Control of adenovirus early gene expression: accumulation of viral mRNA after infection of transformed cells.

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Control of Adenovirus Early Gene Expression:




mRNA After Infection of




Departmentof Microbiology, The Biomedical Center, S- 751 23 Uppsala, Sweden

Received7April1981/Accepted18 June1981

We studiedthe accumulation of viral mRNA in the presence of inhibitors of

proteinsynthesisin anadenovirus type 5-transformed cell line (line293cells). An


of theendogenousviral mRNA's andproteinsrevealed thatonly early (gions1Aand 1Bwereexpressed in uninfected 293 cells. However, viral mRNA's from early regions 2, 3, and 4, as well as mRNA's from early regions 1A and 1B, accumulatedin293cellsafter infection with adenovirus type 2. Cells treated with

anisomycinbefo-.-e infection showed a drastic enhancement of mRNA from early

region 4 compared with drug-free controls. This increase in viral mRNA was

detected by using filter hybridization, Si endonuclease mapping, and in vitro

translation. The rate of transcription of early region 4 nuclear RNA also increased significantly inthe presence of anisomycin. In contrast to the results with early

region 4, the levelsofcytoplasmic mRNA's from early regions 2 and 3 did not

increase in cells treated with inhibitors. These results suggested that multiple

virusencoded controlsoperate ontheearly regions of the adenovirus genome.

Early after adenovirus infection, before the

onset of DNA replication, cytoplasmic RNA is

derived from fiveregionsof the viral genome (3,

24). Since the early proteins encoded in these

regions are expressed at different rates and at

different times after infection, it has been sug-gested that there is a temporal regulation of expressionfrom the viral genome (23, 26).

Nev-ins etal. (19) have shown that the time course

for the appearance oftranscripts isdifferent for

each of the fivegenomic regions.

Studies with adenovirus host range and dele-tion mutants have demonstrated that a major

element ofgenetic control lies within early

re-gion IA (2, 14). In addition, theaccumulation of

cytoplasmic mRNA species encoded by early

regions 1A and lB appears tobe controlled by

complexregulatory patterns (29).Recently,the

control of adenovirus geneexpressionin infected

HeLacellswas examined by using inhibitorsof

protein synthesis from thestartofinfection (16,

22). Theresultswith the mutantsand the

inhib-itors ledtothehypothesisthatviral gene

prod-uctsencodedby earlyregion 1may control the

accumulation of viral mRNA since decreased

levels ofvirus-specific mRNA wereobservedin

cells treatedwith inhibitors before virus

infec-tion (22).

Early region 1 is also of interestsince it

con-tainsthe DNAsequences necessary for

transfor-mation (9,30). Grahametal.(12) have described

ahumanembryonickidney cell line that is

trans-formedby shearedfragmentsof adenovirus type

5 (Ad5) DNA. Thesecells,


293 cells, contain the left 12% and


9% of the viral

genome (1). Thetransformedcells expressboth

RNAs and viral proteinsfromearly



and 1B. Accordingly, infection of293cellswith

mutantsdefectiveinearly region 1A results ina

productiveinfection(2, 14).

Inthisstudy,weanalyzedthe gene


ofwild-type Ad2 in 293 cells to determine the effects of early


1A and 1B



viral RNA accumulation. Using inhibitors of

proteinsynthesis, wefound that

early region


gene products




and accumulation of


viral mRNA from


E4.Our results




regionsofthe viral genome may beunder

sepa-rate controls.

MATERIALS AND METHODS Cells and virus. TheAd5-transformedhuman

em-bryonic kidneycell line293 wasgrown inmonolayers

in Eagle minimal essential medium containing 10% fetal calf serum (12). Early-passage, untransformed

human embryonic kidneycells were purchasedfrom Flow Laboratories andwere propagated inthesame way as the 293 cells. Wild-type Ad2 and Ad5 were

purifiedfrominfected HeLa cellsas described previ-ously (24).

Labelingconditionsand virusinfection. Unin-fected293cellswerelabeled with[35S]methionine(10 1LCi/ml)for 6h inmediumcontaining 1/40 the normal

concentration ofmethionine. After thecellswere la-beled for 3 h with[3H]uridine(50



RNAwas prepared fromuninfected 293 cellsbythe 358

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methodof Brawermannetal.(5). Both 293 cells and untransformed human embryonic kidney cells were

infected with purified Ad2 at a multiplicity of 100

PFU/cell. Cytoplasmic RNAwas prepared from in-fected cells thatwerelabeled from2 to 5h

postinfec-tion with[3H]uridine (50uCi/ml).Nuclear RNAwas

extracted by the method of Smith et al. (27) from infected293 cells labeled for5minwith[3H]uridine (500


at 5hpostinfection.


sulfate-polyacrylamidegelelectrophoresis,and invitro

translation. Theproceduresused for

immunoprecip-itation and translation inarabbitreticulocytecell-free system have been described previously (23). Virus-specific RNAwasselectedonAd2 DNA orpurified

restriction enzymefragments (24) boundto nitrocel-lulosefilters, as described by McGrogan et al. (18).

Eluted RNAwastranslated in themRNA-dependent

reticulocyte cell-free system. Sampleswere analyzed on 13% sodium dodecyl sulfate-polyacrylamide slab gels as described previously (21, 23). The gelswere

analyzed byfluorography(4).

SiendonucleasemappingofmRNA. We used the method originally described byBerk andSharp

(3) and modifiedbyPerssonetal. (22).Briefly,20tig


con-tainingahighconcentration offormamide(6)to1 ug

of Ad2 DNA. Eachsamplewastreated withS1 endo-nuclease (225 U) and separated on a 2.0% alkaline agarosegel.Thegelswereblottedontonitrocellulose sheets (28) andhybridized to3P-labeled nick-trans-lated probes (25).

Filterhybridization. Equalamountsof RNA from thedifferentsampleswerehybridizedfor16hat650C tonitrocellulosefilterscontainingEcoRIfragmentsof Ad2 derivedfrom 10lOgof viral DNA. After

hybridi-zation, the filterswerewashed extensively and then treated with RNase A (20 ug/ml). The amounts of hybridized RNAweremeasured withaBeckman



Viral mRNA and


expressed in


293cells. Each human embryonic kidney cell line (293 cells) transformed with shearedAd5 DNA contains fourtofive copies of the region which includes the leftmost 12% of the Ad5 genome and one copy of the region which includes the rightmost 9% of the viral

genome(1).Weanalyzed the viral proteins

syn-thesized in thiscell line by





anti-Tsera. The anti-Tseraused wereprepared

against either








These antisera

have been shown to react with avariety of early

proteins encodedinearlyregions 1A and 1B (11, 15). Although we observed nonspecific immu-noprecipitation,alloftheantiserawhich we used immunoprecipitateda58,000-daltonprotein and

a setofproteinswithmolecular weightsof


42,000 to 50,000. A 15,000-dalton

proteinwasalso immunoprecipitated by two of

theanti-Tsera (Fig. 1A). Separation of the293

cells into a cytoplasmicfraction and a nuclear

fraction showed that the 15,000-dalton protein predominated in the cytoplasm, whereas the 58,000-dalton proteinwas mostly confined to the nuclear fraction. The 42,000- to 50,000-dalton proteins were equally distributed between the cytoplasm and the nuclear fraction. None of these proteins was immunoprecipitated from [3S]methionine-labeled untransformed human

embryonic kidney cells (datanotshown).

The viral mRNA present in 293 cells was analyzedby using hybridization selectionof 293 cell cytoplasmic RNAtoAd5DNA,followedby in vitro translation. The selectedRNA directed the synthesis in vitro of thesame set of proteins (molecularweights,58,000, 42,000 to 50,000,and

15,000)that wereobserved afterlabelingin vivo.

In someexperiments a 17,000-dalton protein was

alsosynthesized from selectedviralmRNA(Fig.

1B). The cytoplasmic RNA from 293cells was also selectedbyhybridizationtoDNAfragments specific for early regions 1A or 1B. The early

region 1A selected viral mRNAsynthesizedfour



weights, 50,000, 48,000,

44,000, and 42,000), whereastwo proteins

(mo-lecular weights, 58,000 and 15,000)were synthe-sized when


region mRNA wastranslated invitro (Fig. 1B). Theseproteins corresponded in size and map location to the early proteins


identified in


infected HeLa cells from


regions 1A and 1B of Ad5DNA (7,17). Figure 1B also shows thatnofunctional viral mRNA from early region 4 of the viral

genome was detected, indicating that the viral

DNAfrom therightmost end of the genomeis not transcribed into mature mRNA. A more sensitiveassayfor thepresence of

early region

4 mRNA, which involved


of la-beled viral RNAtoa


DNAprobe, con-firmed that

early region

4 was not



293cells.Expression ofearly region4mRNA in


presence ofthe proteinsynthesis inhibitor

ani-somycin (see


Infection of293cells withAd2. The exper-iments described above showed that human em-bryonic




Ad5 DNAonly expressedfunctionalviral mRNA and proteinsfromearly regions1A and 1B. In addi-tion,these geneproductsappearedtobe identi-caltothe products synthesized duringlytic in-fectionwithAd5inhumancells(7). Studieswith

host range mutantshave suggested that early

region1A of theviralgenomeplaysanimportant

role in the accumulation of functional viral

mRNA from other early regions (2, 14). The

addition of


astringentinhibitor of

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r-.-4, -


NJ-- j

-... -ta-.

o .







El b,/ 58K_ z



b/s17K-E 1 b / l 5 K vM



FIG. 1. (A)Immunoprecipitation of[3S],methionine-labeled 293 cellextracts with different anti-Tsera.

Monolayers of293 cells were labeled with [3S]methionine for6 h, and cellextracts wereprepared by

disrupting the cells in RIPA buffer (0.02 Mpotassium phosphate, pH 7.5, 0.1 M NaCl, 0.5% sodium

deoxycholate, 1% Triton X-100, 0.1% sodium dodecyl sulfate, 1 mMphenylmethylsulfonyl fluoride). The antisera usedforimmunoprecipitationareindicatedatthe top,asfollows: normal, preimmuneserumfrom

hamsters; 14b,serumfromhamstersbearingtumorsinducedbytheAd5-transformedcell line14b;Fl7,serum

from ratsinjectedwith theAd2-transformed ratcell line F17; 297, serumfrom hamsters bearingtumors inducedbytheAd5-transformedcell line297-C43; A2T2C4,serumfromratsinjectedwith theAd2-transformed

ratcell lineA2T2C4.Cellline14bcells containtheleft40%oftheAd5genomeand transcribeearly region1

(8).The 297-C43cellline containssequencesfrom0to23mapunits,aswellassequencesfrom32to52map units(31).The F17 cell line containsonlytheleft14%ofthe Ad2genome,whereastheA2T2C4cell line contains

sequencesfromallearlyviralregions (9). Theimmunoprecipitateswereanalyzedon a13% sodiumdodecyl sulfate-polyacrylamide gel, andthegelwasanalyzed byfluorography.Inthis andsubsequent figures,Ad2 denotesa[fSlmethionine-labeledAd2marker virus. (BandC) CytoplasmicRNA waspreparedfrom293 cells and selected by hybridization tofilterscontainingAd5 DNA orrestriction enzymefragments ofAd2 DNA.The selected RNAwastranslated invitro,andthetranslationalproductswereanalyzedon13% sodium

dodecyl sulfate-polyacrylamide gels. TheDNAprobesusedfor hybridizationselectionwere asfollows:lanes

aandb,EcoRIfragmentBofAd5 DNA(84.0to100.0mapunits);lanec,total viral DNAfromAd5. TheRNA used for hybridization selection in lane b wasprepared from 293 cells maintained in thepresence of

anisomycin (100 pM) for5h.Lane-RNA,noRNAaddedtothecell-freesystem, laned,SmaIfragmentJof

Ad2 DNA(0to2.8mapunits); lanee,HindIIIfragmentCofAd2 DNA(7.9to17.0mapunits).


z -c Ca.



L... do







-48K l 42 K |

on November 10, 2019 by guest



protein synthesis, before viral infection in HeLa

cells depresses the accumulation of early viral

mRNA (16, 22). These observations suggested

that aviral protein is involved in the

accumu-lation ofearly viral mRNA. Since 293 cells

ex-pressed early region 1A and 1B proteins, we

infected these cells with Ad2 in the absence and inthepresence ofanisomycin and analyzed the

accumulation of early viral mRNA. The early

region 1A and 1B proteins should have been

present in 293cells even after anisomycin was

added,provided that theywerestable, since they

weresynthesized before the addition of the drug.

To study the stability of these proteins,

[35S]-methionine-labeled 293 cellswere treatedwith

anisomycin for 2 h. Cellextractswereprepared

before and after thistreatment.

Immunoprecip-itationwith anti-Tserarevealednosubstantial

reduction in theamountsof theearly region 1A

42,000- to 50,000-dalton proteins during drug

treatment(datanotshown). This suggested that

the early region 1A proteinswere stable inthe

293 cellsforatleast2h.Therefore, if theearly

region 1Aproteinsareinvolved in the

accumu-lation ofearly viral mRNA, the addition of

ani-somycin before viral infection shouldnotdepress

theaccumulation ofearly viral RNA.

Cytoplasmic RNAwasprepared from 293cells

infected with Ad2 in the absence and in the

presence ofanisomycin. The RNAwas

hybrid-ized to nitrocellulose filters containing DNA

fragmentsspecificfor the differentearly regions.

Table 1 shows that addition ofanisomycin 30

min before viral infection did not depress the

accumulation of early viral RNA from early

region 2, 3, or 4. Instead, filter hybridization

showed thatthe amount of viralRNA fromearly

region2wasincreased about 2-fold and thatthe

amount ofviral RNA from early region 4 was

increasedatleast 10-foldby the drugtreatment.

Identical resultswere obtained in several

inde-pendent experiments. The same results were

obtained whenweusedpuromycin, another

po-tent inhibitor of protein synthesis (data not

shown). To obtain further evidence that the

enhanced accumulation ofearly region 4 RNA

was controlled by viral proteins, cytoplasmic

RNAwasprepared from Ad2-infected

untrans-formed humanembryonic kidneycells.The

ad-dition of anisomycin before viral infection in

untransformed human embryonic kidney cells

did causea slightdecrease inthe accumulation

ofviral RNAs from early regions 2, 3, and 4

(Table 2),as previously demonstrated in HeLa

cells (22).

Rateoftranscription.The enhanced

accu-mulation of viralearly region4RNAcould have

been at thelevel oftranscription or at a

post-transcriptional level. To distinguish between

TABLE 1. Effect of anisomycin on the accumulation of cytoplasmic early viral RNA in Ad2-infected 293


Amt of RNAhybridized (cpm)"

Prepn Early Early Early

region region region 4

2 3

Ad2 459 1,325 652

Anisomycin-treatedAd2b 981 1,220 10,466

aCytoplasmic RNAwasprepared5 hafter infection

with Ad2from293cells maintained in the presence or

absence of anisomycin. The cells were labeled with

[3H]uridine (50 1iCi/ml) from 2 to 5 h postinfection. EquivalentamountsofearlyviralRNA were

hybrid-izedto filterscontaining viral DNA fragments from

different early regions. The Ad2 DNA probes used were asfollows:early region2,EcoRI-B (58.5 to 70.7 mapunits); early region3,EcoRI-D (75.9 to84.0 map

units); early region 4, EcoRI-C (89.7 to 100.0 map

units). Background radioactivity (approximately 40 cpm, asdeterminedby using filterswithout DNA) was

subtracted fromeachvalue.

bAd2-infected293cellswere maintained in the



anisomycin(PfizerLaboratories) from

30minbefore infection until5hafterinfection.

TABLE 2. Effect of anisomycin on the accumulation of cytoplasmicearlyviral RNA inAd2-infected

humanembryonic kidney cells

AmtofRNAhybridized (cpm)a Prepn Early Early Early

region region region

2 3 4

Ad2 166 205 371

Anisomycin-treatedAd2b 105 92 240

aCytoplasmic RNA was prepared from

Ad2-in-fected human embryonic kidney cells labeled with

[3H]uridine (50


from 2 to 5h postinfection.

The RNAwashybridizedtoAd2DNAprobes

repre-senting early regions2, 3,and4;theDNAprobesused weretheonesdescribedinTable1,footnotea.

Back-ground radioactivity (40cpm as determinedbyusing

filters withoutDNA)wassubstractedfrom each value.

bThe cells were treated with 100,LM anisomycin

from30minbeforeinfection until5hafter infection.

these two


293 cells infected with

Ad2 in the absence and in the presence of




5min at 5 h postinfection. The nuclear RNAs

were extracted and


to filter-bound

Ad2 DNAfragments





transcription units. No increase in the rate of transcriptionwasobserved forearly region2 or

3 in thepresence of the drug


3); indeed,

a twofold decrease was


which may

have been duetothe



drug. However, the rate of


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TABLE 3. Rateof transcription in Ad2-infected 293 cells

Amtof nuclear RNA hy-bridized (cpm)a Prepn Early Early Early

region region region

2 3 4

Ad2 728 727 221

Anisomycin-treated Ad2b 362 385 1,119

aAd2-infected 293 cellswerepulse-labeled for5min

with 500,tCiof[3H]uridinepermlat 5hpostinfection. Equivalent amounts of nuclear RNA were hybridized toDNAfiltersasdescribed in thetext.The Ad2 DNA probes used were theonesdescribed in Table 1, foot-note a. Background radioactivity (approximately 40 cpm,asdetenninedby using filters without DNA)was

substracted from each value.

b Ad2-infected293cellsweremaintained in the

pres-enceof100,uManisomycin from30minbefore infec-tion until5hafter infection.

early region4was significantlyincreased ifthe

cells weretreatedwithanisomycin30minbefore

viral infection. Therefore, the enhanced

accu-mulation ofearly region 4RNAmayhave been

caused atleast in part by an increased rate of transcription.

Processing of RNA. The structure of the


region 4 RNA which accumulated in 293 cells infected with Ad2 was studied by the S1 endonucleasetechnique (3).CytoplasmicRNAs prepared from293cells infectedwith Ad2 in the absence and in thepresenceof anisomycinwere hybridized toAd2DNA, treated with


endo-nuclease, separatedon


agarosegels, and blotted onto nitrocellulose sheets. The filters werethen hybridizedto a


restriction fragment representing early region4. Figure 2B shows that addition of anisomycin30minbefore virus infection greatly increased theamountof


region4mRNA. Weobservedno qualita-tive difference between the RNA pattem ob-tained from


which were treated and the pattern obtained from cells which were not treated with anisomycin. Thus, the increased

level ofearly region 4 RNA obtained after

treat-ment with


represented correctly splicedRNAspecies. Early region4mRNA pre-pared from Ad2-infected 293


treatedwith anisomycinat 30 minbeforeinfection and at 2 hafterinfectionaccumulatedtothe same extent.

Viral RNA accumulated from earlyregion 2 was

alsostudied bythistechnique. Addition of

ani-somycin30minbefore infectiondid notincrease

the amount of viral RNAcompared with


nottreated withthedrug (Fig. 2A).

Invitro translation.Todeterminewhether

the viral RNA which accumulated in

Ad2-in-fected 293 cells in the presence ofanisomycin

7 si

FIG. 2. SI endonucleaseanalysis of early region 2 and4RNAs accumulated in293cellsinfectedwith

Ad2. Cytoplasmic RNA waspreparedfrom

Ad2-in-fected293cells maintained in theabsenceorpresence

ofanisomycin(100pM). The RNAwashybridizedto

Ad2 DNA inasolutioncontainingahigh formamide

concentration, treated with Sl endonuclease, and separated on a2.0%o alkaline agarosegel. Thegel wasblottedonto anitrocellulose sheet and hybrid-izedtoEcoRIrestriction endonucleasefragmentBof

Ad2 DNA (58.5to 70.7map units) (A) or toEcoRI fragment C (89.7to 100.0map units) (B). Thefilter waswashedextensivelyandthenanalyzedby

auto-radiography. -30 min, Anisomycin added 30 min

before infection and maintained throughout until

harvestofthe cellsat 5hpostinfection;nodrug,cells

not treated with anisomycin; +2 hrs, anisomycin

added2hpostinfectionand maintained until harvest

at 5hpostinfection; marker, HindIIIdigest of

32P-labeled Ad2 DNA.



this RNAwas isolated by hy-bridizationtovarious viral DNA


and theselectedRNAs weretranslated invitro.

Fig-ure 3showsthat treatmentwith



minbefore infectionor2hafter infection caused

substantial increases in viral early region 4

mRNA's. All


region 4 proteins identified

fromdrug-treated cellswerealso present incells

that were nottreated with the


as shown

by a longer exposure of the film

(Fig. 4).

In addition,allproteinswereenhancedtothesame

extent. In contrast, bothearly region 2mRNA

andearly region3viral mRNAwere


if thedrugwasadded30min beforeinfection.If

the drug was added 2 h


these mRNA


accumulatedtothesame extent

as cytoplasmic RNA prepared from



Addition of anisomycin at 2 h before viral

on November 10, 2019 by guest



-m mF-a -7


IZ-E 4 , 26K,, II

-%Ez /I85 K



I/', ZK.

-4X/ 4.!3 - _aW


5K-E4,/1 K

E 4 E3 E2

-Nrd2 c' -RNA

ad2 a b c a b c a b

IV w WI ..

_ e -E2/75K

_ _ _ _ _ _ s _ _%

a_ a_


FIG. 3. In vitro translation withhybridization-sekectedRNAfromAd2-infected293cells. At 5 h postinfec-tioncytoplasmic RNA wasprepared from Ad2-infected293 cells maintained in the absenceorpresenceof anisomycin. The RNAwashybridizedtofilters containingAd2DNAfragments representing different early regions. Thehybridized RNAwasthentranslated in themRNA-dependent reticulocyte cell-free system. The

translationalproductswereseparatedona13%sodiumdodecylsulfate-polyacrylamide gel, and the gelwas

analyzed byfluorography. The Ad2DNA restrictionenzymefragments usedasprobeswere asfollows: early region4(E4), EcoRI-C(89.7to100mapunits); early region3(E3),EcoRI-D(75.9to 84.0mapunits);early

region2(E2),EcoRI-B(58.5to70.7mapunits).Lanesa,Infectionwithoutdrugtreatment;lanesb, anisomycin (100 uM) added30minbeforeinfection; lanesc,anisomycin(100 pM) added2 hafterinfection. -RNA, No RNA addedtothecell-freesystem.

infection also causedanenhanced accumulation

ofearly region4viral mRNA's compared with

untreated cells (Fig. 4). Under these conditions

early viral mRNA's from regions 2 and 3 were

depressed dramatically. In summary, early

re-gion 4mRNA's were enhanced irrespective of

when the anisomycin was added, whereas the

levels of early region 2 and 3 mRNA's were

depressed unless the drugwasaddedatleast 1

h afterinfection.


Earlyregion1A of the adenovirus genomeis

located between 1.5 and 4.5 map units and is

important for the accumulation of viral RNA

from other early regions of the genome. This

conclusion is based onresults with host range

mutantshaving deletionsorpoint mutations in

thisregion of the genome (2, 14). The deletion

mutants are defective in more than one viral

geneproductsincethey lackall ofearly region

1A. Thehostrangemutantsincomplementation

group I (10) express at least some viral gene

products from early region 1A(7,15). Elsewhere,

wehaveshown thatthe viralgeneproducts that

aredefective in thesemutantsareresponsible in

part forthe accumulation ofearly viralmRNA

(M. G. Katze,H.Persson, and L.Philipson,Mol.

Cell.Biol., inpress).

Provided that the viralgeneproductinvolved

in the accumulation of early viral RNA is a

protein, the addition of inhibitors of protein

synthesis before viral infection should mimic the

situationwith the early region 1Amutants;i.e.,

thistreatmentshouldpreventthe accumulation

of early viral RNAs from regions 2, 3, and 4.

Experiments have suggested that this

hypothe-sis is correct (16, 22) and that the early region

1Ageneproducts controlling mRNA

accumula-tionact ataposttranscriptional level.

The human embryonic kidney cell line 293

transforned by Ad5 DNA contains viral DNA

representing early regions 1A and 1B and

ex-pressesearly viral RNAs from thesetworegions ofthe genome (1). By using

immunoprecipita-tionwith anti-Tseraof[3S]methionine-labeled

293cellsandhybridization selection of

cytoplas-mic RNA followed by in vitro translation, we

demonstrated that viral mRNA's from these

regionsaretranslated intoproteins(Fig. 1A and

B). Furthermore,allproteins whichwedetected

corresponded in size and map location to the

proteins expressed during lytic infection of

hu-man cells with Ad5. Recently similar results

have beendescribed by Jochemsenetal. (13).

Tostudy the effects of the earlyregion1Aand

1Bgeneproductsonviral mRNAaccumulation,

293 cells were infected with Ad2, and the E

VOL. 40,1981 363

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FIG. 4. Effect of prolonged anisomycin treatment

on the accumulation of early viral RNA in

Ad2-infected 293 cells. At 5 hpostinfection cytoplasmic RNAwaspreparedfrom Ad2-infected293 cells main-tained eitherin the absenceof anisomycin (lanes a)

orin thepresenceofanisomycin (100,uM)from2h before infection (lanes b). This RNAwashybridized

toAd2 DNArestrictionenzymefragments

represent-ing early regions 2,3,and 4(E2, E3,andE4,

respec-tively), as described in the legend to Fig. 3. The selected RNA wasthen translated in vitro, and the samples were analyzed on a 13% sodium dodecyl sulfate-polyacrylamide gel, followed by fluorography of the dried gel.

amounts of Ad2 earlyviral RNAs fromregions

2, 3, and 4 were determined. Ifa viralprotein

encoded in early region 1 regulates the

accu-mulation of early viral RNA, the addition of

anisomycin before Ad2 infection of 293 cells

shouldnotdepressthe accumulation of mRNA's

from early regions 2, 3,and 4 sinceearly region


cells. We tested thishypothesis.

An increase in viral RNAfromearly region4

ofmorethan 10-foldwasobservedifanisomycin

wasadded 30min before infection(Table 1 and

Fig. 2) Early region4 RNA alsoaccumulated at

anenhanced level ifanisomycin wasadded 2h

before infection. Inaddition, themRNA which

accumulated was functional, as assayed by in

vitro translation (Fig. 3 and 4). The enhanced

accumulationofearly region4 mRNAwas more

pronounced if anisomycin was added 30 min

before infection than if itwasadded 2 h before

infection (Fig. 3 and4). Thismay be an


of theearly region 1proteinsthataffectmRNA

accumulation. Ourresultsmaybeinterpretedto

indicate that the functional half-life of these

proteinsisatleast2 to 3h,whichis inagreement

with the results obtained from

immunoprecipi-tation studies. Infection of normal human

em-bryonic kidney cells with Ad2 in the presence of

anisomycin didnotresult in an increased

accu-mulation of early region 4 RNA (Table 2). Thus, it is likely that the viral proteins present in the

293 cellsregulate the accumulation of early

re-gion 4 viral mRNA. It may be argued that the

enhanced accumulationof early region 4 mRNA

afterinfection with Ad2 results from the

expres-sion of the right end of the genome, which is

integrated into cellular DNA in 293 cells.

How-ever, we consider this unlikely since no

func-tional viral mRNA from early region 4 was

de-tected in uninfected 293 cells in the absence or

presence ofanisomycin (Fig. 1B).

Our results with early region 2 and 3 viral

mRNA's were similar to the results obtained in HeLa cells; i.e., the addition of anisomycin

be-foreinfectiondid notincrease the accumulation

ofthese mRNA species. This was demonstrated

both by Si endonuclease analysis and in vitro

translation studies. Therefore, the viral proteins

encoded in early region 1 exhibit a differential

control on the expression of other early regions.

Earlyregions 2 and 3 require either more of the

early region 1proteins or some viral protein not

present in 293 cells to accumulate early viral

mRNA. On the other hand, early region 4 is

directlycontrolled by the early region 1 proteins.

The rate of transcription from early region 4

was also increased in Ad2-infected 293 cells in

the presence ofanisomycin (Table 3). This

in-creased rate of transcription may explain the

enhanced accumulation of early region 4 mRNA,

butsincenosuch rateincrease was observed in

HeLa cells infected with Ad2 or its host range

mutants in the presence of anisomycin (22;

Katze et al., in press), amplified early region 1

proteins in 293cells may exert controlling effects

at two levels (at transcription and at mRNA

accumulation). The effect on transcription

can-notbe detected inHeLa cells since the presence

ofanisomycin prevents expression of the early

region 1A proteins. Since the DNA-binding

pro-teinencoded inearly region2 (E2/75K) appears

to repress early region 4 transcription

specifi-cally (20), the enhanced transcription of early

region4whichwe observedprobably would not

have been detected instudies with defined

mu-tants in early region 1. Such mutants would probably show a reduced level of transcription

fromearly region 4 but since the early region 2

also would beexpressed, it would be impossible

todifferentiate betweenanearly region 1 effect

and an earlyregion 2effect. Therefore, studies

with a protein synthesis inhibitor in 293 cells

should produce less biased effect of the early

region 1 proteins sinceall other products are not



on November 10, 2019 by guest



It is interesting that in293cellstreated with anisomycin, preformed early region 1 proteins

appear tohaveaneffect





producing both increased transcription and in-creased accumulation ofmRNA from this re-gion. Early regions 2 and 3 transcribe and

ex-press mRNA in a pattern similar to that

ob-servedpreviously in drug-treated HeLa cells(22; Katzeetal., inpress).These resultssuggestthat proteins from early region 1 primarily control theearly region4transcription unit in the


sequenceof adenovirus


gene expres-sion (19, 23, 26). Whether the early region 4 proteins in turn affect the next transcription unit,early region 3inthe adenovirusearly cas-cade, canonly be revealed with early region4 mutants.

Our results withprotein


inhibitors (22; Katze et al., in press) suggest that early adenovirus gene


is complex and in-volves several cellular and viral elements. Cells probably containa system which is capable of preventing viral mRNA accumulation and is counteractedbyavirus-encodedprotein defec-tive in the hr-i host range mutantfrom


region 1A (Katze et al., in press). In addition, ourresultssuggestthat the


region 1 pro-teins haveaselective effectonearlyregion 4 at the level oftranscription and probably alsoon mRNA accumulation. Anearlyregion 1B prod-uct may


control translation of the ade-novirus mRNA

(22). Next,

sincesomeevidence nowhas beenobtained for the sites of the


it will be necessary to


in vitro

systems to assayfor the






WearegratefultoMauriceGreen,ArnoldLevine,and Eric Blair for providingtheantisera used toimmunoprecipitate

cellextractsfromuninfected293cells. Wethank Britt-Marie Johansson for excellent technical assistance and Christina

SjoholmandMarianneGustafson for expert secretarialhelp.

This work was supported by grants from the Swedish Society againstCancer.M.G.K.wassupported byalong-term

EuropeanMolecularBiology Organizationfellowship.


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FIG.1.Ad2MonolayersfromDNA.hamsters;ratdeoxycholate,disruptingsequencesanisomycindodecylusedaantiserainducedunitssulfate-polyacrylamidecellsdenotes(8) p.3
TABLE 2. Effect of anisomycin on the accumulationof cytoplasmic early viral RNA in Ad2-infectedhuman embryonic kidney cells


Effect of anisomycin on the accumulationof cytoplasmic early viral RNA in Ad2-infectedhuman embryonic kidney cells p.4
TABLE 3. Rate of transcription in Ad2-infected 293cells


Rate of transcription in Ad2-infected 293cells p.5
FIG. 2.Ad2.Ad2fectedAd2fragmentofandseparatedradiography.harvestconcentration,izedwaswasnotaddedatlabeledbefore 5 anisomycin SI endonuclease analysis ofearly region 2 4 RNAs accumulated in 293 cells infected with Cytoplasmic RNA was prepared from Ad2-in- 2
FIG. 2.Ad2.Ad2fectedAd2fragmentofandseparatedradiography.harvestconcentration,izedwaswasnotaddedatlabeledbefore 5 anisomycin SI endonuclease analysis ofearly region 2 4 RNAs accumulated in 293 cells infected with Cytoplasmic RNA was prepared from Ad2-in- 2 p.5
FIG. 3.regionregions.regionanalyzedRNAanisomycin.tiontranslational(100 In vitro translation with hybridization-sekected RNA from Ad2-infected 293 cells
FIG. 3.regionregions.regionanalyzedRNAanisomycin.tiontranslational(100 In vitro translation with hybridization-sekected RNA from Ad2-infected 293 cells p.6
FIG. on a 13% sodiumsulfate-polyacrylamidebeforeingselected gel, followed by fluorographyoftively), Ad2 in thein Ad2- early was the dried infection 4.RNA accumulation 293 eitherortained main- presence Effect preparedto cells
FIG. on a 13% sodiumsulfate-polyacrylamidebeforeingselected gel, followed by fluorographyoftively), Ad2 in thein Ad2- early was the dried infection 4.RNA accumulation 293 eitherortained main- presence Effect preparedto cells p.7