Stabilization of hepatitis C virus RNA by an
Tetsuro Shimakamia,1, Daisuke Yamanea,1, Rohit K. Jangraa,1,2, Brian J. Kempfb, Carolyn Spaniela, David J. Bartonb,
and Stanley M. Lemona,3
aLineberger Comprehensive Cancer Center and Division of Infectious Diseases, Department of Medicine, University of North Carolina at Chapel Hill, Chapel
Hill, NC 27599-7292; andbDepartment of Microbiology, University of Colorado School of Medicine, Aurora, CO 80045
Edited* by Charles M. Rice, The Rockefeller University, New York, NY, and approved December 5, 2011 (received for review July 27, 2011)
MicroRNAs (miRNAs) are small noncoding RNAs that regulate
eukaryotic gene expression by binding to regions of imperfect
complementarity in mRNAs, typically in the 3′ UTR, recruiting an
Argonaute (Ago) protein complex that usually results in transla-
tional repression or destabilization of the target RNA. The trans-
lation and decay of mRNAs are closely linked, competing pro-
cesses, and whether the miRNA-induced silencing complex (RISC)
acts primarily to reduce translation or stability of the mRNA remains
controversial. miR-122 is an abundant, liver-specific miRNA that is
an unusual host factor for hepatitis C virus (HCV), an important
cause of liver disease in humans. Prior studies show that it binds
the 5′ UTR of the messenger-sense HCV RNA genome, stimulating
translation and promoting genome replication by an unknown
mechanism. Here we show that miR-122 binds HCV RNA in associ-
ation with Ago2 and that this slows decay of the viral genome in
infected cells. The stabilizing action of miR-122 does not require
the viral RNA to be translationally active nor engaged in replica-
tion, and can be functionally substituted by a nonmethylated 5′
cap. Our data demonstrate that a RISC-like complex mediates the
stability of HCV RNA and suggest that Ago2 and miR-122 act co-
ordinately to protect the viral genome from 5′ exonuclease activity
of the host mRNA decay machinery. miR-122 thus acts in an un-
conventional fashion to stabilize HCV RNA and slow its decay,
expanding the repertoire of mechanisms by which miRNAs modu-
late gene expression.
RNA decay|viral host factor
mentarity in the 3′ UTR of mRNAs, recruiting an Argonaute
(Ago) protein complex that results in translational repression or
destabilization of the target RNA (1). Although miRNAs regu-
late a majority of genes, an unresolved question is whether the
miRNA-induced silencing complex (RISC) acts primarily to re-
duce translation or enhance decay of the mRNA, two closely
linked, competing processes (2–4). miR-122 is an abundant, liver-
specific miRNA, comprising >50% of mature miRNAs in human
hepatocytes and regulating the expression of numerous hepatic
genes, including those involved in fatty acid and cholesterol
metabolism (5, 6). It is also a very unusual host factor required
for replication of hepatitis C virus (HCV), an important cause of
liver disease in humans (7, 8). Prior studies show that miR-122
binds the 5′ UTR of the positive-strand HCV RNA genome (7,
9), stimulating viral protein expression and promoting viral rep-
lication by a poorly understood mechanism (10, 11).
Although miR-122 does not directly stimulate HCV RNA
synthesis (12, 13), its ability to promote genome amplification is
independent of its regulation of hepatic metabolism (12) and
requires the binding of its “seed sequence” (nucleotides 2–8) to
two conserved sites (S1 and S2) in the viral 5′ UTR (7, 9). Ad-
ditional “supplementary” base-pairing between miR-122 and
HCV RNA sequences upstream of S1 and S2 has also been
recognized recently and shown to be essential for promotion of
genome amplification (14, 15). The miR-122 binding sites are
icroRNAs (miRNAs) typically regulate eukaryotic gene
expression by binding to regions of imperfect comple-
near the 5′ end of the RNA and immediately upstream of an
internal ribosome entry site (IRES) that has high affinity for the
40S ribosome subunit (16). The unusual ability of miR-122 to
stimulate viral protein translation (10, 11) is dependent on where
it binds, because miR-122 suppresses expression of capped re-
porter mRNAs that contain the HCV target sequence in the 3′
UTR (9). Translation enhancement only partially explains the
role of miR-122 in the HCV life cycle, however, because mutant
viral RNAs that are deficient in miR-122 binding are far more
handicapped in their ability to replicate than viral RNAs with
mutations in the IRES that result in quantitatively comparable
defects in translation (11).
Although there has been speculation that miR-122 might pro-
mote genome amplification and viral protein expression by phys-
ically stabilizing HCV RNA (14, 17), previous experimental results
suggest this is not the case and that miR-122 does not enhance
RNA stability (7, 10). Here we present a contrasting view and
show that binding of miR-122 to the 5′ terminus of HCV RNA in
association with Ago2 significantly slows decay of the viral RNA
genome in infected cells. miR-122 thus acts in an unconventional
fashion to stabilize HCV RNA, expanding the repertoire of
mechanisms by which miRNAs modulate gene expression.
We studied how miR-122 influences the stability of synthetic
HCV RNA transfected into human hepatoma cells. Northern
blots demonstrated significant increases in the abundance of
a replication-defective viral RNA (H77S/GLuc2A-AAG, that
contains a lethal mutation in its RNA polymerase) (Fig. 1A)
when it was electroporated into cells together with duplex miR-
122 (Fig. 1B). miR-124, a brain-specific miRNA that does not
bind HCV RNA, had no such effect. Conversely, cotransfection
of viral RNA with 2’O-methyl-modified (2’OMe) or locked
nucleic acid antisense oligoribonucleotides capable of seques-
tering miR-122 reduced the abundance of HCV RNA at 3, 6,
and 9 h after electroporation. These differences were re-
producible in multiple experiments and observed with HCV
RNAs containing 5′ UTR sequence from either genotype 1
(H77) or genotype 2 (JFH1) virus. We estimated the rate of viral
RNA decay by PhosphorImager analysis of Northern blots,
normalizing HCV RNA abundance (HCV RNA/actin mRNA)
to that present at 3 h after electroporation to compare rates of
Author contributions: T.S., D.Y., R.K.J., B.J.K., C.S., D.J.B., and S.M.L. designed research; T.S.,
D.Y., R.K.J., C.S., and B.J.K. performed research; T.S., D.Y., R.K.J., B.J.K., C.S., D.J.B., and S.M.
L. analyzed data; and T.S., D.Y., R.K.J., D.J.B., and S.M.L. wrote the paper.
The authors declare no conflict of interest.
*This Direct Submission article had a prearranged editor.
1T.S., D.Y., and R.K.J. contributed equally to this work.
2Present address: Department of Microbiology, Mount Sinai School of Medicine, New
York, NY 10029.
3To whom correspondence should be addressed. E-mail: firstname.lastname@example.org.
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.
| January 17, 2012
| vol. 109
| no. 3
AAG mutation in NS5B ablates genome amplification. The position at which miR-122 binds the 5′ UTR is shown. (B) Northern blot of H77S/GLuc2A-AAG and
actin RNA (loading control) in Huh-7.5 cells after electroporation of viral RNA together with duplex miRNAs or antisense oligoribonucleotides. (C) Quanti-
tation of HCV RNA relative to actin mRNA by phosphorimaging of Northern blots from five independent electroporation experiments. Data shown represent
the mean percentage RNA remaining (±SEM) relative to that present at 3 h after electroporation under each condition. Data were fit to a one-phase decay
model (R2= 0.87–0.99). The shaded area represents the approximate limit of detection (LOD; 9 h posttransfection data from anti-miR-122–transfected cells
were below the LOD and excluded from analysis). (D) Mean HCV RNA t1/2estimated by fitting the Northern blot data to a one-phase decay model, as shown in
C. Error bars indicate 95% confidence intervals. (E) Northern blot of HCV RNA in persistently infected (>3 wk) cells treated with PSI-6130 (10 μM or >10-fold
the EC50) and transfected with miRNAs or 2’OMe oligoribonucleotides. (F) PhosphorImager quantitation of Northern blots in two replicate experiments
involving PSI-6130 treatment. Results were normalized to RNA abundance in mock-transfected cells at 12 h. See D for the key. (G) Mean HCV RNA t1/2± SD in
cells supplemented with miR-122 vs. miR-124 after PSI-6130 treatment, estimated by fitting PhosphorImager data from four independent experiments to
a one-phase decay model. P = 0.007 by two-sided paired t test. (H) qRT-PCR determination of HCV RNA decay in infected cells treated with PSI-6130 and
supplemented with miRNAs and antisense oligoribonucleotides as in E. Data are from three replicate infected cultures, and represent HCV genome equiv-
alents (GE) per μg total RNA ± SD (left) HCV RNA in cells supplemented with miR-122 or miR-124. Data were fit to a one-phase decay model (R2= 0.86–0.95):
HCV RNA t1/2=30.3 h for miR-122 vs. 19.8 h for miR-124 (P = 0.018). Right: Cells treated with 2’-OMe anti-miR-122 or anti-random (R2= 0.95–0.96): t1/2=18.2 h
for anti-miR-122 vs. 21.9 h for anti-random (P = 0.023). The dashed line in both panels represents the one-phase decay curve in mock-treated cells.
miR-122 stabilizes the HCV RNA genome. (A) Organization of H77S/GLuc2A-AAG RNA that expresses GLuc as part of the HCV polyprotein (31). The
| www.pnas.org/cgi/doi/10.1073/pnas.1112263109Shimakami et al.
decay between 3 and 9 h under different conditions (Fig. 1C).
This allowed for recovery of the cells after electroporation, and
when the data were fit to a one-phase decay model, indicated
that the half-life (t1/2) of HCV RNA in cells cotransfected with
miR-122 was 5.2 h vs. 3.60 h for cells cotransfected with miR-124
(P = 0.0035 by the extra sum-of-squares F test) and 3.3 vs. 2.0 h
for cells transfected with anti-random vs. the anti–miR-122
antagomir (P = 0.0016) (Fig. 1D). Similar differences in rates of
decay were observed when HCV RNA was assayed by quantita-
tive RT-PCR (qRT-PCR) (Fig. S1A). Differences in RNA decay
were matched by differences in viral protein expression (Fig.
S1B), monitored by measuring Gaussia luciferase (GLuc) enco-
ded by sequence inserted into the viral genome (Fig. 1A). Taken
together, these results indicate that miR-122 positively regulates
the stability of transfected HCV RNA. The corresponding in-
crease in protein expression provides a logical explanation for the
enhanced HCV translation reported previously (10, 11).
Because transfected RNA is likely to be subject to different
decay pathways than replicating viral genomes in infected cells,
we determined whether miR-122 also slows degradation of viral
RNA in infected cells treated with PSI-6130, a potent and specific
nucleoside inhibitor that arrests new viral RNA synthesis (18).
Under these conditions, as expected, viral RNA degraded more
slowly than after electroporation (compare Fig. 1 B and E).
However, its rate of decay was reduced when miR-122 was
transfected simultaneously with PSI-6130 treatment (Fig. 1E).
When fit to a one-phase decay model, PhosphorImager data from
replicate experiments (Fig. 1F) indicated a significant difference
in the rate constant for HCV RNA decay, k (k = ln(2)/t1/2), in
cells supplemented with miR-122 vs. miR-124 (P = 0.048 by the
extra sum-of-squares F test). The difference in the t1/2was highly
significant statistically (P = 0.007 by two-sided paired t test) (Fig.
1G). Likewise, increases in the decay rate in cells transfected with
anti-miR-122 vs. the control anti-random oligonucleotide were
also significant (P = 0.014 by F test), whereas decay rate con-
stants were similar in cells receiving anti-miR-124, anti-random,
or mock treatment (P > 0.05). Similar results were observed
when miR-122 was transfected into cells 8 h after the addition of
PSI-6130, which would allow for any potential delay in suppres-
sion of viral RNA synthesis due to the need for phosphorylation
of the inhibitor (Fig. S1 C and D).
In a completely independent set of experiments, we used qRT-
PCR to quantify HCV RNA in infected cells treated with PSI-
6310. The results suggested a longer t1/2for HCV RNA (≈19 h
vs. ≈10 h) in miR-124–treated cells than that determined by
Northern analysis. This is likely to reflect the small size of the
RNA segment detected in the RT-PCR assay (221 bases vs. the
9.7-kb RNA genome detected in Northern blots) and the in-
ability of the RT-PCR assay to discriminate between intact and
partially degraded RNAs. However, we again observed signifi-
cant differences in HCV RNA decay rates in cells supplemented
with miR-122 vs. miR-124, or anti-miR-122 vs. anti-random (Fig.
1H). Although the magnitude of this effect is relatively small (not
unlike the impact of miRNAs on cellular mRNA translation),
these data show collectively that miR-122 reproducibly stabilizes
the viral RNA genome in infected cells.
We next determined whether miR-122 could directly stabilize
RNA in a cell-free system. For this, we compared poliovirus
(PV) RNA and a related RNA (DNVR2) in which the PV 5′
UTR was replaced with the HCV 5′ UTR (Fig. 2A). The sta-
bilities of these RNAs have been compared previously in S10
translation mixtures prepared from HeLa cells (19, 20), pro-
viding a useful context for these experiments. PV RNA is sta-
bilized in these extracts by poly(rC) binding protein 1 (hnRNP1-
E1), which associates with a 5′-terminal cloverleaf RNA structure,
and decays more slowly than DNVR2 RNA (20). However,
DNVR2 RNA stability was increased and approximated that of
PV RNA when duplex miR-122, but not miR-124, was added to
S10 reactions before the viral RNA (Fig. 2 B and C). The sta-
bilization of DNVR2 RNA by miR-122 was reproducible and
statistically significant (Fig. 2 legend). In contrast, neither
miRNA enhanced stability of PV RNA lacking HCV sequence.
Thus, miR-122 directly regulates stability of RNA containing the
HCV 5′ UTR and does not accomplish this indirectly by mod-
ulating cellular gene expression.
Mutations in S1 and S2 that ablate miR-122 binding are lethal
to replication of HCV that has been adapted to growth in cell
culture (HJ3-5 virus) (11). Similarly, Northern blots revealed
that an HCV mutant defective in miR-122 binding at both sites
(S1-S2-p6m; Fig. 3A) (11), as well as polymerase function (GDD
to GND substitution in NS5B), was not stabilized by miR-122
when transfected into hepatoma cells (Fig. 3B, compare lanes 1–
3 vs. 4–6). In contrast, the complementary miR-122 mutant
(miR-122p6) (Fig. 3A) did stabilize S1-S2-p6m RNA (Fig. 3B,
lanes 1–3 vs. 7–9) but not viral RNA with WT S1 and S2
sequences. The differences in HCV RNA abundance apparent in
these blots were reproduced in multiple experiments, statistically
significant (Fig. S2A), and mirrored by differences in GLuc
expressed from these nonreplicating viral RNAs (Fig. S2B).
Collectively, these data indicate that HCV RNA is physically
stabilized as a result of miR-122 binding to its 5′ UTR, a unique
action for a miRNA.
Because mRNA translation and decay are closely coupled
processes (2), we considered the possibility that miR-122 could
stabilize the RNA by promoting its translation. We thus evalu-
ated its ability to slow decay of an RNA containing three con-
secutive base substitutions in an RNA loop within the HCV
IRES [mutant G(266-8)C; Fig. 3C]. These base changes elimi-
nate IRES affinity for the 40S ribosome particle and ablate
translation (11, 16), even in cells supplemented with miR-122
(Fig. S3A). Consistent with the notion that translation and decay
are intrinsically linked (2), Northern blots of cells transfected
with equivalent amounts of WT and G(266-8)C RNA (both
containing an NS5B mutation ablating RNA replication) con-
sistently showed a lower abundance of the G(266-8)C mutant,
suggesting that it was less stable than RNA with a WT 5′ UTR
(Fig. 3 D and E). Nonetheless, decay of the translationally
Structure of DNVR2 and PV RNAs (19), which differ only in 5′ UTR sequence.
(B) miR-122 slows decay of DNVR2 RNA in HeLa S10 lysate. Data shown
represent acid-precipitable α-[32P]-CTP-labeled RNA in HeLa S10 reaction
mixtures (19) containing 1 μM of duplex miRNA. The DNVR2 decay constant
in miR-122– vs. miR-124–supplemented mixtures, estimated by fitting the
data to a one-phase decay model (R2= 0.972–0.989), differed significantly
(P = 0.002). (C) RNA extracted from HeLa S10 reaction mixtures and frac-
tionated by electrophoresis in 0.8% agarose.
miR-122 stabilizes synthetic RNA containing the HCV 5′ UTR. (A)
Shimakami et al. PNAS
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inactive mutant was significantly slowed when the cells were
supplemented with miR-122 (Fig. 3 D and E). These experiments
were carried out in murine embryonic fibroblasts (MEFs) that do
not express detectable endogenous miR-122. Similar results were
obtained both in MEFs and hepatoma cells with another IRES
mutant, G(267)C, that retains affinity for the 40S subunit but is
also translationally dead (11, 16) (Fig. S3 B–E). Collectively these
results show that miR-122 does not stabilize HCV RNA by pro-
moting its translation or enhancing its association with ribosomes.
Active engagement in translation and miR-122 promote stability
of the RNA genome independently, and possibly additively.
Randall et al. (21) reported previously that RNAi-mediated
depletion of any of the Ago proteins (particularly Ago4) or Dicer
inhibited the ability of HCV to infect cells. In contrast, we found
that only Ago2 depletion (Fig. S4A) inhibited HCV RNA rep-
lication in hepatoma cells with previously established infection
(Fig. 4A). Ago2 depletion had no effect on cell growth (Fig. S4B)
but reduced both HCV RNA abundance and protein expression
(Fig. 4A). This was not observed with depletion of other Ago
proteins or Dicer. Although we are uncertain why our results
differ from those of Randall et al. (21), they suggest that Ago2
plays a special role in HCV replication. Two recent studies are
consistent with this: they show Ago2 to be required for miR-122
to promote HCV genome amplification (15, 22). However,
a third study found no impairment in miR-122 modulation of
HCV in Ago2-depleted cells (14).
Consistent with the stabilization of HCV RNA requiring in-
teraction with an miR-122–associated RISC complex, we ob-
served no stabilizing or translation-enhancing effect in hepatoma
cells transfected with single-stranded miR-122 (guide strand
only, rather than the duplex miRNA transfected in previous
experiments) (Fig. S4C). To determine whether miR-122 binds
as a complex with Ago2, we lysed MEFs shortly after electro-
poration with HCV RNA together with duplex miR-122 and
used RT-PCR to interrogate Ago2 immunoprecipitates for the
presence of viral RNA. HCV RNA with WT S1 and S2 sequence
coimmunoprecipitated with Ago2 (Fig. 4B). However, RNA with
point mutations in S1 and S2 that ablate miR-122 binding (Fig.
3A) coimmunoprecipitated with Ago2 only when cells were
supplemented with the complementary miR-122 mutant, miR-
122p6 (Fig. 4B). Transfected HCV RNA was also enriched in
anti-Flag immunoprecipitates from hepatoma cells ectopically
expressing Flag-Ago2 vs. Flag-Ago1 (Fig. S5). Thus, miR-122
binds the HCV 5′ UTR in association with Ago2. Lesser
amounts of Ago1 may also be present in the complex.
To determine whether Ago2 plays a functional role in stabi-
lizing HCV RNA, we compared the ability of miR-122 to slow
the decay of the RNA when electroporated with it into WT or
Ago2-deficient (Ago2−/−) MEFs (23). This revealed a striking
dependence on Ago2, because miR-122 had no effect in Ago2−/−
MEFs, whereas it significantly stabilized the viral RNA and en-
hanced viral translation in matched WT cells (Fig. 4 C and D).
Ectopic expression of human Flag-Ago2 in the Ago2−/−MEFs
restored the ability of miR-122 to positively regulate protein
expression from HCV RNA, further confirming the requirement
for Ago2 (Fig. S6). To ascertain whether it is possible for other
Ago proteins to functionally substitute for Ago2 in stabilizing
HCV RNA, we also overexpressed human Flag-Ago1 in the
Ago2−/−MEFs. This only partially rescued the ability of miR-122
to promote HCV protein expression (Fig. S6B), although
immunoblots with anti-Flag antibody indicated that Flag-Ago1
was overexpressed at almost threefold the abundance of Flag-
Ago2 and well above physiologically relevant levels (Fig. S6A).
Thus, Ago2 is not unique in its ability to support miR-122 en-
hancement of HCV protein expression, but it seems to do this
more efficiently than Ago1. This is consistent with the greater
enrichment of HCV RNA we observed in Flag-Ago2 compared
with Flag-Ago1 immunoprecipitates (Fig. S5). Additional experi-
ments confirmed that Ago2 is the dominant Ago protein in-
volved in miR-122 stabilization of HCV RNA. RNAi-mediated
depletion of Ago2 reduced the ability of miR-122 to stabilize
HCV RNA and promote its translation in HeLa cells (Fig. S7 A–
D). Importantly, the magnitude of this effect mirrored the re-
duction in miR-122–mediated suppression of a reporter mRNA
containing the HCV miR-122 binding sites within its 3′ UTR (9)
These results suggest a functional interaction of Ago2 with
RNA decay machinery. Because HCV RNA lacks a 5′ m7G cap,
one possibility is that miR-122 recruits an Ago2 RISC complex to
the 5′ end of viral RNA that protects it from 5′ exonuclease
activity. If so, this protective action should be rendered re-
dundant by providing the RNA with a 5′ cap. To test this, we
synthesized HCV RNAs with or without a 5′ nonmethylated
guanosine cap analog and compared their stabilities after trans-
fection into MEFs with or without miR-122. As anticipated, miR-
122 had no effect on the rate of decay of the 5′ capped RNA,
whereas it substantially stabilized the uncapped HCV RNA (Fig.
5A). The magnitude of protein expression (GLuc) from capped
RNA was comparable to that from the uncapped RNA in cells
supplemented with miR-122 and was not further increased by
miR-122 (Fig. 5B). Thus, the 5′ cap enhanced stability and pro-
tein expression from HCV RNA, functionally substituting for
miR-122 and providing strong evidence that the miR-122–Ago2
complex protects HCV RNA from 5′ exonuclease. Importantly,
dependent of translation. (A) Upper: miR-122 and mutant miR-122p6 guide-
strand sequences. Lower: 5′ terminal sequence of HCV (HJ3-5/GLuc2A virus),
with S1 and S2 binding sites shown in red. Point mutations (underlined) in
the related S1-S2-p6m-GND mutant (11) are shown above. SL-1 and SL-2 are
putative stem-loop structures in the 5′ UTR. (B) Northern blots of HJ3-5/
GLuc2A-GND and the related S1-S2-p6m-GND mutant RNA after transfection
into Huh-7.5 cells with RNA oligoribonucleotides as in Fig. 1B. (C) Putative
secondary structure of the HCV 5′ UTR, showing the location of stem-loop
IIId and the G(266-8)C IRES mutation that ablates translation. (D) Northern
blot showing HCV RNA abundance in MEFs transfected with HCV RNA (H77S/
GLuc2A-AAG) containing (Upper) the WT 5′ UTR vs. (Lower) the transla-
tionally inactive G(266-8)C mutant, and supplemented with the indicated
miRNAs. (E) PhosphorImager quantitation of HCV RNA in Northern blots of
6-h cell lysates from two independent experiments carried out as shown in D.
Stabilization of HCV RNA requires miR-122 binding and is in-
| www.pnas.org/cgi/doi/10.1073/pnas.1112263109Shimakami et al.
translation of the capped HCV RNA was IRES-dependent, be-
cause the nonmethylated 5′ cap lacked the ability to recruit
eukaryotic initiation factors.
Our results reveal a unique mechanism by which a miRNA in
association with Ago2 regulates the expression of its target RNA,
in this case the HCV genome. Although miR-10a enhances
translation by binding the 5′ UTR of ribosomal protein mRNAs
containing 5′ terminal oligopyrimidine (TOP) motifs (24), miR-
NAs have not been recognized to slow decay or up-regulate
abundance of their RNA targets. miR-122 does this by recruiting
an Ago2 RISC-like complex to the 5′ end of the HCV RNA ge-
nome. As evidenced by the IRES mutants (Fig. 3D and Fig. S3D),
the stabilizing action of miR-122 does not require the target HCV
RNA to be capable of translation and thus does not result from
increased ribosomal loading. Stabilization also does not require
the RNA to be replication competent (Fig. 1 B and C).
Because a nonmethylated 5′ cap analog functionally sub-
stitutes for miR-122 (Fig. 5), the RISC-like complex recruited
by miR-122 is likely to act by protecting the RNA from 5′
exonuclease. Whether this occurs simply as a result of physically
masking the 5′ end of the viral RNA from 5′ exonuclease at-
tack, or whether Ago2 plays a more complex role by influenc-
ing the association of HCV RNA with P bodies (25), sites of
mRNA degradation and storage, remains to be determined.
Binding of a RISC-like complex could also limit recognition of
the 5′ triphosphate of HCV RNA by retinoic acid-inducible
gene I (RIG-I), a ubiquitous innate immune pathogen recog-
nition receptor (26) that is capable of inducing interferons and
interferon-stimulated genes, including RNase L, an endonucle-
ase, and ISG20, a 3′-5′ exonuclease (27). However, subversion of
RIG-I–dependent viral RNA degradation played no role in the
stabilization of HCV RNA in our experiments because the Huh-7.5
cells used are deficient in RIG-I signaling (28).
stabilization. (A) RNAi depletion of Ago2 impairs HCV genome amplification
in persistently infected (HJ3-5/GLuc2A virus) cells. Left: HCV RNA abundance
relative to that in cells transfected with nontargeting siRNA (Ctrl) 72 h after
siRNA transfection. RNA was assayed by qRT-PCR; data are mean ± range of
paired cultures. Right: GLuc activity secreted into media between 48 and
72 h, relative to si-Ctrl-transfected cultures (mean ± range). (B) miR-122 binds
HCV RNA as a complex with Ago2. Lysates were prepared from WT MEFs 6 h
after electroporation with HJ3-5/GND or S1-S2-p6m-GND RNA (Fig. 3A),
mixed with miR-122 or miR-122p6, and immunoprecipitated with anti-Ago2
antibody. After extensive washing, RNA was extracted from the precipitates
and subjected to a one-step HCV-specific RT-PCR (30 cycles). HCV RNA was
enriched in precipitates from HJ3-5/GND–transfected cells supplemented
with miR-122, or S1-S2-p6m-GND cells supplemented with miR-122p6. (C)
Northern blots showing miR-122 does not stabilize HCV RNA in Ago2−/−
MEFs. Cells were electroporated with HCV RNA (H77S/GLuc2A-AAG) to-
gether with miR-122, miR-124, or no miRNA (Mock), then lysed at 3-h
intervals and assayed for HCV RNA abundance. miR-122 stabilized HCV RNA
only in WT MEFs and was without effect in Ago2−/−cells. (D) GLuc activity in
supernatant fluids from MEFs cotransfected with HCV RNA and the indicated
miRNA (mean ± range). Data shown are representative of two or more in-
Ago2 binds HCV RNA in association with miR-122 and is required for
122 in stabilizing HCV RNA. (A) Northern blots of HCV RNA in lysates of MEFs
after electroporation with HCV RNA (H77S/GLuc2A-AAG) (Fig. 1A) synthe-
sized with or without a nonmethlyated G[5′]ppp[5′]G-RNA cap. HCV RNAs
were transfected together with miR-122, miR-124, or no miRNA (Mock). 28S
rRNA is shown as a loading control. (B) GLuc activity in supernatant fluids of
MEFs transfected with capped or uncapped HCV RNAs (mean ± range of two
replicate cultures). Data shown are representative of two or more in-
A nonmethylated 5′ guanosine cap functionally substitutes for miR-
Shimakami et al.PNAS
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The mechanism by which miR-122 stabilizes HCV RNA is Download full-text
distinct from the up-regulation of translation by miRNAs that
target AU-rich elements within the 3′ UTRs of some mRNAs,
because the latter occurs only in quiescent cells arrested at the
G0phase of the cell cycle (29, 30). Moreover, miRNA-mediated
stimulation of translation in quiescent cells has not been linked
to stabilization of the target mRNA, as we show here for miR-
122. Still to be determined is whether the protein composition of
the RISC-like complex that is recruited to the 5′ UTR by miR-
122 differs significantly from miRNA-induced RISC complexes
involved in translational repression.
The stabilization of HCV RNA by miR-122 is likely to be
responsible for the miR-122–induced enhancement of HCV
translation reported previously (10, 11). Mutations in the viral
RNA that prevent binding of miR-122 also ablate replication of
infectious virus (11) and prevent stabilization of the genome by
miR-122 (Fig. 3). Depletion of Ago2 has similar effects on
replication, translation, and RNA stability (15, 22) (Fig. 4). This
makes it difficult to distinguish between these consequences of
miR-122 binding to the 5′ UTR or to determine the primary role
played by miR-122 as a host factor for viral replication. However,
stabilization of the genome is likely to be a key factor in the
promotion of viral replication by miR-122. A recombinant HCV
in which the U3 RNA sequence was inserted in lieu of the S1
binding site in the 5′ UTR was found recently to be less de-
pendent upon miR-122 for replication (17), possibly because the
U3 sequence stabilized the RNA, much as a 5′ cap did (Fig. 5).
Nonetheless, it would not be surprising to find that miR-122 has
other functions in the viral life cycle in addition to its role in
stabilizing the viral RNA genome, perhaps in the initiation of
viral RNA synthesis.
Viral RNA Stability in Transfected Cells. RNA was transcribed in vitro (11) from
pH77S/GLuc2A-AAG, which contains the complete genotype 1a HCV se-
quence with GLuc2A placed in-frame within the polyprotein-coding region
(31) and a lethal GDD to AAG mutation in NS5B. Where indicated, a non-
methylated 5′ guanosine cap was added using the ScriptCap m7G Capping
System (Epicentre Biotechnologies). Viral RNA (20 μg) and miRNA duplexes
(11) or antisense oligoribonucleotides (1 μM) were mixed with 1 × 107Huh-
7.5 cells in a 4-mm cuvettete and pulsed once at 250 V, 950 μF, and 100 Ω in
a Gene Pulser Xcell Total System (Bio-Rad). HeLa cells were electroporated at
300 V, 500 μF, and ∞ Ω, and MEFs at 400 V, 250 μF, and ∞ Ω. Cells were
harvested at intervals and supernatant fluids assayed for GLuc activity (31)
and HCV RNA abundance in cell lysates assessed by Northern blotting (11).
Polyadenylated reporter RNAs encoding firefly or Cypridina luciferase were
cotransfected to monitor transfection efficiency (11).
HCV-Infected Cells. Synthetic HJ3-5 or HJ3-5/GLuc2A RNA (31) was transfected
into 1 × 107FT3-7 cells, which were passaged until >90% positive for core
antigen in an immunofluorescence assay (11). siRNA pools targeting Ago1-4
or Dicer and control siRNA pools (Dharmacon) were transfected using
siLentfect Lipid Reagent (Bio-Rad). miRNA duplexes or single-stranded oli-
goribonucleotides (50 nM) were transfected using Lipofectamine 2000
Additional methods and associated references can be found in SI Methods.
ACKNOWLEDGMENTS. We thank Lucinda Hensley for expert technical
assistance; William F. Marzluff and Scott M. Hammond for helpful dis-
cussions; Alexander Tarakhovsky and Angela Santana, Rockefeller Univer-
sity, for mouse embryonic fibroblasts; Charles Rice, Rockefeller University,
for Huh-7.5 cells; Thorleif Møller, Mirrx Therapeutics, for psi-Check2/Luc-3′
HCV DNA; and Angela Lam and Phil Furman, Pharmasset, Inc., for PSI-6130.
This study was supported in part by the University Cancer Research Fund and
National Institutes of Health Grants RO1-AI095690, U19-AI040035, P20-
CA150343 (to S.M.L.), and AI042189 (to D.J.B.). R.K.J. was supported by a
James W. McLaughlin Fellowship.
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