MIP-T3 Is a Negative Regulator of Innate
Type I IFN Response
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J Immunol 2011; 187:6473-6482; Prepublished online 11
November 2011;
doi: 10.4049/jimmunol.1100719
http://www.jimmunol.org/content/187/12/6473
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References
Ming-Him James Ng, Ting-Hin Ho, Kin-Hang Kok,
Kam-Leung Siu, Jun Li and Dong-Yan Jin
The Journal of Immunology
MIP-T3 Is a Negative Regulator of Innate Type I IFN
Response
Ming-Him James Ng,* Ting-Hin Ho,* Kin-Hang Kok,* Kam-Leung Siu,* Jun Li,† and
Dong-Yan Jin*
M
icrobial infections are the most common cause of death
in humans. While long-term protection from pathogens
can be achieved by adaptive immune response, pathogens are sensed by pathogen recognition receptors (PRRs) during the early phase of infection. Each PRR recognizes relatively
conserved structural motifs that are commonly found in a subgroup of pathogens (1). Activation of PRRs in turn triggers the
production of cytokines including IFNs, which constitute innate
immunity and facilitate the activation of adaptive response. TNF
receptor-associated factor (TRAF) 3 was initially found to be associated with the cytoplasmic tails of CD40 (2, 3), but recent
studies revealed its critical roles in type I IFN production (4, 5).
By interacting with various partners, TRAF3 adapts activation
signals from upstream PRRs, including both TLRs and RIG-I–
like receptors to downstream kinases TBK1 and IKKε that phosphorylate transcription factors IFN regulatory factor (IRF) 3 and
*Department of Biochemistry, Li Ka Shing Faculty of Medicine, The University of
Hong Kong, Pokfulam, Hong Kong, China; and †Department of Biochemistry,
Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou, China
Received for publication March 11, 2011. Accepted for publication October 19,
2011.
This work was supported by the Hong Kong Research Grants Council-National
Natural Science Foundation of China Joint Research Scheme (Grant N-HKU 720/
08), the Association for International Cancer Research (Grant 07-0424), and the
Hong Kong Research Grants Council Collaborative Research Fund (HKU 7/CRF09).
Address correspondence and reprint request to Dr. Dong-Yan Jin, Department of
Biochemistry, The University of Hong Kong, 3/F Laboratory Block, Faculty of
Medicine Building, 21 Sassoon Road, Pokfulam, Hong Kong. E-mail address:
[email protected]
Abbreviations used in this article: GST, glutathione S transferase; HA-Ub, hemagglutinin-ubiquitin; IRF, IFN regulatory factor; ISRE, IFN-stimulated response element; MEF, mouse embryonic fibroblast; poly-IC, polyinosinic-polycytidylic acid;
PRR, pathogen recognition receptor; qPCR, quantitative PCR; RNAi, RNA interference; siRNA, small interfering RNA; TRAF, TNFR-associated factor; VSV, vesicular
stomatitis virus.
Copyright Ó 2011 by The American Association of Immunologists, Inc. 0022-1767/11/$16.00
www.jimmunol.org/cgi/doi/10.4049/jimmunol.1100719
IRF7 (6, 7). TRAF3 acts as a ubiquitin ligase for itself and other
substrates (6–8). In response to different upstream signals, it
also undergoes K48- and K63-linked ubiquitination that is modulated by other ubiquitin ligases and deubiquitinases (8–12). In
line with its positive regulatory role in antiviral response, TRAF3
signaling complexes are targeted by various viral proteins as
countermeasures to inhibit IFN production (13–15); however, it is
not fully understood how the adaptor function of TRAF3 in innate
IFN response is achieved and regulated in the cell.
One way to derive insight into the roles and regulation of TRAF3
is to identify and characterize its interacting partners. As an
adaptor, TRAF3 is known to interact with both upstream inducers and downstream effectors, including MYD88, TRIF, RIG-I,
MDA5, TANK, TBK1, IKKε, and IRF3 (6, 7, 15). Other cellular
and viral interacting partners of TRAF3 have also been identified (16). Among cellular TRAF3-binding proteins, MIP-T3 is
one of only a few proteins that specifically interact with TRAF3
but not other TRAF proteins (17). MIP-T3 interacts constitutively
with TRAF3 and microtubules through a C-terminal coiled-coil
region and a central microtubule interacting domain, respectively
(17). MIP-T3 has been found to localize to the cytoplasm, the
primary cilium, and the centrosome (17–21). Although MIP-T3
is a component of the intraflagellar transport subcomplex B that
plays an important role in primary cilium formation (18–22),
nonciliary functions of MIP-T3, exemplified by the recruitment of
schizophrenia susceptibility factor DISC1 to the centrosome and
an inhibitory role in IL-13 signaling, have also been implicated
(23–25). The biologic relevance of the interaction between
TRAF3 and MIP-T3 remains elusive. Particularly, it is unclear
whether MIP-T3 might modulate type I IFN production through
its direct interaction with TRAF3. We therefore investigated the
influence of MIP-T3 expression on TRAF3-mediated activation
of IFN-b promoter activity. We demonstrated MIP-T3 to be
a negative regulator of type I IFN-dependent innate immune
response. We further suggested a mechanism through which
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TNFR-associated factor (TRAF) 3 is an important adaptor that transmits upstream activation signals to protein kinases that
phosphorylate transcription factors to induce the production of type I IFNs, the important effectors in innate antiviral
immune response. MIP-T3 interacts specifically with TRAF3, but its function in innate IFN response remains unclear. In this
study, we demonstrated a negative regulatory role of MIP-T3 in type I IFN production. Overexpression of MIP-T3 inhibited
RIG-I-, MDA5-, VISA-, TBK1-, and IKK«-induced transcriptional activity mediated by IFN-stimulated response elements and
IFN-b promoter. MIP-T3 interacted with TRAF3 and perturbed in a dose-dependent manner the formation of functional
complexes of TRAF3 with VISA, TBK1, IKK«, and IFN regulatory factor 3. Consistent with this finding, retinoic acidinducible gene I- and TBK1-induced phosphorylation of IFN regulatory factor 3 was significantly diminished when MIP-T3
was overexpressed. Depletion of MIP-T3 facilitated Sendai virus-induced activation of IFN production and attenuated the
replication of vesicular stomatitis virus. In addition, MIP-T3 was found to be dissociated from TRAF3 during the course of
Sendai virus infection. Our findings suggest that MIP-T3 functions as a negative regulator of innate IFN response by preventing TRAF3 from forming protein complexes with critical downstream transducers and effectors. The Journal of Immunology,
2011, 187: 6473–6482.
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MIP-T3 binds to TRAF3 to impede its formation of functional
complexes with downstream transducers and effectors including
VISA, TBK1, IKKε, and IRF3. Finally, the function of MIP-T3
MIP-T3 INHIBITS TYPE I IFN PRODUCTION
was characterized in the context of viral infection. Our findings
might have implications in viral pathogenesis and the design of
new antivirals.
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FIGURE 1. MIP-T3 inhibits RIG-I/MDA5- and TBK1/IKKε-induced activation of ISRE and IFN-b promoter activity. Constant amounts of ISRE-Luc
(A) or IFN-b–Luc (B) reporter plasmid were cotransfected with a fixed amount of activator plasmid and increasing amounts of Flag–MIP-T3 plasmid into
HEK293 cells. The total amount of plasmids transfected was normalized with corresponding empty vectors. pRL-CMV was used as an internal control for
transfection efficiency. Expression of MIP-T3 and other stimulatory proteins was verified by Western blotting (see inset in A for an example of Western blot
analysis of MIP-T3 expression. *MIP-T3; #1, p = 0.010598 by Student t test; #2, p = 0.01745 by Student t test; #3, p = 0.018214 by Student t test; #4, p =
0.000028 by Student t test. C, Effect of MIP-T3 expression on activation of human T cell leukemia virus I long terminal repeats by viral trans-activator Tax.
D, Effect of MIP-T3 on tunicamycin-induced activation of GRP78 promoter. Tunicamycin was added 12 h prior to cell harvest. E, Effect of MIP-T3 on
RIG-I–induced activation of NF-kB. F, Effect of MIP-T3 on poly-IC–induced activation of TLR3. HEK293 cells were transfected with ISRE-Luc, TLR3
expression plasmid, and increasing amounts of expression plasmid for NS1-V5His or Flag-MIP-T3. Forty-eight hours after transfection, cells were
stimulated with 1 mg/ml poly-IC for another 8 h. No ISRE-Luc activity was detected in the absence of TLR3 (data not shown). Results are representative of
three independent experiments. Error bars indicate SD.
The Journal of Immunology
Materials and Methods
Cell culture and reagents
HEK293, HEK293T, and Vero cells were obtained from the American Type
Culture Collection. Primary mouse embryonic fibroblasts (MEFs) were
generated from E13 embryos of wild-type mouse in C57BL/6J background. Cells were maintained in DMEM with 10% FBS (Life Technologies) and antibiotics. Cells were grown at 37˚C in a humidified
atmosphere with 5% CO2. Transfection of plasmid DNA was performed
using Genejuice (Novagen) as described previously (15, 26). Tunicamycin
was purchased from Calbiochem. Recombinant IkB was bought from
Santa Cruz. Recombinant IRF3 fused to glutathione S transferase (GST)
was obtained from Abnova. Sendai virus (Cantell strain) was obtained
from the American Type Culture Collection. Recombinant vesicular
stomatitis virus expressing GFP (vesicular stomatitis virus [VSV]-GFP)
was a gift from Dr. Jacques Perrault (San Diego State University, San
Diego, CA) and Dr. Brian Lichty (McMaster University, ON, Canada)
(27, 28). Viral infection of HEK293 cells and MEFs was performed as
described (29).
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Immunoprecipitation, Western blotting, reporter assay, and
protein kinase assay
Coimmunoprecipitation, Western blotting, dual luciferase assay, and protein
kinase assay were performed as described previously (15, 39). Mouse
monoclonal anti-Flag Ab M2 (F3165) and rabbit polyclonal anti-Flag Abs
(F7425) were purchased from Sigma. Mouse monoclonal anti-V5 Ab (460705) was purchased from Invitrogen. Mouse monoclonal anti-Myc Ab
(sc-40), rabbit polyclonal anti-IRF3 (sc-9082) and anti–MIP-T3 (C20) Abs
were purchased from Santa Cruz Biotechnology. Rabbit monoclonal anti–
phospho-IRF3 (S396) Ab 4D4G (4741) was obtained from Cell Signaling.
For TRAF3 ubiquitination assay, 0.1% SDS was added to the immunoprecipitation buffer and wash buffer.
Primers, small interfering RNAs, and quantitative PCR
FIGURE 2. MIP-T3 interacts with TRAF3, but not with RIG-I, TANK,
TBK1, or IKKε. The indicated proteins were expressed in HEK293T cells.
Western blotting (WB) was performed to probe MIP-T3 in the cell lysate with anti-V5 (a-V5). Immunoprecipitation (IP) was performed with
a-Flag, and the precipitates were probed with a-Flag and a-V5, respectively.
FIGURE 3. Influence of MIP-T3 on TRAF3 polyubiquitination. The
indicated combinations of HA-Ub, Flag-TRAF3, and V5–MIP-T3 were
coexpressed in HEK293T cells. Cell lysates were probed with anti-Flag
(a-Flag) and a-V5 by Western blotting (WB). Immunoprecipitation (IP)
was performed with a-Flag. HA-Ub conjugated Flag-TRAF3 was revealed
by probing immunoprecipitate with a-Flag and a-HA. Lanes 3, 4, 7, and 8
served as negative controls.
Plasmids
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cDNA clones for MIP-T3, VISA, TANK, IKKε were purchased from
imaGenes (Berlin, Germany). Flag-tagged MIP-T3 was created by cloning
MIP-T3 cDNA into pCMV-Tag2B (Stratagenes) using PCR amplification.
It was further subcloned into pcDNA-3.1+ (Invitrogen) with an N-terminal
His-V5 tag. TRAF3 was provided by Dr. Liusheng He and Dr. Peter Lipsky
(National Institute of Arthritis and Musculoskeletal and Skin Diseases,
Bethesda, MD) (30). pIFNb-Luc (p125-Luc) and Flag-RIG-I plasmids
were provided by Dr. Takashi Fujita (Kyoto University, Kyoto, Japan) (31).
TBK1 and IRF3 expression plasmids were gifts from Dr. Genhong Cheng
(University of California, Los Angeles, Los Angeles, CA) (32, 33). pISRELuc and pkB-Luc reporter constructs were purchased from Clontech.
Expression plasmid for human T cell leukemia virus type I oncoprotein
Tax and reporter construct LTR-Luc were described previously (34, 35).
GRP78-Luc was provided by Dr. Kazutoshi Mori (HSP Research Institute, Kyoto Research Park, Kyoto, Japan) (36). Expression vector for
hemagglutinin-ubiquitin (HA-Ub) was a gift from Dr. Dirk Bohmann
(University of Rochester, Rochester, NY) (37). Expression plasmids for
TLR3 and influenza A virus NS1 were described previously (38).
Sequences of human-specific MIP-T3 primers are 59-TCACACAATT
CTGACAATGA AG-39 (MT3-497F) and 59-TCACTTTCTC GAAGTCAAAT TG-39 (MT3-691R). Primers for amplification of GAPDH and
procedures for semiquantitative RT-PCR have been described elsewhere
(40, 41).
Quantitative real-time PCR was performed using Power SYBR Green
Master Mix (Applied Biosystems). Quantitative PCR (qPCR) primers for
mouse MIP-T3 transcript are 59-TGGAGGCTGA CATTTCTGTG-39
(forward) and 59-ATCACCTCCG GCTTATCTTG-39 (reverse). Primers for
mouse IFN-b are 59-CAGCTCCAAG AAAGGACGAA C-39 (forward)
and 59-GGCAGTGTAA CTCTTCTGCA T-39 (reverse). Primers for mouse
GADPH are 59-AGGTCGGTGT GAACGGATTT G-39 (forward) and 59TGTAGACCAT GTAGTTGAGG TCA-39 (reverse). QPCR reaction was
performed with a StepOne Real-Time PCR System (Applied Biosystems).
Target mRNA expression was quantitated with the comparative Ct method.
Relative expression level of target mRNA was calculated from 22DCt.
Sequences for siGFP are 59-GCAAGCUGAC CCUGAAGUUC at39 (sense) and 59-GAACUUCAGG GUCAGCUUGC CG-39 (antisense).
Sequences for si3A and si3B against human MIP-T3 are 59-GAGGAGACGC UGAAAUAAAt t-39 (sense for si3A), 59-UUUAUUUCAG
CGUCUCCUCt t-39 (antisense for si3A), 59-GCAUGAGCCU GAAAGAACAt t-39 (sense for si3B), and 59-UGUUCUUUCA GGCUCAUGCc
c-39 (antisense for si3B). Sequences for si3C and si3D against mouse MIPT3 are 59-GAUGGAGGCU GACAUUUCUt t-39 (sense for si3C), 59-
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AGAAAUGUCA GCCUCCAUCt t-39 (antisense for si3C), 59-GUGCAGAGCA CAAACAGAAt t-39 (sense for si3D), and 59-UUCUGUUUGU
GCUCUGCACt t-39 (antisense for si3D). Transfection of small interfering
RNA (siRNA) into HEK293 cells was performed as described (40) using
Lipofectamine 2000 according to the manufacturer’s protocol. Transfection of siRNA into MEF cells was performed using X-treme-Gene siRNA
transfection reagents (Roche).
ELISA
ELISA was performed using a mouse IFN-b ELISA kit (PBL Biomedical
Laboratories) and the manufacturer’s protocol.
Plaque formation assay
Vero cells were seeded in 12-well plates. When the cells grew to 100%
confluence, they were infected with serial 10-fold dilutions of the virus in
DMEM culture medium for with routine rocking. One hour postinfection,
infection medium was removed and infected Vero cells were then overlaid
with 1:1 DMEM culture medium containing 1% agarose. At 30 h postinfection, cells were fixed with 3.7% paraformaldehyde and stained with
0.05% crystal violet.
Results
To test whether and how expression of MIP-T3 might affect type I
IFN production, we used luciferase reporter constructs that are
driven by canonical IFN-stimulated response elements (ISRE) or
IFN-b promoter. These ISREs are known to be recognized by
IRF3 (42, 43). As expected, luciferase activity from both ISRELuc (Fig. 1A) and IFNb-Luc (Fig. 1B) reporter constructs was
remarkably stimulated by RIG-I, MDA5, VISA, TBK1, IKKε, and
IRF3, which are key activators of type I IFN production (44).
When MIP-T3 was expressed, a significant and dose-dependent
suppression of ISRE-Luc (Fig. 1A) and IFN-b–Luc (Fig. 1B) reporter activity induced by RIG-I, MDA5, VISA, TBK1, IKKε, or
IRF3 was observed in HEK293 cells. For example, when the dose
of MIP-T3 plasmid introduced into cells increased from 50 to
150 ng (see inset in Fig. 1B for a representative Western blot of
MIP-T3), a more than 2-fold reduction of IKKε-induced ISRE-b and
IFN-b promoter-driven luciferase reporter expression was seen
(Fig. 1A, 1B, lower third panel). In the control experiments, MIPT3 did not affect the basal transcriptional activity of ISRE and
IFN-b promoter (Fig. 1A, 1B, upper left panel).
In addition, to exclude the possibility of a general inhibition
effect induced by overexpression of MIP-T3, we showed that MIPT3 did not inhibit Tax-induced activation of a reporter driven by
human T cell leukemia virus type I long terminal repeats (Fig. 1C).
Tax is a retroviral transactivator that activates transcription from
viral and cellular promoters (34, 45, 46). In addition, MIP-T3 had
no effect on tunicamycin-induced activation of GRP78 promoter
(Fig. 1D). GRP78 resided in endoplasmic reticulum is a protein
chaperone activated by endoplasmic reticulum stressors such as
tunicamycin that inhibits glycosylation (36, 47). Thus, MIP-T3
FIGURE 4. MIP-T3 blocks TRAF3 complex formation. The indicated proteins were coexpressed in HEK293T cells. Immunoprecipitation (IP) and
Western blotting (WB) were performed with the indicated Abs. The formation of protein complexes TRAF3–TBK1 (A), TRAF3–IKKε (B), TRAF3–IRF3
(C), TRAF3–VISA (D), TRAF3–TANK (E), and TBK1–IRF3 (F) was examined.
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MIP-T3 inhibits RIG-I/MDA5- and TBK1/IKK«-induced
activation of IFN-b promoter
MIP-T3 INHIBITS TYPE I IFN PRODUCTION
The Journal of Immunology
is not a general repressor of cellular transcription or translation.
Because both IRF3 and NF-kB are activated in innate IFN response (31, 48, 49), we asked whether MIP-T3 might also exert an
inhibitory effect on NF-kB signaling. We found that MIP-T3 had
no influence on RIG-I–induced NF-kB activation (Fig. 1E), suggesting that MIP-T3 acts specifically on IRF3, but not on NF-kB.
However, a strong inhibitory effect of MIP-T3 was observed
when we activated TLR3 with polyinosinic-polycytidylic acid
(poly-IC) to boost ISRE activity (Fig. 1F). The inhibition by
MIP-T3 was even more dramatic than that mediated by influenza
A virus NS1 protein, a known viral antagonist of IFNs (38). Our
results indicate that MIP-T3 specifically suppresses type I IFN
production induced by various activators including RIG-I, MDA5,
VISA, TLR3, TBK1, IKKε, and IRF3.
Interaction of MIP-T3 with TRAF3 and influence of MIP-T3 on
TRAF3 ubiquitination
FIGURE 5. MIP-T3 inhibits TRAF3–IKKε complex formation in a dosedependent manner. Progressively increasing amounts of MIP-T3 plasmid
were cotransfected with TRAF3 and IKKε constructs into HEK293T cells.
Immunoprecipitation (IP) and Western blotting (WB) experiments were
performed as in Fig. 4B.
MIP-T3 prevents TRAF3 from complex formation with VISA,
TBK1, IKK«, and IRF3
We demonstrated that MIP-T3 could mitigate type I IFN production
(Fig. 1), but was unable to counteract TRAF3 ubiquitination (Fig.
3). However, MIP-T3 specifically interacts with TRAF3, but not
with other downstream transducers or effectors in IFN production (Fig. 2), which are normally associated with TRAF3 (16).
These TRAF3-associated transducers and effectors include RIG-I,
MDA5, VISA (50–53), TBK1, IKKε, IRF3 (4, 5), and TANK (33).
The formation of protein complexes comprising TRAF3 and these
downstream proteins is essential for the activation of IFN production (4, 5). Because MIP-T3 was not in the complexes that
contain RIG-I, TANK, TBK1, and IKKε (Fig. 2), we asked whether MIP-T3 and these downstream proteins might be mutually
exclusive in forming a complex with TRAF3. To address this
question, we extensively examined the formation of TRAF3 complexes with different downstream transducers and effectors in
the presence and in the absence of MIP-T3.
We found that MIP-T3 inhibited the formation of TRAF3–
VISA, TRAF3–TBK1, TRAF3–IKKε, and TRAF3–IRF3 complexes
(Fig.4, A–D); neither did MIP-T3 have any influence on the interaction between RIG-I and TRAF3 or between MDA5 and TRAF3
(data not shown). Particularly, when V5–MIP-T3 was expressed,
FIGURE 6. MIP-T3 inhibits RIG-I–induced and TBK1-induced IRF3
phosphorylation in cultured cells without affecting TBK1 kinase activity.
A, IRF3 phosphorylation in cultured cells. Indicated proteins were expressed in HEK293T cells. Phosphorylated IRF3 (pIRF3) was detected
using phospho-IRF3 Ab (Ser-396). Total IRF3 was also probed. B, TBK1
kinase assay in vitro. Indicated proteins were expressed in HEK293T cells.
TBK1 immunoprecipitated from these transfected cells was incubated with
recombinant IkBa or GST-IRF3 and [32P]-labeled ATP for 30 min at 37˚C
in vitro (lanes 1–3). Phosphorylated IkBa and GST-IRF3 were resolved
by SDS-PAGE. Vacuum-dried SDS-PAGE gel was exposed to x-ray film
for detection of radioactivity. Lane 4 contains a-Flag immunoprecipitate
prepared from HEK293T cells transfected with an empty vector.
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Above we showed the inhibition of RIG-I, MDA5, TBK1, and
IKKε activity by MIP-T3 (Fig. 1). To investigate the cause, we
asked whether MIP-T3 might physically associate with these
activators in addition to TRAF3. We coexpressed MIP-T3 and
RIG-I, TANK, TRAF3, TBK1, and IKKε in HEK293T cells and
performed coimmunoprecipitation assay. The target proteins were
efficiently expressed and successfully precipitated (Fig. 2). MIP-T3
was found only in the TRAF3-containing precipitate (Fig. 2,
lane 3), but not in all other precipitates. Thus, MIP-T3 binds specifically to TRAF3, but not to RIG-I, TANK, TBK1, or IKKε. In
other words, the suppressive effect of MIP-T3 on RIG-I, TBK1, or
IKKε was unlikely due to direct interaction.
TRAF3 is a ubiquitin ligase, and several negative regulators of
TRAF3 have been shown to modulate K63- or K48-linked polyubiquitination of TRAF3 (8, 10, 12). In view of this, we next
investigated whether the interaction of MIP-T3 with TRAF3 might
also influence TRAF3 ubiquitination. We coexpressed HA-tagged
ubiquitin and Flag-tagged TRAF3 in HEK293T cells and then
probed the anti-Flag immunoprecipitate with anti-Flag and antiHA (Fig. 3). The ladder of ubiquitinated TRAF3 proteins in the
precipitate was evident (Fig. 3, lane 5). However, the ubiquitination signal of TRAF3 was only slightly reduced upon expression of MIP-T3 (Fig. 3, lane 6). Therefore, MIP-T3 did not
appear to affect TRAF3 ubiquitination dramatically.
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MIP-T3 INHIBITS TYPE I IFN PRODUCTION
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FIGURE 7. siRNA-mediated depletion of MIP-T3 potentiated viral activation of IFN-b promoter. A, Two independent MIP-T3–targeting siRNAs (si3A
and si3B) at a concentration of 50 nM were used to deplete endogenous MIP-T3 in HEK293 cells. siGFP was used as a negative control. At the 24th hour
after knockdown, reporter plasmids p125-Luc and pRL-CMV were cotransfected into the cells. Twenty-four hours later, transfected cells were infected with
Sendai virus (80 hemagglutinating U/ml). Cells were harvested 0 and 24 h postinfection. Results are representative of three independent experiments. Error
bars represent SD. *p = 0.024 by Student t test, #p = 0.041 by Student t test. h.p.i., hours postinfection. B and C, Verification of MIP-T3 knockdown by siRNAs.
RT-PCR was performed to amplify MIP-T3 and GAPDH transcripts. Western blot analysis was performed using anti–MIP-T3 and anti–b-tubulin Abs. D and E,
RNAi depletion of MIP-T3 in primary MEFs augmented the production of IFN-b transcript. MIP-T3 knockdown in MEF cells before and after Sendai virus
infection was verified. MEFs were transfected with siGFP and siRNAs targeting mouse MIP-T3 (si3C and si3D) 48 h before Sendai virus infection. Expression
level of MIP-T3 mRNA was normalized with that of GAPDH transcript. Induction of IFN-b transcript in MEFs infected with Sendai virus was also analyzed.
Error bars represent SD. *p = 0.028 by Student t test, #p = 0.015 by Student t test. F, Depletion of MIP-T3 augmented IFN-b production. Infection
and transfection of MEFs were performed as in D and E. Production of IFN-b was analyzed by ELISA. Error bars denote SD. (Figure legend continues)
The Journal of Immunology
MIP-T3 inhibits phosphorylation of IRF3 in cultured cells
without affecting TBK1 kinase activity in vitro
Above we showed that MIP-T3 inhibits type I IFN production
by inhibiting TRAF3 complex formation (Fig. 4). IRF3 is one major transcription factor regulating IFN-b transcription. Phosphorylation of IRF3 is a critical event in IFN production (54). Phosphorylated IRF3 would dimerize and translocate into the nucleus
to activate the IFN-b promoter (55). To shed light on the mechanism by which MIP-T3 inhibits IFN production, we next explored whether MIP-T3 might affect RIG-I–induced and TBK1induced phosphorylation of IRF3. Phosphorylated IRF3 in cultured cells overexpressing RIG-I or TBK1 was readily detected
with a phosphospecific Ab (Fig. 6A, lanes 1 and 3). Upon overexpression of MIP-T3, the level of phospho-IRF3 induced by RIGI or TBK1 was significantly reduced to undetectable level (Fig.
6A, lanes 2 and 4). Thus, MIP-T3 plausibly perturbs phosphorylation and activation of IRF3 in cultured cells.
To investigate whether the activity of kinases that phosphorylate IRF3 is also inhibited by MIP-T3, in vitro kinase assay was
performed using TBK1 immunoprecipitated from HEK293T cells
expressing increasing levels of MIP-T3 (Fig. 6B). Because TBK1
recovered from MIP-T3–expressing cells could still phosphorylate
recombinant IkBa and GST-IRF3 to a similar extent as the enzyme prepared from cells that did not express MIP-T3 (Fig. 6B,
lanes 1–3), the kinase activity of TBK1 is not inhibited by MIPT3. Similar results were also obtained with another IRF3 kinase
IKKε (data not shown). Thus, the perturbation of IRF3 phos-
FIGURE 8. MIP-T3 is dissociated from TRAF3 during the course of
Sendai virus infection. HEK293T cells were transfected with Flag-TRAF3
and V5–MIP-T3. Forty-eight hours after transfection, cells were challenged with Sendai virus (80 hemagglutinating U/ml) for 0, 2, and 4 h.
Infected cells were harvested, lysed, and immunoprecipitated with antiFlag Ab. Composition of the immunoprecipitated TRAF3 complexes was
analyzed by Western blotting.
phorylation by MIP-T3 is unlikely mediated by direct inhibition of
IRF3 kinase activity.
Depletion of MIP-T3 potentiates viral induction of IFN-b
production
The above experiments were all conducted with overexpression
assays (Figs. 1–6). To verify our findings in a loss-of-function
experiment, we used MIP-T3–targeting siRNAs to deplete endogenous MIP-T3 in HEK293 and MEFs (Fig. 7). To control for
nonspecific effects induced by siRNA in general, an siRNA
against GFP (siGFP) was also used. In addition, to minimize the
nonspecific effects caused by any particular siRNA targeting MIPT3, two independent MIP-T3–depleting siRNAs targeting different regions of MIP-T3 were used in each experiment. siRNAtransfected cells were further challenged with Sendai virus. The
activity of IFN-b–Luc reporter was significantly induced by
Sendai virus (Fig. 7A, column 4 compared with column 1). Notably, this induction was further augmented in cells transfected
with MIP-T3–targeting siRNAs (Fig. 7A, columns 5 and 6 compared with column 4). The augmentation observed was statistically significant (p , 0.05 by Student t test). The two independent
MIP-T3–depleting siRNAs (si3B and si3A) behaved in a similar
manner, lending support to the specificity of the effect. The effectiveness of MIP-T3 knockdown was verified by semiquantitative RT-PCR and Western blotting using MIP-T3 specific primers
and Abs, respectively (Fig. 7B, 7C).
To gain further insight into the physiologic role of MIP-T3 in
normal fibroblasts, we also depleted the expression of MIP-T3 in
MEFs using two siRNAs, which specifically target mouse MIP-T3
transcript. Real-time qPCR analyses indicated that these siRNAs
were highly effective in dampening the expression of MIP-T3
*p = 0.00475 by Student t test, #p = 0.0152 by Student t test. G, Antiviral activity of MIP-T3–targeting siRNAs. Primary MEFs were transfected with
siGFP and siRNAs targeting mouse MIP-T3 (si3C and si3D) 48 h prior to VSV-GFP infection (multiplicity of infection = 1). Sixteen hours postinfection, virus in the culture supernatant was collected, titrated, and quantitated by plaque formation assay in Vero cells. Plaques were stained and
counted 30 h postinfection. The results were representative of two independent experiments. Error bars represent SD. *p = 0.013 by Student t test, #p =
0.022 by Student t test.
Downloaded from http://www.jimmunol.org/ by guest on June 17, 2017
V5-TRAF3 was not detected in the Flag-TBK1 immunoprecipitate obtained with anti-Flag Ab, whereas abundant V5-TRAF3
was found to be associated with the Flag-TBK1–containing
protein complex in cells that did not express V5–MIP-T3 (Fig. 4A,
lane 2 compared with lane 1). Likewise, when V5–MIP-T3 was
overexpressed, V5-TRAF3 was absent from the Flag-IKKε or
Myc–IRF3 immune complex (Fig. 4B, 4C, lane 2 compared with
lane 1), and V5-VISA was almost undetectable in the Flag-TRAF3
precipitate (Fig. 4D, lane 2 compared with lane 1). In sharp
contrast, MIP-T3 appeared to have no influence on TANK-TRAF3
or IRF3–TBK1 interaction (Fig. 4E, 4F). As such, comparable
amounts of V5-TRAF3 were recovered from Flag–TANK–containing immune complex in cells either not expressing or
expressing V5–MIP-T3 (Fig. 4E), whereas similar levels of endogenous IRF3 were found to be associated with TBK1-Flag
immunoprecipitate in V5–MIP-T3-expressing or V5–MIP-T3–
nonexpressing cells (Fig. 4F). That is to say, MIP-T3 did not prevent TRAF3 from interacting with all its partners promiscuously,
nor did it impede all downstream events. Instead, MIP-T3 specifically perturbs the formation of particular TRAF3 complexes.
Using TRAF3-IKKε as an example, we showed that the progressively elevated expression of MIP-T3 in HEK293T cells
correlated with a gradual diminution of IKKε-associated TRAF3
(Fig. 5, lanes 2–4). Likewise, dose-dependent inhibition of
TRAF3-VISA, TRAF3-TBK1, and TRAF3-IRF3 by MIP-T3 was
also observed when similar experiments were performed (data not
shown). Hence, one functional consequence of the interaction between MIP-T3 and TRAF3 is the perturbation of complex formation between TRAF3 and downstream proteins including VISA,
TBK1, IKKε, and IRF3.
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6480
mRNA (Fig. 7D). Concurrently, the levels of IFN-b mRNA and
protein in MIP-T3–compromised cells were escalated (Fig. 7E,
7F), and the escalation was found to be statistically significant
(p , 0.05 by Student t test). Plausibly, this overproduction of type
I IFNs in infected cells would compromise viral replication. To
investigate this, we silenced the expression of MIP-T3 in MEFs
and measured the replication of VSV-GFP. VSV-GFP was used in
this experiment because the titer of live virus could be measured
readily by plaque formation assay in Vero cells. Indeed, RNA
interference (RNAi) depletion of MIP-T3 caused a significant
reduction of the number of VSV-GFP plaques, supporting the
negative regulatory role of MIP-T3 in antiviral response (Fig. 7G).
Consistently, our results demonstrate that compromising the expression of endogenous MIP-T3 in established and primary fibroblasts further potentiates viral induction of IFN production leading to the attenuation of viral replication.
MIP-T3 is dissociated from TRAF3 during the course of Sendai
virus infection
Discussion
Type I IFNs are important components of innate immunity, which
combats microbial infections at the front line (44, 56, 57). To
ensure a rapid but transient response, the production of type I IFN
is tightly regulated. The identification of MIP-T3 as a TRAF3
binding partner was made in 2000 (17). Although there is increasing evidence to indicate a critical role of TRAF3 in the
regulation of innate type I IFN response (4, 5), how TRAF3
function is influenced by its binding proteins remains largely
unknown. In this study, we provided several lines of evidence to
support the conclusion that MIP-T3 functions as a negative regulator of type I IFN production. First, overexpression of MIP-T3
suppressed RIG-I/MDA5- and TBK1/IKKε-induced ISRE and
IFN-b promoter activity (Fig. 1). Second, MIP-T3 interacted
specifically with TRAF3 (Fig. 2) and prevented it from forming
protein complexes with downstream transducer and effector proteins (Figs. 4, 5). Third, MIP-T3 inhibited RIG-I– and TBK1induced IRF3 phosphorylation in cultured cells without affecting
the IRF3 kinase activity (Fig. 6). Fourth, siRNA-mediated depletion of MIP-T3 augmented viral induction of IFN production
and compromised viral replication in normal fibroblasts (Fig. 7).
Finally, MIP-T3 was dissociated from TRAF3 during the course of
Sendai virus infection (Fig. 8).
Negative regulators of type I IFN production have been well
described (58). PIN1 and LGP2 have been suggested to downreg-
ulate type I IFN production at IRF and RIG-I–like receptor levels, respectively (59–61), although a positive regulatory role of
laboratory of genetics and physiology 2 upstream of RIG-I and
MDA5 has also been reported recently (62). Deubiquitinases
DUBA (11) and OTBU1/2 (10) as well as ubiquitin ligases cIAP1/
2 (12) and Triad3A (9) have been shown to be negative regulators
of innate immunity by modulating degradative and nondegradative
polyubiquitination of TRAF3. Other deubiquitinases and ubiquitin
ligases such as A20, TAX1BP1 (63, 64), CYLD (65), and RNF125
(66) have also been thought to inhibit innate immunity by targeting other transducers and effectors such as RIG-I, TBK1, and
IRF3/7. In addition, a newly identified inhibitor of IFN production
named optineurin is also a ubiquitin-binding protein that likely
fulfills its repressive role by modulating ubiquitination (67). However, the formation of functional TRAF3-containing signaling
complexes has been shown to be targeted by various viral proteins
(13–15). In this study, we characterized MIP-T3 to be a cellular
inhibitor of type I IFN production that impedes TRAF3 complex
formation (Fig. 4). MIP-T3 is unique among cellular inhibitors of
innate immunity, because it appears to affect TRAF3 ubiquitination only slightly (Fig. 3) but is capable of preventing TRAF3
from engaging downstream transducers (Fig. 4). This finding
indicates that both cellular and viral inhibitors of IFN production
might function by the same mechanism of modulating the formation of TRAF3 complexes. It will be of great interest to understand
whether MIP-T3 competes with selected TRAF3-interacting proteins by preoccupying the same binding domains in TRAF3 with
higher affinity, speed, or stability.
Although the knockdown of MIP-T3 by RNAi further enhanced
viral induction of IFNs (Fig. 7), the potentiating effect was not as
dramatic as in the case of other known cellular negative regulators
of IFN production such as PIN1 (59), DUBA (11), and mitofusin
proteins (68–70). This finding might be explained by the incomplete knockdown of MIP-T3 as seen in Fig. 7B–D. Alternatively,
for viral activation of IFN production, MIP-T3 might already be
inactivated. Therefore, further knockdown of MIP-T3 might not
have a substantial effect on top of viral induction. Consistent with
this, we demonstrated the dissociation of MIP-T3 from TRAF3
during the course of Sendai virus infection (Fig. 8), which lent
further support to the notion that MIP-T3 is biologically important
in viral activation of IFN production.
MIP-T3 is a ciliary protein required for ciliogenesis and intraflagellar transport (18–21). Particularly, MIP-T3 has been suggested to be a new component of intraflagellar transport subcomplex B (71). Primary cilia are ubiquitous in mammalian cells,
and they fulfill important functions in cell motility in addition to
cell signaling and homeostasis (72, 73). In particular, primary cilia
serve as a platform for integration of the hedgehog signaling
pathway and might also regulate platelet-derived growth factor
receptor-a and Wnt signaling (72, 74). Thus, it will not be too
surprising if the signal transduction process leading to type I IFN
production might also take place at the primary cilia. Of note,
some viruses such as influenza A virus and severe acute respiratory syndrome coronavirus preferentially infected apical ciliated
side of the respiratory and gastrointestinal epithelia (75, 76). It is
therefore intriguing to elucidate whether MIP-T3 and MIP-T3–
binding proteins concentrated in the cilia might facilitate viral infection by compromising innate immunity.
Acknowledgments
We thank Drs. Dirk Bohmann, Genhong Cheng, Takashi Fujita, Dominique
Garcin, Liusheng He, Brian Lichty, Peter Lipsky, Kazutoshi Mori, and Jacques Perrault for reagents and members of the Jin laboratory for critical
reading of the manuscript.
Downloaded from http://www.jimmunol.org/ by guest on June 17, 2017
The effect of MIP-T3 knockdown on viral induction of IFN was
moderate (Fig. 7A, 7E, 7F). This finding led us to the hypothesis
that MIP-T3 might be inactivated during the course of viral infection. To investigate this and to shed further light on the biologic
relevance of MIP-T3 in the context of viral infection, we monitored the dynamic changes in the composition of the TRAF3
complex in Sendai virus-infected HEK293T cells. Interestingly,
the amounts of MIP-T3 coimmunoprecipitated with TRAF3 were
gradually diminished during the course of Sendai virus infection
(Fig. 8, lanes 2 and 3 compared with lane 1). Concurrently and in
keeping with the essential role of TRAF3–IRF3 complex in viral
activation of innate immunity, the relative amounts of both
phosphorylated IRF3 and TRAF3-associated IRF3 increased with
time (Fig. 8, lanes 2 and 3 compared with lane 1). Thus, the
dissociation of MIP-T3 from TRAF3 accompanied by an enhanced association of IRF3 with TRAF3 and enhanced phosphorylation of IRF3 in virus-infected cells might play an important role in viral activation of IFN response.
MIP-T3 INHIBITS TYPE I IFN PRODUCTION
The Journal of Immunology
Disclosures
The authors have no financial conflicts of interest.
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MIP-T3 INHIBITS TYPE I IFN PRODUCTION