Pyrosequencing of small non-coding RNAs in HIV-1 infected cells: evidence for the processing of a viral-cellular double-stranded RNA hybrid.
ABSTRACT Small non-coding RNAs of 18-25 nt in length can regulate gene expression through the RNA interference (RNAi) pathway. To characterize small RNAs in HIV-1-infected cells, we performed linker-ligated cloning followed by high-throughput pyrosequencing. Here, we report the composition of small RNAs in HIV-1 productively infected MT4 T-cells. We identified several HIV-1 small RNA clones and a highly abundant small 18-nt RNA that is antisense to the HIV-1 primer-binding site (PBS). This 18-nt RNA apparently originated from the dsRNA hybrid formed by the HIV-1 PBS and the 3' end of the human cellular tRNAlys3. It was found to associate with the Ago2 protein, suggesting its possible function in the cellular RNAi machinery for targeting HIV-1.
- SourceAvailable from: Zissimos Mourelatos
Article: The microRNA world: small is mighty.[show abstract] [hide abstract]
ABSTRACT: A new paradigm of RNA-directed gene expression regulation has emerged recently, profound in scope but arresting in the apparent simplicity of its core mechanism. Cells express numerous small ( approximately 22 nucleotide) RNAs that act as specificity determinants to direct destruction or translational repression of their mRNA targets. These small RNAs arise from processing of double-stranded RNA by the Dicer nuclease and incorporate with proteins that belong to the Argonaute family. Small RNAs might also target and silence homologous DNA sequences. The immense potential of small RNAs as controllers of gene networks is just beginning to unfold.Trends in Biochemical Sciences 11/2003; 28(10):534-40. · 13.08 Impact Factor
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ABSTRACT: Small interfering RNA (siRNA) and microRNA (miRNA) are small RNAs of 18-25 nucleotides (nt) in length that play important roles in regulating gene expression. They are incorporated into an RNA-induced silencing complex (RISC) and serve as guides for silencing their corresponding target mRNAs based on complementary base-pairing. The promise of gene silencing has led many researchers to consider siRNA as an anti-viral tool. However, in long-term settings, many viruses appear to escape from this therapeutical strategy. An example of this may be seen in the case of human immunodeficiency virus type-1 (HIV-1) which is able to evade RNA silencing by either mutating the siRNA-targeted sequence or by encoding for a partial suppressor of RNAi (RNA interference). On the other hand, because miRNA targeting does not require absolute complementarity of base-pairing, mutational escape by viruses from miRNA-specified silencing may be more difficult to achieve. In this review, we discuss stratagems used by various viruses to avoid the cells' antiviral si/mi-RNA defenses and notions of how viruses might control and regulate host cell genes by encoding viral miRNAs (vmiRNAs).Cell Research 10/2005; 15(11-12):935-46. · 10.53 Impact Factor
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ABSTRACT: microRNAs (miRNAs) are a large family of 21- to 22-nucleotide non-coding RNAs that interact with target mRNAs at specific sites to induce cleavage of the message or inhibit translation. miRNAs are excised in a stepwise process from primary miRNA (pri-miRNA) transcripts. The Drosha-Pasha/DGCR8 complex in the nucleus cleaves pri-miRNAs to release hairpin-shaped precursor miRNAs (pre-miRNAs). These pre-miRNAs are then exported to the cytoplasm and further processed by Dicer to mature miRNAs. Here we show that Drosophila Dicer-1 interacts with Loquacious, a double-stranded RNA-binding domain protein. Depletion of Loquacious results in pre-miRNA accumulation in Drosophila S2 cells, as is the case for depletion of Dicer-1. Immuno-affinity purification experiments revealed that along with Dicer-1, Loquacious resides in a functional pre-miRNA processing complex, and stimulates and directs the specific pre-miRNA processing activity. These results support a model in which Loquacious mediates miRNA biogenesis and, thereby, the expression of genes regulated by miRNAs.PLoS Biology 08/2005; 3(7):e235. · 12.69 Impact Factor
Pyrosequencing of small non-coding RNAs in
HIV-1 infected cells: evidence for the processing
of a viral-cellular double-stranded RNA hybrid
Man Lung Yeung1, Yamina Bennasser1, Koichi Watashi1, Shu-Yun Le2, Laurent Houzet1
and Kuan-Teh Jeang1,*
1Molecular Virology Section, Laboratory of Molecular Microbiology, National Institute of Allergy and Infectious
Diseases, National Institutes of Health, Bethesda, MD 20892-0460 and2Center for Cancer Research Nanobiology
Program, NCI Center for Cancer Research, National Cancer Institute, Frederick, MD 21702, USA
Received March 4, 2009; Revised August 7, 2009; Accepted August 10, 2009
Small non-coding RNAs of 18–25nt in length can
small RNAs in HIV-1-infected cells, we performed
linker-ligated cloning followed by high-throughput
pyrosequencing. Here, we report the composition
of small RNAs in HIV-1 productively infected MT4
T-cells. We identified several HIV-1 small RNA
clones and a highly abundant small 18-nt RNA that
is antisense to the HIV-1 primer-binding site (PBS).
This 18-nt RNA apparently originated from the
dsRNA hybrid formed by the HIV-1 PBS and the 30
end of the human cellular tRNAlys3. It was found to
associate with the Ago2 protein, suggesting its
possible function in the cellular RNAi machinery
for targeting HIV-1.
Small non-coding RNAs of 18–25nt in length are
important in the RNA interference (RNAi) mechanism
for controlling gene expression (1,2). An intermediate
step in the RNAi pathway is the processing of precursor
double-stranded (ds) RNAs into siRNA or miRNA by the
RNase III Dicer (3). Dicer recognizes and cleaves dsRNA
substrates into products of ?18–25nt in length. Dicer
substrates can be long linear dsRNAs or hairpin RNAs
that have either perfectly complementary or imperfectly
complementary stems. The Dicer-cleaved siRNAs can
enter an Ago2-containing RNA-induced silencing complex
(RISC). This si–RISC complex can target and cleave a
mRNA that is recognized by base complementarity to the
guide siRNA. Alternatively, Dicer-produced miRNAs can
associate with RISC to form a mi–RISC complex which
can act to silence the translation of mRNA targets [review
(4) for further detail].
Dicer processes cellular miRNAs (5) and siRNAs (6).
However, several mammalian viruses, including HIV-1
(7–10), encode viral miRNAs which are also processed
by Dicer (11). To characterize small RNAs in HIV-1
infected cells (12,13), we performed small RNA cloning
followed by high throughput nucleotide pyrosequencing.
This approach identified many clones with discrete HIV-1
sequences and also a highly abundant clone containing a
cellular 18-nt non-coding RNA (PBSncRNA) sequence
which is antisense to the HIV-1 primer-binding sequence
(PBS). The latter finding of a PBSncRNA in HIV-1
infected cells is in agreement with two recent reports on
the identification of similar PBS-complementary short
ncRNAs to endogenous retroviruses (14,15). HIV-1
PBSncRNA was found to be associated with an Ago2
protein intracellularly, suggesting that it is potentially
active in the cell’s RNAi pathway against HIV-1.
MATERIALS AND METHODS
HIV-1 latently infected human monocyte cell line, U1 and
human T-cell line, MT4, were cultured in RPMI 1640
medium with 10% fetal calf serum (FCS) and 2mM
L-glutamine. In U1 cells, HIV-1 virus production was
induced by treatment with 1mM phorbol myristate
acetate (PMA). HeLa and 293T cells were propagated at
37?C with 5% CO2 in Dulbecco’s modified Eagle’s
medium (DMEM) supplemented with 10% FCS and
2mM L-glutamine. To generate an AGO2-over expressing
cell line, 293T cells were transfected with either pFLAG-
AGO2 or an empty vector together with pRS (Origene).
Stable transformants (‘293T-AGO2’ for a line stably
expressing pFLAG-AGO2 and ‘293T-control’ for a
control cell line) were selected with 1mg/ml puromycin.
*To whom correspondence should be addressed. Tel: +1 301 496 6680; Fax: +1 301 480 3686; Email: email@example.com
Published online 3 September 2009Nucleic Acids Research, 2009, Vol. 37, No. 196575–6586
? The Author(s) 2009. Published by Oxford University Press.
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/
by-nc/2.5/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
Plasmids and siRNAs
DNA oligonucleotides corresponding to the PBS sequence
TTGTCCCTGTTCGGGCGCCAA-30) were hybridized
and cloned into the Spe I and Hind III sites of pMIR-
REPORT-b-gal (Ambion) encoding b-galactosidase was
used for luciferase assay normalization. PBS siRNA
CCCGAACAGGGACdTdT-30), PBS mut siRNA (50-A
AdTdT-30) (16) and si-control (50-CUUUAAGCUCCCU
AGUG-30) RNAs were synthesized by Invitrogen. The
expression plasmid for FLAG-AGO2 was prepared by
Lipofectamine 2000 (Invitrogen) with PBSncRNA or
PBSncRNA mutant and a pMIR-REPORT-Luc reporter
REPORT-b-gal plasmid was also added to the trans-
fection as a normalization control. Forty-eight hours
after transfection, cells were washed twice with 1?
phosphate-buffered saline and then lysed in 1? luciferase
(Promega) was used according to the manufacturer’s
protocol, and activity was measured in an Opticom II
luminometer (MGM Instruments). Normalization of
luciferase activity was based on b-galactosidase activity
measured with Galacto-Star as described by the manu-
facturer (Tropix, Bedford, MA, USA). All luciferase
values represent averages±SD from at least three
Reverse transcriptase assay and viral infection
Media collected from pNL4-3 transfected HeLa cells, or
293T cells, or from PMA-treated U1 cells were filtered
using 0.45-mm membrane. Virus was quantified by
Reverse transcriptase (RT) assay (18). In total, 106–107
cpm RT units of virus prepared from transfected HeLa
cells were used to infect 5?106MT4 cells. After 2h of
exposure to virus, cells were washed twice with phosphate
buffered saline and resuspended into RPMI and cultured
for 48h. Supernatant RT was quantified, and the cells
were harvested for small RNA isolation using the
mirVana miRNA isolation kit (Ambion).
In vitro transcription
A T7 promoter driven 50-UTR region (from 584 to 710 nts
of pNL4-3) was PCR amplified by Taq polymerase
(Clontech) using DNA primers (T7 sense primer 50-TAA
The T7 promotor sequence is underlined. In vitro
transcription of the PCR product was carried out using
In vitro reverse transcription and in vitro Dicer assays
Ten nanograms of the in vitro synthesized RNA template
(T7PBS) was hybridized with total tRNA (BioS&T) using
conditions described in Beerens and Berkhout (19).
Reverse transcription of the mixture was carried out in
the presence of 20ng reverse primer (described earlier)
and SuperScriptIII (Invitrogen).
hybridization conditions, a mixture containing the total
tRNA and T7PBS was subjected to digestion with 1U
of recombinant Dicer (Genlantis) for 12h at 37?C.
The diced RNA was resolved in 15% acrylamide—8M
Preparation of AGO2-associated small RNA
293T-AGO2 or 293T-control cells were transfected with
pNL4-3 or pUC18 as a negative control. At 2days post-
transfection, cells were harvested with IP buffer [50mM
HEPES, 150mM NaCl, 10% glycerol, 1% NP-40,
1.5mM MgCl2, 0.2mM PMSF, 1mM DTT, protease
inhibitor (complete, Roche) and 100U/ml SUPERase-in
(Ambion)] followed by immunoprecipitation with anti-
FLAG agarose (Sigma), anti-AGO2 (Cell Signaling)
or mouse normal IgG (as control, Zymed laboratories)
with protein G/A agarose (Calbiochem). After six
washes, smallRNAs in
were recovered with mirVana miRNA isolation kit
the production of PBSncRNA, 5mM of the non-nucleoside
reverse transcription inhibitor (nevirapine) was added 2h
before the cells were transfected, and the drug was
maintained throughout the entire transfection process.
Northern blot analysis
RNAs were separated using 15% polyacrylamide–8M
urea gel and electrotransferred for 1h at 200mA to
nylon membranes. After UV crosslinking, membranes
(Ambion) and incubated overnight at 37?C with cognate
probes. Membranes were washed extensively at room
temperature and autoradiographed.
at 68?Cin Ultrahyb
Small RNA cloning
The cloning of small RNAs from HIV-1 infected MT4 cells
was conducted as described by Lau et al. (20). HIV-1 virus
(>1 MOI) derived from transfection of molecular clone
pNL4-3 into 293T cells was used to infect MT4 cells
for 2days. One-hundred micrograms of size-enriched
small RNAs (<200nt) were prepared from the infected
and uninfected cells and used for cloning followed by
sequencing. The classification of small RNA sequences
was based on sequence analysis using the GenBank
6576Nucleic Acids Research, 2009,Vol.37, No. 19
miRNA registry database (http://microrna.sanger.ac.uk/
sequences/), the rRNA database (http://bioinformatics
GtRNAdb/), and the piRNA database (http://research
Quantitative real-time PCR
Small RNAs (<200nt) were isolated using mirVana
miRNA isolation kit (Ambion). miRNA quantification
was as described earlier (21,22). RNA was polyadenylated
with ATP by poly(A) polymerase at 37?C for 1h using
RNA tailing kit (Ambion) and reverse transcribed using
0.5mg of poly(T) adapter primer (Invitrogen). For each
PCR, equal amounts of cDNA (first normalized using
the snU6 RNA) were mixed with SYBR Green PCR
mix (ABI) and 5pmol of forward primer (designed on
the entire tested miRNA sequence) and reverse primer
(based on the adaptor sequence). Amplification was
done under the condition of 15s at 95?C and 1min at
60?C for 55 cycles in an Opticon real-time PCR detection
system (Bio-Rad) or 7300 Real Time PCR System (ABI).
(Supplementary Figure S1). Random pNL4-3 primers
(HIVs3263 and HIVs5543) starting from 3263 nt (50-AT
GGACAGTACAGCCTATAGTGCT-30) and 5543 (50-A
ACTGACAGAGGACAGATGGAA-30) were used as
To quantify luciferase mRNA and HIV-1 genomic
RNA, total RNA from transfected cells was harvested
by using the mirVana miRNA isolation kit (Ambion)
following the manufacturer’s protocol. Fifteen micro-
grams of total RNA were first treated with RQ1 RNase-
free DNase (Promega) at 37?C for 30min followed by
phenol/chloroform extraction and ethanol precipitation.
One microgeam of the resuspended RNA was used for
RT reaction. The final product was then phenol/
chloroform extracted and ethanol precipitated, and 1ng
of the product was used for quantification. The qPCR
reaction was the same as described earlier. To detect
luciferase mRNA, forward primer 50-CTCGGGTGTAA
TCAGAAT-30and backward primer 50-TTGCTAGTAC
CAACCCTA-30were used. To detect HIV-1 genomic
RNA, forward primer 50-CTCTCTGGCTAACTAGGG
AAC-30and backward primer 50-CAAGCCGAGTCCT
GCGTCGAGAGATC-30were used. The results were
normalized to the amount of GAPDH mRNA measured
using forward primer 50-GCTCACTGGCATGGCCTTC
CGTGT-30and backward primer 50-TGGAGGAGTGG
Pyrosequencing of small RNAs in HIV-1 infected T-cells
Viral miRNAs have been reported for several viruses
(23,24). To understand better the small RNA profile
of HIV-1 NL4-3 infected cells, we sequenced size-
fractionated small RNAs from virus infected MT4
T-cells. The MT4 cells were infected with HIV-1; and
2days later, small RNAs were harvested and cloned
(20). In total 47773 discrete clones were sequenced
(1004656nt) and analyzed. Consistent with other studies
(15,25) which identified miRNA as the largest (?60%)
constituent of small RNAs, 52% of our small RNAs
were miRNAs (Figure 1A). In total, 24892 miRNA
clones corresponding to 398 discrete miRNA species
(Supplementary Table S1) were identified. The relative
distribution of our sequenced miRNA species is consistent
with the published literature on miRNAs expressed T-cells
(26). For instance, miRNAs including the miR-17-92
cluster (different members in our library varied between
57 and 1276 clones), miR-21 (9521 clones) and miR-155
(409 clones) were highly represented in our sampling.
Nonetheless, 27% (107/398) of all the discrete miRNAs
identified in our study were cloned only once (Figure 1B).
In other studies, many bona fide cellular miRNAs have
also been reported to be cloned as single copies (27–30).
For example, Berezikov et al. (30) reported that in their
library of 19291 mouse and 23351 human small RNA
clones, 28% of the known miRNAs were detected either
only once or not at all. In a separate respect, our miRNA-
cloning sensitivity may be slightly better than that
reported in some other studies (26) which identified and
cloned from Jurkat T-cell RNAs many fewer discrete
miRNA species [i.e. only 592 Jurkat miRNA clones were
sequenced by Landgraf et al. to reveal 60 individual
miRNAs (26)]. As noted elsewhere, the results from
various studies should be interpreted with caution since
differencescould be due
methodologies, tissue culturing conditions and sample
size (31). Separately, of interest, 7.58% (3618 clones
representing 151 unique sequences) of our small RNAs
were piRNAs (Figure 1A). While piRNAs are generally
considered to be restricted in expression to germ line cells
(32,33), our identification of piRNAs agrees with a report
of Ago2- and Ago3-associated piRNAs in Jurkat cells
(34). The role of piRNAs in somatic cells requires
to dissimilarcell types,
Characterization of HIV-1 small ncRNAs
Amongst our 47773 clones, 125 HIV-1 entities with
sequence lengths of 15–26 were found (Supplementary
Table S2). Like cellular miRNAs, the largest class of
these viral clones encompassed those sequences (113)
that were cloned only once (six others were cloned twice
and one was cloned thrice). Because cloning proficiencies
may be operator-dependent, the functional implications
and quantitative interpretation of sequences that were
cloned singly versus doubly or triply are not clear,
especially since a plurality of authentic cellular miRNAs
was cloned only once by us (Supplementary Table S1). In
the singly cloned HIV-1 sequences, three observations are
noteworthy. First, we cloned a TARncRNA (Figure 2A,
left) that agrees with the reported isolation of up to 17
TAR miRNA clones by others (10). Second, we cloned a
NEFncRNA sequence (Figure 2A, middle) which is
complementary to a previously described NEF miRNA
(hiv1-miR-N367) [(9,35) discussed in more detail below].
Third, wecloneda ncRNA
(Figure 2B, left) which matches as the miRNA* strand
for a previously reported HIV-1 vsiRNA sequence (36).
Nucleic Acids Research, 2009,Vol.37, No. 196577
These three cloned examples, although difficult to
interpret by themselves, appear to independently confirm
earlier published observations on small HIV-1 non-coding
RNAs. We also identified six HIV-1 RNA clones which
share an identical core sequence but diverged slightly in
their end nucleotides (Figure 2B, right, RREncRNA).
vsiRNA* sequence within the highly secondary structured
HIV-1 RRE stem I (Figure 2B). The variations in the
termini of RREncRNA clones may have arisen from
inexact Dicer end processing which has been noted by
others (14). The functional importance of TARncRNA,
NEFncRNA, vsiRNA/vsiRNA* and RREncRNA in
HIV-1 infected cells remains to be investigated.
It is technically challenging to clone low copy small
RNAs. Cloning, per se, may not be a quantitatively
reliable metric. In contrast, sequence specific real time
RT–PCR is a well-established measure of RNA copy
number. To assess better the abundance of HIV-1 small
RNAs, we next employed real-time quantitative RT–PCR
(RT–qPCR) to characterize TARncRNA, RREncRNA
and NEFncRNA (Figure 3). As expected, TARncRNA,
RREncRNA and NEFncRNA were detected in HIV-1
NL4-3 infected MT4, but not in uninfected control cells
(data not shown). As negative detection controls, we
randomly chose HIV-1 primer pairs that would measure
ncRNAs, if any, which corresponded to NL4-3 sequences
located at 3263 and 5543. These control primers produced
no detectable signals in virus infected MT4 cells (data not
shown). In contrast, sequence-specific RT–qPCR did
quantify 4?104copies of TARncRNA, 187 copies of
RREncRNA and 607 copies of NEFncRNA, per 106
copies of cellular small U6 RNA. Based on published
data (37), there are ?105copies of U6 RNA per cell.
Hence, TARncRNA, RREncRNA and NEFncRNA,
normalized to U6, are calculated to be roughly 3?103,
19 and 61 copies per cell, respectively. We note that
several factors may influence the copy number detection
of small RNAs. For instance, it is possible that fewer than
100% of the MT4 cells were infected by HIV-1; this would
Figure 1. Pyrosequencing of small RNAs from HIV-1 infected T cells. (A) A pie chart indicates the distribution of different cellular small RNAs.
(B) Distribution frequency showing the number of times that different miRNAs were cloned.
6578 Nucleic Acids Research, 2009,Vol.37, No. 19
|| 8 18 28 | 38 48
+1Cap site, mRNA start
58 68 78
------ C A UCU CU
GGU UCUCUGGUUAG CCAGA GAGC G
CCA AGGGAUCAAUC GGUCU
| | AG
H S Q R R Q D I L D L W I Y H T Q G Y F P D W Q N Y T P G P8637 8647 8657 8667 8677 8687 8697 8707 8717 ACUCCCAAAGAAGACAAGAUAUCCUUGAUCUGUGGAUCUACCACACACAAGGCUACUUCCCUGAUUGGCAGAACUACACACCAGGGCCAGG V R Y P L T F G W C Y K L V P V E P D K V E E A N K G E N8727 8737 8747 8757 8767 8777 8787 8797 8807GGGUCAGAUAUCCACUGACCUUUGGAUGGUGCUACAAGCUAGUACCAGUUGAGCCAGAUAAGGUAGAAGAGGCCAAUAAAGGAGAGAACA
E = –31.3 kcal/mol
E = –21.7 kcal/mol
A C A
UUGGC G A UAC CA CCAG GG CAG GGGU G GAUCG C U GUG GU GGUU CC GUC CCUA
A A C A CUA
GUGG UCU CCA AGG C
CAUC AGA GGU UCC
A | CG
C UAG CU
E = –22.0 kcal/mol
E = –34.2 kcal/mol
Stem-loop IIBStem-loop IIA
E = –38.0 kcal/mol
U U C
UUCU UGGGUU CCU GUU UCGAGGAU
AAGA ACCUAA GGA CAA AGCUCCUG
Figure 2. Characterization of HIV-1 small non-coding RNAs. (A) TARncRNA (highlighted in yellow) maps to the LTR (Left). The predicted TAR hairpin structure and the location of the
TARncRNA are shown. NEFncRNA, NEFncRNA* (highlighted in yellow and grey) and hiv1-miR-N367 (red) (9,35) are indicated in the Nef coding sequence (top). Two alternate hairpin
structures can be predicted for these sequences. In one structure, NEFncRNA is complementary to the previously cloned NEFmiRNA, hiv-1-miR367. In the second structure, NEFncRNA is in a
hairpin with NEFncRNA* sequence which was also cloned in our small RNA library. Both structures share similar calculated free energies. (B) Mfold (68) prediction of the HIV-1 pNL4-3 RRE
structure (top). RRE stem I is the apparent precursor for a vsiRNA*-vsiRNA (red) (36) duplex and for the RREncRNA (highlighted in yellow). Both vsiRNA* and RREncRNA were cloned in
the current study. Free energies (E) of the vsiRNA*-vsiRNA and RREncRNA structures are ?38.0and ?34.2kcal/mol, respectively.
Nucleic Acids Research, 2009,Vol.37, No. 196579
affect the copy number per cell calculation. Additionally,
the stability of viral RNA structures and the likelihood
that RNAs with secondary structures are bound by
RNA-binding proteins (e.g. TAR- or RRE-binding
proteins) may affect the accessibility of these sequences
to processing factors.
Small RNA processed from a PBS–tRNAlys3 duplex
Curiously, the most individually abundant HIV-1-related
clone (19 clones were isolated) from the infected MT4 cells
was an 18-nt ncRNA (PBSncRNA, Figure 4A) with a
perfectly matched antisense sequence to the viral PBS.
During HIV-1 replication, an RNA duplex is formed
between the PBS and cellular tRNAlys3 (Figure 4B)
with the 30-end of PBS-bound tRNAlys3 used as the
primer for reverse transcription. It is known that tRNA
maturation requires the addition of the CCA nucleotides
to the 30terminus via the action of CCA-adding enzyme
(tRNA nucleotidyltransferase) (38). The detection of CCA
sequence in our PBSncRNA (Figure 4B) is consistent with
its origin from mature tRNAlys. Indeed, the cloning of
the 18-nt PBSncRNA as a discrete moiety suggests that
the PBS–tRNAlys3 hybrid might be processed by an
RNase III enzyme such as Dicer.
It is possible that the 18-nt PBSncRNA could be an
artifactual product of random tRNAlys3 degradation.
Two observations argue against this explanation. First,
the abundance of processed PBSncRNA was correlated
with HIV-1 expression. Thus increasing amounts of
PBSncRNA were detected when we transfected escalating
amounts of HIV-1 molecular clone pNL4-3 into 293T
cells (Figure 4C). This correlation with HIV-1 expression
was also illustrated by the finding that 19 copies of
PBSncRNA were cloned from HIV-1 infected MT4 cells
(Figure 4A) while in the corresponding uninfected MT4
cells only a single PBSncRNA clone was recovered
(data not shown). In northern blot analysis, detection
of PBSncRNA using a sequence-specific probe was
observed in HIV-1 pNL4-3 transfected (but not mock
transfected) HeLa cells and in PMA-induced (but
not mock induced) HIV-1 latently infected U1 cells
(Figure 4D, lanes 2 and 4).
Second, in the sequencing data from our infected MT4
cells, we quantified the copy number of PBSncRNA versus
other similar 18-nt ncRNAs that could have arisen
from nine other comparably abundant tRNA species
(Figure 4E). If the PBSncRNA were a product of
random degradation of tRNAly3, then one might expect
the other 30tRNA 18-nt fragments from comparably
abundant tRNAs to be produced in closely equivalent
copy numbers. However, the tRNAlys3 PBSncRNA was
by far the most abundant moiety in HIV-1 infected MT4
cells, consistent with it arising from a viral PBS–tRNAlys3
dependent processing event (Figure 4E). Indeed, con-
sistent with this notion, two other groups have recently
described the unexpected cloning and pyrosequencing of
an abundant small ncRNA originating apparently from
the processing of an RNA hybrid formed from a tRNA
primer with the PBS from cell endogenous retroviruses
(14,15). For example, Kawaji et al. cloned and identified
in high abundance the discrete 30-end of tRNAGluCUC
CA-30which is wholly complementary to the PBS (50-UG
(ERV1) (15). Similarly, Calabrese et al. (14) identified
an abundantly expressed short ncRNA sequence which is
anti-sense to the PBS of the transposon repeat, commonly
found in the early mouse embryo and ES cells (39–41).
Thus, these collective findings suggest that tRNA-viral
PBS hybrids could be ubiquitously processed into
discrete intracellular ncRNA species perhaps using either
RNAse III dependent or independent paths.
Next, we queried if a PBS–tRNAlys3 duplex could be
an in vitro substrate for Dicer. To address this question,
we performed Dicer assays using a T7 synthesized
32P-radiolabeled HIV-1 RNA that contains the PBS
processing of the T7PBS RNA after annealing with a
primer pool of total cellular tRNAs with or without
added Dicer was monitored. A mostly unprocessed
T7PBS RNA (127bp) was observed when neither tRNA
nor Dicer was added (Figure 5A, lane 3). The addition of
Dicer-alone to the T7PBS RNA was insufficient to
produce a processed 18-nt band (Figure 5A, lane 2).
However, the specific processing of the T7PBS RNA to
an 18-nt RNA occurred when both tRNA and Dicer were
added to the reaction (Figure 5A, lane 1, see * band).
These results suggest that Dicer can recognize and
process a T7PBS–tRNAlys3 hybrid into a ncRNA
product, although further investigation is needed to
elucidate how this processing might occur inside cells.
We also considered the possibility that the PBSncRNA
could be generated by the RNaseH cleavage activity
of the HIV-1 RT protein (42,43). We do not favor this
explanation for the following reasons. First, PBSncRNA
was produced in a single-cycle transfection of the HIV-1
NL4-3 molecular clone into cells (Figure 4C). This type of
assay does not involve a reverse transcription step.
Second, to check more directly that PBSncRNA is not a
result of RNaseH activity during a reverse transcription
step, we added nevirapine, a non-nucleoside reverse
transcriptase inhibitor, before and during the transfec-
tion of pNL4-3 into 293T-AGO2 cells (Figure 5B).
Figure 3. Quantification of HIV-1 small RNAs by real-time RT–PCR.
The HIV-1 small RNAs (TARncRNA, RREncRNA and NEFncRNA)
were verified by quantitative RT–PCR using U6 small RNA for
normalization. Methodology in Supplementary Figure S1.
6580Nucleic Acids Research, 2009,Vol.37, No. 19
Figure 4. Detection of PBSncRNA that is antisense to the HIV-1 PBS. (A) Multiple copies of PBSncRNAs were cloned from HIV-1 infected MT4
cells. The numbering corresponds to pNL4-3 sequence. (B) A schematic diagram showing the duplex formed between the HIV-1 PBS with the 30-end
of tRNAlys3. PBSncRNA sequence is highlighted in grey. (C) Immunoprecipitation of the AGO2 protein followed by primer-specific RT–qPCR of
the AGO2-associated RNA shows that the amount of PBSncRNA quantified in 293T-AGO2 cells is dose-dependent on the amount of transfected
pNL4-3 (pNL). (D) Northern blot analysis demonstrating increased detection of PBSncRNA in PMA-induced HIV-1 latently infected U1 cells, and
in HIV-1 molecular clone (pNL4-3)-transfected HeLa cells (upper panel). Production of HIV-1 from the indicated cells was verified by measuring RT
activity (bottom panel). (E) Quantification of the predicted processing of the 30-end of the indicated tRNAs in HIV-1 infected MT4 cells. The relative
detection of different 30-end—tRNA fragments from the sequencing data is compared. The value of the 18-nt PBSncRNA from Lys(UUU)
(tRNAlys3) was set as 1. tRNA(UUU)=tRNAlys3.
Nucleic Acids Research, 2009,Vol.37, No. 19 6581
The amount of PBSncRNA produced was essentially the
same in the presence or absence of nevirapine suggesting
that its production is RT-independent. Finally, we also
performed siRNA knock down of Dicer. By knocking
down Dicer, we indeed observed a reduction in the
Association of PBSncRNA with Ago2-RISC
What could be a possible fate for the PBSncRNA? Would
it functionally engage intracellular RISC? The Ago2
protein is a central component of RISC (44), and above
results in Figure 5C suggest that PBSncRNA can engage
RISC. To check in greater detail how PBSncRNA
can function with Ago2, a 293T cell line stably over-
constructed. 293T-AGO2 cells were transfected with
anti-FLAG. The presence of PBSncRNA in the immuno-
As a control, transfection of pNL4-3 into the parental
293T cell line was also immunoprecipitated in parallel
Ago2 (293T-AGO2) was
Figure 5. Evidence for the in vitro processing and intracellular RISC-incorporation of PBSncRNA. (A) In vitro Dicer assay of the HIV-1 PBS–tRNA
hybrid (the PBS containing radiolabeled transcript was made as described in Supplementary Figure S2) produced a small RNA of 18nt (asterisk;
lane 1). This small RNA was not observed in the absence of either tRNA (lane 2) or tRNA+Dicer (lane 3). As a positive control, in vitro T7 RNA
polymerase synthesized and then annealed ds GFP RNA (T7dsGFP) was processed in vitro by Dicer, which yielded the expected small RNAs of
?22bp (lane 4). (B) Non-nucleoside RT inhibitor (Nevirapine) does not affect the expression of PBSncRNA in a single-cycle transfection of an HIV-
1 molecular clone. We transfected pNL4-3 into nevirapine treated (+; lanes 5–8) or untreated cells (?; lanes 1–4) and subsequently quantified
PBSncRNA. Similar expression levels of PBSncRNA was detected in nevirapine treated (lane 7) and untreated (lane 3) 293T-AGO2 cells. Controls
include the immunopreciptation using anti-FLAG in control cell line (293T-cont) (lanes 1 and 5) and the attempted detection of a randomly selected
HIV-1 sequence (HIVs3263) (lanes 2, 4, 6 and 8). (C) siRNA knock down of Dicer reduced the expression of PBSncRNA. PBSncRNA was
quantified in 293T-AGO2 cells transfected with control-siRNA (si-cont) or Dicer-siRNA (si-Dicer) and co-transfected with pNL4-3. The amount
of AGO2-associated PBSncRNA was then quantified by RT–qPCR after immunoprecipitation with anti-AGO2 (lanes 1 and 2). Control immunopre-
cipitation was also performed with an irrelevant IgG (lanes 3 and 4). (D) Immunoprecipitation (IP) of FLAG-Ago2 using anti-FLAG or a control
IgG antibody was performed in a 293T-AGO2 cell line or in a control 293T parental cell line as described in the text. Quantitative real-time
RT–qPCR detection of the HIV-1 PBSncRNA or two randomly chosen HIV-1 sequences (HIVs3263 and HIVs5543) from the IP products was
performed. The value of the RT–qPCR result from the IP of 293T-AGO2 transfected with pNL4-3 was set as 1.
6582Nucleic Acids Research, 2009,Vol.37, No. 19
with either anti-FLAG or with an irrelevant IgG
control antibody (Figure 5D; lanes 1–8). A significant
recovery of PBSncRNA from pNL4-3/293-AGO2 cells
was detected by RT–qPCR in the IP with anti-FLAG,
but not in the IP with IgG control antibody (Figure 5D;
compare lane 4–8); an insignificant amount of PBSncRNA
was seen in the immunoprecipitates from control and
mock transfected cells (Figure 5D; lanes 1–3 and lanes
5–7). To check further for specificity of detection,
we selected two random HIV-1 sequences (HIVs3263
and HIVs5543) for use in control RT–qPCR assays
(Figure 5D; lanes 9–24). In both instances, no specific
recovery wasdetected by
immunoprecipitations (Figure 5D; lanes 1, 3, 6 and 7).
Taken together, the results support the interpretation
of an intracellular association of PBSncRNA with
thesesequences in the
Over-expression of PBSncRNA modulated HIV-1
If PBSncRNA-associates with Ago2, could this inter-
action result in an active RISC? We, next, constructed a
reporter plasmid containing a single PBS-target sequence
positioned downstream of a luciferase cDNA (LucPBS;
Figure 6A). Transfection of LucPBS with a synthetic
PBSncRNA at 5nM into HeLa cells produced a 40%
inhibition of luciferase activity (Figure 6B). Increasing
the amount of transfected PBSncRNA to 50nM increased
luciferase inhibition to 80% (Figure 6B). As control,
(PBSncRNA mutant) did not inhibit luciferase expression,
supporting that the observed PBSncRNA-silencing was
To query the role of PBSncRNA-silencing in viral
biology, we examined how it might affect single-cycle
of HIV-1 replication. HIV-1 molecular clone pNL4-3
was co-transfected into 293T cells with PBSncRNA or
PBSncRNA mutant, and virus production was measured
by checking for the release of RT into the culture
luciferase reporter assays (Figure 6B), co-transfection of
the PBSncRNA, but not the PBSncRNA mutant,
produced a dose-dependent inhibition of virus RT
production (Figure 6C). Next, to ask whether the silencing
effect of PBSncRNA is through destabilization of the
targeted RNA or through a mechanism of translational
inhibition, RT–qPCR was performed to quantify HIV-1
RNAwith or without
(Supplementary Figure S3). The HIV-1 RNA copies
were significantly decreased by transfected PBSncRNA
in a dosage-dependent manner (Supplementary Figure
S3). Finally, to ask if cell endogenous PBSncRNA
exhibits a similar anti-HIV-1 effect as observed with
over expressed PBSncRNA, we reasoned that knocking
down the ambient level of PBSncRNA in cells could
make these cells more susceptible to HIV-1 replication.
We employed synthetic RNAs (antagomirs) designed
to knock down PBSncRNA. When these antagomirs
were introduced into cells, replication of HIV-1 in the
transfected cells was notably augmented (Figure 6D).
expressed and physiologically expressed PBSncRNA
can serve modulating influences on HIV-1 replication
the findings indicatethatbothover
Figure 6. Over expressed PBSncRNA or knock down of physiologically
expressed PBSncRNA can modulate viral replication. (A) A schematic
diagram of the LucPBS construct. A single copy of HIV-1 PBS
sequence (TGGCGCCCGAACAGGGAC) was positioned downstream
of the luciferase coding sequence. (B) Functional studies employing
synthetic dsRNA oligonucleotide corresponding to the PBSncRNA
or its mutated form, PBSncRNAmutant. Mutated nucleotides are
boxed. Different concentrations (5 or 50nM) of the PBSncRNA and
construct (described in A) into HeLa cells. The PBSncRNA inhibited
up to ?80% the luciferase expression. (C) Similar experiments were
performed in 293T cells using HIV-1 molecular clone (pNL4-3) in a
single round replication assay (see text). The PBSncRNA effectively
inhibited up to ?80% of HIV-1 replication while no inhibition was
replication by knock down of cell endogenous PBSncRNA using RNA
‘antagomirs’. Co-transfection of pNL4-3 and increasing amount of
antagomirs, complementary to the PBSncRNA, in 293T cells increased
HIV-1 replication as measured by RT activities.
Nucleic Acids Research, 2009,Vol.37, No. 196583
sequencing of cloned RNAs from HIV-1 infected MT4
cells. We sequenced 47773 clones which included 128
viral small RNA entities. In examining the results,
several reasons suggest (although they do not fully
exclude) that most of our viral RNA sequences are
unlikely to be products of random degradation. First,
we employed a small RNA cloning technique which
requires the presence of intact 50phosphate and 30OH
ends (26). Degraded RNAs have non-phosphorylated
50-ends and are unlikely to be cloned by this technique.
Second, degradation products tend to have a wide-
distribution of lengths due to random cleavage. The
lengths ofour clonedsmall
narrowly restricted. Third, the presence of mRNA
sequences in a small RNA clone library can represent
an internal measure of degradation products. In our
library, only 2.41% of the small RNAs were mRNA-
derived (Figure 1A), a number consistent with other
studies, suggesting a limited contribution of degradation
to our cloning. Finally, in the case of PBSncRNA [which
has related examples described by others (14,15)], we
showed an association of this moiety with Ago2. This
type of association is compatible with a functionally
relevant interaction which is unlikely to occur for RNAs
generated by random degradation.
Several studies have reported on the cloning and
sequencing of small RNAs encoded by viruses (23,24).
Small viral RNAs have most prevalently been described
in Herpesvirus-infected cells (45). In HIV-1 research, a
recent study has performed small RNA-cloning and
pyrosequencing using the Ach2 ‘latent’ HIV-1-infected
cell line and found minimal representation of viral
sequences (26). However, the Ach2 ‘latent’ cell results
could be problematic because the HIV-1 provirus in
Ach2 cells contains a mutated TAR which renders the
LTR non-responsive to the viral transcriptional activator,
Tat. Indeed, it was shown previously that when the same
TAR-mutation in Ach2 cells was transferred into an
otherwise infectious molecular clone of HIV-1, the virus
became crippled for replication, and no viral production
was measured (46). Our study differs from the Ach2-work
in employing the productive infection of MT4 cells with
a replication competent HIV-1 virus. As noted earlier,
our cloning of a wider-range of T-cell specific miRNAs
possibly indicate a greater sensitivity than that reported
by Landgraf et al. who sequenced 1098 Ach2-miRNA
clones (26) while we sequenced 24892 MT4-miRNA
clones. Nonetheless, one should be cautious about
interpreting diverse results since each infection system
and cloning method could have inherent constraints
which influence the precision of measurements (47).
Likely, with further improved sensitivity of cloning and
sequencing, small viral ncRNAs could be discovered for
an increasing number of mammalian viruses (48). Indeed,
in a recent small RNA cloning and pyrosequencing study
of four HTLV-1 infected/transformed cell lines, we have
also identified many discrete small viral ncRNAs (Yeung,
unpublished observations). It remains a future challenge
to determine the biological implications of small viral
ncRNAs in infected cells.
Although we do not yet understand the functions of
small HIV-1 ncRNAs, the identities of some clones are
consistent with the expectations from previous findings
(7–9). Bennasser et al. (49) and Omoto et al. (9) had
earlier predicted two potential miRNA-like hairpin
investigators have subsequently confirmed the existence
and cloning of a TAR miRNA (7,8,10), and our cloning
(Figure 2A; Supplementary Table S2) and RT–qPCR
results (Figure 3) are concordant with these findings.
Omoto et al. (9,35) had also cloned one arm of a
Nef-miRNA (hiv1-mir-N367) hairpin (Figure 2A, right).
Interestingly, our NEFncRNA now appears to be the
Nef-miRNA (hiv1-mir-N367) sequence. Provocatively,
NEFncRNA can also assume an alternative hairpin
structure (Figure 2A, right, NEFncRNA-NEFncRNA*);
and the NEFncRNA* strand of this alternate hairpin
was also recovered in our cloning. In our viral RNA
sequences, we further identified a clone for a putative
complementary arm (vsiRNA1*) of a RNA-duplex that
contains the previously reported vsiRNA1 (36). The
vsiRNA1*-vsiRNA1 duplex maps to a highly secondary
structured RRE segment, which apparently also produces
a cloned RREncRNA (Figure 2B, right). Pending a better
comprehension of the roles played by these RNA
sequences, one interpretation is that these HIV-1 small
ncRNAs are products of processing by the cellular
RNAi machinery, and that their presence agrees with
evidence elsewhere that RNAi serves an innate antiviral
defense in mammalian cells (31,50).
Our pyrosequencing study also identified a small
18-nt antisense ncRNA (PBSncRNA) that is fully com-
plementary to the HIV-1 PBS (Figure 4). The abundance
of PBSncRNA is HIV-1 expression dependant, consistent
with a mechanism whereby much of this moiety is
processed from a PBS–tRNAlys3 duplex by Dicer
(Supplementary Figure S4). Although we were initially
surprised by this cloning, a closer inspection of the
literature revealed two recent studies which also described
the abundant cloning of discrete analogous PBSncRNAs
complementary to the PBS of endogenous retroelements
(14,15). Together, these three PBSncRNA findings fit
a general notion that retroviral PBS–tRNA hybrid
duplex may be ubiquitously processed by the cell to yield
ncRNAs. The deliberate over expression of a HIV-1-PBS-
targeting ncRNA [Figure 6; and Han et al. (51)], like
other PBS-targeting molecules (52), can modulate viral
replication. We also observed that the knock down of
physiologically expressed PBSncRNA in cells increased
the replication of HIV-1 in these cells (Figure 6D)
suggesting that ambiently expressed PBSncRNA can
target intracellularly viral genomic sequence (53).
PBSncRNA represent evidence that mammalian cells
employ RNAi to defend exogenous retroviral infection
agrees with the current view that RNAi is utilized by
cells to suppress mammalian endogenous retroviruses
(40,41,54–56). The findings also reconcile accumulating
6584 Nucleic Acids Research, 2009,Vol.37, No. 19
evidence that mammalian viruses including HIV-1 encode
functional RNAi suppressors (which viruses should
encode if cells use RNAi-against viruses). Examples of
these suppressors include hepatitis C virus (HCV) core
and envelope protein 2 (57,58), vaccinia virus E3L (59),
Ebola virus VP35 (60), primate foamy virus Tas (61),
influenza A virus NS1 (59,62,63) and HIV-1 Tat
(36,50,64). Some of these suppressors may serve to
dsRNA (59,62,63,66). Either mechanism could limit the
capacity by the cell to process viral sequences into small
ncRNAs, perhaps in part explaining some of the
challenges in detecting small viral ncRNAs in mammalian
cells. In the big picture, studying the strike-counter
strike interplay in nucleic acid-based RNAi restriction
between viruses and their hosts (67) may shed further
understanding on viral sequence evolution and viral
(36,65), and/or sequester
Supplementary Data are available at NAR Online.
The authors thank Dixie Mager for bringing two
published references to our attention; and Andrew
Dayton and Venkat Yedavalli for critical readings of the
manuscript. pIRESneo-FLAG/HA-AGO2 was a kind gift
from Drs Thomas Tuschl and Gunter Meister of the
NIAID; NCI; IATAP program from the office of the
director, National Institutes of Health. Funding for
open access charge:National
Conflict of interest statement. None declared.
1. Kim,V.N. (2005) MicroRNA biogenesis: coordinated cropping and
dicing. Nat. Rev. Mol. Cell Biol., 6, 376–385.
2. Nelson,P., Kiriakidou,M., Sharma,A., Maniataki,E. and
Mourelatos,Z. (2003) The microRNA world: small is mighty.
Trends Biochem. Sci., 28, 534–540.
3. Gan,J., Tropea,J.E., Austin,B.P., Court,D.L., Waugh,D.S. and
Ji,X. (2006) Structural insight into the mechanism of double-
stranded RNA processing by ribonuclease III. Cell, 124, 355–366.
4. Yeung,M.L., Bennasser,Y., Le,S.Y. and Jeang,K.T. (2005) siRNA,
miRNA and HIV: promises and challenges. Cell Res., 15, 935–946.
5. Saito,K., Ishizuka,A., Siomi,H. and Siomi,M.C. (2005) Processing
of pre-microRNAs by the Dicer-1-Loquacious complex in
Drosophila cells. PLoS Biol., 3, e235.
6. Watanabe,T., Totoki,Y., Toyoda,A., Kaneda,M., Kuramochi-
Miyagawa,S., Obata,Y., Chiba,H., Kohara,Y., Kono,T., Nakano,T.
et al. (2008) Endogenous siRNAs from naturally formed dsRNAs
regulate transcripts in mouse oocytes. Nature, 453, 539–543.
7. Ouellet,D.L., Plante,I., Landry,P., Barat,C., Janelle,M.E.,
Flamand,L., Tremblay,M.J. and Provost,P. (2008) Identification of
functional microRNAs released through asymmetrical processing of
HIV-1 TAR element. Nucleic Acids Res., 36, 2353–2365.
8. Klase,Z., Kale,P., Winograd,R., Gupta,M.V., Heydarian,M.,
Berro,R., McCaffrey,T. and Kashanchi,F. (2007) HIV-1 TAR
element is processed by Dicer to yield a viral micro-RNA
involved in chromatin remodeling of the viral LTR. BMC Mol.
Biol., 8, 63.
9. Omoto,S., Ito,M., Tsutsumi,Y., Ichikawa,Y., Okuyama,H.,
Brisibe,E.A., Saksena,N.K. and Fujii,Y.R. (2004) HIV-1 nef
suppression by virally encoded microRNA. Retrovirology, 1, 44.
10. Klase,Z., Winograd,R., Davis,J., Carpio,L., Hildreth,R.,
Heydarian,M., Fu,S., McCaffrey,T., Meiri,E., yash-Rashkovsky,M.
et al. (2009) HIV-1 TAR miRNA protects against apoptosis by
altering cellular gene expression. Retrovirology, 6, 18.
11. Grassmann,R. and Jeang,K.T. (2008) The roles of microRNAs in
mammalian virus infection. Biochim. Biophys. Acta, 1779, 706–711.
12. Houzet,L., Yeung,M.L., de,L.V., Desai,D., Smith,S.M. and
Jeang,K.T. (2008) MicroRNA profile changes in human
immunodeficiency virus type 1 (HIV-1) seropositive individuals.
Retrovirology, 5, 118.
13. Yeung,M.L., Benkirane,M. and Jeang,K.T. (2007) Small
non-coding RNAs, mammalian cells, and viruses: regulatory
interactions? Retrovirology, 4, 74.
14. Calabrese,J.M., Seila,A.C., Yeo,G.W. and Sharp,P.A. (2007) RNA
sequence analysis defines Dicer’s role in mouse embryonic stem
cells. Proc. Natl Acad. Sci. USA, 104, 18097–18102.
15. Kawaji,H., Nakamura,M., Takahashi,Y., Sandelin,A.,
Katayama,S., Fukuda,S., Daub,C.O., Kai,C., Kawai,J., Yasuda,J.
et al. (2008) Hidden layers of human small RNAs. BMC Genomics,
16. Hutvagner,G., McLachlan,J., Pasquinelli,A.E., Balint,E., Tuschl,T.
and Zamore,P.D. (2001) A cellular function for the RNA-
interference enzyme Dicer in the maturation of the let-7 small
temporal RNA. Science, 293, 834–838.
17. Meister,G., Landthaler,M., Patkaniowska,A., Dorsett,Y., Teng,G.
and Tuschl,T. (2004) Human Argonaute2 mediates RNA cleavage
targeted by miRNAs and siRNAs. Mol. Cell, 15, 185–197.
18. Willey,R.L., Smith,D.H., Lasky,L.A., Theodore,T.S., Earl,P.L.,
Moss,B., Capon,D.J. and Martin,M.A. (1988) In vitro
mutagenesis identifies a region within the envelope gene of the
human immunodeficiency virus that is critical for infectivity.
J. Virol., 62, 139–147.
19. Beerens,N. and Berkhout,B. (2000) In vitro studies on tRNA
annealing and reverse transcription with mutant HIV-1 RNA
templates. J. Biol. Chem., 275, 15474–15481.
20. Lau,N.C., Lim,L.P., Weinstein,E.G. and Bartel,D.P. (2001) An
abundant class of tiny RNAs with probable regulatory roles in
Caenorhabditis elegans. Science, 294, 858–862.
21. Yeung,M.L., Bennasser,Y., Myers,T.G., Jiang,G., Benkirane,M.
and Jeang,K.T. (2005) Changes in microRNA expression profiles
in HIV-1-transfected human cells. Retrovirology, 2, 81.
22. Shi,R. and Chiang,V.L. (2005) Facile means for quantifying
microRNA expression by real-time PCR. Biotechniques, 39,
23. He,S., Yang,Z., Skogerbo,G., Ren,F., Cui,H., Zhao,H., Chen,R.
and Zhao,Y. (2008) The properties and functions of virus
encoded microRNA, siRNA, and other small noncoding RNAs.
Crit. Rev. Microbiol., 34, 175–188.
24. Sullivan,C.S. (2008) New roles for large and small viral RNAs in
evading host defences. Nat. Rev. Genet., 9, 503–507.
25. Bar,M., Wyman,S.K., Fritz,B.R., Qi,J., Garg,K.S., Parkin,R.K.,
Kroh,E.M., Bendoraite,A., Mitchell,P.S., Nelson,A.M. et al. (2008)
MicroRNA discovery and profiling in human embryonic stem cells
by deep sequencing of small RNA libraries. Stem Cells, 26,
26. Landgraf,P., Rusu,M., Sheridan,R., Sewer,A., Iovino,N.,
Aravin,A., Pfeffer,S., Rice,A., Kamphorst,A.O., Landthaler,M.
et al. (2007) A mammalian microRNA expression atlas based
on small RNA library sequencing. Cell, 129, 1401–1414.
27. Sunkar,R., Zhou,X., Zheng,Y., Zhang,W. and Zhu,J.K. (2008)
Identification of novel and candidate miRNAs in rice by high
throughput sequencing. BMC. Plant Biol., 8, 25.
28. Kloosterman,W.P., Steiner,F.A., Berezikov,E., de,B.E., van de,B.J.,
Verheul,M., Cuppen,E. and Plasterk,R.H. (2006) Cloning and
expression of new microRNAs from zebrafish. Nucleic Acids Res.,
Nucleic Acids Research, 2009,Vol.37, No. 196585
29. Takada,S., Berezikov,E., Yamashita,Y., Lagos-Quintana,M.,
Kloosterman,W.P., Enomoto,M., Hatanaka,H., Fujiwara,S.,
Watanabe,H., Soda,M. et al. (2006) Mouse microRNA profiles
determined with a new and sensitive cloning method. Nucleic Acids
Res., 34, e115.
30. Berezikov,E., van,T.G., Verheul,M., van de,B.J., van,L.L., Vos,J.,
Verloop,R., van de,W.M., Guryev,V., Takada,S. et al. (2006)
Many novel mammalian microRNA candidates identified by
extensive cloning and RAKE analysis. Genome Res., 16, 1289–1298.
31. Pedersen,I.M., Cheng,G., Wieland,S., Volinia,S., Croce,C.M.,
Chisari,F.V. and David,M. (2007) Interferon modulation
of cellular microRNAs as an antiviral mechanism. Nature, 449,
32. Klattenhoff,C. and Theurkauf,W. (2008) Biogenesis and germline
functions of piRNAs. Development, 135, 3–9.
33. Chi,Y.H., Cheng,L.I., Myers,T., Ward,J.M., Williams,E., Su,Q.,
Faucette,L., Wang,J.Y. and Jeang,K.T. (2009) Requirement for
Sun1 in the expression of meiotic reproductive genes and piRNA.
Development, 136, 965–973.
34. Azuma-Mukai,A., Oguri,H., Mituyama,T., Qian,Z.R., Asai,K.,
Siomi,H. and Siomi,M.C. (2008) Characterization of endogenous
human Argonautes and their miRNA partners in RNA silencing.
Proc. Natl Acad. Sci. USA, 105, 7964–7969.
35. Omoto,S. and Fujii,Y.R. (2005) Regulation of human
immunodeficiency virus 1 transcription by nef microRNA. J. Gen.
Virol., 86, 751–755.
36. Bennasser,Y., Le,S.Y., Benkirane,M. and Jeang,K.T. (2005)
Evidence that HIV-1 encodes an siRNA and a suppressor of RNA
silencing. Immunity, 22, 607–619.
37. Lim,L.P., Lau,N.C., Weinstein,E.G., Abdelhakim,A., Yekta,S.,
Rhoades,M.W., Burge,C.B. and Bartel,D.P. (2003) The microRNAs
of Caenorhabditis elegans. Genes Dev., 17, 991–1008.
38. Xiong,Y. and Steitz,T.A. (2006) A story with a good ending: tRNA
30-end maturation by CCA-adding enzymes. Curr. Opin. Struct.
Biol., 16, 12–17.
39. Maksakova,I.A. and Mager,D.L. (2005) Transcriptional regulation
of early transposon elements, an active family of mouse long
terminal repeat retrotransposons. J. Virol., 79, 13865–13874.
40. Brennecke,J., Malone,C.D., Aravin,A.A., Sachidanandam,R.,
Stark,A. and Hannon,G.J. (2008) An epigenetic role for maternally
inherited piRNAs in transposon silencing. Science, 322, 1387–1392.
41. Aravin,A.A., Sachidanandam,R., Bourc’his,D., Schaefer,C.,
Pezic,D., Toth,K.F., Bestor,T. and Hannon,G.J. (2008) A piRNA
pathway primed by individual transposons is linked to de novo
DNA methylation in mice. Mol. Cell, 31, 785–799.
42. Ben-Artzi,H., Shemesh,J., Zeelon,E., Amit,B., Kleiman,L.,
Gorecki,M. and Panet,A. (1996) Molecular analysis of the second
template switch during reverse transcription of the HIV RNA
template. Biochemistry, 35, 10549–10557.
43. Auxilien,S., Keith,G., Le Grice,S.F. and Darlix,J.L. (1999) Role of
post-transcriptional modifications of primer tRNALys,3 in the
fidelity and efficacy of plus strand DNA transfer during HIV-1
reverse transcription. J. Biol. Chem., 274, 4412–4420.
44. Hammond,S.M., Boettcher,S., Caudy,A.A., Kobayashi,R. and
Hannon,G.J. (2001) Argonaute2, a link between genetic and
biochemical analyses of RNAi. Science, 293, 1146–1150.
45. Cullen,B.R. (2009) Viral and cellular messenger RNA targets of
viral microRNAs. Nature, 457, 421–425.
46. Emiliani,S., Van,L.C., Fischle,W., Paras,P. Jr, Ott,M., Brady,J. and
Verdin,E. (1996) A point mutation in the HIV-1 Tat responsive
element is associated with postintegration latency. Proc. Natl Acad.
Sci. USA, 93, 6377–6381.
47. Lim,L.P. and Linsley,P.S. (2007) Mustering the micromanagers.
Nat. Biotechnol., 25, 996–997.
48. Parameswaran,P., Fire,A., Jalili,B., gharizadeh,M., Ronaghi,M.,
Lu,R., Ding,S.W., Burgon,T., Jackson,W., Kirkegaard,K. et al.
(2008) Investigating the interplay between viral replication and the
RNAi machinery in vertebrates and invertebrates. Proceedings of
the Keystone Symposium Whistler Resort. Vancouver, Canada.
25-30 March 2008, Poster abstract number 258, 127.
49. Bennasser,Y., Le,S.Y., Yeung,M.L. and Jeang,K.T. (2004) HIV-1
encoded candidate micro-RNAs and their cellular targets.
Retrovirology., 1, 43.
50. Schnettler,E., de,V.W., Hemmes,H., Haasnoot,J., Kormelink,R.,
Goldbach,R. and Berkhout,B. (2009) The NS3 protein of rice hoja
blanca virus complements the RNAi suppressor function of HIV-1
Tat. EMBO Rep., 10, 258–263.
51. Han,W., Wind-Rotolo,M., Kirkman,R.L. and Morrow,C.D. (2004)
Inhibition of human immunodeficiency virus type 1 replication by
siRNA targeted to the highly conserved primer binding site.
Virology, 330, 221–232.
52. Eberhardy,S.R., Goncalves,J., Coelho,S., Segal,D.J., Berkhout,B.
and Barbas,C.F. III. (2006) Inhibition of human immunodeficiency
virus type 1 replication with artificial transcription factors targeting
the highly conserved primer-binding site. J. Virol., 80, 2873–2883.
53. Westerhout,E.M., Ter,B.O. and Berkhout,B. (2006) The
virion-associated incoming HIV-1 RNA genome is not targeted
by RNA interference. Retrovirology, 3, 57.
54. Yang,N. and Kazazian,H.H. Jr. (2006) L1 retrotransposition is
suppressed by endogenously encoded small interfering RNAs in
human cultured cells. Nat. Struct. Mol. Biol., 13, 763–771.
55. Watanabe,T., Takeda,A., Tsukiyama,T., Mise,K., Okuno,T.,
Sasaki,H., Minami,N. and Imai,H. (2006) Identification and
characterization of two novel classes of small RNAs in the mouse
germline: retrotransposon-derived siRNAs in oocytes and germline
small RNAs in testes. Genes Dev., 20, 1732–1743.
56. Carmell,M.A., Girard,A., van de Kant,H.J., Bourc’his,D.,
Bestor,T.H., de Rooij,D.G. and Hannon,G.J. (2007) MIWI2 is
essential for spermatogenesis and repression of transposons in the
mouse male germline. Dev. Cell, 12, 503–514.
57. Wang,Y., Kato,N., Jazag,A., Dharel,N., Otsuka,M., Taniguchi,H.,
Kawabe,T. and Omata,M. (2006) Hepatitis C virus core protein is a
potent inhibitor of RNA silencing-based antiviral response.
Gastroenterology, 130, 883–892.
58. Ji,J., Glaser,A., Wernli,M., Berke,J.M., Moradpour,D. and Erb,P.
(2008) Suppression of short interfering RNA-mediated gene
silencing by the structural proteins of hepatitis C virus. J. Gen.
Virol., 89, 2761–2766.
59. Li,W.X., Li,H., Lu,R., Li,F., Dus,M., Atkinson,P., Brydon,E.W.,
Johnson,K.L., Garcia-Sastre,A., Ball,L.A. et al. (2004)
Interferon antagonist proteins of influenza and vaccinia viruses
are suppressors of RNA silencing. Proc. Natl Acad. Sci. USA, 101,
60. de Vries,W. and Berkhout,B. (2008) RNAi suppressors encoded by
pathogenic human viruses. Int. J. Biochem. Cell Biol., 40,
61. Lecellier,C.H., Dunoyer,P., Arar,K., Lehmann-Che,J., Eyquem,S.,
Himber,C., Saib,A. and Voinnet,O. (2005) A cellular microRNA
mediates antiviral defense in human cells. Science, 308, 557–560.
62. Haasnoot,J., de,V.W., Geutjes,E.J., Prins,M., de,H.P. and
Berkhout,B. (2007) The Ebola virus VP35 protein is a suppressor of
RNA silencing. PLoS Pathog., 3, e86.
63. Bucher,E., Hemmes,H., de,H.P., Goldbach,R. and Prins,M. (2004)
The influenza A virus NS1 protein binds small interfering RNAs
and suppresses RNA silencing in plants. J. Gen. Virol., 85, 983–991.
64. Qian,S., Zhong,X., Yu,L., Ding,B., de,H.P. and Boris-Lawrie,K.
(2009) HIV-1 Tat RNA silencing suppressor activity is conserved
across kingdoms and counteracts translational repression of HIV-1.
Proc. Natl Acad. Sci. USA, 106, 605–610.
65. Chen,W., Zhang,Z., Chen,J., Zhang,J., Zhang,J., Wu,Y., Huang,Y.,
Cai,X. and Huang,A. (2008) HCV core protein interacts with Dicer
to antagonize RNA silencing. Virus Res., 133, 250–258.
66. Bennasser,Y. and Jeang,K.T. (2006) HIV-1 Tat interaction with
Dicer: requirement for RNA. Retrovirology, 3, 95.
67. Haasnoot,J., Westerhout,E.M. and Berkhout,B. (2007) RNA
interference against viruses: strike and counterstrike. Nat.
Biotechnol., 25, 1435–1443.
68. Zuker,M. (2003) Mfold web server for nucleic acid folding and
hybridization prediction. Nucleic Acids Res., 31, 3406–3415.
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