JOURNAL OF VIROLOGY, Feb. 2005, p. 2151–2159
Copyright © 2005, American Society for Microbiology. All Rights Reserved.
Vol. 79, No. 4
Inhibition of Coxsackievirus B3 Replication by Small Interfering RNAs
Requires Perfect Sequence Match in the Central Region of the Viral
Ji Yuan, Paul K. M. Cheung, Huifang M. Zhang, David Chau, and Decheng Yang*
Department of Pathology and Laboratory Medicine, The James Hogg iCAPTURE Centre for Cardiovascular
and Pulmonary Research, University of British Columbia-St. Paul’s Hospital, Vancouver,
British Columbia, Canada
Received 3 May 2004/Accepted 22 September 2004
Coxsackievirus B3 (CVB3) is the most common causal agent of viral myocarditis, but existing drug therapies
are of limited value. Application of small interfering RNA (siRNA) in knockdown of gene expression is an
emerging technology in antiviral gene therapy. To investigate whether RNA interference (RNAi) can protect
against CVB3 infection, we evaluated the effects of RNAi on viral replication in HeLa cells and murine
cardiomyocytes by using five CVB3-specific siRNAs targeting distinct regions of the viral genome. The most
effective one is siRNA-4, targeting the viral protease 2A, achieving a 92% inhibition of CVB3 replication. The
specific RNAi effects could last at least 48 h, and cell viability assay revealed that 90% of siRNA-4-pretreated
cells were still alive and lacked detectable viral protein expression 48 h postinfection. Moreover, administration
of siRNAs after viral infection could also effectively inhibit viral replication, indicating its therapeutic poten-
tial. Further evaluation by combination found that no enhanced inhibitory effects were observed when siRNA-4
was cotransfected with each of the other four candidates. In mutational analysis of the mechanisms of siRNA
action, we found that siRNA functions by targeting the positive strand of virus and requires a perfect sequence
match in the central region of the target, but mismatches were more tolerated near the 3? end than the 5? end
of the antisense strand. These findings reveal an effective target for CVB3 silencing and provide a new
possibility for antiviral intervention.
Coxsackievirus B3 (CVB3) is a member of the genus En-
terovirus, which is within the family Picornaviridae. Epidemio-
logical studies indicated that nearly 50% of North American
clinical myocarditis cases are attributable to picornaviral infec-
tion, with the CVB3 serogroup making up the most significant
portion of such infections (23). The peak age group in which
CVB-induced myocarditis occurs is young adults, primarily
between 20 and 39 years of age (47). Although most CVB3-
caused diseases are mild, some acute infections are severe and
lethal. Clinically, CVB3 infections are associated with different
forms of subacute, acute, and chronic myocarditis (38, 47). The
infections may cause cardiac arrhythmias and acute heart fail-
ure, while in some cases the myocardial inflammation may
persist chronically and progress to dilated cardiomyopathy (5,
26, 32, 39), requiring heart transplantation, or to death.
The CVB3 genome is a positive single-stranded RNA mol-
ecule. It is ?7.4 kb in length and has a single open reading
frame, which is flanked by 5?- and 3?-untranslated regions
(UTR). The 5?UTR contains a highly structured internal ribo-
some entry site that directs viral translation initiation (30, 49).
The 3?UTR contains three stem-loops followed by a poly(A)
tail. The genomic RNA can serve as a template for viral RNA
transcription to synthesize more copies of positive genomic
RNA through a negative-strand intermediate. It can also be
employed directly as an mRNA template for translation of a
single polyprotein that is posttranslationally processed primar-
ily by CVB3-endoded proteases 2A and 3C to produce indi-
vidual structural and nonstructural proteins. The nonstructural
proteins, particularly the RNA-dependent RNA polymerase
3D, are responsible for viral RNA replication, which takes
place with rapid kinetics in the small membranous vesicles of
cytoplasm. The entire replication cycle of CVB3 from entry of
the host cell to release of progeny virus takes approximately 6
to 8 h. Although the life cycle of CVB3 appears clear, there is
no specific drug available to inhibit this viral replication.
Small interfering RNAs (siRNAs) are short, double-
stranded RNA (dsRNA) molecules that can target mRNA of a
specific sequence for degradation via a cellular process known
as RNA interference (RNAi) (1, 12, 16, 43). RNAi is an evo-
lutionarily conserved phenomenon of posttranscriptional gene
silencing that has been described for plants, invertebrates, and
vertebrates (10, 42). In this process, dsRNA is cleaved into
siRNA of 21 to 28 nucleotides (nt) by an RNAseIII-like en-
zyme known as Dicer, followed by incorporation of siRNA into
an RNA-induced silencing complex (RISC) that recognizes
and cleaves the target sequence (11). In mammals, however,
dsRNAs longer than 30 nt can induce a nonspecific interferon
response and, in turn, lead to the shutdown of a number of
gene expressions. This limitation of application in mammals
has been overcome by introduction of synthetic siRNA. They
are short enough to bypass general dsRNA-induced nonspe-
cific interferon response (28, 34) and thus provided a powerful
reverse genetic approach to develop siRNA in gene functional
study and antiviral drug development. To date, several labora-
tories have demonstrated that siRNA can be used as powerful
* Corresponding author. Mailing address: Cardiovascular Research
Laboratory, University of British Columbia, St. Paul’s Hospital, 1081
Burrard St., Vancouver, B.C., Canada V6Z 1Y6. Phone: (604) 682-
2344 ext. 62872. Fax: (604) 806-9274. E-mail: firstname.lastname@example.org.
antiviral agents for different viral infection, such as poliovirus
(18), influenza A virus (17, 33), respiratory syncytial virus (3),
hepatitis B (21, 50), C (27, 37, 41, 46, 51), and D virus (6),
human immunodeficiency virus type 1 (HIV-1) (4, 7, 9, 24, 25,
29, 35, 36), and West Nile virus (33). Therefore, there has been
considerable interest in the development of siRNA as a pos-
sible treatment for CVB3-induced heart diseases.
In this study, we examined the effects of RNAi on CVB3
replication using five siRNAs targeting different regions of
CVB3 genomic RNA. We demonstrated that three of the five
candidates exerted potent antiviral abilities in HeLa cells and
cardiomyocytes. Among them, the most effective siRNA is the
one targeting the viral protease 2A coding region. Further-
more, mutational analysis of the specific interactions between
the siRNA and its target sequences revealed that the antisense
strand of the siRNA plays a critical role in the specific targeting
of siRNA on viral mRNA, and a single nucleotide mutation at
the center or near the 5? end of the antisense strand of siRNA
can eliminate its antiviral activity.
MATERIALS AND METHODS
siRNAs. All siRNA sequences were designed according to the manufacturer’s
recommendations and were subjected to a BLAST search of the National Center
for Biotechnology Information’s expressed sequence tag library to ensure that
they targeted only the desired genes. Five CVB3-specific double-stranded
siRNAs and two controls (21-mer) were synthesized by QIAGEN-Xeragon, and
the siRNAs used for mutational analysis of antiviral activity were synthesized by
Dharmacon. The final concentration of all siRNA was 40 ?M in provided buffer.
Sequences of all siRNAs are shown in Table 1.
Virus, cell culture, infection, and transfection. CVB3 was produced from a
full-length cDNA clone (provided by Reinhard Kandolf, University of Tubingen,
Germany) and amplified in HeLa cells (American Type Culture Collection) by
transfection. Virus titer was routinely determined at the beginning of the exper-
iment by plaque assay. HeLa cells were cultured in Dulbecco’s modified Eagle’s
medium (DMEM) supplemented with 10% fetal bovine serum and 100 ?g of
penicillin-streptomycin/ml (Invitrogen). The HL-1 cell line, a cardiac muscle cell
line established from an AT-1 mouse atrial cardiomyocyte tumor lineage, was a
gift from William C. Claycomb (8). The cells were maintained in Claycomb
medium (JRH Biosciences) supplemented with 10% fetal bovine serum (JRH
Bioscience), 100 ?g of penicillin-streptomycin/ml, 0.1 mM norepinephrine
(Sigma), and 2 mM L-glutamine (Invitrogen).
The transfection of siRNAs was performed under optimal conditions. Briefly,
2 ? 105cells were grown at 37°C overnight. When cells reached 50 to 60%
confluency, they were washed and overlaid with transfection complexes contain-
ing siRNAs and Oligofectamine (Invitrogen) overnight. Following transfection,
cells were washed and infected with CVB3 at the indicated multiplicity of infec-
tion (MOI) for 1 h. The cells were then overlaid with complete medium and were
incubated at 37°C in 5% CO2. At different time points postinfection, superna-
tants and cell lysates were collected and stored in a ?80°C freezer. For thera-
peutic experiments, cells were infected with virus at the indicated MOI for 1 h
and then were transfected with siRNAs.
Western blot. Western blotting was performed by standard protocols as pre-
viously described (45). Equal amounts of protein were subjected to sodium
dodecyl sulfate-polyacrylamide gel electrophoresis and then were transferred to
nitrocellulose membranes. The membranes were blocked with 5% skim milk
containing 0.1% Tween 20 for 1 h. The blots were probed with primary mouse
antibody against CVB3 capsid protein VP1 (DAKO) or ?-actin (Sigma) for 1 h,
followed by incubation with horseradish peroxidase-conjugated secondary anti-
body. Finally, VP1 and ?-actin expression were detected by ECL reagents (Am-
Viral plaque assay. The virus titer was determined by plaque assay as de-
scribed previously (52). Briefly, HeLa cells were seeded into 6-well plates (8 ?
105cells/well) and incubated at 37°C for 20 h. When cell confluency reached
approximately 90%, cells were washed with phosphate-buffered saline and then
overlaid with 500 ?l of diluted supernatants. The cells were incubated at 37°C for
60 min, and the supernatants were removed. Finally, cells were overlaid with 2 ml
of sterilized soft Bacto-agar—minimal essential medium. The cells were incu-
bated at 37°C for 72 h, fixed with Carnoy’s fixative for 30 min, and then stained
with 1% crystal violet. The plaques were counted, and the amount of virus
(PFU/milliliter) was calculated.
In situ hybridization. Intracellular viral RNA was detected by in situ hybrid-
ization as previously described (31). Fixed cells were hybridized with digoxigenin-
labeled CVB3 antisense riboprobes prepared by in vitro transcription. Hybrid-
ized positive signals were visualized using an alkaline phosphatase-conjugated
anti-digoxigenin antibody (Roche) and the color substrate Vector Red (Vector
Cell viability assay. Cell viability was measured by using a 3-(4,5-dimethyl-
(MTS) assay kit (Promega) according to the manufacturer’s instructions. Cells
were incubated with MTS solution for 2 h, and the absorbance was measured at
492 nm using an enzyme-linked immunosorbent assay (ELISA) reader. The
absorbances of sham-infected cells were defined as the values of 100% survival,
and the remaining data, including that for siRNA-treated, nontreated, and con-
trol cells, were converted to the ratio of the sham-infected sample. Morpholog-
ical changes of cells following CVB3 infection were evaluated by phase-contrast
Genotypic analysis of the siRNA-4 target region of CVB3. Viral RNA was
isolated from culture supernatant as described previously (52). Reverse tran-
scription (RT) was conducted according to the manufacturer’s instructions (In-
vitrogen) using 30 ?l of RNA and 1 ?l of 3 ?M hexamer primer, followed by
PCR to amplify CVB3 cDNA (the 2A region; nt 3423 to 3864). The PCR mixture
contained 10 ?l of RT products and 1 ?l of 15 ?M sense and antisense primers,
and the reaction was run for 35 cycles with standard parameters. The PCR
products representing the 2A region were analyzed with a 0.8% agarose gel and
were purified with a QIAquick gel extraction kit (QIAGEN). DNA sequencing
was preformed by the Biotechnology Laboratory, University of British Columbia.
Design of siRNA sequences specific for CVB3. To test which
region of the CVB3 genome is the most effective site for
siRNA targeting, we selected sequences at different locations
that may have important functions in viral replication. These
regions include the 5?UTR, start codon, viral capsid protein
VP1, viral protease 2A, and RNA-dependent RNA polymerase
TABLE 1. Sequences and locations of siRNAs used to target the CVB3 RNA
siRNA Target sequence (5?-3?) Location in CVB3 RNAa
nt 115–133, 5?UTR
nt 733–751, AUG start codon region
nt 3171–3189, VP1
nt 3543–3561, 2A
nt 6312–6330, 3D
nt 608–630, coding regionb
aAccording to the CVB3 genome sequence in the GenBank database, accession no. M33854. Viral genes are indicated.
bAccording to the Homo sapiens nuclear envelope protein lamin A precursor, accession no. X03444 in the GenBank database.
2152YUAN ET AL.J. VIROL.
3D. A total of five siRNAs targeting these regions were de-
signed (Table 1) following published selection criteria (13, 14),
except for siRNA-2, which targets the start codon region of
CVB3. A BLAST search did not find complementation of
these siRNAs with human genes. In addition, the sequences of
lamin A/C siRNA (siRNA-L) (12) and irrelevant siRNA
(siRNA-C) were synthesized by QIAGEN and used as con-
Inhibition of CVB3 replication in HeLa cells. The HeLa cell
line is a widely used in vitro system for studying CVB3 repli-
cation. To verify that chemically synthetic siRNAs could effi-
ciently silence gene expression in this cellular environment,
siRNA-L was chosen as a validated model, as silencing of
lamin A/C by siRNA-L has been reported previously (12).
siRNA-L or siRNA-C was transfected into HeLa cells under
optimal conditions. Two days posttransfection, cell lysates were
collected and examined for lamin A/C protein levels by Western
blotting. The lamin A/C level was barely detectable in siRNA-L-
transfected samples, whereas the irrelevant siRNA-C had no ef-
fect on lamin A/C expression (data not shown). This result con-
firmed that transfection of synthetic siRNAs into HeLa cells is an
effective approach for evaluation of the activity of siRNA in
silencing gene expression in this cellular environment.
We next tested whether CVB3 replication could be inhibited
by introducing various CVB3-specific siRNAs. HeLa cells were
transfected with siRNAs at a final concentration of 300 nM
overnight and were subsequently infected with CVB3 at an
MOI of 10 for 8 h. CVB3-specific viral protein VP1 expression
in cell lysates and infectious viral particles in the supernatants
were analyzed by Western blotting and viral plaque assay,
respectively. As shown in Fig. 1a, viral protein VP1 expression
dramatically decreased in cells treated with siRNA-4, ?3, or
?5, but it did not decrease in cells treated with siRNA-1,
siRNA-2, siRNA-C, or siRNA-L or in mock-transfected cells.
Compared to the mock-transfected control, VP1 expression in
siRNA-1- to -5-treated samples decreased 31, 18, 85, 92, and
FIG. 1. CVB3-specific siRNAs inhibit CVB3 replication in HeLa cells. (a) Western blot analysis of CVB3 capsid protein VP1. HeLa cells were
transfected with siRNAs at a final concentration of 300 nM using Oligofectamine and then were infected with CVB3 at an MOI of 10. At 8 hpi,
cell lysates were collected for VP1 detection by Western blot and supernatants were used for detecting infectious viral particles by plaque assay.
The VP1 expression levels were quantified by densitometry and were normalized to the level of ?-actin, which served as a loading control. The
ratios of VP1 to ?-actin were calculated and are expressed in the graph. (?), mock transfection. (b) Plaque assays of infectious viral particles. The
assay was conducted on HeLa cell monolayers as described in Materials and Methods. Data are presented as log10values of virus titer. (c) In situ
hybridization of CVB3 RNA. After treatments and infections in chamber slides, CVB3 positive-strand RNAs were detected by in situ hybridization
using antisense riboprobes (red). Cell nuclei were counterstained with hematoxylin (blue). Images 1 to 5 and C (control) represent different
siRNAs treatments. Two negative controls were used: cells transfected with siRNA-C and infected with CVB3 were detected by sense probes (C-S),
and cells transfected with siRNA-4 but sham infected with DMEM were detected with antisense probes (4-sham). Magnification, ?200. Data
shown are from one of two independent experiments.
VOL. 79, 2005 INHIBITION OF CVB3 REPLICATION BY siRNAs 2153
65%, respectively, while siRNA-C- and -L-treated samples had
no change. To evaluate the inhibition of infectious viral parti-
cle production, we performed plaque assays to detect the virus
titer in the supernatants. Data demonstrated that virus titer of
all CVB3-specific siRNAs except siRNA-2 decreased around 2
log10compared to that of supernatant treated with control
siRNAs or left untreated (Fig. 1b). The effects of RNAi on
viral replication were further assessed by in situ hybridization.
Consistent with the above results, the cells treated with CVB3-
specific siRNA showed smaller amounts of positive cells and
weaker signal intensity than the controls (Fig. 1c), indicating
that siRNAs specifically inhibited viral replication in HeLa
cells and that their antiviral activities can be ranked, in order,
as siRNA-4 ? siRNA-3 ? siRNA-5 ? siRNA-1 ? siRNA-2.
Considering the clinical setting, siRNAs were further used to
treat wild-type CVB3. As expected, the result obtained was the
same as that obtained from cDNA-derived virus (data not
To further determine the potency of siRNAs, we performed
experiments to study the effect of concentration of siRNA on
its antiviral activity. We selected siRNA-4 as a candidate for
this purpose. HeLa cells were transfected with a graded
amount of siRNA-4 and were followed by CVB3 infection.
Data presented in Fig. 2a and b demonstrated that as the
amount of siRNA-4 increased, virus titer in the supernatants
and VP1 protein in the cells correspondently decreased, even
at concentrations as low as 10 nM. Compared to the levels of
three controls, an approximate 2 log10decrease in virus parti-
cle production was detected in these cultures treated with
different concentrations of siRNA-4. These results indicate
that siRNA-4 is a highly efficient antiviral agent and that it
exerts a potent anti-CVB3 ability in a dose-dependent manner.
To clarify the time course of RNAi, cells were pretreated with
siRNA-4 or siRNA-C, followed by virus infection. At different
times points postinfection, cell lysates and the supernatants
were collected for detecting VP1 expression levels and virus
titers. We hardly detected the difference between siRNA-4 and
-C in the supernatants at 2 and 4 h postinfection (hpi). This
may be because the first virus replication cycle had not finished
yet, as a single replication cycle of CVB3 requires approxi-
mately 6 h (Fig. 2c). However, at 6 and 8 hpi, fewer virus
particles and lower VP1 levels were observed in siRNA-4-
treated samples than in the control-treated cultures (Fig. 2c
Considering clinical application of siRNAs in viral myocar-
ditis, siRNAs must be able to effectively inhibit an ongoing
virus infection. To further evaluate its therapeutic potential,
HeLa cells were infected with CVB3 at an MOI of 0.01 for 1 h
and then were transfected with siRNA-4 or siRNA-C. Viral
protein VP1 expression and virus titer were analyzed at 40 hpi
As shown in Fig. 3a and b, virus titer as well as viral protein
VP1 expression was significantly reduced by siRNA-4 treat-
ment. Thus, administration of siRNAs after viral infection can
also inhibit viral replication effectively.
We next evaluated the long-term effects of various CVB3-
specific siRNAs on CVB3 replication. After transfection of the
FIG. 2. siRNA-4 interferes with CVB3 replication in HeLa cells. (a and b) Dose-dependent inhibition of CVB3 production by siRNA-4. HeLa
cells were transfected with siRNA-4 at a final concentration as indicated and followed by infection with a CVB3 MOI of 10 for 8 h. Supernatants
were used for viral plaque assay (a), and cell lysates were collected for viral VP1 detection by Western blotting (b). ?-Actin served as the loading
control. (c and d) Time course of inhibition of CVB3 replication by siRNA-4. Cells were transfected with siRNA-4 at a final concentration of 300
nM and followed by CVB3 infection at an MOI of 10. Supernatants and cell lysates were collected at the indicated time points for plaque assay
(c) and Western blotting (d), respectively. Data shown are representatives of two independent experiments.
2154 YUAN ET AL.J. VIROL.
cells with each of the five siRNAs or controls, cytopathic effects
were evaluated by phase-contrast microscopy. Figure 4a shows
that mock-transfected cells and control cells were more sus-
ceptible to CVB3 infection. Approximately 80% of cells were
killed by virus, whereas cells treated with CVB3-specific
siRNAs except siRNA-2 were more resistant to infection and
the majority of the cells were still alive. These observations
were further validated and quantified by MTS assay, as shown
in Fig. 4b. Notably, in the siRNA-4-treated cells, 90% of cells
were alive compared to levels of sham-infected controls. The
viral capsid protein VP1 was also detected by using cell lysates
and supernatants. Figure 4c and d demonstrate that VP1 ex-
pression was undetectable in siRNA-3- or siRNA-4-treated
samples. The anti-CVB3 abilities of different siRNAs in long-
term (48 h) experiments were similar to those in the short-term
(8 h) treatments (Fig. 2), which are ranked according to their
potentials, i.e., siRNA-4 is the strongest one, followed by
siRNA-3, -5, -1, and -2.
Inhibition of CVB3 replication in cardiomyocytes. Because
CVB3 is a cardiotropic virus and commonly induces viral myo-
carditis, we further evaluated these siRNAs in the HL-1 mouse
cardiomyocyte cell line. Using optimal transfection conditions,
HL-1 cells were first transfected with siRNAs and then in-
fected with CVB3 at an MOI of 10. Forty-eight hours postin-
fection, the antiviral activities of siRNAs were evaluated by
Western blotting and plaque assay. The inhibition pattern of
the siRNAs in HL-1 cells (Fig. 5a and b) was similar to that
obtained with HeLa cells, demonstrating that siRNA-4 exhib-
ited the greatest potency in inhibiting CVB3 replication, fol-
lowed by the other three siRNAs. However, no significant
reduction of virus titer was observed in siRNA-2-treated car-
diomyocytes compared to that of the controls.
Effects of mismatches on antiviral activity of siRNA. Be-
cause CVB3 RNA replication occurs through a dsRNA inter-
mediate, the issue arises of which strand of the viral RNA is the
site of sequence complementation with siRNA and which
strand of the siRNA guides its targeting to the viral genome.
To address this issue, we performed site-directed mutagenesis
at the center of the sense and/or antisense strands of siRNA-4
(Fig. 6a) and then evaluated its antiviral activity in HeLa cells.
Figure 6b and c demonstrated that one nucleotide mutation in
the middle of the antisense strand (siRNA-4mAS) or of both
strands of siRNA (siRNA-4mSAS) could eliminate RNAi
completely as they lost their specific antiviral activity, whereas
the corresponding mutation on the sense strand (siRNA-4mS)
did not interfere with the RNAi. This suggests that the positive
strand of viral RNA is the target of siRNA, and the central
sequence match between the antisense strand of siRNA and
the viral positive strand is critical for RNAi. To further test
whether mismatches at the ends are better tolerated than mis-
matches at the center, siRNAs with a 1-nt mutation near either
the 5? end (siRNA-4-5?mAS) or the 3? end (siRNA-4-3?mAS)
of the antisense strand of siRNA-4 was applied to inhibit
CVB3 replication (Fig. 6a). Compared to siRNA-4, siRNA-4-
5?mAS exhibited a small degree of silencing, while siRNA-4-
3?mAS still maintained similar inhibitory ability, implying that
the 5? sequence of antisense siRNA is more critical for target
RNA recognition than the 3?-end sequence (Fig. 6d).
No enhanced inhibitory effects on CVB3 replication by co-
transfection of two siRNAs. The enhanced antiviral effect of
multiple siRNAs has been reported in HIV-1 infection (25).
To test whether cotransfection of cells with a combination of
two specific siRNAs targeting different regions of CVB3 RNA
could increase the antiviral effect in our system, two siRNAs
were cotransfected into the HeLa cells and followed by eval-
uation of the antiviral effects. Because we found that 10 and 20
nM concentrations of siRNA-4 were within the linear correla-
tion between the dose and inhibitory effect, we selected these
two concentrations for evaluation by using combinations con-
taining siRNA-4 and each of the four remaining CVB3-specific
siRNAs. The viral replication was measured by detection of
VP1 protein production. As shown in Fig. 7, siRNA-4 at a final
concentration of 20 nM exerted a higher inhibitory capacity
than any other combination of 10 nM siRNA-4 with 10 nM
siRNA-1, -2, -3, -5, or -C, suggesting that there is no additive or
synergistic effect on anti-CVB3 activity between siRNA-4 and
any other individual siRNA tested.
No escape mutants generated upon siRNA treatment. Emer-
gence of escape viral mutants has been reported in previous
studies following siRNA or short hairpin RNA treatment (4,
18). To determine whether mutated CVB3 would occur to
evade siRNA treatment, virus was passaged on newly
siRNA-4- or siRNA-C-transfected cells for four generations.
Viral RNA was extracted from culture supernatant, and the
siRNA-4 target region was PCR amplified and sequenced. The
data indicate that the sequence of the 2A region, even up to
FIG. 3. siRNA-4 inhibits ongoing CVB3 replication in HeLa cells.
Cells were infected with CVB3 at an MOI of 0.01 for 1 h and then were
transfected with siRNA-4 at a final concentration of 300 nM. Forty
hours after infection, supernatants and cell lysates were collected for
detection of virus titer by plaque assay (a) and VP1 by Western blot-
ting (b), respectively. Data shown are representative of two indepen-
VOL. 79, 2005 INHIBITION OF CVB3 REPLICATION BY siRNAs2155
the fourth generation, was identical to the original viral cDNA
(data not shown).
Initially, when we selected the targeting sequences within the
CVB3 RNA, the 5?UTR and the initiation codon regions were
chosen for potential weaker target controls because the UTR-
binding proteins and/or translation initiation complexes may in-
terfere with binding of siRNA and the RISC in these regions
(http://www.rockefeller.edu/labheads/tuschl/sirna.html). As ex-
pected, cells pretreated with siRNA-1 and -2 targeting the
5?UTR and initiation codon region demonstrated poorer an-
tiviral capability than those pretreated with siRNA-3, -4, and -5
targeting the coding regions. However, several studies have
demonstrated excellent protection against various virus infec-
tions by specific siRNAs targeting the 5?UTR (24, 51). These
studies suggest that whether the binding of protein factors at
certain sites of the 5?UTR has a negative or positive effect on
siRNA function may depend on the local sequence character-
istics. For certain regions, protein complex binding may change
the higher ordered structure of the target sequence and, there-
fore, facilitate the access of the siRNA to the target sequence.
On the other hand, if the siRNA and the protein factor com-
pete for the same target, which is the regulatory sequence of
gene expression, the binding of the protein factors may inhibit
the RNAi activity of siRNA.
Among the siRNAs that we tested, siRNA-4, which is di-
rected against the viral protease 2A region, was the most ef-
fective one, followed by siRNA-3, -5, -1, and -2. These results
may be due to different positional accessibility caused by steric
hindrance by a secondary or tertiary structure and/or protein
binding. In this regard, there are inconsistent reports. Several
studies focusing on the relationship between secondary struc-
ture and siRNA effects showed that the secondary structure at
least in part influences the efficiency of siRNAs (22, 44, 51).
Conversely, it has been reported that the secondary structure
of the target mRNA does not appear to have a strong effect on
gene silencing (22, 48). Whether secondary structure plays a
role in siRNA machinery binding is still debated. However, we
believe that binding of cellular proteins on the target sites or
the secondary structure of the mRNA may affect, at least in
part, the efficacies of siRNAs in the cells. Although we are not
clear on the higher ordered structure of the siRNA-4 targeting
sequence in the cellular environment, we believe that it pos-
sesses a more accessible conformation for siRNA. This may
also be related to the thermodynamic instability of the dsRNA
intermediate at this specific locus during CVB3 transcription,
which is evidenced by this segment lacking any continuous two
FIG. 4. siRNAs protect cells against CVB3-induced cytopathic effects. (a) Morphological changes of HeLa cells following infection. Cells were
transfected with each siRNA at a final concentration of 300 nM and then infected with CVB3 at an MOI of 0.01. Cell morphology was observed
under a phase-contrast microscope at 48 hpi (magnification, ?100). S, sham infected; (?), mock transfected. (b) MTS cell viability assay. The assay
was performed as described in Materials and Methods. Cell viability of each sample was expressed relative to that of the sham-infected control,
which was defined as 100% survival. Values shown here are means ? standard deviations of three independent experiments. P ? 0.005. (c and d)
Western blot analysis of CVB3 VP1 in the cell lysates (c) and supernatants (d). Note that cells transfected with siRNA-2 or control, as well as
mock-transfected cells, were dead after CVB3 infection for 48 h. Thus, no intact cells were remaining 48 hpi for preparing cell lysates used for
Western blot analysis (c). Data shown are representatives of three independent experiments.
2156 YUAN ET AL.J. VIROL.
repeating bases at the middle region (Table 1), or this region
may be favorable for binding of RISC required for siRNA
function. In addition to the secondary structure and protein
binding, the inefficacy of siRNA-2 may also be due to the
sequence containing three continuous cytosines, which may
hyperstack and therefore form agglomerates that potentially
interfere in the silencing mechanism. Similarly, the siRNA
containing five continuous guanosines targeted on the 5?UTR
of hepatitis C virus did not show inhibitory ability on virus
replication (51). Therefore, avoiding more than three Gs or Cs
in the siRNA may exclude this problem. Interestingly, the
sequence covering the initiation codon region has been re-
FIG. 5. CVB3-specific siRNAs inhibit CVB3 replication in HL-1
cells. Cells were transfected with each siRNA at a final concentration
of 300 nM by the Oligofectamine method overnight and then were
infected with CVB3 at an MOI of 10. At 48 hpi, supernatants were
used for detecting virus titer by plaque assay (a), and cell lysates were
collected for VP1 detection by Western blot (b). ?-Actin was used as
the loading control. Data shown are representatives of two indepen-
FIG. 6. Effects of mismatches on antiviral activity of siRNA. (a) Sequences of the mutated siRNA-4. The targeting sequence for siRNA-4 is
listed at the top, with the center base boxed. The sequences of wild-type and mutated siRNAs are shown, with mismatched nucleotides underlined.
(b and c) siRNA-4mAS with one point mutation in the center of the antisense strand failed to inhibit virus replication, which was detected by viral
plaque assay (b) and Western blotting (c). (d) siRNA-4 with one nucleotide mismatch near the 5? end of the antisense strand but not with one near
the 3? end partially reduced its antiviral activity. HeLa cells were transfected with wild-type and mutated siRNA-4 at a final concentration of 300
nM, followed by CVB3 infection at an MOI of 10 for 8 h. Virus titer in the supernatants and VP1 protein expression in the cell lysates were
analyzed by plaque assay (b) and Western blot (c and d). ?-Actin served as the loading control. Data shown are representatives of two independent
FIG. 7. Effect of combined siRNAs on antiviral activity. HeLa cells
were cotransfected with siRNA-4 and each of the other four siRNAs at
a common final concentration as indicated, followed by CVB3 infec-
tion at an MOI of 10 for 8 h. Cell lysates and supernatants in cultures
were used for detecting viral protein expression by Western blot. ?-Ac-
tin served as the loading control. Note that no enhanced inhibitory
effect on CVB3 infection was observed by cotransfection of each
siRNA with siRNA-4. Data shown are representatives of two indepen-
VOL. 79, 2005 INHIBITION OF CVB3 REPLICATION BY siRNAs2157
ported to be an effective target for gene knockdown by an
antisense deoxynucleotide (AS-DON) agent (45). Our previ-
ous study of antiviral activity of AS-DONs also confirmed this
in anti-CVB3 replication (45, 52). This inconsistent data re-
garding siRNA targeting the same region may be due to the
distinct mechanism of action for AS-DON. A commonly ex-
ploited antisense mechanism is RNase H-dependent degrada-
tion of the targeted RNA through recognition of a DNA-RNA
heteroduplex (28), while siRNAs bind to targeted RNA by
Watson-Crick base pairing and induce site-specific cleavage of
the RNAs by a specific unknown RNase. This suggests that the
best target sequences for AS-DON may not be the best can-
didate sites for siRNAs. Due to the different targeting sites
used for siRNA and AS-DON, it is hard to compare their
inhibitory effects on CVB3 replication. Overall, under optimal
conditions it seems that CVB3-specific siRNAs are more ef-
fective than AS-ODNs in terms of potency, efficacy, and dura-
tion, which is consistent with other studies (2, 19, 48).
Sequence specificity of siRNA is very stringent, as single
base pair mismatches between the siRNA and its target se-
quence dramatically reduce the silencing capability (3, 12, 14).
However, there are different reports on this issue with different
experimental systems. A detailed siRNA functional anatomy
analysis revealed that RNAi required a perfect match between
cellular mRNA and the antisense strand of siRNA, but several
mutations in the sense strand of siRNAs did not eliminate the
gene silencing (14, 20). On the other hand, in another report
an siRNA with two nucleotide mismatches in the central re-
gion still had partial inhibitory activity (51). The experiments
reported here used a positive single-stranded RNA virus which
can produce a dsRNA intermediate during replication. This
raises the possibility that siRNA may target positive, negative,
or both strands of virus RNA. To clarify this question, we
perform evaluations using a series of siRNA-4 mutants con-
taining point mutations within the sense and/or antisense
strands at different locations. The data suggest that only one
point mutation in the middle of the antisense strand could
eliminate the anti-CVB3 activity, whereas the corresponding
mutation on the sense strand did not interfere with the viral
replication, suggesting that the negative-strand RNAs pro-
duced during viral replication are not the direct target of
siRNA. This result might be explained by the fact that the
replicating negative strands of virus only exist as a double-
stranded form in the vesicles (15), thus, they are less likely to
be accessible to siRNAs. Conversely, the positive strand is the
recognition site for RNAi, which forms complementary base
pairs with the antisense strand of the siRNA. This conclusion
is not only drawn from our study using positive single-stranded
RNA virus but also has been reported recently for a negative
single-stranded influenza virus in which the siRNA targets the
mRNA of virus during replication (17). For the point mutation
closest to the 5? or 3? end of the antisense siRNA, it is likely
that the mismatch near the 5? end has more negative effects on
gene silencing than that near the 3? end, which is consistent
with a previous report (40). However, the molecular mecha-
nism of this phenomenon needs to be further studied.
Cotransfection of cells with two or more siRNAs targeting
different sites on HIV-1 coreceptor CXCR4 mRNA has been
reported to result in enhanced gene silencing compared to that
of each single siRNA (25). This could be explained by specific
binding of certain siRNAs that may change the secondary
structure of RNA and result in more accessible sites for other
siRNA molecules. However, as with a previous report (22), we
did not observe enhancement effects when using any combina-
tions of two agents, including siRNA-4, in our system. These
particular siRNAs probably could not affect the secondary
structure of the targets or open more space to other siRNAs,
or the amount of siRNA-associated proteins was limited for
silencing rather than target accessibility. In general, the rea-
sons for the discrepancy between the studies may be due to
differences in mRNA targets and the evaluation methods. For
antiviral evaluation, although the underlying mechanism of the
enhanced gene silencing with multiple specific siRNAs is not
clear, cotransfection with multiple siRNAs may benefit long-
term treatment, as mutated virus variants may be produced
following infection to escape from protection by siRNA (3, 4,
To investigate whether escaping CVB3 mutants were gener-
ated following siRNA-4 treatment, a series of passages of
CVB3 were challenged with fresh siRNA-4. However, we did
not detect any mutants in this study. The discrepancy between
previous reports (4, 18) and our result could be due to the fact
that (i) exposure time to siRNA was not long enough com-
pared to that of a previous HIV study, which showed emer-
gence of mutation at the target site at 25 days posttreatment
(4); and (ii) a different targeting region was used, as the
siRNA-4 targeting sequence is a critical site for CVB3, because
mutations in this area would markedly impair the fitness of the
virus. Therefore, highly conserved regions should be used as
targets for siRNA design to limit the occurrence of escape
In summary, this in vitro study is the first step to demon-
strate that siRNA technology is a very promising approach to
antiviral gene therapy. The very strong anti-CVB3 activity of
siRNA-4 has indicated attractive new directions for further
investigation of the underlying mechanism and the develop-
ment of siRNA-4 as a prophylaxis and therapy for CVB3 in-
We thank Reinhard Kandolf, University of Tubingen, Germany, and
William C. Claycomb (Louisiana State University) for generously pro-
viding us with CVB3 cDNA and HL-1 cells, respectively. We also
thank Bruce McManus for his critical discussion on experimental de-
sign and data analysis. Special thanks go to Agripina Suarez for her
help with in situ hybridization and to Brian Wong, Elizabeth Walker,
Zongshu Luo, and Jingchun Zhang for their technical assistance and
This work was supported by grants from the Canadian Institutes of
Health Research (MOP-14068) and the Heart and Stroke Foundation
of British Columbia and Yukon (20R20002).
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