EBV infection is found in a small fraction of the non-neoplastic gastric mucosa both in vitro and in vivo [35], suggesting that clonal growth of EBV-infected gastric epithelial cells results in carcinoma rather than augmenting EBV infection of gastric epithelial cells per se [36,37]. DNA methylation machinery is considered to be involved in this process, transforming infected cells into clonal growth [20] and altering mRNA expression, which results in apoptosis inhibition, EMT, and immune evasion [21]. Furthermore, EBV induces exosome secretion by infected epithelial cells to alter the microenvironment in a manner favorable to the tumor [21].

EBVaGC is known to express Epstein-Barr virus-encoded small RNA (EBER) 1 and 2 (EBERs), EBV nuclear antigen 1 (EBNA1), and microRNA BamHI-A rightward transcripts (miR-BARTs), and to express approximately 40% of the latent membrane protein 2A (LMP2A) [35]. Further, the expression of small nucleolar RNA host gene 8 (SNHG8), a long non-coding RNA, is markedly increased in EBVaGC compared with that in EBV-negative gastric cancers or normal gastric mucosa [38,39,40]. Moreover, the knockdown of SNHG8 inhibits cell proliferation and colony formation, arrests the G0/G1 phase in vitro, and suppresses tumor growth in vivo [40]. Although exosomal SNHG8 has not been reported in EBVaGC, it has been found in exosomes obtained from the serum of patients with breast cancer and the breast milk of healthy mothers [41,42]. These genetic materials are believed to contribute to tumorigenesis, and their functions in EBVaGC are summarized in Table 1. In conclusion, exosomes are involved in EBV transmission into epithelial cells and alteration of the microenvironment.

EBV-infected cells actively release exosomes loaded with viral proteins and miRNAs [7,8,9,10,11,12,13] and EBERs are secreted primarily in complex with the protein La via exosomes [89,90,91]. A previous study characterized exosomes released from EBV-infected cells, including latency I and III cell lines [92]. Compared with the detection of a large number of miRNAs in the exosomes of a latency III cell line, miR-BART1 and miR-BART3 were detected at low levels, and no EBV-mRNA was detected in the exosomes of a latency I cell line [92]. Further, EBV infection alters exosome contents [93]. When exosomes from gastric cancer cell lines with and without artificial EBV infection were analyzed, the expression levels of CD63 and CD81 proteins were increased with EBV infection, indicating an increase in exosome delivery to the microenvironment [93]. The expression level of miR-155 in recipient epithelial cells was also increased in the presence of EBV [94].

These genetic materials are incorporated into cells via exosomes, suggesting that they might function through exosomal transfer [95,96]. Furthermore, accumulating evidence suggests that this exosomal transfer affects the phenotype of recipient cells and influences the microenvironment surrounding the infected cells [92,95]. EBV miR-BART15, which targets the NLRP3 3′-UTR, can be secreted from infected B cells via exosomes to inhibit the nucleotide-binding domain-like receptor protein 3 (NLRP3) inflammasome in non-infected cells, suggesting that EBV may trigger inflammasome activity [12]. EBV latent-infected cells also trigger antiviral immunity in dendritic cells through the selective release and delivery of RNA via exosomes [97]; furthermore, exosomes derived from EBV-positive gastric cancer cell lines suppress dendritic cell maturation [93]. EBER induces type I interferons and inflammatory cytokines via retinoic acid-induced gene I (RIG-I) activation, which are transported to exosomes and function similarly in EBV-infected latency III epithelial cells [95]. In oral squamous cell carcinoma, exosomes carrying EBER-1 can induce indoleamine 2,3-dioxygenase (IDO1) expression in monocyte-derived macrophages, with the help of interleukin (IL)-6 and tumor necrosis factor (TNF)-α-dependent mechanisms via the RIG-I signaling pathway [98]. They can further create an immunosuppressive microenvironment that influences the T-cell immune response [98]. IDO1, an effective immunosuppressive enzyme, is upregulated in EBVaGC, indicating the possibility of an immune evasion strategy [99]. Evidence of inflammasome responses to the EBV genome has been obtained, and this seems to be constitutive during latencies I, II, and III in B cells and epithelial cells [100]. Interferon gamma inducible protein 16 (IFI16) and cleaved interleukins have been detected in exosomes from latency I and III epithelial cells, which could be a strategy for EBV to escape host immunity [100]. Excessive stimulation and uncontrolled proliferation of EBV-infected B cells have been suggested to induce T-cell exhaustion, allowing escape from T-cell surveillance [101]. Furthermore, exosomal IL-1β may promote EBV persistence owing to its inability to recruit neutrophils [100]. In summary, EBV may trigger inflammasome activity through exosomal transfer in the pathogenesis of EBVaGC.

A total of 22 miRNA precursors have been found in EBV, which were then processed into 40 mature miRNAs (miR-BARTs); the spliced and polyadenylated exons then form nuclear non-coding RNAs [20,102]. Four additional miRNAs are derived from BamHI fragment H rightward open reading frame 1 (BHRF-1), but these are only expressed during lytic infections [20]. In EBVaGC, most virus-derived polyadenylated transcripts are from BARTs and these are more abundant in epithelial tumor cells than in lymphoid cells [103]. EBV miR-BART can reduce the lytic replication of viruses by regulating expression of the viral genes BZLF1 and BRLF1, suggesting that it may play a regulatory role between lytic and latent cycles [103]. In latent AGS cells, host cell transcription analysis by RNA-seq revealed that many downregulated genes were BART miRNA targets [102]; this was also observed when a full-length cDNA clone of one of the BART isoforms was artificially expressed in EBV-negative AGS cells [102]. EBV miR-BART cluster 1 and miR-BHRF 1–3 transferred via exosomes also silenced target gene expression in uninfected recipient cells, suggesting that exosomal miRNA transfer from EBV-infected cells to uninfected cells acts as a gene-silencing mechanism [9,16]. miR-BART7-3p shows the highest expression levels in EBVaGC tissues [59]. In NPC, miR-BART7-3p is hypothesized to play a role in chemoresistance, EMT, and metastasis by suppressing phosphatase and tensin homolog (PTEN) and suppressor of mothers against decapentaplegic (SMAD) 7 expression [104,105], but there have been no reports on the function of this miRNA in EBVaGC. miR-BART has various forms that either favorably or adversely affect tumors; however, most of these functions work in favor of tumors. This suggests a complex interaction between miR-BARTs and their targeting mRNA [20]. Further, as miR-BART functions only in EBVaGC, further functional studies are needed to show that miR-BARTs transfer via exosomes and induce substantial changes in recipient cells.

As shown in Table 1, several roles of EBNA1 have been revealed in EBVaGC; however, the role of EBNA1 via exosomes is not well understood. High levels of EBNA1 expression have been observed in the exosomes of patients with multiple sclerosis [106], and the inflammatory cascade induced by the EBNA1-DNA complex from apoptotic EBV-positive B cells has been suggested to underlie the pathogenesis of multiple sclerosis [101]. Although further studies are needed in this regard, as EBNA1 is known to play a crucial role in regulating EBV genome persistence and cell division [107], its role via exosomes is expected to be related to this aspect.

The relationship between LMP2A and exosomes is also unclear. LMP2A is secreted in the form of exosomes, and cholesterol is considered to be involved in the trafficking and stability of LMP2A [108]. In a comprehensive proteomic analysis of LMP1 and LMP2A, these proteins were found to affect proteasome subunits, ubiquitin-specific conjugates, peptidase, and vesicle-trafficking proteins, indicating that they are also involved in regulating exosome trafficking [109]. Further, LMP2A in exosomes can be used for diagnosing NPC with high accuracy [110]. Unlike B-cell lymphoma and NPC, EBVaGC does not express LMP1. In one study, co-culture of LMP1-positive and -negative cells using the gastric cancer cell line AGS resulted in a gradual decrease in the number of LMP1-positive cells at each cell passage; further, LMP1-positive cells stimulated proliferation of the surrounding LMP1-negative cells with exosome-mediated epidermal growth factor receptor (EGFR) activation [111]. This could explain the downregulated LMP1 expression in patients with EBVaGC. Although little is known about the relationship between EBV genetic material and exosomes, exosomal miRNA transfer from EBV-infected cells to uninfected cells acts as a gene-silencing mechanism.