42 Hongrui Li et al. DOI: 10.1002/eji.201847603 Eur. J. Immunol. 2019.

49: 42–53
Basic

Innate immunity

Research Article
USP14 promotes K63-linked RIG-I deubiquitination
and suppresses antiviral immune responses
Hongrui Li1 , Zizhao Zhao1 , Jing Ling1 , Linhui Pan1 , Xibao Zhao1,2 ,
Huihui Zhu1 , Juan Yu1 , Bin Xie1 , Jianzhong Shen3 and Weilin Chen1,2

1
Institute of Immunology, Zhejiang University School of Medicine, Hangzhou, Zhejiang, China
2
Department of Immunology, Shenzhen University School of Medicine, Shenzhen, Guangdong
Sheng, China
3
Department of Drug Discovery and Development, Harrison School of Pharmacy, Auburn
University, Auburn, AL

Retinoic acid-inducible gene I (RIG-I) is a critical RNA virus sensor that initiates antiviral
immune response through K63-linked ubiquitination. In this study, we demonstrated
USP14, a deubiquitinating enzyme, as a negative regulator in antiviral responses by
directly deubiquitinating K63-linked RIG-I. USP14 knockdown significantly enhanced RIG-
I-triggered type I IFN signaling and inhibited vesicular stomatitis virus (VSV) replication
both in mouse peritoneal macrophages and THP1 cells. USP14 overexpression in HeLa
cells attenuated RIG-I-triggered IFN-β expression and promoted VSV replication. Besides,
USP14-specific inhibitor, IU1, increased RIG-I-mediated type I IFN production and antivi-
ral responses in vitro and in vivo. In addition, USP14 could interact with RIG-I and remove
RIG-I K63-linked polyubiquitination chains. This article is the first to report that USP14
acts as a negative regulator in antiviral response through deubiquitinating K63-linked
RIG-I. These findings provide insights into a potential new therapy targeting USP14 for
RNA virus-related diseases.

Keywords: Deubiquitination r RIG-I-like receptors r RNA virus sensor r USP14 r Vesicular

stomatitis virus (VSV)

 Additional supporting information may be found online in the Supporting Information section
at the end of the article.

Introduction associated gene 5, go through conformational changes and acti-

Vertebrates are continuously challenged with pathogenic microbes
that induce a variety of damages into host cells. In return, eukary-
otic cells express PRRs that detect microbial PAMPs and acti-
vate immune responses [1]. PRRs, including TLRs, retinoic acid-
inducible gene (RIG-I)-like receptors, NOD-like receptors (NLRs),
and C-type lectin receptors, recognize specific microbes [2, 3].
RIG-I-like receptors, such as RIG-I and melanoma differentiation-
Correspondence: Prof. Weilin Chen
e-mail: cwl@zju.edu.cn
vate mitochondrial antiviral signaling protein (MAVS, also known
as VISA, IPS-1 and CARDIF) [4, 5] when infected by RNA virus.
Subsequently, activated MAVS would recruit TBK1, then inter-
feron regulatory factor 3 (IRF3) or NF-κB to produce type I IFNs
or inflammatory cytokines [6].
Ubiquitin is a small, 76-amino acid protein that regulates pro-
tein stability, activity, and harbors at least seven types (K6, K11,
K27, K29, K33, K48 and K63) of ubiquitination modifications
[7, 8]. RIG-I ubiquitination is indispensable in antiviral immu-
nity [9, 10] and its K63-linked ubiquitination could be catalyzed
by TRIM25 [11, 12], Riplet (RNF135, REUL) [13], TRIM4 [14],


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Eur. J. Immunol. 2019. 49: 42–53
and Mex3c [15] in cellular antiviral responses. Deubiquitination
of RIG-I could be regulated by deubiquitinating enzyme (DUBs).
USP25 was reported to negatively regulate Sendai virus (Sev)-
induced type I IFN signaling by deubiquitinating RIG-I, TRAF2,
and TRAF6 [16]. USP3 inhibits vesicular stomatitis virus (VSV)-
induced type I IFN signaling by deubiquitinating K63-linked RIG-
I [17].
Ubiquitin-specific protease 14 (USP14) belongs to USP fam-
ily of DUBs [18] and deubiquitinates proteasome-bound sub-
strates that were ubiquitinated at multiple sites [19–21]. Until
now, USP14 has been demonstrated to interact with Tau [22],
CXCR4 [23], prion [24], and NLRC5 [25]. Multiple studies illus-
trated that USP14 regulated innate immune response. As an exam-
ple, USP14 promotes dengue virus replication [26] and IL-1β pro-
duction [27, 28].
To hunt DUBs interacting with RIG-I, the binding proteins of
RIG-I were enriched in RIG-I overexpressed HEK293T cells using
co-IP assay. The immunoprecipitates were separated by SDS-PAGE
gel, and then the proteins were cut from the gel and analyzed by
mass spectrometric (MS) analysis. Among the RIG-I-interacting
proteins, we focused on USP14 and the highest frequency of spe-
cific peptides also indicated USP14 to be a candidate for RIG-I
interacting partner (Supporting Information Fig. S1A and Table
S1).In the present study, we demonstrated that USP14 negatively
regulated RNA virus-triggered type I IFN signaling. USP14-specific
inhibitor, IU1, increased RIG-I-mediated type I IFN production and
antiviral responses in vitro and in vivo. As a DUB, USP14 inter-
acted with RIG-I and removed its K63-linked polyubiquitination
chains. In conclusion, our study is the first to confirm USP14 as a
negative regulator in RIG-I-mediated antiviral immune response
through K63-linked RIG-I deubiquitination.
Results
USP14 knockdown increases IFN-β production and
inhibits VSV replication in macrophages
To understand USP14 function in RIG-I signaling, IFN-β produc-
tions were measured in mouse peritoneal macrophages and THP1
macrophages. First, specific siRNAs targeting mouse and human
USP14 genes were designed, and knockdown efficiency was deter-
mined by RT-qPCR and immunoblot analysis separately (Support-
ing Information Fig. S1B and S1C). RIG-I can be activated by
short ssRNA, dsRNA, or RNAs with 5’pp or 5’ppp end [29], such as
VSV, ssRNA virus, and intratransfected polyinosinic:polycytidylic
acid (poly[I:C]) with low molecular weight. IFN-β production was
higher in USP14 knockdown mouse peritoneal macrophage (Mϕ)
than control cells infected with VSV or transfected with poly(I:C)
both in mRNA and protein levels (Fig. 1A and B). USP14 siRNA
also elevated IFN-α mRNA levels and protein expression that is
triggered by VSV (Supporting Information Fig. S1D and S1E).
Similar difference of IFN-β expression was detected in THP1 cells
(Fig. 1C and D). To examine USP14 function on VSV replication,
cell supernatants were collected and analyzed for TCID50 analysis

C 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Innate immunity 43
after being infected by VSV for 12 or 24 h. Data showed that USP14
deficiency attenuated VSV replication in Mϕ (Fig. 1E) and THP1
cells (Fig. 1F). Above Data indicate USP14 deficiency elevates IFN-
β production and promotes cell antiviral ability exhibited by Mϕ
and THP1 cells.
IU1 enhances RIG-I-mediated IFN-β production and
inhibits VSV replication in macrophages
Specific or nonspecific inhibitors targeting USP14 are signifi-
cant to both scientific studies and clinical therapies. Nonspecific
inhibitors include WP1130 (Degrasyn) [19] and b-AP15 [30]. Spe-
cific inhibitors contain VLX1570 [31] and IU1 screened by Lee et
al [32]. IU1 specificity had been confirmed by quite a few stud-
ies [26, 33–37]. IU1 structure is shown in Supporting Information
Fig. S2A. Previous studies showed IU1 inhibited flaviviruses repli-
cation [26]. In this study, we speculated whether IU1 suppressed
RNA virus propagation. First, we chose the optimal concentra-
tions of IU1 by comparing IFN-β mRNA expressions induced by
VSV. RT-qPCR analysis showed that IU1-pretreated cells at the
concentration of 100 μM exhibited the highest IFN-β mRNA lev-
els (Supporting Information Fig. S2B). Cell toxicity of IU1 was
calibrated and exhibited minor influence (Supporting Information
Fig. S2C). In summary, 100 μM was treated as the working con-
centration.
To verify whether IU1 influenced cell antiviral ability, we
treated cells with IU1/DMSO before challenged with VSV or intra-
cellular poly(I:C). IFN-β mRNA production, mRNA and protein
levels, was measured. Except for VSV induced higher levels of IFN-
β in IU1-treated groups, cells transfected with poly(I:C) produced
more IFN-β mRNA, likewise in Mϕ (Fig. 2A) and THP1 cells (Fig.
2C). IFN-β secretion by IU1-treated cells was significantly higher
than DMSO treated ones in Mϕ (Fig. 2B) and THP1 cells (Fig. 2D),
and similar difference was seen in IFN-α expression (Supporting
Information Fig. S2D). Furthermore, to investigate whether IU1
regulated VSV replication, cell supernatants were collected and
analyzed by TCID50. Less VSV replications in IU1-treated cells
were detected in Mϕ and THP1 cells (Fig. 2E). Two more meth-
ods were applied as follows in THP1 cells. Immunofluorescence
assay indicated that IU1-treated groups exhibited less quantity
of green fluorescence (Fig. 2F). THP1 cells were pretreated with
IU1/DMSO followed by VSV-eGFP infection. Flow cytometry anal-
ysis showed that the percentage of GFP positive cells was lower
in IU1-treated THP1 cells than that of control (Fig. 2G, Support-
ing Information Fig. S2E). Data of this part demonstrate that IU1
could enhance IFN-β production and attenuates VSV replication.
USP14 overexpression attenuates IFN-β production
and promotes VSV replication in HeLa cells
HeLa cell is an immortal cell line that had been used in studies
on virus [38–40]. First, USP14 overexpression in HeLa cells was
measured (Supporting Information Fig. S3A). To weigh the role
www.eji-journal.eu
44 Hongrui Li et al. Eur. J. Immunol. 2019. 49: 42–53

Figure 1. Knockdown of USP14 increases RIG-I-mediated IFN-β production and inhibits VSV replication in macrophages. (A–D) Mouse peritoneal
macrophage or THP1 cells were infected with VSV or intracellular poly(I:C) after transfected with scrambled siRNA or USP14 siRNA for 36 h. IFN-β
mRNA levels (A) and protein levels (B) were measured by RT-qPCR or ELISA in mouse peritoneal macrophage (Mϕ). IFN-β mRNA levels (C) and
protein levels (D) were measured by RT-qPCR or ELISA in THP1 cells. (E–F) TCID50 analysis of Mϕ (E) and THP1 cells (F) with VSV infected for 12 to 24
h were conducted in HEK293T cells. (A–F) Data are shown as mean ± SD of three samples per group and are from one experiment representative of
three independent experiments. Significance was calculated in relation to the control group. *p < 0.05, **p < 0.01, ***p < 0.001 (two-sided Student’s
t-test).

of USP14 overexpression in RIG-I mediated antiviral response,
IFN-β mRNA levels were determined and showed a lower level in
USP14 overexpressed HeLa cells infected with VSV or intracellu-
lar poly(I:C) (Fig. 3A). IFN-β secretions were measured by ELISA
and exhibited similar difference between control and USP14 over-
expression cells (Fig. 3B). VSV replications were determined by
three assays. TCID50 analyses showed that USP14 overexpression
increased virus titers (Fig. 3C). HeLa cells undergone fluorescence
microscopy (Fig. 3D) and flow cytometry analysis (Fig. 3E, Sup-
porting Information Fig. S3B) to detect GFP-positive cells after
being infected by VSV-eGFP. Results indicated that USP14 over-
expression increased the percentage of GFP-positive cells. Data
suggest that USP14 is in favor of VSV infection in HeLa cells.
IU1 enhances the antiviral response of mice in vivo
To prove that IU1 influenced antiviral response in vivo, IU1 and
DMSO were preintraperitoneally injected into mice before VSV
infection. Higher IFN-β mRNA levels were detected in IU1-treated
mice than that of control in lung and spleen (Fig. 4A). VSV repli-
cations were significantly reduced in IU1-treated mice compared
to control in lung and spleen (Fig. 4B). IU1-treated mice produced
more IFN-β in serum (Fig. 4C). The H & E staining of lungs indi-
cated that IU1-treated mice exhibited less inflammation (Fig. 4D).
Challenged with the lethal dose of VSV, mice treated with IU1
were more resistant to VSV infection than control in a durable
observation (Fig. 4E). Data demonstrated that IU1-treated mice
gained greater antiviral ability against VSV infection by producing
more IFN-β.
USP14 targets and interacts with RIG-I
Next, we dissected the mechanisms on which USP14 regulated
RIG-I mediated type I IFN production. By knocking down USP14
or IU1 inhibiting in Mϕ, we detected IFN-β mRNA following stim-
ulation with LPS/poly(I:C)/VSV. Data indicated that USP14 had
no significant influence on TLR3- and TLR4-activated IFN-β (Fig.
5A) and IFN-α (Supporting Information Fig. S4) production. To
investigate the role of USP14 in regulating RIG-I mediated sig-
naling, we therefore used VSV to stimulate Mϕ and THP1 cells.
We observed that IRF3 and TBK1 phosphorylation was elevated in
USP14 knockdown cells (Fig. 5B). Next, dual-luciferase reporter
assay was applied to investigate the target of USP14 in type I
IFN signaling pathway. Data showed that USP14 just suppressed
RIG-I-N-activated IFN-β and ISRE luciferase activation, instead
of MAVS, TBK1, and IRF3-5D (Fig. 5C). This illustrated that


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Eur. J. Immunol. 2019. 49: 42–53 Innate immunity 45

Figure 2. IU1 elevated RIG-I-mediated IFN-β production and inhibited VSV replication in macrophages. (A–D) Mϕ and THP1 cells were pretreated
with IU1or DMSO for 2 h followed stimulation by VSV or intracellular poly(I:C). IFN-β mRNA levels (A) and protein levels (B) were measured by
RT-qPCR or ELISA in Mϕ. IFN-β mRNA levels (C) and protein levels (D) were measured by RT-qPCR or ELISA in THP1 cells. (E) TCID50 analysis of Mϕ
and THP1 cells with VSV infected for 12 to 24 h were conducted in HEK293T cells. (F) THP1 cells infected with VSV-eGFP undergone fluorescence
microscopy (left) for 12 (top) to 16 h (bottom) (40-fold), bar = 100 μm, and average fluorescence intensity by histogram (right). (G) THP1 cells
infected with VSV-eGFP for 12 to 16 h were prepared for flow cytometry (left) at MOI = 0.01 (top), 0.025 (middle), and 0.05 (bottom), and green
fluorescence intensity is shown by histogram (right). (A–E) Data are shown as mean ± SD of three samples per group and are from one experiment
representative of three independent experiments. Significance was calculated in relation to the control group. *p < 0.05, **p < 0.01, ***p < 0.001
(two-sided Student’s t-test).


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46 Hongrui Li et al. Eur. J. Immunol. 2019. 49: 42–53

Figure 3. USP14 overexpression attenuated IFN-β mRNA expression and promoted VSV replications in HeLa cells. (A–E) Hela cells were transfected
with Flag-USP14 plasmids (Flag-USP14) or empty vector (control) for 24 h followed by VSV or intracellular poly(I:C) for 0, 4, 8, 12, or 16 h. (A) IFN-β
mRNA was quantified as described in the Materials and methods. (B) IFN-β production was measured by ELISA at 0 and 12 h. (C) TCID50 analysis
of VSV replication in cells infected with VSV for 12 to 24 h. (D) HeLa cells infected with VSV-eGFP for 16 h undergone fluorescence microscopy
examination (40 ×) (left), bar = 100 μm. Images are representative of three independent experiments. Histogram shows the average fluorescence
intensity (right). (E) Cells were infected with VSV-eGFP for 16 h and then analyzed by flow cytometry. Data were analyzed by FlowJo software (left)
and histogram (right) showed green fluorescence intensity. Similar results were obtained from three independent experiments. (A–E) Data are
shown as mean ± SD of three samples per group and are from one experiment representative of three independent experiments. Significance was
calculated in relation to the control group. *p < 0.05, **p < 0.01, ***p < 0.001 (two-sided Student’s t-test).

USP14 participated in antiviral response through RIG-I. For fur-
ther verification, IPs between exogenous Flag-USP14 and Myc-
RIG-I/MAVS/TBK1/IRF3 were conducted. As expected, USP14
interacted with RIG-I (Fig. 5D). Together, these data indicated
that USP14 regulated RIG-I activation when stimulated by RNA
virus.
Next, we examined the interaction between USP14 and RIG-
I with two IP assays. Plasmids expressing USP14 or RIG-I were
cotransfected into HEK293T cells. The co-IP was operated by
anti-Myc antibodies and analyzed by immunoblot, and the inter-
action was validated (Fig. 5E). Endogenous USP14 from Mϕ
lysates infected with VSV for 4 h were immunoprecipitated by
anti-USP14 antibodies. The immunoblot analysis indicated that
USP14 and RIG-I interacted with each other. Additionally, as
time went by, the interaction between USP14 and RIG-I turned
intense (Fig. 5F). USP14 contains two domains, ubiquitin-like
(Ubl) domain, which was responsible for interaction with protea-
some at C-terminal and ubiquitin-specific protease (USP) domain
at N-terminal was responsible for DUB activity. To determine the
domain responsible for the interaction, we generated two deleted
constructs of USP14. HEK293T cells were cotransfected with plas-
mids expressing USP14-FL/USP14-Ubl/ USP14-USP and RIG-I.
Co-IP and immunoblot analysis revealed that the USP domain
of USP14, instead of Ubl domain, interacted with RIG-I (Fig. 5G).
These results show that USP14 regulates RNA virus-induced type
I IFN signaling pathway through interacting with RIG-I by its USP
domain.
USP14 deubiquinates K63-linked RIG-I
We hypothesized that USP14 promotes RIG-I deubiquitination.
To test the hypothesis, IP and immunoblot were conducted in
HEK293T cells. Results showed that USP14 reduced RIG-I ubiq-
uitination (Fig. 6A). Further, we cotransfected HEK293T cells
with plasmids expressing Myc-RIG-I, Flag-USP14, and HA-Ub with


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Eur. J. Immunol. 2019. 49: 42–53 Innate immunity 47

Figure 4. IU1 enhances the antiviral response of mice in vivo. (A–D) Mice (6 weeks old, n = 8) were infected with VSV by intraperitoneal injection
for 12 h after IU1 or DMSO treatment for 2 h. (A) Quantified IFN-β mRNA expression of lungs and spleens as detailed in the Materials and
methods. (B) VSV replications from mice lungs and spleens were quantified by TCID50. (C) ELISA analyzed the concentrations of IFN-β in serum
from IU1- or DMSO-treated mice. (D) Hematoxylin–Eosin staining of lungs (200 ×) from mice in (A), bar = 100 μm. Images are representative of
three independent experiments. (E) Mice (6 weeks old, n = 12) pretreated with IU1 or DMSO for 2 h were injected with a lethal dose of VSV by
intraperitoneal injection, and survival was followed for 60 h. (A–C) Data are shown as mean ± SD of a single experiment from three independent
experiments (eight mice/group/experiment). (E) Data are shown as mean ± SD of a single experiment from two independent experiments (12
mice/group/experiment). **p < 0.01, ***p < 0.001.

WT/K6/K11/K27/K29/K33/K48 or K63 and immunoprecipitated
with anti-Myc antibodies. Immunblot analysis showed that USP14
attenuated K63-linked RIG-I ubiquitination (Fig. 6B and C). To
confirm the discovery above, endogenous experiment was con-
ducted in Mϕ that was infected with VSV. Cell lysates were
immunoprecipitated with anti-RIG-I antibodies. Immunoblot anal-
ysis suggested that USP14 knockdown increased RIG-I K63-linked
ubiquitination (Fig. 6D). USP domain was responsible for the
interaction between USP14 and RIG-I. Further, we examined the
truncated mutants of USP14 on RIG-I ubiquitination. Data demon-
strated that USP domain of USP14 was in charge of K63-linked
RIG-I deubiquitination (Fig. 6E). These results suggest that USP14
promotes K63-linked RIG-I deubiquitination through USP domain.
Discussion
DUBs play crucial roles in the control of immune pathways. For
example, DUBs could regulate innate immunity in TLRs- and
NLRs-mediated immune signaling. USP2a [41], USP10 [42], and
USP20 [43] regulate immune response through targeting TRAF6.
USP4 [44] and USP18 [45] target TAK1, USP25 [46] targets
TRAF3, A20 [47] and OTULIN [48] could target RIPK1/2 [49]. In
antiviral immune response, RIG-I, which mediated type I IFN pro-
duction, is undependable for controlling virus propagation. DUBs
directly regulate RIG-I K63-linked ubiquitination that includes
USP3, USP15, USP21, and CYLD. RIG-I K48-linked ubiquitination
could be regulated by USP4 [50].
Previous studies of USP14 focused mainly on neuron diseases
and cancer. USP14 loss in ax (J) mice causes a deficit in paired-
pulse facilitation at hippocampal synapses [51]. USP14 regulates
presynaptic function independently of its role in deubiquitina-
tion [52]. USP14’s catalytic activity is required for nervous system
structure, function, and neuromuscular junction synaptic trans-
mission [53]. USP14 is also important in cancer. The deficiency
of USP14 inhibits breast cancer cells proliferation, metastasis, and
promotes apoptosis [54]. In esophageal squamous cell carcinoma
cells, proliferation and tumor genesis were inhibited by reduced
expression of USP14 [55].
In innate immune response, when induced by LPS, USP14
increased the production of IL-6 and TNF-α, ERK1/2 phospho-
rylation, and NF-кB activation [56]. But Meng et al. showed
that USP14 negatively regulates NF-кB signaling by removing the
polyubiquitin chains from NLRC5 to enhance NLRC5-mediated
inhibition of NF-κB activation [25]. We discovered that USP14
suppressed VSV-induced RIG-I activation through deubiquitinat-
ing K63-linked RIG-I.
And USP14 promoted IL-6 and TNF-α production triggered
by VSV or transfected poly(I:C). Immunoblotting analysis indi-
cated that phosphorylation of P65 enhanced significantly in USP14
knockdown cells and negligible difference was observed in ERK,
JNK, and P38 phosphorylation. The inconsistency of conclu-
sions from different labs may result from different experiment
conditions.
The present study identified USP14 as a negative regulator in
antiviral response through its ability to bind to and deubiquitinate


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48 Hongrui Li et al. Eur. J. Immunol. 2019. 49: 42–53

Figure 5. USP14 targets and interacts with RIG-I. (A) Mϕ transfected with scrambled siRNA/USP14 siRNA or DMSO/IU1 for 36 h were infected with
poly(I:C) for 4 h, LPS for 4 h, and VSV for 8 h. IFN-β mRNA expression was measured by RT-qPCR as detailed in the Materials and methods. (B) Mϕ
and THP1 cells were transfected with control siRNA or USP14 siRNA for 36 h and then infected with VSV for indicated hours. Cell lysates were
separated by SDS–PAGE and immunoblotted with the indicated antibodies. (C) Dual-luciferase reporter assay was used to analyze the activation of
IFN-β and ISRE by RIG-I-N, MAVS, TBK1, and IRF3-5D with or without USP14 overexpression as detailed in Materials and methods. (D) HEK293T cells
were cotransfected with Flag-USP14 and Myc-RIG-I/MAVS/TBK1/IRF3. Cell lysates were immunoprecipitated with anti-Flag antibodies. (E) HEK293T
cells were cotransfected with Flag-USP14, Myc-RIG-I, and followed by immuneprecipitation with anti-Myc antibodies. (F) Mϕ were infected with
VSV for 0, 4, 8 h, then cell lyslates were immunoprecipitated with anti-USP14 antibodies and immunoblot analyzed the interaction between USP14
and RIG-I. (G) Schematic structure of USP14 and the derivatives are shown. HEK293T cells were cotransfected with Flag-USP14, Flag-USP14-Ubl,
Flag-USP14-USP, Myc-RIG-I, followed by immuneprecipitation with anti-Flag antibodies. (A, C) Data are shown as mean ± SD of three samples per
group and are from one experiment representative of three independent experiments. Significance was calculated in relation to the control group.
*p < 0.05, ***p < 0.001 (two-sided Student’s t-test). (B, D–G) Data are from one experiment representative of three independent experiments.


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Eur. J. Immunol. 2019. 49: 42–53 Innate immunity 49

Figure 6. USP14 removed K63-linked polyubiquitination conjugates from RIG-I. (A) HEK293T cells were co-transfected with Flag-USP14, Myc-RIG-I
and HA-Ub. Cell lyslates were precipitated with anti-Myc antibodies. Immunoblot analyzed the ubiquitination of RIG-I. (B) HEK293T cells were
co-transfected with Flag-USP14, Myc-RIG-I, HA- Ub including wild type (WT), K6, K11, K27, K29, K33, and K48. Cell lyslates were precipitated
with anti-Myc antibodies. Immunoblot analyzed the ubiquitination of RIG-I. (C) Plasmids expressing Flag-USP14, Myc-RIG-I and HA-Ub including
WT, K63 and K48 were co-transfected into HEK293T cells. Immunoprecipitation and immunoblot analyzed the ubiquitination of RIG-I. (D) Mϕ
was transfected with scrambled siRNA or USP14 siRNA for 36 h, then infected with VSV for 0 and 4 h. Cell lyslates were precipitated with
anti-RIG-I antibodies and immunoblot analyzed the ubiquitination of wild type and K63. (E) The structure of USP14 truncates (top). HEK293T
cells cotransfected with Flag-USP14, Flag-USP14-Ubl, Flag-USP14-USP, Myc-RIG-I, and HA-Ub-K63 for 24 h, followed by immunoprecipitation with
anti-Myc antibodies. (A–E) Blots are from one experiment representative of three independent experiments.


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50 Hongrui Li et al.
RIG-I. Knockdown of USP14 by siRNA significantly enhanced
RIG-I-triggered type I IFN signaling and inhibited VSV replication
both in Mϕ and THP1 macrophages. Inhibition of USP14 by IU1
increased RIG-I-mediated type I IFN production and antiviral
responses in vitro and in vivo. USP14 overexpression in HeLa
cells attenuated cell antiviral ability. In addition, we found that
USP14 acted by interacting with RIG-I and removing K63-linked
polyubiquitination chains from RIG-I.
We found that IU1 treatment exhibited stronger influence on
IFN-β production induced by VSV than USP14 knockdown. The
answer may be that IU1 directly inhibits the activity of USP14, but
USP14 siRNA works by reducing USP14 expression that indirectly
prevents the activity of USP14. However, there are some flaws in
this study, i.e. no USP14-null mice were included. In the future,
we wish to create a new mouse line with systemic or tissue/cell-
specific deletion of USP14 to further confirm our findings. In sum-
mary, our study has identified a previously unrecognized role for
USP14 in the negative regulation of antiviral response through
deubiquitinating K63-linked RIG-I. These findings provide insights
into a potential new therapy specifically targeting USP14 for RNA
virus-related diseases.
Materials and methods
Mice and cell culture
C57BL/6J mice (female, 5–6 weeks) were procured from Joint
Ventures Sipper BK Experimental Animals, Shanghai, China. Mice
were raised in pathogen-free conditions. All animal experiments
were preceded according to the National Institute of Health Guide
for the Care and Use of Laboratory Animals with approval of the
Zhejiang University, Hangzhou.
Mϕ extraction and culture were as described before [57].
THP1 cells were cultured in 1640 medium with 10% FBS and
treated with PMA (1 μg/mL) for 24 h to be induced into THP1
macrophages. HeLa cells, HEK293T cells, and Vero cells were cul-
tured in DMEM medium with 10% FBS.
Plasmids and reagents
Myc-tagged RIG-I (Myc-RIG-I), Myc-tagged RIG-I (N) (Myc-RIG-
I-N), Myc-tagged MAVS (Myc-MAVS), Myc-tagged TBK1 (Myc-
TBK1), Myc-tagged IRF3 (Myc-IRF3), Myc-tagged IRF3 (N) (Myc-
IRF3-5D), Flag-tagged USP14 (Flag-USP14), Flag-tagged USP
domain of USP14 (Flag-USP14-USP), and Flag-tagged Ubl domain
of USP14 (Flag-USP14-Ubl) plasmids were constructed by our lab.
HA-Ub, HA-K63-Ub, HA-K48-Ub, HA-K33-Ub, HA-K29-Ub, HA-
K27-Ub, HA-K11-Ub, HA-K6-Ub plasmids were gifts from Prof.
Xuetao Cao (National Key Laboratory of Medical Immunology,
Shanghai, China). IU1 (S7134) was purchased from Selleck.cn
(Shanghai, China).

C 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Immunol. 2019. 49: 42–53
Antibodies and virus
Anti-c-Myc (sc-40), anti-HA (sc-805), and anti-Flag (sc-807) were
purchased from Santa Cruze Biotechnology (Dallas, Tx, USA).
Antibodies specific to p-IRF3 (4947), IRF3 (4302), p-TBK1 (5483),
TBK1 (3504), USP14 (11931), ubiquitin (3936), and K63-linked
ubiquitin (12930) were purchased from Cell Signaling Technology
(Danvers, MA, USA). Antibody against DDX58 (RIG-I) (20056-1-
AP) was purchased from Proteintech (Rosemont, IL, USA). VSV
and VSV-eGFP belong to our lab.
VSV and reagents treatment
MOI of Mϕ, THP1 cells, and HeLa cells were 1, 0.01, and 1 sep-
arately. The regular dose of VSV for mice was 1× 108 PFU/g and
lethal dose was 5 × 108 PFU/g. Dosage of IU1 in cells lines
and mice were 100 μM and 10 mg/kg separately. Intracellular
and extracellular infection of poly(I:C) was 1 μg and 20 μg/mL,
respectively, and LPS was 100 ng/mL.
Cell transfection and dual-luciferase reporter assay
Transfection and dual-luciferase reporter assay were performed
as described previously [58]. Cells transfection was performed
in accordance with jetPEI (Polyplus transfection, Alsace, France)
instructions. Dual-luciferase reporter assay was performed in 96-
well plate with 100 ng of firefly luciferase reporter plasmid and
10 ng of Renilla luciferase reporter plasmid. All samples were set
as triplicate. The folds were calculated relative to baseline control
in each experiment.
RNA interference
Mϕ and THP1 cells were transfected according to Inter-
ferin instructions (Polyplus transfection). siRNAs were syn-
thesized by Gene Pharma (Shanghai, China). Mouse USP14
siRNA 5’-GGUAGAGAGACGGAUUCUUTT-3’, human USP14 siRNA
5’-GGAGAAAUUUGAAGGUGUA-3’, and scrambled siRNA 5’-
UUCUCCGAACGUGUCACGUdTdT-3’.
Mass spectrometry
MS analysis was performed as described by Song et al [59].
HEK293T cells were transfected with control vector, Flag-RIG-I,
or Myc-RIG-I plasmids for 24 h and then infected with VSV for
8 h before collecting. The cell lysates were immunoprecipitated
by antibodies to Flag or Myc tag and undergo MS analysis to iden-
tify RIG-I interacted DUBs.
www.eji-journal.eu
Eur. J. Immunol. 2019. 49: 42–53
RT-qPCR and ELISA
Total RNA of cells was extracted using Trizol, and cDNAs were
synthesized from RNAs according to manufacturer’s instructions
(Takara, Japan). The products were subjected to quantitative RT-
PCR (RT-qPCR) analysis using Hieff qPCR SYBR Green Master Mix
(No Rox) (YEASEN, Shanghai, China) and gene-specific primers
(Supporting Information Table S2). Calculation was conducted
relative to β-Actin by 2−t method with three replicates in each
three samples. The supernatants from infected cells or mouse
serum were subjected to ELISA using commercial mouse IFN-β
kit (Biolegend, San Diego, CA, USA) and human IFN-β kit (PBL
assay science, NJ, USA) with three samples in each experiment.
Immunoprecipitation and ubiquitination analysis
IP and ubiquitination analysis were conducted in HEK293T cells
and Mϕ, as we previously described [57]. HEK293T cells or Mϕ
were seeded into 6 cm dishes for 24 h. HEK293T cells were trans-
fected with plasmids for 24 h, and Mϕ were infected with VSV for
4 to 8 h. Cells then were lysed with NP40 lysis buffer. Lysates were
clarified by centrifugation and incubated with specific antibodies
for 1 h at 4°C followed by interacting with Protein A/G Sepharose
(Santa Cruze Biotechnology) overnight. The immunocomplexes
were recovered by brief centrifugation and washed five times with
lysis buffer and resuspended in 2 × loading buffer. Protein lysates
were subjected to SDS–PAGE and electrophoretically transferred
to nitro cellulose membranes (Bio-Rad, Hercules, CA, USA). And
subsequent immunoblot analysis was performed with indicated
antibodies.
Flow cytometric assay and immunofluorescence assay
Flow cytometric assay were conducted according to the guidelines
[60]. Cells were infected with VSV-eGFP or PBS for 12 or 16
h, and undergone pancreatin digestion following twice cold PBS
washing. Results were analyzed by FlowJo software. Cells were
pleated into 12-well plate and then infected with VSV-eGFP for
12 or 16 h, then the cells were washed twice with PBS, and then
plates were examined by fluorescence microscope.
Statistical analysis
Statistical significances between groups were determined by two-
tailed Student’s t-test and two-way ANOVA test.
Acknowledgements: This work was supported by grants from
Zhejiang Provincial Natural Science Foundation of China under

C 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Innate immunity 51
Grant No. LZ17H100001 and the National Natural Science Foun-
dation of China (31670914, 31870908).
We are grateful to Prof. Xuetao Cao (National Key Laboratory of
Medical Immunology, Shanghai, China) for providing IRF3-5D,
Ub, K48-Ub, and K63-Ub expression plasmids.
Conflict of interest: The authors declare no conflicts of interest.
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Abbreviations: DUB: deubiquitinating enzyme · IRF3: interferon regula-
tory factor 3 · Mϕ: mouse peritoneal macrophage · MAVS: mitochondrial
antiviral signaling protein · NF-κB: nuclear factor-kappa B · NLRs: NOD-
like receptors · poly(I:C): polyinosinic:polycytidylic acid · RIG-I: retinoic
acid-inducible gene I · TBK1: TANK-binding kinase 1 · Ubl: ubiquitin-like
domain · USP14: ubiquitin-specific protease 14 · VSV: vesicular stom-
atitis virus

54 Zhu, L., Yang, S., He, S., Qiang, F., Cai, J., Liu, R., Gu, C. et al., Downregu-
Full correspondence: Prof. Weilin Chen, Institute of Immunology,
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Zhejiang University School of Medicine, Hangzhou 310058, Zhejiang,
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e-mail: cwl@zju.edu.cn
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56 Liu, N., Kong, T., Chen, X., Hu, H., Gu, H., Liu, S., Chen, X. et al., Ubiquitin- Received: 15/3/2018
specific protease 14 regulates LPS-induced inflammation by increasing Revised: 15/10/2018
ERK1/2 phosphorylation and NF-kappaB activation. Mol. Cell. Biochem. Accepted: 19/11/2018
2017. 431: 87–96. Accepted article online: 22/11/2018


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