The Epstein-Barr virus LMP1 interactome: biological implications and therapeutic targets

Abstract

The oncogenic potential of Epstein-Barr virus (EBV) is mostly attributed to latent membrane protein 1 (LMP1), which is essential and sufficient for transformation of fibroblast and primary lymphocytes. LMP1 expression results in the activation of multiple signaling cascades like NF-ΚB and MAP kinases that trigger cell survival and proliferative pathways. LMP1 specific signaling events are mediated through the recruitment of a number of interacting proteins to various signaling domains. Based on these properties, LMP1 is an attractive target to develop effective therapeutics to treat EBV-related malignancies. In this review, we focus on LMP1 interacting proteins, associated signaling events, and potential targets that could be exploited for therapeutic strategies.

2. Introduction

Epstein-Barr virus (EBV) is a human gamma herpesvirus that has established a latent and persistent infection in more than 90% of world population. EBV is known to cause a number of human diseases including nasopharyngeal carcinoma (NPC), gastric carcinoma, and various lymphomas. In addition, EBV is also responsible for infectious mononucleosis and post-transplant lymphoproliferative disorders [1, 2]. There is also some evidence that EBV may contribute to autoimmune disease and neurological conditions [3, 4]. The study of EBV-host interactions is needed to better understand the contributions of EBV to the development and progression of the diseases associated with infection.

LMP1 is the major oncoprotein encoded by the BNLF-1 gene of EBV [1, 5, 6]. LMP1 was first identified as the LT3 transcript of viral mRNA, which encodes a protein with predominant hydrophobic regions in the N-terminal half that incorporate into cellular membranes. Rabbit antiserum raised against the C-terminus of the protein fused to bacterial beta-galactosidase was used for immunofluorescence studies first suggesting that the viral protein associated with membranes [7, 8]. Cell line specific differential expression of LMP1 was revealed between EBV-positive Burkitt’s lymphoma cell lines and EBV-transformed lymphoblastoid cells lines [9, 10]. Expression of LMP1 alone is sufficient to induce cellular transformation, and EBV lacking LMP1 is unable to immortalize and maintain transformation of primary B lymphocytes in culture [11–13]. When over-expressed in EBV-negative and positive B-cell lymphoma lines, LMP1 induces transformation, leading to cells aggregating into clumps with increased expression of cellular adhesion molecules such as intercellular adhesion molecule 1 (ICAM1), leukocyte function-associated molecule (LFA) 1 and −3. These phenotypic changes are in accordance with upregulation of a number of B-lymphocyte activation molecule like CD23, CD30, CD39, CD40, and CD44 [14].

The transforming potential of LMP1 was first identified in NIH3T3 epithelial cells and Rat-1 fibroblasts [15]. When LMP1 is expressed in these cell lines, it alters cell morphology, producing thinner and longer cells that grow faster under low serum conditions compared to the control cells. LMP1 expression also results in loss of contact inhibition and anchorage independent growth, and the formation of tumors in nude mice [16]. These effects are largely achieved by inhibition of apoptotic and differentiation pathways and the promotion of cell growth, proliferation and survival mechanism [17–22]. In vivo studies using transgenic mouse models of LMP1 expression in distinct cell-types also produces tumors. Specifically, mice expressing LMP1 under Keratin 14 promoter exhibit a slight increase in the formation of squamous cell carcinomas, which is further enhanced in double transgenic models expressing both LMP1 and LMP2A [23]. Additionally, LMP1 targeted expression in skin cells results in epidermal hyperplastic dermatosis and expression of the hyperproliferative cytokeratin marker K6, with a possible predisposition to nasopharyngeal carcinoma [5]. Whereas, B lymphocytes expressing LMP1 behind a IgH promoter results in lymphomagenesis [24]. More recent studies, using mouse models demonstrated the opportunistic nature of persistent EBV infection with rapid occurrence of life threatening lymphoproliferation and lymphoma when the regulation of immune system is compromised [25]. A similar result obtained using a transgenic mouse model with B-cell specific conditional LMP1 expression where lymphatic system disorder was evident only when animals were immune-compromised. This is because the T-cells and Natural Killer cells (NK cells) clear most of the infected B-cells, but under immunosuppressive conditions, depletion of T-cells and NK cells results in massive plasmablast outgrowth, organ defects and mortality [26]. Another EBV protein involved in B-lymphocyte transformation is Epstein-Barr virus nuclear antigen 2 (EBNA2) [27]. EBNA2 is highly expressed during latency III and acts as a transactivator for LMP1 and a number of other genes. However, during latency II, LMP1 promotes its own expression most likely by recruiting Nuclear factor kappa beta (NF-ΚB) transcription factors to the promoter. Both LMP1 and the proto-oncogene c-myc are the direct targets of EBNA2 where c-myc is considered a major transcription factor associated with EBV associated phenotypes while LMP1 activates NF-ΚB transcription factors [28–30].

LMP1 hijacks host machineries that maintain cellular homoeostasis and regulate events such as proliferation, apoptosis, migration, and invasion leading to development of various cancers and other health problems like infectious mononucleosis [31, 32]. Therefore, targeting LMP1 is an effective strategy for preventing and eliminating EBV related diseases. In this review, we will discuss LMP1 interaction partners and signaling properties that mediate LMP1-driven phenotypic events and provide potential therapeutic targets.

3. EBV Latency

EBV possess a unique feature of establishing different latency types characterized by the expression of a set of distinct but overlapping genes in resting and proliferating cells [33]. During type I latency, as in Burkitt’s lymphoma cells, the genes expressed are EBNA1, the DNA binding protein gene, the EBER non-coding RNAs, LMP2A/B and the BART (B_amHI A rightward transcript) RNAs. NPC and Hodgkin’s Lymphoma (HL) show type II latency with the expression of LMP-1 (type IIa), EBER1/2 RNA, EBNA-1, LMP-2A/B, or EBNA-2 (type IIb) and BART RNA. Most of the latent genes are expressed during latency III (like EBER1/2 RNA, EBNA-leader protein (EBNA-LP), EBNA-2, EBNA-3ABC, EBNA-1, LMP-2A/B, LMP-1 protein, and BART RNA) as shown in lymphoproliferative diseases and lymphoblastoid cell lines in vitro [33–35]. It is clear from many studies that LMP1 plays a crucial role in maintaining virus latency and transformation of host cells [14, 34].

4. Structure of LMP1

LMP1 is a membrane protein expressed both during latent and lytic stage of EBV infection [34, 36, 37]. The functional protein has six transmembrane domains with a short N-terminal domain of 24 amino acids and a large C-terminal domain comprised of 200 amino acids. Amino acids 25–187 constitutes the hydrophobic transmembrane domains that span the membrane. Activation and signaling of LMP1 requires targeting to detergent resistant membrane domains called lipid rafts, the formation of homo-oligomers by cysteine cross linking and recruitment of intermediate and effector protein machinery into the carboxyl terminal region [31, 38, 39]. LMP1 mimics constitutively active CD40 signaling even in absence of ligand binding through the formation of homo-oligomers driven by transmembrane (TM) domain interactions through a process similar to receptor clustering involving non-receptor tyrosine kinases, like reelin signaling [40, 41]. In the case of LMP1, TM3–6 and a FLWY motif in TM1 are essential for the intermolecular oligomerization [42, 43]. Phenotypes of Epstein-Barr virus LMP1 deletion mutants have shown that the transmembrane regions and amino-terminal cytoplasmic domains are necessary for signal transduction and its phenotypic effects in B-lymphoma cells [44].

Protein targeting and membrane insertion are important for the proper functioning of transmembrane proteins. In the case of LMP1, the cytoplasmic N-terminal domain regulates correct membrane insertion and orientation [45]. Furthermore, the N-terminus is also involved in the interaction with cytoskeletal machinery and protein degradation through ubiquitin-proteasome pathway [46, 47]. To date, no protein binding partners have been assigned to the N-terminal domain of LMP1.

4.1. CTAR domains

The growth and transformation potential of LMP1 is mediated through the cytoplasmic C-terminal tail that engages a multitude of signaling effector molecules (Fig. 1 and 2). Detailed biochemical studies and genetic analyses using deletion mutants have identified different functional domains necessary for the recruitment of various effector proteins [11, 45, 48–50]. Upon oligomerization, LMP1 signals through C-terminal activating regions (CTARs) by recruiting various adaptor proteins. CTAR domains were initially identified and termed as transformation effector sites −1 and −2 (TES-1 and TES-2) as these domains are required for the transforming activity of LMP1. A detailed understanding of signaling underlying LMP1-mediated transformation has come from many studies from different labs [38, 51]. CTAR1 was shown to be essential for transformation of B-lymphocyte and Rat1 fibroblasts while CTAR2 mediates sustained transformation in lymphocytes [48, 52]. The CTAR1 domain constitute amino acids 194 −232 and CTAR2 was mapped between amino acids 351–386. The CTAR3 domain (amino acids 275 to 330) sits between CTAR1 and CTAR2 with fewer known interaction partners [50, 53].

Fig 1. An overview of LMP1 signaling pathways.

LMP1 signaling through C-Terminal Activating Regions (CTARs) located on the cytoplasmic C-terminal domain results in many downstream cellular events. These signaling domains recruit various intermediate proteins leading to the activation of NF-ΚB, MAPK, PI3K-AKT and JNK pathways. Only the interaction partners with known binding domain are shown. Additional interaction partners are shown in figure 2. EV, extracellular vesicle.

MTORC1: Mammalian target of rapamycin complex 1; NIK: NF-kappaB inducing kinase; IKKa: Inhibitor of nuclear factor kappa-B kinase subunit alpha; JAK3: Janus kinase 3; STAT1: Signal transducer and activator of transcription 1-alpha/beta; EGFR: Epidermal growth factor receptor; TPST1; Protein-tyrosine sulfotransferase 1; CXCR-4: C-X-C chemokine receptor type 4; MMP9: Matrix metalloproteinase 9; VEGF: Vascular endothelial growth factor; TRADD: Tumor necrosis factor receptor type 1-associated DEATH domain protein; IRAK: Interleukin-1 receptor-associated kinase 1; ATF2: Cyclic AMP-dependent transcription factor ATF-2; RIP: Receptor-interacting protein; RIPK: Receptor-interacting serine/threonine-protein kinase; TAB: TGF-beta-activated kinase 1-binding protein 1; TAK1: kinase transforming growth factor-b-activated kinase 1; JNK1: c-Jun N-terminal kinase 1; MAP3K3: Mitogen-activated protein kinase kinase kinase 3; SAPK: Stress-activated protein kinase; PKCd: Protein kinase C delta; AKT: Protein kinase B; ERK: Extracellular signal-regulated kinase 1; ATM: Serine-protein kinase ATM; AP3: Adaptor protein complex AP-3; p90Rsk: 90 kDa ribosomal protein S6 kinase 1; hTERT: Human Telomerase reverse transcriptase.

Fig 2. LMP1 interaction partners and signaling.

LMP1 recruits various adaptor molecules that mediate transduction of multiple cellular signaling events that alter cellular morphology and physiology. The CTAR domains are not indicated since for a number of these identified proteins, the specific binding region is not yet mapped. ALIX, Syntenin and CD63 are associated with EV secretion. Glut1 is involved in glycolysis and along with galectin, in immune response regulation. Recruitment of Id1 and RPS27a prevent ubiquitination of LMP1 and enhances its stability. The P85 subunit of PI3K activates IRF4 and promotes transcription of mir155. Interaction of IRF7 with the LMP1 promoter region activates transcription of LMP1. Ubc9, a SUMO conjugating enzyme is recruited to the complex via the CTAR3 domain of LMP1 and facilitates sumoylation of IRF7 and KAP1. FGD4 is a GEF for small GTPase Cdc42, which along with other cytoskeletal associated proteins modulates the cytoskeleton, cell proliferation and EMT. Various kinases like PKD1 and RIPK1/3 are recruited to LMP1 and regulate processes like cell survival. RNF31 interacts with IRF7 and regulates NF-ΚB through NF-ΚB essential modulator (NEMO). Bram1, HOS, A20 and SHP1 negatively regulate LMP1 signaling through various signaling mechanisms. EV, extracellular vesicle.

GM-CSF: Granulocyte-macrophage colony-stimulating factor; IL-s: Interleukins; NLRP3: Nod-like receptor family protein 3; COX2: Cytochrome c oxidase subunit 2; GLUT1: Glucose transporter type 1; K48-U: Lysine48-ubiquitinated; Bram1: Bone morphogenetic protein receptor-associated molecule 1; HOS: Homologue of Slimb; Id1: inhibitor of DNA binding 1; RPS27a: Ubiquitin-40S ribosomal protein S27a; p85: p85 subunit of PI3Kinase; SRC: Proto-oncogene tyrosine-protein kinase Src; IRFs: Interferon regulatory factors; SHP1: Tyrosine-protein phosphatase non-receptor type 6; PKD1: Protein kinase D1; RNF31: E3 ubiquitin-protein ligase RNF31; A20: Zinc Finger Protein A20; PRA1: Prenylated rab acceptor 1; FGD4: FYVE, RhoGEF and PH domain-containing protein 4; Cdc42: Cell Division Cycle 42; TAZ: Tafazzin; Mcl1: Induced myeloid leukemia cell differentiation protein Mcl-1; NEMO: NF-ΚB essential modulator; KAP1:KRAB-associated protein-1. Also see fig. 1.

Tumor Necrosis Receptor Associated Factors (TRAFs) are adaptor proteins that mediate different cellular functions including proliferation, survival, apoptosis and immune response [54]. CTAR1 contains a PXQXT (a.a. 204–208) TRAF binding site which is critical for the recruitment of TRAF1/2 as well as TRAF3/5 heterodimers [55]. Another motif, YYD (a.a.384–386) is essential for recruiting a death domain containing protein called tumor necrosis factor receptor type 1-associated DEATH domain protein (TRADD), the first protein found to interact with CTAR2 directly. Upon binding, TRADD initiates unique and LMP1-specific signaling events with a different cellular outcome compared to receptor mediated TRADD signaling [48, 56]. Another protein identified that signals through CTAR2 is TRAF6. Direct binding has not been demonstrated, but could occur though a PVQLSYY motif or indirect binding through TRADD or BS69 [38, 57]. Recently, the interaction between LMP1 and TRAF6 has been validated using a newly described proximity-based BioID approach [58]. It has only been over the past decade that the EBV community has begun to appreciate the contribution of CTAR3 in EBV related signaling and cellular events. Zhan et al. first reported a phenotypic effect following mutation of the CTAR3 domain. Deletion of amino acids in these region significantly abrogated LMP1 dependent Janus kinase (JAK3) promoter activation and transcriptional upregulation. Furthermore, the colony forming ability of the cells expressing LMP1 with mutated CTAR3 was considerably reduced [59]. The first identified CTAR3 interacting protein was JAK3. The interaction between CTAR3 and JAK3 leads to increased tyrosine phosphorylation of JAK3 and subsequent activation of the STAT3 transcription factor [49]. The Pagano group reported ubiquitin carrier protein 9 (Ubc9), a CTAR3 interacting protein and Sumo conjugating enzyme, plays an essential role in protein sumoylation mediated by LMP1. Ubc9-LMP1 interactions result in altered subcellular localization of proteins and modified DNA binding abilities [60]. Immunoprecipitation experiments revealed an interaction of LMP1 with the enzymatically active form of Ubc9, but not with inactive Ubc9, even in absence of CTAR1 and CTAR2. Specific target of LMP1-dependent Ubc9-mediated sumoylation includes interferon regulatory factor 7 (IRF7) and KRAB-associated protein-1 (KAP1). In the same study, both EBV infected lymphoblastoid cells and cell lines with transient LMP1 expression showed increased sumoylation of IRF7, leading to decreased turn over, increased nuclear retention, decreased DNA binding and limited transcriptional activation. KAP1 was found to be involved in EBV latency maintenance by binding to EBV Orilyt (origin of lytic replication) and immediate early promoters in CTAR3 dependent manner [60–62]. Taken together, these data support the importance of CTAR3 in cellular pathways regulated by LMP1.

5. LMP1 interaction partners and signaling

A number of LMP1 interacting proteins were identified using various techniques like immunoprecipitation, yeast two hybrid assay, microscopy, glutathion S transferase (GST) -pulldown assay, bimolecular fluorescence complementation (BiFC) and mass spectrometry (MS) based proteomics (Table 1). These proteins, along with others, regulate a number of LMP1-associated cellular events like proliferation, survival, transformation, invasion and metastasis. The major biochemical pathways leading to these cellular events are summarized in fig. 1 and 2.

Table 1.

LMP1 interacting proteins^*

No.	Protein	Method	LMP1 domain	Pathway	Reference
1	TRAF1	IP	CTAR1	NF-KB activation	[67]
2	TRAF2	IP	CTAR1	NF-KB activation	[67]
3	TRAF3	IP	CTAR1	Inhibition/activation of NF-KB	[55]
4	TRAF5	IP	CTAR1	LMP1-mediated c-Jun kinase signaling	[197],[198]
5	TRAF6	IP	CTAR2	NF-KB activation, JNK activation	[98],[199]
6	CD63	IP, Microscopy	LMP1	LMP1 exosomal targeting, downregulation of NF-KB	[83]
7	A20	IP	CTAR1/CTAR2	NF-KB &JNK inhibition	[138]
9	Bram1	IP, GST-PD	CTAR2	NF-KB inhibition, not JNK	[139]
10	SCFHOS/bTrcP	IP, In-vitro assay	C-terminal	NF-KB inhibition	[141]
11	PRA1	Y2H, FRET, BRET	TM	NF-KB activation	[79]
12	Galectin	IP, Proteomics	LMP1	Immune response	[88]
13	BS69	Y2H	C-terminal	JNK activation	[98]
14	TNIK	IP, Proteomics	CTAR2	Canonical NF-KB & JNK	[99]
15	RIPK1/3	IP, PLA assay	CTAR2	Suppress necroptosis	[96]
16	TRADD	IP,Y2H	CTAR2	Activate NF-KB, not JNK	[57]
17	RIP	Y2H	CTAR2	Activate NF-KB	[93]
18	IRF7	IP, microscopy		Activates IRF7	[103]
19	Ubc9	IP	CTAR3	Increased sumoylation of proteins	[60]
20	RNF31	IP	CTAR2	Activate NF-KB	[105]
21	Actinin-1	BIFC	CTAR1	Downregulate NF-KB	[123]
22	Actinin-4	BIFC	CTAR1	Downregulate NF-KB	[123]
23	Gelsolin	BIFC, IP	CTAR1	Downregulate NF-KB	[123],[127]
24	Tropomyosin	BIFC	LMP1	May be lipid raft targeting or regulate motility and invasion	[123]
25	Tmem134	BIFC, IP	CTAR1/CTAR2	Activate NF-KB	[125]
26	FGD4	IP	TM	Cdc42 activation, migration	[128]
27	RPS27a	IP, GST-PD	LMP1	Suppress LMP1 degradation	[106]
28	Id1	IP	CTAR1	Suppress LMP1 degradation	[107]
29	GLUT1	IP, IF	CTAR3	MDSC expansion, resistance to host defense	[91]
30	Src	IP	CTAR1	IRF4 activation	[110]
31	PDK1	IP, Microscopy	LMP1	Increased cell survival	[110]
32	PI3K (p85 subunit)	IP	CTAR1	Transformation and survival	[111]
33	vimentin	IP, proteomics	CTAR1	Transformation of fibroblast	[133].

^*Proteins identified in high through-put studies are not included

5.1. TRAFs and NF-ΚB signaling

NF-ΚB proteins are a group of transcription factors involved in regulating multiple cellular processes including cell proliferation, differentiation and apoptosis. Uncontrolled activation of NF-ΚB pathways can contribute to pathological conditions such as inflammation and cancer. The five members include RelA (P65), RelB, c-Rel, NF-ΚB1 (P50), NF-ΚB2 (p52) which form homo- or hetero dimers before moving into the nucleus to regulate gene transcription. Several signaling molecules and agents have been identified to induce activation of NF-ΚB including tumor necrosis factor, cytokines, the bacterial cell wall component lipopolysaccharide, and many viruses like human herpesvirus-8 (HHV-8), human T-cell leukemia virus type 1 (HTLV-1) and EBV [38, 51].

The role of LMP1 mediated NF-ΚB signaling was first reported in 1992 by Laherty et al. The authors showed that LMP1 upregulates transcription of the A20 protein by inducing NF-ΚB factor binding to the A20 promoter region [63]. The A20 protein has been implicated as a key molecule regulating TNF-induced apoptosis. NF-ΚB signals are transduced through both CTAR1 and CTAR2 domains of LMP1. CTAR1 is primarily responsible for activation of non-canonical NF-ΚB signaling whereas CTAR2 was shown to be essential for the canonical pathway [38].

The cellular TRAF proteins were some of the first identified and best characterized LMP1 interacting proteins. TRAFs were originally described as adaptors that are recruited to the cytoplasmic tails of TNF-receptor proteins. Now it has become more evident that TRAFs are widely recruited by a larger array of signaling proteins like toll like receptors, nod like receptors, RIG-I like receptors, T-cell receptors, IL-1, IL-17 receptor families, interferon receptors, TGF-b receptors, etc. Most of these receptors activate NF-ΚB family of transcription factors, MAPK cascades, and interferon regulatory factors [64–66].

Out of the six known members of TRAF family, LMP1 has been shown to physically interact with TRAFs 1, 2, 3, 5, and 6 by virtue of its C-terminal cytoplasmic tail. TRAFs 1, 2, 3, and 5 associate with CTAR1 of LMP1 and activate non-canonical NF-ΚB signaling, leading to nuclear translocation of p52 containing dimers. On the other hand, TRAF2 and TRAF6 were shown to form complex with CTAR2 domain, leading to activation of canonical NF-ΚB pathway involving RelA [67–70].

Studies conducted by Mosialos, G. et al. first identified TRAF1 and TRAF2 as the LMP1 interaction partners. In an attempt to understand role of LMP1 in B-lymphocyte transformation, the authors found that two proteins namely LMP1-associated protein 1 (LAP1) and EBI6 were co-immunoprecipitated with LMP1, which are human homologues of TRAF2 and TRAF1, respectively [71]. TRAF2 and TRAF3 play a crucial role in activating NF-ΚB signaling. TRAF2 is required for LMP1-dependent NF-ΚB signaling through CTAR1 domain but is dispensable for CTAR2 signaling events. This was evident with the increased activation of NF-ΚB when TRAF2 is overexpressed in C33A cells (cervical carcinoma cells) [55]. Conversely, reduced NF-ΚB activation was observed when a dominant negative amino terminal deletion of TRAF2 was expressed or the protein levels were knocked-down [67, 72]. Distinct from epithelial cells, B-lymphocytes mostly depend on TRAF3 for signaling instead of TRAF2. B cells with a TRAF3 deletion using homologous recombination shows defective signaling leading to impaired activation of JNK and NF-ΚB, loss of CD23, CD80 upregulation, and reduced antibody production. However, in TRAF2 knock-out cells, LMP1 signaling results in a modest reduction or remains unaffected [73–75]. However, studies conducted using specimens from patients of lymphoproliferative disorders concluded a positive correlation between LMP1 and TRAF2 expression, and not TRAF3 [76]. TRAF3 also negatively regulates signaling by competing with TRAF1 and TRAF2 for binding to CTAR1 [67, 68]. Upon signaling activation, TRAF3 is selectively removed from CTAR1 in a proteasome independent process (unlike CD40 signaling, where the process is proteasome dependent), leading to downstream signaling. Additionally, TRAF3 recruitment to the cytoplasmic domain may be direct (mediated via CTAR1) or indirect (mediated via CTAR2). TRAF3 also functions as an inhibitor of TRAF1 and TRAF2 recruitment to membrane rafts through CTAR1, limiting their signaling potential and acting as a mediator of the physical interaction between two C-terminal domains of LMP1 [75].

Both B-cells and fibroblasts utilize TRAF6 for LMP1 signal transduction. Luftig et al. used MEF (murine embryo fibroblast) cell lines lacking various components of NF-ΚB signaling pathways to evaluate the effects of individual proteins in the activation process. TRAF6 KO cells were highly deficient in NF-ΚB signaling in MEFs as in the case of IRAK1 in human embryonic kidney 293 (HEK293) cell signaling. The significance of TRAF6-mediated NF-ΚB signaling in HEK293 cells was verified by overexpressing dominant negative TAB2 or Ubc13. In both the cases, LMP1-mediated NF-ΚB activation was adversely affected [73]. TRAF6 has also been shown to play a critical role in LMP1-dependent activation of NF-ΚB signaling in B cells, which unlike CD40 signaling, requires the TRAF6-receptor binding domain. A mouse model generated with B-cell specific TRAF6 deletion demonstrated the role of this adaptor protein in B cell functions mediated by LMP1. In the transgenic mouse the absence of TRAF6 showed impaired abilities in antibody and autoantibody production, as well as defective germinal center formation. However, TRAF6 did not play any significant role in secondary lymphoid organ enlargement, which is a consequence of LMP1 expression [69, 77].

The ring finger domains of TRAF2 and TRAF6 both possess E3 ubiquitin ligase activity which is important in activating NF-ΚB signaling. Using yeast two hybrid assay, Hadweh et al. identified PP4R1, regulatory subunit R1 of protein phosphatase 4, as an interacting partner of TRAF2. PP4R1 dephosphorylates TRAF2 at S11 resulting in the downregulation of NF-ΚB activation. Over expression of PP4R1 not only inhibits TRAF2 dependent events, but also signaling via TRAF6, possibly by interfering with its ubiquitin ligase activity [78].

In addition to its function in NF-ΚB activation, LMP1 signaling mediated through TRAF5 and/or TRAF6 also contributes to the maintenance of EBV latency. Expression of dominant negative mutant TRAFs or the inhibition of downstream effector protein p38 MAP kinase abrogates the origin of replication (oriP) suppression due to LMP1 [70]. Taken together, these findings reveal a unique requirement of TRAF protein engagement that depending on the cell line is critical for the downstream activation of many pathways.

5.2. Trafficking proteins

Prenylated Rab Acceptor 1 (PRA1) is a transport protein that plays a critical role in protein targeting to various cellular compartments and associates with LMP1. Since LMP1 functions depend on its targeting to lipid raft membrane microdomains, the transport functions of PRA1 is significant for proper LMP1 signaling. PRA1 directly interacts with the transmembrane domains of LMP1, promoting LMP1-dependent NF-ΚB signaling. Studies using export mutant PRA1 constructs, or siRNA knock-down of PRA1 showed impaired LMP1 trafficking and subsequent re-distribution to ER [79, 80]. Therefore, PRA1 is likely important for ER to Golgi transport of LMP1.

CD63 a is component of the cellular trafficking machinery involved in endosomal sorting of proteins into multivesiclular bodies (MVBs) and subsequent lysosomal degradation or exocytosis [81]. CD63 belongs to the family of tetraspanin proteins and plays a pivotal role in LMP1 trafficking into exosomes and regulation of intracellular signaling. CD63 and LMP1 have been shown to interact and when CD63 was deleted using CRISPR-Cas9 genome editing technique or knocked-down with shRNAs, LMP1 trafficking into extracellular vesicles (EVs) is considerably reduced. In addition, LMP1-dependent enhancement of small extracellular vesicle production was reduced concomitant with enhanced MAPK, mTOR, and non-canonical NF-ΚB signaling. These data suggest that LMP1 EV trafficking through CD63 is directly linked to LMP1-mediated signaling transduction [58, 82–84].

5.3. Immune response

Galectins are a family of glycoproteins that function in regulating immune responses and homeostasis [85, 86]. Analysis of tumor samples from NPC patients revealed higher expression of galectin in recurrent tumor compared to primary tumor, suggesting a probable role of galectin in tumor recurrence and increased malignancy [87]. Indeed, galectin 9 is a LMP1 interacting protein both in NPC cell lines and EBV transformed LCLs. Although simvastatin was shown to dissociate LMP1 from lipid rafts in B-cells, it had minimal effect on LMP1 and galectin distribution in NPC cell line. Interestingly, this drug showed potent cytotoxicity which was irrespective of presence of LMP1 in the cells [88].

Immune suppression is a critical response in tumor progression and a compromised immune system fails to fight against malignant conditions. Tumor cells are capable of evading the immune system by exploiting various cellular and molecular mechanisms [89]. Cancer cells achieve this in multiple ways by modulating T cell functions with involvement of related immunosuppressive cytokines, nutrient availability in tumor cells and recruiting checkpoint molecules like programmed cell death protein 1 (PD-1) and cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) [90]. Immune suppression can also result from the expansion of myeloid- derived suppressor cells (MDSCs) as observed in many cancers including NPC. The presence of LMP1, glucose transporter (GLUT1), and CD33+ MDSCs in tumor sections suggests a possible role of metabolic regulation in EBV-associated cancers. LMP1 enhances extra-mitochondrial glycolysis by inducing glycolytic genes, including GLUT1. The transcriptional upregulation of these genes results in the activation of multiple signaling events and cytokine production through Nod-like receptor family protein 3 (NLRP3) inflammasome components, COX2, and P-p65. LMP1 brings about these effects by associating with GLUT1 and preventing its K48 ubiquitination and degradation via p62 dependent autolysosomes. Together these biochemical pathways lead to MDSC expansion and tumor immunosuppression [91].

5.4. Apoptosis modulators

Exploitation of TRADD by LMP1 as a signal transducer is an excellent example of how viruses differentially modulate cellular signaling proteins and contribute to oncogenesis. TRADD is a TNF signal transducer that induces apoptosis. Using TRADD deficient human B lymphocytes, Schneider et al. has shown that LMP1 interacts with TRADD and is an indispensable mediator of TRAF6 dependent signaling. Surprisingly TRADD recruitment to LMP1 and downstream events does not result in apoptosis. Later studies mapped out a unique TRADD binding site on C-terminal of LMP1 that confers resistance to apoptosis. This finding was further validated by substituting TNFRs death domain with the TRADD binding domain of LMP1 which switched TNFR signaling from apoptotic to anti-apoptotic [57, 92–94].

Apoptosis is a common host defense response against invading pathogens. However, when this defense system is compromised, cells use alternative mechanisms for programmed cell death. Necroptosis is a regulated cellular process mediated by receptor interacting protein kinase (RIPK) activated independent of caspases as part of host defense to eliminate abnormal cells [95, 96]. Some viruses, including EBV, suppress necroptosis to escape host defense mechanisms. Direct binding between the LMP1 C-terminal region and RIPK1 and RIPK3 regulates K63-mediated polyubiquitination and availability of these proteins, resulting in a cell fate transition from apoptotic to survival. This mechanism helps EBV to bypass host defense pathways and enhance cell survival and tumorigenic properties [96].

5.5. Interactions that activate JNK signaling

LMP1 also activates the JNK pathway by recruitment of multiprotein complex through its CTAR2 region. BS69 was initially identified as an interaction partner of the adenoviral oncoprotein E1A with transcription co-repressor and possible tumor suppressor functions. BS69 is recruited to the signaling complex by binding the PXLXP motif of LMP1 through the MYND domain on BS69 [97]. Thus BS69 bridges LMP1 signaling to JNK activation through an axis consisting of LMP1-BS69-TRAF6-TAB1/TAK1-JNKKs. siRNA mediated gene knock-down revealed an essential role for this protein in activating JNK signaling. Furthermore the disruption of interaction using mutants defective in binding abrogated signaling, while introduction of LMP1 mutant –BS69 chimera restored JNK signaling [98].

Another protein recruited to CTAR2 centered signaling complex (TRAF6-TAK1/TAB2 and IKKb) is the germinal center kinase family member TNIK (TRAF2 and NCK interacting kinase). Distinct from BS69, TNIK activates both canonical NF-ΚB and JNK transduced from LMP1 or CD40, although TNIK utilizes entirely different domains (N-terminus of TNIK for NF-ΚB and the C-terminus for JNK) for signal transduction. Therefore, TNIK plays an essential role in activating cell proliferative and survival mechanisms by bifurcating signals from CTAR2 domain of LMP1 [99]. A recent report on colorectal cancer verified the oncogenic role of TNIK where Masuda et al. used small molecule inhibitor against TNIK and showed reduced levels interstitial tumor and colorectal cancer. This researchers also used mice deficient for TNIK that resulted in reduced levels of cancer stem cells and tumor formations [100].

5.6. Interferon Regulatory Factors

IRF7 is a multifunctional transcription factor that regulates type 1 interferon responses during pathogen infections and is activated by signaling from pathogen recognition receptors (PRR) [101]. EBV-LMP1 activates IRF7 expression, and levels of IRF7 and LMP1 directly correlated in EBV infected latency type II cells. IRF7 localizes to cytoplasm in EBV negative B cells and type I latency, but exhibits more nuclear localization in type III latency. Also, immunoaffinity pull-down showed both proteins form a complex in the cytoplasm (fig. 2). LMP1 triggers IRF7 expression and activation, at the same time, IRF7 binds to the LMP1 promoter region to activate transcription. This positive regulatory circuit of reciprocal regulation of LMP1 and IRF7 is disrupted by over expression of IRF5, an interacting partner of IRF7. This effect was achieved by downregulating IRF7 induction on LMP1 [102, 103].

Receptor interacting protein (RIP) is another protein closely associated with TNFR1 signaling. RIP is stably associated with LMP1 in lymphoblastoid cells, but is not required for NF-ΚB activation [93]. In EBV+ Burkitt lymphoma cells, RIP physically interacts with IRF7 (fig. 2). LMP1 induced activation of IRF7 requires RIP-IRF7 interactions and ubiquitination of both proteins. RIP mediated ubiquitination of IRF7 on lysine 63 ( K63) leads to enhanced modulation of IRF7 functions, but does not induce its proteasomal degradation [104].

In addition to ubiquitination and phosphorylation, sumoylation is a protein modification LMP1 employs to modify and regulate other cellular proteins. This is achieved by recruiting the sumo-conjugating enzyme Ubc-9 through physical interaction of Ubc-9 with CTAR3 of LMP1 (fig. 2). IRF7 is sumoylated at leucine 452 (L452) in a LMP1 dependent manner resulting in decreased degradation, increased nuclear retention, and reduced binding to DNA, minimizing its transcriptional activation. Together these events inactivate IRF7, limiting the antiviral host immune response [61].

Linear ubiquitination mediated by LUBAC (linear ubiquitin chain assembly complex) is another way to regulate LMP1 functions. RING finger protein 31 (RNF31) a major protein in LUBAC, interacts with both IRF7 and LMP1, leading to linear ubiquitination of NF-kappa-B essential modulator (NEMO ) and IRF7 (fig. 2). This process initiates LMP1-mediated NF-ΚB signaling, but negatively regulates LMP1-specific activation of IRF7. In accordance with these data, RNAi mediated knock-down of RNF31 in EBV transformed cells negatively affects LMP1-dependent cellular events including cell proliferation [105].

5.7. Proteasomal targeting

One of the factors contributing to constitutive activation of downstream signaling by LMP1 is its cellular stability achieved by avoiding proteasome degradation. The ribosomal protein, ubiquitin-40S ribosomal protein S27a (RPS27a) was identified as a direct interaction partner of LMP1, both in vitro and in vivo using affinity purification methods. Interaction between RPS27a and LMP1 completely inhibits LMP1 ubiquitination allowing the viral protein to escape from proteasomal targeting and promote increased cellular proliferation and invasion [106]. Similarly, Id1 (inhibitor of DNA binding 1) stably interacts with LMP1 in EBV infected cells, and knock-down of Id1 results in enhanced proteasomal degradation of LMP1 (fig. 2) [107].

5.8. Kinases

Kinases Src, p85 subunit of PI3Kinase (PI3K) and polycystic kidney disease 1 (PKD1) otherwise called protein kinase C mu (PKCμ) have been found to interact with LMP1. LMP1 mediated cell survival is partially transduced through PKD1. LMP1 regulates PKD1 expression and stability by direct protein-protein interaction in B-lymphocytes. This induces myeloid leukemia cell differentiation protein Mcl-1. Mcl-1 is an anti-apoptotic protein belong to Bcl family and is a highly regulated protein using multiple signals [108]. LMP1 upregulates Mcl1 through PKD1 interactions [109]. Src is a non-receptor protein kinase and its interaction with LMP1 is dependent on p85 subunit of phosphoinositide 3-kinase (PI3K). The Src-LMP1 interaction contributes to LMP1 activation of interferon regulatory factor 4 (IRF4), where Src phosphorylate IRF4. Activated IRF4 interacts with interferon regulatory factors binding site of MIR155 Host Gene (B-Cell Receptor Inducible) promoting its expression thereby contributing to increased tumorigenicity [110]. PI3kinase/Akt signaling axis is major pathway activated due to LMP1 expression contributing to actin polymerization, cell transformation and survival. Immunoprecipitation studies revealed that LMP1 interacts with p85 subunit of PI3kinase through CTAR1 domain [111].

5.9. Identification of interaction partners LMP1 Bio-ID

Using the BioID approach combined with traditional immunoaffinity purification, one of the largest studies of the broader LMP1 interactome has recently been published [58]. The Bio-ID method utilizes a bacterial protein biotin ligase, BirA, with a R118G mutation (BirA*) that abrogates its specificity towards natural substrates, but maintains ligase activity [112, 113]. In this study, BirA* was fused to LMP1 either N-terminally or C-terminally, to maximize output, followed by affinity purification using streptavidin magnetic beads. The BioID approach is useful for identifying protein-protein interactions which may be direct or indirect and is based on molecular proximity [114]. One of the greatest advantages of this method is that both weak and transient interaction are preserved since the biotin labelling takes place in the cells prior to lysis. Additionally, interactions from insoluble or inaccessible cellular compartments can also be identified due to the harsh lysis conditions [115]. Moreover, non-specific binding and antibody related issues can be surpassed due to high specificity and extreme affinity between streptavidin and biotin.

Mass spectrometry data from the N- and C-terminally tagged LMP1 constructs detected more than 1000 proteins as potential interaction partners of LMP1. The identified proteins were subjected to bioinformatics analysis using different computational tools including DAVID, Funrich and SAINT [58]. DAVID analysis identified pathways enriched in EBV infection, endoplasmic reticulum (ER) protein processing, endocytosis and proteasome. These results could be expected since LMP1 is an EBV protein synthesized in ER, transported through endocytic pathways, degraded by the proteasome and lysosome, and released from cells in extracellular vesicles [84]. Moreover, a significant number of interacting proteins are part of cell cycle machinery or metabolic pathways. These interactions further validate the role of LMP1 in modifying cell cycle processes leading to the activation of cell survival signaling and inhibition of apoptosis. LMP1 signaling is known to increase cell growth, survival and transformation, with increased metabolism contributing significantly in to these processes [31, 91, 116].

FunRich analysis classified proteins identified in the study into different sub-cellular compartments. As expected, the largest group was cytoplasm with more than 50% belonging to this group. The cellular compartment with un-expected number of identified protein was the nucleus, with nearly 50% of identified proteins in this category [58]. Since LMP1 interacts with a number of transcription factors, adaptors and kinases in the cytoplasm that are known to exert functions in the nucleus, it is possible some of the identified proteins represent true interacting partners [31]. Furthermore, LMP1 exerts its function on nuclear processes. For example, LMP1 regulates Op18/Stathmin pathway by activating cyclin-dependent (CD) Kinase Cdc2 leading to phosphorylation of Op18/Stathmin and polymerization of microtubules, thus facilitating cell division [117]. It was also shown that in T cells, contrary to B-cells, LMP1 localizes to nucleus [118]. Even though nuclear specific localization of LMP1 has not been well studied in epithelial cell lines, LMP1 does exhibit perinuclear localization and may interact with proteins translocating to nucleus [119]. Nevertheless, it is quite possible some of the identified proteins attached non-specifically to the beads. The other compartments like lysosome and exosome showed high enrichment (around 20% each) emphasizing significance of these machineries in LMP1 processing, via protein degradation and exocytosis.

The proteins identified in the experiments mentioned above were subjected to SAINT analysis (Significance Analysis of INTeractome), which rank the identified proteins based on spectral count and reproducibility [120]. Using a cut-off SAINT score of 0.6 (for immunoaffinity pulldown) or 0.8 (for Bio-ID), the authors narrowed down the identified proteins to 485 which were used to construct protein interaction network using FunRich. FunRich analysis yielded signaling nodes with the proteins known to interact with LMP1 or part of signaling cascades regulated by LMP1. In addition, the analysis also revealed signaling clusters centered on newly identified interaction partners which play crucial roles in activating various pathways like MEK1/2, ERK, AKT/PI3K, Wnt/Catenin and hypoxia inducible factor (HIF). Altogether, the study laid a strong foundation with the identification of a number of proximal or direct interacting proteins that contribute to a better understanding of regulation of cellular signaling by LMP1 and its effects in various pathophysiologies [58].

5.10. Cytoskeletal proteins as activator and inhibitors of LMP1 signaling

The actin cytoskeleton plays a critical role in cell motility and migration [121]. It also considered in maturity, structural and functional integrity of membrane lipid rafts [122]. NPC is characterized by highly malignant cells that are notoriously metastatic. NPC cells in culture display increased migration with reduced adhesive properties [31]. These cellular and oncogenic features can be attributed to the modulation of actin cytoskeleton by LMP1. LMP1 likely interacts with a number of actin binding proteins including non-muscle actinin-1 and −4, tropomyosin, transmembrane protein 134 (Tmem134) and gelsolin. These proteins were identified as potential interactors using bimolecular fluorescence complementation (BiFC) technique and further validated by proximity dependent biotinylation assay. Addition of gelsolin, actinin-1, and −4, which were tagged with cyan fluorescent protein (CYFP) to facilitate BiFC experiment, showed decreased NF-ΚB receptor activity compared to the vector control (CYFP-Zip only) in BiFC experiments showing a potential role in LMP1 trafficking, signaling, maturation or exocytosis [123]. Instead, BiFC studies using Tmem134siRNA or over expression CYFP-Tmem134 shows a positive correlation with NF-ΚB activity and Tmem134, when LMP1 was overexpressed. These protein interactions are limited to membrane raft domains and mediated by CTAR1, as CTAR2 mutants still gave high fluorescence comparable to wild type LMP1 in BiFC assays [123–125]. All these cytoskeletal protein interactions occur around lipid raft microdomains, facilitating constant restructuring of cytoskeletal machinery and aiding in cell migration.

Gelsolin is a calcium dependent actin modulating protein, which blocks the monomer exchange by binding to the plus end of actin filaments and severing of actin filament into two [126]. Tafazzin (TAZ) is a nuclear effector of Hippo related pathways and LMP1 induces its expression in NPC cell lines. Interestingly, gelsolin is an inhibitor of TAZ. Hence, another function of LMP1-gelsolin interaction may be to increase TAZ stability by inhibiting serine/threonine-protein kinase LATS1/2 phosphorylation. This is significant as TAZ plays a critical functions in cell proliferation, cellular pluripotency (stemness) and LMP1 mediated epithelial to mesenchymal transition (EMT) [127].

LMP1 additionally modulates the actin cytoskeleton through Cdc42. Cdc42 is a small GTPase belonging to RHO family of GTPases that functions in regulating cytoskeletal structure, and activating signaling events involving NF-ΚB and c-June N-terminal MAP Kinase (JNK). Using active Cdc42 binding domain fused to glutathione S-transferase (GST-CBD) pulldown assay, Cdc42 was identified as an LMP1 interacting protein. The binding of Cdc42 and LMP1 to the GST-CBD was dependent on LMP1 transmembrane domains. In an effort to identify a mediator of these binding events, Liu et al. discovered that FYVE, RhoGEF and PH domain-containing protein 4 (FGD4), a guanine nucleotide exchange factor (GEF) for Cdc42, binds to transmembrane domains in LMP1. Recruitment of FGD4 into the signaling complex activates Cdc42, leading to re-organizations of actin cytoskeleton and enhanced motility of NPC cells [128, 129]. LMP1 dependent signaling events promote induction of various cytokines. These cytokines can activate Cdc42, facilitating cell migration and formation of filopodia. However, these actin dependent migratory phenotype can be suppressed by blocking Cdc42 activation [129].

Early work on LMP1 described a unique localization pattern of LMP1 in B-cells that co-localizes with the cellular protein vimentin [130]. Vimentin is an intermediate filament protein that plays a critical role in many cellular processes and is present in lipid raft microdomains containing LMP1 [131, 132]. Other studies using co-immunoprecipitation and mass spectrometry have confirmed LMP1-vimentin interaction and further described a key role for vimentin in LMP1 dependent signaling events. Inhibition of vimentin function through genetic means (for example, shRNA mediated knockdown and use of dominant negative constructs) or using chemical inhibitors showed the essential role of vimentin in the activation of PI3K and MAPK pathways, as well as transformation of Rat1 fibroblast cells [133].

5.11. Interactions that negatively regulate LMP1 signaling

LMP1 signaling leads to both hyper-proliferation and cell death effects. For example, in B-cells and keratinocytes LMP1 expression induces a hyper-proliferative phenotype while in diffuse large B-cell lymphoma cell lines (DLBCL) and lymphoblastoid cell lines LMP1 expression showed cytotoxic effects [134–137]. Zinc Finger Protein A20 (A20) was one of the first proteins shown to interact with LMP1 and block p53 mediated apoptosis in LMP1 stably expressing epithelial cells. A20 directly interacts with LMP1, competing with TRADD and TRAF1 for binding, and thereby altering LMP1-TRADD-TRAF signaling complex. A20 is induced by LMP1 via NF-ΚB as a negative feedback mechanism to block further activation of both canonical and non-canonical NF-ΚB, and JNK activation [21, 138]. Thus, A20 complements the functions of survivin, an anti-apoptotic protein induced by LMP1 expression [116].

Bone morphogenic protein receptor 1A binding protein (Bram1), formed by alternative splicing of BS69, plays a critical role in tumor suppression. Using biochemical and confocal microscopy, Bram1 was found to interact with LMP1, possibly through its MYND domain and the CTAR2 domain of LMP1. The interaction of Bram1 and LMP1 interfered with the NF-ΚB dependent downstream signaling but surprisingly, JNK signaling remained intact. These data suggest a divergence of NF-ΚB and JNK signaling from CTAR2 through distinct mechanisms. The effects of Bram1 is attributed to its ability to target IkBα and cause vulnerability to TNF-α induced cell toxicity in LMP1 expressing cells [139]. In other signaling pathways like lymphotoxin beta-receptor, Bram1 inhibits signaling by blocking receptor clustering. Since oligomerization is a hallmark of LMP1 signaling, it will be worthy to investigate whether BRAM1 acts through inhibiting the clustering of LMP1 [140]. The Homologue of Slimb (HOS), a receptor for SCFHOS/ betaTrcP (Skp1-Cullin1-HOS-Roc1) ubiquitin protein isopeptide ligase, interacts with LMP1 through a PDL motif and inhibits LMP1 induced NF-ΚB signaling. A mutant LMP1 unable to bind HOS shows increased degradation of inhibitory kappa beta (Ikβ) and transcription of target genes [141, 142]

6. Potential therapeutic strategies targeting LMP1

EBV infection leads to various health problems including several types of cancers. Therefore, developing effective means to prevent EBV infection or to treat post infection pathological outcomes is very critical. Various strategies currently under investigation include vaccination, use of extracts from botanical origin, and the use of pharmacological drugs that target various enzymes. Some of these agents are listed in the table 2–4. Viral infections are hard to control and successful treatment options are not available in most cases. Vaccination is an ideal method to control the incidence and outbreaks of virus-related diseases. Vaccines are being administered for preventive measure as well as in therapeutic applications. Preventive vaccines use neutralizing antibodies against viral particles aiming to prevent occurrence of infection. In therapeutic vaccines, neutralizing antibodies generated are used after viral infection to elicit virus specific cellular responses and destroy or inhibit viral activity following the establishment of persistent infection [143].

Table 2:

Preventive and therapeutic vaccines

	Agent	Targeted health problem	Result	Clinical/non clinical stage	References
1	Vaccinia construct-GP350	NPC	Vaccine was immunogenic	Phase I (May be discontinued)	[148]
2	gp350/AS04 vaccine	Infectious mononucleosis	Safety and immunogenicity assessed. Infectious mononucleosis prevented	Phase II	[148]
3	pcDNA3.1-LMP1	NPC	Tumor free when vaccinated before tumor induction in xenograft model	Pre-clinical	[150]
4	Mytomycin C-Fab fragments	NPC cell line HNE2-LMP1	Increased apoptosis in-vitro Increased tumor suppression in-vivo xenograft model	Pre-clinical	[151]
5	LMP1-TES1/CTAR1 specific antibody	NPC cell line HNE2-LMP1	Inhibited cell growth in-vitro	Pre-clinical	[153]
6	LMP1-specific IgG antibody	ENKTL**	Induced apoptosis	Pre-clinical	[155]

^**extranodal nasal-type natural killer (NK)/T-cell lymphoma (ENKTL)

Table 4:

Sensitizer, DNAzyme and inhibitors

	Agent	Mode of action	Result	Clinical/non clinical stage	Reference
1	Diphenyleneidium	NOX inhibitor	Inhibits LMP1 upregulated NOX. Cytotoxic in NP69 cells	Pre-clinical	[166]
2	Fospeg-PDT	photosensitizer	Enhanced sensitivity of photodynamic therapy. Anti-tumor effect on NPC cell lines	Pre-clinical	[167]
3	PI3K/Akt inhibitors	Inhibitors	Targets a number of different types of tumors	Clinical Phase I,II, and III	[166,168]
4	DNAzyme	Inhibitor	Higher apoptosis, decreased cell proliferation and increased radiosensitivity	Pre-clinical	[174–176]

6.1. Vaccination

The idea of creating vaccine against Epstein-Barr virus was first proposed by the co-discoverer of the virus, Micheal A. Epstein. Even after 40 years since his initial publication highlighting the need for an EBV vaccine, a successful vaccine has yet to be produced commercially [144]. Various attempts have been made to synthesize an EBV vaccine, and the first notable development came in 1995 when researchers used a vaccinia construct capable of over-expressing glycoprotein 350, to vaccinate Chinese children against nasopharyngeal carcinoma. Later in 2007, a similar vaccine was successfully used in a phase II clinical trial to control infectious mononucleosis, which is an EBV-related non-malignant condition [145–148]. A series of attempts continued to limit other EBV related malignancies, like post-transplant lymphoproliferatve disease and other lymphomas [149]. Unfortunately, none of these vaccines transferred to the clinical settings for multiple reasons. The main hurdle was the long incubation period of the virus before the development of any associated disease. This made the evaluation of vaccine in terms of efficacy nearly impossible, despite the fact that in a few cases the vaccines used were highly immunogenic and safe [32].

A promising study was published in 2017 by Lin et al. showing the potential use of a LMP1 vaccine as a therapeutic. They developed a vaccine using pcDNA3.1 construct expressing LMP1 and delivered it into an animal model using helium driven gene gun. The results of these in vivo studies were highly encouraging, with the LMP1 vaccine significantly suppressing tumor growth and metastasis compared to the control group. When the vaccine was administered prior to tumor induction, all the animals in vaccine group were tumor free while the control group developed tumors within two weeks [150]. It is clear from these data that vaccination could help control EBV-associated malignancies.

6.2. Immunotherapy

In addition to vaccination, antibody mediated immunotherapy has been utilized in treating various health problems including cancer. Multiple attempts have been made by different groups aiming to develop a LMP1-specific antibody with high affinity that could be used to suppress tumor growth or induce apoptosis [151–154]. One of the early attempts used mytomycin C (MMC) conjugated human Fab fragments (HLEAFab-MMC) specifically targeting an extracellular domain of LMP1. This was evaluated for its efficiency to inhibit cell proliferation and promote apoptosis. The immunoconjugate inhibited cell proliferation of NPC cell line HNE2/LMP1 up to 76% with correspondingly high level of apoptosis. In vivo, using a xenograft mouse model, NPC tumor growth was inhibited 55% compared to 5.6% HLEAFab group [151]. Furthermore, a higher rate of apoptosis was observed among xenograft tumor cells with combined therapy compared to MMC alone. All these anti-tumor effects may be partially achieved by downregulating VEGF expression, which is an important factor contributing to tumor progression [152]. HLEAFab was also used to reliably detect LMP1 expression using immunohistochemistry on clinical samples obtained from NPC patients [154]. Other attempts to test Fab-based antibodies included the development of a LMP1-TES1/CTAR1 specific antibody that inhibits the growth of HNE2-LMP1 cells [153]. One of the most recent developments was the construction and characterization of a LMP1 specific IgG antibody, which induces apoptosis through antibody-dependent cell-mediated cytotoxicity and complement-dependent cytotoxicity. The antibody blocks LMP1 induced phosphorylation of JAK3 and STAT3 and remains a promising LMP1-dependent therapeutic target of EBV [155].

6.3. Products of natural origin

Various chemicals of natural origin have also been tested for their efficacy in treating EBV related malignancies. These include plant extracts of Chrysanthemum, Trypterygium and Piper longum, Radicicol, and Romidepsin of microbial origin, and the small molecule inhibitor diphenyleneiodonium (DPI). Crude extracts of Chrysanthemum indicum linne showed a profound effect on cell survival and viability of lymphoblastoid cell lines. Treatment with chrysanthemum extract reduced NF-ΚB, IKKα and IKKβ activation in LCLs, but interestingly its effects was negligible on human foreskin fibroblast cells, HeLa and EBV negative Burkitt lymphoma cells [156]. Later, Lupeol, obtained by methylene chloride fractionation was identified as the active compound responsible for anti-lymphoma activity of chrysanthemum extracts [157]. A similar result obtained for Piperlongumine, an active agent obtained from long pepper. This compound showed a concentration dependent reduction in cell proliferation and increased apoptosis in a transgenic mouse model of human Burkitt’s lymphoma cells, by downregulating NF-ΚB and Myc activity and subsequently a number of downstream target genes [158]. Triptolide, obtained from Trypterygium extracts is known to possess anti-cancer and immunosuppressive activities. Like Piperlongumine and Lupeol, Triptolide inhibited EBV-positive B-lymphocyte proliferation, reduced LMP1 transcriptional and protein levels, both in cell lines and nude mice models [159]. Wogonin and Fisetin are two flavanoid chemicals obtained from Scutellaria and Fabaceae family of plants respectively, have also been shown to have anti-tumor characteristics. Non-cytotoxic concentrations of Fisetin inhibited migration and invasion of the NPC cell line expressing LMP1 (CNE-LMP1) and blocked associated molecular changes leading to EMT. This makes Fisetin as a strong candidate for developing an anti-metastatic drug [160, 161]. Another flavonoid, Wogonin, caused increased apoptosis in Raji cells (Burkitt’s lymphoma cell line) by suppressing expression of NF-ΚB through a pathway involving LMP1/mir-155/NF-ΚB /PU.1, resulting in decreased tumor growth, and downregulation of Ki67 and p65 [162, 163]. Romidepsin and Radicicol are natural products of microbial origin which can downregulate LMP1 expression and signaling. Romidepsin, a histone deacetylase inhibitor obtained from bacteria, has been shown to have selective cytotoxic effects on cancer cells. In both DLBCL and in-vivo xenograft tumors, Romidepsin showed cytotoxicity through downregulation of LMP1 and c-myc expression and the activation of EBV lytic cycle genes [164]. Radicicol obtained from fungus Pochonia, and Tanespimycin, a derivative of the antibiotic geldanamycin are potent inhibitors of HSP90, an interacting partner of LMP1. In EBV-positive SNK6 natural killer cells and B- and T-cell lymphoma cell lines these agents caused a reduction in LMP1 expression, decreased cell proliferation, and reduced tumor size highlighting HSP90 as a suitable target to control EBV associated malignancies [165].

6.4. Inhibitors

One of the downstream effectors of LMP1 signaling is p22phox, a regulatory subunit of NAD(P)H oxidase (NOX), which is significantly upregulated in EBV related malignancies through the c-Jun kinase pathway. At cellular level, this results in increased production and accumulation of reactive oxygen species and enhanced glycolytic activity contributing to increased oncogenesis. In light of this pathway, diphenyleneiodonium (DPI), an inhibitor of NOX, could be a potential candidate to develop an anti-cancer therapeutic [166]. Another drug, Fospeg-PDT, which enhances sensitivity towards photodynamic therapy was also shown to have anti-tumor effects on NPC cell lines. Interestingly, the effect of this drug is achieved by up regulating LMP1 expression, both mRNA and protein levels [167], probably through the increased apoptosis due to higher amount of LMP1 than physiological levels [134, 135]. LMP1 increases store-operated Ca2+ Entry (SOCE) causing increased pathogenicity of NPC. Inhibition of LMP1-augmented SOCE activity correlates with decreased cell migration, angiogenesis, and vascular permeabilization in vitro, and vasculature-invasion of circulating cells and metastasis in vivo. Therefore, LMP1-enhcanced Ca2+ signaling is a novel target for developing therapeutics to control cancer cell invasion and metastasis [168, 169]

The PI3Kinase/Akt signaling axis is a major pathway involved in cell proliferation, survival and apoptosis, and is one of the most frequently dysregulated pathways in most of the human cancers [170, 171]. LMP1 activates the PIK3/Akt pathway leading to cellular transformation and increased malignancy of EBV related cancers. In Bio-ID mass spectrometry analysis, LMP1 was shown to interact with both regulatory and catalytic subunits of PI3K. These different subunits and their downstream effectors are found altered in many types of cancers. Therefore, PIK3 stands as a promising therapeutic target for EBV related cancers. Indeed, a number of drugs targeting different subunits of PI3K/Akt are currently in varying stages of clinical trials as a combination therapy, with many of them in phase III [170, 172].

6.5. DNA enzymes

DNA enzymes or DNAzymes are single stranded DNA molecules with catalytic capabilities isolated from a pool of DNA sequences [173]. DNAzyme targeting LMP1 inhibited LMP1 expression and caused deregulation of cell cycle with G1 arrest. This correlated with decreased expression of cyclin D1, E and CDK4. DNAzyme dependent downregulation of LMP1 induced DNA damage and malfunctioning of cell cycle check points resulting in a higher rate of apoptosis and decreased cell proliferation. Injection of DNAzyme increases radiosensitivity and decreases tumor size in xenograft mouse model using NPC cell line C666 [174–176]. This radiosensitivity was achieved by suppressing telomerase expression and thus disrupting telomerase activity. Furthermore, DNAzyme, inhibited LMP1 dependent HIF1/VEGF activity resulting in the formation of defective microtubules in human umbilical cord vein endothelial cells (HUVECs) co-cultured with NPC cells [177, 178]. These results demonstrate DNAzymes are promising agents in designing therapeutics against LMP1. Taken together, LMP1 and its downstream signal transduction targets offer excellent therapeutic targets to combat EBV-associated cancers.

7. Extracellular vesicles

Extracellular vesicles (EVs) constitute the nanoscale particles called exosomes, microvesicles shed from cell membranes, and apoptotic bodies eliminated from the cells undergoing apoptosis [179]. In recent years, these excretory particles, especially exosomes, have achieved greater significance due to their potential to be utilized in many aspects of health science including therapeutic delivery and biomarkers for diagnosis and prognosis. EVs encapsulate different biologically active molecules like lipids, mRNAs, miRNAs, proteins and even DNA, which can modulate cellular physiology in the cells of origin as well as at a distant site [179–182]. Since every bodily fluid examined so far contains EVs and they show cell and tissue specific signatures, these vesicles are valuable tools that can be manipulated for human benefits. Likewise, EVs from normal cells and cancer cells vary in the number, size and contents and are now the focus of extensive research in oncology [183, 184].

LMP1 can trigger increased production of small EVs and manipulate EV cargo and function thereby differentially modifying target cell properties [82, 185]. This cargo alteration may be one of the strategies EBV exploits to eliminate biomolecules that are potentially harmful to the host cells protecting them from apoptosis. One example is LMP1 itself, as increased levels of this viral protein will lead to cell death effects and hence exocytosed in high level [82]. LMP1 targets proteins to EVs through direct physical interactions or by influencing their expression or activity by altering signaling pathways [133, 186]. Adding to this, using proximity dependent Bio-ID, a number of EV proteins which potentially interact with LMP1 have been identified. Broadly, these proteins could be part of the cargo to be exocytosed from the cells or the core export machinery that that are essential for vesicle formation and function [58, 133, 186].

Increased LMP1 signaling through multiple signaling axis not only contributes to increased cell survival and transformation, but can also lead to increased apoptosis of host cells [134, 135]. This cell death specific effect may occur when expressed above-physiological levels leading to hyperactive signaling. Hence LMP1 expression is strictly regulated in EBV infected cells. Therefore, inhibiting exosome/LMP1 release from the cells will augment intracellular LMP1 signaling and result in increased cell death. Since LMP1-modified exosomes can target to distant sites and initiate signaling events that can alter tissue microenvironment, inhibiting exosome release, or neutralizing LMP1 specific exosomes in the circulation may prove to be a strategy to limit progression LMP1 associated cancers [187].

LMP1 is thought to be secreted via endosomal sorting complex required for transport (ESCRT) dependent and independent pathways of exosome release. A number of ESCRT components are identified in LMP1 Bio-ID pull down study, including ALG-2-interacting protein X (ALIX) and syntenin [58]. Therefore, another therapeutic strategy could be inhibiting ESCRT components thereby disrupting the release of EVs packed with LMP1. Indeed, the downregulation of ALIX and syntenin using shRNA in 293 cells was shown to decrease the targeting of LMP1 into EVs, with syntenin showing a more profound effect. A number of LMP1 interacting proteins, including the chaperone protein HSC70 may have a similar effect. Therefore, inhibition of ESCRT machinery with drugs, antibodies or genetic methods would negatively impact the dissemination of LMP1 containing vesicles. Since the ESCRT-independent pathway is mainly driven by ceramide, and enzymes involved in ceramide synthesis are in complex with LMP1, it may be beneficial to inhibit enzymes like ceramide synthase in EBV-associated cancers. Various groups have used the compound GW4869, an inhibitor of neutral sphingomyelinase-2 and ceramide production, to inhibit EV production. In context of cancer, the drug has been found to decrease the metastatic potential of Lewis lung carcinoma cells in a lung cancer mouse model [188]. Similarly, inhibition of channel proteins as well as down regulation of various Ras-related proteins (Rab) also have shown to hinder exosome release and cancer cell metastasis. Interestingly, a number of these proteins are LMP1 interacting partners [58, 189, 190]. Antibodies targeting EV trafficking or biogenesis components offer another intriguing avenue to control EBV associated cancer progression and metastasis. One recent study investigated the protein content of exosomes produced by human NCI-60 cancer cell lines which provided in depth insight towards molecular components involved in exosome biogenesis. In the study, 213 proteins were found to be common to all EV isolates, including many proteins involved in vesicle trafficking or production [191]. Generating neutralizing antibodies or small molecule inhibitors against some of these proteins may be an excellent strategy to block and neutralize LMP1 loading into circulating EVs and their subsequent docking on target tissues.

A number of other LMP1 interacting partners including MAP kinases and Src kinases, various GTPases, GEFs and GAPs, important adaptor proteins, transport proteins, chaperones and metabolic enzymes are also identified. Regulation of some of these proteins using suitable techniques are under investigation to target various malignancies. Others could be potentially utilized as a targets after careful mechanistic evaluation. Novel targets are also likely to come from future studies of the molecular events orchestrated by LMP1 within the cell, the LMP1 interactome, and the mechanisms of LMP1 EV manipulation and secretion.

8. Conclusion

LMP1 is the major oncogene of EBV and is associated with immortalization of human B cells and transformation of rodent fibroblasts. EBV hijacks and regulates major cellular pathways involved in growth and proliferation by means of LMP1 expression. LMP1 constitutively signals mainly through its CTAR regions in the C-terminal domain, by recruiting a multitude of interacting proteins, depending on the downstream effectors and cellular process. Therefore, LMP1 is an attractive target in developing therapeutics against EBV-associated LMP1-positive malignancies. A number of enzymes, including kinases and phosphatases, are recruited to LMP1 signaling complex (Fig. 1 and 2). Targeting these interacting proteins by chemical, immunological and pharmacological means will be an excellent strategy to control distinct EBV-associated diseases. In fact, a number of drugs targeting different kinases are under study, and even though they are not specific to EBV-infected cells will likely be beneficial in treating EBV related diseases. Promising results have been obtained using chemicals of plants and microbial origin, and by use of vaccine and antibody therapy. A large proportion of LMP1 is secreted from the cells via EVs, along with other cargos modified by LMP1 expression. Targeting these LMP1 modified EVs has high potential in developing therapeutics as these vesicles can define and modify future metastatic sites. Further studies are required to understand the detailed biochemistry and enhanced efficiency of these agents. Also, current knowledge of the broader LMP1 interactome will help find additional drug targets with higher efficacy and potency in treating EBV-related diseases.

9. Future perspective

Epstein-Barr virus associated cancers are responsible for nearly 150,000 deaths every year [192]. Some of the EBV-associated cancers are endemic, like NPC in Southeastern China and Burkitt’s lymphoma in Sub-Saharan Africa, resulting in an increased death toll in these areas [193, 194]. Ever since the initial finding of EBV from the Burkitt’s lymphoma samples, extensive research has focused on understanding the role of this virus in triggering multiple malignancies. At the same time, strategies have also been sought to prevent the incidence of the disease, and to treat and cure individuals once affected. Researchers have made tremendous improvements in understanding the detailed genetics and molecular biology of the virus, but the treatment modalities remain ineffective. One main reason for this the virus has established a persistent infection in nearly all of the world’s population and therefore eradication is nearly impossible. Another challenge is the long time period for malignancies to develop and the fact that only a small fraction of those infected will develop EBV-associated pathologies. This makes it hard to predict disease occurrence. These are the main hurdles in developing preventive vaccine against the infection.

A number of therapeutic strategies have been attempted or are currently under investigation, with promising results in inhibiting tumor growth and metastasis. Further conscious attempts are needed to take these findings to clinical settings. Other potential targets to develop therapeutics can be identified by focusing on the signal transduction mechanisms of LMP1 and the nature of interaction partners. A number of ligand specific signaling events, for example in the case of receptor tyrosine kinase signaling, takes place through the process of receptor clustering. Oligermization of LMP1 leads to trans-activation of kinases bound to cytoplasmic domain, initiating signaling. It has been shown that transmembrane domains are essential for dimerization. Therefore, it will worth developing small molecule inhibitors or blocking peptides directed against the critical residues involved in the dimerization process. Recruitment of various adaptor molecules like TRAFs, TRADD and BS69 to the C-terminal cytoplasmic domain is required for transduction of LMP1 signaling. Inhibitors of these adaptors could be another strategy to downregulate LMP1 specific events. For example, CD40-TRAF6 inhibitor 6877002 has been shown to reduce inflammatory responses in the brain cells. This compound may be capable of inhibiting LMP1-TRAF6 interaction and potentially reducing downstream signaling.

The LMP1 Bio-ID study identified a number of kinases, including the already known ones like PI3Kinase, Src and JAK. Among these, PI3kinase is the target of a number of clinical trials utilizing small molecule inhibitors. If successful for other cancers, these should exploited for treatment of EBV-related malignancies as PI3kinase-AKT pathway is hyperactive even in LMP1 expressing tumors. Inhibitors of other kinases may also be effective as some of the identified kinases are likely involved in LMP1 signaling or phenotypic changes to infected cells.

Another interesting area gaining considerable attention over the past few years is EVs. EVs can be utilized as biomarkers for diagnosis and prognosis, as well as for therapeutic interventions. In the case of LMP1 positive EBV-associated malignancies and cancers in general, increased levels of exosome release has been demonstrated. Hyperactivation of LMP1 signaling leads to apoptosis. Therefore, inhibition of LMP1 exosomal targeting might be an effective strategy to induce apoptosis. Additionally, LMP1 positive exosomes released from the cells can predispose future metastatic sites by modifying tissue microenvironment. Hence blocking these circulating exosomes by various means will be helpful in preventing metastasis. Furthermore, exosomes can be used as carriers of various drugs for therapeutic drug delivery. By means of EV engineering, drugs are incorporated into the EVs and can be targeted tissue specifically [195, 196]. Thus, EVs offers high promise in the field of cancer research. Taken together, there are a lot of new promising molecules and pathways to target therapeutically for EBV-associated malignancies. Future studies of LMP1 protein-protein interactions and mechanisms of signal transduction will only continue to advance efforts to control disease caused by the virus.

Table 3:

Plant based agents

	Agent	source	Result	Clinical/non clinical stage	References
1	Crude extract chrysanthemum	chrysanthemum	Reduced cell survival in LCL	Pre-clinical	[156]
2	Lupeol	Chrysanthemum	Anti-lymphoma activity	Pre-clinical	[157]
3	Piperlongumine	Piper longum	Decreased cell proliferation and increased apoptosis in-vivo in Burkitt’s lymphoma base xenograft model	Pre-clinical	[158]
4	Triptolide	Trypterygium	Decreased cell proliferation EBV+ B-lymphocytes in-vitro and in-vivo	Pre-clinical	[159]
5	Fisetin	Scutellariae	Inhibited migration and invasion of NPC cells. Blocked EMT	Pre-clinical	[160, 161]
6	Wogonin	Fabaceae	Increased apoptosis in Raji cells. Decreased tumor growth in-vivo	Pre-clinical	[162, 163]
7	Romidepsin	Bacteria	Cytotoxic on DLBCL cells in-vitro and on in-vivo xenograft models	Pre-clinical	[164]
8	Radicicole and Tanespimycin	Fungus	Inhibitors for HSP90. Reduction in cell proliferation and tumor size	Pre-clinical	[165]

Executive summary.

Abstract, Introduction and EBV latency

• EBV is a human gamma herpes virus which has established a persistent infection more than 90% of world population.

• EBV-associated pathological conditions include nasopharyngeal and gastric carcinomas, various lymphomas, infectious mononucleosis, lymphoproliferative disorders, autoimmunity and neurological conditions.

• LMP1 is the major oncoprotein expressed by EBV during type II and III latency.

• When overexpressed, LMP1 alone is capable of inducing cellular transformation in rodent fibroblasts.

• EBV is an opportunistic virus with higher disease occurence during immunocompromised conditions and organ transplantation.

Structure of LMP1

• LMP1 is a membrane proteins consisting of short N-terminal, six transmembrane domains and a longer C-terminal region.

• LMP1 mimics CD40 signaling through effector protein recruitment leading to constitutive activation of downstream signaling pathways.

• The C-terminal cytoplasmic tail contain CTAR domains which mediate most of the LMP1 mediated signaling events.

LMP1 interaction partners and signaling

• A number of interacting proteins were identified using biochemical, genetic, microscopy and BiFC methods.

• The interacting proteins include modulators of protein trafficking, immune response, interferon response, apoptosis, cytoskeletal modulators, proteasomal targeting and various kinases.

• Some of the recruited proteins function as the activators of LMP1 signaling while some others inhibit LMP1 signaling.

• A recent study using mass spectrometry combined with Bio-ID and affinity purification has identified more than 1000 potential LMP1 interacting proteins.

Potential therapeutic strategies targeting LMP1

• Several researches have attempted vaccination to control EBV-associated pathologies but none of them have been effective in clinical trials mainly due to the long latency period of the virus.

• Immunotherapy stands as promising treatment approach with a number of preclinical studies showing increased apoptosis and decreased tumor growth in mouse xenograft models.

• A number of products obtained from plant extracts and fungal origin, and drugs like diphenyleneiodonium and Fospeg-PDT inhibit cell proliferation, tumor growth and metastasis by downregulating LMP1 signaling.

• A number of drugs targeting various kinases, especially PI3Kinase and AKT are under varying stages of clinical trials.

Extracellular vesicles

• EVs are nanoscale particles including exosomes, microvesicles and apoptotic bodies that carry cell and tissue specific bio-active molecular signatures.

• LMP1 expression leads to increased production of EVs, and altered EV cargo and functions.

• Inhibiting EV release, or blocking exosome docking at target sites using drugs and neutralizing antibodies could be used as a strategy for anti-metastatic therapeutics.