AD-753 289

JAPANESE ENCEPHALITIS VIRUS REPLICATION:A PROCEDURE FOR THE SELECTIVE' ISOLATIONAND CHARACTERIZATION OF VIRAL RNA SPECIES

E., Zebovitz, et al

Naval Medical Research Institute

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17 February 1972

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JAPANESE ENCEPHALITIS VIRUS REPLICATIONt A PROCEDURE FOR THE SELECTIVEISOLATION AND CHARACTERIZATION OF VIRAL RNA SPECIES

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MEDICAL RESEARCH PROGRESS REPORTit av ?PO•miss •Pottl nata. middle Enitial, la5st1 ame)

E. ZEBOVITZe J. K. L. LEONG# AND S. C. DOUGHTY

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Reprinted from: Archiv fur die gesame BUREAU OF MEDICINE AND SURGERY (NAVY)Virusforschung 38, 319-327(1972) DEPARTMENT OF THE NAVY

WASHINGTON , D. C. 20390I I AITkAC TThe viral RNA specier synthesized in a porcine kidney cell fin e, PS(Y-15), by

Japanese encephalitis virus (JEV) are described. Virus titers on these cells rangedbetween 106 to 107 p.f.u/ml at the end of 2 to 3 days incubation at 35% C. Actino-mycin D9AD) mycin D(AD) could not be used to unmask JEV RNA syntehsis since itinhibited virus replication at concentrations necessary to substantially reducehost cell RNA synthesis. Treatment of cells with yig AD/ml and removal prior toinfection permitted good JEV replication, and at the same time strongly suppressedsynthesis of 28 S and 18 S cellular ribosomal RNA. The problem of separatin gviral RNA from non-ribosomal RNA that was still being synthesized by AD pretreat-ment cells was resolved by the isolation of the cytoplasmic membrane fraction ofinfected cells. RNA extracted from the membranes of infected Ad pretreated cells andanalyzed for sedimentation characteristics on sucrose gradients has four RNAspecies not found in uninfected cells. They are, (1) 45 S. single stranged RNAbelieved to be the infectious RNA found in the viron; (2) a 27 S RNA single strandedRNA (3), a 20 S ribonuclease resistant RNA believed to be double stranded and(4) and 8 S RNA species. The RNA species found in JEV infected cells, except forthe 8 S form, have been found in group A arboviruses. The procedure describedutilizing AD pretreatment of host cells and the separation of the cellular cyto-plasmic fraction may well have value for cells and the separati6n of the cellularcyto-plasmic fraction may well have value for the study of the biosynthetic eventsinvolved in the replication of other animal viruses that are inhibited by AD.

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- ~1. Japanese Encophalitit'VirusR . NA Synthesis

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Archiv fir die gic:1tokVirusforschui~g t8, 319-327 (1972)

© by SpringerVrhlag 1972

"J ,pakim ese-E neephslitis Virus Rleplication:A Procedure for the Selectiiv Isolation

and Characterizationf.0Viral RNA Species1

E~. ZiFIIOVITZ,,j. K%. Ld. LEXW1N2, an1d 8. C. lDouGITVDeoartmnentof NMirobiologyv, Nfý'al Medical lecsarich llnstituo,j

National NaV1aVMcdail Cvnltei' Beoitlesda, Moryhiti , U.S.A.

R"eceived Februnary 17, 11972) DQ .

Sitnuuinry

I h11b viral RNA species synthesize.d ill a porcine kidney cell line, PS(Y.15), bytpalvive encepihalitis virus (JEW) arc desoribed. Virus titers on these cells ranged

behowen 100 to I0, pf0.n.1ml at, the enld of 2 to 3' days iuclabation at:351 ( Actino.mychinl) (AlD) could notb e used to unmask .1] IV NA syatthe:is sinNe it, inhinbitedvirus replication at, concentrations nevessar. to i•bstiantially reduce host, cellRNA synthesis, 'l'reatment.of cells with I tig A1)/mil and removal pri-or to infectionpermitted good .1 EV replication, andi at, thesame t estronlgl.,i'illpressed synthesisof 28 8 and 18 89 eelinlar rkliosomal RNA. The iroblemi of separat ing viral RtNAfrom non.rilosomal ICNA tiat. was still being syntlhesized by Al) pretreated cellswas resolved by the isolation of the £'ytoplasminll membranle fraction of infectedcells. ItN1A extracted from the membranes of infected ADI) pretreatecd cells andanalyzed for sedimentation eharacteristices on sucrose gradienlts has four R1NAspecies not. found in uininfeeted cells.l'hlaeyare: (1) 45, singlest randed lRNA believedto be the infectious RNA found in the(, virion; (2) a 27 S RNA single strandedRNA, (3) a 20 8 rilonuioleaso resistant, INA believed to be double stranded and(4) an 8 8 IRNA species. The •NA species fomud in ,1IV infected clls, except. for(lhe 8 S form. have been fonnl in grouip A arbovirtises. 'l'he proce(dirc ese,4ibedutilizing AD) pretreatmeut, of host cells and the separation of thie cellular cyto.plasmnic fraction may well have value for the study of the biosynthetic events ill.v'olved in the replication of other animal viruses that are inhibited by AD).

I Tlbhis work wa, support,(l Iy he I Ilivllu of DleiIieiu icin ido( 8iugeiy, l)epa ii Ilnt oftho Navy, Project. M It 04 1.05.01(00ll II :13(0,I.

Tho opinions or assert ions cotll iiine livivili ire ithe llrivatet ones of lh(' iiiathors filid1110 niot, to eo colst riell ais official or "-flectinig the viows of Ieho Navy l)epari eni, ofilhos iNavall Service at, lillage

"I Post doNioridl Rleseaichi lA moilate. Natioullifl I rl•e-ai Coill, ils upported by thoBureiaui of Medicinl iud tii(lrt'ry, Depairtmiut of tho Navy.

my-SaAMkIK11 0

VPANOU9~3 2~) 0 4E. Z'ssOO'xTz, J.,K. L.LioNo, mid S. C.-Douuiir:

ly......I..... ...... .Soevral Hbnili~cd(N)species synthesized inl vcrlkcbratoceOlls infectedDiSIBIBUT IOYLABILII wiith~yarlous group A-arbovirtmes !utv-p'becn isolated and~chmaractcrfred (6, 8, '15,

Slist. A"iLs'c 6)._IFotr kpOc~ d v~iral RNA have been~scparated-and classified oui tito basis oftheir biolo"gical pro,-ertics, sedimentatioii properties fit sucrose gradients andL.. clectrophbi etic mobilities ill aeryhinide gels: a) a 45 S single stranded RINA whichlit;intfectious anld is founi;441 the mature viri~on, b) a 27S single strand~ed 'RNA of

reiia iuointermnediate aMid (1),a1 remct e'on.Tl prceimo sequence of fkyxlthe-

sNA was t rese t. RApodult in strace munt ifat. ll.

acionla ofuc ain ycin1e(r). ne with tudes hvat er nim RNA n s ipaes eeeinktis viru (J1V, a group 13 arboviruti s iv, to 1) srorngl inhi bitedb thxl8sis (ctg..

Stadm d met beds ofort't3 S yinfectiou l RNA,_ 0S yntltesi S utijiin as) toinhibit.hostceollp A syntheisim could6 not RNA howe ved with 3EV. ob rttveyrs

RNhiws preport. onlysaih ta jrce amunt ifo iniitin all. el 'hoonasythesis l)y petratmenof-iiIovit. of cels gllerie y o d to inecio wrtsist)aan the seetile

searationo vi ral (JEW) f1 romp1 arbovlirus ton~iooa besrg y ihbthed isltino thesdrg

cytop~latsmic membrane fraction.

A lporeiaio kiducy statue cell linio desigautted l1 I's (Y- 15) wa-Is Selected becaluse it, ispartic aularly well Suiited for plaique formnat iou by JEV. (13). COll were growion Ol aiglo'sminimiala eselat jil aileielillu (1E)Supplemennt ed wVitl It10% Calf Seam n faid 0.5%)filet albunilal hyd rolysalt on 6(1i waui t immao cualture a plaites for' 3 to 4 days ait 37' C' in aoiitt aosphleao of caeirb)on d ioxido inl air.

.1 l.V straaina Ynaelidat of unakatownl pa saigo listory was obalitnied froma D~r. Vil lillianA lleni. Viruas seedfs conisist ed of 101,,' lra in sus1peaisioais of Stuckl ing aaaieo inifected iat ix.acerebraailly.

2.3. Vi ria Ti tratijouisCoatfiawait. imnoaolaiyers of VIS (Y15) cells Were iaioeulaitted w%!it Ia0.1 faill vrduuaas of

virais dihatt ed in lolll at p o~plna -ffercd sal iaa (11138) 1)1 7.4 coaltatianinag 0.751% bovaaiosciean11 alhuaaiai. D)ilut ioas Nveai kept. ill illa ice hathI tuntil tas.-id, un was aittSorbed tiacoils for- I to 2 houars aint, 35' C' atud overlaiyedl witha 5 fil of at amediumt coaapogedofMEcoantainiaag 1.00% calf seata aat aad 0.81%i aigarosi. Laalturem wvow hiaacbat ed for 4 duiys at3V~C iaifall iaeiubat or suppl)1ied with 50 o, cial aon d joxido i at air, A siemoud uver'layCoalsimtilig of 5 ailt of I ;10,000 dilutionaa of aicut tail red .1 I .01", Nolli;-'s igna'. wats adlded.

I ~~~~'Nlio platcea wororetoiratad to t la :154 Chinctibittr atod tli. 4rlpara.scounted thu3(oilowiiig (lily.

!;4. In fectiont wtid AD_ WPrvt,trel tihdnat -.

thio~wlit ifdoIi!3ln wqjatrinrolac)'d from tii lf''5 Idilfy n 'elic citni'tr a

tatdfor oo. inil ;te',it 3 fill of I jig/pil AD) in P1381. The (Irig' was Foanove0d bytji)10rlt iota lutd I ho~ c(Ais ashmqd wiwaco with I 113. '('Ito cialttirt's4 wero ilifc'cto('at. a1 om11t i*pieiity~of IpAflaque forrainj_ aiiti (). f.1.) per coil. The v~irum wnts allowed to jidsorb for'

no1'~ h caIdtuvii ýr~$wf)tliuu overlat.evi with,5puil iatsacahni ~~rlalt116itiiliuia (141), pt0118ec front Grand Islanda Iliological Co., soplelol~laiittod AithI 10"Icalf ao tioi find ifjaieoi.etitcc,t3 5* C widi 5%' ciii'oii (liux ido in itir. Girowtht inediitito wamt

1l'0liilov0( (ll -(lo sec0q' filta-iy fill(,[ rt)~epICOeiwit h :1 oallpkdei 111,11 wit hiid, calf micrunbcoititaining 5 1±Ci 311 .15.uaiditio/oda. Caaltt'c~s Were roiiaculaitd eitad 350 C for 2laioui's.Jfl&cells wero-luatrv'o'ed by tscrajpilg into~ the) tliecilit kwith a*Iil'r )~ciiOiidsci

aioten d ' Iby Cc 'iifriftigait io . 'Ili coil pojfi'lt. wias was5l~~liv t ~b Ite w ithS11 . Theli stib'se tacaatI)1ocvdtilreAwer perforini ind -ill tOice b~athI.

2.5. P1'ropaa'ait-ioii~ofCv-t~oplatsaoai,o Meaaibratao Cont atining Fl'itet'ioitThe PBS3 watlslie(Iel weia' stispedncec inl roticulocyto antauidard buttfer (R1351) (0.01 %1

TritAllCi 0.01 it KCI -, .5 x 10-3 ml 1'ilP11,, 1)1 7.4),aifill atliowea to swell for 10 ininittes.4'i'lio cells wore riptutrtol witht 25 ad rok' if at closely fit~ting 1)ounc'o laoingeitii'.er. Laarge

wIac_(-otita',ifolgo([!(MOf(jiolpt1,8iaco)alt. 1t 4$.0Q0 x ggfor s10ýiaadaaitcs.. The:1-s ic'lta1111"x 11t.

dOnsistedc lairgely of cellI ineinaaiies'aiaa u l pr iobaibly inclll(kiiid Infocholdioici'an11(1 850110rifioso-itiek. Th'lis nuataeriad will ha' designaattedi as the ey t bil imi 'c iIniolil-ralao cont ainaing(103C) fratction. Approxiiaiattdy l.e x-107 to :3.0 x 10? cells Wvorued 15( iluthli prepatrat ioolof 0h0 CIC fraactioln.

2.6l. ENtractioia of R1NA front 010 Fract ioinIlailaiceliately aifter its isoha I1(111, (110 CIC 'tract ion was resuspciided inl 201nil of

0.0)2 %1 phlosphlate buhffer coldtatining 10-3 m 11)'A p11'7.4 (5). find extractedl twive withf 20naii of cold waitor-satidratted redcist illed jili101Conicit aininag 0.1!,% s0(hll deldecyl

mialftate. To precipitate the RINA, (lt ~ie a(uaŽti plaiso obtained atter the second plienoiext ractioq was-addevd to 3..5 volitaiaes oef'colc1 95", etllaiaol coniatininlg O.;l' 3)otfssinabfaacot utt an hldO( 111itt - 20' C foi- at ianifinuain of 2 hiours. 'Thle sedineot, wjas collect cd-byceattrifuigat ion, dlissolved~ inl 30 11111111, precipitiatted atgatin aolld Stoared fitt - 20 C.

2.7. Sucerose Doosi ty Grad ieat. AunaysisIt NA derived a; ailaovo froant OMC tract ionu mid pelleted( froilat ileolael by vcitt iifugat.

tion was dissolved ant 2 oil -l'13S, layered omi top of it ý28 fil sucrose gratdient, ((5 to 359,sasucrose iln R8B1 con taining, 0.15 mi NaCI) and ceittri fnged ill i SW 25.1 rot or ait 2:1,000r.p.1m. ill at Model 14 Spineo toir I8 hours. Chick cell RINA wais aidded to (lie CMC IONA(02lprov'idl 28 8 finda 18 8 rihosoanal IINA species as sedinieantat ion maatmkerm. Cradivitt,tutlms were pi'rcee fit ath (limttoll with fil 18 gatlgu nleedl~e; 30 drop fracttionas welocollectedl notd (lilutitil to 1.0 n11l volunies wit It P118. Thae atbseibatace titt 260( not1 tat eatehfraction was detei'iojn1ed to localize the positlo iof at (o clhick cell ribosoanatl RINA peincit.

Acid iitsoltiblo radiioactivit y wats ineatstred by precipitatinig ! Ito tract ioiis in, (aojiresoeiio of 5t)0 jig yealtt IINM its carrier withl 10a!,' trichloroatootio e dl ('VCA). Tiodotiealainlo the posit ioal of t ho rihonticeaso i'csit uitat, (lotble Mat nuded lviii R INA, eachfract ilat wats dlividled into two 0.5 aol aliquaots. Oino itlnitiot was t reated'( with I jigpitacrvaitt1 ribonticleaso tear :30 ainiittttes ait :17' C, ftndl the atcid insolubli, 11NA precipi.fated( its described atbove. The second Ai(iqitI. Wits h)rOd'0&4('dl Withleit rihonlklea1SOtreait iaiat.

'lhi 'lCA insolithie praecipiitaite s were collected ofil tiottibritne filtvirs of 3.0 jian poresize. TIlao moatiout of rtadioatttivit y t rappthed oal dlriedi tilt ers platcead in 10 fili of at iqjoicisciit 11tal. hi ct'slut ionl [4 gilo Oanai itltaur (NOW El'iglnit I NtaeVlla') ill (laR- litero t Oluclkito W1ts(loteri'aaiied With ia a ieeklml Laaa 1 250 I iqtial seiit illatIion spectroetrinc '.

'Sediliicid ntiot Coetffiienelts ~ero elltleftlielt i (It.V n01t 1111(10of 001iA'Rax ftold AMY.s (17)usinag chic'k eel I 28 faind IS8 ri hosonitil 11NA ats sa'd iiientatitt miltatrkers(.

-iik

322 B. ZES.OVITZ, J. K. L.,10146, and-8.1-C. Diiulrry:

31Th Efetof AD on JEV Replloation '

C. -PS(Y.15) cells support. good growth, of JEV and titers rahging between UP':andA07 PJ..fi.nil arm reachoU n6 the oten of 2 to 3 (days incubattion 4- 35* C.

AD lifts a pippounce~linhibitory effect uiion ~tho, replication of~ JEY ý(Fig.. 1).The idtibition is dose-depenident- and virus growth is lnlubioltedJihor than*.I 00%'N~ the presence of 1- tg AD/nil. Sevcral JEV strainis, available in-oIýkr laboratory,'Nakayazna,, Pelkig dtid a Taiwaln ,train 143 were also sengitive, to, AD *lion growni

C. '5

a.'

0 I 2 3,TIME (DAYS)

Flit,. IThioetfert of Al iioonijIW reilicattion o o'meli 15 s1~ i. 5 nil 111,11 iidii~tiite~ ~ii:cnoiomhe deetrod concentration or A I) ~a iins md to whd culture filer Infectioni, 5S(inil"e of jmooled aiii'dtiii1roi,*0loiltcato ciiltiv wer, taken ot diyltervuld and (ilaquo AMR)'. luertoriued as descriliod III MiaterlAts Ani~liidu

-. 0 m-alloiil. G-Di 0.1 RV!1iuIý a-0 1,0 pugfgo

on i'S(Y-l5)cllA.'Thcso results jindicate that -AD) can not'be used to 11tiuiask tJEWRNA syn'thesis since it inhibits vir 'i s replication at. lovoht necesary to olufficienitlyreduco host cell RNA synth6sis. Anothler method is therdlo)rc r~uquired to solebtlyoiyinhibit host cell .RN'A synflhesis.

3.32. f-rotroatmnont of-,Colls with AD)

It was shiowni that treathment of normal cells with Al) for a short Piirfid~ followedby its removal resulted in n-minarkedlly altered pattern of collular RNA 8ynithemis.Ribosoinal RNA synthiefis i, :.particularly 81onsitivo to AD) and renlailus inhi1bitedoven after the drug it; removiod (20). Reduction of the aiotunt. of riboson 'afl RNAnmade~ in infected cells is esential for the detect iou of viral ANA slinco the RNAspecies made in infected cells have sedimentation values very skhflhr to tho 28 Sand 18 S ribosomial RNA species.

To test the effect of pretreatment of 1IS(Y-l5) cells with Al) upon JHV replica.tion and1 cellular ribosoinal RINA stynthesis, the cells were incubated with hicreasinglevels of AD for (J0 minutes, the drug removedl and infected with JEW. Viralgrowth aiter pretreatmenlt with sieveral Al) coneeutiations is shown in F~igure 2.High concentrations of Al) from I to' 10 Rg/mil (lid not adlvemsly affect virus

IJilipanoso Encephialitit; Virus U~NA Simt hesis :323

,,roWffiý and titibis obtained itt 2' days equnalledl that of the unftreated control

I The scdineiitatidih elkaracturistics; of RNA obtained froin uninfected PS(Y-lfl)Jc61l8 treatedl with -1,1giml and 5 [Lg/mn'Al) in the manner descril)be aIov anid

7-

61-

5-

I I riME oQAYS)F'Iig,.~ Te ffectI (if p:rotat e~nt of I'8(y.t Ce3T14 wit I$ A D) liiioo tcrv Ili ofJH IV. (.7l~06%wro I real ed with u(I fieulCoio o-thitlb:: of*A D Iit 0 ml iI)18 for (i) l:Ites. Th'Illi ediluh: on rmiunm mitoit 1114VIIA WuAol~edieII to oih IIJIS befortIftrliol,. Plto Civro oryed %k [1115 "fill 111.11 11141111111 'A Ith 1 0% Calif SerlOWS1111:: I C:14e ~ ro t~Iirwt i:li

izo n iillIt Ittopht 0%r ctQ tketi clly. Viuctlr (0cil' t oyiii pI~Ilaiqto IOOiteomy wdtivalw I lii 310elsltc li ,iwl

incubated with radlioactive iuridiuie for 2 days nre iilohs ,tuisted in Figure:3. Thel AnortAl) treatment~ with I l.±g/In AD resultedl in it partial inhibitioni of 28 S and 18 Sribosoiial XN'A spnthesis and on increaso in the amount, of RINA that sedimenitsill the J5 to t0 $- region, Five jig ARl~ml almo~st comllIetely inhibited ribosomnalRNA Oynthesis, but. the afliount, of s]Qivly se'(illelltilg RNA was-Iltt itwitalsed

V.oota biild ihIjgM.Tepeslc fapeill 11titolnts of 1103).ribosomnal RNA\, 'jiowover, requlired all luldidlinal ;tioeedtite for separait ionl of hlost.

1.3. Isola t)ion of Vivil RiNA fromt -)M C Fraction

in RNA Virus1 repliCaltion (1, 9, 11). Viral protmin, RNA, arid 111N, i polylincilsoWere found -to bu assovialted with thist cell- fraction. JEWI~, like otlieR urboviruses.

dee opson the cytop)iil(,ll"ic mebralIei4 ( 18).Thi associat ion of viral HNA withIth Oytopthttiili ntivhmitriles seemedt to offer thi, emtica of separatiog vi'ir RNA

* from the celhular RNA that. is not. inhibited by short. treatmient with AD.RNA extracted fromn the, 010 fraction of nornal awlt infected cells conitainied

aptproximantely 10 and 20 oj0 respvctively, oft lie n~ifoaet ivity origiititll. ineorporat.

.324 H. ZMHovrTZ, LLPK. L~. Lima~, aid S. 0.'Douqury:

ed into tho intAct cells, These dath inililaed ~btli the-p~rocedifre of isoldtinti .0~0MI fraction prior to RNA extraction renuioives a iýistjt~'m u.dfiaRNA,

28S_

40-

Dc 3C

-, les

10. 1v

00

C0 10 20 30 40FRACTION

Fla. 3. Theo effect of ti~earoint-tiio-f I el WaI %I t1 lli AD l)I tpit lm~imisul IUVA i4'pillifth, CIcII tirw lr~III%lila Al) for tO mitiltt b. JI~i~lttl All %illI r01ui1101,14 by'4140 URA11' %lw it 1111m. Thio IIIxt %ere uliveyel: fi;b '4 -11l111,11 m($lu i(411 li111t 103 Td~Itr*~s o iiiIu~~ i l ( fot'2' flAys ltllxwotiinl RNA 1A 'tmia Irwtri luit ItiIIWI

rei Wilt ro 4'il'Iwn)l 1)) Itho j'rln41ra 41trllIK41 fur C.IV frrnllim %i Mateialsrhal d nijt )llIua.

rad(ioactivity recove'red front-the, control cells will much Itim tMon thaut, of InifectediWit; an no nijrpeakii A Obse lrvedi. Toamiount, fIt is elR Ao~

CO1C froction. Thei mtost, rilpidhly 8edintienting species, 415) S. hit approxltnatcly t hesamo sedimentattion coefficientit s that determined I)3IcmiAsIII d at. tot, the in.

feflious -RNA of JV H W0 ). The secondi major peak sedlinvoits att- 20$8 and isreeistanit to ri'oiuonleaso (Fig. 4 B~) and pmrobably rejpres'inta the dlouble st randedviralI RNA spec~ies. Tile two smoitli butt, (hiztilind peoikpi consitti of it 1271 S UNA,which correspiondcs to -the ono isolated in group A arbovirtmes bi nitwit largeramloutl~s, aund ani 8 S RNA which is; possibly of v'intI origin Initt whoso significancein JBV replication is not-yet- kaown.

AThe piossibility' that- the sepairation of tite 0110 fraction would be wuffideiit- toxsecetively isohitto JEW~ RINA 1trm~itlits front cellulamr ItNA widhout first treatning

4ýp1tnoso Eneophnllitis Virus UNIA Syntheigis, '320.,

Ith ccells wihlvADŽ *0 tv'dScil-diincntation pattern of RNA extracted froin'thoD(BIC-'fraction db introatco el pimaril of the two cpellilar

rib~malRNAspecies. Viral 4\.'A peaks were coinplcetbikiapkecd y. those 6f tho-dollular~RKA. AD.trc~tmcnt. oX,'-. coils prior-to infection is an essential

K>" ýqtpprims rijV(*omaI'RNA synthesi~s.28S 185 2LIS fis

jA 11

67 67 20s;

.4 40 27SU- as4

'3 -3

2

0 IQ 20 50 AO0 0 1'9 20 30 40FRACTI ON

AVW I frecotm 8114 coutrul uiirfcc&evll OV11 icotft'4itu 'A114 1 111 11 1AOsrQ thii~Intell it iS f 4C' !!r 41)' ttaf.%E%ecludiss Jahrlex stu A11 6VI..t~~~ for 0. l"msst. O'W flnw*k1t0'IA i UIrjk~ 6114 £tlaI)'lnt R&enMrik'4 fit

3ItatC11i4 31t h w .~link,L V',im) J1XA (W --- -o)l A ont rol tvl H.%k (a -0) i. It. M$9411rlroW rcthioniu rOl US~A I.'-1

T'he RNA slieceks syttliesized in 1JEW hifcted 18(Y~.15) colls are Aiunilar tot;OCO reported for group A arbovirunit's (0. 8. IS, 20). except tha~t the 1.111okint of'127 8 fiNA In relation to 45 8 and 20 8 flNA'is th0atare its tols com. ausocwlatied will)the 0,11 fracetion appearedl to be much smutaller ¶fbo low levcel of *27. ICXrecoovered inny be the result of the procedlure tnued for-the isolation or ohe vyto.lihtlatmic membranes. ])estrm.-tioni of the, 27 8 INA by~ (lie utiolva"e pre-mit fnt thecell extracts iW possibly not~ the complete e~litustiluti, biecause JINA rojiftid onthe cell memnbranes 6s believed to he redstauit to nuelvaso notion *,' 4 AIR- tit-,45 t9 RNA. a s1ingle atranded HNNA. appearm to be retaniaw4 ititact. fluiel'er, it is

K3.2P R1 Znvoviiz, J.,K. L. Lpo-xa, and S. 0. DoUonTY:

ýIki~ib ietat the 27 S RNA~is loosely boti idi to thlcienibraunc andl somec is releaspdfroiiiit-oariiig the i~olation procedumc Jf'this-is the case this RNA fraction Would(.bc'discArdcdio6r~ciposcd to nuclease digestion.-Attcznpts to detect this RNA in 'the cell extract, after remov.-IiA tile mnenibrane fraction ~havc failed. Finially, it ispossible that the lowiii ofd 27 (1 RNA a rje of J1V. A similar, observationwas mnadle for type 2 denmgue virus b3ySroiL;,Jfe (11. (22) who reported that 20 SRNýA was found inl infected cells inl very' small amounts or not at aill.

S$tudy qfRNA synthesis by JJhX was contplicatcd by the sensitivity of this virustoA).Jf i otuiuei lmt cpct. since a mnumbr of RNA viru.~cs arc known

to be inhibited,iiv AD perhaps for different ,reasons. Unldc cl"erl'oill conditions thlegrowth of somc 81"iiz'i of poliovirus (10, 19),"reovzrus (16),jniumpps virus (7), fowlIplague virufs (2), i.V:Inliocytie choriomneningitis virus (4), An~d- the R(NA tumorviruses (3, 24)-is inhibited by AD.

The effect- of AD) has been to.stedi with only a few members of group 13 nrbci.virusesq. lovols it) the-rauge of 0.01 to 0.03 jig/ul AD) have a slight stimulatingW-efft'lpl d1)11(engue (21) but inhibit virus growth at, concentratiojis inl excess of0.1 nfmnl. Ali enhancement effect by low AD concentrations has also been

t ile inhlibit ionl by-My of interferon qvuintlesis. St. Louis encelpha lit is v;Irus i's rep~ort ed

to repJlicato normially *n 13111C.21113 cells in-the pree'mnce of Al) at 0.5 [.tg/mll anldthle Sytinthe~ls of viral IINA was; studied un1dole condlitions inl which cellular RANAsynthesis was inhibited (2) h'i*sstem JBN1 was inhibited by Al) at, this conl-centrationl.

'rho muochanisnil of inhibit ion~ of J 8'N 6y Al) is not known. The j)ossib'lity thatta ;subtle cellular function or- viris (lif eCted activity may Ile involvedl is nlow b~einginvest igaied.

TheII procedurer t4l'-iVP fill the selective isolat ion-Lf .1 EN? 11NA utilizing Al)pretreatmneit of host, cells, anwilj iv s4epal-at ion of the celhtars {'ytoplastnic fractionmay well have Value for tho -AtIudy of thle biosynthetic evellto finolvedi inl thltreplication of other anlimlal virtwse. that- are inhioiited by AM)

Tihe auithors det-ply aij'plinviale tlmt, cmxelleiit Wtelolical imhlsisteon& of Mr. Hieliard

(Uryt fond )~If. ltodiwty T. Miller.

I. 11 ut.Tmstons. D... It. .1. lEoovinsI. .31. lRNKISI3II, 11311d 1. l'.lM~m: l'OliOVirulS-imulurdlItNA poiymetruir' andl t-fiets of 01-1111 Specifie 111ibiliors oil it product ion. Proe.

2. AiclxV J. M): ThPla VffVtMt of aet anOmycýink 1) 110 1d h1W raiOIle irra'ldialtion Oil 0h0lproudI~tivion st fow~l jplwigmi virus. Vurolog.N 24. 503i- 6011 (196(4).

:1. l1AsuF. It. M'~. md A%. 8. Kvusew luhihibmon of Itatirielier lanrino loukemin virosgrowlvii tii vitro 1hý 1itauct ). Vlr,irt.gv 32, 1 75- -18:1 (1907).

4. lHvvii, L. 1111 ad C.J.. PFA. _ lahuibitioa of lympluoeyluc elloriolaeningitis virusn IIat1)11 li3ne 11t13031113 Crum 1) #tilt; ̀ anummridmv. Virology 37, 698- - 701 (1960).

51. CituoI5,.1. L. 11iuwl .. M1 I Immi r: Factorsf v~t.3Iwlg p~laq~ue formuat lot3 by t he infect ioulsriwluoletatleivid(1 .fir life J*~lIcecli nu s~~.. infect. l31m. 114, 61 68 (I964).

11, 1lutams. P_. wind I.E1'1.lE l'r3Jlrtmits of4218ni.dl t1 S S33(b35viral rilionuelvci

Japaniese Huccplialif i' Virus "NA Synthesis 327

7. E~AST, J. L., anid D. IV. Kinusnuitv: 1tinps virus replication inl chick emlbryo lungcails: Properties of KNA in virions and infected cells. J. Viroi. 8, :,31-173 (1971).

8. Fitixz)MAN, 11.2%., H. 13. Lnvv, a11d W. 13. CARTER: Replication of Seinliki Fores'viruts: Three forms of viral RNNA produced1 during infection. 1'roc. tint.. Acaid. Sci.(Wash.) 56, 440-446 (1900).

9. GIRARD, M.L, D. BAi.TItORP, alld( J. I. D)ARN r,: Tileo poliovirtis replication coin.plox: Site for- Synthesis of polioviruis RNA. J. mimlec. Bioll. 24, 59-74 (1907).

10. GRADe, C., S. FISCERin, an 'd 0. CONTnR.:AS: The ihibibtion by actinoinycin D of

Y, 11 . 'iohoviri an I. 31. Fam'DMAN: Cytopla~smic structures

- ~~12.JOAlRASmi, A., F. FuUKN~AA, an1d K. FUKA!: Sietiinentation charact~oristics ofJapanecso encephalitis viruis RNA. Bikezi J. 7, 111-119 (1904).

13. Iioux, Y. K., and 11. A. YAMiA6A: Clonal lineo of porcine, kidney stable cells forassay of JaIpaneso encejphaitiits viruis. J. ilact. 87, 12:19-1240 (1004),

14. LEVENTOW, L., anld J. .1. IliSuoi: Comparative Aispects of poliovirus replication.J. cell. comp. Physiol. 70, 265-272 (1970).

15. IRVIN, J1. G., and It. M. FitimiAx: Analysis of arbovirus ribonueleic acid formisby polyncrylainido gel olectrophoresis. J. Virol. 7, 504- -.514 (197 1).

10. ],oi, P. C., andl( M. Soraomi: Growth characteristics of reovirtis typo 2: Actino.mnycin 1) and synthesis of viral RINA. Proc. tnat.. Acadi. Sci. (Ws .) 4, 857-803(110m).

17. MARTIN, X. G., anld 11. N. A3IKs: A method for determining thle Sedimeneattionheluwvior of enziymes: Application to p~rotein mixtures. J. b)iol. Client. 236,1372-1:379 (1901).

18. OTA, Z.. Electron micro~scope studly of the dlevelopmienit of Japanese B encephalitisvirus tin porcine kidney stable (P'S) cells. Virology 25, 372-378 (1905).

19. SCHAFFERi, F. L., and 31. GORD ON: Differential inhibitor~y affects of actinozaycin 1)among strains of jpeliovirlls. J. IBact. 91, 2309-2310 (1900).

20. Seunixumm-nnn, A., It. C. HlANIDL, andl( S. CmiAVAwmIC: Actiziou'ycin D): RenewedRN' sytheis fte reova frm ~aznnalan ell. Siene 12, 77-579 (1971).

21. STOidUA, V., TP.31t. STE.VENS, and It. W1. Sell taFslsitu:: Studies onl thle nature ofdengue virutses. 11. Chiaracterizat ion of viral HNNA anzd effects oi inhibitors of RNA

22. iatis Virology 30, 30:3- :311- (1960). Srvix:tuesnthnareo2.STrmIm.An, V., It. IV. ScuLMIN61-an, and V. M. T N:SidCQ1tleial' O

(leiguo viruses. 111. WNA synthei-sS ink Cells infected With type 2 dengne viruses.Virology 33, 650-058 (19167).

*21 Tnnm'r, 19, AV., C. C. Swwsr, andl A, A. Quartsuz: Syntheivs of Saint, Louisonzcepimio'4 viro rbenveiileieactiil inl 131 -2 1/13 cells. J. Virol. 3, 385-41394 (1969).

24. Vmoirit, P., and it. t'.7ffocts of acet inonlycinl 1) anid o" Imuitoinycil C Onl thle(leveloJpinent. of Hoits sarcojnam Virology 23, 511 -- 519 (11)(14).

'25. YAMA~ZAKI, S.: Effect, of endogenous ',iterlemin ýtmd actinemaycin 1) oil growth of'Japanleso encephalitis v'iruzs inl chick emlbryo cells.311i1). J. Mlig-robiol. 12, 17 1---1 78(1130).

26. U1mOVI'rZ, EL., mand A. llnowx : Altered viral rihonueloic, avid synthesis byv a temper.ature-scnsit ive mutant of (vastprii equiiod eneephialit its virus. J. inmoiee. lial. 50,185--198 (1970).

Auithors' addre-ss: D)r. Btnii1i: ZFI1OVmrZ, DIN11arlaaezit of Microbiology, NavalMed~ical Rtesearchl Institute, 13~miat l.111 M) 20014, UJ.S.A.

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