JOURNAL OF VIROLOGY, Feb. 2008, p. 1946–1958
Copyright © 2008, American Society for Microbiology. All Rights Reserved.
Vol. 82, No. 4
Epstein-Barr Virus Latent Membrane Protein 1 Induces Cellular
MicroRNA miR-146a, a Modulator of Lymphocyte
Jennifer E. Cameron,1,2Qinyan Yin,1,2,3Claire Fewell,3Michelle Lacey,4Jane McBride,3Xia Wang,3
Zhen Lin,3Brian C. Schaefer,5and Erik K. Flemington1,2,3*
Louisiana Cancer Research Consortium,1Tulane Cancer Center,2Department of Pathology,3and Department of Mathematics,4
Tulane University, New Orleans, Louisiana, and Department of Microbiology and Immunology,
Uniformed Services University of the Health Sciences Center, Bethesda, Maryland5
Received 27 September 2007/Accepted 20 November 2007
The Epstein-Barr virus (EBV)-encoded latent membrane protein 1 (LMP1) is a functional homologue of the
tumor necrosis factor receptor family and contributes substantially to the oncogenic potential of EBV through
activation of nuclear factor ?B (NF-?B). MicroRNAs (miRNAs) are a class of small RNA molecules that are
involved in the regulation of cellular processes such as growth, development, and apoptosis and have recently
been linked to cancer phenotypes. Through miRNA microarray analysis, we demonstrate that LMP1 dysregu-
lates the expression of several cellular miRNAs, including the most highly regulated of these, miR-146a.
Quantitative reverse transcription-PCR analysis confirmed induced expression of miR-146a by LMP1. Analysis
of miR-146a expression in EBV latency type III and type I cell lines revealed substantial expression of
miR-146a in type III (which express LMP1) but not in type I cell lines. Reporter studies demonstrated that
LMP1 induces miR-146a predominantly through two NF-?B binding sites in the miR-146a promoter and
identified a role for an Oct-1 site in conferring basal and induced expression. Array analysis of cellular mRNAs
expressed in Akata cells transduced with an miR-146a-expressing retrovirus identified genes that are directly
or indirectly regulated by miR-146a, including a group of interferon-responsive genes that are inhibited by
miR-146a. Since miR-146a is known to be induced by agents that activate the interferon response pathway
(including LMP1), these results suggest that miR-146a functions in a negative feedback loop to modulate the
intensity and/or duration of the interferon response.
The Epstein-Barr virus (EBV) infects over 90% of the human
population worldwide. Like other members of the gammaherpes-
virus family, EBV typically establishes a lifelong, predominantly
asymptomatic infection in its host (20). Nevertheless, persistent
EBV infection can result in a number of malignancies, including
Burkitt’s lymphoma, Hodgkin’s lymphoma, nasopharyngeal car-
cinoma, and lymphoproliferative diseases in immunosuppressed
individuals (34). EBV oncogenesis is principally associated with
viral genes is transcribed. Latency gene expression is classified
into three groups. In cells exhibiting latency type I, the EBV
nuclear protein EBNA1 is the only viral protein-coding gene that
is expressed. Latency type II is characterized by expression of
EBNA1 as well as latent membrane proteins (LMPs) 1, 2A, and
2B. Type III latency cells express the entire repertoire of latency-
associated EBV gene products. Six of these encode nuclear pro-
teins (EBNA1, EBNA2, EBNA3A, EBNA3B, EBNA3C, and
EBNA-LP), and three encode membrane proteins (LMP1,
LMP2A, and LMP2B) (20). In addition to these protein-coding
genes, the noncoding RNAs EBER1 and EBER2 and a set of
EBV encoded microRNAs (miRNAs) are expressed in all three
forms of latency (34).
Of the viral latency genes expressed in EBV-associated malig-
nancies, LMP1 is the most highly implicated in tumor formation
(for a review, see reference 42). First, although different EBV-
associated tumors exhibit distinct latency gene expression pat-
terns, LMP1 is expressed in all but Burkitt’s lymphoma. Second,
LMP1 is required for EBV-mediated transformation of lympho-
cytes in vitro, and it has the ability to transform rodent fibroblasts.
Finally, transgenic mice expressing LMP1 from an immunoglob-
ulin promoter develop B-lymphocyte tumors more frequently
than nontransgenic mice (22).
LMP1 is a six-transmembrane, constitutively active signaling
molecule that functionally mimics cellular tumor necrosis factor
receptor (TNFR) family members. While the transmembrane
domain is required for aggregation and constitutive activation of
LMP1 (11, 12, 24), two cytoplasmic domains, referred to as C-
terminal activator regions 1 and 2 (CTAR-1 and CTAR-2), have
(17–19). Together, these signaling domains act through cellular
TNFR-associated factors (TRAFs) and other cell signaling mol-
ecules to activate transcription factors, including nuclear factor
?B (NF-?B), activator transcription factor 2 (via p38 mitogen-
activated protein kinase), and AP-1 (via c-Jun N-terminal kinase)
(3, 6, 8–10, 16–18, 29). Through the hijacking of these cell signal-
ing pathways, LMP1 is able to manipulate host cellular processes
that regulate cell proliferation, migration, and apoptosis and
thereby contribute to cellular immortalization and tumorigenesis.
* Corresponding author. Mailing address: Tulane University Health
Sciences Center, 1430 Tulane Ave., SL79, New Orleans, LA 70112.
Phone: (504) 988-1167. Fax: (504) 988-5516. E-mail: eflemin@tulane
† Supplemental material for this article may be found at http://jvi
?Published ahead of print on 5 December 2007.
Among the pathways regulated by LMP1 is the interferon (IFN)
type I (alpha/beta) pathway, which is induced in part through the
induction of IFN regulatory factor 7 (IRF7) (15, 31).
Recently, a novel class of small (?22-nucleotide) RNA mol-
ecules referred to as miRNAs have been identified as impor-
tant regulators of a broad array of cell processes in mammals,
including proliferation, differentiation, and apoptosis. Micro-
RNAs function predominantly through the inhibition of
mRNA translation via imperfect complementary sequence rec-
ognition in the 3? untranslated region (UTR) of target mRNA
transcripts. The binding of microRNA/protein complexes to
the 3? untranslated regions (and less frequently the 5? untrans-
lated or coding sequence) causes transport to a perinuclear
compartment referred to as GW or P bodies, thereby seques-
tering the mRNA away from the Golgi apparatus (25–27). In
this compartment it is thought that at least some of these
mRNAs can then be subjected to degradation (25, 26).
MicroRNAs have been implicated in regulation of immune
responses. The BIC transcript, which is upregulated in B-cell
lymphomas, including EBV-associated lymphoproliferative
diseases, encodes miR-155. Knockout mice deficient in BIC/
miR-155 were unable to generate a protective immune re-
sponse in a Salmonella postimmunization challenge model and
were impaired in antibody production, class switching, and
production of interleukin-2 (IL-2) and IFN-? when immunized
with tetanus toxin fragment C (35). Interestingly, miR-155
transgenic mice developed B-cell lymphomas (5), suggesting
that some miRNAs that are critical to normal cell functions
may have oncogenic potential when inappropriately expressed.
Regulation of innate immune responses by miRNAs has also
been demonstrated. Toll-like receptor (TLR) signaling in
monocytes induced miR-155 as well as miR-132 and miR-146a
expression (37). Further exploration of the response of miR-
146a in innate immune pathways demonstrated that miR-146a
is induced by multiple TLRs recognizing bacterial stimuli, in-
cluding lipopolysaccharide, but not those that recognize viral
stimuli (37). Interestingly, Taganov et al. identified TRAF6
and IL-1 receptor-associated kinase 1 (IRAK1) as two poten-
tial targets of miR-146a using 3? UTR reporter analysis (37).
Since these two factors are key mediators of TLR and IL-1
signaling pathways, they suggested the possibility that miR-
146a can inhibit endogenous TRAF6 and IRAK1 expression
and thereby feedback to inhibit TLR and IL-1 signaling
LMP1 has been shown to regulate a large number of mRNA
transcripts that likely play roles in the life cycle of the virus (4).
Since LMP1 predominantly influences the cell through activa-
tion of transcription, we assessed whether LMP1 alters the
expression of miRNAs that play a role in LMP1-regulated
pathways. Through microarray analysis, we identified cellular
miRNA species that are regulated by LMP1. While this anal-
ysis revealed the LMP1-mediated induction of miR-146a,
changes in other miRNAs shown previously to be induced by
TLR signaling were not observed, suggesting distinctions
between these signaling pathways leading to microRNA sig-
naling. We also provide evidence that miR-146a negatively
modulates the interferon response pathway, which may be a
component of a negative feedback loop.
MATERIALS AND METHODS
Maintenance of cell lines. Lymphocyte cell lines (IB4, X50-7, Rael, Akata, JY,
JC5, P3HR1, Mutu, and BL30) were maintained in RPMI medium plus 10%
fetal calf serum and 0.5% penicillin-streptomycin. Epithelial cell lines (HEK293
and A549) were maintained in Dulbecco’s modified Eagle’s medium (DMEM)
plus 10% fetal calf serum and 0.5% penicillin-streptomycin. Retrovirally trans-
duced EBNA-1 dominant negative (EBV-negative) Mutu clone 3 cells were
maintained in RPMI medium plus 10% fetal calf serum, 0.5% penicillin-strep-
tomycin, 1 ?g/ml puromycin, and 1 mg/ml gentamicin. The same selective me-
dium that was used for the EBV-negative Mutu clone 3 cells was used for Mutu
clone 3 cells transduced with pEHyg or pEHyg-FLAG-LMP1 retroviruses, with
the addition of 250 ?g/ml hygromycin.
Plasmid cloning. To generate dominant negative forms of EBNA1, full-length
EBNA1 was first excised from the plasmid pCEP4 with ClaI and SacII. Since the
SacII digestion cleaves the last few carboxy-terminal amino acids from the
EBNA1 reading frame, two adaptors were synthesized which together encode
the terminal amino acids from EBNA1 plus a BglII overhang (Adaptor 1-sense,
5?-GGAGGGTGATGACGGAGATGACGGAGATGAA-3?; Adaptor 1-anti-
C-3?; Adaptor 2-sense, 5?-GGAGGTGATGGAGATGAGGGTGAGGAAGG
GCAGGAGTGA-3?; Adaptor 2-antisense, 5?-GATCTCACTCCTGCCCTTCC
TCACCCTCATCTC-3?). The ClaI/SacII EBNA1 fragment was ligated to an
EcoRI- and BglII-cut pMSCV-neo vector in the presence of Adaptor 1 plus
Adaptor 2 and an EcoRI/ClaI adaptor. Ligations were carried out in the pres-
ence of 1 ?l T4 polynucleotide kinase to phosphorylate adaptors during the
ligation reaction. This construction led to the generation of pMSCV-neo-
EBNA1wt. Next, pMSCV-neo-E1dn was generated from this plasmid by digest-
ing pMSCV-neo-EBNA1wt with EcoRI and ApaI to excise the amino-terminal
EBNA1 sequences. The resulting vector plus carboxyl-terminal EBNA1 se-
quences were then ligated to three adaptors which, when linked together, contain
a translation initiation sequence, an hemagglutinin epitope tag, and the simian
virus 40 nuclear localization signal, plus an EcoRI overhang at the 5? end and an
ApaI overhang at the 3? end (DN adaptor 1-sense, 5?-AATTCTGCTGAAGAT
GATGGCCTATCCTTATG-3?; DN adaptor 1-antisense, 5?-CATCATCTTCA
GCAG-3?; DN adaptor 2-sense, 5?-ATGTGCCTGACTATGCCGCCCCAAAG
AAA-3?; DN adaptor 2-antisense, 5?-CATAGTCAGGCACATCATAAGGAT
AGGC-3?; DN adaptor 3-sense, 5?-AAGCGAAAGGTGGCCGGCC-3?; DN
adaptor 3-antisense, 5?-GGCCACCTTTCGCTTTTTCTTTGGGGCGG-3?). Li-
gations were carried out in a standard ligation reaction mixture except that 1 ?l
of T4 polynucleotide kinase was added to phosphorylate adaptors during the
ligation reaction. This strategy generated pMSCV-neo-EBNA1dn. The pMSCV-
puro-EBNA1dn construct was derived from this plasmid by simply excising the
hemagglutinin/nls/EBNA1dn cassette from pMSCV-neo-EBNA1dn and trans-
ferring it to pMSCV-puro using appropriate standard cloning adaptors. All
constructs were verified by sequence analysis.
The pEhyg-FLAG-LMP1 vector was generated by excising FLAG-LMP1 se-
quences from pcDNA3-FLAG-LMP1 (generous gift of Nancy Raab-Traub) with
HindIII and NotI and cloning into XhoI- and NotI-digested pEhyg using a
HindIII/XhoI adaptor (5?-TCGAGGCTGCAGGCA-3? and 5?-AGCTTGCCTG
CAGCC-3?). Cloning was analyzed by diagnostic digest, and the LMP1 insert was
sequenced in its entirety.
The pEhyg-miR-146a vector was generated by amplifying a 452-bp sequence
containing the miR-146a hairpin (nucleotides 4721752 to 4722203 of chromo-
some 5; NCBI reference assembly) from Mutu cell genomic DNA by PCR and
cloned downstream from the hygromycin resistance gene of the vector pEhyg
The miR-146a promoter/luciferase reporter construct was made by amplifying
the human miR-146a promoter extending from ?1153 to ?21 relative to the
start site from Mutu genomic DNA by PCR using the primers 5?-GCAGCTAG
CTTT CGGTCCATGAGCACGT-3? (forward primer) and 5?-GCAAAGCTTA
GCGGTCAAGCGTCTTGG-3? (reverse primer). The isolated fragment was
digested with NheI and HindIII and cloned into NheI- and HindIII-cut
pGL3basic (Promega). The entire promoter region was then sequenced, and no
discrepancies were identified relative to the GenBank genomic sequence.
Mutagenesis of the miR-146a reporter plasmid was carried out using a
QuikChange II site-directed mutagenesis kit (Stratagene) and the following
oligos: 5?-CG GAG AGT ACA GAC CTC GAG CCT GGG GAC CCA G-3?
and 5?-CTG GGT CCC CAG GCT CGA GGT CTG TAC TCT CCG C-3?
(c-Ets); 5?-CAG GCT GCT CCT GAC CTC GAG TGC AAG AGG GTC
CCC-3? and 5?-GGG GAC CCT CTT GCA CTC GAG GTC AGG AGC AGC
CTG-3? (c-Myc); 5?-CAC TGC CAG GCT GGC TCG AGC CAT TCC GGC
CCA G-3? and 5?-CTG GGC CGG AAT GGC TCG AGC CAG CCT GGC
VOL. 82, 2008CELLULAR miRNA AND EPSTEIN-BARR VIRUS ONCOGENESIS1947
AGT G-3? (HSF2); 5?-CGA TAA AGC TCT CGG CTC GAG CCC GCG GGG
CTG CGG-3? and 5?-CCG CAG CCC CGC GGG CTC GAG CCG AGA GCT
TTA TCG-3? (distal NF-?B); 5?-GAG GGA TCT AGA AGG CTC GAG CCA
GAG AGG GTT AGC-3? and 5?-GCT AAC CCT CTC TGG CTC GAG CCT
TCT AGA TCC CTC-3? (proximal NF-?B), 5?-GAA ATG GAA TAA AAG
CCT CGA GAA ATA GGC CTT AGC TG-3? and 5?-CAG CTA AGG CCT
ATT TCT CGA GGC TTT TAT TCC ATT TC-3? (Oct-1); 5?-CCG GCC CAG
CCT CCT CGA GCC TCG CTG TGC C-3? and 5?-GGC ACA GCG AGG CTC
GAG GAG GCT GGG CCG G-3? (PU.1). In each case, the core transcription
factor binding site was replaced with a XhoI restriction site. Mutations were
initially screened by digesting with XhoI and then verified by sequence analysis.
Transfection and luciferase expression analysis. Mutant and wild-type miR-
146a promoter-reporter vectors were cotransfected with pSG5 (control) or
pSG5-LMP1 expression constructs into Mutu EBNA-1 dominant negative clone
3 cells using Lipofectamine reagent (Invitrogen) according to the manufacturer’s
instructions. Cells were harvested 48 h posttransfection and analyzed for lucif-
erase reporter activity using Promega firefly luciferase assay reagents according
to the manufacturer’s protocol.
BLAST analysis of miR-146a promoter versus mouse genome. Two kilobases
of sequence upstream and 190 bp downstream from the miR-146a start site in
humans (reported by Taganov and Baltimore ) was used to analyze with
BLAST the mouse genome, using the NCBI blastN algorithm with default
settings. The two homologies shown in Fig. 5, below, were identified as the only
hits, and they correspond to nucleotides 8703897 to 8703794 and 8703618 to
8703488 of mouse chromosome 11 (NCBI reference assembly).
Retroviral infections. Transient-transfection experiments were performed by
using a modified version of the calcium phosphate precipitation procedure (a
detailed protocol is available at http://www.flemingtonlab.com). Briefly, 106
HEK293 cells were plated onto 100-mm-diameter tissue culture dishes. The
following day, the medium was replaced with 8 ml of fresh supplemented
DMEM; 4 h later, DNA precipitates were generated by mixing 0.5 ml of 1?
HEPES-buffered saline (0.5% HEPES, 0.8% NaCl, 0.1% dextrose, 0.01% anhy-
drous Na2HPO4, 0.37% KCl [pH 7.10]) with a total of 30 ?g of plasmid DNA (10
?g retroviral vector plus 10 ?g G protein of vesicular stomatitis virus expression
vector, plus 10 ?g pVPACK dGI packaging vector). A total of 30 ?l of 2.5 M
CaCl2was added, and samples were mixed immediately. Precipitates were al-
lowed to form at room temperature for 20 min before being added dropwise to
cells. Cells were incubated at 37°C with 5% CO2for 16 h before the medium was
replaced with 10 ml of fresh DMEM (plus 10% fetal bovine serum).
Forty-eight hours later, viral supernatants were collected and subjected to one
round of centrifugation followed by filtration through a 0.45-?m surfactant-free
cellulose acetate membrane filter. Infections were carried out in six-well plates
with 1 ml virus plus 106appropriate B cells suspended in 1 ml DME plus 10%
fetal bovine serum. Polybrene was added to a final concentration of 12 mg/ml,
and the mixture was mixed by gently rocking. Cells were spun in six-well plates
at 1,000 ? g for 1 h followed by a 4-h incubation at 37°C, 5% CO2. Cells were
then collected, spun down, and resuspended in 2 ml RPMI (plus 10% fetal
bovine serum, penicillin, streptomycin, and glutamine) per well. Cells were cul-
tured for 2 days prior to antibiotic selection.
Generation of EBV-negative Mutu clones. Mutu I cells were serially infected
with either a control retrovirus (pMSCV-neo followed by pMSCV-puro) or a
retrovirus containing the dominant negative EBNA1 (pMSCV-neo-E1dn fol-
lowed by pMSCV-puro-E1dn). Infection with the pMSCV-puro-based virus was
done 3 days after pMSCV-neo-based infections. Cells were selected for 7 days,
after which cells were cloned by limiting dilution. EBV-negative clones were
identified by PCR analysis of genomic DNA for the presence of DNA from the
BamHI Z, BamHI R, and BamHI Q fragments. Further verification that clones
were EBV negative was carried out by reverse transcription-PCR (RT-PCR)
analysis of EBNA1 transcripts, Cp-initiated transcripts, Wp transcripts, and
LMP1 and LMP2 transcripts both before and after treatment with 5-azacytidine.
By all accounts, Mutu E1dn Cl.3 cells were found to be EBV negative.
RNA isolation. RNAs used for miRNA array analysis and for assessing mature
miRNAs were generated by a modified TRIzol method. Cells were stained with
trypan blue and counted, and 107cells were pelleted by centrifugation. Cells were
suspended in TRIzol reagent (Invitrogen) and processed as per the manufactur-
er’s protocol up through the addition of isopropanol. Samples were allowed to
precipitate in isopropanol overnight at ?20°C. Samples were then centrifuged at
12,000 ? g for 30 min at 4°C. Isopropanol was decanted, and nucleic acid pellets
were resuspended in 200 ?l nuclease-free water. RNA was then precipitated
again by adding 20 ?l 3 M NaO-acetate and 0.5 ml of 100% ethanol and
incubating overnight at ?20°C. Samples were centrifuged at 12,000 ? g for 30 min
at 4°C, ethanol was decanted, and samples were washed once with 75% ethanol
and allowed to air dry for no more than 10 min. RNA pellets were resuspended
in nuclease-free water, quantitated by UV spectrophotometry, aliquoted, and
stored at ?80°C.
RNAs used for mRNA arrays and for assessing the levels of primary miR-146a
were generated using a Qiagen RNeasy kit. Total RNA was isolated from 107
cells using the RNeasy kit according to the manufacturer’s protocol. The RNA
was eluted from the column using 50 ?l nuclease-free water, quantitated, and
stored at ?80°C.
Real-time qRT-PCR for mature miRNA and primary transcript (pri-miRNA).
To quantitate mature miRNA expression, total RNA isolated by a modified
TRIzol protocol (see “RNA isolation,” above) was polyadenylated and reverse
transcribed using the NCode miRNA first-strand synthesis kit (Invitrogen) ac-
cording to the manufacturer’s instructions. The resulting cDNA was subjected to
real-time quantitative RT-PCR (qRT-PCR) using the NCode universal reverse
primer (Invitrogen) in conjunction with a sequence-specific forward primer for
miR-146a (forward primers for NCode miRNA detection are the exact sequence
of the mature miRNA). Similarly, U6 small nuclear RNA was quantified using
the NCode universal reverse primer and a U6-specific primer (5?-CTCGCTTC
GGCAGCACA-3?). A master mix was prepared for each PCR run, which in-
cluded Sybr green Supermix plus UDG (Invitrogen), fluorescein-NIST traceable
dye, and 100 nM forward and reverse primers. Amplification consisted of 2 min
at 50°C and 2 min at 95°C, followed by 40 cycles of 95°C for 15 s and 60°C for 30 s.
Total RNA prepared by Qiagen RNeasy column extraction was DNase treated
and reverse transcribed using random hexamer primers and SuperScript III
reverse transcription enzyme mix and reagents (Invitrogen) according to the
manufacturer’s instructions. Primary transcript miR-146a expression was quan-
tified using specific forward (5?-TGAGAACTGAATTCCATGGGTT-3?) and
reverse (5?-ATCTACTCTCTCCAGGTCCTCA-3?) primers at a 200 nM con-
centration in a reaction mixture consisting of Sybr green Supermix plus UDG
and fluorescein-NIST traceable dye. Cycling parameters were as follows: 2 min at
50°C and 2 min at 95°C, followed by 40 cycles of 95°C for 30 s and 60°C for 30 s.
Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was quantified similarly;
the primer sequences have been published elsewhere (40).
Melt curve analysis was performed at the end of every qPCR run. Samples
were tested in triplicate. Real-time PCR was performed on Bio-Rad iCyclers
(MyIQ, IQ4, or IQ5), and data analysis was performed using Bio-Rad IQ5 v2
software. Measures taken to prevent and identify PCR contamination included
maintaining a physically separate, nucleic acid-free area with designated equip-
ment for PCR setup and using both no-template controls and no-reverse tran-
scription controls in every experiment. A standard dilution curve, generated by
serially diluting a pooled stock of positive control cDNAs, was included in each
run. Experiments were repeated if controls did not behave as predicted.
Relative expression was calculated for each test sample by the standard curve
method (for formulas, see the ABI Prism 7700 Sequence Detection System User
Bulletin 2: Relative Quantitation of Gene Expression; Applied Biosystems, Fos-
ter City, CA). Relative quantities of each test sample were extrapolated from the
standard curve run concurrently with the sample, and the values were normalized
to U6 small nuclear RNA (mature miRNA NCode assays) or GAPDH (primary
transcript assays). The normalized values were then compared to a calibrator
sample within the run. Data are presented as the expression level relative to the
calibrator, with the standard error of the mean of triplicate measures for each
Cellular miRNA microarray analysis. Small RNA isolation and hybridization
were performed by LC Sciences (Houston, TX). Briefly, small RNA species were
isolated from total RNA by column exclusion. The concentrated small RNAs
were 3?-polyadenylated with poly(A) polymerase. A nucleotide tag was then
ligated to the poly(A) tails. The tagged RNAs were hybridized to a ?Paraflo
superfluidic array chip containing probes for human and viral microRNA se-
quences and were subsequently labeled in a second hybridization reaction with
Cy3 and Cy5 dendrimer dyes. After overnight hybridization, arrays were strin-
gently washed and scanned on an Axon GenePix 4000B laser scanner (Axon
Instruments). Data extraction and imaging were performed using ArrayPro soft-
ware (Media Cybernetics).
Each array compared RNA isolated from control (Mutu plus pEHyg) and
experimental (Mutu plus pEHyg-FLAG-LMP1) cells. Five separate arrays were
performed, utilizing five independent pairs of RNA preparations. The most
up-to-date chip available for analysis was used (containing probes for all human
miRNA listed in the Sanger Institute miRBase, release 9.1, totaling 470 unique
miRNAs). Additional probes (108 unique sequences) for viral miRNA expres-
sion were included on each chip. Each probe was repeated in sextuplicate. Dye
swap experiments were executed to control for labeling bias. Data within each
individual chip were adjusted by subtraction of background fluorescence (calcu-
lated as the median of the lower 5 to 25% of signal intensities) followed by
normalization of the data to the statistical median of all detectable signals.
1948 CAMERON ET AL. J. VIROL.
Variation among arrays introduced by differences in experimental conditions
(i.e., sample preparation, dye labeling, chip quality, and scanning variations) was
corrected by normalization of the data by the cyclic LOWESS (locally weighted
regression) method (2). Intensity values were then log2transformed and further
normalized and centered using the following equation: [(log2intensity value) ?
[(mean of values across all samples for the individual gene)/(standard deviation
of values across all samples for the individual gene)]. The ratio of expression
between control and test samples was normalized for clustering analysis by the
following equation: [log2(ratio)]/[(standard deviation of log2(ratio)]. Statistical
comparison of control (Mutu-pEHyg) and test (Mutu-pEHyg-FLAG-LMP1)
sample miRNA expression levels was performed using t tests, and corresponding
P values were calculated (32). MicroRNAs showing differential expression at the
P levels of ?0.10, ?0.05, and ?0.01 were selected for clustering analysis, which
was performed using a hierarchical method based on average linkage and a
Euclidean distance metric (7). All data calculations were performed by LC
Sciences. Clustering plots were created using TIGR MeV (multiple experimental
viewer) software (The Institute for Genomic Research).
Cellular mRNA microarray analysis. RNAs prepared using Qiagen RNeasy
were from two separate pEhyg infections and two separate pEhyg-miR-146a
infections. Each sample was labeled with Cy3 and with Cy5 to allow for dye swaps
for each pair of pEhyg- and pEhyg-miR-146a-infected cells and hybridized to
Agilent human 4 ? 44,000 60-mer oligonucleotide arrays (Miltenyi Biotech).
Data were processed and normalized using Rossetta Resolver software to iden-
tify genes from each infection pair showing greater-than-twofold differences
and with P values of ?0.01 (see Tables S1 and S2 in the supplemental
material). To ensure that differences were reproducible in separate biological
samples, differentially expressed genes in both infection sets were considered
for further analysis.
Microarray data accession numbers. The raw array data have been published
in the National Center for Biotechnology Information (NCBI) Gene Expression
Omnibus (GEO) and assigned accession numbers GSM254072, GSM254076,
GSM254077, GSM254081, GSM255655, GSM255656, GSM255658, GSM255659,
and GSM255660. The series accession number for this work is GSE10107.
Cellular miRNAs are regulated by LMP1. EBV-negative
derivatives of the Burkitt’s lymphoma cell line, Mutu, were
generated by transducing with a retrovirus expressing a dom-
inant negative form of the EBV episomal replication/mainte-
nance factor EBNA1. A resulting subclone confirmed to be
EBV negative was transduced with either a control retroviral
FIG. 1. EBV LMP1 alters expression profiles of cellular miRNA. Microarray analysis of miRNA expression was performed on RNA isolated
from control (Mutu pEHyg) and LMP1-expressing (Mutu pEHyg-LMP1) EBV-negative Mutu Cl.3 cells. Five separate RNA pairs were hybridized
to miRNA arrays, and the data were combined for analysis. A. Western blot analysis of LMP1 expression in the EBV-positive latency type III cell
lines JY, X50-7, and Jijoye, the type I latency cell line Akata, and in control or LMP1-transduced EBV-negative Mutu cells. B. Cluster analysis
of miRNAs significantly altered by LMP1 at a P level of ?0.01. C. MicroRNAs significantly (P ? 0.01) regulated by LMP1, showing mean signal
intensities and log-transformed ratios.
VOL. 82, 2008 CELLULAR miRNA AND EPSTEIN-BARR VIRUS ONCOGENESIS1949
vector (pEhyg) or an LMP1-expressing retroviral vector
(pEhyg-FLAG-LMP1). Cells were selected for 2 to 4 weeks in
hygromycin prior to harvesting for total RNA preparations.
Little cell death was observed during the selection procedure,
indicating that retroviral transduction efficiency was high and
that the resulting cell populations were highly polyclonal. Five
separate RNA preparations were generated from control and
LMP1-expressing cells, and the RNAs were size selected to
isolate the small RNA fractions. Fractionated RNAs were then
subjected to microRNA array analysis for expression of 470
unique human miRNAs. Data from five independent arrays
were compiled (Fig. 1). Expression levels of 35 miRNAs were
altered by LMP1 at P ? 0.05, and of these, 15 miRNAs were
regulated at the P ? 0.01 level (10 induced by LMP1 and 5
suppressed by LMP1). In all five microarray assays, expression
of miR-146a showed the highest level of differential expression
(12- to 23-fold induction by LMP1), and we therefore focused
on this miRNA for further analysis.
To confirm the results of the miRNA microarray analysis
and to validate induction of the processed miR-146a transcript,
qRT-PCR analysis was performed to assess the levels of ma-
FIG. 2. qRT-PCR analysis of miR-146a expression. A. Mature
miR-146a. Two separate preparations of RNA were isolated from
EBV-negative Mutu-pEHyg and Mutu-pEHyg-LMP1 cells and ana-
lyzed by real-time PCR to assess levels of mature miR-146a. Data were
normalized to U6 small nuclear RNA expression. B. Primary miR-
146a. Total RNA isolated from EBV-negative Mutu-pEHyg and
Mutu-pEHyg-LMP1 cells was reverse transcribed and used in a real-
time PCR assay to detect the primary miR-146a transcript. Data were
normalized to GAPDH expression.
FIG. 3. LMP1 induces miR-146a in the epithelial cell line A549.
Total RNA isolated from A549 lung epithelial cells transduced with
pEHyg or pEHyg-FLAG-LMP1 retroviruses was polyadenylated, re-
verse transcribed, and used in a qPCR analysis of mature miR-146a
expression. Data were normalized to U6 small nuclear RNA expres-
1950 CAMERON ET AL.J. VIROL.
ture miR-146a in EBV-negative Mutu-pEHyg and Mutu-
pEHyg-FLAG-LMP1 cells. As shown in Fig. 2A, quantitative
RT-PCR analysis demonstrated induction of processed miR-
146a. Since the related microRNAs miR-146a and miR-146b
differ by only two nucleotides, analysis of the primary miR-
146a transcript was carried out to more firmly establish LMP1-
mediated regulation of the miR-146a locus. As shown in Fig.
2B, real-time RT-PCR analysis using pri-miR-146a-specific
primers demonstrated elevated expression of pri-miR-146a in
LMP1-expressing cells. Together, these results demonstrate
that LMP1 induces the expression of pri-miR-146a and pro-
cessed (mature) miR-146a.
LMP1 induces miR-146a expression in the epithelial cell
line A549. In addition to its ability to transform B lymphocytes,
EBV has also been linked to epithelial carcinoma of the na-
sopharynx (34). EBV-associated nasopharyngeal carcinomas
display a type II latency expression pattern in which LMP1 is
expressed (34). To determine if LMP1-mediated induction of
miR-146a occurs in nonlymphoid cells, control and LMP1-
expressing retroviruses were transduced into the lung epithe-
lial cell line A549. Induction of mature miR-146a was detected
in LMP1-transduced A549 cells compared to control trans-
duced cells by qRT-PCR (Fig. 3), indicating that induction of
miR-146a is not restricted to lymphoid tissues.
Mature miR-146a and pri-miR-146a are expressed in cells
displaying type III but not type I latency gene expression
patterns. To assess the level of miR-146a in an EBV-positive
context where expression of LMP1 is naturally occurring, a
panel of EBV-positive cell lines was examined by qRT-PCR.
As shown in Fig. 4A, mature miR-146a was not detected in the
type I Burkitt lymphoma cell lines Akata, Rael, and Mutu,
which do not express LMP1. In contrast, high levels of miR-
146a were detected in the type III cell lines JY, IB4, X50-7, and
Jijoye, all of which express comparably high levels of LMP1.
Analysis of the primary miR-146a transcript similarly showed
significant differences between type III and type I latency cells,
indicating that the differential expression levels in this cell
panel are likely due in large part to differences in transcription
The data in Fig. 4 provide evidence that EBV type III la-
tency genes are responsible for the activated expression of
miR-146a. To substantiate that the differential expression ob-
served in type III versus type I cell lines is due to EBV gene
expression and not clonal cell line differences, expression of
FIG. 4. Mature and primary miR-146a transcript analysis in a panel
of cell lines. A. Total RNA isolated from EBV-positive, latency type III
cell lines JY, IB4, X50-7 (lymphoblastoid cell lines), and Jijoye (Bur-
kitt’s lymphoma) and EBV-positive latency I Burkitt’s lymphoma cell
lines Akata, Rael, and Mutu was polyadenylated and reverse tran-
scribed for qPCR analysis of mature miR-146a expression. The West-
ern blot shows LMP1 expression in EBV latency type III cell lines but
not in latency type I cell lines. B. Total RNA isolated from the indi-
cated cell lines was reverse transcribed and subjected to qPCR to
assess the levels of primary miR-146a transcripts. C. Total RNA ex-
tracted from the EBV-negative Burkitt’s lymphoma cell line BL-30 and
BL-30 cells infected with EBV B95-8 virus were reverse transcribed
and assayed for pri-miR-146a expression by qPCR. Mature miR-146a
expression was normalized to U6 small nuclear RNA expression, and
pri-miR-146a expression was normalized to GAPDH expression.
VOL. 82, 2008 CELLULAR miRNA AND EPSTEIN-BARR VIRUS ONCOGENESIS1951
pri-miR-146a transcripts were quantified in the EBV-negative
Burkitt’s lymphoma cell line BL30 and BL30 cells infected with
the EBV strain B95-8 (which exhibit type III latency) (data not
shown). As shown in Fig. 4C, EBV infection of BL30 cells
induces expression of pri-miR-146a. Together, these results
demonstrate that miR-146a is expressed in type III latency
cells and that latency type III gene expression likely mediates
induction of miR-146a.
LMP1 induces miR-146a expression through activation of
two NF-?B elements in the miR-146a promoter. The experi-
ments discussed above implicated a transcriptional mechanism
for LMP1-mediated induction of miR-146a. To further address
this issue, an approximately 1.2-kb region of the miR-146a
promoter region was isolated from Mutu cells and cloned into
a luciferase reporter plasmid. A BLAST search of the mouse
genome using a 2-kb region of the human miR-146a promoter
identified two significant homologies located in an analogous
position relative to the miR-146a hairpin sequence. Visual
inspection and analysis of these homologies using TFSEARCH
several high-probability transcription factor binding sites in the
homologous regions, including two NF-?B sites previously
identified by Taganov et al. (37) as well as potential c-Ets,
PU.1, c-Myc, and Oct-1 sites. Each of these elements was
mutated separately in the context of the miR-146a reporter
plasmid. In addition, a mutant in which both NF-?B sites were
disrupted was also generated. The wild-type or the mutant
reporter plasmids were cotransfected into EBV-negative Mutu
cells with either a control or an LMP1 expression vector. As
shown in Fig. 5, expression of LMP1 enhanced the activity of
the wild-type miR-146a reporter construct, as well as con-
structs containing mutations in the c-Myc, HSF2, and Oct-1
sites. Mutation of the PU.1 site marginally reduced the ability
of LMP1 to induce expression from the miR-146a promoter
(P ? 0.05). Mutation of either NF-?B site substantially re-
duced the LMP1-mediated induction of the miR-146a pro-
moter, and mutation of both NF-?B sites abrogated LMP1-
mediated induction of miR-146a promoter expression (P ?
0.05). These results indicate that LMP1 induces miR-146a
expression primarily through activation of transcription via an
NF-?B-dependent mechanism. Analysis of the Oct-1 site dem-
onstrated that although mutation of this site does not alter the
level of LMP1-mediated induction, the activity of this mutant
is reduced in both control transfected as well as LMP1-trans-
fected cells. This indicates that the Oct-1 site plays a substan-
tive role in facilitating constitutive and induced expression of
the miR-146a promoter and could therefore play a possible
FIG. 5. miR-146a promoter analysis. A. Alignment of major homologies between human and mouse miR-146a promoter regions. Homologies
were identified through a BLAST search using the human miR-146a promoter as described in Materials and Methods. Percentage scores for the
indicated transcription factors were generated by TFSEARCH (http://www.cbrc.jp/research/db/TFSEARCH.html). B. Schematic representation of
the human miR-146a promoter, with putative transcription factor binding sites. C. EBV-negative Mutu cells were cotransfected with pSG5 or
pSG5-LMP1 plus wild-type or mutant miR-146a promoter-reporter vectors and harvested 48 h later. Luciferase expression is represented relative
to luciferase activity of the wild-type promoter in the presence of LMP1. Error bars show the standard errors of the means. Increases for each
reporter are indicated. Deletion of the dual NF-?B sites abrogated the ability of LMP1 to induce the miR-146a promoter.
1952CAMERON ET AL.J. VIROL.
role in mediating cell type-specific differences in miR-146a
MicroRNA miR-146a suppresses interferon-responsive gene
expression. To gain insights into the role that induction of
miR-146a plays in influencing cellular responses, we generated
an miR-146a retroviral expression vector and transduced it
into Akata cells that do not express endogenous miR-146a.
Duplicate infections of both the control retrovirus (pEhyg-
miR-Control) and the miR-146a retrovirus (pEhyg-miR-146a)
were carried out, and RNA was isolated from each infection 18
days later. The pEhyg-miR-146a-infected cells but not pEhyg-
miR-Control-infected cells expressed the mature form of miR-
146a (data not shown), indicating that the miR-146a transcript
generated from this retrovirus undergoes appropriate micro-
RNA processing. RNA preparations were then subjected to
mRNA array analysis using a two-color Agilent 60-mer oligo
4 ? 44,000 platform. Arrays were carried out on the duplicate
infections, and dye swaps were analyzed for each. Genes show-
ing greater-than-twofold differences in both sets of infections
with P values of less than 0.01 were then compiled (see Tables
S1 and S2 in the supplemental material for gene lists for each
infection pair). As shown in Tables 1 to 3, eight genes were
induced while 38 were suppressed by miR-146a. Of the 38
suppressed genes, 16 (42%) were identified as known interferon-
inducible genes (Table 3). This suggests that miR-146a mod-
ulates a critical component(s) of the interferon signaling path-
way. A higher proportion of genes that are not known to be
interferon responsive contained potential 7-mer or better seed
sequences (59%) than in the set of interferon-responsive genes
(25%), indicating that regulation of most of the interferon-
responsive genes likely occurs through a common upstream
mediator (Tables 2 and 3; Fig. 6). The observed changes in
TABLE 1. Transcripts induced in miR-146a-expressing
Akata cells (P ? 0.01)
Name and function
Pair 1Pair 2
GIMAP4 7.47.7GTPase, IMAP family member;
decreased in T and B cells
Stonin 2; involved in
Protein tyrosine phosphatase,
receptor type, G; linked to
familial breast cancer; tumor
Recombination activating gene
1; initiates V(D)J
recombination during B-cell
Oxysterol binding protein-like
1A; likely regulates lipid
Syntrophin; interacts with
aData were derived from array element AK094799.
TABLE 2. Transcripts suppressed in miR-146a-expressing Akata cells (without known response to IFN) (P ? 0.01)
Name and/or function
Pair 1Pair 2
?12.0 8-merDNA polymerase-transactivated protein 6; transactivated by viral
Sterile ?-motif domain-containing 9-like
Epithelial stromal interaction 1; induced in tumor cells cocultivated
Regulator of G-protein signaling 13; may mediate cytokine signaling
in germinal centers; suppressed by EBV LMP1
Heat shock 70-kDa protein 1A, HSP72; antiapoptotic
Bcl2-related protein A1; induced by NF-?B, EBV-LMP1, and
Chemokine (c-c motif) receptor 9; receptor for TECK, involved in
mucosal homing of lymphocytes
Integrin ?-like 1
Proliferation-induced gene 13
9-mer, two 7-mers
Zinc finger and BTB domain-containing 32; transcription factor
involved in osteoblastic differentiation
Transmembrane and tetracopeptide repeat-containing 2
Synaptotagmin XII, or SRG1; involved in brain disorders
LIM domain only 2 (rhombotin-like 1)
aData were derived from the following array elements: EPSTI1 (ENST00000313624), ITGBL1 (ENST00000376155), and C16ORF81 (ENST00000270031).
VOL. 82, 2008CELLULAR miRNA AND EPSTEIN-BARR VIRUS ONCOGENESIS1953
mRNA expression in miR-146a-expressing Akata cells are not
likely due to artifacts generated by expressing RNA of a dou-
ble-stranded nature, since there was little similarity in altered
gene expression patterns in Akata cells transduced with other
miRNA-expressing retroviruses (miR-155 and miR-21) com-
pared to miR-146a-transduced Akata cells (unpublished).
MicroRNAs have been shown to play important regulatory
roles in a wide variety of cellular processes, including cell cycle
progression, differentiation, apoptosis, and signaling. In addi-
tion, a number of groups have reported the altered expression
of miRNAs in various forms of human cancers. Viruses regu-
larly exploit cellular pathways in order to complete the viral life
cycle. Although EBV and other herpesviruses encode their
own set of microRNAs, the extent to which DNA tumor vi-
ruses utilize cellular miRNA pathways during infection has not
been explored. We have shown here that LMP1, the major
oncoprotein of the human tumor virus EBV, alters the expres-
sion profile of several cellular miRNA species, including the
most highly regulated of these, miR-146a. Induction of miR-
146a likely occurs during the natural course of EBV infection,
since miR-146a is highly expressed in EBV-positive cells ex-
hibiting type III latency gene expression (but not type I la-
tency) and infection of EBV-negative cells with EBV induces
the expression of miR-146a.
While regulation of microRNA expression can occur at a
number of different processing levels, it is clear that LMP1
induces the transcription of miR-146a, since LMP1 increases
primary miR-146a transcript levels and activates a reporter
plasmid containing the miR-146a promoter. As expected, in-
duction of the miR-146a promoter occurs to a large extent
through two conserved NF-?B transcription factor binding
sites. These data are in line with a recent report demonstrating
NF-?B-dependent induction of miR-146a expression by TLR
signaling (by TLR-2, -4, and -5 ), emphasizing the impor-
tance of the NF-?B sites in mediating the activation of miR-
146a expression by various signaling molecules. In contrast,
however, although Taganov et al. also demonstrated induction
of two other microRNAs by TLR signaling, miR-155 and miR-
132 (37), our array analysis did not reveal the induction of
either of these, and we have not been able to demonstrate
substantial induction of miR-155 by LMP1 in a number of
systems despite the presence of candidate NF-?B and AP-1
promoter elements in the miR-155 promoter (Q. Yin and E.
Flemington, unpublished data). Although the differential re-
sponses of miR-155 and miR-132 to TLR and LMP1 signaling
al. ) and B lymphocytes (this study), it may on the other
hand suggest that TLR and LMP1 elicit similar but distinct
signaling pathways which result in differential miRNA induc-
In addition to NF-?B, LMP1 induces a number of other
transcription factor pathways (p38/mitogen-activated protein
kinase/activator transcription factor 2, cJUN/c-Jun N-terminal
kinase/AP-1, and phosphatidylinositol 3-kinase/Akt) (reviewed
in reference 23) and is known to induce some genes through
TABLE 3. Transcripts suppressed in miR-146a-expressing Akata cells (known to be IFN responders) (P ? 0.01)
Name and/or function
Pair 1Pair 2
Interferon-induced protein 44-like, or histocompatibility 28
Interferon-induced protein 44; induced during hepatitis virus infection
Myxovirus (influenza virus) resistance 2, or interferon-induced GTP
binding protein MXB
Radical S-adenosyl methionine domain-containing 2, or viperin; antiviral;
induced during TLR signaling
Interferon-induced protein with tetratricopeptide repeats 3
2?-5?-oligoadenylate synthetase-like; binds double-stranded RNA and DNA
Tripartite motif-containing 22, or STAF50; can inhibit viral replication
Interferon-induced protein with tetratricopeptide repeats 1
Interferon-induced protein with tetratricopeptide repeats 5
None Interferon-induced transmembrane protein 1, LEU13, or CD225; regulates
Interferon-induced transmembrane protein 3 IFITM3
Interferon-induced transmembrane protein 4 pseudogene
Interferon regulatory factor 7; transcriptional activator in response to
infection; binds to Qp of EBV EBNA1; involved in TLR signaling;
colocalizes with and regulates EBV LMP1
N-myc (and STAT) interactor; transcription factor induced by interferon
ISG15 ubiquitin-like modifier; targets STAT1, MAPK3, JAK1, etc;
Alpha interferon-inducible protein 27
aData were derived from the array element ENST00000313624.
1954CAMERON ET AL. J. VIROL.
FIG. 6. Down-regulated genes with 7-mer or better miR-146a seed sequences. Transcript sequences were obtained from the website www
VOL. 82, 2008 CELLULAR miRNA AND EPSTEIN-BARR VIRUS ONCOGENESIS1955
c-Ets sites (21). Nevertheless, despite the presence of a poten-
tial c-Ets site within the conserved region of the miR-146a
promoter, we did not see an evident role for this element in
activation of the miR-146a reporter. Additional experiments
using a natural genomic context and/or other cell systems are
required to address this issue more thoroughly.
It is notable that the expression of miR-146a in latency type
III cells is significantly higher than that of EBV-negative cells
infected with an LMP1 retrovirus. It is unclear what may ac-
count for this difference. It is possible that the genetic back-
ground of Mutu cells is less favorable for LMP1 signaling than
the type III latency cell types assessed in our studies. Another
plausible explanation is possible cooperative induction of miR-
146a by LMP1 and other type III latency genes. For example,
EBNA-3C has been shown to activate gene expression through
PU.1 promoter elements (43), and the conserved PU.1 site in
the miR-146a promoter could be influenced by EBNA-3C.
Notably, mutation of this element had a moderate but signif-
icant inhibitory effect on the miR-146a promoter, suggesting
that this element contributes to promoter function.
The miR-146a promoter also contains a highly conserved
Octamer binding site (Oct-1) near the TATA box that contrib-
utes substantially to both basal and induced miR-146a pro-
moter activity. While a number of Octamer binding proteins
have been identified that bind to prototypical Oct-1 elements,
this element was shown early on to confer B-cell-specific ex-
pression of the immunoglobulin genes (28). This element may
therefore play a role in conferring at least some level of lym-
phocyte specificity for miR-146a and may contribute to induc-
tion of miR-146a by various lymphocyte-stimulating agents.
Nevertheless, it is clear that miR-146a can be expressed in
other tissues (such as A549 cells infected with an LMP1 ret-
rovirus), and the Oct-1 site may or may not be required for this
activity. It is notable that the closely related miRNA, miR-
146b, was only marginally induced by LMP1 (less than twofold
induction; P ? 0.05). MicroRNAs miR-146a and miR-146b
have identical 5? ends, which are the regions of microRNAs
that appear to play the predominant role in specific binding
and inhibition of mRNAs (30). Although these two miRNAs
share many of the same targets, it appears that the regulation
of miR-146b is distinct at least in some ways from that of
miR-146a. Such distinct regulatory mechanisms for cognate
microRNAs may be a way to elicit the same downstream re-
sponse in different tissues or under different conditions.
Cellular miRNA expression alters the expression level of
some (but not all) mRNA transcripts directly targeted by
miRNAs. In addition, miRNA modulation of protein expression
also impacts the expression of mRNA transcripts downstream of
the modulated proteins within the biological pathway. Therefore,
we employed mRNA transcriptional microarray profiling to
simultaneously shed light on the direct targets of miR-146a and
the cellular pathway(s) impacted by miR-146a expression. Our
array analysis of miR-146a-expressing Akata cells revealed a
group of miR-146a-suppressed genes that are known to be
responsive to interferon signaling. This indicates that miR-
146a may be part of a negative feedback loop that plays a role
in modulating the interferon response. This could be a means
of fine-tuning the level of interferon signaling and/or could be
a means of providing a shut-off mechanism following stimula-
tion during normal physiological signaling. Interestingly, like
miR-155, elevated miR-146a has been correlated with certain
tumor types (13, 39, 41). It is possible that the inappropriate
expression of miR-146a could lead to suppression of the inter-
feron response pathway in tumors, thereby helping suppress
immune-mediated surveillance through interferon signaling. In
the context of EBV infection, induction of miR-146a could
result in suppression or modulation of the interferon-mediated
antiviral response, protecting the virus from host immunity. It
is interesting that the interferon pathway itself has been shown
to induce cellular miRNAs that inhibit hepatitis C virus repli-
cation, indicating that cellular miRNA induction during viral
infection can be either productive or deleterious to the virus
One of the interferon-related genes found here to be sup-
pressed by miR-146a is the transcriptional activator IRF7,
which has been shown to bind and activate the LMP1 promoter
(31). It is therefore likely that a more direct consequence of
miR-146a expression on interferon signaling is the negative
feedback regulation of LMP1 expression itself. This may be a
means of establishing a refined regulation of LMP1 expression
to avoid some of the known deleterious effects of overex-
Our initial target analysis of miR-146a-regulated genes dis-
cussed above did not reveal any potential direct miR-146a
targets that may play a role in mediating disruption of the
interferon signaling pathway. Although the ability of micro-
RNAs to inhibit translation of target mRNAs is clear, the
contribution of microRNA-mediated degradation of target
mRNAs is much less well understood, and it is thought that
only a subset of mRNAs may be subject to degradation. There-
fore, a key target of miR-146a involved in the interferon sig-
naling pathway may not be revealed by analyzing RNA levels
and may only be detected by assessing protein expression.
Taganov et al. previously demonstrated that reporter plasmids
containing the 3? UTRs of TRAF6 and IRAK1 were sup-
pressed by miR-146a (37), but they did not assess whether
endogenous TRAF6 or IRAK1 (RNA or protein) levels were
influenced by miR-146a. Our array analysis showed no alter-
ation of TRAF6 RNA levels in either miR-146a infection pair;
however, expression of IRAK1 was suppressed in the first
infection set by more than the twofold cutoff, while it was
suppressed in the second infection set by just less than the
twofold cutoff (?1.9) (Fig. 7; see also Tables S1 and S2 in the
supplemental material). It is therefore possible that suppres-
sion of IRAK1 expression could play a role in mediating the
miR-146a feedback mechanism. It is of note that IRF7 resides
downstream of both TRAF6 and IRAK1 in antiviral TLR7/8
signaling (38). Since no direct miR-146a target site was iden-
tified in the IRF7 transcript, it is possible that the regulation of
IRF7 occurs through miR-146a-mediated regulation of IRAK1
and/or TRAF6. The regulation of IRF7 would in turn limit
production of IFN-? (14), thereby limiting the antiviral re-
sponse. We also noted that a more direct mediator of inter-
feron signaling, signal transducer and activator of transcription
1 (STAT1), was similarly missed in our analysis, since it was
suppressed above our threshold in only one experiment while
it was suppressed by 1.8- to 1.9-fold in the second experiment
(Fig. 7; see also Tables S1 and S2 in the supplemental mate-
rial). In addition, we identified an 8-mer miR-146a seed se-
quence in the 3? UTR of STAT1 (ensembl.org transcript) (Fig.
1956 CAMERON ET AL.J. VIROL.
7). While IRAK1 and STAT1 are possible candidates for direct
mediators of interferon suppression, STAT1 is interesting
since it is more directly upstream of activation of interferon-
Despite the possible negative feedback mechanism, it is
likely that miR-146a also targets other pathways. In this con-
text, we note that although LMP1 induces the expression of a
relatively large number of genes, it has also been shown to
suppress the expression of some genes, one of which is RGS13
(4). As shown in Table 2, miR-146a suppresses the expres-
sion of RGS13, suggesting that LMP1 may suppress RGS13
(and perhaps other genes) in part through the induction of
Support for this project was provided by funding from the Tulane
Cancer Center and grants awarded to E. Flemington by the National
Support was also provided by a National Institutes of Health COBRE
grant, P20 RR020152.
We thank Nancy Raab-Traub for providing the CMV-FLAG-LMP1
vector. We also thank Xiaochuan Zhou and LC Sciences for miRNA
microarray expertise and Xiaofeng Hu (Tulane Cancer Genetic Core)
for her expertise with the Agilent microarray studies.
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