Initiation complex dynamics direct the transitions between distinct phases of early HIV reverse transcription

Transcription

1 Initition omplex dynmis diret the trnsitions etween distint phses of erly IV reverse trnsription Shixin Liu 1,6, Bryn T rd 2,6, Jennifer T Miller 3, Sturt J Le rie 3 & Xiowei Zhung 1,4,5 21 Nture meri, In. ll rights reserved. umn immunodefiieny virus (IV) initites reverse trnsription of its virl RN (vrn) genome from ellulr trn 3 primer. This proess is hrterized y slow initition phse with speifi puses, followed y fst elongtion phse. We report single-moleule study tht monitors the dynmis of individul initition omplexes, omprised of vrn, trn nd IV reverse trnsriptse (RT). RT trnsitions etween two opposite inding orienttions on trn vrn omplexes, nd the prominent pusing events re relted to RT inding in flipped orienttion opposite to the polymeriztion-ompetent onfigurtion. stemloop struture within the vrn is responsile for mintining the enzyme predominntly in this flipped orienttion. Disruption of the stem-loop struture triggers the initition-to-elongtion trnsition. These results highlight the importnt role of the struturl dynmis of the initition omplex in direting trnsitions etween erly reverse trnsription phses. s key step in the life yle of IV, reverse trnsription onverts the single-strnded vrn genome into n integrtion-ompetent doule-strnded DN 1. This multistep retion is tlyzed y the virl enzyme RT, whih initites DN synthesis from the terminus of ell-derived trn 3 nneled to n 18-nuleotide (nt) primer inding site (PBS) in the vrn 2. s prerequisite of reverse trnsription, the trn primer, vrn templte nd RT must ssemle into produtive ternry rionuleoprotein omplex, lled the initition omplex 3 5. Vrious hemil nd enzymti proing ssys hve reveled extensive intermoleulr intertions within this omplex, whih re importnt for the effiieny nd speifiity of the initition of minus-strnd DN synthesis 6 1. The erly stges of reverse trnsription omprise slow, distriutive initition phse followed y fst, proessive elongtion phse 11,12. The initition phse is hrterized y slow DN polymeriztion, rpid dissoition of RT nd frequent kineti puses during primer extension. These pusing events result in the umultion of short extension produts, suh s the trn+3 nd trn+5 intermedites. Notly, fter ddition of the sixth nuleotide to the trn primer, reverse trnsription trnsitions to the elongtion phse with mrkedly inresed polymeriztion rte nd proessivity 13. The unique nd omplex nture of the initition proess mkes it n ttrtive trget for ntivirl drugs. Indeed, it hs een shown tht some RT inhiitors nd RT drug-resistne muttions hve different effets on initition s ompred to elongtion 9. Eluidting the mehnism underlying the initition proess ould therefore set the stge for developing novel inhiitors for omting IV infetion. owever, onsensus on the struture of the initition omplex hs yet to e rehed 7,14,15. urthermore, the dynmis of the omplex nd the mehnism underlying the trnsitions etween vrious phses of erly reverse trnsription remin inompletely understood 13, We reently demonstrted tht RT shows remrkle orienttionl nd trnsltionl dynmis on its nulei id sustrtes 2,21. Suh lrge-sle motions filitte vrious stges of reverse trnsription, inluding DN synthesis, RNse levge nd strnd-displement synthesis. In prtiulr, RT n ind its sustrtes in two opposite orienttions 2,22. In one orienttion, the DN polymerse tive site is positioned over the end of the primer, redy to tlyze primer extension. In the other, RT is flipped ~18 suh tht the DN polymerse tive site is physilly seprted from the primer end nd therey unle to support synthesis. ere, we investigted whether suh orienttionl dynmis regulte the initition of reverse trnsription. To test this hypothesis, we pplied single-moleule fluoresene resonne energy trnsfer () 23,24 nd ensemle primerextension ssys to proe the onformtion nd enzymti tivity of the initition omplex. Notly, we found tht this omplex supports oth the polymerse-ompetent nd the flipped RT inding orienttions. urthermore, the equilirium etween these two inding orienttions evolves s RT elongtes the trn primer, nd hnges in the primer extension tivity orrelte with hnges in the enzyme s inding orienttion. In prtiulr, stemloop struture within the vrn djent to the PBS fores RT to ind predominntly in the flipped, polymeriztion-inompetent orienttion, using reverse trnsription puses. Disrupting this struture y strnd-displement synthesis llows RT to ind in the polymerse-ompetent mode nd triggers the trnsition from initition to elongtion. 1 Deprtment of hemistry nd hemil Biology, rvrd niversity, mridge, Msshusetts, S. 2 rdute Progrm in Biophysis, rvrd niversity, mridge, Msshusetts, S. 3 IV Drug Resistne Progrm, Ntionl ner Institute, rederik, Mrylnd, S. 4 Deprtment of Physis, rvrd niversity, mridge, Msshusetts, S. 5 owrd ughes Medil Institute, mridge, Msshusetts, S. 6 These uthors ontriuted eqully to this work. orrespondene should e ddressed to X.Z. (zhung@hemistry.hrvrd.edu). Reeived 15 June; epted 23 Septemer; pulished online 21 Novemer 21; doi:1.138/nsm.1937 nture struturl & moleulr iology VOLME 17 NMBER 12 DEEMBER

2 21 Nture meri, In. ll rights reserved. igure 1 Single-moleule ssy for proing the struturl dynmis of the initition omplex. () The vrn templte (ornge) is leled with eptor (y5, red str) ner the PBS (lue), nneled to trn primer (gry) nd immoilized to the PE-oted surfe vi streptvidiniotin linkge. The surfe-nhored trn vrn sustrtes re immersed in solution ontining RT (yellow) leled with the donor (y3, green str). The fingers nd RNse domins of RT re indited y nd, respetively. luoresene signl from single trn vrn RT omplexes re deteted using TIR mirosope. () nlysis of RT inding events. Top, the fluoresene signls from y3 (green) nd y5 (red) under 532-nm illumintion nd the signl from y5 diretly exited y 635-nm illumintion (purple). Binding of RT to the sustrte (highlighted y yellow) results in n inrese in the totl fluoresene signls from y3 nd y5 under 532-nm illumintion due to exittion of the donor, ut does not ffet the y5 signl from diret exittion y 635-nm light. Middle, vlues during the inding event. Bottom, histogrm of the inding event. () Sequenes of the vrn nd simple RN PBS templtes studied in this work. (d) Sequenes of the nturl trn 3, syntheti trn (syntrn), nd oligorionuleotide (ORN) nd oligodeoxyrionuleotide (ODN) primers. RESLTS Single-moleule ssy for studying initition omplex dynmis To filitte single-moleule mesurements, we leled speifi sites of the initition omplex with donor nd eptor dyes. irst, we onstruted vrn templte using the sequene orresponding to nuleotides of the IV-1 genome (NL4.3 isolte), with the PBS loted t nuleotides It hs een shown tht the sequene within the initition site of the NL4.3 isolte is representtive of 86% known IV-1 isoltes 14 nd tht sequenes outside this region do not ffet the effiieny of reverse trnsription 25. eptor dye (y5) ws site-speifilly tthed to 28 ner the end of the PBS (ig. 1). The nturl trn 3 primer ws then nneled to the PBS. Next, trn vrn omplexes were nhored to slide surfe nd immersed in retion uffer ontining IV-1 RT leled with donor dye (y3) t its RNse domin (ig. 1). Dye leling of RT nd vrn did not lter reverse trnsription kinetis ppreily (Supplementry ig. 1). luoresene emission from individul trn vrn RT ternry omplexes ws monitored using totl-internl-refletion fluoresene (TIR) mirosope. reely diffusing RT ws oserved to ind nd dissoite from the trn vrn sustrtes in rel time. Eh inding event used n inrese in the totl fluoresene signl due to exittion of the donor dye (ig. 1). vlues reorded during the inding event llowed us to determine the inding onfigurtion of the enzyme. In this sheme, dissoition of the enzyme would result in loss of the totl fluoresene signl, wheres vlue hnge without loss of totl fluoresene ould e interpreted s hnge of inding onfigurtion. In ddition to the trn vrn sustrtes, we lso proed the inding onfigurtion of RT on sustrtes omprising vriety of primer nd templte strutures (ig. 1,d). TIR field y3 15 vrn PBS PE Simple PBS templte vrn PBS PBS y5 trn Streptvidin Biotin Reverse trnsriptse Qurtz slide d luoresene intensity 2, 1, RT dopts two opposite orienttions in initition omplexes It hs een shown tht IV-1 RT n ind to simple primer templte sustrtes in two opposite orienttions 2,22 : in the se of DN primer, RT inds lmost exlusively in the polymerse-ompetent orienttion; in the se of rndom-sequene RN primer nneled to simple DN templte, RT inds in the flipped orienttion tht inhiits primer extension. It is thus interesting to sk whih orienttion RT would dopt on the sustrte formed etween two omplex RN strutures (trn nd vrn), whih supports primer extension despite its RN primer omposition. Should RT ind in the polymerse-ompetent orienttion, we expet to oserve high vlue due to the proximity of the donor nd eptor dyes ording to previous rystllogrphi nd footprinting dt In ontrst, the flipped orienttion, with the donor nd eptor seprted y the 18-se-pir trn PBS duplex, should give low vlue of ~.2.3 (ig. 2). Notly, the inding of RT to the trn vrn omplex gve two distint peks entered t ~.9 nd ~.2 (ig. 2), suggesting tht RT inds in oth the polymerse-ompetent nd flipped orienttions on this sustrte. To onfirm the ssignments of the two sttes, we took dvntge of the knowledge tht ognte deoxyrionuleotides preferentilly stilize RT inding in the polymerseompetent orienttion 2. Indeed, when the distriution ws quired in the presene of the ognte nuleotide (over short durtion to void sustntil primer extension), the equilirium shifted towrd the high- stte (ig. 2), supporting the ssignment of this stte to the polymerse-ompetent inding mode. The two inding modes were lso oserved when the trn 3 primer ws nneled to simple RN templte ontining the PBS sequene without ny seondry strutures (ig. 1 nd Supplementry ig. 2), Time (s).14.7 trn 3 p nti-pbs m 1 m D 5 m ψ D 2 m 5 Tm D m 7 D ψ ψ R S ORN syn-trn nti-pbs D ODN TTTT 1454 VOLME 17 NMBER 12 DEEMBER 21 nture struturl & moleulr iology

3 21 Nture meri, In. ll rights reserved. d Binding orienttion K trn trn mm dtp.2 mm dtp trn trn+5 further supporting the notion tht the two sttes originte from different inding onfigurtions of RT on the trn PBS duplex rther thn inding to regions of vrn outside the PBS. Moreover, the time tre showed spontneous trnsitions etween the ~.9 nd ~.2 sttes within individul inding events (ig. 2), inditing tht RT ould dynmilly trnsition etween the two orienttions without dissoition. We next studied the influene of trn struture on RT inding orienttion y testing sustrtes formed y vrious other primers (ig. 1d) nd the vrn templte. irst, we repled the nturl trn 3 with syntheti, unmodified trn (syn-trn) to test the effet of the modified trn ses. The distriution oserved for the syn-trn vrn RT omplexes ws similr to tht of trn 3 vrn RT (Supplementry ig. 3,). The equilirium onstnt (K) etween the high- nd low- orienttions ws.54 ±.6 for nturl trn 3 nd.64 ±.1 for syn-trn, respetively. Next, we used n 18-nt oligorionuleotide (ORN) primer omplementry to the PBS in ple of trn 3. In this se, RT n still trnsition etween the two orienttions, with the equilirium shifting towrd the high- orienttion with K = 1.8 ±.2 (Supplementry ig. 3). It hs een suggested tht the ntiodon loop of the trn se-pirs with the -rih loop upstrem of the PBS, lthough dt on whether suh intertions exist in the NL4.3 isolte re mixed 14,15,29. Beuse suh intertions would not e present in the se of the ORN primer, our results indite tht the ility of RT trn trn trn trn trn+2 trn+3 trn+5 trn+6 1 Time (s) e Polymeriztion rte (s 1 ) trn trn+1 trn trn+3 trn+5 trn+6 igure 2 RN-dependent DN polymerse tivity of RT orreltes with its inding orienttion in the initition omplex. () rtoon illustrting RT ound to the trn vrn omplex in the polymerse-ompetent (left) nd flipped (right) orienttions. () distriution otined when y3-leled RT inds to y5-leled trn+n vrn omplexes in the presene of 2 μm ognte dntp. In the se of trn (n = ), the distriution in the sene of dntp (red) is lso shown. The distriutions for other n vlues in the sene of dntp re shown in Supplementry igure 5. () Representtive time tre of n RT inding event (highlighted in yellow) shows spontneous trnsitions etween the high- nd low- sttes. (d) The equilirium onstnts K etween the polymerse-ompetent nd the flipped inding orienttions of RT in the presene of 2 μm ognte dntp. Error rs re s.e.m. from t lest three independent experiments. (e) Primer extension rtes on the vrn templte. The primer extension rte is highly orrelted with the inding orienttion equilirium K, showing orreltion oeffiient of.94. Error rs re s.d. from t lest three independent experiments. to dopt nd flip etween the two orienttions does not require these intertions outside the PBS. inlly, when n 18-nt oligodeoxyrionuleotide (ODN) primer ws nneled to the vrn templte, RT ound lmost exlusively in the high-, polymerse-ompetent orienttion (Supplementry ig. 3d). In ddition, the verge inding time of RT ws sustntilly longer on the ODN primer (4 s) thn on the ORN (2. s) nd trn (1.6 s) primers. These results indite tht oth the nture of the nuleotide (deoxyrionuleotide versus rionuleotide) nd the seondry struture of the primer re involved in determining RT s inding orienttion. Polymerse tivity orreltes with RT inding orienttion To investigte how RT s inding orienttion hnges during the initition of minus-strnd DN synthesis, we onstruted series of trn-dn himers, trn+n, representing intermedites enountered t vrying primer extension steps (where n denotes the numer of deoxyrionuleotides dded to the trn terminus) (Supplementry ig. 4). y3-leled RT ws then dded to the y5-leled trn+n vrn sustrtes. ll trn+n vrn RT initition omplexes (n = 6) supported oth high- nd low- inding orienttions (ig. 2 nd Supplementry ig. 5). The equilirium onstnt K etween the two sttes inresed when n ws inresed from to 2 (ig. 2,d nd Supplementry ig. 5). Notly, when trn ws further extended y one extr nuleotide (to trn+3), RT inding shifted sustntilly towrd the flipped orienttion, whih remined dominnt until position trn+5 (ig. 2,d nd Supplementry ig. 5). inlly, when sixth nuleotide ws dded to the trn primer, nother mjor shift ourred, resulting in predominnt polymerse-ompetent inding orienttion for the enzyme (ig. 2,d nd Supplementry ig. 5). nlysis of the flipping rte onstnts indites tht the hnge in the flipping equilirium is modulted primrily y the rte t whih RT trnsitions from the polymerse-ompetent to the flipped orienttion, wheres the reverse rte does not vry sustntilly with the length of the DN extension (Supplementry ig. 5). Intriguingly, the positions t whih the inding orienttion equilirium undergoes mjor hnges (trn+3 nd trn+6) orrespond to mjor trnsitions in primer extension kinetis: polymeriztion shows strong puse t trn+3 nd trnsition from the slow initition phse to the rpid elongtion phse t trn+6 (refs. 11,13). These orreltions rise the possiility tht the DN synthesis tivity of RT during initition my e regulted y its inding orienttion. To test this notion, we performed single-turnover nuleotide inorportion ssys to determine the primer extension tivity of RT on ll retion intermedites used in the single-moleule experiments. nture struturl & moleulr iology VOLME 17 NMBER 12 DEEMBER

4 21 Nture meri, In. ll rights reserved. igure 3 The stem-loop struture upstrem of the PBS uses the mjor puses during initition nd governs the initition-toelongtion trnsition. () Left, rtoon of RT ound to trn+3 simple PBS sustrte. Middle, histogrms for RT ound to trn+3 primers nneled to the vrn templte (lue) nd simple PBS templte (red). Right, rtes of single-nuleotide ddition to vrious trn primers on the simple PBS templte. () Left, rtoon of RT ound to trn+3 vrn h sustrte. Middle, histogrms for RT ound to trn+3 primers nneled to wild-type (WT) vrn (lue) nd vrn h (red) templtes. Right, rtes of singlenuleotide ddition to trn+3 primers nneled to WT vrn (lue) nd vrn h (red) templtes. () Left, rtoon of RT ound to trn+6 vrn h+ sustrte. Middle, histogrms for RT ound to trn+6 primers nneled to WT vrn (lue) nd vrn h+ (red) templtes. Right, rtes of single-nuleotide ddition to trn+6 primers nneled to WT vrn (lue) nd vrn h+ (red) templtes. The nuleotides mutted to rete the vrn h nd vrn h+ onstruts re shown in green. Error rs re s.d. from t lest three independent experiments. Indeed, the primer-extension rte ws losely orrelted with the equilirium etween the two inding orienttions (ig. 2d,e). In prtiulr, RT hd the lowest primer extension tivity on the trn+3 primer, where the enzyme preferentilly ound in the flipped orienttion, nd the fstest extension kinetis on trn+6, where the enzyme predominntly ound in the polymerse-ompetent orienttion. These oservtions suggest tht the polymerse tivity of the enzyme is regulted t lest in prt y its inding orienttion. Stem-loop ner PBS uses reverse trnsription pusing Next we investigted the origin of the puses during erly reverse trnsription, nmely the struturl fetures of the initition omplex responsile for the flipped RT inding orienttion nd slow primer extension rte. The most prominent puse during initition is t position +3, for whih two possile explntory senrios my e hypothesized: (i) the 3-nt DN ddition to the trn primer my onfer on it unique struture (or primer PBS duplex struture) tht disfvors RT inding in the polymerse-ompetent orienttion; (ii) lterntively, the stem-loop struture in the vrn templte 3 nt upstrem of the PBS 14,16 my fore RT to ind in the flipped orienttion nd derese DN synthesis tivity. To distinguish etween these possiilities, we ompred the inding onfigurtion nd primer extension kinetis of RT on the simple PBS templte (ig. 1) to those on the vrn templte. lthough RT preferentilly ound in the low- stte on the trn+3 vrn sustrte, it strongly fvored the high-, polymerse-ompetent orienttion on the trn+3 PBS sustrte (ig. 3). Moreover, the sustntil drop in primer extension rte from the trn+2 to trn+3 position oserved on the vrn templte ws not reprodued on the simple PBS templte (ig. 3). s result, unlike in the se of the vrn templte, no sustntil puse ws deteted t position +3 on the simple PBS templte (Supplementry ig. 6,). These results indite tht the strong +3 puse on the vrn templte is not due to n intrinsi struturl property of the trn+3 primer, ut rther origintes from the templte struture. To test the role of the vrn stem-loop struture in regulting RT s inding dynmis, we reted mutnt, vrn h, y ltering T trn+3 simple PBS trn+3 vrn h trn+6 vrn h+ T T T Simple PBS WT vrn vrn h WT vrn vrn h+ WT vrn Polymeriztion rte (s 1 ) trn trn+1 trn+2 trn+3 trn+5 trn+6 the sequene t positions suh tht the 8 se pirs t the se of the stem-loop ould not form (ig. 3). When RT ws dded to the trn+3 vrn h sustrte, the distriution showed single pek t ~.9 (ig. 3), inditing inding predominntly in the polymerse-ompetent orienttion. This result differs drstilly from the ehvior of RT on the wild-type vrn templte ut mimis its intertion with the simple PBS templte. Moreover, the single-nuleotide inorportion ssy showed tht eliminting the stem-loop struture in the vrn h templte inresed the primer extension rte t the +3 position y ~8-fold ompred to the wildtype vrn (ig. 3), onsistent with previous report tht ltering vrn sequenes upstrem of the PBS n eliminte the +3 puse site 16. Overll, the ove oservtions demonstrte tht the seondry struture of the vrn templte strongly influenes the inding onfigurtion of RT: the stem-loop struture upstrem of the PBS reorients RT into flipped, polymerse-inompetent orienttion nd indues pusing t position +3. onfliting results hve een reported onerning whether the sme +3 puse ours when DN primer is used 11,16. Notly, we found tht the ility of the stem-loop to reorient RT vnished when the trn ws repled y n ODN primer. RT ound predominntly in the polymeriztion orienttion on n ODN+3 vrn sustrte despite the presene of the stem-loop struture (Supplementry ig. 6). urthermore, RT showed muh weker pusing t the +3 position when reverse trnsription ws initited from the ODN primer (Supplementry ig. 6d). This oservtion rules out the possiility tht the strong +3 puse seen with the trn primer is due only to the intrinsilly slower rte of strnd-displement synthesis 3 s the enzyme psses through the vrn stem-loop, whih should our for oth trn nd ODN primers, nd further supports the notion tht the flipped RT orienttion on the trn vrn sustrte imposed y the stem-loop ws mjor determinnt of pusing. Disruption of stem-loop triggers trnsition to elongtion Beuse the stem-loop struture indues strong pusing nd onsequently the slow, distriutive initition phse of DN synthesis, we hypothesize tht disssemly of the stem-loop struture might use.12.6 Polymeriztion rte (s 1 ) Polymeriztion rte (s 1 ) trn+3 WT vrn trn+3 vrn h trn+6 WT vrn trn+6 vrn h VOLME 17 NMBER 12 DEEMBER 21 nture struturl & moleulr iology

5 21 Nture meri, In. ll rights reserved. d trn the trnsition to the elongtion phse tht ours etween trn+5 nd trn+6. Supporting this hypothesis, suh trnsition ws not oserved on the simple PBS templte lking the stem-loop struture, on whih the trn+6 nd trn+5 primers showed similr extension kinetis (ig. 3). If unfolding of the stem-loop struture medited the initition-toelongtion trnsition, we ntiipted tht preventing hirpin disssemly would inhiit entry into the elongtion phse. We therefore reted nother mutnt vrn templte ontining hirpin struture lking ulges (vrn h+ ), whih we expeted to e more stle nd diffiult to disrupt. Indeed, this modifition shifted the inding onfigurtion of RT towrd the low- orienttion (ig. 3). In ddition, the trn+6 vrn h+ sustrte showed primer extension rte tht ws sustntilly slower thn tht of wild-type trn+6 vrn ut ws more similr to those of the initition phse (ig. 3). These results suggest tht entry into the elongtion phse requires disssemly of the stem-loop struture. To further test this notion, we diretly proed the stem-loop struture y pling y3 nd y5 on 132 nd 177 of the vrn, respetively (ig. 4). fully formed hirpin would ring the two dyes into proximity nd generte high vlue, wheres n opened hirpin would fore them prt nd yield low vlue. We then nneled the douly leled vrn templte to vrious e igure 5 IV-1 N destilizes the stem-loop struture. () Left, rtoon of the douly leled trn vrn onstrut omplexed with N (green). pper right, distriutions otined from the douly leled trn vrn sustrte in the sene (red) or presene (lue) of 1 μm N. Lower right, representtive time tre in the presene of 1 μm N. () Left, rtoon of RT ound to trn+3 vrn in the presene of N. Right, distriution otined for y3-leled RT ound to y5-leled trn+3 vrn omplexes in the presene of 1 nm N. () Single-nuleotide extension kinetis of trn+3 vrn omplex in the presene of 2 nm RT nd vrious onentrtions of N. Left, the frtion of trn primers extended y single nuleotide. Right, primer extension rte onstnts. trn trn trn extended igure 4 Disruption of the stem-loop struture upstrem of the PBS ours upon ddition of the sixth nuleotide to the trn primer. () Digrm of the douly leled onstrut used to monitor the folding of the stemloop struture. y3 (green str) nd y5 (red str) re tthed to vrn positions 132 nd 177. ( e) The distriutions otined when 1 nm RT ws dded to the douly leled templte nneled to the trn (), trn+3 (), trn+5 (d) nd trn+6 (e) primers. trn+n primers nd inuted it with unleled RT. In the se of n unextended trn primer, we oserved mjor pek t.95 (ig. 4), refleting folded stem-loop struture. Similr distriutions were oserved for the trn+3 vrn nd trn+5 vrn sustrtes (ig. 4,d), inditing tht the stem-loop ws still lrgely intt fter inorportion of up to five deoxyrionuleotides. In ontrst, in the presene of trn+6, the mjority of omplexes showed low vlue of ~.15 (ig. 4e), inditive of opening of the hirpin struture. Presumly, in the trn+6 vrn RT omplex, ompetition from the terminl three nuleotides of trn+6 destilizes the stem-loop nd, together with the ulges in the upper hlf of the stem-loop, mkes this struture reltively unstle. nfolding of the stem-loop struture explins why RT n engge this sustrte predominntly in the polymerse-ompetent mode (ig. 2,d) nd synthesize DN effiiently (ig. 2e). Role of N in the initition phse of DN synthesis The virl nuleopsid (N) protein hs een reported to filitte vrious stges of IV replition, inluding reverse trnsription 31,32. N is smll, si protein tht nonspeifilly inds nulei ids nd possesses nulei id hperon tivity 32,33. In prtiulr, it hs een suggested tht N ould disrupt templte seondry strutures t whih RT puses during the elongtion phse It is thus interesting to sk whether N ould disrupt the stem-loop struture upstrem of the PBS nd inrese the primer extension rte t the +3 T N T [N] (nm) Extension rte (min 1 ) trn vrn Time (s) trn+3 vrn w/n [N] (nm) w/ N w/o N.3.15 nture struturl & moleulr iology VOLME 17 NMBER 12 DEEMBER

6 Dynmi flipping of RT Nuleotide inorportion stilizes polymerse-ompetent inding Stem-loop reorients RT into the flipped orienttion nd slows DN synthesis Stem-loop opens, resulting in the polymerse-ompetent inding orienttion of RT nd high DN synthesis rte TT TT T T Initition phse Elongtion phse igure 6 Struturl dynmis of the IV-1 initition omplex regulte the erly phses of reverse trnsription. RT inds to the initition omplex in two orienttions polymerse-ompetent orienttion nd flipped, polymerse-intive orienttion. RT spends lrge portion of the time ound to the trn vrn sustrte in the flipped orienttion. ddition of the first ouple of deoxyrionuleotides to the trn primer shifts the RT inding equilirium towrd the polymerse-ompetent orienttion nd inreses the DN synthesis rte. The synthesis rte drops mrkedly t position +3, where RT enounters stem-loop struture in the vrn templte tht fores the enzyme to ind predominntly in the flipped orienttion, therey inresing the proility of pusing. Strnd-displement synthesis until the +6 position eventully leds to unfolding of the stem-loop, whih llows RT to reorient into the polymerse-ompetent inding mode nd enter the fst, proessive elongtion phse of DN synthesis. 21 Nture meri, In. ll rights reserved. position. ddition of N to the douly leled trn vrn omplex rodened the distriution onsiderly (ig. 5). tres of individul initition omplexes reveled flututions mong wide rnge of vlues (ig. 5), suggesting N-medited destiliztion of the stem-loop. reent high-throughput footprinting study lso shows tht the stem-loop upstrem of the PBS is t lest prtilly open in the virus prtile, wheres disruption of the N-vRN intertions restores se-piring 15. With the stem-loop struture disrupted, we ntiipted tht RT would ind primrily in the polymerse-ompetent orienttion. Indeed, when dded to the y5-leled trn+3 vrn sustrte in the presene of N, y3-leled RT ound predominntly in the high-, polymerse-ompetent orienttion, in ontrst to wht ourred without N (ompre ig. 5 with the trn+3 pnel in ig. 2). Intriguingly, despite the disruption of the stem-loop, the ddition of N did not result in ppreile inrese in the extension rte of the trn+3 primer (ig. 5), onsistent with previous oservtions 16,19. onsidering tht N inding my redue inding of RT to the primer templte omplex 36 nd inhiit DN synthesis s result of steri hindrne 37, these negtive effets ould ounterlne its disruption of the stem-loop struture. Indeed, we oserved sustntil redution in the inding frequeny of RT to the trn+3 vrn sustrte in the presene of N in singlemoleule tres (dt not shown), nd N hs een previously found to hve oth positive nd negtive effets on reverse trnsription depending on the experimentl onditions It is worth noting tht N is present t n extremely high onentrtion in the virions nd tht suh ondition is diffiult to simulte in vitro euse of protein ggregtion. uture experiments tht proe the virus diretly will e required to further eluidte whether the initition puses re llevited y N. DISSSION Reverse trnsription of the IV genome is initited from ellulr trn primer ound to the virl RN. The speifi struture nd dynmis of the trn vrn omplex result in n initition phse with properties mrkedly different from those of lter stges of reverse trnsription, providing unique opportunities for nti-iv drug design. It is thus importnt to understnd the orreltion etween the struturl dynmis of the initition omplex nd their funtionl onsequenes. By monitoring the dynmis of individul initition omplexes in rel time, we investigted in detil how IV-1 RT interts with trn vrn sustrtes. Our results revel tht RT dopts two orienttions in the initition omplex polymerse-ompetent orienttion nd flipped orienttion in whih the polymerse tive site is pled mny nuleotides from the end of the trn, preluding primer extension. The equilirium etween these two orienttions regultes RT tivity throughout the initition proess (ig. 6). The reltively slow polymeriztion rtes during initition result in prt from the ft tht RT spends lrge portion of the time ound to the trn vrn sustrte in the flipped, polymerse-inompetent orienttion (ig. 6), though the nuleotide nture of the primer (RN versus DN) nd the seondry struture of the templte, whih determines whether RT hs to undergo strnd-displement synthesis, ould lso ffet polymeriztion kinetis diretly 13,16,17. Notly, the ility to support RT inding in the flipped, polymerse-intive orienttion is unique property of the RN primer. When the trn primer is repled with DN primer omplementry to the PBS sequene, RT inds to the primer templte omplex nerly exlusively in the polymerse-ompetent orienttion, nd slow initition phse is not oserved (Supplementry ig. 6). ddition of the first few nuleotides to the trn primer leds to shift of the RT inding equilirium towrd the polymerse-ompetent orienttion nd n inrese in the DN synthesis rte (ig. 6). This rte then drops drmtilly t position +3, where RT enounters stem-loop struture on the vrn templte tht fores the enzyme to ind predominntly in the flipped, polymerse-intive orienttion, inresing the proility of pusing (ig. 6). RT nnot espe this slow mode of synthesis until it hs synthesized enough DN to disrupt the stem-loop t position +6. Disssemly of the stem-loop llows RT to reorient into the polymerse-ompetent inding mode nd triggers trnsition to the fst, proessive elongtion phse of DN synthesis (ig. 6). The single-strnded ulges within the vrn stem-loop pper to promote the initition-to-elongtion trnsition (ig. 3). Tken together, the ove results provide struturl sis for understnding the erly stges of reverse trnsription nd demonstrte how the struturl dynmis of the initition rionuleoprotein omplex regulte RT tivity. onsidering the ritil role of the stem-loop in regulting RT inding orienttion nd tivity, it is interesting to speulte tht IV my hve evolved this struture to t s temporl rke to slow initition of reverse trnsription. Suh mehnism my help prevent reverse trnsription of the RN genome efore virus udding 39,4, whih is detrimentl to virl infetivity 31,41. This speultion is supported y the oservtions tht oth muttions of the virl N protein tht use premture reverse trnsription nd muttions ner the vrn 1458 VOLME 17 NMBER 12 DEEMBER 21 nture struturl & moleulr iology

7 21 Nture meri, In. ll rights reserved. PBS tht inrese the initition effiieny result in virus replition defets onveniently, the N protein hs n ility to unwind the stem-loop (ig. 5), nd evidene suggests tht this stem-loop is prtilly disrupted within the virus prtile 15. Therefore, this mehnism to inhiit reverse trnsription in the initition phse my e prtilly llevited in mtured virion. On the other hnd, the preursor form of N within the g polyprotein (the form found in infeted ells efore virus udding) ppers to hve weker hperone tivity thn the leved form of N present in mture virions 45, whih my men tht the inhiition mehnism is stronger efore virus udding to prevent premture reverse trnsription. lthough N ws not oserved to inrese the primer extension rte during initition here, it is diffiult to mimi the ext effets of N in vitro. uture in virio nd in vivo experiments my help test these hypotheses. These unique properties of the initition omplex ould mke it n ttrtive trget for the development of nti-iv gents 9. Indeed, initition n e suppressed y oth nuleoside-nlog nd nonnuleoside RT inhiitors, some of whih show higher effiy during initition thn elongtion 46,47. The struturl nd mehnisti understnding of the initition proess demonstrted here ould potentilly help the development of novel ntivirl gents, in prtiulr y designing drugs tht regulte the struturl dynmis of the virl RN nd the inding orienttion of RT, two ftors of ritil importne during erly reverse trnsription. Methods Methods nd ny ssoited referenes re ville in the online version of the pper t Note: Supplementry informtion is ville on the Nture Struturl & Moleulr Biology wesite. knowledgments We thnk J. Wu nd E. ondnzieri for helpful disussions nd R. orelik (Ntionl ner Institute (NI), rederik, Mrylnd, S) for providing N proteins. This work is supported in prt y the S Ntionl Institutes of elth (NI; M to X.Z.) nd the Intrmurl Reserh Progrm of the enter for ner Reserh, NI (to S..J.L..). B.T.. ws supported y NI/Ntionl Institute of enerl Medil Sienes Moleulr Biophysis Trining rnt (M8313 to the rvrd Biophysis Progrm). X.Z. is owrd ughes Medil Institute investigtor. TOR ONTRIBTIONS S.L., B.T.. nd X.Z. designed the experiments; S.L. nd B.T.. performed the experiments nd nlyzed the dt; S.L., B.T.. nd X.Z. interpret the dt nd wrote the pper; J.T.M. mde the enzyme nd some of the trn onstruts; S..J.L.. ontriuted to disussion, dt interprettion nd mnusript preprtion. OMPETIN INNIL INTERESTS The uthors delre no ompeting finnil interests. Pulished online t Reprints nd permissions informtion is ville online t reprintsndpermissions/. 1. Telesnitsky,. & off, S.P. Reverse trnsriptse nd the genertion of retrovirl DN. in Retroviruses (eds. offin, J.M., ughes, S.. & Vrmus,.E.) (old Spring ror Lortory Press, 1997). 2. 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Rodríguez-Rodríguez, L., Tsuhihshi, Z., uentes,.m., Bmr, R.. & y, P.J. Influene of humn immunodefiieny virus nuleopsid protein on synthesis nd strnd trnsfer y the reverse trnsriptse in vitro. J. Biol. hem. 27, (1995). 35. Wu, W. et l. umn immunodefiieny virus type 1 nuleopsid protein redues reverse trnsriptse pusing t seondry struture ner the murine leukemi virus polypurine trt. J. Virol. 7, (1996). nture struturl & moleulr iology VOLME 17 NMBER 12 DEEMBER

8 36. Ji, X., Klrmnn,.J. & Preston, B.D. Effet of humn immunodefiieny virus type 1 (IV-1) nuleopsid protein on IV-1 reverse trnsriptse tivity in vitro. Biohemistry 35, (1996). 37. rohmnn, D., odet, J., Mely, Y., Drlix, J.L. & Restle, T. IV-1 nuleopsid trps reverse trnsriptse on nulei id sustrtes. Biohemistry 47, (28). 38. Tnhou, V., us,., Rogemond, V. & Drlix, J.L. ormtion of stle nd funtionl IV-1 nuleopsid omplexes in vitro. J. Mol. Biol. 252, (1995). 39. Lori,. et l. Virl DN rried y humn immunodefiieny virus type 1 virions. J. Virol. 66, (1992). 4. Trono, D. Prtil reverse trnsripts in virions from humn immunodefiieny nd murine leukemi viruses. J. Virol. 66, (1992). 41. Zhng,., Dorndul,., Orenstein, J. & Pomerntz, R.J. Morphologi hnges in humn immunodefiieny virus type 1 virions seondry to intrvirion reverse trnsription: evidene inditing tht reverse trnsription my not tke ple within the intt virl ore. J. um. Virol. 3, (2). 42. ouzet, L. et l. Nuleopsid muttions turn IV-1 into DN-ontining virus. Nulei ids Res. 36, (28). 43. Thoms, J.., Boshe, W.J., Shtzer, T.L., Johnson, D.. & orelik, R.J. Muttions in humn immunodefiieny virus type 1 nuleopsid protein zin fingers use premture reverse trnsription. J. Virol. 82, (28). 44. Beerens, N., root,. & Berkhout, B. Initition of IV-1 reverse trnsription is regulted y primer tivtion signl. J. Biol. hem. 276, (21). 45. Wu, T. et l. undmentl differenes etween the nulei id hperone tivities of IV-1 nuleopsid protein nd g or g-derived proteins: iologil implitions. Virology 45, (21). 46. ooker,.w., Lott, W.B. & rrih, D. Inhiitors of humn immunodefiieny virus type 1 reverse trnsriptse trget distint phses of erly reverse trnsription. J. Virol. 75, (21). 47. Rigourd, M. et l. Inhiition of the initition of IV-1 reverse trnsription y -zido--deoxythymidine. omprison with elongtion. J. Biol. hem. 275, (2). 21 Nture meri, In. ll rights reserved. 146 VOLME 17 NMBER 12 DEEMBER 21 nture struturl & moleulr iology