Deoxyribonucleic Acid Replication in Simian Virus 40-Infected Cells IV. Two Different Requirements for Protein Synthesis During Simian Virus 40 Deoxyribonucleic Acid Replication

Copyright © 1971 American Society for Microbiology Printed in U.S.A.

Deoxyribonucleic Acid Replication in Simian

Virus

40-Infected Cells

IV. Two Different Requirements for Protein Synthesis During Simian

Virus

40

Deoxyribonucleic Acid Replication

H. S. KANG, T. B. ESHBACH, D. A. WHITE, AND A. J. LEVINE Department of Biochemistry, PrincetonUniversity, Princeton, New Jersey 08540

Received forpublication 8 September 1970

The replication of simian virus 40 (SV40) deoxyribonucleic acid (DNA) was inhibited by 99% 2 hr after the addition of cycloheximide to SV40-infected primary

Africangreenmonkey kidney cells. The levels of 25S (replicating) and 21S (mature) SV40 DNAsynthesizedafter cycloheximide treatment were always lower than those

observedinaninfecteduntreated control culture. This is consistent with a require-mentfor aprotein(s) or for protein synthesis at the initiation step in SV40 DNA

replication. Therelativeproportion of 25S DNA as compared with215 viral DNA

increased with increasing time after cycloheximide treatment. Removal of cyclo-heximide from inhibited cultures allowed the recovery of viral DNA synthesis to

normallevelswithin 3 hr. During the recovery period, the ratio of 25S DNA to 21S DNA was 10 timeshigher than that observed after a 30-min pulse with3H-thymidine

with an infected untreated control culture. The accumulation of 25S replicating SV40 DNA during cycloheximide inhibition or shortly after its removal is

inter-pretedto meanthat aprotein(s) orprotein synthesis is required to convert the 25S

replicatingDNAto21Smatureviral DNA. Further evidence of a requirement for proteinsynthesisinthe 25Sto 21S conversion was obtained by comparing the rate of this conversion ingrowingandrestingcells. Theconversion of25S DNA to 21S DNAtookplaceat afaster rate in infected growingcells than in infected confluent

monolayer cultures. A temperature-sensitive SV40 coat protein mutation

(large-plaqueSV40) had no effect on the replicationof SV40 DNA at the nonpermissive temperature.

Infection of primary African green monkey kidney(AGMK) cellswith simian virus 40(SV40) results in the synthesis of viral deoxyribonucleic

acid

(DNA)

and infectious virus (10,

12).

SV40

DNAreplication begins at about 15 hr after in-fection and reachesa maximum rate atabout 30 hr. When infected cells were

pulse-labeled

with 3H-thymidine for shortperiodsof time (less than tO min), littleor no mature viral DNA was de-tected eitherby neutraloralkalinesucrose gradi-ents or by cesium chloride-ethidium bromide

equilibrium centrifugation. Instead,a viral DNA form that sedimented in neutralsucrose

gradients

at25S was observed. This DNAwas

completely

denaturable in alkali and banded at a lighter densitythanclosed circular matureviralDNA in ethidium bromide-cesium chloride equilibrium

gradients. The 3H-labeled 25S DNA could be chased into 21S mature viral DNA when

un-labeled thymidine was added to the culture medium (12).

Electron micrographs of 25S DNA showed

SV40replicating circles (two branchpoints, three

branches, and no visible ends) where90%O of the SV40 moleculehad completedits replication. At any one time, most of the replicating molecules

observed in the infected cell were of this 25S replicating type (75 % of thereplicating molecules were90% completed). This ledto thesuggestion

that therewas a sloworrate-limiting step late in the replication of an SV40 DNA molecule and thatthisresulted inanaccumulation of 255 DNA

(12).

In thepresentstudy,it is shownthat

cyclohexi-mide

(10

lg/ml) inhibits thereplicationof SV40 DNA. Thesedataarein agreementwith previous

studies with SV40 (11) and polyoma virus

(5).

The results indicate that protein synthesis is

re-quiredat twodifferent stages in the

replication

of SV40 DNA: (i) theinitiation ofDNA

synthesis,

and(ii)theconversion ofthe25Sreplicatingform to 215matureviralDNA

(finishing

step).

112

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MATERIALS AND METHODS

Virus. The SV40 large-plaque mutant (16, 17, 19, 20) and wild-type strain (13) wereemployed in thesestudies.

Tissue culture. AGMK cells (Flow Laboratories, Inc.) were cultured in either plastic petri dishes (Falcon, 100 by 20 mm) or plasticflasks (Falcon,75 ml) in Dulbecco's modified Eagle's medium (Grand Island Biological Co.) supplemented with 10% calf

serum.

Infectivity assay. Virus stocks were prepared as describedpreviously (12). Infectiousviruswastitrated

onmonolayer cultures of AGMKor BSC-1 cellsby

useofaplaqueassayprocedure (6).

Measurement of the rate of DNA synthesis. The

rate ofviral DNA synthesis was measured by the addition of3H-thymidine (14 to 18 Ci/mmole, New England Nuclear Corp.) to infected cell cultures. After an appropriate exposure to the isotope, the cells were washed twice with ice-cold phosphate-buffered saline(PBS,0.01 Msodiumphosphatebuffer

at pH 7.2 and 0.15 M NaCl) and lysed with 0.6% sodium dodecyl sulfate (SDS) in 0.01 M sodium phosphate buffer, pH 7.2, 0.15 M NaCl, and 0.01 M disodium ethylenediaminetetraacetic acid (EDTA). Viral DNA wasseparatedfromcellularDNAbythe Hirt procedure (9). A portion of the 1 M NaCl-SDS soluble fraction (small-molecular-weight DNA) was

sedimentedthrougha5 to20%linearsucrosegradient for 3 hrat40,000rev/mininanSW 50.1 rotor.The 3H-labeled component that sedimented at 21S was

used to quantitate the levels of mature viral DNA synthesis. The quantity of 3H-labeled DNA that sedimented at 25S was employed to determine the

amounts ofreplicating SV40 DNA madeduring the

labeling period (12). The quantity of3Hcounts per minute in the 1 M NaCl-SDS precipitable fraction (large-molecular-weight DNA) was employed to

determine the rate of cellular DNA synthesis. Only the fraction ofthe 3H-label that was trichloroacetic acid-precipitableandresistanttoalkalinedegradation (0.3 MKOH,37 Cfor 18hr)wasusedto measurethe

rate of cellular DNA synthesis.

After a 1-hr exposure ofSV40-infected cells with 3H-thymidine, over 90% of the trichloroacetic acid-precipitable radioactive label in the 1 M NaCl-SDS soluble fraction is virus-specific (12). Greater than 90% ofthe 3H-labeled DNA in the 1 M NaCl-SDS precipitable fractioniscellularDNA(12).

Benzoylated-naphoylated diethylaminoethyl cellu-lose(BNC)chromatography. BNCwassynthesizedby the procedure of Gillam et al. (7) as modified by Sedat, Lyon, and Sinsheimer (18). BNC columns were prepared and utilized as described previously (12).

Centrifugationtechniquesandisotopemeasurements.

Sucrose density centrifugation was performed as

previously described (12). The details of each sedi-mentation run aregivenin thefigurelegends. Radio-active samples were prepared and counted in a

Beckman liquid scintillation counter as described previously (12).

Cycloheximide treatment. Cycloheximide was

obtained fromSigmaChemical Co. At the

concentra-tion employed (10 ug/ml), cycloheximide inhibited the incorporation of 3H-leucine (30 to 50 Ci/mmole, NewEngland Nuclear Corp.) into hot trichloroacetic acid-precipitable (100 Cfor45min) materialby96% during the first hour and 99% by the second hour of treatment. Infected and uninfected AGMK cells behaved in an identical manner.

RESULTS

Effectof cycloheximide onSV40 DNA replica-tion. To examine the effects ofcycloheximide on thereplication of SV40 DNA, thefollowing experi-ment was performed. Six monolayer cultures of AGMK cells were infected with SV40 large-plaque mutant, and 30 hr later five of these cultures were treated with 10,ug of cycloheximide per ml. One cycloheximide-treated culture and one untreated control culture were then labeled with 3H-thymidine (1,uCi/ml) for 1 hr at 36 C. A second cycloheximide-treated infected culture was labeled from 1 to 2 hr after theaddition of theantibiotic (31 to 32 hr after infection). Each additional culture was labeled in an identical manner during the 3rd, 4th, or 5th hr after exposure tocycloheximide.At theend of the 1-hr

pulse-labeling period, each culture was washed two times with ice-cold PBS, and the cells were lysed with0.6% SDS as described in the preceding

section. A 0.2-ml sample ofthe 1 M NaCl-SDS

soluble fraction was sedimented through a 5 to 20%linearsucrose gradient for 3 hr at 40,000

rev/

min. Theresults of this experiment are presented inFig. 1.

Theadditionofcycloheximidetovirus-infected

AGMK cells results in the cessation of SV40

DNAreplication. The synthesis ofSV40-specific

DNAwas inhibited by 99 % after 2 hr of exposure to this antibiotic. Cycloheximide inhibited the

production of both 25S (replicating) DNA and 21S (mature) SV40 DNA. However, after an initial 1-hr delay, the ratio of 25S to 21S DNA was observed to increase from 0.3 to 1.0. This

can be seen in the sucrose gradientprofile pre-sented inFig. 1 (compare 1A with 1E or IF).

Levine etal. (12) demonstrated that 25S viral DNAis aprecursor of 21S mature SV40 DNA. Theincreased levels of 25S DNA relative to 21S DNAobservedaftercycloheximidetreatment

sug-gestedthepossibility thatcycloheximidemay act

byblocking the conversion of 25S to 21S DNA

(as wellasinhibiting the initiation ofSV40DNA

synthesis). Because ofdifficulties in quantitating

therelativeamounts of 25S and 21S DNA when sucrose gradient centrifugation was used, a secondprocedure was also employed to separate these two forms ofSV40DNA. BNC chromatog-raphyhas been shown to fractionate replicating

(25S) and mature SV40 DNA (12). The

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KANG ET AL.

X10

Hcpm

Hcpm

X10-2

3H

cpm D

3Hcpm2

X -2

xI

10 20 30 40

FRA1 N UMB R

FIG. 1. Sedimentation ofSV40 DNA synthesized after cycloheximide inhibitioii. Sixmontolayercultures

ofAGMK cells were iitected with SV40, and30 hr

laterfive ofthe cultures received10jig of

cyclohexi-mide per ml. One cycloheximide-treated culture (B)

andone untreatedculture (A) were labeled with

3H-thymidine (1 ,uCi/ml) for1hr.Theotherclulturzeswere

labeled (C) from I to 2hr, (D) from 2 to 3 hr, (E)

from3to4hr,anid(F) from4to5 h-after additionof

the drufg. A sample of the I M NaCI-SDS soluble fraction was sedimented for 3 hr at 40,000 rev/mim

in an SW50.1 rotor. (*) 3H collultspe- miniute; the

arrow represents thepeak of a 21S 92P-labeled SV40

DNA sedimentationi mnar-ker.

periment presentedin Fig. 1 wasrepeated except

that the 1 M NaCl-SDS soluble fraction was

phenol-extracted and chromatographed on BNC columns. Mature viral DNA (21S) eluted from the BNC resin with1 MNaCl. The 25SDNA

re-mained boundto the column under these condi-tionsandwaseluted ata later time with 2% caf-feine in 1 M NaCl. This procedure permit<> an

excellent separation of replicating and matui-e

[image:3.491.63.254.70.403.2]

viral DNA (12).

TABLE 1. Inhibition of S V40 DNA synthesis by cycloheximide

Sucrose gradient BNCb Cyclohex-_{iiodeX |Labeling}

iie timePecetPrcn

concn Per cent of Percent Per cent of Percent control DN2A control DNA2

,.g, nl hr

0 0-1 lOOc 24 lO0c 27

10 0-1 24 22 31 23

10 1-2 7.5 37 9.3 33

10 2-3 1.0 43 1.8 41

10 3-4 0.9 50 1.0 48

10 4-5 0.6 54 1.7 53

aPercentage of total DNA synthesized during

thetimeinterval indicated that sedimented at255

or was eluted with 26/a caffeine from a BNC

column.

bBenzoylated-naphoylated diethylaminoethyl cellulose.

,The100%value for the 3Hcountsperminuteas

measured by sucrose gradients is 29,000 counts/ minandby BNC is215,000counts/min.

Table 1 presents theresults of experiments de-signedto investigate theeffects of cycloheximide

onSV40DNA replication. Boththerateof viral DNA synthesis and the relative proportions of 25S and21S SV40 DNAweremeasured byuseof

BNC chromatography and sucrose gradient analysis. Within the first hour after the addition ofcycloheximide, viral DNAsynthesis decreased

to25to30%3oof thelevel observedin theuntreated control culture. During this time, the relative

amountsof25S and 21SDNAremainedthesame

in the antibiotic-treated and untreated cultures. After a 1-hr exposure to this drug, the rate of viralDNAsynthesis continuedto fall(from 10to

1 %), but an increasing proportion of the SV40

DNA remained in the replicating form (25S or

caffeine-eluted DNA). The percentage of 25S DNA synthesized in a 1-hr pulse-labeling period increased from 25cO in the absence of cyclohexi-mideto50c%Oby3to4 hr after the addition of this drug. The sameresults wereobtained with either sucrose gradient analysis or BNC chromatog-raphy.

Recovery ofSV40DNAsynthesis after the

re-movalof cycloheximide.Theexperimentsdescribed in the preceding section suggest that cyclohexi-mide might be inhibiting both the initiation of viral DNA synthesis and the conversion of 25S

DNA to mature viral 21S DNA. It is difficult

however, to make any conclusions about the

significance of the relative proportions of 255

and 21S DNA when the rate of viral DNA synthesis is only 1% of the normal levels. For

thisreason,itwasnecessarytoinvestigatewhether

114

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viral DNA replication could fully recover from

this cycloheximide treatment and to determine

whathappened during thisrecoveryperiodtothe conversion of 255 to 21S DNA.

Cycloheximide (10,g/ml) was added to five

AGMK monolayer culturesat 36 hrafter

infec-tion. After 3 hr,the cycloheximide was removed

fromfour of thesecultures by washing the cells four times with 10 ml ofwarm (37 C) medium.

Viral DNA synthesis was measured by

pulse-labeling these cultures with 3H-thymidine (1 ,uCi/ ml) for 30 min at different time intervals after the

removal of cycloheximide. The pulse-labeling

periods were stopped by washing the cultures

with ice-cold PBS and the cells were lysedwith

0.6%

SDS. A sample of the 1 M NaCl-SDS soluble fractionwasanalyzed bysucrosegradient centrifugationaspreviously described. The

radio-active sedimentation profiles from these sucrose gradients are presented in Fig. 2. Figure 2Awas

obtained from an infected culture where cyclo-heximidewas notremoved. Figures2B, C,D,and E were obtained by pulse-labeling with 3H-thymidine at 0 to 0.5hr, 0.5 to 1 hr, 1.5 to 2hr, and 3to3.5 hr after the removal of

cycloheximide.

Figure 2F was obtained from a control culture

that neverreceivedcycloheximide. Itcanbeseen thatremoval of thisdrug allowsafull recovery of

therate of viralDNA synthesis. The viral DNA

synthesized

shortly

after the removal of

cyclo-heximide (0to 0.5hrand0.5to1

hr)

was

mostly

25Sreplicating DNA. With

increasing

timeafter

reversal ofcycloheximide

inhibition,

the ratio of

25Sto21SDNA

changed

from 3.0to0.3.

Table2presents theresults ofexperimentsthat

demonstrate the reversal of

cycloheximide

in-hibition of viral DNA synthesis. Both BNC chromatography and sucrose gradient

analysis

were employed to investigate the rate of viral

DNAreplication andthe relativeproportions of

25S and21SviralDNA. After 3 hrofexposureof infected cells to

cycloheximide,

the rate of viral

DNA synthesis was 1%ofthatin anuninfected control culture. By 3.5 hr after the removal of this drug, the rate ofviral DNA

synthesis

was normal (87 to 100% ofthe control value), and the relativeamounts of25S and 21S DNA were

identicaltothoseintheuntreated infected control culture(30to35% of 25SDNA).Between0and 2 hrafter removal ofthisdrug, the rate of SV40 DNA synthesis recovered from 20% to 70% of the untreated control. At that time, the levels of 25S DNAdeclined from 75% to 39 % of the total viral DNA synthesized during the 30-min

pulse-labeling period.

Effects of a "shift-up in cell growth" on SV40 DNAreplication. The results presented in the two previous sections suggest that the conversion of

20 30

FRACTION NUMBER

40

FIG. 2. Sedimentation of SV40 DNA synthesized during recoveryfromcycloheximideinhibition. Cyclo-heximide(10

,ig/ml)

wasaddedtofive infectedAGMK monolayer cultures for 3 hr. At that time, the drug was removed from four of these cultures. Viral DNA synthesis was measured by pulse-labeling with 3H-thymidine (I,uCi/ml)for30-min intervals. (A) Cyclo-heximide not removed; (B-E) cycloheximide was removedand the cultures were labeledat 0 to0.5 hr,

0.5 to Ihr, 1.5 to 2hr, and 3 to3.5hr, respectively,

after reversalof the inhibition. (F) No cycloheximide added. Theprocedures forsucrose gradient

sedimenta-tion are presented in figure 1. (0) 3H counts per minute; thearrow

represenits

the peak ofa 21S 32p_ labeled SV40 DNA sedimentation marker.

25S to 21S DNA requires a protein(s) that is

reduced in amount by cycloheximide treatment. At2 to 3 hrafter removal of thisdrug, the

pro-tein(s)ispresent in sufficientquantitiesto permit

thenormalrateof conversion of25S DNAto21S mature DNA.Thesedata, however, restsolelyon the use of an inhibitor (cycloheximide) and thereforerequireconfirmationbydifferent

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TABLE 2. Recovery of SV40 DNA synthesis from cycloheximide inhibition

Sucrose gradient BNCb

Cycloheximideconcn Labeling time

Per cent Per cent Per cent Percent of control 25SDNAa ofcontrol 25SDNAa

,Ag/ml hr

10 (not removed) 1-1.5c 1.8 49 0.9 52

10 (removed) 0-0.5d 21 74 25 68

10 (removed) 0.5-1 54 60 50 58

10 (removed) 1.5-2 68 39 68 47

10 (removed) 3-3.5 87 35 100 39

0 (notadded) 3_3.5e lOOf 33 lOOf 35

aPercentageoftotalDNAsynthesizedduring the time intervalindicated that sedimented at

25S

or eluted with 2% caffeine from a BNC column.

IBenzoylated-naphoylated diethylaminoethyl cellulose.

cLabeling time 1 to 1.5 hr aftercycloheximidide was added.

dLabeling time 0 to 0.5 hr after removal of cycloheximide.

e Nocycloheximidewaseveradded; 3-to 3.5-hrlabeling time refers to the same time interval as for the sample labeled3 to 3.5 hr after removal ofcycloheximide.

fThe 100% valuefor the 3H counts per minute as measured by sucrose gradients is 16,500

counts/

min andbyBNC is 136,000counts/min.

To determine whether protein synthesis plays arole in the 25Sto 21S conversion, cultures of AGMK cells in two very different physiological

states

(growing

and resting cells) were infected with SV40.Growing AGMK cells(about 40% of

the petri dish surface contained cells) were

in-fected with large-plaque SV40 in 2 ml of

Dul-becco's medium containing 2%calf serum. After 3hrofadsorption, theexcess virus wasremoved,

and 15mlof Dulbecco's medium containing 10% calf serum wasaddedtotheculture. Atthesame

time, monolayer cultures of AGMK cells (100%

of the

petri

dish surface contained

cells)

were

infected with

large-plaque

SV40 in 2ml of Dulbecco's medium containing 2% depleted (21) calfserum. Afterthe 3-hr adsorption period, the

excessviruswasremoved,and 15ml ofDulbecco's mediumcontaining 2% depleted calfserum was

added to the cultures. At 30 hr after

infection,

each of these cultures was

pulse-labeled

with 3H-thymidine

(25,Ci/ml)

for a different

length

of time

(4,

5,

8, 12, 20,

or 25

min) depending

on the experiment. The

pulse-labeling

period was terminated

by washing

thecell cultures with

ice-cold PBS, and the cells were

lysed

with

0.6%

SDS. A 0.2-ml

sample

of the 1M NaCl-SDS

soluble fraction was

adjusted

to 0.1 M NaOH (final

concentration)

and incubated at 37 C for 10min. This

sample

wasthen

centrifuged

through analkaline sucrose

gradient,

and thepercentage

of the total 3H counts per minute that co-sedi-mentedwitha32P-labeledSV40 DNA sedimenta-tion marker was determined

(Fig.

3).

In the in-fected

monolayer

cultures with

depleted

serum

medium, therewas a 7- to8-min

delay

in the

pro-so80h~~~~~~~~~~~~ ~ ~

%ofcpm60+

MATURE

SV40 /

DNA

40L

20t / /

4 8 12 16 20 24

TIME AFTERISOTOPE ADDITION

FIG. 3. Kineticsof formation of3H-labeledmature

viral DNA as afunction ofthelength of exposure to

3H-thymidine. At 30 hr after SV40 infection of

sta-tionaryorgrowingcultures ofAGMKcells, each cul-ture waslabeled with 3H-thymidine for the length of

time (inminutes) indicatedon the abscissa. A sample of the I M NaCI-SDS solublefractionwasadjustedto

0.1 M NaOH and incubatedat37Cfor10min.Alkaline

sucrose gradients (5 to20% sucrose in 0.1 m NaOH

and0.9 M NaCI) were runfor2 hrat40,000rev/min

in an SW 50.1 rotor. The ordinate is plottedas the

percentage oftotal counts per minute that co-sedi-mented witha 32P-labeledSV40 closed circular DNA sedimentation marker.(-,*, *) Stationarycultures; (0, O, i) growing cultures.

duction of 3H-labeled mature viral DNA. This

delayis duetothe time

required

to

complete

one replication ofanSV40 genome

(one doubling

of

a DNA

molecule).

It is

clearly

not due to the

difficulty

of

getting

the

radioisotope

into the

cell,

because the

incorporation

of

3H-thymidine

into viral DNA

(25S

and

21S)

is

approximately

linear

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with timeovertheseshort timeperiods (12). The lag in the production of mature labeled viral DNA has been attributed, therefore,toaslow or ratelimitingstepin thereplicationofSV40DNA (12). This slowstephas beenshowntobe the

con-version of 25Sto21S DNA (12). In the infected growing cultures with 10% calfserumthelagin theproductionof labeledmatureviral DNAwas

4to 5 min (Fig. 3). It canalso beseen that the

rate ofproduction of mature viral DNA in the growing cultures (compare the slopes of thetwo curves) wasgreaterthan inthenongrowingcells. These results demonstrate that in infected growing

cells, where therateofprotein synthesisishigher than innongrowing infectedcells,theconversion of 25Sto215DNA takes less timethan in

mono-layer cultures. This is consistent with the idea that active protein synthesis is required for the 25S to 21S conversion and that this is a

rate-limitingstepin confluentmonolayer cultures.Itis important to point out that even in infected

growing cultures, where the rate ofprotein

syn-thesisisveryhigh,thereisstillalagordelayin the production of labeled mature viral DNA.

Effectsof SV40temperature-sensitive coat

pro-tein mutation on viral DNA replication. The pre-vious resultssuggestedthatprotein synthesiswas

required fortheproductionofmatureviralDNA (21S) from its255precursor.Ifthis is correct, the protein(s) required for this process could be coded forbythevirus,thecell,orboth. The fact thatafunctionalcoat protein is required for the synthesis of single-stranded 4X174 DNA (14) and the maturation of double-stranded linear lambda DNA (15) suggested the possibility that SV40coatproteins mightbeinvolved in the

con-version of 255to21S viral DNA.

TheSV40 large-plaquemutanthasbeenshown to possess a temperature-sensitive mutation in a

coatproteingene(16, 17, 19, 20). Todemonstrate the temperature-sensitive nature of the SV40 large-plaque mutant, the following experiment

was performed. Two cultures of AGMK cells wereinfected withlarge-plaque SV40andanother setoftwocultures with wild-type SV40.

Adsorp-tion wasallowedtoproceed for 3 hr at36C. At the end of that time, one culture infected with

large-plaque SV40 culture infected with wild-type SV40 wereshifted to 40 C. All four infected cell cultures were harvested at 72 hr after infection and the sonically treated celllysatesweretitrated

forinfectious virusat36 C (experiment A, Table 3). Wild-type SV40 replicated almost as wellat

40 C as at 36 C. On the other hand, the large-plaque SV40 was restricted in its replication

(greater than 99.9%) at 40C.

To determine the time course of the SV40

large-plaque temperature-sensitive restriction, the

same experiment was repeated but the infected

cultureswereshiftedupto40 Cat24hrafter in-fection. Infected cell lysates were prepared by

sonic treatment at 10, 24, and 72 hr after infec-tion, and infectious virus was titered at 36C (experiment B, Table 3). Wild-type SV40 was

made innormal quantities after the shiftto 40 C. Little or no increase in infectious virus was

ob-served when the SV40 large-plaque mutant was

shiftedto40 C at24hrafterinfection. This

indi-cates that the temperature-sensitive step occurs

afterviral DNAsynthesis normallybegins (16to

20 hr afterinfection at 36C). These data are in

excellentagreement with those of others (16, 17, 19, 20) and are consistent with the results that

indicate that thelarge-plaque SV40 isa

tempera-ture-sensitive mutant for a late function (coat

protein; 16, 17).

To observe whether the SV40 large-plaque mutation in the viral coatprotein had anyeffect onviral DNAreplication,twomonolayercultures of AGMK cells were infected with large-plaque

SV40 at 36C. After 24 hr ofincubation at the permissive temperature (36C), one of the

in-fectedcellcultureswas shiftedto40 Cfor 24 hr. 3H-thymidine (1,Ci/ml) was then added to a

cultureateachtemperature (36 and 40 C) for 45 min. Atthe end of this labeling period, the cells werewashedwithcold PBS andlysed with 0.6% SDS. Asample of the 1MNaCl-SDSsupernatant

fraction wassedimentedthrough asucrose

[image:6.491.46.440.555.625.2]

gradi-entfor3 hrat40,000rev/min. The results of this

TABLE 3. Temperature-sensitive restriction of large-plaque SV40

ExptA:Gtiter of virusreplicatedat ExptB:btiter at time after infection Virus

36C 40C 10hr 24hr 72 hr

Wild-type SV40... 4.2 X 108 1.2 X

108

2.5 X 104 8.8 X 104 1.8 X

108

Large-plaque

SV40... 1.0 X 109 5.0 X

106

6.0 X 104 1.4 X

106

3.0 X

l0r

a Viruswasadsorbed for 3 hrat36 C and thenshifted to 40 C. Virus production was titrated at 72 hr after infectionat36C.

bViruswasadsorbed andincubated for 24 hr at 36 C. Cultures were then shifted to 40 C, and virus production was titrated at 36 C. Samples were obtained at 10, 24, and 72 hr after infection.

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KANG ET AL.

Hcpm

20-3

H cpm B

X10-3 2 z

10 20 30 40

FRACTIONNUMBER

FIG. 4. Sedimentation of large-plaque SV40 DNA synthesized at 36 or 40 C. Twomoniolayercultures of AGMK cells were infected with large-plaque SV40 at 36 C; 24 hr later, one culture was shifted up to 40 C. At 48 hr afterinfection, each culture was labeled with3H-thymidine (1juCi/ml)for45min. Asample of the IMNaCI-SDS soluble fraction was sedimented in

aneutral sucrose gradient as described in Fig. 1. (A) 40C; (B) 36C. (0) 3H-counts per minute; the arrow represents the peak ofa 32P-labeled 215 SV40 DNA sedimentationmarker.

experiment are presented in Fig. 4. SV40 DNA

was synthesized atboth thepermissive and non-permissive temperatures. About 1.9 times more

viral DNA was made at 40C than at 36C. In

bothcases,however,22 to23 %of theSV40DNA made in a 45-min pulse-labeling period was 25S DNA. The

only

detectable difference between the

two cultures was thatabout twiceasmuch viral

DNA wasmadeat40Cas at36C.Twoadditional

experiments were

performed

todetermine whether this difference was duetothe absence ofa func-tionalcoatproteinat40Cor

just

dueto a

differ-ence in the incubation temperature of the two cultures. First, the

experiment

described above

was

repeated

with the

wild-type

SV40instead of

the large-plaque virus. In this

experiment,

the wild-typeviruswasfoundto

synthesize

1.7times

more viral DNA at 40C than at 36C. This is consistent with the

interpretation

that the

large

plaqueSV40synthesizesmoreviralDNAat40 C thanat36C because ofa temperature difference

and not becauseafunctional viralcoat

protein

is missing. Inasecondtype of

experiment,

six mono-layer cultures of AGMK cellswereinfected with large-plaque SV40 at 36C. At 48 hr after

infec-tion, half of these cultureswereshiftedto40 Cfor

1 hr.

3H-thymidine

was then added to each

in-fectedculture(at36and 40

C),

andthe amountof viral and cellular DNA

synthesized

during 4-,

7.5-, and 8-hr

labeling

periods

wasdeterminedas

described previously. In each case, the rate of viral DNA synthesis was 1.6 to 2.5 times greater at 40C than at 36 C. The rate of cellular DNA

synthesis inthese same cells was 1.5 to 2.3 times greater at 40 C than at 36 C. Since a functional

viral coatprotein is clearlynot required for

cel-lularDNAsynthesis,the higherlevels of host-cell DNA replication at 40 C must be due to the

difference in incubation temperatures. Thus, the rates of cellular and viral DNA synthesis were

increasedby aboutthe same amounts (1.5to2.5 times) at 40C. This is also consistent with the

interpretation that the absence of a functional

viral coatproteinat40 C is notresponsible for the observed increase in the rateof viral DNA syn-thesis.

DISCUSSION

Figure 5 presents amodel forSV40 DNA repli-cation based ontheresults of Levine etal. (12).

The mature SV40closed circular and suprcoiled

DNA is first nicked and replication begins. Be-cause16S, nickedcircularDNAisneverobserved witheither shortor long

pulses

of

3H-thymidine,

replicationmust startvery soon after theopening

ofthe SV40

polynucleotide

strand. At least one

strand ofthe SV40duplexmustbeopenatsome timeduringsynthesistopermitasemiconversative modeofreplicationto occur (8,22). Mostofthe

replicating molecules observed inaninfected cell have completed 90% of their

replication

(25S form; 12). This ledtothe suggestion that step I

ofthis

replication

modelwasthefaststep

(50 sec),

whereas step IIwas slower

(5

to8

min). Thus,

a rate-limitingorslowstep

(II)

occurs atthe endof

thereplication ofanSV40circle

(12).

Mutants of

polyoma

virus

(1-3)

have been isolatedthatdo not

synthesize

viralDNAatthe

nonpermissivetemperature. Ifthese mutantsare

shifted up to the

nonpermissive

temperature during polyoma DNA

synthesis,

then

polyoma

DNA replication stops (4). This shows that the

FAST STEP

(50SEC.)

Nickand replicate

STEPI

SLOWSTEP

(5-8MINUTES)><

-~~~ -~~~2

25S

(CAFFEINE DNA) 21S

STEP E

FIG. 5. Modelfor the replication ofSV40 DNA.

Each line representsadouble helix. The 21SDNA is

closed circular and supercoiled DNA; 25S DNA is

thepredominantformofreplicatingmolecules observed

in aninfectedcell.

118 _J. VIROL.

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[image:7.491.262.453.517.606.2]

presenceofavirus-specific protein is

required

con-currently with the synthesis of

polyoma

DNA. Although it has not been proven, this

protein

is

most likely required for the initiation step of polyomareplication(aviral DNAintermediate is

not accumulated; 4). The addition of

cyclohexi-mide to SV40-infected cells inhibits viral DNA replication (11) by 99% within2 hr. One

might

expect thatcycloheximide inhibitsthe

synthesis

of

a virus-specificprotein (SV40

protein

analogous

to that found with the polyoma

mutants)

that initiates the synthesis of SV40 DNA. As

cyclo-heximide inhibition of SV40 DNA

replication

proceeds (3 to 4hr after the additionofthe

drug),

thereis anincreasein theproportion of25SDNA synthesized (from one-fourth to one-half 25S

DNA) in a 1-hrpulse. Thiswould be expected if

theinitiation step wasinhibited at a slowerrate than thefinishing step (step

II).

This is not ob-served during the first hour of cycloheximide

treatment,indicatingthatforthis timeintervalthe

protein(s) needed for the finishing step is in-hibited or inactivated at the same rate as the

initiator

protein(s).

It is

important

to

point

out that throughout thecycloheximide treatment (or recovery) lower than normal levels of 21S and 25S viralDNA wereobserved. Thesedata indicate that both initiation and finishing steps are

in-hibitedbycycloheximide.

Removal of cycloheximide from infected cells allows the recovery of SV40 DNA synthesis to normal levels. The first viral DNA accumulated (labeled with 3H-thymidine) after removal ofthe

drug

(0

to 0.5

hr)

ismainly 25S

(75%).

This is consistentwith theexpectationthat initiator

pro-tein(s) recover from cycloheximide inhibition at a faster rate than the finishing

protein(s).

The presence of normal levels of finishing protein(s)

canbeobserved

by

3hr after reversal of thisdrug. These data show that finishing

protein(s)

is in-activated or inhibited at a faster rate and re-coversfrom cycloheximide inhibitionat aslower

rate than theinitiator protein(s). By 0.5 to 1 hr after removal of cycloheximide from infected cultures, the rateofviralDNAsynthesisis 50 to

55% ofnormal, and thelevels of25S DNA are 40 to 45 %higherthannormal.

Theuseofaninhibitor likecycloheximide

suf-fersfromtheproblemthattheinfected

cell

ismost

likely in an abnormal state during the inhibition orrecovery stagesofthetreatment. It was

there-foredesirable toobtainanindependent confirma-tionofarequirement forproteinsynthesis for the 25S to 21S conversion step. To do this, stage II of SV40 DNA

replication

was measured in in-fectedgrowing and resting cells. The rate of pro-teinsynthesis wasfoundto bethree to five times

greater in infected growing cells than in the

in-fected confluentmonolayer cells. The conversion of 25S to 21S DNA was also faster in growing

cells. There was both a decrease in the delay of the production of 3H-labeled 21S DNA and an

increase in the rate ofproduction of 21S DNA.

These data demonstratethat atleastaportion of

the rate-limiting step (step II) in SV40 DNA replication can be eliminated by using infected growing cells. It is important to note, however,

that alagordelayin theappearanceof3H-labeled 21SDNAisstillobserved withthese cells (4- to

5-min delay), and therefore some of the factors needed for step II arestillrate-limiting for SV40

DNAreplication.

There are several difficulties in comparing

events in growing and stationary cells. Virus

production and the rate ofincorporation of

3H-thymidineintoviralDNAarefasterin the growing

cell system. It is clear that 30 hr after infection

with growing cells may be analogous to a later

time period in infected monolayer cultures. To

eliminate theseobjections, therateofconversion

of 25Sto21SDNAhas been measuredatdifferent times after lytic infection of stationary cultures. At all timestested (24, 36,48, and60hr),therate

of conversion of 25S DNA to 21S DNA was

about the same (6to 8 min). Since the ratio of

replicating DNA to coat protein is probably differentat24and 48 hrafterinfection,thisresult

is consistent with thesuggestion that SV40 coat

protein has no effect on this step II conversion. Presumably, if one could look

early

enough in infection (when the replicating pool ofDNA is

very

small),

the

delay

in the 25Sto 21S

conver-sion would be eliminated.In

addition,

thesedata eliminate thepossibility that the time after infec-tion thatone measuresthe 25Sto21S conversion

may not be comparable in resting and growing cells.

The experiments presented in this paper sug-gest thata protein(s), orproteinsynthesis, is

re-quired at two stages in the replication ofSV40 DNAmolecules.Avirus-specificinitiator protein

is

probably

requiredto start around of

replica-tion (step I). A second protein, or proteins, ap-pears to beneededeithertofinishthe last10%of

thereplicationor toclose the SV40 polynucleotide

strands,

or to do both. At

least

one SV40 coat

protein, prescribed by the SV40 large-plaque mutation, is not involvedin DNA replication.

ACKNOWLEDGMENTS

Theexcellenttechnical assistance of Angelika K.Tereskyis gratefullyacknowledged.

Thisresearch wassupported byPublicHealth Service grant CA 11 049-02fromtheNational Cancer Instituteandby grant E-591 fromtheAmerican CancerSociety. We thank theWhitehall

VOL. 7, 1971

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Foundation in the Biology DepartmentatPrinceton University for thegenerous useof their equipment.

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