lymphoma, diffuse large B cell lymphoma, NK/T-cell lymphomas, and post-transplant lymphoproliferative disease. This review will focus on oral lymphomas and carcinomas associated with EBV infection.EBV is primarily transmitted through saliva. Early studies showed that infectious EBV is secreted in saliva from patients with infectious mononucleosis and in healthy adults [6]. In healthy individuals, EBV can be detected intermittently over a period of 15 months, with 25% of healthy adults secreting EBV at every sampled time point [7]. The level of viral shedding tends to be stable for days to months and is quickly replaced within 2 min after swallowing [8]. Patients recovering from infectious mononucleosis typically shed higher viral loads for at least 6 months after convalescence [9]. HIV-infected individuals shed higher EBV loads in saliva than HIV-negative individuals. Anti-retroviral therapy to improve the immune status of HIV-infected individuals decreases EBV loads suggesting an additional benefit of anti-retroviral therapy in limiting oral EBV transmission [10]. However, HIV infected individuals on anti-retroviral therapy are at a higher risk of developing an EBV-associated cancer compared to the general population [11].Epithelial cells in the oral cavity are the major producers of EBV found in saliva [8]. EBV infects and replicates in various epithelial tissues in the oral cavity that include the gingiva, tongue, and tonsillar epithelium [12]. EBV can directly infect an epithelial cell or be transcytosed through polarized oral epithelial cells [13]. Entry is mediated by direct fusion at the plasma membrane or internalized by endocytosis [14]. Various binding interactions between viral glycoproteins and cellular receptors allow for entry into epithelial cells include binding of gH/gL to either integrins, ephrinA2 receptor, and non-muscle myosin heavy chain IIA, viral gB with neuropilin 1. BMRF-2, a viral glycoprotein, can bind to αvβ1 integrins on basolateral surface of polarized epithelial cells, and viral gp350/220 can bind to complement receptor 2 (CR2/CD21) in cases when CR2 is expressed on epithelial cells [15]. Following entry and nuclear delivery of the viral DNA, the linear EBV genome circularizes by recombination of its terminal repeats and is maintained as an extrachromosomal DNA that can be amplified through rolling circle replication [16]. The cell type that produces EBV virions alters the tropism of the virus based on HLA class II interactions with gp42 [17]. Epithelial derived virions have higher levels of the gH/gL/gp42 complex than B cell derived virions, which increases the efficiency of B cell infection [17].The importance of epithelial cells in the lifecycle of EBV has proven difficult to study due to challenges associated with in vitro epithelial infection. Infection of primary epithelial cells in monolayer culture typically is not efficient and viral DNA is rapidly lost within days [18]. In addition, a non-permissive EBV infection is observed with growth arrest of infected primary nasopharyngeal epithelial cells [19]. In contrast, primary epithelial cells grown in organotypic raft culture, which model the differentiated and stratified layers of the epithelium, indicate that differentiated epithelial cells support robust EBV replication and spread [20]. Differentiation-induced transcription factors (KLF4 and PRDM1) have been identified in regulating the expression of EBV immediate early genes, BZLF1 and BRLF1 [21]. BZLF1 and BRLF1 encode the Z and R transactivators, respectively, that initiate the productive EBV replication cycle. Infection of carcinoma cell lines requires selective pressure to retain the viral genome achieved by using EBV recombinants that carry antibiotic resistance markers and typically results in a latent EBV infection. EBV-positive carcinomas also exhibit a latent EBV infection, which is not typically observed in normal oral epithelial and may reflect the poor differentiation state of tumors. Laser capture microdissection of tonsillar basal epithelial cells detected latent EBV encoded RNAs (EBERs) by quantitative reverse transcription PCR in the absence of EBV immediate early transcripts, supporting the possibility of transient latency in tonsillar basal epithelial cells [4].EBV infection of circulating resting B lymphocytes in the oral cavity initiates a journey for life-long persistence in the memory B cell compartment. It is also important to note that EBV may directly infect germinal center or memory B cells [22]. Entry into B lymphocytes is mediated by attachment of viral glycoprotein gp350/220 to the complement receptor 2 (CR2/CD21) and interaction of the tripartite glycoprotein complex of gH/gL/gp42 with HLA class II [23]. Endocytosis is required for internalization and gB mediates membrane fusion for viral escape from the acidified endosome. Following viral genome circularization, the viral genome is maintained as an episome [24]. When B cells proliferate and divide, the EBV episome is replicated once per cell cycle by DNA replication licensing at the latent origin of DNA replication, OriP [25]. In the first week after infection, a pre-latent phase is observed with expression of EBV nuclear antigens (EBNAs) and lytic genes [26]. The viral genome is epigenetically silenced using the host epigenetic machinery. DNA methylation and repressive histone modifications deposited on the viral genome restrict viral gene expression [27]. As the EBV-infected B lymphocyte navigates through the B cell maturation/differentiation states, various latency programs are established that support B cell growth and survival of the infected B cells, while avoiding immune surveillance [28]. Infected resting B cells exhibit the growth/latency III program characterized by expression of all EBV nuclear antigens (EBNA-1, -2, -3A, -3B, -3C and -LP), the latent membrane proteins (LMP1, -2A, -2B), and EBV non-coding RNAs: the EBV encoded RNAs (EBERs), BamHI fragment A rightward long non-coding transcripts (BARTs) and BART microRNAs. EBV-infected germinal center B cells express the default/latency II program restricted to EBNA1, LMP1, LMP2, and non-coding RNAs. Infected memory B cells that are dividing express only EBNA1 and viral non-coding RNAs (latency 1), while non-viral gene products are detected in non-dividing memory B cells (latency 0). The infected memory B cells provide a long-lived cellular reservoir for EBV persistence and life-long infection. Terminal differentiation of the memory B cell to a plasma cell in response to antigenic stimulation reactivates the EBV productive lifecycle [29] Expression of differentiation-induced plasma cell transcription factors (PRDM1 and XBP-1) activate the expression of EBV immediate early genes BZLF1 and BRLF1 [30]. 2. Mechanisms of EBV OncogenesisEBV infects over 90% of adults worldwide, and EBV is associated with 1% of all cancers [31]. EBV immortalizes B lymphocytes into lymphoblastoid cell lines (LCLs) [32]. However, intrinsic and extrinsic tumor suppression protects the host from developing EBV-associated cancers. Immunosurveillance, an extrinsic tumor suppressor activity, effectively eliminates EBV-infected lymphoblasts, such that immunocompromised individuals are more prone to developing EBV-associated cancers [33]. In contrast, primary epithelial cells are not readily immortalized by EBV without prior genetic alterations [19]. Such transformed epithelial cell become latently infected with EBV in vitro and acquire oncogenic phenotypes similar to what is seen in EBV-associated carcinomas [34]. Thus, the cellular context of genetic and epigenetic alterations can influence the oncogenic outcome in the presence of EBV. EBV infection not only can initiate oncogenic processes as demonstrated by B cell immortalization, but also could contribute to tumor progression and tumor evolution. Thus, EBV likely exerts distinct oncogenic activities depending on the tumor context.EBV latent infection is a shared feature among EBV-associated malignancies (Table 1). The EBV latent genes have been shown to contribute to various oncogenic phenotypes supported by LMP activation of signaling pathways and by EBNA transcriptional activation that combined act to reprogram host gene expression [35]. In addition to promoting growth, EBV infection and latent proteins contribute to resistance to apoptosis, attenuated responsiveness to differentiation, and enhancement of cellular invasion [36]. Table 2 summarizes known functions encoded by EBV latent genes.High viral loads due to deregulated viral replication can also contribute to the tumorigenic process. Individuals with infectious mononucleosis typically exhibit higher viral loads in blood and saliva for months after diagnosis and are at an increased risk for developing Hodgkin’s lymphoma [37]. Furthermore, elevated EBV antibody titers frequently precede tumor onset by a few years, a phenomenon observed in EBV-associated Burkitt’s lymphoma, Hodgkin’s lymphoma, and nasopharyngeal carcinoma [38,39]. Although not well understood, the elevated EBV antibody titer likely reflects increased viral loads and viral reactivation. In the context of viral opportunism, higher EBV loads likely increase the risk of infecting premalignant or malignant cells with the infection process inducing a rapid tumor progression.EBV infection has been shown to increase genomic instability by various mechanisms [40]. EBNA-1 can induce the production of reactive oxygen species promoting DNA damage. LMP-1 inhibits DNA damage responses and DNA repair. EBNA-3C interferes will mitotic spindle checkpoint allowing DNA damage to propagate into the next generation of cells [41]. In addition, EBV infection induces the activation-induced family of cytidine deaminases/apolipoprotein B mRNA editing catalytic polypeptide-like (AID/APOBEC), enzymes with mutagenic activities. AID is responsible for somatic hypermutation (SHM) of B cell immunoglobulin genes undergoing class switch recombination in the germinal center [42]. EBV induced AID activation has been implicated in the translocation of MYC into the immunoglobulin heavy chain or light chain loci, resulting in the overexpression of the c-Myc oncogene. APOBEC deaminates viral RNA/DNA to restrict viral replication but can alter the host genome as well [43].EBV induces epigenetic alterations that regulate the expression of tumor suppressor expression and apoptotic responses. Nasopharyngeal carcinoma and EBV-associated gastric carcinoma display CpG island hypermethylator phenotypes that silence tumor suppressor gene expression. Common tumor suppressors silenced in NPC and EBV-associated gastric cancer include: RASSF1, CDKN2A/p16, CDH1, and PTEN [44]. Importantly, EBV-associated gastric carcinoma is classified as having an extremely high DNA hypermethylation epigenotype [45]. EBNA-3A and EBNA-3C epigenetically repress BIM, p14, p15, p16, and p18 gene transcription by recruiting polycomb repressive complex 2 [46]. LMP-1 and LMP-2 signaling have been shown to activate the DNA methyltransferases 1, 3A, and 3B, that subsequently leads to tumor suppressor gene silencing [47]. Additionally, LCLs, Burkitt’s lymphoma, and EBV infected B cells display similar tumor suppressor gene silencing due to targeted promoter DNA hypermethylation, despite the observation of global hypomethylation [48]. Alterations in histone modifications are also observed. The repressive histone H3K27 trimethylation mark is elevated in NPC, but not in other EBV associated cancers; while H3K27me3 and H4K20 trimethylation is reduced in EBV-positive lymphoblastoid cells compared to activated EBV-negative B cells [49]. Unlike mutations, epigenetic modification are reversible and identify a potential target for treatment of EBV-associated cancer.

In this review, we will describe various molecular mechanisms utilized by EBV to promote oncogenesis. Specifically, we will direct our attention to EBV-associated lymphomas and carcinomas affecting the tissues and organs of the oral compartment either directly or indirectly. While many of the precise mechanisms remain unclear our goal is to present a current summation of established links between EBV and cancer development and to identify the questions that remain. EBV driven malignancy is a complex process that differs between the various EBV-associated cancer types. It is important to understand both the similarities and differences in the role of the virus across different cancers to prevent and treat EBV-related neoplasms.