USP21 negatively regulates antiviral response by acting as a RIG-I deubiquitinase

16 Pages·2014·3.33 MB·English

Article
USP21 negatively regulates antiviral
response by acting as a RIG-I deubiquitinase
Yihui Fan,1,3 Renfang Mao,2 Yang Yu,1 Shangfeng Liu,3 Zhongcheng Shi,5
Jin Cheng,1 Huiyuan Zhang,6 Lei An,6 Yanling Zhao,1 Xin Xu,1
Zhenghu Chen,3 Mari Kogiso,1 Dekai Zhang,5 Hong Zhang,2,6
Pumin Zhang,7 Jae U. Jung,8 Xiaonan Li,1 Guotong Xu,3,4 and Jianhua Yang1
1Texas Children’s Cancer Center, Department of Pediatrics, 2Department of Pathology and Immunology, Dan L. Duncan Cancer
Center, Baylor College of Medicine, Houston, TX 77030
3Translational Center for Stem Cell Research, Tongji Hospital, Department of Regenerative Medicine; 4Department
of Ophthalmology, Shanghai Tenth People’s Hospital, and Tongji Eye Institute, Tongji University School of Medicine, Shanghai Do
w
200065, China n
lo
5Center for Infectious and Inflammatory Diseases at Institute of Biosciences and Technology, Texas A&M University Health ad
ine 6SDceipeanrctem Ceenntt oefr , PHaothuostloogny, ,T MX 7D7 A0n3d0erson Cancer Center, Houston, TX 77030 ed from
ic 7Departments of Molecular Physiology and Biophysics, Baylor College of Medicine, Houston, TX 77030 http
Med 8LDoesp Aarntgmeelenst, oCfA M 9o0l0e3c3ular Microbiology and Immunology, Keck School of Medicine, University of Southern California, ://rupress.o
rg
al Lys63-linked polyubiquitination of RIG-I is essential in antiviral immune defense, yet the /jem
t /a
en molecular mechanism that negatively regulates this critical step is poorly understood. Here, rticle
m we report that USP21 acts as a novel negative regulator in antiviral responses through its -pd
eri ainbdiluitcye dt oR bIGin-dI ptool yaunbdi qdueiutbiniqautiiotinn aatned RRIIGG--II.– Omveedreiaxtperde sisnitoenr foefr oUnS P(I2F1N )i nshigibniatleind gR, NwAh evrieruass– f/211/2/3
xp deletion of USP21 resulted in elevated RIG-I polyubiquitination, IRF3 phosphorylation, 13/12
E IFN-/ production, and antiviral responses in MEFs in response to RNA virus infection. 124
2
f USP21 also restricted antiviral responses in peritoneal macrophages (PMs) and bone 3/je
o marrow–derived dendritic cells (BMDCs). USP21-deficient mice spontaneously developed m_
al splenomegaly and were more resistant to VSV infection with elevated production of IFNs. 2012
2
n Chimeric mice with USP21-deficient hematopoietic cells developed virus-induced spleno- 84
ur megaly and were more resistant to VSV infection. Functional comparison of three deubiqui- 4.pd
Jo tinases (USP21, A20, and CYLD) demonstrated that USP21 acts as a bona fide RIG-I f by gu
e deubiquitinase to down-regulate antiviral response independent of the A20 ubiquitin- est o
h editing complex. Our studies identify a previously unrecognized role for USP21 in the n 1
T 8
negative regulation of antiviral response through deubiquitinating RIG-I. M
a
rch
2
0
2
The innate immune system, an evolutionarily RIG-I, MDA5, and LGP2, all of which contain 3
CORRESPONDENCE
Guotong Xu: conserved mechanism, is the first line of de- a DExD-box RNA helicase domain, but only
[email protected] fense against viral infection. The hallmark of RIG-I and MDA5 contain N-terminal tandem
OR
antiviral innate immune response is induction caspase recruitment domains (CARDs) that me-
Jianhua Yang:
[email protected] of type I IFNs and proinflammatory cytokines diate downstream signaling (Yoneyama et al.,
(Yoneyama and Fujita, 2009; Takeuchi and 2004; Fujita, 2009). RIG-I is essential for type-I
Abbreviations used: BMDC,
Akira, 2010). Type I IFNs not only locally sup- IFN production in mouse embryonic fibroblasts
BM-derived dendritic cell;
CARD, caspase recruitment press viral infection and proliferation but also (MEFs), conventional dendritic cells (cDCs), and
domain; DUB, deubiquitinating macrophages in response to RNA viruses such
facilitate effective adaptive immune responses.
enzyme; MEF, mouse embry- as Sendai virus (SeV), vesicular stomatitis virus
onic fibroblast; PM, peritoneal Induction of type-I IFN is initiated by detec-
(VSV), hepatitis C virus (HCV), influenza A
macrophage; PRR, pattern tion of viral nucleic acids through the pattern
recognition receptor; RLR,
recognition receptor (PRRs) family, including
RIG-I-like receptor; SeV, Sen- © 2014 Fan et al. This article is distributed under the terms of an Attribution–
dai virus; Ub, ubiquitin; USP21, TLRs, RIG-I–like receptors (RLRs), NOD- Noncommercial–Share Alike–No Mirror Sites license for the first six months
after the publication date (see http://www.rupress.org/terms). After six months
ubiquitin-specific peptidases 21; like receptors (NLRs), and C-type lectin recep- it is available under a Creative Commons License (Attribution–Noncommercial–
VSV, vesicular stomatitis virus. Share Alike 3.0 Unported license, as described at http://creativecommons.org/
tors (CLRs; Akira et al., 2006). RLRs include
licenses/by-nc-sa/3.0/).
The Rockefeller University Press $30.00
J. Exp. Med. 2014 Vol. 211 No. 2 313-328 313
www.jem.org/cgi/doi/10.1084/jem.20122844
virus (Flu), and Japanese encephalitis virus (JEV; Kato et al., (RIG-I-CARD)–induced IRF3 activation, IFN-/ pro-
2005; Kato et al., 2006). Virus-induced IFN production is duction, and antiviral response. Mice lacking USP21 are via-
tightly regulated to prevent persistent IFN production, which ble and fertile, but they present splenomegaly and are more
has been associated with a variety of immunological disorders resistant to VSV infection with elevated IFNs production. The
(Seth et al., 2005; Moser et al., 2009; Ramos et al., 2011). inhibitory function of USP21 in antiviral signaling is inde-
Upon binding to RNA ligands, the conformation of the pendent of A20 ubiquitin-editing complex. Our results reveal
RIG-I protein changes and then the N-terminal tandem the novel physiological function of USP21 in antiviral re-
CARDs trigger interaction with its downstream partner sponse and highlight USP21 as a RIG-I deubiquitinase.
MAVS (Kawai et al., 2005; Meylan et al., 2005; Seth et al.,
2005; Xu et al., 2005). MAVS contains an N-terminal CARD RESULTS
that interacts with the tandem CARDs of RIG-I and a USP21 inhibits RIG-I-CARD–mediated IRF3
C-terminal trans-membrane domain that localizes it to activation and negatively regulates antiviral response
the mitochondrial outer membrane. MAVS then activates Lys63-linked polyubiquitination of RIG-I-CARD plays an
IKK–IKK–NEMO and TBK1–IKK complexes, which in essential role in RNA virus–induced IRF3 activation and
D
turn activate the transcription factors NF-B and IRF3, respec- IFN- production (Gack et al., 2007). However, the molecu- ow
n
tively. Together with other transcription factors, NF-B and lar mechanism of RIG-I-CARD deubiquitination is not fully loa
d
e
IRF3 induce the expression of type I IFNs and other antiviral understood. To explore whether members of the ubiquitin- d
cytokines (Yoneyama and Fujita, 2009; Takeuchi and Akira, specific peptidase (USPs) subclass of DUBs are involved in from
h
2010). Lys63-linked polyubiquitination of RIG-I at Lys-172 the deubiquitination of RIG-I-CARD and down-regulation ttp
catalyzed by TRIM25 is an important step for RIG-I activa- of RIG-I-CARD–mediated IRF3 activation, we screened a ://ru
p
tion (Gack et al., 2007). Another RIG-I E3 ligase, RNF135, library of mammalian expression vectors that encode 37 USPs ress.o
mediates Lys63-linked polyubiquitination of RIG-I at the to assess the effects of overexpression of each USP on RIG-I- rg
/je
C-terminal domain and is essential for RIG-I–dependent im- CARD–induced IFN- luciferase activity. In this screening, m
/a
mune responses (Gao et al., 2009; Oshiumi et al., 2009, 2010). USP18 and USP21 significantly inhibited RIG-I-CARD– rticle
An in vitro reconstitution system of the RIG-I pathway induced IFN- reporter activity, whereas other USPs had no -p
d
showed that unanchored Lys63-linked polyubiquitin chains effect or fewer effects (Fig. 1 A). USP18, an ISG15 deconju- f/21
1
binding to RIG-I is required for RIG-I activation (Zeng gating protease, has been suggested to negatively regulate anti- /2/3
1
et al., 2010). Compared to Lys63-linked polyubiquitination- viral response (Ritchie et al., 2004; Malakhova et al., 2006). 3/1
2
mediated RIG-I activation, the negative regulation of Lys63- USP21 is a highly conserved deubiquitinase and has a rela- 12
4
linked polyubiquitination of RIG-I is less understood. tively high expression in immune organs and cells (unpub- 23
/je
Ubiquitination, a reversible process, can be reversed by lished data). m
_
2
deubiquitinating enzymes (DUBs), which specifically cleave Next, we examined whether the deubiquitinase activity 01
2
the isopeptide bond at the C terminus of ubiquitin (Ub; of USP21 is required for its inhibitory effect. Overexpression 28
4
4
Komander et al., 2009). A20, an ubiquitin-editing enzyme, has of USP21 WT, but not its deubiquitinase-deficient (C221A) .p
d
been shown to negatively regulate antiviral pathways (Wang mutant, blocked the SeV-, RIG-I-CARD–, and TBK1- f by g
et al., 2004; Saitoh et al., 2005; Lin et al., 2006; Parvatiyar et al., induced IFN- reporter activity (unpublished data). Because ue
2010). However, due to the fact that the deubiquitinase activ- IFN- reporter activity is dependent on both NF-B and st o
n
1
ity of A20 is not required for its inhibitory effect in antiviral IRF3 (Honda et al., 2006), we further tested the inhibitory 8
M
signaling, A20 is unlikely to act as a direct RIG-I deubiquitin- effect of USP21 on SeV-, RIG-I-CARD–, and TBK1- arch
ase and the mechanism remains to be clearly defined (Lin et al., induced NF-B and ISRE reporter activities. USP21 WT but 2
0
2
2006; Parvatiyar et al., 2010). Another DUB family protein, not C221A mutant inhibited SeV-, RIG-I-CARD–, and 3
CYLD, has been suggested as a RIG-I deubiquitinase to neg- TBK1-induced NF-B reporter activity (unpublished data).
atively regulate antiviral response (Friedman et al., 2008; However, USP21 only inhibited SeV- and RIG-I-CARD–,
Zhang et al., 2008). However, CYLD has also been shown to but not TBK1-induced ISRE reporter activity (Fig. 1, B–D).
bind to and deubiquitinate TBK1 and IKK (Friedman et al., Consistently, USP21 WT but not C221A mutant inhibited
2008). A recent in vivo study reported that CYLD is required RIG-I-CARD– but not MAVS- or TBK1-induced IRF3
for host defense against VSV infection, suggesting the precise phosphorylation (Fig. 1, E–G). Furthermore, human USP21
mechanism and the direct target(s) of CYLD in antiviral re- inhibited SeV-induced IFN- reporter activities in MEFs
sponse remain to be clarified (Zhang et al., 2011). Therefore, whereas mouse USP21 inhibited SeV-induced IFN- re-
the authentic deubiquitinase of RIG-I remains unclear. porter activities in HEK293T cells (Fig. 1 H and not de-
Using a functional genomic screening, we have identified picted), suggesting that USP21 function in antiviral signaling
USP21 as a bona fide RIG-I deubiquitinase. USP21 inhibits is not limited by cell type and is conserved between human
virus-induced IRF3 activation via binding to and deubiqui- and mouse.
tinating RIG-I. Genetic deletion of USP21 in primary MEFs, Because IRF3 activation is critical for antiviral response,
peritoneal macrophages (PMs), and BM–derived dendritic we determined whether USP21 could inhibit antiviral response.
cells (BMDCs) enhances virus- and RIG-I CARD domain Ectopic expression of USP21 WT but not C221A mutant
314 USP21 negatively regulates antiviral response | Fan et al.
Article
D
o
w
n
lo
a
d
e
d
fro
m
h
ttp
://ru
p
re
ss.o
rg
/je
m
/a
rticle
-p
d
f/2
1
1
/2
/3
1
3
/1
2
1
2
4
2
3
/je
m
_
2
0
1
2
2
8
4
4
.p
d
f b
y g
u
e
st o
n
1
8
M
a
rch
2
0
2
3
Figure 1. USP21 inhibits RIG-I-CARD–induced IRF3 activation and IFN- signaling. (A) RIG-I-CARD was co-transfected with indicated USPs or
control vectors along with IFN- luciferase reporter into HEK293T cells for 36 h. IFN- luciferase activity was measured and normalized with the Renilla
activity. (B) HEK293T cells were transfected with USP21 WT or C221A (CA) mutant along with ISRE luciferase reporter vectors for 24 h, and then infected
with SeV or left uninfected for 12 h before ISRE luciferase was measured. (C and D) RIG-I-CARD or TBK1 were co-transfected with USP21 WT or CA
mutant along with ISRE luciferase reporter vectors into HEK293T cells for 36 h before ISRE luciferase activity was measured. (E–G) FLAG-RIG-I-CARD,
FLAG-MAVS, or FLAG-TBK1 were co-transfected with empty vector or expression vector encoding MYC-USP21 WT or CA mutant into HEK293T cells for
36 h. Cells lysates were immunoblotted with the indicated antibodies. (H) HEK293T cells were transfected with mouse orthologous USP21 WT or CA mutant
along with IFN- luciferase vectors for 24 h, and then infected with SeV or left untreated for 12 h before luciferase was measured. (I and J) HEK293T cells
were transfected with USP21 WT or CA mutant for 36 h, and then infected with SeV at the indicated time points. The mRNA level of IFN- and CCL5 was
measured by real-time RT-PCR. Error bars indicate ±SD in duplicate experiments. Data from A is a screening data from one experiment. Data are repre-
sentative of two (H–J) or at least three (B–G) independent experiments.
blocked SeV-induced IFN- and CCL5 production (Fig. 1, and failed to prevent VSV replication (unpublished data).
I and J). Consistently, ectopic expression of USP21 WT but These results suggest that USP21 is a novel negative regulator
not C221A mutant made cells more susceptible to VSV infection in antiviral response and very likely targets RIG-I.
JEM Vol. 211, No. 2 315
Figure 2. USP21 binds to RIG-I and de-
ubiquitinates RIG-I. (A) HEK293T cells were
transfected with MYC-USP21 WT or CA
mutant along with FLAG-RIG-I-CARD, FLAG-
MAVS, or FLAG-TBK1 for 36 h. Cell lysates
were immunoprecipitated with anti-MYC
antibodies and immunoblotted with anti-
FLAG antibodies. (B) HEK293T cells were
transfected with MYC-USP21 WT or CA
mutant along with FLAG-RIG-I for 24 h, and
then infected with SeV for the indicated time
points. Cell lysates were immunoprecipitated
with anti-MYC antibodies and immunoblotted
with anti-FLAG antibodies. (C) HeLa cells were
infected with SeV for the indicated time
points. Endogenous USP21 in cell lysates were D
o
w
immunoprecipitated with anti-USP21 anti- n
lo
bodies and immunoblotted with anti–RIG-I ad
e
antibodies. (D) HeLa cells were transfected d fro
with GFP-USP21 and FLAG-RIG-I-CARD for m
h
36 h, and then stained by anti-FLAG antibodies ttp
(red). Bar, 10 µm. (E) HeLa cells were trans- ://ru
p
fected with GFP-USP21 for 36 h, and then re
left uninfected or infected with SeV for 8 h. ss.o
rg
immunofluorescent staining was performed /je
m
with anti–RIG-I antibodies (red). Bar, 10 µm. /a
(F) HeLa cells were left uninfected or infected rticle
with SeV for 8 h. Immunofluorescent staining -pd
was performed with anti-USP21 (green) and f/21
1
anti–RIG-I (red). Bar, 10 µm. (G) FLAG- /2/3
1
RIG-I-CARD and HA-Ub were co-transfected 3
/1
into HEK293T cells with control or MYC- 21
2
USP21 WT or CA mutant. Cell lysates were 42
3
heated in the presence of 1% SDS and diluted /je
m
with lysis buffer, and then immunoprecipi- _2
0
tated with anti-FLAG antibodies and immuno- 12
2
8
blotted with anti-HA antibodies. (H) HEK293T 4
4
cells were transfected with control, MYC- .pd
USP21 WT, or CA mutant and cell lysates were f by g
immunoprecipitated with anti-MYC antibod- ue
ies. In the parallel experiments, HEK293T cells st o
n
were transfected with FLAG-RIG-I-CARD and 18
M
HA-Ub. Cells were lysed in the lysis buffer a
only with PMSF. Supernatant containing poly- rch 2
ubiquitinated FLAG-RIG-I-CARD proteins were 02
3
incubated with immunoprecipitated MYC-
USP21 WT or CA mutant for 2 h at 30°C, and
then immunoprecipitated with anti-FLAG
antibodies and immunoblotted with anti-HA antibodies. (I) FLAG-RIG-I and HA-Ub-K63 were co-transfected into HEK293T cells with control, MYC-USP21
WT, or CA mutant. Transfected cells were infected with SeV for the indicated time points. Cell lysates were immunoprecipitated with anti-FLAG antibodies
and immunoblotted with anti-HA antibodies. (J) HEK293T cells were transfected with control, MYC-USP21 WT, or CA mutant for 24 h, and then infected
with SeV for the indicated time points. Cell lysates were immunoprecipitated with anti–RIG-I antibodies and immunoblotted with anti-Ub antibodies.
Data are representative of two (D–F) or at least three (A–C and G–J) independent experiments.
USP21 binds to and deubiquitinates RIG-I MYC-USP21 and FLAG-RIG-I-CARD proteins were also
To examine the targets of USP21 in RIG-I–mediated IRF3 confirmed by immunoprecipitation with anti-FLAG anti-
activation, we transfected MYC-USP21 into HEK293T cells bodies and immunoblotting with anti-MYC antibodies (un-
along with FLAG-RIG-I-CARD, FLAG-MAVS, or FLAG- published data).
TBK1. We found that MYC-USP21 proteins coimmuno- To determine whether the molecular interaction of
precipitated with FLAG-RIG-I-CARD but not with FLAG- USP21 and RIG-I is dependent on virus infection, we trans-
MAVS and FLAG-TBK1 (Fig. 2 A). The binding between fected MYC-USP21 and FLAG-RIG-I plasmids into
316 USP21 negatively regulates antiviral response | Fan et al.
Article
HEK293T cells and treated with SeV for indicated time and ISRE reporter activities. We found that the deubiquitin-
points. MYC-USP21 and FLAG-RIG-I were coimmuno- ase activity of USP21 was required for its strong inhibitory
precipitated regardless of virus infection (Fig. 2 B). To study effect on SeV- and RIG-I-CARD–induced IFN-, NF-B,
the physiological function of USP21, we generated rabbit and ISRE reporter activities (unpublished data). Although
polyclonal antibodies against USP21. Using this antibody, we A20 strongly inhibited SeV- and RIG-I-CARD–induced
found that endogenous USP21 coimmunoprecipitated with IFN-, NF-B, and ISRE reporter activities, its deubiquitin-
RIG-I in HeLa cells regardless of SeV infection (Fig. 2 C). ase activity was not required (unpublished data). Interestingly, the
Consistently, transfected GFP-USP21 or endogenous USP21 inhibitory effect of CYLD is relatively weaker compared with
colocalized with transfected FLAG-RIG-I-CARD and en- USP21 and A20 in these assays (unpublished data). However,
dogenous RIG-I regardless of SeV infection (Fig. 2, D–F). CYLD strongly inhibited TBK1-induced ISRE reporter activity,
These results strongly suggest that USP21 binds to RIG-I. whereas USP21 had no obvious effect on TBK1-induced
The Lys63-linked polyubiquitination of RIG-I plays an ISRE reporter activity (unpublished data). These results sug-
essential role in virus-induced IRF3 activation (Gack et al., gest USP21, A20, and CYLD use quite different mechanisms
2007). We reasoned that USP21 might deubiquitinate RIG-I to regulate antiviral signaling.
D
to inhibit virus-induced IRF3 activation. To test this hypoth- To further dissect the distinct mechanism of these three ow
n
esis, FLAG-RIG-I-CARD and HA-Ub plasmids were co- deubiquitinases (USP21, A20, and CYLD) in antiviral signal- loa
d
e
transfected with control or MYC-USP21 WT or C221A ing, we compared their deubiquitinase activities and binding d
mutant into HEK293T cells. Cell lysates were heated in the affinities to RIG-I-CARD. CYLD failed to deubiquitinate from
h
presence of 1% SDS and diluted with lysis buffer to disrupt non- RIG-I-CARD both in vivo and in vitro (Fig. 3, A and B). ttp
covalent protein–protein interactions. Overexpression of USP21 Although A20 inhibited RIG-I-CARD polyubiquitination ://ru
p
WT but not C221A mutant abrogated FLAG-RIG-I-CARD in vivo, it failed to do so in vitro (Fig. 3, A and B). In contrast, ress.o
polyubiquitination (Fig. 2 G). Next, we analyzed the role USP21 showed a much higher deubiquitinase activity to rg
/je
of USP21 in the deubiquitination of FLAG-RIG-I-CARD RIG-I-CARD both in vivo and in vitro (Fig. 3, A and B). Con- m
/a
in vitro. Because USP21 is unstable when expressed in bacte- sistently, we found USP21 but not A20 or CYLD coimmuno- rticle
ria (Ye et al., 2011) and is difficult to be purified from pro- precipitated with RIG-I-CARD (Fig. 3 C). Together, these -p
d
karyotic system, we immunoprecipitated MYC-USP21 data suggest that USP21 is a bona fide RIG-I deubiquitinase. f/2
1
1
proteins expressed in HEK293T cells with anti-MYC anti- /2
/3
1
bodies and then co-incubated MYC-USP21 proteins with USP21 inhibits TRIM25- and RNF135-mediated 3/1
2
cell lysates containing polyubiquitinated FLAG-RIG-I-CARD. RIG-I polyubiquitination and activation 12
4
The ubiquitination level of FLAG-RIG-I-CARD was signif- Two E3 ligases RNF135 and TRIM25 have been suggested 23
/je
icantly decreased by incubation with USP21 WT but not to target RIG-I for Lys63-linked polyubiquitination and are m
_
2
C221A mutant (Fig. 2 H). We further examined the role of required for antiviral response (Gack et al., 2007; Oshiumi 01
2
USP21 in the inhibition of SeV-induced RIG-I polyubiqui- et al., 2010). To uncover whether USP21 affects RNF135- 28
4
4
tination and found that overexpression of USP21 WT but and TRIM25-mediated RIG-I polyubiquitination and acti- .p
d
not C221A mutant inhibited SeV-induced polyubiquitina- vation, we first tested the effect of USP21 on RNF135-mediated f by g
tion of both FLAG-tagged and endogenous RIG-I (Fig. 2, RIG-I activation. Overexpression of RNF135 alone could not ue
I and J). Together, our results demonstrate that USP21 acts as induce IFN- reporter activity, whereas once co-overexpressed st o
n
1
a RIG-I deubiquitinase. with RIG-I, RNF135 robustly induced IFN- reporter ac- 8
M
The RLRs family includes three members (RIG-I, MDA5, tivity, which was strongly inhibited by USP21 WT but not arch
and LGP2), which have been shown to mediate antiviral re- C222A mutant (Fig. 4 A and not depicted). Furthermore, 2
0
2
sponse to different stimulators (Kato et al., 2006; Satoh et al., USP21 WT but not C221A mutant also strongly inhibited 3
2010). Interestingly, we found that overexpression of USP21 RIG-I and RNF135-induced NF-B and ISRE reporter
WT but not C221A mutant inhibited IFN-, NF-B, and activities (unpublished data). For A20, its deubiquitinase activity
ISRE reporter activities in HEK293T cells in response to was not required for its inhibitory effect on RNF135-mediated
transfected poly(I:C), which was known to be mediated by RIG-I activation (unpublished data). Moreover, USP21 de-
MDA5 (unpublished data), suggesting that USP21 might also ubiquitinated RNF135-mediated RIG-I polyubiquitination
target MDA5. Indeed, overexpression of USP21 WT but not both in vivo and in vitro (Fig. 4, C and D). However, CYLD
C221A mutant inhibited Lys63-linked polyubiquitination of had no significant effect on RNF135-mediated RIG-I poly-
MDA5 and MDA5-mediated IFN-, NF-B, and ISRE re- ubiquitination, whereas A20 inhibited RNF135-mediated
porter activities (unpublished data) and USP21 could also bind RIG-I polyubiquitination in vivo but not in vitro (Fig. 4,
to MDA5 and LGP2 (unpublished data). These findings sug- C and D).
gest that USP21 also acts as a MDA5 deubiquitinase to inhibit Consistent with a previous study (Gack et al., 2007),
MDA5-mediated antiviral response. TRIM25 enhanced SeV- and RIG-I–induced IFN- activ-
To further understand the functional differences of USP21, ity (Fig. 4 B and not depicted). USP21 inhibited TRIM25-
CYLD, and A20 in antiviral signaling, we tested their effect on mediated IFN- reporter activities, which was also inhibited
SeV-, RIG-I-CARD–, and TBK1-induced IFN-, NF-B, by CYLD and A20, but the deubiquitinase activity of A20 was
JEM Vol. 211, No. 2 317
Figure 3. USP21 but not A20 and CYLD
binds to and deubiquitinates RIG-I-CARD.
(A) FLAG-RIG-I-CARD and HA-Ub were co-
transfected with MYC-USP21 WT, CA mutant, MYC-
CYLD, or MYC-A20. The heated cell lysates were
immunoprecipitated with anti-FLAG antibodies
and immunoblotted with anti-HA antibodies.
(B) HEK293T cells were transfected with MYC-
USP21 WT, CA mutant, MYC-CYLD, or MYC-A20.
MYC-tagged proteins were immunoprecipitated
with anti-MYC antibodies. Supernatant contain-
ing polyubiquitinated FLAG-RIG-I-CARD proteins
were co-incubated with the immunoprecipitated
MYC-tagged proteins for 2 h at 30°C, and then
immunoprecipitated with anti-FLAG antibodies
and immunoblotted with anti-HA antibodies. D
o
w
(C) FLAG-RIG-I-CARD was co-transfected into n
lo
HEK293T cells with control, MYC-USP21 WT, ad
e
CA mutant, MYC-CYLD, or MYC-A20. Cell lysates d fro
were immunoprecipitated with anti-MYC anti- m
h
bodies and immunoblotted with anti-FLAG anti- ttp
bodies. Data from A–C are representative of three ://ru
p
independent experiments. re
ss.o
rg
/je
m
/a
rticle
-p
d
f/2
1
1
/2
/3
1
3
/1
2
1
2
4
2
3
/je
m
_
2
0
1
2
not required (Fig. 4 B and not depicted). These results dem- or survival when housed in pathogen-free condition. How- 28
4
4
onstrate that USP21 inhibits TRIM25- or RNF135-mediated ever, the spleens from 9 wk old USP21/ mice were sig- .p
d
RIG-I polyubiquitination and activation to negatively regu- nificantly larger than the ones from WT littermate controls f b
y g
late antiviral signaling. (Fig. 5, E and F). The total splenocyte number was slightly ue
increased in USP21/ mice (unpublished data). Flow cyto- st o
n
Generation of USP21 deficient mice metric analysis of splenocytes revealed an increased percentage 18
M
To further study the function of USP21 in antiviral responses, of macrophages and neutrophils in USP21/ mice (Fig. 5, arch
USP21 knockout mice were generated. We constructed the G and H). However, the distribution of DCs, T cells and 2
0
2
USP21 conditional targeting vector with which the two-loxP B cells in spleens were similar between USP21/ and WT mice 3
and two-frt strategy was used to obtain homologous recombi- (unpublished data). To determine whether USP21/ hema-
nation in embryonic stem (ES) cells (Fig. 5 A). USP21 het- topoietic cells play an important role in splenomegaly devel-
erozygous knockout mice were generated by breeding USP21 opment, we transferred WT or USP21/ BM cells into SCID
conditional knockout mice with Cre transgenic mice to de- mice. Both WT and USP21/ BM cells successfully repopu-
lete USP21 exons 3 and 4, which encode part of essential lated in the recipient mice (Fig. 5 I). Spleens from SCID mice
USP domain (Fig. 5 A). Breeding of USP21 heterozygous transferred with WT or USP21/ BM cells were slightly en-
male and female mice gave rise to a litter of mice with WT larged compared with that from non-transferred mice; how-
(+/+), heterozygous (+/), and homozygous (/) genotypes ever, the sizes of spleens from WT and USP21/ BM transferred
with a Mendel ratio, suggesting that USP21/ mice are via- mice were comparable (Fig. 5, J–L). These data suggest that
ble (Fig. 5 B). RT-PCR and immunoblotting analysis of USP21/ hematopoietic cells are insufficient to cause sple-
USP21 expression in MEFs showed that endogenous WT nomegaly development under pathogen-free condition. No
USP21 transcription and protein expression were abolished significant difference was observed between thymuses isolated
in USP21/ mice (Fig. 5, C and D). from USP21/ and WT littermates (unpublished data).
USP21/ mice are viable and fertile. The young To determine whether USP21 deficiency in mice affect the
USP21/ mice (4 wk old) showed no gross defects in growth basal RIG-I protein level and IFNs production, we examined
318 USP21 negatively regulates antiviral response | Fan et al.
Article
Figure 4. USP21 inhibits TRIM25- and
RNF135-induced RIG-I activation. (A) HEK293T
cells were transfected with indicated plasmids for
24 h, and then left uninfected or infected with
SeV for 12 h before IFN- luciferase activity was
measured. (B) HEK293T cells were transfected
with MYC-USP21 WT, CA mutant, MYC-CYLD, or
MYC-A20 along TRIM25. Transfected cells were
left uninfected or infected with SeV for 12 h be-
fore IFN- luciferase activity was measured.
(C) Expression vector encoding FLAG-RIG-I,
RNF135, and HA-Ub were co-transfected with
control, MYC-USP21 WT, CA mutant, MYC-CYLD,
or MYC-A20. Cell lysates were immunoprecipi-
tated with anti-FLAG antibodies and immuno-
blotted with anti-HA antibodies. (D) HEK293T cells D
o
w
were transfected with expression vectors encod- n
lo
ing MYC-USP21 WT, CA mutant, MYC-CYLD, or ad
e
MYC-A20. MYC-tagged proteins were immuno- d fro
precipitated with anti-MYC antibodies. In the m
h
parallel experiments, HEK293T cells were trans- ttp
fected with FLAG-RIG-I, RNF135, and HA-Ub. ://ru
p
Supernatant containing polyubiquitinated re
FLAG-RIG-I proteins were incubated with immuno- ss.o
rg
precipitated MYC-USP21 WT, CA mutant, MYC- /je
m
CYLD, or MYC-A20 for 2 h at 30°C, and then /a
immunoprecipitated with anti-FLAG antibodies rticle
and immunoblotted with anti-HA antibodies. -pd
Error bars indicate ±SD in duplicate experiments. f/21
1
Data are representative of two (A and B) or three /2/3
1
(C and D) independent experiments. 3
/1
2
1
2
4
2
3
/je
m
_
2
0
1
2
2
8
4
RIG-I protein level in multiple organs and no significant dif- with these results, VSV and SeV induced a stronger IRF3 4.p
d
ference was found between 9-wk-old WT and USP21/ phosphorylation in USP21/ MEFs compared with that in f b
mice (Fig. 5 O). The basal RIG-I protein level in BMDCs WT MEFs (Fig. 6 E). SeV also induced a stronger IKK/ y gu
e
and PMs was also comparable between WT and USP21/ phosphorylation in USP21/ MEFs compared with that in st o
n
mice (Fig. 5 M). Basal IFN-/ production in 9-wk-old WT MEFs (Fig. 6 E). Furthermore, SeV replication was sig- 18
USP21/ mice is similar to that in WT mice (Fig. 5 N). Our nificantly inhibited in USP21/ MEFs (Fig. 6 F). Consis- Ma
results suggested that deletion of USP21 in mice did not tently, endogenous USP21 and RIG-I coimmunoprecipitated rch 2
0
affect the basal RIG-I expression and IFNs production in in MEFs (Fig. 6 G) and SeV induced a stronger RIG-I poly- 23
9-wk-old USP21/ mice with noticeable splenomegaly. ubiquitination in USP21/ MEFs compared with that in
WT MEFs (Fig. 6 H).
Deletion of USP21 enhances antiviral Furthermore, SeV induced a stronger IRF3 phosphory-
response in multiple cell types lation in USP21/ PMs compared with that in WT PMs
To further assess the physiological function of USP21, we iso- (Fig. 7 A). SeV induced much more IFN-/ production in
lated MEFs from WT and USP21/ embryos. SeV infection, USP21/ PMs compared with that in WT PMs (Fig. 7,
transfection of poly(I:C), and RIG-I-CARD overexpression B and C). SeV also induced a stronger IRF3 phosphorylation
resulted in higher IFN- and ISRE reporter activities in and much more IFN-/ productions in USP21/ BMDCs
USP21/ MEFs compared with that in WT MEFs (Fig. 6 A). compared with that in WT BMDCs (Fig. 7, D–F). However,
Consistently, virus-induced IFN- and - expressions were we could not observe any significant effect of USP21 deficiency
much higher in USP21/ MEFs compared with WT MEFs on SeV-induced IRF3 phosphorylation, IFN-/ produc-
(Fig. 6 B). Upon infection with VSV-eGFP, USP21/ MEFs tion in BM-derived macrophages (BMMs; unpublished data).
showed significantly decreased number of VSV-eGFP+ cells It is noteworthy that USP21 expression level was much lower
(Fig. 6 C). USP21/ MEFs were also more resistant to VSV in BMMs compared with MEFs, BMDCs, and PMs (unpub-
infection compared with WT MEFs (Fig. 6 D). Consistent lished data).
JEM Vol. 211, No. 2 319
Figure 5. Generation of USP21 knockout
mice. (A) Generation of USP21 conditional
knockout mice by two-loxP, two-frt strategy.
(B) PCR genotyping of WT and USP21/ litter-
mates generated from USP21+/ mice inter-
crossing. (C) Total RNA was extracted from
MEFs and mRNA level of USP21 was examined
by RT-PCR. (D) Cell lysates from WT and
USP21/ MEFs were immunoblotted by anti-
USP21 antibodies. (E) Representative images
of spleens isolated from 9-wk WT and
USP21/ mice. (F) Quantitative analysis of
the weight of spleens isolated from 9-wk-old
WT (n = 10) and USP21/ (n = 10) mice.
(G) Macrophage and dendritic cell popula-
tions in splenocytes from USP21/ and WT D
o
w
mice were analyzed by FACS using anti- n
lo
CD11b and anti-CD11c antibodies. (H) Neu- ad
e
trophil populations in splenocytes from d fro
USP21/ and WT mice were analyzed by m
h
FACS using anti-Gr1 and anti-CD11b antibod- ttp
ies. (I) SCID mice were transferred with WT or ://ru
p
USP21/ BM cells for 10 wk. T cell popula- re
tions in splenocytes were analyzed by FACS ss.o
rg
using anti–mouse CD3, CD4, and CD8 anti- /je
m
bodies. (J) Representative images of spleens /a
from naive and BM-transferred SCID mice. rticle
(K) Spleens from naive and BM transferred -pd
SCID mice were homogenized. Total spleno- f/21
1
cytes were quantitatively analyzed. (L) Quanti- /2/3
1
tative analysis of the weight of spleens 3
/1
isolated from naive and BM-transferred SCID 21
2
mice. (M) Fresh BM cells were cultured in the 42
3
medium with IL-4 and GM-CSF cytokines /je
m
for 7 d. Cell lysates from BMDCs or PMs were _2
0
immunoblotted by anti–RIG-I antibodies. 12
2
8
(N) Blood was collected from 9-wk-old WT 4
4
or USP21/ mice, and IFN level in sera was .pd
measured by ELISA kit. (O) Cells from different f by g
organs were lysed and immunoblotted by ue
anti–RIG-I antibodies. Error bars indicate ±SD st o
n
in duplicate experiments. ***, P < 0.001 (two- 18
M
tailed paired Student’s t test). Data are repre- a
sentative of two (E–O) or at least three (B–D) rch 2
independent experiments. 02
3
USP21 inhibits antiviral response independent that USP21 function in antiviral response is independent of
of A20 ubiquitin editing complex A20 ubiquitin-editing complex. Next, we examined whether
Because A20 has been suggested to negatively regulate anti- USP21 and A20 can cooperatively inhibit antiviral signaling.
viral response (Lin et al., 2006; Parvatiyar et al., 2010; Saitoh Both USP21 and A20 inhibited SeV- and RIG-I-CARD–
et al., 2005; Wang et al., 2004), we next determined whether induced IFN- reporter activity in a dose-dependent manner.
the inhibitory function of USP21 is dependent on A20 ubiq- The inhibitory effect of co-overexpression of USP21 and A20
uitin-editing complex. USP21 inhibited SeV-induced IFN- was much stronger than individual expression of USP21 or
reporter activity in WT, A20/, ITCH/, and TAXBP1/ A20 (Fig. 8 E and not depicted).
MEFs (Fig. 8, A–D). USP21 also inhibited RIG-I-CARD– Recently, A20 has been suggested to inhibit TNF-induced
induced IFN- reporter activity in WT, A20/, ITCH/, and NF-B activation by promoting Ubc13 and UbcH5C degra-
TAXBP1/ MEFs (unpublished data). These results indicate dation or disrupting Ubc13 or UbcH5C binding with E3
320 USP21 negatively regulates antiviral response | Fan et al.
Article
Figure 6. Knockout of USP21 expression
enhances antiviral response in MEFs.
(A) WT and USP21/ MEFs were transfected
with IFN- or ISRE reporter along with or with-
out RIG-I-CARD for 24 h, and then left unin-
fected or infected with SeV or transfected with
poly (I:C) for 12 h before luciferase activity was
measured. (B) MEFs were treated with SeV for
the indicated time points. The mRNA levels of
IFN-/ were measured by real-time RT-PCR.
(C) MEFs were infected with VSV-eGFP (moi = 0.1)
for 48 h and GFP+ cells were imaged. Bar,
50 µm. (D) MEFs were infected with VSV for
the indicated MOI for 48 h and relative cell
viability was measured by CCK8 kit. (E) MEFs
were infected with VSV or SeV for the time D
o
w
points indicated. Cell lysates were immuno- n
lo
blotted by anti–p-IRF3, anti-IRF3, anti-USP21, ad
e
anti–p-IKK/, anti-IKK, and anti–-actin d fro
antibodies. (F) MEFs were infected with SeV m
h
for the indicated time points. Cell lysates were ttp
immunoblotted by anti-SeV antibodies. ://ru
p
(G) MEFs were infected with SeV for the indi- re
cated time points. Endogenous USP21 were ss.o
rg
immunoprecipitated with anti-USP21 anti- /je
m
bodies and immunoblotted with anti–RIG-I /a
antibodies. (H) MEFs were infected with SeV rticle
for the indicated time points. RIG-I proteins in -pd
the cell lysates were immunoprecipitated with f/21
1
anti–RIG-I antibodies and immunoblotted /2/3
1
with anti-Ub antibodies. Error bars indicate 3
/1
±SD in duplicate experiments. Data are repre- 21
2
sentative of two (B–D and F) or three (A, E, G, 42
3
and H) independent experiments. /je
m
_
2
0
1
2
2
8
4
4
.p
d
f b
y g
u
e
st o
n
1
8
M
a
rch
2
0
2
3
ligase (Shembade et al., 2010). Thus, we examined whether A20 deficiency on SeV-induced IRF3 phosphorylation. Sur-
A20 inhibits antiviral response via the aforementioned mech- prisingly, SeV failed to induce IRF3 phosphorylation at ear-
anism. Overexpression of USP21, CYLD, and A20 failed to lier time points in A20/ MEFs (Fig. 8 F). However, at later
promote Ubc13 and UbcH5C degradation regardless of SeV time points, we did see a weak induction of IRF3 phosphory-
treatment (unpublished data). Consistently, no significant dif- lation (unpublished data). To further confirm the result, we
ference was found in terms of UbcH5C degradation in re- tested the sensitivity of WT and A20/ MEF to VSV infec-
sponse to SeV infection in WT, USP21/, and A20/ MEFs tion and found that A20/ MEFs were more sensitive com-
in the time course examined (unpublished data). pared with WT MEFs (unpublished data). Consistently, SeV
The fact that SeV induced weaker IFN- reporter activ- failed to induce IFN-/ production in A20/ MEFs (Fig. 8 G
ity in A20/ MEFs compared with WT, ITCH/, and and not depicted), although IL-6 expression was comparably
TAXBP1/ MEFs suggests that the role of A20 in antiviral induced in both USP21/ and A20/ MEFs (unpublished
response is far to be fully understood (Fig. 8, A–D). To better data). These results indicate that the physiological role of A20
understand the role of A20 in antiviral response, we infected in antiviral response is very complicated and its function in
WT and A20/ MEFs with SeV and examined the effect of antiviral response needs to be further investigated.
JEM Vol. 211, No. 2 321
D
o
w
n
Figure 7. Knockout of USP21 expression enhances antiviral response in PMs and BMDCs. (A) PMs isolated from WT and USP21/ mice were loa
d
infected by SeV with the indicated time points. Cell lysates were immunoblotted by anti–p-IRF3 and anti-IRF3 antibodies. (B and C) PMs were infected by ed
SeV with the indicated time points. IFN- (B) and IFN- (C) production in cell culture medium were measured by ELISA kit. (D) BMDCs were infected by fro
m
SeV with the indicated time points. Cell lysates were immunoblotted by anti–p-IRF3 and anti-IRF3 antibodies. (E and F) BMDCs were infected by SeV with http
the indicated time points. IFN- (E) and IFN- (F) production in cell culture medium was measured by ELISA kit. Error bars indicate ±SD in duplicate ex- ://ru
periments. Data from A–F are representatives of two independent experiments. pre
ss.o
rg
/je
Deletion of USP21 enhances antiviral compared with that in WT mice (Fig. 9 L). 4-wk-old USP21/ m
/a
immune defense in vivo mice did not show noticeable splenomegaly and basal RIG-I rticle
To investigate the role of USP21 in vivo, WT and USP21/ expression in 4-wk old WT and USP21/ mice was compa- -p
d
mice were injected with VSV through tail vein, and then sera rable (unpublished data). Intraperitoneal injection of VSV in f/21
1
were collected to measure type-I IFN level. We found that tail 4-wk-old USP21/ also induced a stronger IFN production /2/3
1
vein injection of VSV induced a stronger production of IFNs compared with that in 4-wk-old WT mice (unpublished 3/1
2
in USP21/ mice compared with that in WT mice, whereas data). Collectively, our data indicate that USP21 negatively reg- 12
4
the steady-state levels of IFNs without VSV infection were ulates antiviral response in vivo and the hyper-responsiveness 23
/je
similarly very low in both WT and USP21/ mice (Fig. 9, of USP21-deficient mice to VSV is not due to splenomegaly m
_
2
A and B). Consistently, the viral titers in sera were much or increased basal RIG-I expression. 01
2
lower in USP21/ mice than those in WT mice at all time 28
4
4
points examined (Fig. 9 C). USP21 deficiency did not affect DISCUSSION .p
d
VSV entry into the MEFs and mice organs (unpublished data). Excessive production of IFNs or proinflammatory cytokines f by g
These results suggest that USP21 deficiency enhances viral is destructive to hosts, thus a successful immune response ue
clearance caused by a higher level of type-I IFN induction. against viral infections must be tightly regulated. Lys63-linked st o
n
1
To determine whether USP21 deficiency affects the sur- polyubiquitination of RIG-I is a pivotal event in RIG-I– 8
M
vival of mice after VSV infection, mice were infected with mediated antiviral response. USP21 is a highly conserved arch
VSV through tail vein and their survival was monitored. We deubiquitinase and its physiological role is largely unknown. 2
0
2
found that USP21/ mice were more resistant to VSV- Biochemical study and crystal structure showed that USP21 3
induced lethality (Fig. 9 D). At day 8 after infection, 40% of is a highly active deubiquitinating enzyme that can efficiently
WT mice died, whereas no USP21/ mice died until day 10. cleave Lys63-linked ubiquitin chain (Nakagawa et al., 2008;
After 2 wk of VSV infection, splenomegaly in USP21/ Xu et al., 2010; Ye et al., 2011; García-Santisteban et al., 2012;
mice was even more severe than that in naive mice (Fig. 9, Urbé et al., 2012). In this study, we present biochemical and
E and F). SCID mice transferred with USP21/ BM cells genetic evidences that USP21 acts as a RIG-I deubiquitinase
developed splenomegaly after VSV infection and were resis- to negatively regulate RIG-I–mediated antiviral signaling
tant to VSV-induced lethality (Fig. 9, G-I). These results sug- through its deubiquitinase activity. The inhibitory effect of
gest that USP21/ hematopoietic cells are sufficient to cause USP21 on antiviral signaling is independent of A20 ubiquitin-
splenomegaly development in mice infected with VSV and editing complex. USP21 inhibits RNA virus-induced Lys63-
protect mice from VSV-induced lethality. linked polyubiquitination of RIG-I and RIG-I–mediated
Consistent with the results obtained by intravenous VSV downstream signaling. Genetic deletion of USP21 gene in
infection, intraperitoneal injection of VSV also induced a mice enhances RIG-I–mediated antiviral response. Further-
stronger production of IFNs in USP21/ mice compared more, we also found that USP21 binds to two other RLR
with that in WT mice (Fig. 9, J and K). Virus titer detected in family members (MDA5 and LGP2) and deubiquitinates
different organs of USP21/ mice was significantly lower MDA5, suggesting USP21 acts as a major negative regulator
322 USP21 negatively regulates antiviral response | Fan et al.