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$ $$$$$$$]]]$Lymphocyte$development,$Oncogenic$func5on$ IRF*8&(ICSBP)& Repressor/Ac5vator,$Lymphoid$specific$ $$$$$$$]]]$Lymphocyte$development,$Tumor$suppressor]like$ IRF*9&(ISGF3γ)& Ac5vator$(DNA$binding$subunit$of$ISGF3),$Ubiquitous$ $$$$$$$]]]$IFN]signaling$ IRF*3& Ac5vator,$Ubiquitous$ $$$$$$$$]]]$Induc5on$of$IFN]α/β$genes$ IRF*7& Ac5vator,$Ubiquitous,$IFN]inducible$ $$$$$$$]]]$Induc5on$of$IFN]α/β$genes$ IRF*5& Ac5vator?,$IFN]inducible?$ IRF*6& Repressor?$ Studies$in$1990’s Ectopic&Expression&of&IRF*3&Augments&& IFN*α,&*β&Promoter&Ac:vity& 80 60 40 20 0 NDV: cont. IRF-3 cont. IRF-3 cont. IRF-3 cont. IRF-3 cont. IRF-3 p-55Luc p-125Luc pαLuc p-55C1BLuc p-55A2Luc (control) (IFN- β) (IFN- α) (PRDI) 8 (PRDII) 3 . L929 DNA&binding&Complex&Induced&by&Virus&Infec:on . (HA-IRF-3) F5 VA-IRF ISGF3 controlControl$ αAn5$IRF]3$IRFー3 αAn5$HA$HA αAn5$p300$p300 αAn5$CBP$CBP An5$p300+CBPαp300 + αCBP Anitibodies $ NDV F5 (HA-IRF-3) cells VA-IRF ISGF3 control Control$ αAn5$IRF]3$IRFー3 αAn5$p300$p300 αAn5$CBP$CBP An5$p300+CBPαp300 + αCBP HEC-1 (IFNR VA-IRF - ) Time&course&of&polyI:C*induced&Nuclear&Transloca:on&& of&Endogenous&IRF*3&and&NF*κB&in&HeLa&Cells& IRF-3 PolyI:C 0' 10' 20' 30' 60' NF-κB (p50) IFN gene regulation by IRF-3$ vector$IRF]3$ ]$ +$ ]$ +$ :NDV:$ ]$ +$ Western$ dimer$ IRF]3$ IRF]3$ 32P$ monomer$ mock$ NDV$ Na5ve/Western$ Signal p300/CBP IFN genes IRF-3 Crystal$Strucuture$of$IRF]3$ Graduate School of Pharmaceutical Sciences Hokkaido University & CREST/JST, Kawaguchi Kiyohiro Takahasi Nobuo N. Suzuki Masataka Horiuchi Hiroaki Terasawa & Fuyuhiko Inagaki Potential phosphorylation sites in IRF-3 regulatory domain� IRF-3 N C WT:$ LVEMARVGGASSLENTVDLHISNSHPLSLTSDQYKAYLQDLVEGMDFQGPGES 374& 385&386& 427& 386A:$LVEMARVGGASALENTVDLHISNSHPLSLTSDQYKAYLQDLVEGMDFQGPGES →No$ac5va5on$ P 5D:$ LVEMARVGGASSLENTVDLHIDNDHPLDLDDDQYKAYLQDLVEGMDFQGPGES →Cons5tu5ve$ac5ve$ 5A:$ LVEMARVGGASSLENTVDLHIANAHPLALAADQYKAYLQDLVEGMDFQGPGES →Virus]inducible$ac5va5on$ Purifica:on&of&&recombinant&IRF*3&from&E.Coli& (A)$ IPTG� (B)$ +GroE� +Trx� S: 200� I$: M� S� I� S� I� S� I� S� I� 116� 116� 66� GroEL� 66� 42� His - ΔIRF3 � 42� 30� 175-427� His]ΔIRF3$ 30� 17� GroES� $$$$$$175]427$ CBB Trx� 5� 6� 7� 8� 17� CBB αHis His - ΔIRF3 � 175-427� IRF]3$Regulatory$Domain Smad$Regulatory$Domain Quasi&dimer&in&the&crystal& R211 K360 R213 S386 G212 R361 L387 SRR-loop HLR-pocket S385 F209 W345 Ser386 L299 IRF*3& Smad& Ac:va:on&of&IRF*3&and&Smad& at&Low&Resolu:on& TBK]1,$IKK]i(ε)!p]IRF]3,$p]IRF]7$ $ IKK(α, β, γ)!p]IκB$ Regula5on$of$An5viral$Innate$ Immunity$by$RIG]I]Like$Receptors$ Takashi$Fujita$ Ins5tute$for$Virus$Research$ Kyoto$University$ Virus- and IFN-induced Signals Virus dsRNA IFNs IFN Receptor RIG*I cytoplasm IFN-α genes IRF*3 IRF*3 IFN-β gene IFN inducible genes nuclear Antivirus activities Helicase Domain: Conformational Change ? Signaling Domain RNA Binding Domain Conserved helicase motifs CARD 1 a 925 RIG-I: * T55I K270 : ATP Binding 23 35 41 % % % 1025 1 MDA5: 31 % 1 678 LGP2: RNA Binding Domain RIG*I*Like&Receptor&(RLR) [kDa] 116 input polyI:C-pulldown competitor: polyI:C polyA:U polyA dsDNA RIG-I RIG]I$Binds$to$dsRNA$but$not$ssRNA$or$dsDNA$ Ac5va5on$of$RLRExposure$of$CARD CTD CTD 3 OFF 3 OFF 1 2 1 2 CARD CARD ON CARD Analysis&of&RIG*I*/*&and&MDA5*/*&mice& & Kato, H., Sato, S., Yoneyama, M., Yamamoto, M., Uematsu, S., Matsui, K., Tsujimura, T., Takeda, K., Fujita, T., Takeuchi, O., and Akira, S. Immunity 23, 19-28, (2005) Kato, H., Takeuchi, O., Sato, S., Yoneyama, M., Yamamoto, M., Matsui, K., Uematsu, S., Jung, A., Kawai, T., Ishii, K. J., Yamaguchi, O., Otsu, K., Tsujimura, T., Koh, C-S., Reis e Sousa, C., Matsuura, Y., Fujita, T. and Akira, S. Nature 441, 101-105 (2006) IFN$gene$induc5on$by$MEFs$from$KO$mice$ JEV$challenge$to$KO$mice$ EMCV$challenge$to$KO$mice$ Differen5al$Func5ons$of$RIG]I$Family$ Helicases$ NDV,$Sendai$virus,$ RIG]I$ Posi5ve$ Influenza$virus,$VSV,$JEV,$ Regulator$ WN,$etc.$$ MDA5$ Posi5ve$ Picorna$viruses,$WN,$ Regulator$ etc.$$ LGP2$ Posi5ve$Regulator$ EMCV,$VSV$ A B WT kbp 10 RIG-I-/- MDA5-/- 7.7 8 4.2 RIG-I -/- MDA5 -/- * Not detected 1.3 6 (ng/ml) β 4 0.3 IFN- 2 * * * * * * * * * * * * 0 M 0 1 5 10 30 60 M mock 0 60 180 RNaseIII treatment (min) RNaseIII treatment (min) C D WT RIG-I-/- MDA5-/- 100 RNaseA Bal31 hydrolysis 80 8 WT 3 60 MDA5-/- 1.6 (ng/ml) 6 RIG-I -/- β 2 40 IFN- 4 0.8 Relative induction (%) Relative induction 20 1 2 0 0 1 5 10 30 60 0 0 0 RNaseIII treatment (min) Kato,$H.$et$al,$JEM,$2008$ RNA$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$structure$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$$RLR$ pp$ Short$poly$I:C$ pp$ RIG]I$ $$$$(~300$bp)$ $ $ $ pp$ MDA5$ Long$poly$I:C$ pp$ $$$$(>$4$kbp)$ $ $ $ RIG]I$ in$vitro$T7$transcript$ ppp$ $$(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$par5cle$ $ $$$$$$$(copyback)$ ppp$ Uniden5fied$ $ $ $ $ High$molecular$weight$RNA$ MDA5 $$$$$(RNA$web)$ $ ? $ $ Helicase Domain: Conformational Change ? Signaling Domain RNA Binding Domain Conserved helicase motifs CARD Repression 1 a Domain 925 RIG-I: * T55I K270 : ATP Binding 23 35 41 % % % 1025 1 MDA5: 31 % 1 678 LGP2: RNA Binding Domain Repression RIG-I Like Receptors (RLR) Domain CTD 3 1 2 RIG*I CARD dsRNA CARD CARD RIG]I$func5ons$as$double]stranded$RNA$sensor/signaling$switch Intracellular$localiza5on$of$RIG]I$$ and$an5viral$innate$immunity Viral$Transcrip5on RIG6I Viral$RNA$ Viral$Transla5on $ (dsRNA) Viral$Protein Virion$Packaging REPLICATION$ $OF$$ INFLUENZA$VIRUS 1 8 2 6 5 7$Nucleocapsid 4 3 Fields$VIROLOGY Non]structural$Protein$1:$$ $$$$$$$$$$$$$$$$$$$$$$$$$Strong$Antagonist$of$Interferon$System 5’ ppp- 3’ “Panhandle”$Structure$ Natural0Ligand0for0RIG6I Genomic$RNA$of$Influenza$A$virus IAV PR8 IAV deltaNS1 0h 3h 6h 9h 12h 0h 3h 6h 9h 12h IRF-3 RIG-I PKR phospho PKR (Thr446) phospho eIF2α (Ser51) NP actin RIG-I Flu NP TIAR RIG-I Flu NP TIAR 0h 0h 3h 3h 6h 6h 9h 9h 12h 12h IAV PR8 IAV ΔNS1 Stress$Signals$ (Virus$Infec5on,$heat$shock$etc.)$ PKR$(dsRNA)$ GCN2$(aa.$Starva5on)$ HRI$(oxida5on)$ PERK$(ER$stress)$ eIF2α$Phosphoryla5on$(Ser51)$ RLR) TIAR& Forma5on$of$ TIA1& Stress$Granules$ eIF3$ G3BP& HuR$ PABP$ RIG]I G3BP DAPI merge mock IAV$ IFN ⊿NS1$ NaAsO2 NO$IFN Oxida:ve&Stress&Induces&SG&but&not&Interferon vRNA/FISH& NP/IF& Merge& Mock& IAV& ΔNS1& Virus]induced$SG$contains$viral$RNA$$$$$(an5$virus$SG) MDA5 G3BP1 PI DAPI MERGE MOCK EMCV MDA5 TIAR PI DAPI MERGE MOCK EMCV Colocaliza5on$of$MDA5,$SG$markers$and$viral$dsRNA ppp NUCLEUS Influenza$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 Stable Transient Transient Alternate Alternate Different)Paterns)of)SG)forma,on)) By)viral)infec,on) 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 Cells with 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 20 Cells with Distinct Cells with Cells Distinct Cells with Cells Distinct 20 with Cells Distinct 20 20 20 N.D.