Hepatitis C virus infection activates an innate pathway involving IKK-α in lipogenesis and viral assembly

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1 A r t i l s Hptitis C virus intion tivts n innt pthwy involving in lipognsis n virl ssmly Qishng Li, Véroniqu Pèn, Sihrth Krishnmurthy, Hln Ch T Jk Ling npg 213 Ntur Amri, In. All rights rsrv. Hptitis C virus () intrts xtnsivly with host tors to not only stlish proutiv intion ut lso triggr uniqu pthologil prosss. Our rnt gnom-wi sirna srn monstrt tht IkB kins- (IKK-) is ruil host tor or. Hr w sri nw nulr tor kb (NF-kB)-inpnnt n kins-mit nulr untion o IKK- in ssmly., through its 3 untrnslt rgion, intrts with DEAD ox polyppti 3, X-link () to tivt IKK-, whih trnslots to th nulus n inus CBP/p3-mit trnsriptionl progrm involving strol rgultory lmntining protins (SREBPs). This innt pthwy inus lipogni gns n nhns or-ssoit lipi roplt ormtion to ilitt virl ssmly. Chmil inhiitors o IKK- supprss intion n IKK- inu lipognsis, oring proo-oonpt pproh or nw thrputi vlopmnt. Our rsults show tht uss novl mhnism to xploit intrinsi innt rsponss n hijk lipi mtolism, whih my ontriut to high hroniity rts n th pthologil hllmrk o sttosis in intion. intion is ling us o hroni livr iss ssoit with sustntil moriity n mortlity worlwi 1. A prottiv vin is not vill, n th urrnt thrputi rgimn is suoptiml 2. Th virus hs uniqu propnsity to us prsistnt intion n inu progrssiv livr mg 3. xtnsivly xploits host tors, suh s llulr lipi mtoli pthwys, or iint propgtion 4,5. hs n shown to ltr th lipi mtolism o int hptoyts 6, rsulting in uniqu pthologil tur o intion: hpti sttosis. Ativtion o SREBPs, whih r ruil trnsriptionl rgultors o holstrol n tty i mtolism 7, hs n shown in -int hptoyts 8,9. Howvr, th mhnisti sis o thir tivtion rmins unlr. tivts host innt immunity tht untions to limit virl intion. Rognition o virl pthogn-ssoit molulr pttrns (PAMPs) y pttrn rognition rptors (PRRs), suh s RIG-I lik rptors (RLRs), n tivtion o vrious signling pthwys, inluing intrron rgultory tor 3 (IRF3) n NFκB, r rly stps o n intrinsi innt immun rspons tht ls to th susqunt inution o intrrons 1. Th NF-κB pthwy is tightly rgult y th IKK omplx, whih onsists o two tlyti suunits, n IKK-β, n rgultory suunit, NEMO (lso known s IKK-γ) 11. Ativtion o IKK-β n NEMO n susqunt IκB grtion r ruil stps in th tivtion o th nonil NF-κB pthwy 12. prrntilly phosphorylts NF-κB2 rthr thn IκB n ls to th tivtion o p52-rlb htroimrs tht rgult istint sust o NF-κB trgt gns 12. This ltrntiv tion is rrr to s th nonnonil pthwy 13. is rmrkly vrstil molul tht is involv in ivrs signling pthwys to rgult gn xprssion; mny o its tions r inpnnt o NF-κB 12,14. Unlik IKK-β, n shuttl twn th ytoplsm n th nulus 15,16. In th nulus, intrts with CREB ining protin (CBP) n ontriuts to NF-κB mit gn xprssion through phosphoryltion o histon H3 (rs. 15,17,18). Th trnsriptionl trgts o, howvr, rmin lrgly unknown. W rntly prorm gnom-wi RNAi srn to isovr host pnnis. W intii (unr th nm CHUK) 19 s ing importnt or th li yl. Hr w onirm tht is rquisit or proutiv intion n show tht it uprgults lipognsis o host lls or iint virl ssmly through trnsriptionl inution o SREBPs. intion, through th tion o th 3 untrnslt rgion (3 UTR), intrts with to tivt n thry inus lipi roplt iognsis. W provi irt untionl link twn intion, inlmmtion, innt immunity n hpti lipi mtolism. RESULTS IKK- is rquir or proutiv intion W prviously us two-prt virl intion protool to hrtriz host pnnis ssoit with oth th rly (prt-on) n lt (prt-two) stgs o th li yl 19. W onirm tht th t o silning ws mor pronoun in th prt-two (>85% inhiition) thn th prt-on ssy (~6% inhiition) in Huh7.5.1 lls, suggsting tht ts mor uring th lt stg o virl intion (Fig. 1 n Supplmntry Fig. 1). W onirm th t o pltion 19 y tsting our iniviul sirnas o th SMARTpool (Fig. 1 n Supplmntry Fig. 1). Exprssion o sirna-rsistnt mutnt rstor intion in lls trt with sirna trgting (Supplmntry Fig. 1), urthr vliting th phnotyp-spii rol o in intion. Livr Disss Brnh, Ntionl Institut o Dits n Digstiv n Kiny Disss, US Ntionl Instituts o Hlth, Bths, Mryln, USA. Corrsponn shoul rss to T.J.L. (jkl@g1.nik.nih.gov). Riv 29 August 212; pt 9 April 213; pulish onlin 26 My 213; oi:1.138/nm VOLUME 19 NUMBER 6 JUNE 213 ntur miin

2 r t i l s npg 213 Ntur Amri, In. All rights rsrv. Figur 1 Rol o in intion. () Prt-on n prt-two or stining. R, or; lu, ll nuli. Sl rs, 1 µm. Th numrs rprsnt th prntgs o or stining positiv lls (mn ± s.. o triplit xprimnts). Th si prix inits th sirna ginst th init protin;, nontrgting sirna ontrol; ApoE, polipoprotin E. () Eiis o vrious sirnas in silning n rstrining RNA proution. Vlus r normliz rltiv to th ontrol. () Et o pltion on intious proution n srtion ssss y limiting ilution ssy. TCID 5, 5% tissu ultur intiv os. () Et o ovrxprssion o on intion. () Et o ovrxprssion o th HA KM on intion., mok trnstion with HA-tgg mpty plsmi (,). (,g) Ets o wlolton (W.; 3 µm), IKK Inhiitor XII (; 1 µm) n (1 µm) on proution () n virl intivity (g). Sl rs, 1 µm. (h,i) Dos-rspons ts o wlolton n IKK Inhiitor XII on RNA proution n srtion in Huh7.5.1 lls (h) n PHHs (i). Error rs (,g i) rprsnt ± s.. o triplit xprimnts. *P <.5, P <.1 trmin y Stunt s t tst. sirna sustntilly impir th proution n srtion o intious y mor thn 9% (Fig. 1). Ovrxprssion o y trnsting Huh7.5.1 lls with plsmi noing hmgglutinin-tgg (HA ) sustntilly inrs intious proution (Fig. 1). Th untion o rquirs its kins tivity, n th kins-tiv K44M mutnt o (ll hr KM ) hvs s ominntngtiv mutnt 2. Trnstion o th IKKα KM lok propgtion similrly to sirna (Fig. 1). To urthr invstigt th rol o in intion, w tst vrious IKK inhiitors in -int Huh7.5.1 lls n primry humn hptoyts (PHHs) (Fig. 1 i n Supplmntry Fig. 2). Trtmnt o Huh7.5.1 lls with wlolton n IKK Inhiitor XII, whih r hmil inhiitors o oth n IKK-β, mrkly ru or protin stining n intious virl prtil proution (Fig. 1,g). Inrsing onntrtions o oth ompouns l to os-pnnt lin in proution n srtion in oth Huh7.5.1 lls n PHHs tht oul not ount or y minor ytotoxiity t high onntrtions (Fig. 1h,i n Supplmntry Fig. 2,)., n IKK-β spii inhiitor, h littl t on proution (Fig. 1 i n Supplmntry Fig. 2,). Th rol o IKK- in ssmly n lipi roplt ormtion To invstigt th stp o th li yl in whih is rquir, w us multipl virologi ssys. silning prrntilly t xtrllulr lvls in th ll ultur intion systm (Fig. 1). W thror spiilly xmin singl-yl rplition y trnsting gnomi into CD81-iint Huh7 (Huh7.25) lls 21 n oun tht rplition ws not t y silning (Fig. 2). silning h no t Prt on Prt two h Prt on 39.5 ± 12.2 ± sicd81 siapoe si pool si ± ± ± ± ± ± ± 6.1 ±.3 sicd81 i β-tuulin on ssys trgting iniviul stps o th rly li yl, inluing ntry ( psuovirus ssy), trnsltion ( intrnl riosom ntry sit (IRES)-rivn rportr) n rplition (sugnomi rplion) (Fig. 2 n Supplmntry Fig. 3), onsistnt with prominnt rol o in th lt stg o th virl li yl. W thus hypothsiz tht is involv in th ssmly or srtion o intious virl prtils. Th lipi roplt hs n shown to ply ruil prt in ssmly Consistnt with this, w osrv protins, prtiulrly th or n NS5A protins, in los proximity to lipi roplts in -int lls (Fig. 2 n Supplmntry Fig. 4,). Clls int with h mrk inrs in lipi roplt numrs, lipi roplt positiv r, lipi roplt luorsn intnsity n triglyri ontnt, n silning signiintly lok this inrs (Fig. 2 n Supplmntry Fig. 4,). In -int lls trt with sirna, or protin stining ws only mostly ru, ut th ssoition twn or protin n lipi roplts ws iminish sustntilly (Fig. 2 n Supplmntry Fig. 4), suggsting tht is importnt or th ssoition o lipi roplts with protins uring th ssmly pross. Th IKK inhiitors wlolton n Inhiitor XII, ut not, show strong inhiition o lipi roplt ormtion g siapoe * 5. HA β-tuulin.2 Intivity (TCID 5 pr ml) si 2 si 3 si 4 si pool HA Prt two si 1 si 2 si 3 si 4 W ± 12.5 ± 3.5 ± ± HA 1 3 W. 1 W. Huh7.5.1 HA (µm) 1..5 KM Intivity (TCID 5 pr ml) Intivity (TCID 5 pr ml) W. Intivity (TCID 5 pr ml) PHH KM HA KM HA β-tuulin W. (µm) ntur miin VOLUME 19 NUMBER 6 JUNE

3 A r t i l s npg 213 Ntur Amri, In. All rights rsrv. Figur 2 Th untion o in ssmly n -inu lipi roplt ormtion. () Ets o vrious sirnas on JFH-1/ P7-luirs (Lu) RNA rplition in CD81- iint Huh7.25 lls. () Ets o vrious sirnas on sugnomi rplion ssy. Vlus r normliz rltiv to th ontrol, n rror rs rprsnt ± s.. o quintuplit xprimnts (,). () Lipi roplt () ontnts (BODIPY) n or xprssion in Huh7.5.1 lls trt with or si. Th imgs on th right show nlrg vrsions o slt lls (orng oxs) in th imgs irtly to th lt. Blow, th lipi roplt numrs r shown s th mns pr ll ± s.. (>15 lls nlyz), th prntgs o lipi roplt positiv rs r shown s th mns pr ll ± s.. (>15 lls nlyz), n th lipi roplt mn luorsn intnsitis r shown s th mns pr ll ± s.. (>3 lls nlyz) () Lipi roplt numrs, lipi roplt positiv rs n mn luorsn intnsitis (quntii low s th mns pr ll ± s.. (>3 lls nlyz)) o Huh7.5.1 lls trnst with ontrol (trl), WT HA or HA KM plsmis n thn stin or HA xprssion n lipi roplt ontnts. () Et o silning on 3 UTR mit lvtion o lipi roplt ontnts in Huh7.5.1 lls. Sl rs ( ), 2 µm. P <.1 trmin y Stunt s t tst. NS, not signiint. n ololiztion o or protin n lipi roplts (Supplmntry Fig. 4). ovrxprssion lso signiintly inrs th numr, siz n luorsn intnsity o lipi roplts (Fig. 2). Convrsly, trnstion o th HA KM strongly ru lipi roplt ormtion (Fig. 2). Colltivly ths t init tht prominntly ts -inu lipi roplt ormtion n virl ssmly, lthough n itionl t on virion srtion nnot rul out ompltly. 3 UTR tivts IKK- n lipi roplt ormtion To in th mhnism o th -rsponsiv tivtion o n inution o lipognsis, w xmin th rol o th 3 UTR o th virl gnom, whih ontins prviously intii PAMP molul: poly(u/c) squn 25. Trnstion o 3 UTR RNA monstrt strong inution o lipi roplt ormtion, n silning y sirna mrkly iminish th 3 UTR mit inrs in lipi roplt ontnt (Fig. 2 n Supplmntry Fig. 5). Poly(I:C), synthti PAMP, lso nhn lipi roplt ormtion (Supplmntry Fig. 5). Trnstion o HA plsmi n 3 UTR RNA togthr rsult in inrs mounts o phosphorylt, whrs w i not tt phosphoryltion in lls trnst with th HA KM (Supplmntry Fig. 5). Thror intion inus lipi roplt iognsis through virl RNA triggr tivtion o n pnnt pthwy. Th untion o IKK- in intion is NF-kB inpnnt is omponnt o IKK tht ontrols NF-κB tivtion, ut it hs lso n implit in NF-κB inpnnt untions 12. To isst th untion o th NF-κB pthwy in onntion with in Singl yl o rplition (rltiv luirs units) rplion (rltiv luirs units) Numr o s pr ll Anti-HA Anti-HA sipik4ca Huh7.25 NS NS NS siapoe si Huh7.5.1 NS Anti-HA WT KM + + sipik4ca siapoe si WT + KM + Prntg o r pr ll si Numr o s pr ll WT HA HA KM si WT + KM + mn intnsity UTR Control Prntg + r pr ll si si si si + + intion, w irst siln our mjor tors o th NF-κB mily (NF-κB1, NF-κB2, RELA n RELB) n two IKK tlyti units ssntil or NF-κB tivtion (IKK-β n NEMO) 11 (Fig. 3 ). W osrv n inrs in intion n rplition (Fig. 3,), whih irtly ontrsts th rsult o silning. Silning o th ov-mntion NF-κB gns i not t mit inution o lipi roplt ormtion or nhnmnt o propgtion (Fig. 3,). In ition, signls known to tivt th NF-κB pthwy, suh s intrlukin-1β (IL-1β) n lymphotoxin-α n -β (LT-α/β), i not inu lipi roplt ormtion (Fig. 3 n Supplmntry Fig. 5). sirna ginst LT-β (lso ll LTB) or its rptor (LTBR), whih r known to involv in nonnonil tivtion o NF-κB through 12, h littl or no t on rplition or lipi roplt inution (Supplmntry Fig. 5 g). Furthr vin or th NF-κB inpnnt t o is shown in th Supplmntry Rsults. In ition, svrl prviously rport NF-κB inpnnt untions o r not involv in moulting intion (Supplmntry Rsults). Th rol o in ssmly n lipi roplt ormtion W nxt xplor th signling pthwy tht mits th spii t o th 3 UTR on tivtion. RIG-I hs n shown to th ky PRR or th PAMP tht rsis in th 3 UTR 25. Bus RIG-I is nonuntionl in Huh7.5.1 lls 25, w tst th rols o two othr RLRs (MDA5 (lso known s IFH1) n LGP2 mn intnsity 724 VOLUME 19 NUMBER 6 JUNE 213 ntur miin

4 r t i l s npg 213 Ntur Amri, In. All rights rsrv. Prntg intion sinf-κb 1 * * sinf-κb 2 sirela sirelb sinemo sinf-κb 1 sinf-κb 2 sirela sirelb sinemo (lso known s DHX58)), th RLR signling-ptor molul MAVS n mjor ownstrm ytosoli kins, TBK1. Dpltion o ths tors y sirna h littl or no t on -triggr or 3 UTR triggr lipi roplt ormtion (Supplmntry Fig. 6 ). Th ts on rplition wr somwht vril, with MDA5 sirna using minor inrs n LGP2 sirna using most rs (Supplmntry Fig. 6), whih w ttriut to positiv rol o MDA5 n ngtiv rol o LGP2 in RLR signling 26. W thn invstigt whthr DDX1, puttiv PRR tht is pl o snsing oul-strn RNA 27, is involv in 3 UTR mit ts, n show tht DDX1 sirna h no t (Supplmntry Fig. 6 )., nothr DExD/H hlis, hs rntly n implit in innt immunity 28,29 n hs n shown to importnt or intion 3,31. ws lso intii s strong provirl tor in our gnom-wi sirna srn, n similr to, its silning h grtr t on th lt stg o th virl li yl 19. Silning o l to mor sustntil inhiition o xtrllulr thn o intrllulr lvls n signiint impirmnt o virl intivity (Fig. 4), whrs ovrxprssion o inrs th xtrllulr lvl o mor thn th intrllulr lvl (Fig. 4). Dpltion o lso rogt th inution o lipi roplt iognsis mit y th 3 UTR n ru th or lipi roplt ssoition in th ontxt o intion (Supplmntry Fig. 6 ). intrts with 3 UTR n tivts IKK- In 3 UTR trt or -int lls, ristriut to orm spkl-lik ytoplsmi struturs (Fig. 4, n Supplmntry Figs. 7 n 8), lthough its ovrll xprssion rmin unltr (Supplmntry Fig. 8,). ololiz spiilly with trnst Cy3-ll 3 UTR RNA ut not ontrol RNA (Fig. 4 n Supplmntry Fig. 7). 3 UTR RNA, ut not ontrol RNA, orm omplx with in ll lysts (Fig. 4). Trtmnt with th 3 UTR or intion inu th ormtion o ggrgts, whih ololiz xtnsivly with 3 UTR omplxs (Fig. 4, n Supplmntry Figs. 7 n 8). Poly(I:C) lso inu ssoition (Supplmntry Fig. 8,). Gn xprssion (rltiv mrna lvl) Figur 3 Th untion o n th NF-κB pthwy. ( ) Ets o knokown o th NF-κB pthwy on intion. () Ets o NF-κB silning on or-ssoit lipi roplt ontnts n 3 UTR inu lipi roplt ormtion. Sl rs, 2 µm. () lvls (normliz to smpls trt with n ontrol plsmi) in Huh7.5.1 lls trt with th init sirnas n thn trnst with ontrol or HA plsmis or intion with. () Lipi roplt ontnts n in Huh7.5.1 lls inut with IL-1β (.1 µg ml 1 ) or LT-α/β (.1 µg ml 1 ) or 3 min n stin. Sl rs, 2 µm. (,) Vlus rprsnt th mns ± s.. o triplit xprimnts. *P <.5, P <.1 trmin y Stunt s t tst sinf-κb 1 sinf-κb 2 sirela sirelb siikk-β sinemo silkk-β 3 UTR HA sinf-κb 1 sinf-κb sinemo sinf-κb 1 sinf-κb 2 + IL-1β HA LT-α/β In -int lls, w vrii inrs intrtion o with using oimmunopripittion (Fig. 4). Silning o mrkly ru th ololiztion o th 3 UTR with, whrs sirna h no t on omplx ormtion twn th 3 UTR n (Supplmntry Fig. 7), suggsting tht intrtion o th 3 UTR with is rquir or th susqunt rruitmnt n tivtion o. Silning o MAVS, whih hs n suggst to intrt with in th tivtion o innt immunity 28,32, h no t on intrtions mong th 3 UTR, n (Supplmntry Fig. 7). Ths t init tht in -int Huh7.5.1 lls, signls prominntly through th mit lipogni pthwy to ply provirl prt in propgtion. Prvious stuis show tht th or ins n ristriuts to th virl ssmly sits roun lipi roplts; howvr, this intrtion sms to ispnsl or rplition 33. In int lls, ristriut prtilly ololiz with or n lipi roplts (Supplmntry Fig. 8). Trtmnt o Huh7.5.1 lls with th 3 UTR or poly(i:c) lso inu ssoition, ut th omplxs i not loliz to th lipi roplts (Fig. 4g n Supplmntry Fig. 8). To onirm tht th or is not involv in th intrtion twn n IKKα, w stui n mutnt with or mino i sustitution (F24Y) tht olishs its intrtion with 33 n show tht in th sn o or- ining, intrtion is still inu y intion without lipi roplt ssoition (Fig. 4h n Supplmntry Fig. 8g). inus IKK- phosphoryltion n nulr trnslotion W nxt xmin th onsqun o intrtion inu y intion. W int Huh7.5.1 lls with n hrvst thm t vrious tim points (4 72 h) tr intion to trmin gn xprssion. Thr ws no hng in ithr mrna or protin lvls o in th prsn o intion (Supplmntry Fig. 9,). Howvr, w i in signiint inrs in phosphorylt, rprsnting th tiv orm o, ntur miin VOLUME 19 NUMBER 6 JUNE

5 A r t i l s npg 213 Ntur Amri, In. All rights rsrv. Figur 4 Intrtion o with th 3 UTR n its rol in intion. () Et o sirna on proution (lt), intious proution s trmin y prt-two or stining (mil) n intivity (right). () Et o ovrxprssion o on proution. (,) Error rs rprsnt ± s.. o triplit xprimnts. P <.1 trmin y Stunt s t tst. () Cololiztion o n trmin y onol mirosopy in Huh7.5.1 lls trnst with Cy3-ll RNA or 4 h. Th ontrol RNA ws riv rom ngtivstrn trnsript o n nhn GFP plsmi. CR, Prson s orrltion oiint; orng rrows n oxs orrspon to ololiztion nlyss o luorsn intnsitis n r shown nxt to h img. () intion n n ololiztion in Huh7.5.1 lls. () Wstrn lot (WB) nlysis prorm with -spii ntiois o protin lut rom strptviin s tht wr oun to iotinylt RNA n inut with lysts o Huh7.5.1 lls. IP, immunopripittion. () Assoition o n in -int lls trmin y oimmunopripittion. WCL, whol-ll lysts. (g), n lipi roplts in Huh7.5.1 lls trnst with th 3 UTR or 24 h n thn stin. (h) Th or, n in Huh7.5.1 lls trnst with WT or F24Y mutnt or 48 h n thn stin. Sl rs, 2 µm (unlss othrwis init in th igur). t h tr intion (Fig. 5 n Supplmntry Fig. 9)., whn tivt, shuttls rom th ytoplsm to th nulus 15,16. W thror xmin th istriution o in th nulr n ytosoli rtions o -int lls. By 12 h tr intion, th lls show mrk nulr umultion o (Fig. 5). Trtmnt o lls with tumor nrosis tor-α (TNF-α), on i tivtor o 15, signiintly nhn th phosphoryltion n nulr trnslotion o, whrs th ovrll xprssion o rmin unhng (Fig. 5, n Supplmntry Fig. 9). Conol mirosopy onirm th nulr umultion o in -int or 3 UTR trnst lls (Fig. 5 n Supplmntry Fig. 9,), n ths lls show spkl-lik ssoition o n (Fig. 5) n lvt lipi roplt ontnts (Supplmntry Fig. 9,), whrs th mjority o rsi in th ytoplsm o unint lls. Phosphoryltion o is rquir or its nulr trnslotion, s th HA KM i not show nulr loliztion (Supplmntry Fig. 9g). IKK- inus SREBP-mit lipi roplt ormtion W prorm mirorry gn xprssion proiling n intii llulr gns tht r trnsriptionlly rgult y. W trt Huh7.5.1 lls with ithr nontrgting ontrol sirna or sirna in th sn or prsn o intion (our onitions totl). W onirm th knokown iiny o g UTR si 3 UTR CR:.3 RNA RNA CR:.29 CR:.22 CR:.5 Prntg intion µm 5 µm Prt on Prt two si 1 µm Intivity (TCID 5 /ml) β-tuulin h si 4, 3, 2, 1, 5, 1, Distn (nm) 3 UTR 4, 3, 2, 1, 5, 1, Distn (nm) 4, 3, 2, 1, 5, 1, Distn (nm) RNA 1,5 1, 5 5, 1, Distn (nm) WT F24Y si Mok 1 µm 1 µm 3 UTR Biotin-3 UTR Biotin-trl RNA IP Input WT F24Y 1 µm 1 µm n its t on intion y msuring mrna n lvls (Supplmntry Fig. 1,). Lipogni gns, prtiulrly SREBPs n SREBP trgt gns involv in tty i n triglyri synthsis, wr uprgult y, n thir inutions wr rogt in siln lls (Fig. 5 n Supplmntry Fig. 1 i). Ovrxprssion o wil-typ (WT) uprgult, n ovrxprssion o th KM ownrgult, th xprssion o SREBPs (Supplmntry Fig. 1j). Unr our xprimntl onitions, th ts o intion n trnstion tn to unrstimt, s th iinis o intion n trnstion wr only ~4 5% o lls unr th st irumstn. W ompr our mirorry t st with rnt pulition tht intii ll SREBP-1 rgult lipi mtolism n humn hpti gns 34. W show tht th gns rgult y SREBP-1 wr mrkly t y intion or silning (Supplmntry Fig. 1k,l), implying tht hs mjor rol in SREBP-1 rgultion o hpti lipi mtolism gns. W urthr monstrt tht 5 µm 5 µm WB: HA- HA- HA β-tuulin CR:.52 4, 3, 2, 1, 5, Distn (nm) Mok Mok CR:.5 1,5 1, 5 5, 1, Distn (nm) Mok Mok Mok WCL IP: IP: Mok WCL IP: IP: 726 VOLUME 19 NUMBER 6 JUNE 213 ntur miin

6 r t i l s npg 213 Ntur Amri, In. All rights rsrv Rltiv p: rtio IP: nti (h.p.i.) TNF-α p SREBP knokown hs similr t s silning on intion. Atr pltion o SREBP-1 n SREBP-2, thr ws signiint impirmnt o proution (Fig. 5), likly stmming Cytoplsmi Rltiv intnsity /β-tuulin TNF-α (h.p.i.) β-tuulin TFIIB Nulr TNF-α Rltiv intnsity /TFIIB 3 UTR Figur 5 intion, tivtion, SREBP inution n lipi roplt ormtion. () Et o intion or TNF-α trtmnt on th phosphoryltion o (p) s trmin y immunopripittion ollow y wstrn lotting. h.p.i., hours post intion. () Th nulusto-ytoplsm rtio o trmin y iviing th rltiv intnsity o nulr normliz to trnsription tor IIB (TFIIB) ovr tht o ytoplsmi normliz to β-tuulin. () Immunostining or n in Huh7.5.1 lls trt with th 3 UTR or ull-lngth (FL) or 4 h. Whit rrows init nulr trnslotion o in virl RNA trt lls showing ololiztion o n. Sl rs, 2 µm. () SREBP mrna lvls in iint lls msur t 72 h tr sirna trtmnt, 24 h tr intion or tr oth trtmnts n normliz rltiv to -trt smpls in th sn o intion. (,) Ets o SREBP silning in Huh7.5.1 lls on intion () n lipi roplt ormtion (). Sl rs, 2 µm. (g) Ets o vrious IKK inhiitors (wlolton, 3 µm; IKK Inhiitor XII, 1 µm; or, 1 µm) on 3 UTR mit inution o SREBP-1. Error rs rprsnt ± s.. o triplit xprimnts (,,g). *P <.5, P <.1 trmin y Stunt s t tst. NS, not signiint. Rltiv promotr tivity 1..5 SREBP sicbp + sip3 SREBP-2 sicbp sip3 Prntg intion sicbp Prt on Prt two * 1. *.5 HA sisrebp sisrebp sicbp/p3 + + HA sicbp sip3 Prntg DNA input (SREBP-1 promotr) sicbp/p3 sip3 sisrebp-1 sisrebp-2 sisrebp-1+2 FL RNA SREBP-1 mrna NS si * sisrebp-1 sisrebp-2 sisrebp-1+2 SREBP-2 mrna mrna lvl sisrebp-1 sisrebp-2 rom th pprnt iminution o lipi roplt ormtion (Fig. 5). silning n IKK inhiitors (wlolton n Inhiitor XII) lso rogt 3 UTR inu SREBP inution (Fig. 5g 3 UTR IgG Anti Anti-CBP Anti Pol II PAMP RLRs IKK-ε MAVS TBK1 IKK IRF3 NF-κB CBP/p3 Antivirl gns SREBPs Virl ssmly Lipi roplt iognsis Lipogni gns Lipi roplts g SREBP-1 mrna si Control 3 UTR NS NS NS W. Figur 6 Th signling pthwy involv in mit lipogni inution o intion. () Ets o or CBP/p3 sirna on SREBP luirs rportr tivitis. () Lipi roplt ontnts n or xprssion in CBP or p3 sirna trt Huh7.5.1 lls or intion. Sl rs, 2 µm. () Ets o CBP/p3 silning on intion. Lt, intrllulr n xtrllulr lvls; right, or quntiition in prt on n prt two o th ll ultur ssy. A wstrn lot nlysis o CBP n p3 protin lvls is shown in Supplmntry Figur 12. () ChIP ssys prorm with th init ntiois n Huh7.5.1 lls tht wr untrt (trl), int with or trnst with 3 UTR RNA or 48 h. Only t or th SREBP-1 promotr r shown hr, n t or th IL-8 (positiv ontrol) n tin (ngtiv ontrol) promotrs r shown in Supplmntry Figur 12. Dt r shown s th mn ± s.. o quruplit xprimnts. Pol II, polymrs II. () ovrxprssion in -int lls priv o SREBP-1, SREBP-2 or CBP/p3. () A propos mol o th innt ntivirl rspons, -inu lipognsis n lipi roplt ormtion in ssmly. Th thiknss o th rrows rprsnts th puttiv mgnitu o th two pthwys (provirl or ntivirl) in Huh7.5.1 lls. Dsh rrows rprsnt possil rosstlk twn th two prlll pthwys. Error rs rprsnt ± s.. o triplit xprimnts (,,). *P <.5, P <.1 ompr to ontrol trmin y Stunt s t tst. ntur miin VOLUME 19 NUMBER 6 JUNE

7 A r t i l s npg 213 Ntur Amri, In. All rights rsrv. n Supplmntry Fig. 1i). In PHHs, IKK inhiitors ru th mrna lvls o SREBP-1 n SREB-2 (Supplmntry Fig. 1m). Silning o,, SREBP-1 or SREBP-2 in PHHs l to sustntil rution o xtrllulr s ompr to intrllulr lvls (Supplmntry Fig. 11,). IKK- inus xprssion o SREBP through CBP/p3 Ativt hs n shown to phosphorylt CBP n uprgults its tivitis in th nulus 18. CBP/p3 is lso involv in SREBP trnsriptionl tivity 35,36. Ovrxprssion o CBP/p3 mrkly uprgults th xprssion o oth SREBP-1 n SREBP-2 (r. 37). W oun tht oth n CBP/p3 sirnas signiintly ru SREBP promotr tivitis n mrna lvls (Figs. 5 n 6 n Supplmntry Fig. 11,). W lso oun tht xrts its t on SREBP inution n lipognsis through rruiting th CBP/p3 omplx. CBP/p3 pltion y sirna signiintly rs ytosoli lipi roplt ontnt n proution (Fig. 6,). sirnas ginst CBP/p3 mrkly iminish 3 UTR mit SREBP-1 inution n lipi roplt ormtion (Supplmntry Fig. 11,). Using hromtin immunopripittion (ChIP) ssys, w oun tht inings o th SREBP-1 promotr to oth n CBP wr nhn y intion or 3 UTR trnstion (Fig. 6 n Supplmntry Fig. 11). In ition, nhn virl proution y ovrxprssion ws sustntilly ompromis in lls trt with CBP/p3 or SREBP sirna (Fig. 6), initing tht CBP/p3 n SREBPs r involv in th mit provirl t. To show tht th SREBP pthwy is in ruil or proutiv intion, w siln ths gns in omintion n monstrt tht intion ws muh mor ru (>1-ol) thn whn silning th iniviul gns (Supplmntry Fig. 11g). DISCUSSION W monstrt prviously unrogniz untion o in rgulting llulr lipi mtolism n thror ssmly, oth o whih r inpnnt o its rol in intrinsi innt immunity. W xtnsivly vlit n mhnistilly invstigt this untion o. W show tht, through th tion o th virl 3 UTR ontining prviously intii PAMP, spiilly intrts with n tivts to mit n NF-κB inpnnt untion in th nulus. Rnt umulting vin hs init n mrging rol o in innt immunity, lthough th pris mhnism rmins unlr 28,29. Othr mmrs o th DEAD ox mily, inluing th wll-known RLRs n th rntly intii DDX1, DDX21 n DHX36, r importnt intrllulr PRRs or RNA viruss 27. Although hs n suggst to untion s PRR 28, irt vin or this is lking. Hr w show tht spiilly rognizs th 3 UTR s possil PRR. Bining o th 3 UTR to ls to tivtion o. or protin hs n shown to in to 33, ut this intrtion is ispnsl or -triggr tivtion o th pthwy. This pthwy involving n sms to uniqu to, s gnom-wi sirna srns o svrl viruss, inluing HIV-1, Dngu, Wst Nil n inlunz, using th sm srning pltorm i not intiy this pthwy s ruil host tor or ths viruss W thror propos mol tht illustrts th mhnism o tion or n in intion (Fig. 6). Unlik othr tors in th NF-κB tivtion pthwy tht r xlusivly ntivirl gns,, lthough pl o mostly inuing intrronstimult gn (ISG) xprssion (Supplmntry Fig. 12), xrts prominntly provirl t tht trgts th ssmly stg o th li yl y tivting SREBP-mit lipognsis n lipi roplt iognsis. It is worth noting tht vrious protins hv lso n implit in intrting with host lipi mtolism to ilitt ssmly 22,23, ut our t init tht th primry triggr o lipi roplt iognsis is mit y th virl 3 UTR, n virl protins minly hv ownstrm rol in virl ssmly 22,23. RNA viruss sltivly xploit host-spii lmnts to orm spiliz orgnlls in whih llulr lipis r ruil in nhning virl proution. Dngu virus intion lunhs n utophgymit prossing o lipi roplts n triglyris tht is rquir or iint virl rplition 42. Wst Nil virus intion stimults holstrol iosynthsis n ristriuts holstrol to virl RNA rplition mmrns 43. Rntly, intion ws shown to pn on phosphtiylinositol 4-phospht (PI4P) lipi-nrih mmrns n PI4KIII kinss or RNA rplition 44. intion lso inus lipi roplt ormtion or proutiv virion ssmly 24. Cllulr lipognsis is oorint with lipi roplt ormtion n mturtion 45. intion is ssoit with hpti sttosis n insulin rsistn 4, prsumly us o -inu ysrgultion o lipi mtolism. Hpti sttosis ontriuts sustntilly to th progrssion o irosis in ptints with hroni hptitis C 46. Intrstingly, hpti tivtion o IKK-β n NF-κB hs n implit in th tiology o insulin rsistn n typ 2 its 47,48. Trgt ltion or phrmologil inhiition o IKK-β n rstor insulin snsitivity in os mi n humns 49,5. Rnt gnom-wi ssoition stuis hv shown tht singl-nuloti polymorphisms in th CHUK (noing ) gn lous r ssoit with nonloholi tty livr isss 51. As w hr untionlly link to lipognsis in hptoyts, it will intrsting to invstigt th intrtion mong th vrious IKKs in -ssoit sttosis n insulin rsistn, s wll s in gnrl llulr mtolism., s prt o th NF-κB tivtion pthwy, is involv in host rognition n ns ginst pthogn intion. Hr w show tht hs n importnt rol in th onstitutiv mintnn o lipogni gn xprssion n lipi roplt ormtion in hptoyts, n its tivtion y urthr stimults th lipogni pthwys to ilitt virl ssmly. W rson tht n its ssoit pthwys hv ul t on intion n ntivirl t through th NF-κB pthwy n provirl t through th lipogni pthwy with th lttr ing th ominnt pthwy in our xprimntl mols. signling my thror pivotl mhnism whry hroni intion n inlmmtion l to th ysrgult mtolism tht is implit in th vlopmnt o vrious hroni mtoli isorrs 52. Our inings or ruil insights into th inlmmtory origin o mtoli iss n th pthogni onsquns o hroni virl intion 14,53. A omprhnsiv unrstning o th rosstlk twn, host innt rsponss n lipi mtolism my hlp intiy nw n roly tiv ntivirl trgts 54. Th isovry o provirl untion o ors thrputi opportunitis or inhiitors in th trtmnt o intion. Th monstrt iy o ommrilly vill inhiitors o IKK tivitis in loking intion in this stuy provis proo-o-onpt pproh or nw thrputi vlopmnt. W lso prsnt th irst vin, to our knowlg, tht usurps intrinsi innt immun pthwy or its own vntg n survivl. Suh n xploittion o host ntivirl 728 VOLUME 19 NUMBER 6 JUNE 213 ntur miin

8 r t i l s npg 213 Ntur Amri, In. All rights rsrv. ns my unrli th mhnism whry intion prisposs to prsistnt intion spit n tiv host immunity. Mthos Mthos n ny ssoit rrns r vill in th onlin vrsion o th ppr. Assion os. Mirorry t hv n posit in th Gn Exprssion Omnius unr ssion o GSE Not: Supplmntry inormtion is vill in th onlin vrsion o th ppr. Aknowlgmnts W thnk Z. Hu, Y.-Y. Zhng, Y.-M. Li, E. Thoms, V. Gonzlz-Munoz n L. Holz or thnil ssistn, Y.-P. Wu o th Ntionl Institut o Dits n Digstiv n Kiny Disss (NIDDK) Conol Mirosopy or hlping with onol imging n W.-P. Chn o th NIDDK Mirorry Fility or DNA mirorry nlysis. W lso thnk A. Ptl o th Univrsity o Glsgow or proviing th or mutnt (F24Y) virus n C. Ri (Rokllr Univrsity) or th 9E1 NS5A ntioy n othr rgnts. This work ws support y th Intrmurl Rsrh Progrm o th NIDDK, US Ntionl Instituts o Hlth (NIH). Primry humn hptoyts wr provi y th NIH-un Livr Tissu Prourmnt n Cll Distriution Systm (N1-DK-7-4/HHSN26774C, prinipl invstigtor, S. Strom, Univrsity o Pittsurgh). AUTHOR CONTRIBUTIONS Q.L. n T.J.L. oniv n sign th stuy. Q.L., V.P., S.K. n H.C. onut xprimnts. Q.L., V.P. n T.J.L. nlyz t. Q.L. n T.J.L. wrot th ppr with th input rom V.P. T.J.L. suprvis th stuis. COMPETING FINANCIAL INTERESTS Th uthors lr no ompting innil intrsts. Rprints n prmissions inormtion is vill onlin t rprints/inx.html. 1. Ling, T.J., Rhrmnn, B., S, L.B. Hoongl, J.H. Pthognsis, nturl history, trtmnt, n prvntion o hptitis C. Ann. Intrn. M. 132, (2). 2. Ling, T.J. Ghny, M.G. N. Engl. J. M. 368, (213). 3. Rhrmnn, B. Hptitis C virus vrsus innt n ptiv immun rsponss: tl o ovolution n oxistn. J. Clin. Invst. 119, (29). 4. Sy, G.H., Amko, Y. Siiqui, A. Hptitis C virus hijks host lipi mtolism. Trns Enorinol. Mt. 21, 33 4 (21). 5. Hrkr, E. Ott, M. Uniqu tis twn hptitis C virus rplition n intrllulr lipis. Trns Enorinol. Mt. 22, (211). 6. Dimon, D.L. t l. Tmporl protom n lipiom proils rvl hptitis C virus ssoit rprogrmming o hptollulr mtolism n ionrgtis. PLoS Pthog. 6, 1719 (21). 7. Horton, J.D., Golstin, J.L. Brown, M.S. SREBPs: tivtors o th omplt progrm o holstrol n tty i synthsis in th livr. J. Clin. Invst. 19, (22). 8. Wris, G., Flml, D.J., Ngro, F. Siiqui, A. Hptitis C virus inus protolyti lvg o strol rgultory lmnt ining protins n stimults thir phosphoryltion vi oxitiv strss. J. Virol. 81, (27). 9. Lrt, H. t l. Hptitis C virus protins inu lipognsis n tiv triglyri srtion in trnsgni mi. J. Biol. Chm. 284, (29). 1. Bowi, A.G. Untrholznr, L. Virl vsion n suvrsion o pttrn-rognition rptor signlling. Nt. Rv. Immunol. 8, (28). 11. Hyn, M.S. Ghosh, S. Shr prinipls in NF-κB signling. Cll 132, (28). 12. Prkins, N.D. Intgrting ll-signlling pthwys with NF-κB n IKK untion. Nt. Rv. Mol. Cll Biol. 8, (27). 13. Sntln, U. t l. Ativtion y IKKα o son, volutionry onsrv, NF-κB signling pthwy. Sin 293, (21). 14. Psprkis, M. Rgultion o tissu homostsis y NF-κB signlling: implitions or inlmmtory isss. Nt. Rv. Immunol. 9, (29). 15. Anst, V. t l. A nulosoml untion or IκB kins-α in NF-κB pnnt gn xprssion. Ntur 423, (23). 16. Birh, A. t l. Signling moluls o th NF-κB pthwy shuttl onstitutivly twn ytoplsm n nulus. J. Biol. Chm. 277, (22). 17. Ymmoto, Y., Vrm, U.N., Prjpti, S., Kwk, Y.T. Gynor, R.B. Histon H3 phosphoryltion y is ritil or ytokin-inu gn xprssion. Ntur 423, (23). 18. Hung, W.C., Ju, T.K., Hung, M.C. Chn, C.C. Phosphoryltion o CBP y IKKα promots ll growth y swithing th ining prrn o CBP rom p53 to NF-κB. Mol. Cll 26, (27). 19. Li, Q. t l. A gnom-wi gnti srn or host tors rquir or hptitis C virus propgtion. Pro. Ntl. A. Si. USA 16, (29). 2. Zni, E., Rothwr, D.M., Dlhs, M., Hykw, M. Krin, M. Th IκB kins omplx (IKK) ontins two kins suunits, IKKα n IKKβ, nssry or IκB phosphoryltion n NF-κB tivtion. Cll 91, (1997). 21. Akzw, D. t l. CD81 xprssion is importnt or th prmissivnss o Huh7 ll lons or htrognous hptitis C virus intion. J. Virol. 81, (27). 22. Miynri, Y. t l. Th lipi roplt is n importnt orgnll or hptitis C virus proution. Nt. Cll Biol. 9, (27). 23. Hrkr, E. t l. Eiint hptitis C virus prtil ormtion rquirs iylglyrol yltrnsrs-1. Nt. M. 16, (21). 24. MLuhln, J. Lipi roplts n hptitis C virus intion. Biohim. Biophys. At 1791, (29). 25. Sito, T., Own, D.M., Jing, F., Mrotrigino, J. Gl, M. Jr. Innt immunity inu y omposition-pnnt RIG-I rognition o hptitis C virus RNA. Ntur 454, (28). 26. Rothnussr, S. t l. Th RNA hlis Lgp2 inhiits TLR-inpnnt snsing o virl rplition y rtinoi i-inuil gn-i. J. Immunol. 175, (25). 27. Zhng, Z. t l. DDX1, DDX21, n DHX36 hliss orm omplx with th ptor molul TRIF to sns srna in nriti lls. Immunity 34, (211). 28. Oshiumi, H., Ski, K., Mtsumoto, M. Sy, T. DEAD/H BOX 3 (DDX3) hlis ins th RIG-I ptor IPS-1 to up-rgult IFN-β inuing potntil. Eur. J. Immunol. 4, (21). 29. Shrör, M., Brn, M. Bowi, A.G. Virl trgting o DEAD ox protin 3 rvls its rol in TBK1/IKKε-mit IRF tivtion. EMBO J. 27, (28). 3. Ariumi, Y. t l. DDX3 DEAD-ox RNA hlis is rquir or hptitis C virus RNA rplition. J. Virol. 81, (27). 31. Rnll, G. t l. Cllulr otors ting hptitis C virus intion n rplition. Pro. Ntl. A. Si. USA 14, (27). 32. Oshiumi, H. t l. Hptitis C virus or protin rogts th DDX3 untion tht nhns IPS-1 mit IFN-β inution. PLoS ONE 5, (21). 33. Angus, A.G. t l. Rquirmnt o llulr DDX3 or hptitis C virus rplition is unrlt to its intrtion with th virl or protin. J. Gn. Virol. 91, (21). 34. R, B.D., Chros, A.E., Szkly, A.M., Wissmn, S.M. Snyr, M. Gnom-wi oupny o SREBP1 n its prtnrs NFY n SP1 rvls novl untionl rols n omintoril rgultion o istint lsss o gns. PLoS Gnt. 4, 1133 (28). 35. Erisson, J. Ewrs, P.A. CBP is rquir or strol-rgult n strol rgultory lmnt-ining protin-rgult trnsription. J. Biol. Chm. 273, (1998). 36. Olinr, J.D., Anrsn, J.M., Hnsn, S.K., Zhou, S. Tjin, R. SREBP trnsriptionl tivity is mit through n intrtion with th CREB-ining protin. Gns Dv. 1, (1996). 37. Ginomnio, V., Simonsson, M., Gronroos, E. Erisson, J. Cotivtor-pnnt tyltion stilizs mmrs o th SREBP mily o trnsription tors. Mol. Cll Biol. 23, (23). 38. Brss, A.L. t l. Intiition o host protins rquir or HIV intion through untionl gnomi srn. Sin 319, (28). 39. Sssions, O.M. t l. Disovry o inst n humn ngu virus host tors. Ntur 458, (29). 4. Krishnn, M.N. t l. RNA intrrn srn or humn gns ssoit with Wst Nil virus intion. Ntur 455, (28). 41. Brss, A.L. t l. Th IFITM protins mit llulr rsistn to inlunz A H1N1 virus, Wst Nil virus, n ngu virus. Cll 139, (29). 42. Hton, N.S. Rnll, G. Dngu virus inu utophgy rgults lipi mtolism. Cll Host Miro 8, (21). 43. Mknzi, J.M., Khromykh, A.A. Prton, R.G. Cholstrol mnipultion y Wst Nil virus prturs th llulr immun rspons. Cll Host Miro 2, (27). 44. Hsu, N.Y. t l. Virl rorgniztion o th srtory pthwy gnrts istint orgnlls or RNA rplition. Cll 141, (21). 45. Frs, R.V. Jr. Wlthr, T.C. Lipi roplts inlly gt littl R-E-S-P-E-C-T. Cll 139, (29). 46. Asslh, T., Rui-Brnt, L., Mrllin, P. Ngro, F. Sttosis in hroni hptitis C: why os it rlly mttr? Gut 55, (26). 47. Ci, D. t l. Lol n systmi insulin rsistn rsulting rom hpti tivtion o IKK-β n NF-κB. Nt. M. 11, (25). 48. Yun, M. t l. Rvrsl o osity- n it-inu insulin rsistn with sliylts or trgt isruption o Ikkβ. Sin 293, (21). 49. Arkn, M.C. t l. IKK-β links inlmmtion to osity-inu insulin rsistn. Nt. M. 11, (25). 5. Zhng, X. t l. Hypothlmi IKKβ/NF-κB n ER strss link ovrnutrition to nrgy imln n osity. Cll 135, (28). 51. Romo, S. t l. Gnti vrition in PNPLA3 onrs susptiility to nonloholi tty livr iss. Nt. Gnt. 4, (28). 52. Hotmisligil, G.S. Inlmmtion n mtoli isorrs. Ntur 444, (26). 53. Bkr, R.G., Hyn, M.S. Ghosh, S. NF-κB, inlmmtion, n mtoli iss. Cll Mt. 13, (211). 54. Mungr, J. t l. Systms-lvl mtoli lux proiling intiis tty i synthsis s trgt or ntivirl thrpy. Nt. Biothnol. 26, (28). ntur miin VOLUME 19 NUMBER 6 JUNE

9 npg 213 Ntur Amri, In. All rights rsrv. ONLINE METHODS sirna trnstion. sirnas wr trnst into Huh7.5.1 lls t inl onntrtion o 5 nm using rvrs trnstion protool with Oligotmin (Invitrogn) s prviously sri 19. For PHHs (provi y th NIHun Livr Tissu Prourmnt n Cll Distriution Systm, N1-DK-7-4/HHSN26774C), lls wr s on 12-wll plts t 5, lls pr wll n trnst with sirna t inl onntrtion o 5 nm using RNAiMAX (Invitrogn). Unlss othrwis init, urthr trtmnts or ssys wr typilly prorm 72 h tr sirna trnstion, whn gn silning iiny rhs its mximl lvl. or stining uring prt on (rly stg) n prt two (lt stg). For prt on, Huh7.5.1 lls wr trt with th init SMARTpool sirnas t onntrtion o 5 nm or 72 h n thn int with th JFH-1 strin. Atr 48 h, lls wr stin n img or or proution. This tts host tors involv in th rly stgs o th virl li yl, omprising ntry, virl protin trnsltion n RNA rplition. Cultur suprntnts o prt-on lls wr trnsrr n us to int niv Huh7 lls, thus strting prt two, whih tts protins involv in th ltr stgs o virl intion, inluing virion ssmly n rls. sirnas ginst CD81 n ApoE srv s provirl ontrols or prt on n prt two, rsptivly. A til protool o th or stining is sri in th Supplmntry Mthos. In vitro trnsription n lling o n trnstion. Plsmis rrying JFH1, J6/JFH1 or Lu-JFH1/P7 gnomi RNA or sugnomi rplion RNA squns wr linriz with X I n purii y phnolhloroorm-isomyl lohol xtrtion. In vitro trnsription ws prorm using th MEGAsript T7 kit (Amion) oring to th mnuturr s protool. Th qulity n quntity o RNA wr vlut y NnoDrop sptrophotomtr (Thrmo Sintii). Aliquots (22 µl, 1 µg µl 1 ) o RNA wr stor rozn t 8 C until us. 3 UTR RNA ws gnrt rom plsmi hroring th 3 UTR o tht ontins poly(u/c) rgion prviously in s th virl PAMP 25. Cy3 lling o RNA ws onut using th Silnr sirna lling kit Cy3 (Amion). Biotin-ll RNA ws gnrt y inluing iotin-utp (Roh; 1:6 rtio o iotin to UTP) uring th synthsis pross. RNA trnstion ws prorm using DMRIE-C Rgnt (Invitrogn) oring to th mnuturr s instrutions. li-yl ssys. li-yl ssys wr prorm using psuoprtils (or virl ntry) n sugnomi rplions (or virl IRESmit trnsltion n RNA rplition), n singl-yl intion ssys wr onut using CD81-iint Huh7-25 lls trnst with JFH1/ P7-Lu or J6/JFH1 RNA. Dtil protools or th vrious ssys r sri in th Supplmntry Mthos. Immunoluorsn, lipi stining, onol mirosopy n quntiition o imgs. Clls grown on L-TkII orosilit our-wll hmr ovr slips (Nun) wr ix with 4% prormlhy, prmiliz in.3% Triton X-1 n inut with loking solution in PBS ontining 3% BSA n 1% norml got srum (Vtor Lortoris). Clls wr thn ll with th pproprit primry ntiois ilut in PBS with 1% BSA ollow y inution with Alx Fluor 488, 568 or 647 onjugt sonry ntiois (Invitrogn) in PBS with 1% BSA. Th primry ntiois us wr to th ollowing: or (gnrt rom nti-or 6G7 hyriom lls, 1:5), NS5A (9E1, git o C. Ri o th Rokllr Univrsity, 1:1,), NS5B (6541, Am, 1:5), NS3 (6547, Am, 1:2), HA (C29F4, Cll Signling, 1:5), (54626, Am, 1:2) n (A3-474A, Bthyl Lortoris, 1:25). Nuli wr ountrstin with Hohst (Invitrogn) t 1:5, in PBS. Lipi roplts wr stin with BODIPY 493/53 (Invitrogn) ppli t 1 µg ml 1 or 1 h in PBS with 1% BSA. Eh stp ws ollow y thr wshs with PBS. Conol lsr snning mirosopy nlysis ws prorm with n Axio Osrvr.Z1 mirosop quipp with Ziss LSM 5 Liv DuoSn Systm unr n oil immrsion 1.4 numril prtur (NA) 63 ojtiv lns (Crl Ziss). Imgs wr quir using ZEN 29 sotwr (Crl Ziss). Dul or tripl olor imgs wr quir y onsutiv snning with only on lsr lin tiv pr sn to voi ross xittion. Quntiition o lipi roplts ws prorm with ImgJ (US Ntionl Instituts o Hlth), ZNF29 n st o in intnsity thrshols tht wr ppli to ll imgs. Mirorry nlysis. Huh7.5.1 lls wr trnst with nontrgting or sirna or 72 h n thn mok int or int with t multipliity o intion (MOI) o 1. Forty-ight hours ltr, llulr RNA ws xtrt n purii using RNsy Mini Kit (Qign). RNA ws quntii with sptrophotomtr, n RNA qulity ws nlyz with n Agilnt Bionlyzr oring to th mnuturr s instrutions. RNA ws thn mplii with n Agilnt Enzo kit. Amplii omplmntry RNA ws hyriiz to n Aymtrix Humn 133 Plus 2. mirorry hip ontining 54,675 gn trnsripts. Mirorry nlyss wr prorm t th NIDDK Mirorry Fility. Th ioinormtis n sttistil nlyss wr sri prviously 55. A >-ol hng in xprssion omining >95% proility o ing irntilly xprss (P <.5) ws onsir to iologilly signiint. Th intivity o th JFH-1 virus or Huh7.5.1 lls ws tst or th mirorry xprimnt. Gn xprssion ssy. Totl llulr RNA rom rpli xprimnt ws prpr with th RNsy Mini Kit (Qign). DNA ws synthsiz rom totl RNA with th First Strn DNA Synthsis Kit (Roh). Th mrna xprssion lvls o trgt gns wr quntii y quntittiv PCR using gn-spii primrs n pros (IDT) n TqMn Gn Exprss Mstr Mix (Appli Biosystms) on n ABI 75 Rl Tim PCR Systm. Rltiv trnsript lvls wr lult using th CT mtho with 18S riosoml RNA s th normlizing ontrol gn. ChIP ssys. ChIP ssys wr prorm using th SimplChIP Enzymti Chromtin IP Kit (#93, Cll Signling) s sri y th mnuturrs. In ri, tr rosslinking, nuli wr purii, n hromtin ws shr y sonition (thr tims, 2 s h). Chromtin ws inut ovrnight with spii ntiois ginst (4111, Am, 1:5), CBP (2832, Am, 1:5) n RNA polymrs II CTD (MA1-4693, Thrmo Sintii, 1:5). Isotyp IgG ws us s ngtiv ontrol or th immunopripittion xprimnts. Immunopripitt hromtin ws thn inut with protin G mgnti s, wsh n lut. Atr rvrsl o th rosslinks n puriition o DNA, pripitt DNAs wr nlyz y quntittiv rl-tim PCR (4 yls) with spii primrs to th humn tin promotr s ngtiv ontrol, th IL-8 promotr s positiv ontrol 17 n th SREBP-1 promotr (5 -GCTGTCCCGTGTTAGCCCTT-3 n 5 -TCTACCCGGGAGGTAGGGA-3 ). Quntittiv rl-tim PCR ws prorm in uplit on uplit ChIP ssys using 5 nm o th ov oligonuloti primrs n input DNA stnrs ilut in ivol inrmnts rom 5% to.8% with SYBR Grn PCR Mstr Mix (#439155, Appli Biosystms) n th Appli Biosystms 75 Squn Dttion Systm. Th ol inrss o th ssoitions o, CBP n Pol II to th IL-8 n SREBP-1 promotrs wr normliz to tin signls n lult or -int n PAMP RNA trnst lls s ompr to untrt lls. P vlu ws lult using Stunt s t tst. Chmil inhiitor stuis. IKK Inhiitors II (wlolton), XII n III () wr purhs rom Mrk (EMD) Chmils. Sril ilutions o th IKK inhiitors wr m in 1% immitly or th ssy so tht th inl onntrtions o in h rtion wr intil. Clls wr int with JFH-1 t n MOI o.5 or 6 h n thn trt with rsh DMEM ontining with or without IKK inhiitors. Culturs wr inut or 48 h, n ws xtrt rom th ll lysts or ultur mium n quntii y TqMn rl-tim PCR. Th xprimnts wr on in triplit, n h os ws tst t lst twi. Aitionl mthos. Dtil mthoology is sri in th Supplmntry Mthos. 55. Fl, J.J. t l. Hpti gn xprssion uring trtmnt with pgintrron n rivirin: intiying molulr pthwys or trtmnt rspons. Hptology 46, (27). ntur miin oi:1.138/nm.319