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: cusack@embl.fr

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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