Download Cells Double-Stranded RNA in Myeloid Dendritic DHX9 Pairs with

DHX9 Pairs with IPS-1 To Sense
Double-Stranded RNA in Myeloid Dendritic
Cells
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of June 14, 2017.
Zhiqiang Zhang, Bin Yuan, Ning Lu, Valeria Facchinetti and
Yong-Jun Liu
J Immunol published online 28 September 2011
http://www.jimmunol.org/content/early/2011/09/28/jimmun
ol.1101307
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Published September 28, 2011, doi:10.4049/jimmunol.1101307
The Journal of Immunology
DHX9 Pairs with IPS-1 To Sense Double-Stranded RNA in
Myeloid Dendritic Cells
Zhiqiang Zhang, Bin Yuan, Ning Lu, Valeria Facchinetti, and Yong-Jun Liu
The innate immune system is equipped with many molecular sensors for microbial DNA/RNA to quickly mount antimicrobial host
immune responses. In this paper, we identified DHX9, a DExDc helicase family member, as an important viral dsRNA sensor in
myeloid dendritic cells (mDCs). Knockdown of DHX9 expression by small heteroduplex RNA dramatically blocked the ability of
mDCs to produce IFN-a/b and proinflammatory cytokines in response to polyinosine-polycytidylic acid, influenza A, and reovirus.
DHX9 could specifically bind polyinosine-polycytidylic acid via its double-strand RNA binding motifs. DHX9 interacted with IPS1 via the HelicC-HA2-DUF and CARD domains of DHX9 and IPS-1, respectively. Knockdown of DHX9 expression in mDCs
blocked the activation of NF-kB and IFN regulatory factor 3 by dsRNA. Collectively, these results suggest that DHX9 is an
important RNA sensor that is dependent on IPS-1 to sense pathogenic RNA. The Journal of Immunology, 2011, 187: 000–000.
Department of Immunology, Center for Cancer Immunology Research, University of
Texas MD Anderson Cancer Center, Houston, TX 77030
Received for publication May 6, 2011. Accepted for publication August 29, 2011.
Address correspondence and reprint requests to Prof. Yong-Jun Liu, Department of
Immunology, Center for Cancer Immunology Research, University of Texas MD
Anderson Cancer Center, Houston, TX 77030. E-mail address: [email protected]
Abbreviations used in this article: BMDC, bone marrow-derived dendritic cell; DC,
dendritic cell; DSRM, dsRNA binding motif; DUF, domain of unknown function; Flu
A, influenza A; HA, hemagglutinin; HA2, Helicase associated domain; HelicC, helicase C terminal domain; IRF3, IFN regulatory factor 3; mDC, myeloid dendritic cell;
pDC, plasmacytoid dendritic cell; PKR, protein kinase receptor; Pol III, polymerase
III; poly A:U, polyadenylic-polyuridylic acid; poly A, polyadenylic acid; poly dA-dT,
poly deoxyadenylic-deoxythymidylic acid; poly dG-dC, poly deoxyguanylic-deoxycytidylic acid; poly dI-dC, poly deoxyinosinic-deoxycytidylic acid; poly I:C, polyinosine-polycytidylic acid; qPCR, quantitative PCR; shRNA, small heteroduplex
RNA; TAR, transactivation response element.
Copyright Ó 2011 by The American Association of Immunologists, Inc. 0022-1767/11/$16.00
www.jimmunol.org/cgi/doi/10.4049/jimmunol.1101307
suggest that DHX9 plays important roles in the regulation of
immune response. Results from luciferase reporter gene assays
showed that DHX9 enhances the expression of p65 and TNF-a.
Knockdown of DHX9 expression by RNA interference reduced
levels of NF-kB–dependent transactivation by mediating the
transcriptional activity of NF-kB (14). DHX9 is a microbial DNA
sensor that is dependent on MyD88 and triggers NF-kB responses
in plasmacytoid DCs (pDCs) (15). DHX9 was found as an autoantigen in the autoimmune disease systemic lupus erythematosus
(16). DHX9 has two dsRNA-binding motifs. These motifs are
conserved in the cis-acting transactivation response element
(TAR)-binding protein and PKR, a dsRNA sensor. DHX9, TARbinding protein, and PKR are all involved in the regulation of
HIV-1 gene expression through their binding to TAR RNA (17).
Further investigation indicated that DHX9 interacts with PKR and
is identified as a substrate for PKR (18).
In this study, we show that DHX9 acts as an important viral
sensor in myeloid DCs (mDCs). DHX9 could specifically bind
to dsRNA; binding activity required the dsRNA binding motif
(DSRM) of DHX9. DHX9 also interacted with IPS-1; this interaction was dependent upon the helicase-associated domain
(HA2)-domain of unknown function (DUF) domains of DHX9
and the CARD domain of IPS-1. We demonstrate that knockdown
of DHX9 expression by small heteroduplex RNA (shRNA) dramatically blocked the ability of mDCs to produce type I IFN and
proinflammatory cytokines in response to dsRNA and RNA virus.
Taken together, our results suggest that DHX9 is an important
IPS-1–dependent RNA sensor.
Materials and Methods
Reagents
Poly I:C was purchased from InvivoGen. This poly I:C is composed of
a strand of poly (I) annealed to a strand of poly (C). The size of the short poly
I:C is from 0.2 to 1 kb. The size of the long poly I:C is from 1.5 to 8 kb. Poly
deoxyadenylic-deoxythymidylic acid (poly dA-dT), poly deoxyguanylicdeoxycytidylic acid (poly dG-dC), and poly deoxyinosinic-deoxycytidylic
acid (poly dI-dC) were purchased from Sigma-Aldrich. Lipofectamine 2000
was purchased from Invitrogen. The following Abs were used for immunoprecipitation and/or immunoblotting: anti-DHX9 and anti-IFN regulatory
factor 3 (IRF3) (Santa Cruz Biotechnology), anti-MDA5, anti–RIG-I, anti–
IPS-1, anti-p65, anti–phospho-IRF3, and anti–phospho-p65 (Cell Signaling
Technology), anti–b-actin, anti-hemagglutinin (HA)-HRP, and anti–MyCHRP (Sigma-Aldrich). Anti-HA and anti-MyC beads were purchased from
Sigma-Aldrich. NeutAvidin beads were purchased from Pierce.
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T
he innate immune system detects viral infection predominantly by sensing viral nucleic acids. Innate immune
cells, such as macrophages, dendritic cells (DCs), and
epithelial cells/fibroblasts, use at least three types of pattern recognition receptors, including the TLRs (TLR3, 7, 8, and 9) (1–3),
retinoid acid-inducible gene I-like helicases (RLH, RIG-I, LGP2,
and MDA5) (4–7), and dsRNA-activated protein kinase receptor
(PKR) (8, 9), to sense different forms of viral nucleic acids for
inducing IFN responses. Some RNA virus infections generate
dsRNA, which are detected by different types of receptors within
the innate immune system. DCs are in the frontline of the innate
immune system—they can sense viral infection, process viral Ag,
and mount both innate and adaptive immune responses to virus
(10). Although previous studies using gene-targeting technology
demonstrated that myeloid DCs (mDCs) or conventional DCs
use TLR3, RIG-I, and MDA5 to sense viral RNA infection or
synthetic dsRNA polyinosine-polycytidylic acid (poly I:C) that
mimics viral dsRNA to produce type 1 IFN, whether there are
additional dsRNA sensors in mDCs are unknown (7).
DHX9 is a member of the DExDc helicase family and is a homolog of Drosophila maleless protein, which is essential to regulate X-linked gene expression and the survival of male larvae
(11). DHX9 recognizes a structured 59-terminal posttranscriptional control element in retroviruses that regulates translation
(12). The ATPase helicase function of DHX9 is necessary for
efficient HIV-1 RNA translation (13). Several lines of evidence
2
D2SC mDCs cell culture
The murine splenic DC cell line D2SC was maintained in IMDM containing
5% heat-inactivated FCS and 1% penicillin–streptomycin (Invitrogen Life
Technologies). D2SC cells were infected with the lentiviral vector carrying
a helicase-targeting shRNA or a scrambled shRNA. After 1–2 d of growth,
cells were selected by adding puromycin (2 ng/ml) in the medium.
Knockdown efficiency and specificity was confirmed by immunoblot. Cells
were stimulated with one of the following nucleic acids plus Lipofectamine 2000: long poly I:C, short poly I:C (both at 2.5 mg/ml concentration), 1 mg/ml poly dA-dT, and 2 mM CpG A or RNA virus, influenza A
(Flu A), or reovirus, each virus at a multiplicity of infection of 10.
A NEW IPS-1–DEPENDENT dsRNA SENSOR IN DCs
CTAAG-39 (reverse); IFN-b, 59-CCCTATGGAGATGACGGAGA-39 (forward) and 59-TCCCACGTCAATCTTTCCTC-39 (reverse); reovirus, 59CAGTCGACACATTTGTGG-39 (forward) and 59-GCGTACTGACGTGGATCATA-39 (reverse); and GAPDH, 59-ACCCAGAAGACTGTGGATGG-39 (forward) and 59-CAGTGAGCTTCCCGTTCAG-39 (reverse).
Signal transduction
D2SC cells were lysed in radioimmunoprecipitation assay buffer after a 0-,
30-, 60-, and 120-min stimulation with 2.5 mg/ml poly I:C delivered by
Lipofectamine 2000. Lysates were resolved by 4–20% SDS-PAGE and
blotted with Abs recognizing unphosphorylated or phosphorylated p65
or IRF3.
GM-CSF–derived DCs preparation
Single-cell suspensions of bone marrow cells were cultured in RPMI 1640
medium containing 10% FCS, 1 mM sodium pyruvate, HEPES, penicillin,
streptomycin, and 2-ME (RPMI 1640 medium/FCS) supplemented with
murine GM-CSF (50 ng/ml) (R&D Systems). Fresh GM-CSF was provided on days 3 and 5 of culture. Cells were recovered on day 6 for shRNA
knockdown of a targeted helicase or scrambled control. The knockdown
efficiency was detected with western blotting 36 h post-shRNA treatment.
RNA interference
Luciferase reporter gene assay
HEK293T cells were seeded on 48-well plates (1 3 105 cells/well) and then
transfected with 100 ng IFN-b luciferase and 2 ng Renilla luciferase reporter vectors plus 20, 100, or 200 ng DHX9, ΔDSRM, MyD88, or IPS-1
expression vector. Empty control vector was added so that a total of 500 ng
vector DNA was transfected into each well of cells. At 30 h posttransfection, cells were stimulated with 2.5 mg/ml poly I:C delivered by
Lipofectamine 2000. Cells were harvested after 6 h of stimulation. The
luciferase activity in the total cell lysate was detected with the DualLuciferase Reporter Assay.
In vitro pulldown and immunoblotting assay
Lysates from HEK293T cells transfected with HA-tagged DHX9 or DHX36
expression plasmids were incubated with anti-HA beads. Proteins were
eluted from beads after washing six times with PBS. For nucleic acid
pulldown assays, purified HA-DHX9 or HA-DHX36 proteins were incubated for 2 h with biotin-labeled poly I:C, poly dA-dT, poly dG-dC, poly
dI-dC, CpG A, or CpG B. Following incubation with NA beads, bound
complexes were pelleted by centrifugation and analyzed by immunoblot
with anti–HA-HRP Ab. To ascertain DHX9 binding specificity, pulldown
assays were repeated with HA-DHX9 protein plus biotin-labeled poly I:C
in the absence or presence of increasing amounts (0.5, 5, and 50 mg/ml) of
unlabeled CpG B, polyadenylic acid (poly A), polyadenylic-polyuridylic
acid (poly A:U), poly I:C, or CpG A. For DHX9:IPS-1 interaction analysis,
D2SC cells were unstimulated (2) or stimulated (+) with short poly I:C or
long poly I:C for 4 h; whole-cell lysates were prepared from the cells and
then immunoprecipitated with anti-DHX9 Ab; bound proteins were analyzed by immunoblotting with anti-DHX9, anti–IPS-1, or anti–RIG-I Abs.
To map region(s) in DHX9 required for interaction with IPS-1, purified
MyC-IPS-1 was incubated for 1 h with HA-DHX9 or DHX9 truncations
proteins, individually, followed by one more hour of incubation after
adding anti-MyC beads. Bound proteins were pelleted by centrifugation,
and analyzed by immunoblotting with anti–HA-HRP Ab. To map region(s)
in IPS-1 required for interaction with DHX9, purified HA-DHX9 protein
was incubated with purified Myc-IPS-1 or Myc-DCARD protein for 1 h;
anti-HA beads were added and incubated for 1 h. Bound complexes were
pelleted by centrifugation, and analyzed by immunoblotting with anti–
Myc-HRP Abs.
RNA extract and quantitative PCR
Total RNA was isolated from cells using the RNeasy kit (Qiagen), and
treated with DNase (Qiagen). cDNA was synthesized using SuperScript II
(Invitrogen) and random hexamer primers. Quantitative PCR (qPCR)
amplification was performed using TaqMan Universal Master Mix (Applied
Biosystems) on the 7500 Fast Real-Time PCR system (Applied Biosystems). The following primers are used for qPCR: TNF-a, 59-CGTCAGCCGATTTGCTATCT-39 (forward) and 59-CGGACTCCGCAAAGT-
DHX9 senses both short poly I:C and long poly I:C in D2SC
mDCs
The DExD/H helicase family members RIG-I, MDA5, LGP2, and
DICER have been shown to play important roles in sensing dsRNA
and viral infection (4–7, 19). In pDCs, DHX9 is able to sense CpG
mediated by MyD88 (15). In addition, DHX9 regulates HIV-1
gene expression through its binding to TAR RNA, indicating
that DHX9 is also an RNA binding protein (17). We decided to
investigate the function of DHX9 in sensing dsRNA and RNA
virus. We therefore established stable D2SC cell lines expressing
shRNA to knockdown expression of DHX9, MyD88, IPS-1, RIGI, or MDA5. D2SC cells expressing a scrambled shRNA targeting
sequence served as the control. Knockdown efficiency of each cell
line was assessed by immunoblot (Fig. 1A). IFN-a/b, TNF-a, and
IL-6 production was measured after control and knockdown D2SC
cells were stimulated with a short poly I:C (0.2–1 kb). As shown
in Fig. 1B, control D2SC mDCs produced high levels of IFN-a/b,
TNF-a, and IL-6 following stimulation with short poly I:C. This
cytokine response was strongly attenuated in DHX9-, IPS-1–, and
RIG-I–knockdown D2SC mDCs and was not or only slightly affected in MyD88– and MDA5–knockdown D2SC mDCs, confirming the previous report showing that MDA5 only plays an
important role in sensing long poly I:C (4).
We next investigated whether DHX9 also plays a role in sensing
long poly I:C (1.5–8 kb) in D2SC cells. We found that knocking
down DHX9 expression resulted in 60% reduction in type I IFN,
TNF-a, and IL-6 production by D2SC cells in response to long
poly I:C (Fig. 1C). MDA5 and IPS-1 knockdown in D2SC cells
led to 80–90% reduction in type I IFN, TNF-a, and IL-6 production, whereas RIG-I and MyD88 knockdown had little effect
on IFN-a/b production by D2SC cells in response to long poly I:
C. These data suggest that DHX9 plays important roles in sensing
both short (0.2–1 kb) and long (1.5–8 kb) poly I:C in D2SC cells.
Our data also confirms previous studies showing that MDA5/IPS-1
preferentially senses long poly I:Cs and RIG-I/IPS-1 preferentially
senses short poly I:Cs in DCs (20).
To determine whether DHX9 senses other nucleic acids, cytokine production was measured after control and knockdown D2SC
cells were stimulated with B form DNA (poly dA-dT) or CpG. As
shown in Fig. 1D and 1E, DHX9 knockdown, as well as MyD88,
IPS-1, RIG-I, and MDA5 knockdown in D2SC mDCs, had little
effect on IFN-a/b production in response to poly dA-dT. Although
DHX9 or MyD88 knockdown led to 90% reduction in IFN-a/b
production in response to CpG, confirming the previous report
showing that DHX9 senses CpG mediated by MyD88 (15).
DHX9 senses RNA virus in D2SC mDCs
To determine the function of DHX9 in sensing viral infection,
cytokine production was measured after culturing control and
knockdown D2SC cells with reovirus. DHX9- and IPS-1–knockdown D2SC mDCs had 80% reduction in IFN-a/b production in
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shRNA lentiviral vectors were purchased from Open Biosystems: target for
RIG-I, clone TRCN0000103886; target for MDA5, clone V2LHS_202104;
target for IPS-1, clone TRCN0000124770; target for MyD88, clone
TRCN0000077236; and target for DHX9, clone TRCN and clone
TRCN0000071116 (DHX9-a) and TRCN0000071117 (DHX9-b).
Results
The Journal of Immunology
3
response to reovirus (Fig. 1F). Although both RIG-I– and MDA5knockdown D2SC mDCs had 50% reduction in IFN-a/b levels,
MyD88 knockdown in D2SC cells had little effect on IFN-a/b
production in response to reovirus. To further determine whether
DHX9 senses other RNA viruses, cyokine levels were measured
after culturing control and knockdown D2SC cells with Flu A
virus. As shown in Fig. 1G, DHX9 knockdown in D2SC mDCs led
to 50% reduction in IFN-a/b production in response to Flu A. In
contrast, RIG-I- and IPS-1-knockdown D2SC mDCs had almost
80% reduction in IFN-a/b production, whereas knockdown of
MyD88 had little effect on IFN-a/b production by D2SC cells in
response to Flu A. These data indicate that DHX9 plays an important role in mediating DC responses to RNA viruses. In addition, our study suggests that there are different and redundant
sensors for detecting different RNA species during viral infection.
DHX9 knockdown in bone marrow-derived DCs abolishes their
cytokine responses to poly I:C and RNA virus
To determine whether DHX9 senses dsRNA in primary cells, GMCSF-derived mDCs were prepared and treated with shRNA to
knockdown expression of DHX9, IPS-1, RIG-I, or MDA5. Fig. 2A
shows the specificity and efficiency of shRNA knockdown at the
protein level without affecting the expression of nontargeted
proteins in the bone marrow-derived DCs (BMDCs). Control and
knockdown BMDCs were examined for their IFN-a/b response
following stimulation with nucleic acid or virus. As shown in Fig.
2B, DHX9- and IPS-1–knockdown BMDCs produced much reduced IFN-a/b levels in response to short poly I:C, long poly I:C
or reovirus when compared with levels produced by the control
cells. Although RIG-I– and MDA5-knockdown BMDCs had 50%
reduction in IFN-a/b production in response to reovirus, the RIGI–knockdown BMDCs produced reduced levels of IFNa/b in response to short poly I:C and normal levels of IFNa/b in response
to long poly I:C. On the contrary, MDA5-knockdown BMDCs
produced normal levels of IFNa/b in response to short poly I:C
but much reduced levels of IFNa/b in response to long poly I:C.
These data indicate that DHX9 and IPS-1 play a critical role in
sensing both short and long poly I:C in BMDCs.
To determine the function of DHX9 in sensing viral infection
in BMDCs, cytokine production was measured after culturing
control and knockdown BMDCs with reovirus. DHX9- and IPS-1–
knockdown BMDCs had 85% reduction, whereas both RIG-I– and
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FIGURE 1. DHX9 senses poly I:C and RNA virus in mDCs. A, Immunoblot (IB) showing the knockdown efficiency of shRNAs targeting the indicated
proteins in D2SC cells, a mouse mDC cell line. Nontargeting shRNA served as a control (first left lane). Actin blots are shown as loading controls (lower
panel). ELISA of IFN-a/b, TNF-a, and IL-6 production from D2SC cells with the indicated shRNA after a 16-h stimulation with 2.5 mg/ml of short poly I:
C (B), 2.5 mg/ml long poly I:C (C), 1 mg/ml poly dA-dT (D), 2 mM CpG A (E), reovirus (F), or Flu A virus (G). Nucleic acids were delivered to the cells by
Lipofectamine 2000. Viruses were added to the cells at a multiplicity of infection (MOI) = 10 for 2 h. Cells were collected, washed with PBS, and cultured
for an additional 16 h. N-STM, scrambled shRNA-treated D2SC cells without stimulation.
4
A NEW IPS-1–DEPENDENT dsRNA SENSOR IN DCs
MDA5-knockdown BMDCs had 50% reduction in IFN-a/b production in response to reovirus (Fig. 2D). To determine whether
DHX9 plays an important role in antiviral defense, reovirus RNA
replication in BMDCs was measured by qPCR. The replication of
reovirus substantially increased in DHX9- and IPS-1–knockdown
BMDCs (Fig. 2E). These data indicate that DHX9 plays an important role in mediating BMDCs responses to reovirus.
DHX9 is also involved in basic cellular functions to selectively
regulate mRNA through binding of 59-UTRs. To further determine
whether the DHX9 directly affects the cytokine mRNA transcription or stability, the mRNA of TNF-a and the mRNA of IFNb in BMDCs treated with scrambled shRNA or shRNA targeting
DHX9 were measured. As shown in Fig. 2F, no change of TNF-a
mRNA or IFN-b mRNA was observed, excluding the possibility
of DHX9 directly regulating the cytokine production at the transcriptional or translational level.
DHX9 is a poly I:C-binding protein
To determine whether DHX9 directly binds nucleic acids, we
conducted pulldown assays in which a purified HA-tagged DHX9
or DHX36 recombinant protein was incubated with biotin-labeled
poly I:C, poly dA-dT, poly dG-dC, poly dI-dC, CpG A, or CpG B,
followed by pulldown with NA-beads. We found recombinant
DHX9 protein to associate with poly I:C RNA and CpG DNA,
suggesting that DHX9 binds dsRNA directly (Fig. 3A). To map the
dsRNA binding site of DHX9, we repeated pulldown assays using
biotin-labeled poly I:C and truncated versions of DHX9. Results
indicated that the DSRM, of DHX9 was required for poly I:C
binding (Fig. 3C). To confirm that DHX9 was specifically binding
to poly I:C, we performed competition experiments by adding
increasing amounts of unlabeled poly I:C, poly A:U, CpG, and
poly A to HA-DHX9 protein plus biotin-labeled poly I:C, followed by pulldown with NA beads. We found that only unlabeled
poly I:C could block the binding of biotin-labeled poly I:C to
DHX9 (Fig. 3D), supporting the idea that DHX9 directly associates with poly I:C.
Overexpression of DHX9 leads to enhanced IFN-b promoter
activity in response to poly I:C
To further confirm the role of DHX9 in sensing poly I:C, we
conducted transactivation assays to determine whether DHX9
expression was associated with Ifnb promoter activity in response
to poly I:C. MyD88, IPS-1, DHX9, and a DHX9 mutant lacking
the DSRM were overexpressed in HEK293T cells containing
a luciferase reporter controlled by the Ifnb promoter. As shown in
Fig. 4, we found that overexpression of DHX9 and IPS-1 led to
increased Ifnb promoter activation following stimulation with
short or long poly I:C, whereas the DHX9 mutant with deletion of
the DSRM failed to active the Ifnb promoter. Overexpression of
DHX9 and the DHX9 mutant with deletion of the DSRM had little
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FIGURE 2. DHX9 knockdown in BMDCs abolishes their cytokine responses to poly I:C and reovirus. A, Immunoblot (IB) showing the knockdown
efficiency of shRNA targeting the indicated genes in BMDCs. Nontargeting shRNA served as a control (first left lane). b-Actin blots are shown as loading
controls (lower panel). ELISA of IFN-a/b production by BMDCs with the indicated shRNA after 16 h stimulation with 2.5 mg/ml short poly I:C (B), 2.5
mg/ml of long poly I:C (C), or reovirus (D) at a multiplicity of infection (MOI) = 10. Nucleic acids were delivered to the cells by Lipofectamine 2000. E,
qPCR of reovirus copies from BMDCs with shRNA knockdown of indicated protein. BMDCs were incubated with reovirus at MOI of 10 for 2 h. Cells were
collected, washed with PBS, and cultured for an additional 16 h, followed by RNA extract and RT-PCR. F, qPCR of TNF-a and IFN-b mRNA extract from
BMDCs treated with scrambled shRNA (control) or shRNA targeting DHX9. N-STM, scrambled shRNA-treated D2SC cells without stimulation.
The Journal of Immunology
5
effect on the activation of the Ifnb promoter without poly I:C
stimulation, indicating that this activation by DHX9 is poly I:C
dependent. Overexpression of IPS-1 led to increased Ifnb promoter activation without poly I:C stimulation, confirming the
previous report that the transfection of IPS-1 expression plasmid
actives the Ifnb promoter (21). Overexpression of MyD88 had
little effect on the activation of the Ifnb promoter with or without
poly I:C stimulation. These data indicate that the dsRNA-binding
activity of DHX9 is necessary for eliciting the IFN response to
poly I:C.
DHX9 interacts with IPS-1 in D2SC mDCs
Because DCs with knockdown of DHX9 or IPS-1 displayed similar
cytokine responses to short and long poly I:C, we hypothesize that
DHX9 may use IPS-1 as an adaptor molecule for signal transduction. We next investigated the potential interaction between
DHX9 and IPS-1 in mDCs. We performed anti-DHX9 immunoprecipitation experiments in resting or poly I:C-activated D2SC
mDCs. As shown in Fig. 5A, endogenous DHX9 associates with
IPS-1 in both resting and poly I:C-stimulated D2SC mDCs. By
contrast, association between DHX9 and RIG-I was undetectable.
To map the regions of DHX9 and IPS-1 that mediate their interaction, we incubated a recombinant MyC-IPS-1 protein with
FIGURE 4. Overexpression of DHX9 leads to
enhanced Ifnb promoter activity in response to
poly I:C. Fold activation of the Ifnb promoter
in HEK293T cells transfected with IFN-b luciferase reporter (100 ng) plus MyD88, IPS-1,
DHX9, or DHX9-deleted DSRM expression
vectors at 20, 100, or 200 ng concentrations. The
Renilla-luciferase reporter gene (2 ng) was
transfected simultaneously for the internal control. At 30 h posttransfection, cells were without
stimulation or stimulated for 6 h with 2.5 mg/ml
poly I:C delivered by Lipofectamine 2000 and
then harvested.
either a purified full-length or truncated HA-DHX9 protein, followed by pulldown with anti-MyC beads. As indicated in Fig. 5B,
the helicase C-terminal domain (HelicC), HA2, and DUF domains
of DHX9 were required for DHX9 to interact with IPS-1. Likewise, we conducted reciprocal experiments using truncated versions of IPS-1 and full-length DHX9 to identify the IPS-1 binding
site(s) to DHX9. We found that the CARD domain of IPS-1 was
required for IPS-1 interaction with DHX9 (Fig. 5D).
As DHX9 or RIG-I knockdown displayed similar effect on the
cytokine responses to short poly I:C, to further determine whether
the DHX9 signaling is independent on RIG-I signaling, we perform
experiments using RIG-I deficiency D2SC cell line to knockdown
DHX9. The residual response to short poly I:C in RIG-I deficiency
D2SC is existing. This residual response was decreased dramatically after knockdown DHX9 (Fig. 5E), suggesting that DHX9 is
independent on RIG-I to sense poly I:C.
DHX9 and IPS-1 are required for activating the NF-kB and
IFN-response signal transduction pathways upon poly I:C
stimulation
It was previously shown that over-expression of IPS-1 in
HEK293T cells resulted in robust activation of IRF3 and NF-kB
(22). We investigated whether downstream signaling of DHX9 in
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FIGURE 3. DHX9 requires DSRM domain to bind poly I:C. A, Immunoblot using anti-HA Ab of pulldown assays in which purified HA-DHX9 or HADHX36 recombinant proteins were incubated with biotinylated poly I:C, poly dA-dT, poly dG-dC, poly dI-dC, CpG A, or CpG B, followed by addition of
NA beads. B, Schematic representations of full-length and serial truncations of DHX9. HA-DHX9, DHX9 full size; HA-9DDSRM, DHX9-deleted DSRM;
HA-9DDEXDc, DHX9-deleted DSRM and DEXDc; HA-9DHelicC, DHX9-deleted DSRM, DEADc and HelicC; HA-9CDDUF, DHX9-deleted DUF; HA9CDHD, DHX9-deleted HA2 and DUF. Numbers denote amino acid residues. C, Immunoblot using anti-HA Ab of pulldown assays in which purified HAtagged serial truncations of DHX9 were individually incubated with biotinylated poly I:C, followed by addition of NA beads. D, Immunoblot using anti-HA
Ab of pulldown competition assays in which 0.5, 5, or 50 mg/ml CpG B, poly A, poly A:U, poly I:C, or CpG A was added to a mixture of HA-DHX9 protein
plus biotinylated poly I:C, followed by addition of NA beads. DEAD, Asp-Glu-Ala-Asp box motif.
6
A NEW IPS-1–DEPENDENT dsRNA SENSOR IN DCs
D2SC mDCs induced by poly I:C involves IRF3 and NF-kB activation. Scrambled shRNA (control), DHX9- and IPS-1–knockdown D2SC cells were stimulated with poly I:C, total cell extracts
were prepared and analyzed by SDS-PAGE, followed by Western
blotting (Fig. 6). In scrambled shRNA D2SC cells, the phosphorylation of p65 and IRF3 was detected at 60–120 min after
poly I:C stimulation. In contrast, the phosphorylation of p65 and
IRF3 was barely detectable in DHX9- or IPS-1–knockdown D2SC
cells at these time points. These data suggest that DHX9 and IPS-1
are required for activating the NF-kB and IFN-response signal
transduction pathways upon poly I:C stimulation.
Discussion
Over the past decade, many molecular sensors for viral or selfnucleic acids that trigger type 1 IFN or inflammatory cytokine
responses have been identified. PKR, 29,59-oligoadenylate synthetase, TLR3, DDX3, and the RLH family members RIG-I and
MDA5 have been shown to recognize poly I:C or dsRNA with
different subcellular localizations, lengths or structure (4–9, 23–
FIGURE 6. DHX9 and IPS-1 are required for activating the NF-kB and
IFN-response signal transduction pathways upon dsRNA stimulation.
Immunoblot of indicated proteins from lysates of D2SC with indicated
shRNA after stimulation with 2.5 mg/ml poly I:C delivered by Lipofectamine 2000. b-Actin served as loading control.
26). Although MDA5 was found to preferentially sense long form
of poly I:C (.1000 bp), RIG-I was found to preferentially sense
short form of poly I:C (.300 and ,1000 bp) or dsRNA with 59triphosphate (4). Interestingly, RIG-I was also shown to sense
cytosolic B form DNA (poly dA-dT) through RNA polymerase III
(Pol III) to trigger IRF-3–dependent type 1 IFN responses (27,
28). A number of cytosolic DNA sensors, such as Pol III, AIM2,
and IFI16, have recently been identified. AIM2 was shown to
sense cytosolic DNA by activating inflammasome responses via
the adaptor molecule apoptosis-associated-speck-like protein containing a caspase recruitment domain (29–35). More recently, it
was reported that IFI16, an AIM2-like molecule, uses STING for
sensing cytosolic DNA (36). By direct biochemical approaches
using biotin-labeled CpG-DNA or poly I:C, immunoprecipitation
assays and mass spectrometry, we recently identified novel nucleic
acid sensors within the (DExD/H)-box helicase superfamily in
both pDC (15) and mDCs (37). We found that DHX9 and DHX36
were able to sense cytosolic CpG-DNA via MyD88 in pDCs and
a DDX1/DDX21/DHX36 complex was able to sense both short
and long poly I:C via TRIF in mDCs. Our bioinformatics analysis
revealed the presence of at least 60 members of the (DExD/H)box helicase superfamily, including the RLR subfamily (RIG-I,
MDA5, and LGP-2). Our hypothesis is that members of the
DExD/H-box helicase superfamily may play a more important
role in sensing microbial nucleic acids than previously thought.
It was reported that DHX9 regulates translation by recognizing
the posttranscriptional control element of a retrovirus RNA via its
ATPase activity (12, 13). Overexpression of DHX9 leads to increasing expression of p65 and TNF-a genes. In addition, DHX9
is a CpG-DNA sensor in pDCs (15). DHX9 has two dsRNAbinding motifs in the N terminus. Those motifs bind the TAR
element of HIV-1 to active PKR and regulate HIV-1 gene expression (17). Therefore, it is very interesting to investigate
whether DHX9 is able to sense cellular dsRNA or RNA viruses.
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FIGURE 5. HelicC-HA2-DUF domain of
DHX9 and CARD domain of IPS-1 required
for DHX9:IPS-1 interaction. A, Immunoblot
(IB) of indicated proteins precipitated with
anti-DHX9 Ab from whole-cell lysates of
unstimulated (2), short poly I:C-, or long
poly I:C-stimulated (+) D2SC cells. IgG Ab
served as a control; input, D2SC lysates used
in immunoprecipitation assays; B, IB using
anti-HA Ab of pulldown assays in which purified MyC-IPS-1 was incubated with fulllength or truncated HA-DHX9 protein, followed by addition of anti-MyC beads; C,
schematic representations of full-length and
serial truncations of IPS-1. MyC-IPS-1: IPS-1
full size; Myc-DCARD: IPS-1–deleted
CARD. D, IB using anti-Myc Ab of pulldown
assays in which purified HA-DHX9 was incubated with Myc-IPS-1 or Myc-DCARD
protein, followed by addition of anti-HA
beads. E, IB showing the knockdown efficiency of shRNA targeting DHX9 in RIG-I
deficiency D2SC. Non-targeting shRNA served
as a control (first left lane). b-Actin blots
are shown as loading controls (lower panel).
ELISA of IFN-a production by D2SC after
16 h stimulation with 2.5 mg/ml short poly
I:C. CARD, N-terminal caspase recruitment
domain; N-STM, scrambled shRNA-treated
RIG-I deficiency D2SC cells without stimulation.
The Journal of Immunology
Acknowledgments
We thank M. Wentz for critical reading, and we thank all colleagues from
the laboratory at the University of Texas MD Anderson Cancer Center.
Disclosures
The authors have no financial conflicts of interest.
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In this study, we found that the ability of DHX9 in sensing both
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DSRM domain of DHX9 was required for DHX9 binding to poly I:
C, and the HelicC, HA2, and DUF domains of DHX9 and the
CARD domain of IPS-1 were required for DHX9:IPS-1 interaction.
We have demonstrated that the observed association between
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immunoprecipitation data indicate that the DHX9–IPS-1 complex
exists in resting cells and the complex formation does not require
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may have forced the innate immune system to develop different
and redundant sensors for detecting different RNA species derived
from Flu A virus during viral infection. Similar antiviral sensors
may exist for detecting other viral infections as well.
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