Cellular Importin-α3 Expression Dynamics in the Lung Regulate Antiviral Response Pathways against Influenza A Virus Infection.

A icle Cellula Impo in-a3 Exp ession Dynamics in he Lung Regula e An i i al Response Pa hways agains In luenza A Vi us In ec ion G aphical Abs ac Highligh s dImpo in-a3 is he mos abundan ly exp essed iso o m in he mammalian lung dImpo in-a3 is highly conse ed ac oss species dImpo in-a3 is one o he majo nuclea anspo e s o NF-kB dImpo in-a3 ac s as an immune senso o in luenza A i us in ec ions Au ho s Swan je Thiele, S ephanie S anelle-Be am, Sebas ian Beck, ..., Michael Bade , Enno Ha mann, G€ ulsah Gab iel Co espondence guelsah.gab i[email p o ec ed] In B ie Thiele e al. show ha impo in-a3 is one o he majo nuclea anspo e s o NF- kB in he mammalian lung. High-le el TNF-a-inducing HPAIVs inhibi impo in- a3 mRNA ansc ip ion by in e e ing wi h i s p omo e ac i i y. Thus, HPAIVs may e ade an i i al immuni y in he espi a o y ac by gene a ing a bo leneck in impo in-a3 a ailabili y. Nucleoplasm TNF-α TNFR TNF-α TNFR A ian- ype HPAIV PB2 , PB2 Human- ype HPAIV PB2 , PB2 RNP Cy oplasm Nucleoplasm Cy oplasm an i i al genes an i i al genes α3 p50 p65 α3 p50 p65 α3 p50 p65 α3 p50 p65 p50 p65 −+ RNP p50 p65 701D 627E 701N627K Thiele e al., 2020, Cell Repo s 31, 107549 Ap il 21, 2020 ª2020 The Au ho (s). h ps://doi.o g/10.1016/j.cel ep.2020.107549 Cell Repo s A icle Cellula Impo in-a3 Exp ession Dynamics in he Lung Regula e An i i al Response Pa hways agains In luenza A Vi us In ec ion Swan je Thiele, 1 S ephanie S anelle-Be am, 1 Sebas ian Beck, 1 Nancy Mounogou Kouassi, 1 Ma in Zickle , 1 Ma in M€ ulle , 1 Be in Tuku, 1 Pa icia Resa-In an e, 1,12 Debby an Riel, 1,3 Malik Alawi, 4,5 Thomas G€ un he , 5 F anziska Ro he , 6,7 S e anie H€ ugel, 6 Susanne Reime ing, 8 Alice McHa dy, 8 Adam G undho , 5 Wol am B une, 9 Albe Os e haus, 10 Michael Bade , 6,7,11 Enno Ha mann, 7 and G€ ulsah Gab iel 1,2,13, * 1 Vi al Zoonosis – One Heal h, Hein ich Pe e Ins i u e, Leibniz Ins i u e o Expe imen al Vi ology, Hambu g, Ge many 2 Ins i u e o Vi ology, Uni e si y o Ve e ina y Medicine, Hanno e , Ge many 3 Depa men o Vi oscience, E asmus Medical Cen e , Ro e dam, he Ne he lands 4 Bioin o ma ics Se ice Facili y, Uni e si y Medical Cen e Hambu g-Eppendo , Hambu g, Ge many 5 Vi us Genomics, Hein ich Pe e Ins i u e, Leibniz Ins i u e o Expe imen al Vi ology, Hambu g, Ge many 6 Molecula Biology o Pep ide Ho mones, Max Delb € uck Cen e o Molecula Medicine, Be lin, Ge many 7 Ins i u e o Biology, Cen e o S uc u al and Cellula Biology in Medicine, Uni e si y o L€ ubeck, L€ ubeck, Ge many 8 Compu a ional Biology o In ec ion Resea ch, Helmhol z Cen e o In ec ion Resea ch, B aunschweig, Ge many 9 Vi us-Hos In e ac ion, Hein ich Pe e Ins i u e, Leibniz Ins i u e o Expe imen al Vi ology, Hambu g, Ge many 10 Resea ch Cen e o Eme ging In ec ions and Zoonoses, Uni e si y o Ve e ina y Medicine Hanno e , Hanno e , Ge many 11 Cha i e ´-Uni e si a ¨ smedizin, Be lin, Ge many 12 P esen add ess: AIDS Resea ch Ins i u e I siCaixa, Ba celona, Spain 13 Lead Con ac *Co espondence: guelsah.g[email p o ec ed] h ps://doi.o g/10.1016/j.cel ep.2020.107549 SUMMARY Impo in-aadap o p o eins o ches a e dynamic nu- clea anspo p ocesses in ol ed in cellula homeo- s asis. He e, we show ha impo in-a3, one o he main NF-kB anspo e s, is he mos abundan ly ex- p essed classical nuclea anspo ac o in he mammalian espi a o y ac . Impo in-a3 p omo e ac i i y is egula ed by TNF-a-induced NF-kBina concen a ion-dependen manne . High-le el TNF- a-inducing highly pa hogenic a ian in luenza A i- uses (HPAIVs) isola ed om a al human cases ha bo ing human- ype polyme ase signa u es (PB2 627K, 701N) signi ican ly down egula e impo in-a3 mRNA exp ession in p ima y lung cells. Impo in-a3 deple ion is es o ed upon back-mu a ing he HPAIV polyme ase in o an a ian- ype signa u e (PB2 627E, 701D) ha can no longe induce high TNF-ale els. Impo in-a3-de icien mice show educed NF-kB- ac i a ed an i i al gene exp ession and inc eased in luenza le hali y. Thus, impo in-a3 plays a key ole in an i i al immuni y agains in luenza. Li ing he bo leneck in impo in-a3 a ailabili y in he lung migh p o ide a new s a egy o comba espi a o y i us in ec ions. INTRODUCTION Molecula a icking o p o eins be ween he cy oplasm and he nucleus is a undamen al p ocess ha is essen ial o main ain cellula homeos asis (Go ¨ lich and Ma aj, 1996; Imamo o e al., 1995). Dis u bances in hese highly sensi i e egula o y p o- cesses may esul in he disequilib ium o cellula and nuclea p o eins and e en ually cause diseases. Impo in-ap o eins belong o he majo nuclea anspo ac o s in he cell shu ling a ious ca go p o eins con aining a nuclea localiza ion signal (NLS) om he cy oplasm o he nucleus (Gold a b e al., 2004). Impo in-ap o eins a e acidophilic p o eins ha bind as adap- o s o ca go p o eins con aining a basic s e ch o mono- o bipa i e NLS mo i s. This impo in-a/ca go complex o ms a ime ic complex wi h he ecep o p o ein impo in-b 1 , which inally ansloca es h ough he nuclea po e by he in e ac ion o impo in-b 1 wi h nucleopo ins (Hu e al., 1996; Radu e al., 1995; Rexach and Blobel, 1995). In he nucleus, he guanosine iphospha e (GTP)-bound o m o Ran binds o impo in-b 1 and media es he dissocia ion and elease o he ca go p o ein in o he nucleus (Lee e al., 2005; Ma suu a and S ewa , 2005). A e success ul nuclea impo , impo in-abinds o he ecycling ac o cellula apop osis suscep ibili y p o ein (CAS) ha , in conjunc ion wi h RanGTP, anspo s he complex back in o he cy oplasm whe e impo in-ais again a ailable o he nu- clea impo o o he ca go p o eins (Ku ay e al., 1997). Impo in- ap o eins a e highly conse ed h oughou e olu ion (Gold a b e al., 2004). In euka yo es, se en impo in-aiso o ms we e desc ibed, whe eas he budding yeas encodes one impo in- a, and lies and nema odes encode o h ee impo in-aiso o ms. Howe e , all o hese o ganisms encode o only one impo in-b 1 ecep o p o ein. The speci ici y o impo in-aiso o ms o ca go p o eins is de e mined by he complexi y and he h ee-dimen- sional con ex (F ied ich e al., 2006; Gold a b e al., 2004; Sank- hala e al., 2017). Mo eo e , impo in-ap o eins play a key ole in cellula di e en ia ion by egula ing he anspo o ansc ip ion Cell Repo s 31, 107549, Ap il 21, 2020 ª2020 The Au ho (s). 1 This is an open access a icle unde he CC BY-NC-ND license (h p://c ea i ecommons.o g/licenses/by-nc-nd/4.0/). ac o s h ough lineage-speci ic exp ession p o iles in cells (Ya- suha a e al., 2007). Howe e , li le is known abou he exp es- sion p o iles o impo in-aiso o ms in o gans ha could gi e impo an insigh s in o molecula mechanisms in heal h and dis- ease. The e is accumula ing e idence ha unde cellula s ess, such as oxida i e s ess, hea shock, o ul a iole i adia ion, im- po in-ap o eins may accumula e in he nucleus and dis up cellula homeos asis (Fu u a e al., 2004; Kodiha e al., 2004; Ya- suda e al., 2012). In he las decade, a g owing amoun o e i- dence sugges s ha impo in-ap o eins a e u ilized by a ious i uses (e.g., in luenza, Ebola, dengue, hepa i is C, and co ona- i uses) o ci cum en hos de ense mechanisms (Basle and Ama asinghe, 2009; Can on e al., 2018; F ase e al., 2014; F ie- man e al., 2007; Gab iel e al., 2011; Hudje z and Gab iel, 2012; Kopecky-B ombe g e al., 2007; Neu eld e al., 2013; P yo e al., 2007). Li le e idence is a ailable on hei mode o ac ion in speci ic o gans unde in lamma o y condi ions such as i us in ec ions. In his s udy, we iden i ied he ana omic exp ession p o ile o impo in-aiso o ms in he mammalian lung and s udied hei ansc ip ional egula ion and impac on in luenza- i us- induced pneumonia using cell cul u e models, genome-wide ansc ip ion analyses, and ansgenic mouse models. RESULTS Impo in-a3 Is he Mos Abundan ly Exp essed Impo in- aIso o m in he Mammalian Respi a o y T ac In o de o analyze he exp ession pa e n o majo impo in-a iso o ms h oughou he mammalian espi a o y ac (RT), we ana omically dissec ed he human and mu ine RT in o an uppe pa de ined by espi a o y cells o he nasal concha (URT) and a lowe pa de ined by b onchi and al eoli (LRT). Impo in-a1, -a3, and -a5/7 iso o ms (Kpna2,Kpna4, and Kpna1/6, espec i ely) we e abundan ly exp essed in epi helial cells, glands, and mac- ophages o he human (Figu e 1A) as well as he mu ine URT and LRT (Figu e 1B). We hen measu ed he ela i e mRNA exp es- sion le els o impo in-aiso o ms along he mammalian RT. In he human URT and LRT, impo in-a3 was by a he mos abun- dan ly exp essed iso o m, wi h 4–5 imes inc eased mRNA le els compa ed o impo in-a1. A e age exp ession le els o o he iso o ms we e simila o hose o impo in-a1, excep o impo - in-a7, which was he second-mos abundan iso o m (Fig- u e 1C). Simila ly, impo in-a3 was also he highes -exp essed iso o m, ollowed by impo in-a7 in he mu ine URT and LRT. Lase -based mic odissec ion o epi helial cells was used o ensu e homogeneous cell popula ions (Figu e 1D). We u he con i med he exclusi e abundance o impo in-a3 in he mu ine lung, ollowed by impo in-a7 as he second-mos abundan iso- o m also on he p o ein le el, using whole-o gan homogena es (Figu es 1E and 1F). No ably, in immo alized cells (Figu es S1A–S1D) and unlike in p ima y cells, impo in-a1 is he mos abundan iso o m and is he e o e used as a p ognos ic cance ma ke (Ch is iansen and Dy skjø , 2013; Dahl e al., 2006; Kau e al., 2004). Thus, i is pa icula ly impo an o in es iga e he ole o impo in-aiso o ms ei he in p ima y cells o in an in i o animal model. We hen de e mined he exp ession o impo in-a iso o ms in a ious mu ine o gans on he mRNA and p o ein le els and con i med ha he lung (Figu e S1E) is unique in exhib- i ing exclusi ely high impo in-a3 le els, compa ed o o he solid o gans (Figu es S1F–S1K). Mo eo e , compa able dis ibu ion and exp ession le els o impo in-aiso o ms wi hin he human and mu ine RT sugges ha he mouse model may be consid- e ed adequa e o s udy he impac and mode-o -ac ion o hese cellula ac o s in heal h and disease. Nex , we di ec ly compa ed ela i e impo in-aamoun s in lase -mic odissec ed epi helial samples o he URT e sus he LRT (Figu es 1G–1K). Fo all impo in-aiso o ms, mRNA le els we e highes in he al e- ola issue (LRT (A), up o 6 imes) ollowed by epi helial cells o he b onchi and b onchioles (LRT [b onchiola epi helium, BE]). The lowes mRNA le els we e de ec ed in epi helial cells o he nasal concha (URT). Thus, impo in-aiso o ms a e exp essed as a g adien in he RT wi h g adually inc easing mRNA le els om he URT owa d he LRT (Figu e 1L). Howe e , impo in- a3 is he mos abundan ly exp essed impo in-aiso o m in he mammalian RT, unlike in o he solid o gans. Impo in-a3 Media es Nuclea T ansloca ion o TNF- a-Ac i a ed NF-kB NF-kB subuni s (p50, p65) a e ansloca ed in o he nucleus in an impo in-a-media ed manne . He ein, pa icula ly impo in-a3 and i s close ela i e, impo in-a4, we e shown o ac as one o he main iso o ms ha impo s umo nec osis ac o alpha (TNF-a)-ac i a ed p50/p65 he e odime s in o he nucleus ia di ec in e ac ion (Fage lund e al., 2005). Since he mammalian lung mos abundan ly exp esses he impo in-a3 iso o m, we con inued o dissec he physical and unc ional in e ac ion be- ween impo in-a3 and NF-kB. We could con i m ha pu i ied human impo in-a3 p o ein di ec ly in e ac s wi h NF-kB p50 and p65 subuni s as well as wi h i s p ecu so p105 p o ein upon ac i a ion wi h TNF-aunlike impo in-a1 ha was used as a nega i e con ol (Figu es 2A and 2B). Consis en ly, ea - men o mu ine ib oblas s wi h TNF-amos signi ican ly inc eased NF-kB le els in nuclea cell ac ions along wi h impo - in-a3(Figu e 2C). In o de o con i m ha impo in-a3is equi ed o he nuclea impo o NF-kB, we gene a ed mu ine emb yo ib oblas s (MEFs) wi h a dele ed impo in-a3 gene (a3 / ). Nuclea ac iona ion analysis in TNF-a- ea ed a3 / MEFs showed signi ican ly educed nuclea ansloca ion o he NF-kB subuni p65 compa ed o wild- ype (WT) con ol (a3 +/+ ) MEFs (Figu e 2D). Immuno luo escence analysis u he con i med ha nuclea localiza ion o NF-kB p65 was also educed in a3 / MEFs compa ed o a3 +/+ MEFs (Figu e 2E). Con e sely, NF-kB p65 accumula ed in he cy oplasm o a3 / MEFs, albei sligh ly, likely due o he deg ada ion o ine icien ly anspo ed ca go p o eins (Figu e 2E). The ex en o educed nuclea localiza ion o impo in-a3 was dependen on he me hod used, albei he absence o impo in-a3 consis en ly impai ed e icien nuclea localiza ion o TNF-a-induced NF-kB subuni p65. These da a clea ly show ha impo in-a3 ac s as a nuclea ansloca o o TNF-a-ac i a ed NF-kB. Impo in-a3 P omo e Ac i i y Is Regula ed by TNF-a-Ac i a ed NF-kB We u he obse ed an inc ease in gene al impo in-a3 p o ein le els upon TNF-a ea men in o al cell lysa es, including nu- clea and cy oplasmic cell ac ions o mu ine as well as p ima y 2Cell Repo s 31, 107549, Ap il 21, 2020 Human URT A Epi helial cells Glands α1 α3 α5/7 Human LRT (Al eoli) Epi helial cells Mac ophages Human LRT (B onchi) Epi helial cells Impo in α1α3α5/7 Mu ine URT B Epi helial cells Mu ine LRT (Al eoli) Epi helial cells Mac ophages Mu ine LRT (B onchi) Epi helial cells Impo in C Human LRT * ** D Human URT α1 0 1 2 3 4 5 6 7 8 α3 α4 α5 α7 Impo in Rela i e mRNA exp ession le els (N0 (GOI) / N0 (GAPDH)) * ** Mu ine URT Mu ine LRT *** * ** *** ** Impo in-α1 H KJ G URT LRT (BE) LRT (A) 4 6 8 10 12 Impo in-α3 URT LRT (BE) LRT (A) Impo in-α5 URT LRT (BE) LRT (A) Impo in-α7 URT LRT (BE) LRT (A) IImpo in-α4 URT LRT (BE) LRT (A) *** *** *** ** *** ** *** *** *** E 0 1 2 3 4 5 6 7 8 0 1 2 3 4 5 6 7 8 0 1 2 3 4 5 6 7 8 Rela i e mRNA exp ession le els (N0 (GOI) / N0 (GAPDH)) α1 α3 α4 α5 α7 Impo in α1 α3 α4 α5 α7 Impo in α1 α3 α4 α5 α7 Impo in Rela i e mRNA exp ession le els (N0 (GOI) / N0 (GAPDH)) 0 2 4 6 8 10 12 0 2 Rela i e mRNA exp ession le els (N0 (GOI) / N0 (GAPDH)) 4 6 8 10 12 0 2 4 6 8 10 12 0 2 4 6 8 10 12 0 2 URT LRT B onchi Al eoli Impo in-α LNasal Concha _ Rela i e impo in-α p o ein amoun (α /GAPDH) Mu ine LRT 0 5 10 15 20 α1 α3 α5 α7 Impo in * Impo in-α3 GAPDH MW [kDa] 58 37 Mu ine LRT Impo in-α1 Impo in-α5 Impo in-α7 60 58 GAPDH 37 GAPDH 37 Dilu ion Impo in-α FLAG 1:5 1:200 1:5 1:100 1:5 1:50 1:5 1:50 F * ** Figu e 1. Impo in-aExp ession in he Mammalian Respi a o y T ac (A and B) Impo in-a1, -a3, o -a5/7 p o eins we e s ained ( ed) in human (A) o mu ine (B) uppe (URT, nasal epi helial cells) and lowe espi a o y ac (LRT, b onchi and al eoli) sec ions by IHC-P (immunohis ochemis y-pa a in p o ocol) and coun e s ained wi h hema oxylin. The impo in-a7 an ibody c oss- eac s wi h impo in-a5. 4003(URT and b onchi in A) o 10003(al eoli in A and B) o iginal magni ica ion. Scale ba , 10 mm. (C and D) Impo in-amRNA le els in he human (C) and mu ine (D) URT and LRT de e mined by qRT-PCR. Rela i e impo in-a1 exp ession alues we e se o 1 a e no maliza ion agains GAPDH (Glyce inaldehyd-3-phospha -dehyd ogenase). Each da a poin ep esen s an indi idual sample (n = 6–12). (E and F) Impo in-ap o ein amoun s in mu ine LRT. Wes e n blo analyses using impo in-aiso o m-speci ic an ibodies and GAPDH adjus men we e pe o med o de e mine endogenous impo in-ap o ein amoun s (a1, a3, a5, a7) in mu ine o gans. Fo ela i e quan i ica ion (E), s anda d cu es o a ini y-pu i ied, FLAG- agged impo in-ap o eins we e used. Rela i e impo in-a1 p o ein amoun in mu ine LRT was se o 1. Da a shown ep esen means ±SD (n = 3 biological eplica es; echnical eplica es: n = 1–2 pe o gan, n = 1–2 wes e n blo analyses). Rep esen a i e wes e n blo s o he endogenous impo in-aiso o ms and he s anda d cu es a e shown in (F). The gaps in (F) depic c opping o he ele an bands un on he same gel. (G–K) Compa ison o impo in-amRNA exp ession le els be ween mu ine URT and LRT: a1 (G), a3 (H), a4 (I), a5 (J), o a7 (K). A, al eoli; BE, b onchiola epi helium; n = 8–12. (L) Schema ic pa i ion o he RT wi h inc easing impo in-amRNA le els om URT o LRT. *p < 0.05; **p < 0.01; ***p < 0.001. Cell Repo s 31, 107549, Ap il 21, 2020 3 F G K LM D E H IJ N ABC O (legend on nex page) 4Cell Repo s 31, 107549, Ap il 21, 2020 ai way epi helial cells (Figu es 2F and 2G). These indings migh sugges ha impo in-a3 gene exp ession i sel could also be a ec ed by TNF-a ea men . In o de o unde s and how impo - in-a3 gene ansc ip ion is egula ed, we sc eened he p omo e egion o he impo in-a3 gene o he p esence o po en ial an- sc ip ion ac o binding si es. We ound ha he p omo e egion o he impo in-a3 gene con ains a binding si e o NF-kB, as shown by ea lie ch oma in immunop ecipi a ion s udies (Raska- o e al., 2012)(Figu e 2H). Consis en ly, using a epo e cons uc encoding o enilla luci e ase unde he con ol o he impo in-a3 p omo e , we could show ha impo in-a3 gene exp ession is di e en ially a ec ed upon TNF-a ea men in a cell- ype- and dose-dependen manne (Figu es 2I–2K). In clinically ele an p ima y human ai way epi helial cells as well as in gene a ed MEFs he ein, impo in-a3 p omo e ac i i y was highes upon low-dose TNF-a ea men , while high-dose TNF-a ea men esul ed in a educ ion o impo in-a3 gene exp ession (Figu es 2J and 2K). Howe e , mul i-passage cance ous A549 cells p esen ed high esis ance o TNF-a ea - men (Figu e 2I), sugges ing a delayed dose- esponse cu e. T ea ing MEFs wi h o he cy okines, such as in e leukin (IL)-10 o ans o ming g ow h ac o b(TGF-b) did no signi ican ly al e impo in-a3 p omo e ac i i y, unlike TNF-a ea men (Figu es S2A and S2B), albei IL-10 p esen ed a sligh induc ion (Fig- u e S2A). Nex , we wan ed o assess whe he TNF-a-ac i a ed NF-kB is di ec ly in ol ed in egula ing impo in-a3 gene an- sc ip ion. The e o e, we used cells de icien in he NF-kB essen- ial modula o (NEMO), he egula o y subuni o he IKK (IkB ki- nase), which is equi ed o NF-kB phospho yla ion and hus i s ac i a ion. Impo in-a3 mRNA le els we e educed in TNF- a- ea ed NEMO / cells compa ed o un ea ed con ols (Fig- u e 2L). As expec ed, educed impo in-a3 mRNA le els also e- sul ed in educed impo in-a3 p o ein exp ession in NEMO / cells ea ed wi h TNF-aas compa ed o un ea ed cells (Fig- u e 2M). Reduced impo in-a3 exp ession in NEMO / cells could be es o ed upon ans ec ion o a NEMO-exp essing plasmid back in o NEMO / cells (Figu e 2M). Con ol ea men o NEMO / cells wi h IL-6, IL-10, in e e on (IFN)-go TGF-b showed simila ly educed impo in-a3 mRNA exp ession le els as upon TNF-a ea men (Figu e 2N). I is impo an o no e ha IL-6 ansc ip ion is di ec ly dependen on NF-kB, unlike IL-10, IFN-go TGF-b. These indings suppo he concep ha TNF-a-ac i a ed NF-kB is c ucial o impo in-a3 gene exp es- sion. Howe e , o he cy okines migh also educe impo in-a3 exp ession a high, bu no low, cy okine concen a ions (Fig- u e S2C). Ou indings suppo he concep ha impo in-a3 gene exp ession is con olled by TNF-a-ac i a ed NF-kBina cell- and dose-dependen manne . This is in line wi h gene al con ol mechanisms in cells, such as nega i e o posi i e eed- back loops, whe e gene ansc ip ion is egula ed (e.g., ei he by ac i a ing o blocking he p omo e , espec i ely, depending on demand) (Figu e 2O). Howe e , o he , no ye de ined, NF-kB- independen pa hways migh also a ec impo in-a3 gene exp ession. Fu u e s udies a e equi ed o dissec he impo in- a3 p omo o o ansc ip ion ac o binding si es o he han NF-kB. High-Le el TNF-a-Inducing Human-Type HPAIV In ec ions Reduce Impo in-a3 P o ein Exp ession Le els in Human Respi a o y Cells Since he NF-kB anspo ing nuclea impo ac o impo in-a3 was he only iso o m in he lung ha al e ed i s exp ession le els upon TNF-a ea men , we wan ed o know how impo in-a3is egula ed unde espi a o y s ess associa ed wi h cellula cy o- kine changes. The e o e, we used an in luenza in ec ion model. We in ec ed MEFs wi h highly pa hogenic a ian in luenza A i us (HPAIV) mu an s ha we e known o ac as low- o high-le el TNF-ainduce s (Figu e 3A). Then, we in ec ed MEFs ei he wi h he low-le el TNF-a-inducing HPAIV con aining an a ian- ype polyme ase signa u e (SC35-PB2 701D ), common in in lu- enza A i us (IAV) ha ing a low eplica i e i ness in mammals, o i s high-le el TNF-a-inducing HPAIV coun e pa con aining a human- ype polyme ase signa u e (SC35M-PB2 701N ), com- mon in IAV possessing a high eplica i e i ness and i ulence Figu e 2. Molecula Func ion and Regula ion o Impo in-a3 (A) Schema ic o e iew o impo in-a3-media ed NF-kB p65 nuclea ansloca ion. (B) Pull-down o NF-kB om lysa es o un ea ed o TNF-a- ea ed A549 cells wi h Sepha ose-immobilized GST-impo in-a1 and -a3. Exp ession o GST-im- po in-aiso o ms was e i ied wi h Coomassie s aining (uppe panel), and bound NF-kB p105, p65, and p50 we e de ec ed ia wes e n blo (lowe panel). GAPDH p o ein de ec ion se ed as loading con ol. One ep esen a i e expe imen is shown ou o h ee independen expe imen s. (C) E ec o TNF-a ea men on exp ession and localiza ion o NF-kB p65 and impo in-a3 in WT MEFs (n = 3). (D and E) Nuclea ac ion (NF; con ol p84) and cy oplasmic ac ion (CF; con ol GAPDH) localiza ion o NF-kB p65 in a3 / MEFs o WT con ols he eo (a3 +/+ ) a e TNF-a ea men using cell ac iona ion assay (D) o immuno luo escence (E) as well as quan i ica ions he eo (n = 3–5). Scale ba , 20 mm. (F and G) A e seeding, cells we e se um s a ed o 24 h. Subsequen ly, cells we e con ol ea ed (w/o) o ea ed wi h TNF-a. Endogenous impo in-a3 and NF- kB p65 p o ein le els we e de ec ed and quan i ied o whole-cell lysa es (WCLs), CFs and NFs o WT MEFs (n = 3) (F), and WCLs o HSAEpCs (n = 3) (G). Con ol- ea ed samples o each ac ion we e se o 100%. (H) NF-kB binding si es in he p omo e elemen o he human impo in-a3 gene in A549 cells (Raska o e al., 2012). (I–K) E ec o TNF-a ea men on impo in-a3 p omo e ac i i y in HSAEpC (n = 2) (I), MEFs (n = 3) (J), and A549 cells (n = 6) (K) upon con ol (C ) o impo in-a3 p omo e epo e cons uc (a3) ans ec ion. Rela i e impo in-a3 p omo e ac i i y in con ol- ea ed samples was se o 100%. (L) Impo in-a3 mRNA le els in mock o TNF-a- ea ed MEF NEMO / cells (n = 6). Rela i e exp ession alues o impo in-a3 in samples we e no malized o GAPDH, and impo in-a1 mRNA exp ession was se o 1. Each da a poin ep esen s an indi idual sample. (M) MEF NEMO / cells we e ans ec ed wi h emp y plasmid (Mock) o NEMO and un ea ed o ea ed wi h 50 ng TNF-a. Impo in-a3 and NEMO p o ein exp ession le els we e de ec ed (n = 3). GAPDH p o ein de ec ion se ed as loading con ol. (N) MEF NEMO / cells we e ea ed wi h 10 ng TNF-a, IL-6, IL-10, IFN-gand TGF-band impo in-a3 mRNA le els we e measu ed as desc ibed abo e (n = 3) (O) Impo in-a3 gene exp ession is con olled by TNF-a-ac i a ed NF-kB in a dose-dependen manne . Low amoun s o TNF-ainduce ac i a ion o NF-kB and high exp ession o impo in-a3 (uppe panel). In con as , high amoun s o TNF-ainduce high ac i a ion o NF-kB and low exp ession o impo in-a3 (lowe panel). Da a shown ep esen means ±SD o a leas h ee independen biological expe imen s. *p < 0.05; **p < 0.01; ***p < 0.001. Cell Repo s 31, 107549, Ap il 21, 2020 5 SC35- PB2 701D SC35M- PB2 701N 0 500 1,000 1,500 TNF-α concen a ion [pg/ml] TNF-α *** A 0 50 100 150 Rela i e impo in-α3 amoun [(α3/GAPDH) %] SC35- PB2701D SC35M- PB2701N Mock *** * 0 50 100 150 Rela i e impo in-α3 amoun [(α3/NP) %] *** SC35- PB2701D SC35M- PB2701N Mock Mock SC35- PB2701D SC35M- PB2701N Impo in-α3 NP GAPDH MW [kDa] 58 56 37 BC D E F Mock SC35M- PB2701N G α1 0 50 100 150 Rela i e impo in-α amoun [(α/GAPDH) %] α7α5α4 Impo in SC35M-PB2701N L J Time p. i. [h] Vi us i e [log (p. .u. • ml-1)] 024487296 0 2 4 6 8 *** *** *** SC35-PB2701D – MOI 0.1 024 48 72 96 0 2 4 6 8 Vi us i e [log (p. .u. • ml-1)] Time p. i. [h] *** *** *** SC35-PB2701D – MOI 1 Vi us i e [log (p. .u. • ml-1)] Time p. i. [h] 0 24487296 0 2 4 6 8 *** *** *** *** SC35M-PB2701N – MOI 0.01 WT α3-/- α7-/- Vi us i e [log (p. .u. • ml-1)] Time p. i. [h] 024487296 0 2 4 6 8 *** *** SC35M-PB2701N – MOI 0.001 HI WT α3-/- α7-/- WT α3-/- α7-/- WT α3-/- α7-/- K 24 48 α3+/+ α3-/- -TNF-α α3+/+ α3-/- 0 0 2 4 6 8 10 Vi us i e [log (p. .u. • ml-1)] Time p. i. [h] +TNF-α (legend on nex page) 6Cell Repo s 31, 107549, Ap il 21, 2020 in mammalian species. Impo in-a3 p o ein le els we e s ongly educed in human- ype HPAIV compa ed o a ian- ype HPAIV- in ec ed MEFs (Figu es 3B–3E). In con as , p o ein exp ession le els o o he impo in-aiso o ms (a1, a4, a5, and a7) we e no a ec ed (Figu es 3F and 3G). Nex , we wan ed o assess whe he eplica ion o a ian- and human- ype HPAIV is a ec ed in cells de icien in impo in-a3. Cells de icien in impo in-a3 we e ob ained om ib oblas s o mice gene a ed o his s udy wi h a dele ed impo in-a3 gene (a3 / ;a3 / MEFs; Figu es S2D and S2E). MEFs wi h a de icien impo in-a7 gene we e ob- ained om mice wi h a dele ed impo in-a7 gene (a7 / ), desc ibed p e iously (Gab iel e al., 2011). Replica ion kine ics in MEFs e ealed ha a ian- ype bu no human- ype HPAIV eplica ion is impai ed in cells wi h a dele ed impo in-a3 gene (a3 / ), unlike in con ol cells, wi h a dele ed impo in-a7 gene (a7 / ) ha ac s as a posi i e ac o o human- ype HPAIV epli- ca ion in mammalian cells (Hudje z and Gab iel, 2012)(Figu es 3H and 3I). In e es ingly, a ian- ype HPAIV eplica ion in a3 / MEFs is inc eased by >1,000 imes compa ed o WT con ol cells. Human- ype HPAIV eplica ion was compa able be ween WT and a3 / cells (Figu es 3J and 3K). In line, human- ype SC35M-PB2 701N HPAIV eplica ion was no a ec ed in human H1299 a3 +/+ cells upon TNF-a ea men , likely due o he s ong inhibi ion o impo in-a3 p o ein exp ession (Figu e 3L). In human H1299 a3 / cells, SC35M-PB2 701N HPAIV eplica ion sligh ly inc eased, sugges ing some esidual inhibi o y ac i i y o impo in-a3 in human cells (Figu e 3L). These indings show ha low-le el TNF-a-inducing HPAIVs wi h a ian- ype polyme - ase signa u es a e highly sensi i e o impo in-a3-media ed i us es ic ion. In con as , high-le el TNF-a-inducing HPAIVs wi h human- ype polyme ase genes a e esis an o impo in-a3- media ed i us es ic ion. Impo in-a3 Belongs o he Mos Conse ed Nuclea T anspo Fac o s be ween A ian and Mammalian Species Di e en ial egula ion o impo in-a3 exp ession le els upon a ian- and human- ype HPAIV in ec ions p omp ed us o analyze he sequence iden i y o impo in-aiso o ms in a ious a ian and mammalian species. Among all impo in-aiso o ms analyzed, impo in-a1 was he mos di e se nuclea impo ac- o , wi h amino acid homology o 83.52% be ween chicken and humans (Figu e 4A; Table S1). The iso o ms impo in-a3and impo in-a4 displayed he highes amino acid homologies be- ween chicken and humans o 99.04% and 98.08%, espec- i ely (Figu es 4B and 4C; Table S1). Impo in-a5, -a6, and -a7 p esen ed amino acid homologies be ween chicken and humans o 95.91%, 94.22%, and 94.4%, espec i ely (Figu es 4D–4F; Table S1). These indings sugges ha pa icula ly im- po in-a3andimpo in-a4, bo h known nuclea anspo e s o NF-kB(Fage lund e al., 2005), a e highly conse ed ac oss species kingdoms. Howe e , impo in-a3 is exp essed up o ~5- imes-highe le els han impo in-a4in hemu ineand human lung (Figu es 1C and 1D), highligh ing pa icula ly he impo ance o impo in-a3in espi a o yimmune e- sponses. Thus, acqui ing he abili y o down egula e impo in- a3 in he lung migh se e as an impo an basis in a ian- mammalian i us ansmission. In o de o see whe he di e en ial impo in-a3 egula ion is also p esen in human iso- la es, we analyzed human- ype HPAIV s ains ha caused poul y ou b eaks wi h human in ec ions in he Ne he lands (H7N7) o Thailand (H5N1). In ec ion o p ima y lung cul u es wi h cy okine s o m, including TNF-a-inducing H5N1 and H7N7 HPAIV isola es wi h human- ype polyme ase signa u es (PB2 627K ;PB2 701N ), showed signi ican ly educed impo in-a3 exp ession le els compa ed o hei coun e pa s wi h a ian- ype polyme ase signa u es (PB2 627E ;PB2 701D )(Figu es 4G and 4H). These indings highligh he c ucial ole o impo in- a3 as a espi a o y ac o conse ed ac oss animal kingdoms in HPAIV in e species ansmission. Human-Type Polyme ase Signa u es in HPAIV Media e Reduced Impo in-a3 mRNA Exp ession Le els in he Mu ine BE Nex , we wan ed o assess whe he di e en ial egula ion o impo in-a3 can also be de ec ed in a mu ine in ec ion model. The e o e, we assessed impo in-a3 exp ession le els in lung samples o HPAIV-in ec ed WT mice. In ec ion wi h a ian- ype SC35-PB2 701D led o inc eased impo in-a3 mRNA le els in he lung compa ed o unin ec ed con ols on day 3 pos in ec ion (p.i.) (Figu es 5A and 5B). In con as , in ec ion wi h human- ype SC35M-PB2 701N esul ed in signi ican ly educed impo - in-a3 mRNA le els (Figu e 5C). Since he human- ype SC35M HPAIV con ains mainly wo hos adap i e signa u es (PB2 701N and NP 319K), we analyzed ecombinan i uses wi h hese single mu a ions o dissec hei indi idual impac on he dys egula ion o impo in-amRNA ansc ip ion. He ein, he PB2 D701N hos adap i e mu a ion was p edominan ly espon- sible o educed impo in-a3 ansc ip ion, while i s e ec was u he enhanced when combined wi h he NP N319K hos Figu e 3. HPAIV Replica ion Kine ics in Impo in-a3 / Cells (A) TNF-ainduc ion in MEFs in ec ed wi h a ian- ype SC35-PB2 701D o human- ype SC35M-PB2 701N H7N7 HPAIV (MOI = 0.1) measu ed by ELISA (n = 3). (B–E) P o ein ex ac s he eo we e analyzed and quan i ied by wes e n blo a 48 h p.i. Impo in-a3 and i al NP p o ein we e de ec ed (B and D) and quan i ied (C and E) wi h GAPDH (B and C) o NP adjus men (D and E) (n = 4). Dashed lines in (D) depic c opping o he ele an bands un on he same gel. (F and G) Addi ionally, impo in-a1, -a4, -a5, and -a7 and i al NP p o ein we e de ec ed and quan i ied wi h GAPDH adjus men o con i m ha obse ed e ec s we e speci ic o impo in-a3. Con ol- ea ed, unin ec ed samples o SC35-PB2 701D -in ec ed samples we e se o 100%. C oss- eac i i y o impo in-aan i- bodies wi h NP esul ed in a band (#) in (B) and (F). (H–K) G ow h kine ics o a ian- ype SC35-PB2 701D (H and I) and human- ype SC35M-PB2 701N (J and K) H7N7 HPAIV in impo in-a / MEFs (n = 3). WT (black), impo in-a3 / (a3 / , ed), and impo in-a7 / (a7 / , g een) MEFs we e in ec ed wi h low (H and J) o high (I and K) mul iplici ies o in ec ion (MOI). Vi us i e s gi en in plaque- o ming uni s (p. .u.) we e de e mined by plaque assay on MDCKII cells (0, 24, 48, 72, and 96 h p.i.). (L) H1299 a3 +/+ (black) and a3 / ( ed) cells we e in ec ed wi h human- ype SC35M-PB2 701N HPAIV (MOI = 0.001) and eplica ion kine ics measu ed in he p esence o absence o TNF-a(n = 3). Vi us i e s we e de e mined by plaque assay on MDCKII cells (0, 24, 48 h p.i.). Da a shown ep esen means o ela i e impo in-aamoun s o loga i hmic i us i e s ±SD o a leas h ee independen biological expe imen s (*p ˂0.05; ***p < 0.001). Cell Repo s 31, 107549, Ap il 21, 2020 7 adap i e signa u e (Figu es 5D–5F). Compa ed o all o he im- po in-aiso o ms, impo in-a3 con inuously showed he mos p ominen al e a ions. In e es ingly, he mos signi ican al e - a ions in impo in-a3 mRNA le els we e de ec ed a 3 days p.i. compa ed o 1 day p.i. (Figu es S2F–S2K), consis en wi h he concep ha accumula ing cy okine le els h ough mul iple epli- ca ion kine ics is equi ed o a p o ound cy okine-dependen ep ession o impo in-a3 ansc ip ion. A ian- ype SC35- PB2 701D i us eplica ion, which caused an inc ease in impo - in-a3 mRNA le els, was es ic ed o he espi a o y ac (Fig- u es S3A and S3B). In con as , human- ype SC35M-PB2 701N i al eplica ion led o a signi ican educ ion o impo in-a3 mRNA le els in he lung, inc eased i us load in he lung, and sys emic i us sp ead (Figu e S3C). The ecombinan i uses wi h he single o combined human- ype PB2 701N and NP 319K signa u es showed s epwise sys emic sp ead along wi h hei abili y o educe impo in-a3 mRNA le els in he lung (Fig- u es S3D–S3F). These indings u he highligh ha hos 0 100 200 300 400 500 48 h p.i.24 h p.i. 48 h p.i.24 h p.i. Rela i e impo in-α3 amoun [(α3/NP) %] HSAEpC H5N1 H7N7 PB2701D PB2701N PB2627E PB2627K ** *** 0 200 400 600 800 1,000 1,200 Rela i e impo in-α3 amoun [(α3/NP) %] 48 h p.i.24 h p.i. 48 h p.i.24 h p.i. HBEpC H5N1 H7N7 * * * H Anas pla y hynchos Gallus gallus Mus musculus Sus sc o a Macaca ascicula is Homo sapiens Homo sapiens Macaca ascicula is Sus sc o a Mus musculus Gallus gallus Anas pla y hynchos Anas pla y hynchos Gallus gallus Mus musculus Sus sc o a Macaca ascicula is Homo sapiens Homo sapiens Macaca ascicula is Sus sc o a Mus musculus Gallus gallus Anas pla y hynchos Anas pla y hynchos Gallus gallus Mus musculus Sus sc o a Macaca ascicula is Homo sapiens Homo sapiens Macaca ascicula is Sus sc o a Mus musculus Gallus gallus Anas pla y hynchos 85 90 95 100 iden i y AB C Impo in-D1Impo in-D3Impo in-D4 Anas pla y hynchos Gallus gallus Mus musculus Sus sc o a Macaca ascicula is Homo sapiens Homo sapiens Macaca ascicula is Sus sc o a Mus musculus Gallus gallus Anas pla y hynchos D Impo in-D5 Anas pla y hynchos Gallus gallus Sus sc o a Macaca ascicula is Homo sapiens Homo sapiens Macaca ascicula is Sus sc o a Gallus gallus Anas pla y hynchos EImpo in-D6 G Gallus gallus Mus musculus Sus sc o a Macaca ascicula is Homo sapiens Homo sapiens Macaca ascicula is Sus sc o a Mus musculus Gallus gallus 85 90 95 100 iden i y FImpo in-D7 Figu e 4. High Impo in-a3 Homology ac oss Species and I s Role in HPAIV In e species T ansmission om Bi ds o Humans (A–F) Impo in-ahomologies ac oss animal kingdoms. Homolgies o impo in-a1 (A), -a3 (B), -a4 (C), -a5 (D), -a6 (E), and -a7 (F). Pai wise amino acid sequence iden i ies we e calcula ed o impo in-aac oss species. (G and H) P ima y human b onchial (HBEpC; G) o p ima y human small ai way epi helial cells (HSAEpC; H) we e in ec ed wi h clinical a al case A/Thailand/ 1(KAN-1)/2004 (H5N1, MOI = 1) o A/Ne he lands/219/2003 (H7N7, MOI = 10) isola es ha bo ing he o iginal human- ype signa u es (PB2 701N o PB2 627K , espec i ely) o hei ecombinan a ian- ype coun e pa s (PB2 701D o PB2 627E , espec i ely). Impo in-a3 was quan i ied in NP-adjus ed wes e n blo s o HBEpC (H) and HSAEpC (I) in ec ed wi h H5N1 (MOI = 0.1) o H7N7 (MOI = 1). Impo in-a3/NP a ios o human- ype i us in ec ed samples a 24 h p.i. we e se o 100%. Da a shown ep esen means ±SD o a leas h ee independen biological expe imen s. *p < 0.05; **p < 0.01; ***p < 0.001. 8Cell Repo s 31, 107549, Ap il 21, 2020 STAR+METHODS KEY RESOURCES TABLE LEAD CONTACT AND MATERIALS AVAILABILITY Fu he in o ma ion and eques s o esou ces and eagen s should be di ec ed o and will be ul illed by he Lead Con ac , Guelsah Gab iel ([email p o ec ed]). Ma e ials, eagen s and mouse lines o his s udy a e a ailable wi h a comple ed Ma e ial T ans e Ag eemen . REAGENT o RESOURCE SOURCE IDENTIFIER An ibodies impo in-a1 Abcam ab6036; RRID: AB_305245, ab54489; RRID: AB_2249727 impo in-a3 Abcam ab6039; RRID: AB_1977565 impo in-a3 No us Biologicals #NB100-93345; RRID: AB_1237130, eplaced by #NB100-81651; RRID: AB_1146049 impo in-a4 Abcam ab6038; RRID: AB_305247 impo in-a5/7 Uni e si y o L€ ubeck Ko ¨hle e al., 1999 NF-kB p65 Cell Signaling #3987; RRID: AB_2341215 Vi us S ains SC35-PB2 701D Uni e si y o Ma bu g Gab iel e al., 2011 SC35M-PB2 701N Uni e si y o Ma bu g Gab iel e al., 2011 A/Thailand/1(KAN-1)/2004 (H5N1) Uni e si y o Ma bu g Pu ha a hana e al., 2005 A/Ne he lands/219/2003 E asmus Medical Cen e Fouchie e al., 2004 Biological Samples Human biopsy ma e ial om nasal concha and he lung E asmus Medical Cen e Ro e dam, he Ne he lands #MEC-2011-129, MEC 2008-207, MEC 2009-128 Chemicals, Pep ides and Recombinan P o eins Human TNF-aR&D Sys ems #210-TA Mouse TNF-aBioLegends #575204 C i ical Comme cial Assays Dual-Luci e ase Repo e Assay Sys em P omega E1910 Ligh Swi ch Luci e ase Assay Reagen Swi chGea Genomics LS010 Deposi ed Da a Eu opean Nucleo ide A chi e (ENA) Hein ich Pe e Ins i u e, Hambu g h ps://www.ebi.ac.uk/ena/da a/ iew/PRJEB8023 Expe imen al Models: Cell Lines Impo in-a3 / MEFs Max Delb € uck Cen e , Be lin This s udy Impo in-a3 / H1299 Hein ich Pe e Ins i u e, Hambu g This s udy NEMO / MEFs Hein ich Pe e Ins i u e, Hambu g Mak is e al., 2000 HBEpC P omoCell C-12640 HSAEpC P omoCell C-12642 Expe imen al Models: O ganisms/S ains Impo in-a3 / mice Max Delb € uck Cen e , Be lin This s udy Recombinan DNA pLigh Swi ch_P om-impo in-a3 Swi chGea Genomics This s udy pLigh Swi ch_P om-con ol Swi chGea Genomics This s udy So wa e and Algo i hms Muscle e sion 3.8.1551 No applicable Edga , 2004 Clus al Omega e sion 1.2.4 No applicable Sie e s e al., 2011 Cell Repo s 31, 107549, Ap il 21, 2020 e1 EXPERIMENTAL MODEL AND SUBJECT DETAILS Human Subjec s Biopsy ma e ial om human unin ec ed URT (nasal concha) and LRT (lung, mainly consis ing o al eola issue) was ob ained om he E asmus Medical Cen e in Ro e dam, he Ne he lands, om dono s a he E asmus Medical Cen e in Ro e dam. App o al o use he clinical samples o scien i ic use was ob ained om he Du ch Medical E hical Commi ee unde he pe mi numbe s #MEC- 2011-129, MEC 2008-207 and MEC 2009-128. Dono sex and age is unknown. Cells and Vi uses Cell lines o human emb yonic kidney cells (HEK293T, ATCC), human al eola adenoca cinoma cells (A549, H1299, ATCC), a ican g een monkey kidney cells (Ve oE6) and human ce ical adenoca cinoma cells (HeLa, ATCC) we e g own in Dulbecco’s modi ied Eagle’s medium (DMEM, PAA) supplemen ed wi h 10% e al cal se um (FCS; PAA), 1% penicillin/s ep omycin (P/S, PAA), and 1% L-Glu amine (PAA). Mouse emb yonic ib oblas s (MEF, see desc ip ion o hei gene a ion in a sepa a e pa ag aph below) we e g own in DMEM supplemen ed wi h 10% FCS, 1% P/S, 1% L-Glu amine, 1% sodium py u a e, and 1% non-essen ial amino acids (NEAA, PAA). Immo alized NEMO / MEFs (Mak is e al., 2000) we e kindly p o ided by Michael Ka in (Uni e si y o Cali o nia, San Diego). Madin Da by canine kidney cells (MDCKII, ATCC) we e g own in Minimal Essen ial Medium (MEM, PAA) supplemen ed wi h 10% FCS, 1% P/S, and 1% L-Glu amine. P ima y human b onchial (HBEpC, P omoCell, C-12640) and p ima y human small ai way epi helial cells (HSAEpC, P omoCell, C-12642) we e g own in Ai way Epi helial Cell Media o Small Ai way Epi helial Cell Media (P omoCell), espec i ely, supplemen ed wi h 1% P/S. HBEpC cell dono was a 52 yea old emale subjec . HSAEpC cell dono was a 57 yea old emale subjec . Cells we e cul i a ed a 5% CO 2 , 96% H and 37C. Recombinan H7N7 HPAIV i uses SC35-PB2 701D , SC35M-PB2 701N , he single-poin mu an s SC35-PB2 701N and SC35-NP 319K o he double mu an SC35-PB2 701N -NP 319K (Gab iel e al., 2011) as well as ecombinan A/Thailand/1(KAN-1)/2004 (H5N1) wild- ype s ain (H5N1-PB2 701N )(Gab iel e al., 2011; Pu ha- a hana e al., 2005), he a ian- ype single poin mu an he eo (H5N1-PB2 701D ) and H7N7 HPAIV i us isola es A/Ne he lands/219/ 2003 (H7N7-PB2 627K , a al case) o he ecombinan i us he eo (H7N7-PB2 627E )(Czudai-Ma wich e al., 2014; Fouchie e al., 2004) we e used. All expe imen s wi h H5N1 and H7N7 i uses we e conduc ed a he Hein ich Pe e Ins i u e, Leibniz Ins i u e o Expe imen al Vi ology in Hambu g, Ge many, in biosa e y le el 3 acili ies, excep o he SC35M-PB2 701N s ain ha was classi ied as biosa e y le el 2 agen , and app o ed by he ele an Ge man au ho i ies (Gen echnikbeho ¨ de Hambu g). Animals Impo in-a3 / mice in he C57BL/6 gene ic backg ound and WT li e ma es he eo we e b ed and housed unde speci ic pa hogen- ee condi ions a he animal acili y o he Hein ich Pe e Ins i u e, Leibniz Ins i u e o Expe imen al Vi ology, Hambu g, Ge many. We used 6-12 week old emale and male mice. Expe imen al g oups we e ma ched ega ding equal numbe s o emale and male mice as well ma ched in compa able age g oups. Animal expe imen s we e pe o med in s ic acco dance wi h he guidelines o he Ge man Animal Wel a e Regula ions. All animal p o ocols we e app o ed by he ele an Ge man au ho i y (Beho ¨ de € u S ad en wicklung und Umwel Hambu g, license numbe s: 29/09, 54/11). Mice we e humanely killed upon > 25% weigh loss o upon achie emen o de ined body index sco es (e.g., on phys- ical appea ance, clinical signs, beha io , ea ing and d inking e c.) acco ding o he Ge man Animal Wel a e Regula ions. METHOD DETAILS T ans ec ion and Vec o s T ans ec ions we e pe o med using Lipo ec amine 2000 (In i ogen) o je PRIME(Polyplus-T ans ec ion) acco ding o he manu- ac u e ’s ins uc ions. Vec o cons uc s used we e pHW2000-(SC35-PB2 701D , SC35-PB2 627K , SC35-PB1, SC35-PA, SC35-NP, SC35M-PB2 701N , SC35M-PB1, SC35M-PA, SC35M-NP) (Gab iel e al., 2005), pcDNA-impo in-a1-N-FLAG, pcDNA-impo in-a3- N-FLAG, pcDNA-impo in-a5-N-FLAG and pcDNA-impo in-a7-N-FLAG, pPol-I-NP-Luc-human (Gab iel e al., 2005), pRL-TK (P omega), pLigh Swi ch_P om-con ol and pLigh Swi ch_P om-impo in-a3 ( enilla luci e ase epo e cons uc s; Swi chGea Ge- nomics), pFL-TK and pCMVTAG-NEMO (Addgene). The pcDNA-impo in-a-N-FLAG cons uc s we e desc ibed p e iously (Gab iel e al., 2011; Wang e al., 1997). pFL-TK was gene a ed by ampli ica ion o he i e ly luci e ase gene om he pPol-I-NP-luc-human cons uc (Gab iel e al., 2005) using p ime s p5_NheI_Fluc_ o (50-GCGCTAGCCACCATGGAAGACGCCAAAAACATAAAGAA AGGCCCGGC-30) and p3_Fluc_No I_ e (50-GCGCGGCCGCTTACAATTTGGACTTTCCGCCCTTCTTGG-30). Inse ion o he i e ly luci e ase gene in o pRL-TK wi hou he enilla luci e ase gene was pe o med ia he NheI and No I es ic ion si es. An ibodies P ima y an ibodies used o wes e n blo and immunohis ochemical analyses include goa an i-impo in-a1 (Abcam, ab6036), abbi an i-impo in-a1 (Abcam, ab54489), goa an i-impo in-a3 (Abcam, ab6039), goa an i-impo in-a4 (Abcam, ab6038), abbi an i-im- po in-a3 (No us Biologicals, #NB100-93345, eplaced by #NB100-81651), abbi an i-impo in-a3(Ko ¨hle e al., 1997), abbi an i- impo in-a5/a7(Ko ¨hle e al., 1999), abbi an i-NF-kB p65 (Cell Signaling, #3987), abbi an i-LSD1 (Cell Signaling, #2139), abbi e2 Cell Repo s 31, 107549, Ap il 21, 2020 an i-GAPDH (Cell Signaling, #2118), mouse an i-nuclea ma ix p o ein p84 (Abcam, ab487), mouse an i-FLAG M2 (Sigma-Ald ich, #F3165), abbi an i-HA (Sigma-Ald ich, #H908), mouse an i-in luenza A i us nucleop o ein (HB65; Ame ican Type Cul u e Collec- ion, Manassas, VA) an ibodies and abbi an i-FPV se um (Gab iel e al., 2008). An i- abbi -HRP, an i-goa -HRP, an i-mouse-HRP (Sigma-Ald ich, #A8275, #A5420, and #A4416, espec i ely), and IRDye-conjuga ed an i- abbi (Li-Co ) an ibodies we e used as sec- onda y an ibodies o wes e n blo . Impo in-asequence analysis Nucleo ide sequences o he coding egions o impo in-a1, impo in-a3, impo in-a4, impo in-a5, impo in-a6 and impo in-a7 we e downloaded om he NCBI nucleo ide da abase o Homo sapiens (GenBank: NM_001320611.1, NM_002268.5, NM_002267.4, CR456743.1, NM_001366307.2, NM_012316.5), Macaca ascicula is (GenBank: NM_001283904.1, XM_005546256.2, XM_005585866.2, XM_005547987.2, XM_005551660.2, XM_005544119.2), Sus sc o a (GenBank: NM_001163404.1, XM_021069735.1, NM_001193575.1, NM_001163405.1, XM_005654393.3, XM_021095806.1), Mus musculus (GenBank: NM_010655.3, NM_008467.4, NM_008466.5, BC006771.1, NM_008468.4), Gallus gallus (GenBank: NM_001006209.1, NM_001007963.1, NM_001193575.1, NM_001030774.1, XM_015284586.2, NM_001012841.2) and Anas pla y hynchos (GenBank: XM_005012314.3, XM_027464723.1, XM_027471420.1, XM_027449713.1, XM_027454663.1). Fo Mus musculus, no impo in-a6 sequence was a ailable, while he impo in-a7 sequence was missing o Anas pla y hynchos. The nucleo ide sequences we e sub- sequen ly ansla ed in o amino acid sequences using he s anda d gene ic code. Fo each Impo in-a, he sequences we e aligned using Muscle e sion 3.8.1551 (Edga , 2004) wi h s anda d pa ame e s. Pai wise nucleo ide and amino acid sequence iden i ies we e hen calcula ed using Clus al Omega e sion 1.2.4 (Sie e s e al., 2011). Gene a ion o Impo in-a3 / Mice and Impo in-a / -MEFs To gene a e an impo in-a3 a ge ing cons uc , a 900 bp-long sequence ups eam and a 5100 bp-long sequence downs eam o exon 1 o he impo in-a3 gene we e cloned in o a a ge ing ec o desc ibed be o e (Wal he e al., 1998). A e homologous ecom- bina ion in emb yonic s em (ES) cells, exon 1 bea ing he ansla ional s a si e o he impo in-a3 p o ein, was dele ed. ES cells we e elec opo a ed wi h he linea ized cons uc and clones we e picked a e double selec ion wi h neomycin and gancyclo i . Posi i e clones we e iden i ied by PCR and one o hem was chosen o blas ocys injec ion. F om he injec ed ES cell clone, ge mline chi- me as we e ob ained and b ed wi h C57BL/6 mice. Impo in-a3 de icien mice we e backc ossed o eigh gene a ions o he C57BL/6 gene ic backg ound; he colony was main ained by b eeding he e ozygous mice. Fo geno yping, he ollowing p ime s we e used o PCR on genomic DNA o ail o ea biopsies: Impa3 o 5’-CCCTCAGTGTGAATTACTTCC-3’; Impa3 e 5’-GAGTC CAAAGCCACTTCGAG-3’; Impa4neo e 5’-GCCCAGTCATAGCCGAATAG-3’. To con i m he gene dele ion, absence o impo in- a3 mRNA and p o ein was de e mined by RT-PCR and wes e n blo o o gans. Absence o impo in-a3 mRNA wi h b-Ac in as e e ence was e i ied using he ollowing p ime s: Impa3Ex1_ o 50-GACAACGAGAAATTGGACAACC-30; Impa3Ex7_ e 50-CGTTCCAGATGCAATGTTTGTC-30; be a-ac in_ o 50-TACAATGAGCTGCGTGTG-30; be a-Ac in_ e 50-CACAGCCTGGATGGC TAC-30. Wild- ype (WT) and impo in-a / mu ine emb yonic ib oblas s (MEFs) we e p epa ed om mu ine emb yos ha es ed on emb yonic day 13.5 om p egnan emales. To ob ain immo alized cell lines, p ima y MEFs we e passaged a leas 25 imes acco ding o an adap ion o he 3T3 p o ocol (Toda o and G een, 1963). Gene a ion o Impo in-a3 / Cell Lines To gene a e H1299 impo in-a3 / and con ol impo in-a3 +/+ cell lines, he cells we e seeded in 12 wells and ans ec ed using Lip- o ec amine 2000 (In i ogen) wi h ei he a con ol CRISPR-Cas9 plasmid o an impo in-a3 / CRISPR-Cas9 plasmid (San a C uz Bio echnology). 48 hou s pos ans ec ion posi i e cells we e so ed ia FACS in o 96 well pla es wi h help o a g een luo escen p o ein selec ion ma ke . Clones we e es ed ia wes e n blo o impo in-a3 exp ession and posi i e clones we e cul i a ed u he o c yop ese a ion. Animal Expe imen s Mice we e anaes he ized wi h ke amine-xylazine (70 mg/kg and 7 mg/kg, espec i ely) and in anasally in ec ed wi h he espec i e i us dose in 50 ml 1x PBS. WT mice we e in ec ed wi h 6*10 4 p. .u. (100- old MLD 50 o SC35M-PB2 701N in BALB/c mice 9 ) o SC35- PB2 701D , SC35M-PB2 701N , he single-poin mu an s SC35-PB2 701N o SC35-NP 319K o he double mu an SC35-PB2 701N -NP 319K . Con ol g oups ecei ed PBS. On day 3 p.i., i e animals pe ime poin we e anaes he ized wi h an o e dose o ke amine-xylazine (140 mg/kg and 14 mg/kg, espec i ely) and sac i iced a e in aca dial pe usion wi h DEPC- ea ed 1x PBS. Pe used o gans (lung, achea, li e , spleen, b ain) we e emo ed and dissec ed in o pieces (< 5 mm) and subme ged in RNAla e RNA S abiliza ion Reagen (QIAGEN). Mu ine URT samples we e ob ained by decapi a ion o he head abo e he a las. A e skinning and sagi al di ision o he head, he issue o he nasal concha was excised and subme ged in RNAla e . Fo ela i e mRNA-Exp ession le els de e mina ion o an i i al genes unde an NF-kB p omo e by RT-qPCR, WT (n = 5) and im- po in-a3 / (n = 5) mice we e in ec ed wi h 10 6 p. .u. o SC35M. Con ol g oups ecei ed PBS. On day 3 p.i., i e animals we e anaes- he ized wi h an o e dose o ke amine-xylazine and sac i ice. Whole lungs we e emo ed and dissec ed in o pieces and subme ged Cell Repo s 31, 107549, Ap il 21, 2020 e3 in RNAla e . O gans (lung, spleen, b ain) o unin ec ed WT mice (n = 3) o wes e n blo lysa e p epa a ion we e ob ained a e sho inhala i e iso lu ane na cosis ollowed by ce ical disloca ion. A e dissec ion in o small pieces, he o gans we e immedia ely ozen on d y ice. Fo i us i e de e mina ion o o gans, WT (n = 3) and impo in-a3 / (n = 3) mice we e in ec ed wi h 6*10 4 p. .u. o SC35-PB2 701D (~0,1x MLD 50 o SC35-PB2 701D in C57BL/6 mice) o a low and high dose o SC35M-PB2 701N (10 3 p. .u.; ~0,5x MLD 50 o 6*10 4 p. .u.; ~30x MLD 50 o SC35M-PB2 701N , espec i ely). On days 3 and 6 p.i., h ee animals pe ime poin we e sac i iced, o gans (lung, achea, b ain) emo ed, i us i e s de e mined by plaque assay 64 , and he URT and LRT s ained immunohis ochemically agains in luenza i us and impo in-aan igen. Fo his, mu ine o malin- ixed, pa a in-embedded (FFPE) lungs o skinned, decalci ied FFPE heads we e hin sec ioned (3 mm). Heads we e decalci ied in 10% EDTA (pH 7,4) o 8 days, ollowed by sagi al di ision and incuba ion in same solu ion o 3 mo e days. Mouse le hal dose 50 (MLD 50 ) was assessed by in ec ing WT (n = 4-9) and impo - in-a3 / (n = 4-9) mice wi h se ial i us dilu ions o SC35-PB2 701D (10 5 , 5*10 5 , and 10 6 p. .u.) o SC35M-PB2 701N (10 2 ,10 3 , and 10 4 p. .u.). Animals we e moni o ed o weigh and su i al o 14 days p.i. and he MLD 50 was calcula ed. Humane endpoin s we e de ined acco ding o he Ge man Animal Wel a e Regula ions as desc ibed abo e. Immunohis ochemical Analysis (IHC-P) Vi al and impo in-a1, -a3, and -a7 an igen we e de e mined by IHC-P wi h an igen-speci ic p ima y and HRP-conjuga ed seconda y an ibodies. URT and lungs o in ec ed WT and impo in-a3 / mice we e excised on days 1 and 3 p.i.. FFPE hin-sec ions (3 mm) o mu ine samples and human biopsies we e p epa ed as desc ibed (Gab iel e al., 2009). Vi al an igen was s ained using he mouse an i-in luenza A nucleop o ein (HB65; Ame ican Type Cul u e Collec ion, Manassas, VA) o he abbi an i-FPV-se um, a bio in-con- juga ed an i- abbi seconda y an ibody (Jackson ImmunoResea ch, #711-066-152), he A idin/Bio in Blocking Ki SP2001 (Biozol), and he Zy oChemPlus (HRP) B oad Spec um (DAB) Ki (Zy omed) acco ding o he manu ac u e ’s ins uc ions. Fo isualiza ion o impo in-aiso o ms in mu ine and human FFPE samples, he desc ibed abbi an i-impo in-a1, abbi an i-impo in-a3 (No us Biologicals), and abbi an i-impo in-a5/a7 an ibodies we e used oge he wi h a bio in-conjuga ed an i- abbi seconda y an ibody (DAKO; #E0432), a pe oxidase-conjuga ed a idin-bio in-complex- eac ion ki (DAKO), and he HRP subs a e AEC (DAKO). Coun e - s aining was pe o med wi h hema oxylin. Since he impo in-a7 an ibody, c oss- eac s wi h impo in-a5, lung issues o mice wi h a dele ion o ei he he impo in-a5 o he -a7 gene we e addi ionally used as con ols (da a no shown). To exclude alse-posi i e s ain- ing due o c oss- eac i i y o he an ibodies, FFPE hin sec ions we e addi ionally s ained using impo in-aiso o m-speci ic blocking pep ides o impo in-a1 (aa518-529, QVQDGAPGTFNF, GenSc ip ), impo in-a3 (aa509-521, NSSANVPTEGFQF, Abcam, #ab23144), impo in-a5 (aa3-16, TPGKENFRLKSYKN, GenSc ip ) and impo in-a7 (aa3-12, MASPGKDNYR; aa526-536, PEAP- MEGFQL, GenSc ip ) in di e en concen a ions (0.2 mg/ml, 2 mg/ml, and 20 mg/ml) (Table S5). P ima y an ibodies we e incuba ed wi h he espec i e pep ides o 30 min p io o IHC-P. No unspeci ic s aining was obse ed wi h 2 mg/ml and 20 mg/ml o pep ide (da a no shown). Images we e acqui ed using a NIKON AZ100 wide ield ligh mic oscope wi h a NIKON DS-Ri1 high- esolu ion mi- c oscope came a, AZ Plan Fluo 5x and AZ Plan Apo 1x objec i es, and he NIKON Digi al Sigh DS-L2 Imaging Con olle . Image p ocessing was pe o med using AdobePho oshopCS4 Ex ended .11.0.2. Lase Mic obeam Mic odissec ion (LMM) LMM was pe o med on mu ine lung c yosec ions o ob ain b onchiola epi helium and al eola issue samples using he PALM Mic oBeam Sys em (Axio e 200 mic oscope incl. PALM CapMo e ,PALM RoboS age I,PALM RoboSo wa e 2.2.; P.A.L.M. Mic olase Technologies GmbH/ZEISS). A e ho aco omy o WT C57BL/6 mice, lungs we e illed wi h Tissue TekCompound (50% in 1x PBS, Saku a Fine ek) and we e shock- ozen in mel ing isopen ane. C yosec ions (10 mm) we e gene a ed a he C yos a CM3050 S (LEICA), le d y o 5 min a 20C and s ained wi h c esyl iole (2 min 70% e hanol, 20C; 2 s 1% c esyl iole in 50% e hanol, RT; 30 s 70% e hanol, 20C; 30 s 100% e hanol). RNA Isola ion To al RNA om cells o issue samples was isola ed using he innup ep RNA Miniki (Analy ik Jena). Homogeniza ion o ~50 mg o RNAla e - ixed o gans was pe o med in 450 ml lysis bu e RL wi h 10 s e ile, s ainless s eel beads (Ø 2 mm, #22.455.0010, Re sch) a 30 Hz and 4C o 6 min in he mixe mill MM400 (Re sch). RNase ee DNase Se (QIAGEN) was used o on-column DNase I- ea - men . A e elu ion in RNase- ee wa e , 1 U/mlRibolock RNase Inhibi o (The mo Scien i ic) was added. RNA concen a ion and pu i y we e de e mined using he Nanod op 1000 (Peqlab). Mic odissec ed mu ine issue was o exed o 1 min in lysis bu e RL, incu- ba ed o 30 min a RT and o exed again (1 min). A e sho cen i uga ion, he lysa e was s o ed a 80C o e nigh . A e hawing, 1 min o o exing, and cen i uging (8 s), he sample was mixed en imes wi h 700 ml o 70% e hanol using a needle and sy inge. RNA was isola ed acco ding o he innup ep RNA Miniki ’s ins uc ions. To al RNA om human FFPE URT was isola ed om 1-2 sec ions (20 mM) a e xylene depa a iniza ion using he QIAGEN RNeasyFFPE Ki (QIAGEN). Isola ed RNA was ei he p ocessed immedia ely o s o ed a 80C. cDNA Gene a ion cDNA om issues o cells was gene a ed using andom p ime s and he Supe Sc ip III Re e se T ansc ip ase (In i ogen) acco ding o he manu ac u e ’s ins uc ions. Random noname p ime (Gene Link, pd(N)9, 26-4000-06, inal concen a ion: 5 mM) and dNTP e4 Cell Repo s 31, 107549, Ap il 21, 2020 mix (QIAGEN, inal concen a ion: 500 mM each dNTP) we e incuba ed o 5 min a 65C wi h 5 mg o o al RNA in a inal olume o 13 ml and cooled o a leas 1 min on ice. Then, 1x Fi s S and Bu e , DTT ( inal concen a ion: 5 mM), RiboLock RNase Inhibi o (The mo Scien i ic, inal concen a ion: 2 U/ml), and 10 U/ml Supe Sc ip III Re e se T ansc ip ase we e added ( inal eac ion olume: 20 ml). cDNA was gene a ed using he GeneAmpPCR Sys em 9700 (Applied Biosys ems; cycle: 25C o 5 min, 50C o 60 min, 70C o 15 min, 4C o N). cDNA om mic odissec ed samples was gene a ed using he Re e Aid H Minus Re e se T ansc ip ase (The mo Scien i ic). 2 mlo o al RNA we e ea ed wi h 1 U/ml DNase I and 1x DNase I bu e (Sigma-Ald ich, #AMPD1) o 15 min a RT ( eac ion olume: 10 ml). DNA es ic ion was s opped by adding 1.5 ml 50 mM EDTA and incuba ion o 10 min a 70C. Then, 1x Reac ion Bu e , DTT ( inal concen a ion: 500 mM), dNTP-Mix (Peqlab; inal concen a ion: 500 mM each dNTP), 250 ng andom hexame p ime (pd(N)6, Eu o ins MWG Ope on), and 100 U Re e Aid H Minus Re e se T ansc ip ase we e added ( inal eac ion olume: 20 ml). cDNA was gene a ed using he cycle 42C o 50 min, 70C o 15 min, and 4C o N. Fo cDNA om human FFPE URT, 250 ng o o al RNA we e incuba ed wi h Random P ime s (P omega, 500 mg/ml, # C1181; inal concen a ion: 500 ng) and dNTP mix ( inal concen a ion: 500 mM each dNTP) o 5 min a 65C in a olume o 13 ml. A e cooling (a leas 1 min on ice), 1x Fi s S and Bu e , DTT ( inal concen a ion: 5 mM), RNasinRibonuclease Inhibi o (P omega, #N251B; inal concen a ion: 2 U/ml) and 10 U/ml Supe Sc ip III Re e se T ansc ip ase we e added ( inal eac ion olume: 20 ml). cDNA was gene - a ed using he ollowing cycle: 25C o 5 min, 50C o 45 min, 70C o 15 min, and 4C o N. All cDNA samples we e p ocessed immedia ely o s o ed a 20C. Design o RT-qPCR P ime s DNA oligonucleo ides (Sigma-Ald ich; Table S6 o RT-qPCR we e designed using Clone Manage 9 P o essional Edi ion o P ime - BLAST (h ps://www.ncbi.nlm.nih.go / ools/p ime -blas /index.cgi). Real-Time Quan i a i e PCR (RT-qPCR) Gene o in e es (GoI) mRNA exp ession le els we e de e mined using speci ic p ime pai s (Table S6). Singleplex eac ions (10 ml) we e se up manually in Ul aPu e DNase/RNase-F ee Dis illed Wa e (GIBCO) in Mic oAmpOp ical 96-Well Reac ion Pla es (In i- ogen, #4306737): 5 ml Pla inumSYBRG een qPCR Supe Mix-UDG (2x, In i ogen), 0.02 ml ROX Re e ence Dye (25 mM, In i o- gen, inal concen a ion: 50 nM), 300 nM o o wa d and e e se p ime each, and 1 ml cDNA empla e. RT-qPCR uns we e conduc ed on he ABI 7500 Fas Sys em (Applied Biosys ems) in he S anda d 7500 mode wi h endpoin luo escence de ec ion: 2 min a 50C, 3 min a 95C, 50 ampli ica ion cycles (15 s a 95C, 10 s a 65C, and 30 s a 72C). Analysis was pe o med in iplica e o quad u- plica e o each GOI and GAPDH in each sample. Subsequen ly, mel ing cu e analysis was pe o med on he ABI 7500 Fas Sys em (15 s a 95C, 1 min a 60C, 15 s a 95C). Co ec amplicon size was checked by aga ose gel elec opho esis. Da a o eac ions wi h alse p oduc s was excluded om da a analyses. Rela i e exp ession alues we e de e mined using he E -DDCT -me hod (Ramake s e al., 2003; Ruij e e al., 2009). The Rn- alues we e expo ed om he SDS So wa e 1.3.1 (Applied Biosys ems) o Mic oso O ice Excel 2007 and N 0 - alues o he s a ing concen a ion o he ansc ip in he o iginal sample we e ob ained wi h LinReg PCR So - wa e 11.1 (Ruij e e al., 2009). The a e aged N 0 - alue o he GOI (e.g., N 0 (impo in-a3) , n = 3–4 echnical eplica es) was hen no mal- ized wi h he a e aged N 0 - alue o GAPDH (N 0 (GAPDH) ) o he espec i e sample. The ela i e N 0(GOI) /N 0 (GAPDH) -exp ession alues o he biological eplica es a e p esen ed. Fo easie compa ison o di e en ial exp ession, he a e aged ela i e N 0 - alue was se 1. Rela i e mRNA exp ession le els o NF-kB egula ed genes we e de e mined using speci ic p ime pai s (Table S6) o he genes o in e es (GOIs) and he e e ence gene HPRT. Singleplex eac ions (20 ml) we e se up manually in H 2 O PCR g ade (ROCHE) in Ligh Cycle 480 Mul iwell Pla e 96 (ROCHE, #04729692001): 10 ml Fas S a Essen ial DNA G een Mas e (2x, ROCHE), 300 nM o o wa d and e e se p ime each, and 2 ml cDNA empla e. RT-qPCR uns we e conduc ed on he Ligh Cycle 96 Real-Time PCR Sys em (ROCHE) wi h endpoin luo escence de ec ion: 10 min a 95C, 50 ampli ica ion cycles (15 s a 95C, 10 s a 65C, and 20 s a 72C). Analysis was pe o med in iplica e o each GOI and HPRT in each sample. Subsequen ly, mel ing cu e analysis was pe o med (15 s a 95C, 15 s a 60C, 1 s a 95C). Co ec amplicon size was checked by aga ose gel elec o- pho esis. The ela i e N 0(GOI) /N 0 (HPRT) -exp ession alues o he biological eplica es a e p esen ed. P o ein Ex ac ion and Pu i ica ion Whole cell lysa es (WCL) o cells we e ob ained by lysis in HEPES lysis bu e (50 mM HEPES (pH 8.0), 200 mM NaCl, 0.5% IGEPAL, 25% glyce ol, 1 mM PMSF, 0,07 ml/ml b-me cap oe hanol, 1x HALT P o ease and Phospha ase Inhibi o Cock ail, 1x EDTA Solu ion) o samples we e ac iona ed in o cy oplasmic (CF) and nuclea ac ions (NF) using he NE-PER Nuclea and Cy oplasmic Ex ac ion Reagen s (The mo Fishe Scien i ic Inc.). Whole o gan lysa es o unin ec ed WT mice we e ob ained by weighing ~30 mg o issue in o O- ing ubes illed wi h 8 s ainless s eel beads (Ø 2 mm, #22.455.0010, Re sch). A e washing he o gan pieces once wi h 800 ml1x PBS and cen i uga ion a 500 xg and 4C o 5 min, he supe na an was disca ded and 10 ml HEPES lysis bu e pe mg o gan we e added. Homogeniza ion was ca ied ou a 4C and 20 Hz o 2 min ollowed by 30 Hz o 1 min in he mixe mill MM400 (Re sch). The homogena es we e hen incuba ed o 5 min on ice and subsequen ly cen i uged o 20 min a 21,000 xg and 4C. The supe na an was ans e ed in o a esh ube, mixed wi h 4x p o ein loading dye and boiled o 5 min a 95C be o e being subjec ed o wes e n blo analysis. Whole cell lysa es o HEK293T cells ans ec ed wi h 10 mg o each o he pcDNA-impo in-a-N-FLAG cons uc s we e subjec ed o a ini y pu i ica ion using he EZ iew Red ANTI-FLAG M2 a ini y gel (Sigma-Ald ich) and elu ed using a 3x FLAG pep ide Cell Repo s 31, 107549, Ap il 21, 2020 e5 (Sigma-Ald ich) acco ding o he manu ac u e ’s ins uc ions. Thus, pu i ied FLAG- agged impo in-aiso o ms we e ob ained and used o gene a e s anda d cu es o de e mine he a ini ies o he di e en iso o m-speci ic impo in-aan ibodies. The p o ein amoun s o each iso o m we e quan i ied in mu ine o gan homogena es using wes e n blo analyses and s anda dized using he in- di idual s anda d cu es as desc ibed p e iously (Ko ¨hle e al., 2002, 1999). Wes e n Blo Success ul knockdown o impo in-aiso o ms was con i med by wes e n blo analysis using he desc ibed goa an i-impo in-a1, goa an i-impo in-a3, goa an i-impo in-a4, and abbi an i-impo in-a5/a7 an ibodies, espec i ely, wi h no maliza ion o GAPDH using he abbi an i-GAPDH an ibody. The same an ibodies we e used o de ec ion and quan i ica ion o endogenous impo in-a iso o ms in cell o o gan lysa es. Fo de ec ion and quan i ica ion o FLAG- agged impo in-aiso o ms, he mouse an i-FLAG M2 an i- body was used. Amoun s o i al NP p o ein o NF-kB p65 in cell lysa es a e i al in ec ion o TNF-a ea men we e quan i ied using he abbi an i-FPV se um and he abbi an i-NF-kB p65 an ibodies, espec i ely. Fo de ec ion o HA- agged NEMO, he abbi an i- HA an ibody was used. Equal amoun o p o ein was loaded o each sample using GAPDH, p84 o LSD1 as loading con ol. Quan- i ica ion o p o ein was pe o med wi h he Bioimage Image Quan LAS 4000 a non-sa u a ed le els wi h GAPDH o LSD1 adjus - men o WCL, GAPDH adjus men o CF, and LSD1 o p84 adjus men o NF. Al e na i ely, NP le els we e adjus ed and used o no maliza ion. Immuno luo escence WT MEFs as well as MEFs de icien o impo in-a3 we e seeded in glass bo om dishes (#81218-200, Ibidi GmbH) using egula g ow h medium (10% FBS, 1% P/S, 1% L-Glu amine, 1% NEAA, 1% sodium py u a e). A e 8h, he g ow h medium was eplaced wi h s a a ion medium (se um educed o 0.5% FBS) and cells we e s a ed o ano he 16h. Subsequen ly, cells we e ei he con ol- ea ed o ea ed wi h mu ine TNF (10 ng/ml; #575204, BioLegend, Inc) o 15 min a 37C. Then, immuno luo escence s aining was pe o med. All washing s eps we e pe o med wi h phospha e bu e ed saline (PBS), and all incuba ion s eps we e pe o med a oom empe a u e, i no o he wise s a ed. B ie ly, cells we e washed and ixed wi h 4% pa a o maldehyde (in PBS) o 10 min a 37C. Fixed cells we e washed wice and pe meabilized wi h 0.1% T i on X-100 (in PBS) o 20 min. A e washing, cells we e blocked wi h 3% bo ine se um albumin (BSA) solu ion (in PBS) and hen incuba ed wi h a p ima y an ibody ecognizing mu ine NF-kB p65 subuni (1:300 in 3% BSA/PBS; #8242S, Cell Signaling) o 1h. Cells we e washed again and incuba ed o 1h wi h a donkey an i- ab- bi IgG an ibody coupled o Alexa-555 dye (1:500 in 3% BSA/PBS; A31572, Li e Technologies GmbH). This s ep also included s ain- ing o nuclei wi h Hoechs dye (1:1000 in 3% BSA/PBS; #62249, Li e Technologies GmbH). A e u he washing, s ained cells we e s o ed in PBS a 4C. Images we e acqui ed on a con ocal Nikon Eclipse Ti-E spinning disc mic oscope (Nikon, Japan) a 100- old magni ica ion. Image p ocessing was pe o med using Nikon NIS-Elemen s Ad anced Resea ch ( e sion 4.51; Nikon, Japan) and ImageJ so wa e (Na ional Ins i u e o Heal h, NIH). Semi-au oma ic quan i ica ion o nuclea and cy oplasmic NF-kB p65 signals was ca ied ou using a cus om-designed plugin o he ImageJ so wa e which is a ailable on eques . Da a a e based on h ee in- dependen biological eplica es, each pe o med wi h echnical duplica es. HPAIV G ow h Kine ics WT MEFs and MEFs wi h dele ed impo in-a3o -a7 genes we e in ec ed wi h di e en mul iplici ies o in ec ion (MOI) o SC35- PB2 701D (H7N7, MOI 1 o 0.1) o SC35M-PB2 701N i us (H7N7, MOI 0.01 o 0.001). 7*10 5 cells we e inocula ed wi h 2 mL i us dilu ion in DMEM (con aining 1% P/S, 1% L-Glu amine, 1% NEAA, 1% sodium py u a e) and incuba ed o 30 min a 5% CO 2 , 96% H and 37C. A e washing wice wi h acidic incuba ion medium (DMEM, pH 5.0, con aining 1% FCS, 1% P/S, 1% L-Glu amine, 1% NEAA, 1% sodium py u a e), 2 mL o pH-neu al incuba ion medium we e added and he in ec ed cells we e incuba ed a 37C. Vi us i e s o supe na an s aken a 0, 24, 48, 72, and 96 hou s pos in ec ion (p.i.) we e de e mined as plaque o ming uni s pe ml (p. .u./ml) by plaque assay 17 on MDCKII. Fo de e mina ion o impo in-ap o ein le els a e i al in ec ion, WT MEFs we e in ec ed wi h SC35- PB2 701D o SC35M-PB2 701N i us a an MOI o 0.1. o WT MEF. Simila ly, H1299 WT and H1299 wi h dele ed impo in-a3 gene we e ea ed wi h TNFa(10ng/ml) 24h p io o inocula ed wi h SC35M-PB2 701N a MOI 0.001 (see abo e). TNFa ea men was e- applied and supe na an aken a 0, 24 and 48h p.i. Vi us i e s we e de e mined by plaque assay on MDCK cells and gi en in p. .u/ml. Addi ionally, p ima y human lung cells (HBEpC and HSAEpC) we e in ec ed wi h H5N1-PB2 701N and H5N1-PB2 701D (MOI = 0.1 and 1) o H7N7-PB2 627K and H7N7-PB2 627E (MOI = 1 and 10). Cells we e lysed 24 and/o 48 hou s p.i. in lysis bu e (50 mM HEPES (pH 8.0), 200 mM NaCl, 0.5% IGEPAL, 25% glyce ol (In i ogen), 1 mM PMSF, 0,07 ml/ml b-me cap oe hanol, 1x HALT P o ease and Phospha ase Inhibi o Cock ail, 1x EDTA Solu ion (bo h: 100x, Pie ce/The mo Scien i ic). Whole cell p o ein ex- ac s we e subjec ed o wes e n blo analysis. Lib a y P epa a ion and Nex Gene a ion Sequencing Lungs o WT and a3 / mice in ec ed wi h 6*10 4 p. .u. o SC35-PB2 701D o SC35M-PB2 701N (H7N7) we e subjec ed o gene exp es- sion analysis. Con ol mice ecei ed PBS. On day 3 p.i., whole lungs we e pe used, ha es ed and subme ged in RNAla e and o al RNA was isola ed using he innup ep RNA Mini Ki (Analy ik Jena). RNA samples om h ee animals pe mouse s ain and i us we e pooled and used o gene exp ession analysis. Fo each sample, 1mg o o al RNA was used o gene a ing sequencing lib a ies wi h he Illumina T uSeq RNA Sample P epa a ion Ki 2 as ecommended by he manu ac u e (Illumina Inc.). Size and quali y o e6 Cell Repo s 31, 107549, Ap il 21, 2020 he lib a ies we e isualized on a BioAnalyze High Sensi i i y DNA Chip (High Sensi i i y DNA Ki , Agilen Technologies). Dilu ed li- b a ies (2 nM) we e mul iplex-sequenced on he Illumina HiSeq 2500 ins umen . Fo each sample be ween 78 and 113M, pai ed-end eads o 101 bp leng h we e gene a ed. The eads we e aligned o he mu ine e e ence ansc ip ome (UCSC mm10) using Bow ie2 ( 2.2.2) (Langmead and Salzbe g, 2012). DESeq (Ande s and Hube , 2010) was employed o assess di e en ial exp ession based on ead coun s pe gene. Genes we e hie a chically clus e ed acco ding o hei exp ession p o iles using he R unc ion hclus (h p:// www.R-p ojec .o g/). DAVID ( 6.7) (Huang e al., 2009) was employed o unc ionally anno a e he esul ing clus e s. Ne wo ks o p o- ein-p o ein in e ac ions we e compu ed and isualized using Cy oscape (Shannon e al., 2003). TNF-aELISA TNF-ap o ein amoun was de e mined in lung homogena e supe na an s o WT mice in ec ed in anasally wi h 6*10 4 p. .u. o SC35- PB2 701D o SC35M-PB2 701N i uses (H7N7). Whole lungs we e ha es ed on day 1 p.i. and ~50–100 mg o issue homogenized wi h ~300 ml glass beads (RETSCH #22.222.0003) in 1 mL 1x PBS (PAA) in he mixe mill MM400 (Re sch; 20 Hz, 4C, 10 min). A e cen i- uga ion (6000 xg, 4C, 5 min), lung homogena e supe na an s we e s o ed a 80C. Supe na an s o h ee animals pe i us we e pooled. Then, he Enzyme-linked Immunoso ben Assay (ELISA) Ki o mu ine TNF-awas pe o med acco ding o he manu ac- u e ’s ins uc ions (#E90133Mu, USCN Li e Science Inc.). TNF-aT ea men , Impo in-a3 mRNA Exp ession, Subcellula Localiza ion and P omo e Ac i a ion HSAEpC, WT MEFs and NEMO / MEFs we e seeded in 24-well pla es un il con luen o in 10 cm-dishes o 24 h, espec i ely, and se um s a ed o 24 h. Then, cells we e con ol- ea ed (w/o) o ea ed wi h human TNF-a(50 ng/ml; #210-TA, R&D Sys ems) o 6 h o wi h mu ine TNF-a(50 ng/ml; #575204, BioLegend, Inc.) o 6 h o 24 h, espec i ely. WCL, CF, and NF we e subjec ed o wes e n blo analysis o impo in-a3 and NF-kB p65 de ec ion. To al RNA we e isola ed 6h pos ea men o ela i e impo in-a3 exp ession, using RT-qPCR. Fo de e mina ion o he ela i e impo in-a3 mRNA exp ession le els a e cy okine ea men , NEMO / MEFs we e seeded in 10cm cell cul u e dishes (5x10 6 cells pe dish). A e se um s a a ion o 24 h, cells we e con ol- ea ed (mock) o ea ed 10 min wi h 10ng/ml o mu ine TNF-a, IL-6, IL-10, IFN-gand TGF-b. A e 6h pos ea men , cells we e ha es ed, RNA isola ed and cDNA gene a ed be o e RT-qPCR analysis. Fo he de e mina ion o nuclea impo o NF-kB p65 a e TNF-as imula ion, WT MEF and Impo in a3 KO MEF we e seeded in 10cm cell cul u e dishes (1x10 6 cells pe dish). A e se um s a a ion o 18 h, cells we e ea ed wi h 10ng/ml TNF-a. Subsequen ly, cells we e ha es ed by sc aping in ice-cold PBS, cen i uged and a e emo ing he supe na an , he cells we e esuspended in 50ml o Bu e A (10mM T is pH7,9; 1,5mM MgCl 2 ; 10mM KCl). A e adding 6ml o 2% NP-40 and o exing, he samples we e incuba ed o 10min on ice and cen i uged € u 15min a 6,0000 pm a 4C. The supe na an (cy osolic ac ion) was placed in a esh ube, sub- sequen ly he pelle was washed again wi h 100ml o bu e A, cen i uged again o 15min and he supe na an was emo ed. Fo nuclea ac ions, he pelle was esuspended in 50 ml o bu e B (20 mM T is pH 7.9; 10mM KCl; 1.5mM MgCl 2 ; 20% Glyce ol) and o exed; hen 33 ml o bu e C (20mM T is pH 7.9; 1.2M KCl; 1.5 mM MgCl 2 ; 20% Glyce ol) was added and he samples we e incuba ed on a wheel a 4C o 45 min. A e wa d, samples we e cen i uged o 30 min a 12,000 pm a 4C and he supe - na an (nuclea ac ion) was ans e ed o a esh eac ion ube. Bo h ac ions we e mixed wi h 4x p o ein loading dye and boiled o 5 min a 95C be o e being subjec ed o wes e n blo analysis. Fo de e mina ion o impo in-a3 p omo e ac i a ion in MEF cells, cells we e seeded in 96-well pla es (5000 cells/well) o each app oxima ely 60% con luency a e 24h. Then, cells we e ans ec ed using FuGene HD ans ec ion eagen (Swi chGea Geno- mics), ei he wi h pLigh Swi ch-P om-Con ol (Swi chGea Genomics; 100 ng/well) as a con ol, o pLigh Swi ch-P om-impo in- a3 (Swi chGea Genomics; 100 ng/well) o impo in-a3 p omo e ac i i y eadou . A e 24h, ans ec ed cells we e ea ed wi h mu- ine TNF-a, IL-10 o TGF-ba 0, 1 o 50 ng/ml, o 15 min. P omo e ac i i y was measu ed a 3h pos ea men using Ligh Swi ch TM Luci e ase Assay Reagen (Swi chGea Genomics, LS010) acco ding o manu ac u e ’s ins uc ions. Fo de e mina ion o impo in-a3 p omo e ac i a ion, A549 o HSAEpC we e seeded in 96-well pla es o 24 h o un il 80% con- luency, espec i ely, and ans ec ed wi h pLigh Swi ch_P om-con ol (50 ng; Con ol) o pLigh Swi ch_P om-impo in-a3 (50 ng; a3) and pFL-TK (250 ng; i e ly luci e ase ans ec ion con ol) using Lipo ec amine-2000 o je PRIME, espec i ely. 14 h a e ans ec ion, se um s a a ion was ini ia ed o 30 h. Subsequen ly, cells we e ea ed wi h 0, 10, 50 o 100 ng/ml (A549) o wi h 0, 1, 10 o 100 ng/ml (HSAEpC) o human TNF-aand lysed 20 h la e . Fi e ly and Renilla luci e ase ac i i ies we e assessed using he Dual-Luci e aseRepo e Assay Sys em (P omega, E1910). NEMO Complemen a ion Assay NEMO / MEFs we e ans ec ed in suspension wi h 5 mg emp y ec o pcDNA3.1 o pcDNA-HA-human NEMO in 6-well pla es using Lipo ec amine 2000 (In i ogen) o 6 h. A 48 h pos ans ec ion, cells we e se um s a ed o 24 h. Then, cells we e con ol- ea ed (w/o) o ea ed wi h mu ine TNF-a(50 ng/ml; #575204, BioLegend, Inc.) o 6 h. WCL we e subjec ed o wes e n blo analysis o impo in-a3 and NEMO de ec ion. Cell Repo s 31, 107549, Ap il 21, 2020 e7 QUANTIFICATION AND STATISTICAL ANALYSIS P alues o RT-qPCR da a we e ob ained by K uskal-Wallis one-way ANOVA using PAST .2.17c (h p:// olk.uio.no/ohamme /pas /). RT-qPCR da a we e conside ed as ele an when mo e han ±1.5- old change was de ec ed in ela i e mRNA exp ession le els. S a is ical signi icance o animal su i al a es was de e mined wi h G aphPad P ism 5 .5.03 (G aphpad So wa e, Inc.) using he Log Rank (Man el-Cox) es and he Kaplan-Meie su i al cu es. All o he mean alues, s anda d de ia ions (SD), and P alues we e calcula ed wi h G aphPad P ism 5 ( 5.03) o he G aphPad So wa e, Inc. ool (h ps://www.g aphpad.com/quickcalcs/ es 1/?Fo ma =SD) using he unpai ed, wo- ailed S uden ’s es o he one-sample, wo- ailed S uden ’s es when all alues in one g oup we e iden ical, e.g., se 100%. S a is ical signi icance was de ined as p < 0.05 (* p < 0.05, ** p < 0.01, *** p < 0.001). DATA AND CODE AVAILABILITY T ansc ip ome sequence da a epo ed in his publica ion is submi ed o he Eu opean Nucleo ide A chi e (ENA) and is a ailable unde ollowing link: h ps://www.ebi.ac.uk/ena/da a/ iew/PRJEB8023. e8 Cell Repo s 31, 107549, Ap il 21, 2020