JOURNAL OF VIROLOGY, July 2005, p. 9301–9305
Copyright © 2005, American Society for Microbiology. All Rights Reserved.
Vol. 79, No. 14
Cloning and Identification of a MicroRNA Cluster within the
Latency-Associated Region of Kaposi’s
Mark A. Samols, Jianhong Hu, Rebecca L. Skalsky, and Rolf Renne*
Department of Molecular Genetics and Microbiology and UF Shands Cancer Center, University of Florida,
Gainesville, Florida 32610
Received 15 February 2005/Accepted 16 March 2005
MicroRNAs (miRNAs) are small, noncoding regulatory RNA molecules that bind to 3? untranslated regions
(UTRs) of mRNAs to either prevent their translation or induce their degradation. Previously identified in a
variety of organisms ranging from plants to mammals, miRNAs are also now known to be produced by viruses.
The human gammaherpesvirus Epstein-Barr virus has been shown to encode miRNAs, which potentially
regulate both viral and cellular genes. To determine whether Kaposi’s sarcoma-associated herpesvirus (KSHV)
encodes miRNAs, we cloned small RNAs from KSHV-positive primary effusion lymphoma-derived cells and
endothelial cells. Sequence analysis revealed 11 isolated RNAs of 19 to 23 bases in length that perfectly align
with KSHV. Surprisingly, all candidate miRNAs mapped to a single genomic locale within the latency-
associated region of KSHV. These data suggest that viral and host cellular gene expression may be regulated
by miRNAs during both latent and lytic KSHV replication.
MicroRNAs (miRNAs) range from 19 to 24 nucleotides (nt)
in length and are small, noncoding RNA molecules which bind
complementary mRNAs to regulate gene expression at the
posttranscriptional level. In plants, miRNAs bind precisely to
complementary sequences within the 3? untranslated region
(UTR) of a target gene and induce mRNA degradation
through the RNA interference pathway. Most animal miRNAs
have limited complementarity to their target sequences within
the 3? UTR and either degrade mRNA via the RNA interfer-
ence pathway or down-regulate translation by a mechanism not
yet understood. In human cells, over 235 miRNAs have been
identified to date (for review, see references 1 and 4). Targets
and functions of very few miRNAs have been experimentally
determined thus far, yet some molecules, such as human hsa-
miR-14 and hsa-miR-181, are known to have roles in funda-
mental biological processes like apoptosis, cell proliferation,
and hematopoiesis (6, 9).
Most recently, miRNAs have been identified and isolated in
the gammaherpesvirus Epstein-Barr virus (EBV) (21) and pre-
dicted for the human immunodeficiency virus using in silico
methods (5). Kaposi’s sarcoma (KS)-associated herpesvirus
(KSHV), also called human herpesvirus type 8 (HHV-8), is
another gammaherpesvirus. The virus is associated with KS
and two lymphoproliferative diseases: primary effusion lym-
phomas (PELs) and a subset of multicentric Castleman’s dis-
ease (7, 8, 26). In this report, we demonstrate that KSHV, like
EBV, encodes miRNAs.
Cloning of small RNAs from KSHV-infected cells. To deter-
mine whether KSHV encodes miRNAs, we generated small
RNA libraries by positional cDNA cloning from a primary
effusion lymphoma-derived cell line (BCBL-1) undergoing ei-
ther latent or tetradecanoyl phorbol acetate (TPA)-induced
lytic KSHV infection (23). Additionally, we cloned small
RNAs from a telomerase-immortalized endothelial cell line
latently infected with KSHV (TIVE-LTC) (F. Q. An and R.
Renne, data to be published elsewhere). Cloning was per-
formed as described in reference 16, with minor modifications.
Briefly, 600 ?g of total RNA was size fractionated by denatur-
ing polyacrylamide gel electrophoreses (PAGE). The gel area
containing RNA molecules around 24 nt in length was excised,
and RNA was recovered by elution and precipitation. RNA
molecules were dephosphorylated, ligated to a 3? adapter
primer (RNA/DNA hybrid), and size fractionated by PAGE
again. Following recovery, RNAs were phosphorylated and
ligated to a 5? adapter (RNA/DNA) hybrid. Reverse transcrip-
tion was initiated using a primer complementary to the 3?
adapter. Differences between 3? and 5? adapters allowed us to
determine the orientations of the captured RNA inserts. The
resulting cDNA pool was amplified by PCR (20 cycles followed
by 12 cycles) using a second PCR primer pair which introduced
BanI restriction sites. Amplicons were digested with BanI,
concatamerized by ligation, and after size fractionation on
agarose gels inserted into pCRII-Topo (Invitrogen) for
transformation, resulting in thousands of white colonies.
One hundred fifty clones each derived from BCBL-1,
BCBL-1 with 24 h of TPA treatment, and latently infected
TIVE-LTC cells were analyzed by restriction enzyme diges-
tion. Sequencing of 260 clones revealed a total of 634 cap-
tured small RNA sequences.
Identification of 11 KSHV-encoded candidate miRNAs. To
determine the genomic origins of the cloned sequences, three
homology searches were performed. First, sequences were
aligned to known miRNAs within the miRNA registry (15, 20,
24, 25), which contains 235 human miRNA sequences. Next, all
sequences were compared to the human genome and, finally,
* Corresponding author. Mailing address: Department of Molecular
Genetics and Microbiology, University of Florida, Gainesville, FL
32610 Phone: (352) 392-9848. Fax: (352) 368-5802. E-mail: rrenne
the KSHV genome (U75698, U93872) (20, 24) using NCBI
BLAST. Table 1 summarizes our results.
The majority of sequences identified represented rRNA (47
to 57%). Known human miRNAs represented about 14% of all
cloned sequences derived from BCBL-1 cells (66 sequences
representing 17 miRNA species) and 21% from TIVE-LTC
cells (31 sequences representing 15 miRNA species), while
smaller fractions represented other small nuclear RNAs (4%).
The distribution of identified rRNA sequences and miRNA
sequences correlates well with previously reported miRNA
cloning studies (21).
A total of 52 sequences representing 11 unique RNA species
between 19 and 24 bases in length matched with 100% comple-
mentarity to KSHV. Surprisingly, all sequences aligned with a
single region of the KSHV genome: the latency-associated
locus (11, 27). During latency, KSHV gene expression is re-
stricted to a few viral genes, most of which are located within
this region (Fig. 1). These polycistronic latency-associated
transcripts encode the latency-associated nuclear antigen
(LANA/open reading frame 73 [ORF73]), v-cyclin (ORF72),
and v-Flip (ORF71). The kaposin gene is located approxi-
mately 4 kbp downstream and encodes at least three proteins
that are expressed from alternate start codons upstream of
direct repeat 1 (DR1) and DR2 (Fig. 1) (25). Both loci are
highly expressed in KS tumors and PEL-derived cell lines and
play an important role in regulating viral and cellular gene
expression (3, 13, 14, 18, 19, 22, 25). To date, no coding regions
or functional elements have been annotated for the 3.6-kbp-
long intragenic region between the kaposin gene and ORF71.
Strikingly, 10 out of 11 candidate miRNAs are located within
this intragenic region, while one is located in the kaposin locus
at position nt 117992. The genomic location of this newly
identified viral miRNA cluster and their sequences are shown
in Fig. 1A and B.
miRNAs are believed to be RNA polymerase II transcripts.
TABLE 1. Distribution of cloned small RNA moleculesa
Distribution of cloned small RNA molecules (%)
aA total of 450 clones were analyzed by restriction digestion with 260 clones
sequenced resulting in 634 sequences. Genomic loci represent RNA sequences
that match annotated loci in the human genome. “Not matched” sequences are
very short highly repetitive sequences.
FIG. 1. Identification of an miRNA cluster in the intragenic region between K12 and ORF71. (A) Schematic of the latency-associated region
of KSHV. All nucleotide annotations are based on the BC-1 KSHV sequence (24). ORFs, transcripts, DR1 and DR2, and promoters are drawn
as described in references 11, 17, 25, and 27. The miRNA cluster, as well as the proposed kaposin transcripts by Li et al. (17), are shown in orange.
(B) Sequences of cloned candidate miRNA species, nucleotide position, and cloning frequency are indicated. Preliminary names of candidate
miRNAs as submitted to GenBank are given (accession number AY973824).
9302 NOTES J. VIROL.
FIG. 2. Isolated miRNAs fold into hairpin structures of appropriate length. (A) Secondary structure predictions of candidate KSHV miRNA
sequences using mFold (28). Shown are only structures with the lowest free energy. (B) Northern blot analysis for the expression of KSHV miRNAs
3-3p, 4, and 10 and the human miRNA hsa-miR-16 as a positive control. Thirty micrograms of total RNA from BCBL-1, TPA-induced BCBL-1,
TIVE-LTC, and latently infected SLK cells (SLK KS?) was separated on 15% PAGE, blotted to nylon membranes, probed with an end-labeled
antisense oligonucleotide, and detected by phosphorimaging as previously described (16).
VOL. 79, 2005NOTES9303
While some are expressed from miRNA genes, many mamma-
lian miRNAs are expressed as clusters from introns and 5?
UTRs of mRNAs (for review, see reference 10). In our anal-
ysis, all isolated candidate KSHV miRNAs are derived from
the same strand, suggesting that they are expressed from a
single transcript. Within this region, two promoters have been
reported. Sadler et al. reported a transcript initiation site at nt
118758 (25), and Li et al. reported on a 2.3-kbp-long cDNA
encoding kaposin proteins A and C from a promoter located
within ORF73, initiating transcription at nt 123842 (17). The
spliced intron (nt 123594 to 118799) has the potential to en-
compass the entire KSHV miRNA cluster (Fig. 1). Thus,
KSHV encodes a cluster of miRNAs which may be expressed
from an intron and encodes one miRNA expressed within an
exon (KSHV-miRNA-10) (Fig. 1).
Several miRNA clusters have been reported in animal cells
which are expressed from otherwise protein coding mRNA
species; likewise, these KSHV miRNAs might be expressed
coordinately with the kaposin locus. Interestingly, the expres-
sion of kaposin proteins is detectable in latently infected cells
but is also induced by TPA (25). Li et al. have also reported
TPA responsiveness of the newly described kaposin promoter
(17). In congruence with this data, we observed viral miRNA
expression in both latent BCBL-1 cells and TPA-induced
BCBL-1 cells, which suggests a role for miRNA-dependent
gene regulation during both latent and lytic growth of KSHV.
KSHV-encoded candidate miRNAs fold into hairpin struc-
tures. In order to annotate miRNAs, evidence for both the
expression and the biogenesis of a novel sequence should be
provided (2). One strong indicator for biogenesis is the pres-
ence of complementary adjacent sequences that form stable
hairpins. miRNAs are excised from precursor RNAs by a mat-
uration process that entails cleavage by two cellular RNase III
enzymes, namely Drosha and Dicer. These enzymes recognize
specific RNA secondary hairpin structures to process the 5?
and 3? ends of the mature miRNA (1, 4).
To analyze potential precursor structures for the isolated 11
KSHV candidate miRNAs, each sequence including an area of
60 to 80 bases surrounding the sequence was analyzed by
mFold (28), which predicts RNA secondary structure. Figure
2A shows the predicted folding patterns which suggest that all
KSHV miRNAs show stable hairpin formation with long
paired stems and an appropriate distance between the end of
the miRNA and the loop, a feature recognized by Dicer (1, 4).
Hence these predictions further support that the cloned se-
quences represent miRNAs. To further confirm the expression
of KSHV miRNAs, Northern blot analysis using total RNA
isolated from BCBL-1, BCBL-1/TPA, TIVE-LTC, and latently
infected SLK cells was performed. We detected both the im-
mature miRNA precursor and the mature miRNA for KSHV
miRNAs 3-3p, 4, and 10, while we were unable to detect
expression of the remaining miRNAs (Fig. 2B). This further
confirms that KSHV-infected cells of both lymphoid and en-
dothelial origin express viral miRNAs. In summary, we have
identified a microRNA cluster within the latency-associated
region of KSHV between nt positions 119839 and 124426 (Fig.
Four criteria for the existence of this newly identified
miRNA cluster are addressed by our data. (i) We identified 11
unique miRNA species from 52 clones (Table 1). (ii) Observed
miRNA sequences folded into hairpin structures (Fig. 2A).
(iii) All sequences are expressed from a single strand, compat-
ible with miRNA maturation but not with small interfering
RNA maturation, which often involves the formation of larger
double-stranded RNA species generated by bidirectional tran-
scription. (iv) Northern blot analysis demonstrates both pre-
miRNA and mature miRNA forms in infected lymphoid and
endothelial cells. We detected 3 out of the 11 miRNA species,
indicating that although these miRNAs may be expressed from
a common pre-mRNA, differential processing might explain
their relative abundance.
The main and most important question to solve in the future
will be to determine the functional consequences of miRNA
expression in KSHV-infected cells. Most recent computational
predictions have drawn a complicated picture in which each
human miRNA can target many different genes. The same is
true for the KSHV-encoded miRNA. Utilizing a recently pub-
lished algorithm, miRanda, several hundred human and many
viral target genes were predicted even under most stringent
95% cutoff conditions (data not shown) (12). Therefore, it will
require genetic approaches to decipher whether this fascinat-
ing mechanism of posttranscriptional regulation contributes to
KSHV latent and/or lytic replication and will ultimately have
implications in viral pathogenesis.
This work was supported by start-up funding from the University of
Florida Shands Cancer Center. R.R. is supported by grants from the
National Institutes of Health (RO1 CA 88763, R21 CA97939).
ADDENDUM IN PROOF
After this work was accepted, the identification of KSHV-
encoded miRNAs was independently reported by two other
groups (S. Pfeffer, A. Sewer, M. Lagos-Quintana, R. Sheridan,
C. Sander, F. A. Grasser, L. F. van Dyk, C. K. Ho, S. Shuman,
M. Chien, J. J. Russo, J. Ju, G. Randall, B. D. Lindenbach, C.
M. Rice, V. Simon, D. D. Ho, M. Zavolan, and T. Tuschl, Nat.
Methods 2:269-276, 2005; and X. Cai, S. Lu, Z. Zhang, C. M.
Gonzalez, B. Damania, and B. R. Cullen, Proc. Natl. Acad. Sci.
USA 102:5570-5575, 2005).
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