Short-lived minus-strand polymerase for Semliki Forest virus.

JOURNAL OF VIROLOGY, Apr. 1980, p.108-118 Vol. 34, No. 1 0022-538X/80/04-0108/11$02.00/0

Short-Lived Minus-Strand

Polymerase

for Semliki Forest

Virus

DOROTHEA L.SAWICKI* AND STANLEY G. SAWICKI Departmentof Microbiology, MedicalCollege of Ohio, Toledo, Ohio 43699

Semliki Forest virus (SFV)-infected BHK-21, Vero,and HeLa cells

incorpo-rated[3H]uridineinto42S and 26Splus-strandRNA and into viral minus-strand

RNA (complementary to the 42S virion RNA) early in the infectious cycle.

Between3 and 4 hpostinfection, the synthesisof minus-strand RNA ceased in

thesecultures,

although

the

synthesis

of

plus-strand

RNA continuedat amaximal

rate. At thetime ofcessation of minus-strandRNAsynthesis,two

changes

inthe

patternofviralproteinsynthesisweredetected: adecrease in the translation of

nonstructural proteins and anincrease inthe translation ofthe viral structural

proteins. Additionofcycloheximide andpuromycintocultures of SFV-infected

BHKcellsactivelysynthesizing both viral

plus-

and minus-strand RNAresulted

within 15 to 30 min in the selective shutoff of minus-strand RNA synthesis.

Removalof thecycloheximide-containingmedium ledtotheresumptionof

minus-strandsynthesis and toan increased rate of viral RNAsynthesis. We conclude

that the minus-strand polymerase regulates the rate of SFV plus-strand RNA

synthesis by determining the number ofminus-strand

templates

and that the

synthesis of the minus-strand

templates

is

regulated

atthe level of translation

by

amechanismwhichutilizes one or moreshort-livedpolymerase proteins.

Semliki Forest virus

(SFV)

is an

alphavirus

and contains as its genome a single strand of RNA of

plus

polarity having

amolecular

weight

of 4 x 106 to 4.5 x 106 and a sedimentation

coefficient of 42S (8, 12). The

replication

of

alphaviruseshas

recently

been reviewed (9, 18,

29). In orderfor SFV toreplicate, the parental

42S plus-strandRNAmustbe transcribed into

a complementary minus strand that in turn

serves as a

template

for the

synthesis

ofboth

additional 42S

plus-strand

RNA and

subge-nomic 26S mRNA. The 42S plus-strand RNA

serves as a

template

for the synthesis of

non-structural viral proteins and is packaged into

virions.Thesubgenomic26S mRNA serves as a

template for the translation of the structural

viral proteinsand is notfoundinvirions.

In animal viruses that contain a plus-strand

RNA genome, it is generally assumedthat the

RNApolymerasesresponsible for the replication

ofthe viral genome are composed of nonstruc-turalpolypeptides becausetranslation must

oc-curbeforeviral RNAsynthesis commences and

purifiedvirions lack detectable

RNA-polymer-izing activity.Thenonstructural proteins (ns) of

SFV that are repeatedly detected in infected

cells are three in number, having molecular

weights ofapproximately 70,000 (ns7O), 86,000

(ns86), and 72,000 (ns72), all of which are

ini-tially

translated fromthe42S plus-strand RNA

as onepolyprotein (9, 29). The products of the

alphavirus RNA polymerase are three distinct

RNA species: the 42S minus-strand RNA and

the plus-strand 42S and 26S RNA. The SFV

minus-strand RNA, unlike the plus-strand 42S

or 26S RNA, does not accumulate as

single-strandedRNA; rather,it isdetectedonlyaspart of the replicative intermediates (RIs) (26, 27).

Apparently, immediately afteraminus strand is

synthesized, it is used as a template for

plus-strandsynthesis.

Recently, thenatureof thealphavirus

polym-eraseresponsible forthesynthesis of SFVRNA 4 to 8hpostinfection (p.i.) waspartially

eluci-dated. Three laboratories have data which

indi-catethat the viralns7Ois themajor polypeptide

associated with membrane-bound replication

complexes transcribing42S and 26S RNA (4, 19;

P. J. Gomatos,unpublisheddata). Two of these

laboratories also consistently found smaller amountsof ns86 and ns72insuch complexes (19;

Gomatos,unpublisheddata). Theoverallaim of

our studies istocharacterizethe SFV polymer-aseresponsible for thesynthesisof minus-strand RNA. The resultspresented in this paper indi-cate that atrelatively early times in the infec-tious cycle, the synthesis of SFV minus-strand RNA ceases while the synthesis of plus strand 42S and 26S RNAcontinues at a maximal rate; this wasfound tobe true for SFV replicating in three different cell types: BHK-21, Vero, and HeLa. Whereas Brutonand Kennedy (2)

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SFV MINUS-STRAND RNA 109

ously reported a similar observation for

SFV-infectedBHK-21 cells,wealso foundthat

cyclo-heximide andpuromycin treatment of

SFV-in-fected cells which were in the process of

tran-scription of both plus- and minus-strand RNA also resulted in the selective shutoff of

minus-strandtranscription, leaving untouched the

on-going plus-strandRNA synthesis. Although

pre-vious investigators have considered the alpha-virus RNA polymerase to be one species of en-zyme, ourresultssuggest that theremay actually exist two forms of the SFV RNA polymerase: one form responsible for the synthesis of

minus-strandRNA which is detected only early in the

infectiouscycle and whichrequires continuous

protein synthesisfor activity; and a second, more

stable polymerase which is responsible for the

synthesis of 42S and 26S plus-strand RNA

throughout the infectious cycle.

MATERIALS AND METHODS

Cell culture. BHK-21cells, acontinuouscell line derived frombabyhamsterkidneycells, weregrown in plastic petri dishes in Dulbecco-modified Eagle minimal essential medium (DMEM) supplemented with5%fetal bovineserum.HeLa (CCL-2)and Vero (CCL-81) cellswereobtainedfrom the AmericanType Culture Collection andweregrowninDMEM supple-mented with 5% fetal bovineserum.

Virus. Wild-type SFV, obtained from the second

passage ofSFV-infected mouse brain suspension in BHK cells, wasused in these experiments. Growth andpurification of virus and determination of its in-fectivity by plaque assaywere described previously (22).

Preparation of infected-cell extracts. BHK monolayers were infected with wild-type SFV at a multiplicity of infection (MOI) of100 as previously described (22), exceptthatactinomycinD (AMD) at aconcentration of1 ug/mlwaspresent.Thetime of addition of virus is designated zerotime. HeLa and Vero monolayers were similarly infected with wild-typeSFV except that AMDwasadded30minbefore labelingof the infected HeLamonolayersbecause of the sensitivity of HeLa cells to the cytotoxicity of AMD(24).

SFV RNA was labeled with [5,6-3H]uridine at a final concentration of 250

ILCi/ml.

For the protein synthesis inhibition studies, cycloheximide or puro-mycinat afinal concentration of50ug/mlwasadded

to DMEM containing 0.2% bovine serum albumin (BSA)and1

iLg

of AMD per ml.[3H]uridinewasadded

to a freshportionof this medium foranalysisof RNA

synthesizedinthe presence ofeachdrug. Cyclohexi-mideinhibitionwasreversedbyremoval of the drug-containing medium followedbyfoursuccessive washes with37°C DMEM and further incubation in DMEM containing BSA and AMD.

Atthe timesindicated forharvest, the cellswere

washed with ice-cold phosphate-buffered saline, a

small volume of ET buffer (0.01 M EDTA-0.01 M

Tris-hydrochloride,pH 7.4)containing2% sodium

do-decyl sulfate (SDS) was added, the lysed cells were scraped from the dish, and the cellular DNA was sheared by passing the extracts four times through a 27-gauge needle. For analysis of SFV RNA, the total cell extract waslayered directly onto 15 to 30% sucrose gradients in NET buffer (0.1 M NaCl-0.01 M EDTA-0.01 M Tris-hydrochloride, pH 7.4) containing 0.2% SDS and sedimented in an SW27 rotor at 95,400 x g for14.5hat20°C. Fractions of 1 ml werecollected by using a peristaltic pump, and the radioactivity in each was determined by counting a portion in a toluene-based scintillation fluid containing Triton X-100.

Labeling with [3S]methionine was carried out in methionine-free DMEM, using 50 ,uCi/ml per 60-mm petri plate. At thetimes indicated in individual exper-iments, infected BHK cells were exposed for 30 min to 335 mM NaCl (16, 21) in methionine-free DMEM containing0.2% BSA and 1 tgof AMD per ml. This medium was removed, and new medium containing

[3S]methionineand0.1M sucrose was added for the

timesindicated, followed by the addition of DMEM containing 20-fold the normal concentration of methi-onine.Aftera90-min chase period, the cell monolayers werewashed several timeswith ice-cold phosphate-buffered saline and harvested as described above.

Isolation of RF RNA, the RNase-resistant

coresof SFV RIs. The intracellular double-stranded SFV RNA (RIs) was obtained from material sedi-mentingatthe35 to20Sregion ofsucrosegradients of SDS-treated whole-cellextracts.After ethanol precip-itation at -20°C for 18h, the 35 to 20S RNA was dissolved in0.5ml ofdigestionbuffer(0.15 M NaCl-0.02MTris-hydrochloride, pH7.4); 25 ngof pancreatic RNasewasadded; and the samplewasincubatedat

room temperature for 15min. SDS was added to a final concentration of 2%. The digested sample was overlaid onto a 15 to 30%sucrose gradient in NET buffer containing 0.2% SDS. Centrifugation was at

154,000xgfor16h inanSW40rotor.Fractions of0.5

ml were collected; the fractions containing the RF RNA, the RNase-resistant cores of the RIs which sedimented at 20 to15S, werepooled,and the RNA was ethanolprecipitated in the presence of25 tLg of carrierratliver RNA.

Hybridizationof RF RNA with unlabeled 42S viron RNA. The procedures followed were as de-scribedpreviously (23).Briefly,theRF RNA thatwas obtained fromone100-mmpetriplate (approximately

7 x 106 cells)wasdissolved in0.2mlof1mMEDTA, pH 7.4. One-quarter of the dissolved RF RNA was dilutedto3.0ml with1mMEDTAat100°C,heated

at100°Cfor2min,quicklycooledto0°C,and divided into threeequalportions.Oneportionof the RF RNA served as a control for the fraction of the RNA

re-maining double stranded afterheatingandquick cool-ing.Thesecondand thirdportionsweresubjectedto hybridization conditions(0.4MNaClat68to70°Cfor

30minfollowedby30minat roomtemperature),one

inthe absence andthe otherinthe presence of10yg of unlabeled virion RNA.Toone-half of eachsample

wasaddedanequalvolume ofasolutioncontaining

0.3 M NaCl, 0.03 M sodium citrate, and pancreatic RNase(10

i&g/ml),

andthemixturewasincubatedat

370Cfor 30min. Theuntreated and RNase-treated portions were precipitated with trichloroacetic acid VOL. 34, 1980

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110 SAWICKI AND SAWICKI

and theacid-insoluble radioactivity wasdetermined. Theheated andquick-cooledportion and the portion hybridized in the absence of anyunlabeled virion RNA gaveessentially equalRNase resistance of1 to 12%. Thisvaluewassubtracted from fractions of RNase-resistantradioactivity obtained from the correspond-ingportionhybridized in the presence ofan excessof unlabeled virion RNA. This RNase-resistant radioac-tivity was taken as the relative amount oflabeled minus-strand RNA in the RF RNA, since use of RNase-derived RF RNA results in the loss ofsome

nascentminus-strand RNA.

SDS-polyacrylamide gel electrophoresis. Poly-acrylamide slab gels with 8% separation gel and 5% spacergelwereprepared accordingtothe method of Laemmli (11). The gelswerefixed in 20% trichloroace-tic acid for60 min before treatmentwith dimethyl sulfoxide and2,5-diphenyloxazole (PPO) (1).

Materials. [5,6-3H]uridine (41Ci/mmol) was pur-chased from Amersham Radiochemicals (Arlington Heights,Ill.) and New England Nuclear Corp. (Boston, Mass.). [3S]methionine (600Ci/mmol) wasobtained from NewEngland Nuclear Corp. Cycloheximide was purchased from Calbiochem (SanDiego, Calif.) and puromycin was purchased from Boehringer Mann-heim Corp. (Indianapolis, Ind.). All other materials

wereobtainedfrompreviously described sources (22, 23).

RESULTS

Time course of SFV minus-strand RNA

synthesis. Functionalminus-strand SFV RNA

polymerase was detected by assaying for the

synthesis of SFV minus-strand RNA in BHK

cells infected with SFV

(100

PFU/cell)

in the

presence of 1

,tg

of AMD per ml.

Replicate

cultures were labeled with [3H]uridine for 30

min atintervals

starting

1.5h

p.i.

At the end of

thepulse period, the cellsweresolubilizedwith

2% SDS in ET bufferand

centrifuged

on 15 to

30%sucrose

gradients;

the RIs

sedimenting

from 35 to20S werecollected, treated with

pancreatic

RNase, and again

subjected

to rate zonal

cen-trifugation on 15 to30%sucrose

gradients.

The

resulting double-strandedcores (RF RNA)

sed-imentingat 20 to15Swereused in theannealing

reactions instead of the intact RIstoeliminate

any

possible

interference

by

the vast excess of

nascent, labeled plus-strand RNA chains in the

RIs. Figure 1 shows the amount of

[3H]uridine

incorporated intoRF RNA obtained from a

por-tion (one-seventh) of eachsample. LabeledRF

RNA wasreadilydetected 1.5 to 2 h p.i. Between 1.5and 3 hthere was a threefold increase in RF

RNA synthesized in each 30-min period. At

about 3 to 3.5 hp.i., the synthesis of RF RNA became constant. The synthesis of single-stranded SFV RNAalsoincreased between 1.5 and 3 h p.i. and became constant at 3.5 h p.i.

(datanotshown).

The fractionof

[3H]uridine

incorporated into

J. VIROL.

200

100

x

E

50

2C

I0

5

2

total ds

/ minus strand

I'

IJ

I '

I

\

I I

0

0

2 3 4 5

Hours p. i.

FIG. 1. Time course of SFV minus-strand RNA synthesis. BHK-21 cells in100-mmpetri plateswere

infected with SFV(MOI of100) in the presence of AMD.Startingat1.5hp.i.,[3H]uridine(250,uCi/ml)

wasaddedforsequential30-minperiods, followedby preparationofinfected-cellextracts asdescribed in thetext. In allfigures, the amountofRNA synthe-sizedduringapulse is indicatedatthe endof the pulseperiod. [3H]uridine-labeledSFV RNA which sedimentedin the35 to20Sregionof15 to30%sucrose

gradients waspooled and treated with pancreatic RNase, and the RF RNA was resedimented on a

secondsucrosegradient. Anequalportion (one-sev-enth) of the labeled RF RNA (ds RNA)wasanalyzed forits[3H]uridine incorporation (0),and,afterheat denaturation and hybridization in the presenceof

excessunlabeled 42S virionRNA,fortheamountof

[3H]uridine incorporated into minus-strand RNA

(0). The[3H]uridine-labeledRF RNA which

specif-ically hybridizedtounlabeled virion RNA was con-sidered minus-strand RNA (see text).

minus strands of the RF RNA wasdetermined

by hybridization of denatured RF RNA with

unlabeled 42S virion RNA. Incorporation of

[3H]uridine into minus-strand RNA reached a

maximum at 3 hp.i.,afterwhich timeit declined

(Fig. 1).After3.5h, essentiallynominus-strand

synthesiswas detectable, although plus-strand

RNAcontinued to besynthesizedat amaximal rate.

Weinvestigated the time course of SFV

mi-nus-strandRNA transcription in two othercell

types. Monolayers of Verocellsand HeLacells

were infectedwith SFV at an MOI of 100, the

viral RNAwas labeled with

[3H]uridine

in the presence of AMD for sequential 30-min periods, andthe RF RNA was isolated and analyzed as

describedabove for the BHKcellextracts

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SFV MINUS-STRAND RNA 111

ble 1). Both cell types produced viral RNA,

although HeLa cells incorporated only about

one-fourthasmuch

[3H]uridine

into viral RNA

asdid eitherVero or BHKcells.Thesynthesis

of SFV minus-strandRNAdeclined3 to 4 h p.i.

in eachofthese celltypes: Vero cellsexhibited

asimilar ifnotidenticaltimecourse as the

BHK-21cell cultures; inHeLacells,theinhibition was

not asabrupt.

IfSFV minus-strand RNA were synthesized

butrapidly degraded after3hp.i., we might not

have detected synthesis of new minus-strand

RNA after 3 h, using 30-min labeling periods.

Therefore,wedetermined theamountof

incor-poration of [3H]uridine into nascent

minus-strand RNA, using shorter pulse-labeling times.

A 5-min pulse was sufficient to label

minus-strand RNA when thepulsewasgiven at 2 h p.i.

(Fig. 2). This resulted in thevastmajority of the

labeledRNAsedimentingasRIsand only very

smallamounts oflabeled RNA sedimenting as

completed 42S and 26S species (datanotshown).

Pulse times of10to 30min at 2hp.i. resultedin

maximallevels of labeled minus strands in the

RF RNA.However,duringa 5- to30-minpulse

of

[3H]uridine

given toinfected BHK cell

cul-tures at 3hp.i., little or nosynthesis of

minus-strand RNAwasdetected (Fig.2). We conclude

from these results that transcription of

minus-strandRNA, and thusthe activity of the

polym-erase synthesizing the minus strands, ceases

TABLE 1. TimecourseofSFV minus-strand synthesis in threedifferentcelltypesa

[3H]uridine

time % of RF RNA inminus-strand RNA of addition BHK-21

(h p.i.) cells Vero cels HeLacells

2-2.5 46 44 45

2.5-3 24 28 41

3-3.5 4 6 38

3.5-4 0 5 26

4.5-5 0 1 13

5.5-6 0 8

6.5-7 6

7.5-8 6

8.5-9 4

aLabeled viral RNA in total

cell

extractsharvested

atthe end of thepulseperiodwascentrifugedon15to 30%sucrosegradients;RNAsedimentinginthe35to

20Sregionwascollectedby ethanolprecipitation;and the double-stranded RF RNA was prepared as de-scribed in the text. Between 1,000 and 50,000 cpm (approximately0.03

pg)

ofRF RNAwasusedineach annealingreaction with10jugof unlabeled42S virion RNA. Hybridization of SFV RF RNA isolated from infectedBHK-21 cell extracts, which hadbeenlabeled

with[3H]uridinefrom0to4hp.i.,gave valuesranging from38 to47% ofthe total labeled RNA in

minus-strand RNA.

qA

C

-E

50

40

30

20

10

2 hr pi

i

i

L. M. _

_6-3hr pi

* _.n _. _.

-IU LU0 U

Pulse,

minutes

FIG. 2. Presence of nascent SFV minus-strand RNA inRFRNA. SFV-infected BHK-21 cell cultures

wereexposed at2or 3h p.i. to[3H]uridine (250

ACil

ml)for periods of 5 to 30 min, after which time they wereharvestedasdescribed in the text. The RFRNA

was obtained and, after heat denaturation, the

amount of labeled RF RNA in minus-strand RNA

was determinedby hybridization with excess unla-beled virion RNA (see Fig. 1). Symbols: 0, Minus-strand RNA in RF RNA obtained from cultures pulsed at2hp.i.; , minus-strand RNA in RF RNA obtainedfiomculturespulsed at 3 h p.i.

after3h.Furthermore, since the rate of

[3H]-uridine

incorporation

into

newly

synthesized

plus-strand RNA remainedconstantfor1to 2 h

after the cessation of minus-strand synthesis,

previously synthesized minus-strand RNAmust

continuetofunctionastemplate for plus-strand

synthesis andnotbesubjecttodegradation after

3h.

Patternof

virus-specific

proteins.We next

determinedwhether there was a changein the

pattern

of viral

protein synthesis

atthetime of

minus-strand RNA inhibition. At different

times

after infection with

SFV,

BHKcellswerelabeled

with

[3S]methionine

aftertreatmentof the

cul-tures for 30 min with hypertonic medium to

reduce the

background

of hostprotein synthesis.

Figure 3 represents a

fluorogram

of the viral

proteinssynthesized during 30-min intervals

be-tween1and4.5hafter infection.The

synthesis

ofns70 andns86wasdetectable

beginning

at1.5

handwas

maximnal

between 2.5and3

h;

there-after the

synthesis

of ns7O and ns86 declined

precipitously.

On the other

hand,

the

synthesis

of the structural

proteins,

capsid

and the two

envelope

proteins

(El

and

E2),

was first

ob-servedat2h and

only

became maximalat3to

3.5 h p.i. The structural

proteins

became the

predominant

proteins

synthesized by

infected

cells at 3.5 h.

Thus,

the time atwhich

minus-strand

synthesis

ceased was

nearly

the same

time that the infected cell

began

to

synthesize

almost

exclusively

structural

proteins.

Effect of

protein

synthesis

inhibition on

If%

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112

SAWICKI AND SAWICKI

1 1.5 2 2.5 3 3.5 4 X

...g._ ...

* m

__

beginningat 1.5h

p.i.,

cycloheximide

wasadded

to a final concentration of 50

,ug/ml.

Thirty

minutes after the

cycloheximide

wasadded, half

of the cultures were labeled for 30 min with

[3H]uridine.

Theother halfwerelabeled for30

min with

[3H]uridine

at 4.5 h after infection.

Replicate cultures of SFV-infected cells that

re-ceivedno

cycloheximide

alsowerelabeled for30

minwith

[3H]uridine

at0.5-hintervals

beginning

1.5hp.i. Between1.5and2.5h

p.i.,

therewas an

86

exponential

riseintherateof RNA

synthesis

in

SFV-infected cultures (Fig. 4). The addition of

cycloheximide

blocked the increase in the rate

of SFV RNA

synthesis.

Addition of

cyclohexi-nz7O mide to infected cultures after 3 h p.i. had no

effecton the rate of RNA

synthesis.

RF RNA

wasisolated from cellsthateitherweretreated

with

cycloheximide

at 1 or 1.5 h

p.i.

or were

E2

E1

FIG. 3. SFV viralproteins synthesized at various timesafterinfection. BHK-21 cell cultures in 60-mm petri plateswereinfectedwithSFV at an MOI of 100. The timesindicated represent the time of addition of medium containing 0.335 M NaCl (16, 21) to the individual cultures. Startingat 1 hp.i., the cultures

wereexposed to the hypertonic medium for 30 min, followed by a 30-min pulse of[35S]methionine in mediumcontaining0.1Msucrose and a90-min chase period. Whole cells were collected into ET buffer containing 2% SDS, and portions containing equal

amountsof total cell protein were electrophoresed on

an8%polyacrylamide slab gel as described in the

text.At3hp.i.,aduplicate culture was pulsed with

[365]methionine for 7min after synchronization of

translation by hypertonic treatment and chased for

90minin order to label specifically theN-terminal

twopolypeptides translatedfromthe 42Splus-strand RNA, ns7Oandns86(lane X). The portions applied

tothe gelcontained approximately the following cpm: (1 h) 200,000; (1.5 h) 160,000; (2 h) 120,000; (2.5 h) 130,000; (3 h) 90,000; (3.5 h) 90,000; (4 h)120,000. The exposure time was 20 h.

minus-strand synthesis. We then asked

whether the cessation of synthesis of minus-strand RNAwouldoccurwhen protein synthesis wasinhibited before the

time

that the infected

cells began to synthesize predominantly viral

structuralproteins.

Replicate

cultures of BHK

cellswereinfected withSFV. At 0.5-h intervals

20

10

'U,

II55

U a

I.2

I

a

s

1 2 3 4 S

Moore p.1.

FIG. 4. Effectofcycloheximidetreatment onSFV RNAsynthesis.BHK-21 cell cultures in35-mmpetri plates were infected with SFV (MOIof 100). Un-treated cultureswerelabeled in the presenceofAMD for30 minwith [3H]uridine (50 uCi/ml) at 30-min intervals between 1 and5 hp.i. At 0.5-h intervals beginning 1.5 hp.i., replicate cultureswere treated withcycloheximide (50 pg/ml). The arrowsindicate the timeofcycloheximide addition. Half ofthe

cul-tures were labeledfor a30-minperiod with

[3H]-uridinebeginning30minafteradditionofthedrug. The otherhalfwaspulsed with

[3H]uridine

at 4.5 to 5hp.i. Theinfectedcellswereharvestedatthe end ofthepulseperiod by solubilization in ET-SDSbuffer

asdescribed in thetext. Portions induplicatewere

assayedforacid-insolubleradioactivity andfor pro-tein by the methodof Lowryetal. (13).Symbols: 0,

[3H]uridine

SFV RNA in untreated cultures; 0,

[3H]uridine

SFV RNA incycloheximide-treated

cul-tures.

I

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.: oo:.kfw

"Ie.

O' I

I

0

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VOL. 34, 1980

untreated and thatwerelabeledat6h

p.i.

for1

h with [3H]uridine. At 1 to 1.5h

p.i.,

therewas

nodetectable synthesis of 26S SFV RNA (data

notshown) or ofviralstructural proteins (Fig.

3). The presence oflabeled minus-strand RNA

intheRFRNAwasdeterminedbyhybridization

to an excessof unlabeledvirionRNA.Nolabeled

minus-strand RNAwasdetected when the label

was added 5 h after cycloheximide

(data

not

shown). We

conclude, therefore,

that the

syn-thesisofminus-strand, butnot

plus-strand,

RNA

isprevented by

long-term cycloheximide

treat-ment,and that the accumulation of viral

struc-tural proteins is not

required

to shut off the

synthesis

of minus-strand RNA.

A

possible

requirement for continued

transla-tion of the viral nonstructuralproteinsforSFV

minus-strand

transcription

wassuggested from

these results and from those ofFig. 3 andwas

investigated

further. The effect ofcycloheximide

on minus-strand synthesis was monitored at

shortintervals after its addition totheinfected

monolayers. At 2 h p.i., when transcription of

minus-strand RNA was at a maximum,

cyclo-heximide was added to a final concentration

of 50 ug/ml. The cultures were pulsed with

[3H]uridine

for 30-min periods between 2 and

4.5hp.i. The addition of cycloheximide at 2 h

p.i. stoppedthe increase in viral RNA synthesis

(Fig.5A) and the increase in RF RNA (Fig.5B).

Compared with the increasing amounts of

la-beled RF RNAisolated from untreated cultures

between2 and 3.5 h p.i., a constant amount of

labeled RF RNA was obtained from infected

cultures thatwere treated withcycloheximide at

2hp.i. andmaintained in the presence of

cyclo-heximide.

Hybridization ofthe labeled RFRNA with an

excess ofunlabeled virion RNA indicated that

minus-strand RNAsynthesis rapidly ceased in

thepresence of cycloheximide (Fig.

50).

When

infected cells were labeled at 2 h p.i. with

[3H]uridine

for30mininthepresence of

cyclo-heximide, the amount of labeled minus-strand

RNAdetected in theRF RNA wasthe same as

that foundininfected cellsthatwere labeled at

1.5hp.i. for30mininthe absenceof

cyclohexi-mide. When infected

cells

were treated with

cycloheximide

at 2h p.i. and labeledfor 30min

with

[3H]uridine

at2.5 to 3or 3 to3.5h,

essen-tially

no labeled minus-strand RNA was

de-tected inthe RFRNA.Sucrosegradient analysis

demonstratedthat 42S and 26SRNA was

syn-thesized during cycloheximide treatment (data

not shown). Therefore, minus-strand, but not

plus-strand,RNAsynthesis rapidly declined in

thepresenceofcycloheximide.

An identicalexperimentwascarriedout, using

SFV MINUS-STRAND RNA 113

puromycin to inhibitprotein synthesis. At 2 h

p.i., medium containing puromycin (50 ug/ml) wasadded toSFV-infectedcultures whichwere

subsequently labeled with [3H]uridine for

30-min intervals over the next 2 to 3h (Fig. 6A).

Compared with the untreated cultures,

puro-mycin addition resultedin a50% decreaseinthe

amountof SFV RNAsynthesized in the first30

min of

drug

addition. This was followed by a

constant or even decreased level of RNA

syn-thesis for thenext 2h.Similartocycloheximide

treatment, the addition of puromycin stopped

the increase in total RF RNA, and the total

amountoflabeled RF RNApresentin

puromy-cin-treated cultures remained relatively

con-stant.Also similartotheeffect of

cycloheximide,

puromycin addition ledtotheselective and

rapid

shutoff of minus-strand RNAtranscription (Fig.

6B).

A closer examination of minus-strand

tran-scription was undertaken

by

labeling

with

[3H]uridine the SFVRNAsynthesized in5-min

intervalsduring the first30minof

cycloheximide

treatment.Cycloheximidewasaddedat2h

p.i.,

and the cells were given a 5-min

pulse

label

between2 and2.5h p. i.

(Fig.

7A). Therateof

SFV

transcription

increased

during

thefirst 15

min of

cycloheximide

treatment and became

constant by 20 min. The amount of labeled minus-strand RNA in isolated RF RNA

de-creasedata

rapid

ratefor thefirst 15minafter

drug

addition, continuing

to decrease

slowly

thereafter

(Fig.

7B).

Therefore,

labeled minus

strands detected after

cycloheximide

additionin

Fig.5Cwere

synthesized mainly

during

the first

15 minof the30-min

labeling period.

Since the

synthesis

of minus-strand RNA

essentially

ceased 20 min after the addition of

cyclohexi-mide but the

synthesis

of

plus-strand

RNA

con-tinued at a

given

rate, the number of

minus-strand RNA

templates

most

probably

deter-mines the rate of

plus-strand

RNA

synthesis.

Theseresults demonstratethattheSFV

polym-erase

responsible

for the

synthesis

of minus

strands is

short-lived,

whereas the SFV

polym-erase

responsible

for the

synthesis

of

plus

strands is

long-lived.

Reversal of inhibition of minus-strand

RNA

synthesis.

Since our results

implicated

continued

protein

synthesis

as a

requirement

for

minus-strand

transcription,

the

resumption

of

translation afterremoval of the

cycloheximide-containingmediumand

thorough

washing

of the

cell

monolayers

shouldinturnbe

followed

by

a

resumption of minus-strand

transcription.

When

cycloheximide

wasaddedat2hp.i.andremoved

30 or 60 min later and then the

newly

synthe-sized RNA was labeled with

[3H]uridine,

the

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114 SAWICKI AND SAWICKI A

16

2

x

E

0L.

(z

I

12

8

4

J. VIROL.

B

Double stranded RNA

v 20

1

x

E 15

< 10

M,

I 5

E30

0..

< 20

10

I

2 3 4

Ho urs, p. i.

C

Minus Strand RNA

Hours, p. i.

FIG. 5. Effectof cycloheximidetreatment onSFVminus-strand RNAsynthesis.Starting 1.5 h p.i.,duplicate culturesofSFV-infectedBHK-21 cells(100-mm petri plates)werelabeled with[3H]uridine (250pLCi/ml)for sequential30-minperiods. Onesetofcultureswasleftuntreated(0);tothe otherset, mediumcontaining50 pgofcycloheximide(CH)permlwasaddedbeginningat2 hp.i.(0). The cultureswereharvestedatthe end ofthepulseperiodasdescribed in the text. (A) Total acid-insoluble[3H]uridine incorporation into SFV RNA.(B)Total[3HJuridine incorporationinto RF RNA obtainedfromuntreated andcycloheximide-treated

cultures. The RF RNAwasobtainedasdescribed in thetext.(C) Theamountoflabeled RF RNA in minus-strand RNA.TheRFRNAwasdenatured andhybridizedin the presenceofexcessunlabeled virion RNAas described inFig.1.

rate ofsynthesis of total SFV RNA increased afteralag period ofapproximately30min (Fig. 8A). The amount of labeled double-stranded

RNA also increased, and was proportional to

thatobserved for single-stranded RNA. Analysis of thepolarityofthelabeled RNA in the isolated RF RNAconfirmedthe presence of newly

syn-thesized minus strands in these RIs (Fig. 8B).

Transcription of minus-strand RNA began

within the first 30 min of drug removal and

increased substantially during the next 30 min

(drugremovedat3h) to 60 min (drug removed

at2.5h). Interestingly, the transcription of mi-nus strands declined between 4 and 4.5 h in

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SFV MINUS-STRAND RNA

115

thesis ofplus-strand RNA, is temporally

regu-lated and that the polymerase

activity

respon-sible for minus-strand RNA

synthesis

is

short-lived and

disappears quickly

afterinhibition of

protein synthesis with cycloheximide or

puro-mycin.

Our results

strengthen

and extendthose

of Bruton and

Kennedy (2),

who also

demon-strated the

temporal regulation

of minus-strand

RNAsynthesis in SFV-infected BHK cells and

suggested that therateof

plus-strand

RNA

syn-thesismight be correlated with theamount of

minus-strand RNA. We observed that therate

of

plus-strand

RNA

synthesis

becomesconstant

whenminus-strand RNA

synthesis

ceases, and

that blockage of

protein

synthesis,

which

in-hibits

rapidly

the

synthesis

of minus-strand

201

a40 ">

C.

30

E 20 .9,

20~~~~~~

%A 10. \

1 2 3 4 S

HOURS, POST-INFECTION

FIG. 6. Effect ofpuromycinadditiononSFVRNA

synthesis. (A) Total [3H]uridine incorporation into

SFVsingle- and double-stranded RNA during 30-min pulse periods from1.5to4.5hp.i. BHK-21 cell

cultures in 100-mmpetri plates were infected with

SFVatanMOIof100,labeled with [3H]uridine(250

.Ci/ml),

andharvested asdescribed inFig. 5.

Me-diumcontaining puromycin (50pg/ml)wasaddedto

culturesat2 hp.i. (arrow). (B) Percentageof total

[3HJuridine incorporatedintoRF RNA thatwasin

minus-strand RNA. The RF RNA was isolated as

described in thetext, denatured by heating,and hy-bridized in thepresenceofexcess unlabeled virion

RNA. Symbols: 0, [3H]uridine-labeled SFV RNA

obtainedfromuntreatedcultures;0,

[3HJuridine-la-beled SFV RNA obtained from puromycin-treated

cultures.

these cultures, although only 40 to 75% ofthe amountoftotal RF RNApresentin untreated cultures had beenfornedby this time.

DISCUSSION

Ourresults demonstratethat thesynthesisof

SFVminus-strandRNA,in contrast tothe

syn-C

c E

z

cc

to

Sc

l0o

5

2

40 3a 201

10I

A

0

5 10 15 20 25 30 B

N\

[image:8.508.65.247.52.430.2]

5 10 1 5 20 25 30 Minutes after CH addition

FIG. 7. Kineticsofminus-strand inhibitionby

cy-cloheximide. SFV-infected BHK-21 cell cultures in

100-mmpetri platesweretreated withcycloheximide (50 pg/ml) at2 hp.i. andpulsedwith [3HJuridine

(250,Ci/ml) for sequential5-minperiods between2

and 2.5 hp.i. (A)Total[3H]uridine incorporatedinto

acid-insoluble SFV single- and double-stranded

RNA.(B) Percentage ofthe total[3HJuridine

incor-poratedintoRF RNA in minus-strandRNA,

deter-minedasdescribedinFig.1.

VOL. 34, 1980

< 16

E

C)

12

z

,2 8

r-=4 ?.

c0

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116 SAWICKI AND SAWICKI

0

ICH 2-2112

hr

i Ii i

I!

i

Ii

I

1i

,c

CH continuous

75

° 50

x

E

Ca.

z

I25

B

4

CH

2-2I/2

hr

CH 2-3 hr

CH continuous

2 3

4

5

Hours post-infect ion

2 3 4 5

FIG. 8. Resumption ofSFV minus-strand RNAsynthesis afterremovalofcycloheximide.Medium

contain-ing cycloheximide (50 pg/ml)wasadded at 2 hp.i.toall cultures(arrow);in certaincultures,itwasremoved

30min (0) or60min (A) later (see text).[3H]uridine(250 g.Ci/ml) wasaddedfor30minat0.5-hintervals either in thepresence (cultures continuously treated with cycloheximide) orin the absence(cultures from

which the drug hadpreviously been removed) of cycloheximide. The cpm shown represents the total

incorporationobtainedfrom each 100-mmpetri plate. Thecultureswereharvested atthe endofthepulse period. (A)[3H]uridineincorporation into SFVsingle- anddouble-stranded RNA. (B)[3H]uridine-labeled

RF RNA in minus-strand RNA. Between 2 and 4.5 hp.i.,theamountoflabeledminus-strand RNAdetected

in these cultureswasasfollows: (cycloheximide,2to2.5h) 625,000cpm;(cycloheximide,2to3h)356,000cpm;

(untreated)820,000cpm;(cycloheximidecontinuousfrom2h)100,000cpm.

RNA but not ofplus-strand RNA,also results in

a constant rate of RNAsynthesis. Polymerase

proteinspresent in SFV-infected cells that have had theirprotein synthesisarrested continue to function in thesynthesisofplus-strandRNAby transcribing the template minus-strand RNA which was formed before protein synthesis

in-hibition; thus,the number ofminus-strand tem-platesappearstocontrol therateofplus-strand synthesis. Only when minus-strand synthesis

wasallowedtoresumebyremoval ofthe cyclo-heximide didweobserveanincreaseintherate

ofplus-strand synthesis.

Weconclude fromthese results that transcrip-tion of SFV minus-strand RNA is regulatedat

the level of translation bya mechanism which

utilizesone or moreshort-lived polymerase

pro-teins that either are not involved in the

tran-scriptionofplus-strandRNAor aremodified by

cleavage or other posttranslational modifica-tions to form the stableplus-strand polymerase. Thus, there may exist in SFV-infected cells

either two forms of theviralpolymeraseortwo distinctpolymerases.

Nothingis known ofthe location within the SFV-infected cell ofminus-strandpolymeraseor

ofthe replication complex transcribing minus-strandRNA. The recentreportof Dasguptaet

al. (5) indicates that thepresumptive poliovirus minus-strand polymerase (replicase) was

de-tected inasoluble ratherthanmembrane-bound

fraction of theinfected cell.The SFV plus-strand polymerase is associated with a

membrane-boundreplication complex (4,7, 9, 15, 18, 28, 29). Recently, the SFV nonstructural protein ns70 has been shown to be the major viral poly-peptideassociated with thereplication complex (4, 19; Gomatos,unpublished data). In addition, A

16

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2 I.x

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VOL. 34,1980

smalleramountsof ns72 andns86werefoundin

this complex (19; Gomatos, unpublished data). Themigration of ns70 in polyacrylamidegels is always as a broad band, suggesting that this

polypeptide chain may have undergone

post-translational modification. Suchamodification

could account for temporal alteration of

tem-plate preference of the SFV polymerase. Also uncleavedorincompletelycleavedprecursorsof

thenonstructuralproteins could function in al-phavirus minus-strandsynthesis. Insupport of these possibilitiesisthe finding that continued proteinsynthesisisnecessaryfor the replication

ofvesicular stomatitis virion RNA (17, 31) and that there is an apparent correlation between

the cleavage of certain poliovirus proteins and replicase instability in poliovirus-infected cells

(10).

It has been reported previously that the amount but notthe ability of the viral

polym-erase to synthesizeplus-strand RNA in alpha-virus-infected cellscould be varied bythe

addi-tionofpuromycinorcycloheximideatdifferent

timesearly in infection (30). However, by 3to4 h p.i., essentially all of the normal amount of viralpolymeraseresponsible for the synthesis of

plus-strandRNA had been formed and

inhibi-tion ofprotein synthesis after this time didnot affect the amount ofRNA synthesized (6, 25, 30). We found,asdid others (6, 25, 30), that the

SFVplus-strandpolymerase formed before the

addition of cycloheximide or puromycin

re-mained active formanyhours in theabsence of

continuedprotein synthesis. Our results indicate that this is not the case for the synthesis of

minus-strand RNA. The addition of cyclohexi-mide orpuromycintoculturesactively

synthe-sizing both plus- and minus-strand SFV RNA

led veryquicklytothe selective loss of minus-strand transcription. The fact thattwoprotein synthesisinhibitors whose mechanisms of inhi-bitionare differentresult in thesame phenom-enonleadsustoconcludethat this isduetothe inhibition of translation.Thus,wearguethatit is thesynthesisoftheminus-strandpolymerase thatregulatestherate ofplus-strandRNA

syn-thesis by determining the number of minus-strandtemplatesformed in the infected cell.

Our characterization of the synthesis of mi-nus-strand RNAsuggestedthat its cessationwas

correlated with two changes in the pattem of viralprotein synthesis: adecrease in the

trans-lation of nonstructuralproteinsand the

appear-anceoflargeamounts ofstructuralproteins.The

nonstructural proteins are translated from the

42Splus-strandRNAas apolyproteinfollowed

by cleavageinto the individualproteins,whereas

the structural proteins are read from the 26S

mRNA, also initiallyas a polyprotein (2, 9, 18,

SFV MINUS-STRAND RNA

117

29). Therefore, the

ability

toregulate the

pro-ductionof the

nonstructural

proteins

independ-ently of the structural proteins exists for the

alphavirusesbut notfor otherplus-strandRNA

virusessuchaspoliovirus,whichproducesallof

itsviralproteins from thesamepolyprotein(20).

The observed

instability

of the minus-strand

polymerasecoupled with the decreased

transla-tionof

all

nonstructural proteins could be one

explanation

for theselectivecessation of

detect-able minus-strand transcriptionatabout 3 to 4

hp.i.

Weproposethe

following

scheme for the early events Mi SFV replication. The SFV parental genomeisfirsttranslated into the viral

nonstruc-tural

proteins,

someof which

function

to

tran-scribe thissameRNA molecule into

complemen-taryminus strands. For thistooccur,the

minus-strand

polymerase

mustinitiate RNAsynthesis

at the 3' end of the 42S parental plus strand

(22). Because ribosomes bind to the 5' end of

42Splus-strand RNA and failtoproceedpast a

termination signal located approximately

two-thirdsof the distance from the 5' end(9, 18, 29),

the3'terminus of the

parental

plus strand might

be

readily

accessible for recognition by

minus-strand

polymerase.

We have been unable to

detect the

synthesis

of

only minus-strand

RNA;

RIs synthesizing

both

plus

and minus strands

were always present

early

in infection, in

ap-proximately

a5:1 ratio

(see

Fig.

2). At

approxi-mately 3.5 h after

infection,

minus-strand

syn-thesis ceased

although

plus-strand

RNA

contin-ued toaccumulate: 26S on polysomes and42S

in

nucleocapsids.

A

possible

advantage conferred

by thecessation of minus-strand RNA

synthesis

is that of allowing "free" 42S

plus

strands to

accumulate for the fonnation of nucleocapsids;

otherwise, 42S

plus

strandsmight continuetobe

converted into RIs at the expense of progeny

virion

production.

ACKNONLEDGMENTS

Wethank ThomasSawyerforexcellenttechnical asistance and Peter J.GomatosandPaul Lehmann forhelpful discus-sions.

This investigation was supported by funds from Public HealthService Research grant AI-15123 from the National Institute forAllergyand Infectious Diseases and from BRS grant5S07-RR-05700-09awardedbytheBiomedical Research

Support Resources,National Institutes ofHealth.

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