© 2016 Macmillan Publihers Limited, part of Springer Nature. All rights reserved. 2 1 essential are enzymes deubiquitinating and ligases these that strate demon clearly studies these Although TBK1. from chains ubiquitin CYLD prote promoting by response antiviral and activation TBK1 regulate tively (TRIP) protein TRAF-interacting and respectively (LPS), ride lipopolysaccha and RNAto virus response in only of TBK1 tination polyubiqui K63-linked mediate Nrdp1 and MIB2 MIB1, However, polyubiquitination. K63-linked its promoting by TBK1 activate to (ref. MIB2 MIB1, ligases ubiquitin E3 The phosphorylation. and ubiquitination protein ing homeostasis. TBK1 activation is regulated in a variety of ways, includ immune maintain to regulated tightly be must activation its ferons, immunopathology. harmful without clearance viral efficient for critical is production interferon of control precise Thus, disease. inflammatory and/or causes autoimmune production interferon excessive whereas infection, I chronic type induce to IRF7 expression and interferon IRF3 factors transcription the rylates (ref. TBK1 activate to MAVSSTING TRIF, and including adaptors recruit PRRs these acids, nucleic (cGAS) synthase GMP–AMP cyclic as include acids nucleic viral (ref. TLR3 these recognize that PRRs immunity. antiviral innate the initiate to PAMPs efficient are RNA, and DNA (PAMPs) patterns molecular pathogen-associ of ated recognition (PRR) receptor pat requires tern-recognition response immune innate the of Activation invasion. microbial against defense of line host’s first the is immunity Innate for K63-linked and ubiquitination activation of TBK1 and delineated a previously unrecognized function for RNF128. innate antiviral immune responses to RNA and DNA viruses, IRF3 activation and IFN- (PA)protease-associated domain and catalyzed the of K63-linked polyubiquitination TBK1, which led to TBK1 activation, E3 ubiquitin ligase RNF128 as a positive regulator of TBK1 activation. RNF128 directly interacted with TBK1 through its TBK1 is essential for interferon- Chengjiang Gao Guanhua Song immunity through K63-linked ubiquitination of TBK1 E3 ubiquitin ligase RNF128 promotes innate antiviral nature immunology nature Received 27 July; accepted 21 September; published online 24 October 2016; Institute Institute of Basic Medicine, Shandong Academy of Medical Sciences, Jinan, China. should Correspondence be addressed to C.G. ( Department of Immunology and Key Laboratory of Infection and Immunity of Shandong Province, Shandong University the School of Medicine, Jinan, China. Because Because the kinase TBK1 is crucial in the production of type I inter a somal degradation somal of degradation TBK1, and the deubiquitinating enzymes 1 4 and USP2b 2 ), RIG-I ), 1 , 1 2 1 3– 5 , Bingyu , Liu Bingyu inhibit TBK1 activation by cleaving K63-linked 5 9 , MDA5 (ref. (ref. , MDA5 . Insufficient interferon production causes causes production interferon Insufficient . 10

, aDV 1 1 1 b 0 . The E3 ubiquitin ligase DTX4 (ref. (ref. DTX4 ligase ubiquitin E3 The . production. Deficiency of RNF128 expression attenuated IRF3 activation, IFN- ) and Nrdp1 (ref. (ref. Nrdp1 and ) A NCE ONLINE PUBLIC ONLINE NCE b 1 . Viral nucleic acids, including including acids, nucleic Viral . (IFN- 1 6 8 , , Zhihui Li ) and some DNA sensors such such DNA sensors some ) and ). Activated TBK1 phospho TBK1 Activated ). 1 3 7 have been shown to nega to shown been have . Upon recognition of the the of recognition Upon . b ) ) production and innate antiviral immunity. Here we identified the T cell anergy–related 1 1 ) have been found found been have ) 2 A , , Haifeng Wu TION

1 in in vitro 2 doi:10.1038/ni.358 ------) 1 , , Peng Wang THP-1 cells stimulated with poly(I:C) and ISD than in unstimulated unstimulated in than ISD and poly(I:C) with stimulated THP-1 cells Fig. 1a ( macrophages ISD in than unstimulated and poly(I:C) with stimulation after macrophages peritoneal mouse DNA of mRNA Expression RNF128 and (ISD). in protein was higher interferon-stimulatory and poly(I:C) mimics acid nucleic with lated stimu cells (THP-1) leukemia monocytic human and macrophages peritoneal primary mouse in expression RNF128 Wemeasured first expression RNF128 induces infection Virus RESULTS DNA RNA to and viruses. TBK1 of ubiquitination K63-linked for as a RNF128 E3 new ligase As chains. our uitin such, study identified moted TBK1 kinase activity through conjugation of ubiq K63-linked pro and TBK1 with associated RNF128 Mechanistically, 1 (HSV-1). type virus simplex herpes DNA virus the and (VSV) virus stomatitis vesicular virus RNA the with infection to mice wild-type than tible and IFN- activation IRF3 impaired deficiency or knockdown RNF128 virus. positive regulatoressential an of innateis antiviral immunityGRAIL) to as RNA known virus and DNA(also RNF128 ligase ubiquitin E3 immunity innate of regulation the in roles important have to demonstrated been have Itch, and c-Cbl other Cbl-b, as such anergy, Several cell T regulate known. that ligases not E3 is responses immune other in RNF128 anergy cell T of maintenance and polyubiquitination upon DNA-virus infection has not been identified. confirmed Importantly, been ubiquitinresponsibleE3 ligasethe forTBK1K63-linked not have roles their regulation, TBK1 for and In the present study we demonstrated that T cell anergy–related anergy–related cell T that demonstrated we study present the In induction the in involved regulator well-studied a is RNF128 in in vivo ). ). Expression of RNF128 mRNA and protein was also higher in β signaling. Consistently,signaling. 1 8 . . Our study identified RNF128 as an E3 ligase , , Kai Zhao 19– 2 1 . 1 , , Guosheng Jiang Rnf128 16– 1 [email protected] 8 . However, the function of of function the However, . Fig. 1 Fig. −/− b mice were more suscep production and a and 2 , Lei , ZhangLei ticles e l c i rt A Supplementary Supplementary ).

n vivo in 1

&  - - - - .

© 2016 Macmillan Publihers Limited, part of Springer Nature. All rights reserved. To explore the function of RNF128 in the innate antiviral response, response, antiviral innate the in RNF128 of function the Toexplore RNF128 positively regulates IRF3 activation and IFN- immunity. innate antiviral in role a have may RNF128 that indicates which expression, RNF128 induces expres ( sion RNF128 HSV-1-induced and SeV- of also attenuation (siRNA) showed RNA interfering small STAT2-specific and STAT1- ( antibody control a with treated cells to compared expression, RNF128 HSV-1-induced and SeV- of IFN- to antibody an by macrophages peritoneal ( in expression protein RNF128 increased genes interferon-stimulated of transcription IFN- ( cells uninfected to compared as cells, THP-1 and macrophages peritoneal mouse in mRNA of and protein RNF128 expression or HSV-1 (SeV) increased RNA- or DNA-virus Infection with infection. RNA virus Sendai virus and RNA double-stranded for DNA, respectively mimics considered were ISD and THP-1 ( cells results. similar with experiments of 3 independent STAT1-specific (siSTAT1),siRNA siSTAT2 SeV with or for 48 HSV-1h infected (siCtrl) then siRNA or for 8 control h. are Data representative for 2 h then infected with SeV or HSV-1 for 8 h. ( ( ( ( cells THP-1 1 Figure s e l c i rt A  (mRNF128 RNF128 mouse for specific siRNAs three designed we d c Fig. 1 Fig.

) Immunoblot analysis of RNF128 expression in mouse peritoneal macrophages treated with mouse recombinant IFN- recombinant mouse with treated macrophages in peritoneal mouse expression of RNF128 analysis ) Immunoblot ) Immunoblot analysis of RNF128 expression in of RNF128 mouse analysis peritoneal macrophages pretreated with ) antibody IFNAR1-specific Immunoblot or control antibody (IgG) Virus infection induces production of IFN- of production induces infection Virus β Fig. 1 Fig. receptor and leads to the activation of STAT1of STAT2 activation and the to leads and and receptor c

). Furthermore, cells in which IFN- which in cells Furthermore, ). Viral infection induces RNF128 expression. ( expression. RNF128 induces infection Viral d c b a e b ). Together, these data demonstrate that virus infection infection virus that demonstrate data Together, ). these Fig. Fig. 1 ) upon stimulation with poly(I:C) and ISD or infection with SeV with and HSV-1 and ISD or infection for poly(I:C) 0, with 4, 8 or ) control. 12 stimulation upon h. was as used GAPDH loading RNF12 RNF12 Time (h Time (h GAPDH GAPDH RNF12 Time (h GAPDH RNF12 Time (h GAPDH IFNAR Fig. 1a Fig. 3 lgG b , 8 8 2 ) ) 8 and ) 1 8 2 ) , we further measured RNF128 expression after expression RNF128 measured , we further 4 0 + – – β , 4 0 b Supplementary Fig. 1b Supplementary 2 0 receptor 1 (IFNRA1) showed attenuation attenuation showed (IFNRA1) 1 receptor 8 0 and and – + Poly(l:C) SeV Poly(l:C) Supplementary Fig. 1a Fig. Supplementary + i. 1 Fig. 1 8 1 8 8 4 + – + – d 2 2 ). Cells transfected with with transfected Cells ). 8 0 β e ) Immunoblot analysis of RNF128 expression in mouse peritoneal macrophages transfected with transfected macrophages in peritoneal mouse expression of RNF128 analysis ) Immunoblot + – signaling was blocked blocked was signaling HSV-1 2 β 16 3 IFN- . , which binds to the the to binds which , ). Because poly(I:C) poly(I:C) ). Because a – + , b 4 0 4 0 ) Immunoblot analysis of RNF128 protein expression in mouse peritoneal macrophages ( macrophages in peritoneal mouse expression protein of RNF128 analysis ) Immunoblot 24 + – β , b b stimulation stimulation production ). ISD ISD e 1 8 1 8 siSTAT siSTAT RNF12 GAPDH STAT2 STAT1 siCtr - SeV

8 2 1 0 2 2 l transfection ( transfection siRNA2 mRNF128 or siRNA1 by mRNF128 affected not was phages of NF- of Expression macrophages transfected simulation with control upon siRNA ( siRNA2 with mRNF128 poly(I:C) or ISD or infection with SeV or HSV-1, or as compared to siRNA1 mRNF128 with a encodes IFN- only had siRNA3 ( effect mRNF128 minor whereas siRNA2, mRNF128 and expression were attenuated after with siRNA1transfection mRNF128 protein and mRNA RNF128 Endogenous macrophages. peritoneal siRNA1, siRNA2 or siRNA3) and transfected them into mouse primary in mice and humans. and mice in IFN- in RNF128 of function the indicating of not expression the of expression induced SeV-,sion in THP-1poly(I:C)-, ISD- and decreased cells also HSV-1- expres RNF128 human of knockdown siRNA-mediated Similarly, ( sequence scrambled a bearing siRNA trol of expression attenuate on effect negligible a had which ( decreased and + + – – – – Ccl5 κ B signaling but not interferon signaling in peritoneal macro in peritoneal but signaling not interferon B signaling 4 0 + – , which are induced downstream of IFN- of downstream induced are which , aDV Supplementary Fig. 2b Fig. Supplementary Supplementary Fig. 2b Supplementary β 8 4 + + – + Supplementary Fig. 2a Fig. Supplementary – – – Il6 A ) ) was in decreased peritoneal macrophages transfected SeV NCE ONLINE PUBLIC ONLINE NCE SeV and and + 1 8 IL6 Tnf IFNB1 Ifnb1 or , proinflammatory cytokines downstream downstream cytokines proinflammatory , 12 2 siSTAT2 siSTAT1 TNF RNF128 GAPDH , , STAT2 STAT1 HSV-1 , , Cxcl10 siCtrl CXCL10 mRNA ( Rnf128 4 0 A 4 0 ). Notably, mRNF128 siRNA3, siRNA3, mRNF128 Notably, ). β , , ), ), but expression of + – – – TION for 0, 2, 4, 8, 16 or 24 h. Mx1 ). Expression of of Expression ). , , mRNA expression, did not not did expression, mRNA MX1 Supplementary Fig. 2c Fig. Supplementary

or HSV-1 HSV-1 + + – – Supplementary Fig. 2b Fig. Supplementary β Supplementary Fig. 2b nature immunology nature signaling is conserved conserved is signaling Ccl5 and and 1 8 + + + 1 8 – – compared to con compared CCL5 β signaling, was was signaling, + – – 2 2 Ifnb1

Cxcl10 mRNA but but mRNA

(which (which a

) ) and

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Mx1 , d ). ). ), ), ).

- - -

© 2016 Macmillan Publihers Limited, part of Springer Nature. All rights reserved. and peaked at 8 h after infection in wild-type mice, whereas the the whereas mice, wild-type in infection after h 8 at peaked and induced ( ditions in impaired not was mRNA Ccl5 of HSV-1. Expression or SeV with infection or ISD ( mice, from macrophages wild-type from macrophages to Compared littermates. type pared primary peritoneal macrophages from (mean experiments 3 independent control. protein nuclear as served PCNA control. cytoplasmic as served GAPDH HSV-1. or SeV with infected macrophages peritoneal KO and WT in fractions (cyto) cytoplasmic and nuclear in protein p65 and IRF3 of analysis ( stimulation. no Ctrl, control. loading as served GAPDH HSV-1. or SeV with infection or ISD or poly(I:C) with stimulation upon macrophages peritoneal ( h. 24 for RIG-IN or TRIF, for cGAS+STING plasmids expression ( vector. empty Ctrl, h. 24 for RIG-IN or IFN- with transfected then h and 24 for (sihRNF128) siRNA hRNF128 ( HSV-1. or SeV with infected macrophages peritoneal KO or ( or (WT) wild-type from 2 Figure nature immunology nature of expression g b Fig. 2 Fig. a d b ) Immunoblot analysis of IRF3 dimerization with native PAGE in WT and KO peritoneal macrophages upon SeV or HSV-1 infection. ( HSV-1 or infection. SeV upon macrophages peritoneal KO PAGEand WT in native with dimerization IRF3 of analysis ) Immunoblot ) ELISA quantification of IFN- of quantification ) ELISA To confirm the function of RNF128 in IFN- in RNF128 of function the Toconfirm IFN-β Luc activity IFN-β (pg/ml) lfnb1 mRNA (fold) 150 100 Ccl5 mRNA (fold) 200 400 600 mRNA was also decreased ( decreased also was mRNA 20 30 40 50 60 70 10 50 20 30 40 50 60 10 0 0 0 0 a

) and secretion ( secretion and ) Fig. 2 Fig. RNF128 positively regulates IFN- regulates positively RNF128 Ifnb1 Ctrl Ctrl Ctrl Ctrl Poly(l:C) Poly(l:C) Poly(l:C) **

TRIF WT * * expression was detected as early as 4 h after infection infection after h 4 as early as detected was expression a Ifnb1 **

). Time-course studies showed that SeV- or HSV-1- or SeV- that showed studies Time-course ). SeV SeV SeV * *

+STINGcGAS * KO sihRNF128 Rnf128 NC mRNA in in mRNA ** ISD lSD ** Rnf128 lSD ** * RIG-IN HSV-1 HSV-1

HSV-1 Fig. 2 Fig. * ** * aDV * −/− −/− β Rnf128 mice showed lower IFN- lower showed mice A (KO) mice stimulated with poly(I:C) or ISD or infected with SeV or HSV-1 for 8 h. Ctrl, control (no stimulation). stimulation). (no control Ctrl, 8 h. HSV-1 or for SeV with infected or ISD or poly(I:C) with stimulated mice (KO) secretion in WT or KO peritoneal macrophages treated as in in as treated macrophages peritoneal KO or WT in secretion b c NCE ONLINE PUBLIC ONLINE NCE Rnf128 e ) after stimulation with poly(I:C) or or poly(I:C) with stimulation after ) ± lfnb1 mRNA (fold)

s.d. of triplicate assays ( assays triplicate of s.d. IFNB1 mRNA (fold) ll6 mRNA (fold) Cxcl10 mRNA (fold) Fig. 2 Fig. SeV 15 20 25 10 10 20 30 40 50 60 70 10 20 30 40 50 60 70 80 90 10 20 30 40 50 0 5 0 0 0 e −/− ) ) Ctrl Ctrl 8 4 2 0 IFNB1 −/− Ctrl Poly(l:C) a β macrophages in these con these in macrophages Poly(l:C) ). Expression of of Expression ). signaling. ( signaling. macrophages was greatly greatly was macrophages TRIF cGAS+STING * Rnf128 * mRNA expression in HEK293 cells transfected with NC or sihRNF128 and then transfected with with transfected then and sihRNF128 or NC with transfected cells HEK293 in expression mRNA SeV SeV ** β * * sihRNF128 NC signaling, we pre we signaling, lSD lSD ** −/− 12 A a Cxcl10 RIG-IN * TION ) Expression of of ) Expression HSV-1 HSV-1 mice and wild- * 24 d * β a ) Luciferase (Luc) activity in HEK293 cells transfected with control siRNA (NC) or (NC) siRNA control with transfected cells HEK293 in activity (Luc) ) Luciferase expression expression h Il6 ,

b , , – f Mx1 and and ) Immunoblot analysis of phosphorylated (p-) IRF3, p65 and p38 in WT and KO and WT in p38 and p65 IRF3, (p-) phosphorylated of analysis ) Immunoblot d ,

HSV-1 Tnf mRNA (fold) e 20 30 40 50 10 Mx1 mRNA (fold) )) or are representative of 3 independent experiments with similar results ( results similar with experiments 3 independent of representative are or )) h

lfnb1 mRNA (fold) 0 RNF128 and and 10 20 30 40 50 60 70 80 GAPDH 10 20 30 40 β 0 Tnf PCNA

0 Ctrl luciferase reporter together with expression plasmids for TRIF, cGAS+STING TRIF, for cGAS+STING plasmids expression with together reporter luciferase IRF3 Ifnb1 SeV p65 - - Poly(l:C) Ctrl

8 4 2 0

Poly(l:C) WT WT n qa aons cA+TN) r w Ntria caspase- N-terminal two or associated and (cGAS+STING) recruitment domains of RIG-I amounts (RIG-IN) induced IFN- equal in ( in Ifnb1 ( points time these at decreased I-N ad GSSIGidcd IFN- cGAS+STING-induced and RIG-IN- TRIF-, increased markedly cells HEK293 in RNF128 of expression ( effects these attenuated expression ( expression ( activity luciferase promoter–driven , , – – Supplementary Fig. 2e Fig. Supplementary * *P < *P Cxcl10 Transfection of HEK293 cells with TRIF, cGAS and STING mixed TRIF,mixed with STING and cGAS cells of HEK293 Transfection

Rnf128 SeV KO KO * Cyto SeV * , , 0.05; 0.05; Cxcl10

WT lSD + + ** , ,

lSD *

Mx1 HSV-1 −/− KO 12

** HSV-1 **P ** Fig. 2 Fig. macrophages, as compared to wild-type macrophages macrophages wild-type to compared as macrophages, , , , , KO WT WT 24 Ccl5 Mx1 a – – < 0.01 (two-tailed Student’s Student’s (two-tailed < 0.01 . . ( h KO c Nuclei f e , , ) Time-course studies of of studies ) Time-course RNF128 RNF128 and and GAPDH ), and siRNA-mediated knockdown of RNF128 RNF128 of knockdown siRNA-mediated and ), Il6 p-IRF3 p-p38 p-p65 WT IRF3 + p38 p65 and and g RNF128 Ccl5 ). KO + IRF3 Tnf WT , but not not but , Ctrl RNF128 mRNA in peritoneal macrophages macrophages peritoneal in mRNA GAPDH KO KO HSV-1 PCNA Fig. 2 Fig. IRF3 Ctrl p65 WT Poly(l:C) WT Fig. 2d Fig. WT Il6 c – i. 2 Fig. ). LPS-induced expression of of expression LPS-induced ). KO KO and and KO Ifnb1 SeV + – Cyto t -test). Data are from from are Data -test). WT WT , SeV WT e d Tnf ). In contrast, ectopic ectopic contrast, In ). expression in WT in expression ) and and ) β KO KO KO promoter–driven promoter–driven , was also impaired impaired also was , + HSV-1 ticles e l c i rt A h WT WT WT ) Immunoblot ) Immunoblot – – ISD IFNB1 KO Nuclei KO monomer IRF3 IRF3 dimer WT WT f HSV-1 + + mRNA mRNA –

h

). KO KO β 

© 2016 Macmillan Publihers Limited, part of Springer Nature. All rights reserved. to control vector ( vector control to IFN- of activation RNF128 III. MAVS-TRIF-, overexpression increased and and cGAS+STING-induced I PRDs overlapping the to binds IRF3 and tively, IV, and respec II (PRDs) domains regulatory positive to bind AP-1 IRF3, NF- pathways. mediated regulates IFN- 2f Fig. and activity luciferase ( results similar with experiments 3 independent of representative are or cells. HEK293 10 bars, Scale ( 10 bars, Scale 8 h. for SeV with infected then HA-TBK1 and V5-RNF128-WT with transfected or cGAS+STING for plasmid expression vitro in ( times. various HSV-1 or for SeV with infected macrophages peritoneal mouse in TBK1 ( IP, cells. immunoprecipitation. HEK293 in HA-TBK1 or Myc-STING Myc-MAVS, ( h. 24 for plasmid expression RNF128 with along 5D IRF3 or MAVS, TBK1 RIG-IN, TRIF, for cGAS+STING, plasmids expression with transfected then h and 24 for (sihRNF128) siRNA hRNF128 or (NC) siRNA ( vector). (empty control Ctrl, h. 24 for (NC) vector control or V5-RNF128 with along 5D IRF3 or MAVS, TBK1 RIG-IN, TRIF, for cGAS+STING, plasmids expression with transfected 3 Figure s e l c i rt A  IFN- containing reporter of the activation the g

) Immunostaining of primary peritoneal macrophages infected with SeV or HSV-1 for 8 h then immunostained with anti-TBK1 and anti-RNF128. anti-RNF128. and anti-TBK1 with or HSV-1SeV 8 for with immunostained h then infected macrophages peritoneal primary of ) Immunostaining a c h e V5-RNF1 IFNB1 IFNB1 mRNA (fold) Input Input HA V5-RNF1 20 30 40 50 60 70 10 Input 0 HA IP: -TBK1 –translated RNF128 and TBK1. ( TBK1. and RNF128 –translated IP: , g IP: Ctr -TBK1 HA HA ). Together, these data demonstrate that RNF128 positively positively RNF128 that demonstrate data Together, these ). RNF1 RNF128 targets TBK1. ( TBK1. targets RNF128 V5 V5 28 transcription requires binding of the transcription factors factors transcription the of binding requires transcription RNF1 RNF1 My l κ

HA cG V5 V5 28 TBK1 TBK1 TBK1 TRIF B B and AP-1 to its promoter enhancer region

c A

S+S * 28 28 28 + + – µ *P β TING m. m. ( + – production downstream of TLR3-, RIG-I- and cGAS- < 0.05, ** < 0.05, * + + – RIG-IN + β h Supplementary Fig. 3a Fig. Supplementary + + ) Immunoprecipitation and immunoblot analysis of V5-RNF128-WT, V5-RNF128-H2N2 or V5-RNF128- or of V5-RNF128-WT, V5-RNF128-H2N2 analysis immunoblot and ) Immunoprecipitation promoter reporter in HEK293 cells, compared compared cells, HEK293 in reporter promoter * H2N2

+ MA Input V5-RNF1 +

IFNB1 VS * Flag-IRF3 ∆ IP: TBK1 b P PA + ) Expression of of ) Expression < 0.01 (two-tailed Student’s Student’s (two-tailed < 0.01 Flag Flag IRF3 5D * V5 V5 28 RA xrsin ( expression mRNA a ) Expression of of ) Expression cG f S + + – TING AS SeV Ctr f

) V5- and HA-specific immunostaining of HEK293 cells transfected with V5-RNF128-WT, HA-TBK1 and and HA-TBK1 V5-RNF128-WT, with transfected cells HEK293 of immunostaining HA-specific and ) V5- + IFN-β Luc activity l 80 20 40 60 + 0 Ifnb1

). RNF128 also increased increased also RNF128 ). Ctr V5-RNF1 l V5-RNF1 Input

My cG

β TRIF

IFNB1 A promoter PRDs I–III promoter I–III PRDs mRNA and luciferase activity of IFN- of activity luciferase and mRNA c-MA S+S * IP:

My TING 28 My VS * V5 V5 28 c c Supplementary Supplementary RIG-IN mRNA and luciferase (Luc) activity of IFN- of activity (Luc) luciferase and mRNA ** 2 c t 4 + + – ) Immunoprecipitation and immunoblot analysis of V5-RNF128-WT with Flag-IRF3, Flag-IRF3, with V5-RNF128-WT of analysis immunoblot and ) Immunoprecipitation -test). Data are from 3 independent experiments ( experiments 3 independent from are Data -test). . . NF- MA VS * HA V5-RNF1 NC

+ TBK1 κ -TBK1 * B B and IRF3 5D V5-RNF1 My Input c 28 – - c-S h IP: ). b TING My My RNF128 facilitates IRF3 activation, we compared IRF3 activation in activation IRF3 we compared activation, IRF3 facilitates RNF128 dependent manner ( by in overexpression a was RNF128 increased dose- phosphorylation phos ( IRF3 phorylation STING+cGAS-induced and MAVS- TRIF-, increased (ref. IRF3 of end C-terminal the IFN- regulates and PRD IV ( IFN- containing reporters of activation in vector ble difference between the RNF128 expression plasmid and the control (

V5 V5 IFNB1 mRNA (fold) 28 Supplementary Fig. 3a 20 25 30 35 40 15 10 c c 0 5 IRF3 activation requires phosphorylation on multiple sites at at sites multiple on phosphorylation requires activation IRF3 d ) Immunoprecipitation and immunoblot analysis of RNF128 and and RNF128 of analysis immunoblot and ) Immunoprecipitation Mer Ctr + – l

ge cG TRIF

e A

) ) S+S * β + + aDV In vitro In promoter reporter in HEK293 cells transfected with control control with transfected cells HEK293 in reporter promoter TING Supplementary Fig. 3a Supplementary Fig. 3b Supplementary *

β RIG-IN A production mainly through the activation of IRF3. activation the through mainly production d HS Input NCE ONLINE PUBLIC ONLINE NCE g interaction analysis of RNF128 with TBK1 using using TBK1 with RNF128 of analysis interaction SeV * Ctr V- MA IP: 1 Supplementary Fig. 3c Supplementary l VS β RNF1 * RNF1 promoter reporter in HEK293 cells cells HEK293 in reporter promoter TBK1 TBK1 TBK1 ). By contrast, we did not observe an apprecia

IRF3 5D * 28 28 RNF1 28 a 0 , b ; mean ; mean SeV 8 4

2 IFN-β Luc activity 12 14 10 ). These data suggest that RNF128 A 5 20 40 60 80 ). ). Notably, MAVS-induced IRF3 TION 0 0 0 0 ). Overexpression of RNF128 RNF128 of Overexpression ).

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12

28 h - - © 2016 Macmillan Publihers Limited, part of Springer Nature. All rights reserved. RNF128 enhances type I interferon signaling. Transfection of an an of Transfection signaling. interferon I type enhances RNF128 which by mechanisms molecular the determine to sought Wenext TBK1 targets RNF128 IFN- regulate to activation IRF3 lates Takenregu positively that RNF128 data together, demonstrate these in affected not were p65 of translocation nuclear HSV-1-induced and SeV- whereas macrophages, wild-type in lower ( dimerization IRF3 into the nucleus ( infection after in not impaired was p38 and of p65 rylation in was lower phosphorylation IRF3 HSV-1-induced and ISD- SeV-, Poly(I:C)-, macrophages peritoneal from primary results. similar with experiments 3 independent of representative are Data plasmids. HA-ubiquitin-K63 and/or V5-RNF128 with along Myc-TBK1-K30R-K401R or Myc-TBK1-K401R Myc-TBK1-K30R, or Myc-TBK1-WT for plasmids expression with ( control. as used were SeV with infected macrophages and WT from macrophages primary in ubiquitination TBK1 ( UbCH5A. and E1 with ubiquitin-K48 or ubiquitin-K63 ubiquitin-WT, of presence the in TBK1 and RNF128 ( Myc-TBK1-WT. with together plasmids expression HA-ubiquitin-K63 or HA-ubiquitin-K48 HA-ubiquitin-WT, with transfected h then 24 for (sihRNF128) siRNA hRNF128 or siRNA control with transfected cells HEK293 ( plasmids. HA-ubiqutin-K63 or HA-ubiquitin-K48 HA-ubiquitin-WT, and vector ( (UbcH5A). E2 ( IP, immunoprecipitation. plasmid. expression V5-RNF128-H2N2 or V5-RNF128-WT vector, control of presence the in HA-ubiquitin-WT or Myc-TBK1-WT with transfected 4 Figure nature immunology nature RNF128 expression plasmid further increased TRIF-, cGAS+STING-, V5-RNF128-H2N b a Ubiquiti RNF128 UbcH5A RNF128 V5-RNF128-WT TBK1 TBK1 Input HA-ubiquiti E1 Myc-TBK1 n IP: My

Rnf128 RNF128 promotes K63-linked ubiquitination of TBK1. ( TBK1. of ubiquitination K63-linked promotes RNF128 + + My My – – – HA HA V5 c c c n 2 Rnf128 + + + – – 4 3 2 1 c + + – – – – ) Coimmunoprecipitation analysis of TBK1 ubiquitination in HEK293 cotransfected with Myc-TBK1-WT, V5-RNF128-WT or control control or V5-RNF128-WT Myc-TBK1-WT, with cotransfected HEK293 in ubiquitination TBK1 of analysis ) Coimmunoprecipitation −/− 2 Fig. 2 Fig. 6 . Consistent with decreased IRF3 phosphorylation, phosphorylation, IRF3 decreased with . Consistent + – + + + + + – macrophages infected with SeV or SeV HSV-1 with in than infected macrophages Fig. 2 Fig. −/− + + + + – f + + + + macrophages ( macrophages ). IRF3 forms a homodimer and then moves moves then and homodimer a forms IRF3 ).

+ + – b ) ) g aDV In vitro In TBK1 TBK1(Ub)n ) and nuclear translocation ( ) and translocation nuclear IP: My c HA-ubiquiti V5-RNF128 Input A Myc-TBK1 NCE ONLINE PUBLIC ONLINE NCE c TBK1 ubiquitination assay using using assay ubiquitination TBK1 My My HA HA V5 IP: TBK1 f c c n Inpu β Fig. 2 Fig. Rnf128 + + W – – production. t RNF128 – W T TBK1 Rnf128 Se K6 K4 f Rnf128 Ub ). In contrast, phospho ). In contrast, + + V −/− 3 8 T + – – and mice. wild-type + – WT −/− g K4 ) Coimmunoprecipitation analysis of TBK1 ubiquitination in HEK293 cells cotransfected cotransfected cells HEK293 in ubiquitination TBK1 of analysis ) Coimmunoprecipitation A + – – −/− K 8 TION cells ( cells Rnf128 macrophages macrophages + + 48 Fig. 2 Fig. – KO

+ – + Fig. 2 Fig. RNF128 K6 h a HSV-1 −/− + + – ) Coimmunoprecipitation analysis of TBK1 ubiquitination in HEK293 cells cells HEK293 in ubiquitination TBK1 of analysis ) Coimmunoprecipitation TBK1 ) ) were K 3 K4 K6 (KO) mice infected with SeV or HSV-1. I HSV-1. or SeV with infected mice (KO) in vitro in Ub + 63 8 3 h e ). ). ) ) - - – – WT In vitro In Inpu IP: My d –translated RNF128 and TBK1 in the presence of ubiquitin, E1 or E1 ubiquitin, of presence the in TBK1 and RNF128 –translated + + HA-ubiquiti tion between RNF128 and TBK1. When cotransfected into HEK293 HEK293 into cotransfected When TBK1. and RNF128 between tion TBK1. by targeting signaling interferon I type enhance may ( siRNA3 hRNF128 by 5D–induced IRF3 whereas IFNB1 activation, promoter and expression MAVS- RIG-IN-, and TBK1-induced cGAS+STING-, (hRNF128) siRNA3 into HEK293 cells substantially attenuated TRIF-, ( not was activity promoter with an RNF128 expression plasmid, whereas IRF3 5D–induced IFNB1 Similarly, TRIF-,( cGAS+STING-, overexpression RIG-IN-, MAVS-RNF128 by and enhanced TBK1-induced not was 5D) (IRF3 aspartate by and were S404 the phosphomimetic S405 replaced S402, IFNB1 ( cells HEK293 in vector control of transfection to TBK1-induced and MAVS- RIG-IN-, sihRNF128 t Myc-TBK1 d RNF128 To that confirm RNF128 targets TBK1, we investigated the interac c ) Coimmunoprecipitation analysis of TBK1 ubiquitination in ubiquitination TBK1 of analysis ) Coimmunoprecipitation KO TBK1 ubiquitination (TBK1(Ub)n) assay using using assay (TBK1(Ub)n) ubiquitination TBK1 My My HA HA mRNA expression and promoter were activation not impaired promoter activity was increased in HEK293 cells transfected transfected cells HEK293 in increased was activity promoter expression induced by an IRF3 variant in which S396, S398, S398, S396, which in variant IRF3 an by induced expression c c n IP: + – – Input: l κ B MG132 α

WT SeV + + – K48 K6 l l Ub κ κ

WT B B 3 – + + + – + –

K48 + + – Fig. 3 Fig. +

K48 – + Fig. 3 Fig. g HA-ubiquitin-K63 f + + – ) Coimmunoprecipitation analysis of analysis ) Coimmunoprecipitation b IP: My κ ). These data suggested that RNF128 RNF128 that suggested data These ). B Input

K63 V5-RNF128 – α Myc-TBK1 a ubiquitination in primary primary in ubiquitination c ). Transfection of human RNF128 RNF128 human of Transfection ). K63 + + My My HA HA V5 c c IFNB1 RNF128 Ubiquitin e RNF128 UbcH5A TBK-1 TBK1 – – WT E1 expression compared compared expression

WT + – + + + + + – in vitro in WT

WT + + ticles e l c i rt A Fig. 3 Fig. + +

WT K48 + + + + + + –translated –translated IFNB1 + +

K30R K63 + + a ), whereas whereas ),

+ + Fig. 3 Fig.

TBK1 TBK1(Ub)n

K401R mRNA

IFNB1

+ + K30R- K401R a ). ).  -

© 2016 Macmillan Publihers Limited, part of Springer Nature. All rights reserved. eioel arpae ( macrophages peritoneal mouse in TBK1 and RNF128 endogenous between colocalization of and HA-TBK1 ( RNF128 V5- between colocalization increased cGAS+STING of expression ( cells HEK293 into transfected very low degree of colocalization between V5-RNF128 and HA-TBK1 TBK1. and RNF128 between tion ( anti-RNF128 with assays down an 4a Fig. RNF128 and TBK1 was in also detected THP-1 cells ( ( infection SeV HSV-1 or upon enhanced was TBK1 and RNF128 endogenous between interaction the Notably, proteins. two the between interaction tive ( macrophages peritoneal mouse in cells ( MAVS (Myc-MAVS) or Myc-STING in when overexpressed HEK293 coimmunoprecipitate with Flag-tagged IRF3 (Flag-IRF3), Myc-tagged HA-tagged TBK1 (HA-TBK1) ( with coimmunoprecipitated (V5-RNF128) RNF128 V5-tagged cells, ( results similar with experiments 3 independent of representative are or assays) triplicate of plasmid. expression Myc-TBK1-S172A or Myc-TBK1-WT vector, control of presence the in HA-ubiquitin-WT and V5-RNF128-WT with transfected cells HEK293 in ubiquitination TBK1 of ( cells. HEK293 into transfected plasmids Myc-TBK1-S172A or Myc-TBK1-WT with V5-RNF128-WT of analysis and WT in TBK1 TANK or with NEMO of ( plasmids. expression V5-RNF128-WT and Myc-TBK1-K30R-K401R or Myc-TBK1-K401R Myc-TBK1-K30R, ( IP, (bottom). immunoprecipitation. immunoprecipitates TBK1 the in TBK1 (p-) phosphorylated and TBK1 of analysis Immunoblot 6 h (top). HSV-1 for 5 Figure s e l c i rt A  HSV-1or ( SeV with infection after enhanced b

) qRT-PCR analysis of of ) qRT-PCR analysis Immunostaining with V5- and HA-specific antibodies showed a showed antibodies HA-specific and V5- with Immunostaining in in vitro Fig. 3 ). In addition, a mixture of RNF128 and TBK1 generated in in generated TBK1 and RNF128 of mixture a addition, In ).

a IP RNF128 promotes TBK1 activation. ( activation. TBK1 promotes RNF128 TBK1 activity (c.p.m.) protein expression system coimmunoprecipitated in pull in coimmunoprecipitated system expression protein 1 20,000 40,000 60,000 80,000 00,000 c p-TBK1 ). Endogenous RNF128 and TBK1 coimmunoprecipitated TBK1 0 PBS Fig. 3 Fig. Ifnb1 d SeV Input Fig. 3 Fig. * V5-RNF1 My i. 3 Fig. mRNA expression (top) and immunoblot analysis of p-TBK1 (bottom) in in (bottom) p-TBK1 of analysis immunoblot and (top) expression mRNA d IB: c-TBK1 ). An interaction between endogenous endogenous between interaction An ).

IP: TBK1 TBK1 Fig. 3 IP f ). Similarly, we detected a low degree a low degree we Similarly, ). detected Fig. 3 Fig.

28 V5 V5 Fig. 3 Fig. TBK1 activity (c.p.m.) g 200,000 1 p-TBK1 1 ), but colocalization was greatly greatly was colocalization but ), 00,000 50,000 50,000 TBK1 c *P Fig. 3 Fig. WT + – ). However, V5-RNF128 did not Rnf128 f e < 0.05, ** < 0.05, 0 ). Infection of SeV or ectopic ectopic or SeV of Infection ). ), indicating a direct interac a direct indicating ), PBS d a WT WT ), suggesting a constitu a suggesting ), ) TBK1 kinase activity in WT or or WT in activity kinase ) TBK1 −/− Fig. 3 Fig. HS (KO) peritoneal macrophages infected with SeV or HSV-1 for 8 h. ( 8 h. HSV-1 or for SeV with infected macrophages peritoneal (KO) ** V- S1 KO P 1 < 0.01 (two-tailed Student’s Student’s (two-tailed < 0.01 72A – g Supplementary Supplementary ). V5-RNF1 b My S1 G p-TBK1 72A c-TBK1 + APDH TBK1 lfnb1 mRNA level (fold) 28 20 25 30 35 40 45 50 15 10 0 5 – –

e – - - - IP: HA Input V5-RNF1 My WT My -ubiquitin lation of TBK1 activation, we transfected Myc-TBK1 into HEK293 HEK293 into Myc-TBK1 transfected we activation, TBK1 of lation regu the in involved was RNF128 of activity ligase E3 the whether promote the ubiquitination of several substrates to that reported has been ligase E3 ubiquitin is a RNF128 RING-type TBK1 of ubiquitination K63-linked promotes RNF128 PA RNF128. the of domain through TBK1 with these results demonstrate that RNF128 targets and physically interacts ( V5-RNF128-WT to compared element response (ISRE) promoter activation or IRF3 in phosphorylation HEK293 cells interferon-stimulated cGAS+STING-induced V5-RNF128- Consistently, association. RNF128–TBK1 for necessary is PA the domain that ing but V5-RNF128-H2N2, not with V5-RNF128- domain. HA-TBK1 coimmunoprecipitated with V5-RNF128-WT and V5-RNF128- or domain, RING the in tions muta point contains which mutants—V5-RNF128-H2N2, RNF128 two of one or (V5-RNF128-WT) V5-RNF128 wild-type either and HA-TBK1 with cells HEK293 cotransfected we TBK1, to bound that substrates captured of tination ubiqui the facilitates domain finger RING the and ubiquitination, c c-TBK1 + + Rnf128

The PA domain of RNF128 captures target proteins for cytosolic cytosolic for proteins target captures RNF128 of PAdomain The WT ** My My HA V5 28 c c t * -test). Data are from 3 independent experiments ( experiments 3 independent from are Data -test). K30R * WT −/− – – aDV + + (KO) mouse peritoneal macrophages infected with SeV or SeV with infected macrophages peritoneal mouse (KO) K401R a WT – A + –

e K30R- NCE ONLINE PUBLIC ONLINE NCE ). K401R WT Input + + c Input Tbk1 S1 IP: IP: IB: IB: 72A – – NEMO NEMO −/− ∆ T

T TBK1 TBK1 TBK1 TBK1 ANK ANK PA did not increase TRIF-, MAVS- or or MAVS- TRIF-, increase not did PA c MEFs transfected with Myc-TBK1-WT, Myc-TBK1-WT, with transfected MEFs S1 ) Coimmunoprecipitation analysis analysis ) Coimmunoprecipitation 2 e 72A + + – 7 ) Coimmunoprecipitation analysis analysis ) Coimmunoprecipitation Supplementary Fig. 4b,c Fig. Supplementary . To define the domains of RNF128 RNF128 of domains the To . define WT Ctr S1 A d TION 72A + l ) Coimmunoprecipitation ) Coimmunoprecipitation KO

WT ∆ nature immunology nature SeV PA, which lacks the PA the lacks PA,which ∆ 18 PA ( KO , 27– Fig. Fig. 3 2 WT a 9 HS , . . To investigate b ; mean ; mean V- KO 1 h ). Overall, Overall, ). ), ), indicat

± s.d. s.d.

- - - - © 2016 Macmillan Publihers Limited, part of Springer Nature. All rights reserved. down of RNF128 expression in peritoneal macrophages attenuated macrophages peritoneal in expression RNF128 of down in infection SeV after cells ( HEK293 activity kinase TBK1 increased RNF128 of in 5 in decreased greatly was activity kinase or HSV-1 infection ( TBK1 kinase activity was increased in wild-type macrophages upon ubiquitination performed and macrophages SeV peritoneal TBK1 mouse from TBK1 immunoprecipitated we RNF128-induced activation, TBK1 affects whether determine To activation TBK1 promotes RNF128 K401. and at K30 TBK1 of tion these data demonstrate that RNF128 promotes K63-linked ubiquitina ubiquitination induced by ( V5-RNF128-WT Myc-TBK1-K30R, TBK1 low showed Myc-TBK1-K30R-K401R and V5-RNF128-WT. Myc-TBK1-K401R with together cells HEK293 into them and transfected respectively) or Myc-TBK1-K30R-K401R, Myc-TBK1-K401R (Myc-TBK1-K30R, both or K401R K30R, tions muta point with variants Myc-TBK1 constructed we K401, or K30 through promotes TBK1 ubiquitination RNF128 whether investigate activation catalytic for its required is K401 and at K30 of TBK1 antibodies. K63-specific and K48- the of validity the NF- cytoplasmic inhibitory the of polyubiquitination K63-linked not but greatly K48-linked it that increased found and infection SeV of effects the examined we in impaired not Rnf128 were ubiquitination STING HSV-1-induced and ( infection in not but phages, macro wild-type in ubiquitination TBK1 K63-linked in increase an observed We infection. HSV-1 or SeV after macrophages primary TBK1. of ubiquitination ( but not or ubiquitin-K48 ubiquitin-K63 ubiquitin-WT with together TBK1 ubiquitination was induced in the presence of E1, E3 that showed and andubiquitin-K63 binant ubiquitin-WT, ubiquitin-K48 RNF128 HA- ( not ubiquitin-K48 but HA-ubiquitin-K63 or HA-ubiquitin-WT with fected expression trans RNF128 cells in HEK293 ubiquitination TBK1 attenuated substantially of knockdown siRNA-mediated addition, In ( HA-ubiquitin-K48 not but HA-ubiquitin-K63 (HA-ubiqui or tin-WT) HA-ubiquitin wild-type of presence significantly the was in increased TBK1 of polyubiquitination RNF128-mediated HA-ubiquitin-K63). and (HA-ubiquitin-K48 arginine with replaced or were K48 K63 except residues lysine all in which mutants we used ( enzymes conjugating E2 and enzymes activating in TBK1 tinated ( V5-RNF128-WT to as compared cells, in HEK293 or phosphorylation IRF3 activation promoter ISRE cGAS+STING-induced or MAVS- TRIF-, increase polyubiquitination ( plasmid ( expression RNF128 of presence the in increased markedly was tion above. described constructs showed that ubiquitina TBK1 experiments V5-RNF128 Coimmunoprecipitation the with together cells nature immunology nature TBK1 kinase activity after infection SeV ( i. 4 Fig. a ). SeV- and HSV-1-induced TBK1 phosphorylation was also decreased Structural studies have shown that K63-linked polyubiquitination polyubiquitination have K63-linked that shown studies Structural Next, we measured TBK1 ubiquitination in To further investigate RNF128-mediated TBK1 polyubiquitination, Rnf128 e −/− ). These data indicate that RNF128 mediates K63-linked K63-linked mediates RNF128 that indicate data These ). Fig. Fig. 4 macrophages ( macrophages −/− Fig. 4 Fig. arpae ( macrophages Supplementary Fig. Supplementary 5a a Fig. 4 Fig. ). ). Notably, did V5-RNF128-H2N2 not increase TBK1 f in vitro in ). In contrast, SeV-induced MAVS ubiquitination MAVSubiquitination SeV-induced contrast, In ). Supplementary Fig. 4b Supplementary Fig. 4 Fig. 5 Rnf128 d ). ).

ubiquitination assays in the presence of E1 E1 of presence the in assays ubiquitination a In vitro In a aDV Supplementary Fig. 4d Fig. Supplementary ). ). Consistently, V5-RNF128-H2N2 did not ), ), whereas SeV- and HSV-1-induced TBK1 κ −/− B chaperone B I chaperone i. 5 Fig. A macrophages, after SeV or HSV-1 HSV-1 or SeV after macrophages, NCE ONLINE PUBLIC ONLINE NCE ubiquitination assays with recom with assays ubiquitination a ). Furthermore, overexpression overexpression Furthermore, ). ), ), and siRNA-mediated knock Rnf128 in in vitro Supplementary Supplementary Fig. 5b ). RNF128 directly ubiqui ). directly RNF128 κ B Fig. Fig. 4 Rnf128 α −/− ( TBK1 kinase assays. assays. kinase TBK1 Fig. 4 Fig. macrophages( Fig. 4 Fig. g , A ). ). Taken together, e −/− TION ). As a control, control, a As ). f and wild-type ), confirming ), confirming b ).

Fig. 4 Fig. 3 0 ). Fig. Fig. . . To c ). ). ------­

found found that Myc-TBK1 with a S172A substitution (Myc-TBK1-S172A) activity kinase TBK1 for required is which S172, due, ( macrophages of TANK Binding in not was to impaired TBK1 than in wild-type macrophages after SeV or HSV-1 infection ( in lower was TBK1 to NEMO of of and TBK1 (ref. activation in of resulting complexes, TBK1–adaptor tors such as NEMO and TANK to trigger higher-order oligomerization tion is not clear. In one model, K63-ubiquitinated TBK1 recruits adap in expression of and TBK1-K30R-K401R and TBK1-K401R TBK1-K30R, of ylation TBK1-induced and Ifnb1 phosphorylation TBK1 increased further mid expression ( tion in embryonic fibroblasts (MEFs). Myc-TBK1-WT restored TBK1 activa into transfected were Myc-TBK1-K30R-K401R and ( results similar with experiments 3 independent of ( experiments Student’s (two-tailed significant controls. used were mice (WT) wild-type from macrophages Peritoneal HSV-1. or SeV with infected or LPS with stimulated and (siNrdp1) siNrdp1-3 and siNrdp1-2 of a mixture and/or (siMIB1/2) ratio equal at mixed Mib2-2 and Mib1-3 Mib1-1, with in (top) p-TBK1 and IRF3 (p-) phosphorylated of analysis immunoblot and (bottom) ( h. 48 for (1–3) siRNA3 siRNA1– targeted or (NC) siRNA control with transfected macrophages of analysis 6 Figure a

After activation, TBK1 was phosphorylated at a single serine resi serine at single a phosphorylated was TBK1 activation, After How K63-linked ubiquitination of TBK1 leads to TBK1 phosphoryla Myc-TBK1-K401R Myc-TBK1-K30R, Myc-TBK1-WT, Next, Mib1 mRNA (fold) Tbk1 0.2 0.4 0.6 0.8 1.2 b 0 1 siMIB1/2 GAPDH siNrdp1 p-TBK1 expression in in expression p-lRF3 HSV-1 TBK1 NC Tbk1 lfnb1 mRNA (fold) IRF3

LPS SeV −/− 40 60 80 20 WT KO RNF128 is a general regulator of TBK1 activation. ( activation. TBK1 of regulator a general is RNF128 0 * Mib1 MEFs ( MEFs 9 8 7 6 5 4 3 2 1 1 Fig. Fig. 5 −/− + – – – – – – – a 8 7 6 5 4 3 2 1 , * Ifnb1 b MEFs, as assessed by TBK1 phosphorylation and Fig. 5 Fig. + , , Rnf128 – – – – – – ; mean ; mean 3 2 Mib2 b + + – – – – Fig. 5 Fig. were greatly decreased in were the of presence greatly decreased RNF128 ), ), and of transfection the RNF128 expression plas + + + – – – – – – + – – – c Tbk1 and and ). −/− ± b + s.d. of triplicate assays) or are representative representative are or assays) triplicate of s.d. ) qRT-PCR analysis of of ) qRT-PCR analysis b (KO) peritoneal macrophages transfected transfected macrophages peritoneal (KO) Mib2 mRNA (fold) + + – – – – – 0.2 0.4 0.6 0.8 1.2 ). Nrdp1 −/− 0 1 + + – – – – – – ns 3 MEFs ( MEFs ** 1 NC t + + + – – – – -test). Data are from 3 independent 3 independent from are Data -test). ). Consistent with this model, binding model, with this ). Consistent * mRNA expression in mouse peritoneal peritoneal mouse in expression mRNA 1 9 + + + – – – ns Rnf128 ** 1 10 + + + + – – – 1 0 11 *P Fig. 5 Fig. + + 3 2 – – – – – 1 1 < 0.05; < 0.05; 12 −/− ** + + – – – – – 1 2 trans ns 13 peritoneal macrophages macrophages peritoneal b 1 3 + + + – – – – Ifnb1 * ). However, phosphor However, ). ns 14 1 4 + + + + + + – – – – – -autophosphorylation -autophosphorylation b Nrdp1 mRNA (fold) Rnf128 0.5 1.5 15 **P ). + 1 5 – – 0 1 mRNA expression expression mRNA 16 ticles e l c i rt A + + – – – – – – < 0.01; ns, not not ns, < 0.01; 1 6 NC 17 Tbk1 + + – – – – – 1 7 ns 3 −/− 2 ns 18 + + + . Indeed, we we Indeed, . – – – a 1 8 ns * 1 ) ) qRT-PCR peritoneal peritoneal −/− 19 ** + + + – – – – – 2 9 Fig. 5

20 3 2 mouse mouse + + + + – – 0

Ifnb1

c

). ).  ------

© 2016 Macmillan Publihers Limited, part of Springer Nature. All rights reserved. Simultaneous siRNA-mediated knockdown of MIB1, MIB2 and and MIB2 MIB1, of knockdown siRNA-mediated Simultaneous in phosphorylation LPS-induced, decreased HSV-1-induced, or SeV- not but further Nrdp1 of knockdown mediated ( siRNA by down knocked were MIB2 and MIB1 HSV-1-infected, or stimulated ( Rnf128 Ifnb1 for ( used were experiments and subsequent genes target their of expression the attenuated efficiently siRNA3 and siRNA2 Nrdp1 and siRNA2 MIB2 siRNA2, into them on siRNAs and for transfected each three we designed RNF128 activation, to TBK1 relation in Nrdp1 and MIB2 MIB1, of (refs. function the TBK1 of activation and ubiquitination K63-linked promote to reported been have Nrdp1 and MIB2 MIB1, activation TBK1 for regulator a general is RNF128 activation. and phosphorylation TBK1 to leading TBK1, of data these indicate that ubiquitination promotes RNF128 K63-linked Together, of phosphorylation. TBK1 upstream occurs ubiquitination ( cells HEK293 in was not lower than that of of TBK1-WT expression V5-RNF128-WT 5 with V5-RNF128-WT and Myc-TBK1-WT or transfected Myc-TBK1-S172A cells ( HEK293 in coimmunoprecipitation by assessed as TBK1-WT, and TBK1-S172A between similar was RNF128 with ing or rylated unphosphorylated TBK1. We found that the extent of bind phospho targets RNF128 whether investigate to Myc-TBK1-S172A into transfected IFN- induce not did ( experiments × 10 (2 40 bars, Scale group). ( 40 bars, Scale h. 72 for VSV with infected mice WT and KO from ( mice WT and KO from liver and lung in protein HSV-1 or ( VSV with injection intravenous 8 h after mice WT ( units. plaque-forming PFU, 10). HSV-1 or (MOI 0.1) (MOI) infection of (multiplicity VSV with infected mice 7 Figure s e l c i rt A  in Nrdp1 n Fig. 6 Fig. d

= 3 mice per group) infected with HSV-1 for 72 h. ( h. 72 HSV-1 for with infected group) per = 3 mice d ). Furthermore, ). ubiquitination of Furthermore, induced by TBK1-S172A ectopic WT KO a VSV log(PFU/ml) expression and IRF3 and TBK1 phosphorylation were lower in lower were phosphorylation and TBK1 and IRF3 expression 10 15 20 25 0 5 b 7 −/−

PFU/mouse). PFU/mouse). ) and were further attenuated in SeV-infected, but not LPS- not but SeV-infected, in attenuated further were ) and RNF128 positively regulates antiviral response. ( response. antiviral regulates positively RNF128 WT PBS peritoneal macrophages than in wild-type macrophages macrophages wild-type in than macrophages peritoneal Rnf128 * Rnf128 KO a – c , e Tbk1 ; mean ; mean −/− Fig. 5 Fig.

−/− HSV-1 log(PFU/ml) 10 15 20 25 30 0 5 peritoneal macrophages decreased LPS- and and LPS- decreased macrophages peritoneal β µ *P Rnf128 peritoneal macrophages. MIB1 siRNA1 and and siRNA1 MIB1 macrophages. peritoneal VSV m. ( m. −/− production or TBK1 phosphorylation when when phosphorylation TBK1 or production < 0.05, < 0.05, WT e ± ), suggesting that RNF128-mediated TBK1 TBK1 RNF128-mediated that suggesting ), MEFs ( MEFs s.d. of triplicate assays) or are representative of 3 independent experiments with similar results ( results similar with experiments 3 independent of representative are or assays) triplicate of s.d. ** g Fig. 6 Fig. ) Survival of KO and WT mice ( mice WT and KO of ) Survival KO −/− e peritoneal macrophages ( macrophages peritoneal **P a Supplementary Fig. 5c Fig. Supplementary Rnf128 b ). LPS-, SeV- and HSV-1-induced HSV-1-induced and SeV- LPS-, ). Ifnb1 Relative gDNA copy number < 0.01 (two-tailed Student’s Student’s (two-tailed < 0.01 10 12 IFN-β (pg/ml) 0 2 4 6 8 400 100 200 300 0 expression, IRF3 and TBK1 TBK1 and IRF3 expression, WT −/− * macrophages in which which in macrophages PBS n KO KO WT = 3 mice per group) infected with the VSV for 72 h. ( h. 72 for VSV the with infected group) per = 3 mice 10 f ) ICP8-specific immunohistochemistry in the brains of HSV-1-infected mice ( mice HSV-1-infected of brains the in immunohistochemistry ) ICP8-specific ,

Fig. 6 Fig. HSV-1 log(PFU/ml) 1 10 15 0 5 1 a VSV * ). To evaluate evaluate To ). ) VSV and HSV-1 titers in peritoneal macrophages from wild-type (WT) and and (WT) wild-type from macrophages peritoneal in HSV-1 titers and ) VSV n n WT = 3 mice per group). ( group). per = 3 mice = 7 mice per group) after intravenous injection of VSV (4 × 10 (4 VSV of injection intravenous after group) per = 7 mice b ). We used used We ). ). siRNA- ). * Fig. 6 Fig. µ m. ( m. KO t * -test ( -test HSV-1 Fig. e b ) HSV-1 genomic DNA copy number and viral titers in brain of KO and WT mice mice WT and KO of brain in titers viral and number copy DNA ) HSV-1 genomic ). ). g f WT KO - - a – c c wild-type mice with VSV and wild-type HSV-1. IFN- ( macrophages wild-type in than macrophages and HSV-1 replication were higher in in and HSV-1 ( ( VSV increased hRNF128 of knockdown siRNA-mediated Similarly, transfec (control). to siRNA ‘scramble’ a compared with as tion macrophages, peritoneal in replication VSV ( with case V5-RNF128- or the V5-RNF128-H2N2 not cells was in this but vector, than control the with V5-RNF128-WT transfected with transfected cells HEK293 in ( that VSV was sion significantly indicated replication 6a Fig. V5-RNF128- 6a ( vector Fig. (control) empty to compared rep as viral lication, inhibited substantially VSV with infected cells HEK293 tion Type I interferons have critical responses antiviral rolesregulates positively RNF128 in the immune response to viral infec signaling. innate and or DNA-virus-induced RNA-virus- of LPS-, downstream expression ubiquitination TBK1 for regulator general a is RNF128 ubiquitination TBK1 of regulation the in Nrdp1 and MIB2 MIB1, of roles the reported with consistent is which infection, RNA-virus upon and activation ubiquitination TBK1 modulate signal MIB2 LPS and of MIB1 whereas downstream ing, activation TBK1 in mainly role a has ( phosphorylation and TBK1 HSV-1-induced, not but SeV-induced, , e VSV log(PFU/ml) ) or Gehan–Breslow–Wilcoxon test ( test Gehan–Breslow–Wilcoxon ) or To test the function of RNF128 increased mRNF128 of transfection that showed assays Plaque 20 40 60 3 0 3 c . Plaque assays showed that overexpression of V5-RNF128-WT in Supplementary Fig. 6b Fig. Supplementary ) Plaque assay of VSV titers and immunoblot analysis of VSV-G of analysis immunoblot and titers VSV of assay ) Plaque ). Immunoblot analysis of VSV glycoprotein (VSV-G)of VSV expres analysis glycoprotein ). Immunoblot ). In contrast, overexpression of V5-RNF128-H2N2 or or V5-RNF128-H2N2 of overexpression contrast, In ). Lung ** aDV Supplementary Fig. 6e Supplementary ∆

Percent survival 100 A PA did not inhibit viral replication ( replication viral inhibit not did PA 10 20 40 60 80 NCE ONLINE PUBLIC ONLINE NCE 0 , 1 0 1 . Taken together, these results demonstrate that that demonstrate results these together, Taken . Time afterinfection(d) ** Liver 6 4 2 d KO WT ) Hematoxylin–eosin staining of lung sections sections lung of staining ) Hematoxylin–eosin VSV b GAPDH GAPDH Fig. 6 Fig. VSV-G VSV-G ) ) IFN- ) and HSV-1 and ( ) in vivo 1 8 g b β )). Data are from 3 independent 3 independent from are Data )). ) replication ) in THP-1replication VSV cells. ∆ A ). These data suggest that Nrdp1 that suggest data ). These production in sera from KO and and KO from sera in production 0 TION PA ( , we challenged Rnf128 Ifnb1 WT

Percent survival 100 Supplementary Fig. 6a Fig. Supplementary

20 40 60 80 7 β Supplementary Fig. 6d Fig. Supplementary c 0 PFU/mouse) or HSV-1 or PFU/mouse) Supplementary Fig. 6c Fig. Supplementary protein after abundance , nature immunology nature d 6 4 2 0 , expression and IRF3 IRF3 and expression −/− f Fig. 7 Fig. Time afterinfection(d) ). KO WT primary peritoneal n ( ( = 3 mice per per = 3 mice n n Rnf128 Supplementary Supplementary Supplementary Supplementary =7) =7) a HSV-1 P ). KO Rnf128 < lower 0.05) −/− (KO) (KO) −/− Liver Lung Ifnb1 8 and ). - - - - - ) )

© 2016 Macmillan Publihers Limited, part of Springer Nature. All rights reserved. knockdown attenuated antiviral response both or deficiency RNF128 or DNARNA with virus virus; infection upon virus RNA with or infection DNA virus; RNF128 increased upon IRF3 activation and IFN- induced was expression response. RNF128 antiviral innate of regulation the in role essential an has CD4 on focused and adaptive have immunity RNF128 of studies previous Most addition to its well-studied function in the regulation of T cells. T of regulation the in function well-studied its to addition defines RNF128 as a positive regulator of innate antiviral immunity in study our Thus, replication. virus inhibited overexpression RNF128 experiments. knockdown siRNA-mediated with activation of TBK1 regulation on the RNF128 of MIB2 and MIB1, Nrdp1 the specificity in we relation to confirmed activation TBK1 LPS-induced of regulation the in involved Ifnb1 of expression LPS-induced impairs BMDMs in expression Nrdp1 of (ref. TBK1 of ubiquitination K63-linked promote RNA-virus or infection RNA double-stranded by activation TBK1 regulate Cxcl10 MIB2 do not regulate LPS- or DNA-virus-induced expression of SeV-induced impair of expression to found been have (BMDMs) macrophages bone-marrow-derived or MEFs in MIB2 and MIB1 of knockdown siRNA-mediated or Deficiency TBK1. of ubiquitination K63-linked stimulation. LPS and infection RNA-virus infection, DNA-virus of downstream TBK1 of Therefore, RNF128 is a general regulator of K63-linked ubiquitination IFN- LPS-induced replication. virus to inhibit to an inability leading cells, HEK293 in ubiquitination TBK1 or IFN- phosphorylation induce IRF3 duction, not did respectively, domain, PA the lack and and H2N2 RNF128- RNF128- mutants RNF128 The domain. finger RING TBK1 the of through ubiquitination K63-linked the mediated and domain PA the through TBK1 with interacted RNF128 TBK1. of activation and IFN- phosphorylation, impairs RNA- that deficiency RNF128 and IRF3 DNA-virus-induced RNA- in Our results or DNA-virus infection. after TBK1 of ubiquitination K63-linked promotes and TBK1 with interacts RNF128 Here, E3 that ligase the we ubiquitin demonstrated DISCCUSION DNA and viruses. RNA both against responses immune antiviral for regulator positive HSV-1 ( or VSV with infection upon mice wild-type than mortality ( mice wild-type than brain that indicated ICP8 HSV-1 to the protein specific antibody with Immunohistochemistry of brains the in higher significantly were titers DNA viral and genomic of lungs with VSV after infection ( the in observed were injuries severe and in higher nificantly ( VSV or HSV-1 infection was much lower in the sera of nature immunology nature in involved molecules several of degradation and ubiquitination Fig. 7 Fig. Fig. 7 Fig. RNF128 controls T cells anergy and tolerance through K48-linked K48-linked through and tolerance controls T anergy cells RNF128 unresponsiveness cell T of gatekeeper important an is RNF128 promote to found were ligases ubiquitin E3 two Previously, Rnf128 β and and signaling was also impaired in RNF128-deficient cells. cells. RNF128-deficient in impaired also was signaling or or b g ). These These ). ). VSV titers and replication in the lung and liver were sig were liver and lung the in replication and titers VSV ). 1 0 −/− Ccl5 Cxcl10 . The E3 ubiquitin ligase Nrdp1 has also been shown to to shown been also has Nrdp1 ligase ubiquitin E3 The . ie hn n hi wl-ye oneprs ( counterparts wild-type their in than mice Ifnb1 mRNA, suggesting that MIB1 and MIB2 specifically specifically MIB2 and MIB1 that suggesting mRNA, in vivo in mRNA, indicating that Nrdp1 may be specifically specifically be may Nrdp1 that indicating mRNA, , , Rnf128 ∆ Rnf128 Cxcl10 PA, RING domain have which a nonfunctional 17 β

data indicate that RNF128 is an important important an is RNF128 that indicate data production and K63-linked ubiquitination ubiquitination K63-linked and production , 18 Fig. Fig. 7 aDV , −/− 3 and and −/− 4 Fig. 7 Fig. . . Here we provide evidence that RNF128 mice than in wild-type mice ( mice wild-type in than mice A mice had more HSV-1 virions in the the in virions HSV-1 more had mice d NCE ONLINE PUBLIC ONLINE NCE Ccl5 ). ). Similarly, copy numbers of HSV-1 f ). ). mRNA Rnf128 −/− 1 Rnf128 in vitro 0 . However, MIB1 and and MIB1 However, . mice showed higher higher showed mice A 1 −/− TION Rnf128 1 and Rnf128 ). Knockdown Knockdown ). mice showed mice in vivo β

signaling + Fig. 7 Fig. −/− Fig. 7 Fig. −/− 1 T cells cells T 1 β . Here Here . mice mice Ifnb1 pro mice , and e c 1 6 ). ). ), ), - - . ,

4. 3. 2. 1. 10. 9. 8. 7. 6. 5. reprints/index.htm R The authors declare no competing interests.financial and wrote the paper. Z.L. and G.J. provided facility for study.virus G.S. and C.G. analyzed the data H.W., P.W., K.Z. and L.Z. provided reagents and contributed to discussions. and prepared the figures. B.L. performed part of the ubiquitination experiments. C.G. conceived the andstudy. supervised G.S. performed most of the experiments of Shandong, China. of Higher Education of to China (20130131130010) C.G. G.J. is a Taishan Scholar 81273219) and Research the FundSpecialized for the ProgramDoctoral grants from the Natural FoundationScience of China 81471538, (81525012, Medical forSciences) VSV and HSV-1. This work was supported in part by MEFs, and H. Meng (Institute of Basic Medicine, Shandong Academy of Pasteur of Shanghai, AcademyChinese of forSciences) providing TBK1-deficient and TBK1 mutant plasmid S172A, respectively. We thank B. Sun (Institut We thank G.C. Fathman (Stanford University School of Medicine), online version of the pape Note: Any Supplementary Information and Source Data files are available in the the pap the of in version available are references associated any and Methods M infection. viral limit and inflammation excessive prevent to design drug for clues peutic thera some provide our might data diseases, autoimmune and other Given the role pathological of IFN- lates TBK1 activation after both RNA-virus and DNA-virus infection. proteins. target of activation or ubiquitination degradation the to of leading ubiquitination form K63-linked new a delineated mediated by and RNF128. Therefore, RNF128 may mediate both K48- and substrate new identified a TBK1, of ubiquitination K63-linked promotes RNF128 (ref. (ref. (TCR)–CD3 receptor antigen cell T degrades and with interacts RNF128 activation. cell T (Wuhan University) for providing RNF128 expression plasmids, IFN- K.A. Fitzgerald (University of Massachusetts Medical andSchool) H.-b. Shu C AU A eprints and permissions information is available online at at online available is information permissions and eprints ck OM

ethods In summary, we have identified a RING-type E3 ligase that regu that ligase E3 RING-type a identified have we summary, In

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© 2016 Macmillan Publihers Limited, part of Springer Nature. All rights reserved. 50 mM EDTA; 150 mM NaCl; and a protease inhibitor cocktail (Merck). (Merck). cocktail inhibitor protease a and NaCl; mM 150 EDTA; mM 50 7.4; pH Tris-HCl, mM 50 P-40; Nonidet (vol/vol) 1.0% in containing lysed buffer were IP and transfection after h 36 collected were extracts whole-cell (IP), For immunoprecipitation reagent. extraction with equal and were made (Pierce) assay acid bicinchoninic a with measured were extracts the in tions ‘cocktail’. inhibitor a protease with concentra Protein supplemented (Pierce) Reagent Extraction Protein M-PER with lysed analysis. were tissues or cells analysis, immunoblot and Coimmunoprecipitation luciferase. Renilla of by that activity luciferase firefly of division manufacturer’s by the efficiency to transfection for normalized were Data according (Promega). instructions system Assay Reporter Dual-Luciferase activity. luciferase of Assay changes. expression relative expression in each individual sample. The 2 manufacturer’s and instructions, data were by normalized the level of GAPDH Biosystems) using the SYBR Green PCR Master Mix (Toyobo) according to (Applied the System Detection 7300 ABI by amplified were samples of products in listed are assays RT-PCR for used Primers (Invitrogen). instructions manufacturer’s the to according quantification. RNA Systems). (R&D Kits by ELISA measured ELISA. Biotechnology. Santa Cruz from obtained were which siRNA, STAT2 and siRNA STAT1 except are listed in (Genlantis) according to the manufacturer’s protocol. The sequences of siRNAs siRNA into 2 were with Transfectioncells the transfected Geneporter Reagent used (Invitrogen). For macrophages and THP-1 cells, plasmids and duplexes of of plasmids and duplexes of siRNA into HEK293 cells, Lipofectamine 2000 was mids used in this study previously were described plas Other Shu. H.-b. by provided was TBK1-S172A plasmid mutant TBK1 The Fitzgerald. K.A. by provided were plasmids reporter PRDIV and PRDII IFN- The Fathman. G.C. by provided were plasmids expression DNA sequencing. V5-RNF128-WT, V5-RNF128-H2N2 and V5-RNF128- using the KOD-Plus-Mutagenesis kit (Toyobo).generated were TBK1-K30R-K401R) and All constructsTBK1-K401R (TBK1-K30R, were TBK1 confirmed by transfection. and plasmids Sequences, Table 1 Supplementary in provided is study this in used antibodies the about Information Meng. H. by (HSV-1) were provided virus-1 simplex (VSV) and herpes virus stomatitis virus was purchased from China Center for Type Culture VesicularCollection. Sendai U/ml. 1,000 of concentration final a at PeproTech used from and was 10 of concentration final a at used were ISD from and InvivoGen. were poly(I:C) and (PMA) purchased ester phorbol with 10% FCS, 100 U/ml penicillin and 100 colate. The cells were cultured at 37 °C under 5% CO C57BL/6J mice (5–6 weeks old) through intraperitoneal injection with female thiogly from prepared were macrophages peritoneal primary Mouse Sun. B. by provided were MEFs TBK1-deficient Collection. Culture Type American reagents. and Cells University. Shandong of School Medical the of Board Investigation Scientific the of approval conditions the with pathogen-free specific under maintained were and background TGGCAGATACC-3 5 5 5 primer forward primers: with the following and mice was (WT) knockout performed Mice. METHODS ONLINE doi: × 14,000 at min 10 for centrifugation After ′ ′ -GCTGAAGTTAGTACTGCATG-3 -GGGCAAGTCCTGTGTTCTAAAAGC-3 10.1038/ni.3588

Rnf128 The concentrations of IFN- of concentrations The ′ -CCAAAATCGATTCTCGTGGCTCC-3 Supplementary Supplementary Table 2 −/− mice were from Jackson Laboratory. Genotyping of wild-type ′ HEK293, THP-1 and Vero cells were obtained from from obtained were cells Vero and THP-1 HEK293, for for Total RNA was extracted with TRIzol reagent reagent TRIzol with extracted was RNA Total . Rnf128 Supplementary Table 3 Table Supplementary Luciferase activity was measured with the the with measured was activity Luciferase −/− mice. All the mice were on the C57BL/6 C57BL/6 the on were mice the All mice. . . siRNAs were obtained from Genepharma β in culture supernatants and sera were were sera and supernatants culture in ′ and reverse primer 5 primer reverse and µ Mutant expression plasmids for for plasmids expression Mutant µ − g/ml. Mouse recombinant IFN- recombinant Mouse g/ml. ∆ g/ml g/ml streptomycin. ISD, poly(I:C) ∆ ′ g Ct for WT mice; forward primer primer forward mice; WT for , supernatants were collected collected were supernatants , method was used to calculate 3 5 . . For transient transfection 2 in DMEM supplemented ′ . Reverse-transcription Reverse-transcription . and reverse primer primer reverse and For immunoblot immunoblot For ′ -GTCAACAGG β , PRDI-III, PRDI-III, , ∆ PA β - - -

meabilized for 20 min at room temperature with 0.5% Triton X-100. After After X-100. incubation for Tritonblockade of nonspecific binding for 0.5% 30 min, primary antibodies with temperature room at min 20 for meabilized per PFAand 4% with min 10 for fixed were slips cover on cultured phages microscopy. and staining Immunofluorescence changes. histological for light microscopy by examined and solution hematoxylin–eosin with stained sectioned, fixed in into 10% dissected, embedded formalin, phosphate-buffered paraffin, were mice virus-infected or control- from brains or Lungs infection. HSV-1 VSV or after survival for monitored were mice experiments, survival For the and lung liver and the HSV-1 in titers in the brain were titers by determined standard VSV plaque assays. ELISA. by measured was sera the in production 10 × (4 VSV with sex-matched WT and infection Virus ACTTACCC-3 primer: HSV-1-specific an using 5 PCR real-time by determined were bers num copy DNA genomic HSV-1 Verocells. in conducted was assay plaque rocking for 2 h at 4 °C followed by centrifugation for 5 min at 4 °C. Pellets Pellets °C. 4 at min 5 for centrifugation by followed °C 4 at h 2 for rocking was extract cell plus by protein G gentle antibody beads total anti-TBK1 with immunoprecipitated the in protein TBK1 Briefly, manufacturer. the instructions from the to according Promega from System Enzyme Kinase TBK1 assay. kinase TBK1 described as (Promega). with a were Transcription/Translationexpressed TNT Coupled Quick System anti-ubiquitin. with by immunoblot analyzed and anti-TBK1 with cipitated were immunopre extracts or whole-cell HSV-1, SeV then with infected were HA. For of analysis of the ubiquitination TBK1 in macrophages macrophages, were anti- extracts with immunoblot by analyzed whole-cell and anti-Myc with then immunoprecipitated mutants, or V5-RNF128-WT and mutants cells, cells were transfected with Myc-TBK1, HA-ubiquitin-WT or HA-ubiquitin assays. Ubiquitination ( antibodies indicated with blotted then and membranes nitrocellulose onto transferred to SDS–PAGE, and subjected were loaded lysates or whole-cell noprecipitates by eluted with SDS 1% boiling (wt/vol) sample buffer. For immu were analysis, immunoblot Immunoprecipitates buffer. IP with times five washed were reagent 1 with together Immunoprecipitation Plus-Agarose G protein with incubated and lqe sa ad S rpiain ee efre a described as performed were replication VSV and assay plaque VSV MOI). (10 HSV-1 or MOI), (1 SeV MOI), (0.1 VSV with infected were 10 × (2 macrophages assay. peritoneal plaque and infection Viral membrane immunoblotting. for nitrocellulose a onto transferred were proteins After separation. electrophoresis, for gel native 7.5% pre-run 30 a to then applied and was blue), protein total bromophenol 0.1% and glycerol; 30% 6.8; pH HCl, and 2× with diluted native PAGEwas quantified mM (125 sample Tris-buffer × 13,000 at centrifugation After cocktail). Tris-HCl, pH 7.5; 150 mM NaCl; and 0.5% NP-40 containing protease inhibitor 50 with harvested were macrophages In brief, PAGE. Native System. Enzyme assay buffer. TBK1 kinase activity was assayed with kinase in ADP-Glo °C 30 at and min TBK1 30 Kinase for resuspended was pellet washed Each fluoride). pyrophosphate; sodium mM 2.5 mM 1 X-100; NaCl; Triton mM 150 vol/vol 7.4; 1% pH EDTA; Tris-HCl, mM mM 1 (20 buffer lysis with washed were In vitro In ′ -TGGGACACATGCCTTCTTGG-3 β -glycerophosphate; 1 mM Na mM 1 -glycerophosphate; binding and ubiquitination assay. ubiquitination and binding Supplementary Table 1 Supplementary In vitro In 3 5 . The IRF3 dimerization assay was performed as described as performed was assay dimerization IRF3 The ′ . µ g corresponding antibodies. After 6 h of incubation, beads beads incubation, of h 6 After antibodies. corresponding g n vivo in 7 PFU/mouse) or HSV-1 (2 × 10 × (2 HSV-1 or PFU/mouse) interaction and ubiquitination assays were performed performed were assays ubiquitination and interaction TBK1 kinase activity was assayed with ADP-Glo and and ADP-Glo with assayed was activity kinase TBK1 Rnf128 For analysis of the ubiquitination of TBK1 in HEK293 . For For −/− littermate mice were intraperitoneally infected n vivo in 5 ) were plated 24 h before infection. Cells Cells infection. before h 24 plated were ) ). HEK293, THP-1 or mouse primary primary mouse or THP-1 HEK293, 3 VO ′ 5 , virus infection studies, age- and and age- studies, infection virus 4 ′ ; and 1 mM phenylmethylsulfonyl phenylmethylsulfonyl mM 1 and ; -ACCCTTAGTCAGACTCTGTT g for 10 min, supernatant protein protein supernatant min, 10 for µ RNF128 and TBK1 proteins proteins TBK1 and RNF128 l ice-cold lysis buffer (50 mM (50 buffer lysis l ice-cold HEK293 cells and macro and cells HEK293 nature immunology nature 7 PFU/mouse). Cytokine Cytokine PFU/mouse). 3 5 . HSV-1 HSV-1 . µ 3 6 g - - - - - .

© 2016 Macmillan Publihers Limited, part of Springer Nature. All rights reserved. Kaplan–Meier survival curves were generated and analyzed with GraphPad GraphPad with analyzed and generated were curves survival Kaplan–Meier with Student’s with was two-tailed determined significance ments. Statistical analysis. Statistical (Olympus). microscope fluorescence on IX71 Olympus an acquired were Images Scientific). Fisher (Thermo antibodies secondary 488–conjugated Fluor Alexa or 568– Fluor Alexa suitable with fur stained were ther Samples temperature. room at h 2 for incubated and added were nature immunology nature P < 0.05 considered statistically significant. For mouse survival studies, studies, survival mouse For significant. statistically considered 0.05 < All data are presented as data are All mean presented ± s.d. of s.d. 3 or more experi t -test, -test, - -

36. 35. used. was blinding or No randomization study. the from excluded ensure were to mice or size points sample data No of power. adequate estimation for used were studies Pilot 5.0. Prism

Mori, M. Mori, regulates negatively TRIM26 C. Gao, & L. Zhang, K., Zhao, W., Zhao, P.,Wang, 9698–9702 (2004). 9698–9702 activation. determines that phosphorylation inducible for target degradation of nuclear IRF3. nuclear of degradation interferon- et al. et β rdcin n atvrl epne hog pluiutnto and polyubiquitination through response antiviral and production Identification of Ser-386 of interferon regulatory factor 3 as critical as 3 factor regulatory interferon of Ser-386 of Identification PLoS Pathog. PLoS

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