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A Distinct Role of Riplet-Mediated K63-Linked
Polyubiquitination of the RIG-I Repressor Domain in
Human Antiviral Innate Immune Responses
Hiroyuki Oshiumi*, Moeko Miyashita
¤
, Misako Matsumoto, Tsukasa Seya
Department of Microbiology and Immunology, Graduate School of Medicine, Hokkaido University, Kita-ku, Sapporo, Japan
Abstract
The innate immune system is essential for controlling viral infections, but several viruses have evolved strategies to escape
innate immunity. RIG-I is a cytoplasmic viral RNA sensor that triggers the signal to induce type I interferon production in
response to viral infection. RIG-I activation is regulated by the K63-linked polyubiquitin chain mediated by Riplet and
TRIM25 ubiquitin ligases. TRIM25 is required for RIG-I oligomerization and interaction with the IPS-1 adaptor molecule. A
knockout study revealed that Riplet was essential for RIG-I activation. However the molecular mechanism underlying RIG-I
activation by Riplet remains unclear, and the functional differences between Riplet and TRIM25 are also unknown. A genetic
study and a pull-down assay indicated that Riplet was dispensable for RIG-I RNA binding activity but required for TRIM25 to
activate RIG-I. Mutational analysis demonstrated that Lys-788 within the RIG-I repressor domain was critical for Riplet-
mediated K63-linked polyubiquitination and that Riplet was required for the release of RIG-I autorepression of its N-terminal
CARDs, which leads to the association of RIG-I with TRIM25 ubiquitin ligase and TBK1 protein kinase. Our data indicate that
Riplet is a prerequisite for TRIM25 to activate RIG-I signaling. We investigated the biological importance of this mechanism
in human cells and found that hepatitis C virus (HCV) abrogated this mechanism. Interestingly, HCV NS3-4A proteases
targeted the Riplet protein and abrogated endogenous RIG-I polyubiquitination and association with TRIM25 and TBK1,
emphasizing the biological importance of this mechanism in human antiviral innate immunity. In conclusion, our results
establish that Riplet-mediated K63-linked polyubiquitination released RIG-I RD autorepression, which allowed the access of
positive factors to the RIG-I protein.
Citation: Oshiumi H, Miyashita M, Matsumoto M, Seya T (2013) A Distinct Role of Riplet-Mediated K63-Linked Polyubiquitination of the RIG-I Repressor Domain in
Human Antiviral Innate Immune Responses. PLoS Pathog 9(8): e1003533. doi:10.1371/journal.ppat.1003533
Editor: Michael Gale Jr, University of Washington, United States of America
Received January 30, 2013; Accepted June 17, 2013; Published August 8, 2013
Copyright: ß 2013 Oshiumi et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported in part by a grant-in-aid from the Ministry of Education, Science and Culture of Japan, and the Ministry of Health Labour, and
Welfare of Japan, Kato Memorial Bioscience Foun dation. The funders had no role in study design, data collection and analysis, decision to publish, or preparation
of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: oshiumi@med.hokudai.ac.jp
¤ Current address: Hokkaido Pharmaceutical University School of Pharmacy, Katsuraoka-cho, Otaru, Hokkaido, Japan.
Introduction
The innate immune system is essential for controlling virus
infections, and several viruses have evolved strategies to evade host
innate immune responses. Cytoplasmic viral RNA is recognized by
RIG-I-like receptors, including RIG-I and MDA5 [1,2]. The
RIG-I protein comprises N-terminal Caspase Activation and
Recruitment Domains (CARDs), a central RNA helicase domain,
and a C-terminal Repressor domain (RD) [3]. RD consists of C-
terminal RNA binding domain (CTD) and a bridging domain
between CTD and helicase [4]. RIG-I CARDs are essential for
triggering the signal that induces type I interferon (IFN). In resting
cells, RIG-I RD represses its CARDs signaling [3]. After viral
infection, RIG-I RD recognizes 59-triphosphate double-stranded
RNA (dsRNA), which results in a conformational change in the
RIG-I protein [3]. This conformational change leads to the release
of RD autorepression of CARDs, after which CARDs associate
with an IPS-1 adaptor molecule (also called MAVS, Cardif, and
VISA) localized at the outer membrane of mitochondria
[3,5,6,7,8]. IPS-1 activates downstream factors such as TBK1,
IKK-e, and NEMO [9,10,11]. NEMO forms a complex with
TBK1 and IKK-e and has a polyubiquitin binding region [12].
These protein kinases are essential for activating transcription
factors such as IRF-3 to induce type I IFN production [13].
Several ubiquitin ligases are involved in regulating the RIG-I-
dependent pathway, and RIG-I itself is regulated by ubiquitin
chains [14]. Gack MU and colleagues firstly reported that
TRIM25 ubiquitin ligase mediates K63-linked polyubiquitination
of RIG-I N-terminal CARDs, which results in RIG-I activation
[15]. Other groups also detected a RIG-I-anchored polyubiquitin
chain after ligand stimulation or viral infection [16,17]. It was
recently demonstrated that an unanchored polyubiquitin chain but
not ubiquitination is essential for RIG-I activation [18,19].
However, RIG-I anchored K63-linked polyubiquitin chains are
detected after viral infection [15,20].
Another E3 ubiquitin ligase, Riplet, binds RIG-I RD and
mediates the K63-linked polyubiquitination of RIG-I RD [21]. In
contrast, Chen DY and colleagues reported that Riplet (also called
Reul) ubiquitinated RIG-I CARDs [22]. A study that used Riplet
knockout mice showed that mouse Riplet is essential for RIG-I-
PLOS Pathogens | www.plospathogens.org 1 August 2013 | Volume 9 | Issue 8 | e1003533
mediated type I IFN production in response to vesicular stomatitis
virus (VSV), Flu, and Sendai virus (SeV) infections [23]. However,
the functional difference between Riplet and TRIM25 remains
unclear, and the molecular mechanism of how Riplet-mediated
RIG-I ubiquitination activates RIG-I signaling remains unre-
solved.
Hepatitis C virus (HCV) is a major cause of hepatocellular
carcinoma (HCC) worldwide. HCV RNA is primarily recognized
by RIG-I in vitro and in vivo [24]. The HCV protease NS3-4A can
suppress type I IFN production [25]. NS3-4A cleaves IPS-1 to
suppress RIG-I-mediated innate immune responses [7,26]. Hu-
man monocyte-derived dendritic cells recognize HCV RNA
through Toll-like receptor 3, and NS3-4A has the ability to cleave
TICAM-1, which is a solo adaptor molecule of Toll-like receptor 3
[27,28]. In this study, we found that Riplet was another target of
NS3-4A. Here, we demonstrated the molecular mechanisms of
how Riplet-mediated RIG-I polyubiquitination triggered the type
I IFN production signal and showed that this mechanism was
targeted by HCV.
Results
Riplet and TRIM25 ubiquitin ligases play different roles in
RIG-I activation
In mouse embryonic fibroblasts (MEFs), TRIM25 is essential for
type I IFN production in response to SeV infection [15]. As with
TRIM25 knockout, Riplet knockout abolished IFN-a2 and IFN-b
mRNA expressions in response to SeV infection in MEF
(Figure 1A and 1B), which suggested that both ubiquitin ligases
were essential for type I IFN expression in MEFs in response to
SeV infection. Thus, we examined whether these two ubiquitin
ligases played different roles in RIG-I activation.
We performed reporter gene assays with p125luc (IFN-b
reporter) using either RIG-I CARDs fragment or full-length
RIG-I expression vectors. It is known that ectopic expression of
RIG-I CARDs fragment activates the signaling even in the
absence of stimulation with RIG-I ligand [2,3] and that the auto-
activation is observed when full-length RIG-I is ectopically
expressed in HEK293 cell [21,29]. As previously reported,
TRIM25 ectopic expression efficiently increased RIG-I CARDs
fragment-mediated signaling (Figure 1C). However, TRIM25
expression only mildly increased full-length RIG-I signaling
(Figure 1D). In contrast, Riplet ectopic expression efficiently
increased the full-length RIG-I-mediated signaling, although
Riplet failed to increase the RIG-I CARDs-mediated signaling
(Figure 1C and 1D). Interestingly, when Riplet was co-expressed
with TRIM25, ectopically expressed TRIM25 could increase the
full-length RIG-I-mediated signaling (Figure 1F). We observed the
same effects of TRIM25 and Riplet expressions on the reporter
activation in the presence of stimulation with HCV 39 UTR
dsRNA (a RIG-I ligand) or VSV infection, which are recognized
by RIG-I (Figure 1E and 1F). These different effects of Riplet and
TRIM25 on the signaling suggested that Riplet and TRIM25
ubiquitin ligases played different roles in RIG-I activation.
We then investigated whether the two ubiquitin ligases associate
with RIG-I after stimulation. First, we performed immunoprecip-
itation assay and found that endogenous Riplet and TRIM25
bound to endogenous RIG-I after stimulation with HCV dsRNA
(Figure 1G). Second, we investigated subcellular localizations of
the proteins by confocal immunofluorescence microscopy. In
resting cells, RIG-I was barely detectable, whereas RIG-I
exhibited punctate staining in cytoplasm after VSV infection
(Figure 2A), and ectopically expressed Riplet was co-localized with
RIG-I and TRIM25 in perinuclear region in VSV infected cells
(Figure 2A–2C and 2E). This is consistent with a previous
observation that RIG-I and TRIM25 co-localized extensively at
cytoplasmic perinuclear bodies [15]. Pearson’s correlation coeffi-
cient values also suggested the correlation of the colocalizations of
those proteins (Figure 2D and 2F). Recently, it was reported that
RIG-I recognized short polyI:C in stress granules [30]. G3BP is a
marker of the stress granule [30]. In resting cells, G3BP dispersed
in cytoplasm [30], and thus barely detectable (Figure 2G and 2H),
whereas G3BP speckles were detected in cells stimulated with short
polyI:C in the cytoplasm (Figure 2G and 2H). Riplet and TRIM25
localizations within G3BP speckles were detected in the stimulated
cells (Figure 2G and 2H). Taken together, these data indicated that
both Riplet and TRIM25 associated with RIG-I after stimulation.
Next, we assessed the RIG-I regions that bind to the two
ubiquitin ligases using RIG-I fragments. As previously reported,
TRIM25 bound to RIG-I CARDs fragment (Figure 2I). However,
Riplet bound to RIG-I RD (735–925 aa), but not to CARDs
fragment (Figure 2J). Deleting the Riplet binding region (RIG-I-
DCTD) abrogated Riplet effect on RIG-I signaling (Figure 2K).
This was contrast to TRIM25, which affects RIG-I CARDs.
Taken together, our genetic and biochemical data indicated that
the two ubiquitin ligases associated with RIG-I after stimulation
but showed different effects on RIG-I activation. Thus, we next
focused on Riplet specific role in RIG-I activation.
Riplet-mediated RIG-I polyubiquitination is dispensable
for RIG-I RNA binding activity
RIG-I CARDs harbor K63-linked polyubiquitination [15]. As
RIG-I CARDs, RIG-I RD harbored K63-linked polyubiquitina-
tion (Supplemental Figure S1). Riplet expression increased the
polyubiquitination of RIG-I RD but not that of CARDs
(Figure 3A). RIG-I RD has two functions. One is RNA binding
activity and the other is autorepression of its CARDs signaling.
Firstly, we tested whether Riplet affects RIG-I RNA binding
activity. In a pull-down assay using biotin-conjugated dsRNA and
streptavidin beads, we found that both polyubiquitinated and non-
ubiquitinated RIG-I were recovered (Figure 3B), which suggested
that Riplet-mediated polyubiquitination was dispensable for RIG-I
Author Summary
The cytoplasmic viral RNA sensor RIG-I recognizes various
types of pathogenic viruses and evokes innate immune
responses, whereas several viruses have evolved strategies
to escape the host innate immune responses. RIG-I triggers
a signal to induce type I interferon and inflammatory
cytokines. RIG-I activation is regulated by K63-linked
polyubiquitin chains mediated by the ubiquitin ligases
TRIM25 and Riplet; however, the functional difference
between the two ubiquitin ligases remains unclear, and
the molecular mechanism underlying Riplet-mediated RIG-
I activation is unknown. We revealed sequential roles of
the two ubiquitin ligases during RIG-I activation and found
that Riplet-mediated polyubiquitination of the RIG-I
repressor domain released RIG-I autorepression of its N-
terminal CARDs responsible for triggering the signal, which
resulted in an association with TRIM25 ubiquitin ligase and
TBK1 protein kinase. Interestingly, we found that this
mechanism was targeted by hepatitis C virus, which is a
major cause of hepatocellular carcinoma. This result
emphasizes the vital role of Riplet-mediated release of
RIG-I RD autorepression in antiviral responses. Our results
establish that Riplet releases RIG-I RD autorepression and
demonstrated the biological significance of this mecha-
nism in human innate immune responses.
Riplet Releases RIG-I Autorepression
PLOS Pathogens | www.plospathogens.org 2 August 2013 | Volume 9 | Issue 8 | e1003533
RNA binding activity. As RIG-I is known to form homo-oligomers
[3], it is possible that non-ubiquitinated RIG-I was recovered
through ubiquitinated RIG-I by the pull-down assay. To exclude
this possibility, we used RIG-I mutants, RIG-I 5KR and RIG-I
K788R, which were barely ubiquitinated by Riplet (described
below). We compared the binding abilities of the RIG-I mutants to
that of wild-type RIG-I by pull-down assay using cell lysate
isolated from cells without stimulation with RIG-I ligand to avoid
any ubiquitination. The results showed that the RIG-I 5KR and
RIG-I K788R mutant proteins were recovered by pull down assay
as wild-type RIG-I (Figure 3C). This data also indicated that the
polyubiquitination is dispensable for RIG-I RNA binding activity.
Riplet releases RIG-I RD autorepression of CARDs
signaling
Secondly, we investigated whether Riplet expression affects
RIG-I RD autorepression of CARDs signaling. To test this
possibility, we constructed RIG-I mutant proteins (Figure 4A). We
previously showed that Lys to Ala amino acid substitutions at Lys-
849, 851, 888, 907, and 909 of RIG-I (RIG-I 5KA) severely
reduced RIG-I polyubiquitination and activation [21]. However, it
is possible that the substitutions of Lys with Ala affect other
function of RIG-I because the substitution abolishes positive
charge of Lys residues. Thus, we constructed the RIG-I 5KR
mutant protein, in which the five Lys residues were substituted
with Arg, and examined the signal activation ability. The results
showed that the 5KR mutation reduced RIG-I signaling, however
residual activation of RIG-I 5KR was still detected (Figure 4B–
4D). Thus, we assessed other Lys residues within RIG-I RD.
Because Riplet is essential for RIG-I activation, it is expected that
the Lys residues targeted by Riplet are conserved during evolution.
Thus, we tested Lys residues within RIG-I RD conserved among
vertebrate, and found that an RIG-I K788R mutation reduced
RIG-I signaling at a level comparable to that by a K172R
mutation, which abrogates TRIM25-mediated RIG-I activation
[15,19] (Figure 4B–4D). Interestingly, the 5KR and K788R
Figure 1. Riplet promotes TRIM25-mediated full-length RIG-I activation. (A, B) Wild-type and Riplet KO MEFs were infected with SeV at
MOI = 0.2. The mRNA expressions of IFN-b (A) and –a2 (B) at the indicated times after viral infection were determined by RT-qPCR. Results are
presented as mean 6 SD (n = 3). (C, D) The activation of IFN-b promoter was examined by reporter gene assay using a p125luc IFN-b reporter. RIG-I
CARDs (C) or full-length RIG-I (D) expression vectors were transfected into HEK293 cells together with a Riplet and/or TRIM25 expression vector as
indicated. 24 hours after the transfection, the reporter activities were determined. (E, F) The activation of IFN-b promoter was examined by reporter
gene assay using a p125luc IFN-b reporter. Full-length RIG-I expression vectors were transfected into HEK293 cells together with a Riplet and/or
TRIM25 expression vector as indicated. 24 hours after the transfection, the cells were stimulated with 50 ng of HCV 39 UTR dsRNA by transfection or
infected with VSV at MOI = 1 for 24 hours, and then reporter activation was determined. Data are presented as mean 6 SD (n = 3). *p,0.05. (G)
HEK293FT cells were stimulated with 0.8
mg of HCV double-stranded RNA (HCV RNA) using lipofectamine 2000 in 6-well plate. Cell lysates were
prepared at the indicated times, followed by immunoprecipitation with an anti-RIG-I mAb (Alme-1).
doi:10.1371/journal.ppat.1003533.g001
Riplet Releases RIG-I Autorepression
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Riplet Releases RIG-I Autorepression
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mutations reduced the RIG-I polyubiquitination (Figure 4E). RIG-I
polyubiquitination in cells stimulated with HCV dsRNA was also
reduced by the K788R mutation (Figure 4F). Lys-788 is located
within the 55-amino acid region (747–801 aa) of RIG-I essential for
RD autorepression of CARDs signaling [31] (Figure 4A). It is
possible that the reduction of RIG-I ubiquitination by the mutations
was caused by the defect of RIG-I other functions, such as RNA
binding activity. However, Figure 3C showed that the RIG-I 5KR
and K788R mutant proteins efficiently bound to dsRNA as wild-
type RIG-I. This data weakened the above possibility.
Using the RIG-I mutants, we investigated whether Riplet
releases RIG-I RD autorepression. Because Riplet ectopic
expression activated full-length RIG-I signaling even in the
absence of stimulation (Figure 1D), we could assess whether
Riplet can remove RIG-I RD autorepression by Riplet ectopic
expression study. Due to autorepression, full-length RIG-I
expression weakly activates the signaling compared with RIG-I
CARDs expression [31]. Riplet ectopic expression increased full-
length RIG-I signaling to a level comparable to that of RIG-I
CARDs signaling (Figure 5A), suggesting that Riplet released the
autorepression. Moreover, the K788R substitution canceled this
Riplet ectopic expression effect on RIG-I signaling (Figure 5A).
This suggested that the K788R mutation abrogated the release of
RIG-I RD autorepression but not RNA binding activity.
Expression of the RIG-I RD fragment is known to represses full-
length RIG-I-mediated signaling [3]. Interestingly, Riplet ectopic
expression removed the RD fragment repression effect on RIG-I
signaling (Figure 5B). This Riplet expression effect was canceled by
the K788R amino acid substitution within full-length RIG-I
(Figure 5B).
MDA5 C-terminal region does not function as an RD, and thus
ectopically expressed MDA5 induced IFN-b promoter activity
irrespective of viral infection [3]. If Riplet ectopic expression
released RIG-I RD autorepression, it is expected that RIG-I and
Riplet co-expression will induce IFN-b promoter activity irrespec-
tive of viral infection. As expected, Riplet and RIG-I co-expression
induced IFN-b promoter activity irrespective of SeV infection
(Figure 5C). Taken together, these genetic data indicated that
Riplet released RIG-I RD autorepression.
If Riplet is essential for the release of RIG-I RD autorepression, it
is expected that Riplet expression will increase the interaction
between RIG-I and TRIM25, because TRIM25 efficiently activated
RIG-I CARDs but not full-length RIG-I (Figure 1C and 1D). To test
this possibility, we examined the interaction between TRIM25 and
RIG-I in the presence or absence of Riplet ectopic expression and
found that Riplet expression increased the interaction between
TRIM25 and RIG-I (Figure 5D and 5E). Moreover, this interaction
was abolished by the K788R mutation (Figure 5F). These data were
also consistent with our model that Riplet affects RIG-I RD
autorepression rather than RNA binding activity of RIG-I.
If Riplet is essential for the release of RIG-I RD autorepression
leading to the interaction between TRIM25 and RIG-I, it was
expected that Riplet is essential for endogenous RIG-I K63-linked
polyubiquitination that is mediated by both Riplet and TRIM25.
To test this possibility, we investigated endogenous RIG-I K63-
linked polyubiquitination in mouse spleen cells infected with SeV.
Endogenous RIG-I K63-linked polyubiquitination was increased
after SeV infection in wild-type splenocyte, however knockout of
Riplet abrogated the endogenous K63-linked polyubiquitination of
RIG-I after SeV infection (Figure 5G). Recently, it was reported
that knockdown of Riplet strongly reduced endogenous RIG-I
polyubiquitination in response to SeV infection in a mouse cell line
Hepa 1.6 [32]. Based on our genetic and biochemical data in
Figure 3–5, we concluded that Riplet affects RIG-I RD
autorepression rather than the RNA binding activity.
Riplet is required for the formation of a hetero-protein
complex of RIG-I, TBK1, and IKK-
e
Because Riplet-mediated release of RD autorepression in-
creased the interaction between RIG-I and TRIM25, we
investigated whether the release of RD autorepression also
increased the interaction of RIG-I with other factors. Interestingly,
we found that ectopically expressed IKK-e, TBK1, and NEMO
ubiquitin binding region co-immunoprecipitated with RIG-I RD
(Figure 6A–6D), and Riplet expression enhanced the physical
interactions of RIG-I with TBK1, IKK-e, and the NEMO
ubiquitin binding region (Figure 6D–6G). The physical interac-
tions between these proteins were not through IPS-1, as IPS-1 did
not co-immunoprecipitate with RIG-I RD (Figure 6B).
Microscopy analysis showed that the RIG-I was co-localized
with TBK1 or NEMO in the cytoplasm. (Figures 6H and S2A).
RIG-I and TBK1 was detected in the region where there is no
mitochondria (Figure 6I), and the colocalization of RIG-I with
TBK1 was also detected in the region where there is no
mitochondria (yellow stained region in Figure 6J). This was
consistent with our immunoprecipitation results that RIG-I RD
could bind TBK1 without IPS-1. TBK1 is phosphorylated in its
activation loop [33]. Surprisingly, the phosphorylated TBK1 (p-
TBK1) foci were exclusively localized on mitochondria (Figure 6K
and 6L). Co-localization of RIG-I with p-TBK1 was observed
after dsRNA stimulation (Figure S2B and S2C). We next assessed
the role of endogenous Riplet in the interaction between RIG-I
and TBK1. Immunoprecipitation assay showed that Riplet KO
reduced the interaction between endogenous RIG-I and TBK1 in
mouse spleen cells during VSV infection, indicating that Riplet
promoted the interaction between RIG-I and TBK1 (Figure 6M).
These data indicated that the Riplet function increased the
interaction of RIG-I with TBK1 as well as TRIM25.
Hepatitis C virus protease NS3-4A targets the Riplet
protein
Several viruses have evolved strategies to escape the innate
immunity. For instance, NS1 of influenza A virus inhibits
TRIM25 function. This emphasizes the vital role of TRIM25 in
Figure 2. Riplet and TRIM25 ubiquitin ligases associate with RIG-I. (A, B) HeLa cells were transfected with Riplet-HA expression vector (A) or
FLAG-Riplet and TRIM25-HA (B). 24 hours after transfection, the cells were infected with VSV at MOI = 1 for six hours. The cells were fixed and stained
with anti-RIG-I (Alme-1), HA, and/or FLAG antibodies as indicated. Histograms display the measured fluorescence intensity along the white line in the
merged panels. (C–F) Colocalization coefficients of Riplet localization to RIG-I (C) or TRIM25 (E) in mock or VSV infected HeLa cells. Pearson’s
correlation coefficient of Riplet and RIG-I (D) and TRIM25 (F) (mean 6 SD, n.10). (G, H) HeLa cells were transfected with Riplet-HA (G) or TRIM25-HA
(H) expression vector. The cells were stimulated with 100 ng of short polyI:C for six hours. The cells were fixed and stained with anti-G3BP and HA
antibodies. Colocalization coefficient indicates values (mean 6 SD, n.10) of Riplet (G) or TRIM25 (H) localization to G3BP staining region. Histograms
display the measured fluorescence intensity along the white line in the merged panels. (I, J) TRIM25 (I) or Riplet (J) expression vector was transfected
into HEK293FT cells together with FLAG-tagged-RIG-I CARDs or -RIG-I RD expression vectors. Cell lysate was prepared at 24 hours after transfection,
followed by immunoprecipitation with an anti-FLAG antibody. (K) Riplet, RIG-I, and/or RIG-I-DRD, which lacks RD, expression vector was transfected
into HEK293 cell with p125luc reporter. Reporter activation was determined at 24 hours after transfection. Data are presented as mean 6 SD (n = 3).
doi:10.1371/journal.ppat.1003533.g002
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modulating antiviral response [34]. To assess the biological
significance of Riplet-mediated release of RIG-I RD autorepres-
sion in antiviral innate immune response, we investigated whether
viral protein suppresses this mechanism.
The endogenous Riplet protein level was not affected by
polyI:C, HCV dsRNA stimulations, or by VSV infection
(Figure 7A–7C), however the Riplet protein level was severely
reduced in a human hepatocyte cell line with HCV 1b full-length
replicons (O cells) compared with hepatocyte cell line without
these replicons (O curred cells: Oc cells; Figure 7D), suggesting
that viral protein reduced the Riplet protein level. Riplet knockout
abolished the expression of type I IFN, IP-10, and type III IFN in
response to HCV RNA (Figure 7E), indicating that Riplet was
essential for type I IFNs expression in response to HCV RNA.
Because HCV protease NS3-4A suppresses type I IFN
expression in response to viral infection [7,25], we examined
whether NS3-4A could cleave the Riplet protein. N-terminal
FLAG-tagged Riplet or C-terminal HA-tagged Riplet was
expressed with or without NS3-4A, after which the Riplet protein
levels were compared. NS3-4A expression severely reduced the
FLAG-tagged and HA-tagged Riplet protein level but not that of
FLAG-tagged RIG-I, whereas catalytically inactive NS3-4A*
(S139A) failed to reduce the Riplet protein level (Figure 7F, 7G,
and S3A). This suggested that this protease’s activity reduced the
Riplet protein level. Although NS3-4A reduced IPS-1 protein level
as previously reported, NS3-4A did not reduce the TRIM25 and
IKK-e protein levels (Figure 7H, 7I, S3B–D).
Within the Riplet RING-finger domain is a sequence that is
similar to the NS3-4A target consensus sequence (D/E-x-x-x-x-C/T-
S/A; Figure 7J) [35]. NS3-4A cleaves the target just after C/T within
this consensus sequence. Acidic amino acids before the C/T site are
conserved among the NS3-4A cleavage site within HCV polypep-
tide. The acidic amino acids from 16 to 18 aa within the Riplet
sequence were substituted with Ala, and a Riplet-3A mutant protein
was constructed (Figure 7J). The Riplet-3A mutant protein was
resistant to NS3-4A (Figure 7I and 7K). Moreover, the Riplet-3A
protein co-localized with NS3-4A in cytoplasm (Figure 7L). Inter-
estingly, recombinant NS3-4A that was purified from E. coli cleaved
the immunoprecipitated FLAG-tagged Riplet protein and recombi-
nant GST-fused Riplet protein purified from E.coli, and the cleaved
fragments were detected at expected size (Figure 7M and 7N). These
data indicated that NS3-4A directly targeted the Riplet protein.
Although Riplet digestion products were not observed in
HEK293 cell lysate (Figure S3E and S3F), it is known that the
digestion products of TICAM-1 (TRIF) obtained by NS3-4A are
not detectable because these products are unstable [28]. Cys-21 of
Riplet corresponds to the C/T site in the NS3-4A target consensus
sequence. The Cys-21 residue is the first Cys in the RING-finger
motif; thus a C21A substitution causes the disruption of RING
finger domain structure [36]. The Riplet-C21A mutant protein
was unstable and barely detectable (Figure S3G), which suggested
that the loss of Cys-21 destabilized the Riplet protein.
HCV protease NS3-4A abolishes an early step of RIG-I
activation
To determine if NS3-4A abolishes RIG-I activation by
disrupting Riplet, we examined RIG-I ubiquitination and
Figure 3. Riplet function is dispensable for RIG-I RNA binding
activity. (A) Expression vectors encoding Riplet, FLAG-tagged RIG-I
CARDs, and/or FLAG-tagged RIG-I RD were transfected into HEK293FT
cells together with an HA-tagged ubiquitin expression vector. Cell
lysate was pre pared at 24 hours after transfection, follow ed by
immunoprecipitation with an anti-FLAG (RIG-I) antibody. (B) HEK293FT
cells were transfected with expression vectors encoding FLAG-tagged
RIG-I, Riplet, and HA-tagged ubiquitin. Cell lysate was prepared at
24 hours after transfection, and then incubated with biotin-conjugated
(Biotin-dsRNA) or non-conjugated (dsRNA) double-stranded RNA.
Biotin-dsRNA was pull-downed with streptavidin beads. Samples were
subjected to SDS-PAGE, and proteins were detected by western
blotting. (C) HEK293FT cells were transfected with FLAG-tagged wild-
type RIG-I, RIG-I 5KR, or RIG-I K788R expression vector. 24 hours after
the transfection, the cell lysate was prepared. The pull down assay with
biotin-dsRNA was performed as described above.
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interaction between RIG-I and TRIM25. Riplet-mediated
ubiquitination of full-length RIG-I or RIG-I RD was abolished
by NS3-4A expression (Figure 8A and 8B). K63-linked poly-
ubiquitination of RIG-I RD in SeV infected cells were also
reduced by NS3-4A expression (Figure 8C). Moreover, NS3-4A
expression reduced the interaction between TRIM25 and full-
length RIG-I (Figure 8D).
An IPS-1-C508A mutant protein is resistant to NS3-4A
cleavage [7]. A reporter gene assay showed that NS3-4A
expression severely reduced IFN-b promoter activation induced
by RIG-I and Riplet expression even in the presence of the IPS-1
C508A mutant protein in HEK293 cell (Figure 9A), and a
catalytically inactive NS3-4A mutant failed to reduce this signal
(Figure 9B). These data were consistent with our observation that
NS3-4A targeted the Riplet protein. There was endogenous IPS-1
in HEK293 cell, and thus the decrease of IFN-b promoter
induction by NS3-4A in HEK293 cell could be due to the cleavage
of endogenous IPS-1 by NS3-4A. To exclude this possibility, we
next used IPS-1 KO mouse hepatocyte [37]. The IPS-1 C508A
mutant protein was expressed in IPS-1 KO hepatocyte. NS3-4A
expression reduced the reporter activation induced by RIG-I/
Riplet expression or by stimulation with HCV dsRNA even in the
presence of IPS-1 C508A (Figure 9C and 9D). These results
indicated that NS3-4A targeted both IPS-1 and upstream factors
of IPS-1 and were consistent with our observation that NS3-4A
reduced the Riplet protein level. We could not test whether NS3-
4A failed to impair IFN-b promoter activity in the presence of the
Riplet-3A and Riplet C21A mutants because the mutant proteins
were not functional and failed to activate RIG-I (Supplemental
Figure S3H and S3I). Thus, we could not exclude the possibility
that NS3-4A targeted another protein in addition to Riplet and
IPS-1.
Hepatitis C virus abrogates endogenous Riplet function
Endogenous RIG-I exhibited punctate staining in the human
hepatocyte cell line HuH7 and HepG2 cells after simulation with
Figure 4. Lys residues within RIG-I RD were essential for Riplet-mediated K63-linked polyubiquitination. (A) Schematic diagram of RIG-I
mutant proteins. (B–D) IFN-b promoter activation was determined using a p125luc IFN-b reporter gene. HEK293 cells were transfected with 0.1
mgof
expression vectors encoding full-length RIG-I, RIG-I K172R, RIG-I 5KR, and RIG-I K788R in 24-well plate. The transfected cells were stimulated with
0.1
mg of HCV 39 UTR dsRNA using lipofectamine 2000 reagent for eight hours (C) or infected with VSV at MOI = 1 for 24 hours (D), after which
reporter activation was determined. Data are presented as mean 6 SD (n = 3). *p,0.05. (E) The expression vectors encoding HA-tagged Riplet and
FLAG-tagged RIG-I, RIG-I 5KR, and RIG-I K788R were transfected into HEK293FT cells together with Myc-tagged K63-only ubiquitin expression vector,
in which all Lys residues except Lys-63 within ubiquitin were replaced with Arg. At 24 hours after transfection, cell lysates were prepared, followed by
immunoprecipitation with an anti-FLAG (RIG-I) antibody. (F) The expression vectors encoding FLAG-tagged wild-type and mutant RIG-I proteins were
transfected into HEK293FT cells together with HA-tagged ubiquitin expression vector. 24 hours after the transfection, the cells were stimulated with
HCV 39 UTR dsRNA by transfection for eight hours. Then cell lysate was prepared and immunoprecipitation was performed with anti-FLAG antibody.
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HCV RNA (Figure 9E) but not in HuH7.5 cell line (Figure 9E). In
HuH7.5 cells, there is a T55I mutation within endogenous RIG-I
gene that disrupts the interaction between RIG-I and TRIM25
[38]. We investigated whether Riplet is required for RIG-I to
exhibit punctate staining, and found that knockdown of Riplet
decreased RIG-I punctate staining induced by HCV RNA
(Supplemental Figure S4). We next investigated whether HCV
abrogated Riplet-dependent RIG-I punctate pattern in the
cytoplasm. As expected, RIG-I failed to exhibit punctate staining
in O cells with HCV replicons in NS3 positive cells and HuH7
Figure 5. Riplet affects RIG-I RD autorepression of CARDs signaling. (A, B) IFN-b promoter activation was determined using a p125luc IFN-b
reporter gene. Expression vectors encoding RIG-I CARDs, full-length RIG-I, RIG-I K788R, RD, RD-K788, and/or Riplet were transfected into HEK293 cells
as indicated. Reporter activation was determined after transfection. Data are presented as mean 6 SD (n = 3). *p,0.05. (C) IFN-b promoter activation
was determined using a p125luc IFN-b reporter gene. RIG-I and Riplet expression vectors were transfected into HEK293 cells as indicated. 24 hours
after transfection, cells were infected with SeV at MOI = 1 for 24 hours, and the reporter activities were determined. (D–F) HEK293FT cells were
transfected with expression vectors encoding FLAG-tagged RIG-I, HA-tagged TRIM25, HA-tagged Riplet, FLAG-tagged RIG-I K788R as indicated. Cell
lysate was prepared at 24 hours after transfection, followed by immunoprecipitation with an anti-FLAG antibody (RIG-I). Relative band intensity of
immunoprecipitated TRIM25 in panel D was determined (E). (G) Mouse splenocyte was isolated from wild-type and Riplet KO mice spleen. The cells
were infected with SeV, and then cell lysate was prepared. Immunoprecipitation was carried out with anti-RIG-I rabbit mAb (D14G6), and subjected to
SDS-PAGE. Endogenous K63-linked polyubiquitin chain was detected using K63-linked polyubiquitin chain specific antibody.
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cells infected with HCV JFH1 (Figure 9F–9H). We confirmed that
HCV disrupted IPS-1 in our experimental condition (Figure 9G
and 9H).
To further assess whether HCV abrogates endogenous Riplet
function, we observed endogenous K63-linked polyubiquitination
of RIG-I in cells with HCV replicons. Although SeV infection
induced endogenous K63-linked polyubiquitination of RIG-I in
HuH7 cells, HCV replicons failed to increase the polyubiquitina-
tion (Figure 9I). Next, we investigated the association of
endogenous RIG-I with TRIM25 and TBK1, which is promoted
by Riplet as shown in Figure 5 and 6. SeV infection induced the
association of endogenous RIG-I with TRIM25 and TBK1,
whereas HCV replicons failed to induce the association (Figure 9J).
Taken together, these data indicated that HCV abrogated
endogenous Riplet function.
Although NS3-4A cleaves IPS-1, a mutation within endogenous
RIG-I gene increased the permissiveness to HCV infection in
HuH7-derived cells [3], indicating that RIG-I is required for
antiviral response to HCV infection before NS3-4A cleaves IPS-1.
We used siRNA to knockdown endogenous Riplet in HuH7 cells,
and then the cells were infected with HCV JFH1. Interestingly,
Riplet knockdown increased the permissiveness to HCV JFH1
infection (Figure 9K), indicating that endogenous Riplet is
required for antiviral response to HCV infection.
Discussion
RIG-I activation is regulated by two ubiquitin ligases Riplet and
TRIM25 [15,21]. The two ubiquitin ligases are essential for RIG-I
activation [15,23], however the functional difference had been
unclear. It is known that TRIM25 is essential for RIG-I
oligomerization and association with IPS-1 adaptor molecule
[15,19]. Here, we demonstrated that Riplet was essential for the
release of RIG-I RD autorepression of its CARDs, which resulted
in the association with TRIM25. This functional difference
explained the reason why RIG-I requires the two ubiquitin ligases
for triggering the signal.
It has been reported that TRIM25 activates RIG-I signaling
[15,19]. We confirmed that ectopic expression of TRIM25
increases RIG-I CARDs-mediated signaling. However, most
previous studies used a RIG-I CARDs fragment but not full-
length RIG-I [15,19,38]. Unexpectedly, we found that the
increase of full-length RIG-I-mediated signaling by TRIM25
expression was much less than that of the CARDs-mediated
signaling. It is intriguing that Riplet helped TRIM25 to activate
full-length RIG-I. Riplet expression promoted the interaction
between TRIM25 and full-length RIG-I, and this interaction was
abrogated by an RIG-I K788R mutation, which reduced Riplet-
mediated RIG-I ubiquitination. Thus, we propose that Riplet-
mediated polyubiquitination of RIG-I RD is a prerequisite for
TRIM25 to activate RIG-I (Figure 10).
Ectopic expression of Riplet activated RIG-I without stimula-
tion with RIG-I ligand. This is not surprising because ectopically
expressed Riplet bound to RIG-I without stimulation with RIG-I
ligand, whereas endogenous Riplet bound to endogenous RIG-I
after stimulation with RIG-I ligand. RIG-I undergoes its
conformational change after binding to a ligand [3,39]. The
conformational change would allow the access of endogenous
Riplet to RIG-I, which resulted in Riplet-mediated K63-linked
polyubiquitination leading to the release of RD autorepression.
This model is consistent with the observation that TRIM25
ectopic expression did not activate full-length RIG-I without
Riplet expression, because TRIM25 hardly bound to full-length
RIG-I without Riplet.
Previously, we reported that the five Lys residues within RIG-I
RD were important for Riplet-mediated RIG-I ubiquitination. We
constructed the RIG-I 5KR mutant and indicated that the 5KR
mutation reduced RIG-I ubiquitination and activation without loss
of RNA binding activity. This is consistent with our previous
conclusion. However, there is residual ubiquitination of RIG-I
5KR mutation, and we found that K788R mutation showed more
sever phenotype. These data indicated that Riplet targeted the
several Lys residues within RIG-I RD. This is not surprising,
because TRIM25 targets not only Lys-172 but also other Lys
residues within mouse RIG-I CARDs [32].
TBK1 and IKK-e are downstream factors of IPS-1. We found
that TBK1 and IKK-e could bind RIG-I RD. It is possible that
RIG-I associates with TBK1 through IPS-1. However, Hiscott J
and colleagues demonstrated that IKK-e could bind IPS-1 and
that TBK1 did not bind IPS-1 [40]. Moreover, RIG-I RD did not
bind IPS-1, and RIG-I and TBK1 co-localization was detected in
the cytoplasmic region where there are no mitochondria. These
observations weaken this possibility. Our results indicated that
RIG-I RD bound to the NEMO ubiquitin binding region. IRF-3
activation requires the ubiquitin binding domain of NEMO, and
an endogenous K63-linked polyubiquitin chain plays a key role in
IRF3 activation [41]. Thus, we prefer a model in which TBK1
associates with an RIG-I RD-anchored polyubiquitin chain
through NEMO (Figure 10). Although Riplet knockout reduced
the binding of RIG-I to TBK1, residual binding was still
detectable. Thus, there appears to be Riplet-dependent and
independent associations between RIG-I and TBK1. TRAF3 is an
E3 ubiquitin ligase, and is involved in the RIG-I-mediated type I
IFN production pathway [42]. Because there is residual activation
of the type I IFN production pathway even in TRAF3 knockout
cells [41], it is possible that the RIG-I polyubiquitin chain may
compensate for the TRAF3 defect in recruiting TBK1 to
mitochondria. Further studies will be needed to determine the
precise molecular mechanisms. Although TBK1 dispersed in the
cytoplasm, p-TBK1 was exclusively localized on mitochondria.
Considering that TBK1 is phosphorylated in its activation loop
[33], these results suggested that RIG-I RD associated with
Figure 6. The association of TBK1 and IKK-e protein kinases with RIG-I RD is enhanced by Riplet. (A–F) The interaction of RIG-I with TBK1,
IKK-e, and NEMO was examined by immunoprecipitation assay. FLAG-tagged RIG-I or RIG-I RD expression vector was transfected into HEK293FT cells
together with HA-tagged IKK-e (A, B, and E), Myc-tagged TBK1 (C, F), HA-tagged NEMO ubiquitin binding region (D), and/or Riplet (D–F) expression
vectors as indicated. 24 hours after the transfection, cell lysate was prepared, and immunoprecipitation was performed with anti-FLAG antibody.
Asterisk indicates non-specific bands. (G) Relative band intensity of immunoprecipitated NEMO, IKK-e, and TBK1 in D–F was determined. (H–L)
Intracellular localization of endogenous RIG-I (H–J), TBK1 (H–J), phosphorylated-TBK1 (p-TBK1) (K), and mitochondria (I–K) were observed using anti-
RIG-I (Alme-1), TBK1, p-TBK1 mAbs, and mitotracker. HeLa cells were stimulated with HCV dsRNA for six hours using lipofectamine 2000.
Colocalization coefficient of TBK1 localization to RIG-I (H), RIG-I and TBK1 localization to mitochondria (I), and TBK1 and p-TBK1 localization to
mitochondria (L) were determined (mean 6 sd, n.10). Person’s correlation coefficient of RIG-I and TBK1 was determined (H). (M) Splenocytes from
wild type and Riplet KO mouse were infected with VSV at MOI = 10 for eight hours. Immunoprecipitation was performed using an anti-RIG-I rabbit
monoclonal antibody (D14G6), and the immunoprecipitates were analyzed by SDS-PAGE. Endogenous RIG-I, TBK1, TRIM25, and b-actin were detected
using anti-RIG-I, p-TBK1, TRIM25, and b-actin antibodies.
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Figure 7. NS3-4A of HCV targets the Riplet protein. (A–D) Endogenous RIG-I and Riplet protein levels were observed by western blotting. HeLa
cells were stimulated with polyI:C transfection (A), HCV dsRNA transfection (B) or infected with VSV (C). HCV replicon positive (HCV) and negative (-)
cell lysates were prepared from a HuH7-derived cell line O cell that contains HCV 1b full-length replicons and O curred cell (Oc cell) in which HCV
replicons were removed by IFN treatment (D). (E) The response to HCV RNA in wild-type and Riplet KO MEFs was examined by RT-qPCR. Wild type
(WT) and Riplet knockout (KO) MEF cells were transfected with 100 ng of HCV ssRNA and dsRNA. Six hours after stimulation, mRNA expressions of
IFN-a2, IP10, and IFN-l2/3 were measured by RT-qPCR. Data are presented as mean 6 SD (n = 3). *p,0.05. (F–H) FLAG-tagged Riplet and RIG-I (F), HA-
tagged Riplet (G), or HA-tagged TRIM25 (H) expression vectors were transfected into HEK293FT cells together with NS3-4A or NS3-4A* expression
vectors. NS3-4A* mutant protein harbors an amino acids substitution at its catalytic site Ser-139 with Ala. 24 hours after transfection, cell lysate was
prepared and subjected to SDS-PAGE. (I) Band intensity ratio of IPS-1, Riplet, TRIM25, IKK-e, and Riplet-3A with/without NS3-4A expression (mean 6
sd, n = 3). (J) NS3-4A cleavage sites within an HCV polypeptide are compared with a candidate site in the Riplet RING-finger domain. Homologous
amino acids are shown in bold, and identical amino acids are underlined. In Riplet-3A mutant protein, three acidic amino acids, Glu-16, Asp-17, Asp-
18, were substituted with Ala. (K) An expression vector encoding wild-type Riplet or Ripled-3A mutant protein was transfected into HEK293 cells
together with NS3-4A or NS3-4A* expression vectors. Cell lysate was prepared 24 hours after transfection, and subjected to SDS-PAGE. (L) HA-tagged
Riplet-3A and NS3-4A expression vectors were transfected into HepG2 cell. 24 hours after transfection, the cells were fixed and stained with anti-HA
monoclonal antibody (mouse) and anti-NS3-4A polyclonal antibody (goat). (M) N-terminal FLAG-tagged Riplet was expressed in HEK293FT cells, and
immunoprecipitation was carried out with anti-FLAG antibody. Immunoprecipitates were incubated with recombination NS3-4A purified from E.coli
at 37uC for one hour, and samples were subjected to SDS-PAGE analysis. The proteins were detected by western blotting. (N) Purified GST fused Riplet
(1–210 aa) was incubated with or without recombinant NS3-4A (rNS3-4A) at 37uC for 30 min. The proteins were subjected to SDS-PAGE and detected
by western blotting.
doi:10.1371/journal.ppat.1003533.g007
Figure 8. NS3-4A inhibits Riplet-mediated RIG-I polyubiquitination. (A, B) Riplet, NS3-4A, and/or HA-tagged ubiquitin (HA-Ub) expression
vectors were transfected into HEK293FT cells along with either full-length RIG-I (A) or RIG-I RD (B). Cell lysate was prepared 24 hours after transfection,
and subjected to SDS-PAGE. The proteins were detected by western blotting. (C). HEK293FT cells were transfected with Myc-tagged K63-only
ubiquitin, FLAG-tagged RIG-I RD, and/or NS3-4A expression vectors. 24 hours after the transfection, cells were infected with SeV for six hours, and
then cell lysate was prepared. Immunoprecipitation was carried out with anti-FLAG antibody, and the samples were subjected to SDS-PAGE. (D) HA-
tagged TRIM25, Riplet and/or FLAG-tagged RIG-I expression vectors were transfected into HEK293FT cells with or without NS3-4A expression vector.
Cell lysate was prepared 24 hours after the transfection, and immunoprecipitation assay was performed with anti-FLAG antibody. The precipitates
were subjected to SDS-PAGE.
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inactive TBK1 and that TBK1 was activated after loading on to
mitochondria (Figure 10).
HCV is a major cause of HCC and has the ability to evade host
innate immune response [7,43]. HCV RNA is primarily recog-
nized by the cytoplasmic viral RNA sensor RIG-I. Previous studies
showed that the protease NS3-4A cleaves IPS-1 to shut off RIG-I
signaling. However, our results indicated that there was another
target of NS3-4A in RIG-I signaling. First, RIG-I failed to exhibit
punctate staining in cells infected with HCV. Second, NS3-4A
reduced RIG-I signaling even in the presence of an IPS-1-C508A
mutant, which is resistant to the cleavage by NS3-4A. Third, the
endogenous Riplet protein level was severely reduced in cells with
HCV replicons. Fourth, NS3-4A targeted Riplet and abrogated
Riplet-dependent RIG-I ubiquitination and complex formation
with TRIM25 and TBK1. These data support our model that
NS3-4A targets not only IPS-1 but also Riplet to escape host
innate immune responses (Figure 10). Recently it was reported that
NS1 proteins of Influenza A virus inhibited Riplet function [32].
These findings indicated biological importance of Riplet in RIG-I
activation during viral infection.
In general, a ubiquitin ligase has several targets. We have
performed yeast two-hybrid screening using Riplet as bait and
found a candidate clone that encodes a tumor suppressor gene.
Our pilot study showed that Riplet mediated K63-linked
polyubiquitination of this tumor suppressor and suppressed
retinoblastoma (Rb) activity. Thus, Riplet disruption by NS3-4A
might be a cause of liver disease induced by HCV infection.
Materials and Methods
Ethics statement
All animal studies were carried out in strict accordance with
Guidelines for Animal Experimentation of the Japanese Associa-
tions for Laboratory Animal Science. The protocols were
approved by the Animal Care and Use Committee of Hokkaido
University, Japan (Permit Number: 08-0245 and 09-0215).
Cell
HEK293, Vero, and HepG2 cells were cultured in Dulbecco’s
modified Eagle’s medium low glucose medium (D-MEM) with
10% heat-inactivated fetal calf serum (FCS) (Invitrogen). HeLa
cells were cultured in minimum Eagle’s medium with 2 mM L-
glutamine and 10% heat-inactivated FCS. HEK293FT cells were
maintained in D-MEM high glucose medium containing 10% of
heat-inactivated FCS (Invitrogen). Human hepatocyte cell line
with HCV 1b full-length replicons (O cells) and O curred cells (Oc
cells) were kindly gifted from Kato N [44]. O cells were cultured in
D-MEM high glucose with 10% of heat-inactivated FCS, G418,
NEAA, and L-Gln.
Viruses
VSV Indiana strain and SeV HVJ strain were amplified using
Vero cells. To determine the virus titer, we performed plaque assay
using Vero cells. HCV JFH1 was amplified using HuH7.5 cells.
Mice
Gener ation of IPS-1 KO and Ri plet K O mice were des-
cribed previously [23,45]. Splenocyte was isolated from C57BL/6
wild-type and Riplet KO mice. Isolated cells were cul tured in
RPMI1640 containing 10% of heat-inactivated FCS. The
preparations of wild-type and Riplet KO MEFs were described
previously [23]. Preparation of IPS-1 KO mouse hepatocyte
was described previously [37]. All m ice were maintained
under spe cific-pathogen free conditions in the animal facility
of the Hokkaido University Graduate School of Science
(Japan).
Plasmids
Expression vectors encoding for N-terminal FLAG-tagged RIG-
I, N-terminal FLAG-tagged RIG-I CARDs (dRIG-I), FLAG-
tagged RIG-I DRD (RIG-I-dRD), FLAG-tagged RIG-I RD, C-
terminal HA-tagged TRIM25, C-terminal HA-tagged Riplet, and
Riplet-DRING (Riplet-DN) plasmids were described previously
[21,23]. The amino acids substitutions from 16 to 18 with Ala was
carried out by PCR-mediated mutagenesis using primers, Ripelt-
3A-F and Riplet-3A-R and pEF-BOS/Riplet plasmid as a
template. The primer sequence is Riplet-3A-F: TTC CCG TGT
GGC TGG CCG CGG CCG CCC TCG GCT GCA TCA TCT
GCC, and Riplet-3A-R: GGC AGA TGA TGC AGC CGA
GGG CGG CCG CGG CCA GCC ACA CGG GAA. RIG-I
K172R and RIG-I K788R expression vectors was constructed by
PCR-mediated mutagenesis using primers, RIG-I K172R-F, RIG-
I K172R-R, RIG-I K788R-F and RIG-I-K788R-R, and pEF-
BOS/FLAG-RIG-I plasmid as a template. The primer sequences
are RIG-I K172R-F: GGA AAA CTG GCC CAA AAC TTT
GAG ACT TGC TTT GGA GAA AG, RIG-I K172R-R: CTT
TCT CCA AAG CAA GTC TCA AAG TTT TGG GCC AGT
TTT CC, RIG-I-K788R-F: TGC ATA TAC AGA CTC ATG
AAA GAT TCA TCA GAG ATA GTC AAG AA, and RIG-I-
K788-R: CTT GAC TAT CTC TGA TGA ATC TTT CAT
GAG TCT GTA TAT GCA G. RIG-I 5KR expression vectors
were constructed by PCR-mediated mutagenesis using primers,
RIG-I 849 851 RR-F, RIG-I 849 851 RR-R, RIG-I 888R-F,
RIG-I 888R-R, RIG-I 907 909 RR-F, RIG-I 907 909 RR-R, and
Figure 9. HCV abrogated Riplet-mediated RIG-I activation. (A and B) The inhibition of IFN-b promoter activation by NS3-4A was assessed by
reporter gene assays. IPS-1-C508A mutant protein harbors an amino acid substitution at Cys-508 with Ala. 100 ng of IPS-1, IPS-1-C508A, RIG-I, Riplet,
NS3-4A, and/or NS3-4A* expression vectors were transfected into HEK293 cells in 24-well plates with p125luc reporter plasmid. The total amount of
transfected DNA (800 ng/well) was kept constant by adding empty vector (pEF-BOS). 24 hours after the transfection, the reporter activities were
measured. Data are presented as mean 6 SD (n = 3). *p,0.05. (C and D) IPS-1 KO mouse hepatocyte was transfected with IPS-1 C508A, RIG-I, Riplet, and/
or NS3-4A expression vectors together with p125luc and Renilla luciferase plasmids. Transfected cells were stimulated with 50 ng of HCV dsRNA for
24 hours by transfection (D). Data are presented as mean SD (n = 3). *p,0.05. (E and F) Intracellular localizations of endogenous TBK1 and RIG-I were
determined by confocal microscopy. HepG2, HuH7, and HuH7.5 cells were stimulated with 100 ng of HCV dsRNA for six hours by transfection (E).
Stimulated cells (E) and O cells with HCV replicons (F) were stained with anti-RIG-I, TBK1, and/or NS3 antibodies. (G and H) HuH7 (G) and HuH7.5 (H) cells
were infected with HCV JFH1 strain. Seven days after the infection, the cells were stained with anti-RIG-I, IPS-1, and NS3 antibodies. (I) HuH7 cells were
infected with SeV at MOI = 1 for 24 hours. Cell lysates were prepared from mock or SeV infected HuH7 or HuH7 cells with HCV replicons (O cell).
Immunoprecipitation using high salt buffer was performed with anti-RIG-I (Alme-1) antibody. The samples were subjected to SDS-PAGE. Endogenous
K63-linked polyubiquitin chain was detected using ubiquitin K63-linkage specific antibody. (J) HuH7 cells were infected with SeV at MOI = 1 for 24 hours.
Cell lysates were prepared from mock or SeV infected HuH7 or HuH7 cells with HCV replicons (O cell). Immunoprecipitation was performed with anti-
RIG-I (Alme-1) antibody. The samples were subjected to SDS-PAGE. (K) HuH7 cells were transfected with siRNA for mock or Riplet. 48 hours after the
transfection, cells were infected with HCV JFH1 for 2 days. RT-qPCR was performed to determine HCV genome RNA, GAPDH, and Riplet expression.
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pEF-BOS/FLAG-RIG-I plasmid as a template. The primer
sequences are RIG-I 849 851 RR-F: AGT AGA CCA CAT
CCC AGG CCA AGG CAG TTT TCA AGT TTT G, RIG-I
849 851 RR-R: CAA AAC TTG AAA ACT GCC TTG GCC
TGG GAT GTG GTC TAC T, RIG-I 888R-F: GAC ATT TGA
GAT TCC AGT TAT AAG AAT TGA AAG TTT TGT GGT
GGA GG, RIG-I 888R: CCT CCA CCA CAA AAC TTT CAA
TTC TTA TAA CTG GAA TCT CAA ATG TC, RIG-I 907
909RR-F: GTT CAG ACA CTG TAC TCG AGG TGG AGG
GAC TTT CAT TTT GAG AAG, RIG-I 907 909RR-R: CTT
CTC AAA ATG AAA GTC CCT CCA CCT CGA GTA CAG
TGT CTG AAC. HCV cDNA fragment encoding NS3-4A of
JFH1 strain was cloned into pCDNA3.1 (-) vector. The mutation
on catalytic site of NS3-4A S139A was constructed by PCR-
mediated mutagenesis using primers, NS3-4A S139A-F and NS3-
4A S139A-R, and pCDNA3.1 (-)/NS3-4A plasmid as a template.
The primer sequences are NS3-4A S139A-F: TTC GAC CTT
GAA GGG GTC CGC GGG GGG ACC GGT GCT TTG C
and NS3-4A S139A-R: AAG CAC CGG TCC CCC CGC GGA
CCC CTT CAA GGT CGA AAG G.
RT-PCR and Real-Time PCR
Total RNA was extracted with TRIZOL (Invitrogen), after
which the samples were treated with DNaseI to remove DNA
Figure 10. Model for Riplet-mediated RIG-I activation. In resting cell, RIG-I RD represses its CARDs-mediated signaling. When RIG-I CTD
associates with viral RNA, Riplet mediates K63-linked polyubiquitination of RIG-I RD, leading to the association with TRIM25 and TBK1. K63-linked
polyubiquitin chain mediated by TRIM25 induces RIG-I oligomerization and association with IPS-1 adaptor. TBK1 associated with RIG-I is activated on
mitochondria.
doi:10.1371/journal.ppat.1003533.g010
Riplet Releases RIG-I Autorepression
PLOS Pathogens | www.plospathogens.org 15 August 2013 | Volume 9 | Issue 8 | e1003533
contamination. Reverse transcription was performed using High
Capacity cDNA Reverse Transcription Kit (ABI). Quan titative
PCR analysis was performed using Step One software ve2. 0.
(ABI) with SYBER Green Master Mix (ABI). HCV ss and dsRNA
was in vitro synthesized with SP6 and/or T7 RNA polymerase
using 39 UTR of HCV cDNA as template as described previously
[46].
Confocal microscopy
Cells were plated onto microscope cover glasses (matsunami) in
a 24-well plate. The cells were fixed for 30 min using 3%
formaldehyde in PBS and permeabilized with 0.2% Triton X-100
for 15 min. Fixed cells were blocked with 1% bovine serum
albumin in PBS for 10 min and labeled with the indicated primary
Abs for 60 min at room temperature. Alexa-conjugated secondary
Abs were incubated for 30 min at room temperature to visualize
staining of the primary Ab staining. Samples were mounted on
glass slides using Prolong Gold (Invitrogen). Cells were visualized
at a magnification of 663 with an LSM510 META microscope
(Zeiss). Data collected with confocal microscopy were analyzed
with ZEISS LSM Image Examiner software. NS3, RIG-I, TBK1,
IPS-1, and p-TBK1 were stained with anti-NS3 goat pAb (abcam),
anti-RIG-I mouse mAb (Alme-1, ALEXIS BIOCHEMICALS),
anti-NAK (TBK1) rabbit mAb (EP611Y, abcam), anti-MAVS
(IPS-1) rabbit pAb (Bethyl Laboratories Inc), and anti-p-TBK1
rabbit mAb (Cell Signaling Technology),
Reporter gene analysis
HEK293 cells were transiently transfected in 24-well plates
using FuGene HD (Promega) or lipofectamine 2000 (Invitrogen)
with expression vectors, reporter plasmids (IFN-b: p125luc), and
an internal control plasmid coding Renilla luciferase. The total
amounts of plasmids were normalized using an empty vector. Cells
were lysed in a lysis buffer (Promega), and luciferase and Renilla
luciferase activities were determined using a dual luciferase assay
kit (Promega). Relative luciferase activities were calculated by
normalizing the luciferase activity by control. HCV dsRNA (39
UTR polyU/UC region) was synthesized using T7 and SP6 RNA
polymerase as described previously [46].
Pull-down assay
RNA used for the assay was purchased from JBioS. The RNA
sequences are as follows: (sense strand) AAA CUG AAA GGG
AGA AGU GAA AGU G; and (antisense strand) CAC UUU
CAC UUC UCC CUU UCA GUU
U. Biotin was conjugated at
the U residue at the 39-end of the antisense strand (underlined).
Biotinylated dsRNA was phosphorylated by T4 polynucleotide
kinase (TAKARA). dsRNA was incubated for one hour at 25uC
with 10
mg of protein from the cytoplasmic fraction of cells that
were transfected with Flag-tagged RIG-I, Riplet, and/or HA-
tagged ubiquitin expressing vectors. This mixture was added into
400
ml of lysis buffer (20 mM Tris-HCl pH 7.5, 150 mM NaCl,
1 mM EDTA, 10% Glycerol, 1% NP-40, 30 mM NaF, 5 mM
Na
3
VO
4
, 20 mM iodoacetamide, and 2 mM PMSF) containing
25
ml of streptavidine Sepharose beads, rocked at 4uC for two
hours, harvested by centrifugation, washed three times with lysis
buffer, and resuspended in SDS sample buffer.
Immunoprecipitation
Splenocytes (1610
7
) were infected with or without VSV at
MOI = 10 for eight hours, after which cell extracts were prepared
with lysis buffer (20 mM Tris-HCl pH 7.5, 150 mM NaCl, 1 mM
EDTA, 10% glycerol, 1% Nonidet P-40, 30 mM NaF, 5 mM
Na
3
VO
4
, 20 mM iodoacetamide, and 2 mM phenylmethylsulfo-
nyl fluoride). Immunoprecipitation used an anti-RIG-I Rabbit
monoclonal antibody (D14G6, Cell Signaling Technology). To
detect endogenous K63-linked polyubiquitin chain that is ligated
to RIG-I, 6610
7
of mouse splenocyte were infected with SeV at
MOI = 0.2 for 24 hours. Immunoprecipitation was performed
with anti-RIG-I mAb (D14G6). Anti-K63-linkage specific poly-
ubiquitin (D7A11) Rabbit mAb (Cell Signaling) was used for
western blotting. HEK293FT cells were transfected with or
without 0.8
mg of HCV dsRNA in a 6-well plate. HCV dsRNA
(HCV 39 UTR polyU/UC region) was synthesized using T7 and
SP6 RNA polymerase as previously described [46]. Cell lysates were
prepared at the indicated times. Immunoprecipitation was per-
formed with an anti-RIG-I mouse monoclonal antibody (Alme-1).
An anti-FLAG M2 monoclonal antibody (Sigma) was used for the
immunoprecipitation of FLAG-tagged protein. An anti-TRIM25
rabbit polyclonal antibody (abcam), an anti-p-TBK1 rabbit mAb
(Cell Signaling Technology), an anti-NAK (TBK1) rabbit mAb
(EP611Y), and an anti-RNF135 (Riplet) pAb (SIGMA), were used
for western blotting. For ubiquitination assay, immunoprecipitates
were washed three times with high salt lysis buffer ((20 mM Tris-
HCl pH 7.5, 1M NaCl, 1 mM EDTA, 10% glycerol, 1% Nonidet
P-40, 30 mM NaF, 5 mM Na
3
VO
4
, 20 mM iodoacetamide, and
2 mM phenylmethylsulfonyl fluoride) to dissociate unanchored
polyubiquitin chain [21], and then washed once with normal lysis
buffer described above for SDS-PAG analysis. Band intensity was
semi-quantified using Photoshop software.
RNAi
siRNAs for human Riplet (Silencer Select Validated siRNA) and
negative control were purchased from Ambion. siRNA sequences
for Riplet are: (sense) GGA ACA UCU UGU AGA CAU Utt and
(anti-sense) AAU GUC UAC AAG AUG UUC CCac. siRNA was
transfected into cells using RNAiMax Reagent (Invitrogen)
according to the manufacture’s instructions.
In vitro NS3/4A cleavage assay
FLAG-tagged Riplet was expressed in HEK293 FT cells , and
cell l ysate was prepared with the lysis buffer described above.
The protein was immunoprecipitated with anti-FLAG antibody
and protein G sepharose beads, and washed with Buffer B
(20 mM Tr is-HCl pH 7.5, 150 mM NaCl, 10% glycerol, 1%
Nonidet P-40). The samples were susp ended in 50
mlof
Buffer B, and incubated with 400 ng of recombinant NS3-4A
(rNS3-4A) protein at 37uC for one hour, and then subjecte d to
SDS-PAGE analysis. The NS3-4A protein was purchased fr om
AnaSpec Inc (CA). N-terminal GST-fused Riplet (1–210 aa)
(rRiplet) was purchased from Abnova. 500 ng of rRiplet was
incubate d with or without 500 ng of rNS3-4A in 10
mlof
reaction buff er (20 mM Tris-HCl (7.5), 4% Gly cerol, 5 mM
DTT, 150 mM NaCl, 0.1% of Triton-X100, 0.9% polyvinyl
alcohol) at 37uC for 30 min.
Accession numbers
The accession numbers are Riplet (BAG84604), TRIM25
(NP_005073), TBK1 (NP_037386), IKK-e (AAF45307), IPS-1
(BAE79738), RIG-I (NP_055129), and G3BP (CAG38772).
Supporting Information
Figure S1 K63-linked polyubiquitination of RIG-I RD.
HA-tagged ubiquitin and FLAG-tagged RIG-I RD expression
vectors were transfected into HEK293FT cells. 24 hours after
transfection, the cells were infected with VSV at MOI = 1 for six
Riplet Releases RIG-I Autorepression
PLOS Pathogens | www.plospathogens.org 16 August 2013 | Volume 9 | Issue 8 | e1003533
hours. Then, cell lysate was prepared. Immunoprecipitation was
carried out using anti-FLAG antibody. The samples were
subjected to SDS-PAGE, and the proteins were detected by
western blotting using anti-HA, FLAG, and K63-linked poly-
ubiquitin specific antibodies.
(TIF)
Figure S2 Intracellular localization of RIG-I, NEMO,
and p-TBK1 proteins. (A) HeLa cells were transfected with
HCV dsRNA using lipofectamine 2000 reagent. The cells were
fixed six hours after transfection. The microscopic analysis was
performed using anti-RIG-I mAb (Alme-1) and anti-NEMO pAb.
(B) HeLa cells were transfected with HCV dsRNA using
lipofectamine 2000 reagent (Invitrogen). The cells were fixed at
indicated hour. The microscopic analysis was performed using
anti-RIG-I mAb (Alme-1). (C) HepG2 cells were transfected with
HCV dsRNA using lipofectamine 200 reagent. The cells were
fixed six hours after the transfection. The microscopic analysis was
performed using anti-RIG-I (Alme-1) mAb and anti-p-TBK1
mAb.
(TIF)
Figure S3 NS3-4A of HCV cleaves IPS-1 and Riplet but
not IKK- e. (A) HA-tagged Riplet was transfected into HEK293
cells together with NS3-4A. 24 hours after transfection, cell lysate
was prepared and subjected to SDS-PAGE. The proteins were
detected by western blotting and CBB staining. (B, C) HA-tagged
IKK-e (B) or IPS-1 (C) expression vectors were transfected into
HEK293FT cells with or without NS3-4A of HCV expression
vector. 24 hours after the transfection, the cell lysate was prepared,
and analyzed by SDS-PAGE. The proteins were detected by
western blotting using anti-HA or anti-b actin antibodies. (D) HA-
tagged IPS-1 or HA-tagged Riplet expression vector was transfected
into HEK293FT cells with or without NS3-4A expression vectors.
24 hours after transfection, cell lysate was prepared and subjected to
SDS-PAGE. The proteins were detected by western blotting using
anti-HA antibody. (E, F) N-terminal FLAG-tagged Riplet (E) or C-
terminal HA-tagged Riplet (F) expression vector was transfected
into HEK293FT cells with NS3-4A or NS3-4A*. 24 hours after the
transfection, cell lysates were analyzed by SDS-PAGE. (G) HA-
tagged wild-type Riplet or mutant Riplet-C21A expression vector
were transfected into HEK293FT cells with NS3-4A or NS3-4A*.
24 hours after the transfection, the cell lysate was prepared, and
analyzed by SDS-PAGE. The proteins were detected by western
blotting using anti-HA or anti-b actin antibodies. (H, I) RIG-I,
Riplet, Riplet-3A (H), and/or Riplet C21A (I) mutant expression
vectors were transfected into HEK293 cells together with p125luc
reporter and Renilla luciferase. 24 hours after transfection, luciferase
activity was measured.
(TIF)
Figure S4 siRNA for Riplet or control was transfected
into HeLa cells in 24-well plate using RNAi MAX
(Invitrogen) according to manufacture’s protocol.
48 hours after transfection, the cells were transfected with
100 ng of HCV dsRNA. Six hours after transfection, the cells
were fixed and stained with anti-RIG-I mAb (Alme-1) and anti-
mouse Alexa-488 Ab.
(TIF)
Acknowledgments
We thank Dr. Shimotohno K (Chiba institute of Technology), Dr. Fujita T
(Kyoto University), and Dr. Sasai M (Osaka University) for critical
comments, and Suzuki T for help on microscopic analysis. O and Oc cells
were kindly gifted from Kato N [44].
Author Contributions
Conceived and designed the experiments: HO MMi MMa TS. Performed
the experiments: HO MMi. Analyzed the data: HO MMi. Contributed
reagents/materials/analysis tools: HO MMi. Wrote the paper: HO MMi
TS.
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