Synthesis of the adenovirus-coded DNA binding protein in infected cells.

(1)

Synthesis

of the

Adenovirus-Coded

DNA

Binding

Protein

in

Infected

Cells

ZVEE GILEAD,* KENJI SUGAWARA, G. SHANMUGAM, AND MAURICE GREEN

Institute for MolecularVirology,St. LouisUniversity School ofMedicine,St.Louis,Missouri63110

Receivedfor publication 25 November 1975

Synthesis of the 75K (75K indicates a molecularweight of70,000to 75,000)

DNAbinding protein, an early virus-coded proteininadenovirus 2-infected KB

cells, and its regulation were studied by using a radioimmune precipitation

inhibition assay. The protein was first detected at 4 h postinfection and

accumu-lated at an exponential rate. An arrest of further synthesis (accumulation)was

observed at 10 to 11 hpostinfection,coincidingwiththeonsetofsynthesisoflate

virion proteins. In contrast, when the infected cells were treated with25 ug of

arabinosyl cytosine per ml toblockviral DNAreplication, thesynthesisof 75K

protein did not cease but continuedfor up to 36 h postinfection. Thesynthesis of

75K protein incellsafter release from acycloheximide block (2to 9h

postinfec-tion) wasanalyzed. Increased amounts ofearlyadenovirus-specificmRNA

accu-mulate in infected cellsduringacycloheximideblock(Parsons andGreen,1971).

However, cycloheximide treatment did not produce increased levels of75K

pro-tein, and an abrupt arrest of 75K protein formation was again observed at the

time ofsynthesis of late virion proteins. Partition of the 75K protein between the

nuclear and cytoplasmic fractions during the course of infection was studied.

The 75K proteinappeared first in the cytoplasm andthen in the nucleus after a

slight lag.Accumulation of the 75K protein continued both in thecytoplasm and

nucleus, with higherlevelsbeingfoundinthe cytoplasm.

Several virus-specific polypeptides that are

not viron components are synthesized in

hu-mancells infected with adenovirus 2 or 5(Ad2,

Ad5). These new polypeptides were identified

by electrophoresis on sodium dodecyl

sulfate-polyacrylamidegels of extracts ofinfectedcells pulse labeled at early times after infection (1, 2,

9, 14, 15, 20).They range in size from 75,000 to

10,000daltons, based on their migration in gels.

Only one of these early polypeptides with a

molecular weight of 70,000 to 75,000 (referred to

as 75K protein) has been purified to date. It

binds to single-stranded but not double-stranded DNA and has been termed the "DNA

binding protein" (17, 18). The 75K proteins

codedby Ad2 andAd5 have similarproperties

andmolecularweights (13). Use of Ad5

temper-ature-sensitive mutants has indicated that the

75Kprotein is virus coded and isnecessary for

viral DNAreplication (19). The viral gene

cod-ing for ithas been mapped, by the use of

tem-perature-sensitive mutants, in the EcoRl B

fragment of the adenovirus genome (T.

Grod-zicker, C. W. Anderson, and J. Sambrook,

personal communication). Another polypeptide

ofmolecular weight 45,000 elutes from

DNA-cellulosecolumns together with the 75K protein

and appearstobeadegradation product of the

75Kprotein (13).

We have recently shown by radioimmune

precipitation and radioimmune precipitation

inhibition (RIPI) that the Ad2 75K protein is

virus specific (5). The method involved theuse

ofpurified 3H-labeled 75K protein and its

ho-mologous antibody present in sera from

ham-sters immunized with extracts of Ad2-simian

virus 40-induced tumors (4, 5). We have now

developed the RIPI method for the accurate

quantitation of the 75K protein in order to

study its synthesis. The presentcommunication

describes ourfindingson the synthesis of this

proteinduring infection and its modulationby

interferencewithDNAorprotein

syntheses.

MATERIALS AND METHODS

Isolation of labeled 75K protein. Labeled 75K protein was isolated from Ad2-infected KB cells and purified by DNA-cellulosechromatographyas previ-ously described (5, 17) withtwomodifications. We foundthat thetrailingedgeofthe0.6Mpeak elut-ing from the DNA-cellulose column contained only 75Kprotein, whereas the leading edge of the peak contained also the 45Kprotein. Fractions from the trailing edge of the peak only were pooled and used asthesourceof75K protein for the RIPI assay. In 454

on November 10, 2019 by guest

http://jvi.asm.org/

(2)

SYNTHESIS OF DNA BINDING PROTEIN 455

addition, labeling was performed with [35S]methio-nine instead of [3H]leucine, yielding preparations with higher specific radioactivity. Infected cells were incubated with 5 ,uCi of [35S]methionine per ml (210 to 466 Ci/mmol) in methionine-free Eagle minimal essential medium containing 5% horse se-rum.M5S-labeled75K protein wasprepared monthly tominimizeradioactive decay.

Experimental infection of KB cells and prepara-tion of cell extracts.Suspension cultures ofKBcells

wereinfected with purified Ad2 at a multiplicity of 200 PFU/cell aspreviouslydescribed (17). Infected

cellsweremaintainedat aconcentration of 3 x 105 cells/ml in Eagle minimal essential medium con-taining 5% horseserum. To prepare whole-cell ex-tractsfor assay of 75K protein, 9 x 106cells (30 ml) werewithdrawnatspecifiedtimesand washed with Ca2+- and Mg2+-free phosphate-buffered saline (PBS). The cells wereresuspended in 1 ml of PBS containing 1% Triton X-100 and stored at -5C. Before assay, suspensions weredisruptedfor 3 min atfull power inaRaytheon DF-101 sonicoscillator and clarified at 2,000 x g for 10 min at4C. The supernatant fractionswerecollected for assay. For the preparation ofcytoplasmicandnuclear extracts,

washed samplesof 9x 106 cellswereresuspendedin 1 ml ofPBS containing 1% Triton X-100 at room temperature, and after5minnuclei andcytoplasm

were separated by centrifugationat800 xgfor 10 min. Thenucleiweresonicated and clarifiedas de-scribed for whole-cellextracts.

To prepare extractsforcomplement fixation tests, 10-ml samplesofinfected cells (3 x 106cells)were washed withPBS,resuspendedin0.5mlof veronal buffer (16),sonicated for3min,and clarifiedat2,000

x g for 10 min. The supernatant fractions were collected for assay.

RIPI. The double-antibody method for radioim-mune precipitation oflabeledpurified 75Kprotein

and thesourceof antisera have beendescribed (5).

Inthe RIPI assay, 10-,ugaliquotsof immune ham-ster immunoglobulin G (IgG) (supplemented with 70 ,ugof "carrier" normal hamsterIgG)were

prein-cubated with various amountsofextracts of unla-beled infected cellssampledatvarioustimes post-infectionto blockantibody activity. Preincubation wasfor 1 h at 37 C in PBS containing 1% Triton X-100 (vol/vol) and 1% sodium deoxycholate (wt/

vol) inconical 0.75-ml centrifuge tubes (Sarstedt,

Princeton, N.J.).The final reaction volumewas200

A1l.

Labeled 75K protein (10,000 counts/min) was thenadded, and incubation wascontinued for1 h. Finally, 30Al of goatserum antihamsterIgG was

added, and theprecipitatesformed after2 h of in-cubation at 37 C were centrifuged, washed, and counted as previously described (5). Background

precipitationvalues(obtained with80 Agof normal hamster IgG) were subtracted in each determina-tion. The degree of inhibition observed with ex-tractsof unlabeled infected cellswascalculated as percentage of controlby comparisonwith reactions substitutingPBSfor cellextracts.

Titration of virionproteinsin extractsof infected cells by the complement fixation. The

micro-com-plement fixation testofSever (16) was used for the titration of virion proteins as previously described (6). Eight units of goat antibody to purified Ad2 virions wasused with 2 U of complement.

RESULTS

Quantitation of75K protein in extracts of

infected cells by the RIPI assay. The RIPI

assay is one of the few methods available to

accurately measure early proteins present in relatively low concentrations in infected cells.

Wehave developed a RIPI assay for

quantitat-ing intracellular 75K protein usquantitat-ing 35S-labeled

75K proteinanditshomologousantibody from

hamsters immunized with extracts of tumors

induced by an Ad2-simian virus 40 hybrid. To

standardizethe assay, a precipitation reaction

was performed first with increasing amounts of immune hamster IgG and 10,000 counts/min of 35S-labeled 75K protein (Fig. 1A). Ten

micro-grams of immune IgG, which precipitated.

about one-half of the 10,000-counts/min input

(Fig. 1A), was used subsequently in the RIPI

assays as follows.Aliquots of 10 ugof immune

IgG, supplemented with 70 ,ug of normalIgG,

were preincubated withincreasingvolumes of

unlabeled infected cellextractfor 1h at37 C.

35S-labeled 75K protein (10,000 counts/min)was

then added, thereaction mixtures werefurther

incubated, and the assay wascontinued as

de-scribed inMaterials and Methods. A represent-ative inhibition curve obtained with different

volumesof a 20-h infected cell extract is shown

in Fig. 1B. In this instance, 50% inhibition of

radioimmune precipitation was obtained with

1.03 ,tl of cell extract; the volume of extract

causing50%inhibition isdesignated

Vw.

Quan-titation ofthe 75K protein content in cell

ex-tracts is based upon the determination ofV50

from curves obtained for the individual ex-tracts, as describedin Fig. 1B, using different extractvolumes. Since the amount of 75K

pro-teinpresent in the extract isinverselyrelated

to V50, the 75K protein titer is therefore

ex-pressedininhibitoryunits as1/V50 x 100;

mul-tiplication by 100 is used to avoid fractional

values.

The fidelity ofthe RIPI assay was tested by

reconstruction reactions with known amounts

of unlabeled 75K protein. The 0.6 M eluate,

from a DNA-cellulose column, which is the source for labeled 75K protein, contains also

unlabeled hostcomponents (Sugawara, Gilead,

and Green, unpublished data). Unlabeled 75K

protein was therefore purified from the 0.6 M eluatetohomogeneity (Sugawara, Gilead, and

Green, manuscript in preparation). Known

amounts of pure 75Kproteinweremixed with

VOL. 18, 1976

http://jvi.asm.org/

(3)

456 GILEAD ET AL.

~60 j60

0- L 8nVX _

040

"40-20 20

0 0 ii

1 10 100 0.1 1 10

IMMUNOGLOBULININPUT(,g) EXTRACTINPUT(,1)

FIG. 1. Quantitation of the RIPI assay. (A) Standard precipitation curve of35S-labeled 75Kprotein.

Aliquots of 75K protein (10,000counts/min) weremixedwithincreasing amountsofimmunoglobulin from

immunized hamsters (immuneIgG),supplemented withIgGfromnormal hamsters(normalIgG)togive 80

pgoftotal IgG, in reaction mixtures containing PBS, 1 % Triton X-100, and 1% sodiumdeoxycholate.After1h of incubation at 37 C, 30 4 of goat serum anti-hamster IgG was added and precipitates formed after 2 hof

incubation at 37 C werecentrifuged, washed twice with PBS containing 1% Triton X-100 and 1% sodium deoxycholate, dissolved in 0.25 M acetic acid, and counted in a scintillation spectrometer. Background radioactivity due tononspecificprecipitation, measured with 80pgofnormalIgGalone, was subtracted. (B) The RIPIassay. Increasing volumes of a 20-h infected cell extract were preincubated with 70pgof "carrier" normal IgG and 10pgof immuneIgG, shown in (A) to precipitate 50% of the10,000-counts/mininputof 35S-labeled 75K protein, for 1 h at 37 C in the buffer described in (A).35S-labeled75K protein (10,000counts/min)

wasadded, and incubation was continued for an additional 1 h at 37 C. Goat anti-hamster IgG (30pd) was

added,andthe precipitates formed after2 h at37 C weretreated wasdescribedin(A).Thebackground counts per minuteprecipitated with 80pgofnormal IgG was subtracted, and the percentage ofcontrol was calculated by comparison with a reactionmixtureinwhichPBSwassubstituted for the infected-cell extract.

extractsofuninfected KB cells (9x 106cells/ml) preparedasdescribed for infected cells. Aplot

of the concentration of 75K protein in the

ex-tracts versus the inhibitory titer measured in

the RIPItest (1/V5,, x 100) waslinear (Fig. 2), even at low 75K protein concentrations. The smallest detectable titer of 75Kprotein in in-fected cell extract is 0.5 inhibitory units. One unitcorrespondstoaconcentration of 1.4 Ag of

75Kprotein perml ofextract, accordingtothe

plotinFig. 2. However,wehavenotusedthis

plotto convertthe relativeinhibitorytiters into actual 75Kprotein concentrationsinthispaper

sincethe reconstructionmixturesarenot

iden-tical to extracts ofinfectedcells. Purified 75K

protein showsatendencyto aggregateandmay

be more prone to inactivation in the

recon-structedextractsthanintracellular75Kprotein

in extractsofinfectedcells.

Kinetics of synthesis of 75K protein in

in-fectedcells intheabsenceandpresenceof Ara

C. The 75Kprotein isanearlyprotein essential

for viral DNAreplication(19). Itissynthesized

beforetheonsetofviral DNAreplication, and also when viral DNA synthesis is inhibited

with arabinosyl cytosine (Ara C) (17, 18). The kinetics ofaccumulation ofthe 75K protein in

infected KB cellswereexamined(Fig. 3).Itwas

first detected at4hpostinfection, and its

syn-thesis (accumulation)wasexponentialupto 11

hpostinfection,atwhich time itstopped.There

was aslowdecline in theamountof 75Kprotein

after 11h, perhapsduetolysisofsomecellslate

duringproductive infectionortoinactivation of

75Kprotein molecules in vivo. In contrast, the initial rate ofaccumulation of 75K protein in Ara C-treated cellswas much slower, but did

notexhibitthe arrest observedinuninhibited

cultures (Fig. 3). The synthesis of 75K protein continued up to 36 h postinfection, at which time Ara C-treated cells accumulated four timesmore 75K protein than thehighestlevel

observed inuninhibitedcultures.

Synthesis of 75K protein in cells after

release fromacycloheximide block and effect of Ara C. Cells treated early after infection

with cycloheximide do not synthesize viral

DNA (18) and accumulate upto five times as

much early viral mRNAasuntreated infected

cells (12). It was of interest to determine whetherincreasedlevels of viral mRNA would result in an accelerated accumulation of 75K

protein after release from inhibition. Two pairs

of cultures, A and B, were infected. One A

A

1O

80

B

Inn, 1 rwn

http://jvi.asm.org/

[image:3.503.79.403.56.250.2]

(4)

Z50

200

C-,

I-L&J

-9 150

1oo[

50

n

140 120 100 80 60 40 20 0

[image:4.503.49.243.58.317.2]

UNITS(1/V5ox100)

FIG. 2. Linearity of RIPI assay. A series of ex-tracts containing known concentrations of 75K pro-tein was prepared by adding different amounts of

purified unlabeled75Kprotein to an extract of

unin-fectedKBcells (9 x106 cells per ml of extract). The

reconstructedextracts were assayed by the RIPI as-say.The inhibitory titermeasuredinthe assay(1/VW

x100) was plotted against 75K proteinconcentration

(micrograms per milliliter of extract).

culturewastreated withcycloheximide(25

Ag/

ml) at2hpostinfection, and the other A culture

servedascontrol. At 9hpostinfection,both A

cultureswerewashedtwicewithwarmmedium

and incubatedinfresh medium lacking

inhibi-tor. One B culture received25 ,ugof

cyclohexi-mide per ml at 2 h postinfection, while the

other Bculture received25

,g

of Ara C per ml

at 1 h postinfection. Both B cultures were

washed twice at 9 h postinfection with warm

mediumcontaining25

jig

of Ara C per ml and

incubatedinmediumcontaining25 ug of AraC

per ml. Samples of 30 ml (9 x 106 cells) were

removedfrom AandBculturepairsthroughout

theexperiment foranalysisof 75Kprotein. The

A cultures (no Ara C) were sampled also for

assayof late virionproteinsbythecomplement

fixation test. The kinetics ofaccumulation of

75Kproteinin AculturesarepresentedinFig.

4A.Thesynthesis of75Kprotein inthecontrol

Aculture (not treatedwithcycloheximide)

ex-hibitedagain thearrestof 75Kprotein

synthe-sis.Afterreleasefrominhibition,the

cyclohexi-mide-treatedAculture exhibited a morerapid

rateofaccumulation of the75Kproteinas

com-pared with the control A culture. However, a

somewhatfaster onsetofarrestwas

observed,

resulting in a lower final titer. The time of

arrest in both A cultures coincided with the

onsetof synthesis of late virion proteins,

meas-ured by CF. Incontrast, noarrest insynthesis

of the 75K protein wasobserved in the B

cul-ture released from cycloheximide and then

treated with Ara C immediately after release.

Again, the released culture exhibited a

some-whatfasterinitial rate ofaccumulation as

com-paredwith theparallelBculture thatwas not

treated withcycloheximide.

Partition of the75K protein between

cyto-plasm and nucleus. The cytoplasmic fraction

from Ara C-treated infected cells harvestedat

24hpostinfection isroutinely used for the

puri-fication of the 75K protein. Unlike the nuclear

fraction, chromatography of the cytoplasmic

fraction on a DNA-cellulose columnyields

al-most pure labeled 75K protein (17). It was of

interest todetermine the kinetics of synthesis

and the partition of 75K proteininthe

cytoplas-mic and nuclear fractions. Infected cells were

sampled at various times after infection, and

10000

100

HOURS POSTINFECTION

FIG. 3. Synthesis of 75K protein inAd2-infected KB cells in thepresence and absence of Ara C. A suspension culture ofKB cells was infected with 200 PFUof Ad2per cell, divided into two portions, and maintained at 300,000 cellslml in Eagle minimal essential mediumcontaining5%horseserum. Ara C wasadded tofinal concentration of 25pg/mlat 1 h postinfection to one culture. Samples (30 ml) were removed at the indicated times,centrifuged,washed withPBS,andresuspendedin1mlofPBS contain-ing 1% Triton X-100. Cell suspensions were

soni-cated,clarified,andassayedfor75Kprotein bythe RIPIassay. Symbols: 0, infected culture,nodrug;

*,infected culture treated withAraC.

XrX

U- E 2

VOL. 18, 1976

I I A I

http://jvi.asm.org/

[image:4.503.257.446.317.543.2]

(5)

458 GILEAD ET AL.

Z;

I-m

24 0 T

HOURS POST INFECTION

FIG. 4. Synthesis of 75K protein and virion proteins in Ad2-infected KB cells after release from a

cycloheximideblock in the absence(A)andthe presence(B)ofAraC.(A)AsuspensioncultureofKB cells was

infectedand divided into two. One culture received 25pg ofcycloheximidepermlat2hpostinfection,and the other culture served as control. Both cultures werecentrifugedat 9hpostinfection, washedtwicewithwarm

medium,andfurtherincubated at 37 C.Samples (30ml) wereremovedfromeach cultureatthe indicated times, and extracts wereprepared and assayed for 75K protein content by the RIPI assay. Ten-milliliter samples were removedalso for virion protein assay by the complement fixation test. (B) A suspension culture

ofKBcells wasinfected and divided into two portions. One culture received 25 pgof Ara C per mlat1 h postinfection, and thesecond received 25pgofcycloheximide per ml at 2 h postinfection. Both cultures were

centrifugedat 9 hpostinfection, washed twice with warm medium containing 25 pgofAraC perml,and further incubated in medium containing 25 pg ofAraC per ml.Samples(30ml) were removed andextracts were prepared and assayed for 75K protein by the RIPI assay. 75K protein titer: (A) 0, No drug; 0,

cycloheximide treated (2 to 9 h). (B)0,Ara C (1 to 24 h); *, cycloheximide (2 to 9 h), Ara C (9 to24h).Virion proteintiter: A, Nodrugs; A,cycloheximide treated.

thecytoplasmic andnuclear fractionswere

pre-pared as described in Materials and Methods.

Theaccumulation of 75K proteinincytoplasmic

and nuclear fractions of untreated infected cells

(Fig. 5A) and Ara C-treated cells (Fig. 5B)

showed the sameoverall kinetics observed for

whole cells (Fig. 3). Therewas somelaginthe

appearanceof the 75Kproteininthe nucleusas

compared with thecytoplasm, and theamount

of 75K inthe cytoplasmalways exceeded that

present inthenuclearfraction, except for late

timesinAra C-treated cultures.

DISCUSSION

Several investigations have identified early

and lateadenovirus-specific polypeptides in

in-fected human cells by using sodium dodecyl

sulfate-polyacrylamide gel electrophoresis of

extracts of infected cells labeled with [35S]methionine (1,2, 9,14, 15,20).Early

virus-specific protein synthesiswasdetectedstarting

at2.5 to4hpostinfection, andmaximum rates

ofsynthesis occurred at 6 to 12hpostinfection.

A gradual cessation was observed at later

times,anda"switch-over"tolate virionprotein

synthesis occurred at 12 to 16 hpostinfection.

The electrophoretic study of 75K protein,

termed ICSP3 or

E,

by Russel etal. (14, 15) or

Maizel etal. (9, 20), respectively, was

compli-cated by the presence ofa host protein band

thatmigratedclosetothe 75K protein band(9,

20). The present study, measuring 75K protein

synthesis by a different method, is consistent

with the general pattern of early adenovirus

proteinsynthesis thatwaspreviously observed

by gel analyses. The methodusedin ourstudy

hastheadvantagethat it accurately measures

thenetaccumulation ofimmunologically active

proteinmolecules andis notinfluenced by

pos-sible variations in the equilibration and

con-centration of radioactiveamino acid pools and

by the background ofsimilarlymigratinghost

proteins. Byimmunological assay, we observed

anearly synthesis of 75K protein that is

appar-ently influenced by later events in the viral replication cycle. The synthesis of 75K protein

occurred from4 to 11 h postinfection, with an

arrestat 11 h thatcoincided withthe onset of virionprotein synthesis. However, the synthe-sis ofthe 75Kprotein continued without arrest

P

ac

http://jvi.asm.org/

[image:5.503.78.402.60.280.2]

(6)

if the synthesisof viral DNA and consequently

lateproteins wasinhibited. Some investigators

haveobservedby gel electrophoresis a residual

low level of synthesis of 75K protein at late times postinfection. This synthesis could be due

to acertain degree ofasynchrony in infection.

Alternatively, since the RIPI test measures net

accumulation ofimmunologically reactive

mol-ecules rather than the rate of synthesis, we

cannot completely rule out the existence of a

residual level ofsynthesis. The arrest in

accu-mulation of 75K protein observed in the present

studycan, therefore, reflect a steady-state level

whereby residual synthesis is masked by the

inactivation of some existing 75K protein.

Progeny viral DNA synthesis is blocked in

Ad2-infected cells treated with cycloheximide

early after infection (8), leading to the

accumu-lation of five times as muchearly viral mRNA

in uninhibited cells (12). We do not know

whether the viral mRNA that codes for the 75K

protein is increased in amount by this

proce-dure. Release from acycloheximide block

pro-duces an overall decrease in the rate of

synthe-sis of host polypeptides and increase in the

synthesis of several early adenovirus-specific

polypeptides,asmeasuredbygel

electrophore-sisof extractsofcellspulselabeledimmediately

after release from inhibition (M. Harter, G.

Shanmugam, and M. Green, in preparation).

By theRIPI assay, which measures the actual

quantity of 75K protein, we observed an

ini-tially accelerated accumulation of 75K

mole-cules after release fromcycloheximide,but the

accelerated rate was not maintained even in

cells treated with Ara C toprevent arrest.

Thegene coding for the 75K protein is located

in EcoRI restriction fragment B (Grodzicker,

Anderson, and Sambrook, personal

communi-cation). A 19 to20S early mRNA mapped in this

region (3) that is H-strand specific (W.

Buett-ner, S. Veres-Molnar, and M. Green,

manu-scriptinpreparation) has the appropriate

cod-ingcapacity for a75,000-dalton polypeptide and

is a class Iearly mRNA (3) by thenomenclature

of Lucasand Ginsberg (10); i.e., it is absent or

greatly reduced inamount at 18 h postinfection.

The arrest in 75K synthesis cannot be

ex-plainedbytheexpected normal rate of decay of

mRNA, but could be due to (i) more effective

competition for available ribosomes by abun-dant late mRNA species or (ii) inhibition by

late virionorvirus-coded proteins. Inhibition of

viral DNA synthesis by Ara C may allow

con-tinuedtranscription of the mRNA for75K

pro-tein and/or the inhibition ofsynthesis of late

virionproteins may preventcompetitionand/or

regulation.

Regulated synthesis of virus-specific early

proteins that is linked to DNA synthesis has

beendescribedinseveralother DNAvirus

sys-tems. Forexample, the synthesis ofphage

T4-1000

0

10

0 8 16 24 0 8 16 24

HOURS POSTINFECTION

FIG. 5. Partitionof75Kprotein incytoplasmicand nuclearfractions of Ad2-infectedcellsinthe absence (A)andpresence(B)ofAraC.AcultureofKBcellswasinfectedand divided intotwoportions.One culture received25pgofAraC per mlat1 hpostinfection,and thesecond culturewasuntreated.Samples(30ml) wereremovedattheindicatedtimes,washed,andresuspendedin PBScontaining1%Triton X-100. Nuclear and cytoplasmic fractions were separated and assayed for 75Kprotein by the RIPI assay. Symbols: 0,

Cytoplasmic fractions; 0,nuclearfraction. VOL. 18, 1976

http://jvi.asm.org/

[image:6.503.99.392.402.612.2]

(7)

codedearly enzymesceasesat10to12minafter

infection. When phage DNA synthesis is

in-hibited or delayed, such as by infection with

phage T4 amber mutants or UV-irradiated

phage, early enzyme synthesis continues well

beyond10to12min. Theamountof enzymesat

60min after infection may reach levels four to

five times greater than after infection with

wild-typeT4phage(21). Thesynthesisof

poxvi-rus-coded thymidine kinase, an "immediate"

early protein, is also abruptlyinhibited early

after infection. Studies using inhibitors have

suggested thataviralgeneproductsynthesized

afteruncoating of virus (perhaps a "delayed"

early protein)turns off thesynthesisof

thymi-dine kinase (11). Thesynthesis of herpesvirus

type1-specificpolypeptidesiscoordinately

reg-ulated andsequentially ordered. Three classes,

a, f3, andy, weredescribedaccordingto their

timesofsynthesis. Thesynthesisofone ormore

functional a polypeptides is necessary to

"switch on" thesynthesisofif orypolypeptides,

andone or more83 or ypolypeptides"switchoff'

thesynthesis ofapolypeptides (7).

The intracellular localization of virus-specific

earlyproteins is of interest since it may provide

cluesregarding function. Wehave shown that

the 75K protein is present in both the

cyto-plasm andnucleus, and the kinetics of

accumu-lation provide guidelines for the isolation of

maximumyields forfurther studies. However,

conclusionsregardingintracellular localization

are subject to the usual cautions concerning

leakage fromthenucleusduringcell

fractiona-tion. The lag in appearance of 75Kprotein in

thenucleus and theassociation of 75K protein

with rapidlysedimenting structuresinthe

cy-toplasmic andnuclearfractions (Gileadetal.,

unpublisheddata) suggestthatthe

intracellu-larlocalization deduced from biochemical

frac-tionation may be valid. Immunoflourescence

studies withfixed-cell preparationsusing

anti-body to purified 75K protein may resolve this

importantpoint.

ACKNOWLEDGMENTS

Wethank R. Gilden ofFlowLaboratories for generous gifts ofhamster sera, without which these studies could not havebeenundertaken.

Thisinvestigationwassupported by Public Health Ser-vicegrant AI-01725 from theNational Institute of Allergy andInfectious Diseases and by research contractNO1 CP 43359within the Virus Cancer Program of the National CancerInstitute. M.G. is a ResearchCareer Awardee of the NationalInstitutesofHealth(Public Health Service grant 5K6-A14739from theNationalInstitute of Allergy and In-fectiousDiseases).

LITERATURE CITED

1. Anderson, C. W., P. R. Baum, and R. F. Gesteland. 1973.Processingof adenovirus 2-inducedproteins.J.

Virol. 12:241-252.

2. Bablanian, R., and W. C. Russel. 1974. Adenovirus polypeptide synthesisin the presence of

non-replicat-ingpoliovirus.J.Gen.Virol. 24:261-279.

3. Craig,E.A.,S.Zimmer,and H. J.Raskas.1975. Anal-ysisofearlyadenovirus2RNAusingEcoRAR1viral DNAfragments.J.Virol. 15:1202-1213.

4. Gilden,R.V.,J.Kern,A.E.,Freeman,C. E.Martin,

R.C. McAllister,H.C.Turner,and R. J.Huebner. 1968. T andtumourantigensofadenovirusgroup C-infected and transformed cells. Nature (London)

219:517-518.

5. Gilead, Z.,M.Q. Arens,S.Bhaduri, G.Shanmugam,

and M.Green. 1975. Tumourantigen specificityofa

DNAbindingproteinfromcellsinfected with adeno-virus2. Nature(London)254:533-536.

6. Gilead, Z.,and H. S.Ginsberg.1965.Characterization ofatumorlikeantigenintype12andtype 18 adeno-virus-infectedcells.J.Bacteriol. 90:120-125. 7. Honess, R. W., and B. Roizman. 1975. Regulationof

herpesvirus macromolecular synthesis: sequential

transition of polypeptide synthesis requires func-tional viral polypeptides. Proc. Natl. Acad. Sci. U.S.A.72:1276-1280.

8. Horwitz, M. S., C. Brayton, and S. G. Baum. 1973.

Synthesisoftype2adenovirusDNA inthe presence ofcycloheximide.J.Virol.11:544-551.

9. Ishibashi,M.,andJ. V.Maizel,Jr.1974.The

polypep-tides ofadenovirus.VI. Earlyandlate glycopolypep-tides.Virology58:345-361.

10. Lucas,J. J., and H. S. Ginsberg. 1971. Synthesisof

virus-specific ribonucleic acid in KB cells infected withtype2adenovirus.J.Virol.8:203-213. 11. McAuslan,B.R. 1963.The induction andrepressionof

thymidinekinase in thepoxvirus-infectedHeLa cell. Virology21:383-389.

12. Parsons,J.T.,and M.Green.1971.Biochemical studies

on adenovirus multiplication. XVIII. Resolution of early virus-specificRNAspeciesinAd2infectedand transformed cells.Virology45:154-162.

13. Rosenwirth,B.,K. Shiroki,A.J.Levine,and H. Shi-mojo.1975.Isolationandcharacterizationof

adenovi-rustype12DNAbindingproteins. Virology67:14-23. 14. Russel,W.C.,andJ.J.Skehel.1972.Thepolypeptides

ofadenovirus-infectedcells. J.Gen. Virol. 15:45-57. 15. Russel,W.C.,J.J.Skehel,and J. F.Williams.1974. Characterizationof temperature-sensitive mutants of adenovirus type 5: synthesis ofpolypeptides in in-fected cells. J. Gen. Virol.24:247-259.

16. Sever,J. L. 1962. Applicationofa microtechniqueto

viralserologicalinvestigations.J.Immunol. 88:320-329.

17. Shanmugam, G.,S.Bhaduri,M.Arens, and M. Green. 1975.DNAbindingproteinsinthe cytoplasm and ina

nuclearmembranecomplexisolated from uninfected andadenovirus 2 infected cells.Biochemistry 14:332-337.

18. Van derVliet, P. C., andA. J. Levine. 1973. DNA-bindingproteins specificforcells infectedby

adenovi-rus.Nature(London)New Biol. 246:170-174. 19. Van derVliet,P.C.,A. J.Levine, J. M. Ensinger, and

H. S. Ginsberg. 1975. Thermolabile DNA binding proteinsfrom cellsinfected witha temperature-sensi-tive mutant ofadenovirus defective in viral DNA synthesis.J.Virol. 15:348-354.

20. Walter, G.,and J. V.Maizel,Jr.1974.Thepolypeptides ofadenovirus. IV. Detection ofearlyand late virus-inducedpolypeptidesandtheirdistribution in subcel-lularfractions. Virology57:402-408.

21. Wiberg, J. S., M. L. Dirksen, R. H. Epstein, S. E. Luria, and J. M. Buchanan. 1962. Early enzyme synthesis and its control in E. coli infected with

someambermutantsofbacteriophage T4.Proc. Natl. Acad. Sci. U.S.A. 48:293-302.

http://jvi.asm.org/