**4. EBERs associated oncogenesis and cell transformation**

Comparison of EBV-positive and -negative cell clones revealed that the presence of EBV in Akata cells was required for the cells to be more malignant and apoptosis resistant, which underlined the oncogenic role of EBV in the genesis of BL (Komano et al., 1998; Ruf et al., 1999). Subsequent studies revealed that EBERs were responsible for these phenotypes (Komano et al., 1999; Ruf et al., 2005). Transfection of the EBER genes into EBV-negative Akata clones restored the capacity for growth in soft agar, tumorigenicity in SCID mice, resistance to apoptotic inducers, and upregulated expression of bcl-2 that was originally retained in parental EBV-positive Akata cells but lost in EBV-negative subclones. More recently, a new investigation indicated that in vivo expression of a polymerase III driven non-coding viral EBER-1 construct led to the transgenic mouse more inclined to develop tumor. In Repellin's reports, they provided the first evidence by producing ten transgenic mouse lines expressing EBER1 in the lymphoid compartment, and discovered the transgenic mice developed lymphoid hyperplasia, which in some cases proceeded to B cell malignancy (Repellin et al., 2010).

Because EBERs are expressed in large amounts in latently infected cells and virtually all EBV-associated tumors, it had long been speculated that they may play a vital role in the process of transformation. To support this hypothesis, EBER-negative recombinants generated by Yajima in 2005 provided a quantitative advantage in transformation ability. Transformation assays performed with high titres of recombinant EBV generated from the EBER knockout and knock-in strains revealed that the EBER knock-in recombinant possessed approximately 20-fold more transforming ability than the EBER-negative recombinant. Furthermore, growth of the EBER-negative LCLs was impaired compared with that of the revertants under low serum conditions. In contrast, Swaminathan *et al* demonstrated that EBERs were not essential for the immotalization of B lymphocytes or for the replication of the virus. In their experiments, strains of EBV with deletions of the small RNA (EBER) genes were made by homologous recombination using the EBV P3HR-1 strain, which has undergone deletion of the essential transforming gene that encoded the EBV nuclear antigen, EBNA-2, and a DNA fragment that was wild type at the EBNA-2 locus but from which the EBER genes had been deleted. EBER-deleted recombinants transformed primary B lymphocytes into LCLs, which were indistinguishable from LCLs transformed by wildtype EBV. However, they were not able to produce a large quantity of pure EBER-deleted EBV, which may lead to the false negative outcome. To address this issue more specifically, Wu *et al*. (Wu et al., 2007) demonstrated that the transforming ability of recombinant EBVs expressing EBER2 was as high as that of EBVs expressing both EBER1 and EBER2. In contrast, the transforming ability of recombinant EBVs carrying EBER1 was impaired and was similar to that of EBV lacking both EBER1 and EBER2. Gregorovic *et al*. (Gregorovic et al., 2011)recently reported there was little effect of either EBER deletion on the transformation efficiency. This contrasts with the results of a previous study (Wu et al., 2007) where deletion of EBER2 caused a 50-fold reduction of

Intriguingly, to date there has been no investigation with respect to the function of EBERs involved RNP complexes in NPC and whether the same machinery in lymphomas readily

Comparison of EBV-positive and -negative cell clones revealed that the presence of EBV in Akata cells was required for the cells to be more malignant and apoptosis resistant, which underlined the oncogenic role of EBV in the genesis of BL (Komano et al., 1998; Ruf et al., 1999). Subsequent studies revealed that EBERs were responsible for these phenotypes (Komano et al., 1999; Ruf et al., 2005). Transfection of the EBER genes into EBV-negative Akata clones restored the capacity for growth in soft agar, tumorigenicity in SCID mice, resistance to apoptotic inducers, and upregulated expression of bcl-2 that was originally retained in parental EBV-positive Akata cells but lost in EBV-negative subclones. More recently, a new investigation indicated that in vivo expression of a polymerase III driven non-coding viral EBER-1 construct led to the transgenic mouse more inclined to develop tumor. In Repellin's reports, they provided the first evidence by producing ten transgenic mouse lines expressing EBER1 in the lymphoid compartment, and discovered the transgenic mice developed lymphoid hyperplasia, which in some cases proceeded to B cell malignancy

Because EBERs are expressed in large amounts in latently infected cells and virtually all EBV-associated tumors, it had long been speculated that they may play a vital role in the process of transformation. To support this hypothesis, EBER-negative recombinants generated by Yajima in 2005 provided a quantitative advantage in transformation ability. Transformation assays performed with high titres of recombinant EBV generated from the EBER knockout and knock-in strains revealed that the EBER knock-in recombinant possessed approximately 20-fold more transforming ability than the EBER-negative recombinant. Furthermore, growth of the EBER-negative LCLs was impaired compared with that of the revertants under low serum conditions. In contrast, Swaminathan *et al* demonstrated that EBERs were not essential for the immotalization of B lymphocytes or for the replication of the virus. In their experiments, strains of EBV with deletions of the small RNA (EBER) genes were made by homologous recombination using the EBV P3HR-1 strain, which has undergone deletion of the essential transforming gene that encoded the EBV nuclear antigen, EBNA-2, and a DNA fragment that was wild type at the EBNA-2 locus but from which the EBER genes had been deleted. EBER-deleted recombinants transformed primary B lymphocytes into LCLs, which were indistinguishable from LCLs transformed by wildtype EBV. However, they were not able to produce a large quantity of pure EBER-deleted EBV, which may lead to the false negative outcome. To address this issue more specifically, Wu *et al*. (Wu et al., 2007) demonstrated that the transforming ability of recombinant EBVs expressing EBER2 was as high as that of EBVs expressing both EBER1 and EBER2. In contrast, the transforming ability of recombinant EBVs carrying EBER1 was impaired and was similar to that of EBV lacking both EBER1 and EBER2. Gregorovic *et al*. (Gregorovic et al., 2011)recently reported there was little effect of either EBER deletion on the transformation efficiency. This contrasts with the results of a previous study (Wu et al., 2007) where deletion of EBER2 caused a 50-fold reduction of

**4. EBERs associated oncogenesis and cell transformation** 

applies to NPC remains to be seen.

(Repellin et al., 2010).

transformation efficiency. It should be noted that a different EBV strain background was used in the two experiments.

With respect to the role of EBER in the carcinogenesis of NPC, it was reported that EBERs expression may confer an apoptotic-resistant phenotype in immortalized nasopharyngeal epithelial cells. The EBER-expressing NP69 cells attained a higher growth rate compared to cells transfected with control plasmid (pcDNA3). However, the EBER-expressing NP69 cells did not form colonies in soft agar and were non-tumorigenic in nude mice (Wong et al., 2005). Iwakiri, however, reported that EBV infection induces IGF-1 expression in NPC cell lines, and that the secreted IGF-1 acts as an autocrine growth factor. These findings seem to be operative in vivo, as NPC biopsies consistently express IGF-1 (Iwakiri et al., 2005). Recently, in contrast, there are somewhat contradictive observations from Tomokazu. In their experimets, MDCK cells transfected with EBERs-high-expression vector showed an enhanced growth ability in soft agar compared with the MDCK transfected with EBERslow-expression vector-transfected or untransfected MDCK cells. However, they did not show the acquisition of any anti-apoptotic potential against either IFN-α or serum deprivation. Introduction of EBERs-low-expression vector into MDCK cells did not show anchor independent growth characteristics (Yoshizaki et al., 2007). The reasons for these contrary outcomes are not clear and whether EBERs could transform cells or even be tumorigenic are still obscure. It may be attributable to the origin of the cell line. For instance, NPC-KT, the parental cell line of EBV-neg-KT, was derived from NPC, whereas MDCK is derived from normal epitheliumand (Yoshizaki et al., 2007).

It has long been believed that both EBER1 and EBER2 play similar roles in the pathologic process. Microarray expression profiling, however, identified genes whose expression correlates with the presence of EBER1 or EBER2 (Gregorovic et al., 2011). To researchers' surprise, although most emphasis has previously been given to EBER1 because it is more abundant than EBER2, the differences in cellular gene expression were greater with EBER2 deletion. The number of genes and degree to which the regulated genes were unique to EBER1 or EBER2 was further analyzed, showing that the greater number of differences in cell gene expression was observed in EBER2 deletion. To look more specifically at some of the cellular genes whose expression correlated with EBER2 expression, the expression values from individual cell lines were derived. In each case, the expression level in parental and revertant was similar, but the expression in the EBER2 deletion was consistently different. Some additional data from an earlier comparison of del-EBER2 and parental LCLs were consistent. LCL gene expression was modified according to the presence of EBER2. The examples include genes involved in receptor function and signaling (CNKRS3, CXCL12, CXCR3, DACT1, GDF15, GPR125, IGF1, and IL12RB2A), cellular adhesion (IGSF4), a transcription factor (TBX15), an RNA binding protein (MEX3A), and a proposed tumor suppressor gene (SASH1). This comprehensive description of EBER2 related genes indicates many facets of biological process related to EBER2. Especially there seems to be a link between EBER2 and lymphoma invasion and metastasis. Hopefully this microarray analysis may lead to new insight into the EBERs' research in EBV related carcinomas.

#### **5. EBERs participate in cytokine secretion pathway through TLR3 and RIG-I**

Expression of a variety of cytokines and growth factors is enhanced in several types of EBERs-expressing cells. It was demonstrated that IL-10 induced by EBERs acts as an

Pathologic Significance of EBV Encoded RNA in NPC 35

Fig. 2. Modulation of RIG-I signaling by EBERs contributes to EBV-mediated oncogenesis in BL cells. What's more, EBERs bind PKR and inhibit its phosphorylation, which disturb

infections and tissue damage. A well-regulated inflammatory response can also be antitumorigenic and have a role in tumorsuppression (Mantovani et al., 2008). Chronic inflammation, however, is detrimental and, among other deleterious effects, will frequently predispose cells for an oncogenic transformation. Various mechanisms account for the oncogenic role of chronic inflammation. These include induction of genomic instability, increasing angiogenesis, altering the genomic epigenetic state and increasing cell proliferation. Over-production of reactive oxygen and nitrogen species (RONS), aberrant inflammatory cytokine and chemokine expression, increased (COX-2) and NFκB expression are just some of the molecular factors that contribute to inflammation-induced

PKR mediated apoptosis. Adapted from Iwakiri & Takada, 2010.

autocrine growth factor for BL (Kitagawa et al., 2000). It was found that EBV-positive Akata and Mutu cell clones expressed higher levels of interleukin (IL)-10 than their EBV-negative subclones at the transcription level. Transfection of an individual EBV latent gene into EBVnegative Akata cells revealed that EBERs were responsible for IL-10 induction. Recombinanat IL-10 enabled EBV-negative Akata cells to grow in low (0.1%) serum conditions. Likewise, Iwakiri *et al*. reported infection of EBV-negative gastric carcinoma cell lines with EBV led to expression of a limited number of EBV genes including EBERs and was correlated with increased IGF-1 production, as was transfection of EBERs genes (Iwakiri et al., 2003). Using the recombinant virus, Yang *et al*. (Yang et al., 2004) found that a human T-cell line, MT-2, was susceptible to EBV infection, and succeeded in isolating EBV-infected cell clones with type II EBV latency, which was identical with those seen in EBV-infected T cells in vivo. EBV-positive MT-2 cells expressed higher levels of interleukin (IL)-9 than EBVnegative MT-2 cells at the transcriptional level. It was also demonstrated that EBV-encoded small RNA was responsible for IL-9 expression. Addition of recombinant IL-9 accelerated the growth of MT-2 cells, whereas growth of the EBV-converted MT-2 cells was blocked by treatment with an anti-IL-9 antibody. These results suggest that IL-9 induced by EBVencoded small RNA acts as an autocrine growth factor for EBV-infected T cells.

Since EBERs are expected to form dsRNA structure, it's believed they activate RIG (Yoneyama et al., 2004), a specific pattern-recognition receptors (PRR) that specifically recognize pathogen-associated molecular patterns (PAMPs) within microbes and induce interferon induction. Recently this hypothesis was experimentally tested by Samanta *et al* (Samanta et al., 2006). According to their observation, transfection of RIG-I plasmid induced IFNs and IFN-stimulated genes (ISGs) in EBV-positive Burkitt's lymphoma (BL) cells, but not in their EBV-negative counterparts or EBER-knockout EBV-infected BL cells. Transfection of EBER plasmid or in vitro synthesized EBERs induced expression of type I IFNs and ISGs in RIG-I-expressing, EBV-negative BL cells, but not in RIG-I-minus counterparts. (Samanta et al., 2006). EBERs are recognized by RIG-I through the RNA helicase domain, and following recognition, RIG-I associates with the adaptor IPS-1 via CARD. IPS-1 is localized to mitochondria and initiates signaling leading to activation of IRF3 and NF-κB to induce type-I IFNs and inflammatory cytokines. Furthermore, they indicated that EBERs induce an anti-inflammatory cytokine IL-10 through RIG-I-mediated IRF-3 but not NF-κB signaling (FIG. 2) (Samanta et al., 2008).

Toll-like receptors (TLRs) constitute distinct families of PRRs that sense nucleic acids derived from viruses and trigger antiviral innate immune responses through activation of signaling cascades via Toll/IL-1 receptor (TIR) domain-containing adaptors (Akira & Takeda, 2004). Iwakiri et al. (Iwakiri et al., 2009) reported that EBERs were released extracellulary and recognized by TLR3, leading to induction of type-I IFN and inflammatory cytokines. A substantial amount of EBER, which was sufficient to induce TLR3 signaling involving IRF3 and NF-B activation, was released from EBV-infected cells. Thus, EBERs can contribute to the pathogenesis of EBV infection through interaction with RIG-I and TLR3.

TLRs and RIG-I as PRRs could trigger the innate immune response, as first line of defense against pathogens and tissue injury. This response i.e. inflammation, is a complex response to infection, trauma, and other conditions of homeostatic imbalance (Nathan, 2002). An acute inflammatory response is usually beneficial, especially in response to microbial

autocrine growth factor for BL (Kitagawa et al., 2000). It was found that EBV-positive Akata and Mutu cell clones expressed higher levels of interleukin (IL)-10 than their EBV-negative subclones at the transcription level. Transfection of an individual EBV latent gene into EBVnegative Akata cells revealed that EBERs were responsible for IL-10 induction. Recombinanat IL-10 enabled EBV-negative Akata cells to grow in low (0.1%) serum conditions. Likewise, Iwakiri *et al*. reported infection of EBV-negative gastric carcinoma cell lines with EBV led to expression of a limited number of EBV genes including EBERs and was correlated with increased IGF-1 production, as was transfection of EBERs genes (Iwakiri et al., 2003). Using the recombinant virus, Yang *et al*. (Yang et al., 2004) found that a human T-cell line, MT-2, was susceptible to EBV infection, and succeeded in isolating EBV-infected cell clones with type II EBV latency, which was identical with those seen in EBV-infected T cells in vivo. EBV-positive MT-2 cells expressed higher levels of interleukin (IL)-9 than EBVnegative MT-2 cells at the transcriptional level. It was also demonstrated that EBV-encoded small RNA was responsible for IL-9 expression. Addition of recombinant IL-9 accelerated the growth of MT-2 cells, whereas growth of the EBV-converted MT-2 cells was blocked by treatment with an anti-IL-9 antibody. These results suggest that IL-9 induced by EBV-

encoded small RNA acts as an autocrine growth factor for EBV-infected T cells.

IRF-3 but not NF-κB signaling (FIG. 2) (Samanta et al., 2008).

RIG-I and TLR3.

Since EBERs are expected to form dsRNA structure, it's believed they activate RIG (Yoneyama et al., 2004), a specific pattern-recognition receptors (PRR) that specifically recognize pathogen-associated molecular patterns (PAMPs) within microbes and induce interferon induction. Recently this hypothesis was experimentally tested by Samanta *et al* (Samanta et al., 2006). According to their observation, transfection of RIG-I plasmid induced IFNs and IFN-stimulated genes (ISGs) in EBV-positive Burkitt's lymphoma (BL) cells, but not in their EBV-negative counterparts or EBER-knockout EBV-infected BL cells. Transfection of EBER plasmid or in vitro synthesized EBERs induced expression of type I IFNs and ISGs in RIG-I-expressing, EBV-negative BL cells, but not in RIG-I-minus counterparts. (Samanta et al., 2006). EBERs are recognized by RIG-I through the RNA helicase domain, and following recognition, RIG-I associates with the adaptor IPS-1 via CARD. IPS-1 is localized to mitochondria and initiates signaling leading to activation of IRF3 and NF-κB to induce type-I IFNs and inflammatory cytokines. Furthermore, they indicated that EBERs induce an anti-inflammatory cytokine IL-10 through RIG-I-mediated

Toll-like receptors (TLRs) constitute distinct families of PRRs that sense nucleic acids derived from viruses and trigger antiviral innate immune responses through activation of signaling cascades via Toll/IL-1 receptor (TIR) domain-containing adaptors (Akira & Takeda, 2004). Iwakiri et al. (Iwakiri et al., 2009) reported that EBERs were released extracellulary and recognized by TLR3, leading to induction of type-I IFN and inflammatory cytokines. A substantial amount of EBER, which was sufficient to induce TLR3 signaling involving IRF3 and NF-B activation, was released from EBV-infected cells. Thus, EBERs can contribute to the pathogenesis of EBV infection through interaction with

TLRs and RIG-I as PRRs could trigger the innate immune response, as first line of defense against pathogens and tissue injury. This response i.e. inflammation, is a complex response to infection, trauma, and other conditions of homeostatic imbalance (Nathan, 2002). An acute inflammatory response is usually beneficial, especially in response to microbial

Fig. 2. Modulation of RIG-I signaling by EBERs contributes to EBV-mediated oncogenesis in BL cells. What's more, EBERs bind PKR and inhibit its phosphorylation, which disturb PKR mediated apoptosis. Adapted from Iwakiri & Takada, 2010.

infections and tissue damage. A well-regulated inflammatory response can also be antitumorigenic and have a role in tumorsuppression (Mantovani et al., 2008). Chronic inflammation, however, is detrimental and, among other deleterious effects, will frequently predispose cells for an oncogenic transformation. Various mechanisms account for the oncogenic role of chronic inflammation. These include induction of genomic instability, increasing angiogenesis, altering the genomic epigenetic state and increasing cell proliferation. Over-production of reactive oxygen and nitrogen species (RONS), aberrant inflammatory cytokine and chemokine expression, increased (COX-2) and NFκB expression are just some of the molecular factors that contribute to inflammation-induced

Pathologic Significance of EBV Encoded RNA in NPC 37

In light of this model, When nasopharyngeal epithelial cells were infected by EBV, EBERs could initiate NF-κB induced pro-inflammatory cytokines production through PRRs TLR3 and RIG-I. Thus cellular physiological status is shifted in several avenues such as proliferation and apoptosis due to the pro-inflammatory cytokines. Meanwhile, NF-κB could induce oncogenic protein LMP1 transcription, which in turn trigger the NF-κB pathway thus intensify the pro-inflammatory cytokines production. Cooperation between this pro-inflammatory cytokine production and LMP-1 probably potentiate risk of

Akira, S. & Takeda, K. (2004). Toll-like receptor signalling. *Nat Rev Immunol*, Vol. 4, No. 7,

Ambinder, R. F. & Mann, R. B. (1994). Epstein-Barr-encoded RNA in situ hybridization:

Arrand, J. R. & Rymo, L. (1982). Characterization of the major Epstein-Barr virus-specific RNA in Burkitt lymphoma-derived cells. *J Virol*, Vol. 41, No. 2, pp. 376-389 Arrand, J. R., Young, L. S. & Tugwood, J. D. (1989). Two families of sequences in the small

Banati, F., Koroknai, A., Salamon, D., Takacs, M., Minarovits-Kormuta, S., Wolf, H., Niller,

Barletta, J. M., Kingma, D. W., Ling, Y., Charache, P., Mann, R. B. & Ambinder, R. F. (1993).

Bhat, R. A. & Thimmappaya, B. (1983). Two small RNAs encoded by Epstein-Barr virus can

adenovirus 5. *Proc Natl Acad Sci U S A*, Vol. 80, No. 15, pp. 4789-4793 Bhat, R. A. & Thimmappaya, B. (1985). Construction and analysis of additional adenovirus

RNA-encoding region of Epstein-Barr virus (EBV) correlate with EBV types A and

H. H. & Minarovits, J. (2008). CpG-methylation silences the activity of the RNA polymerase III transcribed EBER-1 promoter of Epstein-Barr virus. *FEBS Lett*, Vol.

Rapid in situ hybridization for the diagnosis of latent Epstein-Barr virus infection.

functionally substitute for the virus-associated RNAs in the lytic growth of

substitution mutants confirm the complementation of VAI RNA function by two small RNAs encoded by Epstein-Barr virus. *J Virol*, Vol. 56, No. 3, pp. 750-756 Chang, K. L., Chen, Y. Y., Shibata, D. & Weiss, L. M. (1992). Description of an in situ

hybridization methodology for detection of Epstein-Barr virus RNA in paraffinembedded tissues, with a survey of normal and neoplastic tissues. *Diagn Mol* 

(1996). Expression of Epstein-Barr virus-encoded RNAs as a marker for metastatic

small RNA promoters for driving the expression of fusion transcripts harboring

Chao, T. Y., Chow, K. C., Chang, J. Y., Wang, C. C., Tsao, T. Y., Harn, H. J. & Chi, K. H.

undifferentiated nasopharyngeal carcinoma. *Cancer*, Vol. 78, No. 1, pp. 24-29 Choy, E. Y., Kok, K. H., Tsao, S. W. & Jin, D. Y. (2008). Utility of Epstein-Barr virus-encoded

short hairpin RNAs. *Gene Ther*, Vol. 15, No. 3, pp. 191-202

diagnostic applications. *Hum Pathol*, Vol. 25, No. 6, pp. 602-605

developing NPC.

**6. References** 

pp. 499-511

B. *J Virol*, Vol. 63, No. 2, pp. 983-986

*Mol Cell Probes*, Vol. 7, No. 2, pp. 105-109

*Pathol*, Vol. 1, No. 4, pp. 246-255

582, No. 5, pp. 705-709

carcinogenesis (Schetter et al., 2010). Considering the emerging link between EBERs and TLR and RIG-I in lymphoma and the involvement of PRR in the subsequent inflammation associated cancer, it was intriguing to investigate this potential interaction in other carcinoma. For instance, we demonstrated that EBERs' expression could instantly trigger acute accumulation of inflammation cytokines and this response was impaired in EBERs knock-down NPC cell lines (unpublished data). Furthermore, EBERs induced this inflammation response through TLR3 and RIG-I signaling, as was the scenario in lymphoma. To dissect the exact role of the EBER induced signaling pathway, we have established a positive feedback loop between NF-κB and the EBV encoded oncogenic protein LMP1, which constitute the cascade downstream of EBERs activated NF-κB (Fig. 3).

Fig. 3. EBERs induced inflammation response in NPC is intensified by the feedback loop composed of NF-κB and LMP1. Dotted line represents tentative transcription activation. Refer to main text for details.

In light of this model, When nasopharyngeal epithelial cells were infected by EBV, EBERs could initiate NF-κB induced pro-inflammatory cytokines production through PRRs TLR3 and RIG-I. Thus cellular physiological status is shifted in several avenues such as proliferation and apoptosis due to the pro-inflammatory cytokines. Meanwhile, NF-κB could induce oncogenic protein LMP1 transcription, which in turn trigger the NF-κB pathway thus intensify the pro-inflammatory cytokines production. Cooperation between this pro-inflammatory cytokine production and LMP-1 probably potentiate risk of developing NPC.

#### **6. References**

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

carcinogenesis (Schetter et al., 2010). Considering the emerging link between EBERs and TLR and RIG-I in lymphoma and the involvement of PRR in the subsequent inflammation associated cancer, it was intriguing to investigate this potential interaction in other carcinoma. For instance, we demonstrated that EBERs' expression could instantly trigger acute accumulation of inflammation cytokines and this response was impaired in EBERs knock-down NPC cell lines (unpublished data). Furthermore, EBERs induced this inflammation response through TLR3 and RIG-I signaling, as was the scenario in lymphoma. To dissect the exact role of the EBER induced signaling pathway, we have established a positive feedback loop between NF-κB and the EBV encoded oncogenic protein LMP1, which constitute the cascade downstream of EBERs activated NF-κB

Fig. 3. EBERs induced inflammation response in NPC is intensified by the feedback loop composed of NF-κB and LMP1. Dotted line represents tentative transcription activation.

Refer to main text for details.

(Fig. 3).


Pathologic Significance of EBV Encoded RNA in NPC 39

Iwakiri, D. & Takada, K. (2010). Role of EBERs in the pathogenesis of EBV infection. *Adv* 

Iwakiri, D., Zhou, L., Samanta, M., Matsumoto, M., Ebihara, T., Seya, T., Imai, S., Fujieda,

Kimura, H., Miyake, K., Yamauchi, Y., Nishiyama, K., Iwata, S., Iwatsuki, K., Gotoh, K.,

Kitagawa, N., Goto, M., Kurozumi, K., Maruo, S., Fukayama, M., Naoe, T., Yasukawa, M.,

Komano, J., Maruo, S., Kurozumi, K., Oda, T. & Takada, K. (1999). Oncogenic role of

Komano, J., Sugiura, M. & Takada, K. (1998). Epstein-Barr virus contributes to the malignant

Laing, K. G., Elia, A., Jeffrey, I., Matys, V., Tilleray, V. J., Souberbielle, B. & Clemens, M. J.

synthesis and cell growth regulation. *Virology*, Vol. 297, No. 2, pp. 253-269 Laing, K. G., Matys, V. & Clemens, M. J. (1995). Effects of expression of the Epstein-Barr

Lerner, M. R., Andrews, N. C., Miller, G. & Steitz, J. A. (1981). Two small RNAs encoded by

Mantovani, A., Romero, P., Palucka, A. K. & Marincola, F. M. (2008). Tumour immunity:

Mathews, M. B. (1980). Binding of adenovirus VA RNA to mRNA: a possible role in

Murray, V. & Holliday, R. (1979). Mechanism for RNA splicing of gene transcripts. *FEBS* 

Nanbo, A., Inoue, K., Adachi-Takasawa, K. & Takada, K. (2002). Epstein-Barr virus RNA

1773

1087

*Cancer Res*, Vol. 107, No. pp. 119-136

*Exp Med*, Vol. 206, No. 10, pp. 2091-2099

induction. *Embo J*, Vol. 19, No. 24, pp. 6742-6750

Vol. 73, No. 12, pp. 9827-9831

2, pp. 805-809

9614, pp. 771-783

*Lett*, Vol. 106, No. 1, pp. 5-7

*Embo J*, Vol. 21, No. 5, pp. 954-965

*Virol*, Vol. 72, No. 11, pp. 9150-9156

*Biochem Soc Trans*, Vol. 23, No. 2, pp. 311S

splicing? *Nature*, Vol. 285, No. 5766, pp. 575-577

nasopharyngeal carcinoma-derived cell lines. *Oncogene*, Vol. 24, No. 10, pp. 1767-

M., Kawa, K. & Takada, K. (2009). Epstein-Barr virus (EBV)-encoded small RNA is released from EBV-infected cells and activates signaling from Toll-like receptor 3. *J* 

Kojima, S., Ito, Y. & Nishiyama, Y. (2009). Identification of Epstein-Barr virus (EBV)-infected lymphocyte subtypes by flow cytometric in situ hybridization in EBV-associated lymphoproliferative diseases. *J Infect Dis*, Vol. 200, No. 7, pp. 1078-

Hino, K., Suzuki, T., Todo, S. & Takada, K. (2000). Epstein-Barr virus-encoded poly(A)(-) RNA supports Burkitt's lymphoma growth through interleukin-10

Epstein-Barr virus-encoded RNAs in Burkitt's lymphoma cell line Akata. *J Virol*,

phenotype and to apoptosis resistance in Burkitt's lymphoma cell line Akata. *J* 

(2002). In vivo effects of the Epstein-Barr virus small RNA EBER-1 on protein

virus small RNA EBER-1 in heterologous cells on protein synthesis and cell growth.

Epstein-Barr virus and complexed with protein are precipitated by antibodies from patients with systemic lupus erythematosus. *Proc Natl Acad Sci U S A*, Vol. 78, No.

effector response to tumour and role of the microenvironment. *Lancet*, Vol. 371, No.

confers resistance to interferon-alpha-induced apoptosis in Burkitt's lymphoma.


Clarke, P. A., Schwemmle, M., Schickinger, J., Hilse, K. & Clemens, M. J. (1991). Binding of

Clarke, P. A., Sharp, N. A. & Clemens, M. J. (1990). Translational control by the Epstein-Barr

Dobbelstein, M. & Shenk, T. (1995). In vitro selection of RNA ligands for the ribosomal L22

Fok, V., Friend, K. & Steitz, J. A. (2006). Epstein-Barr virus noncoding RNAs are confined to

Ghadge, G. D., Malhotra, P., Furtado, M. R., Dhar, R. & Thimmapaya, B. (1994). In vitro

Gilligan, K., Rajadurai, P., Resnick, L. & Raab-Traub, N. (1990). Epstein-Barr virus small

Houmani, J. L., Davis, C. I. & Ruf, I. K. (2009). Growth-promoting properties of Epstein-Barr

Hovanessian, A. G. (1989). The double stranded RNA-activated protein kinase induced by

Howe, J. G. & Shu, M. D. (1989). Epstein-Barr virus small RNA (EBER) genes: unique

Howe, J. G. & Steitz, J. A. (1986). Localization of Epstein-Barr virus-encoded small RNAs by in situ hybridization. *Proc Natl Acad Sci U S A*, Vol. 83, No. 23, pp. 9006-9010 Iwakiri, D., Eizuru, Y., Tokunaga, M. & Takada, K. (2003). Autocrine growth of Epstein-Barr

Iwakiri, D., Sheen, T. S., Chen, J. Y., Huang, D. P. & Takada, K. (2005). Epstein-Barr virus-

leukoplakia. *Proc Natl Acad Sci U S A*, Vol. 87, No. 22, pp. 8790-8794 Gregorovic, G., Bosshard, R., Karstegl, C. E., White, R. E., Pattle, S., Chiang, A. K., Dittrich-

lymphoblastoid cell lines. *J Virol*, Vol. 85, No. 7, pp. 3535-3545

interferon: dsRNA-PK. *J Interferon Res*, Vol. 9, No. 6, pp. 641-647

small RNA. *Cancer Res*, Vol. 63, No. 21, pp. 7062-7067

nucleocytoplasmic shuttling. *J Cell Biol*, Vol. 173, No. 3, pp. 319-325

and cDNA-derived RNA libraries. *J Virol*, Vol. 69, No. 12, pp. 8027-8034 Felton-Edkins, Z. A., Kondrashov, A., Karali, D., Fairley, J. A., Dawson, C. W., Arrand, J. R.,

*Chem*, Vol. 281, No. 45, pp. 33871-33880

No. 7, pp. 4137-4151

No. 19, pp. 9844-9853

*Cell*, Vol. 57, No. 5, pp. 825-834

protein kinase DAI. *Nucleic Acids Res*, Vol. 19, No. 2, pp. 243-248

641

Epstein-Barr virus small RNA EBER-1 to the double-stranded RNA-activated

virus small RNA EBER-1. Reversal of the double-stranded RNA-induced inhibition of protein synthesis in reticulocyte lysates. *Eur J Biochem*, Vol. 193, No. 3, pp. 635-

protein associated with Epstein-Barr virus-expressed RNA by using randomized

Young, L. S. & White, R. J. (2006). Epstein-Barr virus induces cellular transcription factors to allow active expression of EBER genes by RNA polymerase III. *J Biol* 

the nucleus, whereas their partner, the human La protein, undergoes

analysis of virus-associated RNA I (VAI RNA): inhibition of the double-stranded RNA-activated protein kinase PKR by VAI RNA mutants correlates with the in vivo phenotype and the structural integrity of the central domain. *J Virol*, Vol. 68,

nuclear RNAs are not expressed in permissively infected cells in AIDS-associated

Breiholz, O., Kracht, M., Russ, R. & Farrell, P. J. (2011). Cellular gene expression that correlates with EBER expression in Epstein-Barr Virus-infected

virus EBER-1 RNA correlate with ribosomal protein L22 binding. *J Virol*, Vol. 83,

transcription units that combine RNA polymerase II and III promoter elements.

virus-positive gastric carcinoma cells mediated by an Epstein-Barr virus-encoded

encoded small RNA induces insulin-like growth factor 1 and supports growth of

nasopharyngeal carcinoma-derived cell lines. *Oncogene*, Vol. 24, No. 10, pp. 1767- 1773


Pathologic Significance of EBV Encoded RNA in NPC 41

Samanta, M., Iwakiri, D. & Takada, K. (2008). Epstein-Barr virus-encoded small RNA

Schetter, A. J., Heegaard, N. H. & Harris, C. C. (2010). Inflammation and cancer:

Schwemmle, M., Clemens, M. J., Hilse, K., Pfeifer, K., Troster, H., Muller, W. E. &

Sharp, T. V., Schwemmle, M., Jeffrey, I., Laing, K., Mellor, H., Proud, C. G., Hilse, K. &

Swaminathan, S. (2010). The role of non-coding RNAs in EBV-induced cell growth and

Toczyski, D. P. & Steitz, J. A. (1991). EAP, a highly conserved cellular protein associated with Epstein-Barr virus small RNAs (EBERs). *Embo J*, Vol. 10, No. 2, pp. 459-466 Toczyski, D. P. & Steitz, J. A. (1993). The cellular RNA-binding protein EAP recognizes a

Wang, Y., Xue, S. A., Hallden, G., Francis, J., Yuan, M., Griffin, B. E. & Lemoine, N. R. (2005).

Wong, H. L., Wang, X., Chang, R. C., Jin, D. Y., Feng, H., Wang, Q., Lo, K. W., Huang, D. P.,

Wu, Y., Maruo, S., Yajima, M., Kanda, T. & Takada, K. (2007). Epstein-Barr virus (EBV)-

Yajima, M., Kanda, T. & Takada, K. (2005). Critical role of Epstein-Barr Virus (EBV)-encoded

EBV-associated tumors. *Cancer Res*, Vol. 65, No. 4, pp. 1523-1531

growth transformation. *J Virol*, Vol. 81, No. 20, pp. 11236-11245

apoptotic stress. *Mol Carcinog*, Vol. 44, No. 2, pp. 92-101

155-166. Caister Academic Press, ISBN 978-1-904455-62-2, Norfolk , UK. Toczyski, D. P., Matera, A. G., Ward, D. C. & Steitz, J. A. (1994). The Epstein-Barr virus

adenovirus VAI RNA. *Nucleic Acids Res*, Vol. 21, No. 19, pp. 4483-4490 Swaminathan, S., Tomkinson, B. & Kieff, E. (1991). Recombinant Epstein-Barr virus with

*Proc Natl Acad Sci U S A*, Vol. 88, No. 4, pp. 1546-1550

pp. 4150-4160

3467

Vol. 13, No. 1, pp. 703-710

79, No. 7, pp. 4298-4307

Vol. 31, No. 1, pp. 37-49

*S A*, Vol. 89, No. 21, pp. 10292-10296

induces IL-10 through RIG-I-mediated IRF-3 signaling. *Oncogene*, Vol. 27, No. 30,

interweaving microRNA, free radical, cytokine and p53 pathways. *Carcinogenesis*,

Bachmann, M. (1992). Localization of Epstein-Barr virus-encoded RNAs EBER-1 and EBER-2 in interphase and mitotic Burkitt lymphoma cells. *Proc Natl Acad Sci U* 

Clemens, M. J. (1993). Comparative analysis of the regulation of the interferoninducible protein kinase PKR by Epstein-Barr virus RNAs EBER-1 and EBER-2 and

small RNA (EBER) genes deleted transforms lymphocytes and replicates in vitro.

transformation, in: *Epstein-Barr Virus: Latency and Transformation,* Erle S. Robertson.

(EBV) small RNA EBER1 binds and relocalizes ribosomal protein L22 in EBVinfected human B lymphocytes. *Proc Natl Acad Sci U S A*, Vol. 91, No. 8, pp. 3463-

conserved stem-loop in the Epstein-Barr virus small RNA EBER 1. *Mol Cell Biol*,

Virus-associated RNA I-deleted adenovirus, a potential oncolytic agent targeting

Yuen, P. W., Takada, K., Wong, Y. C. & Tsao, S. W. (2005). Stable expression of EBERs in immortalized nasopharyngeal epithelial cells confers resistance to

encoded RNA 2 (EBER2) but not EBER1 plays a critical role in EBV-induced B-cell

RNA in efficient EBV-induced B-lymphocyte growth transformation. *J Virol*, Vol.


Nathan, C. (2002). Points of control in inflammation. *Nature*, Vol. 420, No. 6917, pp. 846-

Niller, H. H., Salamon, D., Ilg, K., Koroknai, A., Banati, F., Bauml, G., Rucker, O.,

Orellana, T. & Kieff, E. (1977). Epstein-barr virus-specific RNA. II. Analysis of

Owen, T. J., O'Neil, J. D., Dawson, C. W., Hu, C., Chen, X., Yao, Y., Wood, V. H., Mitchell, L.

Pathmanathan, R., Prasad, U., Chandrika, G., Sadler, R., Flynn, K. & Raab-Traub, N. (1995).

Powell, A. L., King, W. & Kieff, E. (1979). Epstein-Barr virus-specific RNA. III. Mapping of

Repellin, C. E., Tsimbouri, P. M., Philbey, A. W. & Wilson, J. B. (2010). Lymphoid

Ruf, I. K., Lackey, K. A., Warudkar, S. & Sample, J. T. (2005). Protection from interferon-

Ruf, I. K., Rhyne, P. W., Yang, C., Cleveland, J. L. & Sample, J. T. (2000). Epstein-Barr virus

Ruf, I. K., Rhyne, P. W., Yang, H., Borza, C. M., Hutt-Fletcher, L. M., Cleveland, J. L. &

Samanta, M., Iwakiri, D., Kanda, T., Imaizumi, T. & Takada, K. (2006). EB virus-encoded

an effect on apoptosis. *J Virol*, Vol. 74, No. 21, pp. 10223-10228

lymphoma-derived cells. *J Virol*, Vol. 32, No. 1, pp. 8-18

Vol. 25, No. 18, pp. 4207-4214

of PKR. *J Virol*, Vol. 79, No. 23, pp. 14562-14569

RNA, EBER1 of Epstein-Barr virus. *PLoS One*, Vol. 5, No. 2, pp. e9092 Rosa, M. D., Gottlieb, E., Lerner, M. R. & Steitz, J. A. (1981). Striking similarities are

Schwarzmann, F., Wolf, H. & Minarovits, J. (2003). The in vivo binding site for oncoprotein c-Myc in the promoter for Epstein-Barr virus (EBV) encoding RNA (EBER) 1 suggests a specific role for EBV in lymphomagenesis. *Med Sci Monit*, Vol.

polyadenylated viral RNA in restringent, abortive, and prooductive infections. *J* 

E., White, R. J., Young, L. S. & Arrand, J. R. (2010). Epstein-Barr virus-encoded EBNA1 enhances RNA polymerase III-dependent EBER expression through induction of EBER-associated cellular transcription factors. *Mol Cancer*, Vol. 9, No.

Undifferentiated, nonkeratinizing, and squamous cell carcinoma of the nasopharynx. Variants of Epstein-Barr virus-infected neoplasia. *Am J Pathol*, Vol.

DNA encoding viral RNA in restringent infection. *J Virol*, Vol. 29, No. 1, pp. 261-

hyperplasia and lymphoma in transgenic mice expressing the small non-coding

exhibited by two small Epstein-Barr virus-encoded ribonucleic acids and the adenovirus-associated ribonucleic acids VAI and VAII. *Mol Cell Biol*, Vol. 1, No. 9,

induced apoptosis by Epstein-Barr virus small RNAs is not mediated by inhibition

small RNAs potentiate tumorigenicity of Burkitt lymphoma cells independently of

Sample, J. T. (1999). Epstein-barr virus regulates c-MYC, apoptosis, and tumorigenicity in Burkitt lymphoma. *Mol Cell Biol*, Vol. 19, No. 3, pp. 1651-1660 Rymo, L. (1979). Identification of transcribed regions of Epstein-Barr virus DNA in Burkitt

RNAs are recognized by RIG-I and activate signaling to induce type I IFN. *Embo J*,

852

9, No. 1, pp. 1-9

pp. 241

274

pp. 785-796

*Virol*, Vol. 22, No. 2, pp. 321-330

146, No. 6, pp. 1355-1367


Yang, L., Aozasa, K., Oshimi, K. & Takada, K. (2004). Epstein-Barr virus (EBV)-encoded RNA promotes growth of EBV-infected T cells through interleukin-9 induction. *Cancer Res*, Vol. 64, No. 15, pp. 5332-5337

**3** 

*Morocco* 

**Role of the Epstein-Barr Virus ZEBRA** 

Moumad Khalid1,4, Laantri Nadia1, Attaleb Mohammed2, Dardari R'kia1, Benider Abdellatif3, Benchakroun Nadia3,

*1Laboratory of Oncovirology, Institut Pasteur du Maroc, Casablanca, 2Biology and Medical Research Unit, Centre National de l'Energie,* 

*3Service de Radiothérapie, Centre d′Oncologie IBN Rochd, Casablanca, 4Laboratoire de Biologie Moléculaire, Institut Pasteur du Maroc, Casablanca* 

Approximately 15% of all cancers worldwide appear to be associated with viral infections, and several human DNA viruses are now accepted as causative factors of specific malignancies. Human papillomaviruses (HPVs) cause cervical and anogenital cancers (zur Hausen 1999) and is now associated with oral cancers (Gillison & Shah 2001), but, the natural history of oncogenic HPV infections in the oral cavity is poorly understood. Epstein-Barr virus (EBV) causes infectious mononucleosis and is closely associated with Burkitt's lymphoma, nasopharyngeal carcinoma (NPC), and Hodgkin's disease (Raab-Traub 1996). NPC is a malignant tumour that originates within the post nasal space (Pathmanathan et al. 1995). The etiologic factors of endemic NPC include environmental risk factors, genetic susceptibility and viral infection (Yu 1991). Evidence of EBV DNA in almost all NPC cells that were studied supports the association of NPC with EBV, while, HPV has been detected in a variety of head and neck tumours including NPC. Current data suggest that approximately 15–20% of head and neck squamous cell carcinomas (HNSCC) are linked to HPV infection. To date, different degrees of associations between HPV and NPC have been described, yet no conclusive data have been obtained. Given the particular characteristics of NPC in the Moroccan population in terms of incidence, age distribution and the predominance of specific EBV strains, and HPV genotype we describe in this chapter the role of the Epstein-Barr virus ZEBRA protein and HPV in the carcinogenesis

NPC is a malignancy of the head and neck region that arises in the epithelium surface of the posterior nasopharynx, and shows a peculiar geographic and ethnic distribution. The

**1. Introduction**

of NPC.

**1.1 Nasopharyngeal carcinoma** 

**of Nasopharyngeal Carcinoma** 

*des Sciences et Techniques Nucléaires (CNESTEN), Rabat,* 

Ennaji Mustapha4 and Khyatti Meriem1

**Protein and HPV in the Carcinogenesis** 

