**3. Localization and potential function of EBERs involved RNP complexes**

EBERs are believed to be confined in the nucleus by ISH according to several early publications (Barletta et al., 1993; Chang et al., 1992; Howe & Steitz, 1986). In contrast, Schwemmle *et al*. traced EBERs localization in interphase and mitotic phase and they discovered both RNAs were found in the cytoplasm as well as in the nuclei of interphase cells. The cytoplasm distribution of the EBERs was similar to that of the double-stranded RNA-dependent protein kinase, to which these RNAs could bind, and the location was coincident with the rough endoplasmic reticulum. Thus a cytoplasmic location for EBER-1 and EBER-2 in interphase cells is consistent with the evidence for a role for these small RNAs in translational control (Schwemmle et al., 1992). Despite this sole publication in accord with the potential function of EBERs involved RNP complexes, a recent report (Fok et al., 2006) indicated that EBERs are confined to the nucleus. They carried out heterokaryon assays and oocyte assays, the outcomes of which indicated EBERs did not shutter out of the nucleus under any circumstances and speculated that the report of cytoplasmic localization of EBERs (Schwemmle et al., 1992) was probably due to the complement between the probe and regions including conserved polymerase III promoter elements A and B. EBER1 was shown to have a half-life of 25-30 hours, and was more stable than RNAs that did undergo shuttling, indicating that rapid cytoplasmic degradation was not responsible for the inability to detect shuttling.

While the accurate localization of EBERs is still controversial, the scenario of the function of EBERs involved RNP complexes is perhaps more complicated. EBERs, which resemble another virus encoded RNA VA RNAs (adenovirus virus-associated RNAs) , were firstly found to be complexed with La. Although there is no striking nucleotide sequence homology between EBERs and VAs, similarities exist in their size, degree of secondary structure, and genomic organization (Bhat & Thimmappaya, 1983). The many shared features of the two RNA molecules enumerated above and the fact that they bind a common antigenic host protein supports the supposition that these RNPs play similar roles in virusinfected cells. A specific role in the splicing of adenovirus messenger RNAs has been proposed for the VA RNAs (Murray & Holliday, 1979). The demonstration of a direct physical association between the VA RNAs and certain adenovirus late messenger RNAs supports this proposal (Mathews, 1980). Thus, EBERs could well perform comparable functions in splicing of EBV messenger RNAs. Furthermore, VA RNAs play an important role in adenovirus replication by rescuing cells from inhibition of protein translation mediated by the cellular kinase PKR, which is induced by interferon and activated by double-stranded RNAs produced during replication of many viruses (Ghadge et al., 1994;

cells. (Gilligan et al., 1990). Recently, researchers have discovered that EBERs could be used as a sensitive marker to monitor NPC cells at various metastatic sites by techniques of in situ hybridization. In cases of metastatic cancer of unknown origin, it is thus reasonable to consider NPC if EBV is present in the tumor cells (Chao et al., 1996). Kimura has established a novel flow cytometric in situ hybridization assay to detect EBV+ suspension cells using a peptide nucleic acid probe specific for EBERs. With this method, they can not only decide the EBV load but also locate EBV-infected cells, which will be beneficial for diagnosis of Epstein-Barr virus (EBV)–associated diseases and exploration of the pathogenesis of EBV

**3. Localization and potential function of EBERs involved RNP complexes** 

EBERs are believed to be confined in the nucleus by ISH according to several early publications (Barletta et al., 1993; Chang et al., 1992; Howe & Steitz, 1986). In contrast, Schwemmle *et al*. traced EBERs localization in interphase and mitotic phase and they discovered both RNAs were found in the cytoplasm as well as in the nuclei of interphase cells. The cytoplasm distribution of the EBERs was similar to that of the double-stranded RNA-dependent protein kinase, to which these RNAs could bind, and the location was coincident with the rough endoplasmic reticulum. Thus a cytoplasmic location for EBER-1 and EBER-2 in interphase cells is consistent with the evidence for a role for these small RNAs in translational control (Schwemmle et al., 1992). Despite this sole publication in accord with the potential function of EBERs involved RNP complexes, a recent report (Fok et al., 2006) indicated that EBERs are confined to the nucleus. They carried out heterokaryon assays and oocyte assays, the outcomes of which indicated EBERs did not shutter out of the nucleus under any circumstances and speculated that the report of cytoplasmic localization of EBERs (Schwemmle et al., 1992) was probably due to the complement between the probe and regions including conserved polymerase III promoter elements A and B. EBER1 was shown to have a half-life of 25-30 hours, and was more stable than RNAs that did undergo shuttling, indicating that rapid cytoplasmic degradation was not responsible for the inability

While the accurate localization of EBERs is still controversial, the scenario of the function of EBERs involved RNP complexes is perhaps more complicated. EBERs, which resemble another virus encoded RNA VA RNAs (adenovirus virus-associated RNAs) , were firstly found to be complexed with La. Although there is no striking nucleotide sequence homology between EBERs and VAs, similarities exist in their size, degree of secondary structure, and genomic organization (Bhat & Thimmappaya, 1983). The many shared features of the two RNA molecules enumerated above and the fact that they bind a common antigenic host protein supports the supposition that these RNPs play similar roles in virusinfected cells. A specific role in the splicing of adenovirus messenger RNAs has been proposed for the VA RNAs (Murray & Holliday, 1979). The demonstration of a direct physical association between the VA RNAs and certain adenovirus late messenger RNAs supports this proposal (Mathews, 1980). Thus, EBERs could well perform comparable functions in splicing of EBV messenger RNAs. Furthermore, VA RNAs play an important role in adenovirus replication by rescuing cells from inhibition of protein translation mediated by the cellular kinase PKR, which is induced by interferon and activated by double-stranded RNAs produced during replication of many viruses (Ghadge et al., 1994;

infection (Kimura et al., 2009).

to detect shuttling.

Hovanessian, 1989). Considering the resemblance, Bhat and Thimmappaya successfully proved that EBERs can functionally substitute for the VA RNAs in the lytic growth of Ad5 (Bhat & Thimmappaya, 1983, 1985). What's more, EBERs could directly bind PKR and inhibit its activity, then block phosphorylation of eIF2a, thus resulting in the blockage of inhibition of protein synthesis by eIF2a (Clarke et al., 1991; Sharp et al., 1993). When added to reticulocyte lysates at high concentrations, EBER-1 could prevent inhibition of translation by double-stranded RNA (Clarke et al., 1990). However, EBER-1 enhanced overall protein synthesis in the absence of PKR expression (Laing et al., 2002; Laing et al., 1995). In support of EBERs function regardless of PKR, EBER-deleted recombinant EBV transformed primary B lymphocytes into LCLs, which were indistinguishable from LCLs transformed by wildtype EBV in their proliferation, in latency-associated EBV gene expression, and in their permissiveness for EBV replication cycle gene expression (Swaminathan et al., 1991). Especially, another publication indicated EBERs could support replication of the defective adenovirus in vivo but PKR phosphorylation status wasn't influenced (Wang et al., 2005). This difference is likely a result of distinct subcellular compartmentalization of these two molecules, with the EBERs being exclusively nuclear, while PKR is predominately found in the cytoplasm.

Furthermore, it was speculated EBERs could partly restores resistance to both spontaneous and interferon-induced apoptosis (Komano et al., 1999) and PKR probably act as the mediator of the EBER protective effect against apoptosis despite controversial observation provided by Ruf et al. (Ruf et al., 2000). According to Komano et al., 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 oncoprotein that were originally retained in parental EBV-positive Akata cells and lost in EBV-negative subclones. To support this hypothesis, researchers have made it clear that when EBV-negative Akata cells transfected with EBERs were analysed, PKR autophosphorylation *in vitro* was inhibited (Nanbo et al., 2002). However, Ruf reported that EBERs did posses a modest ability to protect the cell against interferon-induced apoptosis, but this process was independent of PKR-eIF-2α activation (Ruf et al., 2005). Thus Swaminathan suggested that EBERs might inhibit apoptosis while it was unlikely that inhibition of PKR was the primary mechanism for this effect (Swaminathan, 2010).

EBER-1 also interacts with the ribosomal protein L22, a componenet of the 60S eukaryotic ribosomal subunit unique to eukaryotes (Dobbelstein & Shenk, 1995; Toczyski et al., 1994; Toczyski & Steitz, 1991, 1993). In EBV-infected BL cells, roughly 50% of the cellular pool of L22 is found in association with EBER-1 ribonucleoprotein (RNP) particles, and a substantial fraction of L22 is physically relocalized from nucleoli to the nucleoplasm. Using the recombinant viruses and novel EBER expression vectors, the nuclear redistribution of rpL22 protein by EBER1 in 293 cells was confirmed (Gregorovic et al., 2011). Binding to 28S rRNA likely serves to target L22 to nucleoli, while binding to EBER-1 RNA likely results in sequestration or retention of L22 in the nucleoplasm. In truth, BL cells expressing mutated EBER-1 RNAs incapable of binding to and relocalizing L22 have significantly reduced capacity to enhance cell growth potential relative to BL cells expressing wild-type EBERs (Houmani et al., 2009), which indicated that the EBER1-L22 complex may be beneficial for lymphoma growth.

Pathologic Significance of EBV Encoded RNA in NPC 33

transformation efficiency. It should be noted that a different EBV strain background was

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

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

used in the two experiments.

derived from normal epitheliumand (Yoshizaki et al., 2007).

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 applies to NPC remains to be seen.
