PI3Kδ activates E2F1 synthesis in response to mrna translation stress

Transcription

1 DOI: 1.138/s w OPEN PI3Kδ tivts synthsis in rspons to mrna trnsltion strss Sivkumr Vivl Gnnsunrm 1, Slovéni Pynih 1, Chrysoul Dskloginni 1, Kt Armfil 2, Krin Nylnr 3, Jonn B. Wilson 2 & Roin Fåhrus 1,3, Th -my onogn stimults riosoml iognsis n protin synthsis to promot llulr growth. Howvr, th pthwy y whih lls sns n rstor ysfuntionl mrna trnsltion n how this is link to ll prolifrtion n growth is not known. W hr show tht mrna trnsltion strss in is triggr y th gly-l rpt squn of Epstin Brr virus (EBV)-no EBNA1, rsults in PI3Kδ-pnnt inution of mrna trnsltion with th onsqunt tivtion of -My n ll prolifrtion. Trtmnt with spifi PI3Kδ inhiitor Illisi (CAL-11) supprsss n -My lvls n uss ll th in EBNA1-inu B ll lymphoms. Supprssion of PI3Kδ prvnts tivtion lso in non-ebv-inft lls. Ths t illustrt n mrna trnsltion strss rspons pthwy for tivtion tht is xploit y EBV to promot ll growth n prolifrtion, offring nw strtgis to trt EBV-rrying nrs. 1 Insrm UMRS1162, Equip Lllisé l Ligu Contr l Cnr, Institut Génétiqu Moléulir, Univrsité Pris 7, Hôpitl St. Louis, 751 Pris, Frn. 2 Shool of Lif Sins, Collg of Mil, Vtrinry n Lif Sins, Univrsity of Glsgow, Glsgow G12 8QQ, UK. 3 Dprtmnt of Mil Biosins, Umå Univrsity, Builing 6M, SE Umå, Swn. 4 RECAMO, Msryk Mmoril Cnr Institut, Zluty kop 7, Brno, Czh Rpuli. Sivkumr Vivl Gnnsunrm n Slovéni Pynih ontriut qully to this work. Corrsponn n rqusts for mtrils shoul rss to R.Fåh. (mil: roin.fhrus@insrm.fr) NATURE COMMUNICATIONS 8: 213 DOI: 1.138/s w 1

2 Cllulr growth n prolifrtion is oorint y E2F trnsription ftors vi multipl ownstrm trgt gns, inluing ylins n -my 1,2. Th E2Fs r rtin in n intiv stt y ining to th rtinolstom protin (pr), p13 n p17 pokt protins 3. Th phosphoryltion of pr y ll yl kins tivity rlss E2Fs n triggrs th xit from G into G 1 n S-phs n llulr prolifrtion 4 6. Th pr E2F intrtion is th trgt of onogni viruss suh s th humn ppillom virus (HPV), Simin virus 4 (SV4) n novirus tht vi E7, Lrg T n E1A, rsptivly, ompt for E2F ining to pr 7 9. To t, tn E2F trnsription ftors hv n sri, with 6 ining th sm DNA onsnsus motif. Whrs 3 r gn tivtors, E2F4 6 r onsir supprssors 1. Downstrm of th -my onogn r numrous trgt gns rgulting most spts of llulr iology, illustrting th omplx homoostsis rquir to oorint llulr growth n ivision 1. Stimultion of riosoml iognsis y -My is n importnt spt of llulr growth 11 ut th pthwys tht sns riosoml tivity n how this fs k n is intgrt with growth n prolifrtiv signlling pthwys r poorly unrstoo. Th nmi form of Burkitt s lymphom is hrtris y trnslotion of -my to th immunogloulin lous n y xprssion of th Epstin Brr virus (EBV)-no EBNA1, whih is th only virl ntign xprss in this nr 12,13. But th rol of -My in ontrolling host ll prolifrtion uring norml EBV inftion is unknown. Two inpnnt trnsgni EBNA1 mous mols show invrs orrltion twn th lvls of EBNA1 xprssion n lymphom inin 14,15. Th phnotyp isrpny twn high n low EBNA1-xprssing nimls hs not n xplin. EBV is lso tightly ssoit with othr humn nrs, most notly nsophryngl rinom n Hogkin s lymphom, ut spit knowing th link twn EBV n humn nrs for ovr hlf ntury, th onogni tivitis of EBV r rly unrstoo 16. EBNA1 rris rpt squn of glyins n lnins (), whih supprsss EBNA1 mrna trnsltion in is in orr to minimis th proution of EBNA1-riv ntigni pptis for th mjor histoomptiility (MHC) lss I pthwy 17,18. Th trnsltion inhiitory pity of th in is mks it uniqu tool for stuying th llulr rspons to ysfuntionl mrna trnsltion 19. Th PI3Kδ longs, togthr with PI3Kα n PI3Kβ, to th lss IA of th PI3K fmily tht us phosphtiylinositol (4,5) isphospht (PI(4,5)P 2 ) s sustrt to gnrt th lipi son mssngr PI(3,4,5)P 3 following tyrosin kins rptor tivtion n th rruitmnt of th p85-typ rgultory suunits in omplx with th p11 tlyti suunits 2. Th PI (3, 4, 5) P 3 tivts AKT-pnnt n AKT-inpnnt ownstrm pthwys tht ontrol ro rng of ll rsponss inluing prolifrtion, mtolism n survivl. Th p11α n p11β suunits r xprss uiquitously whil p11δ is prominntly xprss in luoyts ut is lso tt in tumour lls of soli origin Th tivtion of B ll n ytokin rptors inrs (PI(3,4,5)P 3 ) lvls in p11δ-pnnt fshion n p11δ plys importnt rols in th inflmmtory n llrgni rspons n mutt p11δ is foun in ptints suffring from primry immunofiiny 24. Th omprtmntlistion n homoostsis of th TLR4 is rgult y p11δ inpnntly of mtor. Th immun rgultory pity of p11δ lso inlus T ll-mit immun tolrn, giving it potntil ro rol in nr vlopmnt 26. Drugs suh s Illisi (CAL-11) tht spifilly inhiit p11δ r us to trt hroni lymphoyti lukmi, folliulr B ll Hogkin lymphom n rlps smll lymphoyti lymphom 27. W hv xploit th uniqu pity of th EBNA1 to supprss its own mrna trnsltion in is to show how mrna trnsltion strss is intgrt with growth stimultory pthwys. W show tht EBV, lik th simin, no n humn ppillom viruss lso trgts ut y using uniqu mhnism tht os not involv th pr ut rquirs th tivtion of th PI3Kδ n th inution of synthsis. Th t hlp xplin th onogni tivity of EBNA1 n suggsts tht in ition to supprssing th proution of ntigni pptis, EBNA1- mit mrna trnsltion lso promots ll growth n prolifrtion. Rsults Th stimults -My n ll prolifrtion. Th glyin lnin () rpt of th EBV-no EBNA1 isrupts mrna trnsltion in is n offrs uniqu tool to stuy th ll iologil ffts of isrupt trnsltion of iniviul mrnas tht nnot hiv using hmil ompouns Whn th ws xprss in H1299 humn rinom ll lins, signifint inrs (~35%) in olony formtion ws osrv whn ompr to plsmi ontrol () (Fig. 1 n Supplmntry Fig. 1). Th notion tht th xprssion of th promots ll prolifrtion ws support y fluorsntivt ll sorting (FACS) nlysis showing n vrg 3% inrs of lls in S-phs xprssing th unr norml growth onitions. Th -pnnt inrs in prolifrtion ws furthr nhn (from 11 to 19%) in lls tht h rh onflun n th most notl inrs ws osrv unr onitions of low srum whn th xprssion of th rsult in nr thrfol inrs from n vrg 3.6 to 11.1% (Fig. 1 n Supplmntry Fig. 1). Th hs mor prominnt fft on trnsltion inhiition whn fus to th 5, s ompr to th 3, n of opn ring frms (ORFs) (Fig. 1, lowr pnls). Exprssion of thr iffrnt onstruts rrying th in th N trminus (th hikn ovlumin (Ov), p53 or GFP) inu n inrs of lls in S-phs rnging from 18, 27 n 4%, rsptivly. Howvr, fus to th C trminus h littl fft on ll prolifrtion, spit th -fusion protin ing xprss t high lvls (Fig. 1, uppr grph). Similrly, whn ltr squns wr xprss in whih th trnsltion isrupting pity ws ompromis y hngs in th rpt squn 28, w osrv irt orrltion twn ll prolifrtion n trnsltion inhiition (Supplmntry Fig. 1). Thus, th prsn of th in th lls pr s os not hv n fft on ll prolifrtion n only whn it ttnuts trnsltion. Tkn togthr, ths rsults show link twn -mit supprssion of mrna trnsltion in is n ll prolifrtion. Th link twn EBNA1 n -My is mnifst in Burkitt s lymphom y th trnslotion of -my gn to th immunogloulin (Ig) lous ut th orrltion twn -My n EBNA1 unr norml onitions hs rmin n nigm. Whn w xprss n inrsing mount of full-lngth (.2 1. µg DNA in trnsftion) in H1299 lls, w osrv ospnnt inrs in -My lvls (Fig. 1). W osrv similr inrs in -My following xprssion of in Sos-2 n A549 lls (Supplmntry Fig. 2). Th -my 5 UTR supports p-inpnnt trnsltion n fusion of this RNA squn to th 5 UTR of -Ov (IRES -Ov) is suffiint to ovrom trnsltion inhiition without ltring th oing squn, rsulting in high lvls of -Ov ut without th inution in -My xprssion (Fig. 1) 19,29. Exprssion of DNA onstrut rsult in n pproximtly sixfol inrs in -my mrna lvls t 16 h following trnsftion tht rh pk of out tnfol t 36 h. In prlll, th mrna lvls of th -My trgt gn CAD strt to inrs t 24 h n pk t 2 NATURE COMMUNICATIONS 8: 213 DOI: 1.138/s w

3 Colony forming ssy Numr of olonis (μg) -My Cll popultion in S-phs (%) * * Norml Conflunt Strvtion -Ov Cll popultion in S-phs (%) 15 5 * * 5 -Ov Ov-3 5 -Ov p p p53--3 * 5 -GFP GFP-3 Atin My fol inution Ov -My 64 f Rltiv -my mrna lvls (%) Rltiv mrna lvls (%) g Rltiv -my mrna lvls (%) 15 5 * Atin EBNA1Δ h Rltiv mrna lvls 1, 1 1 * h Ov Ov-3 5 -IRES Ov-3 5 -IRES -Ov-3 EBNA1 WT RPS19 RPL38 45S pr-rrna Cylin 1 Ck4 16 h 24 h 36 h 48 h 6 h 72 h h 16 h 24 h 36 h 48 h 6 h 72 h Fig. 1 -inu mrna trnsltion strss stimults ll prolifrtion vi -My. Colony forming ssy. Th numr of olonis of H1299 lls xprssing th gly-l rpt () of th EBV-no EBNA1, or plsmi ontrol (), wr ount ftr 15 ys in sltion mium n plott. Th grph shows th prntg of prolifrting lls (S-phs) xprssing th or unr init growth onitions s trmin y FACS nlysis. Wstrn lots (lowr pnl) show tht fusing th to th N trminus of th opn ring frms of hikn ovlumin (Ov), (-Ov), p53 (-p53) or GFP (-GFP) supprsss mrna trnsltion, in ontrst to whn insrt t th C-trminl n (Ov- or p53-). Th grph (uppr pnl) shows th rltiv numr of lls in S-phs xprssing th init onstruts. Wstrn lots show th xprssion of -My following xprssion of th init quntitis of DNA in H1299 lls. Th numrs low th lots init -My lvls normlis to tin. Fusing th 5 UTR of -my tht ontins IRES to th 5 of -Ov (IRES -Ov) rogts trnsltion supprssion n vrts -My inution. f Th uppr grph shows RT-qPCR of -my mrna lvls t init tim points following xprssion of th. Th t show th rltiv -my/gapdh mrnas rtio n th highst vlu t 36 h is st to %. Th lowr grph shows th rltiv vlus of -My-inu CAD mrna lvls rltiv to GAPDH. Th highst vlu is st to %. g RT-qPCR nlysis show th rltiv -my mrna lvls normlis to GAPDH in H1299 lls xprssing, EBNA1 WT or EBNA1 Δ. h RT-qPCR nlysis of -my trgt gns (Ck4, ylin 1, 45S pr-rrna, Rps19 n Rpl38) in lls xprssing or. Not th logrithmi sl. FACS nlyss show mn vlus with s.. from t lst thr inpnnt xprimnts. Atin ws us s loing ontrol. For wstrn lots, RT-qPCR n olony formtion ssys rprsnttiv of thr inpnnt xprimnts wr shown with s.. Sttistil signifin ws lult using t tsts (*p <.1, p <.5 n *p <.1) 48 h (Fig. 1f, uppr n lowr grphs). Similrly, whn w xprss EBNA1 WT in H1299 lls, thr ws signifint inution of -My, whrs n EBNA1 onstrut lking th squn (EBNA1 Δ) h no fft, onfirming th importn of th in -My inution (Fig. 1g). W lso osrv signifint inrs in mrna lvls of -My Pol II (Ck4, Cylin D1, Rps19 n Rpl38) n Pol I (45S pr-rrna) trgt gns following xprssion (Fig. 1h). Whn th -My inhiitor F4-58 ws, w osrv n ~8% supprssion of lls in S-phs t 16 µm in ontrol lls, whrs th orrsponing supprssion in -xprssing lls t th sm onntrtion ws out 4% (Supplmntry Fig. 3). Howvr, th supprssion profil of CAD mrna lvls y F4-58 ws similr in lls xprssing th ompr to ontrol lls (Supplmntry Fig. 3). This isrpny twn prvnting ll prolifrtion n -My trgt gns inits tht othr -inu ftors r promoting ll prolifrtion. Th trtmnt with F4-58 i not prvnt th from inuing -My xprssion (Supplmntry Fig. 3). Inution of -my mrna lvls y is mit y. In orr to intify th -pnnt ftor tivting th - my promotr, sris of onstruts tht inlu th P1 n P2 of th -my promotr wr fus to Rnill luifrs rportr gn. Thr onstruts spnning from ( 252 to +34), ( 462 to +34) n ( 17 to +34), ll inu luifrs mrna lvls following xprssion of th full-lngth (Fig. 2). Th inution NATURE COMMUNICATIONS 8: 213 DOI: 1.138/s w 3

4 Rltiv luifrs mrna lvls 1 * * 8 * 6 ( 252/+34) ( 17/+34) ( 462/+34) -my gn Rltiv -my promotr lvls * P1 P2 Exon 1 ( 462) ( 252) ( 17) Exon 2 Luifrs DNA ChIP WT Mut1 Mut2 α- Exon 3 Rltiv luifrs ( 17/+34) mrna lvls α-e2f2 α-e2f3/ Ov - Ov EBNA1 WT EBNA1 Δ IRES -Ov EBNA1 WT EBNA1 Δ Atin Rltiv luifrs mrna lvls f Rltiv mrna lvls WT * Mut1 Mut2 P2 17 -my ( 17/+34) +34 E2F TATA INR E2F ining sit muttions. WT: ( 17/+34) : Mut1: ( 17/+34): Mut2: ( 17/+34): Fig. 2 promots -pnnt inution of -my. RT-qPCR t show th rltiv inution of -my-luifrs (Rnill) rportr onstruts (init in lowr pnl) following xprssion of th, s ompr to ontrol plsmi (). Rltiv RT-qPCR vlus show th inution of -my promotr tivity from th ( 17/+34) luifrs rportr onstrut using init Ov n -Ov (±IRES) DNA onstruts. Rltiv RT-qPCR vlus from th ( 17/+34) luifrs rportr onstruts rrying ithr thr (Mut1) or fiv (Mut2) point muttions in th onsnsus ining sit (init in lowr pnl). DNA hromtin IP (ChIP) ssy using th ( 17/+34) squn of th -my promotr onstruts (WT, Mut1 n Mut2) n ntiois ginst, E2F2 or E2F3 from lls xprssing th or. Wstrn lots showing th lvls in lls xprssing, EBNA1 WT, EBNA1 Δ or with tin us s loing ontrol. f RT-qPCR t of th inution of th trgt gns Cylins E n B following xprssion of. For RT-qPCR, th vlus wr normlis with GAPDH. Th vlus rprsnt th mn t from thr inpnnt xprimnts with s.. Signifin ws lult using t tsts (*p <.1 n p <.5) IRES Ov 8 6 Cylin E Cylin B of th Rnill rportr gn ( 17/+34) i not tk pl whn th trnsltion inhiitory pity of th ws olish y pling th -my IRES in th 5 UTR (Fig. 2). All onstruts inlu th E2F-ining sit n th pity of th to tivt th shortst -my promotr onstrut ( 17 to +34) wr svrly hmpr whn thr nulotis wr mutt (Mut1) in th E2F-ining sit n ompltly olish whn fiv muttions wr introu (Mut2) (Fig. 2). To furthr rss th rol of E2F in th -mit inution of -My, w rri out DNA ChIP ssys on th -my ( 17 to +34) rportr onstruts n w us ntiois ginst, E2F2 or E2F3. Quntittiv PCR rvl n ovr thrfol inrs in ining to th -my promotr following xprssion of th n minor ining with E2F2 n E2F3. This ws prlu whn w us E2F-ining sit mutnt (Mut1 n Mut2) onstruts (Fig. 2). W lso rri out ChIP ssys on th nognous -my promotr n w onfirm n inrs in ining following xprssion (Supplmntry Fig. 4). Th -mit tivtion of th -my promotr ws lso osrv in mous mryo firolst (MEF) lls (Supplmntry Fig. 4). Also, whn w siln using sirna, w osrv rs in -My lvls oth in norml n -xprssing lls (Supplmntry Figs. 4 n 4). Furthrmor, xprssing EBNA1 WT, s ompr to EBNA1 Δ, rsult in signifint inrs in xprssion (Fig. 2 n Supplmntry Fig. 4, f). Nxt, w tst th fft on othr trnsriptionl trgt gns n oul osrv n pproximtly four- n svnfol inrs in th lvls of Cylin E n Cylin B mrnas (Fig. 2f). triggrs post-trnsriptionl inrs of lvls. W nxt st out to unrstn how is tivt y mit trnsltion strss. Quntittiv RT-PCR following xprssion of rsult in n ovr sixfol inution of -my mrna lvls, without ffting th lvls of mrna, suggsting tht tivtion of is post-trnsriptionl (Fig. 3). Virl ftors suh s HPV E7, novirus E1A n SV4 lrg T tivt E2F y prvnting th intrtion with th pr protin. As th xprssion lvls pr s r not importnt for mit inution of it is unlikly tht th woul mimi th funtion of ths virl protins n prvnt th pr intrtion. In lin with this, w osrv tht ovrxprssion of pr rsult in omplt supprssion of -mit tivtion of, s msur y Cylin E mrna lvls. Howvr, whrs.2 µg of pr DNA ws suffiint to inhiit E2F tivity in th ontrol lls, thrfol xss ws rquir to hiv similr fft in lls xprssing th (Fig. 3). Th hypothsis tht th is not trgting th prb E2F intrtion ws furthr support y th osrvtion tht ovrxprssion of HPV E7 rsult in strong inution of E2F tivity in ontrol lls ut rsult in only mrginl furthr inrs in E2F tivity in lls xprssing th -Ov 4 NATURE COMMUNICATIONS 8: 213 DOI: 1.138/s w

5 Rltiv mrna lvls 8 * 6 -my Rltiv Cylin E mrna lvls 5 * μg pr Rltiv Cylin E mrna lvls μg HPV E7 64 GFP -GFP GFP -GFP GFP -GFP C 5 C 5 Rosovitin (μm) h MG132 (2 μm) -GFP μg pr GFP Atin Atin -My Atin f Fig. 3 inus protin lvls. RT-qPCR t show th rltiv lvls of -my n mrna following xprssion. Th fft of inu tivity s trmin y RT-qPCR on Cylin E mrna lvls following inrsing mounts of pr. As in ut with inrsing mounts of th pr ining HPV16-E7. Th fft on -mit inution of -My xprssion following trtmnt with inrsing onntrtions of th ll yl kins inhiitor rosovitin. Wstrn lots showing th lvls of in lls xprssing GFP or -GFP following inrsing mounts of pr. f Th lvls of xprssion following trtmnt with th protsom inhiitor (MG132). Th t prsnt in, n show th mns from thr inpnnt xprimnts with s.. Th mrna lvls wr normlis ginst GAPDH n th rfrn vlu ws st for non-trnsft lls. Sttistil signifin ws lult using t tsts (*p <.1). Wstrn lots rprsnt n 3 n tin ws us s loing ontrol (Fig. 3). During th trnsition from G to G 1, pr oms phosphorylt y ylin-pnnt kins (CDK) tivity, whih rsults in th rls of. Whn w trt ontrol lls with th CDK inhiitor rosovitin, w osrv ru lvls of -My ut this fft ws signifintly lss in lls xprssing th (Fig. 3). Fr tiv hs highr turnovr rt n ths thr osrvtions r onsistnt with highr lvls of n tiv in -xprssing lls. In support of this, w osrv tht n inrs in th lvls of pr rsult in n inrs in protin lvls in lls xprssing th - GFP ompr to lls xprssing GFP lon (Fig. 3). A similr inrs in lvls ws osrv using th protsom inhiitor (MG132) (Fig. 3f). W i not osrv inution whn ws xprss unr -my IRES ontrol (Supplmntry Fig. 4g). Inution of is mit vi its oing squn. An inrs in protin synthsis y th ws osrv using 35 S-mtoli puls llling follow y immunopripittion (Fig. 4). As th mrna lvls of o not hng following xprssion of th, this is onsistnt with th triggring n inrs in th rt of mrna trnsltion. Exprssion of onstruts tht inlu th 5 n th 3 UTRs ( UTR), or th oing squn lon (CDS), rsult in similr -pnnt inrs in xprssion, initing tht th inrs in th rt of mrna trnsltion is not mit y th lssi rgultion of trnsltion tht ts vi th untrnslt squns (Fig. 4). Polysoml profiling follow y RT-qPCR nlysis of mrna show signifint nrihmnt of mrna in th polysoml frtions in lls xprssing th ompr to mpty vtor (), initiv of mor riosoms ing link to th mrna n, thus, highr rt of protin synthsis (Fig. 4). Othr mrnas, suh s BiP n p21, i not show n inrs in polysom frtions (Supplmntry Fig. 5) initing tht th inrs in lvl unr inution is not rsult of glol trnsltion inrs. Furthrmor, ltion mutnts of th CDS trunt from th 5 show tht th first 432 nulotis of th CDS ontin th rsponsiv lmnt (Fig. 4). Intrstingly, Δ(+1 to +324) show low xprssion in th sn of n strong inution, suggsting tht rgultion of synthsis is mit vi two RNA omins n might involv th rls of ngtiv ting ftor. In support of this, w osrv tht th ltions Δ(+1 to +5) n Δ(+1 to +585) rsult in highr lvls of xprssion ut ru inution (Fig. 4). Togthr, ths rsults point towrs mrna strutur motif/s ing th trgt for pnnt inution of mrna trnsltion. mrna trnsltion strss inus vi PI3Kδ. Th inution of synthsis following isruption of mrna trnsltion on -rrying polysoms is likly to rly y signlling pthwy. Som of th sri signlling pthwys known to fft glol p-pnnt mrna trnsltion t vi mtor n th phosphoryltion of 4E-BP1, or vi S6 kins 3. Howvr, 8 h trtmnt with th mtor inhiitor rpmyin, or with AKTinhiitors, h no fft on -mit inution of xprssion (Fig. 5 n Supplmntry Fig. 6). This is in lin with t showing tht th trns-fft of th on mrna trnsltion is mit vi th CDS n not vi squns in th UTRs. W lso tst inhiitors ginst th MAPK, ll yl kinss, DNA-PK, ATM n svrl othr kinss link to growth ontrol ut with littl fft. Howvr, whn w tst two gnrl PI3K inhiitors, wortmnnin n LY92, w osrv omplt inhiition of -mit inution of (Fig. 5 n Supplmntry Fig. 7). Inhiitors of iffrnt PI3Ks tlyti suunits show tht th PI3Kδ-spifi inhiitor CAL-11, ut not inhiitors of p11α, p11β or p11γ, supprss mit inution of (Fig. 5 n Supplmntry Figs. 6 n 7). Wortmnnin ffiintly prvnt phosphoryltion of PI3K ownstrm trgts AKT n th mtor sustrt 4E-BP1 in lls, whr PI3K tivity h n inu following trtmnt with 4 ng/ml of PDGF for 3 min in srum-fr mium. Howvr, CAL-11 h lss fft on phosphoryltion of AKT n littl fft on phosphoryltion of 4E-BP1 unr ths onitions (Fig. 5). In lin with this, thr ws no -pnnt fft on th phosphoryltion of AKT n 4E-BP1, showing th spifiity of PI3Kδ tivtion (Fig. 5 n Supplmntry Fig. 6). A rol for p11δ in th ontrol of xprssion ws furthr support using sirna ginst p11δ. Importntly, supprssion of p11δ xprssion using sirna lso ru lvls in NATURE COMMUNICATIONS 8: 213 DOI: 1.138/s w 5

6 + + CDS UTR GFP -GFP GFP -GFP C 5 C 5 C 5 C 5 MG132 6 h Atin A4 Δ(+1 to + 249) Δ(+1 to +324) Δ(+1 to +432) Δ(+1 to +5) Δ(+1 to +585) 5% Fr Monosoms Polysoms 5% pool A4 Cylin-ining omin DNA-ining omin Dimriztion omin WT Ativtion omin R fmily-ining omin Atin Δ249 Δ324 Δ432 Δ5 Δ585 nts mrna (% of totl) 5% Fr Monosoms Polysoms 5% pool F6 F7 F8 F9 F1 F11 F12 F13 F14 F15 F16 F17 Frtions Fig. 4 -inu mrna trnsltion strss stimults synthsis. Autoriogrph shows th fft of on nwly synthsis lvls following 35 S-Mt puls llling in th prsn of protsom inhiitor (2 µm MG132) follow y immunopripittion. Wstrn lots showing -pnnt inution of from onstruts rrying th untrnslt (UTR) rgions of th mrna ( UTR), or th oing squn lon ( CDS). Polysom profiling n RT-qPCR nlysis of mrna in lls xprssing th or. Th uppr pnls show polysom profils. Th lowr pnl shows th rltiv mrna lvls (%) from totl mrna normlis ginst tin. Th vlus rprsnt th mn t from thr inpnnt xprimnts with s.. Shmti rprsnttion of funtionl omins of lt from th 5 of th CDS. Th +1 init th first AUG of th CDS. Wstrn lots showing th xprssion of ltion mutnts with n without xprssion. Wstrn lots rprsnt n 3, tin ws us s loing ontrol non--trt lls, initing tht th p11δ-pnnt rgultory pthwy is not uniqu to EBNA1 tivity (Fig. 5 n Supplmntry Fig. 7). -pnnt inution of is furthr stimult following trtmnt with gnrl PI3K stimulnt (LPS [1 µg/ml] n MgSO 4 [2 mm]) 31 n ws ompltly supprss y th ition of CAL-11 (1 µm). This ws lso osrv in EBV-inft lympholstoi B95-8 lls (Fig. 5f n Supplmntry Figs. 6 n 7). In lin with this, lvls wr ru signifintly upon CAL-11 trtmnt in H1299 lls xprssing EBNA1 WT s ompr to EBNA1 Δ (Supplmntry Fig. 7). Thr r no pprnt hngs in th xprssion lvls n lolistion pttrn of p11δ in lls xprssing th (Fig. 5 n Supplmntry Fig. 8). PI3Kδ mits EBNA1 onogni tivity. Svrl onogni viruss trgt th pthwy ut this hs not yt n shown for th EBV. Nvrthlss, EBNA1 hs n implit in hving n onogni tivity n trnsgni EBNA1 niml mol in whih EBNA1 trnsgn xprssion ws rivn y th immunogloulinhvyhin(eµebna1) show n invrs orrltion twn EBNA1 xprssion n lymphom phnotyp 14. To tst if th onogni proprtis of EBNA1 r rlt to tivtion of p11δ, wusneµebna1 mous-riv primry lymphom ll lin. Trtmnt with CAL-11 in ultur show os-pnnt inrs in ll th strting t 1 µm (Fig.6). Thr furthr inpnnt primry tumours from iffrnt mi of th sm trnsgni lin (EµEBNA1.26) 14 show CAL-11-mit ll th strting t 2 ys of trtmnt (1 µm) (Fig. 6). EBNA1 trnsgni lls show inrs xprssion of oth -My n ompr to th non-trnsgni wil-typ (WT) lymphoyts (Fig. 6 nsupplmntry Figs. 9 n9) n w osrv loss of oth n -My xprssion ftr 1 y of trtmnt t 1 µm (Figs. 6, n Supplmntry Fig. 9). To tst if CAL-11- mit ll th is link to EBNA1 xprssion, w ompr ll lin riv from n LMP1 n EBNA1 itrnsgni lymphom (59.48) to n LMP1-only ll lin (.415) 14,32. Th LMP1 n EBNA1 i-trnsgni lls r EBNA1-pnnt n trtmnt with CAL-11 t 5 µm rsult in th, whil th EBNA1-inpnnt (.415) ll lin show no inrs in ll th (Fig. 6). W lso trt th EBV-positiv Burkitt s lymphom (BL) Rji lls n EBVngtiv BL41 lls with CAL-11 n w osrv supprssion of in Rji ut with lss fft in BL41 (Fig. 6f). Rji lls rry th BL hrtristi -my gn Ig trnslotion, whrs B95.8 is n EBV-trnsformlympholstoilllin n o not hv th -my trnslotion. Only in th B95.8, n not in th Rji lls, i w osrv CAL-11-pnnt supprssion of -My xprssion (Fig. 6g). Ths rsults support th hypothsis tht EBNA1 xrts n onogni tivity vi -pnnt tivtion of n th onsqunt inution of -My. 6 NATURE COMMUNICATIONS 8: 213 DOI: 1.138/s w

7 Drug/Trgt Inhiitor VII/ AKT Inhiitor II/ DNA-PK SB2358/ P38 MAPK Conntrtion (μm) Atin Atin Atin Drug/Trgt CAL-11/ PI3K p11δ AS-6524/ PI3K p11γ A66/ PI3K p11α Conntrtion (μm) Atin Atin Atin 5 5 Wortmnnin.3 μm 1 μm 5 μm 1 μm CAL-11 P-AKT AKT P-4E-BP1 4E-BP1 Atin 5 5 p11δ AKT p-akt 4E-BP1 p-4e-bp1 Atin Rpmyin/ mtor Rosovitin/ CDKs Atin P-4E-BP1 Atin + + Atin sirna (p11δ) f LPS 1 μg, Mg SO 4 2 mm CAL-11 1 μm Atin ATMi/ ATM Atin Wortmnnin/ PI3K (Pn) Atin Fig. 5 -pnnt inution of is mit vi PI3Kδ. Wstrn lots show th xprssion of in H1299 lls xprssing th or following trtmnt with init rugs for 8 h. Phosphorylt 4E-BP1 (P-4E-BP1) ws us to show mtor tivity. Wstrn lots show xprssion lvls in lls xprssing th or ontrol plsmi (), trt with th init onntrtions of inhiitors of PI3K p11δ (CAL-11), p11γ (mil pnl) n p11α (lowr pnl) suunits. Th fft of wortmnnin n th PI3Kδ-spifi inhiitor (CAL-11) on th phosphoryltion of AKT (P- AKT) n 4E-BP1 (P-4E-BP1) in lls trt with 4 ng/ml of PDGF for 3 min in srum-fr mium. Wstrn lots showing th fft of on th phosphoryltion of AKT n 4E-BP1. Wstrn lot showing th xprssion of following 24 h trtmnt with sirna ginst PI3Kδ. f Wstrn lot showing th xprssion of in lls xprssing or follow y gnrl inution of PI3K tivity using MgSO 4 (2 mm) n LPS (1 µg), with, or without, CAL-11 (1 µm). Wstrn lots rprsnt n 3. Atin ws us s loing ontrol. Disussion By isrupting mrna trnsltion in is using th uniqu proprtis of th gly-l rpt of th EBNA1 w n show tht lls rspon to mrna trnsltion strss y PI3Kδ-pnnt tivtion of synthsis n th inution of -My. W hv lso shown how this pthwy ounts for EBNA1 onogni tivity in trnsgni niml mol. It is wll sri how uprgultion of -My inus riosoml iognsis to promot protin synthsis n ths rsults illustrt fk pthwy from th riosom to -My, whry th ll snss th sttus of th mrna trnsltion n triggrs pthwy im t rstoring ysfuntionl protin synthsis. W propos tht this pthwy is xploit y th EBV to promot ll growth (Fig. 7). Th pool of inu is tiv n not oun to pr. In lin with this, w foun tht ovrxprssion of th ppillom E7 protin h only mrginl fft on tivity in xprssing lls. Th inution of y PI3Kδ is t th lvl of inrs mrna trnsltion n is unusul in th sns tht it is mit vi th oing squn of th mrna, n not vi th mor ommonly us 3 n 5 UTRs. This inits n mrna-spifi rgultion tht hlps to xplin how ro inhiitors of th PI3K tlyti suunits, suh s wortmnnin n LY292, n ffiintly lok -pnnt inution of, whil inhiitors of AKT or mtor tht ontrol gnrl ppnnt trnsltion hv littl, or no fft. W r urrntly trying to intify th PI3Kδ sustrt tht ontrols synthsis n s on th ltion sris of th oing squn it is likly this ftor is n RNA strutur-ining protin. This shows onptul similrity to th inution of p53 synthsis following DNA mg, whih is lso mit y strss-rsponsiv RNA strutur in th oing squn of th p53 mrna 33. Th rol of PI3Kδ in th mrna trnsltion strss rspons ws unxpt s this fmily of kinss is normlly ssoit with xtrllulr growth stimultory ftors n tivtion y tyrosin kins or G- protin-oupl rptors. Howvr, hint tht PI3Kδ might ply rol in th EBV lif yl is suggst y th osrvtion tht ptints who vlop th gin-of-funtion onition, tivt PI3Kδ synrom (APDS), typilly suffr from hroni EBV (n othr hrpsviruss) virmi 34. It is intrsting tht supprssion of PI3Kδ inhiits xprssion lso in lls tht o not xprss th, whih implis ror rol of this pthwy in ontrolling xprssion. It is, thus, likly tht llulr mssgs lso triggr this pthwy n tht high rt of protin synthsis n hlp xplin PI3Kδ tivity lso in soli nrs. PI3Kδ inhiitors hv n fftiv in trting hroni lymphoyti lukmi (CLL) n inolnt Hogkin s lymphom n ths rsults hlp to ttr unrstn th unrlying molulr mhnisms. Hogkin s lymphoms r ssoit with EBV in ~4% of ss n it will intrsting to s if thr is orrltion twn EBV-rrying nrs n Illisi ffiy. In support of this, w osrv tht i-trnsgni lymphom lls pning on EBNA1 r snsitiv to CAL-11 trtmnt. Nsophryngl rinoms tht rry th EBV n xprss EBNA1 in % of th ss is nothr typ of nr in whih this pthwy might of thrputi intrst. Ativtion of is ommon strtgy y onogni viruss to promot ll prolifrtion ut this hs prviously not n monstrt for EBV. Ppillom, novirus n simin SV4 viruss hv vlop strtgis to tivt E2Fs y xprssing ftors tht isrupt th intrtion with pokt protins. Th EBNA1 hs volv iffrnt rout tht inst xploits llulr pthwy tht ims to rstor riosoml tivity vi tivtion of unr onitions of ysfuntionl riosoml tivity. Th sm inhiition of mrna trnsltion in is y th forms NATURE COMMUNICATIONS 8: 213 DOI: 1.138/s w 7

8 Liv ll ount/ml Liv ll ount/ml EμEBNA1.26-riv primry lymphom lls Tim (ys) WT NT NT Trnsgni lymphom ll lins Tim (ys) 5 6 CAL-11 Control μm 5 μm μm 1 μm Dys -My Atin LMP1 ontrol LMP1 LMP1 CAL-11 LMP1-EBNA1 ontrol LMP1-EBNA1 LMP1-EBNA1 CAL-11 Growth rlt to untrt lls EμEBNA1.26-riv primry lymphom lls Tim (ys) CAL-11 CAL-11 g Rji lls (BL lls) B95.8 lls (LCLs) NT CAL NT CAL-11 f Rji lls (EBV+) BL41 lls (EBV ) CAL-11 CAL-11 Dys -My Atin Dys EBNA1 Atin Dys -My Atin Fig. 6, -My xprssion n ll viility r mit vi PI3Kδ in EBNA1 trnsgni mous tumour lls. Primry lymphom lls riv from mi wr ultur (in triplit) with 1 µm CAL-11 or, or no itiv (ontrol) for 6 ys. Rpi ll th orrlts with inrsing onntrtions of CAL-11. Clls from thr iffrnt xplnt primry EBNA1-positiv trnsgni lymphoms wr h ultur in triplit for 4 ys with 1 µm CAL-11 (in.1% ) or.1%. Th rtio of CAL-11 n -trt ll ounts ginst untrt ll ounts is grph., Wstrn lots showing tim ours of th xprssion of n -My in non-trnsgni lls (WT) n using trnsgni lls from two iffrnt primry tumours (ID: [C] n [D]) (s lso Supplmntry Fig. 9). Explnt EµEBNA1 trnsgni tumour lls wr ultur for up to 4 ys, with no trtmnt (NT), or with vhil () or with ily trtmnt of 1 µm CAL-11, s init. n -My xprssion in th EBNA1 trnsgni lymphom lls r mrkly ru y CAL-11 trtmnt. Cll growth ws msur for 7 ys in two mous B ll lymphom ll lins LMP1 positiv (ID:.415) n th i-trnsgni LMP1 n EBNA1 positiv (ID: 59.48). Clls wr ultur with ily ition of CAL-11 to 5 µm, or with vhil () n vil lls ount. f Wstrn lots showing th xprssion of in two Burkitt s lymphom ll lins rrying -my gn trnslotion. Rji is EBV positiv n BL41 is EBV ngtiv. Both wr trt with CAL-11 (1 µm) for init tim. g Wstrn lots showing -My xprssion following trtmnt with CAL-11 (1 µm) in Rji n th lympholstoi ll lin B95.8 (no -my gn trnslotion). Wstrn lots rprsnt n 3; Atin ws us s loing ontrol. Th vlus in, n rprsnt th mn t from thr inpnnt xprimnts with s. 43S 8S ppti EBNA1 mrna PI3Kδ PI3Kδ-inhiitors (Illisi) 43S 8S mrna ylins -my Growth Prolifrtion Fig. 7 mrna trnsltion strss on EBNA1 polysoms tivts PI3Kδ n synthsis. Trnsltion ttnution in is us y th squn of th EBNA1 mrna uss trnsltion strss, whih rsults in tivtion of PI3Kδ n th inution of mrna trnsltion inpnnt of AKT n mtor pthwys. Th nwly synthsis inus ownstrm trgt gns, inluing ylins n -my, rsulting in n inrs in ll growth n prolifrtion prt of th virl strtgy to v th MHC lss I-rstrit ntign prsnttion pthwy. Hn, trgting mrna trnsltion llows th virus to hit two irs with on ston: v th immun systm n t th sm tim support th prolifrtion of inft lls. Dspit ing th only EBV ntign xprss in ll BL n lso onsistntly xprss in ll othr EBV-ssoit tumours suh s nsophryngl rinoms, th onogni tivity of EBNA1 hs rmin n nigm. An invrs orrltion twn EBNA1 lvls n th pntrn of B ll lymphoms ws rport 14,15. Aitionlly, -My ws foun to not plyr in ths EBNA1 tumours, showing uprgult xprssion (Supplmntry Fig. 9). Importntly, w n now show tht n -My xprssion in primry lymphoms riv from th lymphom-pron EµEBNA1 mi r in inhiit following CAL-11 trtmnt. It is, thus, likly tht th rport invrs orrltion twn th xprssion lvls of EBNA1 n th lymphom phnotyp in th EµEBNA1 nimls 8 NATURE COMMUNICATIONS 8: 213 DOI: 1.138/s w

9 n xplin y th inution of y EBNA1-mit mrna trnsltion strss. In lin with this, -My lvls rs ftr CAL-11 trtmnt in EBV-inft lympholstoi lls, ut not in Burkitt s lymphom (BL) lls whr th trnsription of -my is rgult through trnslotion to n immunogloulin lous. Mthos Cll ultur n trtmnts. Unlss mntion othrwis, xprimnts wr prform mostly using H1299 lls (non-smll-ll lung rinom humn ll lin) [NCI-H1299 (ATCC CRL583 )]. Othr ll lins us wr Rji (typ III ltny Burkitt s lymphom), B95.8 lympholstoi ll lin. All ll lins wr ultur in RPMI 164 mium supplmnt with 1% fotl ovin srum, 2 mm L-glutmin, U/ml pniillin, µg/ml strptomyin (Invitrogn) n 5 µg/ml Plsmoin prophylti (Invivogn). Cll lins wr routinly hk for myoplsm ontmintion using PlsmoTst kit (Invivogn). Two mous B ll lymphom ll lins wr us:.415 stlish from n LMP1 rivn trnsgni mous tumour n stlish from n LMP1 n EBNA1-positiv itrnsgni mous tumour 32. Primry tumour lls from lymphoms riv from th EµEBNA1 trnsgni mous lin 26 14, or in vivo pssgs of ths tumours, or wil-typ (non-trnsgni) splnoyts wr xplnt, r loo lls lys n ultur irtly, or frozn for susqunt ultur. Strvtion xprimnt llow th lls to grow for 72 h with omplt mium supplmnt with.1% fotl ovin srum. H1299 stl ll lins wr gnrt through trnsint trnsftion of nomyin gn-ontining pdna3 vtors. Aftr 2 to 3 wks, gntiin rsistnt popultions wr slt n gn stility ws vrifi for t lst tn pssgs. Lymphom lls wr ultur in RPMI (with L-glutmin, 1% FBS) with 5 µm 2ME in primry ulturs. CAL-11 ws issolv in n supplmnt in ulturs from 1 to µm ily. Liv ll ounts n ll viility wr trmin y trypn lu xlusion n ount using th Countss Automt Cll Countr (Thrmo Fishr Sintifi). Plsmi DNAs n sirnas. Th full-lngth DNA xprssion vtor ws isolt from th Epstin Brr virus (strin B95-8)-no EBNA1 n lon nxt to ovlumin DNA or p53 DNA. Th gnrt pdna3 onstruts,, Ov, -Ov, Ov-, p53, -p53, p53-, -my IRES-Ov n -my IRES -Ov, wr sri in rlir rports 17,19. Th DNA ws lon in th pgfp-n1 vtor gnrting -GFP fusion protin with t th NH2 trminl (-GFP). oing squn ws mplifi from th mv- (provi y Dr O. Bishof, Inst. Pstur, Pris) n lon in to pdna3 vtor. CDS ltion onstruts wr rt using pdna3- (CDS) mplifi with orrsponing primrs (list in Supplmntry Tl. 1) n lon into pdna3 vtor. Th prb onstrut ws provi y Dr O. Bishof n CMV-HPV16-E7 is n xprssion vtor for high-risk HPV16-E7 onoprotin tht ws provi y Dr K. Müngr (Brighm & Womn s Hospitl, MA). Th pgl2- Bsi luifrs rportr vtor ontining th humn -My promotr ( 252/ +34), ( 462/+34) n ( 17/+34) wr kinly provi y Profssor LZ Pnn (Toronto Mil Disovry Towr, ON, Cn). Using sit-irt mutgnsis, squntil ltions of th humn -my promotr ( 17/+34) wr prform to gnrt two mutt DNA frgmnts, signt ( 17/+34) Mut1 n ( 17/+34) Mut2. For silning, sirna 1 (297963, Qign), sirna 2(2821, Qign), AllStrs srml sirna (SI365318, Qign), p11δ sirna 1 ( , Qign), p11δ sirna 2 ( , Qign), E2F3 sirna ( , Qign) wr us. For trnsftion of plsmi DNA n sirna, GnJui (EMD Chmils) n INTERFERin (Polyplus-trnsftion) rgnts wr us oring to th mnufturr s instrutions. Wstrn lotting. Clls wr lys in BC lysis uffr ( mm NCl,.2% NP- 4, 1% (v/v) glyrol, 1. mm ithiothritol (DTT), 1. mm EDTA, n mm Tris-HCl, ph 7.8) ontining 1% (v/v) ukryoti prots inhiitor oktil (Cliohm). Equl protin mounts wr lo n rsolv in 4 12% Bis Tris Plus Gls (Thrmo Fishr), trnsfrr on BioTr NT pur nitrollulos lotting mmrn (PALL Corportion) n lok with 5% non-ft ry milk in Trisuffr slin ph 7.6 ontining.1% Twn-2. Protins wr thn pro with orrsponing ntiois; nti- rit pas [1:] (C-2, Snt Cruz), nti- GA rit pas ris ginst th gly-l squn of EBNA1 protin [1:5], ntip53 rit pas (CM-1) [1:], nti-gfp mous mas [1:] (lons 7.1 n 13.1, Roh), nti-hikn gg ovlumin rit pas [1:] (Sigm-Alrih), nti--my mous mas [1:5] (9E1, Snt Cruz n Y69 Am: #3272) n nti-tin mous pas [1:] (AC-15, Sigm-Alrih), nti-p11δ rit pa [1:] (H219 Snt Cruz), nti-akt rit ma [1:] (2938, Cll Signling), nti-p-akt rit ma [1:5] (9271, Cll Signling), nti-4e-bp1 rit ma [1:] (9644, Cll Signling), nti-p-4e-bp1 rit ma [1:] (28, Cll Signling). Unropp lots r vill in supplmntry informtion. Colony formtion ssys. Twnty-four hours post trnsftion, pdna3 (vtor ontrol) or pdna3- trnsft H1299 lls wr trypsiniz n slt in growth mium supplmnt with gntiin. Surviving olonis wr llow to grow for 15 ys n stin with 4% (v/v) Gims stin. Colonis wr thn quntifi using Clono ountr progrmm. Flow ytomtry. Clls xprssing init onstruts wr fix y ovrnight inution in ol 7% thnol. Aftr 3 min inution with U/ml RNs A (Sigm-Alrih) t 37 C, propiium ioi (1 µg/ml) stin lls wr nlys with n LSR flow ytomtr n CllQust softwr (Bton-Dikinson). ChIP ssys. ChIP ssys wr prform using kit oring to th mnufturr s instrutions (Upstt). Th lr smpls wr inut ovrnight with th nti- (H137) nti-e2f2 (s-632) or nti-e2f3 (s-56666) rit polylonl ntiois. Antioy/protin/DNA omplxs wr thn opripitt with th s oring to th kit rommntions n th DNA ws rovr y phnol/hloroform xtrtion n thnol pripittion. Th P2 promotr rgion ( 83/+3) of th humn -my gn ws mplifi y qpcr (s Supplmntry Tl. 1 for primr squns). Quntittiv rvrs trnsription-pcr. Totl RNA ws xtrt with RNsy Mini Kit (Qign) following th mnufturr s instrutions. DNA synthsis ws rri out using th Molony murin lukmi virus rvrs trnsripts n Oligo(T) Primr (Invitrogn). RT-qPCR ws prform on StpOn rl-tim PCR systm (Appli Biosystms) using Prft SYBR Grn FstMix, ROX (Qunt Biosins) (S Supplmntry Tl. 1 for trgt primr squns). Polysom profiling. Fiv fifty prnt wt/vol linr suros grints wr frshly st on SW41 ultrntrifug tus (Bkmnn) using th Grint mstr (BioComp instrumnts) following th mnufturr s instrutions. Forty-ight hours post trnsftion, H1299 lls (with 8% onfluny) wr trt with ylohximi µg/ml for 5 min t 37 C n thn wsh twi with 1 PBS (Dulo moifi PBS, GIBCO) ontining ylohximi µg/ml. Clls wr thn srpp n lys with polysom lysis uffr ( mm KCL, 5 mm HEPES- KOH, 5 mm MgCl 2,.1% NP-4, 1 mm DTT, ylohximi µg/ml, ph 7.4). Lysts wr thn lo on suros grint n ntrifug t g for 2 h t 4 C in SW41 rotor. Smpls wr thn frtiont using Foxy R1 frtion olltor (Tlyn ISCO) t.5 min intrvls 35,36. RNA purifitions from frtions wr prform using Trizol-LS rgnt (Invitrogn) omin with thnol pripittion. Rvrs trnsription for th trgt mrnas wr rri out using qul volum of DNA fr RNA from frtions n thn normlis with tin lvls. Mtoli puls ll. Twnty-four hours post trnsftion, lls wr ultur for 1 h in Dulo s moifi Egl s strvtion mium (Sigm-Alrih) (without mthionin, ystin n L-glutmin supplmnt with 2% ilys fotl ovin srum togthr with 2 µm of protsom inhiitor MG132. Clls wr mtolilly lll for 1 h with 45 µci/ml of EsyTg Exprss 35 S-mthionin Protin Llling Mix (Prkin-Elmr). protin ws thn immunopripitt n th nwly synthsis protin wr rsolv in 4 12% Bis Tris Plus Gls n visulis on utoriogrph. Immunofluorsn stining. Clls wr grown in ovr slips for h (for growing non-hrnt lls, ovr slips wr trt first with.1% poly-l-lysin solution [Sigm]) n fix with 4% prformlhy (for H1299 lls wr fix 48 h post trnsftion), prmiliz with.4% Triton X-,.5% CHAPS in PBS. Clls wr thn lok using 3% BSA,.1% sponin in PBS n inut with nti-pi3kδ mous ma [1:] (s-13632) for 2 h t RT n with ALEXA- 488 nti-mous ntioy [1:5] for 45 min t RT, stin with DAPI n wsh with PBS. Covr slips wr thn ri n mount in sli using DAKO fluorsnt mounting mium. Clls wr thn nlys y Ziss Axiovrt invrt mirosop. Drugs. MG132 ( , Cliohm), ATMi (1185, Cliohm), DNA-PK Inhiitor II (26961, Cliohm), AKT Inhiitor VIII (12418, Cliohm), Rpmyin (321, Cliohm), SB2358 (9389, Cliohm), Wortmnnin (681675, Cliohm), F4-58 (F368, Sigm-Alrih), Rosovitin (R7772, Sigm-Alrih), AS-6524 (S141, Sllk Chmils), CAL-11 (S2226, Sllk Chmils), A-66 (S2636, Sllk Chmils), LY292 (113, Toris Biosin), LPS LipoPolyShri (L288, Sigm-Alrih). Sttistil nlysis. Sttistil signifin ws nlys y ompring t with rfrn point using two-til t tsts, p <.5 ws onsir sttistilly signifint (*p <.1, p <.5 n *p <.1). All sttistil ssssmnt ws prform using th Mirosoft xl progrmm. NATURE COMMUNICATIONS 8: 213 DOI: 1.138/s w 9

10 Dt vilility. Th uthors lr tht t supporting th finings of this stuy r vill within th rtil n supplmntry fil, or vill from th orrsponing uthor on rsonl rqust. Riv: 18 April 217 Apt: 17 Novmr 217 Rfrns 1. Chn, H. Z., Tsi, S. Y. & Lon, G. Emrging rols of E2Fs in nr: n xit from ll yl ontrol. Nt. Rv. Cnr 9, (9). 2. Roussl, M. F., Dvis, J. N., Clvln, J. L., Ghysl, J. & Hirt, S. W. Dul ontrol of my xprssion through singl DNA ining sit trgt y ts fmily protins n E2F-1. Onogn 9, (1994). 3. Clsson, M. & Hrlow, E. Th rtinolstom tumour supprssor in vlopmnt n nr. Nt. Rv. Cnr 2, (2). 4. Winrg, R. A. Th rtinolstom protin n ll yl ontrol. Cll 81, (1995). 5. Hrour, J. W., Luo, R. X., Di Snti, A., Postigo, A. A. & Dn, D. C. Ck phosphoryltion triggrs squntil intrmolulr intrtions tht progrssivly lok R funtions s lls mov through G1. Cll 98, (1999). 6. Chllppn, S. P., Hirt, S., Muryj, M., Horowitz, J. M. & Nvins, J. R. Th E2F trnsription ftor is llulr trgt for th RB protin. Cll 65, (1991). 7. Whyt, P. t l. Assoition twn n onogn n n nti-onogn: th novirus E1A protins in to th rtinolstom gn prout. Ntur 334, (1988). 8. Phlps, W. C., Y, C. L., Mungr, K. & Howly, P. M. Th humn ppillomvirus typ 16 E7 gn nos trnstivtion n trnsformtion funtions similr to thos of novirus E1A. Cll 53, (1988). 9. Flsni, A., Milo, A. M. & Pggi, M. G. Rtinolstom fmily protins s ky trgts of th smll DNA virus onoprotins. Onogn, (6). 1. Ahikry, S. & Eilrs, M. Trnsriptionl rgultion n trnsformtion y My protins. Nt. Rv. Mol. Cll Biol. 6, (5). 11. Iritni, B. M. & Eisnmn, R. N. -My nhns protin synthsis n ll siz uring B lymphoyt vlopmnt. Pro. Ntl A. Si. USA 96, (1999). 12. Tu, R. t l. Trnslotion of th -my gn into th immunogloulin hvy hin lous in humn Burkitt lymphom n murin plsmytom lls. Pro. Ntl A. Si. USA 79, (1982). 13. Hnl, W. & Hnl, G. Epimiologi spts of Epstin-Brr virus (EBV)- ssoit isss. Ann. N. Y. A. Si. 354, (198). 14. Wilson, J. B., Bll, J. L. & Lvin, A. J. Exprssion of Epstin-Brr virus nulr ntign-1 inus B ll noplsi in trnsgni mi. EMBO J. 15, (1996). 15. Kng, M. S. t l. Epstin-Brr virus nulr ntign 1 os not inu lymphom in trnsgni FVB mi. Pro. Ntl A. Si. USA 12, 82 8 (5). 16. Young, L. S., Yp, L. F. & Murry, P. G. Epstin-Brr virus: mor thn 5 yrs ol n still proviing surpriss. Nt. Rv. Cnr 16, (216). 17. Yin, Y., Mnoury, B. & Fhrus, R. Slf-inhiition of synthsis n ntign prsnttion y Epstin-Brr virus-no EBNA1. Sin 31, (3). 18. Murt, P. t l. G-quruplxs rgult Epstin-Brr virus-no nulr ntign 1 mrna trnsltion. Nt. Chm. Biol. 1, (214). 19. Aphr, S. t l. mrna trnsltion rgultion y th Gly-Al rpt of Epstin- Brr virus nulr ntign 1. J. Virol. 83, (9). 2. Thorp, L. M., Yuzugullu, H. & Zho, J. J. PI3K in nr: ivrgnt rols of isoforms, mos of tivtion n thrputi trgting. Nt. Rv. Cnr 15, 7 24 (215). 21. Okknhug, K. Signling y th phosphoinositi 3-kins fmily in immun lls. Annu. Rv. Immunol. 31, (213). 22. Swyr, C. t l. Rgultion of rst nr ll hmotxis y th phosphoinositi 3-kins p11lt. Cnr Rs. 63, (3). 23. Koysu, S. Th rol of PI3K in immun lls. Nt. Immunol. 4, (3). 24. Ali, K. t l. Essntil rol for th p11lt phosphoinositi 3-kins in th llrgi rspons. Ntur 431, (4).. Aksoy, E. t l. Th p11lt isoform of th kins PI(3)K ontrols th sullulr omprtmntliztion of TLR4 signling n protts from notoxi shok. Nt. Immunol. 13, (212). 26. Ali, K. t l. Intivtion of PI(3)K p11lt rks rgultory T-ll-mit immun tolrn to nr. Ntur 51, (214). 27. Furmn, R. R. t l. Illisi n rituxim in rlps hroni lymphoyti lukmi. N. Engl. J. M. 37, (214). 28. Aphr, S., Dskloginni, C., Mnoury, B. & Fhrus, R. Epstin Brr virusno EBNA1 intrfrn with MHC lss I ntign prsnttion rvls los orrltion twn mrna trnsltion initition n ntign prsnttion. PLoS Pthog. 6, 1151 (21). 29. Cool, L. C. t l. Intifition of intrnl riosom ntry sgmnt (IRES)- trns-ting ftors for th My fmily of IRESs. Mol. Cll Biol. 28, 4 49 (8). 3. M, X. M. & Blnis, J. Molulr mhnisms of mtor-mit trnsltionl ontrol. Nt. Rv. Mol. Cll Biol. 1, (9). 31. Su, N. Y., Png, T. C., Tsi, P. S. & Hung, C. J. Phosphoinositi 3-kins/Akt pthwy is involv in miting th nti-inflmmtion ffts of mgnsium sulft. J. Surg. Rs. 185, (213). 32. Hnnign, A. & Wilson, J. B. Evlution of LMP1 of Epstin-Brr virus s thrputi trgt y its inhiition. Mol. Cnr 9, 184 (21). 33. Mlrt-Cols, L. t l. HDMX fols th nsnt p53 mrna following tivtion y th ATM kins. Mol. Cll 54, (214). 34. Lus, C. L., Chnr, A., Njntsv, S., Conliff, A. M. & Okknhug, K. PI3Klt n primry immunofiinis. Nt. Rv. Immunol. 16, (216). 35. Gnin, V. t l. Polysom frtiontion n nlysis of mmmlin trnsltoms on gnom-wi sl. J. Vis. Exp. 87, 514 (214). 36. Guo, H., Ingoli, N. T., Wissmn, J. S. & Brtl, D. P. Mmmlin mirornas prominntly t to rs trgt mrna lvls. Ntur 466, (21). Aknowlgmnts This work ws support y Equip Lllisé l Ligu Contr l Cnr, th Insrm, th RECAMO projts GACR P26/12/G151, MEYS-NPS I-L1413, Cnrforskningsfonn Norr n Cnrfonn S.V.G. ws support y l Ligu Contr l Cnr n S.P. y th ARC n PACRI. W thnk Dr O. Bishof for proviing us mv- n pr onstruts, Dr K. Müngr for mv-hpv16-e7 onstrut n Prof. Dr L.Z. Pnn for pgl2--my promotr-luifrs onstruts. Author ontriutions S.V.G. n S.P. sign n rri out most of th xprimnts. C.D. sign n rri out xprimnts. K.A. rri out xprimnts rlting to trnsgni murin lls n J.B.W. suprvis this spt of th projt n ontriut to n it th mnusript. S.V.G. ssml th MS; R.F. suprvis th projt n ssml th MS togthr with K.N. Aitionl informtion Supplmntry Informtion ompnis this ppr t Compting intrsts: Th uthors lr no ompting finnil intrsts. Rprints n prmission informtion is vill onlin t rprintsnprmissions/ Pulishr's not: Springr Ntur rmins nutrl with rgr to jurisitionl lims in pulish mps n institutionl ffilitions. Opn Ass This rtil is lins unr Crtiv Commons Attriution 4. Intrntionl Lins, whih prmits us, shring, pttion, istriution n rproution in ny mium or formt, s long s you giv pproprit rit to th originl uthor(s) n th sour, provi link to th Crtiv Commons lins, n init if hngs wr m. Th imgs or othr thir prty mtril in this rtil r inlu in th rtil s Crtiv Commons lins, unlss init othrwis in rit lin to th mtril. If mtril is not inlu in th rtil s Crtiv Commons lins n your intn us is not prmitt y sttutory rgultion or xs th prmitt us, you will n to otin prmission irtly from th opyright holr. To viw opy of this lins, visit linss/y/4./. Th Author(s) NATURE COMMUNICATIONS 8: 213 DOI: 1.138/s w