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Basis for Variable Response of Arboviruses to Guanidine Treatment

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Basis for

Variable

Response

of Arboviruses

to

Guanidine Treatment

ROBERT M. FRIEDMAN

Laboratory ofPathology,NationalCancerInstitute, Bethesda,Maryland 20014

Received forpublication7July1970

The effect ofguanidine onthe replication of the group A arboviruses, Sindbis

virus,andSemliki Forest virus (SFV)wasstudied. Guanidine rapidly, but reversibly,

inhibited SFV ribonucleic acid (RNA) synthesis. The synthesis of all species of

viral RNAwasinhibited, but that of ribonuclease-resistant formswasleastaffected.

This inhibition occurred when the drug was added at any point during the log

phase of virus growth. Thegrowth of SFVwasalso markedly inhibited, butSindbis

virus growth was unimpaired. Infection ofguanidine-treated cells with the viruses

together resultedinasignificant inhibition of the yields of both. Itappearsthat,in

thecaseofSindbisvirus, viralRNA is ordinarilyproduced in suchexcessthat

in-hibition of its synthesis doesnotreducevirusyields. In thecaseofSFV, guanidine

alsomarkedly distorts thepattern ofRNAsynthesisby greatly decreasing the

pro-duction of the 26Sinterjacent RNA form. Thismay account forthe observed

in-hibition of SFV growth in thepresence ofguanidine.

Guanidinehas beenemployed extensivelyas an

inhibitorofpicornavirus replication.Some ofthe

data so far published suggest that its inhibitory

action is dueto a block in the initiation of new

viral ribonucleic acid (RNA) chains (2, 3),

al-thoughothermechanisms of action have also been

suggested (1, 11).A recentstudy suggestedthat a

structuralprotein ofpoliovirusis theprimarysite

ofguanidineaction (4). Onlyonereporthas been

published indicating that arboviruses are also

sensitive to

guanidine.

Semliki Forest virus

(SFV,

arborirus group

A) growth

was

reversibly

inhibitedby 30mmguanidine (5),aconcentration

which is20times thatusually employedtoinhibit

poliovirusreplication.

Inthisinvestigation,theeffect ofguanidineon

twogroup Aarboviruses,SFVand Sindbis

virus,

wasstudied insomedetail. Guanidinewasfound

toinhibit SFVreplication,

probably

as aresult of

analteration in thepattern of SFV RNA

produc-tion. On the other hand, despite aprofound

in-hibition of Sindbis virus RNA synthesis in

guanidine-treated cells,

no

significant

inhibition

of virusproductionwasfound.

MATERIALS AND METHODS

Viruses andcells. Primary chick embryo fibroblast

(CEF) cultures and pools ofSFV and Sindbis virus

were prepared and assayed by previously described methods(8, 13).

Proteinand RNAsynthesis. Protein and RNA syn-thesiswere estimated byincorporation of3H-leucine

or 3H-uridine into perchloric acid-precipitable

radio-activity(8).Total protein was estimated by the method

of Lowryetal. (9).

Viral RNA synthesis. RNA was extracted from

virus-infected cells which had been treated with

ac-tinomycin D (1 pg/ml) by a phenol-sodium dodecyl

sulfate method (8). The extracted RNA was analyzed

on a6 to30% sucrosedensity gradient,and the

acid-precipitable radioactivity in each fraction was

esti-mated bypreviously described methods (8).

Polyacrylamide gel electrophoresis. Gels, 7.5 cmin

length and containing 2.2% acrylamide and 0.5%

agarose, werepreparedbypreviously described meth-ods (10). Since the buffer system used contained

0.5%sodiumdodecylsulfate, alloperations were run

atroomtemperature.Electrophoresisat5 ma pergel

wascarriedoutfor 30 min before the addition of the

specimens. Thespecimens (in50,uliters orless) were

layeredonthegels and theelectrophoresis was carried

out at6mapergel for4 hr. Thegelswerethenfixed in 5% trichloroacetic acid and sliced into 1.3-mm

seg-ments. Theslices were prepared foranalysis in a liquid

scintillationcounter aspreviously described (10).

Reagents. Actinomycin D was agift from Merck, Sharp & Dohme. Guanidine was purchased from

Eastman Organic Chemicals. 3H-uridine (20

Ci/m-mole)waspurchasedfrom Schwarz BioResearch, Inc.,

and3H-leucine, (58.2Ci/mmole) from NewEngland

Nuclear Corp.

RESULTS

Inhibitionof SFVgrowth and RNAsynthesisby

guanidine. SFVreplicationwasinhibited by

treat-mentwith 30mmguanidine atthetime of

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VOL. 6, 1970 VARIABLE RESPONSE OF ARBOVIRUSES TO GUANIDINE

tion ofvirus infection (5). It was of interest to determine whether guanidine added during the

course of infection would also inhibit virus

growth. Therefore, CEF monolayers were

in-fected with SFV, and samples of infected cells

werefrozen at various time periods after infection.

At each period selected, guanidine (30 mM) was

added to a duplicate set of monolayers. In all

cases,the cells to whichguanidinehad been added

wereharvested with the last sample taken for the

growth curve, at 8 hr after infection. All

speci-menswere then titered for virus yield.

Theresults (Fig. 1)indicatedthat virusgrowth

wasinhibitedat any time afterinfectionby

guani-dine addition.Theseresults resemble those previ-ously reported in similarly conducted experiments

in which cycloheximideorpuromycin was added

during the growth of SFV (7, 12).

Since guanidine probably blocks picornavirus

replication by inhibiting viral RNA synthesis (1-3, 11) andguanidine has also been shown to

inhibit arbovirus RNA synthesis (5), the

dose-response curve ofthis inhibition wasinvestigated.

Variousconcentrations of guanidine were added

to actinomycin D-treated monolayers of

SFV-infected cells4hrafter infection, early in the log

phase of virus replication. After 10 min,

3H-uridine was added to aconcentration of 10

,uCi/

mlfor 5 min. The cells were washed, and

acid-precipitable radioactivity was determined. The

results (Fig. 2)showed that a 3 mmconcentration

ofguanidine effected only a 50% inhibition of

viral RNAsynthesis. Markedinhibitionwas seen

at 10and 30 mmconcentrations.

Similarexperimentswere

performed

atvarious times after virus infection to determine to what

extent viral RNA synthesis was inhibited. At

eachtime

period,

the

incorporation

of3H-uridine

(10 ,uCi/ml) into acid-precipitable counts was

determined aftera 5-min

pulse

in thepresenceor

absence of 30 mm

guanidine.

In the controls (Fig. 3), thepreviously

reported biphasic

pattern

of RNA

synthesis

inSVF-infected CEFwas

again

observed

(13).

The same pattern was seen in

guanidine-treated cells,

butthe

specific activity

of

the RNAwas

only

about 10%of the control in

allcases. Theseresults indicated that SFV RNA

synthesis,

like virus

replication (Fig. 1),

can be

inhibited

by guanidine

additionatany time

during

the

log

phaseof virus

growth.

The rateatwhich SFV RNA

synthesis

was

in-hibitedwasalsostudied.Guanidine

(30 mM)

was

added to SFV-infected cells. At various

periods

thereafter, monolayers

were

pulse-labeled

for 2

minwith 10,uCiof3H-uridine per

ml,

andtherate

of viral RNA

synthesis

wasestimated. The results

(Fig. 4) indicated that viral RNA

synthesis

was

rapidly inhibited after

guanidine

addition.

Curi-108

Z /7

I.,

U,

z z

0

0

6

105

2 4 6 8

HOURS AFTER INFECTION

FIG. 1. Effect ofguanidine onSemliki forest virus yields. Chick cellswereinfectedwith SFVatavirus-cell multiplicity of 20:1. After I hr ofinfection, the cells were washedfive times, andfresh medium was added.

Onepairofcultureswasfrozenat2, 4, 6, and8hr after infection. Also, at each of these times, guanidine

(30 mxf) was added to ani additional set of cultures

[image:2.489.245.439.60.339.2]

whichwasincubated until 8 hr after infection and then frozen.All cultures were later thawed and assayed for

virus titers.

ously, forup to 60min after

guanidine

addition,

no significant inhibition of virus-directed protein synthesis was observed in several experiments.

At that time after infection, more than 80%

oftheprotein

synthesis

is virus-directed (5).

Theseresultsindicated thatrapid inhibition of

viral RNA

synthesis,

but not of viral protein

synthesis, takes place after guanidine addition.

Inoneotherexperiment

performed

with a

differ-ent lot of

guanidine,

a 30 to 40% inhibition of

virus protein synthesis was observed under the

conditions

employed

in the

experiment

shown in

Fig. 4. The results shown are

probably

more

meaningful becausethey indicate that inhibition

of virus RNA

synthesis

isnot

necessarily

accom-panied by inhibitionof virus

protein synthesis.

In a previous

study,

guanidine

inhibition of

SFV

replication

was shown to be a reversible

phenomenon, if

guanidine

wasadded atthetime of initiation of virus infection

(5).

The reversi-629

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> 80

H

U)

0L v) 60

-j

0 z 0

L_

0 40

z

Li

cr

Li

20

O 3 10 30

[image:3.489.54.242.54.356.2]

GUANIDINE CONCENTRATION(mM)

FIG. 2. Effect ofguanidine conicenltration ont SFV-directed RNA synthesis. Click cells weretreated with

actinomycint D (1 jug/ml) for I hr anid were inzfected

with SFVat a virus-cell multiplicity of 20:1. After 4 hr, pairsof cultures were treated with various concen-trationsofguanidine for10miii;'H-uiridinte(10 j,Ci/ml)

wasthen added forani additio,ial5miii, and thespecific

activity of acid-precipitable radioactivity was deter-minedon each pair. Resultsarepresentedasthe per-centage ofacid-precipitable radioactivity of cultures

nottreatedwithguanidine.

bility of guanidine action at later times was

studied (Fig. 5). Guanidine (30

mM)

was added

tosixmonolayersof chick cellsat 3hrafter SFV

infection andtreatmentwith 1,ug ofactinomycin

Dper ml.Duringthenext 1hr,therateofRNA

synthesis increased in two control cultures not

treated with guanidine. In the guanidine-treated

cells,a marked drop inRNA synthesis was seen

between 3 and 3.5 hr after infection. At 3.5 hr

afterinfection, twoplateswere washed fivetimes

to remove guanidine,and the rate of viral RNA

synthesis in these was estimated 4 hr after

infec-tion. RNA synthesis rose in the washed cultures

but remained at low levels intwo cultures from

which guanidine had not been removed;

there-fore, the inhibitory effect of guanidine on viral RNAsynthesisseemed reversible.

10 P

8 e

C

0

a

O-)

ILI

CE

E

c

-t

ul

6

4

0

() GUANIDINE

I_

2 3 4 5 6 7

HOURS AFTER INFECTION

FIG. 3.Effectofguaiiidineadditio'i at various times afterinfectiononSFV-directed RNAsynthesis.

Actinio-mycinD-treatedchick cells were ilifectedwithl SFV as

previously described. At various times after infectioll,

onepairof cultureswastreatedwithguanidiiie(30 mm) for 10 min. To this and to an additioiialpair of uin-treatedcultures, 3H-uridine (10 iACi/ml) wasadded for

5minandthe specific activity oftheacid-precipitable radioactivity wasdeterminedoiieach pair.

It wasof interesttostudy what effect guanidine

addition had on the species of viral RNA

pro-duced in SFV-infected cells. Cultures were

treated with 30mmguanidine for 10 min, and

3H-uridine (10 ,Ci/ml) was added for 1 hr, a

suffi-cientperiod of time for all virus RNA species to

become tritium-labeled (8). RNA was then ex-tracted from the cells and analyzed on sucrose

density gradients. The results (Fig. 6) showed a

typical pattern of arbovirus RNA synthesis in

control cells (Fig. 6A). Several species of RNA

were seen-42S and 26S single-stranded RNA,

the 18Score of thereplicativeintermediate form,

and the replicative form (6). In the

guanidine-treated cells, however, a different pattern was

evident (Fig. 6B). In addition to a marked

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VOL. 6, 1970 VARIABLE RESPONSE OF ARBOVIRUSES TO GUANIDINE

100

H- 80

C-)

LU

ci 60

J 0

z

0

U-o 40

(9

z

LLI

Cr-0l n11

0

10

C-LLI

0s E)

0

20 40 60

TIME AFTER GUANIDINE ADDITION-MINUTES

FIG. 4. Rate of inhibition by guanidine of SFV-directed macromolecule synthesis. Actinomycin D-treated chick cellswereinfected withSFVaspreviously

described. After 4 hr, pairs of cultures were treated with guanidine (30 mm) for the indicated periods of time, and then 3H-uridine (0 ,iCi/ml) or 3H-leucine

(10 ,Ci/ml) was addedfor 2 min. Acid-precipitable

radioactivity wasdeterminedfor eachpairofsamples.

Resultsarepresentedasspecificactivityofeach sample as apercentageofcontrols which hadnotbeentreated

with guanidine.

tion of viral RNA synthesis (note the change in

scale), the major radioactive species of RNA

present was an 18S form which was quite

re-sistant to ribonuclease treatment. In addition, some42S RNAwas evident. Itwas not possible

todeterminebysucrose density analysis whether

tracesof 26SRNAwerepresent.

To analyze further the RNA forms of SFV

produced in guanidine-treated cells, samples of

the tritiated SFV-RNApreparation used in Fig.

6Bweresubjected toco-electrophoresis on 2.2%

acrylamide gelswith 32P-SFV RNA fromcontrol

cells. The result(Fig. 7)showedincontrolsa com-plexpatternwhich iscurrently being investigated (R. Friedman and J. Levin, unpublished data).

The four RNA formspreviously described were

0

3 4

HOURS AFTER INFECTION

FIG. 5. Reversibilityofguanidine inhibition of

SFV-directed RNA synthesis. Actinomycin D-treated chick cells were SFV-infectedaspreviouslydescribed. After 3or4 hrof inifection, 10 Ci/mlofuridine wasadded

toeachoftwosetsof monolayers for10 min(controls).

At 3 hrafter infection, threepairs ofcultureswerealso

treated with 30 m.i guanidine (guanidine added),and

after3.5 and 4 hrof infection onepaireachof guani-dine-treated cultureswasalsopulse-labeledwith 3H-uri-dine (guanidine remains). The additional set of guani-dine-treated cultureswaswashedfivetimesafter 3.5hr of infection andincubatedinfresh medium until 4 hr after infection, when it too was pulse-labeled with

3Huridine(guanidine removed). Acid-precipitable

radio-activitywastheni determinedonall cultures.

identified: the replicative intermediate, 42S and

265 single-stranded RNA, and the replicative

form. Inacrylamide gels,thereplicativeform has

consistently been shownto separate into atleast

two distinct species (replicative forms). In

addi-tion, two intermediate minor species of

single-stranded RNA (I, and 12) haveconsistently been identified. Finally, small molecular weight RNA

specieswhicharepresentin uninfected cellswere

alsoseen. As in thecaseof thepoliovirus

replica-631

20

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80 _ .

kV,NI

nUL 28S 18S

60

40

x2020

0 ,d b

1- 0

040.000.0

Z 28S 18S

10

B.GUANIDINE

z TREATED

D 0

7/o

2

('Y;>oo-°°°

!

0~

0~~~~~~~~~~ -0

10 20 30

[image:5.489.249.445.60.269.2]

FRACTION NUMBER

FIG. 6. ViralRNAsyiiithesisinthepreseiiceatid ab-seitce ofguanidine. Actintomyciii D-treated cells were

inifected withSFVaspreviously described. After4hr,

2cultures (-4 X 107cells)were treated with

guianidinie

(30mm!)Jor10 miti; twoadditiolialcultures served as

conitrols. 3H-uridine (10

uACilml)

wastheenadded to the

mediumoftheseJburcultures.After 1hr, thecellswere

washed and RNA was extracted from thtem by a

phentol-sodium dodecyl sulfatemethod. The RNA was

layeredovera 6to 30% sucrosedentsity gradient and

sedimented lbr 1 hr at 300,000 X g.

Fractionis

were

collected anidanalyzed for acid-precipitable

radioactiv-ity (-). Partofeachfractioniwas treated with 2

lig

of

ribonulcleaseper ml (37C, 30 min, 0.1 .m NaCl)

anid

also analyzedfor acid-precipitable radioactivity (0). Thetopofthegradientini thisantdothersucrosedentsity

gradient analysis patterns showii is to the right. The

designiations28Satnd18Sintdicatethepositions of

ribo-somal RNA ini the gradienitas determined by optical

denisityreadingsat260Jim.

tive intermediate (11), the replicative

interme-diate of SFV did not enter the gel under the

conditionsemployed.

In the guanidine-treated cells, the major SFV RNAformsappearedto be present (Fig. 7). The

striking change was in their distribution, since themajor speciesseenwerethe replicative forms.

Some replicative intermediate RNA was present. The single-stranded RNA forms (42S, 26S, L,

700

600

3PH -.o 32p

*-400

Er

a-z

D

1

0

o200

100-T t

t

RI RFIs 42S II 12 26S -SW.

10 20 30 40 50 60 70

SLICE NUMBER

FIG. 7. Polyacrylcamide gelelectrophoresis of

SFV-RNA forms. Chiick cells were inifrcted aspreviouisly

described. After4hr, the cells werei,icubatedJbr I hr ill the preseiice of otherwise phosphate-free medium

containiiig 2mCi/ml ofH3,2PO4 for 1 hr. RNA ivas

tlheii extrcicted as previously described. A mixture of

45ul,iters ofthev'iral3H-RNApreparatio'i from

gutaiii-dine-treated cells described ill Fig. 6Bwassubjectedto

co-electrophoresis oni a 2.2% acrylamide gel with 5

ul,iters oftheabove-described3"P-SFVRNA.After4hr

at37C, thegelswerefixed, sliced, aiid couiitedas

de-scribediltMaterials anzdMethiods. Th1especiesofRNA

presenit have been idejitifiedas replicativeinitermediate

form (RI), replicative forms (RF's), 42S RNA, two

silngle-strantded iuiternmediate Jbrms (11 antd 12), 26S

RNA, aiid small molectular weighlt RNA species

(SMW).

and

probably 12)

appeared

to be present, but in

relatively reducedamounts.The 42S RNAformed

in the presenceof

guanidine

maybe

abnormal,

as

its peak lacked the sharpness

consistently

noted

in that of controls

(Fig.

7).

Thefate of viral RNA, the

synthesis

of which

hadalreadybeeninitiatedatthe time of

guanidine

addition, was also studied. Cells were

pulse

labeled for 1 min with 100

,uCi

of 3H-uridine per

mnl.

At that time, as observed

previously

(8),

only the

replicative

intermediate form of the

virus was prominently labeled

(Fig.

8A).

The

cells were then washed five times with cold

medium, and medium

containing

uridine

(3

x

104 M), with or without guanidine (30

mM),

was added to the cultures. The cells were then

incubated for anadditional 1 hr.

In controls (Fig. 8C), additional

radioactivity

over that

incorporated

into the

replicative

inter-mediate of the 1-min pulse

(Fig. 8A)

was found

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VOL. 6, 1970 VARIABLE RESPONSE OF ARBOVIRUSES TO GUANIDINE

2

0

T

B.

GUANIDINE t

Z 28S l8S

O- C.C0NTR0L I

0 'O.10.20. 3

62.5-0

FRACTION NUMBER

FIG. 8. Fate of pulse-labeled .RNA in guan2idin2e-treated cells. Actino0mycinD-treated cells wereinfhected

WithlSFV as previously described. After 4 hr, six culi-tures were treated with 3H-uridine (100 uC&i/ml) for

mini. Th1ecells were quickly washed withl icedEagle's medium, anldRNA wasextracted from two ofthle cul-tures. Warm Eagle's medium con2tainting guanlidinle (30m.vI) anduridinle (3 X JQ-4 M) or only uridine was added to eacht of two other cultures. After htr, thze RNA was also extracted from these cultures. Allth7ree RNApreparationts were thenl antalyzedonsucrose denl-sity gradients as described iii the legenld to Fig. 6. Fractions were treated with ribonuclease as inl Fig. 6. Symbols: (a), acid-precipitable radioactivity; (0), acid-precipitable radioactivity after ribonuclease

treat-menlt.

associated with RNA forms similar to those

shown in Fig. 6A. In cells treated withguanidine

after the 1-mmn pulse (Fig. 8B), additional 3H-uridine incorporation (over that in Fig. 8A)

was also seen. Again, as in the case of Fig. 6B,

a change in the pattern of RNA synthesis was

noted. The 18S species was most prominent and the 42S single-stranded RNA was seen. In addi-tion, at variance with the findings in Fig. 6B, a

26S RNA peak was also consistently evident.

Although pulse-chase conditions could not be

established, this result suggested that RNA

synthesis initiated before guanidine additionmay

becarried out normally in its presence, since all

RNAspecies ofSFVwerenoted in cells treated in

thismanner (Fig. 8B). Incontrast, RNA

synthe-sis initiated after guanidine addition appears to

be distinctly abnormal in that the replicative

forms were the mostheavily labeled forms (Fig. 6B and 7).

Inhibition ofSindbis virus RNAsynthesis inthe

absence of growth inhibition. Since SFV and Sindbis virusareclosely related,itwasanticipated

that the effect ofguanidineonSindbisviruswould

be similartoits effectonSFV. This provedtobe

an incorrect assumption, for, although guanidine

treatmentdid inhibit Sindbis virus RNAsynthesis

(Table 1), and this inhibition was rapid and

reversible,asinthecasewithSFV,guanidine had

no effect on Sindbis virus growth (Table 2). In

the same experiment, SFV growth was inhibited

by40-fold. Thegrowth of SFV inthepresenceof

guanidine was not greatly enhanced in cells also

infected with Sindbis virus, since no significant

portion of the virus producedin co-infected cells

could be neutralized by SFV specific antibody

(Table 2). In fact, a significant inhibition of

Sindbis virus growth was seen in the co-infected

cells.

The inhibition of Sindbis virus growth in the

presence of SFV, the growth of which was

blocked by guanidine treatment, was repeatedly

observed(Table 3).The inhibitionwasnot

signifi-cantly enhanced by preinfecting the cells with

SFV. This wouldappear tobe anintrinsic

inter-ferencephenomenonnotdependentoninterferon

production, since it took place in thepresenceof

actinomycin D atconcentrationshigh enough to

inhibitcompletely interferon production induced

byvirus infection in chick cells(13).

Theability of Sindbisvirustogrowunder

con-TABLE 1. Inhibition ofSiutdhis virus RNA synithesis

byguaniidine

Sample perCounts perjg of protein'min

Control... 6-67

Guanidine-treated. 062

aFour monolayers of chick cells (-8 X 107

cells)weretreatedfor I hrwith1 ,ug of

actinomy-cin D permlandwereinfected with Sindbisvirus at amultiplicity of20 to 40plaque-formingunits/

cell. After 4 hr, two plates were treated with guanidine (30 mM) for 10 min, and then to all

plates 3H-uridine (10 ,uCi/ml) was added for 10

additionalmin.Theplateswerewashed,and

acid-precipitable radioactivity was determined.

633

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[image:7.489.55.247.70.203.2]

TABLE 2. Growth of Sindbis virus and SFV in the presence ofguanidine

Yield(plaque-formingunits/ml) afterinfection with

Sample-Sindbis SFV+

virus SFV ~Sindbi-s

Control... 19X 107 11 X 107 10 X 107

Guanidine-treated.. 15X 107 27 X 105 40 X 106

Guanidine treated +

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anti-SFV antibody

...13 X 107 24X 104 42 X106

aActinomycin D-treated chick cells were

in-fected with SFV, Sindbis virus, or a mixture of

equal titers of the two viruses in the absenceor

presenceofguanidine. Inall cases, infection was atthesamevirus to cellmultiplicity. After 8 hr,

the cultures were frozen, and they were later

assayed under standard conditions. Samples

from cultures of guanidine-treated cells were

treatedwith a1:10 dilution of rabbit anti-Semliki

Forestvirus (anti-SFV)

antibody

for 1 hrat25 C

beforeassay.

TABLE 3. Effectof SFVinfection onyieldofSindbis virusinguanidine-treatedcells

Guanidine TimeofadditionofSFV Sindbisb

concn" virusyieldb

mm

0 None added 40 X 107

30 Noneadded 28 X 107

30 2hr before Sindbis virus 27 X 106 30 At the timeofSindbis 43 X 106

virus

aChick cells were treated with actinomycin D

(1 ,ug/ml) for 1 hr, washed, and divided into four

groups.One setwasinfected with SFV in the

pres-enceofguanidine (30mM) for 2 hr. The cellswere

then infectedwith Sindbis virus. Anotherset was

infected with both SFV and Sindbis virus in the

presenceofguanidine; athird,withSindbis virus in the presence ofguanidine;and thefourth,with Sindbis virus in the absenceofguanidine.After 8 hr of Sindbis virus infection, the cultures were

frozen and thawed, and the culture fluids were assayedfor Sindbis virus yields.

bExpressed as plaque-forming units per

milli-liter.

ditions

inhibitory

to SFV replication was puz-zling. A clue to theexplanation for this

observa-tion wasfound in the effect of guanidine on the

pattern ofSindbisvirusRNAsynthesis.Normally

thispattern isidenticalto that shown in Fig.6A

and 8CforSFV.Inguanidine-treated

cells,

how-ever, under conditions which markedly distorted

theRNApatternof SFV

(Fig. 9A),

thepatternof RNAsynthesisinSindbis-infectedcellswasclose

tonormal

(Fig. 9B)

inthatapeakof26S RNA

was evident and the replicative-form peak was

notdominant.

SindbisvirusRNA

produced

in thepresenceof guanidine (30 mM) was also subjected to

co-electrophoresis

with 32P-SFV RNA (Fig.

10).

Again,

less ofa distortion of the normalpattern

ofviralRNA

synthesis

wasnoted thanwasseen

in the case of SFV-RNA produced under the

same conditions

(Fig. 7).

Although the replica-tiveformsweremuch moreinevidence in guani-dine-treated cells than in normal cells (Fig.

10),

10

5

N

I-z 0

0

z 0 u

O '

28S

A. SFV X

B.SINDBIS

28S 18S

I

I

10 20

FRACTION NUMBER

30

FIG.9.Sindbis virus andSFV-directed RNA synthesis

in the presence ofguanidine. Actinomycin D-treated cells wereinfected with SFV or Sindbis virus as pre-viouslydescribed. After 4 hr, four cultures (two infected with Sindbis virus, two with SFV) were treated with guanidine; four similar cultures served as controls. After 10min,8H-uridine wasadded toeach of the cul-tures,andRNAwasextracted and analyzed on sucrose density gradientsaspreviously described (Figs. 6 and 8). Resultsincultures notexposed to guanidine closely re-sembled those shown in Fig. 6A and 8C. Symbols are

asinFig. 6 and 8.

on November 11, 2019 by guest

http://jvi.asm.org/

(8)

VOL. 6, 1970 VARIABLE RESPONSE OF ARBOVIRUSES TO GUANIDINE

1200 1100

1000 3 0--a

32P

900

700

w

z600 C 500

en

z400

0

300

200 100

10 20 30 40

t t t t t t t

[image:8.489.50.243.66.338.2]

RI RF's 42S I, I2 26S SLICE NUMBER

FIG. 10.Polyacrylamide gel electroph

virusRNAforms. Sindbis virus3H-RN

as describedfor3H-SFVRNA in the1

Itwasco-electrophoresed with thesami

32P-SFVRNAaswasemployedinFig. analyzedaspreviously described(Fig.

tive intermediateform; RF's, replicati

I,, 12,anid 26S indicatesingle-stranded

a large amount of replicative inte

present, together with sharp peal

single-strandedRNA forms (42S,I,

The 26S RNA was the dominant1

activity.

DISCUSSION

The results indicated thatguanidi

reversibly, inhibited SFV andSindl

synthesis atany timeduring logari

of virus growth. Growth of SFV

pressed, but Sindbisvirusgrowth M

Co-infection ofguanidine-treated c and Sindbisvirus resulted insignifi(

of theyields of both viruses.

The mechanism of guanidine

arbovirusRNAsynthesiswasnotc

cidated by these studies. The sy

speciesofviral RNAwasinhibited,

tion of single-stranded RNA fori

significant than that if ribonuc

RNA. The results taken together indicated that,

in thepresenceofguanidine, viralRNA synthesis

is slowed, andnewly formed viralRNA leavesthe

template onwhich itwas synthesized more

slug-gishly than under normal conditions. Since the

number of templates is limited, this situation

would soon lead to an inhibition of viral RNA

synthesis, greater in the case of single-stranded

RNA, which appears to be formed on the

tem-plates, than oftheribonuclease-resistant

replica-tiveform.

The lack of effect of guanidineonSindbis virus

yields, despite its inhibition ofSindbis virusRNA

synthesis and of both RNA synthesis and virus

yields in SFV infection, was surprising. This

result suggests that, under normal conditions,

excess viral RNA is produced during Sindbis

virus infection, since marked inhibition of virus

RNAsynthesis resulted innosignificantdecrease

in virus yield. T. Sreevalsan (personal

communi-cation) has independently confirmed the herein

reported findings on the lack of sensitivity of

Sindbis virus yields todoses of guanidine which

_ t, markedly inhibit SFV replication. At 29 C,

how-50 60 70 ever, both viruses are equally sensitive to the

drug.

The difference noted betweenSFVandSindbis

virus with respect tosensitivity of virusyields to

Aeswas fSendbsd

guanidinewouldappeartobe duetothenatureof

'egendtoFig 7 the RNA produced rather than the actual

epreparationof amount. In thecaseof

SFV,

agrossdistortion of

7. The gelswere the normalpatternof RNAsynthesiswas seenin

7). RI, replica- guanidine-treated cells (Fig. 6 and 9A). Other

ive forms; 42S, forms of RNA were produced, but most of the I RNAforms. RNAwasin the ribonuclease-resistant replicative forms. The 26S RNAform,whichwasoneof the

rmeditate was dominant species in thecontrols, was greatly

de-ks

of all four creased in

guanidine-treated

cells. In cultures 1,

I2,

and26S). pulse-labeled with uridine before addition of

peak ofradio-

guanidine, however,

26S viral RNA was

con-sistently found,evenafter 1 hr ofguanidine

treat-ment(Fig. 8).This suggeststhat SFVRNA, the

synthesis of which was initiated beforeguanidine

nerapidly,but

addition,

may

eventually give

rise to26S

RNA;

bis

virusRNA

however,

during

SFV

infection,

the

synthesis

of

ithmetic

phase 26S RNAwas

greatly

inhibited in RNAinitiated

was also de- after

guanidine

addition. On the other

hand,

in

vas unaffected. Sindbis virus infection in the presence of

guani-oells

with SFV

dine,

the distortion of the normal

pattern

of RNA

cant

inhibition synthesis was not as great as in SFV infection.

Some 26S RNA was

apparent

along

with 42S

inhibition of and ribonuclease-resistant RNA

(Fig.

9B and

10).

ompletely elu- The ribonuclease-resistant

species

were not as

,nthesis of all dominant in Sindbis infection in the presence of

but the inhibi- guanidine as in SFV.

ms was more The results with both SFV and Sindbis virus

-lease-resistant indicate thatinhibition of 26S RNAsynthesis by

635

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http://jvi.asm.org/

(9)

guanidineisrelativelymoremarkedin the case of SFV-infectedcells. This suggests that thisspecies

of RNA is in some way associated with normal

virus maturation leading to production of

infec-tious virions. A similar conclusion was recently

reached as a result of studies on a

temperature-sensitiveSindbisvirus mutant, TS-24(12).

ACKNOWLEDGMENTS

Igratefullyacknowledge the technical assistance ofD.Hughes andM.Myers.

LITERATURE CITED

1. Baltimore, D. 1968. Inhibition ofpoliovirus replication by guanidine, p.340-347.In M. Sanders and E.H.Lennette (ed.), Medical and applied virology. Warren H. Green, St.Louis,Mo.

2. Caliguiri,L.A., and I. Tamm. 1968. Action ofguanidineon

thereplicationofpoliovirusRNA.Virology35:408-417. 3. Caliguiri,L.A., and I.Tamm. 1968.Distribution and

trans-lation ofpoliovirusRNA inguanidine-treatedcells. Virol-ogy36:223-231.

4. Cooper, P. D., B. B. Wentworth, and D. McCahon. 1970. Guanidine inhibition ofpoliovirus:a dependence of viral

RNA synthesisontheconfigurationofstructural protein. Virology 40:486-493.

5. Friedman, R. M. 1968. Structural andnonstructural proteins ofanarbovirus.J.Virol.2:1076-1080.

6. Friedman,R. M.1968.Repicativeintermediateofan arbo-virus. J.Virol. 2:547-552.

7.Friedman, R. M., and P. M.Grimnley. 1969. Inhibitionof arbovirusassemblybycycloheximide. J. Virol. 4:292-299. 8. Friedman, R. M., H. B. Levy, andW.B.Carter. 1966. Repli-cationofSemlikiForestvirus.Threeforms of viral RNA producedduring infection. Proc. Nat. Acad. Sci. U.S.A.

56:440-446.

9. Lowry,0.H., N. J. Rosebrough, A. L. Farr, and R. J. Ran-dall. 1951. Protein measurement with the Folin phenol reagent.J.Biol. Chem.193:265-275.

10. Moss, B., and R. Filler. 1970. Irreversibleeffects of cyclo-heximide during theearlyperiod of vacciniavirus replica-tion. J.Virol. 5:99-108.

11. Noble,J., and J. Levintow. 1970. Dynamics of poliovirus-specificRNAsynthesisand theeffectsof inhibitors of virus replication. Virology40:634-642.

12. Scheele, C. M., and E. R. Pfefferkorn. 1969. Inhibition of in-terjacent ribonucleic acid (26S) synthesis in cells infected bySindbis virus. J. Virol. 4:117-122.

13. Taylor, J. 1965. Studies on the mechanism of action of inter-feron. I. Interferon action and RNA synthesis in chick embryo fibroblasts infected with Semliki Forest virus. Virology25:340-349.

on November 11, 2019 by guest

http://jvi.asm.org/

Figure

FIG.1.frozen.yields.multiplicityinfection.wereOneviruswhich(30 Effect of guanidine on Semliki forest virus Chick cells were infected with SFV at a virus-cell of 20:1
FIG. 2.directedhr,actinomycintcentageactivityminedtrationswithwasnot pairs Effect of guanidine conicenltration ont SFV- RNA synthesis
FIG.4.described.directedasradioactivityResultstreatedtime,withwith(10 a Rate of inhibition by guanidine of SFV-macromoleculesynthesis.ActinomycinD- chick cells were infected with SFV as previously After 4 hr, pairs of cultures were treated guanidine (30 mm
FIG. 6.phentol-sodiumseitce2conitrols.mediumlayeredsedimentedinifectedgradientcollectedalsodesigniationssomaldenisityity(30ribonulcleasewashedThe cultures Viral RNA syiiithesis in the preseiice atid ab- of guanidine
+4

References

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