V IROLOG ICAS IN ICA2014 DO I10 .1007 /s12250 -014 -3459 -1
RESEARCHART ICLE C r i t ica lro leo fDengueV i rusNS1p ro te ininv i ra lrep l ica t ion 1 ,2 1 J ing j ingFan1,2,Y iL iu ,Zh im ingYuan
1 .KeyLabo ra to ryo fAg r icu l tu ra landEnv i ronmen ta lM ic rob io logy , WuhanIns t i tu teo fV i ro logy ,Ch inese Academyo fSc iences , Wuhan430071 ,Ch ina 2 .Un ive rs i tyo ftheCh ineseAcademyo fSc iences ,Be i j ing100039 ,Ch ina Denguev i rus(DENV )nons t ruc tu ra lp ro te in1(NS1 )isah igh lyconse rved46 -kDap ro te intha t con ta ins2g lycosy la t ions i tes(Asn -130and Asn -207 )and12conse rvedcys te ine(Cys )res idues . He re , wepe r fo rmeds i te -d i rec ted mu tagenes istogene ra tesys tema t ic mu tan tso fv i ra ls t ra inTSV01 . Theresu l tso fthesubsequen tana lys isshowedtha tana lan inesubs t i tu t iona tthesecond N l inked g lycan Asn -207in NS1de layedv i ra l RNAsyn thes is ,reducedv i rusp laques ize ,and weakenedthe cy topa th ice f fec t .Th ree mu tan tsa tCyss i tes(Cys -4 ,Cys -55 ,Cys -291 )andaC te rm ina lde le t ion(ΔC ) mu tan ts ign ifican t lyimpa i red RNAsyn thes is ,andconsequen t lyabo l ishedv i ra lg row th , whe reas a lan ine mu ta t ionsa tAsn -130and G lu -173resu l tedinpheno typestha t we res im i la rtothe w i ld type (WT )v i rus .Fu r the rana lys isshowedtha tthe Asn -207 mu ta t ions l igh t lyde layedv i ra lrep l ica t ion . Theseresu l tssugges ttha ttheth reeconse rvedd isu lfidebondsandthesecond N l inkedg lycanin NS1a rerequ i redfo rDENV -2rep l ica t ion . KEYWORDS denguetype2v i rus ;NS1 ;rep l ica t ion ;g lycosy la t ions i te
INTRODUCT ION D engu ev i ru s(DENV )i son eo fs ev e r a lg lob a l lyim po r t an ta r th ropod -bo rn ep a thog en sw i th inth eg enu s F la v i v i ru s. D engu ein f e c t iono c cu r sm a in lyint rop i c a l andsub t rop i c a lcoun t r i e sinSou th e a s tA s i a ,th eP a c ific , andth eAm e r i c a s .Inr e c en td e c ad e s ,th eannu a lav e r ag e in c id en c eo fd engu ein f e c t ion sh a sin c r e a s edd r am a t i c a l ly ; wo r ldw id e ,i ti se s t im a t edth a tth e r ea r eov e r100 m i l l ionc a s e sannu a l ly ,l e ad ingto~500 ,000ho sp i t a l i z a t ion sw i thac a s ef a t a l i tyr a t eo f5%(Ky l eJ ,2008 ; WHO , 2012 ) .Th e r ea r efou ran t ig en i c a l lyd i s t in c ts e ro typ e s (DENV -1 ,-2 ,-3 ,and-4 ) ,andep id em io log i c a ls tud i e s ov e ra20 -y e a rp e r iodh av ed e t e rm in edth a t DENV -2i s th e mo s tp r ev a l en ts e ro typ e(Ho angL ,2010 ;V aughn D , 2000 ) .Un t i lr e c en t ly ,th ep a thog en i cm e ch an i smandth e in t e r a c t ion sb e tw e en DENVandho s tf a c to r sh av eb e en in comp l e t e lyund e r s tood . Rece ived :1 1Ap r i l2014 ,Accep ted :16 May2014 Pub l ishedon l ine :26 May2014 Co r respondence : Phone :86 -27 -87197242 ,Fax :86 -27 -87198120 , Ema i l :yzm@wh . iov .cn
©W IV ,CASandSp r inge r -Ve r lagBe r l inHe ide lbe rg2014
Th eg enom eo f DENVi sapo s i t iv es ing l e s t r and ed RNAo fapp rox im a t e ly1 1kbinl eng th , wh i chcon t a in s as ing l eop enr e ad ingf r am eth a tcod e sfo rth r e es t ru c tu r a lp ro t e in s( c ap s id[C ] ,m emb r an e[M ] ,andenv e lop e [E ] )ands ev ennon s t ru c tu r a lp ro t e in s(NS1 , NS2A /B , NS3 , NS4A /B ,and NS5 ) .Th e NS1p ro t e ini sah igh ly con s e rv ed46 -kD ag ly cop ro t e in ,con t a in ing12h igh ly con s e rv edcy s t e in e(Cy s )r e s idu e sth a tfo rm6in t e rmo l e cu l a rd i su l f id ebond s ,and2 N l ink edg ly co sy l a t ion s i t e s(C h am b e r sT ,1 9 9 0 ) .NS 1i sc o t r a n s l a t i o n a l l yt r a n s lo c a t edin toth eendop l a sm i cr e t i cu lum(ER )lum enby th et r an s -m emb r an es ign a ls equ en c ea tth eC t e rm inu s o fth eEp ro t e in(M a ck en z i eJ ,1996 ;P ryo rM ,1994 ; W e s t aw ayE ,1997 ) .Th eg ly co sy l a t ions t a tu so f NS1 i simpo r t an tfo ri t spo lym e r i z a t ionandd i s t r ibu t ion , wh i cho c cu r sinth eERlum en ,andfu r th e r mod ific a t ion o c cu r r edinth e Go lg in e two rk wh e r eacomp l ex typ e sug a ri sadd edtoth e N130g ly c anbu tth e N207g ly c an i sr e t a in edinth eh igh m anno s e(F a lgou tB ,1995 ;P ryo r M ,1994 ;W ink l e rG ,1988 ) .I th a sb e enfoundth a t NS1 lo c a l i z e sw i th inth eER m emb r an e ,fo rm sahomod im e r wh i chi sr equ i r edfo rt r an spo r tth roughth e Go lg ianda p a r t i a l lyhyd rophob i cn a tu r ek e ep s NS1s t ay m emb r an e a s so c i a t ed . NS1a l soc ans t ay w i th inc e l lu l a rv e s i cu l a r 1
Mu ta t ionso fDENVNS1a f fec tv i ra lrep l ica t ion
comp a r tm en tb e fo r ea t t a ch ingtoth ec e l lsu r f a c eand th enf in a l lyb e ings e c r e t edf rom m amm a l i anc e l l s , p r edom in an t lya sah ex am e r(F l am and M ,1999 ;P ryo r M ,1994 ;W ink l e rG ,1989 ) . NS1c anb ed e t e c t edinth e s e r ao fin f e c t edp a t i en t s(upto50μg /mL ) ,w i thh igh e r l ev e l sb e ingp r e s en tintho s e whod ev e lop edd engu e h emo r rh ag i cf ev e r(DHF )r a th e rth and engu ef ev e r(DF ) , ind i c a t ingth a ts e c r e t ed NS1 m aycon t r ibu t etoth es e v e r i tyo fin f e c t ioninp a t i en t s(A l conS ,2002 ;Y oungP , 2000 ) . Du r ingd engu ein f e c t ion , NS1c ane l i c i tas t rong humo r a limmun er e spon s e ,enh an c ein f e c t ioninh ep a to c y t e s ,a n dp r om o t ee n d o t h e l i a lc e l ld am a g eb yf a c i l i t a t i n g imm u n ec om p l e xf o rm a t i o n(A v i r u t n a nP ,2 0 0 6 ;C h a n gH , 2002 ;L inC ,2003 ;SunD ,2007 ) .I tc ana l soan t agon i z e comp l em en ta c t iv a t ionbyb ind ingf a c to rH ,anda l soC4 andC1 s ,th e r ebyd ep r e s s ingcomp l em en ta c t iv a t ionand p ro t e c t ing DENVf romd i r e c tcomp l em en t -d ep end en t n eu t r a l i z a t ion(A v i ru tn anP ,2010 ;ChungK ,2006 ;Fu ch s A ,2010 ) . Th econ s e rv a t iono f12 Cy sr e s idu e ssugg e s t sth a t th e s er e s idu e sa r eimpo r t an tin NS1 mu l t im e r i z a t ion andfun c t ion .I th a sb e enp ropo s edth a t mu t a t ion so f g ly co sy l a t ions i t e sandcon s e rv edCy sr e s idu e sa r ea s so c i a t ed w i thd e c r e a s edv i ru sg row th ,r edu c edp l aqu e s i z e ,d e l ay edcy top a th i ce f f e c t ,andimp a i r ed NS1s e c r e t ion(P ryo rM ,1993 ;P ryo rM ,1994 ) . How ev e r ,th e e f f e c t so fth e mu t a t ion sth a tw e r eex am in edbyd i f f e r en t g roup sapp e a r edtob ein con s i s t en tinc e l lcu l tu r e(T a j im a S ,2008 ) ;andth ed eg ly co sy l a t iono fN l ink eds i t e sl e ad s tod iv e r s eph eno typ e sw i thr e sp e c tto mou s en eu rov i ru l en c e ,fo l low ingf l av iv i ru sin f e c t ion(Muy l a e r tI ,1996 ; P l e tn evA ,1993 ;P ryo rM ,1998 ; Wh i t em an M ,2010 ) .A eC t e rm in a lendo f p r ev iou ss tudya l soind i c a t edth a tth t h ep r o t e i ni sim p o r t a n ti nNS 1d im e rf o rm a t i o n(P r y o rM , 1993 ) . B e c au s eth ee f f e c to f NS1onv i r a lr ep l i c a t ionand v i ru l en c er em a in sun c l e a r ,w einv e s t ig a t edth efun c t ion s o ftheCy sr e s idu e sando fth eC t e rm in a lo f NS1inth e v i r a ll i f ecy c l e .Inth ep r e s en ts tudy ,w ed emon s t r a t eth a t ana l an in e mu t a t iona tA sn -207d e l ay sv i r a lr ep l i c a t ion andth ecy top a th i ce f f e c t ,andl e ad stor edu c edp l aqu e s i z e .Th r e eCy s s i t e mu t a t ion sandaC t e rm in a ld e l e t ion (ΔC )e a chcomp l e t e lyabo l i shv i r a lg row thandr ep l i c a t ion , wh e r e a s mu t a t ion sa tA sn -130and G lu -173l e adto aph eno typ eth a ti ss im i l a rtoth a to fth e WTv i ru s .Th e r e su l t sp rov id ev a lu ab l ein s igh t sin toth ero l eo f NS1in v i r a lin f e c t ionandcou ldgu id eth ed ev e lopm en to fnov e l an t iv i r a lth e r ap i e sfo rd engu ep a t i en t s . MATER IALSAND METHODS Ce l lsandv i ruses BHK -21(B abyh am s t e rk idn ey )and V e ro(A f r i c an 2
g r e en monk eyk idn ey )c e l l s( Inv i t rog en ,C a r l sb ad ,CA , USA )w e r ep rop ag a t edin Du lb e c co ’ s mod i f i edE ag l e ’ s m ed ium(DMEM ,G ib co ,G r andI s l and , NY ) ,supp l e m en t ed w i th10%(w /v )f e t a lbov in es e rum(FBS )and t 100 Up en i c i l l in s t r ep tomy c in /mL ,in5%(v /v ) CO2a 37 °C .S to ck so f DENV -2v i ru s e sw e r eg rownin V e ro c e l l sw i th2%(w /v )FBS ,andth ev i r a lt i t e rw a sc a l cu l a t edw i thp l aqu ea s s ay sonBHK -21c e l l s . P lasm idcons t ruc t ion DENV -2g enom e l eng thcDNAin f e c t iou sc lon e scon t a in ingv a r iou s mu t a t ion sw e r econ s t ru c t edbyu s inga mod ifi edpACYC -DENV2 -TSV f1v e c to r(L inS ,201 1 ) . Ar e comb in an tR en i l l alu c i f e r a s e r epo r t ingr ep l i con ,pA CYC -TSV -R lu c2A -R ep ,w a su s edtocon s t ru c t mu t a t iv e r ep l i con sfo rv i r a lr ep l i c a t ionan a ly s i s .D r .P e i -Y ongSh i p rov id eda l lo fth eo r ig in a lv e c to r s(Nov a r t i sIn s t i tu t e fo rT rop i c a lD i s e a s e s ,S ing apo r e ) . NS1 mu t a t ion s(C4A , C55A , C291Aand N130A , N207A )andaΔC w e r e ob t a in edbys t and a rdov e r l ap -po lym e r a s ech a inr e a c t ion (PCR ) .Th eov e r l ap -PCRf r agm en t sw e r ein s e r t edin to th e DENV -2TSV01in f e c t iou sc lon eandr ep l i conu s ing twoun iqu er e s t r i c t ionen zym e s ,B s rG I(nu c l eo t id epo s i t ion2103 )and M luI(nu c l eo t id epo s i t ion4571 ) .A l l con s t ru c t sw e r ev e r ifi edbyDNAs equ en c ing . RNAt ransc r ip t ionandt rans fec t ion Th ein f e c t iou scDNAc lon e sw e r el in e a r i z ed w i th C la I .Th eg enom e l eng thandr ep l i con RNA sw e r e p rodu c ed w i thl in e a r i z edcDNAu s inga mMESSAGE mMACH INET7k i t(Amb ion ,TX , USA ) ,a c co rd ing toth em anu f a c tu r e r ’ sin s t ru c t ion s .App rox im a t e ly5μg .8 RNA sw e r et r an s f e c t edin toBHK -21c e l l s(8×106)in0 mLco ldpho sph a t e -bu f f e r eds a l in e(PBS ,pH7 .5 )by e l e c t ropo r a t ionu s ingaG en epu l s e rapp a r a tu s(B io -R ad , H e r cu l e s , CA , USA ) . As e t t ingo f0 .85kVand25μF w a su s ed ,pu l s ingth r e et im e sa t3 sin t e rv a l s ,fo l low ed bya10 -m inr e cov e ryp e r ioda troomt emp e r a tu r e .Th e t r an s f e c t edc e l l sw e r ed i lu t edin25 mL DMEMcon t a in ing10%(w /v )FBS ,and w e r eth enin cub a t edina a t37 °C( J i aF , T -75fl a skfo r5 -7dund e r5%(v /v )CO2, 2013 ) . Ind i rec tImmunofluo rescenceassay( IFA ) App rox im a t e ly3×105V e roc e l l sw e r es e ed edon to s l id e sina6 -w e l lp l a t eandth enin f e c t ed w i th WTand m u t a n tv i r u s e sa tam u l t i p l i c i t yo fi n f e c t i o n(MO I )o f0 . 1 . t37 °C ,th ec e l l s A t24 -hpo s t in f e c t ionin5%(v /v )CO2a w e r ef ix edu s ing5%(v /v )co ld( -20 °C )a c e t i ca c idin m e th ano lfo r10 m in .Th efix edc e l l sw e r ew a sh edth r e e t im e sinPBSandin cub a t ed w i thp r im a ry mono c lon a l an t ibodyag a in s tS t .Lou i sen c eph a l i t i sv i ru s(SLEV ) env e lop ep ro t e in( c ro s s r e a c t iv eto DENV -2env e lop e ; MAB8744 ;M i l l ipo r e ,B i l l e r i c a , MA , USA )a tad i lu t ion V IROLOG ICAS IN ICA
J ing j ingFane ta l
o f1 :250fo r1ha t37 °C .A f t e rb e ingw a sh edth r e et im e s inPBS ,th ec e l l sw e r ein cub a t ed w i thT ex a sR ed– con ju g a t edA f f in ipu r e Go a tAn t i -Mou s eIgG(1 :100d i lu t ed , P ro t e in t e ch )fo r1ha troomt emp e r a tu r e .Th es l id e sw e r e ag a in w a sh edth r e et im e sinPBS , moun t edin95%(v / v )g ly c e ro landth enex am in edund e raL e i c aTCG -SP2 L a s e rS c ann ing Con fo c a lM i c ro s copy(L e i c a ,W e t z l a r , G e rm any ) . P laqueassay S e r i a l1 :10d i lu t ion sw e r em ad eby m ix ing15µL v i r a l s to ck sw i th135µLDMEMcon t a in ing10%(w /v )FBS . Fo re a chd i lu t ion ,100µL w a ss e ed edinanind iv idu a l w e l lo fa24 -w e l lp l a t e ,con t a in ingsub con f lu en tBHK t37 °C ,th e c e l l s .A f t e r1hin cub a t ionund e r5% CO 2a c e l l sw e r eov e r l a idw i thDMEMp lu s2%(w /v )FBS ,and 1%(w /v )m e thy l c e l lu lo s e(Aqu a c id eI I ,C a lb io ch em , S an D i ego , CA , USA ) .Fo l low ing4do fin cub a t ion th ec e l l sw e r ef ix ed w i th3 .7% a t37 ºCund e r5%CO 2, (w /v )fo rm a ld ehyd eands t a in ed w i th1%(w /v )c ry s t a lv io l e t .V i r a lt i t e r sw e r ec a l cu l a t eda sth enumb e ro f p l aqu e fo rm ingun i t s(PFU )p e rµL . RNAex t rac t ionandRea l t imeRT -PCR BHK -21c e l l sw e r et r an s f e c t ed w i th mu t an tand WT RNA s . Th et r an s c r ib ed RNA sf rom DENV2TSV01 cDNAc lon e sw e r ep r ep a r eda sabov e .T od e t e c tth e mu t an t sw i thou tobv iou scy top a th i cin f e c t ion(CPE ) ,th e c e l lsup e rn a t an t sw e r eco l l e c t eda sP0a t96 -hpo s t t r an s f e c t ion(p . t . ) .P0sup e rn a t an t sw e r eu s edtoin f e c tf r e sh BHK -21c e l l sandp a s s ag eda c co rd ing lya f t e r4d ay s . T o t a l RNA sw e r eex t r a c t edf rom200μLv i r a ls to ck s andc e l lsu sp en s ion sw i thaTR I zo lk i t( Inv i t rog en , C a r l sb ad ,CA , USA )a c co rd ingtoth em anu f a c tu r e r ' sin s t ru c t ion s .Th eRNAw a sr e su sp end edin10μLRNA s e f r e ed i s t i l l ed w a t e rands to r eda t-80 °C . RNAcopy numb e r sw e r ec a l cu l a t edonth eb a s i so fcon c en t r a t ion and mo l e cu l a rw e igh t .T en fo ldd i lu t ion so ft r an s c r ib ed 8 cop i e sw e r eu s eda ss t and a rd RNA ,con t a in ing102-10 ina l lr e a l t im equ an t i t a t iv e(qRT ) -PCR s . A On eS t ep SYBRP r im eS c r ip tPLUSRT -PCR K i t(T aK aR a ,Sh ig a , J ap an )w a su s edinth ed e t e rm in a t iono fth eRNAcon c en t r a t iono f DENV -2byqRT -PCR ,w i thth efo rw a rd p r im e r5 ' -ACAGTCTGGAACAGGGTGTGGATT -3 ' , andth er ev e r s ep r im e r5 ' -TTGTTAGCCCAATCAAT GAGCCGC -3 ' ;u s inga B io -R adiQ5 ,th efo l low ing th e rmo cy c l ingcond i t ion sw e r eemp loy ed :95 °Cfo r10 s ;th en40cy c l e sa t95 °Cfo r5s ,60 °Cfo r30sand95 °C fo r1 m in ;fo l low edby95 °Cfo r1 m inandfin a l ly81cy c l e sa t55 °Cfo r30s .
s e ed edin to12 -w e l lp l a t e sanda s s ay edfo rlu c i f e r a s e a c t iv i tya tsp e c ifict im epo in t sp . t . ;t r ip l i c a t ew e l l sw e r e s e ed edfo re a cht im epo in t .A tth eind i c a t edt im e s ,c e l l s w e r ew a sh edw i thco ldPBSandly s edw i th200μLly s i s bu f f e rfo r15 m ina t37 °C .L y s a t e s(20μL )w e r em ix ed w i th100μLa s s ayr e ag en tina96 -w e l lp l a t e ,andth e lu c i f e r a s es ign a l sw e r em e a su r edw i tha Modu lu sm i c ro p l a t e mu l t imod er e ad e r(T u rn e rB io sy s t em s ,Sunnyv a l e , CA ,USA ) . Ana lys iso fNS1p ro te ins BHKc e l l mono l ay e r sw e r et r an s f e c t ed w i th1μg RNA ,m a in t a in edin DMEM w i th2%(w /v )FBS ,and th enh a rv e s t ed wh enCPEb e c am eobv iou sinth e WT in f e c t edBHKc e l l s .A t48 -hp . t . ,c e l l sw e r eh a rv e s t ed , w a sh ed ,andin cub a t edin1×c e l lly s i sbu f f e rfo rw e s t e rnandimmunop r e c ip i t a t ion( IP )(B eyo t im e ,H a im en , Ch in a ) .Th ely s a t ew a sc l a r i f i edbyc en t r i fug a t iona t 12 ,000×gfo r10 m ina t4 °Candth ens to r eda t-70 °C . S amp l e sp r ep a r eda sabov ew e r ed i lu t edinnon r edu c ing s amp l ebu f f e r(62 .5 mmo l /LT r i s /HC l[pH6 .8 ] ,10%[v / v ]g ly c e ro l ,0 .05%[w /v ]b romoph eno lb lu e )a t37 °C ,o r in cub a t ed w i th1×s amp l ebu f f e rcon t a in ingar edu c ing r e ag en t(1%[v /v ]2 -m e r c ap to e th ano l )a t100 °Cfo r4 m in ,and w e r eth ensub j e c t edtoe l e c t ropho r e t i cs ep a r a t ion ,c a r r i edou tu s ing10%(w /v )sod iumdod e cy lsu l f a t e (SDS )po ly a c ry l am id eg e l s .S ep a r a t edp ro t e in sw e r e t r an s f e r r edtopo lyv iny l id en ef luo r id e(PVDF )m em b r an e s(M i l l ipo r e ) , wh i ch w e r eth enb lo ck edbyt r e a t m en tw i th5%(w /v )non f a tm i lkinT r i s -bu f f e r eds a l in e w i thTw e en(TBST )fo r1ha troomt emp e r a tu r e . An an t i -NS1 mou s epo ly c lon a lan t ibody(g en e rou s lyp ro v id edby D r .J i aF an )w a su s eda sth ep r im a ryan t ibody (1 :1000d i lu t ion ) .A f t e rth r e e10 -m inw a sh e sw i thTBST , th eb lo t sw e r ein cub a t edw i ths e cond a ryan t i -mou s eIgG con jug a t edtoho r s e r ad i shp e rox id a s e(1 :5000 ,M i l l ipo r e ) a t37 °Cfo r1h .B lo t sw e r ean a ly z edu s inganIm ag ing D en s i tom e t e r(B io -R ad ,CA ,USA ) . RESULTS
P roduc t ionandcha rac te r iza t iono fNS1 mu tan t v i ruses F igu r e1 show sth es t ru c tu r eo fth er e comb in an t f e c t iou sc lon eandth eam inoa c ids equ en c e DENV-2in o f DENV -2 NS1 .As e r i e so f mu t an t so f NS1( th r e eCy s s i t e s[C4A ,C55A ,C291A ] ,two N l ink edg ly co sy l a t ion s i t e s[N130A , N207A ] ,ag lu t am i ca c ids i t e[E173A ] , andaC t e rm in a lt run c a t edf r agm en t[ΔC ] ) ,w e r econ s t ru c t edandin co rpo r a t edin toth ein f e c t iou s DNEV -2 c lon e . Mu t an tRNA sw e r esyn th e s i z edinv i t r oandt r an s f e c t edin toBHK -21c e l l s . Mu t an t s N130AandE173A Luc i fe raseassay T h et r a n s f e c t e dc e l l sw e r es u s p e n d e di n2 5mLDMEM show ednos ign ifi c an td i f f e r en c e scomp a r edto DENV -2 ec e l lsu sp en s ion(1 mL )w a s TSV01(WT w i th10%(w /v )FBS . Th )v i ru s e s ,bu tC4A ,C55A ,C291A ,andΔC
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Mu ta t ionso fDENVNS1a f fec tv i ra lrep l ica t ion
F igu re1 . Schema t icd iag ramo f DENV2 -TSV01andtheam inoac idsequence(aa807 -1 159 )o fthe NS1p ro te in . S y s tema t i cs i te -d i re c ted mu tagene s i so ftheN -g l y co s y la t ions i te sandC y ss i te s i sshownb ya r row s .TheC te rm ina l reg iono ftheDENV -2NS1p ro te in(aa333 -352 )tha twa sde le tedtogene ra tetheC te rm ina lt run ca ted mu ta t ion(ΔC ) isunde r l ined .Do t tedl inesdeno tethefo rma t iono fd isu lfidebondsbe tweenCysres idues .
e a chf a i l edtoc au s eCPEint r an s f e c t edc e l l sandd idno t g en e r a t eanyv i s ib l ep l aqu e s(F igu r e2A) .Inp a r t i cu l a r , th e N207A mu t a t ion , wh i ch w a sa l t e r eda tth es e cond NS1g ly co sy l a t ions i t e ,p rodu c edf ew e rin f e c t iou sv i r a l p a r t i c l e sandr e su l t edinsm a l l e rp l aqu e s .Twopo s s ib i l i t i e sm igh tb eadv an c edtoa c coun tfo rth eph eno typ e o fnop l aqu eg en e r a t ion :th ef i r s ti sth a t C4A , C55A , C 2 9 1A ,a n dΔCm u t a n tRNAc o u l dn o tg e n e r a t ea n yv i r a l p a r t i c l e s ;th es e condi sth a ta l though mu t an tRNA sw e r e ab l etog en e r a t ein f e c t iou sp a r t i c l e sth a tcou ldno tfo rm p l aqu e s .T ofu r th e rinv e s t ig a t eth ep rodu c t iono fin f e c t iou sv i r a lp a r t i c l e sfo l low ingt r an s f e c t ion ,sup e rn a t an t so lu t ion sh a rv e s t edf romt r an s f e c t edc e l l sw e r eu s edfo r th ein f e c t iono fBHK -21c e l l s , wh i ch w a sth enfo l low ed byimmunofluo r e s c en ta s s ay( IFA )an a ly s i su s inganan t i -E mAba t24 -hpo s tin f e c t ion .Con s i s t en t ly ,nopo s i t iv e IFAr e spon s ecou ldb ed e t e c t edfo rC4A ,C55A ,C291A , igu r e2B) ,d emon s t r a t ingth a ta l lo f o rth eΔC mu t an t(F th e s e mu t a t ion simp a i rth ep rodu c t iono fin f e c t iou sv i r a l p a r t i c l e s .Inadd i t ion , N130A , N207A ,andE173A mu t an t se a chshow edanapp rox im a t e ly5 -20 fo ldr edu c t ion int e rm so fth esp e c ifi cin f e c t iv i tycomp a r edto WTRNA ( T ab l e1) . Fo r mu t an t sN130A ,E173A ,andN207A ,w ea l soan a ly z edg row thk in e t i c sonV e roc e l l s .A sshowninF igu r e 3,nos ign ifi c an td i f f e r en c e sb e tw e enth e mu t an tand WT v i ru s e sw e r eob s e rv edint e rm so fv i r a lt i t e ra t24 ,48 , o r72 -hp . t . On lya t96 -and120 -hp . t .d idth e mu t an t v i ru s e sshows l igh t lys low e rg row thth an WTv i ru s e s . S im i l a rr e su l t sw e r eob s e rv edinqRT -PCRan a ly s e so f th esup e rn a t an tso lu t ion sh a rv e s t edf romc e l l sin f e c t ed w i th mu t an t s N130A ,E173A , N207A ,and w i thth e WT (d a t ano tshown ) . 4
Fo r mu t an t sC4A ,C55A ,C291A ,andΔC ,w ea t t emp t edtos e l e c tfo ranyr ev e r t an t sbyb l indp a s s ag ingo fth e sup e rn a t an tso lu t ionf romt r an s f e c t edc e l l s . How ev e r , ev eninth esup e rn a t an tso lu t ionf romth ef i r s tp a s s ag e , th ein t r a c e l lu l a rv i r a lRNA sw e r eb e lowth el im i to fd e t e c t ion .Th eRNAcop i e sth a tw e r ed e t e c t ab l einth eP0 sup e rn a t an tso lu t ion m ayh av eb e enr e s idu a linpu tRNA s T ab l e2) .Co l l e c t iv e ly ,th e s er e su l t sd emon s t r a t edth a t ( th eC4 ,C55 ,C291 ,andC t e rm in a lr e s idu e so f NS1a r e e s s en t i a lfo rv i r a lr ep l i c a t ion . E f fec to fNS1 mu ta t iononDENVRNAsyn thes is inBHK -21ce l ls T ofu r th e rd e t e rm in e wh e th e rth ed e f e c tinv i r a l r ep l i c a t ion w a sdu etoth eabo l i t iono fRNAsyn th e s i s , alu c i f e r a s e r epo r t ingr ep l i cono f DENV -2(R lu cR ep ) TSV01s t r a in w a su s edtoqu an t i fyv i r a lt r an s l a t ionand RNAsyn th e s i sw i thr e sp e c ttoth e NS1 mu t an t s .A l lth e r ep l i con sg en e r a t edas im i l a rl ev e lo flu c i f e r a s ea c t iv i tya t2 -6hp . t .( F igu r e4) ,ind i c a t ingth a tnon eo fth e mu t a t ion sa f f e c t edth et r an s l a t iono fth einpu tr ep l i con RNAandth a tth eya l lpo s s e s s eds im i l a rt r an s f e c t ione f fic i en c i e s . N130AandE173Ar ev e a l edtyp i c a ltwo -p e ak lu c i f e r a s ep a t t e rn s wh i l e N207Ar ep l i c a t edab i ts low ly wh encomp a r ed w i th WT ,r ep l i c a t iono fo th e r mu t an t s w e r ed e c l in eds ign ific an t lyinc e l l s .T ak entog e th e r ,th i s w a scon s i s t en tw i thth er e su l t sob t a in edu s ingth eg e nom e l eng thRNA s .Th e s er e su l t sind i c a t edth a tth eCy s s i t e sa tC4 ,C55 ,andC291andth eC t e rm in a lr e s idu e s o fNS1a r er equ i r edfo rv i r a lRNAsyn th e s i s . Ana lys iso fNS1in mu tan tv i ruses Th er e su l t sshow edth a t ,a sinth ec a s eo f WTRNA , V IROLOG ICAS IN ICA
J ing j ingFane ta l
F igu re2 .A :P laque mo rpho logyo f mu tan tand WTv i rusesinBHK -21ce l ls ;B :IFAo fVe roce l ls24ha f te rin fec t ion w i th mu tan tand WTv i ruses . Redind ica tesEp ro te ininin fec tedVe roce l ls(N130A ,E173A , N207Aand WT ) .The IFAsca leba rrep resen ts20μm .O the r mu tan ts(C4A ,C55A ,C291AandΔC )gene ra tednov is ib lefluo rescence .
mu t a t ion s N130A ,E173A ,and N207Ae a chg av er i s e toan NS1b andco r r e spond ingtoabou t90kD a ,r ep r e s en t ingth ed im e r i cfo rmo f NS1 .Incon t r a s t ,no NS1 exp r e s s ioncou ldb eob s e rv edinc e l l st r an s f e c t ed w i th C4A ,C55A ,C291A ,o rΔC mu t an t s ,ona c coun to fv i r a lr ep l i c a t iond i s rup t ion .Inth ec a s eo fth e N130Aand N207A mu t an tv i ru s e s ,i tw a sno t ab l eth a tth e NS1d i www .v i ros in .o rg
m e rm ig r a t eds l igh t lyf a s t e rdu r inge l e c t ropho r e s i sth an th e NS1d im e ro fe i th e rth e WTo rE173Av i ru s e s , mo s t l ik e lydu etod eg ly co sy l a t iona tth eco r r e spond ingpo s i t ion s( F igu r e5) .A l thoughth el ev e lo fNS1exp r e s s ionin N207Aw a sl e s sth anin N130A ,E173A ,o r WTv i ru s e s , i ti sc l e a rth a tg ly co sy l a t iona tpo s i t ion s130and207i s no te s s en t i a lfo rv i r a lr ep l i c a t ion . 5
Mu ta t ionso fDENVNS1a f fec tv i ra lrep l ica t ion T ab le2RNAcopyana lys isfo rthe mu tan ts
T ab le1De te rm ina t iono fw i ldtypeanda l l mu tan tst i te r RNA
a t i te r
CPE
N130A N207A E173A C4A C55A C291A ΔC WT
2 .80x107 1 .20x107 5 .45x107 2 tec tab le )
RESEARCHART ICLE C r i t ica lro leo fDengueV i rusNS1p ro te ininv i ra lrep l ica t ion 1 ,2 1 J ing j ingFan1,2,Y iL iu ,Zh im ingYuan
1 .KeyLabo ra to ryo fAg r icu l tu ra landEnv i ronmen ta lM ic rob io logy , WuhanIns t i tu teo fV i ro logy ,Ch inese Academyo fSc iences , Wuhan430071 ,Ch ina 2 .Un ive rs i tyo ftheCh ineseAcademyo fSc iences ,Be i j ing100039 ,Ch ina Denguev i rus(DENV )nons t ruc tu ra lp ro te in1(NS1 )isah igh lyconse rved46 -kDap ro te intha t con ta ins2g lycosy la t ions i tes(Asn -130and Asn -207 )and12conse rvedcys te ine(Cys )res idues . He re , wepe r fo rmeds i te -d i rec ted mu tagenes istogene ra tesys tema t ic mu tan tso fv i ra ls t ra inTSV01 . Theresu l tso fthesubsequen tana lys isshowedtha tana lan inesubs t i tu t iona tthesecond N l inked g lycan Asn -207in NS1de layedv i ra l RNAsyn thes is ,reducedv i rusp laques ize ,and weakenedthe cy topa th ice f fec t .Th ree mu tan tsa tCyss i tes(Cys -4 ,Cys -55 ,Cys -291 )andaC te rm ina lde le t ion(ΔC ) mu tan ts ign ifican t lyimpa i red RNAsyn thes is ,andconsequen t lyabo l ishedv i ra lg row th , whe reas a lan ine mu ta t ionsa tAsn -130and G lu -173resu l tedinpheno typestha t we res im i la rtothe w i ld type (WT )v i rus .Fu r the rana lys isshowedtha tthe Asn -207 mu ta t ions l igh t lyde layedv i ra lrep l ica t ion . Theseresu l tssugges ttha ttheth reeconse rvedd isu lfidebondsandthesecond N l inkedg lycanin NS1a rerequ i redfo rDENV -2rep l ica t ion . KEYWORDS denguetype2v i rus ;NS1 ;rep l ica t ion ;g lycosy la t ions i te
INTRODUCT ION D engu ev i ru s(DENV )i son eo fs ev e r a lg lob a l lyim po r t an ta r th ropod -bo rn ep a thog en sw i th inth eg enu s F la v i v i ru s. D engu ein f e c t iono c cu r sm a in lyint rop i c a l andsub t rop i c a lcoun t r i e sinSou th e a s tA s i a ,th eP a c ific , andth eAm e r i c a s .Inr e c en td e c ad e s ,th eannu a lav e r ag e in c id en c eo fd engu ein f e c t ion sh a sin c r e a s edd r am a t i c a l ly ; wo r ldw id e ,i ti se s t im a t edth a tth e r ea r eov e r100 m i l l ionc a s e sannu a l ly ,l e ad ingto~500 ,000ho sp i t a l i z a t ion sw i thac a s ef a t a l i tyr a t eo f5%(Ky l eJ ,2008 ; WHO , 2012 ) .Th e r ea r efou ran t ig en i c a l lyd i s t in c ts e ro typ e s (DENV -1 ,-2 ,-3 ,and-4 ) ,andep id em io log i c a ls tud i e s ov e ra20 -y e a rp e r iodh av ed e t e rm in edth a t DENV -2i s th e mo s tp r ev a l en ts e ro typ e(Ho angL ,2010 ;V aughn D , 2000 ) .Un t i lr e c en t ly ,th ep a thog en i cm e ch an i smandth e in t e r a c t ion sb e tw e en DENVandho s tf a c to r sh av eb e en in comp l e t e lyund e r s tood . Rece ived :1 1Ap r i l2014 ,Accep ted :16 May2014 Pub l ishedon l ine :26 May2014 Co r respondence : Phone :86 -27 -87197242 ,Fax :86 -27 -87198120 , Ema i l :yzm@wh . iov .cn
©W IV ,CASandSp r inge r -Ve r lagBe r l inHe ide lbe rg2014
Th eg enom eo f DENVi sapo s i t iv es ing l e s t r and ed RNAo fapp rox im a t e ly1 1kbinl eng th , wh i chcon t a in s as ing l eop enr e ad ingf r am eth a tcod e sfo rth r e es t ru c tu r a lp ro t e in s( c ap s id[C ] ,m emb r an e[M ] ,andenv e lop e [E ] )ands ev ennon s t ru c tu r a lp ro t e in s(NS1 , NS2A /B , NS3 , NS4A /B ,and NS5 ) .Th e NS1p ro t e ini sah igh ly con s e rv ed46 -kD ag ly cop ro t e in ,con t a in ing12h igh ly con s e rv edcy s t e in e(Cy s )r e s idu e sth a tfo rm6in t e rmo l e cu l a rd i su l f id ebond s ,and2 N l ink edg ly co sy l a t ion s i t e s(C h am b e r sT ,1 9 9 0 ) .NS 1i sc o t r a n s l a t i o n a l l yt r a n s lo c a t edin toth eendop l a sm i cr e t i cu lum(ER )lum enby th et r an s -m emb r an es ign a ls equ en c ea tth eC t e rm inu s o fth eEp ro t e in(M a ck en z i eJ ,1996 ;P ryo rM ,1994 ; W e s t aw ayE ,1997 ) .Th eg ly co sy l a t ions t a tu so f NS1 i simpo r t an tfo ri t spo lym e r i z a t ionandd i s t r ibu t ion , wh i cho c cu r sinth eERlum en ,andfu r th e r mod ific a t ion o c cu r r edinth e Go lg in e two rk wh e r eacomp l ex typ e sug a ri sadd edtoth e N130g ly c anbu tth e N207g ly c an i sr e t a in edinth eh igh m anno s e(F a lgou tB ,1995 ;P ryo r M ,1994 ;W ink l e rG ,1988 ) .I th a sb e enfoundth a t NS1 lo c a l i z e sw i th inth eER m emb r an e ,fo rm sahomod im e r wh i chi sr equ i r edfo rt r an spo r tth roughth e Go lg ianda p a r t i a l lyhyd rophob i cn a tu r ek e ep s NS1s t ay m emb r an e a s so c i a t ed . NS1a l soc ans t ay w i th inc e l lu l a rv e s i cu l a r 1
Mu ta t ionso fDENVNS1a f fec tv i ra lrep l ica t ion
comp a r tm en tb e fo r ea t t a ch ingtoth ec e l lsu r f a c eand th enf in a l lyb e ings e c r e t edf rom m amm a l i anc e l l s , p r edom in an t lya sah ex am e r(F l am and M ,1999 ;P ryo r M ,1994 ;W ink l e rG ,1989 ) . NS1c anb ed e t e c t edinth e s e r ao fin f e c t edp a t i en t s(upto50μg /mL ) ,w i thh igh e r l ev e l sb e ingp r e s en tintho s e whod ev e lop edd engu e h emo r rh ag i cf ev e r(DHF )r a th e rth and engu ef ev e r(DF ) , ind i c a t ingth a ts e c r e t ed NS1 m aycon t r ibu t etoth es e v e r i tyo fin f e c t ioninp a t i en t s(A l conS ,2002 ;Y oungP , 2000 ) . Du r ingd engu ein f e c t ion , NS1c ane l i c i tas t rong humo r a limmun er e spon s e ,enh an c ein f e c t ioninh ep a to c y t e s ,a n dp r om o t ee n d o t h e l i a lc e l ld am a g eb yf a c i l i t a t i n g imm u n ec om p l e xf o rm a t i o n(A v i r u t n a nP ,2 0 0 6 ;C h a n gH , 2002 ;L inC ,2003 ;SunD ,2007 ) .I tc ana l soan t agon i z e comp l em en ta c t iv a t ionbyb ind ingf a c to rH ,anda l soC4 andC1 s ,th e r ebyd ep r e s s ingcomp l em en ta c t iv a t ionand p ro t e c t ing DENVf romd i r e c tcomp l em en t -d ep end en t n eu t r a l i z a t ion(A v i ru tn anP ,2010 ;ChungK ,2006 ;Fu ch s A ,2010 ) . Th econ s e rv a t iono f12 Cy sr e s idu e ssugg e s t sth a t th e s er e s idu e sa r eimpo r t an tin NS1 mu l t im e r i z a t ion andfun c t ion .I th a sb e enp ropo s edth a t mu t a t ion so f g ly co sy l a t ions i t e sandcon s e rv edCy sr e s idu e sa r ea s so c i a t ed w i thd e c r e a s edv i ru sg row th ,r edu c edp l aqu e s i z e ,d e l ay edcy top a th i ce f f e c t ,andimp a i r ed NS1s e c r e t ion(P ryo rM ,1993 ;P ryo rM ,1994 ) . How ev e r ,th e e f f e c t so fth e mu t a t ion sth a tw e r eex am in edbyd i f f e r en t g roup sapp e a r edtob ein con s i s t en tinc e l lcu l tu r e(T a j im a S ,2008 ) ;andth ed eg ly co sy l a t iono fN l ink eds i t e sl e ad s tod iv e r s eph eno typ e sw i thr e sp e c tto mou s en eu rov i ru l en c e ,fo l low ingf l av iv i ru sin f e c t ion(Muy l a e r tI ,1996 ; P l e tn evA ,1993 ;P ryo rM ,1998 ; Wh i t em an M ,2010 ) .A eC t e rm in a lendo f p r ev iou ss tudya l soind i c a t edth a tth t h ep r o t e i ni sim p o r t a n ti nNS 1d im e rf o rm a t i o n(P r y o rM , 1993 ) . B e c au s eth ee f f e c to f NS1onv i r a lr ep l i c a t ionand v i ru l en c er em a in sun c l e a r ,w einv e s t ig a t edth efun c t ion s o ftheCy sr e s idu e sando fth eC t e rm in a lo f NS1inth e v i r a ll i f ecy c l e .Inth ep r e s en ts tudy ,w ed emon s t r a t eth a t ana l an in e mu t a t iona tA sn -207d e l ay sv i r a lr ep l i c a t ion andth ecy top a th i ce f f e c t ,andl e ad stor edu c edp l aqu e s i z e .Th r e eCy s s i t e mu t a t ion sandaC t e rm in a ld e l e t ion (ΔC )e a chcomp l e t e lyabo l i shv i r a lg row thandr ep l i c a t ion , wh e r e a s mu t a t ion sa tA sn -130and G lu -173l e adto aph eno typ eth a ti ss im i l a rtoth a to fth e WTv i ru s .Th e r e su l t sp rov id ev a lu ab l ein s igh t sin toth ero l eo f NS1in v i r a lin f e c t ionandcou ldgu id eth ed ev e lopm en to fnov e l an t iv i r a lth e r ap i e sfo rd engu ep a t i en t s . MATER IALSAND METHODS Ce l lsandv i ruses BHK -21(B abyh am s t e rk idn ey )and V e ro(A f r i c an 2
g r e en monk eyk idn ey )c e l l s( Inv i t rog en ,C a r l sb ad ,CA , USA )w e r ep rop ag a t edin Du lb e c co ’ s mod i f i edE ag l e ’ s m ed ium(DMEM ,G ib co ,G r andI s l and , NY ) ,supp l e m en t ed w i th10%(w /v )f e t a lbov in es e rum(FBS )and t 100 Up en i c i l l in s t r ep tomy c in /mL ,in5%(v /v ) CO2a 37 °C .S to ck so f DENV -2v i ru s e sw e r eg rownin V e ro c e l l sw i th2%(w /v )FBS ,andth ev i r a lt i t e rw a sc a l cu l a t edw i thp l aqu ea s s ay sonBHK -21c e l l s . P lasm idcons t ruc t ion DENV -2g enom e l eng thcDNAin f e c t iou sc lon e scon t a in ingv a r iou s mu t a t ion sw e r econ s t ru c t edbyu s inga mod ifi edpACYC -DENV2 -TSV f1v e c to r(L inS ,201 1 ) . Ar e comb in an tR en i l l alu c i f e r a s e r epo r t ingr ep l i con ,pA CYC -TSV -R lu c2A -R ep ,w a su s edtocon s t ru c t mu t a t iv e r ep l i con sfo rv i r a lr ep l i c a t ionan a ly s i s .D r .P e i -Y ongSh i p rov id eda l lo fth eo r ig in a lv e c to r s(Nov a r t i sIn s t i tu t e fo rT rop i c a lD i s e a s e s ,S ing apo r e ) . NS1 mu t a t ion s(C4A , C55A , C291Aand N130A , N207A )andaΔC w e r e ob t a in edbys t and a rdov e r l ap -po lym e r a s ech a inr e a c t ion (PCR ) .Th eov e r l ap -PCRf r agm en t sw e r ein s e r t edin to th e DENV -2TSV01in f e c t iou sc lon eandr ep l i conu s ing twoun iqu er e s t r i c t ionen zym e s ,B s rG I(nu c l eo t id epo s i t ion2103 )and M luI(nu c l eo t id epo s i t ion4571 ) .A l l con s t ru c t sw e r ev e r ifi edbyDNAs equ en c ing . RNAt ransc r ip t ionandt rans fec t ion Th ein f e c t iou scDNAc lon e sw e r el in e a r i z ed w i th C la I .Th eg enom e l eng thandr ep l i con RNA sw e r e p rodu c ed w i thl in e a r i z edcDNAu s inga mMESSAGE mMACH INET7k i t(Amb ion ,TX , USA ) ,a c co rd ing toth em anu f a c tu r e r ’ sin s t ru c t ion s .App rox im a t e ly5μg .8 RNA sw e r et r an s f e c t edin toBHK -21c e l l s(8×106)in0 mLco ldpho sph a t e -bu f f e r eds a l in e(PBS ,pH7 .5 )by e l e c t ropo r a t ionu s ingaG en epu l s e rapp a r a tu s(B io -R ad , H e r cu l e s , CA , USA ) . As e t t ingo f0 .85kVand25μF w a su s ed ,pu l s ingth r e et im e sa t3 sin t e rv a l s ,fo l low ed bya10 -m inr e cov e ryp e r ioda troomt emp e r a tu r e .Th e t r an s f e c t edc e l l sw e r ed i lu t edin25 mL DMEMcon t a in ing10%(w /v )FBS ,and w e r eth enin cub a t edina a t37 °C( J i aF , T -75fl a skfo r5 -7dund e r5%(v /v )CO2, 2013 ) . Ind i rec tImmunofluo rescenceassay( IFA ) App rox im a t e ly3×105V e roc e l l sw e r es e ed edon to s l id e sina6 -w e l lp l a t eandth enin f e c t ed w i th WTand m u t a n tv i r u s e sa tam u l t i p l i c i t yo fi n f e c t i o n(MO I )o f0 . 1 . t37 °C ,th ec e l l s A t24 -hpo s t in f e c t ionin5%(v /v )CO2a w e r ef ix edu s ing5%(v /v )co ld( -20 °C )a c e t i ca c idin m e th ano lfo r10 m in .Th efix edc e l l sw e r ew a sh edth r e e t im e sinPBSandin cub a t ed w i thp r im a ry mono c lon a l an t ibodyag a in s tS t .Lou i sen c eph a l i t i sv i ru s(SLEV ) env e lop ep ro t e in( c ro s s r e a c t iv eto DENV -2env e lop e ; MAB8744 ;M i l l ipo r e ,B i l l e r i c a , MA , USA )a tad i lu t ion V IROLOG ICAS IN ICA
J ing j ingFane ta l
o f1 :250fo r1ha t37 °C .A f t e rb e ingw a sh edth r e et im e s inPBS ,th ec e l l sw e r ein cub a t ed w i thT ex a sR ed– con ju g a t edA f f in ipu r e Go a tAn t i -Mou s eIgG(1 :100d i lu t ed , P ro t e in t e ch )fo r1ha troomt emp e r a tu r e .Th es l id e sw e r e ag a in w a sh edth r e et im e sinPBS , moun t edin95%(v / v )g ly c e ro landth enex am in edund e raL e i c aTCG -SP2 L a s e rS c ann ing Con fo c a lM i c ro s copy(L e i c a ,W e t z l a r , G e rm any ) . P laqueassay S e r i a l1 :10d i lu t ion sw e r em ad eby m ix ing15µL v i r a l s to ck sw i th135µLDMEMcon t a in ing10%(w /v )FBS . Fo re a chd i lu t ion ,100µL w a ss e ed edinanind iv idu a l w e l lo fa24 -w e l lp l a t e ,con t a in ingsub con f lu en tBHK t37 °C ,th e c e l l s .A f t e r1hin cub a t ionund e r5% CO 2a c e l l sw e r eov e r l a idw i thDMEMp lu s2%(w /v )FBS ,and 1%(w /v )m e thy l c e l lu lo s e(Aqu a c id eI I ,C a lb io ch em , S an D i ego , CA , USA ) .Fo l low ing4do fin cub a t ion th ec e l l sw e r ef ix ed w i th3 .7% a t37 ºCund e r5%CO 2, (w /v )fo rm a ld ehyd eands t a in ed w i th1%(w /v )c ry s t a lv io l e t .V i r a lt i t e r sw e r ec a l cu l a t eda sth enumb e ro f p l aqu e fo rm ingun i t s(PFU )p e rµL . RNAex t rac t ionandRea l t imeRT -PCR BHK -21c e l l sw e r et r an s f e c t ed w i th mu t an tand WT RNA s . Th et r an s c r ib ed RNA sf rom DENV2TSV01 cDNAc lon e sw e r ep r ep a r eda sabov e .T od e t e c tth e mu t an t sw i thou tobv iou scy top a th i cin f e c t ion(CPE ) ,th e c e l lsup e rn a t an t sw e r eco l l e c t eda sP0a t96 -hpo s t t r an s f e c t ion(p . t . ) .P0sup e rn a t an t sw e r eu s edtoin f e c tf r e sh BHK -21c e l l sandp a s s ag eda c co rd ing lya f t e r4d ay s . T o t a l RNA sw e r eex t r a c t edf rom200μLv i r a ls to ck s andc e l lsu sp en s ion sw i thaTR I zo lk i t( Inv i t rog en , C a r l sb ad ,CA , USA )a c co rd ingtoth em anu f a c tu r e r ' sin s t ru c t ion s .Th eRNAw a sr e su sp end edin10μLRNA s e f r e ed i s t i l l ed w a t e rands to r eda t-80 °C . RNAcopy numb e r sw e r ec a l cu l a t edonth eb a s i so fcon c en t r a t ion and mo l e cu l a rw e igh t .T en fo ldd i lu t ion so ft r an s c r ib ed 8 cop i e sw e r eu s eda ss t and a rd RNA ,con t a in ing102-10 ina l lr e a l t im equ an t i t a t iv e(qRT ) -PCR s . A On eS t ep SYBRP r im eS c r ip tPLUSRT -PCR K i t(T aK aR a ,Sh ig a , J ap an )w a su s edinth ed e t e rm in a t iono fth eRNAcon c en t r a t iono f DENV -2byqRT -PCR ,w i thth efo rw a rd p r im e r5 ' -ACAGTCTGGAACAGGGTGTGGATT -3 ' , andth er ev e r s ep r im e r5 ' -TTGTTAGCCCAATCAAT GAGCCGC -3 ' ;u s inga B io -R adiQ5 ,th efo l low ing th e rmo cy c l ingcond i t ion sw e r eemp loy ed :95 °Cfo r10 s ;th en40cy c l e sa t95 °Cfo r5s ,60 °Cfo r30sand95 °C fo r1 m in ;fo l low edby95 °Cfo r1 m inandfin a l ly81cy c l e sa t55 °Cfo r30s .
s e ed edin to12 -w e l lp l a t e sanda s s ay edfo rlu c i f e r a s e a c t iv i tya tsp e c ifict im epo in t sp . t . ;t r ip l i c a t ew e l l sw e r e s e ed edfo re a cht im epo in t .A tth eind i c a t edt im e s ,c e l l s w e r ew a sh edw i thco ldPBSandly s edw i th200μLly s i s bu f f e rfo r15 m ina t37 °C .L y s a t e s(20μL )w e r em ix ed w i th100μLa s s ayr e ag en tina96 -w e l lp l a t e ,andth e lu c i f e r a s es ign a l sw e r em e a su r edw i tha Modu lu sm i c ro p l a t e mu l t imod er e ad e r(T u rn e rB io sy s t em s ,Sunnyv a l e , CA ,USA ) . Ana lys iso fNS1p ro te ins BHKc e l l mono l ay e r sw e r et r an s f e c t ed w i th1μg RNA ,m a in t a in edin DMEM w i th2%(w /v )FBS ,and th enh a rv e s t ed wh enCPEb e c am eobv iou sinth e WT in f e c t edBHKc e l l s .A t48 -hp . t . ,c e l l sw e r eh a rv e s t ed , w a sh ed ,andin cub a t edin1×c e l lly s i sbu f f e rfo rw e s t e rnandimmunop r e c ip i t a t ion( IP )(B eyo t im e ,H a im en , Ch in a ) .Th ely s a t ew a sc l a r i f i edbyc en t r i fug a t iona t 12 ,000×gfo r10 m ina t4 °Candth ens to r eda t-70 °C . S amp l e sp r ep a r eda sabov ew e r ed i lu t edinnon r edu c ing s amp l ebu f f e r(62 .5 mmo l /LT r i s /HC l[pH6 .8 ] ,10%[v / v ]g ly c e ro l ,0 .05%[w /v ]b romoph eno lb lu e )a t37 °C ,o r in cub a t ed w i th1×s amp l ebu f f e rcon t a in ingar edu c ing r e ag en t(1%[v /v ]2 -m e r c ap to e th ano l )a t100 °Cfo r4 m in ,and w e r eth ensub j e c t edtoe l e c t ropho r e t i cs ep a r a t ion ,c a r r i edou tu s ing10%(w /v )sod iumdod e cy lsu l f a t e (SDS )po ly a c ry l am id eg e l s .S ep a r a t edp ro t e in sw e r e t r an s f e r r edtopo lyv iny l id en ef luo r id e(PVDF )m em b r an e s(M i l l ipo r e ) , wh i ch w e r eth enb lo ck edbyt r e a t m en tw i th5%(w /v )non f a tm i lkinT r i s -bu f f e r eds a l in e w i thTw e en(TBST )fo r1ha troomt emp e r a tu r e . An an t i -NS1 mou s epo ly c lon a lan t ibody(g en e rou s lyp ro v id edby D r .J i aF an )w a su s eda sth ep r im a ryan t ibody (1 :1000d i lu t ion ) .A f t e rth r e e10 -m inw a sh e sw i thTBST , th eb lo t sw e r ein cub a t edw i ths e cond a ryan t i -mou s eIgG con jug a t edtoho r s e r ad i shp e rox id a s e(1 :5000 ,M i l l ipo r e ) a t37 °Cfo r1h .B lo t sw e r ean a ly z edu s inganIm ag ing D en s i tom e t e r(B io -R ad ,CA ,USA ) . RESULTS
P roduc t ionandcha rac te r iza t iono fNS1 mu tan t v i ruses F igu r e1 show sth es t ru c tu r eo fth er e comb in an t f e c t iou sc lon eandth eam inoa c ids equ en c e DENV-2in o f DENV -2 NS1 .As e r i e so f mu t an t so f NS1( th r e eCy s s i t e s[C4A ,C55A ,C291A ] ,two N l ink edg ly co sy l a t ion s i t e s[N130A , N207A ] ,ag lu t am i ca c ids i t e[E173A ] , andaC t e rm in a lt run c a t edf r agm en t[ΔC ] ) ,w e r econ s t ru c t edandin co rpo r a t edin toth ein f e c t iou s DNEV -2 c lon e . Mu t an tRNA sw e r esyn th e s i z edinv i t r oandt r an s f e c t edin toBHK -21c e l l s . Mu t an t s N130AandE173A Luc i fe raseassay T h et r a n s f e c t e dc e l l sw e r es u s p e n d e di n2 5mLDMEM show ednos ign ifi c an td i f f e r en c e scomp a r edto DENV -2 ec e l lsu sp en s ion(1 mL )w a s TSV01(WT w i th10%(w /v )FBS . Th )v i ru s e s ,bu tC4A ,C55A ,C291A ,andΔC
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3
Mu ta t ionso fDENVNS1a f fec tv i ra lrep l ica t ion
F igu re1 . Schema t icd iag ramo f DENV2 -TSV01andtheam inoac idsequence(aa807 -1 159 )o fthe NS1p ro te in . S y s tema t i cs i te -d i re c ted mu tagene s i so ftheN -g l y co s y la t ions i te sandC y ss i te s i sshownb ya r row s .TheC te rm ina l reg iono ftheDENV -2NS1p ro te in(aa333 -352 )tha twa sde le tedtogene ra tetheC te rm ina lt run ca ted mu ta t ion(ΔC ) isunde r l ined .Do t tedl inesdeno tethefo rma t iono fd isu lfidebondsbe tweenCysres idues .
e a chf a i l edtoc au s eCPEint r an s f e c t edc e l l sandd idno t g en e r a t eanyv i s ib l ep l aqu e s(F igu r e2A) .Inp a r t i cu l a r , th e N207A mu t a t ion , wh i ch w a sa l t e r eda tth es e cond NS1g ly co sy l a t ions i t e ,p rodu c edf ew e rin f e c t iou sv i r a l p a r t i c l e sandr e su l t edinsm a l l e rp l aqu e s .Twopo s s ib i l i t i e sm igh tb eadv an c edtoa c coun tfo rth eph eno typ e o fnop l aqu eg en e r a t ion :th ef i r s ti sth a t C4A , C55A , C 2 9 1A ,a n dΔCm u t a n tRNAc o u l dn o tg e n e r a t ea n yv i r a l p a r t i c l e s ;th es e condi sth a ta l though mu t an tRNA sw e r e ab l etog en e r a t ein f e c t iou sp a r t i c l e sth a tcou ldno tfo rm p l aqu e s .T ofu r th e rinv e s t ig a t eth ep rodu c t iono fin f e c t iou sv i r a lp a r t i c l e sfo l low ingt r an s f e c t ion ,sup e rn a t an t so lu t ion sh a rv e s t edf romt r an s f e c t edc e l l sw e r eu s edfo r th ein f e c t iono fBHK -21c e l l s , wh i ch w a sth enfo l low ed byimmunofluo r e s c en ta s s ay( IFA )an a ly s i su s inganan t i -E mAba t24 -hpo s tin f e c t ion .Con s i s t en t ly ,nopo s i t iv e IFAr e spon s ecou ldb ed e t e c t edfo rC4A ,C55A ,C291A , igu r e2B) ,d emon s t r a t ingth a ta l lo f o rth eΔC mu t an t(F th e s e mu t a t ion simp a i rth ep rodu c t iono fin f e c t iou sv i r a l p a r t i c l e s .Inadd i t ion , N130A , N207A ,andE173A mu t an t se a chshow edanapp rox im a t e ly5 -20 fo ldr edu c t ion int e rm so fth esp e c ifi cin f e c t iv i tycomp a r edto WTRNA ( T ab l e1) . Fo r mu t an t sN130A ,E173A ,andN207A ,w ea l soan a ly z edg row thk in e t i c sonV e roc e l l s .A sshowninF igu r e 3,nos ign ifi c an td i f f e r en c e sb e tw e enth e mu t an tand WT v i ru s e sw e r eob s e rv edint e rm so fv i r a lt i t e ra t24 ,48 , o r72 -hp . t . On lya t96 -and120 -hp . t .d idth e mu t an t v i ru s e sshows l igh t lys low e rg row thth an WTv i ru s e s . S im i l a rr e su l t sw e r eob s e rv edinqRT -PCRan a ly s e so f th esup e rn a t an tso lu t ion sh a rv e s t edf romc e l l sin f e c t ed w i th mu t an t s N130A ,E173A , N207A ,and w i thth e WT (d a t ano tshown ) . 4
Fo r mu t an t sC4A ,C55A ,C291A ,andΔC ,w ea t t emp t edtos e l e c tfo ranyr ev e r t an t sbyb l indp a s s ag ingo fth e sup e rn a t an tso lu t ionf romt r an s f e c t edc e l l s . How ev e r , ev eninth esup e rn a t an tso lu t ionf romth ef i r s tp a s s ag e , th ein t r a c e l lu l a rv i r a lRNA sw e r eb e lowth el im i to fd e t e c t ion .Th eRNAcop i e sth a tw e r ed e t e c t ab l einth eP0 sup e rn a t an tso lu t ion m ayh av eb e enr e s idu a linpu tRNA s T ab l e2) .Co l l e c t iv e ly ,th e s er e su l t sd emon s t r a t edth a t ( th eC4 ,C55 ,C291 ,andC t e rm in a lr e s idu e so f NS1a r e e s s en t i a lfo rv i r a lr ep l i c a t ion . E f fec to fNS1 mu ta t iononDENVRNAsyn thes is inBHK -21ce l ls T ofu r th e rd e t e rm in e wh e th e rth ed e f e c tinv i r a l r ep l i c a t ion w a sdu etoth eabo l i t iono fRNAsyn th e s i s , alu c i f e r a s e r epo r t ingr ep l i cono f DENV -2(R lu cR ep ) TSV01s t r a in w a su s edtoqu an t i fyv i r a lt r an s l a t ionand RNAsyn th e s i sw i thr e sp e c ttoth e NS1 mu t an t s .A l lth e r ep l i con sg en e r a t edas im i l a rl ev e lo flu c i f e r a s ea c t iv i tya t2 -6hp . t .( F igu r e4) ,ind i c a t ingth a tnon eo fth e mu t a t ion sa f f e c t edth et r an s l a t iono fth einpu tr ep l i con RNAandth a tth eya l lpo s s e s s eds im i l a rt r an s f e c t ione f fic i en c i e s . N130AandE173Ar ev e a l edtyp i c a ltwo -p e ak lu c i f e r a s ep a t t e rn s wh i l e N207Ar ep l i c a t edab i ts low ly wh encomp a r ed w i th WT ,r ep l i c a t iono fo th e r mu t an t s w e r ed e c l in eds ign ific an t lyinc e l l s .T ak entog e th e r ,th i s w a scon s i s t en tw i thth er e su l t sob t a in edu s ingth eg e nom e l eng thRNA s .Th e s er e su l t sind i c a t edth a tth eCy s s i t e sa tC4 ,C55 ,andC291andth eC t e rm in a lr e s idu e s o fNS1a r er equ i r edfo rv i r a lRNAsyn th e s i s . Ana lys iso fNS1in mu tan tv i ruses Th er e su l t sshow edth a t ,a sinth ec a s eo f WTRNA , V IROLOG ICAS IN ICA
J ing j ingFane ta l
F igu re2 .A :P laque mo rpho logyo f mu tan tand WTv i rusesinBHK -21ce l ls ;B :IFAo fVe roce l ls24ha f te rin fec t ion w i th mu tan tand WTv i ruses . Redind ica tesEp ro te ininin fec tedVe roce l ls(N130A ,E173A , N207Aand WT ) .The IFAsca leba rrep resen ts20μm .O the r mu tan ts(C4A ,C55A ,C291AandΔC )gene ra tednov is ib lefluo rescence .
mu t a t ion s N130A ,E173A ,and N207Ae a chg av er i s e toan NS1b andco r r e spond ingtoabou t90kD a ,r ep r e s en t ingth ed im e r i cfo rmo f NS1 .Incon t r a s t ,no NS1 exp r e s s ioncou ldb eob s e rv edinc e l l st r an s f e c t ed w i th C4A ,C55A ,C291A ,o rΔC mu t an t s ,ona c coun to fv i r a lr ep l i c a t iond i s rup t ion .Inth ec a s eo fth e N130Aand N207A mu t an tv i ru s e s ,i tw a sno t ab l eth a tth e NS1d i www .v i ros in .o rg
m e rm ig r a t eds l igh t lyf a s t e rdu r inge l e c t ropho r e s i sth an th e NS1d im e ro fe i th e rth e WTo rE173Av i ru s e s , mo s t l ik e lydu etod eg ly co sy l a t iona tth eco r r e spond ingpo s i t ion s( F igu r e5) .A l thoughth el ev e lo fNS1exp r e s s ionin N207Aw a sl e s sth anin N130A ,E173A ,o r WTv i ru s e s , i ti sc l e a rth a tg ly co sy l a t iona tpo s i t ion s130and207i s no te s s en t i a lfo rv i r a lr ep l i c a t ion . 5
Mu ta t ionso fDENVNS1a f fec tv i ra lrep l ica t ion T ab le2RNAcopyana lys isfo rthe mu tan ts
T ab le1De te rm ina t iono fw i ldtypeanda l l mu tan tst i te r RNA
a t i te r
CPE
N130A N207A E173A C4A C55A C291A ΔC WT
2 .80x107 1 .20x107 5 .45x107 2 tec tab le )
