**2.1 ZEBRA on the scene**

44 Carcinogenesis, Diagnosis, and Molecular Targeted Treatment for Nasopharyngeal Carcinoma

highest incidence rates of NPC are found among the southern Chinese population and in isolated northern populations such as Eskimos and Greenlanders (30 to 80 cases per 100,000 per year) (Parkin & Muir 1992). Intermediate incidence (8 to 12 cases per 100,000 per year) was reported in the Mediterranean basin, especially among the Arabic populations of North Africa (7-10% of all cancers among men), where NPC is also the commonest tumour of the ear, nose and throat region (Benider et al. 1995). The aetiology of NPC seems to be multifactorial with evidence that genetic susceptibility, environmental factors and viral infection with EBV reactivation and HPV infection are involved together or separately,

The increased risk of NPC in North African population was associated with the consumption of rancid butter and rancid sheep fat. In fact, higher level of N-nitrosamines in rancid fat has not been demonstrated, which suggests some other disease causing chemicals in this population. A possible compound is butyric acid, which is also named n-Butanoic Acid. The glyceride form of butyric acid makes up 3 to 4% of butter, and is released into free butyric acid by hydrolysis when it becomes rancid (Feng et al. 2007). Butyric acid is known to be able to activate EBV in the B-lymphoid cells into lyric cycle (Takimoto et al. 1984), and therefore, could be related to NPC. In addition, Marijuana smoking was associated significantly to high NPC risk independently of cigarette smoking which suggests dissimilar carcinogenic mechanisms between cannabis and

Genetic traits play a significant role in the development of NPC. Specific human leukocyte antigen (HLA) haplotypes have been reported to be associated with high risk for NPC, namely HLA-B13 in Tunisians, HLA-A3, B5 and B15 in Algerians and HLA-B18 allele in Moroccans population. In contrast, HLA-Aw33, -B14 and A9 were associated to low risk of

Retrospectives and prospectives epidemiologic studies have indicated that assocaition between EBV, an ubiquitous human herpesvirus, and the development of different malignancies, such as Burkitt's lymphoma, 40%–50% of Hodgkin's disease, B-cell lymphoma in immunocompromised individuals, and NPC (Rickinson 2002). Undifferentiated NPC is one of the most striking examples of human malignancies that have been found strongly associated with the EBV, and interest in HPV as a cofactor in NPC occurrences has emerged

During primary infection, EBV initially undergoes a brief replication in the epithelial cells of the oropharynx and salivary glands (Young & Rickinson 2004). The virus subsequently infects trafficking B-cells where the virus establishes a lifelong persistence and proceeds periodic spontaneous reactivation, resulting in lytic replication, infectious virus production and transmission (Cohen 2000). Upon reactivation, EBV can productively infect oropharyngeal epithelium, leading to infectious virus production and transmission (Jenkins, Binne & Farrell 2000). In latent infection, EBV genomic DNA exists as an episome, replicating only once during S phase and partitioning accurately into daughter cells during the mitotic phase. In lytic state, the EBV genomic DNA is linear. The initiation of lytic replication process greatly depends on the expression of two EBV immediate-early (IE)

simultaneously or consecutively (Hildesheim & Levine 1993).

NPC in Tunisians, Algerians and Moroccans, respectively.

over the last few years (Punwaney et al. 1999).

**2. EBV life cycle in brief** 

tobacco.

In cancer cells, EBV is also present in a latent state. During latency, EBV is effectively hidden from the immune system but if viral replication is initiated and lytic replication ensues, the cells express EBV genes that are more readily recognized by the immune system. The lytic DNA replication of EBV requires many viral proteins, including ZEBRA, polymerase, polymerase processivity factor, single-stranded DNA binding protein, primase, helicase and primase-associated factor. Among them ZEBRA (also called BZLF1, EB1 and Zta) is a lytic switch transactivator for expression of many early lytic genes and plays a critical role in both viral gene transcription and viral replication (Fixman, Hayward & Hayward 1995). Of all the viral transactivators, ZEBRA is unique in initiation of the ordered cascade of EBV gene expression, resulting in the expression of an estimated over 100 viral replication associated genes including those encoding early antigens, viral capsid antigens and membrane antigens (Baer et al. 1984). Many target genes of ZEBRA, such as BZLF1, BRLF1 and BMLF1 encoding the transactivators, BHRF1 and BHLF1 encoding the viral homologues of Bcl-2 (Marshall et al. 1999), and BMRF1 encoding EBV DNA polymerase accessory protein (Zhang et al. 1997), have been identified in the EBV genome. Through binding to cisacting AP-1 or ZEBRA responsive elements (ZREs) in lytic cycle promoters ZEBRA activates the transcription of the target genes (Lieberman et al. 1990). Recently, some cellular genes modulated by ZEBRA have also been revealed. The products of these cellular genes are fundamentally linked to the viral life cycle, virus-host interactions, hosT-cell environment, cell cycle progression and immunomodulation.

#### **2.2 ZEBRA structure**

ZEBRA or Zta is a member of the family of bZIP transcription factors (Sinclair 2003); it contains adjacent DNA contact (approximately amino acids 175 to 195) and multimerization domains (approximately amino acids 196 to 245) (figure 1) (Sinclair 2006) and can interact directly with specific DNA sequence elements, i.e., ZREs as a multimer . By analogy with other members of the bZIP family, the multimerization interface of ZEBRA has been predicted to fold through a coiled-coil structure (Sinclair & Farrell 1992). Biophysical evidence that this prediction holds true was recently provided (Hicks et al. 2001).

The DNA binding region and dimerization region partly conform to the well-characterized bZIP (basic/leucine zipper) domain that is found in a family of cellular transcription factors such as fos/jun, C/EBPa and GCN4. Interestingly, ZEBRA recognizes a wider range of DNA binding sites than other bZIP members. bZIP proteins are homo- or heterodimers that contain highly basic DNA binding regions adjacent to regions of a-helix that fold together as coiled coils (Sinclair 2006); the interaction with DNA is dependent on dimer formation (Busch & Sassone-Corsi 1990).

Role of the Epstein-Barr Virus ZEBRA Protein

**2.4 Cellular genes induction** 

CDC25A and E2F1.

**Guthrie, et al. 2002)** 

**C/EBPα (Wu et al. 2003) p21 (Wu et al. 2003)** 

chemokines (Hsu et al. 2008).

and HPV in the Carcinogenesis of Nasopharyngeal Carcinoma 47

The EBV lytic transactivator ZEBRA not only initiates expression cascade of viral lytic genes but also induces some cellular genes involved in immune regulation (Cayrol & Flemington 1995; Chen, C., Li & Guo 2009). ZEBRA can turn on gene expression through binding to and activation of the target promoters. Notably, a previous study shows that ZEBRA induces

Table 2 lists a number of genes whose expression is perturbed by ZEBRA. In each case, the regulation occurs in the absence of other viral genes. Regulation at the RNA level implies that ZEBRA may act as transcription factors on the cellular promoters by direct binding or via their associations with other transcription factors. However, the mechanisms of regulation of the cellular genes have not been assessed further as yet. Several of these genes have also been shown to be regulated at the protein level, suggesting relevance. Of special note is the identification of a series of cell cycle regulatory genes such as p21CIP1, p53,

transcription of human interleukin 10 (IL-10) in B cells (Mahot et al. 2003).

**RNA changes Proteins changes TGFβ inh3 (Cayrol & Flemington 1995)** C/EBPα (Wu et al. 2003)

**α1 collagen (Cayrol & Flemington 1995)** p53 (Wu et al. 2003)

**Stem–loop binding protein (MauserHolley-**

**CDC25A (MauserHolley-Guthrie, et al. 2002) Cyclin E (MauserHolley-Guthrie, et al. 2002)** 

IL-10 producing cells, facilitating local immunosuppression.

Table 1. Cell genes regulated by ZEBRA

**TGFβ (Cayrol & Flemington 1995)** p21 (Cayrol & Flemington 1996)

**TKT tyrosine kinase (Lu, J. et al. 2000)** p27 (Cayrol & Flemington 1996)

**IFN-γ receptor (Morrison et al. 2001)** Stem–loop binding protein

**MMP1 (Lu, J. et al. 2000)** E2F1 (MauserHolley-Guthrie, et al. 2002)

**E2F1 (MauserHolley-Guthrie, et al. 2002)** CDC25 (MauserHolley-Guthrie, et al.

In NPC, ZEBRA was found to be a potent inducer of IL-8, increasing IL-8 at both protein and RNA levels and activating the IL-8 promoter suggesting that the EBV lytic infection may contribute to the inflammation-like microenvironment of NPC by the upregulation of

During EBV reactivation in NPC cells, the lytic protein ZEBRA not only induces GM-CSF expression but also upregulates COX-2 that increases production of PGE2 (Dolcetti et al. 2010; Kared et al. 2008). The secreted GM-CSF and PGE2 may cooperatively promote IL-10 production from monocytes (Lee et al. 2011). Thus, through the Zta-induced immunomodulators, EBV lytic infection in NPC cells may drive nearby monocytes into

2002)

2002)

(MauserHolley-Guthrie, et al. 2002)

Cyclin E (MauserHolley-Guthrie, et al.

Fig. 1. Schematic representation of the functional regions of ZEBRA and its structure. The transactivation (TA) domain is shown in grey and the DNA contact region and the dimerization region are shown in green and blue. The amino acid sequence of the DNA binding and dimerization domain is expanded below. The location of the region with homology to leucine zippers (ZIP) and the additional region required for dimerization function (CT) are indicated below the sequence

#### **2.3 Role of ZEBRA during viral replication**

During the lytic phase of the EBV life cycle, the activation of viral DNA synthesis is related to ZEBRA efficient recognition of a large (1 kb) complex intergenic region that serves as the origin of replication. This region, known as oriLyt, consists of essential and auxiliary segments (Hammerschmidt & Sugden 1988).The two essential components of oriLyt, the upstream and downstream elements, together constitute the minimal origin of DNA replication (Rennekamp, Wang & Lieberman 2010). The auxiliary component serves as an enhancer element that augments DNA replication (Cox, Leahy & Hardwick 1990). ZEBRA recognizes the origin of lytic DNA replication (oriLyt) by interacting with seven ZEBRA-binding sites (Schepers, Pich & Hammerschmidt 1993). Mutation of all seven binding motifs in the background of a recombinant virus drastically reduces production of infectious virus particles (Feederle & Delecluse 2004).These ZEBRA binding elements are located in two noncontiguous regions of oriLyt. Four elements are present in the upstream core region of oriLyt and overlap with the promoter of the BHLF1 open reading frame and three additional ZEBRA binding elements located mainly in the enhancer region are dispensable for viral replication (Schepers, Pich & Hammerschmidt 1996).

The current model for the role of ZEBRA in lytic DNA replication suggests that the protein serves as a physical link between oriLyt and core components of the replication machinery. The six core replication factors encoded by EBV are the DNA polymerase (BALF5); the polymerase processivity factor (BMRF1); the helicase (BBLF4); the primase (BSLF1); the primase associated factor (BBLF2/3), and the single-stranded DNA binding protein (BALF2) (El-Guindy, Heston & Miller 2010). The function of tethering replication proteins to oriLyt is not limited to ZEBRA; the transactivation domains of Sp1 and ZBP89 interact with BMRF1 and BALF5 and target them to the downstream region of oriLyt (Baumann et al. 1999). Similarly, ZBRK1, a cellular DNA binding zinc finger protein, serves as a contact point for BBLF2/3 on oriLyt (Liao et al. 2005).
