JOURNAL OF VIROLOGY, Mar. 2009, p. 2357–2367
Copyright © 2009, American Society for Microbiology. All Rights Reserved.
Vol. 83, No. 5
Comprehensive Profiling of Epstein-Barr Virus MicroRNAs in
Katherine Cosmopoulos,1Michiel Pegtel,2Jared Hawkins,1Howell Moffett,3,4Carl Novina,3,4
Jaap Middeldorp,2and David A. Thorley-Lawson1*
Department of Pathology, Tufts University School of Medicine, Jaharis Building, Boston, Massachusetts1; Department of Pathology,
Vrije Universiteit Medical Center, Amsterdam, The Netherlands2; Cancer Immunology and AIDS, Dana-Farber Cancer Institute,
Boston, Massachusetts3; and Department of Pathology, Harvard Medical School, Boston, Massachusetts4
Received 6 October 2008/Accepted 11 December 2008
Epstein-Barr Virus (EBV) establishes a long-term latent infection and is associated with a number of human
malignancies that are thought to arise from deregulation of different stages of the viral life cycle. Recently, a large
number of microRNAs (miRNAs) have been described for EBV, and it has been suggested that their expression may
vary between the different latency states found in normal and malignant tissue. To date, however, no technique has
been utilized to comprehensively and quantitatively test this idea by profiling expression of the EBV miRNAs in
primary infected tissues. We describe here a multiplex reverse transcription-PCR assay that allows the profiling of
39 of the 40 known mature EBV miRNAs from as little as 250 ng of RNA. With this approach, we present a
comprehensive profile of EBV miRNAs in primary nasopharyngeal carcinoma (NPC) tumors including estimates of
miRNA copy number per tumor cell. This is the first comprehensive profiling of EBV miRNAs in any EBV-
However, we confirm the hypothesis that the BHRF1 miRNAs are not expressed in NPC. Lastly, we demonstrate
that EBV miRNA expression in the widely used NPC line C666-1 is, with some caveats, broadly representative of
primary NPC tumors.
Epstein-Barr virus (EBV) is a ubiquitous human herpesvi-
rus. It infects ?90% of the human population, usually during
childhood, and persists for life. Persistent infection is almost
always benign; however, acute EBV infection causes infectious
mononucleosis in some individuals (18, 24). When B cells from
the blood of infected individuals are cultured they can give rise
to lymphoblastoid cell lines which are latently infected with
EBV and express nine latent proteins (latency III) (22, 26).
However, it was discovered that in vivo, latency III is a tran-
sient state the cell passes through on the way to persistence in
resting memory B cells where latent protein expression is ex-
tinguished (latency 0) (reviewed in reference 34). EBV is also
associated with several malignancies, including B-cell lympho-
mas (Burkitt’s lymphoma [BL], Hodgkin’s lymphoma [HL],
and immunoblastic lymphoma in the immunosuppressed) and
carcinomas (nasopharyngeal [NPC] and gastric [GC]) (re-
viewed in references 11 and 33. Viral infection in the tumors is
also latent but the infection is associated with more restricted
forms of latent gene expression (latency I in BL and latency II
in HL and NPC). However, when passaged in culture the
tumor cells tend to drift toward latency III (36, 40). This is
particularly true of BL-derived lines. Thus, the in vitro growth
of in vivo-infected B or tumor cells selects for expression of a
latency transcription program, latency III, that is not represen-
tative of the primary tissue. Therefore, in the EBV field it is
crucial to check whether the viral gene expression patterns
seen in cell lines faithfully represents what is seen in primary
In addition to the latent proteins, there are several untrans-
lated RNAs that appear to be present in all EBV-infected cells
studied thus far. These include two small RNAs (?170 bp),
termed EBER1 and EBER2, and a group of alternatively
spliced RNAs from the BamHI A fragment known as BARTs
(2, 4, 11, 15, 29, 31). The function of EBERs is not well
understood, but they may play a role in cell survival by increas-
ing the apoptotic threshold of infected cells (23). BARTs are
particularly abundant in the EBV-associated carcinomas and
encode a large number of microRNAs (miRNAs) (6, 9, 14, 16,
28, 31, 35).
miRNAs are 19 to 24 nucleotides in length and regulate
posttranscriptional gene expression by blocking translation or
causing the degradation of target mRNAs (reviewed in refer-
ences 12 and 17). These potent gene regulators are thought to
control up to one third of all genes and have received increased
attention due to their roles in a wide range of biological func-
tions, including differentiation, cell growth, and disease, espe-
cially cancer (reviewed in reference 13). With the exception of
ebv-mir-BART2, all of the BART-derived miRNAs map to
two clusters, including one novel miRNA cloned in our lab
named miR-BART22 (Fig. 1A) (6, 16, 28). A cluster of four
miRNAs have also been identified which are derived from the
BHRF1 gene (Fig. 1A) (28). This brings the total of known
mature EBV miRNAs to 40, dramatically increasing the num-
ber and complexity of potentially biologically active molecules
encoded by EBV during latent infection. However, like most
* Corresponding author. Mailing address: Department of Pathology,
Jaharis Building, Tufts University School of Medicine, 150 Harrison
Ave., Boston, MA 02111. Phone: (617) 636-2726. Fax: (617) 636-2990.
† Supplemental material for this article may be found at http://jvi
?Published ahead of print on 17 December 2008.
miRNAs discovered to date, the functions of the EBV
miRNAs are poorly understood. It has been shown that mir-
BART2 targets the EBV DNA polymerase BALF5 for degra-
dation, effectively inhibiting lytic replication (3). Cluster 1
BART miRNAs have been reported to downregulate the ex-
pression of the viral latent membrane protein 1 (LMP1) and
miRNAs from the BHRF1 region have been associated with
replication of the virus and regulation of the chemokine
CXCL-11 (21, 38).
Systematic profiling of cellular miRNAs has become an im-
portant tool in unearthing the roles of these molecules in
human cancer causation and in the development of miRNA-
based screens as diagnostic and prognostic cancer indicators
(20, 30). Given the strong association of EBV with human
cancer, systematic quantitative profiling of the EBV miRNA
expression patterns in EBV-associated diseases may help un-
earth their potential functions and their possible value as prog-
nostic and diagnostic tools.
The studies to date of EBV miRNA expression have used
Northern blotting, which is relatively insensitive and provides a
qualitative measure of expression levels (6, 19, 21, 38). A few
studies have analyzed small subsets of miRNAs in primary
infected cells, but the most detailed studies have used EBV-
infected cell lines to suggest differential expression of EBV
miRNAs in different latency states (6, 7, 10, 16, 19, 21, 27, 38,
39). Specifically, it has been proposed by Cai et al. that the
BART miRNAs are highly expressed in latency II, while the
BHRF1 miRNAs are highly expressed in latency III (6). How-
ever, Edwards et al. have reported that EBV miRNA expres-
sion is not cell type specific (10) and highlight the difficulties
and discrepancies that can arise when using EBV-infected cell
lines. Thus far, these issues have not been confirmed or tested
in a systematic way by comprehensive, quantitative profiling in
primary infected tissue. This is a central issue at this point
given the well-documented and powerful effect of tissue cul-
ture on latent protein expression. In the present study we
FIG. 1. Genomic locations of the reported EBV miRNAs and the PCR technique used to profile EBV miRNAs in NPC. (A) Schematic
representation of EBV miRNA locations within the EBV genome including a novel miRNA (miR-BART22) identified in our laboratory. The
location and promoters for latency genes expressed in NPC are shown in black. The lytic replication-associated gene BHRF1 and its promoter are
shown in white. All other latency genes and promoters are shown in gray. The BART miRNAs are grouped into two clusters based on their
locations. (B) Stem-loop primers specific for the 3? end of all EBV miRNAs are used to generate a pool of cDNA. A fraction of the total cDNA
generated is used to quantify individual EBV miRNAs using miRNA-specific forward primers, a universal reverse primer, and a TaqMan MGB
probe (labeled Q-F).
2358COSMOPOULOS ET AL. J. VIROL.
describe the first systematic profiling of EBV miRNA expres-
sion in a human cancer, as well as quantitative measurements
of their expression levels in biopsy samples and in cell lines.
The present study confirms, in primary tumors, some of the
predictions of Cai et al. (6) and provides the most complete
description of EBV miRNAs in primary samples to date.
MATERIALS AND METHODS
Patient samples. All biopsy samples were provided by the laboratory of Jaap
Middeldorp at Vrije University Medical Center (VUmc) in Amsterdam, The
Netherlands. Nasopharyngeal biopsy samples were collected under endoscope
guidance at the ENT Department of the Cipto Mangunkusumo Hospital
(Jakarta, Indonesia) as part of the routine clinical workup of patients suspected
of having NPC. All biopsy samples were taken before treatment and were
obtained with patients’ informed consent as part of an ongoing diagnostic mon-
itoring study of Marlinda Adham (32) as approved by the Dr. Cipto hospital.
Upon collection, biopsy samples were cut into two parts. One part was snap-
frozen in liquid nitrogen and stored at ?80°C until transport to VUmc. The other
part was fixed in buffered formalin for diagnostic histopathology. NPC diagnosis
was made by the local Pathology department and confirmed independently at
VUmc. EBV-positive undifferentiated NPC was confirmed on paraffin-embed-
ded biopsy material by EBER in situ hybridization with PNA-probes (Dako,
Denmark) and LMP1 immunohistochemical staining using OT21C and/or the
S12 mouse monoclonal antibody (Organon Teknika, The Netherlands).
Cell lines. C666-1 is an EBV-positive NPC-derived cell line. HONE-1 is an
EBV negative NPC-derived cell line that lost EBV after multiple passages.
HONE Akata is derived from HONE-1 by reinfection with the Akata strain of
EBV (HONE-1 and HONE Akata kindly provided by Ronald Glaser). Akata
2A8.1 (kindly provided by Jeffery Sample) and Rael are Burkitt’s lymphoma-
derived cell lines, and AT is a lymphoblastoid cell line derived in our laboratory.
HONE-1 and HONE Akata were cultured in Dulbecco modified Eagle medium
(Gibco). All other cell lines were grown in RPMI 1640 medium. Both media were
supplemented with 10% fetal calf serum, 2 mM sodium pyruvate, 2 mM L-
glutamine, 100 U of penicillin/ml, 100 U of streptomycin/ml, and 10 ?g of
ciproflaxin/ml. Complete Dulbecco modified Eagle medium was also supple-
mented with 2 mg of HEPES/ml (Gibco).
EBV latency gene PCRs. RNA from biopsy samples was isolated using the
RNABee protocol according to the manufacturer’s instructions (Qiagen) and
from cell lines using TRIzol (Invitrogen). The integrity of the RNA was assessed
with an Agilent Bioanalyzer and was considered intact based on the detection of
prominent bands corresponding to 18S and 28S rRNA (see Fig. 4). LMP1,
LMP2, and ENBA1 TaqMan real-time PCR primers and probes were generated
by Applied Biosystems and incorporated into Assays-on-Demand gene expres-
sion kits. All primers and probes are listed below. TaqMan ?-actin control
reagents (Applied Biosystems) were used as a loading control. Reverse tran-
scription (RT) reactions were performed by using the iScript cDNA synthesis
reagents kit (Bio-Rad), and all samples were DNase treated using DNAfree
reagents (Ambion) according to manufacturer’s instructions. The 20-?l PCRs
were incubated on a Bio-Rad MyIQ cycler at 95°C for 3 min, followed by 50
cycles of 95°C for 15 s and 60°C for 1 min. TaqMan PCR primers and probes for
EBV gene expression were as follows: EBER1 Fwd (5?-ACCGAAGACGGCA
GAAAGC-3?), EBER1 Rev (5?-CCTACGCTGCCCTAGAGGTTT-3?), and
EBER1 probe (5?-6FAM-ACAGACACCGTCCTCACCACCCG-TAMRA-3?);
LMP1 Fwd (5?-ACCACGACACACTGATGAACAC-3?), LMP1 Rev (5?-CTA
GAATCGTCGGTAGCTTGTTGA-3?), and LMP1 probe (5?-6FAM-ACTCCC
TCCCGCACCC-MGB-3?); LMP2 Fwd (5?-TTCTGGCTCTTCTGGGAACAC-
3?), LMP2 Rev (5?-GGCTCTTCATTAGATTCACGTTCCT-3?), and LMP2
probe (5?-6FAM-ACCCCACCGAACGAT-MGB-3?); and EBNA1 Fwd (5?-TG
AGTCGTCTCCCCTTTGGA-3?), EBNA1 Rev (5?-CCTTAGTGGGCCAGGT
TGTG-3?), and EBNA1 probe (5?-6FAM-ATGGCCCCTGGACCC-MGB-3?).
Multiplexed stem-loop RT-PCR. All EBV miRNA sequences were obtained
from the Sanger miRNA Registry (http://microrna.sanger.ac.uk/; see also Table
S1 in the supplemental material) and stem-loop RT primers were designed based
on these sequences as previously described (8). (See Table S1 in the supplemen-
tal material for a complete list of the miRNAs used in the present study and their
sequences.) Stem-loop RT primers for all EBV miRNAs were combined so that
the RT reaction contained a final concentration of 12.5 nM of each RT primer.
When a single RT primer was used, the final concentration was 50 nM. TaqMan
MicroRNA RT kit (Applied Biosystems) was used, and the reactions were
incubated according to kit instructions. The volume of the RT reaction was
adjusted according to the number of PCRs to be performed. The final input of
NPC biopsy RNA in RT reactions ranged from 250 to 350 ng. All RT reactions,
including a no-template control, were performed in duplicate. Real-time PCR
primers and probes were designed for each EBV miRNA as previously described
(8). (See Table S2 in the supplemental material for a complete list of the primer
and probe sequences used in the present study.) Each 10-?l miRNA PCR
included 1 ?l of RT product, 5 ?l of 2? IQ Supermix (Bio-Rad), 1.5 ?M forward
primer, 0.7 ?M universal reverse primer, and 0.2 ?M TaqMan probe (Applied
Biosystems). The samples were incubated on a Bio-Rad MyIQ cycler at 95°C for
3 min, followed by 40 cycles of 95°C for 15 s and 60°C for 30 s. All real-time PCRs
were performed from the same batch of RT product for each sample and were
performed in duplicate. TaqMan microRNA assay for RNU6b (Applied Biosys-
tems) was used as a loading control.
Calculation of miRNA copy number per cell. To calculate the copy number of
each miRNA per tumor cell, it is necessary to know how many cells were used to
produce the input RNA used for the PCRs. However, because of their small size
it is not possible to directly count the number of cells in the biopsy samples;
therefore, we derived an approach to estimate this. First, serial dilutions of the
EBV positive NPC cell line C666-1 were made, and RT-PCR for expression of
the cellular ?-actin gene and the two EBV latent genes EBER1 and LMP1 was
performed. From this, we could generate C666-1 calibration curves (cell number
versus RT-PCR signal) for all three genes. In parallel, RT-PCR for ?-actin,
EBER1 and LMP1 was performed on the same biopsy samples used to measure
EBV miRNA copy numbers. These RT-PCR signals and the C666-1 calibration
curves were then used to calculate the number of C666-1 cell equivalents in each
NPC biopsy sample. The actin analysis provides an estimate of the total cell input
into the miRNA PCRs. However, since NPC tumors have a large non-tumor
lymphoid infiltrate, we also used the EBER1 and LMP1 analysis to provide two
independent estimates of the total number of EBV positive, i.e., tumor, cells
used to make the RNA. Finally, we took the total miRNA copy number (see Fig.
5) and divided by the estimated cell input number derived from the C666-1
calibration curves to estimate the average miRNA copy number per cell in the
EBV miRNA profiling with multiplex RT-PCR. Nearly 40
mature miRNAs derived from the BHRF1 and BART regions of
the EBV genome have been described. In addition, we report a
novel, previously unpublished miRNA (miR-BART22) using the
miRNA cloning protocol described by Ambros et al. (1). Based
on the PCR protocol described by Chen et al. for detecting
miRNAs (8), we developed a sensitive and specific multiplex
PCR method to quantify these EBV miRNAs in the small
amounts of material available from clinical biopsy samples. This
technique makes use of a stem-loop primer for RT, followed by
TaqMan real-time PCR using miRNA-specific forward primers
and probes and a universal reverse primer specific for the con-
stant region of the stem-loop RT primer (Fig. 1B). Using these
specifications, we successfully designed specific primers and
probes for the novel EBV BART miRNA discovered in our lab
(miR-BART22) and 38 of 39 of the EBV miRNAs listed in the
Sanger Center miRNA database, miRBase, at the time of this
Sensitivity and specificity of the multiplex assay. To validate
the utility of our assay and discover the limits of its sensitivity,
we performed a number of control experiments. Synthetic oli-
gonucleotides representing all of the miRNAs were mixed at
known copy numbers and subjected to RT. Each miRNA was
analyzed using a fraction of the resulting cDNA. Figure 2
shows the sensitivity of two representative EBV miRNA PCRs.
With the exception of one PCR, miR-BART14, which was
excluded from further analysis, all of the PCR assays detected
miRNAs down to 10 copies of synthetic miRNAs and demon-
strated a linear relationship between copy number and PCR
cycle number down to 100 copies (for example, see Fig. 2A).
This held true whether the miRNAs were tested alone (not
VOL. 83, 2009EBV miRNAs IN NASOPHARYNGEAL CARCINOMA 2359
shown) or in mixtures containing all 39 miRNAs. In addition,
the sensitivity of the assay was not altered when the RT reac-
tion was carried out using pooled miRNA-specific RT primers
(All RT) compared to using a single miRNA-specific RT
primer (single RT) (Fig. 2B). Lastly, we compared the repro-
ducibility of detection when the oligonucleotides were as-
sayed alone or after spiking into a whole extract from EBV-
negative cells. Again, we observed no significant differences
(data not shown) indicating that all of the miRNAs were
efficiently recovered and that the low copy numbers found
for miR-BART2-3p for example, were not simply due to
inefficient recovery of that particular miRNA.
The assay was also highly specific. All PCRs were tested for
cross-reactivity by using the combined RT primers on a pool of
all of the oligonucleotides, including or excluding the synthetic
oligonucleotide for the miRNA to be tested. In all PCRs,
detection of the specific sequence was ?106-fold more sensi-
tive than for nonspecific/cross hybridizing sequences (data not
shown). In other words, a cross-reacting miRNA would need to
be present at a copy number of ?108to generate a cross-
reactive signal equivalent to 100 copies of the specific se-
Direct cloning by many groups has revealed single nucleo-
tide end polymorphisms for many miRNAs (37). Whether
these variants are real or artifacts introduced by the cloning
technique has not yet been determined. We were concerned
that this could affect the sensitivity of our assay, since it has
been shown that the efficiency of the stem-loop real-time PCR
may be decreased for variants (8, 37). We examined the effi-
ciency of the PCR over a range of copy numbers for each of the
nine EBV miRNAs for which variants have been listed in
miRBase. The results for two representative miRNAs are
shown in Fig. 3. In all cases the PCR efficiently detects variants
down to ?102copies. In only two cases, where the new variant
involved the deletion rather than the addition of a single nu-
cleotide, did the assay become less quantitative and then only
at copy numbers of ?103. Not surprisingly, when we examined
the EBV miRNA expression profile in C666-1 using variant
sequences as the standard controls, the profile did not change
significantly (data not shown). Therefore, this is only a poten-
tial concern for miRNAs present in biopsy samples at low copy
numbers such as miR-BART2-3p and miR-BART10*, al-
though at present no variants of these miRNAs have been
EBV miRNA profiles in NPC biopsy samples using multi-
plex RT-PCR. Having established the sensitivity and specificity
FIG. 2. Sensitivity of quantitative multiplex RT-PCR for EBV miRNAs. (A) Synthetic miRNAs were used at copy numbers ranging from 107
to 102to determine the sensitivity of each of the 40 EBV miRNA-specific PCRs and as standard controls for the quantification of the EBV miRNAs
in NPC biopsy samples. (B) PCR was performed using either a single RT primer specific for the miRNA under analysis (Single RT) or all of the
EBV miRNA-specific RT primers (All RT) on pooled oligonucleotides for all of the miRNAs. The figure shows representative results for two
miRNAs: mir-BHRF1-2*(upper panels) and mir-BART1-5p (lower panels).
2360 COSMOPOULOS ET AL.J. VIROL.
of the multiplex RT-PCR, we then used it to perform a com-
prehensive survey of EBV miRNA expression in NPC biopsy
samples. We selected five samples with a confirmed diagnosis
of NPC that demonstrated intact RNA (Fig. 4A) and were
determined to be EBV positive by EBER in situ hybridization
(data not shown) and immunohistochemical staining of LMP1
(Fig. 4B). Of the five biopsy samples, four had sufficient
amounts of RNA to confirm the expected profile of EBV latent
gene expression. We tested these samples for the expression of
the EBV small RNA EBER1 and the latent proteins LMP1,
LMP2, and EBNA1Q-K by RT-PCR analysis (Fig. 4C).
To determine the EBV miRNA copy number in NPC tumor
biopsy samples the RT-PCR signal for each miRNA was com-
pared to a titration of known quantities of the appropriate
synthetic miRNA oligonucleotides. The results for all five bi-
opsy samples were essentially the same, and the data are sum-
marized in Fig. 5. All of the BART miRNAs were detectable in
the NPC biopsy samples, including miR-BART22 cloned in
our lab (Fig. 5B, C, and D). The copy numbers of the BART
miRNAs in each biopsy sample ranged from ?102(miR-
BART2-3p) to ?106(miR-BART17-3p), and we observed a
larger degree of variability within the cluster 2 miRNAs than
within the cluster 1 miRNAs. In contrast, the BHRF1 miRNAs
were not detected in any of the five biopsy samples (Fig. 5A).
As negative controls, we used six EBV-negative biopsy samples
and the EBV-negative NPC cell line HONE-1 and consistently
failed to detect the EBV miRNAs (data not shown).
To compare the EBV miRNA expression profiles between
samples, we normalized the BART miRNA copy numbers in
each sample to the cell-derived U6 snRNA. We then calcu-
lated the amount of each miRNA relative to an arbitrary
miRNA from the profile with a low copy number, miR-
BART10* (Fig. 6). Overall, the profiles of the miRNAs for all
of the NPC biopsy samples were strikingly similar. We con-
clude therefore that there is a consistent EBV miRNA expres-
sion profile associated with NPC tumors characterized by the
absence of the BHRF1 miRNAs and highly variable expression
of the BART miRNAs.
BHRF1-derived miRNAs are absent from NPC tumor cells
but not cell lines. It has been proposed previously, based on
studies in cell lines by Northern blotting, that the BHRF1-
derived miRNAs are absent from NPC (6). Consistent with
this, we found that the BHRF1-derived miRNAs were unde-
tectable by our technique in the NPC biopsy material (Fig. 5
and 6). Our inability to detect the BHRF1 miRNAs in biopsy
material was not due to a failure of our assay, since we were
readily able to detect the BHRF1 miRNAs in collection of cell
lines that we tested (Fig. 7). These included the EBV-positive
NPC-derived epithelial cell lines studied previously (C666-1
and HONE Akata) that express latency II, a B-cell line ex-
pressing latency III (AT), and a B-cell line expressing latency
I (Rael). In passing, it is noteworthy that we observed no
obvious correlation between latency stage and BHRF1 miRNA
expression in the cell lines.
The detection of BHRF1 miRNAs in the NPC cell lines
might suggest that they are not representative of the tumors in
terms of BHRF1 expression. However, estimates of per-cell
expression in the NPC lines indicated that all four BHRF1-
derived miRNAs were present in very low copy numbers (?1
copy per cell, Fig. 7) similar to that of the least-abundant
BART-derived miRNAs. It is unlikely that these miRNAs are
present in sufficient abundance to affect the behavior of at least
the vast bulk of the cells in the C666-1 cell line.
Since there were approximately 103to 104cell equivalents in
the biopsy PCRs (based on EBER or ?-actin levels [see be-
low]) and our PCRs can detect at least 10 copies of the
miRNAs, we can estimate that the BHRF1 miRNAs were
present at ?0.001 to ?0.01 copies per tumor cell in the biopsy
samples. This result highlights the sensitivity and validates the
utility of our approach in allowing us to definitively conclude
that the BHRF1 miRNAs were not present in the five NPC
tumor biopsy samples we have studied. We conclude, there-
fore, that the prediction of Cai et al. (6) that BHRF1 miRNAs
are not expressed in NPC is true for tumor cells but not for the
NPC-derived cell lines.
miRNA expression in the NPC cell line C666-1. The EBV-
positive NPC cell line C666-1 has been widely used in NPC
studies, including analysis of miRNA expression (6, 10, 21).
Therefore, it is important to verify that EBV miRNA expres-
sion in C666-1 cells faithfully represents primary NPC biopsy
samples. We have already shown that, unlike the biopsy sam-
ples, the BHRF1 miRNAs were readily detectable in C666-1,
albeit at low levels (Fig. 7). However, the overall BART
miRNA expression profile in C666-1 was similar to the average
FIG. 3. miRNA polymorphism does not affect PCR efficiency at high copy numbers. The efficiency of PCRs at detecting the sequences for which
the primers and probes were originally designed and their variants was compared. Representative data are shown for the seven known variants that
did not affect the PCR efficiency over the range of copy numbers tested (A) and the two that did at low copy numbers (B).
VOL. 83, 2009 EBV miRNAs IN NASOPHARYNGEAL CARCINOMA2361
profile for the NPC biopsy samples (Fig. 8) with some notable
exceptions. For example, miR-BART2-3p and miR-BART16
were present in C666-1 at ?10% of the level seen in the biopsy
materials. Determining whether these differences have func-
tional implications will have to await description of their func-
tions and their profiling in other infected cell types.
In conclusion, our analysis reveals that that there is a repro-
ducible EBV miRNA profile found in NPC tumors and that for
most, but not all, miRNAs the profile in the C666-1 cell line is
representative of NPC tumors.
Copy numbers of BART miRNAs in NPC biopsy tumor cells.
It has been reported that BART miRNAs are present at high
copy numbers in NPC cell lines compared to B-cell lines (6),
but this observation remains to be confirmed in primary tu-
mors. To calculate miRNA copy number per cell in the pri-
mary tumors, we need to know the number of input tumor cells
used to make the RNA from each biopsy sample. However, it
is not possible to directly determine this due to the small size
and heterogeneity of NPC biopsy samples and the small
amount of RNA obtained. Therefore, we developed an ap-
proach that would allow us to estimate this (see Materials and
Methods for details). Briefly, we used the NPC-derived cell
line C666-1 to generate calibration curves for the total cell
number versus RT-PCR signal for the cellular gene ?-actin and
the two EBV genes EBER 1 and LMP1. We then performed
RT-PCR for EBER1, LMP1, and ?-actin on the biopsy sam-
ples. The resulting RT-PCR signals, combined with the C666-1
calibration curves, were then used to estimate the average
number of input cells (based on beta actin) or tumor cells
(based on EBER or LMP1) from each biopsy sample used in
FIG. 4. RNA integrity and EBV gene expression of NPC biopsy samples. (A) RNA integrity of the five EBV-positive NPC biopsy samples used
in the present study, analyzed with an Agilent Bioanalyzer. The ribosomal bands are labeled. Their presence as strong discrete bands indicates that
the RNA is intact. (B) An example of an NPC biopsy sample, NPC2, that was diagnosed as containing tumor cells by keratin staining and confirmed
as EBV positive with LMP1 staining. (C) The presence of EBV in the biopsy samples was confirmed using RT-PCR for the EBV-encoded small
RNA EBER1 and the latent genes LMP1, LMP2, and EBNA1. The data are expressed as the estimated number of C666-1 cell equivalents used
in the PCRs for each biopsy sample. This was calculated for all four PCRs by comparing the signal from each biopsy sample to those obtained with
known numbers of the NPC-derived cell line C666-1. NPC4 is excluded from this analysis due to insufficient RNA. †, EBNA1 was not detected
2362 COSMOPOULOS ET AL.J. VIROL.
the miRNA PCRs. From this value and the data shown in Fig.
5 we calculated the copy number of each miRNA per cell (Fig.
9). The values were consistently ?10-fold lower for the EBER
than for the LMP1 or actin-based calculations, suggesting a
disparity in the relative copy numbers of EBER and LMP1
RNAs between C666-1 and the tumors. Nevertheless, some
general conclusions can be drawn. First, it was apparent that
the BART miRNAs were present in the biopsy samples in a
wide range of copy numbers with most in the range of ?1 ?
102to ?5 ? 104copies. However, some (e.g., miR-BART2-3p,
miR-BART7*, and miR-BART10*) were only present at
about 1 to 10 copies per cell. Whether this represents a small
copy number in every cell or high copy number in a small
number of cells cannot be resolved by our technique. Second,
it was apparent that the average copy numbers in the biopsy
samples was very similar to that in the C666-1 line, again
confirming that this cell line is generally representative of bi-
opsy samples in terms of BART miRNA expression. However,
when we compared copy numbers to those in a collection of
other cell lines we were unable to discern a trend whereby
some or all of the BART miRNAs were more highly expressed
in NPC (data not shown). The cell lines included a second
EBV-positive NPC-derived epithelial cell (HONE Akata), a
B-cell line transformed in vitro with EBV and expressing la-
tency III (AT), and a B-cell line expressing the EBNA1 only
program characteristically expressed in Burkitt’s lymphoma
In conclusion, our analysis demonstrates that the prediction
of Cai et al. (6) that BART miRNAs are expressed at high copy
numbers in NPC tumors was true for most but not all of the
miRNAs. However, we were unable to confirm that BART
miRNAs are more highly expressed in NPC cell lines than in
infected B-cell lines expressing other latency programs.
Whether this will hold true for infected cells in vivo or the
B-cell tumors themselves requires a comprehensive compara-
tive analysis of miRNA expression in those tissues.
In this study we have established a quantitative multiplex
PCR technique to profile 39 of the 40 mature EBV-encoded
miRNAs known to date and used it to describe the profile for
NPC tumor biopsy samples. This is the first time that a com-
prehensive, quantitative profile of EBV miRNAs has been
described for any EBV-infected cell type and is the first pro-
filing of EBV miRNAs for any EBV-associated cancer. In
addition, we were able to determine the profile with as little as
250 ng of sample RNA. The sensitivity and specificity of this
technique make it ideal for screening the small amounts of
material available from tumor biopsy samples. One concern
with analysis of biopsy material is that the integrity of the RNA
extracted from them may be compromised. In addition to the
five biopsy samples reported here we have tested five other
NPC biopsy samples where the RNA showed varying levels of
degradation and the expression profiles were essentially indis-
tinguishable from those obtained with intact biopsy RNA (data
not shown). Thus, the technique remains robust even with
samples containing highly degraded RNA, a point of consid-
erable practical importance for analysis of biopsy material that
FIG. 5. Quantitative analysis of BART miRNAs in five NPC biopsy samples. BART miRNAs were quantified using synthetic miRNAs as
standard controls. The data shown are the total number of copies in the NPC biopsy RNA used in the PCR. (A) ebv-miR-BHRF1; (B) ebv-miR-
BART2; (C) ebv-miR-BART cluster 1; (D) ebv-miR-BART cluster 2. †, The ebv-miR-BHRF1 miRNAs were not detected in any of the biopsy
samples.*, The dominant miRNA when two are derived from the same stem-loop precursor.
VOL. 83, 2009 EBV miRNAs IN NASOPHARYNGEAL CARCINOMA2363
may have been collected or stored under suboptimal condi-
Previous studies have made predictions about differential
EBV miRNA expression based on studies in cell lines(6). How-
ever, it is well known that the pattern of EBV latent protein
expression is strongly affected by in vitro culture. The classic
example was the demonstration that BL tumors only express
EBNA1 but upon tissue culture drift to express all of the nine
known latent proteins (36, 40). Similarly, with in vitro infection
the virus persists in proliferating blasts that express all of the
latent proteins but in vivo it persists in resting cells that
express no viral proteins (reviewed in reference 34). It is
essential, therefore, to confirm that cell lines are represen-
tative of the primary tissue. In the present study we have
confirmed the hypothesis of Cai et al. (6) that NPC does not
express the BHRF1 miRNAs; however, we could not confirm
their claim that all of the BART miRNAs are present at high
copy numbers. It is likely that their inability to detect BHRF1
miRNAs in NPC cell lines was a function of lack of sensitivity
not because the BHRF1 miRNAs are truly absent.
Profiling of miRNA expression is currently an active area of
research in an attempt to correlate individual miRNAs with
specific biological states or functions and with disease, espe-
cially cancer (13). Several approaches are used, including di-
FIG. 6. EBV miRNA profile in NPC. BART miRNAs were quantified using synthetic miRNAs as standard controls and normalized to
expression of the ubiquitous small nucleolar RNA RNU6b. The level of each miRNA is shown relative to the expression of miR-BART10*.
(A) ebv-miR-BHRF1; (B) ebv-miR-BART2; (C) ebv-miR-BART cluster 1; (D) ebv-miR-BART cluster 2. †, ebv-miR-BHRF1 miRNAs were not
detected in any of the biopsy samples.*, The dominant miRNA when two are derived from the same stem-loop precursor.
FIG. 7. BHRF1 miRNAs are expressed in cell lines. BHRF1 miRNAs were quantified using synthetic miRNAs as standard controls. Copy
numbers of BHRF1 miRNAs per 104cells of the NPC cell lines C666-1 and Hone Akata, the Burkitt’s lymphoma-derived lines 2A8.1 and Rael,
and the lymphoblastoid cell line AT are shown. †, Not detected.
2364 COSMOPOULOS ET AL.J. VIROL.
rect cloning, microarray, and PCR, and each approach has its
strengths and drawbacks. Wu et al. demonstrated that while
each method differed slightly, the general observations made
with all three methods correlated with one another (37). The
main limitation of the stem-loop RT-PCR approach used in
the present study arises due to polymorphisms that may occur
at the 3? end of the miRNAs. The significance of these varia-
tions remains unclear. One possibility is that they are cloning
artifacts, but it has also been suggested that they provide a
mechanism for increasing the diversity of miRNA targets (37).
Until we know more about the targets of the miRNAs and
their variants, we will not know the significance of these end
polymorphisms. One problem with variants is that they could
affect the efficiency of the PCR used in our assay. For the nine
EBV miRNAs for which variants have been listed in miRBase,
decreased efficiency of PCR does not seem to be a concern in
our assays. This provides confidence that the profile we have
described is as accurate as possible given the state of the field.
Furthermore, given the limited amount of material available
from biopsies, our technique presents a practical approach to
comprehensive profiling. Ultimately, the goal would be to con-
firm differences detected by multiplexed PCR either by mi-
croarray or based on functional assignations.
Several studies have used direct cloning and Northern blots
to identify the known EBV miRNAs and to examine EBV
miRNA expression in different cell types (6, 10, 19, 21, 28, 39).
However, Northern blot analysis provides a qualitative mea-
sure of relative levels of miRNA expression, and the high
amount of RNA needed makes it prohibitive for profiling
multiple miRNAs, especially for samples with limited amounts
of RNA such as biopsy samples. Thus, if a miRNA is not
detected using Northern blotting, it is not possible to deter-
mine whether it is truly absent or if it is below the limit of
detection. By comparison the multiplex PCR technique used in
the present study is quantitative over a wide range of miRNA
copy numbers and is highly sensitive. It can routinely measure
as few as 10 copies of any given miRNA and the profile for all
39 miRNAs can be derived from quantities of RNA far lower
FIG. 8. Comparison of BART miRNA expression in NPC biopsy samples and the NPC-derived cell line C666-1. The average BART miRNA
profiles of the five NPC samples from Fig. 6 are shown compared to BART miRNA expression in the NPC cell line C666-1. Analysis was performed
as in Fig. 6.
FIG. 9. Estimated copies of EBV miRNAs per cell in NPC biopsy samples. The average copy number per cell for each BART miRNA was
calculated and compared to the NPC cell line C666-1. RT-PCR was used to estimate EBV-positive (EBER1 or LMP1) or total cells (beta actin)
in each biopsy sample based on the EBER1, LMP1, or actin RT-PCR signals from known numbers of C666-1 cells (for details see Materials and
Methods). The copy numbers for each miRNA (Fig. 5) were then divided by the estimated cell number to derive the estimated copy number per
C666-1 cell equivalents in each NPC biopsy sample. The values obtained for the five NPC biopsy samples were averaged, and the mean values,
with the standard deviations, are plotted.
VOL. 83, 2009EBV miRNAs IN NASOPHARYNGEAL CARCINOMA 2365
than the amounts required for Northern blots. A simple cal-
culation makes this immediately apparent. From an NPC nee-
dle biopsy sample (about the size of a rice grain) one can
typically obtain about 106cells or about 2 ?g of RNA. Typi-
cally, a single Northern blot to detect EBV miRNAs requires
from 5 to 30 ?g of RNA (6, 10). Therefore, at best it might be
possible to perform one blot for a single EBV miRNA with
RNA from a biopsy sample. Stripping and reprobing the blot
might increase this to four miRNAs but in order to profile 39
miRNAs by Northern blot it would require 50 to 300 ?g of
RNA, an impossibility with biopsy material. In comparison, as
described in Materials and Methods, with our technique we
could perform PCRs for all 39 EBV miRNAs in duplicate with
250 to 350 ng of RNA, i.e., significantly less than that available
from a biopsy sample. Furthermore, for the BART miRNAs
we are well in the linear range of detection for our samples,
and so for these we could easily have profiled tenfold less
material, i.e., ?25 ng. Therefore, our technique is at least a
thousandfold more sensitive than Northern blotting. This
makes our approach ideal for small or rare samples. The sen-
sitivity of this assay allowed us to detect miRNAs such as the
BHRF1 miRNAs and miR-BART8 that have previously been
reported to be undetectable by Northern blot in the NPC cell
line C666-1 (6, 10). Because of the quantitative nature of the
technique, we are able to make definitive statements about
miRNA expression levels within samples. Thus, we may con-
clude that the BHRF1 miRNAs were not present in the five
NPC tumor biopsy samples we have studied but miR-BART8
was expressed. In addition, we are able to normalize the
miRNA expression using the internal control U6 and other
markers, making it possible to compare miRNA profiles be-
The different EBV latency states, as defined by variable
patterns of latent protein expression, are associated with dif-
ferent tumors, and it has been hypothesized that this may also
hold true for miRNA expression in vivo. Using the multiplex
approach it is now possible to rigorously test this idea by
performing comprehensive, quantitative EBV miRNA profil-
ing on other EBV-infected normal and malignant tissues. This
may also provide insights into the possible functions of these
There have been limited studies linking EBV miRNAs to
biological functions. It has been claimed previously that the
BHRF1 miRNAs are only readily detectable in B cells infected
with EBV expressing the growth program (5). In addition, it
has been proposed that they are associated with viral replica-
tion, and it has been shown that BHRF1 miRNA expression
increases upon induction of viral replication (39). Our results
demonstrate that these miRNAs are expressed in a wide range
of cell lines, including the Rael (latency I) C666-1 (latency II)
and AT (latency III), but are absent from the five fresh NPC
tumor biopsy samples we have studied here. Since NPC tumors
are known to not express the growth program and to be tightly
latent, the low expression profile of BHRF1 miRNAs in the
C666-1 cell line is likely due either to spontaneous reactivation
of the virus in a small subset of cells or the drifting of a few
cells toward the growth program in culture (25, 39). Either
way, our data show that NPC tumors are indeed very tightly
latent as judged by BHRF1 miRNA expression.
In summary, this work provides a quantitative analysis of the
EBV miRNA expression profile in five biopsy samples of the
EBV-associated cancer NPC. It will be interesting to see the
evolution of the profile as additional data emerge for EBV
miRNA expression using alternative methods, such as microar-
ray analysis. This study is the first step in defining EBV miRNA
profiles in cancer and, as more targets are identified may aid in
the discovery of the role that EBV plays in oncogenesis.
This study was supported by Public Health Service grants R01
CA65883, R01 AI18757, and RO1 AI062989 to D.A.T.-L.
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