JOURNAL OF VIROLOGY, Feb. 2007, p. 1401–1411
Copyright © 2007, American Society for Microbiology. All Rights Reserved.
Vol. 81, No. 3
Retinoic Acid-Inducible Gene I Mediates Early Antiviral Response
and Toll-Like Receptor 3 Expression in Respiratory Syncytial
Virus-Infected Airway Epithelial Cells?
Ping Liu,1,2Mohammad Jamaluddin,1Kui Li,3Roberto P. Garofalo,3,4
Antonella Casola,4and Allan R. Brasier1,2,5*
Departments of Medicine,1Biochemistry and Molecular Biology,2Microbiology and Immunology,3and Pediatrics4and
the Sealy Center for Molecular Medicine,5University of Texas Medical Branch, Galveston, Texas 77555-1060
Received 11 August 2006/Accepted 8 November 2006
Respiratory syncytial virus (RSV) is one of the most common viral pathogens causing severe lower respi-
ratory tract infections in infants and young children. Infected host cells detect and respond to RNA viruses
using different mechanisms in a cell-type-specific manner, including retinoic acid-inducible gene I (RIG-I)-
dependent and Toll-like receptor (TLR)-dependent pathways. Because the relative contributions of these two
pathways in the recognition of RSV infection are unknown, we examined their roles in this study. We found that
RIG-I helicase binds RSV transcripts within 12 h of infection. Short interfering RNA (siRNA)-mediated RIG-I
“knockdown” significantly inhibited early nuclear factor-?B (NF-?B) and interferon response factor 3 (IRF3)
activation 9 h postinfection (p.i.). Consistent with this finding, RSV-induced beta interferon (IFN-?), inter-
feron-inducible protein 10 (IP-10), chemokine ligand 5 (CCL-5), and IFN-stimulated gene 15 (ISG15) expres-
sion levels were decreased in RIG-I-silenced cells during the early phase of infection but not at later times (18
h p.i.). In contrast, siRNA-mediated TLR3 knockdown did not affect RSV-induced NF-?B binding but did
inhibit IFN-?, IP-10, CCL-5, and ISG15 expression at late times of infection. Further studies revealed that
TLR3 knockdown significantly reduced NF-?B/RelA transcription by its ability to block the activating phos-
phorylation of NF-?B/RelA at serine residue 276. We further found that TLR3 induction following RSV
infection was regulated by RIG-I-dependent IFN-? secreted from infected airway epithelial cells and was
mediated by both IFN response-stimulated element (ISRE) and signal transducer and activator of transcrip-
tion (STAT) sites in its proximal promoter. Together these findings indicate distinct temporal roles of RIG-I
and TLR3 in mediating RSV-induced innate immune responses, which are coupled to distinct pathways
controlling NF-?B activation.
Respiratory syncytial virus (RSV) is the most frequent cause
of bronchiolitis and pneumonia in young children requiring
hospitalization worldwide (36). In the United States alone,
lower respiratory tract infection with RSV is responsible for
over 100,000 hospitalizations annually (47). RSV is a single-
stranded RNA virus of the Paramyxoviridae family whose ma-
jor site for productive replication is the epithelial cells in the
airway mucosa (2, 18). Here, RSV replication induces cytokine
and chemokine gene expression networks in a coordinated
manner (6, 14, 43, 48, 56). One important cytokine network is
the expression of type I interferons (IFNs), including alpha
interferon (IFN-?), IFN-?, IFN-?, IFN-ε, and IFN-?, which
are responsible for producing an antiviral state and stimulating
effector arms of cellular immunity (22, 42).
The detection of RNA virus by host cells occurs in a cell-
type- and pathogen-type-specific manner. The sensors for viral
infection primarily involve two kinds of receptors: the cytoplas-
mic pattern recognition receptors, including that for the reti-
noic acid-inducible gene I (RIG-I) and the pathogen-associ-
ated molecular pattern receptors known as the Toll-like
receptors (TLRs) (20, 23, 37, 38). RIG-I is a highly inducible
cytoplasmic RNA helicase that signals antiviral responses after
binding double-stranded RNA (dsRNA), a pathway that has
been implicated in antiviral responses to Sendai virus, vesicular
stomatitis virus, and Newcastle disease virus, as well as the
flaviviruses Japanese encephalitis virus, dengue virus 2, and
hepatitis C virus (7, 24, 45). However, the role of RIG-I is
cell-type dependent; for example, in fibroblast cells, RIG-I is
the major sensor for viral infection, but in plasmacytoid den-
dritic cells, TLRs play a more important role (24, 53). The role
of RIG-I in mediating epithelial cell response to RSV infection
in airway epithelial cells has not been investigated. The TLRs
are a family of membrane-bound pattern receptors expressed
in a cell-type-specific manner that recognize distinct classes of
virus- and bacterium-derived ligands (3, 4). In the case of RSV,
TLRs 3 and 4 are thought to be major mediators of virus-
inducible signaling. TLR3 binds dsRNA, the replication inter-
mediate of RSV and other RNA viruses (10). In fact, others
have recently shown that short interfering RNA (siRNA)-me-
diated TLR3 silencing results in the inhibition of RSV-induced
chemokine ligand 5 (CCL-5) and interferon-inducible protein
10 (IP-10) expression in airway epithelial cells (32). In addi-
tion, although TLR4 binds lipopolysaccharide as its ligand, the
RSV fusion glycoprotein binds TLR4 directly, mediating in-
ducible-cytokine activation in monocytes (26). Among all
TLRs, TLR3 is one of the most abundant isoforms expressed
* Corresponding author. Mailing address: Division of Endocrinol-
ogy, MRB 8.128, University of Texas Medical Branch, 301 University
Blvd., Galveston, TX 77555-1060. Phone: (409) 772-2824. Fax: (409)
772-8709. E-mail: email@example.com.
?Published ahead of print on 15 November 2006.
in airway epithelial cells (40). Moreover, two groups have re-
cently reported that RSV infection induces TLR3 expression
and cell-surface translocation through incompletely under-
stood mechanisms (15, 33). Although these studies suggest that
TLR3 may mediate epithelial responses to RSV infection, its
antiviral role is controversial and the mechanism for its up-
regulation following RSV infection is unknown.
In this study, we examined the roles of RIG-I and TLR3 in
RSV-induced gene expression in transformed human alveolar
epithelial cells (A549). In the UV-cross-linking experiment,
active RIG-I bound RSV transcripts within 12 h of RSV ex-
posure. siRNA-mediated RIG-I silencing inhibited the activa-
tion of both NF-?B and IRF-3, as well as IFN-?, IP-10, CCL-5,
and ISG15 expression, at the early phase of RSV infection (9
h p.i.). Surprisingly, siRNA-mediated TLR3 “knockdown” had
little influence on the early response of RSV-induced genes
but significantly inhibited their late expression. The role of
TLR3 is not related to controlling RSV-induced NF-?B DNA
binding, but rather is required to mediate the RSV-induced
activating RelA at serine residue 276. We further found that
the TLR3 expression was RSV inducible via a transcriptional
mechanism mediated by two IFN-?-responsive ISRE and
STAT binding sites in its proximal promoter. We further found
that siRNA-mediated RIG-I silencing inhibited the upregula-
tion of TLR3 and that the paracrine IFN-? secreted from
infected cells was necessary and sufficient to induce the expres-
sion of TLR3. Together, these data indicate that TLR3 signal-
ing in RSV infection is dependent on early RIG-I signaling and
controls NF-?B/RelA subunit phosphorylation.
MATERIALS AND METHODS
Cell culture. Human A549 pulmonary type II epithelial cells (American Type
Culture Collection [ATCC]) were grown in F12K medium (Gibco) with 10%
fetal bovine serum (FBS), penicillin (100 U/ml), and streptomycin (100 ?g/ml) at
37°C in a 5% CO2incubator. African green monkey kidney Vero cells (ATCC)
were cultured in Eagle’s minimum essential medium with 0.1 mM nonessential
amino acids, 1.0 mM sodium pyruvate, and 10% FBS.
Virus preparation and infection. The human RSV A2 strain was grown in
HEp-2 cells and purified by centrifugation on discontinuous sucrose gradients
(14). The viral titer of purified RSV pools was 8 to 9 log PFU/ml, determined by
a methylcellulose plaque assay. Viral pools were aliquoted, quick-frozen on dry
ice-ethanol, and stored at ?70°C until they were used. For viral adsorption, cells
were transferred into F12K medium containing 2% (vol/vol) FBS and RSV
infected at a multiplicity of infection (MOI) of 1 for 18 h prior to harvest and
siRNA-mediated gene silencing. siRNA against human RIG-I (M-012511-00)
and TLR3 (M-007745-00) and control siRNA (D-001206-13) were commercially
obtained (Dharmacon Research, Inc., Lafayette, CO) and transfected at 100 nM
into A549 cells by using a TransIT-siQuest transfection kit (Mirus Bio Corp.,
Madison, WI) according to the manufacturer’s instructions. Forty-eight hours
after transfection, cells were RSV infected for the times indicated in the figure
legends. The efficiency of siRNA silencing was evaluated using reverse transcrip-
tase PCR (RT-PCR).
Plasmid construction. For the TLR3-Luc reporter, 1.0 kb of the human TLR3
promoter was amplified from A549 cell genomic DNA by PCR using the forward
primer 5?-TCAGAGGATCCGGCATGTTCTTAGGCAAACC-3? and reverse
primer 5?-TCAGAGATATCCTGTTGGATGACTGCTAGCC-3?. The PCR
product was digested with BamHI/SmaI, gel purified, and ligated into the same
sites in the pOLUC plasmid (5). Site-directed mutagenesis was conducted by
rolling circle PCR (18 cycles) to mutate the ISRE1 site (forward primer, 5?-CC
TCCCTAGGTTTCGCGCTCCTAATTTCTCAAA-3?; reverse primer, 5?-TT
TGAGAAATTAGGAGCGCGAAACCTAGGGAGG-3?), the ISRE2 site
(forward primer, 5?-AAGCTTTACTTTCACGATCGAGAGTGCCGTCT-3?;
reverse primer, 5?-AGACGGCACTCTCGATCGTGAAAGTAAAGCTT-3?),
and the STAT site (forward primer, 5?-TTTCTCCCTTTGCCCCCCTTGGAA
TGCACCAA-3?; reverse primer, 5?-TTGGTGCATTCCAAGGGGGGCAAAG
GGAGAAA-3?). The pT1S vector was constructed by removing the cytomega-
lovirus (CMV) promoter from pCDNA6-V5-HisB (Invitrogen) and replacing it
with a fragment containing the cytomegalovirus (CMV) promoter driving the
tetracycline transactivator, followed by the simian virus 40 poly(A) signal and a
tetracycline-responsive element (TRE). pT1S-RIG-I and pT1S-MDA5 were gen-
erated by inserting Flag-tagged RIG-I and Flag-tagged MDA5 into pT1S under
the control of the TRE. Plasmids were purified by ion exchange chromatography
(QIAGEN, Chatsworth, CA), and mutations were sequenced to verify authen-
ticity. The PRDII luciferase plasmid was a kind gift from Michael Gale (12).
Helicase RNA UV cross-linking and immunoprecipitation. The UV-cross-
linking assay is modified from the assay described in reference 30. A549 cells
were transfected with pT1S vector, pT1S-FLAG-RIG-I, or pT1S-FLAG-MDA5
for 48 h, and the transfectants were then infected with RSV (MOI ? 1, 12 h).
Cells were washed with prechilled phosphate-buffered saline (PBS) and UV
irradiated for 5 min with an 8 W germicidal lamp at a 4-cm distance (GS gene
linker; Bio-Rad). Cells were suspended in 500 ?l of immunoprecipitation buffer
(20 mM Tris–HCl, pH 7.4, 0.15 M NaCl, 5 mM EDTA, 4 ?g/ml each leupeptin
and pepstatin, and 1 mM phenylmethylsulfonyl fluoride), and lysed by sonication
for 20 s. The lysates were treated with 20 U of RNase T1and 10 ?g of RNase A
for 30 min at 37°C to remove unbound RNA. Immunoprecipitation was con-
ducted with anti-FLAG M2 antibody (Ab) for 4 h at 4°C and with protein
A–Sepharose for 1 h at 4°C. Total RNA was isolated from half of the immuno-
precipitation using proteinase K, and the other half of the immunoprecipitation
was fractionated by sodium dodecyl sulfate (SDS)-polyacrylamide gel electro-
phoresis for Western immunoblotting. Half of the RNA sample was subjected to
35 cycles of RT-PCR with RSV N protein-specific primers and visualized by
agarose gel electrophoresis.
RT-PCR and quantitative real-time PCR (QRT-PCR). Total RNA was ex-
tracted using acid guanidium phenol extraction (Tri Reagent; Sigma), and 1 ?g
of RNA was reverse transcribed using Moloney murine leukemia virus reverse
transcriptase (New England Biolabs) in a 20-?l reaction mixture. One ?l of
cDNA product was diluted 1:2, and 2 ?l was amplified in a 25-?l reaction mixture
containing 12.5 ?l of SYBR green supermix (Bio-Rad) and 0.4 ?M each of
forward and reverse gene-specific primers (Table 1), aliquoted into 96-well,
0.2-mm thin-wall PCR plates, and covered with optical-quality sealing tape. The
plates were denatured for 90 s at 95°C and then subjected to 40 cycles of 15 s at
94°C, 60 s at 60°C, and 1 min at 72°C in an ABI 7000 thermocycler. After PCR
was performed, PCR products were run on 2% agarose gels to assure a single
amplification product. Duplicate cycle threshold (CT) values were analyzed by
using the comparative CT(??CT) method (Applied Biosystems). The relative
amount of target mRNA (2???C
GAPDH (glyceraldehyde-3-phosphate dehydrogenase) reference and expressed
relative to the amount from uninfected cells.
Electrophoretic mobility shift assay (EMSA). Nuclear extracts (NE) were
prepared as described previously (17). A total of 5 ?g NE was incubated in DNA
binding buffer (5% glycerol, 12 mM HEPES, 80 mM NaCl, 5 mM dithiothreitol,
5 mM MgCl2, 0.5 mM EDTA) with 1.5 ?g of poly(dA-dT) and 0.1 nM IRDye
700/IRDye 800-labeled ds oligonucleotide (Table 2) in a total volume of 10 ?l.
T) was obtained by normalizing to endogenous
TABLE 1. Forward and reverse gene-specific primers
Primer sequence (5?–3?)
1402LIU ET AL.J. VIROL.
Complexes were fractionated by native polyacrylamide gels in 1? TBE buffer (89
mM Tris, 89 mM boric acid, 2 mM EDTA). For competition, unlabeled ds
competitor was added at the time of the binding reaction. Gels were then
scanned in an Odyssey infrared scanner (Odyssey system; Licor Biosciences,
Immunofluorescence microscopy. A549 cells (105) plated on coverslips were
mock or RSV infected (MOI ? 1) for the times indicated in the legend to Fig.
3. The cells were fixed with 4% paraformaldehyde in PBS, pH 7.4. The cells were
then incubated for 60 min at 37°C with anti-IRF-3 Ab or anti-NF-?B/RelA C20
Ab (Santa Cruz) diluted 1:200 in PBS-T (PBS, 0.1% Tween 20). Cells were
washed three times in PBS-T and incubated with secondary fluorescein isothio-
cyanate-conjugated anti-rabbit Ab in PBS-T for 1 h at 22°C. Nuclei were visu-
alized by staining for 15 min with SYTOX orange (Molecular Probes). Confocal
microscopy was performed on a Zeiss LSM510 META system. Images were
captured at a magnification of ?40. For each condition, 10 pictures were taken
and the percentage of cells which showed the nuclear staining for IRF-3 or RelA
was counted and expressed as the percentage of total cells examined.
RSV-CM collection and IFN neutralization. RSV-conditioned medium (RSV-
CM) was prepared by infecting A549 monolayers with RSV (MOI ? 1, 48-h
incubation). The supernatant was collected, centrifuged at 3,000 ? g, exposed to
UV light to inactivate the live virus, quick-frozen, and stored at ?70°C until used.
For IFN neutralization, 20 ?l of RSV-CM was mixed with either 15 ?g of rabbit
anti-human IFN-? Ab (Chemicon International) or 15 ?g of rabbit immuno-
globulin G (IgG) in a total volume of 2 ml of culture medium and incubated for
2 h at 37°C.
Northern blots. Total RNA (30 ?g) was fractionated by electrophoresis on a
1% agarose-formaldehyde gel and transferred to a nylon membrane (Zeta Probe
GT; Bio-Rad). A TLR3 cDNA probe was made using asymmetric PCR. The
membrane was hybridized with 2 ? 106cpm/ml of radiolabeled probe at 60°C
overnight in 5% SDS hybridization buffer (55). The membrane was washed with
1? SSC (1? SSC is 0.15 M NaCl plus 0.015 M sodium citrate) with 0.1% SDS
for 20 min at 60°C. Internal control hybridization was carried out with ?-actin
mRNA. The image was developed and quantified by exposing the membrane to
a Molecular Dynamics phosphorimager cassette.
Western immunoblot. Whole-cell extracts were prepared using modified ra-
dioimmunoprecipitation assay buffer (50 mM Tris-HCl [pH 7.4], 150 mM NaCl,
1 mM EDTA, 0.25% sodium deoxycholate, 1% Nonidet P-40, 1 mM phenyl-
methylsulfonyl fluoride, 1 mM NaF, 1 mM Na3VO4, and 1 ?g each of aprotinin,
leupeptin, and pepstatin/ml). One hundred micrograms protein was fractionated
by 10% SDS-polyacrylamide gel electrophoresis and transferred to a polyvinyli-
dene difluoride membrane by electroblotting. Membranes were blocked in 5%
nonfat dry milk in Tris-buffered saline–0.1% Tween and probed with the primary
Ab indicated in the figure legends. Membranes were washed and incubated with
IRDye 700-conjugated anti-mouse Ab or IRDye 800-conjugated anti-rabbit Ab
(Rockland, Inc.). Finally, the membranes were washed three times with PBS-T
and scanned by infrared scanner. RelA C20 Ab (Santa Cruz), anti-phospho-276
RelA Ab (Cell Signaling), anti-phospho-536 RelA Ab (Cell Signaling), and
Anti-FLAG M2 Ab (Sigma) were commercially obtained.
RSV infection activates NF-?B and IRF-3 in airway epithe-
lial cells. A549 cells are human epithelial cells that retain
morphological features of type II alveolar cells, including sur-
factant secretion, and support productive RSV replication
(27). Previous work has shown that RSV infection activates the
nuclear factor-?B (NF-?B) and IRF-3 pathways (6, 16, 21),
both central mediators of antiviral cytokine expression. To test
their relative kinetics of activation, A549 cells were RSV in-
fected (MOI ? 1); NF-?B pathway activation was measured
using an EMSA (Fig. 1A), and nuclear IRF-3 activation was
measured by Western immunoblotting (Fig. 1B). Consistent
with previous work, the NF-?B binding assay in the EMSA
showed three distinct complexes (Fig. 1A) (14, 21). The less-
mobile complex showed a time-dependent increase in response
to RSV infection; previous work has shown that this species
represents the heterodimer of the 65-kDa RelA-transactivat-
ing subunit and the 50-kDa NF-?B1 DNA binding subunit
(14, 21). Conversely, the middle complex, representing ho-
modimers of the 50-kDa NF-?B1 DNA subunit, was not af-
fected by RSV treatment. The most-mobile band represents
nonspecific DNA binding species. We noted that a significant
increase in NF-?B binding was detected 6 h after RSV infec-
FIG. 1. RSV activates NF-?B and IRF-3 in A549 cells. A549 cells
were infected by the human RSV A2 strain (MOI ? 1.0) for different
times as indicated (in hours), and NE was prepared. (A) EMSA was
performed on 5 ?g NE using 0.1 nM IRDye 700-labeled DNA probe
containing the ?B element. The composition of the bound complexes
is indicated. RelA · p50, RelA-p50 heterodimer; p50, p50 homodimer;
N.S., nonspecific product. (B) Western immunoblotting was conducted
on 50 ?g NE. Top panel, the membrane was stained with anti-IRF-3
Ab; bottom panel, ?-actin was stained as a loading control.
TABLE 2. Sense and antisense EMSA probes
Strand Primer sequence (5?–3?)
VOL. 81, 2007RIG-I/TLR3 IN RSV-INFECTED AIRWAY EPITHELIAL CELLS 1403
tion, which suggested the activation of NF-?B in response to
RSV infection (Fig. 1A).
Similarly, IRF-3 pathway activation was rapid, with an in-
crease of nuclear IRF-3 6 h after RSV infection, peaking after
12 h (Fig. 1B). The multiple bands detected represented dif-
ferent phosphorylation forms of IRF-3, characteristic of its
activation mechanism (6, 41). Together, these data indicate
that RSV activates both NF-?B and IRF-3 translocation rap-
idly, within 6 h after RSV infection.
The RIG-I pathway mediates the early phase of RSV-in-
duced gene expression. Although RSV-induced activation of
the epithelial TLR3 pathway has been discovered recently, the
role of the RIG-I pathway is unclear. RIG-I is a cytoplasmic
helicase that activates signaling when it binds RNA. To inves-
tigate whether there is a physical interaction between RIG-I
and RSV replication products, an RNA helicase UV-cross-
linking experiment was performed. In this assay, eukaryotic
expression vectors encoding nothing, FLAG-tagged RIG-I, or
FLAG-tagged MDA5 were transfected into A549 cells. After
24 h, the transfectants were RSV infected for 12 h and RNA
was UV cross-linked to cellular proteins. To determine
whether RIG-I or MDA5 bound RSV transcripts, epitope-
flagged protein was immunoprecipitated by anti-FLAG Ab,
and the released RNA was assayed by RT-PCR. We found that
a specific 69-bp band corresponding to the RSV N protein
transcript was detected only in immunoprecipitates from RIG-
I-transfected cells (Fig. 2A).
To further understand the roles of RIG-I and TLR3 in
RSV-induced gene expression, A549 cells were transfected
with RIG-I, TLR3, or control siRNA and subsequently RSV
infected for 0, 9, or 18 h, and QRT-PCR was used to measure
the antiviral gene expression. RSV induced RIG-I expression
by 30-fold within 9 h of infection, which, significantly, fell to
20-fold after 18 h (Fig. 2B). Relative to control siRNA-trans-
fected cells, RIG-I siRNA-transfected cells showed significant
inhibition of RIG-I expression at 9 h and less inhibition after
18 h of RSV infection (Fig. 2B). Similarly, RSV induced TLR3
expression 22- and 25-fold at 9 and 18 h, respectively. TLR3
knockdown significantly reduced RSV-induced TLR3 expres-
sion at both times. These data indicate that RSV induced both
RIG and TLR3 expression and that siRNA knockdown was
We next examined the effect of RIG-I knockdown on RSV-
induced IFN-?, IP-10, ISG15, and CCL-5 expression (Fig. 2C).
RSV strongly induced IFN-?, IP-10, and ISG15 production, at
100-fold, 280-fold, and 75-fold, respectively, after 9 h of infec-
tion. By contrast, CCL-5 expression peaked at 2,000-fold 18 h
after RSV infection. In the RIG-I knockdown mutants, basal
expression levels of IFN-? and the IFN-responsive IP-10 and
ISG15 genes were all increased relative to those of the control
siRNA transfectants but were not significantly induced by RSV
infection 9 h later (Fig. 2C). For CCL-5, basal expression was
not detectable and, in the RIG-I knockdown, its expression was
reduced at 9 h as well. In addition, we noted that after 18 h of
RSV infection, levels of IP-10, ISG15, and CCL-5 were en-
hanced significantly in RIG-I-silenced cells, whereas there was
no significant difference in the level of expression of IFN-?
between RIG-I-silenced cells and control cells.
RSV-induced expression of the same genes was then exam-
ined after TLR3 knockdown (Fig. 2D). In the TLR3 knock-
down mutants, the basal expression levels of all investigated
genes were increased, and after 9 h of RSV infection, the gene
expression levels of IP-10 and CCL-5 were not affected. In
contrast to the effects of RIG-I knockdown, expression levels
of both IFN-? and ISG15 were increased, rather than inhib-
ited, 9 h after infection. In this group of genes, expression
levels were inhibited 18 h after RSV infection (Fig. 2D). These
results suggest that the RIG-I pathway is involved in the early
response of host cells to RSV infection. At later phases of RSV
infection, other RIG-I-independent pathways are activated
that mediate downstream gene expression, including that of
The RIG-I pathway mediates early NF-?B/RelA and IRF-3
activation in response to RSV infection. To determine whether
RSV-induced DNA binding of NF-?B or IRF-3 was affected
after RIG-I or TLR3 expression was silenced, DNA binding
activity was measured in NE by EMSA using either double-
stranded NF-?B or ISRE sites. NF-?B-specific DNA binding
activity was abolished in RIG-I-silenced cells at 9 h or 18 h
after RSV infection compared to that in controls, whereas only
a slight decrease in binding occurred in cells after TLR3 si-
lencing (Fig. 3A, left panel). Conversely, siRNA-mediated
RIG-I silencing inhibited specific RSV-inducible IRF-3 bind-
ing to its cognate ISRE element 9 h after RSV infection, but a
significant increase of IRF-3 binding was observed later (18 h
after infection; Fig. 3B). Similar to its lack of effect on RSV-
induced NF-?B DNA binding, siRNA-mediated TLR3 silenc-
ing had little detectable effect on inducible IRF-3 binding at
any time point (Fig. 3B). An EMSA experiment using the
oligonucleotide containing the OCT1 element was conducted,
which confirmed equivalent nuclear protein preparation for
each extract (Fig. 3C). Together, these data indicated that the
RIG-I, but not the TLR3 signal, was required for RSV-induced
NF-?B and IRF-3 DNA binding.
To determine whether RIG-I was required for cytoplasmic-
nuclear translocation, we next examined the subcellular distri-
butions of NF-?B and IRF-3 using confocal microscopy in
RSV-infected cells transfected with control siRNA or RIG-I
siRNA. In control siRNA-transfected cells, 32% of cells
showed nuclear RelA accumulation 9 h after RSV infection;
this number increased to 63% 18 h after infection. By contrast,
in RIG-I-silenced cells, nuclear RelA translocation was signif-
icantly inhibited, with only 13% of cells after 9 h and 22% of
cells after 18 h showing nuclear RelA signals (Fig. 3D and F).
In the case of IRF-3 activation, in control siRNA-transfected
cells, 21% (9 h) and 38% of cells (18 h) showed IRF-3 nuclear
translocation after RSV infection, whereas in RIG-I-silenced
cells, 8% and 29% of cells showed nuclear IRF-3 accumulation
9 and 18 h after infection, respectively (Fig. 3E and F). These
data indicated that RIG-I expression was necessary for nuclear
translocation of both RelA and IRF-3 at early times after RSV
infection (9 h). In addition, we noted that there was no statis-
tically significant difference between the RIG-I siRNA group
and control groups for IRF-3 nuclear translocation 18 h after
RSV infection, and this later increase in IRF-3 binding was
consistent with the preserved RSV-induced IRF-3 DNA bind-
ing detected by EMSA (Fig. 3B), suggesting that, in the ab-
sence of RIG-I, other redundant pathways may mediate IRF-3
activation later during the infection.
1404LIU ET AL.J. VIROL.
The TLR3 pathway regulates the phosphorylation of NF-?B/
RelA at serine 276. Our findings suggested that the TLR3
pathway was not required for inducing NF-?B/RelA or IRF-3
DNA binding activity in response to RSV infection (Fig. 3A
and B), yet it significantly inhibited the expression of genes
which we have found to be NF-?B dependent, including the
CCL-5 and IP-10 genes (48). To examine whether TLR3 sig-
naling affected NF-?B/RelA transcriptional activity, we tested
RSV-inducible NF-?B activity using a luciferase reporter plas-
mid containing the IFN-? PRDII domain in control or TLR3
FIG. 2. RIG-I mediates early innate immune response to RSV infection. (A) RNA helicase UV cross-linking and immunoprecipitation assay.
A549 cells were transfected with 2 ?g pT1S vector as control (Con), pT1S-FLAG-RIG-I (RIG), or pT1S-FLAG-MDA5 (MDA) for 24 h.
Thereafter, cells were infected with RSV for 12 h. UV cross-linking and immunoprecipitation experiments were conducted as described in
Materials and Methods. RT-PCR yielded two bands in the sample corresponding to RIG-I immunoprecipitation. The upper band was confirmed
as RSV N protein RNA by sequencing (top panel); the bottom band is a nonspecific product (N.S.). Molecular sizes in base pairs (bp) are shown
on the left. The bottom panel shows a Western immunoblot of the immunoprecipitates, including RIG-I and MDA5. Specific bands are indicated
by asterisks. Molecular sizes are shown on the left. M, marker. (B) A549 cells were transfected with 100 nM of nonspecific siRNA as control (Con),
RIG-I siRNA (RIG-I), or TLR3 siRNA (TLR3) for 48 h. Cells were infected by RSV for 0, 9, or 18 h, and total RNA was extracted. QRT-PCR
was performed to determine changes in RIG-I (top panel) or TLR3 (bottom panel) expression levels as indicated.*, P is ?0.01 compared to
control siRNA (Student’s t test). (C and D) A549 cells were transfected with control (Con), RIG-I siRNA (RIG-I), or TLR3 siRNA (TLR3) for
48 h and then RSV infected for 0, 9, or 18 h. CCL-5, IP-10, IFN-?, and ISG15 expression levels were determined by QRT-PCR; shown are the
changes (n-fold) relative to unstimulated cells transfected with control siRNA (0 h).*, P is ?0.05 relative to the corresponding group at the same
time point (Student’s t test). (C) Results of QRT-PCR of cells transfected with RIG-I siRNA and (D) cells transfected with TLR3 siRNA. Error
bars indicate standard deviations.
VOL. 81, 2007 RIG-I/TLR3 IN RSV-INFECTED AIRWAY EPITHELIAL CELLS1405
siRNA-transfected cells. Reporter gene expression levels in-
creased four- and eightfold after 9 and 18 h of RSV infection
in the cells transfected with control siRNA, whereas NF-?B-
dependent reporter gene expression was significantly inhibited
in TLR3-silenced cells (Fig. 4A). This suggested that the TLR3
pathway controls NF-?B transcriptional activation.
NF-?B is known to be a nuclear phosphoprotein with acti-
vating sites at serine residues 276 (50, 58) and 536 (8, 35). We
therefore investigated whether RSV induced RelA phosphor-
ylation and, if so, whether it was inhibited by TLR3 silencing.
In control siRNA-transfected cells, RelA phosphorylation on
serine 276 and 536 sites increased 9 and 18 h after RSV infec-
tion. By contrast, in TLR3 siRNA transfectants, serine 276
phosphorylation was significantly inhibited 18 h after RSV
infection (Fig. 4B). Together, these data suggested that the
activation of the TLR3 pathway in airway epithelial cells con-
trols the phosphorylation of RelA at serine 276 as its mecha-
nism for regulating RSV-induced NF-?B-dependent gene
expression at the late phase of infection.
The RIG-I pathway mediates RSV-induced TLR3 upregula-
tion by increasing paracrine IFN-? secretion. Previous studies
(15, 31, 32) and ours (Fig. 2B) have shown that TLR3 expres-
sion is induced by RSV infection. To further investigate the
interaction between the RIG-I pathway and the TLR3 path-
way, we next explored the effect of RIG-I knockdown on TLR3
transcription and expression. QRT-PCR was conducted to
measure endogenous TLR3 mRNA levels after RIG-I expres-
sion was silenced. In the control siRNA group, the TLR3
mRNA levels were increased 6- and 18-fold after 9 or 18 h of
RSV infection. siRNA-mediated RIG-I silencing abolished the
RSV-induced TLR3 induction. These data suggested that the
activation of the epithelial RIG-I pathway is required for RSV-
induced TLR3 upregulation. To initially localize the regulatory
regions in the TLR3 gene, a computational analysis of the
human TLR3 promoter was conducted using position weight
matrices (TRANSFAC) (51). In this analysis, we predicted two
interferon response elements (ISRE1 and ISRE2) and one
STAT site. A 1.0-kb fragment of the human TLR3 promoter
containing these regulatory regions was cloned and inserted
into a luciferase reporter plasmid, generating hTLR3/LUC. To
determine their relative contributions, each ISRE and STAT
site was individually mutated to non-DNA binding sequences
in the context of the 1-kb hTLR3/LUC (Fig. 5B). The wild-type
hTLR3/LUC and its respective site mutants were then trans-
fected into A549 cells and luciferase reporter activity was mea-
sured in the absence or presence of RSV infection. We found
that hTLR3/LUC was induced sixfold by RSV relative to ac-
tivity in the uninfected control (Fig. 5C). In addition, mutation
of the ISRE1 site did not affect RSV-induced reporter gene
expression, but mutation of either the ISRE2 or STAT site
significantly decreased RSV-induced reporter gene activity
(Fig. 5C). Because RSV is known to induce IFN-? secretion by
airway epithelial cells (22, 42, 43) and others have reported
that type I IFNs enhance TLR3 expression (49), we next in-
vestigated whether RSV-induced TLR3 expression is con-
trolled in a paracrine manner by IFN-? secretion. CM from
RSV-infected A549 cells (24 h after infection) was collected,
and naı ¨ve A549 cells were incubated with 2.5% (vol/vol) of
UV-inactivated RSV-CM (UV-RSV-CM). Six and twelve
hours after exposure, RNA was extracted and Northern blot-
ting was conducted to measure TLR3 expression. We found
that UV-RSV-CM induced TLR3 expression in naı ¨ve A549
cells, indicating that paracrine activators of TLR3 expression
were present in infected A549 cell culture supernatants. To
determine whether the paracrine mediator in the RSV-CM
was IFN-?, two other plates were treated with UV-RSV-CM
neutralized with either rabbit IgG or neutralizing anti-IFN-?
Ab. The UV-RSV-CM induction of TLR3 was significantly and
selectively inhibited in the medium after IFN-? was neutral-
ized (Fig. 5D). These data suggested that IFN-? acts in a
paracrine manner to up-regulate TLR3 expression. To further
establish that paracrine IFN-? secretion is necessary for RSV-
induced TLR3 expression, RSV-induced TLR3 expression was
measured in Vero cells, cells deficient in IFN-? expression but
capable of productive RSV replication (44, 54). Although RSV
FIG. 3. RSV activates NF-?B and IRF-3 through the RIG-I path-
way at the early phase of infection. (A) A549 cells were transfected
with 100 nM of nonspecific siRNA as control (Con), RIG-I siRNA
(RIG-I), or TLR3 siRNA (TLR3) for 48 h, followed by RSV infection
for 9 or 18 h. NE from each siRNA treatment were prepared and
assayed by EMSA. Shown are bound complexes on the IRDye 700-
labeled ?B oligonucleotides visualized by infrared scanning (left
panel). A competition experiment was performed using the sample
from the control siRNA-treated group that was infected with RSV for
18 h and incubated with 0, 0.5, or 1 nM unlabeled oligonucleotides
(right panel). ?, none. (B) IRF-3 binding at different times of RSV
infection. EMSA was performed on NE using 0.1 nM IRDye 700-
labeled ISRE binding site (left panel). A competition experiment with
unlabeled probe and mutant probe was conducted (right panel). ?,
none. (C) OCT-1 binding. EMSA was performed using the same NE,
binding 0.1 nM IRDye 800-labeled OCT-1 binding site. (D) A549 cells
were transfected with either control (Con) or RIG-I siRNA (RIG-I)
for 48 h and then RSV infected for 0, 9, or 18 h. The cells were fixed,
incubated with rabbit anti-RelA Ab, and then stained with fluorescein
isothiocyanate-conjugated anti-rabbit secondary Ab (top panels). The
nuclei were stained with Sytox orange (middle panels). The slides were
imaged using confocal microscopy, and colors were merged (bottom
panels). Colocalization of RelA and nuclei is shown by light grey.
White arrows indicate the cells which had RelA nuclear translocation.
(E) A549 cells were treated as in (D), except that rabbit anti-IRF-3 Ab
was used. Colocalization of IRF-3 and nuclei is shown by light grey and
indicated by white arrows. (F) The percentages of cells with nuclei
positive for RelA or IRF-3 at each time point and for each treatment
were calculated based on five randomly photographed fields from two
independent experiments. Asterisks indicate a significant difference
between siRNA groups at the same time point of RSV infection (P ?
0.05, Student’s test). Error bars indicate standard deviations.
1406LIU ET AL.J. VIROL.
VOL. 81, 2007RIG-I/TLR3 IN RSV-INFECTED AIRWAY EPITHELIAL CELLS1407
infection increased TLR3 expression in A549 cells, it did not
induce TLR3 in Vero cells. Importantly, adding UV-RSV-CM
to Vero cells induced TLR3 gene expression (Fig. 5E). These
data indicate that IFN-? is necessary and sufficient for TLR3
RSV is the major etiologic agent of epidemic wheezing and
bronchiolitis in children, leading causes of hospitalization in
children (36). In natural infections, airway epithelial cells are
the primary sites for RSV invasion and these represent the cell
type where productive replication takes place (2). Previous
studies have shown that host cells primarily use two different
classes of “sensors” for viral detection. One is a group of
pattern recognition receptors localized in the cytoplasm that
includes the DExD/H box-containing RNA helicases, RIG-I,
and the melanoma differentiation-associated gene 5 (MDA5),
and the second is a group of membrane-bound pathogen-as-
sociated molecular pattern receptors known as the TLRs (20).
In this study, we found that the expression of both RIG-I and
TLR3 is rapidly induced by RSV infection in alveolar-like
A549 cells. Which of these two mechanisms is used by airway
epithelial cells to detect RSV infection and their interrelation-
ships are not understood.
In this study, we are the first to demonstrate that RIG-I
mediates RSV-induced early signaling events leading to the
activation of NF-?B and IRF-3, two key transcription factors
controlling inflammatory cytokine and chemokine expression
in airway epithelial cells (20). We have shown that RIG-I, but
not the related MDA5 molecule, specifically binds RSV RNA.
In addition, siRNA-mediated RIG-I knockdown significantly
inhibits the activation of NF-?B and IRF-3, especially at the
early phase of RSV infection (9 h p.i.). The mechanisms by
which RIG-I couples to transcription factor activation are par-
tially understood. After RNA virus infection, transcription and
replication of the virus yields RNA intermediates which are
bound by the helicase domain of RIG-I. This event activates
the two amino-terminal caspase-recruiting domains (CARD),
which in turn, are required for binding another CARD-con-
taining molecule, known as mitochondrion antiviral signaling
(MAVS, also known as IPS-1/VISA/Cardif) (25, 29, 39, 52).
Although we have not investigated its role here, MAVS has
been identified as the only downstream adaptor for RIG-I. The
CARD-mediated association between RIG-I and MAVS then
leads to the activation of NF-?B and IRF-3. IRF-3 activation
appears to be mediated by activation of the atypical IKKs,
TBK1/IKK?/ε, that phosphorylate IRF-3, resulting in its dimer-
ization and nuclear translocation.
By contrast, the mechanism for NF-?B activation is not fully
understood. Our previous work has shown that RSV controls
NF-?B nuclear translocation by its effects on I?B? proteolysis
(21); interpreted together with our studies demonstrating that
RIG-I is required for NF-?B nuclear translocation, these data
suggest that RIG-I is upstream of the canonical NF-?B path-
way. We note that a recent publication showed that TNF re-
ceptor-associated factor 3 (TRAF3) was involved in the acti-
vation of the IKK complex through the RIG-I–MAVS pathway
(34). In this regard, it will be of interest to examine whether
TRAF3 is involved in RSV-induced NF-?B activation. Finally,
our immunohistochemistry experiments indicate that the
NF-?B translocation response, as well as the IRF-3 response,
occurs only in a subpopulation (?30%) of RSV-infected cells.
These findings are consistent with other recent studies, which
used an even higher multiplicity of infection (43). These find-
ings indicate that there is significant heterogeneity in the RSV-
induced antiviral cell-signaling response.
The activation of both RIG-I-dependent and RIG-I-inde-
pendent pathways by West Nile virus has also been reported
recently (13). Using RIG-I-null embryonic fibroblasts, it was
found that West Nile virus activated IRF-3 through the RIG-I
pathway at the early phase of viral infection. However, at late
times of infection, West Nile virus was still able to activate
IRF-3 through a RIG-I-independent pathway whose mecha-
nism is unknown (13). These data are consistent with our
findings (Fig. 3B). We suspect that the activation of the RIG-
I-independent pathway may have a higher threshold, signaling
only under conditions of higher levels of viral replication. It
will be of interest to identify the mediators of this RIG-I-
FIG. 4. The TLR3 pathway mediates the phosphorylation of NF-
?B/RelA at serine 276. (A) A549 cells were transfected with control
siRNA (Con) or TLR3 siRNA, and a luciferase reporter plasmid
containing the PRDII domain was cotransfected for 48 h. Cells were
infected with RSV for 0, 9, or 18 h before cell lysis was performed.
Shown is normalized luciferase activity expressed as change (n-fold)
relative to that of uninfected cells.*, P ? 0.05 (Student’s t test). Error
bars indicate standard deviations. (B) A549 cells were transfected with
control siRNA (Con) or TLR3 siRNA (TLR3) and then RSV infected
for 0, 9, or 18 h. Western immunobloting was performed to detect
changes in phospho-Ser276 RelA (top panel), phospho-Ser536 RelA
(middle panel), and RelA (bottom panel) levels using 100 ?g of whole-
1408LIU ET AL. J. VIROL.
independent pathway; these could include protein kinase R,
TLR, or other cytoplasmic RNA helicases not yet identified.
In our data and the work published by others (32), TLR3
pathway inhibition affects the expression of some antiviral
genes, such as CCL-5 and IP-10. However, the exact mecha-
nism underlying this observation was not known previously.
We show here that the TLR3 pathway does not contribute to
the DNA binding activity of NF-?B and IRF-3 following RSV
infection (Fig. 3A and B). Rather, it regulates the transcription
of these genes by modulating the phosphorylation of RelA at
serine residue 276 (Fig. 4B). Serine 276 phosphorylation is
controlled by several protein kinases, including the catalytic
subunit of PKA and the mitogen- and stress-activated protein
kinase-1 (50, 58). Serine 276 phosphorylation is required to
induce intermolecular interaction between RelA and the p300
coactivator, thereby resulting in transcriptional activation (57).
It is not presently known which kinase mediates RelA serine
276 phosphorylation in response to RSV, and we will investi-
gate this in future studies.
The results of our study indicate that the TLR3 pathway
functions only later during the evolution of RSV infection. The
subcellular localization of TLR3 in uninfected cells may deter-
mine its kinetics and role in antiviral response. In particular,
some studies have indicated that TLR3 is localized, at least
partially, in an endosomal compartment in unstimulated cells
(9, 28). Because RSV is a paramyxovirus which enters the cell
directly by pH-independent fusion with the plasma membrane
(19), TLR3 would likely encounter its dsRNA ligand only later
FIG. 5. RSV-induced TLR3 expression depends on RIG-I-induced
IFN-? secreted from infected cells. (A) A549 cells were transfected
with control siRNA (Con) and RIG-I siRNA for 48 h and RSV in-
fected for 0, 9, or 18 h. QRT-PCR was performed using TLR3 probe.
#, P is ?0.01;*, P is ?0.05 relative to control siRNA at the same time
point. Error bars indicate standard deviations. The results shown here
are representative of two independent experiments. (B) Noncontigu-
ous genomic sequence of hTLR3 promoter. The location relative to
the major transcription start site is shown at left. Underlines, two
predicted ISRE sites (ISRE1 and ISRE2) and one STAT site; bold
font, site-directed mutagenesis of each individual regulatory element
was performed by rolling-circle PCR. (C) A549 cells were transfected
with either wild-type hTLR3/LUC reporter gene or different site mu-
tants. Twenty-four hours later, cells were RSV infected and normal-
ized luciferase activity was measured 12 h later. WT, wild type;*, P is
?0.001 relative to wild-type hTLR3/LUC activity at 12 h. Error bars
indicate standard deviations. (D) Naı ¨ve A549 cells were treated with
20% (vol/vol) UV-RSV-CM taken from RSV-infected cells for the
indicated times (in hours). Prior to its addition to A549 cells, UV-
RSV-CM was preincubated with PBS, rabbit IgG (IgG), or neutraliz-
ing anti-IFN-? Ab for 2 h. An autoradiogram from Northern blot
hybridization is shown. Top panel, hybridization using radiolabeled
TLR3 cDNA; bottom panel, hybridization with ?-actin as an internal
control. ?, anti. (E) IFN-?-deficient Vero cells were infected with RSV
for 12 h or were treated with 20% (vol/vol) UV-RSV-CM for 12 h. The
conditioned medium was collected from A549 cells 24 h after RSV
infection. Top panel, 20 ?g total RNA was isolated and Northern blot
hybridization conducted using TLR3 cDNA probe; bottom panel,
?-actin hybridization. ?, absent; ?, present.
VOL. 81, 2007RIG-I/TLR3 IN RSV-INFECTED AIRWAY EPITHELIAL CELLS1409
in the viral life cycle. The implication here is that other viruses
that enter via the endosomal pathway may be able to activate
the TLR3 pathway as a primary event. Although TLR3 is
partially endosomal in unstimulated cells, in response to stim-
ulation, newly synthesized TLR3 distributes to the plasma
membrane (15). The translocation of TLR3 might allow RSV-
infected cells to respond to extracellular dsRNA. Alternatively,
dsRNA released during late RSV infection might be taken up
by cells and transported to the endosomal compartments
where it can be recognized by TLR3. This hypothesis will
require further study.
Recently, two groups have reported the interaction between
RSV and TLR3 (15, 31, 32). The identical phenomenon that
they observed was that the expression of TLR3 was induced by
RSV infection. However, the mechanism of this induction is
not clearly understood. In this study, we found that IFN-?
secreted from RSV-infected epithelial cells is necessary and
sufficient to activate TLR3 expression. Importantly, induction
of TLR3 is not directly the result of cytoplasmic RSV replica-
tion, because TLR3 expression was not induced by RSV infec-
tion in Vero cells. Vero cells are capable of high levels of RSV
replication (44, 54), but are deficient in IFN-? expression (11).
The induction of TLR3 in response to RSV infection was
absent in Vero cells, but when Vero cells were treated with
RSV-conditioned media, rich in IFN-?, TLR3 was induced.
These observations suggest that TLR3 activation is a secondary
paracrine response mediated by local IFN-? secreted by RSV-
infected epithelial cells. The induction of TLR3 expression in
epithelial cells by measles virus and type I interferon has also
been reported recently (46, 49). Like RSV, measles virus is a
negative-sense single-stranded paramyxovirus. Measles virus
infection increased the expression of TLR3 through a tran-
scriptional mechanism involving the ISRE2 binding sites in the
hTLR3 promoter (46). Consistent with this finding, we also
found that the ISRE2 site was essential for TLR3 induction in
response to the IFN-? present in UV-RSV-CM. However, we
found that a proximal STAT site is also required for hTLR3
promoter expression. Here, IFN-? binds to its IFNAR1 recep-
tor, inducing the activation of receptor-associated Jak/Tyk ty-
rosine kinases and phosphorylation of receptor-associated
STATs. This process induces the formation of interferon-stim-
ulated gene factor 3, including STAT1 and STAT2 and IRF-9
(1). Because the hTLR3 promoter contains a functionally im-
portant ISRE and a STAT site, it is uniquely poised to inte-
grate signals from the RSV-IRF and IFN-STAT pathways into
enhanced transcriptional activation. Since IFN-? played an
essential role for inducing TLR3 expression in response to
RSV infection, it was reasonable to find that RIG-I was in-
volved in this process. siRNA-mediated RIG-I silencing abol-
ished the endogenous TLR3 induction in response to RSV.
This result suggests that RIG-I is a primary sensor for RSV
detection in airway epithelial cells and that TLR3 expression is
secondary to RIG-I-signaling action.
In summary, we found that the RIG-I pathway mediates the
early response of airway epithelial cells to RSV infection,
which initiates the innate immune response. The TLR3 path-
way only affects the late-time gene expression, which regulates
the phosphorylation of RelA at serine 276. TLR3 expression is
induced by RSV in a paracrine manner that depends on RIG-
I-induced IFN-? secretion.
This project was supported by NIAID grant R01 AI40218 (to
A.R.B.) and, in part, by PO1 AI062885 (to A.R.B.). Core laboratory
support was funded by NIEHS grant P30 ES06676 (to J. Halpert,
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