JVI Accepts, published online ahead of print on 23 July 2014 J. Virol. doi:10.1128/JVI.01400-14 Copyright © 2014, American Society for Microbiology. All Rights Reserved.
1
Downregulation of miR-526a by Enterovirus Inhibits RIG-Ⅰ-Dependent Innate
2
Immune Response
3
Changzhi Xu,1,2* Xiang He,1* Zirui Zhen g,1,2 * Zhe Zhang,1* Congwen Wei,1 Kai
4
Guan,1 Lihua Hou,1 Buchang Zhang 2, Lin Zhu2, Yuan Cao,3 Yanhong Zhang,1
5
Ye Cao,1 Shengli Ma,1 Penghao Wang,1 Pingping Zhang,3,1 Quanbin Xu,1
6
Youguo Ling,1 Xiao Yang,1,4 and Hui Zhong1, 4
7 8 9 10
1
Beijing Institute of Biotechnology, Beijing, 100850, China.
2
Institute of Health Science, School of Life Sciences, AnHui University, Hefei,
Anhui 230601,China. 3
Department of Laboratory Medicine, General Hospital of Jinan Military
11
Region, Jinan, Shandong, 250031, China.
12
* These authors contributed equally to this work.
13
4
Correspondence should be addressed to H. Z ([email protected]) or X. Y
14
([email protected]).
15
Running title: EV71 3C Targets miR-526a to Inhibit the Innate Immune
16
Response
17
18
Abstract
19
Retinoic acid-inducible gene I (RIG-I) is an intracellular RNA virus sensor that
20
induces type I interferon-mediated host protective innate immunity against viral
21
infection. Although cylindromatosis (CYLD) has been shown to negatively
22
regulate innate antiviral response by removing K-63-linked polyubiquitin from
23
RIG-I, the regulation of its expression and the underlying regulatory
24
mechanisms are still incompletely understood. Here we show that RIG-I
25
activity is regulated by miR-526a-mediated inhibition of CYLD expression. We
26
found that viral infection specifically upregulates miR-526a expression in
27
macrophages via IRF-dependent mechanisms. In turn, miR-526a positively
28
regulates virus-triggered type Ⅰ Interferon (IFN-I) production, thus suppressing
29
viral replication, the underlying mechanism of which is the enhancement of
30
RIG-I K63-linked ubiquitination by miR-526a via suppressing the expression of
31
CYLD.
32
downregulation are blocked by enterovirus 71 (EV71) 3C protein, while ectopic
33
miR-526a expression inhibits the replication of EV71 virus. The collective
34
results of this study suggest a novel mechanism of the regulation of RIG-I
35
activity during RNA virus infection by miR-526a and propose a novel
36
mechanism for the evasion of innate immune response controlled by EV71.
37
Remarkably,
viral-induced
miR-526a
upregulation
and
CYLD
38
Importance
39
RNA virus infection upregulates the expression of miR-526a in macrophages
40
through IRF-dependent pathways. In turn, miR-526a positively regulates
41
virus-triggered type I IFN production and inhibits viral replication, the
42
underlying mechanism of which is the enhancement of RIG-I K-63
43
ubiquitination by miR-526a via suppressing the expression of CYLD.
44
Remarkably, viral-induced miR-526a upregulation and CYLD downregulation
45
are blocked by enterovirus 71 (EV71) 3C protein, cells with overexpressed
46
miR-526a were highly resistant to EV71 infection. The collective results of this
47
study suggest a novel mechanism of the regulation of RIG-I activity during
48
RNA virus infection by miR-526a and propose a novel mechanism for the
49
evasion of innate immune response controlled by EV71.
50
51
Introduction
52
EV71 is a positive-stranded RNA virus, which belongs to the picornavirus
53
family (1) and is the causative agent of hand-foot-and-mouth disease (HFMD)
54
in young children and infants. The genome of EV71 is approximately 7.5 kb in
55
length and contains a single open reading frame encoding a polyprotein
56
precursor, which is processed into structural (VP1, VP2, VP3, and VP4) and
57
nonstructural proteins (2A, 2B, 2C, 3A, 3B, 3C, and 3D) during viral infection
58
(2). Despite the protective role of IFN-I on EV71 infection, EV71 inoculation is
59
unable to elicit their production. Most members of the picornavirus family,
60
including poliovirus, rhinovirus, echovirus, and encephalomyocarditis virus,
61
use strategies to inhibit IFN-Ⅰ induction by interfering with melanoma
62
differentiation-associated gene 5 (MDA-5) and RIG-Ⅰ (3-5) or by restricting
63
IFN-secretion through repressing the cellular secretory pathway (6). Recent
64
studies revealed that the 3C protease of EV71 associated with RIG-Ⅰ and
65
cleaved TRIF (TIR-domain-containing adapter-inducing interferon-β)
66
IRF7 ( interferon regulatory factor 7) (7, 8); moreover, EV71 inhibited
67
IFN-I-induced ISGs (interferon stimulating genes) in host cells by reducing
68
IFNAR1 (type I interferon receptor 1) levels in host cells (9). However,
69
additional work is required to understand the mechanisms for EV71 to escape
70
from innate antiviral responses.
and
71
IFN-I, as the first line of host immune response, is critical in mediating
72
antiviral defense. The host senses viral and bacterial pathogen invasion by
73
recognition of pathogen-associated molecular patterns with pattern recognition
74
receptors, including membrane-bound TLRs (Toll-like receptors) (10, 11) and
75
cytosolic sensory molecules, such as the multi-domain containing NOD
76
(nucleotide-binding oligomerization domain) proteins, RIG-Ⅰ, and MDA-5
77
helicases (12-14). Both RIG-Ⅰ and MDA-5 contain caspase recruitment
78
domains (CARDs) that interact with the CARD domain-containing protein
79
mitochondrial antiviral signaling (MAVS) upon binding to uncapped RNA,
80
resulting in MAVS association with IκB kinase (IKK) proteins. While MAVS
81
association with IKKα/β activates NF-κB (nuclear factor-κ-gene binding), its
82
association with TBK1 (TANK-binding kinase 1 ) as well as IKKε leads to the
83
activation of IRF-3/IRF-7; coordinated activation of NF-κB and IRF pathways
84
further results in the assembly of a multi-protein enhancer complex that drives
85
the expression of IFN-β (interferon-β) and the IFN-mediated antiviral immunity
86
(15-19).
87
RIG-I signaling is negatively regulated at multiple levels. Previous reports
88
showed that the ubiquitination status of RIG-I is controlled by CYLD, a tumor
89
suppressor originally identified as a genetic defect in familial cylindromatosis
90
(20). Indeed, CYLD was shown to interact with the CARDs of RIG-I and to
91
remove K63-linked polyubiquitin chains from RIG-I, which inhibits downstream
92
signaling. DC (Dendritic cells) lacking CYLD constitutively polyubiquitinates
93
RIG-I and shows enhanced activity of TBK1 and IKKε, suggesting that CYLD
94
regulates basal RIG-I activity by modulating its K63-polyubiquitin status (21).
95
CYLD also acts as a negative regulator of NF-κB and Jun N-terminal kinase
96
signaling pathways by removing Lys 63-linked polyubiquitin from NEMO
97
(nuclear factor-kappaa B essential modulator), IKKε, TRAF2 (TNF receptor
98
associated factor 2) or BCL3 (B-cell CLL/lymphoma3) (22-25). These findings
99
thus establish CYLD as a critical regulator of antiviral innate immune response.
100
MiRNAs, an abundant class of highly conserved noncoding RNA
101
oligonucleotides (18-25 nt long), suppress gene expression by binding to the
102
3’-untranslational region (UTR) of target mRNAs. MiRNAs play key roles in the
103
regulation of diverse biological processes. Recently, a role for miRNAs has
104
been proposed in the regulation of innate immune responses in monocytes
105
and macrophages. Direct roles of miRNAs in innate immune response were
106
discovered in a report that identified miR-146a as a negative feedback
107
regulator in RLR signaling by targeting IL-1R associated kinase (IRAK) 1 and
108
TNF receptor associated factor 6 (TRAF6) (26). Further reports showed that
109
both miR-155 and miR-132 were induced in monocyte cell line treated with the
110
TLR4 (Toll-like receptor 4) ligand lipopolysaccharide (LPS) (27). Given the
111
important roles of the RIG-Ⅰ signaling pathway in innate antiviral immune
112
response, identifying more miRNAs that can regulate RIG-Ⅰ-dependent IFN-I
113
production is of vital importance. In fact, many viruses have evolved strategies
114
to interfere with these innate signaling events and hence inhibit IFN-β
115
production. However, to date, there is few report about the regulation of RIG-Ⅰ
116
signaling pathway by miRNAs, especially during EV71 infection.
117
In the present study, we found that miR-526a was significantly
118
upregulated in macrophages upon viral infection in an IRF-dependent manner.
119
Then we demonstrated that miR-526a feedback positively regulated
120
VSV-triggered IRF3 activation by suppressing CYLD expression and
121
subsequent RIG- Ⅰ ubiquitination. Furthermore, we found that miR-526a
122
upregulation was blocked by EV71 3C protease, whereas ectopic miR-526a
123
expression inhibited the replication of EV71. Thus the present study has for the
124
first time demonstrated that miR-526a is a positive feedback regulator of the
125
RIG-Ⅰ signaling and that EV71 targets miR-526a to suppress RIG-Ⅰ-dependent
126
IFN-I production.
127
Materials and Methods
128
Cell Culture and Transfection
129
293T, Vero, RD, MDCK and THP-1 cells were cultured in DMEM and
130
RPMI1640 (Invitrogen) respectively, supplemented with 10% heat-inactivated
131
fetal bovine serum (FBS, Invitrogen), 100 U/mL penicillin and 100 mg/mL
132
streptomycin. Vectors and Epitope Tagging of Flag-tagged IRF3, IRF7, p65,
133
MDA-5, RIG-I, N-RIG-I, MAVS and TBK1 were expressed by cloning the
134
respective genes into the pcDNA3-Flag vector. MiR-526a expression plasmid
135
was expressed by cloning miR-526a into Pires 2-EGFP vector. siRNA oligos
136
were ordered from GenePharma and transfected with Lipofectamine 2000
137
(Invitrogen) according to the manufacturer’s instructions. The sequences of
138
primers for small interference RNAs used were shown in Table 1.
139
miRNA mimics and inhibitors
140
MiR-526a
141
(single-stranded chemically modified oligonucleotides) from GenePharma
142
were used for the overexpression and inhibition of MiR-526a activity
143
respectively. Negative control mimics or inhibitors (GenePharma) were
144
transfected as matched controls.
145
MiR-526a mimics sense: 5’-CUCUAGAGGGAAGCACUUUCUG-3’;
146
anti-sense: 5’-GAAAGUGCUUCCCUCUAGAGUU-3’,
147
MiR-526b mimics sense: 5’-CUCUUGAGGGAAGCACUUUCUGU-3’;
148
anti-sense: 5’-AGAAAGUGCUUCCCUCAAGAGUU-3’,
mimics
(dsRNA
oligonucleotides)
and
MiR-526a
inhibitors
149
control mimics sense: 5’-UUCUCCGAACGUGUCACGUTT-3’;
150
anti-sense: 5’-ACGUGACACGUUCGGAGAATT-3’,
151
miR-526a
152
modified by 2’-Ome),
153
control inhibitor 5’-CAGUACUUUUGUGUAGUACAA-3’.
154
Viruses and Viral Plaque Assay
155
The construction of the GFP-encoding Newcastle disease virus (NDV-GFP)
156
used in this study was previously described (28) and was obtained from Dr.
157
Cheng Cao (Beijing Institute of Biotechnology). Vesicular stomatitis virus (VSV)
158
and herpes simplex virus (HSV) was obtained from Dr. Cheng Cao (Beijing
159
Institute of Biotechnology). VSV and NDV-GFP was propagated in chicken
160
embryo fibroblasts and chicken embryos respectively and titrated in MDCK
161
cells. HSV was propagated and titrated in Vero cells. EV71 viral strain
162
GDV-103 (purchased from China Center for Type Culture Collection, CCTCC)
163
was grown in RD cells and was propagated and titrated in RD cells.
164
Luciferase Reporter Assays
165
CYLD 3’-UTR luciferase reporter assay: Four CYLD 3'-UTR luciferase
166
reporter constructs were made by amplifying the human CYLD mRNA 3'-UTR
167
sequence by PCR and cloning into the pGL3-cm plasmid. The 293T cells were
168
co-transfected with 200 ng of luciferase reporter plasmid (cm-1 to cm-4), 4 ng
169
of pRL-TK-Renilla-luciferase (pRL) plasmid, and the indicated RNAs (final
170
concentration, 20 nM). After 36 h, luciferase activities were measured using
inhibitor
5’-CAGAAAGUGCUUCCCUCUAGAG-3’
(chemically
171
the Dual-Luciferase Reporter Assay System (Promega) according to the
172
manufacturer's instructions. Data was acquired through the ratios of firefly
173
luciferase activity to that of pRL luciferase.
174
IFN-β, NF-κB and IRF3-luciferase reporter assay: 200 ng of luciferase
175
reporter plasmids containing promoter of IFN-β, NF-κB (IFN-β-luc, NF-κB-luc)
176
and IRF3-luciferase plasmids (Gal4-IRF3 and UAS-luc) were cotransfected
177
with 4 ng of pRL plasmid into 293T cells. After indicated treatments, luciferase
178
activities were measured as previously above (29).
179
RNA quantification
180
Total RNA from cells was extracted with TRIzol reagent (Invitrogen) following
181
the manufacturer's instructions. For the quantification of miR-526a and
182
miR-526b, RNA was reverse transcribed (RT) using the Takara microRNA
183
Reverse Transcription Kit and miRNA-specific stem-loop primers (Table 2).
184
Similarly, U6A small nuclear RNA was quantified using its reverse primer for
185
RT reaction (Table 2). Real-time quantitative polymerase chain reaction
186
(RT-PCR) analysis was performed using the Multicolor Real-Time PCR
187
Detection System (IQ5, BioRad) and SYBR RT-PCR kits (Takara). RT-PCR
188
primer sequences for miR-526a, miR-526b and U6A were listed in Table 3 and
189
the relative expression level of miRNAs was normalized to U6A by 2-△△Ct
190
threshold method (30). RT-PCR reactions were incubated in a 96-well plate at
191
94℃ for 2 min, followed by 40 cycles of 94℃ for 20 s and 60℃ for 30 s. All
192
reverse transcriptase reactions, including no-template controls and RT minus
193
controls, were run in duplicate. Sequences of RT-PCR primers for other genes
194
used in the paper were listed in Table 3. Data were normalized by the level of
195
β-actin expression in each sample as described above.
196
Immunoprecipitation and Immunoblot
197
Cells were harvested in cell lysis buffer (50 mM Tris-HCl [pH 7.5], 10 mM
198
sodium fluoride, 10 mg/mL aprotinin, 10 mg/mL leupeptin, 1 mM
199
phenylmethylsulfonyl fluoride, 1 mM dithiothreitol, and 10 mg/mL pepstatin A)
200
containing 1% Nonidet P-40. Whole cell lysates was subjected to
201
immunoprecipitation
202
Immunoblot
203
(Sigma-Aldrich). The prepared samples were detected with anti-VSV G (Santa
204
Cruz Biotechnology), CYLD, IRF3, IRF3-p, IKKα, IKKβ and IRF7 (Epitomic),
205
IKKα/β-p (Cell Signaling Technology), IκB-p and IκB (Sigma-Aldrich)
206
antibodies, MDA-5 and IFNAR1(Abcam) antibodies, respectively.
207
Flow cytometry
208
293T cells were cotransfected with miR-526a mimics or miR-526a inhibitors
209
followed by NDV-GFP infection. At 24 h post-infection, cells were subjected to
210
flow cytometric analysis on a FACS Calibur, and data were analyzed with
211
CellQuest software (both from BD Biosciences). The mean fluorescence
212
intensity and positive percentage rate of green-fluorescing cells were
213
determined.
214
Assay of IFN-β Secretion from THP-1Cells
analysis
with was
anti-Flag
agarose
performed
with
beads anti-HA
(Sigma-Aldrich). and
anti-Flag
215
To assay for IFN-β secretion, THP-1 cells (1×106 cells/ mL) were infected with
216
VSV. The culture medium was then used to quantify IFN-β by AlphaLISA IFN-β
217
Kit following the manufacturer's instructions (PerkinElmer Life Sciences).
218
In vivo Ubiquitination Assay
219
Samples were harvested post transfection and infection, and whole cell lysates
220
(WCL) were prepared in a 1% NP-40 lysis buffer supplemented with 0.1%
221
protease inhibitor cocktail (Sigma-Aldrich) and the deubiquitinase inhibitor
222
N-ethylmaleimide (10 mM; Sigma-Aldrich). Protein-protein interactions were
223
disrupted by sonication (3 pulses of 10 s) using the 550 Sonic Dismembrator
224
(Fisher Scientific Inc) followed by boiling for 10 min in 1% SDS. WCL (250 to
225
500μg)
226
Polyubiquitination was detected using a monoclonal anti-HA antibody
227
(Sigma-Aldrich).
228
Statistical Analysis
229
The difference between two groups was statistically analyzed by a two-tailed
230
Student’s t test. All data points were the average of triplicates, with error bars
231
representing standard deviations. All data were representative of results from
232
at least 3 independent experiments. *, P -0.04
-22.9
DUSP4
> -0.04
-21.8
MARCH5
-0.03
-22.6
BCL11A
-0.03
-22.1
CREB1
> -0.03
-26.8
KSR2
> -0.03
-33.7
HIPK2
> -0.02
-28.8
IL21R
> -0.01
-22.3
786
Table 5. miR-526a targets on EV71 predicted by Vita MEF miR-526a
miR-526b
miR-296
≤-10
98
203
303
≤-15
26
95
115
≤-20
3
7
14
≤-25
0
0
6
≤-10
23
31
85
≤-15
14
19
50
≤-20
3
1
8
≤-25
0
0
0
≤-10
1
2
12
≤-15
0
2
7
≤-20
0
0
4
≤-25
0
0
0
（Kcal/mol） Score≥120
Score≥140
Score≥160
787
1
Downregulation of miR-526a by Enterovirus Inhibits RIG-Ⅰ-Dependent Innate
2
Immune Response
3
Changzhi Xu,1,2* Xiang He,1* Zirui Zhen g,1,2 * Zhe Zhang,1* Congwen Wei,1 Kai
4
Guan,1 Lihua Hou,1 Buchang Zhang 2, Lin Zhu2, Yuan Cao,3 Yanhong Zhang,1
5
Ye Cao,1 Shengli Ma,1 Penghao Wang,1 Pingping Zhang,3,1 Quanbin Xu,1
6
Youguo Ling,1 Xiao Yang,1,4 and Hui Zhong1, 4
7 8 9 10
1
Beijing Institute of Biotechnology, Beijing, 100850, China.
2
Institute of Health Science, School of Life Sciences, AnHui University, Hefei,
Anhui 230601,China. 3
Department of Laboratory Medicine, General Hospital of Jinan Military
11
Region, Jinan, Shandong, 250031, China.
12
* These authors contributed equally to this work.
13
4
Correspondence should be addressed to H. Z ([email protected]) or X. Y
14
([email protected]).
15
Running title: EV71 3C Targets miR-526a to Inhibit the Innate Immune
16
Response
17
18
Abstract
19
Retinoic acid-inducible gene I (RIG-I) is an intracellular RNA virus sensor that
20
induces type I interferon-mediated host protective innate immunity against viral
21
infection. Although cylindromatosis (CYLD) has been shown to negatively
22
regulate innate antiviral response by removing K-63-linked polyubiquitin from
23
RIG-I, the regulation of its expression and the underlying regulatory
24
mechanisms are still incompletely understood. Here we show that RIG-I
25
activity is regulated by miR-526a-mediated inhibition of CYLD expression. We
26
found that viral infection specifically upregulates miR-526a expression in
27
macrophages via IRF-dependent mechanisms. In turn, miR-526a positively
28
regulates virus-triggered type Ⅰ Interferon (IFN-I) production, thus suppressing
29
viral replication, the underlying mechanism of which is the enhancement of
30
RIG-I K63-linked ubiquitination by miR-526a via suppressing the expression of
31
CYLD.
32
downregulation are blocked by enterovirus 71 (EV71) 3C protein, while ectopic
33
miR-526a expression inhibits the replication of EV71 virus. The collective
34
results of this study suggest a novel mechanism of the regulation of RIG-I
35
activity during RNA virus infection by miR-526a and propose a novel
36
mechanism for the evasion of innate immune response controlled by EV71.
37
Remarkably,
viral-induced
miR-526a
upregulation
and
CYLD
38
Importance
39
RNA virus infection upregulates the expression of miR-526a in macrophages
40
through IRF-dependent pathways. In turn, miR-526a positively regulates
41
virus-triggered type I IFN production and inhibits viral replication, the
42
underlying mechanism of which is the enhancement of RIG-I K-63
43
ubiquitination by miR-526a via suppressing the expression of CYLD.
44
Remarkably, viral-induced miR-526a upregulation and CYLD downregulation
45
are blocked by enterovirus 71 (EV71) 3C protein, cells with overexpressed
46
miR-526a were highly resistant to EV71 infection. The collective results of this
47
study suggest a novel mechanism of the regulation of RIG-I activity during
48
RNA virus infection by miR-526a and propose a novel mechanism for the
49
evasion of innate immune response controlled by EV71.
50
51
Introduction
52
EV71 is a positive-stranded RNA virus, which belongs to the picornavirus
53
family (1) and is the causative agent of hand-foot-and-mouth disease (HFMD)
54
in young children and infants. The genome of EV71 is approximately 7.5 kb in
55
length and contains a single open reading frame encoding a polyprotein
56
precursor, which is processed into structural (VP1, VP2, VP3, and VP4) and
57
nonstructural proteins (2A, 2B, 2C, 3A, 3B, 3C, and 3D) during viral infection
58
(2). Despite the protective role of IFN-I on EV71 infection, EV71 inoculation is
59
unable to elicit their production. Most members of the picornavirus family,
60
including poliovirus, rhinovirus, echovirus, and encephalomyocarditis virus,
61
use strategies to inhibit IFN-Ⅰ induction by interfering with melanoma
62
differentiation-associated gene 5 (MDA-5) and RIG-Ⅰ (3-5) or by restricting
63
IFN-secretion through repressing the cellular secretory pathway (6). Recent
64
studies revealed that the 3C protease of EV71 associated with RIG-Ⅰ and
65
cleaved TRIF (TIR-domain-containing adapter-inducing interferon-β)
66
IRF7 ( interferon regulatory factor 7) (7, 8); moreover, EV71 inhibited
67
IFN-I-induced ISGs (interferon stimulating genes) in host cells by reducing
68
IFNAR1 (type I interferon receptor 1) levels in host cells (9). However,
69
additional work is required to understand the mechanisms for EV71 to escape
70
from innate antiviral responses.
and
71
IFN-I, as the first line of host immune response, is critical in mediating
72
antiviral defense. The host senses viral and bacterial pathogen invasion by
73
recognition of pathogen-associated molecular patterns with pattern recognition
74
receptors, including membrane-bound TLRs (Toll-like receptors) (10, 11) and
75
cytosolic sensory molecules, such as the multi-domain containing NOD
76
(nucleotide-binding oligomerization domain) proteins, RIG-Ⅰ, and MDA-5
77
helicases (12-14). Both RIG-Ⅰ and MDA-5 contain caspase recruitment
78
domains (CARDs) that interact with the CARD domain-containing protein
79
mitochondrial antiviral signaling (MAVS) upon binding to uncapped RNA,
80
resulting in MAVS association with IκB kinase (IKK) proteins. While MAVS
81
association with IKKα/β activates NF-κB (nuclear factor-κ-gene binding), its
82
association with TBK1 (TANK-binding kinase 1 ) as well as IKKε leads to the
83
activation of IRF-3/IRF-7; coordinated activation of NF-κB and IRF pathways
84
further results in the assembly of a multi-protein enhancer complex that drives
85
the expression of IFN-β (interferon-β) and the IFN-mediated antiviral immunity
86
(15-19).
87
RIG-I signaling is negatively regulated at multiple levels. Previous reports
88
showed that the ubiquitination status of RIG-I is controlled by CYLD, a tumor
89
suppressor originally identified as a genetic defect in familial cylindromatosis
90
(20). Indeed, CYLD was shown to interact with the CARDs of RIG-I and to
91
remove K63-linked polyubiquitin chains from RIG-I, which inhibits downstream
92
signaling. DC (Dendritic cells) lacking CYLD constitutively polyubiquitinates
93
RIG-I and shows enhanced activity of TBK1 and IKKε, suggesting that CYLD
94
regulates basal RIG-I activity by modulating its K63-polyubiquitin status (21).
95
CYLD also acts as a negative regulator of NF-κB and Jun N-terminal kinase
96
signaling pathways by removing Lys 63-linked polyubiquitin from NEMO
97
(nuclear factor-kappaa B essential modulator), IKKε, TRAF2 (TNF receptor
98
associated factor 2) or BCL3 (B-cell CLL/lymphoma3) (22-25). These findings
99
thus establish CYLD as a critical regulator of antiviral innate immune response.
100
MiRNAs, an abundant class of highly conserved noncoding RNA
101
oligonucleotides (18-25 nt long), suppress gene expression by binding to the
102
3’-untranslational region (UTR) of target mRNAs. MiRNAs play key roles in the
103
regulation of diverse biological processes. Recently, a role for miRNAs has
104
been proposed in the regulation of innate immune responses in monocytes
105
and macrophages. Direct roles of miRNAs in innate immune response were
106
discovered in a report that identified miR-146a as a negative feedback
107
regulator in RLR signaling by targeting IL-1R associated kinase (IRAK) 1 and
108
TNF receptor associated factor 6 (TRAF6) (26). Further reports showed that
109
both miR-155 and miR-132 were induced in monocyte cell line treated with the
110
TLR4 (Toll-like receptor 4) ligand lipopolysaccharide (LPS) (27). Given the
111
important roles of the RIG-Ⅰ signaling pathway in innate antiviral immune
112
response, identifying more miRNAs that can regulate RIG-Ⅰ-dependent IFN-I
113
production is of vital importance. In fact, many viruses have evolved strategies
114
to interfere with these innate signaling events and hence inhibit IFN-β
115
production. However, to date, there is few report about the regulation of RIG-Ⅰ
116
signaling pathway by miRNAs, especially during EV71 infection.
117
In the present study, we found that miR-526a was significantly
118
upregulated in macrophages upon viral infection in an IRF-dependent manner.
119
Then we demonstrated that miR-526a feedback positively regulated
120
VSV-triggered IRF3 activation by suppressing CYLD expression and
121
subsequent RIG- Ⅰ ubiquitination. Furthermore, we found that miR-526a
122
upregulation was blocked by EV71 3C protease, whereas ectopic miR-526a
123
expression inhibited the replication of EV71. Thus the present study has for the
124
first time demonstrated that miR-526a is a positive feedback regulator of the
125
RIG-Ⅰ signaling and that EV71 targets miR-526a to suppress RIG-Ⅰ-dependent
126
IFN-I production.
127
Materials and Methods
128
Cell Culture and Transfection
129
293T, Vero, RD, MDCK and THP-1 cells were cultured in DMEM and
130
RPMI1640 (Invitrogen) respectively, supplemented with 10% heat-inactivated
131
fetal bovine serum (FBS, Invitrogen), 100 U/mL penicillin and 100 mg/mL
132
streptomycin. Vectors and Epitope Tagging of Flag-tagged IRF3, IRF7, p65,
133
MDA-5, RIG-I, N-RIG-I, MAVS and TBK1 were expressed by cloning the
134
respective genes into the pcDNA3-Flag vector. MiR-526a expression plasmid
135
was expressed by cloning miR-526a into Pires 2-EGFP vector. siRNA oligos
136
were ordered from GenePharma and transfected with Lipofectamine 2000
137
(Invitrogen) according to the manufacturer’s instructions. The sequences of
138
primers for small interference RNAs used were shown in Table 1.
139
miRNA mimics and inhibitors
140
MiR-526a
141
(single-stranded chemically modified oligonucleotides) from GenePharma
142
were used for the overexpression and inhibition of MiR-526a activity
143
respectively. Negative control mimics or inhibitors (GenePharma) were
144
transfected as matched controls.
145
MiR-526a mimics sense: 5’-CUCUAGAGGGAAGCACUUUCUG-3’;
146
anti-sense: 5’-GAAAGUGCUUCCCUCUAGAGUU-3’,
147
MiR-526b mimics sense: 5’-CUCUUGAGGGAAGCACUUUCUGU-3’;
148
anti-sense: 5’-AGAAAGUGCUUCCCUCAAGAGUU-3’,
mimics
(dsRNA
oligonucleotides)
and
MiR-526a
inhibitors
149
control mimics sense: 5’-UUCUCCGAACGUGUCACGUTT-3’;
150
anti-sense: 5’-ACGUGACACGUUCGGAGAATT-3’,
151
miR-526a
152
modified by 2’-Ome),
153
control inhibitor 5’-CAGUACUUUUGUGUAGUACAA-3’.
154
Viruses and Viral Plaque Assay
155
The construction of the GFP-encoding Newcastle disease virus (NDV-GFP)
156
used in this study was previously described (28) and was obtained from Dr.
157
Cheng Cao (Beijing Institute of Biotechnology). Vesicular stomatitis virus (VSV)
158
and herpes simplex virus (HSV) was obtained from Dr. Cheng Cao (Beijing
159
Institute of Biotechnology). VSV and NDV-GFP was propagated in chicken
160
embryo fibroblasts and chicken embryos respectively and titrated in MDCK
161
cells. HSV was propagated and titrated in Vero cells. EV71 viral strain
162
GDV-103 (purchased from China Center for Type Culture Collection, CCTCC)
163
was grown in RD cells and was propagated and titrated in RD cells.
164
Luciferase Reporter Assays
165
CYLD 3’-UTR luciferase reporter assay: Four CYLD 3'-UTR luciferase
166
reporter constructs were made by amplifying the human CYLD mRNA 3'-UTR
167
sequence by PCR and cloning into the pGL3-cm plasmid. The 293T cells were
168
co-transfected with 200 ng of luciferase reporter plasmid (cm-1 to cm-4), 4 ng
169
of pRL-TK-Renilla-luciferase (pRL) plasmid, and the indicated RNAs (final
170
concentration, 20 nM). After 36 h, luciferase activities were measured using
inhibitor
5’-CAGAAAGUGCUUCCCUCUAGAG-3’
(chemically
171
the Dual-Luciferase Reporter Assay System (Promega) according to the
172
manufacturer's instructions. Data was acquired through the ratios of firefly
173
luciferase activity to that of pRL luciferase.
174
IFN-β, NF-κB and IRF3-luciferase reporter assay: 200 ng of luciferase
175
reporter plasmids containing promoter of IFN-β, NF-κB (IFN-β-luc, NF-κB-luc)
176
and IRF3-luciferase plasmids (Gal4-IRF3 and UAS-luc) were cotransfected
177
with 4 ng of pRL plasmid into 293T cells. After indicated treatments, luciferase
178
activities were measured as previously above (29).
179
RNA quantification
180
Total RNA from cells was extracted with TRIzol reagent (Invitrogen) following
181
the manufacturer's instructions. For the quantification of miR-526a and
182
miR-526b, RNA was reverse transcribed (RT) using the Takara microRNA
183
Reverse Transcription Kit and miRNA-specific stem-loop primers (Table 2).
184
Similarly, U6A small nuclear RNA was quantified using its reverse primer for
185
RT reaction (Table 2). Real-time quantitative polymerase chain reaction
186
(RT-PCR) analysis was performed using the Multicolor Real-Time PCR
187
Detection System (IQ5, BioRad) and SYBR RT-PCR kits (Takara). RT-PCR
188
primer sequences for miR-526a, miR-526b and U6A were listed in Table 3 and
189
the relative expression level of miRNAs was normalized to U6A by 2-△△Ct
190
threshold method (30). RT-PCR reactions were incubated in a 96-well plate at
191
94℃ for 2 min, followed by 40 cycles of 94℃ for 20 s and 60℃ for 30 s. All
192
reverse transcriptase reactions, including no-template controls and RT minus
193
controls, were run in duplicate. Sequences of RT-PCR primers for other genes
194
used in the paper were listed in Table 3. Data were normalized by the level of
195
β-actin expression in each sample as described above.
196
Immunoprecipitation and Immunoblot
197
Cells were harvested in cell lysis buffer (50 mM Tris-HCl [pH 7.5], 10 mM
198
sodium fluoride, 10 mg/mL aprotinin, 10 mg/mL leupeptin, 1 mM
199
phenylmethylsulfonyl fluoride, 1 mM dithiothreitol, and 10 mg/mL pepstatin A)
200
containing 1% Nonidet P-40. Whole cell lysates was subjected to
201
immunoprecipitation
202
Immunoblot
203
(Sigma-Aldrich). The prepared samples were detected with anti-VSV G (Santa
204
Cruz Biotechnology), CYLD, IRF3, IRF3-p, IKKα, IKKβ and IRF7 (Epitomic),
205
IKKα/β-p (Cell Signaling Technology), IκB-p and IκB (Sigma-Aldrich)
206
antibodies, MDA-5 and IFNAR1(Abcam) antibodies, respectively.
207
Flow cytometry
208
293T cells were cotransfected with miR-526a mimics or miR-526a inhibitors
209
followed by NDV-GFP infection. At 24 h post-infection, cells were subjected to
210
flow cytometric analysis on a FACS Calibur, and data were analyzed with
211
CellQuest software (both from BD Biosciences). The mean fluorescence
212
intensity and positive percentage rate of green-fluorescing cells were
213
determined.
214
Assay of IFN-β Secretion from THP-1Cells
analysis
with was
anti-Flag
agarose
performed
with
beads anti-HA
(Sigma-Aldrich). and
anti-Flag
215
To assay for IFN-β secretion, THP-1 cells (1×106 cells/ mL) were infected with
216
VSV. The culture medium was then used to quantify IFN-β by AlphaLISA IFN-β
217
Kit following the manufacturer's instructions (PerkinElmer Life Sciences).
218
In vivo Ubiquitination Assay
219
Samples were harvested post transfection and infection, and whole cell lysates
220
(WCL) were prepared in a 1% NP-40 lysis buffer supplemented with 0.1%
221
protease inhibitor cocktail (Sigma-Aldrich) and the deubiquitinase inhibitor
222
N-ethylmaleimide (10 mM; Sigma-Aldrich). Protein-protein interactions were
223
disrupted by sonication (3 pulses of 10 s) using the 550 Sonic Dismembrator
224
(Fisher Scientific Inc) followed by boiling for 10 min in 1% SDS. WCL (250 to
225
500μg)
226
Polyubiquitination was detected using a monoclonal anti-HA antibody
227
(Sigma-Aldrich).
228
Statistical Analysis
229
The difference between two groups was statistically analyzed by a two-tailed
230
Student’s t test. All data points were the average of triplicates, with error bars
231
representing standard deviations. All data were representative of results from
232
at least 3 independent experiments. *, P -0.04
-22.9
DUSP4
> -0.04
-21.8
MARCH5
-0.03
-22.6
BCL11A
-0.03
-22.1
CREB1
> -0.03
-26.8
KSR2
> -0.03
-33.7
HIPK2
> -0.02
-28.8
IL21R
> -0.01
-22.3
786
Table 5. miR-526a targets on EV71 predicted by Vita MEF miR-526a
miR-526b
miR-296
≤-10
98
203
303
≤-15
26
95
115
≤-20
3
7
14
≤-25
0
0
6
≤-10
23
31
85
≤-15
14
19
50
≤-20
3
1
8
≤-25
0
0
0
≤-10
1
2
12
≤-15
0
2
7
≤-20
0
0
4
≤-25
0
0
0
（Kcal/mol） Score≥120
Score≥140
Score≥160
787
