Genetic dissection of the transactivating domain of the E1a 289R protein of adenovirus type 2.

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0022-538X/89/041495-10$02.00/0

Microbiology

Genetic

Dissection

of the Transactivating Domain of the

Ela 289R

Protein of Adenovirus

Type

2

M. L. FAHNESTOCKt AND JAMES B. LEWISt*

Fred Hutchinson Cancer ResearchCenter, 1124 Columbia Street, Seattle, Washington 98104, andDepartmentof Pathology, University of Washington, Seattle, Washington 98195

Received 25 October1988/Accepted 8December 1988

A series of linker-scanning, deletion, and frameshift mutations were made in the pm975 variant of the adenovirus type 2 Elagene,whichexpressesonly the largerof thetwomajor Ela proteins. Most of thesewere within the46-amino-acidsegmentuniquetothe larger Ela protein product (the289R protein),whichconfers

onittheabilitytoactivate intranstheexpression of othergenes.The mutationswererecombined into virus and

assayed by in vitro transcription in nuclei isolated from infected cells for their ability to activate the transcription of other viral earlygenesand of the endogenous hsp7Ogene. Mutant Elaproteins from which the

289R-uniquesegmentwasremoved by deletionortruncation didnotcompletely lose theabilitytotransactivate bycomparisonwithavirus which makesnoElaatall, indicating thatsequencesoutside this domainareactive in the positive regulation of transcription. The Ela mutations tested fell into several classes: those that increasedtransactivation of virtually allgenes,thosethat severely depressed transactivation of allgenes, and those thatdepressed transactivation only moderately. Each mutation had similar effectsontheexpression of all

transcription units tested, indicatingacommon processin theirtransactivation. However,somemutantsin the third categorydecreasedtransactivation ofsomeinducedgenes moreseverely than of others. Suchgene-specific

defectssuggesttheexistence of subclasses ofEla-responsive transcription units, consistent with the involvement of diverse proteins in the transactivation of different genes. Two specific structural components of the transactivating domain, a putative metal-binding element and a region with high potential for n-sheet

formation at itscarboxy-terminus,appeartobe importanttothetransactivation function.

The289-amino-acid-residue(289R) protein product of the adenovirus Ela gene initiates the viral infectious cycle by activating in trans (transactivating) the expression of other viralgenesatthelevelof transcription(31, 32, 46, 51). The Ela 289R protein also specifically transactivates a few

cellulargenes during infection andnonspecifically activates the expression of mostgenes with which the Elagene is

cotransfected in transient expression systems (reviewed in reference 3). In addition, Ela-mediated activation of tran-scription has been observed in vitro with extracts of Ela-containing cells(20, 53) and uninfected cellextracts supple-mented with bacterially produced Ela protein (43) and synthetic peptides corresponding to the Ela amino acid sequence (13). The mechanism for thisEla-dependent acti-vation of transcription isnotclear, but a number of

impor-tant parameters have been defined. The enhancement of transcription does not require the continuous presence of Ela protein (53), nordoes itrequire protein synthesis (13, 43). Ela-mediated transactivation is accompanied by an

increasein thenumber oftranscribing complexes rather than by an increased rate ofinitiation, shown by template

com-mitment assays (53) and experiments with inhibitors of reinitiation in in vitrotranscription reactions (25). Nuclease protection assays demonstrate qualitative and quantitative alterations in the interaction of proteins with upstream promotersequences ofsome (but notall) susceptible genes

in the presence of Ela (reviewed in reference 21), but the protein itself does not bind DNA directly (6, 24). These

* Correspondingauthor.

tPresent address: Department of Microbiology, University of

California, LosAngeles,CA 90024.

tPresentaddress: ONCOGEN,3005 FirstAvenue, Seattle, WA

98121.

observations have led to the prediction that the Ela 289R protein indirectly affects transcription by catalyzing the formation of stable transcription complexes, by increasing the availability of or altering the activity of one or more

existing cellulartranscription factors (2).

On the basis of amino acid sequence homology and splicingpatterns among the Elaproteins of various adeno-virusserotypes, threefunctional domains have been identi-fiedinthe289R Elaprotein, termed conserved regions 1, 2, and 3(CR1, CR2, and CR3), each shown by geneticanalysis tobe important forone oranother ofthe multiple activities of Ela(reviewedinreference33).Thepositive effects ofEla

ontranscriptionareassociatedwithCR3,the46-amino-acid

segment unique to its 289R protein product. The smaller, 243-residue (243R) Ela protein, which lacks this segment, does not transactivate (3). The 289R-unique sequence has beenpostulatedtobeafunctionallydistinctdomaincarrying an independent transcriptional effector activity, a

supposi-tion supported by a recent demonstration that a

49-amino-acid synthetic peptide corresponding to the 46 residues unique to the 289R protein plus 3 adjacent amino acids encodedbythesecondexoniscapableofefficiently activat-ing transcription fromanEla-induciblegenefollowing coin-jection into mammalian cells (27) or addition to in vitro transcription reactions (13).

Described here are aseries ofsubstitution, deletion, and

truncation mutations in the transactivating domain ofthe Ela289R protein, constructed with the purpose of investi-gatingthe mechanism by which this viral effector manipu-latescellulartranscriptionsystems.Theeffect of eachonthe Ela-mediated transcriptional activation of five inducible

geneswasmeasured.Itwasfound that all mutationsaffected the transcription of all five target genes coordinately, al-though several affectedtheexpression ofsometargetgenes

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more strongly than ofothers. These results are consistent with thecurrentmodel that transactivation isa

single

activ-ity effected through normal promoter sequences and the

multiple factorswhichbindthem,which differ for each gene

assayed (2, 8, 21). Inaddition, low-level activation ofsome geneswas seen even in the absence ofmost orall ofCR3,

and a protein carrying a mutation outside this segment

transactivated to higher levels than didthe wild-type

poly-peptide.

Thisobservationindicates that sequencesoutside of

the Elatransactivating domainmayalsoaffect

positively

the

transcriptional regulation

ofsomeofallEla-inducible genes. The effects of these mutationsarediscussed intermsof the

possible

structural

organization

ofthe

transactivating

do-main.

MATERIALS AND METHODS

Cells and viruses. HeLa

monolayers

were

propagated

in

Dulbecco modified Eagle medium (DME)

containing

5% fetal bovine serum. HeLa

suspension

cells were grown in Joklik modified minimal essential medium

containing

5% heat-inactivated calf serum, 75 UofpenicillinG perml, and 50 ,ug of

streptomycin

perml. Human 293 cell monolayers

(12)

were

propagated

in DME

containing

10% fetal bovine serum. Penicillin G and

streptomycin

were included to 100

U/ml and 100

,ug/ml, respectively,

in most

experiments.

Adenovirustype2(Ad2), dl309,andpm975werepropagated

on

suspension

HeLa cultures. All other viruses were

prop-agated

on 293 monolayers. Experiments were performed

with virus particles purified by banding in CsCl gradients.

Stocks were characterizedforPFU per milliliterby plaque

assayon293monolayersandforvirusparticlespermilliliter

by measuring

the A260 of sodium dodecyl sulfate

(SDS)-disrupted

virions. All virus stocks used in theseexperiments showed

particle-to-PFU

ratios of 10 to 50. The values

obtained for all mutant stocks fell in the range of 20 to 30

particles/PFU.

Infectionswere

performed

asfollows.From 6x

106

to8x

106

HeLacellswereplatedper100-mm dish 18 hbeforeuse.

Medium was removed, and the monolayers were washed once with DME without serum and inoculated with 0.5 to 0.75ml ofdiluted virus.After

adsorption

for1hat37°C,the

inoculumwas removed, monolayerswere washed with

me-dium,

and 10 mlof freshmediumcontaining 5%fetalbovine

serum (Hyclone) was added back to each. Cultures were returned to the incubator until harvest. A comparison of

multiple

Ad2

preparations

showed that early-gene

expres-sion,

asmeasuredatthe levelofearly protein accumulation,

is mostcomparable from infectiontoinfection if numberof

particles

rather than PFU per milliliter is used as the measure of virus concentration. Either 300 or 1,500 virus

particles

per cell were

used,

as indicated for each

experi-ment.

Generation ofmutations. Linker-scanning mutations (30)

were generated in plasmid pPFpm975, a derivative of

pEKpm975

(31)which encodesonlythe13S Ela mRNA and its 289-residue protein product. To generatepPFpm975, an

1,100-base-pair

(bp)HpaI-EcoRI

fragment containingthe ad-enovirus Elb gene and M13 RF sequences was removed,

leaving

adenovirus sequences from nucleotide positions1to 1569 intact in a pBR322 background, flanked by PstI and EcoRIsites. TwofragmentsofpPFpm975weresubclonedto useassubstratesfordeletion.The1,193-bp fragment

extend-ing

from the left end of the viral chromosome

(PstI)

to an

AhaIll sitein the Ela intronwasligated intovectorpUC19

(48)

togenerate

plasmid

pEA. To prepare

plasmid pCH,

the

ClaI-Hpal fragment encompassingnucleotides 916 to 1569 of theviral chromosomewas

ligated

into

pUC18

(48).

Nested deletion sets weregenerated by progressive exo-nuclease III digestion of adenovirus sequences from the BamHI sites in parentplasmids pEAandpCH.Theplasmids

wererestrictedwith BamHI and

digested

with exonuclease IIIfollowedby S1nuclease. Deleted endswere

repaired

with Escherichia coli DNA

polymerase

I

large fragment

(Kle-now). BamHI 10-mer linkers(BethesdaResearch

Laborato-ries)

were

ligated

tothe deleted

ends,

followed

by

restriction with BamHI and PstI (pEA) or EcoRI (pCH) to release deleted inserts. These were then religated into

BamHI-PstI-cut

pUC19

DNAfor

pEA

derivativesor

BamHI-EcoRI-digested

pUC18

DNAfor

pCH derivatives,

and the

resulting

plasmids

wereusedtotransform E. coliHB101to

ampicillin

resistance. Deletion

endpoints

of viral DNA in the recombi-nants were determined

by

sequence

analysis

on double-stranded

plasmid

DNA. Mutant Ela genes were recon-structed from the

complementary

deletion sets by ligating

appropriate

gel-purified

inserts from

BamHI-EcoRI-re-stricted

pCH

derivatives into

complementary

BamHI-EcoRI-digested

pEA derivatives. The

position

of the linker within each mutantgenewas verified

by

sequence

analysis

on

plasmid

DNA.

Mutant EIA sequenceswererecombined into

H5dl309,

a variantofadenovirus type 5(AdS),

by

the methodsofStow

(45)andof Montelletal.

(31).

Resulting

viruseswere

plaque

purified

and screened

by

restriction

digestion

of viral chro-mosome DNA for the appearance ofa novel BamHI

frag-mentandfor the lossof the

wild-type

Hindlll G

fragment

on double

digestion

with the twoenzymes.

Immunoblotanalysisof viralproteins. Viral

proteins

were detectedintotal cell

lysates by

immunoblot

by

the methods of Palmer et al. (35). To prepare

lysates,

infected and

mock-infected monolayers were washed twice with cold

phosphate-buffered

saline

(PBS)

and then

scraped

into

elec-trophoresis sample

buffer(24a)at aconcentration of

approx-imately 107

cells per ml. Each

lysate

was sonicated with three 15-s

pulses

at 30 W and boiled for 3 min

prior

to fractionation by electrophoresis in 10% (for the

72,000-Mr

DNA-binding protein

[72K DBP]) or 15%(ElA)

polyacryl-amide-SDS

gels

and

electrophoretic

transferof

proteins

to

nitrocellulose

(BA;

Schleicher and

Schuell),

followed

by

reactionwith

antibody

and

1251I-labeled Staphylococcus

au-reus

protein

A. For detection of Ela

proteins,

a 500-fold

dilution ofa

polyclonal

antiserum raised in rabbits

against

a

bacterially produced

Trp-E:ElA fusion

protein

was used

(44). A

polyclonal

rabbit antiserum to the Ad2 72K DBP

(supplied by

Carl

Anderson),

diluted 1:1,000, was used for

detection ofthatviral

product.

Results were

quantitated by

densitometry

of

autoradiograms.

Tofacilitate

comparison

of

data among

experiments,

numerical values for

protein

accu-mulation obtainedby densitometryin eachexperimentwere normalizedtothatforpm975.

Transcriptioninisolated nuclei.

Transcriptional

activation of all viral

early

genes and several cellular genes were

assayed by run-on

transcription

in nuclei isolated from infected HeLa cells. HeLa monolayers were infected as described above. Infections with all viruseswere

begun

and cultures were harvested at

roughly

the same time on the same

day,

and

samples

were

processed

and

analyzed

as a grouponthesame

day.

Atthe timesindicated

postinfection,

cultures were harvested as follows.

Monolayers

were washed twice with cold PBS and scraped into 5 ml of PBS per

plate.

Nucleiwere isolated

by lysis

in 10 mMTris

(pH

7.4)-10mMNaCl-3 mM

MgCl2-3

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RNasin (Promega Biotec) per ml-0.5% Nonidet P-40 for 3

min on ice with occasional agitation. Nuclei were pelleted for 10minat 1,500 rpm(IEC model DPR-6000), washed once with buffer without Nonidet P-40, and finally resuspended in 40 lI of storage buffer (5 mM MgCl2, 10 mM Tris [pH 7.5], 0.5 M sorbitol, 2.5% Ficoll, 0.3 mM spermidine, 1 mM dithiothreitol, 50% glycerol) per plate. All samples were stored at

-70°C

until use.

Transcription reactions were carried out for 20 min at

30°C,

essentially as described by Weinheimer et al. (49). The purified, radiolabeled transcription products were collected by centrifugation, washed once with 70% ethanol, dried under vacuum, and suspended in 1 ml of hybridization buffer (0.3 M NaCl, 100 mM Tris [pH 8], 10 mM EDTA, 100,ugof sheared salmon sperm DNA per ml, 100 ,ug of yeast tRNA per ml, 1x Denhardt solution (29a), 50% formamide [49]). A

107

cpm amount of each sample was diluted to 1.5 ml with hybridization buffer and applied to filters and prehybridized for 6 to 24 h at 42°C in the same buffer, on which DNAs corresponding to genes to be assayed were immobilized. After hybridization for 24 h at42°C with mixing, filters were washed with 2x SET (0.3 M NaCl, 100 mM Tris [pH 8], 5 mM EDTA-0.1% SDS for 0.5 h at room temperature and then for 1 h at60°Cin0.lx SET-0.1% SDS. Following this stringent wash, filters were incubated in RNase A (5,ug/ml)

in 2 x SET for 20min, washed again for 1 h in two changes ofO.lx SET-0.1% SDS, and exposed to preflashed X-ray film at -70°C with an intensifying screen.

DNAs used as probes included the following. For Ela, single-stranded DNA from an M13 derivative containing the right strand of Ad2 DNA from nucleotides 267 to 1530 was used (pHEB4-R). For E1B, phage DNA from an M13 derivative containing the right strand of Ad2 (nucleotides 1767 to 2045) was used (mpSK-ElB). The probe for the E2 gene was phage DNA from an M13 derivative containing the left strand of the Ad2 chromosome fromnucleotides 21338 to 23924 (mpEcoB-EX). The E3 probe was phage DNA from an M13 derivative carrying Ad2 nucleotides 27000 to 28653, right strand only (mpHinH-EH). The probe used for E4 was single-stranded DNA "rescued" by M13 infection from a pBS+ vector (Vector Cloning Systems) into which nucleo-tides 33594 to 34933 had been subcloned(pHinF-KH). M13 DNA carrying nucleotides 6575 to 7262 of the Ad2 right strand was used as probe for the major late promoter (R1131 [10]). VA RNAs were detected with adouble-stranded probe (pVA [7]) containing Ad2 DNA between nucleotides 9831 and 11555. Plasmid pUR-HS70 (40), obtained from Joseph Nevins, was used to detect transcripts from thehsp70 genes. A cDNA to the human,-actin gene (pHF,BA-1 [14]) was used as a control in all experiments.

Densitometry was used to quantitate the intensity of hybridization to the various probes. The image from each slot was scanned three times, at a different position each time. Data for Ela, Elb, and E4 wereobtained from a 36- to 38-h exposure. All other measurements were derived from a 16- to 18-h exposure. M13mpl9single-stranded phage DNA, pBS+ single-stranded DNA, and pBR322 were included in all experiments as controls. No background signal was seen with phage or single-stranded pBS+ probes. pBR322 (and single-stranded DNA obtained by M13 rescue of the pBS-plasmid; not shown) did hybridize to some RNA product from infected cells, especially at later times postinfection. This background noise was subtracted from values obtained by densitometry before the data wereplotted.

Computer analysis of nucleic acid and protein structure. The GenePro program (Riverside Scientific) was used for

nucleotide sequence analysis and prediction ofprotein se-quence. Prediction ofprotein secondary structure was per-formed with both GenePro and PROTLYZE

(Scientific

and Educational Software).

RESULTS

Generationandcharacterization ofviruses mutated in Ela. Avariant Ela gene thatmakesonlythe 13S mRNA

(pm975

[31])wasmutagenizedsothatmutantsof the289-residue Ela protein could be studied in the absence of the 243-residue protein encoded by the 12S mRNA.

Eight

linker-scanning

mutants were generated which

replaced

in combination all but 14 aminoacidsofthe46 thatarepresentin 289R butnot 243R. In addition, a truncation mutantwas made

by

intro-ducingaframeshift in the

protein-coding

sequenceafter the first codonof the 289R-unique sequence, and twoin-frame deletion mutations were

made,

one

removing

42 residues fromCR3 andoneremovingCR2,aconservedsequence that is required forEla-mediated

repression

ofenhancer-driven

transcription (27, 37). The

position

of the linker in each mutant gene was verified

by

DNA sequence

analysis

on

plasmid DNA. Figure 1 shows the

predicted

amino acid

replacementsin the mutantEla

proteins

andtheir

positions

withinthe 289R sequence. Also included are twoadditional

Elavariants, pm1098and

pmlll2

(27),

for

comparison

with

previously described Ela mutants.

Infection was chosen over transfection as the means for

manipulatingmutantgenesfor severalreasons.

First,

viruses are veryreliable vehicles for

introducing

foreign

DNAinto cells. Second, infectionis the best wayto ensure consistent production of sufficient Ela

protein

for

analysis,

as insur-ance that differences in

protein

production

from mutant genes are not

responsible

for the

phenotypes

observed. Third, infection is the

only

practical

meansfor

assaying

the effects of multiple Ela mutations on the

transcription

of multiple target genes in the same

experiment.

Each mutant Ela genewastherefore recombined intothe chromosome of

virus d1309 (38). This virus is

essentially

isogenic

for Ela with Ad2 but isavariant ofa

closely

AdS.The mutant viruses used for the

experiments

describedhere are thus derived from Ad2 for nucleotides 1 to 1339 and from

AdS forthe remainder ofthe viral chromosome. d1309 was

included as a control in all

experiments,

as were Ad2/

5pm975,

the dl309derivative

carrying

the

pm975

gene from which the mutants described here were

generated,

and

d1312,

adl309derivative which does notexpress Ela dueto the deletionofmost of the genefrom the viral chromosome

(38).

Assay ofmutants for activation of viral andcellular genes. Two assay systems wereused to assess theeffects of these mutations on Ela-mediated transactivation. To measure

their effects on the

transcriptional

activation of a series of target genes, the extent of

transcription

of all viral

early

genes and the

endogenous

hsp70

gene, whose

expression

is increased in the presence of Ela

(22),

wasmeasured

directly

by

transcription

in nuclei isolated from cells infected with

wild-type andmutant viruses. In

addition,

the accumulation of the stableprotein

product

of theE2agene, the 72K

DBP,

was measured by immunoblot on

lysates

of infected cells from cultures infected in

parallel

to and harvested at the same timeas those usedfor assays of

transcription.

Results from one setof

experiments

are shown in

Fig.

2. Datafrom quantitation of the

autoradiographic

images

are shown in

Fig. 3 and4.

Direct measurement of

transcriptional

activity

in nuclei isolated from infected cells showed considerable

variability

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CR I CR 2 CR 3

F

.1l

I

108 19

GPV SMPNLVPEVI DLTCHEAGFP PSDDEDEEGE EFVLDYVEHP GHGCRSCHYH RRNTGDPDIM CSLCYMRTCG MFVYSPVSEP w.t. GP .GS DEEGE EFVLDYVEHP GHGCRSCHYH RRNTGDPDIM CSLCYMRTCG MFVYS DL2

E..I GSGIVYS DLI

E AGGYVEHP GHGCRSCHYH RRNTGDPDIM CSLCYMRTCG MFVYS LS1

E EFVLDYVEHP GHGCRSSGSG RRNTGDPDIM CSLCYMRTCG MFVYS LS5 E EFVLDYVEHP GHGCRSCHYH jj3 PDIM CSLCYMRTCG MFVYS LS6 E EFVLDYVEHP GHGCRSCHYH RRNT RIRM CSLCYMRTCG MFVYS LS6A E EFVLDYVEHP GHGCRSCHYH RRNTGDPI CSLCYMRTCG MFVYS LS7

E EFVLDYVEHP GHGCRSCHYH RRNTGDPDIM ISGSGMRTCGMFVYS LS8 E EFVLDYVEHP GHGCRSCHYH RRNTGDPDIM CSLCSG GMFVYS LS9

E EFVLDYVEHP GHGCRSCHYH RRNTGDPDIM CSLCYMRTCTIS LS10 ER GkS&CGAPR ARLQVLSLSP EEYGGPRYYV FALLYEDLWH VCLQ* LSlT

E EFVLDYVEHP GHGCRSCHYH RRNTGDPDIM CSLCYMRTC| MFVYS p.1098 E EFVLDYVEHP GHGCRSCHYH RRNTGDPDIM CSLCYMRTCG MFVY P.1112

FIG. 1. Positions of and predicted amino acid replacements in mutations generated in the 289Rprotein oftheadenovirustype 2 Ela gene. Domain structure, defined by sequence and functional analysis (32), is indicated schematically at the top,withtheamino acidsequenceof domains inwhichmutations were made indicated below. Amino acids substituted by aBamHI linkerareboxed.Dotsindicateresidues that havebeen lost indeletionmutants. Unrelated sequence at thecarboxy terminus of truncation mutantLS1T resulting from translationin an alternatereadingframeafterframe shift is underlined. The nameassigned to each mutant Ela gene and the virus thatcarriesit isindicated totheright of its sequence (w.t., wild type). The positions of the two point mutations in mutantspm1098andpmll2areindicatedbyabox aroundthe amino acid change that each produces.

in the transactivating potential of the various mutant Ela proteins. As expected, little or no expression of viral early genes ortheendogenoushsp7Ogene was seenin the absence of Ela products, represented by infection with dl312, while

wild-type controls dl309 and pm975 appeared to be roughly

equivalentin theirability to stimulate transcription from the genestested. LS9 and DL1consistently stimulated the least transcription from all genes, indicating a severe defect for transactivation in these mutant Ela proteins. Oddly, how-ever,neither of these mutations appeared to be as defective for transactivation as dl312. At the other end of the scale,

LS6and DL2 activated higher levels of transcription from all viral genes than did the wild-type controls. Enhancement of

transactivation of the hsp7Ogene was not seen at thistime

point with either of these mutants, but was noted with mutantDL2 at 6hpostinfection in other experiments (M. L. Fahnestock, Ph.Dthesis, University of Washington, Seattle, 1988).

Allothermutationsreduced Ela-mediated transactivation of the transcription units assayed to various degrees. LS1,

LS7,and LS8 were moderately defective fortransactivation of all target genes, while LS6A generally appeared to be nondefective for transactivation or only slightly so compared with pm975. Other mutations showed gene-specific defects. LS1T appeared to be almost equivalent in defect to LS9 and DL1 but showed a greater ability to activate the E3

tran-scriptionunit. LS5 was somewhat more effective at eliciting transcription from E3 and E4 than were LS9 and DL1, but wasequivalently defective for activation of the E2 andhsp70

genes. LS1O, pm1098, andpmlll2 were exceptionally de-fective for transactivation of E4. The specificity of this defect is underscored by the fact thatpm1098 and pmlll2

were minimally defective for the transactivation of all other genes assayed.

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- m . mm m - m - - . . a. m mm m FIG. 2. Comparisonoftranscriptionalactivityof viral and cellu-lar genes and accumulation of Ela and the E2a DBPs 8 h after infection of HeLa cells with 300 virus particles ofwild-type and mutantviruses per cell.(A) Exposures of immunoblots for Elaand E2aproteins. Thepositionsof both are marked atleft. (B)A 36-h exposureof filters fromanassayoftranscriptioninisolatednuclei. Eachcolumn represents a singlefilter hybridized with RNAfrom cells infected by a single virus, indicated at the top. The genes represented byeach rowofbands areindicatedatleft.MLP,Major late promoter.

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FIG. 3. Graph of numerical data from densitometry of autoradiograms of transcriptionassaysinisolated nuclei shown in Fig. 2. Bar height indicates theintensityof hybridization to the gene indicated during infection by a given virus, identified below. Numbers are the average of data fromtwoexperiments. theline within each barindicatesthe range of values that wereaveraged.

(VA) RNAs was included as a measure of efficiency of

infection bythedifferentviruses. Thetranscriptionunitsfor

these RNAsareactiveatearlytimes(41), and their expres-sion is Elaindependent at early times postinfection in our hands. Hybridization to this probe was quite strong in all

infectedcultures,indicating

successful

infectioninall cases,

and was insensitive to a-amanitin (Fig. 2), consistent with

transcription byRNApolymerase III(50). Hybridizationto all other probes was abolished when a-amanitin was in-cluded in the transcription reaction mix, consistent with theirbeingtranscribed byRNA polymerase II.

As shown in Fig. 4, the amount of E2a DBP detected

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E2a

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C6 - E E

Virus

FIG. 4. Comparison of accumulationofElaproteinsand E2a DBP inwild-type-and mutant-infected cellsat8hpostinfectionwith 300 particlespercell and6 hpostinfectionwith1,500particlespercell.Datafromdensitometricscansof immunoblotswerenormalizedtovalues forpm975. Valuesaretheaverageof results fromtwoexperiments with300particlespercell and threeexperimentswith1,500particlesper cell. The line within eachbarindicates the range of values that wereaveraged.

during these infections varied markedly, mirroring defects

seen in transactivation of the E2 gene at the level of transcription. Additional data from experiments performed

at higher multiplicity with harvest at 6 h postinfection are

included for comparison. LS5, LS9, LS10,DL1, and LS1T all showed very littleaccumulation ofDBPby8 h postinfec-tion. Slightly greater accumulation was detected in cells

infected with LS1, LS7, LS6A, pm1098, andpmlll2. LS6

andDL2showedincreased accumulation ofthe DBPrelative

to pm975. The relative protein accumulation found 8 h after

infection with several mutants (LS6A, LS7, LS8, pm1098,

andpmlll2) at 300 particles per cell was depressed

com-pared with the relatively moderate defect seen above in

transcription rates. This difference might reflect a lag be-tween transcription and processing of an mRNA and its subsequent translation into protein. DBPaccumulationwas

relatively greaterwith mutants LS6A, LS7, and LS8 after

infection with 1,500 particles per cell and harvest at the

earlier time point. Together, these observations confirm a

lesserdefectin mutantsLS6A, LS7, and LS8 such that early

deficits in transcription canbeovercomeby increased time

orgenedosage.pm1098andpmlll2were not included in the assays athigh multiplicity.

The same lysates in which the E2 DBP was quantitated

were also assayed for Ela products to verify that defects in

transactivation couldnotbeattributedtodecreasedlevelsof

theeffectorprotein inmutant-infected cells. Figure4shows

that theamountofElaprotein productspresent atthis time during

infection

differed by no more than threefold among

virusescarrying in-frame mutations whatever the conditions used for infection and that the variation observed did not correlate with the effect of the mutation on transactivation.

Osborne et al. reported that a decrease of 10-fold in wild-type Ela mRNA levels after infection at low multiplicity has no apparent effect on the progress of infection or on focus

formation in assays of transformation (34). Also, other

studieshave shown that inclusion of protein synthesis

inhib-itors during the early phase of infection with wild-type viruses decreases ElaRNA production (and thus

presum-ablythe amountof Ela protein) 10- to 20-fold but does not

decreasethe amounts of RNA transcribed from other early genes (26). These experiments were performed before

anti-sera to the Ela protein products were available, so

corre-sponding information on Ela protein accumulation is lack-ing. However, they support the idea that the level of

expressionofearlygenes is relativelyinsensitive to moder-ate fluctuations in the amount of Ela protein present and thus that the differences in transactivation of viral and cellular genes noted for the mutant Elaproteins assayed in theseexperiments cannotbeattributed to defects in

expres-sion ofthe mutant Elagenes themselves.

DISCUSSION

The simultaneous assay of expression of several

Ela-inducible target genes during the early phase of infection

with wild-type adenovirus and with variants carrying

muta-tions in the transactivating domain ofthe major289R Ela

protein showed a variety of phenotypes with respect to

Ela-mediated transactivation.Allofthemutations described

hereaffectedtheactivity ofthe Elaprotein,shownby either increasesordecreasesin thetranscriptionalactivity of early

viralgenes and the cellularhsp70gene.Removal of42ofthe 46 amino acids ofthe 289R-unique domain by deletion or

truncation produced a severe deficit in transcription ofall genes. This reaffirms the importance of that region to the

activationoftranscription by Ela.

However, no mutant was completelydefective for trans-activationofallgenesbycomparisonwithdl312,suggesting

that sequencesoutsidethetransactivating domain, and

prob-ablyamino-terminal toit, cancontribute topositive

regula-tion of transcription by the Ela protein, at least during

infection. Precedent for such a conclusion exists in a few reports(forexample, reference 6) that sometransactivation activity is associated withthe 243R as well as with the 289R

protein. Somegeneralproperty of the Elapolypeptide may be responsiblefor this phenomenon, such asits acidity (pl

4.5

[16]).

The activating domains of several yeast and mammalian transcriptional regulatory proteins have been

shown to beacidic (39), and acidic translation products of randomly cloned fragments of E. coli DNA can serve as

transcriptional activatorsinSaccharomyces cerevisiae (29). The transactivating domain, on the other hand, is distin-guished by numerous basicamino acidsand twoconserved

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sequencemotifs(see below)thatdiffer

considerably

from the restof the Ela 289R

protein.

Ittherefore seems

unlikely

that sequencesoutside this domain could influence

transcription

by

thesame mechanism.

In

general,

each of the Ela-inducible genes tested was

affected

similarly by

a

given

mutation withinCR3, aswould be

expected

if

CR3-dependent transcriptional

activation of all genes involvedacommonprocess. For

example,

linker-scanning

mutantLS9 showed the least

ability

toactivate all genes

tested,

while LS6 enhanced the induction of

transcrip-tion of all viralgenes. Ini

addition,

thepatternof response of each gene tothe full spectrum ofmutationswas

similar,

as

judged by

visual

inspection

of

Fig.

3. However, in a few cases an Ela mutation affected the

expression

of

specific

genes

preferentially.

Mutant LS5 was

relatively

more defi-cient for activation of E2 and

hsp7O

thanfor activation of the E3 and E4 genes. Alterations near the

carboxy

terminus of the

transactivat'ing

domain

(LS10,

pm1098,

and

pm1112)

substantially

decreased induction of E4 but had little effect on the other genes

assayed.

These observations

-could

be rationalized if Ela-mediated activation of

transcription

in-volved interactions among

multiple

proteins,

with different

sUibsets used for different genes. A

single transcription

"factor" common to all

Ela-susceptible

genes is thus not

required

to

explain

the effect of the viral

protein

on the

activity

of

disparate

promoters transcribed

by

distinct

poly-merases,

only susceptibility

to a common Ela function which is eliminated in the

severely

defective mutants

LS9,

DL1,andLSlTand enhanced inLS6. It is

impossible

tosay from this information whether direct interaction between Ela and

multiple

factors is reflected in these results or the interaction ofsome other constituent of the transactivation

pathway

with

multiple

other

proteins.

The mutants de-scribed here may be useful in

answering

such

questions

in the future.

Two genes, Ela and Elb, conformed to the

general

pattern of increased

expression

withLS6 and DL2 butwere

largely

insensitive to the mutations that decreased transac-tivation of the othertargetgenes.Furthermore,in thecaseof

Ela, the amount of Ela

protein

observed in infected cells

was not

proportional

to

transcription

levels. For

example,

the increased

transcription

seenwithLS6and DL2 wasnot reflected in

higher

levels of Ela

protein.

This suggeststhat the Ela gene is

regulated

in a

complex

fashion at several

levels,

possibly including

posttranscriptional

control at the level oftranslationor

protein stability.

TheBibgene,

although

essentially

inactive in the absence of Ela

expression (during

infection wilth d1312), was little affected in a

negative

sense

by

any of the mutations in the transactivator

protein. Insensitivity

of the Bib gene to mutations in Ela has been

reported

previously

(4, 42). The cause of this

phenomenon

is unknown. It is

interesting

that the structureof itspromoteris

simpler

thanthatof the other viral

early

genes,

consisting

of

only

a

binding

site for

transcription

factor

Spl

andaTATAbox(52). Itcontainsno inducible elements, which are associated with all other adenovirus

early

transcription

units(8, 21). The

peculiarities

of its

regulation by

Ela

might

be duetothis

unique

promoter

structure or

perhaps

to cis effects from its

proximity

to the Ela

transcription

unit andupstreamenhancer elements (17, 19). The latter

possibility

is

supported by

the

especially

dramatic

(threefold)

effect on Bib

transcription

of mutant

DL2, which is defective for enhancer

suppression

(see below).

An additional

layer

of

complexity

in activation of

tran-scription by

Ela isindicated

by

theeffects ofmutantDL2on

that process. Enhanced

transcription,

above levels induced

by

the

wild-type

289R Ela

protein,

from all geneswas seen

during

infection witha virus

carrying

this mutation. CR2, a

domain which is involved in the

negative

regulation

of

metal

binding

beta sheet

140 85

6EEFVLDYVEHPGH

HYHRRNTGDPDIN

NMRTCG.MFVYS

R E L A T V

E

T R A N S

R

p T 0 N

2.50_

2.25_

2.00_

1.75_

1

.50_

1.25_

1.00~

0.

75_

0.

50_

0.25_

0.00

I

I I

975 312 LS1

I I Ia a --- I I a I

LS5 LS6 LS6A LS7 LS8 LS9 LS1O01098 1112

VIRUS

0E3

ME4

FIG. 5. Positions of structural elements within thetransactivating domainasdefinedby sequence andcomputer analysisand assays of transactivation potentialofmutant Elaproteins. The46-amino-acid sequence of the289R-unique segmentisalignedabove agraphof the relativetransactivatingabilities ofmutantscarryinglinker-scanningandpointmutations, sothat the effect of each mutationontranscription is shown below eachposition.Datafortwoof the sixEla-responsivegenesassayedareshown.975,pm975;312, d1312; 1098,pm1098;1112,

pm1112. Transcriptionis shown relativetothat inpm975.

.1.

I I

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Pk A

A

LIAIAMAIi

AV

Ad 7

m3

*Y

C

_. NA-

VI-vX

&IAi-A

v

iAd

AA.

AALMiAdl

12

o J1H'I'. 0

O3

00

0 000OCD

t L

AnA

A A #

cd Ad2

2

8'9

R

AA

nfA

.,. A | Ad

4~~4~ Ad 2

t1 / ~243R| ~~~~ 1

50 100 150 200 250

Amino acid position

FIG. 6. Computer analysisofp-sheetpotentialinthelargeEla proteinsofdivergentadenovirusserotypesand the 243Rproteinof

Ad2. Boxes above each graphindicate sequences with calculated potentialsufficient topredictp-sheetformation. Ahorizontal bar in

eachgraphindicates thepositionof thesegmentuniquetothelarger

Elaproteinineachserotype.Curvedarrowsindicate thepositions

ofthe conservedP-sheetstructure. Astraightarrow indicates the

positionof thesplice junctioninthe Ad2 243Rproteinwhichdeletes

CR3.

enhancer-driven transcription by Ela proteins (27, 37), is deleted inDL2. In transient expression assays, cotransfec-tion with the DL2

Ela

mutant results in greatly enhanced transcriptionfroma reportercatgene drivenby the simian virus 40 enhancer relative to that seen aftercotransfection with thepm975 or LS6variant, consistent with the loss of enhancer repression capabilities with this mutation (Bill Demers,personal communication). Since Elaproteinlevels in cells infected with the DL2 virus were no higher than those seenduringinfectionwithpm975ord1309,theeffect of DL2ontransactivationcannotbeexplained as the resultof hyperexpressionof Ela from reliefofnegative autoregula-tionbyEla geneproducts.Amorelikely explanationfor this phenomenonisthat theexpressionofallgenesassayed here, andtherefore possibly all Ela-sensitive genes, isunder the influence ofan enhancerelement(s). This isconsistent with

a recent report that an enhancer at the left end of the adenovirusgenome positivelyaffects thetranscriptionof all viral genes(18).

Comparison of sequences among the Ela proteins of severaladenovirusserotypesdefinestwostructuralmotifs in CR3 thatare also implicated in transactivation by the

mu-tantsdescribed here.Theirpositionsareindicated inFig.5. The first isaputative metal-bindingdomaincomposedoftwo cysteine pairs separated by13amino acids, many ofwhich

are basic or hydrophobic in nature (1). Mutants carrying

mutations which affect the cysteine pairs and amino acids

immediately carboxy-terminaltothem(LS5, LS9, and LS8)

are highly defective for transactivation. Interestingly, the

Elaproteins produced by LS5 and LS9, shown in Fig. 2, migrate aberrantlyinpolyacrylamide gels, indicating that the sequencessubstituted inthesemutationsmaybeinvolved in formation ormaintenance ofconformation orare sites ofa

modification(s)of the Elaproteinwhichenhancesfunction. Mutations betweenthecysteine pairs (LS6, LS6A,andLS7)

had a negligible effect on transactivation. In fact, one of these mutations (LS6) is an "up" mutation for thatactivity.

Inthismutant, two amino acids (Thr-164 and Gly-165)which may bepresent at the same position in the Ela proteins of all

sIerotypes

(23, 47) are replaced. Its phenotype suggests that these residues may function to down regulate transactiva-tion.

Overlapping this metal-binding segment, extending ap-proximately between amino acids 170 and 184 in Ad2, is a sequence predicted by computer analysis with two programs

bythe rules of Chou and Fasman (5) to have highpotential for

p-sheet

formation. Output from one of these programs (PROTLYZE), showing the position of this structure and its occurrence in the Elaproteins of multiple adenovirus sero-types, isreproducedinFig. 6. Mutationslying solely in this region (LS10, pm1098, and pmlll2) have an especially deleterious effect on transcription of the viral E4 gene.

Although prediction of secondary structure in proteins is

currently an inaccurate process, the presence of a similar structurein multiple divergent proteins is morelikely to be

significant. Theimportance of this sequence to transactiva-tion is underscored by the observation that all defective

in-frame mutations in Ela generated by random methods

whichhave beendescribed in theliteraturefallwithin it(hr3,

hr4, and hr5[11, 15]andpm1098andpmlll2 [27]).

The splicing event which removes the Ela 289R-unique

coding sequence in Ad2 completely deleted the two sub-structuresdescribed above from the Elaproteins of diver-gent serotypes(Adl2, Ad7, and Ad4) (23, 47),shown for the Ad2 243R ElaproteininFig. 6.However, the more

amino-terminalresidues oftheuniquesegment of the Ad2 289R Ela

protein areactually retainedin thesmallerproteinsof other serotypes due toutilization of a splice donor at adifferent

position

in the common Ela precursor RNA (36). If exon structurereflects domain structure, assuggested byGilbert

(9), the probable metal-binding sequence and'

p-sheet

to-gethermayconstituteasingle functional subunit ofthe Ela

protein. However, the example of LS1 indicates that the upstream residues do influence Ela-mediated

transactiva-tionandmightbe a secondfunctionalclusterin the

transac-tivating domain. No conserved functional groups in this

sectionofthe Elaprotein sequence areevide'nt from com-puteranalysis of secondarystructure andchargein the Ela

proteins of multiple serotypes. Furthermutagenesis within thissegmentofthe Ad2transactivating domainisnecessary todefine its contributiontothemechanism oftransactivation

by Ela.

The results

presented

heredemonstrate that

transactiva-tion of viral and cellulargenes by the Ad2 289R Elagene

product

during

adenovirus infection isacomplexfunctionof

severalactivities. Maximal activationrequiresthepresence

of the 46-amino-acid segment

unique

to the larger Ela

protein. However, since mutants which retain

little

orno sequencefromthis segmentoftheprotein retainsomeability

to activate at least some genes, a second, much weaker

positive

regulatoryactivityhasbeenpostulated, mediated by

sequences outside the transactivating domain. The

discov-ery of this activity, which affects the transcription of both viral and cellulargenes, coupled with the information that

Ela-inducible transcription units can be differentially

af-fectedby specific mutations inElaincreases thedifficulty in separating any process inwhich the Elaprotein(s)

partici-patesfrom its

positive

effectsontranscription. Thispointis atodds with theconclusions ofLillieetal. intheir analysis ofthephenotypes ofmutantspm1098andpmlll2(27). From assays ofsteady-state RNAlevels 16 h postinfection, with

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cytosine arabinoside used to prevent replication, thus ex-tending the early phase and amplifying early-gene expres-sion, these authors reported that pm1098 andpmlll2 are extremely (50-fold) defective for transactivation of viral early genes relative to the wild-type Ela 289R protein and yet are equivalent in transforming ability. In the assays reported here, in which transcription was measured directly during the early phase of infection in the absence of drugs, these two mutants were no more than twofold defective for the activation of any gene but E4. They were by no means as defective as the Ela- mutant d1312 or many of the other Ela variants assayed.

The mutations in the Ela 289R protein described here include several phenotypes with respect to transactivation which had not previously been reported or studied in depth. The results of their analysis in assays of transcription point toward more than one mechanism by which Ela can affect gene expression positively and to the possible definition of subclasses of Ela-responsive genes by the gene-specific effects of modification of the Ela 289R protein. Until the underlying mechanisms behind Ela activities can be eluci-dated, direct assays can be developed for them, and the spectrumof genes that they affect can be examined in some detail, correlations drawn or denied between the positive regulation of transcription by Ela and its other functions may represent oversimplifications.

ACKNOWLEDGMENTS

We thank R. Morgan Wain and Kathleen Critchett for expert technical assistance, Carl Anderson for antiserum to theDBP, and Arnold Berk for providing plasmids pEKpm975 andpKS103.

This investigation was supported by Public Health Service grants CA29600 and CA39636 from the National Institutes of Health.

LITERATURE CITED

1. Berg, J. M. 1986. Potential metal-binding domains in nucleic acid binding proteins. Science 232:485-487.

2. Berk, A. J. 1986. Adenovirus promoters and Ela transactiva-tion. Annu. Rev. Genet. 20:45-79.

3. Berk, A. J. 1986. Functions of adenovirusElA.CancerSurveys 5:367-387.

4. Carlock, L. R., and N. C. Jones. 1981.Transformation-defective mutant of adenovirus type 5 containing a single altered ElA mRNA species. J. Virol. 40:657-664.

5. Chou, P. Y., and G. D. Fasman. 1978. Empirical predictions of protein conformation. Annu. Rev. Biochem. 47:251-276. 6. Ferguson, B., B. Krippl, 0. Andrisani, N. Jones, H. Westphal,

and M. Rosenberg. 1985. ElA 13S and 12SmRNA products made in Escherichia coli both function as nucleus-localized transcription activators but do not directly bind DNA. Mol. Cell. Biol. 5:2653-2661.

7. Fowlkes, D. M., and T. Shenk. 1980. Transcriptional control regions of the adenovirus VAl RNA gene. Cell22:405-413. 8. Garcia, J., F. Wu, and R. Gaynor. 1987. Upstream regulatory

regions required to stabilizebinding to the TATAsequencein an adenovirus early promoter. NucleicAcids Res. 15:8367-8385. 9. Gilbert, W. 1985. Genes in pieces revisited. Science 228:

823-824.

10. Gingeras, T. R., D. Sciaky, R. E. Gelinas, J. Bing-Dong, C. E. Yen, M. M. Kelly, P. A. Bullock,B. L. Parsons, K. E. O'Neill, and R. J. Roberts. 1982.Nucleotide sequencesfrom the adeno-virus-2 genome. J. Biol. Chem. 257:13475-13491.

11. Glenn, G. M., and R. P. Ricciardi. 1985. Adenovirus 5 early region1A host range mutants hr3, hr4, and hr5 contain point mutations which generate single amino acid substitutions. J. Virol. 56:66-74.

12. Graham, F. L., J. Smiley, W. C. Russell, and R. Naiva. 1977. Characteristics of ahuman cell linetransformedby DNA from humanadenovirus 5. J. Gen. Virol. 36:59-74.

13. Green, M.,P.Loewenstein, R. Pusztal, and J. Symington. 1988. An adenovirus ElA protein domain activatestranscription in vivo and in vitro in the absence of protein synthesis. Cell 53:921-926.

14. Gunning, P., P.Ponte, H.Okayama,J. Engel, H. Blau, and L. Kedes. 1983.Isolation andcharacterizationoffull-lengthcDNA clonesfor human a-, 3-, and -y-actin mRNAs: skeletal butnot

cytoplasmic actins have an amino-terminal cysteine that is subsequentlyremoved. Mol. Cell. Biol.3:787-795.

15. Harrison, T., F. Graham, and J. Williams. 1977. Host-range mutants ofadenovirus type 5defectiveforgrowth in HeLa cells. Virology77:319-329.

16. Harter, M., and J. Lewis.1978. Adenovirus type 2earlyproteins synthesized invitro andinvivo:identification in infected cells of the38,000- to50,000-molecular-weight protein encodedbythe left endof theadenovirus type 2 genome. J.Virol.26:736-749. 17. Hearing, P., and T. Shenk. 1983. The adenovirus type 5ElA transcriptional control region contains a duplicated enhancer element. Cell33:695-703.

18. Hearing, P., and T. Shenk. 1986. The adenovirus type 5 Ela enhancer contains two functionally distinct domains: one is specific forElAand the other modulatesallearly units incis. Cell 45:229-236.

19. Hen, R., E. Borrelli, P. Sassone-Corsi, and P. Chambon. 1983. Anenhancer element is located 340base pairs upstreamfrom the adenovirus-2 ElA capsite. Nucleic Acids Res. 11:8747-8760.

20. Hoeffier, W. K., and R. G. Roeder. 1985. EnhancementofRNA polymerase IIItranscription bythe ElA gene product of ade-novirus. Cell41:955-963.

21. Jones, N. C., P. W. Rigby, and E. B. Ziff. 1988. trans-Acting protein factors and the regulation ofeukaryotic transcription: lessons from studies on DNA tumor viruses. Genes Dev. 2:267-281.

22. Kao, H.-T., and J. R. Nevins. 1983. Transcriptional activation and subsequent control of the human heat shock gene during adenovirusinfection. Mol. Cell. Biol. 3:2058-2065.

23. Kimelman, D., J.S.Miller, D.Porter, and B. E. Roberts. 1985. Ela regions of the human adenoviruses and of the highly oncogenic simian adenovirus 7 are closely related. J. Virol. 53:399-409.

24. Ko, J.-L., B. Dalie, E. Goldman, and M. Harter. 1986. Adeno-virus-2 earlyregion IA protein synthesized in Escherichia coli extractsindirectlyassociates withDNA.EMBOJ.5:1645-1651. 24a.Laemmli, U. K. 1970. Cleavage of structuralproteinsduringthe assembly of the head ofbacteriophage T4. Nature (London) 227:680-686.

25. Leong, K., and 4. Berk. 1986. Adenovirus early region 1A protein increasesthe numberoftemplate molecules transcribed in cell-free extracts. Proc. Natl.Acad. Sci. USA 83:5844-5848. 26. Lewis, J. B., and M. B. Mathews. 1980. Control of adenovirus early geneexpression: a classof immediateearlyproducts.Cell 21:303-313.

27. Lillie, J. W., M.Green, and M. R. Green. 1986. Anadenovirus Ela protein region required for transformation and transcrip-tionalrepression. Cell 46:1043-1051.

28. Lillie, J. W., P. M. Loewenstein, M. R. Green, and M. Green. 1987. Functional domains of adenovirus type 5 Ela proteins.

Cell 50:1091-1100.

29. Ma, J., and M. Ptashne. 1987. A new class ofyeast transcrip-tionalactivators. Cell 51:113-119.

29a.Maniatis, T., E. F. Fritsch, andJ. Sambrook. 1982. Molecular cloning: alaboratory manual. Cold Spring HarborLaboratory,

ColdSpring Harbor, N.Y.

30. McKnight, S. L., and R. Kingsbury. 1982. Transcriptional

control signals of a eukaryotic protein-coding gene. Science 217:316-324.

31. Montell, C., E. F. Fisher,M. H.Caruthers,andA.J.Berk.1982. Resolving the functions of overlapping viral genes by site-specific mutagenesis at amRNA splice site. Nature (London)

295:380-384.

32. Moran, E., T.Grodzicker,R.J.Robert, M.B.Mathews, andB. Zerler. 1986. Lytic and transforming functions of individual

on November 10, 2019 by guest

http://jvi.asm.org/

(10)

products of the adenovirus ElA gene. J. Virol. 57:765-775. 33. Moran, E., and M. B. Mathews. 1987. Multiple functional

domainsin theadenovirusElAgene.Cell 48:177-178. 34. Osborne, R. F., R. B. Gaynor, and A. J. Berk. 1982. TheTATA

homology and the mRNA 5' untranslated sequence are not required for expression of essential adenovirus ElAfunctions. Cell29:139-148.

35. Palmer, D. K., K. O'Day, M. H. Wener, B. S. Andrews, and R.L. Margolis. 1987. A 17-kD protein (CENP-A) copurifies with nucleosome core particles and with histones. J. Cell Biol. 104:805-815.

36. Perricaudet, M., J.-M.le Moullec, P. Tiollais, and U. Pettersson. 1980. Structure of two adenovirus type 12 transforming poly-peptides and their evolutionary implications. Nature (London) 288:174-176.

37. Schneider, J. F., F. Fisher, C. R. Goding, and N. C. Jones. 1987. Mutational analysis of the adenovirus Ela gene: the role of transcriptional regulation in transformation. EMBO J. 6:2053-2060.

38. Shenk, T., N. Jones, W. Colby, and D. Fowlkes. 1980.Functional analysis ofadenovirus-5 host-range deletion mutants defective for transformation of rat embryo cells. Cold Spring Harbor Symp. Quant. Biol.44:367-375.

39. Sigler, P. 1988. Acid blobs and negative noodles. Nature (London) 333:210-212.

40. Simons, M. C., K. Kitchener, H.-T. Kao, E. Hickey, L. Weber, R. Voellmy, M. Heintz, and J. R. Nevins. 1987. Selective induction of human heat shock gene transcription by the ade-novirus ElA gene products, including the 12S ElA product. Mol. Cell. Biol. 7:2884-2890.

41. Soderlund, H., U. Petterson, B. Vennstrom, L. Philipson, and M. B.Mathews. 1976. A newspecies of virus-coded low molec-ularweight RNA fromcells infected with adenovirus type 2. Cell7:585-593.

42. Solnick, D., and M. A. Anderson. 1982. Transformation-defi-cient adenovirus mutant defective in expression of region 1A butnotregion 1B.J. Virol. 42:106-113.

43. Spangler, R., M. Bruner, B. Dalie, and M. Harter. 1987. Activation of adenovirus promoters by the adenovirus Ela protein in cell-freeextracts. Science237:1044-1046.

44. Spindler, K. R., D.S.E.Rosser,andA.J. Berk. 1984. Analysis of adenovirustransforming proteins from early regions 1A and 1B with antisera to inducible fusion antigens produced in Escherichiacoli. J.Virol. 49:132-141.

45. Stow, N. D. 1981.Cloning ofaDNAfragment from the left-hand terminus of the adenovirus type 2 genome and its use in site-directedmutagenesis. J. Virol. 37:171-180.

46. Svensson, C., and G.Akusjarvi. 1984.Adenovirus2early region ElA stimulates expression of both viral and cellular genes. EMBOJ. 3:789-794.

47. Tokunaga,O., T. Yaegashi, J. Lowe, L. Dobbs, and R. Padman-abhan. 1986. Sequence analysis in the El region of adenovirus type 4 DNA. Virology. 155:418-433.

48. Vieira, J., and J. Messing. 1982. The pUC plasmids, an M13mp7-derivedsystemfor insertion mutagenesis and sequenc-ing withsynthetic universal primers. Gene 19:259-268. 49. Weinheimer, S. P., andS. L. McKnight. 1987. Transcriptional

andpost-transcriptional controls establish the cascade ofherpes simplex virus protein synthesis.J. Mol. Biol. 195:819-834. 50. Weinmann, R., T. G. Brendler, H. J. Rashkas, and R. G.

Roeder. 1976. Lowmolecular weight viral RNAs transcribed by RNA polymerase III during adenovirus 2 infection. Cell 7: 557-566.

51. Winberg, G., and T. Shenk. 1984. Dissection of overlapping functions within the adenovirus type 5 ElA gene. EMBO J. 3:1907-1912.

52. Wu, L., D. Rosser, M.Schmidt, and A. Berk. 1987. A TATA box implicatedin ElAtranscriptional activation of a simple adeno-virus 2 promoter. Nature (London) 326:512-515.

53. Yoshinaga, S.,N.Dean, M. Han, and A. J. Berk. 1986. Adeno-virus stimulation of transcription by RNA polymerase III: evidence foranElA-dependent increaseintranscriptionfactor

IIIC concentration. EMBOJ. 5:343-354.