The$Interferon$System$

Takashi$Fujita$ Ins5tute$for$Virus$Research$ Kyoto$University$ Days$aFer$inﬂuenza$virus$infec5on

Fields$Virology:pp1402$

Fever

$ $ Neutralizing$an5body$ Nasal$wash$

Interferon

Virus Neutralizing$an5body$ Serum

Days Infec5ous$Diseases

Pathogen Human$(Animal) Human$(Animal) Bacteria Parasites Viruses others Virions

Vaccinia Herpes Adeno Papilloma

Reo Sindbis Polio Inﬂuenza VIRAL$ENTRY outside cytoplasm nucleus

Fields$VIROLOGY VIRAL$EXIT nucleus cytoplasm outside

Fields$VIROLOGY Yasuichi$Nagano$ Yasuhiko$Kojima Interfering$Factor$(1954)$

Alick$Isaacs Jean$Lindenmann Interferon$(1957)$ VIRUS$INFECTION

VIRUS$INFECTION INTERFERON0 REPLICATION$$ OF$$ PICORNAVIRUS

dsRNA$ $(Replica5ve$Intermediate)

dsRNA$ $(Replica5ve$Form)

Fields$VIROLOGY ]IFN,$+EMCV$infec5on$ +IFN,$+EMCV$infec5on$

EMCV:&Non*enveloped,&+strand&RNA&virus& CELLS$INFECTED$$ WITH$$ RABIES$VIRUS

VIRION$OF$$RABIES$VIRUS REPLICATION$OF$RHABDOVIRUS

RdRP

dsRNA$

Transla5on

Fields$VIROLOGY 1000$U/ml$Interferon]β Vesicular$Stoma55s$Virus Vesicular$Stoma55s$Virus

An:viral&Eﬀect&of&Interferon How$Interferon$Works? Action of Interferon Interferon

JAK0 JAK0

STAT0 STAT0 STATSTAT0 0 IRF*90 An5viral$State

ISGs0IFN0genes0ISGs0

ISGs:&Interferon&S:mulated&Genes Viruses$

PENETRATION$

UNCOATING$

Antiviral Proteins$ TRANSCRIPTION$ PKR OAS Tetherin Etc.

TRANSLATION$

ASSEMBLY $

RELEASE OF PROGENY VIRUSES$ Antiviral State$

Tetherin/BST]2/CD137 How$Interferon$is$Produced? Production of Interferon

VIRUS$

Interferon

Viral&Replica:on& Viral&RNA& Viral0RNA0Sensors0

IRF63,0IRF670 NF6κB0

Interferon0Genes0 Regulatory&Sequences&& for&IFN&and&IFN*inducible&genes$

IFN Regulatory Factor ATF-2/c-jun (IRF) NF-κB IFN genes ATF/CREB PRDII PRDIII PRDI

-110 -100 -90 -80 -70 -60 IFN-β gene: AAAATGTAAATGACATAGGAAAACTGAAAGGGAGAAGTGAAAGTGGGAAATTCCTCTGAAT

IFN-α1 gene: GAGTGCATGAAGGAAAGCAAAAACAGAAATGGAAAGTGGCCCAGAA -100 -90 -80 -70 GAAAGTGAAAGTG TG

IFN-inducible genes 2’-5’AS : AGGAAA-CGAAACCA PKR : GGGAAAACGAAACAG

GBP-1 : ATGAAACTGAAAGTA

ISG15 : GGGAAACCGAAACTG

ANGAAANNGAAACT G Studies$in$late$1980’s ISRE Interferon&Regulatory&Factors&(IRFs)& DNA&Binding&Domain& N& C&

IRF*1&& Ac5vator,$Ubiquitous$ $$$$$$$$]]]Immunomodula5on,$$Tumor$suppressor]like$ IRF*2& Repressor,$Ubiquitous$ $$$$$$$$]]]$Immunomodula5on,$$Oncogenic$func5on$ IRF*4&(Pip/LSIRF)& Ac5vator,$Lymphoid$speciﬁc$ $$$$$$$]]]$Lymphocyte$development,$Oncogenic$func5on$ IRF*8&(ICSBP)& Repressor/Ac5vator,$Lymphoid$speciﬁc$ $$$$$$$]]]$Lymphocyte$development,$Tumor$suppressor]like$ IRF*9&(ISGF3γ)& Ac5vator$(DNA$binding$subunit$of$ISGF3),$Ubiquitous$ $$$$$$$]]]$IFN]signaling$ IRF*3& Ac5vator,$Ubiquitous$ $$$$$$$$]]]$Induc5on$of$IFN]α/β$genes$ IRF*7& Ac5vator,$Ubiquitous,$IFN]inducible$ $$$$$$$]]]$Induc5on$of$IFN]α/β$genes$ IRF*5& Ac5vator?,$IFN]inducible?$

IRF*6& Repressor?$ Studies$in$1990’s Ectopic&Expression&of&IRF*3&Augments&& IFN*α,&*β&Promoter&Ac:vity&

0 NDV: cont. IRF-3 cont. IRF-3 cont. IRF-3 cont. IRF-3 cont. IRF-3 p-55Luc p-125Luc pαLuc p-55C1BLuc p-55A2Luc (control) (IFN- β) (IFN- α) (PRDI) 8 (PRDII) 3 .. .

DNA&binding&Complex&Induced&by&Virus&Infec:on$ F5 L929 (HA-IRF-3)

. CBP CBP 3 α 3 α ーー IRF HA p300CBPp300Anitibodies + IRF p300CBPp300 + controlControl$ An5$IRF]3$ An5$HA$ An5$p300$ An5$CBP$ An5$p300+CBP α α α α α controlControl$ αAn5$IRF]3$ αAn5$p300$ αAn5$CBP$ αAn5$p300+CBP VA-IRF NDV ISGF3

VA-IRF VA-IRF ISGF3

F5 (HA-IRF-3) cells HEC-1 (IFNR-) Time&course&of&polyI:C*induced&Nuclear&Transloca:on&& of&Endogenous&IRF*3&and&NF*κB&in&HeLa&Cells&

IRF-3

PolyI:C 0' 10' 20' 30' 60'

NF-κB (p50) IFN gene regulation by IRF-3$

vector$IRF]3$ ]$ +$ ]$ +$ :NDV:$ ]$ +$

Western$ dimer$ IRF]3$ IRF]3$ 32P$ monomer$

mock$ NDV$ Na5ve/Western$

Signal

p300/CBP

IFN genes IRF-3 Crystal$Strucuture$of$IRF]3$

Graduate School of Pharmaceutical Sciences Hokkaido University & CREST/JST, Kawaguchi Kiyohiro Takahasi Nobuo N. Suzuki Masataka Horiuchi Hiroaki Terasawa & Fuyuhiko Inagaki Potential phosphorylation sites in IRF-3 regulatory domain� IRF-3 N C

WT:$ LVEMARVGGASSLENTVDLHISNSHPLSLTSDQYKAYLQDLVEGMDFQGPGES 374& 385&386& 427&

386A:$LVEMARVGGASALENTVDLHISNSHPLSLTSDQYKAYLQDLVEGMDFQGPGES →No$ac5va5on$ P 5D:$ LVEMARVGGASSLENTVDLHIDNDHPLDLDDDQYKAYLQDLVEGMDFQGPGES →Cons5tu5ve$ac5ve$

5A:$ LVEMARVGGASSLENTVDLHIANAHPLALAADQYKAYLQDLVEGMDFQGPGES →Virus]inducible$ac5va5on$ Puriﬁca:on&of&&recombinant&IRF*3&from&E.Coli&

(A)$ IPTG� (B)$ +GroE� +Trx� S: 200� I$: M� S� I� S� I� S� I� S� I� 116� 116�

66� GroEL� 66� 42� His - ΔIRF3 � 42� 30� 175-427� His]ΔIRF3$ 30� 17� GroES� $$$$$$175]427$ CBB Trx� 5� 6� 7� 8� 17� CBB αHis His - ΔIRF3 � 175-427� IRF]3$Regulatory$Domain Smad$Regulatory$Domain

Quasi&dimer&in&the&crystal&

R211 K360

R213

S386 G212 R361 L387 SRR-loop HLR-pocket S385 F209 W345 Ser386

L299 IRF*3&

Smad&

Ac:va:on&of&IRF*3&and&Smad& at&Low&Resolu:on& TBK]1,$IKK]i(ε)!p]IRF]3,$p]IRF]7$ $ IKK(α, β, γ)!p]IκB$ Regula5on$of$An5viral$Innate$ Immunity$by$RIG]I]Like$Receptors$

Takashi$Fujita$ Ins5tute$for$Virus$Research$ Kyoto$University$ Virus- and IFN-induced Signals

Virus dsRNA IFNs

IFN Receptor

RIG*I

cytoplasm

IFN-α genes IRF*3 IRF*3 IFN-β gene IFN inducible genes nuclear Antivirus activities Helicase Domain: Conformational Change ? Signaling Domain RNA Binding Domain Conserved helicase motifs CARD 1 a 925 RIG-I: * T55I K270 : ATP Binding

23 35 41 % % % 1025 1 MDA5:

31 % 1 LGP2: 678 RNA Binding Domain

RIG*I*Like&Receptor&(RLR) [kDa] 116

input polyI:C-pulldown

competitor: polyI:C polyA:U polyA dsDNA

RIG-I

RIG]I$Binds$to$dsRNA$but$not$ssRNA$or$dsDNA$

Ac5va5on$of$RLRExposure$of$CARD

CTD CTD 3 OFF 3 OFF 1 2 1 2 CARD CARD ON

CARD Analysis&of&RIG*I*/*&and&MDA5*/*&mice& &

Kato, H., Sato, S., Yoneyama, M., Yamamoto, M., Uematsu, S., Matsui, K., Tsujimura, T., Takeda, K., Fujita, T., Takeuchi, O., and Akira, S. Immunity 23, 19-28, (2005)

Kato, H., Takeuchi, O., Sato, S., Yoneyama, M., Yamamoto, M., Matsui, K., Uematsu, S., Jung, A., Kawai, T., Ishii, K. J., Yamaguchi, O., Otsu, K., Tsujimura, T., Koh, C-S., Reis e Sousa, C., Matsuura, Y., Fujita, T. and Akira, S. Nature 441, 101-105 (2006) IFN$gene$induc5on$by$MEFs$from$KO$mice$ JEV$challenge$to$KO$mice$ EMCV$challenge$to$KO$mice$ Diﬀeren5al$Func5ons$of$RIG]I$Family$ Helicases$

NDV,$Sendai$virus,$ RIG]I$ Posi5ve$ Inﬂuenza$virus,$VSV,$JEV,$ Regulator$ WN,$etc.$$

MDA5$ Posi5ve$ Picorna$viruses,$WN,$ Regulator$ etc.$$

LGP2$ Posi5ve$Regulator$ EMCV,$VSV$ A B WT kbp 10 RIG-I-/- MDA5-/- 7.7 8 4.2 RIG-I -/- MDA5 -/- * Not detected 1.3 6 (ng/ml) β 4

0.3 IFN- 2 * * * * * * * * * * * * 0 M 0 1 5 10 30 60 M mock 0 60 180 RNaseIII treatment (min) RNaseIII treatment (min)

C D WT RIG-I-/- MDA5-/- 100 RNaseA Bal31 hydrolysis 80 8 WT 3 60 MDA5-/- 1.6 (ng/ml) 6 RIG-I -/- β 2 40

IFN- 4 0.8

Relative induction (%) Relativeinduction 20 1 2 0 0 1 5 10 30 60 0 0 0 RNaseIII treatment (min)

Kato,$H.$et$al,$JEM,$2008$ RNA$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$structure$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$RLR$

pp$ Short$poly$I:C$ pp$ RIG]I$ $$$$(~300$bp)$ $ $ $ pp$ MDA5$ Long$poly$I:C$ pp$ $$$$(>$4$kbp)$ $ $ $ RIG]I$ in$vitro$T7$transcript$ ppp$ $$(with$copyback)$ $ $ $ $ $ $ ppp$ (polyU,$UC)n$ RIG]I HCV$RNA$ $ $ $ $ $ $$p$ RNaseL$cleavage$product$ p$ RIG]I $$(RNA$of$host/virus)$ (MDA5)$ $ $ Viral$genomic$RNA$ $ (with$panhandle$structure)$ ppp$ RIG]I $ $ $ $ RNA$of$DI$par5cle$ $ $$$$$$$(copyback)$ ppp$ Uniden5ﬁed$ $ $ $ $ High$molecular$weight$RNA$ MDA5 $$$$$(RNA$web)$ $ ? $ $ Helicase Domain: Conformational Change ? Signaling Domain RNA Binding Domain Conserved helicase motifs CARD Repression 1 a Domain 925 RIG-I: * T55I K270 : ATP Binding

23 35 41 % % % 1025 1 MDA5:

31 % 1 LGP2: 678 RNA Binding Domain Repression RIG-I Like Receptors (RLR) Domain CTD 3 1 2 RIG*I CARD

dsRNA

CARD CARD

RIG]I$func5ons$as$double]stranded$RNA$sensor/signaling$switch Intracellular$localiza5on$of$RIG]I$$ and$an5viral$innate$immunity

Viral$Transcrip5on RIG6I Viral$RNA$ Viral$Transla5on $ (dsRNA) Viral$Protein

Virion$Packaging REPLICATION$ $OF$$ INFLUENZA$VIRUS 1

2 6

7$Nucleocapsid 4 3

Fields$VIROLOGY Non]structural$Protein$1:$$ $$$$$$$$$$$$$$$$$$$$$$$$$Strong$Antagonist$of$Interferon$System 5’ ppp- 3’

“Panhandle”$Structure$ Natural0Ligand0for0RIG6I

Genomic$RNA$of$Inﬂuenza$A$virus IAV PR8 IAV deltaNS1 0h 3h 6h 9h 12h 0h 3h 6h 9h 12h

IRF-3

RIG-I

PKR

phospho PKR (Thr446) phospho eIF2α (Ser51)

actin RIG-I Flu NP TIAR RIG-I Flu NP TIAR

0h 0h

3h 3h

6h 6h

9h 9h

12h 12h

IAV PR8 IAV ΔNS1 Stress$Signals$ (Virus$Infec5on,$heat$shock$etc.)$

PKR$(dsRNA)$ GCN2$(aa.$Starva5on)$ HRI$(oxida5on)$ PERK$(ER$stress)$

eIF2α$Phosphoryla5on$(Ser51)$

RLR) TIAR& Forma5on$of$ TIA1& Stress$Granules$ eIF3$ G3BP& HuR$ PABP$ RIG]I G3BP DAPI merge mock

IAV$ IFN ⊿NS1$

NaAsO2 NO$IFN

Oxida:ve&Stress&Induces&SG&but¬&Interferon vRNA/FISH& NP/IF& Merge&

Mock&

IAV& ΔNS1&

Virus]induced$SG$contains$viral$RNA$$$$$(an5$virus$SG) Colocaliza5on$of$MDA5,$SG$markers$and$viral$dsRNA

EMCV MOCK EMCV MOCK MDA5 MDA5 G3BP1 TIAR PI PI DAPI DAPI MERGE MERGE ppp NUCLEUS Inﬂuenza$genomic$RNA

NS1 PKR& CYTOPLASM

RLR) TIAR$ Forma5on$of$ TIA1$ Stress$Granules$ eIF3$ G3BP$ HuR$ PABP$ RLR)ac,va,on) eIF2α$Phosphoryla5on) $(Ser51)$ Virus&infec:on&and&& & Stress)Granule& Stable Transient Alternate By)viral)infec,on) Diﬀerent)Paterns)of)SG)forma,on)) Alternate Transient Stable A NDV B IAV C D E IAV∆NS1 EMCV SINV **& 100 120 **& 100 100 *&

100 **& 80 80 **& 80 80 60 60 60 60 40 40 40 40

SG Pattern (%) SG Pattern (%) SG Pattern 20 (%) SG Pattern 20 (%) SG Pattern 20 Cells with Cells Distinct 20 with Cells Distinct with Cells Distinct with Cells Distinct N.D. N.D. N.D. N.D. N.D. N.D. N.D. N.D. N.D. N.D. 0 0 0 0 Adeno5∆E1A Adeno5WT F PolioV G SeV H VSV I J 120 120 120 100 120

**& **& **& *& 100 100 100 80 *& 100 80 80 80 80 60 60 60 60 60 40 40 40 40 40

SG Pattern (%) SG Pattern SG Pattern (%) SG Pattern SG Pattern (%) SG Pattern (%) SG Pattern 20 Cells with Cells Distinct Cells with Cells Distinct Cells with Cells Distinct 20 with Cells Distinct 20 20 20 N.D. N.D. N.D. N.D. N.D. N.D. N.D. N.D. 0 0 0 0 0

K TMEV **& Stable formation Transient formation Alternate formation (n time) No formation SG Pattern (%) SG Pattern Cells with Cells Distinct Diﬀerent)Paterns)of)SG)forma,on)) By)viral)infec,on) SG&Induc:on&and&Viral&Replica:on& & & Picornaviridae GFP*G3BP

Minutes$aFer$EMCV$infec5on EGFP]G3BP$

Q325E& cleavage)resistant A& B&

Minutes$aFer$EMCV$infec5on GFP*G3BP&Q325E HeLa

95 Endogenous 72 G3BP1

EMCV 55 MW G3BP1 cp 43 (kDa) 0 2 4 6 8 10 p.h.i IB: α-G3BP1 Mock N.C. 34 116 97 EGFP-G3BP1 (≈ 96kDa) IB: α-GFP 26 EGFP-G3BP1 cp (≈ 80 kDa) 55 IB: α-actin IB: α-actin 45 β-actin 43 Lane 1 2 3 1 2 3 4 5 6 7 8

G/G3BP1G/G3BP1

EMCV

WT Q325E MW

Mock 0 2 4 6 8 10 p.h.i

(kDa) N.C. FLAG-G3BP1 66 IB: α-FLAG Q325E MW" HeLa WT HeLa pLeader pLeader pEmpty pEmpty (kDa) p3C EMCV p3C EMCV IB: α-actin 45 β-actin EGFP-G3BP1 95 IB: α-GFP 1 2 3 4 5 6 7 8 72 G3BP1 cp 55 55 β-actin IB: α-actin 43 Lane 1 2 3 4 5 6

G3BP$is$cleaved$by$$ G3BP$is$cleaved$by$$ EMCV$infec5on 3C$protease A B G3BP1 DAPI MERGE siControl siControl

IB: α-G3BP1 EMCV RNA EMCV IB: α-actin siG3BP1#1+#2 siG3BP1#1+#2 " " ] 6 ] 6 [x10 [x10 EMCV RNA EMCV Virus titer (PFU/mL) titer Virus Virus titer (PFU/mL) titer Virus

MOI: MOI:

G3BP$Q325E$inhibits$$ G3BP$knockdown$enhances$$ EMCV$replica5on EMCV$replica5on A B C HeLa

(EMCV)

EMCV EMCV

**&

**& *& mRNA (Relative) mRNA **& *& β *& IFN- IL-6 mRNA (Relative) IL-6 mRNA

CXCL10 mRNA (Relative) CXCL10 mRNA *& *& *&

Time after infection (hr) Time after infection (hr) Time after infection (hr) D E

EMCV &

IAV∆NS1 MW IAVΔNS1 & (kDa) N.C. 0 4 8 12 16 p.h.i & 116 EGFP-G3BP1 & IB: α-GFP 97 *& mRNA (Relative) mRNA

&β IB: α-actin 45 β-actin *& &IFN- 1 2 3 4 5 6 RANTES mRNA (Relative) RANTES mRNA & &&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&&Inﬂuenza:&Control Time after infection (hr) Time after infection (hr) G3BP$Q325E$enhanced$IFN$and$cytokine$gene$ac5va5on$ Viral&Inhibi:on&of&SG&Forma:on

Inﬂuenza$A$virus:$NS1$ West$Nile,$Dengue$virus:$targets$TIA/TIAR$ Polio$virus,$EMCV:$3C$protease$ Sendai$virus:$C$protein,$trailer$RNA$ Measles$virus:$C$protein$ Semliki$Forest$Virus:$unknown$ Junin$virus:$nucleoprotein,$glycoprotein$precursor$ Rota$virus:$NSP3$ Vaccinia$virus:$E3L$ $ $ Viral&Inhibitors&of&RLR&Signaling

HCV$NS3/4A Inﬂuenza$NS1 SG PKR

dsRNA RLR IPS]1/MAVS/VISA/Cardif

G3BP TBK]1 SFTSV$NS

EMCV$3C$ TMEV$Leader Polio$V$3C IRF]3 $ $ $ $ $ $ $ $ $ Mitochondrion$and$An5viral$ $ $ $ Responses$ $ $ $ $ $ $ $ $ $$$$$$$$$Onoguchi$K.$et$al.,$PLoS$Pathogens$2010 Redistribu5on$of$IPS]1$by$Sendai$Virus$infec5on Mitochondrial&redistribu:on&is&induced&by&diﬀerent&viruses Mi LIVE

MOCK +poly$I:C$ ]]Confocal]] ]]Confocal]] ]]High$resolu5on]] IPS*1 IPS*1 IPS*1 Mitofusin$1

Re]distribu5on$of$IPS]1$on$mitochondrion MFN1&is&necessary&for&IPS*1&redistribu:on&(NDV) MFN 1 positively regulates IFN production induced by virus or 5’pppRNA

cytoplasmic Critical role of MFN1 but not MFN2 in IFN gene activation (KO MEFs: obtained from David Chan’s lab)

Mi CTD Summary 3 1 2 CARD mitochondrion

Mitofusin01

CARD IPS]1 PKR avSG TRAFs TBK− NFκB IRFs CARD CARD

Inﬂammatory$cytokine$genes Type$I$and$III$interferon$genes Mitochondrion

Energy$Produc5on

Regula5on$of$Cell$Death

An5viral$Immunity Plaworm$for$interac5ons$between$signaling$adaptors

Regula5on$of$IPS]1$distribu5on$by$fusion/ﬁssion University&of&Wisconsin*Madison,&USA& Palmenberg,$A$

Kyoto&University& Ins5tute$for$Virus$Research$ Ng,$C]S$ Onomoto,$K$ Onoguchi,$K$ Yoneyama,$M$ Go,$S$ Wakimoto,$M$ Kato,$H$