Simian Virus 40 Deoxyribonucleic Acid Replication

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Copyright @ 1969 AmericanSocietyforMicrobiology Printed in U.S.A.

Simian Virus 40

Deoxyribonucleic

Acid

Replication

I.

Effect

of

Cycloheximide

on

the

Replication

of

SV40 Deoxyribonucleic

Acid

in

Monkey

Kidney Cells and in Heterokaryons of

SV40-transformed

and

Susceptible

Cells

SAULKIT, TAKASHI KURIMURA, RAMON A. DE TORRES, AND DEL R. DUBBS

Department of Virology andEpidemiology, Division of Biochemical Virology, Baylor University College of

Medicine, Houston,

Texas77025

Received for publication 23 September 1968

Infectious deoxyribonucleic acid (DNA) wasextracted from greenmonkey kid-ney (CV-1) cultures atvarious times after the cultures wereinfected with simian

virus 40(SV40)atinputmultiplicities of 0.01 and 0.1plaque-forming unit (PFU)per

cell. Apronounced decrease in infectious DNAwasobserved from 3 to16hrafter virus infection, suggesting that structurally altered intracellular forms may have

beengenerated early in infection. Evidence is also presented thatSV40DNA

syn-thesis requires concurrent protein synthesis. DNA replication was studied in the presenceand absence ofcycloheximidein: (i)SV40-infected and uninfected cultures ofCV-1cells;(ii) culturessynchronized with1-3-D-arabinofuranosylcytosine(ara-C) for 24to30hrpriortotheaddition ofcycloheximide; and (iii) in heterokaryons of SV40-transformed hamster andsusceptible monkey kidney cells. DNA synthesiswas

determined bypulse-labeling the cultures with 3H-thymidineatvarious times from 24to46hr after infection. In addition, thetotal infectious SV40 DNAwasmeasured.

Addition of cycloheximide, even after early proteins had been induced, grossly

inhibited both SV40 and cellular DNA syntheses. The activities of thymidine kinase, DNApolymerase, deoxycytidylate deaminase, and thymidylate kinasewere

measured; theseenzymeactivitiesremained high foratleast9hrinthepresenceof

cycloheximide. SV40 DNA prelabeled with 3H-thymidine before the addition of cycloheximidewasalso relativelystable during the time required for cycloheximide

to inhibit further DNAreplication.

Thekinetics of simian virus 40 (SV40)

deoxy-ribonucleic acid

(DNA) replication

have been studied

by

anumberof methods:

plaque

assayof

infectious

DNA,

radioautography,

and band

centrifugation

or nitrocellulose chromatography of

pulse-labeled

DNA

(16, 17, 19).

Thesestudies haveshown that SV40 DNA

replication begins

at

about 12 to 16hrand continues at a

rapid

rate

until about44 hrafter virus infection.Incontrast, infectious virus

production

commences after a

20-to24-hreclipseperiod and virus titers increase untilabout60 to 72 hrafter infection.Also, infec-tious SV40 DNA was first detected in hetero-karyons of transformed andsusceptible cells at19

hraftercellfusion,butwasnotdetected in trans-formedcells beforefusionorduringthe first 11 hr after fusion (19). Although the timing of SV40 DNAsynthesishasbeenelucidated,themolecular events whichoccur duringreplicationarepoorly understood. Presumably, these eventsinclude: (i) conversion of closed circular, double-stranded SV40 DNA (form I) to a "nicked" circle (form

II);

(ii) formation of complementary copies of

each of the parental DNA strands; and (iii)

joining

ofends ofpolynucleotide chainsto form superhelical (form I) daughter molecules. It is possible thatsome ofthese processes take place atspecific cellular sites, perhapsthenuclear mem-brane (4, 22, 27).

Two facets of SV40 DNA relication are de-scribedin thepresentstudy. First,it is shown that infectious SV40 DNA undergoes an

"eclipse"

soonaftervirusinfection ofgreenmonkeykidney (CV-1) cells. Secondly, we have asked whether synthesisofessentialproteins earlyininfection is a sufficientcondition for the

replication

of SV40 DNA late in infection, or whether there is an additional requirement for the

synthesis

of pro-teins concurrentwith

replication

ofSV40 DNA. Ourdata show that theadditionofcycloheximide eitherto cultures pretreated with

1-f3-D-arabino-furanosylcytosine (ara-C) or to culturesnot pre-treated with ara-C, at timesafter early enzymes have been induced

by

virus

infection,

strongly inhibitsSV40 DNA

replication.

25

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MATERIALS AND METHODS

Cellcultures and SV40 stocks. The cultivation of CV-1 and SV40-transformed hamster (TSV-5) cells hasbeendescribed (6, 17). SV40 virus was propagated and assayed in monolayer cultures of CV-1 cells.

Heterokaryonsof transformed TSV-5 andsusceptible

(CV-1) cells were produced by treating mixtures of cellswithultraviolet light-inactivatedSendai virus, as

described previously (6).

Extractionand assay of infectious SV40 DNA.

In-fectious SV40 DNA was extracted from cultures of infected cells andfromheterokaryonsoftransformed andsusceptible cellswithp-aminosalicylateandphenol (16). The infectivityof theDNA extracts was

deter-mined by plaque titration onmonolayer cultures of

CV-1cells (19).

Incorporation of 3H-thymidine (3H-dT) into DNA and separation of labeled SV40 DNA from cellular DNA.At various times after infection, cultures were

inoculated with0.1or0.2mlof3H-dT(NewEngland Nuclear Corp., Boston, Mass.). The cultures were

incubatedat37Cfor the times indicated in thelegends

to the tables and figures. Noninfected cultures were

treatedsimilarly,except that eithergrowthmediumor

spent medium (medium removed from 4- to 7-day-old cultures) was substituted for the virus prepara-tions. After the 3H-dT labeling period, the cultures wereharvestedas previously described (16), and the total DNAcontentandspecific activity oftheDNA

were determined on portions of the cellsuspensions

(18). Theremainder ofthe cellsweresedimented by centrifugation, washed with saline-glucose solution,

andusedforextraction ofcellular and viral DNA. To determine therelativeamountsof 3H-dTincorporated

intocellularand viralDNA, the nitrocellulose

chro-matography procedure of Kit et al. (16) and band

centrifugation in CsCl gradients (16, 31) were

em-ployed.

Enzyme assays. Assaysfor thymidine kinase, thy-midylatekinase,deoxycytidylate deaminase,andDNA polymerase were performed by standard methods employedinthislaboratory(15, 17, 20, 21).

RESULTS

Decrease in DNA

infectivity

early after SV40 infectionof CV-1 cells.When CV-1 cellswere

pro-ductively

infectedwithSV40

virus,

anincreasein infectiousSV40 DNAwas

observed,

beginning

at

about 12 to 16 hr after

infection,

and maximal titers ofDNA

infectivity

were attained at about 44 hr after infection.Theinitial increase in

infec-tious SV40 DNA took

place

at 12 to 16

hr,

whether cells were infected at

high input

multi-plicities

[10

to50

plaque-forming

units

(PFU)

per cell] orat low

input

multiplicities (0.01

to 0.1 PFU/cell). It

apeared

from

input

multiplicities,

but not at

input

multiplicities

of 1

PFU/cell

or

greater, theinfectious DNAcontentof cells was

less at 7.5 hr thanat2.5 hrorat 20 hr after infec-tion (19).

Theobserved decrease ininfectiousDNA early after SV40 infection suggested that structurally altered intracellular forms might begenerated in virus-infectedcells. As a first step in testing this idea, theearlyinfectionperiod was studied more closely. At low inputmultiplicities, a decreasein SV40 DNAinfectivity was observed by 6 hr after virus infection (Fig. 1). When the input multi-plicity was 0.1 PFU per cell, DNA infectivity diminishedby15.5 hr toalmost one-tenththat at 3 hr after infection. At the input multiplicity of 0.01 PFU/cell, the low levels of DNA infectivity from 6 to 15.5 hr after infection represented the limits of detectableSV40DNA. The data in Fig. 1 demonstrate that, early after virus infection, most of the parental SV40 DNA either is

con-105

2104

>\

D 102

-_j

Z)

UL_ 102

0L

10~ ~ ~ M-0

lo

0 5 10 15 20 25 30 35

HOURS AFTER SV40 INFECTION

FIG. 1. Assay ofinfectious SV40 DNA at various times after infection ofCV-1 cells with intact virus.

Confluent monolayer cultures (107 cells per culture)

wereinfectedwith Imlof SV40atinputmultiplicities

of0.1and 0.01 PFU/cell. Afteran adsorptionperiod of2hr at 37 C, unadsorbedvirus wasremoved,20ml ofnew growth medium was added, and the cultures werefurtherincubatedat37C.Atthetimesindicated,

the cultures were trypsinizedand DNA extracts were preparedfrom the resulting cell suspensions (16, 19).

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vertedto anoninfectious form or to aform not

extractable

by

the

p-aminosalicylate

procedure.

[We

also considered the hypothesis that the loss of SV40DNA

infectivity

may haveresulted from the progressive loss of virus adsorbed at the cell surface which never penetrated andwas pro-gressively detached from the cells. To test this hypothesis, CV-1 cultures were infected with SV40 atinput

multiplicities

of about 20 and 0.2 PFU/cell, respectively. After a 2-hr adsorption period at 37C, the unadsorbed virus was re-moved, the cultures were rinsed four times, and 20mlof growth mediumwasadded. At2, 6, 10, and 16 hr. samples were removed from the me-dium and assayed for SV40. Contrary to the foregoing

hypothesis,

the virus concentration in the supernatant media decreased progressively from 2 to 16 hr after infection. Thetotal PFU/ culture in thesupernatantmediawasineach case less than

1O-3

that extracted from the cells in the culture at 16 hr after infection.]

Effect of cycloheximide on the incorporation of 3H-dTinto DNA of noninfected andSV40-infected CV-1 cells. Tolearn whether concurrentprotein synthesis was required for

incorporation

of 3H-dT into DNA, the effect of cycloheximide was studied in nonsynchronized cultures and in cul-turesin which DNA synthesis was synchronized by pretreatment of cells

with

ara-C. A pulse-labeling experiment involvingara-C pretreatment isshown inFig. 2.SV40-infectedCV-1cells were incubated;with15,ugofara-Cper mlfrom2to24

H3-dT INCORPORATION FOR 2 HRS.

'7r% r% INTO DNA n

z

a

c@

=L

hr after

infection.

"Mock-infected" control cul-tures were

similarly

treated. It has been shown previously (14, 17,

21)

that ara-C inhibits DNA synthesis and SV40

capsid

protein formation,

but not theinduction ofearlyenzymes or SV40-tumorantigen.

Indeed,

the activities of

thymidine

kinase and DNA polymerase were greater in

either uninfected or

SV40-infected

monkey

kidney cultures treated with ara-C than in

un-treated cultures.

Theara-Cinhibition ofDNA

synthesis

canbe reversed by

removing

ara-C and

adding

deoxycy-tidine. After the addition of

deoxycytidine,

a rapid

synthesis

of DNA was initiated

(Fig. 2).

The

synthesis

of

DNA,

asmeasured either

by

the

incorporation

of tritium label for2hr intoDNA or by colorimetric measurement of the total DNA content per culture, was greater in SV40-infected cultures than in noninfected cultures. When

cycloheximide

was added to the cultures atthe time of the

deoxycytidine

reversal of the ara-C

block,

3H-dTwas

incorporated

into

DNA,

butat a

grossly

inhibitedrate.Similarresultswere obtained after

cycloheximide

additiontocultures not treated with ara-C.

ThedatainFig.2suggestedthat

cycloheximide,

an inhibitor of

protein synthesis

(17), severely

curtails DNA

synthesis

within 2 to 4 hr after addition ofthe

drug.

Tolearnmore

precisely

the time required to produce the cycloheximide in-hibition,theexperimentshownin

Fig.

3 was car-ried out. SV40-infected cultures of CV-1 cells

TOTAL ,ug DNA SV40- INFECTED

CV-I CELLS

200_

UNINFECTED _

CV-I CELLS t6_

-J

U120_

\ o _

<

80-TED z

L__---v D40

TED+CH

w

I

+1

u

SV40 -INFECTED CV- CELLS

UN NFECTED+CH INFECTED+CH

Ara-C

28 32 36 40 0 2 24 28 32 36

HOURS AFTER SV40 INFECTION

40

FIG. 2. Effectof cycloheximide (3,g/ml) ontheincorporation,for2hr,of3H-dTintoDNA ofSV40-infected

anduninfectedgreen monkeykidneycultures.Seven-day-old CV-J cells (9.3 X 10 cells/culture) were infected

with SV40at an inputmultiplicity of90PFU/cell. At 2 hrafterinjection, the unadsorbed virus wasremoved, andara-C(15

,g/mI)

wasadded. The cultureswereincubatedat37Cfor24 hrandwashed:50 ,ug of deoxycytidine perml, withorwithout3 ,ugof cycloheximideperml, wasadded. Cultureswereharvestedat the times indicated

in thefigure. At22, 24,29,and 35 hr after infection,0.1 mlof3H-dT(0.5,uc and2,ug/ml) was added to each

bottle.

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z

0

z 1,000

w

a-m

SV40-NFECTED

500

NONINFECTED

NOINEC-T

PLUSCYCLOHEXIMIDE

15 45 75 105 135 165

MINUTES AFTER ADDITION OFH3-dT AND CYCLOHEXIMIDE

FIG. 3. Effect of'cycloheximide (3 jig/ml) on the kinetics of 3H-dT incorporation i)ito DNA of SV40-infected and uninfected CV-1 cell cultutres. Replicate CV-1 cell cultures (11.4 X JQ6 cells/culture) were

infected with SV40 at an inpuit multiplicity of' 50

PFU/cell. At 36 hrafter intkctio,i, 0.2 ml of3H-dT

(2,uc and2 jg/ml) wasadded. Some of the cultures were also treated with cycloheximide. Cultures were

harvestedatthetimesshownonthefigure.

wereincubated for 36 hrto

permit

theinitiationof viralDNA synthesis. 3H-dT wasadded to

repli-cate cultures, either with or without cyclohexi-mide. Cultureswereharvestedatthetimesshown in Fig. 3.

Incorporation

of 3H-dT was severely inhibited within 1 hr of

cycloheximide

treatment

and only limited

incorporation

of 3H-dT into DNA occurred thereafter. The results suggested thataprotein

synthesized

denovoisrate

limiting

for DNA

synthesis

in both uninfected and SV-40-infectedcultures.

Separation oflabeled cellular and SV40 DNA. The

experiments

in

Fig.

3 show thatSV40 stimu-lated DNA synthesis in CV-1 cell cultures and that the synthesis of DNA in either infected or

uninfected cultures was inhibited

by

cyclohexi-mide.Todeterminewhether bothSV40 and cellu-lar DNA syntheses were inhibited

by

cyclohexi-mide treatment, nitrocellulose

chromatography

and bandcentrifugation

experiments

werecarried out(Fig. 4,

5).

Inthenitrocellulose

chromatogra-phy experiment

(Fig. 4),

double-stranded DNA

representsSV40DNAand

single-stranded

DNA

is cellular DNA. As shown

previously

(16),

SV 40infection stimulatescellularDNA

synthesis

in CV-1 cells 36 to 44 hr after infection.

Cyclo-heximide treatment prevents this stimulation of

cellular DNA synthesis and also markedly re-duces SV40 DNA synthesis. The band centrifuga-tion experiment(Fig. 5) further demonstrated the inhibitoryeffect of cycloheximide on both cellular DNA synthesis (fractions 1 to 22) and on SV40 DNA synthesis (fractions 23 to 30). Similar results were obtained when the 3H-dT labeling periods were34 to 38 hr, 40 to 44 hr, or 42 to 46 hr afterinfection.

Stability of "pulse-labeled" SV40 DNA during

[image:4.487.67.256.64.287.2]

a "chase" period with or without cycloheximide. We considered the possibility that SV40 DNA synthesized during cycloheximide inhibition might beunstable. This instability could, perhaps, result from afailure to synthesize essential "maturation" proteins or capsid proteins in the presence of the drug. The "pulse-chase" experiment depicted in Table 1 contradicts this hypothesis. Infected and uninfected cultures of CV-1 cells were labeled with :H-dTfrom 36 to 40 hr after infection. The cultures were then washed, and excess "cold" thymidinewasaddedeither with or without cyclo-heximide. Cultures were further incubated at 37 C and harvested at 44 and 48 hr. The per cent tritium counts in cellular and viral DNA were

800r 700

O 600

w 500

z, 400

Ir-I X. 300

a. z

o o 200 Z Z)

-J 40

H

H3dT CH H3dT+CH

FIG.4. Estimationby nitrocellulose column

chroma-tography ofthe relative amounts ofSV40 DNA and cellular DNA synthesized in uninfected and

SV40-in,fected CV-I cellcultures.DNA samples wereheated

at100Cfor10mm in1 X SSCbeforechromatography. Single-stranded (SS) DNA is the denatured DNA

which requires 0.1 X SSC and 0.01 N NaOH,for

elution. Double-stranded (DS) DNA is that DNA which is not denatured by the heating and is eluted from the columns with 2 X SSC (circular SV40 DNA). The intervalsof3H-dTincorporationintoDNA

are shown below each column. Replicate cultures of

7-day-old CV-1 cells (107 cells/culture) were infected

with SV40 at an input multiplicity of100PFU/cell.

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EFFECT OF CYCLOHEXIMIDE ON SV40 DNA

0

P

400-tz. NONINFECTED, NONINFECTED, SV40-INFECTED, 300 WITHCH NOCH WITHCH

200 36-40hrs.PI 6-40hrs. PI

100

5 9 13 17 21 25 29 33 37 41 43

FRACTION NUMBER

FIG. 5. Sedimentation-velocity analysis (band centrifugation with CsCJ, density 1.503 gcm-3,

dis-solved in 0.01 M tris(hydroxymethyl)aminomethane

buffer, pH 8) ofDNA from uninfected or

SV40-in-fectedCV-1 cellslabeledwith3HdTfrom36to40 hr after infection. DNA samples were from the same

experimentshown inFig. 4. Alamellaof 0.1 ml,

con-taining 3 gg of DNA, was added to each tube. Replicate cultures were treated with 3 jg of cyclo-heximideper mlat the time of 3H-dT addition. The centrifugation was for 150 min at 20Cand100,000 X g in the SW39 rotor of the Spinco L2

cen-trifuge.

determined by nitrocellulose column chroma-tographyand bandcentrifugationinCsCl density gradients. Thetotal 3H-dTin theDNA of both the uninfected and the infected cultures was

about the sameafter the 8-hr chaseperiodasat

40hr.Cycloheximidetreatmentdidnotaccelerate breakdown of either cellularorSV40 DNA. Thus,

the inhibition of 3H-dTincorporation into DNA causedbycycloheximidecannotbe ascribedtoan

enhancement of DNA turnover.

Effect of cycloheximide on the replication of

infectiousSV40 DNA. The experimentsin Fig. 2

through 5 depict the effect of cycloheximide on

the "pulse-labeling" of SV40 DNA. The total

SV40 DNA accumulated can be determined by

measuring the infectious SV40 DNA at various times after infection. Figure 6 shows the effect of cycloheximide treatment on this process. Cultureswerefirst incubated for 25 hrormoreto

permitthe formationof"earlyproteins"andthe initiation of DNA replication. Cycloheximide

was added toreplicate cultures 25, 28, or 34 hr after infection. Cycloheximide treatment in-hibited the formation of infectious SV40 DNA, but did not induce loss of DNA infectivity. Ex-perimentswerealsocarriedoutinwhichCV-1 cell cultures were first treated with ara-C to permit formationof "earlyproteins" andtosynchronize

w

tL3

0L.

z

0

c)

Uf)

0

w

z

io7I

6

In5

CH ADDED NO CH

ADDEDDE

CH ADDED WITH OH

WITH OH

:~~~~~ 0

0 10 20 30 40 50 60

[image:5.487.34.227.66.230.2]

HOURS AFTERSV40 INFECTIONOFCV-I CELLS

FIG. 6.Effectofcycloheximide (CH)on the

forma-tionof infectious SV40 DNAin CV-1cultures. Seven-day-old CV-1 cell cultures (11 X 106 cells/culture)

were infected with SV40 at an input multiplicity of 20 PFU/cell. Cycloheximide (3 ,ug/ml) was added

at 25, 28, and 34 hr, and cultures were harvestedat

thetimesindicatedinthe figure.

TABLE1.Stability ofSV40 DNA labeledfrom36 to 40 hrpostinfection (PI) duringsubsequentincubation

of infectedcellsfrom40 to48 hr PI with and withoutcycloheximide (CH)a

Totalcountsper min perculture

Total counts/min for SV40 DNA Time of cell) Noninfected SV40-infected

NoCH With CH NoCH WithCH NoCH WithCH

40 5 X 105 1.59 X 106 8.6 X 105

44 5.7 X 105 5 X 105 1.66 X 106 1.48 X 106 8.6 X 105 8.6 X 105

48 5.3 X 105 4.8 X 105' 1.27 X 106 1.15 X 106 7.6 X 105 6 X

10'

aSeven-day-oldCV-1 cells (9.4 X 106cells/culture) were infected with SV40 at an input multiplicity of100PFU/cell. Replicatecultures wereincubated with 3H-dT (2 ,uc and 0.25,g/ml) from 36 to 40 hr PI. At 40 hrPI, cultureswerewashed,20,ugof nonradioactive thymidine per ml was added, and cultures

werefurtherincubated at 37C.Cycloheximide (3,ug/ml) was added at 40 hr PI to the infected and un-infected cultures.

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In the preceding experiments, cycloheximidewas added at a time when enzymes in the DNA bio-synthetic pathway had already attained high levels. We considered the hypothesis that one of

NO CH the enzymesmightundergo rapidturnoverduring

the arrest of

protein

synthesis

and that this

en-- CH ADDED WITH CH zyme deficiency might restrict DNA synthesis.

+C______- - ( - Four ofthe enzymes catalyzing nucleotide inter-conversions and DNA replication were therefore studied, namely, thymidine kinase, thymidylate WITH CH> - kinase, deoxycytidylate deaminase, and DNA

/ _-~ polymerase. In some instances, cultures were

pre-treatedfor 30 to 32 hr with ara-C before the addi-tion of cycloheximide. The results of these

experi--

--

ments failed to substantiate the hypothesis that

- l

cycloheximide

initiated a

rapid

loss of enzyme

CH ADDED activities. Thus, in

SV40-infected

cultures treated

-3,ug/ml) with cycloheximide at 36 hr, the activities of

(3,ug/ml)Ithymidine kinase, deoxycytidylate deaminase, and

thymidylate kinase were as high at 42 hr as in culturesnottreatedwith the

drug. Similarly,

when

!O 30 40 50 60 70 80 cycloheximide was added at 38 hr, the activities of

IOURS AFTER FUSION OF TSV-5 a CV-I CELLS thymidine kinase and DNA polymerase were

* 7. Effectofcyclohexinmicle (CH) on thetorma- about as high with cycloheximide as without drug )J infectious SV40 DNA in heterokaryons of treatment at 45 hr after infection. An experiment

9rmed TSV-5 and susceptible CV-1 cells. Mix- in which uninfected and SV40-infected cultures

)f CV-1 (107 cells) antd TSV-5 (5.8 X 106 cells) werepretreated with ara-C for 30 hr before the

treated

wit/i

14,000

hemagglutinlatinig untits

of addition of cycloheximide is shown in Table 2.

olet-inactivated Sendai virus to promote fusion The cultures exhibited high levels of thymidine 4mixture

equivalenit

to2X 101 CV-1 and 1.2 X kinase activity for at least 9 hr after

cyclohexi-V-5 cells was theni seeded inprescriptioni bottles mide was added. anid inzcubatedat 37 Cfbrthe times inidicated.

Cyclo-heximide (3 ,ug/ml) wasadded at 29 or at48hrafter

fusioni.

subsequent SV40 DNA

replication.

Ara-C was

removed and

deoxycytidine

wasadded24 hrafter infection.

Cycloheximide

was added to

replicate

cultures at 28 and 38 hr after infection. In this case, also,

cycloheximide

treatment inhibited infectious SV40 DNA

synthesis.

However, for 8 hr after

cycloheximide addition,

there was no

appreciable

decrease of infectious SV40 DNA. These experiments further demonstrated that SV40 DNA is

relatively

stable for several hours after protein synthesis is inhibited.

Experimentswerealsoperformedonthe forma-tion ofinfectious SV40DNAinheterokaryonsof susceptible CV-1 and transformed TSV-5 cells. Addition ofcycloheximide to the cultures at 29 or 48 hr aftercell fusion also retarded the

rapid

formation of infectious SV40 DNA in these cul-tures (Fig. 7).

Effect of cycloheximide treatment on enzyme activities. The induction of enzymes by SV40 is

first detected at about 12 to 16 hr after virus infection, and maximal enzyme activities are

[image:6.487.61.250.54.310.2]

usuallyobserved

by

about30 hr

(14,

17,

19,

21).

TABLE 2. Thymidinie kiniase activities ofuninlfected

anidSV40-inifectedcultures ofCV-1cells treatedwith ara-Cfrom2 to30 hr PI

anidsubsequenitly treated with

cycloheximide (CH)a

Time Uninfected SV40-infected enzyme

assayed

(hrPI) NoCH withCII No CII W\ithCH

30 11.0 14.6

32 10.2 9.6 13.5 16.1

35 12.2 11.5 18.0 14.3

39 13.2 12.7 19.0 16.0

42 12.3 9.1 19.2 12.6

aEnzyme activity is expressed as picomoles of

3H-deoxyuridinemonophosphate formed from 3H-deoxyuridine per microgram ofprotein in 10 min

at 38 C. Seven-day-old cultures of CV-1 cells (11 X 106 cells/culture) were infected with SV40

at an input multiplicity of83 PFU/cell. Cultures

wereincubated with20,ugofara-C perml from2to

30 hrafterinfection,themediawerechanged,and

50 ug ofdeoxycytidine per ml, and in some cases

3,ug ofcycloheximide per ml,wasadded. Cultures

wereharvestedatthetimes indicated.

104

7

5

3 2

Q 103

J 5

cr

H

a-- 2

z

02

0 T 7

n 5 3 2

lo'l

2 H

FIG tion o

tranisfc

turesc

were I

ultravi( (19).A

106TS

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DISCUSSION

The present experimentshave shown thatSV40 DNA infectivity undergoesaneclipse early after

virus infection of CV-1 cells. This finding is

reminiscent of the observation that phage X

DNA extracted from cells soon after infection

retainsverylittleinfectivityintheKaiser-Hogness transformation assay due to the formation of

structurallyalteredintracellularforms(3, 5, 32). It ispossiblethat the loss ofSV40DNAinfectivity also results from the formation of structurally altered intracellular forms. The DNA of

(pX174

RF, which resemblesSV40DNA in conformation

and molecularweight,is known toundergo

struc-tural changes during replication (22). Parental

spX174-RF molecules are attached to a

fast-sedimenting cell component; the "attached"

RF is foundinpartasRFIIand inpartasRFI.

Only attached RF molecules in the RFIH form

(nicked-circle) replicate, anddaughterRF

mole-culesarelater found free in thecytoplasmofthe

bacterialcell.

We recognize, however, that the loss ofSV40 DNA infectivity may havea less interesting

ex-planation. Perhaps this loss results from

intra-cellular nuclease action which occurs after virus

particles are "uncoated" and while the SV40 DNA is "in transit" to replication sites.

Addi-tional studies, including experiments utilizing labeled virus, arerequired to ascertain the

mol-ecular conformation of the noninfectious SV40 DNA and its intracellular localization, and to

learn whether it is, indeed, an obligatory

inter-mediate inSV40DNAreplication.

Thepresentexperimentshave also shownthat

protein synthesis must occur concurrently with

SV40 DNA replication in productively infected

CV-1 cellsand in heterokaryonsof transformed andsusceptiblecells. It isnot knownwhetherthe

requisite proteins are viral-specific or cellular

proteins. This studyand otherinvestigations (25, 28, 30) have shown that drugs which prevent

protein synthesisinhibitthe acceleration of

cellu-larDNAsynthesis insynchronized cellcultures;

it is conceivablethata specificclass ofhost pro-teins isrequiredfor bothcellular andviral DNA

replication.

From thetemporalpoint ofview, the proteins requiredduring SV40DNAreplicationare"late"

proteins. Thatis, ininfected culturesnot treated withara-C, they arerequired severalhours after

progenySV40DNAandcapsid protein syntheses

have beeninitiated.Also,inculturessynchronized with ara-C, the new class ofproteins is needed after the "early" class of proteins has

accumu-lated. Data which suggest a stoichiometric

rela-tionshipbetween DNA andprotein synthesis have

been obtained in several microbial and mam-maliansystems(23-25, 28). Invirus-infected cells, it has been shown that protein synthesis late in phage development is necessary

for

the produc-tion ofinfectiousX DNA (5). Thesynthesis of a histidine-containing protein is necessary for the replication of the parental replicative form of

spX

DNA

(9).

Evidence

hasbeen

presented

that poxvirus DNA synthesis requires concurrent protein synthesis (1, 13). Moreover, although the enzymes

catalyzing

the

incorporation

of thy-midine intoDNA in cellularextractsfrom pseu-dorabies-infected cellsarestable after addition of puromycin, DNA synthesis in vivo is rapidly inhibited

by

addition of the drug (2).

The nature and functions of the proteins re-quired for SV40DNAreplication havenot as yet been elucidated. Some possibilities that may be considered arethattheproteins are: (i) enzymes

exhibiting

marked

instability

during inhibition of protein synthesis; (ii) proteins which stabilize progenySV40

DNA;

or

(iii)

proteins correspond-ing to the structural and initiator proteins (24, 26) postulated by Jacob et al. (11). Studies on the activities of the thymidine kinase, DNA polymerase,

deoxycytidylate

deaminase, and

thy-midylate

kinase of infected

cells

indicate that these enzyme

activities

are relatively stable for several hours after cycloheximide treatment. It is,therefore, unlikely thatone of these enzymes is the essential protein. However, these results do not

necessarily

excludearoleforanother enzyme

functioning

in DNA biosynthesis. The data in Table 1 and those in Fig. 6 and 7 also suggest that the required protein is not one needed to stabilize SV40 DNA,

since

progeny DNA is relatively stable for several hours after

cyclo-heximide

addition. The third possibility, that protein synthesis is required eitheras astructural componentofanSV40 DNAreplicationmachine oras aninitiator protein, merits furtherstudy (11, 22, 24, 26). Data supportingthe hypothesisthat DNA

replication

mightoccur in direct association withmembranestructureshavebeen obtainedfor the (pX174RF (22) and for microbial(8, 12) and mammaliancell systems (4,27).Therateof

cellu-lar DNA

replication

is about2

pm/min

at 37 C for mammalian cells

(10,

29) and variesfrom 6 to40

,tm/min

for microbial

cells,

dependingupon the growth media (7). Since SV40 DNA is less than 2,umlong,it isprobablethatonce,initiated, around ofreplication requiresonly afew minutes. The time intervals studied in the experiments shown in

Fig.

2and 3aremuchlongerthan this. Thus, the observed inhibitions may reflect the failure to fabricate new viral DNA

replication

complexes, while existing replication complexes

on November 11, 2019 by guest

http://jvi.asm.org/

(8)

may continuetofunction. This may permit con-tinued synthesis of SV40 DNA atreduced rates.

ACKNOWLEDGMENTS

Thisinvestigationwassupportedby National Science

Founda-tion grant GB5917, American Cancer Society grant E-291E,

Robert A. Welch Foundation grant Q-163, and Public Health ServicegrantsCA-06656-06,1-K6-AI-2352,and5-K3-CA-25,797. WethankMarjorie Johnson, CarolynSmith,JudithRotbein,

and Sun Ock Yim for able technical assistance. LITERATURE CITED

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