JOURNAL OF VIROLOGY, July 2011, p. 7296–7311
Copyright © 2011, American Society for Microbiology. All Rights Reserved.
Vol. 85, No. 14
Identification and Expression Analysis of Herpes B
Virus-Encoded Small RNAs?†
Melanie A. Amen1,3and Anthony Griffiths1,2,3*
Department of Virology and Immunology, Texas Biomedical Research Institute, San Antonio, Texas1;
Southwest National Primate Research Center, San Antonio, Texas2; and Microbiology and
Immunology Track of the Integrated Multidisciplinary Graduate Program, University of
Texas Health Science Center, San Antonio, Texas3
Received 11 March 2011/Accepted 25 April 2011
Herpes B virus (BV) naturally infects macaque monkeys and is genetically similar to herpes simplex virus
(HSV). Zoonotic infection of humans can cause encephalitis and if untreated has a fatality rate of ?80%. The
frequent use of macaques in biomedical research emphasizes the need to understand the molecular basis of BV
pathogenesis with a view toward improving safety for those working with macaques. MicroRNAs (miRNAs) are
small noncoding RNAs that regulate the expression of mRNAs bearing complementary target sequences and
are employed by viruses to control viral and host gene expression. Using deep sequencing and validation by
expression in transfected cells, we identified 12 novel BV-encoded miRNAs expressed in lytically infected cells
and 4 in latently infected trigeminal ganglia (TG). Using quantitative reverse transcription-PCR (RT-qPCR),
we found that most of the miRNAs exhibited a high level of abundance throughout infection. Further analyses
showed that some miRNAs could be generated from multiple transcripts with different kinetic classes, possibly
explaining detection throughout infection. Interestingly, miRNAs were detected at early times in the absence
of viral gene expression and were present in purified virions. In TG, despite similar amounts of viral DNA per
ganglion, it was notable that the relative amount of each miRNA varied between ganglia. The majority of the
miRNAs are encoded by the regions that exhibit the most sequence differences between BV and HSV. Addi-
tionally, there is no sequence conservation between BV- and HSV-encoded miRNAs, which may be important
for the differences in the human diseases caused by BV and HSV.
Viruses have evolved numerous mechanisms to regulate
the expression of their genome in an optimal manner. These
include transcriptional and posttranscriptional regulatory mecha-
nisms. Of interest is the capacity of viruses to encode micro-
RNAs (miRNAs), which are small noncoding RNA molecules
that modulate gene expression posttranscriptionally. Cur-
rently, most reports of virus-encoded miRNAs are associated
with herpesviruses. The family of these viruses includes large
enveloped viruses with double-stranded DNA genomes that
cause diseases of great medical and veterinary importance, for
which there are limited treatments and no cures. A hallmark of
herpesviruses is their ability to establish and maintain latent
infections, during which there is limited virus gene expression.
Small RNA molecules are nonantigenic and thus appear ide-
ally suited to be exploited during latency. Herpes simplex vi-
ruses 1 and 2 (HSV-1 and -2) are members of the alphaher-
pesvirus subfamily and are characterized by their ability to
establish a latent infection in neurons, typically in the trigem-
inal ganglia (TG) for HSV-1 or dorsal root ganglia for HSV-2
(28). To date, 16 HSV-1-encoded miRNAs and 17 HSV-2-
encoded miRNAs have been identified and shown to be ex-
pressed during the lytic and latent phases of the virus life cycle
(14, 22, 35, 36, 38).
A close relative of HSV, herpes B virus (BV; Macacine
herpesvirus 1; herpes B) is an alphaherpesvirus that is enzootic
in macaque monkeys (genus Macaca) (44). Infection in its
natural host usually results in only mild localized self-limiting
or asymptomatic infections (44). However, BV can zoonoti-
cally infect humans and is associated with an extremely high
mortality rate (20). Left untreated, BV typically causes enceph-
alitis and paralysis, resulting in ?80% lethality (44). Even with
timely antiviral therapy, BV infection results in ?20% lethal-
ity. Many survivors suffer severe neurological complications
and need to be maintained on high doses of antiviral therapy
Several macaque species, particularly rhesus (Macaca mu-
latta) and cynomolgus (Macaca fascicularis) monkeys, are com-
monly used in biomedical research. According to the USDA
Animal Care Annual Report of Activities for 2007 (41), ap-
proximately 70,000 nonhuman primates were used in research
and the majority of those are likely to have been rhesus ma-
caques. BV is present in most macaque colonies. Thus, animal
care staff are at high risk for BV infection. Moreover, despite
great financial investment to generate BV-free colonies, all
macaque monkeys need to be treated as potentially infected.
Therefore, from a safety and financial viewpoint, BV is a great
impediment for those working with macaques in the research
setting. Due to the highly pathogenic nature of BV, the Cen-
ters for Disease Control and Prevention (CDC) has classified
BV as a Risk Group 4 agent that may only be propagated in a
maximum containment laboratory (Biosafety Level 4 [BSL-4]).
* Corresponding author. Mailing address: Department of Virology
and Immunology, Texas Biomedical Research Institute, P.O. Box
760549, San Antonio, TX 78227. Phone: (210) 258-9557. Fax: (210)
670-3329. E-mail: firstname.lastname@example.org.
† Supplemental material for this article may be found at http://jvi
?Published ahead of print on 4 May 2011.
Additionally, the U.S. Department for Health and Human
Services has designated the virus and viral DNA as select
There is mounting evidence supporting the notion that her-
pesvirus-encoded miRNAs are important for virus pathogen-
esis. Some important regulatory functions of herpesvirus-en-
coded miRNAs are thought to include control of latent/lytic
cycle entry and exit and immune evasion, in addition to cell
survival and proliferation (7). We have reported that BV en-
codes three miRNAs using computational predictions and val-
idation by Northern blot hybridization (6). Based on studies of
HSV and other alphaherpesviruses, we hypothesized that BV
encodes more miRNAs than originally detected and chose to
use ultra-deep sequencing technology to identify additional
BV-encoded miRNAs. We examined RNA from both naturally
infected TG harvested from macaque monkeys and lytically
infected cells. We reasoned that looking throughout the course
of BV infection would provide the maximum opportunity for
complete identification of BV-encoded miRNAs.
MATERIALS AND METHODS
Cells, tissues, and viruses. African green monkey (Vero) cells and HeLa cells
were obtained from the American Type Culture Collection and maintained in
Dulbecco’s modified Eagle’s medium, supplemented with 10% fetal bovine se-
rum, at 37°C and 5% CO2. Cynomolgus macaque TG were obtained at necropsy
from the Southwest National Primate Research Center (SNPRC), immediately
frozen in liquid nitrogen, and stored at ?80°C in BSL-3 containment until
processed. This occurs as rapidly as 20 min following euthanasia. BV strain
E2490 (NCBI reference sequence no. NC_004812) was used for the in vitro
experiments in this study.
Biosafety. BV is classified as a Risk Group 4 agent (CDC) and may only be
propagated in a maximum containment laboratory. All experiments using infec-
tious virus were performed in the Animal Biosafety Level 4 (ABSL-4) laboratory
at the Texas Biomedical Research Institute (certified by the CDC) by trained
personnel wearing protective biosafety suits.
Illumina sequencing and data analysis. cDNA libraries for deep sequencing
were prepared using the small RNA sample prep kit v1.5.0 protocol from Illu-
mina. Between 100 ng and 500 ng small enriched RNA was ligated to adapters,
followed by reverse transcription (RT) and PCR amplification. The amplified
cDNA libraries were resolved using a 6% polyacrylamide Tris-borate-EDTA
(TBE) gel. cDNAs that corresponded to approximately 18 to 30 nucleotides (nt)
were identified by comparison to an SRA ladder (Illumina) and excised from the
gel. The cDNA was eluted from the gel for 4 h with 100 ?l 1? gel elution buffer
(Illumina). Gel debris was removed using Spin-X cellulose acetate filters
(Illumina), and the cDNA was precipitated with 1 ?l glycogen (Ambion), 3 M
sodium acetate (Ambion), and 325 ?l chilled 100% ethanol. After being
washed with 70% ethanol, the cDNA pellet was resuspended in resuspension
Raw sequence data were collapsed into a single file of nonredundant se-
quences with an associated abundance. Adapter sequences were removed, and
the file was converted to FASTA format. The analysis was performed with
Formatdb, Megablast, filter_alignments, excise_candidate, auto_blast, and
miRDeep scripts from the miRDeep package (15), together with the BV
genomic sequence (NC_004812) and miRBase database (release 14). These
algorithms consider pre-miRNA structure, the relative frequency at which a
mature miRNA sequence and star sequence were observed, and likelihood that
the mature miRNA was generated through a Dicer-dependent mechanism. How-
ever, for the very rare reads that are sometimes observed for alphaherpesvirus
miRNAs, considering the relative frequency of mature sequences to star se-
quences can be too stringent. Therefore, reads that failed based on this criterion
were further analyzed manually: a candidate sequence was considered a potential
miRNA when the sequence was present in a hairpin that was predicted as the
lowest free-energy structure using mfold.
Plasmids, small interfering RNAs (siRNAs), and transfections. To generate
plasmid pAMBVgB, the BV virion membrane glycoprotein B (gB; UL27) coding
sequence was amplified from BV strain E2490 DNA by PCR and cloned into
pCR2.1 TOPO using the TOPO TA cloning system (Invitrogen). The PCR
primers were as follows: BV glycoprotein B forward primer (5?-TCCGGGCCC
GGAATGCGGCCCCGCGCC-3?) (IDT) and reverse primer (5?-GCGGCCGC
A portion of the second exon of immediate early protein ICP0 coding se-
quence (between bases 122943 and 123375) was synthesized as a “minigene” by
IDT using the published BV sequence, yielding pIDTSMART-KAN:ICP0. The
minigene was digested with HindIII and XhoI (NEB) to release the ICP0 se-
quence insert, which was then inserted into pCR2.1 TOPO using the TOPO TA
cloning system (Invitrogen) to yield pMAICP0.
To generate miRNA expression plasmids, each miRNA sequence together
with ?200 nt of flanking sequence was amplified from BV strain E2490 DNA by
PCR and first subcloned into pCR2.1TOPO or pCR-Blunt II TOPO using the
TOPO cloning system (Invitrogen). The PCR primers, along with the TOPO
subcloning vectors and enzymes, are listed in Table S1 in the supplemental
material. To generate a cassette-type vector that can accommodate cloned in-
serts from pCR2.1 TOPO or pCR-Blunt II TOPO, regardless of initial insert
orientation, a multiple cloning site was cloned into pSIREN-Retro-Q-ZsGreen1
(Clontech), using a duplex of synthetic oligonucleotides. The oligonucleotides
used were as follows: 5?-GATCCGTTAACGCGGCCGCGGGCCCCCAGTGT
GCTGGTCTAGAT-3? and 5?-AATTATCTAGACCAGCACACTGGGGGCC
CGCGGCCGCGTTAACG-3? (BamHI, HpaI, NotI, ApaI, BstXI, XbaI). The
recognition sequence for EcoRI was destroyed to serve as a diagnostic tool.
Oligonucleotides were annealed, and the resulting duplex was ligated to BamHI-
and EcoRI-digested pSIREN-Retro-Q-ZsGreen1 vector to yield pSIREN-MCS.
miRNA sequences were excised from the TOPO vectors and cloned into
pSIREN-MCS using the appropriate restriction sites for correct orientation
of the insert.
All constructs generated by oligonucleotide cloning or PCR were confirmed to
be correct by sequencing at the Nucleic Acids Core Facility at the University of
Texas Health Science Center, San Antonio (UTHSCSA).
Candidate miRNA sequence-containing plasmids were transfected into HeLa
cells using the Neon transfection system (Invitrogen) according to the manufac-
turer’s recommendations. To confirm that the candidate miRNAs were synthe-
sized in a Drosha-dependent manner, 44 nM Drosha siRNA (RNASEN siRNA
identification no. s26492; Ambion) was also cotransfected; this amount of siRNA
had been determined to reduce Drosha expression by approximately 90%. A
second siRNA (Silencer negative control 2 siRNA; Ambion) was used as a
negative control. Briefly, 2 ? 107HeLa cells were resuspended in 100 ?l of buffer
R (Invitrogen), containing 8 ?g plasmid and 44 nM Drosha siRNA or negative
control siRNA. The electroporation was performed using 2 pulses at 1,400 V for
20 ms. After electroporation, cells were plated on 60-mm dishes in 10% fetal calf
serum (FCS) without antibiotics. Forty-eight hours later, total RNA was har-
vested using the mirVana miRNA isolation kit (Ambion) according to the man-
RNA and DNA extractions. To harvest RNA from infected cells, 60-mm dishes
of 80% confluent Vero cells were infected with BV at a multiplicity of infection
(MOI) of 10. At various times postinfection, cells were scraped from the dishes
and pelleted in a low-speed centrifuge; the supernatant was removed and the
pellet was flash-frozen in liquid nitrogen. Dry pellets were stored at ?80°C until
processed. Both large (?200 nt) and small (?200 nt) RNA were harvested from
infected cells using the mirVana miRNA isolation kit (Ambion) according to the
Nucleic acid isolation from macaque TG began in the BSL-3 laboratory.
Lysis/binding solution (600 ?l; from the mirVana kit) was added to each gan-
glion, and the tissues were homogenized on ice using an RNase-free plastic
pestle and tube. The tissues were processed immediately without allowing the
tissue to thaw, thereby preventing the rupture of cells by ice crystals and
the subsequent release of RNases. miRNA homogenate additive (60 ?l; from
the mirVana kit) was added to the homogenized tissue, and the samples were
incubated on ice for 10 min. Fifty microliters of the homogenized tissue was then
set aside for DNA isolation, and the remainder was used for RNA isolation. An
equal volume of acid phenol-chloroform (Ambion) was added to the homoge-
nate set aside for RNA isolation, and the samples were vortexed thoroughly.
Likewise, an equal volume of phenol-chloroform (Ambion) was added to the
homogenate set aside for DNA isolation, and the samples were vortexed thor-
oughly. Samples were then transferred to the BSL-2 laboratory, where the re-
mainder of the RNA isolation was performed using the mirVana miRNA isola-
tion kit according to the manufacturer’s instructions. RNA concentration was
determined by spectrophotometry.
In the BSL-2 laboratory, the samples set aside for DNA isolation were cen-
trifuged and the aqueous layer was removed. DNA was precipitated by adding
one volume of isopropanol (Sigma-Aldrich) and centrifuged for 1 h at 4°C and
14,000 ? g. The supernatant was removed, and the DNA pellet was washed with
VOL. 85, 2011 HERPES B VIRUS-ENCODED miRNAs7297
70% ethanol, followed by resuspension in sterile water and spectrophotometry to
Drug treatments. Various pharmacological agents were used to investigate
whether miRNA accumulation was affected by blocking expression of genes from
particular kinetic classes. To inhibit BV DNA synthesis, which prevents expres-
sion of true late-class genes, cells were pretreated with 300 ?g/ml phosphono-
acetic acid (PAA) (Sigma-Aldrich) for 30 min before cells were infected with BV
at an MOI of 10 in the presence of the same concentration of the drug. At 18 h
postinfection (hpi), cells were processed as described above. To confirm PAA
activity, RNA was harvested from infected cells and subjected to Northern blot
hybridization probed for the true late-class viral gene UL44 (see Fig. 7B).
To inhibit expression of all but immediate early class genes, Vero cells were
pretreated with 100 ?g/ml of the protein synthesis inhibitor cycloheximide
(CHX) (Sigma-Aldrich) for 30 min before cells were infected with BV at an MOI
of 10 in the presence of the same concentration of drug. At 1 hpi, cells were
scraped from the dishes and pelleted in a low-speed centrifuge, the supernatant
was removed, and the pellet was flash-frozen in liquid nitrogen and stored at
?80°C until processed. To confirm CHX activity, uninfected Vero cells were
treated with and without CHX in the presence of 0.1 mCi/ml of [35S]methio-
nine (PerkinElmer) in parallel to the infected cells. After 1 h, the cell lysates
were resolved by SDS-PAGE and processed for autoradiography (see
To inhibit both viral and cellular transcription, actinomycin D (act D; MP
Biomedical) was used similarly to CHX, but at a concentration of 2 ?g/ml. To
confirm act D activity, RNA was harvested from infected cells and subjected to
Northern blot hybridization probed for the immediate early class viral gene ICP4
(see Fig. 7D).
Purification of virions. Virions were purified by centrifugation through a 20%
sorbitol cushion as described by Stinski (34). Briefly, Vero cells were grown to
80% confluence in 150-cm2flasks and infected at an MOI of 10. The cells were
harvested 24 hpi and centrifuged at 2,000 rpm for 5 min. The cell pellet was lysed
in reticulocyte standard buffer (RSB) (10 mM Tris, pH 7.5, 10 mM KCl, 1.5 mM
MgCl2) containing 0.5% (vol/vol) NP-40. The lysate was incubated on ice for 10
min, and the nuclei was pelleted. Viral particles in the supernatant were recov-
ered by centrifugation at 50,000 rpm for 1 h at room temperature in a SW 55 Ti
rotor through 20% (wt/vol) D-sorbitol (Sigma-Aldrich) in 50 mM Tris-HCl, pH
7.5, 1 mM MgCl2, and 100 ?g/ml bacitracin (Sigma-Aldrich). The pellet was
resuspended in RNase ONE buffer (Promega) containing 9.15 ? 107molecules
of synthetic ath-miR-166 miRNA standard (IDT) and 50 U RNase ONE (Pro-
mega). ath-miR-166 is a plant miRNA that is absent from mammalian cells (26),
including Vero cells (M. A. Amen and A. Griffiths, unpublished observations)
and was used to determine the efficiency of RNase digestion of miRNAs. The
reaction mixture was incubated at 37°C for 30 min. To boost digestion, an
additional 50 U RNase ONE (Promega) was added and the reaction mixture was
incubated for a further 30 min at 37°C. To dissociate aggregates, 5 mM urea
(Fisher) was added and the mixture was sonicated for 10 to 15 s. An equal
volume of phenol-chloroform (Ambion) was added and vortexed thoroughly
before the sample was transferred to the BSL-2 laboratory. Next, the aqueous
phase was harvested and half was devoted to RNA isolation and half to DNA
isolation. RNA isolation was performed using the mirVana miRNA isolation kit
(Ambion) according to the manufacturer’s instructions, and the final RNA con-
centration was determined by spectrophotometry. DNA was precipitated by
adding one volume of isopropanol (Sigma-Aldrich) and centrifuged for 1 h at 4°C
and 14,000 ? g. The supernatant was removed, and the DNA pellet was washed
with 70% ethanol, followed by resuspension in sterile water and spectrophotom-
etry to determine concentration.
Real-time qPCR. To determine if macaque TG contained BV DNA, we used
a previously reported quantitative PCR (qPCR) assay (21). The primers and
probe were designed to detect the conserved glycoprotein B (gB) sequence of BV
and differentiate between BV and other alphaherpesviruses such as HSV. A
standard curve was generated using DNA isolated from uninfected Vero cells
spiked with a known amount of pAMBVgB.
To account for the possibility that an infected TG was supporting lytic cycle
replication at the time of necropsy, a reverse transcription-qPCR (RT-qPCR)
assay was developed to detect expression of the immediate early ICP0 gene.
Large RNA (5 ng/?l) was subjected to reverse transcription using 500 nM ICP0
reverse transcription primer (5?-AACTGGTGCCCCTACCAC-3?) (IDT), 5?
iScript select reaction mix (Bio-Rad), GSP enhancer solution (Bio-Rad), and
iScript reverse transcriptase (Bio-Rad) in a total volume of 20 ?l. RTs were
performed at 42°C for 60 min and 85°C for 5 min. One microliter of the resulting
cDNA was added to 1? iQ SYBR green Supermix (Bio-Rad), 500 nM ICP0
forward primer (5?-CAGACGTGCCTCGCGTA-3?) (IDT), and 500 nM ICP0
reverse primer (5?-TCGACAACGCGTACCCG-3?) (IDT) in a total volume of
25 ?l. A standard curve was generated using large RNA isolated from uninfected
Vero cells spiked with a known amount of ICP0 RNA in vitro transcribed using
an mMESSAGE mMACHINE kit (Ambion) from pMAICP0. Reverse transcrip-
tion and qPCR were performed using the iScript select cDNA synthesis kit
(Bio-Rad) and iQ SYBR green Supermix (Bio-Rad), respectively, according to
the manufacturers’ instructions. To determine if there was equivalent RNA
recovery between samples of large RNA, a previously reported RT-qPCR assay
was utilized to detect RPL13a, a cellular marker shown to be effective for use
with rhesus macaque tissues (1). The sequence of RPL13a for cynomolgus
macaque has several base differences from the rhesus macaque sequence. To
account for these differences, the following forward primer was used: 5?-CTTG
For analyses of relative expression of miRNAs, stem-loop RT-qPCR using a
single RT reaction mixture containing multiple RT primers was used as previ-
ously described (13). Stem-loop RT-qPCR of miRNAs has been shown to be
highly specific for a particular miRNA, even able to differentiate between
miRNAs of different sizes and miRNAs that have single-base substitutions in an
miRNA sequence (11). Briefly, 250 ng small RNA was reverse transcribed with
a final concentration of 12.5 nM of each stem-loop RT primer (IDT) (see Table
S2 in the supplemental material), 1? RT buffer (Applied Biosystems), 0.25 mM
(each) deoxynucleoside triphosphates (dNTPs) (Applied Biosystems), 20 U/?l
RNase inhibitor (Applied Biosystems), and 3.33 U/?l MultiScribe reverse trans-
criptase (Applied Biosystems). The volume of the RT reaction mixture was
adjusted according to the number of PCRs to be performed. The RT reactions
were performed at 16°C for 30 min, followed by 42°C for 30 min and 85°C for 5
min. The efficiencies of two reactions (hbv-mir-B3RC-3P and hbv-mir-B14RC-
3P) were improved by increasing the RT temperature. These RTs were per-
formed at 19°C for 30 min, followed by 48°C for 30 min and 85°C for 5 min. For
real-time PCR, a 20-?l reaction mixture was made containing 1.33 ?l reverse
transcription reaction, 1? TaqMan universal PCR master mix (Applied Biosys-
tems), 0.2 ?M TaqMan probe, 1.5 ?M forward primer, and 0.7 ?M reverse
primer (5?-AGTGCAGGGTCCGAGGTA-3?) (IDT). The reactions were per-
formed in triplicate and incubated in a 96-well plate at 95°C for 10 min, followed
by 40 cycles of 95°C for 15 s and 60°C for 1 min. Detection of the most commonly
sequenced isoform of each miRNA was performed using forward primers (IDT)
and TaqMan probes (Applied Biosystems). The primer and probe sequences are
listed in Table S2 in the supplemental material.
To confirm that Drosha mRNA was knocked down in the presence of the
Drosha siRNA, a Drosha qPCR assay was used (Applied Biosystems,
Hs00294820_m1). An assay to detect 18S rRNA (Hs03928990_g1; Applied Bio-
systems) was used as a control. Briefly, 100 ng/?l total RNA was DNase treated
using a Turbo DNA-free kit (Ambion) according to manufacturer’s instructions.
The DNase-treated samples were reverse transcribed with oligo(dT)20primers
using the SuperScript III first-strand synthesis system (Invitrogen) according to
the manufacturer’s instructions. For the real-time PCR, a 20-?l reaction mixture
was made containing 1 ?l Drosha or 18S TaqMan assay mix, 10 ?l TaqMan
universal PCR master mix, no AmpErase uracil N-glycosylase (UNG) (Applied
Biosystems), and 1 ?l cDNA. The reactions were performed in triplicate and
incubated in a 96-well plate at 95°C for 10 min, followed by 40 cycles of 95°C for
15 s and 60°C for 1 min.
To estimate the number of molecules of each miRNA packaged in virions,
standard curves were generated using small RNA isolated from uninfected Vero
cells spiked with a known amount of synthetic RNA oligonucleotides (IDT)
representing each miRNA sequence. Eighty-five nanograms of small RNA iso-
lated from purified virions treated with and without RNase was used for RT-
qPCR of the miRNAs as described above. To estimate the number of viral
genomes in each sample, 293 ng DNA isolated from purified virions treated with
and without RNase was used for the qPCR of gB as described above.
To determine the changes in levels of individual miRNAs between multiple
samples (time course and multiple TG), we used a relative quantification strat-
egy. To determine if the cellular miRNA Let-7a was an appropriate normaliza-
tion factor for these assays, we measured its expression during infection. Using
absolute quantification we determined that it was expressed at constant levels
between samples harvested at various times postinfection using RT-qPCR (see
Fig. S1 in the supplemental material). This confirmed our previous observation,
using Northern blot hybridization that Let-7a expression was unaffected by virus
infection (6). Briefly, a standard curve was generated using a synthetic RNA
oligonucleotide (IDT) that represents the 22-nt form of Let-7a. Reverse tran-
scription and qPCR was performed as described above (see Table S2 in the
Northern blot hybridization. RNA species of ?200 nt isolated from infected
cell lysates (1 hpi and 18 hpi) were resolved using a glyoxal-agarose gel and
transferred to a nylon membrane using the NorthernMax-Gly kit (Ambion)
7298AMEN AND GRIFFITHS J. VIROL.
according to the manufacturer’s instructions. The RNA was cross-linked to the
membrane using a Stratalinker (Stratagene). The membranes were probed with
DNA oligonucleotides (IDT) end-labeled with [32P]ATP (MP Biomedicals, Ir-
vine, CA). Unincorporated nucleotides were removed using micro Bio-Spin
columns, (Bio-Rad) according to the manufacturer’s instructions. Only probes
with specific activities exceeding 5 ? 106dpm/pmol, as measured using a liquid
scintillation counter, were used. The sequences of the probes are detailed in
Table S3 in the supplemental material. Hybridizations were performed in
ULTRAhyb-Oligo buffer (Ambion) according to the manufacturer’s instructions.
Probed membranes were exposed to phosphor storage screens (GE Life Sci-
ences) and detected on a Storm phosphorimager (GE Life Sciences).
Detection and quantification of viral DNA in naturally in-
fected macaque TG. BV establishes a latent infection in the TG
of macaques (reviewed in reference 44). To identify BV-en-
coded miRNAs expressed during latent infection, we harvested
macaque TG at necropsy. To determine if a particular TG to
be tested contained BV DNA, we employed a real-time PCR
assay previously developed to measure BV DNA in macaque
TG (20). The levels of viral DNA varied only slightly between
all four TG (Fig. 1), estimated to range between 1.4 ? 104and
2.4 ? 105molecules per TG. It was crucial to discount the
possibility that the virus had reactivated in these TG, as we
wished to identify miRNAs expressed during latency. To ad-
dress this, we developed an RT-qPCR assay to detect ICP0
mRNA, an immediate early transcript encoding a protein re-
quired for HSV reactivation from latency (10). The limit of
detection for these assays was determined to be 1.9 ? 102
molecules per TG. No ICP0 transcripts were detected in any
tested TG. Furthermore, using RNA isolated from lytically
infected Vero cells, we determined that 1.7 ? 103molecules of
ICP0 were present per infected cell at 3 hpi. Together, these
data are consistent with absence of lytic replication in all re-
ported TG, thereby supporting the notion that the virus was
present in the latent state.
Identification of BV-encoded small RNAs. cDNAs corre-
sponding to the approximate size of miRNAs were subjected to
deep sequencing. At 3 hpi, 12,595,747 total reads and 3,414,075
unique reads were returned; at 9, 15, and 21 hpi and in the TG
the numbers were 7,289,829 and 1,209,685, 8,303,409 and
1,409,962, 6,679,109 and 1,246,758, and 7,904,359 and 1,907,690,
respectively. Similarly to results in a report describing HSV
miRNAs expressed in human TG (39), the most frequently
observed miRNAs in monkey TG were members of the Let-7
family. The most frequently observed miRNA in all lytically
infected Vero cells was miR-21.
A sequence was considered an miRNA candidate when (i)
the sequence was within a hairpin-like RNA secondary struc-
ture and (ii) the predicted miRNA precursor structure and the
position of the small RNA sequence met the requirements of
a genuine pre-miRNA as described by Ambros et al. (2).
Twenty-eight sequences fulfilled these criteria and were fur-
ther tested using stem-loop RT-qPCR assays to detect the
candidate miRNAs in infected cells. Of those 28 candidate
sequences, 10 were not amplified, and we concluded that they
were not miRNAs. To determine if the remaining candidate
sequences were miRNAs or products of RNA degradation, the
remaining 18 candidate sequences were then cloned into ex-
pression vectors and transfected into Vero cells. As an addi-
tional method to test if a candidate sequence is an miRNA, we
asked if the transfected sequence was processed in a Drosha-
dependent manner. Drosha is an RNase III enzyme that is
responsible for cleaving the primary miRNA (pri-miRNA) into
the characteristic stem-loop precursor miRNA in the nucleus
and is necessary for miRNA biogenesis (23). To this end, an
siRNA targeting Drosha (or a control siRNA) was cotrans-
fected with each miRNA expression plasmid. Small RNAs that
are not miRNAs will be unaffected by the reduction of Drosha.
Only genuine miRNAs will be affected by the reduction of
Drosha. In the presence of the Drosha siRNA, Drosha mRNA
was shown to be present at approximately 15% of the amount
compared to when a control siRNA is transfected (Fig. 2).
Passing all criteria were 13 miRNA sequences resulting from
10 hairpin precursors (Fig. 3). All recovered sequence reads of
the BV-encoded miRNAs, including the number of hits gen-
erated in cells harvested at each time point or in TG, are listed
in Table 1. Several of the miRNA sequences exhibited variable
5? and 3? ends, which are also shown in Table 1. The majority
of the novel miRNAs were located in the repeat region of the
BV genome (Fig. 4). A few miRNAs were located in the
unique long (UL) region, and none were found in the unique
short (US) region (Fig. 4). Similar observations have been
made for HSV-1- and HSV-2-encoded miRNAs (14, 22, 40).
It is interesting to note that the majority of the miRNAs are
encoded within the long repeat (RL) region, in which there is
no sequence homology to HSV or in the short repeat (RS)
region where there is an additional ?1.5 kb of nonhomologous
sequence relative to HSV (Fig. 4) (17). The majority of the
miRNAs detected in the repeat regions map to a locus that in
HSV would encode the latency-associated transcript (LAT),
the only abundantly expressed viral RNA during latency. Three
miRNAs were recovered in the ULregion. The position and
location of all the miRNAs detected are shown in Table 1.
Expression of BV-encoded miRNAs in latently and produc-
tively infected cells. Since the deep sequencing technique was
not quantitative and is a poor predictor of the amount of a viral
miRNA in an infected cell (16), we chose to analyze expression
FIG. 1. Analysis of naturally infected cynomolgus macaque TG.
Detection of BV glycoprotein B (gB) sequences in macaque isolates
from SNPRC. DNA was isolated from the right (R) or left (L) TG
following necropsy and measured by quantitative PCR. A standard
curve was generated using DNA isolated from uninfected Vero cells
spiked with a known amount of pAMBVgB. The limit of detection
(denoted as l.o.d.) for this assay is 35 molecules per TG. Animal 20408
had no detectable levels above the limit of detection of gB and is used
as a negative. The average values are denoted by number of molecules
of viral DNA per TG, with the standard deviations indicated.
VOL. 85, 2011HERPES B VIRUS-ENCODED miRNAs 7299
FIG. 2. Detection of BV-encoded miRNAs using plasmid-expressed pri-miRNAs. Each miRNA sequence, together with ?200 nt of flanking
sequence, was cloned into an expression vector. Vero cells were transfected with each plasmid and a negative siRNA (siNeg) or Drosha siRNA
(siDrosha), and the total RNA was harvested at 48 hpi. RT-qPCR was used to measure the relative expression of each miRNA and is denoted by
a line above the graph. All values are represented as changes in expression relative to an empty vector plasmid after normalization to values for
a cellular miRNA, Let-7a. As a control, Drosha expression was shown to be decreased in the presence of a Drosha siRNA using RT-qPCR and
is depicted to the right of each miRNA. All values are represented as changes in expression relative to cells transfected with a negative siRNA as
denoted by 1 after normalization to values for a cellular RNA, 18S. Samples were assayed in triplicate at least three times, and average values are
displayed, with standard deviations indicated.
of virus-encoded miRNAs by stem-loop RT-qPCR in latently
infected TG and throughout productive infection. This tech-
nique has been shown to have a high level of specificity for the
precise miRNA sequence for which the probes are designed.
Notably, miRNAs with only single-base differences in either
length or sequence are readily differentiated using this tech-
nique (11). Expression levels were normalized to levels of a
cellular miRNA, Let-7a, and values are expressed relative to a
negative control sample of small RNA isolated from a BV
DNA-negative TG or noninfected Vero cells (Fig. 5 and 6).
The expression of Let-7a was shown to be unaltered during
virus infection (see Fig. S1 in the supplemental material) (6).
The use of relative expression permits the comparisons be-
tween the relative abundance of an individual miRNA under
different conditions (e.g., at different times during infection),
but not between the different miRNAs, and has been used in
other studies of virus-encoded miRNAs (24, 38–40). We ana-
lyzed RNA recovered from cells harvested at 0, 3, 6, 9, 15, 18,
21, and 24 hpi, one BV-negative TG, and four BV-positive TG.
We reasoned that several of the miRNAs not detected in the
TG during deep sequencing and located in the region that
encodes the LAT transcript in HSV may still be expressed
during latency; thus, ganglia were tested for all miRNAs in this
The following four BV-encoded miRNAs were detected in
latently infected TG by RT-qPCR: hbv-mir-B8-3P, -B20-5P,
-B22-3P, and -B26-5P (Fig. 5). hbv-mir-B20-5P was readily
detectable in all positive ganglia, but at variable levels (Fig. 5).
The other miRNAs also exhibited inconsistent expression be-
tween TG (Fig. 5). Although identified from TG during deep
sequencing, hbv-mir-B7-5P was not detectable in any TG by
RT-qPCR. This observation suggests that a combination of
techniques may be required to discover all miRNAs encoded in
a given system.
RT-qPCR of BV-infected cells at various times postinfection
was used to analyze the expression of 13 BV miRNAs during
lytic infection (Fig. 6). Surprisingly, most of the miRNAs did
not exhibit the expected regulated cascade-type pattern of al-
phaherpesvirus gene expression. Rather, most of the miRNAs
were abundantly detected at early times, and either continued
FIG. 3. Predicted hairpin structures of BV-encoded pre-miRNAs. The structures of BV-encoded pre-miRNAs were predicted by minimal
free-energy folding using mFold (http://mfold.rna.albany.edu/). The most abundant species of each miRNA are bold.
VOL. 85, 2011 HERPES B VIRUS-ENCODED miRNAs7301
TABLE 1. Recovered sequence reads of BV-encoded miRNAs from deep sequencing
Prevalence (hpi or ganglia)e
UL, 62 nt us UL30
UL, 126 nt ds UL45
UL, 70 nt ds UL45
RS, minor LATd
RS, minor LAT
RS, minor LAT
RL, minor LAT
7302AMEN AND GRIFFITHS J. VIROL.
RL, minor LAT
RL, minor LAT
RL, minor LAT
RS, minor LAT
RL, minor LAT
RL, minor LAT
aBold sequences were used for RT-qPCR.
bPosition noted for internal repeat only if located in repeat regions; ?, forward orientation; ?, reverse orientation.
cus, upstream; ds, downstream.
dInferred from latent expression from hbv-mir-B22 through hbv-mir-B8.
eNumber of times sequence is represented in deep sequencing data.
VOL. 85, 2011 HERPES B VIRUS-ENCODED miRNAs7303
to increase in abundance or remained at constant levels as
infection progressed (Fig. 6). An exception is hbv-miR-B19-5P,
which was first detected at 15 hpi.
The absolute amount of each miRNA was determined at the
time of maximal expression using RT-qPCR assays calibrated
with synthetic standards. The measurements of each miRNA
are in the following format: miRNA, time postinfection as-
sayed, number of molecules per cell. The measurements are as
follows: hbv-mir-B2-3P, 21 hpi, 94; hbv-mir-B3RC-5P, 21 hpi,
20; hbv-mir-B3RC-3P, 15, 94; hbv-mir-B7-5P, 21, 3,994; hbv-
mir-B8-5P, 21, 2,897; hbv-mir-B8-3P, 21, 32,757; hbv-mir-
B14RC-3P, 18, 303; hbv-miR-B19-5P, 21, 88; hbv-mir-B20-5P,
9, 2,048; hbv-mir-B20-3P, 21, 2,127; hbv-mir-B21RC-5P, 21, 57;
hbv-mir-B22-3P, 21, 2,702; hbv-mir-B26-5P, 9, 17,313. Further
work will be required to determine if these levels of expression
are biologically meaningful, particularly the lower values. It
has been proposed by others that expression of ?100 mole-
cules of an miRNA per cell suggests an active miRNA (9).
However, it should be noted that the measurements described
above reflect a steady-state level of miRNA abundance, which
is dependent on both expression and stability. Recent evidence
suggests that miRNA stability is influenced by target abun-
dance; thus, an miRNA may be present at low levels because of
a large amount of target in a cell (3). Further work is also
required to determine the effect of stability on the abundance
of the miRNAs.
Expression analysis of BV-encoded miRNAs using pharma-
cological inhibitors of viral gene expression. Expression of
alphaherpesvirus gene can be grouped into three kinetic classes,
immediate early, early, or late, based on their requirement for
the expression of viral proteins and viral DNA replication (19).
To determine the specific kinetic class of each of the viral
miRNAs, viral infections were performed in the presence of
various pharmacological agents.
PAA inhibits DNA replication. Therefore, BV transcripts
that are expressed in a DNA replication-dependent manner—
true late-class transcripts—are not expressed in the presence of
PAA. The absence of the BV true late gene UL44 (gC) mRNA
in cells infected with BV shows that PAA effectively inhibits
BV-late gene expression (Fig. 7B). In the presence of PAA,
hbv-mir-B2-3P, -B14RC-3P, and -B21RC-5P were not affected,
suggesting they could be generated from immediate early, early,
or leaky late transcripts (Fig. 7A). The remaining miRNAs
appear to fall into two categories: (i) hbv-mir-B7-5P and
hbv-mir-B19-5P were undetectable in the presence of PAA,
suggesting they are generated with true-late kinetics; (ii) hbv-
mir-B3RC-5P, -B3RC-3P, -B8-5P, -B8-3P, -B20-5P, -B20-3P,
-B22-3P, and -B26-5P exhibit intermediate levels of expression
in the presence of PAA, which suggests they may be generated
with leaky late, early, or immediate early kinetics, or some
combination of multiple kinetic classes.
We considered that miRNAs readily detectable at 3 hpi (Fig.
FIG. 4. Genomic location of BV-encoded miRNAs. The prototypic arrangement of the BV genome is depicted. The unique long (UL) and
unique short (US) sequences (thick lines) are surrounded by terminal repeat (TR) and internal repeat (IR) regions (boxes). One copy of the
internal repeat sequences (IRSand IRL) is expanded below the BV genome. The relative sizes, locations, and orientations of the transcripts
encoded by the corresponding region of HSV are denoted by arrows. The transcriptional map of HSV is shown, as this region of BV has yet to
be carefully mapped. Bent lines indicate sequences removed by splicing. Latency-associated transcripts are abbreviated as LAT. Locations of the
miRNAs are denoted by filled and open vertical boxes. Open boxes represent miRNAs that were detected in latently infected TG and lytically
infected Vero cells. Filled boxes represent miRNAs that were only detected in lytically infected Vero cells. Vertical boxes above the line indicate
that pri-miRNAs are synthesized in a left-to-right direction and those below are synthesized in a right-to-left direction. The boxes with a large
checkerboard pattern denote nonhomologous sequence relative to HSV. The box filled with a small checkerboard pattern denotes an ?1.5-kb
additional sequence relative to that of HSV.
7304 AMEN AND GRIFFITHSJ. VIROL.
6) were potentially expressed with immediate early kinetics
(hbv-mir-B8-5P, -B8-3P, -B20-5P, -B20-3P, -B22-3P, and -B26-
5P). Only immediate early class genes are synthesized in the
absence of viral protein synthesis. Cells were infected in the
presence of the protein synthesis inhibitor CHX, and RNA
was harvested at 60 min postinfection. Expression of candidate
immediate early miRNAs was quantified by RT-qPCR and
compared to RNA harvested from infected cells not treated
with CHX (Fig. 7E). miRNAs hbv-mir-B8-5P, -B8-3P, -B20-
5P, -B20-3P, and -B26-5P were expressed at similar levels in
the presence or absence of CHX consistent with immediate
early class expression of their pri-miRNAs (Fig. 7E). hbv-mir-
B22-3P was slightly affected by CHX (Fig. 7E), suggesting that
most of the miRNA detected at 60 min postinfection arose
from an immediate early pri-miRNA, in addition to miRNA
that arose from pri-miRNAs from another kinetic class. As a
control, effective inhibition of de novo protein synthesis was
demonstrated by using a pulse of [35S]methionine (Fig. 7C);
protein from these cells was resolved by SDS-PAGE, and the
gel was stained with Coomassie blue and then exposed to a
phosphor storage screen. Equal loading of both lanes on the
gel was demonstrated by similar Coomassie blue intensities,
and inhibition of protein synthesis was demonstrated by the
absence of incorporated radiolabel in the CHX-treated cells
Several herpesviruses have been reported to package RNA
molecules into virions (5, 8, 12, 18, 30). To investigate the
possibility that BV-encoded miRNAs were detectable in the
absence of viral gene expression—suggestive of incorporation
into the virion—cells were infected in the presence of the
inhibitor of transcription act D, and RNA was harvested at 60
min postinfection. Expression of candidate virion-packaged
miRNAs was quantified by RT-qPCR and compared to RNA
harvested from infected cells not treated with act D. miRNAs
hbv-mir-B8-5P, -B8-3P, -B20-5P, -B20-3P, and -B26-5P were
all detected from cells infected in the presence of act D,
thereby suggesting that miRNAs may be incorporated into
virions (Fig. 7E). As a control, effective inhibition of de novo
viral transcript synthesis was demonstrated by showing that
ICP4, an immediate early class viral gene, is not expressed in
the presence of act D (Fig. 7D). For each miRNA, the amounts
detected were lower than those for RNA harvested from in-
fected cells treated with CHX. The amount of each miRNA
detected in the presence of CHX likely represents newly syn-
thesized miRNAs plus miRNAs brought into the cell with the
virion. Thus, the difference between the amount observed with
CHX and that with act D likely represents only newly synthe-
Analysis of miRNAs harvested from purified virions. To
further investigate the possibility that miRNAs detected in the
presence of act D were packaged into BV virions, we harvested
RNA from purified virions. To reduce the possibility that cy-
toplasmic miRNAs copurified with virions, the virions were
RNase treated. Prior to RNase treatment, the virions were
spiked with a known amount of synthetic ath-mir-166, an
miRNA from Arabidopsis thaliana that is absent from mam-
FIG. 5. BV miRNA expression levels in naturally infected cynomolgus macaque TG. Small RNA was isolated from the right (R) or left (L) TG,
and indicated miRNA expression was then measured by RT-qPCR. As shown in Fig. 1, TG from animal number 20408 is BV negative, and the
remaining TG are BV positive. All values are given as changes in expression relative to those of animal number 20408 after normalization to values
for a cellular miRNA, Let-7a. Samples were assayed in triplicate at least three times, and average values are displayed, with standard deviations
indicated. ND, not detected.
VOL. 85, 2011 HERPES B VIRUS-ENCODED miRNAs7305
FIG. 6. BV miRNA expression determined by RT-qPCR in lytically infected cells. Vero cells were infected with BV at a high multiplicity
(MOI ? 10). Small RNA was isolated at 0, 3, 6, 9, 15, 18, 21, and 24 hpi and measured by RT-qPCR to quantify the expression of indicated
miRNAs during lytic infection. All values are represented as changes in expression relative to non-BV-infected cells after normalization to
values for a cellular miRNA, Let-7a. Samples were assayed from two different infections at least three times, and average values are
displayed, with standard deviations indicated.
FIG. 7. Expression kinetics of BV-encoded miRNAs. BV-infected cells were treated with phosphonoacetic acid (PAA), cycloheximide (CHX),
or actinomycin D (ActD). (A) At 18 hpi in the presence of PAA, small RNA was harvested and subjected to RT-qPCR for miRNAs as indicated.
All values are represented as changes in expression relative to BV-infected cells without drug after normalization to values for a cellular miRNA,
Let-7a. (B) As a control, PAA was shown to effectively inhibit BV true late gene expression. Large RNA was subjected to Northern blot
hybridization and probed for the BV true late-class gene for glycoprotein C (gC). The bottom portion of the gel is ethidium bromide stained and
is the loading control. (C) CHX was shown to be an effective inhibitor of protein synthesis using a pulse of [35S]methionine. The gel was Coomassie
blue stained as a loading control. (D) As a control, act D was shown to effectively inhibit BV transcription. Large RNA was subjected to Northern
blot hybridization and probed for the immediate early gene, ICP4. The bottom portion of the gel is ethidium bromide stained and is the loading
control. (E) At 60 min postinfection, small RNA was harvested and subjected to RT-qPCR. All values are represented as changes in relative
expression to non-BV-infected cells after normalization to a cellular miRNA, Let-7a. Samples were assayed in triplicate at least three times, and
average values are displayed, with standard deviations indicated. ND, not detected.
VOL. 85, 2011 HERPES B VIRUS-ENCODED miRNAs7307
malian cells (26), including Vero cells (M. Amen and A. Grif-
fiths, data not shown). The absolute amount of ath-mir-166 was
determined in RNase-treated samples and non-RNase treated
samples by calibrating the assay using a standard curve. Ac-
counting for 28% recovery of input ath-mir-166 (determined
from knowing the input and mass recovered in the absence of
RNase), the RNase treatment procedure was shown to be effec-
tive and resulted in ?94% degradation of the input ath-miR-166.
Using standard curves generated for each miRNA by using
synthetic RNA oligonucleotides, the number of molecules
of miRNAs hbv-mir-B8-5P, -B8-3P, -B20-5P, -B20-3P, and
-B26-5P harvested from virions were quantified. These values
were normalized to the number of viral genomes harvested
from the virions and ranged from 1 to 28 miRNA molecules
per viral genomic DNA molecule (Table 2). Additionally, the
number of infectious units per DNA genome was determined
to be 51 and 113, from two different viral stocks (Amen and
Griffiths, not shown). Thus, the number of miRNAs per infec-
tious virion is likely to be greater than the estimate per
genomic DNA molecule.
BV-encoded moRNAs. Recent evidence of a new class of small
RNA species, known as miRNA offset RNAs (moRNAs), in
Kaposi’s sarcoma-associated herpesvirus (KSHV) (25) and
HSV (22, 37) prompted us to search our sequencing data for
BV-encoded moRNAs. Manual analysis of the longer precur-
sor stem-loops revealed four BV-encoded moRNAs originat-
ing from three loci (Table 3). The precursor stem-loops are
shown in Fig. 3. As previously reported, these moRNAs were
detected at much lower levels than BV-encoded miRNA levels
(32). The moRNAs were detected later in infection, at 15 and
21 hpi only, and were located within the nonhomologous re-
gions relative to HSV. We assume that only when the precur-
sor stem-loops reach high abundance can the moRNAs be
detected by deep sequencing. We also detected 5? sequence
heterogeneity of hbv-mor-B20-3P, as has been observed for
other moRNAs (32).
Deep sequencing was used to identify a total of 13 BV-
encoded miRNAs. All miRNAs were detected during lytic
infection, and four were detected in naturally infected cyno-
molgus macaque TG.
BV-encoded miRNA sequences are not conserved with other
virus-encoded miRNA sequences. Unlike the high degree of
sequence conservation seen between cellular miRNAs, herpes-
virus-encoded miRNAs are generally not well conserved (17,
33, 42). Until recently, Epstein-Barr virus (EBV) and rhesus
lymphocryptovirus (rLCV) were the only herpesviruses known
to share evolutionarily conserved miRNAs. New evidence has
revealed that HSV-1 and HSV-2 share 11 positional and/or
sequence homologs (22). Three of them share perfectly
homologous seed sequences, while five are similar in 6 of the
7 bases of the seed region (22). These observations suggest
conserved targets for the homologous miRNAs. Since BV is a
close relative to HSV-1 and HSV-2, it is notable that only one
of the BV-encoded miRNAs reported here shared any se-
quence similarity to the HSV-encoded miRNAs. hbv-mir-
B14RC exhibits 68% identity to hsv1-miR-H16, which does not
have a homolog in HSV-2 (22). Interestingly, hbv-mir-B14RC
is located in the RLregion that is nonhomologous to HSV-1
(Fig. 4), while hsv1-mir-H16 is located in the 5? untranslated
region (UTR) of UL33 in the ULregion (22). Both hbv-mir-
B14RC and hsv1-mir-H16 were expressed during the lytic-cycle
(Fig. 6) (22). The miRNAs are not likely to have the same
target sequence, as only 3 of 7 nucleotides of the seed sequence
are identical. There were no sequence similarities between
BV-encoded miRNAs and other virus-encoded miRNAs.
Most BV-encoded miRNAs are located in genomic locations
similar to those of the HSV-encoded miRNAs (Fig. 4) (22).
This is similar to other related herpesviruses, such as KSHV
and rhesus rhadinovirus (RRV) (29, 42). The significance of
positional homology and sequence diversity is unclear, al-
though it may reflect similarities in the expression of the miR-
NAs (e.g., during latency), rather than the targets of the
miRNAs. However, we cannot rule out the possibility that the
BV-encoded miRNAs share homologous viral targets or cel-
lular targets with HSV-encoded miRNAs. Interestingly, 10
BV-encoded miRNAs are located in the region of the BV
TABLE 2. Detection of BV-encoded miRNAs
packaged into virions
No. of molecules
TABLE 3. Recovered sequence reads of BV-encoded moRNAs from deep sequencing
BV moRNASequence Length (nt) Positiona, orientation
Prevalence (hpi or ganglia)c
RS, minor LATb
19 125175–125193, ?
RL, minor LAT
RL, minor LAT
aPosition noted for internal repeat only if located in repeat regions. ?, positive orientation.
bInferred from latent expression of hbv-mir-B22 through hbv-mir-B8.
cNumber of times sequence is represented in deep sequencing data.
7308AMEN AND GRIFFITHSJ. VIROL.
genome that lacks sequence similarity to HSV-1 (Fig. 4). While
the majority of the BV genome is similar to that of HSV, three
areas in the repeat regions have notably low sequence similar-
ity to the corresponding regions of HSV. These regions are the
flanking sequences to ICP0 (which are also shorter than in
HSV) and a region of an additional ?1.5-kb sequence in the
short repeat (Fig. 4) (25). Given the sequence differences, we
are investigating the possibility that the miRNAs encoded in
these regions play important roles in the differing severity of
human disease seen between BV and HSV.
Loci and expression kinetics of BV-encoded miRNAs. The
majority of miRNAs identified in HSV-1 and ?2 are generated
from regions in or proximal to LAT, and many are thought to
be important for latency (12). Like HSV, most of the BV-
encoded miRNAs are located in or proximal to LAT (Fig. 4).
The four miRNAs identified in the TG by deep sequencing are
likely to be generated from LAT (Fig. 4). Although the BV
LATs have not been formally mapped, based on the latent
expression of BV-encoded miRNAs, we infer that LAT begins
upstream of hbv-mir-B22-3P and extends downstream of hbv-
mir-B8-3P (Fig. 4). Thus, it appears that the LAT region is at
least 8.3 kb and includes ?1.5 kb of sequence in the RSregion
that is considered absent in HSV. The two miRNAs in this
region generated from the opposite strand (hbv-mir-B14RC
and -B21RC) were detected only during lytic replication.
There were variable expression levels of the miRNAs in the
TG (Fig. 5), which was surprising given they are likely to be
generated from the same pri-miRNA (LAT). While the signif-
icance of this is unclear, it is interesting to speculate that it
relates to the function of the miRNAs in the different ganglia.
It has been recently shown that miRNA stability is decreased
when the concentration of its target sequence is increased (3).
Thus, it is possible that the BV-encoded miRNAs are acting
upon target mRNAs that have variable expression between the
ganglia, resulting in variable miRNA stability between ganglia.
We investigated the expression of the miRNAs during lytic
infection by examining their changes in expression over time
and their sensitivity to various agents that inhibit expression of
genes in particular kinetic classes. Interestingly, expression of
many of the miRNAs could not be easily categorized into the
traditional immediate early, early, and late (true late or leaky)
classes. It should be remembered that rather than directly
inhibiting the expression of the miRNAs, these inhibitors are
affecting expression of the progenitor RNAs, the pri-miRNAs.
While it is possible that the pri-miRNAs are expressed in a
noncanonical manner, we believe that it is more likely that several
of the miRNAs are expressed from multiple pri-miRNAs from
different temporal classes. This would result in a highly com-
plex BV transcriptome, although it is recently becoming clear
that herpesviruses have a much more complex pattern of tran-
scription than previously recognized (46). The reasons for ex-
pression from multiple pri-miRNAs is unclear, but it is inter-
esting to speculate that this represents a strategy that allows
the virus to express a particular miRNA throughout infection
to maximize the time an miRNA has to perform its function.
BV-encoded miRNAs are packaged into virions. Analyses of
miRNA expression during the course of infection led us to
examine their expression in the absence of de novo gene tran-
scription. Both viral and cellular RNAs have been reported to
be packaged in virions of herpesviruses (8, 18, 30). Packaging
of RNAs in the virion allows for immediate expression upon
virus entry. After the first round of infection, the infected cell
releases new virus particles as well as cell debris and cellular
products, such as cytokines, that may affect the surrounding
cells. RNAs packaged in the virion may be influential in over-
coming the activated host response immediately upon entry
into these uninfected cells. For example, the human cytomeg-
alovirus (CMV) has been shown to package UL21.5 mRNA,
which binds the chemokine receptor RANTES and modulates
the host cell response by blocking its ability to bind to its
receptor (43). This CMV-encoded protein can therefore po-
tentially modify the antiviral response before viral transcrip-
tion occurs. Additionally, viruses that have short replication
cycles may take advantage of RNAs packaged in the virion to
establish productive and persistent infection in a timely man-
ner. While it has been well established that RNAs can be
packaged in virions, it is not widely recognized that viruses
package noncoding RNA molecules, including miRNAs. Cliffe
et al. demonstrated that viral tRNA-like molecules are pack-
aged in murine gammaherpesvirus 68 (MHV-68) virions at
levels detectable by Northern blot hybridization (12), which
suggests that they are present at higher levels in virions than
the BV-encoded miRNAs. While their role in virus patho-
genesis is unknown, their presence in the virion suggests they
have a role early in infection. Similarly, several BV-encoded
miRNAs were packaged into virions. The number of molecules
of hbv-mir-B8-5P, -B8-3P, -B20-5P, -B20-3P, and -B26-5P
ranged from 1 to 28 molecules per viral genome. While these
numbers are fairly low, we recognize that not every genome is
packaged within an infectious particle. The genome infectivity
ratio was determined to be 51 and 113; therefore, these num-
bers are likely an underestimation of the number molecules
per infectious virion. This is the first report showing that
miRNAs are packaged into virions, and further work is re-
quired to understand the biological significance of miRNAs
packaged into virions.
Implications of sequence heterogeneity. Analysis of the ma-
ture miRNA sequences revealed sequence variation at both
the 5? and 3? ends (Table 1). This sequence heterogeneity has
major implications for miRNA function. The 5? end is impor-
tant for miRNA target selection; full sequence complementa-
rity of nt 2 to 8 of the mature miRNA, referred to as the seed
region, is important for mRNA translational inhibition (4).
Therefore, even differences of 1 nt at the 5? end potentially
alter the target mRNAs. miRNA sequence heterogeneity
has been observed in humans (45), Drosophila melanogaster
(31), Caenorhabditis elegans (27), and herpesviruses, including
KSHV (37), HSV (39), and rLCV (27). In all three of these
herpesviruses, it was reported that 3?-end variation occurred
more frequently than 5?-end variation (27, 37, 39). Impor-
tantly, it has been extensively documented that sequence het-
erogeneity observed in mature miRNAs is not due to RNA
degradation or any other artifact (27, 39). The 5? sequence
heterogeneity observed in BV-encoded miRNAs may enable
BV to expand the number of regulated genes, thereby optimiz-
ing the limited capacity within the viral genome. While the 3?
end of miRNAs may have a minor role in target discrimination,
there is recent evidence that they are important for stability
(3). 3?-sequence heterogeneity may result in differing degrees
of stability between the various species. In the context of BV-
VOL. 85, 2011 HERPES B VIRUS-ENCODED miRNAs 7309
encoded miRNAs, the significance of the observed 3?-end het-
erogeneity is unclear; however, it is interesting to consider that
it may afford the virus a mechanism to regulate miRNA activity
at a postexpression level.
BV-encoded moRNAs. A newly recognized class of small
RNA molecules, called miRNA offset RNAs (moRNAs), has
recently been described. They were first discovered in 2009 by
using deep sequencing of small RNAs expressed in the simple
chordate Ciona intestinalis (32) and recently in KSHV (37) and
HSV-1 and -2 (22). While the significance and biogenesis of
this new class of RNA species are unclear, it is understood that
they arise from sequences located adjacent to the predicted
pre-miRNA stem-loop, and Drosha is responsible for cleavage
at one end. It may be possible that these molecules are still
loaded into the RNA-induced silencing complex (RISC) and
could therefore be potential regulatory molecules. Further in-
vestigation of this new class of small-RNA species will be
required to understand their significance in BV biology.
Drawbacks of different identification methods. There are
advantages and disadvantages to all of the methods we have
used to identify miRNAs. For example, it has been noted
previously that there are drawbacks in the use of deep sequenc-
ing for miRNA identification, including its ability to report
certain miRNAs more efficiently than others (22, 39). Further-
more, the number of sequence reads for a single miRNA is not
necessarily proportional to its relative abundance (22). The
order that we addressed each criterion for candidate miRNAs
was dictated by practicality. However, there remains an anom-
aly: hbv-mir-B19-3P did not pass the final criterion to be clas-
sified as an miRNA. Deep sequencing detected this molecule
in abundance; the most prevalent species from deep sequenc-
ing had 86 hits at 3 hpi, 198 at 9 hpi, 454 at 15 hpi, and 489 at
21 hpi. RT-qPCR revealed latent expression of hbv-mir-B19-
3P; however, it was not detected by deep sequencing RNA
from TG. RT-qPCR also revealed it was highly expressed
throughout infection; at 3 hpi, it was expressed at 103-fold over
the background and increased to 104by 24 hpi. We anticipate
that hbv-mir-B19-3P is most likely a real miRNA but was not
detected in transfected cells, perhaps because there is a differ-
ence in the production of the star and mature miRNA as in
infected cells. Further analysis will be required to understand
whether hbv-mir-B19-3P is indeed an miRNA. Taken together,
it appears that a combination of techniques and conditions is
desirable for comprehensive virus-encoded miRNA discovery.
Possible roles of BV-encoded miRNAs in BV biology. Al-
though the list of virus-encoded miRNAs continues to grow,
especially within the herpesvirus family, understanding their
function remains a challenging task. Herpesvirus-encoded
miRNAs have been shown to function during both latency and
productive infection (7). Given the rapid replication cycle of
simplexviruses, it is perhaps easier to imagine a productively
expressed miRNA repressing only viral genes, while a latently
expressed miRNA has sufficient time to repress both viral
and/or cellular genes. However, the observation that miRNAs
are packaged into virions allows the virus to regulate gene
transcription before de novo synthesis occurs, and thus we
anticipate that all herpesvirus-encoded miRNAs may regulate
both viral and cellular genes in both latent and lytic infections.
Latent infections of herpesviruses are characterized by very
low levels of viral protein expression, effectively avoiding de-
tection by the immune system. Thus, viral miRNAs may pro-
vide herpesviruses with a nonimmunogenic method to modify
the cellular environment. During productive infection, virus-
encoded miRNAs may be important for replication and the
production of infectious progeny.
Herein we describe the discovery and expression analysis of
BV-encoded miRNAs using a combination of deep sequenc-
ing, RT-qPCR, and expression in transfected cells. Using these
techniques, a total of 13 miRNAs were detected during lytic
infection, and 4 were detected in latently infected macaque
TG. This is the first report that miRNAs are incorporated into
virions. These data provide the framework for future analyses
of BV-encoded miRNA functions.
We thank Mallory Harden for excellent technical support and crit-
ical reading of the manuscript, S.-J. Gao and Robert Lanford for
advice, Adriana Mejia for generating plasmid pAMBVgB, Jean Pat-
terson and Ricardo Carrion, Jr., for help in the ABSL-4 laboratory,
and Edward Dick for help with necropsies. We also thank Roy Garcia,
Sarah Williams-Blangero, and John Blangero for help with deep se-
quencing. In particular, we are deeply grateful to Tim Anderson and
Shalini Nair for help with the initial stages of deep sequencing.
This work was supported by Texas Biomedical Research Institute
V&I startup funds and a grant from the Southwest Foundation Forum,
P51 RR013986, and was conducted in facilities constructed with sup-
port from the Research Facilities Improvement Program (grant num-
ber C06 RR012087) from the NCRR.
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