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Control of Adenovirus Gene Expression: Cellular Gene Products Restrict Expression of Adenovirus Host Range Mutants in Nonpermissive Cells

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0022-538X/83/040050-10$02.00/0

Copyright ©1983, AmericanSocietyforMicrobiology

Control of

Adenovirus Gene

Expression:

Cellular Gene

Products

Restrict

Expression of Adenovirus

Host

Range

Mutants

in

Nonpermissive Cells

MICHAEL G. KATZE,t*HAKAN PERSSONJBRITT-MARIEJOHANSSON,ANDLENNART PHILIPSON

Departmentof Microbiology, TheBiomedical Centrum, Uppsala, Sweden

Received 15September1982/Accepted 10 December 1982

Adenovirus type 5 (Ad5) host range mutants dl312 and hr-1, with lesions in

region ElA (0 to 4.5 map units) ofthe viral genome, fail to accumulate

virus-specificearlyRNAduring infectionin HeLa cells. Inarecentreport, weshowed

thatthe addition ofanisomycin, a stringent inhibitor ofprotein synthesis,at 1 h

after infection of HeLa cells with hr-1 virus resulted in the accumulation of

properlysplicedand translatable mRNAfromallearlyregions (M. G. Katze, H.

Persson, and L. Philipson, Mol. Cell. Biol. 1:807-813, 1981). Based on these

results we proposed a model in which expression of early mutant RNA was achievedthroughinactivationofacellularprotein normallycausingareduction in theamountofviral RNA. These studies have been extended in thepresentreport, which shows that early viral proteinscanbe detected inAdSdl312-andAdS

hr-i-infected HeLa cellswhich have been treated for several hours withanisomycin

either shortlyafter infection orbefore infection. Apulse ofdrugtreatment also resulted in expression of substantial amounts of adenovirus structural proteins afterinfectionwith both AdShr-1and AdSdl312,whereas indrug-freecontrolsno

lateproteins weredetected. The AdS hr-1 viruspreviously reported tobe DNA

replicationnegativeinnonpermissiveHeLa cellswasfoundtoreplicateitsDNA,

albeitatlow levels, whenanisomycinwas presenteither from1 to5 h

postinfec-tionorfor 5 hbefore infection. Wheninfectiousvirusproductionwasexamined in

mutant-infected cells the titer of AdS dl312 viruswas foundto increaseatleast

500-fold inanisomycin-treated HeLacells. Takentogether, these andour

previ-ous results suggest that the block in gene expression characteristicfor comple-mentation group I Ad5 host range mutants in HeLa cells can beovercome by

inactivating cellular gene products serving as negative regulators ofviral gene

expression.

Permissive infectionof human cells with

ade-novirus is divided into twoclearly

distinguish-ablephasesseparatedbytheonsetof viralDNA replication. Several virus-mediated controls of gene expressionoperate duringthe early phase of virus infection. Evidence for virus-encoded controls of early virus gene expression stems from studies with host range mutants in early region1A(1,11).Thesemutants,referredto as complementationgroup I mutants,either havea definedpointmutation(adenovirustype 5 [AdS]

hr-1)or anextensive deletion of viralDNA(AdS

tPresent address: MolecularBiologyandGenetics Unit of theGraduateSchoolMemorial Sloan-Kettering Cancer Cen-ter, NewYork, NY 10021.

i Presentaddress:DepartmentofGenetics,Harvard Medi-calSchool,Boston,MA02115.

d1312) in early region 1A (7, 10, 12). Both

mutants do not accumulate viral mRNA from anyotherearlyregionof the viralgenome(1,11, 13).Themutationin thehr-1 virus wasrecently showntoaffecta51,000-daltonpolypeptide (27).

The mechanism for this control of early viral gene expression is still unresolved, but studies with inhibitors of protein synthesis have sug-gested an interplay between viral and cellular genes. Inhibition of protein synthesis at 1 h

postinfectionenhanced the accumulation of viral

mRNAfrom allearlyregions(5,23).By measur-ing therateofearly viraltranscription with and without inhibitors of protein synthesis it ap-peared thattranscriptionwasunaffectedby drug treatment(23), suggestingapost-transcriptional control ofearly viralgeneexpression. Accord-ingly, experiments designed to determine the half-lifeofearlyviral mRNAs showed that

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bition of proteinsynthesis prolongs the half-life of earlyviral RNA. It ispossible, therefore,that acontrollingelement in regionElA operatesat thelevel ofmRNA stability (31).

The addition of inhibitors before or at the onset of virus infection, on the other hand, decreasestheaccumulation of early viralmRNA (16, 23), emphasizing that viral proteins are required for accumulation ofviralmRNA. Pre-treatment of cells with inhibitors of protein synthesis didnotdecrease therateof transcrip-tion fromtheearly viralpromoters to anextent thatcould explainthedeficient accumulation of viral mRNA, again suggesting a post-transcrip-tional control. Superinfection ofan Ad2-trans-formedhumanembryonic kidney cellline (293) onlyexpressingthe El products showed, in the absence ofproteinsynthesis,anenhanced accu-mulation ofviral mRNA only from region E4, whereastheamountof mRNA from regions E2 and E3was decreasedcomparedwithdrug-free controls (22). These data suggest that the pro-teinsencodedinearly region1actpreferentially on region E4 and that additional controlling elements presumably from region E4 may be required to control theaccumulation ofmRNA fromregionsE2 and E3.

These results led to a model to explain how the viral proteins encoded in region ElA can enhance accumulation ofviral mRNA from oth-erearly regions. TheElA proteins may inacti-vate a cellular protein which, when present, decreasesthe amountofearly viralmRNA(13, 22, 23). This model isconsistent with all of the results obtained from experiments withprotein synthesis inhibitors and furthermore suggests that the hostrangemutantsfromregionElA fail toinactivate the cellularprotein,which leads to adrastic reductionin the accumulation ofearly viral mRNA. The addition of inhibitors of

pro-tein synthesis shortly after infection should

therefore result in accumulation ofearly viral mRNAfrom mutant-infected cells, since

synthe-sis of the cellularproteinis inhibited.Consistent with this hypothesis, infection of HeLa cells with the Ad5 hr-1 mutantvirus allowed

accumu-lation offunctional early viral mRNAfrom all

early regions in the absence of protein synthesis

(13).

In the present- study we show that virus-specific early and late proteins were expressed inboth Ad5d1312- and hr-i-infected HeLa cells only whenanisomycinwaspresent either before

or shortly after infection. Furthermore, drug

treatmentresulted inenhanced virusproduction in mutant-infected cells. These results, taken together withourpreviousdata, suggestthatthe block in gene expression in d1312- and hr-i-infectedHeLacells couldbeovercomeby inac-tivationof cellular geneproducts.

MATERIALS AND METHODS

Cellsandvirus. HeLacells were grown in suspen-sion atconcentrations of 3x105to 5x105cells per ml in Eagle spinner medium containing 7% calf serum. Wild-typeAd5 was grown and purified from infected cells aspreviously described (26). Ad5 mutants hr-1, hr-6 (10), and d1312 (12) were propagated in the

permissive AdS-transformed 293 cells (9). Both hr-1 andd1312 viruses had titers on HeLa cells104to105

times lower than the titers on 293 cells, whereas hr-6 had approximately 102 times lower titers on HeLa cells.

Infection andlabelingconditions.The same number of HeLa cells were infected with wild-type Ad5 and mutantshr-1 and hr-6 at amultiplicity of infection of 0.5 PFU per cell. Infection with d1312 was at a multiplicity of infection of 2.5 PFU per cell. After

labelingwith [15Simethionine, cell extracts from 107

cells wereimmunoprecipitated.

DNAfor filter hybridization studies was prepared from HeLa cells labeled with [3H]thymidine (10

,uCi/ml)from 16 to 20 hours postinfection.

Preparation of RNA and DNA. Cytoplasmic RNA was preparedfrom HeLa cells as previously described (24). DNA was prepared by treating infected HeLa cells with STEbuffer (0.1 M NaCl, 20 mM Tris [pH

7.6],10mMEDTA)containing 500 ,ugof predigested pronase perml and0.5% sodium dodecyl sulfate (SDS) at roomtemperatureovernight.The DNA was extract-ed with neutralizextract-edphenol-chloroformCl3(50:50) sev-eral times and subjected to RNase (50 ,ug/ml) treat-ment. After additional phenol-chloroform extractions and ethanol precipitation, the DNA was dissolved in TEbuffer (10 mM Tris [pH7.6]1mMEDTA).

Southernblotanalysisof viralDNA. A totalof 1 Fg of DNA was digested withEcoRI or HindIII, and the

fragmentswereseparated on a0.8%agarosegel. The

gelswerethenblotted ontonitrocellulose filters(29), which were subsequently hybridized toAdSDNA32p labeledbynick translation (28).

Filterhybridization.Equivalent amounts of

[3H]thy-midine-labeled DNA were alkali treated, neutralized, andhybridized for 16 h at650Cin 6xSSC (1x SSC is 0.15 MNaCl plus 0.015 M sodium citrate)-3x Den-hardtsolution-0.5%SDS tofilters containing 20 ,ug of

AdSDNAper ml. After hybridization the filters were washed extensively in 0.2x SSC at 50°C, and the amountof labeled DNA hybridized was determined by

liquidscintillationcounting.

Immunoprecipitation, SDS-polyacrylamide gel elec-trophoresis, and in vitro translation. The procedures for immunoprecipitation and translation in a rabbit reticulocyte cell-free system have been described pre-viously (21, 24). Virus-specific RNA was selected on

AdSDNAboundtonitrocellulose filters asdescribed byMcGrogan etal. (18). Samples were analyzed on 13%SDS-polyacrylamide slab gels as described previ-ously (21, 24). The gelswereanalyzed by fluorography (3).

Antisera. Antisera against the Ad2 hexon (polypep-tide II),pentonbase (polypeptideIII),andfiber (poly-peptide IV)wereprepared by the method of Persson et al.(24).Monospecific antisera against theE2/75Kand E3/19K proteins were prepared as previously de-scribed (17, 25). Normal sera were prepared from nonimmunized rabbits.

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RESULTS

Synthesis ofearly proteins in mutant-infected cells. HeLa cells infected with mutant or wild-typeviruses were treated withanisomycinfrom 1 hpostinfection. At 5 hpostinfection the drug was removed, and the cells were pulse-labeled

with[35S]methioninefor 30 min at 8 h

postinfec-tion. Infected cells not treated with the drug were analyzed inparallel. Immunoprecipitation of cell extracts from complementation groupII (hr-6)- orwild type-infected cells with monospe-cific antiseraagainsttheE2/75K and theE3/19K proteins showed that these proteins were syn-thesized both indrug-freeanddrug-treated cells

(Fig. 1). In complementation group I, infected

cells (hr-1 andd1312)theE2/75Kand theE3/19K

proteins were only detected in cells that were

treated with the drug. Similar results were ob-tained when immunoprecipitations were per-formed with antitumor sera; the E1B/58K and

ElB/15K as well as the E4/11K protein were

detectable only in hr-i-andd1312-infected cells treated withanisomycin (data not shown). This result shows that the complementation group I mutants behave differently from group II mu-tants and that early viral proteins can only be detected in AdS hr-i- and AdS d1312-infected

cells treated withanisomycin.

Synthesis oflate viralproteinsin mutant-infect-ed cells. To investigate whether a pulse

treat-ment with anisomycin ofmutant infected cells

also resulted in synthesisof late viral proteins,

cellsweretreatedwithanisomycinfrom1 to5 h

postinfection. The cells were labeled with

[35S]methionine

at 16 h postinfection, and cell extractswereimmunoprecipitatedwith antisera against the structural proteins hexon (II), pen-tonbase (III), and fiber (IV). Wild-type virus-infected cells showed comparable amounts of these late structural polypeptides irrespective

whether the drug was present or absent (Fig.

2D). An enhancement of the synthesis of these

proteinswasdetected in hr-6-infected cells

treat-edwith thedrug comparedwith that indrug-free

controls(Fig. 2C). Cells infected with the ElA mutants showed no synthesis ofany late pro-teins in the absence of thedrug(Fig. 2AandB), but treatment of cells with anisomycin during

theearly phase of virus infection allowed sub-stantial accumulation ofthehexon, pentonbase,

andfiberpolypeptides.

Analysis of viral proteins from mutant-infected cells treated with anisomycin before virus infec-tion. HeLacells were treated with anisomycin

for 5h, thedrugwasremoved bycareful wash-ing, and thecells were infectedwith AdS d1312

orwild-typevirus.At 6 or 16 hpostinfectionthe

cells were pulse-labeled with

[35S]methionine,

and the accumulation of early and late viral

proteins was analyzed by immunoprecipitation. The early proteins (E2/75K and E3/19K) could bedetected in wild type-infected cells both with and withoutdrug treatment (Fig. 3B). The drug treatment did however reduce the amount of early viralproteins synthesized. Large amounts of late viral proteins (hexon, pentonbase, and fiber) were detected in wild-type virus-infected cells both in the presence and absence of the drug. On the contrary, late proteins were only detected in AdS d1312-infected cells that had beenexposed to anisomycin before virus infec-tion (Fig. 3A). The same result was also ob-served withthe early E2/75K proteins, whereas the E3/19K protein was detected at a low level also in the absence of drug treatment. Under these same experimental conditions infection with theAd5 hr-1 mutant gave apattern similar tothatof the Ad5 dl312 virus (data not shown). Theseresults show that complementation group

I viruses do allow accumulation of both early

and latefunctional viral mRNAs if protein syn-thesis is inhibited before virus infection. Since the drug is not present during infection, these

data provide additional evidence that cellular

protein(s) plays a role in the control of viral gene expression.

Accumulation of functional late viral mRNAs in

AdSd1312-infectedcells.Tocorrelate the amount

of late viral protein synthesized in vivo to the amount of late viral mRNAs present in Ad5 d1312-infected cells treated withanisomycin be-fore virusinfection, cytoplasmic RNA was ex-tracted,selectedby hybridization to viral DNA, and translated in vitro. Several late viral pro-teins were detected among the translational products synthesized in response to RNA pre-pared from drug-treated cells, whereas RNA preparedfromdrug-freecells didnotsynthesize

late proteins (Fig. 4). The addition of the drug before infection of cells withwild-typevirusdid not change the amount of late protein synthe-sized in vitro compared with that in drug-free

controls.

Replication of viral DNA in mutant-infected cells. The substantialsynthesisoflateviral pro-teins in complementation group I-infected cells after treatment with anisomycin suggests that viral DNA replication occurs in these cells. DNA wasthereforeextracted at 19 h postinfec-tionfromuntreated cells and cells treated with anisomycinduringtheearlyphase of viral gene expression (1 to5 postinfection). Southern blot analysis of viral DNA digested with EcoRI showed comparable amounts of viral DNA in drug-free and drug-treated, wild type-infected

cells (Fig. 5A). The addition ofanisomycin to complementation group II (hr-6)-infected cells drastically reduced viralDNA replication com-pared with that of thedrug-free control. Onthe

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HR-1

B

c d

-E2/75K

-E3/19K

HR-6

b

c

d

40

-E2/75K

DL 312

a

b

c

d

-

-E2/75K

-E3/19K

D

AD5

a b c

d

_00

a

--E2/75K

o

-E43/19K

[image:4.490.78.412.77.563.2]

-E3/19K

FIG. 1. Synthesis ofadenovirus early proteinsin hostrangemutant-infectedHeLa cells. HeLa cellswere

treated withanisomycin (100 I1M)1 h aftermutantorwild-typeAdS infection. Thedrugwasremovedat5 h

postinfection, thecellswerewashed, andfresh mediumwasadded. At 8 hpostinfectionthe cellswere pulse-labeled with [35S]methionine (20 ,Ci/ml) for 30 min. In controls, virus infection was examined withoutthe

additionofdrug, butotherwise treated identically. Cellextracts wereprepared andimmunoprecipitatedwith eithernormal orAd2-specific antiserum. Theimmunoprecipitates wereanalyzed by SDS-polyacrylamidegel electrophoresisfollowedby fluorography. Lanes:aandb,nodrug added;candd,100,uM anisomycinadded.

Immunoprecipitationswereperformedwithnormal rabbitserum(lanesaandc)andamixture ofmonospecific antiseraprepared againstthe E2/75K and the E3/19Kproteins (lanesb andd).Hereand insubsequent figures,M denotesa W35S]methionine-labeledAd2 virusmarker.

A

a

b

C

M a

mJ-e

m -.

K--1X- *

0

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B DL31 2

L.) c

;a.

_-

r--oe ffl

D. A%ADS

a b am

_0AM

. 0

d

4 -ir

_-Im

_IV

d

am=-Hi

_-nI

--mV

FIG. 2. Synthesis of adenovirus late structural proteins in mutant-infected HeLa cells. HeLa cells were treated asdescribed in the legendtoFig. 1, except that the cells were pulse-labeledat16 hpostinfection for 30 minwith[35S]methionine. Lanes: aandb, no drug added;candd,100,uManisomycin present from 1to5h postinfection.Theimmunoprecipitations were performedwithnormal rabbit serum (lanesa andc)or a mixture ofmonospecific antisera prepared against the hexon(II),pentonbase (III), and fiber (IV) proteins (lanes b andd).

contrary, Ad5 hr-i-infected cells showed viral DNAreplication onlyin cells treated with aniso-mycin.However, the level of viral DNA replica-tion in AdS hr-i-infected, anisomycin-treated cells was substantially less than in wild type-infected cells. Thesameresultwasobtainedby

analyzing viral DNA replication in cells that

were maintained in the presence ofanisomycin before virus infection (Fig. SB). Replication of AdS hr-1 DNA was only observed in

drug-treatedcells,whereaswild-typevirusreplicated its DNA withorwithoutdrug treatment.

To quantitate viral DNA replication in the

Ad5hr-i-infectedcells treated withanisomycin, the cellswerelabeled with[3H]thymidinefor4 h

at 16 h postinfection, and the amount of viral

DNA synthesized was measured by filter

hy-bridization (Table 1). Cells treated with aniso-mycin before AdS hr-1 infection showed levels of viral DNA replication corresponding to

Awl, -1 -!..

114.ft U.r

i.IV

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A

M

a

nf-VrA-.

rIY

-

l1

W-

o

vu-4b

DL312

b

c

d

e

f

g

h

--

-

wE2/75K

j-E3/19

K

VII[-t

IXI-t

AD5

b

c

d

e

-40

-M

f9

h

-E

2/75

K

-E3/

19K

FIG. 3. Synthesis ofearlyand lateadenovirusproteins inHeLa cells treated withanisomycinbefore AdS

d1312andwild-type infection. HeLa cellsweretreated withanisomycin (100ILM)for5h,thedrugwasremoved,

and thecells wereinfectedwithAd5d1312(A)orAd5wild-typevirus(B). Ascontrols,thesameviruseswere

usedtoinfectuntreated cells(lanesathrough d)andcellstreated withanisomycin (lanesethrough h).Infected cellswerepulse-labeledat6 hpostinfectiontodetectearly protein (lanesa,b,e,andf)and 16 hpostinfectionto detectlateproteins (lanesc,d,g,andh).Cellextractswerepreparedandimmunoprecipitatedwithnormal rabbit sera(lanes a, c, e,andg),anantiserum mixtureagainstthe E2/75K and E3/19Kproteins (lanesbandf)or an antiserummixture againstthe hexon, pentonbase, and fiber(lanes d andh). The immunoprecipitations were analyzed by SDS-polyacrylamide gel electrophoresisfollowedby fluorography.

around 10% of the value obtained from wild type-infected cells pretreated with the drug. As

expected from the Southern blot analysis, no

detectable viral DNAreplication was observed

inuntreated, Ad5 hr-i-infected cells.

Virusproductioninmutant-infectedcells. The

synthesis of large amounts of structural viral

proteins and the replication of viral DNA in

complementation group I-infected cells treated

with anisomycin infer that drugtreatment may

also support virus production innonpermissive HeLa cells. Mutant-infected cell extracts

pre-B

-

vm--M

a

A.

VIE-.

W-

a

'[C-

^b

f

l-b

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M

I.I.-l

F11-a

-I.

fv

X- _

WIX

AD5 DL312 Z

a b a b

I 0 m_ l_

AI"

.al

_=

*

-FIG. 4. In vitrotranslation with hybridization-se-lectedRNApreparedfrom AdS d1312- and Ad5-infect-ed HeLa cells.CytoplasmicRNAwaspreparedat16 h postinfectionfrom HeLa cells infected withwild-type Ad5ord1312virus.Cellsweretreated withanisomycin

(100 ,uM)for5h before infection(lanes a)oruntreated (lanes b). The RNAwashybridizedtofilters contain-ingAd5 DNA, and the elutedRNAwastranslated ina

reticulocyte cell-free system. The translational

prod-ucts wereanalyzed by SDS-polyacrylamide gel

elec-trophoresis followed by fluorography. -RNA, No RNA addedtothe cell-freesystem.

pared from HeLa cells 48 h postinfection were

therefore titrated both on permissive 293 cells

and nonpermissive HeLacells. Wild-type virus analyzedas acontrol showedathree- to fourfold

reduction in virus production when the cells

were pretreated with anisomycin for 5 h before

infection compared with that ofdrug-free

con-trols (Table 2). Both the Ad5 hr-1 and the AdS d1312 viruses showed anenhanced titer on 293

cells when prepared from HeLa cells treated with anisomycin before infection. The enhanc-ing effect of drugtreatment onvirusproduction

was more pronounced for AdS d1312 than for

[image:7.490.75.222.59.277.2]

AdS hr-1 virus and in the formercaseresulted in

TABLE 1. Effect of anisomycin onDNA replication in wild type- and mutant-infected HeLa cells

Virus DNA hybridized

Wild-typeAd5 98,025

Wild-type Ad5 with 20,487

anisomycinb

hr-1 Nondetectablec

hr-I withanisomycinb 1,987

aEquivalent weight amounts of DNA were hybrid-ized to filters containing AdS DNA as described in the text. Background values obtained from hybridization to filters without DNA have been subtracted from

each value.

bHeLa cells were maintained inthe presence of 100 FM anisomycin for 5 h beforeinfection.

cValues above background levels could not be observed.

a 500-fold increase in virus production. Both mutantviruses obtained afterdrugtreatmentdid notyield virus foci onHeLacells, thusproving that the mutant phenotype of the virus was replicatedafterdrugtreatment.

DISCUSSION

Studies with AdS host range mutants d1312 and hr-1 have demonstrated that an important controlin adenovirusgeneexpression resides in early region ElA of the viral genome. These mutants fail to accumulate early viral mRNA after infection of HeLa cells. The mutation in Ad5 hr-1 virus was recently defined by DNA sequence analysis and appears to effect the translation of the ElA/13S mRNA encoding a polypeptide of 51,000 daltons(27).Inadditionto the13S mRNA, early region 1A encodes a12S mRNA (2, 6). Site-specific mutagenesis that effects only the ElA/12SmRNAdoesnot reduce theaccumulation of viralmRNAfrom the other early regions (19), indicating that the ElA/13S mRNA is critical in the control of viral gene expression. However, additional transcriptsand virus-coded protein(s) coded by the leftward reading strand of the transforming region of the adenovirusgenome have recentlybeen detected

TABLE 2. Effect ofanisomycinoninfectious virusproduction in wild type-andmutant-infected HeLa cells

Fluorescentfocus-formingunitspermla

Virus 293 cells HeLa cells

-Anisomycin +Anisomycin -Anisomycin +Anisomycin

Wild-typeAd5 3.0 x 109 8.6 x 10" 5.8 x 108 2.0 x 108

hr-1 7.6 x 106 4.2 x 107 <1.0 x i05 <1.0 x 105

d1312 2.5 x i05 1.6 x 108 <1.0 x 105 <1.0 x 105

aInfectious viruswasharvested fromHeLacells maintained in the absenceorpresence of 100 ,uManisomycin for5hbefore infection. The virussamples werethen titratedonmonolayers of either 293orHeLacells.

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CONTROL OF ADENOVIRUS GENE EXPRESSION

AD5

HR-6

Hi

a

b

a

b

a

A;

40

4d

Br

44

C__

B

R-1

b

AD5

a

b

HR-1

a

b

A-B-h C-4

F-

H-FIG. 5. Effect of anisomycin treatment on DNA replication in mutant-infected HeLa cells. DNA was

extracted fromAd5 hr-i-, hr-6-, and wild type-infected HeLacellsasdescribed in thetext. Afterrestriction

enzymedigestion, the DNAwaselectrophoresedon a0.8%agarosegel,blottedontoanitrocellulosefilter,and subsequently hybridizedtonick-translated, 32P-labeled Ad5DNA.(A)Infected HeLa cellsweretreatedwith 100 ,M anisomycin from 1 to 5 h postinfection. The drugwasremoved, and fresh mediumwas added. At 16 h

postinfectionthe cellswereharvested,andthe DNAwasextracted.Lanes:a, cellstreated withanisomycin; b, drug-free controls. TheDNAwasrestricted withEcoRI, and the lettersontheleftrefertothe EcoRIrestriction fragments. (B) HeLacells were treated withanisomycinfor 5 h,thedrugwasremoved, and thecells were

infected with AdS hr-1 and wild-type virus. Cellswereharvested 16hpostinfection, and the DNAwasextracted. Lanes:a, cellstreated with 100,uM anisomycin; b, drug-freecontrols. The DNAwasrestricted withHindIII,and the lettersonthe left refertothe restrictionfragments.

(14). Therefore, the exact identification of the

virus-encoded products responsible for gene

regulation requires further analysis.

Studies with inhibitors of protein synthesis have provided information on the possible mechanisms ofgene controlthat reside inearly region 1A. If the regulatory element encoded in

ElA is a protein, the addition of inhibitors of

protein synthesis shortly before infection should effect the accumulation of early viral mRNA.

Studies fromourlaboratory and others(13, 16, 22, 23) have tested this hypothesis, and the accumulation of viral mRNAwasreduced under these conditions. However, the addition ofthe

drugat1 hpostinfectionorlater has theopposite effect, i.e., early viral mRNA accumulation is

enhanced(5, 23).Theseresultswereinterpreted

to mean that the ElA regulatory protein is

formedduringthe firsthourofadenovirus

infec-tion, consistent with the early appearance of

viral transcripts from this region. We further postulated that the viral regulatory protein does

not control early gene expression directly, but

does soby inactivating acellulargene product that normally decreases the accumulation of early viral mRNA (13, 23). Results consistent with this modelwerealso presented by Nevins

(20). The complementation group I mutants

whicharedefective in ElAgene products have

provided another modelto testourhypothesis.

We recently showed that the defect in early mRNAaccumulation in AdShr-i-infected HeLa cells could beovercome bytreatmentof HeLa cells with anisomycin (13). Thus, although the

group I mutants lack the ElA regulatory ele-ment, inactivation of the cellulargene product

by drugtreatment allowedviralearly mRNAto

accumulate to high levels. Additional evidence forourmodel is provided inthe presentstudy.

Itwas first established that theearly mRNA

A

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accumulating in drug-treated, hr-i-infected HeLacells was translatable invivo, since signif-icant amounts of early viral proteins were de-tectable afterreversion of the anisomycin treat-ment. Complementation group II mutants such as hr-6with a lesion in earlyregionE1B (8, 10) have been shown to accumulate early viral mRNA to thesame extent aswild-type virus (1). In addition, hr-6 has been showntosynthesize

viral DNA and late proteins in infected HeLa cells (15). Our in vivo studies have confirmed theseobservations since Ad5hr-6,likewild-type

virus, expressed early protein irrespective of

drug treatment of HeLa cells (Fig. 1). Thus,

additional evidence of an ElA location for a viral regulatory elementisprovidedby the stud-ies withAdS hr-6.

Our modelsuggestedthatthenegative

regula-tionofviral gene expressionis provided bythe cell. Toobtain further evidenceforthis hypothe-sis and to rule out thepossibilitythatanisomycin

treatment allowed mRNA accumulation by in-hibiting synthesis of a viral protein servingasa negative regulator, anisomycin wasadded for 5

hbeforeviralinfection. Thedrugwasremoved,

and thecellswere infected withmutant or wild-type virus. The viral products were then ana-lyzed at bothearly and late times afterinfection

(Fig. 3). Bothearly and late proteinswere

syn-thesized in AdS d1312-infected cells pretreated

with the drug, whereas little synthesis was ob-served withoutanisomycin. These datasuggest

that expression and accumulation of mutant

mRNA aredependenton inhibition ofacellular protein since the drugwas removed before mu-tantvirus infection.

Thesubstantialincrease in late mRNA

detect-ed in vitro and late protein synthesizedin vivo

suggestedthatreplication ofmutant viral DNA

had occurred in cells pretreated with anisomy-cin. Southern blotanalysisshowed that

replica-tion of mutant viral DNA in drug-treated cells

did occur, whereas no viral DNA replication

was observed in drug-free controls. The same results were obtained irrespective of whether anisomycin was present before or during the early stage of viral infection(Fig. 5). However, therate ofAd5 hr-1 DNAreplicationwas only

around 10% ofthe wild-type level (Fig. 5 and Table 1). This suggeststhatonlyaminor fraction of viral DNA serves as template for late tran-scription. If thisexplanationiscorrect,the10%

level of viralDNAreplicationobserved in

drug-treated, mutant-infected cells maybeenoughto provide the number oftemplates necessary to accumulate high levels of late viral mRNAs. However,wecannot rule out thepossibilitythat

lateexpressionandDNAreplication are

uncou-pledeventsand that latetranscriptionmayoccur withoutDNAreplication, althoughevidencehas

been presented against this hypothesis (30). A recently constructed mutant in early region 1A

effects only the EiA 13S mRNA, but it is defective in viral DNA replication even though viral RNA accumulates from all early regions (4). The direct involvement of the ElA/13S mRNA in viral DNAreplication may explain our finding that drug treatment of mutant-infected cells only yielded around10%of DNA synthesis comparedwith wild type-infected cells.

The datapresented here are consistent with the involvement of a cellular protein in the control of early adenovirus gene expression.

Studies on theinteraction between cellular and

earlyviralproteinsand theinduction of cellular

proteins in virus-infectedcells should be helpful in further elucidating control mechanisms for gene expression in mammalian cells.

ACKNOWLEDGMENTS We thank Marianne Remy for secretarial help.

This work was supported by grants from the Swedish Societyagainst Cancer and the Swedish Medical Research Council. M.G.K.was supported by a long-term postdoctoral fellowshipfrom the European Molecular Biology Organisa-tion.

LITERATURECITED

1. Berk, A. J., F. Lee, T.Harrison,J.Willams,and P. A. Sharp. 1979. Pre-earlyadenovirus 5geneproduct regu-lates synthesis of early viral messenger RNAs. Cell 17:935-944.

2. Berk, A. J., and P. A. Sharp. 1977. Structure ofthe adenovirus 2 early mRNAs.Cell 14:695-711.

3. Bonner,W.M., and R.A.Laskey.1974.Afilmdetection method for tritium labelledproteinand nucleicacids in polyacrylamide gels.Eur. J.Biochem.46:83-88. 4. Carlock, L.R., and N. C. Jones. 1981. Transformation

defectivemutantof adenovirustype 5containingasingle alteredEla mRNAspecies.J.Virol. 40:657-664. 5.Eggerding, F., and H. J. Raskas. 1978. Effect of protein

synthesis inhibitorsonviralmRNAssynthesized early in adenovirus type 2infection.J. Virol. 25:453-458. 6.Esche, H., M. B. Mathews, and J. B. Lewis.1980.Proteins

andmessengerRNAsofthetransforming region of wild-typeandmutantadenoviruses.J. Mol.Biol.142:399-417. 7. Frost, E., and J. Williams.1978. Mapping temperature-sensitive andhost-rangemutations of adenovirus type 5 bymarker rescue.Virology 91:39-50.

8.Galos, R. S., J.Williams,T.Shenk, and N. Jones. 1980. Physicallocation ofhost-rangemutations of adenovirus type 5; deletion and marker rescue mapping. Virology 104:510-513.

9. Graham, F. L., J. Smiley, W. C.Russeil,and R.Nairu. 1977.Characteristics of a human cell line transformed by DNAfrom human adenovirus type 5. J. Gen. Virol. 36:59-72.

10.Harrison, T., F. Graham, and J.Williams. 1977. Host-rangemutantsof adenovirus type 5 defective forgrowth in HeLa cells.Virology 77:319-329.

11.Jones, N., and T. Shenk. 1979. An adenovirus type 5early gene function regulates expression of otherearly viral genes. Proc. Natl. Acad. Sci. U.S.A.76:3665-3669. 12.Jones, N., and T. Shenk. 1979. Isolationof adenovirus

type 5host-range deletionmutantsdefective for transfor-mation ofratembryocells. Cell17:683-689.

13. Katze, M. G., H. Persson, and L. Philipson. 1981. Control ofadenovirus gene expression: posttranscriptional con-trol mediatedby both viral and cellular gene products. Mol.Cell.Biol. 1:807-813.

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http://jvi.asm.org/

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CONTROL OF ADENOVIRUS GENE EXPRESSION 59

14. Katze, M. G., H.Person, and L. Phllipson. 1982. A novel mRNA and a low molecular weightpolypeptide encoded inthetransforming region ofadenovirus DNA. EMBO J. 1:783-789.

15. Lassam, N.J., S. T. Bayley, and F. L. Graham. 1978. Synthesis of DNA, late polypeptides, and infectious virus byhost-rangemutants ofadenovirus S in nonpermissive cells.Virology 87:463-467.

16. Lewis, J. B., and M. B. Mathews. 1980. Control of adenovirusearly gene expression, a class of immediate early products.Ccli21:303-313.

17. Linn,T., H.Jornvall,and L.Pbllipson.1977. Purification andcharacterization of the phosphorylated DNA-binding protein from adenovirus type 2 infected cells. Eur. J. Biochem. 76:481-491.

18. McGrogan,M., D. J. Spector, C. J. Goldenberg, D. Ha-bert, and H. J. Raskas. 1979. Purification of specific adenovirus 2 RNAs by preparative hybridization and selective thermal elution. Nucleic Acids Res. 6:593-607. 19. Moutell,C., E. F.FYsher,M. H.Caruther,and A. J. Berk. 1982. Resolvingthe functions ofoverlapping viral genes bysite-specificmutagenesis at amRNA splice site. Na-ture(London)295:380-384.

20. Nevins, J. R. 1981. Mechanismofaction of early viral transcriptionby the adenovirusEIA gene product. Cell 26:213-220.

21. Persson, H., M. Jansson, and L.Philipson. 1980. Synthesis andgenomicsite for an adenovirus type 2 early glycopro-tein. J.Mol.Biol. 136:375-394.

22. Persson, H., M. G. Katze, and L.Phllipson.1981.Control ofadenovirusearlygeneexpression: accumulation of viral mRNA after infection of transformed cells. J. Virol.

40:358-366.

23. Persson, H., H.-J. Monstein, G.Aku4Arvl,andL. Philip-son.1981. Adenovirusearly gene products may control viral mRNAaccumulation and translation in vivo. Cell 23:485-496.

24. Persson, H., U. Petterson, and M. B. Mathews. 1978. Synthesis ofastructural adenovirus polypeptide in the absence of viral DNA replication.Virology 90:67-79. 25. Person,H., C.Slgnis,and L.Phlipson.1979. Purification

and characterization of an earlyglycoprotein from adeno-virus type 2infected cells. J. Virol. 29:938-948. 26. P on, V., C.Tlbbetts, and L. Phlipson. 1976.

Hy-bridization maps of early and late mRNA sequences on the adenovirus type 2 genome. J. Mol. Biol. 101:479-502. 27. Rkciardl,R.P., R. L. Jones, C. L. Cepko, P. A. Sharp, and B. E. Roberts.1981. Expressionofearlyadenovirus genesrequires a viral encoded acidic polypeptide. Proc. Natl.Acad. Sci. U.S.A. 78:6121-6125.

28. Rigby, P. W., M.Dkinana, C. Rhodes, and P. Berg. 1977. Labelling deoxyribonucleic acid to high specific activity in vitro by nick translation with DNApolymerase I.J.Mol. Biol.113:237-251.

29. Southern, E. M. 1975. Detection ofspecific sequences among DNAfragmentsseparated by gel electrophoresis. J.Mol. Biol. 98:503-517.

30. Thomas, G. P., and M. B. Mathews. 1980. DNA replica-tionand theearly to latetrnsitioninadenovirus infec-tion.Cell22:523-533.

31. WHson, M. C., J. R. Nevlns, J. M. Blanchard, H. S. Ginsberg, andJ.E. Darnell. 1979. The metabolism of mRNA fromthe transforming regionof adenovirustype 2. ColdSpringHarborSymp. Quant.Biol.44:447-455.

VOL.46,1983

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Figure

FIG. 1.antiseradenotesadditioneitherImmunoprecipitationslabeledtreatedelectrophoresispostinfection, Synthesis of adenovirus early proteins in host range mutant-infected HeLa cells
FIG. 2.oftreatedpostinfection.min monospecific Synthesis of adenovirus late structural proteins in mutant-infected HeLa cells
FIG. 3.detectcellsd1312antiserumanalyzedusedandsera Synthesis of early and late adenovirus proteins in HeLa cells treated with anisomycin before AdS and wild-type infection
FIG. 4.lectededpostinfectionAd5(100(lanesreticulocyteinguctstrophoresisRNA In vitro translation with hybridization-se- RNA prepared from AdS d1312- and Ad5-infect- HeLa cells
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