Cancers, Vol. 14, Pages 5780: Functional Implications of Epstein-Barr Virus Lytic Genes in Carcinogenesis

EBV has developed mechanisms to regulate and evade immune detection, and this is believed to be a critical component of its oncogenic capability. For example, reactivation of the EBV lytic cycle is accompanied by decreased expression of MHC class I and class II molecules at the cell surface, protecting cells harboring replicating EBV from recognition and lysis by virus-specific cytotoxic T cells (CTLs) [64,65]. There is convincing evidence showing that an impaired MHC class I antigen processing pathway during the lytic cycle is an important mechanism to compromise the recognition of EBV-infected cells by CD8+ T cells [65,66,67]. A number of lytic genes are thought to contribute to these immunoregulatory effects. 4.1.1. BNLF2aIt was shown in 2005 that the CD8+ T cell recognition of EBV-infected B cells undergoing the lytic cycle was dramatically reduced with progress through the lytic cycle, accompanied by a reduction in transporter associated with antigen processing (TAP) function and surface expression of HLA class I [64,67]. Screening of EBV gene products for their involvement in this process identified BNLF2a, which efficiently disrupted CTL-mediated cell lysis through HLA-A, -B, and -C alleles [66]. This study also showed that BNLF2a down-regulated levels of MHC class I at the cell surface and significantly blocked TAP function. TAP is a member of the family of ABC transporters that translocate peptides from the cytosol into endoplasmic reticulum (ER) for binding to newly synthesized MHC class I molecules and for subsequent presentation to CD8+ T-cells [68]. TAP is composed of two subunits, TAP1 and TAP2, that form a structure consisting of a C-terminal domain mediating ATP binding. The transport of peptides across the ER membrane by TAP depends on ATP hydrolysis [69]. BNLF2a directly interacted with the TAP complex and inhibited both its peptide- and ATP-binding functions, resulting in the failure of TAP to transport peptides into ER lumen [66]. Subsequent experiments showed that BNLF2a is an early lytic protein which co-localizes with TAP primarily in the ER [53]. More specifically, BNLF2a was characterized as a tail-anchored (TA) protein where a cellular protein called Asna1 was responsible for integration of BNLF2a into the ER membrane, and thereby required for BNLF2a-mediated HLA-I down-regulation [70]. The knockout of BNLF2a led to better CTL recognition of immediate early and early antigens, but not the late antigens, suggesting that BNLF2a expression is stage-specific and predominantly hampers the presentation of immediate early and early proteins [71].Several research groups have independently showed that BNLF2a is expressed in the early phase of the lytic cycle in B cells, and that its expression is regulated by Zta [53,71,72,73]. Unexpectedly, Strong and colleagues demonstrated that BNLF2a is expressed in a substantial proportion of EBVaGCs and that this is independent from lytic gene expression [54]. This was the first report describing BNLF2a expression along with EBV latent genes and prompted the researchers to propose a new form of the type II latency program, latency IIc (EBNA1, LMP2 and BNLF2a). Subsequently, BNLF2a has also been shown to be expressed in primary NPC biopsies and patient-derived NPC xenografts [12,17,74]. Somatic aberrations in genes related to innate and adaptive immunity were identified through whole genome profiling of primary NPC [74]. Interestingly, a trend for mutual exclusivity between BNLF2a expression and these somatic alterations was observed, suggesting a host-viral cooperation for NPC cells to avoid immune detection. In accordance with these observations, single-cell transcriptomic analysis also identified a strong correlation between the expression of BNLF2a/2b and the host genes involved in immune responses [17]. By analyzing publicly available RNAseq datasets, 4 out of 1017 non-small cell lung cancer were found to be EBV-positive [75]. Notably, high expression of BNLF2a was detected in the sample with the highest EBV read number in the absence of other lytic gene expression. In addition to carcinomas, BNLF2a was also expressed in extra nodal natural killer T cell lymphoma (ENKTCL) and angio-immunoblastic T cell lymphoma (AITL) that exhibit latency IIc [6,13,16]. Taken together, these data strongly implicate BNLF2a in the oncogenic process aside from its contribution to the immune evasion of lytically-infected cells. 4.1.2. BGLF5A novel nuclease activity mapping to BGLF5 gene was originally reported in a Burkitt lymphoma (BL) cell line, P3HR-1 [76], with a subsequent study identifying the BGLF5 gene product as an alkaline exonuclease [77]. Following the discovery that Kaposi sarcoma herpesvirus (KSHV) ORF37 encodes a host shutoff function [78,79,80], BGLF5 was also found to inhibit cellular protein synthesis [81]. Viral host shutoff (vhs) is an important mechanism exploited by viruses to curb cellular protein production thereby favoring the synthesis of viral proteins. Herpesviruses use this mechanism to down-regulate surface expression of MHC class I and II molecules, perturbing T cell recognition to achieve immune evasion [82]. Indeed, BGLF5 was capable of causing a widespread shutoff of cellular gene expression by enhanced mRNA degradation, including HLA class I and class II molecules [81]. Consequently, a marked impairment of HLA class I-restricted CD8+ T cell recognition was evident [83]. Although the shutoff function of BGLF5 was originally found to be genetically separable from its exonuclease activity [83], subsequent mutational analysis showed that its DNase and RNase activities share a catalytic site, and some mutations were more selectively affecting either DNA degradation or shutoff [84]. It was also shown that BGLF5 expression leads to nuclear relocalization of cytoplasmic poly(A) binding protein (PABPC), a protein involved in mRNA stabilization, thereby augmenting its shutoff phenotype [84]. Further detailed investigations revealed that BGLF5 is a component of virus-induced nodular structures (VINORCs) that contribute to the vhs-associated blockade of nuclear export of cellular mRNA while facilitating selective processing and export of viral mRNAs [85].Cross-talk between the innate and adaptive immune system is required for the successful elimination of most pathogens. Upon the recognition of pathogen-associated molecular patterns, toll-like receptors (TLRs) activate intracellular signaling networks to either achieve direct antiviral activity or orchestrate the adaptive immune response [86]. Notably, BGLF5 was found to downregulate TLR9 levels through RNA degradation, a mechanism by which EBV could obstruct the host’s innate response [87]. Knockdown of BGLF5 in reactivated Akata BL cells restored the levels of several immunologically relevant molecules, such as TLR2, HLA class I/II molecules and CD1d [88]. In addition to its ability to facilitate immune evasion, BGLF5 also directly contributes to viral DNA replication and virion assembly [89]. It was found that knockout of BGLF5 resulted in a significant reduction of virus production by impairing virus nucleocapsid maturation, reducing primary egress and viral DNA synthesis. BGLF5 is expressed early in the viral replicative cycle [81,83]. Antibodies against BGLF5 were detected in sera from NPC patients [90] and BGLF5 protein expression, as well as nuclease activity, have been documented in both NPC biopsies and transplanted tumor lines [91]. In support of these observations, recent RNAseq analysis also revealed BGLF5 expression in primary NPC tissues [12], but the precise levels and proportion of tumor cells expressing BGLF5 remain to be determined. It is noteworthy that so far, there is no report describing BGLF5 expression in other EBV-associated malignancies. It remains to be determined whether the expression of BGLF5 is restricted to EBV-associated epithelial malignancies. 4.1.3. BILF1BILF1 was initially identified as a putative G protein-coupled receptor (GPCR) based on its homology with the equine herpesvirus 2 E6 viral GPCR gene [92], but its functional role as a GPCR was not examined until 2005 [93,94]. These studies revealed that BILF1 is a constitutively active GPCR which signals through Gαi, classifying it as an “orphan” receptor. BILF1 contains seven transmembrane (TM) helices and displays conserved cysteine residues in the N-terminus and in the extracellular loops (ECLs). However, unlike most rhodopsin-like GPCRs (Class A GPCRs) which comprise a DRY (aspartic acid, arginine, tyrosine) motif in TM-III that is essential for receptor signaling, BILF1 has an alternative DRY motif, EKT (glutamic acid, lysine, threonine) [94]. Functioning as a ligand-independent GPCR, BILF1 is capable of modulating a number of intracellular pathways, including NF-kB and cAMP-response element-binding protein (CREB) pathways [93,94]. BILF1 was originally identified as an early lytic gene [95], but its expression has also been detected in various primary tumor biopsies alongside the predominant latent mode of EBV infection [9,11,12,15,16,17,18,96,97].The contribution of BILF1 to immune evasion was initially suggested by the observation that its expression resulted in the reduction of RNA-dependent protein kinase (PKR) activation [93]. This interferes with the cellular antiviral defense system in which PKR functions by shutting down host protein synthesis and inducing apoptosis of infected cells to halt virus spread [98]. Subsequently, BILF1 was found to down-regulate MHC class I molecules on the surface of host cells, making it the third EBV lytic gene identified to subvert the antigen presentation pathway [99]. BILF1 physically interacts with MHC-I molecules through its C-terminal tail that leads to accelerated internalization and lysosomal degradation, resulting in reduced levels of MHC-I at the cell surface and consequently abrogation of T-cell recognition [95,99]. The underlying mechanism for this effect is rather complex, but the conserved residues in both the EKT motif and ECL domains appear to play important roles [100,101]. Significantly, BILF1 can selectively reduce cell surface levels of HLA-A, -B, and -E alleles, while only marginally affecting HLA-C [95]. Through this refined modulation, BILF1 could interfere with HLA-A and –B presentation to CD8+ T-cells while enabling the infected cells to retain the inhibitory effect of HLA-C on NK cells, evading both adaptive and innate immune responses. In addition to the enhanced endocytosis of surface MHC-I molecules, BILF1 also diverts newly synthesized MHC-I/peptide complexes during exocytosis, resulting in a significant reduction of processed target peptides presented to CD8+ T-cells [100].There is evidence to show that heterodimerization of two different GPCRs can alter the functional features of the individual partners, including signaling and trafficking [102]. Interestingly, BILF1 has been shown to form heterodimers with several human chemokine receptors [103], a strategy that EBV might use to hijack cellular communication for its own benefit. In particular, BILF1 hetero-oligomerizes with human CXCR4 to disrupt binding of CXCL12 to CXCR4 in a constitutive manner, resulting in the impairment of CXCL12-mediated CXCR4 signaling [104]. Whist the functional significance of this effect has yet to be explored, the authors postulated that the migration of BILF1-expressing plasma B cells to CXCL12 gradients could be suppressed, and this might provide an advantage for EBV by homing to sites that are most optimal for viral replication and dissemination. Nonetheless, it is noteworthy that CXCR4 has been shown to cause a reduction of MHC class I levels at the cell surface [105]. The involvement of CXCR4 in BILF1-mediated down-regulation of MHC-I warrants further investigation.In the context of oncogenesis, another intriguing property of BILF1 is its function as an oncogene [106]. BILF1 was shown to induce foci in NIH3T3 cells in vitro, as well as tumor formation in vivo through EKT-dependent Gαi signaling. BILF1 also stimulated vascular endothelial growth factor (VEGF) secretion in a constitutively active manner [106]. The contribution of VEGF to tumor angiogenesis is well-defined, and its up-regulation has been reported in many tumors, including NPC and non-Hodgkin lymphoma [107,108]. In support of its tumor-promoting properties, BILF1 has been demonstrated to up-regulate intercellular adhesion molecule-1 (ICAM-1) [109]. The up-regulation of ICAM-1 has been described in various types of cancer [110] and can promote cancer metastasis [111]. Site-directed mutagenesis of the NF-kB binding sites on the ICAM-1 promoter significantly diminished the ability of BILF1 to up-regulate ICAM-1, suggesting BILF1 exerts its function through a mechanism involving the NF-kB pathway [109]. Further experiments demonstrated that BILF1 decreased the cellular levels of IkBα (an inhibitory molecule of NF-kB), and this likely resulted in the translocation of NF-kB from cytoplasm to nucleus. Taken together with the detection of BILF1 in EBV-associated tumors, these results imply a significant role for this protein in the pathogenesis of these malignancies.During EBV replication, CD8+ T cell responses to EBV immediate-early and some early antigens are more frequently observed than responses to late antigens. This hierarchy of immunodominance seems to correlate with the phase-specific efficiency of antigen presentation that is modulated by the viral immuno-evasins. Knockdown experiments in LCLs have shown that BNLF2a impairs antigen presentation with decreasing efficiency as the lytic cycle progresses while subversion by BILF1 increases with progression through lytic cycle [112]. In contrast, BGLF5 has a relatively minor effect on CD8+ T cell recognition of antigens expressed in any phase of lytic cycle [112]. 4.1.4. BCRF1The BCRF1 gene encodes viral interleukin-10 (vIL-10), a human homolog of interleukin-10 (hIL-10) that is commonly regarded as an immunosuppressive cytokine [113,114]. BCRF1 was originally thought to be expressed only during the late phase of the lytic cycle, however, its expression has also been detected immediately after the infection of primary B cells [73,115]. A viral pre-initiation complex (vPIC) comprising several early lytic proteins (also known as “late gene regulators”) is required for the transcription of late genes [116]. Interestingly, BCRF1 and another late gene, BPLF1, are transcribed independently of the vPIC [117], a mechanism different from that regulating late genes encoding structural proteins. Notably, the BRLF1-encoded transcription activator (Rta) selectively binds to eight late promoters, including BCRF1, suggesting the expression of BCRF1 is likely regulated by BRLF1 [117]. Significantly, an early in vitro study showed that vIL-10 was present in EBV-transformed cell lines and that BCRF1 antisense oligonucleotides inhibited B cell transformation, pointing to a critical role for BCRF1 in EBV tumorigenesis alongside the well-established latent genes [115]. However, a study published around the same time reported that vIL-10 was not essential for B cell transformation in vitro [118]. Nonetheless, the expression of BCRF1 was reported in primary tumors of NK/T lymphoma and BL [31,119] and is now evidenced in various types of EBV-associated malignancies (Table 1). Notably, Badya and colleagues [16] reported that most AITL patients co-express BNLF2a and BCRF1, supporting a previous in vitro study showing that co-expression of these two genes facilitated immune evasion during the early phase of EBV lytic infection [73].The immunosuppressive properties of vIL-10 were revealed by a number of studies in the 1990s. In a murine model, infection with a recombinant vaccinia virus expressing vIL-10 resulted in reduced NK and CTL responses, suggesting that BCRF1 may facilitate the establishment of latent infection in B lymphocytes [120]. These results were supported by subsequent experiments, showing that vIL-10 protected EBV-infected B cells from NK cell-mediated killing [73]. Similar to hIL-10, vIL-10 functions as a cytokine synthesis inhibitory factor that blocks interferon-γ (IFN-γ) synthesis by activated lymphoid cells [121]. Furthermore, vIL-10 was shown to interfere with the antigen presentation pathway by down-regulating TAP1 to hamper the transport of peptide antigens into the ER, which in turn reduced the surface levels of MHC-I levels, thereby contributing to the evasion of T cell recognition [122]. In addition, vIL-10 significantly inhibited antigen-specific CD4+ T-cell activation by impairing the antigen-presenting capacity of monocytes through down-regulation of MHC class II expression as well as adhesion/costimulatory molecules ICAM-1, CD80, and CD86 [123,124]. In accordance with these observations, vIL-10 has been shown to abrogate the ability of autologous T cells to inhibit EBV-induced B cell transformation [125]. This effect appeared to be mediated through an augmentation of the growth of infected cells and the suppression of IL-2 and IFN-γ production induced by activated T cells [125]. In addition, vIL-10 prevented the secretion of anti-viral cytokines and diminished CD4+ effector T cell functions [73]. Collectively, these results indicate that EBV appears to have captured a human cytokine gene and retained activities necessary to enhance cell transformation during initial infection and to suppress host immune responses triggered by subsequent viral reactivation.

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