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Molecular Cell, Volume 62
Supplemental Information
Structural Analysis of dsRNA Binding
to Anti-viral Pattern Recognition Receptors
LGP2 and MDA5
Emiko Uchikawa, Mathilde Lethier, Hélène Malet, Joanna Brunel, DenisGerlier, and Stephen Cusack
Structural analysis of dsRNA binding to
anti-viral pattern recognition receptors LGP2 and MDA5
Emiko Uchikawa1,2
, Mathilde Lethier1,2
, Hélène Malet1,2
,
Joanna Brunel3-7
, Denis Gerlier3-7
and Stephen Cusack1,2#
1European Molecular Biology Laboratory, Grenoble Outstation, 71 Avenue des Martyrs, CS
90181, 38042 Grenoble Cedex 9, France
2University Grenoble Alpes-Centre National de la Recherche Scientifique-EMBL Unit of
Virus Host-Cell Interactions, 71 Avenue des Martyrs, CS 90181, 38042 Grenoble Cedex 9,
France.
3CIRI, International Center for Infectiology Research, Université de Lyon, Lyon, France;
4Inserm, U1111, Lyon, France;
5CNRS, UMR5308, Lyon, France;
6Ecole Normale Supérieure de Lyon, Lyon, France;
7Université Lyon 1, Centre International de Recherche en Infectiologie, Lyon, France
#Corresponding author:
Stephen Cusack
European Molecular Biology Laboratory, Grenoble Outstation, Grenoble Cedex 9, France.
Tel. (33)476207238, Email: [email protected]
Supplemental Information
Supplementary Figure Legends S1-S7
Supplementary Figures
Supplementary Table S1
Supplemental Experimental Procedures
Supplemental References.
Supplementary Figure Legends.
Figure S1 Sequence alignment of chicken and human LGP2 and MDA5 and duck and
human RIG-I (excluding the CARDs). Related to Figures 1 and 4.
The secondary structure of chLGP2 and chMDA5 are at the top and bottom
respectively. Outline boxes in green, yellow, cyan, red and orange delimit the Hel1, Hel2i,
Hel2, Pincer and CTD domains. Helicase conserved motifs are indicated in black on the
bottom and important loops such as the Hel2i, Hel2, end-binding and capping loops are
highlighted with a green, yellow, cyan, magenta or orange background respectively. Figure
made with ESPript (Gouet et al., 1999).
Figure S2. ATP analogue binding and ATP hydrolysis by chLGP2. Related to Figure 1.
(A) Diagram showing interaction network between chLGP2 and ADP:AlF4:Mg2+
. Residue
colours are as in Figure1C with polar interactions (blue) and hydrophobic interactions
(black).
(B) Ligand electron density for ADP:AlF4:Mg2+
in the chLGP2 5′p 10-mer dsRNA
complex with Mg2+
(magenta), Al (grey), F (pale blue) and coordinated water
(marine). Final 2Fo-Fc electron density is contoured at 2.0 σ.
(C) Comparative RNA-dependent ATP hydrolysis activity by LGP2, RIG-I and MDA5
showing initial reaction velocities versus ATP concentration. The concentration of
proteins and dsRNAs are 0.125 M dRig-I and 0.5 M dsRNA, 0.25 M chLGP2 and
1 M dsRNA, 0.5 M chMDA5 and 2 M dsRNA. Plotted values are mean ±SD
(n3).
(D) ATPase activity of RLHs as a function dsRNA length using 18-mer hairpin (5′-
GGGCGGCUUCGGCCGCCC-3′), 24-mer (same dsRNA as for crystallization), 32-
mer hairpin (5′-pppGGGCGAGCGUGCGCUUCGGCGCACGCUCGCCC-3′), or 38-
mer (5′-GGGACGUAGCAUCCGAUGUACAUCGGAUGCUACGUCCC-3′). The
graph compares the amount of hydrolysis after 16 minutes. The reaction conditions
were 1 µM RLH, 4 µM dsRNA and 2 mM ATP. Poly(I:C) is polyinosinic-
polycytidylic acid (Amersham Biosciences).
(E) Multiple sequence alignment of Motif I of RLHs
(F) Multiple sequence alignment of Motif VI of RLHs
Figure S3. Sequence alignment of LGP2 from representative vertebrates. Related to
Figure 1.
Sequences aligned include those from birds (chicken, Muscovy duck), mammals (human,
mouse), reptile (alligator), amphibians (frog) and fish (zebrafish). Annotation is similar to
Figure S1.
Figure S4. 5′ tri-phosphate conformation and summary of RNAs used. Related to Figures
2, 3 and 5.
(A) Schematic diagram showing the interactions with the 5′ tri-phosphate in the
chLGP2_10ppp structure.
(B) Schematic diagram showing the interactions with the 5′ tri-phosphate in the
chLGP2_3′ovg structure.
(C) RNAs used in various experiments.
Figure S5. Comparative binding of RLHs to dsRNA. Related to Figure 2.
(A) Electron density showing residues from Hel2i helix α10 interacting directly or via
water molecules with nucleotides from the 3′ (violet) and 5′ (yellow) strands in the
chLGP2 5′p 10-mer dsRNA complex. Final 2Fo-Fc electron density is contoured at
1.5 σ.
(B) Representative curves of fluorescence anisotropy changes measured by titrating full-
length hRIG-I (upper panel), chLGP2 (middle panel) or chMDA5ΔCARD (bottom
panel) to FAM labelled 5′OH, 5′ppp, 3′ overhang and 5′ overhang 12-mer dsRNA
with or without various nucleotides.
Figure S6. Comparison of helicase opening in known RLH crystal structures and
comparison of the closed and semi-closed states of chLGP2. Related to all Figures.
(A) Disposition of helicase Hel1 (green) and Hel2 (cyan) domains for known RLH crystal
structures (with PDB entry code indicated) after superposition of Hel1 of each
structure onto that of the closed state of chLGP2, used as reference. ATP analogues
(slate blue) and Mg2+
(magenta) are shown with spheres and the dsRNA back bone
(orange). Other domains are coloured as in Fig 1A. According to the relative
displacement of Hel1 and Hel2, the structures are classified closed, semi-closed, semi-
open and opened (see Table S1 for quantification).
(B) Comparison of the structures of the closed and semi-closed states of chLGP2 after
superposition of the Hel1 domain. The Hel1, Hel2, Hel2i and CTD domains are
coloured green (pale green), cyan (pale cyan), yellow (sand) and orange (pale orange)
for respectively and the dsRNA 5′ strand violet (pale violet) and 3′ strand yellow
(sand) for the closed (semi-closed) states respectively.
(C) Schematic diagram comparing interactions made in the closed (left) and semi-closed
(right) states of chLGP2 to the dsRNA illustrating the shift of Hel2 interactions (red
box) one phosphate down the 3′ strand.
Figure S7 . Co-operativity of LGP2 with MDA5 in human cells. Related to Figure 7.
(A) Expression of chLGP2, chLGP2 variants and hLGP2 in chicken DF1 cells (top) and
expression of hLGP2 and hLGP2 variants in human Huh7.5 cells (bottom). The
protein expression of other MDA5 constructs used in this work has been previously
determined and found to be comparable to that of their wt counterpart (Louber et al.,
2015).
(B) RIG-I and MDA5-mediated endogenous response to dsRNA in human HEK293 (in
blue) and Huh7.5 (in red) cells. Activation of huIFN promoter by RIG-I agonist
5′ppp-dsRNA (61mer), MDA5 agonist 6 bacteriophage 2.9, 4.1 and 6.4 kbp long
dsRNA, and RIG-I/MDA5 agonist poly(I:C). Note the lack of detectable endogenous
response observed in Huh7.5 cells and the neutral effect of the pCG-duF vector (noted
“F”) used as a complement loading DNA to ensure identical amounts of DNA in the
experiments with expression vectors as a source of exogenous MDA5 and/or LGP2.
(Related to Figure 7B)
(C) Enhancing effect of exogenously supplied hLGP2 on endogenous response to
poly(I:C) in human HEK293 cells with the unit 1 corresponding to 5.5 ng LGP2
DNA/well. (Related to Figure 7A).
(D) Lack of signalling ability of exogenous hLGP2 upon activation with poly(I:C) or 5′
ppp-dsRNA (left) and dose-response enhancement by hLGP2 of the activation of
exogenous hMDA5 by poly(I:C) (right) in human Huh7.5 cells. Note the high
constitutive activation by MDA5_1 (corresponding to 5.5 ng DNA/well) (as
previously reported see (Louber et al., 2015) and references therein, see also panel E
below, upper left histogram, condition 0 RNA) that is neither enhanced by
exogenously added hLGP2 (compare histograms with no RNA (0) nor upon
transfection of RIG-I agonist 5′ppp
dsRNA with or without exogenous hLGP2 (compare
histograms with 0 and 5′ppp
dsRNA conditions). (Related to Figure 7B).
(E) Enhancing effect of hLGP2 on exogenous hMDA5 is observed only when transfected
in limited amount and/or activated by a sub-optimal amount poly(I:C) in human
Huh7.5 cells. Note the loss of dose-dependent enhancing effect of hLGP2 (with the
unit 1 corresponding to 5.5 ng LGP2 DNA/well) in the presence of 0.05 ng versus
0.005 ng of poly(I:C) in the presence of 0.55, 1,67 and 5.5 ng/well of hMDA5 DNA
(MDA5_0.1, MDA5_0.3, MDA5_1 at the upper right, bottom left and bottom right,
respectively) and the reduction of this enhancing effect with increasing amount of
hMDA5 (compare luciferase signal gradation according to hLGP2 amounts for a same
amount of poly(I:C) in the presence of hMDA5 (MDA5_0.1, MDA5_0.3, MDA5_1)).
Note also that the enhancing effect of hLGP2 on hMDA5 is restricted to poly(I:C) and
is not observed with another MDA5 RNA agonist, the 6 bacteriophage 2.9, 4.1 and
6.4 kbp long dsRNA, while hMDA5 do respond also to this agonist in the absence of
huLGP2 (compare 6 and the no (0) conditions in the upper left panel for each
amount of transfected hMDA5). (Related to Figure 7B).
(F) RNA binding deficient hLGP2 mutants (K138E/R492E or hel°, K650E/K651E or
CTD°, K138E/R492E/K650E/K651E or hel°CTD°) but not ATPase-deficient E132Q
mutant (see Figure 1E for ATPase activity) no longer exhibit enhancing activity for
hMDA5 activated by poly(I:C) in human Huh7.5 cells (Related to Figure 7B).
(G) RNA binding deficient hLGP2 mutants (K138E/R492E or hel°, K650E/K651E or
CTD°, K138E/R492E/K650E/K651E or hel°CTD°) but not ATPase-deficient E132Q
mutant no longer exhibit enhancing activity for the homopolymerisation-defective
hMDA5 IE/KR mutant activated by poly(I:C) in human Huh7.5 cells. Cells were
transfected with a mixture of LGP2 (27.5 ng DNA/well) and MDA5 (1.67 ng
DNA/well), i.e. at 5:0.3 ratio (Related to Figure 7C).
chLGP2 356 F TR L L G G MT Q F L T VL E F R IV K A WL T IR HSN G VI R G NL S Q H QPPGSS. G T QS HS LS QD AGLCGQH AAV T S QAK QNE QD TL .Y EL FS
hLGP2 358 F TR L L G G MT Q F L T VL F S R II R A WL Q IR SS VI G NL VA S QRQ SS NSP. G T QS HS LL QQ QGLQTVD AQL I A N QSTH QRD QE QK Q.D TL
dRig-I 619 F TR L L G G MT Q F L T VL D Y N R LL K V M N IK R G VL K L IA S D A RY PQT. T A AL SA KKC EE PILN..Y PGV M R RDQTT LPS KG DA TSKDNR
hRig-I 618 F TR L L G G MT Q F L T VL E Y N IL K V WI N LK KTN G IL K G NI IA S Q E HL PET.IT V AL DA KN EG PKLS..F PGI T R QNT LPA KC DA AS DH
hMDA5 708 F TR L L G G MT Q F L T VI E Y T R II K A WI N VK HSS P VI R G NL IA T M Q TR EESA G T QS YA SQ TE EKFAEVG AHH I A EFK QNE KE SK .T KI
chMDA5 683 F TR L L G G MT Q F L T VL E F T R II K A WI N IK HNS P VI R G NL IA T M E TK EEP. G T QS LA YH MD PKFEEVG AHF I A ETK QNE RE DK .G SI
.
TT TT
chLGP2 439 A EG DI CN V Y N M Q GR RA S E N M Q KI E L I V M I V A LA S V R L L AI V Y L PE R GL T E A Q A QN MYSV KAN RE Y Q ESLVG ER RA AMPERK RL VE
hLGP2 441 A EG DI CN V Y N M Q GR RA S E N M Q KI E L V V L I V AR VA S L R I E L AV V Y L PH R GL T E S A DQ VYAF TEG RE K L EAL T EQ AA KMDQAE QA RD
dRig-I 701 A EG DI CN V Y N M Q GR RA S E N M Q KI D I L V I VR LV T V E M AV I F L VQ L EYSG VTK G AG K.CI TSK EV EN KC RYK E NK EK KWDEET AK HN
hRig-I 700 A EG DI CN V Y N M Q GR RA S E N M Q KI D I L I V I I R LL I K I E M I L F I AQ L EY G V K T G RG K.CF TSNAGV E Q MYK K NDS LR TWDEAV RE LH
hMDA5 792 A EG DI CN V Y N M Q GR RA S E N M Q KI E L I I V I V AR VA S I H V E M AI V Y L KE R GL T E A A DE TYVL HSG GV E T DFR K YK HC NMKPEE AH LE
chMDA5 766 A EG DI CN V Y N M Q GR RA S E N M Q KI E L I I V I V AR VA S V R V E M AI V Y KE R GL T E A A DE TYAL SSG GA E D IFR N YK RR EMPPEE LN QDF
TT
TTT
chLGP2 524 Q C C D H F VK R VAVC IR VE HV I Y W I RNAVLSWQ EARSSER QLHDPDDVYFH VN N RGS T AM N NPN RFY TVSSGKIHFERTFRD EPGCR
hLGP2 526 Q C C D H F R A R L VAV LR VE HV V Y W I QAALTK A QAAQRENQ QQFPVEHVQL IN M GHGS K GT N NPN SNY NVSRDPVVINKVFKD KPGGV
dRig-I 785 Q C C D H F R LR K L A AC IR I HI L Y F M MK...E V DSRRKEI PKVVEGQKNL GK K Y STD I KDS V GEA KER TTKPHKK..PMQFDG EKKSK
hRig-I 784 Q C C D H F K IR K L ALAC VR IE L F F I TH...E F DSQEKP. PVPDKENKKL RK K YTA V EC YTV GDA KEC VSRPHPK..PKQFSS EKRAK
hMDA5 877 Q C C D H F K MK H L VLAC IH IE HV M Y Y I MQSIME K TKRNIAK YKNNPSLITF KN S SGE V KM N TPE KEL IVRENKA.LQKKCAD QINGE
chMDA5 851 Q C C D H F K MK L LIC I IE HV V Y Y I LQSIVE Q AKRDQRKTYKKNPSLITF KN HK SGE QV NM S KKD QHL HKRENRT.LQDKHAD QTNVE
TT TTT
T..TT TT . TT
chLGP2 609 C C WG P KW M YR L L IKNFVV Y V F V S..E RQE ME I NVT. I S VTPDEKKK... K ST TFPIEE SYLEYCSSTQDE.SL.
hLGP2 611 C C WG P KW M YK L LKVRS LL V FD S R..N GEV LQ I SVK. V M ETPQGRIQ...AK SR PFSVPD FLQHCAENLSDLSLD
dRig-I 865 C C WG P KW V Y L IKIKSFVM F I FD Y RNNN QHD IT K LTFDN V ESTATGTQMD. Q KS NSSLKN VEEMSNLYPPF....
hRig-I 863 C C WG P KW V YK I IKI SFVV Y FD F ARQN SHD IH K TFE. V E EDIATGVQTL. S KDFHFEKIP PAEMSK.........
hMDA5 961 C C WG P KW M K L LKIRNFVV Y L D I K... GQA TM VH GLD. C VFKNNSTKKQ. K VE PITFPNL YSECCLFSDED....
chMDA5 935 C C WG P KW M YR L LKIRNFVV F L FD I K..D GQV NM V GLD. C AFEDKKTTKEI K GE PIIFPD YASHCPSSDED....
T..TT TT .
chLGP2 .....................................................................................
hLGP2 .....................................................................................
dRig-I 1 M...TADEKRS.....L.........Q........C.......YRRYIERSLNPV.YVLGNMTDWLPDELRERIRKE.E.ERGVS
hRig-I 1 MTTE...QRRS..LQAFQDYIRKTLDPTYILSY.......MAPWFREEE.VQYIQ.AEKNNKGPMEAA.......TLFLKFLLE.
hMDA5 1 MSNGYSTDENFRYLI...SCFRARVKMYIQVEPVLDYL.T.....F..........LPAEVKEQIQRTVATSGNMQAVELLLSTL
chMDA5 1 MS.EEC.RDERFLYMISCFRPRLKRCIRV...QPVLDWL.P........SLSAEEKDKVRAAALQRGEVEGAEELLCAVERGRRD
chLGP2 .....................................................................................
hLGP2 .....................................................................................
dRig-I 51 GAAALFLDAVLQ.LEARGWFRGMLDAMLAAGYTGLAEAIENWDFSKL....EKLELHRQLLKRIEATMLE.VDPAA......LIP
hRig-I 64 LQEEGWFRGFLDALDHAGY..........SGLYEAIESWD.FKKI......EKLEEYRLLLKRLQPEFKTRI.......IPTDII
hMDA5 67 EKGVWHLGWTREFVEALRRTG.SPLAARYMNPEL.....TDLPSPSFENAHDEYLQLLNLLQPTLV......DKLLVRDV...LD
chMDA5 72 PGWFTEFL....LALKKGG.CDLAACYVNP..SQLPSPQEEDDHDLCVHLVQLLHGTLV.....DNMQTRQV...AEKC......
chLGP2 .....................................................................................
hLGP2 .....................................................................................
dRig-I 124 YISTCL.....IDRECEEIQQISENRSKAAGITKLIECLCRSDKEHWPKSLQLALDTTGYYRASELWDIREDNAKDVDSEMTDAS
hRig-I 125 SDLSECL...INQECEEILQICSTKGMMAGAEKLVECLLRSDKENWPKTLKLALEKE.RNKFSELW.IVEKGIKDVETEDLEDK.
hMDA5 137 KCMEEELLTIEDRNRIAAA.ENNGNESGVRELLKRIVQ..KENWFSAFLNVLRQTGNNELVQELTGSDCSESNAEIENLSQVDGP
chMDA5 136 ..LELGIFQEEDLVGIETVIESRGNRDGARELLSRIVQ..KKDWFSQFLVALRETQHESLADDLSGNTGGTEDKDYELKNNT.GK
chLGP2 .....................................................................................
hLGP2 .....................................................................................
dRig-I 204 ...EDCLEASMTYSEEAEPDD.........................................NLSENLGSAAEGIGKPPPVYETK
hRig-I 204 ...METSDIQIFYQEDPECQ..........................................NLSENSCPPSEV..SDTNLYSPF
hMDA5 219 QVEEQLLSTTVQPNLEKEVWGMENNSSESSFADSSVVSESDTSLAEGSVSCLDESLGHNSNMGSDSGTMGSDSDEENVAARASPE
chMDA5 216 KTEAASQPVYVTEDLKQQENLDDSFVRESSVLETSVGKNSVISES.VAVGDASVS.NSNENLGQSSTT..SDSGEDEAEGRASPE
TT .
chLGP2 1 YQ E PA G N PTG GKT H V Q F V LH L AV L R IV L A V V L R A LVNKV L F ..ME G A R S W A R A H CRR EGRRG....G V H VQ HLEKE H.VLRDA. K
hLGP2 1 YQ E PA G N PTG GKT H V Q F V LR VI L K II L A A V L K V LVNRV L W ..ME S W M E I W A R A Y AKR ETVDG....A V H VT HG.EE RRMLDGR. T
dRig-I 245 YQ E PA G N PTG GKT H V Q F V AR I LA I K LI A V I I K V LATKV V Y ...K S Q N A C S F S L CEH FQNMPA.GRKA F P YE QK.NV KHHFERQG S
hRig-I 242 YQ E PA G N PTG GKT H V Q F V R L LA M K II A V L I L K V AN I V Y ...KP N L K T C C F S L CEH KKFPQ.GQKG FF Q P YE QK.SV SKYFERHG R
hMDA5 304 YQ E PA G N PTG GKT H V Q F V LR M VA L K II L V V I L K I LVNKV L Y PELQ P Q E I C S R A Y AKD DKKKKASEPG V L VE LFRKE QPFLKKW. R
chMDA5 297 YQ E PA G N PTG GKT H V Q F V LR M VA L II L V V I L K I LVNKV L Y PDLT D K N E I C S R A Y TKD DKKRKASEQG V P VE HLRKE NPFLKHW. Q
TT
T..TT
chLGP2 78 SG D T L L F L DECH T YN M YL K V DS LA VVI QI N L T S LV QK I L TA SHKCFFGQ KGS C A Q A LSGEE..EARVE D I H EAV K LS QK SGQR..
hLGP2 78 SG D T L L F L DECH T YN M YL K L D A LA LLI L L T S IV K I L TT MGPR GFGH RCH C AE QMA TSPEE..EEHVE V V H H DTV V SQ EL QRAQ..
dRig-I 325 SG D T L L F L DECH T YN M YL K I EN V VI IIV QI N S T MI T L QG FSN SVEK EDS V P V SFEDGTL.....TS I F N GNHP V TR EQ FNSAS..
hRig-I 322 SG D T L L F L DECH T YN M YL K I T V IV III QI N L S T MI SK I L TG A AEN PVEQ ENN L P V N KKGTI.....PS I F N QHP M FN DQ GGSSG.
hMDA5 388 SG D T L L F L DECH T YN M YL K L DT I VV III QI N L S S II NK I L IG QLK SFPE KSC S A E S LNLENGEDAGVQ D I H EAV N RH MQ KNNRLK
chMDA5 381 SG D T L L F L DECH T YN M YL K L DS I VV VII QI N L S S II QK I I IG ELK SFPE KRY C A E S LNATE.EDESVR D I H EGV N RR KE KNRKQA
TT
chLGP2 159 PQ GLTAS G G C LD I L P F L IL A EHI I AN SA A V K L R DP L I.......D P T GETSFEG V LQ TEV A QEH QH QSH PQ T QYD CQE EQ GQR KK
hLGP2 159 PQ GLTAS G G C LD I L P F L VL A A HV L AN S K L R DP L L.......P P T G SKLDG IN LQ TWC M PQNCCPQ QEHSQQ C QYN CHR SQ GDL KK
dRig-I 403 PQ GLTAS G G C LD I L P F L IL A EHI L TV I M L R P I L.......Q V V N KNIEETI CS SY IQA S REN QE QRF NK EIDVR VKR IHN AAI SN
hRig-I 401 PQ GLTAS G G C LD I L P F L VI A A D I L AS TV L V K R D I L.......P V V D KNTDE L Y CK ASV A KHN EE EQV YK Q FFRKVES IS K KYI AQ
hMDA5 473 PQ GLTAS G G C LD I L P F L IL A A EHI L AN TV L I K I DP L IKENKPVIP P V G TKQAK E LK AFT K KEN DQ KNQ QE C KFA ADATRE KEK LE
chMDA5 465 PQ GLTAS G G C LD I L P F IL A A EHI I AN TV A V K I K DP I IKENKPLIPQ P V G RSNSK E LK ACR M KEH SQ KNQ KE F KTV ADD RR RER IE
TT
chLGP2 237 M FG Q YE HL YN AL I R DA M L T Q IV A R V RR D ND V MM AQIQEHM.....E PE PQ....N V R ELENRA ERFC.......... KTR CAL L T
hLGP2 237 M FG Q YE HL YN AL I R DA M LS T Q VV A V RR D D V AV DQIHDHL.....E PE R....K M Q KLSEAA LAGL..........QEQR YAL L H T
dRig-I 481 M FG Q YE HL YN AL I R DA V LS T IV R I RK D SE A II SETEALMRTI.YS DT QNSKKD N HW VTQRKCRLLQLEDKEEESRIC ALF CTE I D
hRig-I 479 M FG Q YE HL YN AL I R DA L LS T Q IV M K L RK D SE A M RDTESLAKRICKD EN QIQNRE K W TVQKAC VFQMPDKDEESRIC ALF YTS I H K
hMDA5 558 M FG Q YE HL YN AL I R DA M MS T Q AI A R V RK E ND I MI TRIQTYC.....Q SP .....D P W QMEKKA KEGN.......... KER CAE Q T
chMDA5 550 M FG Q YE HL YN AL I R DA L S S Q VI A R V KK D ND I MV QDIQKYC.....Q YPK .....E P W REERRA KEEK.......... KER CAE Q T
chLGP2 303 L F L F L N L L Y R E E L E R FQC QQ ADK DTKD................................PT RF ATT E NRAT QA AGDQRY P SK EEI
hLGP2 303 L F L F L N L L Y H E D L E K LAA QD HRE VTKTQIL.............................CA RR LAL D RKNE AH ATHGP. P EM EKI
dRig-I 565 L F L F L N L L F K E E L E K LSY TE TNV NGPYT...............................EL QH TAK Q KEPE IA SKDETN P EE VCI
hRig-I 564 L F L F L N L L F R E E V E K LDY KD SNV AAGFD...............................EI QD TQR E KLQE ES SRDPSN P ED CFI
hMDA5 623 L F L F L N L L Y K D E L E K YTH ET NEE DKKFAVIEDDSDEGGDDEYCDGDEDEDDLKKPLKLDET RF MTL F NNKM KR AENPEY E TK RNT
chMDA5 615 L F L F L N L L Y K D L D K YNH NN KEL RRKTA...............ESDDDEEPLVS..KQDET EF MRL HAKKKQ KE ARKPEY E MK RNT
Hel2-loop
Hel2i loop
Motif IV Motif IVa
Motif Va Motif VI
Capping loop
α10 extension
Endbindingloop
α1
α20
α19α18α17
α16α15
α14α13α12
α11α10
α9α8α7
α6α5α4
α3α2β1
β13β12
β11β10
β5β4
β6
β9
β8β7
β3
β2
β16β15β14
β20
β19β18β17
β23β22β21
α1 α3α2β1 β2
α6α5α4 β5β4β3
α9α8α7β6 β8β7
α11α10
α14α13α12
α16α15 β11β10β9
α19α18α17 β13β12
β16β15β14 β19β18β17
β20 β23β22β21
Motif Q Motif I Motif Ia
Motif Ib Motif Ic Motif II Motif IIa
Motif III
MotifV
chLGP2 structure
chMDA5 structure
chLGP2 structure
chMDA5 structure
chLGP2 structure
chMDA5 structure
chLGP2 structure
chMDA5 structure
chLGP2 structure
chMDA5 structure
chLGP2 structure
chMDA5 structure
chLGP2 structure
chMDA5 structure
chLGP2 structure
chMDA5 structure
chLGP2 structure
chMDA5 structure
Figure S1
OO
OO
P
O
O
P
O
O
O
ON
N
N
N
N
Al
FF
F
2+
F
Gly27(I)
Thr31(I)
Asp444(Va) Glu67(α3 helix)
Gln7(Q)6
7
1
9 3
Arg32(I)
His4(Q)
Glu2(Q)
5
Gly29(I)
2 2
1 1
3
Ala28(I)
Lys30(I)Arg469(VI)
1
2
3
4
Glu132(II)
3 2
(NE2,OE1) (O)
αβ
(N,NH2)
(OE2)(OD1,OD2)
(N)
(NZ,N)
(OG,N)
(NE1,NE,NH2)(NH1,NH2)
(OD,N)
4
(N)
Asp131(II)(OD1,OD2)
Asp441(Va)
Gly442(Va)
Ala168(III)
Arg465
Leu166(III)
Arg471(VI)
(N)(O)
(N,O) (NE2,OE1,O)
(N)
Mg
(O)
chLGP2
chLGP2
chMDA5
dRIG-I
dRIG-I
ATP concentration (mM)
(μM/min)
ν0
18-m
er ha
irpin
32-m
er ha
ipin_
p
32-m
er ha
ipin_
ppp
24ds
38ds
poly
IC
1 1 0
Homo_sapiens/1-925 A P T G G K T V F C Mus_musculus/1-926 A P T G G K T V F C Sus_scrofa/1-940 A P T G G K T V F C Pteropus/1-945 A P T G G K T V F C Cairina_moschata/1-934 A P T G G K T V F S Alligator/1-928 A P T G G K T V F S Anolis/1-781 A P T G G K T V F S Chrysemys/1-932 A P T G G K T V F S Xenopus/1-966 A P T G G K T V S L Cyprinus_carpio/1-946 A P T G G K T V C I Danio_rerio/1-937 A P T G G K T V C I
1 1 0
Gallus_gallus/1-1001 L P T G S G K T R VHomo/1-1025 L P T G S G K T R VMus_musculus/1-1025 L P T G S G K T R VSus_scrofa/1-1023 L P T G S G K T R V[Pteropus/1-1024 L P T G S G K T R VCairina/1-1003 L P T G S G K T R VAlligator/1-986 L P T G S G K T R VAnolis/1-1006 L P T G S G K T R Vfrog/1-998 L P T G S G K T R VChrysemys/1-1010 L P T G S G K T R VCyprinus_carpio/1-993 L P T G S G K T R VDanio_rerio_zebra�sh_/1-997 L P T G S G K T R V
1 1 0
Homo_sapiens/1-925 M Q R G R G R A I T RGMus_musculus/1-926 M Q R G R G R A I T RDSus_scrofa/1-940 M Q R G R G R A I T RGPteropus/1-945 M Q R G R G R A I T RGCairina_moschata/1-934 M Q R G R G R A I V AGAlligator/1-928 M Q R G R G R A I V EGAnolis/1-781 M Q R G R G R A I V KYChrysemys/1-932 M Q R G R G R A I V KDXenopus/1-966 M Q R G R G R A I V KDCyprinus_carpio/1-946 M Q R G R G R A V V EGDanio_rerio/1-937 M Q R G R G R A V V QG
1 1 0
1 M Q A R G R R A V A DE4 M Q A R G R R A V A DE5 M Q A R G R R A V A DE6 M Q A R G R R A V A DE7 M Q A R G R R A I A DE2 M Q A R G R R A L A DE8 M Q A R G R R A V A DE9 M Q A R G R R A V A DE10 M Q A R G R R A V A DD11 M Q A R G R R A V A DE12 M Q A R G R R A I ED G 13 M Q A R G R R A I ED G
1 1 0
Chicken L P T G G K T R A A Human L P T G G K T R A A Mouse L P T G G K T R A A Wild_boar L P T G G K T R A A Bat L P T G G K T R A S Muscovy_duck L P T G G K T R A A Alligator L P T G G K T R A A Lizard L P T G G K T R A A Frog L P T G G K T R A A Turtle L P T G G K T R A A Carp L P T G G K T R A G Zebra�sh L P T G G K T R A G
1 1 0
Chicken Q A G R A R MV A N Q Q Human Q A G R A R MV R A Q D Mouse Q A G R A R MV R A Q G Wild_boar Q A G R A R MV R A Q S Bat Q A G R A R MV R A NEMuscovy_duck Q A G R A R MV R A N E Alligator Q A G R A R MM R A N E Lizard Q A G R A R MM R A N P Turtle Q A G R A R MV R A N E Frog Q A G R A R MV R HEDCarp Q A G R A R A NQQ S L Zebra�sh Q A G R A R A NQQ S S
24 33 322 331 264 273
LGP2LGP2
463 474 790 801 724 735
MDA5 RIG-IMDA5 RIG-I
A
B
C D
E FMotif VIMotif I
12dsRNA
24dsRNA
24dsRNA
24dsRNA
ADP
Mg²+
AlF4
0 5 100
5
10
15
12dsRNA
0
5
10
15
20
25
30
35
40
45
50
hRig-IchLGP2chMDA5
noRNA
phos
phat
e co
ncen
tratio
n (µ
M)
Figure S2
TT .
Chicken 1 M L YQ E AL G N I WLPTG GKTRAA V HLE V VN VHLV QH EF L E H AVAP R V A V R R R AVL K H V F .. G L R S H CR G RGG Q LEK . RDA K
Human 1 M L YQ E AL G N I WLPTG GKTRAA V HLE V VN VHLV QH EF L E R VIMP K I A AY R T K VVL R R M W .. S W E I AK VDGA T .GE R DGR T
Mouse 1 M L YQ E AL G N I WLPTG GKTRAA V HLE V VN VHLV QH EF L E R VILP K I A AF R T K VVL R R M W .. P W E I AK VDRG S .AE R DKH T
Muscovy_duck 1 M L YQ E AL G N I WLPTG GKTRAA V HLE V VN VHLV QH EF L E R AVAP R V A V K SR R AVL K H V F .. G H R C H CW RDG E AKE . QGS R
Alligator 1 M L YQ E AL G N I WLPTG GKTRAA V HLE V VN VHLV QH EF L D VIMP I A VY R TR K AVL K V F .. YD W E Q I CK ARA D FSK G. RSS Q
Frog 1 M L YQ E AL G N I WLPTG GKTRAA V HLE V VN VHLV QH EF L E H VI P K I A LY R K K LM K H Y .. D W G E I AM M RNA C D FSN PH KDK K
Zebrafish 1 M L YQ E AL G N I WLPTG GKTRAA V HLE V VN VHLV QH EF L E R VV I VY K TK K AVL K Y LR P E QA R E S G AE ANA D YMK GHY RHK R
TTT
Chicken 77 SGD F ICTA L AL E VE F L DECHHT K VYN Y K VT V S K G LAK SDVV QI N S E E R LTD S LVI E KIM L K LS A SH CF Q G Q L G A Q A LS Q
Human 77 SGD F ICTA L AL E VE F L DECHHT K VYN Y K VT L R G LAR DLL L TS E E H LT S IVV D IM L LQ T MGP AG H CH E QM P E V H T V SQ EL
Mouse 77 SGD F ICTA L AL E VE F L DECHHT K VYN Y K VT L R G MAR DLL L NS E D H L E S IVV D IL L L T MGS AG L SH E QL S E R H T T SR EQ K
Muscovy_duck 77 SGD F ICTA L AL E VE F L DECHHT K VYN Y K VT I S K G AVK SDVV QI N S E D R LTD S LVI E KIM L R LS A SH CF Q R H L R T Q A LH Q
Alligator 77 SGD F ICTA L AL E VE F L DECHHT K VYN Y K VT I S K VVK N VV QI N SS E E H LTD S LVI E KIM L R LS P DQ FI SH N H H S M H S QD Q
Frog 78 SGD F ICTA L AL E VE F L DECHHT K VYN Y K V I T K LV NDVI QI N SS E H LTD T LII D KLM L R IT VA EH CF AE QN Q SS I H G EG E
Zebrafish 80 SGD F ICTA L AL E VE F L DECHHT K VYN Y K I I S K G LVR SDLV QI N NN D D H ITD T LVI E KIM V K V KA SE DF R V E M K N S WR E R
TT
Chicken 156 LPQ LGLTASPGTG A H LQ CANLD I S L P K Y R DPFG K I G TS EG VE I I T A VPQ Q DL RL GQRD E F EV AQEHAQH QSH T CQE EQ Q
Human 156 LPQ LGLTASPGTG A H LQ CANLD I S L P K Y R DPFG K V G S DG I V L T M Q Q L L RAQP A KL N WC PQNCCPQ QEHSQ C N CHR SQ DL
Mouse 156 LPQ LGLTASPGTG A H LQ CANLD I S L P K Y R DPFG K V G T G ID I L T M P Q DL I KAEP A KLQ CH PKNCYSQ LMHN K C CQR AQ DL
Muscovy_duck 156 LPQ LGLTASPGTG A H LQ CANLD I S L P K Y R DPFG K I G TS EG VE V I T VPQ Q DL RL GRRD K F EV T AQEHVQH QSQ R CQE AW Q
Alligator 156 LPQ LGLTASPGTG A H LQ CANLD I S L P K Y R DPFG K I G S G D I I T M VPQ Q DI KL GQQN AL FK K AK SQKHHSH EAC K PHE TQ R
Frog 157 LPQ LGLTASPGTG A H LQ CANLD I S L P K Y R DPFG K I TS E E I I T M A Q Q DL KL QKGK RA F K E WR AEVHRED EAK K N VTE PR D
Zebrafish 159 LPQ LGLTASPGTG A H LQ CANLD I S L P K Y R DPFG K I G S D VE V I S V VP DI HL KEGR NK L K .K TKNYTPM QNF K K E VER DK D
Chicken 235 M I GTQ YE V L A HLR YND L DT R DA L Y I Q M P L F Q I E A R TRVC L R A LI V MM F K A QEH EM E PQ...N V R E NRA ERFC K N FQC QQ
Human 235 M I GTQ YE V L A HLR YND L DT R DA L Y L Q H L P L F Q V A QRV L R A LI V AV DF K D DH EM E SR...K M Q K SEAA LAGLQE Y H LAA Q
Mouse 235 M I GTQ YE V L A HLR YND L DT R DA L Y L Q H L P L F Q V K A QRV L R A I V A DF K N QQ EM D KQ...Q M Q Q C DA EAGLQE Y F H R LDM Q
Muscovy_duck 235 M I GTQ YE V L A HLR YND L DT R DA L Y I Q M L F Q V EK A R TRAC L K A LI V MV F K A QQH GTAG PQ...D T Q E RA ERFC K N FQH QQ
Alligator 235 M I GTQ YE V L A HLR YND L DT R DA L Y L Q H L P V F Q V EK A R TRLC M K A LI V MV EF E A QK NV S SQ...D T Q L TG ESFC K N VNY D
Frog 236 M I GTQ YE V L A HLR YND L DT R DA L Y L T H L F Q V EK A R R C L K LV V MM DY E K EY RTTDFCE..SD L K E EG VEAN MK T S H YEL D
Zebrafish 237 M I GTQ YE V L A HLR YND L DT R DA L Y M H M P V V EK V R C L K A LI I MV EF S LM EF P. T SRGLREL E AD E AG KVNN LIAQ N FRV E
Chicken 311 L F L LA ENP L L L F RGI F TR L W R K D E F A EENR Y R E I E Q SS V TK QSAHS LQADK DT ... PT R TT AT QA GDQR SK E Q H PPG LS
Human 311 L F L LA ENP L L L F RGI F TR L W EH K E L L DD K K E I Q S S I TR QSAHS LQHR VT TQILCA RR A R NE AH TH.GP EM K QR SSN P LL
Mouse 311 L F L LA ENP L L L F RGI F TR L W ER K E W L L DD K K E I Q T I TR QTA S L DR TT TQMVRA S K H NV GQ AR.GP EM R LK GSPGH S LL R
Muscovy_duck 311 L F L LA ENP L L L F RGI F TR L W ER K D E F A EENR Y R E I EN Q SS V TK QSAHS LQAT DM ... PT R TT AS LA GCQR SK E R PQG LS
Alligator 311 L F L LA ENP L L L F RGI F TR L W EK K D F M L DENK Y K E I DQ Q SS I TK QSAH IQQQ AM FLP VIWH T ST LA RNLE SK K Q ALD A HK
Frog 313 L F L LA ENP L L L F RGI F TR L W EK R D D F I L D NR F K E I DQ Q S I TR QS HS ISQQ VI KQN PT A Q G AR LE QDVR RK E R FSSG T HN
Zebrafish 315 L F L LA ENP L L L F RGI F TR L W D D F L DEN Y K E Q NS I SK H V NSRS..SKLL GT I QG SLE KH SDAR AQ QSR L E .DT RGT C ND K
TT
Chicken 387 L G G MTQ Q I F G LL TSVAEEGLDI CN VVRYGL TNEI Q L IRA V T SNQ NE DV R LN S P I M MV DTAG CGQH A S H AKG Q TL Y E F E A
Human 389 L G G MTQ Q I F G LL TSVAEEGLDI CN VVRYGL TNEI Q LQ V IRA L A SSQS H D EV LN P V L MV QQQG T D Q I N T R Q QK QD T VA H S
Mouse 389 L G G MTQ Q I F G LL TSVAEEGLDI CN VVRYGL TNEI Q LQ V IK M A TSQS H D EV R LS V L MV QQPC T G PQ I N T K Q QE D I VA AQ S
Muscovy_duck 387 L G G MTQ Q I F G LL TSVAEEGLDI CN VVRYGL TNEI Q LS IRA V T A SNQT H NE DV R LN S P I M MV DAAE GQH A Y K Q RQ K D F K A
Alligator 390 L G G MTQ Q I F G LL TSVAEEGLDI CN VVRYGL TNEI Q LT L IKA V T A SNQT H NE DV R IN S P V M MM GMPS Q G A Y K M KN K D F E A
Frog 392 L G G MTQ Q I F G LL TSVAEEGLDI CN VVRYGL TNEI Q Q M VK T A SNQS H NE E R LN S P I M MV SKHSF I G TAP Y K R T EM K Q I Q S
Zebrafish 391 L G G MTQ Q I F G LL TSVAEEGLDI CN VVRYGL TNEI Q LQ V I A I T A TE V R LN S P L L TNRE R N T G NGA..NN KS SH Q Y I E AQQ
TT
Chicken 466 A GRAR S Y A E E NE L L A VQ M LQ A N M S L A SR V R L M R I A Y KI E AVL V AR RRQ Q Q V K N Y Q S VG E R A PERK RL V RN SWQ KE SSE L
Human 468 A GRAR S Y A E E NE L L A VQ M LQ R A Q V V SR L R I E M V A EY KI D AL A A R RQ D AF TEG K L A T EQ A K DQA QA R QA TKRA QA Q ENQ Q
Mouse 468 A GRAR S Y A E E NE L L A VQ M LQ R A Q V S L SR M R E M K V A EF KI D LV A AHR G F TEG K LT A V E A K DPD KA R QAS KRA RA EIQQGQ
Muscovy_duck 466 A GRAR S Y A E E NE L L A VQ M LQ R A N V S L A SR V R L M K I A EY KI E AVI V AK RR E V K N S L N VE Q Q A PER RL A RN GWQ KE ISE RL
Alligator 469 A GRAR S Y A E E NE L L A VQ M LQ R A N S L A SK V R L E M R I EY I E AII I V R K Q E T V K N S Q T M T KD A PEQ QHQ G KA S.Q MK D VQ Q L
Frog 471 A GRAR S Y A E E NE L L A VQ M LQ R S L I K I R E M R I A EY KI E VI V AKR KRN HED C F K GG R ET T G K E R PEQ QK K EES ARK KQ DQ T
Zebrafish 468 A GRAR S Y A E E NE L L A VQ M LQ A N V S V V R V K V E R I DF KV E AVV KR KKQ S S V D GG R L Y D TA DE R SPV RH F KT IRMEAER DA R
TTT TTT
Chicken 545 V C C V G D R E H VN N F Y DW PG C C WG PD VN VA S I V H I P R Y VS I R FR I EMIYHD D YFH N CR T AM N F T SGK HFE T E CR V SE RQE M
Human 547 V C C V G D R E H VN N F Y DW PG C C WG E IN VA S L V H V P Y VS V K FK I MIYFPV H QLL M GH K GT N SN N RDP VIN V K GV S RN GEV LQ
Mouse 547 V C C V G D R E H VN N F Y DW PG C C WG PE IN VA S L V H V P Y S V K FK I MIYFL H QLL M GY K GT N SV TT QNP VIN V R GT R SN GEV FQ
Muscovy_duck 545 V C C V G D R E H VN N F Y DW PG C C WG P VN VA S I V H I P R Y VS I R FK V EMIYHD GD YFY N CC T GM N C T LGK SFP T E CR M SG RQE M
Alligator 547 V C C V G D R E H VN N F Y DW PG C C WG PD L A S L V R I P K Y AS V R FK I EMIFHD T CFY H NE CH I KM Y H R NNP EIP T K SI S RK GQA M
Frog 550 V C C V G D R E H VN N F Y DW PG C C WG PE A I I R Y V L K I EMIYFY Q RFY RC SQ AH D F T GT Y SD I E CSPP DFG KMV T GK R L. GQD F
Zebrafish 547 V C C V G D R E H VN N F Y DW PG C C WG P S A I I H V K V V R F I EI FYS GQ QLQ R F S CS G K NS TE NH K G.DQ NME T E E RI S RK KKD F K
TT . TT
Chicken 624 V P TP W V F R L IL IKNFVV KK YKK S I EF YL CS D L N T. S V DE K T T P E S EY STQ. ES ....
Human 626 V P TP W V F K L VL VRS LLE R KK S V DFDFL C D L S K. K M QG IQA R P S P QH AENLS LS ...D
Mouse 626 V P TP W V F K L VL I S LLE K KK S I FD L CT E L S T. K G I RG IQA R P S PV I QD QSLS LS ...D
Muscovy_duck 624 V P TP W V F H L IL IKNFVVE KR YKK S I EFDYV CS D H K. C DE R G T P K EY STQE ESF....
Alligator 626 V P TP W V F R L L I NFVVE RK YKK S I DFDYV CQ D S K. N S E DM K K T V K EY SLN. .......
Frog 628 V P TP W V F K AI VKNFVVE KR Y R V E YV E V H N.F S EI P A KD P P D LN QHVRIHP. LL HFED
Zebrafish 625 V P TP W V F K L L IKSF K YKK V EFDFI S D L K AI C K SFN KET P KD E Q T EYM CRFP LD ..SD
Hel2-loop
Motif IV
Motif IVa Motif Va
Motif VI
Capping loopEndbindingloop
α1
α20
α19α18
α17α16
α15α14α13
α12α11α10
α9α8α7
α6α5α4
α3α2β1
β13
β12β11β10
β5β4
β6
β9
β8β7
β2
β16β15β14 β20β19β18β17
β23β22β21
Motif Q Motif I Motif Ia
Motif Ib Motif Ic Motif II Motif IIa
Motif III
MotifV
chLGP2 structure
chLGP2 structure
chLGP2 structure
chLGP2 structure
chLGP2 structure
chLGP2 structure
chLGP2 structure
chLGP2 structure
chLGP2 structure
406
67
595●
●
●
Figure S3
G(NZ)Lys634
1
P
PTrp602Phe595
Ser407(N)
Asn408(N,ND2)
His406(ND1)(OG)Ser632
βα
W
PP
WW
γ
Mg 2+
G(NZ)Lys634
1
P
PTrp602Phe595
Asn408(N,ND2)
His406(ND1)(OG)Ser632
β
α W
PP
W
γ Arg607(NH2)Ser407(N)
A B
GGGG
GCC CA AA
UGG
G CC CCC A U
5’-3’- -5’
-3’UU
(FAM)
ppp
GGGG
GCC CA AA
UGG
G CC CCC A U
5’-3’- -5’
-3’UU
(FAM)
OH
GG G G GCC CA AAUGGG CCCCC AAA U
5’-3’- -5’
-3’(FAM)
OH
GG G G GG
CGCG
CCCCCC
A AAAAU
A U5’-3’-
-3’-5’(FAM)
OH
OH
12 dsRNAOH
12 dsRNAppp
3’ overhangOH
5’ overhangOH
GGG GC CA AA
UG
G CCCC A U
5’-3’- -5’
-3’U
Up
p
GGG GC CA A
AU
GG CCCC A U
5’-3’- -5’
-3’U
UCGGC
p
p
CA UGG G GG
GC
GC
GCG
CG
CG C
AUC
AUC
5’-3’-
ppp
10 dsRNAp
10 dsRNAppp
12 dsRNAp
5′-ppp and 3' two nucleotide (GG) overhang hairpin RNA duplex
3’-CA GG CGGG CG CG CCAU C5’- A UU
-5’-3’
CAG GC G G GC GC CA UC AU Up
pC GU U A A
UUAACGGC
3’-CA GG CGGG CG CG CUCAU C5’- A UU
-5’-3’
CAG GC G G GC GC GC CA UC AAU Up
p
C G G C C GG C C G G C
3’-CA U GG CGGG CG CG CCAU C5’- A A UU
-5’-3’
CAUG GC G G GC GC GC CA UC AAU Up
p
GCCG24 dsRNAp
26 dsRNAp
27 dsRNAp
GGG GC CA AA
UG
G CCCC A U
5’-3’- -5’
-3’U
Up
p10 dsRNAp
Anisotropy
chLGP2 crystallisation
chMDA5 crystallisation
C
RNA
Figure S4
GGG GC CA AA
UG
G CCCC A U
5’-3’- -5’
-3’U
Uppp
ppp
Q260E264
G9
G10*
A
B
C10
α10 helix
0 .001 0 .01 0 .1 1 1 00 .00
0 .05
0 .10
0 .15
0 .20
0 .25
hRig-I concentration (µM)
∆ A
isot
ropy
0 .01 0 .1 1 1 0 1 000 .00
0 .05
0 .10
0 .15
chMDA5 concentration (µM)
∆ A
isot
ropy
chLGP2 concentration (µM)
no NTP 12dsRNAOH K648E K649E
no NTP 12dsRNAOH K138E R490E K648E K649E
no NTP 12dsRNAOH H406A
∆ A
isot
ropy
NTP RNA Protein
NTP RNA Protein Hill coeficient
NTP RNA Protein Hill coeficient
12dsRNAOHATP
12dsRNAOHADP:AlF4
12dsRNAOHno NTP
ADP:AlF4 12dsRNAppp
ATP 12dsRNAppp
ADP 12dsRNAppp
no NTP 12dsRNAppp
ATP 12dsRNAOH
no NTP 12dsRNAOH
ADP:AlF4 12dsRNAOH
no NTP 12dsRNAOH
ATP 12dsRNAOH
ADP 12dsRNAOH
no NTP 12dsRNAppp
3’ overhang no NTP
5’ overhang no NTP
no NTP 12dsRNAOH K138E R490E
1.19 ± 0.098
1.14 ± 0.078
1.12 ± 0.168
0.96 ± 0.092
1.18 ± 0.058
1.35 ± 0.120
0.96 ± 0.104
0.68 ± 0.047
1.47 ± 0.064
1.38 ± 0.121
1.50 ± 0.224
0 .001 0 .01 0 .1 1 1 00 .00
0 .05
0 .10
0 .15
0.97 ± 0.090
Figure S5
wt
wtwtwt
wt
wt
wt
wt
wt
wt
wt
wt
wt
wt
wt
wt
chMDA5∆CARD
chMDA5∆CARDchMDA5∆CARD
dRIG-I (4A2W) hRIG-I (4AY2) hRIG-I (3TMI) dRIG-I (4A36)
chMDA5 chLGP2
hMDA5 (4GL2)
chLGP2
open semi-open semi-closed closed
E139(IIa)K138(IIa)Q137(IIa)
5’ end 3’ endF599F595
G82 (Ib)T103(Ic)V57 (Ia)S404(IVa)G403(IVa)T436(V)R375(IV)
PPPPP
PPPPP
PP
PP
2
3
4
5
6
7
1*
2*
3*
4*
5*
6*
7*R285(Hel2i)
Q409E139(IIa)K138(IIa)Q137(IIa)
5’ end 3’ endF599F595
G82(Ib)T103(Ic)V57(Ia)
S404(IVa)G403(IVa)T436(V)R375(IV)
PPPPP
PPP
PP
PP
PP
1
2
3
4
5
6
7
1*
2*
3*
4*
5*
6*
7*R285(Hel2i)
A
B
Hel2i
Hel2
Hel1
CTD
α16α15
α12
1
closed semi-closed
Figure S6
C
MDA5 IE/KR Ø
MDA5 IE/KR 0,05ng/µL Poly(I:C)0.05 ng/μL Poly(I:C)
Ø
hLGP2wt E132Q hel° CTD° hel°CTD°Ø
hMDA5 IE/KR
G
hIF
Nβ
prom
oter
act
ivat
ion
(RLU
)
104
103
102
A
Ø wt E132Q
hel°K138ER492E
CTD°K650EK651E
hel°CTD°K138ER492EK650EK651E
α-GAPDH
α-Flag
hLGP2
wt
hel°K138ER490E
CTD°K648649E
hel°CTD°K138ER490EK648649E wt
α-GAPDH
α-Flag
chLGP2 hLGP2 chMDA5250
130
100
70
HumanHuh7.5 cells
ChickenDF1 cells
RNA ng/µl
HEK293 + Ø + Poly(I:C) HEK293 + Ø + Φ6HEK293 + Ø + 5'ppp-dsRNA HEK293 + F + Poly(I:C)HEK293 + F + Phi6 HEK293 + F + 5'ppp-dsRNAHuh7.5 + Ø + Poly(I:C) Huh7.5 + Ø + Phi6Huh7.5 + Ø + 5'ppp-dsRNA Huh7.5 + F + Poly(I:C)Huh7.5 + F + Phi6 Huh7.5 + F + 5'ppp-dsRNA
0 0.001 0.01 0.1 1
B
hIF
Nβ
prom
oter
act
ivat
ion
(RLU
)
3x103
3x102
3x101
Dh
β
IFNβ
prom
oter
act
ivat
ion
(RLU
)
3x103
3x102
3x101
00.20.515
+ hLGP2hMDA5_1Ø + hLGP2
0 0.005 0.05
Poly(I:C) 5’ppp dsRNA0.005 0.05ng 0 0.005 0.05
Poly(I:C)0.005 0.05
Human Huh7.5 cells
Ø 0,005 ngµµ/µL Poly(I:C)0.005 ng / l Poly(I:C)
C
hLGP2
hIF
Nβ
prom
oter
act
ivat
ion
(RLU
)
0 0.2 0.5 1 5 6
Human HEK 293 cells3x103
3x102
3x101
hIF
Nβ
prom
oter
act
ivat
ion
(RLU
)
3x103
3x102
3x101
3x103
3x102
3x101
0 0.005 0.05Poly(I:C) Φ6
0.005 0.05 0 0.005 0.05Poly(I:C) 6
0.005 0.05ng
+ hLGP2hMDA5_1+ hLGP2hMDA5_0.3
Ø + h +5ADM hLGP2hMDA5_0.1E 0
0.20.515
Human Huh7.5 cells
hIFN
βpr
omot
er a
ctiv
atio
n (R
LU)
4x103
4x102
4x101
Ø (I:C)Ø (I:C)Ø (I:C)Ø (I:C)Ø (I:C)wt E132Q hel° CTD° hel°CTD°
hLGP2
F0.20.5
15
0
Φ6
Φ6
Φ6
µ
Figure S7
Φ
5’ppp dsRNA
Table S1. Quantification of the degree of opening (rotation angle and center of mass
distance) of the Hel1 and Hel2 helicase domains in published RLH structures. Related to
Figures 1, 4 and S6.
RLH
Structure
PDB ID RNA Bound
Nucleotide
Angle
(degree)
Distance*
(Å)
State
chLGP2
(Reference)
5JAJ 10 base pair 5′p ADP:AlF4 0.0 0.0 closed
dRIG-I 4A36 19 base pair 5′OH ADP:AlF4 2.5 1.3 closed
hMDA5 4GL2 12 base pair 5′OH AMPPNP 8.0 3.3 semi-
closed
chMDA5 5JC3 10 base pair 5′p ADP 10.1 3.9 semi-
closed
hRIG-I 5E3H
(3TMI)
14 base pair 5′OH ADP:BeF3 12.1 5.3 semi-
closed
chLGP2 5JBJ 12 base pair 5′p - 13.1 5.4 semi-
closed
hRIG-I 4AY2 20-mer hairpin 5′ppp ADP 39.3 14.9 semi-
open
hRIG-I 2YKG 10 base pair 5′OH SO4 40.3 15.9 open
dRIG-I 4A2W(A) no RNA - 51.8 18.5 open
dRIG-I 4A2W(B) no RNA - 59.8 19.2 open
mRIG-I 3TBK no RNA AMPPNP 68.2 24.4 open
Supplementary Experimental Procedures Protein preparation
chMDA5. Chicken (Gallus gallus) MDA5 constructs (Genbank: NP_001180567) full-length
chMDA5 (residues 1-1001), chMDA5_CARD (residues 298-994), chMDA5CARD-Q
(residues 298-994 with E436Q mutation) were cloned into pETM11 (EMBL) using a
synthetic gene (GeneArt). All constructs were expressed in E. coli Rosetta 2 (Novagen) and
cells were harvested and lysed by sonication in buffer containing 50 mM Tris pH 7.6, 500
mM NaCl, 10 % glycerol, 20 mM imidazole and 1 mM DTT. After centrifugation the lysate
was applied to nickel-NTA-Superflow resin (QIAGEN). The protein was dialyzed against
dialysis buffer (20 mM Hepes pH 7.5, 100 mM NaCl, 5% Glycerol, 1 mM DTT) at 4°C for
16 hours. The N-terminal 6-histidine-tag was cleaved with his-tagged Tobacco Etch Virus
(TEV) protease during dialysis and cleaved protein repurified on nickel-NTA resin. chMDA5
constructs were further purified with a cation exchange column (HiTrap SP, GE Healthcare)
and size-exclusion chromatography (Superdex 200, GE Helthcare) in a buffer containing 20
mM Hepes pH 7.5, 150 mM NaCl and 2 mM DTT.
chLGP2. A synthetic gene (GeneArt) comprising the coding sequence of chLGP2 (Genbank:
AEK21509) with an added N-terminal GGGGS linker was cloned into pETM11SUMO
(EMBL). Full-length chLGP2 full length was expressed in E.coli Rosetta 2 as a Sumo fusion
protein and cells were harvested and lysed by sonication in sonication buffer (50 mM
Na2HPO4 pH 8, 300 mM NaCl, 5% glycerol, 50 mM arginine, 5 mM β-mercapto-ethanol).
After centrifugation the lysate was applied to nickel-NTA-Superflow resin. The protein was
dialyzed against sonication buffer at 4°C for 16 hours. The 6-histidine-tag and Sumo-tag was
cleaved with TEV protease during dialysis. Cleaved protein was further purified with anion
exchange column (HiTrap Q GE Healthcare) and size-exclusion chromatography (Superdex
200, GE Healthcare) in a buffer containing 20 mM Hepes pH 7.5, 200 mM NaCl and 250 µM
TCEP (tris(2-carboxyethyl)phosphine).
hLGP2. Full-length hLGP2 (Genbank NM_024119.2) was cloned into the pETM11SUMO
vector (EMBL) and expressed in E.coli and purified as for chLGP2. The chromatograph of a
HiTrap Q purification shows two peaks, corresponding to hLGP2 monomers or dimers (the
majority), as deduced from size-exclusion chromatography.
RIG-I. Full-length hRIG-I was cloned into pFastBAC and expressed in HiFive insect cells.
Cells were harvested and lysed by sonication in buffer (50 mM Tris pH7.6, 500 mM NaCl,
10% glycerol, 20 mM Imidazole, 1 mM DTT). After centrifugation the lysate was applied to
nickel-NTA-Superflow resin and further purified as for chLGP2. Full-length dRIG-I was
purified from insect cells as described (Kowalinski et al., 2011).
Crystallization
For protein crystallization, 576 different conditions were initially screened in vapour
diffusion sitting drop format using a Cartesian robot. After screening, the crystal conditions
were optimized in 24-well hanging drop crystallization plates.
chMDA5 1:1 complex: Directly after size exclusion chromatography chMDA5ΔCARD-Q
was mixed with 10 bp dsRNA in a 1:1 molar ratio and incubated for 30 minutes on ice. The
complex was concentrated using an Amicon Ultra concentrator to around 10 mg/ml and 2
mM AMPPNP (adenosine 5′-(β,γ-imido)triphosphate lithium salt hydrate ) was added.
Sample and reservoir buffer (0.025 M Bis-Tris pH 6.5, 0.075 M succinic acid pH 7.0, 12-
14% PEG 3350, 2% sucrose) were mixed in a 2:1 ratio. Three hours after setup, cover glasses
with drops were transferred from a reservoir containing 12-14% PEG 3350 to one containing
8% PEG 3350. Crystals grew in one week at 20°C and were harvested in cryo-protectant
solution (0.025 M Bis-Tris pH 6.5, 0.075 M succinic acid pH 7.0, 25% 3350, 10% ethylene
glycol) before flash freezing with liquid nitrogen.
chMDA5 2:1 complexes: Directly after size exclusion chromatography chMDA5ΔCARD-Q
was mixed with 24, 26 or 27 bp dsRNA in a 0.5:1 molar ratio and incubated for 30 minutes
on ice. The complexes were concentrated with an Amicon Ultra concentrator to around 10
mg/ml and 2 mM AMPPNP and 200 mM NDSB211 (Dimethyl(2-hydroxyethyl)ammonium
propane sulfonate, Hampton Research) were added.
chMDA5ΔCARD-Q 24-mer dsRNA-AMPPNP was mixed with reservoir buffer (0.1 mM
Hepes pH 7.5, 11-12% PEG 3350, 2-4% sucrose) in a 2:1 ratio. Crystals grew in three days at
20 degree and were harvested in cryo-protecting solution (0.1 mM Hepes pH 7.5, 25% 3350,
10% ethylene glycol) before flash freezing with liquid nitrogen.
chMDA5ΔCARD-Q : 27-mer dsRNA-AMPPNP was mixed with reservoir buffer (0.1 mM
Hepes pH 7.5, 4-6% PEG 6000, 2-4% sucrose) in a 2:1 ratio. Crystals grew in three days at
20°C. The cover glasses of crystal-containing drops were transferred to a reservoir containing
cryo-protecting solution (0.1 mM Hepes pH 7.5, 10% PEG 6000, 25% ethylene glycol) 12
hours before harvesting and flash freezing with liquid nitrogen.
chLGP2: chLGP2, directly after size exclusion chromatography, was mixed with dsRNA in a
1:1 ratio and incubated for 30 minutes on ice. The complexes were concentrated with an
Amicon Ultra concentrator to around 10 mg/ml and then 2 mM ADP:AlF4 (adenosine-5′-
diphosphate: aluminium fluoride) and 2 mM MgCl2 were added. All chLGP2 crystals were
harvested in the reservoir buffer containing 25% ethylene glycol and flash frozen with liquid
nitrogen.
chLGP2 12-mer dsRNA-ADP:AlF4 complex was mixed with reservoir buffer (0.1 M Bis-
Tris propane pH 6.5, 0.05-0.1 M magnesium formate) in a 2:1 ratio. Crystals grew in three
days at 4°C.
chLGP2 5′ monophosphate 10-mer dsRNA-ADP:AlF4 complex was mixed with reservoir
buffer (0.1 M tri-sodium citrate dihydrate pH 6.1, 0.04-0.08 M ammonium dihydrogen
phosphate) in a 2:1 ratio. Crystals grew in one week at 4°C.
chLGP2 5′ triphosphate 10-mer dsRNA-ADP:AlF4 complex was mixed with reservoir
buffer (0.1 M Mg formate, 20% PEG3350M) in a 2:1 ratio. Crystals grew in one week at 4°C.
chLGP2 5′ triphosphate, 3′-GG overhang hairpin RNA-ADP:AlF4 complex was mixed
with reservoir buffer (0.1 M citric acid pH 5.0, 0.8 M ammonium sulphate) in a 1:1 ratio.
Crystals grew in one day at 4°C from a sitting drop 96 well plate.
Crystallography
Diffraction data were collected at 100 K on beamlines ID23-1 or ID29 equipped with
a Pilatus 6M detector or ID23-2 equipped with a Pilatus 2M-F (DECTRIS), at the European
Synchrotron Radiation Facility (ESRF), Grenoble, France. Data were processed with the
XDS suite and scaled with XSCALE (Kabsch, 2010). All further analysis was done with the
CCP4 suite (Winn et al., 2011) and refinement performed with REFMAC5 (Murshudov,
1997) or PHENIX (Adams et al., 2002). Models were built using COOT (Emsley and
Cowtan, 2004), structure validation by MOLPROBITY (Chen et al., 2010) and structure
figures drawn with PYMOL (DeLano, 2002). Buried surface are calculations performed with
PISA (http://www.ebi.ac.uk/pdbe/prot_int/pistart.html) (Krissinel and Henrick, 2007).
Crystallographic and refinement statistics are given in Tables 1 and 2.
The chMDA5 structure was initially determined by molecular replacement with
PHASER (McCoy et al., 2007) using the hMDA5 structure (PDB 4GL2). The chLGP2
structure was initially determined with PHASER using the hLGP2 CTD (PDB: 2W4R) and
RIG-I helicase sub-domains as search models.
Crystals of MDA5 with 10- and 24-mer dsRNAs were of highly variable quality,
requiring testing of many to obtain usable data. For the 10-mer complex, several related
space-groups were obtained with two (P21 or P212121) or four (large P21) 1:1 complexes in
the asymmetric unit. Both 10- and 24-mer crystals were generally twinned (probably lattice
translocation disorder), this being manifested by weak but significant residual extra density
after refinement that could be explained by the same model but referred to a different origin.
One 10-mer crystal in the P21 form was not twinned and gave a cleaner map and lower R-
factors (Table 2).
RNA preparation
The palindromic 10 bp dsRNA with 5′ mono-phosphate or 5′ tri-phosphate (5′-
pGGUACGUACC-3′ or 5′-pppGGUACGUACC-3′), 12 bp dsRNA with 5′ mono-phosphate
(5′-pGGUAGCGCUACC-3′), 24 bp dsRNA with 5′ mono-phosphate (5′-
pGGGACGUCAUGCGCAUGACGUCCC-3′) and 27 bp dsRNA with 5′ monophosphate (5′-
pGGGCACGUGCAGGACCUGCACGUGCCC-3′) and 5′ triphosphate 3′-GG overhang 26-
mer hairpin RNA (5′-pppGGAGCGUGCCGUACGGCACGCUCCGG-3′) were prepared by
in vitro T7 transcription. Five times excess of GMP over GTP was added to the reaction for
the 5′ mono-phosphate RNAs. RNA was purified by excising the band from denaturing urea-
PAGE. A summary of RNAs used is shown in Figure S4.
Electron microscopy
Φ6 RNA (Thermo Scientific) is a mixture of 2948 bp (S segment), 4063 bp (M), and
6374 bp (L) dsRNAs. To form complexes, full-length chMDA5 or chLGP2 and Φ6
bacteriophage dsRNA were incubated during 5 min at 20°C in a buffer containing 20 mM
Hepes pH 7.5, 150 mM NaCl, 2 mM ATP and 2 mM MgCl2. Different protein:RNA ratios
were used with a 1:1 ratio of MDA5/LGP2:dsRNA corresponding to one MDA5/LGP2
molecule for 15 bp of dsRNA. Where indicated, ATP was replaced by ADP, ADP:AlF4 or no
NTP analogues in order to test their influence.
For each condition tested, 4 μl of sample was applied to the clear side of carbon on a
carbon-mica interface and stained with 1% (wt/vol) uranyl acetate. Images were recorded
under low-dose conditions with a JEOL 1200 EX II microscope at 100 kV with a nominal
magnification of 15,000×.
Cryo-EM grids were prepared by mixing 4 μl of MDA5 or LGP2 at 5 μM with
dsRNA in a 1:1 ratio. The sample was applied onto a glow-discharged quantifoil grid 400
mesh 3.5/1 (Quantifoil Micro Tools GmbH, Germany), the excess solution was blotted for 2 s
with a Vitrobot (FEI) and the grid frozen in liquid ethane. Data collection was performed on a
FEI Polara microscope operated at 300 kV. Fifty cryoEM micrographs were manually
collected on a K2 summit electron direct detector (Gatan) at a magnification of 20,000×,
giving a pixel size of 0.97 Å. Movies of 6 frames were collected with a total exposure time of
6 s and a total dose of 30e-/Å
2. The contrast transfer function (CTF) for each micrograph was
determined with CTFFIND3 (Mindell and Grigorieff, 2003) and showed defocuses between
2.2 and 3.8 µm. A total of 7185 overlapping segments of MDA5 and 3153 overlapping
segments of LGP2 (400 pixels long) were picked using EMAN boxer (Ludtke et al., 1999)
with a shift of 64 pixels between adjacent segments. Corresponding segments were suggested
to 2D classification in Relion1.3 (Scheres, 2012). Power spectra were calculated from
masked 2D class averages.
Fluorescence polarisation anisotropy
dsRNAs used for anisotropy experiments were formed by annealing the following separate
strands for 1 min at 95 °C and cooling down on ice in a buffer containing 20 mM Hepes, 2
mM EDTA at pH 6.8 (see also Figure S5 for RNA sequences):
12-mer dsRNA : 5′-FAMCAUGUGGAGCCC-3′ and 5′-OHGGGCUCCACAUG-3′,
12-mer dsRNAppp : 5′-FAMCAUGUGGAGCCC-3′ and 5′-pppGGGCUCCACAUG-3,
3′ overhang dsRNA : 5′-OHGGCCAGUGCGAA-3′ and 5′-FAMCGCACUGGCCAA-3′,
5′ overhang dsRNA : 5′-OHAACCGGUCACGC3′ and 5′-OHAAGCGUGACCGGFAM-3′.
Anisotropy measurements were performed at room temperature using a multimodular
fluorometer (Quantamaster QM4CW; Manufacturer: Photon Technology International).
RNA concentration was adjusted to 2.5 nM (12-mer 5′ppp-dsRNA) or 5 nM (12-mer 5′OH-
dsRNA, 3′ 2 nt overhang dsRNA and 5′ 2 nt overhang dsRNA for LGP2 measurements), 13
or 50 nM (12-mer 5′OH-dsRNA for MDA5 measurements) by mixing in the cuvette with
buffer (20 mM Hepes pH 7.5 150 mM NaCl, 200 µM TCEP, 4 mM MgCl2). Protein was
titrated into the cuvette and incubated for 5 minutes before measurements. The total volume
of titrated protein was less than 10% of the final volume. Anisotropy measurements were
made with excitation wavelength 495 nm and emission wavelength 515 nm for 100 seconds.
Experiments were performed more than three times per system. Binding data was analysed by
Graph Pad Prism software and fitted with the following equation (eq. 1).
𝑦 =(R+𝑃𝑛+𝐾𝑑𝑛)−√(𝑅+𝑃𝑛+𝑘𝑑𝑛)2−4𝑅𝑃𝑛
2𝑅× 𝑏 + (𝑚 − 𝑏) (eq. 1)
The experimental anisotropy (y), Initial anisotropy (b), the maximum anisotropy (m), the
RNA concentration (R), and the protein concentration (P), equilibrium binding constant (Kd)
and hill coefficient (n).Rig-I measurement curves are fitted with n=1 and the value of n are
written in Figure S4. The error bars represent the S.D. value of at least three individual
experiments.
ATPase activity assays
ATPase reactions were monitored using a Malachite green assay kit (Bioassays) over
0.5-30 min time courses. All proteins were pre-incubated with 4-fold molar excess of dsRNA
for 10 minutes at 28 °C in ATP hydrolysis buffer (20 mM Hepes pH7.5, 100 mM NaCl, 1%
glycerol, 2 mM DTT, 4 mM MgCl2). The reaction was initiated by adding 2 mM ATP. For
determination of kinetic parameters, the ATP concentration was varied from 0.125 to 10 mM.
The reactions were quenched at 5 time points between 30 seconds to 4 minutes. The 10 µl
reaction aliquots were quenched by mixing with 10 µl of quenching buffer (20 mM Hepes pH
7.5, 100 mM NaCl, 1% glycerol, 2 mM DTT, 100 mM EDTA). 80 µl of 5 times diluted
Malachite green solution was added and developed for 30 minutes at room temperature. The
absorbance at 622 nm was measured with a plate reader. For each ATP concentration, the
initial velocity was plotted and Km and kcat calculated using the Michaelis–Menten equation
(eq. 2) in Graph Pad software Prism.
Y=Et*kcat*X/(Km+X) (eq. 2)
X is the substrate concentration, Y is enzyme velocity and Et is the concentration of enzyme.
Cellular assays.
Myc-hMDA5 and Flag-hRIG-I constructs in pEF-BOS vector have been described
previously (Kowalinski et al., 2011; Louber et al., 2015; Louber et al., 2014) and chMDA5,
chLGP2, hLGP2 tagged at the N terminus with Flag peptide were sub-cloned into pCDNA3
(chicken) and pEF-BOS (human) vector as detailed elsewhere (Louber et al., 2015; Louber et
al., 2014).
Chicken DF1 (Himly et al., 1998) and human Huh7.5 (Sumpter et al., 2005) cells
were maintained in Dulbecco’s Minimum Essential Medium culture medium supplemented
with 10% foetal calf serum, 10 mM HEPES, 2 mM L-glutamine, 10 mg/ml gentamycin and
1% non-essential amino acids for Huh7.5 cells at 37°C and 5% CO2. Being of chicken origin,
DF1 cells naturally lack endogenous RIG-I (Barber et al., 2010), but express functional
endogenous MDA5, LGP2 and type I IFN system (Karpala et al., 2011; Liniger et al., 2012).
Huh7.5 cell line is defective in MDA5 and IFN receptor expression and expresses inactive
T55I RIG-I (Binder et al., 2011; Eguchi et al., 2000; Keskinen et al., 1999; Li et al., 2005;
Sumpter et al., 2005), a phenotype verified experimentally by the inability of these cells to
respond to both RIG-I and MDA5 RNA agonists (Figure S7 and our previous data (Louber et
al., 2015)).
Protein expression was analysed after lysis of DF1 and Huh7.5 cells (6-well plate,
6x105 cells/well) transfected the day before with 2.5 µg DNA using TransIT®-LT1
Transfection Reagent (Mirus Bio) (7.5 µl/well), separation by polyacrylamide gel
electrophoresis and visualisation by western immunoblotting using anti-Flag (1:1,000; M2;
Sigma, St. Louis, MO, USA), anti-C-Myc (1:50) 9E10, anti-GAPDH (1:2,000; Millipore,
Billerica, MA, USA) monoclonal antibodies
The human IFNβ promoter luciferase assay was performed essentially as described
(Louber et al., 2015; Louber et al., 2014). Briefly, Huh7.5 cells seeded one day before (96
wells, 2 x104 cells/well) were co-transfected with MDA5 and LGP2 expressing vectors (33
ng of DNA/well to which was added 17 ng of DNA/well of pHRL-tk-Renilla-Luc (Promega))
together with the reporter pβIFN-fl-lucter (King and Goodbourn, 1994) (50 ng of DNA/well),
using TransIT®-LT1 Transfection Reagent (Mirus Bio) (0.3 µl /well), followed 24 hours later
by transfection of either poly(I:C) (Amersham Biosciences), Φ6 dsRNA (Thermo Scientific)
or T7-transcribed 61-mer 5′ppp-dsRNA (Marq et al., 2011) in complex with
Oligofectamine™ Transfection Reagent (Invitrogen) (0.6 µl/well). The chicken IFNβ
promoter luciferase assay was performed following exactly the same procedure, but using
chicken adapted material, i.e. DF1 cells, pCDNA3 based expression vectors for MDA5 and
LGP2, pHRL-CMV-Renilla-Luc (Promega) instead of pHRL-tk-Renilla-Luc, the reporter
pGL3-P_chIFNβ-Luc (Liniger et al., 2012). For both human and chicken assay, control
experiments were included by transfecting the same amount of a neutral plasmid (pCG-duF)
(Cathomen et al., 1995). This plasmid was also used to complement transfected DNA so as to
reach total DNA amount of 100 ng per well. Data were expressed as mean +/-SD of
normalized luciferase activity. Statistical analysis (Student t test) was done on data from three
independent experiments, each done in independent triplicates.
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