Pathologic Significance of EBV Encoded RNA in NPC

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Pathologic Significance of EBV
Encoded RNA in NPC
Zhi Li, Lifang Yang and Lun-Quan Sun
Center for Molecular Medicine, Xiangya Hospital, Central South University, Changsha,
China
1. Introduction
The EBV-encoded RNAs (EBERs) are the most abundant EBV transcripts (about 107 copies
per cell) during latent infection by EBV in a variety of cells. Owing to its expression
abundance and universal existence in all of the 3 forms of latent infection, EBERs have been
under intensive studies since they were discovered by Lernar (Lerner et al., 1981) for the
first time. Looking back over the past 30 years, great efforts have been made to unveil the
accurate role of EBERs in the latency and transformation process, the definite secondary
structure and the signaling pathways they participate in. Despite significant achievements
were achieved in these fields, most pioneer work was conducted in lymphoma cells. Bearing
this in mind, we explore the similarities between lymphoma and carcinoma to fill the gaps
in our knowledge of EBERs’ roles in nasopharyngeal carcinoma (NPC). However, it remains
to be clarified whether the same scenario accurately applies to the pathological significance
of EBERs in NPC.
Epstein-Barr virus (EBV) is consistently detected in NPC from regions of both high and low
incidence. In EBV infected cells, there exist some polyribosomal virus-specific RNAs which
are the most abundant RNAs (Rymo, 1979). Initial transcription mapping studies by Kieff
and colleagues indicated that polyribosomal virus-specific RNA was encoded primarily by
the internal repeat region of EBV DNA and, to a lesser extent, by certain other regions of the
genome (Orellana & Kieff, 1977; Powell et al., 1979). Making use of cloned restriction
endonuclease fragments of EBV, Arrand discovered that the major cytoplasmic RNA in
these cells was specified by part of the EcoRI J fragment, which was consistent with Rymo’s
observation (Arrand & Rymo, 1982). Meanwhile, there were reports that revealed SLE
antibodies anti-La, but not the other sera tested, identified two new small RNAs, which
corresponded to the most actively transcribed portion of EBV DNA in Rymo’s investigation
and they were termed EBERs for the first time. In the following 1980’s, emphasis were put
on the structure, transcription regulation and the function of EBER-La complex. After these
preliminary explorations, intensive research was focused on the role of EBERs in the
oncogenesis of lymphoma, the involvement of EBERs in the process of lymphoblastoid cell
line (LCL) transformation and the potential anti-apoptosis response triggered by EBERs.
With these inspiring achievements, some scholars were intrigued by the autocrine growth of
several tumor cells and successfully discovered the link between cytokine induction and
EBERs in B and T lymphocyte, gastric carcinoma and nasopharyngeal carcinoma in the

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following decade. More recently, our knowledge has been deepened by unveiling the TLR3
and RIG-I signaling pathways induced by EBERs, which are responsible for the autocrine
growth of lymphomas and some EBV associated pathogenesis (Iwakiri et al., 2005; Samanta
et al., 2008). However, the accurate role of EBERs in the pathogenesis of NPC is still obscure.
There have been some contradictory reports with respect to the contribution of EBERs to the
oncogenesis of NPC and the relationship between EBERs and anti-apoptosis response. What
makes these dilemmas more complicated is the existence of EBERs in various stages of NPC.
Interestingly, expression of the EBERs seems to be down-regulated during differentiation.
Thus examples of NPC that have differing degrees of differentiation lack EBER expression
in differentiated areas (Pathmanathan et al., 1995). The EBERs are also not detected in the
permissive EBV infection, hairy leukoplakia, and are downregulated during viral replication
(Gilligan et al., 1990). Collecting the previous data together, despite that the EBERs have
been studied over 3 decades and some observations indicate they may play important roles
in the transformation of lymphoma (Yajima et al., 2005) and NPC (Yoshizaki et al., 2007), the
exact function of EBERs in NPC are still controversial.
2. Structure, transcription and clinical significance of EBERs
EBERs, the most abundant cytoplasmic RNA species identified in five lymphoid cell lines
and a Burkitt lymphoma biopsy, are encoded by the right-hand 1,000 base pairs of the EcoRI
J fragment of EBV DNA (Rosa et al., 1981). EBER1 is 166 (167) nucleotides long and EBER2 is
172 ± 1 nucleotides long with the heterogeneity resides at the 3' termini (Fig. 1). Striking
similarities are apparent both between the EBERs and the two adenovirus-associated RNAs,
VAI and VAII, and between the regions of the two viral genomes that specify these small
RNAs (Arrand et al., 1989). The EBER genes are separated by 161 base pairs and are
transcribed from the same DNA strand. Both EBER genes carry intragenic transcription
control regions A and B boxes which can be transcribed by RNA polymerase III (pol III).
However, both EBER1 and 2 contain upstream elements and TATA-like sequences typical of
polⅡ promoters including Sp1 and ATF binding sites (Howe & Shu, 1989). Within 1 kilo
base EBER region, 10 single base changes which group the strains into two families (1 and 2)
have been identified. The EBER1 sequences are completely conserved, two base changes are
within EBER2-coding sequence and eight are outside the coding regions (Arrand et al.,
1989). EBV has been shown to induce the cellular transcription factors TFIIIB and TFIIIC
(leading to induction of general pol III-mediated transcription) and the typical pol II
transcription factor ATF-2, that enhance expression of EBER1 and EBER2 (Felton-Edkins et
al., 2006), which may account for the low expression of transfected EBERs plasmids in EBV-
negative cells (Komano et al., 1999). To elucidate transcription regulation of EBERs more
exactly, Thomas J Owen discovered that transient expression of EBNA1 in Ad/AH cells
stably expressing the EBERs led to induction of both EBER1 and EBER2 through
transcription factors used by EBER genes, including TFIIIC, ATF-2 and c-Myc (Owen et al.,
2010). To shed more light on the transcription of EBERs, Hans Helmut Niller analyzed
protein binding at the EBER locus of EBV by genomic footprinting electrophoretic mobility
shift, reporter gene assay, and chromatin immunoprecipitation in a panel of six B-cell lines.
With these methods, 130 base pairs upstream of the EBER1 gene, contains two E-boxes
providing a consensus sequence for binding of the transcription factor and oncoprotein c-
Myc to the EBV genome. Translocated and deregulated c-myc directly activates and
maintains the antiapoptotic functions of the EBER locus in a single EBV-infected B cell

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which is undergoing the germinal center (GC) reaction. This single translocated and
surviving cell is the founder cell of an endemic BL, which accounts for the oncogenic role of
EBV in lymphoma (Niller et al., 2003). What’s more, Ferenc Banati found that in vitro
methylation blocked binding of the cellular proteins c-Myc and ATF to the 50-region of the
EBER-1 gene, which indicated a complicated transcription regulation of EBERs (Banati et al.,
2008). With the special transcription elements of EBERs, Choy had devised shRNA plasmid
to silence gene expression, which achieves better effect in some cases (Choy et al., 2008).

Fig. 1. Potential secondary structures of EBERs. The arrows indicate alternate 3' termini.
(A) EBER 1; (B) EBER 2. Adapted from Rosa et al. (1981)
EBER in situ hybridization is considered the gold standard for detecting and localizing
latent EBV in tissue samples (Ambinder & Mann, 1994). After all, EBER transcripts are
consistently expressed in virtually all the EBV positive tumors, and they are likewise
expressed in lymphoid tissues taken from patients with infectious mononucleosis, and in the
rare infected cell representing normal flora in healthy virus carriers. The only EBV-related
lesion that lacks EBERs is oral hairy leukoplakia, a purely lytic infection of oral epithelial

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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
infection (Kimura et al., 2009).
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 virus-
infected 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;

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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.