Specific Sindbis virus-coded function for minus-strand RNA synthesis.

(1)

0022-538X/81/080348-11$02.00/0

Specific

Sindbis Virus-Coded Function for Minus-Strand RNA

Synthesis

DOROTHEA L.SAWICKI,`* STANLEY G. SAWICKI,' SIRKKAKERANEN,2ANDLEEVI

KAARIAINEN2

Departmentof Microbiology, MedicalCollegeofOhio, Toledo, Ohio 43614,1 and Department of Virology, University of Helsinki, 00290Helsinki 29,Finland2

Received29January1981/Accepted20April1981

Thesynthesis of minus-strand RNAwasstudiedincell cultures infected with

the heat-resistant strain of Sindbis virus and with temperature-sensitive (ts)

mutants belonging to complementation groups A, B, F, and G, all of which

exhibitedanRNA-negative(RNA-) phenotype when infectionwasinitiated and maintainedat390C,thenonpermissivetemperature.When infected cultureswere

shiftedfrom28°C (thepermissive temperature)to39°Cat 3hpostinfection,the

synthesis of viral minus-strand RNA ceased in cultures infected withts mutants

ofcomplementationgroupsB andF, but continued in cultures infected with the parental virus and mutants ofcomplementation groups A and G. In cultures infected with tsll of complementation group B, the synthesis of viral minus-strand RNA ceased, whereas the synthesis of 42S and 26S plus-strand RNAs continued for atleast5 hafter theshift to39°C. However, when tsll-infected cultureswerereturnedto280C 1haftertheshiftto39°C, the synthesis of viral minus-strand RNA resumed, and therateof viral RNA synthesis increased. The

recovery of minus-strand synthesis required translation of new proteins. We

conclude thatatleastoneviralfunction isrequired for alphavirus minus-strand synthesis that isnotrequired for plus-strand synthesis. Incultures infected with

ts6 of complementation group F, the syntheses of both viral plus-strand and

minus-strand RNAswere drastically reduced after the shift to 390C. Since ts6

failed tosynthesize both plus-strand and minus-strand RNAs after the shiftto

390C,

at least one common viral component appears to be required for the

synthesis ofbothminus-strand andplus-strandRNAs.

As one of the initial steps in the infectious

cycle of the alphaviruses Sindbis virus (SIN)

and Semliki Forest virus, the parental42S ge-nomeRNAistranslated toyield aviral

RNA-dependentRNA polymerase whichutilizes the parental42Splus-strandRNA as atemplate for the synthesis of 42S minus-strand RNA. The 42Sminus-strandRNA in turn serves as a

tem-plate for the synthesis ofmore42S plus-strand

RNA and ofsubgenomic 26S mRNA,which is

translated into the viralstructuralproteins (15,

22, 29). During thefirst 3 hpostinfection (p.i.),

the rate ofsynthesisofplus-strand and

minus-strand RNAs increases; however, at

approxi-mately 3 to 3.5 h p.i., the synthesis of minus

strands ceases abruptly, whereas the synthesis

of both42S and 26S plus strandscontinues at a

constantratethroughout the infectious cycle (3,

25). During the early period of infection, the

synthesis of plusstrands occurs in about a

five-fold excess relative to minus-strand RNA

syn-thesis (25).

The RNA-dependent RNA polymerase

re-sponsibleforthesynthesisof 26S and 42S

plus-strand RNAsmustbestable,since theaddition

ofinhibitors ofproteinsynthesis after3h

post-infection does not significantly interfere with

plus-strand RNA synthesis (13, 25, 26, 30). We

haverecently shown that thesynthesis of minus-strand RNA in Semliki Forest virus-infected

cellsceaseswithin 15to20minafter the addition

of inhibitorsofprotein synthesis(25). Thus, the polymeraseresponsibleforminus-strand

synthe-sisdiffers from the viralpolymerasesynthesizing

plus-strandRNAbyhavingashorthalf-life.We have suggested (25) thatalphaviruses regulate

the rateoftranscription ofviral RNAby

regu-lating the number of minus-strand templates

and that thesynthesisofminus-strand RNA is

regulatedatthelevel of translationbya

mech-anism which utilizes one or more short-lived

polymerase proteins. One possible mechanism could involve an unstable or rapidly

turning-overpolymerase proteinthat isrequired onlyfor

minus-strandsynthesis.Wereport in this

com-municationtheidentification ofa temperature-348

on November 10, 2019 by guest

http://jvi.asm.org/

(2)

sensitive(ts) mutantof the heat-resistantstrain

of SIN (SIN HR) that selectively ceased

minVs-strand syntheis upon shiftingtothe

nonpermis-sivetemperature.

MATERIALS AND METHODS Cell cultures. Secondarycultures of chicken em-bryofibroblast (CEF) cells were prepared from pri-mary cultures of 10-day-old embryos obtained from the eggs ofleukosis-free flocks and were grown in plastic petri dishes inDulbecco modified Eagle mini-mal essential medium (DMEM) supplemented with 5% fetal bovine serum and 5% tryptose phosphate broth. BHK-21 cells, a continuous cell line derived from baby hamster kidneycells, were grown in plastic petri dishes in DMEM supplemented with 5% fetal bovineserum.

Virus. SIN HR and theRNA-negative (RNA-) ts mutantsused in thisstudywerethe generousgift of E. R. Pfefferkorn(Dartmouth MedicalSchool, Hano-ver,N.H.) and have been characterized previously (5, 6, 22,29).Growth andpurification of each virus stock anddetermination of its infectivity by plaque assayat both the permissive (280C) and the nonpermissive

(390C)temperatureswere aspreviouslydescribed(16). Infection and RNAlabeling.In all of the experi-mentspresented, with the exception of those in Fig.1 andTable 1,weemployedCEFmonolayers in 50-mm petri dishes infected with either SIN HRor oneof the mutants at a multiplicity of infection (MOI) of50. After themonolayerwaswashed with Hanks balanced salt solutionat 28°C, the viruswasadsorbedto the CEF cells for1hat280Cinaninoculum of0.5 to 1.0 ml ofcompleteDMEM (DMEM containing0.2% bo-vine serum albumin, 22 mM HEPES

[N-2-hydroxy-ethylpiperazine-N'-2-ethanesulfonic acid, pH 7.4],and

1 ug of actinomycin D per ml). At the end of the adsorption period, thevirus-containing medium was removed,complete DMEM at280C wasadded,and the cultures were incubated at 280C for the times indicated in thetext.Toaccomplishthe shiftto390C,

themonolayerswerewashed withHanks balanced salt

solution at 390C before the addition of complete

DMEMat390C.When culturesat390Cwerereturned

to280C,theywerewashedoncewithHanks balanced salt solution at 280C, followed by the addition of complete DMEMat280C.SIN RNAwaslabeled with

[5,6-3H]uridineat afinalconcentration of250,uCi/ml

per 50-mm petri dish, unless otherwise indicated in thetext.

Sinceourstudies involved the incubation of infected cellsat twodifferent temperatures,wedetermined the kinetics ofSIN RNAsynthesisatthesetwo tempera-tures. At various times after infection, SIN HR-in-fectedcells were labeled for60min with[3H]uridine, and the cultures were solubilized with 2% sodium

dodecylsulfate in ET buffer(0.01 MEDTA,0.01 M

Tris-hydrochloride [pH 7.4]). DNA was sheared by

passageseveraltimesthrougha27-gauge needle. Sam-ples were taken for protein determination by the method ofLowryetal.(17) and for total acid-insoluble radioactivity. Wefound, ashave others (15, 16, 22), that theentirereplicationcycletakesapproximately

twiceaslong when incubationisat280Cthanwhen it

is at 390C. When the maximum number of transcrip-tion complexes had formed and RNA synthesis was at aconstant rate(by 4 to 5 h at390Cand by 8 to 9 h at 280C), [3H]uridine incorporation into RNA during a 1-hpulse-label in cultures maintained at390Cwas twice that obtained from cultures maintained at280C(data not shown). Therefore, to adjust for the effect of temperature on the observed rate of transcription, a pulse of 60 min was given to cultures to be labeled at

280C,and a 30-min pulse was used for cultures labeled

at390C.

Isolation of SIN RF RNA. At the times indicated, the infected cells were washed once with ice-cold phosphate-buffered saline,followed by the addition of 2ml of lysis buffer (0.15 M NaCl, 0.01 M Tris-hydro-chloride [pH 7.4], 1.5 mM MgCl2) containing 0.65% Triton X-100. The cells were scraped from the surface of the petri dish and transferred to 15-ml conical centrifuge tubes; the solution was vigorously blended inaVortex mixer before being centrifuged at 250 x g for10minat40Ctopellet the nuclei. The cytoplasmic supernatantwasremoved and adjusted to 1% sodium dodecyl sulfate. The solution was extracted twice with phenol, followed by two extractions with chloroform-isoamyl alcohol (96:4). RNA was precipitated from the aqueousphase by the addition of LiCl to 0.2 M and ethanol to 70%. After overnight storage at-200C, RNAwascollectedby centrifugation in an SW41 rotor at98,000xgfor1hat0°C. The dried precipitate was dissolved in0.2ml ofdigestion buffer (0.15 M NaCl, 0.05 M Tris-hydrochloride [pH 6.8], 1 mM EDTA) anddigested with0.1 ugof pancreatic RNase perml atroomtemperaturefor15min.A solution of 0.05 M Tris-hydrochloride (pH 6.8) and 1 mM EDTA was addedtolower theNaCl concentration to 0.1 M, and ethanolwasthen addedto afinal concentration of 35% (vol/vol). The samples were immediately applied to columns of CF-11cellulose by the procedure of Frank-lin(11)for isolation ofreplicative form (RF) RNA.

CF-11 cellulose columnswerepoured in 3-ml plastic syringes (2.5 ml of packed CF-11cellulose) andwere washed first with modifed STE buffer (0.1 M NaCl, 0.05 MTris-hydrochloride [pH 6.8], 10 mMEDTA) containing 1%beta-mercaptoethanol, followedby ex-tensivewashing with STE buffer (0.1 M NaCl,0.05M Tris-hydrochloride[pH6.8],1mMEDTA) containing 35%ethanol. Thesampleswereappliedtothecolumns and washed with10 to 15mlof STE buffercontaining

35% ethanol, followed by an equal volume of STE

buffercontaining 15%ethanol;RFRNAwaseluted in STEbuffer. We routinely recovered 2 to5% of the

applied radioactivityasRF RNA. RF RNAwas

col-lectedby ethanolprecipitationinthe presence of0.2 MLiCl and25 Lgof carrierratliver RNA.

Thisprocedure quantitatively recovered RF RNA from cellsinfected with SIN HR or tsmutantsof SIN HR. Thesameamountof RF RNAwasobtainedby either thesucrose gradient method used previously (24, 25) or the CF-11 cellulose method of Franklin (11). Furthermore, bothofthesepreparationsofRF RNAcontainedthesameproportionoflabeled minus-strand RNA andsedimentedat15Sto20Sinsucrose

gradients. RFRNAisolatedbytwocyclesofsucrose

gradientcentrifugation (24, 25)boundtoCF-11

cellu-lose, and 100% ofappliedRF RNAwasrecoveredin

http://jvi.asm.org/

(3)

350

the STE buffer fraction. Less than 0.1% of labeled

single-stranded viral RNAwasfound to elute in the

STE buffer fraction after treatment with this concen-trationofpancreatic RNase.CellularDNA eluted in STEbuffercontaining15%ethanol and thus could be efficiently removed from the RF RNA fraction.

Quantitationof minus-strand RNAsynthesis.

Minus-strandRNAwasmeasuredby determiningthe

amountof[3H]uridineincorporatedintoRF RNA that hybridized to unlabeled 42S plus-strand RNA. The conditions of hybridization were identical to those reported byuspreviously (24, 25).Labeled RNA that

wasprotected frompancreatic RNasedigestionwas

takentobelabeled minus-strandRNA. Itshould be noted that this minus-strand RNArepresentedthat whichremainedaftertreatmentof thereplicative in-termediates with low levels of pancreatic RNase to obtain RF RNA.Such RNasedigestionwouldremove

any single-stranded, nascent minus-strand or

plus-strand RNA in thereplicativeintermediates. Materials. [5,6-3H]uridine (53 Ci/mmol)was pur-chased from Amersham Radiochemicals, London, England. Cycloheximide was obtained from Calbi-ochem, LaJolla,Calif.,andCF-11 cellulose was ob-tained from Whatman, Inc., Clifton, N.J. All other materialswerefrompreviouslydescribedsources (23-25).

RESULTS

Blocking of minus-strandRNAsynthesis

in SIN HR-infected cells by inhibition of

proteinsynthesis.Figure1showsthe effectof

blocking translation on the synthesis of viral

RNA(Fig.1A) and minus-strand RNA(Fig. 1B)

incells infected with SIN HR. In untreatedcells,

the overallrateofviralRNAsynthesisincreased

exponentially beginning at 1 to 1.5 h p.i. and

continued until3.5 to4hp.i., afterwhichtime it becameconstantorslowlydecreased(Fig.1A).

The rateofsynthesisof minus-strand RNA

in-creasedatthesametime that the overallrateof viral RNAsynthesis increased and ceasedatthe time that theoverallrateofviral RNAsynthesis becameconstant(Fig. 1B). When cycloheximide

wasaddedduring theperiodin which therates

of synthesis of viral plus-strand and

minus-strand RNAswereincreasing,theoverallrateof synthesis of viral plus-strand RNA stopped in-creasing and became constant or slowly declined

(Fig. 1A), and the synthesis of minus-strand

RNA ceased within 30 min aftertheaddition of

cycloheximide (Fig. 1B). Cycloheximidehad no

effectontheoverallrateofviral RNAsynthesis

whenit wasadded after the nornal cessation of

minus-strand synthesis had occurred and after

the overall rate of viral RNA synthesis had

become constant (data not shown). These

re-sults,which werethesameregardlessofwhether

CEForBHK-21cellswereusedfortheinfection,

demonstrated thatminus-strandtranscriptionin

SIN-infected cells was shut off selectively by

to

0

E

oU

z

2 /

1

E-II 2 _

en I I I I I I

-J B

WU120

a

10

8

64

6 CL

2

HOURS p.1.

FIG. 1. Rate of SIN HR RNA synthesis in the presence and absence of cycloheximide. BHK-21 monolayers in50-mmpetridisheswereinfected with SIN HR(MOI, 50) andweremaintainedat37°Cin the presenceofactinomycin D. At2hp.i., medium containingcycloheximide (100pg/mi)wasadded to one setofcultures(arrow). Bothsetsof cultures were

pulsedwith[3H]uridine(250yiCi/ml)at37°Cfor

30-minperiodsbetween2and5.5hp.i. and were

har-vestedimmediatelyafter the pulse. (A) Total [3H]-uridineincorporationinto viral RNA. Acid-insoluble radioactivitywasdetermined by counting a sample

aftertrichloroacetic acidprecipitation. The values

shown represent the total

[3Hluridine

in each

sam-ple; duplicate experiments gave similar results. (B)

Total[3H]uridine incorporation into minus-strand RNA. RF RNA in each sample was isolated by CF-11cellulosechromatography as described in the text, denaturedby heatingat1000C,and allowed to rean-neal in the presenceof an excess of unlabeled SIN HR 42S virion RNA. RNase-resistant,radiolabeled

RNAformedduring the hybridization reaction was

taken as labeled minus-strand RNA. Symbols: 0, untreatedcultures;0,cultures treated with cyclohex-imide.

cycloheximidein amanner identical to that

ob-served in BHK-21,HeLa, or Vero cells infected

withSemlikiForest virus (25). Thesynthesisof

alphavirus minus-strand RNA, therefore,

ap-pears torequirethecontinued synthesis of

http://jvi.asm.org/

[image:3.503.275.430.66.372.2]

(4)

SINDBIS VIRUS MINUS-STRAND 351

teins. To determine whether aviral protein is

responsible for minus-strand transcription, we

screenedseveraltsmutantsof SIN HRto

iden-tifymutantspossessingatsdefect thatmanifests

itself in the failure to synthesize minus-strand

RNA, butnotplus-strand RNA, atthe

nonper-missivetemperature.

Screening for SIN HRts mutantsthatare

defective in minus-strand RNA synthesis.

We screened sevenrepresentatives of SIN HR

tsmutantsthathavebeenassignedtofour

com-plementationgroupsthat giveanRNA-

pheno-type. These RNA- mutants fail to synthesize

detectableamountsofviral RNAwhen the

in-fection isinitiated and maintainedat390C,the

nonpermissive temperature. To identify ts

mu-tantsofSIN HR thatareselectively unableto

synthesize minus-strand RNAatthe

nonpermis-sive temperature, the cells mustbe infected at

the permissive temperature. The infected cells

arethenshiftedtothenonpermissive

tempera-ture at a time when minus-strand RNA

tran-scription is detectable. Thus, the RNA-mutants

canbecharacterizedby their abilityorinability

to continue synthesizing plus-strandor

minus-strand RNA. Nascent viralRNAand all

minus-strand RNAsarefoundaspartofthe replicative

intermediates. During apulse-label with

[3H]-uridine,theproportion of radiolabel incoporated

intominus-strand RNA relativetothat in

plus-strand RNA in the replicative intermediates

in-dicates the relative level ofsynthesis of

minus-strand RNA. During the period in which minus

strandsarebeingaccumulated, 40to50% of the

[3H]uridineincorporated into RF RNA derived

from replicative intermediates is in minus

strands, and this proportion isconstantforpulse

periods of 15 minto4 h(24, 25).

Monolayers of CEF cells were infected with

either SIN HR orRNA- tsmutantsand were

maintainedat280Cfor 3 h beforebeing shifted

to39°C.After30minat390C,

[3H]uridine-con-tainingmediumwasadded to thecultureswhich

weremaintainedat390Cforanadditional 2.5 h

before harvest.Viraldouble-stranded RFRNA

wasisolated, RNAwasdenaturedby heatingat

1000C, and the proportion of labeled

minus-strand RNA in RF RNA was determined by

hybridization in the presence of an excess of

unlabeled SIN HRvirion RNA.Table 1 shows

three tsmutantsthatappearedtobe unableto

continue synthesis of minus-strand RNA after

the shift to 390C: ts4, ts6, and tsll. RF RNA

isolated from tsll-infected cells contained the

least proportion of labeled minus-strand RNA

(about 6%);RFRNA isolated from ts4- and

ts6-infected cells containedonlyabout 10%labeled

minus-strand RNA. Theseresultswere in

con-trastwiththoseobtainedwith RF RNA isolated

TABLE 1. Analysis of SIN RNA- mutants for defects in minus-strandsynthesisa

Complementa- RN

ineedR

Virus

°tiopn

grmoeunp

RNA in

mu'nus-tionroup strand RNA

SIN HR 40.4

ts4 A 9.8

ts6 F 8.8

ts7 G 20.3

tsll B 6.2

tsl5 A 24.6

tsl8 G 31.6

ts24 A 36.2

aMonolayers of CEF cells in 50-mm petri dishes

wereinfected with either SIN HR or the ts mutants asdescribed in the text. An MOI of 50 was used for the B and G group mutants, and an MOI of 100 was used for the A and F group mutants. SIN HR was tested at both MOIs and gave equivalent results for minus-strand synthesis. The different MOI was used to maximize incorporation into ts4 and ts6 RNAs because of the reported effect of the shift to390Con RNAsynthesis (16). At 3 h p.i., the monolayers were shifted to 39°C and were further incubated at 39°C in fresh medium. Beginning 30 min after the shift to

390C,[3H]uridine-containingmedium was added (500

,uCi/2ml per petri dish), and the incubation continued for2.5h.Theinfectedcellextracts wereprepared, and RNase-resistant RF RNA was isolated and hybridized to an excessof unlabeled 42S virion RNA. The hy-bridization values shown represent the average of from two to five determinations. A control preparation of RF RNA from SIN HR-infected cells labeled with

[3H]uridinefrom1 to 5hp.i.at370Ccontained 49% of

thelabeled RNA in the RFs in minus-strand RNA.

from theremainingmutants (20to36% labeled

minusstrands) and SINHR(40%labeled minus strands).

A closer examination of these mutants was

undertaken.Pulses of[3H]uridineweregivenfor

30min atthe time oftheshiftto390Candat

30-minintervalsthereaftertocultures infected with

SIN HRmutantsts4,ts6,tsll,andtsl5and with

theparentalSINHR. Therateof RNA

synthe-sis inSIN HR-infected cells increased

approxi-mately 20-foldoverthe 3-hperiodafter the shift

to390C (Fig.2). However, inallof the

mutant-infected cultures, raising the temperature to 390C at3 hp.i.resulted inafailuretoincrease

substantially the rate of RNA synthesis. The

rate of RNAsynthesisintsll-infected cultures

increasedonlytwo-tothreefoldoverthis

period,

remained essentially constant in ts4- and

tsl5-infected cultures, and slowly decreased in

ts6-infected cultures.Weexamined theother

RNA-mutantsshown inTable1,and allof them failed

toincreasesignificantlytherateofRNA

synthe-sisafterthe shiftto390Cat3 hp.i.;their rate of

synthesis either was constant or slightly

de-creased over the 3-h period after the shift to

http://jvi.asm.org/

(5)

352

20

15

L-I

2n

I0

5

3 4 5 6

[image:5.503.64.226.55.402.2]

Hours,

post-infection

FIG. 2. Relative RNA synthesis in SIN HR- and mutantvirus-infected culturesat39°C. CEFcells in 50-mmpetridisheswereinfected with SINHRor one

ofthets mutants atanMOIof50andmaintainedat

28°Cuntil 3h p.i.,atwhich timetheywereshiftedto

39°C. [3H]uridine incorporation intoRNAat39°C

wasdetermined with pulses of [3H]uridine(250 ,iCi/ ml) for30min between3and 6 h p.i. The cellswere

harvestedasdescribed inthetext,and sampleswere

takenfor acid-insolubleradioactivity. Theamountof incorporation observedin cultures pulsedbetween3 and3.5hp.i. (shownbelow in parentheses)wastaken

as1.0, andtheamountof incorporationobserved in thesubsequentpulse periods foreachmutantand the parental virus is expressedrelativeto thisamount.

During the infectious cycle, [3H]uridine incorpora-tion intoSINHR RNAduringa30-minpulse-label

increasedbyanaverageof 22-fold (fiveexperiments ranging from 9- to 33-fold). Cultures wereinfected

with: A,ts4(133, 181 cpm);A,ts6(183, 848 cpm);0,

tsll(273,600cpm);x,ts15(230,854cpm);*,SIN HR

(241, 270 cpm); and EL SINHR incubated in the

presenceofcycloheximidefrom the timeof the shiftto

39°C (241,029cpm).

390Cat3hp.i. (datanotshown).The inability

ofthe RNA- mutants to increase significantly

thelevel ofviral RNA synthesisafter the shift

to 390C at 3 h p.i. was not unexpected. The

failuretoincrease therateof viral RNA

synthe-siscouldhave resulted fromafailure to

synthe-sizeadditionalminus-strandtemplates,afailure

to accumulate functional polymerase

compo-nents,and theinactivation ofpreviously formed

polymerase components that are temperature sensitive inthese mutants. Therefore, the rela-tive level of minus-strand RNA synthesis was

determinedfromRF RNA isolated from each of

the datumpointsshown in Fig. 2

(Fig.

3).

Be-tween 40 and 50% of RF RNA

synthesized

in

SIN HR-infected cells between1 and 2 hafter

theshiftto390Chybridizedtounlabeled virion RNA anddecreasedtoless than 1%by3h after

theshift.Therefore,theseresults

again

showed that theincreasingrateof SIN HR

plus-strand

RNAtranscriptioncorrelated with thesynthesis

ofminus-strand RNA. Cells infected with SIN

tsl5 of theAcomplementiongroup continuedto

synthesizeminus-strandRNAforanadditional

3hafter the shiftto390C(Fig. 3).Similar results

havebeen obtained for cells infected with SIN

ts18 of the G complementation group and for

SINts24of theA

complementation

group

(data

notshown). Unlike the resultsobtained forSIN

HR,tsl5, ts18,andts24,

only

asmall

proportion

60

5 50

40

3

40

E

C - 20

E

u 0

aI 0 A

0

3 4 5 6

[image:5.503.276.423.358.580.2]

Hours, post- i nfection

FIG. 3. Minus-strand RNA synthesis in infected CEF culturesshifted to390C.RF RNA wasisolated fromextractsof thecultures shown in Fig. 2 byCF-I1 cellulosechromatography. The percentage of [3H]-uridineincorporatedintoRF RNA thatwas in minus' strands was determined by hybridization as de-scribedin thetext. RF RNA was isolated from cul-turesinfected with: *, SIN HR;A,ts4;A, ts6;0,tsll; U, tsl5.

http://jvi.asm.org/

(6)

SINDBIS VIRUS MINUS-STRAND MUTANT 353

oflabeled RF RNA obtained from ts4-,ts6-,and

tsll-infected cells after the shift to390Cwasin

minusstrands(Fig. 3). Thus, minus-strand

syn-thesis ceased in ts4-, ts6-,and tsll-infectedcells

soon after the shift to the nonpermissive

tem-perature.

Analysisofplus-strandRNAsynthesis in

toll-infected cells.Wefocused our studies on SIN HRtsll,thesolemember of the B

comple-mentation group, that is, the only mutant in

whichminus-strand synthesisappears to be

se-lectively temperature sensitive. Although

mi-nus-strandsynthesis failed to continue at 390C

ineither ts4- orts6-infectedcells (Fig. 3), the ts

defectdoes not appear toberestricted to

minus-strand synthesis, but also directlyaffects

plus-strand RNA synthesis. Ineither ts4- or ts6-in-fected cells, thesynthesis of42Sand 26S

plus-strand RNAs is reducedafter the shift to390C atlate timesintheinfectiouscycle when

minus-strand transcription has already ceased (16).

This is incontrast totsll-infected cells,in which

42S and 26Splus-strandsynthesescontinueafter

the shiftto390C atlate timesin theinfectious cycle (16). We analyzed the pattem and the

amountsof42Sand26Splus-strandRNAs

syn-thesized in tsll-infected cells after the shift to 390Cat atime (3hp.i.) intheinfectious cycle

whenminus-strand synthesiswasjustbeginning. CulturesofCEF cellswereinfectedat280Cwith

tsll andwere either maintained at

280C

(Fig.

4A) or shiftedat 3hp.i. to390C (Fig. 4B) and

labeled with[3H]uridine.At bothtemperatures,

full-length42S and 26Splus-strandRNAs were

synthesized. The cultures that hadbeenshifted

to390C incorporated 18% as much [3H]uridine

into42S and26S RNAsasdid the culture

main-tained at 280C, but an amount equal to that

obtained in a culture maintained at 280C but

labeled at 3 h p.i. (data not shown).

Further-more,the ratio oflabeled 26S RNAto42S RNA

did notchangesignificantly withashift in

tem-perature; at280Cthe ratiowas1.7,andat390C

the ratio was 1.2. Therefore, plus-strand RNA

continued to be synthesized at

390C

in

tsll-infectedcells, although minus-strand

transcrip-tion ceased when the cultures were shifted to

thenonpermissivetemperature at 3h p.i. Analysis of viral RNA synthesis at the

permissivetemperature. Wenextdetermined

whethertheregulationofminus-strand

synthe-sis inSINHRtsll-infectedcellswasnormalat

the permissive temperature. Cultures of CEF

cells were infected with tsll at280C and were

pulsedfor 60 min with

[3H]uridine

atintervals

throughout infection (Fig. 5A). An increasing

amount of

[3H]uridine

incorporation into RNA

wasdetected between2and 6 hp.i.,followedby

several hours of a more orlessconstant rateof

x

E 8

Z 6

wc

E 4

t'l 2

As"s.

2

B 42 265

IU0 U0 U0 U0 30

IRACTION NUMBER

FIG. 4. Sucrosegradientanalysis of RNA synthe-sized intsll-infectedcultures at thepermissive and nonpermissive temperatures. Monolayers of CEF cellswereinfected withtsllat28°C. Certain of these culturesweremaintainedat28°C,receivinga60-min pulse of [3H]uridine atvarious intervals; other cul-tures wereshiftedto39°Cat3 hp.i. andwere main-tained at39°C. Cellswereharvestedafter the pulse into ETbuffercontaining 2% sodium dodecyl sulfate, and theextracts werelayeredonto15 to30%sucrose gradients in NETbuffer (0.1 MNaCI,0.01M EDTA, 0.01 MTris-hydrochloride[pH 7.4]) containing 0.2% sodiumdodecyl sulfate and sedimented inanSW27.1 rotor at 93,000xgfor18hat20°C. Samples of 0.5 ml were collected, and the radioactivity in each was determinedby counting the entirefraction afteracid precipitation and collectionon aglassfiberfilter. (A) tsll-infected culture maintainedat28°C and pulsed with [3H]uridine between 7and8 hp.i.; (B) tsll-infected culture shifted to 39°C at 3 hp.i., subse-quently maintainedat39°C, andpulsed with [3H]-uridine between 7.5 and8hp.i.

incorporation.RF RNAwasisolated and showed asimilarpatternofsynthesis(Fig.5B).As shown inFig.5C,theperiodofincreasingRNA synthe-siswasassociatedwithanincrease in the

incor-poration of radiolabel into minus-strandRNA.

The insert to Fig. 5C shows the proportion of

the totalincorporation in RF RNA thatwasin

minus strands. The synthesis of minus-strand

RNA occurred at an increasing rate at 280C

until after 4 h p.i., at which time it declined:

between 2 and 3 h and 3 and 4 hp.i.,about 50%

of the radiolabel in RF RNA was in minus

strands;by6p.i.,less than 20% of labeled RNA

was in minus strands; and only 3% was found

between 6and 7hp.i. Thus, atthepermissive

temperature, the synthesis of minus-strand

RNA intsll-infected cellswastemporally

reg-ulatedandwassimilartothatseenin the

paren-tal SIN HR-infected cells (Fig. 1). Since

plus-strand RNA continuedtobesynthesizedfor at

least 5 h after the shift to 390C (Fig. 4), the

previously formed minus strands continued to

function astemplates for

plus-strand synthesis

I11--1 -Ld baft..AgnoL1-D -AL

http://jvi.asm.org/

[image:6.503.262.460.58.224.2]

(7)

354 SAWICKI ET AL.

0

E

aL

z

-J

z

CO) 0

E

0

E

,'so

_

,so

h.0

I *E20

.1

0

3 4 5 * 7 9

hours p.i.

HOURS p.j.

FIG. 5. Rateof[3H]uridine incorporation into SINtsll RNA at28°C andaftertheshiftto39°C. CEF

monolayers were infected with tsll (MOI, 50) andmaintained at28°C. Beginningat2hp.i., apulseof

[3HJuridinewasgiven for60-minperiodsat28°C. Duplicate cultureswereshiftedto39°Cat 3hp.i. andwere

pulsed with[3HJuridinefor 30-min periods beginningatthe timeof the shift. (A) Total incorporation into viraltsllRNA; (B) total incorporation into RF RNA thatwasisolatedfromthe extract of each ofthe samples shown in(A); (C) total incorporation intotsll minus-strand RNA. RF RNA was denatured and allowedto reannealin the presenceofan excessof unlabeled 42S virion RNAasdescribed in the text. The insert shows thesameresultsas those shown in(C) expressedas apercentageof the total labeled RF RNA that is in minus-strand RNA. Symbols: 0, cultures maintainedat28°C; 0, cultures shiftedto39°Cat3hp.i. and maintainedat39°C.

http://jvi.asm.org/

[image:7.503.152.372.70.559.2]

(8)

after tsll-infected cellswere shifted from 28 to

390C

at 3 h p.i. For a direct comparison with

events at280C, duplicatecultures wereshifted

to 390C at 3 h p.i. and similarly analyzed. An

initial two- tothreefold rise in plus-strand

syn-thesiswasfollowed by a slowdecline, so that 7

h after the shift, 40% of the initial rate was

observed (Fig. 5A).Aconstant amount of

incor-porationwasdetectedin RF RNA (Fig.5B),and

as seenpreviously(Fig. 3), minus-strand

synthe-sisceased about 60minafter the shift to 390C

(Fig. 5C).

Resumption of minus-strand synthesis.

Since raising thetemperature to390C resulted

in the cessation of minus-strand synthesis in

tsll-infected cells, we next asked whether

mi-nus-strandsynthesis wouldresume after a return tothepermissivetemperature. The requirement

fortranslation ofnew viralproteins for the

re-sumption of minus-strand transcriptionupon a

shift down from39 to

280C

wasinvestigated to

distinguish between the reactivation of previ-ously formed proteins and the requirementfor

newly synthesized ones. Cultures that were

shiftedup to390C at 3 h p.i.werereturned to 280C 1 hlaterandincubatedin thepresence or

absence of cycloheximide (100 ,ug/ml). Viral

RNAsynthesized inthese cultureswas labeled

during 60-min pulse periods with [3H]uridine,

and the amountsoflabeled viral RNA andRF

RNA andtheproportion of labeled RF RNAin

minus strandsweredetermined.Areturn to the

permissivetemperature inthe absenceof

cyclo-heximide ledtothe resumption ofanincreasing

rateof RNAsynthesis that eventuallyreached

about 60%of that in infected cultures thatwere

maintainedat280C (Fig. 6). However, areturn

to280Cinthepresenceofcycloheximide ledto

only a small increase over the rate of RNA synthesis occurringat390C.After thereturn to

280C,

the amount oflabeled RF RNA in the untreated culturesincreased, but essentiallyno

increase withtime inthe amountof labeled RF RNA occurredinculturestreated with cyclohex-imide(Fig. 6B).Correspondingto anincreasein

synthesis of totalRNAand RF RNA afterthe

return to280C,therewas aresumption of

minus-strandsynthesis(Fig.6C).Asimilarresumption of minus-strandtranscription aftertheshift back down to280C didnot occur inthe presence of cycloheximide.Active minus-strandpolymerase

musthave been present in all ofthesecultures

at the time of the shift to the nonpermissive

temperature, since minus strands were

accumu-lating at 3 h p.i. at

280C.

Since minus-strand

polymerase activity in SIN HR-infected cells

wasnormally short-lived,as wasdemonstrated

byitsrapid disappearanceafter the inhibition of

T

x=

CE

-C

II

x E

Vb

a

E

z

CK

Hours. post-infection

FIG. 6. Effect of cycloheximide addition on the

temperature-dependent resumption ofminus-strand

synthesis. tsll-infectedcultureswereshiftedto39°C

at3hp.i.,and RNAsynthesiswasmonitoredby the

addition of[3H]uridinefor 30-minpulses at 39°C (x). Two setsof these cultureswerereturnedto28°C at 4hp.i. Oneset wasincubatedcontinuouslyin the presence of100pgofcycloheximideperml (A); the secondset was notfurthertreated and servedasthe 28°C control(0).Incorporationof radioactivityinto theacid-insolubleform bythese cultureswas deter-minedafterthe culturesweregiven60-minpulsesof

[3H]uridinebeginningfromthe timeofthereturnto

28°C.Inaddition,severalcultureswhichwere main-tainedat28°C throughouttheexperiment (0) were

pulsed for60-minperiodswith[3Hluridinebetween

2and3hp.i.andbetween 8and9hp.i. tomonitor theamountofRNAsynthesis occurring in infected

culturesnotsubjectedtothesetemperatureshifts. (A) Totalincorporation into viralRNA; (B) incorpora-tion into RFRNA;(C)percentageoftotal labeled RF RNAinminus strands.

http://jvi.asm.org/

[image:8.503.284.435.70.448.2]

(9)

356

proteinsynthesis (Fig. 1), the failure to

demon-strate aresumption of minus-strand RNA

syn-thesis in SIN HR tsll-infected cells that had

been shifted to390C at 3 h p.i. and then returned

to 280C 1 h later in the presence of

cyclohexi-mide couldhave resulted fromeither therapid

degradation or therapidfunctional inactivation

at390C ofaviral polypeptide required for mi-nus-strand RNA synthesis. In either case, the

minus-strand polymerase activity that

reap-pearedin SIN HRtsll-infected cellsupon the

return to28°Crequiredthesynthesis of proteins.

Cultures shifted to 39°C at 3 hp.i. were

re-turned to28°C atvarious times (Fig. 7). In all

cases, there was an immediate increase in the

[3H]uridineincorporationascompared with that detected in infected cultures not returned to

280C.Increasing thetime atthenonpermissive

temperaturebefore thereturntothepermissive

temperaturedecreased theextent towhichtotal

RNAsynthesisrecovered, but, nevertheless, mi-nus-strand synthesis resumed in tsll-infected cultures if the cultures were shifted back to

280C.

DISCUSSION

Previously,we have shown that minus-strand

transcription in Semliki Forest virus-infected

cellsisshut off selectivelywhen protein

synthe-sis isinhibited with puromycinor cycloheximide

(25).The data presented in this study extended

this observation to include SIN and demon-strated that when SIN HR tsll-infected cells

10

x 8

caE

6

C=4

4

I2

2 3 4 5 6 7 8 9 10 11

Hours, post-i nfection

FIG. 7. Temperature-dependent increase in tsll RNA synthesis. CEF monolayers infected with tsll

wereshiftedto39°Cat3hp.i. At the indicatedtimes, certainof these cultureswerereturnedto28°Cand labeled with[3H]uridineat28°C for 60-minperiods; culturesmaintainedat39°Cwerepulsed for30-min

periods. Cultureswereharvestedinto ET buffer with

2% sodium dodecyl sulfate immediately after the pulse, and theamountof incorporation into the acid-insolubleform was determined. Symbols: 0, 39°C; 0,280C.

wereshifted to the nonpermissivetemperature

during the early phase of the viralreplication

cycle, when therateof minus-strand RNA

syn-thesis was increasing, minus-strand transcrip-tion ceasedselectively. Temperature sensitivity

of a component of the viral RNA-dependent

RNA polymerase that is required for minus-strand transcription would account for the

RNA-phenotype of SINmutant tsll.The

prod-ucts ofatleast four cistrons (complementation

groups A, B, F,andG) of SIN HR arerequired

forviral RNA synthesis (5, 6, 22, 29). SINHR tsllistheonlymember of theB

complementa-tiongroup(22, 28,29). The results ofourstudies

on ts mutantsofSINHR suggestthat the

prod-uctof theBcistronof SIN HR isapolypeptide

thatfunctionsinminus-strandtranscriptionand demonstrated that thermal inactivation of

mi-nus-strand transcription occurred in tsll-in-fected cellswithoutinactivation ofplus-strand

transcription.

Minus-strand RNA synthesis was also ob-servedtobe temperature sensitive in SIN HR

ts4 (complementation group A)-infected cells

and SIN HR ts6 (complementation group

F)-infected cells. This observation was consistent withthe results reported by Pfefferkorn et al. (21) andPfefferkorn andBurge (20), who dem-onstrated that inputts4andts6viral RNAsare notconverted intoanuclease-resistant, double-stranded form at the nonpermissive

tempera-ture. However,since we (16;unpublished data)

and others (22) have shown that ts6 has

diffi-cultysynthesizingplus-strandRNAatthe

non-permissive temperature, we conclude that the

product of the F cistron is required for both

plus-strand and minus-strand RNA syntheses. Cells infected withts4of theAcomplementation

groupsynthesize viralRNApoorly atboth the

permissive and nonpermissive temperatures,

suggesting atemperature-independent defectin

the viral RNApolymerase (16). Another mem-ber of the Acomplementation group, tsl5, did not stop synthesizing minus-strand RNA upon the shift to the nonpermissive temperature.

Most ts mutants of the A complementation

group show an alteration in the ratio of 26S-to-42S plus-strand synthesis after the shift to the

nonpermissivetemperature(29), with the

excep-tion ofts4,which does notsignificantlychange

the ratio of 26S-to-42S plus-strand synthesis

(16). Therefore, we conclude that only tsll of

the Bcomplementationgroupunequivocally

ap-pears to be defective selectively in minus-strand RNAsynthesis. Ashift of tsll-infectedcellsto the nonpermissive temperature at a late time

afterinfection does notaffectplus-strand RNA

synthesis (16). When tsll-infected cells were

shifted to the nonpermissive temperature at

http://jvi.asm.org/

[image:9.503.77.220.413.550.2]

(10)

early times in the infectious cycle, plus-strand RNA synthesis continued atthe rate that was

occurringatthetime of the shift.

Our results with SIN HRtsll suggest that the

product of theBcistron isa polypeptide which

is required selectively for minus-strand RNA

synthesis. Because minus-strand, but not

plus-strand, RNA synthesis is short-lived and

re-quires continualprotein synthesis, it is not

pos-sible fromourresultstodetermine whether the

productof the B cistron istemperature sensitive

in tsll orwhetherthe cleavageof the

polypep-tide encodedby theBcistronis defective at the

nonpermissive temperature. It has been

sug-gested (29) from the results reported by Waite

(30) that one ofthe polypeptide intermediates inthe processing of the SINnonstructural

pro-teins accumulates in tsll-infected cells at the

nonpermissivetemperature.The isolationof

ad-ditionalts mutantsbelongingtotheB

comple-mentationgroupwhichalsoshare the phenotype

of SIN HR tsll is necessary to conclusively demonstrate thatthe Bcistron is responsible for

minus-strand synthesis.

To date, only three SIN nonstructural

pro-teins have been identified (4, 14). Their gene

order has been determined to be 5'-ns6O-ns89-ns82-3' (4). These proteins are translatedfrom

the 42SpolycistronicmRNAwith a single

initi-ation site(1, 4, 15). A recent study byFullerand

Marcus (14) with increasing doses ofUV

irradia-tion, which resultsinthe blockingof translation

of polypeptides beyond the site of damage on

the mRNA, confirmed the gene order of the

nonstructural proteins of SINand also assigned twoof theseproteinstoparticular complemen-tationgroups;theN'-ns6O proteinwasidentified

astheproduct of the G complementationgroup,

and thens89proteinwasidentifiedas the prod-uctoftheAcomplementationgroup. The third

protein (ns82) could not be assigned to either

the B or F groups because of the verysimilar dose of UV irradiationresulting in loss of

com-plementation withmutantsof boththe F(tsllO)

and theB(tsll)groups.The authorsconsidered

thepossibility that the fourth complementation

group represents anoncoding region of the

ge-nomeRNA and thusfunctionsatthe nucleotide level rather thanatthepolypeptide level.Ifthe

Bcistronrepresentssuchanoncoding regionof

the genome, its ability to function in

minus-strandtranscription wouldnotbeexpectedtobe

sensitive to protein synthesis inhibition. After

the return to

280C

ofSINtsll-infectedcellsthat

were shifted to

390C

earlyininfection,the

syn-thesis of minus-strand RNA recovered only in

the absence ofcycloheximide.Wewould suggest

that, if there areonly three nonstructural

pro-teins,the ns82proteincontainstwofunctionally

distinctdomains,oneof which functionsin

mi-nus-strand RNA synthesis and thus can be

in-dependently mutated, resulting in theB andF

complementationgroups.

Thataspecific function notneededforviral

plus-strand transcription is involved in

minus-strandsynthesis isnotsurprising. The initiation of minus-strand synthesis may be unusual in

that the5'-initiating nucleotidemay be a

pyrim-idine: 5'-ppUp and 5'-pUp residues have been detected in a polyuridylate sequence which is

locatedatthe5' terminus of Semliki Forest virus

minus-strand RNA (23). A polyuridylate

se-quencehas beenfound alsoinSIN HR

minus-strand RNA (12).Althoughasimilar 5'-terminal polyuridylate sequence is presentin poliovirus minus-strand RNA(27, 33, 34), its 5' terminus is covalently coupled to a viral protein (10, 18). Since alphavirus plus-strand RNA synthesis probably initiates with apurine residue (9, 19,

32),anadditional functionmayberequired for pyrimidine initiation. Also, the ability to

regu-late theamountof minus-strandRNA, which is only needed inatemplate capacity and is stable

once synthesized (3, 24-26, 31), confers the

added advantage of the efficient utilization of

available substrates for single-stranded virion

RNA and mRNAformationoncesufficient

mi-nus-strand templates areformed. Since regula-tion ofthe initiationofminus-strand transcrip-tionwould controldirectly the number of

tem-platesproduced, translational control of the

pro-teinrequired for this initiation would bea

sen-sitiveregulatorymechanism.Theactualprocess

of initiation mayrequire the function ofmore

than asingle protein. Therole ofoneviral and

several host cell proteins in QB and f2 RNA phage transcription and replication has been

recently reviewed (1), and in vitro evidence

in-dicates that several picornaviruses, e.g.,

polio-virus (7) and encephalomyocarditis virus (8), mayutilizeahostcellproteininadditiontoviral proteins fortranscriptionon aplus-strand

tem-plate,possiblyfor the initiation oftranscription

(7). Whether this is true for alphaviruses also remainstobedetermined.

ACKNOWLEDGMENTS

We thankRaija Lahdensivu and Ritva Rajala for excellent technical assistance. WeareendebtedtoH.vonBornsdorffor hisgenerousgiftofpurifiedSIN.

Thisinvestigationwassupported byfundsfrom theSigrid Juselius Foundation andfrom the Finnish Academy,from PublicHealthServiceresearchgrantAI-15123from the Na-tionalInstitute ofAllergyandInfectiousDiseases,and from Biomedical Research Support grant 5S07-RR-05700-09 awardedbytheNationalInstitutes of Health.

LITERATURE CITED

1. Blumenthal, T., and G. G. Carmichael. 1979. RNA replication: function and structure of QB-replicase.

Annu.Rev. Biochem. 48:525-548.

http://jvi.asm.org/

(11)

358 ET AL.

2. Bracha, M., A. Leone, and M. J. Schlesinger. 1976. Formation of a Sindbis virus nonstructural protein and its relation to42S mRNA function. J. Virol. 20:612-620. 3. Bruton, C. J., and S. I. T. Kennedy. 1975. Semliki Forest virusintracellular RNA: properties of the mul-tistranded RNA species and kinetics ofpositive and negativestrand synthesis. J.Gen.Virol. 32:413-430. 4. Brzeski, H.,and S. I. T.Kennedy. 1977.Synthesisof

Sindbis virus nonstructuralpolypeptidesinchicken em-bryofibroblasts. J. Virol.22:420-429.

5. Burge, B.W., and E. R. Pfefferkorn. 1966.Isolation and characterizationofconditional-lethal mutants of Sindbis virus. Virology 30:204-213.

6. Burge, B. W., and E. R.Pfefferkorn. 1966. Comple-mentation between temperature-sensitive mutants of Sindbis virus.Virology 30:214-223.

7. Dasqupta,A.,P.Zabel,and D.Baltimore. 1980. De-pendenceof theactivity of thepoliovirus replicaseon ahostcell protein. Cell 19:423-429.

8. Dmitrevia, T. M., M. V.Schleglova, and V. I. Agol. 1979.Inhibition ofactivity ofencephalomyocarditis vi-rus-induced RNA polymerase by antibodies against cel-lular components.Virology 92:271-277.

9. Dubin, D. T., K. Timko, S.Gilies,andV.Stollar. 1979. The extreme5'-terminal sequences of Sindbis virus 26S and 42S RNA.Virology98:131-141.

10.Flanigan, J. B., R. F. Pettersson,V.Ambros, M. U. Hewlett,and D. Baltimore.1977.Covalentlinkageof aprotein to a defined nucleotide sequence at the 5'-terminus of virion and replicativeintermediate RNAs ofpoliovirus. Proc. Natl. Acad. Sci. U.S.A. 74:961-965. 11.Franklin, R. 1966. Purification and properties of the replicative intermediate of the RNA bacteriophage R17. Proc. Natl. Acad. Sci. U.S.A. 55:1504-1513.

12.Frey, T. K., and J. H. Strauss. 1978. Replication of Sindbis virus. VI.Poly(A) and poly(U) in virus-specific RNAspecies.Virology 86:494-506.

13.Friedman,R. M., and P. M.Grimley. 1969. Inhibition of arbovirus assembly bycycloheximide. J. Virol. 4: 292-299.

14.Fuller, F. J.,and P.I. Marcus. 1980. Sindbis virus. I. Gene order of translation in vivo.Virology107:441451. 15.Kaariainen,L, and H. Soderlund. 1978. Structure and replication of alphaviruses. Curr. Top. Microbiol. Im-munol. 82:15-69.

16.Keranen, S., and L.Kaariainen.1979.Functional de-fects ofRNA-negative temperature-sensitive mutants ofSindbis andSemliki Forest viruses. J. Virol. 32:19-29.

17. Lowry,0.H., N. J. Rosebrough, A. L. Farr, and R. J. Randall. 1951. Protein measurementwith the Folin phenolreagent.J. Biol. Chem. 193:265-275.

18. Nomoto, A., B. Detjen, R. Pozzatti, and E. Wimmer. 1977.The isolation of thepolio genome protein in viral RNAs anditsimplication for RNA synthesis. Nature (London) 268:208-213.

19. Pettersson,R.F., H. Soderlund, andL.Kaariainen. 1980.Nucleotide sequence of the 5'-terminal Tl-oligo-nucleotideof Semliki Forest virus 42S and 26S RNAs isdifferent. Eur. J. Biochem.105:435-443.

20. Pfefferkorn, E.R., and B. W.Burge. 1968. Morpho-genic defects in thegrowth oftsmutantsof Sindbis virus.Perspect. Virol. 6:1-14.

21. Pfefferkorn, E.R., B. W.Burge, and H.M. Coady. 1967.Intracellularconversion of the RNA of Sindbis virus into adouble-stranded form. Virology 33:239-249. 22. Pfefferkorn, E. R., andD.Shapiro.1974.Reproduction oftogaviruses, p. 171-230.In H.Fraenkel-Conrat and R. R.Wagner. (ed.), Comprehensive virology, vol.2. Plenum Publishing Corp., New York.

23. Sawicki,D.L.,and P. J. Gomatos.1976.Replicationof Semliki Forest virus:polyadenylateinplus-strand RNA andpolyuridylate inminus-strand RNA. J. Virol 20: 446-464.

24. Sawicki,D.L.,L.Kaariainen, C., Lambek,and P. J. Gomatos. 1978. Mechanism for control of synthesis of Semliki Forest virus 26S and 42S RNA. J. Virol. 25:19-27.

25. Sawicki,D.L., and S.G. Sawicki. 1980. Short-lived minus-strand polymerase forSemlikiForest virus. J. Virol. 34:108-118.

26. Scheele,C.M.,and E. R. Pfefferkorn.1969.Inhibition ofinterjacent ribonucleic acid (26S) synthesis in cells infectedby Sindbis virus. J. Virol. 4:117-122. 27. Spector,D.H.,and D. Baltimore. 1975.Polyadenylic

acid on poliovirus RNA. IV. Poly(U) in replicative intermediate anddouble-stranded RNA.Virology67: 498-505.

28. Strauss,E.G.,E. M.Lenches,and J. H.Strauss.1976. Mutants ofSindbis virus. I. Isolation andpartial char-acterization of89 newtemperature-sensitivemutants. Virology 71:154-168.

29. Strauss, J. H., andE. G. Strauss. 1980. Mutants of alphaviruses:geneticsandphysiology,p.393-425. InR. W. Schlesinger (ed.), Togaviruses, biology, structure, replication. AcademicPress,Inc.,New York. 30. Waite,M. R.F. 1973.Proteinsynthesisdirected byan

RNA-temperature-sensitive mutantof Sindbis virus. J. Virol. 11:198-206.

31. Wengler, G., andG. Wengler. 1975. Studies on the synthesis of viralRNApolymerase-templatecomplexes inBHK-21 cells infected with Semliki Forest virus. Virology 66:322-326.

32. Wengler,G.,G.Wengler,and H. J.Gross.1979. Rep-licativeformofSemliki Forest virus RNA containsan unpaired guanosine.Nature(London)282:754-756. 33. Yogo, Y., M. Y.Teng,and E. Wimmer.1974.Poly(U)

in poliovirus minus-strand RNA is5'-terminal. Bio-chem.Biophys.Res.Commun.61:1101-1109. 34. Yogo,Y., and E. Wimmer.1973.Poly(A) and poly(U)in

poliovirus double-stranded RNA. Nature (London) NewBiol.242:171-174.

http://jvi.asm.org/