**2. Arguments of the pathogenic role of lytic infection in EBV-associated malignancies**

EBV establishes a lifelong infection in B lymphocytes achieved through a highly regulated viral gene expression program. Numerous studies have been focused on the contributions of EBV latent infection in the pathogenesis of EBV-induced malignancies. These studies have revealed that the membrane LMP1 protein is the major essential oncoprotein [1, 16]. In recent years, the viral lytic cycle was shown to play an essential role in carcinogenesis through several potential mechanisms. EBV lytic infection may increase the total number of latently infected cells by enhancing transmission of the virus from cell to cell and thus constitutes an essential aspect of viral pathogenesis. A small subset of lytically infected cells was detected in biopsies of EBV-associated malignancies [13, 17–22], suggesting a potential role of viral lytic infection in promoting tumor growth in vivo. Furthermore, several studies have indicated that the viral lytic cycle in a fraction of B cells promotes the transformation of B lymphocytes in vitro [23] and growth of B-cell lymphoma in vivo [24, 25] through the release of paracrine growth factors and angiogenic factors [26]. Focusing on the study of NPC, Wu et al. revealed that recurrent EBV reactivation promotes genome instability, invasiveness, and tumorigenesis of NPC cells. Another Wu et al. finding was that the contribution of the lytic cycle is more marked than that of the latent infection [27, 28]. Additionally, lytic replication enhances in NPC cells the secretion of vascular endothelial growth factor (VEGF), a factor contributing to angiogenesis and subsequent nasopharyngeal carcinoma's

**99**

*The Role of the Epstein-Barr Virus Lytic Cycle in Tumor Progression: Consequences in Diagnosis…*

(NPC) metastasis or relapse after remission [29]. Clinical and epidemiological studies have revealed that individuals with elevated plasma EBV DNA load and antibody titers against the lytic viral capsid antigen (VCA) and early antigen (EA) display a high risk of NPC [30]. These studies have also shown that fluctuation of EBV antibody titers occurs prior to the onset of NPC [31–34]. Attention has also focused on the diagnostic and prognostic value of IgG antibody against the IEA ZEBRA (see further) in patients with NPC [33–37] and in Hodgkin disease (HD) patients as well [38]. More recently we reported that the replicative form of EBV, as investigated using anti-ZEBRA IgG titers, correlates with poorer outcomes in women with breast cancer [39]. All in all, these studies investigating serological lytic EBV markers have emphasized the prominent role importance of the lytic infection in EBV malignancies. Recently, the identification by mass spectrometry of 44 different EBV proteins in Burkitt lymphoma cells undergoing EBV replication has increased the knowledge base on EBV lytic replication, possibly highlighting different targets for future

**3. The role of some lytic EBV proteins in the tumorigenesis and focusing** 

The role of the lytic EBV proteins was highlighted in some recent reviews [13, 14]. Overall, the authors clearly demonstrated the role of these lytic EBV proteins in tumorigenesis. They also showed that EBV reactivation may aid virus transmission within the tumor microenvironment to establish latency and drive cellular proliferation. Having said that, the likely predominant role of the EBV lytic cycle is likely to provide the necessary paracrine, anti-apoptotic, and immunomodulatory signals required for tumorigenesis (**Table 1**). From this point on, we will focus on the critical role of the IE ZEBRA *trans*-activator, because this protein has a key role in the balance between the EBV latent and lytic cycles. The switch from latent to lytic infection is mediated by the IE protein ZEBRA (Orf BZLF1) and R (Orf BRLF1) [6, 41–45]. ZEBRA and R proteins are transcription factors that activate one another's promoters, as well as their own promoters [8]. In combination, ZEBRA and R induce expression of all early (E) lytic viral proteins, thereby enabling the viral replication. The ZEBRA protein belongs to the bZIP family of transcription factors and is homologous to c-jun and c-fos which bind to the consensus AP1 motif as well as to atypical AP1-like motifs known as Z-responsive elements (ZREs) [42, 44, 46]. The EBV genome is not methylated in virions. However, in cells with long-term latent infection, the majority of the EBV genome proves to be highly methylated [47–51]. DNA methylation, which plays a critical role in modulating the expression of both cellular and viral genes, induces transcriptional repression through multiple different mechanisms including prevention of transcription factor binding to DNA [48, 52–56]. Surprisingly, while DNA methylation of the EBV IE BRLF1 promoter (Rp) inhibits its activation through cellular transcription factors, it enhances ZEBRA's ability to activate the R [48]. This unusual effect of Rp methylation on ZEBRA activation is due to the enhanced ability of ZEBRA to bind to the methylated, versus unmethylated, forms of two atypical CpG-containing Rp ZRE sites and requires serine residue 186 in the basic DNA domain of ZEBRA [48, 49]. Several properties of ZEBRA are listed in **Table 2**. ZEBRA was shown to inhibit antiviral cytokine signaling [57, 58] and disrupt T-cell recognition of MHC-II molecules [59]. As ZEBRA is able to activate host cellular genes (i.e., the immunomodulatory genes IL-10 and IL-13 [60, 61]), it was also found that EBV reactivation may contribute to the growth of latently infected cells [25, 62] by promoting the release of B-cell growth factors [26].

*DOI: http://dx.doi.org/10.5772/intechopen.88607*

therapeutic strategies [40].

**onto the Zta/ZEBRA IE transactivator**

*The Role of the Epstein-Barr Virus Lytic Cycle in Tumor Progression: Consequences in Diagnosis… DOI: http://dx.doi.org/10.5772/intechopen.88607*

(NPC) metastasis or relapse after remission [29]. Clinical and epidemiological studies have revealed that individuals with elevated plasma EBV DNA load and antibody titers against the lytic viral capsid antigen (VCA) and early antigen (EA) display a high risk of NPC [30]. These studies have also shown that fluctuation of EBV antibody titers occurs prior to the onset of NPC [31–34]. Attention has also focused on the diagnostic and prognostic value of IgG antibody against the IEA ZEBRA (see further) in patients with NPC [33–37] and in Hodgkin disease (HD) patients as well [38]. More recently we reported that the replicative form of EBV, as investigated using anti-ZEBRA IgG titers, correlates with poorer outcomes in women with breast cancer [39]. All in all, these studies investigating serological lytic EBV markers have emphasized the prominent role importance of the lytic infection in EBV malignancies. Recently, the identification by mass spectrometry of 44 different EBV proteins in Burkitt lymphoma cells undergoing EBV replication has increased the knowledge base on EBV lytic replication, possibly highlighting different targets for future therapeutic strategies [40].

## **3. The role of some lytic EBV proteins in the tumorigenesis and focusing onto the Zta/ZEBRA IE transactivator**

The role of the lytic EBV proteins was highlighted in some recent reviews [13, 14]. Overall, the authors clearly demonstrated the role of these lytic EBV proteins in tumorigenesis. They also showed that EBV reactivation may aid virus transmission within the tumor microenvironment to establish latency and drive cellular proliferation. Having said that, the likely predominant role of the EBV lytic cycle is likely to provide the necessary paracrine, anti-apoptotic, and immunomodulatory signals required for tumorigenesis (**Table 1**). From this point on, we will focus on the critical role of the IE ZEBRA *trans*-activator, because this protein has a key role in the balance between the EBV latent and lytic cycles. The switch from latent to lytic infection is mediated by the IE protein ZEBRA (Orf BZLF1) and R (Orf BRLF1) [6, 41–45]. ZEBRA and R proteins are transcription factors that activate one another's promoters, as well as their own promoters [8]. In combination, ZEBRA and R induce expression of all early (E) lytic viral proteins, thereby enabling the viral replication. The ZEBRA protein belongs to the bZIP family of transcription factors and is homologous to c-jun and c-fos which bind to the consensus AP1 motif as well as to atypical AP1-like motifs known as Z-responsive elements (ZREs) [42, 44, 46]. The EBV genome is not methylated in virions. However, in cells with long-term latent infection, the majority of the EBV genome proves to be highly methylated [47–51]. DNA methylation, which plays a critical role in modulating the expression of both cellular and viral genes, induces transcriptional repression through multiple different mechanisms including prevention of transcription factor binding to DNA [48, 52–56]. Surprisingly, while DNA methylation of the EBV IE BRLF1 promoter (Rp) inhibits its activation through cellular transcription factors, it enhances ZEBRA's ability to activate the R [48]. This unusual effect of Rp methylation on ZEBRA activation is due to the enhanced ability of ZEBRA to bind to the methylated, versus unmethylated, forms of two atypical CpG-containing Rp ZRE sites and requires serine residue 186 in the basic DNA domain of ZEBRA [48, 49]. Several properties of ZEBRA are listed in **Table 2**. ZEBRA was shown to inhibit antiviral cytokine signaling [57, 58] and disrupt T-cell recognition of MHC-II molecules [59]. As ZEBRA is able to activate host cellular genes (i.e., the immunomodulatory genes IL-10 and IL-13 [60, 61]), it was also found that EBV reactivation may contribute to the growth of latently infected cells [25, 62] by promoting the release of B-cell growth factors [26].

*Human Herpesvirus Infection - Biological Features, Transmission, Symptoms, Diagnosis...*

egress of infectious virions [1].

disease (LPD) [11, 12].

**malignancies**

under certain physiological conditions, both viruses undergo lytic reactivation leading to expression of the full lytic gene complement, followed by the assembly and

Lytic replication is mediated by the virally encoded DNA polymerase using the oriLyt replication origin and results in the release of infectious viral particles [9]. In contrast, during latent viral infection, only a viral gene subset is expressed. The genome is replicated once per cell cycle using the cellular DNA polymerase and the oriP replication origin, and the progeny virus is not released. Latent EBV infection allows the virus to persist for the life of the host and to avoid detection by the immune system [1]. In immune-competent hosts, the outgrowth of EBVimmortalized B cells is prevented through a robust expansion of EBV-specific memory T cells directed toward both lytic and latent antigens [10]. Thus, EBVpositive individuals with either congenital or acquired immune deficiencies are highly susceptible to viral reactivation and malignant transformation. Furthermore, EBV-seropositive patients treated with immunosuppressive medications following organ/stem cell transplantation are at significant risk of developing posttransplant lymphoproliferative disorder (PTLD), an often fatal B-cell lymphoproliferative

Importantly however, EBV (as other gammaherpesviruses, e.g., KSHV) can also undergo abortive lytic reactivation, resulting in the expression of early lytic genes without subsequent virion assembly and cell lysis [13]. Although numerous efforts to understand these disorders' molecular basis have focused on viral latency, EBV lytic cycles are now widely accepted as major contributors to oncogenesis and could be important targets in the development of new therapeutic or diagnostic approaches [14, 15]. Thus, in this chapter, we will discuss how lytic replication, particularly via the immediate-early/early antigens or IEAs/EAs, augments the pathogenesis of EBV-associated malignancies, especially in the lymphoma setting or with respect to the treatments that potentially target the lytic replication cycle.

**2. Arguments of the pathogenic role of lytic infection in EBV-associated** 

EBV establishes a lifelong infection in B lymphocytes achieved through a highly regulated viral gene expression program. Numerous studies have been focused on the contributions of EBV latent infection in the pathogenesis of EBV-induced malignancies. These studies have revealed that the membrane LMP1 protein is the major essential oncoprotein [1, 16]. In recent years, the viral lytic cycle was shown to play an essential role in carcinogenesis through several potential mechanisms. EBV lytic infection may increase the total number of latently infected cells by enhancing transmission of the virus from cell to cell and thus constitutes an essential aspect of viral pathogenesis. A small subset of lytically infected cells was detected in biopsies of EBV-associated malignancies [13, 17–22], suggesting a potential role of viral lytic infection in promoting tumor growth in vivo. Furthermore, several studies have indicated that the viral lytic cycle in a fraction of B cells promotes the transformation of B lymphocytes in vitro [23] and growth of B-cell lymphoma in vivo [24, 25] through the release of paracrine growth factors and angiogenic factors [26]. Focusing on the study of NPC, Wu et al. revealed that recurrent EBV reactivation promotes genome instability, invasiveness, and tumorigenesis of NPC cells. Another Wu et al. finding was that the contribution of the lytic cycle is more marked than that of the latent infection [27, 28]. Additionally, lytic replication enhances in NPC cells the secretion of vascular endothelial growth factor (VEGF), a factor contributing to angiogenesis and subsequent nasopharyngeal carcinoma's

**98**


**101**

*The Role of the Epstein-Barr Virus Lytic Cycle in Tumor Progression: Consequences in Diagnosis…*

ZEBRA is able to bind to the methylated DNA Virus Activation of EBV replication [48]

system

Immune system

Animal model

Host tissue

Host tissue

Virus ZEBRA and Rta induce expression

Host cell ZEBRA contributes to the growth of

Immune subversion [57]

EBV homeostasis and immune control of EBV replication activation

Detection of specific IgG antibodies in EBV-associated diseases [34, 38, 75,

ZEBRA might be a prominent factor in the development of EBV-associated

Pathogenesis and tumor progression

Diagnostic and prognostic value [84]

Host cell Toxoid effect [82–84]

[60, 61]

[67–69]

115, 116]

malignancy [24, 62]

in patients [13, 17, 94, 96]

of all early (E) lytic viral proteins, allowing the virus to replicate [45]

latently infected cells by promoting the release B-cell growth factors

**Functions of ZEBRA protein Target Effect**

Scid/hu mice proved to be a good model to study the pathogenesis of posttransplant

B-cell lymphomas [63–65]. Several recent studies emphasized the role of EBV lytic proteins (including ZEBRA) in promoting B-cell transformation and lymphomagenesis in a humanized NOD/LtSz-scid/IL2Rγnull mouse model of an EBV-related disease [24]: The authors modeled a humanized NOD/LtSz-scid/IL2Rγnull mouse that was reconstituted with CD34+ human hematopoietic progenitor cells isolated from fetal liver and also simultaneously xenotransplanted with fetal liver and thymus tissues [hNSG(thy)]. The hNSG(thy) mice were infected with EBV developing CD20-positive diffuse large B-cell lymphomas (DLBCL). To understand the contribution of the lytic cycle in lymphomagenesis, the hNSG(thy) mice were infected with a BZLF1 knockout EBV. Although no significant differences were apparent in the number of infected cells and in their tissue distribution, lymphomas were found in 6/11 (54.4%) of the mice infected with the wild-type virus and in only 2/14 (14.3%) of mice infected with the mutant virus. These data along with other studies support that the lytic cycle directly helps tumorigenic mechanisms rather than the lateral spread of viruses, thereby increasing the number of latently infected cells [66]. In line with this observation, lymphomas developing in the mice infected with wild-type viruses sometimes expressed ZEBRA and BMRF1 lytic proteins but never gp350/220 structural protein. Thus, these data support the participation of the abortive lytic cycle in lymphomagenesis (see below). Hong et al. made a similar observation [62]: This research group reported that when lymphoblastoid cell lines (LCLs) derived from wild type and virus

*DOI: http://dx.doi.org/10.5772/intechopen.88607*

ZEBRA mediates the switch from latent to lytic infection, in combination with the second

ZEBRA exhibits cell-penetrating properties, like

Activation of immunomodulatory genes IL-10 and

High immunogenicity: ZEBRA elicits robust B- and T-cell responses that dominate the early immune

ZEBRA promotes B-cell transformation and lymphomagenesis in a humanized NOD/LtSz-scid/ IL2Rγnull mouse model of EBV-related disease

ZEBRA expression (mainly through abortive cycles) was shown in EBV-associated tumors, including Hodgkin disease and PTLD

ZEBRA is also released in the extracellular milieu (as HIV-1 Tat) and detectable in the serum of transplant patients, particularly those with PTLD

*Characteristics of the ZEBRA transactivator.*

Inhibition of antiviral cytokine signaling Immune

HIV-1 Tat and dengue virus NS1

transactivator Rta

responses in patients

(PTLD)

**Table 2.**

IL-13

*EBV lytic proteins and tumorigenic functions (summarized from Ref. [14]).*

*The Role of the Epstein-Barr Virus Lytic Cycle in Tumor Progression: Consequences in Diagnosis… DOI: http://dx.doi.org/10.5772/intechopen.88607*


#### **Table 2.**

*Human Herpesvirus Infection - Biological Features, Transmission, Symptoms, Diagnosis...*

**100**

**EBV lytic** 

**Cellular** 

**Lytic function**

**Mechanisms of oncogenesis**

**Oncogenic function**

**Clinical and diagnostic relevance (Ref)**

Expression in PTLD, B-cell lymphomas, Hodgkin

lymphomas, NPC, detection of s-ZEBRA in PTLD

patients' serum, specific antibodies in NPC/HD/

PTLD patients [84]

**homologs**

**gene**

ZEBRA

c-Fos, c-Jun

IEA, transactivator in

Promotes secretion of proangiogenic

Angiogenesis,

B-cell growth,

immunomodulation

factors, VEGF and cytokines (IL-6,

IL-8, IL-10, IL-13), toxoid protein

cooperation with Rta

(Zta,

BZLF1)

BHRF1

Bcl-2

EA, delay cell death

Inhibition of apoptosis

Cell survival

and

BALF1

BILF1

G proteincoupled

EA immunoevasin

MHC class I downregulation

receptor

BNRF1 BALF3 BGLF4 BARF1

C-fms

receptor

BGLF5 BcRF1

**Table 1.**

*EBV lytic proteins and tumorigenic functions (summarized from Ref. [14]).*

Il-10

Immunoevasin

Downregulates interferon gamma

*EA, early antigen; IEA, immediate-early antigen; HD, Hodgkin disease; NHL, non-Hodgkin lymphoma; NPC, nasopharyngeal carcinoma; PTLD, posttransplant lymphoproliferative disorder.*

Cell survival

Host cell shutoff (DNase),

DNA damage

Genome instability

TLR-9 downregulation

Terminase

EA DNA synthesis and

DNA damage

incorporation into virions

EA viral DNA replication

DNA damage

and nuclear import

EA immunomodulator

Modulates expression genes involved

Cell survival

Most abundant EBV transcripts in NPC, detection

of s-BARF1 in the NPC patients' serum [110] and

specific antibodies [111]

Expression in NPC biopsies [112]. Specific

antibodies in NPC [113] patients

Soluble vIL10 in NHL patients' serum [114]

in apoptotic signaling

EA nucleocapsid

Interacts with centromeres to cause

Genome instability

Genome instability

Genome instability

centrosome overduplication

trafficking

*Characteristics of the ZEBRA transactivator.*

Scid/hu mice proved to be a good model to study the pathogenesis of posttransplant B-cell lymphomas [63–65]. Several recent studies emphasized the role of EBV lytic proteins (including ZEBRA) in promoting B-cell transformation and lymphomagenesis in a humanized NOD/LtSz-scid/IL2Rγnull mouse model of an EBV-related disease [24]: The authors modeled a humanized NOD/LtSz-scid/IL2Rγnull mouse that was reconstituted with CD34+ human hematopoietic progenitor cells isolated from fetal liver and also simultaneously xenotransplanted with fetal liver and thymus tissues [hNSG(thy)]. The hNSG(thy) mice were infected with EBV developing CD20-positive diffuse large B-cell lymphomas (DLBCL). To understand the contribution of the lytic cycle in lymphomagenesis, the hNSG(thy) mice were infected with a BZLF1 knockout EBV. Although no significant differences were apparent in the number of infected cells and in their tissue distribution, lymphomas were found in 6/11 (54.4%) of the mice infected with the wild-type virus and in only 2/14 (14.3%) of mice infected with the mutant virus. These data along with other studies support that the lytic cycle directly helps tumorigenic mechanisms rather than the lateral spread of viruses, thereby increasing the number of latently infected cells [66]. In line with this observation, lymphomas developing in the mice infected with wild-type viruses sometimes expressed ZEBRA and BMRF1 lytic proteins but never gp350/220 structural protein. Thus, these data support the participation of the abortive lytic cycle in lymphomagenesis (see below). Hong et al. made a similar observation [62]: This research group reported that when lymphoblastoid cell lines (LCLs) derived from wild type and virus

knockout of BZLF1 and BRLF1 IE genes were transplanted into scid mice, both mutant LCLs were shown unable to induce lymphoproliferative disease, supporting a critical role of the lytic cycle in the EBV-induced lymphoproliferation [62].

It appears clearly that ZEBRA expression is critical for viral activation, persistence, and disease pathogenesis. From an immunologic standpoint, ZEBRA expression is thus likely to represent a prime target of adaptive immune responses (T-cell and humoral responses) (**Table 2**). ZEBRA, highly immunogenic, elicits robust T-cell responses [10, 67–72] that dominate the early immune responses in patients [70]. B-cell epitopes were additionally described; to illustrate, the DNA-binding domain of ZEBRA (basic region, including the so-called RAK epitope) is a major target antigen for IgM antibody response in EBV primary infection (45), whereas the N-terminus part (activation domain) is mainly recognized by IgG in patients with EBV reactivation [35, 73–75].

## **4. ZEBRA, a deleterious protein acting as a "toxoid"**

ZEBRA appears to be a multifunctional protein controlling its own expression, virus replication, cell cycle arrest, and DNA damage response in the host cells [76, 77–80]. The structure of the 245-amino acid ZEBRA protein has been resolved in its DNA-bound form [81]. Based on this finding, the protein appears divided into an N-terminal *trans*-activation region and a basic DNA-binding domain flanked by a coiled-coil dimerization region (zipper) [81]. The C-terminal domain interacts with the zipper region by forming intra- and intermolecular interactions, resulting in a hydrophobic pocket. These complex interactions are unique among the bZIP members and result in the stabilization of the ZEBRA dimer when bound to DNA [81] (**Figure 1**).

In 2005, we demonstrated that this multifunctional protein not only binds DNA and controls virus replication but also has the ability to penetrate lymphoid cells [82]. Moreover, we also demonstrated that ZEBRA possesses a transduction domain (C-terminus), crosses the outer membrane of live cells, and accumulates in the nucleus of lymphocytes. The protein transduction mostly occurs via direct translocation through the lipid bilayer rather than by endocytosis [83]. Soluble ZEBRA has additionally been detected in the sera of posttransplant lymphoproliferative disease patients, suggesting a possible secretion of this protein into the circulating

#### **Figure 1.**

*Schematic representation of the ZEBRA protein. The cell-penetrating peptide is located at the C-terminus (aa 178–220), enabling the cell transduction (see Ref. [83]). Two mAbs AZ125 and AZ130 specific for ZEBRA protein (recognizing the transactivation domain and the DNA-binding domain, respectively) were employed in a sandwich ELISA throughout the whole procedure for quantifying captured s-ZEBRA [84]. The DNA-binding domain of ZEBRA (basic region, including the so-called RAK epitope) is targeted by AZ130 mAb-derived scFv and could be used as possible neutralizing therapeutic drug.*

**103**

**Figure 3.**

*suitable for immunotherapeutic approach.*

**Figure 2.**

*The Role of the Epstein-Barr Virus Lytic Cycle in Tumor Progression: Consequences in Diagnosis…*

blood, with further evidence toward a "toxoid" role for this lytic protein [84]. This unusual property must be compared with those of other nonstructural viral proteins like HIV-1 Tat and dengue virus (DV) NS1 [85, 86]. Therefore, we hypothesized that ZEBRA is to be involved in tumorigenesis and tumor progression, not only as an essential protein for EBV replication activation but also as a "toxin" released in the extracellular milieu. We hypothesized that early abortive replication associated with fully lytic cycles may occur within the tumor or its environment, along with eventual ZEBRA release in the bloodstream (**Figure 2**). In the end, this phenomenon could lead to the secretion of cytokines and angiogenesis-promoting factors, further

aggravating the immunosuppressive environment (**Figure 3**) [26, 87].

*Schematic representation of different possibilities of ZEBRA's release from the EBV-infected cells.*

*Multiple effects of the ZEBRA protein on tumorigenesis and tumor progression. At any level, ZEBRA is likely to be expressed and released in the circulation as s-ZEBRA. The expression of ZEBRA and EBV replication activation may be modulated by T-cell and B-cell responses in the context of lytic/abortive cycles. There is possibility to neutralize s-ZEBRA by IgG antibodies, and ZEBRA protein could be a novel EBV-specific antigen* 

*DOI: http://dx.doi.org/10.5772/intechopen.88607*

*The Role of the Epstein-Barr Virus Lytic Cycle in Tumor Progression: Consequences in Diagnosis… DOI: http://dx.doi.org/10.5772/intechopen.88607*

blood, with further evidence toward a "toxoid" role for this lytic protein [84]. This unusual property must be compared with those of other nonstructural viral proteins like HIV-1 Tat and dengue virus (DV) NS1 [85, 86]. Therefore, we hypothesized that ZEBRA is to be involved in tumorigenesis and tumor progression, not only as an essential protein for EBV replication activation but also as a "toxin" released in the extracellular milieu. We hypothesized that early abortive replication associated with fully lytic cycles may occur within the tumor or its environment, along with eventual ZEBRA release in the bloodstream (**Figure 2**). In the end, this phenomenon could lead to the secretion of cytokines and angiogenesis-promoting factors, further aggravating the immunosuppressive environment (**Figure 3**) [26, 87].

#### **Figure 2.**

*Human Herpesvirus Infection - Biological Features, Transmission, Symptoms, Diagnosis...*

role of the lytic cycle in the EBV-induced lymphoproliferation [62].

**4. ZEBRA, a deleterious protein acting as a "toxoid"**

with EBV reactivation [35, 73–75].

(**Figure 1**).

knockout of BZLF1 and BRLF1 IE genes were transplanted into scid mice, both mutant LCLs were shown unable to induce lymphoproliferative disease, supporting a critical

It appears clearly that ZEBRA expression is critical for viral activation, persistence, and disease pathogenesis. From an immunologic standpoint, ZEBRA expression is thus likely to represent a prime target of adaptive immune responses (T-cell and humoral responses) (**Table 2**). ZEBRA, highly immunogenic, elicits robust T-cell responses [10, 67–72] that dominate the early immune responses in patients [70]. B-cell epitopes were additionally described; to illustrate, the DNA-binding domain of ZEBRA (basic region, including the so-called RAK epitope) is a major target antigen for IgM antibody response in EBV primary infection (45), whereas the N-terminus part (activation domain) is mainly recognized by IgG in patients

ZEBRA appears to be a multifunctional protein controlling its own expression, virus replication, cell cycle arrest, and DNA damage response in the host cells [76, 77–80]. The structure of the 245-amino acid ZEBRA protein has been resolved in its DNA-bound form [81]. Based on this finding, the protein appears divided into an N-terminal *trans*-activation region and a basic DNA-binding domain flanked by a coiled-coil dimerization region (zipper) [81]. The C-terminal domain interacts with the zipper region by forming intra- and intermolecular interactions, resulting in a hydrophobic pocket. These complex interactions are unique among the bZIP members and result in the stabilization of the ZEBRA dimer when bound to DNA [81]

In 2005, we demonstrated that this multifunctional protein not only binds DNA and controls virus replication but also has the ability to penetrate lymphoid cells [82]. Moreover, we also demonstrated that ZEBRA possesses a transduction domain (C-terminus), crosses the outer membrane of live cells, and accumulates in the nucleus of lymphocytes. The protein transduction mostly occurs via direct translocation through the lipid bilayer rather than by endocytosis [83]. Soluble ZEBRA has additionally been detected in the sera of posttransplant lymphoproliferative disease patients, suggesting a possible secretion of this protein into the circulating

*Schematic representation of the ZEBRA protein. The cell-penetrating peptide is located at the C-terminus (aa 178–220), enabling the cell transduction (see Ref. [83]). Two mAbs AZ125 and AZ130 specific for ZEBRA protein (recognizing the transactivation domain and the DNA-binding domain, respectively) were employed in a sandwich ELISA throughout the whole procedure for quantifying captured s-ZEBRA [84]. The DNA-binding domain of ZEBRA (basic region, including the so-called RAK epitope) is targeted by AZ130 mAb-derived scFv* 

*and could be used as possible neutralizing therapeutic drug.*

**102**

**Figure 1.**

*Schematic representation of different possibilities of ZEBRA's release from the EBV-infected cells.*

#### **Figure 3.**

*Multiple effects of the ZEBRA protein on tumorigenesis and tumor progression. At any level, ZEBRA is likely to be expressed and released in the circulation as s-ZEBRA. The expression of ZEBRA and EBV replication activation may be modulated by T-cell and B-cell responses in the context of lytic/abortive cycles. There is possibility to neutralize s-ZEBRA by IgG antibodies, and ZEBRA protein could be a novel EBV-specific antigen suitable for immunotherapeutic approach.*
