The genetic driver of Acute Necrotizing Encephalopathy, RANBP2, regulates the inflammatory response to Influenza A virus infection

Domain: neuroscience
Published: 2026-02-06
Editorial outcome: major_revision

Abstract
Influenza virus infections can cause severe complications such as Acute Necrotizing Encephalopathy (ANE), which is characterised by a rapid onset of pathological inflammation following febrile infection. Heterozygous dominant mutations in the nucleoporin RANBP2/Nup358 predispose to influenza-triggered ANE1. The aim of our study was to determine whether RANBP2 plays a role in IAV-triggered inflammatory responses. We found that the depletion of RANBP2 in a human airway epithelial cell line increases IAV genomic replication by favouring the import of the viral polymerase subunits, PB1, PB2, and PA, following viral transcription and translation. Additionally, RANBP2 knockdown enhances the cytoplasmic export of viral genomic RNA (vRNA) and disrupts segment stoichiometry, which is associated with elevated production of the pro-inflammatory chemokines CXCL8, CXCL10, CCL2, CCL3, and CCL4 in human primary macrophages. Using CRISPR-Cas9 knock-in for the ANE1 disease variant RANBP2-T585M, we further demonstrate that this point mutation causes a loss-of-localisation phenotype that excludes RANBP2 from the nuclear envelope, which phenocopies RANBP2 knockdown by increasing IAV replication and driving pro-inflammatory cytokine expression following infection. Together, our results reveal that RANBP2 regulates influenza RNA replication and nuclear export, thereby restraining virus-induced hyperinflammation, and further suggest that ANE1 pathogenesis results from the impaired localisation of RANBP2 at the nuclear envelope.

Introduction
Influenzaviralinfectionsareresponsibleforseasonalepidemicswithrespiratorymanifestationsbutcanalsoleadtoseverecomplications.Among these, Acute Necrotizing Encephalopathy (ANE), also called ANEofChildhood(ANEC),isaseverereactiontofebrileinfectionthatis characterized by rapid progression and very poor prognosis1–4. Influenzavirusesarethoughttoberesponsiblefor~50%ofallANEepisodesandareassociatedwithhighmorbidityandmortality4–9. Althoughthepathogenesisisunclear,ahyperinflammatoryresponsetriggeredbythevirusisproposedastheunderlyingmechanism4,10,11, andearlyanti-inflammatorytherapymayhelpmitigatethedisease8,12,13. InfluenzavirusesbelongtotheOrthomyxoviridaefamilyandareclassifiedintotypes,withInfluenzaAandBbeingthemostprevalentonesassociatedwithseasonalflucases.InfluenzaAisfurtherdividedintosubtypesbasedontheirsurfaceglycoproteins,haemagglutinin(HA)andneuraminidase(NA).TheH1N1andH3N2strainsareresponsibleformajoroutbreaksandepidemics,andinthe2024-2025winterseason,therehasbeenanunprecedentedsurgeinH1N1cases,leadingtoanunusualspikeinhospitalizationsandANEinchildren5. TheInfluenzaAvirus(IAV)isanenvelopedparticlecontaining8negative-sensesingle-strandedgenomicviralRNA(vRNA)segments,eachpackagedbynucleoprotein(NP)intoaviralribonucleoprotein(vRNP)complex,andassociatedwiththeviralpolymerasecomplex(PB1,PB2,andPA).Unlike most RNA viruses, IAV replicates in the nucleus and hijacks the nuclear pore complex (NPC) machineryfor four critical steps of its replication cycle. (i) After endocytosis and uncoating,vRNPsaretraffickedtothenucleus,wheretheviralpolymerasetranscribestheviralgenome.(ii)CappedandpolyadenylatedmRNAsareexportedintothecytoplasmfortranslationbycytoplasmicribosomes.(iii)Thenewlytranslatedviralproteins,includingNP,PB1,PB2,andPA,areimportedbackintothenucleus,whichallowstheviralpolymerasetoreplicatetheviralgenomeintocomplementaryRNAs(cRNA)andgenomicvRNA.(iv)Lastly,newvRNPs,assembledwithM1andNS2/NEP,areexportedbackfromthenucleusforincorporationintobuddingvirions14–17. Manyviralandhostfactorsarethoughttocontributetosevereinfluenza,andgeneticsusceptibility,particularlyingenesthatregulateinnatesignallingandantiviralresponse,hasbeenreported18–20.However, the mechanisms that drive severe influenza-triggered ANE in previously healthychildrenarenotknown.Onecluetounderstandingthe pathogenesis of influenza-associated ANE comes from genetic susceptibilityinfamilial,recurrentcases,knownasANE1(orADANE)7. PatientswithANE1presentwithautosomaldominantmissensemutations in RANBP2 and have a significantly increased lifetime risk of developingANE21.MutationsinRANBP2increasetheriskofrelapseandrecurrence8,10 and may be associated with greater morbidity and mortality. RANBP2 (Nup358) is a cytoplasmic fibril (CF) nucleoporin that regulates nucleocytoplasmic transport (NCT) by interacting with nuclear transport receptors such as Karyopherin α/β and CRM1/Exportin122,23.AdditionalfunctionshavebeenattributedtoRANBP2attheNPC,manyofwhicharelinkedtoitsC-terminaldomain(CTD)E3 SUMO ligase activity, and away from the NPC, namely at annulate lamellae, mitochondria-endoplasmic reticulum junctions and in the nucleus24.Moreover,atleast4proteinisoformshavebeenidentifiedforRANBP2,however,itisnotknownifthesehavedistinctfunctionsorsubcellularlocalisations25.ThepredominantANE1-associatedmutation(c.C1754TonthecodingDNAreferencesequence,leadingtop.T585Mattheproteinlevel),andmostotherpathogenicmutationsinRANBP2,allcluster intheN-terminal domain(NTD),which isresponsibleforanchoringtoNPCs6,26.ThissuggeststhatthelocalizationofRANBP2toCFofNPCmaybephysiologicallyessential,althoughnochangesinlocalisationcouldbedemonstratedfollowing the overexpressionofdiseasevariants27,28. PreviousworkshowedthatRANBP2canmodulateviralinfectionofseveralviruses,suchasAdenovirus29,Herpessimplexvirustype1(HSV-1)30,31 and Human immunodeficiency virus type 1 (HIV-1)32–35, howevernoworkhasuncoveredaroleforRANBP2inInfluenzavirusinfection.Infact,althoughsomestudiesshowedthatNPCcomponentscanimpactthereplicationandtranscriptionofInfluenzavirus,suchasNup62,Nup98,andNup153,RANBP2wasnotidentifiedasahostfactorforInfluenzavirus36–38. Previous work also showed that RANBP2 can modulate innate immunesignalling,howeverthisactivitywasconsistentlyattributedtoits E3 SUMO ligase activity or Cyclophilin-homology motif in the CTD27,39–41,andnoworkhasyetdemonstratedarolefortheNTDwhereANE1mutationsareclustered. TheaimofourstudywastodeterminehowRANBP2contributestoorregulatesinflammatoryresponsestoinfectionbyInfluenzavirus. WeshowthatRANBP2impactsthereplicationofgenomicvRNAofIAVintocRNAbycontrollingthere-importofnewlytranslatedviralpolymerase complex. In addition, RANBP2 knockdown enhances cytoplasmic vRNA export and disrupts segment stoichiometry, thereby enhancing IAV-triggered inflammation. Notably, the predominant ANE1-associated variant T585M causes relocation of RANBP2 away fromthenuclearenvelope,whichphenocopiesknockdownofRANBP2byincreasingvRNAreplicationandexport,andtriggeringhyperinflammation,whichcouldprovideafirstclueinunderstandingthepathogenesisofANE.

Results
KnockdownofRANBP2stimulatesnon-productiveInfluenzavRNAreplicationininfectedcells
To investigate the role of RANBP2 in IAV infection, RANBP2 knockdown(RANBP2-KD)wasinducedinA549humanalveolarcellsbylentiviralvector(LV)transductionofshorthairpinRNAs(shRNAs)beforeinfectionwithA/WSN/1933(H1N1)virusatMOI0.5.Supernatantswereharvestedat10hpost-infection(hpi)toinvestigatetheproductionofinfectious viral particles by TCID50 titration. No differences were observedbetweenthecontrolandRANBP2-KDcells,suggestingthatRANBP2 had no effect on the production of infectious particles(Figs.1a,S1a,b).
However,anunexpectedphenotypewasobservedforthequantificationofinfluenzavirusRNAsinbothA549andthemonocyticcelllineTHP-1. Infection kinetics were performed, and viral RNA levels weredeterminedatearly(2h)andlate(6,8,16,and24h)timepointsusing qPCR primers recognizing M1 on segment 7. In A549 cells(Fig.1b)andinthemonocyticcelllineTHP-1(Figs.1c,S1c),asignificantincreaseinM1RNAwasinducedinRANBP2-KDcells.Theincreaseinviral RNAs was confirmed in A549 cells with another IAV segment,encodingtheNP(Fig.1d),andappearedtobespecificsincenoincreasewasobservedforanyofthehousekeepinggenesthatweremeasured(Fig.1e).Together,thesedatasuggestedthatRANBP2isinvolvedincontrolling the synthesis of influenza RNAs but that this does nottranslateintoincreasedproductiveinfection.
Because different types of viral RNAs are produced during the IAVlifecycle,weinvestigatedwhichonesareimpactedbythedepletionofRANBP2. Sequence-tagged RT primers were used to specificallyrecognizethenegative-sensegenomicvRNAsequenceorthepositive-sensecRNAsequenceforreversetranscription(RT)(Fig.S2a).First,thespecificityofeachprimerwasvalidatedbyamplifyingonlyvRNAaftervRNA-specific RT, or only cRNA after cRNA-specific RT, while notamplifyinganytargetinnon-infectedcells(Fig.S2b).InRANBP2-KDcells, we observed a significant increase of both vRNA and cRNA(Fig.1f),thusshowingthatRANBP2isinvolvedinIAVRNAreplication.Although this was unexpected given the absence of phenotype interms of viral production, the marked increase in vRNA and cRNAlevelssuggestedanenhancementinviralgenomereplication.
HavingobservedanincreaseingenomicvRNA,wetestedifthiscorrelatedwithmorenewlyproducedvRNPs.Aspreviously,A549cellsweretransducedwithcontrol-orRANBP2-shRNAstheninfectedwithIAV (MOI 0.5). At 8 hpi, the distribution of NP was investigated byimmunofluorescence.Althoughexportisongoingineverycondition,RANBP2-KDcellsshowedastrikingincreaseincytoplasmicNPcomparedtocontrol(Figs.1g,h,S3).ThisphenotypewasconfirmedusingapreviouslypublishedRANBP2-targetingshRNA(DiNunzioetal.,2012)and only in cells expressing the shRNA (Fig. S4a–d). In order todetermineifsignalcorrespondedtovRNPsorfreeNPinthecytoplasm,weperformedco-stainingswithothercomponentsofvRNPs(i.e.PB1andPA).Weobservedaco-localisationofcytoplasmicNPwithPB1andwithPA,withaPearsoncoefficient>0.8(Figs.1i–k,S5a–d),indicatingthat RANBP2-KD induces an increase in vRNPs exported to thecytoplasm.

IncreasedIAVreplicationdoesnotresultfromenhancedviralentryintocellsorintothenucleus
TodeterminehowvRNAisincreasedinRANBP2-depletedcells,wefirsttested whether RANBP2 regulates IAV infection by modulating viralentryintocellsand/ornuclei,therebyincreasingthequantityofIAVRNAtobecopied.
ToassesswhetherRANBP2regulatescellornuclearentry,controlorRANBP2-KDcellswereinfectedwithIAV(A/WSN/33)athighMOIof4,inordertoincreasesignaldetectionduringearlystepsoftheviralcycle,andNPlocalizationwasstudiedbyimmunofluorescenceat1hpi to assess internalization, and at 2h to monitor NP nuclear entry.Results indicate that depletion of RANBP2 from A549 cells affectsneitherinternalization(Fig.1l,m)nornuclearimport(Fig.1n,o).
Tofurthervalidatethesefindings,weadaptedanAlpha-Centauriprotein-complementationassaypreviouslydevelopedtomonitorthenuclearimportofHIV-142.Briefly,theNanoLucproteinwasexpressedastwounequalnon-luminescentfragments.ThesmallerfragmentwasfusedtoPB2inIAVparticles(IAV-α),whilethelargerfragment(Centauri) was fused to a NLS and expressed in A549 cells by LV transduction(Cen-NLS)(Figs.1p,S6a,b).UponvRNPnuclearimport,thetwofragmentsreconstituteafunctionalNanoLuc,generatingameasurable signal upon addition of substrate (Fig. S6c). No significantdifferences in luminescence were detected between control andRANBP2-KDcellsat1and2hpi,confirmingthatRANBP2isnotessentialfor vRNP nuclear import (Figs. 1q, S6d), thus demonstrating thatRANBP2doesnotregulateIAVvRNPnuclearimport.

RANBP2knockdownfacilitatesthenuclearimportoftheneosynthesisedviralpolymerasecomplexproteinsPB1,PB2andPA
SinceRANBP2doesnotimpactthequantityoftemplateavailableforreplication, we investigated whether it modulates the quantity orlocalisationofthepolymerasecomplexsubunits.First,thetotallevelsofPB1,PB2,PAandNPwereassessedbywesternblotat8hpi,however,nodifferenceswereobservedbetweenRANBP2-KDcellsandcontrolcells(Fig.2a,b),suggestingthatRANBP2doesnotimpactthetranslationoftheviralpolymerasecomplex.
Being an NPC component, we hypothesised that RANBP2 maycontrolthereplicationofvRNAbyregulatingtheimportoftheviralpolymerase after translation. Therefore, we tested the impact ofRANBP2onthenuclearimportofnewlysynthesisedPB1,PB2,andPAsubunits.Tofocusspecificallyonimport,weinhibitedvRNPexportusingapharmacologicalinhibitorofCRM1,KPT-330/Selinexor.First,weconfirmedthatKPT-330inhibitsvRNPexport(Fig.S7a),withnegligible toxicity (Fig. S7b) and that inhibition operates from 5 hpionwards(Fig.S7c).Thenweperformedsubcellularfractionationfollowedbywesternblottingat6hpitodeterminethelocalisationofthedifferentpolymerasesubunits.ResultsrevealedanincreaseinPB1,PB2,and PA protein levels in the nucleus (Fig. 2c, d), confirmed by theimmunofluorescencedetectionofnuclearPB1(Fig.2e,f),indicatingthattheimportoftheneo-synthesisedpolymeraseisfacilitatedintheabsenceofRANBP2.

RANBP2depletionfavourstheexportofvRNAsegmentstothecytoplasmanddisruptsthesegmentstoichiometry
Having determined that RANBP2 controls IAV replication in thenucleusbyregulatingtheimportofthenewlysynthesisedviralpolymerase, we asked why the increase in vRNA and vRNP does nottranslateintoanincreasedproductionofinfectiousparticles(Fig.1a).
Toaddressthis,weperformedstrand-specificRTtodistinguishvRNAandcRNA,aspreviously(Fig.S2a),thenquantifiedthe8IAVsegmentsbyqPCRtoinvestigatesegmentstoichiometry(Fig.2g,S8a,b).Aftersub-cellularfractionation,analysisofinfluenzavirusRNAlocalizationrevealedthat,whileRANBP2knockdownledtoanoverallincreaseofallcRNA segments inthe nucleus (Fig. S9), it alsoled to anoverallincreaseinthenuclearexportofvRNAsegments(Fig.2h,i).Thiseffectdisproportionallyaffectedindividualsegmentsacrossexperiments,asreflectedbyahigherstandarddeviation(Fig.2j).Thesefindingsindicatethat,whilethedepletionofRANBP2increasesthereplicationofallvRNA segments, it also promotes their uneven export to the cytoplasm,therebydisruptingsegmentstoichiometry.
Taken together, our results indicated that the knockdown ofRANBP2perturbsinfectionbyIAV,firstbyfacilitatingthere-importofpolymeraseintothenucleus,whichincreasescRNAandvRNA,secondbydisproportionatelyfavouringtheexportofvRNAsegmentsintothecytoplasm. The dysregulation of both these steps may cause anabnormal accumulation of some vRNA segments in the cytoplasm,thus constituting potential pathogen-associated molecular patterns(PAMPs)thatmaybesensedbytheinfectedcell.

RANBP2knockdownexacerbatestheinflammatoryresponsetoIAVinfectioninprimaryhumanmacrophages
To investigate whether the abnormal cytoplasmic accumulation ofvRNA, observed in RANBP2-depleted cells, might associate withincreasedinflammation,wemeasuredtheexpressionofinflammatorytranscriptsbyqPCR.InbothA549andU2OScells,RANBP2depletionled to an upregulation of inflammatory transcripts, which was significantinbothcelltypesforIL-6andIL-1β(Fig.3a,b,S10a,b).ThesechangesmirroredtheincreaseinvRNA(Fig.S10c)andwereconfirmedusing an independent RANBP2-targeting shRNA (Fig. S11). Interestingly,amodestincreaseinsomeofthesetranscriptswasalsoobservedin uninfected cells (Fig. 3a, b), likely reflecting low level activationtriggeredbylentiviralvectortransduction,butthiswasnotsignificant.
To confirm this at the protein level, primary monocytes wereisolated from the PBMCs of healthy donors and differentiated intogranulocyte-macrophagecolony-stimulatingfactor(GM-CSF)-derivedmacrophages. After 7 days of differentiation, primary monocyte-derivedmacrophages(MDM)wereCD3-CD14+CD16+CD11b+HLA-DR+CD80-(Fig.S12).WefirstdeterminedthatIAVcaninfectMDMs(Fig. S13a), and stimulate an effective immune response withoutadditional pre-stimulation, triggering the synthesis of pro-inflammatorycytokinessuchasIL-6andIL-1β(Fig.S13b).MDMwerethentransducedwithLVscodingforcontrolorRANBP2shRNA,andinfectedwithIAV(Fig.S14a,b).Supernatantswerecollectedat24hpiand the secretion of pro-inflammatory mediators was analysed byMultiplex Luminex assay using the manufacturer’s pre-definedinflammatorypanel.Strikingly,theknockdownofRANBP2inthepri-mary macrophages from two donors led to an exacerbated inflammatoryresponsetoIAVinfection,withastrongerinductionofthepro-inflammatory chemokines CXCL8, CXCL10, CCL2, CCL3 andCCL4(Fig.3c).
Together,thesefindingssuggestthatRANBP2playsacriticalroleinmodulatingtheinflammatoryresponsetoIAVinfectionbyregulating vRNA replication, nucleocytoplasmic trafficking of viral polymerasesubunits,andselectivevRNAexport.Resultssuggestthattheabnormalaccumulationofviralcomponentsinthecytoplasmamplifies inflammatory signalling, highlighting a potential mechanismunderlyingthepathogenesisofANE.

TheRANBP2-T585MANE1variantdrivesIAVreplicationandhyperinflammationfollowinginfection
Having identified a role of RANBP2 in controlling the IAV-triggeredinflammation,weinvestigatedifthisfunctioniscompromisedbythepredominant ANE-associated mutation, c.C1754T, p.T585M. Heterozygousmutations in RANBP2 are associated withincreased susceptibility to ANE, however, it is not known how these affect proteinfunction.Indeed,previousworkshowedthatANE1mutationsdonotalter the structure of RANBP228, and that exogenously-expressedRANBP2-T585Mstilllocalisestothenuclearenvelope27,28.
Weintroducedthepredominantmutation(c.C1754T,p.T585M)byCRISPR-Cas9knock-inofU2OScells(Fig.4a).Atotalof47cloneswereisolated after puromycin selection during 3 weeks. Mutations wereconfirmedbyallelicqPCR(Figs.4b,S15a–c),usingapreviouslypublishedprotocol43,andbySangersequencingofthecDNA(Fig.4c,d).
Formanyclones,wenotedthatCRISPRrepairhadoccurredusingthehighly homologous RGPD sequences present on the samechromosome24,ratherthantherepairplasmid,thereforeonlyclonesthatdidnotrecombinewithRGPDwereselected(Fig.S15b).Intotal,two wild-type clones (C4 and C15, referred to as WT/WT), oneheterozygously-mutated(C10,WT/C1754T)andthreehomozygously-mutated(C6,C9andC14,C1754T/C1754T)cloneswereisolated.
CRISPR-knock-in clones wereinfected with IAV,and replicationwasassessedbyquantifyingviralNPat8hpi,aspreviously(Fig.1g,h).
Strikingly, cytoplasmic NP was significantly increased in all ANE1CRISPRclonescomparedwithwild-typeclones,regardlessofwhetherthemutationwasheterozygousorhomozygous(Figs.4e,f,S16).qPCRanalysis confirmed that both heterozygous and homozygous ANE1genotypesexhibitedagreaterincreaseinIAVreplicationovertime,withsignificantlyhighervRNAlevelsat24hpicomparedtoWTclones(Figs.4g,S17).Moreover,IAVinfectioninducedanoverallupregulationofinflammatorycytokinesandchemokines,includingCXCL10,IL-6,IL-1β,CXCL8,andCCL3,withsignificantlyhigheroverallexpressioninbothheterozygousandhomozygousANE1genotypescomparedtoWTclones(Figs.4h,S18).Together,theseresultsindicatethattheANE1-associated T585M mutation phenocopies RANBP2 knockdown byenhancingIAVreplicationandassociatedhyperinflammation.

InCRISPR-Cas9T585Mknock-incells,RANBP2mislocalisesawayfromnuclearpores
Although previous studies reported that exogenously-expressedRANBP2-T585M localizes tothe NPC27,28,its expression and localizationhavenotbeenexaminedingeneticallyeditedcelllines.Overall,wedidnotobserveastatisticallysignificantvariationintheproteinlevelsofRANBP2(Fig.S19a,b).WenextexaminedthesubcellularlocalisationofRANBP2byimmunofluorescenceintheclonesexpressingRANBP2-T585M heterozygously and homozygously using a RANBP2-specificantibodyrecognisingtheCTD.Asexpected,RANBP2localisedtothenuclearenvelope in WT clones. In contrast, all three clones homozygousforRANBP2-T585Mlostthecharacteristicnuclearrimstaining,indicatingthatthemutationdisruptsRANBP2retentionatthenuclearenvelope(Fig.4i,4j).Despitethis,nuclearporesappearedintact,sincethelabellingofFG-repeatNupswiththeMAb414antibodywascomparableacrossallclones(Fig.4i).Inheterozygousclones,RANBP2didlocalise at the nuclear envelope, although quantification revealed amodest but significant decrease compared to control clones, suggestinganintermediaryphenotype(Fig.4k).
Inconclusion,ourfindingssuggestthatRANBP2localizationatthenuclearenvelopeiscriticaltosafeguardcellsfromthepathologicalinflammationfollowingviralinfection.InANE1,mutationsintheNTDofRANBP2arespecificallyassociatedwithhyperinflammation.

Methods
Ethicsstatement
Noanimalexperimentswereconductedinthisstudy.HumanPBMCs were isolated from de-identified blood samples obtained from theEtablissementFrançaisduSang(EFS)Occitanieuponsignedinformedconsentfromdonors.

Viruses,lentiviralvectorsandcells
InfectionswithIAVwerecarriedoutwiththeH1N1/A/WSN/1933strain in all experiments, except for the Alpha-Centauri assay, where thissamestrainwasengineeredtoexpressafragmentoftheNanoLuc(IAV-α),asdescribedbelow.Lentiviralvectors(LV)wereproducedinHEK-293Tcells(ATCC)bycalciumphosphatetransfectionusingaVSV-Genvelopeplasmid(pVSVg;15µgina70–80%confluent175cm2flask),aGag-Polencapsidationplasmid(p8.74;30µg),andthetransferplasmidofinterest(30µg;mCherry-shRNA-control,mCherry-shRNA-RANBP2,or Cen-NLS; see Table S1). After 48h, vectors were harvested andultracentrifugedfor1hat82,000×g(OptimaXE-90,BeckmanCoulter)at4°C.A549cells(ATCC)andCRISPR-engineeredU2OSclones(generated in-house) were cultured in Dulbecco’s Modified EagleMedium (DMEM, Gibco) supplemented with 10% of Foetal bovineserum(FBS,Serana)and1×penicillin/streptomycin(PS,Gibco)at37°Cwith5%CO2.THP-1cellsandmonocyte-derivedmacrophages(MDM)wereculturedinRoswellParkMemorialInstitute(RPMI-1640,Gibco)mediumsupplementedwith10%FBSand1×ofPS.MDCKcells(ATCC)were cultured in Minimum Essential Medium (MEM, Gibco) supplementedwith5%FBSand1×ofPS.

Lentiviraltransduction
Cellswereseededintoplatesonedaypriortotransduction.AfteraPBSwash,lentiviraltransductionwasperformedatMOI20to40(transducingunits/cell)inasmallvolumeofculturemediumsupplementedwith2%FBS,tofacilitateviraladsorption.After2hofincubationat37°C, 10% FBS culture medium was added. Transduced cells wereculturedfor48–72hpriortoviralinfectionstoallowefficientknockdown.Allexperimentswerecompletedby56–96hpost-transduction.

Viralproduction
H1N1/A/WSN/1933viralstockswereproducedbyamplificationatanMOIof10−4onMDCKcells,culturedinMEMcontaining2%-FBSand1μg/mLofTPCK-treatedtrypsinfor3daysat37°C.Viralsupernatantswereharvestedandcentrifugedfor5minat2000gtoremovecellulardebris.Aliquotswerestoredat−80°C.IAVtitresweredeterminedbyTCID50assayasdescribedbelow.

IAVinfections
Cells wereinfected with IAV atMOI 0.5 for all experiments, exceptwhenmonitoringtheearlystepsoftheviralcycle(i.e.internalizationandnuclearimport),whereMOI4wasusedtoincreasesignaldetection.CellswereexposedtoIAVfor1hinasmallvolumeof2%FBSculturemediumtopromoteviraladsorptionatthecellsurfacebeforereplacingtheinoculumwithwarmculturemediumandincubatingat37°Cwith5%CO2.

RNAextractionsandRTqPCR
Intracellular RNA was extracted using the RNeasy mini kit (Qiagen) accordingtothemanufacturer’sprotocol.RNAyieldandpuritywereassessed by spectrophotometry (NanoDrop 2000c, ThermofisherScientific). Reverse transcription (RT) using oligo(dT) and randomprimerswasperformedusingthePrimeScriptRTReagentKit(PerfectRealTime,TakaraBioInc).Strand-specificRTforIAVRNAwasperformedusingtaggedprimersthatbindeithertotheconsensus3’-endsequence of vRNA or cRNA of all 8 segments, with Superscript III(Invitrogen, Thermofisher Scientific) in the presence of 0.1M DTT(Invitrogen,ThermofisherScientific),10mMdNTP(Invitrogen,ThermofisherScientific),andRNAsin(Promega).SegmentdistinctionwasthenachievedbyqPCRusingspecificprimerpairs.RANBP2-specificRTwasperformedusingaprimer(1µM)bindingtoaregionabsentfromRGPDs, as described in ref. 43 and the PrimeScript RT Reagent Kit.Real-time quantitative PCR was performed using the Power Up kit(Applied Biosystems, Thermofisher Scientific) on the ViiA7 thermocycler (Life technologies, Thermofisher Scientific). All RT and qPCRprimersarelistedinTableS2.

Immunofluorescence
CellswerewashedwithPBSandfixedwith4%paraformaldehyde(PFA,ThermofisherScientific)for10minatroomtemperature(R/T).After3PBSwashes,remainingPFAwasquenchedwithPBSNH4Cl50mMfor10minatR/T.CellswerethenpermeabilizedusingPBS0.5%Triton-100X(SigmaAldrich)for15minatR/T,andsaturatedinPBS2%Normalgoat serum (NGS, Invitrogen, Thermofisher Scientific) with 2 PBSwashesbetweeneachstep.Incubationwithprimaryantibodiesfor1hatR/Twasfollowedby5PBSwashesandincubationwithsecondaryantibodiesfor30minatR/T(TableS3).Afterfurtherwashing,cells-were stained with Hoechst (1:10,000 dilution in PBS; ThermofisherScientific) for 5min at R/T, washed, and mounted in ProlongDiamond Antifade mounting medium (Invitrogen, ThermofisherScientific). Images were acquired on a Zeiss LSM880 Airyscanconfocal microscope at the Montpellier Ressources Imagerie(MRI) facility and processed using Fiji (2.14.0). For visualisation,Alexafluor-488 signal corresponding to NP was pseudocoloured(“Orange Hot”) and brightness was increased uniformly acrossconditions.

Alpha-Centauriassay
CloningofIAV-α.ADNAfragmentcodingforαNluc(GVTGWRLCERILA)flankedbyNotIandNheIoverhangswasobtainedbyannealingtwooverlappingoligonucleotides:
NotI-αNLuc-NheI-F GGCCGCAGGGGGAGTGACAGGGTGGAGACTATGCGAAAGAATACTTGCATAAG
NotI-αNLuc-NheI-R CTAGCTTATGCAAGTATTCTTTCGCATAGTCTCCACCCTGTCACTCCCCCTGC
ThereversegeneticsplasmidpPolI-SL-PB2-αNlucwasgeneratedbysubcloningthisfragmentbetweentheNotIandNheIsitesofthepPolI-SL-PB2-Nanoluc58.
Particle production by reverse genetics. The eight pPolI-WSNplasmids (-PB2-αNluc, -PB1, -PA, -HA, -NP, -NA, -NS, -M), and fourexpression plasmids (pcDNA3.1-WSN-PB2, -PB1, -PA, -NP; 0.5μg ofeach)wereco-transfectedintoaco-cultureof293TandMDCKcells(seededina6-wellplateat4×105and3×105cells,respectively)using10μL of FuGENE® HD transfection reagent (Promega). After 24h at35°C,cellswere washed twicewithDMEM andincubatedin DMEMcontaining 1μg/mL of TPCK-treated trypsin for 48h. RecombinantPB2-αNluc viruswas amplified atanMOI of10−4 on MDCKcells for3daysat35°C.ViralstocksweretitratedonMDCKbyplaqueassaysandsequencedtoconfirmthepresenceoftheαNluccodingsequence.
CellsweretransducedwithLVCen-NLSfor3days,followedbyLVshRNA-controlorLVshRNA-RANBP2atMOI15for4days.Cellswere-theninfectedwithIAV-αatMOI4fortheindicatedtimes.LuminescencewasmeasuredusingNanoGloLiveCellsubstrate(Promega)andanInfiniteMPlexspectrophotometer(Tecan).

Nucleo-cytoplasmicfractionation
NuclearandcytoplasmicfractionswereseparatedusingtheNE-PERKit(Thermofisher Scientific) according to the manufacturer’s protocol.Briefly,1–2×106cellswerewashedinPBS,lysedincoldCERIbuffer,vortexed15s,andincubatedonicefor10min.ColdCERIIbufferwas-then added, followed by brief vortexing and incubation on ice for1min.After5svortexingandfullspeedcentrifugation,supernatants(cytoplasmic fraction) werecollected and stored at −80°C. Nuclear-pelletswereresuspendedincoldNERbufferandsubjectedto4cyclesof15svortexingthen10minincubationonice.Afterfullspeedcentrifugation,supernatants(nuclearfraction)werecollectedandstoredat−80°C.

Westernblot
Celllysatesweresonicatedfor20cycles(30son/off;BioruptorPico,Diagenode)andmixedwith4XLaemmlibuffer(250mMTris-HClpH7,8% sodium dodecyl sulphate (SDS), 40% glycerol, 10% β-mercaptoethanol,and0.005%bromophenolblue).Afterdenaturationat95°Cfor5min,samplesunderwentSDSpolyacrylamidegelelectrophoresis(SureCast Gel Handcast System,Thermo Fisher Scientific)and weretransferred to a 0.45µm nitrocellulose membrane (Sigma Aldrich).MembranesweresaturatedinPBScontaining0.05%Tween20(Sigma)and10%milkpowderfor30min,incubatedwithprimaryantibodiesovernightat4°C,washed3timesinPBS0.05%Tween20,andincubated with HRP-conjugated secondary antibodies for 1h at R/T(TableS3).SignalsweredetectedwithImmobilonForteWesternHRPsubstrate (Merck) or Immobilon ECL Ultra Western HRP Substrate(Merck)onaChemidocimagingsystem(Bio-Rad).

TCID50assay
MDCK cells were seeded in FBS-free MEM medium (Gibco) andinoculatedwithserialdilutionsofviralsupernatantsin2%FBSmediuminoctuplicates.After3daysat37°Cand5%CO2,cellswerewashedtwiceinPBSandfixedwith4%PFAfor10minatR/Tandstainedwithcrystalviolet.TCID50titrewerecalculatedas:
TCID50/mL = x0 + d(xi/n - 0.5)
wherex0isthelogoftheinitialdilutionfactor,dthelogoftheserialdilution factor, xi the number of positive events, n the number ofreplicates,andνthelogoftheinoculumvolume(mL).

Peripheralbloodmononuclearcell(PBMC)isolationandmonocyte-derivedmacrophagesdifferentiation
BuffycoatsfromhealthydonorswereobtainedfromtheÉtablissementFrançaisduSang(EFS).Bloodsampleswerediluted1:1withPBSandlayered over an equivalent volume of Lymphoprep (Stemcelltechnologies)atR/T.Sampleswerecentrifugedat800gfor30minat20°C (no brake). The PBMC ring was collected using a 5mL pipet,washed3timesinafinalvolumeof50mLPBS(1200rpm,5min,20°Cwith brakes on), and resuspended in 30mL of 10% FBS RPMI-1640medium. PBMCs were counted manually using counting chambers(Kova).
ForMDMdifferentiation,2×107cellswereseededperwellinto6-well plates, allowed to adhere for 45min, at 37°C with 5% CO2,washed 3 times with 10% FBS RPMI-1640 medium directly on thecell layers to remove loosely attached cells, and cultured in RPMI-1640 medium with 10% FBS and 50ng/mL GM-CSF (Gentaur)for8days.

Flowcytometry
After8daysofMDMdifferentiation,cellswerestainedandanalysedonaFortessaflowcytometer(BDBiosciences)usingthefollowingantibodies: CD3-BV421 (clone UCHT1, Biolegend 300433), CD14-PerCP-Cy5.5(clone HCD14, Biolegend 325621), CD16-Alexa700 (clone 3G8,Biolegend302026),CD11b-APC-Cy7(cloneM1/70,Biolegend101225),HLA-DR-FITC(REA805,Miltenyi130-111-941),andCD80-BV650(clone2D10,Biolegend305227)(seeTableS3).

MultiplexLuminexassay
Thepresenceofinflammatorymediatorswasassessedinsupernatantsof stimulated MDMs using the ProcartaPlex Human inflammationPanel 20-plexkit(Invitrogen,ThermoFisherScientific).First,asubsetofsampleswascentrifugedanddiluted1:10or1:50todeterminetheoptimaldilutionfortheassay.A1:50dilutionwasselectedinordertodetectthestronglyinducedmediators(e.g.CXCL8)withintherangeofthestandardcurves suppliedinthe kit.Buffersand standards werepreparedaccordingtothemanufacturer’sprotocol.Briefly,theCapture Bead mix was vortexed and added to the plate, followed bywashingwithWashbuffer,thensamplesandstandardswereadded.The plate was incubated for 1h atR/T on a shaker.After 2 washes,BiotinylatedDetectionAntibodymixwasaddedfor30minatR/Tonashaker.After2washes,Streptavidin-PEwasaddedfor30minatR/Tonashaker.After2morewashes,theplatewasreadontheMAGPIXflowcytometer(Luminex).

EstablishmentofU2OSclonesexpressingRANBP2T585MbyCRISPRknock-in
TheplasmidpUcIDTAmp(synthetizedbyIntegratedDNATechnologies) containing the donor sequence (see below) and the plasmidpSpCas9(BB)−2A-Puro(PX459;Addgene#62988)modifiedtocontainthe guide sequence (5’-GGCAGAATGCCTTCAGAAAA-3’) were co-transfected in equal amounts into human osteosarcomaU2OS cellsusingLipofectamine3000(Invitrogen,ThermoFisherScientific),following the manufacturer’s instructions. Two days post-transfection,cellswereselectedwithpuromycin(Gibco)at2µg/mlforfourdays.Single-cellcloneswereobtainedbylimitingdilution.Atotalof198cellsweredistributedin576wellscontaining10%FBSDMEMmedium.After3 weeks, 47 clones were successfully established and analyzed byRANBP2 allele-specific RTqPCR as described above and Sangersequencing(Eurofins).
DonorDNAsequence:
(GCGGTTTGTACTCTGATTCACAGAAAAGCAGTgtaagtagtaaaacaa aaatattgctttcacttagtgcgtaggttttaccggggatttaatcctcatgtgaagatttaatttgt catgtgacccattaacatatatgtatgtaagccctgaactgtgtatttagaaagcaattttagtaaa ttgaactattttttagACCTGGAAACGTAGCAAAATTGAGACTTCTAGTTCAG CATGAAATAAACACTCTAAGAGCCCAGGAAAAACATGGCCTTCAACC TGCTCTGCTTGTACATTGGGCAGAgTGtCTgCAGAAAAtGgtgagttttaaa gtataagcatttttaaagaacattaccttaattttttaaaatcatgaactttttattgaaagtttttttg ttctgaaaacagcagcttggtcacattatgacagatgtgttttttattgctgcaaaatagttaatgt agttaaatataagcacttagaggagcaatgcctggcacacagtgaatgttacatattagctgagc tgttactgttattccttaataattaagttctgataattattcagcctgaaaattaaaaaaa)
Capitalletters:exon
Lowercase:intron
Underlined:mutations
Boldlowercase:c.1754C>Tmutation

Generationofhomozygous/heterozygousRANBP2-expressingcontrolcellsforallelicqPCR
HEK-293T cells were transfected with plasmids encoding mCherry-RANBP2-shRNAandeitherGFP-RANBP2-WT,GFP-RANBP2-T585M,orboth(TableS1)incalciumphosphate.After48hofculture,cellsweredetachedusingtrypsin(Gibco)andsortedontheARIAIIucytometer(BectonDickinson)attheMRIfacilitytoisolatethemCherry-positivecells. Sorted cells were lysed and total RNAs were extractedusingtheRNeasyminikit(Qiagen)accordingtothemanufacturer’sprotocol.

MTTcellviabilityassay
CellviabilitywasassessedusingtheMTT[3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay (Sigma). Following treatment, the culture medium was carefully removed, and cells werewashes once with PBS to eliminate any residual medium or serumcomponents. A stock solution of MTT was prepared at 5mg/mL inDMEMwithoutphenolred(Gibco).Subsequently,100µloftheMTTsolutionwasaddedtoeachwellandincubatedfor3hat37°Cwith5%CO2to allow the formation of formazan crystals by metabolicallyactive cells. After incubation, 100µL of acidified isopropanol (1:200HCl0.05N)wasaddedtoeachwelltodissolvetheformazancrystalscompletely.Theabsorbancewasmeasuredat570nmusinganInfiniteMPlexspectrophotometer(Tecan).

Statisticalanalysis
StatisticalanalysiswasperformedusingtheGraphPadPrismsoftware(v10.2.3).ForqPCRexperiments,statisticalanalyseswereperformedon ΔCt or ΔΔCt values, as indicated. ΔCt values correspond to themeanoftechnicalreplicatesnormalisedforthehousekeepinggene,and ΔΔCt values are normalised to the control condition. Unlessotherwisestated,biologicalreplicatesarerepresentedassingledotsinbargraphs(mean+/-SEM).Allstatisticaltestsarespecifiedinthefigurelegends.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "Desgraupes et al. addressed the molecular basis of how a clinically relevant mutation in RANBP2 can promote the induction of acute necrotizing 1 encephalopathy. Although RANPB2 knockdown in A549 cells had no effect on overall IAV propagation, it resulted in a significant increase of viral negative-sense genomic vRNA and positive-sense cRNA sequences. An increase in cytoplasmic NP and the co-localisation of cytoplasmic RANPB2 with cytoplasmic NP with PB1 further indicated that RANBP2 knockdown induces an increase in the export of vRNPs to the cytoplasm. Interestingly, RANBP2 knockdown neither affected internalization nor nuclear import of IAV, but it facilitated the import of newly synthesised polymerase. Analysis of the stoichiometry of viral segments indicated that depletion of RANBP2 increases the replication of all vRNA segments, whereas it disproportionately affects their export back to the cytoplasm. Knockdown of RANBP2 in monocyte-derived macrophages resulted in enhanced proinflammatory cytokine and chemokine responses. U2OS cells carrying the RANBP2-T585M mutation homozygously showed enhanced CXCL10 expression following IAV infection. A similar effect was detected upon transfection of a constitutively active RIG-I variant. In WT clones, RANBP2 localised to the nuclear envelope, whereas clones homozygous for RANBP2-T585M lost the characteristic nuclear rim labelling indicating that the RBABP2 mutation disrupts RANBP2 retention at the nuclear envelope, whereas the integrity of nuclear pores seemed not be affected. In conclusion, the clinically relevant RANBP2 mutation facilitates the re-import of polymerase into the nucleus, which increases viral cRNA and vRNA, and selectively exports vRNA into the cytoplasm. The dysregulation of both processes results in an unusual accumulation of vRNA segments in the cytoplasm, that serve like pathogen-associated molecular patterns that trigger enhanced cytokine responses.\nThe authors present a precisely planned study. The manuscript guides the ready through a series of logically designed experiments, of which each one reveals interesting results. In the end, a rather anti-intuitive mechanism is concluded how a mutation in RANPB2 can cause detrimentally enhanced cytokine responses. The paper is a logical exercise at its best. The manuscript is very carefully written by using precise and easily understandable language.\nUnfortunately, the figures and figure captions are not as carefully prepared and need some improvement.\nMinor points:\nWhen the predominant ANE1-associated mutation c.C1754T: p.T585M is mentioned first, the nomenclature should be briefly introduced. Amongst the broad readership of the journal, not everybody is familiar how genomic alterations are identified. In Fig. 4C details are nicely illustrated, but insufficient explanations are given. This should be done ideally in the figure legend or in the main text.\nWithin Figure 1, many different font sizes were used for labelings. This is impairing the readability of the figure.\nFig. 1A-C: What do single dots mean? Do they represent single values or single experiments? How frequently the experiments were repeated?\nFig. 1C: It is difficult so see where the notion comes from that in THP-1 cells an up to 40-fold increase of M1 RNA was detected.\nFig. 1G: NTD presumably stands for N terminal domain. This abbreviation was introduced in the introduction, but not in the figure legend. This should be corrected.\nFig. 1I: Colocalization in the shRNA Control and RanBP2 groups look very similar. Some other way of visualization should be provided.\nFig. 1J: It is not detailed neither in the results section nor in the figure caption what is shown in the upper panel.\nIn the caption of Figure 1, a typo should be correct (J).\nIn Fig. 3A it looks as if the analysis of XCXL10 transcripts in monocyte-derived macrophages has been performed only twice. How can statistics be performed with 2 values? This needs re-evaluation. The same is true for the analysis of TNFalpha in monocyte-derived macrophages. How exactly the experiment was carried out? When for the IL-1beta analysis in monocyte-derived macrophages three values were analyzed, then the samples should be basically available for the analysis of the other cytokine RNAs. In the experiment with THP-1 cells, treatment with control shRNA was also carried out only twice, whereas the experiment with RanPB2 was performed three times. This is no basis to perform statistics. On a first glance, only for the studies with A549 cells enough data points were retrieved to perform reasonable statistics. It is recommended that a professional statistician cross-checks the calculations.\nFor Fig. 3D more details should be provided. Was the experiment carried out only once?\nIn Fig. 4A the caption should say: Schematic depiction of … and in the figure this text should be deleted. Within this figure part, only one font size should be used. The labelling within the box is so small that is it hardly readable.\nFig. 4D: Sanger sequencing goes back to Fred Sanger, therefore it should be written with a capital first letter.\nIn the legend of Fig. 4 reference is given to (I), but (I) is not shown the figure. This should be corrected.\nIn Fig. 4 too many different font sizes are used. In the end, every label is important, otherwise it would not have been shown. Therefore, the authors should consider using only one font size.", "reviewer_2": "Summary:\nIn this work, entitled ‘The genetic driver of Acute Necrotizing Encephalopathy, RANBP2, regulates the inflammatory response to Influenza A virus infection’, Desgraupes et al. describe that loss of RANBP2 leads to increased IAV vRNA replication and increased localization of vRNPs to the cytoplasm. This is accompanied with an increased pro-inflammatory cytokine response. CRISPR-edited cells expressing RANBP2 T585M, the SNP that is observed in ANE patients, also show increased CXCL10 expression in response to IAV infection or after stimulation with RIG-I 2CARD. Additionally, the T585M mutation disrupts the localization of RANBP2 at the nuclear pore. This study shows interesting insights into the function of RANBP2 in influenza virus infection, and how the RANBP2-T585M mutation could contribute to ANE. However, it is not clear if the observed increased inflammatory response in the absence of functional RANBP2 is caused by increased viral RNA replication or by exacerbated signaling downstream of RIG-I.\nMajor comments:\n1. All experiments shown in Figures 1-3 were done using only one shRNA. To exclude potential off-target effects of the used shRNA, key experiments need to be repeated with additional shRNAs and/or including rescue experiments (expression of shRNA resistant RANBP2).\n2. Figure S1: The western blot shows the knockdown at protein level in A549 cells at 72h post lentiviral transduction. Is this the time point that was used for infection with IAV? I could not find this information in the method section. The authors should add this information and potentially provide a western blot for the time point of infection. The western blot showing RANBP2 reduction at protein level should also be added for MDM cells (where currently only a qPCR at 24h is shown).\n3. Fig 1B and Fig 3A: The data showing the knockdown efficiency of shRNA-transduced THP1 cells is missing. The authors should provide a western blot showing RANBP2 levels like for the A549 cells in Figure S1B.\n4. The authors state that nuclear export of segment 1, 2, 3, 7 and 8 is increased by RANBP2 KD. I find it difficult to see this in the data shown in Figure 2F. For all segments, the cytoplasmic levels seem to be increased to at least some extend, and the nuclear levels are either equal or slightly increased compared to the control. The increase of RNA levels in the cytoplasm in RANBP2 KD compared to control is between 1.5-3-fold for all segments, which is a difference of 2-fold in-between the different segments. In a qPCR assay, this change corresponds to only 1 Cycle difference. I think that from this data it is difficult to conclude that the different segments are actually affected differently by RANBP2 KD. Additionally, I wonder why there is no decrease in the nuclear RNA levels observed if nuclear export is increased.\n5. While all virological assays were done in A549 cells, the experiments investigating the effect of RANBP2-T585M (Figure 4), were done in U2OS cells. It is not clear why the authors switch to a different cell line. I assume this is for technical reasons, as it might be difficult to do CRISPR editing in A549 cells. I think the authors should reproduce some of the shRNA KD data (increased cytoplasmic export of vRNPs and increased cytokine production in RANBP KD conditions) in U2OS cells to show if this cell model shows the same phenotype as A549 cells. Additionally, the increase in cytokine induction appears much stronger in U2OS RANBP2-T585M cells (Figure 4E) compared to A549 RANBP2 KD cells (Figure 3A). Is this due to differences in the cell lines, or does the T585M mutation increase cytokine production compared to RANBP2 KD?\n6. In Figure 4, the authors show an effect on CXCL10 with RIG-I 2CARD expression in RANBP2-T585M cells. Does this mean that the phenotype observed in cells expressing RANBP2-T585M is a different one than observed in the KD cells, caused downstream of RIG-I sensing and not by increased viral RNA levels or changed vRNA localization? To clarify this, I would suggest to test if import of viral polymerase and vRNP export are affected in U2OS RANBP2-T585M cells. And vice versa, could the authors repeat the experiment shown in Figure 3A with RIG-I 2CARD transfection instead of IAV infection?\n7. Are the protein levels of RANBP2-T585M the same as WT RANBP2 in U2OS cells? It would be important to show this, to exclude that the observed phenotype of the T585M mutation is not caused by reduced protein levels.\nMinor comments\n- What is the unit of the MOIs stated in the manuscript? PFU/cell or TCID50/cell?\n- I think the qPCR primers used for the validation of strand-specific RT shown in Fig S2 are not listed in the supplement. If I understood correctly, these primers are different from the qPCR primers used in the other assays, since they are specific for the tags added during the RT?\n- Figure 1J: From which condition is the magnified example image and the analysis? Could the authors provide this analysis showing the signal distribution for all conditions? Could the authors additionally provide a quantification of the colocalization (e.g. pearson coefficient) for all conditions?\n- Number of biological replicates is not stated for the experiments. Especially for the western blots shown in Fig 2A and 2B, how often was this experiment done?\n- Figure 2E: It is not clear to me why the authors analyze nuclear export of cRNAs. As far as I know, cRNAs would not need to be exported to the cytoplasm during the IAV life cycle, since they serve as template for vRNA synthesis that occurs in the nucleus.\n- Figure 2E and F: How many replicates were done of this experiment? Does each dot represent a technical replicate from the same experiment or a biological replicate from a different experiment? Please add this information to the figure legend. Additionally, the statistical analysis for this experiment should be added.\n- In the qPCRs in Figure 2E and F, the data was normalized in different ways. In 2E (cRNA), normalization is relative to the nuclear fraction of the control, which means that nuclear and cytoplasmic fractions can be compared to each other, and it is apparent that most of the cRNA is located to the nucleus. In Figure 2F, the same experiment is shown for vRNA, but here the fractions are normalized to the control condition within each fraction. Thus, it is not apparent how much RNA is exported to the nucleus at the observed time point. It would be better to do the normalization in the same was as for Figure 2E.\n- Line 253: ‘In particular, RANBP2 was not involved in the initial export of viral transcripts, which involves the general mRNA export pathway via NXF1:NXT’ – Did the authors test this? Please show data on viral mRNAs, or remove this statement from the discussion.\nComments on Methods part.\nThe following information missing in the methods part:\n- The amounts of plasmids transfected to produce lentiviral particles.\n- Detailed description of how A549 cells were transduced with the lentiviral particles. It is not clear from the manuscript if there was a selection step and if stable cell lines were produced, or if the A549 cells were freshly transduced and immediately infected with IAV in each experiment.\n- Information on IAV-WSN stock production and stock titration\n- Line 315: ‘For experiments with NF-kB canonical pathway inhibition, cells were pre-treated with 2% FBS culture medium containing 20 μM of PS1145 (MedChemExpress) for 1 h at 37°C’. It is not clear which experiments were performed with inhibition of NF-kB.", "reviewer_3": "The submitted study by Desgraupes et al. aims to understand the role of RANBP2 during influenza A virus infection. Through their interesting work, these authors were able to show that RANBP2 is able to directly influence the inflammatory response of epithelial and immune cells during infection. The manuscript is extremely well-written and their experimental work is very close-knit. This study provides significant groundwork towards the understanding of the exact mechanisms of RANBP2, especially in the context of patients with the genetic predisposition to developing ANE.\nMajor comments:\n- Lines 183-185, there is an inherently large jump here from the previous section. Some improvement for the motivation here to align the two sections is missing. Moreover, it was not established if the difference in vRNAs and vRNPs dynamics were also observed in these MDMs, before looking into the expression of proinflammatory genes.\n- The selection of the respiratory epithelial cell line A549 cells (adenocarcinoma) for the initial investigation makes sense. However, this reviewer would like to see the dynamics of proinflammatory gene expression after infection in upper respiratory cells to explicitly show the effect of the RANBP2 in IAV appropriate target cells. This would make an interesting comparison with the effect of infection of MDMs in this study (epithelial vs. immune proinflammatory responses).\n- Figure 3, the authors show that there are differences in the gene expression pattern of these proinflammatory markers after IAV infection after an overnight infection strategy. This reviewer thinks that a variable timelapse analysis may be necessary to clearly say that the differences in gene expression are a result of the cell types. It could be possible that some gene expression changes had happened a lot earlier in some cells and later in others. A time-resolved infection strategy with a qRT-PCR readout would help here.\n- Have the authors looked into interferon stimulated genes (ISGs) and the influence of RANBP2 here?\nMinor comments:\n- In line 105, the figures do not match the cell lines in the text.\n- Lines 178-180, well-written but must be revised to be more explicit that this is still heavily speculative at this point.\n- Figure 4D, please add the clone number on the traces to make it easier to read.\n- Line 765, please use the proper β symbol for IL-1β"}, "author_response": "Answers to the reviewers’ comments\nWe thank all reviewers for their thorough evaluation of our work and constructive comments.\nTo address the reviewers’ comments, we replicated the key experiments using alternative\nassays or shRNA, and we performed new experiments. In particular, following the reviewer 2’s\nsuggestion to better characterize the RANBP2-T585M CRISPR clones, Figure 4 now includes\nevidence that the RANBP2-T585M point mutant increases IAV vRNA and innate immune\nsignaling similarly to RANBP2 knockdown.\nOverall, the revised manuscript includes 41 new or substantially revised panels (Figs. 1B–C,\n1I–K, 2A–E, 2H–J, 3A–B, 4E–H, 4J, S1A–C, S4A–D, S5A–D, S9, S10A-C, S11, S14A–B, S16, S17A-\nB). The author order has been updated to reflect the contributions made during the revision\nperiod with A. Decorsière moved to second author position.\nReviewer #1 (Remarks to the Author):\nDesgraupes et al. addressed the molecular basis of how a clinically relevant mutation in\nRANBP2 can promote the induction of acute necrotizing 1 encephalopathy. Although RANPB2\nknockdown in A549 cells had no effect on overall IAV propagation, it resulted in a significant\nincrease of viral negative-sense genomic vRNA and positive-sense cRNA sequences. An\nincrease in cytoplasmic NP and the co-localisation of cytoplasmic RANBP2 with cytoplasmic NP\nwith PB1 further indicated that RANBP2 knockdown induces an increase in the export of vRNPs\nto the cytoplasm. Interestingly, RANBP2 knockdown neither affected internalization nor\nnuclear import of IAV, but it facilitated the import of newly synthesised polymerase. Analysis\nof the stoichiometry of viral segments indicated that depletion of RANBP2 increases the\nreplication of all vRNA segments, whereas it disproportionately affects their export back to the\ncytoplasm. Knockdown of RANBP2 in monocyte-derived macrophages resulted in enhanced\nproinflammatory cytokine and chemokine responses. U2OS cells carrying the RANBP2-T585M\nmutation homozygously showed enhanced CXCL10 expression following IAV infection. A\nsimilar effect was detected upon transfection of a constitutively active RIG-I variant. In WT\nclones, RANBP2 localised to the nuclear envelope, whereas clones homozygous for RANBP2-\nT585M lost the characteristic nuclear rim labelling indicating that the RBABP2 mutation\ndisrupts RANBP2 retention at the nuclear envelope, whereas the integrity of nuclear pores\nseemed not be affected. In conclusion, the clinically relevant RANBP2 mutation facilitates the\nre-import of polymerase into the nucleus, which increases viral cRNA and vRNA, and\nselectively exports vRNA into the cytoplasm. The dysregulation of both processes results in an\nunusual accumulation of vRNA segments in the cytoplasm, that serve like pathogen-associated\nmolecular patterns that trigger enhanced cytokine responses.\nThe authors present a precisely planned study. The manuscript guides the ready through a\nseries of logically designed experiments, of which each one reveals interesting results. In the\nend, a rather anti-intuitive mechanism is concluded how a mutation in RANPB2 can cause\ndetrimentally enhanced cytokine responses. The paper is a logical exercise at its best. The\nmanuscript is very carefully written by using precise and easily understandable language.\nUnfortunately, the figures and figure captions are not as carefully prepared and need some\nimprovement.\nWe have reviewed all the figure captions and harmonized the fonts and sizes, particularly\nregarding the graph axes.\nMinor points:\nWhen the predominant ANE1-associated mutation c.C1754T: p.T585M is mentioned first, the\nnomenclature should be briefly introduced. Amongst the broad readership of the journal, not\neverybody is familiar how genomic alterations are identified.\nThe mutation nomenclature has been introduced as follows:\n(lines 75-76): “c.C1754T on the coding DNA reference sequence, leading to p.T585M at the\nprotein level”\nIn Fig. 4C details are nicely illustrated, but insufficient explanations are given. This should be\ndone ideally in the figure legend or in the main text.\nExplanation has been added in the figure legend.\n(lines 765-766): “This mutation on DNA (c.C1754T) leads to the T585M mutation on the\nRANBP2 protein (p.T585M)”\nWithin Figure 1, many different font sizes were used for labelings. This is impairing the\nreadability of the figure.\nThis has been corrected.\nFig. 1A-C: What do single dots mean? Do they represent single values or single experiments?\nHow frequently the experiments were repeated?\nAll experiments were performed 3 times. In all graphs, dots represent technical replicates from\nall independent experiments. This was added to the figure legend as follows:\n(line 699-700): “For panels A-F, dots represent technical replicates from all independent\nexperiments.”\nFig. 1C: It is difficult so see where the notion comes from that in THP-1 cells an up to 40-fold\nincrease of M1 RNA was detected.\nThe original THP-1 panel was a mean of 2 experiments, therefore we replicated these\nexperiments. Results in Figure 1C, which are the mean of 3 independent experiments, no\nlonger show a 40-fold increase but rather an average of 10-fold. Therefore, this sentence was\nremoved from the results section.\nFigure 1C: THP-1 cells were transduced with LV shRNA-control\nor -RANBP2 or non-treated (NT) then infected with influenza A\nvirus (A/WSN/1933) at MOI 0.5. At the indicated time points,\ncells were lysed and total intracellular RNAs were extracted.\nViral RNAs encoding the Matrix 1 (M1) protein were amplified\nby RTqPCR.\nFig. 1G: NTD presumably stands for N terminal domain. This abbreviation was introduced in\nthe introduction, but not in the figure legend. This should be corrected.\nThis has been amended. Now NTD refers to N-terminal domain and NT refers to non-\ntransduced/non-treated.\nFig. 1I: Colocalization in the shRNA Control and RanBP2 groups look very similar. Some other\nway of visualization should be provided.\nIn both conditions, viral RNPs are exported, leading to the colocalization of cytoplasmic PB1\nwith NP. However, in the RANBP2-depleted cells, the cytoplasmic levels of NP are increased.\nThis experiment aimed to determine whether the observed increase in NP corresponds to\nassembled RNPs or to unassembled, free NP protein.\nPearson’s correlation coefficients were calculated across all experiments to quantify the\ndegree of colocalization between NP and PB1 signals. As expected, these analyses confirmed\ncolocalization. The figure panel has been updated to include colocalization curves, Pearson’s\ncoefficients determined across all three experiments, and zoomed-in views of the\ncolocalization areas for improved clarity (see Figure 1I-K, S5A-D).\nFig. 1J: It is not detailed neither in the results section nor in the figure caption what is shown\nin the upper panel.\nThe upper panel showed an enlargement of the colocalization signal in the RANBP2-depleted\ncells. In the updated figure, the panel now includes zoomed-in regions of each condition, as\nwell as Pearson’s correlation coefficients. The figure legend has been revised as follows:\n(lines 703-705): “(J) Colocalization curves and Pearson’s correlation coefficients between PB1\nand NP signals were quantified using the Fiji software. (K) Enlargements of colocalization fields\nfrom panel I.”\nIn the caption of Figure 1, a typo should be correct (J).\nThe typo has been corrected.\nIn Fig. 3A it looks as if the analysis of XCXL10 transcripts in monocyte-derived macrophages\nhas been performed only twice. How can statistics be performed with 2 values? This needs re-\nevaluation. The same is true for the analysis of TNFalpha in monocyte-derived macrophages.\nHow exactly the experiment was carried out? When for the IL-1beta analysis in monocyte-\nderived macrophages three values were analyzed, then the samples should be basically\navailable for the analysis of the other cytokine RNAs. In the experiment with THP-1 cells,\ntreatment with control shRNA was also carried out only twice, whereas the experiment with\nRanPB2 was performed three times. This is no basis to perform statistics. On a first glance, only\nfor the studies with A549 cells enough data points were retrieved to perform reasonable\nstatistics. It is recommended that a professional statistician cross-checks the calculations.\nExperiments were replicated and Figure 3A now includes A549 and U2OS cells. As the degree\nof activation varied between experiments, paired Student’s t tests were performed (*P < 0.05,\n**P < 0.002, ***P < 0.0002, ****P < 0.0001, ns: non-significant).\nFor Fig. 3D more details should be provided. Was the experiment carried out only once?\nFigure 3D does not exist. All assays were performed as three independent experiments, except\nthe Luminex assay, which was conducted using primary cells from two donors due to cost\nconstraints.\nIn Fig. 4A the caption should say: Schematic depiction of … and in the figure this text should\nbe deleted. Within this figure part, only one font size should be used. The labelling within the\nbox is so small that is it hardly readable.\nThe figure legend for 4A was revised and font sizes in the panel have been increased and\nhomogenized.\nFig. 4D: Sanger sequencing goes back to Fred Sanger, therefore it should be written with a\ncapital first letter.\nSanger is now written with a capital letter on the figure.\nIn the legend of Fig. 4 reference is given to (I), but (I) is not shown the figure. This should be\ncorrected.\nThis is indeed an error that appeared during rewrites of the manuscript. The legend of figure\n4 has been corrected.\nIn Fig. 4 too many different font sizes are used. In the end, every label is important, otherwise\nit would not have been shown. Therefore, the authors should consider using only one font size.\nThis has been corrected.\nReviewer #2 (Remarks to the Author):\nSummary:\nIn this work, entitled ‘The genetic driver of Acute Necrotizing Encephalopathy, RANBP2,\nregulates the inflammatory response to Influenza A virus infection’, Desgraupes et al. describe\nthat loss of RANBP2 leads to increased IAV vRNA replication and increased localization of\nvRNPs to the cytoplasm. This is accompanied with an increased pro-inflammatory cytokine\nresponse. CRISPR-edited cells expressing RANBP2 T585M, the SNP that is observed in ANE\npatients, also show increased CXCL10 expression in response to IAV infection or after\nstimulation with RIG-I 2CARD. Additionally, the T585M mutation disrupts the localization of\nRANBP2 at the nuclear pore. This study shows interesting insights into the function of RANBP2\nin influenza virus infection, and how the RANBP2-T585M mutation could contribute to ANE.\nHowever, it is not clear if the observed increased inflammatory response in the absence of\nfunctional RANBP2 is caused by increased viral RNA replication or by exacerbated signaling\ndownstream of RIG-I.\nIn the original manuscript, we included a single experiment using constitutive RIG-I, which\nsuggested that the RANBP2 mutation increased CXCL10 (but not TNF) expression\ndownstream of RIG-I. Similar effects can be observed with other artificial innate immune\ntriggers (LPS, R848, LV transduction). Indeed, in the absence of IAV, RANBP2 knockdown\ncaused a modest yet significant increase in TNFα and IL-1β levels (Figure 3A-B), suggesting that\nRANBP2 depletion may induce a mild pro-inflammatory state independently of viral replication\n(possibly due to LV transduction). This effect is low in magnitude and may only be observed in\nsome cell types (e.g. U2OS). In contrast, IAV replication triggered a robust pro-inflammatory\nresponse in all tested cells (up to 12-14 fold increases in secreted CXCL8, CXCL10 and CCL3\nproteins by primary macrophages), demonstrating RANBP2’s role in controlling IAV-induced\ninflammation. Notably, unstimulated CRISPR clones did not show basal activation, unlike LV-\ntransduced U2OS cells (Figure 3A, Reviewer figure 1).\nAccordingly, we removed the RIG-I panel from the revised manuscript and replicated all key\nexperiments using viral infection only. We also include a note in the text to acknowledge this\nobservation:\n(lines 196-199): “Interestingly, a modest increase in some of these transcripts was also\nobserved in uninfected cells (Figure 3A-B), likely reflecting low level activation triggered by\nlentiviral vector transduction, and suggesting that RANBP2 knockdown may induce a mild pro-\ninflammatory state independently of its effect on IAV replication”.\nReviewer Figure 1: Pro-inflammatory cytokine levels in unstimulated CRISPR clones.\nCRISPR clones were lysed and total intracellular RNAs were extracted. Cytokine transcripts were amplified by\nRTqPCR.\nMajor comments:\n1. All experiments shown in Figures 1-3 were done using only one shRNA. To exclude potential\noff-target effects of the used shRNA, key experiments need to be repeated with additional\nshRNAs and/or including rescue experiments (expression of shRNA resistant RANBP2).\nIn order to exclude potential off-target effects, we replicated all key experiments using an\nindependent second shRNA targeting RANBP2 (position 225, Figure S4A) previously validated\nby Di Nunzio et al. (2012). Efficient RANBP2 knockdown was confirmed (Figure S4B). Consistent\nwith the first shRNA, RANBP2 depletion caused cytoplasmic accumulation of NP, specifically in\nRANBP2-shRNA-expressing cells (white arrow heads, Figure S4C and S4D) and significantly\nincreased CXCL10 (70-fold), TNF (4-fold) and IL-6 (7-fold) following IAV infection, confirming\nits role in regulating the inflammatory response to IAV infection (Figure S11).\nThese results have been added to supplementary Figures S4 and S11, and the following\nsentences have been added in the results part:\n(Lines 130-131): “This phenotype was confirmed using a previously published RANBP2-\ntargeting shRNA (Di Nunzio et al., 2012; Figure S4A-D).”\n(Lines 193-195): “RANBP2 depletion led to a significant upregulation of CXCL10, IL-6, TNF-\nand IL-1 (Figure 3A-B, Figure S10A-B), which mirrored the increase in vRNA (Figure S10C) and\nwas confirmed using an independent RANBP2-targeting shRNA (Figure S11)”\nFigure S4: Increase in cytoplasmic NP in RANBP2-KD cells at 8 hpi, using a second shRNA\n(A) Schematic depiction of the hybridizing sites of the two RANBP2-specific shRNAs. While the shRNA-RANBP2\n#1 binds in the 3’UTR sequence, the shRNA-RANBP2 #2 binds at position 225 of RANBP2 RNA. (B) A549 cells were\ntransduced with LV shRNA-control or shRANBP2 for 48h, and RANBP2 knock-down was verified by RTqPCR and\nWestern Blot. (C) Transduced A549 cells were infected with influenza A virus (IAV) (A/WSN/1933) at MOI 0.5. At\n8h post-infection, cells were fixed and stained for the nucleoprotein (NP). Scale bar: 20µm. (D) Cytoplasmic\nfluorescence was quantified using the Fiji software. Each dot represents the cytoplasmic fluorescence of a cell.\nTwo-tailed unpaired Student’s t test. ****P < 0.0001.\nFigure S11: Increase in cytokine transcripts in\nRANBP2-KD cells, using a second shRNA\nA549 cells were transduced with LV shRNA-\ncontrol or shRANBP2 for 48h and infected with\ninfluenza A virus (IAV) (A/WSN/1933) at MOI\n0.5. At 8h post-infection, cytokine transcripts\nwere assessed by RT-qPCR. Results were\nnormalized on RPL13a and on the stimulated\ncontrol condition. Results are presented as\nmean +/- SD. Two-tailed unpaired Student’s t\ntest. *P < 0.05, **P < 0.002, ***P < 0.0002,\n****P < 0.0001, ns: non-significant.\n2. Figure S1: The western blot shows the knockdown at protein level in A549 cells at 72h post\nlentiviral transduction. Is this the time point that was used for infection with IAV? I could not\nfind this information in the method section. The authors should add this information and\npotentially provide a western blot for the time point of infection.\nFor experiments in A549, infection was initiated at 48 h post-transduction but all experiments\nwere ended between 56-96 h post-transduction, which is why we showed knockdown at 72 h.\nThe revised figure now includes western blots at both 48 h and 72 h post-transduction with\nquantification of 3 independent blots (Figure S1A-B). We also included this information in the\nmethod section:\n(lines 348): “All experiments were completed by 56-96 h post-transduction.”\nFigure S1: Assessment of RANBP2 knock-\ndown in A549 cells\nA549 cells were transduced with LV shRNA-\ncontrol or shRNA-RANBP2 at MOI 20. (A)\nKnock-down efficacy was assessed by western\nBlot at 48 h and 72 h post-transduction in A549\ncells. (B) Quantification of band intensity was\ndone using the ImageLab software. Results are\nnormalized on the vinculin signal and on the\ncontrol condition.\nThe western blot showing RANBP2 reduction at protein level should also be added for MDM\ncells (where currently only a qPCR at 24h is shown).\nRANBP2 protein levels were assessed in MDMs at 72 h post-transduction. A representative\nblot is now included, together with a quantification from 3 independent experiments (Figure\nS14B).\nFigure S14B: Transduction with LV shRNA-RANBP2 leads to an\nefficient down-regulation of RANBP2 in MDM cells. MDM\ncells were differentiated from primary monocytes and\ntransduced with LV shRNA-Control or shRANBP2 for 72 h.\nRANBP2 knock-down was verified by Western Blot.\nQuantification of band intensity was done using the ImageLab\nsoftware. Results are normalized on the vinculin signal and on\nthe control condition.\n3. Fig 1B and Fig 3A: The data showing the knockdown efficiency of shRNA-transduced THP1\ncells is missing. The authors should provide a western blot showing RANBP2 levels like for the\nA549 cells in Figure S1B.\nRANBP2 protein levels were assessed in THP-1 cells at 72 h post-transduction (Figure S1C).\nFigure S1C: Transduction with LV shRNA-RANBP2 leads to an efficient down-\nregulation of RANBP2 in THP-1 cells. THP-1 cells were transduced with LV\nshRNA-Control or shRANBP2 for 72 h. RANBP2 knock-down was verified by\nWestern Blot.\n4. The authors state that nuclear export of segment 1, 2, 3, 7 and 8 is increased by RANBP2\nKD. I find it difficult to see this in the data shown in Figure 2F. For all segments, the cytoplasmic\nlevels seem to be increased to at least some extend, and the nuclear levels are either equal or\nslightly increased compared to the control. The increase of RNA levels in the cytoplasm in\nRANBP2 KD compared to control is between 1.5-3-fold for all segments, which is a difference\nof 2-fold in-between the different segments. In a qPCR assay, this change corresponds to only\nsegments are actually affected differently by RANBP2 KD. Additionally, I wonder why there is\nno decrease in the nuclear RNA levels observed if nuclear export is increased.\nWe agree that the evidence for differential vRNP segment nuclear export needed to be\nstrengthened and required clearer presentation. During the revision, we repeated the\nexperiment multiple times and consistently observed increased vRNA export upon RANBP2\nknockdown, though the specific segments affected varied between replicates. The revised data\n(Figures 2H-J) now show that RANBP2-KD causes over-export of most vRNAs, unevenly\naffecting individual segments and thereby altering segment stoichiometry. Consistent with\nthis, nuclear vRNA levels decrease when export is increased (Figure 2H).\nThe manuscript text was also revised as follows:\n(lines 178-183): “RANBP2 knockdown … led to an overall increase in the nuclear export of vRNA\nsegments (Figure 2H-I). This effect disproportionally affected individual segments across\nexperiments, as reflected by a higher standard deviation (Figure 2J). These findings indicate\nthat, while the depletion of RANBP2 increases the replication of all vRNA segments, it also\npromotes their uneven export to the cytoplasm, thereby disrupting segment stoichiometry.”\nFigure 2: (H) Transduced A549 cells were infected with IAV at MOI 0.5. At 6h post-infection, nuclear and\ncytoplasmic fractions were separated and total intracellular RNAs were extracted. vRNAs were reverse\ntranscribed by strand-specific RTqPCR and for each segment, the cytoplasmic and nuclear abundances were\nexpressed as percentages of their sum. Technical triplicates from a representative experiment are presented. (I)\nPercentage of vRNAs exported to the cytoplasm (all segments pooled), from two independent experiments. Each\ndot represents the mean of three technical replicates per segment. Paired analysis was performed using the\nKolmogorov-Smirnov test. **P < 0.002. (J) Same data as (I), shown as mean +/- range. Standard deviation values\nwere determined for each condition. Kolmogorov-Smirnov test. ****P < 0.0001.\n5. While all virological assays were done in A549 cells, the experiments investigating the effect\nof RANBP2-T585M (Figure 4), were done in U2OS cells. It is not clear why the authors switch\nto a different cell line. I assume this is for technical reasons, as it might be difficult to do CRISPR\nediting in A549 cells. I think the authors should reproduce some of the shRNA KD data\n(increased cytoplasmic export of vRNPs and increased cytokine production in RANBP KD\nconditions) in U2OS cells to show if this cell model shows the same phenotype as A549 cells.\nAdditionally, the increase in cytokine induction appears much stronger in U2OS RANBP2-\nT585M cells (Figure 4E) compared to A549 RANBP2 KD cells (Figure 3A). Is this due to\ndifferences in the cell lines, or does the T585M mutation increase cytokine production\ncompared to RANBP2 KD?\nTo reproduce the virological assays in the same cell line used for CRISPR editing, we depleted\nRANBP2 in U2OS cells (Figure S10A-B) and assessed both the viral phenotype (Figure S10C)\nand cytokine production (Figure 3B). RANBP2 knockdown increased viral RNA levels, and\nelevated CXCL10, TNF, IL-6 and IL-1 transcripts, confirming the phenotype observed in A549\ncells. Moreover, although stimulation of both cell types induced similar cytokine levels in\ncontrol cells, higher induction was observed in RANBP2-depleted U2OS cells, suggesting that\nthe higher increase observed in CRISPR clones likely reflects U2OS-specific responses rather\nthan the T585M mutation itself.\nFigure S10: RANBP2 knockdown in U2OS cells leads to increased IAV RNA\nTransduced A549 cells were infected with IAV at MOI 0.5. RANBP2 knockdown was quantified by (A) RTqPCR and\n(B) western blot, at 72 h post-transduction. (C) Total intracellular RNAs were extracted and viral RNAs encoding\nthe Matrix 1 (M1) protein were amplified by RTqPCR. Results are normalized on RPL13a and on the control\ncondition (mean +/- SD). Each dot represents a technical replicate from three independent experiments. Two-\ntailed unpaired Student’s t test, ****P < 0.0001.\nFigure 3B: U2OS cells were transduced with\nLV shRNA-control or shRNA-RANBP2 at MOI\n30 and stimulated with influenza A virus\n(IAV) (A/WSN/1933) at MOI 0.5 overnight.\nCytokine transcripts were assessed by RT-\nqPCR and results were normalized on RPL13a\nand on the unstimulated control condition.\nResults are presented as mean +/- SD. Three\nindependent experiments. Two-tailed paired\nStudent’s t tests. *P < 0.05, **P < 0.002, ***P\n< 0.0002, ****P < 0.0001, ns: non-\nsignificant.\n6. In Figure 4, the authors show an effect on CXCL10 with RIG-I 2CARD expression in RANBP2-\nT585M cells. Does this mean that the phenotype observed in cells expressing RANBP2-\nT585M is a different one than observed in the KD cells, caused downstream of RIG-I sensing and not\nby increased viral RNA levels or changed vRNA localization? To clarify this, I would suggest to\ntest if import of viral polymerase and vRNP export are affected in U2OS RANBP2-T585M cells.\nAnd vice versa, could the authors repeat the experiment shown in Figure 3A with RIG-I 2CARD\ntransfection instead of IAV infection?\nAs mentioned above (see comment to Summary above), the use of synthetic agonists such as\nconstitutive RIG-I can lead to low level background activation. Indeed, in the absence of IAV,\nRANBP2 knockdown causes a modest yet significant increase in TNFα and IL-1β levels (revised\nFigure 3B), suggesting that RANBP2 depletion may induce a mild pro-inflammatory state\nindependently of viral replication (either through an additional mechanism or possibly as a\nresult of lentiviral vector transduction). Nevertheless, despite this mild basal activation, IAV\nreplication triggers a considerable outburst in pro-inflammatory cytokines, clearly highlighting\nthe role of RANBP2 in controlling IAV-induced inflammation.\nIn contrast, RANBP2-T585M did not induce higher basal levels of pro-inflammatory cytokines\nin unstimulated clones (see Reviewer Figure 1 below), suggesting a specific mechanism\ntriggered by IAV in ANE1 mutant cells.\nReviewer Figure 1: Pro-inflammatory cytokine levels in unstimulated CRISPR clones.\nCRISPR clones were lysed and total intracellular RNAs were extracted. Cytokine transcripts were amplified by\nRTqPCR.\nRegarding viral RNP export, we have assessed whether this process is impacted in mutant\nCRISPR clones. Remarkably, while vRNPs remained nuclear in WT clones, cells harboring the\nT585M mutation of RANBP2 displayed increased cytoplasmic NP localization (Figure 4E-F),\naccompanied by increased vRNA replication (Figure 4G).\nThese data have been included in Figure 4 of the revised manuscript and the text has been\nadapted accordingly in the result section.\n(Lines 232- 236): “CRISPR-knock-in clones were infected with IAV, and replication was assessed\nby quantifying viral NP at 8 hpi, as previously (Figure 1G-H). Strikingly, cytoplasmic NP was\nsignificantly increased in all ANE1 CRISPR clones compared with wild-type clones, regardless\nof whether the mutation was heterozygous or homozygous (Figure 4E-F, Figure S16). This was\naccompanied by elevated vRNA levels, which was statistically significant at 24 hpi (Figure 4G).”\nFigure 4E-G: The RANBP2 T585M mutation leads to increased NP export and IAV RNA levels.\nCRISPR clones were stimulated by IAV (A/WSN/1933) at MOI 0.5. (A) At 8h post-infection, cells were fixed and\nstained for the nucleoprotein (NP). Scale bar: 20µm. (B) Cytoplasmic fluorescence was quantified using the Fiji\nsoftware. Multiple Welch’s t tests. *P < 0.05, **P < 0.002, ***P < 0.0002, ****P < 0.0001, ns: non-significant. (C)\nAt 1h, 8h and 24h post-infection, total intracellular RNAs were extracted and viral RNAs encoding the Matrix 1\n(M1) protein were quantified by RTqPCR. For each clone, results were normalized on RPL13a and on the early\ntime point (1h). One-way ANOVA. *P < 0.05, **P < 0.002, ***P < 0.0002, ****P < 0.0001, ns: non-significant.\n7. Are the protein levels of RANBP2-T585M the same as WT RANBP2 in U2OS cells? It would\nbe important to show this, to exclude that the observed phenotype of the T585M mutation is\nnot caused by reduced protein levels.\nWe determined RANBP2 protein levels by western blot in CRISPR clones and did not observe\nany statistically significant variations.\nThis data has been added to supplementary Figure S17 and the following sentence has been\nadded in the text: (Lines 245-246): “Overall, we did not observe a statistically significant\nvariation in the protein levels of RANBP2 (Figure S17A-B).”\nFigure S17: RANBP2 protein levels\nin CRISPR clones\n(A) CRISPR clones were lysed and\nprotein levels of RANBP2 were\ndetermined by western blot. (B)\nQuantification of band intensity\nwas done using the ImageLab\nsoftware. Results are normalized\non the vinculin signal and on the C4\nWT clone. One-way ANOVA. ns:\nnon-significant.\nMinor comments:\n- What is the unit of the MOIs stated in the manuscript? PFU/cell or TCID50/cell?\nThe MOI is indicated as TU/cell. The information has been added in material and methods\nline 345.\n- I think the qPCR primers used for the validation of strand-specific RT shown in Fig S2 are not\nlisted in the supplement. If I understood correctly, these primers are different from the qPCR\nprimers used in the other assays, since they are specific for the tags added during the RT?\nThese primers were indeed overlooked. They have now been added to the primer table (Table\nS2) of the material and method section. Additionally, we noticed that the 5’/3’ endings were\ninverted on the scheme of supplementary figure S2 (for cRNA, lower box), this has been\ncorrected on the figure.\n- Figure 1J: From which condition is the magnified example image and the analysis? Could the\nauthors provide this analysis showing the signal distribution for all conditions? Could the\nauthors additionally provide a quantification of the colocalization (e.g. pearson coefficient) for\nall conditions?\nThe upper panel (Former Figure 1J) showed an enlargement of the colocalization signal in the\nRANBP2-depleted cells. In the updated figure, the panel now includes zoomed-in regions of\neach condition, as well as Pearson’s correlation coefficients (Updated Figure 1I-J-K and Figure\nS5A-D).\nIn both conditions, viral RNPs are exported, leading to the colocalization of cytoplasmic PB1\nwith NP. However, in the RANBP2-depleted cells, the cytoplasmic levels of NP are increased\n(Figure 1G-H). This experiment aimed to determine whether the observed increase in NP\ncorresponds to assembled RNPs or to unassembled, free NP protein.\nPearson’s correlation coefficients were calculated across all experiments to quantify the\ndegree of colocalization between NP and PB1 signals. As expected, these analyses confirmed\ncolocalization. The figure panel has been updated to include colocalization curves, Pearson’s\ncoefficients, and zoomed-in views of the colocalization areas for improved clarity.\nFigure 1I-J-K: RANBP2 depletion leads to an increase in viral RNP export.\nA549 cells were transduced with LV shRNA-Control or shRANBP2 and infected with influenza A virus (IAV)\n(A/WSN/1933) at MOI 0.5. (I) Transduced A549 cells were infected at MOI 0.5 and co-stained for the NP and PB1\nproteins at 8h post-infection. Scale bar: 20µm. (J) Colocalization curves and Pearson’s correlation coefficients\nbetween PB1 and NP signals were quantified using the Fiji software. Welch’s t test. *P < 0.05, **P < 0.002, ***P\n< 0.0002, ****P < 0.0001, ns: non-significant. (K) Enlargements of colocalization fields from panel I.\nFigure S5: Increase in cytoplasmic vRNP in RANBP2-KD cells at 8 hpi\nTransduced A549 cells were infected at MOI 0.5 and co-stained for the NP and PB1 (A) or for the NP and PA (C)\nproteins at 8h post-infection. Scale bar: 20µm. (B-D) Pearson’s correlation coefficients between both signals were\nquantified using the Fiji software.\n- Number of biological replicates is not stated for the experiments. Especially for the western\nblots shown in Fig 2A and 2B, how often was this experiment done?\nWestern blots in Figure 2A-B were performed and quantified in three independent\nexperiments. Western blots in Figure 2C-D were performed five times. Nuclear fractions were\npure in all experiments (quantifications were made across all five experiment), but cytoplasmic\nfractions were successfully purified twice.\nThe information about the number of replicates has been added to all figure legends.\n- Figure 2E: It is not clear to me why the authors analyze nuclear export of cRNAs. As far as I\nknow, cRNAs would not need to be exported to the cytoplasm during the IAV life cycle, since\nthey serve as template for vRNA synthesis that occurs in the nucleus.\nTo meet this reviewer’s comment, the cRNA data has been moved to supplementary figures\n(revised Figure S9). However, we have retained this data in the manuscript, as it provides\nrelevant biological information regarding the nuclear increase of all cRNA segments and\nconstitutes an important control of the fractionation efficacy.\n- Figure 2E and F: How many replicates were done of this experiment? Does each dot represent\na technical replicate from the same experiment or a biological replicate from a different\nexperiment? Please add this information to the figure legend. Additionally, the statistical\nanalysis for this experiment should be added.\nIn the revised manuscript, these panels have been changed. Figure 2E is now Figures 2H-J and\nFigure 2F is now Figure S9.\nTo address this reviewer’s concern, we repeated the experiment multiple times and\nconsistently observed increased vRNA export upon RANBP2 knockdown, though the specific\nsegments affected varied between replicates. Since vRNP segment nuclear export required\nclearer presentation, panel 2H shows technical triplicates from a representative experiment,\nwhile panels 2I, 2J and S9 are the mean of technical triplicates from two or three independent\nexperiments.\nWe have added the information about the number of replicates and statistical analysis to all\nfigure legends.\n- In the qPCRs in Figure 2E and F, the data was normalized in different ways. In 2E (cRNA),\nnormalization is relative to the nuclear fraction of the control, which means that nuclear and\ncytoplasmic fractions can be compared to each other, and it is apparent that most of the cRNA\nis located to the nucleus. In Figure 2F, the same experiment is shown for vRNA, but here the\nfractions are normalized to the control condition within each fraction. Thus, it is not apparent\nhow much RNA is exported to the nucleus at the observed time point. It would be better to do\nthe normalization in the same was as for Figure 2E.\nThis panel was not retained in the revised manuscript (see comment above).\n- Line 253: ‘In particular, RANBP2 was not involved in the initial export of viral transcripts,\nwhich involves the general mRNA export pathway via NXF1:NXT’ – Did the authors test this?\nPlease show data on viral mRNAs, or remove this statement from the discussion.\nThe export of influenza virus messenger RNAs has been extensively documented (Refs 1-4\nbelow), and is known to rely on the NXF1:NXT1 pathway. Consistent with this, in our\nexperiments, inhibiting CRM1 did not impact the translation of PB1, PB2 and PA (data not\nshown), suggesting that viral mRNA export is not affected by RANBP2-KD. We have\nreformulated the text as follows:\n(Line 273): “and did not seem to affect the initial export of viral transcripts, which involves the\ngeneral mRNA export pathway via NXF1:NXT1.”\n(1) Bhat, P., Aksenova, V., Gazzara, M. et al. Influenza virus mRNAs encode determinants for nuclear export via\nthe cellular TREX-2 complex. Nat Commun 14, 2304 (2023). https://doi.org/10.1038/s41467-023-37911-0\n(2) Zhao L, Liu Q, Huang J, Lu Y, Zhao Y, Ping J (2022) TREX (transcription/export)-NP complex exerts a dual effect\non regulating polymerase activity and replication of influenza A virus. PLoS Pathog 18(9): e1010835.\nhttps://doi.org/10.1371/journal.ppat.1010835\n(3) Ke Zhang, Tolga Cagatay, Dongqi Xie, Alexia E. Angelos, Serena Cornelius, Vasilisa Aksenova, Sadaf Aslam, Zhiyu\nHe, Matthew Esparza, Ashley Vazhavilla, Mary Dasso, Adolfo García-Sastre, Yi Ren, Beatriz M.A. Fontoura,\nCellular NS1-BP protein interacts with the mRNA export receptor NXF1 to mediate nuclear export of influenza\nvirus M mRNAs, Journal of Biological Chemistry, Volume 300, Issue 11, 2024, 107871, ISSN 0021-9258,\nhttps://doi.org/10.1016/j.jbc.2024.107871\n(4) Pereira CFRead EKCWise HMAmorim MJ, Digard P2017. Influenza A Virus NS1 Protein Promotes Efficient\nNuclear Export of Unspliced Viral M1 mRNA. J Virol91:10.1128/jvi.00528-17. https://doi.org/10.1128/jvi.00528-\nThe following information missing in the methods part:\n- The amounts of plasmids transfected to produce lentiviral particles.\nThe information has been added in the material & methods section:\n(lines 333-334): “Lentiviral vectors (LV) were produced in HEK-293T cells (ATCC) by calcium\nphosphate transfection using a VSV-G envelope plasmid (pVSVg, 15 µg in a 70-80% confluent\n175cm2 flask), a Gag-Pol encapsidation plasmid (p8.74, 30 µg), and the transfer plasmid of\ninterest (30 µg; mCherry-shRNA-control, mCherry-shRNA-RANBP2 or Centauri-NLuc-NLS; see\nTable S1).”\n- Detailed description of how A549 cells were transduced with the lentiviral particles. It is not\nclear from the manuscript if there was a selection step and if stable cell lines were produced,\nor if the A549 cells were freshly transduced and immediately infected with IAV in each\nexperiment.\nA549 cells were freshly transduced and we do not use selection since transduction efficiencies\nare consistently >95% in cell lines. This information has been added to the material &\nmethods section.\n(Lines 343- 348): “Cells were seeded into plates one day prior to transduction. After a PBS\nwash, lentiviral transduction was performed at MOI 20 to 40 (transducing units/cell) in a small\nvolume of culture medium supplemented with 2% FBS, to facilitate viral adsorption. After 2 h\nof incubation at 37°C, 10% FBS culture medium was added. Transduced cells were cultured for\n48-72 h prior to viral infections to allow efficient knock-down. All experiments were completed\nby 56-96 h post-transduction”.\n- Information on IAV-WSN stock production and stock titration.\nThis information has been added to the material & methods section.\n(Lines 349- 353): “H1N1/A/WSN/1933 viral stocks were produced by amplification at an MOI\nof 10-4 on MDCK cells, cultured in Minimal Essential Medium (MEM, Gibco) containing 2%-FBS\nand 1 μg/mL of TPCK-treated trypsin for 3 days at 37°C. Viral supernatants were harvested and\ncentrifuged for 5 min at 2,000 g to remove cellular debris. Aliquots were stored at -80°C. IAV\ntiters were determined by TCID50 assay as described below.”\n- Line 315: ‘For experiments with NF-kB canonical pathway inhibition, cells were pre-treated\nwith 2% FBS culture medium containing 20 μM of PS1145 (MedChemExpress) for 1 h at 37°C’.\nIt is not clear which experiments were performed with inhibition of NF-kB.\nThese experiments were not included in the original manuscript and this sentence has been\nremoved.\nReviewer #3 (Remarks to the Author):\nThe submitted study by Desgraupes et al. aims to understand the role of RANBP2 during\ninfluenza A virus infection. Through their interesting work, these authors were able to show\nthat RANBP2 is able to directly influence the inflammatory response of epithelial and immune\ncells during infection. The manuscript is extremely well-written and their experimental work is\nvery close-knit. This study provides significant groundwork towards the understanding of the\nexact mechanisms of RANBP2, especially in the context of patients with the genetic\npredisposition to developing ANE.\nMajor comments:\n- Lines 183-185, there is an inherently large jump here from the previous section. Some\nimprovement for the motivation here to align the two sections is missing.\nThe transition between the two sections was re-written as follows.\n(Line 192-194): “To investigate whether the abnormal cytoplasmic accumulation of vRNA,\nobserved in RANBP2-depleted cells, might associate with increased inflammation, we\nmeasured the expression of inflammatory transcripts by qPCR.”\nAnd in the following paragraph (Lines 200-201): “To confirm this at the protein level, primary\nmonocytes were isolated from PBMCs from healthy donors and differentiated into pro-\ninflammatory M1-like macrophages.”\nMoreover, it was not established if the difference in vRNAs and vRNPs dynamics were also\nobserved in these MDMs, before looking into the expression of proinflammatory genes.\nWhile we agree that demonstrating the vRNA and vRNP dynamics in MDMs would strengthen\nthe manuscript, our attempts to do so using the same conditions applied to A549, THP-1 and\nU2OS cells were not successful. Primary MDMs exhibited high background in imaging assays,\nand infection efficiency at the single-cell level was more variable across donors than in\nimmortalized lines, preventing reproducible quantification of vRNA abundance and vRNP\nlocalization. In addition, RANBP2 knockdown in MDMs, although satisfactory at the\ntranscriptional level (~60% inhibition, Figure S14A), was modest at the protein level (~40%\ninhibition, Figure S14B). This is likely due to the non-cycling nature of MDMs, which limits the\nturnover of existing protein pools and leads to increased RANBP2 half-life and nuclear pore\nresidency. Despite these technical constraints, MDMs displayed a robust increase in pro-\ninflammatory gene expression after IAV exposure (Figure 3C), consistent with the phenotype\nobserved in all the other cell types examined. Similarly to the WT/T585M heterozygous clone,\nwhere subtle alterations in RANBP2 localization at the nuclear rim (Figure 4K) produce major\nchanges in the inflammatory response (Figure 4H), these findings indicate that even minor\nperturbations in RANBP2 can lead to substantial IAV-induced inflammatory effects, underlining\nthe relevance of these results to the ANE1 physiopathology.\n- The selection of the respiratory epithelial cell line A549 cells (adenocarcinoma) for the initial\ninvestigation makes sense. However, this reviewer would like to see the dynamics of\nproinflammatory gene expression after infection in upper respiratory cells to explicitly show\nthe effect of the RANBP2 in IAV appropriate target cells. This would make an interesting\ncomparison with the effect of infection of MDMs in this study (epithelial vs. immune\nproinflammatory responses).\nWe appreciate the reviewer’s suggestion to include experiments using primary upper\nrespiratory tract models. In vitro studies of the respiratory epithelium can indeed be\nperformed using air-liquid interface (ALI) cultures or 3D organoids. However, due to budgetary\nand time constraints, we were unfortunately unable to invest in these approaches. More\nimportantly, while analyzing proinflammatory gene expression in upper respiratory cells could\nprovide additional insight into local host responses to IAV infection, this approach is not\ndirectly relevant to the ANE1 pathology. ANE1 is a neurological disorder that primarily affects\nthe brain, leading to acute encephalopathy and hallmark bilateral lesions of the thalami.\nCurrent evidence suggests that the neurological damage is not driven by local effects at the\nprimary site of infection but by downstream effects on the central nervous system.\n- Figure 3, the authors show that there are differences in the gene expression pattern of these\nproinflammatory markers after IAV infection after an overnight infection strategy. This\nreviewer thinks that a variable timelapse analysis may be necessary to clearly say that the\ndifferences in gene expression are a result of the cell types. It could be possible that some gene\nexpression changes had happened a lot earlier in some cells and later in others. A time-\nresolved infection strategy with a qRT-PCR readout would help here.\nAs noted by Reviewer 1, this panel lacked sufficient experimental replicates for some cell types\n(see comment above “In Fig. 3A it looks as if the analysis of XCXL10 transcripts in monocyte-\nderived macrophages has been performed only twice.”). We have therefore removed this\npanel from the revised manuscript. Instead, Figure 3A-B now include triplicate experiments\nfrom A549 and U2OS cells, which show similar results, thus arguing against a cell-type-\ndependent effect.\n- Have the authors looked into interferon stimulated genes (ISGs) and the influence of RANBP2\nhere?\nAlthough we have not specifically analyzed interferon-stimulated genes (ISGs), our data do not\nsupport a strong involvement of RANBP2 in the interferon (IFN) pathway. First, RANBP2\nexpression itself is not responsive to IFNα stimulation (see Reviewer Figure 2), suggesting it is\nnot an ISG. Second, we did not observe major alterations in key components or outputs of IFN\nsignaling following RANBP2 knockdown. For example, IFNα induction in MDMs was modest\n(Figure 3C), and IFNβ levels are not increased in RANBP2-depleted cells upon IAV infection\n(Reviewer Figure 3). Together, these observations indicate that, under our experimental\nconditions, RANBP2 depletion does not markedly influence IFN production or signaling\nresponses.\nReviewer Figure 2: RANBP2 expression is not modified by IFNα\nstimulation.\nJurkat, CEM or THP-1 cells (treated or not with 100 ng/mL PMA)\nwere stimulated with 100 U/mL IFNα for 16 h. Total intracellular\nRNAs were extracted and RANBP2 expression was quantified by\nRTqPCR.\nReviewer Figure 3: RANBP2-depletion does not modify IFNβ expression.\nA549 cells were transduced with LV shRNA-control or shRANBP2 for 48h and\ninfected with influenza A virus (IAV) (A/WSN/1933) at MOI 0.5. At 16h post-\ninfection, cytokine transcripts were assessed by RT-qPCR. Results were\nnormalized on RPL13a and on the stimulated control condition. Results are\npresented as mean +/- SD. Two-tailed unpaired Student’s t test. *P < 0.05, **P <\n0.002, ***P < 0.0002, ****P < 0.0001, ns: non-significant.\nMinor comments:\n- In line 105, the figures do not match the cell lines in the text.\nThe A549 and THP-1 cells were inadvertently inverted. This has now been corrected as follows\n(line 111): “In A549 cells (Figure 1B) and in the monocytic cell line THP-1 (Figure 1C, Figure\nS1).”\n- Lines 178-180, well-written but must be revised to be more explicit that this is still heavily\nspeculative at this point.\nThe title for this results section has been revised to be more explicit as follows:\n(line 171-172): “RANBP2 depletion favours the export of vRNA segments to the cytoplasm and\ndisrupts the segment stoichiometry”.\n- Figure 4D, please add the clone number on the traces to make it easier to read.\nThe modification has been made on the figure.\n- Line 765, please use the proper β symbol for IL-1β.\nThis has been amended (line 775)."}, {"round": 2, "reviewer_comments": {"reviewer_1": "Desgraupes et al. followed many of the reviewers‘ recommendations and implemented several changes in the revised version of their manuscript. New controls were included, and experiments were repeated where needed in order to be able to provide statistics. Thanks to the extended discussion, more context is provided. However, in all figures still different font sizes are used which makes it difficult to read the figures. Since many readers certainly will want to verify some of the conclusions by inspection of the actual data, the authors should try to present the data in an easily readable manner.\nMinor points:\nAbbreviations in context of RNA are inconsistently used. In line 47 of the revised manuscript, it is defined that vRNA stands for “negative-sense single-stranded viral RNA”. However, many times later in the manuscript the authors again refer to „viral RNA“. Do they use “vRNA” and “viral RNA” in a synonymously manner? If this is the case, the authors should better stay with the abbreviation vRNA throughout the manuscript after introduction the abbreviation.\nshRNA is not defined in the manuscript. Considering that it is mentioned more than 100 times in the text, the authors should introduce the abbreviation, which presumably stands for “short” or “small hairpin RNA”.\nThe expression „pro-inflammatory M1-like macrophages“ can be misleading. It is recommended that the authors better refer to “GM-CSF macrophages”.", "reviewer_2": "In the revised version of the manuscript ‘The genetic driver of Acute Necrotizing Encephalopathy, RANBP2, regulates the inflammatory response to Influenza A virus infection’ the authors improved the initial manuscript. Importantly, they added RANBP2 knockdown data in U2OS cells, the cell line which is also used to characterize the ANE-associated T585M mutation, and confirmed the observed phenotype using a second shRNA for RANBP2 knockdown.\nThe authors removed the RIG-I data, which was rather weak in the previous version. In the revised manuscript, the authors do not show a link between increased cytoplasmic vRNA and RIG-I sensing, but I feel it is justified to make this connection from the literature.\nIn the revised version of the manuscript, the authors addressed all of my concerns appropriately. I have two comments regarding the presentation of the data:\n- It is difficult to compare the data in Fig 3A and Fig S11, which show cytokine expression for RANBP2 knockdown with two different shRNAs in A549 cells, since the normalization of the data was performed differently. Especially for CXCL10, it looks like with shRNA#1 (Fig 3A), the induction is ~2 fold (IAV infected, control vs. RANBP2 shRNA), while with shRNA#2 (Fig S11), it is more than 50-fold. Is there a way to improve the visualization to make it easier to compare the two different shRNAs?\n- Fig 2D: Figure legend states n = 5, but for PA only 4 data points are shown. Can the 5th experiment also be included?", "reviewer_3": "The revised study by Desgraupes et al. is a significant improvement from the prior submission. This reviewer thanks the authors for the additional data they have provided (in the manuscript and those in the rebuttal only).\nMajor comment:\nThere is only one point that this reviewer would like to raise which is the confidence on the inflammatory effect of RANBP2.\nThe hint of its presence is there but the wide spread of measurements in Fig3A and Fig4G can raise some concerns.\nFig 3A qPCRs - measurements for control and RANBP2 have a high spread and most significant changes are being driven by 1 measurement point. Have these been tested for outliers or maybe the experiment needs to be repeated?\nFig4G- needs the dots on the box whisker plots. The spread of these measurements are extremely high. The hint of the effect is there but additional confirmation of this would be nice. Similar to Fig3A, test for outliers and a potential repeat of the experiment might be necessary.\nFig3C - LUMINEX assays do not show TNFa, IL6 and IL1b increase and only for CXCL10 which is different from what was found in A549 cells and U2OS. Any point of discussion here?"}, "author_response": "REVIEWER COMMENTS\nReviewer #1 (Remarks to the Author):\nDesgraupes et al. followed many of the reviewers‘ recommenda?ons and implemented several\nchanges in the revised version of their manuscript. New controls were included, and experiments\nwere repeated where needed in order to be able to provide sta?s?cs. Thanks to the extended\ndiscussion, more context is provided. However, in all figures s?ll different font sizes are used which\nmakes it difficult to read the figures. Since many readers certainly will want to verify some of the\nconclusions by inspec?on of the actual data, the authors should try to present the data in an easily\nreadable manner.\nWe thank the reviewer for their construc?ve feedback and have harmonised the font sizes across\nfigures (main and supplemental) to improve readability. All labels - including axes, schema?cs,\nstainings, sta?s?cs, etc – are now in font 9.\nMinor points:\nAbbrevia?ons in context of RNA are inconsistently used. In line 47 of the revised manuscript, it is\ndefined that vRNA stands for “nega?ve-sense single-stranded viral RNA”. However, many ?mes later\nin the manuscript the authors again refer to „viral RNA“. Do they use “vRNA” and “viral RNA” in a\nsynonymously manner? If this is the case, the authors should beWer stay with the abbrevia?on vRNA\nthroughout the manuscript aXer introduc?on the abbrevia?on.\nIn this manuscript, the abbreviation vRNA (viral RNA) refers exclusively to the negative-sense\ngenomic RNA, while “viral RNAs” (plural, non-abbreviated) denotes all RNA species derived from the\nvirus, including vRNA, cRNA and mRNA. We have checked the manuscript and confirm that the\ncorrect terms and abbreviations are used throughout.\nshRNA is not defined in the manuscript. Considering that it is men?oned more than 100 ?mes in the\ntext, the authors should introduce the abbrevia?on, which presumably stands for “short” or “small\nhairpin RNA”.\nThis was introduced line 102.\nThe expression „pro-inflammatory M1-like macrophages“ can be misleading. It is recommended that\nthe authors beWer refer to “GM-CSF macrophages”.\nThis change was made line 201.\nReviewer #2 (Remarks to the Author):\nIn the revised version of the manuscript ‘The gene?c driver of Acute Necro?zing Encephalopathy,\nRANBP2, regulates the inflammatory response to Influenza A virus infec?on’ the authors improved\nthe ini?al manuscript. Importantly, they added RANBP2 knockdown data in U2OS cells, the cell line\nwhich is also used to characterize the ANE-associated T585M muta?on, and confirmed the observed\nphenotype using a second shRNA for RANBP2 knockdown.\nThe authors removed the RIG-I data, which was rather weak in the previous version. In the revised\nmanuscript, the authors do not show a link between increased cytoplasmic vRNA and RIG-I sensing,\nbut I feel it is jus?fied to make this connec?on from the literature.\nIn the revised version of the manuscript, the authors addressed all of my concerns appropriately. I\nhave two comments regarding the presenta?on of the data:\n- It is difficult to compare the data in Fig 3A and Fig S11, which show cytokine expression for RANBP2\nknockdown with two different shRNAs in A549 cells, since the normaliza?on of the data was\nperformed differently. Especially for CXCL10, it looks like with shRNA#1 (Fig 3A), the induc?on is ~2\nfold (IAV infected, control vs. RANBP2 shRNA), while with shRNA#2 (Fig S11), it is more than 50-fold.\nIs there a way to improve the visualiza?on to make it easier to compare the two different shRNAs?\nWe thank the reviewer for this comment. In A549 cells, RANBP2 knockdown consistently leads to a 2-\nto 5-fold increase in TNFα, IL-6, and IL-1β following IAV infection, whereas CXCL10 changes range\nfrom 2-fold to 50-fold. This may be explained by variable basal levels of CXCL10 between cell batches,\ncell passages, or slight variability in IAV inoculum, as CXCL10 mRNA is highly responsive to viral\nreplication and interferon signaling. Thus, while we can confidently conclude that the effect on\ninflamma?on is RANBP2-specific, the difference in CXCL10 induc?on between the two shRNAs likely\nreflect experimental variability rather than mechanis?c differences.\nFor this reason and because, in the previous revision, reviewers asked to see uns?mulated cells,\nwe\nwould prefer to retain the current representa?on of Figure 3A.\n- Fig 2D: Figure legend states n = 5, but for PA only 4 data points are shown. Can the 5th experiment\nalso be included?\nWe thank the reviewer for no?cing this. The 5th data point has now been added to the figure (Fig 2D).\nReviewer #3 (Remarks to the Author):\nThe revised study by Desgraupes et al. is a significant improvement from the prior submission. This\nreviewer thanks the authors for the addi?onal data they have provided (in the manuscript and those\nin the rebuWal only).\nMajor comment:\nThere is only one point that this reviewer would like to raise which is the confidence on the\ninflammatory effect of RANBP2. The hint of its presence is there but the wide spread of\nmeasurements in Fig3A and Fig4G can raise some concerns.\nFig 3A qPCRs - measurements for control and RANBP2 have a high spread and most significant\nchanges are being driven by 1 measurement point. Have these been tested for outliers or maybe the\nexperiment needs to be repeated?\nFig4G- needs the dots on the box whisker plots. The spread of these measurements are extremely\nhigh. The hint of the effect is there but addi?onal confirma?on of this would be nice. Similar to Fig3A,\ntest for outliers and a poten?al repeat of the experiment might be necessary.\nWe appreciate the reviewer’s observation regarding the variability in qPCR measurements. While\nthere is indeed a wide spread, we believe the increased inflammatory effect observed upon IAV\ninfection in RANBP2 knockdown cells is robust. In all experiments performed with RANBP2\nknockdown, we consistently observed an increase in pro-inflammatory cytokine synthesis - across\ndifferent cell types (A549, U2OS, THP-1, and primary macrophages), different experimenters,\ndifferent len?viral stocks, and supported by statistical analysis. The reviewer’s impression likely arises\nfrom the fact that the magnitude of cytokine induc?on varies between experiments in the control\ncondi?ons (see reviewer Figure 1 below), which makes the pooled data appear less striking. This\nbiological variability can be explained by:\n(i) Differences in basal cytokine levels between cell batches or passages. Since data are normalized to\nthe control condition, low starting expression can make fold changes appear larger (see comment to\nReviewer 2).\n(ii) Differences in cell density. Previous work has demonstrated that cell activation and cytokine\nexpression vary considerably with cell density, not only at the time of plating but also based on the\ncumulative history of density during passaging, thus creating heterogeneity in cytokine expression\nbetween experiments (Muldoon et al., Nat Commun 2020, PMID: 32054845; Binan et al., Cell 2025,\nPMID: 40081369). Generally, cytokine expression increases with confluency, a trend we have also\nobserved for IL-1β and IL-6 (data not shown). Therefore, variations in cell density at the time of RNA\nextraction, as well as throughout passaging and preparation, are likely to account for the observed\nvariability.\nTherefore, we prefer not to remove any outliers, as they do not represent technical errors but rather\nbiological variability, and still allow the reader to see the effect of RANBP2-KD.\nReviewer figure 1: Reproducible increase of cytokine expression in the RANBP2-KD condition\nacross experimental replicates. A549 cells were transduced with LV shRNA-control or shRANBP2 for\n48h and infected with influenza A virus (A/WSN/1933) at MOI 0.5 overnight. Cytokine\ntranscripts were assessed by RT-qPCR. Results were normalized on RPL13a and on the uns?mulated\ncontrol condi?on (mean +/- SD). Single experiments from Figure 3A are presented (N1-N4), with each\ndot represen?ng a technical replicate.\nFig3C - LUMINEX assays do not show TNFa, IL6 and IL1b increase and only for CXCL10 which is\ndifferent from what was found in A549 cells and U2OS. Any point of discussion here?\nWe appreciate the reviewer’s observation regarding the Luminex results.\nSeveral factors may contribute to these differences, including the fact that secreted protein levels do\nnot necessarily mirror transcriptional output, and that different cell types produce distinct cytokine\nprofiles. Fig 3C presents fold changes rela?ve to the infected shControl condi?on. Accordingly, our\ndata in non-immune and immune cell types show increases in different cytokines, yet consistently\nconfirm the reproducible effect of RANBP2 on inflammation.\nIn response to this reviewer’s point, we have added a sentence to the discussion to acknowledge\nthis (line 289-293): “Although there was considerable variability in the magnitude of this effect across\nexperiments, as well as differences in the cytokines induced, par?cularly when comparing non-\nimmune cell lines (A549, U2OS) with primary immune cells (macrophages), the global increase in\ninflammatory ac?va?on upon RANBP2-KD was consistently observed.”"}, {"round": 3, "reviewer_comments": {"reviewer_3": "This reviewer would like to thank the authors for their detailed responses regarding the concerns of all reviewers. No further comments or edits the careful phrasing of line 289-293 is sufficient."}, "author_response": ""}]

Decision letter