Cessation of reentry of simian virus 40 DNA into replication and its simultaneous appearance in nucleoprotein complexes of the maturation pathway.

0022-538X/81/070255-08$02.00/0

Cessation

of

Reentry of Simian Virus

40

DNA into Replication

and Its

Simultaneous

Appearance in

Nucleoprotein

Complexes

of the

Maturation Pathway

HWA-TANG WANG AND ANN ROMAN*

Departmentof Microbiology and Immunology, Indiana University School of Medicine, Indianapolis,

Indiana46223

Received 11 February1981/Accepted 15 April 1981

Newly synthesized SV40DNA isused as a template for further DNA synthesis

(reenters replication) or as a substrate in the assembly of virions (maturation

pathway). The timecoursesofreentry intoreplicationandprogressionalongthe

maturationpathwaywereboth determined onidentical samples. DNA,

synthe-sizedduringa20-minpulse, reentered replicationover aperiod of several hours

and thenwasremovedfromthepoolofmolecules availableforreplication. The

cessation ofreentrycoincided with the maturation of this DNA from the

chro-matinformtoprevirion and virion forms. Morereentryand less maturation was

observedat 24hpostinfection thanat42hpostinfection.Thedataareconsistent

withthe hypothesis that the factor(s) responsibleforcessation of reentry is also

responsible for initiation ofthematuration pathway.

The stepsin the maturation (encapsidation)

pathway of simian virus40

(SV40)

arecurrently

underinvestigation in several laboratories (1,3,

4, 6-8, 11). New extraction procedures have

re-vealed that (i) the majority of newly synthesized

SV40

DNA isencapsidated and that (ii) several

species of nucleoprotein complexes (NPCs)can

be isolated (1,3,6-8). Pulse-chase experiments

suggest that labeled SV40 DNA is converted

from chromatin

(NPC-I)

toprevirions(NPC-II)

tomaturevirions(1, 3, 6-8). NPC-I iscomposed ofDNA andcellular histones

Hi,

H2a, H2b, H3,

and H4 (3, 7). It is used for biosynthetic

pro-cessesand has associated with it SV40Tantigen

(13, 19, 23), the DNA-binding

protein

involved

in initiation of viralDNAsynthesis (14, 24, 25,

27) and

regulation

ofviral

transcription

(18, 20,

26), and cellular

proteins

involved in these

pro-cesses

(for

examples,

seereferences 5,

15).

NPC-IIis aheterogeneousset of

complexes

grouped

together because they sediment more

rapidly

than NPC-I under neutral conditions and yet

are salt labile. NPC-II is

composed

of

DNA,

histones withahigher

degree

of

acetylation

than

NPC-I, and viral structuralproteins (3, 4, 11).

Maturevirionsaresaltstable and containDNA, allhistones except

Hi,

and viralstructural

pro-teins (3, 12, 16). The order of addition ofviral

proteinstoanNPChasnotbeendetermined.

Althoughthese studies shed

light

onthe steps

involvedin virus

assembly,

littleis known of the

factor(s)

determining

the fate of

newly

synthe-sized DNA. Romanand Dulbecco

(22)

showed

thatnewly synthesizedpolyomaDNA can serve

as atemplate forfurtherDNAsynthesis(reenter

replication)overaperiodof2to 3 h and then is

removed from the pool of molecules available

for replication. Green and Brooks (9) reported

similarkinetics ofreentryofnewly synthesized

SV40 DNA into

replication.

Recently, Roman

reported that polyoma DNA labeled at 20 h

postinfection (p.i.) reentered replication at a

greater ratethanmolecules labeledat 28hp.i.

and that agreater proportion of molecules

la-beled at 20h

p.i.

reentered

replication

than at

28hp.i. (21). These datasupportthehypothesis

of Roman and Dulbecco that therateofreentry

is a reflection of the availability of

proteins

required for initiation of DNA synthesis (22).

Regardless

of thetime

p.i.

examined,

however,

progenymolecules

appeared

tobe removed from

the

pool

available for

replication

about3hafter

their initial

synthesis.

A role for maturation

proteins was

postulated

by Roman and

Dul-becco(22) and Roman(21).

Similarly,

Garberet

al.(8) and

Baumgartner

etal.(1)

compared

their

determinations on the rate of

encapsidation

of

DNAwith the

published

dataonthe kinetics of

reentryof DNA into

replication

(9, 21, 22),and

postulatedanassociationbetween thetwo

path-ways.

Theresearchreportedherewasconductedto

determine, onidentical

samples,

thekinetics of

reentryofnewly

synthesized

DNAinto

replica-tionand the kinetics ofmaturationof thissame

DNAto establish the

temporal

relationship

of

255

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256 WANG AND ROMAN

the twopathways toeach other. Since Roman had previously reported that the kinetics of

reen-try were dependent upon the time in the

infec-tiouscycle examined(21), the kinetics of thetwo

pathways were also determined at24 and 42h p.i. Cessation ofreentryintoreplication of DNA

labeledduringa 20-minpulsewasfound to

co-incide with the maturation of this DNA from

chromatin to previrions and virions. The rate

and extent ofreentry decreased with time p.i.

whereas the rate ofencapsidation increased.

MATERIALS AND METHODS

Cellsand virus. BSC-1cells,agiftfrom M.Singer,

weregrowninDulbecco modifiedEaglemedium

con-taining 10%calfserum and50,ugofgentamycinper

ml. SV40,agift from W.Eckhart,wasplaque purified

three times andpropagatedat alow(0.05)multiplicity

of infection. Virus stockswerepreparedfrom infected

cells by threecyclesoffreezingandthawingfollowed

by low speedcentrifugation andtheywere storedat

-200C.

Labeling and extraction of viral DNA and

NPCs.ConfluentBSC-1 cellswereinfected withSV40

at multiplicity of infection of 10 PFU per cell. At

different times after infection, the cells wererinsed

twotimes with TDbuffer(150mMNaCl,5mMKCI,

0.7 mM Na3HPO4, and 20 mM Tris, pH 7.4), and

labeled for20minwith75or150,uCiof[3H]thymidine

(specificactivity, 40 to 60Ci/mmol) per ml in

Dul-beccomodified Eagle medium containing 2% serum

and25mMHEPES

(N-2-hydroxyethylpiperazine-N'-2-ethanesulfonicacid) (pH7.0). Cellswerethenrinsed

twicewith TDbuffer,covered with Dulbeccomodified

Eaglemediumcontaining106 Mthymidine,2%serum

and25mMHEPES for30min,and then chasedwith

bromodeoxyuridine(BUdR) chasemedium[5x 10-'

M5-bromodeoxyuridine, 2 x 10-5 M

5-fluorodeoxyu-ridine(28),9x10-6Mdeoxycytidine (2, 17),2% serum,

and25mMHEPES in Dulbecco modified Eagle

me-dium].

After variouslengthsofchase,DNAwasextracted

from half of theplatesby the method of Hirt (10). The

Hirt supernatantwasusedto determine thekinetics

of reentry.

NPCs were extracted from infected cells by the

method of Fernandez-Munoz et al. (7). Cells were

rinsed twice with coldTBS buffer (TD buffer

contain-ing 0.9 mMCaCl and 1mMMgCl2)and scraped into

cold hypotonic buffer (25 mM Tris, pH 7.9, 1 mM

MgCl2,and 0.4 mMCaCl2).Thecellswere broken by

homogenization (30 strokes) inatightly fitting glass

Douncehomogenizer. The lysates were centrifuged at

2,000 rpmfor5min.The NPC-containing supernatant

was used to determine the kinetics of maturation.

Similar results were obtainedifthecellswererinsed

with TD buffer or extracted with hypotonic buffer

containing0.5 Mdithiothreitol and

phenylmethylsul-fonylfluoride.

Theefficiency of extraction of3H-labeledDNA in

the form of NPCswasdetermined by comparing the

total 3H countsper minute (cpm) in unfractionated

NPCstothe total3Hcpm inDNAextracted by the

J. VIROL.

method of Hirt. Theefficiency of extraction of NPCs

wasapproximately 70% that of DNA. During the 6-h

chase,nodecrease in the 3H cpmwasobserved,

sug-gesting that essentially all of the prelabeled DNA

remained cell associated.

Analysis of viral DNA. Form I DNA was

sepa-rated fromform II andreplicative-intermediate DNAs

bylayering Hirtsupernatantsonto5to20%alkaline

sucrose gradients containing 0.75 M NaCl, 1 mM

EDTA, and 0.3 M NaOH(pH12.6). Thesampleswere

centrifuged inanSW41 Tirotor at39,000 rpm for 2.6

h. Fractions were dripped from the bottom of the

centrifuge tubes, and theradioactivityinasamplewas

determined afterprecipitationwith 5% trichloroacetic

acid. Fractions containingform I were pooled,

neu-tralized, and mixed with CsCl in TE buffer (10 mM

Tris,pH 7.4,1 mMEDTA)togiveafinaldensityof

1.72g/ml. Thesampleswerecentrifugedinatype40

rotor at33,000 rpmfor48htoseparateform I DNA

with BUdR substitution inonestrand(HL DNA)from

unsubstituted form I DNA(LL DNA).The percentage

of 3H-labeled HLformIDNA[(HLDNA cpm+ HL

DNAcpm+LLDNAcpm)x 100]wasdetermined.

In someexperiments, thesameprotocolwasused

to determine the percentageof labeled HL DNA in

theslowlysedimenting DNA (formII, RI,cellular)of

theinitial alkalinesucrosegradient. Whenmentioned,

the percentage of labeled HL DNA in the Hirt

super-natantwasalso determined inCsCl.

Analysis ofSV40 NPCs. The NPC supernatants

werelayeredonto 10 to40%sucrosegradients

contain-ing 2 mM Tris(pH7.4) and spun inanSW41 Tirotor

at37,000 rpm for90min. Fractionsweredrippedfrom

the bottom and processed as described above. The

percentagesofNPC-I and of NPC-II+Vwere

deter-minedby the formulas %(NPC-I) = (NPC-I cpm +

total NPCscpm) x 100and%(NPC-II+V) =

(NPC-II+V cpm + total NPCs cpm) x 100, respectively.

Fractions from theNPC-II+V regionofthegradient

were pooled, and salt-labile NPC-II was separated

from salt-stable virions by density gradient

equilib-riumcentrifugation inCsCl (p =1.34g/ml in TBS).

Thesampleswere spun in atype40rotor at 33,000

rpmfor40h. Fractions were dripped from the bottom

andprocessedasdescribed above. The percentage of

virionswascalculatedby theformula(V cpm+

NPC-II+Vcpm)x%(NPC-II+V).Similarly,thepercentage

ofNPC-II was equal to (NPC-II cpm + NPC-II+V

cpm) x %(NPC-II+V). Where noted, the different

NPCs werepooled, and the DNA was extracted by the

method of Hirt (10) andsubjected to density gradient

equilibrium centrifugation as described above to de-termine the percentage of HL DNA in each NPC.

RESULTS

Location of NPCs.Several laboratories have

reported the

sedimentation

and buoyant density

characteristicsof chromatin (NPC-I), previrions

(NPC-II), andvirions (1, 3, 6-8). Initial

experi-mentswereconducted to locate these complexes in sucrose and CsCl gradients. SV40-infected

BSC-1 cellswere labeled for 20minwith

[3H]-thymidine at 33 h p.i. NPCs wereextracted, at

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the end ofthe pulse or after a 6-h chase with

10'

Mthymidine, andseparated on 10 to 40%

sucrose gradients (Figure 1A and 1B,

respec-tively). With a 12-min pulse, only the slower

sedimenting species of NPC was labeled (data

not shown). Thelabeling kinetics and location

of thisspecies in the gradientidentify it as

NPC-I (1, 3,6-8). The more rapidlysedimenting

spe-cies contained NPC-II+V.This was verified by

poolingthe fractions (Fig. 1A and B) and

sub-jecting the complexestoequilibriumdensity

gra-dient centrifugation in CsCl(p = 1.34g/ml) (Fig.

1C andD). Whereas the NPCs located in the

601

50-40

f

E

a

0u

0~

30

20

10

0 80

70

60

50

E

v.

40 30

20

10

NPC-II+V region aftera20-minpulsewere

pre-dominantly salt-labile NPC-II, approximately

50% were salt stable after a 6-h chase. The

sucrose gradients were used to quantitate the

distribution oflabelbetweenNPC-I and

NPC-II+V. When necessary, NPC-II andvirionswere

further separated on CsCl. It should be noted

that, on the CsCl gradients, a new NPC was

detected which had a greater buoyant density

thanmaturevirions. Thiscomplexwasseparable

from bothNPC-II and mature virions and

ap-pearedtobeanintermediate in the maturation

pathway andnot abreakdownproduct of virions

5 10 15 20 25 5 10 15 20 25

FRACTION NUMBER

FIG. 1. Separation of NPCsonsucroseandCsClgradients.SV40-infectedBSC-1 cellswerelabeledfor20

minat33hp.i.with75

gCi

of[3HJthymidineperml(ca.40to60Ci/mmol). NPCswereextractedfromsome

cellsafterthepulse (AandC), fromothersaftera6-hchasein10-6 M thymidine(BandD).NPC-containing

supernatants weresedimented on 10to40%sucrosegradients containing2 mMTris (pH 7.4) (A andB).

Gradientswerecentrifugedfor90minat37,000rpm inanSW41Tirotor.Sedimentation isfrom righttoleft.

Fractionswerepooledasshown andbandedtoequilibriuminCsCI(p=1.34g/ml)inatype40rotorat33,000

rpmfor40h(CandD).Thearrowrepresentstheposition ofthemajority ofcomplexeslabeled ina12-hpulse. Thedensity ofthismaterialis 1.34g/ml.

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258 WANG AND ROMAN

(data notshown);it was namedNPC-III. Inthe

quantitation ofthe percentage ofnewly

synthe-sized DNA inchromatin, previrions, and virions,

thecpminNPC-IIIwereaddedtoNPC-II, since

bothareprevirions.

Effect of BUdR substitution on

encapsi-dation. The protocol for following reentry of

newlysynthesized DNA into replication involves

the use of BUdR. Thus, it was necessary to

determinethe effect of BUdRontheprocessof

encapsidation. At 36 h p.i., 4 plates of

SV40-infected BSC-1 cells were labeled for 20 min

with [3H]thymidine and chased with 10-6 M

thymidine for30min.NPCswereextracted from

twoplates.Athirdwasrinsed andcovered with

Dulbecco modified Eagle medium containing

BUdRchase medium.Theremaining platewas

rinsed, and Dulbecco modified Eagle medium

with 106Mthymidine was added. NPCswere

extracted from the latter two

plates

6 h later.

Thepercentageof NPC-II+V extracted from all

fourplateswasdeterminedon10 to40%sucrose

gradients. BUdRsubstitution didnotaffect the

extentof accumulation ofNPC-II+Vduring the

6-h chase

(Table

1). Thesetwo

species

ofNPCs

were further separated on CsCl. Virions

accu-mulatedto asimilarextentunder thetwochase

conditions(Table 1).

Comparison of the kinetics of

maturation

and kinetics ofreentry. Todirectlycompare

thetimecourseof maturationtothat ofreentry, at 33 h p.i., SV40-infected BSC-1 cells were

pulse-labeled

for20minwith

[3H]thymidine

(75

,uCi/ml),

chased for30minwith 106M thymi-dine, and chased again with the BUdR chase

medium for different lengths of time.The

kinet-ics of maturation of this pulse-labeledDNA was

determined by plotting the percentages of

la-TABLE 1. Effect ofBUdRontheextentof

encapsidationof viral chromatin

%(NPC- %Wt

Chase medium

II+V)'

at: %(V)bat:

Oc 6 0 6

BUdR 42 89 7 45

10-6 Mthymidine 43 91 8 46

aThe percent ofNPC-II+V wasdetermined from

separationon 10to40%sucroseby using the formula

(NPC-II+V cpm + total cpm of NPCs)x 100.

bThe percent of virions (V) wasdetermined after

the separation of NPC-II from V by CsCl density

equilibrium centrifugation by using the formula

%(NPC-II+V) x(V cpm + NPC-II+V cpm).

cChase time in hours. SV40-infected BSC-1 cells

were labeled for 20 min with [3H]thymidine and

chasedfor30minwith10-6 M thymidine. NPCs were

either extractedatthis time(0)oraftera6-hchasein

the mediumshown(6).

beledNPC-I,NPC-II, andvirionsversusBUdR

chase time (Fig. 2).At theend ofthe

pulse

and

30-min chase with 106 M thymidine (T = 0),

the majority of

[3H]DNA

was in NPC-I; very

little was in virions. As the BUdR chase time

increased,thepercentage of[3H]DNAin

NPC-I decreased, with the rate of decrease being

greatestduring the first2 to 3h after initiation

of the BUdR chase. Therewas a concomitant

increase in the fraction of

[3H]DNA

appearing

in virions. The fractionof

[3H]DNA

in NPC-II

remained

relatively

constant during the chase

period (Fig. 2). The level of the NPC-II pool

varied fromexperimenttoexperiment (compare

Fig. 2A and B; similar variations in levels of

NPCshavebeenseenby others [6]). Withinany

one experiment, the level of NPC-II was the

samein thepresenceorabsence ofdithiothreitol

andphenylmethylsulfonylfluoride. Therefore, it

seems

unlikely

that this variation is due to

breakdown of virions.

Todetermine the reentry kinetics for newly

synthesized

DNA

during

the BUdRchase,DNA

wasextractedby theHirtmethod, formIDNA

was isolated onalkalinesucrosegradients, and

HL DNAandLL DNAwereseparated by CsCl

densityequilibrium gradient centrifugation.

3H-labeled HL DNA represented DNAthat

repli-catedduring the short

3H

pulse andwas

subse-quentlyused astemplateforfurther DNA

syn-thesis(reentered replication).Thepercentage of

HLDNAplottedversuschasetime with BUdR

showed the kinetics of reentry of the

pulse-la-beledDNAinto

replication (Fig.

3). It should be

noted that only one round ofreentry was

de-tectedby the

protocol

since form I DNA with

BUdRsubstitution in both strands (HH DNA)

wouldnotbelabeled. LL DNAreentered

repli-cation(wasconvertedtoHLDNA)over aperiod

of2 to3h. After thislengthofchase,either the

pulse-labeledDNAceasedtoreenterreplication

or the rate at which it reentered greatly de-creased.

Also plotted in Fig. 3, for direct comparison

with the reentry

kinetics,

is the rate atwhich

[3H]DNA left the NPC-I pool, calculated from

the data in Fig. 2. A good correlation may be

seenbetween the time thatpulse-labeledDNA

wasremoved from thepool of molecules

avail-able forreplicationand thetimeit left the

NPC-Ipool.

The NPC data presented in Fig. 2 represent thefate of all pulse-labeledDNA, whereas the reentry data in Fig. 3represent only the fate of

forn

I DNA (containing 85 to 89% of the

3H

cpm) in a Hirt

supernatant.

The reentry kinetics

was,therefore, determined directly on a sample

ofaHirt

supernatant

and inparallelonthe form

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[image:4.493.53.244.465.528.2]

z

a I

LL

0

0 2 4 6 0 2 4

LENGTH BUDR CHASE-HRS

FIG. 2. Relationship between the length of BUdR chase and percentage of

[3H]DNA

inNPC-I, NPC-II,

and virions. (A and B) Results of representative experiments. NPCs were isolated as described in the text.

Symbols:0,percentageof NPC-I; A, percentage ofNPC-II; A, percentage of virions.

<: zo 00

I

_0n

cL I

UU

0a.

zz

u>

=u-i

:L-0 2 4 6

*1UO

z 0

-j

20 m

0 2 4 6

[image:5.493.104.397.64.296.2]

LENGTH BUDR CHASE- HRS

FIG. 3. Relationship between the fraction of the initial[3H]DNAinNPC-I leaving the NPC-Ipool and the

percentageof 3H-labeledHL DNA (kinetics of reentry) with increasingchasetime in BUdR. (A and B) Data

from thesameexperiments showninFig.2A and B.To calculate thefraction of[3H]DNA leaving the

NPC-Ipoolduring the B UdR chase, thetotalpercentageof NPC-Iatthe timeBUdR was added (e.g., 51% atT=

0;Fig. 2A)was setequalto1.0.Thefraction of[3H]DNAleaving the NPC-Ipool is determined bytheformula

1-[o(NPC-IT=

x+

%(NPC-IT=-q],in which X and0 arethelengthsof the BUdR chase. The percentage of

3H-labeled HL DNAwasdeterminedafterseparation offormIHL and LL DNA moleculesby CsCl density

equilibriumcentrifugationasdescribed in thetext.

I DNAisolated from thatsupernatant. The

ki-netics ofreentrywasqualitativelysimilartothat

determined for form I DNA (Table 2). The

percentage of HL DNA, however, was lower

than that found when only form I DNA was

analyzed.Thiswasexpected,since thenon-form

I DNAisolatedonalkalinesucroseandanalyzed

on CsCl contained only

3H-labeled

LL DNA

(datanotshown).

Distribution of 3H-labeled HL DNA in

NPC-I, NPC-II, and virions. To determine

more precisely the relationship of reentry to

maturation, the percentages of 3H-labeled HL

DNA in NPC-I and in NPC-II+V were deter-259

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260 WANG AND ROMAN

mined. SV40-infected cells were pulse-labeled and chased for various lengths of time as de-scribed above. NPC-I and NPC-II+Vwere

sep-arated on 10 to40% sucrose gradients, and the percentage of HL DNAwasdetermined on each pool. In parallel, the percentage of HL DNA in form I isolated from a Hirt supernatant was

determined. Figure4showstheaccumulation of HL DNA inNPC-I, NPC-II+V, and form I. 3H-labeled HL DNA accumulated in NPC-I for a period of 2 to 3 h then ceased toaccumulate, a pattern qualitatively similar to the kinetics of reentrydetermined on form I DNA. 3H-labeled HL DNA accumulated more slowly and to a lesser extent inNPC-II+V. It is possible to cal-culate the reentry kinetics curve for total NPCs

bytakinginto accountthe relative contribution

of each NPC pool tothe totalNPCs extracted at each time. This curve isvery similar to that foundby analyzing form I DNA (Fig. 4).Thus, the kinetics ofreentry establishedbyanalysisof form I DNA isdirectlycomparabletothe kinet-ics ofmaturation.

Comparisonof thekineticsofreentry and

maturation at 24 hp.i.versus 42 hp.i.

Ro-man(21)demonstratedthat thekineticsof

reen-try ofpolyoma DNA was dependent upon the time in the infectious cyclethat wasexamined.

Earlyin thereplicationcycle, reentry occurred

more rapidly and to a greater extent than at

latertimes. If the cessation ofreentry is related to astep in the maturationpathway,thekinetics

ofmaturationshould alsobedifferentat

differ-enttimes.Therefore,thekineticsofreentry and

maturationweredetermined at 24and 42 hp.i.

(Table 3). More extensive reentry was seen at

24 h p.i. than at 42 h p.i. When cells were

harvested aftera20-minpulseand30-min chase

(T=0),thepercentageoftheNPC-II+V at42 hp.i.wastwice that seen at 24 h p.i.Therefore,

pulse-labeledDNAproceedsalongthe

matura-tion pathway more rapidly later in infection.

TABLE 2. Kineticsof reentry determinedonisolated

formIDNAorunfractionated Hirtsupernatantsa

% of HL DNA at: HL DNA source

ob 2 6

Form I 0 17 26

Hirt supernatant 0 15 24

aAt36h

p.i.,

SV40-infected

cells

werepulse-labeled

with [3H]thymidine, chased with 10' M thymidine,

and chasedagain for different lengths of time with a BUdR chase mixture. DNA was isolated by the Hirt

method (10). Eitherasamplefrom the Hirt

superna-tantwasdirectly analyzed by CsCldensity equilibrium

centrifugation,orformI DNAisolatedfrom the Hirt

supernatantwasanalyzed.

bChase time in hours.

100

z

6

bU -o

- 50- o

40 /

302g

20-10~~~~~~~

1-

0-0 1 2 3 4 5 6

LENGTH BUDR CHASE-HRS

FIG. 4. Percentage of 3H-labeled HL DNA in

NPC-I, NPC-II+V, total NPC, and form I DNA.

Poolsof NPC-I andNPC-II+V,asshown inFig. 1,

weredeproteinized, put into CsCl gradients (p=1.72

g/ml) andcentrifuged for48hat33,000 rpm inatype

40 rotor to separate 3H-labeledHL DNA from

3H-labeled LL DNA.Symbols:0, Percentage ofHL DNA

inNPC-I; O, percentageof HL DNA inNPC-II+V;

0, percentage of HL DNA inform I;E, calculated

percentageof HL DNA in total NPC. The percentage

of HL DNA in total NPC at each timepointwas

calculatedby the formula[%(HL DNA inNPC-I)x

(NPC-I cpm . total NPC cpm)]+ [%(HL DNA in

NPC-II+V)x (NPC-II+Vcpm +totalNPC)J.

TABLE 3. Kinetics of reentry and maturation of

DNApulse-labeledat 24and42hp.i.

Timep.i. %of HL DNAat: % ofNPC-II+Vat:

(h) oa 2 6 0 2 6

24 0 28 45 25 51 80

42 0 16 31 50 64 80

aChase time in hours.

The percentage of NPC-II+V was the same (80%) after a 6-h BUdR chase at the two times; the20% of[3H]DNAwhichremained in NPC-I may be eitherunavailable for packaging or

in-efficiently packaged.

DISCUSSION

Experiments were undertaken to determine

whether the cessation ofreentry of newly syn-thesized DNA into replication coincided with theprogression of that DNAalong the matura-tion pathway. SV40-infected BSC-1 cells were labeled for a short period with [3H]thymidine

andthen chased with BUdR. Theuse ofnewly

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synthesized DNA as the template for further DNA synthesis (reentry into

replication)

was

measured by the conversion of H-labeled LL

DNA to3H-labeled HL DNA (Fig. 3, Table 3). In parallel, its progression along the maturation pathwaywasmonitored by determining the per-centageof 3H cpm in different NPCs (Fig. 2 and 3; Table 3). The time course of appearance of

[3H]DNAinNPC-I,NPC-II, and virions (Fig. 2)

wasconsistentwith previous reports that NPC-Iprogressed to virions through the intermediate NPC-II (1, 3, 6-8).

Two facts emerge from this study. First, ces-sation of reentry and maturation are closely linked intimne.There do not appear to be factors that terminate reentry for anysignificant period oftime before maturation. Cessation of reentry

coincides with the maturation of DNA from

NPC-ItoNPC-IIorvirions (Fig.3).Thetime of

cessation of reentry and of the conversion of

NPC-I toNPC-II+V varies somewhat from

ex-perimenttoexperiment, butthe two arealways

coincident (Fig. 3). Second, newly synthesized DNA becomes encapsidated more rapidly late in the infectious cycle. This is consistent with

theobservationthat therateand extent of

reen-trydecreaseswith timep.i. (Table3).After a

6-hBUdR chase, 80% of the pulse-labeled DNA hasmaturedtoNPC-II+V,whether the experi-ment wasinitiated at24 or42 hp.i. (Table 3). However, when thepool of NPC-II+V was

sep-aratedonCsCl,24and 59%ofthe

[3H]DNA

was

in the virionfractionat 24and 42 hp.i.,

respec-tively (datanotshown). This suggests that not

only isthere a slower progressionofNPC-I to

NPC-II+V early in infection but also that the

progressionfromNPC-IItovirions isslower.

We propose that the fate of a newly

synthe-sized SV40 moleculeis determined by whether

it isboundfirstbyaproteinorproteins involved

inreplication (or transcription) or byaprotein

or proteins involved in maturation. This

com-petition,between thebindingoflargeTantigen

(andperhapsotherproteinsinvolved in DNAor

RNA synthesis) and a structural protein(s),

could beaffected

by

therelative abundance of

earlyversuslate

proteins.

The

affinity

of

differ-entproteins for the NPC-I

template

would also

affect the outcome of the

competition.

Coca-Pradosetal. (4)

recently proposed

that whether

a DNA molecule was used for replication or

maturationmight dependontheextent of

ace-tylation of histones, with structural proteins

having a higher affinity for histones with a

greaterlevel ofacetylation.

In summary, the data presented suggest a

close correlation betweenremoval of DNA from the pool ofmolecules available for replication

and progression of this same DNA

along

the

maturationpathway. Thesimultaneousanalysis

of the kinetics of reentry and maturation, using

severalmutantsofSV40 defectivein known viral

proteins,should result in a detennination of viral proteins involved in one or both of these pro-cessesandreveal essential details of their

inter-dependence.

ACKNOWLEDGMENTS

WethankRichard Bockrath, Howard Edenberg,andSteve

Larsenfor helpful suggestions during thepreparation of the

manuscript.

Thisinvestigationwassupported by PublicHealthService

grants CA-19708 and CA-29318 from the National Cancer InstituteandbyaProgramDevelopmentProject grant from the IndianaUniversitySchool of Medicine.

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