Functional diversity of E1A gene autoregulation among human adenoviruses.

JOURNAL OF VIROLOGY, June 1992,p. 3833-3845 0022-538X/92/063833-13$02.00/0

Copyright©) 1992, American SocietyforMicrobiology

Functional Diversity

of ElA Gene Autoregulation

among

Human

Adenoviruses

JOY D. COGAN,t

STEPHEN

N.

JONES,:

ROBERT K. HALL,§ AND CLARKTIBBETTS*

Departmentof

Microbiology

andImmunology, Vanderbilt University Schoolof Medicine, Nashville, Tennessee 37232

Received 16 July 1991/Accepted 21 February 1992

Autoregulationof the adenovirus EIAgeneinvolves its constitutive expression and positively and negatively

regulated transcription. Dissection of this process will identify basal-level cis elements and autoregulatory

targetsoftheElApromoterand functional domains within the trans-acting ElAgeneproducts. In thisreport,

the DNAsequenceofthe human subgroupB adenovirustype3 (Ad3)ElAgeneis presented and compared with

that of theEIAgenesof similar and distantly related human adenoviruses. The cDNAforms ofthe Ad3 ElA gene, corresponding to two majorearlymRNA species, are cloned, sequenced, and subcloned into plasmid

expression vectors. Cotransfections ofcell cultures are performed with Ad5 or Ad3 ElA gene expression

plasmidsandareportergeneunder controlof theAd5orAd3ElApromoter.TheAd5 and Ad3 ElApromoters

aresimilarly repressed by either serotype's 12S cDNAgeneproducts.The Ad3 ElApromoterresponds much

morestrongly thantheAdSEIApromoter totransactivationby13ScDNAgeneproducts. Incontrast,the 13S cDNAgeneofAd5 hasgreatertransactivationactivitythanthat ofAd3.Experimentswith missense mutations of the Ad5 ElAgeneindicatethattransactivationof theAd5EIA promoterisweak, just reversingorbalancing

negativeautorepression. Single amino acidsubstitutions in theconserved, repressive functionaldomain 2 of the ElAgenemodulate transactivating activity thatis usuallyassociatedwith the separate and distal conserved

functional domain 3. These results suggestastrongstructure-function relationshipinfluenced bythevariable sequencesseparating theseconserved domains.

Natural variation among similar viruses or strains of virusesprovidesmanyexamplesofevolutionary constraints,

based upon the structure or function of essential gene

products. In a complementary fashion, the immune re-sponses of infected host organisms impose divergent pres-sures oncertain viralgenes.Lateviralgenesareoften cited as examples of these counterpoised forces in viral gene

evolution.Essentialearly viralgenes mayalsobesubjectto complementaryconstraints and divergentpressures.

Theadenovirus EIA earlygene is thefirstcomponent of viralgenomeexpression in the nucleus early after infection.

The intimate associations of ElA gene products with the

cellularmechanics oftranscription regulation (3, 4, 8, 36, 41, 55) create a set of functional constraints on this essential early gene. A role for ElA antigens as targets for cell-mediatedimmunosurveillance(1, 2, 32)maybeanalogousto

the immunological basis for antigenic drift of late-gene capsid antigens.

The levels and the kinetics of ElAgeneexpressionin cells

infected by adenovirus type 5 (AdS) or adenovirus type 3 (Ad3) are remarkably different (51). We attribute this to

evolved differences ofpositive and negative autoregulation of thisessential viralgene's expression (18, 51).Adenovirus ElAgeneexpressioninvolvesautoregulatoryrepressionand transactivation oftranscription (13, 18, 23, 25, 28, 37, 46,

* Correspondingauthor.

t Present address: Department ofPediatrics, Division of

Genet-ics, Vanderbilt University School of Medicine, Nashville, TN

37232.

tPresent address: InstituteforMolecular Genetics, Baylor

Col-legeofMedicine, Houston,TX 77030.

§Present address: Department of Molecular Physiology and

Biophysics,Vanderbilt University School ofMedicine, Nashville, TN 37232.

51). These opposed levels of control are mediated by two

early ElA gene products, distinguished by their 5' donor

sites for the RNAsplicingofacommonElAgenetranscript.

Two functional domains (14, 30, 33) are associated with

enhancer-targeted repression, and these are present in the translation products of both ElA mRNA species. A third functional domain (14, 30, 33) is associated with transacti-vation of transcription, and this domain is found in the translation products of the longer 13S mRNA. The EIA promoter contains elements that represent targets for the regulatoryactivities of the ElAgeneproducts (18), although suchtargets maybeaccessed indirectly, through intermedi-ate cellular factors associated with developing or active

transcription complexes.

The ElAgeneis alsonoted for thevarietyof cellulargenes

whichrespondtoElA-mediatedpositive and negative

regu-lation oftranscription (14, 16, 17, 34, 35,44, 45, 47,48,53). Evolutionary balances of functional constraints and anti-genic pressureson the ElAgene mayhave determined the variation ofmorecomplex propertiesofadenoviruses,such

as tissue tropism and oncogenicity. The variety of such

virus-host cell-host animal interactions and the involvements of the ElAgene are seminal featuresofthelarge family of (human) adenovirusserotypes (42, 57).

The different intensity and kineticsofAdS andAd3 ElA

geneexpression earlyafterproductive infectionare

recapit-ulated in transfections ofthe same host cells with plasmid expression vectors. Our earlier studies (18, 51) were done

withvectors expressingcDNA forms of the AdS ElAgene

or genomic forms of the AdS and Ad3 EIA genes. These

ElA gene constructs are cotransfected into cells with a

reporter gene (chloramphenicol acetyltransferase [CAT]) expressed under control of either the AdS or Ad3 ElA promoter. The ElA promoter of Ad3 responds more to

transactivation by the 13S cDNA of AdS than does the 3833

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3834 COGAN ET AL.

homologous AdS ElA promoter. The Ad5 ElA promoter responds more strongly than the Ad3 ElA promoter to

autorepression mediated by the 12S cDNA form of theAdS ElA gene.

In this report, we present the DNA sequence of the Ad3 ElA gene and describe the successful cloning and

expres-sion of its 12S and 13S cDNAs. New plasmid expression

vectors enable cotransfectionswith the ElA cDNAgenesof

both Ad3 and AdS and the CAT reporter gene, expressed under control of the homologous or heterologous Ad3 and

AdS ElA promoters. The repressive activities of the 12S

cDNAgeneproductsof Ad3 andAdSareverysimilar.There

isa significant difference between the activities ofAd3 and

Ad5ElA 13S cDNAgene productsin transactivation of the Ad3 ElApromoter.Experimentswith missense mutationsin

functional domains2 and 3 of theAd5ElAgene address the

remarkably different responses of the AdS and Ad3 ElA promoterstotransactivationbyElA products.The variable region separating conserved functional domains 2 and 3

appearsto be importantin the role of both domains in ElA

trans autoactivation.

MATERIALSAND METHODS

Cells and viruses. HeLa cell cultures were maintained in

110-mm dishes in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% calf serum, antibiotics (100 U ofpenicillin per ml and 100 ,ug ofstreptomycin per

ml), andanantimycoticagent(1 ,uMp-hydroxybenzoicacid butyl ester). Adenovirus type 3 (strain G.B.)wasoriginally

obtained from the American Type Culture Collection and waspropagatedandpurified by proceduresusedroutinelyin

this laboratory (50, 51).

Enzymes.Restriction endonucleases,T4 DNAligase, and the Klenowand T4 DNApolymeraseswerepurchasedfrom

New England Biolabs. Chemically modified T7 DNA

poly-merase, Sequenase, was purchased from United States Biochemical Co. Enzymes were used with the buffers and

incubation conditionsspecified bythe vendors.

Plasmid expression vectors. The construction and use of

plasmids expressing the CAT gene under the control of adenovirus ElA promoterswere described in earlier publi-cations (18, 51). In the present study, we usedan Ad3 ElA

promoter-CAT plasmid (51)and theAd5/3 chimeric promot-er-CAT plasmid described byJones and Tibbetts (18). The latter CAT expression vector has upstream ElA promoter

sequences derived from Ad5, joined at a common PvuII restriction site (-50 bp) tothe identical transcription initia-tion region of the Ad3 ElA CAT plasmid. Construction of the plasmids expressing the genomic form and the 12S and 13S cDNA forms of theAd5ElAgenewasdescribedearlier (18).

We modifiedouroriginal plasmidvector,whichexpresses

the genomic form of the Ad3 ElAgene (51). This plasmid DNA was first restricted at the unique sites for ClaI and SphlI, followedby blunting andpolishing ofthe noncomple-mentarystickyends with T4 DNApolymeraseand blunt-end ligationwith T4 DNAligase.The modifiedplasmidwasthen cloned by transformation of Eschenichia coli competent cells, screening for ampicillin-resistant colonies with plas-mids about 540bp smaller than theoriginal Ad3 ElAgene

expressionvector. Thesequences deletedin this procedure

remove a remaining portion of the tetracycline resistance

gene of the original pBR322 vector without affecting the expressionof theclonedgenomicform of the Ad3ElAgene

in transfected human cells. Deletion of these plasmid

se-quences removes the

pBR322-derived

BamHI

and

HindIII

restriction sites from the Ad3 ElA

expression

vector,

pro-moting

the

remaining,

Ad3-specific

BamHI

and

HindIII

sites to

unique

status. This facilitatesour

procedure,

which sub-stitutes cDNA sequences for

genomic

DNA sequences be-tweenthesetworestriction

sites,

asdescribed ina

following

section.

Genomic

expression

vectors,

corresponding

to missense

mutations hr3 and hr4 of theAd5 ElAgene, as described in

an earlier

publication

(9),

were obtained from

Gary

Glenn

and Robert Ricciardi of the Wistar Institute. The missense mutations of these mutants debilitate the transactivation

activity

of theAd5ElA

(13S) products,

consistent with the loci of the mutations in functional domain 3 of the ElA gene.

Expression

vectors

corresponding

to the

13S

cDNA of

Ad5 ElA mutants

pm936

and

pm953,

as described in an

earlier

publication

(29),

were obtained from Jim Lillie and

Michael Green of Harvard

University.

Themissense muta-tions of these mutants debilitate

enhancer-targeted

repres-sion of theAdSElA

(12S

and

13S) products,

consistentwith the loci of the mutations in functional domain 2 of theElA

gene.

DNA

sequencing

of the Ad3ElAgene.The DNA sequence of the

left-end,

1,565-bp

BglII

K

fragment

of the Ad3 genome

was determined from viral DNA

(21, 23, 24,

52)

and from viral DNA

fragments

derived from a

functional,

plasmid-cloned Ad3ElAgene

(51).

A

variety

of

partial

and

complete

MspI

and MboI restriction

fragments

of viral DNA were

cloned into the AccIorBamHI site of

phage

M13mp18

DNA and then

subjected

to routine

dideoxynucleotide

sequence

analysis (43). Sequences

from the left-end

region

of

Ad3,

corresponding

to the inverted terminal

repetition

and ElA

promoter of

Ad3,

wereobtained

by

Maxam-Gilbertchemical

DNA

sequencing

protocols

(31),

as

reported

earlier

(21,

24,

52).

Theexon-intron

junctions,

corresponding

tothe 12S and 13S ElA mRNA

species

of

Ad3,

have been confirmed

by

fluorescenceautomated

sequencing

(38)

of Ad3

ElA-specific

cDNA

clones,

described in a

following

section.

Infection of cells and

early

viral RNA

preparation.

HeLa cell cultures were infected with Ad3 at 1.5 x

1010

virion

particles

per 5 x 106cells per

plate

(cells

were about 80%

confluent).

Mediumwasremovedfrom thecells before virus

wasadded in avolume of 0.5 ml per

plate,

using

phosphate-buffered saline with2%calfserum.After 30 minat

37°C,

the virusinoculum fluidwas removed and

replaced

with 10 ml per

plate

of DMEM culture medium

containing

5% calf serum,

antibiotics,

and

antimycotic.

Six hours after

infection,

the cultures were washed with

phosphate-buffered

saline

prior

toextraction of RNA

by

the method of

Chirgwin

et al.

(5).

Theinfected cellswere

lysed

by

addition of 0.8 ml of4M

guanidine isothiocyanate-25

mM sodiumacetate

(pH 6)-120

mM

3-mercaptoethanol

per

plate.

Lysates

were forced

through 18-gauge

syringe

needles to reduce

viscosity,

then

layered

over a1.2-mlcushion of 5.7M

CsCl,

and

centrifuged

at

37,000

rpm for 16 h at

20°C

witha

DuPontAH50rotorand

OTD75

ultracentrifuge

to

pellet

the RNA. The supernatant was

removed,

and the total RNA

pellet

was

resuspended

in 300 ,ul of 10 mM Tris-HCl

(pH

7.5)-i

mM EDTA.

Polyadenylated

RNAwas isolated from total RNA

by

oligo(dT)-cellulose

(Collaborative

Research)

column

chromatography.

Samples

were

applied

tocolumns in 0.5 MNaCl-10mMTris-HCl

(pH

7.5)-i

mMEDTA-0.5% sodium

dodecyl

sulfate. Columns were washed with the

application

buffer and then with 0.1 M NaCl-10 mM

Tris-HCI

(pH

7.5)-i

mM EDTA. The

polyadenylated

RNA fractionwasrecovered

by

elution with 10 mM

Tris-HCI

(pH

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DIVERSE ADENOVIRUS ElA GENE AUTOREGULATION 3835 7.5)-i mM EDTA, and the concentration was estimated

from the A260 measured with a microcell with a Beckman DU70 spectrophotometer. The eluted polyadenylated RNA fractions were ethanol precipitated and resuspended in di-ethylpyrocarbonate-treated water at a concentration of 0.5 ,ug/,ul.

Synthetic DNA oligomers. The DNA sequencing primer (-40, M13 universal forward primer) was obtained from New England Biolabs or United StatesBiochemical Co. We are now preparing oligonucleotide primers for DNA se-quencing in this laboratory with a Du Pont Generator single-column DNA synthesizer with standard phosphoramidite reagents (NEN-Du Pont). The two oligomers used in our Ad3 ElA cDNA cloning protocol were synthesized with a SAM-1 instrument, phosphoramidite reagents, and solvents from BioSearch: oligomer 365,5'-TGATTTAGAGATAGAC GGGCCGGAG-3'; and oligomer 366, 5'-TACTAAGGTCCA AAGGTCCATCCCC-3'. The DNA sequences of these two oligomers flank, on opposite strands, the unique BamHI and HindlIl restriction sites in the 5' and 3' exons of the Ad3 ElA gene.

Ad3 ElA-specificcDNA. First-strand cDNA synthesis was performed with the polyadenylated fraction of RNA isolated early after infection of HeLa cells by Ad3 and the compo-nentsof a cDNA synthesis kit from Promega. Priming of the first-strand cDNA was done with 1 ,ug of oligomer 366 (above) and 2 ,ug of polyadenylated RNA; it was annealed by heating and then cooled in a total volume of 10 ,ul. DNA synthesis proceeded for 60 min at 42°C in a 25-,ul reaction mix containing 50 mM Tris-HCl (pH 8.3), 75 mM KCI, 10 mMMgCl2, 0.5 mM spermidine, 10 mM dithiothreitol, 4 mM sodium

PPi,

1 mM each dATP, dCTP, dGTP, and dTTP, 25 U ofRNasin, and 30 U of avian myeloblastosis virus reverse transcriptase. The reaction mixtures (and products) were frozen and stored at -20°C until the next step of cDNA

amplification by polymerase chain reaction (PCR). These

first-strand cDNA products represent the noncoding strand ofthe Ad3 ElA gene from the 3' exon position flanking the

HindIll site (oligomer 366 sequence) towards the 5' exon and

siteofElA transcription initiation.

Amplification and cloning of Ad3 EIA cDNA sequences. Thecomponents of a PerkinElmer-Cetus GeneAmp PCR kit and the vendor's procedures were used, with some

modifi-cations, to synthesize a second-strand cDNA from the

first-strand cDNA of the Ad3 ElA gene (above) and then to amplify the cDNA segments prior to cloning.

First-strand cDNA products (2

RI

of the reaction mix described above) were combined with 10

RI

of lOx PCR buffer(0.1 MTris-HCl [pH8.3], 0.5 M KCl, 15 mM MgCl2, 0.1% gelatin), 18 nmol each of dATP, dCTP, dGTP, and dTTP, 100 pmol of oligomer 365, 90 pmol of oligomer 366, 64 ,u of H20, and 2.5 U of Taq DNA polymerase in a final volumeof 100

[lI.

Samples were covered with a layer of light mineral oil and amplified in a Perkin Elmer-Cetus thermal cycler. The initial denaturation step was 94°C for 150 s, followed by 45 amplification cycles of three steps: 60 s at 94°C, 120s at 47°C, and 180 s at 72°C. The finalextension step was 10 min at 72°C. The sample was removed from beneath the oil overlay and ethanol precipitated. DNA amplification products were analyzed by agarose gel electro-phoresis.

The PCR amplification products, generated as described above with Ad3 early mRNA and cDNA primers 366 and 365,weredigested with BamHI and HindIll attherestriction sitesjust inside theprimer DNAsequences. TheDNAwas ligated to BamHI- and HindIll-restricted pEMBL18 (6)

vector DNA and transformed intoE. coli UT481 (F' bearing) competent cells, using the ampicillin resistance marker for plasmid selection and white colonies to indicate an inter-rupted lacZ gene.

Recombinant phagemid DNA was isolated for DNA se-quencing to identify the intron junctions associated with the 12S and 13S cDNA species and to screen for possible adventitious mutations resulting from the cDNA synthesis or PCR amplification steps prior to cloning. Thefl phage strain IR1 was used as the helper for propagation and packaging of the recombinant phagemid DNA. The universal M13 se-quencing oligomer was used for sese-quencing of the single-stranded phagemid DNA, with a fluorescence automated Du Pont Genesis 2000 DNA sequencer (38). The sequences of the cDNA clones were compared with established sequences ofthe wild-type (wt) Ad3 ElA gene. Eight candidate clones of the 12Sand the 13S cDNAs were analyzed, with only one apparent (missense) mutation found in one 13S cDNA clone. Later subcloning of this mutant's cDNA in an expression vector revealed modestly diminished transactivation of the Ad3ElA-CAT reporter plasmid. The four 12S and four 13S clones each have intron junctions which agree precisely with the junctions reported for the closely related ElA gene of adenovirus type 7 (7).

The cDNA sequences of the eight cDNA candidates were excised from the pEMBL18 vectors by restriction with BamHI and HindIll for substitution into the corresponding BamHI-HindIII region of the modified genomic Ad3 ElA expression plasmid (described above). The restricted plas-midand cDNAfragments were mixed, ligated with T4 DNA ligase, and used to transformE. coli, with selection based on the plasmid ampicillin resistance gene. The desired plasmids wereselected by agarose gel electrophoresis on the basis of reduced sizes compared with the genomic Ad3 EIA expres-sion vector. The expresexpres-sion and activities of the cloned 12S and 13S Ad3ElA genes were evaluated in cotransfections of HeLa cellcultures withElA promoter-CAT gene expression plasmids. The four 12S candidate clones were indistinguish-able incotransfection functional assays (18, 51). Three of the four 13S candidate clones were also indistinguishable from one another in such assays.

Transfections of HeLa cells and analysis of ElA gene expression. HeLa cells were prepared for DNA transfections from cultures at approximately 50 to 70% confluence on plates. Transfections generally followed the procedures of Graham and Van der Eb (11), Gorman et al. (10), and Tibbetts et al. (18, 51) with modifications. Cells were re-moved fromplates bytrypsinization, pooled, and dispensed asequal aliquots into separate tubes for individual transfec-tions insuspension. Each tube of cells (4 ml per transfection in DMEM-5% calf serum) received 200 ,u of a transfection mixture containing 20 ,ug of plasmid DNA (carrier plus expression vector) and 5 ,ug of sheared, denatured salmon sperm DNA inHEPES-buffered phosphate (125 mM NaCl, 5 mMKCl, 0.6 mM Na2HPO4, 5 mM dextrose, 20 mM HEPES

[N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid, pH

7.05]). CaCl2 was added to the transfection mixtures at 125 mMforprecipitation with DNA, 30 minprior to transfection. Following 3 h of gentle rotation at 37°Cin the CO2 tissue culture incubator, the transfected cells were mixed with equalvolumes of cold 20% glycerol in DMEM and immedi-atelycentrifuged for 5min at 2,500 rpm. The cell pellets were resuspended in 10 ml of DMEM-10% serum-antibiotics-antimycotic and returned to new culture plates for posttrans-fection incubation at 37°C.

Transfected cells were harvested at various times after VOL. 66,1992

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AATATACCTT CTGTGTGGTG b GACGTGACTT c GAGGTGTGGT TAGTTTTGGG TGAATTTCTG ACTTTGACCG GGTGTCAAAG ACGAGTTCCG GCAAGTCAGT AAGATGGAAT ATTGGCTGCG ATGTGGGAGG TTGAACACGG CGGATGCAAG g AGTCATTTCG TTTACGTGGA TCCTGTGTTT TCAAGAGGCC TCTGCGCTTT TCCAGTGAGA CCGGGASCGA CAGTGCAGCC 1 CTGCTAGCTG GGGGTGGTTG ATGGACTTGC GAGCAGTCCA TTGGACTGTC CA.GGAATAAC ACTGGAATGA n CCACTTTATT TACA< GTAAGT TAAACTGTTG AATGGTAGAT CTGLTGALGG GTTGTTATGA TCCATACCGC CGG&GCTGCC GTATTTAAGT TTATGTTTTT GGTGCCAACA a GGGTTAACGG AGTTATGTTG AAGTAGACAG TGAAAATTCT CGGTTATGCC GGTTTCGATT TTACGTAGGT ACTCTTGAGT GAAA ATGAGA AATACTGGAG TTTCGATCCA CALTGAGGAA ATTGGACATA CATAGGTGGG AGAGGGTTTT AGTAAATGAG TGGA.CATGGC TGTAAATGAG CTAAAAGGGG CAAGTTATTA TTTTCCCACG d CCATTTTCGC AGGGTGGAGT ACCGTGTTTT GTCAGCTGAT GCCAGCGAGA CACCTGCGCT TTTGTGGTAA CCTACGCTGC GCTGTGAATG AACCCCTC AAAAAATTGC CCTCCCAGTG GGAGTAAAAG m T< GTAA,GTCTT TGCTATATGA GAAATTTAAA GGAATAGTGT 0 TTCTTGCGAT TTTTTGTAG

CAMCTTCTCC TGATTCA&CT ACCTCACCTC

TATGC&LGCC CLTTCCTGTG ALGCCThLGC q

AGGACTTGTT GGAGGG 2t;GQ T r

ALTGa s

GTGCC CTGCAGCTGT GTTTATTTAA

v

TGCTGAGTGT TTTATTACTT CTTGGGTGGG 1569 CTGAAATTCA CTGGGAAACG _[:I&CZAL GTAATTTAAA 60 CGGCGCGACC 120 CGGTAAATGT 180 CTTACTGACA 240 e GCGAAAACTA 300 ATTTGCCGAG 360 TCACCTAAAT 420 h CGTCAGGGTA 480 AGAGTTTTCT 540 TCCTGCCACL 600 ATACCCTAAT 660 ACGATCTGTA 720 GGTTTTTTAC 780 CTGLGCLCT 840 CTGATCTGGG 900 ATGLTGaaGa 960 CTGCCAGCGL 1020 GTGAATTTCA 1080 GAATGCA.CTG 1140 AGCTGTTTAA 1200 TCCTGTGTCT 1260 GGCGCCCGCA 1320 CCCTGCTGTG 1380 _.Z_CCCGGLAA 1440

TGTGACGTCA TGTAATAAAA 1500 GACTTGGATA TATAAGTAGG 1560

[image:4.612.172.455.55.424.2]

FIG. 1. DNA sequence of the Ad3BglIIKfragment,spanningtheElAgene.This sequence hasbeenreportedinU.S.patent4,920,211. Coding regionsareindicatedinboldface. Numbersindicate basepairs alongtheElAgene-encodingrstrand,proceeding from the leftend of the viral genome. Lowercase italic letters above the DNA sequence highlight landmarks, whichare identified in Table 1. Sequences correspondingtooligomers 365 and366,usedin cDNAcloningprocedures(seeMaterials andMethods), areunderlined.

TABLE 1. Landmarksin the DNA sequenceof the Ad3ElA gene'

Description

HpaI restriction site

End ofleft-end invertedterminalrepeat (47)*

-300 ElA(distal) enhancerelement (49)*

E2Fbinding motif,T'llCGCG (58)* E2Fbinding motif, CGCGAAA (58)* -200ElA(proximal) enhancer element (49)* E2Fbinding motif, T'TlCGCG (58)* ElAgeneTATAelement,TAT'TTA (39, 42)*

5'end ofElAtranscripts(39, 42)*

ElAtranslationstartcodon(39, 42)* Sequenceofoligomer365 (underlined) BamHIrestriction site

12SElAmRNA5'splice donor site 13SElAmRNA5'splice donor site

Common ElAmRNA3'splice acceptorsite

HindIII restrictionsite

Complementofoligomer 366(underlined) ElAtranslationstopcodon (39, 42)* PstIrestriction site

Polyadenylation signal, AATAAA (39, 42)* ElAmRNA3'polyadenylation site (39, 42)* E1BgeneTATAelement, TATAA (39, 42)* BglII restriction site

transfectionbyscrapingthecells from eachplateinto 1.2 ml

of Versene (0.5 mM EDTA in phosphate-buffered saline).

Thesuspensionswerecentrifugedin 1.5-mlmicrocentrifuge

tubes for approximately 2min at 12,000rpm, and the cells were then resuspended in 150

[lI

of 0.25 M Tris-HCl (pH

7.5).Thecellsuspensions weresubjected tothreecycles of

freeze-thawing between -80 and 37°C and then centrifuged in the microcentrifuge for 10 min at about 12,000 rpm. Cellular extractswere assayedforCAT enzyme activity in 100-,u reaction mixes with 80 nmol of acetyl coenzyme A and 2 to 2.5 nmol of

['4C]chloramphenicol,

as described

previously (51). One unit of CAT activity corresponds to

acetylation of 1% of substrate (20 nmol) in 15 minat 37°C

underourstandardassayconditions. Sampleextracts(50

pul

of the

100-pul

reactionmix)areroutinely diluted in assaysto provide results in the range of1to50%substrateacetylation. Higher levels ofacetylation are not linear with the concen-tration of CAT in the extract. Activities of CAT in the

aFeaturesofAd3ElAgenetopographyareidentifiedaslandmarks which correspond tolowercaseitaliclettersabovetheDNAsequenceinFig. 1. A schematicmapof the Ad3 ElAgeneispresentedinFig.2. Features inferred

by comparisons withpublished results for human Ad7 orotherwork are indicatedby asterisksand referencecitations,respectively.

3836 1 CTATCTATAT AAAGTGCGCG GTGGGAAAAT GACGTAAAAC GGATATGAGG f AATGAGGAAG GGCCGAGTAT TTCCGCGTAC TTTAAACCTG CCTCCGCGCC GGGGTTATC k

TGAT TL TTCC

TGATTCTATG TGTTACCCCA AGCAGCTGAA TGGGGCAACT TGTTTTTAAG AAGAACTATT GTGCTCGCTT GLTGATGAGT CCTGCAAACG p GATAAGCTTG CTGCCAAGGC u TTATGTCAGC w AGCAGATCT Land-mark a b c d e

f

g h i k I m n 0 p q r s t U V w Position (bp) 95 135 210 286 295 310 327 479 511 575 721 745 1061 1155 1250 1385 1408 1453 1461 1495 1514 1551 1565

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[image:4.612.67.309.496.731.2]

DIVERSE ADENOVIRUS ElA GENE AUTOREGULATION 3837 extract are calculated to takc into account the extent of

extract dilution (if any). Average levels of CAT activity recovcred from transfected cell cultures are presented plus orminus the standard crror of the mean.

RESULTS

DNA sequence of the Ad3 EIA gene. The DNA sequence of the 1,569-bpBglIl K restriction fragment of human Ad3 is presented in Fig. 1. This figure represents new data and previously published data for the entire length of both complementary strands. Landmarks within the DNA se-quence include the genomic inverted terminal repetition of Ad3 (52), some of the cis clemcnts of the Ad3 EIA promoter (12, 18, 21), and sites associated with transcription and mRNAprocessingofthe EIA gene (7, 54). These landmarks and others are described in Table 1. Figure 2 provides a schematic mapof the Ad3 EIAgenc and its two major early mRNAspccies.

The DNAsequenccof the Ad3 EIA gene is verysimilarto that determined for the closelyrelated subgroup B serotype Ad7 (7). The EIAgcnc-coding regionsof Ad3 and Ad7 differ by only10of 786 bp.Their13S mRNA translation products differby only3 of 262 amino acids, as I for V, V forI, and I for R (single-letter amino acid code). The 12S- and 13S-specific5' splicingdonor sitesand common 3' splice accep-tor sitesofAd3, detcrmined asdescribed later,are identical to thosc specificd byAd7. The 13S intron and the 5' and 3' noncoding DNA sequences of these two viruses differ by only 19 of 779 bp. The enhancer elements (12) and other idcntificd cis elcmcnts oftheirElA promoters are identical DNA sequenccs.

The close relationship of the subgroup B Ad3 and Ad7 EIA genes is reminiscent of that of the EIA genes of subgroup C serotypes Ad2 and Ad5 (54). Aligned DNA sequences ofAd3 and Ad7 or ofAd2 and Ad5 reveal 98 to 99%5 sequcnce identity. Aligned DNA sequences represent-ingthe twodifferent subgroups, suchasAd5and Ad7, show only

52%,c

sequence identity through the EIA gene region. Conscrved features in the EIA regions of subgroup B and subgroup Cadenoviruses includeaninverted terminalrepeat regionand left-end origin of DNAreplication,transcriptional enhancer elements at -200 and -300bp,aTATA element of the EIA promoter as TATTTA, 40 bp of nearly identical DNA sequcncc about the EIA5' RNA cap site, conserved functional domains 1,2, and 3 of the EIAcodingsequences, and anuclcarhoming signal atthecarboxyterminus of EIA

proteins.

We and others (14, 18, 25, 54) have observed differences amongfunctional components of subgroup B and subgroup C EIA genes. Thcsc distinguishing features include the number and arrangement of E2F binding sites in ElA promoters, diffcrent 12S EIA splicc donor sites, and DNA sequence and separationof conscrved functional domains 2 and 3. A schematic comparison of the Ad5 and Ad3 EIA gene structures, similar to the format of Fig. 2, was pre-sented in ourprevious report (Fig. 1 ofrefercnce 18).

Cloning andexpression of Ad3ElA-specific cDNA. Diffcr-cnt levcls ofAd5 and Ad3 EIA genc expression early after infcctionwith wtand mutantviruses,have beenattributcdto diffcrent responses of their EIA promotcrs and also to different activitics of EIA gene products (18, 25, 51). Our rccent workwith cDNA exprcssion cloncs of the Ad5 EIA gcnelocalizcd dcterminantsofthesedifferentautoregulatory

rcsponses in the upstrcam DNA scquences of the Ad5 and Ad3EIA promotcrs. Inordertoanalyzepossible differences

ConservedFunctional Domains

..12S

12S

Ad3 ElA mRNA AUGS

(EIA 13S

_ (EIPrombr

Left End of Genome

i

3 n

ESHIE I PE.-1P!I tUGA .iI.ISISS

BfmHI HIndII1

I I

I

I

I I 0-

I

I

0 500 1000 1500

Basepairs fromleftend

FIG. 2. Map of the left end of the Ad3 viral genome, including the EIA gene and its 12S and 13S mRNA species. The thick horizontal lines above the double-stranded DNA genome (left-end segment) represent thetwomajor early mRNA species of the ElA gene. The thin lines in the middle of the mRNAscorrespondtothe introns removed by RNA splicing of the primary EIA transcript. The shaded areas surrounding the DNA and mRNA sequences correspond to the three highly conserved functional domains of adenovirusElAgenes(30-32). The very thinarea nearthe 3' end of the gene, labeled n, corresponds to the conserved pentapeptide region which has been associated with nuclear targeting of ElA proteins from the cytoplasmic site of mRNA translation (44). The unique sites for restriction endonucleasesBamHI and Hindlllare shown in the 5' and3' exons of the gene, respectively. The AUG

startcodon andUGA stop codon of the mRNAspeciesareindicated

by the vertical doublearrows.Thelarge black wedge highlights the nonconservedregionof variable length and DNA sequence between conserved functional domains 2 and 3. This region may be of importance in modulating the negative and positive transcriptional regulation activities associated primarily with the conserved do-mains(25). An alignment of these landmarks of the Ad3ElAgene withcorresponding features of the more familiarAdSEIA gene was presentedasFig. 1 inour recent report(18).

in Ad3 and Ad5 EIAgene productactivities, itwas neces-sary to constructcDNAexpression clones correspondingto the 12Sand 13S mRNAspecies oftheAd3ElA gene.

Several conventional protocols forsynthesis and cloning ofElAcDNAexpression clones wereunsuccessful, in our laboratory andothers. The successful procedure described in Materials and Methods involvedsynthesis ofa segmentof first-strandcDNA withmRNApurified from cells early after Ad3infection, reverse transcriptase, andanoligonucleotide primer complementarytothe 3' exon sequence of Ad3 ElA mRNA. Asecondprimer, representingthe 5'exonsequence of the Ad3 ElA mRNA, Taq DNA polymerase, and an additional quantity of the first primer were used in a PCR procedure to synthesize the second-strand cDNA andthen amplifythe corresponding cDNAsequence.

Ananalysis of thecDNAsynthesis-amplification products

by agarose gel electrophoresis ispresented in Fig. 3. More than90%of the totalproductsrepresentspeciessmallerthan 500bp, and these do not appear torepresent the Ad3 EIA gene. Inspiteofthespecificoligonucleotide primersusedfor

cDNA synthesis and amplification, an identical pattern of

these abundant, smaller products is obtained with mRNA isolated from uninfected HeLa cells. These

spurious,

if perhaps significant, products appearwitha wide

variety

of reaction conditions, includingdifferent

durations,

tempera-la!

!

m

I all

I I I I I I t

1N

T

of

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3838 COGAN ET AL.

6

7

8 9

Control

500bp

-1

2

3

4

pB R322- Hpa ll markers

-622bp

-4-52 bp

404bp

[image:6.612.78.287.77.486.2]

* 3/ hp

FIG. 3.

Analysis

of the

products

ofcDNA

synthesis

and PCR amplification in 1.8% agarose gels (3 V/cm for 16 h in 0.1 M

Tris-borate [pH 8.3]-1 mM Na,H2EDTA-0.5 ,ugofethidium

bro-mide per ,ul). Theproducts ofthe cDNA PCR amplification

reac-tions are visualized by ethidium fluorescence on longwave UV

transillumination. (Top) Lane 1 correspondsto the 500-bp control

productDNAprovided with the PCR GeneAmp kit. Lanes2, 3, 8,

and9show PCRproducts amplified fromHeLa cellmRNA

prepa-rationswith oligomers 365 and366, as described inMaterials and

Methods.Lanes 8 and9showproducts frommRNAisolated from

uninfected HeLa cells. Theseextraneous products also appear in

lanes 2 and3,whichwere derived from Ad3-infected HeLa RNA.

Lanes 3and 9 showthesameproductsasinlanes 2 and 8following

digestionwithBamHI andHindIIIrestriction enzymes.Ad3

E1A-specific products, susceptible tocleavage by BamHI andHindIII,

appearasthelargest speciesinlanes 2 and3 and correspondtothe 13S cDNAproduct. A slightly smaller and less abundant species,

the 12ScDNAproduct,is alsopresentbut difficulttoseeunless the

gels are overloaded with products. The empty lanes 4 and 5

representnegativecontrols for mock reactions lacking onlyreverse

transcriptase,andlanes 6 and 7representnegative controlsfor mock

reactionslacking only theoligonucleotide primers.(Bottom) In this

overloaded gel, lanes 1, 2, and3 correspondto lanes 1, 2, and 3 in

the toppanel, respectively. Lane4 shows length markers derived fromapBR322DNAHpaIl restriction digest. ThetwoAd3 E1A-specificcDNAproductsappear as630- and535-bp speciesin lane 2.

The smallerspecies corresponds to 12S Ad3 ElA cDNA product

turesand repetitions of the thermal cycling, and concentra-tionsof monovalent and divalent saltsorTaqDNA polymer-ase.

The products of reactions initiated with mRNA from Ad3-infected cells containtwoadditional species, about630 and 535bp. The ratio ofyields of the larger and the smaller products, estimated by ethidium fluorescence in the gel, is typically about 10:1. The lengthsarethoseexpectedforthe Ad3 ElA cDNA products, predicted from the Ad3 DNA sequence, the specific oligomer primers, and the intron

junctionsdescribed for Ad7 ElA mRNA (7).Treatment with

BamHI and HindlIl reduces the lengths of the two Ad3-specific products by about 75 bp each. The sizes of the nonspecific products do not appear to change following treatmentwiththese restriction enzymes. Apparently, only the two Ad3mRNA-specific products have theBamHIand HindlIl restriction sites associated with the amplified re-gions of the 5' and 3' exons of the Ad3 ElA gene.

The BamHI- and HindlIl-treated cDNA products from Ad3-infected mRNApreparationswereligatedwith BamHI-andHindIII-restricted pEMBL18(6)vectorDNAforcloning of the tentatively identified Ad3 cDNA sequences. Several transformants with inserts of 550 or457 bp were selected. DNA sequence analysis confirmed these to be cDNA frag-ments corresponding to the Ad3 ElA gene 13S and 12S mRNAspecies, respectively. Ad3 ElA 12S and 13S cDNA insertswereexcised from the pEMBL18 vectors and substi-tuted for corresponding genomic DNA sequence between theBamHI and HindIII sites of the modified Ad3 ElA gene expression plasmid (51) (see also Materials and Methods). These newAd3ElAcDNA constructs were thenevaluated for transcriptional regulation activities by cotransfection analysis of transient-gene expression with CAT reporter gene expression plasmids. Ad3 ElA-CAT reporter gene

expressionwasanalyzed incells cotransfected with the 12S

or 13S Ad3ElA cDNAexpression clone (Fig. 4).

Significantlylowerlevels of CAT geneproduct,29% of the

controlvalues,wererecovered from cells cotransfected with the 12S cDNA clone than from cells transfected with the Ad3 ElA-CATplasmid alone. This is consistent with

enhancer-targeted repression of transcription, expected for the 12S

cDNAspecies and theAd3 ElA promoter used for expres-sion of the CAT gene. The activities of four different constructs of the Ad3 ElA 12S cDNA expression plasmid

wereindistinguishable in similar cotransfection assays with the repressible Ad3 ElA-CAT, Ad5 ElA-CAT, and pSV2-CAT(10)reporterplasmids(resultsof thesecomparisonsnot shown).

Transactivation of the Ad3 ElA-CAT plasmid is evident in cotransfections with the Ad3 ElA 13S cDNA expression clone(Fig. 4). Three different constructs of the Ad3ElA 13S

cDNA expression plasmid showed similar transactivation

activities, 20-fold stimulation over controls, in cotransfec-tion assays under these conditions. A fourth 13S cDNA clone hadtwo- tothreefold-lowertransactivation activity in such assays, but itwaslaterdeterminedtohave sustained a single base change

(C1142-

T), a missense mutation (H--Y) near the right end of conserved functional domain 3 (results not shown). This mutation is likely to have been introduced early during the rounds of DNA synthesis and

and istypically recovered in amounts lessthan 10%of the larger, 13S-specific cDNA product. Each is reduced by about 75 bp following cleavage byBamHIand HindIII (lane 3).

1

2 3 4 5

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DIVERSE ADENOVIRUS ElA GENE AUTOREGULATION 3839

a

-

I-4c

Uc

1000

100

10

Alone +12S cDNA +13S cDNA

FIG. 4. CATgeneexpression in cell cultures cotransfectedwith Ad3 ElA-CAT (10 ,ugperplate) and the 12Sor13ScDNA clone of

the Ad3 ElAgene (3 ,ugperplate). Repressive activity ofthe12S cDNAclone isevident from the lower level of CATgene product

harvested 42 h after cotransfection. The average amount ofCAT product recovered from these cultureswas only 25to30% ofthat recovered from cells transfected by Ad3 ElA-CAT alone. Transac-tivationactivity of the 13S cDNA clone appears aslevels of CAT

product 20 timesgreaterthan in cells transfected by Ad3 ElA-CAT alone. Error bars indicate intervals 3 standard errors above the

averagelevels of CAT (6to15 samplesperaverage).

amplification with avian myeloblastosis virus reverse tran-scriptase andTaq DNApolymerase.

ElAgeneautoregulationwith 12S and13S cDNAclones of Ad5 and Ad3.The availability of 12S and 13S cDNA clones of both theAd5 and Ad3 ElAgenesenablesanalysis ofthe

positive and negative autoregulation ofthetransient

expres-sion ofthesegenesintransfectedcells. Parallel experiments

permit comparisonsof ElApromoterresponsesand effects

of ElA gene products in homologous and heterologous

cotransfections. Thedata summarized in Table 2 represent

multiple transfection experiments performed with several different batches of HeLa cells. Each set of transfections

wasperformed with either the Ad5orAd3 ElApromoterfor CAT gene expression, at 10 ,ug of ElA-CAT plasmid per

plate, alone or cotransfected with the 12S or 13S cDNA

clone of the Ad5 orAd3 ElAgene at 3 ,ugperplate. CAT

gene product activity was assayed in extracts from the cultures 40to44 hafter transfection.

Theaverage levelsofCAT fromtransfected cell cultures

presentedinTable 2 havelarger variances than would have been the case if the experiments were performed as

repli-cates with a single batch preparation of HeLa cells for

transfection. Our otherdose-response and time course

ex-periments (Fig. 5 and 6) (reference 18 and unpublished results) indicate that thisparticularsetof conditions-batch ofcells, inputsofreporterplasmid (10 ,ugperdish)andEIA

geneplasmid (3 ,ugperdish), glycerolshocktreatment,and time aftertransfection-typicallyunderestimates the poten-tial levels for ElA autotransactivation. With 10to100 times less ElA (13S cDNAor genomic) plasmid input, transacti-vation often approaches 500-fold-higher expression of the Ad3 ElA-CATgene. Nevertheless,the resultspresentedin Table 2 indicate that the differentresponsesof Ad3 andAd5 ElApromoters are statistically significant (P < 0.05).

Sta-tistically significant differences are also observed for the transactivatingactivities of the Ad3 andAd513SElA cDNA

genes.

Homologous cotransfection experiments, in which the ElA-CAT and the E1A-cDNA clones have the same

pro-moters, generate CAT product which should reflect the levels oftranscription from that ElApromoterin the

trans-fected cells. With this consideration, the data in Table 2 supportthe followingconclusions.

TABLE 2. ElA-CAT gene expression in cells transfected alone orwith homologous and heterologousElA12S

and 13S cDNAclones"

Cotransfecting No. of CATactivity

Transfecting plasmid plasmid Independent (U/plate)

Single transfections

Ad3 ElA-CAT 15 6.3± 0.3

Ad5ElA-CAT 15 6.7± 1.2

Homologous cotrans-fections

Ad3 ElA-CAT Ad3 12S cDNA 6 1.8 ±0.2 AdS ElA-CAT Ad5 12ScDNA 6 0.6 ±0.1 Ad3ElA-CAT Ad3 13S cDNA 9 113.7± 13.0 AdSElA-CAT AdS 13ScDNA 9 16.2 ± 1.9 Heterologous

cotrans-fections

Ad3ElA-CAT AdS 12S cDNA 3 1.6± 0.1 AdS ElA-CAT Ad3 12S cDNA 6 1.2± 0.5 Ad3 ElA-CAT AdS13S cDNA 9 221.7± 37.7 AdSElA-CAT Ad3 13S cDNA 9 2.9 ± 0.6

a HeLa cell cultures were transfected by the procedure described in Materials and Methods. Each culture dish of HeLa cells received 10Vgofthe

indicated ElA-CAT reporter plasmid and, if cotransfected, 3 FLg of the

indicatedElAcDNAexpression plasmid.The number ofindependent trans-fections isspecifiedwith the mean and standard error for CATactivityper

plate,recovered 40 to 44 hafter transfection.

(i) Similar levels of ElA-CAT transcription are supported by the Ad3 and Ad5 ElA promoters in the absence of ElA

cDNAexpression clones.

(ii) Lower levels oftranscription from the Ad3 and Ad5 ElA promoters occur in cells homologously cotransfected with 12S ElA cDNA expression vectors. This autorepres-sion assay led tolevels ofCATproduct whichwere 9 and 29%, respectively, of those expressed by the Ad5 and Ad3 ElA-CAT vectors alone. These data suggest, as reported earlier(18),that theAd5 ElA promoter ismore sensitiveto autorepression than the Ad3EIApromoter.

(iii) The autoactivated expression of CAT from the Ad3 ElA promoter with the homologous 13S cDNA of Ad3 ElA isatleastseventimes greater than the homologous transac-tivation of the AdS ElA promoter with the 13ScDNAofAd5 ElA.

Comparisons of the results ofhomologous and

heterolo-gouscotransfectionexperiments leadtoadditional

observa-tions, includingthe following.

(iv) Transcription of Ad3 and AdS ElA promoters is

repressed by expression of either the Ad3orAd5 12ScDNA gene. The levels ofrepressioneffectedbytheAd3 and Ad5 12S cDNA clonesare notstatistically distinguishable.

(v) The Ad3 ElA promoterresponds muchmorestrongly

to transactivation bythe Ad3 or AdS 13S ElAcDNAthan does the Ad5 ElA promoter, as reported earlier

(18).

The Ad5 13ScDNAgene isatleast two times more effective than the Ad3 13S cDNA cloneas atransactivator of the Ad3 ElA promoter.

The responses of the Ad3 and Ad5 ElA promoters to

homologous or heterologous autorepression are

similar,

al-though the Ad5 ElA promoter appears to be

slightly

more sensitiveto repression. The

autorepressive

activities of the Ad3 and Ad5 ElA 12S cDNA genes appear to be very

similar. VOL.66, 1992

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3840 COGAN ET AL.

0.4 0.8 1.2 1.6 2.0 2.4 2.8 3.2 _{0.4 0.8 1.2 1.6} 2.0 2.4 2.8 3.2 pg

Co-transfecting

EIAPlasmid

yg

Co-transfecting ElA Plasmid

FIG. 5. Effects of Ad5 hr4 and hr3 mutants (domain 3, transactivation)on the cotransfection CATgene expressionfrom Ad3 andAd5 ElA-CATvectors.(Left) Dose-response analysisofcotransfectingAd5 ElAplasmidsforCATgeneexpressionunder control of theAdSElA promoter. Thegenomic (gen) form of thewtAdS ElAgeneisweaklystimulatingtoCATgeneexpressionatverylowinputandrepressive

athigherinputscomparedwithexpression in cells transfected with theAdS ElA-CATplasmidalone. Therepressive12S cDNA ofwtAdS strongly decreases the level ofexpression from the CAT reporter vector, as is also the case in cells transfected with genomic clones representing the domain 3mutantshr4 andhr3.Themutantsappeartoabrogatewhat littletransactivating activityis revealedbythisassay

with the poorly activated AdS ElA promoter. (Right) Corresponding experiments performed with the strongly activated Ad3 ElA-CAT

vector.Thechangein ordinate scale reflects theveryhighlevels of transactivation obtained in cells cotransfected with thegenomicAdS ElA

gene. Again, the hr4 and hr3mutants are severelydebilitated in their capacitytotransactivatethe Ad3 EIApromoter,although the data indicate that the hr4 lesion is somewhat less deleterious.

Indistinctcontrast, the AdS 13SElAcDNAgeneproduct

appears to have about 15-fold-higher transactivation activ-ity, with the transactivation-responsive Ad3 ElA promoter

target, than does the homologous Ad3 13S cDNA gene

product. We draw this conclusionfrom acomparisonof the results from different cotransfections. Lower levels of AdS than Ad3 EIA 13S cDNA gene expression take place in cotransfected cells, as judged from the relative levels of

homologous ElA-CAT expression. Nevertheless,

two-times-higher levels of Ad3 ElA-CAT transactivation are

observed in cells cotransfected with the Ad5 than with the Ad3 13S cDNA gene. The results from different

heterolo-gous and homologous cotransfection experiments, using single batches of HeLa cells and lower inputs of the 13S cDNA expression plasmids, suggest that this difference in Ad5 and Ad3 13S ElA transactivation activities may be

significantly greater than maybe inferred from the data in

Table 2alone.

We acknowledge that alternative explanations of these results canbe framed on the basis of variations in the ElA

protein levels (stability, turnover, translatability,

compart-mentalization) in cells transfected with the various ElA expressionmutants. Lackingreagentsand data pertainingto

the differential stability of variant EIA proteins, we prefer

the plausible assumption that levels of transcription,

re-flected by the relative accumulations of the CAT reporter

gene product, are the primary determinant of variation in

EIA protein levels. A second concern arises in our use of

ElA genes under the control of diffcrent ElA-responsive

ElApromoters. Todate, however, noeukaryoticpromoter which is quantitatively refractile to the positive and/or negative transcriptional regulation effected by ElA gene

products has been described.

PositiveElAgeneautoregulation and conserved functional domain 3. It is plausible that the conserved functional domains of the adenovirus ElAgene, associatedwith posi-tive and negative regulation of the transcription of other early viral and cellular genes (14, 30, 33), act in similar fashion in the transcriptional autoregulation of the ElA

gene. Well-characterized plasmids bearing missense

muta-tions in functional domains 2 and 3 ofthe AdS ElA gene

were generously made available to usto test this

assump-tion. In addition, these new cotransfection experiments provided further insight into the different balances of posi-tive andnegative autoregulation which characterize the ElA

genesofAd5 and Ad3.

Figure 5 presents the results ofcotransfections of HeLa cells with the Ad3 and Ad5 ElA-CAT expression vectors

and EIA genes of the Ad5 mutants hr3 and hr4. These missensemutations, characterized by the work of Glenn and Ricciardi (9), represent single-base changes in functional domain 3 of theAd5ElAgene. They compromise

transac-tivation ofElA-dependent earlypromoters, suchasthose of

the adenovirus E2, E3, and E4genes.The leftpanel of Fig. 5 showsthedose-responsecurvesofAd5 ElA-CATreporter

gene expression with increasing amounts ofcotransfecting ElA expression plasmids. Genomic wt Ad5 ElA provides limited transactivation of the Ad5 ElA promoter, but CAT 15

a.

U

I-U

*0

c 10

51

0

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[image:8.612.90.535.74.322.2]

DIVERSE ADENOVIRUS ElA GENE AUTOREGULATION 3841

#-M

0.0CD 200-0

:'E

o 100

I-LA0

S 0

0._

U

co

I-U

-000 0.0

1-. 0

25000

*-3

* 1500.

-1000'

Uc

0.4 0.8 1.2 1.6 2.0 2.4 2.8

0.4 0.8 1.2 1.6 2.0 2.4 2.8

yjg Co-transfecting

El APlasmid

FIG. 6. Effects of theAd5pm936 andpm953mutations(domain2,

repression), ascotransfecting 13S cDNA,on CATgeneexpression

from thepSV2-CATand ElA-CAT vectors. (Top)The 12S cDNA,

and to a lesser extent the 13S cDNA, of the wt Ad5 EIA gene

products do appear at higher levels than in the cotransfec-tions with the repressing wt Ad5 12S cDNA clone. The genomic clones expressing the mutant hr4 and hr3 ElA genes lead torepression levels similar to that of the wt 12S cDNAclone. This is consistentwith the loss of autotrans-activation activity duetothemutationsinfunctionaldomain 3 of the Ad5 ElAgene.

The right panel of Fig.5shows the corresponding analysis performed as cotransfections with the Ad3 ElA-CAT re-porterplasmid.The very different ordinate scalesofthe two panels reflect the much greater response of the Ad3 ElA promoter to transactivation by the wtAd5 EIA gene. The level of Ad3 ElA-CAT expression is much lower in cells cotransfected withthe mutanthr4and hr3ElAgenes. In the caseof the hr4mutant, thisexperiment suggestsdiminished but not entirely abrogated transactivation activity.

Negative EIA gene autoregulation and conserved functional domain 2.Point mutations infunctional domain2oftheAd5 EIA gene shouldcompromise the enhancer-targeted repres-sion activity ofboth 12S and 13S cDNAclones of the Ad5 ElA gene. Such mutations are represented by the Ad5 missensemutantspm936 and pm953, characterized by Lillie andGreen (29). Figure6 presents an analysis ofCATgene expressionfrom three different promoters incotransfections withwt and mutant formsofthe Ad5 EIAgene.

The toppanel of Fig.6shows the resultsobtained with the SV2-CAT (10) reporter gene. The early simian virus 40 (SV40) promoter has an enhancer element (72-bp repeat) which is a target for repression mediated by Ad5 EIA gene products. Thispromoterisnotgenerally recognizedasbeing responsive totransactivation by adenovirus ElA products. Except at the lowest inputs of cotransfecting 13S wt AdS cDNA, both cDNA forms of the wt Ad5 ElAgene repress transcription from the SV2 promoter. In cotransfections with 13S cDNA clones representing mutants pm936 and pm953, there appears to be no repression of the SV2 promoter but rather a modestly elevated level of CAT expression, only twice thatrecovered fromcells transfected with SV2-CAT alone.

Expression of the Ad5 ElA-CATreportergene was eval-uated with the same set of cotransfecting Ad5 ElA genes (Fig. 6, middle panel). The results areremarkably similarto

repressestheenhancer-dependent transcription from the earlySV4t)

promoterin theplasmid pSV2-CAT (41). The dose-responsecurves

revealno repression by the 13ScDNAclonescorrespondingtothe twomissense mutations of the Ad5EIA gene.(Center)Remarkably similar resultsobtained whenAd5ElA-CAT is usedasthereporter.

Again, the 12S cDNA of the wt Ad5 ElA gene represses the

expressioncontrolledby the Ad5EIA promoter. Thisrepressionis compromised by the missense mutations. The transactivation

ex-pected incotransfection with thewtAd5 13S cDNAgene isweak. The overallpatternsofresults with theSV40earlypromoterandthe

Ad5 ElA promoter are remarkably similar, includingthe ordinate

scales of CATactivity. (Bottom)Effects of the AdS EIAdomain 2 lesions on transactivation oftranscription from the Ad3 ElA

pro-moter. In this case, the 13S cDNA clonesof the wt Ad5 and the

mutants are all transactivating the reporter gene through its Ad3

ElApromoter.TheAd5 12ScDNAcontrolrepresses theAd3ElA

promoter. Theplasmidswith eithermutationofdomain2,however,

have lesstransactivation activitythan thewtAd5 13ScDNAgene.

This suggests modulating effects of mutations in one domain on

complementary activities associated with another domain in the sameprotein. Noticeagainthechangeinordinate scalein thispanel

toreflect thesignificantly higherlevelsofCATgeneexpressionfrom the transactivation-responsive Ad3 EIA promoter.

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3842 COGAN ET AL.

thoseobtainedwith SV2-CAT described above. Cotransfec-tion ofAd5 ElA CAT with the wt Ad5 13S cDNA shows onlyslightly greateractivation than cotransfections with the AdS 12S cDNA. The 13S cDNA clones of point mutants pm936 andpm953 donotappeartobecapable of repressing the AdS ElA promoter, but the levels of transactivation effectedby these mutants' ElA genes(as13ScDNA)arenot significantly greater than observed in the SV2-CAT

experi-ment.Theseresultswerealso confirmed inreplicate cotrans-fectionsperformed under the conditions described in Table 2, footnote a (results not shown). The Ad5 ElA promoter thus appears tobe intrinsically weak in its response to 13S ElA-mediated transactivation. Autorepression doesnot ap-pear to be a functional block to an otherwise stronger response to transactivation by the 13ScDNAgenesofAd5 wt, pm936, or pm953.

The lastexperiment of this serieswasdone with theAd3 ElA-CAT reporter gene (Fig.6,bottom).The ordinate scale inthis panel again reflects theintrinsically high response of the Ad3 ElA promoter to activation by the 13S cDNA products of the Ad5 ElA gene. wt Ad5 and the mutants pm936 and pm953 all lead to strong transactivation of the Ad3 ElA promoter when cotransfected as 13S cDNAs. A surprising result in this experiment is the lower level of transactivationactivityassociated with bothmutantforms of the Ad5 13S cDNA. The locus of each mutation is within functional domain 2, associated with enhancer-targeted re-pressionactivity. Apparentlyeven asubtlechange, suchas a missense mutation in one functional domain of the ElA protein,canmodulate other functions associated with

adja-cent ordistal domains.

Theapparently lower transactivation of Ad3 ElA CATby the Ad5pm936 andpm953ElA genes, shownin thebottom panel ofFig. 6, may reflect differences associated with the heterologous Ad3ElA promoteras atarget for transactiva-tion. This isnot particularly surprising, since the Ad5ElA promoter, usedto express the Ad5wtand mutant 13S ElA cDNAgenes inthese experiments, doesnotrespondwellto transactivation.

DISCUSSION

DNA sequencing of similar genes from closely related

organismsis often the firstpointof entry intoproblemareas

associated with prior and continuing evolution of gene structures and functions. Alignments of similar DNA or RNA sequences readily reveal a molecular topography of conserved and nonconserved domains in the gene of inter-est,reflectinga crosssection of variation of that gene within a contemporary population. Establishing consensus se-quences may prompt entertaining speculation about the derivation of contemporary variants from a putative com-mon precursor. The conclusions drawn from such analysis mayevenbe correct, ifthe contemporarypool of variants is derived from a relatively homogenous founder population. In general, however, this type of analysis is suspect, to the extent that one discounts the likely heterogeneity of the gene's precursors and temporal recombining intermediates. A more immediate andproductive application of compar-isons of DNA sequences is correlation of conserved or nonconserved regions of the gene with specific functions of the gene products. Such mapping of functional domains

develops by assay of activities associated with thegene as

derived fromwild-type strains, prevalent allelic variants, or spontaneous and induced mutants. There are risks associ-ated in studiespreoccupied with singularmodels of a

partic-ular gene's structure-function

relationships.

There are also

risks to be managed with the notion that the most

highly

conserved of variant DNA sequences must be associated with domains of essential function.

Structuraland functional dissection of the adenovirusElA gene. Consideration of the adenovirus ElA gene and its

autoregulation,asrevealed from studies of the Ad3 and Ad5

models, leads to several ironies. The ElA promoters of

subgroup B and subgroup C adenoviruses have identical

duplicate EIA enhancer elements

(12, 21, 40, 54).

Sponta-neous or induced deletions of the enhancer

region

cripple

Ad5 EIA gene

expression

and Ad5

infections,

but similar deletions in the Ad3 genome

impair

neither ElA gene

expression nor the course of

productive

infection. What

distinguishes these

highly

conserved elements as either

essential or dispensable for the

expression

of the Ad5 and

Ad3 ElAgenes?

Dissection of the adenovirus ElA promoter has revealed

multiple arrays of othercis

elements,

in

particular,

known

motifs for interactions with cellular

transcription

factors

such as E2F, CREB/ATF, and

enhancer-binding

factors.

Brute-force deletions of selected

regions

of the ElA pro-moter canpromote

ordinarily

subordinate elementsto more

dominant roles in the basal-levelor

autoregulated

expression

of the ElA gene. The

design

of assays, the selection ofa

particular model

adenovirus,

and the nature of

specific

mutageniclesions may confound

interpretation

of

structure-function relationships. Forexample, separate reports have asserted both DNA sequence

independence

and

specific

DNAsequence determinants involved in

positive

and nega-tive

autoregulation

of theElA promoter

(13, 18). Similarly,

conflictingreportshaveappearedwith respecttothe role of

promoter targets of other

ElA-dependent

transcription

units

(20,

22, 26,

27,

44).

Ad3 EIAgeneDNA sequence. TheDNA sequence of the Ad3 EIA gene,

presented

in

Fig.

1 and Table

1,

is very similar to that of the

closely

related Ad7 ElA gene. This

anticipated

result

provides

a limited set of sequence differ-enceswhich may be

predicted

tohave minimal

impact

onthe

autoregulated

expression

of the ElAgenes of these similar

viruses.Alignmentofthe Ad3ElA gene sequence with that ofAd5 follows the

precedents

established in the literature

(14, 30, 33,

54)

and prompts

generalizations

with

regard

to

the coincidence of functional domains

(generally mapped

in Ad2 or AdS) with the

highly

conserved domains of DNA sequences. The DNAsequence of the Ad3 ElA gene sup-portsourselection of this serotypeasa

representative

ofthe

B subgroupwith ourpresent

emphasis

on functional

diver-sityofElA gene

autoregulation

insubgroupBand

subgroup

C adenoviruses.

Diverged patterns of promoter and product roles in EIA

geneautoregulation. The results of

homologous

and

heterol-ogous cotransfections presented in this report suggest that extensivefunctionaldiversityhasdeveloped inthe autoreg-ulation of the Ad3 andAd5ElAgenes.Atthesimplest level,

this diversity isreflected bylower expression levels of the

Ad5 ElA gene than of the Ad3 ElA gene (51). Ourrecent report (18) suggested that a primary determinant of this difference intheautoregulated ElAgeneexpressionof Ad3 andAd5 liesin theupstreamDNAsequences,whichspecify

different responses ofElA promotersto 13S ElA-mediated transactivation. The resultspresented in Table 2and

Fig.

6 further support this conclusion. The Ad5 ElA promoter behavesmoreliketheSV40earlypromoter, which is

gener-ally

thoughttobe

repressed

andnot transactivatedatall

by

the action of adenovirusElA gene

products.

The Ad3ElA J. VIROL.

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DIVERSE ADENOVIRUS ElA GENE AUTOREGULATION 3843 promoter can be repressed by 12ScDNA products, but it is

remarkable for its strong response to transactivation by 13S EIA gene products.

These results suggest a complementary coevolution of the ElA promoters and their autoregulatingEIAgene products. The poorly transactivated Ad5 ElA promoter supports expression of a 13S cDNA product with greater transacti-vating activity. The opposite situation is found in the Ad3 ElA gene. The strongly transactivated Ad3 ElA promoter supports expression of a 13S cDNA product with lower transactivating activity.

Theperspective on ElA gene autoregulation and comple-mentarypatternsofpromoter-product coevolution provokes interest in the regulated expression of other early genes of Ad3 andAd5.Adenoviral early-gene expression is generally recognized to be ElA dependent. In the case of Ad5, early-gene expression takes place in a milieu of limited quantities of the apparently strong Ad5 13S EIA transacti-vator. Basal-level expression ofAd5 early genes appears to besufficienttoevadeabortive infection (at high multiplicity)

with Ad5 d1312, a mutant which lacks the AdS ElA gene

altogether.

Inthe case of Ad3, the early genes are expressed in the presence of very large quantities of what appears to be a weaker 13S EIA transactivator product. The ElA autoreg-ulation mutant Ad3 hrlS (23, 25) invites comparison with

Ad5d1312. Although Ad3 hrl5 encodes wt Ad3 EIA

prod-ucts, the result is apparent shutdown of further EIA tran-scription. Limited expression of the ElA gene supported by the mutant promoter is lethal; infection is aborted, with minimal cytocidal effects even at very high multiplicity. This mayimply that theexpression of other Ad3 early genes has coevolved to a state of greater dependence on the ElA transactivation function.

Another provocative problem is presented by the differ-ences in ElA product activities and responses of the co-evolved EIAgene promoters. Many host genes of adenovi-rus-infected or -transformed cells respond to positive or negative regulation by adenovirus ElA gene products. The promoters of these cellular genes donot typically resemble the particular arrays of enhancer elements or E2F motifs which characterize ElA promoters. We should anticipate that functional divergence of adenovirus ElA genes, re-vealed in our studies of autoregulation, is likely to be manifested in highly variable regulation of cellular genes, which are not constrained to coevolvewith the adenovirus ElA gene. As suggested in the introduction, such variation may play determinant roles in the diverse tissue tropisms

andpathoepidemiology of humanadenovirus serotypes and

subgroups.

Interdependenceoffunctional domainsand activitiesofEIA proteins. A structural relationship of conserved domains 2 and 3 appears to mutually influence their complementary functions intranscriptional regulation.The resultspresented

in Fig. 6 illuminate this point. The missense mutations in

Ad5pm936

and hr953compromise enhancer-targeted

repres-sionactivity, associated with their loci in conserved domain

2. However, the assayswiththe transactivation-responsive

Ad3 ElA-CAT reporter gene showsignificantlylower trans-activation with these mutant Ad5 ElA genes. The single

amino acidchanges corresponding to their missense muta-tions appear to change the overall structure of the mutant 13S cDNAproducts and thereby compromise (in

quantita-tiveterms) the function of the distal transactivation domain. ThisleadsustoconsiderthattheDNAsequencebetween conserved domains2and3(black wedgeinFig. 2)isaregion

of the EIAgene which is balanced in coevolution with the upstream, transactivation-responsive elements of the

EIA

promoter. This is reminiscent of the structure-function rela-tionshipassociated with mutant Ad3 hrlS and its second-site revertants during passage of viral stocks in this laboratory (23, 25). The level of EIA gene expression in Ad3 hrlS represents effective transcriptional shutdown of the gene, while the same promoter overexpresses the ElA genes which bear reverting deletions in the variable region between conserved functional domains 2 and 3. Perhaps this mutant and its revertants functionally recapitulate aspects of the evolution of ElA gene autoregulation among adenovirus serotypes in the field. These aspects are the apparent reiter-ation and divergence of cis elements in the ElA promoter and deletions (or insertions) in variable regions~separating conserved, functional domains in the ElA gene-coding re-gion.

ACKNOWLEDGMENTS

This investigation was supported by Public Health Service grant CA-34126 from the National Cancer Institute. R.K.H. and S.N.J. were also supported in part by Training Program grant CA 09385 from the National Cancer Institute. Oligomers 365 and 366 were synthesized in the laboratory of R. Stephen Lloyd, Center for MolecularToxicology, Vanderbilt University School of Medicine, supported in part by U.S. Public Health Service grant ES 00267. Fluorescent dideoxyterminators, other sequencing reagents, the Genesis 2000 automated DNA sequencer, and Generator oligonu-cleotidesynthesizer were made available to our laboratory as part of the Collaborative Research Agreement between E. I. du Pont de Nemours & Co., Inc., andVanderbilt University School of Medi-cine. We thank Ed Chait, Richard Baxter, and Paul Levy for the important roles they have played in the initiation and support of that program.

We gratefully acknowledge the Ad5 ElA gene expression plas-mids(missense mutants) provided by Robert Ricciardi of the Wistar Institute and Michael Green ofHarvard University (currently Uni-versity ofMassachusetts School of Medicine, Worcester). We also thank Ricciardi, Green, and Lloyd for thoughtful comments on reading early drafts of themanuscript. During these investigations, expert technical assistance was provided by Nancy Love, Patrick Adams, and SusanGlass.

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