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Molecular Properties and Therapeutic Targeting of the EBV-Encoded Receptor BILF1

Simple Summary The Epstein–Barr virus (EBV) is a γ-herpesvirus residing in over 90% of adults worldwide. Besides causing a benign glandular fever (infectious mononucleosis), EBV is also associated with a wide range of different types of cancers. This review will present these malignancies, current therapies, and summarize the present knowledge on an EBV-encoded oncogenic protein called BILF1. As a member of class A G protein–coupled receptors that are intrinsically successful drug targets, BILF1 will be discussed for its potential as future target in EBV-associated diseases. Finally, ongoing development of novel EBV-specific therapeutics is briefly outlined. Abstract The γ-herpesvirus Epstein–Barr Virus (EBV) establishes lifelong infections in approximately 90% of adults worldwide. Up to 1,000,000 people yearly are estimated to suffer from health conditions attributed to the infection with this virus, such as nasopharyngeal and gastric carcinomas as well as several forms of B, T and NK cell lymphoma. To date, no EBV-specific therapeutic option has reached the market, greatly reducing the survival prognoses of affected patients. Similar to other herpesviruses, EBV encodes for a G protein–coupled receptor (GPCR), BILF1, affecting a multitude of cellular signaling pathways. BILF1 has been identified to promote immune evasion and tumorigenesis, effectively ensuring a life-long persistence of EBV in, and driving detrimental health conditions to its host. This review summarizes the epidemiology of EBV-associated malignancies, their current standard-of-care, EBV-specific therapeutics in development, GPCRs and their druggability, and most importantly consolidates the findings of over 15 years of research on BILF1 in the context of EBV-specific drug development. Taken together, BILF1 constitutes a promising target for the development of novel EBV-specific therapeutics.


Introduction
This review on the Epstein-Barr virus (EBV) and its G protein-coupled receptor (GPCR) BILF1 coherently summarizes the knowledge gained within the last years of research. Considering the great impact of EBV on global health with up to 1,000,000 EBVassociated disease cases per year (Table 1), it is of great need to consolidate findings which could drive the development of novel EBV-specific treatments. Additionally, as GPCRs have proven to be highly druggable proteins [1], a significant effort has been put into elucidating the structure, function and druggability of virally encoded GPCRs. First discovered over 15 years ago [2,3], EBV-BILF1 has meanwhile been thoroughly investigated, leading to the revelation of its, among others, oncogenic and immunoevasive properties making it a potential drug target.
Primary infection with EBV, mostly during childhood or adolescence, is known to cause infectious mononucleosis (IM), a generally mild and self-containing glandular fever, in a minority of infected people [5]. More concerning, latent infections with EBV are connected to a large number of cancers in immunodeficient as well as in immunocompetent individuals. As mentioned above, EBV is the first identified oncogenic virus. It is linked to a plethora of health conditions; mostly cancers of B, T or NK cells (Burkitt's lymphoma (BL), Hodgkin's lymphoma (HL), post-transplant lymphoproliferative disorders (PTLD) and Mature T-and NK-cell neoplasms (MTNKL)/Peripheral T-cell Lymphoma (PTCL)) [5,24,25]. Some EBV-associated cancers are of epithelial (nasopharyngeal carcinoma (NPC), EBVassociated gastric carcinomas (EBVaGC), lymphoepithelioma-like carcinomas) and, very rarely, of mesenchymal (leiomyosarcoma) origin [5,24]. Moreover, a causative link between EBV and multiple sclerosis (MS) [26][27][28], systemic lupus erythematosus (SLE) [29][30][31] and breast cancer [32][33][34][35] is currently under investigation. Judging by global incidence, EBV-associated gastric carcinomas and nasopharyngeal carcinomas affect most people worldwide with both over 100,000 cases per year (Table 1). Furthermore, if a clear causality between EBV and breast carcinomas can be established, over 500,000 patients yearly could potentially be treated with more efficacious EBV-specific therapeutics (assuming an average of 26% prevalence of EBV in breast carcinomas [32]). Taken together, up to around 1,000,000 people worldwide are diagnosed with diseases with possible EBV involvement every year. Figure 1 summarizes EBV-associated malignancies, while Table 1 covers these diseases in detail. It should be noted that the stated rates under 'Prognosis' (Table 1) mostly stem from single studies with a limited number of test subjects and not from meta-analyses of multiple studies. Hence, the given values inherently are not representative of every patient and are solely listed to provide a notion on the severity of the respective malignancies. Nonetheless, the OS and PFS found in the literature indicate great room for treatment improvement and immense potential to save lives. Table 1. Description of malignancies in terms of their respective latency program, their EBV-association, the EBV-association depending on the type of the respective disease, the global incidence with EBV-association, the geographical distribution of the disease, the cellular background of the disease, the localization of the disease in the body and the prognosis through the overall survival rate (OS), the progression free survival (PFS), the objective response rate (ORR) and complete remission rate (CRR) where applicable. Inspired by [36].

EBV-Associated Malignancy EBV Association EBV Association-Dependent Upon Type
Incidence with EBV Association Geography Cellular Background Localization Prognosis

Basics
In the following, the basics on G protein-coupled receptors (GPCRs), also known as seven transmembrane receptors (7TM receptors), are outlined. GPCRs are an essential type of eukaryotic membrane proteins, importantly involved in signal transduction across the cell membrane. According to the International Union of Pharmacology (IUPHAR), GPCRs are divided into five main families: Rhodopsin-like (class A), Secretin (class B), Glutamate (class C), Frizzled/Taste (class F) and Adhesion GPCRs [116]. These receptors consist of an extracellular N-terminus, seven hydrophobic transmembrane helices, joined by three intracellular (ICL) and extracellular loops (ECL), and a cytosolic C-terminus. Class A receptors are the most thoroughly studied receptors and are the focus of this review. They contain a conserved disulfide bridge between transmembrane helix 3 (TM-3) and ECL-2 [117,118], and display a highly conserved DRY-motif essential for signaling at the cytosolic end of TM-3 [118,119]. A schematic depiction of the Class A GPCR structure is presented in Figure 2A. Depending on the specific GPCR, the activating stimuli range from endogenous neurotransmitters, metabolites, hormones or chemokines (chemotactic cytokines) over natural exogenous stimuli such as ions, light and odors to synthetic stimuli such as specific medications [1,117,120,121]. In fact, around 34% of all FDA-approved drugs act on GPCRs [1].
for signaling at the cytosolic end of TM-3 [118,119]. A schematic depiction of the Class A GPCR structure is presented in Figure 2A. Depending on the specific GPCR, the activating stimuli range from endogenous neurotransmitters, metabolites, hormones or chemokines (chemotactic cytokines) over natural exogenous stimuli such as ions, light and odors to synthetic stimuli such as specific medications [1,117,120,121]. In fact, around 34% of all FDA-approved drugs act on GPCRs [1]. While remaining in an equilibrium of active and inactive conformations in the absence of a ligand (basal or constitutive activity), a GPCR's active conformation can be stabilized through an agonistic ligand (increased activity to 100% for full agonists and less for partial agonists) [117,120,121]. This allows the GPCR to promote GTP for GDP exchange in the Gα subunit of its cognate heterotrimeric G protein, assembled by a Gα, Gβ and Gγ subunits. Subsequently, the G protein is released from the GPCR and Gα dissociates from the Gβγ dimer, in order to separately initiate amplified intracellular signaling cascades. On the contrary, inverse agonists push the active/inactive conformation equilibrium of the GPCR to the side of inactivation, resulting in a decreased activity compared with the baseline. Additionally, neutral antagonists block the agonist-binding pocket and thus retain basal activity of the receptor and inhibit activation or inactivation through other ligands. Finally, receptors that are active in absence of a ligand are considered constitutively active GPCRs [120,121].
For G proteins, the class of α subunit determines its specificity toward downstream effectors, where Gαi inhibits adenylyl cyclase, Gαs activates adenylate cyclase (triggering the cyclic adenosine monophosphate (cAMP)-dependent pathway), Gαq activates phospholipases C (initiating inositol trisphosphate/diacylglycerol (IP3/DAG) pathway) While remaining in an equilibrium of active and inactive conformations in the absence of a ligand (basal or constitutive activity), a GPCR's active conformation can be stabilized through an agonistic ligand (increased activity to 100% for full agonists and less for partial agonists) [117,120,121]. This allows the GPCR to promote GTP for GDP exchange in the Gα subunit of its cognate heterotrimeric G protein, assembled by a Gα, Gβ and Gγ subunits. Subsequently, the G protein is released from the GPCR and Gα dissociates from the Gβγ dimer, in order to separately initiate amplified intracellular signaling cascades. On the contrary, inverse agonists push the active/inactive conformation equilibrium of the GPCR to the side of inactivation, resulting in a decreased activity compared with the baseline. Additionally, neutral antagonists block the agonist-binding pocket and thus retain basal activity of the receptor and inhibit activation or inactivation through other ligands. Finally, receptors that are active in absence of a ligand are considered constitutively active GPCRs [120,121].
The general concepts of GPCR signaling are depicted in Figure 3.
and Gα12/13, among others, interacting with Ras and Rho [123][124][125]. The Gβγ dimer is known to interact with phospholipases, receptor kinases and ion channels [121]. Finally, /GPCRs can recruit arrestins through phosphorylation of their C-terminus by G protein-coupled receptor kinases (GRKs). This inhibits G protein signaling (desensitization), promotes internalization of the GPCRs by clathrin-vesicles and modulates G protein-independent downstream signaling networks [117,121]. Some ligands might initiate a G protein response, while others are more biased toward generating an arrestin response [117]. The general concepts of GPCR signaling are depicted in Figure 3.

General
BILF1 is a glycosylated viral GPCR (vGPCR) of around 50 kDa (33 kDa unglycosylated) encoded by EBV. This receptor has been shown to mainly associate to the cell membrane of EBV-infected cells [2]. This receptor ticks several boxes of GPCRs, such as signaling through G proteins, containing seven transmembrane helices and displaying  BILF1 is a glycosylated viral GPCR (vGPCR) of around 50 kDa (33 kDa unglycosylated) encoded by EBV. This receptor has been shown to mainly associate to the cell membrane of EBV-infected cells [2]. This receptor ticks several boxes of GPCRs, such as signaling through G proteins, containing seven transmembrane helices and displaying conserved cysteine residues in ECLs and the N-terminus. However, BILF1 has an alternative DRY motif at the intracellular end of TM-3. Instead of the well-conserved triad of aspartic acid, arginine and tyrosine (DRY), known among many as rhodopsin-like GPCRs (Class A GPCRs), BILF1 presents an alternative, while similar, DRY-motif (EKT), which maintains the respective charges of the residues [2].
In 2015, 21 orthologs of BILF1 were identified in primate and ungulate γ1-herpesviruses. Among these orthologs, several conserved regions, most intriguingly in the extracellular loops (ECLs) and predominantly in ECL-2, were found [127]. Likewise, these orthologs contain conserved cysteines in ECL-2 and on top of TM-3 (GPCR bridge), and in the Nterminus and the top of TM-7 (chemokine receptor/CKR bridge), both of which are known to form a disulfide bridge in rhodopsin-like 7TM receptors [118,128]. Similarly, NF-κB activation and Gαi signaling are conserved among EBV-BILF1 and studied primate BILF1 orthologs. Moreover, the mentioned study on BILF1 orthologs [127] and a recent study from 2020 [129] also generated data strongly indicating constitutive internalization and recycling of BILF1.

Structure
Unlike other vGPCRs binding CC or CXC chemokines [131] and potentially acting as chemokine scavengers, no endogenous ligand for BILF1 has been found [2,3]. This has led to the general classification of BILF1 as an "orphan" receptor. However, recent studies on elucidating its structure suggest a molecularly evolved ligand-independency of BILF1 [137]. In contrast to common chemokine receptors, this vGPCR displays an unusual conformation. In fact, BILF1 structurally resembles lipid GPCRs rather than chemokine receptors. Here, ECL-2 binds into its own extracellular vestibule, forming a lid. Together with an inward-facing ECL-3, this blocks access of potential ligands to the extracellular binding pocket. In the light of acting as a "self-antagonist", the previously mentioned conserved regions of ECL-2 among various BILF1 orthologs [127] could prove essential for this lid-forming ability of BILF1. Consequently, the expression "orphan receptor" does not adequately represent the hypothesis that BILF1 "willingly" does not require any ligand for activation. Rather than relying on soluble ligands for activation, it appears that this receptor has acquired several mutations in typical class A GPCR motifs, leading to robust and constitutive activation and signaling [137]. In addition to the previously outlined topologic particularities of BILF1, this study revealed a unique Gαi-binding interface with higher specificity in comparison to endogenous Gαi-binding GPCRs. This could facilitate BILF1 s putative G-protein scavenging properties.

Expression Patterns
BILF1 has been detected in various EBV + cell lines and tissue samples in lytic and latent programs but is mainly considered a lytic protein [3,17,122,[138][139][140][141]. Table 3 displays this matter in more detail by listing malignancies with (not ubiquitously) detected BILF1 expression. This receptor has been described as an early lytic protein, though, curiously, BILF1 was shown to progressively interfere with MHC-I antigen presentation throughout the lytic cycle [16,17,138,139].

Cellular Effects
BILF1 has been shown to modulate several systems of the host cell in order to persist in the host. As an initial evasion mechanism, this receptor has been associated to the inhibition of phosphorylation of the RNA-dependent Protein Kinase (PKR) [3]. This interferes with a cellular antiviral defense mechanism, which normally intends to stop protein synthesis and initiate apoptosis of infected cells, hence minimizing virus spread.
As indicated above, BILF1 is able to interfere with host defense mechanisms against EBV through various distinct mechanisms. One major mechanism besides inactivating PKR is the downregulation of MHC-I receptors on the surface of host cells, effectively avoiding a strong CD8 + T-lymphocyte (cytotoxic T-cell, CTL) response and, hence, evading quick and efficient eradication of EBV by the host immune system [15,16,136]. In several investigations, it has been indicated that BILF1 must somehow, through its intracellular C-terminal tail, physically interact with the HLA-I molecule (more specifically its heavy chain), as ∆C-terminus mutated versions of BILF1 were not able to downregulate MHC-I surface presentation [16,136]. This downregulation is proposed to occur through BILF1directed degradation of MHC-I molecules in lysosomes, as lysosomal inhibitors were able to prevent degradation [15]. Even though a first study did not deem signaling ability (intact EKT motif) important for MHC-I downregulation [15], subsequent studies have suggested signaling-independent importance of an intact EKT motif for MHC-I internalization [16,142]. Additionally, besides triggering internalization and degradation of HLA-I molecules, BILF1 also diverts freshly synthesized MHC class I peptide complexes during exocytosis, causing a decrease in membrane-bound MHC-I and proteasome-derived peptide presentation [16]. The mechanism for this effect seems to be independent of the EKT motif and C-terminus.
Furthermore, recent mutational studies on the roles of conserved amino acids (AA) in the extracellular loops (ECLs) revealed that these residues are also directly or indirectly essential for surface downregulation of MHC class I molecules [142]. Interestingly, BILF1 significantly reduces the presentation of HLA-A, HLA-B and HLA-E classes, while having negligible effect on HLA-C subtypes of MHC-I molecules [136]. Through this, EBV could be able to evade strong CTL responses (mainly mediated through HLA-A and -B) while preventing activation of HLA-C-binding NK-cells [136]. Figure 4 summarizes the aforementioned BILF1-associated MHC-I downregulation, while also illustrating constitutive internalization and Gαi signaling through BILF1.
Cancers 2021, 13 16 of 29 for MHC-I internalization [16,142]. Additionally, besides triggering internalization and degradation of HLA-I molecules, BILF1 also diverts freshly synthesized MHC class I peptide complexes during exocytosis, causing a decrease in membrane-bound MHC-I and proteasome-derived peptide presentation [16]. The mechanism for this effect seems to be independent of the EKT motif and C-terminus. Furthermore, recent mutational studies on the roles of conserved amino acids (AA) in the extracellular loops (ECLs) revealed that these residues are also directly or indirectly essential for surface downregulation of MHC class I molecules [142]. Interestingly, BILF1 significantly reduces the presentation of HLA-A, HLA-B and HLA-E classes, while having negligible effect on HLA-C subtypes of MHC-I molecules [136]. Through this, EBV could be able to evade strong CTL responses (mainly mediated through HLA-A and -B) while preventing activation of HLA-C-binding NK-cells [136]. Figure 4 summarizes the aforementioned BILF1-associated MHC-I downregulation, while also illustrating constitutive internalization and Gαi signaling through BILF1.  Besides its proposed physical interaction with MHC-I, BILF1 has been described to form heteromers with endogenous chemokine receptors [143,144]. Especially, the interaction between BILF1 and CXCR4 has been surveyed, wherein co-expression of these receptors resulted in impaired CXCL12 binding to CXCR4 [144]. This effect seems to be (BILF1) signaling-dependent and is thought to be achieved through the physical stabilization of an un-inducible CXCR4 conformation within the BILF1:CXCR4 heteromer and/or through BILF1-associated constitutive G protein scavenging, possibly leading to the absence of allosteric modulation of the agonist-binding site. Furthermore, BILF1 assembled into heteromers with the histamine receptor H 4 R, which did not hinder histamine binding, but EKT motif-dependently eliminated H 4 R signaling to CREB. Taken together, it seems more plausible that BILF1 actively inhibits endogenous receptors through its persistent scavenging of Gαi proteins. A BILF1-induced reduction of plasma B cell migration toward organs with high CXCL12 expression has been discussed [144]. This could be advantageous for EBV replication and dissemination and thus could be part of the virus's sophisticated survival mechanisms [144].

Oncogenesis
In addition to decreasing surface presentation of MHC-I, a study by Lyngaa et al. revealed convincing tumor-promoting effects of BILF1, making BILF1 an oncogene [122]. In this study, Gαi signaling-dependent transformation of NIH-3T3 cells was observed in vitro and in vivo in a mouse xenograft model. In vitro foci formation assays with BILF1 + NIH-3T3 cells expressing the wild type (EKT-motif) or one of two mutations (DRY and EAT) showed strong foci induction, which correlated with the amount of constitutive signaling. While the wild type with strong signaling profile induced foci formation with the highest frequency, the DRY mutant (intermediate activity) formed foci with a minimal but significant frequency, and the EAT mutant (no activity) did not produce a significant number of foci compared to the negative control. Another in vitro experiment in this study demonstrated that only the BILF1 wild type, but not the EAT mutant, was able to overcome NIH-3T3 cell contact inhibition, stimulate cell transformation and signalingdependently increase vascular endothelial growth factor (VEGF) secretion. Correlating to EBV-associated malignancies, increases in VEGF secretion have been linked to both non-Hodgkin's lymphomas [145] and nasopharyngeal carcinomas [146]. Intriguingly, the bilateral injection of wild type BILF1 + cells in nude mice provoked tumor development in 100% of the mice and 87.5% of injection sites, whereas only 60% of the mice and 40% of injections sites resulted in tumor development in mice injected with the EAT mutant of BILF1. The fact that the signaling-deficient EAT mutant was able to induce tumor formation led to the conclusion that the oncogenic properties of BILF1 are not exclusively associated with constitutive signaling through Gαi. Figure 2B displays an exemplary image of a BILF1-induced tumor in this nude mouse model.
Linked to NF-κB activation, BILF1 has recently been found to upregulate the intercellular adhesion molecule-1 (ICAM-1) [139]. As ICAM-1 upregulation has been connected to various types of cancer and might promote cancer metastasis [147], a causative link between BILF1 expression and ICAM-1 upregulation may be valuable to elucidate mechanisms of EBV oncogenicity. Being an NF-κB dependent gene, mutational studies on the NF-κB-binding sites of the ICAM-1 promoter were undertaken, which revealed a significant disruption of the BILF1-linked upregulation of this promoter. Moreover, the cellular level of the endogenous NF-κB inhibitor protein, IκBα, decreased BILF1-dependently, likely resulting in the translocation of NF-κB from the cytoplasm into the nucleus [139].
Summarizing, the picture that BILF1 is painting in terms of association to diseases is becoming quite clear-its role in immune evasion and oncogenesis has been elucidated extensively. The current data suggests that targeting BILF1 with a novel therapeutic might be a way to treat several EBV-associated malignancies. This and the targeting of other GPCRs will be the topic of the next chapter.

Druggability of GPCRs
As GPCRs constitute a large family of receptors imperative for cell signaling, malfunctions can lead to a manifold of different diseases. Consequently, many drugs have been developed to modulate certain GPCRs. Currently, around 34% (481) of all FDA-approved drugs target 107 unique GPCRs [1]. In fact, 69 new drugs have been FDA-approved within the last 5 years, and, as of 2017, 320 drugs, of which 114 are novel drugs acting on 64 novel GPCRs, were under investigation in clinical trials. The diseases and GPCRs in focus are extremely diverse: Metabolic disorders such as hyperparathyroidism (calcium-sensing receptor) [148] and diabetes type 2 (GLP-1 receptor and GPR119 among others) [149]; psychiatric disorders such as schizophrenia (dopamine receptor D 2 ) [150]; central nervous system-related diseases such as multiple sclerosis (sphingosine 1-phosphate phosphate receptor 1) [151]; several types of cancer [1,152,153]; and viruses like HIV-1 (CCR5) [154].
Based on the link between previously presented vGPCRs (including BILF1) and tumorigenesis, the general druggability of GPCRs in the context of cancer is briefly outlined [152,153]. Research has revealed several mechanisms through which GPCRs can promote oncogenesis:

•
Excess of circulating agonists driving GPCR signaling, which promotes tumor progression (e.g., neuropeptides in small cell lung cancer) [155]; • Mutations in GPCRs or Gα subunit leading to aberrant signaling (e.g., G stimulatory protein (gsp), thyroid-stimulating hormone receptor (TSHR) [156]); • Overexpression of certain GPCRs (e.g., among many others, CXCR4, CCR7 or CXCR1) resulting in increased cancer metastasis, proliferation, cell survival or angiogenesis [157] The latter mechanism is exploited by the monoclonal antibody ulocuplumab, as it blocks CXCR4 and induces apoptosis in a chronic lymphocytic leukemia (CLL) model [157]. To date, it is not yet FDA-approved. GPCR-targeting anti-cancer drugs with FDA approval are mainly small molecules (sonidegib, vismodegib, cabergoline, raloxifene, brigatinib), but peptides (lanreaotide, degarelix, leuprolide) and mAbs (mogamulizumab, erenumab) are also currently in use [152,153]. The fact that there are already therapeutics targeting GPCRs in cancer-with many more to come-illustrates the general feasibility of GPCR targeting in this context, which could likely also be translated to vGPCR-positive malignancies.
In fact, this is not a new idea: In 1999, Rosenkilde et al. constructed a Zn 2+ binding double-mutant Kaposi sarcoma herpes virus (KSHV) GPCR ORF74 in order to study the effects of a potential ORF74-specific small molecule drug [158]. Intriguingly, incubation with Zn 2+ blocked the constitutive signaling of ORF74 with an EC 50 of 1 µM. This observed inverse agonism indicated the possibility of targeting ORF74 extracellularly with a small molecule ligand, which could potentially interfere with ORF74-driven oncogenesis.
In a different approach, KSHV has also successfully been targeted through immunotoxins binding to lytic glycoproteins and thus killing lytically infected cells in a selective manner [159,160]. Though this can be of benefit in a productive infection, the latent reservoir of this herpesvirus remains untouched, allowing the virus to persist in the host.
Additionally, within the last 20 years, a lot of effort has been put into developing human cytomegalovirus (HCMV) GPCR US28-specific therapeutics, most of which are small molecules [161][162][163][164]. These small molecules displayed inverse agonistic and neutral antagonistic properties, while reaching EC 50 values in the lower micromolar range. In a recent effort to find additional US28-binding small molecules, over 12 million molecules from the ZINC database were screened in silico, resulting in a library of 98 potential candidates [165]. After conducting inositolphosphate (IP) accumulation and Ca 2+ mobilization assays, two promising compounds, ZINC36408696 and ZINC38535746, with respective agonistic and inverse agonistic properties, decent potency (0.95 µM and 1.76 µM, respectively) and limited cross-reactivity on other receptors but without CX3CL1 displacing capabilities were identified. In a follow-up study, a new library of commercially available small molecules containing 78 potential US28 modulators was assembled based on the structures of the previously identified compounds [166]. IP accumulation and binding assays revealed several molecules with improvements regarding efficacy and potency compared with the "original" inverse agonist identified in the previous study. Moreover, competitive binding of many of these molecules with CCL2 and CCL4, but not CX3CL1, was observed. These first-in-class studies lay the basis for future development in US28 targeting and modulating small molecules.
Most recently, a research group from the Vrije Universiteit Amsterdam published three highly interesting papers on US28 involvement and targeting in glioblastoma [167][168][169]. Starting chronologically, the first study describes the successful development of a US28specific single-domain antibody (sdAb), also called nanobody (Nb) or variable heavy fragment (VHH), with sub micromolar affinity [167]. The bivalent version of this nanobody specifically inhibited ligand-dependent and constitutive US28 signaling and hence interfered with US28-driven glioblastoma growth in vitro and in vivo in an orthotopic xenograft mouse model. In a subsequent study [168], the group selected another US28-specific, highaffinity (k D of 2 nM) nanobody and conjugated this with a photosensitizer (IRDye700DX) in order to be used in targeted photodynamic therapy. In vitro binding assays showed improved displacement of CX3CL1 and killing assays on US28-expressing glioblastoma cells generated compelling data in 2D cultures and 3D spheroids. Finally, in the latest study (preprint, not yet peer-reviewed), the previous monovalent nanobody was developed into a bivalent version [169]. This increased the binding affinity to US28 once again (by 10-fold, to 0.2 nM) and retained the ability to inhibit constitutive US28 signaling by 50%, which makes this new construct a partial inverse agonist to US28. Through this inhibition, CMV was partially reactivated in latently infected primary CD14 + monocytes and expressed IE genes with only marginal expression of immunoevasins. In the clinic, this could make CMV more detectable to the host immune system, which in turn could promote eradication of the virus.
Similarly, targeting and exploiting a vGPCR with constitutive internalization, a chemokine-ExoA based immunotoxin against HCMV-US28 has been developed [170][171][172]. In these studies, the preferred chemokine to bind US28, CX3CL1, was linked to an ExoA moiety. Additionally, in order to obtain selectivity between US28 and the endogenous CX3CL1 receptor (CX3CR1), a mutated F49A-CX3CL1 was engineered. Due to the constitutive internalizing nature of US28, it has proven to be an ideal target for this kind of treatment, allowing the FTP to efficiently piggyback into the cell. This resulted in a highly potent and selective immunotoxin targeting lytically as well as latently infecting cells, as US28 is expressed in both cycles of CMV. Examples such as the latter clearly indicate the potential of targeting constitutively internalizing viral GPCRs in next-generation antivirals.
The results of all presented studies emphasize the high druggability of the vGPCR US28 and serve as starting points for future development of anti-HCMV medication and as role models for therapeutics targeting of other vGPCRs.
Addressing the need of EBV-specific therapeutics, research on EBV-targeting immunotoxins for nasopharyngeal carcinoma (NPC) has been published [173,174]. These immunotoxins have been shown to selectively kill LMP-1 or LMP-2 expressing NPC cells, respectively, in both in vitro assays and in vivo mouse models. The drawback here is also the incomplete eradication of the virus. As lytic cells do not generally express the latent membrane proteins, these cells would largely remain untouched during the immunotoxin treatment.
BILF1 is the focus of this review and has been suggested as a potential drug target, although research data on BILF1 druggability are scarce. Investigating novel ways to exploit BILF1 in future therapeutics and being aware of the absence of any known endogenous ligands, a research group in 2015 engineered EBV-BILF1 to contain a metal ion binding site through mutations in two transmembrane regions [142], based on the previously mentioned proof-of-concept for ORF74 [158]. This mutated version of BILF1 and the wild type receptor were subsequently incubated with phenanthroline (ZnPhe) or bipyridine (ZnBip) in a Zn 2+ complex and examined for Gαi signaling activity in an IP3 accumulation assay in HEK-293 cells co-transfected with Gα qi4my . The chimeric G protein Gα qi4my couples to Gαi binding GPCRs as a Gαi but signals similar to Gαq, leading to accumulation of IP3 through CRE activation by phospholipase C. While the wild type BILF1 was not affected, ZnBip and ZnPhe decreased the constitutive activity of the double mutant by around 30% with a respective EC 50 of 2 and 1 µM. Beside the observed inverse agonism, both metal chelators increased the surface expression of the double mutant by 30-40%, while, solely in the case of ZnBip, also promoting surface expression of wild type BILF1 by 15% at 10 µM. Finally, the MHC-I downregulation by wild type BILF1 was inhibited by 10%, and by 15% for the double mutant. In conclusion, this proof-of-concept demonstrated the potential druggability of BILF1 through small molecules, effectively acting as inverse agonists and, hence, possibly inhibiting BILF1 s role in EBV pathology.

EBV Drug Pipeline
Tables 4-7 contain a snapshot of the EBV drug pipeline, extracted from Global-Data.com in 2020. As seen in Tables 5 and 6, most drugs in development are based on cellular immunotherapy or small molecules. Additionally, three respective mAb therapeutics (Table 4) and vaccines (Table 7) are presently being developed.
The antibody pipeline mostly focusses on checkpoint inhibitors against the programmed cell death protein 1 (PD-1) and cytotoxic T-lymphocyte-associated Protein 4 (CTLA-4), but an antibody targeting the thymidine kinase 1 (TK1) is also being researched. In terms of cellular immunotherapy, several different autologous and allogeneic EBVspecific cytotoxic T-lymphocyte treatments targeting several EBV-derived epitopes (EBNA1, LMP1, LMP2, BARF-1) are under development. The majority of the investigated vaccines encode for EBV glycoproteins, which ideally should prevent EBV infections. Interestingly, one vaccine candidate is targeted toward the latent proteins EBNA1 and LMP2, as it is aimed to be used in patients with persistent, recurrent or metastatic nasopharyngeal carcinoma. While the aforementioned therapeutics almost exclusively focus on EBV-derived proteins as targets, the small molecule pipeline aims to interfere with various endogenous proteins, though EBV-specific molecules modulating EBNA1 and BZLF1 are also being investigated.
The fact that several different groups are looking into novel therapeutics to tackle EBV-associated malignancies shows the great need of efficacious and selective strategies and the fact that investing in the EBV-therapeutics market might be a financially attractive opportunity, despite still being in its infancy.

Conclusions
EBV ubiquitously and persistently infects the grand majority of adults worldwide, and a large number of patients suffer from EBV-associated malignancies every year. Currently, these patients do not have access to EBV-specific therapeutics. Rather, they are treated with common regimens of surgery, chemotherapy, radiotherapy and, potentially, immunotherapy. It is therefore of high interest to discover and develop alternative and EBV-specific treatment strategies. As outlined in this review, there is significant ongoing development of many new and promising drug and vaccine strategies for EBV-associated diseases. Nonetheless, more options are needed. Given the general druggability of GPCRs and the previously successful targeting of another vGPCR (US28), an EBV-encoded GPCR could prove a viable drug target. BILF1 has the potential to be considered a target as such, due to its role in EBV pathology, its internalizing nature and its expression in several EBV-malignancies. Hence, BILF1 should be considered a future drug target for the treatment of EBV-mediated diseases. In order to confirm its potential, more research on BILF1 expression patterns (especially in EBV-malignancies) and druggability must be conducted.