JOURNAL OF VIROLOGY, Dec. 1982, p. 907-921 Vol. 44, No. 30022-538X/82/120907-15$02.00/0

Expression of Adenovirus-2 Early Region 4: Assignment ofthe Early Region 4 Polypeptides to Their Respective mRNAs,

Using In Vitro TranslationMICHAEL A. TIGGESt AND HESCHEL J. RASKAS*

Department of Pathology, Washington University School of Medicine, St. Louis, Missouri 63110

Received 9 April 1982/Accepted 20 August 1982

Adenovirus-2 early region 4 (E4; map positions 91.3 to 99.1) encodes six 5' and3' coterminal, differently spliced mRNAs, which are 2.5, 2.1, 1.8, 1.5, 1.2, and 0.8kilobases (kb) long. Hybridization selection with five cloned viral DNA fragmentsthat hybridize with subsets of E4 mRNAs was used to purify these six mRNAsand a previously unreported 3.0-kb mRNA from virus-infected cells. E4 mRNAswhich were purified by hybridization selection with cloned EcoRI fragment C(map positions 89.7 to 100) were also fractionated by size. The purified mRNAswere then translated in rabbit reticulocyte or wheat germ lysate systems. The fullcomplement of E4 mRNAs specified as many as 16 different polypeptides, withmolecular weights ranging from 24,000 (24K) to 10K. The most abundant E4mRNA, which was 2.1 kb long, specified an 11K polypeptide. The 1.5-kb mRNA,which differed from the 2.1-kb mRNA only by deletion of a second intron from the3' untranslated region, also specified an 11K polypeptide. The second mostabundant mRNA, which was 1.8 kb long, and the 1.2-kb mRNA, which had anintron deleted from the 3' untranslated region, specified a 15K polypeptide. Thispolypeptide was labeled more intensely with [5,6_3H]leucine than with [35S]methi-onine. The 3.0- and 2.5-kb mRNAs specified four polypeptides (24K, 22K, 19K,and 17K). Translation of E4 mRNAs with a mean size of 0.8 kb, whichaccumulated preferentially in the presence of cycloheximide, yielded at least 10polypeptides that migrated in polyacrylamide gels with apparent molecularweights ranging from 21,800 to 10,000. On the basis of translation in wheat germlysates and the distribution of polypeptides encoded by size-fractionated mRNAs,we concluded that the 0.8-kb mRNA size class includes a heterogeneous mixtureof mRNAs which are probably formed as the result of utilization of alternatesplice acceptor and donor sites during removal of the second intron. Ourpolypeptide assignments for the 2.1-, 1.8-, 1.5-, and 1.2-kb mRNAs are compatiblewith locations of two open coding regions in the DNA sequence (Herisse et al.,Nucleic Acids Res. 9:4023-4042, 1981). The relationship between the fourpolypeptides encoded by the 3.0- and 2.5-kb mRNAs and the two open codingregions is discussed. The production of multiple polypeptides from a heteroge-neous mixture of mRNAs in the 0.8-kb size class is compatible with two largeopen coding regions in that part of the sequence. Thus, nearly all of the potentialcoding information in the leftward strand of E4 is expressed in translatable formduring infection. Moreover, alternate splicing of the 0.8-kb mRNA size class canproduce multiple polypeptides with common amino-terminal and different car-boxy-terminal amino acid sequences, which may have the same function butdifferent specificities.

Early after adenovirus-2 infection, before the leftward direction from a promoter located at mpbeginning of DNA replication, RNA is tran- 99.1 (2, 37). Transcription is apparently termi-scribed from six promoters (early region la nated near the polyadenylation site at mp 91.3[Ela], Elb, E2, E3, E4, and the major late (3, 4, 12). Previous studies have identified apromoter) (15). E4, which is located within map complex mixture of 5' and 3' coterminal cyto-positions (mp) 91.3 to 99.1, is synthesized in the plasmic RNAs derived from the E4 nuclear

precursor by removal of one or two introns (Fig.t Present address: Department of Microbiology and Immu- 1). Three of the E4 species have a single intron

nology, University of California, Berkeley, CA 94720. removed (Fig. 1C, species a through c) and are

907

908 TIGGES AND RASKAS

A Smo G'

H2 H3I I Ins

5 H4

Dir I

B Hi

D3 BSl l r i IH7= 1|2741r- I- D11I1

Eco RIC " JD'& Loll

C 2.5Kb -

2.1 -b

1.8 * _c

1.5 _ d

1.2 -

0.8

2.3 - 9

1.6 *4- h

I I I I I I I I ' I I * I

90 91 92 93 94 95 96 97 98 99 100FIG. 1. E4 mRNAs and cloned viral DNA fragments used in this study. (A) Cloned viral DNAs used to detect

E4 RNAs in Northern blots. Plasmid DNAs were labeled with [32P]dCTP by nick translation. (B) Viral DNAplasmids used to purify E4 mRNAs by hybridization selection (28). (C) Structure of E4 mRNAs. The positions ofthe splice sites were determined by Sl nuclease analysis (species a through c) (5) and electron microscopy(species b through h) (10). The 5' and 3' termini of E4 mRNAs are based on DNA sequence data (19). The mostabundant E4 RNAs that accumulate in the presence of cycloheximide are indicated by the heavy lines (10; thisstudy).

formed by splicing a short 5' leader to threedownstream acceptor sites. These species havebeen detected by S1 nuclease or exonucleaseVII digestion of RNA-DNA hybrids, followedby electrophoresis of the resistant DNA frag-ments (5). Five additional E4 mRNA specieshave been identified by electron microscopy ofRNA-DNA heteroduplexes (10). In addition tospecies b and c detected with S1 nuclease (Fig.1), two species with the leader spliced to theacceptor sites and a second intron (mp 92.4 to94.4) removed have been observed (Fig. 1, spe-cies d and e). A sixth species is formed when theleader is spliced to an acceptor site furtherdownstream and a second intron is removed(Fig. 1, species f). Two rare species that retainsequences 3' to the leader and have one or twointrons removed have been detected by electronmicroscopy (Fig. 1, species g and h).The E4 region is also of interest because of the

complex manner in which its expression is regu-lated. At early times after infection, E4 RNAconstitutes 6% of the viral or 0.05% of thecytoplasmic polyadenylic acid [poly(A)]-con-taining RNA (5, 11). Initiation of E4 transcrip-tion requires a functional product of the ElA13S mRNA (8, 29, 35). Transcription reaches a

maximum rate between 4 and 6 h and thendeclines to 10 to 20% of the maximum rate by 10h when DNA synthesis is inhibited (31). Treat-ment of infected cells with cycloheximide from 1to 7 h after infection results in a greater-than-10-fold increase in the abundance of E4 RNAs (10,14, 31). Although cycloheximide alters the rela-tive amounts of the E4 RNAs, all species ob-served are also produced in the absence of thisdrug (10). Cells infected with a mutant thatproduces a defective 72,000-dalton (72K) DNA-binding protein (adenovirus-5 ts125) overpro-duce E4 RNAs due to a failure to suppress E4transcription (9, 32). Thus, the expression of E4is under the control of at least two other earlyviral genes.E4 21K, 19K, 17K, and 11K proteins have

been identified by using in vitro translation ofRNAs purified by hybridization to EcoRI frag-ment C (mp 89.7 to 100) and have been correlat-ed by size with proteins labeled in vivo (18, 24,25). In another study, E4 mRNAs were reportedto specify six polypeptides (35K, 23K, 22K,21K, 18K, and IlK) (26). This latter group ofpolypeptides was correlated with in vivo pro-teins isolated by immunoprecipitation with seraagainst adenovirus-2-transformed rat cells (7).

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VOL. 44, 1982

In this study, we extended and refined theanalysis of the in vitro translation products ofE4mRNAs purified by hybridization with clonedviral DNA fragments bound to nitrocellulosefilters. The RNAs were separated from eachother by hybridization with cloned restrictionfragments that selected a subset of the E4 se-quences or by hybridization and subsequent sizefractionation in sucrose-formamide gradients.We found that previous studies had underesti-mated the complexity of the region, as E4mRNAs specify as many as 16 polypeptides.These 16 polypeptide products are compatiblewith the open coding regions available in theleftward coding strand of the E4 transcriptionunit (19).

MATERIALS AND METHODSCells and virus. KB cells were maintained in suspen-

sion cultures in Joklik modified minimal essentialmedium containing 5% horse serum (KC Biologicals).Growth, purification, and plaque assays of adenovi-rus-2 stocks have been described previously (13, 16).

Virus infections and RNA isolation. Infections wereperformed as previously described (27). When cyclo-heximide (Sigma Chemical Co.) was used to inhibitprotein synthesis, 25 ,ug of cycloheximide per ml wasadded 1 h after infection, and the cells were harvestedat 7 h. In some experiments, cytosine arabinoside(araC; 20 F,g/ml; Sigma) was added 1 h after infection,and the cells were harvested at 5 h. In all experiments,RNA was labeled with [5,6_3H]uridine (2.5 to 10 ,Ci/ml; 50 Ci/mmol; New England Nuclear Corp.) added 2h before the cells were harvested. The proceduresused for isolating poly(A)-containing cytoplasmicRNA have been described elsewhere (27).When RNA was to be isolated from polysomes,

infected cells were first treated with cycloheximideessentially as described previously (18). The infectedcells were incubated in medium containing cyclohexi-mide until 5 h after infection, washed three times withprewarmed medium containing 20 p.g of araC per ml,and incubated for an additional 1 h in medium contain-ing araC before preparation of a cytoplasmic extract.The extract was divided in half, and poly(A)-contain-ing RNA was isolated. The remaining extract wasdivided in half. To one fraction 10 mM cyclohexanediethylaminetetraacetic acid (CDTA; Sigma) was add-ed. Each fraction was layered over cushions (3.5 mleach) of 1.0 and 1.8 M sucrose solutions containing 40mM PIPES [piperazine-N,N'-bis(2-ethanesulfonicacid)] (pH 7.4) and 1 mM CDTA in SW41 ultracentri-fuge tubes (36). The samples were centrifuged in anSW41 rotor (Spinco) at 25,000 rpm for 15 h. Thepolysome pellets were dissolved in the buffer ofHolmes and Bonner (20), the RNA was extracted withphenol-chloroform-isoamyl alcohol, and the poly(A)-containing RNA was purified by oligodeoxythymidylicacid cellulose chromatography (1).

Purification of E4 RNA. E4 RNA was purified byhybridization of poly(A)-containing cytoplasmic RNAisolated from 1.8 x 108 to 2.0 x 108 cells to clonedviral DNA fragments bound to nitrocellulose filters ina 150-pl reaction mixture containing 50o formamide,100 mM Tris-hydrochloride (pH 7.4), 0.75 M NaCI, 2

E4 POLYPEPTIDES 909

mM EDTA, 0.5% sodium dodecyl sulfate, and 200 pLgof wheat germ tRNA per ml (28). The hybridizationmixtures were incubated at 37°C for 16 to 24 h andnon-specifically bound RNA was removed by repeatedwashes at 55°C until the amount of label eluted in eachwash remained constant. The E4-specific RNA waseluted at 85°C, further purified by oligodeoxythymidy-lic acid cellulose chromatography, and concentratedby ethanol precipitation.E4 RNA purified by hybridization with EcoRI frag-

ment C (mp 89.7 to 100) was separated according tosize in 5 to 20%o sucrose-98% formamide gradients aspreviously described (42). Ethanol precipitates of E4RNA were dissolved in 250 pl of 80%o buffered forma-mide (40 mM PIPES, pH 7.4, 1 mM CDTA), heated at85°C for 7.5 min, and layered on 11.5-ml preformedgradients. The gradients were centrifuged at 4°C in anSW41 rotor at 30,000 rpm for 48 h. Fractions (340 pul)were collected, 10 pg of wheat germ tRNA (Sigma)and 0.2 M ammonium acetate were added, and theRNA was precipitated with 2.5 volumes of ethanol.

Gel electrophoresis and transfer and hybridization ofRNA. RNA samples were prepared for electrophoresisby treatment with 1 M glyoxal (Fisher Scientific Co.),subjected to electrophoresis in 1.2% agarose horizon-tal slab gels for 7 h at 90 V, and transferred tonitrocellulose filters (type BA85; Schleicher & SchuellCo.) as described elsewhere (38). In one experiment,glyoxalated RNAs were subjected to electrophoresisin a vertical 4% acrylamide gel (ratio of acrylamide tobisacrylamide, 40:1) buffered with 10 mM sodiumphosphate (pH 7.0) at 90 V for 3 h with recirculation.The RNA was transferred to diazobenzyloxymethyl(DBM) paper (Transa-Bind; Schleicher & Schuell)prepared according to the instructions of the supplier.On occasion, RNA was transferred to DBM paperfrom agarose gels.The blots were prehybridized and hybridized with

32P-labeled DNA probes by using previously describedprocedures (39). The plasmid DNA or total adenovi-rus-2 DNA probes were labeled with [a-32P]dCTP(>4,000 Ci/mmol; Amersham) by using a nick-transla-tion kit (Amersham). The instructions of the supplierwere modified slightly in that 0.1 ,ug of DNA wasadded to a 20-p.l reaction mixture containing 10 ,uCi oflabel. The reaction mixtures were incubated at 15°Cfor 7 to 9 h. Using this method, we routinely achievedspecific activities of 5 x 108 to 10 x 108 cpm/,ug. Thefilters were hybridized for 16 to 24 h at 42°C and thenwashed with two changes of 2x SSPE (1x SSPE is0.36 M NaCI plus 20 mM sodium phosphate [pH 7.0]plus 2 mM EDTA) containing 0.1% sodium dodecylsulfate at room temperature (15 min each) and twochanges of 0.1 x SSC (1x SSC is 0.15 M NaCI plus0.015 M sodium citrate) containing 0.1% sodium dode-cyl sulfate at 51°C (30 min each). The filters wereblotted dry, covered with plastic wrap, and exposed toKodak ARP-5 film with a Cronex Lightning Plusintensifying screen (Du Pont) at -80°C. Molecularweights were estimated by using adenovirus-2 DNAcut with SmaI and denatured with 1 M glyoxal in 50%dimethyl sulfoxide. RNA abundance was estimatedfrom densitometer traces of the films. The traces werecopied, and the peaks were cut out and weighed.

Cell-free translation in reticulocyte and wheat germlysates. Nuclease-treated rabbit reticulocyte lysateswere prepared by established procedures (33). The

910 TIGGES AND RASKAS

lysates were optimized for translation of cytoplasmicRNA isolated 7 h after infection in the presence ofcycloheximide or 22 h after infection in the absence ofdrugs. E4 RNA (10 to 100 ng dissolved in 1 to 2 RIu ofsterile distilled water) was translated in 25-,u reactionmixtures as described previously (17). The reactionmixtures contained 3 ,1 of [35S]methionine (700 to1,000 Ci/mmol; 9.8 to 12.5 ,uCi/,l; Amersham) or[3H]leucine (153 Ci/mmol; concentrated by lyophiliza-tion to 10 ,uCi/,l; Amersham). Wheat germ translationkits (Bethesda Research Laboratories) were used ac-cording to the instructions of the supplier.

Gel electrophoresis of polypeptides. The translationreactions were terminated by adding 1 ,ul of 250 mMCDTA, and 2.5 ,Il was removed for precipitation withtrichloroacetic acid to determine label incorporation,as described previously (17). Samples (2 pul) wereremoved from the reaction mixtures and added totubes containing 80 ,ul ofTEN (20 mM Tris-hydrochlo-ride, pH 7.4, 20 mM NaCl, 2 mM EDTA) and 20 ,ul of5% unlabeled methionine. The proteins were precip-itated from 3.5 volumes of acetone (-20°C for 30 min),washed twice with acetone, dried, and dissolved in 25pIl of electrophoresis sample buffer (100 mM Tris-hydrochloride, pH 6.8, 2.5% sodium dodecyl sulfate,2.5% glycerol, 0.7 M 2-mercaptoethanol, 0.025% bro-mophenol blue). The samples were subjected to elec-trophoresis in a modified Laemmli polyacrylamide gelsystem (22). The resolving gel was a linear 10 to 20%oacrylamide gradient gel (ratio of acrylamide to bisacry-lamide, 30:0.174; 16 cm long by 0.15 cm thick) that wasstabilized with a 10 to 18% sucrose gradient andformed by pumping the acrylamide solution in fromthe bottom, as described previously (21). The resolv-ing gel was topped with a 1- to 1.5-cm 5% acrylamidestacking gel. Electrophoresis was at 22.5 mA (constantcurrent) for 30 min and at 10 mA for 21 h. The gelswere prepared for fluorography by using previouslydescribed procedures (6, 23). Molecular weight deter-minations were based on the "4C-labeled proteins(Bethesda Research Laboratories) listed in the legendto Fig. 3. Polypeptide abundance was estimated fromdensitometer traces as described above.

Preparation of DNA and DNA filters. All of the viralDNA fragments used in this study were cloned inpBR322 plasmids. The cloning and plasmid isolationprocedures will be described elsewhere (M. A. Tigges,M. Leonardo, and H. J. Raskas, submitted for publica-tion). Plasmid DNA (200 pug-equivalents of viral se-quence; 25 to 40 ,ug of plasmid DNA) was bound tonitrocellulose filters (type BA85; Schleicher &Schuell) as described previously (28, 34). Nicks weregenerated in the plasmids by five cycles offreezing andthawing, followed by heating the mixture to boiling for10 min.

RESULTSPurification of E4 mRNA. In this study, the

amplification effect of cycloheximide was ex-ploited to facilitate the isolation of E4 RNAs forin vitro translation. For purposes of comparison,E4 RNA was also purified from infected cellsthat were confined to early viral gene expressionby treatment with araC, an inhibitor of DNAsynthesis.

J. VIROL.

The size classes of E4 RNAs synthesizedearly in infection were identified by gel electro-phoresis, followed by blotting and hybridizationwith 32P-labeled viral DNA probes (Fig. 2).Poly(A)-containing cytoplasmic RNA isolatedfrom cells infected in the absence of drugs ortreated with cycloheximide or araC was treatedwith 1 M glyoxal, subjected to electrophoresis in1.2% agarose gels, and transferred to DBMpaper. The blot was hybridized with 32P-labeledplasmid DNA which contained viral DNA se-quences from mp 89.7 to 91.6 (Fig. 1A, regionH2), a region homologous to the 3' 190 nucleo-tides of E4 mRNA (Fig. 2, lanes a through c), sothat band density would be directly proportionalto RNA species abundance.As expected from previous studies, six E4

RNA species with sizes between 2.5 and 0.8kilobases (kb) were observed (Fig. 2, lanes athrough c). An additional 3.0-kb cytoplasmicspecies not previously reported was also ob-served. The relative amounts of the E4 RNAswere estimated from densitometer traces of thefilms (Fig. 2, lanes a and c). The sum of the peakweights of the E4 species isolated 7 h afterinfection from cells treated with cycloheximidewas approximately 50-fold greater than the sumof the peak weights of the E4 species isolated 5 hafter infection from an equivalent number ofcells infected in the absence of drugs. In addi-tion, the relative abundance of the 0.8-kb spe-cies was more than 20 times greater in infectedcells when protein synthesis was inhibited (Ta-ble 1). Although cycloheximide did not result inuniform increases in the amounts of all E4species, inhibition of protein synthesis did notappear to result in the production of RNA spe-cies that were not present in drug-free infections(Fig. 2, lanes a and d). To confirm that all sevenE4 RNAs functioned as mRNAs in vivo, thepresence of these RNAs in polysomes was de-termined. Poly(A)-containing RNA was isolatedfrom polysomes formed after treatment withcycloheximide (18). To correct for differences inyield, samples containing 1 ,ug of poly(A)-con-taining RNA from the polysome preparationsand from cells infected without inhibitors weresubjected to electrophoresis, transferred to ni-trocellulose filters, and hybridized with 32p_labeled EcoRI fragment C-containing plasmidDNA (mp 89.7 to 100) (Fig. 2, lanes d through g).All of the E4 species produced in cyclohexi-mide-treated cells served as mRNAs in vivo andwere present in polysomes in the same relativeamounts as in the cytoplasm as a whole (Table1). To assure that the polysomes were notcontaminated with cosedimenting ribonucleo-protein, a portion of the cytoplasmic extract wastreated with 10 mM CDTA before sedimentationof the polysomes. The yield of E4 RNA was

E4 POLYPEPTIDES 911

M a b c d e f 9 h

7 ':5 22-

4,24-

2.93-246-3

F~~

i.33-

-30

-L~5-21-.

i.-1 8

I-0

- .8

FIG. 2. Blot analysis of E4 RNAs. Samples of cytoplasmic poly(A)-containing RNA were treated with 1 Mglyoxal, subjected to electrophoresis in 1.2% agarose gels, and transferred to DBM paper (lanes a through c and hthrough k) or nitrocellulose filters (lanes d through g). The samples were then hybridized with plasmid DNAcontaining viral DNA fragment H2 (lanes a through c), EcoRI-C (lanes d through g), or SmaI-G' (lanes h throughk) labeled with [132P]dCTP by nick translation. The genome segments contained in the probes are diagramed inFig. 1. Lane M, Adenovirus-2 DNA cut with SmaI hybridized with nick-translated viral DNA; lane a, 1.0 ,ug ofRNA isolated from 2 x 106 cells infected in the presence of cycloheximide; lane b, 9.4 ,ug of RNA isolated 5 hafter infection from 2 x 10' cells infected in the presence of araC; lane c, 4.6 p.g of RNA isolated 5 h afterinfection from 2 x 10' cells infected in the absence of inhibitors; lane d, 1 p.g ofRNA isolated from 4 x 106 cellsprepared as described above for lane c (for comparison with lanes e through g). For lanes e through g cells wereinfected and treated with cycloheximide from 1 to 5 h after infection. The inhibitor was removed, and the cellswere incubated for 1 h to allow the formation of polysomes. Samples for electrophoresis contained 1 ,ug ofRNAto correct for differences in yield from the different preparations. Lane e, Cytoplasmic RNA (3 x 106 cells); lanef, polysomal RNA (107 cells); lane g, polysomes pretreated with 10 mM CDTA (the sample contained 0.1 ,ug ofRNA). For lanes h through k poly(A)-containing RNA was isolated at 7 h after infection in the presence ofcycloheximide from 2 x 10i cells. Specific RNA was purified by hybridization selection with plasmid DNAscontaining viral DNA fragments D3 (lane h), Dl (lane i), BS (lane j), and B274 (lane k).

reduced approximately 100-fold without enrich-ment of the pellet with the less abundant spe-cies, which would have been the result if asignificant fraction of these species were non-polysomal ribonucleoproteins.We wanted to confirm that the fragments to be

used for preparative hybridization selected theRNAs expected from the structural analyses(Fig. 1). Therefore, blots of cytoplasmic RNAfrom cells infected in the presence of cyclohexi-mide were hybridized with the various 32P_labeled plasmid DNAs shown in Fig. 1A (datanot shown). The results were as expected fromprevious studies and our data in Fig. 2. The sizesof the E4 RNAs detected in the blots are indicat-ed to the left of the RNAs diagramed in Fig. 1.The 2.3-kb RNA (Fig. 1, species g) was ob-served only in the blot probed with fragment H5because it was obscured by the strong 2.1-kb

band. The previously reported (10) minor 1.6-kbE4 RNA (Fig. 1, species h) was not observed inthe blot probed with the H5 fragment. Becausethe 3.0-kb species hybridized with all of theprobes shown in Fig. 1, it was probably theunspliced precursor to the other E4 mRNAs.

In vitro translation of E4 mRNAs purified byhybridization selection. On the basis of the map-ping results, four of the viral fragments shown inFig. 1B (D3, Dl, BS, and B274) were used topurify E4 RNAs by preparative hybridizationselection (28). Poly(A)-containing cytoplasmicRNA was isolated from infected cultures treatedwith cycloheximide and labeled with [5,6,-3H]uridine (2.5 to 10 t±Ci/ml) for 2 h before thecells were harvested at 7 h. The RNA washybridized for 16 to 24 h with plasmid DNAbound to nitrocellulose filters. After the unhy-bridized RNA was removed by washes at room

VOL. 44, 1982

912 TIGGES AND RASKAS

TABLE 1. Relative amounts of E4 RNAsa

Relative amt Withb: Relative amt with RNARNA size from':

(kb) heximide inhibitor Cytoplasm Polysomes

3.0 0.01 NDd 0.02 0.032.5 0.08 0.09 0.13 0.142.1 1.00 1.00 1.00 1.001.8 0.47 0.20 0.74 0.341.5 0.39 0.03 1.18 1.191.2 0.21 ND 0.91 0.740.8 0.91 0.04 1.21 1.27a Estimates of abundance were made from densi-

tometer traces of the films shown in Fig. 2 or shorterexposures. When the band densities exceeded thelinear response of the film, the peak maxima wereextrapolated. The weights were normalized to the 2.1-kb RNA species.

b RNA was isolated from cells infected in the pres-ence of cycloheximide (Fig. 2, lane a) or without drugs(Fig. 2, lane c).

I RNA was isolated after treatment with cyclohexi-mide and removal of the drug. Abundance estimateswere made for cytoplasmic RNA (Fig. 2, lane e) andpolysomal RNA (Fig. 2, lane f).

d ND, Not detected.

temperature, non-specifically bound RNA waseluted by extensive washes at 55°C. E4 RNAwas eluted at 85°C and further purified by oligo-deoxythymidylic acid cellulose chromatogra-phy. Samples of the purified E4 RNA weresubjected to electrophoresis, blotted, and hy-bridized with 32P-labeled EcoRI fragment C (mp89.7 to 100) (Fig. 2, lanes h through k). ThemRNAs expected on the basis of the RNAmapping experiments were selected by using theD3, Dl, BS, and B274 fragments, except for thesmall amount of 1.8-kb mRNA selected by theDl and BS fragments (mp 96.2 to 98.3 and 95.6to 98.3, respectively) (Fig. 2, lanes i and j).Previous structural studies (10) have shown thata larger intron is removed to form the 1.8-kbmRNA (Fig. 1, species c). Moreover, moredetailed structural studies (Tigges and Raskas,submitted for publication) have confirmed thatthe leader splice acceptor site in the 1.8-kbmRNA (Fig. 1, species c) lies to the left of mp95.6. A possible explanation for this observationis discussed below.E4 RNAs purified by hybridization selection

were used to direct polypeptide synthesis innuclease-treated rabbit reticulocyte lysates (33).Between 10 and 100 ng of E4 RNA was translat-ed in two nuclease-treated reticulocyte prepara-tions by using [35S]methionine as the label. Thepolypeptides were subjected to electrophoresisin 10 to 20% polyacrylamide gels, and the la-beled polypeptides were detected by fluorogra-phy (Fig. 3). Seven prominent polypeptides

(24K, 22K, 21K, 19K, 17K, 15K, and 11K) weresynthesized in mixtures containing all of the E4mRNAs from cycloheximide-treated cells (Fig.3, lanes c, e, 1, and m). At least three lessabundant polypeptides (18.5K, 18K, and 16K)were also observed (see below). A 13K polypep-tide was also present but was difficult to detectas it comigrated with globin from the lysate.When RNA from uninfected cells was subjectedto hybridization selection with EcoRI fragmentC and added to the reticulocyte lysate, no poly-peptides were synthesized (data not shown).The correlation of polypeptides with E4

mRNAs from cycloheximide-treated cells waspartially achieved by hybridization with five E4probes. The D3 fragment (mp 92.7 to 93.9)selected E4 mRNAs that retained the secondintron sequence (i.e., the 3.0-, 2.5-, 2.1-, and 1.8-kb RNAs) (Fig. 1). When these mRNAs weretranslated, all of the prominent polypeptidesspecified by the full complement of E4 mRNAswere observed, although the 21K polypeptidewas present in only trace amounts (Fig. 3, lanek). In longer exposures (data not shown), no18K, 16K, and or 13K polypeptides were detect-ed. Translation of E4 mRNAs purified with theB274 fragment (map 94.8 to 95.6), which did notinclude the 0.8-kb species, yielded a patternsimilar to the pattern obtained for D3-selectedmRNAs, although slightly more 21K polypep-tide was present (Fig. 3, lanes f and h). Selec-tions with fragment H7 (mp 93.0 to 93.2; 160base pairs) were approximately 10-fold less effi-cient than selections with fragments larger than250 base pairs. Thus, only the most abundantmRNA, the 2.1-kb species, was recovered inamounts sufficient to yield a detectable transla-tion product, the 11K polypeptide (Fig. 3, laned). The 3.0-, 2.5-, 2.1-, and 1.5-kb RNAs wereselected with the Dl (mp 96.2 to 98.3) and BS(mp 95.6 to 98.3) fragments. When translated,the mRNAs yielded the 11K polypeptide as themajor product and trace amounts of the 24K,22K, 19K, and 17K polypeptides (Fig. 3, lanes iand j).

Translation of E4 mRNAs isolated from araC-treated cells and purified by hybridization toEcoRI fragment C (mp 98.7 to 100) yielded 11K,24K, and 19K polypeptides (Fig. 3, lane b). Asapproximately fivefold less E4 mRNA was add-ed to the translation mixture in this samplecompared with the E4 mRNAs from cyclohexi-mide-treated cultures, failure to detect the lessabundant polypeptides was not unanticipated.Because the 0.8-kb mRNA was 20-fold lessabundant in infected cells when protein synthe-sis was not inhibited, the missing 21K band inFig. 3, lane b, is consistent with the hypothesisthat the 0.8-kb mRNA encodes the 21K polypep-tide.

J. VIROL.

E4 POLYPEPTIDES 913

K_I v a h c d e f g h

20OK- 11

68K- - W

43K- pnR:

._

4aa

m

*M_am

a__.....V._..

a,-_ o_- I_

I

_tJr

- '2 7 K24K

Ii-K

7n

FIG. 3. Translation of hybridization-purified E4 mRNAs in a reticulocyte lysate system. Purified E4 mRNAswere translated in two nuclease-treated preparations of a reticulocyte lysate from a single animal (lanes a throughffrom the first preparation and lanes g through n from the second preparation). Polypeptides were labeled with[35S]methionine. Samples taken from the translation mixtures were precipitated with acetone and subjected toelectrophoresis in 10 to 20%o polyacrylamide gels, and the gels were prepared for fluorography as described in thetext. The lines between lanes f and g align the prominent E4 polypeptides in separate gel runs. Lane M, 14C-labeled molecular weight markers myosin (200K), phosphorylase b (92.5K), bovine serum albumin (68K),ovalbumin (43K), a-chymotrypsinogen (25.7K), 1B-lactalbumin (18.4K), and cytochrome c (12.3K); lane v,[IS]methionine-labeled adenovirus-2 virion proteins (17); lanes a and n, no RNA added; lane b, E4 mRNAisolated 5 h after infection in the presence of araC and purified with EcoRI fragment C. Cytoplasmic poly(A)-containing RNA was isolated 7 h after infection in the presence of cycloheximide; lane g contained totalcytoplasmic poly(A)-containing RNA. E4 RNA was purified by hybridization with the following fragments:EcoRI-C (lanes c, 1, and m); H7 (lane d); Hi (lane e); B274 (lanes f and h); BS (lane i); Dl (lane j); and D3 (lane k).

The relative amounts of the prominent poly-peptides synthesized from hybridization-puri-fied E4 mRNA templates were estimated fromdensitometer traces (Table 2). The polypeptidescould be divided into the following three abun-dance classes: (i) the 11K polypeptide; (ii) the21K and 19K polypeptides, which, when pre-sent, were approximately one-half as abundantas the l1K polypeptide; and (iii) the 24K, 22K,17K, and 15K polypeptides, the relativeamounts of which were variable. With the ex-ception of the H7-selected sample, one or moreof the third group of polypeptides were observedupon translation of all of the E4 mRNA prepara-tions. Common to these preparations were the3.0- and 2.5-kb mRNAs. Correlation of polypep-tide abundance in Table 2 with mRNA abun-dance in Table 1 allowed tentative assignment ofthe 11K polypeptide to the 2.1-kb mRNA, of the21K and 19K polypeptides to the 0.8-kb mRNAspecies, and of the 24K, 22K, 17K, and 15K

polypeptides to the 3.0-, 2.5-, and 1.8-kbmRNAs.Two additional polypeptides, 32K and 44K

(Fig. 3, arrows), were observed in all transla-tions of E4 RNA purified from cycloheximide-treated cultures. It is likely that the mRNAs thatencode these polypeptides share homology withother regions of the adenovirus genome in addi-tion to E4, as most of the mRNA was eluted inthe low-temperature (55°C) washes after hybrid-ization. The small amounts of 1.8-kb RNA ob-served in samples selected with the Dl and BSfragments (Fig. 2, lanes i and j) may haveconsisted of the species that eluted primarily at550C.In vitro translation of size-fractionated E4

mRNAs. The data from translation of mRNAsselected with cloned fragments were insufficientfor making unambiguous assignments of the E4polypeptides to their respective mRNAs. Todemonstrate directly that the 21K and 19K poly-

257K-e_

l8.4K-fl12 3K-_ ---S

VOL. 44, 1982

914 TIGGES AND RASKAS

TABLE 2. Relative amounts of E4 polypeptideslabeled with [35S]methioninea

Relative amt with the following selecting fragmentsb:Poly-pep-

EcoRI-C D3.IV! ^

B274tide Gradi- (Fig. 3, (Fig. 3, (Fig. 3, (itg. 3, (Fg 3,

ent lane b) lane c) lane 1) lane k) lane h)24K 0.19 0.15 0.33 0.56 0.28 0.6522K 0.11 0.01 0.14 0.23 0.06 0.1421K 0.69 0.01 0.58 0.74 0.01 0.0819K 0.46 0.01 0.34 0.65 0.06 0.0717K 0.17 0.06 0.18 0.24 0.03 0.0715K 0.08 0.01 0.19 0.18 0.03 0.0111K 1.00 1.00 1.00 1.00 1.00 1.00

a E4 RNAs purified with different DNA fragmentswere translated in the reticulocyte lysate system andlabeled with [35S]methionine. Estimates of polypep-tide abundance were made from densitometer traces ofthe films shown in Fig. 3 or shorter exposures. Whenthe band densities exceeded the linear response of thefilm, the peak maxima were extrapolated. In eachinstance, weights were normalized to the 11K poly-peptide.

b E4 RNAs were purified by hybridization selectionwith filter-bound plasmid DNA containing the viralfragments shown in Fig. 1. The Fig. 3 lanes refer to thelanes from which densitometer traces were made.Estimates were made from the films shown in Fig. 5,and the weights (gradient) of each polypeptide bandwere summed across the gradient.

peptides were encoded by the 0.8-kb mRNA, toresolve the products of the 3.0-, 2.5-, and 1.8-kbmRNAs, and to identify the products of the 1.5-and 1.2-kb mRNAs, E4 mRNAs were fractionat-ed by size. Cytoplasmic poly(A)-containingRNA was isolated from cells infected in thepresence of cycloheximide. E4 mRNA was puri-fied by hybridization with EcoRI fragment C(mp 89.7 to 100) and then fractionated by sedi-mentation in 5 to 20% sucrose-98% formamidegradients. Samples from gradient fractions con-taining the larger mRNAs (fractions 11 and 17through 24) were subjected to electrophoresis ina 1.2% agarose gel, and samples from fractionscontaining the smaller mRNAs (fractions 25through 36) were subjected to electrophoresis ina higher-resolution 4% polyacrylamide gel. TheRNA was transferred to DBM paper and hybrid-ized with 32P-labeled EcoRI fragment C-contain-ing plasmid DNA (Fig. 4). Samples from thesame fractions were translated in the reticulo-cyte lysate system, and the polypeptides wereresolved in polyacrylamide gels (Fig. 5). Therelative amounts of the RNAs and polypeptidesin each fraction were determined and are plottedin Fig. 6.These results confirm the assignment of the

11K polypeptide as the product of the 2.1-kb E4mRNA. The 1.5-kb RNA also appeared to en-code an 11K polypeptide; the secondary peak inthe abundance profile of the 11K polypeptide

(fraction 24) coincided with the enrichment forthe 1.5-kb mRNA in that fraction. Fractions 17through 23 contained the 2.1- and 1.8-kbmRNAs and trace amounts of the 3.0- and 2.5-kbmRNAs, which, when translated, yielded 24K,22K, 19K, 17K, and 15K polypeptides in additionto the 11K polypeptide encoded by the 2.1-kbmRNA. Assignment of these five polypeptidesto individual mRNAs could not be made fromthese data.The 0.8-kb mRNA size class was truly unusu-

al, as 10 polypeptides were resolved in the threefractions (fractions 27 through 29) enriched withmRNA of this size. In addition to the 21Kpolypeptide inferred from the hybridization se-lection results, a prominent 19K polypeptide andless abundant 21.8K, 18.5K, 18K, 17K, 16K,15K, 13K, and 10K polypeptides were ob-served.

Translation of E4 mRNAs in wheat germ ly-sates. The multiple polypeptides translated frommRNAs enriched with the 0.8-kb mRNA sizeclass (Fig. 6) might have arisen from partiallydegraded mRNAs or from proteolytic activitiesin the lysate. Because hybridization-selected E4mRNAs were bound to oligodeoxythymidylicacid cellulose before translation, the only likelysource of mRNA degradation would have beenresidual micrococcal nuclease activity from thenuclease treatment. To test this possibility, E4mRNAs were translated in the wheat germ ly-sate system. Moreover, if proteolysis were oc-curring in the reticulocyte lysate, it was unlikelythat the same activity would be present in thewheat germ. Alternatively, the multiple poly-peptides might have arisen as a result of multiplesplice acceptor and donor sites at the ends of thesecond (mp 92.6 to 94.4) intron in the 0.8-kbmRNA size class (see Fig. 9). E4 RNA waspurified from a cycloheximide-treated culture byhybridization selection with plasmids containingEcoRI fragment C (mp 89.7 to 100) and Hi (mp93.4 to 96.3), which selected the full complementof mRNAs, and with B274 (mp 94.8 to 95.6),which did not hybridize with the 0.8-kb mRNAs.In addition, E4 RNA was purified from cytoplas-mic RNA isolated from cells infected in thepresence of araC. The RNAs were translated ina wheat germ lysate system by using [35S]me-thionine. The polypeptides synthesized wereresolved in a polyacrylamide gel (Fig. 7A). The21.8K, 21K, and 19K polypeptides encoded bythe 0.8-kb mRNAs in reticulocyte lysates werereadily observed in the wheat germ translations,although the high background, which was stimu-lated by the addition of small amounts of select-ed RNA, precluded unambiguous identificationof the less abundant polypeptides. The samplesof E4 RNA selected with H7 and with EcoRIfragment C from the araC-treated culture, which

J. VIROL.

E4 POLYPEPTIDES 915

U T 1I 1 7 18 189 t 21 2 23 24 2 5 26 27 28 29 30 31 32 34 35 30

_'

S-

:.Y

_1.5-1.2

o.B.

FIG. 4. Fractionation of E4 mRNA in sucrose gradients. Cytoplasmic poly(A)-containing RNA was isolated 7h after infection in the presence of cycloheximide. E4 mRNA was purified by hybridization to EcoRI fragment C.The RNA was fractionated by sedimentation in a 10 to 20%o sucrose-98% formamide gradient, and samples fromthe fractions were subjected to electrophoresis in a 1.2% agarose gel (lanes M and T and fractions 11 through 24)or a 4% polyacrylamide gel (fractions 25 through 36). The RNAs were transferred to DBM paper and hybridizedwith 32P-labeled EcoRI fragment C. Lane M, Adenovirus-2 DNA cut with SmaI; lane T, unfractionatedcytoplasmic poly(A)-containing RNA. The sizes of the E4 mRNAs (in kilobases) are indicated. The numbers atthe top are fraction numbers.

contained 5- to 10-fold less E4 RNA, served asadditional controls for endogenous mRNAs. Un-selected cytoplasmic RNA was included to as-sure that larger mRNAs were translated effi-ciently. Although larger mRNAs were translatedefficiently in the wheat germ lysate, the mRNAsthat encoded the 24K and 22K polypeptideswere not (see below).E4 mRNAs were also translated in a nuclease-

treated wheat germ lysate in order to reduce thecontribution of endogenous mRNAs to the back-ground in the untreated lysate. In addition, thepolypeptides translated from the E4 mRNAswere labeled with [5,6_3H]leucine to eliminatenonspecific labeling of proteins with [35S]me-thionine (R. Beachy, personal communication).E4 mRNAs were translated in parallel reactionmixtures in the reticulocyte lysate system con-taining [5,6-3H]leucine and [35S]methionine. Inthis experiment, the full complement of E4mRNAs was selected with EcoRI fragment C,and E4 mRNAs lacking the 0.8-kb mRNAs werepurified by selection with the D3 plasmid (mp92.5 to 93.9). E4 mRNAs purified from an araC-

treated culture served as an additional back-ground control, and unselected RNA from cy-cloheximide-treated infected cells providedassurance that larger polypeptides were beingsynthesized. As shown in Fig. 7B, the back-ground was sufficiently reduced to allow detec-tion of the 21.8K, 21K, 19K, 18K, and 17Kpolypeptides encoded by the 0.8-kb mRNAs.These results suggest that neither mRNA degra-dation nor proteolytic activity in the reticulocytelysates plays a major role in the production ofthe multiple polypeptides specified by the 0.8-kbmRNAs.The use of the [3H]leucine label had a fortu-

itous additional result; a 15K polypeptide thatwas more intensely labeled with [5,6-3H]leucinethan with [35S]methionine was observed (Figure7B, lanes c, d, f, and g). This polypeptide wasequally abundant in the sample containing all ofthe E4 mRNAs (Fig. 7B, lane f) and the samplethat did not contain the 0.8-kb mRNAs (Fig. 7B,lane g). These results suggest that the 15Kpolypeptide was encoded by the 3.0-, 2.5-, or1.8-kb mRNA. To test this possibility, E4

uk kb

L--

?i P

pt.5-

iyf

VOL. 44, 1982

916 TIGGES AND RASKAS

m :. 9 ~ k.? -L

...:. 11.

"!el

.K _ S

.'*;AK. ._

FIG. 5. Translation of size-fractionated E4 mRNA in a reticulocyte lysate system using [35S]methioninelabeling. E4 mRNAs from the gradient fractions shown in Fig. 4 were translated and analyzed as described in thelegend to Fig. 3. Lane M, 14C-labeled molecular weight markers; lane T, unfractionated E4 mRNA. The numbersat the top are fraction numbers. The arrows in fraction 28 indicate the 21.8K and 10K polypeptides. The arrowsin lane T indicate the polypeptides encoded by the mRNAs eluted at 55°C (see text).

mRNAs were selected with the EcoRI fragmentC-containing plasmid and fractionated by size.Samples from gradient fractions containing thelarger E4 mRNAs and a sample enriched withthe 0.8-kb mRNAs were assayed in blots andtranslated in reticulocyte lysates by using [5,6-3H]leucine (Fig. 8). Because the fractions en-riched with the larger mRNAs (Fig. 8, fractions17 through 19) contained the highly labeled 15Kpolypeptide, whereas in the fraction enrichedwith the 0.8-kb mRNAs the 15K polypeptidewas less prominent than the 17K polypeptide(Fig. 5 and 8, fraction 27), it is likely that twodifferent 15K polypeptides are specified by E4mRNAs. Moreover, the greatest relative amountof the 15K polypeptide occurred in the fractionsenriched with the 1.8-kb mRNA, whereas the24K, 22K, 19K, and 17K polypeptides weredetected in the fractions containing the 3.0-,2.5-, and 2.1-kb mRNAs, although these poly-peptides are not visible in the photographs.

DISCUSSION

We used in vitro translation of E4 mRNAsseparated according to sequence and size toassign the polypeptides synthesized to their re-spective mRNAs. In a separate communication,we will report the precise locations of splicejunctions in E4 mRNAs (Tigges and Raskas,submitted for publication). Combined with thepreviously reported DNA sequence of E4 (19),these results allowed the construction of a de-tailed map of the translated regions of E4mRNAs (Fig. 9). The expected amino acid com-positions of polypeptides encoded by four opencoding regions not interrupted with splice junc-tions are listed in Table 3. The polypeptideassignments most consistent with these data areas follows.

3.0- and 2.5-kb RNAs. The 3.0- and 2.5-kbminor species were present among the mRNAsselected by the D3, B274, Dl, and BS fragments.

J. VIROL.

11..:% I, K

,; A

iM

IRW

E4 POLYPEPTIDES 917

When the mRNAs were translated in reticulo-cyte lysates, 24K, 22K, 19K, and 17K polypep-tides were produced in addition to the 1lK and15K polypeptides encoded by the 2.1- and 1.8-kb mRNAs, respectively (Fig. 3). The 24K, 22K,19K and 17K polypeptides were encoded bymRNAs with sizes ranging from 3.0 to 1.5 kbafter fractionation by size (Fig. 5 and 8). As the3.0 kb mRNA appears to be unspliced, transla-tion is likely to begin with the first AUG codonin open coding region 1 (Fig. 9), yielding ahydrophobic product that is rich in valine andphenylalanine (Table 3). The structure of the2.5-kb mRNA suggests that open coding region 2

V)z

UJ

Il-zLUI

LUA

FRACTIONFIG. 6. Relative amounts of E4 mRNAs and poly-

peptides plotted versus gradient fraction. The relativeamounts of the E4 mRNAs shown in Fig. 4 and thepolypeptides shown in Fig. 5 were estimated fromdensitometer traces of the films. Xerox copies weremade of the traces, and the peaks were cut out andweighed. (A) Major polypeptides. Symbols: 0, IIK;0, 24K; A, 22K; 0, 21K; *, 19K. (B) Minor polypep-tides. Symbols: 0, 15K;0, 17K; 0, 13K; , 16K; A,18K; A, 18.5K; *, 21.8K; V, 10K; 0, 19K. Note thechange in scale compared with (A). (C) E4 mRNAs.Symbols: 0,2.1 kb;0, 1.8 kb; 0,1.5kb; U, 1.2 kb; A,0.8 kb.

A a b c d e f g h68K -

43K-

2 5 7 K - -

184K-

12.3K-

B a b c

- 21 8K- 21K- 19K

- IlK

d e f g

_*. - 24K

- 21K-19K

1 8K

__ Z i_ - ~~15K

- -I8iFIG. 7. Translation of E4 mRNAs in wheat germ

lysates and comparison of [35S]methionine labelingwith [3H]leucine labeling. Purified E4 mRNAs weretranslated in wheat germ, reticulocyte, and nuclease-treated wheat germ lysate systems and analyzed asdescribed in the legend to Fig. 3. (A) E4 mRNAstranslated in a wheat germ lysate system using[35S]methionine labeling. Lane a, 14C-labeled molecu-lar weight markers; lane b, unfractionated cytoplasmicpoly(A)-containing RNA from cells infected in thepresence of cycloheximide; lane c, no RNA added;lane d, E4 mRNA purified with EcoRI fragment Cfrom cells infected in the presence of araC; lanes ethrough h, E4 mRNA purified from cells infected inthe presence of cycloheximide and hybridized to frag-ments EcoRI-C, H7, HI, and B274, respectively. (B)E4 mRNAs were translated in reticulocyte lysates(lanes a through g) or nuclease-treated wheat germlysates. Polypeptides were labeled with [35S]methio-nine (lanes a through d) or [5,6_3H]leucine (lanes ethrough k). Lane a, No RNA added; lane b, E4mRNAs purified from araC-treated cultures by hybrid-ization with EcoRI fragment C; lanes c through k, E4mRNAs purified from cycloheximide-treated culturesby hybridization with EcoRI fragment C (lanes c, f,and j), D3 (lanes d, g, and k), no RNA (lanes e and h),and cytoplasmic RNA (lane i).

is translated from this species (Fig. 9). Bothopen coding regions encode polypeptides withrelatively high proline contents (Table 3), whichmay account for the lower mobilities of thesepolypeptides in the acrylamide gels.Although the 3.0- and 2.5-kb mRNAs and the

additional minor 2.3- and 1.6-kb species (10)which apparently have 5' ends identical to the 5'ends of the 3.0-kb mRNA (Fig. 9) would beexpected to encode two polypeptides, four poly-

VOL. 44, 1982

918 TIGGES AND RASKAS

,

s

FIG. 8. Fractionation of E4 mRNAtranslation of size classes in reticulocyt[3H]leucine labeling. (A) E4 mRNAs Fbridization with EcoRI fragment C wei

by size. Samples were subjected to eand transferred to a nitrocellulose filtcwere detected by hybridization with a iEcoRI fragment C probe. Lane T,RNA. The numbers at the top are frac(B) E4 mRNAs from the fractions shovtranslated in a reticulocyte lysate[3H]leucine labeling and subjected to elLane N, No RNA added; lane C, unfimRNA; lane T, total cytoplasmic polyRNA.

peptides were observed in reticulctions of sucrose gradient fractionsthem (Fig. 8). When samples conspecies were translated in wheat jthe 24K and 22K polypeptides wewere produced in reduced amountaddition, in one reticulocyte lysat4the 22K polypeptide was lackingscured by endogenous methionine-]rial (Fig. 3, lanes c and 1). These rethat the 24K and 22K polypeptidresulted from post-translational mthe 19K and 17K polypeptides, anwas present in the reticulocyte lystranslation of selected E4 mRNAsh through m) and size-fractionated5 and 8). Alternatively, the stru(minor E4 mRNA species might bplex than presently known. It is olin the DNA sequence open coding2 are in the same reading frame a

rupted by a single UGA codon follcately by an AUG codon that begin

region 2 (19). Thus, a microsplice that removesthe stop codon while preserving the readingframe would allow translation of both regions. Itis also conceivable that in some reticulocytelysate preparations there were conditions thatallowed read-through of the stop codon.

2.1- and 1.5-kb mRNAs. The 2.1-kb mRNA isthe most abundant species, and an 1lK polypep-tide is the most prominent product of purified E4mRNAs. The 2.1- and 1.5-kb mRNAs were theprominent species selected with the Dl and BSfragments (Fig. 1 and 2, lanes i and j). Transla-tion of E4 mRNAs selected with these fragmentsresulted in most of the label being incorporated

,;; in the 11K polypeptide. As shown in Fig. 6, the* : ':profile of 11K polypeptide abundance is bimod-

al, with the peaks centered in those fractions3;W _ containing the greatest amounts of 2.1-kb

mRNA (Fig. 5, fraction 20) or 1.5-kb mRNA(Fig. 5, fraction 24). Structural studies havedemonstrated that the 5' ends of these mRNAs

Ls by size and consist of the leader spliced to the same accep-e lysates using tor site; the 1.5-kb mRNA is produced by dele-3urified by hy- tion of the second intron (10). Open codingre fractionated region 3 (Fig. 9) is located adjacent to the leaderalectrophoresis acceptor site and encodes a polypeptide of 116er. The RNAs amino acids enriched in acidic residues suchnick-translated that, at neutral pH, a net charge of -10.6 isunfractionated expected (Table 3). The 11K in vitro producttion numbers. probably corresponds to an acidic 11K polypep-vn in (A) were tide observed in two-dimensional gels containingectrophoresis. polypeptides labeled in vivo (18, 26).ractionated Ei 1.8- and 1.2-kb mRNAs. The 1.8- and 1.2-kb(A)-containing mRNAs are structural analogs of the 2.1- and

1.5-kb pair (Fig. 9). In the DNA sequence, openreading region 4 contains a single AUG codon atthe 5' end and five leucine codons (19). Compar-

)cyte transla- ison translations in which [35S]methionine orenriched for [3H]leucine was used demonstrated that a 15Kitaining these polypeptide is labeled more intensely withgerm lysates, [3H]leucine than with [35S]methionine (Fig. 7).re lacking or Translations of E4 mRNAs fractionated by sizets (Fig. 7). In in which [3H]leucine was used as the labele preparation demonstrated that the 1.8-kb mRNA encodesor was ob- the 15K polypeptide (Fig. 8). Because gradient

labeled mate- fractions containing 1.2-kb mRNA also con-asults suggest tained 0.8-kb mRNA (Fig. 4), which encodes aes may have 15K polypeptide as well, the existence of a 15Kodification of polypeptide encoded by the 1.2-kb mRNA wasactivity that not confirmed directly. However, the 1.2-kbates used for mRNA appears in polysomes in amounts com-(Fig. 3, lanes parable to the cytoplasmic concentration (Fig. 2,mRNAs (Fig. lanes j and k), suggesting that the 1.2-kb mRNActures of the also encodes the relatively leucine-rich 15Koe more com- polypeptide.f interest that 0.8-kb mRNA. Of the abundant E4 RNAs,regions 1 and production of the 0.8-kb species is affected mostnd are inter- profoundly by inhibition of protein synthesis)wed immedi- (Table 1); 20-fold more accumulates at 7 h in theopen coding presence of cycloheximide than at 5 h in the

-- ----

'W-I - -a.-

'AW U-Nw

J. VIROL.

_.AMI

E4 POLYPEPTIDES 919

kb3.02.31.6

13.9K24, 22K .419, 1 7K

- 25

14.6K

-21- 1.511K -

15K

13.2 K

-1.8-'2

13.4 K

21, 19K --

21.8, 18.5KK18, 17K -16, 15K13, 10 K

-0 8

4 31

Ni IN *IEN Ul llA I-

II Iz.. Jj......

7 6 5 2 1I IIIfil 'III II NI 115I I

5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.51

90 91 92 93 94 95 96 97 98 99 100FIG. 9. Correlation of E4 polypeptides synthesized in vitro with individual E4 mRNA species. E4 mRNAs

are grouped according to the open coding regions utilized during translation. The sizes of the mRNAs areindicated at the 5' ends of the mRNAs, and the apparent molecular weights of the translation products areindicated at the 3' ends. Diagramed below each mRNA group are the open coding region and the molecularweight of the polypeptides predicted from the DNA sequence. Because the open reading frame of the 0.8-kbmRNA is split by deletion of the intron, the extents of open coding sequences in all three reading frames areshown at the 3' side of the intron. Diagramed below the mRNAs is pertinent information from the two readingframes in the adenovirus-2 genome which contain open reading regions for E4 (19). The third reading frame isclosed over the length of this region. The marks above the lines indicate AUG codons, and the marks below thelines indicate stop codons. The open coding regions (heavy lines) are numbered from 5' to 3' in order ofoccurrence.

absence of drugs. Translation of 0.8-kb mRNAresults in 10 polypeptides (Fig. 5, fractions 26through 30, and Fig. 8, fraction 27). The transla-tion products of the 0.8-kb mRNAs migrate inpolyacrylamide gels with larger apparent molec-ular weights than predicted from the structuresof the mRNAs and the DNA sequence (Fig. 9),possibly because of the large number of prolinecodons in open coding region 5 (19). Polypep-tides containing 9 to 13% proline and 9 to 15%arginine are expected, which may account foranomalous migration in polyacrylamide gels.To determine whether mRNA degradation or

post-translational modification played a majorrole in the appearance of multiple polypeptides,we translated hybridization-purified E4 mRNAsin wheat germ lysates (Fig. 7). Although thelower translational efficiency of wheat germlysates precluded detection of all of the polypep-tides synthesized in reticulocyte lysates, the21.8K, 21K, 19K, 18K, and 17K polypeptideswere observed. Furthermore, polypeptideamounts were not distributed symmetricallyabout the peak of 0.8-kb mRNAs in the sucrose

gradient (Fig. 6). The 21K and 19K polypeptideswere most abundant in fraction 27, whereas the18.5K polypeptide was most abundant in frac-tion 28. These data suggest that the 0.8-kbspecies is actually composed of a number ofRNAs that differ by virtue of heterogeneoussplicing to delete the second intron. The DNAsequence in this case is characterized by a largeopen coding region spanning the intron (Fig. 9,region 5). Thus, if more than one donor splicesite is utilized during processing, mRNAs con-taining different amounts of the coding regionwould be produced. Depending on the numberand positions of the acceptor sites, translationcould continue in phase or be shifted into theother two reading frames (Fig. 9). We haveexplored the possibility of heterogeneous splic-ing and have demonstrated that indeed the 0.8-kb species consists of multiple differentlyspliced mRNAs (Tigges and Raskas, submittedfor publication).

Effect of cycloheximide on the accumulation ofE4 RNAs. Previous studies have demonstratedthat inhibition of protein synthesis releases the

VOL . 44, 1982

920 TIGGES AND RASKAS

_ 1, in IC-E4 promoter from regulatory constraints anden -io

,~eo>stabilizes the transcripts in the cytoplasm (14,- - 30, 31, 40, 41). We examined the effect of

X<00 o Wc tn inhibition of protein synthesis by cycloheximide- -- > ^on the steady-state levels of cytoplasmic E4

RNAs in KB cells by using Northern blot analy-sis. The accumulation of E4 RNAs at 7 h after

--'> * >infection in the presence of cycloheximide was50-fold greater than at 5 h in the absence of the

r3aso-f- o drug. In addition, however, inhibition of proteino F ^ ^ >synthesis had a profound effect on splicing of E4

e. F ofi - RNAs; more extensive and more frequent spliceo el deletions occurred. The result was a 20-foldincrease in the relative abundance of the 0.8-kbsize class at 7 h after infection in the presence of

- ,, cycloheximide compared with the amount pre-vdCO > > ~ ; sent at 5 h after infection in the absence of drugs

(Fig. 2 and Table 1).X~ luon > SoImplication of E4 structure. Six open reading

regions are found in the leftward strand of._x>^ > e adenovirus-2 DNA in E4 (19). Our in vitro3.X0x*~xa)translation results demonstrate that nearly all of

z 0 OO0 these sequences occur as translated regions inE4 mRNAs. These observations partially ex-

.*0°°fsi aplain the complexity of E4 mRNAs; complexX ^- o o S gprocessing is necessary to position the five open

D0 Xmreading regions for translation. It is likely thata,3en Dsi i; E4 specifies at least five different functions.

-- , 0 Moreover, the multiple polypeptides synthe-0Q > * - > , = sized from the 0.8-kb mRNAs probably share

common amino-terminal but different carboxy-Q ,.7 x > , > terminal amino acid sequences, suggesting that-Q: z these polypeptides have a common function but

4o.6 > o O different specificities. We have observed that, inareo o> el; the absence of inhibitors, one to three different

.o 46 a ~ OO > E =members of the 0.8-kb mRNA size class appearin the cytoplasm at any one time during infec-tion; as many as seven different species have

o Z < x o < . wc been observed (Tigges and Raskas, submitted0r _ e ! for publication). The family of polypeptides en-

60 W)OOtn

x41

coded by the 0.8-kb mRNAs may serve a regula-oZ-o _ bi ^ tory function with changing specificities duringiv_tON3D the lytic cycle.,,<̂*.-^_xmACKNOWLEDGMENTS

[X1X _2 * W 20 This work was supported by research grant MV-31G fromm

' '<" _̂~̂ ' U the American Cancer Society and by Public Health Service<<,,. grant CA-16007 from the National Cancer Institute. CellEe o=- culture media were prepared in a Cancer Center facility

,so1.o .0en funded by Public Health Service grant CA-16217 from the- > >3National Cancer Institute. This study was also supported by

cdoo CY,'IO'* i Brown & Williamson Tobacco Corp., Philip Morris Inc., R. J.r..: civo w o |Reynolds Tobacco Co., and United States Tobacco Co.

0 a:3LITERATURE CITEDZ~ W) ..,Do I A 1. Aviv, H., and P. Leder. 1972. Purification of biologically0 < vactive globin mRNA by chromatography on oligothymidy-

lic acid-cellulose. Proc. Natl. Acad. Sci. U.S.A. 69:1408-1412.-

en V' 2. Baker, C. C., and E. B. Ziff. 1981. Promoters and hetero-c)

Igeneous 5' termini of the mRNAs of adenovirus serotype2. J. Mol. Biol. 149:189-221.

3. Berk, A. J., F. Lee, T. Harrison, J. Wliams, and P. A.Sharp. 1979. Pre-early adenovirus 5 gene product regu-

J. VIROL.

E4 POLYPEPTIDES 921

lates synthesis of early messenger RNAs. Cell 17:935-944.4. Berk, A. J., and P. A. Sharp. 1977. Ultraviolet mapping of

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