phosphorylation-dependent feedback inhibition of rig-i by ... · poly(i:c) poly(i:c) sev sev...
TRANSCRIPT
![Page 1: Phosphorylation-Dependent Feedback Inhibition of RIG-I by ... · poly(I:C) poly(I:C) SeV SeV 5’ppp-dsRNA 5’ppp-dsRNA relative mIFIT1 mRNA level relative mIFN β 100 mRNA level](https://reader035.vdocuments.fr/reader035/viewer/2022071018/5fd1632d8d6f055ae91e5fb3/html5/thumbnails/1.jpg)
Molecular Cell, Volume 65
Supplemental Information
Phosphorylation-Dependent Feedback
Inhibition of RIG-I by DAPK1 Identified
by Kinome-wide siRNA Screening
Joschka Willemsen, Oliver Wicht, Julia C. Wolanski, Nina Baur, Sandra Bastian, Darya A.Haas, Petr Matula, Bettina Knapp, Laurène Meyniel-Schicklin, Chen Wang, RalfBartenschlager, Volker Lohmann, Karl Rohr, Holger Erfle, Lars Kaderali, JosephMarcotrigiano, Andreas Pichlmair, and Marco Binder
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Supplemental information Willemsen et al.
Figure S1.Protein-Protein-Interaction Network Based Analyses of Primary Hit Candidates,
Related to Figure 1 (A) Using a curated human interactome network, direct protein-protein-
interactions between primary hit candidates were analyzed. Besides several heterotypic one-on-
one interactions (lower panel), one contiguous network centered around the AKT1 protein was
STK39
MAP2K2
ELK1
JUN
MAP2K7
RIOK3
MAPK9
CDC42BPG
ATF2
SGK1
TRAF3 MAPK3
MAP2K6
MAPK1
MAPK14IKBKE
NFKB2 MAPK8
IKBKG
MAP3K7TRADDNFKBIAMAP3K8
IKBKB
IFNAR1
NFKBIB
RELB
STAT1FOS
RELA
NFKB1
CHUK
NFKBIE
IRF5
IFIH1
AURKA
EIF2AK2
AKT1
RPS6KA5
ERN1
MAP2K4
EIF2AK3PRKCD
CHEK1
TLR7
TLR2
CSK TMEM173
TANKTBK1
DDX58
IRAK2TICAM1
MAVS
ZBP1
IRF3
AZI2
MAP2K1
MAP2K3
IRF1
CASP8
IRF7
FADDTRAF6
RIPK1IRAK1
RET
TLR9
PFKL
ATM
PRKD1
JAK1
STAT2
TAB1
TRAF2
TAB2TAB3
IRF9
BMPR1A
KSR2
KDRMKNK2
RPS6KA2
WNK1DAPK1
CDK9 TRIB3IFNAR2
TYK2
TIRAP
MYD88
CASP10
IRAK4
TLR3
TICAM2
TLR4
C
A B
IRAK2
LATS2GSK3AATMINAURKAIP1 RIPK4
SGK1
IRAK1
IRAK4
AURKA
MAP2K7
PRKD1
MAP2K4
MAPK8
PRKCDATM AURKB
KDR
ATR
CDK9
CHEK1
SKP2
AKT1
TRIB3
WINK1
Interactions of primary hit candidates with members of the interferon network
Direct interactions within primary hit candidates Subnetwork of DAPK1 and indirectly interacting hit candidates
PPP2R2A
UNC5C MAPK3
UNC5AKLHL20
RPS6KA2
BECN1
MAPK1
MKNK2
PIK3C3
RETSGK1
MAPK7
SKP2
AKT1
ATM
IKBKG
MARK2
IRAK1
RIPK3
DAPK1
FADD
PDCD6
CUL3
TSC2
MIB1
TRADD
UNC5B
TNFRSF1A
PRKCD
STX1A
TESK1
YWHAB
PFKL
![Page 3: Phosphorylation-Dependent Feedback Inhibition of RIG-I by ... · poly(I:C) poly(I:C) SeV SeV 5’ppp-dsRNA 5’ppp-dsRNA relative mIFIT1 mRNA level relative mIFN β 100 mRNA level](https://reader035.vdocuments.fr/reader035/viewer/2022071018/5fd1632d8d6f055ae91e5fb3/html5/thumbnails/3.jpg)
identified. Homotypic interaction (homodimerization) were ignored. (B) Network analysis of the
high-confidence hit DAPK1. All direct interaction partners represented in the interactome are
displayed and labeled. Further hit candidates that interact with at least one of these DAPK1-
interactors are displayed in turquoise and labeled. (C) Graphical representation of the interferon-
related protein-protein-interaction network. Four hit candidates are found to be members of this
network (colored turquoise). Using a curated full human interactome, further primary hit
candidates that interact with members of the interferon network were identified and arranged in a
circle around the interferon network (turquoise), with their direct interaction partners colored
orange.
![Page 4: Phosphorylation-Dependent Feedback Inhibition of RIG-I by ... · poly(I:C) poly(I:C) SeV SeV 5’ppp-dsRNA 5’ppp-dsRNA relative mIFIT1 mRNA level relative mIFN β 100 mRNA level](https://reader035.vdocuments.fr/reader035/viewer/2022071018/5fd1632d8d6f055ae91e5fb3/html5/thumbnails/4.jpg)
Figure S2. Validation of 22 Hits in Secondary Screen, Related to Figure 1 and 2. Fifty-eight
candidate genes from primary screen were subjected to three rounds of secondary validation
screening with two siRNA per gene (Qiagen siRNA ID indicated in figure); details in STAR
Negati
ve
ALPK3BMX
CDC42SE2
CDKL3
DAPK1DDR1
DGKD
EPHA2
FASTKGK2
IRAK1
IRAK2
IRAK4
ITPKC
MARK2MATK
RIOK3
RIPK4
RPS6KA2
SH3BP4
SKP20%
50%
100%
rela
tive
mR
NA
leve
ls
siRNA
0.05
0.10
0.20
0.50
1.00
2.00
5.00
unst
imul
ated
Hs_
SH
3BP
4_5
MA
VSH
s_FA
STK
_8H
s_R
IOK
3_6
Hs_
PIP
5KL1
_5IR
AK
3H
s_C
DK
L3_5
Hs_
GK
2_3
Hs_
RIP
K1_
6H
s_FA
STK
_5H
s_P
IK3C
D_5
Hs_
CD
KL3
_8H
s_E
PH
B1_
6H
s_IT
PK
C_5
Hs_
IRA
K1_
6H
s_D
AP
K1_
6H
s_S
TK6_
5H
s_D
DR
1_10
Hs_
CS
NK
1A1L
_5H
s_R
IOK
3_5
Hs_
CH
EK
1_9
Hs_
RN
AS
EL_
6H
s_R
PS
6KA
2_10
Hs_
LATS
2_10
Hs_
SK
P2_
5H
s_C
9orf9
8_5
Hs_
TXK
_3H
s_P
IP5K
L1_8
Hs_
EP
HB
1_5
Hs_
CS
F1R
_6H
s_R
IPK
4_5
Hs_
TRIB
3_6
Hs_
CH
EK
1_13
Hs_
CD
C42
SE
2_3
Hs_
MA
GI1
_6H
s_IR
AK
4_6
Hs_
LATS
2_5
Hs_
PIK
3C2G
_6H
s_W
NK
1_5
Hs_
CD
K8_
5H
s_M
AG
I1_5
empt
yC
DK
15H
s_P
IK3C
D_6
Hs_
SH
3BP
4_4
Hs_
TXK
_4H
s_S
KP
2_8
Hs_
RP
S6K
A5_
9H
s_E
PH
A2_
5H
s_M
AP
2K4_
8H
s_C
aMK
IINal
pha_
6H
s_A
KA
P7_
6H
s_C
DC
42S
E2_
2ne
gativ
eH
s_P
RK
R_6
Hs_
STY
K1_
4H
s_N
RB
P_5
Hs_
PR
KC
M_2
Hs_
IRA
K2_
6H
s_B
UB
1B_6
Hs_
MA
TK_1
0H
s_K
SR
2_6
Hs_
BM
X_5
Hs_
EIF
2AK
3_6
Hs_
MA
PK
8_13
Hs_
RE
T_9
Hs_
RIP
K1_
5H
s_TY
K2_
7H
s_C
DK
8_6
Hs_
PR
KC
D_1
1P
ER
KH
s_R
PS
6KA
5_8
Hs_
PR
KC
D_8
Hs_
CS
NK
1A1L
_6H
s_R
IPK
4_6
Hs_
ITP
KC
_7H
s_C
9orf9
8_8
Hs_
KS
R2_
5H
s_S
TYK
1_3
Hs_
STK
6_6
Hs_
RE
T_10
Hs_
CS
NK
1D_5
Hs_
TYK
2_5
Hs_
TRIB
3_5
Hs_
MA
P2K
4_10
Hs_
CS
NK
1D_6
Hs_
EP
HA
2_7
Hs_
OS
RF_
4IR
AK
4H
s_D
DR
1_9
Hs_
WN
K1_
6H
s_C
SF1
R_7
AA
K1
Hs_
HK
2_6
Hs_
DG
KD
_5H
s_N
RB
P_6
Hs_
ALP
K3_
7H
s_M
ATK
_9H
s_M
AP
K8_
12H
s_G
K2_
5O
SR
FH
s_D
GK
D_6
Hs_
PIK
3C2G
_5H
s_B
UB
1B_5
IRA
K2
Hs_
CaM
KIIN
alph
a_5
Hs_
HK
2_5
IRA
K1
Hs_
PR
KD
1_5
Hs_
IRA
K2_
5H
s_IR
AK
3_6
Hs_
ALP
K3_
8H
s_D
AP
K1_
5H
s_B
MX
_6H
s_M
AR
K2_
7P
KR
RN
Ase
LH
s_A
AK
1_6
Hs_
RP
S6K
A2_
9H
s_M
AR
K2_
6
Validation screen 1 – 293TRIG-I cells, stimulated with VSV vRNA
IRF3
repo
rter a
ctiv
ity (f
old
nega
tive)
0.05
0.10
0.20
0.50
1.00
2.00
MA
VSH
s_FA
STK
_8H
s_E
PH
A2_
5H
s_E
PH
B1_
6H
s_S
H3B
P4_
5IR
AK
2H
s_IR
AK
1_6
Hs_
CD
C42
SE
2_2
Hs_
CS
F1R
_7H
s_R
PS
6KA
5_8
Hs_
PIK
3CD
_5H
s_IT
PK
C_5
Hs_
TXK
_4H
s_R
IPK
4_5
Hs_
PR
KC
D_1
1H
s_P
IK3C
D_6
Hs_
LATS
2_5
Hs_
DD
R1_
9H
s_P
IP5K
L1_8
CD
K15
Hs_
DA
PK
1_6
Hs_
RIO
K3_
6H
s_M
AG
I1_6
Hs_
CD
C42
SE
2_3
Hs_
MA
TK_1
0H
s_FA
STK
_5H
s_S
TK6_
6H
s_R
ET_
10H
s_M
AR
K2_
6H
s_P
RK
R_6
Hs_
RN
AS
EL_
6H
s_E
IF2A
K3_
6H
s_G
K2_
3R
NA
seL
Hs_
C9o
rf98_
5em
pty
Hs_
MA
PK
8_13
Hs_
RP
S6K
A2_
10H
s_LA
TS2_
10H
s_TY
K2_
5H
s_S
KP
2_8
Hs_
RE
T_9
IRA
K3
Hs_
AK
AP
7_6
OS
RF
Hs_
TXK
_3H
s_A
LPK
3_7
Hs_
PIP
5KL1
_5H
s_B
UB
1B_5
Hs_
RIO
K3_
5H
s_S
KP
2_5
Hs_
CS
F1R
_6H
s_S
TYK
1_4
Hs_
PIK
3C2G
_5H
s_M
AR
K2_
7H
s_C
HE
K1_
13H
s_M
AP
2K4_
8H
s_IR
AK
2_6
Hs_
ITP
KC
_7H
s_S
TYK
1_3
IRA
K4
Hs_
RIP
K1_
6ne
gativ
eH
s_C
9orf9
8_8
Hs_
DG
KD
_6H
s_M
AP
K8_
12H
s_C
DK
8_6
Hs_
CaM
KIIN
alph
a_5
Hs_
PIK
3C2G
_6H
s_H
K2_
6H
s_C
DK
8_5
Hs_
PR
KC
M_2
AA
K1
Hs_
WN
K1_
6H
s_B
MX
_6H
s_D
AP
K1_
5H
s_C
aMK
IINal
pha_
6H
s_E
PH
B1_
5H
s_D
DR
1_10
Hs_
NR
BP
_6H
s_K
SR
2_6
Hs_
PR
KC
D_8
Hs_
OS
RF_
4H
s_TY
K2_
7IR
AK
1P
ER
KH
s_TR
IB3_
5H
s_R
PS
6KA
5_9
Hs_
CS
NK
1D_5
Hs_
RIP
K4_
6H
s_C
DK
L3_8
Hs_
HK
2_5
Hs_
NR
BP
_5H
s_B
UB
1B_6
Hs_
RIP
K1_
5H
s_IR
AK
3_6
Hs_
STK
6_5
PK
RH
s_C
SN
K1A
1L_5
Hs_
KS
R2_
5H
s_IR
AK
4_6
Hs_
EP
HA
2_7
Hs_
WN
K1_
5H
s_A
LPK
3_8
Hs_
MA
GI1
_5H
s_M
ATK
_9H
s_C
SN
K1A
1L_6
Hs_
SH
3BP
4_4
Hs_
CH
EK
1_9
Hs_
RP
S6K
A2_
9H
s_C
SN
K1D
_6H
s_D
GK
D_5
Hs_
AA
K1_
6H
s_M
AP
2K4_
10H
s_P
RK
D1_
5H
s_B
MX
_5H
s_G
K2_
5H
s_C
DK
L3_5
Hs_
IRA
K2_
5H
s_TR
IB3_
6
0.05
0.10
0.20
0.50
1.00
2.00
Validation screen 2 – 293TRIG-I 2CARD cells
Cell viability upon siRNA silencing
Silencing efficiency
IRF3
repo
rter a
ctiv
ity (f
old
nega
tive)
A
B
C D
E
CDKL3
FASTKSKP2
DAPK1
RIOK3
DDR1IR
AK4
ALPK3
CDC42SE2
SH3BP4
ITPKCDGKD
BMX
EPHA2MATK
RIPK4GK2
Negati
veIR
AK2
MARK2
RPS6KA2
IRAK1
siRNA
%ce
llvi
abili
ty
0%
50%
100%
150%Significant and consistent
in primary, RVFV and atleast one other validation
Consistent in all threevalidations, significant in
at least two validations
Significant inall four screens
123456789
1011121314
15161718
19202122
RIOK3CDC42SE2
DDR1RIPK4
EPHA2MATKIRAK4IRAK1
DAPK1DGKD
CDKL3GK2
ALPK3MARK2
AAK1IRAK2
SKP2BMX
RPS6KA2
SH3BP4FASTK
ITPKC
-6 0 6Z-score (RVFV validation)
signalingactivators
signalinginhibitors
*
0.05
0.05
0.001
0.0011p-value
0
![Page 5: Phosphorylation-Dependent Feedback Inhibition of RIG-I by ... · poly(I:C) poly(I:C) SeV SeV 5’ppp-dsRNA 5’ppp-dsRNA relative mIFIT1 mRNA level relative mIFN β 100 mRNA level](https://reader035.vdocuments.fr/reader035/viewer/2022071018/5fd1632d8d6f055ae91e5fb3/html5/thumbnails/5.jpg)
Methods . (A) Validation screen 1 was performed in 293TRIG-I cells, stimulated by transfection of
VSV vRNA and read out by IRF3 activity luciferase reporter. (B) Validation screen 2 was
performed in constitutively signaling 293TRIG-I 2CARD cells. Read out was IRF3 activity luciferase
reporter. Validation screen 3 was performed in A549Ago2 cells that were infected with Renilla
luciferase expressing Rift Valley fever virus (RVFV∆NSs), which triggers the RIG-I response
very efficiently and at the same time is highly sensitive to interferon. Results are displayed in
figure 1D of the main text. (A,B) From nine biological replicates, the most extreme of each
siRNA was removed and results were normalized to non-targeting siRNA. Significance was
tested against non-targeting siRNA by Student’s t-test and p-values were color-coded, scale
included in figure. (C) Twenty-two candidate genes were validated in secondary screening by
fulfilling the hit calling criteria indicated in the figure (see also STAR methods). The magnitude of
the effect (Z-score) of their knockdown in validation round 3 (RVFV) is color-coded, scale
included in panel. Hits can be classified according to the direction of their effect: genes whose
knockdown increased IRF3 activation upon RIG-I stimulation are considered signaling inhibitors
(i.e. their presence inhibits RIG-I/IRF3 signaling); genes whose knockdown decreases IRF3
activation are considered signaling activators. For each gene, the siRNA yielding the biggest
effect in validation screening was chosen for further characterization of the respective gene (see
figure 2). Cytotoxic effects (D) and silencing efficiency (E) were measured by ATP quantification
and gene-specific qRT-PCR, respectively.
![Page 6: Phosphorylation-Dependent Feedback Inhibition of RIG-I by ... · poly(I:C) poly(I:C) SeV SeV 5’ppp-dsRNA 5’ppp-dsRNA relative mIFIT1 mRNA level relative mIFN β 100 mRNA level](https://reader035.vdocuments.fr/reader035/viewer/2022071018/5fd1632d8d6f055ae91e5fb3/html5/thumbnails/6.jpg)
Figure S3. Knockdown of DAPK1 in Different Cell Types and With Different siRNAs,
Related to Figure 3. DAPK1 was silenced by siRNA in three different human cell lines: Huh7.5
(primary screen), 293TRIG-I cells (A, and main figure 3D) and A549 cells (B). To assess species-
specificity of the effect, primary mouse fibroblasts from lung tissue were generated and
transfected with mouse-specific DAPK1 siRNAs as indicated (C). Cells were either non-
stimulated (left bars) or stimulated with the indicated stimulus (right bars), read-out was IFIT1- or
IFNβ-mRNA levels, measured by gene- and species-specific qRT-PCR. (D) Comparison of three
further siRNAs (from a different vendor) targeting human DAPK1 was performed in A549 cells
stimulated with Sendai virus (SeV). Figure shows one representative of two (panel C) or three
(panels A, B and D) independent experiments. Values represent mean +/- SD of triplicate
values.
primary mouse lung fibroblastsA549 cells293TRIG-I cells
CA B
0
100
200
300
rela
tive
IFNβ
mR
NA
leve
l
rela
tive
IFNβ
mR
NA
leve
l
rela
tive
IFNβ
mR
NA
leve
l
rela
tive
IFNβ
mR
NA
leve
l
0
50
100
150re
lativ
e IF
IT1
mR
NA
leve
l
0
100
200
300
400
rela
tive
IFNβ
mR
NA
leve
l
0
50
100
150
200
rela
tive
IFIT
1 m
RN
A le
vel
poly(I:C) poly(I:C) SeV SeV 5’ppp-dsRNA 5’ppp-dsRNA
rela
tive
mIF
IT1
mR
NA
leve
l
rela
tive
mIF
Nβ
mR
NA
leve
l
0
50
100
150
0
2000
4000
6000
D
0
50
100
150
200
250
0
50
100
150
200
250
0
50
100
150
200
250
A549 cells
SeV SeV SeV
siNegsiNeg
siDAPK1
siNeg
siDAPK1siNeg
siDAPK1
siNeg
siDAPK1_2siNeg
siDAPK1_2
siNeg
siDAPK1_3siNeg
siDAPK1_3
siNeg
siDAPK1
siNeg
siDAPK1siNeg
siDAPK1
siNeg
siDAPK1siNeg
siDAPK1
siNeg
si_mDAPK1_1
si_mDAPK1_2siNeg
si_mDAPK1_1
si_mDAPK1_2
siNeg
si_mDAPK1_1
si_mDAPK1_2siNeg
si_mDAPK1_1
si_mDAPK1_2
0,09 * ****
** ** **
****
**
*****
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Figure S4. DAPK1 Domain Mapping Expression Controls, Related to Figure 4. (A, B)
Truncated DAPK1 variants were expressed in 293TRIG-I cells and checked for their effect on
RIG-I induced IRF3 activation. (A) To rule out artifacts owing to a lack of expression, protein was
detected using anti-HA immunoblotting. Calnexin was stained as a loading control. (B) In order
to monitor kinase activity of the tested DAPK1 mutants cells were treated with 100nM of the
phosphatase inhibitor Calyculin A for 30min and phosphorylation at S308 was detected by
immunoblotting along with total protein amounts by HA staining und calnexin as a loading
control. Figure shows one representative out of at least three independent experiments.
pcD
NA
DA
PK
1 full-length
∆CaM
∆RO
C
∆DD
∆(A
nk|R
OC
|CO
R)
pcD
NA
DA
PK
1 full-length
Kin
ase
dom
ain
Kin
|CaM
Kin
|CaM
|Ank
Ank
yrin
repe
ats
pcD
NA
DA
PK
1 full-length
Kin
|CaM
|Ank
Kin
|CaM
|Ank
S30
8D
Kin
|CaM
|Ank
S30
8A
Calnexin
HA
Calnexin
HA
pS308-DAPK1
DA
PK
1 full-length
Kin
|CaM
|Ank
Kin
|CaM
|Ank
K42
A
Kin
|CaM
|Ank
S30
8D
Kin
|CaM
|Ank
∆1-
73
pcD
NA
A
B
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Figure S5. DAPK1 Activation upon Virus Infection and IFNβ Production upon DAPK1
Silencing, Related to Figure 5. (A) A549 cells were infected with Sendai virus (SeV) for the
indicated amounts of time (or treated with the protonophore CCCP as a positive control) and
then lysed and analyzed for DAPK1 phosphorylation at site S308 and DAPK1 total levels.
Calnexin was stained as a loading control. SeV infection activated DAPK1 similarly to the
positive control and similarly to RIG-I stimulation by 5’ppp-dsRNA (main figure 5). (B) A549 cells
stimulated with 100 IU/ml IFNα for the indicated amount of time and then lysed and analyzed for
phosphorylation of DAPK1 at S308. RIG-I was stained as an indicator for IFN stimulation and
calnexin as a loading control. (C–H) Analysis of mock- or DAPK1-silenced (siDAPK1_1) A549
cells stimulated by transfection of 5’ppp-dsRNA for the indicated time span (0-24 h). IFNβ
induction was detected by immunoblotting from cell lysates (C) (densitometric quantification of
IFNβ signal normalized to GAPDH is shown in panel D) or by ELISA from culture supernatants
after the indicated time (G, accumulated levels in pg/ml) or from supernatants that had been
changed to fresh medium 1 h prior to the indicated time point (H, secretion rate in pg/ml/h). IFNβ
and IFIT1 mRNA levels were analyzed by qRT-PCR (E and F). Figure shows one representative
out of three (A, C-H) or two (B) independent experiments.
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Figure S6. Co-Precipitation of RIG-I Pathway Members with DAPK1, Related to Figure 6.
293TRIG-I cells were transfected with HA-tagged DAPK1 or YFP for 48 h. Cell lysates were used
for HA-immunoprecipitation and analyzed by immunoblotting with antibodies against the
indicated proteins. The co-precipitation with IRF7 could not be reliably reproduced in repetitions
of the experiment; RIG-I co-precipitation was the strongest and only reliable interaction detected.
Figure shows one representative out of three independent experiments.
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Figure S7. Phosphorylation Mutants of RIG-I, Related to Figure 7. RIG-I residues
phosphorylated by DAPK1 were replaced with phosphomimetic glutamic acids and checked for
0
10
20
30
40
A 5'ppp-dsRNA
0,30 1,19
83,814,7
2,38 0,95
62,534,1
73,9 5,76
9,7310,6
0,56 0,44
69,429,6
0,100 0,080
61,338,6
70,6 14,8
9,734,91
B
2,17 0
097,8
0,19 0
099,8
0,48 0
099,5
0,44 0,015
0,01599,5
0,15 0
099,8
0,20 0
099,8
0
-103
103
104
105
0-103 103 104 105
0
-103
103
104
105
0-103 103 104 105
0
-103
103
104
105
0-103 103 104 105
0
-103
103
104
105
0-103 103 104 105
0
-103
103
104
105
0-103 103 104 105
0
-103
103
104
105
0-103 103 104 105
C
FLUAV NP
IFIT
1
FLUAV NP
IFIT
1
0
-103
103
104
105
0-103 103 104 105
0
-103
103
104
105
0-103 103 104 105
0
-103
103
104
105
0-103 103 104 105
0
-103
103
104
105
0-103 103 104 105
0
-103
103
104
105
0-103 103 104 105
0
-103
103
104
105
0-103 103 104 105
empty RIG-I WT RIG-I T667E
RIG-I T667/671E RIG-I Cluster I E RIG-I S8E
empty RIG-I WT RIG-I T667E
RIG-I T667/671E RIG-I Cluster I E RIG-I S8E
unin
fect
edFL
UAV
infe
cted
IRF
3 r
eport
er
activity
pcD
NA
RIG
-IW
T
S8
T6
67
/T6
71
T667
RIG-I S/T A mutants
pcD
NA
S8+
CI+
CII
RIG
-IW
T
Clu
ste
r I
IRF
3 r
eport
er
activity
5'ppp-dsRNA
HA
Calnexin
0
5
10
15
20
RIG-I S/T A mutants
HA
β-actin
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their capacity to mediate antiviral signaling in response to 5’ppp-dsRNA (main figure 7A). (A) As
a control, the same residues (indicated) were mutated into alanines and checked upon
transfection of 293T cells. Mutation of S8, T667 and T667+T671 did not impact the functionality
of RIG-I. Replacing all identified phosphosites or all sites of cluster I (see figure 6F) by alanines
abrogated signaling. Results of one representative out of three experiments are given. (B,C)
A549RIG-I KO cells were reconstituted with empty vector or the indicated wildtype or
phosphomimetic variants of RIG-I and infected with FLUAV at MOI=0.01 (analogous to main
figure 7F) (B) or left uninfected (C). FLUAV NP protein as a marker for infection and IFIT1 as a
marker for active antiviral signaling were stained and analyzed in flow cytometry.
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Name Tag Promoter ORF library
(Sub-) Cloned
Gift from
ALPK1 Flag CMV Dr. Alexey Ryazanov, Robert wood Johnson Medical School New Jersey
BMX HA CMV Prof. Olli Silvennoinen, university of Tampere CDC42SE2 V5 CMV X
CDKL3 V5 CMV X DAPK1 Flag CMV X Prof. A. Kimchi Weizmann Institute of
Science DDR1 V5 CMV XX Prof. Friedemann Kiefer Max-Planck-
Institute for Molecular Biomedicine DGKD 3xFlag CMV Dr. Fumio Sakane Chiba University Japan
EPHA1 V5 CMV X FASTK V5 CMV X
GK2 V5 CMV X IRAK1 V5 CMV Prof. Alexander Weber, University of
Tübingen IRAK2 V5 CMV Prof. Alexander Weber, University of
Tübingen IRAK4 V5 CMV Prof. Alexander Weber, University of
Tübingen ITPKC GFP CMV Dr. Marcus Nalaskowski,
Universitätsklinikum Hamburg-Eppendorf MATK V5 CMV X
MARK2 V5 CMV X RIOK3 V5 CMV X RIPK4 V5 CMV X
RPS6KA2 V5 CMV X SKP2 V5 CMV X
SH3BP4 GFP CMV Prof. Pier Paolo Di Fiore, IFOM-IEO Campus RIG-I HA
Myc Flag
x
IFIT1 promoter lucif. reporter
pGL3B/561, kind gift of Dr. Ganes Sen (Cleveland)
NFkB luciferase reporter
pGL4.32[luc2P/NFκB-RE/Hygro] (Promega)
Table S1. Overexpression Constructs, Related to STAR Methods. Table lists expression constructs for the indicated transgenes (name) with their respective tags and promoters. The cDNAs were either taken from the ORFeome library (“X” in column “ORF library”), were kind donations of the indicated colleagues (“gift from”) or were cloned by RT-PCR from Huh7 mRNA (“X” in column “(Sub-)cloned”). DDR1 was subcloned from a provided plasmid (“XX” in column “(Sub-)cloned”).
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Name sequence provider Hs_ALPK3_8 TCGGTGCACCATCCACAATGA Qiagen
Hs_BMX_5 CCCAATATGACAACGAATCAA Qiagen Hs_CDC42SE2_2 AAGGGAGGTTATGGAGGTGGA Qiagen
Hs_CDKL3_5 TGGGCAGATAGTGGCCATTAA Qiagen Hs_DAPK1_5 AAGCATGTAATGTTAATGTTA Qiagen Hs_DDR1_10 CAGGAATGATTTCCTGAAAGA Qiagen Hs_DGKD_5 CAGCAGATTCTCTTCTATGAA Qiagen
Hs_EPHA2_5 AAGGAAGTGGTACTGCTGGAC Qiagen Hs_FASTK_8 CAGCAGCAAGgTGGTACAGAA Qiagen
Hs_GK2_5 TAGTAACTTCGTCAAGTCTAA Qiagen Hs_IRAK1_5 CCGGGCAATTCAGTTTCTACA Qiagen Hs_IRAK2_5 CAGCAACGTCAAGAGCTCTAA Qiagen Hs_IRAK3_6 GGAUGUUCGUCAUAUUGAATT Qiagen Hs_IRAK4_6 ATCCTATTAGTCATATATTTA Qiagen Hs_ITPKC_5 CAGAAGGAGCCTGTCCCTCAA Qiagen
Hs_MARK2_6 CACCTCTAATTCTTACTCTAA Qiagen Hs_MATK_9 ACGGATTCTAAGGACTCTAAA Qiagen Hs_RIPK4_5 AAGCCTGATGACGAAGTGAAA Qiagen
Hs_RPS6KA2_9 CCGAGTGAGATCGAAGATGGA Qiagen Hs_SH3BP4_5 CACCACGAATAGCACTGGCAA Qiagen
Hs_SKP2_5 AAGTGATAGTGTCATGCTAAA Qiagen All star negative not revealed by the company (SI03650318) Qiagen
RIG-I AACGUUUACAACCAGAAUUUA MWG MAVS CCCACAGGGUCAGUUGUAU MWG
Mouse_DAPK1_1 AAGGATTGACGTCCAGGATAA (Mm_Dapk1_2)
Qiagen
Mouse_DAPK1_2 AAGCCTAAAGACCACCCAACAA (Mm_Dapk1_4)
Qiagen
DAPK1 AAGCATGTAATGTTAATGTTA (Hs_DAPK1_5) Qiagen DAPK1_2 not revealed by the company (SIHK0538) Sigma-Aldrich DAPK1_3 not revealed by the company (SIHK0539) Sigma-Aldrich
Table S2. Sequence or Ordering Information of siRNAs, Related to STAR Methods. SiRNAs were ordered from commercial vendors. Where sequence information was not revealed, the table lists ordering numbers. Names of Qiagen siRNAs starting with “Hs_” are also direct product identifiers.
Target Gene F Primer R Primer GAPDH GAAGGTGAAGGTCGGAGTC GAAGATGGTGATGGGATTTC
IFIT1 GAAGCAGGCAATCACAGAAA TGAAACCGACCATAGTGGAA IFNB CGCCGCATTGACCATCTA GACATTAGCCAGGAGGTTCTC
dsRNA CTTTCTCTTGGTTGGGCCAC TCCTGGCCTGTGAGTTCTTG
Table S3. Primer Sequences of qRT-PCR Primers, Related to STAR Methods. Table lists sequences for forward (F) and reverse (R) primers used for quantitative RT-PCR.
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Name sequence RIG-I cloning
S8E F: ggggacaagtttgtacaaaaaagcaggcttcatgaccaccgagcagcgacgcgagctgcaagccttc R: ggggaccactttgtacaagaaagctgggtctcatttggacatttctgctggatc
S8A F: ggggacaagtttgtacaaaaaagcaggcttcatgaccaccgagcagcgacgcgcgctgcaagccttc R: ggggaccactttgtacaagaaagctgggtctcatttggacatttctgctggatc
cluster I to E F:ggattgaaggaaatcctaaactcgagtttctaaaacctggcatattgactggacgtggcaaagaaaatcagaacgaaggaatggagctcccggcacagaagtgtatattgg R: aattttttaaagcgtccacaagtgc
cluster I to A F:ggattgaaggaaatcctaaactcgcttttctaaaacctggcatattgactggacgtggcaaagcaaatcagaacgcaggaatggccctcccggcacagaagtgtatattgg R: aattttttaaagcgtccacaagtgc
cluster II to E F: tgatgaatgacgagattttacgccttcaggaatgggacgaagcagtatttag R: ttttttctttgtacatgtttatttgttc
cluster II to A F: tgatgaatgacgctattttacgccttcaggcatgggacgaagcagtatttagg R: ttttttctttgtacatgtttatttgttc
T667E + T671E F: catattgactggacgtggcaaagaaaatcagaacgaaggaatgaccctcccggcac R: ccaggttttagaaaactgag
T667A + T671A F: catattgactggacgtggcaaagcaaatcagaacgcaggaatgaccctcccggcac R: ccaggttttagaaaactgag
T667E F: catattgactggacgtggcaaagagaatcagaacacaggaatgaccctc R: ccaggttttagaaaactgag
T667A F: catattgactggacgtggcaaagcgaatcagaacacaggaatgaccctc R: ccaggttttagaaaactgag
T671E F: catattgactggacgtggcaaaacaaatcagaacgagggaatgaccctc R: ccaggttttagaaaactgag
T671A F: catattgactggacgtggcaaaacaaatcagaacgcgggaatgaccctc R: ccaggttttagaaaactgag
DAPK1 cloning full-length DAPK1 F: ggggacaagtttgtacaaaaaagcaggcttcatgaccgtgttcaggcagg
R: ggggaccactttgtacaagaaagctgggtctcaccgggatacaacagagc kinase domain (KD) F: ggggacaagtttgtacaaaaaagcaggcttctacgacaccggcgaggaacttgg
R: ggggaccactttgtacaagaaagctgggtctcagatccagggatgctgcaaac KD + CaM F: ggggacaagtttgtacaaaaaagcaggcttctacgacaccggcgaggaacttgg
R: ggggaccactttgtacaagaaagctgggtctcaatcgcttctggcaacactc ∆ DD F: ctgaacctcctcactcggaggtctgtgttcaaaatcaacctgg
R: ccaggttgattttgaacacagacctccgagtgaggaggttcag ∆ Ank + ROC +
COR F: gatgttaaccaacccaacaagcaggtccgcggcctggagacgg R: ccgtctccaggccgcggacctgcttgttgggttggttaacatc
∆ ROC domain F: gatgggagccagcgttgaggcgaagctgaagaacccactccaag R: cttggagtgggttcttcagcttcgcctcaacgctggctcccatc
∆ CaM F: gaagagaatgacaattgatactctggatgaggaag R: cttcctcatccagagtatcaattgtcattctcttc
KD + CaM + Ank F: ggggacaagtttgtacaaaaaagcaggcttcatgaccgtgttcaggcagg R: ggggaccactttgtacaagaaagctgggtctcacgcctcaacgctggctcccatcag
Ank domain F:ggggacaagtttgtacaaaaaagcaggcttcatggatactctggatgaggaagactcctttg R:ggggaccactttgtacaagaaagctgggtctcacgcctcaacgctggctcccatcagac
KD CaM Ank K42A F: gcctccagtatgccgccgcattcatcaagaaaag R: cttttcttgatgaatgcggcggcatactggaggc
KD CaM Ank S308D
F: gaaaaaatggaaacaagacgttcgcttgatatc R: gatatcaagcgaacgtcttgtttccattttttc
KD CaM Ank S308A
F: gaaaaaatggaaacaagccgttcgcttgatatc R: gatatcaagcgaacggcttgtttccattttttc
KD CaM Ank ∆1-73
F: ggggacaagtttgtacaaaaaagcaggcttcatgcccaatgtcatcaccctgcacg R: ggggaccactttgtacaagaaagctgggtctcacgcctcaacgctggctcccatcag
Table S4. Primer Sequences of Cloning Primers, Related to STAR Methods. The listed forward (F) and reverse (R) primers were used to generate the indicated RIG-I or DAPK1 constructs by standard cloning procedures (see STAR Methods).
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Supplemental data file 1 - Screening results.xlsx, Related to Figure 1. The data file contains two tabs, one holding the data (p-values and enrichment scores) of the primary screen, one holding the data for all validation screens. The latter tab furthermore contains Boolean columns representing the hit calling criteria described in the STAR Methods.
Supplemental data file 2 - Functional annotation clustering.xls, Related to Figure 1. This data table contains the results of a DAVID functional annotation clustering, using DAVID version 6.8 (beta). For the analysis, all primary hit genes were included (i.e. each gene that had at least one siRNA with a p-value of <0.05; in total 102 DAVID IDs). All genes represented in the siRNA library were defined as the background for the analysis (720 unique GeneIDs, see tab “Primary screen genes”). For selected functional annotation clusters, information at the single gene level are given in separate tabs.
Supplemental data file 3 - PPI and virus screens.xlsx, Related to Figure 1. This data file contains the results of the protein-protein-interaction (PPI) analyses performed on the list of screening hits, as well as overlap analyses with previously published virus replication screens. For more information, refer to the STAR Methods section.