Rhinovirus multistranded RNA: dependence of the replicative form on the presence of actinomycin D.

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JOURNALOF VIROLOGY, June 1976, p.926-932 Copyright©3 1976 AmericanSociety forMicrobiology

Vol. 18,No. 3 Printed in U.SA.

Rhinovirus Multistranded RNA: Dependence of the

Replicative

Form

on

the Presence of

Actinomycin D

MALCOLM R. MACNAUGHTON,'* JONATHAN A. COOPER, AND NIGELJ. DIMMOCK

Department ofBiological Sciences, University of Warwick, CoventryCV4 7AL,England Receivedfor publication 15December 1975

The multistranded and double-stranded RNAs synthesized in HeLa cells infected with rhinovirus in thepresence and in the absence ofactinomycin D have been characterized by polyacrylamide gel electrophoresis and hybridiza-tion studies. The replicative form is only foundin infected cells treated with actinomycin D, whereas the replicative intermediateis found in both the

pres-enceand the absenceof thedrug.Thesignificanceof these results isdiscussed.

The

genome

of picornaviruses consists of

one

molecule of

single-stranded RNA that is

infec-tious

under appropriate

conditions (6).

How-ever, RNA

complementary

tothegenome is not

infectious

(4, 5, 26).

The

genome

of

picornavi-ruses is present in

polysomes

of

infected

cells

and

has been shown

to

function

as

mRNA (16,

24, 29).

It has been suggested that the

input

genome

RNA

is

replicated

in a two-step proc-ess,

namely, the formation of

a

complementary

template that

is

then

preferentially copied

to

produce

an excess

of genomic

RNA

(SS RNA)

(6).

Two

replicative

structures,

the replicative

in-termediate

(RI) and the

replicative form (RF),

have been obtained

from human cells infected

with

rhinovirus

in

the

presence

of actinomycin

D

and

characterized

on

polyacrylamide

gels

(17).

The

RI has been shown to be

a

multi-stranded

structure,

and

the RF has

been

shown to

be

a

double-stranded

structure (17). In our

study

we

investigated the effect of actinomycin

Don

rhinovirus multiplication.

We

found that

although the

virus

multiplies with

very

similar

kinetics

in

the

presence or in

the

absence of

actinomycin D, RF

is

only found

in

cells treated

with actinomycin

D.

The inference that RF

does

notexist under

natural conditionb

Jf

infec-tion is discussed.

MATERIALS AND METHODS

Propagation of cells and viruses. Monolayers of HeLa cells were grown in Eagle medium supple-mented with 10% calfserum, 0.1% sodium bicarbon-ate, and antibiotics. The cells were infected with rhinovirus type2 at 0.1PFU/cell. Unlabeled infec-tious virus waspreparedfrom these cells as previ-ouslydescribed (17).

Preparation of radioactive rhinovirus-specified

'Present address: Division of Communicable Diseases, Clinical Research Centre, Harrow, Middlesex HAl 3UJ, England.

RNA. Monolayers of cells were infected with 10 PFU/cell of virus at 33 C. After 1 h the virus was

removed andreplaced with medium containing2%

calfserumand, when appropriate,1 ittgof actinomy-cin D per ml was added. After incubation for the required time, 200

,uCi

of [3H]uridine (specific activ-ity, 24 Ci/mmol) (Radiochemical Centre, Amer-sham) in2mlof mediumcontaining2%calfserum

wasadded.The RNA was extracted with phenol in the presence of sodium dodecyl sulfate (17) and then precipitated with2volumes of ethanolat -20 Cfor16

h.

Chromatography of RNAon cellulose columns. Rhinovirus RNA specieswerefractionatedby chro-matography on Whatman CF11 cellulose, which separatessingle-stranded RNA from multistranded RNA (10). Columns (11 by 1.5 cm) were packed under gravity inTNE buffer solution (0.1M

NaCl-0.05 MTris-0.001 MEDTA [disodiumsalt] adjusted

topH7.0 at 20 C withHCl) containing 0.1% sodium dodecyl sulfate plus35% (vol/vol) ethanol. The

col-umnswereloaded with RNA at concentrations not

more than 0.5 mg/ml, and during elution at room temperature the flow rate was maintained at 1 ml/

min.RNA specieswereeluted by stepwise washings ofthecolumns with0.1%sodiumdodecyl sulfatein

TNE buffer solution containing decreasing concen-trationsof ethanol (i.e., 35%ethanol, 15% ethanol, and 0% ethanol). Fractions (1 ml) were collected,

and aliquots were assayed for radioactivity. The RNA specieswereconcentrated by precipitation at

-20Cwith100,ugof tRNA as carrier and 2 volumes

ofethanol.

Polyacrylamide gel electrophoresis of RNA.

Polyacrylamide gels (2.2%) supported by 0.5%

aga-rose were made by the procedure of Loening (18) with certainmodifications (20). Thegelswere poly-merized in 10-cm tubeswith internal diameters of

0.7cm.Afterapre-electrophoresis of30min at 50V, up to 60,mg ofRNA wasloadedonto eachgeland

subjectedtoelectrophoresis for3 to5hat 50V.After electrophoresis the gelswereextruded andanalyzed

on a Gilfordgel scanner for materialabsorbing at 260 nm. The gels were frozen, sliced into 1-mm

disks, and solubilized, and the radioactivity was

measuredaspreviously described (17).

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Hybridization. The hybridization method used

wasessentially that of Avery (1). Radioactively

la-beled RNA preparations were dissolved in 0.02x

SSC (0.15M NaClplus0.015Msodium citrate, pH

7.0)and denaturedby heatingat98Cfor4minand then rapidly brought to 6x SSC and 65C. When appropriate,excessunlabeled virus RNAwasadded

at this stage. Samples were diluted after 4 h of

incubation inwaterat37Ctobring thesalt

concen-trationto1x SSC. Pancreatic andT, ribonucleases

(Sigma Chemical Co. Ltd., London)wereaddedto50

,ug/ml and170units/ml,respectively, and digestion

was allowed to proceed for 20 min at 37C. The reaction was stopped by the addition of250 ,mg of

yeast RNA and an equal volume of ice-cold 10%

trichloroacetic acid. Other samples were

precipi-tated without priorenzymedigestion to determine the total acid-precipitable radioactivity.

Precipi-tateswerecollectedoncelluloseacetatefilters (Mil-lipore Ltd., London) andwashed twice with cold5%

trichloroacetic acid and ether. The filtersweredried

underaninfraredlamp andassayed for

radioactiv-ity.

Infectivity titrations. The infectivity of virus

sampleswastitratedbyplaqueassayonmonolayers

of HeLacellsseeded into petri dishes (5cmin

diam-eter)(31). The disheswereinoculatedwith 0.2mlof

virusdiluted in Eagle medium containing4%calf serum,which wasallowed toadsorb for 90min at

33C. The inoculum was removed, and the dishes were incubated with 5 ml ofan overlay medium

containing0.5% (wt/vol) agarose (30) at33C for4

days. The cells were fixed with formal saline, the agar wasremoved, andthe cellswerestainedwith 1%gentian violetin 20%ethanol.

Inhibition of protein synthesis in rabbit

reticulo-cytelysatesby double-strandedRNA. Protein syn-thesisin rabbitreticulocyte lysateswas measured

according to established procedures (9).

Double-stranded and multiDouble-stranded RNAs from8x 106 cells

wereseparated by cellulose CF11 chromatography. RNAfrom thepeak fractionwasseriallydiluted in

100mMKCI,and

5-j.l

aliquotswereaddedto35,ul

ofreticulocyte lysate containing 25 JLMhemin.

As-says were preincubated at 30 C for 30 min before adding 10 ,IA of a solution containing 2.5 ,iCi of

[3H]leucine (specific activity, 2.0 Ci/mmol) (Radi-ochemical Centre, Amersham) and other

compo-nentsneeded forprotein synthesis (9). Aftera

fur-ther 30min ofincubation,

10-IAI

samples were

re-moved, hydrolyzed with alkali, precipitated with

trichloroacetic acid, and assayed for radioactivity. Incubationsinthe absenceofaddedRNA incorpo-rated 39,000 counts/min for a 10-1A sample. The

dilutionof RNAinhibitingprotein synthesis by50%

wascalculatedbyinterpolationonastandardcurve.

The kinetics of inhibition were characteristic of

double-stranded RNA. Under thesame conditions a preparation of polyriboinosinic-polyribocytidylic

acidhada50%inhibitoryconcentration of3 x 10-10

g/ml.

RESULTS

Effect ofactinomycin Donrhinovirus

mul-tiplication.

Certain strains of

poliovirus

have been shown to be sensitive to

actinomycin

D

during

their

multiplication

cycle in

human

cells

(7, 13, 28).

HeLa

cells were infected with

rhinovirus

to see

if actinomycin

D

affected the

production

of virions. Table 1 shows

the

effect of

actinomycin

D

on the

production of infectious

virus

in

HeLa

cells during

the period

of

expo-nential increase (from

5 to 8 h

postinfection).

Actinomycin

D (1

,ug/ml) has

only a

slightly

inhibitory effect during this time on the

appear-ance

of infectious virus. However, its rate of

production or final yield after 12 h of infection

was not

affected.

Not

only

does

actinomycin

D

have

little effect

on

the production of infectious virus, but

it

also

has a

negligible effect on the total amount of

viral RNA species synthesized during infection.

However, the relative amounts of RI and

RF

produced under these conditions were difficult

to

calculate, as these species were obscured by

cellular RNAs, presumably heterogeneous

nu-clear

RNAs and rRNA precursors, when no

actinomycin

D

was present

during infection. To

analyze RI and RF further, double-stranded

and

multistranded RNAs

were

separated from

single-stranded RNAs by cellulose

CF1l

chro-matography

(17).

Double-stranded and multistranded RNAs

synthesized

in

the presence and in the absence

of

actinomycin

D.

Figure

1

shows

polyacryl-amide

gel

profiles of

[3H]uridine-labeled

dou-ble-stranded and multistranded RNAs obtained

from

infected or noninfected HeLa cells

incu-bated

in

the presence or absence of actinomycin

D.

The

cells

were

labeled from

6 to 8

h during

TABLE 1. Productionof infectiousrhinovirus in HeLacells in the presence and absenceof

actinomycin Da

Incubation

Treatment time(hours PFU/ml(x 102)1

postinfection)

No actino- 5 9.8

mycin D 6 29

7 340

8 3,100

Actinomy- 5 6.7

cin D (1 6 20

,Lg/ml)

7 250

8 1,400

a Monolayers wereinfectedat amultiplicity of10

PFU/cell for 1 h at 33C. The inoculumwasremoved bywashingthe cell sheet threetimes,andthe incu-bation was continued inEaglemedium containing 2% calf serum and appropriate amountsof actino-mycin D. At the times indicatedcellswerescraped

intothe medium anddisruptedbefore thetotal

in-fectivitywasassayed.

bThese results areaverages ofduplicatesamples.

Asimilarexperimentproducedsimilar results.

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928

MACNAUGHTON, COOPER, AND DIMMOCK

the

exponential period of increase of infectious

virus

(17).

All

the manipulations

were

done

at

the same time under identical

conditions,

and

each

profile

is

derived from the

same

number of

HeLa cells. RNA was

extracted,

and

then

dou-ble-stranded and multistranded RNAs were

separated from single-stranded RNAs on

cellu-DNA

o

-*A

A

4

-

~

RF

3

RI

2

'C

c

DNA

o

C5

lose

CF11 columns by elution with different

concentrations

of ethanol (19).

Single-stranded

RNAs were

eluted with 15%

ethanol, and

mul-tistranded

RNAs were eluted with 0% ethanol.

The elution patterns

of RNAs from cells treated

with and

without actinomycin D were

the

same.

Treatment of the fractions of

Fig.

1

with

Fractions

FIG. 1.Polyacrylamidegelelectrophoresis ofdouble-stranded and multistranded RNAs extractedfrom rhinovirus-infected anduninfected HeLa cells. These RNAswereseparatedfrom single-strandedRNAson

cellulose CFI1 columns. (A) RNAfrom rhinovirus-infected cells labeled with[3H]uridinefrom 6 to8 h postinfection in the presence of actinomycin D; (B) RNA fromrhinovirus-infected cells labeled with 13H]-uridinefrom6 to 8h postinfection in the absence of actinomycin D; (C) RNAfromuninfected cells labeled

with

[3H]uridine

for2h in the presence of actinomycin D; (D) RNAfromuninfected cells labeled with

PH]-uridinefor2h in the absence ofactinomycinD. Thearrows are taken from the absorbance at 260nmtrace indicatingthe position of added"marker"'HeLa DNA.

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RHINOVIRUS

MULTISTRANDED RNA

929

10

,ug

of DNase per ml for

1

h

at room

tempera-ture

did

not

alter the

profiles

on

polyacrylamide

gels.

Both RI

and RF

were found

in

infected cells

treated with actinomycin

D

(Fig.

1A), but

no

RF

was

found

in

cells infected

in

the

absence of

actinomycin

D,

and the

only multistranded

spe-cies

had

a

mobility similar

to

RI

(Fig. 1B). We

have called the latter species putative RI (pRI).

It is interesting to note

that this pRI appears to

be

larger

in

size

than the RI obtained from

infected cells treated with actinomycin

D.

These

profiles from infected

cells

were

com-pared with

parallel profiles

of multistranded

RNAs

obtained from noninfected

cells

incu-bated with (Fig. 1C) or without (Fig. 1D)

actino-mycin

D.

Some

multistranded RNAs were

ob-tained under both conditions, and

a

greater

amount was

obtained from the culture that did

not

receive

the drug.

This RNA appeared

to

be

very

large,

as

it

only

just

entered

the

gels, and

itmay

be

heterogeneous nuclear RNA, which is

known

to

be

partly

double stranded (15, 27). In

both

cases

the

profiles of

the

peaks

were

differ-ent

from those

from infected

cells,

but the

back-ground radioactivity

was

identical

in

infected

and

noninfected cultures.

Similar

results

showing

that RF is

undetect-able in the

absence

of

actinomycin

D

(M. R.

Macnaughton,

unpublished data)

have

been

ob-tained

with rhinovirus-infected human

embryo

lung cells.

Both RI and RF species are infectious (6), and

we

have shown that RI

+

RF

and pRI from

infected

HeLa cells are infectious to similar

degrees when titrated by plaque assay on

mon-olayers

of HeLa

cells.

This result shows that

pRI

is at least partially virus specified. The

following experiments were performed to show

that

RI

and pRI are essentially the same

spe-cies.

Treatment

of pRI with pancreatic RNase (50

,ug/ml)

and

T,

RNase (170 units/ml), which

di-gest

single-stranded but not double-stranded

RNAs (1), converted pRI into a double-stranded

RNA species with a mobility similar to RF (Fig.

2). A similar result is obtained on treating RI

with RNase (17). This result is consistent with

the view that

pRI

is like RI,

which comprises a

template molecule of the

same size as

the

ge-nome

and is partly

hydrogen

bonded to nascent

molecules

that have single-stranded "tails."

These tails are removed by RNase treatment,

leaving a double-stranded structure of the same

size

as

RF

consisting of one

discrete and one

segmented

molecule (6).

Characterization

of

pRI by hybridization.

Multistranded

and double-stranded RNAs

pre-pared

by CF11 cellulose chromatography

from

HeLa

cells infected

in the presence or absence

of

actinomycin D were used

in

these studies.

We

know by polyacrylamide gel electrophoresis

that the preparations from actinomycin

D-treated

cultures contain only RI +

RF'

and

1E

RI 2

20 40 20 40

Fractions

FIG. 2. Polyacrylamidegelelectrophoresis

of

double-stranded and multistranded RNAs extracted

from

rhinovirus-infectedHeLa cells withoutactinomycinDtreatment. These RNAswere

separated

oncellulose CF11 columnsfromsingle-strandedRNAs. The RNAswerelabeled with[3H]uridine

from

6to8 h

postinfec-tion intheabsenceofactinomycin D.(A)UntreatedRNA;(B)RNAincubated with 50

pg

ofpancreaticRNase per ml and170 unitsof T,RNase per mlinI xSSCat37Cfor20min.Thearrows aretakenfromthe

absorb-ance at260 nm traceindicatingthepositionofadded marker HeLa DNA. VOL. 18, 1976

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930 MACNAUGHTON, COOPER, AND DIMMOCK

those from the untreated culturescontainonly pRI. Table 2 shows the percentages of 3H-la-beled RI + RF and pRI that self anneal or anneal withalarge (50-fold)excessofunlabeled virion RNA. The data show that about37% of RI + RF and about 21% ofpRI hydridized to excess virion RNA and hence consist ofviral complementary RNA. Thus, at least this pro-portion of the multistranded pRI consists of virus-specified RNA.

Assuming that the amountoflabeled virion-typeRNApresentin themultistranded species exceeds the complementary RNA (see above), then, bydefinition, theamount of complemen-taryRNApresentis half the value that results from self annealing (RNase resistance counts per minute). One can thus derive the ratio of self-annealed RNA to the independent meas-urement ofcomplementary RNA obtained by annealing with cold virion RNA. This value comesin allexperiments close tothe expected value of 2. This result shows that essentially all RI + RFand pRIare viral RNAspecies, since

any contamination with cellular RNAs would haveresulted in less annealing with unlabeled virion RNA and hencearatio of selfannealing toannealing withunlabeled virion RNA of less than 2.

The proportion of RNA complementary to virion RNA in RF is 50% (Macnaughton,

un-published data). Ourpreparation ofmulti-and double-stranded RNAs from actinomycin D-treatedcells containabout equalproportions of RIand RF.Byannealingthecombined RI+ RF

withvirionRNA,wefind that themixture

con-tains 37% complementary RNA. Since this fig-ureis less than50%weseethatthe majority of

RNA inthe RI synthesized inthe presence of actinomycin Dis of thesamepolarity asvirion RNA. Thisresult also argues that the propor-tion ofviral complementary RNA in pure RI

mustbe less than 37%. Henceour

figure

of21%

viralcomplementary RNA in pRI is consistent with it

being

a structure consisting largely of RNA ofthe same

polarity

as virion RNA. Our figure is closeto the value of 19%

complemen-tary RNA

reported

for

poliovirus

RI (21).

Inhibition of

protein

synthesis in reticulo-cyte lysates by double-stranded RNA. We have estimated theamountofdouble stranded-nessinrhinovirus RI and RI + RFby

measur-ing

theinhibition ofprotein synthesis in

reticu-locyte lysates (9).

Table 3 shows that there is considerablymoreinhibition ofprotein

synthe-sis

by

RI compared to double-stranded RNA from uninfected HeLa cells, although this inhi-bition is less than for a mixture of RI + RF. Assuming that this inhibition is a measure of double strandedness (9, 14), then there is con-siderable double strandedness in RI.

DISCUSSION

During infection with picornaviruses, RI has beenshowntobe the intermediateprecursorof

progeny virus SS RNA. In vivo, it is the first

RNAspecies tobe labeled by briefexposure to

radioactive precursors (3, 17), implyingthat it is the site ofnascent RNA. Similarly, in

vitro,

using virus polymerase preparations, RI has beenidentified as theprecursorofprogenySS RNA (12, 25). However, the role of RF in the synthesis of picornavirus RNA has neverbeen

readily understood, although it is now gener-ally regarded as an end product ofreplication (2, 3, 12, 17), and evidence has accumulated showing that RF isaconsequenceofreplication ratherthananintermediate thereof(3,22, 23). Nevertheless, RF has been shownto be infec-tious (6).

Our results show that in rhinovirus-infected cellsnottreated with actinomycin D, the only

TABLE 2. Annealing of 3H-labeled double-stranded and multistranded RNAs fromrhinovirus-infected HeLa cellsa

Annealing

withexcess RNAcom- Self-anneal- Self-an-Total counts/ unlabeledvir- plementary ingRNasere- nealing

Treatment mininviral ion RNA: tovirion sistance RNase re- Ratio(b/c) RNA RNaseresist- RNA(%) (counts/min) sistance

(a) ance(counts/ (c/a) (b) (%)

min) (c)

Incubation withac- 14,621 5,760 39.4 10,311 70.5 1.8

tinomycin D (RI 34,079 11,849 34.8 22,579 66.3 1.9

+ RF) 28,089 10,725 38.2 18,960 67.5 1.8

Incubation without 17,414 4,009 23.0 7,791 44.7 1.9

actinomycin D 10,772 1,991 18.5 3,943 36.6 2.0

(pRI) 52,386 11,762 20.5 20,861 39.8 1.9

aThe RI + RF andpRIwereobtained from infected cells labeled6 to8 hpostinfection

with

[3H]uridine

andseparatedfrom single-stranded RNAsbychromatographyonCF11cellulose.

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RHINOVIRUS

TABLz 3. Inhibition ofproteinsynthesis in reticulocyte lysates by double-stranded and multistrandedRNAsfrom rhinovirus-infected

and noninfected HeLa cells

ReciprocalofRNA Total dilutionproducing counts/ 50%inhibitionof RNAspeciea *nin un- proteinsynthesis

diluted

RNAb Uncor- Normal-rected izedc RI + RF from infected

cells treated with

ac-112

70 128

tinomycin D (1

Zgg

1,126 720 1,280 ml)

RI from infected cells

not treated with acti- 3,916 820 420 nomycin D

Double-stranded RNA

fromuninfected cells 168 NDd ND

treated with actino-mycin D (1

jig/ml)

Double-stranded RNA

from uninfected cells

8,414

280 70 nottreatedwith

acti-nomycin D

a RNA ineach case was extracted from 8 x

106

cells.

bAll double-stranded and multistranded RNAs wereseparated fromsingle-strandedRNAsby chro-matography on cellulose CF11 columns. Only the peak fractionfrom0% ethanol eluatewasused in theassay.

cNormalization toarbitraryunitsassumingthe

samespecificactivityofuridine ineachsample.

dND, Not done. Toolittle double-stranded RNA wasavailable for an accurate determination of the RNA dilution producing 50% inhibition of protein synthesis.

virus

replication

structure

found

is

RI.

Al-though the

actionof actinomycin D on the

repli-cation

of the viral RNA

is not yet

understood,

wesuggest

that

in

rhinovirus replication RI

is

the

only

true

replication

structure

and that RF

is

an

artifactual

structure

derived from RI

as a

result of actinomycin D

treatment.

It is

clear that

two

RIs

must

be

involved

in

the

production

of

large quantities of genomic

RNA from

a

single molecule of

genome

RNA

(6).

In

the first

RI, the input virus

genome

RNA

is

transcribed

to

form

complementary RNA,

and

this

RI

would

contain an excess

of

comple-mentary or negative RNA

strands.

Thisspecies is

called

negative RI.

The

complementary viral

RNAs

mustthen

be

replicated

to form a

large

number

of

genomic

or

positive RNAs.

This process

presumably

occurs in a

positive

RI

con-taining

an excessof

genomic

RNA

strands. Our

hybridization data

suggest

that under

our

con-ditions

of

infection and

labeling

the

majority of

RI consists of genomic strands, and thus the greater part of this RI consists of positive RI. At present, we are trying toidentify negative RI, which we would expect to be found during ear-lier stages of rhinovirus replication.

Oninfecting HeLa cells with poliovirus type 1 (strain LSc 2ab) in the presence and absence of actinomycin D (1

Ag/ml),

we have observed

that poliovirus

RF exists in the

infected cells

whether or not the cells had been treated with

actinomycin D.

Thus,

in

this

case

RF

is not an

artifactual

structure

caused by

actinomycin D. However,

the effect

of actinomycin D on

poliovirus multiplication is known

to

vary

with

the strain of poliovirus used

(7, 13, 28). Thus,

although

we are suggesting

that

during

natu-ral infections with rhinovirus

type 2 no

detecta-ble

RF is

produced, this hypothesis

appears not to

hold for all

picornaviruses.

Our results

may

have implications for

cer-tain aspects

concerning

the multiplication

of

other viruses,

since

double-stranded RNA

mol-ecules

are

suggested

to

have specific effects,

such

as

the

induction of interferon

(32),

cyto-pathic changes

(11, 30),

and inhibition of

pro-tein

synthesis

(8, 9, 14). We have shown

by

inhibition of

protein

synthesis in

reticulocyte

lysates with double-stranded RNA that there

is

considerable double strandedness

in

RI,

and

this

may

be

enough

to

induce the effects

cur-rently

ascribed

toRF.

However,

before

considering the implications

of these results

further,

we

need

to

know how

general

are

these

phenomena and

in

particular

to

what

extent

the RFs

of other

picornaviruses

depend

on

actinomycin

D

for their existence.

ACKNOWLEDGMENTS

Thanks are dueCheryl Penny for valuable technical assistanceduringpartof this workandtoS. I. Koliais for usefuldiscussions. ActinomycinDwas agiftfromMerck,

Sharpand Dohme ResearchLaboratories.

This workwassupported bya grantfrom the Medical ResearchCouncil, London,England.

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2. Baltimore,D. 1968.Structure of thepoliovirus replica-tiveintermediate RNA. J.Mol.Biol. 10:359-368. 3. Baltimore, D.,and M.Girard.1966.Anintermediatein

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Figure

TABLE 1. Production of infectious rhinovirus inHeLa cells in the presence and absence of

TABLE 1.

Production of infectious rhinovirus inHeLa cells in the presence and absence of p.2
FIG.1.postinfectionwithcelluloseuridineuridinerhinovirus-infectedindicating Polyacrylamide gel electrophoresis of double-stranded and multistranded RNAs extracted from and uninfected HeLa cells
FIG.1.postinfectionwithcelluloseuridineuridinerhinovirus-infectedindicating Polyacrylamide gel electrophoresis of double-stranded and multistranded RNAs extracted from and uninfected HeLa cells p.3
FIG.1Eperancerhinovirus-infectedCF11tion 2. Polyacrylamide gel electrophoresis of double-stranded and multistranded RNAs extracted from HeLa cells without actinomycin D treatment
FIG.1Eperancerhinovirus-infectedCF11tion 2. Polyacrylamide gel electrophoresis of double-stranded and multistranded RNAs extracted from HeLa cells without actinomycin D treatment p.4
TABLE 2. Annealing of3H-labeled double-stranded and multistranded RNAs from rhinovirus-infected HeLacellsa

TABLE 2.

Annealing of3H-labeled double-stranded and multistranded RNAs from rhinovirus-infected HeLacellsa p.5