Characterization of a temperature-sensitive defect of enterovirus 70: effect of elevated temperature on in vitro transcription.

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0022-538X/84/070192-07$02.00/0

Characterization of

a

Temperature-Sensitive Defect of

Enterovirus

70:

Effect of Elevated Temperature

on

In

Vitro Transcription

KIKUKO MIYAMURA,* NAOKAZU TAKEDA, AND SHUDO YAMAZAKI

Central Virus Diagnostic Laboratory, National Institute of Health, Musashimurayama, Tokyo 190-12, Japan

Received 22 December 1983/Accepted 21 March 1984

A crudereplication complex prepared from enterovirus 70-infected cellswasusedtostudy the

temperature-sensitivecharacteristic of the virus. The complex showedatemperaturesensitivity in the in vitro incorporation ofradiolabeled ribonucleoside triphosphate. The endonuclease itself did not account for therestricted RNA synthesisatthenonpermissivetemperature. Analyses of the in vitro products by both gel electrophoresis and sucrose density gradient centrifugation showed that the complex synthesized three typesof viral RNA only when incubated forashortperiod of timeatthe nonpermissivetemperature.When thereplication complexwas

treated with a detergent (deoxycholic acid), incorporation of ribonucleoside triphosphate into RNA at the permissive temperaturewasreduced to the level of thatatthenonpermissive temperature. In addition, the in vitro RNA synthesis by the enterovirus 70 replication complexatthepermissivetemperaturerequiredahigher

concentration of ATP than of other ribonucleoside triphosphates, whereas suchapreference for ATPwasnot

found in the reaction atthe nonpermissive temperature. The results indicatethat the initiation stepof RNA synthesis by the complex is blocked at the nonpermissive temperature. The possible implications ofthese findings arediscussed.

Enterovirus 70(EV70), acute hemorrhagic conjunctivitis

virus, is a naturally occurring temperature-sensitive (ts) virus, which suddenly appeared in 1969 among people in a

pandemic fashion(14, 18, 20). Itis interestingtoinvestigate

the possible mechanism by which the virus appeared so

suddenly and with such a unique pathogenicity as that

causingconjunctivitis.Sofar,onlytwotypesofpicornavirus

are known to be majorcauses ofconjunctivitis, i.e., EV70

and avariant of coxsackievirus A24(19). Since thets nature of the virus is reasonably consideredtofacilitateits growth inconjunctiva, westudiedthemechanism by which the virus

exhibits the tscharacteristic in its growth and attempted to locate the site of the ts lesionin viralreplication.

Inprevious studies (22, 25), the following were found. (i) Novirus-specific RNAwas synthesized in the

EV70-infect-ed cells at anonpermissivetemperature. (ii)Inthe presence

of preformed viral mRNA, synthesis and the subsequent cleavage of virus-specific proteins occurredat a

nonpermis-sive temperature. (iii) Early translation ofviral mRNA at a

nonpermissivetemperaturewasdemonstratedby viral

poly-meraseactivity in theinfected cells. From theseresults, we

concluded that the ts lesion of the virus resided in its transcriptionalstage. Inthe presentstudy,RNAsynthesis of

EV70 at anonpermissive temperature was analyzed in the

cell-free system by using a crude replication complex

pre-paredfromEV70-infected cells.

MATERIALS AND METHODS

Chemicals. BovinepancreaticRNase(type1A), unlabeled

ribonucleoside triphosphates, and deoxycholic acid (DOC)

were purchased from Sigma Chemical Co., St. Louis, Mo. Trichloroaceticacid,sodiumdodecylsulfate(SDS),andTris were from Wako Pure Chemical Industries, Osaka, Japan.

Phosphoenolpyruvateand pyruvate kinasewerefrom

Boeh-ringerMannheimBiochemicals, Mannheim,WestGermany. Dithiothreitolwasfrom SeikagakuKogyo, Tokyo.

[3H]GTP

(10 Ci/mmol),

[3H]CTP

(22 Ci/mmol), and

En3Hance

were from New England Nuclear Corp., Boston, Mass. Seakem

* Correspondingauthor.

Agarose wasfrom FMC Corp., Marine Colloids Div., Rock-land, Maine. Actinomycin D was from Makor Chemicals,

Jerusalem, Israel.

Cells and viruses. The viruses used were the prototype

J670/71 strain ofEV70 and its warmth-adapted clone

artifi-cially induced by serialpassagesofthe prototypeatelevated temperatures (10 passages at each of 37 and 39°C and

another10 passagesat40°C);theclonegrewbetterat39 than at 33°C, and its optimaltemperature for in vivogrowthwas

38°C. The poliovirus included was the Mahoney strain of

wild-type 1. The viruses were propagated in LLC-MK2 cell monolayers with Eagle minimum essential medium.

Preparation of crude replication complex. The procedure wasessentially the same as thatof Yin and Knight (30). The cells were infectedwith the virus at amultiplicityofca. 40

PFU/cell. The EV70-infected cells were incubated at 33°C,

whereas the cellsinfected with thewarmth-adaptedstrain or

polioviruswereincubated at37°C for5 h.The infected cells werescraped,pelletedby low-speedcentrifugation(600x g)

for 5 min, and stored at -70°C. Approximately 4 x 108 frozen cellsweresuspended in 10 ml of cold buffer (0.01 M

Tris, 0.01 M NaCl, 0.0015 M MgCl2, pH 7.4)to be swollen

for10minonice andwereruptured by15strokes inaglass

Dounce homogenizer. The unbroken cells and the nuclei

wereremoved bycentrifugationat800x gfor5min, and the supernatant fraction was centrifuged at 30,000 x g for 20 min.Thepelletwassuspendedin4mlof buffer(0.05MTris,

0.01 MNaCl, pH 8.0)and storedat -70°C. This

suspension

contained ca. 1 mgofproteinper ml and servedasthe viral

replication complex unlessotherwise indicated.

Assay for polymerase activity. A 50-,ul protein of the

replication complex was mixed with an equal volume ofa standard assay mixture (double concentration of the final reaction mixture) containing the following constituents un-less otherwise indicated: 100 mM Tris buffer (pH 8.0); 40 mM KCl; 10mM

MgCI2;

2.5mMphosphoenolpyruvate; 13 mMdithiothreitol; 10

jg

ofactinomycinD perml;50 ,ugof pyruvate kinase per ml; 300 ,uM each of ATP, CTP, and

UTP; and 20 FtM

[3H]GTP

(20

p.Ci/ml).

The mixtures were incubated in a water bath, and the temperatures were

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maintained within an error of less than ±0.05°C. After a

desired period, the incubation was terminated by spotting

the mixture onto DEAE-cellulose disks (Whatman DE 81; Whatman, Inc.,Clifton,N.J.), whichwerethensoaked for 5 min at room temperature in a2.5% Na2HPO4 solution. The wash was repeated four more times in Na2HPO4 and twice each in waterand ethanol. Thedried disks werecounted in toluene-based scintillation fluid.

Sucrose density gradient analysis ofin vitro RNA product. The enzyme reactionwasterminated by the addition to the

reactionmixture ofan equal volume ofTNEbuffer (0.01 M

Tris, 0.1 M NaCl, 0.001 M EDTA, pH 7.4) containing 1% SDS.

RNA was extracted three times, each with an equal

volume of a phenol mixture (50 parts phenol,

48

parts

chloroform, and 2 partsisoamylalcohol,saturated with TNE buffer andcontaining 1 mgof 8-hydroxyquinoline perml) at roomtemperature.The aqueousphasewasadjustedto0.2 M

sodium acetate-70% ethanol, and RNA was precipitated overnight at -20°C. RNA was collected by centrifugation

(10,000 x g, 15 min), dried in vacuo, dissolved in acetate

buffer (0.1 M sodium acetate [pH 5.3], 0.1% SDS, 0.1 M

NaCl, 0.001 M EDTA), and cast on 12 ml ofa 5 to 25% (wt/wt) linear sucrose density gradient in acetate buffer.

Centrifugation wascarriedout at200C for15 hat50,000x g in a Hitachi RPS 40T rotor. Thegradient was collected in

0.5-mlfractions from the bottom. Eachfractionwasdivided

into two equal portions. One portion of each fraction was

diluted with10volumes of 2x SSC(lx SSC is 0.15 M NaCl

plus0.015 M sodium citrate, pH 7.4) and treated with 40

p.g

of bovinepancreatic RNase A per mlat37°Cfor30 min. The other portion remained untreated. RNA from each portion wasprecipitatedwith5%trichloroacetic acid in the presence

ofbovine serum albumin and filtered through a glass-fiber

7

6

-2

A..

filter (WhatmanGF/C)before counting with a liquid scintilla-tion counter.

Agarose gel electrophoresis. Electrophoresis was carried outunder nondenatured conditions asdescribed by Hewlett et al. (13). Agarosewasmelted in Ebuffer (40 mM Tris, 10 mM acetate, 1 mM EDTA, pH 7.3) containing 10% (vol/vol) glycerol. Agarose gel (1%, wt/vol) wascast in a horizontal slab gel apparatus (130 by 130 by 1 mm) and prerun in E buffer at 70 V for 1 h. The ethanol precipitates of RNA

species werewashed with95% ethanol, driedin vacuo,and

dissolved in 10 ,ul of 0.1 M Tris(pH 7.5) containing0.15 M NaCl and0.5%SDS. Thesamples which weremixedwith 5 ,ulof 60% (wt/vol) sucrose containing 0.2% (wt/vol)

bromo-phenolbluewereeachlayeredontothegel under therunning buffer. Electrophoresis wascarried out at 75 Vfor 4.5 h at room temperature. Forfluorography,gel slabs were

impreg-natedwithEn3Hance,dried in vacuo,and exposed toSakura

X-ray filmfor 4daysat -70°C.

RESULTS

Temperature dependency of viral RNA synthesis. EV70 does not synthesize virus-specific RNA in infected cells at 39°C. The effects of temperature on viral RNAsynthesis in

thecell-free system were studied from the kinetics of

incor-porationof[3H]GMP intoRNAproducts(Fig. 1). The EV70 warmth-adapted strain and the Mahoney strain of poliovirus type 1 were included in the experiment as controls; both

viruses can replicateat 39°Cin vivo.

The incorporation of

[3H]GMP

by the polymerase

com-plexextractedfromJ670/71-infected cellswas almostlinear

at 33°C for 60min. At 37 and 39°C, the reactionduring the

first 15 min proceeded similarly to that at 33°C but was

severely impaired thereafter. The incorporation entirely

ceasedat 39°Cafter incubation for 30 min.Thus,the pattern

0 30 60 90 120 0 30 60 90 120 0 30 60 90 120

incorporation

( min )

FIG. 1. Effect of temperature on [3H]GMP incorporation. LLC-MK2 cell monolayers were infected with strain J670/71, its warmth-adaptedstrain, or the Mahoney strainofwild poliovirus1andincubated for 5 h. The infected cells were collected and disrupted to make the replication complex. Portionsof reaction mixture (100,ul)containing50,udof each of thereplication complexand thestandard assaymixture wasincubatedat 33, 37, or39°C.Samplesweretaken at 15-minintervals,and the totalradioactivity incorporatedinto the RNAproductwas

determined. Theradioactivity ofthecomplexat0hwasconsideredtobenonspecific bindingandwassubtractedfrom each value.(A)J670/71, (B)warmth-adapted strain, (C) poliovirus. Incorporationat33(0), 37(A), and39°C (0).

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of the incorporation curve at each temperature seemed to faithfully mimic thekinetics of RNA synthesis as well as of viral growth in the infected cells (21, 25). In viral RNA synthesis by the replication complex preparedfrom warmth-adapted and poliovirus-infected cells, the linear incorpo-ration curves of [3H]GMP which were similar to that of the J670/71 complex were obtained at 330C. The inhibition of the incorporation at both 37 and 39°C, however, was much less than that of the J670/71 complex. Actually, the reaction proceeded progressively even at 390C, although the incorpo-ration was markedly reduced.

Since the incorporation by the EV70 complex entirely ceased at 39°C after incubation for 30 min as described above, the following experiment was carried out to deter-minewhether the replication complex contained a thermola-bile component or whether a cellular endonuclease was preferentially activated at 39°C, resulting in the digestion of RNA synthesized during the reaction. The complex pre-pared from J670/71-infected cells was heated for 60 min at 39°C. The heated complex was then mixed with a reaction mixture and assayed for the heat-resistant transcriptional activity from [3H]GMP incorporation for 30 min at the permissive temperature. The complexes of the warmth-adapted clone and the poliovirus were also tested as con-trols. When incubated at 390C for 60 min, the J670/71 complex retained40% of the [3H]GMPincorporation of the unheated control. At the same time, incorporation by the complexof the warmth-adapted clone also was reduced to a similar extent (to 38% of the unheated control) and that of the poliovirus was even more severely impaired (to29% of the unheated control) after heating at 39°C for 60 min (data notshown). Thus, the temperature defect of EV70 replica-tion could not simply be explained by the thermolability of itspolymerase or by activation of the cellularendonucleases at the elevated temperature.

Analysis of RNA products of the replication complex. It has beendemonstrated that crude replicationcomplexes derived from thepoliovirus-infected cells synthesize three speciesof viralRNA, i.e., single-stranded RNA, replicative intermedi-ate, and replicative form (RF) (12). The products of the EV70 replication complex synthesized in the cell-free sys-tem at both permissive and nonpermissive temperatures were analyzed by agarose gel electrophoresis. Figure 2 shows the comparative gel patterns of RNA products syn-thesized in 30, 60, and 120min at both 33 and 39°C; for the product at 33°C, all three species of viral RNA are demon-strated. The longer the incubation period was, the higher

was theyield. The products at390C also gave clear RF bands with similar intensities to those at 33°C. However, the replicative intermediate or 35 S RNA wasbarelydiscernible

in the products incubated at 39°C. The results were also

confirmed by acrylamide gelelectrophoresis of the products (data not shown).

Tocharacterize the in vitro RNA products more quantita-tively, sucrose gradient analysesof the productssynthesized in vitroat both permissive and nonpermissive temperatures were carried out(Fig. 3). In the products produced at 330C,

increased amounts of viral RNA ofboth 35S and 20Sspecies weredemonstrated, confirming the results shown by agarose gel electrophoresis. The products at 39°C also contained both 35S and 20S RNA. However, the amounts of RNA collected from 60- and 120-min incubations were quite

similar to that produced from 30-minincubations,indicating

that synthesis of all RNA species stopped shortly after the initiation ofincubation.

Effect of DOC on polymerase. The treatment ofthe

cyto-33tC

39'C

30' 60' 120' 30' 60' 120'

-RI

___ _mm Em --RF

-35S

FIG. 2. Agarose gel electrophoresis of the products of the J670/71replication complex synthesized at permissive and nonper-missive temperatures. Portions (1 ml) of the reaction mixture containing 500 ,ul of the J670/71 replication complex and 10 ,uM [3H]GTP (30

VtCi/ml)

wereincubated at 33 or 39°C. The reaction was terminated after various periods of incubation by the addition of TNEbuffer containing 1% SDS, andRNAwasextracted from each reaction mixture. Electrophoresis was performed on1%agarosegel slabs for4.5 h at 75 V, andthegel was fluorographed. The viral RNAinthegel waslocated by coelectrophoresis of in vivo labeled viral RNA. The temperature and the incubation period (in minutes) foreach productareshown inthefigure.

plasmic membrane fraction of the infected cells with a

detergent such as DOC orNonidet P-40

effectively

dissoci-ates the virus-induced

RNA-polymerase

complex

(9). It has been suggested that in vitro viral RNA

synthesis by

a

detergent-solubilized complex

might

belimitedto

elongation

of the RNA chains already started (3, 10), lacking the

initiation of transcription. Therefore, further evidence was soughtbyanalyzing the effect ofanelevated temperature on the reaction by the complex treated with a

detergent.

The

addition of 0.25% DOC to the

replication

complex

of the

J670/71strain, followed

by

heating

for 3 minat

33°C,

reduced the incorporation ofGMP at

33°C

to the level of

incorpo-ration at 39°C, thus

entirely

abolishing

the

preferential

incorporation ofGMP at a

permissive

temperature

(Fig. 4).

The result indicates that a certain functional factor or

structure required for complete RNA

synthesis

ata

permis-sive temperature is

destroyed

by

the

detergent.

Atthe same

time, the result suggests that the

elongation

process is not

prevented at an elevated temperature.

Requirement of ATP for in vitro transcription. In the

procaryotic

DNA-dependent

RNA

polymerase

system, the two events during transcription, initiation and

subsequent

chain elongation,havebeendifferentiated

by

kinetic

analysis

of the initial

velocity

of the reaction at different substrate concentrations (1).Thedifferentiation has beenbasedon

the

facts that initiation ofin vitro

transcription

demonstrates a

stringent preferenceforpurine ribonucleoside

triphosphates

(rNTPs) at a high concentration and, in contrast, that the

subsequent RNA chain elongation

requires

low levels of

purinerNTPs. Consequently, the KmforATP atinitiation is much higher than that for the same nucleoside

during

elongation. The same

principle

has been used to

study

in vitro RNAsynthesis in thevesicular stomatitis virus system (29) and the rotavirus system

(24).

In the present

study,

we

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rf 3

0

X

0--I Z:

xL

_...

3

2

C

28S 18S

D

5 10 15 20 5 10 15 20

Fraction Fraction

FIG. 3. Sucrosedensity gradient analysis of products of the J670/71 replication complex. The reaction mixtures in 1-mlvolumeseach containing 500 ,ul of the J670/71 replication complex and 10 ,uM[3H]GTP (20 ,uCi/ml)wereincubatedat33 (A,B) and39°C (C, D) for various periodsof time.RNA wasextracted and fractionatedon a15 to30%sucrosegradient. One half ofeachfractionwastreatedwith40,ugof RNaseper ml (BandD), and allfractions were determined for acid-insoluble radioactivity. Sedimentation was from right to left. Arrows indicate theposition of 18S and 28SrRNApeaks. Products of incubation for30min( ),60mi ( ), and 120mi ( ).

alsousedkineticanalysistodetermine whether theinitiation eventis involved in the reaction in theEV70system atboth

permissive and nonpermissivetemperatures.

The dependence of the rate of reaction on the rNTP

concentration was examined for each of four rNTPsduring

the first 30 min of incubation, and the apparent K,, was

determined fromdouble-reciprocal plots. The ATP

concen-tration required for in vitro transcription by the EV70

replication complex showedbiphasic double-reciprocal plots (Fig. 5). At a low rNTP concentration, the apparent Km

valueswerealmostidentical forallfourrNTPs, i.e., 9 to 12 FiM. There was, however, a sharp increase in the rate of transcriptionwhen the concentration of ATPwasincreased to ca. 20 ,uM or more in the reaction mixture, whereas no

changewasobserved in the apparentKmfor the other three rNTPs at any concentration tested. Consequently, the Km

forATPattheseconcentrations wasapproximately fourfold

greater, i.e., 43 ,uM, than thatforthe other three rNTPs or

for ATP at concentrations less that 20 ,uM. The results indicate thatanincreasedfrequency of initiationwas expect-ed tooccur at such high ATPconcentrations.

To examine the effect ofan elevated temperature on the

reaction, the apparent Km at 39°C was detertmined. The reaction was carried outfor 10 min, duringwhichperiod of

timeidenticalratesof rNTPincorporation wereobservedat 33 and 39°C (Fig. 1). No increase in the velocity of the reaction was observed at 39°C, even at a higher ATP

concentration (Fig. 6). The result indicates that an

ATP-dependent process, probably initiation, of viral RNA

tran-scription was inhibited atthe nonpermissive temperature.

DISCUSSION

The results reported here show that the transcription in thecell-freesystembyacrudereplication complex prepared

-CL)

B

0 30 60 0 30 60

IncorPoration

( min )

FIG. 4. Effect ofDOCtreatment onJ670/71replication complex. The J670-71complexwas mixed with anequalvolume ofTris buffer (pH 8.0) containing 5% DOCand then heatedfor3min at33'C.The DOC-treated complex was mixed with an equal volume of the standardassaymixture,andincorporation of

[3H]GMP

at33(-)and 39°C

(0)

wasdetermined asdescribedin thelegendforFig. 1. (A) Untreatedcomplex, (B) DOC-treatedcomplex.

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II I I

-100 -50 50 100

l/rNTP (

r-l1

)

FIG. 5. Effectof the rNTPconcentrationon the EV70 RNApolymerase activity.The 100-,u reaction mixtureseach contained50,u1 ofthe EV70 replication complex and the fourrNTPs. [3H]GTPwas added at 10 ,uM(820cpm/pmol), except when GTPdependence was to be measured. ForGTP dependence,[3H]CTPwasused at 10,uM (870cpm/pmol). OtherrNTPs wereincludedat150p.Mexcept XTPwhichwas varied(5to 500p.M). The reaction mixtures wereincubated at33'C for30min.Theradioactivity incorporated intothe RNA product was assayed and expressed as a double-reciprocal plot ofthe rate ofreaction (picomoles of [3H]GMP incorporated/30min) versus the XTP concentration.TheapparentKm values obtainedfor each XTP were asfollows:ATPreactions(0),K,, = 42p.MinthereactionathigherATP concentrations than20p.Mand12p.MatlowerATPconcentrations than20,uM; GTP reactions(A),K,, = 11 FiM;CTPreactions (A), K,, =10 ,uM;UTPreactions (0),K,, = 9 AM.

from EV70-infected cells mimicsfaithfully the in vivo tran-scription, confirming our previous findings that the ts event of the virus ingrowth resides in its transcription. Thus, the system has proved to be useful in studying the ts nature of thevirus.

To analyze the transcriptional events, four identifiable steps have been considered: binding of polymerase to a template, initiation of transcription, elongation of the RNA strand, and its termination. Although itis well known that thepolymerase of picornavirus synthesizes its RNA in a cell-free system, little has been reportedon the involvement of theinitiationstep in itstranscription. Recently, Takegamiet al. (26) statedthat acrudemembrane fraction isolated from

poliovirus-infected cells synthesizes a nucleotidyl

protein,

,_a-p

-Ia- 0

i.e., the 5' termini of a nascent RNA strand. In this report, wealso presentedexperimental evidence which supports the

involvement of the initiation step in transcription by the

crude replication complex prepared from EV70-infected

cells.

First, from analyses ofthe in vitro products by sucrose

gradient centrifugation andby gelelectrophoresis, all types

ofviral RNAwerefound to be continuously synthesizedat the same rate during the test period, suggesting de novo initiation of RNAtranscription. Second, another support for

involvement of the initiation was provided by the kinetic study onthe

requirement

ofATPin thereaction. The study

was based on the fact that the two eventsduring

transcrip-tion, invitro initiationandsubsequent chain elongation,are

-75 -25 10 20 30 40 50 60

l/ATP ( mm-l )

FIG. 6. Effect of elevatedtemperature onapparentK,, forATPof the EV70RNApolymeraseactivity.Thereactionmixtures in 100-p.l quantitieseachcontaining50p.loftheEV70replication complex,10p.M

[3H]GTP

(728cpm/pmol),150p.Meach ofCTPandUTP,andvarious concentrations(5to500

p.M)

ofATPwereincubatedfor 10 minat33 and39°C.Theapparent K,, valuesweredeterminedasdescribed in the legendforFig. 5.Reaction at33°C(0),K,,=40p.Min thereactionat ahigherATPconcentration and 12p.Mat alower ATPconcentration;

reactionat39°C(0),K,,, = 13 p.M.

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successfullydifferentiatedbytheirrequirementsfor different ATP concentrations, such as those in a procaryotic

DNA-dependent RNApolymerase system(1)and in the vesicular stomatitis virus system (29). In our experiments, the Km for ATP at ahigher ATP concentration in in vitro EV70 tran-scription was nearly fourfold greater than that for the other three rNTPs, suggesting the occurrence of the initiation of

transcription on the template. Interestingly, at lower ATP

concentrations, the Km was reduced to an almost identical

leveltothatforthe other rNTPs.Thisbiphasicpatternofthe

transcriptionrateondifferent levels of ATP concentration is

quitesimilartothatreported byTestaand Banerjee(29)with

the preinitiated complexof vesicularstomatitis virus. Such

resemblance inthepatternofthetranscriptionratebetween

the two complexes was consistent with the fact that the EV70 replication complex consisted largely ofendogenous template-binding polymerase preformedinthe infectedcells, comparableto that in the

preinitiated complex

ofvesicular

stomatitis virus.

The results of the

comparative experiments

ontheeffects

of different temperatures strongly indicated the lack of

initiation oftranscriptionatthe

nonpermissive

temperature. In contrast tothe continuous

synthesis

ofthree RNA

mole-cules atthe permissive temperature, RNA

synthesis

at the

nonpermissivetemperatureceased inashortperiod of time, andthepreferential requirementfor ATP in the reactionwas not observed at the nonpermissive temperature. Further-more, basedon the

suggestion

ofother

investigators

(3, 10,

17) that the detergent-treated complex supports only chain

elongation, identicalRNAsynthesisatbothtemperaturesby

theDOC-treatedcomplexprepared from EV70-infectedcells

indicates that the

elongation

process is not

prevented

at an

elevated temperature.

Fromthese results, in vitro RNA synthesis bythe EV70

complex at the

nonpermissive

temperature could be

ex-plained as follows. During a short period of time after incubation, the RNA chain elongation,

having

already

start-ed, continuedtoproceed, and nascent

single-stranded

RNA

dissociated from thetemplate,

producing

35SRNA. Howev-er,thesubsequently

elongating

strand couldnotbe released from the template, possibly due to base pairing with the

template bysome yet unknown mechanism.Thus, all

repli-cative intermediates turned to RF, having no newgrowing

strand onit, resultingin thecompleteblockofRNA synthe-sis. Many in vivo and in vitro studies of

picornavirus

replication have shownthat single-stranded RNA synthesis

is extremely susceptible to such factors as an elevated temperature (4), detergents (10, 16, 17), guanidine (28), hydroxybenzimidazol (7), and the antibody directed against

thehost cellfactor(5, 8). In most cases,

synthesis

ofRFwas not affected. Thus block ofsingle-stranded RNA synthesis

and accumulation of RF in viral transcription, as seen in

EV70 replication at the nonpermissive temperature, might be a phenomenon rather frequently observed under the

condition in which RNA synthesis of picornavirus is im-paired.

Recent advances in studies on theinitiationstepof

picor-navirus RNA replication in vitro have been focused on the roles of the two factors which are associated with de novo

synthesisof RNAreplicase.Oneis a hostfactor(s)which has been shown to be necessary for the initiation of RNA

synthesis by the purified poliovirus replicase (2, 6, 8). The

factor was found to bind to viral polymerase in vitro (5). Thus it might be possible to inhibit the transcription if the

interactionof the hostfactorwith another component of the

EV70-replication complex is susceptible to an elevated tem-perature.

Another factor which is considered to play a role in

initiation is a small, virus-coded protein, VPg, covalently bound to the 5' termini of picornavirus RNA (11, 15).

Nomoto et al. (23) proposed the following model in which VPgfunctionsas aprimer forRNA synthesis. DuringRNA

strandelongation,theRNA-linked VPg remains attachedto

thepolymerase, preventing the nascentstrandfrom anneal-ing to the complementarytemplate RNA. When the

RNA-linked VPg dissociates from polymerase after the initiation ofRNA synthesis, the nascent strand may anneal to

tem-plate RNA, generating double-stranded RNA, RF. Accord-ing to this model, the effect ofan elevatedtemperature on EV70 transcription may be explained as follows. If the linkage between VPg and polymerase is susceptible to an

elevated temperature, VPg

might

dissociate from

polymer-ase during elongation at a nonpermissive temperature,

re-sultingin thegeneration ofRF.Another

possibility might

be adefectin thecleavage oftheprecursor

peptide

ofVPgat an

elevated temperature.

Takegami

et al. (26, 27) recently presenteda model in which VPg is present in the initiation complex as part ofa precursor

polypeptide

and is cleaved

from it after the initiation ofRNA synthesis. Thefailure of this cleavage at an elevated temperature could inhibit the

formation ofafunctional form of VPg.

Further clarification is required for a conformational as well as afunctional relationship of VPgand thepolymerase of EV70 with other components present in the initiation complex, i.e., functional templates andhostfactor(s).

ACKNOWLEDGMENTS

We aregratefultoT.Yamashita, Daiichi College of Pharmaceuti-calSciences, for his useful advice forexperimental procedures.

This workwassupportedinpartbyaGrant-in-Aidfor Scientific Research from the Ministry of Education, Science and Culture, Japan.

LITERATURECITED

1. Anthony,D.D., C.W.Wu,and D. A.Goldthwait. 1969. Studies with the ribonucleic acid polymerase. II. Kinetic aspects of initiation andpolymerization.Biochemistry8:246-256. 2. Baron,M.H.,and D.Baltimore. 1982. Purification and

proper-ties ofahost cell protein requiredfor poliovirus replicationin vitro.J. Biol. Chem. 257:12351-12358.

3. Baron,M.H.,and D. Baltimore. 1982. Invitrocopyingofviral positive strand RNA by poliovirus replicase. J. Biol. Chem. *257:12359-12366.

4. Cooper,P.D.,D.Stantek,and D. F.Summers. 1970.Synthesis of double-stranded RNA by poliovirus temperature-sensitive

mutants. Virology40:971-977.

5. Dasgupta,A. 1983.Antibodytohostfactorprecipitates

poliovi-rusRNApolymerase frompolio-infectedHeLa cells. Virology 128:252-259.

6. Dasgupta, A.,P.Zabel,and D. Baltimore.1980. Dependenceof theactivityofthepoliovirus replicaseon ahost cellprotein.Cell 19:423-429.

7. Dmitrieva, T.M.,andV. I. Agol. 1974. Selective inhibition of thesynthesis ofsingle-strandedRNAofencephalomyocarditis virus by 2-(a-hydroxybenzyl) benzimidazole in cell-free

sys-tems.Arch. Gesamte Virusforsch. 45:17-26.

8. Dmitrieva, T. M., M. V. Shcheglova, and V. I. Agol. 1979. Inhibition of activity of encephalomyocarditis virus-induced RNA polymerase by antibodies against cellular components. Virology92:271-277.

9. Ehrenfeld, E., J.V.Maizel,andD. F.Summers. 1970. Soluble RNApolymerase complexfrompoliovirus-infectedHeLacells. Virology40:840-846.

10. Etchison, D.,and E.Ehrenfeld. 1981.Comparisonofreplication complexessynthesizing poliovirusRNA. Virology111:33-46. 11. Flanegan, J. B.,R. F.Pettersson,V.Ambros,M.J. Hewlett,and

D.Baltimore. 1977. Covalent linkage ofaprotein to adefined nucleotide sequenceatthe5'-terminus of virionandreplicative

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intermediate RNAsof poliovirus. Proc. Natl. Acad. Sci. U.S.A. 74:961-965.

12. Girard, M. 1969. In vitro synthesis of poliovirus ribonucleic acid: roleofthereplicative intermediate. J. Virol. 3:376-384. 13. Hewlett, M. J., S. Rozenblatt, V. Ambros, and D. Baltimore.

1977. Separation and quantitation of intracellular forms of poliovirus RNA by agarose gel electrophoresis. Biochemistry 16:2763-2767.

14. Kono, R., A. Sasagawa, K. Ishii, S. Sugiura, M. Ochi, H. Matsumiya, Y. Uchida, K. Kameyama, M. Kaneko, and N. Sakurai. 1972. Pandemic of new type ofconjunctivitis. Lancet i:1191-1194.

15. Lee, Y. F., A. Nomoto, B. M. Detjen, and E. Wimmer. 1977. A protein covalently linked to poliovirus genome RNA. Proc. Natl. Acad. Sci. U.S.A.74:59-63.

16. McDonnel, J. P., and L. Levintow. 1970. Kinetics ofappearance oftheproducts ofpoliovirus-inducedRNApolymerase. Virolo-gy 42:999-1006.

17. Meyer, J., R. E. Lundquist, and J. V. Maizel, Jr. 1978. Structur-al studies of the RNAcomponent of thepoliovirus replication complex. II.Characterization byelectronmicroscopy and auto-radiography. Virology 85:445-455.

18. Mirkovic, R. R., R. Kono, M. Yin-Murphy, R. Sohier, N. J. Schmidt, and J. L. Melnick. 1973. Enterovirus type 70: the etiologic agentofpandemicacute haemorrhagicconjunctivitis. Bull.W.H.O. 49:341-346.

19. Mirkovic, R. R., N. J. Schmidt, M. Yin-Murphy, and J. L. Melnick. 1974. Enterovirus etiology of the 1970 Singapore epidemic of acuteconjunctivitis. Intervirology4:119-127. 20. Miyamura, K., A. Sasagawa, E. Tajiri, and R. Kono. 1976.

Growth characteristics of acute hemorrhagic conjunctivitis (AHC)virusinmonkeykidneycells. 11.Temperaturesensitivity oftheisolates obtainedatvariousepidemicareas.Intervirology

7:192-200.

21. Miyamura, K., S. Yamazaki, E. Tajiri, and R. Kono. 1974. Growth characteristics of acute hemorrhagic conjunctivitis (AHC) virus in monkey kidney cells. I. Effectof temperature on viral growth. Intervirology4:279-286.

22. Miyamura, K., S. Yamazaki, E. Tajiri, and R. Kono. 1978. Growth characteristics of acute hemorrhagic conjunctivitis (AHC) virusinmonkeykidneycells. III.ViralRNAsynthesisat permissive and nonpermissive temperatures. Intervirology 9:206-213.

23. Nomoto, A., B. Detjen, R.Pozzatti, and E. Wimmer. 1977. The location of the polio genome protein in viral RNAs and its implication forRNA synthesis.Nature (London) 268:208-213. 24. Spencer, E., and M. L. Arias. 1981. In vitro transcription

catalyzed by heat-treated humanrotavirus. J.Virol.40:1-10. 25. Takeda, N., K. Miyamura, R. Kono, and S. Yamazaki. 1982.

Characterization ofatemperature-sensitive defect of enterovi-rustype 70. J.Virol. 44:98-106.

26. Takegami, T., R. J. Kuhn, C. W. Anderson, and E. Wimmer. 1983. Membrane-dependenturidylylation of thegenome-linked protein VPg of poliovirus. Proc. Natl. Acad. Sci. U.S.A. 80:7447-7451.

27. Takegami, T., B. L. Semler, C. W. Anderson, and E. Wimmer. 1983.Membranefractionsactiveinpoliovirus RNAreplication contain VPg precursorpolypeptides. Virology 128:33-47. 28. Tershak, D. R. 1982. Inhibition of poliovirus polymerase by

guanidineinvitro. J. Virol. 41:313-318.

29. Testa, D., and A. K. Banerjee. 1979. Initiation of RNAsynthesis in vitro byvesicular stomatitis virus. J. Biol.Chem. 254:2053-2058.

30. Yin, F. H., and E. Knight, Jr. 1972. In vivo and in vitro synthesis of human rhinovirustype 2ribonucleic acid.J. Virol. 10:93-98.