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Characterization of Simian Virus 40 tsA 58 Transcriptional Intermediates at Restrictive Temperatures: Relationship Between DNA Replication and Transcription


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Copyright©1977 American Society for Microbiology PrintedinU.S.A.


of Simian Virus 40 tsA58










and Transcription


Laboratory ofBiologyof Viruses, NationalInstitute ofAllergyandInfectious Diseases,National Institutesof Health,Bethesda,Maryland 20014

Received for publication 24September1976

When nuclei from simian virus40 (SV40)-infectedcellsarelysed with Sarko-syl and the chromatin is pelleted, the supernatant fluid containsanucleoprotein complex capable ofsynthesizing viral RNA (Lauband Aloni, Virology

75:346-354,1976;Gariglio andMousset,FEBS Lett.56:149-155,1975).The level of activ-ityofthe RNApolymeraseinthecomplex increasedduring infectioninparallel

withtheamountofviral DNA that had beensynthesized. If cells infectedat3300

withthe SV40 mutant tsA58 wereshifted to the nonpermissive temperature of 40°0

at any time between 18 and 48 h postinfection, no viral DNA replication was detectedafter45minandno newrounds ofsynthesiswereinitiated after 20to30

min. However, afterthis shift, polymerase activity associatedwith the nucleo-protein complex did continue to increase for 5 h, at which time it reached a

plateau. Therewas anincreaseof RNAsynthesizedfromboth theearly (E)and late (L) SV40 DNA strands, and there was athreefold increase in the ratio of early-to-late RNA species after the shift. Incomparable experiments with cells infected with wild-type virions, no increase in polymerase activity occurred because of the temperature change alone. At330C,the relativeamountof RNA transcribed from the wild-type E-strand was less thantsA 58at330Cand didnot

increase after a shift to 400C. The tsA58 transcriptional complexes extracted fromcells grown at


sedimented heterogeneouslyin sucrosegradients,with

a peak near 26S. There were no detectable alterations in the sedimentation propertiesof the complexes when tsA58-infected cellswereshiftedto


for2h. Weconclude that continued synthesis of viral DNA isnot anobligatory prereq-uisite for maintenanceof late viral transcriptionnoristhesedimentation of the transcriptional complex at 26S relatedtoactively replicating DNA molecules servingastemplates for transcription. Further,anincreaseinlatetranscription

can occur under conditions where reinitiation of viral DNA synthesis is

pre-vented. The increase inthe synthesis of early and late RNAatthe restrictive temperature withoutconcurrent DNAsynthesisis discussed inrelationshipto

thefunctionof the A gene product.

Lyticinfection of African green monkey kid-ney cells by simian virus 40 (SV40) produces

two classes of viral mRNA. The first is early

mRNA, which is made throughout the

infec-tious cycle and is complementary to

approxi-mately 50% of the early (E) strand of viral

DNA. Early mRNA is translated into the A

protein, T-antigen, and it has been proposed

thatitssynthesis is regulated by the A protein through a negative-feedback mechanism (14, 20). The second class of viral mRNA is late mRNA, which appears afterinitiation of viral

DNA replication and is complementary to

ap-1Presentaddress: Institut de Recherches Scientifiques surleCancer,Villejuif, France.

proximately41% of the late (L) strand of viral

DNA (1, lla). Late transcription and DNA

replication are detectedonlyifthe early viral gene (geneA)isexpressed (2). There may be a directrelationship between viralDNA

replica-tion and late transcription, since "replicating-like" viral DNAmolecules have been reported

to be associated with nascent viral RNA (4). However, once late transcription has begun,

late mRNA synthesis continues when

expres-sionofthe A geneis blocked(2). The

hypothe-sis thatreplicating molecules serve as templates

for late transcription is difficult to reconcile

with thefindingthat maintenance of a pool of

replicatingmolecules requires continued

expres-sion ofgeneA.


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Enzymatically active SV40 nucleoprotein

complexes extracted from isolated nuclei by

Sarkosyl are capable of synthesizing viral RNA

in vitro andarebelieved tobe derived from in

vivotranscriptional intermediates of the virus

(5, 9, 13). In this study we have characterized

the transcriptional intermediates of the SV40

gene A mutant, tsA 58, and measured the RNA

polymerase activity contained in tsA58 tran-scriptional intermediates extracted from

in-fected cells grown at 330C and subsequently shifted to the nonpermissive temperature of 400C. These experiments provide further

in-sightintoregulation ofviraltranscriptionand how it is related to the gene A product and DNA replication.


Tissue culture and virus. SV40 mutant virus tsA58, providedby Peter Tegtmeyer, and SV40 wild-typestocks were propagated in African green

mon-key kidneyBSC-1 cells grown in plastic tissue

cul-ture flasks (150 cm2; Costar) and infected atinput multiplicities of 0.01 PFU/cell. All experimentsused BSC-1 cellsgrown in plastic petri dishes (60 mm for DNAreplication studies and 150 mm for

transcrip-tionstudies;FalconPlastics). Confluentmonolayers

grown inEaglemedium with 2 mM glutamine, 1.35 mgof streptomycin perml, 0.62 mg of penicillin per ml,0.20mgof auromycin perml,500Uof mycosta-tin perml,and10%fetalcalf serum weretrypsinized


90% confluent, were infected with SV40 tsA58 or

SV40wild-typevirusat aninputmultiplicityof1to

5 or 20PFU/cell, respectively,at33°C.Infected cells werethen grown in the same medium except that it contained 2% fetal calf serum. Cells were shifted from 33 to 40°Cby floating the petri dishes onwater ina40°C incubator.

Extraction of transcriptional complexes. The

procedure for extraction of transcriptional

com-plexes is thesame aspreviouslydescribed (13), with certainmodifications. The 150-mmpetridisheswere

removed from the incubator andimmediatelyplaced

onto an icebath. The cells, extracts, and reagents werekept at4°C duringthe isolationprocedure.The medium was removed, and 2.0 ml of calcium-and magnesium-free phosphate-buffered saline with trypsin (1:250; Difco Laboratories), 0.5mg/ml, and EDTA, 0.2 mg/ml, were addedto each petri dish. The cells werethen gently scraped from the petri dish with a rubberpolicemanandpelletedat500xg for 5 min. The nuclei were isolated as previously described (16) and resuspendedin 0.15 ml of 0.05 M

Tris-hydrochloride (pH 7.9), 0.05MKCl, and0.0005 Mdithiothreitol (Rebuffer) per petri dish. Then, 1

volume of 0.5% Sarkosyl NL 97 (recrystallized in 95% ethanol) in 0.8 M NaCl was added, and the resultant nuclearlysate was centrifugedin polyal-lomercentrifuge tubesinaBeckman SW50.1 rotor at21,000 rpm for30mintopellet the chromatin. The resulting supernatant fluid was poured out of the centrifuge tube and is referred to as the Sarkosyl

supernatant. The number of petri dishes used to prepareeach Sarkosyl supernatantvaried from 2 to 6.

Assay of RNA polymerase activity. The assayof RNA polymerase activity was performed as previ-ously described (18),with several modifications.The standard assay mixture contained, in 0.125 ml: 0.05

,umol ofATP, GTP, andCTP;


amountsof UTP and [3H]UTP (specific activity, 27.6 to 41.5Ci/ mmol) or[a-32P]UTP (specific activity, 200 Ci/mmol) asdescribed below; 5 ,umol of Tris-hydrochloride, pH 7.9; 5 ,umol of (NH4)2SO4; 0.2 ,umol of MnCl2; 0.05 ,imol of EDTA; 0.5,umol of dithiothreitol; and 0.075 ml of Sarkosylsupernatant. The reactions were run at low UTP concentrations (0.12 to 0.43 nmol per standard assay mixture), where the UTP concentra-tion was rate limiting, or athigh UTP concentration (4.20nmol per standard assay mixture), where the enzyme velocity approached Vmax. Several experi-ments were run inparallel using high and low UTP concentrations, and similar data were observed ex-ceptfor the expected increase in enzyme velocity at high UTP concentrations. For each experiment

pre-sented, the UTP concentration in the assay is

stated. The reaction mixture was incubated at 21°C for1hunless otherwisestated, and the reaction was terminated by adding 2 to 3 ml of 5% trichloroacetic acid in 0.01 M sodium pyrophosphate at 4°C unless

RNA hybridization was done, in which case 10%

sodium dodecyl sulfate was added to give a final concentration of 1%. To measure the amount of [3H]UMP incorporated, the [3H]UMP-labeled RNA wascollected onWhatman GF/C glass-fiber filters, washed under suction with100mlof 2.5% trichloro-aceticacid in0.01M sodiumpyrophosphate, rinsed with5 mlof 95% ethanol, dried under an infrared heat lamp, and counted in 7 ml of toluene-based scintillant{toluene with 4g of2,5-diphenyloxazole and 50 mg of 1,4-bis[2-(5-phenyloxazolyl)]benzene

perliter}. Background counts were determined by

addingthe5%trichloroacetic acidin 0.01Msodium

pyrophosphate immediately after addition of the

Sarkosylsupernatant to the reaction mixtures and

weresubtracted from the counts measured after in-cubation at 21°C. For enzyme activityexpressedas

[3H]UMPcounts permicrogram of protein, the


determined as describedbyLowryetal. (10). RNA extraction andhybridization.The RNA po-lymerase reaction mixture after 1h of incubation wasdiluted with 10volumes of0.01 M

Tris-hydro-chloride, pH7.4, containing0.05 MNaCl, 0.006M

EDTA, 1% sodium dodecyl sulfate, and 200 ,ug of

carrier RNA. Anequalvolume ofa25:25:1mixture

ofphenol, chloroform,andisoamylalcoholwasthen

added. This extraction mixture was shaken

mechan-ically atroomtemperature for5minandcentrifuged

at5,000 rpm for5min in aSorvallHB-4 rotor. The RNAand DNAintheaqueousphasewere precipi-tated overnight with 2 volumes of95% ethanol at

-20°C and thencentrifuged at 10,000 rpm for30min

inaSorvallHB-4rotor. Thepelletwasdriedunder vacuum, dissolved in 1.0 ml of 0.01 M

Tris-hydro-chloride,pH 7.6, containing0.01 MNaCl and0.01M

MgCl2, anddigested with 50 ,ugof DNaseIper ml

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(RNase free; Worthington Biochemicals Corp.) for 30 min at room temperature. After DNase treat-ment, the mixture was again extracted with the

phenol, chloroform, and isoamyl alcohol solution,

and the RNA was precipitated by ethanol as

de-scribed above.

To determine what fraction of the RNA

synthe-sizedwasSV40specific, [3H]UMP-labeled RNAwas

hybridized to SV40 DNA immobilizedonfilters as

previously described(12).To determinethe relative amounts of RNA synthesized from early and late strands, [32P]UMP-labeled RNA washybridized to separated strands of SV40 DNA immobilized on

strips ofnitrocelluloseasdescribed by Michael

Bot-chan (personal communication). Separated strands ofSV40 DNAwereprepared by denaturing 5 to10 jigof thetwoDNAfragmentsproduced by BamI and HpaIIrestrictionenzymecleavagein 0.17MNaOH

for 10 min at room temperature. The denatured fragmentswerethensubjectedtoelectrophoresisin

a1.4%agaroseslabgel128mmwide, 150mmlong,

and3mmthick. Electrophoresiswasat20mAfor20 h at21°C usingthe buffer systemofHayward (6).

Theseparated strands of each fragmentwerethen

transferredto sheets of nitrocellulose as described

by Southern(17), and thedried sheetswerecutinto

strips5mmwide and70mmlong.[32P]UMP-labeled

RNA was hybridized to the SV40 DNA on these

stripsin0.8mlof6x SSC(0.15MNaClplus 0.15M

sodium citrate) at 68°0 for 18 h. The strips were

treated with RNase, washed, and dried as

previ-ously described (12). Autoradiography was per-formedusingKodakRoyal X-Omat film.

Autoradi-ograms werescannedinaGilfordspectrophotometer at 560 nm to obtain a densitometric tracing. The areasunderthepeaksweredeterminedand usedto calculate thepercentage of RNA hybridizingtothe separated strands of each DNA fragment. 3H-la-beled complementary RNA transcribed from the early strand by Escherichia coli RNA polymerase

wasalsohybridized under thesameconditions,

ex-cept the nitrocellulose strip wasmomentarily

im-mersed in toluene containing 2,5-diphenyloxazole (20%,wt/vol) and air-dried before autoradiography. Sedimentation oftranscriptional intermediates. Samples (0.2 ml)ofSarkosyl supernatantwere

lay-ered onto 3.6-ml linear 5 to 20% (wt/wt) sucrose

gradientsinRebuffer,0.3MNaCl, and0.25% Sar-kosyl. 14C-labeledSV40, form I, isolated fromvirions

propagatedinBSC-1 cells andpurified by isopycnic bandingincesiumchloride-ethidiumbromide(3, 15), wasaddedasamarker. Thegradientswere

centri-fugedinaBeckmanSW60Tirotorat54,000rpmfor 100minat2°C. Tubeswerepuncturedatthe bottom

and 0. 16-ml fractionswerecollected.Samples (0.075

ml)fromeach fractionwereassayed forRNA polym-eraseactivity asdescribed above. Thestandard

re-action mixture contained 0.18nmolof[3H]UTP (spe-cificactivity, 41.5 Ci/mmol). Thereactionwas ter-minated after 2 h at 21°C, and the number of [3H]UMP counts incorporated was determined as


ViralDNAreplication.Tomeasuretheamountof viral DNA replication at 33°C, [3H]thymidine (1

mCi/ml) insterilewater(specific activity, 40to 60

Ci/mmol)wasaddedtoeach 60-mm petridish

con-training 3 ml of mediumtogive a final concentration

of5,uCi/mlfor1h. Tomeasuretheamountof viral

DNAreplication at varioustimesaftershiftingcells

to400C, [3H]thymidine was addedto afinal

concen-tration of10


for 15min. Atthe end ofeach pulse-labelingperiod,viral DNAwasselectively

ex-tracted by the 0.6% sodium dodecyl sulfate-1.0 M

NaClmethod of Hirt (7).Aliquots from theresulting Hirt supernatant fluid were spotted on Whatman GF/C filters, washed in 5% trichloroacetic acid at

40C,dried, and countedin 7ml of thetoluene-based scintillant described above.Mock-infected cellswere

usedinparallel as a control for each measurement. RESULTS

Characterization of infection by tsA58, WhentsA58wasgrown in BSC-1 cells at 33CC,

viral DNAsynthesiswas firstdetectedat 18 to

24 h after infection, and the rate of synthesis

increased in alinear fashion until a maximal

rate wasachievedatabout48hafter infection

(data not shown). After a rapid temperature

shift from 33 to 400C at 33 h after infection, replicating DNA molecules were chased into

form I within 45min. Fromthis, itwas calcu-lated that no new rounds of DNA

synthe-sis wereinitiatedafter20 to 30 min. These

prop-erties are characteristic of complementation group A mutants (19).

Characterization of the tsA58 Sarkosyl

su-pernatantand RNApolymerasereaction. The Sarkosyl extraction procedure used in this study yielded greater than 90% of the RNA polymerase activitysynthesizing SV40 RNAin

theSarkosylsupernatant(M. Shani, E. H.

Bir-kenmeier, E. May, and N. P. Salzman, J.

Virol., in press), in agreement with previous findings of Mousset and Gariglio (13). In the RNApolymerasereaction the kinetics of

incor-poration of[3H]UMP during1hofincubationis

shown in Fig. 1 and is identical to that seen

withwild-typevirus (5; Shani etal., inpress).

After shifting tsA58-infected cells at


RNA continued to be synthesized by the Sar-kosylsupernatant. Becauseofvariations inthe number ofnuclei in each Sarkosyl extraction, the protein concentration in the Sarkosyl

supernatant varied from 2.3 to 4.8 mg/ml, but

showed no apparentrelationship to time after

infection or time at


For this reason

enzyme activity inSarkosyl supernatants was

measured as [3H]UMP counts per minute

in-corporated per microgram of protein duringa 1-hincubationat21°C unlessstated otherwise.

The relative amount of[3H]UMP incorporated

into SV40-specific RNA variedfrom 40 to 80%

for each experiment, presumably due to

vary-ing amounts ofshearedhostchromatin

remain-ing in the Sarkosyl supernatant. In several

experiments the labeled RNA was hybridized

to SV40 DNAimmobilized onfilters, andonly

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- 20 2


u-0 10 20 30 40 50 60


FIG. 1. Pattern of incorporation of[3H]UMP by transcriptional intermediates. At 42 h afterinfection with tsA58at330C, cells wereeither shifted to400C

orallowedtoremainat33°C for 2 h.Sarkosyl super-natants werethen prepared from cells ateach temper-ature,and the RNA polymerase activitywasassayed

asdescribedinthetext.The reaction wasterminated after 15, 30,or 60minof incubationat21°C, and the [3H]UMP countsincorporatedateach time were de-termined.Each standard reactionmixturecontained 0.18 nmol of[3H]UTP (specific activity, 27.6 Cil mmol) and 0.25 nmolof unlabeled UTP.

hybridizable counts were used in calculating

enzyme activity. Since labeled SV40 RNA is

the major species of RNA synthesized in the

extracts, the results obtained by this

pro-cedurewere notsignificantlydifferent from the overall pattern obtained when activity was

expressedas [3H]UMPcountsincorporated. Velocity sedimentation of transcriptional intermediates in sucrose gradients. tsA58-in-fected cellsweregrown at33°Cfor46h.Atthat time, one-half of the cells wereshiftedto 4000

while the remainderstayedat


After 2 h

(48 hafterinfection), cells ateachtemperature

were harvested and Sarkosyl supernatants wereprepared. The transcriptional

intermedi-atesineachsupernatant weresedimentedina

sucrose gradient, and the fractions from the

gradients were assayed for RNA polymerase activity. Figure2showsthat apeak ofenzyme

activityis obtained from both the 33 and4000

supernatants, andtheprofiles for each

temper-ature areidentical. The activity from each

su-pernatant sedimented in abroad band witha

peak near 26S. This sedimentation pattern is identicalto thatobtained withwild-type virus

(5, 9; Shanietal., in press). Enzyme activities determinedwith theremainderof the original

33 and 40'C supernatants were 7.7 and 9.0

hybridizable [3H]UMPcountsincorporatedper


ofprotein,respectively. Inboth supernatants

64%of the totalcountsincorporatedhybridized

toSV40 DNAonfilters. Weconcludethat there

is no change in the velocity sedimentation of

tsA58transcriptional intermediates after

shift-ing to the nonpermissive temperature for 2 h

and thatsedimentationnear26Sis notrelated

to actively replicating molecules serving as

templates fortranscription.

Maintenance of RNA polymerase activity after shiftto 400C. At different times during infection with tsA58at330C, cellswereshifted

to40'Cfor2h; thenaSarkosylsupernatant was

prepared and assayed for enzyme activity. A

Sarkosyl supernatant was also prepared from cellsat330Cbefore the 2-h shift. Table 1shows that transcriptional activity continues for at




b 9


x 7



5 4

10 20




FIG. 2. Sucrose sedimentation analysis of tran-scriptional intermediates. Sarkosyl supernatants

wereprepared from cells infected with tsA58 for 46 h

at33°C and then either shifted to 40°C or allowed to

remain at33°C for2h.Eachsupernatant was centri-fugedin alinear5 to 20%(wtlwt) sucrose gradient, fractions were collected, and each fraction was

as-sayed forRNApolymerase activityas described in

the text. Each standard reaction mixture contained

0.18 nmol of [3H]UTP (specific activity, 41.5 Ci/ mmol) and was incubated for2hat 21 °C. '4C-labeled SV40DNA Iisolatedfrom virions was added to each gradient asa21S marker. Since the gradients were centrifuged simultaneously and the marker was at thesamepositionin eachgradient, the RNA

polym-erase activities in both gradients were plotted

to-gether. The sedimentation oftheDNA I markerin

the gradient with the 33°C supernatant is also presented. Sedimentation isfrom righttoleft.


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[image:4.501.] [image:4.501.257.445.326.488.2]


least 2 h aftershiftingthe cells to the

nonper-missive temperature, irrespective of the time

after infection orlevelof transcriptional

activ-itybefore theshift. Infact, the RNA

polymer-ase activity canincreaseby twofoldduring the

2-h shift. Weconclude that viral transcription

intsA58-infectedcells ismaintainedat40'Cat a level greaterthanthat observed at330C be-fore theshift, eventhoughnoactively

replicat-ing viral DNA moleculesaredetectable inthe


Increase ofRNA polymerase activity after shift to40'C. Tofurther examine the increase

inRNApolymeraseactivitythat occurred

dur-ingthe2-hshiftto40'C(seeTable1),wild-type SV40-infected cellsgrownat330C for24hwere

shifted to 40'C for varying times, andthe

en-zyme activity intheSarkosyl supernatantswas

comparedto activityinSarkosyl supernatants

from cells remaining at 330C. Figure 3 shows

that for 2.5 h after the shift only a slight

in-crease ofenzyme activity occurs at 40'C, and

the activity at40'Cis less than the activity at

330C forat least 5h afterthe shift. The lower activity at40'C forthe first5hmaybearesult of the cells responding to rapid temperature

change. Thermal perturbation of cellular

proc-esseshas been reported in relation to protein

synthesis (11). However, by 11 h at 40'C, the

enzyme activitywas greaterthan the activity

measured inSarkosyl supernatants from cells

at330C.Thisdelayedincreasemayberelatedto an increase ofSV40 DNAsynthesisinthe cell

at 40'C.

The level oftranscriptional activity in

wild-typevirus-infected cellsat 24.5hafter infection

at33°Cis 2.21


of protein (Fig. 3) andis

comparabletothe level of transcriptional

activ-TABLE 1. Transcriptional activityaftershiftto


Time of shift Enzyme activity

(h afterinfection) Before shift After shift

18 0.09 0.43

21 0.24 0.54

24 0.53 1.00

36 2.52 5.07

48 8.75 10.51

a Cells were infected with tsA58 and grown at

33°C. At various times after infection, cells were either shiftedto 40°C for2h or harvested without shifting. Sarkosylsupernatants wereprepared from cellsbeforeand after theshift, and theRNA

polym-erase activity was measured as [3H]UMP counts


in the text. Each standard reaction mixture

con-tained0.12nmolof[3H]UTP (specific activity, 41.5



z /

0 /

0-230 /


20 _



0 24 30 35 40 45 50



FIG. 3. Effect of temperature on transcription of wild-type virus. Cells infected with wild-type virus for 24 h at33°C were eithershifted to 40°C or allowed to remain at 33°C for 0.5, 2.5, 5, 11, and 24 h. Sarkosyl supernatants were prepared from cells at each temperature for each time period, and RNA polymerase activity was measured as [3H]UMP counts incorporated permicrogram of protein as de-scribed in the text. Each standard reaction mixture contained 0.12 nmol of[3H]UTP (specific activity, 41.7Ci/mmol).

ity of 2.52cpm/,ug of protein intsA58-infected

cellsat36 h after infection(Table 1). Therefore,

tsA58-infectedcellsgrown at33°C wereshifted

to 40°C at 36 h after infection, and Sarkosyl

supernatants were prepared at various times

after the shift. An increase ofRNApolymerase activity synthesizing SV40-specificRNAinthe Sarkosyl supernatants occurred between 0.5

and 5h after the shift (Fig. 4). After 5h, the

enzyme activity remainedconstantfor atleast

another6h at40°C.Also,it isimportanttonote

that cells infected with tsA58 and maintained

at33°C tookapproximately50%longertoreach

the maximumenzymeactivityachievedby the

cells at 40°C in 5 h, even though the total

amountof viral DNA inthecells at33°C

contin-ued to increase. Results similar to these were

also obtained when the temperature shift

was carried out at 24 and 48 h postinfection,

and polymerase activity remained constant evenifcellswereat40°Cfor 24 h.

RNA synthesized by transcriptional

com-plexes. Tocharacterizethe RNAsynthesized by

tsA58transcriptionalcomplexes, tsA58-infected

cellsweregrownat33°C for36h,and thenone

half wereshiftedto40°C for5h.The remainder

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FIG. 4. Rateof increase ofRNApolymerase

activ-ity aftertheshift to40°C. Cells infected with tsA58 for36 hat33°Cwereeithershiftedto40°Corallowed toremainat33°C for 0.5, 2.5, 5,and 11 h.Sarkosyl supernatants wereprepared fromcells ateach tem-peratureforeach timeperiod,and RNApolymerase activity was assayedas described in thetext. Each

standard reaction mixture contained 0.45 nmol of [3H]UTP (specific activity, 41.4 Cilmmol) and 3.75 nmolofunlabeledUTP,which caused thevelocity of

theenzymetoapproachVmax., After1 hof incubation, a sample fromeach reaction mixture was removed,

and the numberof [3H]UMPcountsincorporated in SV40 RNA was determined by hybridizing the

newly synthesizedRNA toSV40DNAonfilters, as

described in the text. The enzyme activity is

mea-suredashybridizable [3H]UMPcountsincorporated per microgram of protein. The number ofcounts hybridizedaftera0.5-hshiftwere229cpmat33°C

and 359 cpm at40°C. At 11 h after the shift the counts hybridizedwere1,668cpm at33°C and 756


stayedat3300 for 5 h. Inaparallel experiment,

cells infected withwild-type virusatan

identi-cal input multiplicity were shifted to 40°C or

remained at 33°C for 5 h. Sarkosyl extracts

werepreparedand RNAwassynthesizedinthe

standard reaction mixture containing

[a-32P]UTPtolabel the RNA. The RNAwasthen

isolated, and anequal number ofcounts from

each sample was hybridized to separated

strands of theearlyandlate regions of the SV40

genomeimmobilizedonstripsof nitrocellulose.

Figure 5showsanautoradiogram of the strips

from which adensitometric tracingwas made

and therelative amountsof RNA in each band

quantitated (Table 2). Seventy-eightpercentor

more of the RNA synthesized by the wild-type

or tsA58 transcriptional complexes before or

afterthe shiftto40'Cwastranscribed fromthe

Strand, and 24to 34% of this late RNA was from the earlyregion ofthe genome, i.e., was

antisense early RNA. The RNA transcribed from the E-strand was almost entirely from the early region of the genome; i.e., nosignificant

amount of antisense late RNA was detected.

With wild-type infection either at 330C or shifted to 40'C, no more than 2 to 4% of the

newly synthesized RNA was complementary to the E-strand. However, with tsA58 the relative

amountof early transcription at330C was twice

that seen with wild type, and shifting to400C causedanadditionaltwo- to threefold increase

in the percentage of RNA transcribed from the E-strand. Thus, whereas the ratio of early to late RNA synthesis was lower in wild-type-infected cells and did not increase after the shift, the ratio ofearlyto lateRNA synthesis

with tsA58 increased approximately threefold

after the shiftto 40'C.


When cellsareinfected and maintainedat a

restrictive temperature with theA gene

tem-perature-sensitive mutant tsA30, viral DNA

synthesisand late viral mRNAsynthesisdonot

occur. When these cells are infected at a

per-missive temperature and then shifted to a

re-strictive temperature after DNA and late mRNAsynthesis have begun, late mRNA

syn-thesis continues for at least 20 h, but DNA synthesis isinhibited within1 h(2).

Wehave examined cells infected with theA

genetemperature-sensitive mutant tsA58 and have determined the level of viral transcrip-tional activity after Sarkosyl extraction. The Sarkosyl extraction procedure yields SV40 transcriptional complexes that contain bound RNA polymerase B and a small number of otherproteins. When cells infected with tsA58 virions are shifted to the restrictive

tempera-ture atanytimeafter viral DNAsynthesishas

begun, DNA synthesis iscompletely inhibited

in 45 min. The behavior of tsA58 transcrip-tional intermediates after a shift in

tempera-ture to


is similar to the behavior of

in-fected cellsinvivo;i.e., transcriptional activity

is maintained in extracts prepared from cells that wereheld for anextended time period at

the restrictive temperature. This is true

irre-spective of the time after initiation of viral DNAsynthesis that the cellsareshiftedto


or the level oftranscriptional activity before

the shift. These findings strongly support the

idea that transcriptional complexes contained

inaSarkosyl extract arederivedfrom the

com-plexes thatgenerate viralRNAinvivo. When 7






'33 C


5_ / X



4 /

_ / /~~~~~~~

/0 / 3


OL I. , * * I


I I~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~

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FIG. 5. Autoradiogram of RNA hyb separated strands of the early and latereg

SV40genome.Cellsinfected with wild-tyj

tsA58 virusfor 36 hat339Cwereeithershi, orallowed to remain at330Cfor5 h. Sc tractswerethenprepared, and RNAwass

inastandard reaction mixture containing

of [a-32P]UTP (specific activity, 200Cilm RNAsynthesizedwashybridizedtoseparate

of the early (A) and late (B) regions of

genome immobilizedonstripsof nitroceli

labeledcomplementary RNAwasalsohyb aseparatestriptolocate the positionsof th indicated in the figure. Although it is c

discern the E-strand, early region ban photograph, they could be detected by visu( tionof theoriginal autoradiogram andga peaks in the densitometric tracings. T. order is(a) WTat330C; (b) WTat400C, at33C; (d) tsA58at40°C. The bandsare:

strand, early region; LA -late strand,ea

LB-late strand, lateregion;and EB-ea)

late region.

therateof viral mRNA synthesis isI

after ashiftto 4000,notonlyis itma

but there is an increase in the in v

scriptional activity, whichreachesa

is two- to threefoldhigher than theI

existed priortothetemperature shift

crease in transcriptional activity occi

5-h period, andthen theactivityrema

high level for atleast an additional



of tsA58 and wild-type transcription

tiesafteratemperatureshiftdemonstrates that

theincrease inactivityis relatedtothe

temper-ature sensitivity of the mutantA gene and is

not the result of the temperature shift alone.

Wild-type-infectedcells shifted to40'Cinitially

shownostimulation oftranscriptional activity

-o E A under conditions where viral DNA synthesis is


Since no [3H]thymidine is incorporated into

L A SV40replicatingmoleculesaftertsA58-infected

cellsareat40'Cfor 45min andtranscriptional

complexescontinuetoproducelate RNA for at

least 5 hat400C, actively replicatingmolecules

cannot be serving as templates for late

tran-scription at 40'C. The identical sedimentation

L B of transcriptional complexes from cells at 33



notidentical, templates. Weconcludethat

con-tinuedsynthesisof viral DNA isnotan

obliga-toryprerequisite for maintenance of late viral

transcription nor is the sedimentation of the

transcriptional complexesat26Srelatedto

ac-tively replicating viral DNA molecules serving astemplatesfor latetranscription.


to Therearethreewaystoexplainthe increase ridized to ofenzymeactivityobserved after the






the increase in

polym-fted to40C erase


may result from more RNA po-arkosyl ex- lymerase molecules on each template at400C.

-ynthesized We believe this to be unlikely since the sedi-0.28nmol mentationpatternoftranscriptionalcomplexes

imol). The doesnotchangeafter theshift, and addition of tedstrands only one RNA polymerase molecule per

tem-the SV40 plate with its associated nascent RNA would




the molecular


of the

complex by


over0.5 x 106 (8). In




difficult to increase isseenwhen



ds in this complexes arecomparedat 33 and400C.


observa-yedistinct TABLE 2. Hybridization of32P-labeledRNA 'he sample synthesized by wild-type (WT)and tsA58

;(c) tsA58 transcriptional complexesat330Candaftera5-h

EA-early shiftto400Ca

rly region;


measured intained, itro tran-level that level that L. This in-ars over a


20 h. The


comparison ial

activi-HybridizedRNA (%)

comple-mentarytoeach DNAfragment

DNA fragment

WT, WT, tsA58, tsA58, 33'C 400C 33'C 400C

E-strand, early region 4 2 9 22

(0.16 to 0.735 mapunit)

L-strand, early region 34 34 24 29 (0.16 to 0.735 map unit)

L-strand, late region 62 64 67 49

(0.735 to 0.16 map unit)

E-strand, late region _ -(0.735 to 0.16 map unit)

aThe autoradiogram in Fig. 5 was scanned and a densito-metrictracing was obtained. The percentage of the total areaundereachpeak was calculated and is presented in the table.

bNomeasurable amountwas transcribed.


.r.- '. I.

on November 10, 2019 by guest




The increase in RNA polymerase activity may alsoreflect an increase in the number of templates. These additional templates can

comefrom (i) the pool of replicatingmolecules

present in the cell prior to the shiftup,(ii) DNA

Imolecules that are still able to enter the

repli-cative pool during the first 20 to 30 min after

the shift up before the block in initiation of DNA synthesis was complete, or (iii) DNA I molecules present in the cell prior to the shift to 40'C, which must undergo processing before they can be used as templates for transcription. Thereplicating molecules would complete rep-lication within 40 min after the shift up. Since the newly synthesizedDNA Imoleculescannot

initiate new rounds of DNA synthesis, they could preferentiallybe shunted into the pool of

molecules beingtranscribed and cause an accel-erated increase in transcriptional activity.

A third explanation is that additional

smaller-molecular-weight proteins or even the Ageneproduct itself have either been added to

orremoved from the complexes after the shift and thus affect the rate of movement of RNA polymerasealong the DNA template in vitro or the initiation of transcription in vivo and the species of mRNA that are made. Reed et al. (14) have reported thatafter shiftingtsA58-infected CV-1 cells from 32 to 41'C at 72 h postinfection,

therateofsynthesis of RNAcomplementaryto

the E-strandisabout 15 times greaterthan in

cellsinfected with wild-type virus. These find-ings are consistent with Tegtmeyer's

sugges-tion (20)that T-antigen regulates its own

syn-thesis, andthe rateof synthesis increasesatthe

restrictive temperature. Similarly, in this

study we have observed that in tsA 58-infected BSC-1 cells, the ratio ofearlytolate RNA syn-thesized by transcriptional complexes increases 3-foldafter the shift and is about 12-fold higher than seen in wild-type infection shifted to 400C.

Since DNA synthesis and late viral mRNA synthesis are both inhibited when tsA30-in-fected cells are infected and maintained at a

restrictive temperature, it has been suggested thatthe A gene hasadirect roleinthe

regula-tionof both of these functions. The firstevent in

thesynthesisof viral DNA involves the

synthe-sis ofan RNA primer, and so DNA synthesis andtranscription share a common mechanism

ofinitiation. Further, the DNA initiation site

(0.67 map unit) may be closeto the promoter

site for early and late viral mRNA. In this

studywehave observed thatalthoughthere is a

large increase in therelative amount of early

RNAsynthesized by tsA58 transcriptional

com-plexes at40TC, 70% of the increase in the total

transcriptional activity measured in vitro

dur-ing a 5-h shift is late mRNA. Thus, the two

events, late transcription and replication, can, in a temporal way, be separated. Viral DNA synthesis is blocked completely within 45 min of the time cells are shifted to the restrictive temperature. However, an increase in

tran-scriptional activityoccursovera5-hperiod. We conclude that theoneknown function of the A geneproductisindirectly required for late

tran-scription becauseof its roleininitiation of viral DNAsynthesis, whichallowsapool of replicat-ingand newlysynthesized DNA I moleculesto



We areindebted toMichael Botchanfor demonstrating to us his technique of separating SV40 DNA strandsin agarosegels.


1. Acheson, N. H. 1976. Transcription duringinfection withpolyomavirusandsimian virus40.Cell 8:1-12. 2. Cowan, K., P. Tegtmeyer,and D. D. Anthony. 1973. Relationship ofreplication and transcription of sim-ian virus 40. Proc. Natl. Acad.Sci. U.S.A. 70:1927-1930.

3. Fareed, G.C., E. D. Sebring, and N. P.Salzman.1972.

Cleavageofreplicating intermediatesof simian virus 40deoxyribonucleic acidbythe restriction

endonucle-aseofEscherichia coli B. J. Biol. Chem. 247:5872-5879.

4. Girad, M., L. Marty, and S. Manteuil. 1974. Viral

DNA- RNAhybridsincells infected with simian vi-rus:the simian virus 40transcriptional intermedi-ates.Proc.Natl. Acad. Sci.U.S.A.71:1267-1271. 5. Green,M.H., andT. L. Brooks. 1976.Isolation oftwo

forms of SV40nucleoprotein containingRNA

polym-erase from infectedmonkey cells. Virology 72:110-120.

6. Hayward,G. S. 1972. Gelelectrophoretic separationof the complementary strands ofbacteriophage DNA. Virology 49:342-344.

7. Hirt, B. 1967. Selective extraction of polyoma DNA from infected mouse cell cultures. J. Mol. Biol. 26:265-269.

8. Kedinger, C., F. Gissinger, and P. Chambon. 1974. AnimalDNA-dependentRNApolymerases. Eur.J. Biochem. 44:421-436.

9. Laub, O., and Y. Aloni.1976. Transcriptionof simian virus 40: SV40 DNA-RNA polymerasecomplex

iso-latedfromproductively infectedcellstranscribedin vitro.Virology75:346-354.

10. Lowry,0.H., N.J. Rosebrough, A. L.Farr,and R. J.

Randall. 1951. Proteinmeasurementwiththe Folin

phenolreagent.J. Biol.Chem. 193:265-275. 11. McCormick, W.,andS. Penman. 1969.Regulation of

protein synthesisin HeLacells: translationat ele-vated temperatures. J. Mol.Biol. 39:315-333. 11a.May, E., J. V. Maizel, and N. P. Salzman. 1977.

Mapping of transcription sites ofsimian virus 40-specific late 16S and 19S mRNA by electron mi-croscopy. Proc.Natl.Acad. Sci. U.S.A. 74:496-500. 12. May, E., P. May, andR. Weil.1973.Early

virus-spe-cific RNA may contain information necessary for chromosomereplication andmitosisinducedby sim-ian virus40. Proc. Natl. Acad. Sci. U.S.A. 70:1654-1658.

13. Mousset, S., and P.Gariglio.1975.Sarkosylextraction of an active SV40transcription complex. INSERM 47:67-74.

22, 709

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14. Reed, S. I., G. R. Stark, and J. C.Alwine. 1976. Auto-regulation of simian virus 40 gene Aby T antigen. Proc. Natl. Acad. Sci. U.S.A. 73:3083-3087. 15. Sebring, E. D., T. J. Kelly, M. M. Thoren,and N. P.

Salzman. 1971. Structure of replicating simian virus 40deoxyribonucleic acid molecules. J. Virol. 8:478-490.

16. Shmookler, R. J., J. Buss, and M.Green. 1974. Proper-tiesof the polyoma virus transcription complex ob-tained from mouse nuclei. Virology 57:122-127. 17. Southern, E. M. 1975. Detection of specific sequences

among DNA fragments separated by gel electropho-resis. J. Mol. Biol. 98:503-517.

18. Sudgen, B., and W. Keller. 1973. Mammalian deoxyri-bonucleic acid-dependent ribonucleic acid polymer-ases. J.Biol. Chem. 248:3777-3788.

19. Tegtmeyer, P. 1972. Simian virus 40 deoxyribonucleic acid synthesis: the viral replicon. J. Virol. 10:591-598.

20. Tegtmeyer, P., M. Shwartz, J. K. Collins, and K. Run-dell. 1975. Regulation of tumor antigen synthesis by simian virus 40gene A. J. Virol. 16:168-178.

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FIG.1.[3H]UMPoraftertranscriptionalnatantsature,as0.18withtermined.mmol) allowed described Pattern of incorporation of [3H]UMP by intermediates
FIG. 3.forpolymerase41.7Sarkosylcountscontainedeachscribedtowild-type remain Effect of temperature on transcription of virus
FIG. 4.peraturepertofor[3H]UTPsuredcountscpmandactivitystandardadescribedsupernatantsanditySV40hybridizednmolthenewly sample remain Rate of increase ofRNA polymerase activ- after the shift to 40°C
TABLE 2.transcriptional Hybridization of 32P-labeled RNAsynthesized by wild-type (WT) and tsA58 complexes at 330C and after a 5-hshift to 400Ca


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