The role of capsid maturation on adenovirus priming for sequential uncoating

The Role o Capsid Ma u a ion on Adeno i us P iming o Sequen ial Uncoa ing * □ S Recei ed o publica ion, June 8, 2012, and in e ised o m, July 11, 2012 Published, JBC Pape s in P ess, July 12, 2012, DOI 10.1074/jbc.M112.389957 Ana J. Pe´ ez-Be na´ ‡1 , Al a o O ega-Es eban §2 , Rosa Mene´ndez-Coneje o ‡3 , Dennis C. Winkle ¶ , Ma ga i a Mene´ndez 储 , Alasdai C. S e en ¶ , S. Jane Flin **, Ped o J. de Pablo § , and Ca men San Ma ín ‡4 F om he ‡ Depa men o Mac omolecula S uc u e, Cen o Nacional de Bio ecnología, and § Depa men o Physics o Condensed Ma e , Uni e sidad Au o´noma de Mad id, 28049 Mad id, Spain, he 储 Ins i u o de Química Física Rocasolano and CIBER de En e medades Respi a o ias, 28006-Mad id, Spain, he ¶ Labo a o y o S uc u al Biology, NIAMS, Na ional Ins i u es o Heal h, Be hesda, Ma yland 20892, and he **Depa men o Molecula Biology, P ince on Uni e si y, P ince on, New Je sey 08544 Backg ound: Adeno i us p o eoly ic ma u a ion is equi ed o co ec uncoa ing in he cell. Resul s: Ma u a ion makes he i ion me as able and acili a es pen on and pe iphe al co e p o ein elease, as well as coope - a i e genome ejec ion. Conclusion: P ecu so p o eins ac as sca olds a o ing assembly. Ma u a ion p imes adeno i us o uncoa ing. Signi icance: Iden i ying he molecula de e minan s o i us s abili y and uncoa ing is key o unde s anding he in ec ious cycle. Adeno i us assembly concludes wi h p o eoly ic p ocessing o se e al capsid and co e p o eins. Imma u e i ions con aining p ecu so p o eins lack in ec i i y because hey canno p ope ly uncoa , becoming apped in ea ly endosomes. S uc u al s ud- ies ha e shown ha p ecu so s inc ease he ne wo k o in e ac- ions main aining i ion in eg i y. Using di e en biophysical echniques o analyze capsid dis up ion in i o, we show ha imma u e i ions a e mo e s able han he ma u e ones unde a a ie y o s ess condi ions and ha ma u a ion p imes adeno- i us o highly coope a i e DNA elease. C yoelec on omog- aphy e eals ha unde mildly acidic condi ions mimicking he ea ly endosome, ma u e i ions elease pen ons and pe iphe al co e con en s. A highe s ess le els, bo h ma u e and imma- u e capsids c ack open. The i us co e is comple ely eleased om c acked capsids in ma u e i ions, bu i emains con- nec ed o shell agmen s in he imma u e pa icle. The ex a s abili y o imma u e adeno i us does no equa e wi h g ea e igidi y, because in nanoinden a ion assays imma u e i ions exhibi g ea e elas ici y han he ma u e pa icles. Ou esul s ha e implica ions o he ole o p o eoly ic ma u a ion in ade- no i us assembly and uncoa ing. P ecu so p o eins a o assembly by es ablishing s able in e ac ions wi h he app op i- a e cu a u e and p e en ing p ema u e ejec ion o con en s by igh ly sealing he capsid e ices. Upon ma u a ion, co e o ga- niza ion is loose , pa icula ly a he pe iphe y, and in e ac ions p ese ing capsid cu a u e a e weakened. The capsid becomes b i le, and pen ons a e mo e easily eleased. Based on hese esul s, we hypo hesize ha changes in co e compac ion du ing ma u a ion may inc ease capsid in e nal p essu e o igge p ope uncoa ing o adeno i us. Adeno i us is a mild pa hogen o humans bu can become clinically ele an in immunocomp omised pa ien s, and i is widely examined as a he apeu ic ec o (1, 2). The la ge (⬃950 Å) nonen eloped, pseudo T ⫽25 icosahed al capsid is assem- bled om a leas 11 di e en ypes o p o eins. The gene al icosahed al a chi ec u e can be desc ibed as wo di e en sys- ems o iles. Nine ime s o he majo coa p o ein, hexon, o m hecen alpla eo each ace ,knownas heG oupo Nine (GON). 5 The i epe ipen onalhexon ime s, oge he wi h he pen on base, o m he second ile sys em, known as G oup o Six (GOS) (3). T ime ic ibe s p o ude om each e ex. Mino coa p o eins IIIa, VI, VIII, and IX a e equi ed o co ec capsid assembly and occupy speci ic posi ions in he capsid (3). Polypep ide IX has an ex ended s uc u e and o ms a so o hai ne on he ou e su ace o he i ion, keeping oge he he hexon ime s in each GON and binding GONs o GONs ac oss he icosahed al edges. Each GON is u he s a- bilized by copies o polypep ide VIII loca ed a ound he icosa- hed al 3- old symme y axis inside he capsid. Also on he inne capsid su ace, he N- e minal domain o polypep ide IIIa media es he in e ac ion be ween pen on base and he pe ipen- onal hexons o keep each GOS oge he . Finally, IIIa and VIII coope a e o bind each GOS o i s i e su ounding GONs. The *Thiswo kwassuppo ed,in wholeo in pa ,byNa ional Ins i u eso Heal h G an s GM037705 and AI1058172 ( o S. J. F.). This wo k was also suppo ed by Minis y o Science and Inno a ion o Spain G an s BFU2010-16382/ BMC ( o C. S. M.), MAT2008-02533, PIB2010US-00233, and FIS2011-29493 ( o P. J. P.), FIS2010-10552-E and FIS2011-16090-E ( o C. S. M. and P. J. P.), and BFU2009-10052 ( o M. M.), and by Local Mad id Go e nmen G an P2009/MAT-1467 ( o P. J. P.). □ S This a icle con ains supplemen al Figs. S1–S6, Table S1, and Mo ies S1–S7. 1 Recipien o Juan de la Cie a Pos doc o al Con ac JCI-2009-05187 om he Minis y o Science and Inno a ion o Spain and ecipien o addi ional suppo om Spain CSIC T a el G an PA1002892. 2 Recipien o FPU p edoc o al ellowship om he Minis y o Educa ion o Spain. 3 Recipien o P edoc o al Fellowship FI08/00035 om he Ins i u o de Salud Ca los III o Spain. 4 To whom co espondence should be add essed: Cen o Nacional de Bio ec- nología. Da win 3, 28049-Mad id, Spain. Tel.: 34-91-5855450; Fax: 34- 91-5854506; E-mail: [email p o ec ed]. 5 The abb e ia ions used a e: GON, G oup o Nine; DSC, di e en ial scanning calo ime y; BIR, b eakage/inden a ion a io; GOS, G oup o Six; AVP, ade- no i us p o ease; p, i us pa icles; PI, p opidium iodide; AFM, a omic o ce mic oscopy; EF, ex insic luo escence; N, new on. THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 287, NO. 37, pp. 31582–31595, Sep embe 7, 2012 Published in he U.S.A. 31582 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 287•NUMBER 37•SEPTEMBER 7, 2012 This is an Open Access a icle unde he CC BY license. emaining mino capsid p o ein, polypep ide VI, could no be aced in his s udy, bu i has been assigned o densi y wi hin he in e nal ca i y o each hexon ime (4, 5). The adeno i us co e con ains a single copy o he dsDNA genome and o e 25 MDa o p o ein, including ou DNA-binding i al p o eins: polypep idesV,VII, ␮ ,and he e minal p o ein. A ew copies o he adeno i us p o ease (AVP) a e also packaged (6). The dis- posi ion o DNA and p o eins in he co e is unclea . Adeno i us cell en y has been ex ensi ely s udied using g oup C human adeno i uses; hese a e mos commonly used o ec o de elopmen , in pa icula he highly homologous ypes 5 (HAdV-5) and 2 (HAdV-2) (7, 8). Adeno i us uncoa ing in he cell p oceeds in a s epwise manne (9). The i us s a s o disassemble a he plasma memb ane, whe e upon binding o i s ecep o some ibe s a e eleased (10), and he pen on base unde goes a con o ma ional change ha migh esul in weak- ening i s in e ac ions wi h he es o he capsid (11). The i us is hen in e nalized, and disassembly con inues in he ea ly endosome wi h elease o some in e nal componen s, such as mino coa p o eins IIIa, VI, VIII, and co e polypep ide V (9, 12). This is a c ucial s ep o in ec ion, as polypep ide VI plays he key ole o al e ing he endosomal memb ane o acili a e i us escape o he cy osol (13). The pa ially disassembled i ion does no p oceed o la e s ages o he endocy ic pa hway bu escapes he ea lyendosome(14).A his poin he emus be a ce ain deg ee o dsDNA exposu e o ini ia e in lamma o y esponses (15), bu he i ion is s ill s able enough o su i e anspo along mic o ubules o he nuclea po e (16, 17). The e, he inal phase o uncoa ing akes place. Upon comple e disman ling o he weakened capsid, he i us DNA, oge he wi h majo co e p o ein VII, en e s he nucleus (18–21). Co ec adeno i us uncoa ing is igh ly linked o ma u a ion. Clea age o se e al s uc u al p o eins by AVP is equi ed o yield he ma u e in ec ious i ion (22). Imma u e i ions con- ain he p ecu so e sions o coa p o eins IIIa, VI, and VIII and o co e p o eins VII, ␮ , and e minal p o ein. An expe i- men al model o imma u e adeno i us is he HAdV-2 s1 mu an (23). When g own a he nonpe missi e empe a u e o 39 °C, HAdV-2 s1 does no package AVP (24) and p oduces capsids con aining he unp ocessed p o ein p ecu so s. Vi al genomepackagingisunimpai ed,bu he i usisno in ec ious. I has been shown ha he de ec in in ec i i y is linked o a de ec in uncoa ing. Imma u e s1 a aches o he hos cell and ollows he same in e naliza ion p ocess as he wild- ype (WT) i us bu ails o escape he endosome and is ecycled o he memb ane o deg aded in lysosomes (14, 25). C yo-EM s uc u al s udies showed ha in he imma u e i us, he p esence o unclea ed p ecu so s ein o ces he ne - wo k o in e ac ions holding he i ion oge he a h ee le els. Fi s , he i us co e is mo e compac and s able due o he condensing ac ion o unp ocessed polypep ides pVII and p e- ␮ ; second, a “molecula s i ch” o med by pIIIa and pVIII unde pins he bounda y be ween GOS and GONs (5, 26); and hi d, he e is a mo e o de ed in e ac ion be ween pVI and he inne ca i y o each hexon in he capsid (4, 5). To u he in es- iga e how he p esence o unclea ed p ecu so s ansla es in o a de icien uncoa ing pheno ype, we ha e compa ed he in i o s abili y and disassembly o s1 and WT i ions unde di e en ypes o s ess: he mal, chemical, o mechanical. We epo he di e ences be ween ma u e and imma u e i ions and discuss hei implica ions o assembly and uncoa ing. EXPERIMENTAL PROCEDURES Vi us P oduc ion and Pu i ica ion—We used as wild- ype con ol o ma u e i ions he E1-dele ed HAdV-5 a ian Ad5GL (27), p opaga ed in HEK293 cells. Ad5GL is comple ely WT o all s uc u al polypep ides. Imma u e i us was ob ained by p opaga ing he HAdV-2 s1 mu an in HeLa cells a 39.5 °C, as desc ibed (5). Pa icles we e pu i ied by equilib- ium cen i uga ion in CsCl g adien s, desal ed on a Bio-Rad 10 DC column, and s o ed in 20 mMHepes, pH 7.8, 150 mMNaCl plus 10% glyce ol a ⫺70 °C. Vi us i e s we e 5 ⫻10 12 i us pa icles ( p)/ml o Ad5GL, and 1 ⫻10 13 p/ml o s1. Di e en ial Scanning Calo ime y—DSC measu emen s we e pe o med using a Mic ocal VP-DSC ins umen (Mic o- cal, Inc., No hamp on, MA) a a hea ing a e o 60 °C/h and unde an ex a cons an p essu e o 2 a m o p e en degassing du ing he scan. The VP- iewe package was used o da a acquisi ion. Samples o WT and s1 a ⬃5–7 ⫻10 12 p/ml we e dialyzed agains 8 mMNa 2 HPO 4 ,2mMKH 2 PO 4 , 150 mMNaCl, and 0.1 mMEDTA, pH 7.4. The e e ence cell was loaded wi h bu e om he las dialysis, which was also used o un he bu e -bu e baseline. Appa en excess hea capaci y cu es we e ob ained a e sub ac ion o he bu e -bu e baseline om he expe imen al hea capaci y p o iles and di ision by hexon mola concen a ion. The O igin-DSC so wa e package (7.0 e sion) was used o da a analysis. The excess hea capaci y p o iles we e heo e i- cally analyzed in e ms o independen , i e e sible wo-s a e ansi ions (N i 3D i ) acco ding o Equa ion 1 (28), ⌬Cpex共T兲⫽ 冘 i⫽1 6⌬Hi appEi app RTmi 2⫻exp 冉 Ei app共T⫺Tmi兲 RTmi 2 冊 ⫻exp 冉 ⫺exp 冉 Ei app共T⫺Tmi兲 RTmi 2 冊冊 (Eq. 1) whe e ⌬Cp ex (T) is he excess hea capaci y alue a empe a- u e T;T mi is he empe a u e o he maximum o ansi ion i; ⌬H i app and⌬E i app a e heappa en calo ime ic en halpy change and he ac i a ion ene gy o he same ansi ion, and Ris he gas cons an . Fluo escence Spec oscopy—Ex insic luo escence spec a we e ob ained om i us p epa a ions subjec o di e en kinds o s ess: high empe a u e, py idine, o acidic pH. Ma u e and imma u e i us samples (5 ⫻10 10 p/ml) we e incuba ed in di e en bu e s, depending on he expe imen . The mal dis up ion expe imen s we e ca ied ou in 8 mM Na 2 HPO 4 ,2mMKH 2 PO 4 ,150 mMNaCl, and 0.1 mMEDTA, pH 7.4. Fo pH dis up ion expe imen s, he sample was dilu ed and subsequen ly dialyzed o e nigh a 4 °C agains bu e s a he app op ia e alues o pH: HCl/KCl in he 1–2.5 pH ange, ci ic acid/sodium ci a e a pH 3–6, and Na 2 HPO 4 /KH 2 PO 4 a pH ange 6.5–7.5, always wi h 150 mMNaCl and 0.1 mMEDTA. Fluo escence emission spec a we e ob ained employing a Hi achi Model F-2500 FL spec opho ome e equipped wi h a How Ma u a ion P imes Adeno i us o Uncoa ing SEPTEMBER 7, 2012•VOLUME 287•NUMBER 37 JOURNAL OF BIOLOGICAL CHEMISTRY 31583 Pel ie empe a u e con ol de ice. 1 mMp opidium iodide (PI) (Molecula P obes) was added o he sample ha was hen allowed o equilib a e o 5 min be o e da a acquisi ion. Sample olumes o 0.150 ml we e used in sealed qua z cu e es. The sample was exci ed a 535 nm, and luo escence emission was moni o ed om 580 o 700 nm, using exci a ion and emission sli wid hs o 8 nm. Raw spec a we e co ec ed by sub ac ion o he PI spec um a each es ed condi ion. The a io o PI luo escence o he ini ial emission (I/I 0 ) a he maximum posi- ion ( ␭ max ⫽607 nm) ⫾S.E. o h ee independen expe imen s was plo ed as a unc ion o s ess condi ion alues. Fluo es- cence in ensi y changes we e i ed o a sum o sigmoids using he O igin so wa e package, acco ding o Equa ion 2, I/I0⫽A1⫹B1⫺A1 1⫹exp 冉 C1⫺x C1D1 冊 ⫹ 冘 i⫽2 n 冢 Bi⫺1⫹Bi⫺Bi⫺1 1⫹exp 冉 Ci⫺1⫺x CiDi 冊 冣 (Eq. 2) whe e A i and B i a e he lowe and uppe pla o m alues o each sigmoid (no ice ha s a ing om he second sigmoid, he lowe pla o m A i is o ced o coincide wi h he p e ious uppe pla o m B i⫺1 ); C i is he ansi ion midpoin ; and D i is he slope. The numbe o sigmoid cu es in he summa o y (up o n⫽3) was he minimum necessa y o i he expe imen al cu es, based on bes R 2 alues. The luo escence he mal-dis up ion cu e was also i ed in e ms o he model used o analyze he calo ime ic cu es using Equa ion 3, I/I0共T兲⫽IF⫺共IF⫺1兲⫻ 冘 i⫽1 3 i ⫻exp 冉 ⫺exp 冉 Ei app共T⫺Tmi兲 RTmi 2 冊冊 (Eq. 3) whe e I F is he inal alue o I/I 0 , and i is he ela i e con ibu- ion o ansi ion i o he o al a ia ion o PI luo escence (29). Nega i e S aining Elec on Mic oscopy—Fo imaging o dis- assembly p oduc s, samples ea ed as desc ibed o luo es- cence spec oscopy we e adso bed, a a concen a ion o 5 ⫻ 10 11 p/ml, on o glow-discha ged, collodion/ca bon-coa ed EM g ids, nega i ely s ained wi h 2% u anyl ace a e, and obse edinaJeol 1200EX-II ansmission elec on mic oscope. Pe cen ages o in ac , damaged, and collapsed capsids we e quan i ied by coun ing he numbe o pa icles o each ype pe mic og aph. E o ba s e e o di e ences be ween di e en mic og aphs. C yoelec on Tomog aphy—Vi al samples subjec o di e - en kinds o s ess we e mixed a a concen a ion o 1 ⫻10 12 p/ml wi h 10-nm colloidal gold pa icles (AURION, Wagenin- gen, The Ne he lands) and i i ied as desc ibed (30). A Tec- nai-12 elec on mic oscope (FEI, Hillsbo o, OR) ope a ing a 120 kV wi h a LaB6 sou ce and equipped wi h an ene gy il e (GIF 2002; Ga an, Pleasan on, CA) was used o collec il se ies a ⬃4- ␮ m unde ocus, co e ing he ange ⫾70° in 2° inc e- men s. The da a we e acqui ed using he Se ialEM package (31), o a o al dose o ⬃70–80 elec ons/Å 2 pe se ies. Images we e eco ded on a 2048 ⫻2048-pixel CCD came a (Ga an, Pleasan on, CA) a ⫻38,500 magni ica ion (7.8 Å/pixel). Da a we e p e-p ocessed and aligned using he IMOD so wa e package (32), wi h gold pa icles as iducial ma ke s. The inal aligned il se ies we e no malized and econ- s uc ed using he simul aneous i e a i e econs uc ion ech- nique implemen ed in Tomo3D (33). Indi idual i us pa icles we e ex ac ed om omog ams using IMOD. Tomog ams we e denoised by 100 i e a ions o aniso opic nonlinea di u- sion (34). The in-plane esolu ion o he ull omog ams was in he 43–55-Å ange, as es ima ed using he Bso p og am TOMORES (35). Dihed al angles we e measu ed om cen al slices o single i ion omog ams using he angle measu emen ool in Adobe Pho oshop. A i ion was conside ed o conse e ibe s i a leas one ibe was clea ly isible in he denoised omog am. The p esence o absence o pen ons was de e mined manually by obse a ion o single i ion omog am slices a e alignmen wi h an icosahed al h ee-dimensional map as a e e ence, and compa ison wi h model maps o ull and pen onless i ions. Radial p o iles we e calcula ed om a e age i us omog ams calcula ed a e aligning each indi idual map wi h an icosahe- d al e e ence. Icosahed al symme y was conside ed o align- men bu no imposed in he inal a e aged map o in he indi- idual aligned maps, excep o supplemen al Fig. S6. Alignmen was ca ied ou using maximum likelihood p oce- du es o omog aphy as implemen ed in XMIPP (36, 37). Nanoinden a ion Assays—S ocks o WT and s1 i us in HBS bu e (20 mMHepes, 150 mMNaCl, pH 7.8) we e dilu ed in a solu ion o NiCl 2 in HBS o ob ain a inal solu ion wi h 5 mMo Ni 2⫹ , and adso bed on o eshly clea ed Musco i e mica (V-1 quali y). A d op o 20 ␮ l o he inal solu ion, con aining i us pa iclesa concen a ions be ween 1.5and 2 ⫻10 12 p/ml,was deposi ed on he subs a e and incuba ed o 30 min a 4 °C be o e washing wi h 5 mMNiCl 2 in HBS. The AFM ip was p ewe ed wi h 30 ␮ l o he same bu e . The mica was placed on he AFM sample holde and imme sed in 500 ␮ l o bu e . The AFM (Nano ec Elec o´nica S.L., Mad id, Spain) was ope - a ed in Jumping Mode Plus (38) in liquid using ec angula can- ile e s RC800PSA and Biole e s (BL-AC40TS) (Olympus, Tokyo, Japan) wi h nominal sp ing cons an s o 0.05 and 0.03 N/m, espec i ely. Can ile e sp ing cons an s we e ou inely calib a ed by using he Sade me hod (39). AFM explo a ion a o ces o ⬃100 pNindica ed a andom popula ion o i ions on he mica su ace. Single i us pa icles we e de o med by he AFM ip by pe - o ming indi idual inden a ion ( o ce e sus z-piezo displace- men (FZ)) expe imen s. To ensu e ha he inden a ion is pe - o med a he op o he i us, he i us shell is zoomed in con inuously by educing he x-y scanning size un il he bump o he e y op is unde he whole piezo scan. Then a la ge o ce inden a ion was execu ed a he op o he pa icle. Finally, an image o he i us was aken o con i m i s dis up ion. The maxi- mum o ce applied du ing each inden a ion was high enough o How Ma u a ion P imes Adeno i us o Uncoa ing 31584 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 287•NUMBER 37•SEPTEMBER 7, 2012 ensu e ha he i uspa iclesa edes oyed.The FZ speed is abou 60 nm/s (40). Images we e p ocessed using he WSxM so wa e (41). RESULTS The mal Capsid Dis up ion—To di ec ly de ec he ene gy equi ed o dis up hecapsid and o moni o he s abili y o he in e media es o med along he hea -induced disassembly p ocess, DSC was used. DSC scans o ma u e (WT) and imma- u e ( s1) adeno i us a physiological ionic s eng h and neu al pH a e shown in Fig. 1A. As expec ed o a la ge, complex mac- omolecula specimen, he mal dena u a ion o bo h WT and s1 adeno i us was i e e sible upon hea ing abo e 90 °C. Bo h he - mog ams show h ee well de ined ansi ions (deno ed as T2, T3, andT4inTable1)p ecededbyab oadendo he mbe ween36and 65 °Cin he s1 mu an and h eedisc e epeaksinWT ha ex end o e a simila ange o empe a u e (Fig. 1A,inse ). Indeed, decon- olu iono he hea capaci y p o iles showed ha heo e allendo- he m o bo h WT and s1 adeno i us can be desc ibed in e ms o six i e e sible wo-s a e ansi ions whose appa en ansi ion en halpies, ac i a ion ene gies, and T m alues a e summa ized in Table 1. The wolas peaks o he he mog ams(T m aluesa 71.7 and 78.4 °C o WT and 72.8 and 81.0 °C o s1) ha e p e iously been assigned o dena u a ion o he pu i ied hexon p o ein (42), which is he mos abundan componen (⬎60%) o he p o ein shell. The mino ansi ion (T 2 ) appea ing a 67.3 °C o WT and 68.9 °C o s1 seems o be ela ed o hexon s abiliza- ion upon GON o ma ion, as i is absen in polypep ide IX- dele ed ecombinan i uses (42). The h ee low empe a u e ansi ions (deno ed as T 1A ,T 1B , and T 1C in Table 1) may co - espond o capsid disassembly and e lec changes unde gone by adeno i us du ing uncoa ing in he cell. Howe e , whe he hey a e di ec ly due o dis up ion o hexon in e ac ions o e lec dena u a ion o o he i ion componen s canno a p esen be asce ained.All ansi ionsoccu eda highe empe a u es o s1 han o WT, wi h di e ences in he 1–2 °C ange o all T m alues excep T 1A and T 1B , whe e di e ences we e as high as 5 °C. These di e ences e eal an inc eased he mal s abili y o hexon and o he i ion s uc u es in he imma u e capsid con ex . FIGURE 1. The mal capsid dis up ion. A, he mog ams showing WT and s1 he mally induced e en s upon i us hea ing a a cons an a e o 60 °C/h (10 mM phospha ebu e , 150mMNaCl,0.1 mMEDTA,pH 7.4). Thesolid line is he heo e ical i o heexpe imen al en elopes oi e e sible wo-s a e ansi ionsusing pa ame e s gi en in Table 1. The inse shows he decon olu ion o peaks likely co esponding o capsid disassembly. B, ex insic luo escence measu emen s showingDNA exposu e o sol en uponhea ing. A e age alues ande o ba s co esponding o he s anda dde ia ion o h eeindependen expe imen sa e plo ed. Long dashed lines co espond o he i in e ms o he model used o he calo ime ic cu es. Sho dashed lines co espond o he i acco ding o a mul iple sigmoid unc ion. C, his og am showing s uc u al e ec s o hea ing on he i us pa icles, as analyzed by nega i e s aining. Ca ego ies a e as ollows: I, in ac i ions; D, damaged pa icles, including a ious deg ees o dis up ion bu e aining some deg ee o cu a u e; F, la ened capsids. E o ba s indica e s anda d de ia ion in he pa icle coun s be ween di e en mic og aphs. The o al numbe o pa icles in he da ase , N, is indica ed a he op igh co ne . A–C,blacksymbols andlines e e oWT i us;g ay symbols andlines e e o s1.D, galle yshowing exampleso in ac ,damaged,and la enedcapsids. Thescale ba co esponds o 100 nm. A ows indica e missing e ex pen ons; a owheads indica e missing GOSs. How Ma u a ion P imes Adeno i us o Uncoa ing SEPTEMBER 7, 2012•VOLUME 287•NUMBER 37 JOURNAL OF BIOLOGICAL CHEMISTRY 31585 To de ine he condi ions in which he pa icle unde goes pa ial disassembly and he DNA is exposed, we measu ed he DNA accessibili y o sol en by using he DNA in e cala ing dye PI. PI luo escence is quenched un il he i us capsid is comp omised and enhanced upon binding o DNA when i becomes accessible (43). As we ha e shown p e iously (5), sim- ila deg ees o DNA exposu e occu ed a highe empe a u es o s1 han o WT (Fig. 1B). EF measu emen s we e i ed o mul iple sigmoidal cu es wi h pa ame e s shown in Table 2. WT p esen s a luo escence bu s a 47.5 °C. The sha p slope o he cu e a his empe a u e indica es a highly coope a i e p ocess, also obse ed o WT o ms o HAdV-2 and HAdV-5 a ⬃45 °C (5, 44, 45). In con as , all ansi ions showed low coope a i i y in s1. EF da a we e also analyzed using he i e e sible ansi ion model used o he DSC cu es (Fig. 1B), and he bes i ing pa ame e s a e compa ed wi h hose de i ed om he hea capaci y p o iles in Table 3. The T m alues ob ained om EF expe imen s o WT closely ag eed wi h hose deduced om DSC (Table 3), indica ing ha capsid p o ein dena u a ion (DSC) and DNA exposu e (EF) e en s a e co ela ed. T ansi- ion o e lapping in he imma u e i us impai ed a eliable anal- ysis o he EF cu e, as a ound 90% o he o al emission a ia- ioncanbeappa en lydesc ibedbya single ansi ion wi h a T m alue o 57.0 °C. In oduc ion o a hi d ansi ion app oached he T m alues o he DSC es ima es bu inc eased signi ican ly he e o s o he he mal pa ame e s wi hou imp o ing he R 2 alue. S uc u alchanges due o he maldisassemblywe e ollowed by EM. We had p e iously obse ed ha WT capsids we e comple ely dis up ed and hei con en s eleased a 47 °C, whe eas he s1 i ion only los e ex s uc u es (pen ons and pe ipen onal hexons, o GOS) and ex uded pa o i s con en s as a single nucleop o ein ilamen (5). He e, we epo a quan- i a i e desc ip ion o capsid dis up ion pa e ns a di e en empe a u es. WT and s1 p epa a ions we e imaged by nega i e s aining EM a e hea ing a he empe a u es co esponding o T m al- ues de i ed o WT. To in e p e he EM obse a ion, we g ouped he obse ed pa icles in o h ee ca ego ies (Fig. 1C): in ac i ions (I); damaged capsids, s ill e aining a sphe ical a angemen (D); and collapsed, la ened capsids (F). Fig. 1D shows examples o pa icles in he h ee ca ego ies (see also supplemen al Fig. S1). A he T 1A ansi ion empe a u e (40 °C), mos s1 i ions we e in ac , whe eas a conside able p opo ion (⬃30%) o WT capsids showed small de ec s con- sis en wi h loss o pen ons (Fig. 1D). The ac ha he in e io o hese capsids does no appea da k indica es ha hey s ill con ain s ain-excluding ma e ial (p o ein/DNA), in ag eemen wi h hemodes inc ease inDNAaccessibili y oPI indica edby EF. A he T 1B ansi ion empe a u e (47 °C), only 10% o WT i ions e ained hei s uc u al in eg i y, wi h he es showing di e en deg ees o damage. In pa icula , abou 40% o he WT i ions appea as collapsed la s uc u es, wi h he icosahe- TABLE 1 The mal capsid dis up ion pa ame e s om decon olu ion o DSC endo he ms 1 ⌬Huni s a e kilocalo ies/mol o hexon monome (720 hexon monome s/ i ion). TABLE 2 EF analysis o he mal capsid dis up ion Fi ing o he mal dis up ion EF da a o mul iple sigmoid cu es. Bold ace indica es ansi ions wi h high coope a i i y. WT s1 Midpoin Slope Midpoin Slope °C °C Sigmoid 1 38 ⫾2 2.8 ⫾0.5 41 ⫾1 1.1 ⫾0.1 Sigmoid 2 48 ⴞ1 170 ⴞ10 54 ⫾18⫾4 Sigmoid 3 58 ⫾34⫾1 65.0 ⫾0.4 4.6 ⫾0.6 R 2 0.99482 0.99483 How Ma u a ion P imes Adeno i us o Uncoa ing 31586 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 287•NUMBER 37•SEPTEMBER 7, 2012 d ons spli open, and la ge de ec s a he e ices consis en wi h loss o comple e GOS. Imma u e s1 i ions, howe e , a e no la ened; hey e ain he capsid sphe ical a angemen al hough hey ha e los comple e GOS. A 57 °C, he as majo i y (⬃80%) o WT capsids a e la ened, while mos s1 i ions s ill e ain cu a u e, e en when ex ensi ely damaged. Al hough capsid la ening is mos likely an a i ac induced by he nega i e s aining p epa a ion, he di e ences ound be ween WT and s1 i ions sugges ha capsid p ecu so s s eng hen he seams a he icosahed al edges, p ese ing cap- sid cu a u e. A empe a u es beyond 57 °C, only isola ed cap- some s o small capsid agmen s a e obse ed o bo h WT and s1. Chemical Capsid Dis up ion—Nex , we subjec ed he adeno- i us ma u e and imma u e capsids o di e en sou ces o chemical s ess. Imma u e s1 i ions showed highe s abili y han WT bo h in he p esence o dena u an compounds and, mos in e es ingly, upon acidi ica ion. A c i ical poin in adeno i us uncoa ing in i o is acidi ica- ion wi hin he ea ly endosome, whe e he luminal pH anges be ween 6.5 and 6 (46). Upon acidi ica ion, he inc ease o PI luo escence o WT, indica i e o DNA accessibili y, p oceeds in a biphasic way (Fig. 2A). The i s highly coope a i e ansi- ion is cen e ed a pH 6.1 and s a s as he medium acidi y app oaches he endosomal ange o pH (Table 4). In con as , he imma u e i us wi hs ood mo e se e e alues o pH (Fig. TABLE 3 Compa ison be ween capsid he mal dis up ion pa ame e s ob ained om DSC and calcula ed om EF spec oscopic cu es using he same i e e sible ansi ion model T 1A T 1B T 1C T m1A E 1A app 1A T m1B E 1B app 1B T m1C E 1C app 1C °C kcal/mol °C kcal/mol °C kcal/mol WT DSC 40.0 ⫾0.3 130 ⫾30 47.4 ⫾0.2 61 ⫾6 57.2 ⫾0.3 55 ⫾6 Spec oscopy 40 ⫾625⫾10 0.23 47.8 ⫾0.1 ⬎10 3 0.30 57 ⫾238⫾10 0.47 s1 DSC 45.1 ⫾0.4 57 ⫾5 52.3 ⫾0.7 55 ⫾10 60 ⫾140⫾10 Spec oscopy R 2 ⫽0.998 (42 ⫾40) (21 ⫾10) (0.15) (55.9 ⫾0.4) (90 ⫾40) (0.44) (60 ⫾10) (40 ⫾70) (0.41) R 2 ⫽0.997 39 ⫾339⫾20 0.1 57.0 ⫾0.2 48 ⫾4 0.9 R 2 ⫽0.996 56.8 ⫾0.2 40 ⫾1 1.0 FIGURE 2. Chemical capsid dis up ion: e ec o acidi ica ion. A, ex insic luo escence measu emen s showing DNA exposu e o sol en upon acidi ica ion. A e age alues and e o ba s co esponding o he s anda d de ia ion o h ee expe imen s a e plo ed. Dashed lines co espond o he i acco ding o a mul iple sigmoid unc ion. The shadowed a ea indica es he pH ange in he ea ly endosome. B, his og am showing s uc u al e ec s o acidi ica ion on he i us pa icles, as analyzed by nega i e s aining. Ca ego ies a e as ollows: I, in ac i ions; D, damaged pa icles, including a ious deg ees o dis up ion bu e aining some deg ee o cu a u e; F, la ened capsids. E o ba s indica e s anda d de ia ion in he pa icle coun s be ween di e en mic og aphs. The o al numbe o pa icles in he da ase , N, is indica ed a he op igh co ne .Aand B,black symbols and lines e e o WT i us; g ay symbols and lines e e o s1. C, galle y showing examples o in ac , damaged, and la ened capsids. Pa icles whe e he s aining agen pene a es a e indica ed wi h a s a . Pa icles lacking la ge capsid agmen s a e indica ed wi h a ows. The scale ba co esponds o 100 nm. How Ma u a ion P imes Adeno i us o Uncoa ing SEPTEMBER 7, 2012•VOLUME 287•NUMBER 37 JOURNAL OF BIOLOGICAL CHEMISTRY 31587 2A) and he s1 cu e showed, appa en ly, only a b oad ansi- ion cen e ed a pH 4.1, which is nea he midpoin o he sec- ond WT ansi ion. A pH alues below 3, bo h i uses p esen he same beha io . A highly coope a i e ansi ion leading o almos comple e DNA exposu e was also obse ed upon WT capsid dis up ion by he dena u an agen py idine (supple- men al Fig. S3 and supplemen al Table S1). EM analysis (Fig. 2Band supplemen al Fig. S2) indica ed ha a pH 6 o e 80% o he s1 pa icles we e in ac bu mo e han 70% WT i ions al eady showed damage. Again, comple e col- lapse and la ening we e a ely obse ed o s1 and WT. The same endency ega ding la ening was obse ed o dis up- ion by py idine (supplemen al Fig. S3). Acidi ica ion damage p oduced capsids wi h da k cen e s, indica ing pene a ion o he s aining agen . Howe e , no e i- den gaps indica ing loss o pen ons we e obse ed, meaning ha he capsid de ec allowing DNA exposu e o PI and u anyl ace a e is small and o low occu ence in he icosahed al ne - wo k. Addi ionally, new kinds o disassembly in e media es we e obse ed: pa icles wi h issu es and pa icles ha had los capsid agmen s o di e en sizes and shapes (“chunks”) bu s ill e aining he polyhed al cu a u e (Fig. 2C). These kinds o images a e consis en wi h he capsid eleasing he s ess imposed by acidi ica ion ia c acks a hexame ic posi ions, ins ead o by eleasing e ex s uc u es (47). In e es ingly, 74% o WT damaged pa icles a pH 6 (55% o e o al numbe o pa icles) we e s ain- illed capsids. In s1, howe e , dis up ion ia issu es and chunk elease was mo e abundan : 56% (46% o he o al) o s1 damaged pa icles co - esponded o his ca ego y a pH 4.5, and 60% (58% o he o al) a pH 3.5. This di e ence in dis up ion pa e ns sugges s ha he de ec in he WT capsid ha allows s ain pene a ion is he physiologicalpa h o pa ialuncoa ingin heendosome, a he han hedis up ion ia c acks.Howe e , om henega i e s ain images, i was no clea exac ly wha his de ec was, as no clea pa e n o los capsome s (e.g. e ices) was obse ed. C yoelec on Tomog aphy o Disassembly In e media es— DSCand EFp o idebulkin o ma ion on changesunde goneby he specimen, bu hey do no e eal changes in indi idual i i- ons. Imaging disassembled i ions by nega i e s ain p o ided single i ion images; howe e , in e ac ion wi h he ca bon sup- po and he acidic s aining agen , plus d ying and la ening, may esul in addi ional changes no di ec ly ela ed o he s ess condi ions being es ed. Fu he mo e, in nega i e s ain images only a wo-dimensional p ojec ion o he h ee-dimen- sional i ion is ob ained, and i is no possible o obse e he in e nal s uc u es o he pa icle. To ob ain a be e pic u e o he s uc u al changes occu ing du ing capsid dis up ion, we used c yoelec on omog aphy. Fi s , we in es iga ed he changes in he s uc u e o WT i uses a he s esso condi ions whe e he i s ansi ion was obse ed in DSC o EF assays and whe e s1 i ions emained mos ly unal e ed (Figs. 1–3 and supplemen al Mo ies S1–S3): a 40 °C o pH 6.0. In bo h cases, omog ams showed ha cap- sids conse ed o a la ge ex en hei s uc u al in eg i y, including a ached ibe s (Fig. 3Aand supplemen al Fig. S5). Howe e , he aspec o he co e di e ed be ween con ol and s essed i uses. Incon olcondi ions (25 °C,pH7.4), he i ion in e io p esen ed a smoo h, la densi y wi h g ay le els simila o hose o he capsid shell. In i uses incuba ed a pH 6, he co e densi y appea ed mo e he e ogeneous and sligh ly weake han ha o he capsid. This same di e ence, some imes mo e p onounced,was obse edin he co eo i useshea ed a 40 °C. A adial a e age p o ile calcula ed om aligned indi idual i us omog ams indica ed ha , o bo h ypes o mild s ess, he densi y o he ou e egion o he co e was lowe han in con ol i uses (Fig. 3B). This change in elec on densi y is compa ible wi ha elaxa ion o he DNA-p o ein complex o loss o pe iph- e al co e ma e ial. No dec ease in co e densi y was obse ed o s1 i ions a pH 6 (supplemen al Fig. S4). Nex , we sea ched o capsid de ec s ha would accoun o he inc ease in DNA exposu e obse ed in EF assays. Slice-by- slice analysis o indi idual i ion omog ams indica ed ha bo h mild hea ing and acidi ica ion esul ed in loss o pen ons (Fig. 3Cand supplemen al Fig. S5). Howe e , he deg ee o loss was di e en depending on he kind o s ess; acidi ied i uses los only one o wo pen ons, whe eas hea ing p oduced he elease o h ee o ou pen ons (Fig. 3D). Concomi an ly wi h a la ge loss o pen ons, ewe ibe s we e obse ed in hea -dis up ed WT i ions, al hough upon dis up ion a pH 6 mo e han 70% i ions s ill conse ed ibe s (supplemen al Fig. S5), i.e. mild acidi ica ion by i sel is no causing massi e ibe elease. Occasionally, bo h a 40 °C o a pH6,asho (25–30nmlong and 4–5nmwide)s e ch o DNA was seen o p o ude ou o he missing pen on posi ion (sup- plemen al Fig. S6). Howe e , his was an ex emely a e e en (less han 5% o analyzed i uses unde mild s ess condi ions). Tomog ams o WT and s1 i ions subjec o high s ess con- di ions (hea ing a 47 °C o acidi ica ion a pH 5; also pH 4 o s1) we e also ob ained. A 47 °C, mos WT i ions appea ed as emp y b oken shells wi h missing GOS (Fig. 4A,supplemen al Fig. S5 and supplemen al Mo ies S4 and S5). Only in a ew cases some loose co e s uc u es emained a ached o capsid ag- men s. Mos imma u e s1 i ions had also los GOS and in some cases we e c acked open; howe e , he co e emained a ached o c acked capsids and in a compac ed s a e. We ha e p e iously epo ed ha s1 i ions dis up ed a 47 °C ex ude a nucleop o ein ilamen (5). Howe e , no such ilamen s we e obse ed in c yoelec on omog ams, al hough hey a e com- mon occu ences in nega i e s aining (5) o pla inum/ca bon shadowing p epa a ions (da a no shown). We conclude ha ilamen ex usion is induced by he EM p epa a ion condi- ions,namelyacombina ion o adso p ion o hesuppo , s ain- ing wi h low pH u anyl sal s o shadowing, and d ying. TABLE 4 EF analysis o capsid dis up ion induced by acidi ica ion Fi ing o EF da a o mul iple sigmoid cu es. Bold ace indica es ansi ions wi h high coope a i i y. WT s1 Midpoin (pH uni s) Slope Midpoin (pH uni s) Slope Sigmoid 1 6.1 ⴞ0.1 48 ⴞ54.4 ⫾0.3 7.4 ⫾0.3 Sigmoid 2 4.1 ⫾0.4 5.4 ⫾0.9 R 2 0.99951 0.99916 How Ma u a ion P imes Adeno i us o Uncoa ing 31588 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 287•NUMBER 37•SEPTEMBER 7, 2012 A conside able p opo ion o s1 capsids emained in ac a low pH alues (mo e han 90% a pH 6 and up o 60% a pH 4; supplemen al Fig. S5). No ably, ibe s (which a e no di ec ly in ol ed in p o eoly ic p ocessing) emain a ached o imma- u e i ions e en unde condi ions as ha sh as pH 4 (supple- men al Fig. S5). Upon high acidi ica ion, WT shells had gaps consis en wi hlosso GOSandwe eonly a elyseenincon ac wi hanyco e emains. Con e sely, s1 capsidsonly los pen ons and c acked open, whe eas co es emained a ached o shell agmen s and in a condensed s a e (Fig. 4Band supplemen al Mo ies S6 and S7). Unlike o nega i e s aining EM, no la ened capsids we e obse ed in c yoelec on omog aphy images, con i ming ha la ening was a p epa a ion a i ac . Howe e , measu emen o dihed al angles in omog ams indica ed ha in high s ess con- di ions he cu a u e o dis up ed capsids depa ed om he icosahed al alue (Table 5 and Fig. 4C) in bo h WT and s1 bu wi h a mo e no iceable e ec in WT. We conclude ha la - ened capsids obse ed by nega i e s aining in WT co espond in solu ion o emp y de o med capsids wi h educed cu a u e ein o cemen s ha become la upon adso p ion o ca bon s aining and d ying. In s1, loss o cu a u e is no so p o- nounced, and capsids a e less p one o la ening du ing he nega i es aining p ocedu e. In e es ingly,cu a u echa ac e - is ics a e p ese ed in s1 e en in condi ions whe e la ge e ex s uc u es (GOS) a e los (47 °C). This obse a ion sugges s ha he igh e in e ac ions be ween capsid and compac ed co e es ablished by p ecu so p o eins play a ole in de e mina- ion and main enance o capsid cu a u e du ing assembly. Fo bo h WT and s1, a small p opo ion o pa icles ha appea ed as in ac capsids was ound o lack some pen ons a e close inspec ion (supplemen al Fig. S5, class “P”). When hese FIGURE 3. C yoelec on omog aphy analysis o s uc u al changes induced by mild s ess in WT adeno i us. A, ep esen a i e sec ions o omog ams showing WT i us in con ol condi ions, 40 °C o pH 6, as indica ed. The ba ep esen s 100 nm. Some isible ibe s a e labeled wi h a owheads.B, adial densi y p o iles o a e age maps calcula ed om 22 indi idual i us omog ams o each case. The cen al sec ion o each a e age map is shown a igh .C, example sec ions o indi idual i us omog ams showing pen on loss. Equi alen sec ions o a model lacking pen ons, and o he ull i ion icosahed al map, a e shown o compa ison. Model maps a e low pass- il e ed a 50 Å esolu ion. Red dashed ci cles indica e he posi ion o he e ex. D, his og am showing numbe o pen ons los pe i ion. No pen on loss was obse ed in an equi alen da ase o con ol i uses. 33 indi idual i ion omog ams (396 e ices) we e analyzed o each condi ion. How Ma u a ion P imes Adeno i us o Uncoa ing SEPTEMBER 7, 2012•VOLUME 287•NUMBER 37 JOURNAL OF BIOLOGICAL CHEMISTRY 31589 pa icles we e aligned and a e aged, adial p o iles indica ed ha he WT co e densi y had dec eased no only in he pe iph- e y bu also in he cen e o he i ion, i.e. co e elaxa ion o componen loss inc eased wi h inc easing s ess le els. Con- e sely, co e densi y in s1 i ions lacking some pen ons emained unal e ed, e en a pH 5 o 47 °C (supplemen al Fig. S4). Mechanical Capsid Dis up ion—AFM nanoinden a ion assays allow explo ing i us s abili y unde mechanical s ess. A e loca ing an indi idual in ac i us on he su ace (Fig. 5A), he can ile e pushes he i ion a he e y op o he pa icle. Can- ile e bending is eco ded as a unc ion o zdisplacemen , hus elici ing he i us de o ma ion as a unc ion o he e ical o ce (48). Du ing he i s s ages o inden a ion, he i us pa icles unde go a linea de o ma ion ha is e lec ed by a sus ained climbing in he o ce inden a ion cu e (Fig. 5, Cand D) and p o ides he sp ing cons an o he i us k (49). A a ce ain poin , he cu e endency sha ply changes, and he o ce sud- denly s a s dec easing due o capsid ailu e. The poin whe e endency changes de e mines he b eaking o ce. Finally, an image o he i us is aken o con i m i s dis up ion (Fig. 5B). In he expe imen al condi ions used he e, i uses p e e en ially a ached o he mica in a 3- old o ien a ion. We eco ded da a om single nanoinden a ions on 25 WT (Fig. 5C) and 28 s1 (Fig. 5D) pa icles. F om he esul s ob ained in condi ions o he mal o chemical s ess, we would ha e expec ed o ind he ma u e capsid mo e agile and so e han he imma u e one. On he con a y, WT i ions p esen ed bo h highe b eaking o ce (3.3 ⫾0.2 nN) and s i ness (0.46 ⫾ 0.02 N/m) han s1 pa icles (2.3 ⫾0.2 nN and 0.38 ⫾0.04 N/m). Fu he analysis o inden a ion cu es can supply ex a in o ma ion abou he co e. F om he expe imen al cu es in FIGURE 4. C yoelec on omog aphy analysis o s uc u al changes induced by high s ess. A, ep esen a i e sec ions o omog ams showing WT and s1 i us a 47 °C; B, a pH 5. The ba ep esen s 100 nm. Black a ows indica e missing GOSs; s a s indica e co e emnan s in open capsids; whi e a ows indica e capsid-co e con ac s; black a owhead indica es a angen ial iew o a capsid c ack. C, analysis o capsid cu a u e. Top panel, schema ics showing examples o dihed al angles on cen al sec ions o indi idual i us omog ams. Middle and bo om panels, his og ams o angle alues o WT and s1 con ol, in ac i ions; and o dis up ed capsids a 47 °C o pH 5. TABLE 5 S a is ics o dihed al angles in dis up ed capsids unde high s ess A o al o 30 indi idual i us omog ams we e examined o each condi ion. The numbe o angle measu emen s n o each case is indica ed. Condi ion WT s1 Pe ec icosahed on 138° 138° Con ol 137 ⫾3° (n⫽180) 136 ⫾3° (n⫽180) 47 °C 125 ⫾13° (n⫽160) 133 ⫾8° (n⫽170) pH 5 132 ⫾14° (n⫽168) 134 ⫾11° (n⫽169) How Ma u a ion P imes Adeno i us o Uncoa ing 31590 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 287•NUMBER 37•SEPTEMBER 7, 2012