SUMOylation of RIG-I positively regulates the
type I interferon signaling
Zhiqiang Mi1,2, Jihuan Fu2, Yanbao Xiong2, Hong Tang2✉
1Center for Molecular Immunology, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, China
2Key Laboratory of Infection and Immunity of Chinese Academy of Sciences, Institute of Biophysics, Beijing 100101, China
✉ Correspondence: firstname.lastname@example.org
Received December 29, 2009Accepted January 21, 2010
Retinoic acid-inducible gene-I (RIG-I) functions as an
intracellular pattern recognition receptor (PRR) that
recognizes the 5'-triphosphate moiety of single-stranded
RNA viruses to initiate the innate immune response.
Previous studies have shown that Lys63-linked ubiqui-
tylation is required for RIG-I activation and the down-
stream anti-viral type I interferon (IFN-I) induction. Herein
we reported that, RIG-I was also modified by small
ubiquitin-like modifier-1 (SUMO-1). Functional analysis
showed that RIG-I SUMOylation enhanced IFN-I produc-
tion through increased ubiquitylation and the interaction
with its downstream adaptor molecule Cardif. Our results
therefore suggested that SUMOylation might serve as an
additional regulatory tier for RIG-I activation and IFN-I
RIG-I, SUMOylation, type I interferon,
Type I interferons (IFN-I) play a key role in mediating antiviral
innate immune. Mammalian cells have developed two distinct
pathways to recognize the viral nucleic acids and trigger the
production of IFNs. One is mediated by Toll-like receptors
(TLRs) and mainly recognizes extracellular viral RNA. The
other utilizes the retinoic acid-inducible gene I (RIG-I)-
like helicases (RLHs), including RIG-I and melanoma
differentiation-associated gene 5 (MDA5, also referred as
helicard or IFNH1), to recognize the intracellular viral RNA
(Yoneyama et al., 2005; Kato et al., 2006; Meylan et al.,
2006). Both RIG-I and MDA5 consist of two N-terminal
caspase-recruiting domains (2CARD), a central DExD/H box
RNA helicase domain, and a C-terminal regulatory domain
(Kang 2002, 2004; Kato et al., 2005) with distinct substrate
preference to different viruses (Kato et al., 2006). It seems
that RIG-I is a sensor of short dsRNA as well as 5’ppp ssRNA,
while MDA5 is activated by long dsRNA (Gitlin et al., 2006;
Hornung et al., 2006; Yoneyama and Fujita, 2008). The
2CARD domain interacts with Cardif/IPS-1/MAVS/VISA to
initiate the IFN-I signaling cascade (Kawai et al., 2005;
Meylan et al., 2005; Seth et al., 2005; Xu et al., 2005). In this
process, ubiquitylation at Lys172 of RIG-I 2CARD by an E3
ligase Trim25 is required for RIG-I activation, because
targeting Trim25 inactivates RIG-I and increases replication
of Sendai virus (SeV) and Vesicular Stomatitis virus (VSV) in
fibroblast cells (Gack et al., 2007). Due to the essential roles
in anti-viral innate immune response of the host cells,
ubiquitylation of RIG-I is finely regulated by cellular factors
to safeguard an appropriate activation. For example, both
A20 (Lin et al., 2006) and RNF125 (Arimoto et al., 2007)
negatively regulate RIG-I ubiquitylation. Atg5 or Atg12
conjugation also downregulates IFN-I production by direct
association with RIG-I and Cardif (Jounai et al., 2007).
Similar to ubiquitylation, SUMOylation is a multi-step
reaction that covalently conjugates a 12-kDa small ubiquitin-
like modifier (SUMO) to target proteins by a single E1-
activating enzyme (Aos1/Uba2, also called SAE1/2), a unique
E2 conjugating enzyme (Ubc9) and an array of different E3
ligases (e.g., PIAS family and RanBP2), so as to regulate
their activity, stability and subcellular localization (Hershko
and Ciechanover, 1998; Hay, 2005). In contrast to ubiquityla-
tion, Ubc9 can directly attach SUMO to its substrate in the
absence of E3 ligase (Desterro et al., 1997). SUMOylation
can also antagonize other post-translational modification,
such that SUMOylation stabilizes IκBα through blocking its
ubiquitylation at the same ubiquitin acceptor site (Desterro
et al., 1998). SUMOylation is believed to regulate IFNs
signaling pathway, in that virus-mediated SUMOylation of
IRF3 and IRF7, which are two transcription factors for RIG-I-
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Protein Cell 2010, 1(3): 275–283
Protein & Cell
regulated IFN-I production, attenuates the activation of IFNs
(Kubota et al., 2008).
Bioinformatic analysis suggested that RIG-I is abundant in
lysine residues and some of them match the consensus
SUMO acceptor sites. However, whether RIG-I can be
SUMOylated and which role(s) of this modification might
play in IFN-I signaling is still unclear. In the present work, we
demonstrated that both exogenously expressed and endo-
genous RIG-I were modified by SUMO-1. RIG-I SUMOylation
increased its Lys63 ubiquitin modification and the inter-
molecular interaction between RIG-I and Cardif. Reporter
assays showed that modulation of Ubc9 levels altered the
SUMO-1 modification of RIG-I, which well correlated with the
RIG-I-driven IFN-β production. These results implied that
SUMOylation provided an additional regulation of RIG-I
activation, which might crosstalk to ubiquitylation of RIG-I to
orchestrate the cellular anti-viral response.
RIG-I is modified by SUMO-1
Bioinformatic analysis (SUMOplot Analysis Program,
ABGENT) predicted that nine lysine residues in RIG-I could
be potential SUMO acceptors (supplemental Fig. 1A), bearing
the consensus motif of ψKxE or non-consensus motif with
high modification frequency (Song et al., 2004; Schwamborn
et al., 2008; Xu et al., 2008). To validate these predicted
SUMOylation sites, Flag-RIG-I, HA-SUMO-1 and Myc-Ubc9
were overexpressed in HEK293Tcells. The immunoblotting of
the whole cell lysate with anti-Flag antibody showed several
characteristic band shifts with higher molecular weights,
indicating the existence of post-translational modification
(Fig. 1A, left panel). When the cell lysates were subjected to
immunoprecipitation with anti-Flag antibody, these character-
istic bands could be probed by anti-SUMO-1 antibody
(Fig. 1A, right panel). In physiological condition, the expres-
sion level of RIG-I is trivial in cells (Cui et al., 2004; Imaizumi
et al., 2004), but viral infection, such as by SeV, can cause
massive production of RIG-I. To enhance the SUMOylation
signal of the endogenous RIG-I, SeV was used to infect
HEK293Tcells that had been overexpressed with HA-SUMO-
1 and Myc-Ubc9. Co-immunoprecipitation with anti-SUMO-1
antibody showed that the endogenous RIG-I was present in
the immunoprecipitated complex and a small fraction of RIG-I
was modified by SUMO-1 (Fig. 1B). The apparently unmodi-
fied RIG-I by anti-SUMO-1 antibody was also detected in the
complex, which is due to the dynamic nature of reversible
pCMV-HA-SUMO-1, pcDEF-Myc-Ubc9 and pEF-Flag-RIG-I, and cell lysates were immunoprecipitated with anti-Flag M2 mAb and
immunoblotted with anti-Flag or anti-SUMO-1 (D-11, Santa Cruz) mAb. (B) Endogenous RIG-I was SUMOylated. HEK293T cells
(2×106) were co-transfected with pCMV-HA-SUMO-1 and pcDEF-Myc-Ubc9. Twelve hours post-transfection, cells were infected
with 20 HAU SeV for 1.5h, and excessive virus was washed off. Cells were then changed with fresh medium and subsequently
cultured for 36h. Cell lysates were immunoprecipitated with anti-SUMO-1 antibody or mouse IgG1 as a negative control, and
proteins were resolved in SDS-PAGE for immunoblotting with mouse anti-RIG-I antibody (Alme-1, Alexis). (C) RIG-I was
SUMOylated in in vitro enzymatic assay. Recombinant RIG-I was incubated with purified SUMO-1-GG and E1/E2 enzymes for 3h.
Then the reaction mixtures were separated by SDS-PAGE and immunoblotted with mouse anti-RIG-I or anti-SUMO-1 antibody.
RIG-I was modified by SUMO-1. (A) Overexpressed RIG-I was SUMOylated. HEK293Tcells were co-transfected with
276 © Higher Education Press and Springer-Verlag Berlin Heidelberg 2010
Zhiqiang Mi et al.
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SUMOylation of the protein. Such intracellular SUMOylation
of RIG-I was further confirmed by in vitro enzymatic assays,
where we used purified recombinant enzymes (SAE1/SAE2
and Ubc9) and substrates (SUMO-1-GG and RIG-I) from
bacteria. As indicated in Fig. 1C, in the presence of E1 and E2
enzymes, purified RIG-I was readily conjugated with SUMO-1
with the typical band shifts (Fig. 1C, left panel) that
corresponded to oligomeric or polymeric SUMO-1 attachment
(Fig. 1C, right panel).
RIG-I interacts with Ubc9 and its SUMOylation is SUMO-1
SUMOylation requires direct interaction between Ubc9 and
target proteins in order to transfer SUMO moiety from E1
enzyme. The inter-molecular interaction between RIG-I and
Ubc9 was detected by co-immunoprecipitation in the pre-
sence of SUMO-1 (Fig. 2A). Furthermore, there are at least
four SUMO genes (SUMO1–4) in human, with SUMO1–3
ubiquitously expressed and SUMO-4 mainly in kidney, lymph
node and spleen (Guo et al., 2004). SUMO-2 and SUMO-3
are 97% identical, but share only 50% sequence identity with
SUMO-1. SUMO-1 and SUMO-2/3 have distinct functions, as
they are conjugated to different target proteins in vivo, while
the role of SUMO-4 remains enigmatic (Geiss-Friedlander
and Melchior, 2007). Up to date, a large number of target
proteins were found to be modified by SUMO, but most of
them were substrates of SUMO-1. SUMOplot Analysis
Program predicted the potential SUMO-1 acceptation site in
RIG-I. Therefore, we first investigated whether RIG-I could be
modified by SUMO-1, and we also determined whether
SUMO-2 and SUMO-3 are involved in RIG-I modification. In
our assays, only SUMO-1 overexpression gave rise to the
characteristic higher molecular weight bands, suggesting that
RIG-I was a SUMO-1 acceptor (Fig. 2B).
SUMOylation enhances the interaction between RIG-I and
Although highly reversible and dynamic, a small proportion of
SUMO conjugation results in significant function alteration of
substrate protein through inter- or intra-molecular interaction
(Geiss-Friedlander and Melchior, 2007). It has been pre-
viously shown that Lys63-linked ubiquitylation disrupts the
auto-inhibitory conformation of RIG-I, which is essential for
IFN-I signaling (Saito et al., 2007). In the situation of
overexpression, RIG-I can be ubiquitylated by Lys63-linked
ubiquitin in the absence of viral infection (Gack et al., 2007)
Fig. 1A were immunoprecipitated with anti-c-Myc antibody (9E10, Zymed) and immunoblotted with anti-Flag or c-Myc antibody.
(B) pcDEF-Myc-Ubc9 and pEF-Flag-RIG-I were co-transfected with pCMV-HA-SUMO-1, or pcDEF-GST-SUMO-2, pcDEF-Flag-
SUMO-3, respectively, for 48h. 2 ´105cells were boiled in SDS-PAGE loading dye for 5min before loaded into 10% SDS-PAGE.
Mobility shifts of RIG-I was detected by immunoblotting with anti-Flag antibody. Expression levels of Ubc9, SUMO-1, SUMO-2 and
SUMO-3 were immunoblotted with anti- c-Myc, -HA, -GSTor-Flag antibody.
RIG-I interacted with Ubc9 for SUMO-1 conjugation. (A) RIG-I and Ubc9 co-immunoprecipitated. Cell lysates from
© Higher Education Press and Springer-Verlag Berlin Heidelberg 2010277
RIG-I SUMOylation on type I interferon signaling
Protein & Cell
and drive the production of IFN-β. A small fraction of RIG-I
pool shows activation, and even in the absence of viral
infection, the signaling might facilitate the maintenance of the
basal level of interferon signal transduction that contributes to
the rapid and massive interferon production when pathogen
invades (Taniguchi and Takaoka, 2001, 2002). Change of
RIG-I SUMOylation by overexpression or siRNA knockdown
of Ubc9 increased or decreased RIG-I ubiquitylation, respec-
tively (Fig. 3A and 3B). This result suggested that SUMOyla-
tion likely alters the protein folding to assist RIG-I
ubiquitylation. We then determined whether RIG-I SUMOyla-
tion might affect its interaction with the adaptor Cardif. Co-
immunoprecipitation assays showed that overexpression of
Ubc9 significantly enhanced the interaction between RIG-I
and Cardif (Fig. 3C), whereas RNAi knockdown of Ubc9
(Fig. 3D). The effect of Ubc9 knockdown was verified by
RT-PCR (supplemental Fig. 1B) and this also resulted in
overall reduction of SUMOylation of cellular proteins (supple-
mental Fig. 1C).
RIG-I SUMOylation increases IFN-β production
Although RIG-I exists as a monomer in resting cells due to an
auto-inhibitory domain (Meylan et al., 2005), the viral infection
or overexpression promotes its self-association. Thus,
overexpression of RIG-I can potentially initiate certain
ubiquitylation of RIG-I. HEK293T cells were transfected as described in Fig. 1A except that HA-Ubi expression vector was
included. The cell lysates were immunoprecipitated with anti-c-Myc and immunoblotted with anti-HA mAb. The star (★) indicated
non-specific bands. (B) Knockdown of Ubc9 reduced ubiquitylation of RIG-I. HeLa cells (2 ´106) were pre-infected with 2 MOI
lentiviral vector, lentivirus containing Ubc9-shRNA or scrambled Ubc9-shRNA for 12h. Cells were then co-transfected with pEF-
Flag-RIG-I and pRK5-HA-Ubi-K63 for 48h. Co-immunoprecipitation was performed as described in Fig. 1A for detection of
ubiquitylation with anti-HA antibody. The star (★) indicated non-specific bands. (C) SUMOylation enhanced inter-molecular
interaction of RIG-I and Cardif. HEK293T cells were co-transfected with indicated plasmids, and cell lysates were
immunoprecipitated with anti-Flag mAb and immunoblotted with anti-c-Myc mAb. The input was measured with anti-Flag or anti-
Myc mAb. (D) Knockdown of Ubc9 disrupted RIG-I/Cardif interaction. Endogenous Ubc9 was knocked down by lentivirus-based
shRNA as described in (B). HeLa cells were then co-transfected with pCMV-Myc-Cardif for 36h. Interaction of endogenous RIG-I
with Cardif was detected by co-immunoprecipitation as described in (C).
SUMOylation increased interaction between RIG-I and Cardif. (A) SUMOylation increased Lys63-linked
278© Higher Education Press and Springer-Verlag Berlin Heidelberg 2010
Zhiqiang Mi et al.
Protein & Cell
downstream signaling(s) in cells (Saito et al., 2007).
SUMOylation of RIG-I enhanced ubiquitylation and the
subsequent inter-molecular interaction with Cardif, which
inevitably would result in RIG-I activation and IFN-I induction.
To prove this hypothesis, the IFN-β reporter activities were
measured in the presence of overexpressed SUMO-1 and
Ubc9. Intriguingly, RIG-I-, but not Cardif-driven IFN-β reporter
activity was augmented by Ubc9 (Fig. 4A). Because RIG-I
functions in the upstream of Cardif in IFN-β signaling
cascade, this result suggested that Ubc9 is specifically
involved in RIG-I SUMOylation. This was further confirmed
by RNAi knockdown of endogenous Ubc9, where RIG-I- but
notCardif-driven IFN-β reporter activities were reduced
(Fig. 4B). Previous reports have demonstrated that SeV
infection induces IFN-I production through RIG-I activation
(Gack et al., 2007). Small RNA interference of Ubc9 in HeLa
cells showed that downregulation of Ubc9 caused reduced
IFN-β reporter activities (Fig. 4C), which led to enhanced viral
replication (Fig. 4D). Therefore, our results strongly sug-
gested that SUMOylation actively regulates RIG-I activation,
which modulates IFN-I production and resistance to viral
HEK293Tcells were co-transfected with pIFN-luc,pCMV-renilla, pCMV-HA-SUMO-1, pcDEF-Myc-Ubc9, pEF-Flag-RIG-I (left panel)
or pCMV-Myc-Cardif (right panel) by calcium phosphate participation. Forty-eight hours post-transfection, cells were collected and
the dual-luciferase activities were measured. (B) RIG-I- but not Cardif-driven IFN signaling was affected by Ubc9 knockdown. HeLa
cells were pre-infected with lentiviral vectors to knockdown endogenous Ubc9 as described in Fig. 3B. Cells were then co-
transfected with pIFN-luc, pCMV-renilla, pEF-Flag-RIG-I (left panel) or pCMV-Myc-Cardif (right panel) by jetPEI reagent for 36h. (A)
and (B). The luciferase activity was determined and normalized by renilla luciferase activity. Data were presented as fold induction
through dividing the luciferase activity for each sample by that for the control sample with empty vector only. Data represented the
average of three independent experiments (mean±SD). (C) Knockdown of Ubc9 inhibited SeV-responsive IFN production.
Endogenous Ubc9 was knocked down by small RNA interference as described in (B), and HeLa cells were then co-transfected with
pIFN-luc and internal control pCMV-renilla for 24h. SeV (20HAU) was used to infect cells for 12h before measuring luciferase
activities. The luciferase activity was determined and normalized by renilla luciferase activity. Fold induction represents the ratio of
SeV infection to mock infection. Data from three independent experiments were shown as mean±SD. (D) Knockdown of Ubc9
facilitated SeV replication. After Ubc9 knockdown as described in (C), cells were infected with SeV (20HAU) for 12h. Virus
replication was measured by quantitative real-time RT-PCR analysis of NP gene copy numbers. Data represented the average of
SeV NP gene copy number per 100 β-actin copies (mean±SD) from three independent experiments.
SUMOylation increased RIG-I-driven IFN production. (A) Ubc9 enhanced RIG-I-responsive IFN production.
© Higher Education Press and Springer-Verlag Berlin Heidelberg 2010 279
RIG-I SUMOylation on type I interferon signaling
Protein & Cell
As a pivotal sensor of RNA viruses in IFN-I signaling, RIG-I
activation has to be tightly regulated to ensure effective
eradication of pathogens with minimal excessive inflamma-
tion. For example, to maintain the homeostasis, ubiquitylated
RIG-I by Trim25 (Gack et al., 2007) needs to be down-
regulated by either RNF125-recruited proteasomal degrada-
tion (Arimoto et al., 2007, 2008) or ISG15 conjugation (Zhao
et al., 2005; Arimoto et al., 2008; Kim et al., 2008). In the
present work, we provided evidence that SUMOylation,
however, might serve as an additional positive regulator in
RIG-I activation, which facilitates the ubiquitylation of RIG-I
and inter-molecular interaction with its mitochondrial adaptor
Cardif. This positive role of RIG-I SUMOylation in IFN-I
production is particular interesting, provided current evidence
that SUMOylation is involved in inhibition of IFN-I signaling.
For example, SUMOylation of IRF3 and IRF7 upon VSV
infection inhibits IFN-I transcription activation (Kubota et al.,
2008). SUMOylation of IRF2 by SUMO E3 ligase PIASy
inactivates transcription of IFN-I-responsive genes (Han
et al., 2008). Our finding that SUMOylation enhanced RIG-I-
driven but not Cardif-driven IFN-β reporter strongly indicated
that SUMOylation in the upstream molecules can overcome
its effect on the downstream IRFs, with SUMOylation of RIG-I
becoming dominant in control of IFN-I production.
The existence of an auto-inhibitory conformation of RIG-I is
a useful tactics for host cells since it is activated only after
cells sense the invading
(Saito et al., 2007) and after ubiquitylation triggers the
conformational unfolding (Gack et al., 2007). Interestingly, we
observed that SUMOylation increased Lys63-linked ubiquity-
lation of RIG-I, suggesting that SUMOylation would occur
upstream of ubiquitylation of RIG-I for its activation. Further-
more, RIG-I mutant (K172R) defective in ubiquitylation (Gack
et al., 2007) was also SUMOylated (supplemental Fig. 2A),
implying that these two types of modification did not compete
for the same lysine sites. This was inconsistent previous
reports that SUMOylation and ubiquitylation interfere with
each other (Desterro et al., 1998; Comerford et al., 2003;
Huang et al., 2003; Lin et al., 2003; Steffan et al., 2004).
According to the characteristic band shift pattern, RIG-I might
investigating the potential SUMO acceptors by mutagenesis,
however, we found that these sites were not essential in
mediating RIG-I modification (supplemental Fig. 2B),
because SUMO modification still occurred in these RIG-I
mutants. This could be explained by the variation of flanking
amino acids that do not fit with the consensus motif (Hay,
2005; Anckar and Sistonen, 2007). Whereas we speculate
that the SUMO acceptor sites in RIG-I might not agree with
the consensus acceptor site, further mass spectrometric
analysis is required to prove this hypothesis.
Post-translational and induced modifications of RIG-I by
small molecule (e.g., Ubi, SUMO-1 and ISG15) present a fine
regulatory network for innate cellular response to pathogens.
In terms of SUMOylation, although the E2 enzyme is
universal, the poll of E3 ligase is rather diverse and the
modification is rather target specific (Melchior et al., 2003;
Hay, 2005). It remains interesting to identify other E3 ligase
that would be involved in RIG-I SUMOylation after our
analysis of PIAS family E3 ligases on RIG-I modification.
MATERIALS AND METHODS
Viruses, cells, plasmid constructs and transient tranfection
Sendai virus was from Wuhan Institute of Virology, Chinese Academy
of Sciences. Sendaiviruswas propagatedin10-day-oldembryonated
chicken eggs from specific-pathogen-free flocks (Beijing MERIAL
Ltd.) as previously described (Mattana and Viscomi, 1998). The
hemagglutination titers were measured with 1% hamster blood cell
(Beijing MERIAL Ltd.).
HeLa cell and HEK293Tcell were routinely maintained in minimal
essential medium (MEM) supplemented with 10% fetal bovine serum
(PAA), 100unit/mL penicillin and 100μg/mL streptomycin (HyClone)
and cultured at 37°C with 5% CO2. Cells were transiently transfected
with calcium-phosphate precipitation and jetPEI (Polyplus transfec-
pEF-Flag-RIG-I (Yoneyama et al., 2004) was kindly provided by
Prof. S. Akira (Osaka University, Japan), and its K/R mutant
derivatives were generated by site-directed mutagenesis (Zheng
et al., 2004). pCMV-Myc-RIG-I was constructed by add-on PCR to
insert in between SalI and NotI sites of pCMV-Myc vector (Clontech).
pcDEF-Flag-SUMO-1, pcDEF-Myc-Ubc9, pcDEF-Myc-SAE1/SAE2,
pcDEF-GST-SUMO-2 and pcDEF-Flag-SUMO-3 were kind gifts from
Dr. X. Peng (Chinese Academy of Medical Sciences, Beijing). pGEX-
6P-1-RIG-I was constructed by add-on PCR to insert RIG-I gene in
between SmaI and XhoI sites of pGEX-6P-1 vector (Amersham).
pGEX-4T-1-SUMO-1, Ubc9, SAE1 and SAE2 were constructed by
add-onPCR toinserteachencodinggeneinbetweenEcoRIand XhoI
sites of pGEX-4T-1 vector (Amersham). Ubiquitin expression vector
pRK5-HA-Ubi-K63 containing arginine substitutions except position
63 (Lim et al., 2005) was from Dr. K. Lim (National Neuroscience
Institute, Singapore). Interferon beta reporter (IFN-luc) (Guo and
Cheng, 2007) were kindly provided by Prof. G. Cheng (UCLA, USA).
pCMV-Flag-Ubc9 was constructed by inserting Ubc9 in between
EcoRI and XhoI restriction sites of pCMV-Tag2 (Stratagene). pCMV-
HA-SUMO-1, pCMV-HA-Ubc9, pCMV-Myc-Cardif were constructed
by cloning of each encoding gene in between EcoRI and XhoI, XhoI
and KpnI, and SalI and NotI restriction sites of pCMV cassette vectors
Immunoprecipitation and immunoblotting
Routinely, 2 ´106cells were lysed in 250μL ice-cold immunopreci-
pitation buffer (50mM Tris-HCl, pH 7.5, 150mM NaCl, 1% Nonidet P-
40,0.5% sodium deoxycholate) freshly supplemented with 2mM N-
ethylmaleimide (NEM) (Sigma), 1mM DTT (Sigma) and complete
protease inhibitor cocktail (Roche). Cell lysates (100μg proteins)
were then immunoprecipitated with indicated antibodies, and proteins
280 © Higher Education Press and Springer-Verlag Berlin Heidelberg 2010
Zhiqiang Mi et al.
Protein & Cell
were separated with 7.5% SDS-PAGE for immunoblotting and
visualized by a chemiluminescence reagent (Pierce).
RIG-I and the SUMOylation enzymes were expressed in E.coli BL21
(DE3) individually and purified to homogeneity as previously
described (Boggio et al., 2004). For the conjugation assay, 1μg
SAE1/SAE2, 2μg Ubc9, 2μg SUMO1-GG and 0.5μg RIG-I in 15μL
reaction buffer (20mM HEPES, pH 7.5, 5mM MgCl2) in the presence
of 2mM ATP, 1mM DTTand 2mM NEM were incubated at 30°C for
3h. The reaction was terminated by adding 15μL 2×SDS-PAGE
loading buffer and boiled at 95°C for 5 min.
The target sequence of Ubc9 (5'-GGGAAGGAGGCTTGTTTAAAC-
3') or its scrambled control sequence in a lentiviral vector LTV1 (Sui
and Shi, 2005) was kindly provided by Prof. G. Sui (Wake Forest
University School of Medicine, USA). Packaging lentiviruses were
prepared as previously described (Rubinson et al., 2003). The
lentiviral infection of HeLa cells was performed in the presence of
8μg/mL polybrene (Sigma) for 4h and the knockdown efficiency was
measured 48h post infection.
Total RNA was isolated according to manufacturer’s instruction with
Trizol reagent (Invitrogen) and RT-PCR was performed using RT-
PCR kit (Promega) according to manufacturer’s manual with the
following primers: hUbc9 forward, 5'-CGGAATTCTATGTCGGGGAT-
CTCCCTC-3'; hUbc9 reverse, 5'-CGGGGTACCTTATGAGGGCG-
CAAACTTC-3'; hGAPDH forward, 5'-AAGCGCACGGGCATGGCC-
TT-3', hGAPDH reverse, 5'-AGGAGACCACCTGGTGCTCAG-3'.
Quantitative real-time PCR reactions in a MyiQ cycler (Bio-Rad,
USA) using SYBR Green I (Molecular Probes, USA) were
performed exactly as described previously (Doyle et al. , 2002)
5'-GCGGGAAATCGTGCGTGACATT-3'; human β-actin reverse, 5'-
GATGGAGTTGAAGGTAGTTTCGTG-3' (Lenz et al., 1994); SeV NP
forward, 5'-TGCTGCCAAAGTTCACGAT-3'; SeV NP reverse, 5'-
We thank Drs. S. Akira (Osaka University, Japan), G. Sui (Wake
Forest University School of Medicine, USA), M. Gack (Friedrich-
Alexander University, Germany), G. Cheng (UCLA, USA) and X.
Peng (Chinese Academy of Medical Sciences, China) for kindly
providing us with various plasmid constructs, and our colleagues (G.
Chen, D. Zheng, J. Jiang and X. Yang) for their critical reading and
stimulating suggestions. This research was in part supported by
grants from Chinese Academy of Sciences (Grant No. KSCX1-YW-
10), and the Ministry of Science and Technology (Grant Nos.
2009CB522506) to H.T. The authors have no conflicting financial
CARD, caspase recruitment domain; Cardif, CARD adaptor inducing
IFN-β; IFN, interferon; PRR, pattern recognition receptor; RIG-I,
retinoic acid-inducible gene I; MDA5, melanoma-differentiation-
associated gene 5; MEM, minimal essential medium; PAA, fetal
bovine serum; RLH, RIG-I-like helicase; SeV, Sendai virus; SUMO,
small ubiquitin-like modifier; TLR, Toll-like receptor; Ubi, ubiquitin;
VSV, vesicular Stomatitis virus.
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